Eval Of Reprod Of Steelhead proposal 200305000

FY07-09 proposal 200305000

Section 1. Administrative

Proposal title	Eval Of Reprod Of Steelhead
Proposal ID	200305000
Organization	University of Washington
Short description	Evaluate the individual reproductive success of naturally spawning hatchery steelhead relative to that of native wild steelhead using genetic tools and methods.
Information transfer	Published in a Journal, reported at professional meetings
Proposal contact person or principal investigator

Contacts

Contact	Organization	Email
Form submitter
Todd Seamons	University of Washington	seamonst@u.washington.edu
All assigned contacts
Tom Quinn	University of Washington	tquinn@u.washington.edu
Todd Seamons	University of Washington	seamonst@u.washington.edu

Section 2. Locations

Province / subbasin: Mainstem/Systemwide / Systemwide

Latitude	Longitude	Waterbody	Description
463330N	1233554W	Stream	Forks Creek, Willapa River

Section 3. Focal species

primary: Other Anadromous
Additional: Steelhead

Section 4. Past accomplishments

Year	Accomplishments
2005	Began sampling for the year
2004	Completely eliminated remaining backlog of samples. Generated age data from scales. Successfully continued sampling. Performed parentage analysis for adult offspring. Produced and submitted two manuscripts for publication.
2003	Built MS Access Database. Transfered data to database. Eliminated backlog for adult samples. Eliminated part of backlog for smolt samples. Successfully continued sampling. Produced manuscript on artificial selection in the hatchery.

Section 5. Relationships to other projects

Funding source	Related ID	Related title	Relationship
BPA	200306300	Repro Success Abernathy Creek	See attached narrative
BPA	200306000	Eval Repro Success Snake Rvr C	See attached narrative
BPA	200305400	Repro of Steelhead In Hood Riv	See attached narrative

Section 6. Biological objectives

Biological objectives	Full description	Associated subbasin plan	Strategy
Reproductive success of wild/hatchery steelhead	1) quantify the breeding pattern (i.e., selection of fish for spawning) in a production hatchery, (2) directly determine the realized RS of the individual hatchery fish by DNA parentage analysis of the adults returning over 3-4 complete generations (3) directly measure the effective population size (Ne) of the hatchery population over multiple generations (4) determine whether these was any loss of genetic diversity in the hatchery population over two generations, (5) determine the realized heritability of key life history traits for hatchery fish released to the sea, (6) determine the realized reproductive success of wild and hatchery-wild hybrid parents spawning naturally, and compare these levels to those of the first generation of hatchery steelhead spawning naturally.	None	[Strategy left blank]

Section 7. Work elements (coming back to this)

