© 2013 The Japan Mendel Society Cytologia 78(2): 125–132

First Cytogenetic Study of Malayan Snail-eating , macrocephala (Testudines, ) 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 . Standard T-lymphocyte cell culture at 26°C for 96 h was ap- plied. The mitotic chromosomes were harvested by colchicine-hypotonic-fixation-air drying tech- nique. 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 macrochromo- somes were 4 metacentric, 8 submetacentric, 6 acrocentric, 4 telocentric chromosomes, and 28 mi- crochromosomes. 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 of che- lonians are endangered. The Malayan snail-eating turtle (Malayemys macrocephala), popularly known as the slider, belongs to the order Testudines, suborder , family Geoemydidae, and subfamily (Ernst and Barbour 1989). Brophy (2004) recently reviewed the sys- tematics of the 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 tur- tles. 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 con- ventional 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 . 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 telo- centric chromosomes, and 26 microchromosomes. Elsewhere, Bickham (1981) showed the karyo- type 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 and evolutionary relationships. Moreover, the data provide useful basic information for conservation and on breeding practices as well as analyses of the chro- mosomal evolution of this species of .

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% metha- nol 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 chro- mosomes 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 macro- chromosomes 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, the chromosome number in the order Chelonia ranges from 2n=26 in dumer- iliana (Ayres et al. 1969) to 2n=96 in Platemys platycephala (Bull and Legler 1980, Bickham et al. 1985). Moreover, while karyotypic studies have frequently been published for turtles from the sub- order Cryptodira, information about Pleurodires is scarce and fragmented and mainly based on con- ventional staining techniques (Noleto et al. 2006). The types of macrochromosomes were two large metacentric, two large submetacentric, two medium acrocentric, two small metacentric, six small submetacentric, four small acrocentric, four

Fig. 2. Idiogram showing the lengths and shapes of chromosomes of the Malayan snail-eating turtle (Malayemys macrocephala), 2n=50 by conventional staining technique. 2013 First Cytogenetic Study of Malayan Snail-eating Turtle, in Thailand 129 small telocentric chromosomes, and 28 microchromosomes. It is not consistent with the report of Killebrew (1977), which revealed that the chromosomes of M. macrocephala are as following; 16 metacentric, six submetacentric, and four telocentric chromosomes. No sex chromosome heteromorphism was observed. The most common chromosome number in chelonians is 2n=52, with species presenting from 2n=50 to 2n=60 (Bickham 1981). Bickham and Baker (1976) reported chromosome numbers of 2n=50, 52, and 56, for another 10 species be- longing to the suborder Cryptodira. By making reference to the same study, it appears that the karyotypes of M. macrocephala investigated here are similar to those of crassicolis and Chrysemys terrapin. GTG-banding revealed that number of GTG-bands on one set of haploid chromosomes, which includes macrochromosomes and microchromosomes, is 99 bands (Fig. 3). The GTG-banding tech- nique provides clear chromosome bands which are represented as black (dark band) and white (light band) regions on chromosomes. However, the banding on some chromosomes shows varia- tion, preventing clear identification. As above, the chromosome band scoring is represented by the approximate bands that appear. Studying 48 species of turtles belonging to 29 genera and eight families in the suborder Cryptodira, Bickham (1981) determined a minimum number of chromosome rearrangements using a cladistic analysis on GTG-banding patterns. According to the author, some chromosomes seem to

Fig. 3. Metaphase chromosome plates and karyotypes of male (A) and female (B) Malayan snail-eating turtle (Malayemys macrocephala), 2n=50 by GTG-banding technique, scale bars 10 μm. 130 P. Siripiyasing et al. Cytologia 78(2)

Table 1. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), and total arm chromo- somes (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosomes in 20 cells of the Malayan snail-eating turtle (Malayemys macro- cephala), 2n=50.

