Original Article

Cytogenet Genome Res Published online: January 26, 2019 DOI: 10.1159/000496379

ZZ/ZW Sex Chromosomes in the Endemic Puerto Rican Leaf-Toed Gecko (Phyllodactylus wirshingi)

a c a a, b, d Stuart V. Nielsen Juan D. Daza Brendan J. Pinto Tony Gamble a b Department of Biological Sciences, Marquette University, and Milwaukee Public Museum, Milwaukee, WI, c d Department of Biological Sciences, Sam Houston State University, Huntsville, TX , and Bell Museum of Natural History, University of Minnesota, Saint Paul, MN , USA

Keywords ed sex chromosomes – but can also be used to identify which Gekkota · Lizard · Phyllodactylidae · RADseq · Reptile · chromosomes in the genome are the sex chromosomes. We Squamata here identify a ZZ/ZW sex chromosome system in P. wir- shingi. Furthermore, we show that 4 of the female-specific markers contain fragments of genes found on the avian Z Abstract and discuss homology with P. wirshingi sex chromosomes. Investigating the evolutionary processes influencing the or- © 2019 S. Karger AG, Basel igin, evolution, and turnover of vertebrate sex chromosomes requires the classification of sex chromosome systems in a great diversity of species. Among amniotes, squamates (liz- Investigating the number and directionality of transi- ards and snakes) – and gecko lizards in particular – are wor- tions among sex-determining systems is a vital prerequi- thy of additional study. Geckos possess all major vertebrate site for studying sex chromosome evolution. This in- sex-determining systems, as well as multiple transitions volves not only determining whether a species has a het- among them, yet we still lack data on the sex-determining erogametic male (XX/XY) or female (ZZ/ZW) sex systems for the vast majority of species. We here utilize re- chromosome system, but also identifying which chromo- striction-site associated DNA sequencing (RADseq) to iden- somes in the genome are the sex chromosomes. However, tify the sex chromosome system of the Puerto Rican endem- we still lack basic knowledge of sex-determining mecha- ic leaf-toed gecko (Phyllodactylidae: Phyllodactylus wir- nisms for many species [Bachtrog et al., 2014], let alone shingi), in order to confirm a ZZ/ZW sex chromosome system the genomic homology of said sex chromosomes. Cyto- within the genus, as well as to better categorize the diversity genetic methods, like karyotyping, have long been the within this poorly characterized family. RADseq has proven principal means of identifying an organism’s sex chromo- an effective alternative to cytogenetic methods for deter- some system, yet most vertebrate species possess mor- mining whether a species has an XX/XY or ZZ/ZW sex chro- phologically indistinguishable sex chromosomes [Devlin mosome system – particularly in taxa with non-differentiat- and Nagahama, 2002; Matsubara et al., 2006; Stöck et al.,

