Chromosome Res DOI 10.1007/s10577-017-9555-y

ORIGINAL ARTICLE

Cytogenetic comparison of heteromorphic and homomorphic sex chromosomes in Coccinia () points to sex chromosome turnover

Aretuza Sousa & Jörg Fuchs & Susanne S. Renner

Received: 11 January 2017 /Revised: 17 February 2017 /Accepted: 27 February 2017 # Springer Science+Business Media Dordrecht 2017

Abstract Our understanding of the evolution of in male cells of all three species accumulate some of the sex chromosomes is increasing rapidly due to high- very same repeats that are enriched on the C. grandis Y throughput sequencing data and phylogenetic and chromosome, pointing to either old (previous) sex chro- molecular-cytogenetic approaches that make it possible mosomes or incipient (newly arising) ones, that is, to sex to infer the evolutionary direction and steps leading from chromosome turnover. A 144-bp centromeric satellite homomorphic to heteromorphic sex chromosomes. Here, repeat (CgCent) that characterizes all C. grandis chro- we focus on four species of Coccinia,agenusof25 mosomes except the Y is highly abundant in all centro- dioecious species, including ,thespe- meric regions of the other species, indicating that the cies with the largest known plant Y chromosome. Based centromeric sequence of the Y chromosome diverged on a phylogeny for the genus, we selected three species very recently. close to C. grandis to test the distribution of eight repet- itive elements including two satellites, and several plastid Keywords Coccinia species . genome size . FISH . and mitochondrial probes, that we had previously found histone modification . repetitive DNA . plant sex to have distinct accumulation patterns in the C. grandis chromosomes genome. Additionally, we determined C-values and per- formed immunostaining experiments with (peri-)cen Abbreviations tromere-specific antibodies on two species (for compar- BAC Bacterial artificial chromosome ison with C. grandis). In spite of no microscopic chro- FISH Fluorescent in situ hybridization mosomal heteromorphism, single pairs of chromosomes PCR Polymerase chain reaction rDNA Ribosomal DNA Responsible Editor: Hans de Jong. SDR Sex-determining region

