Spring 2018

Spring2018

FIELDNoTES

insightsfromStudent Ecol ogists

Editor'sNote Welcome to FieldNotes!

This project began as an idea between a few emerging ecologists who saw a need for a platform to showcase the awe-inspiring environmental research performed by undergraduates at the University of Washington. FieldNotes has since expanded to include perspectives and insights from the human side of the environment and highlight student photography.

Our small team of four has grown to include over 25 diverse contributors each with a unique perspective and passion for the environment. Over the past few months this project has instilled a deep commitment in us to effectively communicate environmental science and human reliance on natural ecosystems. Join us as we navigate the intersections between academic research and local interests throughout Puget Sound.

This first issue follows undergraduate researchers as they examine oyster parasites native to Washington and analyze symbiosis between beaver dam restoration and salmon migration. Our community features authors delve into the impacts of ocean acidification on Pacific Northwest municipalities and explore the UW College of the Environment?s commitment to bring underrepresented communities into environmental science.

We hope you enjoy our first issue and will continue following FieldNotes in the quarters to come!

- FieldNotes Editorial Board

- Editorial BoardEt h en Wh at t am

Rach el Fr icke

Community Features

Research

Alan n a Gr een e

M egh an Wir t h

Photography

Design

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Fiel dNotesTeam Editors Andrew Chin Jonathan Huie Abigail Ames Hyejoo Roo Colin Piwtorak Sam Kleinfinger

Authors Shruti Parikh Heather Lopes Lena Wilson Bridger Machus Marina Bambans Sammi Bushman Claire Malarkey

Design Kaelyn Thede

Photography Max Podhaisky Ellen Ng Katie Peterson Thomas Herrmann Taylor Bayly Tiara Adler

Photo by : Thomas Herman 3

Res ea r c h .

- The Effects of Pond-Leveler Devices on Salmon Migration through Restored Riverine Beaver Pond Complexes........ 6 - Morphological and molecular identification of shell boring mud worms from WA........... 11 - Using Endophytes to Increase Plant Salt Tolerance.............. 15

5 C o m m u n it y .

- When Oysters Die, The World Listens.................... 22 - The Cold Desert: Reflections of a Young Botanist.......... 29 - The Future of Diversity........................... 34

21 42

Ph o t o g r a p h y .

- Phtoto Feature by Max Podhaisky...................... 43 - Photo Feature by Taylor Bayly............................. 44 - Exploring the San Juan Islands............................ 46

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RESEARCH

Photo By: Tiara Adler 5

TheEffectsof Pond-Level er Deviceson Sal monMigrationthroughRestored RiverineBeaver PondCompl exes Written by Bridger Machus & Helena Wilson Edited by Johnathan Huie Abstract Through their dam-building, the North American beaver (Castor canadensis) can stabilize hydrologic regimes, increase stream complexity, and increase the biodiversity of riparian systems.

Beaver dams, however, can also create conflicts with human land-use and infrastructure, such has causing flooding on private property and disrupting restoration efforts. One way to reduce human-beaver conflict is the use of pond-leveler devices, which reduce flooding and allow beavers to persist onsite, but little is known about how these devices affect migratory fish movement. In this study we evaluated whether migrating salmon can pass beaver dams fitted with pond-leveling devices. Methods included doing fish surveys as well as doing hydrological and precipitation measurements and observations at the site. During the 2017 migration period at Big Spring Creek, over-dam stream velocity and through-dam velocity (i.e. within the pipe) did not exceed maximum adult salmonids swimming capacities. Beaver dams were passable during all periods of observation leading us to conclude that pond leveler devices are not passage barriers in most low-order streams under normal flow conditions.

Introduction The Pacific Northwest is home to the iconic North American Beaver (Castor canadensis) and Pacific salmon (Oncorhynchus spp.). Beavers are considered to be a keystone species because they have a disproportionately large effect on their environment relative to their abundance (Paine 1966). Beavers can alter the hydrology of riparian and wetland ecosystems by building dams in streams, which create beaver pond.

Beaver ponds increase landscape heterogeneity, producing a diversity of habitats that can support increased species richness and abundance (Rosell et al. 200). Fish often rely on beaver ponds for rearing, refugia during high and low flow, and for overwintering.

Certain salmon species, such as the Puget

Sound Fall Chinook, are threatened within Washington State and habitat restoration has become a conservation priority. Salmon require habitat connectivity for upstream migration to spawning grounds. As salmon return from the ocean and swim upstream to their places of origin, both natural and man-made barriers can impede migration.various barriers can impede salmon migration, increase mortality and prevent them from spawning, these include; logs, vertical drops, dams culverts,and roads.

The effects of barriers on salmon can vary greatly by species and life history stage. Certain species swim faster and longer than others, while juveniles are typically slower than adults (Bell, M. C. 1990). Restoring stream heterogeneity, habitat complexity, and

Figure 1. Diagram of a pond-leveler device with pipe going through the beaver dam. This is the same style device used on our site (adapted from WDFW 2018.)

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provide beavers with food and dam-building materials that may have been absent for a century or more. The long-term benefits of having beavers in the landscape include, increased hydrologic stability and ecosystem resilience (Law et al. 2016).

Non-lethal beaver management techniques, such as pond-leveler devices, are becoming more widely used, especially in areas where conservation or restoration is a focus.

Pond-leveler devices are commonly used in small streams to reduce flooding caused by dams (Taylor and Singleton 2014). A leveler consists of a large flexible pipe running through a beaver dam, allowing water to flow through and decrease the dam?s water holding capacity (Figure 1). The inlet of the pipe, which is located upstream of the dam in the beaver pond, has a cage placed around it to prevent the beavers

connectivity prove to be beneficial for increasing salmon survival rates, especially in the earlier life stages (Anlauf-Dunn et al. 2014).

Restoration projects intended to create salmon habitat often create beaver habitat as well. These restoration projects have unintentionally attract beavers that can create ponds and cause flooding through the construction of their dams (Naiman et al.

1988). Flooding increases the risk of restoration failure and likelihood of unmet design goals due to dramatic changes within the restoration sites. Salmon habitat restoration often focuses on improving fish passage, instream structures, floodplain and off-channel habitat, riparian improvement, sediment reduction, and flow augmentation (Roni et al. 2014). Projects that restore riparian vegetation, such as planting cottonwoods and willows, will consequently

Figure 2. Big spring Creek research site showing visible beaver ponds above dam 1 and 2, along with Newaukum Creek and 244th ave SE for global reference.

Table 1. Types of salmon migration observations implemented.

from detecting flow and plugging the pipe. In stream systems with higher discharge volumes, levelers can use more than one pipe to increase conveyance.

The scientific literature has demonstrated natural beaver dams are usually passable for migrating salmon and other fish (WDFW. 2009) (Bouwes, N, 2016) (ODFW, 1997), but there has been little focus on the study of fish passage at 7

Discussion

Figure 3. Maximum water velocities observed during the 2018 fish migration period (panel A) compared with the range of achievable salmon swim speeds (panel B). Sustained swim speeds can be sustained indefinitely while prolonged and burst ranges are limited by species swim endurance.

dams where flow control devices have been installed. Pond-leveler devices are a low impact and humane beaver management strategy that allow beavers to remain in place, retaining many of the ecological benefits that they create.

However, concerns about leveler devices impeding fish passage have resulted in controversies surrounding their widespread use.

Normally, salmon are known to jump over beaver dams, maneuver themselves around them, or go right through them. During high-flow winter conditions and when the water level overtops the dam, migrating salmon are provide with easy passage. Our objective was to characterize the effects of pond-levelers on flow regime and determine the effects this has on the passage of migrating salmon.

Methods We conducted surveys and monitored Big Spring Creek within the Green River Watershed on the western side of the Cascade Mountains.

The King County Department of Natural Resources and the US Army Corps of Engineers have collaborated on restoring this site as part of the Green-Duwamish Ecosystem Restoration Project (Figure 2). Due to an increase in beaver populations and activity, five pond-leveler devices were installed in 2016 to help stabilize water levels. However, we only monitored Dams 1 and 2 under the assumption that if salmon cannot migrate past the first barriers they will not surpass the ones further upstream.

The site was visited twice per week from

Figure 4. Daily Stream Discharge over duration of observation. This includes the total discharge from both upper and lower pipes 8

October 10, 2017 to November 28, 2017; fish were monitored for a minimum of two hours per visit. We conducted fish surveys which included species identification, abundance, location, carcass count, and behavioral observations.

When observers were not on site, we used three wildlife motion detecting cameras to capture images of any fish. Two cameras were placed at leveler device 1, one aimed at the top of the dam and the other at the face of the dam. A single camera was placed at Dam 2 facing downstream. All three cameras were active 24/ 7, equipped with flash, and took three second videos every time motion was detected.

We also conducted a single daytime snorkel survey on December 13, 2017, to assess adult fish presence upstream of levelers during the late 2017 coho run. We sampled from the confluence of Big Spring Creek and Newaukum Creek to the first leveler device in pond 1 as well as the stream section between leveler device 1 and 2. We recorded the size and class of all the fish and salmonid species observed including adults, juveniles, and carcasses.

