HERPETOLOGICAL JOURNAL, Vol. 9, pp. 147-151 (1999)

SQUAMATE RELATIONSHIPS BASED ON C-MOS NUCLEAR DNA SEQUENCES

1 D. J. HARRIS1 , E. A. S!NCLAIR1 , N. L. MER CADER2, J. C. MAR SHALL AND K. A. CRANDALL3

1 Department of Zoology, Brigham Young University, 574 Widtsoe Building, Provo, Utah, 84602. USA

2 Departament de Biologia , Universitat de Barcelona, Avinguda Diagonal 645. Barcelona 08028, Sp ain

3 Department of Zoologyand Monte L. Bean Museum. Brigham Young University. 574 Widtsoe Building. Provo. Utah. 84602. USA

Relationships among squamate families have classically been difficult to establish, with morphological characters being interpreted to give many diffe rent topologies. Here we combine new C-mos nuclear DNA sequence data with those already published to assess relationships of 19 families within the . Monophyly of all the families examined is upheld. Many relationships between families are estimated, although it appears there may have been rapid cladogenesis associated with the origins of the Squamata.

Key words : Phylogeny, squamates, C-mos

INTRODUCTION METHODS Squamate relationships have remained contentious The additional examined were: F. since Camp's (1923) "Classification of the ". Cordylidae: Cordy/us cordylus; F. : Despite extensive analyses based on morphological Bunopus tuberculatus, doriae; F. characters many relationships remain unknown. Most Jguanidae: Dipsosaurus dorsalis, Iguana iguana; F. widely accepted are the relationships suggested by Lacertidae: Acanthodactylus scute!latus, Lacer/a Estes et al. (! 988), although the analysis has been criti­ kulzeri, Podarcis hispanica; F. Trogonophidae; cized (Kluge, 1989), and alternative suggestions for Diplometophon zarudnyi; F. Xantusidae: Lepidophyma relationships have been made using different morpho­ gaigae, Xantusia vigilis. These were selected to cover logical characters (Presch, 1988). Surprisingly, the five families not included by Saint et al. ( 1998), and to advent of DNA sequence data has had little impact on extend the number of the family Gekkonidae examined our understanding of squamate relationships. Although from one to three. many studies have examined inter-familial relation­ Total genomic DNA was extracted from small (I or 3 ships (e.g. Hedges et al., 1991; Harris et al., 1998), 2 mm ) pieces of tail tissue. The material was fi nely these have been limited due to their use of mitochon­ diced and digested with proteinase K (Kocher et al., drial DNA sequences, which are typically saturated 1989). Purification was by phenol/chloroform extrac­ before the divergence times necessary to estimate rela­ tions (Sambrook et al., 1989), followed by centrifugal tionships across squamates. dialysis through a Centricon 30000 MW membrane Recently Saint et al. (! 998) used a fragment of the (Amicon). Polymerase Chain Reaction (PCR) primers nuclear gene C-mos to investigate relationships of Aus­ used in both the amplification and the sequencing were tralian relative to their overseas relatives. They G73 and G74 (Saint et al., 1998). PCR conditions were showed that C-mos was likely to be a single copy gene the same as those used by Saint et al. ( 1998). Successful in squamates, had no introns, and that a fragment of PCR products were purified using a Qiaex II kit about 400 base pairs could be amplified across many (Qiagen), and sequenced from both strands on an Ap­ squamate families. Graybeal (1994) had already shown plied Biosystems DNA Sequencing System. that C-mos might be phylogenetically informative among taxa that had diverged up to 400 mya. To esti­ SEQUENCE ANALYSIS mate relationships across squamates, we have extended Genbank accession numbers are AF1 48702 to the number of families included, and compared the esti­ AF148712. The sequences were aligned by eye to the mates of phylogeny produced from Maximum previously published sequences (Genbank AF039462 Parsimony (MP) and Maximum Likelihood (ML) with to AF039482) of Saint et al. (1998). The aligned se­ those previously derived from morphological charac­ quences were 375bp long. The codon reading frame ters. was infered by comparison with the published se­ quences. Of the new sequences, all the lacertids and the two had a deletion of seven codons, and the Correspondence: D. J. Harris, Campus Agrario de Vairao,R. Monte-Crasto, 4480 Vila do Conde, Portugal. E-mail: Diplometophon had an eight codon deletion. These [email protected] were in the same region (bp 727-768 of human C-mos 148 D. J. HARRIS ET AL.

