Contemporary Herpetology  1999 Number 3 8 June 1999	ISSN 1094-2246
	PHYLOGENY OF SOME MIDDLE AMERICAN PITVIPERS BASED ON A CLADISTIC ANALYSIS OF MITOCHONDRIAL 12S AND 16S DNA SEQUENCE INFORMATION Steven D. Werman1 (swerman@mesastate.edu), Brian I. Crother2 (bcrother@selu.edu) and Mary E. White2 (mwhite@selu.edu)

¹ Department of Biology, Mesa State College, Grand Junction, Colorado 81502 USA
² Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana 70402 USA

Abstract. The cladistic relationships of several Middle American pitvipers representing the genera Bothrops (sensu stricto), Bothriechis, Cerrophidion, Lachesis and Porthidium were determined using mitochondrial 12S and 16S DNA sequence information. Maximum parsimony analyses were performed using PAUP on aligned sequences that included published information for related taxa. Two sets of analyses were conducted: one disregarding gaps in the aligned matrix, and another with gaps treated as a fifth base. When gaps were excluded resolution declined, although the general arrangement of the taxa changed little. A consistent relationship was the grouping of ((Porthidium, Bothriechis) Lachesis). The placement of Lachesis, as nested within other bothropoid genera, is only partially supported by results of other authors. The arrangement of Crotalus, Bothrops and Cerrophidion was ambiguous when gaps were discounted. In both trees, Agkistrodon was basal to the New World forms. The remaining genera, Trimeresurus (Protobothrops), Vipera, Azemiops, and Coluber, were uniformly distant to the former taxa. Also of interest is the lack of close relationship, based on the DNA data here and elsewhere, between Bothrops and Porthidium. This is in striking contrast to results based on morphologic and allozymic analyses of previous studies. It is concluded that additional DNA sequence information from a larger sample of taxa will be necessary to better assess the phylogenetic relationships among Middle American and related pitvipers.

The bothropoid pitvipers comprise a diverse and widespread assemblage of venomous snakes distributed from southern Mexico to southern Argentina. In the last decade, progress from systematic studies of pitvipers has led to descriptions of new species and the recognition of several new generic arrangements for those species formerly assigned to Bothrops (sensu lato). Detailed species accounts can be found in Campbell and Lamar (1989), whereas the most recent generic arrangement of New World pitvipers can be found in Campbell and Brodie (1992), and summarized in Campbell and Lamar (1992).

The phylogenetic relationships among Neotropical pitvipers remain problematic. Although several studies using phenotypic character information (Crother et al., 1992; Werman, 1992; Gutberlet, 1998) and molecular data (Knight et al., 1992; Kraus et al., 1996; Cullings et al., 1997; Salomão et al., 1997; Vidal et al., 1997; Wüster et al., 1997) have been completed, many inconsistencies of phylogenetic inference persist (see Werman, 1998). Herein, we present a cladistic analysis of novel mitochondrial 12S and 16S DNA sequence information for some Middle American pitviper species, in conjunction with published sequences for related genera.

Of primary concern is the relationship of Lachesis to the other New World pitviper genera and the relationship of Bothrops (sensu stricto) to Porthidium. Among DNA studies that include Lachesis (Kraus et al., 1996; Cullings et al., 1997; Vidal et al., 1997) there is no clear agreement as to the position of this genus relative to other pitviper genera. Lachesis is either a somewhat basal lineage, or is found nested among other bothropoid genera (see Werman, 1998). Regarding Bothrops and Porthidium none of the present DNA analyses place these together as sister lineages. This is curious because Werman (1992), based on a cladistic analysis of phenotypic information, regarded them as a terminal clade of recent divergence among the New World pitvipers.

MATERIALS AND METHODS

Tissue samples of five pitviper species, representing several Middle American genera, were used as DNA sources. Total genomic DNA samples (that included mitochondrial DNA) were isolated and purified from liver and/or skeletal muscle tissue using standard digestion (SDS-Proteinase K) and extraction (phenol-chloroform) techniques. Microgram quantities of genomic DNAs were obtained for the following taxa: Bothrops asper, Bothriechis rowleyi, Cerrophidion godmani, Lachesis muta, and Porthidium nasutum. Locality data and voucher information are available on request from the authors.

