An unusual origin of a ZW sex chromosome system

doi:10.21203/rs.3.rs-2129351/v1

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An unusual origin of a ZW sex chromosome system

https://doi.org/10.21203/rs.3.rs-2129351/v1

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Transitions in the heterogamety of sex chromosomes (e.g. XY to ZW or vice versa) are rare and fundamentally alter the genetic basis of sex determination. Although in an XY->ZW transition the W is more likely to arise from the X because they both carry feminizing genes and the X is expected to harbor less genetic load than the Y, here we show that both the W and the Z have arisen from the Y in willows (Salix). The new Z chromosome shared multiple homologous putative masculinizing factors with the ancestral Y, whereas the new W lost these masculinizing factors and gained feminizing factors. The origination of both the W and Z from the Y was permitted by an unexpectedly low genetic load on the Y and indicates that the origins of sex chromosomes during homologous transitions may be more flexible than previously considered.

dioecy

sex chromosome evolution

Salicaceae

sex determination

cytokinin response regulator

Salix exigua genome

Transitions in sex chromosomes are more common than once thought, and they span the range of possibilities^1–3. Most studies of the genomic changes during these transitions have focused on homogametic transitions, which are defined as those that maintain the same heterogametic sex (XY to XY, or ZW to ZW)^4–14. Heterogametic transitions (XY to ZW, or ZW to XY) are less common than homogametic transitions^15–19, and are often more genetically complex because they require a shift in the relative dominance of the masculinizing and feminizing chromosomes. Tracing the fate of the old chromosome pairs into the new pair allows extraordinary insight into the changes in genetic components and is a key step towards understanding how and why they occur.

Sex chromosome transitions also can be categorized by whether they changed location during the transition. During a heterologous transition, wherein the sex determination region moves to a new chromosome or a new linkage group within the same chromosome, the old sex chromosome (or linkage group) converts back to an autosome. However, during a homologous transition, wherein a new sex determination allele (e.g. W) arises in the same location as the ancestral sex determination alleles (e.g. XY), a three allele polymorphism (e.g. X, Y and W chromosomes) evolves into a two allele polymorphism (e.g. ZW). During these homologous transitions, it would make sense that the Z is derived from Y (or vice-versa) because both chromosomes carry masculinization genes and that the W is derived from the X because both are feminizing chromosomes²⁰. This pattern is supported in the hybrid populations of Japanese soil frogs where the neo-W chromosome originated from the X and the Z arose from the Y^21,22. However, in other taxa such as coenophilid snakes, which exhibit ZW to XY transitions, shared ancestry among several genes on the X, Y, and Z chromosomes suggested that both X and Y were evolved from Z and the W was lost during the transition²³. Finally, recent studies in Populus section Populus indicate that a complex series of events during the shift from ZW to XY resulted in the W converting into the Y, but the origin of the X remains unclear^25,26.

The two largest genera in the Salicaceae, Populus and Salix, within which all but one species is dioecious²⁷, have recently been discovered to harbor multiple sex chromosome transitions in both directions between XY and ZW^28,29. In Populus, XY sex determination systems have been reported on both chromosomes 14 and 19^30–33 and different ZW systems have been identified on chromosome 19 in two species within the same subgenus^25,26. In Salix, XY systems have been identified on chromosomes 7 and 15, and a ZW system was identified on chromosome 15, but the ancestral states and direction of change remain in dispute^28,34−37. In Populus, the genetic basis of sex determination was recently discovered to be affected by complex interactions between a cytokinin response regulator (RR17) and small interfering RNAs (siRNAs) that are generated by partial repeats of RR17 (hereafter ‘partial duplicates’)^26,33. In XY species, complete RR17 gene copies in the pseudo-autosomal regions on both the X and the Y chromosomes act as a feminizing factor²⁶. Partial duplicates are only present on the Y and generate siRNAs that block the expression of the intact copies of RR17 in males via RNA-directed DNA methylation, thereby acting as a dominant masculinizing factor²⁶. In ZW species of Populus, however, the feminizing intact RR17 genes are only present on the W chromosome, and their absence in ZZ results in male phenotypes²⁶. The details of sex determination appear to vary between Populus and Salix, although the fundamental associations of RR17 with feminization is present in Salix, suggesting common origins of the sex determination mechanism throughout both genera^26,29 (But see³⁸).

