Herpetology Notes, volume 11: 1083-1088 (2018) (published online on 19 December 2018)

New karyotype of Lygosoma bowringii suggests cryptic diversity

Artem Lisachov1,*, Nikolay Poyarkov2, Parinya Pawangkhanant3, Pavel Borodin1,4, and Kornsorn Srikulnath5,6,*

Abstract. In the family Scincidae, karyotypes of most species have diploid numbers of 28–32 chromosomes. They are bimodal, i.e. there are two distinct size classes of chromosomes: microchromosomes and macrochromosomes. Previous karyotypic studies of the common supple skink, Lygosoma bowringii, showed the karyotype comprising 18 macro- and 14 microchromosomes (2n=32), which is common for the family. Here, we described a new derived unimodal karyotype of L. bowringii with 2n=24, observed in individuals from Bangkok (Thailand). A difference between the newly and previously described karyotypes of this species suggests the possibility of cryptic intraspecific chromosomal diversity.

Keywords. bimodal karyotype, macrochromosome, microchromosome, chromosomal rearrangements, synaptonemal complexes

Introduction is termed bimodal (Morescalchi, 1977). In birds, the microchromosomes are generally less than 20 Karyotypes of most species of reptiles are Mb in size (Axelsson et al., 2005). However, they characterized by having two distinct size groups of are an indispensable part of the genome and differ chromosomes, called macro- and microchromosomes. from supernumerary B-chromosomes. By contrast, Microchromosomes are very small “dot-shaped” karyotypes showing few or no microchromosomes are chromosomes whose centromere positions are called unimodal. These are observed in all crocodiles undetectable under conventional light microscopic and several families of squamate reptiles (Olmo and analysis of metaphase plates. This karyotypic structure Signorino, 2005). Interestingly, among squamates, unimodal karyotypes are found in lacertid lizards (Lacertidae) and geckos (Gekkota) but rarely in other groups (Srikulnath et al., 2014, 2015). Compared with mammals, which exhibit higher 1 Institute of Cytology and Genetics, Russian Academy of karyotypic diversity between related species and Sciences, Siberian Branch, Novosibirsk 630090, Russia. even within species (Dobigny et al., 2017), birds and 2 Department of Vertebrate Zoology, Biological faculty, M. V. most non-avian reptiles generally tend to have more Lomonosov Moscow State University, Leninskiye Gory, conserved karyotypes. It is likely that in squamate Moscow 119234, Russia. reptiles karyotypic features are usually conserved 3 Division of Fisheries, School of Agriculture and Natural at least at the family or even suborder level with Resources, University of Phayao, Phayao 56000, Thailand. 4 Novosibirsk State University, Novosibirsk 630090, Russia. relatively small variation (Srikulnath et al., 2009, 5 Laboratory of Animal Cytogenetics and Comparative 2013; Pokorná et al., 2011, 2015). Karyotypes of Genomics (ACCG), Department of Genetics, Faculty most skinks (Scincidae) are bimodal and have diploid of Science, Kasetsart University, 50 Ngamwongwan, numbers of 28–32 chromosomes (Olmo and Signorino, Chatuchak, Bangkok 10900, Thailand. 2005). Comparative chromosome painting for five 6 Center for Advanced Studies in Tropical Natural Resources, species of this family revealed that linkage groups of National Research University-Kasetsart University chromosomes are highly conserved among species, (CASTNAR, NRU-KU), Kasetsart University, Bangkok 10900, Thailand. with several chromosomal rearrangements differing * Corresponding authors. AL: E-mail: [email protected]; Scincinae and Lygosominae, and less variation within KS: E-mail: [email protected] the subfamilies (Caputo et al., 1994; Giovannotti et 1084 Artem Lisachov et al. al., 2009, 2010). The results of chromosome painting followed standard techniques (Moorhead et al., 1960). suggest that the ancestral karyotype of Scincidae is Briefly, the testes were minced and incubated in present in the subfamily Scincinae, namely the genera Eagle’s culture medium (ThermoFischer, Waltham, Scincus Laurenti, 1768 and Eumeces Wiegmann, 1834, Massachusets, USA) with 300 ng/ml colcemid which were studied by Giovannotti et al. (2010). (ThermoFischer, Waltham, Massachusets, USA) for The common supple skink Lygosoma bowringii 2 hours at 30°C. Testes portions were then placed in (Günther, 1864) is distributed throughout Southeast 0.56% KCl for 30 minutes at room temperature. The Asia (Uetz et al., 2018). Previous karyotypic studies cells were collected by centrifugation and then fixed of L. bowringii showed that its karyotype is composed with 3:1 methanol/acetic acid. Cells in suspension of 16 pairs of chromosomes (2n=32), including nine were dropped onto clean wet glass slides and air-dried macrochromosome pairs and seven microchromosome at 50°C. The slides were counterstained using 1.5 µg/ pairs (Aranyavalai et al., 2013). This karyotypic feature ml DAPI (4′,6-diamidino-2-phenylindole) in antifade is commonly found in Scincidae (Olmo and Signorino, medium Vectashield (Vector Laboratories, Burlingame, 2005; Giovannotti et al., 2010). California, USA). In this study the karyotypes of L. bowringii from Synaptonemal complex (SC) spreads were prepared Bangkok (Thailand) were extensively examined from the testes as described previously (Peters et al., using immunofluorescent synaptonemal complex 1997). Immunostaining was then performed following (SC) analysis which affords multiple chromosomal Anderson et al. (1999) using the primary antibodies: spreads per specimen (up to hundreds), and provides rabbit polyclonal anti-SYCP3 (protein of the lateral better resolution due to low degree of chromatin element of the SC) (1:500, Abcam, Milton, Cambridge, condensation at the pachytene stage, allowing a more UK), mouse monoclonal anti-MLH1 (protein which accurate determination of relative size and morphology marks mature recombination nodules) (1:50, Abcam), of the chromosomes (Lisachov and Borodin, 2016). and human anticentromere (ACA) (1:100, Antibodies Karyotype variation among L. bowringii individuals is Inc., Davis, California, USA). All antibodies were also discussed. diluted in PBT (Phosphate Buffered Saline and Tween 20: 3% bovine serum albumin and 0.05% Tween 20 in Materials and methods 1X PBS). A solution of 10% PBT was used for blocking Three male specimens of L. bowringii were found non-specific antibody binding. Primary antibody severely injured in parks in Bangkok, and were incubation was performed overnight in a humid chamber euthanized soon after. Morphological identification of at 37°C. Secondary antibodies were subsequently the species was performed as described by (Geissler carried-out using Cy3-conjugated goat anti-rabbit et al., 2011). Detailed information on the sampled (1:500, Jackson ImmunoResearch, West Grove, PA, individuals is presented in Table 1. All experimental USA), FITC-conjugated goat anti-mouse (1:50, Jackson procedures using the animals conformed to the ImmunoResearch), and FITC-conjugated donkey anti- guidelines established by the Animal Care Committee, human (1:100, Jackson ImmunoResearch) antibodies for National Research Council (NRCT) and the Animal 1 hour at 37°C. Finally, the slides were counterstained Experiment Committee, Kasetsart University, Thailand using 1.5 µg/ml DAPI with antifade medium Vectashield (approval no. ACKU59-SCI-006). (Vector Laboratories). Fluorescence signals were Conventional analysis of meiotic chromosomes captured using a CCD camera (CV M300, JAI) mounted

Table 1. DataTable types 1. Dataobtained types from obtained the three from Lygosoma the three bowringii Lygosoma specimens. bowringii specimens.

