Project Title: Mitochondrial DNA Analysis for Species Identification of Snappers (Pisces: Lutjanidae) from Caribbean waters – Final Report.
Completion Report submitted to the Sean Grant Program as a requirement for grant awarded as part of the Omnibus 2000- 2002.
Juan C. Martínez-Cruzado, Ph.D.
Department of Biology, University of Puerto Rico-Mayagüez
Jorge R. García-Sais, Ph.D.
Department of Marine Sciences, University of Puerto Rico-Mayagüez
Aurea E. Rodríguez-Santiago, Ph.D. student
Department of Marine Sciences, University of Puerto Rico- Mayagüez
Mitochondrial DNA Analysis for Species Identification of Snappers (Pisces: Lutjanidae) from Caribbean waters
Juan C. Martínez-Cruzado, Ph.D.
Department of Biology, University of Puerto Rico-Mayagüez
Jorge R. García-Sais, Ph.D.
Department of Marine Sciences, University of Puerto Rico-Mayagüez
Áurea E. Rodríguez-Santiago, Ph.D. student
Department of Marine Sciences, University of Puerto Rico- Mayagüez
Introduction
Most coral reef fishes have a pelagic larval stage that differs morphologically from juveniles and adults, making the identification keys based on adult fish meristics useless. Taxonomic identification of coral reef fish larvae to the species level still represents one of the main bottlenecks in our understanding of the early life cycle of many reef fishes. Such information is
of vital relevance in assessment of marine protected areas (MPA) as management options to restore the declining stocks of commercially exploited coral reef fishes.
Fisheries studies require accurate identification of subject species. Identification of the developmental stages of fishes is complicated by the small size of the specimens, their fragility, and the relatively great changes in their structure and pigmentation. Experience has shown that major changes can occur over very small growth increments and these can only be documented by a continuous growth series (Moser et al. 1984). Traditionally identification of larval fishes was based on morphological characteristics. Character sets, whether morphological or molecular, are the primary data collected to study fish systematics or population structure and dynamics. Each character type has its inherent advantages and disadvantages (Hillis and Moritz 1990; Sarich et al. 1989). Morphological character sets require little equipment to gather and can be very informative when used to address population problems suited to their level of sensitivity. Molecular characters have proven to be particularly informative in studies of closely related taxa (Graves et al. 1990; Bembo et al. 1995; Chow & Inoue 1993).
During the past decade, the use of mitochondrial DNA (mtDNA) to study the phylogeny and population genetics of fishes, amphibians and reptiles has greatly proliferated, and mtDNA has become the standard molecule of choice among most ichthyologists and herpetologists doing comparative molecular genetics. Animal mitochondria have a number of properties, which make them attractive to work with. They are independent units and numerous within a cell thus can be easily extracted from the cell and separated from genomic DNA (Avise et al., 1987). The gene order is highly conserved within phyla (Moritz et al. 1987; Meyer 1993). Their relatively small size in vertebrates (usually around 16,000 bp.) makes them quite manageable. Different mitochondrial genes evolve at different rates, enabling researchers to work at various taxonomic levels. Work using mitochondrial genes has been done to study interrelationships of populations, families and even orders (Moritz et al. 1987; Lamb et al. 1994). Mitochondrial genes in animals are not interrupted; there are few gene replications and intergenic sequences (Avise et al. 1987; Moritz et al. 1987; Meyer 1993). Mitochondria are passed on from generation to generation directly from mother to offspring. That is, there is no recombination or mixing of sequences between individuals. Thus, mitochodria provide a direct chain of ancestry across generations (Avise et al. 1987; Meyer 1993). Finally, because the selective pressures that act on nucleotides seem to be of a different nature than those which act on morphological characters, DNA sequences provide a very valuable additional data set which could aid in resolving situations in which morphological characters sets are simply unresolvable due to homoplasy.
