Sexually dimorphic acetyl-CoA biosynthesis and utilization in response to drought and exogenous acetic acid.

Female willows exhibit greater drought tolerance and benefit more from exogenous acetic acid (AA)-improved drought tolerance than males. However, the potential mechanisms driving these sex-specific responses remain unclear. To comprehensively investigate the sexually dimorphic responsive mechanisms of willows to drought and exogenous AA, here, we performed physiological, proteomic, Lys-acetylproteomic, and transgenic analyses in female and male Salix myrtillacea exposed to drought and AA-applicated drought treatments, focusing on protein abundance and lysine acetylation (LysAc) changes. Drought-tolerant females suffered less drought-induced photosynthetic and oxidative damage, did not activate AA and acetyl-CoA biosynthesis, TCA cycle, fatty acid metabolism, and jasmonic acid signaling as strongly as drought-sensitive males. Exogenous AA caused overaccumulation of endogenous AA and inhibition of acetyl-CoA biosynthesis and utilization in males. However, exogenous AA greatly enhanced acetyl-CoA biosynthesis and utilization and further enhanced drought performance of females, possibly determining that AA improved drought tolerance more in females than in males. Interestingly, overexpression of acetyl-CoA synthetase (ACS) could reprogram fatty acids, increase LysAc levels, and improve drought tolerance, highlighting the involvement of ACS-derived acetyl-CoA in drought responses. In addition, drought and exogenous AA induced sexually dimorphic LysAc associated with histones, transcription factors, and metabolic enzymes in willows. Especially, exogenous AA may greatly improve the photosynthetic capacity of S. myrtillacea males by decreasing LysAc levels and increasing the abundances of photosynthetic proteins. While hyperacetylation in glycolysis, TCA cycle, and fatty acid biosynthesis potentially possibly serve as negative feedback to acclimate acetyl-CoA biosynthesis and utilization in drought-stressed males and AA-applicated females. Thus, acetyl-CoA biosynthesis and utilization determine the sexually dimorphic responses of S. myrtillacea to drought and exogenous AA.

Reference-based genome assembly and comparative genomics of Calamus Brandisii Becc. for unveiling sex-specific genes for early gender detection.

Calamus brandisii Becc. is an endangered rattan species indigenous to the Western Ghats of India and used in the furniture and handicraft industries. However, its dioecious nature and longer flowering time pose challenges for conservation efforts. Developing markers for early gender detection in seedlings is crucial for maintaining viable populations for in-situ and ex-situ conservation. Currently, no sex chromosomes or gender-specific genes have been reported in the species. We report the first comprehensive comparative genomics study between the male and female genomes of C. brandisii to identify polymorphisms and potential genes for gender determination. Reference-based assembly was conducted and the male and female genomes were predicted to contain 43,810 and 50,493 protein-coding genes respectively. The haploid genome size was ∼691 Mb and ∼884 Mb for male and female genomes respectively. Comparative analysis revealed significant genetic variation between the two genomes including 619,776 SNPs, 73,659 InDels, 212,123 Structural variants (SVs) and 305 copy number variations (CNVs). A total of 5 male-specific and 11 female-specific genes linked to the sex determining region was predicted. The genomic variants identified between the two genomes could be used in development of markers for early gender identification in C. brandisii for restoration programs. The gender-specific genes identified in this study also provide new insights into the mechanisms of sex determination and differentiation in rattans.

One-factor sex determination evolves without linkage between feminizing and masculinizing mutations.

The evolution of separate sexes from cosexuality requires at least two mutations: a feminizing allele to cause female development and a masculinizing allele to cause male development. Classically, the double mutant is assumed to be sterile, which leads to two-factor sex determination where male and female sex chromosomes differ at two loci. However, several species appear to have one-factor sex determination where sexual development depends on variation at a single locus. We show that one-factor sex determination evolves when the double mutant develops as a male or a female. The feminizing allele fixes when the double mutant is male, and the masculinizing allele fixes when the double mutant is female. The other locus then gives XY or ZW sex determination based on dominance: for example, a dominant masculinizer becomes a Y chromosome. Although the resulting sex determination system differs, the conditions required for feminizers and masculinizers to spread are the same as in classical models, with the important difference that the two alleles do not need to be linked. Thus, we reveal alternative pathways for the evolution of sex determination and discuss how they can be distinguished using new data on the genetics of sex determination.