© 2019 S. Karger AG, Basel Tony Gamble Department of Biological Sciences, Marquette University P.O. Box 1881 E-Mail [email protected] Milwaukee, WI 53201-1881 (USA) www.karger.com/cgr E-Mail tgamble @ geckoevolution.org Downloaded by: Iowa State University 129.186.138.35 - 1/26/2019 5:51:04 PM 2011; Gamble and Zarkower, 2014; Otto, 2014]. Further- Table 1. Phyllodactylus wirshingi samples used in this study more, such data do not address which chromosomes in ID Sex Locality the genome are the sex chromosomes in a comparative framework. However, recent advances in both cytogenet- TG2007 Male Isla de Caja de Muertos, Puerto Rico ics and DNA sequencing techniques have facilitated the TG3210 Male Bosque Estatal de Guánica, Puerto Rico identification of sex chromosomes in many additional TG3219 Male Bosque Estatal de Guánica, Puerto Rico species, spawning a renewed interest in ascertaining and TG3220 Male Bosque Estatal de Guánica, Puerto Rico TG3221 Male Bosque Estatal de Guánica, Puerto Rico classifying the sex chromosome systems of previously in- TG3223 Male Bosque Estatal de Guánica, Puerto Rico tractable taxa [Pokorná et al., 2011; Deakin et al., 2016; TG3225 Male Bosque Estatal de Guánica, Puerto Rico Gamble et al., 2017, 2018; Nielsen et al., 2018]. TG3228 Male Bosque Estatal de Guánica, Puerto Rico Even within a group as varied as squamates, the geck- TG2016a Male Bosque Estatal de Guánica, Puerto Rico a os (Squamata: Gekkota) are a stand-out clade worthy of TG3208 Male Bosque Estatal de Guánica, Puerto Rico more detailed study. Geckos are a species-rich (>1,700 TG2004 Female Isla de Caja de Muertos, Puerto Rico species [Uetz et al., 2017]), near globally distributed clade TG2008 Female Isla de Caja de Muertos, Puerto Rico of lizards, who possess all major vertebrate sex-determin- TG2009 Female Isla de Caja de Muertos, Puerto Rico TG2385 Female Bosque Estatal de Guánica, Puerto Rico ing systems, as well as multiple transitions among them TG3209 Female Bosque Estatal de Guánica, Puerto Rico [Moritz, 1990; Ezaz et al., 2009; Gamble, 2010; Gamble et TG3222 Female Bosque Estatal de Guánica, Puerto Rico al., 2015a]. The high diversity of geckos makes them an TG3224 Female Bosque Estatal de Guánica, Puerto Rico ideal vertebrate model to study the origins and evolution TG3226 Female Bosque Estatal de Guánica, Puerto Rico of sex chromosomes. However, huge swaths of the gecko TG3227 Female Bosque Estatal de Guánica, Puerto Rico TG2017a Female Bosque Estatal de Guánica, Puerto Rico phylogeny lack any information about sex-determining systems, and fewer than 3% of gecko species have a sex- a These samples were used for PCR validation only. determining system known with high confidence [Gam- ble et al., 2015a]. Despite this paucity of data, roughly one-half to two-thirds of all observed transitions among Table 2. PCR primers used to validate female-specific RADseq squamate sex-determining systems are in geckos [Gam- markers in Phyllodactylus wirshingi ble et al., 2015a]. Fostering an enhanced knowledge con- cerning gekkotan, and thus squamate, sex-determining Primer ID Sequence (5′→3′) Annealing systems will increase their utility as a model clade to study temperature the origins and evolution of sex-determining systems Pw84-F CAGAAGGCATGAGACTGGAGAG 57°C [Janzen and Krenz, 2004]. Therefore, a concentrated ef- Pw84-R CAAATCTCCAGGAGCAGAGTGG fort to identify sex chromosome systems in additional Pw116-F CGATTCCCTTGCCTTAATCGGT 56°C clades will serve to fill crucial gaps in our knowledge, and Pw116-R AGATTCTGACCCAGGAAGAGGA permit more comprehensive hypothesis testing. Pw186-F ACTTTCCACTAAGGTGATCCCC 56°C The Phyllodactylidae are distributed throughout the Pw186-R GGGCCAAGGACTATGACTTGAA New World, North Africa, Europe, and the Middle East. Pw187-F GACTGAGGAGGGTCTGCTCT 56°C Of the more than 135 species, only 14 have published Pw187-R GTCTTCTGGGCTCTGACTGG karyotypes and just 2 species, Phyllodactylus lanei and Thecadactylus rapicauda, exhibit evidence of heteromor- phic sex chromosomes [King, 1981; Pellegrino et al., 2004, 2005, 2010; Olmo and Signorino, 2005; Murphy et al., seq) methodology [Gamble and Zarkower, 2014], another 2009; Schmid et al., 2014]. However, in both species, the population of T. rapicauda from Trinidad and Tobago story is complex. For example, in P. lanei, different karyo- was found to also have ZZ/ZW sex chromosomes [Gam- typic formulas between sexes were indicative of a ZZ/ZW ble et al., 2015a]. In addition to ZZ/ZW sex chromosomes, sex chromosome system, yet more recent work could not a number of species in the genus Tarentola show strong replicate these findings [Castiglia et al., 2009]. Similarly in evidence of environmentally determined sex (i.e., temper- T. rapicauda, the authors conclude that sex chromosomes ature-dependent sex determination or TSD) [Nettmann are in a “nascent state of differentiation” as not all sampled and Rykena, 1985; Hielen, 1992]. Thus, there are some populations were heteromorphic. Using a recently devel- interesting inferences one can draw within this family. oped restriction-site associated DNA sequencing (RAD- First, we know very little about the diversity of sex chro-

