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Home , Acrochordoidea, Apostolepis, Argyrogena, Booidea, Cacophis, Demansia

Molecular Evolution and Phylogeny

Cytogenet Genome Res 2009;127:128–142 Published online: March 10, 2010 DOI: 10.1159/000295789

Cytogenetics and Molecular Data in : A Phylogenetic Approach

a a b N. Oguiura H. Ferrarezzi R.F. Batistic a b Instituto Butantan, Lab. de Ecologia e Evolução, Instituto Butantan, Lab. de Herpetologia, São Paulo,

Key Words Snakes are important for evolutionary and develop- ,Evolution ؒ Mitochondrial genome ؒ Phylogeny ؒ Serpentes ؒ mental research, as well as for their ecological role and - karyotypes ؒ Snake venoms ؒ Toxins from the medical standpoint, due to perspectives for bio prospecting of the biomolecules present in their venoms. Characterization of snake genomes is essential to under- Abstract standing the overall diversity and evolution of the subor- Snakes are among the most successful groups of , der Serpentes. In addition, recent molecular data has ad- numbering about 3,000 extant . In spite of centuries vanced knowledge about snake relationships since classi- of comparative anatomical and morphological studies, many cal morphological classification. aspects of snake systematics remain unsolved. To better un- Snake monophyly and its lepidosaurian membership derstand the evolution and diversity of genomic character- have long been recognized. The same morphological fea- istics in Serpentes, we analyzed online sequence data of mi- tures that make snakes such a distinctive group (i.e., over- tochondrial and nuclear genes, as well as cytogenetic data all structural reductions and simplification of the body and reviewed other genomic characteristics such as toxin plan) may also account for many difficulties and uncer- genes. After the analysis of the whole-genome and chromo- tainties concerning kinships to which a group of somal organization, we find that: (1) cytogenetic compari- is their closest relative, and also relationships among sons could provide a useful tool to investigate intergeneric snake lineages. and tribal relationships within the extremely diverse neo- Systematic biology provides a single information sys- tropical xenodontine snakes; (2) toxin genes could also help tem that connects all extant and extinct organisms by to understand snake evolution if special care is taken to means of their inherited attributes (morphology and de- choose the sequences because of the difficulty in avoiding velopment, physiology and its extensions) or, ultimately, paralogs; (3) snake phylogeny based on mitochondrial ge- the genes regulating the expression of all this (without nome sequences is largely consistent with the relationship forgetting epigenetic process). Evolutionary theory pro- obtained using nuclear genes. vides an explanation for such recurrence of patterns. The Copyright © 2010 S. Karger AG, Basel advancement and testing of these ideas about snake evo- lution depend on an important subject: constraints im-

© 2010 S. Karger AG, Basel Nancy Oguiura 1424–8581/09/1274–0128$26.00/0 Instituto Butantan, Lab. de Ecologia e Evolução Fax +41 61 306 12 34 Av. Dr. Vital Brasil, 1500, São Paulo, SP, 05503-900 (Brazil) E-Mail [email protected] Accessible online at: Tel. +55 11 3726 7222, ext. 2073, Fax +55 11 3726 7222, ext. 2014 www.karger.com www.karger.com/cgr E-Mail nancyoguiura @ butantan.gov.br Table 1. H igher level classification of snakes used herein

Suborder Serpentes Linnaeus, 1758

Infraorder Cope, 1864 Colubroidea incertae sedis: Superfamily Anomalepidoidea Taylor, 1939 Homalopsidae Bonaparte, 1845 Family Taylor, 1939 Epifamily Pareatoidae Romer, 1956 Superfamily Typhlopoidea Merrem, 1920 Family Pareatidae Romer, 1956 Family Stejnejer, 1891 Epifamily Viperoidae Laurenti, 1768 Family Merrem, 1920 Family * Laurenti, 1768 Infraorder Hoffstetter, 1955 Subfamily Azemiopinae Liem, Marx & Rabb, 1971 Parvorder Amerophidia Vidal, Delmas & Hedges, 2007 Subfamily Crotalinae Oppel, 1811 Family Aniliidae Stejnejer, 1907 Subfamily Viperinae Laurenti, 1768 Family Brongersma, 1951 Epifamily Elapoidae Boie, 1827 Parvorder Family Boie, 1827 Superfamily Uropeltoidea Muller, 1831 Subfamily Elapinae Boie, 1827 Family Cylindrophiidae Fitzinger, 1843 Subfamily Bungarinae Eichwald, 1831 Family Müller, 1831 Subfamily Hydrophiinae Fitzinger, 1843 Subfamily Anomochilinae Cundall, Family Fitzinger, 1843 Wallach & Rossman, 1993 Subfamily Dowling, 1967 Subfamily Uropeltinae Muller, 1831 Subfamily Pseudaspidinae Dowling, 1975 Superfamily Pythonoidea Fitzinger, 1826 Subfamily Günther, 1858 Family Xenopeltidae Bonaparte, 1845 Subfamily Pseudoxyrhophiinae Dowling, 1975 Family Loxocemidae Cope, 1861 Subfamily Lamprophiinae Fitzinger, 1843 Family Fitzinger, 1826 Epifamily Colubroidae Oppel, 1811 Superfamily Gray, 1825 Family * Oppel, 1811 Family Calabaridae Subfamily Calamariinae Bonaparte, 1838 Family Gray, 1825 Subfamily Grayiinae Meirte, 1992 Subfamily Erycinae Bonaparte, 1831 Subfamily Oppel, 1811 Subfamily Boinae Gray, 1825 Family Natricidae* Bonaparte, 1838 Subfamily Ungaliophiinae McDowell, 1987 Subfamily Bonaparte, 1838 Alethinophidia incertae sedis: Subfamily Rhabdophiinae Mahendra, 1984 Superfamily Bolyerioidea Hoffstetter, 1946 Subfamily Hydraethiopsinae Dowling, 1978 Family Xenophidionidae Wallach & Günther, 1998 Family Pseudoxenodontidae McDowell, 1987 Family Hoffstetter, 1946 Family Dipsadidae Bonaparte, 1838 Parvorder Hoffstetter, 1939 Subfamily Carphophiinae Zaher et al., 2009 Superfamily Acrochordoidea Bonaparte, 1831 Subfamily Bonaparte, 1838 Family Acrochordidae Bonaparte, 1831 Subfamily Bonaparte, 1845 Superfamily Xenodermatoidea Gray, 1849 Family Xenodermatidae Gray, 1849 Superfamily Colubroidea Oppel, 1811

