Accepted on 1 November 2013 © 2013 Blackwell Verlag GmbH J Zoolog Syst Evol Res doi: 10.1111/jzs.12055 Short Communication 1Department of Ecology and Evolution, University of Colorado at Boulder, Boulder CO, USA; 2Department of Biology, Northeastern Illinois University, Chicago IL, USA; 3Department of Biology, The Graduate School and University Center, The City University of New York, New York NY, USA; 4Department of Biology, The College of Staten Island, CUNY, Staten Island NY, USA; 5Museo de Biologıa, Facultad Experimental de Ciencias, La Universidad del Zulia, Maracaibo Venezuela; 6Sam Noble Museum and Department of Zoology, University of Oklahoma, Norman OK, USA; 7Instituto de Biodiversidad Tropical, Uvita Puntarenas, Costa Rica Finding arboreal snakes in an evolutionary tree: phylogenetic placement and systematic revision of the Neotropical birdsnakes 1,2 3,4 5 6 7 ROBERT C. JADIN ,FRANK T. BURBRINK ,GILSON A. RIVAS ,LAURIE J. VITT ,CESAR L. BARRIO-AMOROS and 1 ROBERT P. GURALNICK Abstract The genus Pseustes Fitzinger, 1843 is composed of three recognized species, Pseustes poecilonotus, P. shropshirei and P. sulphureus, which may be the largest sized colubrid snake in the New World. The group has a complex systematic history that has yet to be untangled using modern molecular phylogenetic approaches. The systematic position, within-group diversity and distribution are therefore uncertain. We obtained samples of four species from multiple specimens across their distribution and analysed one nuclear and two mitochondrial genes to determine the phylogenetic placement of the genus and infer relationships among Pseustes lineages. We find strong support for the paraphyly of Pseustes with respect to the monotypic genus Spilotes, both of which are nested within a clade of at least 23 other New World Colubrinae genera. Based on our results, we formally revise the tax- onomy of P. poecilonotus and P. sulphureus, resurrecting the taxon P. polylepis for populations of P. poecilonotus from South America and allocating P. sulphureus to the genus Spilotes which renders both genera monophyletic. Additionally, we identify two lineages that are putatively new and currently unrecognized species. Finally, the placement of P. sulphureus, the type species of Pseustes, in the genus Spilotes, requires the allocation of the senior synonym Phrynonax be considered for the remaining Pseustes taxa. Key words: Colubridae – phylogeny – Phrynonax – Pseustes – Serpentes – Spilotes Introduction To date, evolutionary relationships within this genus have not been assessed, rendering their evolutionary relationships and spe- Origin and evolution of the Neotropical fauna remains of great cies boundaries poorly understood. Additionally, accurate taxon- interest, especially given the complex geological history of the omy of Pseustes remains questionable as species in the genus area, habitat heterogeneity and high levels of endemicity of the have been classified as belonging to numerous other genera, region (Cracraft and Prum 1988; Duellman 1999; Daza et al. including Ahaetulla, Chironius, Coluber, Dipsas, Herpetodryas, 2009). However, well-resolved phylogenies are still lacking for Natrix, Phrynonax, Spilotes, Synchalinus, Thamnobius and many organisms, and thus, the evolutionary histories as well as Tropidodipsas (Uetz, 2013). Recently, Pyron et al. (2013) found spatial and ecological distributions remain poorly known for a support that Pseustes sulphureus is the sister taxon to Spilotes majority of taxa (Daza et al. 2009; Schargel et al. 2010). Despite pullatus, both included within the Colubrinae. However, the esti- recent advancements in our understanding of evolutionary rela- mated phylogenetic position of this species was based on a sin- tionships of Neotropical colubrid snakes, which represent a gle 12S gene fragment as part of a large, multigene concatenated majority of snake taxa in the New World (e.g. Pyron et al. phylogenetic analysis of 4161 species of squamate reptiles. 2011), most taxa, including several genera, have yet to be exam- Therefore, increased individual, taxonomic and gene sampling is ined using molecular phylogenetic approaches. Here, we begin to warranted to confirm this finding. assemble one piece of that puzzle by focusing on the Neotropical We utilize samples collected from multiple individuals of snake genus Pseustes, commonly referred to as ‘birdsnakes’ or Pseustes across Central and South America in order to conduct a ‘puffing snakes’. Three species are currently recognized in the molecular phylogenetic analysis of this genus to (i) infer the phy- genus (Rivas et al. 2012): Pseustes poecilonotus Gunther€ 1858; logenetic position of Pseustes within the subfamily Colubrinae, P. shropshirei Barbour and Amaral 1924; and