2 Cytogenet Genome Res Nielsen/Daza/Pinto/Gamble DOI: 10.1159/000496379

Downloaded by: Iowa State University 129.186.138.35 - 1/26/2019 5:51:04 PM male female novel gene

A Fig. 1. A–D PCR validation of 4 female-spe- TRABD2A cific RADseq markers in Phyllodactylus LIX1 wirshingi . A Pw84. B Pw116. C Pw186. D Px187. Primers amplified in a female- B specific manner in all examined male and female samples (see Table 1), generating a TRPM3 single (presumably W-specific) band in all but one locus, Pw186. The latter produced C both Z- and W-specific bands. Specimen ID numbers are listed below each lane. E Cytogenetic map of the Gallus gallus Z D chromosome depicting the location of 4 E genes identified by BLAST of P. wirshingi TG3220 TG3221 TG3210 TG3219 TG3223 TG3225 TG3228 TG3208 TG2007 TG2016 TG3224 TG3209 TG3222 TG3226 TG3227 TG2008 TG2009 TG2017 TG2385 female-specific RAD contigs (Table 3).

mosome systems within Phyllodactylidae, and given the with high-fidelity Sbf I restriction enzyme (New England Biolabs). diversity observed in other gekkotan groups, data from Individually barcoded P1 adapters were ligated to the SbfI cut site additional species are essential before more definitive for each sample. We pooled samples into multiple libraries, soni- cated, and size-selected into 200–500-bp fragments using magnet- conclusions can be drawn. Second, based on our limited ic beads in a PEG/NaCl buffer [Rohland and Reich, 2012]. Librar- evidence, there is at least 1 transition among sex-deter- ies were blunt-end repaired and dA-tailed before ligating a P2 mining systems within Phyllodactylidae – between TSD adapter containing unique Illumina barcodes to each pooled li- and female heterogamety. Lastly, both T. rapicauda and brary. We amplified libraries via PCR (16 cycles) with Q5 high- P. lanei possess ZZ/ZW sex chromosome systems, but are fidelity DNA polymerase (New England Biolabs) and cleaned/size- selected a second time into 250–600-bp library fragments using their sex chromosomes homologous? magnetic beads in PEG/NaCl buffer. Libraries were pooled and Here, we used RADseq to discover the sex chromo- sequenced using paired-end 125-bp reads on an Illumina some system of the Puerto Rican endemic leaf-toed gecko HiSeq2500 at the Medical College of Wisconsin. ( Phyllodactylus wirshingi), to confirm the ZZ/ZW sex We demultiplexed, trimmed, and filtered raw Illumina reads chromosome system within the genus, as well as to better using the process_radtags function in STACKS (v1.4.8) [Catchen et al., 2011]. We applied RADtools (v1.2.4) [Baxter et al., 2011] to categorize sex chromosome diversity within the Phyllo- generate candidate alleles for each individual and candidate loci dactylidae. By utilizing paired-end Illumina sequencing, across all individuals from the forward reads employing previous- we generated large RADseq contigs and successfully iden- ly described parameters [Gamble et al., 2015a, 2017]. We identified tified 4 genes putatively linked to the sex chromosomes. putative sex-specific markers from the RADtools output using a We discuss sex chromosome homology within Gekkota custom python script [Gamble et al., 2015a]. This script also pro- duced a second list of “confirmed” sex-specific RAD markers, and among amniotes, with particular attention paid to which are a subset of the initial list of sex-specific RAD markers synteny between the newly discovered P. wirshingi ZZ/ that excludes any sex-specific marker that also appears in the orig- ZW chromosomes and the avian Z. inal raw read files from the opposite sex from further consideration [Gamble and Zarkower, 2014; Gamble et al., 2015a]. We assembled forward and reverse reads from the confirmed sex-specific RAD markers into sex-specific RAD contigs using Geneious ® (v10) Materials and Methods [Kearse et al., 2012]. We then used these confirmed RAD contigs to design sex-specific PCR primers, also in Geneious (v10), and Using the Qiagen® DNeasy Blood and Tissue Kit, we extracted validated the sex specificity of a subset of the confirmed female- genomic DNA from tail clips, or liver, from 8 adult male and 9 specific markers by PCR (Table 2 ). adult female P. wirshingi collected near Guánica in Puerto Rico We attempted to assess synteny between the newly identified and the island of Caja de Muertos ( Table 1 ). RADseq libraries were sex-specific RAD markers in P. wirshingi with chicken chromo- constructed following a modified protocol from Etter et al. [2011] somes. The chicken genome is well annotated and widely used as as described in Gamble et al. [2015a]. Genomic DNA was digested a reference for comparative genomics in nonavian reptiles [Inter-

