Chromosoma
DOI 10.1007/s00412-016-0576-7
ORIGINAL ARTICLE
Discovery of the youngest sex chromosomes reveals first case
of convergent co-option of ancestral autosomes in turtles
E. E. Montiel 1 & D. Badenhorst 1 & J. Tamplin 2 & R. L. Burke 3 & N. Valenzuela 1
Received: 15 December 2015 / Revised: 21 January 2016 / Accepted: 22 January 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract Most turtle species possess temperature-dependent
sex determination (TSD), but genotypic sex determination
(GSD) has evolved multiple times independently from the
TSD ancestral condition. GSD in animals typically involves
sex chromosomes, yet the sex chromosome system of only 9
out of 18 known GSD turtles has been characterized. Here, we
combine comparative genome hybridization (CGH) and BAC
clone fluorescent in situ hybridization (BAC FISH) to identify
a macro-chromosome XX/XY system in the GSD wood turtle
Glyptemys insculpta (GIN), the youngest known sex chromosomes in chelonians (8–20 My old). Comparative analyses
show that GIN-X/Y is homologous to chromosome 4 of
Chrysemys picta (CPI) painted turtles, chromosome 5 of
Gallus gallus chicken, and thus to the X/Y sex chromosomes
of Siebenrockiella crassicollis black marsh turtles. We tentatively assign the gene content of the mapped BACs from CPI
chromosome 4 (CPI-4) to GIN-X/Y. Chromosomal rearrangements were detected in G. insculpta sex chromosome pair that
co-localize with the male-specific region of GIN-Y and encompass a gene involved in sexual development (Wt1—a putative master gene in TSD turtles). Such inversions may have
mediated the divergence of G. insculpta sex chromosome pair
Electronic supplementary material The online version of this article
(doi:10.1007/s00412-016-0576-7) contains supplementary material,
which is available to authorized users.
* N. Valenzuela
nvalenzu@iastate.edu
1
Department of Ecology, Evolution and Organismal Biology, Iowa
State University, Ames, IA 50011, USA
2
Department of Biology, University of Northern Iowa, Cedar
Falls, IA, USA
3
Department of Biology, Hofstra University, Hempstead, NY, USA
and facilitated GSD evolution in this turtle. Our results illuminate the structure, origin, and evolution of sex chromosomes
in G. insculpta and reveal the first case of convergent cooption of an autosomal pair as sex chromosomes within
chelonians.
Keywords Evolution of genome and sex chromosome
organization . Genotypic and temperature-dependent sex
determination . Comparative genome and BAC in situ
hybridization molecular cytogenetics . Turtle reptile
vertebrates . Convergent ancestral reconstruction . Wt1
chromosomal rearrangement
Introduction
The diversity of sex-determining mechanisms (SDMs) across
the tree of life is quite complex (Bachtrog et al. 2014) with
certain branches exhibiting remarkably conserved SDMs
(e.g., mammals, birds, snakes) while others are extremely labile (e.g., fish, some reptiles). Many vertebrates utilize environmental sex determination (ESD), where sexual fate is decided by external factors such as temperature (TSD)
(Valenzuela and Lance 2004). In other species, sex determination depends on genotypic constitution alone (GSD), when
a master gene(s) located in a sex chromosome(s) commits the
developing embryo to its sexual fate. Our understanding of the
evolution of this diversity remains incomplete because SDM
has been studied in only a small fraction of existing species
(Bachtrog et al. 2014). Modern molecular cytogenetic and
genomic methods are accelerating the pace of discovery of
SDM in groups that are traditionally difficult to study such
as many reptiles whose sex chromosomes are cryptic because
they are virtually homomorphic (Badenhorst et al. 2013; Ezaz
et al. 2005; Ezaz et al. 2006; Gamble et al. 2015; Kawai et al.
Chromosoma
2007), adding critical data to understand the evolution of
SDM and associated traits.
