© 2017 The Japan Mendel Society Cytologia 82(1) Special Issue: 67–74 Cytogenetic Study of Three Microhylid Species (Anura, Microhylidae) from Thailand Wiwat Sangpakdee1, Sumalee Phimphan2, Bundit Tengjaroenkul3,7, Krit Pinthong4, Lamyai Neeratanaphan5,7 and Alongklod Tanomtong6,7* 1 Program in Biology, Faculty of Science, Udon Thani Rajabhat University, Udon Thani 40001, Thailand 2 Program in Public Health, Faculty of Science and Technology, Surindra Rajabhat University, Surin 32000, Thailand 3 Department of Veterinary Clinical Medicine, Faculty of Veterinary Medicine, Khon Kaen 40002, Thailand 4 Program in Biology, Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Surin 32000, Thailand 5 Department of Environmental Science, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand 6 Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand 7 Research Group on Toxic Substances in Livestock and Aquatic Animals, Khon Kaen University, Khon Kaen 40002, Thailand Received December 6, 2015; accepted April 29, 2016 Summary The cytogenetics of the ornamented pygmy frog (Microhyla fissipes), painted chorus frog (M. pul- chra) and narrow-mouthed frog (M. heymonsi) were studied in the aspect of chromosome numbers, morphology and nucleolus organizer region (NOR) locations. For this present study, we provided the karyotype and idiogram of these three species by conventional and Ag-NOR staining techniques. The 10 male and female samples of those three species were collected in Northeast Thailand. The metaphase chromosome preparations were pre- pared from the bone marrows by the standard protocol. To stain the chromosomes, 10% Giemsa’s and 50% sil- ver nitrate were applied. The results showed that all three species had the same diploid chromosome number of 2n=24, and fundamental numbers (NF) were 48 in both males and females. The chromosomes of M. fissipes were comprised of 16m+6sm+XY (X and Y were metacentric chromosomes). M. pulchra had chromosomes consist- ing of 12m+12sm and M. heymonsi had chromosomes comprising of 18m+6 with no obvious difference in size of sex chromosomes for the two species. M. fissipes and M. pulchra had the same NORs adjacent to the subcen- tromeric on the long arm of chromosome pair 9, while M. heymonsi had the NOR location on the short arm of chromosome pair 2 close to the centromere. For this case, we suggested that chromosome morphology and the NOR location is still a useful cytological character for taxonomic and evolutionary studies. Key words Microhyla fissipes, M. pulchra, M. heymonsi, Chromosome, Karyotype. Microhylidae is a large anuran family comprising 8% narrow-mouthed frog (M. hermonsi) (Fig. 1) are species of all frogs (519 species). Members of Microhyla show a of pygmy frogs that can be found in Northeastern India, wide distribution across Asia from the Ryukyu Archi- Southern China, and Southeast Asia south to at least pelago in Japan and China to the north, through India Thailand but possibly as far south as Malaysia and Sin- to Sri Lanka to the southwest, and through Southeast gapore. It is a common species in this suitable habitat, Asia to Sumatra, Borneo, Java, and Bali to the southeast but it is not often seen because it is cryptic and seasonal. (Hasan et al. 2014). The ornamented pygmy frog (Mi- It typically occurs near forest edges. crohyla fessipes), painted chorus frog (M. pulchra) and For the family Microhylidae, there were some cytoge- Fig. 1. General characteristics of the ornamented pygmy frog, Microhyla fissipes (A), painted chorus frog, Microhyla pulchra (B) and narrow-mouthed frog, Microhyla heymonsi (C); scale bars indicate 1 cm. * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.82.67 68 W. Sangpakdee et al. Cytologia 82(1) Special Issue Table 1. Review of cytogenetic publications of family Microhylidae (genus Microhyla). Species 2n Karyotype formula NF NOR banded Sex chro. Reference M. berdmorei 24 10m+14sm 48 ̶ ̶ Supaprom and Baimai (2002) M. butleri 22 16m+6sm 44 ̶ ̶ Baorong et al. (1990) 22 18m+4sm 44 2 ̶ Zheng and Fuwu (1995) 22 14m+8sm 44 ̶ ̶ Supaprom and Baimai (2002) M. fissipes 24 18m+6sm 48 2 XY Present study M. heymonsi 24 18m+6sm 48 2 ̶ Present study M. ornata 24 20m+4sm 48 ̶ ̶ Zheng and Fuwu (1995) 24 22m+2sm 48 ̶ ̶ Qian and Zhu (2000) 24 16m+8sm 48 2 ̶ Joshy and Kuramoto (2011) M. pulchra 24 20m+4sm 48 ̶ Zheng and Fuwu (1995) 24 12m+12sm 48 2 ̶ Supaprom and Baimai (2002) 24 12m+12sm 48 2 ̶ Present study M. rubra 26 12m+2sm+12a 52 ̶ ̶ Kasturi Bai (1956) Remarks: 2n=diploid chromosome number, NF=fundamental number (number of chromosome arms), m=metacentric chromosome, sm=submetacentric chromosome, a=acrocentric chromosome, chro.