Research Article
Print
Research Article
Cytogenetic characterisation and chromosomal mapping of microsatellite and telomeric repeats in two gecko species (Reptilia, Gekkonidae) from Thailand
expand article infoWeera Thongnetr, Surachest Aiumsumang§, Rodjarin Kongkaew|, Alonglod Tanomtong|, Chatmongkon Suwannapoom, Sumalee Phimphan§
‡ Mahasarakham University, Maha Sarakham, Thailand
§ Phetchabun Rajabhat University, Phetchabun, Thailand
| Khon Kaen University, Khon Kaen, Thailand
¶ University of Phayao, Phayao, Thailand
Open Access

Abstract

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) = Lsm1 + Lsm2 + Lt3 + Mm1 + Mt4 + Sm2 + Sa2 + St5 while that of C. doisuthep was 2n (34) = Lsm3 + Lm2 + Lt3 + Mm1 + Mt2 + Sm4 + Sa1 + St1. 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.

Keywords

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

Introduction

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.

Material and methods

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.

Results

The diploid chromosome number and fundamental number

The diploid numbers in C. jarujini and C. doisuthep, were 40 and 34, respectively (Fig. 1C, E), whereas NF was 56 in both species (Fig. 1G, I). The type chromosomes of metacentric, submetacentric, acrocentric and telocentric were 8-4-4-24 and 14-6-2-12. There are no sex-related chromosomal heteromorphisms in the two species here studied.

The karyological characteristics

The karyotype of C. jarujini consists of two large metacentric, four large submetacentric, six large telocentric, two medium metacentric, eight medium telocentric, four small metacentric, four small acrocentric and ten small telocentric chromosomes. The karyotype formula for C. jarujini is as follows: 2n (40) = Lm2 + Lsm4 + Lt6 + Mm2 + Mt8 + Sm4 + Sa4 + St10 or 2n (40) = 8m + 4sm + 4a + 24t. The karyotype of C. doisuthep comprises four large metacentric, six large submetacentric, six large telocentric, two medium metacentric, four medium telocentric, eight small metacentric, two small acrocentric and two small telocentric chromosomes. The karyotype formula for C. doisuthep is as follows: 2n (34) = Lm4 + Lsm6 + Lt6 + Mm2 +Mt4 + Sm8 + Sa2 + St2 or 2n (34) = 14m + 6sm + 2a + 12t.

Ag-NOR banding

This technique highlighted active NORs on pairs 13 and 14 of C. jarujini (Fig. 1D, H) and pairs 9 and 13 of C. doisuthep (Fig. 1F, J).

Microsatellite pattern

Microsatellites (A)20, (TA)15, (CAG)10, (CGG)10, (GAA)10 and (TA)15 abundantly distributed in some chromosomes, usually in telomeric regions of both species studied. FISH with the telomeric probe TTAGGG revealed hybridization signals on each telomere of all chromosomes (Fig. 2).

Figure 2.

Karyotypes of two geckos presenting the patterns of microsatellite (A)20, (CAG)10, (CGG)10, (GAA)10, (TA)15 and TTAGGG; C. jarujini (A–F), C. doisuthep (G–L). Scale Bars: 10 µm.

Discussion

Karyological data of the genus Cyrtodactylus

The species in the Cyrtodactylus exhibited a variable chromosome number, ranging from 34 to 42, however, the most frequent numbers were 40 and 42. The present study showed that the chromosome numbers of C. jarujini and C. doisuthep were 40 and 34, respectively. The fundamental number was 56 in both species. These results showed difference and accordance with others Cyrtodactylus that have been reported (Table 1). The karyological characteristics of C. jarujini and C. doisuthep obtained in the present study are the first report of chromosome sizes and the chromosome types in these species. In different species of Cyrtodactylus, different karyological characteristics can be found. However, overall, of these karyotypes of C. jarujini and C. doisuthep resemble those of other Cyrtodactylus species and other gekkonids, which comprised many mono-armed (telocentric) and few bi-armed chromosomes (meta- or submetacentric). For those gekkonid chromosomes which have been reported previously, most species showed that the karyotype comprises of many mono-armed chromosomes and few bi-armed chromosomes. The present results of C. jarujini and C. doisuthep agreed with the chromosomal evolution line hypothesis within the gekkonid group (Trifonov et al. 2011). The karyotype of C. jarujini and C. doisuthep showed the gradient of most telocentrics, while comprising of a few bi-armed chromosomes. These features conform to the hypothesis of re-arrangement from ancestral karyotype by Robertsonian fissions, fusions or pericentric inversions (Gorman 1973; King 1987).