The application of the Principle of Coordination (Article 36 of prove subordination and to maintain the stability of some the International Code of Zoological Nomenclature, 1999), allied long-standing important clades such as the Colubroidea. Even- to the convention of phyletic sequencing (Nelson, 1973), is suffi- tual disagreements found in phylogenetic hypotheses (see text for cient to inform phylogenetic relationships without the parapher- discussion) were properly expressed by incertae sedis and *sedis nalia of creating new names (as done in some other recent propos- mutabilis annotations (Wiley, 1979). als). The epifamily category has been introduced in order to im-

posed by our current knowledge on systematic patterns explicit about the classification scheme followed by us. of snake relationships. Table 1 presents a summary of the present state of snake Since snake systematics has undergone a major revi- classification for the family-group categories and higher sion recently, with a proliferating number of classifica- ones. The higher level follows a consensus of tion proposals, and given that the present review refers to current phylogenetic hypothesis [Lawson et al., 2005; Lee many outdated taxonomic works, it is important to be et al., 2007; Vidal et al., 2007a, b, 2008, 2009; Wiens et al.,

Cytogenetics and Molecular Data in Cytogenet Genome Res 2009;127:128–142 129 Snakes 2008; Wüster et al., 2008; Kelly et al., 2009; Zaher et al., Moreover, fusions and fissions clearly happened in many 2009]. cases. However, some general features seem to be valid for Evolution cannot be separated from systematics and the whole suborder. Typical karyotypes present: (a) Mac- evolution is a population genetic process governed by 4 ro- (M) and microchromosomes (m). (b) 2n = 36 in al- fundamental forces: natural and sexual selection, muta- most all of the families studied, normally of the 16M + tion, recombination, and random genetic drift [Lynch M, 20m . (c) Heterochromatin (visualized by C-band- 2007]. We reviewed some points of the snake genomes in ing) predominantly centromeric or pericentromeric, and this manuscript; some are not directly related to the con- telomeric or subtelomeric. Interstitial C-bands were sel- struction of relationships among the snakes but are useful domly reported. (d) AgNORs and rDNA predominantly to understand it. The genome size of snakes is small, 2.21 on microchromosomes. (e) Morphologically differenti- pg/nucleus in average [Olmo, 2008], and GC content pro- ated sex chromosomes are of the type ZZ/ZW (or multi- files are characterized by a great variability among the ple chromosomes in some cases) or not morphologically snakes from 0.4 to 3.6 mg/cm3 [Hughes et al., 2002]. One differentiated in some groups. important event in differentiation of snakes was the non- Ancestral reconstruction within Serpentes was done synonymous mutations in the homeobox gene HOXA-13 by parsimony optimization using the diploid number that may be responsible for the absence of legs [Kohlsdorf plus numbers of macro- and microchromosomes avail- et al., 2008]. One important genetic marker are microsat- able in the literature [main source: Olmo and Signorino, ellites, which are not directly involved in the evolution of 2005]. These data were complemented with original ob- snakes, however useful for understanding the genetic servation [Batistic and Ferrarezzi, unpublished] for the processes [Hille et al., 2002; Lukoschek et al., 2008]. We Tropidophiidae and Elapomorphini. Note that our Tropi- cannot forget to add the Horizontal Gene Transfer to dophis paucisquamis (2n = 26) report consists of the first these genetic mechanisms. In this context, many trans- and single account for the whole family [Batistic et al., posable sequences were described in snakes as Bov-B 2002], since that currently ascribed as Tropidophiidae in LINE [John et al., 1994; Nakashima et al., 1995; Kordis the literature (from ) actually belongs to the Un- and Gubensek, 1998; Zupunski et al., 2001], Sauria-SINE galiophiinae, here included in Charininae, Boidae. Al- [Piskurek et al., 2006], CR1-like [Nobuhisa et al., 1998], though we are aware this is an oversimplification of a and Ty1-like [Flavell et al., 1995]. complex character set, it was possible to trace a few pre- To better understand the evolution and diversity of ge- liminary remarks about the pattern that arises. These re- nomic characteristics in Serpentes, we elected 3 points to sults are presented in a reduced form in figure 1 . consider in this manuscript. So we have analyzed se- The 2n = 36 (16M + 20m), although not exactly identi- quences of available mitochondrial genome (comparing cal in the morphology of the chromosomes for all taxa the results with those based solely on nuclear loci), rein- coded as such, is sufficiently similar to be considered a terpreted previous cytogenetic data with an up-to-date shared homology among most snake groups. This puta- phylogenetic hypothesis and reviewed some aspects of tive ancestral configuration is notably conservative (re- toxin genes. maining apparently unchanged in many extant species) and showing a pervasive distribution throughout the snake tree branches, from which a number of other con- S n a k e K a r y o t y p e s figurations have been derived. It is suggested to be prim-

The cytogenetic age, although it provides substantial information for comparative purposes, has not resulted in improvements of snake systematics. Beçak [1965, Fig. 1. Ancestral reconstruction of karyotype configurations 1966], Beçak and Beçak [1969], Singh [1972] and Olmo (diploid number plus the number of macro- and microchromo- [1986] conducted massive studies in an attempt to better somes) under parsimony optimization on a phylogenetic hypoth- understand chromosome evolution in this group and its esis of snakes (adapted from various sources). The 2n = 36 with 16 relation to systematics, hence providing a general picture macro- and 20 microchromosomes (green branches) is indicated of the chromosomal constitution in the group. as a shared ancestral homology throughout most of the family tree, and from which many other karyotypes have been derived at The chromosome complement of snakes is relatively different taxonomic levels. Sexual chromosome (ZW) heteromor- conserved, but this does not mean that translocations, phism is a putative synapomorphy of the superfamily Colubroi- inversions, duplications and deletions did not occur. dea.

130 Cytogenet Genome Res 2009;127:128–142 Oguiura /Ferrarezzi /Batistic

Anomalepididae Leptotyphlops Rhinotyphlops Inferred ancestral configuration: Ramphotyphlops 2n = 36: 16 macro + 20 microchromosomes punctatus isomorphic ZW Typhlops jamaicensis-richardi Aniliidae Tropidophiidae Pythonidae Charinini Boa - cooki Corallus caninus Bolyeriidae Achalinus Pareas Crotalinae Echis Daboia Macrovipera Vipera gr.berus ZW heteromorphism* Vipera gr.aspis Homalopsidae Lamprophis Pseudaspis Malpolon Micrurus Aspidelaps Walterinnesia Hemachatus Naja-Boulengerina Bungarus Dendroaspis Ophiophagus Laticauda Demansia Oxyuranus Pseudonaja Pseudechis Cacophis Furina Simoselaps Vermicella Acanthophis-Echiopsis Denisonia Notechini Hemiaspis Elapognathus Rhinoplocephalus Aipysurus-Emydocephalus Disteira-Kerilia Pelamis Enhydrina-Praescutata Hydrophis-Microcephalophis Limnophis Amphiesma Macropisthodon Rhabdophis-Xenochrophis Sinonatrix Trace character Natrix Storeria-Virginia Character: Diploid number: Macro + micro Thamnophis-Nerodia Parsimony reconstruction (Unordered) Regina [Steps: 75] Pseudoxenodon Thermophis 24: 16M 8m Carphophiinae 26: 20M 6m Hydromorphus Dipsadini 28: gradually decreasing to 8–12m Leptodeirini 30: gradually decreasing to 14–16m Elapomorphus 32 gradual Apostolepis Phalotris 32: 14M 18m Tachymenini 32: 16M 16m Tropidodryas 32: 20M 12m Helicops Hydrodynastes 34 Sordellina 34: 14M 20m Oxyrhopus 34: 16M 18m Pseudoboini Philodryas 34: 18M 16m Liophis-Erythrolamprus 36 gradual Xenodon-Waglerophis 36: 16M 20m Ahaetulla Colubrini 36: 18M 18m Hierophis-Platyceps 36: 20M 16m Oligodon 38: 18M 20m Oxybelis Sonorini 38: 20M 18m Trimorphodon 40: 16M 24m Chironius - 40: 20M 20m Pseustes Zaocys-Argyrogena 42: 16–18M 24–26m Elaphini 3n = 42: 21M 21m Bogertophis 42: 22M 20m Lampropeltini Chrysopelea 44: 22–24M 20–22m Dendrelaphis 46: 16M 30m Boiga Telescopus 50: 14M 36m Dinodon Lycodon

