doi: 10.1111/j.1420-9101.2008.01641.x

A sex-linked SCAR marker in (), a dioecious with XY sex-determination and homomorphic sex chromosomes

R. K. OYAMA, S. M. VOLZ & S. S. RENNER Department of Biology, Ludwig-Maximilians-Universita¨t, Munich, Germany

Keywords: Abstract Bryonia; Genetic crosses between the dioecious Bryonia dioica (Cucurbitaceae) and the Cucurbitaceae; monoecious B. alba in 1903 provided the first clear evidence for Mendelian population structure; inheritance of dioecy and made B. dioica the first organism for which XY sex- sex chromosome; determination was experimentally proven. Applying molecular tools to this Y-chromosome. system, we developed a sex-linked sequence-characterized amplified region (SCAR) marker for B. dioica and sequenced it for individuals representing the full geographic range of the species from Scotland to North Africa. For comparison, we also sequenced this marker for representatives of the dioecious B. cretica, B. multiflora and B. syriaca, and monoecious B. alba.In no case did any individual, male or female, yield more than two haplotypes. In northern Europe, we found strong linkage between our marker and sex, with all Y-sequences being identical to each other. In southern Europe, however, the linkage between our marker and sex was weak, with recombination detected within both the X- and the Y-homologues. Population genetic analyses suggest that the SCAR marker experienced different evolutionary pressures in northern and southern Europe. These findings fit with phyloge- netic evidence that the XY system in Bryonia is labile and suggest that the genus may be a good system in which to study the early steps of sex chromosome evolution.

in five genera – Cannabis, Humulus, Rumex, Silene and Introduction Coccinia – are known to have morphologically distinct Sexually specialized life cycle stages that are only male sex chromosomes (Ming et al., 2007). Although there is or only female evolved many times among land . no evidence that all organisms with separate sexes are In flowering plants, however, sexual specialization of destined to acquire morphologically distinct sex chro- the sporophyte is rare, with only 6% of the 240 000 mosomes (Lynch, 2007), theory predicts the assortative species being dioecious (Renner & Ricklefs, 1995). In accumulation of sex-determining genes and sexually spite of this limited overall success, dioecy has evolved antagonistic genes, accompanied by suppression of thousands of times, implying the parallel evolution of recombination on one of the gonosomes (in the case different forms of sex determination. The sex determi- of XY sex determination, on the Y-chromosome), nation mechanism that is most common in animals, culminating in the physical differentiation of the two namely heteromorphic XY or WZ sex chromosomes, in sex chromosomes (Bull, 1983; Charlesworth, 1991; plants evolved exceedingly rarely. Only a dozen species Charlesworth, 2002a,b). An early-stage in sex chromo- some evolution would thus involve a chromosome with Correspondence: Ryan K. Oyama, Menzinger Str. 67, 80638 Munich, a recombination-suppressed region that contains key Germany. sex-determining genes. Tel.: +49 (89) 17861 194; fax: +49 (89) 172638; e-mail: [email protected] The first experimental system for the genetics of sex Sequence data from this article have been deposited with the GenBank determination was the Cucurbitaceae genus Bryonia Data Library. The accession numbers are provided in Tables S1 and S2. (Correns, 1903, 1907; Rheinberger, 2000). In experiments

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 214 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Sequence variation of a sex-linked marker in Bryonia (Cucurbitaceae) 215

