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Genetic Structure and Diversity in Wild Populations of the Light-Footed Ridgway’s Rail Reflect 20 Years of Augmentation Through Captive Breeding and Release

Amy G. Vandergast, Julia G. Smith, Anna Mitelberg, Dustin A. Wood, Kimberly A. Sawyer, Courtney J. Conway · U.S. Geological Survey

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Ecosystems Mission Area—Species Management Research Program Prepared in cooperation with U.S. Fish and Wildlife Service, Carlsbad Fish and Wildlife Office

Genetic Structure and Diversity in Wild Populations of the Light-Footed Ridgway’s Rail Reflect 20 Years of Augmentation Through Captive Breeding and Release

Open-File Report 2025–1011

U.S. Department of the Interior U.S. Geological Survey

Cover. A Ridgway’s Rail in pickleweed at Tijuana Slough National Wildlife Refuge. Photograph by Julia G. Smith, U.S. Geological Survey, June 21, 2021.

Genetic Structure and Diversity in Wild Populations of the Light-Footed Ridgway’s Rail Reflect 20 Years of Augmentation Through Captive Breeding and Release By Amy G. Vandergast, Julia G. Smith, Anna Mitelberg, Dustin A. Wood, Kimberly A. Sawyer, and Courtney J. Conway

Ecosystems Mission Area—Species Management Research Program Prepared in cooperation with U.S. Fish and Wildlife Service, Carlsbad Fish and Wildlife Office

Open-File Report 2025–1011

U.S. Department of the Interior U.S. Geological Survey

U.S. Geological Survey, Reston, Virginia: 2025

For more information on the USGS—the Federal source for science about the Earth, its natural and living resources, natural hazards, and the environment—visit https://www.usgs.gov or call 1–888–392–8545. For an overview of USGS information products, including maps, imagery, and publications, visit https://store.usgs.gov/ or contact the store at 1–888–275–8747. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Although this information product, for the most part, is in the public domain, it also may contain copyrighted materials as noted in the text. Permission to reproduce copyrighted items must be secured from the copyright owner. Suggested citation: Vandergast, A.G., Smith, J.G., Mitelberg, A., Wood, D.A., Sawyer, K.A., and Conway, C.J., 2025, Genetic structure and diversity in wild populations of the Light-footed Ridgway’s Rail reflect 20 years of augmentation through captive breeding and release: U.S. Geological Survey Open-File Report 2025–1011, 24 p., https://doi.org/10.3133/ofr20251011. Associated data for this publication: Mitelberg, A., Wood, D.A., Smith, J.G., and Vandergast, A.G., 2025, Microsatellite genotypes for Light-footed Ridgway’s Rail (Rallus obsoletus levipes) sampled in southern California: U.S. Geological Survey data release, https://doi.org/10.5066/P14CYDJC. ISSN 2331-1258 (online)

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Acknowledgments This research was supported by the U.S. Fish and Wildlife Service (USFWS), Carlsbad Fish and Wildlife Office, U.S. Geological Survey Ecosystems Mission Area, and contributions from the San Diego Foundation. This project could not have been completed without the efforts of many individuals and organizations. We thank Heather Parsons, Eamon Harrity, and Kathryn Sliwa from the University of Idaho; Kylie Curtis and Claire Andrews from the U.S. Geological Survey; and Hans Sin and Kyle Rice from the California Department of Fish and Wildlife for their contributions to fieldwork. Fieldwork was made possible with assistance and site access from the following people and organizations: Loni Byer and Joyce Sisson (U.S. Naval Base Coronado); Martin Ruane (U.S. Naval Air Station Point Mugu); Jon Rishi and Cristan Caviel (U.S. Army Corps of Engineers Santa Ana River Mainstem Salt Marsh); Rob Haskell (Surf Cup), Shawna Anderson (San Diego River Park); Amanda Swanson, Melissa Borde, Hans Sin, Gabriel Penaflor and Kyle Rice (California Department of Fish and Wildlife: Upper Newport Bay, Bolsa Chica, Buena Vista and Agua Hedionda); John Villa (Huntington Beach Wetlands Conservancy’s Lagoon); Mark Berninger and Sara Allen (City of San Diego: Los Penasquitos Creek and San Diego River); Cara Stafford, (California State Parks: Los Penasquitos Marsh); Doug Gibson and Tito Marchant (The Nature Collective: San Elijo Lagoon); Isabelle Kay (University of California Kendall-Frost Mission Bay Marsh Reserve); Edward Owens, Justyn Stahl and Jill Terp (USFWS: Sweetwater and Tijuana Slough National Wildlife Refuge). Aiyana Reissman and her team at the Living Coast Discovery Center were key collaborators in allowing access to captive rails. We thank Justin Brackett and Todd Glazebrook from SeaWorld for providing a crucial blood sample. Rob Fleischer and the Smithsonian Institute provided 1989 rail blood and deoxyribonucleic acid (DNA) samples. Philip Unitt (San Diego Natural History Museum), Kevin Burns (San Diego State University), and Catherine Zeeman (USFWS) provided additional rail samples. Sandra Hamilton (USFWS) provided studbook data and assisted greatly with our questions throughout the project. Steve Bogdanowicz at Cornell University assisted with marker development and bioinformatics. We especially thank Richard Zembal for sharing his experience, annual report data, and assisting with site access. Finally, we especially thank Brian Collins (USFWS, retired), for supporting collaborations that led to the inclusion of genetic research in the rail recovery program.

Contents Acknowledgments��iii Abstract��1 Introduction��1 Purpose and Scope��3 Methods��4 Field Sampling��4 Sampling of Captive-Bred Rails��5 1989 Baseline Samples��5 Marker Development��5 DNA Extraction, Amplification and Sequencing��5 Bioinformatics��6 Population Genetic Dataset��6 Population Structure and Gene Flow��6 Decision Framework for Genetic Rescue��7 Comparing Coancestry and Inbreeding Coefficients from Studbook and Genetic Data��7 Results and Discussion��9 Recent Population Structure��9 Comparisons with Historical Samples��12 Genetic Diversity and Effective Population Size��12 Genetic Rescue��12 Managing Genetic Diversity in the Captive Program��14 Wetland Restoration��14 Preliminary Conclusions and Future Research Objectives��16 References Cited��16 Appendix 1. Supplementary Tables��19

Figures 1. Map showing locations of wetlands where Light-footed Ridgway’s Rails were sampled in southern California for this study between 2020 and 2022 and in 1989��2 2. Graph showing annual pair counts of Light-footed Ridgway’s Rails over time between 2001 and 2024 summed by region��3 3. Graphs showing the number of Light-footed Ridgway’s Rails hatched and released from the captive breeding program between 2001 and 2023 by wetland and grouped into geographic regions��4 4. Graphs showing results of STRUCTURE analyses of Light-footed Ridgway’s Rails supporting three genetic clusters��9 5. Graph showing individual assignment plot for three clusters estimated with STRUCTURE��10 6. Graphs showing principal component analysis plots of major axes of all contemporary sampled Light-footed Ridgway’s Rails��11 7. Graphs showing principal component analysis plots of historical baseline and recent samples of Light-footed Ridgway’s Rails colored by wetland��13

Tables 1. Number of Light-footed Ridgway’s Rails sampled per wetland and regional clusters and corresponding genetic diversity statistics allelic richness rarified to 10 gene copies, private allelic richness, rarified to 10 gene copies, observed heterozygosity, unbiased expected heterozygosity and average pairwise relatedness among individuals��8 2. Estimated gene flow rates among regional populations of Light-footed Ridgway’s Rails��11 3. Tests for differences in genetic differentiation relatedness, allelic richness and unbiased expected heterozygosity in populations of Light-footed Ridgway’s Rails by period��12 4. Linkage disequilibrium estimates of genetic effective population size of Light-footed Ridgway’s Rails populations assuming a monogamous breeding system and using alleles with a frequency of greater than 1 percent��13 5. Genetic rescue decision table for source populations of Light-footed Ridgway’s Rails��13 6. Recent breeding pairs in the captive breeding program of Light-footed Ridgway’s Rails, including hatch years, number of offspring produced, pedigree and genetic-based coancestry/relatedness and individual inbreeding coefficients��15

vii

Conversion Factors International System of Units to U.S. customary units

Multiply

To obtain

Length kilometer (km)

0.6214

mile (mi)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: °F = (1.8 × °C) + 32.

Datum Horizontal coordinate information is referenced to the North American Datum of 1983 (NAD 83).

Supplemental Information Concentrations are given in nanograms per microliter (ng/µL).

viii

Abbreviations >

greater than

less than

confidence interval

DNA

deoxyribonucleic acid

inbreeding coefficient

FST

genetic differentiation

HWE

Hardy-Weinberg equilibrium

effective population size

NWR

National Wildlife Refuge

PCA

principal component analysis

PCR

polymerase chain reaction

USGS

U.S. Geological Survey

USFWS

U.S. Fish and Wildlife Service

Genetic Structure and Diversity in Wild Populations of the Light-Footed Ridgway’s Rail Reflect 20 Years of Augmentation Through Captive Breeding and Release By Amy G. Vandergast,1 Julia G. Smith,1 Anna Mitelberg,1 Dustin A. Wood,1 Kimberly A. Sawyer,2 and Courtney J. Conway1

Abstract Captive breeding and release programs aimed at recovery of rare species can be informed by genetic data to help select high-diversity source populations, make pairing decisions to minimize inbreeding, and manage release strategies. We developed a set of 54 microsatellite loci to assess genetic structure and diversity across the United States range of the Light-footed Ridgway’s Rail (Rallus obsoletus levipes), a federally endangered marsh bird for which populations have been augmented by a captive breeding program annually since 2001. We identified three regional genetic clusters, with the highest genetic diversity reported in the central cluster, which included all sampled wetlands in north San Diego County. Recent (2019–24) captive-breeding adults all clustered within the northernmost cluster (Orange and Ventura Counties), which was expected given that this cluster included the source wetland for the captive breeding program. Gene flow rates, which approximate the proportions of individuals in a population originating from other populations, were relatively high among clusters (4–24 percent) and may have been enhanced through the release of captive-bred rails. Based on the genetic data analyzed in a genetic rescue decision framework, sourcing new breeding birds from the north San Diego County cluster could provide the greatest genetic diversity benefits. The northernmost cluster, which included Mugu Lagoon and all sampled Orange County wetlands, was considered the most in need of genetic rescue. Recent breeding pairs in the captive breeding program have comparatively low diversity and high interrelatedness. Sourcing birds from wetlands with high genetic diversity and population sizes, 1U.S. Geological Survey. 2Idaho Cooperative Fish and Wildlife Research Unit, University of Idaho, Moscow, Idaho.

assessing genetic relatedness before pairing, and focusing releases in areas that have low estimates of genetic diversity could improve the distribution of genetic diversity across wild populations in the future.

