This is an exemplary, innovative project. We thank the proponents for submitting a revised proposal as well as a point-by-point response addressing all of the topics identified in our preliminary comments. In our preliminary comments, we requested responses on the following topics:

1. Compatibility with NOAA status assessment
2. MPG and ESU identification
3. Alternative approaches

The proponents added three appendix tables (A1-A3) in response to our request for more detail about how data generated by this project contribute to the abundance, productivity, or diversity measures used by NOAA for hierarchical assessment of ESU viability.

Table A1 highlights the extent to which this project supports assessments in a variety of other projects by genetically distinguishing sex and the hatchery origin of unmarked fish (that would otherwise be assumed to be natural origin). These and other data contributed by the project are clearly important for improving estimates of pHOS, natural spawner abundance, and indices of genetic and life history diversity.

Tables A2 and A3 support the ISRP’s preliminary comments that the genetic stock groupings to which fish can be assigned by this project often do not exactly match the TRT populations of steelhead and Chinook salmon, and in some cases even the MPGs of Chinook salmon.

Moreover, the proponents’ response does not indicate how estimates of abundance or productivity measured at Lower Granite Dam for genetic stocks comprising mixtures of TRT populations are (or might be) adjusted to provide the spawner-to-spawner data typically required for NOAA's VSP and ESU status assessments. We therefore conclude that the proposal has somewhat overemphasized the utility of this project for the NOAA viability assessments of TRT populations. That said, this project also supports a basinwide effort to implement a variety of approaches for monitoring status and trends at multiple hierarchical levels. To maximize efficiency and effectiveness of that overall effort, it remains important to periodically review and refine the approaches being developed in this and other projects. The ISRP urges the proponents to consider, in future work plans, whether a greater focus on SNPs associated with adaptive traits might improve the capability to resolve TRT populations and MPGs.

Preliminary ISRP report comments: response requested

Response request comment:

We commend the proponents for a clearly written, well-organized and well-justified proposal. The standardization and application of accurate methods of parentage-based tagging (PBT) and genetic stock identification (GSI) are remarkable achievements by this project (in collaboration with project 200890700). The resulting single nucleotide polymorphism (SNP) baselines for Snake River steelhead and Chinook salmon have enabled (1) cost-effective and routine monitoring of spatial and temporal trends in diversity and genetic structure of natural-origin Snake River populations; (2) estimation of stock composition of harvests in mainstem fisheries and escapements past Lower Granite Dam; and (3) evaluation of proportionate natural influence (PNI) of integrated hatchery programs in Idaho.

The proposal states that the project contributes significantly to NOAA and other investigators by providing data for viability assessments of independent populations defined by the Technical Recovery Teams (“TRT populations”), major population groups (MPG), and evolutionarily significant units (ESU). However, the ISRP was unable to understand how, or the extent to which, this project informs these status assessments. The ISRP requests the proponents to address the following points in a revised proposal, and to provide a brief point-by-point response to explain how and where each issue is addressed in the revised proposal:

1. Compatibility with NOAA status assessment. Clarify how estimates from this project are used in the NOAA status assessments. In many cases, it seems that data generated by this project could only be used to estimate abundance and productivity of multi-population groupings returning to Lower Granite Dam. It seems that such estimates would not be adequate for viability assessments that typically rely on estimates of spawner abundance and spawner-to-spawner productivity for TRT populations.
2. MPG and ESU identification. List the steelhead and Chinook salmon populations by MPG and ESU for which this project provides data that are used by NOAA for viability status assessments.
3. Alternative approaches. If the project enables an alternative approach to viability assessment of abundance and productivity, then explain the alternative approach and compare it with the NOAA approach to demonstrate its utility.

Q1: Clearly defined objectives and outcomes

This project addresses management issues and uncertainties that are highly relevant to the Council's 2014 Fish and Wildlife Program, 2017 Research Plan, and High-Level Indicators as well as numerous subbasin plans. However, it is less clear how the project supports and contributes to ESA Recovery Plan objectives.