designed to reveal the mode of inheritance of dioecy, B. multiflora and B. syriaca, and an individual of the Correns carried out numerous reciprocal pollinations monoecious B. alba. The results are the first molecular between the dioecious B. dioica and the monoecious data on sex-determination in this group and represent B. alba. He grew almost 1000 F1 offspring, and from the the first step in re-introducing B. dioica as a system for sex ratio of crosses that involved B. dioica as the father, studying the evolution of dioecy and sex chromosomes. inferred that half the pollen grains of B. dioica carried a ‘female tendency,’ the other half a ‘male tendency.’ In Materials and methods doing so, he provided the first experimental evidence for genetic sex-determination. Because of the importance of Background on study system this discovery, Bateson (1909) and Heilbronn (1948) repeated Correns’s experiments, with the same results. Of the species sampled here, B. dioica occurs throughout Later studies used B. dioica and B. aspera (under the Western Europe and into North Africa; B. alba from synonymous name B. macrostylis) as an additional dioe- Central Europe to Kazakhstan; B. cretica in Greece, cious ⁄ monoecious pair for reciprocal crossing (Heilbronn Crete, Cyprus, Turkey, and south to Egypt and Libya; & Basarman, 1942; Heilbronn, 1953; summarized in B. multiflora in Turkey, Iran, and Iraq; and B. syriaca in Westergaard, 1958). These experiments confirmed XY Lebanon, Israel, and Syria. Information about all sex-determination, with the male the heterogametic sex. sequenced accessions is provided in Table S1. In terms Numerous cytological investigations of Bryonia; however, of genome ploidy, B. alba, B. dioica, B. multiflora, and found no evidence of a dimorphic chromosome pair B. syriaca are diploid (2n =2x = 20), whereas B. cretica is (Strasburger, 1910; Meurman, 1925; Volz & Renner, hexaploid (2n =6x = 60) (Volz & Renner, 2008). All 2008). species of Bryonia are pollinated by pollen- and nectar- In light of current theory on the evolution of sex gathering bees, and the are berries that are chromosomes, the lack of a cytologically distinct pair of dispersed by birds (Kugler, 1981; Westrich, 1989; Schro¨ - chromosomes combined with the clear evidence for an der & Lunau, 2001). One of the pollinators of Bryonia XY system of sex-determination suggests that sex chro- species is the Bryonia pollen specialist Andrena florea, mosome evolution in Bryonia may be at an early stage. which has a foraging range of up to 1 km (Edwards & Support for this hypothesis comes from a phylogeny for Williams, 2004). the seven dioecious and three monoecious species of Bryonia, which reveals at least two switches between DNA extraction monoecy and dioecy within the genus (Volz & Renner, in press). Molecular-clock dating has yielded an age of 8– Total DNA was extracted from silica gel-dried young leaves 9 Myr for the deepest divergence within Bryonia and of sexed plants using NucleoSpin kit (Machery- divergence times on the order of probably a few million Nagel, Du¨ ren, Germany) according to the manufacturer’s years for most of the nine species within core-Bryonia. protocol. DNA concentration and quality was checked The sister genus of Bryonia is Ecballium, which comprises on 1.5% agarose gels by electrophoresis using Lambda a single species that also has XY sex-determination, with DNA as a reference. If required, the concentration was the male again being the heterogametic sex and the adjusted to approximately 150 ng lL)1. chromosomes again monomorphic, but phenotypic sex is environmentally influenced (Gala´n, 1946; Westergaard, Male-linked AFLP marker construction and screening 1958). The sister clade to Bryonia and Ecballium is the Australian genus Austrobryonia, which consists of four We first created DNA bulk samples for each sex by monoecious species (Schaefer et al., 2008). Taken pooling the genomic DNA extractions of up to eight together, these observations point to an evolutionary individuals of B. dioica from northern and southern lability of monoecy and dioecy in the Bryonia clade, and Europe. A restriction digest of these bulks and a ligation suggest that the sex chromosomes of B. dioica may be of the amplified fragment length polymorphism (AFLP) relatively young. adapter ends were performed in a single step, using Here we report on the relative nucleotide divergence in the AFLP library construction kit (Invitrogen GmbH, the X- and Y-homologues of a sex-linked SCAR Karlsruhe, Germany). Approximately 600 ng of DNA (sequence-characterized amplified region) marker within were combined with 1· buffer NEB2 (New England B. dioica, the species that was the focus of Correns’s work Biolabs [NEB], Ipswich, MA, USA), 1· T4 DNA ligase on the inheritance of dioecy. Bryonia dioica occurs from buffer, 1· BSA, 0.8 units of the restriction enzyme MseI, Sweden and Scotland south through Germany, France 4 units of EcoRI, 0.75 units T4 DNA ligase, 30 pm and Spain, to Algeria and Morocco in northern Africa. preannealed MseI+ and MseI) adapter, and 3 pm prean- Thirty-three male and female individuals in this study nealed EcoRI+ and EcoRI) adapter. The adapters serve to represent most of this geographical range. For compar- modify the restriction enzyme recognition site to prevent ative analyses, we also included individuals of the repeated cutting by the enzyme. This solution was then geographically more restricted dioecious species B. cretica, incubated at 22 C overnight and diluted with water to a

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 216 R. K. OYAMA ET AL.

