Gillett and Burton – Molecular Identification of ...the individuals in a given sample. Traditional larval fish identification is done via microscopy of preserved specimens. This

Gillett and Burton – Molecular Identification of Ichthyoplankton

PROJECT TITLE: MOLECULAR IDENTIFICATION OF LARVAL NEKTON INHABITING THE WATERS OF THE SOUTHERN CALIFORNIA BIGHT

PRINCIPAL INVESTIGATORS:

Dr. David Gillett – Scientist, Biology Department, Southern California Coastal Water Research Project

Dr. Ronald Burton – Professor, Marine Biology, Scripps Institution of Oceanography, UC San Diego

ASSOCIATE INVESTIGATORS:

Dr. Eric Stein – Department Head, Biology Department, Southern California Costal Water Research Project

FUNDING REQUESTED

2016-2017: $57,708 Federal/State $0.00 Match $95,733 2017-2018: $50,505 Federal/State $0.00 Match $34,325

STATEMENT OF PROBLEM:

The Southern California Bight (SCB) is the quintessential urban ocean, exposed to a variety stressors at local (e.g., urban runoff, habitat loss, wastewater discharge) and regional (e.g., climate change, ocean acidification) scales. The SCB is also the home to a myriad of ecologically, economically, and culturally important organisms; ranging from benthic infauna to migratory marine mammals. Given the importance of this diverse ecosystem, a variety of programs have been developed to monitor the condition of these biological resources relative to a range of ambient stressors.

However, the tools that are presently available to assess the condition of the SCB’s resources are limited in scope. Regional water quality and natural resource managers have developed robust monitoring programs to assess benthic invertebrates (Ranasinghe et al. 2012; CSD 2014a; LACSD 2014a; OCSD 2014a), demersal fishes (Allen et al. 2011; CSD 2014b; LACSD 2014b; OCSD 2014b), and rocky reef/kelp associated fishes (Craig and Pondella 2006; Claisse et al. 2012; Pondella et al. 2012). Pelagic fishes, especially in coastal habitats, are a conspicuous gap in assessment of the condition of the SCB ecosystem.

In recognition of this gap, the regional water quality and resource management communities have expressed a desire to start monitoring pelagic fishes in concert with the demersal and rocky reef fish assemblages – looking at the nekton of the SCB holistically instead of only as disparate components. Comprehensive community analysis would provide a more complete understanding of the condition of specific areas and their ability to support a diverse nektonic community. One approach to efficiently survey the entire nekton community is to sample eggs

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and larvae (i.e. ichthyoplankton) before they segregate as juveniles to their respective habitats (e.g., hard bottom, soft sediments, or water column) (Reviewed in Auth and Brodeur 2013).

A significant obstacle to the regular monitoring of ichthyoplankton is the difficulty in identifying the individuals in a given sample. Traditional larval fish identification is done via microscopy of preserved specimens. This process requires time and a high degree of taxonomic specialization. Furthermore, the process is complicated by the lack of detailed dichotomous keys for a number species, many of which have only subtly different morphology as larvae (e.g., Sebastes spp.) (Gray et al. 2006).

Molecular taxonomic methods, such as DNA barcoding, offer a potential solution to improve the efficiency and utility of ichthyoplankton assessment by increasing the speed, accuracy, and resolution of a sample’s taxonomic composition. DNA barcoding of individual organisms has been demonstrated to provide reliable identifications for taxonomically challenging organisms like ichthyoplankton (e.g., Gleason and Burton 2012) and other taxa (e.g., Pawlowski and Holzmann 2014; Lejzerowicz et al. 2015). However, despite its utility in improving the identification of problematic species, single organism barcoding is not practical for application in a large-scale monitoring programs given the time needed to process hundreds of individuals in dozens of samples. However, with a reliable DNA reference library, metabarcoding – the extraction and sequencing of all the DNA in sample at one time – represents a promising way to create an ichthyoplankton monitoring program across the SCB based upon molecular identification of samples. Furthermore, if successfully applied to ichthyoplankton, similar metabarcoding techniques could be applied to other biological components of the ecosystem in the future.

INVESTIGATORY QUESTION:

o The primary question this work is centered around is, “Can metabarcoding techniques provide comparable (or better) data to traditional morphological methods of ichthyoplankton identification?”

o As part of this question, we will also be able to address whether metabarcoding is as sensitive to detecting rare taxa as morphological identification and if the costs and time associated with the metabarcoding are amenable to their inclusion in a regional monitoring program.

o The secondary question this work will address is “Does metabarcoding provide similar taxonomic diversity to single organism identification using traditional (Sanger-based) barcoding methods?”

MOTIVATION:

Understanding the larval ichthyoplankton population dynamics along the shelf addresses several management questions/needs. First, it is essential to understanding the potential condition of the entire adult nekton community of the SCB. Second, detailed spatial information on rocky reef associated larval communities would be valuable to managers of the recently established marine protected area network in Southern California. These data could provide insight into the

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effectiveness of the reserves and their productivity value to adult assemblages outside of the reserves. Third, larval nekton monitoring is an ideal approach for early detection of invasive taxa before a population of adults becomes established.

Molecular taxonomic methods, such as DNA barcoding, offer a potential solution to improve the efficiency and utility of ichthyoplankton assessment. Traditional specimen-by-specimen barcoding has been shown to provide valuable taxonomic details beyond those obtained in standard surveys. More recently, new methods are being developed for metabarcoding of bulk environmental samples (i.e. analysis of all barcodes present in an ambient water or sediment sample). This approach holds promise in increasing the speed and taxonomic breadth of an assessment (e.g., Gray et al. 2006; Loh et al. 2014; Hubert et al. 2015). Although already proven effective in concept, this method still requires refinement to allow it to be easily integrated into routine monitoring and assessment programs. Specifically, it needs to be evaluated in the context of routine monitoring and assessment programs and the results need to be directly compared to alternative methods such as microscopy and single-specimen barcoding.

The southern California continental shelf provides a perfect opportunity to refine metabarcoding methods for routine application. First, many species of nekton associated with the different continental shelf habitats of Southern California have been collected as adults, morphologically identified (considerably easier as adults vs. larvae), and had their mitochondrial CO1 (i.e., the DNA barcode) and 16S ribosomal genes sequenced (largely as part of a California Sea Grant project, P. Hastings and R Burton PIs, "Establishing a DNA Sequence Database for California Marine Fishes," (2004-2007). Consequently, an extensive reference library already exists that can be used to match validated species identifications to sequences generated from the sampled larvae. Second, there is existing infrastructure among the local sanitation districts (i.e., City of Los Angeles, Los Angeles County, Orange County, and the City of San Diego), the Southern California Bight Regional Survey, and CalCOFI programs to support routine sample collection. Third, there are important management questions regarding the condition of the local environment relative to discharge locations that could be addressed through application of these new methods.

Beyond providing a spatially integrated profile of the larval stages of the SCB nekton community, this work will provide an approach to improve current nekton monitoring efforts via modernization of laboratory techniques, while also likely increasing the taxonomic breadth of these surveys. Additionally, the regular use of metabarcoding techniques should shorten the time it takes to collect data, analyze samples, and provide information to key decision makers.

Improving the quality, efficiency, and geographical/ecological scope of these data will provide the underlying support for the management and monitoring of coastal nekton communities across the SCB; will support the needs of our research partners, and are consistent with the goals of the CA Sea Grant program. As highlighted in the 2014-17 Strategic Plan, our proposed work will help addresses Healthy Coastal Ecosystems Goal 2: “Ecosystem-based approaches are used to manage land, water and living resources”, specifically short term outcomes 2.2 “and 2.3. The benefits this work would fulfill the stated desire of many of the regional agencies across SCB to integrate molecular-based taxonomy into their programs and to expand the scope of their programs to include ichthyoplankton.