ZZ/ZW Sex Chromosomes in Cytogenet Genome Res 3 Phyllodactylus DOI: 10.1159/000496379 Downloaded by: Iowa State University 129.186.138.35 - 1/26/2019 5:51:04 PM Haemodracon 2n = ? ; 2 sp., 0 karyo., 0 het.

Asaccus 2n = ? ; 18, 0, 0

Thecadactylus 2n = 42-44; 3, 1, 0

Ptyodactylus 2n = 40; 10, 1, 0

Tarentola 2n = 42; 31, 2, 0

Gymnodactylus 2n = 38-40; 5, 3, 0

Phyllopezus Fig. 2. A time-calibrated genus-level phy- 2n = 38-40; 4, 2, 0 logeny of the Phyllodactylidae, modified from Gamble et al. [2015a]. Sex chromo- some systems, if known, are indicated by Garthia colored circles to the left of taxon names. 2n = ? ; 2, 0, 0 Series of numbers under taxon names indi- cate diploid (2n) chromosomal comple- ment (when known), the number of de- Homonota scribed species within the genus, the subset 2n = 40*; 12, 1, 0 that have been karyotyped, and the number that exhibit heteromorphic sex chromo- somes. Karyotype data from Olmo and Si- Phyllodactylus gnorino [2005], Pellegrino et al. [2010], 75 50 25 0 mya 2n = 32–41; 51, 3, 1 and Gamble et al. [2015a]. * The 2n num- ber for Homonota is currently unpub- Sex Chromosome System lished. Photographs of Asaccus and Theca- Unk. TSD ZZ/ZW XX/XY dactylus by T. Pierson and A. Snyder, re- spectively.

Table 3. The 4 hits from the BLAST of the 539 female-specific Phyllodactylus wirshingi RAD contigs against chicken genes demonstrat- ing synteny with avian sex chromosomes

Query Gene name Transcript ID Chickena E value Hit start Hit end

PwF_429 TRPM3 ENSGALT00000024411 Z 1.21e–56 3,246 3,446 PwF_381 LIX1 ENSGALT00000024678 Z 3.87e–44 241 388 PwF_203 Novel gene ENSGALT00000028259 Z 1.25e–44 442 683 PwF_37 TRABD2A ENSGALT00000043645 Z 2.51e–47 809 656

a The location of these genes on the chicken Z chromosome is shown in Figure 1.

