Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(1): 23-28, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-248 Citation: W. Thongnetr, A. Tanom- tong, S. Prasopsin, N. Maneechot, K. Pinthong, I. Patawang (2019) Cytoge- netic study of the Bent-toed Gecko (Reptilia, Gekkonidae) in Thailand; I: Chromosomal classical features and NORs characterization of Cyrtodactylus kunyai and C. interdigitalis. Caryologia 72(1): 23-28. doi: 10.13128/cayolo- gia-248 Received: 19th May 2018 Accepted: 19th October 2018 Published: 10th May 2019 Copyright: © 2019 W. Thongnetr, A. Tanomtong, S. Prasopsin, N. Manee- chot, K. Pinthong, I. Patawang. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Cytogenetic study of the Bent-toed Gecko (Reptilia, Gekkonidae) in Thailand; I: Chromosomal classical features and NORs characterization of Cyrtodactylus kunyai and C. interdigitalis Weera Thongnetr1,2, Alongklod Tanomtong1,3, Suphat Prasopsin4, Nuntiya Maneechot5, Krit Pinthong5, Isara Patawang6,7,* 1 Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen, Thailand 2 Walai Rukhavej Botanical Research Institute, Mahasarakham University, Kantharawi- chai, Maha Sarakham, Thailand 3 Toxic Substances in Livestock and Aquatic Animals Research Group, Khon Kaen Univer- sity, Muang, Khon Kaen, Thailand 4 Research Academic Supports Division, Mahidol University, Kanchanaburi Campus, Sai- yok, Kanchanaburi, Thailand 5 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Muang, Surin, Thailand 6 Department of Biology, Faculty of Science, Chiang Mai University, Muang, Chiang Mai, Thailand 7 Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Muang, Chiang Mai, Thailand * Corresponding author: isara.p@cmu.ac.th Abstract. This study analysed the karyotype of Cyrtodactylus kunyai and C. interdigi- talis from Loei Province in Northeastern Thailand. The metaphase and meiotic chromo- some preparations were obtained by squash technique from bone marrow and testes, respectively. The chromosomes were stained by Giemsa staining and Ag-NOR-banding techniques. The results showed diploid chromosome number (2n) of 40 for C. kunyai and 42 for C. interdigitalis. The chromosome types of metacentric, submetacentric, acro- centric and telocentric chromosomes were 8-4-0-28 and 4-2-4-32, respectively. The Ag- NORs banding technique provides the pair of nucleolar organizer regions (NORs) of both two species at telomeric region of the long arm of the pair 12, metacentric type in C. kunyai and telocentric type in C. interdigitalis. There are no sex differences in karyo- types between males and females of both two species. We found that during metaphase I on meiosis of C. kunyai and C. interdigitalis, the homologous chromosomes showed synapsis of 20 and 21 bivalents, respectively. Moreover, the meiotic phase on prophase II exhibited 20 and 21 haploid chromosome number (n) as respective diploid species. Their karyotype formulas is as follows: C. kunyai (2n = 40): Lm2 + Lsm4 + Lt4 + Mt6 + Sm6 + St18, and C. interdigitalis (2n = 42): La4 + Lt14 + Mt2 + Sm4 + Ssm2 + St16. Keywords. Cyrtodactylus, Cyrtodactylus kunyai, Cyrtodactylus interdigitalis, Karyotype stasis. 24 Weera Thongnetr et al. INTRODUCTION The bent-toed gecko, genus Cyrtodactylus Gray 1827, belong to the class Reptilia, order Squamata, suborder Lacertilia, and family Gekkonidae. The Cyrtodactylus is the most diverse gekkonid genus with ~250 species (Uetz et al. 2018). The species distribution of Cyrtodacty- lus found in mainland of Asia, from boundary between middle east and India to southeast Asia, Archipelago of southeast Asia and Pacific to Australia (Shea et al. 2011; Hartmann et al. 2016). In Thailand, there are about 24 species of Cyrtodactylus that were reported: including C. angularis, C. auribalteatus, C. brevipalmatus, C. chan- homeae, C. consobrinus, C. dumnuii, C. erythrops, C. feae, C. interdigitalis, C. intermedius, C. jarujini, C. lekaguli, C. macrotuberculatus, C. oldhami, C. papilionoides, C. peg- uensis, C. phuketensis, C. pulchellus, C. quadrivirgatus, C. sumonthai, C. surin, C. thirakhupti, C. tigroides, and C. variegatus (Chuaynkern and Chuaynkern 2012). Among the Gekkonidae population in Thailand, the