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GOALS AND OBJECTIVES:

A. Overall Goals: The primary goals of this research are to determine if ichthyoplankton monitoring data derived from metabarcoding methods produces comparable or better quality data to traditional morphological methods and if so, develop basic operating procedures for incorporation into monitoring programs of our management partners

B. 2015-2016 Objectives: 1 – Develop measures of ichthyoplankton community composition across the SCB using morphological identification of samples 2 – Develop similar measures of community composition, but using individually sequenced DNA barcodes (CO1 and 16S)

2016-2017 Objectives 3 – Develop measures of ichthyoplankton community composition across the SCB using bulk sequenced metabarcoding techniques (CO1 and 16S) 4 – Compare the overall taxonomic composition, sensitivity to rare dominant taxa, and effort associated with processing samples among the three methods of identification (morphological, individual barcoding, metabarcoding) 5- Develop basic operating procedures for incorporating metabarcoding methods into routine monitoring programs.

METHODS:

Sample Collection – Sample work flow for the proposed work from collection to assignment of species names/operation taxonomic unit (OTUs) is summarized in Figure 1. Ichthyoplankton samples will be collected from across the Southern California Bight by field crews from the region’s large publicly owned treatment works (POTW) (i.e., Los Angeles County Sanitation District, Orange County Sanitation District, and the City of San Diego, [see attached letters of support]). Samples will be collected with a 150-μm mesh pairovet net, using a vertical tow following CalCOFI sampling protocols (https://swfsc.noaa.gov/textblock.aspx?Division=FRD&id=1343). Samples will be collected along similar sampling grids to those used by the POTWs for their demersal fish trawl sampling (e.g., CSD 2014b; LACSD 2014b; OCSD 2014b). Upon removal from the net, samples will be labeled and placed in 95% ethanol. Samples will be drained and placed in fresh 95% ethanol within 48 hrs of collection.

Sample Identification – After preservation, samples will be processed along one of two approaches: 1.) morphological → single barcode → metabarcode or 2.) morphological →metabarcode. In an effort to benchmark results generated by metabarcoding to more traditional single organism barcoding, a subset of samples will be identified based upon morphology, then by barcoding of individual organisms, and then composited for bulk

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https://swfsc.noaa.gov/textblock.aspx?Division=FRD&id=1343


metabarcoding. All other samples will be identified based upon morphology and then bulk metabarcoding.

For the first identification approach, fish eggs and larvae will be sorted from detritus and zooplankton, enumerated, and identified by taxonomists at the NOAA Southwest Fisheries Science Center. Vouchers of each unique taxon will be kept for archiving purposes. Standard QA/QC procedures for the sorting, enumeration, and identification will be done following modifications to Kramer et al. (1972). From these identified samples, ~ 800 individuals will be removed for individual DNA barcoding. Standard DNA extraction and amplification protocols will be used. Tissue samples of approximately 2-3 mm3 will removed from each organism, placed in 96-well plates with lysis solution. DNA will be extracted and collected from the tissue samples by centrifugation onto glass fiber plates (Ivanova et al. 2006). Mitochondrial cytochrome oxidase subunit 1(CO1) will be targeted using forward primer COI VF1 (5’-TTCTCAACCAACCACAAAGACATTGG-3’) and the reverse primer COI VR1 (5’-TAGACTTCTGGGTGGCCAAAGAATCA-3’ (Ivanova et al. 2006; Ward et al. 2005) and 16S mitochondrial 16S ribosomal DNA using the forward primer 16Sar (5’-CGCCTGTTATCAAAAACAT-3’) and the reverse primer 16Sbr (5’-CCGGTCTGAACTCAGATCACGT-3’) yielding an amplicon of approximately 570 bp (Palumbi 1996). Taq polymerase and standard polymerase chain reaction (PCR) protocols will follow Ivanova and Grainger (2006) and Gleason and Burton (2012).

Upon successful amplification, the PCR amplicons will be sequenced bi-directionally by Sanger sequencing with a capillary DNA analyzer (e.g., Stein et al. 2014). Forward and backward sequences will be aligned and checked for appropriate size and structure (Tamura et al. 2011). Valid DNA sequences will then be assigned names based upon matches from custom DNA libraries of Southern California nekton, as well the larger public genomic databases (e.g., BOLD, GENBANK).

After the Sanger sequencing (i.e., individual barcoding), the individuals from which the tissue was collected will be composited into a new “virtual-sample” with known composition. These virtual-samples will be homogenized for metabarcoding of the bulk sample. An aliquot of each homogenized sample will be archived at -80°C . The DNA from the remainder of the sample will be extracted multiple times using commercial lysing and extraction kits (e.g., Nucleospin, Machercy-Nagel inc.). The sequential extraction s will be combined and COI and 16S DNA sequences will be targeted using a mix of universal primers (e.g., see above) and amplified using Taq polymerase and PCR.

Upon successful amplification, the DNA will be sequenced using an Illumina Mi-Seq high throughput sequencer following Kozich et al. (2013). Produced forward and reverse sequences (of approximately 200-300 bp) will be aligned and verified for size and structure, to insure purity. Valid sequences will be assigned names based upon matches from custom DNA libraries of Southern California nekton, as well the larger public genomic databases (e.g., BOLD, GENBANK).

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Figure 1. Work flow diagram summarizing our experimental design for identifying ichthyoplankton by morphological, single barcode, and metabarcoding

Sample collection

Samples to lab

Change ethanol

Clean sample of detritus and zooplankton

Morphological ID

Sanger sequencing

Composited into virtual sample

Bulk DNA extraction

Next-Gen sequencing

Morphological ID

Clean sample of detritus and zooplankton

Bulk DNA extraction

Next-Gen sequencing

OTU assignment

OTU assignment

OTU assignment Metabarcoding

Single barcode

Morphological Key to identification

approaches

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For the second identification approach, samples will undergo morphological identification as described above. Once the contents of the sample are identified, the sample will be re-constituted and homogenized for metabarcoding. DNA extraction, amplification, and sequencing will be done as described above for the virtual samples (Figure1).

Data Analysis – Similarity in taxonomic composition of each sample using the three identification methods (i.e., morphological, individual barcoding, and bulk metabarcoding) will be compared using a series of multivariate methods (e.g., nMDS, Permanova, etc). Taxa that create dissimilarity in the results among the different methods will be highlighted and further analyzed to ascertain the reason(s) for their failure to be detected by all three methods (i.e., molecular problems or morphological problems).

RELATED RESEARCH:

No current or past USC Sea Grant projects have focused on developing or advancing metabarcoding for analysis of ichthyoplankton communities. However research over the past ten years has led to dramatic advances in the application of molecular methods, such as barcoding and metabarcoding for species identification and community analysis (Jackson et al. 2014, Stein et al. 2014). The use of standardized DNA sequence markers – DNA barcodes – has become a common, standard practice in many areas of biodiversity assessment (Hajibabaei et al. 2007a, 2007b). Customized, public databases of DNA barcodes and other marker gene sequences (e.g., BOLD, GenBank) contain representative DNA barcodes for hundreds of thousands of animal, plant, fungal, and microbial taxa. Comparison of DNA barcodes recovered from unidentified specimens can be used to provide species-level identification for a wide range of organisms.

