8urn:lsid:arphahub.com:pub:A71ED5FC-60ED-5DA3-AC8E-F6D2BB5B3573urn:lsid:zoobank.org:pub:C8FA3ADA-5585-4F26-9215-A520EE683979Comparative CytogeneticsCCG1993-07711993-078XPensoft Publishers10.3897/CompCytogen.v9i4.53715371Research ArticleAnimaliaCellular & Organismal geneticsGeneticsAmericasSouth AmericaCytogenetic analyses of five amazon lizard species of the subfamilies Teiinae and Tupinambinae and review of karyotyped diversity the family TeiidaeCarvalhoNatália Dayane Mouranathydayane@gmail.com1AriasFederico José2da SilvaFrancijara Araújo1SchneiderCarlos Henrique1GrossMaria Claudia1Universidade Federal do AmazonasManausBrazilUniversidade de São PauloSão PauloBrazil
Corresponding author: Natália D. Moura Carvalho (nathydayane@gmail.com)
Academic editor: L. Kupriyanova
20150710201594625644FFE6FF80-E804-217B-FFCE-FFE3E7308A4CAC61F170-41B4-407A-BD49-D6E7891DE50F5755462605201507092015Natália Dayane Moura Carvalho, Federico José Arias, Francijara Araújo da Silva, Carlos Henrique Schneider, Maria Claudia GrossThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://zoobank.org/AC61F170-41B4-407A-BD49-D6E7891DE50F
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 Ameivaameiva (Linnaeus, 1758), Cnemidophorus sp.1, Kentropyxcalcarata (Spix, 1825), Kentropyxpelviceps (Cope, 1868) and Tupinambisteguixin (Linnaeus, 1758) collected in the state of Amazonas, Brazil. Ameivaameiva, K.calcarata and K.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. Tupinambisteguixin 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.
Carvalho NDM, Arias FJ, da Silva FA, Schneider CH, Gross MC (2015) Cytogenetic analyses of five amazon lizard species of the subfamilies Teiinae and Tupinambinae and review of karyotyped diversity the family Teiidae. Comparative Cytogenetics 9(4): 625–644. doi: 10.3897/CompCytogen.v9i4.5371
Background
The family Teiidae is composed of lizards formerly known as macroteiids that are restricted to the New World (Giugliano et al. 2007, Harvey et al. 2012). Harvey et al. (2012) recently divided Teiidae in three subfamilies: (1) Teiinae, including the genera Ameiva (Meyer, 1795), Ameivula (Spix, 1825), Aurivela (Bell, 1843), Aspidoscelis (Fitzinger, 1843), Contomastix (Dumésil and Bibron, 1839), Cnemidophorus (Wagler 1830), Dicrodon (Dumésil and Bibron, 1839), Holcosus (Cope, 1862), Kentropyx (Spix, 1825), Medopheos (Bocourt, 1874) and Teius (Merrem, 1820); (2) Tupinambinae, including the genera Crocodilurus (Spix, 1825), Dracaena (Daudin, 1801), Salvator (Dumésil & Bibron, 1839) and Tupinambis (Daudin, 1802); and (3) Callopistinae, which contains the single genus Callopistes (Gravenhorst, 1837) (Harvey et al. 2012). However, the phylogenetic hypothesis of Teiidae based on molecular data (Reeder et al. 2002, Giugliano et al. 2007) differs substantially from the hypothesis proposed by Harvey et al. (2012).
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).
We did a cytogenetic study of five species in the family Teiidae (Ameivaameiva (Linnaeus, 1758), Cnemidophorus sp.1, Kentropyxcalcarata (Spix, 1825), Kentropyxpelviceps (Cope, 1868) and Tupinambisteguixin (Linnaeus, 1758)) using classical as well as molecular cytogenetic markers (conventional staining, heterochromatin patterns, NOR locations and chromosomal physical mapping of 18S rDNA sequences). Karyotype organization in the family is discussed.
