Diverse mating consequences of the evolutionary breakdown of the sexual polymorphism heterostyly.

Reproductive systems of flowering plants are evolutionarily fluid, with mating patterns changing in response to shifts in abiotic conditions, pollination systems, and population characteristics. Changes in mating should be particularly evident in species with sexual polymorphisms that become ecologically destabilized, promoting transitions to alternative reproductive systems. Here, we decompose female mating portfolios (incidence of selfing, outcross mate number, and intermorph mating) in eight populations of Primula oreodoxa, a self-compatible insect-pollinated herb. This species is ancestrally distylous, with populations subdivided into two floral morphs that usually mate with each other (disassortative mating). Stages in the breakdown of polymorphism also occur, including "mixed" populations of distylous and homostylous (self-pollinating) morphs and purely homostylous populations. Population morph ratios vary with elevation in association with differences in pollinator availability, providing an unusual opportunity to investigate changes in mating patterns accompanying transitions in reproductive systems. Unexpectedly, individuals mostly outcrossed randomly, with substantial disassortative mating in at most two distylous populations. As predicted, mixed populations had higher selfing rates than distylous populations, within mixed populations, homostyles selfed almost twice as much as the distylous morphs, and homostylous populations exhibited the highest selfing rates. Populations with homostyles outcrossed with fewer mates and mate number varied negatively with population selfing rates. These differences indicate maintenance of distyly at low elevation, transition to monomorphic selfing at high elevation, and uncertain, possibly variable fates at intermediate elevation. By quantifying the earliest changes in mating that initiate reproductive transitions, our study highlights the key role of mating in promoting evolutionary divergence.

Community composition of phytopathogenic fungi significantly influences ectomycorrhizal fungal communities during subtropical forest succession.

Ectomycorrhizal fungi (EMF) can form symbiotic relationships with plants, aiding in plant growth by providing access to nutrients and defense against phytopathogenic fungi. In this context, factors such as plant assemblages and soil properties can impact the interaction between EMF and phytopathogenic fungi in forest soil. However, there is little understanding of how these fungal interactions evolve as forests move through succession stages. In this study, we used high-throughput sequencing to investigate fungal communities in young, intermediate, and old subtropical forests. At the genus level, EMF communities were dominated by Sebacina, Russula, and Lactarius, while Mycena was the most abundant genus in pathogenic fungal communities. The relative abundances of EMF and phytopathogenic fungi in different stages showed no significant difference with the regulation of different factors. We discovered that interactions between phytopathogenic fungi and EMF maintained a dynamic balance under the influence of the differences in soil quality attributed to each forest successional stage. The community composition of phytopathogenic fungi is one of the strong drivers in shaping EMF communities over successions. In addition, the EMF diversity was significantly related to plant diversity, and these relationships varied among successional stages. Despite the regulation of various factors, the positive relationship between the diversity of phytopathogenic fungi and EMF remained unchanged. However, there is no significant difference in the ratio of the abundance of EMF and phytopathogenic fungi over the course of successions. These results will advance our understanding of the biodiversity-ecosystem functioning during forest succession. KEY POINTS: •Community composition of both EMF and phytopathogenic fungi changed significantly over forest succession. •Phytopathogenic fungi is a key driver in shaping EMF community. •The effect of plant Shannon's diversity on EMF communities changed during the forest aging process.

Genomic evidence supports the genetic convergence of a supergene controlling the distylous floral syndrome.

Heterostyly, a plant sexual polymorphism controlled by the S-locus supergene, has evolved numerous times among angiosperm lineages and represents a classic example of convergent evolution in form and function. Determining whether underlying molecular convergence occurs could provide insights on constraints to floral evolution. Here, we investigated S-locus genes in distylous Gelsemium (Gelsemiaceae) to determine whether there is evidence of molecular convergence with unrelated distylous species. We used several approaches, including anatomical measurements of sex-organ development and transcriptome and whole-genome sequencing, to identify components of the S-locus supergene. We also performed evolutionary analysis with candidate S-locus genes and compared them with those reported in Primula and Turnera. The candidate S-locus supergene of Gelsemium contained four genes, of which three appear to have originated from gene duplication events within Gelsemiaceae. The style-length genes GeCYP in Gelsemium and CYP734A50 in Primula likely arose from duplication of the same gene, CYP734A1. Three out of four S-locus genes in Gelsemium elegans were hemizygous, as previously reported in Primula and Turnera. We provide genomic evidence on the genetic convergence of the supergene underlying distyly among distantly related angiosperm lineages and help to illuminate the genetic architecture involved in the evolution of heterostyly.

