Molecular phylogeny of advanced snakes ( Serpentes , Caenophidia ) with an emphasis on South American Xenodontines : a revised classification and descriptions of new taxa

We present a molecular phylogenetic analysis of caenophidian (advanced) snakes using sequences from two mitochondrial genes (12S and 16S rRNA) and one nuclear (c‐mos) gene (1681 total base pairs), and with 131 terminal taxa sampled from throughout all major caenophidian lineages but focussing on Neotropical xenodontines. Direct optimization parsimony analysis resulted in a well‐resolved phylogenetic tree, which corroborates some clades identified in previous analyses and suggests new hypotheses for the composition and relationships of others. The major salient points of our analysis are: (1) placement of Acrochordus, Xenodermatids, and Pareatids as successive outgroups to all remaining caenophidians (including viperids, elapids, atractaspidids, and all other “colubrid” groups); (2) within the latter group, viperids and homalopsids are sucessive sister clades to all remaining snakes; (3) the following monophyletic clades within crown group caenophidians: Afro‐Asian psammophiids (including Mimophis from Madagascar), Elapidae (including hydrophiines but excluding Homoroselaps), Pseudoxyrhophiinae, Colubrinae, Natricinae, Dipsadinae, and Xenodontinae. Homoroselaps is associated with atractaspidids. Our analysis suggests some taxonomic changes within xenodontines, including new taxonomy for Alsophis elegans, Liophis amarali, and further taxonomic changes within Xenodontini and the West Indian radiation of xenodontines. Based on our molecular analysis, we present a revised classification for caenophidians and provide morphological diagnoses for many of the included clades; we also highlight groups where much more work is needed. We name as new two higher taxonomic clades within Caenophidia, one Volume 49(11):115-153, 2009 Museu de Zoologia, Universidade de São Paulo, Caixa Postal 42.494, 04218‐970, São Paulo, SP, Brasil. E‐mail: hzaher@usp.br Laboratório de Biologia Genômica e Molecular, PUCRS, Porto Alegre, RS, Brasil. Programa de Pós Graduação em Zoologia, UNESP, Rio Claro, SP, Brasil. Department of Herpetology, California Academy of Sciences, Golden Gate Park, San Francisco, CA 94118, USA. Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, Toronto, Canada. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, P.R. China. Museu de História Natural Capão da Imbuia, and Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brasil. 1.

. Some of these contributions were designed to evaluate higher-level relationships, while others focus on more restricted assemblages (e.g., homalopsines, xenodontines, pseudoxyrhophiines, elapids, psammophiines, lamprophiines).The principal molecular phylogenetic studies examining broader relationships among caenophidians are summarized in Table 1.All of these efforts have resulted in increasing consensus on the content of many snake clades and the relative branching order among some of them.Improved knowledge of morphology is helping diagnose and characterize clades at all levels of their evolutionary history.However, there is as yet little compelling evidence supporting any particular branching order among many caenophidian clades.The family Colubridae, long suspected to be paraphyletic, has especially defied partition into well defined and strongly supported clades and a nested hierarchy of their evolution, although molecular data in particular have been especially helpful in understanding the evolution of this group.
Both molecular and morphological data sets will ultimately be necessary to develop a comprehensive phylogeny of snakes and each data source can make a unique contribution.On one hand, molecular methods can provide large quantities of phylogenetically
More recently, molecular studies have provided new insights on the higher-level phylogeny of caenophidians, corroborating some long-held views and suggesting new hypotheses for evaluation (e.g., Alfaro et al., 2008;Cadle, 1984aCadle, ,b, 1988Cadle, , 1994;;Crother, 1999a,b;Glaw et al., 2007a,b;Gravlund, 2001; informative data.Although data have been plentiful, colubroid molecular phylogenies have been unstable due to their inherent sensitivity to taxon sampling (Kelly et al., 2003;Kraus & Brown, 1998).On the other hand, only few morphological complexes have been analyzed thoroughly within snakes, and the paucity of broadly sampled morphological characters has prevented the compilation of a large morphological data matrix.We prefer a combination of the two data sources.Zaher (1999) synthesized available morphological evidence, primarily from hemipenes, and allocated all "colubrid" genera into subfamilies, based in part on lists published by Dowling & Duellman (1978), McDowell (1987), Williams & Walach (1989), and Meirte (1992).Zaher (1999) recognized the putatively monophyletic Atractaspididae and an ostensibly paraphyletic Colubridae including twelve subfamilies: Xenodermatinae, Pareatinae, Calamariinae, Homalopsinae, Boodontinae, Psammophiinae, Pseudoxyrhophiinae, Natricinae, Dipsadinae, and Xenodontinae.In Zaher's taxonomy, Xenodermatinae, Homalopsinae, Boodontinae, and Pseudoxyrhophiinae were explicitly recognized (using enclosing quotation marks) as possibly non-monophyletic working hypotheses requiring validation.The other subfamilies were supported by at least one putative morphological synapomorphy.Kraus & Brown (1998), in one of the earliest comprehensive studies of snakes employing DNA sequences, provided molecular evidence for the monophyly of the Viperidae, Elapidae, Xenodermatinae, Homalopsinae, Pareatinae, Thamnophiini, Xenodontinae, Colubrinae, and Boodontinae.They were the first to recognize the basal rooting of the Xenodermatinae on the basis of molecular data, although various authors (e.g., Boulenger, 1894) had long recognized their relative basal position within caenophidians.Corrections and modifications to Zaher's (1999) generic arrangement followed in several molecular studies, which concentrated on the "boodontine" and psammophiine lineages, and in the placement of the North American xenodontines (Pinou et al., 2004;Lawson et al., 2005;Vidal et al., 2007Vidal et al., , 2008)).Most importantly, the paraphyletic family Colubridae was redefined as a much more restrictive group, and most of the subfamilies recognized by Zaher (1999) were rearranged among various families and superfamilies (Pinou et al., 2004;Lawson et al., 2005;Vidal et al. 2007Vidal et al. , 2008)).Lawson et al. (2005) revised the allocation of many genera based on a molecular phylogeny of 100 caenophidians representing all subfamilies rec-ognized by Zaher (1999).They recognized families Colubridae, Elapidae, Homalopsidae, Pareatidae, and Viperidae, and resolved Acrochordus as the sister taxon of all other caenophidians.However, their maximum parsimony analysis (MP) did not resolve well supported deeper nodes among the five "colubroid" families, apart from Pareatidae, which was the sister taxon of a clade including the remaining four.Within that clade, Viperidae + Homalopsidae was the sister clade of Colubridae (their Clade B, including Calamariinae, Colubrinae, Natricinae, Pseudoxenodontinae, Xenodontinae) + Elapidae (their Clade A, including Atractaspidinae, Boodontinae, Elapinae, Hydrophiinae, Psammophiinae, Pseudoxyrhophiinae, and Oxyrhabdium).Subsequently, Pinou et al. (2004) applied the resurrected name "Elapoidea" to a clade comprising Atractaspis + Elapidae."Elapoidea" has subsequently been used for "Clade A" of Lawson et al. (2005) in several molecular phylogenetic studies (Vidal et al., 2007(Vidal et al., , 2008;;Kelly et al., 2009; see also our results below).Clade B of Lawson et al. (2005) was referred to as "Colubroidea" by Pinou et al. (2004) and subsequent authors.Vidal et al. (2007Vidal et al. ( , 2008) ) studied broad patterns of phylogenetic relationships among caenophidians based on an analysis of sequences from approximately 25-30 taxa, primarily from Africa, and revised some of the taxonomy of snakes based on their analyses.However, we feel that some of their formally recognized taxa are only weakly supported by their molecular data, or receive conflicting phylogenetic signals in different data sets.These authors made little attempt to analyze the effects of taxon sampling and long branch attraction (Felsenstein, 1978) or repulsion (Siddall & Whiting 1999) in small molecular data matrices, problems that were acknowledged by Kraus & Brown (1998) and Kelly et al. (2008), and supported by simulation and other studies (e.g., Goertzen & Theriot, 2003;Salisbury & Kim, 2001).Vidal et al. (2007) argued that the problem of long branch attraction (and repulsion) in more basal nodes was better addressed through gene sampling rather than taxon sampling, but this will only partially solve the issue.Increasing gene sampling in a reduced taxon sample can actually reinforce long branch attraction (or repulsion), and increasing the taxon sampling density will at least help reveal unstable clades within a phylogenetic analysis.We comment in more detail on certain aspects of their analyses and taxonomy at appropriate points in our discussion below.
In this study we address the phylogenetic relationships of caenophidians with an increased taxonomic sample over all previous studies (131 species).
In particular, we emphasize the vast radiation of South American "xenodontine" snakes.Although this analysis forms the most comprehensive sampling of caenophidian species analyzed thus far, ours has the same deficiency of other studies: a small sample for most previously recognized colubroid lineages, with the exception of the South American xenodontines (77 species representing most major groups within this radiation).Nonetheless, we believe it represents a significant advance to our present knowledge of caenophidian snake relationships, particularly xenodontines.
Based on our phylogenetic analysis, we revise the classification of caenophidians, paying special attention to morphological diagnoses for particular clades.
Although we are able to provide diagnostic morphological characters for most clades (see exceptions below), the characters diagnosing some of the clades are few in number.We believe this reflects the lack of a broad comparative morphological perspective for snakes, rather than weak support for any particular clade (some of the clades that have weak morphological support are strongly supported by molecular data).This should serve to highlight areas needing additional research.