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' - 01°55'S; 58°50' - 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).
52C002F5-7A37-5984-95AB-7863B90B8880
Satellite image of the Amazon basin showing the three different geographical areas; 1 = São Sebastião do Uatumã; 2 = Santa Isabel do Rio Negro; 3 = Tapauá; 4 = Manaus.
https://binary.pensoft.net/fig/59597
Species of the Teiinae and Tupinambinae subfamilies: collection sites, number and the analyzed animals and voucher specimens (lots) are listed. AM
: Amazonas
.
Subfamily
Species
Collection sites
Number and sex the analyzed animals
Voucher specimens (lots)
Ameivaameiva
São Sebastião do Uatumã, AM Santa Isabel do Rio Negro, AM Tapauá, AM
11 (four males; three females; four without sex identification)
INPA H33213
Cnemidophorus sp.1
Manaus, AM
13 (five males; eight females)
INPA H35018
Teiinae
Kentropyxcalcarata
São Sebastião do Uatumã, AM
4 (three males; one females)
INPA H31712
Kentropyxpelviceps
Tapauá, AM
3 (three females)
INPA H34841
Tupinambinae
Tupinambisteguixin
São Sebastião do Uatumã, AM Tapauá, AM
3 (two females; one without sex identification)
INPA H34791
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 MgCL2, 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 microscope 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) (Lowe and 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 Ameivaameiva, Kentropyxcalcarata and Kentropyxpelviceps 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). Tupinambisteguixin 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, Kentropyxcalcarata and Kentropyxpelviceps and in pair 2 in Tupinambisteguixin (Figs 2e, i, m and 3a). No differentiated sex chromosomes were observed in the analysed species.
6B7CD053-DEBB-550C-A4A3-36DDB98C6959
Karyotypes of species belonging to Teiinae: a, e, i, m in conventional Giemsa staining b, f, j, n Regions of heterochromatin evidenced by C-band technique c, g, k, o highlight the nucleolar pair impregnated with AgNO3d, h, l, p highlighted in the chromosome pair bearing the site of 18S rDNA (red) and chromosomes were counterstained with DAPI. m = Macrochromossome, mi = microchromossome. Scale bar = 10 µm.
Karyotype of Tupinambisteguixin: a in conventional Giemsa staining b Regions of heterochromatin evidenced by C-band technique c highlight the nucleolar pair impregnated with AgNO3d highlight the chromosome pair bearing the site of 18S rDNA (red) and chromosomes were counterstained with DAPI. m = Macrochromossome, mi = microchromossome. Scale = 10 µm.
https://binary.pensoft.net/fig/59599
Constitutive heterochromatin was observed in the centromeric and terminal regions in most chromosomes of Ameivaameiva, Cnemidophorus sp.1, Kentropyxcalcarata and Kentropyxpelviceps (Figs 2b, f, j, n). In Tupinambisteguixin, 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 Ameivaameiva (Fig. 2c). In Cnemidophorus sp.1, Kentropyxcalcarata and Kentropyxpelviceps, NORs were seen in the distal region of the long arms of pair 1 and in pair 2 in Tupinambisteguixin, 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 varying 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 Ameivaauberi (Cocteau, 1838) to 2n=54 in Teiusoculatus (D’orbigny & Bibron, 1837) and Teiusteyou (Daudin, 1802), besides the presence of sex chromosomes of XX/XY in Aspidocelistigristigris (Baird & Girard, 1852) and Ameivulalittoralis (Rocha, Bamberg Araújo, Vrcibradic, 2000). Some Aspidoscelis species show triploid numbers such as Aspidoscelistessalatus (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).
Basic cytogenetic data compiled from the literature for the Teiidae family.
Diploid number
(2n),
karyotypic formula
(KF),
fundamental number
(FN). Three descriptions of karyotypic formulas: (a) number of biarmed chromosomes, number of uniarmed chromosomes and number of microchromosomes; (b) chromosomes that show a gradual series of acrocentric chromosomes; (c) macrochromosome chromosomes (M) and microchromosomes (mi). For data not included in the literature, “-” is indicated.
