Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(3): 47-52, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2868 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Phimphan, S., Aiumsumang, S., Khoomsab, K., Tangsuwan, I. & Tanomtong, A. (2024). Karyomorphology and microsatellites characterization of Limnonectes gyldenstolpei: first report from Thailand. Caryologia 77(3): 47-52. doi: 10.36253/caryologia-2868 Received: Jul 14, 2024 Accepted: Sep 30, 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. Karyomorphology and microsatellites characterization of Limnonectes gyldenstolpei: first report from Thailand Sumalee Phimphan1,*, Suracheat Aiumsumang1, Kan Khoomsab2, Itsara Tangsuwan3, Alongklod Tanomtong4 1 Biology Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 2 Education Science Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 3 Natural Resources and Environmental Management Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 4 Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding author. E-mail: Sumalee.phi@pcru.ac.th, joodoof@gmail.com Abstract. In the present investigation, karyotype and microsatellites pattern in the chromosome of Gyldenstolpe’s Frog (Limnonectes gyldenstolpei) have been analyzed. The aspect of chromosome numbers, morphology, nucleolus organizer region (NOR) locations and microsatellites pattern [d(CA)15, d(CGG)10, d(GC)15, d(TA)15]. We pro- vided the karyotype and idiogram of this species by conventional staining, Ag-NOR banding and Fluorescence in situ hybridization techniques. For the study, five male and female samples collected from northern Thailand, were used. The metaphase chromo- some preparations were prepared from the bone marrows by the standard protocol. The result shows that L. gyldenstolpei had the diploid chromosome number (2n) was 26 and the fundamental number (NF) were 56 in both males and females. The karyotype is composed of 4 large metacentric, 4 large submetacentric, 2 medium metacentric, 14 small metacentric and 2 small submetacentric chromosomes. The NORs bearing chro- mosome were in close to the telomere region on chromosome pair 1. In addition, the microsatellite d(CGG)10 and (GC)15 hybridization results confirmed the NOR region. The in situ localization pattern of d(CA)15 microsatellites was positive on all telomere chromosome, while microsatellites d(TA)15 have no signal on chromosome. Here we provide a classical and some molecular genetics information for L. gyldenstolpei useful as a species specific marker. Keywords: Limnonectes gyldenstolpei, karyotype, chromosome, microsatellites. INTRODUCTION Limnonectes gyldenstolpei is a species of frog in the Dicroglossidae fam- ily. It has been recorded throughout much of Thailand, northeastern Lao, https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-2868 https://doi.org/10.36253/caryologia-2868 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode mailto:Sumalee.phi@pcru.ac.th mailto:joodoof@gmail.com 48 Sumalee Phimphan et al. southwestern Cambodia, and central Vietnam. It has recently also been recorded from the Phong Nha-Kẻ Bàng National Park in central Vietnam (Luu et al., 2013). The members of the genus Limnonectes have a broad distribution in Asia from eastern and southern China, eastwards to Japan, throughout Indochina and southwards to Malaysia, Indonesia, Philippines, and New Guinea (Frost, 2016). Limnonectes is one of the most diverse groups amphibians with 69 currently spe- cies recognized and 15 of which have been described in the last ten years (Frost, 2016). The gross chromosome numbers of 1,000 amphib- ian species were reported by Kuramoto (1990). But only 837 of the 3,521 anuran species have been analyzed chro- mosomally (King, 1991). The amphibian fauna in Thai- land comprises of 176 species in 8 families and 3 orders (Khonsue and Thirakhupt, 2001). In the genus Limnon- ectes reported 11 species (Niyomwan, et al., 2019). The list shows chromosome number variation occurs in most of the seven families of anuran amphibians classified with 33 genera. The typical karyotype of the family Dicroglossidae is diploid chromosome number (2n)=22, 24 and 26. For the genus Limnonectes, there were some cytogenetic stud- ies reported the diploid number was 