Among the most popular genes used in phylogenetic systematics and population biology are the mitochondrial ribosomal RNA (rRNA) genes. These are the 12S gene also known as the small subunit (819-975 bp), and the 16S gene also known as the large subunit (1571-1640 bp) (Meyer 1993). Both are highly conserved among taxa, although not as much as the nuclear ribosomal RNA genes (Moritz et al. 1987). Hillis and Dixon (1991) have suggested these genes can be used to sort out relationships between taxa, which diverged as much as 100 million years ago. The use of the mitochondrial rRNA genes seems ideal for phylogenetic studies at lower taxonomic levels, such as for resolving interrelationships of genera and species (Moritz et al. 1987; Hillis and Dixon 1991; Orti and Meyer 1997).
Restriction endonuclease analysis to detect restriction fragment length poylymorphism (RFLP) of mtDNA has been used to demonstrate intra and interspecific genetic relationships among and within various marine and freshwater fishes (Ward 1994). This technique provides an additional tool for the investigation of fish larval identities to the species level. Original molecular protocols were costly, time consuming and, generally not applicable to the small amount of tissue available with early-life-history forms (Whitmore 1992). Analyses of DNA restriction fragment length polymorphism (RFLP) are becoming a popular method to investigate variation and relationships between fish species (Billington and Hebert 1991). Although conventional mitochondrial DNA (mtDNA) methods are powerful for detecting variation in RFLPs, intensive DNA analysis would be difficult especially on very small samples, such as fish embryos or larvae, which offer a very small amount of DNA (Saiki et al. 1988; Avise 1994). The relative recent development of the polymerase chain reaction (PCR), which can amplify DNA sequences more than 10-million fold (Mullis and Faloona 1987; Saiki et al. 1988) solves this problem. One of the more striking advantages of the PCR, which makes it useful for fish larvae analyses, is that it requires as a template only minute quantities of undegraded DNA, as has been obtained from unlikely sources such as single feathers (Taberlet and Bouvet 1991), hairs (Morin et al. 1992), animal excrement (Höss et al. 1992), and fish scales (Whitmore 1992).
In order to manage fisheries resources, it is essential to know the recruitment mechanisms of a given species. Lutjanidae (snappers) is one of the largest teleostean families, comprising 4 subfamilies, 17 genera, and 103 species (Allen 1985). There are over 100 species of snappers found in tropical and subtropical waters largely confined to continental and insular shelves (Anderson 1987). In the western Atlantic there are eighteen species distributed within five genera (Robins and Ray 1986). Considerable concern has been focused on the need to understand recruitment in order to manage these reef species (Huntsman 1981; Doherty 1983; Roberts and Polunin 1991). Taxonomic structure, abundance and distribution patterns of fish larvae were examined across a neritic-oceanic gradient off La Parguera (Ramírez and García, in press). From a total of 35,024 larval fish collected in this study Lutjanids (snappers) presented a neritic distribution. Such distribution is not unique for La Parguera, since pre-flexion larvae were also found in high concentrations within the shelf at Guayanilla Bay (García et al. 1995). This distribution pattern suggests a strong potential for self-recruitment.
Analysis of the distribution of eggs and larvae for life history and recruitment studies of the fishery has been hampered by the inability to identify eggs and larvae of most snapper species. The close morphological similarity of larvae among lutjanid species has made identification of larvae a difficult task (Richards 1985; Leis 1987; Richards and Lindeman 1987). Most of the Lutjanid larvae in their pre-flexion stages have been unidentifiable at the species level, using the conventional morphological observations. Very small snapper larvae may be confused with other percoid families and show substantial geographical variability in reproduction (Richards et al. 1994). In addition, meristic characters are very similar within the family making the life history of this group very difficult to study. Eggs, larvae, and early juveniles are only known for a few species (Richards et al. 1994). Richards et al. (1994) assembled information for the identification of early life history of snappers, but diagnoses for many species are still incomplete.
In a previous study restriction endonuclease analysis of mitochondrial DNA amplified by polymerase chain reaction was applied to snapper species (the genera Lutjanus, Ocyurus and Rhomboplites), in order to assess reliability of this method for genetic species and stock identification studies (Chow et al. 1993). They reported amplification of two mitochondrial genes (cytochrome b and 12S rRNA) using fresh and ethanol preserved embryos and larvae, and alcohol preserved museum samples of thirteen western Atlantic snapper species. It is highly probable that some snapper species contain a large number of maternal lineages within the population, which can be detected by PCR-RFLP analyses as demonstrated in this study. Results from Chow et al. (1993) indicate that PCR-RFLP analysis is much simpler and less expensive than conventional mtDNA and nucleotide sequence analyses. With this method, not only are quantitative analyses of species composition at early life stages possible, but also intensive genetic analysis. Since relatively little is known about the spawning habits, dispersal, and migration of these snappers species this study aims to describe diagnostic motifs that can be used to identify fish larvae of the Lutjanidae family. The approach will be to compare genetic structures between adults and their early life stages.