Table 1.

Karyotype reviews in the genera Cyrtodactylus, Gekko Laurenti, 1768 and Hemidactylus Goldfuss, 1820 (Gekkonidae, Squamata).

Species 2n NF Karyotype formula NORs Location Reference
Cyrtodactylus consobrinus (Peters, 1871) 48 50 2bi-arm+46t Malaysia Ota et al. (1992)
C. doisuthep Kunya et al., 2014 34 56 14m+6sm+2a+12t P9, 13 Thailand Present study
C. interdigitalis Ulber, 1993 42 52 4m+2sm+4a+32t P12 Thailand Thongnetr et al. (2019a)
C. jarujini Ulber, 1993 40 56 8m+4sm+4a+24t P13, 14 Thailand Present study
C. kunyai Pauwels et al., 2014 40 52 8m+4sm+6a+22t P12 Thailand Thongnetr et al. (2019a)
C. pubisulcus Inger, 1958 42 44 2bi-arm+40t Malaysia Ota et al. (1992)
C. saiyok Panitvong, 2014 42 42 42t P15 Thailand Thongnetr et al. (2019b)
Gekko chinensis Gray 1842 40 46 6bi-armed+34uni-armed China Lau et al. (1997)
G. gecko (Linnaeus, 1758) 38 50 12bi-armed+26uni-armed Cohen et al. (1967)
38 50 Lm2+Lsm4+Lt4+Mt6+Sm4+Sa2+St16 P4 Thailand Patawang et al. (2014)
G. hokouensis Pope, 1928 38 56 4m+6sm+20t+8bi-armed P(L)19 China Chen et al. (1986)
G. monarchus (Schlegel, 1836) 38 46 Malaysia Ota et al. (1990)
G. petricolus Taylor, 1962 38 54 Ota (1989)
G. shibatai Toda et al., 2008 38 58 4m+8sm+18t+8bi-armed P(L)19 Japan Shibaike et al. (2009)
G. tawaensis Okada, 1956 38 58 4m+8sm+18t+8bi-armed P(L)19 Japan Shibaike et al. (2009)
G. taylori Grossmann et Ulber, 1990 42 Thailand Ota and Nabhitabhata (1991)
G. vertebralis Toda et al., 2008 38 62 4m+14sm+14t+6bi-armed P(L)19 Japan Shibaike et al. (2009)
Hemidactylus brookii Gray, 1854 40 44 4bi-armed+36t Bhatnagar (1962)
H. flaviviridis Rüppell, 1835 40 60 20bi-armed+20t Asana and Mahabale (1941)
46 46 46t Makino and Momma (1949)
40 52 12bi-armed+28t Branch (1980)
H. frenatus Schlegel, 1836 46 46 46t Makino and Momma (1949)
40 54 14bi-armed+26t P3 King (1978)
40 46 6bi-armed+34t Darevsky et al. (1984)
H. mabouia (Moreau de Jonnès, 1818) 42 56 14bi-armed+28t Becak et al. (1972)
42 54 12bi-armed+30t McBee et al. (1987)

Active NOR sites

Nucleolus organiser regions (NORs) are chromosome sites which contain the 18S and 28S ribosomal RNA genes. If these regions were active during the interphase prior to mitosis, they can be detected by silver nitrate staining (Howell and Black 1980). In the present study, the chromosome markers of both Cyrtodactylus are determined by using the Ag-NOR banding technique as shown in Table 1. C. jarujini had the acrocentric chromosome pair 13 and metacentric chromosome pair 14, which were the NOR-bearing chromosome. Pair 13 NORs were located on the short arm near the telomere (telomeric NOR) and the pair 14 NORs were located on the short arm near the centromere (centromeric NOR). C. doisuthep had the metacentric, two chromosome pair 9 and pair 13 which were the NOR-bearing chromosomes. Pair 9 NORs were located on the arm near the telomere (telomeric NOR) on both sides and the pair 13 NORs were located on the arm near the telomere (telomeric NOR).

