Studies of chromosomes of Cyrtodactylus jarujini Ulber, 1993 and C. doisuthep Kunya et al., 2014 to compare microsatellite and TTAGGG sequences by classical and molecular techniques were conducted in Thailand. Karyological typing from a conventional staining technique of C. jarujini and C. doisuthep showed diploid chromosome numbers of 40 and 34 while the Fundamental Numbers (NF) were 56 in both species. In addition, we created the chromosome formula of the chromosomes of C. jarujini showing that 2n (40) = L^sm₁ + L^sm₂ + L^t₃ + M^m₁ + M^t₄ + S^m₂ + S^a₂ + S^t₅ while that of C. doisuthep was 2n (34) = L^sm₃ + L^m₂ + L^t₃ + M^m₁ + M^t₂ + S^m₄ + S^a₁ + S^t₁. Ag-NOR staining revealed NOR-bearing chromosomes in chromosome pairs 13 and 14 in C. jarujini, and in chromosome pairs 9 and 13 in C. doisuthep. This molecular study used the FISH technique, as well as microsatellite probes including (A)₂₀, (TA)₁₅, (CGG)₁₀, (CGG)₁₀, (GAA)₁₀, (TA)₁₅ and TTAGGG repeats. The signals showed that the different patterns in each chromosome of the Gekkonids depended on probe types. TTAGGG repeats showed high distribution on centromere and telomere regions, while (A)₂₀, (TA)₁₅, (CGG)₁₀, (CGG)₁₀, (GAA)₁₀ and (TA)₁₅ bearing dispersed over the whole genomes including chromosomes and some had strong signals on only a pair of homologous chromosomes. These results suggest that the genetic linkages have been highly differentiated between the two species.

Ag-NOR, Cyrtodactylus doisuthep, Cyrtodactylus jarujini, FISH microsatellite, karyotype

Bent-toed geckos (genus Cyrtodactylus Gray, 1827) in Thailand have been classified into approximately 24 species (Chuaynkern and Chuaynkern 2012). Cyrtodactylus jarujini ranges from Nong kai, Bueng Kan and Nakhon Phanom Provinces, Thailand. More recently, Sumontha et al. (2008), found it in two caves on two sandstone hills, Phu Sing and Phu Thok, where it remained by day on the walls and crevices and emerged from the caves at night. Both in Phu Sing and Phu Thok, syntropy was found with the cave-dwelling agamid Mantheyus phuwuanensis (Manthey and Nabhitabhata 1991). It has also been recorded from central and northern Laos (Stuart 1999), but the exact identity of the Lao populations has to be re-evaluated (Fig. 1A). In contrast C. doisuthep is known only from Doisuthep in the Doi Suthep-Pui Range, Mueang District, Chiang Mai Province, northern Thailand (Fig. 1B).

Figure 1.

The C. jarujini specimen (A), metaphase chromosome plate and karyotypes (A–G) by conventional technique, (D–H) by Ag-NOR banding technique. The C. doisuthep specimen (B), metaphase chromosome plate and karyotypes (E–I) by conventional technique, (F–J) by Ag-NOR banding technique. Arrows indicated Ag-NORs regions. Scale Bar: 5 µm.