Electronic supplementary material The online version of this article (doi:10.1007/s10577-017-9555-y) contains supplementary material, which is available to authorized users. Introduction A. Sousa (*) : S. S. Renner (*) Department of Biology, University of Munich (LMU), 80638 Munich, Germany In flowering , morphologically distinct e-mail: [email protected] (heteromorphic) sex chromosomes are known from 19 e-mail: [email protected] species in 4 families, while homomorphic sex chromo- J. Fuchs somes are known from at least 20 species in 13 families, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), including both X/Yand Z/W systems (Ming et al. 2011; Gatersleben, 06466 Stadt Seeland, Germany Charlesworth 2016). Their patchy phylogenetic Sousa A. et al. distribution implies that most angiosperm sex chromo- chromosomes of the distant papaya relative some systems evolved independent of each other. The Vasconcellea parviflora, while some of the same BACs few systems for which densely sampled phylogenies are applied in Jacaratia spinosa, another distant relative of available further have shown that even species in the papaya, were located at regions showing perfect pairing same genus, such as Silene colpophylla and Silene otitis, at the pachytene stage without any loop or sign of which last shared a common ancestor 10–11 million heteromorphism. years ago (Slancarova et al. 2013), have non- Here, we analyze whether any Coccinia grandis Y homologous sex chromosomes, the first in an XY sys- chromosome-enriched sequences can be used to identify tem and the second in a ZW system (Mrackova et al. (potential) homomorphic sex chromosomes in its close 2008;Maraisetal.2011; Slancarova et al. 2013). Spe- relatives. In C. grandis, the accumulation of repetitive cies with homomorphic sex chromosomes presumably elements (as well as plastid and mitochondrial DNA) on have small sex-determining regions (SDRs), as inferred the Y chromosome causes a male/female difference of for Vitis vinifera (Fechter et al. 2012;Picqetal.2014), 100 Mbp/2C (Sousa et al. 2013), which raises the ques- Populus trichocarpa and P. tremuloides (Geraldes et al. tion if the same elements are already abundant in related 2015), Actinidia chinensis (Zhang et al. 2015), Carica species (Piednoel et al. 2012). To date, there are no papaya (Wang et al. 2012; Lappin et al. 2015), and experimental or other studies on the sex determination Fragaria chiloensis (Tennessen et al. 2016). The sex system in any of the species of Coccinia other than chromosomes in these species may be between 18 and 2 C. grandis. The latter species is native to tropical million years old (Wang et al. 2012;Picqetal.2014; and India and a member of a small, entirely dioecious Geraldes et al. 2015;Lappinetal.2015; Tennessen et al. Cucurbitaceae genus of 25 species (Holstein and Renner 2016), but most these estimates assume that the respec- 2011;Holstein2015). So far, C. grandis is the only tive sex chromosomes are as old as the genus or subge- species in the genus known to have heteromorphic sex nus to which the focal species belongs, which may be a chromosomes. The Y chromosome is so large that the risky assumption. The small SDRs of homomorphic sex male C. grandis genome is 10% larger than the female chromosomes may result from occasional XY recombi- (Sousa et al. 2013). The sex chromosomes of C. grandis nation and from a high rate of turnover, meaning that sex are about 3 Ma old (Holstein and Renner 2011) and are chromosomes are replaced before they had time to de- unusual among sex chromosomes in the cay (Tennessen et al. 2016). For example, the SDR of Y being heterochromatic (Sousa et al. 2013, 2016). P. trichocarpa sits on the subtelomeric region of chro- Using a phylogeny that includes most of the 25 species mosome 19 (Geraldes et al. 2015)andthatof of Coccinia,weselectedC. hirtella, C. sessilifolia,and P.tremuloides on the pericentromeric region of the same C. trilobata as closely related to C. grandis and then chromosome (Kersten et al. 2014), which suggests applied molecular cytogenetic methods, as well as C- restructuring of the sex chromosomes in these closely value comparisons of male and female genomes. The related species. questions that we wanted to answer were as fol- Most studies of homomorphic sex chromosomes lows: (i) what is the extent of variation in chro- have focused on the construction of genetic maps in mosome numbers and/or genome sizes among silico for the identification of X-Yor Z-W-linked homo- close relatives of C. grandis? (ii) Are potential logues (Asparagus officinalis: Telgmann-Rauber et al. chromosome number deviations accompanied by 2007; Deng et al. 2012; Harkess et al. 2015; Silene variations in the number of ribosomal DNA otites: Slancarova et al. 2013; A. chinensis:Fraser (rDNA) loci or the presence of interstitial et al. 2009 ; Fragaria virginiana: Spigler et al. 2008; telomeric sequences? (iii) Do any chromosome Tennessen et al. 2016; Populus hybrids: Yin et al. 2008; pairs differentially accumulate repeats (a question Pistacia vera: Kafkas et al. 2015). Such markers have for which we focused on those repeats accumulat- rarely been tested in situ, except in homomorphic sex ed on the heteromorphic sex chromosomes of chromosomes of C. papaya (Zhang et al. 2008). Taking C. grandis) or have an extended centromere- advantage of 29 bacterial artificial chromosomes specific histone marker accumulation, as found in (BACs) developed to cover the papaya sex- the C. grandis Y chromosome? And (iv) is the determining region, Iovene et al. (2015) detected a 144-bp-long centromere satellite that in C. grandis minute heteromorphic region in pachytene meiotic hybridizes to the centromeres of all chromosomes Heteromorphic and homomorphic sex chromosomes except the Y also detectable in other species of generate CocA1, and its purified PCR product was Coccinia? Rapid divergence of sex chromosome labeled with digoxigenin-11-dUTP (Roche) by nick centromeres is known from primates in which the translation. Fluorescence in situ hybridization, image X chromosomes of closely related species can capture, and editing were performed as Sousa et al. differ in just their centromeric position (Ventura (2016). et al. 2001). Chromosome preparation for FISH