Introduction Genetic monitoring is frequently used along with ecological monitoring tools to assess and manage populations of endangered species (Schwartz and others, 2007; Antao and others, 2011). Genetic diversity data can be particularly informative for managing captive breeding and release programs aimed at restoring declining species. The maintenance of genetic diversity can reduce the potential for inbreeding depression and improve fitness in the short term (a few generations; Reed and Frankham, 2003; Spielman and others, 2004; Markert and others, 2010), and preserve adaptive potential in the long term (many generations; Kardos and others, 2021). For these reasons, measuring the amount and distribution of genetic diversity among wild populations can help to identify appropriate source populations and release sites to manage for diversity. In addition, genetic monitoring pre- and post-release can be used along with mark-recapture, telemetry, and other techniques to assess survival and integration of released individuals, and their genes, into wild populations (Bubac and others, 2019). Finally, genetic relatedness information can be incorporated into studbook management to help guide pairing decisions in captive settings to ensure that inbreeding is minimized and that multiple family lineages are consistently represented in captive populations (Ivy and Lacy, 2010).

2 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release 2020). These trends vary regionally, with an apparent increase in north San Diego County marshes but an apparent decline in Orange County (fig. 2); although changes in pair counts over time were not tested statistically (Zembal and others, 2024). In 1989, genetic samples were obtained from four consistently occupied populations (at that time) throughout the rails’ range (Mugu Lagoon, Ventura County, Seal Beach and Newport Bay, Orange County, and Tijuana Slough National Wildlife Refuge (NWR), San Diego County; fig. 1). Genetic analyses of these samples (Fleischer and others, 1995; Nusser and others, 1996), reported low genetic diversity within populations and suggested that movement of individual rails from larger populations into smaller ones could be a possible management strategy.

Light-footed Ridgway’s Rails (Rallus obsoletus levipes; hereafter rails) are restricted to coastal wetlands within a small geographic range spanning from Ventura County, California, to Ensenada, Baja California, Mexico (fig. 1; Eddleman and Conway, 2020). The subspecies was listed as federally endangered in 1969 (Secretary of the Interior, 1969), state endangered in California in 1971, and was added to the official list of at-risk species in Mexico in 2002 (Secretaría del Medio Ambiente y Recursos Naturales, 2002). Annual call-broadcast surveys throughout the subspecies’ U.S. range began in 1980 and have continued to the present (Zembal and others, 2024). During this period, total pair counts have fluctuated from year to year, but have increased slightly since range-wide counts began (U.S. Fish and Wildlife Service,

119° Oxnard

118° Thousand Oaks Los Angeles

MUG

34°

117°

Long Beach

Anaheim Santa Ana

SLB

BOC HUB

UNB SAA Camp Pendleton Oceanside

EXPLANATION MUG 33°

Wetland and identifier1

PACIFIC OCEAN

AGH

BVL

SEL

SDL

Historical sampling location

LPM

Sampling location MUG, Mugu Lagoon; SLB, Seal Beach; BOC, Bolsa Chica Ecological Reserve; HUB, Huntington Beach; SAA, Santa Ana River; UNB, Newport Bay; BVL, Buena Vista Lagoon; AGH, Agua Hedionda; BAT, Batiquitos Lagoon; SEL, San Elijo Lagoon; SDL, San Dieguito Lagoon; LPM, Los Penasquitos Marsh and Creek; KEF, Kendall-Frost Mission Bay Marsh Reserve; SDR, San Diego River; SBM, San Diego Bay National Wildlife Refuge South Bay Unit; SWE, Sweetwater Marsh; TSN, Tijuana Slough National Wildlife Refuge. 1

Escondido

BAT

KEF

SDR

San Diego

SWE

SBM TSN

0 0

10 10

STATES UNITED MEXICO Tijuana

40 KILOMETERS

40 MILES

Figure 1. Locations of wetlands where Light-footed Ridgway’s Rails (Rallus obsoletus levipes) were sampled in southern California for this study between 2020 and 2022 (blue points) and in 1989 (Historical sampling location; yellow points).

Introduction 3 Starting in 2001, a captive-release program was initiated with founders (birds and eggs) sourced from Newport Bay. Subsequently, eggs from Newport Bay have been brought in to replenish the breeding program (maintained at 3–6 pairs annually) about every 2–3 generations. Juvenile rails from this program have been released annually as part of species recovery efforts, with over 600 individuals released across southern California marshes between 2001 and 2023 (fig. 3). All breeding birds in the captive program were either taken from the Newport Bay wild population or from descendant captive offspring. All released birds can be traced to 76 wild founders through their pedigree between 2001 and 2023 (table 1.1).

350

Pair counts of Light-footed Ridgway’s rails

300

250

200

150

100

Purpose and Scope

Captive breeding program year 2001 (year 1) to 2024 (year 24) EXPLANATION Ventura County (Mugu Lagoon)

North San Diego County

Orange County

South San Diego County

Figure 2. Annual pair counts of Light-footed Ridgway’s Rails (Rallus obsoletus levipes) over time between 2001 (year 1) and 2024 (year 24) summed by region (data taken from Zembal and others, 2024). Points represent total pair counts, and lines are locally weighted (LOESS) smoothers. The Orange County region has declined, whereas north San Diego County has increased. Mugu Lagoon (the only population in Ventura County) has remained relatively low in comparison to all other regions.

Although counts have been completed annually at most occupied wetlands since the 1980s, monitoring of movement and survivorship of released juvenile rails had not occurred until very recently (Zembal and others, 2017; Sawyer, 2024; Sawyer and Conway, in press). In addition, genetic monitoring of wild populations and genetic assessment of captive birds have been lacking until this study. Therefore, little is known about the cumulative effects of releases on population genetic structure and diversity of recipient populations. To address these uncertainties, we developed a set of microsatellite markers to allow for genetic monitoring of wild and captive rails. We evaluated the recent (2020–22) genetic population structure and diversity of rail populations throughout their U.S. range. We also compared the recent genetic structure to the pre-augmentation structure by comparing recent blood samples with blood samples available from the initial 1989 genetic surveys. Moreover, we examined genetic connectivity and diversity across the subspecies’ U.S. range to identify extant populations with high genetic diversity that could be considered for future captive-rearing sources.

4 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release 40

Number of Light-footed Ridgway’s rails hatched and released from the captive breeding program

Ventura County (109)

Orange County (96)

North San Diego County (202)

South San Diego County (224)

Captive breeding program year 2001 (year 1) to 2023 (year 23)

EXPLANATION Wetland Carpinteria Salt Marsh Reserve

AGH—Agua Hedionda

MUG—Mugu Lagoon

BAT—Batiquitos Lagoon

SLB—Seal Beach

SEL—San Elijo Lagoon

BVL—Buena Vista Lagoon

LPM—Los Peñasquitos Marsh and Creek

KEF—Kendall-Frost Mission Bay Marsh Reserve SDR—San Diego River Paradise Marsh

SBM—San Diego Bay National Wildlife Refuge South Bay Unit TSN—Tijuana Slough National Wildlife Refuge

SWE—Sweetwater Marsh

Figure 3. The number of Light-footed Ridgway’s Rails (Rallus obsoletus levipes) hatched and released from the captive breeding program between 2001 (year 1) and 2023 (year 23) by wetland (colored bars) and grouped into geographic regions. Numbers in parentheses are the total number of releases in each region.

Methods Field Sampling We visited and captured wild individuals at 17 wetlands throughout the U.S. range for genetic sampling and banding between 2020 and 2022. Sites were visited during the breeding season, roughly between April and September of each year. We used carpet traps (Harrity and Conway, 2020) with a broadcast of Ridgway’s Rail vocalizations to lure rails to the carpet traps (Pickens and King, 2013; Harrity and Conway, 2020). We removed rails from carpet traps immediately after capture, and we measured, weighed, photographed, and

attached a federal leg band (Smith, 2013) to each rail. We collected blood samples from each captured rail via metatarsal venipuncture using a sterile, 26-gauge needle and transferred to a GenSaver 2.0 (AHLSTROM, Escondido, California, cat no. 8.566.0002.B-N) blood card with a non-heparinized capillary tube (Thermo Fisher Scientific, Waltham, Massachusetts, cat no. 22-260943). We then released rails at the capture location. All fieldwork was authorized following guidelines specified in Federal and State permits held by C. Conway (Federal Endangered Species Permit TE039466; Bird Banding Permit #22524; California Memorandum of Understanding (SCP-S-193610002-20008-001), and as approved by the University of Idaho Institutional Animal Care and Use Committee (2015-51).

Methods 5

Sampling of Captive-Bred Rails Beginning in 2019, we collected blood samples from all captive-bred and released rails, and, when available, breeding adults. Blood samples were not regularly taken from captive rails before 2019. Rails were sampled before release, using metatarsal venipuncture, as described in the “Field Sampling” section; we attached a federal leg band (Smith, 2013) to each released bird.