MAtERIAL And MEtHodS terminal taxa and Genes Sampled
Our molecular matrix comprised 132 terminal taxa and sequences for two mitochondrial and one nuclear gene: 12S, 16S and c-mos respectively (Table 2).We used sequences deposited in GenBank and combined them with our own sequences to sample broadly among caenophidians (Table 2).The caenophidian tree was rooted using a boine, Boa constrictor, as an outgroup.184 sequences were downloaded from GenBank (68 sequences for 12S, 69 for 16S, and 47 for c-mos) and 180 sequences were generated by us (63 sequences for 12S, 60 for 16S and 57 for c-mos); the sequences we generated were primarily from Neotropical xenodontines since these were the lineages of most immediate interest.A list of voucher specimens for the new sequences we present is available from the authors.In all cases our taxon selection was based on the criterion of completeness of gene sequence data; only a few species that represent distinctive and phylogenetically unknown groups were included with fewer than three genes.
Our 180 sequences represent most of the molecular data for the 93 species of Xenodontinae from North, Central, and South America in our matrix, comprising the principal clades (tribes) for this taxon.We sampled 10 species (representing 7 genera) for Central American xenodontines (Dipsadinae) and 77 species (representing 40 genera) for South American xenodontines (Xenodontinae sensu stricto).
We assume the monophyly for the specific category to construct our matrix, so we combined sequences from different specimens to compose our specific terminals (Table 2).Only in two taxa we combined two different species as terminals (Table 2), these are: Calamaria pavimentata (c-mos) + C. yunnanensis (12S and 16S) as one terminal taxon, and D. rufozonatum (12S and c-mos) + D. semicarinatus (16S) as another terminal taxon.

dnA extraction, amplification and sequencing
DNA was extracted from scales, blood, liver or shed skins, following specific protocols for each tissue (Bricker et al. 1996;Hillis et al. 1996).
Amplicons were purified with shrimp alkaline phosphatase and exonuclease I (GE Healthcare) and sequenced using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (GE Healthcare) in a MegaBACE 1000 automated sequencer (GE Healthcare) following the manufacturer's protocols.Chromatograms were checked and, when necessary, were manually edited using Bioedit version 7.0.9.0 (Hall, 1999).

Alignment and phylogenetic approach
Phylogenetic analyses of the sequence data were conducted using the method of direct optimization (Wheeler, 1996), as implemented in the program POY, version 4 (Varón et al., 2008).This approach simultaneously estimates the nucleotide alignment and the phylogenetic tree based on the algorithm described by Sankoff (1975).Homologies among base pairs are inferred as a dynamic process in which the alignment is optimized upon a tree and the best alignment and tree are chosen by the same optimality criterion.Our criterion for direct optimization was Maximum Parsimony (Varón, et al., 2008).Parsimony analysis under direct optimization is distinct from most molecular phylogenetic analyses of snakes done so far, which have used model-based analyses (e.g., maximum-likelihood and Bayesian inferences).
For the non-coding sequences (rRNAs) we conducted a pre-alignment step using the default parameters implemented in Clustal X (Thompson et al. 1997).After that, we identified the regions which were unambiguously homologous (probably the stem regions) by virtue of having high levels of sequence similarity and without insertions and deletions.These regions were used to split both sequences (12S and 16S) into six fragments, each of them comprising approximately 100 base pairs and acting as regions of homology constraint for the alignment search.
On the other hand, for the coding gene (c-mos) we used the retro-alignment approach, which permits the inclusion of the biological information in codon triplets.We used the information on translation sequence available in NCBI GenBank and the frameshift of the sequences to define the starting position for the codon according to which we translated all DNA sequences to amino-acid sequences.Aminoacid sequences were aligned with Clustal X, using the standard parameters of the Gonnet series matrix.These were subsequently retro-translated to DNA in order to be analyzed in the POY search as static homology matrix.

Search strategy and support indexes
Our search strategy involved three routines designed to explore the space of hypotheses for trees and alignments: 1 -We constructed 200 Random Addition Sequences (RAS) followed by branch swapping using the Tree Bisection Reconnection algorithm (TBR).
All best trees and suboptimal trees with fewer than five extra steps were stored.These stored trees were submitted to a round of tree fusing with modified settings for swapping, in which a consensus tree was constructed based on the trees stored in memory, and used as a constraint for the following rounds.After that, the best tree was perturbed using 50 interactions of ratchet with a re-weighing of 20% of the data matrix using a weight of three.One tree per interaction was stored and an additional step of tree fusing was conducted; 2 -Based on previous taxonomies and hypotheses of relationships among taxa, we constructed ten predefined trees as starting trees, thus guaranteeing that these topologies were evaluated, after that we followed the same steps used in routine one; 3 -The last routine was a step of TBR, followed by a tree fusing using the resultant trees from both previous routines as starting trees.
Finally, we conducted a round of TBR using an interactive pass algorithm (Wheeler, 2003), which applies the information of the three adjacent nodes to perform a three dimensional alignment optimization for the target node.The resultant dynamic homologies were transformed into static homologies and the implied alignment was exported in Hennig86 format.The phylogenetic results were then checked using the TNT (Tree analysis using New Technology, version 1.1) software (Goloboff et al., 2008).For TNT Maximum Parsimony search we used the "new technology" algorithms, mixing rounds of TBR, SPR (Sub-tree Pruning and Regrafting), Drift, Ratchet, Sectorial search, and tree fusing.Searches were stopped after the consensus was stabilized for five rounds.To access the corroboration values and support values (sensu Grant & Kluge, 2003) for clades in our best tree, we conducted 1000 site re-sampling in POY, with a static approximation transformed matrix for bootstrap, and we used all visited trees for our analysis routine to infer Bremer support.

Sequence characterization
The implied alignment of the 12S and 16S rRNA sequences resulted in 492 and 688 sites, respectively, whereas the c-mos sequences comprised 501 sites (for a total of 1681 sites among the three genes).Our c-mos sequences had an indel of three base pairs at positions 272-274 in Acrochordus, Bitis, Calamaria, Colubrinae, Natricinae, Pseudoxenodon, and Xenodontinae; this indel is equivalent to that reported in these same groups by Lawson et al. (2005).However it is a deletion of an arginine AA, in an area of the sequence that frequently shows three consecutive arginine, rendering it difficult to determine whether Acrochordus and Bitis show a deletion at the same site as the other monophyletic group (Calamaria, Colubrinae, Natricinae, Pseudoxenodon, Xenodontinae) or a deletion at one of the subsequent arginines.An additional indel of three base pairs at positions 266-268 was found in the sequence of Pseudoeryx.This deletion is one additional arginine indel that occurred in the same three-arginine region.
We found a frame-shift mutation, a deletion of one nucleotide, at position 299 for the monophyletic group Lystrophis hystricus, Lystrophis dorbignyi and Waglerophis merremi (Xenodon neuwiedi was not sequenced for c-mos).In L. hystricus we found one additional indel, an insertion of five nucleotides at position 373-377.To deal with these frame-shift mutations in our alignment approach we conducted the alignment using AA sequences in Clustal X, without this monophyletic group.After that, we retro-translated to DNA and aligned the sequences for this group over the aligned matrix using the default parameters in Clustal X.We do not have a clear explanation for this frameshift mutation, because the first deletion inserts a stop codon at position 101 (AA sequence), probably disabling the c-mos protein.However, mechanisms such as post-transcriptional modifications and RNA editing (Brennicke et al., 1999), could be involved to correct the frame changing of the RNA sequence before translation.This type of frame-shift mutation was also found in snakes for the ornithine decarboxylase gene (ODC, Noonan & Chippindale, 2006).Another possible explanation is the amplification of a paralogous gene for this group of species.However, the sequence trace did not show any signal that could indicate a pseudogene contamination (sequence ambiguities, double peaks, noise, etc).Therefore, more studies are needed to completely understand this new mutational event in such a broadly employed gene as the c-mos.