Materials and methods tips of C. hirtella and C. sessilifolia were collected from potted plants and pretreated in 70 ppm of cyclo- Plant material heximide (Roth) in 2 mM 8-hydroxyquinoline for 5 h at 18 °C to obtain higher numbers of metaphase cells Tubers of C. hirtella, C. sessilifolia,and (Tlaskal 1980). Root tips were fixed in freshly prepared C. trilobata, all of them dioecious climbers, were 3:1 (v/v) ethanol/glacial acetic acid at room temperature collected by N. Holstein in Northeastern Tanzania, for 2 h, transferred to 70% ethanol, kept at room tem- Africa, in 2009. In Munich, we are cultivating these perature overnight, and afterwards stored at −20 °C until plants in the greenhouses of the Botanical Garden, use. Cell suspensions for dropping were obtained fol- and vouchers have been deposited in the herbarium lowing the protocol of Aliyeva-Schnorr et al. (2015) of Munich (official acronym M; for voucher list, see with minor modifications as in the study by Sousa Holstein (2015;hisTable2)). Pistillate or staminate et al. (2016). To make meiotic slides of C. trilobata, flowers were used to identify female or male plants. we used the squashing technique as described in Sousa et al. (2013). FISH probes and conditions Immunofluorescence Arabidopsis-type telomeres and 5S and 45S rDNA sites were visualized according to Sousa et al. (2013), with Immunostaining experiments were carried out on the addition of an ethanol series of 70-90-100%, 2 min C. hirtella and C. sessilifolia. Root tips were pretreated each, after the final washes. To analyze the distribution as previously described, followed by steps of fixation in of repetitive and organellar DNA, we relied on a series paraformaldehyde, enzymatic digestion, and washes as of probes specific for C. grandis (Sousa et al. 2016). in the study by Sousa et al. (2016). The following Purified PCR products were labeled with digoxigenin- primary antibodies were used: rabbit anti- 11-dUTP (Roche) by nick translation. We also designed phosphorylated histone H2AThr120 (diluted 1:150) a probe for a likely homeologous chromosome pair in and monoclonal mouse anti-phosphorylated H3Ser10 C. hirtella, C. sessilifolia, C. trilobata,andC. grandis, (diluted 1: 500, Abcam). Cy3-conjugated anti-rabbit which we named Coccinia autosome 1 (CocA1, IgG (Dianova) and FITC-conjugated anti-mouse IgG KY402258). This probe was obtained after observation (Dianova) were used as secondary antibodies (Houben of the hybridization signals of the satellite CL97, which et al. 1999; Demidov et al. 2014). Finally, preparations in C. grandis labeled the Y chromosome along its were counterstained with 4′,6-diamidino-2- length as well as the subterminal region of one chro- phenylindole hydrochloride (DAPI) mounted in mosome pair and the pericentromeric regions of all Vectashield (Vector). chromosomes. In male nuclei of the other three spe- cies, CL97 always labeled only one chromosome pair. Genome size measurement After sequencing PCR products of CL97 of C. grandis and C. sessilifolia males, we detected 82% identity Nuclei were isolated from young of one male and among sequences and developed primers (R: three female individuals of C. sessilifolia, two male GGTGGAAGGTCGGAATGAAG; F: TGCTTGGG individuals of C. hirtella, and one male of C. trilobata CGAGAGTAGTAC) to amplify the region of the and flow cytometrically measured as described by Sou- monomer shared among these species. These primers sa et al. (2013). Each individual was measured at least and DNA of C. sessilifolia males were used to four times using Glycine max, cv. Cina 5202 ‘Vo ra n’ Sousa A. et al.