Amplifications were carried out in a Thermolyne Amplitron thermal cycler utilizing established protocols with modifications specific for pitvipers (Knight and Mindell, 1993). Symmetrical amplification of the 12S sequences was accomplished with the L strand primer, 5'-AAACTGGGATTAGATACCCCACTAT-3', and the H strand primer, 5'-GTACACTTACCTTGTTACGACTT-3'. The 16S sequences were amplified with the L strand primer, 5'-CGCCTGTTTATCAAAAACAT-3', and the H strand primer, 5'-CCGGTCTGAACTCAGATCACGT-3' (Knight and Mindell, 1993). The cycle parameters for the 12S and 16S sequences were: 85º C, 5 min, followed by 30 cycles of 94º C, 35 sec; 50º C, 35 sec; 72º C, 1 min; with an extension on the last cycle of 72º C, 5 min, then 4º C, dwell/soak. The amplifications resulted in approximately 960 bp for the combined 12S and 16S gene fragments.

Amplification products were either purified or ligated directly into Invitrogen pCR II TA cloning vectors following Invitrogen specific protocols. Ligation products were transformed into Invitrogen "One Shot" competent cells and plated on LB/Amp/X-gal media for colony selection. Plasmid DNAs were isolated from positive colonies using 5 Prime-3Prime "Perfect prep" miniprep kits. Purified plasmid DNAs were digested with EcoRI, to release the inserts, and size fractionated on 2% agarose gels. Plasmids positive for 12S and 16S inserts were collected and stored at -70º C.

Sequencing effort included standard dideoxynucleotide termination methods (Sanger et al., 1977; Hillis et al., 1996) using CircumVent thermal cycle DNA sequencing kits (New England Biolabs) in combination with chemiluminescent Phototope detection kits (NEB). In addition, sequences were also determined with an automated Applied Biosystems Inc. (ABI) Prism DNA sequencer. In both cases, standard M13 forward and reverse sequencing primers were used. Sequences were determined by comparing both forward and reverse sequencing reactions. Approximately 410 nucleotide positions of the 12S gene and 550 nucleotide positions for the 16S gene were scored (e.g., 410 and 548 for P. nasutum 12S and 16S, respectively).

Partial 12S and 16S DNA sequences for Coluber constrictor, Vipera ammodytes, Azemiops feae and Agkistrodon bilineatus were obtained from Knight and Mindell (1993). 12S sequence information for Trimeresurus (Protobothrops) mucrosquamatus was obtained from Genbank, accession # D31613 (Eguchi, unpublished, 1994). 12S and 16S sequences for Crotalus aquilus were also obtained from Genbank under the accession numbers L14373 and L14374, respectively (Knight et al., 1993). These six taxa were used for outgroup comparisons and rooting purposes in the cladistic analyses.

Sequence alignments were accomplished by eye using a color coding scheme specific for each nucleotide. Insertions and deletions in the sequence matrix generated 975 total characters. Aligned sequences were subjected to phylogenetic analysis using PAUP, version 3.1.1 (Swofford, 1993). Maximum parsimony (MP) analyses were performed using random addition of sequences, tree-bisection-reconnection branch swapping, the MULPARS option with ACCTRAN optimization. Gaps were either treated as a fifth base or as missing information in the data matrix. Characters were not weighted. Unknown nucleotide information was designated as "n" and treated as missing information. Strict consensus trees were retained for multiple MP solutions. Bremer support values (Bremer, 1994), that identify how many extra steps on a particular branch are necessary to collapse the branch in a consensus tree of proximate parsimonious solutions, were used to assess nodal stability.Two analyses were performed: the first with gaps included as character information and a second where gaps were discounted. The rationale for these analyses was to determine the effect of gaps on cladogram resolution.

