On the Monophyly of the Agamid Genus Gonocephalus Kaup, 1825 (Reptilia: Squamata): a Chromosomal Perspective

On the Monophyly of the Agamid Genus Gonocephalus Kaup, 1825 (Reptilia: Squamata): A Chromosomal Perspective

CHEONG-HOONG DIONG1, MAY-HON LOW1, ENE-CHOO TAN2, HOI-SEN YONG3, TSUTOMU HIKIDA4, AND HIDETOSHI OTA5*

1 Division of Biology , School of Science, Nanyang Technological University, 469 Bukit Timah Road, SINGAPORE 258756 2 Institute of Cell and Molecular Biology , National University of Singapore, SINGA- PORE 117609 3 Institute of Biological Sciences , University of Malaya, 50603, Kuala Lumpur, MALAYSIA 4 Department of Zoology , Graduate School of Science, Kyoto University, Kitashiraka- wa, Sakyo-ku, Kyoto 606-8502, JAPAN 5 Tropical Biosphere Research Center , University of the Ryukyus, Nishihara, Okinawa 903-0213, JAPAN

Abstract: We karyotyped five species of the agamid genus Gonocephalus, G. chamaeleontinus, G. liogaster, G. bellii, G. grandis (from Peninsular Malay- sia), and G. robinsonii. Of these, karyotypes of the first four species had several chromosomal characteristics exclusively shared with the previously reported karyotypes of G. miotympanum and G. grandis (from Borneo), such as the diploid chromosome number (42) and the presence of 22 biarmed macrochromosomes. This seems to support the monophyly of those four species and G. miotympanum, probably along with some other species of the genus not yet karyotyped. This hypothesis is premised on our finding of distinct chromosomal characteristics that are indicative of highly derived states in the agamid karyotypes. The karyotype of G. robinsonii, while remarkably differ- ent from other congeneric karyotypes in exhibiting much smaller diploid (32) and biarmed macrochromosome numbers (12), share these and other chromosomal characteristics with several Australian species. It seems unlikely for the karyotype of G. robinsonii to directly emerge from other congeneric karyotypes or vice versa. We conclude that the inclusion of this species in Gonocephalus would render the genus paraphyletic.

Key words: Reptilia; Agamidae; Gonocephalus belli; G. chamaeleontinus; G. liogaster; G. grandis; G. robinsonii; Karyotype; Monophyly

INTRODUCTION * Corresponding author. Tel: +81-98-895- 8937; Fax: +81-98-895-8966. The agamid genus Gonocephalus Kaup, E-mail address: [email protected] (H. 1825, is a group of moderate-sized to large Ota) lizards. Darlington (1957) argued that the 72 Current Herpetol. 19 (2) 2000 genus is zoogeographically exceptional, be- et al. (2000) examined only one, two, four, cause it was then considered to occur on and one species, respectively. Considering both sides of Wallace's Line, a zoogeo- that a thorough definition of Gonocephalus graphic border between the Oriental and (sensu stricto) depends only on a few exter- Australian faunas. Based on the micro- nal characters (Moody, 1980; Manthey and chromosome numbers, Witten (1983) also Grossmann, 1997), the monophyly of the postulated that the Australian species as- genus is obviously yet to be examined. signed to Gonocephalus at that date Ota et al. (1992) reported that G. grandis represent recent dispersals from Southeast and G. miotympanum, both from Borneo, Asia across Wallace's Line. share characteristic chromosomal arrange- In his unpublished Ph. D. dissertation, ments that are obviously in highly derived Moody (1980), on the basis of phylogenetic states. This suggests that the karyological analysis of morphological characters, as- approach may be an effective way to exa- serted that the Gonocephalus species east of mine the monophyly of the genus. There- Wallace's line were derived from radiations fore, in this study, we karyotyped four ad- of the Australian stock, and that they are ditional species of Gonocephalus including phylogenetically distant from the Southeast its type species, G. chamaeleontinus, as well Asian species. He further argued that the as G. grandis from the Peninsular Malaysia. genus Hypsilurus Peters, 1867, once syn- onymized to Gonocephalus by Boulenger

