Herpetologica, 58(2), 2002, 270-275 ? 2002 by The Herpetologists'League, Inc. SNAKE RELATIONSHIPS REVEALED BY SLOWLY-EVOLVING PROTEINS: FURTHER ANALYSIS AND A REPLY RICHARD HIGHTON', S. BLAIR HEDGES2, CARLA ANN HASS2, AND HERNDON G. DOWLING3 'Department of Biology, University of Maryland, College Park, MD 20742, USA 2Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA 3Rendalia Biologists, 1811 Rendalia Motorway, Talladega, AL 35160, USA ABSTRACT: A reanalysis of our allozyme data (Dowling et al., 1996) for four slowly-evolving loci in 215 species of snakes by Buckley et al. (2000) concluded that because of ties in genetic distances our published UPGMA tree had "little resolution, indicating that these data are highly ambiguous regarding higher-level snake phylogeny." They also concluded that "the high degree of resolution in the published phenogram is an analytical artifact." Our study was intended to obtain information on lower-level relationships for the snake species that we had available, and it provided support for some current hypotheses of snake relationships at that level. Buckley et al. (2000) reached their conclusions because in their analysis they used only strict consensus trees and did not randomize the order of their input data. By randomizing data input order and using a majority-rule consensus tree, we show that there is considerable phylogenetic signal in our data. Key words: Allozymes; Genetic distances; Phylogeny; Serpentes; UPGMA trees FIVE years ago, we published an allo- tle phylogenetic signal in our data set. zyme study of 215 species of snakes, about Thus, they claimed that the allozyme data 8% of living species (Dowling et al., 1996). are "highly ambiguous regarding higher- We selected four protein loci that ap- level snake taxonomy" and that the "high peared to be evolving relatively slowly be- degree of resolution in our published cause they had the fewest alleles (25-43) phenogram is an analytical artifact" of our among the loci that we initially surveyed. failure to consider alternate trees "implied With only four loci, our primary objective by tied distance values". We disagree with was not to obtain a robust phylogeny of these claims and will show that there is snakes (that would be a remarkable feat phylogenetic information in the allozyme for any allozyme data set surveying 215 data set. species). Instead, our purpose was to ob- In their reanalysis, Buckley et al. (2000) tain information on lower-level relation- removed 92 species because, in their ships among the species that we had avail- words, "identical taxa (D = 1, I = 0) [they able. Because of this, we entitled our study meant D = 0, I = 1] provide no additional a "preliminary survey". Although we point- information about the structure of the ed out a number of protein similarities tree." Actually, adding or deleting identical among higher-level taxa, we concluded taxa may affect tree topology. If identical that the interfamily-level topology of our OTUs are removed from a study, the re- UPGMA tree was "not well supported". maining OTUs may join more basal We also noted that we were unable to dis- branches in a different order. Moreover, tinguish relationships at the species level. the removal of 43% of our species resulted However, at the levels of genera, tribes, in the deletion of the most important re- subfamilies, and families, this approach sults of our study, which focused primarily provided some valuable information. We on lower-level relationships, not on higher- discussed the many cases of concordance level relationships as claimed by Buckley with current snake taxonomy at those lev- et al. (2000). Closely related species (and els. perhaps closely related genera) might be Recently, Buckley et al. (2000) reana- expected to be similar to each other at lyzed our data, constructed strict consen- slowly evolving loci because few substitu- sus trees, and concluded that there was lit- tions that result in changes in electropho- 270 June 2002] HERPETOLOGICA 271 retically detectable proteins are expected ogy. If Buckley et al. (2000) were interest- among recently diverged species. The re- ed in the effect of ties on the topology of sults confirmed our expectations. At the UMPGA trees based on our snake data lowest taxonomic level, there was no with- set, randomly reordering the taxa would in-group variation in 19 groups ranging in have clearly revealed whether or not the size from 2-30 species (identical alleles at tree topology was influenced by the pres- all four loci), and many other closely-re- ence of ties. lated species had identical genotypes at The majority-rule consensus method three of the four loci. However, all species should have been used instead of the strict were not clustered within their own lower- consensus method. As its name implies, level phylogenetic group; we pointed out the strict consensus method only resolves that at least five species were likely mis- groups that appear in all sampled trees placed on the tree (Dowling et al., 1996). (Nei