Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(3): 3-10, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2961 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Supanuam, P., Jantarat, S., Kraiprom, T., Buathip, S., Jumrustha- nasan, S., Kaewsri, S., Donbundit, N., Buasriyot, P., Thongnetr, W., Phim- phan, S., & Tanomtong, A. (2024). The genome of the southern short-horned tree dragon Acanthosaura merid- iona Trivalairat, Sumontha, Kunya & Chaingkul, 2022 (Squamata, Draconi- nae) was analyzed using classical and molecular techniques to identify and study its chromosomal and repetitive elements. Caryologia 77(3): 3-10. doi: 10.36253/caryologia-2961 Received: Sep 12, 2024 Accepted: Nov 1, 2024 Published: March 25, 2025 © 2024 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. 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. ORCID PS: 0000-0002-2979-9245 SJ: 0009-0006-0376-0808 TK: 0009-0004-7741-7151 PB: 0000-0003-0821-7629 WT: 0009-0000-2598-3144 SP: 0000-0002-4781-1009 AT: 0009-0007-3424-2971 The genome of the southern short-horned tree dragon Acanthosaura meridiona Trivalairat, Sumontha, Kunya & Chaingkul, 2022 (Squamata, Draconinae) was analyzed using classical and molecular techniques to identify and study its chromosomal and repetitive elements Praween Supanuama, Sittisak Jantaratb,*, Thaintip Kraipromb, Somsak Buathipb, Sarun Jumrusthanasanc, Sarawut Kaewsric, Nattasuda Don- bunditd, Phichaya Buasriyote, Weera Thongnetrf, Sumalee Phimphang, Alongklod Tanomtongd aBiology Program, Faculty of Science, Ubon Ratchathani Rajabhat University, Ubon Rat- chathani, Thailand; bProgram of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani Campus, Thailand; cBiology Program, Faculty of Science, Buriram Rajabhat University, Buriram, Thailand; dDepartment of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand; eDepartment of Health Sciences, Faculty of Science and Technology, Rajamangala University of Technol- ogy Suvarnabhumi, Nonthaburi, Thailand; fDivision of Biology, Department of Science, Faculty of Science and Technology, Rajamangala University of Technology Krungthep, Bangkok, Thailand; gBiology Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun, Thailand. *Corresponding author: sitthisak.j@psu.ac.th Abstract. The cytogenetics of the southern short-horned tree dragon (Acanthosaura meridiona) are not reported yet. This study describes the karyotype of Acanthosaura meridiona Trivalairat, Sumontha, Kunya & Chaingkul, 2022 from southern Thailand. We using Giemsa staining, Ag-NOR banding, and fluorescence in situ hybridization (FISH) techniques using microsatellites d(CA)15, d(TA)15, d(CGG)10, and d(CAA)10 probes to analyze the chromosome. The karyotype of the A. meridiona is 2n = 34 chromosomes (fundamental number of 46), of which 5 pairs were large metacentric chromosomes, 2 pairs small metacentric chromosomes, and 20 microchromosomes (chromosome formula: 2n=34=Lm 10+ Sm 4+20mi). There are no sex differences in karyo- types between males and females. The NORs loci were on pair 5 of the large metacen- tric macrochromosomes. The FISH technique showed d(CA)15 and d(CGG)10 repeats on specific regions microchromosomes, while signals of d(TA)15 and d(CAA)10 repeats interspersed on macro- and microchromosomes. This study is significant for enhances our comprehension of the evolutionary mechanism of agamid lizards and promotes the conservation of biodiversity in tropical rainforests. Keywords: Acanthosaura meridiona, chromosome marker, fluorescence in situ hybrid- ization (FISH), microsatellite pattern, Draconinae. https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-2961 https://doi.org/10.36253/caryologia-2961 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0002-2979-9245 https://orcid.org/0009-0006-0376-0808 https://orcid.org/0009-0004-7741-7151 https://orcid.org/0000-0003-0821-7629 https://orcid.org/0009-0000-2598-3144 https://orcid.org/0000-0002-4781-1009 https://orcid.org/0009-0007-3424-2971 mailto:sitthisak.j@psu.ac.th 4 Praween Supanuam et al. INTRODUCTION The agamid