Subfamily
Genus
Species (sensu [2])
Species (initial description)
2n
Type of KF and description
FN
Reference
Callopistinae
Callopistes
Callopistesflavipunctatus
Callopistesflavipunctatus
2n=38
c (12M+26m)
50
2
Callopistesmaculatus
Callopistesmaculatus
2n=38
c (12M+26m)
26, 50
2, 8
Teiinae
Ameiva
Ameivaameiva
Ameivaameiva
2n=50
a (0: 26: 24) b (gradual series of acrocentric chromosomes)
a (5: 19: 24) a (5: 19: 22) b(gradual series of acrocentric chromosomes)
53 51 -
14 9 18
Aspidoscelis
Aspidoscelisangusticeps
Cnemidophorusangusticeps
2n=44, 46
a (6: 20: 18), a (2: 24: 20)
50, 48
3, 16
Aspidoscelisburti
Cnemidophorusburti
2n=46
a (2: 24: 20)
48
3
Aspidosceliscalidipes
Cnemidophoruscalidipes
2n=46
a (2: 24: 20)
48
3
Aspidoscelisceralbensis
Cnemidophorusceralbensis
2n=52
-
-
4
Aspidosceliscommunis
Cnemidophoruscommunis
2n=46
a (2: 24:20)
48
3
Aspidosceliscostatus
Cnemidophoruscostatus
2n=46
a (2: 24:20)
48
3
Aspidosceliscozumelae
Cnemidophoruscozumelae
2n=49, 50
a (0: 28: 21), a (11: 19: 20)
49, 61
3, 16
Aspidoscelisdeppei
Cnemidophorusdeppei
2n=50, 52
a (0: 26: 24), a (0: 28: 24)
50, 52
3, 16
Aspidoscelisexsanguis
Cnemidophorusexsanguis
3n=69*
-
-
3, 10
Aspidoscelisflagellicaudas
Cnemidophorusflagellicaudas
3n=69*
-
-
3
Aspidoscelisgularis
Cnemidophorusgularis
2n=46
a (2: 24: 20)
48
3
Aspidoscelisguttatus
Cnemidophorusguttatus
2n=52
a (0: 28: 24)
52
3
Aspidoscelishyperythrus
Cnemidophorushyperythrus
2n=52
a (0: 28: 24)
52
3
Aspidoscelisinoratus
Cnemidophorusinoratus
2n=46
a (2: 24: 20)
48
3, 10
Aspidoscelislaredoensis
Cnemidophoruslaredoensis
2n=46
a (2: 24: 20)
48
4
Aspidoscelislineatissima
Cnemidophoruslineatissima
2n=52
a (0: 28: 24)
52
3
Aspidoscelismarmoratus
Cnemidophorusmarmoratus
2n=46
a (0: 22: 24)
46
11
Aspidoscelismaslini
Cnemidophorusmaslini
2n=47
a (14: 13: 20)
49
3
Aspidoscelismexicana
Cnemidophorusmexicana
2n=46
a (2: 24: 20)
48
3
Aspidoscelismotaguae
Cnemidophorusmotaguae
2n=46
a (2: 24: 20)
48
3
Aspidoscelisneomexicanus
Cnemidophorusneomexicanus
2n=46
a (4: 20: 22)
50
3,10
Aspidoscelisopatae
Cnemidophorusopatae
3n=69*
-
-
3
Aspidoscelisparvisocius
Cnemidophorusparvisocius
2n=46
a (2: 24: 20)
48
3
Aspidoscelisrodecki
Cnemidophorusrodecki
2n=50
-
-
1
Aspidoscelissacki
Cnemidophorussacki
2n=46
a (2: 24: 20)
48
3
Aspidoscelissptemvittatus
Cnemidophorussptemvittatus
2n=46
a (2: 24: 20)
48
3
Aspidoscelissexlineatus
Cnemidophorussexlineatus
2n=46
a (2: 24: 20), a (8: 18: 20)
48, 54
3, 5
Aspidoscelissonorae
Cnemidophorussonorae
2n=46, 3n=69*
a (4: 20: 22)
48
2, 3, 10
Aspidoscelistesselatus
Cnemidophorustesselatus
2n=46, 3n=69*
a (4: 20: 22)
50
3, 10, 15
Aspidoscelistigristigris
Cnemidophorustigristigris
2n=46(XX/XY)
a (6: 16: 24)
52
2, 10
Aspidoscelistigrisaethiops
Cnemidophorustigrisaethiops