Chromosome Chromosome Chromosome Ls Ll LT RL±SD CI±SD pairs size type

1 52.24 80.36 132.61 0.121±0.004 0.605±0.005 Large Submetacentric 2 45.42 56.76 102.19 0.094±0.005 0.554±0.016 Large Metacentric 3 13.31 62.38 75.70 0.069±0.001 0.825±0.011 Medium Acrocentric 4 13.62 46.49 60.11 0.055±0.001 0.776±0.013 Small Acrocentric 5 0.00 54.53 54.53 0.050±0.002 1.000±0.000 Small Telocentric 6 12.36 41.01 53.37 0.049±0.002 0.770±0.020 Small Acrocentric 7 19.89 31.32 51.22 0.046±0.001 0.611±0.008 Small Submetacentric 8 17.88 29.82 47.70 0.043±0.001 0.625±0.012 Small Submetacentric 9 16.47 26.83 43.31 0.039±0.002 0.620±0.010 Small Submetacentric 10 18.47 22.74 41.22 0.037±0.002 0.551±0.009 Small Metacentric 11 0.00 35.80 35.80 0.032±0.001 1.000±0.000 Small Telocentric 12 – – 31.51 0.028±0.000 – Microchromosome 13 – – 31.03 0.028±0.000 – Microchromosome 14 – – 30.28 0.027±0.000 – Microchromosome 15 – – 30.15 0.027±0.000 – Microchromosome 16 – – 29.84 0.027±0.000 – Microchromosome 17 – – 29.33 0.026±0.000 – Microchromosome 18 – – 28.80 0.026±0.000 – Microchromosome 19 – – 28.16 0.025±0.001 – Microchromosome 20 – – 27.17 0.024±0.000 – Microchromosome 21 – – 26.61 0.024±0.000 – Microchromosome 22 – – 25.97 0.023±0.001 – Microchromosome 23 – – 25.12 0.022±0.001 – Microchromosome 24 – – 24.37 0.022±0.001 – Microchromosome 25 – – 23.42 0.021±0.001 – Microchromosome

Fig. 4. Idiogram showing the lengths and shapes of chromosomes of the Malayan snail-eating turtle (Malayemys macrocephala), 2n=50 by GTG-banding technique. 2013 First Cytogenetic Study of Malayan Snail-eating Turtle, in Thailand 131 have been conserved for 200 million years. The first chromosome of the A group and some others were identical in all species of the eight families studied and do not seem to have changed since the suborder Cryptodira appeared 200 million years ago, prompting Bickham to propose that an identi- cal pattern of GTG-banding was maintained for a long period of time. The GTG-banding permitted the visualization, especially in the macrochromosomes, of a pat- tern of bands that enabled better identification and pairing of the chromosomes as well as the con- struction of an idiogram. Such a pattern is similar, but not identical, to that observed in other Cryptodiran turtles, due to the presence and absence of some bands when compared to the patterns found by Bull and Legler (1980) in Pelomedusoid turtles. This variation in the GTG-banding pat- tern in Cryptodiran turtles establishes a different karyotypic evolution from that identified for Pleurodiran turtles. Previous reports have suggested there to be genomic stability in Cryptodiran turtles, in which both the banded chromosome morphology (Bickham 1981) and the DNA se- quences inside the chromosomes have remained unchanged for millions of years (Muhlmann-Díaz et al. 2001). The M. Macrocephala demonstrated that the chromosome marker is the chromosome pair 1, which is the largest submetacentric chromosome. The important karyotype feature of M. Macrocephala is the asymmetrical karyotype, which was found in four types of chromosomes (metacentric, submetacentric, acrocentric, and telocentric chromosomes). The largest chromosome is six times larger than the smallest chromosomes. Figures 2 and 4 shows the idiograms from con- ventional staining and GTG-banding techniques. The karyotype formula could be deduced as:

m sm a m sm a t 2n (50)=L2 +L2 +M2+S2 +S6 +S4+S4+28 microchromosomes

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.

