As an obligate salt marsh species, Seaside Sparrows (Ammospiza maritima) are vulnerable to numerous threats including climate change, coastal erosion, sea-level rise, and both natural and anthropogenic disasters. Of the 9 recognized subspecies, 2 are extinct and 1 is endangered. Previous genetic analyses of mitochondrial DNA (mtDNA) and microsatellite loci showed that current taxonomy does not accurately reflect underlying genetic diversity, with possible consequences for the distribution of conservation resources. To further inform Seaside Sparrow management, we comprehensively describe genetic structure among 24 range-wide sampling locations that include all extant subspecies. We inferred population structure from several thousand single-nucleotide polymorphisms collected from 272 individuals via restriction-site-associated DNA sequencing. Principal components, pairwise FST values, and clustering approaches suggest that Seaside Sparrows on the Atlantic and Gulf Coasts are distinct and consist of at least 5 genetic clusters: 1 in southern Texas, 1 ranging from Aransas County, Texas, to Mississippi; 1 in western Florida; and 2 or 3 genetic groups intermixed along a gradient on the Atlantic Coast. These genetic clusters are not consistent with current subspecies taxonomy and could be used as distinct population segments (DPSs) to inform the most efficient allocation of resources to Seaside Sparrow conservation. Our results regarding the endangered subspecies, A. m. mirabilis, from southern Florida are inconclusive due to low sample size, but indicate that it is distinct and may represent a sixth DPS. Based on our genetic results, we recommend additional song and morphometric analyses in western Florida and a closer study of the boundary between the breeding distributions of A. m. maritima and A. m. macgillivraii to ensure the proper identification of DPSs.

Museum specimens collected prior to cryogenic tissue storage are increasingly being used as genetic resources, and though high‐throughput sequencing is becoming more cost‐efficient, whole genome sequencing (WGS) of historical DNA (hDNA) remains inefficient and costly due to its short fragment sizes and high loads of exogenous DNA, among other factors. It is also unclear how sequencing efficiency is influenced by DNA sources. We aimed to identify the most efficient method and DNA source for collecting WGS data from avian museum specimens. We analyzed low‐coverage WGS from 60 DNA libraries prepared from four American Robin (Turdus migratorius) and four Abyssinian Thrush (Turdus abyssinicus) specimens collected in the 1920s. We compared DNA source (toepad versus incision‐line skin clip) and three library preparation methods: (1) double‐stranded DNA (dsDNA), single tube (KAPA); (2) single‐stranded DNA (ssDNA), multi‐tube (IDT); and (3) ssDNA, single tube (Claret Bioscience). We found that the ssDNA, multi‐tube method resulted in significantly greater endogenous DNA content, average read length, and sequencing efficiency than the other tested methods. We also tested whether a predigestion step reduced exogenous DNA in libraries from one specimen per species and found promising results that warrant further study. The ~10% increase in average sequencing efficiency of the best‐performing method over a commonly implemented dsDNA library preparation method has the potential to significantly increase WGS coverage of hDNA from bird specimens. Future work should evaluate the threshold for specimen age at which these results hold and how the combination of library preparation method and DNA source influence WGS in other taxa.

Museum specimens collected prior to cryogenic tissue storage are increasingly being used as genetic resources, and though high throughput sequencing is becoming more cost-efficient, whole genome sequencing (WGS) of historical DNA (hDNA) remains inefficient and costly due to its short fragment sizes and high loads of exogenous DNA, among other factors. It is also unclear how sequencing efficiency is influenced by DNA source. We aimed to identify the most efficient method and DNA source for collecting WGS data from avian museum specimens. We analyzed low coverage WGS from 60 DNA libraries prepared from four American Robin (Turdus migratorius) and four Abyssinian Thrush (Turdus abyssinicus) specimens collected in the 1920s. We compared DNA source (toepad versus incision-line skin clip) and three library preparation methods: 1) double-stranded, single tube (KAPA); 2) single-stranded, multi-tube (IDT); and 3) single-stranded, single-tube (Claret Bioscience). We found that the multi-tube ssDNA method resulted in significantly greater endogenous DNA content, average read length, and sequencing efficiency than the other tested methods. We also tested whether a predigestion step reduced exogenous DNA in libraries from one specimen per species and found promising results that warrant further study. The ~10% increase in average sequencing efficiency of the best performing method over a commonly implemented dsDNA library preparation method has the potential to significantly increase WGS coverage of hDNA from bird specimens. Future work should evaluate the threshold for specimen age at which these results hold and how the combination of library preparation method and DNA source influence WGS in other taxa.

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