2n=22-26, NF=44-52, including L. kuhlii and L. blytthii (Supaprom, 2003), L. pileatus (Supaprom, 2003; Supaprom and Baimai, 2004), L. gruniens and L. modestus (Nasaruddin, 2009), L. blyt- thii (Donsakul and Rangsiruji, 2005; Phimphan et al., 2020) and L. taylori (Phimphan and Aiumsumang, 2019). All previous knowledge demonstrated that there are sev- eral patterns of chromosomes (number, type, size). This is the first report describing the molecular cytogenetic and karyotype study of chromosome size, standardized idiogram, karyotype formula and meiotic cell division of the L. gyldenstolpei species. The molecu- lar data, microsatellite probes are used to detect if there is some specific hybridization pattern in L. gyldenstol- pei has not been studied yet. The results obtain can be fulfilled to the basic knowledge. In addition, our knowl- edge advances cytogenetic information for further study on taxonomy relationship. Moreover, we provide useful basic information for the conservation and chromosome evolution study of this frog. MATHERIAL AND METHODS Field surveys were conducted in rainy season from northern (16.42°N 101.16°E), Thailand. Five males and five females of L. gyldenstolpei were mature obtained dur- ing. The frogs were transferred to the laboratory and were kept under standard conditions for 3 days before the experimentation. Experiments were performed in accord- ance with ethical protocols (Ref No. U1-04498-2559). The chromosomes were prepared in vivo with slight adapta- tions as follows (Sangpakdee et al., 2016). The colchicine was injected into the frogs’ abdominal cavity. Then, the frogs were left in a box for eight hours and then killed. The bone marrow was collected by cutting the head and the end of femurs and tibias, and then a syringe was used to inject 0.075 M KCl into the marrow to drive out the bone marrow tissue or cells into the plate. We gently cut the tissue to pieces as small as possible. We transferred 8 mL of cell sediments to a centrifuge tube and incu- bated it for 30 min at 37 °C. After centrifugation at 1500 rpm for 8 min, the KCl was discarded. Cells were fixed in fresh cool fixative up to 8 mL by gradually adding it before being centrifuged again at 1500 rpm for 8 min. The fixation was repeated until the supernatant was clear, usually three times. Finally, the pellet was mixed with 1 mL fixative (depending on the amount of cell). The mix- ture was dropped onto a clean and cold slide by a micro- pipette, and then the air-dry technique was applied. Conventional staining was done using 10% Giemsa’s solution for 10 min (Phimphan and Aiumsumang, 2019). Ag-NOR banding was performed (Howell and Black, 1980) by applying two drops of 2% gelatin on the slides, fol- lowed with four drops of 50% silver nitrate. The slides were then covered with a cover slip and incubated at 60°C for 5 min or until the slide changed brownish. After that the slides were dipped in distilled water to remove the cover glass and air-dried on the slide. The microsatellites (CA)15, (CGG)10, (GC)15, and (TA)15 were synthesized according to (Kubat etal., 2008; Supiwong et al., 2014). These sequenc- es were directly labeled with Cy3 at the 5’terminus during synthesis by Sigma (St. Louis, MO, USA). Chromosome counting was performed on mitot- ic metaphase cells under a light microscope. Twenty clearly observable and well-spread chromosomes of each male and female were selected and photographed. The length of the short arm chromosome (Ls) and the length of the long arm chromosome (Ll) were measured, and the length of the total arm chromosome (LT, LT = Ls+Ll) calculated. The relative length (RL), the centro- meric index (CI) and standard deviation (SD) of RL and CI were estimated. The CI (q/p+q) between 0.50–0.59, 0.60–0.69, 0.70–0.89 and 0.90–0.99 were described as metacentric, submetacentric, acrocentric and telocentric chromosomes, respectively (Levan, 1964). The funda- mental number (number of chromosome arm, NF) was obtained by assigning a value of two to metacentric, sub- metacentric and acrocentric chromosomes and one to telocentric chromosome. All parameters were used in karyotyping and idiograming. 