Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 109-117, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1514 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Suphat Prasopsin, Nawarat Muanglen, Sukhonthip Ditcharoen, Chatmongkon Suwannapoom, Along- klod Tanomtong, Weera Thongnetr (2022) First Report on Classical and Molecu- lar Cytogenetics of Doi Inthanon Bent- toed Gecko, Cyrtodactylus inthanon Kunya et al., 2015 (Squamata: Gek- konidae) in Thailand. Caryologia 75(2): 109-117. doi: 10.36253/caryologia-1514 Received: November 30, 2021 Accepted: November 30, 2021 Published: September 21, 2022 Copyright: © 2022 Suphat Prasopsin, Nawarat Muanglen, Sukhonthip Ditch- aroen, Chatmongkon Suwannapoom, Alongklod Tanomtong, Weera Thong- netr. 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, distribution, and reproduction in any medium, 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. First Report on Classical and Molecular Cytogenetics of Doi Inthanon Bent-toed Gecko, Cyrtodactylus inthanon Kunya et al., 2015 (Squamata: Gekkonidae) in Thailand Suphat Prasopsin1, Nawarat Muanglen2, Sukhonthip Ditcharoen3, Chatmongkon Suwannapoom4, Alongklod Tanomtong5, Weera Thong- netr6,* 1 Research Academic Supports Division, Mahidol University, Kanchanaburi Campus, Sai- yok, Kanchanaburi, Thailand 2 Department of Fisheries, Faculty of Agricultural Technology, Sakon Nakhon Rajabhat University, Sakon Nakhon, Thailand 3 Division of Biology, Faculty of Science and Technology, Rajamangala University of Tech- nology Thanyaburi, Khlong Luang, Pathum Thani, Thailand 4 Department of Fishery, School of Agriculture and Natural Resources, University of Phayao, Muang, Phayao, Thailand 5 Program of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen, Thailand 6 Walai Rukhavej Botanical Research Institute, Mahasarakham University, Kantharawi- chai, Maha Sarakham, Thailand *Corresponding author. E-mail: weeraatah@hotmail.com Abstract. This study analyzed the karyotype of Cyrtodactylus inthanon Kunya et al., 2015 from Doi Inthanon, Chiang Mai Province, northern Thailand. The metaphase and meiotic chromosome preparations were obtained by squash technique from bone marrow and testes, respectively. The chromosomes were stained by Giemsa staining, Ag-NOR banding and molecular cytogenetics with fluorescence in situ hybridization (FISH) using microsatellites d(CA)15, d(GC)15, d(CAG)10 and d(GAA)10 as probes. The results showed the diploid chromosome number (2n) of 40. The chromosome types of metacentric, submetacentric, acrocentric and telocentric chromosomes were 12-4-2-22, respectively. The Ag-NORs banding technique provides the pair of nucleolar organ- izer regions (NORs) on the telomeric region of the long arm of acrocentric pair 12. There are no sex differences in karyotypes between males and females. We also found that during metaphase I on meiosis of C. inthanon, the homologous chromosomes appeared synapsis of 20 bivalents. The microsatellite d(CA)15 signals were located on the sub-centromeric region of the metacentric pair 10, whereas the d(GC)15, d(CAG)10 and d(GAA)10 repeats are highly accumulated throughout almost all entire chromo- somes. The karyotype formula is as follows: C. inthanon (2n = 40), Lm2 + Lsm4 + Lt6 + Mm2 + Mt8 + Sm8 + Sa2 + St8. Keywords: Bent-toed Gecko, Cyrtodactylus inthanon, Ag-NOR staining, karyotype, FISH. 110 Suphat Prasopsin et al. INTRODUCTION Cyrtodactylus is a genus of bent-toed geckos which is widely distributed across South Asia to Melanesia (Wood et al. 2012, Grismer et al. 2020, 2021). This genus is the most species group of gekkotans, with approximately 306 species currently recognized (Uetz et al. 2021). The Gekkonidae found in Thailand are highly diverse and include approximately 90 species. Among these species, 38 species of bent-toed geckos (Cyrtodactylus) are the most well-documented and show wide distribution in Thailand (Uetz et al. 2021). Although a high number of Cyrtodactylus species have already been discovered and many more explorations are anticipated based on the rapid