1989 Baseline Samples We received archived blood and genomic deoxyribonucleic acid (DNA) samples from the Smithsonian Museum which were used in previous population genetic analyses (Fleischer and others, 1995; Nusser and others, 1996). These samples were collected in the fall of 1989, before the start of the captive breeding program from four wetlands across the subspecies’ U.S. range: Mugu Lagoon, Seal Beach, Newport Bay, and Tijuana Slough NWR (fig. 1; table 1.2). Although the number of available historical samples per wetland was small by contemporary standards, these samples represent the best available baseline dataset for comparison to recent genetic structure and diversity metrics. All samples were sent to the Western Ecological Research Center’s San Diego Field Station genetic laboratory for extraction and amplification.

Marker Development Microsatellite libraries were developed for R. obsoletus at Cornell University’s Evolutionary Genetic Core Facility using genomic DNA extracted from four individuals. The Evolutionary Genetic Core Facility sequenced a tetrameric, enriched genomic library on an Illumina MiSeq with paired 250 base-pair reads (Nali and others, 2014), used SeqMan NGen (version 11, DNAStar, Madison, Wisconsin) to generate a de novo assembly from the paired fastq files (raw data), and used the program msatcommander 1.0.8_beta (Faircloth, 2008) to scan for candidate microsatellite loci and design primer pairs. To design a panel of highly multiplexed microsatellite markers, we randomly selected and evaluated approximately 500 candidate microsatellite loci (500 forward primers tagged at the 5-prime end with the sequence TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG, and 500 reverse primers, tagged at the 5’ end with the sequence GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG)

for multiplex polymerase chain reaction (PCR) suitability using Multiple Primer Analyzer (MPA; Thermo Fisher Scientific, Sunnyvale, California). We used the MPA output in the package igraph (Csárdi and Nepusz, 2006) to cluster the loci into an arrangement that would minimize primer-dimer formation. This process resulted in four multiplexes, composed of 30–40 loci each. These loci were individually amplified in two individuals, and only loci with successful amplification in both samples (as confirmed by gel electrophoresis) were retained in the final multiplexes. All samples were genotyped using the resulting panel of 108 loci (table 1.3) arranged into four multiplexes (Mpx1-4), as described in the following section.

DNA Extraction, Amplification and Sequencing We extracted genomic DNA from blood cards or capillary tubes using the Puregene kit (QIAGEN, Germantown, Maryland) according to the manufacturer’s protocol, with minor modifications including the addition of Proteinase K to cell lysis with an overnight incubation at 58 degrees Celsius (°C), and final resuspension in 100 microliter (µL) Tris Low ethylenediaminetetraacetic acid (EDTA; TLE) buffer (10 millimolar [mM] Tris, 0.1 mM EDTA, pH 8.0). We quantified extractions using Qubit Broad Range (Thermo Fisher Scientific) and standardized to 10–40 nanograms per microliter (ng/µL) before amplification with the Type-it Microsatellite PCR Kit (QIAGEN). We amplified loci by using four primer cocktails (Mpx1-4; table 1.3), with each primer at a 1.6 micromolar (µM) concentration in the primer cocktail. Each of four 10 µL PCR reactions contained 5 µL 2X Type-it Master Mix, 1 µL of Mpx1, Mpx2, Mpx3 or Mpx4, and 15–60 ng/µL DNA. Amplifications included 30 cycles of 95 °C for 5 minutes, 94 °C for 30 seconds, 56 °C for 1.5 minutes, 72 °C for 1.5 minutes, followed by a 12 °C hold. Upon completion, the four multiplexed PCRs per sample were pooled together, and the pooled PCR product was barcoded using Nextera N5/600 and N7/800 indexes to produce individual dual-indexed amplicon libraries for each sample. Individual sample libraries were then pooled together into one tube per 96-well plate and bead-cleaned to remove primer dimers. Pooled and bead-cleaned libraries from each plate of sample libraries were combined in equimolar proportions and sent for sequencing at MedGenome, Inc. (Foster City, California) on the NovaSeq 6000 (Illumina, San Diego, California), using the Illumina SP 300 cycle reagent kit v1.5.

6 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

Bioinformatics We used the Python script amplicon (https://bitbucket.org/cornell_bioinformatics/amplicon) to extract reads from the Illumina runs and assign them to the appropriate locus and individual. Specifically, the script (1) trims adapters and low-quality reads, (2) creates contigs from overlapping reads (for paired-end sequencing), (3) identifies reads corresponding to each locus, (4) collapses identical reads for each individual, and (5) identifies the top two haplotypes for individuals at all loci (in other words, their diploid genotypes). We used the default options except for the following parameters: -c 1 (minimum number of samples per haplotype), -a 0.001 (minimum minor allele frequency), -l 75 (minimum haplotype length), -r 5 (maximum read count ratio between the two alleles in each sample). We then calculated the total number of reads per locus per individual, whether a locus was heterozygous or homozygous (and if heterozygous, the minimum number of minor allele reads per locus per individual). We scored loci as missing data if the total number of reads was less than (<) 200. Heterozygous loci were recoded as homozygous if the minor allele read count was low (<300), or if the total number of reads was low (<500). Before population genetic analyses, we used the R packages adegenet v. 2.1.10 (Jombart, 2008) and poppr v. 2.9.3 (Kamvar and others, 2014) to assess the quality of loci and samples using several filters. First, we removed any locus with greater than 10 percent missing data, and then removed any individual samples with greater than 10 percent missing data. Next, we applied a minor allele frequency cutoff (MAF=0.01) to identify monomorphic or uninformative loci. Once these loci and samples were removed, we used the R package genepop v. 1.2.2 (Rousset, 2008) to evaluate the dataset for linkage disequilibrium using the exact test for genotypic linkage disequilibrium with 10,000 dememorizations and 5,000 iterations; the significance of linkage disequilibrium was confirmed for loci with p-values below 0.0001. We also used the method described by Brookfield (1996) to estimate the frequency of null alleles for each locus with the R package popgenreport (Adamack and Gruber, 2014). We retained loci with null allele frequencies less than 0.2, following the recommendations of Dakin and Avise (2004).

Population Genetic Dataset During field sampling, we captured and sampled hatch-year and adult rails. However, we removed hatch-year birds from the population structure and diversity analyses to avoid biases resulting from unequal sampling of family groups and to focus on the adult breeding populations present at the time of sampling. We included captive adults used in

the breeding program to represent the captive “population” (hereafter “captive breeders”). We separated the captive breeders into two temporal groups: (1) parents of the captive offspring released before and during the wild sampling period (2019–21; group I), and (2) captive breeders held in the breeding program at the time of this report (2023–24; group II). Group I birds were included along with wild birds in structure and gene flow analyses to help evaluate the influence of the breeding program on genetic structure and diversity. Diversity metrics were calculated for groups I and II to provide information relevant to the breeders in captive breeding facilities at the time of this report. We analyzed the 1989 baseline samples separately from recent samples to compare population structure and diversity pre- and post-augmentation. Loci were screened for deviations from Hardy-Weinberg equilibrium (HWE) at four wetland sites with greater than 20 samples (Newport Bay, Batiquitos Lagoon, San Elijo Lagoon, Tijuana Slough NWR) using an exact test based on 1,000 Monte Carlo permutations of alleles (Guo and Thompson, 1992) and applying the Benjamini and Yekutieli (2001) correction for multiple tests. We removed loci if they deviated significantly (corrected p-value less than 0.05) from HWE at three or more sites.

Population Structure and Gene Flow We used multiple methods to assess population structure. First, we used STRUCTURE (Pritchard and others, 2000) to determine the supported number of genetic clusters (K) that conform to populations in genetic equilibrium. We specified a range for the maximum number of clusters that individuals could be assigned (K=1–10) and completed 10 replicate runs per K using 500,000 iterations of the Markov chain Monte Carlo algorithm following a burn-in of 500,000 iterations to verify consistency across chains. The optimal K was inferred by comparing the results from the maximum mean log-posterior probability for K estimated by STRUCTURE and the change in K (∆ K) criterion (Evanno and others, 2005). Second, we used principal component analysis (PCA) to visualize genotypes in multidimensional space with adegenet v2.1.10 (Jombart, 2008), in R v4.1.2 (R Core Team, 2018). We used the program PopCluster (Wang, 2022a) to estimate gene flow among populations. PopCluster provides estimates of recent gene flow rates (last 3 generations) from an admixture model. We first evaluated up to 10 clusters (K) with 20 replicate runs. After selecting the optimal K, we ran the PopCluster model with migration for 20 replicate runs to estimate gene flow rates among clusters from the individual admixture estimates.

Methods 7 We calculated allelic richness (Ar), private allelic richness (PAr), observed heterozygosity (Ho) and unbiased expected heterozygosity (He), and inbreeding coefficients across marsh sites, and clusters and groups of captive breeders. There was some geographic overlap between cluster assignments in Mission Bay (Kendall-Frost Mission Bay Marsh Reserve and San Diego River). For the purpose of reporting genetic diversity indices by cluster, we grouped these two wetlands in the south San Diego County cluster. The effective population size (Ne) was estimated in NeEstimator v2 (Do and others, 2014) for each cluster and period. We used the linkage disequilibrium method with monogamy and a minimum allele frequency of 0.02, and calculated 95-percent confidence intervals (CI) of point estimates by jackknifing across samples. We compared genetic differentiation (FST), relatedness (R), allelic richness (Ar), and unbiased expected heterozygosity (He) between the baseline and recent sample periods by using group comparisons in FSTAT v2.9.4 (Goudet, 2001), with p-values derived from 10,000 permutations. We restricted our analysis to the three wetlands that were sampled in both periods (Mugu Lagoon, Newport Bay, Tijuana Slough NWR). During the time of our field sampling, only a handful of birds were observed in Seal Beach; we did not pursue sampling there to avoid disturbing the remaining rails. We also ran a PCA across paired wetlands to visualize any changes in genetic clustering over time.

