Karyotype and Relationships of Anolis desechensis

—We determined the karyotype of Anolis desechensis and compared it with the known karyotypes of other members of the Anolis cristatellus series. The diploid (2N) number of chromosomes of two male A. desechensis was 27, with six pairs of large metacentric macrochromosomes, six pairs of microchromosomes of gradually decreasing size, and sex chromosome heteromorphism (three sex chromosomes, X1X2Y). This chromosome complement is identical to that of A. cristatellus and A. scriptus, thus providing additional evidence of a close relationship to these taxa. The evolution of chromosome number within the cristatellus series appears to have involved minimal homoplasy and therefore contains useful phylogenetic information. Historically, karyotype data have played a significant role in inferring the phylogenetic relationships of Caribbean Anolis lizards (e.g., Gorman et al., 1968, 1983; Gorman and Atkins, 1969; Gorman and Stamm, 1975), and they continue to be used in recent phylogenetic analyses of the entire Anolis clade (Poe, 1998, 2004) as well as various subclades (e.g., Creer et al., 2001, for the roquet series; Brandley and de Queiroz, 2004, for the cristatellus series). Within the cristatellus series, a clade of anoles that inhabit the Puerto Rican Island Bank, St. Croix, and the southern Bahamas, several relationships implied by karyotypes were corroborated (or at least not decisively contradicted) by the results of a combined morphological (including karyotypic), allozyme, and DNA phylogenetic analysis (Brandley and de Queiroz, 2004). These are (1) a close relationship between the bimaculatus, distichus, and cristatellus series (all of which exhibit sex chromosome heteromorphism and reductions in chromosome number relative to the ancestral 2N 5 36); (2) a sister relationship between the bimaculatus and cristatellus series (which share a reduction of 2N to , 33/34, though the combined analysis weakly favors a sister relationship between the bimaculatus and distichus series); and (3) a close relationship between Anolis scriptus and Anolis cristatellus (2N 5 27/28), as well as possibly Anolis ernestwilliamsi and Anolis desechensis (karyotypes previously unknown). Thus, despite increasing reliance on DNA sequence data, karyotypes remain informative characters for phylogenetic inference. Karyotype data have been reported for 11 of 13 currently recognized species of the A. cristatellus series (Gorman et al., 1983). Data are lacking for A. ernestwilliamsi and A. desechensis—two species for which Brandley and de Queiroz (2004) inferred close phylogenetic affinities to A. cristatellus and A. scriptus (the four species together forming the cristatellus superspecies). Here we report the karyotype of A. desechensis and discuss its implications concerning the phylogenetic relationships of that species and karyotype evolution in the cristatellus series. MATERIALS AND METHODS Two male specimens of A. desechensis (USNM 561839 and 561840), collected in the vicinity of Puerto de los Botes, Isla Desecheo, Puerto Rico, on 28–29 December 1998, were karyotyped. Each specimen was treated with 0.05% colchicine (approximately 0.15 cc/g body weight) four hours before being sacrificed. Tissues (bone marrow, liver, gut, and testes) were minced in 0.8% sodium citrate and allowed to sit for 20 min, strained, and the cell-bearing suspension centrifuged. Thereafter methods followed Patton (1967). Multiple dividing cells in mitosis, and first and second meiosis, were examined. Mitotic chromosome pairs were matched by eye and characterized in terms of the total number of chromosomes and relative size. Karyotypes were characterized in terms of the total number of chromosomes as well as the numbers of chromosomes in different categories based on morphology (e.g., metacentric versus acrocentric) and relative size. Although differential staining of chromosome segments (chromosome banding) provides additional information about chromosomal homologies, it was not employed in the present study. Standard banding techniques have generally proved unsuccessful on the chromosomes of ectothermic reptiles (but see Kasahara et al., 1987; YonengaYassuda et al., 1988) and data on banding patterns are not available for other members of the A. cristatellus series. To estimate karyotype evolution in the cristatellus series, the chromosome number was coded as a discrete character and optimized using the parsimony criterion implemented by MacClade 4.07 (W. P. Maddison and D. R. Maddison, Sinauer Associates, Sunderland, MA, 2000) on the combined 12S and cytochrome b mtDNA phylogeny of Brandley and de Queiroz (2004: fig. 8), except that the relationships among the bimaculatus, cristatellus, and distichus series were left unresolved because of weak support (bootstrap proportion , 50%). Karyotype data for species other than A. desechensis were obtained from Gorman and Atkins (1966, 1969), Gorman et al. (1968), Gorman (1973), Gorman and Stamm (1975), T. P. Webster in Williams (1977), and Gorman et al. (1983). 136 2 Corresponding Author. RESULTS AND DISCUSSION Nine mitotic spreads were identified. The karyotypes of the two male specimens of A. desechensis were identical, consisting of 2N 5 27 chromosomes, with six pairs of metacentric macrochromosomes (the last two pairs of similar size but smaller than the first four pairs), and six pairs of microchromosomes of gradually decreasing size, the largest two pairs of which were identifiably metacentric (Fig. 1). Three chromosomes do not form pairs of equal size and thus presumably represent the male sex chromosomes (X1X2Y). This interpretation is supported by 15 first meiotic division spreads in which we observed a trivalent (Fig. 2) and in 17 second meiotic division spreads where we consistently identified either seven or eight microchromosomes as expected with X1X2Y sex chromosome heteromorphism. The karyotype of A. desechensis is, within the limits of resolution of this study, indistinguishable from that of A. cristatellus and A. scriptus (Gorman et al., 1968). A recent phylogenetic analysis of the cristatellus series using morphological (including karyotypic), allozyme, and mtDNA data (Brandley and de Queiroz, 2004) inferred a strongly supported (1.00 Bayesian ‘‘posterior probability’’; 0.89 likelihood bootstrap proportion) clade composed of A. desechensis, A. ernestwilliamsi, A. cristatellus, and A. scriptus (Fig. 3). The karyotype of A. desechensis contributes further evidence in support of this close phylogenetic relationship. Anolis desechensis exhibits the same derived chromosome number (2N 5 27 in males) found in A. cristatellus and A. scriptus with similar relative chromosome sizes and morphologies, as well as X1X2Y male sex chromosomes. No females were examined, but given the species’ close relationship to A. cristatellus and A. scriptus, we can predict that A. desechensis females will possess similar karyotypes to those of female A. cristatellus and A. scriptus (2N 5 28, X1X1X2X2). Sex chromosome heteromorphism occurs in all members of the bimaculatus, cristatellus, and distichus series that have been karyotyped to date. The only other reported case of obvious sex chromosome heteromorphism in anoles is in Anolis biporcatus (Gorman, 1973), a distantly related (Nicholson, 2002; Poe, 2004) Central American ‘‘beta’’ (Norops) anole. This condition can therefore be inferred to have evolved in the ancestor FIG. 1. A karyotype of a male Anolis desechensis (USNM 561840), 2N 5 27, X1X2Y. Autosomal chromosome pairs are arranged in order of decreasing size from upper left to lower right, followed by the sex chromosomes. FIG. 2. First meiotic division spread of a male Anolis desechensis (USNM 561839). The putative trivalent is indicated by the arrow. SHORTER COMMUNICATIONS 137