Specimen Locality Synaptonemal Conventional meiotic Nucleotide complex analysis configuration sequencing 1 Lumpini Park + - - 2 Chatuchak Park + - - 3 Chatuchak Park - + +

1 New karyotype of Lygosoma bowringii suggests cryptic diversity 1085 on a ZEISS Axioplan 2 microscope and analyzed using 2017). PCR products were purified by ExoSAP-IT MetaSystems ISIS v.5.2.8 software (MetaSystems, reagent (Affymetrix) and sequenced bidirectionally Alltlussheim, Germany). Brightness and contrast of all using the ABI 3130 Automated Capillary DNA images were enhanced using Corel PaintShop Photo Pro Sequencer (Thermo Scientific, Waltham, MA, USA). X6 (Corel Corp, Ottawa, ON, Canada). BLASTn and BLASTx programs (http://blast.ncbi. To perform the molecular identification of the species, nlm.nih.gov/Blast.cgi) were used to search nucleotide partial sequences of the mitochondrial genes for COI sequences in the National Center for Biotechnology and 16S rRNA were compared with those previously Information (NCBI) database to confirm identity of the deposited in the databases for this species. Whole amplified DNA fragments. Results were also compared genomic DNA was extracted from muscle tissue with sequences obtained previously using the BOLD following the standard phenol-chloroform technique v.4 database (Ratnasingham and Hebert, 2007; http:// as described previously (Sambrook et al., 1989), boldsystems.org/). Sequences generated were deposited and used as a template for polymerase chain reaction in the National Center for Biotechnology Information (PCR). Primers and PCR conditions were previously (NCBI). described (Melnikov et al., 2015; Poyarkov et al.,

Figure 1. Meiotic configurations of spermatocytes of Lygosoma bowringii: synaptonemal complexes (SCs at pachytene stage) (a); diakinesis (b); metaphase I (c); metaphase II (d). Immunoflourescent signals of SYCP3 (red), ACA and MLH1 (green); DAPI (blue). Scale bars represent 10 μm. 1086 Artem Lisachov et al.

