Castiglia et al.

Zoological Studies 2013, 52:60

http://www.zoologicalstudies.com/content/52/1/60

RESEARCH Open Access

Pattern of chromosomal changes in ‘beta’ Anolis

(Norops group) (Squamata: Polychrotidae)
depicted by an ancestral state analysis
Riccardo Castiglia1*, Oscar Flores-Villela2, Alexandra Maria Ramos Bezerra3,4, Antonio Muñoz5
and Ekaterina Gornung1

Abstract
Background: Neotropical lizards, genus Anolis (Polychrotidae), with nearly 380 species, are members of one of the
most diversified genera among amniotes. Herein, we present an overview of chromosomal evolution in ‘beta’ Anolis
(Norops group) as a baseline for future studies of the karyotypic evolution of anoles. We evaluated all available
information concerning karyotypes of Norops, including original data on a recently described species, Anolis
unilobatus. We used the phylogeny of Norops based on DNA sequence data to infer the main pattern of
chromosomal evolution by means of an ancestral state analysis (ASR).
Results: We identified 11 different karyotypes, of which 9 in the species had so far been used in molecular studies.
The ASR indicated that a change in the number of microchromosomes was the first evolutionary step, followed by
an increase in chromosome numbers, likely due to centric fissions of macrochromosomes. The ASR also showed
that in nine species, heteromorphic sex chromosomes most probably originated from six independent events.
Conclusions: We observed an overall good correspondence of some characteristics of karyotypes and species
relationships. Moreover, the clade seems prone to sex chromosome diversification, and the origins of five of these
heteromorphic sex chromosome variants seem to be recent as they appear at the tip nodes in the ancestral
character reconstruction. Karyotypic diversification in Norops provides an opportunity to test the chromosomal
speciation models and is expected to be useful in studying relationships among anole species and in identifying
cryptic taxa.
Keywords: Anole; Anolis; Chromosomal evolution; Cytogenetics; Norops; Sex chromosomes; Systematics

Background and mainland Americas from Texas to tropical South

The genus Anolis includes nearly 380 species of neotrop- America (reviewed in Losos 2009), where they exhibit a
ical lizards and is the most species-rich amniote genus wide array of ecological and morphological specializa-
(Uetz 2013). Anolis was recently the subject of a system- tions (Etheridge 1959; Williams 1983; Losos et al. 1998;
atic revision that resulted in it being split into eight dif- Köhler 2000). Anoles have been thoroughly investigated
ferent genera (Nicholson et al. 2012). However, this in various ecological, behavioral, and evolutionary stud-
systematic arrangement has been vigorously criticized ies (reviewed in Losos 2009). One species, the green
(e.g., Poe 2013), and in this paper, we adopted the clas- anole lizard Anolis carolinensis, was the first reptile gen-
sical view that considers anole lizards as being com- ome to be sequenced. This achievement may consider-
prised of a single genus (Losos 2009; Uetz 2013). Anole ably advance research outcomes on various biological
species are distributed throughout the Caribbean islands aspects of anoles (Alföldi et al. 2011).
Based on a single osteological character, i.e., the pres-
ence/absence of caudal transverse processes, Etheridge
* Correspondence: [email protected]
1
Dipartimento di Biologia e Biotecnologie ‘Charles Darwin’, Università di
(1959) divided Anolis into major sections: ‘alpha’ anoles,
Roma ‘La Sapienza’, via A. Borelli 50, Rome 00161, Italy which lack this morphological trait, and ‘beta’ anoles
Full list of author information is available at the end of the article