We also surveyed the flow and depth of the stream to evaluate the hydraulic conditions experienced by migrating salmon at high and low flow events. We used a velocity meter to measure water velocity all around the dam and the leveler device. We measured water depth at all the same locations calculate and track discharge as it related to weekly precipitation and stream stage. Flow conditions were later compared with maximum swimming velocities to determine if water flow is a potential barrier for migrating salmon.

Results During the monitoring period we did not observed any migrating coho at Big Spring Creek, but did document one male chinook carcass at the start of the study above dam 2. The wildlife cameras did not record any fish passage or attempts. We also did not observe any migrating adults or juveniles during our snorkel survey.

Machus and Wilson in full dry suits for conducting a snorkel study of the beaver ponds at Big SPring Creek in December.

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Big Spring Creek?s near-flat topography made it very prone to bank overtopping and flooding given sufficient precipitation. During and following heavy precipitation, the stream?s wetted width expanded greatly due to flooding, at which point we could not accurately measure stream width. As precipitation increased, the amount of water overtopping the dam and the frequency of overtopping increased. Bank overtopping occurred for multiple weeks when precipitation was at its highest between November 16th to November 28th and was occurred during our snorkel survey on December 13th. Peak discharge in 2017 reached approximately 0.14 m^3/s.

Velocity measurements showed several precipitation and stage-dependent trends. Daily stream discharge ranged from a low of .077 m3/s to a peak of 0.12 m3/s, but maintained a relatively constant rate(Figure 4) after hitting the lowest discharge recorded on October 24th.

The flow of water through the leveler pipes often decreased as stream discharge increased and water overtopped the dam. Maximum velocity recorded at Dam 1 was 0.85 m/s within pipes, and 0.64 m/s for overtopping flows. At Dam 2 the pipes reached 1.2 m/s with no overtopping flows.

Discussion Fish presence was lacking at Big Spring Creek during this study, but it did not prevent us from recovering several notable insights.

While we did not directly observe fish migration, we were able to characterize streamflow conditions throughout the fish migration period, which provide evidence for the lack of physical constraints.

The comparisons between observed overtopping dam velocities and known swimming speeds suggest that stream velocity is not a barrier for migrating fish. Both maximum (i.e. burst) and prolonged fish swim speeds for all fish species were greater than the maximum overtopping dam velocity observed during this study. The leveler pipe velocities were slightly greater, but the water flow still did not likely prevent fish from passing through.

Although, the increased pipe velocities were often too swift for juveniles during high flow, but it seemed unlikely that juveniles would be traveling downstream during high flow events.

Thus, the pipes are not a barrier to juveniles at that time.

During high flow events, overtopping discharge increased and causing flooding around the dams, which created alternative swim routes for migrating fish. This was likely

caused by an increase in backwater pressure placed on the leveler outlets at the dam. This resulted in a decrease in leveling-pipe velocities as stream stage increased. As more water flowed over the dam, fish passability increased as they could swim around the dam during these conditions rather than jump over or swim through the leveler device. Bank overtopping and dam submergence also allowed fish to bypass the dams completely, disregarding any potential impairments caused by pond-leveler devices. The conditions observed during our research are considered normal for the site and was recorded during previous years as within range of our results.

At no point during this study would salmon have needed to achieve burst speeds to overcome stream velocity. Additionally the maximum stream velocities never exceeded the sustained swimming velocities of adult steelhead and cutthroat. Other remaining species faired similarly, requiring only a small percentage of their swimming to fall within the prolonged swim velocity range. Moreover, the juvenile salmon are much slower, and are not always capable of swimming up the pipes.

Conclusion Big Spring Creek experienced typical conditions (e.g. precipitation, discharge, velocities, etc.) of ~2nd order streams in the Puget Sound lowlands during times fall salmon migrations. Velocities seen on Big Spring Creek would be categorized as low in comparison to other waterways in the watershed, such as in Newaukum Creek or the Green River. Within our recorded velocities at no point would the fish not have the swimming capabilities to migrate up Big Spring Creek through the levelers. Other migration barriers including the beaver dam

Big Spring Creek near Dam 2 with pond-levelers underwater supported by rebar, fenced cage (pipe inlet) seen in background.

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itself and the structure of the pipes were negligible. For a majority of the research period the dam was completely submerged during high flow events, making these obstacles easily passable by fish. Based on these factors, we conclude that the implementation of non-lethal pond leveler devices in Big Spring Creek do not hinder salmon migrations. With this new information about the interactions between salmon and pond-leveler devices the implications for beaver management can be impacted greatly with greater wide spread use of pond leveler devices instead of other methods that involved beaver extraction or removal.

Acknowledgements This research was supported by the King County Department of Natural Resources and the University of Washington. We thank our colleagues from King County, Joshua Latterell, Jennifer Vanderhoof, and Dan Lantz, who provided insight and expertise that greatly assisted our research. We also thank Benjamin Dittbrenner from the University of Washington for his advisement during this process. We thank Joshua Latterell for his comments that greatly improved our manuscript, along with generously providing a port ion of t he necessary equipment for this project. The access to our research site was in thanks to him and his team. We would also like to show our gratitude to the University of Washington School of Environmental and Forest Sciences who helped make our research possible.

For references see page 48

Morphol ogical andmol ecul ar identificationof shel l boring mudworms(Polychaeta: Spionidae) fromWashington

Written by:

Heather Lopes Edited by:

Abigail Ames

Abstract Some spionid polychaetes can burrow into the shells of bivalves, creating unsightly mud blisters. Because they are unappealing to consumers and can burst, fouling oyster flesh, these blisters are an economic burden on affected oyster half-shell industries. Historical invasions by the spionid Polydora websteri have resulted in the collapse of aquaculture operations in Australia, New Zealand, and Hawaii, USA. Recent sightings of mud blisters on Pacific oysters (Crassostrea gigas) in Puget Sound, Washington, USA suggest that the area might be experiencing a spionid polychaete invasion. To determine whether P. websteri is the polychaete creating the mud blisters recently observed in Puget Sound, we obtained 24 Pacific oysters from two locations and examined for blisters and burrows. Polychaetes were extracted from the shells and vouchered for molecular analyses. We used mitochondrial (cytochrome c oxidase I [COI] and cytochrome b) and nuclear (18SrRNA) genes for species-level identification. Positive identification of P. websteri found in the mud blisters of Pacific oysters is the first confirmed sighting in Washington, USA.

Introduction Recent sightings of mud blisters on Pacific oysters (Crassostrea gigas; Figure 1) in Puget Sound, Washington suggest that the region might be experiencing a spionid polychaete invasion. Past invasions by spionid polychaete Polydora websteri in Hawaii, New Zealand, and Australia have resulted in aquaculture farm collapses. In Washington, a formal report of shell-boring polychaete has not been made.

Globally, economically important mollusks are damaged by infestations of spionid polychaetes, also called ?mud worms? (Lauckner 1983). Mud worms are parasitic polychaetes from the genus Polydora (Blake 1971) that bore into the shells of mollusk hosts (Blake 1969).

After the worm?s pelagic larval stage, the larvae will settle on to a mollusk?s shell (Nell 2007).

Once inside the shell, the worm forms a U-shaped burrow at the edge of the shell with two exterior openings (Loosanoff and Engle

1943, Hopkins 1958). Within this burrow, the worm collects mud, detritus, and worm feces (Lunz 1941, Haigler 1969). Burrows that breach the surface of the inner valve cause the host to produce brittle, dark calcareous layers to cover the burrows; this deformity is called a mud blister (Handley and Bergquist 1997). These blisters compromise the presentation of oysters served on the half-shell (Morse et al.

2015), and if a blister is burst during oyster shucking, the debris are emptied onto the oyster meat, making it unmarketable (Shinn et al. 2015). Heavy infections of mudworms can impact shell integrity, growth, and survivorship (Sato-Okoshi et al. 2016) Of the spionid polychaetes, Polydora is the most damaging to global commercial aquaculture industries and native species (Lauckner 1983). The possible introduction of Polydora in translocated oysters caused oyster

Figure 1. Crassostrea gigas collected from Oakland Bay, WA displaying pathology associated with mud worm infection.

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identification have been recently developed by Williams et al. (2017) used in this study. There is a need to molecularly identify these worms to species to begin to mitigate the risks Polydora could have on oyster aquaculture in Washington.

Oyster collections We examined 24 cultured Pacific oysters (Crassostrea gigas) purchased from shellfish retail farms in Washington State, USA. Culture location was obtained from Seafood Harvest Tags, where 14 oysters came from Totten Inlet, Washington and 10 oysters came from Oakland Bay, Washington (Figure 2).

Figure 2. We collected oysters from two sites, Oakland Bay (N=10), and Totten Inlet (N=14) where we then extracted worms from infected oysters.

beds of New South Wales, Australia to disappear in the 1860s, and these beds have yet to recover (Whitelegge 1890, Roughley 1922, Roughley 1925, Nell 2007, Ogburn et al. 2007). Oyster transplants brought in P. websteri to Kakuku, Hawaii and caused the collapse of a successful aquaculture operation (Bailey-Brock and Ringwood 1982, Bailey-Brock 1987). Oyster farms on the East coast of the United States have been plagued with Polydora infestations since the 1940s, resulting in oyster farm losses (Lunz 1940, 1941, Loosanoff and Engle 1943). Despite polydorid infestations being observed by oyster growers in Washington, an official documentation has never been reported.