Phylogenetic analysis: broad patterns of relationships
Direct optimization parsimony analysis of the data set using POY resulted in one most parsimonious tree with 5130 steps (Fig. 1).Further independent analysis of the results from POY was obtained by analyzing the optimal implied alignment in TNT, which identified 53 optimal topologies of 5124 steps, one of which is identical to our Figure 1.The strict consensus of the 53 trees generated by TNT produced a polytomy at node 19 (Fig. 1) including clades Colubridae, (Xenodontinae + Dipsadinae), Carphophiinae, (Natriciteres + Rhabdophis + Xenochrophis), Heterodon, Calamaria, Pseudoxenodon, Sinonatrix, Natrix, and Farancia.The remaining topology of the strict consensus was completely concordant with the best tree found in POY.We further used the pruned tree method in TNT to resolve the polytomy at node 19 and found that the position of Pseudoxenodon is the principal cause of different trees found in TNT.Only one gene sequence, c-mos, was available for Pseudoxenodon and this may be responsible for the lability of its position in different trees.Using the 53 parsimony trees as starting trees in one more round of TBR, tree fusing and Ratchet in POY did recover the same most parsimonious tree shown in Figure 1, which is consistent with our results in POY.Thus Figure 1 represents our preferred tree that will be discussed below.
In discussing our results we use informal designations for clades that follow generally recognized familial or subfamilial categories for caenophidians (e.g., subfamilies, as in Lawson et al., 2005).For example, 'viperids' and 'elapids' refer to the classically recognized families Viperidae and Elapidae, whereas 'homalopsines', 'pareatines', and 'colubrines' refer to Homalopsinae, Pareatinae, and Colubrinae, respectively.Discussion of the application of these names in our new taxonomy is deferred to the section on classification.In our discussion we refer to individual clades by the identifying numbers at each node of our tree (Fig. 1).
The broad pattern of relationships indicated by our analysis includes the following main points.Clade 1 (Fig. 1) corresponds to the clade equivalent to the Colubroidea, as used in most recent literature for the caenophidian sister clade to Acrochordus and containing viperids, elapids, and all 'colubrid' groups (e.g., Lawson et al., 2005; but see discussion of this name in the classification section); this clade is robustly supported (bootstrap 94%; Bremer 14).There is strong support for the successive positioning of Acrochordus, xenodermatids, and pareatids as    (Clades 25,29,34), these clades are generally well-supported, as measured by bootstrap and Bremer support (Fig. 1).
Our study thus indicates strong support for the non-monophyly of Colubridae in the classical sense of caenophidians that are not viperids or elapids.Viperids are nested within the successive outgroups of pareatines and xenodermatines, whereas elapids are nested higher in the tree among some primarily-African 'colubrid' clades.