© 2013 Castiglia et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
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(Norops), which carry the trait. In a recent revision and A. unilobatus, was newly studied and included in this sur-
in a previous report, based on molecular phylogenetics vey. One male and three female A. unilobatus were col-
(Poe 2004; Nicholson et al. 2012), Norops was recog- lected in dry tropical forest located in the Reserva da
nized as a monophyletic group that includes 169 species. Biosfera Sepultura, Chiapas State, Mexico. Field numbers
Karyological studies in anoles were very intensive in cited in the text (RCMX) belong to Riccardo Castiglia. Ani-
the 1970s and 1980s, especially studies that were con- mals were managed following all applicable institutional
ducted by Gorman and coworkers who defined karyo- animal care guidelines, and all required state and federal
types of numerous species (Gorman 1973). Since then, permits were obtained.
scientific interest has shifted to other fields of study, and Femurs were taken from individuals after an intraperito-
the chromosomal evolution of anoles has not been fur- neal injection of vinblastine sulfate, Velbe (Lilly, Italy)
ther investigated. Indeed, only a few papers in this re- (1:1,000) for 1 h. Samples were then processed following
gard were published from 1980 to the present (Blake procedures described in Castiglia et al. (2010). Pictures of
1986; Brandley et al. 2006; Castiglia et al. 2010). How- metaphases were collected using a Photometrics Sensys
ever, anoles exhibit remarkable inter- and intraspecific 1600 digital camera (Roper Scientific Photometrics, Tucson,
variations in chromosome number and morphology as AZ, USA).
well as in the presence/absence of heteromorphic sex DNA was extracted from tissues preserved in 100%
chromosomes. For this reason, investigating speciation ethanol using the QIAmp tissue extraction kit (Qiagen,
of anoles as driven by chromosomal changes remains Hilden, Germany). The mitochondrial ND2 gene, five
challenging. Moreover, chromosomal variability is ex- transfer (t) RNAs, the origin of light-strand replication,
pected to be useful in studying relationships among and a portion of the cytochrome oxidase subunit 1
anole species and in identifying cryptic taxa. (CO1) gene were amplified with primers designed by
Herein, we present an overview of the chromosomal Macey et al. (1999) as follows: L4160 ND1 5′-
evolution in Norops as a baseline for future studies of CGATTCCGATATGACCARCT-3′, H4980 ND2 5′-
the karyotypic evolution of anoles. Previous phylogenetic ATTTTTCGTAGTTGGGTTTGRTT-3′, L4437 tRNA-
reconstructions of anoles, which also included chromo- Met 5′-AAGCTTTCGGGCCCATACC-3′, and H5934a
somal data, applied them in the same matrix together CO1 5′-AGRGTGCCAATGTCTTTGTGRTT-3′.
with molecular and morphological characters (Poe 2004; We used a Bayesian algorithm to construct a phyl-
Brandley et al. 2006). In contrast, our approach consists ogeny based on mitochondrial DNA sequences (NDH2,
of inferring the pattern of chromosomal evolution in a 5 tRNAs, the origin of light-strand replication, and a
phylogeny purely based on molecular data. A similar ap- portion of the CO1 gene) that included all 62 species for
proach was recently used for an ancestral state recon- which mtDNA sequences were available in GenBank
struction (ASR) of chromosomal characters in some (mainly published by Nicholson et al. 2005), plus A. uni-
other reptilian clades, such as the Phrynosomatidae lobatus. The final alignment consisted of 1,406 bp.
(genus Sceloporus) (Leaché and Sites 2009), Gekkonidae Data were partitioned by the codon position with a
(Gamble 2010), and Serpentes (Oguiura et al. 2009). For separate partition for each gene. The general time re-
this study, we reviewed all available data on karyotypes versible substitution model with gamma-distributed rate
of Norops and also included original data on one heterogeneity and the proportion of invariant sites were
recently described species, A. unilobatus (Köhler and used for each partition. For the Bayesian analysis, two
Vesely 2010). We used the phylogeny of Norops based independent Monte Carlo (MCMC) analyses were run
on DNA sequence data to infer the main pattern of with MrBayes 3.2 (Ronquist et al. 2012). We used 3 ×
chromosomal evolution by means of an ASR. ASR ana- 106 generations, four chains, and a burn-in of 10% of
lysis allowed us to test whether karyotypic diversity the generated tree. Anolis cristatellus (AF528724) and A.
matched phylogenetic relationships among species and distichus (AF528725) were used as outgroups (Nicholson
enabled us to track the direction of chromosomal et al. 2005).
change. We also tested a hypothesis of an ancient com- The consensus tree was pruned so as to eliminate species
mon origin of heteromorphic sex chromosomes vs. their for which chromosomal data were unknown. The resulting
independent origin in different species. tree, including 31 Norops species, was used for the ASR
analysis that was performed with Mesquite 2.74 software
Methods (Maddison and Maddison 2011). A maximum likelihood
Materials (ML) reconstruction method was used to reconstruct the
We examined published karyotypes of Norops species for ancestral states. We used the one-rate MK1 model (Lewis
the shape of macrochromosomes, the number of micro- 2001), the only model in Mesquite allowing multistate char-
chromosomes, the presence/absence of sex chromosomes, acters, and proportional likelihood (PL) values were used to
and their size and shape (Table 1). One additional species, determine which state was the most likely. Preliminarily,
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Table 1 Karyological data of Norops species