To confirm anecdotal reports of polydorid worms in Puget Sound, we sampled oysters from two oyster farms in Washington, recovered worms from blisters and burrows within those oysters? shells, and used morphological characteristics and sequences of mitochondrial COI gene and nuclear 18S gene to identify these worms to species. Our goal was to assess the identity of these boring polychaetes. Our results constitute the first formal report of any boring polychaete from Puget Sound, and the first report of the notorious invader Polydora websteri. We briefly discuss the implications of the presence of this invader for oyster aquaculture.

Methods Many species within the Polydora genera display morphology that is indistinguishable from one another (Radashevsky and Pankova 2006, Rice et al. 2008). Species identification using morphological characteristics of Polydora alone can lead to taxonomic confusion (Radashevsky and Williams 1998). Waka Sato-Okoshi and Hirokazu Abe (2013) describe the variation in palp pigmentation and morphology of the pygidium, making identification difficult. Primers for mitochondrial COI gene used for molecular 12

Sampling and processing of worms We shucked each oyster and extracted any polydorid worms present, either by teasing worms out with a probe or forcep or by fracturing shells with a hammer to expose worms in their burrows. Once extracted from the shell, the morphological characteristics of the worms were observed under a stereo-microscope (Leica M125 C) to determine if they matched P. websteri based on the description made by James A. Blake (1971).

Specimens were photographed using with a camera (Leica DMC6200) mounted to the microscope. Vouchered specimens (n =24) were fixed 95% ethanol.

DNA extraction, PCR amplification, and sequencing DNA was extracted using DNeasy 96 Blood & Tissue Kit (Qiagen, Valencia, CA) following the manufacturer?s instructions. Two genes were used for molecular identification: 18S rRNA and cytochrome c oxidase I [COI].

For 18S rRNA gene, three primer pairs were amplified (18S-1F1/ 18S-1R632, 18S-2F576/ 18S-2R1209, and 18S-3F1129/ 18S-R1172 (Nishitani et al. 2012) ).

For COI, one primer pair was used (Dorid_COI.3F/ Dorid_COI.1R, Williams et al.

2017). We used polymerase chain reaction (PCR) to amplify DNA using a C1000 Touch (Bio-Rad, Hercules, CA) thermocycler at the Molecular Ecology Lab, University of Washington, Seattle. PCR reactions consisted of 5.0 µM of each primer, 2.0 µl of template DNA, 5 µl of 2X PCR buffer (Phusion® Hot Start Flex, Thermo Scientific, Foster City, CA), and 0.2 µl MgSO4 in a 10 µl reaction. 18S rRNA PCR conditions included an initial activation step of three minutes at 98°C, followed by 35 cycles through four steps: denaturation (30 seconds at 98°C), annealing (30 seconds at 54°C), extension (30 seconds at 72°C), with a final extension step (10 minutes at 72°C), and holding stage (10 minutes at 10°C). COI PCR conditions included an initial activation step of 98°C, followed by 30 cycles through four steps:

denaturation (30 seconds at 98°C), annealing (30 seconds at 45°C), extension (60 seconds at 72°C), with a final extension step (five minutes at 72°C), and holding stage (10 minutes at 10°C). PCR amplification results were checked with 1.5 % agarose gel electrophoresis using SYBR® Green (Molecular Probes, Eugene, OR).

PCR product was sent to Molecular Cloning Laboratories (San Francisco, CA) for sequencing and PCR clean up.

DNA Analysis Forward and reverse complementary full-length sequences of 18S and COI genes were combined using Geneious (version 11.0.5). Sequences were then trimmed to 660-615bp and partial sequences of 18S and COI genes were aligned with sequences of similar species obtained from GeneBank. We created phylogenetic trees using maximum-likelihood method based on Tamura 3- parameter model with Molecular Evolutionary Genetics Analysis (MEGA version 7.0.26) .

Results Morphological identification We viewed three identifying features during our sampling (Figure 3) including a bilobed, incised prostomium, modified fifth setiger, and branchiae beginning on the seventh setiger described by James A. Blake (1971).

Gene analysis Initially, the 18S rRNA sequences were 660 bp in length. We manually reduced the sequences to 615 bp for analysis. COI sequences were initially 680 bp in length. We manually reduced the sequences to 540 bp for analysis.

Phylogenetic trees Of the 10 worms sequenced with 18S rNA

from Oakland Bay, nine of them clustered with P. websteri, while one worm clustered with Boccardiella hamata. This phylogeny is supported by high bootstrap values from the maximum likelihood analysis (Figure 4). Of the nine worms sequenced with COI from Oakland Bay, five clustered with P. websteri.

Worms sampled from Totten Inlet were diverse in their species identities. None of the worms we sampled from Totten Inlet, clustered with P. websteri, but of the 14 worms sequenced with 18S rRNA, 11 clustered monophyletically with P. websteri (Figure 4).

Samples 13 and 14 from Totten Inlet sequenced from COI clustered with P. cornuta (Figure 5).

Some sampled worms remain to be resolved. The cluster containing Oakland Bay 10 and Totten Inlet 12 were consistent for both 18S rNA and COI genes, where a successful identity has yet to be determined. Weak bootstrap values were also noted for the phylogeny of COI sequences in both sample sites.

Discussion We successfully identified Polydora websteri as one of the mud worms found in C. gigas, providing the first formal identification of any shell-boring polydorid in Washington State.

Our results also suggest that, in addition to P. websteri, the closely related shell-boring polychaetes Boccardiella hamata and P. hoplura and the mud-dwelling polychaete P. cornuta may be responsible for the mud blisters found on the inner valves of C. gigas.

All worms from Oakland Bay, with the exception of one, were identified as P. websteri.

The polydorid fauna of Totten Inlet was more diverse, with worms identified as P. websteri, P. cornuta, and P. hoplura. Brown et al (2012) suggests that P. websteri is sensitive to salinity, which may contribute to the distribution of

Figure 3. Defining morphological features of polychaete Polydora websteri described by James A. Blake (1971).

Features include ia bilobed, incised prostomium, modified fifth setiger, and branchiae beginning on the seventh setiger.

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Figure 4. Maximum likelihood phylogeny based on Tamura 3-parameter method using trimmed 18SrRNA sequences (500 replicates). Phyogeny consists of 10 sequenced worms from Oakland Bay, WA, and 14 sequenced worms from Totten Inlet, WA. Pseudopolydora dayii was used as an outgroup. Species accompanied with accession number were acquired from GeneBank.

polydorids.

Further research on the environmental variables for Polydora is needed to confidently explain these differences.

Unresolved phylogeny of our sampled worms requires more analysis. Replicates of our current samples may help resolve phylogeny. Additional samples from our sites may also help support our current analysis where our sample sites harbor different worm species.

The pathology caused by shell-boring mud worms causes unsightly blisters. These blisters

Figure 5. Maximum likelihood phylogeny based on Tamura 3-parameter method using trimmed COI sequences (500 replicates).

Phylogeny consists of nine sequenced worms from Oakland Bay, WA, and six sequenced worms from Totten Inlet, WA (bold text). Pseudopolydora dayii was used as an outgroup.

Species accompanied with accession number were acquired from GeneBank.

For references see page 49 14

reduce the market value of infected oysters, especially those served on the half-shell. Past invasions by spionid polychaete Polydora websteri in Hawaii, New Zealand, and Australia have resulted in aquaculture farm collapses.

The presence of this invader as the potential threaten the valuable oyster aquaculture operations in Washington, and with a positive identification we can move forward to finding a way to reduce any negative effects.

UsingBacterial EndophytestoIncreasePl ant Salt Tol erance Written By: Shruti Parikh Edited By: Andrew

Chin

Abstract Plants are affected by various abiotic and biotic stressors such as pollution, high salinity, drought, and pathogens which can inhibit growth or cause disease. High salinity, a type of abiotic stress that decreases crop productivity and quality, is especially widespread, with nearly half of the world?s cropland facing salinity stress in 2010. This is problematic because there are very few crop plants that can tolerate high levels of salt, and remediation is water and energy intensive.

Endophytes, the beneficial bacteria that live within plants, which have been shown to help plants tolerate these stressors by directly interacting with the stressor or indirectly by modulating the plant?s response to the stress, could be a potential alternative. The main goal of this study was to isolate and test endophytes from Salicornia sp. (pickleweed) and Aaegilops sp. (wild wheat) growing in a marine estuary to determine whether they can colonize other plants and help these plants tolerate high salinity conditions. A total of fifteen salt tolerant endophytes were isolated. Of these strains, four were then inoculated into Phaseolus vulgaris (bush beans) to test whether increased salt tolerance is being conferred. After exposure to the salt treatment, bush beans inoculated with endophyte strains SSP14 and SSP15 had significantly greater biomass and water content and qualitatively appeared more vigorous in comparison to the control. By improving plant growth under abiotic stress, these bacteria have potential to promote sustainable agricultural practices.

Background Plants, just like any other organism on the planet, are affected by environmental stressors.

These stressors can include drought, a lack of nutrients, or pollution, and can limit a plant's ability to survive. Salt is an important abiotic stressor in plants. Although it is a necessary soil compound for plants and microbes, it can be detrimental at high concentrations (Bloodknick 2017). More specifically, salt affects plant growth by reducing the plant?s ability to take up water, causing wilting, loss of structure, and stunted leaf growth (Negrao et al. 2016). It also reduces soil water retention which further reduces the plant?s ability to maintain water (Erysian et al.