(IPK genebank accession number SOJA 392; selected eight repetitive sequences including two satel- 2C = 2.23 pg; Borchert et al. 2007) as internal reference lites and several plastid and mitochondrial probes (next standard. The absolute DNA amounts were calculated section) for the experiments presented here. Seven of the based on the values of the G1 peak means. eight repeats were previously found to accumulate on the Y chromosome and the last one at the centromeres in C. grandis (Sousa et al. 2016). The Ty3/Gypsy/Athila element CL9 in C. grandis Results accumulates on both arms of the Y (Fig. 1i, m). In C. trilobata, this element has subtelomeric positions, Cytology, genome sizes, telomeres, rDNA, repetitive but one chromosome pair in male cells of C. hirtella DNA, and centromere immunostaining distinctly accumulates CL9 (Fig. 1e, arrows; Table 2). The Ty1/Copia/Angela element CL10 in C. grandis pro- The chromosome numbers of C. hirtella and vides a marker for the identification of the Y chromatin C. sessilifolia are 2n = 24 (confirming light domain in interphase nuclei (Fig. 1j), while in the other microscopy counts of Holstein 2015)andthatof three species, it is distributed mostly pericentromerically C. trilobata is 2n =20(Table1). Of the remaining (Fig. 1b, f, b; also Figs. S1f and S3b for C. grandis). The species so far counted, Coccinia grandiflora has Ty3/Gypsy/Ogre/Tat element CL19 is scattered on all 2n =24andCoccinia rehmannii 2n =20(Holstein chromosomes (Fig. 1c,g,o;Fig.S3c,gforC. grandis). 2015). Genome sizes are shown in Table 1; the species Signals of a long interspersed nuclear element (LINE) with the lowest chromosome number, C. trilobata,has CL44 are mostly restricted to the pericentromeric regions the largest genome (1.263 pg/2C). Telomere signals (Fig. 1d,h,p;Fig.S1d,h:inC. grandis), except in one are restricted to chromosome ends (Fig. S1a,d,g,j), chromosome pair of male C. trilobata (Fig. 1l,p;Table2) and the presence of only one 5S locus, located at the where this sequence is highly enriched. Interestingly, this subterminal region of a chromosome pair, is conserved chromosome pair also accumulates CL10 (Fig. 1n,p, among species (Fig. S1b,e,h,k).Coccinia hirtella arrow). The Ty3/Gypsy/Chromovirus element CL86 is and C. sessilifolia each have three loci of 45S rDNA enriched on one chromosome pair of male C. trilobata (Fig. S1c, f, i), while C. grandis and C. trilobata each (Fig. 2j, arrow), on a few chromosomes of C. sessilifolia have two (Fig. S1l). (Figs. 2a and S4a), and two chromosomes of male As seen in the molecular phylogeny (from Holstein C. hirtella (Fig. 2d, arrows) that also accumulate CL97 and Renner 2011; our Fig. S2), C. hirtella and (Table 2). The repetitive element CL260, which is abun- C. sessilifolia are closest to each other, followed by dant in C. grandis (Fig. 2i), accumulates on two chromo- C. grandis,withC. trilobata sister to all three. From somes of male C. sessilifolia (compare Fig. 2c,arrows) probes developed for C. grandis based on high- that also accumulate CL97 (compare Fig. 2b,c,arrows). throughput sequence data (Sousa et al. 2016), we Lastly, CL1, a 144-bp-long satellite that is extremely abundant in all C. grandis centromeres except that of the Y chromosome (Fig. S5c), is abundant in Table 1 Chromosome numbers (2n) and DNA content of male and female Coccinia plants C. sessilifolia males and females and C. hirtella males (Fig. S5a, b) and sometimes spreads over the Species 2n DNA content SD pericentromeric region, occasionally covering entire (pg/2C) arms. In C. trilobata, signals were more apparent at C. grandisa Male 24 0.943 0.005 interphase nuclei than at chromosomes in prophase I of meiosis, where we detected weak signals in only some Female 0.849 0.005 bivalents (data not shown). C. hirtella Male 24 0.988 0.044 Immunostaining with antibodies against the his- C. sessilifolia Male 24 0.984 0.005 tone modifications H2AThr120ph and H3Ser10ph Female 0.998 0.004 revealed signals on the (peri-)centromeric regions C. trilobata Male 20 1.263 0.004 of all chromosomes of C. sessilifolia and SD standard deviation C. hirtella (Figs. S6a–d and S7), but only the Y a Values from Sousa et al. (2013) chromosome of C. grandis showed an extended, Heteromorphic and homomorphic sex chromosomes

