Original Article

Cytogenet Genome Res 2016;148:292–304 Accepted: April 4, 2016 DOI: 10.1159/000447478 by M. Schmid Published online: July 16, 2016

Cytogenetic Insights into the Evolution of Chromosomes and Sex Determination Reveal Striking Homology of Sex Chromosomes to Amphibian Autosomes

a a a a Eugenia E. Montiel Daleen Badenhorst Ling S. Lee Robert Literman b a Vladimir Trifonov Nicole Valenzuela a b Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa , USA; Institute of Molecular and Cellular Biology SB RAS, Novosibirsk , Russian Federation

Key Words niotes revealed that the sex chromosomes of several , 18S rDNA · C-bands · Evolution · Homology · Interstitial as well as mammals and some lizards, are homologous to telomeres · Karyotype evolution · NOR · · Sex components of Xenopus tropicalis XTR1 (carrying Dmrt1 ). chromosome · Sex determination · Turtle Other turtle sex chromosomes are homologous to XTR4 (car- rying Wt1 ). Interestingly, all known turtle sex chromosomes, except in , evolved via inversions around Dmrt1 Abstract or Wt1 . Thus, XTR1 appears to represent an amniote proto- Turtle karyotypes are highly conserved compared to other sex chromosome (perhaps linked ancestrally to XTR4) that vertebrates; yet, variation in diploid number (2n = 26–68) gave rise to turtle and other amniote sex chromosomes. reflects profound genomic reorganization, which correlates © 2016 S. Karger AG, Basel with evolutionary turnovers in sex determination. We evalu- ate the published literature and newly collected compara- tive cytogenetic data (G- and C-banding, 18S-NOR, and telo- The compartmentalization of genomes into chromo- mere- mapping) from 13 spanning 2n = 28–68 somes alters genome function, because the regulatory to revisit turtle genome evolution and sex determination. environment of genes is influenced by their synteny or Interstitial telomeric sites were detected in multiple lineages lack thereof [Ahituv et al., 2005], and co-expressed genes that underwent diploid number and sex determination turn- tend to be clustered in the genome [de Wit and van overs, suggesting chromosomal rearrangements. C-banding Steensel, 2009]. Thus, discerning the causes and conse- revealed potential interspecific variation in centromere com- quences of genome compartmentalization is key to un- position and interstitial heterochromatin at secondary con- derstanding genome function, because chromosome fu- strictions. 18S-NORs were detected in secondary constric- sion or fission events alter the number of chromosomes tions in a single chromosomal pair per species, refuting pre- and the synteny of the genes they contain. Reptilian ge- vious reports of multiple NORs in turtles. 18S-NORs are linked nomes vary in their size, organization, and composition to ZW chromosomes in and and to X (not Y) in . Notably, comparative genomics across am- E.E.M. and D.B. contributed equally to this work.

© 2016 S. Karger AG, Basel Nicole Valenzuela 1424–8581/16/1484–0292$39.50/0 Department of Ecology, Evolution and Organismal Biology Iowa State University E-Mail [email protected] Ames, IA 50011 (USA) www.karger.com/cgr E-Mail nvalenzu @ iastate.edu Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM Fig. 1. Ancestral reconstruction of diploid chromosome numbers duced at intermediate temperatures and females at lower and high- for turtle species with known diploid numbers and sex determina- er values. Three-letter acronyms are shown for the 13 target species tion mechanisms. Evolutionary transitions between temperature- whose cytogenetic data were collected during this study. Branch dependent sex determination (TSD) and genotypic sex determina- color denotes ancestral state reconstruction by parsimony follow- tion (GSD) are indicated in the branches where they were recon- ing Valenzuela and Adams [2011], whereas numbers at key nodes structed to have occurred by maximum likelihood [Valenzuela denote reconstruction by maximum likelihood using the ace func- and Adams, 2011]. GSD = GSD with uncharacterized sex chromo- tion of ape version 3.4 [Popescu et al., 2012] in R [R Core Develop- some system. TSDIa = TSD where males are produced at lower and ment Team, 2012]. females at higher temperature. TSDII = TSD where males are pro-

Turtle Sex Chromosome Homology to Cytogenet Genome Res 2016;148:292–304 293 Amphibian Autosomes DOI: 10.1159/000447478 Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM Fig. 2. G-banded karyotypes of 13 target species examined in this study and their phylogenetic relationships. Taxonomic families are indicated at the corresponding nodes and sex-determining mechanisms are color coded. Green arrowheads denote the chromosomal location of the NORs.