P. sulphureus (ii) assess the relationship between Spilotes and Pseustes, (iii) Wagler 1824; which is among the largest sized colubrid snake in determine whether species of the genus Pseustes form a mono- the New World (Perez-Santos and Moreno 1988). Other species phyletic group, (iv) infer phylogenetic relationships within Pseus- of Pseustes mentioned in Uetz (2013), such as P. cinnamomeus tes and (v) assess species-level diversity to resolve historical Wagler 1824 and P. sexcarinatus Wagler 1824; are considered taxonomic debates. We provide a solid foundation for under- in the synonymy of Chironius scurrulus Wagler, 1924 and standing the evolutionary history of the genus Pseustes, which C. quadricarinatus Boie 1827; respectively (Dixon et al. 1993; will aid future work on this and other understudied groups of Giraudo and Scrocchi 2002). Neotropical snakes. Corresponding author: Robert C. Jadin ([email protected]) Methods Contributing authors: Frank T. Burbrink ([email protected]), Molecular sampling Gilson A. Rivas ([email protected]), Laurie J. Vitt ([email protected]), Cesar L. Barrio-Amoro ([email protected]), Robert P. Guralnick Genomic DNA was isolated from muscle tissue of 17 specimens of ([email protected]) Pseustes and Spilotes using a Qiagen DNeasy extraction kit and protocol. J Zoolog Syst Evol Res (2014) 52(3), 257--264 258 JADIN,BURBRINK,RIVAS,VITT,BARRIO-AMOROS and GURALNICK Two mitochondrial [NADH dehydrogenase subunit 4 (ND4) and cyto- (Swofford 2002), which recovered the GTR + I + Γ model for all three chrome b (cyt b)] and one nuclear oocyte maturation factor Mos (c-mos) codon positions of ND4 and cyt b while finding models HKY + Γ for the gene fragments were independently PCR-amplified using GoTaqâ Green first and second position of c-mos and GTR + Γ for the third position. master mix by Promega, Madison, WI, USA. Protocols for amplification We inferred phylogenetic relationships using Bayesian inference crite- were carried out as described in Arevalo et al. (1994), Burbrink et al. rion implementing MrBayes v3.0b4 (Ronquist and Huelsenbeck 2003). (2000), de Queiroz et al. (2002) and Lawson et al. (2005) incorporating Two simultaneous runs were conducted (with the default Markov chain the primer pairs ND4 + LEU, L14910 + H16064 and S77 + S78 and Monte Carlo [MCMC] settings), for a total of 10.0 9 106 generations annealing temperatures 48, 46 and 55°C, respectively. Sequencing was per run, sampling trees and parameters every 100 generations. We used performed in both forward and reverse directions using the PCR primers potential scale reduction factor values (output by MrBayes), together with on a Beckman Coulter automated capillary sequencer, and sequence chro- plots of cold-chain likelihood values and parameter estimates visualized matographs were edited using Sequencher 4.2, Gene Codes Corporation, in Tracer v1.5.4 (Rambaut and Drummond 2009) to confirm stationarity Ann Arbor, MI, USA. Sequences for each gene fragment were aligned and convergence of MCMC runs. Based on this evaluation, the first separately, first automatically using the program MUSCLE (Edgar 2004), 2.5 9 106 generations from each run were discarded as burn-in. and then manually rechecked using Se-Al v2.0a11 (Rambaut 2002). No Using the same partitioning scheme described above, we inferred the internal stop codons were found in these protein-coding gene fragments. ML tree using RAxML 7.2.8 under the GTRCAT model and assessed Previously published sequences of snakes within the family Colubridae tree support with the rapid-bootstrapping algorithm using 1000 nonpara- were downloaded from GenBank (see Appendix 1) and were combined metric bootstraps (Stamatakis 2006; Stamatakis et al. 2008). Additionally, with new sequence data generated in this study (GenBank accession we performed the SHL test (Shimodaira and Hasegawa 1999; Anisimova numbers KF669664–KF669710, Table 1). and Gascuel 2006) in RAxML 7.2.8 to provide another maximum-likeli- Our preliminary analyses based on taxa throughout the family Colubri- hood measure of support. Support for the SHL test is measured as 1 - p, dae found Pseustes to have a close affinity to members the subfamily Co- where p is equivalent to the probability of obtaining a particular test sta- lubrinae, and we therefore incorporated taxa in that subfamily in our final tistic under the null hypothesis that the maximum-likelihood estimate of analyses. Grayia tholloni, Pseudorabdion oxycephalum, Scaphiodontophis the branch is not significantly more likely than any nearest-neighbour annulatus and Storeria dekayi were used as outgroup taxa to root our Co- rearrangements of that branch. lubrinae phylogenetic tree of 95 species. Results Phylogenetic analyses Our phylogenetic analyses are very similar