Materials and methods
Sample collection
Adult specimens from fourteen species (Lutjanus griseus, Lutjanus apodus, Lutjanus cyanopterus, Lutjanus jocu, Lutjanus vivanus, Lutjanus bucanella, Lutjanus synagris, Lutajnus analis, Lujanus mahogoni, Ocyurus chrysurus, Rhomboplites aurorubens, Pristipomoides macrophtlamus, Etelis oculatus and Apsilus dentatus were obtained from catches of local fishermen at Parguera and Rincón fisherman villages either fresh or frozen, depending on availability. From one to two grams of muscle or liver from each specimen were brought to the laboratory in separate bags on ice. Once in the laboratory 25 mg were used for DNA extraction and the remaining of the samples were stored at –80° C.
Larvae were obtained from plankton tows using a 202 µm mesh net from various samplings efforts. Larvae from a sampling effort to determine potential trajectories of early stages of Mutton snapper after massive spawning aggregations at La Parguera (René Estevez Master’s thesis) were obtained. These samplings were done from March to May 2003, transects from the coast to ocean were made in order to observe possible trajectories. Most samples were fixed with 4% formalin in seawater but others were only preserved with Dimethylsulfonate (DMSO) or ethanol 95% in seawater because samples fixed in formalin showed difficulties to get DNA for downstream applications.
Sorting and Morphological measurements of larvae
Entire samples were sorted for fish larvae, identified to the family level (Lindeman et al 2005) and morphologically characterized using a dissecting microscope with a digital camera. Standard measurements for fish larvae were performed; including body length (standard Length), head length, eye diameter, body depth, pre anal length, snout length following the identification guide by Leis and Carson 2000.
DNA extraction
Total genomic DNA was extracted and purified using the QIAamp DNA Mini Kit from QIAGEN, Inc. from all samples.
Amplification of mitochondrial 12S rRNA
The pair of primers used to target 12S RNA were abbreviated forms of those described by Kocher et al. (1989). The nucleotide sequences of each set of primers were as follows: 12S rRNA, 5’-TCAAACTGGGATTAGATACCCCACTAT-3’ and 5’-TGACTGCA
GAGGGTGACGGGCGGTGTGT-3’.
Polymerase chain reaction was carried in a final volume of 25 µl in a reaction mixture described by Kocher et al. (1989). This reaction mixture was preheated at 94° for 2.5 minutes followed by 30 cycles of amplification (94°C for 30 sec., 45°C for 1.5 min., and 72° for 1.5 min.).
Endonuclease digestion for amplified DNA fragments
Eleven restriction endonucleases used were: Alu I, Bfa I, Hae III, Hin f I, Msp I, Nla I, Taq I, Hha I, Dpn II, Mse II and Ava II all recognizing symmetric 4-base pair sequences. One unit of each enzyme was applied to 11 µl of the amplified PCR product in a final reaction volume of 15µl. The digested samples were electrophoresed through 3% agarose gels. After electrophoresis and staining with ethidium bromide DNA bands were visualized and photographed.
Sequencing
Amplified PCR products of the +/- 455 bp 12S rRNA DNA fragment were sequenced for one specimen of each fourteen adult species.
Results and discussion
Morphological Observations
Three types of larvae have been differentiated morphologically (Figures 2 - 4). We consistently found a morphologically distinct type of lutjanid larva, apparently not previously described in the revised literature, which molecular haplotype and occurrence might be compatible with Lutjanus apodus, whose early stages have not been described. This larva was designated as Type 2 (Figure 3).
Preliminary description includes: Myomeres (vertebrae): 24
Fin Ray counts:
Dorsal fin: X-12 (ten spines, twelve soft rays)
Spines:
Anal fin: III-7
Third dorsal spine longer non serrated.