Decision Framework for Genetic Rescue Following the decision framework presented in Frankham and others (2017), we assessed whether populations met certain criteria indicating genetic erosion and whether genetic rescue could improve genetic diversity in local populations. We calculated the mean inbreeding coefficient (F):

Hinbred F = 1 − _ Houtbred

(1)

where Hinbred Houtbred

is the average heterozygosity of the receiver (inbred) population, and is the average heterozygosity of the donor (outbred) population.

F values greater than 0.1 indicate that genetic diversity is sufficiently higher in donor population(s) to benefit the receiver population (Frankham and others, 2017). We used our estimates of expected heterozygosity to calculate F for each regional cluster in relation to the following donors: (1) captive breeders, (2) Orange County cluster, (3) north San Diego County cluster, and (4) south San Diego County cluster.

Comparing Coancestry and Inbreeding Coefficients from Studbook and Genetic Data We calculated pedigree-based coancestry and inbreeding coefficients for captive breeders using the R package kinship2 (Sinnwell and others, 2014). We then estimated the genetic-based relatedness and inbreeding coefficients for captive breeders in the software EMIBD9 (Wang, 2022b). The EMIBD9 software implements a likelihood expectation maximization (EM) method, updating allele frequencies and identity-by-descent coefficients for each pair of sampled individuals until convergence. The EM method estimates relatedness and allele frequencies simultaneously from a small sample of genotypes, in contrast to traditional methods, which rely on unbiased allele frequencies obtained from a large sample of unrelated genotypes (for example, relatedness presented in table 1). We then compared the productivity, pedigree-based metrics and genetic-based metrics for recent breeding pairs.

BVL, Buena Vista Lagoon

10 10

Captive Breeders Group I (2019–22)

Captive Breeders Group II (2023–24)

SWE, Sweetwater Estuary 5

SDR, San Diego River

KEF, Kendall-Frost Mission Bay Marsh Reserve

TSN, Tijuana Slough NWR

South San Diego County

SBM, San Diego Bay NWR South Bay Unit

4 49

LPM, Los Peñasquitos Marsh

North San Diego County

UNB, Newport Bay

SDL, San Dieguito Lagoon

SAA, Santa Ana River

SEL, San Elijo Lagoon

HUB, Huntington Beach

BOC, Bolsa Chica ER

SEB, Seal Beach NWR

BAT, Batiquitos Lagoon

—

Orange County

AGH, Agua Hedionda Lagoon

5 20

MUG, Mugu Lagoon

—

N (current)

Ventura County

Cluster/wetland

1.8

1.79

2.03

2.08

2.3

2.24

2.3

2.07

2.19

2.28

1.95

—

1.99

1.78

—

0.01

0.08

0.04

0.05

0.02

0.03

0.02

0.22

0.01

—

0.03

—

PAr

0.308

0.281

0.377

0.302

0.369

0.407

0.39

0.396

0.427

0.373

0.394

0.351

—

0.355

0.318

—

0.302

0.28

0.339

0.347

0.348

0.378

0.385

0.396

0.374

0.371

0.389

0.323

—

0.332

0.279

—

0.209

0.221

0.078

0.068

0.058

0.011

0.033

0.04

0.158

0.094

0.028

0.102

—

0.094

0.274

—

N (1989)

—

2.03

—

2.03

—

2.02

—

2.07

2.11

—

0.08

—

0.08

—

0.02

—

0.06

0.12

—

PAr

—

0.318

—

0.318

—

0.345

—

0.377

0.407

—

0.329

—

0.329

—

0.338

—

0.355

0.344

—

0.081

—

0.082

—

0.124

—

0.1

0.154

—

[Diversity statistics were not calculated (NC) for wetlands with fewer than five individuals sampled. Abbreviations: ER, Ecological Reserve; NWR, National Wildlife Refuge; —, no samples were taken]

Table 1. Number of Light-footed Ridgway’s Rails (Rallus obsoletus levipes) sampled (N) per wetland and regional clusters and corresponding genetic diversity statistics including allelic richness (Ar) rarified to 10 gene copies, private allelic richness (PAr), rarified to 10 gene copies, observed heterozygosity (Ho), unbiased expected heterozygosity (He) and average pairwise relatedness among individuals (R; Lynch and Ritland, 1999).

8 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

Results and Discussion 9

Results and Discussion

(fig. 6A) along axes 1 (7 percent of the total genetic variation) and 2 (6 percent of the variation), while axis 3 (5 percent of the variation) separated individuals within marsh sites, particularly within the Orange County cluster (fig. 6B). PopCluster also supported three clusters and estimated recent gene flow rates among clusters ranging from 4.1 percent (from north San Diego County to Orange County) up to 23.5 percent (from Orange County to north San Diego County; table 2). Recent gene flow estimates among clusters were high, especially into the north San Diego County cluster. However, natural versus augmented levels of gene flow are difficult to separate in this system given the approximately 20 years (10–20 generations) of captive breeding and releases before genetic monitoring efforts. Higher rates of recent gene flow (last 3 generations) from the Orange County cluster (the source of the captive program) into the other two clusters could reflect these captive release efforts. Recent telemetry data indicate rail movement is usually localized. In a group of transmittered wild (N=42) and captive released (N=46) hatch year rails, only one captive rail moved between wetlands (from Tijuana Slough NWR to the San Diego Bay NWR South Bay Unit, about 4 kilometers [km]); all other rails stayed close to the initial capture locations (Sawyer, 2024). Similar average distances are reported from earlier studies, although occasional long-distance movements of up to 258 km have been recorded (U.S. Fish and Wildlife Service, 2020). The structuring of individual wetlands into three broader genetic populations that appear to be connected by moderate levels of gene flow provides important context for population management, supporting an inclusive regional approach consistent with genetic structure, rather than focused on individual wetlands as independent populations.

During locus and sample evaluations, we identified 17 loci with greater than 10 percent missing data and 24 additional loci that were monomorphic. We also identified nine loci that deviated significantly from HWE, four loci with significant linkage disequilibrium, and no loci with null allele frequencies greater than 0.2. These 54 loci were removed before further analysis. The final dataset retained the remaining 54 loci and included 143 wild adult birds sampled from 17 wetlands (table 1). We included 10 captive parents of the offspring released just before and during the sampling period (group 1; 2019–21) and 10 breeding adults in the captive breeding program at the time of this report (group II; 2023–24). We also included 27 baseline samples from four wetlands sampled in 1989, before augmentation efforts (table 1). Genotype data are available as a USGS data release in Mitelberg and others (2025).

Recent Population Structure Structure analyses best supported three genetic clusters across the range (fig. 4) that roughly corresponded to sampled regions ([1] Orange County plus Mugu Lagoon and the captive breeders, [2] north San Diego County plus San Diego River, and [3] Kendall-Frost Mission Bay Marsh Reserve and south San Diego County; fig. 5). Individuals of mixed assignment were reported in all clusters, indicating recent or ongoing dispersal and gene flow occur directly among wetlands, or that gene flow is facilitated through the efforts of the captive breeding program. Principal component analysis also grouped individuals into three regional clusters

A. Delta K

B. L(K)

200 –10,500

EXPLANATION

L(K), unitless

Delta K, unitless

150

100

–10,750 Mean (plus or minus one standard deviation) –11,000

–11,250

0 1

Genetic cluster (K)

Figure 4. Results of STRUCTURE analyses of Light-footed Ridgway’s Rails (Rallus obsoletus levipes) supporting three genetic clusters (K=3). A, Delta K (Evanno and others, 2005) for 1 to 9 clusters (K). B, mean log-posterior probability of K (L[K]) from STRUCTURE (Pritchard and others, 2000) for 1 to 10 clusters.

Proportion of cluster

10 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

0.75

0.25

CAP

MUG BOC

UNB

SAA/ BVL HUB

AGH

BAT

SEL-E

SEL-W

SDL

LPM KEF

SDR

SBM SWE

TSN

Wetland1 EXPLANATION Cluster 1

Cluster 2

Cluster 3

1 CAP, captive breeders group I; MUG, Mugu Lagoon; BOC, Bolsa Chica Ecological Reserve; UNB, Newport Bay; SAA/HUB, Santa Ana River and Huntington Beach; BVL, Buena Vista Lagoon; AGH, Agua Hedionda; BAT, Batiquitos Lagoon; SEL-E, San Elijo Lagoon (east of I–5); SEL-W, San Elijo Lagoon (west of I–5); SDL, San Dieguito Lagoon; LPM, Los Peñasquitos Marsh and Creek; KEF, Kendall-Frost Mission Bay Marsh Reserve; SDR, San Diego River; SBM, San Diego Bay National Wildlife Refuge South Bay Unit; SWE, Sweetwater Marsh; TSN, Tijuana Slough National Wildlife Refuge.

Figure 5. Individual assignment plot for three clusters estimated with STRUCTURE. Light-footed Ridgway’s Rails (Rallus obsoletus levipes) from Ventura County (Mugu Lagoon), Orange County, and the captive breeders were mainly assigned to Cluster 1 (blue). Cluster 2 (green) was mainly reported in north San Diego County wetlands. Birds from south San Diego County were mostly assigned to Cluster 3 (purple). Mixed assignments indicate genetic exchange across clusters, and the effect of the captive breeding program.