Cytogenetics and Molecular Data in Cytogenet Genome Res 2009;127:128–142 131 Snakes itive to most family-group taxa, with possible exception rafim et al., 2007]. Serafim et al. [2007] formulated the of the Typhlopidae, Tropidophiidae, and Xenodermati- hypothesis that the chromosome number in the dae (for which just a few or a single species have been tends to increase in and decrease in Cen- karyotyped) and possibly also the Elapidae plus Lam- tral America, changes that especially involve the micro- prophiidae clade and the xenodontine dipsadids, both re- chromosomes in and the macrochro- sulting in equivocal optimization due to variation among mosomes in South America, probably due to fusion and and within terminal taxa. fission processes. The high chromosome variability is Diversification occurred from this ancestral karyo- also true for Bungarus and Naja and other species in the type and from then on possibly all species have suffered family. The members of the Hydrophiinae also show con- at least some modifications in them, altering or not the siderable variation in the number of chromosomes [see diploid number. As new tools for chromosomal investiga- Olmo and Signorino, 2005]. In contrast, the subfamily tions become available, in addition to the visualization of Crotalinae presents very conservative karyotypes, all karyotypes by standard and banding staining, the incor- with 2n = 36 (16M + 20m) and similar morphology, the poration of molecular techniques (FISH and others) 4th pair being the sex chromosomes, with female hetero- made it possible to ‘see’ inside the chromosomes, to local- gamety. Minor differences are observed in the morphol- ize genes such as the crotamine gene mapped in Crotalus ogy of the autosomes from one species to another. Also durissus terrificus by Rádis-Baptista et al. [2003], the the W is variable in size and morphology from species to RABSA , DMRT1 and SOX9 genes to the sexual chromo- species, but always well differentiated from the Z chro- somes by Matsubara et al. [2006], and others. Chromo- mosome. The small morphological differences in the somes may undergo many alterations, with or without macrochromosomes of different species in the subfamily any perceptible change in their morphologies. Chromo- suggest that translocations and inversions still happen somes may also undergo no changes at all in their mor- but possibly without disrupting the main linkage groups. phology or content, as seen in the cryptodiran turtles Good evidence of these changes was given in the complex known for their notable karyotypic stability [Olmo, 1986; of species Bothrops neuwiedi by Trajtengertz et al. [1995], Olmo et al., 2002], further corroborated by Mühlmann- showing a translocation of rDNA genes to a macrochro- Diaz et al. [2001]. These authors used a whole chromo- mosome, but apparently still in the process of fixation. some-1-specific probe from a cryptodiran turtle (Trache- Contrary to Elapidae and many Xenodontinae, the Cro- mys) that resulted in specific hybridization of chromo- talinae seem to have achieved an ideal condition in the some 1 on 4 other turtles, revealing a cytogenetic assemblage of many linkage groups of genes, possibly stability of this chromosome during the past 66–144 mil- maintained, at least partially, by the absence of changes lion years. The authors point out that in one third of that in the chromosome number. This seems to fit well with time, various hominid species underwent extensive chro- the ‘canalization model’ and the achievement of the ‘op- mosomal rearrangements. timum karyotype’ proposed by Bickham and Baker In Typhlopidae, a poorly sampled family based on [1979]. However, it must be stressed that if this is the case, chromosomes, 5 out of 6 species present 2n = 32 and this optimum karyotype was achieved by modifications 2n = 34 with 16M, as in a ‘typical’ 36 karyotype. The without changing the chromosome number and that the variation in number is due to a diminishing number of sex chromosomes must have been already present in the microchromosomes, possibly because of translocations ancestor that gave rise to the family Viperidae. Those and fusion events. However, in Typhlops punctatus , the snakes present a wide geographic distribution (absent ba- variation involves macro- and microchromosomes [Olmo sically in Africa, Europe, Australia and Antarctica), sug- and Signorino, 2005; García and Hernando, 2007]. This gesting that the relative stability of the chromosomal set is also the case for Elapinae, where the karyological al- did not interfere with the dispersal of the genus and its terations involve macro- and microchromosomes. Mi- adaptation to new environments, accompanied by minor crurus from Central America varies its chromosome visible rearrangements that might be a consequence of number from 2n = 26 to 34 [Graham, 1977; Gutiérrez and speciation or part of the process. Bolaños, 1979; Gutiérrez et al., 1988; Luykx et al., 1992]. The opposite situation is found in Elapidae and many Contrarily, in South America M. surinamensis surina- Xenodontinae. Their ability to occupy new environments mensis has 2n = 38 [Gutiérrez et al., 1988], M. lemniscatus is accompanied by karyotypical alterations. The more carvalhoi presents 2n = 42 [Beçak and Beçak, 1969], M. conspicuous case is found in the tribe Elapomorphini corallinus has 2n = 40 and M. ibiboboca has 2n = 42 [Se- where the increase in morphological complexity is ac-