Pigmentation:
Pigments present on articular jaw and branchiostegal rays. Melanophore on dorsal part of the body just below the 1st and 2nd dorsal fins. Melanophore at the end of the anal fin, over the caudal peduncle. Internal melanophore over the lateral line (approximately over myomeres 7-8 counting from notochord to head). Pigments on first dorsal and pelvic fins.
Gene amplification
The pair of primers used successfully amplified 455±5 bp fragment of the 12S rRNA gene for all adult specimens. There were no apparent differences in the fragment size among species. For larvae only those preserved in alcohol or DMSO and processed within a week amplified successfully. After many attempts trying to amplify DNA from larvae initially fixed in formalin or preserved in DMSO for a long period, without success, we decided to design new samplings where ethanol 3% and seawater was used as preservative in the field. Samplings were and are been performed at three locations known as spawning aggregation sites for the mutton snapper and other species. Amplification is been successful for those larvae and were sent for sequence analysis. Sequence data was not available for this report.
Restriction fragment analysis
For the 455 bp 12S rRNA fragment, six enzymes (Bfa I, Hae III, Hha I, Nla I, Taq I and Ava II) had no restriction sites in any of the species. Dpn II and Msp I had restriction site(s) in all species examined, without apparent size difference in restricted fragments between species. Alu I produced two different cutting profiles. Hin f I had the same restriction sites for Lutjanus vivanus, Lutjanus bucanella, Lutjanus analis and Ocyurus chrysurus. Mse I digestion produced two different cutting profiles, with Ocyurus chrysurus, Lutjanus analis and Lutjanus bucanella designed as pattern 1. Lutjanus apodus, Lutjanus cyanopterus and Lutjanus jocu as well as the unknown larvae # 2 showed a cutting profile assigned as pattern 2. Mse I did not cut that fragments from Lutjanus vivanus, Lutjanus synagris and Lutjanus griseus as well as for unknoun larvae #1. One of the unknown larvae (#1) haplotype matched with that of Lutjanus griseus, being similar in shape to type 1 larvae (Figs. 1 and 2). Restriction patterns were summarized for haplotype designation for species discrimination. Seven haplotypes were observed for the species examined after digestion with eleven enzymes (Table 1). Further analysis is necessary to validate these results as well as for intraspecific polymorphism for complete discrimination.
Table 1. Summary of haplotypes for lutjanids
Sequencing
From the amplified product we obtained sequences of 457 bp, after editing we used a fragment of 369 bp which contains those differences. Sequence data for thirteen species belonging to the Lutjanidae family showed differences between them, validating and strengthening the information gotten by restriction enzyme analyses.
12s, 0 bases, 0 checksum. CCCGGG.GAC TAC.GAGCAT CAGCTTGAAA CCCAAAGGAC
003_714_003_71_4_Entry_1, 457 CCCGGG.GAC TAC.GAGCAT CAGCTTGAAA CCCAAAGGAC
004_716_004_71_6_Entry_1, 457 CCCGGG.GAC TAC.GAGCAT CAGCTTAAAA CCCAAAGGAC
005_719_005_71_9_Entry_1, 457 CCCGGG.GAC TAC.GAGCAT CAGCTTGAAA CCCAAAGGAC
006_71B_006_71_B_Entry_1, 457
007_71E_007_71_E_Entry_1, 457
008_710_008_7_10_Entry_1, 457
009_712_009_7_12_Entry_1, 457
011_715_011_7_15_Entry_1, 457
019_717_019_7_17_Entry_1, 457
066_719_066_7_19_Entry_1, 457
067_71C_067_7_1C_Entry_1, 457
068_71E_068_7_1E_Entry_1, 457
070_721_070_7_21_Entry_1, 457
073_723_073_7_23_Entry_1, 457
CCCGGG.GAC TAC.GAGCAT CAGCTTGAAA CCCAAAGGAC
CCCGGG.GAC TAC.GAGCAT CAGCTTAAAA CCCAAAGGAC
