Cytogenetic Study of Northeastern Butterfly Lizard, Leiolepis

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Cytogenetic Study of Northeastern Butterﬂy Lizard, Leiolepis reevesii rubritaeniata (Squamata, Agamidae) in Northeast Thailand

Sumalee Phimphan1, Alongklod Tanomtong1*, Isara Patawang1, Sarawut Kaewsri2, Sitthisak Jantarat3, and La-orsri Sanoamuang1

1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Program in Applied Biology, Department of Science, Faculty of Science, Buriram Rajabhat University, Muang, Buriram 31000, Thailand 3 Biology Program, Department of Science, Faculty of Science and Technology, Prince of Songkhla University (Pattanee), Muang, Pattanee 94000, Thailand

Received June 19, 2012; accepted February 18, 2013

Summary The present study aims to construct the karyotype and idiogram of the Northeastern butterﬂy lizard (Leiolepis reevesii rubritaeniata). Specimens were collected from Khon Kaen Province, Northeast Thailand. Lizard chromosome preparation was conducted by the squash technique using bone marrow and testis. Conventional staining and NOR-banding techniques were applied to stain the chromosomes with Giemsa’s solution. The results showed that the number of diploid chromosome is 2n=36, while the fundamental number (NF) is 24 in both males and females. The types of macrochromosomes were six large metacentric, four medium metacentric, two small metacentric, and 24 microchromosomes. Nucleolar organizer regions (NORs) are located at the secondary constriction of long arm chromosomes, near the telomeres of the largest metacentric chromosomes. The karyotypes are not different for the sex chromosomes of both males and females. The karyotype formula is as follows:

m m m 2n (36)=L6 +M4 +S2 +24 microchromosomes

Key words Leiolepis reevesii rubritaeniata, Chromosome, Karyotype, Thailand.

The Northeastern butterfly lizard, Leiolepis reevesii rubritaeniata (Fig. 1), is a member of class Reptilia, order Squamata, suborder Lacertilia, family Agamidae, and subfamily Leiolepidinae (Chan-ard et al. 1999). The butterfly lizards of the genus Leiolepis, commonly known as butterfly lizards, are a group of agamid lizards of which very little is known. They are native to Thailand, Burma, Laos, Cambodia, Indonesia, and Vietnam (Mertens 1961). They are terrestrial lizards and prefer to live in arid, open regions. The genus Leiolepis Cuvier (1892), found in southeast Asia, in- cludes four bisexual diploids (2n), L. belliana Hardwick & Gray (1827); L. guttata Cuuvier (1829); L. reevesii Gray (1831) and L. peguensis Peters (1971), and two unisexual triploids (3n), L. triploida Peters (1971) and L. guentherpetersi Darevsky and Kupriyanova (1993). However, the one putatively unisexual diploid is L. boehmei Darevsky & Kupriyanova, 1993 (Hall 1970, Peter 1971, Böhme 1982, Darevsky et al. 1985, Darevsky and Kupriyanova 1993, Schmitz et al. 2001, Grismer and Grismer 2010). Peter (1971) presumed an autotriploid (3n, AAA) nature of the unisexual L. triploida, which he argued could have originated from the reorganization of the initial diploid karyotype in one of

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.78.133 134 S. Phimphan et al. Cytologia 78(2) the bisexual ancestor forms, most probably L. belliana. This is in particular deduced from the fact that there are no unisexual diploid species in the area of the Malayan Peninsula which could have contributed to a possible hybrid origin of L. triploida. Moreover, Böhme (1982) described a likely diploid unisexual population of butterfly lizards from Malaysia. He proposed that L. triploida could have originated from an interspecific hybridization between diploid chromosome parthenogenetic females and males of the sympatric bisexual L. belliana. A similar origin is supposed for the majority of the other presently known triploid unisexual lizard species. There have been few previous literature reviews about the chromosome analysis of lizards in the genus Leiolepis, as undertaken by Satrawaha and Ponkanid (1988), Darevsky and Kupriyanova (1993), Wongwattana et al. (2001), Aranyavalai et al. (2004) and Srikulnath et al. (2009). The aim of the present study is to report the karyotype analysis and chromosomal characteristics of the nucleolar organizer regions (NORs) of L. reevesii rubritaeniata from northeast Thailand. In the future, basic knowledge on and the cytogenetics of L. reevesii rubritaeniata could be applied to research in several fields and in particular be of reference to protect from extinction.