Results and Discussion 11 B

SAA

SWE

KEF SDR LPM SDL AGH SEL

MUG CAP

UNB SAA BOC

PCA 1 (7.28 percent)

TSN SBM

BVL

BAT

BOC MUG CAP

UNB

LPM AGH SDL TSN BATSEL BVM KEF SDR SBM SWE

Eigenvalues

PCA 3 (4.38 percent)

PCA 2 (5.58 percent) EXPLANATION Wetland site code AGH—Agua Hedionda BAT—Batiquitos Lagoon BOC—Bolsa Chica Ecological Reserve BVL—Buena Vista Lagoon CAP—captive breeders group KEF—Kendall-Frost Mission Bay Marsh Reserve

LPM—Los Peñasquitos Marsh and Creek MUG—Mugu Lagoon SAA/HUB—Santa Ana River and Huntington Beach SBM—San Diego Bay National Wildlife Refuge South Bay Unit; SWE, Sweetwater Marsh

SEL—San Elijo Lagoon SDL—San Dieguito Lagoon SDR—San Diego River SWE—Sweetwater Marsh TSN—Tijuana Slough National Wildlife Refuge UNB—Newport Bay

Figure 6. Principal component analysis (PCA) plots of major axes of all contemporary sampled Light-footed Ridgway’s Rails (Rallus obsoletus levipes). Points representing individuals are colored by wetland with standard ellipses around wetlands. A, PCA axes 1 and 2 roughly group wetlands into three overlapping regional clusters (Orange County plus Mugu Lagoon and the captive breeders; north San Diego County; south San Diego County). Inset histograms show the proportions of variance explained by each vector, with plotted vectors shaded black; B, PCA axis 3 (plotted with PCA axis 1) appears less geographically informative, and further separates some individuals, particularly in the Orange County cluster. Table 2. Estimated gene flow rates among regional populations of Light-footed Ridgway’s Rails (Rallus obsoletus levipes). [Columns denote the source populations and rows the receiver populations. Proportions to and from the same populations represent non-migrant sources. Sums greater than 1 indicate overall source populations]

From Orange County

From north San Diego County

From south San Diego County

Orange County1

0.865

0.041

0.094

North San Diego County

0.235

0.656

0.11

South San Diego County

0.11

0.083

0.807

Sum

1.21

0.78

1.011

1Includes all birds sampled within Orange County and all birds sampled at Mugu Lagoon in Ventura County.

12 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

Comparisons with Historical Samples Comparing our recent samples to the 1989 baseline samples across the same set of wetlands suggests that genetic differentiation has declined slightly, relatedness has increased, and allelic richness has decreased over time (table 3). Although none of these changes were statistically significant, the direction of these respective measures is consistent with a small decline in overall genetic diversity in rail populations, which is noteworthy given the increase in sample size in two of three wetlands. The slight decline in FST may reflect the effect of the captive-bred individuals being sourced from a single site (Newport Bay) and released throughout the range or could reflect an increase in naturally occurring dispersal and gene flow among regions, facilitated by the increased population sizes in the center of the range. The PCA of baseline and contemporary samples separated sites spatially along the primary axis (fig. 7A). The second axis separated the temporal sampling periods at Tijuana Slough NWR and the third separated the temporal sampling periods within Mugu Lagoon and Newport Bay (figs. 7A, B). Mugu Lagoon seems to be the most distinctive over time, with non-overlapping point clouds (fig. 7B). Genetic differentiation over time is consistent with genetic drift (loss of genetic diversity over time), which is more extreme in smaller and more isolated populations.

Genetic Diversity and Effective Population Size By all measures, north San Diego County has the highest genetic diversity of all sampled regions, followed by south San Diego County, Orange County and, having the lowest genetic diversity, Mugu Lagoon (table 1). Low genetic diversity at Mugu Lagoon could reflect its position at the northern range edge and consistently low survey numbers. Despite augmentation attempts with more than 100 captive-reared birds between 2001 and 2009, the maximum number of pairs observed at Mugu Lagoon during the last two decades was Table 3. Tests for differences in genetic differentiation (FST), relatedness (R), allelic richness (Ar) and unbiased expected heterozygosity (He) in populations of Light-footed Ridgway’s Rails (Rallus obsoletus levipes) by period. [P-values were all greater than 0.1 and were considered not statistically significant. Abbreviations: MUG, Mugu Lagoon; TSN, Tijuana Slough National Wildlife Refuge; UNB, Newport Bay]

Group

FST

Baseline (MUG, UNB, TSN)

0.081

0.053

1.834

0.335

Current (MUG, UNB, TSN)

0.071

0.196

1.756

0.329

P-value

0.366

0.118

0.125

0.331

in the low 20s, and only a handful of pairs were observed during the past few years (fig. 2; Zembal and others, 2024). Newport Bay also appears to have relatively low levels of genetic diversity compared with its baseline sample. Counts during annual surveys have rapidly declined since 2017 in Newport Bay and in surrounding wetlands in Orange County. Declining numbers here have been attributed to increasing tidal inundation (Zembal and others, 2024). We could not estimate effective population size (Ne) at Mugu Lagoon due to low sample size. Among the other three regions, the contemporary Ne point estimate was lowest in Orange County and highest in north San Diego County (table 4), which is consistent with all other diversity metrics. Contemporary Ne point estimates were lower than baseline samples, although CIs overlapped (table 4). General guidelines suggest Ne should be greater than 50–100 to avoid inbreeding, and greater than 500–1,000 to preserve allelic richness and long-term adaptive potential (Frankham and others, 2014). Orange County may be at or below these lower thresholds (upper 95-percent CI=113), whereas north San Diego (upper 95-percent CI=486) and south San Diego County (upper 95-percent CI=559) may be at or below the upper thresholds.

Genetic Rescue Frankham and others (2017) provides decision tables for determining whether a population could benefit from genetic rescue and state that appropriate source populations should have higher heterozygosity than the receiver population (F>0.1). Because it was estimated to have greater heterozygosity than the other genetic clusters, the north San Diego County cluster could be a genetically beneficial source for all other regions examined, producing F>0.1 in the receiver populations (table 5). Mugu Lagoon, with the lowest heterozygosity of any site, could benefit from genetic rescue from any other source (table 5). Finally, because the captive breeders have low diversity when compared to wild populations, augmentation results in negative F for all sites except for Mugu Lagoon (table 5). New source populations could improve diversity in the captive breeding program (see the “Managing Genetic Diversity in the Captive Program” section). Whether or not wild regional clusters could benefit from genetic rescue can also be assessed with information about population size and isolation. Although Mugu Lagoon and the Orange County cluster have low or declining survey numbers, low effective population sizes, and are geographically more isolated, the north and south San Diego County clusters have larger survey numbers based on recent call-broadcast surveys (fig. 3) and higher effective population sizes. Although gene flow estimates among clusters were high, augmentation through the captive release program could account for some of this, and rates were lowest into the Orange County cluster.

Results and Discussion 13 B

TSN

MUG.HIST MUG UNB.HIST

UNB

TSN.HIST Eigenvalues

UNB.HIST

PCA 1 (7.28 percent)

MUG.HIST

TSN.HIST TSN

UNB MUG Eigenvalues

PCA 3 (5.33 percent)

PCA 2 (6.59 percent) EXPLANATION Wetland site MUG—Mugu Lagoon current

TSN—Tijuana Slough National Wildlife Refuge

UNB—Newport Bay current

MUG.HIST—Mugu Lagoon historical

TSN.HIST—Tijuana Slough National Wildlife Refuge historical

UNB.HIST—Newport Bay historical

Figure 7. Principal component analysis (PCA) plots of historical baseline and recent samples of Light-footed Ridgway’s Rails (Rallus obsoletus levipes) colored by wetland. A, PCA axes 1 and 2 separate the northern and southern sites and historical and contemporary samples in Tijuana Slough National Wildlife Refuge (TSN and TSN.HIST); B, PCA axis 3 separates baseline and recent samples from Mugu Lagoon and Newport Bay, respectively. Table 4. Linkage disequilibrium estimates of genetic effective population size (Ne) of Light-footed Ridgway’s Rails (Rallus obsoletus levipes) populations assuming a monogamous breeding system and using alleles with a frequency of greater than 1 percent. [Corresponding 95-percent confidence intervals (CI) were jackknifed across samples. Estimates for Mugu Lagoon could not be calculated (NC) because of low sample size. An upper CI of infinity (INF) indicates that there is not enough information in the dataset to estimate the upper bound. This can occur when sample sizes are small or when Ne is large. Abbreviation: —, no data]

Current Ne (95-percent CI)

Baseline Ne (95-percent CI)

Ventura County (Mugu)

Orange County

45 (25–113)

140 (49– INF)

North San Diego County

235 (148–486)

—

South San Diego County

115 (55–559)

915 (32– INF)

Region

Table 5. Genetic rescue decision table for source populations of Light-footed Ridgway’s Rails (Rallus obsoletus levipes). [In all cases, birds sourced from the north San Diego County Cluster could provide the greatest potential improvements to genetic diversity. Abbreviations: F, inbreeding coefficient; He, unbiased expected heterozygosity]

F by Source

Region

Orange Captive County

Ventura (Mugu 0.279 Lagoon)

0.004

Orange County 0.332 North San Diego County South San Diego County

Is the population isolated (no or low gene flow)?

Is the population very small or small for multiple generations?

10.2

Yes

North South San San Diego Diego County County

10.16

10.285

−0.185

10.16

0.389

−0.391

−0.173

0.348

−0.348

−0.05

10.106

0.047

Yes

−0.118

1Combinations of source and receiver populations with F-values greater than 0.1 indicate an improvement in genetic diversity.