CCCGGG.GAC TAC.GAGCAT CAGCTTAAAA CCCAAAGGAC
CCCGGG.GAC TAC.GAGCAT TAGCTTGAAA CCCAAAGGAC
CCCGGG.GAC TAC.GAGCAT CAGCTTGAAA CCCAAAGGAC
CCCGGG.GAC TAC.GAGCAT CAGCTTAAAA CCCAAAGGAC
CCCGGG.GAC TAC.GAGCNT CAGCTTAGAA CCCAAAGGAC
CCCGGGCTAC TACCGAGCAT TAGCTTAAAA CCCAAAGGAC
CCCGGG.TAC TAC.GAGCAT TAGCTTAAAA CCCAAAGGAC
CCTGGG.AAC TAC.GAGCAT CAGCTTGAAA CCCAAAGGAC
CCCGGG.GAC TAC.GAGCAT CAGCTTGAAA CCCAAAGGAC
45 55 65 75
12s, 0 bases, 0 checksum. TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
003_714_003_71_4_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
004_716_004_71_6_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
005_719_005_71_9_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
006_71B_006_71_B_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
007_71E_007_71_E_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
008_710_008_7_10_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
009_712_009_7_12_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
011_715_011_7_15_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
019_717_019_7_17_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
066_719_066_7_19_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
067_71C_067_7_1C_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
068_71E_068_7_1E_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
070_721_070_7_21_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG 073_723_073_7_23_Entry_1, 457 TTGGCGGTGC TTTAGATCCA CCTAGAGGAG CCTGTTCTAG
85 95 105 115
12s, 0 bases, 0 checksum. AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTTC
003_714_003_71_4_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTTT
004_716_004_71_6_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTCT
005_719_005_71_9_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTTT
006_71B_006_71_B_Entry_1, 457
AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTTC
007_71E_007_71_E_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTCT
008_710_008_7_10_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTCC
009_712_009_7_12_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTCC
011_715_011_7_15_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTCC
019_717_019_7_17_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTCT
066_719_066_7_19_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTTT
067_71C_067_7_1C_Entry_1, 457
AACCGATAAC CCCCGTTCAA CCTCACCTTT TCTTGTTTAA
068_71E_068_7_1E_Entry_1, 457 AACCGATAAC CCCCGTTCAA CCTCACCTTT TCTTGTTTAA
070_721_070_7_21_Entry_1, 457 AACCGATAAC CCCCGTTCAA CCTCACCTTT CCTTGTTTTC 073_723_073_7_23_Entry_1, 457 AACCGATTAC CCCCGTTCAA CCTCACCTTT CCTTGTTTCC
125 135 145 155
12s, 0 bases, 0 checksum. CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
003_714_003_71_4_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
004_716_004_71_6_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
005_719_005_71_9_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
006_71B_006_71_B_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
007_71E_007_71_E_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
008_710_008_7_10_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
009_712_009_7_12_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
011_715_011_7_15_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGGAGG
019_717_019_7_17_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
066_719_066_7_19_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
067_71C_067_7_1C_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
068_71E_068_7_1E_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
070_721_070_7_21_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