Work element name	Work element title	Description	Start date	End date	Est budget
Manage and Administer Projects	Prepare for contract renewal	All PIs and the postdoctoral research associate will meet on a regular basis to discuss progress and further work. As indicated below (“Key personnel”), our project includes three faculty members with distinct but overlapping areas of expertise. Thomas Quinn is responsible for overall project management, Lorenz Hauser and Kerry Naish are responsible for the molecular genetic lab, with special emphasis of assignment tests and parentage (Hauser) and quantitative genetics (Naish). Day to day project management and supervision of lab work will be the responsibility of the post-doc, Todd Seamons, who will also take the lead on papers on reproductive success of naturally spawning fish. In addition, we will continue to collaborate closely with staff at the WDFW Forks Creek hatchery to ensure appropriate sampling.	10/1/2009	11/30/2009	$8,366
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Produce Annual Report	Reproductive Success of Wild & Hatchery Steelhead Annual Report	[Work Element Description Not Entered]	12/1/2007	11/30/2009	$14,625
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Produce Status Report	Progress reports	[Work Element Description Not Entered]	12/1/2007	11/30/2009	$7,895
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Produce/Submit Scientific Findings Report	Prepare manuscripts	Reports detailing our findings will be submitted regularly to the BPA. We will also publish our results in the peer-reviewed literature.	12/1/2007	11/30/2009	$196,628
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Analyze/Interpret Data	Analyze and interpret data	1. Quantify the breeding pattern (i.e., selection of fish for spawning) in a production hatchery, We will continue to use the methods implemented previously and described by McLean et al. (2005) to examine the effects of hatchery selection practices on life history traits in the hatchery population. We will determine whether these practices deviate from methods intended to ensure “selective neutrality” as advocated by Campton (2004). (2) Directly determine the realized RS of the individual hatchery fish by DNA parentage analysis of the adults returning over four complete generations Parentage will be assigned through exclusion using the software package WhichParents (version 1.0 alpha; Bodega Marine Lab, UC Davis). Putative parent-offspring groups will be further analyzed by direct genotype comparisons. One parent will be assigned if an allele matches with the offspring at each locus; two parents will assigned if their genotypes together could have produced the offspring genotype. Subsequent statistical analyses do not include offspring that are not assigned at least one parent. The reproductive success of hatchery parents will be estimated over four complete generations to determine whether spawning protocols in hatcheries result in unequal family sizes and family-biased selection in hatchery populations. We assume that fish returning to the hatchery are representative of the entire population and that we provide an accurate assessment of family-specific survival. The data collected to date support this assumption, because we have been very successful in assigning offspring to parents. (3) Directly measure the effective population size (Ne) of the hatchery population over multiple generations and (4) determine whether these was any loss of genetic diversity in the hatchery population over two generations, Our detailed analysis of variance in RS among individuals amounts to a direct measurement of the current (variance) effective population size (Ne) of the population. Our microsatellite assays will also permit indirect estimates of current Ne based on temporal variance in allele frequencies (Waples 1989; Waples 1990a, b; Waples and Teel 1990) and linkage disequilibrium (Bartley et al. 1992), and we will be able to compare our direct observations of Ne with these commonly used (but rarely validated) indirect approaches. Ne is a parameter of fundamental importance in population and conservation biology; hence these comparisons will be of significant interest and will provide a measure of the rate of loss of genetic diversity in a hatchery population, and the potential for hatchery populations to reduce the effective size of wild populations should they interbreed. (5) Determine the realized reproductive success of wild, hatchery and hatchery-wild hybrid parents spawning naturally, and compare these levels to those of the first generation of hatchery steelhead spawning naturally. Observed and expected heterozygosity, probability tests of Hardy-Weinberg equilibrium, tests of linkage disequilibrium, and genetic differentiation estimates between the hatchery and wild populations will be calculated using the GENEPOP (version 3.0) software package (Raymond and Rousset 1995). This information will be used to assign returning adult offspring in the third and fourth generations, and smolts, to their population of origin following methods described in Hauser et al. (in review). Parentage analyses will be performed as described above. These data will be used to track the fate of hatchery-origin fish and their hybrids in the wild over at least four generations since their release and speed and extent of recovery following the assumed cessation of that release. We will also analyze variability in annual survival of hatchery and wild fish, the rate of introgression, the population viability of hatchery-wild complexes and ultimately, assess the risk that a steelhead production hatchery poses to wild populations.	12/1/2007	11/30/2009	$151,825
