Relationships of West Indian Anolis (Sauria: Iguanidae): an Approach Using Slow-Evolving Protein Loci

Caribbean Journal of Science, Vol. 26, No 1-2, 7-30, 1990 Copyright 1990 College of Arts and Sciences University of Puerto Rico, Mayaguez Relationships of West Indian Anolis (Sauria: Iguanidae): An Approach Using Slow-Evolving Protein Loci K RISTI L. BURNELL AND S. BLAIR H EDGES Department of Zoology, University of Maryland, College Park, Maryland 207421 ABSTRACT . – Protein variation in 49 West Indian species of the iguanid lizard genus Anolis was examined by sequential electrophoresis at 12 slow-evolving loci. The use of this technique nearly doubled the total number of alleles detected (121 before, 233 after). Genetic distance and parsimony analyses identified intra-island radiations on Cuba, Jamaica, Hispaniola, and Puerto Rico, and found little evidence of close inter-island relationships. In most cases, these island radiations (series) defined by protein data were supported by other data (morphology, immunology, chromosomes). No support was obtained for previously defined higher-level groupings (sections and subsections) within the genus. A revised classification is proposed that recognizes 21 series of West Indian Anolis (131 spp.) with distributions centering on the following islands or island groups: Cuba (alutaceus, argillaceous, carolinensis, equestris, lucius, and sagrei), Jamaica (grahami), Hispaniola (chlorocyanus, christophei, cuvieri, cybotes, darlingtoni, distichus, hen- dersoni, monticola, semilineatus, and sheplani), the Puerto Rican Bank (cristatellus, occultus), the northern Lesser Antilles (bimaculatus), and the southern Lesser Antilles (roquet). No categories above the level of series are recognized due to conflicting evidence for higher-level relationships. Although the existence of Anolis on the North Island (Hispaniola) in the Eocene or Oligocene indicates an early arrival of the genus in the West Indies, molecular dating suggests that mid-Tertiary dispersal and not early-Tertiary vicariance best explains the present distribution of the group. Extensive intra-island radiation occurred during the late-Tertiary (Miocene-Present) with relatively little inter-island dispersal among the Greater Antilles. The iguanid lizard genus Anolis (sensu The subsections are based on the shape of Williams, 1976a, b) comprises over 300 de- the interclavicle, which is arrow-shaped scribed species. In addition to being the (punctatus subsection) or T-shaped (caroli- largest amniote genus, it has figured prom- nensis subsection). Subsections contain se- inently in the ecological, ethnological, and ries that are defined, again, by osteological systematic literature. However, the phy- characters as well as by karyological and logeny of this group has been a continuous scale characters. Series are further broken challenge to systematists since the initial into subseries, species groups and work done by Etheridge (1960). Williams subgroups. Most of the systematic work on (1976a, b) published the first comprehen- Anolis has involved West Indian taxa (131 sive taxonomy of these animals. His clas- spp.). sification relied primarily on osteology and Albumin immunological data presented divided the genus into two sections, alpha by Wyles and Gorman (1980a) and Shochat and beta. These sections originally were and Dessauer (1981) did not support the established by Etheridge (1960) and are de- alpha-beta dichotomy. They found that fined osteologically by the absence (alpha) members of the grahami series (beta sec- or presence (beta) of transverse processes tion) and cristatellus series (alpha section) on posterior caudal vertebrae. Williams clustered more closely with each other than further divided the alpha section into two either did with any other series within their subsections, also osteologically defined. own sections. Shochat and Dessauer erect- ed the “Central Caribbean series complex” to include series of alpha and beta anoles 1 Current address: Department of Biological Sci- that appeared to form a monophyletic ences, University of California, Santa Barbara, Cali- fornia 93106, USA (KLB); and Department of Biology, group. 208 Mueller Lab, Penn State University, University Guyer and Savage (1986) proposed a re- Park, Pennsylvania 16802, USA (SBH). vised classification of Anolis after reana- 7 8 K. L. BURNELL AND S. B. HEDGES lyzing several studies. These studies in- locus. However, it was shown recently in cluded karyological, immunological and a study of West Indian frogs of the genus osteological data, but the final phylogeny Eleutherodactylus (Hedges, 1989) that this was based primarily on osteology. Wil- constraint can be avoided by simply using liams (1989) provides a detailed critique of only slow-evolving loci. In that case, 84 Guyer and Savage in which many serious species were examined in a single study. errors are discussed. Additional problems The specific loci to be used are chosen by with the data and methods of analysis are first running all species at a suite of loci discussed by Cannatella and de Queiroz and then choosing those that