073_723_073_7_23_Entry_1, 457 CCCGCCTATA TACCACCGTC GTCAGCTTAC CCTGTGAAGG
165 175 185 195
12s, 0 bases, 0 checksum. ACTCATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
003_714_003_71_4_Entry_1, 457 GCTCATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
004_716_004_71_6_Entry_1, 457 GCTGATAGTA AGCAAGATTG GCATAGCCCA AAACGTCAGG
005_719_005_71_9_Entry_1, 457 ACTCATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
006_71B_006_71_B_Entry_1, 457 ACTCATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
007_71E_007_71_E_Entry_1, 457 ACTTATAGTA AGCAAGATTG GCATAGCCCA AAACGTCAGG
008_710_008_7_10_Entry_1, 457 ACTTATAGTA AGCAAGATTG GCATAGCCCA AAACGTCAGG
009_712_009_7_12_Entry_1, 457 ACTCATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
011_715_011_7_15_Entry_1, 457 ACTTATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
019_717_019_7_17_Entry_1, 457 ACTTATAGTA AGCAAGATTG GCATAGCCCA AAACGTCAGG
066_719_066_7_19_Entry_1, 457 ACTAATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
067_71C_067_7_1C_Entry_1, 457 CCTCATAGTA AGCAAAATTG GCACAGCCCA AAACGTCAGG
068_71E_068_7_1E_Entry_1, 457 CCTCATAGTA AGCAAAATTG GCACAGCCCA AAACGTCAGG
070_721_070_7_21_Entry_1, 457 CCTTATAGTA AGCAAAATTG GCACAGCCCA AAACGTCAGG
073_723_073_7_23_Entry_1, 457 ACTCATAGTA AGCAAGATTG GCACAGCCCA AAACGTCAGG
205 215 225 235
12s, 0 bases, 0 checksum. TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
003_714_003_71_4_Entry_1, 457 TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
004_716_004_71_6_Entry_1, 457 TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
005_719_005_71_9_Entry_1, 457 TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
006_71B_006_71_B_Entry_1, 457
007_71E_007_71_E_Entry_1, 457
008_710_008_7_10_Entry_1, 457
009_712_009_7_12_Entry_1, 457
011_715_011_7_15_Entry_1, 457
019_717_019_7_17_Entry_1, 457
066_719_066_7_19_Entry_1, 457
067_71C_067_7_1C_Entry_1, 457
068_71E_068_7_1E_Entry_1, 457
TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGTATGGAA GGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGTATGGAA GGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGTATGGAA GGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGAATGGAA AGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGTATGAAA AGGGAAGAAA TGGGCTACAT
TCGAGGTGTA GCGTATGAAA AGGGAAGAAA TGGGCTACAT
070_721_070_7_21_Entry_1, 457 TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
073_723_073_7_23_Entry_1, 457 TCGAGGTGTA GCGTATGGAA AGGGAAGAAA TGGGCTACAT
245 255 265 275 12s, 0 bases, 0 checksum. TCCCTAA.TA TAGTGAA.AT ACGAACGATA CACTGAAATA 003_714_003_71_4_Entry_1, 457 TCCCTAA.CA CAGTGAA.AT ACGAACGATA CACTGAAATA 004_716_004_71_6_Entry_1, 457 TCCCTAA.CA TAGTGAA.AT ACGAACGATG CACTGAAATA 005_719_005_71_9_Entry_1, 457 TCCCTAA.TA TAGTGAA.AT ACGAACGATA CACTGAAATA 006_71B_006_71_B_Entry_1, 457 TCCCTAA.TA CAGTGAA.AT ACGAACGATA CACTGAAATA 007_71E_007_71_E_Entry_1, 457 TCCCTAA.CA CAGTGAA.AT ACGAACGATG CACTGAAATA 008_710_008_7_10_Entry_1, 457 TCCCTAA.CA TAGTGAATAT ACGAACGATG CACTGAAATA 009_712_009_7_12_Entry_1, 457 TCCCTAA.TA TAGTGTA.AT ACGAACGATA CACTGAAATA 011_715_011_7_15_Entry_1, 457 TCCCTAA.TA TAGTGAA.AT ACGAACGATA CACTGAAATA 019_717_019_7_17_Entry_1, 457 TCCCTAA.CA CAGTGAA.AT ACGAACGATG CACTGAAATA
066_719_066_7_19_Entry_1, 457 TCCCTAG.CA TCGGGCATAT ACGAACGATA CACTGAAATA
067_71C_067_7_1C_Entry_1, 457 TCTCTAA.TA CAGAGAA..T ACGAACGATA CGCTGAAACA
068_71E_068_7_1E_Entry_1, 457 TCTCTAA.TA CAGAGAA..T ACGAACGATA CGCTGAAACA
070_721_070_7_21_Entry_1, 457 TCCCTAACTA TAGAGAA..T ACGAACGATA CACTGAAATA
073_723_073_7_23_Entry_1, 457 TCCCTAA.TA TAGTGAA.AT ACGAACGATA CACTGAAATA
285 295 305 315
