CICIMAR Oceánides Vol. 30 (1) 2015 by CICIMAR Oceánides

ISSN 1870-0713

Volumen 30(1)

Junio 2015

DIRECTORIO INSTITUTO POLITÉCNICO NACIONAL ENRIQUE FERNÁNDEZ FASSNACHT Director General MIGUEL ÁNGEL ÁLVAREZ GÓMEZ Secretario Académico JOSÉ GUADALUPE TRUJILLO FERRARA Secretaria de Investigación y Posgrado

CENTRO INTERDISCIPLINARIO DE CIENCIAS MARINAS MARÍA MARGARITA CASAS VALDEZ Directora SERGIO AGUÍÑIGA GARCÍA Subdirector Académico y de Investigación FELIPE NERI MELO BARRERA Subdirector de Servicios Educativos e Integración social LUZ DE LA PAZ PINALES SORIA Subdirectora Administrativa

CONSEJO EDITORIAL DAVID A. SIQUEIROS BELTRONES (Editor)

ELISA SERVIERE ZARAGOZA

CICIMAR-IPN MÉXICO

VOLKER KOCH DGIZ/BIOMAR - MÉXICO RAFAEL ROBAINA

CIBNOR MÉXICO

TANIA ZENTENO SAVÍN CIBNOR MÉXICO

FRANCISCO ARREGUÍN SÁNCHEZ

U. DE LAS PALMAS DE GRAN CANARIA ESPAÑA

CICIMAR-IPN MÉXICO

CHRISTINE JOHANNA BAND SCHMIDT

MARK S. PETERSON

CICIMAR-IPN MÉXICO

ESTADOS UNIDOS U. SOUTHERN MISSISSIPPI

ERNESTO A. CHÁVEZ ORTIZ CICIMAR-IPN MÉXICO

RUBÉN ESCRIBANO V.

JOSÉ DE LA CRUZ AGÜERO

U. CONCEPCIÓN DE CHILE

CICIMAR-IPN MÉXICO

SANTIAGO FRAGA

INSTITUTO ESPAÑOL DE OCEANOGRAFÍA ESPAÑA

FERNANDO GÓMEZ

MARIE SYLVIE DUMAS LEPAGE CICIMAR-IPN MÉXICO

MARÍA CHANTAL DIANE GENDRON LANIEL CICIMAR-IPN MÉXICO

UNIVERSIDAD DE VALENCIA- ESPAÑA

PABLO MUNIZ MACIEL

SERGIO GUZMÁN DEL PRÓO

ALAN GIRALDO LÓPEZ

VÍCTOR M. GÓMEZ MUÑOZ

DOMENICO VOLTOLINA

JAIME GÓMEZ GUTIÉRREZ

CICIMAR-IPN MÉXICO

U. DE LA REPÚBLICA DE URUGUAY

CICIMAR-IPN MÉXICO

UNIVERSIDAD DEL VALLE - COLOMBIA CIBNOR MÉXICO

CICIMAR-IPN MÉXICO

BERTHA LAVANIEGOS ESPEJO

JUAN GABRIEL DÍAZ URIBE

CICESE MÉXICO

INAPESCA MÉXICO

HELMUT MASKE

CARLOS MÁRQUEZ BECERRA UABC MÉXICO OSCAR UBISHA HERNÁNDEZ ALMEIDA

CICESE MÉXICO

ARMANDO TRASVIÑA CASTRO CICESE MÉXICO

UNIVERSIDAD AUTÓNOMA DE NAYARIT MÉXICO

AXAYÁCATL ROCHA OLIVARES CICESE MÉXICO

PRODUCCIÓN

RUBÉN E. GARCÍA GÓMEZ. Editor técnico MIREYA G. LUCERO ROMERO Asistente editorial

CICIMAR Oceánides Editor Científico: David A. Siqueiros Beltrones

N° Certificado Reserva de Derechos al Uso Exclusivo del Título: 04-2013-021913491400-102. N° Certificado de Licitud del Título: 12987. N° Certificado de Licitud de Contenido: 10560. ISSN: 1870-0713 Distribuida por: CICIMAR-IPN, Ave. IPN s/n, Col. Playa Palo de Sta. Rita, 23096 La Paz, B.C.S., Tels: (612)123-03-50, (612)123-46-58. Fax: (612)122- 5322.

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In memoriam

M. en C. Ignacio Sánchez Rodríguez (1958-2015)

El Maestro en Ciencias Ignacio Sánchez Rodríguez, cursó sus estudios de Nivel Medio Superior en la escuela Vocacional 6 del IPN, Miguel Othón de Mendizábal, en la especialidad de Técnico Laboratorista Clínico. Sus estudios superiores los cursó en la carrera de Biólogo Marino, en El Centro Interdisciplinario de Ciencias Marinas (1977-1982) dependiente del IPN. Fue contratado como Profesor-Investigador en el CICIMAR en 1983, donde trabajo toda su vida. En su labor docente del CICIMAR colaboró en los cursos de Ecología del Bentos, Temas Selecto de Algas Marinas y Sistemas de Producción en Acuacultura. Pertenecía a la Sociedad de Egresados del IPN, Profesionistas del IPN, Sociedad Botánica de México, Sociedad Ficológica de México, ahora Sociedad Mexicana de Ficología y la Sociedad Ficológica de América Latina y del Caribe. Durante su carrera, participó en 40 proyectos de Investigación en diversos temas relacionados con las algas marinas, como evaluación de poblaciones (Sargassum sinicola y Macrocystis pyrifera), elenco sistemático de algunas bahías del Golfo de California y la zona Pacífico. Colaboró en el grupo de química de algas. También incursionó en estudios de biogeoquímica de elementos traza y algas en fuentes hidrotermales. Desarrolló técnicas de cultivo para las algas verdes Enteromorpha y Ulva. Participó en investigaciones sobre el uso de algas como alimento de camarón, para ganado caprino y ovino, entre otros. Participó activamente en la difusión de la ciencia impartiendo 11 seminarios en CICIMAR, 10 programas de radio y televisión, en empresas como radio Universidad (UABCS), Radio fórmula, Canal 10 y canal 8 de La Paz, BCS, así como Televisa y TV Azteca. Ignacio fue un apasionado del Instituto Politécnico Nacional; llevaba puestos los colores guinda y blanco, no solo en sus ropas, sino en el alma. Siempre lo recordaremos como el líder para dirigir en público la porra del IPN, añadiendo el nombre de nuestro Centro al final: Huelum, Huelum, gloria, a la cachi, cachi porra, a la cachi, cachi porra, pin pon porra, Politécnico, CICIMAR, gloria!!! GHC JULIO 29 DE 2015

SEMBLANZA Dr. Jaime Gómez Gutiérrez quien visitaba La Paz como miembro de un comité de tesis de maestría. El Dr. Brinton fue un reconocido experto de la taxonomía, biología y ecología de krill a nivel mundial, quien se convertiría en su amigo y mentor por muchos años hasta su fallecimiento (1924-2010). Jaime Gómez también tuvo la fortuna de tener la amistad y asesoría de Margaret Knight (19252014) y Annie Townsend (SIO) expertas en taxonomía de krill. El Dr. Jaime Gómez Gutiérrez nació en la Ciudad de México el 15 de Diciembre de 1964. A los 17 años, después de una agitada adolescencia y sin conocimiento de su madre, decidió emigrar a estudiar la preparatoria técnica con especialidad en Acuacultura (Centro de Estudios Tecnológicos del Mar, 1982-1985) en la Ciudad de La Paz, Baja California Sur. Inicialmente sin apoyo familiar tuvo siempre que estudiar y trabajar simultáneamente. En su tercer año de preparatoria tuvo la oportunidad de llevar un curso de identificación de larvas de peces, que sin saberlo, desviaría su curiosidad al estudio del plancton desde entonces hasta la actualidad. Con este rápido adiestramiento fue contratado como técnico separador de huevos y larvas de peces en el CICIMAR-IPN continuando durante los próximos 6 años con el apoyo de numerosos investigadores del Departamento de Plancton y Ecología Marina. Durante 19851990 estudió la licenciatura en Biología Marina en la Universidad Autónoma de Baja California Sur donde se graduó con la tesis de distribución y abundancia de larvas de la langostilla Pleuroncodes planipes en la costa occidental de la península de Baja California (Director Carlos Sánchez Ortiz, UABCS). Durante la maestría en Manejo de Recursos Marinos del CICIMAR (1990-1992, Director Dr. Sergio Hernández Trujillo) enfocó su atención al estudio de la biología y ecología del krill (eufáusidos), donde tuvo la oportunidad de conocer al Dr. Edward Brinton (en ese entonces profesor recién retirado de Scripps Institution Oceanography)

En 1992 fue galardonado con la Presea Lázaro Cárdenas del IPN entregada por el entonces Presidente de la Republica Lic. Carlos Salinas de Gortari que tuvo como consecuencia su posterior contratación en CICIMAR como profesor-investigador desde 1993 a la fecha. Miembro del Sistema Nacional de Investigadores desde 1993, actualmente es nivel II desde el 2003. Durante esta época la planctología en México, casi sin excepción, se avocaba a estudiar los patrones de distribución y abundancia estimada con redes Bongo, desde Diciembre 1993, en colaboración con el Dr. Virgilio Arenas Fuentes y el Dr. Carlos Robinson Mendoza, del Instituto de Ciencias del Mar y Limnología de la Universidad Nacional Autónoma de México, iniciaron investigaciones pioneras en el uso de técnicas de hidroacústica para inferir patrones de la distribución vertical y comportamiento gregario de peces pelágicos pequeños y zooplancton en México a bordo del B/O El Puma en cruceros en ambas costas de la Península de Baja California. En 1994 hizo una estancia en el Virginia Institute of Marine Science (Virginia, EUA) con el Dr. Jeffrey Shields para describir un nuevo género y especie de parásito de krill (isópodo Dajidae Oculophyxus bicauslis), lo que inicio su pasión por los parásitos convirtiéndose en su pasatiempo científico desde entonces. Durante un simposio internacional en hidroacústica aplicada a pesquerías y plancton (Aberdeen, Reino Unido, Junio 1995) conoció a quien posteriormente seria su director de tesis doctoral, el Dr. William T. Peterson (Bill). En 1997 Jaime decidió realizar una estancia

de investigación de dos meses en Newport, Oregon EUA con Bill Peterson, para aprender a realizar estimaciones de producción secundaria de zooplancton. A su regreso realizó en colaboración con investigadores del CICIMAR las primeras estimaciones de producción secundaria de zooplancton (copépodos) en México (en Bahía Magdalena, BCS), lo que daría inicio a la línea de investigación de estimar tasas vitales mediante incubaciones de zooplancton marino en condiciones de laboratorio; estas permiten inferir qué tan productivos son realmente los ecosistemas marinos mexicanos, en su mayoría exagerados dada la previa falta de estimaciones precisas. Esta línea de investigación motivó su pasión por la microfotografía tomando fotos de zooplancton vivo a bordo de buques oceanográficos. En verano 1998 él y su esposa se mudaron a Corvallis, Oregón, EUA donde en Diciembre del 2003 obtuvo el doctorado en el College of Earth, Ocean, and Atmospheric Sciences de Oregon State University (CEOAS-OSU) bajo la tutoría del Dr. William T. Peterson y el Dr. Charles B. Miller (ambos expertos en biología y ecología de krill y copépodos). Jaime llegó a CEOAS en el momento adecuado cuando OSU y NOAA obtuvieron un transcendental apoyo económico de investigación por el programa internacional Global Ocean Ecosystem Dynamics (GLOBEC) para comprender la función y modelación numérica del plancton en los ecosistemas marinos del mundo. Él enfocó su investigación doctoral a comprender aspectos básicos de la biología y ecología del krill en la costa de Washington, Oregon y California, EUA. Durante su investigación doctoral descubrió un evento de mortalidad masiva de krill (epizootia) a 300-600 m de profundidad en el cañon submarino de Astoria ocasionada por ciliados parasitoides de krill (parásitos que deben matar a su hospedero para completar su ciclo de vida); esto lo publicó en la prestigiosa revista Science y posteriormente describió esta nueva especie (Pseudocollinia oregonensis). En su tesis puso en contexto biológico y adaptativo el proceso de eclosión de especies de krill con desove interno y externo; descubrió y describió taxonómicamente los primeros ciliados exuviotróficos del krill (Gymnodinoides pacifica), investigó la producción de huevos de las dos especies de krill más abundantes de la región e investigó los cambios costa-océano de la distribución, abundancia y sucesión de la estructura

de la comunidad del krill en una serie de tiempo mensual realizada en 1970-1972, publicando eventualmente 13 artículos internacionales producto de su investigación de tesis doctoral. A su regreso a México en el CICIMARIPN en Febrero 2014, el Dr. Jaime Gómez ha impartido cursos de posgrado de Oceanografía Biológica y Ecología del zooplancton; desde entonces ha estado interesado en estudiar la productividad secundaria, la biomasa, el comportamiento social de copépodos y krill, la ecología parásito-hospedador y las interacciones depredador-presa de los animales en el ecosistema marino epipelágico. Ha colaborado con investigadores de varias instituciones y disciplinas, incluyendo estudios hidroacústicos, parasitología, microbiología, fisiología, embriología, carcinología, pesquería y ecología de plancton y los mamíferos marinos para comprender las interacciones biológicas entre los organismos que van desde las bacterias hasta las ballenas azules. En colaboración con investigadores de la UNAM realizaron algunas de las primeras estimaciones de abundancia y distribución vertical mediante métodos hidroacústicos de peces pelágicos pequeños en el Golfo de California y calamar Doscidicus gigas. La pesquería de calamar en Guaymas y Santa Rosalía ha disminuido a niveles sin precedentes desde 2010 y la comprensión de los factores ambientales que afectan ese recurso pesquero son de un particular interés de este grupo de investigación. En 2009 fue galardonado con el Premio de Investigación del Instituto Politécnico Nacional por liderar un grupo de investigación en estudios de ecología de zooplancton en el Golfo de California financiado por CONACyT (20042007). En 2009-2010 realizó una estancia de investigación en el Australian Antarctic Division (Tasmania, Australia) con la colaboración del Dr. Steve Nicol y Dr. So Kawaguchi, en la cual escribió dos publicaciones sobre parásitos de krill y realizó una revisión de parásitos de krill a nivel mundial. Con este conocimiento en los últimos años se ha dedicado a investigar la compleja interacción de parásitos-hospederos de zooplancton y sus depredadores. Actualmente ha descrito cinco especies, dos géneros y actualmente está interesado en colaboraciones con investigadores de distintas disciplinas como ecología alimentaria y parasitología de cetáceos marinos, ecología de crustáceos y abanicos de mar en arrecifes rocosos del Golfo de California. Sus más recientes inves-

tigaciones son el establecimiento de una serie de tiempo semanal de zooplancton con énfasis en genética de huevos y larvas de peces en el Parque Nacional Cabo Pulmo (en colaboración con Dr. Octavio Aburto, SIO) y la investigación de la biología y ecología de krill tropical en Cabo Corriente, Jalisco (en colaboración con Dra. Carmen Franco-Gordo y el Dr. Israel Ambriz Arreola, Universidad de Guadalajara). El Dr. Jaime Gómez actualmente forma parte del consejo editorial de dos revistas internacionales de su especialidad: Journal of Plankton Research (UK desde 2012 a la fecha) y Marine Ecology Progress Series (Alemania, desde 2013 a la fecha) y de la revista institucional CICIMAR-Océanides (1996-2010 y 2014 a la fecha) y ha fungido como árbitro en más de 27 revistas nacionales e internacionales y ha

publicado cerca de 80 artículos científicos. La mayor parte de su tiempo está asesorando estudiantes (no es raro que este involucrado entre 15-20 comités de estudiantes de posgrado) por su inmensa interés de aprender de otros temas de investigación ajenos a su especialidad. Tiene la forme convicción que la ciencia es una filosofía de vida y no hay nada más interesante que hacer en esta vida que estudiar la vida misma. Interesado en todo y por ende experto en nada, está consciente de la complejidad de comprender los procesos naturales en el planeta; por esta razón siempre se ha motivado con el pensamiento de su mentor Edward Brinton (SIO): “We will never fully understand the variability of life in the oceans. But please don´t fault us for trying”.

CICIMAR Oceánides, 2015

Vol. 30 no. 1

ISBN 1870-0713

Contents Endogenous polyamine response in epiphytic Bonnemaisonia hamifera (Bonnemaisoniales: Rhodophyta) due to interaction with its host. VERGARARODARTE, M. A., J. I. MURILLO ÁLVAREZ & R. ROBAINA ROMERO

Macrobenthic communities in a temperate urban estuary of high dominance and low diversity: Montevideo Bay (Uruguay). MUNIZ, P. & N. VENTURINI

Survival of juvenile white mullet Mugil curema (Mugilidae) in a coastal lagoon. QUIÑONEZ-VELÁZQUEZ, C., J. R. LÓPEZ-OLMOS & C. I. PÉREZQUIÑONEZ

Dry weight, carbon, C/N ratio, hydrogen, and chlorophyll variation during exponential growth of selected microalgae species used in aquaculture. PÉREZMORALES, A., A. MARTÍNEZ-LÓPEZ & J. M. CAMALICH-CARPIZO

Zooplankton functional groups from the California current and climate variability during 1997-2013. Lavaniegos, B. E., O. Molina-González & M. Murcia-Riaño

New records of the distinctive benthic dinoflagellate genus Cabra (Dinophyceae). GÓMEZ, F. & R. M. LOPES

NOTA Benthic diatoms from shallow environments deposited at 300 m depth in a southern Gulf of California basin. ROCHÍN BAÑAGA, H., D. A. SIQUEIROS BELTRONES & J. BOLLMANN

CICIMAR Oceánides 30(1): 1-8 (2015)

ENDOGENOUS POLYAMINE RESPONSE IN EPIPHYTIC Bonnemaisonia hamifera (BONNEMAISONIALES: RHODOPHYTA) DUE TO INTERACTION WITH ITS HOST Vergara-Rodarte, M. A.*1, J. I. Murillo Álvarez2 & R. Robaina Romero3

, Departamento de Desarrollo de Tecnologías, Centro Interdisciplinario de Ciencias Marinas-IPN, 23096 La Paz, Baja California Sur, México. 3Departamento de Biología, Facultad de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Canary Islands E-35017, Spain. email: vrm491@hotmail.com 1 2

ABSTRACT.-The endogenous polyamine (Pas) content of free and bound-acid soluble fractions was assessed in the epiphyte Bonnemaisonia hamifera under the presence of its host Gelidium arbuscula and of extracts obtained from G. arbuscula and from G. robustum. In the presence of fresh thalli of G. arbuscula the free putrescine (PUT) content decreased (P ‹ 0.05), while spermine and spermidine (SPD) showed a slightly increased concentration. The content of free PUT also decreased in all treatments with the different extracts and the dose. Bound-soluble PAs showed a different trend, particularly with the highest dose of ethanolic extract of G. robustum, PUT and SPD bound-soluble PA content increased significantly. Based on our results we infer that PAs are involved in the epiphytic relationship between macroalgae, and that PUT produces the greatest response of B. hamifera in the interaction with host and its extracts. The potential causes of its variation and its use for the production of secondary metabolites are discussed.

Keywords: Polyamine, seaweed, extracts, biotic relationships, epiphyte.

Respuesta de una poliamina endógena en el alga epifita Bonnemaisonia hamifera (Bonnemaisoniales: Rhodophyta) debido a interacciones con su hospedero RESUMEN.- Se evaluó el contenido endógeno de poliaminas, tanto de la fracción libre como de la conjugadaácido soluble en la macroalga epifita Bonnemaisonia hamifera en presencia de su hospedero Gelidium arbuscula y de extractos obtenidos de G. arbuscula y G. robustum. En presencia de talos vivos de G. arbuscula, el contenido de putrescina libre disminuyó (P ‹ 0.05) y la espermina y espermidina tuvieron un ligero incremento sin diferencia significativa. En los tratamientos con los distintos extractos y dosis, el contenido de putrescina libre también disminuyó. La fracción de poliaminas conjugadas mostró una tendencia diferente, particularmente con la dosis más alta del extracto etanólico de G. robustum donde putrescina y espermidina conjugadas-solubles se incrementaron significativamente. Los resultados obtenidos sugieren que las poliaminas juegan un papel en el establecimiento de las relaciones epifito-hospedero, y que la putrescina es la que presenta la mayor respuesta en B. hamifera en la interacción con su hospedero y en presencia de los extractos. Se discute la variación de las poliaminas y las posibles causas

Palabras clave: Poliamina, macroalga, extractos, relacion biótica, epifita. Vergara-Rodarte M. A., J. I. Murillo Álvarez & R. Robaina Romero. 2015. Endogenous polyamine response in epiphytic Bonnemaisonia hamifera (Bonnemaisoniales: Rhodophyta) due to interaction with its host. CICIMAR Oceánides, 30(1): 1-8.

INTRODUCTION Seaweeds and many other marine organisms are under strong biotic and abiotic interactions due to the changing nature of their environment. The effects of environmental factors as light and nutrients availability on the secondary metabolism of seaweeds have been extensively studied, particularly in the production of polyphenols (Paul & Van Alstyne, 1992; Cronin & Hay, 1996a; 1996b; Puglisi & Paul, 1996; Pavia et al., 1997; Pavia & Toth, 2000; Swanson & Druehl, 2002; Pansch et al., 2009). Biotic relationships are as important as the abiotic ones. Herbivorism and epiphytism can be considered natural enemies of terrestrial plants and seaweeds. These interactions create stressful conditions that probably have played a key role in the natural selection of these organisms (Bakus 1971). There are metabolic and proteomic markers that are used to measure said stress (Shulaev & Oliver, 2006). Endogenous polyamines (PAs) have been used for many years as stress indicators related to ecological relationships. Fecha de recepción: 20 de noviembre de 2014

This is due to the diverse functions that PAs play in plant metabolism, where free putrescine (PUT), spermidine (SPD), and spermine SPM are the most important PAs found in living beings (Groppa & Benavides, 2008). In free form the PAs are positive charged, and they react with negatively charged molecules such as DNA, RNA, proteins and phospholipids. Therefore PAs are involved in the regulation of the physical and chemical properties of membranes and the structure of nucleic acids, and enzymatic activity (Galston & Sawhney, 1990; Tiburcio et al., 1993; Bachrach, 2010). Currently in terrestrial plants there are a many investigations where the correlation between the change in the endogenous PA content under different environmental abiotic stress sources is reported, such as osmotic stress and drought, heat, chilling, oxidative stress, hypoxia, ozone, UV radiation, nutrients, mechanical wounding, heavy metal toxicity and herbicides (Alcazar et al., 2006; 2010; Liu et al., 2007; Groppa & Benavides 2008; Kusano et al., 2008). Also, in biFecha de aceptación: 26 de noviembre de 2014

VERGARA-RODARTE et al.

otic relationships, PAs are involved in the establishment of simbiotic relationships with fungus (Bais et al. 2000; Niemi et al., 2007; Cheng et al. 2012), in response to viral infections (Belles et al., 1993; Yoda et al., 2009; Sagor et al., 2013) and with other plants in cell cultures (Cvikrova et al. 2008).

to the laboratory where the host G. arbuscula and its epiphyte B. hamifera were separated. Samples of G. robustum for the extraction were collected in the Pacific coast of the Baja California peninsula, México. Samples were sun dried and transported in plastic bags.

Polyamine related research on macroalgae is far from what it has been achieved with terrestrial plants. There are reports of their physiological role as growth regulators (García-Jiménez et al., 1998; Cohen et al., 1984; Marián et al., 2000), the reproductive implications in the development and maturation of the cystocarp and sporulation (GuzmánUrióstegui et al., 2002; Sacramento et al., 2004), and the stress response caused by abiotic factors such as salinity in Grateloupia doryphora (GarcíaJiménez et al., 2007) and Ulva fasciata (Lee 1998;). More recently it was reported that PAs in Ecklonia maxima have a seasonal variation, with maximum values during periods of stress caused by high wave activity (Papenfus et al., 2012). In addition to the direct participation of PAs in the stress response, the PAs are precursors of a wide variety of alkaloids and are related with other metabolic pathways. For example, PUT is the precursor of pyrrolidine, tropane and calystegines alkaloids (Ghosh 2000; Kumar et al., 2006; Bhattacharya & Rajam,2007). Spermidine and PUT are also important precursors for the production of lunarine and pyrrolizidine alkaloids (Graser & Hartmann, 2000; Bhattacharya & Rajam, 2007). These types of PAs derivatives have medical applications, and have increased the interest on biotechnological investigation.

The ethanolic extracts were obtained from the alga G. arbuscula and the non-native macroalgae G. robustum. The algae were dried and milled and soaked with ethanol 100%. The ethanol was then filtrated and replaced with new ethanol every 3 days three times. The extracts were concentrated to dryness at 40° C and low pressure in a rota-evaporator (Yamato re500)

Epiphytism is a common phenomenon in macroalgal communities. However, the chemical response in biotic interactions between host and epiphyte has been scarcely investigated. We are also interested in determining if PAs are involved in biotic relationships in macroalgae, in order to get a better understanding of the factors that affect their endogenous content. Thus, we also expect that PAs can be used as stress markers in epiphytic relationships. This is the case of the association between the epiphytic red algae Bonnemaisonia hamifera P. Hariot (Bonnemaisoniales: Rhodophyta) a small filamentous alga in sporophytic phase that can be found living in most of the samples of its host Gelidium arbuscula (Bory de Saint-Vincent ex Børgesen). In this research, we studied the alteration in PAs levels of B. hamifera when cultivated in the presence of fresh thalli of G. arbuscula, and with extracts from this alga and from Gelidium robustum. MATERIALS AND METHODS Seaweed Collection and Culture. Both G. arbuscula and B. hamifera (Fig. 1) were collected from a rocky, intertidal shore during low tide in Las Palmas de Gran Canaria, Spain. Samples were stored in plastic bags and immediately transported

Sporophyte stock cultures of B. hamifer were established from filaments excised from the thallus of collected G. arbuscula. The epiphyte B. hamifera was cultivated in the laboratory in IMR culture medium (Paasche et al., 1996). The stock cultures were maintained in the laboratory at 22±2ºC, and 18:6 light:dark photoperiod for up to two months, until enough samples were obtained to carry out the experiments. Treatments designed for this assay are described in table 1. The assays had a duration of 48 h, 3 plates per treatment, and were maintained in the conditions described above. However, G. arbuscula could not be cultivated; thus, fresh thalli were collected one day before the beginning of the experiment. Polyamine Analysis. The extraction method used was the same as in Sacramento et al. (2004), but with a modification in the weight of the sample, because of the small amount available of B. hamifera. The extract was dissolved again in 0.5 mL of acetonitrile. In short, for the extraction, samples (25-30 mg) were powdered in a mortar with liquid nitrogen and then added 1.5 mL of 5% percloric acid (PCA), and pounded in the mortar until a homogeneous paste was obtained. This was centrifuged at 9000 g for 20 minutes at 6° C. The supernatant was divided into two parts: one to measure free acid-soluble PAs (260 µL), which was frozen for further dansylation, while the other portion was used to determine the bound acid-soluble PAs (300 µL) after it was digested in sealed vials, using 300 µL of HCl (12 M, HPLC grade) overnight at 100° C. The samples were then filtered, vacuum dried, and re-dissolved in 260 µL of 5% PCA. Both free and bound-soluble PAs samples were dansylated following the following method. Briefly, 40 µL of diamino heptane (HTD, 0.05 mM) was added as internal control of dansylation, plus 200 µL of a saturated solution of sodium carbonate (NaCO3), and 400 µL of dansyl chloride (5 mg mL-1, in 2 acetone). It was then incubated overnight in the dark and at room temperature (24° C ±3). Then, 100 µL of proline (100 mg mL-1, in water) were added to re-

ENDOGENOUS POLYAMINE RESPONSE IN Bonnemaisonia hamifera

Figure 1.- Bonnemaisonia hamifera and Gelidium arbuscula in culture plates. A) Amount of thalli of B. hamifera used for all treatments (25-30 mg); B). Amount of fronds of G. arbuscula (around 300 mg) used for the treatments with B. hamifera; C) Both algae together for the treatment; D) Morphology of B. hamifera showing filament cells and vesicles.

move the dansyl chloride excess. Finally, 500 µL of toluene was added and mixed in a vortex. Then 400 µL of the upper decanted phase were vacuum dried and re-dissolved in acetonitrile. All samples were filtered with syringe filters of 0.45 µm membrane before HPLC analysis. Chromatographic analysis was carried out in a Varian system integrated by a fluorescence detector

(Varian ProStar 363) at 365 (excitation) and 510 nm (emission), a 5 µm reverse phase column (Varian C-18), auto sampler (Varian ProStar 410) and pump (Varian 9002). Solvents used were (A) acetonitrile and (B) water. The elution protocol was as follows: 0-3 min, 70% of A; 3-12 min, 100% of A; 12-20 min, 70% of A. The flow rate was 1 mL min-1 and the injection volume was 20 µL. The approximate retention times for the polyamines under these con-

Table 1. Treatments and controls for the experiment Treatments Description IMR

B. hamifera in IMR medium (control)

LGA

B. hamifera in IMR with live G. arbuscula

GR1

B. hamifera in IMR with G. robustum ethanolic extract [0.01 mg mL-1]

GR0.5

B. hamifera in IMR with G. robustum ethanolic extract [0.005 mg mL-1]

GA1

B. hamifera in IMR with G. arbuscula ethanolic extract [0.01 mg mL-1]

GA0.5

B. hamifera in IMR with G. arbuscula ethanolic extract [0.005 mg mL-1]

VERGARA-RODARTE et al.

ditions were as follows: PUT-7.1 min, SPD-8.1 min, SPM-9.4, and the internal control HTD-7.8 min. Statistical Analysis. Values were subjected to one way ANOVA or Kruskal-Wallis test, depending on whether the data showed normality and homoscedasticity. Post-hoc analysis was applied in case of a significant difference among treatments; a Tukey test was carried out with ANOVA, and with a one to one comparison using the Mann-Whitney U for Kruskal-Wallis. RESULTS In the presence of live G. arbuscula (LGA; Fig. 2), the free-soluble PUT in B. hamifera showed a significant decrease (P ‹ 0.05), while SPM and SPD had a slight increase compared with the control (IMR). —Statistical significance was observed for SPM (P ‹ 0.05)—. Bound-soluble PAs showed no significant differences. Significant differences between treatments with extracts and control were found. Free PUT had a decrease of over 90% in treatments GR0.5, GA1 and GA0.5 (P ‹ 0.05), and SPD of about 80% in these same treatments. While in GR1 treatment SPD increased three times its content (P ‹ 0.05). Figure 3A shows the difference in PUT and SPD (P ‹ 0.05) between GR1 treatment and the others.