4 Cytogenet Genome Res Nielsen/Daza/Pinto/Gamble DOI: 10.1159/000496379

Downloaded by: Iowa State University 129.186.138.35 - 1/26/2019 5:51:04 PM national Chicken Genome Sequencing Consortium, 2004; Alföldi et al., 2011; Pokorná et al., 2011; O’Meally et al., 2012]. We per- XY / Z,2,3, Ornithorhynchus anatinus* formed BLAST of the assembled female-specific RAD contigs to 12,13,16 & 17 Mammals the chicken transcriptome (using Ensembl [Zerbino et al., 2018]), XY / 4 & 1 Mus musculus* implemented in Geneious (v10) [Kearse et al., 2012] with a maxi- XY / 4 Monodelphis domestica* mum E-value cutoff of 1e–50 and word size of 15 bp. Birds ZW / Z Gallus gallus*

ZW / 15 Pelodiscus sinensis Results Turtles XY / Z Staurotypus triporcatus

XY / 5 & 26 Glyptemys insculpta Output from the RADtool analysis recovered 143,649

XY / 5 Siebenrockiella crassicollis RAD markers with 2 or fewer alleles including 1 male- specific RAD marker and 574 female-specific RAD mark- XY / ? Lialis burtonis Geckos ers. Of these, we identified zero confirmed male-specific XY / ? Coleonyx elegans RAD markers and 539 confirmed female-specific RAD ZW / Z Phyllodactylus wirshingi markers. “Confirmed” sex-specific markers, as described

Lizards & ZW / ? Christinus marmoratus above, are a subset of the total number of sex-specific Snakes RAD markers that excludes RAD markers which oc- ZW / Z Gekko hokouensis curred in the raw read files of the opposite sex and likely XY / ? Scincus scincus are false positives. From this pool of confirmed female- ZW / 4 & 17 Takydromus sexlineatus* specific RAD contigs, we designed 11 primer pairs, 4 of

ZW / 2 & 27 Thamnophis sirtalis* which amplified in a sex-specific manner (Fig. 1 ). These Snakes loci produced either a single band in each of the female Python molurus XY / 2 & 27 samples with no amplification in male samples (Pw84, XY / 11 Boa constrictor Pw116, and Pw187; i.e., a RAD marker presumably on the ZW / ? Varanus komodoensis* W chromosome) or 2 bands in females and a single band

XY / 15 Anolis carolinensis* in males (Pw186; i.e., different-sized Z and W alleles). The combined results – an excess of female-specific RAD Pogona vitticeps ZW / 17 & 23 markers and PCR amplification of a subset of these mark-

