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