Figure 2.- Concentrations of free and bound-soluble PAs of B onnemaisonia hamifera (control, IMR) ad cultured in the presence of live Gelidium arbuscula (LGA). Data correspond to three replicates and are represented as mean (±SE). PUTwhite, SPD- gray, SPM-dark. Different letters show significant differences between treatments in each PA according to the statistical and post-hoc tests (P ‹ 0.05). Treatments are described in Table 1.

In the bound-soluble fraction (Fig 3B) only the GR1 treatment had a significant increase (P ‹ 0.05) in PUT and SPD in relation to all other treatments and control. DISCUSSION Endogenous content of PAs varies greatly among species. In vascular plants PA content is high, ranging from 0.01 to 9 µmol g-1 fresh weight (fw) for PUT, from 0.006 to 1.72 µmol g-1 fw for SPD, and from 0.005 to 0.74 µmol g-1 fw for SPM (Altman, 1989). In macroalgae there is a large variation among species (Table 2). Endogenous content of PAs in B. hamifera was in this order PUT>SPD>SPM which coincide with the overall trend observed for macroalgae and vascular plants and its values were within the range described above. The results suggest that B. hamifera “senses” the presence of living Gelidium or even its extracts. In this regard, in terrestrial plants the typical response to external factors, mainly abiotic, is by increasing the endogenous content of PAs, which is considered a resistance characteristic (Alcázar et al., 2010; Groppa & Benavides, 2008). The importance of PAs in this process is such that in stressed plants the PUT content may represent up to 1.2% dry weight, which is equivalent to 20% of the total nitrogen content (Galston & Sawhney, 1990). While in the opposite case, the decrease in the PAs content suggests sensitivity to the stress factor.

Figure 3.- Concentrations of endogenous PAs of B onnemaisonia hamifera in the treatments with extracts. Mean values (+SE). A) Free-soluble PAs content; B) Bound-soluble fraction; PUT- white, SPD- gray; SPM-dark. Different letters show significant differences between treatments in each PA by the statistical and post-hoc tests (P ‹ 0.05).

ENDOGENOUS POLYAMINE RESPONSE IN Bonnemaisonia hamifera

For macroalgae it has been reported that PA response under saline conditions is based on the accumulation of the free fraction of PUT, SPD and SPM (Lee, 1998; García-Jiménez et al., 2007) which correlates with a decrease in transglutaminase activity and the increase of arginine decarboxylase activity in a salt-tolerant macroalgae (García-Jiménez et al., 2007). While in terrestrial plants, this topic was studied extensively considering mainly environmental factors, such as drought, salinity, temperature, mineral nutrition, wounding, UV treatment, metal, and oxidative and osmotic stress. All these are shown in tolerant plants in which the induced over expression and silencing of genes for PA biosynthetic enzymes are common (Basu & Ghosh, 1991; Groppa & Benavides, 2008; Alcázar et al., 2010). The plant response will depend on the particular species, therefore it is not possible to generalize or predict the PAs variation, but what it can be expected is that PUT are responsible of short term stress responses and that may occur in a lapse of hours. However, the action mechanism of the stress response is still unclear (Lefèvre et al., 2001; Zacchini & de Agazio, 2004; Bassard et al., 2010). The epiphyte B. hamifera senses the presence of live Gelidium and reacts by decreasing significantly the PUT content after 48 h. For vascular plants it was reported that a decrease in PUT levels is regulated by elevated levels of ethylene (Kumar et al., 1996), and osmotic stress due to saline conditions (El-Shintinawy, 2000; Zapata et al., 2004; Tang et al., 2007). The decrease in PUT content may be caused by: a) an increase in the catabolic degradation or excretion; b) the use of PUT for the production of secondary metabolites; c) an increase in the synthesis of SPD; d) conjugation of free PUT with other cellular compounds (bound-soluble); e) a de-

crease in di novo synthesis. This raises the question, where is the free PUT going? Regarding option c, only the GR1 treatment showed an increase of SPD, although this was not significant (P › 0.05). Regarding option d, four treatments showed values close to the control without a significant difference (P › 0.05). Therefore, these two options are unlikely. Moreover, in terrestrial plants it is reported that PAs derivatives are involved in the establishment of biotic relationships with fungus (Bais et al., 2000; Niemi et al., 2007; Nogales et al., 2009; Cheng et al., 2012), viral infections (Belles et al., 1993; Yoda et al., 2009; Sagor et al., 2013), and with other plants in cell suspension cultures (Cvikrova et al., 2008). For example, with viral infections, PAs can be used as a source of hydrogen peroxide, catalyzed by diamine and PA oxidases (Yoda et al., 2003; 2009). It has also been reported that the addition of fungal elicitors (culture filtrates) promotes the production of coumarines, by modifying the endogenous content of PAs (Bais et al., 2000), with PUT closely involved in this process (Bais et al., 1999). Phenolamides are other PA derivatives that form a large class of secondary metabolites in plants, and they are considered as a link between phenolic and nitrogen metabolism (Morant et al., 2007; Bassard et al., 2010). Regarding the ecological role of phenolamides, they are part of a defensive strategy against plant pathogens (Martin-Tanguy, 1985; von Röpenack et al., 1998), or an insect deterrence activity (Tebayashi et al., 2007), and are involved in the plant response to abiotic stress, because of their antioxidant and radical scavenging activity due to the nature of its phenolic and PA constituents (Edreva et al., 2007). The differences between extracts suggest that

Table 2. Endogenous PA content (µmol g-1 fresh weight) reported in other studies for marine macroalgae and in the present study (modified from Guzmán-Uriostegui et al., 2003) PUT

SPD

SPM

Free

Ulva reticulata

1.36

0.13

0.10

0.05

0.014

0.006

Ulva lactuca

1.36

0.51

0.15

0.01

0.014

0.001

Ulva fasciata

0.25

0.03

0.09

0.019

0.001

Chaetomorpha crassa

0.47

0.68

0.04

0.017

0.029

0.008

Valoniopsis pachynema

0.56

0.22

0.11

0.13

0.004

0.003

Dictyota dichotoma

10.43

0.015

0.005

0.02

0.044

Gelidium canariensis Grateloupia doryphora

7.94

0.03

0.014

Gracilaria cornea

4.42

0.01

0.008

0.018

0.006

Ecklonia maxima

0.68

0.148

0.06 ± 0.03

0.03 ± 0.001

0.01 ± 0.001

0.004 ± 0.001

0.008 ± 0.002

0.01 ± 0.003

Bonnemaisonia hamifera

VERGARA-RODARTE et al.

the addition of the nonnative G. robustum extract induces a singular response. The higher dose was significantly different from all others, suggesting that B. hamifera response to this extract is dose-dependent. The research on the chemistry of Gelidium species resulted in only one report on secondary metabolites, the gelidene, a cyclic polychlorinated monoterpen isolated from G. sesquipedale (Aazizi et al., 1989) but nothing is known about its biological activity. Additional and recent information obtained by our research group with different chromatographic methods (unpublished data), suggests that terpenoids are an important part of G. robustum extracts. Our study indicates that the epiphytic relationship between macroalgae affect the production of PAs and mostly free PUT. This biotic interaction could be considered as an important source of stress, according to studies conducted on the endogenous content of PAs. As reported for terrestrial plants, we also found that in macroalgae PUT is the responsible for the short term stress response. These results generate many other questions and show the need of further investigation. For example, studying the activation of PA biosynthetic enzymes and genes in response to the stress, could help to explain the mechanisms in which the endogenous PAs fluctuate. We also suggest doing research on the possible effect of Gelidium extracts in the production of PA derivatives by B. hamifera. ACKNOWLEDGMENTS This work was financed by the Ministerio de Economía y Competitividad-Plan Nacional Español de Ciencia y Tecnología (BFU2010-17248). Mario Vergara acknowledges the support of CONACYT scholarship for PhD Program. JIMA thanks the Instituto Politécnico Nacional, COFAA, and Secretaría de Investigación y Posgrado for their support. REFERENCES Aazizi, M.A., G.M. Assef & R Faure. 1989. Gelidene, a new polyhalogenated monocyclic monoterpen from the red marine algae Gelidium sesquipedale. J. Nat. Prod., 52: 829-831. Alcazar, R., F. Marco, J.C. Cuevas, M. Patron, A. Fernando, P. Carrasco, A.F. Tiburcio & T. Altabella. 2006. Involvement of polyamines in plant response to abiotic stress. Biotechnol. Lett. 28: 1867-1876. Alcázar, R., T. Altabella, F. Marco, C. Bortolotti, M. Reymond, C. Konez, P. Carrasco & A Tiburcio. 2010. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta. 231: 1237-1249. Altman, A. 1989. Polyamines and plant hormones. 121-145, In: Bachrach U, Heimer YM (eds),

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CICIMAR Oceánides 30(1): 9-20 (2015)

MACROBENTHIC COMMUNITIES IN A TEMPERATE URBAN ESTUARY of high dominance and low diversity: MONTEVIDEO BAY (URUGUAY) Muniz, P. & N. Venturini

Sección Oceanografía y Ecología Marina, Facultad de Ciencias. Iguá 4225, Montevideo 11400-Uruguay. email: pmmaciel@fcien.edu.uy

ABSTRACT. The macrobenthic subtidal community was studied between April 1997 and April 1998 in Montevideo bay, an urban estuary located in the fluvio marine system of the Río de la Plata (Uruguay) that receives a variety of industrial and sewage inputs. Monthly surveys were carried out at ten sampling stations where sediment samples were taken with a manual corer and analysed for granulometric parameters, organic matter content, chlorophyll a and phaeopigments content, redox potential, and macrobenthic fauna. The area presented high organic matter content in its sediments and several regions of the bottom were anoxic during a large part of the sampling period. The benthic macrofauna was dominated, both in numbers as well as in biomass, by the small surface deposit-feeder gastropod Heleobia cf. australis. Cluster analysis, Multidimensional Scaling and Canonical Correspondence Analysis revealed that the study area could be divided in two well-defined regions with different environmental characteristics and different faunal composition. The dissolved oxygen content in the bottom water and variables related to it were the most important factors in explaining the patterns of the benthic communities. At the phylum level, the meta-analysis of “production” showed a high disturbance status for all stations. The inner region, the most affected by anthropogenic activities, was the most compromised environmentally and biologically, and was characterised by a very low diversity and abundance, reduced conditions in the sediments and low oxygenated bottom water. In more external places of the bay, on the other hand, perhaps due to their location at a greater distance from the sources of organic material and in a region with higher hydrodynamic energy, the conditions for the development of benthic fauna were more favourable. Spatial and temporal faunistic patterns observed and their possible causes are analysed and discussed in relation to the natural and anthropogenic factors that act in this coastal ecosystem.

Keywords: Macrobenthic communities, soft-bottom, estuary, Montevideo bay, Río de la Plata.5

Comunidades macrobentónicas en un estuario urbano templado de alta dominancia y baja diversidad: Bahía de Montevideo (Uruguay) RESUMEN. Una comunidad macrobentónica submareal fue estudiada entre abril de 1997 y abril de 1998 en la Bahía de Montevideo, un estuario urbano localizado en el sistema marino fluvial del Río de la Plata (Uruguay) que recibe una variedad de descargas industriales y de alcantarillado. Se llevaron a cabo muestreos mensuales en diez estaciones donde se tomaron muestras de sedimento utilizando un nucleador y a estas les fueron analizadas los parámetros granulométricos, el contenido de materia orgánica, el contenido de clorofila a y de feopigmentos, potencial redox y fauna macrobentónica. El área presentó un alto contenido de materia orgánica en sus sedimentos y muchas regiones del fondo mostraron ser anóxicas durante una gran parte del mismo período. La fauna macrobentónica fue dominada en número y biomasa por los pequeños gasterópodos Heleobia cf. australis. Los análisis de conglomerados, de escala multidimensional y de correspondencia canónica revelaron que el área de estudio podría ser dividida en dos regiones bien definidas con diferentes características ambientales y composiciones faunísticas diferentes. El contenido de oxígeno disuelto en agua de fondo y las variables realcionadas con ella fueron los factores más importatntes en explicar los patrones de las comunidades bentónicas. Al nivel phylum, los meta análisis de la “producción” mostraron un alto estatus de perturbación y biológica que fue caracterizada por baja diversidad y abundancia, condiciones reducidas y agua de fondo poco oxigenada. Por otra parte, en los sitios más externos de la bahía, debido quizas a su localización a una gran distancia de las fuentes de materia orgánica con una alta energía hidrodinámica, las condiciones para el desarrollo de la fauna bentónica fueron favorables. Los patrones faunísticos espaciales y temporales observados y sus posibles causas fueron analizados y discutidos en relación con los factores naturales y antropogénicos que actúan sobre este ecosistema costero.

Palabras Clave: comunidades macrobentónicas, fondos blandos, estuarios, Bahía de Montevideo, Río de la Plata. Muniz, P. & N. Venturini. 2015. Macrobenthic communities in a temperate urban estuary of high dominance and low diversity: Montevideo bay (Uruguay). CICIMAR Oceánides, 30(1): 9-20.

Introduction

in the bottom waters.

In recent years the ecological integrity of many estuarine and coastal systems has been stressed by activities of growing human populations and associated land use. These stresses are usually expressed through a broad category of responses that are termed eutrophication and are associated with excess production of organic matter (Rosenberg, 1985) and the consequent deficiency of dissolved oxygen Fecha de recepción: 28 de enero de 2015

The effects of point source discharges on the structure and composition of nearshore benthic macroinvertebrate fauna has been well documented and extensively reviewed (e.g. Pearson & Rosenberg, 1978; Warwick, 1988). Studies to determine the effects of diffuse pollution in estuaries are often complicated by the presence of inputs of different composition from multiple sources, and discharge to

Fecha de aceptación: 17 de marzo de 2015

MUNIZ & VENTURINI

an area of naturally variable physico-chemical conditions, with the consequent loss of pollutant and effect gradients. It is necessary to evaluate the condition of such estuaries to determine areas of concern and establish effective and economical monitoring systems to record the effects of pollution on the fauna. Benthic organisms are used extensively as indicators of estuarine environmental status and trends, due to numerous studies which have demonstrated that they respond predictably to many kinds of natural and human induced stress (López-Jamar, 1985; Dauer, 1993; among others Ritter & Montagna, 1999). Many characteristics of benthic associations make them useful indicators. Exposure to hypoxia is typically greatest in near-bottom waters and anthropogenic contaminants often accumulate in sediments where benthos lives. The limited mobility of the majority of adult macrobenthic organisms has advantages in environmental assessment because, unlike most pelagic fauna, their assemblages reflect local environmental conditions (Gray, 1979; Muniz et al., 2013). Benthic communities are part of any marine ecosystem, and the analysis of their structure is an important tool to describe changes in space (with application to point source pollution monitoring) and time (with application to the description of changes in the state of the marine system) (Heip, 1992). Although such coastal systems in temperate and high latitudes are well described (Pires, 1992), the Uruguayan coastal zone is poorly understood and studies describing the soft-bottom benthic subtidal system are scarce (Scarabino et al., 1975; Demichelli, 1984; 1986; Venturini et al., 2004; Muniz et al., 2011). Montevideo bay is an urban estuary in Uruguay receiving a variety of industrial and sewage inputs (Muniz et al., 2002; 2004; Muniz & Venturini, 2011, García Rodríguez et al., 2010; Venturni et al., 2012; 2015). The present results are part of an integrated program designed to determine the effects of the different pollutant sources on the aquatic biota and to establish effective and economical monitoring systems. It describes, for the first time, the temporalspatial structure of the macrobenthic subtidal communities over one year of study. Although some new and important advances were developed since the sampling campaigns of the present study, these results are new and important for the knowledge of the studied system. Material and methods Study area Montevideo bay (Figure 1) is located in the fluvio marine system of the Río de la Plata, (Montevideo, Uruguay) between 34º52’-34º56’ S and 56º10’56º15’ W, and has an approximate area of 10 km2 and a mean depth of 5 m. Three streams flow into

it, the Miguelete Stream, the Pantanoso Stream and the Seco Stream which flows through a pipe. These streams carry wastes of many different industries and urban centres, as well from a great number of sewage pipes. The bay also harbours the ANCAP refinery, the Batlle steam water plant (UTE) and an active port: The Port of Montevideo. The Bay is protected from the South winds, which are infrequent but very strong, by two breakwaters built at the beginning of the century: the Sarandí breakwater and the Oeste breakwater. The entrance to the port is a channel 9.3 km long and the port’s main structures are in the southern area. There is a special dock (La Teja Dock) where oil tankers load and unload, in the northern area between the mouths of the Pantanoso and Miguelete Streams. This dock communicates with the outer anchorage by a channel 9 m deep. The bay has a great importance not only for the city, but also for the whole country. Even though its principal use is as the physical structure supporting the Port of Montevideo, there are other uses associated with it: activities pertaining to the port (those related to the piers, dockyards, warehouses etc.), as a water source for cooling of industrial processes (UTE and ANCAP), rowing, recreational fishing, sailing and other secondary uses. At present, it is not possible to use the bay for recreational activities that involve direct contact with the water, mainly due to the input of wastes coming from the streams and the sewage pipes that drain directly into the bay. Predominant winds are from NE and W-SW, being very important in determining water circulation at low depths (Moresco & Dol, 1996), which is mainly clock-wise. Data collection and laboratory methods Monthly surveys were carried out from April 1997 to April 1998 at ten sampling stations distributed across the Bay according to the oxygen content in the bottom water (Figure 1). At each station, 14 replicate sediment samples were taken with a manual corer of 4.5 cm of internal diameter. Of them, 10 replicates were washed through a 0.4 mm sieve mesh and the material retained was preserved in 70% ethanol for the quantitative analysis of the benthic macrofauna. The sorting and identification were made under a stereoscopic microscope. Then the biomass was estimated by means of dry weight (70º C until constant weight) for individual species. Also 10 minutes dredging at 2 knots constant velocity was done at each station. This did not detect important differences in species richness and therefore for this paper these data are not presented. Nearly 100 g of the first of the other four replicates of the corer sediment samples were submitted to the standard dry-sieve and pipette method (Suguio, 1973) and parameters described by Folk &

MACROBENTHIC COMMUNITIES IN MONTEVIDEO BAY

59°

50°W 31ºS

Brazi South America

Uruguay

Argentina

N 34º

Pantanoso Stream Bizcochero Island

ANCAP Refinery

Miguelete Stream

Water Plant

La Teja Dock

Harbour

Oeste Breakwater

Sarandí

J Breakwater

34º

Rio

Plata

56º

biomass, an agglomerative classification analysis in “Q mode” was performed. The similarity matrix was constructed using the Bray-Curtis similarity index (Bray & Curtis, 1957) and grouped by the Unweighted Pair-Group Method using arithmetic Averages (UPGMA) (Romesburg, 1984). The 4th root transformation was used to reduce contributions to similarity by abundant species, and therefore to increase the importance of the less abundant species in the analyses. n-MDS ordinations (Kruskal & Wish, 1978) using the same similarity matrices were undertaken to complement the classification analyses. Species data were also aggregated into phyla and combined to produce a production matrix according to the formula suggested by Warwick & Clarke (1993), P = (B/A)0.73 x A, where A is the abundance and B the biomass. This matrix was made with the 50 standard sites used by these authors and included the 10 sample sites of Montevideo Bay.

Libertad Island

Bajo Humpreys Island

34º

C Batlle Steam

37º

56º

Figure 1. Map of Montevideo bay and region with the 10 sampling stations (black diamonds).

Ward (1957) were calculated for sedimentological data. The second replicate was used to determine the photosynthetic pigments of surface sediment according to Lorenzen (1967). With the third replicate we determined the redox potential of the sediment column using the standard solution (buffer) of Zobell (1946). The last corer sample was used to obtain the organic matter content of the surface sediment by means of the calcination technique according to Byers et al. (1978). Bottom water samples were obtained with “Hydro-Bios” bottles to measure temperature and determine dissolved oxygen content by the Winkler titration method according to Grasshoff (1983). With a “YSI” multi-parameter salinity and pH were determined and the depth was measured with a “Humminbird” echo-sounder. Data analysis Univariate and multivariate methods (classification and ordination) were used. The former included: density as number of individuals per unit area, abundance of individuals per species and species richness per unit area for each month. Diversity was estimated by the Shannon-Wiener index (Shannon & Weaver, 1963) using natural logarithms, and evenness with Pielou’s index (1975). In order to define groups of stations with similar attributes according to species abundance and

The relationships between multivariate community structure and environmental variables considered in this study were examined using the BIO-ENV procedure with Spearman’s weighted coefficient of correlation (Clarke & Ainsworth, 1993). This procedure determines the combination of variables that “best” explain the observed biological patterns, according to the level of correlation between the biotic matrix’s ranking of similarity and the abiotic matrix’s ranking of similarity. The environmental similarity matrix was calculated using the normalised Euclidean Distance, that is, before distances were calculated data were normalised as (x-m)/s, which consists of subtracting from each value (x) the mean value (m) and dividing by the standard deviation (s). This homogenises the variables and solves the problem of different scales and units that they have. The biotic similarity matrix was the same as that used in the classification and ordination analyses. All the above statistical analyses were carried out with the PRIMER software package. A Canonical Correspondence Analysis (CCA) was also applied to the same biological and environmental data using the CANOCO program (ter Braak, 1986; 1988). This makes more restrictive assumptions about the form of the data and the inter-relationships between biological and environmental data but makes for an interesting comparison of techniques. Also it allows the species to be arranged on an environmental basis, offering in a single diagram the direct interpretation of possible relationships between species, stations and environmental variables (Nielsen & Hopkins, 1992). The environmental variables were selected through the forward option in the CANOCO program. The relationship between the species and environmental variables was tested by the Monte-Carlo permutation test (ter Braak, 1990).

MUNIZ & VENTURINI

Results Environmental conditions In Montevideo bay the organic matter content of the surface sediment was higher than in other estuaries of other regions (e.g. Seys et al., 1994; Ieno & Bastida, 1998) and was spatially as well as temporally very variable. The concentrations of chlorophyll a and phaeopigments were lower but very variable throughout the study area, with the highest contents occurring generally at the shallowest stations. Bottom water was alkaline and pH did not show great variations (Table 1). Spatially the results showed that Montevideo bay could be divided in two regions. Stations A, B, C, D and E (inner region) were characterised by their relative shallowness, higher temperature and lower salinity than the rest of the stations. In addition to this, they presented very low bottom oxygen concentrations (Table 1 and Table 2) and the highest chlorophyll a concentrations in superficial sediments. The results of the redox analysis showed that this area of the bay always displayed reduced conditions in the water-sediment interface. These stations also showed the highest percentage of organic matter except C and E. These stations possessed low organic matter content but an organic matter/clay ratio similar to that of stations A, B and D. Stations A, B and D were dominated by the silt-clay fraction while C and E, had a higher contribution of sand, reaching an annual mean of 65.38 % and 70.12 % respectively.

The outer region of the bay, comprising stations F, G, H, I and J, was characterised by a greater depth, higher bottom salinity and higher bottom dissolved oxygen content than the inner region. The chlorophyll a content of the superficial sediments was lower. Stations H and J were situated in the access channels to the Montevideo Port, which are frequently dredged. The organic matter content was lower here than in the inner region but Sts F, G and I presented the lowest values in the study area. The silt-clay fraction was the predominant one. The fauna The total mean abundance determined was of 30118 individuals belonging to the Phyla Arthropoda, Nematoda, Mollusca and Annelida. Because of their abundance and high frequency of occurrence Heleobia cf. australis, Nephtys fluviatilis, Erodona mactroides, Heteromastus similis, and Alitta succinea stand out, as well as unidentified Nematode and Ostracode (Table 3). Density was very variable at each station monthly. Overall, stations the highest values occurred in May, June and July 1997 and the lowest in March 1998 (Figure 2a). In general, stations B, C and D presented a lower number of individuals per unit area than the remaining stations. Ignoring unidentified nematodes, ostracods and barnacles a total of 9 species were recorded. Maximum species richness occurred between April and August 1997 (Figure 2b). Stations F and G showed the highest species richness, and the lowest values were found at stations B, C and D. Species richness was low

Table 1. Environmental variables measured in the Montevideo Bay. Values are annual means Âą one standard deviation (SD). Sts A B C D E F G H I J

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Depth (m) 1,82 0,49 2,34 0,87 1,46 0,33 4,66 0,90 1,64 0,32 3,39 0,95 4,48 0,70 5,59 0,74 6,00 0,63 9,91 1,59

T (ÂşC) 18,87 4,66 18,83 4,36 19,46 4,54 17,22 4,16 18,70 4,22 17,67 4,29 17,51 4,30 17,28 4,29 16,81 3,87 16,59 3,71

Sal (psu) 5,44 3,69 6,72 4,18 5,05 3,49 10,36 6,28 4,45 1,96 5,44 2,42 6,11 5,46 10,80 6,35 12,80 7,82 14,79 8,64

O2 (mg/L) 2,80 0,75 1,18 0,53 2,95 1,42 1,55 1,36 3,88 1,45 7,04 2,12 8,16 1,62 6,49 1,70 6,55 1,31 6,64 1,76

pH 7,22 0,53 7,23 0,21 7,26 0,20 7,37 0,35 7,37 0,21 7,49 0,19 7,61 0,23 7,50 0,20 7,54 0,33 7,67 0,26

Chlor a (mg/m2) 10,97 4,32 9,64 10,03 33,68 27,87 15,38 4,52 8,40 5,38 8,42 7,26 3,91 3,10 5,93 3,98 3,88 3,57 7,58 6,74

%OM 7,77 2,46 9,50 3,16 3,36 3,93 11,17 1,52 5,21 6,89 5,50 1,97 6,36 2,01 7,95 2,45 5,62 2,03 7,33 2,32

Eh (mV) 177,33 119,79 92,33 73,48 147,75 84,83 113,29 47,82 179,00 103,87 229,00 89,28 189,00 62,65 199,00 58,55 184,00 76,95 210,11 61,43

%sand 18,71 9,84 18,44 10,69 65,38 38,49 12,80 12,30 70,12 21,13 26,23 9,61 17,85 26,18 4,46 1,49 15,15 9,37 5,92 6,25

%silt 68,82 10,93 57,81 21,56 26,25 30,22 64,01 11,68 26,04 19,84 64,04 15,38 71,40 23,82 79,49 5,12 72,31 12,31 78,78 9,99

%clay 12,47 7,48 23,84 16,25 8,374 13,85 23,19 12,55 3,874 2,17 9,72 8,52 10,75 5,67 16,05 4,75 12,54 10,90 15,30 8,32

Md(mm) 0,036 0,01 0,032 0,02 0,185 0,14 0,033 0,02 0,134 0,09 0,030 0,01 0,036 0,04 0,023 0,00 0,022 0,01 0,038 0,06

MACROBENTHIC COMMUNITIES IN MONTEVIDEO BAY

Table 2. Hypoxia events registered at the bay during the study period. Station

Date

OD (mg/l)

05- 97

0.09

06- 97

1.45

07- 97

0.36

08- 97

0.45

09- 97

0.01

10- 97

0.88

12- 97

1.31

01- 98

1.84

02- 98

1.56

04- 98

0.62

04- 98

1.22

throughout the study. A higher number of individuals did not always correspond to a higher number of species. Shannon’s diversity was also very low, reflecting the high dominance of Heleobia cf. australis, a very small gastropod that occurs frequently at very high abundance in several regions of the bay. The maximum diversity was registered in September

Table 3. Frequency of occurrence of macrobenthic species and groups recorded in Montevideo Bay and mean density (ind.\0.0016 m2) of each one during the sampling period. Between () are assigned the species code for the figure 5. Organisms Frequency (%) Mean density Nematoda (1) 36.15 7 Polychaeta 6.92 2 Neanthes succinea (2) 26.92 3 Nephtys fluviatilis (3) 20 2 Heteromastus similis (4) 1.53 1 Goniadides sp. (5) 0.76 1 Glycera sp. (6) 0.76 1 Sigambra cf. grubii (7) Gasteropoda 85.38 218 Heleobia cf. australis (8) Bivalvia 25.38 4 Erodona mactroides (9) Ostracoda (10) 12.3 3 Isopoda (11) 4.61 1 Balanus (12) 0.76 1

1997 at station G (1.63) and the minimum at station B (0) where only nematodes were present. Between April and August 1997 the highest diversity values were registered in the majority of the sampling stations. In November of the same year diversity decreased notably and in February 1998 it increased again (Figure2b). As for abundance, biomass was also variable in all stations during the period of study. The highest biomass values of the dominant species Heleobia cf. australis were occasionally exceeded by those corresponding to the second most abundant species, the bivalve Erodona mactroides. The highest biomass value was recorded in July 1997 and the lowest in March 1998 (Figure 2a). Multivariate analyses Cluster analysis of abundance and biomass data (annual arithmetic mean of pooled data) showed two groups of stations at approximately 60% of similarity (Figure 3). One group was composed of the mostinner stations B, C and D, and the remaining stations constituted the other. The same two groups appeared in the n-MDS ordinations (Figure 3). The meta-analysis (Figure 4) showed that stations of Montevideo bay appeared grouped at the right end of the diagram, that is to say, at the end of the pollution gradient showed and described by Warwick & Clarke (1993b). Table 4 presents the data set of Montevideo Bay used to perform the meta-analysis; these data were added to table ´2´ of Warwick & Clarke (1993).

Figure 2. a) Mean monthly values of density and biomass of macrobenthic fauna for Montevideo bay. b) Mean monthly values of diversity and species richness of macrobenthic fauna for Montevideo Bay.

