Appendix A. Hydrologic methods and classification.

Hydrologic methods and classification

Appendix B. Life history and hydrologic metric relationships.

Life history and hydrologic metric relationships

Genetic Differentiation, Isolation-by-Distance, and Metapopulation Dynamics of the Arizona Treefrog (<i>Hyla wrightorum</i>) in an Isolated Portion of Its Range

<div><p>Population attributes such as diversity, connectivity, and structure are important components of understanding species persistence and vulnerability to extinction. <i>Hyla wrightorum</i>, the Arizona treefrog, is native to the southwestern United States and Mexico, and an isolated group of populations exists in the Huachuca Mountains and Canelo Hills (HMCH) of southeastern Arizona, USA. Due to concerns about declining observations of the species within the isolated HMCH portion of its range, the HMCH group is currently a candidate for federal protection under the U.S. Endangered Species Act. We present results of a genetic study examining population diversity, structure, and connectivity within the HMCH region. We sampled DNA from <i>H</i>. <i>wrightorum</i> larvae and adults from ten distinct locations, 8 of which were breeding sites and 4 of which were previously undescribed localities for the species. We developed and genotyped 17 polymorphic microsatellite loci and quantified genetic diversity, population differentiation, and landscape influences on population genetic structure. We found evidence of larger than expected effective population sizes, significant genetic differentiation between populations, and evidence of distance being the primary driver of genetic structure of populations with some influence of slope and canopy cover. We found little evidence of recent genetic bottlenecks, and individual-based analyses indicate admixture between populations despite significant genetic differentiation. These patterns may indicate that the breeding sites within the Huachuca Mountains constitute a metapopulation. We suggest that the HMCH region may contain larger and more connected breeding populations than previously understood, but the dynamics of this system and the limited geographic extent of the HMCH group justify current concern for the persistence of the species in this region. Efforts to ensure availability of high-quality breeding habitats and control for local threats such as effects of invasive predators may be critical to the persistence of these unique populations of <i>H</i>. <i>wrightorum</i>.</p></div

GeneClass2 results for <i>H</i>. <i>wrightorum</i>.

<p>GeneClass2 results for <i>H</i>. <i>wrightorum</i>.</p

<i>F</i>_<i>ST</i>, lin<i>F</i>_<i>ST</i>, <i>D</i>_<i>ps</i> values for <i>Hyla wrightorum</i> populations.

<p><i>F</i>_<i>ST</i>, lin<i>F</i>_<i>ST</i>, <i>D</i>_<i>ps</i> values for <i>Hyla wrightorum</i> populations.</p

Bottleneck test results for <i>Hyla wrightorum</i>.

<p>Bottleneck test results for <i>Hyla wrightorum</i>.</p

Structure results for <i>Hyla wrightorum</i>.

<p>Numbers correspond to sampling locations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160655#pone.0160655.g001" target="_blank">Fig 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160655#pone.0160655.t001" target="_blank">Table 1</a>). Each vertical bar represents one individual. Colors indicate the most likely genetic cluster assignments. Black vertical bars denote individuals from the same sampling locations. Each cluster was hierarchically analyzed for nested structure; nested structure results are shown directly below the original cluster. Hierarchical analyses were repeated until terminal clusters (<i>K</i> = 1) were reached. Note that results for <i>K</i> = 2 (upper-most Structure plot) and <i>K</i> = 3 (just below <i>K</i> = 2) clusters are shown for all individuals, because both had similar support (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160655#pone.0160655.s007" target="_blank">S6 Table</a>). Hierarchical analyses are shown following <i>K</i> = 3 for all individuals. Nested, colored outlines on the map correspond to population clusters.</p

Mixed-effects models and landscape genetic results for <i>Hyla wrightorum</i>.

<p>Mixed-effects models and landscape genetic results for <i>Hyla wrightorum</i>.</p

<i>Hyla wrightorum</i> range map and sampling locations.

<p>The known range extent of <i>H</i>. <i>wrightorum</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160655#pone.0160655.ref006" target="_blank">6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160655#pone.0160655.ref009" target="_blank">9</a>] (A, B), and <i>H</i>. <i>wrightorum</i> sampling locations in the Huachuca Mountains and Canelo Hills region (C). Symbols indicate 2014 sampled locations (blue) or unsampled locations at which <i>H</i>. <i>wrightorum</i> were observed in 2015 (white). Symbols for sampled locations are proportional to sample size, and color indicates life stage of sampled individuals (dark blue = adults; light blue = larvae). Population numbers are shown in black font, and information for each sampling location included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160655#pone.0160655.t001" target="_blank">Table 1</a>.</p

<i>Hyla wrightorum</i> individuals and breeding sites.

<p>(A) <i>H</i>. <i>wrightorum</i> breeding pair at Site 1; (B) Site 10; (C) Site 2; (D) Site 4; and (E) <i>H</i>. <i>wrightorum</i> larvae sampled at Site 4. Sites in B, C, and D are all new localities for this species.</p