Genomic analyses provide insights into sex differentiation of tetraploid strawberry (Fragaria moupinensis).

The strawberry genus, Fragaria, exhibits a wide range of sexual systems and natural ploidy variation. Nearly, all polyploid strawberry species exhibit separate sexes (dioecy). Research has identified the sex-determining sequences as roughly conserved but with repeatedly changed genomic locations across octoploid strawberries. However, it remains unclear whether tetraploid wild strawberries evolved dioecy independently or shared a common origin with octoploid strawberries. In this study, we investigated the sex determinants of F. moupinensis, a dioecious plant with heterogametic females (ZW). Utilizing a combination of haplotype-resolved genome sequencing of the female F. moupinensis, k-mer-based and coverage-based genome-wide association studies (GWAS), and transcriptomic analysis, we discovered a non-recombining, approximately 33.6 kb W-specific region on chromosome 2a. Within this region, only one candidate sex-determining gene (FmoAFT) was identified. Furthermore, an extensive resequencing of the entire Fragaria genus indicated that the W-specific region displays conservative female specificity across all tetraploid species. This observation suggests that dioecy evolved independently in tetraploid and octoploid strawberries. Moreover, employing virus-induced gene silencing (VIGS), we knocked down the expression of the FmoAFT homologue transcript in cultivated strawberries, revealing its potential role in promoting female functions during early carpel development. We also applied DNA affinity purification sequencing (DAP-seq) and yeast one-hybrid assays to identify potential direct targets of FmoAFT. These insights shed new light on the genetic basis and evolutionary history of sex determination in strawberries, thereby facilitating the formulation of strategies to manipulate sex determination in breeding programs.

Sex-specific phosphorus acquisition strategies and cycling in dioecious Populus euphratica forests along a natural water availability gradient.

Soil phosphorus (P) availability affects plant growth and distribution. However, it is still unknown how sex-specific variation in functional traits affects plants' P acquisition and soil P transformation. On wet sites, female poplars had a greater specific root length (SRL), and a higher diversity of arbuscular mycorrhizal fungi (AMF) and phosphate-solubilizing bacteria (PSB). Male poplars living on wet sites increased the abundance of AMF and PSB communities and enhanced moderately labile and highly resistant organic P mineralisation via increased phosphatase activity. In contrast, on the dry site, the abundance and diversity of AMF and PSB communities increased in females, enhancing moderately labile and highly resistant organic P mineralisation via elevating phosphatase activities. Males maintained greater SRL and promoted Ca-P mobilisation via the release of root carboxylic acids and rhizosphere acidification on the dry site. The AMF community diversity followed a similar pattern as that of the PSB community when altering the P availability of different-sex plants. Our results indicated that organic P and Ca-P are the major sources of plant-available P in natural P. euphratica forests. Seasonal shifts and geographic locations affected the share of organic and inorganic P pools, and AMF and PSB diversities, ultimately altering sex-specific P acquisition strategies of plants.

Identification of genes associated with sex expression and sex determination in hemp (Cannabis sativa L.).

Dioecy in flowering plants has evolved independently many different times, and thus the genetic mechanisms underlying sex determination are diverse. In hemp (Cannabis sativa), sex is controlled by a pair of sex chromosomes (XX for females and XY for males). In an attempt to understand the molecular mechanism responsible for sex expression in hemp plants, we carried out RNA-Seq of male and female plants at different developmental stages. Using a pipeline involving differential gene expression analysis and weighted gene co-expression network analysis, we identified genes important for male and female flower development. We also demonstrate that sex-biased expression is already established at very early vegetative stages, before the onset of reproductive development, and several genes encoding transcription factors of the REM, bZIP and MADS family as candidate sex determination genes in hemp. Our findings demonstrate that the gene regulatory networks governing male and female development in hemp diverge already at a very early stage, leading to profound morphological differences in male and female hemp plants.