The NORs in both species of genus Cyrtodactylus exhibited at the telomeric region on the long arm and short arm and are similar to the previous reports of the gekkonids for the Gekkonidae family by King (1978) and Moritz and King (1985). The NORs of Dixonius siamensis (Boulenger, 1898), G. gecko, G. hokouensis, G. shibatai, G. tawaensis, G. vertebralis, H. frenatus and H. platyurus were found at all regions on the short arm and that agrees with those previous reported (Asana and Mahabale 1941; Makino and Momma 1949; Bhatnagar 1962; Cohen et al. 1967; Becak et al. 1972; King 1978; Branch 1980; Darevsky et al. 1984; Chen et al. 1986; McBee et al. 1987; Ota 1989; Ota et al. 1990; Ota and Nabhitabhata 1991; Lau et al. 1997; Ota et al. 2001; Shibaike et al. 2009; Patawang et al. 2014; Trifonov et al. 2011; Trifonov et al. 2015).

Microsatellite pattern

Microsatellites or simple sequence repeats (SSRs) are oligonucleotides of 1–6 base pairs in length, forming excessive tandem repeats of usually 4 to 40 units (Tautz and Renz 1984; Ellegren 2004; Chistiakov et al. 2006). They show abundant distribution throughout eukaryotic genomes, being dispersed or clustered both in euchromatin or heterochromatin. They are highly polymorphic regarding copy number variations (Ellegren 2004). In our present study both species exhibited the same general hybridisation pattern for some applied probes with the motif TAAGGG repeat showing abundance at the telomeric ends of all chromosomes (Fig. 3), corroborating findings from other gekko groups studied to date (Srikulnath 2015). Otherwise, the dinucleotides (A)20, (CAG)10, (CGG)10, (GAA)10 and (TA)15 accumulated exclusively in telomeric and subtelomeric chromosomal regions. However, 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).

Figure 3.

Idiograms represent the (A)20, (CAG)10, (CGG)10, (GAA)10, (TA)15 and TTAGGG mapping on the chromosomes of C. jarujini (A) and C. doisuthep (B).

Conclusions

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.

Acknowledgements

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).