132 Cytogenet Genome Res 2009;127:128–142 Oguiura /Ferrarezzi /Batistic companied (or is it its cause?) by karyological alterations dae [Ferrarezzi, 1994], the diploid number varying from especially involving centric fissions of macrochromo- 2n = 34 to 2n = 56 or more. The increase in the number somes and the increase in the number of microchromo- of chromosomes is accompanied by an increase in mor- somes probably also as a result of centric fissions. Apos- phological derivations [Batistic and Ferrarezzi, unpub- tolepis , the genus showing the greatest number of mor- lished]. Olmo [2008] argues that the translocation of mi- phological evolutionary novelties in the tribe, has a crochromosomes to macrochromosomes could result in diploid number of 56 or higher [Batistic and Ferrarezzi, an increase in R-band numbers. As a consequence an in- unpublished]. In the Pseudoboini, a common diploid crease in recombination levels and a high rate of chromo- number is 50, with all macrochromosomes being telocen- some change would have taken place bringing about a tric, except for the sex chromosomes: the Z is meta- or reduction in the size of genomes and chromosomes. This submetacentric and the W is telocentric, normally the could have caused a further increase in the recombina- biggest one in the karyotype. Sordellina puctata has 2n = tion level and an increase in the chromosome change. 52 [Beçak et al., 1990], but there are no evidences of close Genes thus far mapped for the microchromosomes are relationships between it and Pseudoboini; moreover, un- the rDNA sequences, detected by silver staining [Traj- published molecular data indicate that they belong to dif- tengertz et al., 1995] and some other genes recovered ferent groups [F. Grazziotin, personal communication]. from cDNA libraries mapped by FISH [Matsuda et al., The possible homology detected by parsimony optimiza- 2005; Matsubara et al., 2006]. The rDNA sequences found tion in ancestral reconstruction may be an artifact due to may be located in the macro- or in the microchromo- incomplete taxon sampling between these two. If so, we somes or in both at the same time. The Ag-NOR pattern can infer that the fission process occurred independently, in the neuwiedi group of Bothrops (2n = 36) varied in dif- repeatedly in the phylogeny of snakes, the above exam- ferent populations (some considered as , and ples are just some of them. nowadays as full species) and within a same population. The active rDNA clusters were always present in 2 chro- Microchromosomes mosomes, as follows: 2 macrochromosomes number 6 or A common feature in Reptilia is the presence of mac- one macrochromosome number 6 and one or 2 micro- ro- and microchromosomes, probably due to karyologi- chromosomes [Trajtengertz et al., 1995]. This was the cal ancestral derivation from forms that already present- first case of the presence of rDNA genes in macrochro- ed this attribute. Could microchromosomes be advan- mosomes in Viperidae and a very clear example of a tageous to the owners? Genes present in microchromo- translocation of this gene from micro- to macrochromo- somes are less subject to be bounded in large linkage somes. Despite the translocation, the organization of the groups than genes in macrochromosomes, segregating rDNA repeats analyzed by Southern blot showed to be independently. As a result, there is an increase in the vari- highly conserved for the subspecies. In Liophis poecilogy- ability of the genomes. C-banding showed that not all mi- rus shotti (2n = 32) as well as in B. jararacussu (2n = 36) crochromosomes are heterochromatic. Moreover, even the AgNORs are located in 2 microchromosomes [Traj- microchromosomes that are apparently totally hetero- tengertz et al., 1995]. In C. durissus terrificus, Svartman chromatic may have some euchromatin in them. This et al. confirmed by FISH the mapping of rDNA to micro- condition may be or not advantageous, depending on en- chromosomes [personal communication]. The more vironmental conditions [Olmo and Signorino, 2005; common situation is the presence of one pair of chromo- Olmo 2005, 2008; unpublished observations of RF Batis- somes with rDNA, but 2 pairs are not uncommon. As tic]. Hydrodynastes bicinctus bicinctus has 2n = 24, a very mentioned above, in B. insularis (2n = 36) 2 pairs of mi- small chromosome number for snakes and no micro- crochromosomes show Ag-NORs, one pair near the cen- chromosomes [Beçak and Beçak, 1969], which may be in- tromere and one pair in the distal region, at the telomere terpreted as a formation of favorable linkage groups. This or near it, since 2 dots are clearly visible in 2 microchro- karyotype is clearly a derived one, when other species of mosomes [Batistic, unpublished]. Porter et al. [1991], us- the family are analyzed. In the other extreme, the Apos- ing molecular in situ hybridization with biotinylated tolepis genus presents the highest diploid number so far probes, determined the location of the 28S ribosomal se- found in Serpentes and the highest number of micro- quences in 4 species of Caenophidia. In the viperid Cro- chromosomes, some of them very small [Batistic and Fer- talus viridis and in the colubrid Mastophis flagellum , rarezzi, unpublished]. This genus belongs to the tribe El- both with 2n = 36 and similar karyotypes, the first pre- apomorphini, subfamily Xenodontinae, family Dipsadi- sented rDNA sequences on 2 pairs of microchromosomes

Cytogenetics and Molecular Data in Cytogenet Genome Res 2009;127:128–142 133 Snakes and the second in only one pair. In the natricids Nerodia banding staining methods. They found C. vipera with a fasciata and Thamnophis marcianus , both with 2n = 36 2n = 36 (16M + 20m) and no sex chromosome differen- (34M + 2m), the hybridization occurred only in one pair tiation. The 3 species of Vipera presented a 2n = 42 karyo- of macrochromosomes (long arm of pair 1 or pair 2). type (22M + 20m) and in the examined females of V. aspis Those observations are in agreement with our own ob- atra and V. aspis aspis C-banding pointed to the existence servations in many different species [Batistic, unpub- of a differentiated sex chromosome pair. In this case the lished] using silver staining. Microchromosomes were W chromosome was almost totally heterochromatic, al- found even in a translocation with sex chromosomes in a though morphologically indistinguishable from the Z. population of Bungarus caeruleus from West Bengal re- Mimophis , part of the fauna of Madagascar, is the sulting in a multiple sex chromosome constitution of only psamophiine Lamprophiidae [Vidal et al., 2008] Z 1Z 1Z 2Z 2/Z 1 Z 2 W. In the same females, the W suffered a karyotyped to date. Its karyotype (2n = 44: 24M + 20m) dissociation resulting in multiple W chromosomes: W1 revealed the 4th pair as the heteromorphic sex chromo- and W 2. A predominance of polymorphic females over somes: the Z chromosome is biarmed, and the W is uni- the females carrying the original chromosome constitu- armed. The W element was totally heterochromatic (C- tion led the authors to suggest that polymorphic bearers banding) except for an interstitial euchromatic region had better adaptive flexibility and higher fecundity [Sing [Aprea et al., 2003]. et al., 1979]. The Viperidae and Elapidae show a well differentiated W chromosome [Beçak, 1964, 1966; Ray-Chaudhuri et Sex Chromosomes al., 1971]. A multiple sex chromosome system Z1 Z 1Z 2Z 2 / Morphologically differentiated sex chromosomes are Z1 Z 2 W was first described in vertebrates in Bungarus cae- spread all over the Metazoa. In vertebrates they occur in ruleus (Elapidae) [Singh et al., 1970]. The males were all classes but not in all groups within a class. In non- 2n = 44 and the females 2n = 43 and the W condensed avian reptiles they occur in all Orders but not homoge- chromatin could be observed in the interphasic nuclei. neously. In Serpentes they are present in some families Later another case of multiple sex chromosomes was and not in others. Within families they may be present in found in Hydrophis fasciatus fasciatus (Hydrophiidae) of some species and absent in others, possibly due to differ- the type ZZ/ZW1 W 2 . Also in this case, the interphase nu- ent stages of morphological differentiation. Therefore, clei presented in some tissues 2 kinds of heteropycnotic snakes are a good model for the study of sex chromo- groups, corresponding to the W1 and the W 2 [Ray-Chaud- somes. huri and Singh, 1972]. The great majority of South American Boidae presents What is the first step in the differentiation of the no morphological differentiation of sex chromosomes in homomorphic autosome that harbors sex-determining standard staining [Beçak, 1966]. However, genes for mas- genes? Beçak [1964] suggested that the first step was a culinization or feminization may have accumulated in pericentromeric inversion in one of the homologs, which one or more pairs of chromosomes during evolution prevented crossing-over in the region and the accumula- without morphological differentiation [Beçak, 1965; tion of sex-determining genes in the W. In a second step Singh et al., 1968]. Ray-Chaudhuri et al. [1970] did not this region could undergo heterochromatinization and find W chromatin in interphasic nuclei in a number of differentiation in morphology [Beçak, 1983]. On the oth- tissues of many Boidae species. Acrantophis dumerili was er hand, Ray-Chaudhuri et al. [1971] argued that the first found to have a differentiated W chromosome: the size of step for this differentiation could be the heterochromati- the Z and the W were similar but the former is metacen- nization of one chromosome that would later undergo tric and the latter is acrocentric. This was the first report morphological modifications. Minor satellite DNA frac- of differentiated sex chromosomes in Boidae [Mengden tions were found in great concentrations in the W chro- and Stock, 1980], indeed the only known case in non- mosome, an evidence that crossing-over may have ceased Caenophidian snakes. In the Colubroidae, and especially prior to morphological differentiation between the Z and in the Natricidae and Dipsadidae, different stages of dif- the W chromosomes [Singh et al., 1976; Jones and Singh, ferentiation of the W chromosome occur: they may differ 1985]. Another possibility is that both hypotheses are from the Z by the centromere position or by size or by true, depending on the case under consideration. Mat- both [Beçak and Beçak, 1969; Beçak et al., 1990]. Aprea et subara et al. [2006] compared the chromosomes of 3 al. [2006] examined 3 species of the Vipera aspis complex snakes with different stages of sex chromosome differen- and Cerastes vipera of the Viperinae using standard and tiation: molurus bivittatus (Boidae) with no ap-

