Cytogenetics of the Skinks (Reptilia, Scincidae) from Thailand; IV: Newly Investigated Karyotypic Features of Lygosoma Quadrupes and Scincella Melanosticta

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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics

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Cytogenetics of the skinks (Reptilia, Scincidae) from Thailand; IV: newly investigated karyotypic features of Lygosoma quadrupes and Scincella melanosticta

Isara Patawang, Yodchaiy Chuaynkern, Praween Supanuam, Nuntiya Maneechot, Krit Pinthong & Alongklod Tanomtong

To cite this article: Isara Patawang, Yodchaiy Chuaynkern, Praween Supanuam, Nuntiya Maneechot, Krit Pinthong & Alongklod Tanomtong (2017): Cytogenetics of the skinks (Reptilia, Scincidae) from Thailand; IV: newly investigated karyotypic features of Lygosoma quadrupes and Scincella melanosticta, Caryologia, DOI: 10.1080/00087114.2017.1402249 To link to this article: https://doi.org/10.1080/00087114.2017.1402249

Published online: 18 Dec 2017.

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Download by: [Chiang Mai University] Date: 18 December 2017, At: 22:17 Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 2017 https://doi.org/10.1080/00087114.2017.1402249

Cytogenetics of the skinks (Reptilia, Scincidae) from Thailand; IV: newly investigated karyotypic features of Lygosoma quadrupes and Scincella melanosticta

Isara Patawanga,b, Yodchaiy Chuaynkernc , Praween Supanuamd, Nuntiya Maneechote, Krit Pinthonge and Alongklod Tanomtongc,f

aDepartment of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand; bCenter of Excellence in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Chiang Mai, Thailand; cDepartment of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand; dProgram of Biology, Faculty of Science, Ubon Ratchathani Rajabhat University, Ubon Ratchathani, Thailand; eDepartment of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Surin, Thailand; fToxic Substances in Livestock and Aquatic Animals Research Group, Khon Kaen University, Khon Kaen, Thailand

ABSTRACT ARTICLE HISTORY This study analyzed the karyotype of Lygosoma quadrupes and Scincella melanosticta from Received 22 August 2017 northeastern Thailand. Mitotic chromosomes were prepared directly from intestinal epithelial Accepted 2 November 2017 cells of three L. quadrupes and nine S. melanosticta. The chromosomes were stained by KEYWORDS conventional staining technique. The karyotype was determined for each species based on Lygosoma quadrupes; at least 20 well-spread cells. Both L. quadrupes and S. melanosticta showed the same diploid Scincella melanosticta; skink; number of 2n = 30, but differed in identical chromosome morphology. No sex chromosome karyotype; chromosome heteromorphisms were evident in L. quadrupes; in contrast, S. melanosticta showed the ZZ/ZW sex determination system. The respective karyotype formulae of L. quadrupes and S. melanosticta were deduced as: n m sm m sm 2 (30)=L6 + L2 + S10 + S2 + 10 microchromosomes, and n m sm m sm t m m 2 (30)=L4 + L2 + S4 + S2 + S2 + Z(L )W(L )+14 microchromosomes

Introduction et al. (2013) reported the karyotypes of three Lygosoma in Thailand: L. bowringii, L. khoratense, and L. quadru- Lygosoma quadrupes and Scincella melanosticta are pes; these showed the same diploid number of 2n = 32 squamate reptiles that belong to the family Scincidae. comprised of 18 macrochromosomes and 14 microchro- The family Scincidae, skinks or scincid lizards, contains mosomes. In contrast, Bhatnagar (1962) reported that L. more than 1200 species that range from temperate to punctata had a diploid number of 24, with a karyotype tropical areas on all continents (Zug et al. 2001). Greer that included 12 bi-armed chromosomes, two mono- (1970), based on external and osteological characteris- armed chromosomes, and 10 microchromosomes. tics, divided the family into four subfamilies: Acontinae, Aranyavalai et al. (2013) found three Lygosoma species Feylininae, Scincinae, and Lygosominae. However, a