Barcoding has proven to be a useful method for providing more complete understanding of fish communities. For example, Valdez-Moreno et al. (2010) used barcoding to examine over 1390 specimens including adults, juveniles, larvae and eggs off the coast of Mexico. Barcoding results improved overall taxonomic identification, revealed major range extensions and overlooked taxa, new information about spawning locality and time. More recently, a dual marker system consisting of the mitochondrial genes cytochrome oxidase I (COI) and 16s rRNA have been used together to improve species identification from mixed samples and to enhance the ability to detect hybridization and separate closely related species (Kochzius et al. 2010). Locally, Gleason and Burton (2012) have validated the use of the dual gene approach using COI and 16s markers to identify fish that commonly occur off the California coast.

From a fisheries management perspective, there is great interest in assessing ichthyoplankton communities as a way of improving understanding of issues such as spawning locations and seasonal migration patterns and quantification of population levels or biomass of fished species (Teletchea 2009). Although DNA barcoding has been successfully used to identify pelagic fish larvae and eggs (Kawakami et al. 2010), traditional specimen-by-specimen analysis can be cumbersome, inefficient, and challenging due to inadequate taxonomic keys

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Over the past few years, metabarcoding methods have emerged that take advantage of advances in sequencing technology to rapidly identify many specimens from mixed (or bulk) environmental samples (Ji et al. 2013, Gibson et al. 2015). Laboratory testing of these methods have shown that they can provide a powerful and practical means for generating millions of DNA sequences across broad phylogenetic groups from bulk environmental samples as opposed to single specimen analysis (Hajibabaei et al. 2011; Shokralla et al. 2012; Gibson et al. 2014; Shokralla et al. 2014). Such methods are currently being applied by the Los Angeles Museum of Natural History to help survey terrestrial insects through their BioScan project (http://research.nhm.org/bioscan/about.html).

Metabarcoding has the potential to greatly expand our ability to assess ichthyoplankton communities by improving taxonomic accuracy and resolution and increasing the efficiency and rapidity of sample analysis. Many past studies have provided “proof of concept” for metabarcoding. This study would advance the science and practice by answering practical questions associated with applying metabarcoding analysis in real-world monitoring and assessment programs along the California coast.

BUDGET-RELATED INFORMATION

A. Budget Justification • Year 1 (2016-17)

o $16,513 for 0.75 months of Gillett’s time (salary, benefits, and indirect) to coordinate sample collection and exchange, facilitate two meetings among the participants, manage data, and prepare interim report.

o $10,917 for 0.3 months of Stein’s time (salary, benefits, and indirect) to assist in meeting facilitation and preparation of the interim report.

o $3,459 for 0.3 months of a SCCWRP part-time employee” time (wage, benefits, and indirect) to provide support towards sample preparation, transportation of samples, and data management.

o $2000 in expendable supplies. Purchase of denatured ethanol, sample containers, and field/lab supplies for sample collection and preservation at SCCWRP.

o $300 for travel support to transport samples among different laboratory locations and to cover travel to meetings held at SCCWRP offices in Costa Mesa, CA by project PI’s and partners.

o $720 for two meetings (catering, coffee service, etc) among technical and supervisory staff of project participants. Meetings to be held at SCCWRP offices in Costa Mesa, CA.

o $27,908 in subcontract to UC San Diego. The contract covers 3 months ($15,555 – salary) of a SIO technician’s time to process samples and sequence DNA in the Burton lab, purchase of reagents and supplies

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($2,000) for single specimen (i.e., Sanger) sequencing in Burton lab, $450 in project costs, and $9,903 in indirect costs (55% rate).

• Year 2 (2017-18) o $22,017 for 1.0 month of Gillett’s time (salary, benefits, and indirect) to

manage data, analyze results, prepare manuscripts, and facilitate a meeting among project participants.

o $7,798 for 0.25 months of Stein’s time (salary, benefits, and indirect) to assist in meeting facilitation and preparation of manuscripts

o $2,935 for 0.2 months of a SCCWRP part-time employee (wage and indirect)

o $3200 for travel support of PIs to scientific conferences to present study results and travel of project participants to project meeting held at SCCWRP offices in Costa Mesa, CA.

o $400 for one meeting (catering, coffee service, etc) among project participants and regional water quality and resource managers to communicate the results of the study and promote adoption of new techniques into the regional monitoring programs.

o $17,100 in subcontract to UC San Diego. The contract covers 2 months ($10,732 – salary and benefits), $300 in project costs, and $6,068 in indirect costs.

B. Matching Funds • Year 1 (2016-17)

o $31,377 for 1 month of Burton’s time (salary, benefits, and indirect) for coordinating sample processing at SIO and assisting with data analysis and interim report preparation

o $17,500 in ship and staff time ($3,500/day) from Orange County Sanitation District for sample collection in year 1

o $29,356 in ship and staff time, plus purchase of sampling gear from Los Angeles County Sanitation District for sample collection in year 1

o $17,500 in ship and staff time ($3,500/day) from City of San Diego for sample collection in year 1

• Year 2 (2017-18) o $34,325 for 1 month of Burton’s time (salary, benefits, and indirect) for

assisting with data analysis and report/manuscript preparation

ANTICIPATED BENEFITS

In addition to advancing the science and application of environmental metabarcoding, this project targets benefits for three end-user communities, as indicated by the attached letters of support.

The first beneficiary is the ocean discharge community, consisting primarily of the large wastewater treatment agencies (i.e. City of San Diego, Orange County Sanitation District, City

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of Los Angeles Bureau of Sanitation, and Los Angeles County Sanitation Districts). These agencies are required to demonstrate that their discharges do not adversely affect marine communities. Historically, these agencies have relied on assessment of benthic communities as the main assessment endpoint. However, as discharge practices affect the composition of their effluent (e.g. contaminants of emerging concern) and the behavior of the discharge plumes (e.g. changing density due to increase reclamation) they are being asked to consider potential effects on pelagic communities. Ichthyoplankton assessment will likely become an important part of their ability to answer emerging questions about environmental effects of their discharges. The dischargers recognized these points and they approached SCCWRP to help them develop an approach for sampling ichthyoplankton (this actually ended up being the genesis of this proposal and other similar work we are presently undertaking with our partners). The methods developed through this study would not only help institutionalize ichthyoplankton assessment, but can be applied to improve current practices for assessment of benthic infauna as well.

The second beneficiary is the Marine Monitoring Enterprise. This group is responsible for assessing the effectiveness of marine protected areas at achieving desired fisheries and environmental health goals. As discussed above, analysis of ichthyoplankton communities has the potential to provide important information about spawning, recruitment, and dispersal patterns. When paired with traditional assessments of adult fish communities, it will provide important linkages to understand relative effects of fishing pressures and ocean discharge on overall food web integrity. The methods developed through this study will provide critical tools to support these assessments.

The third beneficiary is major regional monitoring programs in the SCB (e.g. southern California Bight regional survey, Santa Monica Bay Restoration Commission), which (in part) encompasses the first two beneficiaries. These programs provide relatively unique abilities to holistically assess condition and trends of the SCB as a way of providing insight into the relative influence of natural cycles and patterns (e.g. upwelling effects, El Nino) and anthropogenic activities (e.g. fishing, discharges) on ocean health. There has been a long-standing desire to incorporate ichthyoplankton assessment into these programs (for many of the same reasons cited above). However, issues such as limitations on taxonomic capacity and accuracy, time necessary to produce results, and cost have limited the analysis of larval and egg composition. This project will directly address these issues by working with these two monitoring programs to produce operational approaches for application of metabarcoding and to test the ability to apply it in through routine assessments.