discovery rate of new Cyrtodactylus species, sev- eral are defined as endemic including C. sanook, C. sai- yok and C. phuketensis (Panitvong et al. 2014, Pauwels et al. 2013, Sumontha et al. 2012). Recently, C. Inthanon, Doi Inthanon bent-toed gecko was discovered as a new species from Doi Inthanon region, Chiang Mai Prov- ince, northern Thailand by Kunya et al. (2015). It was a novel reptile endemic to Doi Inthanon strengthening the high significance of this mountain in terms of biodiver- sity preservation. In terms of endemic species, they are at higher risk of extinction because their habitat is lim- ited or unique. Additionally, they have been reported in recent years that the rate of destruction of Cyrtodactylus habitat has increased due to both forest land encroach- ment and wildfires, especially in Southeast Asia where they are mainly distributed (Chomdej et al. 2021). However, cytogenetic studies are still quite scarce within Cyrtodactylus, mostly restricted to classical pro- tocols. Studies applying molecular cytogenetic approach- es (i.e. chromosomal mapping of microsatellite sequenc- es) where done only in two species of C. jarujini and C. doisuthep (Thongnetr et al. 2021). Up to date only sev- en species from over 306 recognized species have been cytogenetically examined (Table 1). The diploid number among Gekkonid lizards ranges from 2n=16 to 2n=46 with most of the karyotypes composed of 28-46 chro- mosomes (Gorman 1973, Schmid et al. 1994). The typi- cal karyotype consists of a gradual series of acrocentric chromosomes which there is no difference between mac- ro and microchromosomes (Molavi et al. 2014). The occurrence of techniques related to DNA are encouraging for the advance of the comprehension of the animal genome structure and evolution (Martins et al. 2011; Barreto et al. 2021). Microsatellites are repetitive (in tandem) DNA sequences of one to six nucleotides found in all eukaryotic organisms (Cioffi and Bertollo 2012; López-Flores and Garrido Ramos 2012). In several species, these sequences can be found in long repetitions associated with heterochromatic regions (Martins 2007; Cioffi et al. 2011). This information on chromosomes is considered important along with other information for the identification of the species (Campiranont 2003). The cytogenetic analysis of the higher molecular chro- mosome structure can provide invaluable insight for the management of threatened species, where DNA alone could not address all genetic risks and threats to popu- lations (Potter and Deakin 2018). The distribution of microsatellites on chromosomes could help on the eluci- dation of evolutionary processes that lead to a karyotyp- ic macrostructure differentiation and even to the origin of sex chromosomes systems (Cioffi et al. 2011), where the investigation of the sex-determining system was fin- ished only one species, the Bornean endemic Cyrtodac- tylus pubisulcus through traditional cytogenetics (Ota et al. 1992; Keating et al. 2021) Accordingly, the present study is the first cytoge- netic study on C. inthanon from Thailand accomplished with classical and molecular cytogenetic techniques. Table 1. Karyotype reviews in the genus Cyrtodactylus. Species 2n NF Karyotype NOR Locality Reference C. consobrinus 48 50 2bi-arm+46t - Malaysia Ota et al. (1992) C. doisuthep 34 56 14m+6sm+2a+12t P9, 13 Thailand Thongnetr et al. (2021) C. interdigitalis 42 52 4m+2sm+4a+32t P12 Thailand Thongnetr et al. (2019a) C. inthanon 40 58 12m+4sm+2a+22t P12 Thailand Present study C. jarujini 40 56 8m+4sm+4a+24t P13, 14 Thailand Thongnetr et al. (2021) C. kunyai 40 52 8m+4sm+6a+22t P12 Thailand Thongnetr et al. (2019a) C. pubisulcus 42 44 2bi-arm+40t - Malaysia Ota et al. (1992) C. saiyok 42 42 42t P15 Thailand Thongnetr et al. (2019b) Remarks: 2n = diploid chromosome number, NORs = nucleolus organiser regions, NF = fundamental number (number of chromosome arms), bi-arm = bi-armed chromosome, m = metacentric, sm = submetacentric, a = acrocentric, t = telocentric chromosome, P = chromo- some pair and - = not available. 