2n=46
a (6: 16: 24)
52
3
Aspidoscelist.estebanensis
Cnemidophorust.estebanensis
2n=46
a (6: 16: 24)
52
3
Aspidoscelist.gracilis
Cnemidophorust.gracilis
2n=46
a (6: 16: 24)
52
3
Aspidoscelist.marmoratus
Cnemidophorust.marmoratus
2n=46
a (6: 16: 24)
52
3
Aspidoscelist.maximus
Cnemidophorust.maximus
2n=46
a (6: 16: 24)
52
3
Aspidoscelist.septentrionalis
Cnemidophorust.septentrionalis
2n=46
a (6: 16: 24)
52
3
Aspidoscelisubiparens
Cnemidophorusuniparens
3n=69*
-
-
3, 10
Aspidoscelisvelox
Cnemidophorusvelox
3n=69*
-
-
3
Cnemidophorus
Cnemidophorusarenivagus
Cnemidophorusarenivagus
2n=50
a (2: 24: 24)
52
9, 13
Cnemidophorusarubensis
Cnemidophorusarubensis
2n=50
a (2: 24: 24)
52
9, 13
Cnemidophoruscryptus
Cnemidophoruscryptus
2n=50
-
52
9
Cnemidophorusgramivagus
Cnemidophorusgramivagus
2n=50
-
52
9
Cnemidophoruslemniscatus
Cnemidophoruslemniscatus
2n=50
a (2: 24: 24)
52
2, 3
Cnemidophorusmurinus
Cnemidophorusmurinus
2n=50
a (2: 24:24)
52
4, 3
Contomastix
Contomastixlacertoides
Cnemidophoruslacertoides
2n=50
a (0: 26: 24)
52
6, 17
Kentropyx
Kentropyxborckiana
Kentropyxborckiana
2n=50
a (0: 26: 24)
50
12
Kentropyxcalcarata
Kentropyxcalcarata
2n=50
b(gradual series of acrocentric chromosomes)
50
12, 19
Kentropyxstriata
Kentropyxstriata
2n=50
a (0: 26: 24)
50
12
Kentropyxpaulensis
Kentropyxpaulensis
2n=50
b(gradual series of acrocentric chromosomes)
50
18
Kentropyxpelviceps
Kentropyxpelviceps
2n=50
b(gradual series of acrocentric chromosomes)
50
20
Kentropyxvanzoi
Kentropyxvanzoi
2n=50
b(gradual series of acrocentric chromosomes)
50
18
Teius
Teiusoculatus
Teiusoculatus
2n=54
a (8: 28: 18)
62
17
Teiusteyou
Teiusteyou
2n=54
a (8: 22: 24)
62
2
Crocodilurus
-
Crocodiluruslacertinus
2n=34
c (12M+22m)
46
2
Crocodilurusamazonicus
Crocodilurusamazonicus
2n=34
c (12M+22m)
46
19
Dracaena
Dracaenaguianensis
Dracaenaguianensis
2n=38
a (10:2:26)
48
2
Tupinambis
-
Tupinambisnigropunctatus
2n=36, 38
a (10: 2: 24), c (16M+22m)
46, 54
2, 7
Tupinambisquadrilineatus
Tupinambisquadrilineatus
2n=38
c (12M+26m)
-
19
Tupinambisteguixin
Tupinambisteguixin
2n=38, 36
a (10: 0: 28), (12M+24m)
48
7, 19
Salvator
Salvatormerianae
Tupinambismerianae
2n=36, 38
a (10: 0: 26), c (12M+26m)
48, 50
7, 17, 19
Polyploidy in triploid form (3n). 1 - Fritts 1969; 2 - Gorman 1970; 3 - Lowe et al. 1970; 4 - Robinson 1973; 5 - Bickham et al. 1976; 6 - Cole et al. 1979; 7 - de Smet et al. 1981; 8 - Navarro et al. 1981; 9 - Peccinini-Seale and Almeida 1986; 10 - Ward and Cole 1986; 11 - Porter et al. 1991; 12 - Cole et al. 1995; 13 - Markezich et al. 1997; 14 - Rocha et al. 1997; 15 - Walker et al. 1997; 16 - Manriquen-Moran et al. 2000; 17 - Veronese et al. 2003; 18 - Santos et al. 2007; 19 - Santos et al. 2008; 20 - Present work.