Relationships within clades
Our sampling within clades apart from xenodontines is not dense relative to the diversity within these clades, but the following relationships are indicated in our tree (Fig. 1).
Within Viperidae (Clade 6) Causus appears as the basal-most viperid genus while Bitis and Azemiops are the two successive sister-taxa to a well-supported crotaline clade represented by Bothriechis and Agkistrodon (bootstrap100%; Bremer 9).All nodes within Viperidae are supported by high bootstrap values.
Within elapids (Clade 13; bootstrap 98%, Bremer support 9), our results show strong support for the monophyly of Australopapuan terrestrial elapids (here represented by Notechis) + sea snakes (represented by Laticauda) (bootstrap 97%, Bremer support 7) relative to other Old-and New World elapids (Naja, Micrurus, Bungarus).Support for a monophyletic Elapinae for the last group (bootstrap 81%, Bremer support 3) is less but we recognize our limited sampling within this group.
Clade 15 (bootstrap 94%, Bremer support 6) comprises three genera whose relationships have been controversial (Homoroselaps, Atractaspis, and Aparallactus).These represent an extended "atractaspidine" or "aparallactine" clade (Bourgeois, 1968;McDowell, 1968;Underwood & Kochva, 1993) (5-7).We have not sampled intensively within either the North American or Central American groups, but we note in passing that within the last group, our results show moderate support for a Leptodeirini (Clade 32; Leptodeira + Imantodes) and a Dipsadini (Dipsas, Sibynomorphus, Sibon, but also including the selected species of Ninia and Atractus).However, no internal nodes within Dipsadini are strongly supported.The nesting of Ninia and Atractus within Dipsadini is novel, and suggests that additional work with denser taxonomic sampling should be carried out within this group (see also Mulcahy, 2007).
Within South American xenodontines (Clade 34), our results show a series of dichotomous basal branches that receive poor support (Clades 37,39,42,47,49), whereas many of the internal clades toward the tips of the tree are more strongly supported.alsophis: Alsophis has included a large assemblage in the West Indies, one species in mainland western South America, and several species in the Galapagos Islands (Maglio, 1970;Thomas, 1997).Our results show that Alsophis is polyphyletic, with the species of western Peru (A. elegans) a basal lineage (Clade 35), only remotely related to West Indian species of Alsophis (Clade 64).Within the West Indian radiation, Alsophis antillensis + A. antiguae are a sister group to a clade including species of Darlingtonia, Antillophis, Ialtris, Alsophis, Arrhyton, and Hypsirhynchus.liophis and Xenodontini: Liophis is an assemblage of more than 60 species, making it one of the most diverse genera of South American colubrids.A core of species has been associated with the tribe Xenodontini (see Myers, 1986) but the genus has also been a repository for generalized colubrids whose affinities with other snakes are unclear (e.g., Myers, 1969Myers, , 1973)).Consequently, its taxonomic history has been subject to considerable fluctuation.Our results show that Liophis is polyphyletic, with Liophis amarali, a species of southeastern Brazil, a sister taxon (Clade 45) to Pseudoboini.Within Xenodontini (Clade 55), Liophis is paraphyletic with respect to Erythrolamprus and to a clade (Clade 59) containing Waglerophis, Xenodon, and Lystrophis.Our results are not surprising given the complicated taxonomic history of these snakes.
Clade 59 (Waglerophis + Xenodon + Lystrophis) is strongly supported (bootstrap support 95%, Bremer support 6).The two species of Lystrophis we examined (histricus and dorbignyi) are strongly supported as a clade, but as a terminal clade nested within successive outgroups of Xenodon and Waglerophis as represented by the two species of those genera included here (see further discussion in the section on classification).
West Indian Xenodontines: Clade 60 includes all of the West Indian alsophiines we examined and has moderately strong support (bootstrap support 89%, Bremer support 4).Within that clade, Uromacer (Clade 61) and a clade containing Cuban species of Arrhyton (Clade 63) are successive sister groups to Clade 64, which contains all remaining West Indian alsophiines (Alsophis, Darlingtonia, Antillophis, Ialtris, Jamaican species of Arrhyton, and Hypsirhynchus).Several clades within the West Indian radiation receive strong support from both bootstrap and Bremer measures of support: Uromacer (Clade 61), one clade of Cuban Arrhyton (procerum-tanyplectum-dolichura), Guadeloupe-Antigua Alsophis (Clade 65), Bahamas-Cuban Alsophis (vudii-cantherigerus), Jamaican Arrhyton (Clade 68), and Hypsirhynchus (Clade 69).Most other internal nodes within the West Indian radiation have strong Bremer support but poor support from bootstrap measures.dISCuSSIon Many of our results corroborate those found in earlier molecular studies, but it should be noted that some of our results were based on the same sequences used in earlier studies (those obtained from GenBank; Table 2).Our results corroborate Lawson et al. (2005) in positioning Acrochordus as the sister group to all other caenophidians.A sister-group relationship between Acrochordus and other caenophidians is a wellsupported hypothesis in all recent morphological phylogenetic analyses (Tchernov et al., 2000;Lee & Scanlon, 2002;Apesteguía & Zaher, 2006), as well as other molecular studies and combined molecular/ morphological analyses (Gravlund, 2001;Lee et al., 2004; and references therein).In contrast, Kelly et al. (2003) and Kraus & Brown (1998) found Acrochor-dus to cluster with Xenodermus-Achalinus (Xenodermatinae); in addition, Kraus & Brown (1998) found their Acrochordus-xenodermatine clade to cluster well within other caenophidians.We suspect that these differences between Kelly et al. (2003) and Kraus & Brown (1998) and other molecular/morphological studies are due to taxonomic sampling issues, as all studies with greater representation of clades within caenophidians support a basal position for Acrochordus.We fully expect that this topology with respect to Acrochordus will be recovered as sampling improves.Nonetheless, an association between Acrochordus and xenodermatines is an old hypothesis, as, for example, expressed in Boulenger (1894).
The Xenodermatinae (Clade 2; represented by Xenodermus and Stoliczkia) is a basally diverging clade among caenophidians in our study, as well as Kelly et al. (2003), Vidal &Hedges (2002a,b), andVidal et al. (2008).Some other molecular studies (e.g., Lawson et al., 2005;Kelly et al., 2009) found a radically different phylogenetic position for xenodermatines based on molecular sequences for Oxyrhabdium, which is typically included within this group.Xenodermatinae is supported by a putative synapomorphy: a concave nasal shield that accommodates the nostril (McDowell, 1987).This character is only weakly developed in Oxyrhabdion and does not unambiguously support its relationship to other xenodermatines.Thus, rather than indicating an ambiguous phylogenetic placement for Xenodermatinae, the molecular and morphological data for Oxyrhabdium suggest to us only that this genus is not phylogenetically associated with other Xenodermatinae (as represented by Xenodermus and Stoliczkia in our study and Vidal et al., 2008, and, in addition, by Achalinus in Kelly et al., 2003), which is a basally-diverging clade in several studies.
Within Viperidae the basal position of the genus Causus has been suggested by many workers (e.g., Haas, 1952;Bourgeois, 1968;Marx & Rabb, 1965, and Groombridge, 1984, 1986) on the basis of comparative morphology of the venom apparatus and head circulatory systems.Azemiops is consistently placed as the sister-group of the Crotalinae in most molecular studies (Cadle, 1992;Knight & Mindell, 1993;Parkinson, 1999).Our results are consistent with these studies on both Causus and Azemiops.Kelly et al. (2003) and Pinou et al. (2004) found topological relationships within vipers different from ours and other studies.In particular, these authors found Causus nested within Viperinae (as represented by Bitis and Vipera).Azemiops was a sister clade to Viperinae in the study of Kelly et al. (2003), whereas it was a sister group to Viperinae + Crotalinae in the study of Pinou et al. (2004).We suspect that differences among these studies reflect differences in taxonomic and gene sampling, and different methods of tree construction.Resolving the differences among these studies will require more comprehensive samples for all major lineages within vipers, which was not an objective in this study.
Homalopsines (Clade 8) are a strongly supported clade in all molecular studies, and this clade is usually positioned basally among a large assemblage containing most "colubrids" + elapids (Clade 9 in our study; Clades A + B of Lawson et al., 2005: Fig. 1; Kelly et al., 2003: Figs. 4 and 5;Vidal et al., 2007: Fig. 1).In our study homalopsines are strongly supported as a sister clade to Clade 9 (Fig. 1).We found no support for a sister group relationship between homalopsines and Homoroselaps (Kelly et al., 2003), nor with viperids (Gravlund, 2001); however, these associations were not strongly supported in either of these last studies.
Clade 9, representing crown-group caenophidians, is well supported in our analysis (bootstrap 98%; Bremer 4), and was recovered (with a reduced taxonomic sample) by Pinou et al. (2004) and by Lawson et al. (2005).We are unaware of any characters that diagnose this clade morphologically.Within Clade 9, our phylogeny recovered two major groups (Clades 10 and 19) that include the most diverse assemblages of caenophidians.Clade 10 is supported by a high bootstrap value (85%) but a low Bremer value (2).This is mostly due to the fact that the position of the psammophiines (Clade 11; Psammophis + Rhamphiophis) is unstable, being sometimes the sister-group of Clade 19 and sometimes clustering with Clade 13 (Elapidae) in suboptimal trees.Clade 10 was recovered in the albumin immunological data of Cadle (1988Cadle ( , 1994)), although the lineages in Clade 19 were an unresolved polytomy (Cadle, 1994: Fig. 2).Clades Our analysis found strong support for the monophyly of all of these subfamilies, as well as for Clade 13, which corresponds to the traditional family Elapidae (including Hydrophiinae) (bootstrap 98%, Bremer 9), and Clade 14, which includes Atractaspidinae, Lamprophiinae, and Pseudoxhyrophiinae (bootstrap 91%, Bremer 3).
Snakes in Clade 15 (Aparallactus, Atractaspis, Homoroselaps), usually referred to as "aparallactines" or atractaspidids, have had among the most controversial relationships of any caenophidians (see reviews and references in Underwood &Kochva, 1993, andCadle, 1994).This clade is moderately supported in our analysis (bootstrap 84%, Bremer support 6), and several other studies have shown some unity to this group.The taxonomically most comprehensive studies of this group, Nagy et al. (2005) and Vidal et al. (2008) (both studies based on the same sequences) recovered two monophyletic sister groups, Aparallactinae (Macrelaps, Xenocalamus, Amblyodipsas, Aparallactus, Polemon) and Atractaspidinae (Atractaspis, Homoroselaps).This result is consistent with the placement of Aparallactus, Atractaspis, and Homoroselaps in our study with respect to one another.However, neither Nagy et al. (2005), Vidal et al. (2008), nor our study was able to link Aparallactinae + Atractaspidinae to other clades of caenophidians with strong support.This is reflected in low support values in all three studies and conflicting placements for the entire assemblage with respect to other major caenophidian clades (sister group to Elapidae in Nagy et al., 2005; sister group to Pseudoxyrhophiinae + Lamprophiinae in our study and that of Vidal et al., 2008).
For xenodontines sensu lato (Clade 25) we defer many of our comments to the section on classification.However, we note that virtually all molecular and morphological studies since Cadle (1984a,b;1985) have recovered evidence for three main clades within this group, although the degree of support for these clades varies, as indicated in Results: a North American clade (Clade 26), a Central American clade (Clade 31), and a South American clade (Clade 34); see especially Pinou et al., 2004, Vidal et al. (2000), and Zaher (1999).The topological relationships for major clades within each of these groups are broadly concordant among these studies insofar as clades that are strongly supported.However, as ours is the taxonomically most comprehensive study of these groups, the placement of many taxa is here elucidated for the first time.In particular, we call attention to the placements of Alsophis elegans and Psomophis (Clades 35 and 36), Taeniophallus (Clade 44), Liophis amarali (Clade 45), and the polyphyly of Arrhyton, Also-phis, and Antillophis within the West Indian radiation (Clade 60; see Results).These taxa clearly require further taxonomic revision, which we initiate and discuss in our classification.

CLASSIFICAtIon oF AdVAnCEd SnAkES our approach to caenophidian classification
Prior to presenting our classification of advanced snakes, we make some preliminary comments regarding our approach to formal recognition of clades represented by our phylogeny, and on several recent "readjustments" to the classification of caenophidians.We fully recognize that there are still many details of snake phylogeny to be resolved, that results for particular taxa can conflict with one another in different studies, and that branches in a phylogenetic tree may receive no significant support for various reasons.Many taxa are of uncertain relationships, either because of disagreements among studies due to analytical or sampling issues, unstable phylogenetic position in multiple most parsimonious trees, or simple lack of data.
All of these factors have influenced the manner in which we translate the information contained in our phylogeny into a classificatory scheme.As a first principle, we recognize as formal taxonomic categories those clades that have received broad support from either morphological or molecular phylogenetic studies.In general, these are clades that appear repeatedly in different studies directed at the appropriate level, an example being Caenophidia.In many cases, these are clades with strong statistical support in a particular study, given sufficient taxonomic sampling (specific details given below).Secondly, we do not give formal names to clades whose composition varies widely among different trees or which receive poor support in a phylogeny.We have resisted giving formal names to taxa solely because their phylogenetic position cannot be estimated with any precision or robustness.Instead, we prefer to simply list these taxa as incertae sedis within the least inclusive taxon with which they appear to be associated.This approach simultaneously reduces the unnecessary proliferation of formal taxonomic names and flags these taxa for further study.Finally, we prefer to integrate morphological data into our taxonomy insofar as possible.However, morphological data for caenophidians are scant for many taxa and in general is widely scattered.Morphological diagnoses for taxa can highlight areas for research, predict relationships in the absence of molecular analyses, and complement molecular data.
With these working approaches, we recognize that our classification includes a few named clades which we expect will require modification with additional study.An example is Atractaspididae, for which we feel that the morphological evidence adduced is weak (primarily due to taxonomic sampling issues), and for which molecular studies conflict to some extent and often (as ours) have limited taxonomic sampling.We have retained a few such named taxa because they have some currency in usage.We provide commentary where necessary to highlight some of the problems.However, we do not create new formal taxa for such controversial groups, preferring instead to leave them unnamed.