BIO-ENV results for all data pooled showed that the environmental variable that “best” explains the structure of this benthic community is dissolved

MUNIZ & VENTURINI

oxygen concentration of bottom water (OD) with a correlation coefficient of 0.667 (Table 5). The following were the best combinations of two variables, (OD-chlor a 0.559, temp-OD 0.493, OD-MO 0.482) but these showed no better explanation power than the variable OD alone. Figure 5 shows the resulting ordination diagram obtained by the CCA. Basically, the same groups were obtained as with the other techniques applied. The correlation of macrobenthic species to environmental data was approximately 0.51 for the first axis and 0.76 for the second one. The MonteCarlo test showed a significant relation (p< 0.001) between the species and dissolved oxygen content in the bottom water, content of chlorophyll a in the sediment, organic matter and Eh of the surface sediment. Therefore, only the first two canonical axes were interpreted, representing 28.2 % of the variation between species and environmental data. In this model, only the environmental variables that presented significant relation (p < 0.001) were included in the forward selection procedure. In a general way the CCA analysis corroborated the trend shown by the other statistical techniques applied to the data, showing the same two groups of stations and the environmental variables that were best correlated with the biological data. Discussion and conclusions Although Montevideo bay is a semi-enclosed

Clyde (c1...c12) Linnhe (L63...L73) Eil (E63...E73) Oslofjord (OA...OG) Morlaix (M77...M81) Skagerrak (S1, S3) Northumberland (N) Carmarthen (CR) Kiel (K) Montevideo Bay (MA... MJ)

Stress = 0.16

S1 M77

L71 M78

L70

c8 OD L72 E70

OC E71

E69 S3 c9 L69 M79 c3 OE c2 M81 M80 E68 CR c4 K E72 E65 c10 E66 E63 L66 E67 L63 L68 L73 E64 L65 OA L67 c11 c1 OG L64 c12

E73 c7

c5 MA MG MC MJ ME MF MH MI

Figure 4. n-MDS ordination of phylum level “production” data from all the 50 sites of the NE Atlantic studied by Warwick and Clarke (1993) and the 10 samples from Montevideo Bay.

protected environment it shows a great variability. As a component of the Río de la Plata estuary, it is affected by the mixture of oceanic with fresh water which produces great variations in environmental parameters such as salinity, temperature, dissolved oxygen concentration, turbidity and so on (Framiñan & Brown, 1996; Guerrero et al., 1997). Natural variability, which can be a major source of stress to organisms, and a high nutrient concentration determine that estuaries are very productive but low diversity environments (Wilson, 1994). Low hydrodynamic energy in certain zones, deposition of organic matter on the sediment by natural eutrophication, in addition to anthropogenic inputs and industrial and dock activities, can produce in the fauna changes different from those expected due to natural variability of the system alone. Shallow coastal habitats and estuaries are considered dynamic environments, characterised by great fluctuations in abiotic parameters and subject to continuous disturbance. These processes do not permit the normal development of communities towards stable stages, except as a mean condition on a large temporal-spatial scale (Turner et al., 1995).

Figure 3. Dendrograms and n-MDS ordinations diagrams showing the results of grouping the sample stations (Qmode). (a) by abundance. (b) by biomass. Sampling stations A to J.

Individuals’ density was very variable between the sampling stations and over the year of study, however it was not significantly correlated with the environmental parameters measured. This could simply be natural variability or it could be reflecting that in Montevideo bay other factors affect the structural patterns of the benthic communities, perhaps due to the anthropogenic activities. Species richness was very low throughout, and high abundances did not always correspond to a high species number, indicating that certain species are well adapted to the environmental conditions prevailing in the bay. The general low diversity and high abundance of a single species were previously reported in other estuaries

MACROBENTHIC COMMUNITIES IN MONTEVIDEO BAY

Table 4. “Production” data of the 10 stations of Montevideo bay utilized in the meta - analysis. MA to MJ = sample stations. The nomenclature of the phyla are the same used by Warwick & Clarke (1993). MA MB MC MD ME MF MG MH MI MJ

Cnid 0 0 0 0 0 0 0 0 0 0

Plat 0 0 0 0 0 0 0 0 0 0

Neme 0 0 0 0 0 0 0 0 0 0

Nema 1.1 52.9 48.7 58.3 2.6 0.9 3.0 1.1 0.5 1.3

Pria 0 0 0 0 0 0 0 0 0 0

Sipu 0 0 0 0 0 0 0 0 0 0

of similar characteristics, near to Montevideo bay (Olivier et al., 1972; Ieno & Elias, 1995; Benvenuti, 1997; Ieno & Bastida, 1998), but high dominance of an annelid polychaete (Heteromastus similis) which in our study was not very abundant, has also been reported. According to Tenore (1972), the low diversity was a result of salinity conditions and the sediment instability in the Pamlico River estuary of North Carolina (USA). In Montevideo bay the range of salinity variation was also high but unlike other regions or estuaries this high variability is the common feature not an exceptional one. For that reason and because salinity did not showed any relationship with the faunistic patterns observed, the anthrophogenic effects of the activities developed in the area could be the principal factor causing the very low diversity and species richness and high dominance of the small gastropod H. cf. australis, which is acting as an opportunistic, surface deposit-feeder, tolerating the high organic load in the sediment (Danulat et al., 2002; Venturni et al., 2004). Although H. cf. australis tolerates the high organic content of the sediments, this single variable was not correlated either with its density or its biomass as could be expected for this type of species. Thus, another important factor could be influencing its presence in the bay. The decline in number of H. cf. australis during some months of the study year also is reflecting that it is a short-lived species. The relation between the increase of organic matter, the reduction in the species number, in diversity and the enlargement of the single abundance of one or two species of small size have been well reported in previous studies (see for example Pearson & Rosenberg, 1978; Méndez et al., 1998; Oug et al., 1998; Sánchez-Mata et al., 1999). These species are generally considered as indicators of organically enriched sediments. In such communities, perturbed by organic contamination, the frequency of disturbance is higher than the recovery rate, thus opportunistic species of small size and short lifetime will be favoured and could colonise such habitats with any type of biological competition. For this reason such species can be adapted to a high frequency of continuous disturbance. However, although Heleobia cf. australis was the most abun-

Anne 2.7 0 0.3 0 10.8 4.4 12.9 1.6 11.5 13.9

Chel 0 0 0 0 0 0 0 0 0 0

Crus 0 0 0 3.4 0.7 1.2 14.2 2.8 2.9 2.8

Moll 96.1 47.1 51.0 38.3 83.9 93.5 70.0 94.6 85.2 82.0

Phor 0 0 0 0 0 0 0 0 0 0

Echi 0 0 0 0 0 0 0 0 0 0

Hemi 0 0 0 0 0 0 0 0 0 0

Chor 0 0 0 0 0 0 0 0 0 0

dant (80% of the total abundance) and dominant macrobenthic species, many of the other species, especially the polychaetes Nephtys fluviatilis, Alitta succinea, Heteromastus similis and Goniadides sp., have been reported from organic enriched environments elsewhere (e.g. Dauer & Conner, 1980; Amaral et al., 1998; Arasaki et al., 2004). The high frequency of occurrence of these species in addition to the presence of large-bodied nematodes species retained on a 0.4 mm sieve would be related to the high organic content of the sediments. The lowest densities and species richness were always recorded in stations B, C and D (inner region of the bay). Besides the high organic content and chlor a content of these localities, reduced conditions in their surface sediments were recorded and their bottom water oxygen concentrations were very low (Table 1 and 2). Near these stations are the ANCAP refinery of hydrocarbons and the mouths of the Miguelete and Seco streams which constantly discharge immense amounts of wastes directly to water without any treatment García-Rodríguez et al., 2010). There is no doubt that the effects of such discharges combined with the poor hydrodynamic conditions of this portion of the bay have a great influence over bottom communities. On the other hand at stations. E, F, G, H and I species richness and faunal densities were higher than in the rest of the stations. St. E was at the mouth of the Pantanoso stream, where bottom currents are always high (personal observation of the authors) and the sediment has a large percentage of sand. Table 5. Environmental variables that showed highest Spearman’s correlation coefficients with the abundance patterns of organisms. Variable or combination of variables O2 (mg/l) O2 (mg/l) Chlor. a (mg/m2) O2 (mg/l) T ( 0C) O2 (mg/l) % M. Org O2 (mg/l) Z (m) O2 (mg/l) % clay

Spearman’s 0.667 0.559 0.493 0.482 0.45 0.441

MUNIZ & VENTURINI

Figure 5. Ordination diagram obtained from the canonical correspondence analysis (CCA), showing the main groups formed, the percentage of the total variation of the first two axes are indicated. A to Z = sampling stations; 1 to 12 = macrobenthic species (see code in table 3); OM = organic matter content; Chlor a = chlorophyll a content; Eh = redox values; Oxyg = dissolved oxygen content of the bottom water.

Possibly, because of these characteristics, the sediments were well oxygenated and so the macrobenthic species were under lesser stressful conditions than in the inner northeastern region of the bay. Because of the prevailing circulation patterns (Plata et al., 1992; Moresco & Dol, 1996), which are clockwise, it is reasonable to suppose that currents could transport waste discharges of the Pantanoso stream and deposit these wastes in other inner portions of the bay where the hydrodynamic energy is low. At stations F, G and I (outer southeast part of the bay) redox potential results showed that the sediment surface layer was aerobic, the bottom water was also well oxygenated and the organic matter content of the sediment was lower than in the inner part. Also this area had higher hydrodynamic exchange than the inner bay and according to Plata et al. (1992) the bay water is renewed through this entrance of the bay. These stations are probably better environments for the development of benthic organisms in the bay. St J was one of the most variable in the sampling period in terms of density and species richness, probably because it is located in the access channel to the port. The frequent dredging of this channel is a continuous source of disturbance for the bottom fauna, constantly altering its structure. On a temporal scale, the highest diversity, species richness and densities were recorded in autumn and winter 1997. Due to the increase in the hydro-

dynamic forces in these seasons the sediment column presented high levels of oxygenation and less organic content, making possible the better development of the benthic populations and the establishment of new species which were absent the rest of the year, such as Goniadides sp., Sigambra grubii and Glycera sp. Although diversity, density and species richness were variable through out the sampling year the dominance of the very abundant species, H. cf. australis, was clear and constant over the period of study. According to Turner et al. (1995), this fact could be reflecting community stability even when exposed to an important and significant disturbance, such as the constant increase of organic matter in an ecosystem. In shallow environments, changes in abundance and diversity can also be produced by climate factors. Storms produce the movement of bottom sediments, erosion and deposition, which are very important causes of infauna mortality (Posey et al., 1996). In Montevideo Bay this could be determining the variability observed in the subtidal community, however, storms in this area are not very severe, rather, they are more likely to be disturbances representing a positive factor contributing to the health and cleaning of the ecosystem. It is relevant to emphasise that, a large number of replicates were used in this study, in attempt to overcome criticisms

MACROBENTHIC COMMUNITIES IN MONTEVIDEO BAY

of this type of temporal analysis, that comparisons among the same station over time may be distorted by small-scale spatial variations, because the samples will not necessarily come from the same type of patch at each time of sampling (Morrisey et al., 1992 a; b). Although the bay was a very variable system it was possible to differentiate, by means of the cluster analyses and n-MDS ordination, discrete faunal associations, in regions with particular environmental characteristics. The cluster formed by stations B, C and D that showed less abundance and biomass of benthic organisms, corresponds to the inner part of the bay where environmental conditions are very unfavourable. Water circulation is limited; there is a high percentage of organic matter in sediments and a tendency to the presence of reduced sediments. The other cluster formed by the remaining stations A, E, F, G, H, I and J corresponds to regions of the bay which are heterogeneous but in general have more favourable environmental characteristics than in the inner region. At stations F and G particularly, the high water circulation and oxygenation of the sediment column and the smaller percentage of organic matter may be responsible for the great abundance and biomass of benthic organisms found. The advantage of ordination methods over cluster analysis is that the former shows the inter-relationships that exist between samples on a continuous scale while the latter tries to group samples in discrete clusters and is most appropriate to define groups of stations with a distinguishable community structure, in cases with strong pollution gradients. In this case the ordination method showed the same two groups obtained by the cluster analysis, which clearly confirms that the benthic community studied has, in other parts of the bay, a structure different from that in the most polluted inner part. This trend was also verified by the CCA analysis, in which basically the same two groups of stations were obtained. The four environmental variables identified in this analysis explained almost 28.2% of the variance in species data. Such a figure does not imply that the ordination was a poor representation (ter Braak, 1990). â&#x20AC;&#x153;Noiseâ&#x20AC;? in species data sets typically accounts for 10-50% of the total information (Gauch, 1982). As both the BIO-ENV procedure and CCA identified basically the same environmental variables influencing the benthic distributions, the results can be viewed with a reasonable degree of confidence. One should not forget, as was pointed out by Etter & Grassle (1992) and Clarke & Ainsworth (1993), that there can be no guarantee that highly correlated environmental variables are causative. Moreover, we can see clearly that largebodied nematodes were preferentially found in the group formed by the inner stations, in which the organic matter and chlorophyll a content was high, and also presented the highest bottom temperatures. On the other hand, we can distinguish two subgroups

within the second group formed in the cluster and MDS diagrams. One consists of stations I and J, characterised by the polychaetes Glycera sp. and S. grubii, and also by ostracods, isopods and balanus. In the other subgroup, that included stations. A, H, G, F and also E, N. fluviatylis, A. succinea, Goniadides sp., Erodona mactroides and H. similis were more abundant. The gastropod H. cf. australis showed more preference for the second group of stations but it was nearly in the centre of the CCA diagram. When compared to the training data set (Warwick & Clarke, 1993), all stations of Montevideo Bay appeared in the right extreme of the diagram, near stations C6 and C7 of Clyde sewage-sludge dump-ground. According to the authors, these two sites are situated close to the dump centre and showed signs of gross pollution. Most of the stations of Montevideo Bay are situated in the diagram at the same position of the stations mentioned above (C6 and C7), and two of them (B and D) are situated more to the right. This suggests that the bay could be a more severe and polluted environment than the most extreme sites used for making the training data set. The fact that the n-MDS configuration was not identical to the original in Warwick and Clarke (1993) could be reflecting that Montevideo Bay is also under the effects of other contaminants different from organic ones. In summary, the benthic fauna of Montevideo Bay has patterns of very low diversity and high dominance of a single species when compared to those estuaries of equal characteristics at similar latitudes. Although the only environmental variable measured that relate to anthrophogenic activities was organic matter content in bottom sediment, this and other factors appeared to be related to the distribution patterns of the benthic fauna and contributed to this general trend of low diversity and high dominance of the gastropod H. cf. australis. Spatially the Bay can be clearly divided in two regions with different faunal and environmental characteristics. These two regions are reflecting the difference in the degree of stress under what the Bay is subjected. Findings obtained in this investigation can be a basis for future studies in the region and emphasise the urgent necessity to establish an environmental monitoring system in the area. Since the concentration of dissolved oxygen was one of the critical variables detected in the present study and the organic load is very high, one of the first things that could be taken into account is to attempt to diminish the organic waste discharged into the bay, to allow a rise in oxygen concentration in the sediments, which would promote the settlement of more macrofauna. Finally, the Phylum level meta-analysis seems to be a good tool for assesses the pollution status of a coastal area, especially because it represents an important cost reduction in this type of research programs.

MUNIZ & VENTURINI

Acknowledgements This study was part of an integrated research project of the Sección Oceanología of Facultad de Ciencias supported by IMM (Intendencia Municipal de Montevideo) and CSIC (Comisión Sectorial de Investigación Científica). Authors thank ANII-SNI (National Agency) and the Dive Grou (GRUBU) of the Uruguayan Army for their support in the field sampling. Especial thanks are also due to M. Gómez, M. Rodríguez, G. Lacerot, A. Martínez, A. Carranza, B. Yanicelli and K. Sanz of the Facultad de Ciencias for their invaluable help in the field sampling and laboratory work. We wish to further acknowledge the help of Dr. Bob Clarke, who commented and made useful suggestions about this paper. Special thank to D. Siqueiros for his kind invitation to contribute to this volume of CICIMAR Oceánides. References Amaral, A.C.Z., E.H. Morgado & L.B. Salvador. 1998. Poliquetas Bioindicadores de Poluição Orgânica em Praias Paulistas. Revista Brasileira de Biologia, 58: 307-316. Arasaki, E., P. Muniz & A.M.S. Pires-Vanin .2004. A functional analysis o the enthic macrofauna of the São Sebastião Channel (southeastern Brazil). Marine Ecology, 25: 249 -263 Benvenuti, C.E., 1997. Benthic invertebrates, 43-46. In: Subtropical convergence marine ecosystem. The coast and the sea in the warm temperate southwestern atlantic, (ed.) U. Seeliger, C. Odebrecht and J.P. Castello, New York: Springer Verlag, Heidelberg. Bray, J.R. & J.T. Curtis. 1957. An ordination of the upland forest communities in southern Wisconsin. Ecological Monographs, 27,:325-349. Byers, S.C., E.L. Mills & P.L. Stewart. 1978. A comparison of methods to determining organic carbon in marine sediments, with suggestion for a standard method. Hydrobiologia, 58, 43-57. Clarke, K.R. & M. Ainsworth: 1993. A method of linking multivariate community structure to environmental variables. Marine Ecology Progress Series, 92: 205-219.

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CICIMAR Oceánides 30(1): 21-32 (2015)

SURVIVAL OF JUVENILE WHITE MULLET Mugil curema (MUGILIDAE) IN A COASTAL LAGOON Quiñonez-Velázquez, C., J. R. López-Olmos & C. I. Pérez-Quiñonez

Centro Interdisciplinario de Ciencias Marinas. Av. Instituto Politécnico Nacional S/N, Col. Playa Palo de Santa Rita, A.P. 592. La Paz, B.C.S, México. C.P. 23096. email: cquinone@ipn.mx

ABSTRACT. The hatching date frequency distribution (HDFD) of juvenile white mullet (Mugil curema) sampled at monthly intervals in the coastal lagoon Ensenada de La Paz, B.C.S., México from May 1997 to May 1998 was reconstructed for different age intervals and corrected for differences in the accumulated mortality. The ratio of the HDFD at a given age to the HDFD at an earlier age was used as an index of the relative survival of juveniles grouped into 14-day hatch-date cohorts. The results show that the white mullet spawns during the whole year in Bahía de La Paz, with the highest survival of larvae from October to February. Variations of the relative survival of the age groups were correlated significantly to the variations in growth. A fast growth resulted in high survival while a slow growth yielded low and high survival. The latter was observed when the juveniles used the lagoon after 40-days-old, indicating that the area is used mainly as a refuge. The return to the coastal area can explain the decrease of juveniles older than 80 days in the lagoon.

Keywords: Baja California Sur, Mugil curema, juveniles, otolith, hatch date frequency distribution.

Supervivencia de juveniles de lisa Mugil curema (Mugilidae) en una laguna costera

RESUMEN. A partir de la edad calculada en días se reconstruyó la distribución de frecuencias de fechas de nacimiento (HDFD por sus siglas en Inglés) para diferentes intervalos de edad de juveniles de lisa (Mugil curema) recolectados a intervalos mensuales en la laguna costera Ensenada de La Paz, BCS (México), de mayo 1997 a mayo 1998. Las HDFD fueron corregidas por diferencias en la mortalidad acumulada. El cociente de la HDFD a una edad determinada entre la HDFD a una edad previa se utilizó como índice de supervivencia relativa de juveniles agrupados en cohortes de 14 días de fechas de nacimiento. Los resultados muestran que la lisa desova durante todo el año en la Bahía de La Paz, con la mayor supervivencia larval de octubre a febrero. Las variaciones de la supervivencia relativa de los grupos de edad se correlacionaron significativamente con las variaciones en el crecimiento. Un rápido crecimiento genera una elevada supervivencia y un lento crecimiento genera baja y/o alta supervivencia. Eso se observó cuando los juveniles permanecen en la laguna después de 40 días de edad, lo que sugiere que la zona se utiliza principalmente como un refugio. El regreso a la zona costera se relacionó con la disminución de juveniles mayores a 80 días de edad en la laguna.

Palabras clave: Baja California Sur, Mugil curema, juveniles, otolito, distribución de frecuencias de fechas de nacimiento. Quiñonez-Velázquez, C., J. R. López-Olmos & C. I. Pérez-Quiñonez. 2015. Survival of juvenile white mullet Mugil Curema (Mugilidae) in a coastal lagoon. CICIMAR Oceánides, 30(1): 21-32.

INTRODUCTION The white mullet Mugil curema (Valenciennes, 1836) is distributed on both coasts of the American continent. In the Pacific it is found from Magdalena Bay on the western coast of the Baja California Peninsula and from the Gulf of California to Chile (Harrison, 1995). Reproduction takes place off the coast and the juvenile enter estuaries and coastal lagoons following turbid gradients (Blaber, 1987; Trape et al., 2009). Sometimes they enter rivers and coral reefs (Lozano-Cabo, 1978). Bays, coastal lagoons, inlets, and swamp areas are used by various species as refuge and nursery areas where the organisms remain for several months (Odum, 1972; Yánez-Arancibia, 1976; González-Acosta, 1998; Ross, 2003). In Ensenada de La Paz, a coastal lagoon on the southwest coast of the Gulf of California (Fig. 1), the white mullet is a common species all year, showing a bimodal length distribution (GonzálezAcosta, 1998) and suggesting two recruiting groups. Growth in this species is rapid, achieving 25-cm length in one year (Martin & Drewry, 1978). An Fecha de recepción: 11 de febrero de 2015

important step in the study of fish larvae and juvenile growth and mortality was the discovery of daily growth increments in the otoliths (Pannella, 1971). The daily nature of the growth increments was confirmed for the first time by Brothers et al. (1976) in the northern anchovy (Engraulis mordax). The daily deposition of the growth increments has since been described for many fish species (Wild & Foreman, 1980; Brothers & McFarland, 1981; Campana & Neilson, 1985; Jones, 1986; Stevenson & Campana, 1992). Radtke (1984) validated the deposition of daily growth increments in otoliths of the striped mullet (Mugil cephalus) and we assume that this is also true for Mugil curema. The analysis of the otolith microstructure has shown its utility in the determination of age, identification of periods of stress, assessing individual growth, and estimation of the birth date of fish larvae (Wright & Bailey, 1996). A great portion of the fish larvae cohort does not survive the first year (Gulland, 1965). In consequence, studies of the abundance variations in fish populations have Fecha de aceptación: 13 de abril de 2015

QUIÑONEZ-VELÁZQUEZ et al.

concentrated on the first months of life (Campana, 1996; Quiñonez-Velázquez, 1997). The differences between individuals that survive up to different stages of their ontogenetic development can be identified through the birth date frequency distribution of the survivors; this allows to study the conditions associated with favorable periods of growth and survival (Yoklavich & Bailey, 1990). The white mullet spawning season in Bahía de La Paz takes place during several months in spring-summer (Chávez, 1985). Because of this long spawning period, the larvae will be born under different environmental conditions (feeding and predation) that will influence the growth rate and the probability of survival. By using the age in days and the capture date it is possible to estimate the birth date of the larvae and surviving juveniles at different stages of development. Also, by knowing the biotic and abiotic conditions during hatching periods, we can characterize the most favorable periods for the larval survival (Methot, 1983). The objective of this study is to identify the favorable periods for growth and survival of the white mullet (Mugil curema) larvae captured in El Conchalito inlet, located within Ensenada de La Paz, from May 1997 to May 1998, based on the juvenile hatch dates frequency distribution (HDFD). MATERIAL AND METHODS Juvenile white mullet (Mugil curema) were captured over 13 monthly samplings in El Conchalito inlet located at the southeast end of the Ensenada de La Paz (24°07’53” N and 110°21’00” W) (Fig. 1). Samplings were made from May 1997 to May 1998 during the highest tide of the month. Overall, 1019 specimens were collected. In order to catch the greatest size interval of juveniles in the lagoon, three types of nets were used: a fixed Flume Net 30-m long, 1.5-m high with a central cod-end 4-m long (6-mm mesh); and two seine nets, 15-m long, 1.5-m high with a central cod-end 2-m long (6- and 3-mm mesh). During each sampling, water surface temperature and salinity were recorded using a Horiba U-10 portable analyzer. Sampled fish were put on ice inside labeled polyethylene bags until transported to the laboratory where the white mullet were separated from the rest of the fish and preserved in 96% alcohol. Fish were identified at species level using a morphological key (Harrison, 1995; Trape et al., 2009). To assess a potential effect of alcohol on the juvenile size, a subsample was selected to measure their standard length (SL ± 0.01 mm) using an electronic vernier caliper, and were then preserved individually in 96% alcohol until the measurement of the whole fish sample.

Figure 1. Location of study area. El Conchalito inlet is part of Ensenada de La Paz, which is on the southwest margin of the Gulf of California.

From the total of juveniles sampled monthly a subsample was selected at random choosing up to three juveniles of each 0.5 mm SL interval. The otoliths (sagittae) of the selected fish were extracted for age determination. Both otoliths were mounted on the same glass slide using instant glue. For age determination, the right otolith was used. To make evident the growth increments in the otoliths (Fig. 2), we grinded them on the sagittal plane to the nucleus using paper of 0.2 to 12 µm grit size. Growth increments were counted by two readers. The maximum error among readings was 6% and the average was used to assign the age (Suthers & Sundby, 1993). We assumed that the hatching mark is the first formed and the daily deposition growth increments according to Radtke (1984) for the striped mullet (Mugil cephalus). The same approach was used by Marín-Espinoza (1996). With the purpose of transforming the size structure of juveniles in age structure, an age-length key per month was constructed. Thus, age was assigned to juveniles for which otoliths were not read. The hatching dates of juvenile white mullet were obtained by subtracting the age from the date of capture. The juvenile fish hatched within 5-day intervals were grouped into cohorts and the absolute frequency for each cohort (number of juveniles) was calculated. The HDFD was constructed for all the juveniles and for age-group intervals of 20-40, 4060, 60-80, and >80 days old. The total mortality rate (Z) was estimated using the catch curve in linear form (Sparre & Venema, 1995) at constant time intervals (5 days), LnC(t1,t2) = g – Zt, where LnC(t1,t2) = natural logarithm of the total catch (frequency of juveniles) in

SURVIVAL OF JUVENILE Mugil curema

the age interval t1 to t2; g = constant and is the intercept to the origin of the regression line, Z = slope of the line and total mortality, and t = upper limit of the age interval. This mortality rate was used for each 5-day cohort of the different age groups. For a given age interval, the over representation of late births in the HDFD was corrected by reduction of the 5-day cohort abundance in proportion to the average mortality that should have occurred during the time between the age average of the juvenile in the 5-day cohort and the upper limit of the age interval considered. The survival of a 5-day cohort was defined as the change of the number of juveniles of the cohort of an age-group from the same cohort of the previous age-group (rate between the corrected frequencies). When the result was >1 (late incorporation to the lagoon), a 1 was assigned. To obtain an estimate of the cohort mortality, the value of survival was subtracted from one.

was not necessary to correct the length after alcohol preservation. The white mullet juveniles ranged from 16 to 42 mm SL and 22 to 109 days old (Table 1). The subsample of juveniles selected for age determination was 254 and represent 25% of the total captured. The otolith microstructure showed opaque and hyaline zones and, according to Radtke (1984) these represent a daily growth increment (Fig. 2). The average water surface temperature ranged between 17.9 to 31.8 °C. Maximum temperature values coincided with the juvenile fish that had the longest lengths (29 to 37 mm SL) and greatest ages (67 to 90 days).

RESULTS

The monthly length-frequency distribution depicts two fish groups that show an increasing trend in average length (Fig. 3). The first group covers from May to September 1997 and the second one from October 1997 to May 1998. The average range of length of the fish in the groups are similar (KStest, P > 0.05), 24-34 mm SL and 27-33 mm SL. The monthly age frequency distribution shows the same trend as the length frequency distribution, integrating two fish groups between 20 and 100 days of age (Fig. 4). The younger fish were observed in July 1997 and from December 1997 to April 1998, matching the smaller juvenile white mullet in the lagoon.

The fresh length (range 16-34 mm SL) and length after the preservation in alcohol of 101 juveniles were compared to evaluate the effect of the alcohol during preservation. The potential effect was explored through the regression SLf =a+b*SLp, where SLp is the preserved length and SLf is the fresh length. The slope was 0.99 and is not significantly different from 1 (P > 0.01). In consequence, it

The juvenile hatch dates frequency distribution (HDFD), indicates that white mullet spawn throughout the year in Bahía de La Paz (Fig. 5). The maximum hatch frequency was detected in autumnwinter. In other periods frequencies were < 20 juveniles. A good relationship was observed between the HDFD and temperature (Fig. 5A) (n = 11, P < 0.05, Spearman-Correlation) and with tidal height

For the comparison between the growth and the mortality of the juvenile white mullet in Ensenada de La Paz, the hatch date intervals were extended to 14 days. The purpose of extending the interval was to reduce the empty periods in the distributions of the age groups of 20-40, 40-60, and 60-80 days.

Table 1. Range and average standard length and age of the white mullet (Mugil curema) captured in the inlet El Conchalito, May 1997 to May 1998. Date Temp Juvenile Range Average Age subSL Age SL Age (°C) Total sample (mm) (days) (mm) (days) 23 May 97

25.8

24.7-40.8

55-76

28.1

29 Jun 97 20 Jul 97 19 Aug 97 17 Sep 97 18 Oct 97 15 Nov 97 13 Dec 97 13 Jan 98 11 Feb 98 12 Mar 98 14 Apr 98 12 May 98 Total

29.4 30.4 31.5 31.8 28.1 24.4 17.9 18.9 19.5 21.0 19.0 26.1

21 27 5 31 46 177 148 48 310 87 85 1019

11 15 5 13 13 33 35 23 35 29 24 254

16.6-36.9 22.9-37.6 29.1-36.9 24.8-32.3 22.8-30.5 15.9-34.1 16.1-34.2 17.7-29.9 18.4-42.1 19.2-39.3 25.5-41.4

28-90 43-86 67-91 52-75 45-70 22-78 26-69 29-74 29-109 28-94 51-98

25.9 29.1 33.9 28.0 26.3 27.2 26.0 22.8 31.0 30.6 32.3

56 66 81 64 61 60 55 47 69 67 74

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Figure 2. Microstructure of the right sagittae of a white mullet juvenile (Mugil curema) 22 mm SL and 45 days old, at 400X (a) and 200X (b) magnifications; with transmitted light.