111First Report on Classical and Molecular Cytogenetics of Cyrtodactylus inthanon in Thailand Data provided here will increase our knowledge of cytogenetic information which can be used as a basis to comprehensively examine the taxonomy and evolution- ary relationship of Cyrtodactylus species and other gek- konids. MATERIALS AND METHODS Sample collection and chromosome preparation Five male and five female specimens of C. intha- non were collected from the Doi Inthanon reinforces, Chiang Mai Province, northern Thailand (Fig. 1). All bent-toed geckos were transferred to the laboratory and kept under standard conditions for one day prior to the experimentation. Chromosomes were directly prepared in vivo (Ota et al. 1990) by 0.1% colchicine were injected into the geckos’ intramuscular and abdominal cavity and then left for 8-10 hours. Bone marrow (in male and female) and testis (male) were cut into small pieces and then mixed with 0.075 M potassium chloride (KCl). After discarding all large cell pieces, 15 ml of cell suspension was transferred to a centrifuge tube and incubated for 30-40 minutes, then centrifuged at 3,000 rpm for 8 min- utes. The cell suspension was fixed in fresh cool fixative of methanol:glacial acetic acid (3:1) and gradually made up to 8 ml before centrifuging again at 3,000 rpm for 8 minutes, whereupon the supernatant was discarded. Fix- ation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. Giemsa’s staining, Ag-NOR banding technique and Chro- mosome analysis A drop of the mixture was added to a clean and cold slide by micropipette followed by the air-dry technique. The slide was conventionally stained with 20% Giemsa solution for 30 minutes (Patawang et al. 2014). Then, the slides were rinsed thoroughly with running tap water to remove excess stain. Two drops of each 50% silver nitrate and 2% gelatin were droped on slides, respectively. Then it was sealed with cover glasses and incubated at 60 °C for 5-10 min- ute. There after that it was soaked in distilled water until cover glasses are separated. (Howell and Black 1980). Ten clearly observable metaphase cells with well spread chromosomes of each male and female were selected and photographed. The length of short arm chromosome (Ls) and long arm chromosome (Ll) were measured and the length of total arm chromosome (LT, LT = Ls+Ll) was calculated. The relative length (RL), the centromeric index (CI) and standard deviation (SD) of RL and CI were analysed according to the chromo- some classification of Chaiyasut (1989) and Turpin and Lejeune (1965). Chromosome types were described as metacentric (m), submetacentric (sm), acrocentric (a) and telocentric (t) chromosomes, respectively. The Fun- damental Number (NF, number of chromosome arms) was obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to telocentric chromosomes. All parameters were used in karyotyping and idiograming. Fluorescence in situ Hybridization (FISH) The use of microsatellite probes described by Kubat et al. (2008) was followed here with slight modifications. The microsatellite probes: d(CA)15, d(GC)15, d(CAG)10 and d(GAA)10 were directly labelled with Cy3 at the 5 -́terminal during synthesis by Sigma (St. Louis, MO, USA). Fluorescence In Situ Hybridization (FISH) was performed under highly stringent conditions on mitotic chromosome spreads (Pinkel et al. 1986). After denatur- ation of chromosomal DNA in 70% formamide/2×SSC (saline sodium citrate) at 70 °C, spreads were incubated in 2×SSC for 4 minutes at 70 °C. The hybridization mix- ture (2.5 ng/μL each probe, 2 μg/μL salmon sperm DNA, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides and the hybridization was per- formed overnight at 37 °C in a moist chamber contain- ing 2×SSC. The post hybridization wash was carried out with 1× SSC for 5 minutes at 65 °C. A final wash was performed at room temperature in 4×SSCTween for 5 minutes. Finally, the chromosomes were counterstained Figure 1. General characteristic shape of the Cyrtodactylus Intha- non, the most prominent feature in the top view. 112 Suphat Prasopsin