Higher-level phylogeny

As in previous studies, we find very strong support (SHL = 100) for the clade Serpentes [15,23,36,42,48,81]. In Fig 1 we display a summary of the full ML tree (lnL = -919390.188) to exhibit relationships above the genus-level and present the full species-level tree in Figs 2–10, made available in Newick format in S2 File. Overall, more than half of the nodes in the full species-tree received strong support (73.45% of nodes with SHL values > 85). In the following section we largely compare our tree to Pyron et al [15], since they provide a recent detailed comparison to preceding publications and because theirs is the only other clade-wide species-level tree (but see [23]). In general, we substantiate many of the higher-level relationships reported in Pyron et al [15]; however, several differences also exist. Support for monophyly for each family and subfamily was above 88%, except for Gerrhopilidae (SHL = 48), and Cylindrophiidae was paraphyletic with Anomochilidae [23,35,53].

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larger image TIFF original image Download: Fig 1. Abridged phylogeny on final dataset of 1652 snake species and seven outgroup taxa displaying higher-level relationships. Maximum-likelihood phylogenetic estimate based on 10 concatenated genes. Tips represent families and sub-families. Commonly recognized higher-level clades are labeled in all caps and bold. Species classified as Lamprophiidae incertae sedis are also shown since they did not place within a subfamily. Node values represent SHL support values. Skeleton of the species tree is displayed on the left, colored and labeled as they appear in Figs 2–10. https://doi.org/10.1371/journal.pone.0161070.g001

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larger image TIFF original image Download: Fig 2. Species-level phylogeny on final dataset of 1652 snake species. Maximum-likelihood phylogenetic estimate based on 10 concatenated genes. Node values represent SHL support values. Seven outgroup taxa are not shown. Colors of clades indicate their position in the overall tree, shown at left. Newly sequenced taxa are highlighted in bold. Skeleton of the species tree is displayed on the left with displayed subfamilies/families highlighted. Letters denoted by i and ii represent parts of the tree where external branches do not connect to the part of the tree immediately preceding it. A) Anomalepididae, Epictinae, Leptotyphlopinae, Gerrhopilidae, Xenotyphlopidae, and Typhlopinae. B) Asiatyphlopinae I, Afrotyphlopinae; Madatyphlopinae, and Asiatyphlopinae II. https://doi.org/10.1371/journal.pone.0161070.g002

Scolecophidia. Similar to many prior examinations, we find relationships within Scolecophidia unresolved [15,23,25,31,32,41,42,46–48,82–85], with studies showing either Scolecophidia [25,31,84,85], Anomalepididae [15,41] or Leptotyphlopidae + Typhlopoidea [23,42,46,47,48] as sister to all snakes. Morphology also reveals uncertainty surrounding Scolecophidia (reviewed in [84]), but based on the presence of vestigial supratemporal and ectopterygoid bones, absent in other scolecophidians, Anomalepididae may be the most basal scolecophidian [85]. We believe future work will lead to a reclassification of Scolecophidia, but until then relationships within the infraorder remain problematic. In addition, we find weak support for the placement of Asiatyphlopinae, Afrotyphlopinae, and Madatyphlopinae within Typhlopidae as in previous studies [15,23,29,31,50,86]. The issue appears to lie primarily with the placement of Argyrophis [50] and Xerotyphlops [15,23,50], which together formed Asiatyphlopinae I. Xerotyphlops is represented by two species, one occurring in the eastern Mediterranean and the other on Socotra Island [86], and Argyrophis is distributed from western Asia to Southeast Asia [29,86]. Discordance in topology therefore appears associated with these two genera being intermediate in distribution between African and Asian typhlopids, which may show affinities to clades from both regions.

Henophidia. As mentioned above, Cylindrophiidae is paraphyletic with Anomochilidae. Difficulty in resolving this relationship is likely due to the representation of Anomochilus by one species and two genes (12S and 16S), and Cylindrophis by two species with greater gene coverage. Both of these families were formerly shown as part of or paraphyletic with Uropeltidae [41,42,47,48]. Based on the history of paraphyly between these families, Burbrink and Crother [84] recommended synonymizing Cylindrophiidae and Anomochilidae with Uropeltidae to resolve these families. However, we recommend retaining the current classification until more species are sampled (Table 1) on the grounds that Cylindrophiidae + Anomochilidae share morphological features not present in Uropeltidae [35,84] and since strong support has been shown distinguishing them from Uropeltidae [15,23,32,41]. For boids, our analysis validates the taxonomic changes made in Pyron et al [30], but differs in topology from previous assessments in the placement of Calabariidae, Candoiidae, and Sanziniidae [15,23,53]. Although the relationship Erycidae + Boidae is recovered in all studies [15,23], except one [53], support for this relationship is low. Thus, the only node we can have confidence in is the one joining Charininae and Ungaliophiinae [15,23,53].