Downloaded by [Chiang Mai University] at 22:17 18 December 2017 with the same karyotype, with metacentric macrochro- recent report on skink systematics proposed combining mosome in the first to eighth pairs and telocentric chro- all skinks into seven families (Hedges 2014). Thailand mosome pair 9 of L. quadrupes. In addition, secondary has 54 species of skinks, including 10 Lygosoma and constrictions were evident on the short arms of the first seven Scincella (Chuaynkern and Chuaynkern 2012). pair, the long arms of the second pair, or both in the In Thailand, many skink chromosome studies in many three Lygosoma species. species from various genera have been reported, includ- Chromosomal data have been reported for only three ing: Eutropis (2n = 32, 34, 38), Jarujinia (2n = 30), Lipinia of the 35 currently recognized species (Hosek 2008) of (2n = 28), Lygosoma (2n = 32), and Sphenomorphus (2n the genus Scincella – S. assata (diploid number 28), S. = 30) (Ota et al. 2001; Aranyavalai et al. 2013; Kaewsri lateralis (30), and S. cherriei (30) (Wright 1973; Hedin et et al. 2014; Patawang et al. 2017; Prasopsin et al. 2017). al. 1990; Castiglia et al. 2013). However, Wright (1973) Chromosomal studies have been reported for only and Hedin et al. (1990) reported that male S. lateralis four of the 30 Lygosoma species, i.e. L. bowringii, L. from a population in Edwards Plateau, Texas, USA have khoratense, L. quadrupes and L. punctata. Aranyavalai

a diploid number of 29 and a X1X2Y sex chromosome from Muang District (14°47′05.5ʺN 103°31′09.3ʺE), system. This X1X2Y chromosomal system, seen only in Surin Province, Thailand. They were transferred to the male S. lateralis from Edwards Plateau, was hypothe- laboratory and kept under standard conditions for one sized to have been derived from the more widespread XY day prior to the experiments. male condition of 2n = 30 by the fusion of the ancestral Y chromosome to a metacentric macrochromosome Chromosome preparation and staining (Wright 1973). Chromosomes of the two species were prepared in vivo (Ota and Lue 1994; Patawang et al. 2017) using Materials and methods the following method. The skinks were intraperitone- ally injected with 0.1% (w/v) colchicine solution for 18 Sample collection h. Intestinal samples were cut into small pieces, then The two male and one female L. quadrupes specimens squashed and treated with 0.075 M hypotonic KCl for (Figure 1(a) and 1(b)) used in this study were gathered one hour. Cells were fixed in fresh cool Carnoy’s fixative. from Muang District (16°08′17.0ʺN 103°25′22.7ʺE), The chromosomes of both species were conventionally Maha Sarakham Province, Thailand; the five male and stained using 20% Giemsa’s solution for 30 min (Rooney four female S. melanosticta (Figure 1(c)) were collected 2001).

Downloaded by [Chiang Mai University] at 22:17 18 December 2017 Figure 1. (a, b) Female Lygosoma quadrupes and (c) male Scincella melanosticta.

Table 1. Mean length (L) of the short arm chromosome (Ls), long arm chromosome (Ll), and total arm chromosome (LT), centromeric index (CI), relative length (RL) and standard deviation (SD) of CI, RL from metaphase chromosomes in 20 cells of male and female Lygosoma quadrupes, 2n = 30. Chromosome pairs Ls Ll LT CI ± SD RL ± SD Types Sizes 1 6.100 9.850 15.950 0.618 ± 0.005 0.184 ± 0.007 sm L 2 6.090 7.730 13.820 0.559 ± 0.004 0.159 ± 0.006 m L 3 5.400 5.770 11.170 0.517 ± 0.004 0.129 ± 0.007 m L 4 5.230 5.700 10.930 0.522 ± 0.003 0.126 ± 0.007 m L 5 2.920 3.530 6.450 0.547 ± 0.004 0.074 ± 0.008 m S 6 2.250 2.520 4.770 0.528 ± 0.005 0.055 ± 0.006 m S 7 1.940 2.500 4.440 0.563 ± 0.004 0.051 ± 0.008 m S 8 1.320 2.440 3.760 0.649 ± 0.006 0.043 ± 0.007 sm S 9 1.380 1.630 3.010 0.542 ± 0.003 0.035 ± 0.007 m S 10 1.290 1.590 2.880 0.552 ± 0.003 0.033 ± 0.008 m S 11 ‒ ‒ 2.070 ‒ 0.024 ± 0.005 microchromosome 12 ‒ ‒ 2.060 ‒ 0.024 ± 0.005 microchromosome 13 ‒ ‒ 2.050 ‒ 0.024 ± 0.006 microchromosome 14 ‒ ‒ 1.820 ‒ 0.021 ± 0.004 microchromosome 15 ‒ ‒ 1.600 ‒ 0.018 ± 0.004 microchromosome CARYOLOGIA: INTERNATIONAL JOURNAL OF CYTOLOGY, CYTOSYSTEMATICS AND CYTOGENETICS 3