Here we trace the fate of the old XY chromosome pairs on chromosome 15 into the new ZW sex determination in the same locations in Salix. We start by documenting the chromosomal location and heterogamety of the sex determination system in S. exigua using a new reference genome built for this species. We show that S. exigua is in a key grade on the phylogeny, which confirms the direction of the 15XY to 15ZW shift that occurred during diversification of the genus. We then characterize one large contig as unique to the Y chromosome, which carried several small partial repeats of the RR17 gene that are unique to the S. exigua male genome. Finally, using a half-sib family, we identify a suite of polymorphic homologs that are present on the X and Y of S. exigua and the Z and W of S. purpurea, resulting in the ability to trace the loss of the X chromosome during this transition. These results provide unprecedented detail into chromosomal changes during a homologous shift in the heterogametic status of sex chromosomes allowing illumination of the factors influencing these transitions.

After genotyping 148,325 SNPs in 24 males and 24 females of S. exigua, we identified 20 SNPs that were strongly and significantly associated with sex using the male S. exigua reference genome (#SE967M; Fig. 1a). All 20 SNPs were located in a 100kb region between 1.8 and 1.9 Mb on Chr15 (Fig. 1a,c; Table S1). For these 20 loci, average heterozygosity in males was 91.6% and average homozygosity in females was 96.6%, indicating a male heterogametic sex determination system (XY) on Chr15 (Fig. 1b). Using the same raw data for the 48 individuals, we identified 194,250 SNPs when using the S. purpurea reference genome with the 15W (and without the 15Z chromosome), and 66 SNPs were significantly associated with sex after Bonferroni adjustment of the critical value (α < 0.05). These 66 SNPs were located in a 3.3MB region (4.3MB-7.6 MB) on Chr15W of the S. purpurea reference, which was overlapped with the sex determination region (SDR) in S. purpurea³⁴(Fig. 1d).

To identify the order of evolutionary transitions among the 15XY sex chromosome and the previously described 7XY²⁸ and 15ZW^35,39 sex chromosomes, we built a phylogeny that included two species with each sex chromosome type (Fig. 2). Relationships among species within the phylogeny were strongly supported (all bootstraps 100%), and the nested structure of the phylogeny indicated that the 15ZW chromosome was derived from an ancestor with the 15XY chromosome (Fig. 2).

A likely hemizygous portion of the sex determination region with low SNP coverage in females was identified on chromosome 15 (Fig. 3a). Contig 511 from the male nanopore raw assembly also uniquely assembled to this same portion of the sex determination region between 0.3 MB and 1.2 MB on the unphased Chr15 male Hi-C assembly (Fig. 3c; Table S2; Fig. S2). We found no synteny between contig 511 and any portion of the female S. exigua (XX) genome assembly. Since the preponderance of evidence indicated that contig 511 is male specific and in the SDR, we assigned it as a Y-specific region of S. exigua. Contig 511 exhibited partial synteny with a large region in the sex determination region of S. purpurea Chr15Z and synteny with only small segments of S. purpurea Chr15W (Figs. 3b, 5; Table S3).

Two full length homologs to the type A cytokinin response regulator 17 (RR17) were identified in both the male and female S. exigua genomes and were mapped to Chr19 in our complete male Hi-C assembly. In addition, we identified 5 small partial duplicates of the RR17 gene on contig 511 (Table S4) that were also absent from the female genome. When these 5 partial duplicates were compared to 14 previously-identified partial duplicates from the S. purpurea Chr15 Z²⁹, three of the homologs, Se2, Se4, and Se5, were the exact same lengths and had at least 86% similarity with two partial duplicates found on S. purpurea Chr15Z (Fig. 4a). The two other partial duplicates differed between S. exigua and S. purpurea in their start and end positions but had at least 77% similarity in the regions of overlap (Fig. 4a; Table S5). The direction and arrangement of these five shared partial duplicates was the same in both the Y (S. exigua) and Z (S. purpurea) chromosomes (Fig. 4b). The evolutionary histories of these partial duplicates revealed that Y and Z pairs of partial duplicates are more closely related to each other than to the full-length copies of RR17 on Chr15W (Fig. 5c). Interestingly, two of the identical copies (Se4-Sp6 & Se5-Sp7) exhibited high divergence from the ancestral full-length copy, whereas the other 3 pairs of partial duplicates (Se1-Sp2, Se2-Sp4, & Se3-Sp5) exhibited less divergence from the ancestral full-length copy (Fig. 4c).