Turtles possess diverse SDM, including the predominant
and ancestral TSD and evolutionarily derived GSD systems
of XX/XY and ZZ/ZW sex chromosomes (Tree of Sex 2014;
Valenzuela and Adams 2011). The SDM of only 26 % of turtle
species is currently known [87 out of 335 recognized species
(van Dijk et al. 2014)], of which 79 % have TSD (69 species)
and 21 % have GSD (18 species) (Tree of Sex 2014;
Valenzuela and Adams 2011). GSD is known in all studied
members of the families Chelidae and Trionychidae and coexists with TSD in the families Emydidae, Bataguridae, and
Kinosternidae (Badenhorst et al. 2013; Ewert et al. 2004; Ezaz
et al. 2006; Kawai et al. 2007; Martinez et al. 2008). The SDM
of many GSD turtles was described based on incubation experiments (Ewert et al. 2004), and the sex chromosome system has been characterized in only nine species (Badenhorst
et al. 2013; Bull et al. 1974; Carr and Bickham 1981; Ezaz
et al. 2006; Kawai et al. 2007; Martinez et al. 2008; McBee
et al. 1985; Sharma et al. 1975). Of these, the sex chromosome
system in turtle families with multiple known GSD members
is concordant, such as XX/XY in Chelidae and Kinosternidae,
and ZZ/ZW in Trionychidae. The striking exception is the
family Bataguridae where two GSD species evolved opposing
heterogamety—Siebenrockiella crassicollis possesses a macro-XX/XY system (Carr and Bickham 1981) and Pangshura
smithii a micro-ZZ/ZW (Sharma et al. 1975). The lack of
resolution of the cytogenetic techniques employed in the past,
as well as the presence of numerous micro-chromosomes in
the chromosomal complement of turtles, hindered the ability
to discriminate the sex chromosomes in some species.
However, the use of high resolution molecular cytogenetic
techniques such as comparative genome hybridization
(CGH) allowed the identification of sex chromosomes in several turtles, including the micro-sex chromosomes of
Chelodina longicollis (Ezaz et al. 2006), Pelodiscus sinensis
(Kawai et al. 2007), and Apalone spinifera (Badenhorst et al.
2013), and the slightly differentiated macro-sex chromosomes
of Emydura macquarii (Martinez et al. 2008).
The wood turtle, Glyptemys insculpta, is a member of the
family Emydidae, and based on incubation experiments, it is
known to exhibit GSD (Bull et al. 1985). An earlier cytogenetic study did not reveal discernible sex chromosomes
(Bickham 1975). Wood turtles inhabit regions of the northeast
and upper Midwest of the USA and southeastern Canada and
are listed as endangered by the International Union for
Conservation of Nature Red List (IUCN 2013) due to loss of
habitat, habitat fragmentation, high mortality rates suffered by
eggs and juveniles, their slow sexual maturation, and popularity in the pet trade (Ernst and Lovich 2009). Here, we use
CGH molecular cytogenetics to characterize the sex chromosomes of G. insculpta by the identification of sex-specific
regions to assess its system of heterogamety. Additionally,
by mapping multiple BAC clones from Chrysemys picta to
the sex chromosomes of G. insculpta, we identify chromosomal rearrangements between the two homologs of the sex
chromosome pair that are responsible for their subtle heteromorphism. We discuss our findings in the context of sex chromosome evolution in reptiles, including the homology of
G. insculpta’s sex chromosomes with other species.
Material and methods
Animal and tissue sampling
Skin biopsies were obtained from captive animals held by
Cold Spring Harbor Fish Hatchery and Aquarium or confiscated by Iowa’s DNR and local Humane Society. Equal number of males and females were sampled from each population.
Cell culture and chromosomes preparations
Fibroblast cell cultures were established from collagenase
(Sigma) digests of skin biopsies of five males and five females
and cultured using a medium composed of 1:1 RPMI
1640:Leibowitz media supplemented with 15 % fetal bovine
serum, 2 mM L-glutamine, and 1 % antibiotic–antimycotic
solution (Sigma). Cultures were incubated at 30 °C without
CO 2 supplementation. Four hours prior to harvesting,
10 μg/ml colcemid (Roche) was added to the cultures.