[Olmo, 2008]. Genome evolution has received increased changed for over 200 million years [Bickham, 1981]. Yet, attention as new genomic resources are developed for the wide variation in chromosome number across turtles , a group that holds an important key to under- ( fig. 1 ) point to drastic changes in genome organization standing vertebrate evolution [Wang et al., 2006; Janes that took place during this time, and these rearrange- et al., 2008; Alfoldi et al., 2011; Castoe et al., 2013; Shaf- ments are curiously associated with evolutionary transi- fer et al., 2013; Vonk et al., 2013; Wang et al., 2013; tions in sex-determining mechanisms [Valenzuela and Green et al., 2014]. Adams, 2011]. Here, we provide new cytogenetic data Within reptiles, turtles have been considered to pos- from selected species on the evolution of turtle karyo- sess highly conserved karyotypes [Olmo, 2008; Pokorná types and review our knowledge of chromosomal sex de- et al., 2011] as some chromosomes appear to be un- termination in turtles.

294 Cytogenet Genome Res 2016;148:292–304 Montiel/Badenhorst/Lee/Literman/ DOI: 10.1159/000447478 Trifonov/Valenzuela

Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM Methods ronym hereafter. A summary of new and previously pub- lished data is presented in table 1 . While standardized sys- Cell Culture, Chromosome Preparation, and Chromosome tems of chromosome nomenclature exist for some organ- Banding Chromosome preparations were obtained from fibroblast cell isms, including humans, domestic , and some cultures established from embryonic or muscle tissue following plants [e.g., Linde-Laursen et al., 1997; Cribiu et al., 2001; standard procedures as previously described [Valenzuela, 2009; Simons et al., 2013], no such system exists for turtles. This Badenhorst et al., 2013, 2015; Valenzuela et al., 2014; Montiel et al., is partly due to the scarcity of uniform banding informa- 2016]. New samples were obtained from private collections or pet trade specimens. Briefly, fibroblast cell cultures were established tion across taxa, which limits our understanding of turtle from collagenase (Sigma) digests and cultured using a medium genome macroevolution [Olmo, 2005]. Here, we adopted which was composed of 50% RPMI 1640 and 50% Leibowitz media the organization of chromosomes by decreasing size and supplemented with 15% fetal bovine serum, 2 m M L-glutamine, centromere position as is commonly used today. Namely, and 1% antibiotic-antimycotic solution (Sigma). Cultures were in- G-banded karyotypes (fig. 2 ) were ordered by approxi- cubated at 30 ° C with no CO2 supplementation [Badenhorst et al., 2013]. Four hours prior to harvesting, 10 μg/ml colcemid (Karyo- mate size. In general, smaller microchromosomes were MAX® , Invitrogen) was added to the cultures, which were har- nearly indistinguishable from each other by shape and vested after hypotonic exposure and fixed in 3: 1 methanol:acetic centromere position, and thus, they were ordered by ap- acid following standard procedures [Ezaz et al., 2006; Martinez et proximate size and G-banding pattern where possible. al., 2008; Badenhorst et al., 2013]. G- and C-banding followed con- The diploid number in turtles ranges from 2n = 26 in ventional protocols [Seabright, 1971; Sumner, 1972]. Peltocephalus dumerilianus [Killebrew, 1975a; Ventura et NOR Mapping by 18S rRNA FISH al., 2014] to 2n = 68 in Carettochelys insculpta [Bickham The distribution of NORs was investigated by FISH using a et al., 1983] with an average of 2n = 52 given our current turtle-specific 18S DNA fragment labeled by nick-translation and knowledge of turtle chromosomes [Olmo and Signorino, co-precipitated with salmon sperm DNA [Badenhorst et al., 2013]. 2005] (fig. 1 ). Our comparative cytogenetic data ( fig. 2 ) Telomere FISH Mapping illustrate a general characteristic of turtle karyotypes that A telomeric probe containing the repeat motif (TTAGGG)n is shared by birds and squamates, namely, the presence of was constructed and labelled with biotin or DIG-dUTP by PCR 2 distinct components, macro- and microchromosomes following Ijdo et al. [1991]. The probe was hybridized to meta- [Olmo et al., 2002]. The presence of microchromosomes phase chromosomes of all target species following Badenhorst et appears to be the ancestral condition to amniotes as it is al. [2013]. Briefly, chromosome spreads and the DNA probe were also found in ancestral fish and amphibian lineages, and denatured together in a sealed slide on a hot plate at 65 ° C for