final volume of 50 lL. We then amplified this via PCR, system (Promega, Madison, WI, USA). The cleaned PCR using M02 and E01 primers (preselective amplification). product was cloned using the TEasy-System (Promega), Products were checked on 1.5% agarose gels and diluted and eight colonies were picked. The plasmid product 1 : 50. To reduce the number of bands to a manageable was isolated from the cultures with the Sigma mini-prep number, a second amplification step was applied, using system (Sigma-Aldrich, Munich, Germany) and primers with three additional bases on the 3¢ end sequenced, using the SP6 and T7 primers with BIG DYE (selective amplification). Oligonucleotide nomenclature TERMINATOR 3.0 chemistry (ABI). Sequence assembly follows Keygene guidelines (http://www.keygene.com), and editing were carried out in SEQUENCHER 4.6 (Gene- and all oligos were obtained from MWG Biotech AG, Codes, Ann Arbor, MI, USA) and checked against the Ebersberg, Germany. GenBank database via BLAST. Preamplification was performed in a total volume of 25 lL with 1· PCR buffer (NEB), 0.15 mM dNTP, 12.5 ng SCAR marker development and testing primer M02 and E01 respectively, 0.5 lL Taq polymerase (NEB), 4 lL of the restriction ⁄ ligation product containing Sequences of AFLP markers with the appropriate length approximately 48 ng DNA, and run at 72 C for 2 min, were manually aligned and used to design SCAR primers. followed by 20 cycles of 10s denaturation at 94 C, 30s These primers comprised the restriction-enzyme recog- primer annealing at 56 C and 2min elongation at 72 C, nition site, the three to five selective bases, and 12–20 followed by 30 min at 60 C. Selective amplification was additional sequence-specific bases. As the SCAR primers performed in a total volume of 15 lL with 1· PCR buffer do not comprise the artificial adapter sequences of the (NEB), 0.15 mM dNTP, 12.5 ng selective M-primer (with AFLP primers, the expected SCAR fragment is shorter three selective bases) and fluorescence 6FAM-labelled than the AFLP fragment it amplifies. E-primer (also three selective bases), 0.25 lL Taq poly- Sex specificity of the SCAR primers was tested with merase (NEB), and 3 lL of preamplification product, genomic DNA of 20 male and seven female plants of diluted 1 : 50. PCR conditions were 2 min at 94 C, B. dioica. We used touchdown PCR with 10 lM of primers, 10 cycles of 10 s at 94 C, 30 s at 65 C, 2 min at 25 lM MgCl2,1 lL Q solution (Qiagen, Hilden, Germany), 72 C, followed by 25 cycles of 10 s at 94 C, 30 s 1.25 lM of each dNTP, 2.5 lLof10· PCR-buffer, 0.5 lLof at 56 C, 2 min at 72 C, followed by terminal 30 min Taq DNA polymerase (Qiagen), and 10–50 ng of template at 60 C. If necessary, additional PCR reactions with four DNA per 25 lL of reaction volume. The SCAR primers or five selective nucleotides on the M-primer were were also tested on other species of Bryonia under relaxed performed to reduce the overall amount of fragments. PCR conditions. All resulting bands were gel-cleaned and The AFLP products from the selective PCR were run on sequenced. If the sequence was not clean, the PCR product a 3100 Avant capillary sequencer (Applied Biosystems was cloned and multiple clones were sequenced to assess [ABI], Foster City, CA, USA) alongside a LIZ-labelled the number of alleles. ladder (ABI) and evaluated with GENEMAPPER 3.7 [ABI] to identify AFLP markers linked to the male sex. Because Phylogenetic and population genetic analyses the initial screening was performed with pooled DNA from multiple individuals, the primary difference An alignment of all sequences was created in MacClade between the two samples was the absence or presence 4.08 (Maddison and Maddison, 2003) and adjusted by of a Y-chromosome. Potentially male-linked bands iden- eye. A best maximum-likelihood (ML) tree and corre- tified in the bulk samples were verified by repeating the sponding bootstrap support values were calculated using procedure in several individuals of known sex. the RAxML Blackbox with the default GTR + I + G model of substitution (Stamatakis et al., 2008). Trees were rooted with a sequence from B. alba, the only AFLP fragment isolation and sequencing nondioecious species in our sample. A haplotype net- AFLP markers that were male-linked were isolated and work was also constructed of the B. dioica sequences sequenced. Four parallel amplification reactions were using TCS 1.21 (Clement et al., 2000), treating gaps as pooled, concentrated, re-suspended in 10 lL of water missing characters or as a fifth character. and separated on a Spreadex Mini Gel (Elchrom Scien- Because the distribution of B. dioica is along a mostly tific, Cham, Switzerland) at 100 V for 240 min using the North–South axis from Sweden to North Africa, we SEA 2000 electrophoresis chamber (Elchrom Scientific). grouped the individuals into northern European (i.e. Gels were then stained in ethidium bromide and the North of the Alps) and southern European (i.e. South of targeted fragment was excised and incubated in 50 lLof the Alps and northern Africa) populations. Our northern 10 mM Tris solution at 70 C for 30 min. The supernatant Europe sample consisted of 11 males and six females, the was then used as template DNA to set up a PCR reaction southern Europe sample of 13 males and three females. using the same primers. That reaction was run out on a Sequences from Bavaria were assigned to the southern 2.5% agarose gel, and the target band was excised and population (see Results). Calculations of Waterson’s h purified with the Wizard SV genomic DNA purification (Waterson, 1975), p, Tajima’s D (Tajima, 1989) and FST

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Sequence variation of a sex-linked marker in Bryonia (Cucurbitaceae) 217

