1. Objectives

The previous proposal in Taurus lists three objectives with corresponding hypotheses: (a) evaluate population differentiation and migration (gene flow) among reservoirs; (b) determine relatedness, mean productivity, and number of spawners within each reservoir; and (c) characterize broodstock by identifying origins (i.e., reservoir or population) and degree of relatedness among candidate broodstock fish. More recently, the proponents have developed two additional objectives involving whole genome sequencing to: (d) quantify adaptive variation within and among the sampled populations, and (e) search for a sex-linked marker that can be used to sex sturgeon at any age. Hypotheses were not developed for the two new objectives.

Overall, the goals and hypotheses are well defined, measurable, testable, and relevant to the Fish and Wildlife Program. No specific milestones or timelines are associated with any of the objectives, but it is clear from the narrative and project reports that steady progress is being achieved.

2. Methods

White sturgeon present challenges for genetic analysis because of their tetraploid ancestry, longevity, and the difficulty of obtaining adequate sample sizes. The proponents are to be commended for their skills in developing cost-effective methods (e.g., GT-seq) to screen genotypes at hundreds of DNA loci (SNPs) for application in parent-based tagging and other tools of genetic analysis. Sampling and analytical protocols seem appropriate and are clearly documented in Annual Reports, which include useful links to onlineresourcemonitoring.org websites.

Standard methods are being used and statistically reliable results are being produced. Measures of effect size (e.g., confidence intervals) are provided to support most conclusions. An exception is Figure 3 (i.e., comparing haplotype frequencies between pooled samples representing Snake and Lower Columbia stocks) and the AMOVA results (p.9) in the 2017 Annual Report that lack statistical tests and sample sizes, so the strength of the evidence for differences among stocks is unclear. Questions also remain regarding sample sizes needed to adequately characterize populations in individual reservoirs, and to clarify the influence of sturgeon movements among reservoirs, especially the downstream movement of fry and young-of-year fish.

Two very recent papers by C.D. Waters and colleagues seem highly relevant to identifying and quantifying adaptive variation among white sturgeon populations (objective d). In case these papers are unfamiliar to the proponents, the references are:

Waters C.D., Hard J.J., Brieuc M.S.O., Fast D.E., Warheit K.I., Knudsen C.M., Bosch W.J., and Naish K.A. 2018. Genomewide association analyses of fitness traits in captive-reared Chinook salmon:         Applications in evaluating conservation strategies. Evolutionary Applications 11: 853-868.

Brieuc, M.S.O., Waters C.D., Drinan D.P., and Naish K.A. 2018. A practical introduction to Random Forest for genetic association studies in ecology and evolution. Molecular Ecology Resources 18: 755-766.

3. Results

A major strength of this project is the successful development of a novel suite of 117 SNP markers and the use of the GT-seq high throughput sequencing method. Prior to 2017, genetic monitoring of white sturgeon had been investigated with a small published set of 13 microsatellite markers. The set of SNP markers developed in 2017 is currently being expanded, which will likely improve our knowledge of population structure and facilitate the application of parent-based tagging and other genetic tools.

It is reassuring that preliminary analyses of population structure based on the new SNP markers are corroborating previous results based on microsatellite loci. The metapopulation structure of white sturgeon is characterized by low genetic diversity and small effective population size. The low diversity and relatively small individual pairwise genetic distances likely stem from small numbers of families and/or few effective spawners within several impoundments. Broodstocks from the Yakama hatchery that are used for supplementation in the Middle Columbia River are genetically similar to the wild populations from which they are derived, which justifies their continued use. Gene flow has been highly restricted by barriers in the hydrosystem. The strong pattern of isolation by distance evident in genetic data supports the hypothesis that white sturgeon movement is largely downstream through the hydrosystem. In contrast, white sturgeon in the Lower Fraser and lower Columbia appear genetically similar; these populations have unimpeded access to the Pacific Ocean and may continue to intermix to some degree.

The proponents are now undertaking to sequence the genome of a single female white sturgeon, primarily to develop a practical genetic technique for non-lethally differentiating males and females at any age. The project is also making significant progress toward developing a parentage database that would allow juvenile fish to be assigned to parents spawning in the wild or to identify hatchery-origin fish without the need for other types of identifying marks.

