BPA Fish and Wildlife FY 1997 Proposal

Section 1. Administrative
Section 2. Narrative
Section 3. Budget

see CBFWA and BPA funding recommendations

Section 1. Administrative

Title of project
Genetic Analyses of Oncorhynchus Nerka (ESA)

BPA project number   9009300

Business name of agency, institution or organization requesting funding
Univ/ID, subcontractor WSU

Sponsor type   ID-Consultant

Proposal contact person or principal investigator

BPA technical contact   Rick Westerhof, EWI 503/230-5061

Biological opinion ID   NMFS

NWPPC Program number   7.5A.1

Short description
Provide biological and genetic information on O. nerka samples collected throughout the Snake and Columbia River Basins to be used in the overall recovery effort of the Snake River Sockeye Salmon.

Project start year   1990    End year   

Start of operation and/or maintenance   

Project development phase   Implementation

Section 2. Narrative

Related projects
Projects 91071, 91072 and 92040. All relate to recovery actions for Redfish Lake sockeye. Project 90093 provides the DNA analysis for all the other projects, especially the captive broodstock spawning matrix (sockeye x sockeye).

Project history
Activities have included rearing Redfish Lake kokanee to examine inter-year temperature unit variability for egg incubation and behaviorally for downstream migration volitionally out of circular tanks. Primarily work has focused on defining a technique and regions of DNA base sequences useful for separating the life history forms of O. nerka in Redfish Lake. Costs have included renovation of experimental wet laboratory facilities, equipment for genetic laboratory, salaries and supplies. Benefits are that regions useful for diagnostic purposes have been identified for the anadromous versus non-anadromous components of Redfish Lake O. nerka and a wet lab that can be used for small scale incubation and rearing projects.

Biological results achieved

Annual reports and technical papers
Genetic Analysis of Oncorhynchus Nerka - Annual Progress Reports FY 1991, FY 1992, Genetic Analysis of O. Nerka: Life History and Genetic Analysis of Redfish Lake O. Nerka, Completion Report FY 1993-1994. Monthy Progress Reports 1991 to 12/1996 and Stanley Basin Sockeye Technical Oversight Committee Meeting Notes 1993-12/1995.

Management implications
Our objectives are directed at resolving the origins and phylogeographic relationships of existing O. nerka stocks in the Columbia Basin. Additionally, we wish to determine what level of gene flow exists between populations and what genetic contributions are made by anadromous and resident populations to reciprocal migratory forms. The results of this research will influence management decisions regarding the uniqueness of various sockeye stocks and their designation as evolutionary significant units (ESU). This work has far reaching implications regarding the Endangered Species Act as well as future conservation efforts for non-endangered populations of trout and salmon.

Specific measureable objectives
1) FY 96:
2) Identify more comprehensively the genetic structure of Redfish Lake O. nerka outmigrant-originating populations.
3) Define the relatedness of populations of O. nerka in Redfish, Stanley, and Snake River lakes and the Columbia Basin lakes.
As stated above, these objectives are directed at resolving the origins and genetic relationships of existing stocks of O. nerka in the Columbia Basin with the goals of determining what level of divergence exists between populations and what genetic contributions are made by anadromous and resident populations to reciprocal migratory forms.

Testable hypothesis
The ongoing analysis and proposed future work will be used to test the following hypotheses:
1) Anadromous, Redfish Lake sockeye salmon are a distinct population of O. nerka and represent an evolutionarily significant unit (ESU) recognized under the Endangered Species Act and are following an independent evolutionary trajectory as evidenced by reduced gene flow relative to a sympatric kokanee population.
2) Beach spawning, resident sockeye are an intermediate form between anadromous sockeye and kokanee but, most closely resemble anadromous sockeye in a taxonomic sense leading to their inclusion in the Redfish Lake ESU.
3) The sympatric kokanee population present in Redfish Lake is not significantly contributing to the numbers of outmigrating anadromous sockeye and thus the Redfish Lake ESU.
Populations of O. nerka within the Columbia and Snake River basins form genetically distinct stocks due to their high degree of philopatry and thus may give rise to additional situations where a sockeye populations become reduced in number and have to be considered for protection under the ESA.

Underlying assumptions or critical constraints
A critical assumption to each hypothesis is that the samples analyzed for each population are representative and unbiased. The chance that this problem will occur can be minimized by increasing the number of individuals tested from each population. At present, we are continuing to expand the data base to include where possible, 60 individuals from each population under study. Statistically, the distribution of mitochondrial haplotypes found among each population will then fall within 95% confidence limits for haplotypes that are not considered “rare.” (i.e. with a sample size of 60, there is a 95% chance of observing every haplotype in a population that occurs with 5% or greater frequency)

Methods
Two types of DNA analysis are currently employed. The first involves the development and use of polymorphic mitochondrial gene loci. The second is the development and use of nuclear DNA markers resulting from polymorphisms within mini- and microsatellite loci.

For mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) analysis, isolated mtDNA is amplified using the polymerase chain reaction (PCR) and primers specific for four separate gene regions; ND1, ND2, ND5/6, and Cytochrome b. The amplified regions of DNA are digested using restriction endonucleases. The resulting banding patterns are then evaluated using numerical taxonomy programs such as NTSYS-pc or clustering programs such as REAP.

For nuclear genome analysis, total genomic DNA is isolated from non-destructively collected tissue samples and digested with one of several restriction endonucleases. The digested DNA is electrophoresed in an agarose gel then transferred via Southern blotting to a nitrocellulose or nylon membrane. The transferred DNA is fixed to the membrane and subsequently hybridized with a non-radioactively labeled fragment of DNA used as a marker. The resulting banding patterns, documented using x-ray film, are then scored and analyzed using various phylogenetic inference and numerical taxonomy programs.

Brief schedule of activities
1) Continue DNA analysis on samples of the outmigrant assemblages to assess contributions from kokanee on subsequent year classes.
2) Continue DNA analysis on samples of progeny from the captive broodstock and returning adults so as to characterize the nature of successful outmigrant contributions.
3) Expand the existing data base by obtaining 60 individual samples for each population.
4) Develop additional DNA markers using mini-and microsatellite analysis to differentiate regional populations of sockeye salmon and kokanee.
5) Verify the presence of pseudogenes within the nuclear genome and develop them as potential markers to eliminate any bias in conclusions regarding genetic relatedness and variation based on maternally inherited (mtDNA) polymorphisms alone.
6) Continue genomic DNA analysis to characterize allele frequencies at Spe8.1 HP-0.8 locus for additional Stanley Basin O. nerka populations.
7) Continue to describe haplotype differences and substructure among early and late spawning O. nerka populations.
8) Use mixed DNA fingerprint analysis to examine the relatedness between Deschutes and Okanogan, Wenatchee and Redfish Lake O. nerka.
Additional outmigrant samples will be examined to further investigate the possible loss of haplotype diversity in different brood years.

Biological need
The populations of sockeye salmon present in the Snake river have been considered unique in that their genetics and life history patterns are divergent, to a sufficient degree, from other stocks of sockeye salmon. Under the tenets of the U. S. Endangered Species Act, these fish are now protected. However, the recovery of these fish populations to sustainable levels requires genetic monitoring because long term survival will be influenced not only by the restoration and preservation of their habitat but, also by their ability to genetically contend with their historically stochastic environment.

Critical uncertainties
Please refer to the section “Underlying Assumptions…” for a discussion on the use of larger sample sizes. It should be noted that an assumption has been made regarding the long term survival of Snake River sockeye and their genetic adaptability. We have assumed, until proven otherwise, that sufficient genetic variation exists within the anadromous, beach spawning, and/or sympatric kokanee population to address that organism’s natural environmental challenges to survival. The level of genetic variation needed, changes with the organism under study and with the stability of its environment. Some organisms thrive under conditions of little genetic diversity, but others do not. Consequently, their risk of extinction can be very high. At this point, the level of genetic variation needed in the Snake River sockeye population for good probability of long term survival is unknown. Unfortunately, it is also uncertain whether a sufficient level of genetic variation still exists. The genetic analyses undertaken in this project will help to facilitate an answer to both those questions.

Summary of expected outcome
This project will result in a comprehensive data base and genetic profile from which the immediate and long term genetic risks to Snake River sockeye as well as other populations or stocks of sockeye within the Columbia River Basin can be addressed.

Dependencies/opportunities for cooperation
Since the Snake River sockeye listed under the endangered Species Act are anadromous, their protection and recovery fall under the jurisdiction of the National Marine Fisheries Service. The broodstock program is administered through the Idaho Department of Fish and Game. Our objectives are commensurate with the responsibilities and objectives of the fore mentioned agencies. Thus far this has led to a successful, cooperative, interdisciplinary effort toward the conservation of this endangered species. Handicaps to the future progress of the genetics work is not anticipated.

Risks
There is no known risk associated with non-destructive sampling and genetic analysis of O. nerka populations.

Monitoring activity
Project results are discussed monthly at the Stanley Basin Sockeye Technical Oversight Committee Meeting. All pertinent parties are present to evaluate the findings and develop new programs. Results are used by NMFS and IDFG in the captive brood stock program and also by the recovery team. COTR attends Committee Meetings on a periodic basis and receives monthly progress reports and financial invoices to review.

Section 3. Budget

* For most projects, Authorized is the amount recommended by CBFWA and the Council. Planned is amount currently allocated. Contracted is the amount obligated to date of printout.

Funding recommendations

CBFWA funding review group   Snake River

Recommendation    Tier 1 - fund

Recommended funding level   $140,000

BPA 1997 authorized budget (approved start-of-year budget)   $134,000