values were made using DNASP 4.10.9 (Rozas & Rozas, males, two females), one B. multiflora female, two 1999). B. syriaca females and one individual of monoecious To test whether the variation observed in our sex- B. alba. The resulting 61 sequences have been submitted linked marker was significantly different from expected to GenBank; GenBank accession numbers and voucher variation under a neutral model, we used the program details are provided in Table S1. An alignment of all HKA by Jody Hey (http://lifesci.rutgers.edu/~heylab) sequences was 405 characters long, including gaps. (Hudson et al., 1987). We tested the X- and Y-linked BLAST searches of GenBank with these sequences did sequences from both northern and southern Europe not yield any significant hits, and stop codons exist in all against chloroplast sequences from the same individu- three reading frames. als (see Table S2), using B. multiflora and B. syriaca as Sequences of SCAR-278 from B. dioica males that the outgroup. We adjusted the test for the different matched the original male-linked AFLP marker (AFLP- effective population sizes of the X- and Y-chromo- 278) were classified as Y because the species is known to somes, and of the chloroplast genome. Although the have XY sex-determination (Introduction). All other chloroplast genome may experience selective sweeps, sequences recovered from B. dioica were assigned X or thus violating the assumption of neutral evolution Y status based on the presence (Y) or absence (X) of a (Muir & Filatov, 2007), chloroplast spacer and intron large indel seen between the AFLP-278 marker and the sequences are a suitable yardstick against which to X-homologues (see Fig. 1). Whether the length differ- compare the nuclear SCAR marker sequences. A ence between X- and Y-homologues represents an nonsex-linked nuclear marker, the Leafy gene, has insertion or a deletion cannot be ascertained at this time. been less densely sampled but is congruent with the Our species-level sampling of SCAR-278 sequences is chloroplast phylogeny where the accessions overlap insufficient to reconstruct the evolution of this character (Volz & Renner, 2008). on the available phylogenies for the genus. The Y-sequences of SCAR-278 from B. dioica were Results named BdY1 and the X-sequences BdX1 following the system developed for Silene sex-linked regions (Filatov & Charlesworth, 2002). Sequences from the other species SCAR marker included were not assigned X or Y status at this point Six AFLP markers were unambiguously linked to the because B. alba is monoecious and the sex-determination male sex. We developed one of these markers, AFLP-278, systems of B. multiflora, B. syriaca and B. cretica have not into a SCAR marker (henceforth SCAR-278), following been investigated. However, sequences from these other the procedure summarized in Fig. 1. The SCAR-278 species were similar to the B. dioica X-sequences in that primers were F-gaattcacatcgtggggtcc and R-ttaacacattattt- they also lacked the deletion relative to the original male- caagcaaataagttcc. Using these primers, we obtained PCR linked AFLP-278 marker. products from 33 individuals of B. dioica (24 males, nine All Y-sequences recovered from males in northern females) as well as four individuals of B. cretica (two Europe (n = 10) were identical to each other and to the

0 (bp) 100 200 300 400 500

(a) AFLP-278 (278 bp, including AFLP adaptors)

SCAR-278 (BdY1) (208 bp, excluding SCAR primers)

(b) BdY1 (208 bp)

BdX1 (405 bp)

= AFLP adaptor = Site of indel between homologous X and Y sequences of BdX/Y1

= SCAR primer

Fig. 1 Schematic representation of the products at different stages of sequence characterized amplified region (SCAR) marker development. (a) The relationship between the male-linked AFLP marker and the SCAR marker. (b) The relationship between the male-linked region (BdY1) amplified with the SCAR primers and the homologous sequence assumed to be on the X chromosome (BdX1). The indel event is represented by the white box in the BdX1 sequence.

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 218 R. K. OYAMA ET AL.

original male-linked AFLP maker (AFLP-278). In con- Southern and northern populations behaved inversely trast, there was variation among the nine Y-sequences for Tajima’s D (Table 1). Thus, the X- and Y-sequences in from southern European males (and one southern southern Europe had positive values, whereas the X in European female) and in the length of the deletion northern Europe had negative values (Tajima’s D could relative to the X-sequences. In no case did any individ- not be calculated for the Y-sequences in northern Europe ual, male or female, yield more than two haplotypes. because of the absence of any variation). Although our sample lacks significant genetic structure based on FST values (Table 2), the relatively most divergent groups Phylogenetic analysis were the northern European X- and Y-sequences, with A ML phylogeny of the sequences from B. dioica plus the Y-sequences from northern Europe being the most those from B. alba, B. cretica, B. multiflora and B. syrica is divergent overall. The HKA tests revealed a significant shown as Fig. 2. Sequences from two B. syriaca females departure from neutrality for the X- and Y-sequences in clustered together, whereas two sequences (presumably northern Europe, due to the lower than expected alleles) from a single B. multiflora female did not. Instead, number of segregating sites (S) observed (Table 1). Tests one sequence placed as sister to the B. syriaca clade, the of recombination (Hudson & Kaplan, 1985) found other as sister to the B. dioica ⁄ B. cretica clade (Fig. 2). evidence for at least one recombination event within To better illustrate the relationships among the B. dioica the X- and Y-homologues from southern Europe (see sequences, a pruned version of Fig. 2, including only Table 1). We also tested for recombination after pooling B. dioica sequences and rooted on one of the B. multiflora the X- and Y-sequences for northern and southern alleles, is shown in Fig. 3. The sequences of B. cretica were populations respectively. As expected, we found no excluded from this analysis because this taxonomic entity evidence for recombination among the northern is hexaploid and of hybrid origin, with B. dioica as one of sequences but, somewhat surprisingly, also no evidence the contributing genomes (Volz & Renner, 2008). The for recombination among the southern sequences. This resulting phylogeny shows the Y-sequences from result for the southern sequences is most likely because northern Europe forming a strongly supported clade, of the fact that the southern Y-sequences are extremely indicative of their distinctness (Fig. 3). The homologous heterogeneous in their length, with some having an even X-sequences from these males, together with larger deletion than the northern Y-sequences, resulting X-sequences from northern European females, also form in sequences that are only 159 bp in length. This level of a clade. Sequences from southern European accessions variability in the number of gap characters reduces the behaved differently, with five males (SV006, SV054, power of the test to detect recombination events. SV112, SV123, SV153) yielding two X-sequences and no Y-sequence. Males from Denmark (SV147), Italy (SV136, Discussion SV137), and Morocco (SV107) yielded only Y-sequences, and thus it is unknown where in the phylogeny their Results presented here are the first molecular investiga- homologous X-sequences would place. In addition, one tion into the evolution of sex chromosomes in B. dioica, female from southern Europe yielded an X- and a a classical system for studying the inheritance of dioecy. Y-homologue for the SCAR marker (SV146). Our newly developed sex-linked SCAR marker, which we named BdX1 ⁄ Y1, is perfectly linked to sex in northern Europe and supports the finding of XY sex-determination Haplotype network in B. dioica from northern Europe by Correns (1903, The structure of the haplotype network recovered using 1907). His experimental crosses of the dioecious B. dioica TCS was sensitive to whether gaps were treated as missing and the monoecious B. alba provided the first proof of XY data or as a fifth character (data not shown). With gaps sex-determination in any organism and played an treated as a missing character, two haplotype networks important role in the early understanding of the genetics were found (Fig. 4). One consisted of the Y-sequences of sex (Bateson, 1909; Rheinberger, 2000). However, from Italy and Croatia. The remaining X- and Y-sequences when sequences of BdX1 ⁄ Y1 from southern Europe are formed a second network, and this included all of the included in the analyses, the picture becomes more Y-sequences identical to the original AFLP-278 as well as complicated because the linkage of our SCAR marker the Y-sequences from Spain and Morocco. The haplotype with sex is weaker in southern than in northern network differs from the tree in that the Y-sequences have Europe. Perceptions of molecular variability on the at least two, possibly three, origins (because of the Y-chromosome of B. dioica are thus dependent on placement of two Y-sequences in Group H in Fig. 4). geographic sampling. This suggests that the sex chromo- somes of B. dioica may be at an early stage in their evolution. Analysis of genetic variation The phylogeny obtained from the sex-linked SCAR Among both the X- and Y-sequences, the highest marker sequenced here is congruent with phylogenies pairwise diversity (p) is found in southern Europe. from chloroplast and nuclear loci, to the extent that