49Karyomorphology and microsatellites characterization of Limnonectes gyldenstolpei: first report from Thailand RESULTS AND DISCUSSION The results showed L. gyldenstolpei had diploid chro- mosome number of 2n=26 and fundamental number (NF)=52, the karyotype comprised four large metacen- tric, four large submetacentric, two medium metacen- tric, 14 small metacentric and two small submetacentric chromosomes. The karyotype formula of L. gyldenstol- pei is 2n(26)= Lm 4+Lsm 4+Mm 2+Sm 14+Ssm 2 in both males and female, while sex chromosomes were cytologically indistinguishable (Fig. 1A). The average lengths of each chromosome including short and long arm length, total length, relative length, and centromeric index were cal- culated and presented in Table 1. The previous relevant literatures have been reported that the numbers of dip- loid chromosome and fundamental number in Lim- nonectes studied herein are 2n=22-26 and NF=44-52 including, L. kuhlii and L. blytthii (Supaprom, 2003), L. pileatus (Supaprom, 2003; Supaprom and Baimai, 2004), L. gruniens and L. modestus (Nasaruddin, 2009), L. blytthii (Donsakul and Rangsiruji, 2005; Phimphan et al., 2020) and L. taylori (Phimphan and Aiumsumang, 2019). (Table 2). Comparison to closely related species, L. gyldenstolpei had diploid chromosome number simi- lar to L. gruniens and L. modestus (2n=24), but is higher than that in L. taylori (2n=22) and lower than L. kuhlii and L. pileatus (2n=24). This result was the first report on L. gyldenstolpei. These characteristics are consistent with the theory that reorganization from the original karyotype resulted from Robertsonian fissions, fusions, or pericentric inversions (Gorman 1973; King 1978). Our results confirmed 2n for L. gyldenstolpei species but with differences in the diploid chromosome number. This incongruence reflects probably the number of the Lim- nonectes chromosomes, especially those of polyploids. After Ag-NOR staining, these regions produce numerous gene expressions and contain more non-his- tone protein than others regions on the chromosome. Accordingly, the dark band (NOR-positive) is induced by the reduction of organic silver by these proteins that change from silver to dark (Sharma et al., 2002). If these regions were active during the interphase prior to mito- sis, they can be detected by silver nitrate staining (How- ell and Black 1980). The NOR could be detected to near telomeric region on long arm chromosome pairs 1 (Fig. 1B). We found one pair of Ag-NOR sites in all of the samples examined. However, the results were similar to the previous report on L. kuhlii and L. blytthii (Supa- prom, 2003), L. pileatus (Supaprom, 2003; Supaprom and Baimai, 2004), L. gruniens and L. modestus (Nasaruddin, 2009), L. blytthii (Donsakul and Rangsiruji, 2005; Phim- phan et al., 2020) and L. taylori (Phimphan and Aium- sumang, 2019). The most striking variation is seen in the morphology of the secondary constrictions. Gener- ally, one major nucleolar organizer region is present per genome (n), which may vary in its position between spe- cies. However, closely related and often morphologically very similar species share the same type and location of their nucleolar organizer regions, which can therefore provide an effective taxonomic. Here the first molecular cytogenetic study in meta- phase chromosomes stained by FISH. The in situ hybrid- ized localization of microsatellites d(CA)15, d(CGG)10, d(GC)15, and d(TA)15. Microsatellites, also known as simple sequence repeats, consist of very short motifs (1-6 nucleotides in length) repeated in tandem arrays. Gen- erally, they are located in the heterochromatic regions (telomeres, centromeres and in the sex chromosomes) of genomes, where a significant fraction of repetitive DNA is expected to be localized (Supiwong et al., 2013). The Figure 1. Metaphase chromosome plates and karyotypes of Limnon- ectes gyldenstolpei, 2n=26 by conventional staining [A.], Ag-NOR banding [B.], d(CA)15 [C.], d(CGG)10 [D.] d(GC)15 [E.] and d(TA)15 microsatellite probe [F.]. Note scale bars indicate 10 micrometers. 