To further understand the chromosomal changes during the shift from XY to ZW, we mapped sequence capture data of a S. exigua mother and 48 of her half-sib progeny to the S. purpurea genome that included only the 15W chromosome (and not 15Z). 198 Y-alleles were identified in S. exigua that mapped to the sex determination region of S. purpurea between 6.1 and 8.1MB. Of these, 30 loci were also associated with sex when the S. exigua sequence capture loci were mapped to the S. purpurea reference genome with the 15W chromosome (Fig. 1d, Table S6). Genotypes of these same 30 loci from 10 S. purpurea males (which are ZZ) indicated that 93% (28 of 30) of Y-alleles were identical among the Y, Z, and W chromosomes (W from the reference), whereas 0 of the 30 X-alleles from S. exigua were found on the 15Z and 15W of S. purpurea. Among these 30 SNP loci, all of them were located in the SDR of the S. purpurea 15W and 15Z (Fig. 5). These results indicate that the Z and W haplotypes in the SDR of S. purpurea were derived from an ancestral-Y chromosome and the X-haplotypes were lost during the transition from XY to ZW. Further supporting this conclusion, 151bp regions centered on the 30 SNPs in S. exigua displayed patterns of synteny with both the S. purpurea 15Z and 15W chromosomes. The different relative locations of these regions indicated a complex history of transversions, inversions, insertions, and deletions (Fig. 5). The SNPs that were found outside the 15W-SDR were clustered together and may have been moved outside of the SDR during or after the XY to ZW transition by a large inversion between 15Z and 15W of S. purpurea.

Based on our new phylogeny of multiple individuals with both 15XY and 15ZW sex chromosomes, we confirmed an ancestral transition from a male heterogametic (XY) to a female heterogametic (ZW) sex determination system in willows. Our results showed that during this transition, both the W and Z chromosomes were derived from an ancestral Y chromosome, and the X was lost. Many components in the sex determination region were retained during this transition. Our model for these chromosomal changes is detailed in Fig. 6a. Prior to the transition, at least five duplicates of small segments of the RR17 gene (‘partial duplicates’) were present on the Y chromosome. The function of these RR17 partial duplicates in Salix is still under investigation^26,34,36; however, inverted repeats of RR17 partial duplicates regulate sex determination via sRNA intermediates in Populus tremula^26,40, which is the sister genus to Salix, and RR17 partial duplicates in Salix arbutifolia (15XY) also generate sRNAs^29,40,41. Interestingly, there is currently no evidence of siRNA expression of RR17 partial duplicates in S. purpurea, so this masculinizing component of the XY system may have been lost or modified in the transition to ZW³⁸. After the divergence of ancestors of S. exigua and S. purpurea, a new W chromosome evolved in the S. purpurea lineage coincident with four complete copies of RR17 being duplicated and translocated from Chr19 to the sex determination region of Chr15³⁴. A set of shared alleles among Y, Z, and W and corresponding lack X-specific alleles on the Z or W confirm that the new Z and W chromosomes originated from an ancestral Y. A previous collinearity analysis between Z and W in S. purpurea showed that the four full-length copies of RR17 are located within two palindromes on the W chromosome in a region that differs from the Z by a large inversion³⁴ and indicates a large-scale chromosomal rearrangement during the evolution of the W chromosome. Although RR17 does not exhibit sex-biased expression in S. purpurea, it has been hypothesized that the new sex determination system incorporates dosage mechanism associated with multiple RR17 repeats^34,38,42. After the W chromosome arose, a polymorphism with W, X, and Y chromosomes was segregating in an ancestral population (Fig. 6a). This polymorphism was not stable, as the X chromosome was ultimately lost, and a new ZW female heterogametic system spread to fixation.