(1885), should be resurrected to accommo- MATERIALS AND METHODS date species from the Australian Region. Results of more recent immunogenetic Except for two male Gonocephalus (Baverstock and Donnelan, 1990; King, robinsonii, all lizards, collected from 1990), karyological (Ota et al., 1992), elec- Peninsular Malaysia and Pulau Tioman tron-microscopic (Ananjeva and Mat- (Table 1), were transported to the laborato- veyeva-Dujsebayeva, 1996), and molecular ry where they were injected intraperitoneal- studies (Honda et al., 2000) favored Moo- ly with 0.1ml of colchicine solution dy's (1980) view. All recent authors, with (2mg/ml) per gram of body weight. Six- the exception of a few who have obviously teen to 18h after injection, they were overlooked these works (e.g., Urban, 1999), anesthesized with diethyl ether and were restrict the application of the generic name, dissected to remove femur bones. Bone Gonocephalus, to the Southeast Asian spe- marrow were flushed out from the bones cies (Welch et al., 1990; Manthey and with Hanks balanced buffer solution. For Grossmann, 1997). each sample, the cell suspension was left to In all those works addressing the stand for 10min before it was centrifuged at phylogeny of Gonocephalus (sensu lato), 2000rpm for 5min. Bone marrow cells however, the species assemblage on the were then treated with hypotonic KCl solu- western side of Wallace's Line (i. e., tion (0.06mole/l) at room temperature (26- Gonocephalus [sensu stricto]), though con- 28°C) for 1h, followed by fixation in a 1:3 stituting no less than 16 species (Welch et glacial acetic acid: absolute methyl alcohol al., 1990; Manthey and Grossmann, 1997), mixture. Mitotic chromosome slides were was represented by very few species. For prepared by the splash technique, air-dried, example, Moody (1980) examined osteologi- and were stained in 6% Gurr Giemsa (BDH) cal specimens for only five species. Fur- solution. Mitotic cell slides for the remain- thermore, Baverstock and Donnellan ing two male G. robinsonii were prepared in (1990), Ota et al. (1992), Ananjeva and the field following Ota (1989a), and were Matveyeva-Dujsebayeva (1996), and Honda stained in 2% Giemsa solution. DIONG ET AL. -PHYLOGENY OF AGAMID LIZARDS 73

TABLE 1. Localities, sizes, and sexual compositions of samples of five Gonocephalus species examined in this study.

Karyotypes were determined for each in- ment of Biological Sciences, National dividual lizard on the basis of 8-20 well- University of Singapore (ZRC) and Her- spread metaphase cells. Selected cell petological Collection, Department of Zool- spreads were photographed with a Nikon ogy, Kyoto University (KUZ). Optiphot 2 Photomicrography camera using Kodak TMAX ASA 100 film. Individ- RESULTS ual chromosome pairs were arranged in decreasing size. For the calculation of arm In all Gonocephalus species examined, ratio for each chromosome pair, the lengths karyotypes consisted of chromosomes of chromosome arms were measured with a forming large and smaller size-groups that CALCOM digitizer. Terminology for are referred to here as macrochromosomes chromosomal descriptions follows Green and microchromosomes, respectively (Table and Sessions (1991), and the karyotype for- 2). Of these, macrochromosomes were all mula follows Peccinini-Seale (1981). bi-armed, whereas detailed morphology Voucher specimens were deposited in the remained undetermined for most micro- Zoological Reference Collection, Depart- chromosomes. No sex chromosome hetero-