Development of a reliable suite of genetic markers for white sturgeon is a critical step in understanding ecological and evolutionary dynamics of populations in the Columbia Basin and for future genetic monitoring of stocks in the basin. The ultimate utility of this program rests on its ability to track a population of long-lived fish over many years to fully ascertain some key population parameters. Translocation (i.e., stocking) of hatchery juveniles into upper Columbia River areas might be a useful strategy to compensate for the lack of recruitment or loss of productivity and diversity in blocked areas. But first, it will be necessary to examine the genetic population structure of white sturgeon in the impoundments between Priest Rapids Dam and Grand Coulee Dam and to investigate the conservation implications of possible introgression between remnant wild populations and hatchery fish derived from non-local broodstocks.

Collaboration and sharing of information among partners appear excellent. Annual reporting has been timely with satisfactory detail. However, in future reports, the ISRP would like to see more interpretation of results relative to stated objectives and hypotheses, and more discussion of how data from this project will be used in adaptive management strategies for population management and supplemental stocking programs.

4. 2017 Research Plan uncertainties validation

There is a mismatch between the list/numbering in the Research Narrative versus the Uncertainties Database or 2017 Research Plan. The ISRP concludes that this project indirectly (rather than directly) addresses the three critical uncertainties (CUs) listed by the proponent.

The Uncertainties Database indicates that six questions are being directly addressed by the project, but of these, it seems that only two (F1.4 and F3.2) are directly addressed, and the others are indirectly addressed.

As stated in the 2015 CUs review, "it is unclear how results obtained thus far and those anticipated in the future will be used help make decisions needed for sturgeon hatcheries and stock recovery. A critical uncertainty for these fish is: are observed genetic differences among fish from different reservoir pools indicative of adaptations to specific areas or just the result of recent segregation of putative stocks and thus not of great importance to managers?"

An adequate response will address several ISRP recommendations found in Sections 1 and 2 and listed below. 1. Provide a more robust technical justification for the proposed genetic analyses, including: (a) a review of past and ongoing genetic assessments of white sturgeon in the Columbia River Basin, as well as relevant studies in other rivers outside the Basin, (b) justification for use of microsatellite DNA versus other techniques (SNPs, mitochondrial DNA), (c) an evaluation of potential pitfalls in meaningful interpretation of the results, for example, if migration of individuals interferes with distinguishing fish from specific reservoirs, (d) the rationale for analysis of fish of different ages, and (e) a discussion of the sample sizes required to make interpretations under various assumptions about the breeding population sizes in particular reservoirs. 2. Provide specific details on the relations and coordination between the proposed project and other white sturgeon projects in the Basin: (a) #1986-050-000 (types of data collected, number of suitable fish, and how data for the proponent’s Objective (2) will complement the current effort); (b) the Yakima Sturgeon Management Project (# 2008-455-00), and (c) other projects (e.g. Kootenai Tribe’s genetics work) or additional avenues available to collect sturgeon samples. 3. Include the necessary first step that is missing from the proposal, that is, optimizing the amplification of DNA, genotyping the microsatellite DNA in Columbia River white sturgeon, and confirming that sufficient genetic variation can be detected. 4. For both Objectives (1) and (2), provide a better description of the samples available for analysis and provide evidence that the sampling of fish is consistent with a robust analysis of the genotypic data. 5. For Objective (1), clarify how sampling of various fish will provide complete coverage of potential contributing populations and that the approach to the analysis will be able to sort out migrant individuals. 6. For Objective (2), the experimental design (parentage analysis) and statistical analysis (relatedness analysis) appear to be for two different objectives. Provide an experimental design and statistical analysis for both. Discuss potential limitations of the proposed designs and analyses, for example, in the case that analyses are based only on young-of-the-year genotypes. 7. Provide justification that proposed sample sizes (up to 1000 fish per year for ten years) will be sufficient to yield useful results and interpretations of results. 8. To each of the objectives, add a sufficiently detailed description of potential outcomes of uses of project data that will result in measurable benefits to Columbia Basin fish and wildlife, more specifically white sturgeon. This project has a reasonable likelihood of aiding management and conservation of white sturgeon in these lower Columbia River reservoirs. The Sturgeon Strategic and Hatchery Master Plan document that is currently being prepared should guide this project. It is somewhat surprising that the proponents did not make note of the sturgeon workshop conducted under the auspices of Project #2007-155-00, scheduled to take place the first week of December 2009. The work conducted for the current project needs to support the Master Plan and the analysis conducted under this MOA should reflect uncertainties that are documented in the Master Plan. Once the Master Plan is completed and preliminary data are available on genetic diversity of sturgeon in the mid-Columbia, more robust experimental designs for both fish collections and data analysis should be developed and peer reviewed.