Commentary on recent use of the names Colubroidea, Prosymnidae, Pseudaspididae, and Grayiinae
Several recent studies have addressed the classification of caenophidians based on molecular studies (reviewed in the Introduction).In virtually no case has any attempt been made to integrate morphological data into the classification schemes.We disagree with portions of the taxonomies used in some of these studies and here comment on the nature of our disagreements, and why we do not use a few previously named taxa in our classification.
Colubroidea: The name "Colubroidea" has a long history in snake classificatory literature as the name applied to the sister clade of Acrochordidae within Caenophidia.In other words, "Colubroidea" has had long-standing use as the name of the clade comprising viperids, elapids, and all "colubrid" snakes and their derivatives (hydrophiines, atractaspidids, etc.).We were surprised to find that this widely used and universally understood name was applied in an entirely new way, without so much as a comment, in a much more restrictive sense by Dowling & Jenner (1988) and Pinou et al. (2004).These authors applied "Colubroidea" to a clade (Pinou et al., 2004: Fig. 1) that included only a few lineages of "colubrid" snakes, namely colubrines, natricines, and North American and Neotropical xenodontines (Dipsadinae + Xenodontinae of some authors, e.g., Zaher, 1999).Other than a strongly supported clade in their molecular phylogeny, neither Pinou et al. (2004) nor Dowling & Jenner (1988) attempted to diagnose their concept of "Colubroidea"; in fact, they did not even mention their entirely novel use of the name and its contravening years of historical precedent!Subsequent to Pinou et al. (2004), Vidal et al. (2007Vidal et al. ( , 2008) ) used "Colubroidea" as a name for the same clade, with the addition of Pseudoxenodon.Again, these authors attempted no diagnosis or definition of the group.
This new application of a long-standing taxonomic name clouds an already murky and confusing taxonomy, particularly as it was seemingly done very casually.Examples of works using "Colubroidea" in its near-universally understood sense, but by no means an exhaustive list, include the following : Cadle, 1988;Cundall & Greene, 2000;Cundall & Irish, 2008;Dowling & Duellman, 1978;Ferrarezzi, 1994a,b;Greene, 1997;Kelly et al., 2003;Kraus & Brown, 1998;Lawson et al., 2005;Lee et al., 2004;McDiarmid et al., 1999;McDowell, 1986McDowell, , 1987;;Nagy et al., 2005;Rieppel, 1988a,b;Romer, 1956;Smith et al., 1977;Vidal, 2002;Vidal & Hedges, 2002a,b;and Zaher, 1999.A radical shift in the meaning of a wellestablished taxonomic name, in our view, should be explicit and not simply implicit in the presentation of results of a phylogenetic analysis.It is also true that the name Colubroidea has had several meanings since Oppel (1811) first erected the family-group name Colubrini (for Bungarus and Coluber).Fitzinger (1826) explicitly used "Colubroidea" as a family-group name almost in its modern sense.Romer (1956) formally recognized Colubroidea as a superfamily and his use was followed in most subsequent works.
Nonetheless, we recognize that some names will require changes in definition with improved knowledge of phylogeny, particularly among "colubroid" snakes (sensu Romer, 1956).When making taxonomic changes we maintain current usage of names as far as possible and opted for conservative adjustments to meanings of long-standing names.In any case, when we change the meaning of long-standing names, we provide commentary about the change and our reasons for doing so.Although we do not fully adopt the philosophy and procedures elaborated by Frost et al. (2006: 141-147), we do share some of their concerns about names and ranks.Consequently, for names above the family-group, which are unregulated by the International Code of Zoological Nomenclature, we do not incorporate an explicit concept of rank but we maintain ranks (and comply with the Code's rules for name formation) at the family-group and below.Thus, we apply the name Colubroides new name as a formal taxonomic name above the family level for the sister taxon to Acrochordidae within Caenophidia; this new name replaces Colubroidea Oppel as the name for this clade.We use and re-define Colubroidea Oppel for a reduced clade comprising natricines, calamariines, pseudoxenodontines, colubrines, and xenodontines sensu lato, as explained below.
Prosymnidae and Pseudaspididae: Kelly et al. (2009) proposed new names for several "clades" within Elapoidea (see below).They recognized a new family, Prosymnidae, including only the genus Prosymna based on the fact that Prosymna appeared in all their analyses "at the same hierarchical level as other major clades" and thus should be accommodated in a distinct family.They used a similar argumentation for recognizing a family Pseudaspididae (including Pseudaspis and Pythonodipsas).On the other hand, Vidal et al. (2008) considered Prosymna, Pseudaspis, Pythonodipsas, Buhoma, Psammodynastes, Micrelaps, and Oxyrhabdium to represent elapoid lineages with unresolved affinities, and suggested that additional sequencing was needed to better resolve their affinities.Indeed, Prosymna falls into radically different phylogenetic positions in the studies of Vidal et al. (2008), in which it clusters with Atractaspididae + Pseudoxyrhophiidae + Lamprophiidae, and Kelly et al. (2009), in which it is nested within the Psammophiidae + Pseudoxhyrhophiidae.In neither analysis does the position of Prosymna receive significant support.Similarly, although Kelly et al. (2009) provided strong support for a clade (Pseudaspis + Pythonodipsas), the relationship of that clade to other elapoids was ambiguous.In the taxonomically broader phylogenetic analysis by Lawson et al. (2005), the strict consensus parsimony tree shows Prosymna + Oxyrhabdium as a sister clade to the Elapidae; Psammodynastes as the sister group of Atractaspis; and Pseudaspis + Pythonodipsas as a clade more closely related to the Lamprophiidae than to any other elapoid group.
The conflicting results among these studies might be due to the different strategies of outgroup and ingroup sampling used in these analyses.However, none of these hypotheses show significant statistical support.For these reasons we prefer not to recognize Prosymnidae and Pseudaspididae.Rather, we consider Prosymna, Pythonodipsas, and Pseudaspis as well as Buhoma, Psammodynastes, and Oxyrhabdium as Elapoidea incertae sedis.
Grayiinae Meirte, 1992: Vidal et al. (2007) erroneously thought they were erecting a new family-group name, Grayiinae, but this name should actually be attributed to Meirte (1992).Both Meirte (1992) and Vidal et al. (2007) included only the genus Grayia Günther, 1858 in this taxon.We did not include Grayia in our analysis but its phylogenetic affinities have been found to lie with the Colubrinae by Cadle (1994), Pinou et al. (2004), andVidal et al. (2007), and with the Natricinae by Kelly et al. (2009).The genus was associated with Colubrinae in the maximum parsimony tree of Lawson et al. (2005), although with no significant statistical support, essentially forming a basal polytomy with both Natricinae and Colubrinae.Since there seems to be no compelling evidence that would support an unambiguous position of Grayia within Colubroidea, we here refrain to include the genus in a separate subfamily and place it in Colubridae incertae sedis.

taxonomy of caenophidians, with a focus on xenodontines
The present taxonomic arrangement refers only to the "colubroid" radiation, with special emphasis on the "New World xenodontine" radiation of snakes.We recognize taxonomically all clades that can be characterized morphologically and display either a high bootstrap value (more than 70%) or a high Bremer support (superior to 5).We avoided suggesting new taxonomic arrangements for nodes that are poorly supported in our molecular analysis and that lack any putative morphological synapomorphy.However, in a few cases we recognize a clade taxonomically for which no morphological synapomorphies are known; we discuss these at the appropriate places in the text.
Before each diagnosis we parenthetically present the bootstrap support (expressed as a percentage) and Bremer support for each node discussed.For example, the first clade discussed (Clade 1) is denoted by "(94%, 19)", which reflects a bootstrap value of 94% and a Bremer support of 19.An asterisk (*) denotes bootstrap support < 70%.All clade numbers refer to those indicated in Fig. 1.A few named taxa in our taxonomic hierarchy (e.g., Calamariinae) are represented by only a single terminal taxon in our study.For these, we denote their placement in the tree (Fig. 1) by the name of the terminal taxon rather than a node number (these consequently lack "node support" statistics).
The following summarizes our classification to tribe level as an aid in following the text.We also note here the new higher taxa and genera described (certain genera are placed incertae sedis in many of the higher taxa, as explained below): Colubroides new taxon is equivalent to a clade long recognized by the name "Colubroidea" for the clade of all Caenophidia exclusive of Acrochordidae (see above discussion for application of the name Colubroidea).
Diagnosis: (100%, 33).Putative synapomorphies for the group are: maxilla suspended, in part, from a lateral process of the palatine; loose ligamentous connection between maxilla and prefrontal; and vertebral zygapophyses and neural spines with broad lateral expansions (Bogert, 1964;McDowell, 1987;Ferrarezzi, 1994a,b).(Bogert, 1964).We are not convinced by the few morphological characters adduced by Dowling & Pinou (2003) for a greatly expanded Xenodermatidae.In their concept, the Xenodermatidae comprises "more than 20 genera (…) distributed from Japan, China, and India to Australia, Africa, and South America" (Dowling & Pinou, 2004: 20).Although the reader is referred to a "Table 1" that apparently lists these genera, no such table exists in the published paper.However, at least some of the genera they mention as xenodermatids (Mehelya, Pseudaspis, Xenopholis) are shown in other works to have phylogenetic affinities elsewhere.We expect Xenodermatidae will ultimately prove to be a much more restricted clade than conceived by Dowling & Pinou (2004).Vidal et al. (2007) erected a superfamily Xenodermatoidea including only the family Xenodermatidae, so these terms carry redundant information.
Comments: Some of the morphological characters of the jaw apparatus are convergent between Pareatidae and Dipsadini (Brongersma 1956(Brongersma , 1958;;Peters 1960), probably because many synapomorphies of both groups are associated with a specialized diet of gastropods.Vidal et al. (2007) erected a superfamily Pareatoidea including only the family Pareatidae, so these terms carry redundant information.