2005).

Salt is naturally deposited in soils through the weathering of rocks (Ahmad 2013). Heavy wind and rain also displaces salt from seawater (Ahmad 2013). Over time, the salinity concentration in the soil increases as salt that was previously leached into the groundwater rises up with the water table with the occurrence of a high precipitation event (Leeuw et al. 1991). After the water evaporates, or is transpired by plants, the salt becomes more concentrated in the soil (Leeuw et al. 1991).

Although this is a natural process, and the occurrence of salt is only detrimental to plants at high levels, with climate change, and increasing trends in temperature, precipitation, and 15

extreme weather events, soil salinity will continue to rise (Tubiello et al. 2007).

This issue is more prevalent along the coast and in agricultural areas as increasing soil salinity affects crop productivity and quality (Chinnusamy et al. 2006). In 2010, about half of the world?s cropland faced salinity stress (Mei et al. 2010). In addition, soil salinity in coastal areas such as Bangladesh was modeled to increase 39% by 2050 due to the impacts of climate change (Dasgupta et al. 2015). There are also anthropogenic factors that increase the concentration of salt in soil such as urban and agricultural land use, irrigation, and the use of fertilizers (Ahmad 2013). In many instances, the impacted land is abandoned, such as in California?s Central Valley where over 400,000 acres of land have been retired (Erysian et al. 2005). This can be problematic because there are very few plants that can tolerate high levels of salt, and the process of remediation is water intensive, requiring irrigation to be set up (Payo et al. 2017). An alternative is needed that can remediate the land, promote increased salt tolerance in plants, or both.

Endophytes, or beneficial microbes colonizing the internal tissues of plants, which have been shown to increase host-plant tolerance to a wide range of environmental

stressors are a potential alternative (Reinhold-Hureket et al. 2011). They are able to increase tolerance directly by degrading or sequestering the compound (Kang et al. 2014), or indirectly by promoting plant growth in order to better prepare plants to combat the stressor (Khan et al. 2016).

The goal of this study was to isolate salt tolerant endophytes from Salicornia sp. (pickleweed) and Aaegilops sp. (wild wheat) growing in Port Susan Bay and to test their ability to confer increased salt tolerance to Phaseolus vulgaris (bush beans), a salt sensitive crop plant. We hypothesize that plants growing in this estuarine environment may have endophytes that help them adapt and survive in this high saline environment that can then be inoculated into new host plants to help them survive in the same environmental conditions.

Methods Extraction and Isolation of Endophytes Pickleweed and grass samples were collected from Island County Park on the Iverson Preserve on Camano Island by Dr. Doty.

Samples were washed and surface sterilized using the protocol described in Kandel et al.

(2017). Murashige and Skoog (MS) broth, a plant based minimal growth medium, was added to the sample, and the plant tissue was ground using a mortar and pestle. Dilutions of the extract were plated on 3% NaCl MS agar plates. Individual strains were then isolated from the agar plates and used in this study.

Pickleweed Endophyte Selection via Poplar In addition to isolating extract based on tolerance to 3% NaCl media, plants were allowed to naturally select for the salt tolerant endophytes. Sterile poplar were placed in the pickleweed extractions for 2 days. Following the inoculation, the vessels were washed thoroughly and 10 mL of 25mM NaCl of MS was added. This was then increased to 50 mM NaCl until the control started showing signs of stress.

After 60 days, the poplar was surface sterilized and ground. The extraction was diluted using sterile water (1:10, 1:100, and 1:1000) and plated on 3% NaCl MSplates.

Endophyte Identification Using 16S rRNA Sequencing The colonies were streak purified and isolated. 16S colony PCR was performed using the 8F (5? -AGAGTTTGATCCTGGCTCAG-3?) and 1492R (5? - GGTTACCTTGTTACGACTT-3? ) universal primers to amplify the 16S rRNA gene.

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The samples were sent to GENEWIZ in Seattle, WA and sequenced using Sanger Sequencing.

Sequences were then compared to the GenBank database of 16SrRNA genes.

Assessment of Salt Tolerance of Inoculated Bush Beans Beans were germinated in a wet paper towel for 72 hours. After germination, the beans were planted in Sunshine Mix #4 (Sungro Horticulture) in quarter-gallon pots and received 12 hours of light daily for the extent of the experiment. Rhizobium. tropici CIAT899 was grown in cell suspension in Mannitol Glutamate/ Luria (MG/ L) broth. The strain was washed using sterile water, and the OD600 was adjusted to 0.1 using a spectrophotometer (UV-1700, Shimazu America Inc., Columbia, MD, United States). To account for the variation in legume seeds that naturally had the rhizonium present, 1 mL of R. tropici CIAT899 was added into the hole with the seedling.

After allowing the R. tropici to colonize for one week, each of the strains from Figure 1 were taken from 3% NaCl MS plates and grown in cell suspension in a 3% NaCl MS solution for 48 hours. The strains were washed and diluted to an OD600 of 0.1. Each paired treatment (stress and no stress) consisting of ten plants was watered to saturation with 150 mL of the inoculum. A paired control consisting of ten plants was ?mock? inoculated with 150 mL of water without endophytes.

After a three-day inoculation period, the plants were watered with a 100mM NaCl solution every other day for one week. Control plants were watered with regular tap water on the same days. After one week, the salt treatment was stopped and the plants were watered with tap water as required for two weeks. At the end of the three weeks, wet and dry above-ground biomass was recorded.

Statistical Analysis The results were analyzed with RStudio version 3.2.2 (R Development Core Team, 2015). A one-way ANOVA was run on each parameter followed by a Tukey post-hoc analysis to identify which treatment pairs were significantly different.

Results Plant Extractions/ Poplar Selection A total of 15 salt tolerant endophytes were isolated. Seven were isolated from the pickleweed, five from the marine estuary

Figure 1. Endophytes used for this study

grass, and three from the poplar screen. Four of these endophytes, SSP1, SSP7, SSP14, and SSP15 were the focus of the assessment of the salt tolerance of bush beans (Figure 1).

Assessment of Salt Tolerance of Inoculated Bush Beans The control-no stress plants weighed significantly greater than the control-stress plants (p=0.009)(Figure 2). The difference between the no stress and stress groups for SSP7 and SSP15 were also significant (p=0.015 and p=0.009 respectively). There was no significant difference between the stress and no stress groups for plants inoculated with SSP1 and SSP14. Overall trends in the figure also show that the average wet masses of all the inoculated groups were greater than the wet mass of the control-stressed group (p=0.002).

The Tukey analysis results showed that the stressed plants inoculated with SSP14 and

SSP15 were significantly different (p=0.044 and p= 0.038 respectively). At a 90% level of confidence SSP1 was also significantly different from the control-stressed group (p=0.068).

The water content was found by taking the difference between the wet above-ground biomass and dry above-ground biomass. The difference represents the water content of the plant at the end of the experiment. The difference in average water content between control-stress and control no-stress was significant (p=0.012) (Figure 3), showing that the plants not exposed to stress retained more water. The difference between the no stress and stress groups for SSP7 and SSP15 were also significant (p=0.020 and p=0.006 respectively). There was no significant difference between the stress and no stress groups for plants inoculated with SSP1 and SSP14. Overall trends in the figure also show

Figure 2. Average above ground biomass (wet) of each bean control and treatment group at the end of the experiment prior to drying.

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Figure 3. Average water content at the end of the experiment, which was calculated by subtracting wet mass from dry mass. that water content in the inoculated plants was greater than the control plants (p=0.001). The Tukey analysis results showed that the stressed plants inoculated with SSP14 and SSP15 were significantly different from the control-stress plants (p=0.039 and p= 0.036 respectively). At a 90% level of confidence SSP1 was also significantly different from the control-stressed group (p=0.070). These results are also qualitatively reflected in Figures 4 and 5.

Discussion In this study, salt tolerant endophytes were extracted from pickleweed and wild

wheat to test the hypothesis that plants growing in an estuarine environment may have endophytes that help them adapt to this high salinity environment and to test whether they can confer the same tolerance to a new host plant. Four of the isolated strains, SSP1, SSP7, SSP14, and SSP15 were tested for their ability to confer increased salt tolerance to bush beans.

In order to quantify the effect of the salt treatment on plants, upper biomass and water content measurements were taken. There was a significant difference in mass between the control-stress and control-no stress; thus, the salt treatment inhibited plant growth. A

Figure 4. These photos show the control-stress (left) and control-no stress (right) treatments after the 3 week treatment period. The stressed treatment is wilting, dry, and less green while the no stress looks healthier.

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Figure 5. These photos show the control and inoculated treatments under stress.

Note how the control treatment (top left) looks much browner and more wilted than the inoculated treatments. significantly higher biomass was found in plants inoculated with SSP14 and SSP15 compared to the stressed control. The greater biomass in the inoculated plants means the strains promoted growth in the bush beans under salt stress.

Both strains also had a significantly higher water content compared to the control-stress; thus, helping the bush beans retain more water, through the overall increased biomass or a direct mechanism. A potential mechanism that could allow endophytes to help plants tolerate high salinity is by accumulating salt and triggering water to leave its cell (Kharbikar et al.