Materials and methods

The L. reevesii rubritaeniata samples were obtained from a local market in Khon Kaen Province, Northeast Thailand. The lizards, five males and five females, were transferred to the lab- oratory and were kept under standard conditions for 7 d prior to the experiments. Chromosome preparation was conducted by the squash technique, from bone marrow and testis. The chromosomes were stained with 10% Giemsa’s for 30 min and NORs were identified through Ag-NOR staining (Howell and Black 1980). The length of short arm (Ls) and long arm (Ll) chromosomes

Fig. 1. General characteristics of the Northeastern butterﬂy lizard (Leiolepis reevesii rubritaeniata).

Table 1. Review of butterﬂy lizard cytogenetic reports in the genus Leiolepis.

Chromosome types Species 2n NF References m sm a t mi L. reevesii 2n=36 10 2 – – 24 48 Satrawaha and Ponkanid (1988) 2n=36 10 2 – – 24 48 Srikulnath et al. (2009) 2n=36 12 – – – 24 48 Present study L. belliana 2n=36 10 2 – – 24 48 Satrawaha and Ponkanid (1998) 2n=36 10 2 – – 24 48 Wongwattana (2001) 2n=34 mac=12 24 48 Aranyavalai et al. (2004) L. guentherpetersi 3n=54 mac=18 36 72 Darevsky and Kupriyanova (1993) L. triploida 3n=54 mac=18 36 72 Darevsky and Kupriyanova (1993)

Remarks: 2n=diploid chromosome number, 3n=triploid chromosome number, m=metacentric chromosome, sm= submetacentric chromosome, a=acrocentric chromosome, t=telocentric chromosome, mi=microchromosome, mac= macrochromosome, NF=fundamental number. 2013 Cytogenetic Study of Northeastern Butterﬂy Lizard, in Northeast Thailand 135

Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) Northeastern butterﬂy lizards (Leiolepis reevesii rubritaeniata), 2n=36, by conventional staining technique, scale bars 10 μm.

Table 2. Mean length of the short arm chromosome (Ls), long arm chromosome (Ll), and total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosomes in 20 cells of the Northeastern butterﬂy lizard (Leiolepis reevesii rubritaeniata), 2n=36.

Chromosome Chromosome Chromosome Ls Ll LT RL±SD CI±SD pairs sizes types

1* 2.016 2.314 4.330 0.158±0.013 0.533±0.011 Large Metacentric 2 1.532 2.254 3.786 0.140±0.008 0.592±0.018 Large Metacentric 3 1.511 1.684 3.195 0.118±0.009 0.525±0.013 Large Metacentric 4 1.381 1.549 2.930 0.108±0.007 0.527±0.015 Large Metacentric 5 1.129 1.275 2.404 0.088±0.005 0.530±0.011 Medium Metacentric 6 0.812 0.909 1.721 0.062±0.006 0.528±0.012 Small Metacentric 7 – – 0.998 0.037±0.005 – Microchromosome 8 – – 0.945 0.035±0.005 – Microchromosome 9 – – 0.879 0.032±0.004 – Microchromosome 10 – – 0.836 0.031±0.004 – Microchromosome 11 – – 0.801 0.029±0.004 – Microchromosome 12 – – 0.762 0.028±0.003 – Microchromosome 13 – – 0.723 0.027±0.003 – Microchromosome 14 – – 0.695 0.026±0.003 – Microchromosome 15 – – 0.661 0.024±0.003 – Microchromosome 16 – – 0.621 0.023±0.003 – Microchromosome 17 – – 0.579 0.021±0.003 – Microchromosome 18 – – 0.511 0.019±0.002 – Microchromosome