Content: Adenorhinos
Intra-viperid relationships have been studied by numerous workers and we have little to add to these other works given our deliberate de-emphasis on this group other than its placement broadly within Caenophidia.Because the relationships of New and Old World viperids are under active investigation, we expect revisions to the taxonomy to proceed apace.A recent checklist (McDiarmid et al., 1999) recognized four subfamilies: Causinae (Causus only), Azemiopinae (Azemiops only), Crotalinae (pitvipers), and Viperinae (Old World pitless vipers).Subclades within the last two subfamilies have been recognized as tribes.
Comprehensive summaries and reviews of some of this literature can be found in McDiarmid et al. (1999), Schuett et al. (2002), and Thorpe et al. (1997).
The rattlesnakes, Crotalus and Sistrurus, recently underwent a taxonomic revision by Hoser (2009).Hoser largely used the molecular phylogeny of Murphy et al. (2002) to resurrect older names from synonomies and designate a number of new genera and subgenera.In doing so, he recognized nine genera including three new genera.Some taxonomic arrangements are certainly in error.For example, genus Cummingea Hoser 2009 contains three species, none of which have been included in a phylogenetic study and at least one of which we now know is incorrectly placed in this group (Murphy, unpublished data).Bryson, Murphy et al. (unpublished data) have DNA sequence data for several hundred specimens of the triseriatus complex of Klauber (1972); the phylogenetic relationships among these taxa changed substantially as a consequence of far greater sampling.Hoser placed Sistrurus ravus in a new monotypic genus and thus obscured its phylogenetic relationships.Until a well-supported phylogeny is obtained, we recommend against recognizing Hoser's new taxonomy.

FAMILY HoMALoPSIdAE Bonaparte, 1845 (Clade 8)
Homalopsina Bonaparte, 1845.Comments: The level of generality of the character "viviparity" is unclear, as it has evolved repeatedly among snakes (Blackburn, 1985) and is present widely in the immediate outgroup to endoglyptodonts (Viperidae).
The derived hemipenial feature cited herein as a synapomorphy of the family Homalopsidae is also homoplastically present in several Madagascan genera (Zaher, 1999;Cadle, 1996).Vidal et al. (2007) erected a superfamily Homalopsoidea including only the family Homalopsidae, so these terms carry redundant information.We follow McDowell (1987)  Comments: Dromophis Peters, 1869 was recently synonymized with Psammophis (Kelly et al., 2008).Hypapophyses have been lost repeatedly in the evolution of caenophidians but all immediate outgroups to Psammophiidae retain them on the posterior trunk vertebrae.De Haan (1982, 2003a,b) identified some peculiarities in the infralabial glands associated with a rubbing ("polishing") behavior in Dromophis, Malpolon, Mimophis, and Psammophis, as well as parietal pits (perhaps sensory in nature) in the same genera (see also Steehouder, 1984).If these features are discovered more generally in psammophiids, they may provide additional morphological and behavioral corroboration for the monophyly of this clade.
Comments: Spinulate flounce-like structures have been confirmed only in Polemon, Macrelaps, Amblyodipsas, and most Aparallactus (not present in Atractaspis fallax); they are yet to be confirmed in the other genera.This character is also present in Psammodynastes, which has been shown to be closely related to the Atractaspididae in one molecular phylogenetic study (Lawson et al., 2005).A similar character exists in some Lamprophiidae, but in this case the flounces extend to the hemipenial body.The atractaspidid hemipenis differs from the lamprophiid hemipenis by the condition of the sulcus spermaticus (centripetal in the former and centrifugal in the latter).
The content and relationships of Atractaspididae has been among the most controversial of any clade within advanced snakes (for reviews, see Cadle, 1988, andUnderwood &Kochva, 1993), and we recognize its composition here primarily as one of convenience and historical legacy.The hemipenial synapomorphies we list have appeared in very similar form elsewhere within caenophidians.Furthermore, most of the morphological characters adduced for this group (e.g., Underwood & Kochva, 1993) are in reality only found in particular subsets of taxa within it.Even the derived venom apparatuses of two of the included taxa (Atractaspis and Homoroselaps) show trenchant differences that are difficult to reconcile with one another and with those of less-derived members of the assemblage.

