Ancient heteromorphic sex chromosomes are common in mammals, but not in plants. Sex chromosomes in the plant family Cannabaceae, which includes species like hops and hemp, were identified a century ago because of their obviously heteromorphic XYs. However, we know very little about their structure, nor their role in the development of the economically, medicinally, and culturally important flowers. Here we assembled genomes, with phased sex chromosomes, for three XY male hops (Humulus sp.), two XY male Cannabis, and two monoecious Cannabis individuals. Most of the non-recombining regions evolved prior to the divergence between Cannabis and Humulus around 47 MYA supporting this sex chromosome origin is among the oldest known in flowering plants. The Y chromosomes are highly-degenerate, with very few genes remaining, and show substantial rearrangements, while the X also shows faster rates of molecular evolution. The Cannabaceae sex chromosomes contain critical genes for flower development and the production of key compounds like bitter acids and cannabinoids, which are important traits for the plants. The sex chromosomes thus have played a vital role in the domestication and breeding of these species, and more generally, the Cannabaceae family. ### Competing Interest Statement D.V. is a board member of the 501(C)3 non-profit Agricultural Genomics Foundation and the sole owner of the company CGRI, LLC

Premise of research. The prevalence of sexual dimorphisms, which evolve due to contrasting strategies to maximize reproductive success in males and females, is variable among dioecious plant species. In the Salicaceae, many traits have been assessed across many studies, but direct or indirect associations between these traits and reproductive allocation are often neglected. Given the dynamic evolution of sex determination systems and the strong interest in wood production in the family, we wondered whether sexual dimorphisms related to reproduction may have gone unreported. Here, we assess sexual dimorphism in reproductive traits in two species of Salix. Recognition of reproductive sexually dimorphic traits will contribute to understanding the evolution of sex determination systems in the Salicaceae. Methodology. We conducted observational studies in natural populations to assess the presence of sexual dimorphisms in early spring bud density, catkin number, and flower number per catkin across four sampling periods in Salix exigua. We also analyzed flower number and catkin number per flower in Salix nigra. Pivotal results. We observed no sexual dimorphism in preseason buds per branch in S. exigua but did find that males produced more flowers per catkin and more catkins per branch than females in both S. exigua and S. nigra. Conclusions. Higher flower numbers in males compared to females are consistent with expectations from intrasexual selection among males. The presence of reproductive sexual dimorphisms in Salix suggests that sexual selection may affect the evolution of mating strategies in Salix species and the evolution of the sex determination system within this genus.

AbstractPremise of the ResearchThe prevalence of sexual dimorphisms, which evolve due to contrasting strategies to maximize reproductive success in males and females, is variable among dioecious plant species. In theSalicaceae, many traits have been assessed across many studies, but direct or indirect associations between these traits and reproductive allocation are often neglected. Given the dynamic evolution of sex determination systems and the strong interest in wood production in the family, we wondered whether sexual dimorphisms related to reproduction may have gone unreported. Here, we assess sexual dimorphism in reproductive traits in two species ofSalix. Recognition of reproductive sexually dimorphic traits will contribute to understanding the evolution of sex determination systems in the Salicaceae.MethodologyWe conducted observational studies in natural populations to assess the presence of sexual dimorphisms in early spring bud density, catkin number, and flower number per catkin across four sampling periods inSalix exigua. We also analyzed flower number and catkin number per flower inSalix nigra.Pivotal ResultsWe observed no sexual dimorphism in pre-season buds per branch inS. exiguabut did find that males produced more flowers per catkin and more catkins per branch than females in bothS. exiguaandS. nigra.ConclusionsHigher flower numbers in males compared to females is consistent with expectations from intra-sexual selection among males. The presence of reproductive sexual dimorphisms inSalixsuggests that sexual selection may affect the evolution of mating strategies inSalixspecies, and the evolution of the sex determination system within this genus.

Transitions in the heterogamety of sex chromosomes (e.g., XY to ZW or vice versa) fundamentally alter the genetic basis of sex determination, however the details of these changes have been studied in only a few cases. In an XY to ZW transition, the X is likely to give rise to the W because they both carry feminizing genes and the X is expected to harbour less genetic load than the Y. Here, using a new reference genome for Salix exigua , we trace the X, Y, Z, and W sex determination regions during the homologous transition from an XY system to a ZW system in willow (Salix). We show that both the W and the Z arose from the Y chromosome. We find that the new Z chromosome shares 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 this indicates that the origins of sex chromosomes during homologous transitions may be more flexible than previously considered.