The current proposal combines objectives from two previous BPA projects to test the feasibility of using PBT and GSI to help manage hatchery and wild Snake River steelhead and Chinook salmon. The eight revised objectives are clearly specified, and most meet SMART criteria. An exception is Objective 2, which is not quantitative. Instead of just saying “discover new SNPs,” we suggest indicating more precisely the number of new SNPs, or the attributes of new SNPs, that need to be discovered for the project to achieve this objective.

In future proposals, it would be advantageous for the proponents to include additional objectives related to adaptive management and reporting, as this would serve to highlight other very successful outcomes from the project.

The project now comprises a well-established annual cycle of activities that is expected to continue for the foreseeable future.

Q2: Methods

The proposal includes a succinct but comprehensive overview of methods, which is organized appropriately by objectives, clearly explains the rationale for various approaches, and provides convenient links to details elsewhere (e.g., monitoringresources.org and references to associated projects and the primary literature). Sampling and analytical protocols are documented in more detail in the annual report for 2019. Standard methods are being used for statistical analyses, and confidence intervals or significance test probabilities are provided to support most conclusions. The proponents also use state-of-the-art techniques that were peer-reviewed in their primary publications, and hence, are considered scientifically appropriate.

A notable weakness in the methods section is the lack of detail about how data generated by the project contribute to the abundance, productivity, or diversity measures (i.e., viable salmonid population (VSP)) parameters used by NOAA for hierarchical assessment of ESU viability. VSP parameters used to assess the viability of TRT populations are typically based on multi-generational adult spawner-to-spawner data. This proposal does not describe how estimates of abundance or productivity measured at the Lower Granite Dam for stocks comprising mixtures of TRT populations are adjusted to provide the spawner-to-spawner data typically required for NOAA's VSP and ESU status assessments. It seems that in many cases, the scale of the abundance and productivity estimates provided from this project would not align sufficiently with that needed for assessments at the TRT population, MPG, or ESU level. In summary, the methods section should indicate more precisely which data generated by this project are actually used by NOAA for viability assessment and describe more clearly how these data are adjusted to meet (or circumvent) the requirement for spawner-to-spawner abundance and productivity estimates for TRT populations and the specific populations for which data are generated.

Q3: Provisions for M&E

A brief paragraph refers to a history of periodic adjustments to genetic marker panels, genotyping platforms, and statistical tools through regular meetings with genetic collaborators to evaluate results and discuss new proposals. Significant adjustments to improve cost-effectiveness include transitioning to:

• Absorptive chromatography paper that can hold 50–100 samples per sheet and occupies much less space than an equivalent number of ethanol-filled vials;
• 300-400 GTseq SNP genotyping panels for both Chinook and steelhead that provide near zero false-positive and false-negative rates at less cost than the original 96 samples X 96 SNP loci system;
• A 5-year rotating schedule for resampling populations to update GSI baselines;
• Whole-genome sequencing of pools of individuals (Pool-seq) to more cost-effectively estimate allele frequencies across the genome at the population scale; and 
• The (proposed) addition of microhaplotypes (multiple, tightly linked SNPs that exhibit contrasting allele frequencies across populations) to the existing GTseq SNP panels in hope of further improving GSI accuracy for steelhead.

The proposal states that project results are regularly evaluated and discussed during meetings with collaborators. Although this project adjustment process appears to be working well, the ISRP would like to see more explanation, in future proposals or the next annual report, of the decision process by which the proponents allocate effort and resources among objectives, such as finding new SNPs and updating and expanding baselines.

The project has excelled at sharing information and providing information to support management decision processes. Monitoring and research results are presented in annual IDFG and BPA reports, at various meetings (e.g., LSRCP, IDFG Anadromous Meeting, Steelhead Workshop, Coastwide Salmonid Genetic Conference), and in the primary scientific literature. Impressively, the proponents have authored or co-authored over 20 papers on work undertaken in this project. The Gantt chart (Fig. 19) clearly indicates the annual cycle of activities, and the table in section 8 (Relationships to other projects) helps to clarify roles and responsibilities in collaborations with six other projects.