The evolution of a W chromosome from an ancestral-Y has not been documented in other heterogametic sex chromosomes transitions. Theoretically, there are fewer barriers to the transition when the W is derived from the X than from the Y. For instance, both the X and W are feminizing chromosomes in each sex determination system, whereas the Y chromosome includes masculinizing factors (or may be associated with the default sex)⁴³. Also, for a Y to develop into a W requires three changes: 1) the masking or loss of masculinizing factors, 2) the gain of feminizing factors, and 3) the development of dominance over the Y chromosome. Notably, this is made simpler when the first two changes result from the same mutation⁴³. Alternatively, for an X to develop into a W, only one change, from recessive into a dominant action over the Y chromosome, is required. The origination of the W from the X was concluded in Japanese soil frogs, where the X and W share a female-determining gene and nearly identical structure²². Another impediment to the W being derived from the Y is that the Y chromosome usually has a lower effective population size and higher genetic load compared to the X²⁰. During the transitional stage with an X, Y (= Z), and W polymorphism, if the W is derived from the Y, more deleterious homozygous genotypes will be expressed in the YW individual, causing lower fitness, than if the W is derived from the X (Fig. 6b). Interestingly, both overcoming multiple mutational effects and the potentially higher genetic load in the Y were seemingly evaded during the XY to ZW transition in Salix. In support, in S. arbutifolia, which also is 15XY²⁹, the SDR on the Y chromosome did not harbor more genetic load than either the SDR on the X or the pseudo-autosomal region (PAR) of chromosome 15.

We confirmed an XY to ZW transition within the same pair of sex chromosomes (homologous transition) in Salix. It appears that the ZW sex determination system in most of the subgenus Vetrix (shrub willows) have been derived from this transition because all of the studied species in this subgenus have a 15ZW sex chromosome system^35,37,39. Combined with the previous discovery of an XY to ZW transition between lineages of P. tremula and P. alba, shifts in heterogamety occurred at least twice in Salicaceae²⁶. In other dioecious angiosperm clades, shifts in heterogamety are rare⁴⁴. One exception can be found in Silene section Otites. Here, sex chromosome systems transitioned from ZW to XY and then back to ZW, which was perhaps permitted by the lack of strong degeneration among the sex chromosomes⁴⁵. A similar lack of strong degeneration in Populus and Salix sex chromosomes may also contribute to the frequency of shifts in Salicaceae.

The mechanism driving the XY to ZW shifts in Salix remains unresolved, but no current model seems to strongly align with our observations in Salix. Models based on linkage of the sex determination genes with sexual antagonistic loci are possible, but more difficult to imagine in plants than in animals. In cichlid fish, for instance, color polymorphisms are sexually antagonistic, because females do not benefit from the same color patterns that males do¹². However, in animal pollinated dioecious plants like willows, one would hypothesize that male and female flowers must both attract the same pollinators to assure pollination, limiting the extent of sexually antagonistic dimorphisms associated with pollinator attraction and mating⁴⁶. Nonetheless, the emissions of volatile herbivore defense compounds between is strongly dimorphic between male and female flowers of S. purpurea⁴⁷. Mechanisms related to the hot-potato model²⁴ also do not appear to have strongly contributed to the heterogametic transition in Salix. In this model, heterogametic transitions are unlikely because strong selection against the mutation-loaded Y hinders the spread of an epistatically dominant female sex-determining locus. For example, in true frogs, where build-up of genetic load on the Y is thought to influence sex chromosome transitions, 11 out of 13 transitions preserved the ancestral male heterogamety¹³. Meiotic drive can also induce sex chromosome transitions when a new sex determining gene restores a 1:1 progeny sex ratio⁴⁸. In this process, genetic load is expected to develop when strong meiotic drive persists, which, we have already noted, was not observed on the 15Y chromosome of a close relative of S. exigua²⁹. Also, homologous heterogametic transitions may be less likely induced by meiotic drive because the loci to restore the sex ratio need to arise on chromosomal regions unlinked to the sex determination region⁴⁸. Notably, however, female sex ratio bias has been reported in several species in subgenus Vetrix^49–53. Finally, the heterogametic transition in Salix may have resulted from solely from genetic drift, although under this model XY to ZW shifts are less common than XY to XY shifts unless extreme polygyny is present⁵⁴.