3 min. Hybridization took place in a humid chamber at 37 ° C for they appear to have fused among themselves in crocodil- 2 nights. Post-hybridization washes consisted of a first wash in ians leading to their absence in this group [Uno et al., 0.4× SSC/0.3% Tween 20 for 2 min at 60 ° C, followed by a second 2012]. Our data also underscore that most variation in wash in 2× SSC/0.1% Tween 20 for 1 min at room temperature. turtle diploid number is due to differences in the number The probe-detection solution comprised 4XT/relevant antibody of microchromosomes (fig. 2 ). conjugated with a fluorochrome in a final volume of 250 μl at 37 ° C for 20–45 min. Slides were subsequently washed 3 times in 4XT Our cytogenetic data confirmed previously reported at 37 ° C, counterstained with 4′ ,6-diamidino-2-phenylindole diploid numbers for all species examined (fig. 2 , table 1 ). (DAPI) (6 μl DAPI, 2 mg/ml in 50 ml 2× SSC), and mounted using However, while the numbers of macro- and microchro- an antifade solution (Vectashield). Signals were assigned to spe- mosomes were in agreement with previous reports for cific chromosomes based on their morphology, size, and DAPI banding. (TSC), Chrysemys (CPI), (GIN),

and (EMA) (all 26: 24), Pelodiscus (PSI) (16: 50),

and Apalone (ASP) (18: 48) ( table 1), they differed in Ster-

notherus (SOD) (22: 34) from previous reports (26: 30 re-

Results and Discussion ported by Killebrew [1975b]), Staurotypus (STR) (22: 32)

(26: 28 reported by Killebrew [1975b]), (CSE)

Evolution of Turtle Karyotypes (22: 30) (28: 24 reported by De Smet [1978]),

Here, we present newly collected comparative cytoge- (COB) (24: 30) (22: 32 reported by Bull and Legler netic data (G- and C-banding, 18S rRNA FISH mapping [1980]), Carettochelys (CIN) (18: 50) (16: 52 reported by

(18S-NOR), and telomere FISH mapping) for 13 turtle Bickham et al. [1983]), Pelomedusa (PSU) (24: 12) (26: 10 species spanning the full spectrum of diploid numbers reported by De Smet [1978]), and (PUN)

(2n = 28–68) except for the lowest 2n = 26 (figs. 1, 2 ). We (22: 6) (10: 18 reported by Olmo and Signorino [2005]). refer to these species by their names or 3-letter ac- These discrepancies in the number of macro- and

Turtle Sex Chromosome Homology to Cytogenet Genome Res 2016;148:292–304 295 Amphibian Autosomes DOI: 10.1159/000447478 Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM Martinez et al. [2008] Stock [1972]; Killebrew [1975a]; De Smet [1978] [1976]; De Smet [1978]; Haiduk and Bickham [1982] Matsuda et al. [2005]; Kawai et al. [2007] Bull and Legler [1980] Killebrew [1975a]; Kawagoshi et al. [2014] De Smet [1978] Kasai et al. [2012] salvinii)

(S. ; Bickham and salvinii)

Stock [1972] Killebrew [1975a] Bickham et al. [1983]Killebrew [1976] Bickham et al. [1983] Bull and Legler [1980]Huang and Clark [1969]; Bull and Legler [1980]; De Smet [1978]; Bull and Bull and Legler [1980] Legler [1980] Confirmed reports Countered reports Stock [1972] Bickham and Baker Sato and Ota [2001]; Kawai et al. [2007] Ayres et al. [1969]; Huang and Clark [1969]; Gunski et al. [2013] Rogers [1985] Sites et al. [1979] (S. Killebrew [1977]; Badenhorst et al. [2015] Sato and Ota [2001]; Badenhorst et al. [2013] Cleiton and Giuliano-Caetano [2008] Bickham [1975]; Montiel et al. [2016] blished reports. Chromosome pairs with ITS none 8, 9, 11 1, 2, 3, 4, 5, 6, 7 1, 2 none none none 4 1, 2, 3, 5, micro none 2micro none none micro3 none none 18S FISH NOR X 14 9 Micro- chromosomes 30 32 34 50 30 12 61 Macro- chromosomes 22 22 22 18 24 24 22 number PSI 66 16 50PSU 36 ZW EMA 50 26PUN 24 28 CPI 50 26 24 14 STR 54 COB 54 SOD 56 ASP 66 18 48 ZW TSC 50 26 24 14 CIN 68 CSE 52 GIN 50 26 24 insculpta

odoratus

subrufa triporcatus unifilis

scri pta picta insculpta

oblonga

sinensis

macquarii serpentina

spinifera

Pelodiscus Pelomedusa Staurotypus Emydura Chelodina Podocnemis Species Acronym Diploid Chrysemys Apalone Trachemys Carettochelys Chelydra Glyptemys Trionychidae Pelomedusoidea suprafamily Carettochelyidae Summary of cytogenetic results from 13 target turtles examined in this study Bold font = Novel data not published before; underlined counter to reports; plain matching pu Table 1. Suborder or