References

  • Asana JJ, Mahabale TS (1941) Spermatogonial chromosomes of two Indian lizards, Hemidactylus flaviridis Ruppell and Mabuya macularia Blyth. Current Science 11: 494–495.
  • Branch WR (1980) Chromosome morphology of some reptiles from Oman and adjacent territories. Journal of Oman Studies Special Report 2: 333–345.
  • Campiranont A (2003) Cytogenetics. Bangkok: Department of Genetics, Faculty of Science, Kasetsart University, 306 pp. [In Thai]
  • Chistiakov DA, Hellemans B, Volckaert FAM (2006) Microsatellites and their genomic distribution, evolution, function and applications: a review with special reference to fish genetics. Aquaculture 255: 1–29. https://doi.org/10.1016/j.aquaculture.2005.11.031
  • Chaiyasut K (1989) Cytogenetics and Cytotaxonomy of the Genus Zephyranthes. Bangkok: Department of Botany, Faculty of Science, Chulalongkorn University. [In Thai]
  • Chuaynkern Y, Chuaynkern C (2012) Checklist of reptiles in Thailand. Journal of Wildlife in Thailand 19(1): 75–162.
  • Cohen MM, Huang CC, Clark HF (1967) The somatic chromosomes of 3 lizard species: Gekko gecko, Iguana iguana and Crotaphytus collaris. Experientia 23(9): 769–771. https://doi.org/10.1007/BF02154168
  • Darevsky IS, Kupriyanova LA, Roshchin VV (1984) A new all-female triploid species of gecko and karyological data on the bisexual Hemidactylus frenatus from Vietnam. Journal of Herpetology 18: 277–284. https://doi.org/10.2307/1564081
  • Gorman GC (1973) The chromosome of the Reptilia, a cytotaxonomic interpretation. In: Chiarelli AB, Cappana E (Eds) Cytotaxonomy and Vertebrate Evolution. Academic Press, New York, 349–424.
  • Howell WM, Black DA (1980) Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36(8): 1014–1015. https://doi.org/10.1007/BF01953855
  • Kawai A, Ishijima J, Nishida C, Kosaka A, Ota H, Kohno S, Matsuda Y (2009) The ZW sex chromosomes of Gekko hokouensis (Gekkonidae, Squamata) represent highly conserved homology with those of avian species. Chromosoma 118: 43–51. https://doi.org/10.1007/s00412-008-0176-2
  • King M (1978) A new chromosome form of Hemidactylus frenatus (Duméril and Bibron). Herpetologica 34: 216–218.
  • Kubat Z, Hobza R, Vyskot B, Kejnovsky E (2008) Microsatellite accumulation in the Y chromosome of Silene latifolia. Genome 51: 350–356. https://doi.org/10.1139/G08-024
  • Lauhajinda W, Taksintum W (2006) Principles of Animal Taxonomy. Kasetsart University Press, Bangkok, 256 pp. [In Thai]
  • Lau MW, Ota H, Bogadek A (1997) Chromosome polymorphism and karyotype of Gekko chinensis (Gekkonidae: Reptilia) from Hong Kong. Journal of Herpetology 31(1): 137–139. https://doi.org/10.2307/1565344
  • Matsubara K, Knopp T, Sarre DS, Georges A, Ezaz T (2013) Karyotypic analysis and FISH mapping of microsatellite motifs reveal highly differentiated XX/XY sex chromosomes in the pink-tailed worm-lizard (Aprasia parapulchella, Pygopodidae, Squamata). Molecular Cytogenetics 6: e60. https://doi.org/10.1186/1755-8166-6-60
  • McBee K, Biekham JW, Dixon JR (1987) Male heterogamety and chromosomal variation in Caribbean geckos. Journal of Herpetology 21: 68–71. https://doi.org/10.2307/1564380
  • Moritz C, King D (1985) Cytogenetic perspectives on parthenogenesis in the Gekkonidae. In: Grigg G, Shine R, Ehrmann H (Eds) Biology of Australasian Frogs and Reptiles. Royal Zoological Society of New South Wales, Sidney, 327–337.
  • Ota H (1989) Japalura brevipes Gressitt (Agamidae: Reptilia), a valid species from high altitude area of Taiwan. Herpetologica 45(1): 55–60. https://doi.org/10.2307/1445482
  • Ota H, Hikida T, Matsui M, Mori A (1990) Karyotype of Gekko monarchus (Squamata: Gekkonidae) from Sarawak, Malaysia. Japanese Journal of Herpetology 13(4): 136–138. https://doi.org/10.5358/hsj1972.13.4_136