lizards belonging to the genus Acan- thosaura Gray, 1831, possess spinose scales with spines on heads and above eyes, along with a prominent spiky crest down their spine (Grismer 2011). All of these spe- cies are active during the day and live in trees of South- east Asia’s forested areas, including Myanmar, Thailand, Cambodia, Laos, Vietnam, Yunnan, the Indochinese and Thai-Malay Peninsula, Sumatra, and the Anambas and Natunus Archipelagos (Ananjeva et al. 2008; Manthey 2008; Grismer 2011). The genus Acanthosaura currently contains 20 species (Ananjeva et al. 2020; Liu et al. 2020; Trivalairat et al. 2022; Liu et al. 2022; Uetz and Haller- mann. 2024). Currently, there are seven species of Acanthosaura in Thailand, namely, A. armata, A. aurantiacrista, A. car- damomensis, A. crucigera, A. lepidogaster, A. meridiona, and A. phuketensis (Uetz and Hallermann. 2024). Acan- thosaura meridiona is present in Trang Province, Krabi Province, Nakhon Si Tammarat Province, Songkhla Province, Surat Thani Province, Satun Province, Thai- land. Acanthosaura meridiona is similar to A. crucig- era and was previously regarded as an identical species. Wood et al. (2010) found that the southern population of A. crucigera exhibited different characteristics that were not present in the true A. crucigera population from western Thailand. Nevertheless, A. cf. crucigera from the southern population has undergone separation into A. meridiona (Trivalairat et al. 2022), with the Phuket mountain range acts as a barrier separating the two spe- cies. Acanthosaura is possible that the extent of vari- ety within this genus is still underestimated. Therefore, cytogenetic research on agamid lizards must achieve greater precision in species differentiation. Information about karyotypes in Acanthosaura only concerns one report in A. armata with conventional tech- nique. The karyotypes of Draconinae vary from 2n=32 to 2n=46, with both macrochromosomes and microchromo- somes, and absence of sex chromosomes (Ota and Hikida 1989; Sharma and Nakhasi 1980; Li et al. 1981; Ota 1988; Solleder and Schmid 1988; Kritpetcharat et al. 1999; Diong et al. 2000; Ota et al. 2002; Singh and Banerjee 2004; Zongyun et al. 2004; Patawang et al. 2015). This study looks at the cytogenetic points of view that constitute useful tools of genetic sex chromosome systems, different evolutionary lineages and to delineate evolutionary trends in a great number of taxa (Mezzasal- ma et al. 2021 and Mezzasalma et al. 2024). This paper first describes Acanthosaura meridiona’s chromosomal features, using conventional staining, Ag-NOR banding, and fluorescence in situ hybridization techniques. MATERIALS AND METHODS Five adult male and five female specimens of barred gliding lizard (Acanthosaura meridiona) were collected from Ban Wang Sai, Mae Wat subdistrict, Than To Dis- trict, in Yala Province, Thailand. The agamid lizards were transferred to the laboratory and identified according to the morphological criteria (Chan-Ard et al. 2015; Das 2015). Experiments were performed in accordance with ethical protocols, as approved by the Ethics Committee of Prince of Songkla, Pattani Campus (Ref.AI001/2024). Chromosomes were directly prepared in vivo (Pata- wang et al. 2018) as follows. Metaphasic and meiotic chromosomes were obtained from bone marrow and tes- tis, according the colchicine-hypotonic-fixation-air dry- ing technique (provide references). The chromosomes were stained with 20% Giemsa’s for 30 minutes, Ag- NOR staining was conducted according to Howell and Black (1980). Chromosomal checks were performed on mitotic metaphase cells under light microscope. FISH experiments were performed with microsatel- lite sequences, specifically (TA)15, (CA)15, (CAA)10, and (CGG)10 using high stringency conditions (Yano et al. 2017). The sequences were directly labeled by Cy3 at the 5’end (Sigma, St. Louis, MO, USA) as described by Kubat et al. (2008). FISH was performed under stringent conditions and hybridization in a moist chamber at 37 °C overnight (Sassi et al. 2023). Chromosomes were