RESULTS

The MP analysis for all taxa, with gaps included, resulted in a single parsimonious solution (Figure 1) with a tree length of 403 steps and a consistency index (CI) of 0.556, homoplasy index (HI) of 0.444, retention index (RI) of 0.399 and a rescaled consistency index (RC) of 0.222. Two terminal sister clades included ((Porthidium, Bothriechis) Lachesis) and ((Bothrops, Crotalus) Cerrophidion) with proximal and distal lineages being Agkistrodon and Trimeresurus, respectively. These taxa are rooted proximally by a clade composed of Azemiops and Vipera and distally by Coluber. Bremer support values were strongest for the Porthidium-Bothriechis grouping (9) and the relationship of Lachesis to these two taxa (6).

When gaps were discounted as characters, an analysis of all taxa resulted in five equally parsimonious solutions all with 343 steps, a CI of 0.528, HI of 0.472, RI of 0.377 and a RC of 0.199. One of these solutions as well as a strict consensus tree of the five solutions is shown in Figure 2. At the terminus of this tree is a clade composed of ((Porthidium, Bothriechis) Lachesis) with sister relations to a trichotomy that includes Bothrops, Cerrophidion and Crotalus. Below this group and proximal to a polychotomy of Trimeresurus, Azemiops, Vipera and Coluber, a lineage represented by Agkistrodon diverges. Bremer support values are strongest for the Porthidium-Bothriechis grouping (5) and the sister group relation of Lachesis (4) to these former taxa.

DISCUSSION

In spite of several recent attempts to ascertain the phylogenetic relationships of pitvipers many species relationships remain unresolved (reviewed in Werman, 1998). Character information that includes morphology (Werman, 1992; Crother et al., 1992; Gutberlet, 1998), allozymes (Werman, 1992; Crother et al., 1992) and several mitochondrial gene sequences (Kraus et al., 1996; Cullings et al., 1997; Salomão et al., 1997), exists for many taxa, but a stable phylogeny for pitvipers remains elusive (Werman, 1998). In spite of the general lack of congruence among diverse studies conducted previously, some patterns are emerging to which our present analyses offer support.

It is clear from Figure 1, Figure 2, and Knight and Mindell (1993) that Coluber, Vipera and Azemiops are basal to the pitvipers. However, in our analysis, when gaps are included as character information, Azemiops clusters with Vipera, and Coluber represents the earliest divergent lineage. This arrangement is not in complete agreement with that of Knight and Mindell (1993). They suggested that Azemiops is a sister lineage to Agkistrodon , relative to Vipera and Coluber, and thus may well represent the sister group to all pitvipers. Kraus et al. (1996) found Azemiops to be basal to the pitvipers, although in some cases Causus was a closer sister group to the crotalines. In the present study, when gaps are removed from consideration, these genera are not resolved based on a consensus for equally parsimonious solutions (Figure 2). In addition, there remains little doubt that Trimeresurus (Protobothrops) is outside the New World pitviper radiation. This conclusion is supported by several other studies (e.g., Kraus et al., 1996; Salomão et al., 1997; Vidal et al., 1997). Furthermore, our study (Figure 1 and Figure 2) indicates that New World Agkistrodon is a basal lineage among the New World genera. This was also reported by Werman (1992) although conflicting interpretations based on DNA evidence exist (Kraus et al., 1996; Vidal et al., 1997).

Within the Middle American genera, the 12S and 16S sequence information suggests that Porthidium and Bothriechis are sister taxa (Figure 1 and Figure 2). This arrangement, based on Bremer support values, is robust. In addition, Lachesis emerges as a sister lineage to the former two genera. This is a curious result because no other gene sequence analysis to date supports a close relationship between Porthidium and Bothriechis ; also Lachesis has rarely been placed close to the former two taxa. For example, Werman (1992) showed a monophyletic relationship among Bothriechis, Porthidium and Bothrops , but depicted Lachesis as a lineage of early divergence in relation to Middle American pitvipers. Consequently, the relationship of Bothriechis to Porthidium represents yet another systematic hypothesis to be tested by future study. The relationship of Lachesis to other New World pitvipers remains problematic and both previous and present DNA analyses offer little to resolve the position of this interesting genus.

The relationships of Bothrops , Cerrophidion and Crotalus are unclear, although they appear basal in divergence among New World pitvipers. They either comprise a sister group to the above pitviper genera (Figure 1) or an unresolved trichotomy when gaps are treated as missing data (Figure 2). The basal placement of Cerrophidion is supported by Knight et al. (1992) and Werman (1992), whereas a basal placement for Crotalus is supported by Werman (1992) and Salomão et al. (1997).