(Fig. 5B) (n = 26, P < 0.01, Spearman-Correlation). These two variables showed an inverse seasonal pattern. When the temperature was at a minimum during December-January, the tidal height was at a maximum. During the same months, the HDFD also showed maxima. This HDFD includes the overrepresentation of juveniles that were born close to the sampling dates. These juveniles have been exposed to causes of mortality (changes in the food availability, predation) for a smaller period than juveniles born earlier. To reduce this effect of overrepresentation, we estimated a decreased rate (mortality) in relation to age (Fig. 6). The age structure of the total juvenile catch had a normal distribution (P = 0.15). To estimate the mortality rate, the decreasing part of the age distribution was used from 70 days being obtained a value of 0.04 for mortality at each 5-days interval. The white mullet juveniles were divided into four age groups (20-40, 40-60, 60-80, >80 days) and their corrected HDFD was obtained (overrepresentation) (Fig. 7). The HDFD of juvenile age-group 20-40 days is different from the HDFD of juveniles

40-60 days. The latter age-group is similar to the HDFD of juvenile of 60-80 days. The HDFD of juveniles > 80 days is different from the previous agegroups and was little represented. These results suggest the mortality of the juvenile stabilizes after the juveniles were incorporated into the lagoon, on the average at an age of 30 days. The form of the HDFD of the juveniles > 80 days we infer is the result of the differential return of the juveniles to the coastal area and not caused by mortality. Few juvenile fish survivors were born during spring-summer 1997 in all four age groups. This suggests that the survival conditions were more favorable during autumn 1997 and winter 1997-98 for these young juveniles. The juveniles from 40-60 days (n = 233) and 60-80 days (n = 457) had similar HDFDÂ´s (representing the same 5-days cohorts). Also, this suggests juveniles of these ages were vulnerable to sampling during the year. In the age group > 80 days (n = 236), the hatch dates were the same as in the two previous age groups, but their small representation is the result of an asynchronous exit from the lagoon, to about 80 days. To explore the

SURVIVAL OF JUVENILE Mugil curema

Figure 3. Monthly distribution of length frequency (%) of white mullet juvenile (Mugil curema) captured in Ensenada de La Paz, May 1997 to May 1998.

relationship between growth and mortality, the juveniles were regrouped into 14-day hatch intervals (Fig. 8). Two important events were noted. The first is a significant relationship between growth rate and mortality (r = 0.60, P < 0.05). Juveniles with high growth rates had low mortality. The second is that the values to the left side of the broken line (> 40 days) correspond to the juveniles during their stay in the lagoon, mainly juvenile fish between 40 and 60 days old. These have a lower growth rate and low and high values of mortality. This suggests that the fish that enter the lagoon are those of rapid growth during their early life in the coastal zone (< 20 days) and a decrease in growth during their residency in the lagoon (after 40 days) with variable survival. This suggests white mullet juveniles use the lagoon mainly as a refuge area. DISCUSSION As a survival strategy, the white mullet enters estuaries and lagoons thus avoiding potential predators in the coastal area. The lengths best represented during the study in Ensenada de La Paz were 29-30 mm SL. However, in Bahía de La Paz the lengths

best represented were 70-90 mm SL (Chávez, 1985). This suggests that the presence of juveniles <50 mm SL in the inlet is a consistent pattern and those juveniles will have greater possibilities of survival in protected areas such as lagoons than in the coastal zone. This was most evident for juveniles between 40 and 60 days of age by increasing survival during residence in the inlet, however episodes of high mortality are also present. Individuals >80 days (45-50 mm SL) were little represented in the lagoon because of having begun their return to the coastal zone. This is reinforced by the results of Chávez (1985) who recorded a minimum frequency of fish < 50 mm SL and a significant number > 50 mm SL. The migration of Mugil curema towards the coastal area should happen after metamorphosis (Yánez-Arancibia, 1976; Marín-Espinoza, 1996). The structures of length and age analyzed suggest spawning occurs outside of the coastal zone, and juveniles recently metamorphosed require 20 to 40 days to enter Ensenada de La Paz. We assume white mullet juveniles, with ages between 40 and 80 days are permanent residents in the bay and show

QUIÑONEZ-VELÁZQUEZ et al.

Figure 4. Monthly distribution of age frequencies (%) of white mullet juvenile (Mugil curema) captured in Ensenanda de La Paz, May 1997 to May 1998.

seasonal changes in abundance. Also, juveniles captured in El Conchalito inlet appropriately represent the fish group inside the bay. The smallest sizes of white mullet were captured in December 1997 and January 1998 and were in the 16 mm SL interval. Ditty and Shaw (1996) recorded for the north of the Gulf of Mexico shelf white mullet larvae and juvenile with lengths between 2-26 mm SL. We assume the white mullet juveniles move toward bays and lagoons after metamorphosis, [7.0 to 7.2 mm SL, Martin & Drewry, 1978] and on average they will take 30 days during which they will grow until reaching 16 mm SL. Ditty and Shaw (1996) also mention the presence of eggs of white mullet that demonstrates that the species spawns outside of the coastal zone. The presence of defined groups of juveniles with 30 mm SL and 60 days on average during MaySeptember 1997 and from October 1997 to May 1998 suggests the existence of two reproduction peaks per year, similar to that found along the coasts of Venezuela by Marín-Espinoza (1996). In our work, the biggest catch of white mullet juveniles in Ensenada de La Paz was during

autumn-winter. However, González-Acosta (1998) mentions that the abundance of white mullet juveniles < 50 mm SL decreases in winter. This difference could have been caused by the increase of the sea surface temperature from El Niño1997-98 with a potential increase in the development of eggs and yolk-sac larvae, because the temperature has a direct effect on the development time of the embryo and of the yolk-sac larvae (Ahlstrom & Moser, 1976). This potential increase in the development rate during the larval stage could increase survival, previous to metamorphosis, with an increase of the number of juveniles <60 days of age from December 1997 to April 1998. In this sense, Houde (1987) notes that an increase in the growth rate during the larval stage allows the larvae to avoid a larger number of potential planktonic predators, increasing survival because of a reduction of the time the larvae are available to predators. Another possible cause of the increase of juvenile survival in December 1997 to January 1998 is the presence of the highest tides during the year, increasing the volume of water flooding the lagoon and thus increasing the available habitat. The interannual differences of the winter tides define the re-

SURVIVAL OF JUVENILE Mugil curema

Figure 5. Hatch date frequency distribution of the white mullet (Mugil curema) captured in Ensenada de La Paz, May 1997 to May 1998, relationship to the seasonal change of the water surface temperature (A), and to the tide-height (B).

cruitment of the white mullet in El Conchalito inlet. Victor (1983) recorded a positive relationship of the number of juveniles of Thalassoma bifasciatum entering the reef area at Isla San Blas, Panama with the periods of full moon. The biggest recruitment to the reef was during October-November 1980. According to the above M. curema has a permanent presence of juveniles in Ensenada de La Paz (González-Acosta, 1998). Specifically, based on the HDFD, the spawning of white mullet in Bahía de La Paz occurs throughout the year. This is similar to that reported by Marín-Espinoza (1996) for the same species along the coasts of Venezuela. This author found the maximum hatching periods during December-March, coinciding with cool temperatures, as a consequence of a moderate upwelling event. In our work, the maximum HDFD also coincides with the lowest seasonal temperature and with maximum tidal height. Reproduction all year round in tropical species is common (Bond, 1979; Lagler et al., 1984) and the spawning of many species related to upwelling events has also been documented (Cury & Roy, 1989; Suthers & Sundby, 1993; Marín-Espinoza, 1996).

Turbidity of the water causes low intensity of light at shallow depths. This has been noted as an important ecological factor in the survival of fish that use estuaries and lagoons during their early life (Blaber & Blaber, 1980). Blaber (1987) noted, for the Africa coast, that to avoid predators mullets enter and leave the lagoons along the bottom, taking advantage of the tidal currents that increase water turbidity and reduce light penetration. Because Ensenada de La Paz is shallow, the action of tides associated with winds will create currents that generate much turbidity. In consequence, the entrance and exit of juvenile fish associates to the tidal pattern. This mechanism will maximize survival in winter when the highest tides occur increasing the flooded area and the available habitat. In Ensenada de La Paz, turbidity of the water and variations in temperature are caused by tides. Tides are the most important factor in the hydrological dynamics of the inlet, allowing the formation of a specific habitat (González-Acosta, 1998). The turbidity during the ebb of the tide creates a refuge area against fish predation (Blaber & Blaber, 1980;

QUIĂ&#x2018;ONEZ-VELĂ ZQUEZ et al.

Figure 6. Age structure of the total number of white mullet juvenile (Mugil curema) captured in Ensenada de La Paz, May 997 to May 1998.

Figure 7. Correction for difference in the accumulated mortality for 5-day cohorts and for age groups. The sum of the empty and filled bars are the frequency without correction and the filled bar represents the corrected frequency. The values above the histograms are the age average for the cohort.

SURVIVAL OF JUVENILE Mugil curema

Figure 8. Relationship of the growth and mortality rates for age groups of the white mullet (Mugil curema) captured in Ensenada de La Paz, May 1997 to May 1998.

Blaber, 1987), because predation is a function of the distance at which the prey will be detected visually and of predator size (Folkvord & Hunter, 1986). These conditions and the results of growth and survival in the present study suggest that Ensenada de La Paz is used mainly as a refuge by juvenile Mugil curema ranging from 20 to 80 day old. Acknowledgements We thank the collaboration of Gustavo de la Cruz Agüero and Adrián González Acosta during the sampling, Minerva G. Torres Alfaro for her support in the programming, sampling, and in the separation of the material in laboratory. We sincerely appreciate the effort of an anonymous reviewer who made many important suggestions that improved the current version of the manuscript. CQV is member of COFAA and EDI of the Instituto Politécnico Nacional. REFERENCES Ahlstrom, E. H. & H. G. Moser. 1976. Eggs and larvae of fishes and their role in systematic investigations and in fisheries. Rev. Trav. I.S.T.P.M., 40: 379-398. Blaber, S.J.M. 1987. Factor influencing recruitment and survival of mugilids in estuaries and coastal waters of Southeastern Africa. 507-518. In: Dadswell, M., R. Klauda, C. Saunders, R. Rulifson & J. Cooper (eds.), Common strategies of anadromous and catadromus fishes. Proceedings of an International Symposium held in Boston. Massachusetts, USA. March 9-13, 1986.

Blaber, S.J.M. & T.G. Blaber. 1980. Factors affecting the distribution of juvenile estuarine and inshore fish. J. Fish Biol., 17: 143-162. Bond, C. E. 1979. Biology of fishes. Saunders College Publishing, Philadelphia, U.S.A. 514. Brothers, E. B., C. P. Mathews & R. Lasker. 1976. Daily growth increments in otoliths from larval and adult fishes. Fish. Bull. U.S., 74: 1-8. Brothers, E. B. & W. N. McFarland. 1981. Correlation between otolith microstructure, growth, and life history transitions in newly recruited French grunts [Haemulon flavolineatum (Demarest), Haemulidae], 369-374. In: R. Lasker and K. Sherman (eds.), The early life history of fish. Rapp. R.-v. Réun. Cons. int. Explor., Mer. 178. Campana, S.E. 1996. Year-class strength and growth rate in young Atlantic cod Gadus morhua. Mar. Ecol. Prog. Ser., 135: 21-26. Campana, S. & J.D. Neilson. 1985. Microstructure of fish otoliths. Can. J. Fish. Aquat. Sci., 42: 1014-1032. Chávez, H. 1985. Aspectos biológicos de las lisas (Mugil spp.) de Bahía de La Paz, B.C.S., México, con referencia especial a juveniles. Inv. Mar. CICIMAR, 2(2): 1-112. Cury, P. & C. Roy. 1989. Optimal environmental window and pelagic fish recruitment success in upwelling areas. Can. J. Fish. Aquat. Sci., 46: 670-680.

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Ditty, J.G. & R.F. Shaw. 1996. Spatial and temporal distribution of larval Striped Mullet (Mugil cephalus) and White Mullet (M. curema, Family: Mugilidae) in the northern Gulf of Mexico, with notes on Mountain Mullet, Agnostomus monticola. Bull. Mar. Sci., 59: 271 - 288. Folkvord, A. & J. R. Hunter. 1986. Size-Specific vulnerability of northern anchovy, Engraulis mordax, larvae to predation by fishes. Fish. Bull.U.S., 84: 859-869. González-Acosta, A. 1998. Ecología de la comunidad de peces asociada al manglar del estero El Conchalito, Ensenada de La Paz, Baja California Sur, México. M.Sc. thesis CICIMAR, La Paz, B.C.S., México. 126 p. Gulland, J. A. 1965. Survival of the youngest stages of fish, and its relation to year class strength. ICNAF Spec. Bull., 6: 363-371. Harrison, I. J. 1995. Mugilidae. Lisas. 1293-1298, In: W. Fischer, F. Krupp, W. Schneider, C. Sommer, K.E. Carpenter and V. Niem (eds.) Guia FAO para Identification de Especies para los Fines de la Pesca. Pacífico Centro-Oriental. 3 Vols. FAO, Rome. Houde, E. D. 1987. Fish early life dynamics and recruitment variability. Am. Fish. Soc. Symp. 2: 17-29. Jones, C. 1986. Determining age of larval fish with the otolith increment technique. Fish. Bull.U.S., 84: 91-103. Lagler, K. F., J. E. Bardach, R. R. Miller & D. R. May-Passino. 1984. Ictiología. AGT Editor. S. A. México. 489p. Lozano-Cabo, F. 1978. Oceanografía, Biología Marina y Pesca. Tomo II Cuarta parte: La flora y la fauna marinas. Paraninfo. Madrid. 391p. Marín-Espinoza, B. J. 1996. Transport et recrutement du muge argenté, Mugil curema, dans une lagune côtière tropicale. Doctorate thesis. Laval University, Quebec, Can. 120 p. Martin, F. D. & G. E. Drewry. 1978. Development of fishes of the Mid-Atlantic Bight. An atlas of eggs, larval and juvenile stages. VI. Stromateidae through Ogcocephalidae. Power Plant Project. Biological Services Program. Fish and Wildlife Service, U. S. Departament of the Interior. 346 p. Methot, R.D. 1983. Seasonal variation in survival of larval Northern Anchovy, Engraulis mordax, estimated from the age distribution of juveniles. Fish. Bull. U.S., 81: 741-750.

Odum, W. E. 1972. Utilization of the direct grazing and plant detritus food chains by the striped mullet, Mugil cephalus. 222-240. In: Steele, J.H. (ed.) Marine Food Chains. Univ. Calif. Press. Berkeley. Pannella, G. 1971. Fish otoliths: daily growth layers and periodical patterns. Science, 173: 11241127. Quiñonez-Velázquez, C. 1997. Survie relative et trajectoires de croissance de la ponte à la métamorphose chez la goberge Pollachius virens et l’aglefin Melanogrammus aeglefinus du Plateau Néo-Ecossais. Doctorate thesis, Laval University, Québec, Canadá. 136 p. Radtke, R. L. 1984. Formation and structural composition of larval striped mullet otoliths.Trans. Amer. Fish. Soc. 113: 186-191. Ross, S. W., 2003. The relative value of different estuarine nursery areas in North Carolina for transient juvenile marine fishes. Fish. Bull., 101: 384–404. Sparre, P. & S. V. Venema. 1995. Introducción a la evaluación de recursos pesqueros tropicales. Parte 1. FAO, Documento Técnico de Pesca 306.1 Rev.1, 440 p. Stevenson, D.K. & S.E. Campana [ed.]. 1992. Otolith microstructure examination and analysis. Can. Spec. Publ. Fish. Aquat. Sci., 117-126. Suthers, I. M. & S. Sundby. 1993. Dispersal and growth of pelagic juvenile Arcto-Norwegian cod (Gadus morhua) inferred from otolith microstructure and water temperature. ICES J. Mar. Sci., 50: 261-270. Trape, S., J. D. Durand, F. Guilhaumon, L. Vigliola & J. Panfili. 2009. Recruitment patterns of young-of-the-year mugilid fishes in a West African estuary impacted by climate change. Estuar. Coast. Shelf S., 85: 357–367. Victor, B. C. 1983. Recruitment and population dynamics of a coral reef fish. Science, 219: 419420. Wild, A. & T. J. Foreman. 1980. The relationship between otolith increments and time for yellowfin and skipjack tuna marked with tetracycline. Inter-Am. Trop. Tuna Comm. 17: 509-597. Wright, P.J. & M.C. Bailey. 1996. Timing of hatching in Ammodytes marinus from Shetland waters and its significance to early growth and survivorship. Marine Biology. 126: 143-152.

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Yánez-Arancibia, A. 1976. Observaciones sobre Mugil curema Valenciennes en áreas naturales de crianza, México. Alimentación, crecimiento, madurez y relaciones ecológicas. An. Centro Cienc. del Mar y Limnol., Univ. Nac. Autón. Méx., 3: 92-124. Yoklavich, M. M. & K. M. Bailey. 1990. Hatching period, growth and survival of young walleye pollock Theragra chalcogramma as determined from otolith analysis. Mar. Ecol. Prog. Ser., 64: 13-23.

CICIMAR Oceánides 30(1): 33-43 (2015)

Dry weight, carbon, C/N ratio, hydrogen, and chlorophyll variation during exponential growth of selected microalgae species used in aquaculture Pérez-Morales, A.1,2, A. Martínez-López2 & J. M. Camalich-Carpizo3

Instituto de Ciencias Marinas y Pesquerías, Universidad Veracruzana. Calle Hidalgo No. 617, Col. Río Jamapa, C.P. 94290. Boca del Río, Veracruz, México. Telephone and Fax: + (52) (229) 956-70-70 . 2 Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, Departamento de Plancton y Ecología Marina. Av. IPN s/n, C.P. 23096. La Paz, Baja California Sur, México. 3 IMARES Wageningen UR, PO Box 167, 1790AD, Den Burg (Texel), The Netherlands. email: alfredperezmorales@gmail.com 1

ABSTRACT. Microalgae are commonly used as food source in aquaculture, mainly for shellfish and larvae of crustacean and fish. All hatcheries need an excellent inoculum to produce high-quality microalgae when cultured outdoor in extensive systems, and this depends largely on the health of the microalgae cultured under laboratory conditions as a primary step. Therefore, the aim of this work was to assess variations of dry weight, carbon, C/N ratio, hydrogen and chlorophylls as physiological indicators of nutrients uptake and growth rate during exponential growth of Isochrysis galbana, Chaetoceros calcitrans and Dunaliella tertiolecta, using f/2 as culture medium. Chaetoceros calcitrans and D. tertiolecta had higher carbon content (~30 pg cell-1). The C/N ratio varied widely, gradually decreasing on I. galbana. Chlorophyll a varied among the three microalgae tested, ranging from <0.05 to >0.25 pg cell-1. Growth rate was higher in I. galbana (K’ 0.83) followed by D. tertiolecta and C. calcitrans. Results showed that nutrient incorporation by cell change when cell density increases; this information provides new insights in the physiology of marine microalgae and confirms that nutrient uptake dynamics is different in each microalga species. Finally, this study indicates that using one culture medium is not equally efficient for all microalgae used in aquaculture since each species has specific nutritional requirements.

Keywords: Carbon, C/N ratio, Chlorophyll, Growth rate, Microalgae.

Variación de peso seco, carbono, relación C/N, hidrógeno y clorofilas durante el crecimiento exponencial de especies selectas de microalgas utilizadas en acuacultura RESUMEN. Las microalgas son comúnmente utilizadas como fuente de alimento en acuacultura, principalmente para cultivo de moluscos y para las fases larvarias de crustáceos y peces. Los criaderos de larvas necesitan un excelente inóculo para producir microalgas de alta calidad cuando se cultivan al exterior en sistemas extensivos; esto depende principalmente de la salud de las microalgas cultivadas bajo condiciones de laboratorio como primer paso. Por lo tanto, el objetivo de este trabajo fue evaluar variaciones de peso seco, carbono, relación C/N, hidrógeno y clorofilas como indicadores fisiológicos de la asimilación de nutrientes y tasa de crecimiento durante el crecimiento exponencial de Isochrysis galbana, Chaetoceros calcitrans y Dunaliella tertiolecta, usando f/2 como medio de cultivo. Chaetoceros calcitrans y D. tertiolecta presentaron el mayor contenido de carbono (~30 pg cél-1). La relación C/N varió ampliamente, decreciendo gradualmente en I. galbana. La clorofila a fue la que más varió entre las tres microalgas evaluadas, en el intervalo de <0.05 a >0.25 pg cél-1. La tasa de crecimiento fue mayor en I. galbana (K’ 0.83) seguido por D. tertiolecta y C. calcitrans. Los resultados mostraron que la incorporación de nutrientes por célula cambia cuando la densidad celular se incrementa; esta información provee nuevo conocimiento sobre la fisiología de microalgas marinas y confirma que la dinámica de incorporación de nutrientes es diferente en cada especie de microalga. Por último, este estudio indicó que el uso de un solo medio de cultivo no es igualmente eficiente para todas las microalgas usadas en acuacultura, debido a que necesitan requerimientos nutricionales específicos.

Palabras clave: Carbono, Clorofila, Microalgas, Relación C/N, Tasa de crecimiento. Pérez-Morales, A., A. Martínez-López & J. M. Camalich-Carpizo. 2015. Dry weight, carbon, C/N ratio, hydrogen, and chlorophyll variation during exponential growth of selected microalgae species used in aquaculture. CICIMAR Oceánides, 30(1): 33-43.

INTRODUCTION Microalgae play a key role in aquaculture development, they are the main food source for many farm-raised marine fish, crustaceans, and shellfish, as well as for hatchery-raised zooplankton (rotifers, copepods, cladocerans, and brine shrimp) that serve as food for the larvae of farm-raised marine animals (Brown et al., 1999; Muller-Feuga, 2000; PérezMorales, 2006). All hatcheries need an excellent inoculum to produce high-quality microalgae when cultured outdoor in extensive systems, and this depends largely Fecha de recepción: 02 de marzo de 2015

on the health of the microalgae cultured under laboratory conditions as a primary step (Abu-Rezq et al., 1999; Lourenço et al., 2002; Banerjee et al., 2011). Several factors, such as temperature, salinity, pH, light intensity, photoperiod and aeration can alter the health and the quality of a microalgae culture, but the most important factor depends largely on the appropriate media culture with an adequate amount of nutrients and minerals for each microalgae species (Gopinathan, 1986; Keller et al., 1987; Geider & La Roche, 2002; Lananan et al., 2013). The most popular media used for the culture of Fecha de aceptación: 23 de abril de 2015

PÉREZ-MORALES et al.

marine microalgae are Artificial Sea Water, Conway, ESM, Erd-Schreiber, f/2, K, L1, Miquel’s, and Walne’s medium (Miquel, 1890; Guillard & Ryther, 1962; Okaichi et al., 1982; Keller & Guillard, 1985; Kaplan et al., 1986; Keller et al., 1987; Guillard & Hargraves, 1993; Tompkins et al., 1995). These media show significant variations in mineral composition such as nitrogen or phosphorus compounds, incorporation of trace metals, vitamins, and several inorganic and organic salts; it is worth noting that in hatcheries, culture medium is used routinely unspecified, most of the time using the same for several microalgae species.

thiamine•HCl (B1) (0.1), biotin (B7) (0.5), and cobalamin (B12) (0.5), following Guillard & Ryther (1962).

In aquaculture, microalgae cultures should involve a complete knowledge of their particular nutritional requirements; hence each microalgae species will be assessed through the elemental composition such as carbon, hydrogen, nitrogen and phosphorous content, C/N ratio as well as the chlorophyll content, because these indicators can be quantified, and related to healthy microalgae cultures (Ríos et al., 1998; Geider & La Roche, 2002; Roopnarain et al., 2015).

Dry weight was determined daily, filtering 30 mL of each microalgae culture through pre-weighed fiberglass filters (Ø 25 mm and nominal pore size of 0.7 µm; Whatman GF/F, Whatman International, Kent, UK). Samples were dried in a convection oven at 60 °C for 24 h, and then combusted at 400 °C in the muffle furnace for 2 h. Samples were weighed three times in succession at room temperature with an analytical balance (marca) (±0.00001 g).

In marine hatcheries, diatoms, flagellates and chlorophytes are important groups of phytoplankton commonly used as food sources, most often, Isochrysis galbana (Prymnesiophyceae), Chaetoceros calcitrans (Bacillariophyceae), and Dunaliella tertiolecta (Chlorophyceae). Therefore, the aim of this work was to experimentally assess daily variations of dry weight, carbon, C/N ratio, hydrogen, and chlorophyll (a, b, and c) as physiological indicators of nutrient uptake and growth rate during exponential growth of three microalgae (Isochrysis galbana, Chaetoceros calcitrans, and Dunaliella tertiolecta), using f/2 as medium culture. MATERIAL AND METHODS Algal culture Microalgae strains of Isochrysis galbana (3-5 µm), Chaetoceros calcitrans (8-10 µm), and Dunaliella tertiolecta (10-12 µm) were cultured under laboratory conditions at a temperature of 23 ± 1 °C, salinity of 36 ± 1 ups, pH of 8.0 ± 0.1, photoperiod of 12:12 h light:dark cycle, and artificial illumination of 150 μmol m-2 s-1. Light was provided by fluorescent lamps (cool white) placed behind the culture bottles. The microalgae cultures were continuously aerated with gentle influx of air. Seawater was collected from the Gulf of California and filtered under a low vacuum on Whatman GF/F filters, autoclaved and enriched with f/2 medium containing (in µg L-1): NaNO3 (883), NaH2PO4•H2O (36.3), Na2SiO3•9H2O (54), EDTA•Fe (11.7), FeCl3•6H2O (11.7), CuSO4•5H2O (0.04), ZnSO4•7H2O (0.08), CoCl2•6H2O (0.05), MnCl2•4H2O (0.9), Na2MoO4•2H2O (0.03),

Triplicate cultures of I. galbana, C. calcitrans and D. tertiolecta were initiated with a cell density of 2 x 105 cell mL-1 in bottles of 19 L. Measurement and characterization of microalgae strains were done every 24 h during five days of culture, which correspond to the late exponential growth phase, according to previous work done by Pérez-Morales (2006). Dry weight

Carbon, hydrogen and nitrogen After determining dry weights of each microalgae species, the content of carbon, hydrogen, and nitrogen were determined by combustion with an elemental analyzer (440HA model, Exeter Analytical, Coventry, UK) using a pneumatic auto-sampler. The filters were placed in aluminum capsules, and incinerated at high-temperature (1,000 °C). The helium (99.9 %) carrier gas pressure was set to 1.2 bars with a flow rate of ~100 mL min-1. For combustion, oxygen (99.9 %) was set at 1.9 bars, and air (no water or oil present) at 2.4 bars. Three empty tin capsules were folded, and sealed in the same manner; these were used as blanks to calibrate the standard, and samples. Combustion products (CO2, N2, and H2O) were separated by selective chemical traps, and measured with three pairs of detectors of highprecision thermal conductivity, and simultaneously determining C, H, and N as weight percentages. Chlorophylls To quantify chlorophylls, microalgae samples of known volume were filtered under low vacuum on Whatman GF/F filters, as described earlier. The filters were frozen, and analyzed following the method given by Parsons et al. (1984). Chlorophyll a, b, and c concentration were estimated using equations proposed by Jeffrey & Humprey (1975). Algal growth Cell densities were estimated during the exponential growth phase by direct counting, using a Neubauer haemocytometer. The growth rate (K’) was estimated following Fogg & Thake (1987), where K’ = Ln (N2 / N1) / (t2−t1), and N1 and N2 are biomass at time 1 (t1), and time 2 (t2). Once K’ is

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known, was calculated division per day, where Div day-1 = K’ / Ln2 and generation time = 1 / (Div day1 ). Statistical analysis Data were tested with standard process for homoscedasticity (Levene’s test), and normality (Shapiro-Wilk test). All data were then statistically tested to quantify the differences among the treatments using one-way statistical analyses of variance (ANOVA, p<0.01) with Tukey post-hoc tests, also the Pearson Correlation Coefficient was measured between cell density, and carbon content for each microalgae culture (Sokal & Rohlf, 1981). RESULTS Dry weight The greatest change in dry weight of cells occurred in C. calcitrans (Fig. 1), reaching at day 5 about twice (~90 pg cell-1) that observed on day 3. Isochrysis galbana slightly decrease on days 3 and 4 (~10 pg cell-1). Dunaliella tertiolecta cells at day 1 were heavier (~80 pg cell-1), and decreased to ~60 pg cell-1. Dry weight by cell of each microalgae exhibited significant differences over time (Table 1). Carbon Chaetoceros calcitrans showed the greatest change in cellular carbon content, reaching ~30 pg cell-1 on day 5 (Fig. 2); this was about twice carbon

content that was found in days 2 and 3. Isochrysis galbana had similar carbon content at the beginning and end of the trial (~8 pg cell-1); the maximum content occurred on day 2, and it steadily decreased until day 4. Dunaliella tertiolecta carbon content was similar over days 1 to 5, with a slight decrease from day 1 to day 2. The carbon content in microalgae showed significant differences during the bioassay period (Table 1). Correlation between cell density and carbon content was higher in D. tertiolecta (r= 0.98) compared to I. galbana or C. calcitrans (r= 0.9, and 0.8, respectively). C/N ratio Highest values of C/N ratios were observed in C. calcitrans and D. tertiolecta at day 1 (35 and 26, respectively). Isochrysis galbana showed steadily decline from day 1 until day 5 (Fig. 3). The C/N ratio in C. calcitrans and D. tertiolecta declined after day 1, remaining almost constant on days 2 until 4. Major differences were observed with I. galbana, where statistical analysis demonstrated significant differences in C/N ratio (Table 1). Hydrogen The highest concentration of hydrogen (>7 pg cell-1) was present at day 1 in D. tertiolecta, falling to ~4.5 pg cell-1, and remained constant in the following days without significant differences (Table 2). The most remarkable changes in hydrogen content were observed in C. calcitrans (Fig. 4),

Figure 1. Dry weight by cell (pg/cell ± SE) of a) Isochrysis galbana, b) Chaetoceros calcitrans, and c) Dunaliella tertiolecta cultured on f/2 medium (n=3). For each variable, data carrying similar alphabet are not statistically significant (p>0.01, Tukey test).

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Table 1. Results of one-way analysis of variance performed on dry weight, carbon content, and C/N ratio by cell of Isochrysis galbana, Chaetoceros calcitrans, and Dunaliella tertiolecta subjected to time (5 days). Source of Variation DF SS MS F p Dry weight Isochrysis galbana Between groups 4 505.67 126.42 9.7 0.002 Residual 10 130.54 13.05 Chaetoceros calcitrans Between groups 4 5,078.82 1,269.70 17.2 <0.001 Residual 10 739.62 73.96 Dunaliella tertiolecta Between groups 4 1,443.95 360.99 2.3 0.129 Residual 10 1,561.75 156.17 Carbon Isochrysis galbana Between groups 4 79.89 19.97 147.8 <0.001 Residual 10 1.35 0.135 Chaetoceros calcitrans Between groups 4 566.61 141.65 27.3 <0.001 Residual 10 51.92 5.19 Dunaliella tertiolecta Between groups 4 95.91 23.98 4.6 0.023 Residual 10 52.49 5.25 C/N ratio Isochrysis galbana Between groups 4 152.45 38.11 1429.6 <0.001 Residual 10 0.27 0.03 Chaetoceros calcitrans Between groups 4 1,801.85 450.46 107.1 <0.001 Residual 10 42.06 4.21 Dunaliella tertiolecta Between groups 4 985.38 246.35 95.7 <0.001 Residual 10 25.75 2.58 DF: degrees of freedom; SS: sum of squares; MS: mean square; F: F-ratio

starting with >6 pg cell-1 on day 1, dropping to half this level on days 2 and 3, and returning to the initial level on days 4 and 5. Isochrysis galbana reached a maximum (>2 pg cell-1) on day 2, and decreasing until day 4; this change was significant (Table 2).

til day 3 (1.22 x 106 cell mL-1). From the beginning of the bioassay, D. tertiolecta slightly increased in cell density until day 5 (1.36 x 106 cell mL-1). Chaetoceros calcitrans and D. tertiolecta had similar K′, div day-1, and generation time (Table 3).

