Phylogeography of the Rough Greensnake, Opheodrys aestivus (Squamata: Colubridae), using multilocus Sanger sequence and genomic ddRADseq data

The Rough Greensnake, Opheodrys aestivus, is a moderately-sized, semi-arboreal snake broadly distributed throughout eastern North America. While numerous taxa with similar distributions have been shown to be comprised of multiple species, O. aestivus has yet to be examined in a detailed phylogeographic context. Here, we use Sanger-sequence data of one mitochondrial and three nuclear loci for samples from throughout the distribution of O. aestivus to elucidate phylogeographic patterns in this species. We combine this with ddRADseq data for a subset of samples to test patterns on a more genomically comprehensive scale. In both datasets, we find strong support for three deeply divergent clades within O. aestivus: peninsular Florida, central Texas, and a main clade comprising the rest of the distribution, with the Florida clade the earliest diverging lineage of the three. Estimates of divergence time suggest that the central Texas and main clades diverged approximately 1.34 million years ago (Mya), while the peninsular Florida clade diverged from other lineages approximately 2.94 Mya, and these lineages diverged from the sister taxon, O. vernalis, approximately 6.43 Mya. These results also suggest that the historically recognized Florida subspecies, O. a. carinatus, could be elevated to species status. While the divergence of peninsular Florida or central Texas populations is not unique among squamates, nor is low levels of divergence from the Atlantic coast to eastern Texas, this combination of patterns is unusual, and yields important insight into the biogeography of North American biota. Further, our approach helps illustrate how dense geographic sampling with limited genomic sequencing can be used as a guide for the selection of samples to test phylogeographic patterns comprehensively.

Methods
We obtained 47 samples of Opheodrys aestivus from throughout the distribution of the species, four samples from the sister taxon O. vernalis, and one sample from each of the more distantly related colubrid species Cemophora coccinea, Lampropeltis getula, Pantherophis guttatus, Pituophis melanoleucus, and the natricid Nerodia sipedon (Appendix 1). Whole genomic DNA was extracted for all samples using a Qiagen DNeasy Blood and Tissue kit and quantified using a Qubit 2.0 fluorometer with the High Sensitivity kit (Invitrogen, Inc.). we targeted one mitochondrial (cytochrome b, cytb) and three nuclear loci (LAT clone, LAT; neurotrophin 3, NT3; and prolactin receptor, PRLR). We used maximum likelihood (ML) and Bayesian inference (BI) methods to infer the phylogenetic relationships among populations of O. aestivus. To further evaluate putative species boundaries and clades recovered in the Sanger sequencing analyses in a more genomically comprehensive context, we also used a double-digest restriction associated DNA sequencing (ddRADseq) for a small subset of the samples, representing all clades identified from Sanger-sequence analyses. A subsample of 13 samples of O. aestivus, spanning the distribution of the species and all lineages recovered in Sanger-data analyses, along with a single sample the sister species, O. vernalis.

粗鳞绿蛇（Opheodrys aestivus）是一种体型中等的半树栖蛇类，广泛分布于北美东部地区。尽管诸多分布范围相似的分类群已被证实包含多个物种，但粗鳞绿蛇尚未在精细的系统地理学框架下开展过研究。本研究利用覆盖该物种全分布范围的样本的1个线粒体基因座与3个细胞核基因座的桑格测序（Sanger-sequence）数据，以阐明该物种的系统地理学格局；同时结合部分样本的双酶切限制性位点相关DNA测序（ddRADseq）数据，在更全面的基因组尺度下验证上述格局。

在两组测序数据中，我们均获得了强烈支持的结果：粗鳞绿蛇内部存在3个深度分化的演化支——佛罗里达半岛支系、德克萨斯中部支系，以及包含剩余分布范围的主干演化支，其中佛罗里达支系是3个支系中最早分化的支系。分化时间估算结果显示，德克萨斯中部支系与主干演化支的分化时间约为134万年前（百万年前，Mya），佛罗里达半岛支系与其余支系的分化时间约为294万年前，而上述支系与姊妹类群绿蛇（Opheodrys vernalis）的分化时间约为643万年前。本研究结果还表明，历史上被认定的佛罗里达亚种（O. a. carinatus）可被提升为独立物种。

尽管佛罗里达半岛或德克萨斯中部种群的分化在有鳞目动物中并非独有，大西洋沿岸至德克萨斯东部的种群分化程度较低也并非罕见，但这种格局的组合却较为特殊，这为理解北美生物群落的生物地理学特征提供了重要见解。此外，本研究的方法还阐明了如何通过高密度地理采样结合有限的基因组测序，为全面验证系统地理学格局的样本选择提供指导。

研究方法
本研究共采集了覆盖粗鳞绿蛇全分布范围的47份样本、姊妹类群绿蛇（Opheodrys vernalis）的4份样本，以及亲缘关系较远的游蛇科物种猩红游蛇（Cemophora coccinea）、普通王蛇（Lampropeltis getula）、玉米锦蛇（Pantherophis guttatus）、北美松蛇（Pituophis melanoleucus），以及水游蛇亚科的北方水蛇（Nerodia sipedon）各1份样本（详见附录1）。所有样本均采用Qiagen DNeasy血液与组织试剂盒提取全基因组DNA，并使用Qubit 2.0荧光计搭配高灵敏度试剂盒（Invitrogen公司）进行定量。本研究靶向扩增1个线粒体基因（细胞色素b，cytochrome b，cytb）与3个细胞核基因座（LAT克隆，LAT；神经营养因子3，NT3；催乳素受体，PRLR），并采用最大似然法（maximum likelihood，ML）与贝叶斯推断法（Bayesian inference，BI）解析粗鳞绿蛇种群间的系统发育关系。

为在更全面的基因组尺度下进一步评估桑格测序分析中得到的潜在物种分界与演化支，本研究还针对代表桑格测序分析中所有已鉴定演化支的部分样本开展了双酶切限制性位点相关DNA测序（ddRADseq）。本次亚采样共选取13份覆盖粗鳞绿蛇全分布范围且涵盖桑格数据鉴定出的所有支系的样本，以及1份姊妹类群绿蛇（Opheodrys vernalis）样本。