Species Karyotype Macrochromosomes Microchromosomes Sex chromosomes Source
Anolis mestrei 28 14 M/SM 14 - Gorman and Atkins (1968)
Anolis homolechis 28 14 M/SM 14 - Gorman and Atkins (1968)
Anolis quadriocellifer 28 14 M/SM 14 - Gorman and Atkins (1968)
Anolis rubribarbus 28 14 M/SM 14 - Gorman and Atkins (1968)
Anolis sagrei 29-30a 18 M/SM Male 11 Female 12a X1X2Y - X1X1X2X2 a De Smet (1981)
(type-B)
Anolis lineatopus 30 (type-A) 14 M/SM 16 - Gorman (1965, 1969)
Anolis valencienni 30 (type-A) 14 M/SM 16 - Gorman and Atkins (1968)
Anolis garmani 30 (type-A) 14 M/SM 16 - Gorman and Atkins (1968)
Anolis lineatus 30 (type-A) 14 M/SM 16 - Gorman and Atkins (1968)
Anolis nitens = Anolis 30 (type-A) 14 M/SM 16 - Gorman (1973)
chrysolepis
Anolis auratus 30 (type-A) 14 M/SM 16 - Gorman et al. (1967),
De Smet (1981)
Anolis aquaticus 30 (type-A) 14 M/SM 16 - Gorman unpublished
(in Lieb 1981)
Anolis conspersus 30 (type-B) 14 M/SM 16 XY (seventh) (similar Gorman and Atkins (1966)
sizes)
Anolis opalinus 30 (type-B) 14 M/SM 16 XY (seventh) (similar Gorman (1969)
sizes)
Anolis onca 30 (type-B) 14 M/SM 16 XY (seventh) (similar Gorman (1969)
sizes)
Anolis grahami 30 ~ 37 14 M/SM-10 M/SM, 9A, ST 16-18b - De Smet (1981)
Anolis biporcatus 29-30 Male: 13 M/SM, 1 A; female: Male 15, female 16 X1X2Y - X1X1X2X2 Gorman and Atkins (1966)
(type-A) 12 M/SM, 2 A
Anolis nebulosus 30 (type-C) 14 M/SM 16 XY (different sizes) Lieb (1981), Castiglia et al.
(2010)
Anolis quercorum 30 (type-C) 14 M/SM 16 XY (different sizes) Lieb (1981)
Anolis gracilipes 36 8 M/SM; 12 A 16 - Gorman (1973)
Anolis liogaster 38 6 M/SM; 16 A 16 XY (different sizes) Lieb (1981)
Anolis omiltemanus 38 6 M/SM; 16 A 16 XY (different sizes) Lieb (1981)
Anolis gadovii 36 8 M/SM; 12 A 16 XY (different sizes) Lieb (1981)
Anolis unilobatus 40 20 SM/ST, 4 M 16 XY (different sizes) Present data
Anolis tropidolepis 40 24c 16 - Gorman (1973)
Anolis fuscoauratus 40 24c 16 - Gorman (1973)
Anolis humilis 40 24c 16 - Gorman (1973)
Anolis cupreus 40 4 M/SM; 20 A 16 - Gorman (1973)
Anolis polylepis 40 24c 16 - Gorman (1973)
c
Anolis tropidonotus 40 24 16 - Gorman (1973)
Anolis capito 40 24c 16 - Gorman (1973)
c
Anolis limifrons 40 24 16 - Gorman (1973)
Anolis lionotus 40 24c 16 - Gorman (1973)
Anolis tropidogaster 40 4 M/SM; 20 A 16 - Gorman (1973)
Anolis intermedius 40 24c 16 - Gorman unpublished
(in Lieb 1981)
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Table 1 Karyological data of Norops species (Continued)

Anolis dunni 40 4 M/SM; 20 A 16 XY (different sizes) Lieb (1981)
Anolis taylori 40 4 M/SM; 20 A 16 XY (different sizes) Lieb (1981)
Anolis nebuloides 42 ND ND XY (different sizes) Lieb (1981)
M, metacentric; SM, submetacentric; A, acrocentric; ST, subtelocentric; ND, no distinction was made between macro- and microchromosomes. aInferred from the
male karyotype; bthe presence of inter-individual variation of microchromosomes; cthe morphology of macrochromosomes was not studied.