Ameivaameiva and Kentropyxcalcarata, 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 Ameivaameiva and Kentropyxcalcarata 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 Kentropyxpelviceps, whose cytogenetic characteristics are revealed for the first time in the present study.
Furthermore, karyotypic formulae composed of biarmed chromosomes, uniarmed chromosomes and microchromosomes has been described for Ameivaameiva and Kentropyxcalcarata and in the other species genera of the subfamily Teiinae (Lowe and Wright 1966, Gorman 1970, Beçak et al. 1972, Peccinini-Seale and Almeida 1986, Schmid and Guttenbach 1988, Sites et al. 1990, Veronese et al. 2003). These data show that some differences may result from different classification parameters adopted by several authors in their chromosomal analyses.
Currently, the genus Cnemidophorus is divided into four morphological groups: (1) Cnemidophoruslemniscatus including the species Cnemidophorusarenivagus (Markezich, Cole & Dessauer, 1997), Cnemidophorusarubensis (Lidth de Jeude, 1887), Cnemidophoruscryptus (Cole & Dessauer, 1993), Cnemidophorusflavissimus (Ugueto, Harvey & Rivas, 2010), Cnemidophorusgramivagus (Mccrystal & Dixon, 1987), Cnemidophoruslemniscatusespeuti (Boulenger, 1885), Cnemidophoruslemniscatusgaigei (Ruthven, 1924), Cnemidophoruslemniscatuslemniscatus (Linnaeus, 1758), Cnemidophoruslemniscatussplendidus (Markezich, Cole & Dessauer, 1997), Cnemidophoruspseudolemniscatus (Cole & Dessauer, 1993), Cnemidophorussenectus (Ugueto, Harvey & Rivas, 2010) and Cnemidophorus sp. B.; (2) Cnemidophorusnigricolor including the species Cnemidophorusleucopsammus (Ugueto & Harvey, 2010), Cnemidophorusnigricolor (Peters, 1873), Cnemidophorusrostralis (Ugueto & Harvey, 2010) and Cnemidophorus sp. A; (3) Cnemidophorusmurinus including the species Cnemidophorusmurinus (Laurenti, 1768) and Cnemidophorusruthveni (Burt, 1935) and (4) Cnemidophorusvanzoi including the species Cnemidophorusvanzoi (Baskin & Williams, 1966) (Harvey et al. 2012). It is noteworthy that several new species of this genus have been described, showing that the taxonomy of this genus has not yet been elucidated, which emphasizes the need for morphological and molecular studies in this genus. Cytogenetically, some species of Cnemidophorus have 50 chromosomes, composed of biarmed chromosomes, uniarmed chromosomes and microchromosomes (Table 2, Peccinini-Seale and Almeida 1986). However, the karyotype of Cnemidophorus sp.1 from Manaus, in Amazonas state, differs from those described for other species of the genus. This species has 2n = 48 chromosomes with the absence of a pair of microchromosomes (Table 2, present study). Non-robertsonian chromosomal rearrangements may be associated with chromosomal evolution of this genus, which favored changes in diploid number (reduction in diploid number). Another population in Amazonas state (county Manacapuru) identified as belonging to Cnemidophoruslemniscatus group has the expected diploid number of 50 chromosomes with the presence of biarmed chromosomes and uniarmed microchromosomes (0:26:24) (Sites et al. 1990). Our results show that the specimens we sampled from Manaus are karyotypically distinct from specimens we sampled from Manacapuru so Cnemidophorus sp.1 (Cnemidophoruslemniscatus group) could represent a new species.