Differential growth rate, water use efficiency and climate sensitivity between males and females of Ilex aquifolium in north-western Spain.

BACKGROUND AND AIMS: Dioecious plant species, i.e., those in which male and female functions are housed in different individuals, are particularly vulnerable to global environmental changes. For long-lived plant species, such as trees, long-term studies are imperative to understand how growth patterns and their sensitivity to climate variability differentially affect the sexes. METHODS: Here, we explore long-term intersexual differences in wood traits, namely radial growth rates, water use efficiency quantified as stable carbon isotope abundance of wood cellulose, and their climate sensitivity in Ilex aquifolium trees growing in a natural population in NW Spain. KEY RESULTS: We found that sex differences in secondary growth rates were variable over time, with males outperforming females in both radial growth rates and water use efficiency in recent decades. Summer water stress significantly reduced the growth of female trees in the following growing season, while the growth of male trees was primarily favoured by cloudy and rainy conditions the previous fall and winter combined with low cloud cover and warm conditions in summer. Sex-dependent lagged correlations between radial growth and water availability were found, with a strong association between tree growth and cumulative water availability in females at 30 months and in males at 10 months. CONCLUSIONS: Overall, our results point to greater vulnerability of female tress to increasing drought, which could lead to sex-ratio biases threatening population viability in the future.

Young evolutionary origins of dioecy in the genus Asparagus.

PREMISE: Dioecy (separate sexes) has independently evolved numerous times across the angiosperm phylogeny and is recently derived in many lineages. However, our understanding is limited regarding the evolutionary mechanisms that drive the origins of dioecy in plants. The recent and repeated evolution of dioecy across angiosperms offers an opportunity to make strong inferences about the ecological, developmental, and molecular factors influencing the evolution of dioecy, and thus sex chromosomes. The genus Asparagus (Asparagaceae) is an emerging model taxon for studying dioecy and sex chromosome evolution, yet estimates for the age and origin of dioecy in the genus are lacking. METHODS: We use plastome sequences and fossil time calibrations in phylogenetic analyses to investigate the age and origin of dioecy in the genus Asparagus. We also review the diversity of sexual systems present across the genus to address contradicting reports in the literature. RESULTS: We estimate that dioecy evolved once or twice approximately 2.78-3.78 million years ago in Asparagus, of which roughly 27% of the species are dioecious and the remaining are hermaphroditic with monoclinous flowers. CONCLUSIONS: Our findings support previous work implicating a young age and the possibility of two origins of dioecy in Asparagus, which appear to be associated with rapid radiations and range expansion out of Africa. Lastly, we speculate that paleoclimatic oscillations throughout northern Africa may have helped set the stage for the origin(s) of dioecy in Asparagus approximately 2.78-3.78 million years ago.

Widespread male sterility and trioecy in androdioecious Mercurialis annua: Its distribution, genetic basis, and estimates of morph-specific fitness components.

PREMISE: Angiosperms range in sexual system from hermaphroditism through gynodioecy and androdioecy to dioecy. Trioecy, where females and males coexist with hermaphrodites, is rare. Recently, trioecy was documented in hexaploid populations of the wind-pollinated herb Mercurialis annua in Spain. METHODS: We surveyed the frequency of males, hermaphrodites, and females in M. annua across its distribution in the Iberian Peninsula, tracked sex-ratio variation in several populations over consecutive generations, and assessed evidence for pollen limitation. In a common garden, we estimated male, female, and hermaphroditic fitness. We used controlled crosses to infer the genetic basis of male sterility. Finally, we compared predictions of a deterministic model with the distribution of observed sex ratios in the field based on our fitness estimates and the inferred genetics of sex determination. RESULTS: Trioecy is widespread in Spanish and Portuguese populations of M. annua. Males are determined by a dominant (Y-linked) allele, and female expression results from the interaction between cytoplasmic male sterility and multiple nuclear male sterility restorers partially linked to the male determiner. Male pollen production is approximately 12 times that of hermaphrodites, while female seed production is less than 1.12 times the observed hermaphroditic levels. The distribution of sex ratios in natural populations conforms with predictions of our deterministic simulations. CONCLUSIONS: Our study documents and accounts for a clear case of trioecy in which sex is determined by both maternally and biparentally inherited genes.