300 250 200 150 100 50 0 mya ers only in females – is indicative of a ZZ/ZW sex chro- mosome system within Phyllodactylus ( Fig. 2 ). BLAST queries of the 539 female-specific RAD contigs against chicken genes resulted in 4 hits ( Table 3 ). All 4 Fig. 3. Phylogenetic distribution and chromosomal synteny of the avian Z with the sex chromosomes of a selection of taxa spanning matching genes are on the chicken Z chromosome, re- the amniote tree of life. Boxes at the tips of the phylogeny indicate vealing homology between the avian and the P. wirshingi the sex chromosome system, followed by sex chromosome synte- ZZ/ZW sex chromosomes (Fig. 3 ). ny, if known, with birds. For example, the ZZ/ZW sex chromo- somes of the turtle, Pelodiscus sinensis, are homologous with chick- en ( Gallus gallus) chromosome 15. Rectangular boxes share sex chromosome synteny with birds while curved boxes do not. XY Discussion and ZW indicate male and female heterogamety, respectively. Sev- eral taxa have unknown sex chromosome synteny (indicated by The discovery of ZZ/ZW sex chromosomes in P. wir- “?”), but comparative FISH experiments have shown that their sex shingi makes it the second member of the genus with fe- chromosomes do not share homology with the avian Z [Pokorná male heterogamety, the other being P. lanei [King, 1981]. et al., 2011; Matsubara et al., 2014]. When indicated by an asterisk, the pattern is reflective of the broader clade (e.g., Takydromus sex- The exact identity of the species King [1981] examined, lineatus and all other lacertid lizards examined to date share a ho- however, is up for debate since subsequent karyotypes of mologous ZW sex chromosome system). For more details on sex P. lanei revealed different chromosomal arrangements chromosome synteny data, we refer the reader to the original data and no heteromorphic sex chromosomes [Castiglia et al., sources [Matsubara et al., 2006; Graves, 2008; Veyrunes et al., 2008; 2009]. Because King [1981] apparently did not keep Kawagoshi et al., 2009, 2012, 2014; Kawai et al., 2009; Alföldi et al., 2011; Deakin et al., 2016; Rovatsos et al., 2016a, b; Montiel et al., vouchered specimens from his study, the exact identity of 2016; Gamble et al., 2017]. Phylogeny modified from Anderson the species examined remains unknown. Castiglia et al. and Wiens [2017]. [2009] obtained their samples from Jalisco, Mexico, while

ZZ/ZW Sex Chromosomes in Cytogenet Genome Res 5 Phyllodactylus DOI: 10.1159/000496379 Downloaded by: Iowa State University 129.186.138.35 - 1/26/2019 5:51:04 PM King’s [1981] samples were from Guerrero, Mexico. Pre- liminary genetic data suggest that P. lanei is likely a spe- Correlophus ciliatus Underwoodisaurus milii cies complex composed of multiple, undescribed species Delma butleri [Blair et al., 2015]. Thus, differences between studies may Aprasia parapulchella Lialis burtonis reflect karyotypic variation among cryptic taxa. Further Lialis jicari Coleonyx elegans complicating matters, 5 Phyllodactylus species (P. del- Eublepharis macularius campoi , P. bordai , P. tuberculosus , P. papenfussi , and P. Hemitheconyx caudicinctus Aristelliger expectatus lanei ) all occur in Guerrero where King’s samples origi- Euleptes europaea Gonatodes humeralis nated [Dixon and Kluge, 1964; Murphy et al., 2009; Gam- Sphaerodactylus macrolepis ble, 2010], and it is possible that King’s samples were mis- Sphaerodactylus nicholsi Thecadactylus rapicauda identified. Phyllodactylus wirshingi Phyllodactylus lanei The large number of female-specific RAD markers Tarentola mauritanica identified in P. wirshingi