Metaphase chromosomes were harvested and fixed in 3:1
methanol/acetic acid. Cell suspensions were dropped onto
glass slides and air dried. Conventional protocols were used
for chromosome G- and C-banding (Seabright 1971; Sumner
1972). A minimum of 10 complete metaphases were analyzed
for each specimen. Images were taken with a Photometrics
CoolSnap ES2 Digital Monochrome camera attached to an
Olympus BX41 fluorescent microscope and analyzed using
CytoVision® cytogenetic analysis system (Applied Imaging/
Genetix).
Comparative genome hybridization
Genomic DNA (gDNA) from three male and three female
G. insculpta from the northern Iowa population (different
from those whose metaphases were analyzed) was extracted
and labeled with digoxigen-dUTP or biotin-dUTP using a
nick translation kit (Abbott Molecular) following the manufacturer’s instructions. CGH was performed as described by
Valenzuela et al. (2014). Briefly, chromosome slides were
hardened at 65 °C for 2 h, denatured for 2 min at 70 °C in
70 % formamide/2× SSC, and dehydrated through an ethanol
series and air dried. For each slide, a 15-μl mixture containing
250–500 ng of digoxigen-dUTP female and biotin-dUTP
male was co-precipitated with 5–10 μg boiled gDNA from
Chromosoma
male or female (as competitor) and 20 μl glycogen (as carrier)
and was hybridized to one slide at 37 °C for 3 days. As the
homogametic sex was not known at the onset of the study,
reciprocal experiments were done using the gDNA of both
sexes as competitor and each sex-by-color combination was
tested. Post-hybridization washes were done first in 0.4× SSC/
0.3 % Tween 20 for 2 min at 60 °C, followed by 1 min at room
temperature in 2× SSC/0.1 % Tween 20. A 250-μl solution of
4XT/relevant antibody was used for fluorochrome detection at
37 °C for 45 min. Slides were washed three times in 4XT at
37 °C and counterstained with 1.4 mM 4′,6-diamidino-2phenylindole (DAPI).
BAC FISH mapping
Sets of clones from a C. picta BAC library (library VMRC
CHY3 produced by the Joint Genome Institute) (Badenhorst
et al. 2015) were mapped to G. insculpta metaphases, and
BACs that localized to the sex chromosomes were chosen
for further study. BAC DNA (∼1 μg) was extracted and labeled by standard nick-translation (Abbott Molecular) using
either fluorescein, biotin or digoxingenin dUTPs (Roche) and
co-precipitated with human cot-1 DNA and C. picta cot-1
DNA. FISH was carried out as previously described for
CGH. Combinations of three BACs at a time were hybridized
to determine their relative position within chromosomes and
their hybridization pattern compared to that in C. picta
(Badenhorst et al. 2015) to identify chromosomal rearrangements between species.
Results
Karyotype of G. insculpta
The karyotype of five males and five females was analyzed in
the present study by G-banding, C-banding, and DAPI staining. The chromosome number was 2n = 50 in all cases. The
karyotype consists of 13 pairs of large size chromosomes
(macrochromosomes) and 12 pairs of small and hardly distinguishable chromosomes (microchromosomes), congruent
with earlier data (Bickham 1975). Unlike previously reported
(Bickham 1975), we detected differences between male and
female karyotypes. Namely, the fourth largest chromosome
pair in males showed a subtle but discernible heteromorphy,
with a subtelocentric chromosome and a slightly larger submetacentric chromosome (Fig. 1), whereas the fourth pair in
females was always subtelocentric and homomorphic (Fig. 1),
indicating the presence of an XX/XY system in G. insculpta.
Besides these differences in size and centromere position, we
detected a homomorphic G-banding pattern in the presumptive X chromosomes in females with an intense C-band in the
centromere, while males exhibited an intense C-band on the
short arm and more numerous G-bands in the presumptive Y
chromosome than in the X (Fig. 1).