we also performed an ASR by maximum parsimony (MP) The number of microchromosomes was then coded as
(not shown) and altered the assumption of an equal cost a five-state character: state 0, 14 microchromosomes;
for state changes building different step matrices in Mes- state 1, 12 microchromosomes (female condition in
quite. The results obtained with the ML and MP methods A. sagrei); state 2, 16 microchromosomes; state 3, 18
were highly congruent. Thus, we decided to show only the microchromosomes; and state 4, the absence of micro-
results obtained with ML because one problem with the chromosomes (only observed in A. nebuloides). For
parsimony ASR is the fact that supports at nodes are micro- and macrochromosomes, the outgroup was
usually represented as unambiguous, often obscuring the coded as uncertain due to the observed variability in the
actual degree of support for alternative character states number of chromosomes in the outgroup species (Olmo
(Cunningham 1999). and Signorino 2005; Brandley et al. 2006).
We coded different karyotypic characteristics as cat- Finally, we analyzed the emergence of heteromorphic
egorical characters (as for Serpentes in Oguiura et al. sex chromosomes. We identified three different patterns
2009). In fact, both the chromosomal number (2n) and of sex chromosomes in Norops (Table 1) and coded
the number of chromosomal arms (FN) may discretely them in four states: state 0, the absence of hetero-
change by several simultaneous chromosomal rearrange- morphic sex chromosomes; state 1, heteromorphism at
ments. The presence/absence of sex chromosomes can macrochromosome pair 7 (same size); state 2, multiple
be used as a straightforward categorical character. sex chromosomes X1X1X2X2/X1X2Y; and state 3, hetero-
The choice of an outgroup is a crucial step in the morphism at one macrochromosome pair (different
ASR. According to Nicholson et al. (2005), the sister sizes). Character states 2 and 3 included species with
group of Norops is the clade of anoles composed of spe- slightly different heteromorphic sex chromosomes. Add-
cies that belong to the ‘bimaculatus’, ‘cristatellus’, and itional separation using more states was not feasible. We
‘distichus’ groups. Unfortunately, there is uncertainty also coded 11 species as uncertain in view of the fact
about the identity of the basal karyotype in this highly that the macrochromosomes in those species have not
diversified clade (Olmo and Signorino 2005; Brandley been studied in sufficient detail. The outgroup was
et al. 2006). All of these species are characterized by a coded as state 2 because of the presence of multiple sex
heteromorphic XY or an X1X1X2X2/X1X2Y sex chromo- chromosomes in the outgroup species.
some system. Therefore, the outgroup was considered
uncertain with the exception of the constitution of sex
chromosomes. We first performed an ASR by consider- Results and discussion
ing the karyotype as a single character with nine differ- Results
ent states corresponding to different karyotypes. The karyotype of A. unilobatus
We then separately considered some karyotype charac- Karyotypes were made from metaphase chromosomes of
teristics. First, we coded the different karyotypes in Nor- three females (RCMX70, RCMX71, and RCMX91) and
ops as a two-state character: state 0, karyotypes with 7 ~ one male (RCMX90) of A. unilobatus. All samples
9 pairs of macrochromosomes (28 < 2n < 30), and state 1, showed 2n = 40 with 24 macrochromosomes and 6
karyotypes with 12 pairs of macrochromosomes (2n = microchromosomes, so they are designated because they
40). The karyotype of A. nebuloides (2n = 42) was coded markedly differed in size (Figure 1).
as state 1, even though it does not show a distinction be- The macrochromosomes of A. unilobatus included
tween macro- and microchromosomes, because the dip- two pairs of metacentric, three pairs of submetacentric,
loid number is similar to that observed in species with a and seven pairs of subtelocentric/acrocentric chromo-
high number of macrochromosomes. The polymorphic somes (Figure 1). The shape of the microchromosomes
karyotype in A. grahami (30 < 2n < 37) was coded as was not easily discernible, but some biarmed elements
state 1 since polymorphic states are not allowed under were observed in more elongated metaphases. In the
the likelihood ASR in Mesquite. However, in the prelim- male, the smallest metacentric chromosomes (pair 11)
inary analysis with the parsimony ASR, A. grahami was were heteromorphic in size and likely corresponded to
coded as polymorphic. sex chromosomes of the XY type.
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Figure 1 Karyotype of Anolis unilobatus (2n = 40). Female (above) and male (below). Note the heteromorphic pair 11 in the male karyotype.