Remark: *=Satellite macrochromosomes/NOR pair 1. 136 S. Phimphan et al. Cytologia 78(2) were measured and calculated for the length of total arm chromosomes (LT, LT=Ls+Ll). Relative length (RL) and centromeric index (CI) were estimated. CI was also computed to classify the types of chromosomes according to Chaiyasut (1989). All parameters were used in karyotyping and idio- graming.

Results and discussion

All specimens karyotype possessed a standard karyotype of 2n=36, with six metacentric pairs of macrochromosomes and 12 pairs of apparently acrocentric microchromosomes (Fig. 1). The haploid chromosome number and morphology of macrochromosomes were similar to those of other diploid species, L. belliana and L. guttata (Kupriyanova 1984, Shoubai et al. 1987, Solleder and Schmid 1988, Schmitz et al. 2001, Srikulnath et al. 2009) (Table 1), and of the triploid L. triploida (Hall 1970). Meanwhile, the haploid karyotype of L. boehmei differs from those of other conge- netic species in possessing fewer microchromosomes (11 versus 12) (Aranyavalai et al. 2004). The karyotypes are not different for the sex chromosomes of both males and females, as also seen in the karyotypes of other Leiolepis species (Shoubai et al. 1987, Darevsky and Kupriyanova 1993, Aranyavalai et al. 2004, Srikulnath et al. 2009). The chromosomes of ﬁshes, birds, and some reptile groups are highly variable in terms of size and morphology, and are characterized by bimodal or asymmetric karyotypes composed of macrochromosomes and microchromosomes (Noleto et al. 2006). The Leiolepis species show a great va- riety of karyotypes and sexual systems. Bisexualism has been identiﬁed in L. belliana belliana (2n=36), L. belliana ocellata (2n=34), L. reevesii reevesii (2n=36), L. reevesii rubritaeniata (2n=36), and L. guttata (2n=36) (Kupriyanova 1984, Shoubai et al. 1987, Solleder and Schmid

Fig. 3. Idiogram showing macrochromosomes and microchromosomes of the Northeastern butterﬂy lizard (Leiolepis reevesii rubritaeniata), 2n=36, by conventional staining technique. The arrow indicate satellite macrochromosomes/NOR pair 1. 2013 Cytogenetic Study of Northeastern Butterﬂy Lizard, in Northeast Thailand 137

Fig. 4. Metaphase chromosome plates and karyotypes of male (A) and female (B) Northeastern butterﬂy lizard (Leiolepis reevesii rubritaeniata), 2n=36, by Ag-NOR banding technique. The arrows indicate satellite macrochromosomes/NOR pair 1 (scale bars 10 μm).

Fig. 5. Idiogram showing the lengths and shapes of chromosomes of the Northeastern butterﬂy lizard (Leiolepis reevesii rubritaeniata), 2n=36, by Ag-NOR banding technique. The arrows indicate satellite macrochromosomes/NOR pair 1. 138 S. Phimphan et al. Cytologia 78(2)

1988, Schmitz et al. 2001, Srikulnath et al. 2009). Unisexual triploids have been reported for L. triploida (2n=54) and L. guentherpetersi (2n=54) (Hall 1970, Darevsky and Kupriyanova 1993). There has also been a report of a putatively unisexual diploid for L. boehmei (2n=34) (Aranyavalai et al. 2004). More than 40 diploid (2n) and triploid (3n) pathenogenetic lizard species are known at present. For most, it has been shown with total certainty that they resulted from natural hybridization. As for the triploid unisexual species, they appear in the second stage of this process of hybridization as a result of back crossing between diploid pathenogenetic females and males of their bisexual parental forms. Theoretically, the formation of triploid species is enabled by autopolyploidy, as a conse- quence of spontaneous reorganization of an originally diploid karyotype (Darevsky et al. 1985, Dessauer and Cole 1989, Moritz et al. 1989, Darevsky and Kupriyanova 1993).