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Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM neck suborder Pleurodira appears to have taken place in AB Pelomedusoidea ( + ), whereas within the hidden-neck suborder Cryptodira a dramatic increase in chromosome number took place in the family Trionychidae ( fig. 1 ). In Pleurodira, Pelome- dusoidea has low diploid numbers and few microchro- mosomes (2n = 26–36), whereas Chelidae is character- ized by higher diploid numbers and the presence of nu- merous microchromosomes (2n = 50–58), a characteristic similar to the karyotypes found in Cryptodira [Bull and CD Legler, 1980] (fig. 2 ). The distribution of heterochromatin exhibits variation among chelonians. The C-positive material correspond- ing to constitutive heterochromatin is largely restricted to the centromeric regions in almost all chromosomes in all species examined except in Pelomedusoidea (fig. 3 ; on- line suppl. fig. 1, for all online suppl. material, see www. karger.com/doi/10.1159/000447478). The variation in centromere C-banding (some centromeres are strongly C-positive, other only faintly, and few are C-negative) EF may reflect true differences in the composition of turtle centromeric sequences as occurs in other organisms [Steiner and Henikoff, 2015], perhaps even differences in their origin. Further research is needed to uncover com- positional differences among turtle centromeres that might exist to test this hypothesis. In addition, some mi- crochromosomes showed a heterochromatic p arm in all emydids. Furthermore, interstitial C-positive hetero- chromatin exists in the p arm of chromosomes GIN9, GINY, SOD5, CSE6, the q arm of PSI5, and some micro- Fig. 3. C-banding metaphases of Glyptemys (GIN), Staurotypus (STR), Sternotherus (SOD), Carettochelys (CIN), Pelomedusa chromosomes of Sternotherus , Staurotypus, Chelydra , (PSI), and Podocnemis (PUN). Red arrowheads denote intersti- and Pelodiscus (fig. 3 ; online suppl. fig. 1). Distal C-posi- tial C-positive regions that co-localize with the NORs. Scale bars = tive signals were detected in the q arm of SOD1, STR1, 10 μm. COB4, COB6, and in the telomere region of PSI1, PSI3, and ASP1 (online suppl. fig. 1G). COB4 was heteromor- phic in size and G-banding of the p arm (the largest ho- molog of the pair exhibits an extra distal G-band on the p microchromosomes reflect differences in the ability to arm). Interestingly, C-banding results in a more intense identify chromosomal homologs for the smaller chromo- interstitial band than the centromeric bands in Pelome- some pairs during various studies, and also differences in dusoidea. Interstitial heterochromatin is more limited the classification criteria employed by researchers. This is in Pelomedusa (PSU1 and PSU5), while in Podocnemis it due to the difficulty presented when chromosomes are encompasses 6 chromosome pairs (PUN1–PUN5 and less elongated in the metaphase spread preparations, PUN8; fig. 3 E, F). In all these cases in Podocnemis , this which renders some smaller chromosomes harder to dis- interstitial heterochromatin corresponds with secondary tinguish from microchromosomes. constrictions (1 in PUN3, PUN5, and PUN8 and 2 in Ancestral reconstruction of chromosome number by PUN1, PUN2, and PUN4). Interstitial heterochromatin parsimony, maximum likelihood (fig. 1 ), or using Bayes- is also visible in one microchromosome pair in Trache- ian models [Organ et al., 2008] identified 2 drastic evolu- mys, Chrysemys, Glyptemys, Sternotherus, and one mac- tionary changes in each of the 2 turtle suborders. Namely, rochromosome pair in Carettochelys (CIN2; fig. 3 D), a radical reduction in diploid number within the side- Staurotypus (STRX), and one homolog of the NOR pair

Turtle Sex Chromosome Homology to Cytogenet Genome Res 2016;148:292–304 297 Amphibian Autosomes DOI: 10.1159/000447478 Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM in Emydura (EMA14; online suppl. fig. 1F). This intersti- AB tial C-positive material corresponds to the NOR in Chry- semys [Badenhorst et al., 2015], as well as Sternotherus , Staurotypus , and Emydura [this study], Podocnemis (one of the multiple sites only), and to the NOR-bearing region in Trachemys , Glyptemys, Carettochelys, as detailed be- low. None of the interstitial C bands correspond to the NOR in Pelomedusa.