  • Ota H, Hikida T, Nabhitabhata J, Panha S (2001) Cryptic taxonomic diversity in two broadly distributed lizards of Thailand (Mabuya macularia and Dixonius siamensis) as revealed by chromosomal investigations (Reptilia: Lacertilia). The Natural History Journal of Chulalongkorn University 1(1): 1–7.
  • Patawang I, Tanomtong A, Jumrusthanasan S, Kakampuy W, Neeratanaphan L, Pinthong K (2014) Chromosomal characteristics of NORs and karyological analysis of tokey gecko, Gekko gecko (Gekkonidae, Squamata) from mitotic and meiotic cell division. Cytologia 79(3): 315–324. https://doi.org/10.1508/cytologia.79.315
  • Pinkel D, Straume T, Gray J (1986) Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proceedings of the National Academy of Sciences of the United States of America 83: 2934–2938. https://doi.org/10.1073/pnas.83.9.2934
  • Pokorná M, Kratochvíl L, Kejnovský E (2011) Microsatellite distribution on sex chromosomes at different stages of heteromorphism and heterochromatinization in two lizard species (Squamata: Eublepharidae: Coleonyx elegans and Lacertidae: Eremias velox). BMC Genetics 12: e90. https://doi.org/10.1186/1471-2156-12-90
  • Shibaike Y, Takahashi Y, Arikura I, Iiizumi R, Kitakawa S, Sakai M, Imaoka C, Shiro H, Tanaka H, Akakubo N, Nakano M, Watanabe M, Ohne K, Kubota S, Kohno S, Ota H (2009) Chromosome evolution in the lizard genus Gekko (Gekkonidae, Squamata, Reptilia) in the East Asian islands. Cytogenetic and Genome Research 127: 182–190. https://doi.org/10.1159/000303334
  • Srikulnath K, Uno Y, Nishida C, Ota H, Matsuda Y (2015) Karyotype reorganization in the Hokou Gecko (Gekko hokouensis, Gekkonidae): The Process of Microchromosome Disappearance in Gekkota. PLoS ONE 10(8): e0134829. https://doi.org/10.1371/journal.pone.0134829
  • Stuart B (1999) Amphibians and Reptiles. In: Duckworth JW, Salter RE, Khounboline K (Eds) Wildlife in Lao PRD: 1999 Status Report. Vientiane: IUCN – The World Conservation Union – Wildlife Conservation Society Centre for Protected Areas and Watershed Management, 43–67.
  • Sumontha M, Kunya K, Pauwels OSG (2008) Took-kae-pa Jarujin (Cyrtodactylus jarujini) nai thammachat [Jarujin’s Slender-toed Gecko (Cyrtodactylus jarujini) in the wild]. Ecological Notes 2(1): 22–23. [In Thai]
  • Thongnetr W, Tanomtong A, Prasopsin S, Maneechot N, Pinthong K, Patawang I (2019a) Cytogenetic 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. https://doi.org/10.13128/cayologia-248
  • Thongnetr W, Tanomtong A, Prasopsin S, Patawang I (2019b) Karyotype of the Sai Yok Bent-toed Gecko, Cyrtodactylus saiyok Panitvong et al., 2014 (Reptilia, Gekkonidae). 50 years Mahasarakham University: Public Devotion is a Virtue of the Learned. Mahasarakham University, Thailand, 655–667. [In Thai]
  • Tautz D, Renz M (1984) Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res Acids Research 12: 4127–4138. https://doi.org/10.1093/nar/12.10.4127
  • Trifonov AV, Giovannotti M, O’Brien PCM, Wallduck M, Lovell F, Rens W, Parise-Maltempi PP, Caputo V, Ferguson-Smith AM (2011) Chromosomal evolution in Gekkonidae. I. Chromosome painting between Gekko and Hemidactylus species reveals phylogenetic relationships within the group. Chromosome Research 19: 843–855. https://doi.org/10.1007/s10577-011-9241-4
  • Trifonov AV, Paoletti A, Barucchi CV, Kalinina T, O’Brien CMP, Ferguson-Smith AM, Giovannotti M (2015) Comparative Chromosome Painting and NOR Distribution Suggest a Complex Hybrid Origin of Triploid Lepidodactylus lugubris (Gekkonidae) PLoS ONE 10(7): e0132380. https://doi.org/10.1371/journal.pone.0132380
  • Turpin R, Lejeune J (1965) Les Chromosomes Humains. Gauther Villars, Paris, 535 pp.
  • Ulber T (1993) Bemerkungen über cyrtodactyline Geckos aus Thailand nebst Beschreibungen von zwei neuen Arten (Reptilia: Gekkonidae). Mitteilungen aus dem Zoologischen Museum in Berlin 69(2): 187–200. https://doi.org/10.1002/mmnz.19930690202