This study exemplifies how studies of sex chromosomes in plants can illuminate a broader understanding of the changes in genomic architecture induced by sex chromosome turnovers⁵⁵. As more transitions are studied in additional systems, it will be instructive to understand whether W chromosomes originating from the Y is a rare event. Homologous transitions inevitably require crossing a different set of evolutionary barriers than heterologous transitions, yet current models describing homologous and heterologous transitions do not provide sufficient detail regarding the evolutionary mechanisms driving these processes⁵⁶. Interestingly, genetic load is often considered to be a key factor in the evolution of sex chromosomes. However, during the transition from Y to W chromosomes, genetic load must be very limited or avoided to ensure that the transition is not counteracted by selection. In the future, understanding how sex chromosomes maintain low genetic load or how genetically loaded chromosomes are retained during heterogametic transitions will provide insight into the sex chromosome transition processes.

Salix exigua genome assembly - Fresh frozen leaf tissues were collected for genome sequencing and assembled from one male (SE967M) and one female (SE916F) that originated from the Rio Bonito population in eastern New Mexico. High molecular weight DNA was extracted and sequenced by Nextomics Biosciences (Wuhan, China) using a MinIon platform (Oxford Nanopore Technologies, Oxford, UK). For sequencing error correction, 150bp pair-ended Illumina short-read sequencing was also performed by Nextomics Biosciences (Wuhan, China) on these same individuals using MiSeq 2000 (Illumina, Inc., San Diego, CA, US). Finally, for scaffolding Hi-C libraries were constructed for the male individual and sequenced on the MiSeq 2000 platform (Illumina, Inc., San Diego, CA, US).

The Oxford nanopore reads were filtered for average read quality > = 7 using Guppy (Oxford Nanopore Technologies, Oxford, UK), and adapters were trimmed by Porechop v0.2.4 using default parameters⁵⁷. The raw assembly was completed using Flye v2.9-b1778 with no scaffolding mode⁵⁸ and polished with Illumina NGS reads for 4 rounds using Nextpolish v1.3.1 ⁵⁹. In total, 122.5 X coverage (49 Gb) of long reads were used to assemble the raw genome of S. exigua, and 427.5 X coverage (171 Gb) short reads from Illumina were used for polishing. Hi-C reads were aligned with the nanopore raw assembly contigs using Juicer v1.5⁶⁰, and contig scaffolding into chromosomes was finished by 3d-dna (version 180922)⁶¹ using diploid mode and correction for 10 rounds. An all-to-all alignment using lastz v1.1.13 between our S. exigua assembly and the S. purpurea 94006 PacBio assembly v5.1 (https://phytozome-next.jgi.doe.gov/info/Spurpurea_v5_1) was conducted to confirm assembly quality using the following parameter settings: --chain --gapped --transition --maxwordcount = 4 --exact = 100 --step = 20⁶². Alignments were plotted using last-dotplot⁶². Chromosomes were numbered according to the S. purpurea 94006 PacBio assembly v5.1³⁴.

The initial Oxford nanopore assembly for the Salix exigua male resulted in an N50 of 351k. After scaffolding with Hi-C reads, we anchored 89.5% of the nanopore contigs into 19 chromosomes and 2,258 additional scaffolds. The BUSCO score of final assembly was 98.1% and synteny with the S. purpurea genome was high, indicating a reliable reference genome (Fig. S1). The female genome, which was assembled using only Nanopore and Illumina sequencing, consisted of 1,650 contigs with an N50 of 665k.