Rethinking pathways to the dioecy-polyploidy association: Genera with many dioecious species have fewer polyploids.

PREMISE: Numerous studies have found a positive association between dioecy and polyploidy; however, this association presents a theoretical conflict: While polyploids are predicted to benefit from self-reproduction for successful establishment, dioecious species cannot self-reproduce. We propose a theoretical framework to resolve this apparent conflict. We hypothesize that the inability of dioecious species to self-reproduce hinders their establishment as polyploids. We therefore expect that genera with many dioecious species have fewer polyploids, leading to a negative association between polyploidy and dioecy across genera. METHODS: We used three publicly available databases to determine ploidy and sexual systems for 131 genera and 546 species. We quantified (1) the relationship between the frequency of polyploid species and the frequency of dioecious species across genera, and (2) the proportion of polyploids with hermaphroditism and dioecy across species, adjusting for phylogenetic history. RESULTS: Across genera, we found a negative relationship between the proportion of polyploids and the proportion of dioecious species, a consistent trend across clades. Across all species, we found that sexual system (dioecious or not) was not associated with polyploidy. CONCLUSIONS: Polyploids are rare in genera in which the majority of species are dioecious, consistent with the theory that self-reproduction favors polyploid establishment. The low frequency of polyploidy among dioecious species indicates the association is not as widespread as previously suggested. Our findings are consistent with previous studies identifying a positive relationship between the two traits, but only if polyploidy promotes a transition to dioecy, and not the reverse.

Gender specific SNP markers in Coscinium fenestratum (Gaertn.) Colebr. for resource augmentation.

BACKGROUND: Unregulated extraction of highly traded medicinal plant species results in drastic decline of the natural resources and alters viable sex ratio of populations. Conservation and long-term survival of such species, require gender specific restoration programs to ensure reproductive success. However, it is often difficult to differentiate sex of individuals before reaching reproductive maturity. C. fenestratum is one of the medicinally important and overexploited dioecious woody liana, with a reproductive maturity of 15 years. Currently, no information is available to identify sex of C. fenestratum in seedling stage while augmenting the resources. Thus, the current study envisages to utilize transcriptomics approach for gender differentiation which is imperative for undertaking viable resource augmentation programmes. METHODS AND RESULTS: Gender specific SNPs with probable role in sexual reproduction/sex determination was located using comparative transcriptomics approach (sampling male and female individuals), alongside gene ontology and annotation. Nine sets of primers were synthesized from 7 transcripts (involved in sexual reproduction/other biological process) containing multiple SNP variants. Out of the nine primer pairs, only one SNP locus with no available information of its role in reproduction, showed consistent and accurate results (males-heterozygous and females-homozygous), in the analyzed 40 matured individuals of known sexes. Thus validated the efficiency of this SNP marker in differentiating male and female individuals. CONCLUSIONS: The study could identify SNPs linked to the loci with apparent role in gender differentiation. This SNP marker can be used for early sexing of seedlings for in-situ conservation and resource augmentation of C. fenestratum in Kerala, India.

A CLAVATA3-like Gene Acts as a Gynoecium Suppression Function in White Campion.