Werman (1992) found a close affinity between Bothrops and Porthidium in a cladistic analysis of morphology and allozymes. He hypothesized that these taxa constituted a clade of recent divergence, relative to other New World pitviper genera with similar and presumably derived cranial features (Werman, 1992, 1998) and allozymes (Werman, 1992, 1997). This sister relationship was further supported by an in-depth phylogenetic analysis of morphological characters of many bothropoid genera by Gutberlet (1998). The close relationship of Bothrops and Porthidium is not supported by the present analysis or other DNA studies (e.g., Kraus et al., 1996). Although addressed in detail in Werman (1998), it is apparent that the analyses of morphologic and allozymic information are discordant with respect to the DNA analyses. Either the phenotypic data are strongly convergent or the DNA sequence data are not rigorous nor voluminous enough to allow for accurate phylogenetic reconstructions. Surely, more DNA sequence information is necessary to resolve the relationship between Bothrops and Porthidium, as well as the relationships among other New World pitviper genera. However, we suspect that the incongruences among data sets may simply reflect the disjoint evolutionary nature of mitochondrial DNA with respect to the nuclear genome. Perhaps an exploration of the latter will allow for further resolution of the relationships among these interesting snakes.

ACKNOWLEDGMENTS

This work was supported by a NSF-ROA in conjunction with NSF-RUI 9207751 to B. I. C. and M. E. W. and a Faculty Development grant to S. D.W. from the Mesa State College Foundation. We thank Paul Chippendale and Jonathan Campbell, University of Texas at Arlington, for providing tissue samples for DNA sources. Jonathan Weist and Marshall Anderson, formerly of the St. Mary's Cancer Research Institute, as well as Christopher Bacot, of the Florida State University DNA Sequencing Facility, were most helpful in the generation of automated DNA sequence information. Novel sequences determined in this study will be deposited into GenBank subsequent to the acceptance and publication of this document.

LITERATURE CITED

BREMER, K.

1994. Branch support and tree stability. Cladistics 10: 295-304.

 

CAMPBELL, J.A. AND E. D. BRODIE, JR.

1992. Biology of the pitvipers. Selva, Tyler, Texas.

 

CAMPBELL, J. A. AND W. W. LAMAR.

1989. The venomous reptiles of Latin America. Comstock Publ. Assoc. Cornell Univ. Press, Ithaca, New York.

1992. Taxonomic status of miscellaneous Neotropical viperids, with the description of a new genus. Occasional Papers Museum Texas Tech University 153: 1-31.

 

CROTHER, B. I., J. A. CAMPBELL  AND D. M. HILLIS.

1992. Phylogeny and historical biogeography of the palm-pitvipers, genus Bothriechis: Biochemical and morphological evidence. Pages 1-20 in Biology of the pitvipers. (Campbell, J. A. and E. D. Brodie, Jr. eds). Selva, Tyler, Texas.

 

CULLINGS, K.W., D.J. MORAFKA, J. HERNENDEZ  AND J.W. ROBERTS.

1997. Reassessment of phylogenetic relationships among pitviper genera based on mitochondrial cytochrome b gene sequences. Copeia 1997: 429-432.

 

GUTBERLET, JR., R.L.

1998. The phylogenetic position of the Mexican black-tailed pitviper (Squamata: Viperidae: Crotalinae). Herpetologica 54: 184-206.

 

HILLIS, D.M., B. K MABLE, A. LARSON, S. K. DAVIS AND E. A. ZIMMER.

1996. Nucleic acids IV: Sequencing and cloning. Pages 321-381 in Molecular systematics. (Hillis, D. M., Moritz, C. and B. K. Mable, eds). 2nd ed. Sinauer Associates Inc. Sunderland, MA, USA.

 

KNIGHT, A.,  L. D. DENSMORE III. AND E. D. RAEL.