Results and discussion number was 50 in both sexes and no sex chromosome heteromorphisms were evident (Table 1 and Figure 2(a– Chromosome study of Lygosoma quadrupes c)). This result differed from Aranyavalai et al. (2013), Lygosoma quadrupes had a diploid number of 30. The who reported the karyotypes of population of L. quadru- karyotype comprised six large metacentric, two large pes from Chanthaburi, Bangkok, Suphanburi, Chai Nat, submetacentric, 10 small metacentric, two small subme- and Kanchanaburi, Thailand, all with a diploid number tacentric, and 10 microchromosomes. The fundamental of 32; a karyotype comprised of 16 metacentric, two Downloaded by [Chiang Mai University] at 22:17 18 December 2017

Figure 2. Conventionally stained somatic metaphase complement and karyotypes of (a) male and (b) female Lygosoma quadrupes, 2n = 30, and (c) its idiogram (scale bars = 10 μm). 4 I. PATAWANG ET AL. Downloaded by [Chiang Mai University] at 22:17 18 December 2017

Figure 3. Conventionally stained somatic metaphase complement and karyotypes of (a) male and (b) female Scincella melanosticta, 2n = 30, and (c) its idiogram (scale bars = 10 μm).

telocentric, and 14 microchromosomes; and no evident submetacentric, four small metacentric, two small subme- sex chromosome heteromorphisms. Our results also dif- tacentric, two small telocentric, two sex-chromosomes, fered from another Lygosoma species (Table 2). However, and 14 microchromosomes. ZZ sex chromosomes were ours is the first chromosome study ofL. quadrupes pop- detected in the males; ZW chromosomes in the females. ulations from northeastern Thailand. Both Z and W chromosomes were of the same type, large metacentric; nevertheless, the W chromosome was Chromosome study of Scincella melanosticta smaller than Z chromosome (Table 3 and Figure 3(a–c)). The meiosis features of male S. melanosticta showed S. melanosticta had a diploid number of 30 and a fun- bivalent synapsis between each homologous chromo- damental number of 44 in both sexes. The chromosome during late diakinesis and metaphase I (Figure somal types include four large metacentric, two large 4(d) and 4(e)). Moreover, both macrochromosome and CARYOLOGIA: INTERNATIONAL JOURNAL OF CYTOLOGY, CYTOSYSTEMATICS AND CYTOGENETICS 5

Table 2. Comparative chromosome studies between the genera of Lygosoma and Scincella (Scincidae, Squamata).