The ocean dischargers, the marine protected area community, and the regional monitoring programs have all expressed a desire to integrate molecular-based taxonomy into their programs and to expand the scope of their programs to include ichthyoplankton and have therefore expressed support for this project. SCCWRP and SCIO’s scientific reputation and relationship with the management community will facilitate incorporation of this new technology once our proposed demonstration of the concepts is completed

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COMMUNICATION OF RESULTS

The communication and outreach aspects of this project will have two target audiences: the management community and the scientific community. The technical staff of local (the POTWs) and federal (NOAA SWF) management agencies will be directly involved the project and they will be learning the new techniques for metabarcoding while helping us refine them for future application in their respective monitoring programs. Their direct involvement in the project will facilitate knowledge transfer and ultimate incorporation of the new methods into routine monitoring and assessment. We will engage with the upper level managers of these agencies – the decision makers who set their monitoring program agendas –as well as those from CalCOFI and the MPA Monitoring Enterprise, via the presentation of our final results at a meeting at SCCWRP. In addition, we will provide them copies of all reports and manuscripts produced by this work. SCCWRP has a long history of transitioning new technologies into our management partners. Based upon our history and the management community’s interest in metabarcoding technology, communication will be key to the technology transfer. We will be interacting with the wider scientific community by presenting the overall results and the comparison of morphological, individual barcoding, and metabarcoding-based identification methods at scientific conferences (e.g., CERF, ASLO) and via publication of manuscripts in peer reviewed journals.

In compliance with NOAA’s Directive on Data Management, all of the molecular data (sequences, primers used, assigned species names), as well as their accompanying spatial data, will be posted as publicly accessible and searchable in the Barcode of Life Database (BOLD) (http://www.boldsystems.org/) upon publication of project results. For analytical purposes, sample composition data measured from all three methods, sample location data (e.g., latitude, longitude, water depth), sample collection data (e.g., tow duration, sea conditions, crew identity), and any concurrently collected environmental data will managed in a relational database. Upon completion of the project and publication of the results, this database will be made available to USC Sea Grant.

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Los Angeles County Sanitation Districts. 2014b. Chapter 6 Invertebrate and Fish Trawls. In: Joint Water Pollution Control Plant biennial receiving water monitoring report 2012-2013. 27p. Los Angeles County Sanitation Districts, Ocean Monitoring and Research Group, Technical Services Department, Whittier, CA.

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Orange County Sanitation District. 2014a. Chapter 5 Macrobenthic invertebrate communities. In: Ocean Monitoring Annual Report Year 2012-2013. 38p. Orange County Sanitation District Marine Monitoring, Fountain Valley, CA.

Orange County Sanitation District. 2014b. Chapter 6 Trawl Communities and Organism Health. In: Ocean Monitoring Annual Report Year 2012-2013. 36p. Orange County Sanitation District Marine Monitoring, Fountain Valley, CA.

Pawlowski, J., and M. Holzmann. 2014. A plea for DNA barcoding of foraminifera. Journal of Foraminiferal Research, 44: 62-67.

Pondella, D., J. Williams, J. Claisse, R. Schaffner, K. Ritter, and K. Schiff. 2012. Southern California Bight 2008 Regional Monitoring Program: V. Rocky Reefs. 113p. Southern California Coastal Water Research Program, Costa Mesa, CA.

Ranasinghe, J.A., K.C. Schiff, C.A. Brantley, L.L. Lovell, D.B. Cadien, T.K. Mikel, R.G. Velarde, S. Holt, S.C. Johnson. 2012. Southern California Bight 2008 Regional Monitoring Program: VI. Benthic Macrofauna. 89p. Southern California Coastal Water Research Program, Costa Mesa, CA.

Shokralla, S., Gibson, J.F., Nikbakht, H., Janzen, D.H., Hallwachs, W. and Hajibabaei, M. 2014. Next-generation DNA barcoding: using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens. Molecular Ecology Resources, 14: 892-901.

Shokralla, S., Spall, J.L., Gibson, J.F. and Hajibabaei, M. 2012. Next-generation sequencing technologies for environmental DNA research. Molecular Ecology, 21: 1794-1805.

Stein, E.D., B.P. White, R.D. Mazor, J.K. Jackson, J.M. Battle, P.E. Miller, E.M. Pilgrim, B.W. Sweeney. 2014. Does DNA Barcoding Improve Performance of Traditional Stream Bioassessment Metrics? Freshwater Science. 33(1):302–311. DOI: 10.1086/674782

Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar. 2011; MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biological Evolution, 28: 2731-2739.

Teletchea, F. 2009. Molecular identification methods of fish species: reassessment and possible applications. Reviews in Fish Biology and Fisheries 19(3):265-293. doi 10.1007/s11160-009-9107-4

Valdez-Moreno, M., Vásquez-Yeomans, L., Elías-Gutiérrez, M., Ivanova, N. V., and Hebert, P. D. N. 2010. Using DNA barcodes to connect adults and early life stages of marine fishes from the Yucatan Peninsula, Mexico: potential in fisheries management. Marine and Freshwater Research, 61: 655–671

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Ward, R.D., T.S. Zemlak, B.H.Innes, P.R. Last, and P.D.N. Hebert. 2005. DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 360: 1847-1857.

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WORK SCHEDULE

Task Feb '16 Mar '16 Apr '16 May '16 Jun '16 Jul '16 Aug '16 Sep '16 Oct '16 Nov '16 Dec '16 Jan '17Organizational meeting Sampling SOP creation/refinementSample collectionMorphological ID of samplesIndividual specimen (Sanger) sequencingInterim Report

Feb '17 Mar '17 Apr '17 May '17 Jun '17 Jul '17 Aug '17 Sep '17 Oct '17 Nov '17 Dec '17 Jan '18Bulk sample (Nex Gen) sequencingAnalysis of resultsPreperation of final report and manuscriptsPresentation of results to management groups

Year 1

Year 2

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OMB Control No. 0648-0362Expiration Date 1/31/2018

SEA GRANT BUDGET FORM 90-4

GRANTEE: David J. Gillett GRANT/PROJECT NO.:

DURATION (months): 24

12 months 1 Yr.A. SALARIES AND WAGES: man-months

1. Senior PersonnelNo. of People

Amount of Effort Sea Grant Funds Matching Funds

a. (Co) Principal Investigator: 1 0.75 4,935b. Associates (Faculty or Staff): 1 0.30 3,572

Sub Total: 2 1.05 8,507 0

2. Other Personnela. Professionals:b. Research Associates:c. Res. Asst./Grad Students:d. Prof. School Students:e. Pre-Bachelor Student(s):f. Secretarial-Clerical:g. Technicians:h. Other: 1 0.30 881

Total Salaries and Wages: 3 1.35 9,388 0

B. FRINGE BENEFITS: 52.6% 4,938 0Total Personnel (A and B): 14,325 0

C. PERMANENT EQUIPMENT:

D. EXPENDABLE SUPPLIES AND EQUIPMENT: 2,000

E. TRAVEL:1. Domestic 3002. International

Total Travel: 300 0

F. PUBLICATION AND DOCUMENTATION COSTS:

G. OTHER COSTS:1 Meeting support 7202 UCSD subcontractor 27,908 31,3773 Orange County Sanitation District 17,5004 Los Angeles County Sanitation District 29,3565 City of San Diego 17,50067

Total Other Costs: 28,628 95,733

TOTAL DIRECT COST (A through G): 45,253 95,733

INDIRECT COST (86.94% on wages/benefits only): 2385.66667 12,454 0INDIRECT COST (Off campus % of $ ):

Total Indirect Cost: 12,454 0

TOTAL COSTS: 57,708 95,733

Year One

PRINCIPAL INVESTIGATOR: David J. Gillett

BRIEF TITLE: Molecular Identification of Ichthyoplankton

17


OMB Control No. 0648-0362

Expiration Date 1/31/2018SEA GRANT BUDGET FORM 90-4

GRANTEE: David J. Gillett GRANT/PROJECT NO.:

