Southeastern Naturalist L.M. Hardy, L.R. Raymond, and S. Harris 2017 Vol. 16, No. 3 326 2017 SOUTHEASTERN NATURALIST 16(3):326–330 The Karyotype of Plestiodon anthracinus (Baird, 1850) (Sauria: Scincidae): A Step Toward Solving an Enigma Laurence M. Hardy1, *, Larry R. Raymond1, and Shannon Harris1 Abstract - The cosmopolitan lizard genus Eumeces was first revised in 1936 and consisted of 15 species-groups comprising a total of 50 species. Nine species in North America were later recognized as belonging to the genus Plestiodon and all contained the diploid chromosome number of 26. Modern cladistic techniques indicated that Plestiodon anthracinus (Coal Skink) was near the ancestral form for the fasciatus group. We employed the hypotonic citrate method to study chromosomes of 5 Coal Skink specimens from Louisiana and Arkansas and found them to have a diploid number of 24 (12 macrochromosomes, 12 distinctly smaller chromosomes, all biarmed) and a fundamental number of 48. The diploid number of 24 is probably derived by some chromosome rearrangements in the evolution of Plestiodon and of the Plestiodon anthracinus group. Introduction Taylor (1936) recognized 15 species-groups within the cosmopolitan genus Eumeces (= Plestiodon), which he regarded as monophyletic. Within Eumeces, Taylor (1936) identified the anthracinus group, which contained 3 species: Plestiodon anthracinus Baird (Coal Skink), P. copei (Taylor), and P. septentrionalis Baird (Prairie Skink), all endemic to North America. Dixon (1969) removed copei from the anthracinus group and placed it in the brevirostris group, based on several morphological characters. Lieb (1985) upheld removing P. copei from the anthracinus group, but added P. tetragrammus Baird (Four-lined Skink) to P. anthracinus and P. septentrionalis in this group. Schmitz et al. (2004) reported that the laticeps species-group includes the obsoletus and anthracinus species-groups in a clade that comprises laticeps, inexpectatus, fasciatus, obsoletus, and septentrionalis. However, they determined that “E.” anthracinus is not part of this group, because it is consistently placed outside of the latter clade and is mostly recovered as a sister species to “E.” egregius (Schmitz et al. 2004). This view was supported by Brandley et al. (2012) who placed P. anthracinus as the sole member of the anthracinus species-group within the fasciatus species series. Plestiodon tetragrammus was included in the fasciatus species-group (Brandley et al. 2012). Multiple studies have concluded that Eumeces (sensu lato) is, in fact, polyphyletic (Brandley et al. 2005, Caputo, et al. 1993, Griffith et al. 2000, Schmitz et al. 2004), with the east Asian/ North American species of “Eumeces” being designated as Plestiodon by Brandley et al. (2005). Brandley et al. (2012) included P. anthracinus as the sole member of the anthracinus group in their revised phylogeny and placed the anthracinus group 1Museum of Life Sciences, Louisiana State University in Shreveport, Shreveport, LA.*Corresponding author - lhardy@lsus.edu. Manuscript Editor: Kristen Cecala Southeastern Naturalist 327 L.M. Hardy, L.R. Raymond, and S. Harris 2017 Vol. 16, No. 3 as a sister taxon to the fasciatus group (their Clade C5). The mtDNA data (Brandley et al. 2012) provides strong support for the sister relationship of P. anthracinus with P. egregius and P. reynoldsi (C4 clade) and the exclusion of P. septentrionalis from close relationship with P. anthracinus. The cosmopolitan lizard genus Eumeces (sensu lato) contains at least 25 valid species (according to ITIS 2016), including 8 species referred to Plestiodon as invalid. We follow Brandley et al. (2005:388) and Brandley et al. (2012:182) for the use of Plestiodon. Despite the large number of species in the family Scincidae, the karyotypes of this family are comparatively poorly known (Giovannotti et al. 2009). All of the species that have been karyotyped share the characteristics of having a relatively low diploid number, with the first 4 pairs of chromosomes being metacentric and larger than the remainder of the chromosome complement (e.g., Gionvannotti et al. 2009). We had access to several live specimens of the uncommon Coal Skink in northwestern Louisiana and Arkansas, which allowed us to compare the karyotype of Plestiodon anthracinus to other species in the genus. We tested the hypothesis that the diploid number of chromosomes in P. anthracinus is 26, the known diploid number reported for other species of Plestiodon (Dowling 1975). Materials and Methods We euthanized specimens by an intraperitoneal injection of 10% Nembutal and processed for analysis of mitotic and meiotic cells sampled from bone marrow from crushed vertebrae and/or testes. We prepared chromosomes by the hypotonic citrate method of Patton (1967) and used the modification by Cole and Leavens (1971). We made an intraperitoneal injection of 10% Velban as a mitotic inhibitor, instead of colchicine. We examined chromosomes under a Leitz Dialux microscope and photographed appropriate Giemsa-stained images with a 10.16 cm x 12.70 cm (4” x 5”) black and white, high-contrast film. We prepared the karyotype from a scanned image (positive) of the 10.16 cm x 12.70 cm (4” x 5”) negative. Chromosome terminology follows Cole (1970). Specimens examined: LSUS 4545, female, Caddo Parish: 4.02 km (2.5 mi) W, 1.61 km (1.0 mi) S Blanchard, 14 September 1980 (LMH 8933); LSUS 5742, male, Caddo Parish: Walter Jacobs Nature Park (T18N, R15W, Sec 7), 26 March 1981 (LRR 929); LSUS 5743, female, Caddo Parish: Walter Jacobs Nature Park (T18N, R15W, Sec 7), 12 March 1980 (LRR 712); LSUS 5744, male, Caddo Parish: Walter Jacobs Nature Park (T18N, R15W, Sec 7), 27 February 1980 (LRR 706); LSUS 8911, male, Arkansas, Polk County, 2.90 km (1.8 mi) S, 2.57 km (1.6 mi) W Big Fork (Ouachita Mountains Biological Station), 21 July 2003 (LMH 12954). Results Our chromosome analysis of 4 specimens (2 males, 2 females) of P. anthracinus from northwestern Louisiana and 1 specimen (male) from the Ouachita Mountains of Arkansas indicated a diploid number of 24 chromosomes, including 12 macrochromosomes and 12 microchromosomes. The macrochromosomes include, Southeastern Naturalist L.M. Hardy, L.R. Raymond, and S. Harris 2017 Vol. 16, No. 3 328 from largest to smallest, 1 metacentric to submetacentric, 1 submetacentric, and 4 metacentrics. The microchromosomes are metacentrics and submetacentrics. No chromosomes are telocentric and no secondary constrictions or satellites were observed. Twelve pairs of chromosomes are shown in metaphase (Fig. 1). Both macrochromosomes and microchromosomes are biarmed, and the fundamental number is 48. We observed no obvious heteromorphic sex chromosomes. We counted chromosomes from more than 380 cells, using both mitotic and meiotic preparations; in all cases, the total diploid number was 20–26. We assume that the cells containing 20 or 22 chromosomes were incomplete cells because those numbers were recorded in specimens that also showed the modal number of 24. The 2 cells with 26 chromosomes were part of 384 cells analyzed that included 336 cells containing 24 chromosomes from 1 specimen (LSUS 5744). This result indicates that the correct diploid number for our sample is 24. In addition, the reduction in chromosome number from 26 seen in all other species of Plestiodon to 24 in Plestiodon anthracinus is due to a reduction of the microchromosome number (from 14 to 12) and not a reduction of the macrochromosome numb er. Discussion Nakamura (1931a, 1931b) reported a diploid number of 26 chromosomes for E. latiscutatus, a member of the fasciatus group and, according to Taylor (1936), the only species from Japan. Talluri (1968) reported a diploid number of 32 chromosomes for Eumeces algeriensis (see Caputo et al. 1993) a member of the North African/Southeast Asia clade of Brandley et al. (2005). Deweese and Wright (1970) reported chromosome data for 6 New World species of Plestiodon (all with a diploid number of 26), including P. copei from Morelos, Mexico, which is 1 of 3 species of Plestiodon placed by Taylor (1936) in the P. anthracinus species group, but later removed from this group (Brandley et al. 2012, Dixon 1969, Lieb 1985). Dowling (1975) listed diploid chromosome numbers for 9 species of Plestiodon, all Figure 1. Karyotype of an adult male Plestiodon anthracinus (LSUS 5744); 2n = 24; bar = 0.01 mm. Southeastern Naturalist 329 L.M. Hardy, L.R. Raymond, and S. Harris 2017 Vol. 16, No. 3 of which had a diploid number of 26. A review of chromosomal data for Eumeces included 7 North American species (not P. anthracinus); all of which have a diploid number of 26 (Caputo et al. 1994). The karyotype of Plestiodon anthracinus differs from that of P. copei studied by DeWeese and Wright (1970) by having a diploid number of 24 (only 6 pairs of microchromosomes, not 7 as in P. copei and all other species of Plestiodon for which karyotypes have been published). Chromosome change during evolution can be a useful indicator of phylogenetic relationships, especially when used together with molecular and morphological data (Giovannotti et al. 2009). However, we do not know the significance of the loss of a microchromosome, which appears to indicate the loss of genetic material. We do know that microchromosomes contain genetic material and are probably important to the genome. For example, a microchromosome is, in some species, one of the sex chromosomes and, therefore, very important for that species (Cole et al. 1967). Microchromosomes may be translocated onto a macrochromosome and, therefore, easily misinterpreted as a chromosome loss; however, it is not lost and the genetic material is still present and probably functional. Scincidae have highly conserved karyotypes (Giovannotti et al. 2009) and all Plestiodon in the North American clade have a diploid number of 26, except for P. anthracinus. By having a unique karyotype for the genus Plestiodon, based on present knowledge, P. anthracinus is somewhat of an enigma. Its biogeographic location would not suggest any particular evolutionary event to explain this, and it has not previously been associated with any more-derived species group. However, in the results of Brandley et al. (2011, 2012), P. anthracinus appeared in one of the more divergent positions of the cladogram, and was nested between the fasciatus and egregius groups. The most conservative conclusion suggests that the chromosome rearrangement to a diploid number of 24 in P. anthracinus occurred after the divergence of P. anthracinus from the fasciatus-species group, and the P. anthracinus karyotype is independent of all of the North American Plestiodon containing a diploid number of 26. Acknowledgments We thank Charles J. Cole and several anonymous reviewers for advice and constructive comments on the manuscript. Literature Cited Brandley, M.C., A. Schmitz, and T.W. Reeder. 2005. Partitioned Bayesian analyses, partition choice, and the phylogenetic relationships of Scincid lizards. Systematic Biology 54:373–390. Brandley M.C., Y. Wang, X. Guo, A.N. Montes De Oca, M. Feria-Ortiz, T. Hikida, and H. Ota. 2011. Accommodating heterogenous rates of evolution in molecular-divergence dating methods: An example using intercontinental dispersal of Plestiodon (Eumeces) lizards. Systematic Biology 60: 3–15. Brandley, M.C., H.O. Fls, T. Hikida, A.N. 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