suggests significant differentia- Tarentola boettgeri Phelsuma dubia tion between the Z and W chromosomes. Indeed, com- Phelsuma madagascariensis Phelsuma guimbeaui pared to other squamate species, using similarly prepared Phelsuma guentheri Sbf I RADseq libraries, the 539 sex-specific P. wirshingi Christinus marmoratus Paroedura stumpfii RAD markers are second only to the 855 female-specific Paroedura lohatsara Paroedura oviceps RAD markers identified in the ZZ/ZW gecko Christinus Paroedura masobe marmoratus [Gamble et al., 2015a]. Because C. marmora- Dixonius siamensis Heteronotia binoei tus has heteromorphic sex chromosomes [King and Rofe, Gekko gecko Gekko japonicus 1976], it is probable that P. wirshingi also has cytogeneti- Gekko hokouensis cally differentiated Z and W chromosomes. However, Cyrtodactylus pubisulcus Hemidactylus frenatus since it is not clear whether there is a direct relationship Hemidactylus mabouia Hemidactylus turcicus between the sequence similarity of sex chromosomes and ZZ/ZW Gehyra mutilata the degree of chromosomal heteromorphism [Vicoso et Gehyra australis XX/XY Gehyra nana al., 2013; Gamble et al., 2014], this would need to be veri- TSD Gehyra purpurascens fied cytogenetically. Using sex-specific RAD markers to identify a species’ 120 80 40 0 mya sex chromosomes typically requires additional genomic resources because determining chromosomal synteny in- volves comparing gene identity and order among species. Fig. 4. Evolution of sex-determining mechanisms in geckos. Col- Although most RAD markers map to noncoding frag- ored circles at the tips of the phylogenetic tree indicate sex-deter- ments, and thus provide little information as to their ge- mining systems of selected species. Sex chromosomes in Phyllo- nomic location absent in a sequenced genome, a small dactylus wirshingi and Gekko hokouensis (highlighted in gray) both number of RAD markers – usually less than 15% of RAD share homology with the avian Z chromosome. Given their phylo- contigs – may overlap with a gene or other coding region genetic placement, it is likely that these have evolved independent- ly. Sex chromosome data were taken from multiple sources [Gam- [Amores et al., 2011; Baxter et al., 2011; Chutimanitsakun ble, 2010, and citations therein; Gornung et al., 2013; Gamble et al., et al., 2011; Bruneaux et al., 2013]. Sequencing paired-end 2015a, 2018, and citations therein; Rovatsos et al., 2016a]. Phylog- reads, as we have done here, will generate larger RAD eny modified from Gamble et al. [2015b]. contigs, which in turn increases the probability of identi- fying genes [Amores et al., 2011; Baxter et al., 2011; Gam- ble and Zarkower, 2014]. Sex-specific RAD markers with gene fragments can be used to query genomic assemblies at least 5 times ( Fig. 3): in birds; in the ZZ/ZW gecko, of related species to identify the sex chromosomes [Bru- Gekko hokouensis ; in an XX/XY clade of turtles, Stauroty- neaux et al., 2013; Gamble and Zarkower, 2014; Fowler pus salvinii and S. triporcatus ; as part of the multiple sex and Buonaccorsi, 2016; Qiu et al., 2016]. This is the ap- chromosomes in monotremes; and finally, the ZZ/ZW proach we successfully applied here to identify synteny sex chromosomes of P. wirshingi [Veyrunes et al., 2008; between the avian Z and the P. wirshingi ZZ/ZW. Kawai et al., 2009; Kawagoshi et al., 2014]. Comparative Among amniotes, the ancestral autosome that became analyses, including cytogenetics and genome-scale data- the avian Z has been recruited into a sex-determining role sets, have identified the sex chromosomes in at least 8

