First Cytogenetic Study of Malayan Snail-Eating Turtle, Malayemys Macrocephala (Testudines, Geoemydidae) in Thailand

© 2013 The Japan Mendel Society Cytologia 78(2): 125–132 First Cytogenetic Study of Malayan Snail-eating Turtle, Malayemys macrocephala (Testudines, Geoemydidae) in Thailand Pornnarong Siripiyasing1, Alongklod Tanomtong2*, Sarun Jumrusthanasan2, Isara Patawang2, Sumalee Phimphan2, and La-orsri Sanoamuang2 1 Major of Biology, Faculty of Science and Technology, Mahasarakham Rajabhat University, Muang, Mahasarakham 44000, Thailand 2 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand Received June 19, 2012; accepted February 17, 2013 Summary The first cytogenetics of the Malayan snail-eating turtle (Malayemys macrocephala) from the Chi river basin, Khon Kaen Province, Thailand, were studied. Blood samples were taken from two male and two female turtles. Standard T-lymphocyte cell culture at 26°C for 96 h was applied. The mitotic chromosomes were harvested by colchicine-hypotonic-fixation-air drying technique. Conventional staining and GTG-banding techniques were applied to stain the chromosomes with 20% Giemsa’s solution. Results showed that the number of diploid chromosomes was 2n=50, while the fundamental number (NF) was 40 in both males and females. The types of macrochromosomes were 4 metacentric, 8 submetacentric, 6 acrocentric, 4 telocentric chromosomes, and 28 microchromosomes. The GTG-banding technique showed that each chromosome pairs could be clearly differentiated and the numbers of bands in the M. macrocephala was 99. There is no observation of strangely size chromosomes related to sex. The karyotype formula is as follows: m sm a m sm a t 2n (50)=L2 +L2 +M2+S2 +S6 +S4+S4+28 microchromosomes Key words Malayan snail-eating turtle, Malayemys macrocephala, Karyotype, Idiogram. Chelonians are found in rivers, lakes, seas, swamps, deserts, and forests. Many species of chelonians are endangered. The Malayan snail-eating turtle (Malayemys macrocephala), popularly known as the slider, belongs to the order Testudines, suborder Cryptodira, family Geoemydidae, and subfamily Geoemydinae (Ernst and Barbour 1989). Brophy (2004) recently reviewed the sys- tematics of the genus Malayemys and argued for the presence of two taxonomically distinct species. Analyses of head-stripe and shell characters revealed a clear pattern of geographic variation that was consistent with the topography of Southeast Asia and the poor dispersal abilities of these turtles. Turtles from the Mekong River Basin retained the name M. subtrijuga (Schlegel & Müller 1844), whereas those from the Chao Phraya and Mae Klong River basins, coastal areas of south- eastern Thailand, and the Malay Peninsula were assigned the name M. macrocephala (Gray 1859). The M. macrocephala is a small geoemydid turtle reaching a maximum size of 22 cm carapace length (Srinarumol 1995). This species has pronounced sexual dimorphism, with females exhibiting larger overall body sizes, proportionally wider carapaces, and shorter, narrower tails. Populations of M. macrocephala can be found in virtually all lowland areas of the Chao Phraya River basin in cen- tral Thailand, where it is the most common turtle (Ernst and Barbour 1989, Srinarumol 1995). * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.78.125 126 P. Siripiyasing et al. Cytologia 78(2) Information about karyotypes in turtles is scarce and fragmented, and usually based on conventional staining technique; only a few studies have been published, all of them recent (Ayres et al. 1969, Bickham 1975, 1981, Bickham and Baker 1976, Bull and Legler 1980, Bickham et al. 1985). This fact is probably due to the difficulty in obtaining samples for cytogenetic analysis in some species, or to problems in obtaining metaphase cells by cell culture induction. Lymphocyte culturing provides an alternative method for turtle cytogenetics that can generate better samples if compared to cell culture induction (Cleiton and Giuliano-Caetano 2008). Moreover, cytogenetic studies using conventional staining technique provide valuable information on the great karyotype diversity shown by these animals. Analyses of cytogenetic markers, including the number and karyotype formula, sex determination, B chromosomes, number and location of nucleolar organizer regions (NORs), heterochromatin distribution, G-banding and R-banding, treatments with base- specific fluorochromes and, more recently, in situ hybridization techniques, allowed the cytogenetic characterization of populations, species, and supra-specific groups (Vitturi et al. 2000, Swarça et al. 2001, Carvalho et al. 2002, Azevedo et al. 2003, Affonso and Galetti, Jr. 2005). There are two previous reports