A literature survey of Norops karyotypes Gorman 1969), we can say that the
We found 11 different karyotypes, of which 9 were from macrochromosomes were identical to those
species previously used in molecular studies. They are observed in the 2n = 28 karyotype. The only
briefly described below. Ideograms are shown for the difference between the 2n = 28 and 2n = 30 (type-A)
eight karyotypes for which the morphology of the karyotypes was the presence of one additional pair
macrochromosomes was studied in detail and molecular of microchromosomes. The 2n = 30 (type-A)
data were available (Figure 2). The karyological informa- karyotype was found in seven species (Table 1).
tion for each species is summarized in Table 1:  2n = 30 (type-B). Three species, A. opalinus, A.
conspersus, and A. onca, had a karyotype identical to
 2n = 28. This karyotype is composed of seven pairs the 2n = 30 (type-A), except for the smallest pair of
of biarmed macrochromosomes and seven pairs of macrochromosomes (number 7) which was in a
microchromosomes. The morphology of the heteromorphic condition (submetacentric and
macrochromosomes seemed to be conserved acrocentric homologues were of similar size), thus
throughout the group, even if we could not exclude matching the XY sex chromosome system.
small differences in the arm ratio. The chromosomes Unfortunately, no females of these species were
were all meta-submetacentric and unevenly decreasing studied.
in size. The shape of the microchromosomes was not  2n = 29–30 (type-A). Anolis biporcatus had a
classifiable in major published karyotypes. However, in karyotype that resembled the 2n = 30 (type-A) but
A. rubribarbus and A. mestrei, one or two pairs of showed an X1X1X2X2/X1X2Y multiple sex
microchromosomes were distinctly metacentric chromosome system (2n = 29 in males, 2n = 30 in
(Gorman and Atkins 1968). The 2n = 28 karyotype females; Gorman and Atkins 1966). X1 is an
was found in four species: A. mestrei, A. homolechis, acrocentric chromosome and X2 is a
A. quadriocellifer, and A. rubribarbus (Table 1). microchromosome. The Y chromosome is
 2n = 30 (type-A). This karyotype is composed of metacentric and is similar in size to X1.
seven pairs of biarmed macrochromosomes and  2n = 29–30 (type-B). Males in A. sagrei showed a
eight pairs of microchromosomes. Examining peculiar karyotype composed of 9 pairs of
published karyotypes (Gorman and Atkins 1968; macrochromosomes and 11 pairs of
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Figure 2 Ideograms of eight different karyotypes found in Norops males. Dark areas indicate centromeric regions. Asterisks denote that the
karyotype with 2n = 32 is one of the karyotypes found in A. grahami, characterized by a 2n variation. See text for explanation.

microchromosomes (De Smet 1981). The shape of insufficient data to discuss variations of
the macrochromosomes, however, differed from macrochromosomal complements within the 2n = 40
both the 2n = 28 and 2n = 30 karyotypes. In addition, karyotype because the morphology of the
there were two X chromosomes and one Y macrochromosomes so far has been studied in only
chromosome (X1X2Y) in males, all of which were three species: A. dunni, A. taylori (in Lieb 1981),
microchromosomes. The female karyotype has not and A. unilobatus (this work). The first two species
been studied yet, but it is expected to be 2n = 30 shared a very similar 2n = 40 karyotype with one
with four X chromosomes (X1X1X2X2). large metacentric autosomal pair. However, they
 2n = 40. This karyotype has 12 pairs of have not been studied by molecular methods, and
macrochromosomes and 8 pairs of therefore, it is not possible to place them in a
microchromosomes. Unfortunately, there were phylogenetic context. In addition, Gorman (1973)
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reported a similar, although haploid, karyotype for A. Phylogenetic tree

