First molecular evidence for the phylogenetic placement of the enigmatic snake genus Brachyorrhos (Serpentes: Caenophidia)

Molecular Phylogenetics and Evolution 61 (2011) 953–957

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Short Communication

First molecular evidence for the phylogenetic placement of the enigmatic snake genus Brachyorrhos (Serpentes: Caenophidia) John C. Murphy a,⇑, Mumpuni b, Kate L. Sanders c a

Division of Amphibians and Reptiles, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, IL 60605-2496, USA Center for Research in Biology, Museum of Zoology, LIPI, Bogor, Indonesia c School of Earth and Environmental Sciences, University of Adelaide, Adelaide, Australia b

a r t i c l e

i n f o

Article history: Received 29 April 2011 Revised 20 July 2011 Accepted 9 August 2011 Available online 22 August 2011 Keywords: Homalopsidae Indonesia Terrestrial–aquatic transition Snakes

a b s t r a c t Brachyorrhos Schlegel, 1826a is a terrestrial–fossorial snake genus endemic to eastern Indonesia that has been assigned to six different families and subfamilies within Colubroidea (advanced snakes) over the past 200 years. Here we report the first molecular sequences for Brachyorrhos and use them to test the position of the genus within snake phylogeny. Our Bayesian and Maximum Likelihood analyses of three mitochondrial and one nuclear gene strongly resolve Brachyorrhos within the rear-fanged semiaquatic Homalopsidae (Colubroidea), as the sister taxon to all other genera and sampled species. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Knowledge of the eastern Indonesian snake Brachyorrhos albus has been limited to physical descriptions, distribution records, and speculation about its phylogenetic affinities. It has been assigned to diverse families and lower taxonomic ranks within the superfamily Colubroidea (advanced snakes sensu Lawson et al., 2005 and Pyron et al., 2011), including: Coronellae (Schlegel, 1826b), Brachyophes (Fitzinger, 1843), ‘‘les Leptognathiens’’ (Duméril et al., 1854), Calamaridae (Günther, 1858), Rabdosominae (Jan, 1862 , 1882 who also assigned it to the Calamaridae), Colubridae (Boulenger, 1893), and Natricinae (Dowling and Duellman, 1974). In a review of snake systematics, McDowell (1987, p. 35) proposed that despite its terrestrial–fossorial life style and absence of fangs, Brachyorrhos probably belongs in the mostly aquatic, rear fanged family Homalopsidae, because of their shared viviparity and similar hemipenes, vertebrae and skull. Molecular studies have since shown Homalopsidae to be monophyletic (Alfaro et al., 2008) and to represent a relatively early phylogenetic divergence from other colubroids (Lawson et al., 2005; Pyron et al., 2011). In a review of the Homalopsidae, Murphy (2007) considered Brachyorrhos to be incertae sedis in the clade, and was skeptical of its relationship to the homalopsids given its narrow, pointed snout,

⇑ Corresponding author. Address: 15824 Weather Vane Way, Plainfield, IL 60544, USA. E-mail addresses: [email protected] (J.C. Murphy), [email protected] (K.L. Sanders). 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.08.013

laterally placed eyes, anteriorly directed nostrils, nasal scales separated by the rostral, and other morphology not shared with recognized members of the Homalopsidae. However, homalopsids are morphologically very diverse and despite efforts to identify synapomorphies, none have been found. Here, we present the first molecular data for Brachyorrhos and provide compelling evidence for its phylogenetic position within Homalopsidae. A complete treatment of the nomenclature and systematics of the genus will be published elsewhere. 2. Methods and materials 2.1. Taxon sampling and laboratory methods We sampled DNA from three B. albus specimens collected by the authors in Ternate, North Maluku (eastern Indonesia) in 2009. Because preliminary analyses (using Geneious software: Drummond et al., 2010) indicated a close affinity of Brachyorrhos to the Homalopsidae, data for 19 species representing all genera and major clades of homalopsids were obtained from GenBank. Nine additional outgroup taxa were also obtained from GenBank to test the placement of Brachyorrhos within snake phylogeny. These taxa spanned most major lineages of colubroids, plus Acrochordus, two representative henophidians (boas, pythons and relatives) and one scolecophidian (blind snake). Specimen information and GenBank numbers are given in Table 1. Standard protocols were used to extract genomic DNA and to amplify three mitochondrial fragments and one nuclear fragment

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Table 1 Locality information and GenBank accession numbers for specimens used in the phylogenetic analyses. Higher grouping