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Sequence variation of a sex-linked marker in Bryonia (Cucurbitaceae) 219

Fig. 2 Maximum-likelihood (ML) phylog- eny of all SCAR-278 sequences. Numbers above branches are ML bootstrap values. Support values for most clades within the Bryonia dioica ⁄ B. cretica group are shown in Fig. 3. Sequence labels are organized in the order: Species ⁄ accession-number ⁄ local- ity ⁄ allele. Abbreviations of species names are: alb = B. alba, cre = B. cretica, dio = B. dioica, mul = B. multiflora, syr = B. syriaca. Country abbreviations fol- low the ISO 3166 (3-alpha) system. See supplementary Table S1 for full information on accessions. Sequences from dioecious taxa other than B. dioica were not assigned X or Y status because the basis for sex determina- tion in these taxa has not been investigated. species sampling overlaps (Volz & Renner, 2008). evidence all suggest that, at the very least, this sex-linked Phylogenies that include all species of Bryonia, indicate marker is not highly conserved across species within the that there were at least two evolutionary transitions genus. between dioecy and monoecy within the genus, which The pattern of sequences from the 11 males and six makes it unlikely that all dioecious Bryonia species females of B. dioica sampled across northern Europe fit inherited sex-determining chromosomes from a common the expectation of XY sex-determination. No female from ancestor (Volz & Renner, 2008). This conclusion is northern Europe yielded a Y-sequence and, with two supported by the phylogeny presented here in that the exceptions, all males yielded both an X- and a sequences of the sex-linked SCAR-278 locus group by Y-sequence. The two exceptions were accession SV172 species, rather than by their X or Y status (Fig. 2). The from Austria, from which we only recovered an primers for BdX1 ⁄ Y1 also failed to work in some of the X-sequence, and SV147 from Denmark, from which we seven dioecious species of Bryonia, such as the Canary only obtained a Y-sequence. Although we would expect Island endemic B. verrucosa, which is sister species to the to find, respectively, a Y and an X from these males as nine remaining species of Bryonia. These multiple lines of well, these results do not contradict the findings of

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 220 R. K. OYAMA ET AL.

Fig. 3 A maximum-likelihood (ML) phy- logeny of just the Bryonia dioica BdX ⁄ Y1 sequences to allow the relationships to be seen more clearly. Numbers above branches are maximum-likelihood bootstrap values. Accessions in bold italics are from southern Europe, all other B. dioca accessions are from northern Europe.

Correns as PCR could have failed for a variety of reasons. observed both an X- and a Y-sequence from four males Most importantly, all of the BdY1 sequences from and only an X from one female in southern Europe. northern Europe were identical to the original male- However, from the remaining accessions, the picture is linked AFLP marker (AFLP-278) and grouped together in mixed, with five males yielding only a Y-sequence, four the phylogeny (Fig. 3). Thus, BdY1 and the male-deter- males yielding two X-sequences and one female yielding mining locus appear to be strongly linked in northern an X- and a Y-sequence. Thus, only half of the accessions European B. dioica. from southern Europe yield SCAR-278 sequences that In contrast to northern Europe, sequences of BdY1 segregate as expected, and five yield sequences that from southern Europe have weaker linkage disequilib- appear to contradict the finding of XY sex-determination rium to the male-determining locus. In keeping with our by Correns. In addition, phylogenetic analyses place most expectation for an XY sex-determination system, we of the southern X- and Y-sequences in a paraphyletic