Chlorophylls

DISCUSSION

For all microalgae tested, the main changes in chlorophyll content occurred in chlorophyll a (Fig. 5). Isochrysis galbana had the lowest content (<0.05 pg cell-1), C. calcitrans exhibited a significant increase from day 2 to day 4 (0.07 to 0.14 pg cell-1), whilst in D. tertiolecta the most notable increase occurred from day 2 to day 3 (0.18 to 0.28 pg cell-1). Chlorophyll c was present in low amounts in I. galbana, and D. tertiolecta, whereas with C. calcitrans was slightly higher remained constant from days 1 until day 5. Dunaliella tertiolecta had lower content (<0.05 pg cell-1) of chlorophyll b (pigment restricted to Chlorophyta). Statistical analyses indicated significant differences in chlorophyll a and c for all microalgae evaluated (Table 2).

Microalgae represent the main source of food in the initial ontogeny of many marine animals in the open sea, for this reason the microalgae culture is very important for hatchery production of the larval forms of farmed crustaceans, finfish and shellfish. The microalgae are also prey for zooplankton, which later become prey for larger larval fish stages. Thus, healthy microalgae cultures are essential for aquaculture production (Abu-Rezq et al., 1999; Brown et al., 1999; Muller-Feuga, 2000).

Algal growth Regarding cell densities, Isochrysis galbana increased in density until day 4 (3.9 x 106 cell mL1 ), decreasing on day 5 (Fig. 6), with a high growth rate and div day-1 (K′ of 0.58, and 0.83, respectively). Cell density of C. calcitrans increased un-

Physiological indicators of dry weight, carbon, C/N ratio, hydrogen, and chlorophyll content were consistent with healthy cultures reported by other authors (Estep & Hoering, 1981; Kaplan et al., 1986; Timmermans et al., 2001; Sebastien & Klein, 2006; Raghavan et al., 2008; Lananan et al., 2013; Roopnarain et al., 2015). In this study, dry weight by cell varied between species (because I. galbana cells are smaller, dry weight was less than C. calcitrans and D. tertiolecta cells), and growth phases. Regarding to variations in growth phases, some authors have reported that the increase in cell bio-

VARIATION OF NUTRIENTS UPTAKE IN MICROALGAE

Figure 2. Carbon content by cell (pg/cell ± SE) of a) Isochrysis galbana, b) Chaetoceros calcitrans, and c) Dunaliella tertiolecta cultured on f/2 medium (n=3). For each variable, data carrying similar alphabet are not statistically significant (p>0.01, Tukey test).

mass is influenced by availability of nutrients; most phytoplankton store nutrients and increase their cell volume during central growth phase (Banerjee et al., 2011; Roleda et al., 2013; Roopnarain et al., 2015). Variations in dry weight observed in this work, suggest that I. galbana, C. calcitrans and D. tertiolecta process accumulated nutrients into the cells at different times, assimilating these later when needed for growth. Hence microalgae cells could change their cell volume and weight during this process, which is a typical daily cycle when binary fission occurs; once finished the cells start again to accumulate and store nutrients changing with this their sizes over the time (Tantanasarit et al., 2013). Carbon content and C/N ratios of the three microalgae were consistent with values reported by others authors (Bienfang & Harrison, 1984; Cuhel et al., 1984; Brutemark et al., 2009). Variations in carbon content in different phases of cell growth in nutrient-enriched batch cultures are affected by available carbon source, because cell growth is based on different inorganic carbon sources (CO2, H2CO3, HCO3–, CO32–), and are transformed into complex molecules. Moreover, it has been reported that these variations differ between phytoplankton species, and depend of their exponential and stationary growth phase as well as of the nutrients sources (Brutemark et al., 2009; Roleda et al., 2013). In this study, the best correlation (r= 0.98) between cell density and carbon content was shown in D. tertio-

lecta, indicating the best nutrients incorporation into the cell to increase the population. The decline of C/N ratio during the culture time was consistent in all the three species, suggesting that stored carbon in the initial days was gradually converted into biochemical components such as carbohydrates (mainly β-glucose for cell wall formation), lipids, proteins, and nucleic acids which are primary products of photosynthesis (Bienfang & Harrison, 1984; Cuhel et al., 1984). In this work I. galbana showed a marked gradual decrease in C/N ratio (Fig. 3a), which has been related to a high growth rate and to the increase in the proportion of proteins into the cell (Ríos et al., 1998). Otherwise, C. calcitrans and D. tertiolecta showed values of C/N ratio slightly greater than the Redfield ratio of 6.6 from day 2 to 5, but this is commonly observed in nutrient repleted phytoplankton cultures (Geider & La Roche, 2002). Further, some authors have reported an inverse relationship between C/N ratio and nutrient concentration, showing that higher C/N ratios (11 to 7) are present when nitrogen is low (0.5 to 2 mmol N/L), but when nitrogen is high (4 to 16 mmol N/L) the C/N ratio stabilizes around 6 (Fábregas et al., 1995). Roleda et al. (2003) found that cell growth and carbon assimilation can be affected by pH variations and determined that C/N ratio is directly related to cell growth, showing a positive correlation between lipid increase and C/N ratio decrease. Besides, it has

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Table 2. Results of one-way analysis of variance performed on hydrogen, and chlorophyll (Chl a, and c) content by cell of Isochrysis galbana, Chaetoceros calcitrans, and Dunaliella tertiolecta subjected to time (5 days). Source of Variation DF SS MS F p Hydrogen Isochrysis galbana Between groups 4 2.46 0.62 78.5 <0.001 Residual 10 0.078 0.0078 Chaetoceros calcitrans Between groups 4 27.06 6.77 22.8 <0.001 Residual 10 2.96 0.30 Dunaliella tertiolecta Between groups 4 18.44 4.61 20.7 <0.001 Residual 10 2.22 2.22 Chl a Isochrysis galbana Between groups 4 0.00046 0.00011 45.7 <0.001 Residual 10 0.000025 0.0000025 Chaetoceros calcitrans Between groups 4 0.0085 0.0021 28.9 <0.001 Residual 10 0.00073 0.000073 Dunaliella tertiolecta Between groups 4 0.028 0.0069 19.3 <0.001 Residual 10 0.0036 0.00036 Chl c Isochrysis galbana Between groups 4 0.000069 0.000017 21.3 <0.001 Residual 10 0.000008 0.0000008 Chaetoceros calcitrans Between groups 4 0.000387 0.000097 16.4 <0.001 Residual 10 0.000059 0.0000059 Dunaliella tertiolecta Between groups 4 0.00014 0.000034 9.1 0.002 Residual 10 0.000038 0.0000038 DF: degrees of freedom; SS: sum of squares; MS: mean square; F: F-ratio

been documented that variations in carbon content and C/N ratio by cell are functions of cell nutrient quota of each microalgae species and are directly related to the increase in cell density (Cuhel et al., 1984; Cloern et al., 1995). Hydrogen cell content was observed in high quantities in both C. calcitrans and D. tertiolecta cultures at first day, whilst I. galbana cultures had slight variations. These values showed a direct relation between hydrogen content, and chlorophyll a content for all microalgae evaluated. Variations in hydrogen content have been associated with the formation of chlorophyll, and essential molecules (carbohydrates, lipids, and proteins). Higher values of hydrogen content by cell are directly related with both photosynthesis process, and high respiration rates, which indicate good nutrient uptake (Estep & Hoering, 1981). Concentration of chlorophylls observed in this study were consistent with their corresponding phyTable 3. Growth rate (K’), divisions per day (Div/day), and generation time (Gen. t) of Isochrysis galbana, Chaetoceros calcitrans, and Dunaliella tertiolecta cultured on f/2 medium (n=3). Species K’ Div/day Gen. t Isochrysis galbana Chaetoceros calcitrans Dunaliella tertiolecta

0.58 0.35 0.38

0.83 0.51 0.55

28 h 44 min 47 h 38 min 43 h 23 min

toplankton group (Fogg & Thake, 1987; Lourenço et al., 2002). Chlorophyll production over time is related to use and availability of nutrients in batch cultures, which is an indicator of growth efficiency, this due to that the instantaneous photosynthetic rate is limited by either, light energy or a nutrient (Cuhel et al., 1984; Cloern et al., 1995). Cell densities and growth rates of microalgae evaluated in this study differ from those reported by other authors. In this work, I. galbana showed higher cell density (3.9 x 106 cell mL-1) than occurs with Erd-Schreiber medium (3.2 x 106 cell mL-1) or Walne’s medium (~1 x 106 cell mL-1) (Gopinathan, 1986; Sebastien & Klein, 2006). Roleda et al. (2013) reported that I. galbana cultured with f/2 medium have a low growth rate of K’ = 0.45, whilst this study was of K’ = 0.58. Kaplan et al. (1986), state that temperature influences performance of microalgae cultures because the optimum temperature for achieving highest algal yield in I. galbana was found at 27 °C. Several reports claim that f/2 medium is a good promoter for rapid growth in I. galbana, principally because the microalga requires thiamine (B1), and cobalamin (B12), as well as Fe3+, one of the main trace metals that enhance the growth rate under laboratory conditions (Kaplan et al., 1986; Brown et al., 1999). Results in this work showed lower values than ob-

VARIATION OF NUTRIENTS UPTAKE IN MICROALGAE

Figure 3. Carbon/nitrogen (C/N) ratio by cell of a) Isochrysis galbana, b) Chaetoceros calcitrans, and c) Dunaliella tertiolecta cultured on f/2 medium (n=3). For each variable, data carrying similar alphabet are not statistically significant (p>0.01, Tukey test).

Figure 4. Hydrogen content by cell (pg/cell Âą SE) of a) Isochrysis galbana, b) Chaetoceros calcitrans, and c) Dunaliella tertiolecta cultured on f/2 medium (n=3). For each variable, data carrying similar alphabet are not statistically significant (p>0.01, Tukey test).

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Figure 5. Chlorophyll content (Chl a, b, c) by cell (pg/cell ± SE) of a) Isochrysis galbana, b) Chaetoceros calcitrans, and c) Dunaliella tertiolecta cultured on f/2 medium (n=3). For each variable, data carrying similar alphabet are not statistically significant (p>0.01, Tukey test).

Figure 6. Cell density (cell/mL ± SE) of Isochrysis galbana, Chaetoceros calcitrans, and Dunaliella tertiolecta cultured on f/2 medium (n=3). For each variable, data carrying similar alphabet are not statistically significant (p>0.01, Tukey test).

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served with Artificial Sea Water medium (ASW) or Conway medium (5 x 106 cell mL-1) during the same time of culture (Kaplan et al., 1986; Lananan et al., 2013). The main differences observed in this study with f/2 medium could be related to the level of sodium bicarbonate (NaHCO3), potassium nitrate (KNO3) or some trace metal present in both medium cultures, e.g. boric acid (H3BO3), as well as different forms of copper (CuCl2), and zinc (ZnCl2) which could be more suitable for I. galbana cells compared with copper (CuSO4) and zinc (ZnSO4) forms in f/2 medium. Cell densities of C. calcitrans in Conway medium (3.5 x 106 cell mL-1) and Walne’s medium (2.5 x 106 cell mL-1) were higher compared to the results quantified in this work (1.2 x 106 cell mL-1) (Raghavan et al., 2008; Banerjee et al., 2011; Lananan et al., 2013). This may indicate that C. calcitrans required certain nutrients in quantities different to those offered by f/2 media to increase growth. Moreover, some authors have established that low iron, low light, and addition of carbon dioxide are more adequate conditions for enhancing growth of C. calcitrans to ensure a higher content of important macromolecules, such as carbohydrates, lipids, and proteins (Timmermans et al., 2001; Raghavan et al., 2008). Lananan et al. (2013) state that f/2 is a better media for culturing chlorophytes, leading to high cell densities, such as 4 x 106 cell mL-1 in Dunaliella sp., compared with Conway medium that yields 3 x 106 cell mL-1. Roleda et al. (2013) also find higher growth rate (K′ = 0.74) in D. tertiolecta cultured with f/2; in this study, were observed lower densities (~1.4 x 106 cell mL-1) and growth rate (K′ = 0.38), which are consistent with those recently reported by Barakoni et al. (2015). However, differences in cell densities between cultures using the same culture medium may be due to light intensity and photoperiod. Cuhel et al. (1984) and Roleda et al. (2013) reported that light intensity influence the growth of Dunaliella tertiolecta, since batch cultures improves their growth with lower light intensity (100 μmol m-2 s-1) and longer photoperiod (16:8 light to dark); both conditions may improve D. tertiolecta cultures assessed here. Differences in cell densities and growth rates for each microalga evaluated in this work were mainly dependent of nutritional conditions, although some of these variations have been related to intrinsic characteristics for each strain (Fogg & Thake, 1987; Abu-Rezq et al., 1999). Whether other culture media, addition of trace elements and/or vitamins may improve the growth of microalgae strains evaluated here remains to be tested. Studies of nutrients uptake in microalgae for aquaculture purposes are catered rarely, this because in aquaculture systems a routine management is per-

formed using the same culture medium for several microalgae species in order to reduce costs, which may result in a lower nutritional quality of cultures. In summary, the most important aspects observed in this work were daily variations in nutrient uptake during exponential growth, which was reflected in the elemental composition (dry weight, C/N ratio, carbon, hydrogen and chlorophyll a content) of microalgae cells. This information provides new insights about physiology of marine microalgae and confirms that nutrient uptake dynamics is different in each microalga species. These results further demonstrate that to study nutrient uptake in microalgae, daily assessments are more efficient than experiments of few hours, this due to variations over time. Lastly, this study indicates that using one culture medium is not equally efficient to all microalgae used in aquaculture since each species has specific nutritional requirements. ACKNOWLEDGEMENTS For this study APM received fellowships from PIFI-IPN and Consejo Nacional de Ciencia y Tecnología (CONACYT, Grant #103374). AML is COFAA-IPN and EDI-IPN fellow. Microalgae strains were obtained from the Culture Collection of Microalgae at the Centro Interdisciplinario de Ciencias Marinas-IPN, La Paz, B.C.S., Mexico. The authors thank the analytical laboratory staff of the Institute of Marine Sciences at the University of California, Santa Barbara for CHN analyses. Authors also thank Laura G. Flores-Montijo for technical assistance. REFERENCES Abu-Rezq, T. S., L. Al-Musallam, J. Al-Shimmari & P. Dias. 1999. Optimum production conditions for different high-quality marine algae. Hydrobiologia, 403: 97-107. Banerjee, S., W. E. Hew, H. Khatoon, M. Shariff & M. Md.Yusoff. 2011. Growth and proximate composition of tropical marine Chaetoceros calcitrans and Nannochloropsis oculata cultured outdoors and under laboratory conditions. Afr. J. Biotechnol., 10(8): 1375-1383. Barakoni, R., S. Awal & A. Christie. 2015. Growth performance of the marine microalgae Pavlova salina and Dunaliella tertiolecta using different commercially available fertilizers in natural seawater and inland saline ground water. J. Algal Biomass Utln., 6: 15-25. Bienfang, P. K. & P. J. Harrison. 1984. Co-variation of sinking rate and cell quota among nutrient replete marine phytoplankton. Mar. Ecol. Prog. Ser., 14: 297-300. Brown, M. R., M. Mular, I. Miller, C. Farmer & C. Trenerry. 1999. The vitamin content of micro-

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Keller, M. D. & R. R. L. Guillard. 1985. Factors significant to marine diatom culture. In: Anderson DM, White AW, Baden DG (eds.). Toxic Dinoflagellates. Elsevier, New York, USA, pp. 113116. Keller, M. D., R.C. Selvin, W. Claus & R. R. L. Guillard. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol., 23: 633-638. Lananan, F., A. Jusoh, N. Ali, S. S. Lam & A. Endut. 2013. Effect of Conway medium and f/2 medium on the growth of six genera of South China Sea marine microalgae. Biores. Technol., 141: 75-82. Lourenço, S. O., E. Barbarino, J. Mancini-Filho, K. P. Schinke & E. Aidar. 2002. Effects of different nitrogen sources on the growth and biochemical profile of 10 marine microalgae in batch culture: an evaluation for aquaculture. Phycologia, 41(2): 158-168. Muller-Feuga, A. 2000. The role of microalgae in aquaculture: situation and trends. J. Appl. Phycol., 12: 527–534. Miquel, P. 1890. De la culture artificielle des diatomees. Diatomiste, 1: 93-99. Okaichi, T., S. Nishio & Y. Imatomi. 1982. Collection and mass culture. 22-34, In: Japanese Fisheries Society (eds.). Toxic phytoplankton - occurrence, mode of action and toxins. Japan Fisheries Society, Tokyo, Japan. Parsons, T. R., Y. Maita & C. M. Lalli. 1984. A manual of chemical and biological methods for seawater analysis. Pergamon press, UK, 173 p. Pérez-Morales, A. 2006. Efecto de diferentes microalgas en las tasas vitales de Euterpina acutifrons (Dana, 1848) (Copepoda: Harpacticoida) en condiciones controladas. MSc. thesis. Centro Interdisciplinario de Ciencias MarinasInstituto Politécnico Nacional. 67 p. Raghavan, G., C. K. Haridevi & C. P. Gopinathan. 2008. Growth and proximate composition of the Chaetoceros calcitrans f. pumilus under different temperature, salinity and carbon dioxide levels. Aquac. Res., 39: 1053-1058. Ríos, A. F., F. Fraga, F. F. Pérez & F. G. Figueiras. 1998. Chemical composition of phytoplankton and particulate organic matter in the Ría de Vigo (NW Spain). Sci. Mar., 62(3): 257-271.

VARIATION OF NUTRIENTS UPTAKE IN MICROALGAE

Roleda, M. Y., S. P. Slocombe, R. J. G. Leakey, J. G. Day, E. M. Bell & M. S. Stanley. 2013. Effects of temperature and nutrient regimes on biomass and lipid production by six oleaginous microalgae in batch culture employing a two-phase cultivation strategy. Biores. Technol., 129: 439449. Roopnarain, A., S. D. Sym & V. M. Gray. 2015. Time of culture harvest affects lipid productivity of nitrogen-starved Isochrysis galbana U4 (Isochrysidales, Haptophyta). Aquaculture, 438: 12-16. Sebastien, N. Y. & V. L. M. Klein. 2006. Efeito do meio Erd Schreiber no cultivo das microalgas Dunaliella salina, Tetraselmis chuii e Isochrysis galbana. Acta Sci. Biol. Sci., 28: 149-152. Sokal, R. R. & F. J. Rohlf. 1981. Biometry: The principles and practice of statistics in biological research. Freeman, W.H. and Company, New York, USA, 859 p.

Tantanasarit, C., A. J. Englande & S. Babel. 2013. Nitrogen, phosphorus and silicon uptake kinetics by marine diatom Chaetoceros calcitrans under high nutrient concentrations. J. Exp. Mar. Biol. Ecol., 446: 67-75. Timmermans, K. R., M. S. Davey, B. Van der Wagt, J. Snoek, R. J. Geider, M. J. W. Veldhuis, L. J. A. Gerringa & H. J. W. De Baar. 2001. Co-limitation by iron and light of Chaetoceros brevis, C. dichaeta and C. calcitrans (Bacillariophyceae). Mar. Ecol. Prog. Ser., 217: 287-297. Tompkins, J., M. M. Deville, J. G. Day & M. F. Turner. 1995. Culture collection of algae and protozoa. Catalogue of strains. Ambleside, UK, 204 p.

CICIMAR Oceánides 30(1): 45-62 (2015)

ZOOPLANKTON FUNCTIONAL GROUPS FROM THE CALIFORNIA CURRENT AND CLIMATE VARIABILITY DURING 1997-2013 Lavaniegos, B. E.1, O. Molina-González1 & M. Murcia-Riaño1,2

Centro de Investigación Científica y Educación Superior de Ensenada. Carretera Ensenada-Tijuana No. 3918, Zona Playitas, 22860. Ensenada, Baja California, México. 2Instituto de Investigaciones Marinas y Costeras, Calle 25 No. 2-55, Playa Salguero, Santa Marta, Colombia. email: berlav@cicese.mx; magnolia.murcia@invemar.org.co

ABSTRACT. Zooplankton plays an important role in recycling matter and energy trough the pelagic ecosystem. The California Current is one of the large marine ecosystems with high productivity and bio-physical variability at multiple time scales. An interannual scale or longer periods requires data series sufficiently long to ensure reliable averages of zooplankton abundance in order to estimate their low frequency variability. Here, tendencies in physical and biological variables are presented for the period 1997-2013 with data obtained from IMECOCAL cruises in the Mexican sector of the California Current. The area was divided into four regions, two oceanic (off North and Central Baja California) and two neritic (Vizcaino bay and Gulf of Ulloa). Sea surface temperature (SST) and El Niño Oceanic Index (ONI) showed correlation in all areas, while extratropical indices (PDO and NPGO) exhibited different tendencies among the regions. The PDO had significant correlation with SST only in the central and Vizcaino bay regions. The NPGO was not correlated with temperature but presented significantly strong correlation with sea surface salinity in all regions, which has been attributed to changes in large-scale circulation of the north Pacific subtropical gyre. In spite of a significant influence of the El Niño Southern Oscillation (ENSO) in SST, the correlation between ONI and zooplankton abundance was limited to gelatinous herbivorous (tunicates) from the North region. Local influence was remarkable in Vizcaino bay where the tunicates showed a period of negative abundance anomalies (2000-2004) followed by increasing positive anomalies between 2005 and 2013 associated with positive upwelling index anomalies. Geometric mean abundance of salps (per oceanographic cruise) averaged in Vizcaino bay 33.3 ind m-3 during 2005-2013 compared to 1.4 ind m-3 in 2000-2004. Salps partially displaced crustacean herbivores since they compete for feeding particles; copepods decreased from 88.2 ind m-3 during 2000-2004 to 59.7 ind m-3 in 2005-2013; and euphausiids from 16.1 ind m-3 to 10.4 ind m-3. In the oceanic domain a period of saline stratification during 2002-2006 was associated with positive anomalies of all trophic groups (crustaceans, tunicates and carnivores). Alternation of particular taxa of tunicates and carnivores is discussed. The increase of gelatinous organisms associated to higher stratification in the oceanic region and enhanced upwellng in the coastal shelf appears to be in detriment of crustaceans, though the time-series are short to outline a more defined trend. That tendency is particularly disturbing in Vizcaino bay affecting the availability of food for fishes and other predators.

Keywords: Baja California, ENSO, salps, copepods, euphausiids

Grupos funcionales de zooplancton de la Corriente de California y variabilidad climática durante 1997-2013

RESUMEN. El zooplancton juega un papel fundamental en el flujo de materia y energía en el ecosistema pelágico. La Corriente de California es uno de los grandes ecosistemas marinos con elevada productividad y amplia variabilidad físico-biológica a múltiples escalas temporales. A escala interanual y de mayor periodo es necesario contar con series de datos lo suficientemente extensas temporalmente que permitan calcular promedios robustos de la abundancia del zooplancton y poder estimar la variabilidad de baja frecuencia. En el presente estudio se muestran las tendencias en variables físicas y biológicas del periodo 1997-2013 de los datos obtenidos por los cruceros IMECOCAL en el sector mexicano de la Corriente de California. El área fue dividida en cuatro regiones, dos oceánicas (frente a Baja California, Norte y Central) y dos neríticas (Bahía Vizcaíno y Golfo de Ulloa). En todas las regiones la temperatura superficial del mar (TSM) estuvo correlacionada con El Niño Oceanic Index (ONI). Los índices extratropicales (PDO y NPGO) mostraron diferentes tendencias entre regiones. El PDO tuvo fuerte correlación con la TSM solo en la región central y en Bahía Vizcaíno. El NPGO no se correlacionó con la temperatura pero presentó correlación significativa con la salinidad superficial del mar en todas las regiones, lo cual ha sido atribuido a cambios en la circulación a gran escala del giro subtropical del Pacífico norte. A pesar de una influencia significativa del ENSO en la TSM, la correlación entre el ONI y la abundancia del zooplancton estuvo limitada a los herbívoros gelatinosos (tunicados) de la región Norte. La influencia local fue notable en Bahía Vizcaíno donde los tunicados mostraron un periodo de anomalías negativas (2000-2004) seguido por un periodo con anomalías positivas de creciente amplitud entre 2005 y 2013 asociadas con anomalías positivas del índice de surgencias. La abundancia expresada mediante medias geométricas de salpas (por crucero) mostró en Bahía Vizcaíno 33.3 ind m-3 durante 2005-2013 comparada con 1.4 ind m-3 en 2000-2004. Las salpas desplazaron parcialmente a los crustáceos herbívoros puesto que ambos compiten por las partículas de alimento; los copépodos disminuyeron de 88.2 ind m-3 durante 2000-2004 a 59.7 ind m-3 en 2005-2013; los eufáusidos disminuyeron de 16.1 ind m-3 a 10.4 ind m-3. En el dominio oceánico un periodo de estratificación salina durante 2002-2006 estuvo asociado con anomalías positivas de todos los grupos tróficos (crustáceos, tunicados y carnívoros). Se discute la alternancia de taxa particulares de tunicados y carnívoros. El incremento de organismos gelatinosos asociado a una mayor estratificación en la región oceánica y a la intensificación de las surgencias en la plataforma costera parece ir en detrimento de los crustáceos, aunque las series de tiempo son cortas para establecer una tendencia definida. Dicha tendencia es particularmente perturbadora en Bahía Vizcaíno al afectar la disponibilidad de alimento para peces y otros depredadores.

Palabras clave: Baja California, ENSO, salpas, copépodos, eufáusidos

Lavaniegos, B. E., O. Molina-González & M. Murcia-Riaño. 2015. Zooplankton functional groups from the California current and climate variability during 1997-2013. CICIMAR Oceánides, 30(1): 45-62.

Fecha de recepción: 26 de marzo de 2015

Fecha de aceptación: 23 de abril de 2015

LAVANIEGOS et al.

INTRODUCTION Zooplankton abundance is highly variable in space and time and in greater extent in advective marine ecosystems as the California Current (CC). The Mexican region off Baja California has received increasing attention thanks to the intensive plankton monitoring by the California Current Mexican Investigations program (IMECOCAL, Spanish acronym) (Fig. 1). The better studied zooplankton at species level in the IMECOCAL region are copepods (Jiménez-Pérez & Lavaniegos, 2004; Lavaniegos & Jiménez-Pérez, 2006), euphausiids (Lavaniegos & Ambriz-Arreola, 2012), amphipods (Lavaniegos, 2014; Lavaniegos & Hereu, 2009), salps (Hereu et al., 2006), and fish larvae (Funes-Rodríguez et al., 2006; Jiménez-Rosenberg et al., 2007, 2010). However, communities as a whole remain elusive due to the arduous and time consuming work required to identify species from multiple taxonomic groups in subtropical regions; which are particularly diverse as has been shown for amphipods (Lavaniegos & Hereu, 2009). An alternative way to address community structural changes is through functional diversity, namely the abundances of organisms with different morphology and trophic function in the ecosystem (Walker, 1992). Functional groups are relatively easy to identify and may be counted from one fraction of the sample (Ohman & Lavaniegos, 2002).

Figure 1. Sampling area showing oceanographic stations and the 200 m isobath. Oceanic and coastal stations are indicated with black squares and white circles, respectively. Dashed line is the boundary of north and central regions used in this study.