Species

Museum #

Incertae sedis

Brachyorrhos albus Brachyorrhos albus Brachyorrhos albus Bitia hydroides Cantoria violacea Cerberus australis Cerberus microlepis Cerberus rynchops Enhydris bocourti Enhydris chinensis Enhydris enhydris Enhydris innominata Enhydris subtaeniata Enhydris longicauda Enhydris matannensis Enhydris plumbea Enhydris punctata Erpeton tentactulatus Fordonia leucobalia Gerarda prevostiana Homalopsis buccata Myron richardsonii Xenochrophis vittatus Dinodonrufozonatum Pareas carinatus/macularius Calamariapavimentata Bungarus fasciatus Atheris nitschei Acrochordus granulatus Xenopeltis unicolor Leptotyphlops humilis

T075 T099 T128

Homalopsidae

Colubridae; incertae sedis Colubridae; Colubrinae Colubridae; Pareatinae Colubridae; Calamariinae Elapidae Viperidae Acrochordidae Henophidia; Xenopeltidae Scolecophidia; Leptotyphlopidae

using HotMaster Taq reagents (Perkin Elmer/Applied Biosystems). Mitochondrial fragments were 1100 bp (base pairs) of cytb (cytochrome b), 981 bp of 12S rRNA (12S small subunit ribosomal RNA), and 510 bp of 16S rRNA (16S small subunit ribosomal RNA). The nuclear fragment was 750 bp of c-mos (oocyte maturation factor). Double-stranded sequencing was outsourced to the Australian Genome Research Facility Ltd. (AGRF) in Brisbane, Australia. Cytb primers were: Forward Elapid Cytb Lb (50 -GGA CAA ATA TCA TTC TGA GCA GCA ACA G-30 ) and Reverse Elapid Cytb H (50 TTG TAG GAG TGA TAG GGA TGA AAT GG-30 ) (Lukoschek and Keogh, 2007). 12S primers were: Forward L-12Shom (50 -ATA CCC ATA CAT GCA AGC CTC-30 ) and Reverse H-12Shom (50 -CAC ACT TTC CAG TAC GCT TAC C-30 ) (Alfaro et al., 2008). 16S primers were: Forward M1272 (50 -CGC CTG TTT ATC AAA AAC AT-30 ) and Reverse M1273 (50 -CCG GTC TGA ACT CAG ATC ACG T-30 ) (Kocher et al., 1989). C-mos primers were: Forward G74 (50 -TGA GCA TCC AAA GTC TCC AAT-30 ) and Reverse G303 (50 -ATT ATG CCA TCM CCT MTT CC-30 ) (Saint et al., 1998; Hugall et al., 2007). Geneious Pro v5.1.7 (Drummond et al., 2010) was used to generate consensus sequences from forward and reverse reads, and to perform alignments, which were manually adjusted.

2.2. Phylogenetic analyses MrBayes v.3.1.2 (Ronquist and Huelsenbeck, 2003) was used to reconstruct trees for each locus (c-mos and the concatenated mitochondrial fragments) separately, and for the full nuclear and mitochondrial matrix. The data were partitioned according to Bayes factors (see Ronquist et al., 2005) and assigned best-fit models of nucleotide evolution using the Akaike information Criterion (AIC) in MrModeltest v. 2.3 (Nylander, 2004) and PAUP (Swofford, 2002): c-mos first and second codon positions (GTRg), c-mos third positions (HKYig), cytb first and second positions (GTRig), cytb third positions (GTRig), rRNA (GTRig). Values for model parameters

GenBank accession number 12S

16S

cytb

c-mos

EF395872 EF395873 EF395874 EF395875 EF395876 EF395877 EF395878 EF395879 EF395880 EF395881 EF395882 EF395883 EF395884 EF395887 EF395888 EF395889 EF395891 EF395892 EF395893 EF395871 AF233939 AF544773

EF395847 EF395848 EF395849 EF395850 EF395851 EF395853 EF395854 EF395855 EF395856 EF395857 EF395858 EF395859 EF395860 EF395863 EF395864 EF395865 EF395867 EF395868 EF395869 EF395846 HM439980 AF544802

EU547135 AY223650 AB177879 AF512735 GQ469228

EU547184 AY223663 AB177879 AF512735 GQ469228

EF395896 EF395897 EF395898 EF395899 EF395900 EF395902 EF395903 EF395904 EF395905 EF395906 EF395907 EF395908 EF395909 EF395912 EF395913 EF395914 EF395916 EF395917 EF395918 EF395895 AF471063 AF471082 AF471081 EU547086 AF471070 AB177879 AY121369 AY099991