Sequence capture and SDR mapping - Salix exigua leaf tissue from 24 males and 24 females were collected near the Rio Bonito in Fort Stanton-Snowy River Cave National Conservation Area, Lincoln County, Ruidoso, New Mexico in the spring of 2017. Tissues were dried in silica gel prior to DNA extraction. Sampled individuals were > 25 m apart to minimize sampling from the same genotype. Sex was scored by the presence of male or female flowers on catkins. To phase sex-linked haplotypes, we grew 48 half-sibs from seeds collected from the same S. exigua female that was used for genome assembly (SE916F). Leaf tissues were collected for DNA libraries construction and then merged into the same sequence capture pipeline for the wild population. Finally, leaves were sampled for DNA extractions from a diverse set of 10 male S. purpurea in the germplasm collection of the willow breeding program at Cornell University in Geneva, NY.

For S. exigua samples, DNA was extracted from leaves following the protocol from the Qiagen Plant DNA Extraction Kit (Qiagen, Hilden, Germany), but modified with 2 extra hours of precipitation. Bar-coded libraries were made for each individual using the NEB Next Ultra II Library preparation Kit (New England Biolabs, Ipswitch, MA, USA). Library size and concentration were confirmed using Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), and 8 libraries were pooled prior to hybridization during the sequence capture. 60,000 RNA baits were designed from coding regions of S. purpurea 94006 PacBio assembly as described in Sanderson et al. 2021 and were used to enrich 16580 target genes in library sample ²⁸ using standard protocols provided by Arbor Biosiences myProbes protocol v 3.0.1. After hybridization, 48 hybridized libraries were pooled and sequenced to 60x on Illumina HiSeq 5000 Genome Analyzer (Illumina, Inc., San Diego, CA, USA). Leaf samples were also collected from ten unrelated male S. purpurea samples and DNA was extracted using a modified Qiagen protocol that used 500 uL of buffer AP1 instead of 400uL, and included three, 65uL elution steps, followed by an ethanol precipitation to purify DNA. Library preparations was conducted using SparQ enzymatic DNA fragmentation and illumina compatible iTru adapters. Libraries were sequenced using an illumina paired-end P3 300 cycle flow cell platform (Illumina, Inc., San Diego, CA, USA).

Paired-end reads were mapped to the S. purpurea version 5 genome assembly³⁴ with the W chromosome (without Chr15Z) using bwa mem v0.7.17-r1188⁶³. S. exigua population were additionally mapped to S. exigua genome assembly to examine the position of SDR as well as S. purpurea version 5.1 genome assembly with W chromosome for analyzing homologies among X, Y, Z, and W. Mapping output was converted into BAM files following the Broad Glod Standard methods for variant processing⁶⁴.⁶⁵..

Variant calling was completed with the GATK variant calling pipeline⁶⁶. HaploCaller module from GATK was employed to call variants from mapping results of 48 individuals under parameters of allowing minimum heterozygosity 0.015 for SNP and 0.01 for indels. The raw VCF file which includes genotypes was generated by GATK GenotypeGVCFs module. Mapping information including QualByDepth (QD), Depth (DP), FisherStrand (FS), StrandOddsRatio (SOR), RMSMappingQuality (MQ), MQRankSum and ReadPosRankSum was screened to determine the hard filtering criteria. To remove low quality data, initial filtering was performed under criteria of MQ < 15, SOR > 4, QD < 20, FS > 60, MQRankSum < -10, ReadPosRankSum < -2, ReadPosRankSum > 2. Individual DP values were also checked to avoid the removal of good quality variant calls from a few individuals. Final genotype filtering kept variants with DP between 6 to 100.

SNP loci with at least 75% genotypes in filtered VCF files of each species were used for downstream analyses. Sex was included and tested as a binary phenotype using the GWAS pipeline using PLINK v1.90b4⁶⁷. GWAS results were shown as p values for each SNP loci. Lower p values indicate large distinction found between males and females. Manhattan plot was generated in R⁶⁸ using ggplot2 package⁶⁹ based on the position and negative log-transformed p values of each SNP to show the boundary of SDR. After transformation, SNPs with high values on plot from upper outliers with a Bonferroni-adjusted significance threshold of P < 0.05 were selected out for screening allele preference in males and females. Genotype matrix of SNPs with high p-value using Bonferroni multiple testing correction were output from vcftools v0.1.1⁷⁰. Output of genotypes was reinterpreted as heterozygosity level on sex-associated SNP loci. Sex determination systems were determined by whether heterozygous happens on male (XY) or female (ZW).