et al. with DAPI (1.2 μg/mL), mounted in antifading solution (Vector, Burlingame, CA, USA) and analyzed in fluores- cence microscope Nikon ECLIPSE. RESULTS Diploid chromosome number (2n), fundamental number (NF) and karyotype The diploid number and NF in C. inthanon are 40 and 58, respectively. The karyotype of C. inthanon is composed of 12 metacentrics, 4 submetacentrics, 2 acro- centrics and 22 telocentrics There are exhibited no sex differences in karyotypes between males and females. (Table 2 and Fig. 2A-D). Nucleolar organizer region and meiotic cell characteristics The determination of chromosome marker for this species is firstly obtained by using the Ag-NOR band- ing technique. The nucleolar organizer regions (NORs) are observed on the telomeric region of the acrocentric chromosome pair 12 both male and female (Fig. 2 E-H). The meiotic cell of C. inthanon reveals the diplotene phase (Fig. 3), which shows synapsis between two the homologous and compacted chromosomes. The meta- phase I (meiosis I, reductional division) can be defined as the 20 bivalents Patterns of microsatellite The microsatellite d(CA)15 presents the signals high- ly accumulated at the interstitial subcentromeric region on short arms of metacentric chromosome pair 10 (Fig. 4A) whereas, the microsatellite d(GC)15, d(CAG)10 and d(GAA)10 are distributed weak signals throughout the whole chromosomes (Fig. 4C-D). DISCUSSION From the previous reports, the chromosome exhibited various number in the Cyrtodactylus, ranging from 34 to 48, however, the most frequent numbers were 40 and 42. The present study showed that the chromosome number of C. inthanon were 40. This result revealed accordance with other species that have been reported, such as C. jarujini and C. kunyai (Thongnetr et al. 2019a, 2021). Moreover, the diploid chromosome number (2n) differed from C. Table 2. Mean length of short arm chromosome (Ls), length of long arm chromosome (Ll), length of total chromosomes (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) from 10 metaphases of male and female Cyrtodactylus inthanon, 2n=40. Chr. pair Ls Ll LT CI±SD RL±SD Chr. size Chr. type 1 3.568 4.695 8.263 0.567±0.026 0.101±0.008 Large metacentric 2 2.325 4.177 6.503 0.640±0.019 0.079±0.003 Large submetacentric 3 2.059 3.799 5.858 0.648±0.023 0.072±0.002 Large submetacentric 4 0.000 5.921 5.921 1.000±0.000 0.072±0.005 Large telocentric 5 0.000 5.894 5.894 1.000±0.000 0.072±0.003 Large telocentric 6 0.000 5.330 5.330 1.000±0.000 0.065±0.004 Large telocentric 7 0.000 4.827 4.827 1.000±0.000 0.059±0.003 Medium telocentric 8 0.000 4.302 4.302 1.000±0.000 0.052±0.002 Medium telocentric 9 0.000 3.994 3.994 1.000±0.000 0.049±0.002 Medium telocentric 10 1.688 2.476 4.164 0.595±0.021 0.051±0.002 Medium metacentric 11 0.000 3.786 3.786 1.000±0.000 0.046±0.002 Medium telocentric 12* 1.069 2.431 3.500 0.705±0.070 0.043±0.003 Small acrocentric 13 0.000 2.785 2.785 1.000±0.000 0.034±0.003 Small telocentric 14 0.000 2.707 2.707 1.000±0.000 0.033±0.001 Small telocentric 15 0.000 2.334 2.334 1.000±0.000 0.028±0.004 Small telocentric 16 1.202 1.698 2.900 0.584±0.035 0.036±0.004 Small metacentric 17 0.000 2.166 2.166 1.000±0.000 0.027±0.003 Small telocentric 18 1.087 1.512 2.599 0.582±0.025 0.032±0.003 Small metacentric 19 0.916 1.284 2.200 0.586±0.016 0.027±0.003 Small metacentric 20 0.796 0.999 1.795 0.557±0.075 0.022±0.001 Small metacentric Remark: Chr. = chromosome; * = satellite chromosome (nucleolar organizer region, NOR). 