Comments:
Although it has a poor bootstrap and Bremer support, this clade is diagnosed by a significant hemipenial feature.Our clade 16 has also been retrieved again with poor support by Vidal et al. (2008).Alternatively, Kelly et al. (2009) retrieved a poorly supported clade that includes pseudoxyrhophiines and psammophiids.
Comments: Spinulate flounce-like structures are also present on the hemipenial lobes of some atractaspidid genera (Zaher, 1999), and might represent a synapomorphy uniting this family with the Lamprophiinae.However, flounce-like spinulate structures on the hemipenial body are unique to the Lamprophiinae.
The broader phylogenetic analyses of Lawson et al. (2005) and Kelly et al. (2009) demonstrated convincingly that Duberria and Amplorhinus were more closely related to the Pseudoxyrhophiinae than to any other elapoid or colubroid lineage; a similar relationship of Amplorhinus (but not Duberria) to pseudoxyrhophiids was previously suggested by Cadle (1994).Bourquin (1991) suggested, on the basis of skull morphology, that Montaspis is closely related to the Pseudoxyrhophiidae.We recognize both Stenophis and Lycodryas as valid, but the systematics of these snakes needs revision (Cadle, 2003(Cadle, : 1000(Cadle, -1001)); furthermore, Kelly et al. (2009)  Comments: Zaher (1999) discussed the variation regarding the sulcus spermaticus in colubroid snakes.
Well-developed calyces on the hemipenial lobes are considered to be lost secondarily by the Natricidae.See above discussion on the new use of this name.
Diagnosis: Frontals and sphenoid forming ventral border of the optic foramen (excluding entirely, or nearly so, the parietals); hemipenial body nude; hemipenial body bearing a pair of longitudinal ridges (Zaher, 1999).Comments: Use of the name "Colubridae" for this clade is a much more restricted use of this name than its long-standing use in the literature on caenophidian systematics, in which "Colubridae" generally referred to all caenophidians that were not acrochordids, elapids, or viperids.The single sulcus spermaticus of colubrids and natricids is considered to have derived from a centrifugally divided sulcus, but in different ways in the two groups (McDowell 1961).On unilobed organs of colubrids the sulcus extends centrolineally to the distal end of the hemipenis, whereas on some distally bilobed organs the sulcus always extends to the right lobe.On the other hand, in natricids when the sulcus extends to only one of the lobes of a bilobed organ, it is always to the left lobe (see also Rossman & Eberle, 1977;and Zaher, 1999: 25-26).Lawson et al. (2005) have shown that Macroprotodon lies within the family Colubridae, but without clear affinities within that group.The phylogenetic affinities of Scaphiophis Peters, 1870 has been disputed (Zaher, 1999;Vidal et al., 2008).Recently, Kelly et al. (2008) included the genus in their molecular analysis, in which it appears nested within colubrines.For this reason, we include this genus in the family Colubridae.
Diagnosis: Hemipenis deeply bilobed, with each lobe separately calyculate on the distal half and nude on the medial half; fringes of large papillae separating the nude region from the calyculate area (Zaher, 1999).
Diagnosis: (89%, 12).Sulcus spermaticus single and highly centripetal, forming a nude region on the medial surfaces of the hemipenial lobes; hemipenial calyces absent (evolutionary loss).Comments: Among Natricidae, the New World natricids are a monophyletic tribe (Thamnophiini) supported by molecular and morphological evidence (Rossman & Eberle 1977;Alfaro & Arnold 2001;De Queiroz et al. 2002).Relationships among African and Eurasian species are largely unresolved.See Comments under Colubridae concerning differences between the simple sulci spermatici of natricids and colubrids.
Comments: The diagnosis we give here for Dipsadidae includes those synapomorphies previously considered for the more restricted group Xenodontinae (sensu Zaher, 1999).We present them here for Dipsadidae because the North American Farancia and Heterodon also have these characters.Thus, these characters could have separately evolved in Farancia and Heterodon, and South American xenodontines (with subsequent loss in Carphophis, Contia, and Diadophis); or, the interpretation we adopt here, the characters could be synapomorphic at the level of Dipsadidae, with subsequent transformations (losses) in the clade including Carphophis, Contia, and Diadophis on one hand, and in Dipsadinae on the other.This question must be resolved with further research.In any case, we note that there is evidence from the present study and from the immunological comparisons of Cadle (1984a,b,c) for three major clades within the Dipsadidae as we conceive it, namely a North American clade, a Dipsadinae clade, and a Xenodontinae clade (see also Pinou et al., 2004).However, Pinou et al. (2004) found the North American xenodontines (their North American relicts) paraphyletic with respect to dipsadines, xenodontines, and natricids.The monophyly of the North American xendontines was also unstable in the present analysis, with a low bootstrap support on Clades 23, 25, and 30 due to the variable positions of Heterodon and Farancia with respect to these nodes in suboptimal trees.Thus, further revisions on that issue may be warranted.On the other hand, Carphophis, Contia, and Diadophis form a well-supported clade (Clade 29; 88%, 4) corroborated by putative hemipenial synapomorphies.Those synapomorphies also support the clade Dipsadinae (Clade 31; 74%, 7) and are here viewed as having evolved homoplastically in these two groups.The optimization of these characters on the tree depends on a better understanding of the position of Heterodon and Farancia that are here included in Dipsadidae incertae sedis.
The genus Xenopholis Peters, 1869, not included in the present analysis, has been recently associated with the Xenodermatidae by Dowling & Pinou (2003).However, its dipsadid hemipenial morphology, the presence of a well-developed septomaxillary-frontal articulation, and previous immunological studies do not support the latter hypothesis (Cadle, 1984a), suggesting dipsadid affinities instead (see also discussion above in Xenodermatidae).Since the position of Xenopholis within the Dipsadidae is still unknown, we opted to include it in the family as incertae sedis, but we have no reservations at all about its placement within this group.We also assume, following Zaher (1999), that the other Neotropical genera Crisantophis, Diaphorolepis, Emmochliophis, Enuliophis, Enulius, Hydromorphus, Nothopsis, Rhadinophanes Synophis, and Tantalophis which have a dipsadid hemipenial morphology, belong within Dipsadidae, and we place them here incertae sedis.Guo et al. (2009) and He et al. (2009) have shown convincingly that the genus Thermophis Malnate, 1953 is more closely related to the Dipsadidae than it is to any other colubroid clade.However, a more thorough analysis of the phylogenetic affinities of Thermophis is still needed in order to clearly place this genus in respect to the Dipsadidae.Meanwhile, we include Thermophis Malnate, 1953 in the Dipsadidae as incertae sedis.Finally, the poorly known genera Cercophis, Lioheterophis, Sordellina, and Uromacerina that present a dipsadid hemipenial morphology and were considered by Zaher (1999) as being Xenodontinae incertae sedis are here included in the Dipsadidae incertae sedis.
Comments: Because Carphophis, Contia and Diadophis form a strongly supported clade that is also corroborated by derived hemipenial evidence, we here include them in a new subfamily Carphophiinae.
Whether Farancia and Heterodon belong to this subfamily is a question that needs further investigation (see also comments under Dipsadidae).The hemipenial morphology of Carphophiinae new subfamily ressembles the one of Dipsadinae, but differs in an important detail, namely the lack of capitation on the lobes.
For the sake of stability of the shark family name Heterodontidae Gray, 1851, the name Heterodontinae Bonaparte, 1845, used by Vidal et al. (2007) for the North American xenodontines (including Heterodon and Farancia), should be avoided (Rossman & Wilson, 1964).
Diagnosis: (74%, 7).Hemipenes unilobed or with strongly reduced bilobation; hemipenes unicapitate; sulcus spermaticus dividing distally, either at the base of, or within, the capitulum (Myers, 1974;Cadle, 1984b;Zaher, 1999).Comments: Hemipenial morphology varies among this diverse group and the level of generality of the hemipenial synapomorphies we cite should be reviewed as more taxa are surveyed (see Zaher, 1999 for discussion).A simple sulcus spermaticus is present in some dipsadines as a further derived condition.
We refrain from defining tribes within Dipsadinae in the present analysis since we have sampled little of the diversity within this large group.However, there are indications from both molecular (Cadle, 1984b;Mulcahy, 2007) and morphological (Peters, 1960;Myers, 1974;Cadle, 1984bCadle, , 2007;;Oliveira et al., 2008;Vidal et al., 2000) data for a monophyletic Leptodeirini including at least the genera Leptodeira and Imantodes and a monophyletic Dipsadini including at least Dipsas, Sibon, Sibynomorphus, and Tropidodipsas.However, much more work will be required to confidently resolve the relationships among the other species of this diverse group (> 200 species).Comments: The clade Xenodontinae (Clade 34) is here recognized tentatively, in spite of its poor measures of support (only 60% and 5) for three main reasons: 1) we still do not have a strong case with respect to the exact optimization of the hemipenial characters here associated with Dipsadidae (Clade 25, see above discussion), that might turn over to be synapomorphies of Clade 34 as suggested previously by Zaher (1999); 2) the name Xenodontinae Bonaparte, 1845 has a long standing association with this group of snakes and therefore is widely understood as such; 3) not recognizing Xenodontinae for the mainly South American xenodontine radiation would require the allocation of its constituent monophyletic subgroups to a higher taxonomic level, i.e., subfamily, thus greatly changing the well-established taxonomic hierarchy for this group.Such reallocation might be needed in the future, although it still needs further research and clarification on the higher-level interrelationships between these parts.

SuBFAMILY
Our analysis reveals very strong support for several previously known Xenodontinae tribes (Zaher, 1999): Elapomorphini (86%, 6), Tachymenini (92%, 9), Pseudoboini (99%, 21), Philodryadini (93%, 6); Hydropsini (97%, 8), Xenodontini (100%, 10), Alsophiini (89%, 4).These tribes are here formally recognized.However, except fot the sister group relationship between Xenodontini and Alsophiini that shows some measure of support (69%, 4), interrelationships between well established tribes are highly unstable, showing no significant measure of support in our analysis.We thus refrain to further comment on these nodes (Clades 37,39,42,47,49).Alsophis elegans and Liophis amarali fall in our analysis well outside their generic allocation and have been here assigned to new tribes and genera.Additionally, the genera Psomophis, Tropidodryas, Taeniophallus, Conophis, and Hydrodynastes are here placed in separate new tribes due to their isolated phylogenetic position in the tree, clustering only weakly with well-supported tribes for which they have no known morphological affinities.Conophis and Hydrodynastes form a monophyletic group in our analysis (Clade 51) that shows a high bootstrap (90%) but a low Bremer support (3).However, similarly to our reasoning above for the recognized tribes, we decided to allocate these two genera in separate tribes because they do not share any known morphological synapomorphy.

tRIBE SAPHEnoPHIInI new tribe (terminal taxon: Alsophis elegans)
Diagnosis: Reduction or loss of ornamentation on the asulcate and medial surfaces of the hemipenial lobes; papillate ridge on medial surface of hemipenial lobes in a lateral-to-medial orientation from proximal to distal, and confluent proximally with the enlarged lateral spines (Zaher, 1999).