counterstained with 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1.2 μg/ml) mounted in antifade solution (Vector, Burlingame, CA, USA,) (Aiumsumang et al. 2021; Patawang et al. 2022; Prasopsin et al. 2022; Thongnetr et al. 2022a). At least 20 metaphase spreads per individual were analyzed to confirm the diploid number, karyotype structure, NORs and FISH data. Chromosomes were classified according to centromere position as metacen- tric (m), submetacentric (sm), acrocentric (a), and telo- centric (t) (Turpin and Lejeune 1965). For the chromo- somal arm number (NF; fundamental number); m, sm and a were scored as bi-armed while t as mono-armed. The microchromosomes are chromosomes that are 5 times less long than the largest pair of chromosomes (Patawang et al. 2016; 2017; 2018). RESULTS AND DISCUSSION Mitotic chromosome features from Giemsa staining Agamidae includes 585 species, of which 94 have been karyologically investigated (Mezzasalma et al. 2024). Karyotypes are discontinuous with a variable chromosome number of macro- and/or micro-chromo- 5Genome Acanthosaura meridiona analyzed using classical and molecular techniques somes, namely macrochromosomes ranging from 10 to 28, and microchromosomes from 0 to 24 (Mezzasalma et al. 2024). In Draconinae, based on 21 species reports, karyotypes range from 2n=32- to 46 (Table 2). Howev- er, so far the present study first reports the karyotype of Acanthosaura meridiona, loci of NORs, and by Fluo- rescence in situ hybridization the distribution of (TA)15, (CA)15, (CAA)10, and (CGG)10 microsatellites. The results revealed that the chromosome number of A. meridiona was 34 (14 macrochromosomes, and 20 microchromosomes). The karyotype comprised ten large metacentric chromosomes, four small metacentric chromosomes, and 20 microchromosomes (Table 1 and Figure 1). This result differs with from that of A. arma- tus of 2n=32 with 12 metacentric macrochromosomes, 20 microchromosomes. The fundamental number (NF) of A. meridiona and A. armatus was 46 and 44, respec- tively. It is possible that the different macrochromosome numbers may have been caused by an event of tandem fusion and centromere deletion involving the chromo- some number and NF variation. A similar process of autonomous reduction in total chromosomal number through autosome translocation has been reported in other lizard families, including Anguidae, Scincidae, Iguanidae, Gekkonidae, and Phrynosomatidae (Adegoke and Ejere 1991; Trifonov et al. 2015).  There is no evidence of differentiated sex chromo- somes in this species which agreeable with all species of Draconinae (Ota and Hikida 1989; Sharma and Nakha- si 1980; Li et al. 1981; Ota 1988; Solleder and Schmid 1988; Kritpetcharat et al. 1999; Diong et al. 2000; Ota et al. 2002; Singh and Banerjee 2004; Zongyun et al. 2004; Patawang et al. 2015). Squamates exhibit a con- siderable degree of variability in their chromosome sex determination systems. Various families exhibit diverse sex-chromosome systems, which can be either simple or multiple, and include either male (XX/XY) or female (ZZ/ZW) heterogamety. These systems encompass all hypothesized stages of heterogametic sex chromosomes, including homomorphic and pseudo-autosomal to heter- omorphic and completely heterochromatic chromosomes (Alam et al. 2018 and Mezzasalma et al. 2021). Nucleolar organizer region from Ag-NOR banding Ag-NOR banding, a species-specific marker, primar- ily identifies karyotypes. Silver staining, on the other hand, only detects the nucleolar organizer areas that are actively involved in transcription (Silva et al. 2008). The improvement of the Ag-NOR staining method has been very important in comparing NOR variation because it Table 1. 