Molecular Phylogenetics and Evolution 134 (2019) 61–65 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Short Communication Hiding in the lianas of the tree of life: Molecular phylogenetics and species T delimitation reveal considerable cryptic diversity of New World Vine Snakes ⁎ Robert C. Jadina, , Christopher Blairb,c, Michael J. Jowersd, Anthony Carmonaa, John C. Murphye,1 a Department of Biology, Northeastern Illinois University, 5500 North St., Louis Avenue, Chicago, IL 60625-4699, USA b Department of Biological Sciences, New York City College of Technology, The City University of New York, 285 Jay Street, Brooklyn, NY 1120, USA c Biology PhD Program, CUNY Graduate Center, 365 5th Ave., New York, NY 10016, USA d CIBIO/InBIO (Centro de Investigação em Biodiversidade e Recursos Genéticos), Universidade do Porto, Campus Agrario De Vairão, 4485-661 Vairão, Portugal e Science and Education, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA ARTICLE INFO ABSTRACT Keywords: The Brown Vine Snake, Oxybelis aeneus, is considered a single species despite the fact its distribution covers an Bayesian estimated 10% of the Earth’s land surface, inhabiting a variety of ecosystems throughout North, Central, and Biodiversity South America and is distributed across numerous biogeographic barriers. Here we assemble a multilocus mo- Oxybelis lecular dataset (i.e. cyt b, ND4, cmos, PRLR) derived from Middle American populations to examine for the first RASP time the evolutionary history of Oxybelis and test for evidence of cryptic lineages using Bayesian and maximum Reptilia likelihood criteria. Our divergence time estimates suggest that Oxybelis diverged from its sister genus, Leptophis, Serpentes approximately 20.5 million years ago (Ma) during the lower-Miocene. Additionally, our phylogenetic and species delimitation results suggest O. aeneus is likely a complex of species showing relatively deep species-level di- vergences initiated during the Pliocene. Finally, ancestral area reconstructions suggest a Central American origin and subsequent expansion into North and South America. 1. Introduction deciduous forests, and tropical rainforest (Keiser, 1982; Van Devender et al., 1994). Due to the extensive distribution and morphological New World vine snakes of the genus Oxybelis are long, slender variation in O. aeneus, various populations were described as distinct snakes that possess a dramatically elongated head and are specialized numerous times (e.g. Dryophis vittatus Girard, 1854; O. microphthalmus for arboreality (Fig. 1). These species are diurnal with excellent vision Barbour and Amaral, 1926; O. potosiensis Taylor, 1941). However, in for hunting small lizards. Currently there are four species of Oxybelis, all spite of the many geographic barriers (e.g. Isthmus of Tehuantepec, with populations in Central America (Kӧhler, 2008; Uetz et al., 2018). Isthmus of Panama, Mexican Volcanic Belt, Andes Mountains and the Oxybelis wilsoni Villa and McCranie, 1995 is known only from Isla de Amazon River) occurring across its range and morphological diversity, Roatán, Honduras while O. brevirostris (Cope, 1861) and O. fulgidus the Brown Vine Snake is currently considered a single, extremely (Daudin, 1803) occur in Central and South America. However, the variable species (Keiser, 1974, 1982). Brown Vine Snake, O. aeneus (Wagler, 1824) has a distribution far ex- Although Oxybelis aeneus and O. fulgidus have recently been in- ceeding its congeners extending from southern Arizona southward cluded in broad molecular phylogenetic analyses to assess evolutionary through Central America and into South America to southeastern Brazil. histories among snake genera, the current phylogenetic position of This distribution covers more than 58° of latitude, a distance of more Oxybelis within the Colubrinae is unresolved (e.g. Pyron et al., 2013; than 9000 km on a north-south axis, and approximately 15 million Figueroa et al., 2016) and relationships within the genus have not been square kilometers (∼10% of the Earth’s land surface), making it one of investigated. In this study, we implement multiple phylogenetic ana- the most widespread snake species on the planet (Keiser, 1982). Oxy- lyses using mitochondrial (mtDNA) and nuclear (nDNA) DNA datasets belis aeneus has an elevational range extending from sea level to at least to assess the evolutionary history and timing of diversification of the 2500 m and populations inhabit semi-desert, tropical savanna, seasonal genus Oxybelis. More specifically, we conduct the first phylogeographic ⁎ Corresponding author at: Department of Biology, University of Wisconsin Eau Claire, Eau Claire, WI 54702, USA. E-mail address: [email protected] (R.C. Jadin). 