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Sequence variation of a sex-linked marker in Bryonia (Cucurbitaceae) 221

Fig. 4 A haplotype network of all BdX1 ⁄ Y1 sequences from Bryonia dioica.

grade below their respective northern counterparts. although none are significant. That the highest FST Together, these results suggest geographical structure values were found in the pairwise comparisons with among the sequences of BdX1 and BdY1. BdY1 from northern Europe suggests that this may be the The suggestion of geographical structure based on most isolated group, even though the comparisons were phylogenetic analysis is supported by population genetic not significant. The complete lack of variation among the analyses. The southern European population has positive BdY1 sequences from northern Europe, meanwhile, Tajima’s D values for both X- and Y-homologues, and the suggests either a selective sweep or a bottleneck event northern European population has negative values, for the Y-homologue in the north, although the existence

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 222 R. K. OYAMA ET AL.

Table 1 Intra-population variation among the SCAR-278 sequences (BdX ⁄ Y1) recovered from Bryonia dioica.

HKA

2 Population nS nH S phw DRv P

BdX1-all 32 12 12 0.0119 0.0121 )0.0521 1 5.5024 0.064 X – North 16 6 7 0.0037 0.0058 )1.2687 0 6.5686 0.037* X – South 17 10 10 0.0138 0.0120 0.5618 1 5.1005 0.078 BdY1-all 19 5 15 0.0380 0.0296 0.7656 2 3.3916 0.183 Y – North 10 1 0 0 0 – 0 10.9690 0.004* Y – South 9 5 15 0.0502 0.0381 1.1453 2 3.4946 0.174 nS, number of sequences; nH, number of haplotypes; S, variable sites; D, Tajima’s D; R, minimum number of recombinations. The HKA test was performed using chloroplast sequences as the second marker and, as the outgroup, two haplotypes of B. syriaca and two of B. multiflora. *, significant P-value.

Table 2 Values of p (lower left half) and FST (upper right half) from observed in Y-linked markers of Silene (Ironside & pairwise comparisons of SCAR-278 sequences (BdX ⁄ Y1) from each Filatov, 2005). However, the high diversity within population of Bryonia dioica. southern European BdX1 and BdY1 differs from the situation in Silene, where most of the polymorphic sites F ST were fixed within geographical areas (Ironside & Filatov, p X – north X – south Y – north Y – south 2005). In addition, the population structure of X- and Y-linked genes within the five species of Silene that have X – north 0.5340 0.9067 0.5883 sex chromosomes may be influenced by ongoing gene X – south 0.0116 0.4630 0.0549 Y – north 0.0295 0.0284 0.5613 flow among these species (Ironside & Filatov, 2005; Muir Y – south 0.0321 0.0228 0.0380 & Filatov, 2007; Prentice et al., 2008). In contrast, ongoing hybridization is unlikely to be an issue in B. dioica. Although ancient hybridization with B. cretica of variation among BdY1 sequences in the south shows in mainland Greece is a possibility, these species do not that there has not been a species-wide selective sweep. co-occur today nor do the two other dioecious species The HKA tests support this interpretation of different included here (see Materials and methods for the evolutionary histories between northern and southern geographic range of each species). populations for both BdX1 and BdY1, with the signifi- The different levels of sequence variation in northern cantly lower than expected number of segregating sites and southern Europe may be due to different levels of among the northern BdX1 ⁄ Y1 sequences again suggesting linkage disequilibrium between BdY1 and the male- either background selection or a population bottleneck in determining locus (i.e. maleness) in the two populations. the evolutionary past. A bottleneck is congruent with If we assume that BdY1 is strongly linked to the male- phylogeographic analyses of chloroplast and leafy data, determining locus of the Y-chromosome in the north and which suggest that B. dioica recolonized northern Europe only weakly linked in the south, then this leads to from the south after the last glacial maximum (Volz & predictions that are met by our data. As recombination Renner, in press). Nevertheless, this demographic narra- on the Y-chromosome should only be suppressed in (or tive alone does not explain the lower levels of variation near) the male-determining locus, one would expect to in northern Europe relative to the chloroplast dataset, as find recombination in our marker from populations where seen in the HKA tests. the two are weakly linked and no recombination in the Geographic structure and the absence of variation strongly linked populations. And indeed, we detected among BdY1 sequences from northern Europe (from a recombination events in southern BdX1 and BdY1, but geographic sample that spans Scotland, France, none in the north (Table 1). In southern populations, the Germany, the Netherlands and Denmark) are both correlation between the possession of a Y-homologue of consistent with findings in other organisms. Thus, studies our marker and maleness should be blurred, resulting in across European Silene latifolia (Filatov et al., 2000; males with two X-sequences and females with a Laporte et al., 2005) and European domestic horses Y-sequence. In the southern populations, we indeed (Lindgren et al., 2004) found no variation among the found four males with two X-homologues and no Y-linked sequences. Indeed, in 10 species of animals for Y (SV006, SV054, SV112 and SV123), and a female with which data are available, levels of variation on the an X- and Y-sequence (SV146). Where differently Y-chromosome are undetectable or substantially lower behaving populations come into contact, one would than those on either the X or the autosomes (Lynch, expect to find individuals with a mix of southern and 2007; Table 12.2). Background selection and a local, northern X- and Y-sequences, which we did (SV047, rather than species-wide, selective sweep have also been SV050).