2018). Water content within the plant is important to study because a high salt concentration in the soil causes an influx of ions to enter and water to leave the plant (Meena et al. 2017). However, if the endophyte accumulates salt within its cell, it allows the plant to retain water by limiting osmosis between the plant and soil (Kharbikar et al.

2018).

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There was a significant difference in biomass and water content between the stress and no stress plants inoculated with SSP7 and SSP15; therefore, despite the presence of the strains, the plant growth was inhibited by the stress. SSP15, however, still performed better than the control stress. Even with the inhibited growth, the endophyte still helped the plant produce more biomass and retain more water than the control plants.

There are many salt tolerant strains that exist, but the goal is not just to find strains that grow in NaCl, but to find strains that are compatible with the plant. The plants themselves may naturally select for endophytes that help them tolerate the condition better, whether it?s by increasing root growth through auxin production or some other plant growth promoting mechanism. SSP7 was isolated from the poplar inoculated with the pickleweed extract. Since the addition of this endophyte had no effect on bush beans, the

poplar-selected endophyte could be specific to helping poplar tolerate high salinity.

There was no significance in biomass or water content between the stress and no stress plants inoculated with SSP1 and SSP14. This means that SSP1 and SSP14 allow bush beans to tolerate a high concentration of salt to retain water and produce the same amount of biomass that they would without the presence of salt.

Since SSP14 also significantly performed better than the control under stress, this strain effectively mitigates the effect of salt. SSP1 was not significantly different from the control stress. However overall trends, significance at a 90% confidence level, and qualitative observations (Figure 5) show that there was increased growth and a higher water content in the plant, and thus will be studied further with more replicates to account for variation.

Qualitative results reflect the same response to the salt treatment as seen in the graphs. The control-stress is wilting and brown, showing clear signs of salt stress compared to the control-no stress that looks much healthier (Figure 4). In addition, the SSP1, SSP14, and SSP15 stressed treatments are comparatively more vigorous than the control-stress (Figure 5).

It was observed that many of the plants in each treatment were experiencing outside stress that caused an extra loss of leaves. It is possible that they lost a significant amount of biomass due to this, which could have impacted the results. However, it is important to note that the inoculated plants still produced significantly greater biomass.

The mechanisms of how SSP14 and SSP15 promote growth under high NaCl concentrations are unknown, however past studies on various endophytes do suggest potential hypotheses. A study on Brassica juncea (Indian mustard) showed that Trichoderma harzianum increased oil in response to NaCl which improved nutrient uptake and enhanced antioxidant and osmolyte uptake, thereby decreasing Na+ uptake (Ahmad et al. 2015). Another study showed that Trichoderma produces ACC-deaminase which lowers plant ethylene levels to decrease the stress response (Brotman et al. 2013).

Pseudomonas sp. and Acinetobacter sp. also enhance ACC-deaminase and IAA (an auxin) in a study done on barley and oats (Chang et al.

2014). These mechanisms will be tested in future studies.

With the increase in global warming, there will be an increase in extreme weather events which can significantly impact agricultural and

coastal lands (Tubiello et al. 2017). About 800 million people in Africa and Asia alone are undernourished and, according to the UN, a 60% increase in world food production over the next 20 years is necessary in order to sustain the population (Jewell et al. 2010). In order to address this concern, arable land is needed.

However, only about 20% of the land in these countries is currently arable due to the impacts of drought and salinity (Jewell et al. 2010).

With this, there is an increasing demand for a way to make crop plants more tolerant to salt.

The results in this study show that SSP14 and SSP15 significantly promote growth and water retention under high salinity conditions and SSP1 and SSP14 have the potential to mitigate the effects of high salinity on bush beans.

Conclusion The experimental data and photos showed that SSP1, SSP14, and SSP15 have the potential to confer increased salt tolerance to bush beans. This study will be repeated with these strains under improved experimental conditions ensuring plants are not experiencing stress unrelated to salt. If the experimental trends continue, the endophytes will be characterized for other beneficial traits such as hormone production, N-fixation, phosphate solubilization, and antifungal activity. For phytoremediation and biofuel applications, the strains will be tested to see whether they promote increased salt tolerance in poplar.

Proline content will also be tested in the next study as a potential mechanism for alleviating salinity stress.

Acknowledgements I want to express my sincere gratitude to Dr. Sharon Doty for the research opportunity and constant guidance. I also want to express my appreciation to Pierre Joubert for his mentorship, Will Braks for assisting with experimental setup, and Andrew Chin & Brookelyn Parsons for helping with manuscript preparation.

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For references see page 49

COMMUNITY FEATURES

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Written by: Claire Malarkey Edited by: Ethen Whattam

WhenOystersDie, Theworl dListens Under a pressing timeline, scientists, policy makers, and farmers have joined forces to raise awareness for finding solutions to counter the effects of ocean acidification on Puget Sound oysters.

Adult oysters at Taylor Hatchery, Photo by Max Podhaisky

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On a bright afternoon in April, sun-struck by the Olympic Mountains?rain shadow, the Taylor Shellfish Hatchery is an idyllic facility. The farm, one of three owned by Taylor, is located on Dabob Bay, a little over ten miles from the town of Quilcene, Washington. The long stretch of beach adjacent to the farms is layered with deep, wet sediment dotted with lavender-tinted bivalve shells. Oysters grow naturally out there, according Dave Pederson, an employee at Taylor Shellfish for over two decades, but not enough to make a business out of it.

Harvesting shellfish at Dabob Bay has never been consistent enough annually to generate enough revenue as an industry itself, and any extra oysters collected by Taylor Shellfish is considered a bonus for the company. Taylor Shellfish has been operating in Quilcene since the 1970s as one of the leading shellfish growers in Washington, which has the largest shellfish industry in the nation. For Quilcene and other small, rural towns along the Puget Sound, oyster farming is their livelihood.

Dave Pederson holds juvenile oysters, Photo by Max Podhaisky

Many farms have been part of the industry for over a century including Taylor Shellfish, which started in 1890. Since, it has grown into three separate farms that produce oysters, geoducks, and clams, as well as bivalve ?seed? that are distributed internationally. But ten years ago, the hatchery production collapsed. The annual production between 2008 and 2009 was off 75% from historic levels, and there were no oyster seeds for the farms. Taylor Shellfish was not alone; this phenomenon proliferated throughout oyster hatcheries in the Puget Sound. Only in hindsight could this event be viewed positively, as it launched the Pacific Northwest into the limelight for fighting the global issue of ocean acidification.

Research on ocean acidification (OA) is not new; the simple chemical reaction from adding excess carbon to saltwater has long been known and there was no dispute that it should be happening. The National Oceanic and Atmospheric Administration (NOAA) has long been observing the carbon cycle in marine environments, as part of their mandate for understanding global nutrient cycles.

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These datasets provided the foundation for the discovery of the phenomenon of OA. At the beginning of this century, oceanographers started conducting research on cruises in the Puget Sound, where they observed OA and began to understand the severity of this issue locally.

Crucial to this understanding were time-series that NOAA had generated for decades that have been used to recently refute claims about the ?natural? influx of CO2 saturation in the Puget Sound. Two factors (unrelated to human activity) combine to cause higher-than-average-levels of CO2 in the Pacific Northwest. Upwelling occurs frequently along the coast, which is where cold water (that retains gases better than warmer water) from deeper depths in the ocean rises to the surface due to winds near the coast, dragging up stored CO2 with it. Additionally, generated by differences in heat and density, the ocean acts as a large conveyor belt in which water sinks and rises as it moves around the globe? a cycle called thermohaline circulation which

Written by Claire Malarkey

Photography by Max Podhaisky

Edited by Ethen Whattam

Geoducks grown on Dabob Bay, Photo by Max Podhaisky

takes around 1,000 years to cycle. The North Pacific is the final destination for this water before it sinks at the pole and begins the cycle again, water near this area has collected CO2 from all of the respiring organisms it has encountered for a millennium.

Upwelling and thermohaline circulation in addition to anthropogenic factors makes the Puget Sound much closer to crossing the threshold than many other places on earth.

The ?threshold? that is referred to frequently is 1. More specifically, an aragonite saturation state which is less than 1 is where organisms tend to be in distress.

Aragonite is often used to track ocean acidification, as it is related to carbonate ion concentration. When excess carbon dioxide enters the ocean, the reaction with the water results in carbonic acid. Carbonic acid makes carbonate ion less available, which can be detrimental to corals, clams, oysters, various plankton, and other organisms that use carbonate ions to form their shells or skeletons. This causes lack 24

of reproduction, or death. Organisms in larval stages are particularly affected by lower aragonite saturation states, but more developed organisms can also be affected This is what occurred in Pacific Northwest oysters.

In 2008, when Pacific Northwest oyster hatcheries collapsed, local farmers eliminated the usual factors that would normally cause a failure of larval development? bacteria, temperature increase, etc. Confounded by the dismal yield that season, they met with marine biologists and oceanographers in Seattle, who had recently returned from the cruises documenting carbon levels. Having just observed the severity of OA, there was no question that the issue should begin affecting the farms. In a simple experiment, scientists buffered the water to change the pH levels and observed whether the oyster larvae survived. They did. Now the acidity of the water that oyster larvae are hatched in is heavily controlled, and larvae survival rates have returned to normal. What began that year was a vital partnership between NOAA and UW scientists with the local shellfish industry.