134 Cytogenet Genome Res 2009;127:128–142 Oguiura /Ferrarezzi /Batistic parent sex chromosome differentiation, Elaphe quadri- tween ZW and XY systems have occurred many times virgata (Colubridae) with intermediate differentiation, during the course of evolution in fish, amphibians and and Protobothrops flavoviridis (Viperidae) with well dif- reptiles [Ezaz et al., 2006; Janes et al., 2008]. Further in- ferentiated ones. E. quadrivirgata and P. f lavoviridis, both vestigations are needed to give some definitive answers presenting the short arm of the W chromosome exten- on the subject. sively degenerated, retain the homology between Z and W only in the telomeric regions. Moreover, in E. quadri- virgata , a region near the centromere on the long arm of T o x i n G e n e s the W chromosome is partially homologous. They ad- vance the hypothesis that the differentiation of sex chro- The venom systems in snakes consist of glands that mosomes began at the distal region on the short arm of a produce toxic substances with or without an injection protosex chromosome in a common ancestor through system. If Viperidae snakes have the most efficient system the occurrence of a sex differentiator on one of the homo- of venom accumulation and injection, Elapidae snakes logs, favoring chromosomal rearrangements with a con- show the most toxic venoms [Kochva, 1987]. The venom sequent cessation of meiotic recombination. Supposedly, system presents many levels of complexity: from those this favors the accumulation of mutations, of euchro- undifferentiated serous glands, through many forms ar- matin deletions and heterochromatinization with accu- tificially taken as intermediaries and assembled as Du- mulation of repetitive DNA sequences. This process pro- vernoy’s glands, to the most developed venom gland gresses from the short to the long arm of the W chro- in Elapidae and Viperidae. The evolutionary homology mosome, as in the E. quadrivirgata and T. f lavoviridis among venom glands is supported by similarities in em- lineages, where the process would have continued inde- bryonic development of the dental, Duvernoy’s, and true pendently after their divergence. Their hypothesis seems venom glands [Kochva, 1978; Kardong, 2002; Jackson, to agree with Beçak [1964], although they do not explain 2003]. On the other hand, McDowell [1986] argues that how and why this first differentiation occurs. the true venom glands derived from the rictal gland, a Have the sex chromosomes of all snakes had the same small oral gland that has muscles bound to them, while origin? The study of Matsubara et al. [2006] strongly sug- the Duvernoy’s gland would have a different origin. Ac- gests that all Alethinophidia share the same Z chromo- cording to dentition, the snakes can be aglyphous (with somes, based on gene content, but nothing is so far known no specialized teeth), opisthoglyphous (rearward grooved in this respect about the Scolecophidia. The other ques- fang), proteroglyphous (forward grooved fang) and sole- tion is: have the sex chromosomes of snakes had the same noglyphous (forward pipe grooved fang). After studying origin as those of other vertebrates? For instance, were all the dental ontogeny, Vonk et al. [2008] observed that the originated from the same ancestral autosome pair? Many front and rear fangs are homologous. Given that the true research groups are trying to answer these questions by venom glands (of Viperidae and Elapidae) and the Duver- combining cytogenetics and molecular approaches. Mat- noy’s glands (of the other Colubroidea) originate from suda et al. [2005] constructed comparative cytogenetic different oral glands, the toxins that are part of their ar- maps of a turtle ( Pelodiscus sinensis ), and of a snake ( Ela- senal could have different origins, even though they have phe quadrivirgata ) using cDNA clones of reptile func- the same protein structure and/or similar biological ac- tional genes. They concluded that the homology between tivity. Moreover, it is known that the venom toxins are turtle and chicken chromosomes are highly conserved highly polymorphic in all taxonomic levels even within a but not as high with snake chromosomes. Interestingly, single species [Chippaux et al., 1991]. however, turtle chromosome 6q and snake 2p represent The toxin genes have undergone an accelerated evolu- conserved synteny with the chicken Z chromosome. tion that has permitted the endogenous proteins to ac- Their results point to the conclusion that avian and snake quire toxic activities, and after to enlarge their range of sex chromosomes were derived from different autosomes activities and targets. This accelerated evolution of se- in a putative common ancestor. Later on, also Matsubara quences was shown by Nakashima et al. [1995] for PLA2 et al. [2006] and Kawai et al. [2007] using molecular tech- of Protobothrops, where exons showed rates of synony- niques associated to cytogenetics came to the same con- mous and non-synonymous mutations larger than the in- clusions. However, some observations challenge the view trons. In the PLA2 genes from Vipera palaestinae, this ac- that sex chromosomes have evolved independently in celerated evolution is limited to the third exon [Kordis et vertebrates, because there is evidence that transitions be- al., 1998]. The accelerated evolution was also observed for