Comparative genome hybridization
CGH was performed in five males and five females of
G. insculpta using as probes the gDNA of three males and
three females grouped into three DNA pairs. gDNA from each
sex was labeled with different fluorochromes and female or
male gDNA was used as competitor. At least 10 metaphase
spreads were studied per individual, per pair of probes, and
per competitor DNA, and the results revealed sex-specific
signals in males. Namely, three male-specific regions were
detected on the submetacentric fourth largest chromosome in
males, which corresponds with the heteromorphic chromosome pair detected above by morphology and G-/C-banding.
These male-specific regions were evident using all three combinations of probes and in all spreads examined in males. No
differential signal was observed in any of the female metaphase spreads. The male-specific regions correspond with
the three positive G-bands, one of them localized in the short
arm and the other two in the long arm of the submetacentric
homolog of the pair (Fig. 2). Combined, our results provide
strong evidence of the existence of an XX/XY system in
G. insculpta, with a subtelocentric X chromosome and a submetacentric Y containing three peri-centromeric and Gpositive male-specific regions.
BAC FISH
To further characterize the content and evolution of GIN XY
chromosomes, we hybridize seven BAC clones from C. picta
using zoo-FISH that had been mapped to CPI chromosome 4
(CPI-4) and whose sequence homology with other vertebrates
had been established previously (Badenhorst et al. 2015). All
seven BACs mapped to GIN-X and GIN-Y. Based on the gene
content of these C. picta BACs (Badenhorst et al. 2015) and
assuming that the content of these genomic regions is conserved between C. picta and G. insculpta, we tentatively assign these genes to the X and Y of G. insculpta
(Supplementary Table), but this hypothesis requires direct
validation.
Notably, the relative position of five of the seven BACs
differed between the X and Y chromosomes in G. insculpta
(Fig. 3). Specifically, BACs 94H12 and 85H12 showed the
same position relative to the centromere in the p arm, and
BACs 6H12 and 36H12 overlapped in their position in the q
arm of GIN-X and GIN-Y, which is identical to their pattern in
CPI-4 [denoted (6H12 + 36H12) hereafter]. However, on
GIN-Xq, the BAC order was (6H12 + 36H12)-88H1263H12-106H12 from the centromere distally towards the telomere, while on GIN-Yq the order was 88H12-(6H12 +
36H12)-106H12-63H12 (Fig. 3b), suggesting that at least
Chromosoma
Fig. 1 G- and C-banded
Glyptemys insculpta chromosome
spreads. G-banded metaphase
karyotype of male (a) and female
(d) individuals show autosomes
and sex chromosomes ordered by
size. Sex chromosomes in male
(b) and female (c) spreads present
differences in C bands. Boxes
contain the enlarged sex
chromosomes from each spread.
Scale bar = 10 μm
two inversions occurred between the X and Y in G. insculpta
(Fig. 3b). A comparison with the relative position of these
BACs in CPI-4 [88H12-(6H12 + 36H12)-63H12-106H12
(Badenhorst et al. 2015)], and assuming that C. picta represents the ancestral autosomal condition from which the X and
Y evolved in G. insculpta, suggests that these chromosomal
rearrangements may have occurred at or after the split of
Glyptemys from their common ancestor (Fig. 3b). One inversion occurred between the BAC 63H12 and 106H12 regions
in the Y. The second inversion rendered the position of the
88H12 region more distal from the centromere relative to the
overlapping (6H12 + 36H12) region and is found in GIN-X
exclusively.