on genus Malayemys cytogenetics. Killebrew (1977) demon- strated with conventional staining technique that the karyotype of M. subtrijuga is 2n (diploid) = 52. The macrochromosomes (26 pairs) are composed of 16 metacentric, 6 submetacentric, 4 telocentric chromosomes, and 26 microchromosomes. Elsewhere, Bickham (1981) showed the karyotype of M. Subtrijuga to be 2n=50. The present study is the first report on the chromosomal charac- teristics of M. macrocephala determined using conventional staining and GTG-banding techniques. The results enhance the level of cytogenetic information available and enable future comprehensive studies to be conducted on taxonomy and evolutionary relationships. Moreover, the data provide useful basic information for conservation and on breeding practices as well as analyses of the chromosomal evolution of this species of reptile. Materials and methods Blood samples and cell cultures Blood samples from two males and two female M. macrocephala living in the Chi River basin, Khon Kaen Province, Thailand were collected from blood veins using the aseptic technique, and kept on ice in 5 ml vacuum tubes coated with heparin to prevent blood clotting. 0.5 ml of whole blood was cultured in 5 ml RPMI 1640 medium, supplemented with 2% phytohemagglutinin (PHA) as a mitogen at 26°C, 5% CO2. The cultured bottle was loosely capped and regularly shaken two times a day, in the morning and evening. After 72 h of incubation, colchicine was introduced and mixed before a further incubation for 30 min. Cell harvest and chromosome staining After colchicine incubation, the blood mixture was centrifuged at 1,200 rpm for 10 min. After discarding the supernatant, the cells were treated with 10 ml of hypotonic solution (0.075 M KCl) and incubated at 26°C for 30 min. Then the cells were centrifuged and the supernatant was dis- carded. Fresh cool fixative (3 methanol : 1 acetic acid) was used to fix the cells by gradually added up to 8 ml. After centrifugation, the fixation was repeatedly conducted until the supernatant was clear. The cells were added to 1 ml fixative by dropping the cells onto clean cold slides and then drying the slides by air-dry technique. Next, chromosomes were stained using the GTG-banding technique (Rooney 2001). Well-dried slides were soaked in 0.025% trypsin EDTA and incubated at 37°C. After washing with 10% fetal calf serum (FCS) or PBS, FCS was eliminated by 50% methanol and slides were stained with 10% Giemsa’s solution for 30 min. 2013 First Cytogenetic Study of Malayan Snail-eating Turtle, in Thailand 127 Chromosome checks Chromosome counting was performed on mitotic metaphase cells under light microscope. Twenty clearly observable and well-cells spread chromosomes of each male and female were se- lected and photographed. The length of short arm chromosomes (Ls) and the length of long arm chromosomes (Ll) were measured and calculated to establish the total length of arm chromosomes (LT, LT=Ls+Ll). The relative length (RL), the centromeric index (CI) and standard deviation (SD) of RL and CI were estimated (Chaiyasut 1989). CI (q/p+q) values between 0.50–0.59, 0.60–0.69, 0.70–0.89, and 0.90–0.99 were described as indicative of metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively. The fundamental number (number of chromosome arm, NF) was obtained by assigning a value of two to metacentric, submetacentric, and acrocentric chromosomes and of one to telocentric chromosomes. All parameters were used in karyotyping and id- iograming. Fig. 1. Metaphase chromosome plates and karyotypes of male (A) and female (B) Malayan snail-eating turtle (Malayemys macrocephala), 2n=50 by conventional staining technique, scale bars 10 μm. 128 P. Siripiyasing et al. Cytologia 78(2) Results and discussion The Geoemydidae is a large family (24 genera) of turtles and, until now, no study has investigated the karyotype of M. macrocephala. Furthermore, this is the first report on cytogenetic characterization to use conventional staining and GTG-banding techniques for this species. For M. macrocephala, the results indicated a diploid chromosome number of 2n=50 in all studies samples, of which 11 pairs were macrochromosomes and 14 pairs were microchromosomes (Fig. 1). This diploid chromosome number agrees with the study of Bickham (1981), which investigated M. subtrijuga. It differs, however, from previous studies by Killebrew (1977), which show M. subtrijuga to have 2n=52. 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. Turtle karyotypes show two general tendencies based on the presence or absence of microchromosomes, but still there is much variation between groups. For example,

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