Turtle NOR Evolution The NOR contains the genes for the 18S, 5.8S, and 28S C D ribosomal RNAs [Shaw and McKeown, 2011]. NORs are frequently located on a secondary constriction of chro- mosomes but not always [Bickham and Rogers, 1985; Pri- eto and McStay, 2008], and this condition has been pro- posed as a conserved trait in turtles [Cleiton and Giulia- no-Caetano, 2008]. Turtle NORs have been investigated in a number of turtles, mostly using the secondary con- striction method (scNOR) [e.g., Huang and Clark, 1969; De Smet, 1978] or silver staining (AgNOR) [e.g., Bickham and Rogers, 1985; Martinez et al., 2009; Fantin and dos E F Santos Monjelo, 2011; Badenhorst et al., 2015], which ex- poses the active NOR by dyeing the proteins associated with rDNA activity [Howell, 1977]. However, not all sec- ondary constrictions correspond to NORs such that this method can be misleading [Bickham and Rogers, 1985]. Also, because genomes may retain inactive copies of the NOR after translocations have occurred, silver staining reveals only a partial view of NOR’s evolutionary histo- ry. Thus, we also used 18S rDNA FISH (18S-NOR) as pre- viously applied to a few turtle taxa [Kawai et al., 2007; Fig. 4. NOR mapping using 18S FISH in Glyptemys (GIN), Stauro- Cleiton and Giuliano-Caetano, 2008; Badenhorst et al., typus (STR), Sternotherus (SOD), Carettochelys (CIN), Pelomedu- 2013, 2015; Kawagoshi et al., 2014] to detect potentially sa (PSI), and Podocnemis (PUN). Arrowheads point to the NORS. inactive NOR copies that may have passed undetected in Scale bars = 10 μm. some of the turtles previously examined using AgNOR or scNOR techniques. Our 18S FISH data of the 13 target taxa includes the first reports of 18S-NORs for 7 of them (GIN, CSE, SOD, CIN, EMA, COB, and PSU) ( table 1 ). method. Namely, 3 NOR-bearing chromosomes were Our combined results and published data indicate that all identified in Trachemys dorbigni and 2 in trivit- turtles thus far studied possess a unique NOR located on tata based on secondary constrictions [De Smet, 1978], a single chromosome pair (fig. 4 ; online suppl. fig. 2). This but later AgNOR and 18S FISH studies demonstrated that is the most common condition in reptiles [Olmo and Si- the T. dorbigni NOR is located on a single chromosome gnorino, 2005], amphibians [King et al., 1990; Schmid [Cleiton and Giuliano-Caetano, 2008] as in T. scripta and Steinlein, 2015], and fish [Gornung, 2013], although studied here (fig. 4, table 1 ; online suppl. fig. 2). Similarly, some species in these lineages possess multiple NORs, as 7 NOR-bearing chromosomes were reported in Podocne- do humans, other mammals [Prieto and McStay, 2008; mis expansa and P. unifilis based on secondary constric- Farley et al., 2015], and birds [de Oliveira Barbosa et al., tions [Huang and Clark, 1969], but our data indicate the 2013]. Importantly, existing data indicate that previous existence of a single NOR in PUN1 (fig. 4 , table 1 ). Thus, reports of multiple NORs in turtles are in error, likely be- the reports for P. expansa and B. trivittata based on the cause they were based on the secondary constriction assumption that all secondary constrictions are indicative

