Cytogenetic Study of Three Microhylid Species (Anura, Microhylidae) from Thailand

Home , Microhyla, Microhyla heymonsi, Microhyla pulchra, Rhacophoridae

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. pulchra) 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 prepared from the bone marrows by the standard protocol. To stain the chromosomes, 10% Giemsa’s and 50% silver 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: 2 n=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 composed 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), centromeric 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.

(Ll) were measured and the length of the total arm chro- 1989). The CI (q/p+q) between 0.50–0.59, 0.60–0.69, mosome (LT, LT=Ls+Ll) was calculated. The relative 0.70–0.89, and 0.90–0.99 were described as metacentric, length (RL), the centromeric index (CI), and standard submetacentric, acrocentric and telocentric chromo- deviation (SD) of RL and CI were estimated (Chaiyasut somes, respectively (Chooseangjaew et al. 2017). All 2017 Cytogenetic Study of Three Microhylid Species (Anura, Microhylidae) from Thailand 71

Table 3. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL), centromeric index (CI), and standard deviation (SD) of RL, CI from 20 metaphase cells of painted chorus frog (Microhyla pulchra), 2n=24.

Chro. pair Ls Ll LT CI±SD RL±SD Chro. size Chro. type

1 10.883 12.278 23.161 0.532±0.017 0.138±0.008 Large Metacentric 2 8.945 11.726 20.672 0.568±0.029 0.124±0.006 Large Metacentric 3 7.482 11.955 19.437 0.611±0.043 0.116±0.006 Large Submetacentric 4 6.690 10.737 17.427 0.615±0.026 0.104±0.005 Large Submetacentric 5 6.621 9.190 15.812 0.579±0.038 0.095±0.003 Large Metacentric 6 6.324 7.122 13.445 0.529±0.012 0.081±0.003 Medium Metacentric 7 4.424 6.566 10.990 0.595±0.033 0.066±0.004 Small Metacentric 8 4.280 5.996 10.276 0.582±0.018 0.062±0.003 Small Metacentric 9* 4.030 5.836 9.866 0.593±0.025 0.059±0.002 Small Metacentric 10 4.187 5.151 9.338 0.556±0.030 0.056±0.003 Small Metacentric 11 3.926 4.993 8.918 0.564±0.036 0.054±0.003 Small Metacentric 12 2.748 4.862 7.611 0.638±0.041 0.046±0.005 Small Submetacentric

Remarks: *=NOR-bearing chromosome and Chro.=chromosome.

Table 4. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL), centromeric index (CI), and standard deviation (SD) of RL, CI from 20 metaphase cells of narrow-mouthed frog (Microhyla heymonsi), 2n (diploid)=24.

Chro. pair Ls Ll LT CI±SD RL±SD Chro. size Chro. type

1 10.162 11.140 21.303 0.523±0.008 0.141±0.001 Large Metacentric 2* 7.554 10.740 18.295 0.587±0.004 0.121±0.003 Large Metacentric 3 5.435 9.342 14.778 0.632±0.005 0.098±0.002 Large Submetacentric 4 6.342 10.686 17.028 0.628±0.010 0.113±0.002 Large Submetacentric 5 6.024 7.909 13.933 0.568±0.003 0.092±0.003 Large Metacentric 6 5.728 6.680 12.408 0.538±0.001 0.082±0.002 Medium Metacentric 7 4.608 5.704 10.313 0.553±0.001 0.068±0.002 Medium Metacentric 8 3.791 5.231 9.022 0.601±0.002 0.060±0.001 Small Submetacentric 9 3.850 5.178 9.028 0.604±0.003 0.060±0.001 Small Submetacentric 10 3.930 4.779 8.709 0.619±0.005 0.058±0.001 Small Submetacentric 11 3.563 4.664 8.227 0.627±0.001 0.054±0.004 Small Submetacentric 12 3.756 4.222 7.978 0.529±0.001 0.053±0.007 Small Metacentric

Remarks: *=NOR-bearing chromosome and Chro.=chromosome. data were used in karyotyping and idiograming. chromosomes (Fig. 2a, b). The karyotype of M. pulchra was composed of metacentric chromosomes in Results and discussion pairs 1, 2, 5–7, 12 and submetacentric chromosomes in pairs 3, 4, 8–11 (Fig. 3a, b). Meanwhile, M. heymonsi The diploid chromosome number in the Microhyla showed metacentric chromosomes in pairs 1–2, 5–11 species varies between 22–26 chromosomes. M. fis- and submetacentric chromosomes in pairs 3–4, 12 and sipes, M. pulchra and M. heymosi herein showed the no obvious difference in size of sex chromosomes were same diploid number and fundamental number having found (Fig. 4a, b). The type and number of chromo- 2n=24 (NF=48). These results are in concordance with somes of M. pulchra herein coincide with those previ- previous cytogenetics data (Zheng and Fuwu 1995, Qian ously reported (Supaprom and Baimai 2002). From all and Zhu 2000, Supaprom and Baimai 2002, Joshy and parameters in Tables 2–4, we can conclude that the Kuramoto 2011), with the exception of M. butleri which karyotype of M. fissipes, M. pulchra and M. heymonsi m sm m sm m had 2n=22 (NF=44) (Baorong et al. 1990, Zheng and could be deduced as: L5 +L2 +M5 +S4 +S8 (XY sys- m sm m sm m m sm m m sm Fuwu 1995, Supaprom and Baimai 2002) and M. rubra tem), L6 +L4 +M4 +S8 +S2 and L6+L4 +M2 +S10+S2 , which had increased 2n=26 chromosomes (NF=52) respectively. The standardized idiogram of the three Mi- (Kasturi Bai 1956), the latter of which is similar to the crohyla species are shown in the Fig. 5a–c. Phyllomedusa species exhibiting a conservative chromo- For the sex chromosome, this is the first report for some number of 2n=26 (Haddad et al. 1994, Morando M. fissipes. Cytological evidence that was supportive of and Hernando 1997, Kasahara et al. 2007, Bruschi et al. male heterogamety (XX/XY sex chromosome) has been 2012). published by Iturra and Veloso (1981) in a leptodactylid The karyotype of M. fissipes showed metacentric frog, Eupsophus migueli. Another species, Rana escu- chromosomes in pairs 1, 2, 4–5, 8–11, and submetacen- lenta, demonstrated the existence of XX/XY sex chro- tric chromosomes in pairs 3, 6–7 and X, Y metacentric mosome by BrdU-Hoechst Giemsa’s technique (Schempp 72 W. Sangpakdee et al. Cytologia 82(1) Special Issue