Fig. 1 a–p Distribution of repeats in species of Coccinia using species, they point to chromosomes or bivalents that have accu- fluorescence in situ hybridization. Compare Table 2 for probe mulated certain repeats (discussed in the text). The sex of each names; an asterisk stands for Ty3/gypsy/ and a section sign stands karyotype is indicated in the upper right-hand corner. Bars corre- for Ty1/copia/. Results for C. grandis are from Sousa et al. (2016). spond to 5 μm Arrows in C. grandis point to its Y chromosome, while in the other

unusual distribution of H2AThr120ph and chromosomes, especially surrounding the centromeres. H3Ser10ph markers (Fig. S6e, f). The plastid small single-copy (SSC) and inverted repeat (IR) regions are abundantly accumulated on all chromo- Mitochondrial and plastid sequences in the nuclear somes of C. grandis (Fig. 3g, h). Among the other three genomes species, only C. sessilifolia showed strong hybridization signals for both regions on most chromosomes, espe- In C. grandis, our mitochondrial probe hybridized most- cially in male cells, sometimes covering an entire arm ly on the Y chromosome (Fig. 3i), while in (Fig. 3a,b),whileinC. hirtella, only the SSC region C. sessilifolia, C. hirtella,andC. trilobata (Fig. 3c,f, yielded a strong signal (compare Fig. 3a,b,d,e)andin l), it yielded strong diffuse signals on most C. trilobata only the IR region (compare Fig. 3j,k). Sousa A. et al.

Table 2 Repeat clusters selected for fluorescence in situ hybridization (FISH), with their numbering from the RepeatExplorer pipeline, which was used to name them

Clusters Annotation Y–C. grandis Chromosome pair in

C. sess. C. hirt. C. tri.

CL1 Centromere satellite – *** CL9 Ty3/gypsy/Athila * – * – CL10 Ty1/copia/Angela * * – * CL19 Ty3/gypsy/Ogre/Tat * ––– CL44 LINE * ––* CL86 Ty3/gypsy/chromovirus * – ** CL97 Satellite * * * * CL260 Unclassified * * Plastid and mitochondrial probes Plastid IR_Cg * ––* Plastid SSC_Cg * ––– CL173 Mitochondria seq. * –––

An asterisk (*) indicates that FISH signals accumulated on the Y chromosome of C. grandis and also on a chromosome pair in the respective species LINE long interspersed nuclear element, IR inverted repeat, SSC small single copy, Cg Coccinia grandis, C. sess. C. sessilifolia, C. hirt C. hirtella, C. tri. C. trilobata

Discussion expected that C. hirtella and C. sessilifolia would cyto- genetically resemble C. grandis, while C. trilobata We applied fluorescent in situ hybridization (FISH) in might have a more divergent karyotype. Indeed, three close relatives of C. grandis, the only species of C. trilobata turned out to have a lower chromosome Coccinia with heteromorphic sex chromosomes, to test number and larger genome size, but in the accumulation if any of them would show an accumulation of the of repetitive elements, it showed no consistent differ- repeats and/or plastid and mitochondrial sequences that ences from the other three species. we had previously found to accumulate on the As found in many other angiosperms (e.g., C. grandis Ychromosome(Sousaetal.2016). In none Oyama et al. 2008; Chung et al. 2012; Pellicer of the three species did we detect a single chromosome et al. 2014; Rockinger et al. 2016; this study), with unique repeat accumulation (the potential Y chro- chromosome number is unrelated to genome size, mosome), but our dual-probe FISH analyses revealed and the species with the lower chromosome num- strong accumulation of repeats on single chromosome ber (2n = 20 instead of 24), C. trilobata,hasthe pairs in male nuclei of all three species (CL10, CL97, largest genome size (Table 1). Interstitial telomere CL260 in C. sessilifolia; CL9, CL86, CL97 in repeats (ITRs) have been used as footprints of C. hirtella; CL10, CL44, CL86, CL97 in C. trilobata). (evolutionarily recent) chromosome rearrange- The accumulation of the very same repeats that are ments, such as fusions in angiosperms and gym- enriched on the C. grandis on single pairs of chromo- nosperms (Fuchs et al. 1995; Sousa et al. 2013; somes in the related dioecious species points to either Sousa and Renner 2015; Rockinger et al. 2016), old (previous) sex chromosomes or incipient (newly although their absence does not preclude rear- arising) ones, that is, to sex chromosome turnover. rangement events. In the species studied here, all However, only C. sessilifolia exhibited sex differences, telomere signals were restricted to chromosome with females, but not males, accumulating the gypsy/ ends, and no interstitial telomeric signals were chromovirus element CL86. Based on their relative detected in C. trilobata,whichhoweverhadfewer phylogenetic proximity to C. grandis (Fig. S2), we 45S rDNA loci, perhaps because of chromosome Heteromorphic and homomorphic sex chromosomes