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Collect/Generate/Validate Field and Lab Data	Collect and process new samples	Sampling Methods The hatchery has spawned steelhead since winter 1995-96 (Brood Year 1996 by our designation; Table 1) and we have obtained samples (length, weight, scales for age determination and fin clip for DNA, and egg size and fecundity from females) from almost all hatchery fish. A weir across the creek guides salmon and steelhead into a deep concrete channel filled with water from the hatchery. This weir functions well except under very high water conditions. Hatchery staff remove steelhead from the channel, which are examined each week. The fish are allowed to remain and mature, spawned, sacrificed, or allowed upriver to spawn in the river, depending on whether they are wild or hatchery, fully mature or not, and whether the hatchery’s capacity has been reached. The hatchery rears steelhead for one year before releasing them to sea and most of the steelhead spend two full years at sea before returning (inferred from scale examination, size frequency analysis, and DNA parentage). Thus in winter 1999-2001 most of the hatchery-produced adults returning to Forks Creek were the progeny of adults that we sampled in 1996 and 1997 (i.e., the first generation), and in winter 2001-2004, we sampled the F2 (grandchildren). In winter 2005, we initiated sampling of the F3 (Figure 1). We allow hatchery staff to select and spawn the fish according to their standard practice or decision of the moment, and then sample the fish. Fecundity of females is estimated by weighing the entire mass of eggs, and weighing and counting a subsample of eggs. We can thus determine how much of the variation in RS can be attributed to fecundity, body size or spawning date. In addition to our sampling of hatchery fish and the patterns of breeding there, we have also systematically sampled the naturally spawning fish since 1996. All fish allowed upriver were identified to origin in 1996 and 1997 (all hatchery fish have the adipose fin removed). Subsequently, only wild-origin fish have been allowed upstream. The sex, and length of all fish sampled since 1996 are recorded, scales are removed for age determination and fin tissue is collected for DNA analysis. We already know from parentage and population assignment that the late returning wild fish are captured at a low efficiency at the trap, because spring floods in April make the weir passable without human intervention. This incomplete sampling of late returning fish complicates parental assignments and thus the estimation of RS, and improvement of the trap is one of our top priorities for the forthcoming funding cycle. We have also operated a smolt trap from mid-late April to early June each year. This fan trap catches a very large fraction of the smolts (essentially all, except on high water events when a panel must be removed to prevent damage). All steelhead smolts are measured and weighted and a fin clip removed, and these data are recorded with the date of capture. In addition, the trap catches a number of steelhead kelts and this augments our sampling of upstream migrants. Genetic Methods We have determined the parentage (and other forms of kinship) for steelhead in our study by genotyping microsatellite loci. The attributes of microsatellites as genetic markers have been reviewed extensively (e.g., Wright and Bentzen 1994; O’Reilly and Wright 1995; McConnell and Wright 1997). Briefly, they consist of 1-5 base pair (bp) repeats that form tandem arrays < 300 bp in length, and exhibit high levels of allelic variation in repeat number. Polymorphism exhibited by specific microsatellites is readily detected by amplification of the microsatellite through the use of oligonucleotide primers specific to the non-repetitive regions that flank the repeat array, in combination with the polymerase chain reaction (PCR). Allelic variation is scored by gel electrophoresis of the PCR products, most commonly on automated systems facilitating running and scoring of genotypes. Microsatellites have been widely implemented in kinship analyses (reviewed in Hughes 1998; Marshall et al. 1998) because the large numbers of alleles and high heterozygosities provide the power for discriminating parent-offspring combinations from unrelated individuals. Power increases dramatically with increasing expected heterozygosity (HE) (Blouin et al. 1996). For loci with HE ? 80% the average exclusion probability is > 0.999 for nine unlinked loci. We have sampled almost all of the Forks Creek hatchery spawners and so anticipate a high success rate in identifying offspring. Genomic DNA extractions and PCRs were performed as outlined in McLean et al. (2003, 2004). A 96-well capillary system Molecular Dynamics MegaBACE 1000 (Amersham Scientific) was used to size fractionate the eight microsatellite loci examined. Electropherograms were analyzed using Genetic Profiler software version 1.1 (Molecular Dynamics).	12/1/2007	11/30/2009	$390,650
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Create/Manage/Maintain Database	Maintain current database	All data gathered in this study will be added to the Microsoft Access database which we designed and implemented during the first year of support from BPA. The database includes all phenotypic and genetic data collected for each individual sampled, as well as the parentage and population assignments. The development of this database was in part a response to the comment from reviewers to all proposals for this BPA program that database management should be a high priority. Accordingly, in addition to development of our database, we also purchased an external hard drive (at a remote location) and have automatic weekly backup of the entire database and raw data. This database has proven to be a great asset in merging phenotypic and genotypic data for analysis.	12/1/2007	11/30/2009	$15,330
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Install Fish Monitoring Equipment	Construct smolt trap	Build or purchase smolt trap	7/1/2007	9/30/2007	$15,000
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics
Install Fish Monitoring Equipment	Upgrade current fish weir	Upgrade the current fish weir to include a resistance board fish weir	7/1/2007	9/30/2007	$40,000
Biological objectives Reproductive success of wild/hatchery steelhead			Metrics