References

Affonso, P. R. A. M., and Galetti, Jr., P. M. 2005. Chromosomal diversification of reef fishes from genus Centropyge (Perciformes, Pomacanthidae). Genetica 123: 227–233. Ayres, M., Sampaio, M. M., Barros, R. M. S., Dias, L. B., and Cunha, O. R. 1969. A karyological study of turtles from the Brazilian Amazon region. Cytogenetics 8: 401–409. Azevedo, M. F. C., Foresti, F., Ramos, P. R. R., and Jim, J. 2003. Comparative cytogenetic studies of Bufo ictericus, B. par- acnemis (Amphibia, Anura) and an intermediate form in sympatry. Genet. Mol. Biol. 26: 289–294. Bickham, J. W. 1975. A cytosystematic study of turtles in the genera Clemmys, and . Herpetologica 31: 198–204. Bickham, J. W. 1981. Two-hundred-million-year-old chromosomes: Deceleration of the rate of karyotypic evolution in turtles. Science 212: 1291–1293. Bickham, J. W., and Baker, R. J. 1976. Chromosome Homology and Evolution of Emydid Turtles. Chromosoma 54: 201– 219. Bickham, J. W., Tucker, P. K., and Legler, J. M. 1985. Diploid-triploid mosaicism: An unusual phenomenon in side-necked turtles (Platemys platycephala). Science 227: 1591–1593. Brophy, T. R. 2004. Geographic variation and systematics in the south-east Asian turtles of the genus Malayemys (Testudines: Bataguridae). Hamadryad 29: 63–79. Bull, J. J., and Legler, J. M. 1980. Karyotypes of side-necked turtles (Testudines, ). Can. J. Zool. 58: 828–841. Carvalho, B. A., Oliveira, L. F. B., Nunes, A. P., and Mattevi, M. S. 2002. Karyotypes of nineteen marsupial species from Brazil. J. Mammal. 83: 58–70. Chaiyasut, K. 1989. Cytogenetics and Cytotaxonomy of the Family Zephyranthes. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok. 132 P. Siripiyasing et al. Cytologia 78(2)

Cleiton, F., and Giuliano-Caetano, L. 2008. Cytogenetic characterization of two turtle species: dorbigni and Trachemys scripta elegans. Caryologia 61: 253–257. Ernst, C. H., and Barbour, R. W. 1989. Turtles of the world. Smithsonian Press, Washington. Killebrew, F. 1977. Mitotic chromosomes of turtles. IV. The . Tex. J. Sci. 29: 245–253. Muhlmann-Díaz, M. C., Ulsh, B. A., Whicker, F. W., Hinton, T. G., Congdon, J. D., Robinson, J. F., and Bedford, J. S. 2001. Conservation of chromosome 1 in turtles over 66 million years. Cytogenet. Cell Genet. 92: 139–143. Noleto, R. B., Kantek, D. L. Z., Swarça, A. C., Dias, A. L., Fenocchio, A. S., and Cestari, M. M. 2006. Karyotypic charac- terization of tectifera (Testudines, Pleurodira) from the upper Iguaçu River in the Brazilian state of Paraná. Genet. Mol. Biol. 29: 263–266. Rooney, D. E. 2001. Human cytogenetics: constitutional analysis. Oxford University Press. Oxford. Srinarumol, N. 1995. Population biology of the Malayan snail-eating turtle Malayemys subtrijuga (Schlegel & Müller, 1844). M.Sc. thesis, Chulalongkorn University, Bangkok. Swarça, A. C., Giuliano-Caetano, L., Vanzela, A. L. L., and Dias, A. L. 2001. Polymorphism of rRNA genes in Pinirampus pirinampu (Pisces: Pimelodidae) detected by in situ hybridization. Cytologia 66: 275–278. Vitturi, R., Colomba, M. S., Gianguzza, P., and Pirrone, A. M. 2000. Chromosomal location of ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n telomeric repeats in the Neogastropod fasciolaria lignaria (Mollusca: Prosobranchia). Genética 108: 253–257.