14 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

Managing Genetic Diversity in the Captive Program Adjustments to captive rearing source populations and release strategies, informed by new empirical estimates of population genetic diversity and structure, could help preserve genetic diversity. The pool of captive breeders has lower genetic diversity than all wild genetic clusters except for Mugu Lagoon. Therefore, the recently released hatch year birds likely added little or no genetic diversity benefit to the receiver populations into which they have been released (table 5). Two factors may contribute to this. First, breeding birds for the captive program have been consistently sourced from one wetland across the range (Newport Bay). However, this wetland has recently declined in size and has low genetic diversity and low effective population size, suggesting it may benefit from genetic rescue itself (tables 4, 5). Second, the captive breeding program is small, composed of up to six pairs annually. Some of the recent breeding birds have high inbreeding coefficients and some pairs have elevated (non-zero) genetic relatedness, despite efforts to minimize pairings between known relatives based on the pedigree (table 6). These genetic estimates could indicate non-zero relatedness among the wild ancestors. Given the small number of breeding birds in the captive program at any one time, efforts to rotate in wild birds more frequently could help to incorporate new genetic diversity. Retaining later generations of offspring in the breeding program could increase relatedness, depending on pairings. Large differences in productivity among breeding pairs may also skew the genetic makeup of captive-released cohorts. This could be reduced by limiting the number of clutches produced by each captive pair each season. Limiting breeding windows, especially to the beginning of the season, may also help increase the probability of survival for captive-released juvenile rails. Analysis of telemetry data indicated that captive rails released early in the summer had higher survival rates than those released later (Sawyer, 2024; Sawyer and Conway, in press). Finally, ensuring receiver sites receive a mix of clutches produced by unrelated pairs could decrease the overall relatedness of birds released at a single site and season. Wetlands in north San Diego County have the highest heterozygosity, allelic richness, and private allelic richness across all surveyed regions and the lowest relatedness. Sourcing birds or eggs from the larger wetlands within the north San Diego County cluster could provide the greatest increase to the genetic diversity and representation within the captive breeding population for future population augmentation (table 4). Because relatedness values were generally higher within than among wetlands even within the

same regional clusters, pairing birds sourced from different wetlands instead of a single wetland could also help reduce the chances of including closely related birds in the captive program. Finally, genotyping all candidate parents could directly estimate genetic relatedness and suggest pairings to minimize inbreeding.

Wetland Restoration Given that wetlands in north San Diego County appeared largely unoccupied before the mid-2000s, it could be possible that a combination of habitat restoration coupled with captive releases (fig. 3) are responsible for the increase in numbers of pairs and high genetic diversity in north San Diego County. In addition, given estimated gene flow rates of 8–11 percent between south and north San Diego County genetic clusters, it is possible that natural dispersal of wild birds may be sufficiently high to maintain genetic diversity and connectivity across this part of the subspecies’ range. Although opportunities to restore wetlands may be rare throughout the northern part of the subspecies’ range, restored wetlands could provide more stepping stones for increased connectivity. In the more immediate time frame, our genetic analyses suggest that Orange County and Mugu Lagoon clusters could benefit from augmentation and genetic rescue from a higher-diversity source population. Another important factor in maintaining high diversity is retaining large populations to minimize the erosion of local genetic diversity. Habitat management and restoration can assist in maintaining large populations and could become even more critical given predicted sea-level rise, which may threaten wetland habitat in areas without sufficient upland habitat for marsh retreat (Osland and others, 2022), and may already be affecting the population at Newport Bay (Zembal and others, 2024). Models of California wetland vulnerabilities to sea-level rise, including three marshes occupied by rails (Newport Bay, Sweetwater, and Tijuana Slough NWR) predicted significant loss of high and middle marsh habitat by 2050 and between 50- and 100-percent conversion to bare mudflats by 2100 under moderate to high sea-level rise scenarios (Thorne and others, 2018). Survival of juvenile rails is affected by elevation, and the timing and water level at high tide (Sawyer, 2024; Sawyer and Conway, in press). The abundance of raptors may also have a negative effect on survival, especially for captive-released rails (Sawyer, 2024; Sawyer and Conway, in press). A recent 5-year study of mortality in California Ridgway’s Rails in San Francisco Bay indicated that avian predators accounted for most of the observed mortalities (Casazza and others, 2016).

2023

2021–22

2022–23

2023

In progress

8322, 8392

8383, 8251

8903, 8392

7941, 9263

8933, 9075

2020–22

7511, 7612

2021–22

2018–19

6801, 6761

8172, 8482

2018–19

8122, 8222

2018–20

6751, 6791

Offspring hatch years

6781, 6811

Breeding pair studbook numbers (sire and dam)

In progress

Number of offspring produced

0.03125

0.125

Pedigree-based coancestry of breeding pair

0.0286

0.0742

0.0032

0.0061

0.0074

0.0481

0.0433

Genetic relatedness of breeding pair

0.125

Pedigree-based inbreeding coefficient of sire

0.0701±0.0242

0.0036±0.0036

0.0171±0.0085

0.1169±0.0169

0.0283±0.0099

0.0287±0.0141

0.0792±0.0237

0.0534±0.0130

0.0683±0.0175

Mean genetic inbreeding coefficient of sire

Pedigree-based inbreeding coefficient of dam

0.0438±0.0125

0.0498±0.0120

0.1360±0.0252

0.3799±0.0182

0.1360±0.0252

0.0234±0.0159

0.1370±0.0170

0.0207±0.0076

0.0403±0.0148

Mean genetic inbreeding coefficient of dam

[Pedigree-based statistics were calculated in kinship2 and genetic statistics were calculated in EMIBD9. Superscripts following sire and dam studbook numbers denote the maximum number of generations the individual is removed from wild ancestors. Abbreviations: ±, plus or minus; NC, not calculated because a genetic sample was not available]

Table 6. Recent breeding pairs in the captive breeding program of Light-footed Ridgway’s Rails (Rallus obsoletus levipes), including hatch years, number of offspring produced, pedigree and genetic-based coancestry/relatedness and individual inbreeding coefficients (±standard deviations).

Results and Discussion 15

16 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

Preliminary Conclusions and Future Research Objectives In collaboration with researchers from Mexico, USGS has received samples from the southernmost part of the range in Ensenada, Baja California, Mexico (Estero de Punta Banda and Bahía de San Quintín). Genetic and genomic analyses of these samples can help characterize genetic diversity across the full subspecies range. A larger, genome-wide set of single nucleotide polymorphisms (Peterson and others, 2012) may provide greater sensitivity to discern any additional structure among sampled wetlands, could help assess genomic diversity, and may better resolve effective population sizes given small sample sizes (Andrews and others, 2016). Nevertheless, results to date suggest that the microsatellite loci described and analyzed here identified regional patterns in genetic diversity in wild populations and estimates of genetic relatedness and inbreeding of captive rails. These markers could provide a cost-effective tool to monitor genetic diversity in the breeding program moving forward.

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Zembal, R., Hoffman, S.M., and Konecny, J., 2017, Light-footed Ridgway’s (Clapper) Rail in California—2017 season: Final Report to U.S. Fish and Wildlife Service and California Department of Fish and Wildlife, 39 p.

Secretaría del Medio Ambiente y Recursos Naturales, 2002, Protección ambiental—Especies nativas de México de flora y fauna silvestres—Categorías de riesgo y especificaciones para su inclusión, exclusión o cambio—Lista de especies en riesgo: Diario Oficial de la Federación, Segunda sección, p. 1–85. Sinnwell, J.P., Therneau, T.M., and Schaid, D.J., 2014, The kinship2 R package for pedigree data: Human Heredity, v. 78, no. 2, p. 91–93. [Available at https://doi.org/10.1159/000363105.] Smith, G.J., 2013, The U.S. Geological Survey Bird Banding Laboratory—An integrated scientific program supporting research and conservation of North American birds: U.S. Geological Survey Open-File Report 2013–1238, 88 p. [Available at https://doi.org/10.3133/ofr20131238.] Spielman, D., Brook, B.W., and Frankham, R., 2004, Most species are not driven to extinction before genetic factors impact them: Proceedings of the National Academy of Sciences, v. 101, no. 42, p. 15261–15264. [Available at https://doi.org/10.1073/pnas.0403809101.]

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Appendix 1. Supplementary Tables 19

Appendix 1. Supplementary Tables Table 1.1. Founder contribution to the release program by region and overall, expressed as percentage of the 655 Light-footed Ridgway’s Rails (Rallus obsoletus levipes) released in southern California wetlands between 2001 and 2024.

Founder

Year of first offspring released

Year of last offspring released

Mugu Lagoon

Orange County

North San Diego County

South San Diego County

Percent total contribution

WILD8

2001

2005

0.88

0.38

0.88

0.53

2.67

WILD7

2001

2005

0.88

0.38

0.88

0.53

2.67

WILD1

2001

2005

0.88

0.38

0.88

0.46

2.60

WILD2

2001

2005

0.88

0.38

0.88

0.46

2.60

WILD3

2001

2006

1.35

0.11

0.50

0.39

2.35

WILD5

2001

2006

1.35

0.11

0.50

0.39

2.35

WILD4

2001

2006

1.35

0.11

0.50

0.39

2.35

WILD6

2001

2006

1.35

0.11

0.50

0.39

2.35

WILDSB2

2003

0.00

0.53

0.00

0.53

WILDSB1

2003

0.00

0.53

0.00

0.53

WILD9

2003

2012

1.35

0.15

0.73

0.28

2.51

WILD10

2003

2012

1.35

0.15

0.73

0.28

2.51

WILD14

2004

2008

0.11

0.00

0.11

WILD13

2004

2008

0.11

0.00

0.11

WILD11

2004

2010

0.08

0.19

0.23

0.34

0.84

WILD12

2004

2010

0.08

0.19

0.23

0.34

0.84

WILD18

2005

2007

0.08

0.00

0.04

0.19

0.31

WILD17

2005

2007

0.08

0.00

0.04

0.19

0.31

WILD24

2006

2013

0.84

0.36

0.88

0.43

2.51

WILD23

2006

2013

0.84

0.36

0.88

0.43

2.51

WILD27

2006

2014

0.23

0.61

0.97

1.11

2.91

WILD28

2006

2014

0.23

0.61

0.97

1.11

2.91

WILD19

2007

2009

0.11

0.00

0.11

0.08

0.31

WILD20

2007

2009

0.11

0.00

0.11

0.08

0.31

WILD22

2007

2012

0.27

0.19

0.34

0.31

1.11

WILD21

2007

2012

0.27

0.19

0.34

0.31

1.11

WILD25

2007

2012

0.11

0.00

0.11

0.08

0.31

WILD26

2007

2012

0.11

0.00

0.11

0.08

0.31

WILD15

2008

2009

0.04

0.08

0.00

0.11

WILD16

2008

2009

0.04

0.08

0.00

0.11

WILD32

2008

2012

0.27

0.19

0.31

0.11

0.88

WILD31

2008

2012

0.27

0.19

0.31

0.11

0.88

WILD30

2008

2018

0.15

1.11

2.18

2.30

5.74

WILD29

2008

2018

0.15

1.11

2.18

2.30

5.74

WILD36

2009

2012

0.00

0.19

0.00

0.08

0.27

WILD35

2009

2012

0.00

0.19

0.00

0.08

0.27

20 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release Table 1.1. Founder contribution to the release program by region and overall, expressed as percentage of the 655 Light-footed Ridgway’s Rails (Rallus obsoletus levipes) released in southern California wetlands between 2001 and 2024.—Continued Year of first offspring released