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Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM AB fig. 3). This observation is consistent with reports that telomeres are crucial for maintaining chromosomal sta- bility as they protect chromosome ends during cell divi- sion, repair of double-strand breaks, and ensure the via- bility of chromosomes products from fission events [Flint et al., 1994; de Lange, 2002; Bolzan and Bianchi, 2006; Gomes et al., 2010]. However, non-terminal TTAGGG signals were detected (interstitial telomeric sites, ITS) in several of our target species, including the centromeric region of 7 chromosome pairs in Chelydra (CSE1, 2, 3, 5, C D 8, 9, 11) and Podocnemis (PUN1, 2, 3, 4, 5, 6, 7), 2 pairs in Staurotypus (STR1, 2), and 1 pair in Glyptemys (GIN9) ( fig. 5 , table 1 ; online suppl. fig. 3). The ITS in Podocne - mis do not co-localize with the secondary constrictions where C-positive interstitial heterochromatin was detect- ed ( fig. 5 F). ITS are commonly found in fish [Ocalewicz, 2013], birds [Nanda et al., 2002], humans [Azzalin et al., 2001], and other mammals [Finato et al., 2000; Ruiz-Her- rera et al., 2005, 2008; Castiglia et al., 2007], and unlike terminal telomeres, these repetitive regions can be linked E F to chromosomal instability and rearrangements that may lead to genome reorganization and speciation [Ishii et al., 2008; Brown and O’Neill, 2010]. Indeed, telomeric re- peats correlate with chromosomal evolutionary break- points [Flint et al., 1994; Azzalin et al., 2001; Ruiz-Herre- ra et al., 2002, 2005, 2008]. The observation that changes in chromosome number occurred in the turtle lineages where ITS were found (fig. 1 ; CSE 2n = from 54 to 52, PUN 2n = from 38 to 28, GIN 2n = from 52 to 50, STR 2n = from 56 to 54) suggests Fig. 5. Telomere FISH mapping in Glyptemys (GIN), Staurotypus that some of these turtle ITS perhaps derive from chro- (STR), Sternotherus (SOD), Carettochelys (CIN), Pelomedusa mosomal fusions [Azzalin et al., 2001] which will de- (PSI), and Podocnemis (PUN). Arrowheads point to ITS. Scale crease chromosome number. But whether these turtle ITS bars = 10 μm. constitute such ghosts-of-fusions-past requires further testing, as fusion ITS may be characterized by stretches of tandem telomeric repeats that are oriented head-to-head of NORs may also be mistaken. Therefore, current data [Azzalin et al., 2001]. An alternative explanation is that support the notion that the turtle NOR has changed its turtle ITS represent sites where telomeric repeats were chromosomal location among taxa, and yet, no inactive inserted during the repair of double-strand breaks [Az- copies of NORs exist in any of the examined chelonians zalin et al., 2001] and thus represent the relics of evolu- as revealed by 18S FISH ( fig. 4 ; online suppl. fig. 2). Thus, tionary genome reorganization events other than fusions when the NOR moved to a new chromosome from its an- [Ruiz-Herrera et al., 2005]. Also, telomeric sequences are cestral location in some turtle lineages, it did so by a com- sometimes found interspersed in the rRNA gene cluster, plete translocation without evidence in any chromosome which may also represent fragile sites prone to fission of a NOR duplication event having occurred. [Rousselet et al., 2000] that would increase chromosome number. However, this alternative can be ruled out in our Turtle Telomere Evolution target taxa, since no ITS co-localized with the NORs in As expected, our telomeric (TTAGGG)n probe [Ijdo any of species examined here. Finally, telomeres appear et al., 1991] hybridized to the termini of all the chromo- to be the evolutionary precursors of eukaryote centro- somes in the species investigated ( fig. 5 ; online suppl. meres [Villasante et al., 2007; Mendez-Lago et al., 2009;

Turtle Sex Chromosome Homology to Cytogenet Genome Res 2016;148:292–304 299 Amphibian Autosomes DOI: 10.1159/000447478 Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM Fig. 6. Homology of some turtle sex chromosome systems to chick- sinensis , ASP = Apalone spinifera , GGA = Gallus gallus , GHO = en, some lizards, human, and Xenopus tropicalis. Arrows denote Gekko hokouensis , ACA = Anolis carolinensis , TSE = Takydromus chromosomal inversions. X. tropicalis homology to chicken is sexlineatus, HSA = Homo sapiens, XTR = Xenopus tropicalis , based on Brelsford et al. [2013] and Warren et al. [2010]. CPI = Wt1 = Wilm’s tumor supressor gene 1, Dmrt1 = doublesex and Chrysemys picta , GIN = Glyptemys insculpta , SCR = Siebenrocki- Mab-3 related transcription factor 1. ella crassicollis, STR = Staurotypus triporcatus, PSI = Pelodiscus