1992. Molecular systematics of the Agkistrodon complex. Pages 49-70 in Biology of the pitvipers. (Campbell, J. A. and E. D. Brodie, Jr., eds). Selva, Tyler, Texas.

 

_____ AND D. P. MINDELL.

1993. Substitution bias, weighting of DNA sequence evolution, and the phylogenetic position of Fea's viper. Syst. Biol. 42: 18-31.

 

_____, STYER, D. S., S. PELIKAN, J. A. CAMPBELL, L. D. DENSMORE III  AND D. P. MINDELL.

1993. Choosing among hypotheses of rattlesnake phylogeny: A best-fit rate test for DNA sequence data. Systematic Biology 42: 356-367.

 

KRAUS, F., D. G. MINK  AND W. M. BROWN.

1996. Crotaline intergeneric relationships based on mitochondrial DNA sequence data. Copeia 1996: 763-773.

 

SALOMÃO, M. G., W. WÜSTER,  R. S. THORPE, J-M. TOUZET   AND BBBSP.

1997. DNA evolution of South American pitvipers of the genus Bothrops (Reptilia: Serpentes: Viperidae). Pages 89-98 in Venomous snakes: Ecology, evolution and snakebite. (R. S., Thorpe, Wüster, W. and A. Malhotra, eds). Symposia  Zoological Society of London No. 70., Oxford University Press.

 

SANGER, F. NICKLEN AND A. R. COULSON.

1977. DNA sequencing with chain-termination inhibitors. Proceedings of the National Academy of Sciences U.S.A. 74: 5463-5467.

 

SWOFFORD D. L.

1993. PAUP: Phylogenetic Analysis Using Parsimony, Version 3.1. Formerly distributed by Illinois Natural History Survey, Champaign, Illinois.

 

VIDAL, N., G. LECOINTRE, J.C. VIE AND J.P GASC.

1997. Molecular systematics of pitvipers: Paraphyly of the Bothrops complex. C.R. Acad. Sci. Paris, Sciences de la vie/Life Sciences 320: 95-101.

 

WERMAN, S.D.

1992. Phylogenetic relationships of Central and South American pitvipers of the genus Bothrops (sensu lato): Cladistic analyses of biochemical and anatomical characters. Pages 21-40 in Biology of the pitvipers (Campbell, J. A. and E. D. Brodie, Jr., eds). Selva, Tyler, Texas. 

1997. Systematic implications of lactate dehydrogenase isozyme phenotypes in Neotropical pitvipers (Viperidae: Crotalinae). Pages 79-88 in Venomous snakes: Ecology, evolution and snakebite. (R. S., Thorpe, Wüster, W. and A. Malhotra, eds). Symposia  Zoological Society of London  No.70, Oxford University Press.

1998. Molecular phylogenetics and morphological evolution in Neotropical pitvipers: An evaluation of mitochondrial DNA sequence information and the comparative morphology of the cranium and palatomaxillary arch. Kaupia, Darmstadter Beitrage zur Naturgeschichte (in press).

 

WÜSTER, W.,  M. G. SALOMÃO, R. S. THORPE, G. PUORTO, M. F. D. FURTADO, S.A. HOGE, R. D. G. THEAKSTON AND D. A. WARRELL.

1997. Systematics of the Bothrops atrox complex: New insights from multivariate analysis and mitochondrial DNA sequence information. Pages 99-113 in Venomous snakes: Ecology, evolution and snakebite. (R. S., Thorpe, Wüster, W. and A. Malhotra, eds). Symposia  Zoological Society of London No. 70, Oxford University Press.

APPENDIX

Character matrix of aligned (L-strand) mitochondrial 12S and 16S DNA sequence information. The following abbreviations are: C- Coluber constrictor, AZ- Azemiops feae, V- Vipera ammodytes, AG- Agkistrodon bilineatus, N- Porthidium nasutum, R- Bothriechis rowleyi, L- Lachesis muta, CE- Cerrophidion godmani, AS- Bothrops asper, CR- Crotalus aquilus, T- Trimeresurus mucrosquamatus. Asterisks indicate the start of the 12S and 16S sequences. Missing information is designated by "n", gaps by "-".

Submitted: Monday 23 November 1998 09:39:57 +1000 (EST)