EF395921 EF395922 EF395923 EF395924 EF395925 EF395927 EF395928 EF395929 EF395930 EF395931 EF395932 EF473654 EF395933 EF395935 EF395936 EF395937 EF395939 EF395940 EF395941 EF395920 AF471163 AF471150 AF471103 AF544732 AF471125 HM234057 AF544689 AY099979

were unlinked, i.e. allowed to vary independently across partitions. MCMC analyses were repeated at least five times using four chains and from different starting seeds (with default heating parameters and branch-length priors). The final analysis was run for 5000,000 generations and sampled every 1000 generations. The first 30% of sampled trees were excluded as burn-in. Convergence was assessed by comparing the split posterior probabilities from different runs in MrBayes, and by examining effective sample sizes (ESS values) and likelihood plots through time in TRACER v. 1.4.1 (Rambaut and Drummond, 2007). Partitioned Maximum Likelihood (ML) analyses were performed on the full nuclear and mitochondrial alignment using RAxML version 7.0.3 (Stamatakis, 2006). The GTR substitution model (with gamma distributed rate heterogeneity and a proportion of invariable sites) was applied to the same partitions as in the full MrBayes analysis. Node support was evaluated with 2000 nonparametric bootstrap pseudoreplications. All analyses used the scolecophidian Leptotyphlops as an outgroup to root the trees; there is extensive morphological and molecular evidence that Scolecophidia is the sister taxon to all other extant snakes (e.g. Lee et al., 2007) and that Brachyorrhos is included within the latter group (McDowell, 1987). We opted not to date the tree because our data matrix contains only 750 nuclear sites; whereas mitochondrial data provides sufficient variation to resolve colubroid phylogeny, it is prone to substitutional saturation at the deeper nodes for which reliable fossil calibrations are available (compression of basal branches causes overestimation of dates for nodes above the calibration points: Gatesy et al., 2003; see also Sanders and Lee, 2008).

3. Results The final data matrix comprises 31 terminals (22 homalopsids and 9 outgroups) and 3300 sites and is 96% complete (only five se-

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quences missing from a possible total of 124). The composite mitochondrial (cytb, 12S and 16S) and nuclear c-mos data contain 847 and 155 polymorphic sites, respectively. Bayesian runs for each locus and for the full alignment yield effective sample sizes (ESS values) above 500 for all parameters. Based on the full alignment, the Bayesian all compatible consensus tree and the ML tree (Fig. 1) are identical in topology and mostly consistent with recent molecular snake phylogenies in topology, branch lengths and node support (e.g. Lawson et al., 2005; Alfaro et al., 2008; Vidal et al., 2007; Pyron et al., 2011). All analyses (Bayesian and ML for each locus and the full alignment) recover Brachyorrhos as the sister lineage to a clade containing all genera of rear-fanged homalopsids. The full alignment yield strong support for the Brachyorrhos-homalopsid node (posterior probability of 1.0 and nonparametric bootstrap value of 100 for Bayesian and ML analyses, respectively). The basalmost node within rear fanged homalopsids is strongly supported in the Bayesian tree (posterior probability of 0.99) and moderately supported in the ML tree (bootstrap value of 72). 4. Discussion 4.1. Taxonomic recommendations Among the great many conflicting hypotheses for the origins of Brachyorrhos, our results support McDowell’s (1987) placement of the genus within the colubroid family Homalopsidae. Given strong molecular support for the monophyly of Brachyorrhos and the rear fanged homalopsids, and their close sister-taxon relationship to the exclusion of other colubroids, we suggest expanding Homa-

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lopsidae to include Brachyorrhos. This group lacks morphological synapomorphies to our knowledge but shares viviparity and the following traits: valvular, crescent-shaped nares; small eyes; vertical pupils; usually dorsal eyes; hypapophyses throughout vertebral column; hemipenes divided for about half their length, distal region finely calyculate, cups shallow with blunt spines, sulcus forked, flounced spines; shallow rostral notch; and downwardprojecting tongue (McDowell, 1987; Murphy, 2007). For the remainder of the discussion, we refer to Homalopsidae as the group inclusive of Brachyorrhos, and use ‘rear-fanged homalopsids’ for the clade excluding Brachyorrhos. 4.2. Ancestral ecology and biogeography of the Homalopsidae Brachyorrhos (Fig. 2) is unusual among the primarily aquatic, fish-crustacean diet specialist homalopsids in being fangless, terrestrial–fossorial and vermivorous (worm-eating) (Voris and Murphy, 2002; Murphy, 2007). Assuming that Brachyorrhos closely resembles the ancestral lineage that produced the rear fanged homalopsids, their common ancestor may have been fangless, fossorial–terrestrial and vermivorous, albeit viviparous. Alternatively, the absence of fangs in Brachyorrhos may be derived, as a result of its vermivorous diet. While the rear fanged homalopsids are substantially divergent morphologically, their diets are perhaps less diverse (Voris and Murphy, 2002). Species with known dietary habits feed mostly on fish, but clade C (Alfaro et al., 2008) specializes in crustaceans (Cantoria, Fordonia and Gerarda). Clade A contains a highly aquatic species (undescribed Lake Touwti species) as well as some of the most terrestrial homalopsids (‘Enhydris’