How do separate sexes originate and evolve? Plants provide many opportunities to address this question as they have diverse mating systems and separate sexes (dioecy) that evolved many times independently. The classic "two-factor" model for evolution of separate sexes proposes that males and females can evolve from hermaphrodites via the spread of male and female sterility mutations that turn hermaphrodites into females and males, respectively. This widely accepted model was inspired by early genetic work in dioecious white campion (Silene latifolia) that revealed the presence of two sex-determining factors on the Y-chromosome, though the actual genes remained unknown. Here, we report identification and functional analysis of the putative sex-determining gene in S. latifolia, corresponding to the gynoecium suppression factor (GSF). We demonstrate that GSF likely corresponds to a Y-linked CLV3-like gene that is specifically expressed in early male flower buds and encodes the protein that suppresses gynoecium development in S. latifolia. Interestingly, GSFY has a dysfunctional X-linked homolog (GSFX) and their synonymous divergence (dS = 17.9%) is consistent with the age of sex chromosomes in this species. We propose that female development in S. latifolia is controlled via the WUSCHEL-CLAVATA feedback loop, with the X-linked WUSCHEL-like and Y-linked CLV3-like genes, respectively. Evolution of dioecy in the S. latifolia ancestor likely involved inclusion of ancestral GSFY into the nonrecombining region on the nascent Y-chromosome and GSFX loss of function, which resulted in disbalance of the WUSCHEL-CLAVATA feedback loop between the sexes and ensured gynoecium suppression in males.

Comparative transcriptomic analysis of male and females in the dioecious weeds Amaranthus palmeri and Amaranthus tuberculatus.

BACKGROUND: Waterhemp (Amaranthus tuberculatus (Moq.) Sauer) and Palmer amaranth (Amaranthus palmeri S. Wats.) are two dioecious and important weed species in the world that can rapidly evolve herbicide-resistance traits. Understanding these two species' dioecious and sex-determination mechanisms could open opportunities for new tools to control them. This study aims to identify the differential expression patterns between males and females in A. tuberculatus and A. palmeri. Multiple analyses, including differential expression, co-expression, and promoter analyses, used RNA-seq data from multiple tissue types to identify putative essential genes for sex determination in both dioecious species. RESULTS: Genes were identified as potential key players for sex determination in A. palmeri. Genes PPR247, WEX, and ACD6 were differentially expressed between the sexes and located at scaffold 20 within or near the male-specific Y (MSY) region. Multiple genes involved with flower development were co-expressed with these three genes. For A. tuberculatus, no differentially expressed gene was identified within the MSY region; however, multiple autosomal class B and C genes were identified as differentially expressed and possible candidate genes. CONCLUSIONS: This is the first study comparing the global expression profile between males and females in dioecious weedy Amaranthus species. Results narrow down putative essential genes for sex-determination in A. palmeri and A. tuberculatus and also strengthen the hypothesis of two different evolutionary events for dioecy within the genus.

Structural variation of a sex-linked region confers monoecy and implicates GATA15 as a master regulator of sex in Salix purpurea.

The Salicaceae, including Populus and Salix, are dioecious perennials that utilize different sex determination systems. This family provides a useful system to better understand the evolution of dioecy and sex chromosomes. Here, a rare monoecious genotype of Salix purpurea, 94003, was self- and cross-pollinated and progeny sex ratios were used to test hypotheses on possible mechanisms of sex determination. To delimit genomic regions associated with monoecious expression, the 94003 genome sequence was assembled and DNA- and RNA-Seq of progeny inflorescences was performed. Based on alignments of progeny shotgun DNA sequences to the haplotype-resolved monoecious 94003 genome assembly and reference male and female genomes, a 1.15 Mb sex-linked region on Chr15W was confirmed to be absent in monecious plants. Inheritance of this structural variation is responsible for the loss of a male-suppressing function in what would otherwise be genetic females (ZW), resulting in monoecy (ZWH or WWH ), or lethality, if homozygous (WH WH ). We present a refined, two-gene sex determination model for Salix purpurea, mediated by ARR17 and GATA15 that is different from the single-gene ARR17-mediated system in the related genus Populus.

Allelochemicals and soil microorganisms jointly mediate sex-specific belowground interactions in dioecious Populus cathayana.