Species 2n Karyotype NOR Population Reference Genus Lygosoma L. bowringii 32 16 m+2sm+14mi 1p or 2q C, N, E, W, S, NE of TH Aranyavalai et al. (2013) L. khoratense 32 16 m+2sm+14 mi 1p or 2q Nakhon Ratchasima, TH Aranyavalai et al. (2013) L. quadrupes 32 14m+2sm+2t+14mi 1p or 2q C, E, W of TH Aranyavalai et al. (2013) 30 16 m+4sm+10mi ‒ Maha Sarakham, TH Present study L. punctata 24 12bi+2 mono+10mi ‒ ‒ Bhatnagar (1962) Genus Scincella S. assata 28 12 m+2st+14mi ‒ Chiapas, MX Castiglia et al. (2013) S. cherriei 30(♂) 12m+2st+16mi ‒ Chiapas, MX Castiglia et al. (2013) S. lateralis 30 12mac+16mi+2sec (XX/XY, mac) ‒ SE of USA Wright (1973) 29(♂)/30(♀) 10mac+16mi+3sec (X1X2Y, mac)‒ ‒ Texas, USA Wright (1973) 30(♂) 12mac+16mi+2sec (XY, mac) ‒ Sabine County, Texas, USA Hedin et al. (1990) 29(♂) 10mac+16mi+3sec (X1X2Y, mac) ‒ Edwards Plateau, Texas, USA Hedin et al. (1990) S. melanosticta 30 ‒ Surin, TH Present study Abbreviations: 2n, diploid number; NOR, nucleolar organizer region; m, metacentric; sm, submetacentric; st, subtelocentric; t, telocentric; mac, macrochromosome; mi, microchromosome; bi, bi-armed chromosome; mono, mono-armed chromosome; sec, sex-chromosome; p, short arm; q, long arm; C, central; E, eastern; W, western; N, northern; S, southern; NE, northeastern; SE, southeastern; TH, Thailand; MX, Mexico; ‒, not available. Downloaded by [Chiang Mai University] at 22:17 18 December 2017

Figure 4. Meiotic cell division of the male Scincella melanosticta 2n (diploid)= 30 on (a) leptotene, (b) zygotene, (c) diplotene, (d) late diakinesis, (e) metaphase I, and (f) metaphase II. (Scale bars indicates 10 µm.)

microchromosome presented suitable synapsis of the The diploid number of 30 for Scincella that we found homologous chromosome. ZZ bivalent and Z univalent was similar to previous reports (Wright 1973; Hedin et chromosomes, with the univalent chromosome appear- al. 1990; Castiglia et al. 2013), except S. assata and male ing in metaphase II, also presented suitable synapsis at S. lateralis from Edwards Plateau. Nonetheless, the ZW late diakinesis and metaphase I (Figure 4(d) and 4(e)), sex chromosomes system of S. melanosticta we found

and was the second largest of all macrochromosome differed from the XY or 1X X2Y system of S. lateralis pairs (Figure 4(d–f)). In prophase I, we also found lep- (Table 2). The ZZ/ZW chromosomes (Figure 3(a–c)) totene (Figure 4(a)), zygotene (Figure 4(b)) and diplo- from metaphase and ZZ bivalent synapsis (Figure 4(d) tene (Figure 4(c)) by conventional staining techniques. and 4(e)) from late diakinesis and metaphase I of the 6 I. PATAWANG ET AL.

Table 3. Mean length (L) of the short arm chromosome (Ls), long arm chromosome (Ll), and total arm chromosome (LT), centromeric index (CI), relative length (RL) and standard deviation (SD) of CI, RL from metaphase chromosomes in 20 cells of male and female Scincella melanosticta, 2n = 30. Chromosome pairs Ls Ll LT CI ± SD RL ± SD Types Sizes 1 7.900 12.700 20.600 0.617 ± 0.006 0.161 ± 0.006 sm L 2 6.900 8.000 14.900 0.537 ± 0.005 0.116 ± 0.006 m L 3 5.800 7.000 12.800 0.547 ± 0.006 0.100 ± 0.007 m L 4 4.500 5.400 9.900 0.545 ± 0.005 0.077 ± 0.005 m S 5 3.400 4.600 8.000 0.575 ± 0.005 0.062 ± 0.006 m S 6 2.100 3.700 5.800 0.638 ± 0.006 0.045 ± 0.005 sm S 7 0.000 3.800 3.800 1.000 ± 0.000 0.030 ± 0.006 t S Z 8.000 11.100 19.100 0.581 ± 0.005 0.149 ± 0.005 m L W 5.900 6.500 12.400 0.524 ± 0.004 0.097 ± 0.004 m L 9 – – 3.500 – 0.027 ± 0.004 microchromosome 10 – – 3.200 – 0.025 ± 0.004 microchromosome 11 – – 3.100 – 0.024 ± 0.003 microchromosome 12 – – 3.000 – 0.023 ± 0.004 microchromosome 13 – – 2.800 – 0.022 ± 0.004 microchromosome 14 – – 2.700 – 0.021 ± 0.003 microchromosome 15 – – 2.600 – 0.020 ± 0.004 microchromosome

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