DURATION (months): 24

12 months 2 Yr.A. SALARIES AND WAGES: man-months

1. Senior PersonnelNo. of People

Amount of Effort Sea Grant Funds Matching Funds

a. (Co) Principal Investigator: 1 0.25 6,778b. Associates (Faculty or Staff): 1 1.00 3,066

Sub Total: 2 1.25 9,844 0

2. Other Personnela. Professionals:b. Research Associates:c. Res. Asst./Grad Students:d. Prof. School Students:e. Pre-Bachelor Student(s):f. Secretarial-Clerical:g. Technicians:h. Other: 1 0.20 604

Total Salaries and Wages: 3 1.45 10,448 0

B. FRINGE BENEFITS: 52.6% 5,496 0Total Personnel (A and B): 15,944 0


D. EXPENDABLE SUPPLIES AND EQUIPMENT:

E. TRAVEL:1. Domestic 3,2002. International

Total Travel: 3,200 0


G. OTHER COSTS:1 Meeting support 4002 UCSD subcontractor 17,100 34,32534567

Total Other Costs: 17,500 34,325


INDIRECT COST (86.94% on wages/benefits only): 1458.33333 13,861 0INDIRECT COST (Off campus % of $ ):

Total Indirect Cost: 13,861 0

TOTAL COSTS: 50,505 34,325

Year Two

BRIEF TITLE: Molecular Identification of Ichthyoplankton

PRINCIPAL INVESTIGATOR: David J. Gillett

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Curriculum Vitae

David James Gillett Ecologist Phone: (714) 755-3249 Southern California Coastal Water Research Project Fax: (714) 755-3229 Costa Mesa, California, 92626 Email: [email protected]

EDUCATION:

Ph.D., Marine Science, The College of William and Mary, Williamsburg, VA - 2010 M.S., Marine Biology, University of Charleston, Charleston, SC - 2003 B.S., Cum Laude, Marine Science, Eckerd College, St. Petersburg, FL - 1997

AREAS OF EXPERTISE:

Dr. Gillett is an ecologist who specializes in studying the influence of anthropogenic disturbances and habitat quality on the community structure and ecosystem functioning of marine, estuarine and freshwater benthic communities. His research has focused primarily on ecosystem-scale field studies and experiments, but also incorporates biogeochemical and toxicological information into ecological field data to better understand biotic structure-function relationships. Dr. Gillett is leading the efforts to develop a causal assessment/stressor ID framework for California’s freshwater and coastal habitats in order to better understand and characterize the sources of impacts to biotic resources in these systems. He is also involved with the development of habitat assessment tools using both traditional monitoring information and newly emerging, genetic/molecular-based information.

PROFESSIONAL EXPERIENCE:

Scientist, Southern California Coastal Water Research Project. Costa Mesa, CA. 2010-Present Graduate Research Fellow, Chesapeake Bay National Estuarine Research Reserve of Virginia. Gloucester Point, VA. 2006-2008 Graduate Fellow, Virginia Institute of Marine Science. Gloucester Point, VA. 2003-2006; 2008-2010 Research Assistant, South Carolina Department of Natural Resources. Charleston, SC. 1999-2003 Teaching Assistant, College of Charleston. Charleston, SC. 1998-1999

HONORS AND AWARDS:

National Estuarine Research Reserve Graduate Research Fellowship (2006-2008) Program Chair, Atlantic Estuarine Research Society (2006-2007) Best Student Poster Presentation, Atlantic Estuarine Research Society Spring Conference (2006)

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mailto:[email protected]


Outstanding Student Presentation, Estuarine Research Federation Conference (2005) Outstanding Graduate Student Award, University of Charleston (Spring 2003) Virginia Institute of Marine Science Graduate Fellowship (2003-2005)

PROFESSIONAL SOCIETIES:

Association for the Sciences of Limnology and Oceanography California Estuarine Research Society Coastal and Estuarine Research Federation Society of Environmental Chemistry and Toxicology Southern California Association of Marine Taxonomists

PUBLICATIONS:

Ode, P.R., A.C. Rehn, R.D. Mazor, K.C. Schiff, E.D. Stein, J.T. May, L.R. Brown, D.B. Herbst, D. Gillett, K. Lunde, and C.P. Hawkins. In press. Evaluating the adequacy of a reference site pool for the ecological assessment of streams in environmentally complex regions. Freshwater Science.

Mazor, R.D., A.C. Rehn, P.R. Ode, M. Engeln, K. Schiff, E. Stein, D. Gillett, D. Herbst, and C.P. Hawkins. In press. Bioassessment in complex environments: designing an index for consistent meaning in different settings. Freshwater Science.

Gillett, D.J., S.B. Weisberg, T. Grayson, A. Hamilton, V. Hansen, E.W. Leppo, M.C. Pelletier, A. Borja, D. Cadien, D. Dauer, R. Diaz, M. Dutch, J.L. Hyland, M. Kellog, P.F. Larsen, J.S. Levinton, R. Llansó, L.L. Lovell, P.A. Montagna, D. Pasko, C.A. Phillips, C. Rakocinski, J.A. Ranasinghe, D.M. Sanger, H. Teixeira, R.F., Van Dolah, R.G. Velarde, and K.I. Welch. 2015. Effect of ecological group classification schemes on performance of the AMBI benthic index in US coastal waters. Ecological Indicators 50:99-107.

Gillett, D.J., K.C. Schiff, D.J. Pondella II, J. Freiwald, J.E. Casselle, C. Shuman, and S.B. Weisberg. 2012. Comparing volunteer and professionally collected monitoring data from the rocky subtidal reefs of southern California, USA. Environmental Monitoring and Assessment 184:3239-3257.

Gillett, D.J. and L.C. Schaffner. 2009. Benthos of the York River. Journal of Coastal Research SI57:80-98. Gillett, D.J., A.F. Holland, and D.M. Sanger. 2007. On the ecology of oligochaetes: Variation in community composition and environmental characteristics of two South Carolina tidal creeks at monthly scales. Estuaries and Coasts 30:238-252. Gillett, D.J., A.F. Holland, and D.M. Sanger. 2005. Secondary production of a dominant oligochaete (Monopylephorus rubroniveus) in the tidal creeks of South Carolina and its relation to ecosystem characteristics. Limnology and Oceanography 50:566-577.