Species are better valuable indicators of climate variability, particularly those adapted to narrow temperature ranges. Functional groups are considered relatively less sensitive but they may also respond to different types of environmental perturbations (Lavaniegos & Ohman, 2007; Lavaniegos, 2009; Lavaniegos et al., 2010). One example of this ecological sensitivity is the increase in chaetognaths abundance during the strong El Niño 19971998, while salps increased during the transition to La Niña conditions with swarms of these organisms covering a large area off Baja California (Lavaniegos et al., 2002; Hereu et al., 2006). Long-term changes are also reflected by functional groups as it has occurred with the biomass declination of salps during the warm regime of 1977-1998 off southern California (Lavaniegos & Ohman, 2003). Lavaniegos (2009) analyzed the interannual variability of zooplankton groups by trophic levels in the Mexican sector off the California Current during the period 1997-2007, and detailed time-series for zooplankton major taxa were offered in Lavaniegos et al. (2010). The most remarkable events were El Niño 1997-1998, followed by La Niña 1998-2000, and the subarctic water intrusion in 2002-2003. This last atmospheric and oceanographic event produced sequential events in the pelagic ecosystem causing high chlorophyll-a concentrations (Gaxiola-Castro et al., 2010), apparently not consumed due to a drop in zooplankton biomass at the end of 2002. Furtherly, the zooplankton biomass followed a progressive recovery between 2003 and 2007 (Lavaniegos, 2009). The increasing abundance trend was observed in herbivores (crustaceans and tunicates) and carnivores (chaetognaths, siphonophores, medusae, ctenophores, and heteropods) suggesting a general increase in secondary production. A significant correlation of zooplankton biomass and North Pacific Gyre Oscillation prompted to think in a basin scale forcing more than regional mechanisms (Lavaniegos, 2009). However, the influence of two weak El Niño events (2004-2005 and 2006-2007) and La Niña 2005-2006 were not specifically discussed by Lavaniegos (2009). El Niño events have been weak since the beginning of the 21st century presenting moderate sea surface temperature (SST) anomalies complicating their forecast (Fedorov et al., 2003; Lee & McPhaden, 2010). These weak El Niño events are currently considered a new type of El Niño, with high warm anomalies limited to the central equatorial Pacific flanked by anomalously cooler SST to its east and west, what is named El Niño Modoki or Central Pacific (CP) El Niño (Ashok et al., 2007; Kug et al., 2009). This anomalous SST pattern is different from that observed during typical El Niño events or Eastern Tropical Pacific (EP) El Niño with propagation of warm SST anomalies from central to eastern equatorial Pacific. Atmospheric differences

ZOOPLANKTON GROUPS AND CLIMATE VARIABILITY

are also observed with a western displacement of the main rainfall center during CP El Niño (Yeh et al., 2009). Yeh et al. (2009) confirmed a higher incidence of CP El Niño since 1990 which could be associated to global warming. In the present study we re-analyse of the biophysical coupling between zooplankton and climatic indices including ENSO with updated time series (1997-2013) to infer if the weak El Niño events produced detectable changes in the abundances of zooplankton identified into major taxa or these were only caused by the strong events (1997-1998). MATERIAL AND METHODS The study area is located in the Mexican sector of the California Current, downstream along the Baja California peninsula (Fig. 1). This region was sampled between September 1997 and May 2013 by quarterly cruises on the R/V Francisco de Ulloa. The total number of cruises performed was 55 with only 8 missing cruises (see Appendix Table 1 for dates of cruises). At each station hydrographic casts were done using a Seabird CTD. Zooplankton was collected with a bongo net of 0.5 mm mesh-width by performing oblique tows in the upper 210 m to the surface (from 10 m above the bottom at shallow stations). The diameter of the net was 61 cm before October 2001, and later it was replaced by one of 71 cm. The volume of water strained was measured with a digital flow-meter fixed in the mouth of the net. Samples were preserved with 4% formalin and sodium borate. In the laboratory, the zooplankton was counted from a fraction of the original sample, between 1/8 and 1/32, depending on the amount of plankton. Major taxa were counted under a stereoscopic microscope. Taxa considered in this study were crustaceans herbivores/omnivores (copepods and euphausiids), herviborous tunicades (appendicularians, doliolids, salps, and pyrosomes), and carnivores (chaetognaths, siphonophores, medusae, ctenophores, and heteropods). Other taxonomic groups usually had low abundances and were neglected. The zooplankton was counted only from samples collected during night in the oceanic regions (bottom depth >200 m) in order to reduce the well known day-night variability due to zooplankton vertical migration and daytime visual zooplankton net avoidance. In the coastal shelf all samples were analyzed regardless of the hour of sampling given the low number of coastal stations (Fig. 1, Appendix Table 1). Zooplankton taxa abundances were standardized to ind m-3. In order to normalize the data these were transformed to logarithms (log x + 1). Subsequently, anomalies of zooplankton abundances were calculated removing the long-term seasonal means for the period 1997–2013 in four separate ecoregions (Fig. 1): two oceanic (north and central) and two coastal (Vizcaino bay and Gulf of Ulloa). Sever-

al cruises did not cover all four sampling regions or had few data in some region, and therefore were discarded in the calculation of abundance anomalies. Anomalies of environmental variables were estimated in the same form described for zooplankton data. Cruises with only one datum of physical variables for any particular region, as in biological variables, were discarded for anomalies calculation. Several oceanographic cruises had a time lag compared to usual sampling months, introducing uncertainty in calculations of sea surface temperature anomalies. This is particularly critical in summer and autumn when surface temperature typically changes 1-2°C from one month to the next (or even larger changes in the coastal shelf, see Appendix Figure). For example, cruises 9908 and 0708 had 20 and 38 days of delay respectively in relation to the average performance of the rest of summer cruises. Due to the climatological mean is more representative of July would result in overestimation of anomalies in cruises done in August. The bias introduced by time lags was corrected adding (or subtracting) the monthly increment of temperature based in information from CALCOFI data from the period 1951-1966. This correction to mean temperature in such cruises out of date was applied only when the time lag was >15 days and if the monthly increment represented >0.5°C (see Appendix Table 1 for date cruises and Table 2 for temperature values). Spearman correlations were done between zooplankton taxa abundances and environmental variables (Table 1). Regional environmental variables were sea surface temperature (SST), sea surface salinity (SSS), and upwelling Index (UI). The measurement at 10 m was used to represent SST and SSS to avoid diurnal variations and ensuring the stabilization of CTD sensor. In oceanic stations thermal and saline stratification (dT and dS respectively) were also included as the difference between values at 10 and 200 m depths. Large-scale climatic indices were also correlated with local variables: El Niño Oceanic Index (ONI), the Pacific Decadal Oscillation (PDO), and the North Pacific Gyre Oscillation (NPGO). Source for each environmental variable is shown in Table 1. Monthly values of the PDO, NPGO, and UI anomalies were converted to quarterly means in correspondence with the zooplankton data frequency for further Spearman correlation analysis. This was done averaging values for winter (December to February), spring (March to May), summer (June to August), and autumn (September to November). RESULTS Large scale Pacific indices El Niño index for the oceanic region 3.4 (ONI) is based on surface temperature and during the study period the highest positive values (>2) were associated with El Niño 1997-1998 (Fig. 2a). The rest of El

LAVANIEGOS et al.

Table 1. Environmental variables used in correlation analyses. Variables Code Source Local 10 m temperature anomalies SST IMECOCAL 10 m salinity anomalies SSS IMECOCAL Thermal stratification anomalies (10-200 m) dT IMECOCAL Saline stratification anomalies (10-200 m) dS IMECOCAL Upwelling Index UI NOAA Pacific Fisheries Environmental Laboratory station 30°N, 119°W (http://www.pfel.noaa.gov/products/PFEL/modeled/indices/PFELindi(used for north and Vizcaino bay regions) ces.html) station 27°N, 116°W Indicate water transport due to wind stress with units of m3 s-1 per 100 m of coastline. Monthly means were calculated from daily values. Nega(used for central and Ulloa regions) tive index indicate downwelling and positive for upwelling (from –270 to +442 and –241 to +607 in the selected locations). Large-scale El Niño Oceanic Index 3.4 ONI NOAA Climate Prediction Center (region 5°N-5°S, 120°-170°W) (http://www.cpc.ncep.noaa.gov/products/analysis_monitoring/ensostuff/ ensoyears.shtml). Index based on sea surface temperature anomalies and is defined as the three-month running-mean SST departures NOAA ERSST data. El Niño is characterized by ONI ≥ +0.5 C, and La Nina ≤ -0.5 C in 5 consecutive 3-months running mean. Pacific Decadal Oscillation Index PDO (http://jisao.washington.edu/pdo/PDO.latest) Derived as the leading principal component of monthly SST anomalies in the north Pacific Ocean, poleward to 20°N. Value ranged from –3.6 to +3.5 during 1900-2013 North Pacific Gyre Oscilllation NPGO (http://eros.eas.gatech.edu/npgo/data/NPGO.txt) Second dominant mode of sea surface height variability (2nd EOF SSH) in the Northeast Pacific. Assuming that anomalies of SSH reflect changes in geostrophic flow, positive values indicate strengthening of the North Pacific Current and in consequence in the California Current. Value range is -3 to +3.

Niño events presented lower values with short peaks just over +1 anomaly only in three events (20022003, 2006-2007, and 2009-2010). In contrast, the cool phase of ENSO showed a persistence of two years in three La Niña events (1998-2000, 20072009, and 2010-2012), and only La Niña 2005-2006 was less than two years.

The North Pacific decadal oscillation showed a pattern relatively similar with the ONI (Fig. 2b). Major shifts of PDO occurred at the end of the years 1998, 2002, and 2007. Further, between 2007 and 2013 values remained negative excepting one short period in 2009-2010. The North Pacific Gyre Oscillation changed from negative to positive in the winter of 1998 remaining positive until the end of 2004 (Fig. 2c). After a short period of two years with negative values, the NPGO changed again observing a long period of positive values (2007-2013). The PDO and NPGO varied inversely for long periods though there was an interval in 2002-2004 in which both indices (and also the ONI) were positive. Environmental variables Anomalies of temperature, salinity, and upwelling index off Baja California presented different long-term tendencies (Figs. 3-5). In consequence, the correlations with large scale indices were also variable and not always coherent among sampling regions (Table 2). The ONI showed significant positive correlations with SST anomalies from all regions (Figs. 3a, 4a, 5a, c) suggesting that the ENSO

influence reached the study region. However, the low correlation coefficients between ONI and SST (Table 2) are also indicative of local factors influence. SST positive anomalies followed well the positive anomalies of the ONI except during 2002 when negative anomalies occurred off Baja California and were particularly strong in October 2002 for the central region (Fig. 3a). These negative anomalies corresponded with a subarctic water intrusion (Durazo et al., 2005), apparently causing a delay in the warming due to El Niño 2002-2003; that effect was finally observed as a shift from negative to positive anomalies in local temperature in the beginning of 2003. Another difference between ONI and regional temperatures was the magnitude of La Niña events. Following the ONI, three cool events of similar magnitude may be traceable (1999-2000, 20072008, and 2010-2012; see Fig. 2a). In contrast, oceanic regions off Baja California recorded lower intensity for the first two events compared to La Niña 2010-2012 (Figs. 3a, 4a). Similar observations were found from Vizcaino bay (Fig. 5a). In the Gulf of Ulloa SST anomalies (Fig. 5c) had more negative values during 1999 (-1 to -2°C) compared to Vizcaino bay, and could be even more negative but there were gaps in the first part of the time-series when the warm phase of the ENSO took place. La Niña 20072008 showed weak and fleeting anomalies, coherent in the coastal shelf as in oceanic regions compared to the strength and endurance depicted by the ONI.

ZOOPLANKTON GROUPS AND CLIMATE VARIABILITY

Thermal stratification (dT) anomalies in the oceanic regions (Figs. 3b, 4b) were similar to SST indicating that the interannual warming (cooling) is less intense at 200 m during El Niño (La Niña) and therefore the difference with the SST produced a higher (lower) gradient. However dT anomalies were significantly correlated with ONI only in the Central region (Table 2). The upwelling index was inversely correlated with ONI (Table 2) showing negative UI anomalies roughly coincident with El Niño events (Figs. 3e, 4e). However, differences between ONI and UI were also evident as the predominance of negative anomalies in 2000-2004, followed by a period of positive anomalies only interrupted by a short interval of strongly negative anomalies between October 2009 and April 2010. This period of relaxed UI is consistent with a shift from El Niño 2009-2010 to La Niña 2010-2012. Therefore, the UI pattern ap-

pears to be opposite to that observed during the 1997-2000 ENSO cycle which presented light positive anomalies during the warm phase and close to zero in the cool phase. Positive significant correlations were found between the PDO and local temperature, similar to those observed with ONI, as the variability pattern of PDO and ONI were quite similar (Fig. 2a,b). Negative PDO values were roughly consistent with La Niña while positive values with El Niño. A different pattern was obtained for the sea surface salinity (SSS) without correlation with the ONI. Instead, the SSS anomalies in the four regions (Figs 3c, 4c, 5b, d) were significantly correlated with the NPGO (Table 2). According to Di Lorenzo et al. (2008) NPGO index captures changes in the strength of the North Pacific Current implying that positive values observed most of the time during 1997-2013 could lead to reinforce the CC. Sign anomaly reversions

Figure 2. Climatic indices: (a) El Niño Oceanic Index from region 3.4 (5°N-5°S, 120°-170°W), (b) Pacific Decadal Oscillation, and (c) North Pacific Gyre Oscillation.

LAVANIEGOS et al.

Table 2. Spearman correlation matrix between environmental variables (anomalies) and climatic indices in four regions off Baja California: oceanic (North and Central) and coastal (Vizcaino bay and Gulf of Ulloa). Significant coefficients are highlighted: p<0.001 (***), p<0.01 (**), and p<0.05 (*). For complete name of variables see Table 1. Variable

ONI

PDO

North Region SST

0.343 **

NPGO

0.159

−0.145 −0.046

0.154

0.039

SSS

−0.184

−0294 *

0.247

0.307 *

−0.265 *

−0.250 *

0.619 *** −0.598 *** −0.099

Central Region SST

0.560 ***

0.448 ***

SSS

0.136

0.447 ***

−0.144

0.475 ***

−0.091

−0.094

0.511 ***

−0.048

−0.015

−0.518 ***

−0.409 ***

−0.443 ***

−0.046

SST

0.505 ***

0.400 **

−0.036

SSS

0.067

SST

0.527 ***

SSS

0.294

Vizcaino bay −0.136

0.458 ***

Gulf of Ulloa

appear to be gradual excepting the winter of 20042005 which means drastic weakening of the North Pacific Current (Fig. 2c). In coincidence, local SSS anomalies during 2002-2003 experienced a gradual decrease in magnitude changing from positive to negative and reaching the most negative value in February 2004, more clearly observed in the North oceanic region (Fig. 3c). Shortly after, the inverse process took place reaching positive SSS anomalies in 2007. The oscillation in SSS anomalies was not entirely gradual during the second period of positive NPGO (2007-2013), showing a decreasing pulse in 2010-2011. In the oceanic domain, inverse correlations were found between saline stratification anomalies (dS) and NPGO. The inverse pattern of dS anomalies (Figs. 3d, 4d) and SSS anomalies (Figs. 3c, 4c), reversing signs in the water column, could be attributable to a more saline subsurface water mass y a strengthening of the California Undercurrent at 200 m. Therefore, the period of positive dS anomalies recorded in 2004-2007 indicate a strong saline stratification coincident with the negative NPGO (Fig. 2c). Correlations between PDO and salinity anomalies were found only in the north region, positive for SSS and inverse to dS (Table 2). Correlation coefficients were lower to those observed for the NPGO and with reverse signs due the PDO is inversely cor-

0.020 −0.065

0.039 0.528 ***

related with NPGO (r = -0.29, p = 0.022). Trophic zooplankton groups Abundances of the trophic zooplankton groups presented high seasonal and interannual variability (Figs. 6-7). Crustaceans (herbivore/omnivores) were the most abundant taxonomic group in the oceanic regions followed by carnivores, and tunicates in third place (Fig. 6a, b). In the north region, all trophic groups presented similar patterns of abundance anomalies (Fig. 6c-e) with a predominance of negative anomalies between 1998 and 2004 and positive anomalies between 2004 and 2010. However, some differences in magnitude may be observed as a higher positive anomaly of carnivores compared to herbivore groups in the winter of 1998, highest positive anomalies of tunicates in the summers of 2001 and 2002, as well as slight time offsets in the shift from positive to negative anomalies at the end of the time-series. All the northern trophic groups were inversely correlated with the NPGO while positive correlations with ONI were restricted to tunicates and carnivores, and none correlation was observed with the PDO (Table 3). The lack of correlation with local variables is noteworthy, except for tunicates and ds that indicate increasing abundances with saline stratification. The proportion of doliolids was higher in most of the 1999-2002 cruises while during the years of strongest dS anomalies (2003-2006) the

ZOOPLANKTON GROUPS AND CLIMATE VARIABILITY

Figure 3. North region off Baja California: (a) sea surface temperature, (b) thermal stratification in the upper 200 m, (c) sea surface salinity, (d) saline stratification in the upper 200 m, and (e) Upwelling Index from 30째N, 119째W. Asterisks indicate corrections made to mean temperature due to seasonal bias in sampling, before to estimate the long-term seasonal mean and anomalies.

LAVANIEGOS et al.

Table 3. Spearman correlation between zooplankton abundance anomalies with environmental variables (anomalies) and climatic indices in four regions off Baja California: oceanic (North and Central) and coastal (Vizcaino bay and Gulf of Ulloa). Significant coefficients are highlighted: p<0.001 (***), p<0.01 (**), and p<0.05 (*). For complete name of variables see Table 1. Variable n Crustaceans Tunicates Carnivores North Region ONI 54 0.163 0.448 *** 0.282 * PDO 54 0.089 0.214 0.061 NPGO 54 −0.366 ** −0.391 ** −0.511 *** SST 54 −0.154 0.120 0.003 dT 54 −0.249 0.067 −0.140 SSS 54 −0.130 −0.203 −0.176 dS 54 0.239 0.281 * 0.267 UI 54 −0.051 −0.014 0.157 Central Region ONI 53 0.153 0.131 0.297 * PDO 53 −0.026 0.225 0.302 * NPGO 53 −0.074 −0.254 −0.305 * SST 53 −0.048 0.122 0.374 ** dT 53 −0.118 0.091 0.288 * SSS 53 0.140 0.028 0.116 dS 53 0.048 −0.090 −0.027 UI] 53 0.385 ** 0.193 0.095 Vizcaino bay ONI PDO NPGO SST SSS UI Gulf of Ulloa ONI PDO NPGO SST SSS UI

52 52 52 52 52 52

0.130 0.341 * −0.109 −0.026 −0.036 −0.005

−0.176 −0.215 −0.188 0.146 0.043 0.286 *

−0.078 0.194 −0.402 ** 0.066 −0.056 0.194

43 43 43 43 43 43

0.169 0.134 −0.124 −0.076 −0.070 0.072

0.115 0.184 −0.313 * 0.000 −0.087 0.019

0.072 0.238 −0.310 * −0.240 −0.112 0.234

community was characterized by predominance of appendicularians and salps (Fig. 8b). Anomaly patterns of the trophic groups maintained a relative temporal coherence in the central region (Fig. 6f-h), albeit less harmonized compared to the northern zooplankton. The group of tunicates from central region showed strong anomalies during 1998 and 2010 probably associated to El Niño events but no significant correlation was observed with ONI (Table 3). In contrast the carnivores from the central region were correlated with the three Pacific indices (ONI, PDO, and NPGO), and also with local variables (SST and dT), suggesting that thermal stratification was favorable for the proliferation of these organisms. Chaetognaths were the main proportion of carnivores during El Niño 19971998 with a mean of 65% (Fig. 8f). Furtherly, chaetognaths continued as the most abundant carnivores but during the stratified period of 2003-2006 represented a lower proportion (55%), gained by siphonophores (38%). Crustaceans showed correlation only with UI anomalies without evident changes in the proportion of copepods and euphausiids through the time-series (Fig. 8d). Tunicates were not correlated with any environmental variable (Table 3). Trophic groups from the coastal shelf presented stronger seasonal and interannual variability (Fig.

7a, b) compared to oceanic zooplankton. Crustaceans were the most abundant group while abundances of tunicates and carnivores were relatively similar. In Vizcaino bay, contrasting long-term trends were observed in abundance anomalies of crustaceans and gelatinous herbivores, with the first decreasing while the second increased (Fig. 7c, d). Patterns of abundance anomalies were of the same sign in carnivores (Fig. 7e) and tunicates (Fig. 7d) but only the latter presented amplified anomalies toward the end of the time-series. None of the trophic groups in coastal habitat were correlated with ONI (Table 3). Instead, correlations with other indices were observed, crustaceans with PDO and carnivores with NPGO. Dominance of negative anomalies for crustaceans during 2007-2013 coincided with negative values of the PDO and euphausiids appeared to be more affected after 2010 (Fig. 8g). However, absolute abundances of both copepods and euphausiids showed a strong decrease in Vizcaino bay between 2007-2013, with abundances of 40 and 7 ind m-3 respectively, implying a decrease of 48 and 43% in comparison with 1998-2006 period. Carnivores were inversely correlated with NPGO in Vizcaino bay (Table 3). During the stratified period (2003-2006) the highest proportion of carnivores were siphonophores (66%) while chae-

ZOOPLANKTON GROUPS AND CLIMATE VARIABILITY

Figure 4. Central region off Baja California: (a) sea surface temperature, (b) thermal stratification in the upper 200 m, (c) sea surface salinity, (d) saline stratification in the upper 200 m, and (e) Upwelling Index from 27째N, 116째W. Asterisks indicate corrections made to mean temperature due to seasonal bias in sampling, before to estimate the long-term seasonal mean and anomalies..

LAVANIEGOS et al.

Figure 5. Coastal shelf properties at Vizcaino bay (a-b) and the Gulf of Ulloa (c-d). Anomalies of sea surface temperature (a, c) and sea surface salinity (b, d) are shown. Asterisks indicate corrections made to mean temperature due to seasonal bias in sampling, before to estimate the long-term seasonal mean and anomalies. Anomalies from the Gulf of Ulloa must be taken with caution due to a deficient sampling coverage.

tognaths averaged only 25% (Fig. 8i). The tunicates were correlated with UI anomalies (Table 3). Appendicularians reaching a mean of 72% of the tunicates numerically predominated during the period with mostly negative UI anomalies (2000-2004) (Fig. 8h). In contrast during 2005-2013, when UI anomalies were mostly positive, appendicularians represented only 31% of the tunicates. As appendicularians decreased their abundances, salps increased from 24 to 60 % during 2005-2013. Slightly different patterns were found in the Gulf of Ulloa for all the trophic groups (Fig. f-h). Contrary to observations in Vizcaino bay, crustaceans from the Gulf of Ulloa showed several positive anomalies during 2010-2013 (Fig. 7f), tunicates had mainly moderate positive anomalies between 2005 and 2010 shifting further to negative (Fig. g), and carnivores shifted to moderated negative anomalies since 2008 (Fig. 7h). The only significant correlations were with NPGO for the groups of tunicates and carnivores (Table 3). A low number of data available for the Gulf of Ulloa probably precluded significant correlations for carnivores with environmental variables. The proportion of appendicularians was higher during 2000-2004 (Fig. 8k) as it occurred in Vizcaino bay (Fig. 8h). However, the relative abundance of doliolids was higher in the Gulf of Ulloa, particularly during 2005 (Fig. 8k). The incidence of salps increased mainly after 2008. However, mean absolute abundances for salps in the Gulf of Ulloa during 2008-2013 were considerably lower (8 ind m-3) than abundances from Vizcaino bay (44 ind m-3). DISCUSSION The period covered in the present study (19972013) was complicated by the simultaneous occurrence of diverse atmospheric and oceanographic processes. Among these were: 1) the ENSO with

two different flavors (Canonical and Modoki) forcing the ecosystem from the equatorial Pacific; 2) extra-tropical oscillations related to geo-position of atmospheric pressure cells (PDO), and the strength of the North Pacific gyre (NPGO); 3) local upwelling intensity fueled forced by global warming. ENSO effects Following the ENSO signal in the study region was particularly difficult due to problems with SST anomalies. These are the basis for identifying the propagation of the thermal signal from the tropics but require robust seasonal means not only in the number of years involved but also in the month of sampling schedule. This problem was evident with several IMECOCAL cruises mainly for the summer. Fortunately, monthly temperatures for Baja California waters are available from the historic CALCOFI cruises (1951-1966) performed on a monthly basis that represent a strong baseline to understand seasonal variability of temperature in this region. These were useful to adjust in situ temperatures with timelags during the study period (1997-2013). After temporal correction, a correlation was found between ONI and SST, which was particularly strong in the oceanic central region (r = 0.560). The same result was found by Herrera-Cervantes et al. (2014) using satellite derived sea surface temperature off Punta Eugenia. While it is true that El Ni単o influence was detected, differences in magnitude between ONI and local SST anomalies are intriguing, as well as differences in SST within regions. For the event of 2002-2003 Lavaniegos (2014) suggested a blocking of the poleward propagation of El Ni単o during summer 2002 due to a large eddy of subarctic water located offshore to Punta Eugenia. Mesoscale eddies that are recurrent in the vicinity of Punta Eugenia (Soto-Mardones et al., 2004) could be enhanced

ZOOPLANKTON GROUPS AND CLIMATE VARIABILITY

Figure 6. Trophic zooplankton groups mean abundances (± 95% confidence interval) from north (a) and central (b) oceanic regions. Anomalies are also shown for the north (c-e) and central (f-h) regions in separated insets for crustaceans (c, f) tunicates (d, g), and carnivores (e, h).

during other weak El Niño events, avoiding the spread northward to Punta Baja of SST anomalies, inasmuch a significant correlation between SST and ONI was not found in the north region during 2002. The advection of water and the propagation of SST anomalies has become an issue in El Niño theoretical discussions (Yeh et al., 2009; Lee & McPhaden, 2010). However, the period of 2002-2007 with frequent weak El Niño events (mostly Modoki type) presented other influential perturbations in the water column as was higher thermohaline stratification in 2003-2006. Stronger thermal and saline gradients could affect to vertical migrating organisms as was observed in copepods by Lougee et al. (2002); but in contrast it could be favorable for gelatinous zooplankton which efficiently maintains an osmotic balance with seawater (Sanders & Childress, 1995). Decadal Variability Peterson and Schwing (2003) considered the occurrence of a climate shift in 2002-2003 as the PDO reversed sign, and a higher numerical dominance of cold water copepods was observed. In the present study, isolated significant correlations were

observed for PDO, crustaceans from Vizcaino bay and carnivores from the central region. A strong decrease of abundance of crustacean (copepods and euphausiids) is consistent with negative values of PDO in 2002-2003 and again during the 2007-2013 period (Figs. 2b and 7c). This could indicate that subtropical dominant species such as Calanus pacificus and Nyctiphanes simplex could be affected by cold water. The response of the euphausiid N. simplex as a function of the PDO was documented with an increased abundance during warm regime for southern California (Brinton & Townsend, 2003). However, changes observed in the zooplankton community in the present study joint with salinity were better correlated with the NPGO. The shift in 2002-2003 was marked with a northward movement of the North Pacific Current from 42° to 51°N (Freeland & Cummins, 2005) increasing the volume of subarctic water in the California Current (Bograd & Lynn, 2003; Huyer, 2003; Durazo et al., 2005). The subarctic water intrusion promoted high productivity at first, but after 2002 a strong decrease in chlorophyll-a concentration followed and remained

LAVANIEGOS et al.

Figure 7. Trophic zooplankton groups mean abundances (± 95% confidence interval) from Vizcaino bay (a) and the Gulf of Ulloa (b). Anomalies are also shown for Vizcaino bay (c-e) and and the Gulf of Ulloa (f-h) in separated insets for crustaceans (c, f) tunicates (d, g), and carnivores (e, h).

low until 2006 (Gaxiola et al., 2010), which could be related with stratification of the water column as the pattern was similar to SSS and dS anomalies reported in the present study (Figs. 3-4). Therefore, the variability of chlorophyll-a concentration and salinity appears to be more related to NPGO than to El Niño events. Satellite derived surface chlorophyll-a off Punta Eugenia did not show correlation with the multivariate El Niño index (HerreraCervantes et al., 2014). Tunicates showed a significant correlation with dS in the north region (Table 3), meaning that low chlorophyll-a concentrations during high stratification in 2003-2006 (Gaxiola et al., 2010) were favorable for these gelatinous herbivores. These observations are consistent with the incidence of salps and doliolid blooms reported in mesotrophic conditions which are more suitable to their fine mucous filters (Deibel et al., 2009). The size of phytoplankton particles could also play a part in the formation of large aggregations of tunicates. This last statement is suggested by the remarkable high proportion of appendicularians (Fig. 8b), which are known to feed on nanophytoplankton (Acuña et al., 1996).

Upwelling effects The correlation between SST anomalies and ONI was significant but coefficients were relatively low (r < 0.6). Other influences are affecting the pelagic ecosystem as it is reflected in the SST anomalies; for example the low magnitude for negative anomalies during La Niña 2007-2008 (Figs. 3a, 4a, 5a, c). Climate change could be behind the inconsistencies in magnitude of temperature anomalies since has been documented a persistent warming of the word ocean since 2006 (Roemmich et al., 2015). This also could explain the intensification of coastal upwelling since 2005, induced by enhanced alongshore winds by differential land-ocean heating due to greenhouse effect (Bakun et al., 2015; Wang et al., 2015). While strong upwelling may lead to enhanced nutrient enrichment, hypoxic events will be prone to occur and ocean acidity will rise. Hypoxic events are already underway in Vizcaino bay where fishermen from Isla Natividad reported unusual high mortality of abalone, sea urchins, and other benthic organisms during the spring 2009 and summer 2010, associated with shoaling of hypoxic waters (Micheli et al., 2012) similar to other events reported

ZOOPLANKTON GROUPS AND CLIMATE VARIABILITY

Figure 8. Percentage structure of the trophic groups based in geometric means of the zooplankton taxa (staked bars) from the four regions: North (a-c), Central (d-f), Vizcaino bay (g-i) and the Gulf of Ulloa (j-l). Trophic groups are crustaceans (a, d, g, j), tunicates (b, e, h, k), and carnivores (c, f, i, l).

in northern coastal areas of the California Current (Connolly et al., 2010). Low oxygen concentration is also contributed by turbulence as the subsurface eddy recorded off north Baja California during July 2004 (Jeronimo & Gómez-Valdés, 2007). El Niño has also changed in the global warming scenario, with higher incidence and intensity of CP El Niño in the last 30 years (Lee & McPhaden, 2010). Based in models, Yeh et al. (2009) concluded that the ratio of EP-El Niño/CP-El Niño could increase fivefold at the end of 21th century. In conclusion, to the question whether weak El Niño events that occurred during the study period produced any detectable changes in the abundances of zooplankton major taxa, the response would be negative, despite the temperature signal indicating a link with ENSO. In the present study, the main factor influencing structural changes in zooplankton community were associated to stratification in oceanic regions and upwelling enhancement in the coastal shelf. Stratification of the water column appears to be linked to geostrophic circulation (NPGO) mainly in the oceanic region. The notable increase

of gelatinous organisms associated to these processes appears to be in detriment of crustacean plankton though the time-series are still short to outline a more defined trend. That tendency is particularly disturbing in Vizcaino bay with a drastic decrease of grazing crustaceans which in turn nourish fish larvae and adults of sardines and anchovies and, in turn, are being foraged by large predators. It appears that global warming may be behind the enhancement of coastal upwelling but the link between NPGO and global warming or with ENSO requires future investigation. AKNOWLEDGEMENTS José Luis Cadena assisted in zooplankton counting. Thanks are given to all the people involved in the performance of the IMECOCAL cruises. We are especially grateful for the critical insights of Jaime Gómez Gutiérrez to the manuscript. Financial support was from CONACYT (G0041T, G35326T, 42569, 23947, 99252) and SEMARNAT-CONACYT (23804). There were additional funds for curing the zooplankton collection from CONACYT (47044, 129611) and UCMEXUS (CN07-125).