12s, 0 bases, 0 checksum. CGTAT.CCGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
003_714_003_71_4_Entry_1, 457 CGTAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
004_716_004_71_6_Entry_1, 457
005_719_005_71_9_Entry_1, 457
006_71B_006_71_B_Entry_1, 457
007_71E_007_71_E_Entry_1, 457
008_710_008_7_10_Entry_1, 457
009_712_009_7_12_Entry_1, 457
011_715_011_7_15_Entry_1, 457
019_717_019_7_17_Entry_1, 457
CGCAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
CGTAT.CCGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
CGTAT.CCGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
CACAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
CGCAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
CGTAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
CGTAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
CACAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
066_719_066_7_19_Entry_1, 457 CGTAT.CCGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
067_71C_067_7_1C_Entry_1, 457 CGTATACTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
068_71E_068_7_1E_Entry_1, 457 CGTATACTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
070_721_070_7_21_Entry_1, 457
CGTAT.CCGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
073_723_073_7_23_Entry_1, 457 CGTAT.CTGA AGGAGGATTT AGCAGTAAGC AGAAAATAGA
325 335 345 355
12s, 0 bases, 0 checksum. GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
003_714_003_71_4_Entry_1, 457 GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
004_716_004_71_6_Entry_1, 457 GCGTTCTGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
005_719_005_71_9_Entry_1, 457 GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
006_71B_006_71_B_Entry_1, 457 GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
007_71E_007_71_E_Entry_1, 457 GCGTTCTGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
008_710_008_7_10_Entry_1, 457 GCGTTCTGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
009_712_009_7_12_Entry_1, 457 GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
011_715_011_7_15_Entry_1, 457 GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
019_717_019_7_17_Entry_1, 457 GCGTTCTACT GAAACCGGCC CTGAAGCGCG CACACACCGC
066_719_066_7_19_Entry_1, 457 GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
067_71C_067_7_1C_Entry_1, 457 GCGTTCCGCT GAAATCGGCC CTGAAGCGCG CACACACCGC
068_71E_068_7_1E_Entry_1, 457 GCGTTCCGCT GAAATCGGCC CTGAAGCGCG CACACACCGC
070_721_070_7_21_Entry_1, 457 GCGTTCCGCT GAAATCGGCC CTGAAGCGCG CACACACCGC 073_723_073_7_23_Entry_1, 457 GCGTTCCGCT GAAACCGGCC CTGAAGCGCG CACACACCGC
365 375 385 395 12s, 0 bases, 0 checksum. CCGTCACCC. ..........
003_714_003_71_4_Entry_1, 457 CCGTCACCC. .......... .......... .......... 004_716_004_71_6_Entry_1, 457 CCGTCACCC. .......... .......... .......... 005_719_005_71_9_Entry_1, 457 CCGTCACCC. .......... .......... .......... 006_71B_006_71_B_Entry_1, 457 CCGTCACCC. .......... .......... .......... 007_71E_007_71_E_Entry_1, 457 CCGTCACCC. .......... .......... .......... 008_710_008_7_10_Entry_1, 457 CCGTCACCC. .......... .......... .......... 009_712_009_7_12_Entry_1, 457 CCGTCACCC. .......... .......... .......... 011_715_011_7_15_Entry_1, 457 CCGTCACCC. .......... .......... .......... 019_717_019_7_17_Entry_1, 457 CCGTCACCC. .......... .......... .......... 066_719_066_7_19_Entry_1, 457 CCGTCACCC. .......... .......... .......... 067_71C_067_7_1C_Entry_1, 457 CCGTCACCC. .......... .......... .......... 068_71E_068_7_1E_Entry_1, 457 CCGTCACCC. .......... .......... ..........
070_721_070_7_21_Entry_1, 457 CCGTCACCC. .......... .......... .......... 073_723_073_7_23_Entry_1, 457 CCGTCACCC. .......... .......... ..........