..----Sphenodon punctalus

] Lacertinae Lacertidae Acan thodactylus scut•llatus J Eremiainae

Amphisbaenidae Diplome tophon mrudnyi Trogonophidae ..--_,1 �00_ _, Xantusia vigilis 100 J Xantusidae Lepidophyma gaigae ] T1 lrquascm caidts Egerma lucluosa Scinadae L1pm1JJ noctua

Sphenonwrphus leptofascUJtus

Ablepharus pannon1cus

78 Cordy/us cordylus 73 Cordylidae Iguana ig uana

Dipsosaurus dorsalis ] Iguanidae

Sceloporus grammicus Phrynosomatidae Ctenophorus decresii J Agamidae Physignathus coeincinus

'------Chamaeleosp. Chamaeleonidae

Elgaria multicarinata Anguidae 55 '------Varanus rosenbergi Varanidae

Python reticu latus Pythonidae Candoia carinata Boidae J Boo idea �-- Rmnphotyphlops auslralis Typhlopidae

100 J Gekkonidae 84

Eublepharidae

J Pygopodidae

Cnemidophorus tigris Te iidae Chelodina rugosa

Elseya denlala

�---- Crocodylus poros us FIG. 1. Single most parsimonious tree derived from an analysis of C-mos nucleotide sequence. Numbers above branches indicate bootstrap support (1000 replicates). Numbers below branches indicate boostrap support from an MP analysis based on the amino acid sequence, with all changes weighted equally. See text for details. The tree was rooted using the Crocodylus, Chelodina and Elseya sequences. sequence), and overlapped deletions also found in the neity exists within the data set; and (5) there is no sig­ skink Lipinia noctua and the teiid Cnemidophorus nificant proportion of invariable sites. Once a model of tigris. They were therefore treated as missing data in evolution was chosen, it was used to estimate a tree us­ the analyses. ing maximum likelihood (Felsenstein, 1981) , using The data were analysed using PAUP* (Swofford, random sequence addition and a heuristic search with 1998). When estimating phylogenetic relationships 10 replicates. Also an MP analysis was performed.Two among sequences, one assumes a model of evolution hundred and nine of the 375 characters were parsi­ regardless of the optimality criteria employed. Deter­ mony-informative. A 10 replicate heuristic search was mining which model to use given the data is a statistical carried out, and support fornodes was estimated using problem (Goldman, 1993). We used the approach out­ the bootstrap (Felsenstein, 1985) technique, with 1 OOO lined by Huelsenbeck & Crandall (1997) to test replicates. A furtherMP analysis was carried outon the alternativemodels of evolution, employing PAUP* and translated amino acid sequences. All changes were Modeltest (Posada & Crandall, 1998). A starting tree weighted equally. was obtained using neighbour-joining. With this tree, likelihood scores were calculated forvarious models of RESULTS evolution and then compared statistically using a chi­ Using MP, 209 of the 375 characters were parsi­ square test with degrees of freedom equal to the mony-informative. A 10 replicate heuristic search difference in free parameters between the models being found one MP tree with 892 steps. (Cl= 0.46, HI= tested. The null hypotheses tested in this way included: 0.54). Support for nodes was estimated using the (1) nucleotide frequenciesare equal; (2) transition rates bootstrap (Felsenstein, 1985) technique, with 1000 rep­ are equal to transversion rates; (3) transition rates are licates (Fig. 1). In the translated amino acid sequence, equal and transversion rates are equal; (4) rate homoge- 60 characters were informative. A ten replicate heuris- PHYLOGENY OF SQUAMATES 149

TABLE I. Tests of hypotheses relating to the model of evolution appropriate forphylogeny reconstruction (Huelsenbeck and Crandall, 1997). ?-values were obtained using the computer program Modeltest (Posada & Crandall, 1998). Due to the perfo�ance of multiple tests, the significance level of rejection of the null hypothesis should be adjusted via the Bonferroni correction to a= 0.01.