Xenophidiidae and Bolyeridae. Perhaps the most notable difference from the topology of Pyron et al [15] was the placement we recovered for Xenophidiidae + Bolyeridae (SHL = 91). Earlier studies showed them as sister to various clades within Henophidia [23,32,38,41,42], but we found very strong support (SHL = 100) for them as sister to Caenophidia (SHL = 100), as also shown in other studies [53,85]. In addition, these snakes possess morphological characters, particularly within the palate, bolstering their close relationship with Caenophidia and not to Henophidia [85]. Pyron et al [15] is the only study showing a disassociation between these families placing Xenophidiidae as sister to Alethinophidia, with the exception for Aniliidae + Tropidophiidae, and Bolyeridae as sister to Booidea. Currently, both clades are represented by one species and Xenophidiidae by only one gene (cyt-b). Both clades contain two species; for Xenophidion, both species are known only from one specimen each, and for Bolyeridae, Bolyeria is extinct, and Casarea is rare [38], so obtaining additional sequences for either clade is unlikely. If this placement is retained, then Caenophidia should be redefined to include Xenophidiidae and Bolyeridae, or they should be given their own taxonomic grouping.

Caenophidia. Pyron et al [22] recently reviewed and attempted to resolve several problematic issues within Caenophidia. The major problems hindering resolution of this clade are 1) placement of Xenodermatidae inside or outside of Colubroidea; 2) placement of Homalopsidae; 3) topology of Lamprophiidae; and 4) topology of Colubridae. Previous studies have placed Xenodermatidae as sister to Acrochordidae [15,37] or as basal in Colubroidea [23,27,40,42,47,87], have placed Homalopsidae as sister to Lamprophiidae + Elapidae [15,27,40] or as sister to (Lamprophiidae + Elapidae) + Colubridae [23,32,39,42,45,47], and have shown conflicting topologies for the subfamilies within Lamprophiidae and Colubridae [15,23,27,28,37,40,45,47]. Pyron et al [22] used seven methods to examine these relationships showing Xenodermatidae as basal in Colubroidea with varying support and Homalopsidae as sister to (Lamprophiidae + Elapidae) + Colubridae with strong support. However, they expressed little confidence in resolving the topology within Lamprophiidae and Colubridae since several divergences were defined by low support. We confirm their findings that Xenodermatidae is sister to the rest of Colubroidea (SHL = 100) and that relationships within Lamprophiidae and Colubridae remain unresolved, but our findings for the placement of Homalopsidae contradicted theirs, as we recovered strong support (SHL = 91) for Homalopsidae + Lamprophiidae, and found Elapidae to be nested within Lamprophiidae. Typically, Lamprophiidae and Elapidae are recovered as distinct clades [15,22,28,39,40,41,64], but we found strong support (SHL = 96) for Elapidae + Buhoma depressiceps as sister to Pseudoxyrhophiinae (SHL = 99), shown previously only in Pyron and Burbrink [32]. The topology of Lamprophiidae is complicated by the presence of several incertae sedis taxa (see Lamprophiidae [28,32,39,41]), but Elapidae remains nested within Lamprophiidae even when these taxa are removed (S1 Fig). In addition, we found the placement of Pareatidae and Viperidae within Colubroidea unresolved. Pareatidae is consistently placed as sister to Viperidae, which is sister to Colubridae, Elapidae, Homalopsidae, and Lamprophiidae [15,22,23,27,32,41,42]. A possible explanation for this is that our dataset includes the greatest sampling of pareatids, adding seven additional species previously not included in higher-level relationships, two we sequenced and five from You et al [58].