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). Tupinambisteguixin 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.
Cytogenetic banding data compiled from the literature for the differential Teiidae family.
Nucleolar organizer regions
(NORs),
constitutive heterochromatin
(CH),
fluorescent in situ hybridization
(FISH). Locality:
Amazonas
(AM),
Bahia
(BA),
United States
(USA),
Espírito Santo
(ES),
Goiás
(GO),
Mato Grosso
(MT),
Minas Gerais
(MG),
Pará
(PA),
Rio de Janeiro
(RJ),
Rio Grande do Sul
(RS),
Rondônia
(RO),
São Paulo
(SP),
Sergipe
(SE),
Tocantins
(TO). For data not included in the literature, “-” is indicated.
Terminal region of the long arms of pair 8 Multiple NORs (not indicated pairs) Terminal region of the long arms of pair 5
- - Centromeric and terminal regions
- - -
7 5 8
Cnemidophorusarenivagus
Cnemidophorusarenivagus
-
Terminal region of the long arms of pair 1
-
-
3
Cnemidophoruscryptus
Cnemidophoruscryptus
-
Terminal region of the long arms of pair 1
-
-
3
Cnemidophorusgramivagus
Cnemidophorusgramivagus
-
Terminal region of the long arms of pair 1
-
-
3
Cnemidophoruslemniscatus
Cnemidophoruslemniscatus
-
Terminal region of the long arms of pair 1
-
-
3
Cnemidophorus sp.1
-
AM
Terminal region of the long arms of pair 1
Centromeric and terminal regions
18S rDNA (pair 1)
Present work
Contomastixlarcetoides
Cnemidophoruslarcetoides
RS
-
Centromeric region
-
6
Kentropryxcalcarata
Kentropryxcalcarata
BA, TO, MT
Distal region of the long arms of pair 1
-
-
8
Kentropryxcalcarata
Kentropryxcalcarata
AM
Distal region of the long arms of pair 1
Centromeric and terminal regions
18S rDNA (pair 1)
Present work
Kentropryxpaulensis
Kentropryxpaulensis
SP
Distal region of the long arms of pair 1
Centromeric and terminal regions
-
8
Kentropyxpelviceps
Kentropyxpelviceps
AM
Distal region of the long arms of pair 1
Centromeric and terminal regions
18S rDNA (pair 1)
Present work
Kentropryxvanzoi
Kentropryxvanzoi
RO
Distal region of the long arms of pair 1
-
-
8
Teiusoculatus
Teiusoculatus
RS
Multiple NORs (not indicated pairs)
-
-
6
Tupinambinae
Crocodilurusamazonicus
Crocodilurusamazonicus
PA
Distal region of the long arms of pair 2
Pericentromeric region
-
9
Salvatormeriane
Tupinambismerianae
TO, SP, ES
Distal region of the long arms of pair 2
Pericentromeric region
-
9
Tupinambisquadrilineatus
Tupinambisquadrilineatus
GO, TO
Distal region of the long arms of the pair 2
Centromeric, pericentromeric, interstitial, proximal and terminal regions
-
9
Tupinambisteguixin
Tupinambisteguixin
GO, TO
Distal region of the long arms of pair 2
-
-
9
Tupinambisteguixin
Tupinambisteguixin
AM
Distal region of the long arms of pair 2
Centromeric and terminal regions
18S rDNA (pair 2)
Present work
1 - Bickhan et al. 1976; 2 - Bull 1978; 3 - Peccinini-Seale and Almeida 1986; 4 - Porter et al. 1991; 5 - Rocha et al. 1997; 6 - Veronese et al. 2003; 7 - Peccinini-Seale et al. 2004; 8 - Santos et al. 2007; 9 - Santos et al. 2008
The heterochromatin patterns for Cnemidophorus sp.1, Kentropyxcalcarata, Kentropyxpelviceps and Tupinambisteguixin 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 (Crocodilurusamazonicus (Spix, 1825), Salvatormerianae (Duméril & Bibron, 1839) and Tupinambisquadrilineatus (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).