A key entity in this partnership is Washington Sea Grant. Meg Chadsey, who acts as a liaison for the organization, describes Sea Grant as a ?two-way pipeline? that not only allows businesses to utilize data from federal and university research, but also

Tubs of algae is cultivated and fed to growing oysters via pipes, Photo by Max Podhaisky

allows these businesses to ask questions that will shape and direct the sort of research performed. It stays neutral in order to act as an unbiased broker for information in order to facilitate practical and applied research about on-the-ground problems that need attention. Chadsey holds another liaison role at NOAA Pacific Marine Environmental Laboratory (PMEL) in order to facilitate communication between scientists and those in need of scientific information.

A recent example of Sea Grant funding is UW scientist Carolyn Friedman?s investigation on the effects of oyster and eelgrass co-cultivation. Eelgrass Wasting 25

Oysters a couple weeks old spiral in tubes filled with circulating water, Photo by Max Podhaisky

Disease is currently a serious problem facing eelgrass, but, it is hypothesized that oysters reduce the known pathogens that cause it. Meanwhile, eelgrass? a photosynthesizing organism? absorbs CO2 and performs better in a CO2 rich environment, as carbonate ions are unnecessary for its functions. The eelgrass, whose health may be aided by the diminished pathogens, reduces the CO2 (and therefore acidity) in the surrounding waters and could support oyster growth.

Studying how the multipartite relationships between eelgrass, oysters, and Eelgrass Wasting Disease work, as well as how a changing ocean (both acidification and rising temperatures) affects these relations is a complicated yet important example of questions that Sea Grant is helping scientists answer. Washington Sea Grant has also partnered with Taylor Shellfish to aid in research that develops oyster strains that are more resistant to the effects of OA, a study which concluded in 2017. Another project with Carolyn Friedman and Taylor that concluded in 2015 studied transgenerational effects of OA exposure.

A greater question that others are attempting to answer is, ?What?s the effect on people?? Kevin Decker, Washington Sea Grant?s economist, tries to quantify this. There are many factors, however, that prevent an easy answer. For example, due to upwelling, the specific location of a hatchery along the Puget Sound will cause a monetary loss directly due to ocean acidification to vary. Larger companies like Taylor Shellfish move a significant amount of their seed production to other regions, such as Hawaii, before shipping the oysters back to Washington when they are less vulnerable. Smaller farms in South Puget Sound regulate the pH themselves instead.

However, in Decker ?s 2015 report to the state1, the shellfish industry adds $270 million to the state?s GDP. Ocean acidification jeopardizes $110 million annually for the industry, which is quite detrimental to the small towns that rely on the oyster industry as drivers of their local economy. Even more negatively affected are tribal communities that harvest oysters, and to which there is not only economic but also cultural significance tied to shellfish. A study2 on the social vulnerability of human 26

communities related to shellfish decline across the United States at first glance provides a relatively positive outlook for the Pacific Northwest compared to the rest of the country. In other places where shellfish collection is prevalent, namely East Coast states like New Jersey and Massachusetts, and certain regions in the South, social vulnerability is extremely high compared to the overall moderate social impact on the Pacific Northwest. However, due to upwelling and thermohaline circulation, the timeframe in which those regions will feel the severe impacts is roughly 100 years, whereas communities in the Pacific Northwest are expected quantifiable economic hardships within a few decades.

In part due to the hatchery collapse and in part because of NOAA and UW researchers, Washington State has become a global leader in addressing OA and the ecological and economic issues that accompany it. Chadsey says that, because the shellfish industry here is so vulnerable, ?[OA?s] effects were noticed first because we were paying attention.? She gives a lot of credit to Bill Dewey, who has elevated this issue from an internal crisis within Taylor Farms to an international platform. Dewey is extremely positive about the power of oysters as a catalyst for evolving policies. ?What?s fascinating is how our story has drawn international attention?, he says. ?I?m asked to tell our story at forums all over the world.?

This is true. Dewey served the Washington State Blue Ribbon Panel on Ocean Acidification which, in 2012, was the first in the nation to consist of a coalition of scientists, federal agencies, and other organizations to seriously address OA in Washington. Currently he serves on the state Marine Resources Advisory Council, appointed by Governor Jay Inslee, to carry forth the recommendations of the Blue Ribbon Panel. Beyond that, he has spoken at international panels on ocean conservation, a State Department conference in front of world leaders, and three times at a United Nations forum. Earlier that morning, before talking to FieldNotes, he was on a conference call to discuss a new shellfish industry entity of The Nature

Building of Taylor Hatchery, Photo by Max Podhaisky 27

Conservancy. Taylor Shellfish is one of seven founding members of the Shellfish Growers Climate Coalition, which uses the ?shellfish story? to educate consumers and lawmakers in order change policy. ?It?s cool,? says Dewey. ?You?re telling your story to people who can actually make a difference in carbon policy.?

Perhaps nothing better sums up Dewey?s unique opportunity for science communication than when asked why oysters specifically are provocative. He says, ?It?s people, it?s jobs, it?s food? food people can relate to. A lot of the other effects people have trouble relating to.? He notes that many other organisms that currently are being directly impacted by OA are low in the food chain and therefore many people do not react to them, using pteropods (planktonic snails) as an example of animals that humans do not care about because we do not eat. ?But salmon do,? he adds.

For all the research currently being done on OA and shellfish, the farmers themselves have run experiments since the inception of their businesses, whose successes are a series of ?trial-and-error ?, according to Dave Pederson at Taylor Farms. They work to determine how to optimize growing seasons, having only last year installed heaters to extend the growing season of algae. Taylor Farms grows all the feed for their shellfish; huge teal and viridian cylinders containing diatoms and flagellates (different types of plankton) are the sole limiting factor on annual yields. In an ongoing effort to maximize growth for the oysters, they experiment with different combinations of algae feed and alter the ratios over time.

Dave, who knows better than anyone how sensitive oysters are to changing ocean chemistry, is grateful for the nearby organic farms for reducing fertilizer runoff and for the community for its ongoing effort to replant trees and wiring off streams.

But even an extremely conscientious community like Quilcene has yet to find ways of preventing the blooms of bright coccolithophore phytoplankton that turn Dabob Bay water into a Caribbean-like blue every several years. Few oysters survive the rapid influx of algal growth. Dave explains that many environmental factors cause oyster death, but they are exacerbated by OA, like a weakened immune system is to us.

Increased acidification will weaken the organism?s ability to cope with various stressors.

He brought up a sobering fact towards the end of the tour; due to the timescale of upwelling, the added CO2 causing oyster decline is from about 60 years ago. The increased concentration of carbon currently put into the atmosphere is already stored underwater and waiting to resurface in another 60 years. To what extent oysters? and the many other marine organisms impacted by OA? will have survived that far into the future to feel the increased concentration is unknown.

There is an unidentified orange bucket lying by one of the larvae tanks in the hatchery. Like large, silver ears, several shells piled upon one another glisten under the water. When asked during the tour about an unlabeled bucket full of bivalves, Dave replies, ?That?s someone?s dinner.? Before or after work, employees at Taylor Shellfish harvest shellfish along Dabob Bay, as if no time had passed at all.

Thanks to Meg Chadsey, Kevin Decker, Shallin Busch, Carolyn Friedman, Bill Dewey, and Dave Pederson for contributing to this story.

For references see page 49 28

THECOLD DESERT Reflectionsof aYoungBotanist By: MarinaBambans EditedBy: EthenWhattam

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View from Wild Horse Campground, Nevada.

June 2017. Photo by Marina Bambans

The natural world is a big mystery ? one that entrances most of us, and one that we know can never fully be solved. If we use the clues it gives us, maybe we will be able to understand it, even if it reveals only a fraction of its wonder.

In Washington, we travel through conifer-covered landscapes dripping with Lichen, and lush waters flowing into the Pacific. The amount of biodiversity is evident through the thousands of fauna, flora and fungi species living here. We can camp among the giants of the temperate rainforests and hike the shrub steppe east of the Cascades. Thee range of environments provide many favorable growing conditions for flora, sustaining and supporting a multitude of ecosystems.

One of the greatest things about witnessing nature is that it is not limiting or selective. Anyone can take note of the world around them. For me, after dropping out of college at 19 and returning a few years later, a course called Pacific Northwest Biology did not seem so intimidating like classes in my past. There were no looming math tests on the horizon but rather invitations to explore the environments of Washington.

The class at North Seattle College provided a pathway into identifying the plants and animals of the Pacific Northwest. Between our hours in the field and reading we had timed identification tests, where we would use field guides and memorization to identify the biota. Plants of the Pacific Northwest by Jim Pojar and Andy Mackinnon and The Audubon Society?s Field Guide to the Pacific Northwest were resources that allowed us to navigate through the beautiful, complex, and sometimes confusing flora of the Pacific Northwest. When the quarter ended I could not stop; every day I would walk through the neighborhood and try to identify the plants starting with Bergenia (Pigsqueak) and Iberis (Candytuft) in my neighbor ?s rockery. The observations were elementary in the botanical world, yes, but that was the point: you have got to start somewhere.