and Kumar, 2000). Thus, if the par- Ties can be a problem with some meth- ticular group of species clustered at a node ods of phylogenetic analysis,including the is different in only one tree out of 10,000, one that we used (UPGMA). Tree-building the strict consensus tree will reflect this algorithms often break ties arbitrarilyand disagreement, resulting in a polytomy for therefore the order in which taxa occur in all involved branches. We believe that a the input file of the data used to calculate rare occurrence should not completely ne- the tree may influence the topology of the gate an overwhelming agreement among tree (Hedges et al., 1992; Hendy et al., the majority of the trees. For example, 1988). Buckley et al. (2000) acknowledged among the 39 species of natricines in our the possibility of this taxon-orderbias, yet study, I-values vary from 1.0 to 0 with they did not randomize the input order of many ties at all values (1.0, 0.75, 0.50, taxa. Instead they used a taxon order that 0.25, 0). The same is true for the reduced was similar to the one in our study to cal- number of eight species of natricines that culate their 9999 "equivalent"trees, the Buckley et al. used, except that there are maximum number for their software. no I-values of 1.0 since they removed all Their method did not alwaysdetect ties, species that were identical at all four loci. even with 9999 trees. An example is the By using their method of analysis, the na- arrangementof three genera (Carphophis, tricines form a polytomy with most or all Diadophis, and Farancia) on their consen- of the remaining groups of snakes in both sus trees. These genera clustered together of their consensus trees (70 of 73 groups in our tree in the arrangement (Carpho- in their Fig. 2 and all 31 groups in their phis (Diadophis, Farancia)) and in the Fig. 3). In contrast, UPGMA trees will al- same way in of all their 9999 UPGMA most always cluster all 39 species of natri- trees (their Fig. 2) since they do not show cines (or their reduced sample of eight a polytomy for the group. In their Fig. 3, species) as a monophyletic group. This is their 9999 trees all had a different topol- because all natricines in our study have an ogy ((Carphophis, Diadophis) Farancia) I-value to at least one other natricine indicating the input order had been re- -0.75, while none has an I-value to any versed (the I-value between the pair Car- non-natricine snake in the study >0.50 (all phophis and Diadophis is the same as that natricines differ from all species in other between Diadophis and Farancia). Using groups at two or more of the four loci). their methodology,they did not find an in- However, by using a strict consensus tree, dication of the tie involved in either of if only one tree in 10,000 clusters a single their two sets of 9999 trees in spite of the species of natricine in some other group fact that each of the two arrangements (or a single species of another group with- would be expected half the time. In our in the natricines), both groups will be majority-rule consensus tree (discussed placed in a polytomy, indicating no reso- below), the tie was no longer present be- lution of the groups. cause we included two species of Faran- Buckley et al. (2000) noted that many of cia; thus all 50 trees had the same topol- their trees agreed well with "traditional 272 HERPETOLOGICA [Vol. 58, No. 2 Clharina bottae Tropidophis haetianus Arrhyton funereus* Dartirsgtonia haetiana Tihamnodynastes strigilis Phitodryas burmeisteri Philodryas viridis Lamprophis fuliginosus 8 4 - | - Alsophis cantherigerus Antillophis parvidons 8 0 Arthyton exiguum Alsophis portoricensis Hydrodynastes gigas HHelico s angulatus _ 9 0 1 00 Helicops leopardinus 8 0 | -- Arrhyton taeniatum 84 so -C= Arrhytoncalli/aemum 58 100 Arrhytonlandoi Dipsas catesbyi Rhadinaeaflavlata _ 7 4 Liophis miliaris 100 F- ~~~~~~~~~~~~~Waglerophismerrami Lys/rophis dorbingi 74 n Xenodonseverus 10 Hypsirhynchusferox 1 8 4 _ ~ -- -- Uromacercatesbyi b _ 10 0 Uromacer frenatus la/tris dorsalis 1 0 0 t .. ,,- -Uromacer catesbyi a A - -- ------Liophis poecilogyrus 84 Clelia rustica 9 0 1- - - Liophisviridis 90 Micruroides euryxanthus Naja naja 100 Ophiophagus hannah Bungarus fasciatus 100 Micrurus annellatus Micrurus dias/ema Dendrelaphis caudolineata Amphiesma sto/ata Reginarigida Reginaseptemvittata i 00^ w v - ~~~~~ ~Sinonarrix- annularis Chlonophis kirtlandii Nerodia cyclopion 100 Nerodia erythrogaster 100 Nerodia fasciata Nerodia harteri Nerodia compressicauda Nerodia rhombifer Nerodia sipedon Nerodia taxispilota Regina alleni Seminatrix pygaea Storeria occipitomaculata Thamnophis brachystoma Thamnophis but/eri Thamnophis chrysocepha/us 100 Thamnophis couchi Thamnophis elegans 62 Thamnophis eques Thamnophis marcianus Thamnophis melanogaster Thamnophis mendax Thamnophis ordinoides Thamnophis proximus Thamnophis radix Thamnophis rufipunctatus Thamnophis sauritus Thamnophis sirtalis Thamnophis sumicrasti Virginia striatula Virginia va/eriae Rhabdophis chrysargus Rhabdophis sp.