Null Hypothesis Models Compared -lnL0 -lnL1 df p Equal base frequencies H0: JC69, H1 : F8 1 5010. l 5005.5 3 0.012 Equal ti/tv rates H0: JC69, H, : K80 5010.1 4763.8 1 <0.001 Equal ti and equal tv rates H0: K80, H1 : GTR 4763.8 4761.6 3 0.340 Equal rates among sites H0: K80, H1 : K80+G 4763.8 4582.3 I <0.001 Proportion of invariable sites H0: K80+G, H1 : K80+G+invar 4582.3 4568.9 <0.001 Molecular clock H0: no rate heterogeneity, 4635.8 4568.9 34 <0.00 1 H1 : rate heterogeneity

Sphenodon punctatus Podarcishispanica Acanthodactylus sc�tellatus Lacertidae Lacertkulzeri J Bipesbiporus Amphisbaenidae Diplomdophon zarudnyi Trogonophidae J Amphisb aenia �---1 Xantusiauigilis Ltpidophymagaigae J Xantusidae Tiliquascincuides Egem ialu ct11 osa

Lipinianoctua Scincidae Sphenomorphus leptofasciatus J Ablepharus pannonicus Cordy/us cordylus Cordylidae lgu1111a iguana ] Dipsosaurus dorsalis Iguanidae Sceloporusgrammic us Phrynosomatidae Ctenophorus decresii ] Physignathus cocincinus Agamidae Chamaeleo sp. Chamaeleonidae Python reticu/atus Pythonidae Candoiacarinata Boidae J Booide a Ramphotyphus australis Ty phlopidae Elgaria luctuosa Anguidae . . J Angwmorpha Varanus rosenbergi Vararudae Phyllodactylus mannotus Bunopus tubercu/atus Gekkonidae Stenodactylus doriae J Eublepharis macularius Eublepharidae Carphodactylus /aevis

L..__,-- Strophorus intermedius Pygopodidae Delma fraseri J '------Cn emidaphorus tigris Te iidae Chelodina rugosa Elseya dentata - 0.05 substitutions/site ____ ...... _ Crocodylus porosus

FIG. 2. Single maximum likelihood tree, derived using the K80 model with estimation of the proportion of invariant sites and a discreet approximation of the gamma distribution. See text fordetai ls. tic search found three equally parsimonious trees of DISCUSSION 376 steps (CI=0.61, Hl=0.39). Support for nodes was again estimated using 1 OOO bootstrap replicates (Fig. Analysis of our extended data set supports many of 1). With ML, using Modeltest (Posada & Crandall, the conclusions drawn by Saint et al. (1998). The analy­ 1998) we concluded that the Kimura 80 model (transi­ ses based on C-mos sequences support the monophyly tion/ transversion ratio = 2.6584), with a gamma of the squamates, and that the closest living relative is Sp henodon punctatus. Within the squamates, all the distributed rate heterogeneity model (a= 3.0825), and an estimated proportion of invariable sites (0.2905) was superfamilies and familieswhere multiple species were the most appropriate model of evolution forthese data. sampled came out as monophyletic groups - Agamidae The data did not fit a molecular clock (Table 1 ). A ten (99% bootstrap support from MP tree), Amphisbaenia replicate heuristic search using random sequence addi­ (100%), Booidea (99%), Gekkonidae (95%), Iguanidae tion with this model produced a single maximum (100%), Lacertidae (100%), Pygopodidae (95%), likelihood tree of score -In 4568.9 (Fig. 2). Scincidae (99%) and Xantusidae (100%). In the analy- 150 D. J. HARRIS ET Al.