Year of last offspring released

Mugu Lagoon

Orange County

North San Diego County

South San Diego County

Percent total contribution

WILD33

2009

2018

0.00

0.31

0.98

1.01

2.29

WILD34

2009

2018

0.00

0.31

0.98

1.01

2.29

WILD39

2010

0.00

0.08

WILD40

2010

0.00

0.08

WILD37

2010

2014

0.00

0.03

0.05

0.18

0.25

WILD38

2010

2014

0.00

0.03

0.05

0.18

0.25

WILD47

2011

0.00

0.08

0.00

0.15

WILD48

2011

0.00

0.08

0.00

0.15

WILD51

2011

2012

0.00

0.19

0.08

0.00

0.27

WILD52

2011

2012

0.00

0.19

0.08

0.00

0.27

WILD49

2012

0.00

0.08

0.00

0.08

WILD50

2012

0.00

0.08

0.00

0.08

WILD41

2012

2015

0.00

0.08

0.31

0.08

0.46

WILD42

2012

2015

0.00

0.08

0.31

0.08

0.46

WILD_L

2012

2016

0.00

0.04

0.69

0.11

0.84

WILD_K

2012

2016

0.00

0.04

0.69

0.11

0.84

WILD46

2012

2016

0.00

0.04

0.69

0.11

0.84

WILD45

2012

2016

0.00

0.04

0.69

0.11

0.84

WILD56

2014

0.00

0.08

0.00

0.08

WILD54

2014

0.00

0.08

0.00

0.08

WILD55

2014

0.00

0.08

0.00

0.08

WILD53

2014

0.00

0.08

0.00

0.08

WILD57

2014

2019

0.00

0.15

0.00

0.15

WILD58

2014

2019

0.00

0.15

0.00

0.15

WILD_I

2018

2019

0.00

0.18

0.20

0.38

WILD_J

2018

2019

0.00

0.18

0.20

0.38

WILD66

2018

2023

0.03

0.34

0.94

2.39

3.71

WILD65

2018

2023

0.03

0.34

0.94

2.39

3.71

WILD64

2018

2023

0.03

0.34

0.86

2.39

3.63

WILD63

2018

2023

0.03

0.34

0.86

2.39

3.63

WILD69

2018

2024

0.13

0.76

0.64

1.34

2.87

WILD68

2018

2024

0.13

0.84

0.56

1.34

2.87

WILD67

2018

2024

0.13

0.84

0.56

1.34

2.87

WILD70

2018

2024

0.13

0.76

0.64

1.34

2.87

UNK_B

2019

0.00

0.08

UNK_C

2019

0.00

0.08

WILD_A

2020

2024

0.13

0.27

0.41

0.94

1.75

WILD_B

2020

2024

0.13

0.27

0.41

0.94

1.75

WILD_H

2022

2024

0.10

0.27

0.10

0.29

0.74

WILD_G

2022

2024

0.10

0.27

0.10

0.29

0.74

All founders

2001

2024

17.25

16.49

30.99

35.27

100.00

Founder

Appendix 1. Supplementary Tables 21 Table 1.2. Baseline blood and DNA samples of Light-footed Ridgway's Rails (Rallus obsoletus levipes) collected in 1989 and provided by R. Fleischer, Smithsonian Institution. [DNA, deoxyribonucleic acid; ID, identification; NWR, National Wildlife Refuge]

Sample ID this study

Smithsonian sample ID

Sample type

Site

County

TSN_001

486

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_002

487

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_003

488

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_004

490

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_005

491

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_006

492

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_007

493

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_008

494

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_009

495

Blood (capillary)

Tijuana Slough NWR

San Diego

TSN_082

498

Blood (capillary)

Tijuana Slough NWR

San Diego

UNB_001

496

Extracted DNA

Newport Bay

Orange

MUG_001

497

Extracted DNA

Mugu Lagoon

Ventura

UNB_002

601

Extracted DNA

Newport Bay

Orange

UNB_003

602

Extracted DNA

Newport Bay

Orange

UNB_004

605

Extracted DNA

Newport Bay

Orange

UNB_005

606

Extracted DNA

Newport Bay

Orange

UNB_006

607

Extracted DNA

Newport Bay

Orange

UNB_007

608

Extracted DNA

Newport Bay

Orange

MUG_002

609

Extracted DNA

Mugu Lagoon

Ventura

MUG_003

613

Extracted DNA

Mugu Lagoon

Ventura

MUG_004

614

Extracted DNA

Mugu Lagoon

Ventura

UNB_008

616

Extracted DNA

Newport Bay

Orange

SEB_001

622

Extracted DNA

Seal Beach

Orange

SEB_002

623

Extracted DNA

Seal Beach

Orange

SEB_003

624

Extracted DNA

Seal Beach

Orange

SEB_004

626

Extracted DNA

Seal Beach

Orange

Mpx1

Mpx2

RAOB_1332

RAOB_14777

RAOB_17999

RAOB_2350

RAOB_23910

RAOB_25163

RAOB_2774

RAOB_3020

RAOB_32277

RAOB_34562

RAOB_36470

RAOB_4239

RAOB_4254

RAOB_4555

RAOB_4746

RAOB_4853

RAOB_5114

RAOB_5737

RAOB_6796

RAOB_7107

RAOB_7389

RAOB_10511

RAOB_1164

RAOB_1329

RAOB_13848

RAOB_13860

RAOB_1556

RAOB_1639

RAOB_20271

RAOB_22344

RAOB_22510

RAOB_2335

RAOB_24304

RAOB_2524

RAOB_25393

RAOB_2565

RAOB_2664

Multiplex

RAOB_12508

Locus

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTAGGCAATGCTTCTTTCTGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTCCAAGACTCAGTGACATC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGTTTGGCTCATCTCTGTAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGATAAGAAGCATGGAGGAAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGCTGCCATGGAAGGATG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTACACTAGGAAGGCTGCTTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTGTGATGGTGTGATGCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTACTTGCCATACTTCAGAAGCG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGTCTGTGCACTTGTTCTCTAC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAACCTTGACACTGAGATAGCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACAGTCTGGACCATGGATG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCATCAGTGCAGTGTTCAGAAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAGATGTGCCTTTGTTACATGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACACAAATCTTTAAAGCTGGGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAGCAAGATCTTACCAGTCCTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGCCCTGCTCATATTGTCAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAAACTGTTGCGTTCGCTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTAGTACAGTCTGCCCTTAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGCTCATATGTAGAGAAAGTCCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAGGGAGTAGGTTGTGTATTGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTTTGCCTTTGATCTCCAGAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTTCAGAGAAGCACATGGAAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTATGACTAGGAAGCTGGGAGTC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCACTTGGCCAAACAAATGTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTGTGAGGATGTGAATGAGGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTCCTGCAGAGCGTGTTTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGCAGAAATCCATAGAGAAGCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTACTCTGGTTCTTGAGTGTGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTCCAGGTGAGCTGAGTTAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAGCAAAGAAAGTGAGGGCATC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTGCATGAGTGTTGGATACC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTCCTCCTGTTACTAGGAAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGATTCATTCCCACACCTACAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCCAGAATTTCGATACACATGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTACTTCACACCCAGAATCAACC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTCTTCTCCCATCACCTCTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGACTTTACTCTTCCTCGTTTC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAAGGCCCATAATGCTTTGAAG

Forward primer sequence

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGGCAGATCTTAGGAAACAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTACAAATCCCACTACCAGCAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTAGGAGAGGACCCATGAAAGAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGGTTTAGGAGGTGTTTAGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTTGGTCTTCTTGTTACCAGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATCTAGCCATCAATTCCCAGTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATGGGTGCTTGGAGGTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTGCACACATGAAACTGGAAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTATCCAGGTCATTCATCCACC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCAGGGAGAGCCTTATATTGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTACTTGCAAACTCCCACCTAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTGTTCATTGCTTTCACCACC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTAGATGAGCGGGTACTTAGAGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTATTCCACTATTCCAGCAAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGAGAAATGAGGATCAGCTTGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGAATTCAAGGTGGGTGCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGAAGGAGGGAGACACTACAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGAGCTCTGACATTAACTGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATGCAATTTACACTTGCTCAAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTTGAGTTTGGAAGGTTTGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTGCCGCTTCCATCCATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACAATGCCACTTTCTTGAGAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCTTAGTGCTTGGAGTTCTTTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTACCACCAACAGAAGACAATTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTAACCAAGGGCACAGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCAGCTCAGAGGTATCAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGGCTCCTCTAGAAGTATTCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCAAGGACAGGCAGCAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTGACCAGATGATTACAGAGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCTGCTTCTGGTTTGGTTTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGCAAACCATCAATGTTTAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCTTTACCATCTTGCACTCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGCAGCATGAACTTTAAGGTTTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCGTTCGGTTTAGAAGAATCATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTCACAGGCTGTTCTATCACTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTCTTCTTGGAAAGGCAAATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATTGCTGGTACTGGTCACATG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCCCATCATCAGGGTCTG

Reverse primer sequence

Table 1.3. Microsatellite primers and multiplex mixes designed for Light-footed Ridgway’s Rails (Rallus obsoletus levipes). All primer sequences are presented 5’ to 3’.