Fig. 1. MRBAYES Bayesian consensus tree showing all compatible partitions for Brachyorrhos and other colubroid snakes (Acrochordus, henophidian and scolecophidian outgroups not shown). Support values >70% are shown for Baysian and Maximum Likelihood analyses above and below nodes, respectively. The photographs illustrate the morphological diversity of the homalopsids. In order from the top: Cantoria violacea, a coastal dwelling crustacean specialist from Indochina and the Sunda Shelf; Erpeton tentaculatus, a freshwater, piscivorous, Indochinese species that rarely leaves the water; Cerberus australis, a coastal piscivorous species; Enhydris enhydris, a freshwater, piscivorous species that takes advantage of human modified habitats, such as rice paddies; Enhydris plumbea, a semi-terrestrial frog and fish-eating species from Indochina and China; and Brachyorrhos sp., the terrestrial–fossorial, worm-eating species discussed here. Photographs by JCM and KLS.

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Fig. 2. A Brachyorrhos sp. from the island of Ternate. Brachyorrhos shows morphology typical of many fossorial snakes, including reduced dorsal scale rows, fused head scales, small eyes, and a short tail. Photograph KLS.

plumbea Group) that are known to feed on frogs, tadpoles, and fish. Dietary habits of some homalopsids remain unknown; two species of uncertain phylogenetic affinity within the family (Enhydris indica and Enhydris alternans) show morphological traits (gracile bodies, small heads, short tails and wide ventrals) that suggest at least partial adaptation to a fossorial life style and therefore possible candidates for being vermivores. A fossorial, worm-eating ancestor for the Homalopsidae may also help to explain the rear fanged species’ preferences for muddy substrates and small prey. Alfaro et al. (2008) dated the most recent common ancestor of extant rear fanged homalopsids at 21.8 million years ago (mya), and this timescale is consistent with several other studies that used long nuclear sequences and many of the same fossil calibrations to date colubroid divergences (e.g. Daza et al., 2009; Kelly et al., 2009; Sanders et al., 2010). The short internode distance between Brachyorrhos and the rear-fanged homalopsids, while allowing for some saturation effects in the mitochondrial data, suggests that the Brachyorrhos lineage is probably also of early Miocene – late Oligocene origin. This finding (a relatively old lineage endemic to eastern Indonesia) adds to evidence from other phylogenetic studies suggesting that several terrestrial vertebrate lineages have had long histories of persistence and diversification in the sub-aerial landmasses to the north of the Australian plate and east of the Asian plate (see Oliver, 2011). These studies have prompted a reconsideration of the biogeographic role of the region as representing more than merely a filter bridge for terrestrial Asian and Australasian faunas or a sink for colonists from continental source biotas (Jønsson et al., 2011; Oliver, 2011). B. albus is distributed throughout North and South Maluku; molecular sampling of island populations would provide valuable insights into their history of colonization and diversification in the region. Homalopsidae is spread throughout South and Southeast Asia, Indo-China, northern Australia and New Guinea (Alfaro et al., 2008). It remains uncertain whether Brachyorrhos is a relic of dispersal from mainland or Southeast Asia or a lineage that represents an eastern Indonesian center of origin for the Homalopsidae. However, the current distribution of Brachyorrhos, and its sister-taxon relationship to the remaining Homalopsidae, supports an Indochinese or Southeast Asian origin of the family followed by westward dispersal (Alfaro et al., 2008), as opposed to a South Asian origin and subsequent eastward dispersal. Acknowledgments We are grateful to the Indonesia Institute of Sciences (LIPI) for granting us permission to conduct fieldwork in Ternate. At the Field Museum (FMNH) we would like to thank Harold K. Voris, Alan Resetar, and Kathleen Kelly for lab space and logistical support. For the loan of specimens we thank Colin McCarthy at the British Museum of Natural History (BMNH), Fred Kraus at the Bernice Pauahi

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