50 Sumalee Phimphan et al. result of L. gyldenstolpei analyzed was being abundantly distributed on all telomere chromosomes such as, the accumulation of (CA)15 in long arm chromosomal pair 1 (Fig 1C), while (CGG)10 and (GC)15 detected subtelomeric region on long arm chromosomal pair 1 (Fig. 1D, 1E) and (TA)15 sequences are not present in the all chromo- some (Fig. 1F). However, an intriguing feature exclusive for L. gyldenstolpei was the strong accumulation of all microsatellites at the regions of specific chromosomal pair, indicating that these microsatellites may be used as chromosomal markers in this frog species. In the frog genomes, microsatellites are usually abundant in the telomeric and centromeric regions, Otherwise, the dinu- cleotides (CA)15, (GC)15 and (CGG)10 accumulated exclu- sively in telomeric and subcentromeric chromosomal regions, corroborating findings from other frog groups studied to date (Phimphan, et al. 2021a; 2021b). These molecular cytogenetics data could also be a substantial prerequisite for future frog genome projects. This study discovered that the cytogenetic maps of L. gyldenstolpei allowed us to map out the steps involved in this species’ chromosomal rearrangement. This is the first report on the Fluorescence in situ hybridization (FISH) study of this species in Thailand. The present study on the meiotic cell division of L. gyldenstolpei found that during interphase, nucleolus 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 standart deviation (SD) from 20 metaphases of male and female Limnonects gyldenstolpei, 2n (dip- loid)=26. Chromosome pairs Ls Ll LT CI±SD RL±SD Chromosome size Chromosome type 1* 6.162 7.628 13.790 0.552±0.012 0.161±0.005 Large metacentric 2 4.166 6.849 11.015 0.622±0.014 0.128±0.004 Large submetacentric 3 3.469 5.991 9.460 0.633±0.019 0.110±0.004 Large submetacentric 4 3.786 5.168 8.954 0.580±0.022 0.105±0.002 Large metacentric 5 3.700 4.503 8.203 0.550±0.018 0.096±0.003 Medium metacentric 6 2.179 2.966 5.145 0.577±0.014 0.060±0.002 Small metacentric 7 1.866 2.892 4.758 0.606±0.032 0.056±0.002 Small submetacentric 8 1.876 2.799 4.675 0.597±0.037 0.054±0.002 Small metacentric 9 1.766 2.590 4.356 0.595±0.017 0.051±0.002 Small metacentric 10 1.799 2.475 4.274 0.577±0.021 0.050±0.002 Small metacentric 11 1.609 2.387 3.996 0.596±0.015 0.047±0.001 Small metacentric 12 1.505 2.260 3.765 0.598±0.018 0.044±0.001 Small metacentric 13 1.486 1.967 3.452 0.572±0.024 0.040±0.002 Small metacentric * NORs bearing chromosomes (satellite chromosome). Table 2. Review of cytogenetic publications of family Dicroglossidae (genus Limnonectes). Species 2n Karyotype formula NF NORs FISH Reference L. gruniens 24 24m 48 - - Nasaruddin et al. 2009 L. modestus 24 20m+4t 44 - - Nasaruddin et al. 2009 L. kuhlii 26 8m+14sm 52 2 - Supaprom 2003 L. pileatus 26 16m+10sm 52 2 - Supaprom 2003 26 16m+10sm 52 2 - Supaprom and Baimai 2004 L. taylori 22 16m+6sm 44 2 - Phimphan and Aiumsumang 2019 L. blythii 24 10m+12sm+2a 48 - - Donsakul and Rangsiruji 2005 24 20m+4sm 48 2 + Phimphan et al. 2021a L. gyldenstolpei 26 20m+6sm 52 2 + Present study 2n diploid chromosome number, NF=fundamental number (number of chromosome arms), m metacentric, sm submetacentric, a acrocen- tric, t telocentric chromosome, NORs Ag-NOR banding, FISH Fluorescence in situ hybridization, + positive and - not available. 51Karyomorphology and microsatellites characterization of Limnonectes gyldenstolpei: first report from Thailand could be clearly seen, while chromatins were absent. In prophase, metaphase I (meiosis I) the homologous chromosomes showed synapsis, which can be defined as the 13 bivalent and 13 haploid chromosomes at metaphase II as diploid species. It is confirmed for this species had 2n=26 in similar to previous reports. The largest metacentric chromosome pair 1 is the largest bivalent. We found that L. gyldenstolpei had the dis- tinct character of the observable leptotene (initiation of chromosome shrinking), pachytene (completion of chromosome synapsis) and diakinesis (terminalization) according to Patawang (Patawang et al., 2013) (Fig. 2). In conclusion, this study provides the first chromo- some, molecular cytological details and Ag-NOR mark- er for L. gyldenstolpei from Thailand. The results sup- port the karyotype of genus Limnonectes are conserved among several other species. However, the chromo- somal morphology may be slightly different depending on populations of L. gyldenstolpei present in different countries. 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