tropidogaster. Heteromorphic X and Y (different-sized) The phylogenetic tree based on mtDNA genes including
chromosomes were described for A. dunni, A. taylori, all Norops species was fully congruent with previously
and A. unilobatus. published phylogenies (Nicholson et al. 2012). The
 2n = 30 (type-C). Anolis nebulosus showed one phylogenetic analysis placed A. unilobatus (GenBank
more 2n = 30 karyotype, but the chromosome arm accession number KF796613) close to A. sericeus and
ratios in the macrochromosomal complement A. isthmicus (with 100% Bayesian posterior probabil-
differed with respect to the abovementioned ities). The latter two species were not included in the
‘ordinary’ 2n = 30 (type-A) and (type-B). In some tree (Figure 3) because their karyotypes were not yet
specimens, there were at least two pairs of available. The affinities found with the phylogenetic ana-
chromosomes in a heterozygous condition of an lysis concurred with those using morphological data. In
unknown nature (Castiglia et al. 2010). A similar fact, A. unilobatus was only recently distinguished from
karyotype may be that found in A. quercorum. A. sericeus and currently belongs to the same ‘species
These two karyotypes share an apparently similar complex’ sensu Köhler and Vesely (2010). The resulting
macrochromosomal constitution, and both have topology, once pruned by eliminating species the karyo-
XY chromosomes that are similar in shape and types of which were not available, is depicted in Figure 3,
size (Lieb 1981; Castiglia et al. 2010). 4, 5, and 6. Some basal nodes were numbered from 1
 2n = 30 ~ 37. Anolis grahami was characterized by (the ancestor of all Norops) to 5 to facilitate the descrip-
extensive chromosomal polymorphism with tion of ancestral states.
chromosome numbers (2n) ranging 30 ~ 37 due to
multiple fission events within the Ancestral state reconstruction
macrochromosomal complement as well as by The ASR of anole karyotypes under ML is shown in
eight or nine pairs of microchromosomes (Blake Figure 3. It shows ambiguity at node 1, corresponding to
1986). Only the karyotype with 2n = 32 is shown the basal karyotype of all Norops, either 2n = 28 (PL = 0.59)
as an ideogram in Figure 2. or 2n = 30 (type-A; PL = 0.36) (Figure 3). The 2n = 40
 2n = 42 (not figured). Anolis nebuloides showed a karyotype was more derived and characterized a monophy-
very distinctive karyotype with the highest diploid letic group of 11 species (node 5).
number among Norops (Lieb 1981). Except for The ASR for macrochromosomes (Figure 4) showed a
the four large meta-submetacentric chromosomes, monophyletic group including all species characterized
the autosomes had an uncertain morphology. It is by a high chromosome number (2n = 40; node 5). The
worth noting that this karyotype was unique only exceptions were A. nebuloides (2n = 42) and A. gra-
among Norops because chromosomes could not hami (polymorphic), in which high diploid numbers oc-
be divided into macro- and microchromosomes. curred independently.
Moreover, the heteromorphic XY chromosomes For microchromosomes, the ASR retrieved a tree with
were of very different sizes. The X was a large uncertainty in the ancestral state of all Norops (node 1;
metacentric chromosome, and the Y was a Figure 5). Thus, states 0 (14 microchromosomes; PL =
medium-sized subtelocentric one. 0.63) and 2 (16 microchromosomes; PL = 0.35) were the
 2n = 36 (not figured). Anolis gracilipes had a 2n = 36 most likely to have been retrieved.
karyotype with four pairs of metacentric and six pairs In the ASR for the origin of heteromorphic sex chro-
of acrocentric macrochromosomes and 16 pairs of mosomes (Figure 6), the ancestral node for all Norops
microchromosomes (reported by Gorman (1973) as was coded without heteromorphic sex chromosomes
‘Gorman unpublished’). This karyotype is very similar (PL = 0.99). Six independent occurrences of hetero-
(or identical) to that observed in A. gadovii (Lieb 1981). morphic sex chromosomes were observed. It is worth
In that species, Lieb (1981) observed heteromorphic noting that the same types of sex chromosomes, the het-
sex chromosomes as two metacentrics of different eromorphism of macrochromosome 7 and multiple sex
sizes. Molecular data are lacking for these 2n = 36 chromosomes, resulted from independent origins in
species, so it is impossible to properly place them in a groups of three (A. conspersus, A. opalinus, and A. onca)
phylogenetic context. and two species (A. sagrei and A. biporcatus), respect-
 2n = 38 (not figured). Anolis liogaster and A. ively. Heteromorphic sex chromosomes of different sizes
omiltemanus (Lieb 1981) had a karyotype similar resulted at the origin of the monophyletic group com-
to that found in A. taylori (2n = 40) but with two posed of A. nebuloides, A. nebulosus, and A. quercorum.
pairs of large biarmed chromosomes instead of It is not clear when these heteromorphic chromosomes
one. No molecular data are available for these arose. Their formation could have occurred at node 4
species. (PL = 0.27) but was more likely to have occurred in the
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Figure 3 Ancestral state reconstruction of Norops karyotypes. The outgroup is coded as uncertain. Numbers 1 ~ 5 indicate basal nodes as
discussed in the text. All nodes are supported by Bayesian posterior probabilities of 90% ~ 100% with the exception of nodes marked with an
asterisk, for which values were 50% ~ 89%.

Figure 4 Ancestral state reconstruction of macrochromosomes. The out-group was coded as uncertain. Numbers 1 ~ 5 indicate basal nodes
as discussed in the text. Nodes are supported as described in Figure 3.
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Figure 5 Ancestral state reconstruction of microchromosomes. Numbers 1 ~ 5 indicate basal nodes as discussed in the text. Nodes are
supported as described in Figure 3.

ancestor of the three species (PL = 0.84). Node 5, basal accomplished by a change in chromosomal numbers
to 2n = 40 species, was uncertain under ML. Under MP, from 2n = 30 to 2n = 40. The increase in diploid num-
this node was coded equally as state 0 (absence of het- bers may be explained by a series of five centromeric fis-
eromorphic XY) or 1 (heteromorphic XY) (not shown). sions that occurred in five pairs of metacentric
macrochromosomes in the 2n = 30 chromosome
Discussion complement, which produced chromosomes of the 2n =
Karyotype evolution in Norops 40 karyotype. This is supported by the comparison of
We observed an overall good correspondence of some the shape of the macrochromosomes in the two karyo-
characteristics of karyotype and species relationships. typic forms. Indeed, macrochromosomes are meta-
The state change in the number of macrochromosomes submetacentric in karyotypes with a low diploid number
and microchromosomes, which altered the diploid num- (2n = 28 ~ 30), while in karyotypes with a high diploid
ber, marked monophyletic groups of species. This cor- number (2n = 40 ~ 42), macrochromosomes are almost
respondence can most likely be ‘predictive’ of the all acro-submetacentric (Table 1). Moreover, some other
phylogenetic position of other species for which molecu- differences between the 2n = 28 ~ 30 and 2n = 40 karyo-
lar data are not available. For example, two of the spe- types are evident. In fact, the presently studied A.
cies with 2n = 40 studied by Lieb (1981), A. dunni and unilobatus had several submetacentric chromosomes
A. taylori, can possibly be placed within the monophy- probably obtained by pericentric inversions that followed
letic group that was derived from the ancestor at node 5, chromosomal fissions. Interestingly, one species, A. gra-
characterized by 2n = 40. hami, showed an extensive chromosome number poly-
To summarize the results of the ancestral state ana- morphism (2n = 30 ~ 37), probably due to multiple
lysis, the first step during chromosomal evolution of Robertsonian fission events in metacentric macrochro-
Norops consisted of the emergence or disappearance of mosomes (Blake 1986). The high chromosomal number
a pair of microchromosomes (from node 1 to 2). These found in A. nebuloides (2n = 42) also seems to be due to
events were almost equally probable under the ML fission events (Figure 4). In fact, the common ancestor
reconstruction. of A. nebuloides and A. quercorum had a PL = 0.94 for a
The second main event took place between nodes 4 low number of macrochromosomes. The present obser-
and 5, where an increase in the number of macrochro- vations concur with the hypothesis that chromosomal
mosomes from 7 ~ 9 to 12 pairs took place, fission is a characteristic trait of Norops. Other similar
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Figure 6 Ancestral state reconstruction of the origin of heteromorphic sex chromosomes. Numbers 1 ~ 5 indicate basal nodes as
discussed in the text. Nodes are supported as described in Figure 3.

studies should be planned to understand whether biporcatus, A. conspersus, A. nebuloides, A. nebulosus, A.