Q4: Results - benefits to fish and wildlife

The proponents have worked collaboratively with CRITFC staff (project 200890700) to develop and standardize SNP panels for steelhead and Chinook salmon that cost-effectively integrate application to both PBT and GSI, and identify the genetic sex of each species. The ISRP noted an apparent inconsistency between pages 15 and 27 of the proposal which state the current Columbia-basin-wide panel contains “390 SNPs for steelhead and 299 SNPs for Chinook Salmon” versus “368 SNPs for steelhead and 343 SNPs for Chinook Salmon,” respectively, with Hess et al. (2020) cited in both instances.

On average, 4,900 steelhead and 12,000 Chinook salmon broodstock are sampled each year to create PBT baselines comprising all steelhead and Chinook salmon broodstock used in hatcheries throughout the Snake River basin. This effort allows the Snake River PBT program to genetically “tag” about 95% of 20 million steelhead and Chinook salmon smolts released annually.

Since 2018, the steelhead GSI baseline has represented 23 TRT populations and all 6 MPG. These steelhead collections are pooled to create 45 “GSI populations” for stock composition analysis and 10 “genetic stock” groups for reporting mixture proportions. The Chinook salmon baseline represents 31 of 41 TRT populations and all 5 MPG. The Chinook salmon collections are pooled to create 30 GSI populations for analysis and 6 genetic stocks for reporting.

PBT has now superseded coded-wire tagging (CWT) to estimate the harvest of hatchery Chinook salmon in the Snake River basin, although CWTs are still used to monitor ocean and downriver harvests, and to assess and compare alternative hatchery rearing and release strategies. Multiple year results have shown that PBT and CWT methods provide similar accuracy, but PBT can provide greater precision because of the larger number of “tags” available.

The proponents have used GSI and PBT in combination (working collaboratively with projects 199005500, 199107300, 198335003, and 201800200) to estimate abundance and stock composition of wild steelhead, spring/summer Chinook salmon, and fall Chinook salmon passing Lower Granite Dam. Abundance, productivity, and measures of genetic diversity are provided to NOAA as part of requirements to review the listing classification of Snake River steelhead and Chinook salmon at least once every five years.

Incorporating PBT with GSI has significantly improved the accuracy of wild escapement estimates at Lower Granite Dam by detecting untagged hatchery-origin fish that would otherwise be mistaken for wild fish, resulting in a significant overestimation of natural abundance. PBT analysis identified that, on average from 2014-2018, 19.6% of Chinook salmon and 8.3% of steelhead adults passing Lower Granite Dam were hatchery-origin, despite having no physical or mechanical marks. Similarly, a comparison of stock-specific abundance estimates for hatchery Chinook salmon returning in 2016 to 2019 revealed that the in-season PIT-tag method accounted for only 65% (averaged across all release groups) of the total detected by PBT.

The project also demonstrated that PBT should be used as part of long-term monitoring of proportionate natural influence (PNI) for integrated hatchery programs in Idaho. Analyses based on physical marks consistently overestimated PNI by overestimating the proportion of natural fish in both the natural spawning and broodstock components of the hatchery program compared to analyses based on PBT.

The proponents have fully addressed previous ISRP recommendations:

• Genetic data from PBT and GSI projects in the Columbia River basin (and throughout the Pacific Coast of North America) are now stored in FIshGen (McCane et al. 2018) and available to any lab running PBT projects;
• PBT data for PIT-tagged fish are now being linked to life history data from scale sampling at Lower Granite Dam (i.e., age at maturity) and migration behavior from PIT-tag detections at in-stream arrays (IPTDS). This step allows VSP metrics to be assessed at the scale of some TRT populations. However, because in-stream array coverage is not complete across the landscape, the method cannot be applied to all TRT populations.

