Assay Design
We used a pair of avian universal primers (L14080 and H15049; Sorenson 2003) to sequence a region of the ND5 gene within the mitochondrial genome of 10 Rallidae species, including R. obsoletus (Table 1). Resulting sequences were used for assay design. To begin, we extracted DNA from tissues supplied by natural history museums (Table 1) using Qiagen DNeasy Blood and Tissue kits. Extractions then underwent PCR amplification for ND5 using Invitrogen Platinum Taq and associated manufacturer-suggested reaction volumes (at a total volume of 50 µL, with 2 µL of DNA template) and thermal cycler programs (with an annealing temperature of 55 °C and 40 PCR cycles). Resulting PCR amplicons underwent FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) clean-up before undergoing Sanger sequencing on an Applied Biosystems (ABI) 3500XL using ABI’s BigDye Terminator v 3.1 sequencing chemistry. Resulting sequences were assembled and aligned in Geneious Prime v 2019.2.3, then trimmed to approximately 679 bp before submission to Genbank (NCBI Accession # ON241255 – ON241264). Using this ND5 alignment and Primer3 (Rozen & Skaletsky 2000), we designed two qPCR assays for use in US-based R. obsoletus eDNA surveys: RIRA-ND5-1 and RIRA-ND5-2.
Table 1. Tissues from Rallus obsoletus (Ridgway’s rail) and 9 non-target, potentially co-occurring species used in eDNA assay development, acquired from the Florida Museum of Natural History (FLMNH) and the San Diego Natural History Museum (SDNHM; in conjunction with the San Diego State University Museum of Biodiversity). Voucher origin represents the state from which the specimen was collected: California (CA), Florida (FL), and Louisiana (LA).
Species
common name
|
Species
scientific name
|
No. of tissues
|
Loaning institution: tissue accession #
|
Voucher origin
|
Target
|
|
|
|
|
Ridgway’s rail
|
Rallus obsoletus
|
2
|
SDNHM: 52750, 53162
|
CA
|
Non-target
|
|
|
|
|
King rail
|
Rallus elegans
|
1
|
FLMNH: 92758
|
LA
|
Clapper rail
|
Rallus crepitans
|
1
|
FLMNH: 92827
|
FL
|
Virginia rail
|
Rallus limicola
|
1
|
SDNHM: 54296
|
CA
|
Sora
|
Porzana Carolina
|
1
|
SDNHM: 54148
|
CA
|
Common gallinule
|
Gallinula galeata
|
1
|
SDNHM: 52890
|
CA
|
American coot
|
Fulica Americana
|
1
|
SDNHM: 54256
|
CA
|
Purple gallinule
|
Porphyrio martinica
|
1
|
FLMNH: 96637
|
FL
|
Yellow rail
|
Coturnicops noveboracensis
|
1
|
FLMNH: 91692
|
FL
|
Black rail
|
Laterallus jamaicensis
|
1
|
FLMNH: 71852
|
FL
|
Assay Validation
We next undertook in silico, in vitro, and in situ testing of designed assays (Goldberg et al., 2016). Both assays were first assessed in silico for specificity to R. obsoletus using Primer-BLAST (Ye et al. 2012), as provided online by the National Center for Biotechnology Information (NCBI). Primer-BLAST was executed with no taxonomic or genome locus exclusions, and with the nr database selected (date of query: April 2022).
In vitro qPCR tests for assay specificity were conducted against DNA samples from the species listed in Table 1. All qPCRs had total volumes of 20 μL containing 1X TaqMan Environmental Master Mix, 0.5 μM of each primer, 0.125 μM of the probe, and 1 ng of DNA template. The qPCR cycling program began with an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 62 °C for 1 min (60 °C for RIRA-ND5-1). All reactions were run on an ABI ViiA 7 Real-Time PCR System.