113First Report on Classical and Molecular Cytogenetics of Cyrtodactylus inthanon in Thailand Figure 2. Metaphase chromosome plates and karyotypes of male and female Cyrtodactylus inthanon, 2n=40 by conventional staining (A-D) and Ag-NOR banding technique (E-H). Scale bars indicate 5 micrometers. The arrows indicate nucleolar organizer regions/NOR. 114 Suphat Prasopsin et al. doisuthep (2n=34), C. interdigitalis, C. pubisulcus, C. sai- yok (2n=42) and C. consobrinus (2n= 48) (Ota et al. 1992, Thongnetr et al. 2019a, 2019b, 2021). The different Cyrto- dactylus species underwent an extremely diversified karyo- type evolution, considering the numerical and structural aspects of their complements, with the NF that varied from 42 to 58. The karyotype of C. inthanon is composed of 12 metacentric, 4 submetacentric, 2 acrocentric and 22 telocentric chromosomes. The karyological characteristics of C. inthanon obtained in the present study is the first report of chromosome sizes and the chromosome types. In other species of Cyrtodactylus, the different karyological structure can be found as well. However, the karyotype of C. inthanon resemble other Cyrtodactylus and other gek- konids, which comprised many gradient mono-armed (tel- ocentric) and few bi-armed chromosomes (meta- or sub- metacentric). The proximity of chromosome number and karyotype feature within genus Cyrtodactylus represents a close evolutionary line in the group. (Trifonov et al. 2011). This analysis was performed to highlight the combined importance of the different chromosome rearrangements in the evolutionary modeling of their karyotypes, such as Robertsonian rearrangements or centric fission (Ueno and Takai 2000) fusion and especially, pericentric inversions (Jacobina et al. 2011). The nucleolar organizer regions (NORs) represent the location of genes that have a function in ribosome synthesis (18S and 28S ribosomal RNA). NORs of C. inthanon exhibited a single pair of Ag-NORs located on the telomeric region on the long arm of the acrocentric and are similar to the previous reports of the genus Cyr- todactylus, e.g., C. interdigitalis, C. kunyai (Thongnetr et al. 2019a, 2021). The metaphase I (meiosis I, reductional division) was found in C. inthanon, which showed as the 20 biva- lents (Fig. 3). No metaphase I cell with partially paired bivalents, which are speculated to be male heteromor- phic sex chromosomes in this species. From this result, the behavior and number of chromosomes in metaphase I confirmed of each other’s accuracy and also verified the accuracy of diploid chromosomes in somatic cells. Microsatellites or simple sequence repeats (SSRs) are oligonucleotides of 1-6 base pairs in length, form- ing excessive tandem repeats of usually 4 to 40 units (Tautz and Renz 1984, Ellegren 2004, Chistiakov et al. 2006). They showed abundant distribution throughout eukaryotic genomes, being dispersed or clustered both in euchromatin or heterochromatin. They are highly pol- ymorphic regarding copy number variations (Ellegren 2004). In our present study in C. inthanon exhibited the microsatellite d(CA)15 was revealed that the sig- nals highly accumulated at the interstitial subcentro- meric region on short arms of metacentric chromosome pair 10 (Fig. 4A), whereas, the microsatellite d(GC)15, d(CAG)10 and d(GAA)10 were distributed weak signals throughout the whole chromosomes (Fig. 4C-D). In pre- vious study, microsatellite pattern (the dinucleotides d(A)20, d(CAG)10, d(CGG)10, d(GAA)10 and d(TA)15) on C. jarujini and C. doisuthep accumulated exclusively in telomeric and subtelomeric chromosomal regions bear- ing dispersed over the whole genomes including chro- mosomes and some had strong signals on only a pair of homologous chromosomes (Thongnetr et al. 2021). How- ever, the results clearly indicate that the microsatellite repeats are in high copy number on some chromosome pairs, according to previous reports on reptile groups (Pokorná et al. 2011; Matsubara et al. 2013). The first cytogenetic study of the C. inthanon has enabled us to delineate the process of chromosomal reorganization in this group chromosome system and Figure 3. Metaphase I chromosome plate and karyotypes of Cyrtodactylus inthanon, n=20 by conventional staining technique. Scale bars indicate 5 micrometers. 115First Report on Classical and Molecular Cytogenetics of Cyrtodactylus inthanon in Thailand FISH mapping. The results obtained here can be used to support the further investigation on taxonomy, biodiver- sity conservation and evolutionary relationship among the genus Cyrtodactylus and others. 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