Discussion
The evolution of the diversity of SDMs has as yet defied
scientific explanation, partly because of the paucity of data
on SDMs in groups with labile sex determination, such as
reptiles. Here, we identify the system of heterogamety in
G. insculpta, characterize the structure of the sex chromosomes, assign their partial putative gene content, and infer
chromosomal rearrangement that accompanied the evolution
of GSD in this endangered species. Previous incubation experiments with G. insculpta had established that it possesses
GSD (Bull et al. 1985), but an earlier cytogenetic study
(Bickham 1975) found no heteromorphic sex chromosomes,
leaving open the question of whether it had evolved male or
female heterogamety. The combination of G-banding, Cbanding, and CGH used here provides strong evidence of a
heteromorphic XX/XY sex chromosome system in
G. insculpta.
Research on sex determination in non-avian reptiles has
undergone a renaissance in the last decade with the discovery
of sex chromosomes in numerous species (Badenhorst et al.
2013; Gamble et al. 2015; Matsubara et al. 2013; Pokorná
et al. 2010; Pokorna et al. 2014; Quinn et al. 2010; Wang
et al. 2015), and significant effort has been devoted to understanding their origin and evolution (Matsubara et al. 2014a, b;
Chromosoma
Fig. 2 CGH on female (a, c, e, g)
and male (b, d, f, h) Glyptemys
insculpta chromosome spreads.
Boxes contain the enlarged sex
chromosomes which are indicated
by arrows in each spread. Female
DNA was detected with antidigoxigenin-rodhamine (red) and
male DNA with FITC avidin
(green). Scale bar = 10 μm
Rovatsos et al. 2014a, b; Valenzuela and Adams 2011;
Vicoso et al. 2013). Despite these advances, a large fraction
of taxa remain unstudied, particularly in groups with labile
SDM, such as lizards and turtles. This study contributes an
important new piece to the puzzle of sex determination in
Testudines; of the 18 turtle species known to exhibit GSD,
10 are now known to possess sex chromosomes (Badenhorst
et al. 2013; Bull et al. 1974; Carr and Bickham 1981; Ezaz
et al. 2006; Kawai et al. 2007; Martinez et al. 2008; McBee
et al. 1985; Sharma et al. 1975). The XX/XY of G. insculpta
is a macrochromosome system, as are the XX/XY systems
found in Staurotypus tripocarpus and Staurotypus salvinii
(Bull et al. 1974), Siebenrockiella crassicollis (Carr and
Bickham 1981), and Emydura macquarii (Martinez et al.
2008) turtles. In contrast, Chelodina longicollis (Ezaz et al.
2006) has a microchromosome XX/XY system. The Y chromosome of G. insculpta is larger than the X chromosome, a
condition also observed in E. macquarii turtles (Martinez et
al. 2008), plus a variety of other organisms, including some
vertebrates, invertebrates, and plants (some of which exhibit
female heterogamety with a larger W than the Z) [e.g.,
(Lewis and John 1963; Matsubara et al. 2014a, b;
Matsunaga and Kawano 2001; Sato and Ikeda 1992; Solari
1994)]. Using CGH, we detected at least three male-specific
regions in the Y that together account for this increase in
size. This condition is unlike E. macquarii, where the
male-specific regions that render the Y larger than the X
are located terminally in the p arm of the Y, probably due
to a translocation of an ancestral micro-Y to an autosome
(Martinez et al. 2008; Matsubara et al. 2015).
Chromosoma
Fig. 3 Chromosomal locations of
Chrysemys picta BAC clones in
Glyptemy insculpta. a Multi BAC
FISH on male G. insculpta
chromosome spread. Probes
labeled with biotin-dUTP are
yellow, with digoxegen-dUTP are
red and fluorescein-dUTP are
green. Boxes show enlarged
pictures of BAC hybridization
signals in X and Y chromosomes.
