Cytogenetic analyses of five amazon lizard species of the subfamilies Teiinae and Tupinambinae and review of karyotyped diversity the family Teiidae

Abstract Lizards of the family Teiidae (infraorder Scincomorpha) were formerly known as Macroteiidae. There are 13 species of such lizards in the Amazon, in the genera Ameiva (Meyer, 1795), Cnemidophorus (Wagler, 1830), Crocodilurus (Spix, 1825), Dracaena (Daudin, 1801), Kentropyx (Spix, 1825) and Tupinambis (Daudin, 1802). Cytogenetic studies of this group are restricted to karyotype macrostructure. Here we give a compilation of cytogenetic data of the family Teiidae, including classic and molecular cytogenetic analysis of Ameiva ameiva (Linnaeus, 1758), Cnemidophorus sp.1, Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868) and Tupinambis teguixin (Linnaeus, 1758) collected in the state of Amazonas, Brazil. Ameiva ameiva, Kentropyx calcarata and Kentropyx pelviceps have 2n=50 chromosomes classified by a gradual series of acrocentric chromosomes. Cnemidophorus sp.1 has 2n=48 chromosomes with 2 biarmed chromosomes, 24 uniarmed chromosomes and 22 microchromosomes. Tupinambis teguixin has 2n=36 chromosomes, including 12 macrochromosomes and 24 microchromosomes. Constitutive heterochromatin was distributed in the centromeric and terminal regions in most chromosomes. The nucleolus organizer region was simple, varying in its position among the species, as evidenced both by AgNO3 impregnation and by hybridization with 18S rDNA probes. The data reveal a karyotype variation with respect to the diploid number, fundamental number and karyotype formula, which reinforces the importance of increasing chromosomal analyses in the Teiidae.

Most chromosome data for teiid lizards refer only to the determination of diploid numbers and karyotype formulae (Fritts 1969, Gorman 1970, Lowe et al. 1970, Robinson 1973, Cole et al. 1979, de Smet et al. 1981, Navarro et al. 1981, Ward and Cole 1986, Cole et al. 1995, Markezich et al. 1997, Rocha et al. 1997, Walker et al. 1997, Manriquen-Moran et al. 2000, Veronese et al. 2003. Some species of this family have, however, been analyzed in detail with respect to their chromosomal structure and organization, as revealed by differential staining techniques, such as the detection of heterochromatin and nucleolar organizer regions (NORs), as well as chromosomal physical mapping of DNA sequences (Bickham et al. 1976, Bull 1978, Peccinini-Seale and Almeida 1986, Porter et al. 1991, Rocha et al. 1997, Veronese et al. 2003, Peccinini-Seale et al. 2004, Santos et al. 2007, Santos et al. 2008. The family Teiidae can be divided into two chromosomal groups: the Dracaena group (currently the subfamily Tupinambinae), which has a karyotype with 34-38 chromosomes and a clear distinction of macrochromosomes (M) from microchromosomes (mi), and the Ameiva group (currently the subfamily Teiinae), which has a diploid number ranging from 46-56 chromosomes, with no distinction between macrochromosomes and microchromosomes (Gorman 1970).

Methods
Thirty-three specimens belonging to the subfamilies Teiinae and Tupinambinae were collected in the state of Amazonas, Brazil, in the following localities: the riverside forests of the Jatapu river, the city of São Sebastião do Uatumã (0°50'E 01°55'S; 58°50'E 60°10'W), the Darahá and Ayuanã rivers, both in the city of Santa Isabel do Rio Negro (0°24'24"N; 65°1'1"W), the city of Manaus (3°07'13.03"S; 60°01'440"W) and the Purus riverside in the city of Tapauá (5°42'115"S; 63°13'684"W). All of the collections were conducted with permission from the Brazilian Environmental Protection Agency (ICMBio/SISBIO 41825-1). The collection sites are located in public lands (Table 1, Figure 1). The animals were euthanized soon after capture in the field with a lethal dose of the anesthetic sodium thiopental to avoid being deprived of food or water. This research was approved by the Ethics Committee for Animal Experimentation of the Fundação Universidade do Amazonas / Universidade Federal do Amazonas (UFAM) (number 041/2013). No endangered or protected species were used in this research study. The animals underwent cytogenetic procedures and were then fixed with 10% formaldehyde (injected in the coelom and digestive tract), preserved in 70% alcohol. Voucher specimens were deposited in the Herpetological Collection of the Instituto Nacional de Pesquisas da Amazônia (INPA H31712, 33213, 34791, 34841, 35018).