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Sequence variation of a sex-linked marker in Bryonia (Cucurbitaceae) 223

The evidence for recombination within BdX1 and BdY1 Charlesworth, B. 1991. The evolution of sex-chromosomes. in southern Europe suggests crossing over between Science 251: 1030–1033. X- and Y-chromosomes, although recombination Charlesworth, B. 2002a. The evolution of chromosomal sex between two Y-chromosomes cannot be ruled out. determination. Genetics and Biology of Sex Determination. According to theory for the evolution of sex-determining pp. 207–224. Novartis Foundation Symposium, Wiley. Charlesworth, D. 2002b. Plant sex determination and sex chromosomes, suppression of recombination should chromosomes. Heredity 88: 94–101. spread gradually along the Y-chromosome as male-linked Charlesworth, D., Charlesworth, B. & Marais, G. 2005. Steps in genes accumulate there, with morphological differentia- the evolution of heteromorphic sex chromosomes. Heredity 95: tion of the X and Y being the last step (Charlesworth, 118–128. 2002a,b; Charlesworth et al., 2005). As the sex chromo- Clement, M., Posada, D. & Crandall, K.A. 2000. TCS: a computer somes of B. dioica are not morphologically distinguish- program to estimate gene genealogies. Mol. Ecol. 9: 1657–1660. able, the pseudo-autosomal region (PAR) of the Correns, C. 1903. U¨ ber die dominierenden Merkmale der Y-chromosome should still be relatively large. One Bastarde. Ber. Dtsch. Bot. Ges. 21: 133–147. consequence of this would be that recombination would Correns, C. 1907. Die Bestimmung und Vererbung des Gesch- still be expected to occur between much of the X- and lechtes, nach Versuchen mit ho¨ heren Pflanzen. Verh. Ges. deutscher Naturforscher und A¨ rzte 1907: 794–802. Y-chromosomes. This also leads to an explanation for the Edwards, M. & Williams, P. 2004. Where have all the bumble- different levels of linkage disequilibrium of our marker bees gone and could they ever return? Br. Wildl. 2004: 1–11. and sex between northern and southern Europe. Filatov, D.A. & Charlesworth, D. 2002. Substitution rates in the Namely, that in northern Europe the nonrecombining X- and Y-linked genes of the plants, Silene latifolia and S. dioica. region may be larger, either encompassing BdY1 or being Mol. Biol. Evol. 19: 898–907. close enough to result in strong linkage to the male- Filatov, D.A., Moneger, F., Negrutiu, I. & Charlesworth, D. 2000. determining locus, whereas in southern Europe BdY1 is Low variability in a Y-linked plant gene and its implications well inside the PAR, and thus weakly linked to maleness. for Y-chromosome evolution. Nature 404: 388–390. In this study, we bring modern molecular tools to bear Gala´n, F. 1946. Sur la ge´netique de la monoecie et la dioecie on the sex chromosomes of B. dioica, the classical plant zygotique chez Ecballium elaterium Rich. C. R. des Se´ances de l’Acad. Sci. de Paris 222: 1130–1131. system for investigating the inheritance of dioecy and the Heilbronn, A. 1948. U¨ ber die Genetik von Mono¨ zie und first organism in which XY sex-determination was Getrenntgeschlechtlichkeit bei Bryonia Bastarden. Proc. 8th experimentally proven. Although the limited nucleotide Int. Congr. Genet. Hereditas Suppl. 590–591. sampling requires cautious interpretation, geographic Heilbronn, A. 1953. U¨ ber die Rolle des Plasmas bei der sampling of B. dioica is representative, and the results Geschlechtsbestimmung der Bryonien. Rev. Fac. Sci. Univ. can be interpreted with the help of an existing phyloge- Istanbul Ser. B Sci. Nat. 18: 205–207. netic framework that includes all species in the genus. Heilbronn, A. & Basarman, M. 1942. U¨ ber die F2 der Bryonia The main patterns found, especially the extremely low Bastarde und ihre Bedeutung fu¨ r das Problem der Geschlechts- level of polymorphism of the Y-linked marker compared realisation. Rev. Fac. Sci. Univ. Istanbul, Ser. B: Sci. Nat. 7: 138–144. to the X-linked sequences in northern Europe, are Hudson, R.R. & Kaplan, N. 1985. Inferring the number of recombination events in the history of a sample. Biometrics 41: consistent with the original interpretation of Correns 572. that B. dioica has XY sex-determination. The data from Hudson, R.R., Kreitman, M. & Aguade, M. 1987. A test of southern Europe, meanwhile, point to the as yet incom- neutral molecular evolution based on nucleotide data. Genetics pletely understood complexity of sex chromosome evo- 116: 153–159. lution in Bryonia. Ironside, J.E. & Filatov, D.A. 2005. Extreme population structure and high interspecific divergence of the Silene Y chromosome. Genetics 171: 705–713. Acknowledgments Kugler, H. 1981. Zur Besta¨ubung von Bryonia dioica Jacq. Ber. The authors thank F. Blattner, M. Erben, A. Kocyan, and Dtsch. Bot. Ges. 94: 287–290. Laporte, V., Filatov, D.A., Kamau, E. & Charlesworth, D. 2005. F. Schuhwerk for collections; F. Blattner, Institut fu¨ r Indirect evidence from DNA sequence diversity for genetic Pflanzengenetik, Gatersleben for guidance with AFLP degeneration of the Y-chromosome in dioecious species of the marker generation; and Grischa Brokamp for his diligent plant Silene: the SlY4 ⁄ SlX4 and DD44-X ⁄ DD44-Y gene pairs. work in the lab. This project was supported by a grant J. Evol. Biol. 18: 337–347. to SSR from the Deutsche Forschungsgemeinschaft Lindgren, G., Backstrom, N., Swinburne, J., Hellborg, L., (603 ⁄ 4-1). Einarsson, A., Sandberg, K., Cothran, G., Vila, C., Binns, M. & Ellegren, H. 2004. Limited number of patrilines in horse domestication. Nat. Genet. 36: 335–336. References Lynch, M. 2007. The Origins of Genome Architecture. Sinauer Associates, Inc., Sunderland, MA. Bateson, W. 1909. Mendel’s Principles of Heredity. Cambridge Maddison, W. & Maddison, D. 1992. MacClade: Analysis of University Press, Cambridge. Phylogeny and Character Evolution. Sinauer, Sunderland, Mas- Bull, J.J. 1983. Evolution of Sex Determining Mechanisms. Benja- sachusetts. min ⁄ Cummings, Menlo Park, CA.