Scale bar = 10 μm. b
Comparative map of BAC clone
locations in C. picta chromosome
4 and G. insculpta sex
chromosomes. Green bands
indicate the location of the malespecific regions of the Y detected
by CGH
These CGH bands correspond to DAPI-positive and Gpositive bands, indicating that the male-specific regions of
GIN-Y are AT-rich, perhaps gene poor (Comings et al. 1973;
Holmquist 1992), and presumably composed of a large number of repeated sequences that have accumulated or expanded,
leading to the larger size of the Y compared to the X chromosome. Similarly, the male-specific region of E. macquarii Y
also corresponds with a heterochromatin region likely caused
by the expansion of repeated sequences (Martinez et al. 2008),
including microsatellites (Matsubara et al. 2015). Such expansion of repeated sequences is usually associated with young
sex chromosomes in early stages of differentiation as seen in
other eukaryotes, including animals [e.g., (Cioffi et al. 2012;
Devlin et al. 1998; Nanda et al. 2000; Palacios-Gimenez
et al. 2015a, b) and plants (Kejnovsky et al. 2013; Kubat
et al. 2008; Wang et al. 2012)]. This repeat expansion can
result from a lack of recombination in certain regions of
the sex chromosomes that hides mutations from purifying
selection and permits their accumulation (Charlesworth
et al. 1994).
Phylogenetic comparative analyses support temperaturedependent sex determination (TSD) as the ancestral condition
in turtles, from which GSD evolved multiple times independently, including the transition in the lineage of G. insculpta
(Valenzuela and Adams 2011). Several hypotheses exist to explain the transition from environmental sex determination (such
as TSD) to GSD, and the most accepted model starts with the
acquisition of a dominant masculinizing mutation or a polygenic
system of sex determination under strong enough selection to
favor reduced recombination in the sex-determining region
(Bachtrog et al. 2011; Charlesworth et al. 2005). This reduction
of recombination is facilitated by chromosomal rearrangements
such as translocations and inversions (Charlesworth et al. 2005).
Consistent with this model, our BAC clone mapping results
revealed evidence of at least two inversions between the X
and Y of G. insculpta that could have contributed to a reduction
of recombination. Inversions between sex chromosomes are often reported, and examples in vertebrates include the human Y
with at least two inversions (Bachtrog 2013), the gecko Gekko
hokouensis, where a pericentromeric inversion on the Z chromosome may have been followed by other successive inversions on the W chromosome (Kawai et al. 2009), and
P. sinensis softshell turtles with a paracentromeric inversion on
the W chromosome (Kawagoshi et al. 2009). The sex chromosomes of G. insculpta are the youngest known in turtles, having
arisen either ∼20 Mya at the split of Glyptemys from its sister
clade (which exhibits the ancestral TSD condition), or ∼8 Mya
at the split of G. insculpta from Glyptemys muhlenbergii (whose
SDM is unknown). In contrast, the sex chromosomes of
P. sinensis and its co-familial Apalone spinifera arose at least
∼95 Mya (Valenzuela and Adams 2011). Given the purportedly
low rate of chromosomal changes accrued in turtles (Bickham
Chromosoma
1981; Olmo 2008) and the young age of G. insculpta XX/XY
system (Valenzuela and Adams 2011), the presence of inversions in the sex chromosomes of P. sinensis and G. insculpta
may reflect their key role in the evolution of GSD in these
lineages. While results from our comparative BAC mapping
uncovered intrachromosomal rearrangements between
G. insculpta and C. picta, data from additional species are needed to determine the ancestral condition, order of occurrence, and
directionality of these rearrangements. The inversion involving
BAC 88H12 and the overlapping BACs (6H12 + 36H12) is
particularly interesting from a functional standpoint, because
BAC 88H12 maps to a genome scaffold in C. picta that contains
Wt1 (Supplementary Table), a putative master TSD gene
(Valenzuela 2008). But whether a chromosomal rearrangement
encompassing Wt1 is functionally relevant and facilitated the
evolution of GSD in G. insculpta remains to be tested. Also
unclear is whether this inversion between BAC 88H12 and the
overlapping (6H12 + 36H12) BACs occurred early in the protosex chromosomes and was followed by a second inversion in
GIN-Y that recovered the original relative position of these two
regions with respect to their position in C. picta, explaining why
the inversion with respect to C. picta is found in the GIN-X and
not GIN-Y (Fig. 3b). This inversion co-localizes with one of the
male-specific regions of the GIN-Y detected by CGH (Fig. 3b),
giving some support to the double inversion scenario described
above.