have the few- (1989). Both critiques recommended rejec- est alleles, up to a number that can be re- tion of the phylogenetic conclusions and solved accurately on a gel. Sequential elec- classification proposed by Guyer and Sav- trophoresis also can be used to reduce allelic age. Because no new data were presented convergence by detecting “hidden” vari- in Guyer and Savage, and given the serious ation (Coyne, 1982). It involves using a problems associated with their paper, their succession of electrophoretic conditions revised classification will not be used here, (usually different gel and electrode buff- and their study will not be discussed fur- ers) on the same samples. As much as 85– ther. 100% of the actual amino acid sequence Previous molecular studies of West In- variation can be detected using this meth- dian Anolis have focused primarily on rel- od (Lewontin, 1985). atively small groups of species from Puerto This study examines the phylogenetic Rico and the Lesser Antilles (Gorman and relationships of West Indian Anolis using Dessauer, 1965, 1966; Gorman and Atkins, sequential electrophoresis of slow-evolv- 1969; Yang et al., 1974; Gorman and Kim, ing loci. Forty-nine species from all four 1975, 1976; Gorman et al., 1980a, b, 1983; Greater Antillean islands, Guadeloupe in Wyles and Gorman, 1980a). Those studies the Lesser Antilles, and North America have been useful in clarifying the rela- were compared. These new molecular data tionships of eastern Caribbean Anolis. provide further insight into the relation- However, two-thirds of West Indian Anolis ships of this large genus. occur on Cuba and Hispaniola (42 and 39 species, respectively; Schwartz and Hen- M ATERIALS AND M ETHODS derson, 1988). Aside from population stud- Forty-nine species of West Indian Anolis ies of one or a few species (Webster and were collected (Appendix 1): all seven Ja- Burns, 1973; Webster, 1975; Perez-Beato and maican species, 26 of the 38 Hispaniolan Berovides, 1982; Case and Williams, 1984) species, 9 of 13 from the Puerto Rican Bank, and the immunological distance data for 1 of 20 from the Lesser Antilles, and the cybotes (Wyles and Gorman, 1980b), no mo- single North American species. Cuban lecular studies have been published con- Anolis were attainable only through Guan- cerning the phylogenetic relationships of tanamo Bay Naval Station, thus only 5 of species on these two major islands. The the 42 Cuban species were collected. Cha- only comprehensive molecular study of maelinorops barbouri from Hispaniola was West Indian Anolis relationships (Shochat included to provide a root for the parsi- and Dessauer, 1981) examined two Cuban mony trees (see below). Because electro- and two Hispaniolan species, none of phoretic differences between species and which was represented by antisera. As stat- species groups usually are complete with ed by Gorman et al. (1984), the definition no shared alleles (Avise, 1975; Gorman and of the finer divisions within Anolis (series, Renzi, 1979), only one individual per species groups) remains an unfinished species was used. Errors that might result taxonomic task. from this sampling strategy (e.g., missing Electrophoretic studies rarely involve some shared alleles) would most likely af- more than 25 species (Avise and Aquadro, fect close relationships and not the com- 1982) because of the technical aspect of position of species groups or larger clus- comparing a large number of alleles at a ters. RELATIONSHIPS OF WEST INDIAN ANOLIS 9 TABLE 1. Protein loci and electrophoretic conditions Electrophoretic Enzyme conditions Commission Protein Locus Number a 1234 Stainc Alcohol dehydrogenase Adh 1.1.1.1 5 3 Aspartate aminotransferase Aat 2.6.1.1 5 2 Carboxylesterase-D Esd 3.1.1.1 5 1 Glucose-6-phosphate isomerase Gpi 5.3.1.9 547 2 Lactate dehydrogenase Ldh-1 1.1.1.27 31 3 Lactate dehydrogenase Ldh-2 1.1.1.27 3127 3 Lactoyl-glutathione lyase Lgl 4.4.1.5 36 1 Phosphoglucomutase Pgm 5.4.2.2 1235 2 Protein 1 Pt-1 — 432 3 Protein 2 Pt-2 — 421 3 Protein 3 Pt-3 — 46 3 Pyruvate kinase Pk 2.7.1.40 51 1 a Nomenclature Committee of the International Union of Biochemistry (1984). b (1) Tris-citrate pH 8.0, 130 v, 6 h; (2) Tris-citrate pH 6.7, 150 v, 6 h; (3) Poulik, 300 v, ca. 5.5 h; (4) Lithium hydroxide, 350 v, ca. 7 h; (5) Tris-versene-borate, 250 v, 6 h; (6) Tris-HCl, 250 v, 4 h; (7) Tris-citrate EDTA, 300 V, 6 h. c (1) Harris and Hopkinson (1976); (2) Selander et al. (1971); (3) Hedges (1986). Lizards were transported live to the lab- Differences and similarities in electro- oratory for processing or were processed phoretic mobility were confirmed in com- in the field. Blood was collected and tissue parison runs. By alternating samples that samples (heart, liver, kidney, and leg mus- presumably represented the same allele on cle) were returned to the laboratory in liq- the same gel, very small differences in mo- uid nitrogen. Samples were prepared for bility were detected. This procedure was electrophoresis following the methods of repeated for all pairs of samples repre- Hedges (1986, 1989).

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