Cyrtodacty- lus is the most diverse group than other gekkonid genera and there is continually new species discovered. Karyological analyses in bent-toed gecko thus far has differentiated species based on mitotic metaphase chro- mosomal morphology while sporadic reports have based the species differentiation based on meiotic metaphase chromosomal morphology. Thus the basic diploid num- ber of the bent-toed gecko is in the range of 42-48 (Ota et al. 1992). Example of chromosome study of other gekko- nid that have been reported such as; Gekko: diploid num- ber ranging from 38-42 and mostly 38 (Ota 1989; Shiba- ike et al. 2009; Trifonov et al. 2011; Patawang et al. 2014; Patawang and Tanomtong 2015a), Hemidactylus: diploid distance from 40-56 and mostly 40 or 46 (De Smet 1981; Patawang and Tanomtong 2015b), Gehyra: mostly 44 (King 1984), Ptychozoon: 2n = 34 and 42 (Ota and Hikida 1988), Paroedura: diploid number ranging from 31-38 and mostly 36 (Aprea et al. 2013; Koubová et al. 2014), Phelsuma: 2n = 36 (Aprea et al. 1996), and Dixonius: 2n = 42 (Ota et al. 2001). Most gekkonid chromosome complements consist of acrocentric or telocentric chromosomes which gradually decrease in size, and karyotype evolution within the group is accompanied by Robertsonian fissions, fusions and pericentric inversions (Gorman 1973). The report of King (1987) presented eight putative ancestral karyomorphs (2n = 32, 34, 36, 38, 40, 42, 44, and 46 all acrocentric or telo- centric chromosomes) and assigned the genus Cyrtodacty- lus to a group sharing an ancestral karyomorph consisting of 2n = 42 uni-armed chromosomes. In Thailand, there are no studies of Cyrtodactylus’s chromosome or karyotypic analyses. The present study of the cytogenetic of two Cyrtodactylus provides the first report of both two species, overall on the conven- tional Giemsa, Ag-NOR banding, and meiotic cell divi- sion. Data provided here will increase our knowledge of cytogenetic information which can be used as a basis to comprehensively examine the taxonomy and evolu- tionary relationship of Cyrtodactylus species and other gekkonid. MATERIALS AND METHOD Five male and four female specimens of C. kunyai (Figure 1a) and five male and six female of C. interdigi- talis (Figure 1b) were collected from Puan Phu Sub-dis- trict, Nong Hin District, Loei Province, Northeastern Thailand. Chromosome preparation was conducted by the squash technique, from bone marrow for mitotic cell and testis for male meiotic cell, and followed with colchicine-hy potonic-f i xation-air-dr y ing technique (Patawang et al. 2014). The chromosomes were stained with 10% Giemsa for 30 min and NORs were identi- fied through Ag-NOR staining (Howell and Black 1980; Rooney 2001). The length of short arm (Ls) and long arm (Ll) chromosomes were measured and calculated for the length of total arm chromosomes (LT, LT = Ls+Ll). Relative length (RL, RL = LT/∑LT) and centromeric index (CI, CI = Ll/LT) were estimated. CI was also com- puted to classify the types of chromosomes (Turpin and Lejeune 1965; Chaiyasut 1989). All parameters were used in karyotyping and idiograming. RESULTS AND DISCUSSION Mitotic chromosome features from Giemsa staining Karyomorphology of the C. kunyai and C. interdigi- talis revealed that the diploid chromosome number (2n) Fig. 1. General characteristic of male Cyrtodactylus kunyai (a) and female C. interdigitalis (b) (scale bar = 2 cm). 25Cytogenetic study of the Bent-toed Gecko (Reptilia, Gekkonidae) in Thailand is 40 and 42, respectively. The karyotype of C. kunyai composed of 8 metacentric, 4 submetacentric, and 28 telocentric (Table 1 and Figures 2a-b), while the karyo- type of C. interdigitalis comprised 4 metacentric, 2 sub- metacentric, 4 acrocentric, and 32 telocentric (Table 2 and Figures 2c-d). Both two species exhibit no sex differ- ences in karyotypes between males and females (Figures 2a-d). Chromosome sizes of C. kunyai have pairs 1st to 5th to be large, pairs 6th to 8th to be medium, and pairs 9th to 20th to be small (Figure 3a). For the size of C. interdigitalis included large size in pairs 1st to 9th, medi- um size in the 10th, and small size in pairs 11th to 21st (Figure 