1. Technical Justification, Program Significance and Consistency, and Project Relationships The need for the monitoring of hatchery salmon and steelhead performance once released is an essential feature of management obligations to evaluate the efficacy of the artificial production programs in terms of their yields to fisheries and impacts to natural population. The ISAB/ISRP, as well as the Pacific Salmon Commission technical team, recently concluded that Parental Based Tagging (PBT) has the potential to provide important data for cohort analysis of salmon and steelhead populations. The project proponents (IDFG) propose to genotype the entirety of Chinook salmon and steelhead broodstock from 14 Snake River hatcheries to empirically test and validate whether PBT can be used to replace coded-wire tags (CWT) for estimating stock contributions of Snake River steelhead and Chinook salmon to fisheries, run-timing, straying, etc., of adult salmon in the Snake/Columbia River basins. The technical justification of using PBT as a replacement for CWT-based assessment of hatchery production in harvest, straying, and returns to hatcheries (life-cycle survival) is adequately described. The ISRP notes, however, that successful implementation of PBT will not eliminate all problems associated with CWTs, mass marking, and selective fisheries. For example, there will still be a need to handle and mass mark juvenile hatchery fish with an adipose fin clip or some other highly-visible mark for selective fisheries. The technological development to bring PBT to pilot scale trial is consistent with the BiOp, Fish and Wildlife Program, and Lower Snake River Compensation Plan. Relationships with other projects in the Basin are sufficiently described, and evidence is provided that the proponents are familiar with other PBT trials in Puget Sound, Washington and the Sacramento River, California. The proposed project involves coordination with the Columbia River Intertribal Fish Commission (CRITFIC) and other Genetics Analysis of Pacific Salmon (GAPS) and Stevan Phelps Allele Nomenclature (SPAN) laboratories to develop a coastwide Single-Nucleotide Polymorphism (SNPS) baseline. One concern is that the ISRP could not determine whether this proposed project unnecessarily duplicates work already being performed or proposed by other BPA-funded projects. For example, the proposed work is contingent upon the success (efficiency, precision, and accuracy) of efforts associated with BPA Project #200890700, “Genetic Assessment of Columbia River Stocks.” Moreover, the project will "identify SNP loci" for Chinook salmon and steelhead. Presumably, BPA Project #200890700 should be far enough along to inform these choices now without significant new work. Finally, the proposed project will not analyze future harvested or escaped (from fishery) Snake River salmon and steelhead except as a test (objective 5). The ISRP does not understand why BPA Proposal No. 201002600: “Chinook and steelhead genotyping for genetic stock identification (GSI) at Lower Granite Dam,” which is contingent on the results of this proposed project, was submitted as a separate proposal. As a result, is there unnecessary overlap or duplication of some work elements between the two proposals? 2. Project History and Results The proposed project is new, but efforts to begin collecting tissue for genetic analysis are already underway, e.g., samples from broodstock at most Snake River hatcheries were obtained in 2008 and 2009. Since this project is closely linked with the CRITFC project developing SNP assays for Columbia River salmon ESUs (BPA Project #200890700), a more detailed description of the number of SNPs identified to date and how many of those have been polymorphic in the populations of interest would have been useful. 3. Objectives, Work Elements, and Methods The objectives are relatively straightforward. The overall work plan and strategy to collect tissue samples; develop SNPs; identify a SNP panel for both genetic stock identification (GSI) and PBT; and then test that panel using progeny from known populations; and finally cross check with harvested fish that carry CWTs is sound. One difficulty for ISRP reviewers not expert in the field of salmon genetics was that proponents used lots of technical jargon without defining their terminology. In addition, publications and computer software packages were frequently cited for methods without providing a concise summary or explanation of these methods in the proposal. The ISRP suggests that for objective 4, 157...SNP Genotyping (juveniles) - a number of non-hatchery wild juveniles originating from major population groups (MPGs) or reporting groups should be tested against the parents used to establish the PBT baseline. They should not assign to any of the hatcheries. It was not apparent from the objectives and methods that this testing was included. One final concern is that the high throughput requirements for analysis of 16.5K samples per year plus any future samples from fisheries, etc., may quickly overwhelm the system. Given the genetic laboratory's other demands, proponents need to verify in contracting that results can be provided in accordance with proposed schedules.