Cytogenetics and Molecular Data in Cytogenet Genome Res 2009;127:128–142 135 Snakes other snake toxins such as other PLA2 s [Moura-da-Silva viperid snakes because the PLA 2 toxins have different or- et al., 1995; Chuman et al., 2000; Fujimi et al., 2002], dis- igins and structures [Fry and Wüster, 2004]. Lynch VJ integrins [Moura-da-Silva et al., 1996, 1997; Juárez et al., [2007] built evolutionary trees of groups I and II sepa- 2008], serine proteases [Deshimaru et al., 1996] and 3-fin- rately, and he found the same phylogenetic relationships ger toxins [Fry et al., 2003; Tamiya and Fujimi, 2006]. of the group I PLA 2 s in elapid to that found by Slowinski Tracing the phylogenetic history among the major et al. [1997] using different methods of phylogenetic anal- groups or between species of snakes using toxins is a com- ysis. This success should be consequence of the single or- plex task. There are abounding doubts about snake tax- igin of elapid PLA2 s and a lack of further events of gene onomy in the data source [Fry et al., 2003], largely because duplication. the systematics of these has been neglected in the Fry and Wüster [2004], with a large number of toxin biomedical literature [Wüster and Harvey, 1996]. The sequences, amino acids or cDNAs, confirm the conclu- main difficulty is to define whether the genes are orthol- sion of Strydom [1973] that the toxins were recruited ogous or paralogous, in order to properly examine the from existing proteins in snakes. Phylogenetic analysis of phylogenetic relationships. This is particularly important toxin sequences made by Fry and Wüster [2004] showed for understanding the evolution of snake venom proteins, that they were not recruited at the same time, despite for which an exceptionally high amount of copies have showing similar structures; from 8 families examined, 5 been reported, even in one individual [Fujimi et al., 2004; (Kunitz protease inhibitors, CRISP toxins (cysteine-rich Oguiura et al., 2009]. A third is the lack of information secretory proteins), GBL toxins (galactose-binding lec- about colubrid toxins due to the difficulty in obtaining tins), M12B peptidase toxins and NGF toxins (nerve the venom. This is currently being solved with studies of growth factor)) were recruited to the poison before the transcriptome of Duvernoy’s glands of Philodryas olfersii separation of Viperidae and Elapidae families, and toxins [Ching et al., 2006] and cDNAs from other colubrids [Fry as lectin-like, PLA 2 and natriuretic peptides are clearly et al., 2008]. the result of 2 independent recruitment events. The 3FTx The family of phospholipase type A2 (PLA2 ) toxins il- (3-finger toxin) family is inferred to have been recruited lustrates the complexity of the toxin world and the at- before the split between the elapid and colubrid lineages, tempt to use their sequences for phylogeny. The PLA 2 tox- and after divergence of the Viperidae, since they are ab- ins are classified into 2 groups according to their amino sent in the latter [Fry et al., 2008]. Another example is the acid sequence and pattern of disulfide bridges: group I, disintegrins; the dimeric disintegrins are widely distrib- PLA 2s from the pancreatic juice of mammals and the uted in Viperinae and Crotalinae, whereas short disinte- venom of the Elapidae, and group II, non-pancreatic grins appear to be restricted to African and Asian Echis PLA2 s of mammals and of the Viperidae. The group I and Eristicophis genera. This fact indicates that the emer- PLA2 s is divided into 2 subgroups: IA that is produced in gence of dimeric disintegrins represents an early evolu- the venom gland and IB, non-toxic which is produced in tionary event predating the Viperinae-Crotalinae split, the pancreas [Danse et al., 1997]. whereas short disintegrins have evolved much more re- The gene structures of group II PLA2 may be divided cently after the radiation of Viperinae [Juárez et al., 2008]. into 2 types according to the number of exons and in- The sarafotoxins appear to be unique to the genus Atrac- trons. PLA2 genes from Vipera ammodytes are organized taspis as are the small basic myotoxins, such as crot- as non-pancreatic PLA2 of humans and mice and contain amine, present only in the venom of the rattlesnakes Sis- 5 exons and 4 introns. The PLA 2 genes of Crotalinae trurus and Crotalus. Both toxins were recruited from en- snakes ( Crotalus and Protobothrops ) are organized as dogenous proteins such as endothelins [Kochva et al., pancreatic PLA2 of humans and dogs and contain 4 exons 1993] and beta-defensins [Torres and Kuchel, 2004; Rá- and 3 introns. Another difference between Viperinae and dis-Baptista et al., 2004]. However, the extension of the Crotalinae genes is the presence of Bov-B-LINE sequence processes from these recruitments and the distinction in introns [Gubensek and Kordis, 1997]. between the independent and common events remain a Davidson and Dennis [1990] analyzed evolutionary vast research field to be explored, from an evolutionary trees for all PLA2 s with the amino acid sequences de- as well as from a systematic point of view. We assumed scribed, but this approach was successful only in the clas- that the construction of phylogeny using toxin data would sification of PLA 2s in groups I and II, but not in the taxo- not be useful in a discussion of higher level groups of nomic classification. Indeed, the recruitment of these 2 snakes as we did here. toxins might have occurred independently in elapid and