This initial interest had now morphed into a botanical obsession.

Door into UW/ Burke Herbarium, Seattle. August 2016. Phtoto by Marina Bambans.

Seven years later, I found myself in a brightly-lit basement with no windows or people in sight, staring at a green door with faded plant posters and a sign that read ?Herbarium?. With my hand on the handle I slowly opened the door and entered into a botanist?s dream. The room was dressed in plant pressings and jars scattered across tables? it was a hidden library, a place full of specimens.

But, the most important specimens lay beyond the door where professional botanists held the knowledge of the diversity and intricacies of these plants. Within this flora cavern on the edge of the University of Washington?s campus, was a community of people who shared my interests. Suddenly the university felt less cold 30

to a transfer student like me.

The University of Washington?s Herbarium is a part of the Burke Museum, but resides across campus in Hitchcock Hall. The building is named after former UW biology professor Charles Leo Hitchcock (who also co-wrote the locally famous Flora of the Pacific Northwest with professor Arthur Cronquist). The herbarium has a database of 660,000 plants with over 400,000 available for viewing online.

I went to the herbarium to meet with Dr. David Giblin, the UW Herbarium Manager, and active botanist, who at the time had helped organized an aquatic plant workshop through the Washington Native Plant Society (Plants and water, what could be better?). While talking with Dr. Giblin, he mentioned a class called Plant Identification and Classification. A formal botany class, one that sounded much more encompassing than field guides, became my new botanical challenge. Next spring, I enrolled.

The Herbarium members travel each year on forays to collect and record plant data. Dr. Giblin explained, ?The specimens gathered provide important, novel information about diversity on public lands, new populations of rare plant species, and the spread of invasive species? he says.

The trips are undertaken by a collection of undergraduates, graduates, community members, professional botanists and other science professionals. When else would I be able to travel to the desert with botanists? Thinking about the invitation to Nevada I wondered, can I keep up? Am I ready? We were preparing to

Cypripedium montanum patch in Emigrant Springs Campground, Meacham, Oregon June 2017. Photo by Marina Bambans.

Elaeagnus in Bruneau Sand Dunes, Mountain Home, Idaho June 2017. Photo by Marina Bambans.

head into somewhere new. I was scared, but I knew that feeling well. So, I took a breath and jumped in.

The benefits of the trip were not solely educational, but also personal for me with the ability to interact with a community that I admired. The array of ages and experience levels supported a progressive learning environment because the experienced botanist provided me with new-found knowledge The opportunity to be a part of witnessing the mysteries of the natural world, through plant clues, was something I could not pass up. Two months later, we were on our way to the cold desert, a section of northern Nevada where the expansive mountain ranges and higher elevations makes it colder than other deserts.

We left Seattle one evening, traveling to Oregon where we stayed the night at Emigrant Springs Campground. Before leaving the next morning to continue the 31

journey south, we noticed a patch of Cypripedium montanum (Mountain Lady Slipper Orchids) growing on part of the grounds. Driving on the highway, the landscape changed from wet to dry quickly, and as we drove through the dry southern Idahoan landscape, we ventured off the comforts of the paved roads to the shifting sands of the Bruneau Sand Dunes. Surprisingly, there was a wetland bordered by tall Juncus and the silvery-blue foliage of Elaeagnus (Russian Olive) swaying in the wind.

Hours later, twisting down the highways of Nevada, with views of Artemisia tridentata (Sagebrush) hillsides and eerie backyard clotheslines, we arrived to our campground in the Humboldt National Forest near Jarbidge. The area was patched with Populus tremiloides (Black Cottonwood), Juniperus (Juniper), and dotted with a few Pinus (Pines). It was a comforting surprise to see rivers, so I set up my tent near the black cottonwoods while the drone of insects and rattlesnakes drowned out the thoughts of doubt in my head.

The blazing sun came through the tent and woke me up the next morning. The Nevada sun was bright and as we gathered around the picnic table covered in field guides and plant parts, the botanists laid out a large map. They divided us into groups to target specific areas; For me, day one partially consisted of climbing a rocks with Dr. Giblin, where we stumbled upon Opuntia (Prickly Pear Cactus), Alliums (Wild Onions) and Lewisia (Bitterroot). During our lunch hour we were swarmed by cattle and by late afternoon we were ankle deep collecting yellow Castilleja (Indian Paintbrush). It came to me, one of those moments where you realize your love for something is bigger than your doubts: I was in rattlesnake country during high noon, on top of an ant hill, collecting dozens of tiny Mimulus (Monkey Flower) and I could not have been happier.

The wind had picked up by mid-day, blowing the specimens and newspapers across the field. At that point we thought fast. I sat in the back of the van, balancing the cardboard and newspaper layers above and below each specimen all while staying out of the breeze.

Each day we had to switch our collection groups, giving everyone the opportunity to collect for a different lead botanist. This educated us on differences in techniques of plant collecting.

One day we were advised to collect according to plant color, the next it was by botanical family or topographical area. Nonetheless it gave us the ability to understand the multiple ways to approach plant collecting.

Opuntia, Elko County, Nevada June 2017. Photo by Marina Bambans.

Lewisia, Elko County, Nevada June 2017. Photo by Marina Bambans.

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Beetle, Elko County, Nevada June 2017.

Photo by Marina Bambans.

Mertensiana longiflora Elko County, Nevada June 2017. Photo by Marina Bambans.

Plant Press, Elko County, Nevada June 2017. Photo by Marina Bambans.

Pediocactus in Elko County, Nevada, June 2017. Photo by Marina Bambans

Taking in the views, it was clear that Nevada was abundant with mountainsides of electric blue Penstemon (Beardstongue) along with ultraviolet Delphinium (Larkspur), yellow Wyethia (Mule Ear ?s), and fragrant wafts of Lupinus (Lupine). Beetles appeared at our feet and antelope galloped in the distance while we searched. Near a portion of the top of Tennessee mountain, there were Pediocactus (Barrel Cactus) and unforgettable Mertensia longiflora (Long Blue Bells), whose shades of blue and yellow blossoms splattered the landscape.

Thinking about the whole experience, specifically about the plants ? I realized that these specimens were going to live in the herbarium database forever. A very humbling and emotional thought; finally, I had created a part of my academic legacy I was truly proud of. Even more so was the possibility of these collections having a positive impact on the health and sustainability of our ecosystems. ?The approximately 12-15,000 specimens we have collected in all of the forays have helped tremendously to better understand the distribution of plant diversity in the Pacific Northwest? Dr. Olmstead details.

Driving out of Nevada, it was a burning sunset. The kind that shines through the car windshield with such unwavering intensity that you can?t help but squint your eyes. It was miles on miles of empty road with no end in sight. Was I still scared? Of course, but it felt different - it felt more like empowerment and peace.

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Th e Fu t u r e of Diver sit y?

UW?s College of t h e En vir on m en t pion eer s STEM -w ide in it iat ives t o br in g u n der r epr esen t ed com m u n it ies in t o scien ce Au t h or : Sam an t h an Bu sh m an Edit or : Et h en Wh at t am Ph ot ogr aph er : M ax Podh aisk y, Ellen Ng

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Lif e as a Ph D st u den t can be t im e con su m in g. In bet w een lon g days spen t h u n ch ed over dat a an d sm all br eak s dr eam in g of f ieldw or k , Er ica Escajeda of t en t h in k s abou t t h e pr ivilege sh e h as.

Getting money to work in the Arctic was no easy task. Spending time in the Bering Strait with whales is considered by many to be a marine biologist?s dream. But for some, the bigger challenge isn?t funding. It?s not even getting into school, or picking what part of the environment they want to study.

For underrepresented communities, even learning about the outdoors can be hard. Public lands, and the STEM field in general, have historically not been representative of the communities that populate them. Escajeda and colleagues hope to help change this narrative.

Diversity initiatives at UW have long spent time supporting those enrolled in an attempt to provide equal opportunities among the arts and sciences. The College of the Environment wants to push further in the coming months with a series at initiatives targeted at storytelling and open discussions on the state of minority affairs.

Terryl Ross is spearheading the campaign for more equal representation from an institutional perspective. As the College of the Environment's first assistant dean of diversity, equity, and inclusion, he has developed these initiatives designed to tell stories and bring awareness to those that have been lost in the waves.

As a graduate researcher, Escajeda doesn?t have a lot of time on her hands.

Shuffling between thesis writing and working with the Society for Advancement of Chicanos/Hispanics and Native Americans in Science (SACNAS), most of her school energy is dedicated to work and helping others.

Diversity can be defined as a lot of things. It?s race, ethnicity, gender, and economic status among many others. At UW, the focus has mainly centered on bringing more native and african americans, latinx, black, and other populations that are underrepresented into the fold.

Together as students and staff, members of the UW and more broadly the STEM community are actively trying to change the narrative of who is a scientist. They connect students to each other and attempt to bridge the gap between identity and vocation.

Wh at it m ean s: Statistically, the College of the Environment is still behind. In 2018, a report highlighted that while the number of caucasian individuals studying the environment was proportionally greater than those enrolled in UW as a whole, all other racial and ethnic backgrounds trailed.

Perhaps the starkest difference was in the african american population. While 3.7% of students at UW 36

Erica on a research cruise in the Arctic

A young scientist participating in Native Scholars on the Rise

identified as black, less than 1% of the college of the environment shared that view.For hispanics, 2% less studied nature at a STEM level.