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 20 metaphases of male and female of the southern short-horned tree dragon (Acanthosaura meridiona) 2n=34. Chro. Pair Ls (µm) Ll (µm) LT (µm) RL±SD. CI±SD. Chro. Size Type 1 2.022 2.772 4.794 0.165±0.014 0.575±0.029 Large metacentric 2 1.634 1.736 3.371 0.117±0.006 0.517±0.021 Large metacentric 3 1.411 1.794 3.205 0.110±0.007 0.556±0.031 Large metacentric 4 1.371 1.684 3.055 0.106±0.005 0.551±0.026 Large metacentric 5* 1.270 1.509 2.779 0.096±0.004 0.543±0.025 Large metacentric 6 1.059 1.136 2.195 0.076±0.003 0.516±0.021 Small metacentric 7 0.982 1.082 2.064 0.071±0.004 0.524±0.022 Small metacentric 8 0.000 0.813 0.813 0.029±0.002 1.000±0.000 microchromosome 9 0.000 0.762 0.762 0.027±0.003 1.000±0.000 microchromosome 10 0.000 0.761 0.761 0.027±0.005 1.000±0.000 microchromosome 11 0.000 0.762 0.762 0.027±0.003 1.000±0.000 microchromosome 12 0.000 0.699 0.699 0.024±0.003 1.000±0.000 microchromosome 13 0.000 0.679 0.679 0.024±0.003 1.000±0.000 microchromosome 14 0.000 0.632 0.632 0.022±0.002 1.000±0.000 microchromosome 15 0.000 0.573 0.573 0.020±0.003 1.000±0.000 microchromosome 16 0.000 0.540 0.540 0.019±0.003 1.000±0.000 microchromosome 17 0.000 0.469 0.469 0.016±0.003 1.000±0.000 microchromosome * = NORs bearing chromosomes, Chro. = Chromosome. 6 Praween Supanuam et al. lets us find the metaphase chromosomal locations that are linked to NOR. The present study, the chromosome markers of A. meridiona observable NORs on the telo- meric region of large metacentric macrochromosome pair 5th (Figure 2). Similarly, the previous report of NOR position in Draconinae was located on telomeric region of q-arm in 4 species of Calotes consisting of C. cristate- llus, C. emma, C. mystaceus, and C. versicolor (Solleder and Schmid 1988; Patawang et al. 2015). However, find- ings from both traditional and molecular cytogenetics suggest that the location of NOR loci on microchromo- somes is usually thought of as an ancestral trait in most families and genera (Mezzasalma et al. 2021; Waters et al. 2021; Deakin and Ezaz 2019). Microsatellite pattern Microsatellites, also known as simple sequence repeats (SSRs), are short DNA sequences consisting of Table 2. Comparative chromosome studies of subfamily Draconinae. Species 2n Karyotype NOR Locality References Acanthosaura armata 32 12m+20mi - Malaysia Ota et al. (2002) A. meridiona 34 14m+20mi 5qter Thailand This study Bronchocela cristatella 34 14m+20mi - Singapore Ota et al. (2002) 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) Calotes emma alticristatus 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) 34 12m/sm+22mi - Thailand Kritpetcharat et al. (1999) 34 12m+22mi - Malaysia Ota et al. (2002) 34 - - India Singh and Banerjee (2004) C. jerdoni 34 12m/sm+22mi - India Sharma and Nakhasi (1980) 34 - - India Singh and Banerjee (2004) C. mystaceus 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) 34 12m/sm+22mi - Thailand Kritpetcharat et al. (1999) 34 - - India Singh and Banerjee (2004) 34 10m+2m+22mi 2qter Thailand Patawang et al. (2015) C. versicolor 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) 34 12m/sm+22mi - Thailand Kritpetcharat et al. (1999) 34 12m+22mi Singapore Ota et al. (2002) 34 12m/sm+22mi 2qter Thailand Patawang et al. (2015) C. vultuosus 32, 34 - - India Singh and Banerjee (2004) 34 12m/sm+22mi - India Sharma and Nakhasi (1980) Draco cornutus 34 16m+18mi - D. haematopogon 34 16m+18mi - Malaysia Ota and Hikida (1989) D. quinquefasciatus 34 16m+18mi - Diploderma splendidum 34 12m+22mi - China Zongyun et al. (2004) D. swinhonis 36 40 46 10bi+26a 6bi+34a 46a - - - Central Taiwan Central Taiwan Northern Taiwan Ota (1988) Gonocephalus chamaeleontinus 42 22m+20mi - G. liogaster 42 22m+20mi - G. bellii 42 22m+20mi - Malaysia Diong et al. (2000) G. grandis 42 22m+20mi - G. robinsonii 32 12m+20mi - Japalura variegata 34 - - India Singh and Banerjee (2004) J. varcoae 34 12m+22mi - China Li et al. (1981) Ptyctolaemus gularis 34 12m/sm+22mi - India Sharma and Nakhasi (1980) Note: 2n: diploid chromosome number, m: metracentric, sm: submetracentric, a: acrocentric, bi: biarms, mi: microchromosome, and qter: long arm of chromosome. 