3b). The diploid number of C. kunyai (2n = 40) and C. interdigitalis (2n = 42), both showed difference and accordance with others Cyrtodactylus that have been reported (Table 3). However, overall of these karyotypes of C. kunyai and C. interdigitalis resemble to other Cyr- todactylus and other gekkonid, which comprised many gradient mono-armed (telocentric) and few bi-armed chromosomes (meta- or submetacentric). Proximity of chromosome number and karyotype feature within genus Cyrtodactylus represents a close evolutionary line in the group. Nucleolar organizer region and meiotic cell characteristics The objective of the Ag-NOR banding technique is to detect nucleolar organizer regions (NORs) which represent the location of genes that have a function in ribosome synthesis (18S and 28S ribosomal RNA). The first cytogenetic study of C. kunyai and C. interdigitalis performed by Ag-NOR banding technique was obtained from this research. We found the clearly observable NORs on the region adjacent to telomere of long arm of the metacentric chromosome pair 12th (Figures 4a-b) for C. kunyai and on the region adjacent to telomere of long arm of the telocentric chromosome pair 12th (Figures 5a-b) for C. interdigitalis. Compared with other geckos, most showed two NORs appearing near telomeric region of small bi-armed or small mono-armed chromosome. An example of the previous reports of the geckos’ NOR Fig. 2. Conventionally stained somatic metaphase complement and karyotypes of male (a) and female (b) of Cyrtodactylus kunyai, 2n = 40, and male (c) and female (d) of C. interdigitalis, 2n = 42, (scale bars = 10 μm). Table 1. Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total chromosomes (LT), centromeric index (CI), relative length (RL) and standard deviation (SD) of CI and RL from 20 metaphases of male and female Cyrtodactylus kunyai, 2n = 40. Ch.p Ls Ll LT CI±SD RL±SD Ch.s Ch.t 1 4.306 6.392 10.698 0.597±0.005 0.106±0.005 L m 2 2.753 5.639 8.392 0.672±0.036 0.083±0.000 L sm 3 2.911 5.120 8.031 0.637±0.042 0.080±0.003 L sm 4 0.000 7.179 7.179 1.000±0.000 0.071±0.003 L t 5 0.000 6.838 6.838 1.000±0.000 0.068±0.003 L t 6 0.000 6.452 6.452 1.000±0.000 0.064±0.002 M t 7 0.000 5.837 5.837 1.000±0.000 0.058±0.003 M t 8 0.000 5.443 5.443 1.000±0.000 0.054±0.003 M t 9 0.000 5.210 5.210 1.000±0.000 0.052±0.002 S t 10 0.000 4.799 4.799 1.000±0.000 0.048±0.003 S t 11 0.000 4.208 4.208 1.000±0.000 0.042±0.004 S t 12* 1.946 2.147 4.093 0.525±0.017 0.041±0.003 S m 13 0.000 3.319 3.319 1.000±0.000 0.033±0.002 S t 14 0.000 3.300 3.300 1.000±0.000 0.033±0.003 S t 15 0.000 3.189 3.189 1.000±0.000 0.032±0.003 S t 16 1.570 1.580 3.150 0.547±0.030 0.035±0.003 S m 17 0.000 2.793 2.793 1.000±0.000 0.028±0.003 S t 18 0.000 2.654 2.654 1.000±0.000 0.026±0.002 S t 19 0.000 2.443 2.443 1.000±0.000 0.024±0.003 S t 20 1.136 1.156 2.292 0.552±0.020 0.025±0.002 S m Abbreviations: Ch.p, chromosome pair; Ch.s, chromosome size; Ch.t, chromosome type; *, nucleolar organizer region; L, large size; M, medium size; S, small size; m, metacentric; sm, submetacentric; t, telocentric. 26 Weera Thongnetr et al. localization included in the genus Gehyra (King 1983), Gekko (Chen et al. 1986; Shibaike et al. 2009; Patawang et al. 2014), Hemidactylus (Patawang and Tanomtong 2015b), and Lepidodactylus (Trifonov et al. 2015). These previous studies showed the NOR appearing near termi- nal region of one homologous small chromosome. The present study on male meiotic cell division in C. kunyai and C. interdigitalis found the late interphase to early prophase that the cell of each species showed two signals of nucleolus by positive silver staining (Figures 4c, 5c). The characteristics of two nucleolus structure at early phase of cell division supported the appearing of two NORs on one homologous at metaphase chro- mosome in both two Cyrtodactylus species. We found diplotene phase in meiotic cell of both two species (Fig- ures 4d, 5d), which showed synapsis between two of homologous and compacted chromosome. The meta- phase I (meiosis I, reductional division) was found in two species, which can be defined as the 20 bivalents for C. kunyai (Figure 4e) and 21 bivalents for C. inter- digitalis (Figure 5e). No metaphase I cells with partially paired bivalents, which are speculated to be male het- eromorphic sex chromosomes in both two Cyrtodactylus species. Moreover, n = 20 in C. kunyai (Figure 4f) and n = 21 in C. interdigitalis (Figure 5f ) were found at meta- phase II (meiosis II, equational division) of spermatid cells. Form these results, behavior and number of chro- mosome in metaphase I and metaphase II confirmed of each other’s accuracy and also verified the accuracy of diploid chromosome in somatic cells. Overview of chromosomal feature of the two Cyrtodactylus Gekkonid chromosome that has been reported in the past, most species show the gradient karyotype, which comprising of many mono-armed chromosomes Table 2. Mean length of short arm chromosome (Ls), long arm chro- mosome (Ll), total chromosomes (LT), centromeric index (CI), rela- tive length (RL) and standard deviation (SD) of CI and RL from 20 metaphases of male and female Cyrtodactylus interdigitalis, 2n = 42. Ch.p Ls Ll LT CI±SD RL±SD Ch.s Ch.t 1 2.715 6.857 9.572 0.716±0.012 0.090±0.005 L a 2 2.193 6.374 8.567 0.744±0.018 0.081±0.003 L a 3 0.000 8.136 8.136 1.000±0.000 0.077±0.004 L t 4 0.000 7.822 7.822 1.000±0.000 0.074±0.003 L t 5 0.000 7.505 7.505 1.000±0.000 0.071±0.005 L t 6 0.000 6.513 6.513 1.000±0.000 0.062±0.002 L t 7 0.000 6.066 6.066 1.000±0.000 0.057±0.003 L t 8 0.000 5.874 5.874 1.000±0.000 0.056±0.003 L t 9 0.000 5.677 5.677 1.000±0.000 0.054±0.002 L t 10 0.000 5.337 5.337 1.000±0.000 0.050±0.003 M t 11 0.000 4.539 4.539 1.000±0.000 0.043±0.004 S t 12* 0.000 4.031 4.031 1.000±0.000 0.038±0.003 S t 13 0.000 3.808 3.808 1.000±0.000 0.036±0.003 S t 14 1.824 1.830 3.654 0.501±0.027 0.035±0.004 S m 15 1.102 2.235 3.337 0.670±0.041 0.032±0.004 S sm 16 0.000 3.113 3.113 1.000±0.000 0.029±0.003 S t 17 0.000 2.856 2.856 1.000±0.000 0.027±0.003 S t 18 0.000 2.820 2.820 1.000±0.000 0.027±0.001 S t 19 1.399 1.410 2.809 0.502±0.038 0.027±0.003 S m 20 0.000 2.017 2.017 1.000±0.000 0.019±0.003 S t 21 0.000 1.712 1.712 1.000±0.000 0.016±0.002 S t Abbreviations: Ch.p, chromosome pair; Ch.s, chromosome size; Ch.t, chromosome type; *, nucleolar organizer region; L, large size; M, medium size; S, small size; m, metacentric; sm, submetacentric; a, acrocentric; t, telocentric. Table 3. Review of cytogenetic study of the genus Cyrtodactylus. Species 2n NF Karyotype NOR Locality Reference C. consobrinus 48 50 2bi-armed+46t - Sarawak, Malaysia Ota et al. (1992) C. interdigitalis 42 52 4m+2sm+4a+32t Loei, Thailand Present study C. kunyai 40 52 8m+4sm+28t Loei, Thailand Present study C. pubisulcus 42 44 2bi-armed+40t - Sarawak, Malaysia Ota et al. (1992) Abbreviations: 2n, diploid chromosome; NF, fundamental number; NOR, nucleolar organizer region; bi-armed, bi-armed chromosome; m, metacentric; sm, submetacentric; a, acrocentric; t, telocentric. Fig. 3. Standardized idiogram of Cyrtodactylus kunyai (a) and C. interdigitalis (b) by conventional staining. 27Cytogenetic study of the Bent-toed Gecko (Reptilia, Gekkonidae) in Thailand and few bi-armed chromosomes. Present results of C. kunyai and C. interdigitalis agree with chromosomal evolution line hypothesis within the gekkonid group. The karyotype of C. kunyai and C. interdigitalis showed the gradient of most telocentric while comprised of a few bi-armed chromosome, 12 chromosomes in C. kunyai and 10 chromosomes in C. interdigitalis. These features conform to the hypothesis of rearrangement from ances- tral karyotype by Robertsonian fissions, fusions or peri- centric inversions (Gorman 1973; King 1987). ACKNOWLEDGEMENTS This research was financially supported by shar- ing from the Research Fund for Supporting Lecture to Admit High Potential Student to Study and Research on His Expert Program Year 2016, the Toxic Substanc- es in Livestock and Aquatic Animals Research Group, Khon Kaen University, and the Royal Thai Government Scholarship National Science and Technology Develop- ment Agency (NSTDA). The project was approved by the Institute of Animals for Scientific Purpose Development of National Research Council of Thailand (Resolution U1-02740-2559). 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