The college has made a lot of progress in the past few years. By bumping the proportion of native americans enrolled to be greater as a whole within the college and increasing numbers overall, they have proved that outreach and connecting with the community works. ?We have a better percentage of females here than all the other stem fields on campus,? Ross said. But there?s still a long way to go. People are not just statistics. ?There's a very important relationship between the health of people the environment by which they live,? Ross said. ?A kind of social justice?? it?s always been connected.?

Escajeda noted that in her artic field research, almost all of the scientists representing native communities were white. The humpback whales and other marine mammals she studies are resources for natives. Yet, as the environment changes, the communities remain disconnected to those changes and potential solutions are many times lost due to a lack in representation among scientists in academia simply because they do not have a strong presence in academia. ?My ultimate goal is to bring the science to communities [to] have them participate,?

Escajeda said. ?There?s very little representation of the Inuit indigenous communities in Gray Whale diving in the Arctic ocean. Photo by Erica Escajeda these meetings.?

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Allison Burdick, an employee at Klondike National Historical Park and former environmental studies undergrad who focuses on bringing underrepresented communities outside agrees that locally outside school, this is an issue. ?There is a disconnect between diverse communities and what we see in our national parks,?

Burdick said.

Listening in on the final projects

Inequalities can be traced back through American history to a variety of sources.

These include but are not limited to the whitewashing of academia, racial and economic segregation, and majorly today, a simple lack of access and outreach. ?We?re part of this world, and this country and we need to be included in all the parts that make up this country,? MESA UW president Martha Florez Perez said.

Ross acknowledged that diversity shifts over time. A room full of people each representing a different ethnic group is not always going to be true to what the population of a city or a school looks like.

In that context, goals change. Staff and students recognized that there was no one perfect equilibrium when it came to accurate representation. It was about working together to find the best way to get there. To help all students understand that they can and should be a voice for the cultures they represent. ?As collaborative as we should be we're still siloed, and the environment really doesn't discriminate,?

Ross said. ?people discriminate, but the environment doesn't, and the environment always wins.?

Explor in g pr ivilege an d per son al con n ect ion s: For Arianna Gomez, being a part of SACNAS has given her a way to connect with those that share her own identity and give back. Now a grad student, she got involved in diversity initiatives as an undergrad participant. ?It was really nice to be able to like meet with people who are in field related to mine

A gr ou p of em er gin g scien t ist s pr esen t in g t h eir f in al pr oject at Nat ive Sch olar s on t h e Rise h ost ed by SACNAS

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and like make friends and also like be involved in the community,? Gomez said.

As a child, Escajeda attended marine biology camp off the coast of California. She was lucky. Her parents gave her the opportunity to explore what she was passionate about before having to make the choice to pursue it professionally. Others, she said, have never even seen the ocean.

The idea that people should be educated about the environment at an early age is a shared sentiment among privileged advocates for diversity, who have used their acquired knowledge to help drive change.?I was so fortunate to grow up with a family that really appreciated the outdoors,? Burdick said.

Ross asserted there was power in this idea. In round table meetings, he found the majority of participants were white women. While discussing inclusion, they couldn?t help point out the irony of their own lack of representation. ?The only way we're gonna change things is with your help,? Ross said. ?So please, don't apologize for who you are.?

Th e Pow er of You t h : Within student and community organizations, the priority is largely being placed on children. Programs like UW Mathematics, Engineering, and Science Achievement (MESA) and SACNAS target kids to jump start an interest in science at an early age.

At Native Scholars Day, students from around the Pacific Northwest assembled in the lobby of the Fisheries building with their UW SACNAS counterparts. They marvelled at the comprehensiveness of their campus tour, highlighting time in the lab as a favorite experience of the day. ?It's really fun to get their input and like for them to be so wide eyed about like the whole college experience,? Gomez said.

MESA leads programs in elementary, middle, and high schools. Saturday

SACNAS members leading the group through a project on ecology in the Pacific Northwest

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Young scholars presenting their final project

Academy, a monthly program brings students to UW for science themed workshops.

Florez Perez said she also teaches weekly lessons to classrooms in a variety of STEM centered topics.

Beyond that, the organization provides parent and teacher funding, summer classes, and scholarships to students in their target communities. UW SACNAS has similar goals to that of MESA, promoting STEM development for chicanos/hispanics and Native American youth.

For educators, native scholars day was a success. ?It means a lot to us, this is special this is something that we?re not going to get anywhere else.? Sarah Sense-Wilson said.

At Klondike Gold Rush National Historical park, the A Place in the Park program employs young people to supplement historically retired age volunteers, who have more time to give after they stop working. Employee Kelsey Johnson said that now, a lot more young, diverse people are coming in. ?We?re focusing on the underrepresented and underserved communities in Seattle, which is primarily South Seattle, which happens to have the most diverse zip codes,? Burdick said.

In it iat ives in act ion : Escajeda, Ross, and others recognized that there is a long way to go when it comes to achieving STEM wide diversity. ?We need to get rid of this idea of Ivory Tower,? Escajeda said.

One way to do that, she suggested, is by devoting more research-ships and grants to underrepresented communities and those that would otherwise be driven away from science due to the cost. Escajeda also wished employers would stay away from unpaid internships.

On an even more basic level, she suggested classrooms try and do better to recognize all the different perspectives that already exist. The university needs to capitalize on the diversity it already has to bring viewpoints into the spotlight.

Escajeda, Perez Florez, and Ross constantly reiterated the power of bringing young 40

people into the folds.

While UW has a plethora of resources for underrepresented communities and minorities, it is also a very big place. Sense-Wilson appreciated the smaller events hosted by SACNAS because it allowed her students to get more individualized and targeted attention. It is a multifaceted issue that cannot be solved on a short timeline or with one initiative.

Ross has high hopes for his time at UW. Pulling from years of experience and connections, he wants to use multimedia storytelling to do this, bringing attention to those who have historically have had none. ?I want you to be able to talk to people that are different than you and and to be able to to have authentic conversations with people,? Ross said. ?I want you too be able to lead teams with people that are different than you.? ?From the very beginning UW students have been our first interns, our first students driving this program,? Johnson said. ?UW comes through with awesome students who are not only dedicated to their studies but really giving back to the community.? ?It can be very hard adjustment time for people to come come here from those small tight-knit communities,? Escajeda said. While the sciences may be different, their motivations remain relatively the same. Diversity outreach may be addressed by Ross and ?My ultimate goal is to bring the science to communities have them participate,? Escajeda said. ?There's a real problem in environmental science that I think we can only work on to a certain point at university level," Escajeda said, "There needs to be more work done to connect to the community to look for those barriers and remove them.?

The final project created by all of the young scientists participating on Native Scholars on the Rise

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PH O TO GRAPH Y

Otis Douglas Hyde Herbarium, May 2018. Phtoto by Thomas Herrmann.

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P h o t o Fe a t u r e

Max Podhaisky is an emerging nature photographer from UW studying Geology and DXARTS. His photos capture the drama and dichotomy of the environment.

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Komodo National Park

INTERNATIONAL PROTECTION

In this photo essay, Taylor Bayly depicts the diversity of places that are protected throughout the world.

Tanzania 44

Indonesia

Table National Park 45

Th e exf oliat ed r ock s of t h e Lopez com plex. San d Besides h ou sin g st u den t s, f acu lt y an d r esear ch er s, an d m u d deposit s h ave been h ar den ed in t o UW?s Fr iday Har bor Labs ar e also h ost t o deer t h at . r ock t h en com pr essed in t o sh eet s. gr aze on t h e f acilit y gr ou n ds.

Expl oring the SanJuan SIsluciaand Isl ands Ech elon f r act u r es sh ow w h er e t h e r ock w as sh ear ed in t h e past .

Her e, t h e r igh t side of t h e r ock w as m ovin g dow n w ar ds r elat ive t o t h e lef t side, cau sin g t h e f r act u r es t o split open in t h e r ock .

SanJuan Isl and

ESS st u den t s an d pr of essor s obser vin g t h e r ock s at Iceber g Poin t in t h e r ain .

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Lopez Isl and 4

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(Lef t ) Gr aded beddin gs in t h e r ock s on t h e sou t h w est side of Lopez Islan d.

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(Top) Glacial st r iat ion s lef t on t h e r ock s at Wash in gt on Par k .

Fossils f r om t h e Nan aim o Gr ou p spot t ed by GeoClu b m em ber s. Fossil collect in g is pr oh ibit ed on t h e islan d.

By El l enNg

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(Top) ?Hon eycom bs?ar e pr evalen t on Su cia Islan d. Con t in u ou s w et t in g by salin e seaw at er an d dr yin g u n der t h e su n allow s salt cr yst als t o w edge apar t m in er al gr ain s in t h e r ock , leadin g t o cavit ies. Film s of algae t h at f or m on t h e in n er w alls pr ot ect t h e h on eycom bs f r om expan din g in t o on e an ot h er m u dst on e.

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(Lef t ) Look in g Nor t h f r om Rosar io Head.

Of t h e t w o ou t cr ops in t h e f or egr ou n d, t h e lef t com pr ises ch er t an d t h e r igh t , m u dst on e.

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