Fig. 6. Meiotic cell division of the Northeastern butterﬂy lizard (Leiolepis reevesii rubritaeniata), 2n=36, on interphase (A and B), leptotene (C), zygotene (D), pachytene (E), diplotene (F), early diakinesis (G), late diakinesis (H), metaphase I (I), metaphase II (J), two genome doubling, 4n of diakinesis (K), and two genome doubling, 4n of metaphase I (L). Scale bars indicate 10 μm. 2013 Cytogenetic Study of Northeastern Butterﬂy Lizard, in Northeast Thailand 139

Fig. 7. Evolutionary diagram of Leiolepis species including L. boehmei (2n=34), L. belliana (2n=36), L. reevesii (2n=36), L. guentherpetersi (2n=54) and L. triploida (4n=54). Remarks: mac=macrochromosome and mi=microchromosome.

In this species, silver nitrate staining (Ag-NOR banding technique) showed simple NORs located interstitially in the long arm of one largest metacentric chromosome pair 1 (Fig. 4). We found one pair of Ag-NOR sites in all of the samples examined. However, the results were similar to the previous report on L. reevesii rubritaeniata (Srikulnath et al. 2009). NOR location can describe chromosome evolution. Interstitial NORs probably originated by tandem fusion between NOR- bearing chromosomes and other chromosomes, but in L. reevesii rubritaeniata these NORs might have been produced through pericentric inversion or even through centric fusion of two telocentric chromosomes, one of which had telomeric NORs. The present study on the meiotic cell division of L. reevesii rubritaeniata found that during metaphase I (meiosis I) the homologous chromosomes showed synapsis, which can be deﬁned as the 18 bivalent (six bivalents of macrochromosomes and 12 bivalents of microchromosomes), and 18 haploid chromosomes at metaphase II as diploid species. The largest metacentric chromosome pair 1 is the largest bivalent. No diakinesis and metaphase I cells with partially paired bivalents, that are speculated to be heteromorphic X (Z) and Y (W) chromosomes, and no metaphase II cells with condensed chromosomes, that are speculated to be the Y (W) chromosome, were detected. In prophase I (meiosis I), we found that L. reevesii rubritaeniata had the distinct character of the observable leptotene (initiation of chromosome shrinking), zygotene (initiation of chromosome synapsis), pachytene (completion of chromosome synapsis), diplotene (chiasma and crossing over), and diakinesis (terminalization). We also found that the use of colchicines caused genome doubling from 2n to two genome doubling, 4n of diakinesis and two genome doubling, 4n of metaphase I (Fig. 6). The important chromosome marker of L. reevesii rubritaeniata is the symmetrical karyotype that was found in only metacentric macrochromosomes. The largest and smallest chromosomes show size differences (approximately 8 folds). The chromosome markers are chromosome pair 1 which is the largest metacentric chromosomes. Data of the chromosomal checks on mitotic metaphase cells of L. reevesii rubritaeniata are shown in Table 2. Idiograms by conventional staining and Ag-NOR banding techniques are shown in Figs. 3 and 5, respectively. Figure 7 shows an evolutionary diagram of Leiolepis species including L. boehmei (2n=34), L. belliana (2n=36), L. reevesii (2n=36), L. guentherpetersi (2n=54) and L. triploida (4n=54). The karyotype formula of L. reevesii rubritaeniata is as follows:

m m m 2n (36)=L6 +M4 +S2 +24 microchromosomes 140 S. Phimphan et al. Cytologia 78(2)

Acknowledgments

This work was supported by the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant; ATRC-R5304 and by a grant from the Faculty of Science, Khon Kaen University.

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