Cellular suspensions were obtained from the bone marrow was removed soon after the euthanasia of animals in the field using an in vitro colchicine treatment (Ford and Hamerton 1956). Constitutive heterochromatin (CH) was detected using barium hydroxide (Sumner 1972) and the NORs were detected using silver nitrate staining (Howell and Black 1980). Genomic DNA was extracted from muscle tissue using a phenol-chloroform protocol (Sambrook and Russell 2001) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). 18S rDNA was amplified by polymerase chain reaction (PCR) using primers 18Sf (5'-CCG CTT TGG TGA CTC TTG AT-3') and 18Sr (5'-CCG AGGACC TCA CTA AAC CA-3') (Gross et al. 2010). PCR reactions were performed on a final volume of 15 µL, containing genomic DNA (200 ng), 10× buffer with 1.5 mM of MgCL 2 , Taq DNA polymerase (5 U/µL), dNTPs (1 mM), forward and reverse primers (5 mM) and Milli-Q water. The amplification cycles followed these steps: 1 min at 95 °C; 35 cycles of 1 min at 94 °C, 1 min at 56 °C, 1 min 30 s at 72 °C and 5 min at 72 °C.
The PCR product of the 18S rDNA was labeled with digoxigenin-11-dUTP (Dig-Nick Translation mix; Roche), by nick translation according to the manufacturer's instructions. The antibody anti-digoxigenin rhodamine (Roche) was used for probing the signal. Homologue (DNA probes from the same species) and heterologue (probes of one species hybridized to the chromosome of another) hybridizations were made under stringency conditions of 77% (2.5 ng/µL of 18S rDNA, 50% formamide, 10% dextran sulfate, and 2× SSC at 37 °C for 18 h) (Pinkel et al. 1986). The chromosomes were counterstained with DAPI (2 mg/ml) in VectaShield mounting medium (Vector). The chromosomes were analyzed using an Olympus BX51 epifluorescence mi- croscope and the images were captured with a digital camera (Olympus DP71) using Image-Pro MC 6.3 software. Mitotic metaphases were processed in Adobe Photoshop CS4 software and were measured using program ImageJ software. Chromosomes were organized by decreasing size, and chromosome morphology was determined based on the arm ratio for metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a) chromosomes (Levan et al. 1964). The karyotype formula was determined according to chromosomes that show a gradual series of acrocentric chromosomes, number of biarmed chromosomes, number of uniarmed chromosomes and number of macrochromosomes (M), and microchromosomes (mi) Wright 1966, Peccinini-Seale 1981). Macrochromosomes and microchromosomes are chromosomes that can be differentiated according to size; macrochromosomes are large and have one or two chromosome arms; microchromosomes are small (0.5-1.5 µm), puntiform and do not have any specific chromosome morphology.

Results
The diploid number for all specimens of Ameiva ameiva, Kentropyx calcarata and Kentropyx pelviceps was 50 chromosomes, and the karyotypic formula was classified by a gradual series of acrocentric chromosomes (Fig. 2a, i and m). Cnemidophorus sp.1 had 48 chromosomes with 2 biarmed chromosomes, 24 uniarmed chromosomes and 22 microchromosomes (Fig. 2e). Tupinambis teguixin had 36 chromosomes with 12 macrochromosomes (M) and 24 microchromosomes (mi). Pairs 1, 3, 4 and 5 of the macrochromosomes were metacentric and pairs 2 and 6 were submetacentric chromosomes (Fig. 3a). A secondary constriction was observed in the distal region of the long arms of pair 1 in Cnemidophorus sp.1, Kentropyx calcarata and Kentropyx pelviceps and in pair 2 in Tupinambis teguixin (Figs 2e,i,m and 3a). No differentiated sex chromosomes were observed in the analysed species. Constitutive heterochromatin was observed in the centromeric and terminal regions in most chromosomes of Ameiva ameiva, Cnemidophorus sp.1, Kentropyx calcarata and Kentropyx pelviceps (Figs 2b,f,j,n). In Tupinambis teguixin, heterochromatic blocks were located in the centromeric region of all the macrochromosomes. However, tenuous blocks were observed in the terminal regions in macrochromosomes and microchromosomes (Fig. 3b).
The NORs were located in the terminal region of the long arms of pair 7 in Ameiva ameiva (Fig. 2c). In Cnemidophorus sp.1, Kentropyx calcarata and Kentropyx pelviceps, NORs were seen in the distal region of the long arms of pair 1 and in pair 2 in Tupinambis teguixin, coincident with the secondary constriction present in the karyotypes of these species (Figs 2g, k, o and 3c, respectively). Fluorescent in situ hybridization (FISH) with an 18S rDNA probe revealed a chromosome pair bearing this site, coincident with the NOR sites in all of the five analyzed species (Figs 2d, h, l, p and 3d).