Results (Aranyavalai et al., 2013). Our karyotype analysis was performed on three individuals from Bangkok, indicating Molecular identification was performed to confirm the that this karyotype is not an anomaly. Absence of morphological identification of the species. We obtained microchromosomes was indicated as the smallest SCs partial sequences of 16S rRNA (MG930820) and COI of L. bowringii were close to 7 μm, whereas reptile and (MG930821) genes of a specimen of L. bowringii from bird microchromosomes commonly exhibit 2–4 μm in Bangkok. These markers are the most commonly used length at the SC spreads (Malinovskaya et al., 2018). for reptile DNA barcoding (Vences et al., 2012). They The karyotype of L. bowringii from Bangkok was corresponded to the respective sequences of L. bowringii very similar to the karyotype of L. punctata (Gmelin, deposited previously in GenBank and BOLD databases 1799) (2n=24) from India which also consisted of 12 (best match for 16S rRNA was 99% and for COI was pairs of macrochromosomes (Bhatnagar, 1962). Within 95%). This result confirmed that the animals used in this the same subfamily Lygosominae, the species of the study were L. bowringii. African lineage exhibited bimodal karyotypes (2n=30 All meiotic chromosome spreads of three individuals of in Lepidothyris fernandi (Burton, 1836) and Mochlus L. bowringii showed 12 bivalents of macrochromosomes sundevalli (Smith, 1849) (De Smet, 1981; Olmo and (or univalents at metaphase II stage), corresponding to Signorino, 2005)). Molecular phylogenetic studies 2n=24 (Fig. 1). Results of immunostaining clearly showed suggest that the genus Lygosoma Hardwicke & Gray, that the karyotype contained four large metacentric 1828 is paraphyletic and L. punctata is more closely and submetacentric chromosome pairs, seven small related to the latter lineage than to L. bowringii (Datta- metacentric chromosome pairs, and a small acrocentric Roy et al., 2014, Karin et al., 2018). This suggests chromosome pair (Fig. 2). No microchromosomes were independent formation of similar karyotypes in L. found in the analyzed L. bowringii karyotypes. The size punctata and L. bowringii. However, considering the of the smallest SCs of L. bowringii was about 7 μm, and small number of karyotypes described in the Lygosoma the average total SC length in fifty spreads from two lineage, this question should be studied in more detail. specimens was 197.4±21.4 μm. Srikulnath et al. (2015) asserted that in squamate reptiles repeated fusions of microchromosomes may have Discussion occurred independently between microchromosomes The karyotype of all common supple skink individuals and/or macro- and microchromosomes in each lineage, collected from Bangkok (2n=24) differed from previously leading to the disappearance of microchromosomes described karyotype of an individual from Chanthaburi and appearance of small-sized macrochromosomes. (2n=32 comprising macro- and microchromsomes) This suggests the possibility that the karyotypes of L. bowringii and L. punctata described here resulted from repeated fusion processes occurring in the ancestral karyotype. At least four fusions might have occurred from the ancestral karyotype with 2n=32 to the derived state with 2n=24. Comparative chromosome mapping data is required to discuss the karyotype evolution in the lygosomine lineage in more detail. Intraspecific chromosomal diversity in squamate reptiles is rare. Several remarkable cases of chromosome races were described in Sceloporus Wiegmann, 1828 (Hall, 2009). In the Sceloporus grammicus complex, chromosome number variation was observed from 2n=32 to 2n=46. In American iguanian lizards of the genus Sceloporus, several species groups showed consequent steps of transition from the ancestral 2n=32 to 2n=22 in the “S. formosus/S. undulatus” group (Leaché et al., 2016). Also intraspecific chromosomal differences were Figure 2. Idiogram of synaptonemal complexes (SCs) of described in the lacertid lizard genus Acanthodactylus Lygosoma bowringii, 2n=24. Y-axis shows mean length of Wiegmann, 1834 (Giovannotti et al., 2017). Considering each SC (μm). X-axis shows size rank. the large distribution range of L. bowringii, this species New karyotype of Lygosoma bowringii suggests cryptic diversity 1087 may represent a complex of cryptic species with full karyotypes of 36 lizard species (Lacertilia, Reptilia) belonging reproductive isolation (Geissler et al., 2011). In the to the families Teiidae, Scincidae, Lacertidae, Cordylidae and phylogenetic study by Karin et al. (2018), L. bowringii Varanidae (Autarchoglossa). Acta Zoologica et Pathologica Antverpiensia 76: 73–118. is represented by two highly divergent lineages. This Dobigny, G., Britton-Davidian, J., Robinson, T.J. (2017): suggests the possibility of karyotypic differences within Chromosomal polymorphism in mammals: an evolutionary the cryptic species complex of L. bowringii. perspective. Biological Reviews of the Cambridge Philosophical Finally, it is possible that the specimen described Society 92: 1–21. as “L. bowringii” by Aranyavalai et al. (2013) was Geissler, P., Nguyen, T.Q., Phung, T.M., Devender, R.W., misidentified. The authors did not provide molecular Hartmann, T., Farkas, B., et al (2011): A review of Indochinese data for the studied animal. To clarify the discrepancy skinks of the genus Lygosoma Hardwicke & Gray, 1827 (Squamata: Scincidae), with natural history notes and an between the two karyotype descriptions, both molecular identification key. Biologia (Bratisl.) 