In the meantime, important insights about the origin of
GIN-XY pair can be gained by combining our results from
comparative BAC mapping between G. insculpta and C. picta
with previous data on the homology between C. picta chromosomes and those of chicken (Badenhorst et al. 2015). The
seven BAC clones that mapped to GIN-X/Y mapped to CPI-4,
and all but one map to C. picta genome scaffolds containing
genes homologous to chicken chromosome 5 (CHICKEN-5)
almost exclusively, whereas BAC 94H12 showed homology
to CHICKEN-26 (Badenhorst et al. 2015). Thus, our BAC
mapping results suggest that the GIN-X/Y are also homologous to CHICKEN-5, which has been shown to share homology with the XY sex chromosomes of S. crassicollis
(Kawagoshi et al. 2012), a turtle in the same superfamily
Testudinoidea, and PELODISCUS-6. Consistently,
SIEBENROCKIELA-X/Y exhibit similar morphology to
GIN-X/Y. In combination, these data suggest that the XX/
XY sex chromosomes of G. insculpta and S. crassicollis originated independently from the same pair of ancestral autosomes. In contrast, a single evolutionary event is needed to
explain the XX/XY systems of Staurotypus tripocarpus and S.
salvinii in the family Kinosternidae, which are homologous to
CHICKEN-Z/W (Kawagoshi et al. 2014). Similarly, a single
event explains the evolution of ZZ/ZW systems of P. sinensis
and A. spinifera in the family Trionychidae (Badenhorst et al.
2013) which are homologous to CHICKEN-15 (Kawagoshi
et al. 2009). Furthermore, within the family Bataguridae,
Pangshura smithii exhibits a ZZ/ZW system (Sharma et al.
1975) that contrasts the XX/XY system of S. crassicollis (Carr
and Bickham 1981). Whether PANGSHURA-Z/W is homologous to CHICKEN-5, as are SIEBENROCKIELA-XY and
GIN-XY, remains to be tested.
In summary, this study reveals the sex chromosome system
of the wood turtle, G. insculpta, sheds light on their structure,
putative gene content, and evolutionary origin. Results reveal
the first case of convergent co-option of an ancestral pair of
autosomes as sex chromosomes within turtles, as represented
in G. insculpta and S. crassicollis. Notably, the appearance of
G. insculpta sex chromosomes corresponds to a period of
global warming before the ice age of the Miocene Epoch
(Valenzuela and Adams 2011; Zachos et al. 2001). It is tempting to speculate that the increasing temperature in this period
may have favored the transition from TSD to GSD to counterbalance female-biased sex ratios produced under such global warming and which would have favored a masculinazing
mutation(s) that would help restore such balance. The occurrence of the inversions identified here, before or soon after the
appearance of such sex-determining mutation(s), would have
helped reduce recombination, thus favoring the divergence of
G. insculpta sex chromosomes following classic models of
sex chromosome evolution. Finally, when placed in a comparative framework, the novel data obtained here indicate that sex
chromosomes have arisen independently and multiple times
from different ancestral chromosomes in turtles and that some
have been co-opted repeatedly for this function, a phenomenon that has occurred among distant vertebrates lineages as
well (e.g., Chen et al. 2014). Thus, it appears that more than
one pair of chromosomes in turtles are Bgood at sex^
(O’Meally et al. 2012).
Acknowledgments We thank Steve DeSimone (director of the Cold
Spring Harbor Fish Hatchery and Aquarium) for the access to specimens
for sampling and R. Literman for the help with DNA extractions. This
work was funded in part by NSF grant MCB 1244355 to NV.
Compliance with ethical standards All procedures were approved by
the IACUC of Iowa State University and were carried out under appropriate local permits. This article does not contain any studies with human
participants.
Conflict of interest The authors declare that they have no competing
interests.
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