Fig. 2 a–l Distribution of repeats in species of Coccinia using chromosome, while in the other species, they point to chromo- fluorescence in situ hybridization. Compare Table 2 for probe somes or bivalents that have accumulated certain repeats names; the asterisk stands for Ty3/gypsy/. Results for C. grandis (discussed in the text). The sex of each karyotype is indicated in are from Sousa et al. (2016). Arrows in C. grandis point to its Y the upper right-hand corner. Bars correspond to 5 μm rearrangements that resulted in its lower chromo- (Bock and Timmis 2008: review). In tobacco, the some number. plastid-to-nuclear gene transfer differs even among Chloroplast probes were as abundant in cells of a tissue (Bock and Timmis 2008). In Rumex C. sessilifolia genomes as in C. grandis (Fig. 3), acetosa, both the Y1 and Y2 chromosomes have while in C. hirtella and C. trilobata, one of the accumulated plastid DNA; in Silene latifolia,pieces plastid regions (IR or SSC) was less abundant. This of the IR region are found on the Y chromosome and non-phylogenetic distribution (also of the mitochon- pieces of the SSC region in the centromere drial signals) points to a random transfer of organellar (Kejnovsky et al. 2006; Steflova et al. 2014), but in DNA to the nucleus. Genomic studies suggest that Coccinia, plastid sequences are present on most chro- such transfer is a continuous process and variable mosomes (Sousa et al. 2016; this study). All these among and even within species, depending on the results confirm the independent degree of insertions nuclear genome size, level of polyploidy, number of of organellar DNA into the nuclear genome of plant plastids per cell, and number of genomes per plastid species. Sousa A. et al.

Fig. 3 Distribution of plastid and mitochondrial sequences in they point to chromosomes or bivalents that have accumulated mitotic and meiotic metaphase chromosomes of Coccinia certain repeats. Compare Table 2 for probe names; IR stands for sessilifolia (a–c), C. hirtella (d–f), and C. trilobata (j–l). Results inverted repeat and SSC for small single copy region. The sex of for C. grandis (g–i) are from Sousa et al. (2016). Arrows in each karyotype is indicated in the upper right-hand corner. Bars C. grandis point to its Y chromosome, while in the other species, correspond to 5 μm

Centromeres centromeres of all C. grandis chromosomes except those of Y chromosomes, also labeled all centromeres Centromeres are often composed of tandem repeats, but of C. sessilifolia and C. hirtella (weak signals were relatively few of these repeats have been characterized observed in some bivalents of C. trilobata). Immuno- (Nagaki et al. 2009, Han et al. 2009), among them those staining with antibodies against two cell cycle-regulated of two species with heteromorphic sex chromosomes, histone modifications revealed signals on the S. latifolia and C. grandis. In both, the Y chromosome (peri-)centromeric regions of all chromosomes of centromeres appear to be different from those of the X C. sessilifolia and C. hirtella, but no signal expansion and the autosomes (Cermak et al. 2008; Sousa et al. throughout chromosome arms as observed on the Y 2016). The satellite CgCent (CL1), which labels the chromosome of C. grandis. Heteromorphic and homomorphic sex chromosomes

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