TABLE 2. Karyotypes of species of the genus Gonocephalus. M=macrochromomes; m= microchromosomes. 74 Current Herpetol. 19 (2) 2000 morphisms or secondary constrictions were types. evident in any karyotypes. In the family Agamidae, two karyo- Karyotypes of G. chamaeleontinus, G. morphs are typical: (1) 2n = 34 or 36 chro- liogaster, G. bellii, and G. grandis consisted mosomes, including six pairs of metacentric of 2n=42 chromosomes, including 22 or submetacentric macrochromosomes and macrochromosomes (pairs 1-11) and 11 or 12 pairs of microchromosomes; and 20 microchromosomes (pairs 12-21). The (2) 2n=46 or 48 chromosomes, all telocen- macrochromosomes of G. chamaeleontinus tric chromosomes without a distinct size- and G. liogaster were all metacentric (Fig. break (Bickham, 1984; King, 1981; Moody 1). From these, macrochromosomes of G. and Hutterer, 1978; Olmo, 1986; Ota and bellii and G. grandis differed in including Hikida, 1989; Solleder and Schmid, 1988; submetacentric elements in pairs 1, 4 and Witten, 1983). One of these karyomorphs 10, and pairs 2, 5, 7 and 9, respectively (Fig. is considered to be derived from the other 2). Thus, the arm numbers in macro- through a series of Robertsonian rearrange- chromosomes were 44 in all of the four ments of macrochromosomes, sometimes karyotypes. With respect to the micro- accompanied with addition or deletion of chromosomes, the largest pair (i. e., pair 12) one microchromosome pair (Bickham, of the G. bellii karyotype was distinctly en- 1984; King, 1981). Judging from the fact larged compared to the chromosome pair that both of the two karyomorphs some- immediately following, thus obscuring the times occur in a single genus or closely size-gap difference between the macro- and related genera (Gorman and Shochat,1972; microchromosomes. In contrast, chromo- Ota, 1988, 1989b) and that there are so few some pair 12 was almost as small as pair 13 karyotypes representing intermediate states in karyotypes of G. chamaeleontinus, G. between the two extremes, such chro- liogaster, and G. grandis, resulting in a mosomal rearrangements may proceed more prominent size-gap difference between rapidly when they are once triggered (King, the two groups of chromosomes. 1981). The karyotype of G. robinsonii differs It is obvious that the karyotypes of G. remarkably from those of the other four chamaeleontinus, G. bellii, G. liogaster, G. Gonocephalus species in having substantial- grandis, and G. miotympanum could not be ly fewer (2n=32) chromosomes. Of the derived from either of the two typical diploid chromosomes, 12 (pairs 1-6) were agamid karyomorphs merely through metacentric macrochromosomes, whereas Robertosonian rearrangements of macro- the remaining 20 (pairs 7-16) were microchromosomes and slight numerical changes chromosomes (Fig. 3). Therefore, the arm of microchromosomes, because these number in macrochromosomes of this karyotypes include a much larger number of karyotype equaled 24. biarmed macrochromosomes (22) than those of any other agamid karyotypes hitherto reported, and at the same time, ex- DISCUSSION hibit a greater diploid number (42) than the Karyotypes of G. chamaeleontinus, G. agamid karyomorph (1). Moreover, their bellii, G. liogaster, and G. grandis from fundamental number (NF, >64) is con- Peninsular Malaysia and Pulau Tioman siderably greater than those with karyo- share similar chromosomal features with morph (2) (NF=46 or 48). We thus con- those of G. miotympanum and G. grandis sider karyotypes of the five Gonocephalus from Borneo (Ota et al., 1992). In con- species to represent a highly derived state, trast, the karyotype of G. robinsonii differs and that the chromosomal features exclu- strikingly from other congeneric karyo- sively shared among these species support DIONG ET AL. -PHYLOGENY OF AGAMID LIZARDS 75

FIG. 1. Karyotypes of (A) Gonocephalus chamaeleontinus, and (B) G. liogaster. Bars equal 5μm. 76 Current Herpetol. 19 (2) 2000

FIG. 2. Karyotypes of (A) Gonocephalus bellii, and (B) G . grandis. Bars equal 5μm. DIONG ET AL. -PHYLOGENY OF AGAMID LIZARDS 77

FIG. 3. Karyotype of Gonocephalus robinsonii. Bar equals 5μm. their monophyly, presumably along with G. robinsonii actually represents dispersals some other species of the genus not yet stu- from the Australian Region into Southeast died karyologically. Asia like Physignathus cocincinus (see Hon- The karyotype of G. robinsonii is similar da et al., 2000). However, it is also proba- to karyomorph (1), and considering ble that the karyotype of G. robinsonii is remarkable differences in the diploid num- derived from karyomorph (1), exhibited by ber of chromosomes or the arm number of a number of other agamid species including macrochromosomes between this and other several from Southeast Asia (see Ota and congeneric karyotypes (Table 2), it is un- Hikida [1989]), through deletions of one or likely that the karyotype of G. robinsonii two microchromosome pairs. More com- directly arose from other congeneric karyo- prehensive analyses using biochemical and types or vice versa. Thus, we conclude that molecular approaches are needed to deter- the inclusion of this species in Gonocepha- mine the relationship of this enigmatic spe- lus would render the genus paraphyletic. cies with certainty. The number of microchromosomes (20) in the G. robinsonii karyotype is smaller ACKNOWLEDGMENTS than that in the typical agamid karyomorph (1)(22 or 24: Bickham, 1984; King, 1981). We thank the Institute of Biological Such a chromosomal arrangement (i.e., Sciences, University of Malaya, Kuala 12M+20m) is exclusively shared with Lumpur, for provision of facilities for ex- several agamid species that are supposedly periments. We are also much indebted to derived from the Australian endemic radia- J. A. Schulze for commenting on an early tion (Witten, 1983). It is thus probable that version of the manuscript. H. Ota and T. 78 Current Herpetol. 19 (2) 2000

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