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CURRICULUM VITAE Ronald S. Burton Work Address: Marine Biology Research Division 0202 Scripps Institution of Oceanography University of California, San Diego La Jolla, CA 92093-0202 Phone: 858 822 5784 Fax: 858 534 7313 Email: [email protected] Education: Degrees: Stanford University B.S. Biological Sciences, 1976 (1972-1976) M.S. Biological Sciences, 1976 Stanford University Ph.D. Biological Sciences, 1981 (1977-1981) Current Position: Professor of Marine Biology, Scripps Institution of Oceanography (SIO), 1995-present. Head, Biology Section, SIO/UCSD, 2015-present Professional Experience: Director, Marine Biology Research Division, SIO, UCSD, 2000-2012. Head, Biology Section, SIO, UCSD, 2004-2012. Associate Professor, Marine Biology Research Division, SIO, UCSD 1992-1995. Associate Professor of Biology, University of Houston, 1987-1992. Assistant Professor of Biology, University of Pennsylvania, 1981-1987. Professional Societies: AAAS, ASLO, Society for the Study of Evolution Honors and Awards: 1976 Phi Beta Kappa, B.S. Degree with Departmental Distinction, Stanford University. 1990 Outstanding Educator (Teaching Award), University of Houston. 2003 Presidential Lecture Series, Florida International University. 2005 Vice Presidential Symposium, American Society of Naturalists. 2007. Mentor Recognition Award, UCSD 2011 Plenary Speaker: Ocean Acidification Principal Investigators’ Meeting, Woods Hole. 2012 University of British Columbia, Zoology Annual Symposium, Student Invited Speaker. 2013 Distinguished Speaker: Smithsonian Tropical Research Institute, Panama. 2013 Elected Fellow: American Association for the Advancement of Science (AAAS) Professional Activities and Outreach: • Advisory Panel: White Abalone (endangered species) Recovery Team, National Marine Fisheries Service, 2002- 2008. • Associate Editor: EVOLUTION (1999- 2001), J. Exp. Mar. Biol. Ecol. (1995-2005). • Editorial Advisor: Marine Ecology - Progress Series (1991- present). • NSF Panel Member, nine panels in five different programs (1989-2015). • Scientific advisor/partner with the Ocean Discovery Institute of San Diego since 2007. • I developed a hands-on molecular biology exercise that is now delivered by ODI staff to over 500 fifth-grade students in underserved San Diego schools each year. • Public lecture at the Birch Aquarium at Scripps, March 2007. Shown on university TV throughout the country (on demand at http://www.uctv.tv/ with over 150,000 views) Selected Publications (of 100 total publications): Burton, R. S. and B.-N. Lee 1994. Nuclear and mitochondrial gene geneologies and allozyme

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polymorphism across a major phylogeographic break in the copepod Tigriopus californicus. Proc. Nat. Acad. Sci. USA. 91:5197-5201. Burton, R.S. 1998. Intraspecific phylogeography across the Point Conception biogeographic boundary. Evolution 52:734-745. Hellberg, M.E., Burton, R.S., Neigel, J.E., Palumbi, S.R. 2002. Genetic assessment of connectivity among marine populations. Bull. Mar. Sci. 70(1) Suppl:273-290. Flowers, J.M., S.C. Schroeter, and R.S. Burton. 2002. The recruitment sweepstakes has many winners: genetic evidence from purple sea urchins. Evolution 56:1445-1453. Gruenthal, K.M. and R.S. Burton. 2005. Genetic diversity and species identification in the endangered white abalone (Haliotis sorenseni). Conservation Genetics 6:929-939. Ellison, C.K. and R.S. Burton. 2008. Interpopulation hybrid breakdown maps to the mitochondrial genome. Evolution 62:631-638. doi:10.1111/j.1558-5646.2007.00305.x Burton, R. S. 2009. Molecular markers, natural history and conservation of marine animals. BioScience 59:831-840. Barreto, F. S., G.W. Moy and R.S. Burton. 2011. Interpopulation patterns of divergence and selection across the transcriptome of the copepod Tigriopus californicus. Molecular Ecology 20:560-572. Gleason, L.U. and R.S. Burton. 2012. High-throughput molecular identification of fish eggs using multiplex suspension bead arrays. Molecular Ecology Resources 12: 57–66. Burton, R. S. and F.S. Barreto 2012. A disproportionate role for mtDNA in Dobzhansky- Muller incompatibilities? Molecular Ecology 21: 4942–4957. Schoville S.D., F.S. Barreto, G.W. Moy, A. Wolff and R.S. Burton 2012. Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus. BMC Evolutionary Biology 2012, 12:170 Barreto, F.S., and R.S. Burton. 2013. Evidence for compensatory evolution of ribosomal proteins in response to rapid divergence of mitochondrial rRNA. Molecular Biology and Evolution 30:310-314. DOI: 10.1093/molbev/mss228 Barreto, F. S., and R.S. Burton. 2013. Elevated oxidative damage is correlated with reduced fitness in interpopulation hybrids of a marine copepod. Proceedings of the Royal Society B 280: 20131521. http://dx.doi.org/10.1098/rspb.2013.1521 Fisch, K.M., J. A. Ivy, R. S. Burton and B. May 2013. Evaluating the performance of captive breeding techniques for conservation hatcheries: A case study of the delta smelt captive breeding program. Journal of Heredity 104: 92-104. Burton, R.S., R. J. Pereira and F. S. Barreto. 2013. Cytonuclear genomic interactions and hybrid breakdown. Ann. Rev. Ecol. Evol. Syst. 44:281-302. DOI: 10.1146/annurevecolsys- 110512-135758 Pereira, R.J., F. S. Barreto and R.S. Burton. 2014. Ecological novelty by hybridization: experimental evidence for increased thermal tolerance by transgressive segregation in Tigriopus californicus Evolution 68:204-215. doi:10.1111/evo.12254. Barreto, F.S., R.J. Pereira, and R.S. Burton 2015. Hybrid dysfunction and physiological compensation in gene expression. Molecular Biology and Evolution 32: 613- 622 doi:10.1093/molbev/msu321. Barreto, F.S., S.D. Schoville and R.S. Burton 2015. Reverse genetics in the tidepool: Knockdown of target gene expression via RNA interference in the copepod Tigriopus californicus. Molecular Ecology Resources 15: 868–879

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Eric D. Stein Principal Scientist Phone: (714) 755-3233 Southern California Coastal Water Research Project Fax: (714) 755-3299 Costa Mesa, California, 92626 Email: [email protected] PROFESSIONAL PREPARATION: D.Env. Environmental Science and Engineering, University of California, Los Angeles, 1995 M.Ed. Science Education, University of California, Los Angeles, 1988 B.S. Biology, University of California, Los Angeles, 1987 PROFESSSIONAL EXPERIENCE: 2002 – Present Principal Scientist - Southern California Coastal Water Research Project 1998 – 2002 Adjunct Associate Professor - California State University, Los Angeles, Department of Geography and Urban Analysis 1998 – 2002 Principal Ecologist, Associate Principal - PCR Services Corporation 1993 – 1998 Biologist, Senior Project Manager - U.S Army Corps of Engineers, Los Angeles District SELECTED RELATED PEER-REVIEWED PUBLICATIONS: Stein, E.D., M.C. Martinez, S. Stiles, P.E. Miller, and E.V. Zakharov. 2014. Is DNA Barcoding Actually Cheaper

and Faster Than Traditional Morphological Methods: Results from a Survey of Freshwater Bioassessment Efforts in the United States? PLoS ONE. 9(4): e95525. doi:10.1371/journal.pone.0095525

Mazor, R.D., E.D. Stein, P.R, Ode, and K Schiff. 2014. Integrating intermittent streams into watershed assessments: Applicability of an index of biotic integrity. Freshwater Science. DOI: 10.1086/675683

Stein, E.D., B.P. White, R.D. Mazor, J.K. Jackson, J.M. Battle, P.E. Miller, E.M. Pilgrim, B.W. Sweeney. 2014. Does DNA Barcoding Improve Performance of Traditional Stream Bioassessment Metrics? Freshwater Science. 33(1):302–311. DOI: 10.1086/674782

Jackson, J.K, J.M. Battle, B.P. White, E.M. Pilgrim, E.D. Stein, P.E. Miller, and B.W. Sweeney. 2014. Cryptic biodiversity in streams - a comparison of macroinvertebrate communities based on morphological and DNA barcode identifications. Freshwater Science. 33(1):312–324. DOI: 10.1086/675225.