=chromosome and ̶=not available. Fig. 2. Metaphase chromosome plates and karyotypes of ornamented pygmy frog (Microhyla fissipes), male (A) and female (B), by conventional staining technique, and male (C) and female (D) by Ag-NOR banding technique. The karyotype was composed of 2n=24 chromosomes. Arrows indicate nucleolar organizer regions (NORs) (Scale bars=5 µm). netical studies reported about Microhyla. Perhaps due to of 2n=24 and had a fundamental number (NF) of 48 its body being very small, there is a lack of cytogenetics (Zheng and Fuwu 1995, Qian and Zhu 2000, Supaprom data compared with other members of the anuran fam- and Baimai 2002, Joshy and Kuramoto 2011). How- ily. According to the compilation of cytogenetics for this ever, some species such as M. butleri showed the diploid genus, the majority of the species had a diploid number number 2n=22 (NF=44) (Baorong et al. 1990, Zheng 2017 Cytogenetic Study of Three Microhylid Species (Anura, Microhylidae) from Thailand 69 Fig. 3. Metaphase chromosome plates and karyotypes of painted chorus frog (Microhyla pulchra), male (A) and female (B), by conventional staining technique, and male (C) and female (D) by Ag-NOR banding technique. The karyotype was com- posed of 2n=24 chromosomes. Arrows indicate nucleolar organizer regions (NORs) (Scale bars=5 µm). and Fuwu 1995, Supaprom and Baimai 2002), while M. the tissue to pieces as small as possible. We transferred rubra showed an increase with having 2n=26 chromo- 8 mL of cell sediments to a centrifuge tube and incubat- somes (NF=52) (Kasturi Bai 1956) (Table 1). Thus, the ed it for 30 min at 37°C. After centrifugation at 1500 rpm objective of this study was to provide a cytogenetical for 8 min, the KCl was discarded. Cells were fixed in characterization on M. fissipes, M. pulchra and M. hey- fresh cool fixative up to 8 mL by gradually adding it be- monsi. This present study is also the first report describ- fore being centrifuged again at 1500 rpm for 8 min. The ing the cytogenetics of M. fissipes and M. heymonsi. We fixation was repeated until the supernatant was clear, also found that M. fissipes had the XY sex-chromosome usually three times. Finally, the pellet was mixed with system. 1 mL fixative (depending on the amount of cell). The mixture was dropped onto a clean slide and air-dried. Materials and methods Conventional staining was done using 10% Giemsa’s solution for 10 min (Chooseangjaew et al. 2017). Ag- Ten males and females of M. fissipes, M. pulchra and NOR banding was performed (Howell and Black 1980) M. heymonsi were collected in Northeast Thailand (Fig. by applying two drops of 2% gelatin on the slides, fol- 1). The chromosomes were prepared in vivo (Schmid lowed with four drops of 50% silver nitrate. The slides 1978) with slight adaptations as follows. The colchicine were then covered with a cover slip and incubated at was injected into the frogs’ abdominal cavity. Then, the 60°C for 5 min or until the slide changed brownish. After frogs were left in a box for eight hours and then killed. that the slides were dipped in distilled water to remove The bone marrow was collected by cutting the head and the cover glass and air-dried on the slide. the end of femurs and tibias, and then a syringe was used Twenty clearly well spread chromosomes of each male to inject 0.075 M KCl into the marrow to drive out the and female were photographed. The length of the short bone marrow tissue or cells into the plate. We gently cut arm chromosome (Ls) and the long arm chromosome 70 W. Sangpakdee et al. Cytologia 82(1) Special Issue Fig. 4. Metaphase chromosome plates and karyotypes of narrow-mouthed frog (Microhyla heymonsi), male (A) and female (B), by conventional staining technique, and male (C) and female (D) by Ag-NOR banding technique. The karyotype was composed of 2n=24 chromosomes. Arrows indicate nucleolar organizer regions (NORs) (Scale bars=5 µm). Table 2. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL), cen- tromeric index (CI), and standard deviation (SD) of RL, CI from 20 metaphase cells of ornamented pygmy frog (Microhyla fissipes), 2n=24. Chro. pair Ls Ll LT RL±SD CL±SD Chro. size Chro. type 1 3.255 2.422 5.677 0.144±0.004 0.527±0.004 Large Metacentric 2 2.660 2.616 4.275 0.128±0.001 0.593±0.004 Large Metacentric 3 1.050 2.437 3.487 0.092±0.006 0.695±0.027 Large Submetacentric 4 1.441 1.978 3.419 0.089±0.010 0.575±0.032 Medium Metacentric 5 1.352 1.654 3.006 0.079±0.006 0.549±0.019 Medium Metacentric 6 0.905 1.381 2.286 0.060±0.004 0.603±0.029 Small Submetacentric 7 0.842 1.279 2.121 0.056±0.004 0.602±0.022 Small Submetacentric 8 0.804 1.160 1.964 0.063±0.004 0.590±0.036 Small Metacentric 9* 0.798 1.084 1.882 0.060±0.004 0.577±0.016 Small Metacentric 10 0.801 1.031 1.832 0.058±0.003 0.561±0.040 Small Metacentric 11 0.757 1.025 1.781 0.058±0.007 0.576±0.017 Small Metacentric X 1.609 2.103 3.712 0.116±0.007 0.563±0.030 Large Metacentric Y 1.193 1.541 2.734 0.071±0.008 0.563±0.026 Medium Metacentric Remarks: *=NOR-bearing chromosome and Chro.=chromosome.