Garavis et al., 2013], and telomeric DNA may give rise to heterogamety (figs. 1, 6 ), 3 in the suborder Pleurodira neocentromeres [Villasante et al., 2007; Ishii et al., 2008] which are all XX/XY [McBee et al., 1985; Martinez et al., as reported in plants, , and mammals [Fu- 2008; Ezaz et al., 2009], and 7 in Cryptodira, 3 of which kagawa and Earnshaw, 2014; Steiner and Henikoff, 2015]. are ZZ/ZW [Sharma et al., 1975; Kawai et al., 2007; Baden- But whether some of the observed turtle ITS represent horst et al., 2013] and 4 are XX/XY [Bull et al., 1974; Carr evolutionary neocentromeres remains to be tested. and Bickham, 1981; Montiel et al., 2016]. Of these 10 cas- es, 3 involved microchromosomes and 7 macrochromo- Evolution of Turtle Sex Determination somes. Relatively little is known about the molecular con- Of the 335 recognized turtle species [van Dijk et al., tent of turtle sex chromosomes, although information is 2014], 87 have a known sex-determining mechanism increasing [Kawagoshi et al., 2009, 2012, 2014; Montiel et (SDM) established based on either incubation experi- al. 2016]. Molecular cytogenetic data from comparative ments or cytogenetic evidence [Tree of Sex Consortium, genome hybridization (CGH) has been used to identify 2014; Montiel et al., 2016]. Phylogenetic analyses recover the extent of the male- or female-limited region of the temperature-dependent sex determination (TSD) as the turtle sex chromosomes [Ezaz et al., 2006; Kawai et al., ancestral SDM in turtles, and most evolutionary transi- 2007; Martinez et al., 2008; Badenhorst et al., 2013; Mon- tions involved the evolution of genotypic sex determina- tiel et al., 2016] where recombination should be reduced tion (GSD) [Janzen and Krenz, 2004; Valenzuela and or absent. These data suggest that the pseudoautosomal Adams, 2011], with 2 potential reversals back to TSD in- region (segments of sex chromosomes that do recombine ferred by maximum likelihood analysis [Valenzuela and in XY males or ZW females [Bachtrog et al., 2011]) is Adams, 2011] (fig. 1 ). Of the 18 turtles known to possess miniscule in Chelodina longicollis [Ezaz et al., 2006] which GSD, 10 have a cytogenetically characterized system of possesses micro sex chromosomes, potentially larger in