Chow et al. (1993) were able to distinguish between 8 of 13 Lutjanidae species, including 6 of 9 species that were studied in this project, using nine restriction endonucleases for two fragments of the mitochondrial DNA (12S rRNA and cyt b. Of those nine enzymes, five coincide with the ones we used (Hae III, Msp I, Alu I, Taq I and Hinf I). As for them our results show that Hae III did not produced length variation of the restricted fragments between species. We found a diagnostic restriction profile for Lutjanus vivanus in the fragment digested by Alu I, they observed intraspecific polymorphism for some species using this enzyme but not for L. vivanus. They reported that Taq I digestion produced a straightforward diagnostic restriction profile in the 12S rRNA fragment of Lutjanus griseus but we did not detected any restriction sites for Taq I in our specimens. They observed intraspecific polymorphism for Msp I for L. analis, L. cyanopterus and L. jocu, we did not observed length variation in the restricted fragments in our subject species. The 12S rRNA fragment has been widely used for phylogenetic studies among distantly related species (Stock et al. 1991; Lecointre 1996). In fact, the cyt b fragment provided more useful information than the 12S rRNA fragment in the Lutjanidae study (Chow et al. 1993). The 12S rRNA and the cyt b fragments were found in this study to possess low and medium restriction variability, respectively. Thus, we suggest the use of a 650 bp of the ATPase subunit 6 gene that might be more informative in terms of intraspecific polymorphism because it has been found to be highly variable in humans (Cann et al. 1984). Finally, many studies of mammalian mtDNA have focused on the major noncoding region often called the control region. It has already been useful in fish evolution studies (Lee et al. 1995). Because of its rapid rate of evolution (Hoelzel et al. 1991), this region of the mt DNA may be useful for intraspecific polymorphism studies.
So far, we were able to characterize thirteen species for the adult specimens by means of the sequence of the 12S rRNA fragment of the mtDNA of adult specimens: and one larva as Lutjanus griseus. Further analyses are necessary to validate these results as well as for intraspecific polymorphism for complete discrimination of species. In conclusion, the procedure that we are undertaking is producing results from which we will be able to identify snapper species from the southwest Puerto Rico area, as well as their unknown larvae.
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Appendix 1.
A. Thesis the project is producing: Aurea E. Rodriguez – Santiago dissertation.. Mitochondrial DNA analyses on Caribbean Snapper Larvae for species identification. Expected graduation date: May 2007.
B. Presentations associated to the project:
Gulf and Caribbean Fisheries Institute (GCFI) annual conference, November 7-11, 2005, San Andres, Cololmbia - Description of a distinct snapper larvae and species identification based on mitochondrial DNA analyses (talk).
Sea Grant Program First Annual Symposium on Coastal and Marine Applied Research, September 2, 2005, University of Puerto Rico – Mayagüez - Species Identification of snapper larvae (Family Lutjanidae) based on mitochondrial DNA analyses (talk).
First AGEP Island Wide Conference, March, 2005.
Hotel Boquemar, Boquerón, Puero Rico - Mitochondrial DNA analyses on Caribbean Snapper Larvae for species identification (poster).
V Congreso de la Biodiversidad Caribeña, January 25-28, 2005, Santo Domingo, República Dominicana - Species Identification of Snapper larvae (Family: Lutjanidae) based on mitochondrial DNA analyses. (talk).
Emerge Workshop, April 21-25, 2003, Atlanta, Georgia - Mitochondrial DNA Analyses on Caribbean Snappers (Subfamily: Lutjaninae) for Species Identification (poster).
V Larval Biology Meeting, September 16-20, 2002, Vigo, Spain - Mitochondrial DNA Analyses of various snapper larvae (Subfamily: Lutjaninae) for species identification (poster).
Latin American and Caribbean Biotechnology Congress November 23, 2002, Mayagüez, Puerto Rico - Analyses of the 12S rRNA Mitochondrial DNA as a method for species identification of Caribbean Snappers larvae (talk).
Gulf and Caribbean Fisheries Institute annual conference, November 12-16, 2001, Turks and Caicos, Providenciales - Mitochondrial DNA Analysis for Species Identification of Snappers (Pisces: Lutjanidae) from Caribbean waters (poster).
C. Plans for publication:(Titles are not final):
i. Description of a distinct snapper larvae and species identification based on mitochondrial DNA analyses. To be sent to Fisheries Bulletin.
ii. Caribbean snappers (Family: Lutjanidae) phylogeny based on 12S rRNA and cytb mtDNA. To be sent to: Fisheries Bulletin or Marine Biology
iii. Species Identification of Snapper larvae (Family: Lutjanidae) based on mitochondrial DNA analyses, implications on dispersal and connectivity. To be sent to: Fisheries Bulletin or Marine Biology