ses of the nucleotide sequences, the teiid shared derived character of the muscle encircling the Cnemidophorus tigris comes out basal to all other external ear opening. C-mos sequences support this, squamates, although this is not the case when the amino with the Diplodactylinae being sister group to the acid sequence is analysed. Based on morphological Pygopodidae (Delma) in both the ML and MP analysis characters, teiids are usually regarded as the sister taxa (95% bootstrap support). to lacertids (e.g. Estes et al., 1988). The basal position One diffe rence between the MP and ML analyses in this analysis could be due to the presence of a was in the placement of Eublepharis macularius. ML paralogous sequence in teiids, or it could be due to an analyses associate it with the Gekkonidae, while the artifact in the data such as long branch attractions MP analysis places it as sister taxon to the (Felsenstein, 1978), or due to massive convergence in Pygopodidae. Morphological characters suggest that it the morphological characters. Long branch attraction is basal to a clade of these two groups (Grismer, 1988). could be due to rate variation or inadequate sampling. While C-mos sequences clearly group Gekkonidae with Taxon sampling should not be a problem, as we have Pygopodidae and Eublepharidae, the exact relationship included C-mos sequences of lacertids, which are between these three groups remains unresolved. thought to be closely related to teiids (Estes et al., Most of the other intra-familial relationships are ex­ 1988). Rate variation cannot be ruled out, as the data do tremely weakly supported, as shown by very short not fita molecular clock (Table 1 ), and it is clear from internal branches in the ML analysis. As suggested by the ML analysis (Fig. 2) that Cnemidophorus has the Saint et al. (1998), this could be the result of rapid longest external branch of all the squamates sampled. cladogenesis, or simply a result of the limitations of us­ Since its position is only weakly supported (55% ing only one gene region to examine relationships. bootstrap in MP tree), and since the branches immedi­ Only the inclusion of more sequence data will help to ately above its position are extremely short, it cannot be resolve this, although it is clear that C-mos is an ex­ placed with much confidence byth is data set. tremely useful gene for examining many aspects of Based on morphological characters, the Scincomor­ squamate relationships. pha is thought to include Scincidae, Cordylidae, ACKNOWLEDGEMENTS Xantusidae, Lacertidae, Teiidae and Gymnophthalmi­ dae (Estes et al., 1988), with some authors suggesting We thank E. N. Arnold,R. L. Bezy and J. W. Sites that the amphisbaenians should be included (e.g. Sch­ Jr. for supplying us with some of the tissues used. This wenk, 1988). Excluding Gymnopthalmidae, which was work was supported by NSF IBN-97-02338 and the Al­ not sampled, and Cnemidophorus tigris, these taxa are fred P. Sloan Foundation (to KAC). We also thank an also associated by the MP analysis, with the Xantusidae anonymous reviewer who gave constructive criticisms being the sister taxon to the Scincidae, and with the of an earlier draft of this paper. next closest relative being the Cordylidae. These are the REFERENCES same relationships suggested by Presch (1988) based on morphological characters. The two amphisbaenians Camp, C. L. ( 1923 ). Classification of the lizards. Bulletin included, Bipes biporus and Diplometophon zarudny i, of the American Museum of Natural History 48, 289- are strongly grouped as monophyletic (100%), and ap­ 481. pear to be the sister taxa to the Lacertidae. Evidence Estes, R., de Que iroz, K. & Gauthier, J. (1988). from amphisbaenian fossils also suggests they may be Phylogenetic Relationships within Squamata. In members of the Scincomorpha (Wu et al., 1996). With­ Phylogenetic Relationships of the Families. in the Lacertidae, the monophyly of the subfamily Estes, R. & Pregill, G. (Eds.). Stanford: Stanford

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