300 Cytogenet Genome Res 2016;148:292–304 Montiel/Badenhorst/Lee/Literman/ DOI: 10.1159/000447478 Trifonov/Valenzuela

Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM and Staurotypus as it is in Apa lone [Baden- and zebra finch in turn shows high homology with chick- horst et al., 2013] and Pelodiscus sinensis [Ka wai et al., en (GGA) chromosomes [Warren et al., 2010] such that 2007], and more extensive in Emydura [Martinez et al., XTR1 contains genomic blocks homologous to GGA4, 2008] and Glyptemys [Montiel et al., 2016], but these pre- GGA15, GGA26, GGA28, and GGAZ. Notably, compar- dictions require direct testing. ative genomics revealed that the XY chromosomes of Turtle sex chromosomes may be the carriers of the Staurotypus are homologous to GGAZ [Kawagoshi et al., NOR ( Staurotypus [Sites et al., 1979; Bickham and Rog- 2014] as are the lizard Gekko hokouensis ZW [Kawai et al., ers, 1985]; Pelodiscus [Kawai et al., 2007], this study, fig. 4 ; 2009]. The ZW of Pelodiscus and Apalone are homolo- Apalone [Badenhorst et al., 2013], this study, fig. 4 ), as it gous to GGA15 [Badenhorst et al., 2013; Kawagoshi et al., also occurs in diverse taxa [reviewed in Literman et al., 2009] as is the Anolis X [Gamble et al., 2014; Rovatsos et 2014] such as some reptiles, mammals, amphibians, and al., 2014], and the p arm of Glyptemys GINX/Y maps to fish [Goodpasture and Bloom, 1975; Hsu et al., 1975; GGA26 [Badenhorst et al., 2015; Montiel et al., 2016]. Schmid et al., 1983, 1993; Porter et al., 1994; Born and Moreover, the block of XTR1 that is homologous to Bertollo, 2000; Wiley, 2003; Abramyan et al., 2009; Take- GGA4 is in turn homologous to human X and the lizard hana et al., 2012], as well as some invertebrates [e.g., Takydromus sexlineatus X [Rovatsos et al., 2016], while Monti et al., 2011; Montiel et al., 2012]. The NOR in the XTR1 block homologous to GGA28 and CPI22 maps ASPW and PSIW is much larger than the Z-NOR in these to the human X [Badenhorst et al., 2015] ( fig. 6 ). On the turtles, a dimorphic trait that can be leveraged to diagnose other hand, most of GINXY and SCRXY the genotypic sex in these turtles using molecular tech- (which evolved independently from each other) exhibit niques [Literman et al., 2014]. In contrast, we found that homology with GGA5 [Montiel et al., 2016] and XTR4. in STR the 18S-NOR is located on the X chromosomes Thus, these observations reveal that the sex chromosomes exclusively and not on the Y, as previously described in of Staurotypus, Pelodiscus, Apalone, and Glyptemys (in the congener Staurotypus salvinii [Sites et al., 1979; Bick- part) are homologous to XTR1, while the sex chromo- ham and Rogers, 1985]. Loss of a NOR by a deletion in somes of Siebenrockiella and most of Glyptemys are ho- one homolog of the chromosomal pair harboring them mologous to XTR4 ( fig. 6 ). Interestingly, XTR1 carries may explain the evolutionary origin of the lack of Y- Dmrt1, a gene co-opted for a sex-determining function in NORs observed in STR, as a single NOR was detected in birds and various other vertebrates and whose molecular one individual examined of the TSD turtle evolution is linked to evolutionary turnovers of sex deter- subrubrum and in 1 out of 4 individuals studied of the mination in reptiles [Janes et al., 2014]. Further, XTR4 XX/XY turtle Siebenrockiella crassicollis whose NORs are carries Wt1, another important regulator of gonadal autosomal [Bickham and Rogers, 1985]. Alternatively, formation and putative master TSD gene in Chrysemys the lack of a NOR in STRY may be the result of the degen- [Valenzuela, 2008]. Moreover, the evolution of turtle sex eration of the Y in STR as part of the evolutionary dynam- chromosome systems in all these cases involved chromo- ics of sex chromosomes [Bachtrog et al., 2011], or the re- somal inversions, and in all but Trionychid turtles, these sult of the translocation of the NOR to the X chromosome inversions encompassed Dmrt1 or Wt1 [Kawagoshi et al., exclusively (and not to the Y) as occurred in Hyla femo- 2009, 2012, 2014; Montiel et al., 2016]. The evolutionary ralis [Wiley, 2003]. However, X- and Y-linked NORs origin of the Pangshura smithii ZZ/ZW system [Sharma were detected in S. triporcatus and S. salvinii by Kawa- et al., 1975] in the same turtle family as goshi et al. [2014] using a combined 18S-28S probe, sug- Siebenrockiella, as well as the origin of the sex chromo- gesting that instead, the STRY retains 28S rRNA genes somes of chelid turtles ( Emydura , Chelodina , and Acan- but lost 18S rRNA genes. Further research is needed to thochelys radiolata ), remains unknown. test these alternatives. Thus, current data support the hypothesis that multi- Recent studies showed that chromosome 1 of Xenopus ple turtle chromosomes have been co-opted as sex chro- tropicalis (XTR1) is homologous to the sex chromosomes mosomes (and sometimes repeatedly) during the inde- in 3 divergent anurans and birds [Brelsford et al., 2013], pendent evolution of GSD in various lineages [Montiel et and existing data indicate that this deep homology ex- al., 2016], and it appears that this process may have been tends to turtles as well, as it does to other amniotes includ- facilitated by their particular gene content, which seems ing lizards and humans (fig. 6 ). Namely, XTR1 contains to make them good at determining sex [O’Meally et al., genomic blocks homologous to zebra finch (TGU) chro- 2012]. Furthermore, the proclivity of these genomic re- mosomes TGU4, TGU15, TGU26, TGU28, and TGUZ, gions to take on a role as sex chromosomes and master

Turtle Sex Chromosome Homology to Cytogenet Genome Res 2016;148:292–304 301 Amphibian Autosomes DOI: 10.1159/000447478 Downloaded by: Iowa State University 129.186.138.35 - 4/11/2017 12:17:39 AM activators of the sex determination cascade seems to em- Statement of Ethics anate from their deep shared homology to what appears All procedures were approved by the IACUC of Iowa State Uni- to be a proto sex chromosome in non-amniotic tetrapods. versity.

Acknowledgements Disclosure Statement

We thank J. Iverson, B. McCord, G. Rivera, as well as C. Innis The authors have no conflicts of interest to declare. and New England Aquarium for kindly donating samples, R. Stan- yon for his generous advice on lab procedures, and J. Iverson for editorial comments. This work was funded in part by NSF grants MCB 0815354 and MCB 1244355 to N.V.

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