chromosomal fission is common throughout the genus onca, A. opalinus, A. quercorum, A. sagrei, and A. unilo-
Anolis. At the moment, it was suggested to occur in at batus. Furthermore, in A. carolinensis, sex microchro-
least one more species of anole, Anolis monticola mosomes (XY) were identified by means of in situ
(Webster et al. 1972). hybridization as two identical microchromosomes
According to Nicholson et al. (2005), mainland species (Alföldi et al. 2011). Thus, the absence of evident hetero-
of Norops originated from a West Indian ancestor. In morphic sex chromosomes does not exclude the possi-
our tree, mainland species all arose from node 3. bility of genotypic sex determination.
Colonization of the mainland almost certainly was car- Our survey showed that there are basically three types
ried out by a 2n = 30 (type-A) ancestor (PL = 0.99 at of heteromorphic sex chromosomes in the Norops
node 3, in Figure 3). This colonization was followed by group. Thus, in males of A. conspersus, A. onca, and A.
an important change in the chromosomal number and a opalinus, the smallest pair of macrochromosomes (num-
high level of karyotypic diversification. Moreover, karyo- ber 7) is heteromorphic. In this pair, one chromosome is
typic diversity within the mainland 2n = 40 karyotype metacentric, while its homologue is acrocentric and of
could possibly have been underestimated because, for similar size. Interestingly, A. opalinus and A. conspersus
many species, macrochromosomes have not yet been are phylogenetically close, as they fall in the same
studied. One can speculate that chromosomal evolution monophyletic group with A. grahami and A. garmani.
during adaptive radiation can be driven by adaptation to However, the ASR indicated likely independent origins
new environments (the ‘canalization model’ of Bickham of sex chromosome heteromorphism in A. opalinus and
and Baker (1979)). However, no empirical data are cur- A. conspersus (Figure 6). Furthermore, according to
rently available to support this issue, and more data on Pokorná and Kratochvíl (2009), sex-determining mecha-
island Norops species are required to properly assess the nisms in Squamata evolved by one-way transition, i.e.,
rate of karyotypic diversification between continental from the absence to the emergence of sex chromosomes;
and island anoles. therefore, heteromorphic sex chromosomes must have
been absent in the common ancestor of the four species
Sex chromosome evolution of the clade. This implies a motivating hypothesis that
Sex chromosome heteromorphism was described in sex chromosomes arose independently in A. opalinus
nine species of Norops included in this analysis: A. and A. conspersus. Considering A. onca, which is not
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phylogenetically related to the two aforementioned spe- some characteristics of karyotypes (number of macro- and
cies, the analogous heteromorphism of chromosome pair microchromosomes) and species relationships. However,
7 seems to be another independent event. detailed comparative analysis of the morphology of macro-
The second type of sex chromosomes with X1X2Y chromosomes is still lacking, and here, we were the first to
males and X1X1X2X2 females was described in A. bipor- tentatively categorize karyotypes according to macrochro-
catus (Gorman and Atkins 1966) and A. sagrei (De Smet mosome morphology and diploid number. Moreover, the
1981). Yet, the sex chromosomes in these two species clade seems prone to sex chromosome diversification, and
obviously differ because one of the X chromosomes and the origins of five heteromorphic sex chromosome variants
the Y chromosome are medium-sized in A. biporcatus, seem to be recent, as they appear at the tip nodes in the an-
while in A. sagrei, all sex chromosomes are shaped as cestral character reconstruction. The observed karyotypic
microchromosomes (Gorman and Atkins 1966; De Smet diversification in Norops provides an opportunity to test
1981). Moreover, A. sagrei and A. biporcatus are unre- the chromosomal speciation models and is expected to be
lated species (Figure 6), and their sex chromosomes useful in studying relationships among anole species and in
might thus have occurred independently. The presence identifying cryptic taxa.
of multiple sex chromosomes is usually due to
Competing interests
autosome-sex chromosome translocations (Ashley 2002). The authors declare that they have no competing interests.
The occurrence of such rearrangements has not been
studied in Anolis, but our review indicates that the genus Authors’ contributions
RC conceived the research and performed the analysis. RC, AMRB, and AM
may be prone to this kind of rearrangement. collected the specimens. RC, AMRB, and EG performed the laboratory work.
The third sex chromosome constitution was observed in RC and EG wrote the first version of the manuscript; OFV and AMRB critically
A. nebulosus, A. quercorum, A. nebuloides, and A. uniloba- read the manuscript and made modifications. All authors read and approved
the final manuscript.
tus, in which heteromorphic X and Y chromosomes are of
different sizes (Lieb 1981; Castiglia et al. 2010). This condi- Acknowledgements
tion might have easily occurred in the common ancestor of We thank Gunther Köehler for his help in identifying Anolis unilobatus and
Flavia Annesi for sequencing. Secretaria de Medio Ambiente y Recursos
A. nebulosus, A. quercorum, and A. nebuloides (Figure 6).
Naturales (SEMARNAT) granted licenses (no. FOV540110) for specimen
The ancestor at node 5 had an uncertain state because collection. This work was supported by funds from ‘Progetti di Ricerca, di
many species have not been studied in detail. Other species Università’ for RC, and AMRB received a postdoctoral fellowship and an
‘Atração de Jovens Talentos’ researcher fellowship from Conselho Nacional
of Norops with 2n = 40, albeit not studied phylogenetically,
de Ciência e Tecnologia (CNPq).
showed sex chromosomes of different sizes (Lieb 1981).
Therefore, we cannot exclude that all descendants from Author details
1
Dipartimento di Biologia e Biotecnologie ‘Charles Darwin’, Università di
node 5 have this type of heteromorphic sex chromosomes.
Roma ‘La Sapienza’, via A. Borelli 50, Rome 00161, Italy. 2Museo de Zoología,
Additional chromosomal studies of 2n = 40 species are Facultad de Ciencias, Universidad Nacional Autónoma de México, A.P.
needed to elucidate this issue. 70-399, Coyoacán, Mexico, DF 04510, Mexico. 3Departamento de Zoologia,
Universidade de Brasília, IB, Brasilia, DF 70910-900, Brazil. 4Current address:
Considering the 31 species of Norops, the chromosome
Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios,
morphology and molecular DNA data of which are known, IOC-FIOCRUZ, Rio de Janeiro CEP 21045-900, Brazil. 5El Colegio de la Frontera
a minimum of six independently originated sex chromo- Sur - San Cristóbal de las Casas Carr. Panamericana y Av. Periférico Sur s/n
29290, San Cristóbal de las Casas, Chiapas, Mexico.
some constitutions are possible. It looks like the clade is
prone to sex chromosome diversification. Moreover, the or- Received: 1 February 2013 Accepted: 8 October 2013
igins of five of these heteromorphic sex chromosome vari- Published: 13 December 2013
ants seem to be recent as they appear at the tip nodes in
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