Little is known about how sex differences in root zone characteristics, such as contents of allelochemicals and soil microbial composition, mediate intra- and intersexual interactions in dioecious plants. We examined the processes and mechanisms of sex-specific belowground interactions mediated by allelochemicals and soil microorganisms in Populus cathayana females and males in replicated 30-yr-old experimental stands in situ and in a series of controlled experiments. Female roots released a greater amount and more diverse phenolic allelochemicals into the soil environment, resulting in growth inhibition of the same sex neighbors and deterioration of the community of soil microorganisms. When grown with males, the growth of females was consistently enhanced, especially the root growth. Compared with female monocultures, the presence of males reduced the total phenolic accumulation in the soil, resulting in a shift from allelopathic inhibition to chemical facilitation. This association was enhanced by a favorable soil bacterial community and increased bacterial diversity, and it induced changes in the orientation of female roots. Our study highlighted a novel mechanism that enhances female performance by males through alterations in the allelochemical content and soil microbial composition. The possibility to improve productivity by chemical mediation provides novel opportunities for managing plantations of dioecious plants.

Genomic data provide a robust phylogeny backbone for Rhodiola L. (Crassulaceae) and reveal extensive reticulate evolution during its rapid radiation.

The Tibetan Plateau and adjacent mountain regions (TP; including the Tibetan Plateau, Himalaya, Hengduan Mountains and Mountains of Central Asia) harbor great biodiversity, some lineages on which may have undergone rapid radiations. However, only a few studies have investigated the evolutionary pattern of such diversification in depth using genomic data. In this study, we reconstructed a robust phylogeny backbone of Rhodiola, a lineage that may have undergone rapid radiation in the TP, using Genotyping-by-sequencing data, and conducted a series of gene flow and diversification analyses. The concatenation and coalescent-based methods yield similar tree topologies, and five well-supported clades were revealed. Potential gene flow and introgression events were detected, both between species from different major clades and closely related species, suggesting pervasive hybridization and introgression. An initial rapid and later slowdown of the diversification rate was revealed, indicating niche filling. Molecular dating and correlation analyses showed that the uplift of TP and global cooling in the mid-Miocene might have played an important role in promoting the rapid radiation of Rhodiola. Our work demonstrates that gene flow and introgression might be an important contributor to rapid radiation possibly by quickly reassembling old genetic variation into new combinations.

Gone with the wind: Negative genetic and progeny fitness consequences of habitat fragmentation in the wind pollinated dioecious tree Brosimum alicastrum.

PREMISE: Habitat fragmentation negatively affects population size and mating patterns that directly affect progeny fitness and genetic diversity; however, little is known about the effects of habitat fragmentation on dioecious, wind pollinated trees. We assessed the effects of habitat fragmentation on population sex ratios, genetic diversity, gene flow, mating patterns, and early progeny vigor in the tropical dioecious tree, Brosimum alicastrum. METHODS: We conducted our study in three continuous and three fragmented forest sites in a Mexican tropical dry forest. We used eight microsatellite loci to characterize the genetic diversity, gene flow via pollen distances, and mean relatedness of progeny. We compared early progeny vigor parameters of seedlings growing under greenhouse conditions. RESULTS: Sex ratios did not deviate from 1:1 between habitat conditions except for one population in a fragmented habitat, which was female biased. The genetic diversity of adult trees and their offspring was similar in both habitat conditions. Pollen gene flow distances were similar across habitat types; however, paternity correlations were greater in fragmented than in continuous habitats. Germination rates did not differ between habitat conditions; however, progeny from fragmented habitats produced fewer leaves and had a lower foliar area, total height, and total dry biomass than progeny from continuous habitats. CONCLUSIONS: Changes in mating patterns because of habitat fragmentation have negative effects on early progeny vigor. We conclude that negative habitat fragmentation effects on mating patterns and early progeny vigor may be a serious threat to the long-term persistence of tropical dioecious trees.

Comparative floral development in male and female plants of Palmer amaranth (Amaranthus palmeri).