Comments:
The papillate ridge on the hemipenial lobes in Saphenophiini is here considered nonhomologous to a ridge in a similar position in Alsophiini (see below).The non-homology of the two structures is indicated by their different orientations proximal to distal.See also Comments under Pseudoboini.
Alsophis elegans is clearly set apart from the other species of the genus Alsophis in our analysis, being more closely related to the genus Psomophis (although with a low Bootstrap support of 71% and Bremer of 3) than to any of the West Indian xenodontine snakes.Zaher (1999) pointed out important hemipenial differences between Alsophis elegans and species of West Indian Alsophis, suggesting that its affinities would lie with the Galapagos species of xenodontines, allocated by Thomas (1997) to the genera Philodryas (P.hoodensis), Alsophis (A. occidentalis, A. biserialis), and Antillophis (A. slevini, A. steindachneri).Zaher (1999) also elevated all the subspecies of Galapagos snakes recognized by Thomas (1997) to species status.The Galapagos snakes have a hemipenial morphology that is not only closer in most respects to that of Alsophis elegans, but it also departs significantly from the hemipenial patterns shown by the West Indian species of Alsophis and the genera Philodryas and Antillophis.On the other hand, the Galapagos xenodontines and Alsophis elegans share with the Ecuadorian genus Saphenophis a characteristic hemipenial morphology (see Zaher, 1999).Based on this hemipenial evidence and in order to render the genera Alsophis, Philodryas, and Antillophis monophyletic, we allocate Alsophis elegans and the Galapagos xenodontine species in a new genus.The Galapagos species are presently under study and will be dealt in more detail elsewhere.
Diagnosis: Hemipenis generally deeply bilobed, bicalyculate, semicapitate, with a forked sulcus spermaticus dividing on the proximal half of the body, with branches extending centrolineally until the base of the capitula, here it takes a centrifugal position on the lobe, ending in the distal region; intrasulcar region mostly nude, without spines; enlarged lateral spines of moderate size and numerous; capitula formed by diminutive papillate calyces and are most restricted to the sulcate side; asulcate and medial surfaces of the lobes almost completely nude, except for the presence of a medial papillate and inflated crest or ridge that runs from the lobular crotch to the distal edge of each capitulum; vestigial body calyces along all the internal region of the lobes.
Comments: Viviparity and male-biased sexual dimorphism have evolved repeatedly in colubroids, but are here considered derived characters of Tachymenini.These characters are otherwise rare in Xenodontinae.Ferrarezzi (1994b) questioned the autorship of this Tribe, probably due to the inexistence of a formal diagnosis for the group in the Bailey's paper (1967).However, as pointed out by Franco (1999), Bailey (1967) characterized adequately the group, justifying thus its authorship of the tribe.Bailey's (1967) attribution of oviparity to this group is an obvious misprint, which he corrected in Bailey (1981).Diagnosis: (99%, 21).A pair of pigmented spots on the palate; posterior region of the palatine bone longer than dental process, behind vomerian process; dorsal region of the vomer with a distinct process in which the ligament of the muscle retractor vomeris is attached; distinct maxillary process of the prefrontal forming a well defined articular area; lateral (nasal) process of the prefrontal hook-like; hemipenis bicalyculate and bicapitate; large lateral spines on the lobular crests; presence of a pair of calycular pockets within the lobular crotch of the hemipenis; enlarged lateral spines of hemipenis extending onto the lobular crests; lobular crests inflated (Zaher, 1994b(Zaher, , 1999)).
Our analysis confirmed the polyphyletic nature of the genus Clelia already suggested by Zaher (1994b;1999).We thus describe the new genus Mussurana to accommodate Clelia bicolor and two closely related species previously assigned to Clelia (Zaher, 1994b).
Diagnosis: Presence of ontogenetic changes in color pattern; juveniles with a brick red color, a black longitudinal vertebral band, and an uniformly creamish venter.Adults with dorsum entirely black; Hemipenis with a unique row of larger papillae on the internal face of the lobes; postero-ventral tip of the nasal gland longer than wide; dorsal wall of Duvernoy gland reduced along all its dorsal surface (Zaher, 1994b;1999) (Zaher, 1999).
Comments: Although not present in our analysis, the genus Manolepis is included here in Conophiini due to its hemipenial similarities with Conophis (Zaher, 1999).
Furthermore, our analysis revealed that the genera Erythrolamprus, on the one hand, and Waglerophis and Lystrophis on the other hand, are nested within the genera Liophis sensu stricto and Xenodon, respectively.Morphological support for the inclusion of the genera Waglerophis and Lystrophis within Xenodon are compelling and have been described and discussed by Zaher (1999), Moura-Leite (2001), and Masiero (2006).Therefore, in order to retrieve monophyly of these genera, we synonymize Lystrophis Cope, 1885 andWaglerophis Romano &Hoge, 1972 with Xenodon Boie, 1826.
Erythrolamprus appears firmly nested within Liophis in our analysis, being strongly supported by a bootstrap of 100% and Bremer support of 17 in Clade 58 and appearing as the sister-group of Liophis typhlus (bootstrap 85%, Bremer 6).Although there is no apparently known morphological evidence supporting this grouping, we here synonymize the genus Erythrolamprus Boie, 1826 with Liophis Wagler, 1830 in order to retrieve a monophyletic Liophis Boie, 1826.However, Liophis is a highly speciose and diverse group of snake and we expect a more comprehensive sampling than ours within the whole diversity of Liophis will provide more stable support for the taxonomic decisions taken here.
Content: Alsophis Fitzinger, 1843;Antillophis Maglio, 1970;Arrhyton Günther, 1858 Comments: See Comments under Saphenophiini.Our study, as well as earlier molecular studies (e.g., Cadle, 1984aCadle, , 1985;;Vidal et al., 2000;Pinou et al., 2004), retrieves a monophyletic Alsophiini including all endemic West Indian genera of Xenodontinae (our study used many of the same sequences as the study by Vidal et al., 2000, but our other reference taxa were very dissimilar).The molecular evidence, along with the unusual morphological synapomorphy of this group (Zaher, 1999), strongly supports the monophyly of this clade relative to mainland xenodontines (for a contrary view, see Crother, 1999a,b).We also exclude from Alsophiini the mainland South American species "Alsophis" elegans and the snakes of the Galapagos Islands (contra Maglio, 1970;Thomas, 1997) (see Saphenophiini).
Within Alsophiini, the hierarchy of relationships we find are strongly supported by morphological evidence presented by Zaher (1999).Examples are, Clade 63 (Cuban Arrhyton), Clade 68 (Jamaican Arrhyton), Clade 65 (the primarily Lesser Antillean Alsophis), and, within Clade 66, a polyphyletic Antillophis and a clade of primarily Greater Antillean Alsophis.We therefore name the following new, redefined, and resurrected genera to reflect these relationships:
Diagnosis: Lobular crotch and medial surface of hemipenial lobes ornamented with well-developed, horizontally directed papillate flounces; asulcate surfaces of lobes completely nude and bearing a large overhanging edge of the capitulum; expanded papillate circular area present on the lobular crotch.
Etymology: Named after Albert Schwartz, who made significant contributions to knowledge of West Indian herpetology; gender masculine.
Diagnosis: Asulcate surfaces of hemipenial lobes completely nude except for a row of two to three enlarged papillae aligned vertically on the lobular crotch and proximal region of the lobes; hemipenes long and slender (hemipenial body at least four to five times as long as the lobes).
Etymology: Caraiba, in allusion to the "mar das Caraibas," a Portuguese designation of the Caribbean region, gender feminine.
Diagnosis: Long lobes ornamented with spinulate calyces on the sulcate surface; enlarged, transverse papillate flounces on the asulcate surface; papillate flounces decrease in size proximal to distal.

FIGuRE 1 :
FIGuRE 1: Best Phylogenetic tree based on molecular matrix (12S, 16S and c-mos) found by Directed optimization under Maximum Parsimony analyses (implemented in POY 4.1).Numbers above branches are bootstrap support values; numbers below branches are Bremer supports.The asterisk (*) corresponds to nodes with bootstrap values less than 60%.

tABLE 2 :
List of taxa and sequences analyzed in this study.
found that the two species of Stenophis they examined were not monophyletic relative to other pseudoxyrhophids.Species and generic level taxonomy of pseudoxyrhophids needs more research.
of the name "Pseudoboini" was meant to be informal ("I call informally a tribe, Pseudoboini"), he nonetheless defined the original concept of the tribe in a table on page 158 (without Saphenophis and Tropidodryas, which were included in this group by Jenner & Dowling, but which are not closely related; seeMyers & Cadle Dowling et al. (1983;owling et al. (1983; see Jenner & Dowling,  1985).AlthoughBailey's (1967: 157; see also Bailey 1940) use .
Diagnosis: (93%, 6).Hemipenial body much longer than the lbes (more than twice the length), with the aulcate side of the hemipenial body covered with two parallel rows of enlarged body calyces on most or all its surface.