White, B.P., E.M. Pilgrim, L.M. Boykin, E.D. Stein, R.D. Mazor. 2014. Comparison of four species-delimitation methods applied to a DNA barcode data set of insect larvae for use in routine bioassessment. Freshwater Science. 33(1):338–348. DOI: 10.1086/674982

Lackey, L.G., and E.D. Stein. 2014. Selecting the optimum plot size for a California design-based stream and wetland mapping program. Environmental Monitoring and Assessment. 186:2599–2608. DOI 10.1007/s10661-013-3563-y

Fetscher, A.E., R. Stancheva, J.P. Kociolek, R.G. Sheath, E.D. Stein, R.D. Mazor, P.R. Ode, and L.B. Busse. 2014. Development and comparison of stream indices of biotic integrity using diatoms vs. non-diatom algae vs. a combination. Journal of Applied Phycology. 26(1):433-450. DOI 10.1007/s10811-013-0088-2

Stein, E.D. 2013. Using Regional Stormwater Monitoring Programs to Provide Reference Data for Wetland Mitigation Performance Evaluation. National Wetlands Newsletter. 35(4):13-14

Stein, E.D., M.R. Cover, A.E. Fetscher, C. O’Reilly, R. Guardado, and C.W. Solek. 2013. Reach-scale geomorphic and biological effects of localized stream bank armoring. Journal of the American Water Resources Association. 49(4):780-792. DOI: 10.1111/jawr.12035

Lackey, L.G. and E.D. Stein. 2013. Evaluation of design-based sampling options for monitoring stream and wetland extent and distribution in California. Wetlands. 33:717–725. DOI 10.1007/s13157-013-0429-6

Stein, E.D., J.S. Brown, T.S. Hogue, M.P. Burke, and A. Kinoshita. 2012. Storm water contaminant loading following southern California wildfires. Environmental Toxicology and Chemistry. 31(11):2625-2638.

Hawley, R.J., B.P. Bledsoe, E.D. Stein, and B.E. Haines. 2012. Channel Evolution Model of Response to Urbanization in Southern California. Journal of the American Water Resources Association 48(4):722—744. 2

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Bledsoe, B.P., E.D. Stein, R.J. Hawley, and D.B. Booth. 2012. Framework and tool for rapid assessment of stream susceptibility to hydromodification. Journal of the American Water Resources Association 48(4):788-808.

Solek, C.W., M.A. Sutula, E.D. Stein, C. Roberts, R. Clark, K. O’Connor, and K.J. Ritter. 2012. Determining the Health of California’s Coastal Salt Marshes Using Rapid Assessment. Wetland Science and Practice 29(1):8-28

Dark, S., E.D. Stein, D. Bram, and J. Osuna. 2012. Historical Ecology as a Living Resource for Informing Urban Wetland Restoration. Urban Coast. 3(1):54-60.

Solek, C.W., E.D. Stein, and M.A. Sutula. 2011. Demonstration of an Integrated Watershed Assessment Using a Three-tiered Assessment Framework. Wetlands Ecology and Management. 19(5):459-474

Stein, E.D. and D.B. Cadien. 2009. Ecosystem Response to Regulatory and Management Actions: the Southern California Experience in Long-term Monitoring. Marine Pollution Bulletin 59:91-100.

Lyon, G.S. and E.D. Stein. 2009. How Effective Has the Clean Water Act Been at Reducing Pollutant Mass Emissions to the Southern California Bight over the Past 35 Years? Environmental Monitoring and Assessment 154(1):413-426.

Stein, E.D. and V.K. Yoon. 2008. Dry Weather Flow Contribution of Metals, Nutrients, and Solids from Natural Catchments. Water Air and Soil Pollution 190:183-195.

Ackerman, D. and E.D. Stein. 2008. Evaluating the Effectiveness of Best Management Practices Using Dynamic Modeling. Journal of Environmental Engineering 134(8):628-639.

Stein, E.D. and B. Bernstein. 2008. Integrating Probabilistic and Targeted Compliance Monitoring for Comprehensive Watershed Assessment. Environmental Monitoring and Assessment 144:117-129.

SYNERGISTIC ACTIVITIES: - Associate Editor, Wetlands: Journal of the Society of Wetland Scientists (2009-Present) - Southern California Wetlands Recovery Program Science Advisory Panel (1999-Present) - Santa Monica Bay Restoration Commission Technical Advisory Committee (2013-present) - USEPA, Environmental Law Institute (ELI), Nature Conservancy (TNC) - watershed approach (2012 – present). - California Wetland Monitoring Workgroup, Co-chair (2008 – present) - California Healthy Streams Partnership (2011 – present) - USDA-NRCS National Easement Assessment Project – technical team (2011-present) - U.S. Army Corps of Engineers Workgroup: Quantifying Significance of Aquatic Ecosystems (2010) - NOAA Water Quality Synthesis & Assessment (SAM) Technical Advisory Committee (2006-2009) - Society of Wetland Scientists, Western Chapter President (2006-2010) - Society of Wetland Scientists, Wetland Concerns Committee (2006- present) - National Wetlands Awards, Selection Committee (2007, 2008, 2012) - California State Stream and Wetland Protection Policy Science Advisory Team (2009 – present) - US Army Corps of Engineers – National Workgroup on Arid Stream Assessment (2007) - NOAA National Estuary Eutrophication Workgroup, S. Pacific Coast Coordinator (2007) - California State Stream and Wetland Protection Policy Science Advisory Team (2009-present) RECENT COLLABORATORS: B. Bledsoe (Colorado State University), Erik Pilgrim (USEPA), Mehrdad Hajibabaei (University of Ontario), D.

Booth (UCSB), D. Carlisle (USGS), R. Ambrose (UCLA), G.M. Kondolf (UCB), M. Cover (CSU Stanislaus), B. Jones (USC), T. Longcore (USC), S. Dark (CSU Northridge), T. Hogue (UCLA), J. Warrick (USGS), J.H. Dorsey (Loyola Marymount University)

THESIS ADVISORY & POSTGRADUATE-SCHOLAR SPONSOR: S. Lopez (UCLA), L. Lackey (UCLA), L. Fong (UCLA), M. Schliebe (CSULB), B. White (CSU Fullerton), I. Irvine

(UC Irvine), S. Eberhart (Colorado State University), B. Hawley (Colorado State University), B. Haines (Colorado State University), V. Yoon (UCLA), S. Lee (UCLA), D. Cummings (CSULA), L. Morales (CSULA)

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SUMMARY PROPOSAL FORM

PROJECT TITLE: Molecular Identification of Larval Nekton Inhabiting the Waters of the Southern California Bight

OBJECTIVE: The primary goals of this research are to determine if ichthyoplankton monitoring data derived from metabarcoding methods produces comparable or better quality data to traditional morphological methods. If so, we will help develop basic operating procedures for incorporation of these molecular methods into monitoring programs of the water quality and resource management community across the Southern California Bight.

METHODOLOGY: Ichthyoplankton samples will be collected from across the Southern California Bight by field crews from the region’s large publicly owned treatment works (POTW) (i.e., Los Angeles County Sanitation District, Orange County Sanitation District, and the City of San Diego). Samples will be labeled and placed in 95% ethanol. From ethanol preserved specimens, fish eggs and larvae will be sorted from detritus and zooplankton, enumerated, and identified by taxonomists at the NOAA Southwest Fisheries Science Center. After morphological identification, individual samples will be homogenized and the DNA will be extracted using standard protocols. CO1 and 16S DNA will be sequenced from the bulk extraction using Illumina Mi-Seq high throughput sequencer (i.e., metabarcoding). Produced forward and reverse sequences (of approximately 200-300 bp) will be aligned and verified for size and structure, to insure purity. Valid sequences will be assigned names based upon matches from custom DNA libraries of Southern California nekton, as well the larger public genomic databases (e.g., BOLD, GENBANK).