PREMISE: Characterizing the developmental processes in the transition from hermaphroditism to unisexuality is crucial for understanding floral evolution. Amaranthus palmeri, one of the most devastating weeds in the United States, is an emerging model system for studying a dioecious breeding system and understanding the biological traits of this invasive weed. The objectives of this study were to characterize phases of flower development in A. palmeri and compare organogenesis of flower development in female and male plants. METHODS: Flower buds from male and female plants were dissected for light microscopy. Segments of male and female inflorescences at different stages of development were cut longitudinally and visualized using scanning electron microscopy. RESULTS: Pistillate flowers have two to three styles, one ovary with one ovule, and five obtuse tepals. Staminate flowers have five stamens with five acute tepals. Floral development was classified into 10 stages. The distinction between the two flower types became apparent at stage four by the formation of stamen primordia in staminate flowers, which developed female and male reproductive organs initially, as contrasted to pistillate flowers, which produced carpel primordia only. In staminate flowers, the putative carpel primordia changed little in size and remained undeveloped. CONCLUSIONS: Timing of inappropriate organ termination varies across the two sexes in A. palmeri. Our study suggests that the evolution of A. palmeri from a cosexual ancestral state to complete dioecy is still in progress since males exhibited transient hermaphroditism and females produced strictly pistillate flowers.

Development differs between independently evolved staminode whorls in the same flower.

PREMISE: Staminodes are commonly studied in hermaphroditic flowers, in which a fraction of the androecium evolves into infertile structures, but few studies have addressed the evolution of staminodes as they occur through the loss of stamen function in carpellate flowers. Plants of Paronychia (Caryophyllaceae) are monoecious with hermaphroditic flowers with one staminodial whorl, except for the dioecious P. chartacea and P. minima. Dioecious species have carpellate flowers that evolved an additional whorl of staminodes, providing an exceptional opportunity to study a second origin of staminodes in the same flower. METHODS: Using scanning electron microscopy, we observed the development of carpellate and staminate flowers to determine whether the developmental pathway of the staminodes in hermaphroditic flowers was co-opted during the evolutionary transition to unisexual flowers. RESULTS: In carpellate flowers, antesepalous staminodes initiate as sterile anthers that develop similar to functioning stamens, but arrest before full development, leaving a rudimentary anther with lateral lobes that correspond to thecae. After antesepalous staminodes arrest, alternisepalous staminodes initiate as structures that correspond with filaments, as they do in staminate and hermaphroditic flowers. CONCLUSIONS: The second origin of staminodes in carpellate flowers evolved using a different developmental pathway than what had previously evolved in the alternisepalous whorl. The two androecial whorls in the same flowers are serialogous as members of the androecium, but are paralogous as staminodes on the basis of structural and developmental differences.

Sexual dimorphism in a dioecious species with complex, specialist-pollinated flowers.

PREMISE: Pollinators with flower constancy and long nectar-feeding organs should favor less or no sexual dimorphism in the individual flowers of dioecious plants. This hypothesis is deduced because such pollinators can discriminate between intersexual flower size differences, and morphological differences between male and female flowers often diminish pollen transfer. METHODS: We compared floral traits and pollinator behavior between male and female flowers in the hawkmoth-pollinated species, Trichosanthes cucumeroides. In field studies, we removed petal fringes on both sexes and observed pollinators to assess the role of elaborate petal fringes in pollinator attraction and pollination success for each flower sex. RESULTS: Female flowers had a similar front flower size and fringe extension as male flowers, supporting our hypothesis. In contrast, females allocated fewer resources to floral biomass. Additionally, they had smaller and narrower petal lobes, lower fringe density, shorter tubes with inferior nectar rewards, and lower display size than males, which is inconsistent with the hypothesis. Nocturnal hawkmoths prefer flowers with long fringe extensions. Fringe removal significantly decreased hawkmoth visitations to both female and male flowers but reduced success only in females. A literature survey indicated that female flowers of specialist-pollinated species are similar in size or larger than the males and thus tend to attract more pollinators compared with female flowers of generalist-pollinated species. CONCLUSIONS: Female flowers have evolved fringe extensions that are similar to those of male flowers, likely increasing pollinator attraction even slightly, and had less biomass in other floral parts and produced less nectar compared with male flowers. Our findings imply that female-biased resource limitation and flower-size sensitivity of pollinators together exert sex-specific selection of floral traits in T. cucumeroides.