66: 1159–1176. and cytogenetic traits of the representatives of L. Giovannotti, M., Nisi Cerioni, P., Caputo, V., Olmo, E. (2009): bowringii from different localities throughout the wide Characterization of a GC-rich telomeric satellite DNA in distribution range of this species or species complex Eumeces schneideri Daudin (Reptilia, Scincidae). Cytogenetic must be characterized. and Genome Research 125: 272–278. Giovannotti, M., Caputo, V., O’Brien, P.C.M., Lovell, F.L., Acknowledgements. We thank Victor Spangenberg (Institute of Trifonov, V., Cerioni, P.N., et al. (2010): Skinks (Reptilia: General Genetics RAS) for his help with meiotic chromosome Scincidae) have highly conserved karyotypes as revealed by preparation, Eugenia Solovyeva (Zoological Museum of chromosome painting. Cytogenetic and Genome Research 127: Moscow University) for her help with the molecular work, and 224–231. the Microscopic Center of the Siberian Branch of the Russian Giovannotti, M., Nisi Cerioni, P., Slimani, T., Splendiani, A., Academy of Sciences for granting access to microscopic Paoletti, A., Fawzi, A., Olmo, E., Caputo Barucchi, V. (2017): equipment. This work was supported by the Federal Agency Cytogenetic characterization of a population of Acanthodactylus of Scientific Organizations via the Institute of Cytology and lineomaculatus Duméril and Bibron, 1839 (Reptilia, Lacertidae) Genetics (project # 0324-2018-0019), research grant # 16-04- from Southwestern Morocco and insights into sex chromosome 00087 from the Russian Foundation for Basic Research, Russia, evolution. Cytogenetic and Genome Research 153: 86–95. and partially supported by Science Research Fund (ScRF) Hall, W.P. (2009): Chromosome variation, genomics, speciation (No. ScRF-S19–2558) from the Faculty of Science, Kasetsart and evolution in Sceloporus lizards. Cytogenetic and Genome University, Thailand. Research 127: 143–165. Karin, B.R., Freitas, E.S., Shonleben, S., Grismer, L.L., Bauer, References A.M., Das, I. (2018): Unrealized diversity in an urban rainforest: A new species of Lygosoma (Squamata: Scincidae) from western Anderson, L.K., Reeves, A., Webb, L.M., Ashley, T. (1999): Sarawak, Malaysia (Borneo). Zootaxa 4370: 345–362. Distribution of crossing over on mouse synaptonemal complexes Leaché, A.D., Banbury, B.L., Linkem, C.W., de Oca, A.N.M. using immunofluorescent localization of MLH1 protein. (2016): Phylogenomics of a rapid radiation: is chromosomal Genetics 151: 1569–1579. evolution linked to increased diversification in North American Aranyavalai, V., Lertpanich, K., Chulalaksananukul, W. (2013): spiny lizards (genus Sceloporus)? BMC Evolutionary Biology Distribution and chromosomal variation in the scincid lizard 16: 63. genus Lygosoma (Reptilia: Squamata) in Thailand. Natural Lisachov, A.P., Borodin, P.M. (2016): Microchromosome Science 5: 993–996. polymorphism in the sand lizard, Lacerta agilis Linnaeus, 1758 Axelsson, E., Webster, M.T., Smith, N.G.C., Burt, D.W., Ellegren, (Reptilia, Squamata). Comparative Cytogenetics 10: 387–399. H. (2005): Comparison of the chicken and turkey genomes reveals Malinovskaya, L., Shnaider, E., Borodin, P., Torgasheva, A. a higher rate of nucleotide divergence on microchromosomes (2018): Karyotypes and recombination patterns of the common than macrochromosomes. Genome Research 15: 120–125. swift (Apus apus Linnaeus, 1758) and eurasian hobby (Falco Bhatnagar, A.N. (1962): Chromosome cytology of two lizards, subbuteo Linnaeus, 1758). Avian Research 9: 4. Riopa punctata Gmelin and Hemidactylus brooki Grey. Melnikov, D., Śmiełowski, J., Melnikova, E., Nazarov, R., Caryologia 15: 335–349. Ananjeva, N.B. (2015): Red’n’blues: a new species of Caputo, V., Odierna, G., Aprea, G. (1994): A chromosomal study Pseudotrapelus (Agamidae, Sauria) from Sudan, Africa. of Eumeces and Scincus, primitive members of the Scincidae Russian Journal of Herpetology 22: 53–60. (Reptilia, Squamata). Bollettino di Zoologia Italiana 61: 155– Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. T., 162. Hungerford, D. A. (1960): Chromosome preparations of Datta-Roy, A., Singh, M., Karanth, K.P. (2014): Phylogeny of leukocytes cultured from human peripheral blood. Experimental endemic skinks of the genus Lygosoma (Squamata: Scincidae) Cell Research 20 (3): 613–616. from India suggests an in situ radiation. Journal of Genetics 93: Morescalchi, A. (1977): Phylogenetic aspects of karyological 163–167. evidence. In: Hecht M.K., Goody P.C., Hecht B.M. (Eds.), De Smet, W.H.O. (1981): Description of the orcein stained Major Patterns in Vertebrate Evolution. Springer, Boston, MA, 1088 Artem Lisachov et al.