22 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

Mpx2

Mpx3

RAOB_3540

RAOB_3642

RAOB_3754

RAOB_4634

RAOB_4709

RAOB_4768

RAOB_5228

RAOB_5271

RAOB_5797

RAOB_6334

RAOB_6838

RAOB_6945

RAOB_13269

RAOB_13477

RAOB_1383

RAOB_1475

RAOB_15280

RAOB_15740

RAOB_1743

RAOB_1786

RAOB_2253

RAOB_2268

RAOB_24572

RAOB_2982

RAOB_3322

RAOB_3577

RAOB_35986

RAOB_3965

RAOB_45183

RAOB_4686

RAOB_4758

RAOB_4933

RAOB_5623

RAOB_5805

RAOB_6407

RAOB_7609

Multiplex

RAOB_3316

Locus

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTCAGTGATGAGTAGGTGGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCAACACAAAGCAGCTGTAAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAAATGGAGTCTTTGGCTCTAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCACTTGTCTCGCTTTGATGTTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGATGGAAAGTCTGGTGATCTC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTAACATGGGAATTCAGCTGGTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTTGAGGCTTTGACAGGTTTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTATGAAGAAAGGATGGGTGGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGCCAAATGACAACTGAAAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTATGGCACTGGGAAGGAAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAGAGGGCTGATAGATGATGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCAGACCCACACAAATGAAGAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTCATCAGGGCTTCTTTGTTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTAGGTGGAAAGGTCAGATGTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCACAGATGCAAAGACAGGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCAGGATGTGGATTTGTAGGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGTGTAGTTATCAAGCCAGGTC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTTGAGTTGGGAAGTAGGGAAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTCTCCTCCAAGTCTGTTTGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACAGAGGAAGGGAAGGGAAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCAAATGGAGGTTCAACTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGCCACTCTCCTTCACAATATC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTTCACGTGGCAAGTCAC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAACCTTACCCATGAGCATTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGCTGGAAACCACCCTCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGTGTCTGGGAGATGCTAAC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAAGAGAGAGTTGTTGGTAGGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGAAAGCTGTGAGAAAGGAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCTTGTGTTTGTTCTTCCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTCGGTGTTGTGGGTTGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTGGAGATATGGTTTAGCAGCG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTAGGTCCATATGCAGACAAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGGAGGAGTTACTAGGGTTGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCAGTGTTCTTGCTTACTTGTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGGCCTACAAATGGGTCAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACAGTAATGCAGGAAGTCATGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAGGTCAGGGTGTTACTTCTTG

Forward primer sequence

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTCTCTTGGTGTATTATGGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACCGTGAGTTACCCAAAGATTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTGACAATGGCAAAGTGTTTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTGTTAACCAAGCAACTGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGCGTAGTTACAAAGACAGAGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGGTTGCAGCACATTCAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCACAACAGGGTCACATGAATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACGACTATTTGTGCAAGACC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTGCTCCTGGGTTTAGCTCTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTGCTGGTGAAGACTGAGTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAACCTTCAGCTCCTCCAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAACTTCAGGGAACGAGGTATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATGTTTAGAAGGGTGATTGCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAACCCACAGAACCAAGGAAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTCTCCATCTAGCCCTCAAAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCAAAGGACAAGTTAGGAGTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTTGAAATGGAATGGAAATTGAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAAGGCTCGAGATAAGGACAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCTGTGTAAGGCTGTAAGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCTTCTTAGCATTTCTGAGGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGGAATAGCAAGGTCTGGATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGTTCTGGTTAGCTGCTGTTAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGATGAGATGCACAGAGAAGAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCACAGAGGGATGGCAAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGCTTGATCGTTATGGGAAAGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATAGCTATTCATTCCACAGTTAAAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTGAGACCTGTGACTTGTTCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATCATGGGTCAAGGTAAAGGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTGGAAGCCTGTGAATCAAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCAAGTGGAGCTGTTGTGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTCATGTTTGTATGCTCTCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGAGGTATCAGTATTGTGGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGCAGGACTATGTTGTACAGTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAATGATGAAGCAGTAGGGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACAGTGCAGGGAAACATCATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGAGCAAGAGTAGGTTTGGTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAAGTGTAGTGATTGCATGGC

Reverse primer sequence

Table 1.3. Microsatellite primers and multiplex mixes designed for Light-footed Ridgway’s Rails (Rallus obsoletus levipes). All primer sequences are presented 5’ to 3’.—Continued

Appendix 1. Supplementary Tables 23

Mpx3

Mpx4

RAOB_9050

RAOB_11545

RAOB_1299

RAOB_1423

RAOB_1474

RAOB_1492

RAOB_1552

RAOB_1748

RAOB_1837

RAOB_1950

RAOB_19980

RAOB_2122

RAOB_2211

RAOB_2246

RAOB_2325

RAOB_26068

RAOB_28243

RAOB_28586

RAOB_29614

RAOB_3740

RAOB_40351

RAOB_4473

RAOB_554

RAOB_653

RAOB_6556

RAOB_6812

RAOB_7093

RAOB_746

RAOB_807

RAOB_854

RAOB_8994

RAOB_9014

Multiplex

RAOB_8569

Locus

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCTTTGCAGAGTCACAAGTC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCCTACCTCCACATTGTAAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTAGGCTGCATGGAGTTCAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAAGAAAGCCATATTTAGAAGGTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTTGGAGATTCTTGTGTTGGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAGAACAAGCAGGATGACC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAGCATTGTCTGTCCTGGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTGAGAGATTTCCTGAGGGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCAGGTGTTCTTAGTTCTGAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCAGATGTGAGGTTTGCAGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTGCTCCTTCTAATTTGCCTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGCAACAGAGGGTCTAATCAC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCTTCCAGATTCCCAACTC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTATCCTCTTACATTTGGCCACC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATACAGATTGATGGCATCTTGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAGCCGCCGTCAATCAC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCCTGAATTACCCTCTTCCAC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCAGTAGTAGAAAGCCAGACTC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACACTCAAGCAACAATAAACCTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTTCTGGGCTGAGATTTCCTAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTACAGGAAGACAAAGGAGCAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAAGAAGTAACGTGTAGGTCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAGGAGTTGGACTTGATGATCC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTCCTTCCTCCTCAACAATGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAAGGCAGAATAGGGCAGAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTACATCTTCCTGGAGTTCACC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTAAGCAGATGAGACCAGACCAG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACAGGAAGGTGCAGCAGG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTGAAGAAGGAGGCAGAGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTGAGTGGGTTAGGCTCTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTCAAATCCCTCCTGAATGCTG

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGGATGCAGTCGTGGC

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGCGTAGTTACAAAGACAGAGC

Forward primer sequence

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCACCTGGTCATAGAAAGACC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACAGCGACTACCCTGAAGATAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAGGAACAAGAGCAGTTGGAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACCAGGTTCCCAGCTTATC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATCCATCCTTCTGTCCTCCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTGTGTGTTGGAGCAGAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAGCTGCAGTCTATTCAGAGAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAAATTCAAGAGGTCATGTGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATTTGGATCCAGTTGCCTAGTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTGTTAGGCATGCATTCATTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGCCATTTGTGGTCATGTTAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGAGTTGAATCACTCGCTATCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGAACTAAAGGACAAAGGAGCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCCTATCAGTGCATTATGTCC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATACAGGGCTTTGGTGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCATCTGTGCCATGGACAGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCCATGCCTCCTTCTAATAAAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAACGTTATCAGACATACCAGGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACATTTGGAGGTGAAGAGCTAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGGCACAATTCCACAAGTAAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTAGCCCAAAGTCTCAATGGAAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTCAGCATCTTCATAGCCCTG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCATGGGCAAAGTTTGGTAGATG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTAAAGCCAGAGGAAATAGGTGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTACTCTTCCCTCAACCACTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGAGCACGTAAACAACAGAAGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACAGGTAAGAAGCAAAGAAATGAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAGCATCCAATACATCCAGGG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGTGCTTGCTCCTCTCCAG

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTTCCACCCATCCACTTGTC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCAGAGGAAGAGAAGGGC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTGCTGCTTGTCGAGGTAC

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGATGGAAAGTCTGGTGATCTC

Reverse primer sequence

Table 1.3. Microsatellite primers and multiplex mixes designed for Light-footed Ridgway’s Rails (Rallus obsoletus levipes). All primer sequences are presented 5’ to 3’.—Continued

24 Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release

For more information concerning the research in this report, contact the Director, Western Ecological Research Center U.S. Geological Survey 3020 State University Drive East Sacramento, California 95819 https://www.usgs.gov/centers/werc Publishing support provided by the Science Publishing Network, Sacramento Publishing Service Center

Vandergast and others—Genetic Structure, Diversity in Wild Populations of Light-Footed Ridgway’s Rail, 20 Years of Captive Breeding and Release— OFR 2025–1011

ISSN 2331-1258 (online) https://doi.org/10.3133/ofr20251011

Genetic Structure and Diversity in Wild Populations of the Light-Footed Ridgway’s Rail Reflect 20 Years of Augmentation Through Captive Breeding and Release

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