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Ohman, M. D. & B. E. Lavaniegos. 2002. Comparative zooplankton sampling efficiency of the ring net and bongo net with comments on pooling of subsamples. Cal. Coop. Ocean. Fish. Invest. Rep, 43: 162-173. Peterson W. T. & F. B. Schwing. 2003. A new climate regime in northeast pacific ecosystems. Geophys. Res. Lett., 30, doi:10.1029/2003GL017528. Roemmich, D., J. Church, J. Gilson, D. Monselesan, P.Sutton & S. Wijffels. 2015. Unabated planetary warming and its ocean structure since 2006. Nat. Clim. Change, 5: 240-245. Sanders N. & J. Childress. 1995. Nitrogen and buoyancy in marine organisms. 51-62, In: Walsh P.J. & P. Wright (Eds.), Nitrogen metabolism and excretion, CRC Press, Inc., Boca Ratón, Florida, 341 p. Soto-Mardones, L., A. Parés-Sierra, J. Garcia, R, Durazo & S. Hormazaba. 2004. Analysis of the mesoscale structure in the IMECOCAL region (off Baja California) from hydrographic, ADCP and altimetry data. Deep Sea Res II, 51: 785798. Walker, B. H. 1992. Biodiversity and ecological redundancy. Conserv. Biol., 6(1): 18-23. Wang, D., T. C. Gouhier, B. A. Menge & A. R. Ganguly. 2015. Intensification and spatial homogenization of coastal upwelling under climate change. Nature, 518: 390-394. Yeh, S. W., J. S. Kug, B. Dewitte, M. H. Kwon, B. P. Kirtman & F. F. Jin. 2009. El Niño in a changing climate. Nature, 46: 511-515.

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APPENDIX Table 1. Cruise dates and number of zooplankton samples used in taxonomic identification. All nighttime samples were used with additional daytime samples from coastal stations. (*) Cruises with >15 days of time-lag from the mean sampling day. Cruise Name Start date End date Nighttime samples Daytime samples 9710 28 Sep 1997 6 Oct 1997 18 4 9801 25 Jan 1998 11 Feb 1998 33 2 9807 15 Jul 1998 30 Jul 1998 17 5 9810 28 Sep 1998 1 Nov 1998 32 3 9901 14 Jan 1999 31 Jan 1999 26 4 9904 30 Mar 1999 16 Apr 1999 22 7 9908 * 8 Aug 1999 22 Aug 1999 35 5 9910 10 Oct 1999 22 Oct 1999 41 2 0001 14 Jan 2000 1 Feb 2000 46 4 0004 4 Apr 2000 21 Apr 2000 31 3 0007 10 Jul 2000 30 Jul 2000 36 8 0010 10 Oct 2000 29 Oct 2000 38 3 0101 16 Jan 2001 3 Feb 2001 37 4 0104 5 Apr 2001 12 Apr 2001 8 1 0107 * 26 Jun 2001 16 Jul 2001 29 6 0110 3 Oct 2001 23 Oct 2001 43 8 0201 19 Jan 2002 6 Feb 2002 35 3 0204 19 Apr 2002 7 May 2002 27 3 0207 12 Jul 2002 01 Aug 2002 40 6 0210 23 Oct 2002 9 Nov 2002 41 0 0302 30 Jan 2003 19 Feb 2003 48 4 0304 4 Apr 2003 22 Apr 2003 31 3 0307 07 Jul 2003 27 Jul 2003 35 7 0310 10 Oct 2003 29 Oct 2003 47 4 0402 30 Jan 2004 18 Feb 2004 36 7 0404 18 Apr 2004 6 May 2004 32 6 0407 09 Jul 2004 29 Jul 2004 36 6 0410 9 Oct 2004 27 Oct 2004 45 5 0501 21 Jan 2005 10 Feb 2005 51 8 0504 14 Apr 2005 5 May 2005 39 7 0507 14 Jul 2005 04 Aug 2005 41 8 0510 13 Oct 2005 27 Oct 2005 41 3 0602 5 Feb 2006 25 Feb 2006 39 6 0604 19 Apr 2006 1 May 2006 19 1 0607 7 Jul 2006 25 Jul 2006 33 4 0701 23 Jan 2007 10 Feb 2007 51 3 0704 26 Apr 2007 6 May 2007 10 0 0708 * 25 Aug 2007 13 Sep 2007 42 9 0801 23 Jan 2008 11 Feb 2008 35 5 0804 16 Apr 2008 1 May 2008 21 7 0807 14 Jul 2008 02 Aug 2008 41 8 0810 14 Oct 2008 26 Oct 2008 29 4 0904 9 Apr 2009 23 Apr 2009 31 4 0911 * 30 Oct 2009 13 Nov 2009 38 5 1004 29 Mar 2010 17 Apr 2010 38 5 1008 29 Jul 2010 7 Aug 2010 16 1 1010 4 Oct 2010 18 Oct 2010 28 1 1101 20 Jan 2011 6 Feb 2011 39 3

ZOOPLANKTON GROUPS AND CLIMATE VARIABILITY

Table 1. Continued. Cruise Name

Start date

End date

Nighttime samples

Daytime samples

1104 * 1107 1110 1202 1203 * 1302 * 1305 *

19 Apr 2011 10 Jul 2011 4 Oct 2011 25 Jan 2012 8 Mar 2012 8 Feb 2013 23 May 2013

9 May 2011 27 Jul 2011 25 Oct 2011 11 feb 2012 24 Mar 2012 27 Feb 2013 7 Jun 2013

32 34 42 49 41 14 26

6 6 5 5 7 0 8

Table 2. Temperature corrections in cruises out of phase performed previously to estimation of climatologic means and anomalies. The correction was done with AMT = MT • TL • DTC; where (MT) is the mean temperature, (AMT) adjusted mean temperature, (TL) are days before (negative) or after (positive) the mean seasonal sampling date, and (DTC) is daily temperature change which was based in monthly means from the period 1951-1966 (see Appendix Figure 1). Only AMT with differences higher than 0.5°C were considered in the estimation of climatologic means and anomalies (highlighted in bold). Region NORTH

Mean seasonal sampling date

Out-ofphase Cruise 1302

Temperature difference (1951-1966) Jan‒Feb ‒0.23

MT (°C)

AMT (°C)

‒0.008

14.73

14.88

Spring (Apr 16)

9904 1104 1203 1305

‒15 18 ‒36 39

Mar‒Apr 0.37 Apr‒May 0.54 Feb‒Apr 0.24 Apr‒Jun 1.10

0.012 0.018 0.004 0.018

14.89 15.94 14.82 16.93

15.08 15.62 14.96 16.22

Summer (Jul 21)

9908 0107 0708

20 ‒22 38

Jul‒Aug 1.05 Jun‒Jul 1.15 Jul‒Sep 2.07

0.035 0.038 0.035

18.60 18.35 19.34

17.90 19.19 18.03

Autumn (Oct 13)

0911

Oct‒Nov ‒0.84

‒0.028

18.72

19.25

1302

Feb‒Mar ‒0.56

‒0.019

15.55

15.90

Spring (Apr 23)

1203 1305

‒34 44

Mar‒Apr ‒0.04 Apr‒Jun 0.73

‒0.001 0.012

15.65 18.22

15.60 17.68

Summer (Jul 29)

9908 0107 0708

19 -20 40

Jul‒Aug 1.55 Jun‒Jul 2.04 Jul‒Sep 2.27

0.052 0.068 0.038

19.46 20.36 22.00

18.48 21.72 20.49

Autumn (Oct 21)

9710 0911

‒20 19

Sep‒Oct ‒0.35 Oct‒Nov ‒0.27

‒0.012 ‒0.009

24.40 21.29

24.17 21.46

1302

Feb‒Mar ‒0.84

‒0.028

14.34

14.93

1203 1305

‒33 38

Mar‒Apr 0.21 Apr‒Jun 0.45

0.007 0.008

14.81 15.47

15.04 15.19

9908 0107 0708 9710 0911

20 ‒21 38 ‒17 18

Jul‒Aug 1.10 Jun‒Jul 1.86 Jul‒Sep 3.00 Sep‒Oct ‒1.23 Oct‒Nov ‒0.31

0.037 0.062 0.050 ‒0.041 ‒0.010

17.61 17.14 19.75 24.62 20.25

16.88 18.44 17.85 23.92 20.44

Winter (Jan 27)

CENTRAL Winter (Feb 6)

VIZCAINO Winter (Feb 3) BAY Spring (Apr 21)

Summer (Jul 26)

Autumn (Oct 19)

TL (d)

DTC (°C d-1)

LAVANIEGOS et al.

Table 2. Continued. Region

Mean seasonal sampling date

Out-ofphase Cruise

TL (d)

GULF OF ULLOA

Spring (Apr 23)

1203 1305

‒31 43

Summer (Aug 1)

9908 0107 0708 1107

Autumn (Oct 25)

0911

Temperature difference (1951-1966)

DTC (°C d-1)

MT (°C)

AMT (°C)

Mar‒Apr ‒0.96 Apr‒Jun ‒0.20

‒0.032 ‒0.003

15.54 15.22

14.55 15.36

19 ‒19 39 ‒18

Jul‒Aug 4.66 Jun‒Jul 2.44 Jul‒Sep 6.64 Jul‒Aug 4.66

0.155 0.081 0.111 0.155

20.17 18.32 21.90 17.45

17.22 19.87 17.58 20.25

Oct‒Nov ‒0.79

‒0.026

24.27

24.72

Figure 1. Seasonal mean temperature at 10 m depth for the period 1951-1966 in oceanic and coastal shelf regions (mean ± standard deviation). Data generated by the California Cooperative Oceanic Fisheries Investigations program (http://www.calcofi.org/ new.data/index.php/publications/calcofi-data-reports/archived-data-reports).

CICIMAR Oceánides 30(1): 63-70 (2015)

New records of the distinctive benthic dinoflagellate genus Cabra (Dinophyceae) Gómez, F. & R. M. Lopes

Laboratory of Plankton Systems, Oceanographic Institute, University of São Paulo, Praça do Oceanográfico 191, Cidade Universitária, Butantã, São Paulo 05508-900, Brazil. email: fernando.gomez@fitoplancton.com

ABSTRACT. The benthic dinoflagellate genus Cabra is reported for the first time in the Mediterranean Sea and the South Atlantic Ocean, with additional records in the Caribbean Sea and the eastern Asian coasts. Cabra aremorica is reported for the first time after the original description. However, these records should be considered cautiously because the distinction between Cabra aremorica and C. reticulata is difficult based on routine light microscopy observations. It is uncertain whether there is a high intraspecific morphological variability or several co-occurring undescribed species. Cabra levis, a species recently described, is reported for first time beyond the type locality.

Keywords: benthic Dinophyta, epiphytic Dinoflagellata, microphytobenthos, psammophilic, Dinophyceae, sand-dwelling dinoflagellate.

Nuevos registros del distintivo género de dinoflagelado bentónico Cabra (Dinophyceae) Resumen. El dinoflagelado bentónico del género Cabra se describe por primera vez en el Mar Mediterráneo y el Océano Atlántico Sur, con registros adicionales en el Mar Caribe y las costas orientales de Asia. Cabra aremorica se cita por primera vez después de la descripción original. Sin embargo, estos registros deben ser considerados con cautela porque la distinción entre Cabra aremorica y C. reticulata es difícil basándose solo en observaciones rutinarias de microscopía óptica. No está claro si existe una alta variabilidad morfológica intra-específica o si existen varias especies no descritas que coexisten. Cabra levis, una especie recientemente descrita, se describe por primera vez más allá de su localidad tipo.

Palabras clave: Dinoflagelado bentónico, dinoflagelado epifítico, microfitobentos, Dinophyceae psammofílico, dinoflagelado arenícola. Gómez, F. & R. M. Lopes. 2015. New records of the distinctive benthic dinoflagellate genus Cabra (Dinophyceae). CICIMAR Oceánides, 30(1): 63-70.

Introduction Cabra matta Sh. Murray et D.J. Patterson is the type species of a distinctive genus of marine benthic dinoflagellates described in sand habitats of eastern Australia (Murray & Patterson, 2004). Cabra reticulata Chomérat et Nézan and C. aremorica Chomérat, Couté et Nézan were further described from sandy sediments of the coasts of north-western France (Chomérat & Nezán, 2009; Chomérat et al., 2010). The cells were highly laterally compressed with a more or less pentagonal outline, asymmetrical with different right and left sides. The hypotheca showed one dorsal and three antapical pointed flanges. Beyond the original descriptions in the Australian and French Atlantic coasts, further records were restricted to the Gulf of Mexico (Okolodkov et al., 2007, 2014), Russian coasts of the Japan Sea (Selina & Levchenko, 2011) and Korea (Shah et al., 2013). Recently, a new species, Cabra levis Selina, Chomérat et Hoppenrath has been described from Russian coasts (Selina et al., 2015). It is characterized by a rounded shape and lacking pointed flanges. This study reports the first records of Cabra in the Mediterranean Sea and the South Atlantic Ocean, and additional records in the Caribbean Sea and the eastern Asian coasts. These observations constitute the first record of C. aremorica beyond the type locality. Specimens with different morpho-logies Fecha de recepción: 04 de mayo de 2015

co-occurred in the samples from Puerto Rico and Brazil. This raises a question on the delimitation of the known species: is there a high intraspecific morphological variability and/or several undescribed species?: whether there is a high intraspecific morphological variability and/or several undescribed species. The recently described species C. levis is reported for the first time beyond the type locality. These new records are discussed within the context of an increase in the reports of invasive and nonindigenous species. Materials and methods The study in the Mediterranean Sea was focused on the planktonic dinoflagellates. However, during surveys near pier walls at Marseille and Valencia numerous epiphytic dinoflagellates were accidentally collected (i.e., Coolia Meunier, Ostreopsis Johannes Schmidt). One specimen of the genus Cabra was collected with a bucket from the pier of the Station Marine d’Endoume at Marseille, France (43° 16’ 48.05” N, 5° 20’ 56.22” E, bottom depth ~3 m) on August 11th, 2008. A strainer of 20 µm mesh size was used to concentrate the sample. The ‘plankton’ concentrate was examined in a composite settling chamber at ×100 with an inverted microscope (Nikon Eclipse TE200, Nikon Inc., Tokyo, Japan). The cell was photographed at ×200 or ×400 with a digital camera (Nikon Coolpix E995). Another specFecha de aceptación: 12 de mayo de 2015

GÓMEZ & LOPES

imen of Cabra was collected using a phytoplankton net (20 µm mesh size) at the port of Valencia, Spain (39° 27’ 38.13” N, 0° 19’ 21.29” W, bottom depth ~4 m) on November 18, 2011. The sample was examined in a composite settling chamber with an inverted microscope (Nikon Eclipse T2000) and photographed with a digital camera (Olympus DP71, Olympus, Tokyo, Japan). In the coasts of the Caribbean Sea, the epiphytic dinoflagellates attached to the marine plant Thalassia testudinum Banks et Sol. ex K.D. Koenig, were examined. Seagrass leaves were collected by snorkeling (2–3 m depth) around the pier of Magüeyes Island (17° 58’ 11.80” N, 67° 2’ 46.56” W) at La Parguera, Puerto Rico, during ten surveys in February and March 2012. The leaves were placed in PVC bottles containing 200 ml of seawater and stirred vigorously to detach the epiphytic cells. The suspended material was sieved through a 100 µm mesh to remove large particles. The samples were examined in a composite settling chamber with an inverted microscope (3030 Accu-scope, Commack, New York) and photographed with a digital camera. In the South Atlantic Ocean, epiphytic dinoflagellates on macroalgae were investigated from the coasts of São Paulo State, Brazil. At São Sebastião (23° 49’ 34.54” S, 45° 25’ 18.26” W), macroalgae were collected from tidal pools (<1 m depth) during the low tide according to the procedure described above, in sporadic surveys between March and December 2013. Samples were examined in a composite settling chamber at ×200 with an inverted microscope (Nikon Diaphot-300) and photographed with a digital camera mounted on the microscope’s eyepiece (Cyber-shot DSC-W300, Sony, Tokyo, Japan). At Ubatuba (23° 30’ 3.16” S, 45° 7’ 6.78” W), macroalgae were collected from rocky surfaces during the low tide (<1 m depth) according to the procedure described above, in sporadic surveys between January 2014 and December 2014. Samples were examined in a composite settling chamber with an inverted microscope (Nikon Eclipse TS-100F) and photographed with a digital camera mounted on the microscope’s eyepiece (Cyber-shot DSC-W300). In the north-western Pacific Ocean, one macroalgae sample was collected at Dongshan Island, China (23° 35’ 25.64” N, 117° 26’ 4.24” W) on August 15th 2014. The sample was examined in a composite settling chamber with an inverted microscope (Nikon TS-100F). Results The distinction between the type species, C. matta, and the other two described species with pointed flanges (C. aremorica and C. reticulata) was relatively easy because the dorsal margin of the hypotheca of C. matta is more or less rounded, while it is polygonal in the other described species. However, it is more difficult to distinguish between C.

reticulata and C. aremorica because the diagnostic characters used for species delimitation (i.e., thecal ornamentation) were not easy to discern based on routine light microscopy observations. The two specimens observed from the Mediterranean Sea corresponded to Cabra aremorica or C. reticulata (Fig. 1A–B, E–F). The original illustrations of C. aremorica (Fig. 1C–D) and C. reticulata (Fig. 1H–J) were reproduced here in order to facilitate the comparisons. The specimen from Marseille was 42 µm long and 37 µm deep (as dorso-ventral diameter) (Fig. 1E). The specimen from Valencia was 40 µm long and 31 µm deep (Fig. 1F). Consequently, the hypotheca of the latter specimen was relatively more elongated, in agreement with the original description of C. reticulata (Fig. 1H). The relative distance from the dorsal margin of cingulum to the pointed flange of the plate 3’’’ was higher in the specimen from Marseille. It is assumed that a slight difference in size when compared to the original description is a poor diagnostic character. Based on the cell shape, the specimens from Marseille and Valencia have been designated as C. aremorica and C. reticulata, respectively. However, we have to be cautious because the delimitation between C. aremorica and C. reticulata is difficult based on light microscopy. To the best of our knowledge, these are the first records of the genus Cabra in the Mediterranean Sea. While the Mediterranean observations were restricted to two specimens accidentally collected in plankton samples, the surveys in Puerto Rico and Brazil were intended for the study of the epiphytic dinoflagellates. In Puerto Rico, each seagrass sample showed specimens of Cabra with different morphologies. A single specimen showed different shapes according to the viewing angle (Fig. 1J–K). The records could be divided into two groups: specimens with a polygonal dorsal margin (Fig. 1J–L) as in C. aremorica and C. reticulata, and a second group for specimens with a rounded dorsal margin of the hypotheca. The latter group included specimens with pointed flanges, as in C. matta (Fig. 1M–O), and rounder specimens lacking the pointed flanges (Fig. 1P–R). The morphology of some specimens did not match with the three described species. Figures J–K show two views of a single specimen. In any view, the relative distance between the dorsal end of the cingulum and the pointed flange of the plate 3’’’ was higher in the Puerto Rican specimens (Fig. 1J–K) than in the Mediterranean specimens (Fig. 1E–F) and in the original descriptions (Fig. 1C–D, H–I). The specimens with polygonal hypotheca were 36–41 µm long and 28–35 µm deep. With doubts whether these specimens correspond to C. aremorica, C. reticulata or undescribed species, we designated the specimens as C. aremorica (Fig. 1J–K). Co-occurring with these specimens, the samples contained specimens with a rounded dorsal margin of the hypotheca that has been identified as C. matta (Fig. 1M-O). Other specimens with a round

NEW RECORDS OF Cabra (DINOPHYCEAE)

cell shape showed a relative larger epitheca and semi-circular hypotheca. The antapical flanges were lacking or scarcely visible (Fig. 1P-R). The cells were 33–35 µm long and 30–32 µm deep (Fig. 1P-

R). These specimens were identified as Cabra levis. Cabra morphological diversity was similar on macroalgae epiphytes from Brazil. A group of

Figure 1. Light micrographs of Cabra spp.: A-B, E-G. Specimens from the western Mediterranean Sea; J-R. Specimens from the Caribbean Sea; S-AD. Specimens from the South Atlantic Ocean; A–B. Right lateral view of C. aremorica from Marseille, France; C–D. Line drawings of C. aremorica redrawn from Chómerat et al. (2010); E. Left lateral view of C. aremorica from Marseille, France; F–G. Left lateral view of C. reticulata from Valencia, Spain. H–I. Line drawings of C. reticulata redrawn from Chómerat et al. (2010); J–L. C. aremorica from La Parguera, Puerto Rico; M-O. C. matta from La Parguera, Puerto Rico; P–R. Cabra levis from La Parguera; S–U. Left lateral view of C. aremorica from São Sebastião, Brazil; V–Y. C. matta from São Sebastião; Z–AB. Cabra levis from São Sebastião; AC–AD. Left lateral view of C. reticulata from Ubatuba, Brazil. Note the yellow-brownish food bodies in the middle of the cells. Scale bars, 20 µm.

GÓMEZ & LOPES

specimens with a polygonal dorsal margin of the hypotheca has been tentatively designated as C. aremorica (Fig. 1S–U) and C. reticulata (Fig. 1AC– AD). Other group of specimens showed a rounder hypotheca (Fig. 1V–AB). This group included specimens with a rounded dorsal margin, and the dorsal margin with an irregular outline, more or less dentate (Fig. 1V–W), and other specimens with an ellipsoidal hypotheca and smooth dorsal outline (Fig. 1X–Y). The cells were 32-36 µm long, and about 27 µm deep. These specimens were designated as C. matta (Fig. 1V–Y). Other specimens showed a rounded cell shape with a diameter of about 31 µm, slightly longer than deep. In some angles of view, at least two short antapical flanges were visible (Fig. 1Z–AA); while in other specimens the flanges were not observed (Fig. 1AB). These specimens were identified as Cabra levis. These are the first records of the genus Cabra in the South Atlantic Ocean. In the Chinese coasts at Dongshan Island, we observed three specimens from a single sample that corresponded to the morphology of C. matta. Discussion Historical records of Cabra The morphology of the known species of Cabra is very distinctive, and here we show that they are a relatively common epiphyte on macrophytes in different biogeographic regions. However, the records are scarce and surprisingly the genus had not been described until the last decade (Murray & Patterson, 2004) (Fig. 2). Benthic dinoflagellates have been undersampled for decades until Japanese researchers discovered that some epiphytic dinoflagellates (i.e., Gambierdiscus Adachi et Fukuyo) were responsible for the ciguatera disease (Yasumoto et al., 1977). Carlson (1984) investigated the epiphytic dinoflagellate assemblage in a ciguatera endemic area in the U.S. Virgin Islands, near Puerto Rico. He illustrated a specimen as a new genus, Thecadinium sp. (Carlson, 1984). However, he did not provide a formal description and Chomérat & Nézan (2009) considered that Carlson’s Thecadinium sp. corresponded to C. reticulata. Ballantine et al. (1988) investigated the epiphytic dinoflagellates at La Parguera, Puerto Rico. They did not report Cabra probably because their study was exclusively focused on the potentially toxigenic species (Gambierdiscus, Ostreopsis, Coolia, Prorocentrum Ehrenb.). The present study, based on only ten surveys from the same location, revealed that Cabra spp. are common epiphytes on the seagrass Thalassia testudinum. Okolodkov et al. (2007, 2014) reported T. testudinum as substrate for epiphytic dinoflagellates. It should be noted that the leaves of Thalassia are also substrate for algal turfs. Other studies in the Caribbean Sea that focused on epiphytic dinoflagellates on T. testudinum have not recorded species of Cabra (i.e., Rodríguez et al., 2010). Jacobson (1999, p. 378) illustrated a speci-

men of Cabra, but with no information on the figure legend or the text. According to this author, the specimens were collected in Vineyard Sound, near Woods Hole, USA. He did not describe them due to the paucity of specimens and the difficulties to interpret the thecal plate pattern (D.M. Jacobson, personal communication). The shape of his specimens resembles C. aremorica, with a dentate dorsal margin of the hypotheca with 3-4 projections or spines (Jacobson, 1999, p. 378). These spines are lacking in the original description of the known species of Cabra. In the present study, an irregular outline of the dorsal margin of the hypotheca was observed in some specimens (Fig. 1V). In the case of the heterotrophic genus Cabra, the paucity of specimens represents a challenge for studies on intraspecific morphological variability or even molecular analysis. Cabra is one of the few benthic genera described in the last decade with no molecular information. Carlson (1984) realized that it was a new genus, but he did not describe his specimens. Jacobson (1999) did not describe the genus due to the difficulties to interpret the tabulation (D.M. Jacobson, personal communication). The interpretation of the tabulation is challenging and varied between Murray & Patterson (2004) and Chomérat et al. (2010). For these reasons, Cabra was not described until recently from sandy sediments in Australia (Murray & Patterson, 2004). The original descriptions of the flange-bearing species were based on specimens collected in sandy sediments (Murray & Patterson, 2004; Chomérat & Nezán, 2009; Chomérat et al., 2010). However, most records of Cabra are as epiphyte on macroalgae (Carlson, 1984; Okolodkov et al., 2007, 2014; Selina & Levchenko, 2011; Shah et al., 2013). Selina & Levchenko (2011) reported that C. matta was found fairly often on macrophytes (with occurrence up to 35% of the examined samples). Cabra did not show a clear preference for a substrate, being found as epiphyte of several species of Rhodophyta, Phaeophyta and Chlorophyta in the cold waters of the western Pacific (Selina & Levchenko, 2011). We have not observed Cabra as a sand-dwelling dinoflagellate (Gómez & Artigas, 2014). However, according to our observations Cabra is relatively common as epiphyte on macrophytes. Although the flange-bearing species of Cabra were described as sand-dwelling dinoflagellates, we should consider that macrophyte surfaces are the preferential habitat. There is a high tradition of taxonomical studies on dinoflagellates in the Mediterranean Sea. That basin represents a small percentage of the world’s oceans (<1%). However, about 88% of the dinoflagellate genera have been reported in the Mediterranean Sea (Gómez, 2006). In the last decade, the Mediterranean Sea has been subjected to intensive monitoring of epiphytic dinoflagellates, especially after reports of human health problems attributed

NEW RECORDS OF Cabra (DINOPHYCEAE)

to Ostreopsis proliferations (e.g., Del Favero et al., 2012). In the present study, Ostreopsis, Coolia, Amphidinium Clap. et J. Lachm., and some benthic species of Prorocentrum were the most common dinoflagellates co-occurring with Cabra. The Mediterranean records of Cabra corresponded to specimens accidentally detached from the macroalgae, collected from the water column near piers. It is expected that dedicated studies on the epiphytic dinoflagellates, including heterotrophic species, will provide a more realistic estimate of Cabra distribution and abundance in the Mediterranean Sea, and other ocean basins. Dinoflagellates reported as new records in Mediterranean Sea are often categorized as newcomers, invasive species or biological indicators of global warming (Gómez, 2008). These first Mediterranean records of Cabra are explained because this genus has been overlooked in the past. One Mediterranean specimen was observed in November, when water temperature was low, suggesting that Cabra is not a biological indicator of warming. Despite its common occurrence in the tropical waters of the Caribbean Sea and Brazil, the warm-water affinity of the genus Cabra is unclear. Three of the four species were first described from the cold waters of northwestern France (Chomérat et al., 2010) or the cold Pacific waters (Selina et al., 2015). In the South Atlantic Ocean, the studies on the benthic dinoflagellates in the South American and African coasts are almost inexistent, and any observation reveals new records of benthic dinoflagellates. We also observed three specimens of C. matta from a single sample of macroalgae epiphytes near Xiamen, China. Cabra matta has been recently cited in the eastern coasts of Asia (Selina & Levchenko, 2011; Shah et al., 2013; Selina et al., 2015) (Fig. 2). Consequently, we have

to be cautious on the consideration of the species of Cabra as a newcomer, invasive species or as a biological indicator of warming. Obviously, the species of Cabra have been overlooked in the past and its biogeographic range has been underestimated. Difficulties in species delimitation While the genus Cabra is highly distinctive, identification at the species level is problematic due to difficulties in the distinction between C. reticulata and C. aremorica based on routine light microscopy observations. In addition, we cannot discard that records attributed to these species corresponded to undescribed taxa. The three flange-bearing species of the genus Cabra were described based on detailed observations of the thecal plate pattern and ornamentation by scanning electron microscopy (Murray & Patterson, 2004; Chomérat & Nezán, 2009; Chomérat et al., 2010). However, these studies included few light microscopy pictures and there is no information on the intraspecific morphological variability. The records of Cabra in the Gulf of Mexico are an example of the confusion. Before the descriptions of C. reticulata and C. aremorica, Okolodkov et al. (2007) provided a micrograph of Cabra, which they identified tentatively as C. matta. More recently, Okolodkov et al. (2014) reported the species as Cabra cf. aremorica. In a recent book by the authorities who described the flange-bearing species of Cabra, the Mexican records have been attributed to C. reticulata (Hoppenrath et al., 2014, p. 74). This evidences the difficulties to discern between C. aremorica and C. reticulata. Both species more or less overlay in size, and we can observe that the length or shape of the dorsal and antapical flanges are variable, as well as the relative distance between the cingulum and the dorsal pointed flange.

Figure 2. Records of Cabra spp. in the world’s oceans. Square: C. matta; Triangle: C. aremorica; Hexagon: C. reticulata; Circle: Cabra levis. Sources: 1. Carlson (1984); 2. Jacobson (1999); 3. Murray & Patterson (2004); 4. Chomérat & Nezán (2009), Chomérat et al. (2010); 5. Okolodkov et al. (2007, 2014); 6. Selina & Levchenko (2011); 7. Shah et al. (2013); 8. Selina et al. (2015); 9. This study (red symbols).