136 Cytogenet Genome Res 2009;127:128–142 Oguiura /Ferrarezzi /Batistic M i t o c h o n d r i a l G e n o m e s incompleteness of some of these regions in the partial ge- nomic sequences used for a few but important taxa. Gene There are 13 protein-coding genes known in snake mi- order information was compiled from the same species, tochondrial genomes, 2 ribosomal RNAs genes, and 22 after alignment of the whole mitochondrial sequences us- transfer RNAs genes [Kumazawa et al., 1996]. The base ing Mauve Genome Alignment Software [Darling et al., compositions are biased in snakes as in other vertebrates 2004]. The Mesquite program [Maddison and Maddison, with predominance of A-T over G-C base pairs, and a 2009] was used to edit the character matrix. greater A+C content in the gene-rich strand [Asakawa et The results of mitogenomic analysis are from the raw al., 1991; Yan et al., 2008]. Other features of the complete empirical dataset using parsimony analysis ( fig. 2 ). Our sequences can be summarized as follows: size ranging results were then compared, in terms of taxonomic con- from 16,218 bp in Leptotyphlops humilis to 18,905 bp in gruence, with the most comprehensive phylogenetic , but can fairly exceed in 3 Dipsadinae, analyses of snake higher taxa, which is based exclusively mainly due to extensive control and/or repeated regions, on nuclear loci [Wiens et al., 2008]. Although the 2 inde- reaching an extreme of 23,038 bp in Leptodeira septen- pendent data sets are similar in alignment length and trionalis . Alethinophian snakes in general have longer se- variable positions (nuclear: 13,322 bp, 6,783 variable and quences due to the duplicated control region, exhibiting mitochondrial: 11,539 bp, 7,958 variable), on the one 2 identical copies per genome. These have been main- hand, the number of nuclear genes employed is larger (20 tained stable since their origin and may function as an vs. 13), but on the other, the number of parsimony infor- additional origin of heavy strand replication [Kumazawa mative positions is higher in the mitochondrial set (7,133 et al., 1996; Jiang et al., 2007; Yan et al., 2008]. vs. 4,766). However, the most important differences re- Since the sequencing of the first snake ( Dinodon semi- gard the sampling of characters and taxa. The nuclear set carinatus ) mitochondrial complete genome by Kuma- [Wiens et al., 2008] was previously designed for an analy- zawa at al. [1996], the comparative mitogenomics of sis of higher level snake relationships (18 of the 20 genes the group have been studied by a number of authors, used were newly sequenced for this purpose) with a taxon mainly with phylogenetic purposes [Kumazawa et al., sampling more homogeneously distributed among the 1996; Kumazawa, 2004; Dong and Kumazawa, 2005; snake family-group categories; whereas the mitogenomic Jiang et al., 2007]. Although the subject has recently been set has been accumulated in a public database during the reviewed, the fast increasing sampling of new taxa pro- entire last decade, resulting in the efforts of different au- vides a continuously exciting field of research. thors with different goals, from evolutionary questions to Because the last 2 phylogenetic analyses, published by taxonomy and, more recently, phylogeographic purposes Yan et al. [2008] and Castoe et al. [2009], included 14 and [Mulcahy and Macey, 2009]. 15 snake species representing 7 and 10 families, respec- Thus, the mitogenomic taxon sampling is highly un- tively, we carried out here a cladistic analysis of all avail- balanced, including many duplicated species, with some able snake mitochondrial genomes with a complete cod- families overrepresented, while entirely lacking others. ing region (a data set comprising 51 sequences from 41 On the other side, the character sampling is complete for species, representing 15 families). all terminal taxa with virtually no missing data in the Methods of genome sequence alignments and cladistic mitogenomic set, whereas in the nuclear set there are analysis used here are the following. Protein-coding- genes many genes differentially lacking for most taxa (so, a were extracted from GenBank mitochondrial sequences higher proportion of missing data). Notwithstanding the using the software PEGA 0.99a [Patané, 2009], which pars- considerable differences and a small number of species in es a GenBank flat file with multiple accessions, outputting common, the nuclear and mitogenomic data sets share a only the specified sequence (DNA or protein) of interest number of suprageneric taxa (e.g. families and subfami- from each accession to a multi-FASTA file. Alignment was lies) whose monophyly has been decisively supported in done with RevTrans [Rasmus and Pedersen, 2003], then previous studies, so that both sets can be effectively com- adjusted manually, edited and concatenated using BioEdit pared at this level ( fig. 3 ). [Hall, 2007]. Maximum Parsimony Analysis was conduct- The taxonomic congruence obtained between the nu- ed using TNT [Goloboff et al., 2000], treating the few in- clear and mitogenomic datasets is surprising, showing dels as missing data. The 22 transfer RNAs and the 2 ribo- only 2 incongruent clades, concerning the position of Xe- somal RNAs were not included for 2 reasons: multiple nodermatidae as sister to Acrochordoidea or to Colubroi- alignment ambiguities due to an abundance of indels, and dea and the position of Homalopsidae as sister to Elapidae

Cytogenetics and Molecular Data in Cytogenet Genome Res 2009;127:128–142 137 Snakes Caenophidia Henophidia Colubroidea Scolecophidia Boidae Viperidae Elapidae Colubridae Dipsadidae:Dipsadinae Aniliidae Pythonidae Xenopeltidae Incertae sedis Homalopsidae Tropidophiidae Cylindrophiidae Acrochordoidea Xenodermatoidea Hypsiglena ochrorhyncha nuchalata EU728581 Hypsiglena ochrorhyncha klauberiHypsiglena EU728582 ochrorhyncha Hypsiglena chlorophaea deserticolaHypsiglena chlorophaea EU728587 EU728593 chlorophaea Hypsiglena chlorophaea Hypsiglena chlorophaea chlorophaea EU728585 chlorophaea Hypsiglena chlorophaea EU728577 chlorophaea Hypsiglena chlorophaea Hypsiglena ochrorhyncha klauberiHypsiglena EU728589 ochrorhyncha Hypsiglena ochrorhyncha ochrorhyncha EU728578 ochrorhyncha Hypsiglena ochrorhyncha Hypsiglena sp. DGM2008 EU728580 Hypsiglena sp. Hypsiglena torquata EU728591 Hypsiglena torquata Hypsiglena EU728592 jani texana Gloydius blomhoffi brevicaudus EU913477 blomhoffi Gloydius Pseudoleptodeira latifasciata EU728579 Pseudoleptodeira Hypsiglena slevini EU728584 Ovophis AB175670 okinavensis Ramphotyphlops braminus DQ343649 Bungarus multicinctus EU579522 Agkistrodon piscivorus DQ523161 piscivorus Agkistrodon Bungarus fasciatus EU579523 Agkistrodon piscivorus EF669477 piscivorus Agkistrodon Ramphotyphlops australis AM236346 Naja atra EU921898 Naja atra EU913475 Naja naja DQ343648 Leptodeira septentrionalis polysticta septentrionalis EU728590 Leptodeira Imantodes cenchoa EU728586 Imantodes cenchoa Ophiophagus hannah EU921899 nebulatus EU728583 Pantherophis guttatus AM236349 Pantherophis Pantherophis slowinskii DQ523162 Pantherophis Viridovipera stejnegeri stejnegeri FJ752492 stejnegeri stejnegeri Viridovipera Elaphe poryphyracea GQ181130 Deinagkistrodon acutus DQ343647 Deinagkistrodon acutus EU913476 Dinodon semicarinatus AB008539 zhaoermii GQ166168 Thermophis Enhydris plumbea DQ343650 Enhydris Daboia russellii EU913478 Achalinus meiguensis FJ424614 Achalinus 8 11 10 12 Typhlops reticulatus EU747730 reticulatus Typhlops Acrochordus granulatus AB177879 granulatus Acrochordus Python regius AB177878 Python regius Xenopeltis unicolor AB179620 unicolor Xenopeltis

Typhlops mirus AM236345 Typhlops 9 Boa constictor imperator AM236348 Boa constictor imperator Boa constictor AB177354 Eunectes notaeus AM236347 Cylindrophis ruffus AB179619 Cylindrophis haetianus FJ755181 Tropidophis scytale FJ755180 Leptotyphlops humilis AB079597 Leptotyphlops

7 6 4 Mitogenomic cladogram (length 60,364) 3 13 loci: 11,539 characters 7,958 variable 7,133 parsimony informative

2 5 2 1 1 = duplication of control region; 2 = translocation of tRNA-Leu (between 16S/ND2); 3 = translocation of tRNA-Gln (between Trna-Trp/Ala); 500 4 = translocation of tRNA-Lys (between ATP8/ATP6); 5 = loss of Light Strand replication origin; 6 = degradation of tRNA-Pro(3’ of CR1); nucleotide substitutions 7 = duplication of tRNA-Pro; 8 = duplication of tRNA-lle; 9 = extension of control region; 10 = extreme extention of repeat region on control region; 11 = insertion of repeat region (between ND5/ND6); 12 = insertion of long novel repeat region (between tRNAs-Cys/Tyr