Although the five species in the family Teiidae analyzed in the present study present a conserved karyotype macrostructure, some chromosomal characteristics differentiate the karyotype of these species. In Cnemidophorus sp.1, Kentropyxcalcarata, Kentropyxpelviceps and Tupinambisteguixin, the presence of a secondary constriction localized in the distal region of pairs 1 and 2 was observed. The secondary constriction is absent in Ameivaameiva.
Secondary constrictions are typically present in a single chromosomal pair and are very common in several lizard species (Bertolloto et al. 1996, Kasahara et al. 1996, Bertolloto et al. 2002, Srikulnath et al. 2009a). This region contain genes that produce ribosomal RNA and these regions may hold nucleoli proteins during the entire process of cellular division (Guerra 1988). In such secondary constrictions, NORs are usually placed and they are identified, indirectly, by silver nitrate impregnation of the chromosomes. Such impregnation marks only nucleoli proteins involved in the transcriptional activity of ribosomal genes of the 45S family. NORs may be located in a single chromosomal pair, a basal characteristic already reported for different lizard species (Porter et al. 1991).
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 Teiidae, such as Kentropyx (Kentropyxcalcarata, Kentropyxpaulensis (Boettger, 1893) and Kentropyxvanzoi Gallagher & Dixon, 1980), Crocodilurus (Crocodilurusamazonicus), Cnemidophorus (Cnemidophorusarenivagus, Cnemidophoruscryptus, Cnemidophorusgramivagus and Cnemidophoruslemniscatuslemniscatus), Salvator (Salvatormerianae) and Tupinambis (Tupinambisquadrilineatus and Tupinambisteguixin). Localization of the NORs is important for characterizing species and evolutionary studies among teiid lizards (Santos et al. 2007, 2008).
Tupinambisteguixin 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, Ameivaameiva, Cnemidophorus sp.1, Kentropyxcalcarata and Kentropyxpelviceps, also have simple NORs, but they are located in distinct chromosomal pairs. In Cnemidophorus sp.1, Kentropyxcalcarata and Kentropyxpelviceps, a secondary constriction was seen in pair 1 while in Ameivaameiva occurred in pair 7. The NOR data analyzed for Ameivaameiva and Kentropyxcalcarata 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 Kentropyxpelviceps they are new data.
Two populations of Ameivaameiva 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 Ameivaameiva 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 Ameivaameiva is a specie complex, as other teiids like Ameivulaocellifera (Spix, 1825) (Arias et al. 2011) or Cnemidophoruslemniscatus (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, Terencio 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 Ameivaauberi (Cocteau, 1838). In Aspidoscelismarmorata (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.
Acknowledgments
This work was supported by the Universidade Federal do Amazonas (UFAM), the graduate program of INPA Genética, Conservação e Biologia Evolutiva, Rede BioPHAM (CNPq/FAPEAM grant number: 563348/2010-0); Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (grant number: 474617/2013-0); FAPEAM (020/2013); CAPES (Pró-Amazônia – grant number 23038.009447/2013-45, 3295/2013). Species were collected with a permit issued by the Chico Mendes Institute for Biodiversity Conservation (ICMBio/SISBIO license number 41825-1). 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.
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