GÓMEZ & LOPES

Other difficulty is that some specimens cannot be assigned to a known species. Selina & Levchenko (2011) reported rounded cells lacking the pointed flanges, which they designated as Cabra cf. matta, and recently described as C. levis (Selina et al., 2015). Their pictures corresponded to the same morphology here illustrated from Puerto Rico and Brazil (Figs 1P–R, 1Z–AB). This implies a wide distribution of C. levis which co-occurred with C. matta and C. aremorica/reticulata. The co-occurrence of congeneric species is a common feature in other heterotrophic benthic dinoflagellates, such as Amphidiniopsis Wolosz. (Gómez et al., 2011; Gómez & Artigas, 2014). The absence or reduced extension of the flanges and the rounded cell shape are distinctive features for congeneric species. However, the pointed flanges and the polygonal shape are distinctive characters of the genus Cabra. Consequently, the less distinctive member of Cabra species such as C. levis could be mistaken with other benthic dinoflagellates, such as large specimens of Aduncodinium glandula (Herdman) N.S. Kang, H.J. Jeong et Moestrup, or Durinskia agilis (Kof. et Swezy) Saburova, Chomérat et Hoppenrath. In the last decade, almost all new species of benthic dinoflagellates have been described including their molecular data. This is not the case of the four species of Cabra. The study of the intraspecific morphological variability, life stages from cultures, or single-cell PCR could help to discern whether there are several species or a single species with a high intraspecific morphological variability. We are unable to demonstrate an affinity of Cabra to any other known dinoflagellate. In the species descriptions, Cabra has been related to the family Podolampadaceae (i.e., Podolampas F. Stein; Blepharocysta Ehrenb.) (Murray & Patterson, 2004; Chomérat et al., 2010). A close relation between benthic and plankton species has been demonstrated in species such as Amphidiniopsis or Herdmania J.D. Dodge (Gómez et al., 2011; Yamaguchi et al., 2011). The sanddwelling genus Roscoffia Balech has been placed in Podolampadaceae (Saldarriaga et al., 2003; Gómez et al., 2010). The phylogenetic relation between Cabra and Podolampadaceae remains unresolved. Concluding remarks The records of species of Cabra indicate that the genus is relatively common as epiphyte on macrophytes in the examined locations. The genus Cabra is a good example that: 1) despite the distinctive morphology, a genus could remain undescribed due to the difficulties in the interpretation of the tabulation and paucity of specimens for the standard requirements for benthic dinoflagellate descriptions. In fact, Cabra was too rare to be described before. Cabra was first described as a sand-dwelling dinoflagellate when it is really more common as an epiphytic dinoflagellate. Taxonomists working on epiphytic dinoflagellates are more focused on the photosynthetic cultivable species, especially the

toxigenic genera, and the surrounding assemblage of heterotrophic dinoflagellates did not receive attention. In contrast, taxonomists working on sanddwelling dinoflagellates have more expertise on species descriptions based on fewer specimens and taxa with difficult interpretations of the plate pattern; 2) the delimitation of the species Cabra aremorica and C. reticulata is unclear based on routine light microscopy observations; and 3) the records of Cabra will spread to other ocean regions in the next years. Benthic dinoflagellates, especially the heterotrophic species, were undersampled and overlooked in the past. This should be taken into account when categorizing benthic dinoflagellates as newcomers, invasive species or biological indicators of warming. Acknowledgements We thank E. Otero and B.M. Soler for the hospitality extended during the sampling in Puerto Rico. F.G. was supported by the Ministerio Español de Ciencia y Tecnología (contract no. JCI-201008492). This research is supported by the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant numbers BJT 370646/2013-14 to F.G., and 402759/2012-5 and 311936/2013-0 to R.M.L.). References Ballantine, D.L., T.R. Tosteson & A.T. Bardales. 1988. Population dynamics and toxicity of natural populations of benthic dinoflagellates in south-western Puerto Rico. J. Exp. Mar. Biol. Ecol., 119: 201–212. Carlson, R.D. 1984. Distribution, periodicity, and culture of benthic/epiphytic dinoflagellates in a ciguatera endemic region of the Caribbean. PhD thesis. Southern Illinois University, Carbondale, U.S.A. Chomérat, N. & E. Nézan. 2009. Cabra reticulata sp. nov. (Dinophyceae), a new sand-dwelling dinoflagellate from the Atlantic Ocean. Eur. J. Phycol., 44: 415–423. Chomérat, N., A. Couté & E. Nézan. 2010. Further investigations on the sand-dwelling genus Cabra (Dinophyceae, Peridiniales) in South Brittany (north-western France), including the description of C. aremorica sp. nov. Mar. Biodiv., 40: 131–142. Del Favero, G., S., Sosa M., Pelin, E., D’Orlando, C., Florio, P., Lorenzon, M., Poli & A. Tubaro. 2012. Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: A multidisciplinary scientific approach. Annali dell’Istituto Superiore di Sanita, 48: 407–414. Gómez, F. 2006. Endemic and Indo-Pacific plankton in the Mediterranean Sea: A study based on dinoflagellate records. J. Biogeogr., 33: 261–270.

NEW RECORDS OF Cabra (DINOPHYCEAE)

Gómez, F. 2008. Phytoplankton invasions: Comments on the validity of categorizing the nonindigenous dinoflagellates and diatoms in European Seas. Mar. Poll. Bull., 56: 620–628. Gómez, F., D. Moreira & P. López-García. 2010. Molecular phylogeny of the dinoflagellates Podolampas and Blepharocysta (Peridiniales, Dinophyceae). Phycologia, 49: 212–220.

paceae and the taxonomic position of the genus Roscoffia. J. Phycol., 39: 368–378. Selina, M.S. & E.V. Levchenko. 2011. Species composition and morphology of dinoflagellates (Dinophyta) of epiphytic assemblages of Peter the Great Bay in the Sea of Japan. Rus. J. Mar. Biol., 37: 23–32.

Gómez, F., P. López-García & D. Moreira. 2011. Molecular phylogeny of the sand-dwelling dinoflagellates Amphidiniopsis hirsuta and A. swedmarkii (Peridiniales, Dinophyceae). Acta Protozool., 50: 255–262.

Selina, M.S., N. Chomérat & M. Hoppenrath. 2015. Morphology and spatial distribution of Cabra species (Dinophyceae, Peridiniales) from Peter the Great Bay (northwestern Sea of Japan), including the description of C. levis sp. nov. Eur. J. Phycol., 50: 80–91.

Gómez, F. & L.F. Artigas. 2014. High diversity of dinoflagellates in the intertidal sandy sediments of Wimereux (NE English Channel, France). J. Mar. Biol. Assoc. U.K., 94: 443–457.

Shah, M.M.R., S.-J. An, & J.-B. Lee. 2013. Presence of benthic dinoflagellates around coastal waters of Jeju Island including newly recorded species. J. Ecol. Environment, 36: 347–370.

Hoppenrath, M., S. Murray, N. Chomérat & T. Horiguchi. 2014. Marine benthic dinoflagellates –unveiling their worldwide biodiversity. Kleine Senckenberg-Reihe 54. Stuttgart: Schweizerbart’sche Verlagsbuchhandlung.

Yamaguchi, A., M. Hoppenrath, V. Pospelova, T. Horiguchi & B.S. Leander. 2011. Molecular phylogeny of the marine sand-dwelling dinoflagellate Herdmania litoralis and an emended description of the closely related planktonic genus Archaeperidinium Jörgensen. Eur. J. Phycol., 46: 98–112.

Jacobson, D.M. 1999. A brief history of dinoflagellate feeding research. J. Eukaryotic Microbiol., 46: 376–381. Murray, S. & D.J. Patterson. 2004. Cabra matta, gen. nov., sp. nov., a new benthic, heterotrophic dinoflagellate. Eur. J. Phycol., 39: 229–234. Okolodkov, Y.B., G. Campos-Bautista, I. GárateLizárraga, J.A.G. González-González, M. Hoppenrath & V. Arenas. 2007. Seasonal changes of benthic and epiphytic dinoflagellates in the Veracruz reef zone, Gulf of Mexico. Aquat. Microb. Ecol., 47: 223–237. Okolodkov, Y.B., F.C. Merino-Virgilio, J.A. AkéCastillo, A.C. Aguilar-Trujillo, S. EspinosaMatías & J.A. Herrera-Silveira. 2014. Seasonal changes in epiphytic dinoflagellate assemblages near the northern coast of the Yucatan Peninsula, Gulf of Mexico. Acta Bot. Mex., 107: 121–151. Rodríguez, E.A., J.E. Mancera Pineda & B. Gavio. 2010. Survey on benthic dinoflagellates associated to beds of the Thalassia testudinum in San Andrés Island, seaflower biosphere reserve, Caribbean Colombia. Acta Biol. Colomb., 15: 229–246. Saldarriaga, J.F., B.S. Leander, F.J.R. Taylor & P.J. Keeling. 2003. Lessardia elongata gen. et sp. nov. (Dinoflagellata, Peridinales), Podolam-

Yasumoto, T., I. Nakajima, R. Bagnis & R. Adachi. 1977. Finding of a dinoflagellate as a likely culprit of ciguatera. Bull. Jap. Soc. Scient. Fish., 43: 1021–1026.

CICIMAR Oceánides 30(1): 71-76 (2015)

NOTA Benthic diatoms from shallow environments deposited at 300 m depth in a southern Gulf of California basin

Diatomeas bentónicas de ambientes someros depositadas a 300 m de profundidad en una cuenca del sur del Golfo de California

RESUMEN. Se registraron diatomeas bentónicas recolectadas con una trampa Technicap modelo PPS-3/3 con una abertura de 0.125 m2, la cual consta de un carrusel programable motorizado con doce botellas de 250 mL instalada a 300 m de profundidad en Cuenca Alfonso, Bahía de La Paz. Se contrastó la hipótesis de que diatomeas depositadas en la trampa de sedimento estarían relacionadas a ambientes de manglar de la Bahía de La Paz. Así, con el objetivo de identificar las diatomeas bentónicas e inferir su procedencia, se revisó una muestra del período diciembre de 2011 a enero de 2012. La identificación se realizó a partir de imágenes tomadas con un microscopio electrónico de barrido Zeiss Supra Vp55. Se identificaron 38 taxa de diatomeas, 32 de las cuales fueron formas bentónicas entre las que se incluyen las especies: Actinoptychus vulgaris, Halamphora coffeaeformis, Delphineis surirella, Fragilariopsis doliolus y Nitzschia amabilis. Como éstas, las diatomeas observadas corresponden principalmente a taxa bentónicos comúnmente representadas en hábitats de las zonas intermareal y submareal someras. Aunque la hipótesis fue respaldada, la composición de especies de diatomeas no permitió mayor precisión sobre el ambiente de procedencia. Rochín Bañaga, H.1, D. A. Siqueiros Beltrones2 & J. Bollmann3. 1Departamento de Geología Marina, Universidad Autónoma de Baja California Sur. Carretera al Sur km 5.5, C.P. 23080, La Paz, B.C.S., México. 2Dpto. Plancton y Ecología Marina, Instituto Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas. Av. IPN s/n, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23050. 3Department of Geology, Earth Sciences Centre, University of Toronto, 22 Russell Street, Toronto, Ontario, Canada M5S 3B1. email: hrochin09@gmail.com.

Rochín Bañaga, H., D. A. Siqueiros Beltrones & J. Bollmann. 2015. Benthic diatoms from shallow environments deposited at 300 m depth in a southern Gulf of California basin. CICIMAR Oceánides, 30(1): 71-76.

Studies using sediment traps from different environments have been focused on understanding the different processes that affect settled particles, and variations of total masses fluxes and their components (Thunell et al., 2007; Silverberg et al., 2014). In a recent study using sediment traps deployed at a 300 m depth on Alfonso basin, Bahía de La Paz, benthic foraminifera were observed in samples from September 2011 to September 2012, showing highest fluxes in the December 2011-January 2012 sample (Rochín-Bañaga, 2014). Along with these, benthic diatom taxa were also observed in the sample from December 2011 to January 2012. These were identified in order to develop a reference for the recorded benthic foraminifera and the transport of particles in the area. The record of benthic diatoms on marine sediments may provide evidence of displacement of Fecha de recepción: 23 de febrero de 2015

shallow-water material or, when said diatoms are autochthonous, they may indicate the depth of the sea floor at the time of sediment deposition (Martínez López et al., 2004). For this reason, the aim of this study was to identify the benthic diatoms represented in the trap, and to infer their most likely provenance assessing the feasibility of transport from their original location to Alfonso basin. Previous data suggested that in the winter 2011 the highest fluxes of benthic foraminifera collected at a 300 m depth could suggest re-depositing of material in Alfonso basin, related to the period of strongest gust of wind promoting the suspension of material, as well as to transporting by streams (Rochín-Bañaga, 2014). According to this, our hypothesis proposes that benthic diatoms deposited in the sediment traps are related to sediments from shallow mangrove ecosystems found in Bahía de La Paz and thus, the main taxa represented are expected to be species of Actynopthychus, Amphora, Diploneis, Lyrella, previously identified (Siqueiros Beltrones, 2002; 2006) as common elements of the diatom assemblages in the region. Method. Alfonso Basin (2635 km2) is located in the northern part of Bahía de La Paz, (24º 39’ N and 110º 36’ W), and reaches a depth of 420 m (Fig. 1). The communication with the Gulf of California occurs mainly through Boca Grande located north of the bay where depths vary between 220 and 320 m. To the south, communication is through the San Lorenzo Channel with depths no greater than 20 m (Cruz Orozco et al., 1996; Obeso Nieblas et al., 2008; Salinas González et al., 2003). A combination of diurnal currents from 1851 (cm/s)2 at a depth of 20 m, to 42 (cm/s)2 at a depth of 30 m (Zaytsev et al., 2010), and the more sluggish currents over the sea floor make it a natural depositional environment. One sample from the Dec 2011 to Jan 2012 period was analyzed in order to identify the benthic diatoms and infer their origin, and to assess the feasibility for their transportation from a close by location to Alfonso basin. Settling particles were collected with a Technicap model PPS-3/3 sediment trap with a 0.125 m2 opening, and a programmable motor-driven carousel containing twelve 250 mL bottles. The sample bottles contained a 4% formaldehyde solution with filtered (0.45 μm) seawater to which high purity NaCl was added to a practical salinity of 40, dense enough to limit exchange with the ambient seawater (Silverberg et al., 2014). The resolution of the sample was 30 days. The instrument was installed approximately at 24º39′ N, Fecha de aceptación: 30 de marzo de 2015

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Table 1. Diatom taxa collected in the sediment trap set in Alfonso basin at a depth of 300 m from December 2011 to January 2012. Planktonic taxa commonly observed in benthic samples*. 1. Actinoptychus aster 2. Actinoptychus vulgaris 3. Alveus marinus

Brun Schumann (Fig. 2G) (Grunow) Kaczmarska & Fryxell 4. Amphora proteus var. contigua Cleve (Fig. 2C) Gregory 5. Amphora spectabilis (Salah) Simonsen (Fig. 6. Amphora wisei 2A) T. Nagumo & H. Kobayasi 7. Amphora holsaticoides 8. Asteromphalus heptactis 9. Azpeitia nodulifera 10. Cocconeis cf. distans 11. Cocconeis cf. californica 12. Cyclotella striata 13. Delphineis cf. minutissima 14. Delphineis surirella 15. Diploneis littoralis 16. Diploneis papula 17. Diploneis smithii 18. Fallacia vittata Figure 1. Bathymetry of Bahía de La Paz and location of the sediment trap mooring over Alfonso basin. After Silverberg et al. (2014)..

19. Fragilariopsis doliolus 20. Halamphora coffeaeformis 21. Haslea spicula

110º36′ W, at a depth of 300 m, and 100 m above the sea floor (Aguirre-Bahena, 2007; Silverberg et al., 2014). The bulk sample (without swimmers) was divided into 10 subsamples (splits) using a rotary splitter. Organic matter was removed according to Bairbakhish et al. (1999). The sample was then filtered through 0.8 μm Nucleopore membranes (Bollmann et al., 2002). Images were automatically taken using a Zeiss Supra Vp55 scanning electron microscope (Bollmann et al., 2004), with a magnification of 1500x. Benthic diatoms (and others) were identified based on regional studies, regarding the particular species composition of the represented habitats: Siqueiros Beltrones & Hernández Almeida (2006), López Fuerte & Siqueiros Beltrones (2006), López Fuerte et al. (2010), Siqueiros Beltrones (2002); Siqueiros Beltrones (2006); Siqueiros Beltrones et al. (2014), Siqueiros Beltrones & Argumedo Hernández (2014).

22. Mastogloia cf. mauritiana 23. Mastogloia sp. 24. Navicula cf. pennata 25. Navicula diserta 26. Navicula sp. 27. Neodelphineis cf. pelagica 28. Nitzschia bicapitata 29. Nitzschia amabilis 30. Nitzschia sicula

Observations. Overall, 38 diatom taxa were identified, including 32 benthic and 6 planktonic forms. The recorded number of identified benthic diatom taxa falls within the typical interval for benthic diatom samples (Siqueiros Beltrones, 2002) which commonly includes also planktonic forms, and usually depicts a specific area and date. In accordance with our hypothesis all diatom taxa from table 1 have been previously recorded in floristic studies of benthic diatoms carried out in the surrounding areas. The identified taxa (Table 1; Figs. 2-3) are commonly represented in assemblages from the intertidal and shallow subtidal habitats (planktonic forms included), comprising various benthic substrata, such

31. Nitzschia sp. 32. Odontella aurita 33. Perissonoë cruciata

(Brébisson) Ralfs * (AWF Schmidt) GA Fryxell & PA Sims* (Fig. 3H) Gregory Grunow (Fig. 2F) (Kützing) Grunow (Hustedt) Simonsen (Ehrenberg) Andrews (Donkin) Cleve (AWF Schmidt) Cleve (Brébisson) Cleve (Cleve) DG Mann (Fig. 2E) (Wallich) Medlin & Sims (Agardh) Levkov (Hickie) L. Bukhtiyarova (Fig. 3I) Brun (Fig. 3J) A. Schmidt Hustedt (Fig. 2D) Takano Cleve Suzuki (Castracane) Hustedt (Fig. 3K)

(Lyngbye) C. Agardh * (Janisch & Rabenhorst) Andrews & Stoelzel 34. Psammodictyon cf. constric- (Gregory) DG Mann tum (Roper) Grunow* (Fig. 35. Roperia tesselata 3L) 36. Stephanopyxis cf. palmeriana (Greville) Grunow* (Ehrenberg) Cleve* 37. Thalassiosira eccentrica W. Smith 38. Tryblionella acuminata

as rock, sediments and macroalgae (López Fuerte & Siqueiros Beltrones, 2006; López Fuerte et al., 2010; Siqueiros Beltrones, 2002; 2006; Siqueiros Beltrones & Argumedo Hernández, 2014). Alfonso Basin is located more than 10 km away from the shoreline. This is a considerable distance for particles such as diatoms, with an approximate density of 2.1 g cm-3, to reach into the trap considering physical aspects such as particle sedimentation. However, it is feasible that when the speed of tidal currents and greater wind gusts, which can exceed 5 m s-1 (Silverberg et al., 2014), the displacement of particles such as foraminifera and diatoms among

BENTHIC DIATOMS AT A 300 m DEPTH

Figure 2. Benthic diatoms collected in Alfonso basin, La Paz bay at a depth of 300 m (December 2011 - January 2012). A) Amphora wissei, B) Halamphora coffeaeformis, C) Amphora proteus var. contigua, D) Navicula sp., E) Fallacia vittata, F) Cocconeis cf. californica.

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Figure 3. Benthic diatoms collected in Alfonso basin, La Paz bay at a depth of 300 m (December 2011 - January 2012). G) Actinoptychus vulgaris, H) Azpeitia nodulifera, I) Haslea spicula, J) Mastogloia sp. (top left), K) Nitzschia sicula, L) Roperia tesselata.

BENTHIC DIATOMS AT A 300 m DEPTH

others into the basin may occur. Highly productive diatom mats on the continental shelf have been detected down to water depths of 40 m, caused by the successful establishment of benthic diatoms transported from inshore sediments (Cahoon et al., 1990); these grow well even under low light levels, <0.03% surface incident radiation, possibly down to depths of 200 m to where the potential limits of benthic primary production extends (MacGee et al., 2008). However, no record of benthic diatoms existed hitherto for La Paz bay from depths of 100-200 m, although seven taxa from the first 100 m depth in Alfonso Basin have been recorded earlier using light microscopy by Villegas-Aguilera (2009). Results from laboratory experiments on re-suspension of benthic diatoms by Delgado et al. (1991) show that the presence of a diatom film increases sediment stability, thereby suppressing re-suspension of sediment and diatoms; however, if the sediment contains many fine particles and much detritus, sediment stability decreases, leading to increased re-suspension. Therefore, turbulent water currents generated by winds greater than 5 m s-1 govern the re-suspension, particularly in shallow environments, and consequently the displacement of microphytobenthic populations. The presence of benthic diatoms in the sediment trap, such as those depicted in table 1 and figures 2-3, most likely indicates a re-deposition process from shallow environments to the deeper portion of the bay. In a similar study off Bahía Magdalena, B.C.S. (Martínez López et al., 2004), the recorded species assemblage also included species such as Actinoptychus vulgaris, Halamphora (Amphora) coffeaeformis, Delphineis surirella, Fragilariopsis doliolus and Nitzschia (laevis) amabilis, all of which were found to be components of assemblages found in mangrove environments, albeit also in nearby rocky coastlines. Lateral transport of benthic diatoms from the coastal zone was suggested to be an important phenomenon at that site. Likewise, the assemblage from the Alfonso basin traps could be related to those found in mangrove environments from different localities in La Paz bay (Siqueiros Beltrones & Morzaria Luna, 1999; Siqueiros Beltrones & Sánchez Castrejón, 1999; Siqueiros Beltrones, 2006). However, the species composition found in the sediment trap does not allow to pin-point any precise location or specific environment. This requires an ex profeso sampling design. We can assert only that the taxa are common in the currently studied lacunar systems of the southern coasts of the Baja California peninsula. Thus, it is still uncertain if all of the benthic diatoms were from the bay, or if some diatoms came from more distant localities. ACKNOWLEDGMENTS We thank F. Aguirre-Bahena for his support through a research financed by grants from the Instituto Politécnico Nacional (project SIP-20130358)

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Rochín-Bañaga H., 2014. Contribución de cocolitóforos y foraminíferos al flujo de carbonato de calcio en Cuenca Alfonso, B.C.S. MSc. Thesis, Centro Interdisciplinario de Ciencias Marinas - Instituto Politécnico Nacional, La Paz, Baja California Sur, 87 p. Salinas Gonzáles, F., O. Zaytsev & V. Makarov. 2003. Formación de la estructura termohalina del agua en la Bahía de La Paz de verano a otoño. Ciencias Marinas, 29: 51-56. Silverberg, N., F. Aguirre-Bahena & A. Mucci. 2014. Time-series measurements of settling particulate matter in Alfonso Basin, La Paz Bay, southwestern Gulf of California. Continental Shelf Research, 84: 169-187. Siqueiros Beltrones, D. A. 2002. Diatomeas bentónicas de la Península de Baja California; diversidad y potencial ecológico. Oceánides/ CICIMAR-IPN/UABCS. 102 p. Siqueiros Beltrones, D. A. 2006. Diatomeas bentónicas asociadas a trombolitos recientes registrados por primera vez en México. CICIMAR Oceánides, 21 (1, 2): 113-143. Siqueiros Beltrones, D. A. & U. Argumedo Hernández. 2014. Particular structure of an epiphytic diatom assemblage living on Ploclamium cartilagineum (Lamoroux) Dixon (Rhodophyceae: Gigartinales). CICIMAR-Oceánides, 29(2): 1124.

Siqueiros Beltrones, D.A. & E. Sánchez Castrejón. 1999. Association structure of benthic diatoms from a mangrove environment in a Mexican subtropical lagoon. Biotropica. 31(1): 48-70. Siqueiros Beltrones, D.A. & H.N. Morzaria Luna. 1999. New records of marine benthic diatom species for the northwestern Mexican region. Oceánides. 14(2): 89-95. Siqueiros Beltrones, D. A., U. Argumedo Hernández, J. M. Murillo Jiménez & A. J. Marmolejo Rodríguez. 2014. Diversidad de diatomeas bentónicas marinas en un ambiente ligeramente enriquecido con elementos potencialmente tóxicos. Revista Mexicana de Biodiversidad, 85: 1065-1085. Thunell, R. C., C. Benitez Nelson, R. Varela, Y. Astor & F. Muller Karger. 2007. Particulate organic carbon fluxes along upwelling-dominated continental margins: rates and mechanisms. Global Biogeochemical Cycles, 21, GB1022(htt). Villegas-Aguilera, M. 2009. Fitoplancton silíceo de la zona eufótica, como señal de la productividad primaria en Cuenca Alfonso, Golfo de California. MSc. thesis, CICIMAR-Instituto Politécnico Nacional, La Paz, 112 p. Zaytzev, O., A. B. Rabinovich, R. Thomson & N. Silverberg. 2009. Intense diurnal surface currents in the bay of La Paz, Mexico. Continental Shelf Research, 30: 608–619.

INSTRUCCIONES A LOS AUTORES CICIMAR Oceánides publica trabajos originales de investigación científica del ámbito marino, v. gr., tópicos de: Biología y Ecología Marina, Geología Marina, Oceanografía Física y Química, así como Meteorología, Pesquerías y Acuicultura. Las contribuciones podrán ser: Artículos, informes in extenso de investigaciones acerca de los temas propios de la revista. Notas, contribuciones originales de corta extensión que contengan resultados parciales o hallazgos que merezcan ser dados a conocer en el corto plazo. Todos ellos serán considerados siempre que su contenido tenga carácter teórico, técnico, o metodológico y no hayan sido sometidos simultáneamente en ninguna otra revista. Si el manuscrito ya ha sido enviado y rechazado en una revista científica, se solicitan los comentarios de los árbitros y un resumen de las modificaciones más relevantes realizadas. Asimismo, se publicarán revisiones y ensayos que hagan aportaciones al desarrollo de una rama de la ciencia y artículos por invitación formal del Consejo Editorial. También, se aceptan réplicas a trabajos publicados en CICIMAR Oceánides. En todos se exigirá claridad y congruencia entre el título, el problema y su planteamiento, así como con la(s) hipótesis del estudio. CICIMAR Oceánides acepta críticas de libros, que se refieran a la trascendencia de una obra determinada. La revista está incluida en el sistema de resúmenes ASFA (Aquatic Sciences and Fisheries Abstracts), Ecological Abs-tracts, Oceanography Literature Review, BIOSIS (Zoological Record), Periódica y Thompson Master Journal list. Los trabajos deberán remitirse al Editor de Cicimar Oceánides por vía electrónica en formato Microsoft Word (doc) o Adobe Acrobat (pdf) incluyendo en un solo archivo el manuscrito completo con tablas y figuras. Los manuscritos serán arbitrados por pares de su especialidad; el autor podrá sugerir revisores, proporcionando su correo electrónico, dirección de contacto y área de experiencia científica (preferentemente internacionales de reconocido prestigio). Asimismo, se podrá solicitar el descarte de árbitros inadecuados para revisar el manuscrito por conflicto de intereses. El Editor devolverá a los autores, sin evaluarlos, los manuscritos que no caigan dentro del ámbito de CICIMAR Oceánides. Aquellos que no se adapten a los requerimientos de las presentes instrucciones serán también devueltos para una corrección previa a su evaluación. Se enviarán pruebas de imprenta (en formato PDF) a los autores, quienes serán responsables de la corrección de errores y de devolverlas en un término de dos semanas desde su recepción. En caso contrario, el Consejo Editorial podrá efectuar la corrección o aplazar la publicación del trabajo.

Se requerirá un Abstract y su versión en Español (máximo 300 palabras), donde incluirán en un sólo párrafo objetivos, resultados y conclusiones, mencionando lo esencial del nuevo conocimiento aportado; sólo se aceptarán referencias bibliográficas cuando sean indispensables. Deberá contener 2-3 frases de introducción al tema, el contenido del trabajo y estado del arte; 2-3 frases donde se resalten los resultados en un contexto general que demuestren un avance en el tema y un postulado con las conclusiones principales. Deberá indicarse un máximo de cinco Palabras Clave en Español y en Inglés que permitan identificar el contenido del trabajo en bases de datos electrónicas. Las Tablas y Figuras, junto con sus leyendas, deberán presentarse consecutivamente con numeración arábiga y en hojas aparte. Las leyendas deben ser breves y explicativas. Deberán evitarse repeticiones de información entre tablas y figuras. Tablas y figuras se citarán en el texto por su número (las figuras en forma abreviada i.e., Fig.). Las tablas serán diseñadas de forma que se ajusten al formato de la página impresa. No se usarán líneas verticales y solo líneas horizontales en el primer renglón y en el último renglón. Los Agradecimientos, se referirán a la colaboración de personas o instituciones que hayan hecho aportaciones sustanciales al trabajo, incluyendo financiamiento, en todo o en parte. En las Referencias bibliográficas deberá existir una correspondencia entre las citas en el texto (incluidas las tablas y figuras) y la lista de este apartado. Sólo se podrán incluir en las Referencias trabajos publicados o “en prensa” (i.e., aceptados para publicación; ello deberá certificarse adjuntando copia de la carta de aceptación). Expresiones como “com. pers.”, “in litt.“, “datos no publ.”, etc., sólo serán permitidas en el texto, en donde se usará el sistema de citas por autor y año. Las referencias bibliográficas en el texto deberán indicarse mediante el apellido del autor, o autores si son dos, seguido del año de publicación separado por una coma; todo entre paréntesis. Si el nombre del autor forma parte de la redacción del escrito, sólo el año se pondrá entre paréntesis. Cuando sean más de dos autores se escribirá el apellido del primer autor seguido de et al. La lista bibliográfica se presentará por orden alfabético. Se tendrá en cuenta la cronología sólo cuando exista más de una referencia con idénticos autores. Las referencias deberán incluir el título completo y los números de las páginas. Las abreviaturas de revistas serán según la WORLDLIST OF SCIENTIFIC PERIODICALS. Deberán subrayarse (para cursivas) todas las abreviaturas de revistas y títulos de libros.

Los manuscritos deberán escribirse a doble espacio con letra Arial 12, incluidos los pies de figura y leyendas de tablas, en tamaño CORRESPONDENCIA: Se dirigirá al Editor: carta con márgenes de 2.5 cm. Las páginas se numerarán de for- Av. IPN s/n, Col. Playa Palo de Sta. Rita, 23096, La Paz, B.C.S., al Apartado Postal 592, La Paz, B.C.S., México, o al email: ocema corrida. anide@ipn.mx. Se recomienda someter sus manuscritos en idioma Inglés, aunNOTA: LAS VERSIONES ELECTRÓNICAS A COLOR PUEDEN que también son aceptados aquellos en Español. Las noENCONTRARSE EN LA SIGUIENTE DIRECCIÓN tas serán publicadas en Inglés. Se recomienda que los artíhttp://sistemas.cicimar.ipn.mx/ojs/ culos se dividan en: Título, Resumen, Introducción, Material y Métodos, Resultados, Discusión, Agradecimientos y Referencias. Las palabras a imprimir en cursiva deberán subrayarse en el manuscrito (específicamente nombres latinos de organismos y símbolos matemáticos en el texto). Las medidas se expresaránen unidades SI, usando las abreviaturas de la International Standards Organization (ISO) La localización geográfica se expresará según latitud y longitud como E, W, N, S. El Título deberá presentarse tanto en Inglés como en Español; se prefiere el descriptivo (informativo) sobre los indicativos o temáticos; se incluirá un título abreviado que no exceda de 40 caracteres. A continuación se indicarán el nombre, institución y dirección del autor (incluyendo su E-mail) y coautores, diferenciando las instituciones de los participantes con números arábigos consecutivos.

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