1 Present address: 2564 E. Murdoch Ct., Green Valley, AZ 85614, USA. https://doi.org/10.1016/j.ympev.2019.01.022 Received 8 October 2018; Received in revised form 6 December 2018; Accepted 25 January 2019 Available online 01 February 2019 1055-7903/ © 2019 Elsevier Inc. All rights reserved. R.C. Jadin, et al. Molecular Phylogenetics and Evolution 134 (2019) 61–65 Chironius carinatus Area B O. fulgidus 1 100 D O. wilsoni 1 A Lineage 1 1 C 100 Lineage 2 O. aeneus 0.97 E 93 Lineage 3 0.54 67 F 3.0 Lineage 4 Oligocene Miocene Plio. Pleis. 3 5 3 0 2 5 2 0 1 5 1 0 5 0 Ma Fig. 1. Phylogenetic relationships and divergence times within Oxybelis based on coalescent-based species tree analysis in StarBEAST2 (cyt b, PRLR, cmos; total of 2112 bp). ‘Species' were defined based on posterior probability support obtained from multiple BPP runs. Values above branch indicate posterior probability values for relationships, whereas values below nodes represent bootstrap support values from SVDquartets. Plio. = Pliocene; Pleis. = Pleistocene. Locality map representing populations of Oxybelis sampled (Table 1). Colored areas represent geographic regions used for DEC ancestral area reconstruction: (A) Sierra Madre Occidental Pine- oak forest, Sierra Madre del Sur Pine Forest, and Sinaloan Dry Forest (O. aeneus Lineage 1, sea green); (B) Central American Moist Forest (O. fulgidus, green); (C) Central American Montane Forest (O. aeneus Lineage 2, gold); (D) Isla de Roatán, Honduras (O. wilsoni, aqua star); (E) Isthmian-Pacific Moist Forest (O. aeneus Lineage 3, sepia); and (F) Trinidad and Tobago Moist Forest (O. aeneus Lineage 4, olive). Insert is an in-life photograph of O. aeneus from Tobago (J.C. Murphy). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) assessment of the Brown Vine Snake, O. aeneus, throughout much of its deposited in GenBank (MK497173-MK497235) and combined with northern range and assess if biogeographic barriers found to affect several other Oxybelis sequences, along with different colubrid se- other Middle American taxa influence Oxybelis. Finally, we estimate quences previously published on GenBank (Supplemental Appendix). ancestral areas to investigate the historical biogeography of Oxybelis. PRLR sequences were generated in this study to assess intrageneric relationships within Oxybelis and were unavailable on GenBank for 2. Materials and methods other taxa. Therefore this gene was not used for the analysis to de- termine the phylogenetic position of Oxybelis within the Colubridae. 2.1. Molecular data Likewise, ND4 sequences were generated in only a few Oxybelis speci- mens to evaluate the phylogenetic status of Oxybelis among numerous Full details for all methods and analyses are provided in the sup- genera, for which ND4 sequences are prevalent, but was not sequenced plementary materials. Genomic DNA was extracted from tissues of in enough Oxybelis specimens to determine relationships within Oxy- Oxybelis aeneus and O. fulgidus (Table 1) using a Qiagen DNeasy ex- belis and therefore ND4 was excluded from those analyses. We selected traction kit and protocol. Two mitochondrial [Cytochrome b (cyt b) and ingroup taxa based on recent studies that found Oxybelis in a clade of NADH dehydrogenase subunit 4 (ND4)] and two nuclear [oocyte ma- New World colubrids (Pyron et al., 2013; Jadin et al., 2014; Figueroa turation factor mos (cmos) and prolactin receptor (PRLR)] gene frag- et al., 2016). ments were independently amplified using GoTaq Green master mixby Promega, (Madison, WI, USA) with the primer pairs: L14910 + H16064 2.2. Phylogenetic analyses (cyt b), ND4 + LEU (ND4), S77 + S78 (cmos), and PRLR_f1 + PRLR_r3 (PRLR). Annealing temperatures were 48, 46, 55, and 50 °C, respec- To determine the phylogenetic placement of Oxybelis within colu- tively. Sequencing was performed in both forward and reverse direc- brids, we conducted three independent analyses using both maximum tions using the PCR primers on a Beckman Coulter automated capillary likelihood (ML) and Bayesian (BI) criteria. Both unpartitioned and sequencer, and sequence chromatographs were edited using Geneious partitioned (by locus) ML analyses of the concatenated cyt b, ND4, and R6 6.1.6. Gaps in alignments were treated as missing data and no in- cmos data (2319 bp) were performed in RAxML v.8.2.11 (Stamatakis, ternal stop codons were found. Novel sequences from this study were 2014) under a GTR+GAMMA model of nucleotide substitution (-f a 62 R.C. Jadin, et al. Molecular Phylogenetics and Evolution 134 (2019) 61–65 Table 1 Genbank numbers for DNA sequences analyzed in this study. Abbreviations of institutions and individuals