Fig. 5. Idiogram showing lengths and shapes of chromosomes of ornamented pygmy frog, Microhyla fissipes (A); painted chorus frog, Microhyla pulchra (B) and narrow-mouthed frog, Microhyla heymonsi (C) by conventional staining technique. and Schmid 1981), as well as Eupsophus insularis (Cue- replicated synchronously. vas and Formas 1996), R. tagoi and R. sakuraii from M. fissipes and M. pulchra showed the same loca- Tokyo (Ryuzaki et al. 1999). Additionally, the XX/XY tion of the nucleolar organizer regions (NORs) on the sex chromosome system was found in the genus Bufo long arm of chromosome pair 9 metacentric and sub- (Siripiyasing et al. 2008) and also in rice frogs, Fejer- metacentric chromosome, respectively (Figs. 2c, d, 3c, varya limnocharis (Patawang et al. 2014). The existence d), as the idiogram also shows in Fig. 6a, b. This result of the highly differentiated XX/XY sex chromosome was according to the cytogenetics data reported on M. has been reported in Gastrotheca riobambae by Schmid pulchra by Supaprom and Baimai (2002). Meanwhile, et al. (1983), in G. ovifera and G. walkeri by Schmid M. heymonsi exhibited the NORs on the short arm of et al. (1988), in G. pseustes by Schmid et al. (1990), and chromosome pair 2 (Fig. 4c, d); the idiograms are shown in Eleutherodactylus maussi by Schmid et al. (1992). For in Fig. 6c. These three Microhyla species herein have this study on M. pulchra and M. heymonsi, we proposed chromosomes with slight differences in morphological that the X and Y sex chromosomes were the same in patterns. The use of additional NOR-banding techniques size. The previous study on R. esculenta (Schempp and also showed some differences in their karyotypes, sup- Schmid 1981) exhibited that the Y chromosome had no porting the idea that one of them (M. heymonsi) are size difference with X but differed by possessing a small species-specific. Moreover, there were other Microhyla extremely late replicating region in the middle region of species reported; M. ornata showed the NORs on chro- the long arm, while in the females, both X chromosomes mosome pair 11 (Joshy and Kuramoto 2011), and M. 2017 Cytogenetic Study of Three Microhylid Species (Anura, Microhylidae) from Thailand 73

Fig. 6. Idiogram of ornamented pygmy frog, Microhyla ﬁssipes (A); painted chorus frog, Microhyla pulchra (B) and narrow- mouthed frog, Microhyla heymonsi (C) by Ag-NOR banding technique. Arrows indicate nucleolar organizer region (NOR). butleri had the NORs on chromosome pair 8 (Zheng of Microhyla butleri Boulenger and the karyotype evolution. and Fuwu 1995). The difference of the NOR position in Journal of Fujian Normal University (Natural Science Edition) 62: 88–91. homologous chromosomes at interspeciﬁc levels is not Bruschi, D. P., Busin, C. S., Siqueira, S. and Recco-Pimentel, S. M. infrequent and this may be important to characterize 2012. Cytogenetic analysis of two species in the Phyllomedusa individual species with the same karyotypes (Silva et al. hypochondrialis group (Anura, Hylidae). Hereditas 149: 34–40. 2000). We suggested that chromosome morphology and Chaiyasut, K. 1989. Cytogenetics and Cytotaxonomy of the Family the NOR location are still useful cytological characters Zephyranthes. Department of Botany, Faculty of Science, Chul- alongkorn University, Bangkok. for taxonomic and evolutionary studies in the anurans. Chooseangjaew, S., Tanyaros, S., Maneechot, N., Buasriyot, P., Getle- kha, N. and Tanomtong, A. 2017. Chromosomal characteristics Acknowledgements of the tropical oyster, Crassostrea belcheri Sowerby, 1871 (Os- treoida, Ostreidae) by conventional and Ag-NOR banding tech- This study was supported by the Research Group niques. Cytologia 82: 3–8. Cuevas, C. C. and Formas, J. R. 1996. Heteromorphic sex chromo- on Toxic Substances in Livestock and Aquatic Animals, somes in Eupsophus insularis (Amphibia; Anura: Leptodactyli- Khon Kaen University. dae). Chromosome Res. 4: 467–470. Haddad, C. F. B., Pombal, J. P. Jr. and Batistic, R. F. 1994. Natural hybridization between diploid and tetraploid species of leaf-frogs References genus Phyllomedusa (Amphibia). J. Herpetol. 28: 425–430. Baorong, G., Jianmin, G. and Mingzhang, C. 1990. The karyotype Hasan, M., Islam, M. M., Kuramoto, M., Kurabayashi, A. and Sumi- 74 W. Sangpakdee et al. Cytologia 82(1) Special Issue

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