Section 8. Budgets

Itemized estimated budget

Item	Note	FY07	FY08	FY09
Personnel	[blank]	$101,116	$104,486	$106,577
Fringe Benefits	[blank]	$22,674	$23,450	$27,137
Supplies	[blank]	$4,750	$4,750	$4,750
Travel	[blank]	$1,753	$1,753	$1,753
Capital Equipment	[blank]	$67,000	$0	$0
Overhead	[blank]	$87,282	$89,583	$92,789
Other	Professional meetings	$1,782	$1,782	$1,782
Other	Utilities	$500	$500	$500
Other	Services	$24,690	$24,690	$24,690
Other	Tuition	$8,900	$8,900	$0
Totals		$320,447	$259,894	$259,978

Total estimated FY 2007-2009 budgets

Total itemized budget:	$840,319
Total work element budget:	$840,319

Cost sharing

Funding source/org	Item or service provided	FY 07 est value ($)	FY 08 est value ($)	FY 09 est value ($)	Cash or in-kind?	Status
Totals		$0	$0	$0

Section 9. Project future

FY 2010 estimated budget: $259,978
FY 2011 estimated budget: $259,978

Comments: We intend for the project to continue until the issues surrounding hatcheries are resolved and as long as we can positively contribute to the discussion

Future O&M costs:

Termination date:
Comments:

Final deliverables:

Section 10. Narrative and other documents

Reviews and recommendations

FY07 budget	FY08 budget	FY09 budget	Total budget	Type	Category	Recommendation
NPCC FINAL FUNDING RECOMMENDATIONS (Oct 23, 2006) [full Council recs]
$320,447	$259,894	$259,978	$840,319	Expense	Basinwide	Fund
NPCC DRAFT FUNDING RECOMMENDATIONS (Sep 15, 2006) [full Council recs]
$320,447	$259,894	$259,978	$0		Basinwide

ISRP PRELIMINARY REVIEW (Jun 2, 2006)

Recommendation: Fundable

NPCC comments: In many ways, this is a model project and proposal that tackles critical uncertainties with a clear design, modest budget, and a stellar publication record. The technical section of the proposal provides an excellent review of the issue of hatchery-wild fish interaction that summarizes the available and relevant literature and presents the project in context to issues of concern within the Columbia and elsewhere. Previous and positive past ISRP comments apply. Results from the project are applicable systemwide, thus the work is recommended as fundable, highest priority. A benefit includes the fact that the project is well underway and already has two to three generations of pedigree and fitness data available for analysis. The region should take advantage of this opportunity. It will provide data much sooner than other proposals that are in planning stages. A thorough summary of the history of this project, including bumps along the way, was provided. Monitoring efforts have improved continually and further evaluation has occurred. This is a rare opportunity to evaluate hatchery spawning effects on a wild steelhead population, and of particular significance to Washington steelhead culture and segregation projects. Continued work is justified, and most work to date is published, in press, or shall be submitted to peer-reviewed reports. This proposal offers opportunities to monitor essentially all the genetic issues raised in regard to hatcheries. This project is out-of-basin but addresses the specific objectives on relative fitness of wild and hatchery steelhead, thus is highly relevant to the Columbia River Basin with notable strengths and applicability. Objectives are to evaluate relative reproductive success, and the proponent's publication record indicates that the information will be useful to subbasin plans with similar supplementation experiments or interests. The proposal also describes how sponsors have reached out to other supplementation researchers to foster an ongoing dialogue. We support this and encourage its continuation. The project was compared to others in the basin (e.g., Hood, Abernathy) and outside the basin (Minter, Hamma Hamma; no mention of Keogh), and an integration of studies has begun (the proponent should provide a report of the workshops). Project documentation would benefit by a letter from WDFW Forks Creek facility documenting their continued support of and participation in this project. No doubt they do.

ISRP FINAL REVIEW (Aug 31, 2006)

Recommendation: Fundable