Discussion
Since the 1970s, cytogenetic analysis of the family Teiidae has shown that individuals could be categorized into two groups: the Ameiva group, with diploid number vary-  ing from 30-56 chromosomes, with no distinction between macrochromosomes and microchromosomes, and the Dracaena group, with a karyotype varying from 34-38 chromosomes, with a clear distinction between macrochromosomes and microchromosomes (Gorman 1970). By the end of the 1980s, several osteological and morphological studies corroborated the chromosomal data, thus supporting these two groups, which were subsequently considered subfamilies (Estes et al. 1988): Teiinae (Ameiva group) and Tupinambinae (Dracaena group).
Most karyotype data comes from species of the subfamily Teiinae, with descriptions of diploid numbers for 63 species. The karyotypes reveal a diploid number varying from 2n=30 in Ameiva auberi (Cocteau, 1838) to 2n=54 in Teius oculatus (D'orbigny & Bibron, 1837) and Teius teyou (Daudin, 1802), besides the presence of sex chromosomes of XX/XY in Aspidocelis tigris tigris (Baird & Girard, 1852) and Ameivula littoralis (Rocha, Bamberg Araújo, Vrcibradic, 2000). Some Aspidoscelis species show triploid numbers such as Aspidoscelis tessalatus (Say, 1823) with 69 chromosomes. Interspecific hybridization has been observed in some species of the genus Aspidoscelis, which were previously placed within the genus Cnemidophorus (Lowe et al. 1970, Walker et al. 1997, Lutes et al. 2010, Manriquez-Morán et al. 2000. Although the Ameiva group proposed by Gorman (1970) corresponds to the subfamily Teiinae, some species have a distinction between macrochromosomes and microchromosomes, while most chromosomes are acrocentric. This finding is contrary to what was proposed by Gorman (1970) as a cytogenetic feature of the Ameiva group (Table 2).
Ameiva ameiva and Kentropyx calcarata, which belong to Teiinae, have the same diploid number (2n=50 chromosomes). This result corroborates the available data for these species from different localities (Gorman 1970, Beçak et al. 1972, Peccinini-Seale and Almeida 1986, Schmid and Guttenbach 1988, Sites et al. 1990, Veronese et al. 2003, Santos et al. 2007. However, in present study Ameiva ameiva and Kentropyx calcarata present a gradual series of acrocentric chromosomes characterized by absence of distinction between macrochromosomes and microchromosomes, similar to the results described by Cole et al. (1995) and Santos et al. (2007). The same finding is observed for Kentropyx pelviceps, whose cytogenetic characteristics are revealed for the first time in the present study.
Seven species from the subfamily Tupinambinae, have had their karyotypes analyzed, with diploid numbers varying from 2n=34-38 chromosomes, with the presence of both macrochromosomes and microchromosomes (Santos et al. 2008, present study). No sex chromosome system has been documented in the subfamily (Gorman 1970). Tupinambis teguixin has 2n=36 chromosomes (12M+24m) ( Table 2) the same number and karyotype formula was found by other authors (Gorman 1970, de Smet et al. 1981, Santos et al. 2008. Beçak et al. (1972) described a diploid number of 38 chromosomes (12M+26m) for T. teguixin, with an additional pair of microchromosomes.
In the family Teiidae, heterochromatic blocks are located in the centromeric and terminal regions of almost all chromosomes. In some chromosomes, heterochromatic blocks are present in the pericentromeric, interstitial and terminal regions (Table 3). In the five species of the family Teiidae analyzed in this study, we observed a significant number of heterochromatic blocks in the centromere and terminal regions in the most of the chromosomes, which is consistent with similar patterns described in the literature.
The heterochromatin patterns for Cnemidophorus sp.1, Kentropyx calcarata, Kentropyx pelviceps and Tupinambis teguixin are described for the first time in this study. The heterochromatin distributional pattern is similar among the analyzed species, suggesting a common pattern for species in the family Teiidae. Three species in the subfamily Tupinambinae (Crocodilurus amazonicus (Spix, 1825), Salvator merianae (Duméril & Bibron, 1839) and Tupinambis quadrilineatus (Manzani & Abe, 1997), however, show species-specific heterochromatin patterns, with heterochromatic blocks in the centromeric, pericentromeric, interstitial and proximal regions of most chromosomes (Santos et al. 2008). The existence of such a distinctive pattern can likely be attributed to the addition of heterochromatin or the heterochromatization process during the evolution of these species. Heterochromatic regions are rich in repetitive DNA sequences usually located in the centromeric or terminal regions of chromosomes. This has often been considered important species-specific or population markers (Carvalho et al. 2012, Schneider et al. 2013. Even though heterochromatin may be located on the same chromosome region in different species, this does not mean it has the same genetic composition, which may differ in the amount of repetitive DNA sequences in the chromosomes (Chaiprasertsri et al. 2013).