We next investigated the performance of our assays using 46 water samples (in situ testing) collected in October 2019 from 36 sampling points spread among four different locales (Suppl. File, Table S1). Locales included two national wildlife refuges (NWRs) with contemporary R. obsoletus satellite-tracking observations (Harrity et al. 2020): Imperial NWR, AZ and Sonny Bono Salton Sea NWR, CA. Other locales were positioned within Department of Defense (DoD) lands. The first, Naval Weapons Station Seal Beach in Anaheim Bay, CA (NWSSB), has contemporary survey records of R. obsoletus. Additionally, a small number of samples were taken from Vandenberg Space Force Base, CA (SFB; formerly Vandenberg Air Force Base), which is situated approximately 150 km north of the known northernmost extent of southern coastal populations of R. obsoletus and approximately 300 km south of the known southern extent of northern coastal populations of R. obsoletus. At the two NWRs, samples were taken from within or along the edges of thick stands of reeds or rushes, and guided by observed individual R. obsoletus site use observation (satellite-tracking data). At NWSSB, samples were taken from near-shore waters along reaches expected to be used by R. obsoletus (R. Schallmann, personal communication). On Vandenberg SFB, samples were taken from points proximal to riparian, emergent vegetation (e.g., rushes).
When collecting water filtrates we followed protocols published by the US Forest Service’s National Genomics Center for Wildlife and Fish Conservation (Carim et al. 2016). For these filtrate samples, water from suspected R. obsoletus habitat was pumped through one or more 0.2 µm cellulose nitrate (CN) filters. Water turbidity and filter clogging rates were variable, and sample volumes ranged from 0.15 – 5 L. For a majority of samples (14 of 23), ≥ 4.75 L was filtered. Upon completion of sample collection, filters were preserved in silica beads until DNA extraction. Water grab samples were taken at a subset of sites, including some sites at which filtrate samples were also procured (Suppl. File, Table S1). Grab samples were collected using up to five 50 mL screw-top polypropylene tubes, for total volumes of 200 – 250 mL (Suppl. File, Table S1). At NWSSB, filtering was extremely difficult due to rapid clogging and only grab samples were obtained at the majority of these sites. Overall, 50% of our samples were filtrate samples and 50% were grab samples (Suppl. File, Table S1). All samples were kept at 4 °C over the course of the sampling effort (4 days), then shipped overnight on ice to our laboratory facilities, after which they were stored at -20 °C until extraction.
DNA was extracted from all samples (filtrate and grab) using a modified CTAB protocol (cetyltrimethyl ammonium bromide; Doyle and Doyle 1987; Guan et al. 2019). For those sampling points where more than one filter was employed, all associated filters were combined into a single CTAB extraction. An extraction negative control was included with the sample CTAB extractions. Three qPCRs were run for each sample with each assay, following the thermal cycling parameters described previously. Each 384-well qPCR plate included the extraction negative control and a qPCR negative control (i.e., no template control). A sample was considered positive for R. obsoletus eDNA if one or more of its replicate qPCRs resulted in fluorescence above the critical threshold of detection (CT). The CT was automatically set by ViiA 7 software. Amplicons from a subset of positive qPCRs were sequenced to verify accuracy to the intended target. Following qPCR, amplicons were cleaned using EDTA/Tris-EtOH precipitation, and then sequenced in both directions using an ABI 3500XL genetic analyzer and ABI BigDye Terminator v 3.1 Cycle Sequencing Kit.
Assay Sensitivity and Efficiency
The limits of detection (LOD) and limits of quantification (LOQ) for validated assays were assessed using serial dilutions of synthetic DNA templates (gBLOCKs Gene Fragments; Integrated DNA Technologies). Methods used to determine the LOD and LOQ followed Klymus et al. (2020) and Guan et al. (2019). Briefly, for LOD, qPCRs were run with each assay, using 24 replicate solutions for each of the following template concentrations: 16, 8, 4, and 2 copies per reaction. LOD was delineated by the template concentration at which a 95% detection rate was observed. For LOQ, qPCRs were run with each assay, using 8 replicate solutions for each of the following template concentrations: 128, 64, 32, 16, 8, 4, 2, and 1 copies per reaction. LOQ was identified as the template concentration at which the observed coefficient of variation (CV) among replicates was ≤ 35%. The amplification efficiency of each assay was determined by the ViiA 7 software and was based on a standard curve generated using the following synthetic DNA template concentrations: 31250, 6250, 1250, 250, 50, and 10 copies per reaction.