pp. 149–167. A., Suputtitada, S., Apisitwanich, S., Matsuda, Y. (2009): Olmo, E., Signorino, G. (2005): Chromorep: a Reptile Karyotypic evolution in squamate reptiles: Comparative gene Chromosomes Database. http://chromorep.univpm.it [accessed mapping revealed highly conserved linkage homology between 26 January 2018] the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Peters, A.H., Plug, A.W., van Vugt, M.J., de Boer, P. (1997): Lacertilia) and the Japanese four-striped rat snake (Elaphe A drying-down technique for the spreading of mammalian quadrivirg). Chromosome Research 17: 975–986. meiocytes from the male and female germline. Chromosome Srikulnath, K., Uno, Y., Nishida, C., Matsuda, Y. (2013): Karyotype Research 5: 66–71. evolution in monitor lizards: Cross-species chromosome Pokorná, M., Giovannotti, M., Kratochvíl, L., Kasai, F., Trifonov, mapping of cDNA reveals highly conserved synteny and gene V.A., O’Brien, P.C.M., et al. (2011): Strong conservation of order in the Toxicofera clade. Chromosome Research 21: 805– the bird Z chromosome in reptilian genomes is revealed by 819. comparative painting despite 275 million years divergence. Srikulnath, K., Matsubara, K., Uno, Y., Nishida, C., Olsson, M., Chromosoma 120: 455–468. Matsuda, Y. (2014): Identification of the linkage group of the Z Pokorná, M.J., Trifonov, V.A., Rens, W., Ferguson-Smith, sex chromosomes of the sand lizard (Lacerta agilis, Lacertidae) M.A., Kratochvíl, L. (2015): Low rate of interchromosomal and elucidation of karyotype evolution in lacertid lizards. rearrangements during old radiation of gekkotan lizards Chromosoma 123: 563–575. (Squamata: Gekkota). Chromosome Research 23: 299–309. Srikulnath, K., Uno, Y., Nishida, C., Ota, H., Matsuda, Y. Poyarkov, Jr. N.A., Van, D.T., Orlov, N.L., Gogoleva, S.S., (2015): Karyotype reorganization in the Hokou gecko (Gekko Vassilieva, A.B., Nguyen, L.T., et al. (2017): Molecular, hokouensis, Gekkonidae): the process of microchromosome morphological and acoustic assessment of the genus disappearance in Gekkota. PloS One 10: e0134829. Ophryophryne (Anura, Megophryidae) from Langbian Plateau, Uetz, P., Freed, P., Hošek, J. (2018): The Reptile Database, http:// Southern Vietnam, with description of a new species. ZooKeys www.reptile-database.org, accessed [accessed 8 May 2018] 672: 49–120. Vences, M., Nagy, Z.T., Sonet, G., Verheyen, E. (2012): DNA Ratnasingham, S., Hebert, P.D. (2007): BOLD: The Barcode of barcoding amphibians and reptiles. In: DNA Barcodes. Humana Life Data System (http://www. barcodinglife.org). Molecular Press, Totowa, NJ, pp. 79–107. Ecological Research 7: 355–364. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989): Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Srikulnath, K., Nishida, C., Matsubara, K., Uno, Y., Thongpan,

Accepted by Spartak Litvinchuk