Only 13% of gekkonid species have been karyotyped (Olmo and Signorino 2005) and were studied with conventional cytogenetic methods, including routine staining, as well as R-, NOR- and C-banding (Moritz 1983; Olmo and Signorino 2005; Shibaike et al. 2009). However, a small number of species were studied by molecular cytogenetic techniques (Kawai et al. 2009). The diploid number amongst gekkonid lizards ranges from 2n = 28 to 46 with most of the karyotypes composed of 28–46 chromosomes (Gorman 1973; Olmo 1986; Schmid et al. 1994). There are five karyotyped Cyrtodactylus species: C. consobrinus 2n = 48, NF = 50, C. pubisulcus 2n = 42, NF = 44 (Ota et al. 1992); C. interdigitalis 2n = 42, NF = 52 and C. kunyai 2n = 40, NF = 52 (Thongnetr et al. 2019a); C. saiyok 2n = 42, NF = 42 (Thongnetr et al. 2019b). The typical karyotype consists of a gradual series of telocentric chromosomes (sometimes with a few metacentric) and there is no distinction between macro- and microchromosomes, the centromere often being subterminal (Gorman 1973). Karyotype evolution within the group is accompanied by fissions and fusions and pericentric inversions (Gorman 1973; Olmo and Signorino 2005). This information on chromosomes is considered important along with other information for identification of the species (Campiranont 2003), especially the identification of related species, because of similarity of shape, appearance and other phenotypic expressions that are presumed to be associated with the genotype. Information from sequences of DNA allows us to understand the creation of a phylogenetic tree (dendrogram), because these characteristics often have a particular pattern. Information on chromosomes can be used to identify the phylogenetic relationship between species and population of animals (Lauhajinda and Taksintum 2006). Therefore, it is necessary to study the karyology of this group. In addition, geckos could be affected by the actions of humans in their use of household objects and agricultural chemicals. Thus, the gecko is one of the important groups of animals that can serve as a model for studying the environmental impact from human actions in the future.

The samples of C. jarujini and C. doisuthep were collected from the Phu Wua, Ban Phaeng District, Nakhonphanom Province and Doi Suthep-Pui Range, Mueang District, Chiang Mai Province, Thailand, (permission from an ethical committee ID U1-04498-2559). Chromosomes were directly prepared in vivo (Ota et al. 1990) by 0.1% colchicine were injected into the animals’ intramuscular and abdominal cavity and left for 8–10 hours. Bone marrow, liver 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 30–40 minutes, then centrifuged at 3,000 rpm for 8 minutes. Cells were 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. Fixation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. Using conventional Giemsa staining, 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. Afterwards, the slides were allowed to air-dry at room temperature. Ag-NOR banding was analysed following the method of Howell and Black (1980). Two drops each of 50% silver nitrate and 2% gelatine solutions were added to slides, respectively. Then, they were sealed with cover glasses and incubated at 60 °C for 5–10 minutes. Afterwards, they were then soaked in distilled water until the cover glasses were separated. Finally, the slides were allowed to air-dry at room temperature and observed under microscope. Metaphase figures were analysed according to the chromosome classification of Chaiyasut (1989) and Turpin and Lejeune (1965). Chromosomes were classified as metacentric (m), submetacentric (sm), acrocentric (a) and telocentric (t). The Fundamental Number (NF: number of chromosome arms) is obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to acrocentric chromosomes. The use of microsatellite probes described by Kubat et al. (2008) was followed here with slight modifications. These sequences 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 denaturation of chromosomal DNA in 70% formamide/ 2×SSC at 70 °C, spreads were incubated in 2×SSC for 4 min at 70 °C. The hybridization mixture (2.5 ng/μL probes, 2 μg/μL salmon sperm DNA, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides, and the hybridization was performed overnight at 37 °C in a moist chamber containing 2×SSC. The post hybridization wash was carried out with 1×SSC for 5 min at 65 °C. A final wash was performed at room temperature in 4×SSCT for 5 min. Finally, the chromosomes were counterstained with DAPI (1.2 μg/mL), mounted in antifading solution (Vector, Burlingame, CA, USA), and analyzed in fluorescence microscope Nikon ECLIPSE.

In this study, the comparison of the cytogenetic maps of two Cyrtodactylus species (C. jarujini and C. doisuthep) enabled us to delineate the process of chromosomal re-organisation in this group. This is the first report in Thailand for the study of cytogenetics of both species. Therefore, the cytogenetic data obtained can be used to benefit cytotaxonomy and the study of evolution of geckos, as well as being an essential prerequisite for future genome projects of gecko groups.

This research project was financially supported by Mahasarakham University, Phetchabun Rajabhat University and The Unit of Excellence 2020 on Biodiversity and Natural Resources Management, University of Phayao (UoE63005).