Lamprophiidae. Part of the issue with resolving the topologies within Lamprophiidae, and within Colubridae, is that they exemplify rapid radiations manifested by the presence of short internodes [22]. Yet another major issue hindering progress within Lamprophiidae is the presence of several incertae sedis taxa, not identified as rogue taxa by RogueNaRok. These taxa constantly show contrasting phylogenetic placement between studies [15,23,28,39,40,64,87]. We are reluctant in placing any confidence in the topology between subfamilies recovered for Lamprophiidae, despite high support values. However, the topology after all rogues and incertae sedis taxa were pruned remained essentially the same (S1 Fig) adding supplementary support for this topology. Nonetheless, our topology differs from earlier studies. Previous studies have consistently recovered the sister relationship between Aparallactinae + Atractaspidinae [15,22,28,32,39,40,41,64]; however, we found this relationship unresolved, likely due to the strong placement (SHL = 95) of Atractaspis irregularis as sister to these two clades, and this taxon is represented by only one gene. The topology recovered here was Psammophiinae + ((B. procterae + Prosymninae) + (Pseudaspidinae + (Atractaspidinae + Aparallactinae) + (O. leporinum + Lamprophiinae)) + (((Ditypophis sp. + M. bicoloratus) + Pseudoxyrhophiinae) + (B. depressiceps + Elapidae)))). All nodes received strong support (SHL > 88), except for subclades B. procterae + Prosymninae and Ditypophis sp. + M. bicoloratus. Pyron et al [15] had augmented the definition of Pseudaspidinae to include Buhoma and Psammodynastes. With added sampling of Psammodynastes, we recovered this genus as paraphyletic with Rhamphiophis oxyrhynchus (SHL = 100) within Psammophiinae, making Rhamphiophis paraphyletic (Fig 5A). Buhoma, on the other hand, was split with B. procterae sister to Prosymninae and B. depressiceps sister to Elapidae. Oxyrhabdium leporinum was sister to Lamprophiinae and Micrelaps bicoloratus was placed within Pseudoxyrhophiinae. In all preliminary and final analyses, Psammodynastes constantly occupied the same phylogenetic position; however, placement of the other four species was erratic and always differed. Therefore, we tentatively include Psammodynastes as part of Psammophiinae. Due to their perpetual variable placement, we continue recognizing Buhoma, M. bicoloratus, and O. leporinum as Lamprophiidae incertae sedis.

Colubridae. For Colubridae, we recovered the following four subclades: i) Sibynophiinae + Natricinae (SHL = 80); ii) Pseudoxenodontinae + Dipsadinae (SHL = 82); iii) Grayiinae + Calamariinae (SHL = 70); and iv) Ahaetuliinae subfam. nov. + Colubrinae (SHL = 95). The nodes between these subclades all received very strong support (SHL > 97). The only consistently recovered clade among these is subclade ii [22,27,32,40,41]; although other studies do not recover this subclade [15,23,65]. Several studies also regularly recovered the subclade Natricinae + (Pseudoxenodontinae + Dipsadinae) [22,27,32,40], but we do not uncover that relationship here. Instead, Natricinae formed a subclade with Sibynophiinae, also reported in [41]. The subfamily Sibynophiinae was only recently included in molecular analyses, originally grouped with Calamariinae [27], then subsequently placed as sister to Grayiinae + Colubrinae [15,23], and to Calamariinae + (Colubrinae + Grayiinae) [22]. The subfamily Grayiinae was also recently described [45] and grouped with Calamariinae in that study, also recovered in Pyron and Burbrink [32]. However, Grayiinae has most frequently been grouped with Colubrinae [15,22,23,27,39–41]. Dipsadinae is exclusively a New World family, but recent placement of Stichophanes and Thermophis as sister to Dipsadinae [15,88,89] expanded its distribution into the Old World. Pyron et al [15] did not include Stichophanes, and they mentioned that Thermophis may even warrant its own subfamily. However, our results do not uphold this view since we show Stichophanes + Thermophis (SHL = 96; Fig 7B) as placed within Dipsadinae. Wang et al [89], on the other hand, supported Stichophanes + Thermophis as sister to Dipsadinae, but their dataset was not as extensive and did not include T. zhaoermii. Until now, the basal node of Colubrinae has remained ambiguous. Pyron et al [15] suggested that monophyly of Ahaetulla, Chrysopelea, and Dendrelaphis at the base of Colubrinae, may warrant recognition as a distinct subfamily, but support for division of these taxa in their study was low. Due to increased sampling, and the inclusion of Dryophiops, we established strong support for recognizing these taxa as a new subfamily, using the name proposed by Pyron et al [15], Ahaetuliinae subfam. nov.