Phasing sex-linked haplotypes in S. exigua - The same mapping and SNP calling pipeline was used on 48 half-sibs from S. exigua. First, sexes of each half-sib were determined using sex associated SNPs identified in our mapping study (see above). Alleles on the Y chromosome (hereafter Y-alleles) in the S. exigua SDR could be identified for some loci by subtracting the mother alleles from half-sib progeny genotypes. When the mother was 0/0, Y-alleles could be identified when male progeny were 0/0, 0/1, or 0/2; and when the mother was 0/1, Y-alleles could be identified when male progeny were 0/0, 1/1, 1/2, & 0/2. Y-alleles were confirmed only if all males in the half-sib progeny elicited one of these genotype patterns. X-alleles were identified as the alternate alleles in the female half-sib progeny at the same loci where the Y-alleles were identified. X- and Y-alleles were assigned as sex-linked if they were sex-linked in our GWAS analysis.

Flanking regions (150bp) of Y-linked SNPs that mapped onto the S. purpurea 15W chromosome were extracted to align in S. purpurea 15Z and S. exigua Chr15 to decide the movement of sex-linked region among different sex chromosomes using BLASTN⁷¹. Plotting the positions and connection between pairs of flanking regions were performed using a custom Visual Basic application (Balena, F., & Fawcette, J., 1999).

Identification of Y-specific contigs - Y-specific contigs from our S. exigua genome assembly were identified by comparing sequence coverage on Chr15 and all unassembled scaffolds between males and females. Y-specific regions are absent from females. Candidates for sex-specific regions were used to identify and extract specific contigs from the Nanopore assemblies. Additional alignments were performed against the genome assembly of the opposite sex to confirm that the Y-specific regions were absent from females. Synteny maps between candidate and sex chromosomes were generated using lastz⁶² and perl tools.

Phylogeny reconstruction - Eight species in Salicaceae were selected to reconstruct a phylogeny (Idesia polycarpa, Populus trichocarpa, S. nigra, S. chaenomeloides, S. arbutifolia, S. purpurea, S. exigua, S. viminalis). Each sample was hybridized against a sequence capture array targeting 1126 genes (Sanderson et al. 2020) and sequenced. Reads of all species were mapped to reference genes and assembled using Hybpiper⁷². All assembled genes in each species were concatenated into supermatrices. Species tree were inferred by IQTree under parameters of –bb 1000 -alrt 1000 -bnni -m MFP^73–75.(Johnson et al., 2016). All assembled genes in each species were concatenated into supermatrices. The phylogenetic tree was inferred using IQTree under parameters of –bb 1000 -alrt 1000 -bnni -m MFP^73–75.

Identifying ARR17 partial duplicates in S. exigua and S. purpurea - RR17 gene (P. trichocarpa: XM_002325453.3) copies were queried at both the chromosome and contig level in our S. exigua assembly, in S. purpurea Chr15 Z and Chr15W, and on Chr19 of both S. exigua and S. purpurea using BLASTN with -wordsize 8⁷¹. Different partial duplicate copies on Chr15Y of S. exigua and Chr15Z of S. urpurea, and the full-length RR17 genes from Chr 15W of S. purpurea and Chr19 of P. trichocarpa (an outgroup) were aligned by ClustalW, respectively⁷⁶. Phylogenies of each of the partial duplicates were reconstructed by MEGA11 using Maximum Likelihood with 100 bootstraps⁷⁷.

Acknowledgements

We thank Ashmita Khanal, Ken Keefover-Ring, and Matt Johnson for helpful discussion and comments. This research was supported by grants from the US National Science Foundation (1542509, 1542599, 1542479, 1542486) and the National Natural Science Foundation of China (31561123001).

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An unusual origin of a ZW sex chromosome system

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