The apple cultivar 'Honeycrisp' has superior fruit quality traits, cold hardiness, and disease resistance, making it a popular breeding parent. However, it suffers from several physiological disorders, production, and postharvest issues. Despite several available apple genome sequences, understanding of the genetic mechanisms underlying cultivar-specific traits remains lacking. Here, we present a highly contiguous, fully phased, chromosome-level genome of 'Honeycrisp' apples, using PacBio HiFi, Omni-C, and Illumina sequencing platforms, with two assembled haplomes of 674 Mbp and 660 Mbp, and contig N50 values of 32.8 Mbp and 31.6 Mbp, respectively. Overall, 47,563 and 48,655 protein-coding genes were annotated from each haplome, capturing 96.8-97.4% complete BUSCOs in the eudicot database. Gene family analysis reveals most 'Honeycrisp' genes are assigned into orthogroups shared with other genomes, with 121 'Honeycrisp'-specific orthogroups. This resource is valuable for understanding the genetic basis of important traits in apples and related Rosaceae species to enhance breeding efforts.

Abstract 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.

The genus Solidago represents a taxonomically challenging group due to its sheer number of species, putative hybridization, polyploidy, and shallow genetic divergence among species. Here we use a dataset obtained exclusively from herbarium specimens to evaluate the status of Solidago ulmifolia var. palmeri, a morphologically subtle taxon potentially confined to Alabama, Arkansas, Mississippi, and Missouri. A multivariate analysis of both discrete and continuous morphological data revealed no clear distinction between S. ulmifolia var. palmeri and Solidago ulmifolia var. ulmifolia. Solidago ulmifolia var. palmeri’s status was also assessed with a phylogenomic and SNP clustering analysis of data generated with the “Angiosperms353” probe kit. Neither analysis supported Solidago ulmifolia var. palmeri as a distinct taxon, and we suggest that this name should be discarded. The status of Solidago delicatula (formerly known as Solidago ulmifolia var. microphylla) was also assessed. Both morphological and phylogenic analyses supported the species status of S. delicatula and we suggest maintaining this species at its current rank. These results highlight the utility of the Angiosperms353 probe kit, both with herbarium tissue and at lower taxonomic levels. Indeed, this is the first study to utilize this kit to identify genetic groups within a species.

PREMISE The successful application of universal targeted sequencing markers, such as those developed for the Angiosperms353 probe set, within populations could reduce or eliminate the need for specific marker development, while retaining the benefits of full‐gene sequences in population‐level analyses. However, whether the Angiosperms353 markers provide sufficient variation within species to calculate demographic parameters is untested. METHODS Using herbarium specimens from a 50‐year‐old floristic survey in Texas, we sequenced 95 samples from 24 species using the Angiosperms353 probe set. Our data workflow calls variants within species and prepares data for population genetic analysis using standard metrics. In our case study, gene recovery was affected by genomic library concentration only at low concentrations and displayed limited phylogenetic bias. RESULTS We identified over 1000 segregating variants with zero missing data for 92% of species and demonstrate that Angiosperms353 markers contain sufficient variation to estimate pairwise nucleotide diversity (π)—typically between 0.002 and 0.010, with most variation found in flanking non‐coding regions. In a subset of variants that were filtered to reduce linkage, we uncovered high heterozygosity in many species, suggesting that denser sampling within species should permit estimation of gene flow and population dynamics. DISCUSSION Angiosperms353 should benefit conservation genetic studies by providing universal repeatable markers, low missing data, and haplotype information, while permitting inclusion of decades‐old herbarium specimens.

The reduced cost of high‐throughput sequencing and the development of gene sets with wide phylogenetic applicability has led to the rise of sequence capture methods as a plausible platform for both phylogenomics and population genomics in plants. An important consideration in large targeted sequencing projects is the per‐sample cost, which can be inflated when using off‐the‐shelf kits or reagents not purchased in bulk. Here, we discuss methods to reduce per‐sample costs in high‐throughput targeted sequencing projects. We review the minimal equipment and consumable requirements for targeted sequencing while comparing several alternatives to reduce bulk costs in DNA extraction, library preparation, target enrichment, and sequencing. We consider how each of the workflow alterations may be affected by DNA quality (e.g., fresh vs. herbarium tissue), genome size, and the phylogenetic scale of the project. We provide a cost calculator for researchers considering targeted sequencing to use when designing projects, and identify challenges for future development of low‐cost sequencing in non‐model plant systems.