In the present study, the localization of the NORs was revealed as an genus marker and this information has already been discussed for some genera in the family  (Santos et al. 2007(Santos et al. , 2008. Tupinambis teguixin has a simple NOR, as evidenced by the secondary constriction of the long arm of pair 2. A common characteristic among species the subfamily Tupinambinae is the presence of such a secondary constriction in pair 2 (Gorman 1970). Four species of the subfamily Teiinae, Ameiva ameiva, Cnemidophorus sp.1, Kentropyx calcarata and Kentropyx pelviceps, also have simple NORs, but they are located in distinct chromosomal pairs. In Cnemidophorus sp.1, Kentropyx calcarata and Kentropyx pelviceps, a secondary constriction was seen in pair 1 while in Ameiva ameiva occurred in pair 7. The NOR data analyzed for Ameiva ameiva and Kentropyx calcarata in the present study corroborate previous data (Schmid and Guttenbach 1988, Cole et al. 1995, Veronese et al. 2003, Santos et al. 2007), but for Cnemidophorus sp.1 and Kentropyx pelviceps they are new data.
Two populations of Ameiva ameiva from the eastern Amazon showed multiple NORs involving pairs 1, 2, 6, 16, 18, 19 and some small chromosomes (Peccinini-Seale and Almeida 1986). Some authors suggest that the inter-individual variation observed in Ameiva ameiva may be related to the identification of active NOR sites, once the silver nitrate binds to acid nucleoli proteins involved with the transcriptional activity of the ribosomal genes (Miller et al. 1976, Howell and Black 1980, Boisvert et al. 2007. Such variability may also result from impregnation of CH regions rich in acid residues, in which the nitrate impregnates both the NORs and heterochromatic regions not bearing ribosomal sites, thereby not revealing the exact number of NORs (Sumner 2003). Moreover, this variation may be suggesting that Ameiva ameiva is a specie complex, as other teiids like Ameivula ocellifera (Spix, 1825) (Arias et al. 2011) or Cnemidophorus lemniscatus (Harvey et al. 2012).
Using 45S ribosomal DNA probes and FISH, it is possible to understand the organization of the NORs and to elucidate questions concerning the chromosomal organization and karyotypic evolution. The FISH technique is a more refined method than silver nitrate impregnation to locate 45S rDNA sequences in mitotic chromosomes (Carvalho et al. 2012, Schneider et al. 2013. However, for the species analyzed in the present study, the fluorescent in situ hybridization of the 18S ribosomal gene corroborated the results obtained with silver nitrate impregnation, confirming the existence of this ribosomal site in a single pair of chromosomes. This same pattern was identified in other species in the family Teiidae, supporting the sites seen in a microchromosome pair in Ameiva auberi (Cocteau, 1838). In Aspidoscelis marmorata (Baird & Girard, 1852), the same pattern was located in a macrochromosome pair (Porter et al. 1991). Furthermore, it was possible to observe a size heteromorphism of the sites between the homologue chromosomes in the four analyzed species, a fact also described for other lizard species (O'Meally et al. 2009, Srikunath et al. 2009b, Srikunath et al. 2011. Such a size heteromorphism is likely associated with unequal crossing-over mechanisms, rearrangements such as transpositions, deletions and/or duplications or variations in the number of rDNA copies present in such regions that would entail some changes in ribosomal sites (Gross et al. 2010, Ribeiro et al. 2008).

Conclusion
Our present data and those from the literature show that teiid lizards have karyotype variation with respect to diploid number, fundamental number and karyotype formula. This, reinforces the importance to increase the number of chromosomal analyses in the family Teiidae. Studies are currently underway with the chromosomal physical mapping of repetitive DNA sequences in three species of Amazonian teiids that are essential for the understanding of genome organization and karyotype evolution in this group of lizards. NDMC received funding from the Fundação de Amparo a Pesquisas do Estado do Amazonas. The authors are grateful to Sergio Marques de Souza for revision of the manuscript and Dra. Eliana Feldberg for the epifluorescence microscope. American Manuscript Editors reviewed this paper.