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 224 R. K. OYAMA ET AL.

Meurman, O. 1925. The chromosomee behavior of some Tajima, F. 1989. Statistical method for testing the neutral dioecious plants and their relatives with special references to mutation hypothesis by DNA polymorphism. Genetics 123: sex chromosomes. Soc. Sci. Fenn. Comm. Biol. 2: 1–105. 585–595. Ming, R., Wang, J., Moore, P.H. & Paterson, A.H. 2007. Volz, S.M. & Renner, S.S. 2008. Hybridization, polyploidy, and Sex chromosomes in flowering plants. Am. J. Bot. 94: 141– evolutionary transitions between monoecy and dioecy in 150. Bryonia (Cucurbitaceae). Am. J. Bot. 95: 1297–1306. Muir, G. & Filatov, D. 2007. A selective sweep in the chloroplast Volz, S.M. & Renner, S.S. in press. Phylogeography of the DNA of dioecious Silene (Section Elisanthe). Genetics 177: ancient Eurasian medicinal plant genus Bryonia (- 1239–1247. ceae) inferred from nuclear and chloroplast sequences. Taxon. Prentice, H.C., Malm, J.U. & Hathaway, L. 2008. Chloroplast Waterson, G.A. 1975. On the number of segregating sites in DNA variation in the European herb Silene dioica (red genetical models without recombination. Theor. Popul. Biol. 7: campion): postglacial migration and interspecific introgres- 256–275. sion. Plant Syst. Evol. 272: 23–37. Westergaard, M. 1958. The mechanism of sex determination in Renner, S.S. & Ricklefs, R.E. 1995. Dioecy and its correlates in dioecious flowering plants. Adv. Genet. 9: 217–281. the flowering plants. Am. J. Bot. 82: 596–606. Westrich, P. 1989. Die Wildbienen Baden-Wu¨ rttembergs Volume 1 Rheinberger, H.J. 2000. Mendelian inheritance in Germany and 2. Eugen Ulmer, Stuttgart. between 1900 and 1910. The case of Carl Correns (1864– 1933). C. R. Acad. Sci. Ser. III. Sci. Vie ⁄ Life Sci. 323: 1089– 1096. Supporting Information Rozas, J. & Rozas, R. 1999. DnaSP version 3: an integrated Additional Supporting Information may be found in the program for molecular population genetics and molecular online version of this article: evolution analysis. Bioinformatics 15: 174–175. Table S1 List of SCAR-278 sequences used in this study Schaefer, H., Telford, I.R.H. & Renner, S.S. 2008. Austrobryonia (Cucurbitaceae), a new Australian endemic genus, is the and their accession information. closest living relative to the Eurasian and Mediterranean Table S2 Accession information for chloroplast sequ- Bryonia and Ecballium. Syst. Bot. 33: 125–132. ences used in HKA analyses. Schro¨ der, S. & Lunau, K. 2001. Die oligolektische Sandbiene Please note: Wiley-Blackwell are not responsible for Andrena florea und die Rote Zaunru¨ be Bryonia dioica – Schnit- the content or functionality of any supporting materials tstelle zweier spezialisierter Fortpflanzungssysteme. Mitt. Dtsch. supplied by the authors. Any queries (other than missing Ges. Allg. Angew. Entomol. 13: 529–533. material) should be directed to the corresponding author Stamatakis, A., Hoover, P. & Rougemont, J. 2008. A rapid for the article. bootstrap algorithm for the RAxML web-servers. Syst. Biol. 57: 758–771. Received 26 May 2008; revised 25 September 2008; accepted 3 October Strasburger, E. 1910. U¨ ber geschlechtsbestimmende Ursachen. 2008 Jahrb. Wiss. Bot 48: 427–520.

ª 2008 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 214–224 JOURNAL COMPILATION ª 2008 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY