INTRODUCTION
Ecotypic variation within a species often corresponds to selection pressures that give rise to local adaptation, and studies of local adaptation are important for understanding how selection may lead to evolution in natural systems. These kinds of population-level ecotypic differences have been commonly described in freshwater fishes (stream and lake stickleback, Gasterosteus aculeatus ; Hendry et al., 2002), coastal and inland annuals (Mimulus guttatus ; Lowry et al., 2008), and island finches (large- and small-billed Geospiza fortis ; Hendry et al., 2009). Among vagile, widely distributed, continental birds, however, local adaptation at a broad scale is less commonly described (but see Alcaide et al., 2014). Specifically, in avian taxa, geographic variants—described variably as populations, races, or subspecies depending on the magnitude of differentiation—are often expressed through plumage or song, with a less obvious connection to their environments (Toews et al., 2016; Toews, Taylor, et al., 2016). These phenotypic differences can be further obscured by overlap with respect to ecological affinities and have presented significant debate to their contribution to biodiversity and conservation science (Mayr, 1982; Zink, 2004).
Wood warblers of North, Central, and South America (Parulidae) are particularly notable among bird species for lacking strong ecological disparities: the classic study of niche partitioning by MacArthur (1958) was in fact initiated because “ecologists studying them have concluded that any differences in the species’ requirements must be quite obscure”. Indeed, although MacArthur observed fine-scale habitat partitioning, it is still generally appreciated that some of the most distinct differences among warbler species occur within plumage and song (Kent et al., 2021; Price et al., 2000). Moreover, any morphological and ecogeographic differences within most species are not discrete, but clinal in nature, such as the gradual increase in body size with increasing latitudes (Jones et al., 2003; Youtz et al., 2020).
One exception to the general pattern—where there appears to be discrete, non-clinal morphometric variation within a warbler species—occurs within the black-throated green warbler (Setophaga virens ). This Neotropical-Nearctic migrant is a common breeder in upland, mixed-hardwood and evergreen forests of the eastern USA and much of Canada (Morse & Poole, 2020). Its occurrence is much rarer in several disjunct breeding locations scattered across the southern periphery of its breeding range (Haggerty, 2009; Mumford et. al., 1984; Rodewald, 1997), including the south Atlantic Coastal Plain where one formerly recognized subspecies, S. virens waynei , was first described (Bangs, 1918). Colloquially referred to as Wayne’s warbler, S. v. waynei is uniquely associated with swamp and non-riverine wetland habitats (Sprunt, Jr., 1953; Watts et al., 2011; Worm & Carpenter, 2021) and separated by more than 400 km (and up to 1200 m elevation) from the closest conspecifics in the southern Appalachian Mountains. While plumage differences from the nominate form may be subtle, S. v. waynei  is reported to be smaller, especially in bill dimensions, even when compared to individuals from the same latitude (Morse & Poole, 2020). Despite these reported differences in morphology and habitat use—as well as continued recognition ofS. v. waynei as a Species of Greatest Conservation Need in three U.S. state Wildlife Action Plans (North Carolina Wildlife Resources Commission, 2015; South Carolina Department of Natural Resources, 2014; Virginia Department of Game and Inland Fisheries, 2015)—this population is now considered “non-diagnosable” (Morse & Poole, 2020), leaving the status of S. v. waynei as a meaningful subspecies in doubt.
Genetic diagnosability is important for conservation to appropriately delineate management units and prioritize limited resources; however, differences between S. virens and S. v. waynei as reflected in the genome remain unknown. This uncertainty may delay critical conservation action for S. v. waynei, which has experienced recent declines (Watts et al., 2011) likely related to loss and degradation of its breeding habitat (Davis et al., 1997; Richardson, 1983).
To address this taxonomic debate and fill a critical knowledge gap, we apply whole-genome resequencing to quantify, for the first time, genetic difference across the S. virens complex, including samples obtained from putative S. v. waynei individuals. Our goals are to determine: 1) whether S. v. waynei cluster independently from other S. virens populations, 2) if there are genetic differences among the groups, including the extent and distribution of the divergence (i.e., is differentiation clustered into regions of the genome), and 3) approximately when and where S. v. wayneiseparated from the nominate form. This information is essential to better understand the unique evolutionary history of the species (Bermingham et al., 1992) and to increase our ability to conserve and manage a putative subspecies of concern within the complex.