Fig. 2. Most parsimonious cladogram obtained from a cladistic are number 1–12. Those 4 from the basal dichotomy were polar- analysis of the nucleotide mitogenomic data set consisting of the ized by means of outgroup comparison with genomes of the other concatenate alignments of the 13 protein coding genes (total Lepidosauria in general, but especially the Anguimorpha. Branch alignment length: 11,539 bp, of which 7,958 are variables and 7,133 lengths are proportional to the number of nucleotide substitu- are parsimony informative) for 51 terminal taxa. Inferred synapo- tions (scale bar in the lower left). morphies and autapomorphies regarding the gene order features

or to a group including Elapidae plus other colubroids. have used incorporates no evolutionary models attempt- Such degree of congruence clashes with some previous ing to correct these problems. Even under such extreme assumptions that the mitochondrial DNA evolves too fast condition, the mitogenomic approach performs very and, consequently, the amount of sequence saturation well, but the same is not true with regards to the perfor- might be too high to give confident phylogenetic recon- mance of the separate genes. Our results reinforce the structions at the level of deeper ancient branches. utility and importance of continuing sequencing of whole Yan et al. [2008] concluded that: ‘The among-lineage mitochondrial genomes to address important questions and among-gene variation in rate dynamics observed in about the phylogeny and classification of snake higher snakes is the most extreme thus far observed in taxa (e.g. at and above the family level). genome’. This point is especially emphasized here, con- It would be desirable to place some taxon sampling pri- sidering that the method of phylogeny reconstruction we orities in doing so. Of particular interest at higher level

138 Cytogenet Genome Res 2009;127:128–142 Oguiura /Ferrarezzi /Batistic Anomalepididae Leptotyphlopidae Typhlopidae Aniliidae Tropidophiidae Pythonidae Loxocemidae Xenopeltidae Uropeltinae Cylindrophinae Bolyeriidae Fig. 3. Taxonomic congruence between Calabaria Eryx nuclear and mitogenomic data sets, at the Boinae higher level snake systematics. The left Ungaliophiinae tree was adapted from Wiens et al. [2008], Acrochordidae who analyzed 20 nuclear loci, and the right Xenodermatidae one from our fig. 2. Dots on the nodes de- Pareatidae Crotalinae note the congruent clades recovered in Azemiopinae common by both data sets; terminal dots Viperinae also denote monophyletic congruent Homalopsidae 20 loci: Elapidae 13 loci: groups, but a different sampling of sub- 13,322 characters Lamprophiidae 11,539 characters 6,783 variable Natricidae 7,958 variable component taxa used in each tree; open 4,766 parsimony informative 7,133 parsimony informative Colubridae squares denote incongruent clades. The Nuclear gene Dipsadidae Mitogenomic character counts presented are from the cladogram Thermophis cladogram original complete data sets.

snake systematics would be the completion of genomes for techniques to the classical cytogenetic methodologies will those family group taxa still unrepresented: Anomalepi- provide better answers to the great questions about snake didae, Uropeltidae, Loxocemus , Calabaria, and Casarea. It evolution, thus far not properly answered. is also crucial to investigate some additional boid samples, To overcome the difficulties encountered in the con- like the erycines/ungaliophines, as well as the indopacific struction of phylogenetic trees from toxin genes, we have taxa, which have been either poorly sampled or unsatisfac- to pay attention to some points. First, the toxinologists torily resolved using nuclear loci and for which there are who produce the majority of toxin sequence data, in ad- exciting historical biogeographical puzzles to be solved. dition to specifying the snake species (whose taxonomic With regard to the advanced snakes, priority should be status often have a transitory application), inform about determined on the same grounds, in order to maximize its origin, and provide museum collection numbers that the covering of phylogenetic diversity: the Pareatidae, were sources of toxin sequences for future validation and Lamprophiidae, Natricidae, and Pseudoxenodontidae. identification as well as for other important deductions (for example, different homologous sequences from a same specimen can be easily interpreted as paralogous Conclusions copies). It is important not only in phylogeny but also in medical care for the treatment of snake bites. Second, The most extensive karyotype diversification and di- understanding organismal phylogeny also provides the vergence from the postulated 2n = 36 generalized config- means to identify paralogous from orthologous gene cop- uration are found within the Elapidae, the Natricidae, and ies and to study the events of gene duplications and losses the xenodontine dipsadids. Information regarding Anom- (independent from cladogenesis) that, as a consequence alepididae, Aniliidae, Uropeltidae, and Bolyeriidae are in of differential lineage sorting, results in incompatibilities prior want to complement the scheme of snake karyotype between gene and species trees. Third, the emerging field evolution proposed here. Given the variation and a pattern of phylogenomics enables predicting gene function for of taxonomic distribution in apparent consistency with sequences obtained in snake transcriptomes of venom current groupings, cytogenetic comparisons may provide glands as well as studying the origin and evolution of a useful unexploited tool to investigate intergeneric and these toxic proteins, i.e., their gene-specific history. tribal relationships within the extremely diverse neotrop- In general, nuclear and mitochondrial genes without ical xenodontine snakes. The incorporation of molecular multiple copies are elected to be used in phylogenetic re-

Cytogenetics and Molecular Data in Cytogenet Genome Res 2009;127:128–142 139 Snakes construction studies. For the Serpentes, the c- mos and ment. The sequencing of whole mitochondrial genome is RAG1 are the most extensively sampled examples, but also increasing and the phylogenetic trees obtained with more than 20 new nuclear loci have been recently sam- these data are convergent to nuclear data. In addition, we pled and analyzed for a limited, but representative, num- found a different performance in the construction of phy- ber of snake taxa [Wiens et al., 2008] providing a real logenetic trees using mitogenomic or separate gene ap- improvement on the available character evidence at the proaches; therefore, we reinforce the usefulness and im- family level and above. Mitochondrial genes, although portance of continuing to sequence the whole mitochon- extensively sampled, such as cyt-b and ND4, and rRNAs, drial genome. are also very useful, but often biased for having hundreds The accelerated evolution detected in toxin genes and of sequences for a single or a few related species but none mitochondrial genome architecture, combined with the or an insufficient representation for many important genetic variety that microchromosomes can provide, higher taxa. Of particular interest at higher level snake could explain the extraordinary physiology and metabol- systematics is the completion of genomes for those fam- ic flexibility of snakes that enable the snake’s adaptation ily group taxa that are still unrepresented: Anomalepidi- in such diverse habitats and conditions. dae, Uropeltidae, Loxocemus , Calabaria , and Casarea as well as Pareatidae, Lamprophiidae, Natricidae, and Pseu- doxenodontidae, which will permit an equivalent com- Acknowledgements parison regarding the taxonomic congruence between nuclear and mitochondrial genomic datasets. This work was supported by funds of the INCTTOX PRO- GRAM of CNPq, Brazil and FAPESP, SP, Brazil. We are grateful The advantage in using these sequences is the quan- to the anonymous reviewers for the valuable suggestions which tity of data available in number of sequences and number improved this manuscript and to English Consulting (B.V. Young) of taxa and the possibility of easily concatenating infor- for their proofreading services. mation from different sources in a single original align-

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