The proponent proposes to use single nucleotide polymorphism (SNPs) based genetic stock identification (GSI) for establishing the composition of various Snake River salmon and steelhead populations passing the Lower Granite Dam (LGD). Based on suites of genetic characters, GSI models the most likely composition of populations contributing to the fish passing. The modeling methods have a long history and this proposal builds on these to fit an advertised need to better estimate salmon populations in the Snake River basin. This methodology can be applied at Lower Granite Dam to evaluate populations that may not be amendable to analysis by PIT tagging. The proposed project has the potential to provide important data to understand the life-history and population dynamics of Snake River steelhead and Chinook salmon. 1. Technical Justification, Program Significance and Consistency, and Project Relationships This project will develop (and test) standardized SNP baselines for fine-scale GSI of Snake R. Basin stocks as adults migrate past Lower Granite Dam (LGD). The general explanation of life history variation in steelhead in the Snake River basin, and justification that a GSI approach can contribute to estimating abundance of MPGs and reporting groups (aggregates of spawning tributaries) is well summarized. Estimates of population components crossing LGD for one year are included in the problem statement. The project is greatly interdependent, in part, on project #201003100 which creates the reference baseline used to decompose the population mixture. The proposed project is consistent with BiOp RPAs, the regional monitoring strategy, and recommendations in the ISRP/AB tagging report. This and related projects provide an alternative tool for mark and recapture (referenced in our tagging report Section A.6 pp. 67-73). The project relationships are summarized and appear extensive. This project will use fish collected by other projects, and these are adequately summarized. At least a portion of the SNP development effort will involve collaboration with the Hagerman Laboratory operated by CRITFC, specifically project 200890700 Genetic Assessment of Columbia River Stocks, that will be using GSI to estimate stock proportions at Bonneville Dam. Section I. Key Personnel indicates that the CRITFC lab will be a subcontractor to develop SNPs and genotype fish. Geneticists throughout the Pacific Northwest have collaborated on development of microsatellite gene markers and standardized genes, alleles, and methods of analysis. The collaboration on SNP development and standardization of genotyping and analysis platforms is appropriate. The ISRP does believe a more thorough clarification of the roles of the different projects in the field, laboratory, and analysis is needed. 2. Objectives, Work Elements, and Methods The objectives are straight forward and build on previous or currently proposed work. The methods for developing SNP assays, evaluating the distribution of SNP alleles in Snake basin populations, and establishing the utility of a set of SNPs for both GSI of wild steelhead and Chinook at LGD and Parental Based Tagging of hatchery steelhead are consistent with practices and methods in the discipline. It is less clear that methodologies for using abundance estimates from LGD, partitioned into reporting groups in VSP analysis for BiOp monitoring and ESA delisting, etc are developed. The rationale for sampling and genotyping 2000 fish at LGD needs to be justified.

The RME Workgroup comments agreed with three project BiOp RPA associations (50.5, 62.5 and 64.2), questioned one (50.7) and suggested that three be deleted (55.8, 63.2, 64.1).  For 50.7 that asked to confirm that the scope of work proposed is for 100% marking of fish (visible or non visible) and that if the project involved marking fish for the hatchery, that we specify the hatchery name and populations affected.  The hatchery stocks are listed below for both spring/summer Chinook salmon and steelhead.  Responses to the three RPAs suggested for deletion are also provided below. 

 50.7- Fund marking of hatchery releases from Action Agencies funded facilities to enable monitoring of hatchery-origin fish in natural spawning areas and the assessment of status of wild populations. (Annually) 

Response:  BPA project will permanently genetically mark all hatchery spring/summer Chinook salmon and steelhead in the Snake River basin (Idaho, Oregon and Washington).  These populations are summarized in Table 1 and 2 along with the number of broodstock spawned in 2008 and 2009. 

 Table 1.  Total number of Snake River steelhead hatchery broodstock sampled in 2008 and 2009.  *Early egg-takes were missed.  **ODFW hatcheries were not sampled in 2008.

 Table 2.  Total number of Snake River Chinook salmon hatchery broodstock sampled in 2008 and 2009. 

 55.8-Evaluate new tagging technologies for use in improving the accuracy and assessing delayed or indirect hydro effects on juvenile or adult fish.