7Genome Acanthosaura meridiona analyzed using classical and molecular techniques 1–6 base pairs. They are distinguished by the presence of repetitive units, which can range from 4 to 40 repeats in a sequence (Tautz and Renz 1984; Ellegren 2004; Chistiakov et al. 2006). They appear either dispersed or clustered in euchromatin and heterochromatin regions, widely distributed across eukaryotic genomes. They show a significant variation in the number of copies of genet- ic material (Ellegren 2004). Microsatellite repeat pat- terns of A. meridiona indicated the presence of specific regions on microchromosomes, including pair 13 and 15 with d(CA)15 repeats, and pair 15 and 16 with d(CGG)10 repeats. While d(TA)15 and d(CAA)10, showed cumu- lative signals dispersed throughout the chromosomes (Table 3, Figure 3). The microsatellite loci exhibited a significant level of evolutionary advancement. Thus, it is common for many species to have diverse patterns of repeated sequences. Most of them and scattered them A . B . C . Figure 1. Metaphase plates and standardized karyotypes of male (A.), female (B.) and Idiogram (C.) of the southern short-horned tree dragon, Acanthosaura meridiona, 2n=34 by conventional staining. A . B . C. Figure 2. Metaphase plates and standardized karyotypes of male (A.), female (B.) and Idiogram (C.) of the southern short-horned tree dragon, Acanthosaura meridiona, 2n=34 by Ag-NOR banding, arrows indicate NORs. A. D. C. B. Figure 3. Metaphase plates and hybridization patterns with micro- satellite probes d(CA)15 (A.), d(CGG)10 (B.), d(TA)15 (C.), and d(CAA)10 (D.) (red signals) on metaphase plates of the southern short-horned tree dragon, Acanthosaura meridiona, 2n=34, chro- mosomes were counterstained with DAPI (blue). Table 3. The hybridization patterns with microsatellite probes d(CA)15, d(CGG)10, d(TA)15, and d(CAA)10 of the southern short- horned tree dragon (Acanthosaura meridiona). Probe Signal d(CA)15 Pair 13 & 15 d(CGG)10 Pair 15 & 16 d(TA)15 Throughout genome (weak) d(CAA)10 Throughout genome (strong) 8 Praween Supanuam et al. across the genome (Thongnetr et al. 2019; 2022a; 2022b; Khawporntip et al. 2024). However, in certain species, they may localize into specific regions (Srikulnath et al. 2009; Alam et al. 2021). Interestingly, signals of the d(CA)15 probe specifically is on a chromosome of pair 13, a finding that is unclear and remains unexplained. This suggests that using Fluorescence in situ Hybridisation (FISH), the process of mapping cDNA or BAC clones to the chromosomes of southern short-horned tree dragon has successfully addressed certain constraints (O’Meally et al. 2009; Alföldi et al. 2011; Srikulnath et al. 2015; Young et al. 2013; Deakin et al. 2016; Badenhorst et al. 2015). By integrating data from several species, one can obtain intra-sequence information that enhances our understanding of the evolution of chromosome in lizards. In conclusion, we first present the karyotype, NORs and microsatellite d(CA)15, d(TA)15, d(CGG)10, and d(CAA)10 patterns on the chromosomes of the southern short-horned tree dragon. Acanthosaura meridiona has 2n=34 chromosomes (14 macrochromosomes, and 20 microchromosomes), NF=46. The karyotype consisting of 5 pairs of large metacentric chromosomes, 2 pairs of large metacentric chromosomes, and 20 microchromo- somes. NORs were located on the telomeric region of large metacentric macrochromosome pair 5th. Micros- atellite repeat patterns indicated the presence of specific regions on microchromosomes, including pair 13 and 15 with d(CA)15 repeats, and pair 15 and 16 with d(CGG)10 repeats. While d(TA)15 and d(CAA)10, showed cumula- tive signals dispersed throughout the chromosomes. This study is valuable for improving our understanding of the evolutionary process of agamid lizards and advo- cating for biodiversity protection in tropical rainforests. Moreover, we suggest that more species be studied using cytogenetics and that techniques be investigated further to gain a deeper understanding of chromosomal diver- sity and evolution within this genus. ACKONOWLEDGEMENT This research was supported by National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (Ref. No. SAT6701179S). REFERENCES Adegoke JA, Ejere VC. 1991. 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