Additionally, ~ 800 individuals will be removed from a subset of samples for individual DNA barcoding. Standard DNA extraction and amplification protocols will be used and PCR amplicons will be sequenced bi-directionally by Sanger sequencing with a capillary DNA analyzer. Valid DNA sequences will then be assigned names based upon matches from custom DNA libraries of Southern California nekton, as well the larger public genomic databases (e.g., BOLD, GENBANK). Taxonomic assignments from the morphological, Sanger sequenced DNA, and metabarcoded DNA will be compared for taxonomic composition and similarity.

RATIONALE:

Understanding the larval ichthyoplankton population dynamics along the shelf addresses several management questions/needs including general adult population structure, larval production/success of marine protected areas, and early detection of invasive taxa before a population of adults becomes established. A significant obstacle to the regular monitoring of ichthyoplankton is the difficulty in identifying the individuals in a given sample. Traditional larval fish identification done via microscopy of preserved specimens requires time and a high degree of taxonomic specialization. Molecular taxonomic methods, such as DNA barcoding, offer a potential solution to improve the efficiency and utility of ichthyoplankton assessment. However, despite its utility in improving the identification of problematic taxa, single organism

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barcoding is not practical for application in a large-scale monitoring programs given the time needed to process hundreds of individuals in dozens of samples. However, with a reliable DNA reference library, metabarcoding – the extraction and sequencing of all the DNA in sample at one time – represents a promising way to create an ichthyoplankton monitoring program across the region based upon molecular identification of samples.

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UNITED STATES DEPARTMENT OF COMMERCE National Oceanic and Atmospheric Administration NATIONAL MARINE FISHERIES SERVICE Sout hw est Fisher ies Science Cent er 8604 La Jo lla Shores Dr ive La Jo lla, CA 92037-1508

July 9, 2015 Ms. Ruth Dudas, Contract and Grants Coordinator USC Sea Grant 3616 Trousdale Pkwy, AHF 253 Los Angeles, CA 90089-0373 RE: Letter of Support for “Molecular Identification of Larval Nekton Inhabiting the Waters of the Southern California Bight” Project Dear Ms. Ruth Dudas: I am pleased to provide this letter supporting the Southern California Coastal Water Research Project’s (SCCWRP) grant application for “Molecular Identification of Larval Nekton Inhabiting the Waters of the Southern California Bight”. The work proposed in this grant would develop a new approach and application to sample processing that will allow us to increase the geographic scope and utility of an ecologically important component of the coastal ocean ecosystem across the Southern California Bight (SCB). Monitoring of larval fishes from nekton samples in California over the past 70 years has provided valuable information regarding the responses of fish populations and communities to perturbation. However, identification of larvae based on morphological characteristics requires extensive expertise and thus limits the capacity of many programs to conduct this sampling. Although advances in molecular technology to identify nekton have been made in many individual laboratories worldwide, these new techniques have yet to be adopted by the broader management community in the United States. We are excited to work with SCCWRP to refine tools and sampling protocols that will facilitate the integration of emerging technologies in next-generation DNA sequencing and taxonomic identification into existing field programs. Beyond our conceptual support for this project, we intend to provide in-kind support towards this project via partial support of a post-doctoral investigator focusing on the bioinformatics and sequencing aspects of this work, a laboratory technician focusing the processing/sorting of samples, and taxonomic expertise in the identification of the sorted larvae. Thank you for your consideration and I appreciate your favorable consideration of SCCWRP’s application to fund this project. Sincerely, Andrew Thompson Ph.D., Research Fisheries Biologist, Ichthyoplankton Ecology

OMB Control No. 0648-0362 Expiration Date 1/31/2018

SEA GRANT BUDGET FORM 90-4 GRANTEE: The Regents of the University of California GRANT/PROJECT NO.:

BRIEF TITLE: MOLECULAR IDENTIFICATION OF LARVAL NEKTON INHABITING DURATION (months): 12 2/1/2016 - 1/31/2017

PRINCIPAL INVESTIGATOR: Burton 12 months 1 Yr.

A. SALARIES AND WAGES: man-m

1. Senior Personnel No. of People Amount of Effort Sea Grant Funds Matching Funds

a. (Co) Principal Investigator: 1 1.0 0 20,243 b. Associates (Faculty or Staff):

Sub Total: 1 1.0 0 20,243

2. Other Personnel

a. Professionals:

b. Research Associates:

c. Res. Asst./Grad Students:

d. Prof. School Students:

e. Pre-Bachelor Student(s):

f. Secretarial-Clerical:

g. Technicians: 1 3.0 15,555 0 h. Other:

Total Salaries and Wages: 2 4.0 15,555 20,243

B. FRINGE BENEFITS: 0.0% 0 0 Total Personnel (A and B): 15,555 20,243


D. EXPENDABLE SUPPLIES AND EQUIPMENT: 2,000

E. TRAVEL:

1. Domestic

2. International

Total Travel: 0 0


G. OTHER COSTS:

Project specific costs 450

2

3

4

5

6

7

Total Other Costs: 450 0


INDIRECT COST (On campus 55% ): 37.5 9,903 11,134 Total Indirect Cost: 9,903 11,134

TOTAL COSTS: 27,908 31,377 OMB Control No. 0648-0362

Expiration Date 1/31/2018 SEA GRANT BUDGET FORM 90-4

GRANTEE: The Regents of the University of California GRANT/PROJECT NO.:

BRIEF TITLE: MOLECULAR IDENTIFICATION OF LARVAL NEKTON INHABITING DURATION (months): 12 2/1/2017 - 1/31/2018

PRINCIPAL INVESTIGATOR: Burton 12 months 1 Yr.

A. SALARIES AND WAGES: man-m

1. Senior Personnel No. of People Amount of Effort Sea Grant Funds Matching Funds

a. (Co) Principal Investigator: 1 1.0 0 22,145 b. Associates (Faculty or Staff):

Sub Total: 1 1.0 0 22,145

2. Other Personnel

a. Professionals:

b. Research Associates:

c. Res. Asst./Grad Students:

d. Prof. School Students:

e. Pre-Bachelor Student(s):

f. Secretarial-Clerical:

g. Technicians: 1 2.0 10,732 0 h. Other:

Total Salaries and Wages: 2 3.0 10,732 22,145

B. FRINGE BENEFITS: 0% 0 0 Total Personnel (A and B): 10,732 22,145


D. EXPENDABLE SUPPLIES AND EQUIPMENT:

E. TRAVEL:

1. Domestic

2. International

Total Travel: 0 0


G. OTHER COSTS:

Project specific costs 300

2

3

4

5

6

7

Total Other Costs: 300 0


INDIRECT COST (On campus 55%): 65% 6,068 12,180 Total Indirect Cost: 6,068 12,180

TOTAL COSTS: 17,100 34,325 Scripps PI Burton will provide 1.0 month of effort each year serving as matching funds for the project. Funds requested from USC Sea Grant are for 5 months of technician time (SRA I, 3 months in year 1 and 2 months in year 2). Salary recharge rates are calculated for actual productive time only (except for nonfaculty academic sick leave). The rates include components for employee benefits, provisions for applicable merit increases and range adjustments in accordance with University policy, except postdoc rates which do not include components for downtime, so those rates are calculated for all working hours. Staff overtime or remote location allowance may be required in order to meet project objectives, and separate rates are used in those cases.

Supplies are budgeted at $2,000 in year 1 based on Burton lab experience in obtaining ~800 sequences. Project specific costs that include research telephones, tolls, voice and data communication charges, photocopying, faxing and postage are requested. Supply and expense items, categorized as project specific, and computer and networking services are for expenses that specifically benefit this project and are reasonable and necessary for the performance of this project.

Documents

Gillett and Burton – Molecular Identification of ...the individuals in a given sample. Traditional larval fish identification is done via microscopy of preserved specimens. This