Response:  It appears that the new tagging technology that RPA 55.8 references primarily involves acoustic tags.  The Pacific Ocean Shelf Tracking (POST) program is a BPA funded project (2003-114-00) that is developing electronic tagging technology and methods to determine delayed or indirect effects of hydro passage by tracking salmon and steelhead in the estuary below Bonneville Dam and the ocean environment off the Pacific coast.  While our current project goals do not include sampling hatchery salmon and steelhead in these areas, it easy to see how PBT technology ultimately could be used to address many of the same types of questions that acoustic tag technology is attempting to address.  These questions are outlined on the POST website (http://www.coml.org/projects/pacific-ocean-shelf-tracking-project-post) and includes:

• ·         Do different salmon stocks form stock-specific aggregations at sea, or do they share common migration pathways and foraging areas? If such aggregations or pathways occur, where are the boundaries and how long do salmon remain in these areas?
• ·         Do distributions and migration patterns fluctuate with annual and long-term changes in ocean productivity, such as spring bloom cycles and El Niño/La Niña conditions?
• ·         How do different salmon species respond to changes in ocean conditions?
• ·         What are the winter movements and habitat usage of juvenile salmon?
• ·         What differences exist between the movement patterns and habitat use of wild and hatchery salmon?
• ·         Do shelf migration behaviors correlate with changes in annual stock success?
• ·         How do distribution and habitat use differ among species and stocks?
• ·         Is there stock-specific, life-stage specific or location-specific mortality in the ocean?

The hatchery offspring that are genetically tagged as part of our project represents all of the Snake River hatchery steelhead and spring/summer Chinook salmon produced each year and  roughly 50% of the entire production of hatchery steelhead and spring/summer Chinook salmon that migrate out of the Columbia River basin annually. 

 63.2- Determine the effect that implemented hatchery reform actions have on the recovery of targeted salmon and steelhead populations.

Response:  The effectiveness of hatchery reform measures will require monitoring genetic divergence between hatchery and wild stocks and making assessments of hatchery straying.

For integrated hatchery programs, actions will be implemented to minimize genetic divergence between the hatchery broodstock and the wild population and to maximize the proportion of natural influence (PNI).  This project can provide annual estimates of the degree of genetic divergence between every hatchery stock within the Snake River basin and associated wild stocks.  Successfully managing segregated hatchery programs will require enhancing harvest opportunities and minimizing straying between hatchery and wild populations.  Parentage Based tagging (PBT) of hatchery stocks in the Snake River basin will allow managers to more efficiently evaluate the effects of different release strategies (where and when fish are released, size and age at release, etc.) on increasing harvest and minimizing straying.  We will demonstrate through a paired coded-wire tag (CWT) and PBT recovery experiment, as part of existing hatchery evaluations in Idaho, that PBT technology can provide the same information as CWT (information on stock and age) for harvest evaluations.  This project’s initial goals are also to demonstrate that any hatchery adult that has found to have strayed into natural production areas (either collected alive or as a carcass) could be sampled and its sex, stock and exact age be determined.  For hatchery steelhead in the upper Salmon River basin, we also intend to determine whether straying adults were from an on-site versus off-site juvenile release.  In the near future, with the completion of a collaborative hatchery rearing and release database module, we should also be able to identify the release location of any hatchery steelhead collected/sampled in a wild production area in the upper Salmon River basin.

 55.8-Continue to estimate the relative reproductive success (RSS) of hatchery-origin salmon and steelhead compared to reproductive success of their natural-origin counterparts.

 Response:  Throughout the Snake River basin Chinook salmon and steelhead hatcheries will be implementing hatchery reform measures to support sustainable fisheries while conserving natural spawning populations.  For stocks designated to be part of integrated programs, effectiveness monitoring will be necessary to assess the reproductive contribution of hatchery fish spawning in the wild and the number of adult recruits produced per hatchery spawner.  In coordination with this project (2010-031-00), IDFG, Tribal and federal co-managers in the Snake River basin are currently collecting genetic samples from all natural and hatchery fish passed above weirs associated with specific hatchery programs.   Because single, standardized SNP genetic marker sets are being developed to address genetic issues for both wild and hatchery stocks, this program will be able to contribute hatchery parental genotypes to any RRS study throughout the Snake River basin.