6 Cytogenet Genome Res Nielsen/Daza/Pinto/Gamble DOI: 10.1159/000496379

Downloaded by: Iowa State University 129.186.138.35 - 1/26/2019 5:51:04 PM other amniote lineages, which were recruited from ances- most any kind of gene can become a sex-determining tral autosomes not homologous with the avian Z [Ma- gene and every chromosome a sex chromosome [Hodg- tsubara et al., 2006; Veyrunes et al., 2008; Graves, 2008; kin, 2002]. Thus, sex chromosome recruitment should be Kawagoshi et al., 2009, 2012, 2014; Kawai et al., 2009; random, and there should be no biases when sex chromo- Alföldi et al., 2011; Deakin et al., 2016; Montiel et al., some synteny is examined in a phylogenetic context. Dif- 2016; Rovatsos et al., 2016b; Gamble et al., 2017]. Further- ferentiating between these 2 alternatives is not straight- more, comparative FISH analyses using fluorescently la- forward because we only know the sex chromosomes of a beled chromosome paints have shown that the sex chro- small number of taxa. Indeed, the sex chromosome iden- mosomes of additional geckos, Lialis burtonis (XXXX/ tity of nearly two-thirds of amniote sex chromosome sys- XXY), Coleonyx elegans (XXXX/XXY), and C. marmora- tems remains unknown (Fig. 3 ). To resolve this requires tus (ZZ/ZW), are not homologous to the avian ZZ/ZW an approach that integrates phylogenetic, cytogenetic, [Pokorná et al., 2011; Matsubara et al., 2014]. Because P. and genomic analyses, and exploits species-rich verte- wirshingi and G. hokouensis possess ZZ/ZW sex chromo- brate model clades in which many transitions among sex somes that are homologous with the avian Z, it is worth chromosome systems have occurred, e.g., amniotes and/ asking whether their ZZ/ZW sex chromosomes are de- or squamates. Only after identifying the sex chromo- rived from a common ancestor with “avian ZW” sex somes in most or all of the relevant lineages, we can say chromosomes [Ezaz et al., 2017]. While not impossible, it with any certainty whether there have been biases in appears unlikely in this case. The phylogenetic distribu- which chromosomes get recruited into a sex-determining tion of these taxa, coupled with the numerous transitions role. The current study makes an incremental step in among sex chromosome systems across geckos more gen- achieving this objective. erally, strongly suggests that the P. wirshingi and G. ho- kouensis ZZ/ZW systems derived independently (Fig. 4 ). The repeated recruitment of the avian Z as a sex chro- Acknowledgements mosome in amniotes can inform the search for factors that determine which ancestral chromosome will become The authors thank D. Martino, S.A. Ramírez-Lohner, D. Zar- kower, and J. Bernstein for help in the field, as well as T. Pierson a sex chromosome. There are several competing hypoth- and A. Snyder for the use of their excellent photographs (of Asac- eses to address this. For example, it has been hypothesized cus and Thecadactylus, respectively). We also thank G. Mayol and that one or more of these extant sex chromosome systems M.T. Chardon (The Department of Natural and Environmental are ancestral and thus exist in multiple lineages due to Resources of Puerto Rico) for facilitating fieldwork at Caja de inheritance from a common ancestor [Graves and Peichel, Muertos. Fieldwork and sequencing was supported by the U.S. Na- tional Science Foundation NSF-IOS1146820 to D. Zarkower; Mar- 2010; O’Meally et al., 2012; Ezaz et al., 2017]. As men- quette University startup funds to T.G.; and additional funding tioned above, this seems unlikely, at least in geckos. How- from Villanova University and Sam Houston State University to ever, there remain 2 other hypotheses that we can con- J.D.D. B.J.P. was supported by NSF-DEB1657662. sider here. First, some autosomes, because of their gene content, may be “better” at being sex chromosomes than others. These chromosomes may host genes playing a role Statement of Ethics in the sex-determining pathway that can be co-opted into controlling sex determination [Graves and Peichel, 2010; All experiments were carried out in accordance with animal use protocols at University of Minnesota (0810A50001 and O’Meally et al., 2012]. The avian Z, for example, contains 1108A03545) and Marquette University (AR279 and AR288). DMRT1, a member of a gene family involved in sex deter- Samples were collected under Puerto Rico permits DRNA: 2013- mination and sexual differentiation in all animals, and the IC-006, 2014-IC-042, and 2016-IC-091. likely sex-determining gene in birds and several other vertebrates [Raymond et al., 1998; Matsuda et al., 2002; Nanda et al., 2002; Yoshimoto et al., 2008; Smith et al., Disclosure Statement 2009; Matson and Zarkower, 2012; Chen et al., 2014; The authors declare no conflicts of interest. Hirst et al., 2017]. Under this scenario, those chromo- somes that are “better at being sex chromosomes” should be preferentially recruited into a sex-determining role in different lineages. Second, laboratory experiments with the roundworm Caenorhabditis elegans suggest that al-

ZZ/ZW Sex Chromosomes in Cytogenet Genome Res 7 Phyllodactylus DOI: 10.1159/000496379 Downloaded by: Iowa State University 129.186.138.35 - 1/26/2019 5:51:04 PM References

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