Drivers of foliar 15 N trends in southern China over the last century.

Foliar stable nitrogen (N) isotopes (δ15 N) generally reflect N availability to plants and have been used to infer about changes thereof. However, previous studies of temporal trends in foliar δ15 N have ignored the influence of confounding factors, leading to uncertainties on its indication to N availability. In this study, we measured foliar δ15 N of 1811 herbarium specimens from 12 plant species collected in southern China forests from 1920 to 2010. We explored how changes in atmospheric CO2 , N deposition and global warming have affected foliar δ15 N and N concentrations ([N]) and identified whether N availability decreased in southern China. Across all species, foliar δ15 N significantly decreased by 0.82‰ over the study period. However, foliar [N] did not decrease significantly, implying N homeostasis in forest trees in the region. The spatiotemporal patterns of foliar δ15 N were explained by mean annual temperature (MAT), atmospheric CO2 ( P CO 2 ), atmospheric N deposition, and foliar [N]. The spatiotemporal trends of foliar [N] were explained by MAT, temperature seasonality, P CO 2 , and N deposition. N deposition within the rates from 5.3 to 12.6 kg N ha-1  year-1 substantially contributed to the temporal decline in foliar δ15 N. The decline in foliar δ15 N was not accompanied by changes in foliar [N] and therefore does not necessarily reflect a decline in N availability. This is important to understand changes in N availability, which is essential to validate and parameterize biogeochemical cycles of N.

Development of the first microsatellite markers using high-throughput sequencing for a hexaploid coastal species, Tournefortia argentea L. f. (Boraginaceae).

BACKGROUD: Tournefortia argentea L. f. is a hexaploid shrub or tree species with ecological and evolutionary significances, which forms the fringe of vegetation closest to the sea on tropical coral islands. Previous studies have never addressed on genetic information, and thus genomic resources remain scarce. METHODS AND RESULTS: We used nine individuals from different islands to identify polymorphic microsatellites of T. argentea by Illumina high-throughput sequencing. Thirty-five polymorphic microsatellite markers were developed. Characteristics of each locus were tested using 48 individuals collected from three populations of T. argentea. A total of 320 alleles were found across the 35 microsatellite loci. The number of alleles per locus ranged from 5 to 15, with an average of 9.1. Observed and expected heterozygosities in each locus per population varied from 0.000 to 1.000 and from 0.000 to 0.893, respectively. CONCLUSIONS: In this study, we report the development of 35 polymorphic microsatellite markers based on Illumina high-throughput sequencing. These markers will facilitate the investigations of genetic diversity, population structures and evolutionary history of T. argentea.

Evolution of CYCLOIDEA-like genes in Fabales: Insights into duplication patterns and the control of floral symmetry.

Cycloidea-like (CYC-like) genes are the key regulatory factors in the development of flower symmetry. Duplication and/or reduction of CYC-like genes have occurred several times in various angiosperm groups and are hypothesized to be correlated with the evolution of flower symmetry, which in turn has contributed to the evolutionary success of these groups. However, less is known about the evolutionary scenario of CYC-like genes in the whole Fabales, which contains four families with either symmetric or actinomorphic flowers. Here we investigated the evolution of CYC-like genes in all the four families of Fabales and recovered one to nine CYC-like genes (CYC1, CYC2, and CYC3) depending on which lineages, but the CYC3 genes were most likely lost in the ancestor of Leguminosae. Phylogenetic analysis suggested that the CYC-like genes could have undergone multiple duplications and losses in different plant lineages and formed distinct paralogous/orthologous clades. The ancestor of the Papilionoideae and Caesalpinioideae may possess two paralogs of CYC1 genes but one of them was subsequently lost in Papilionoideae and was retained only in several species of Caesalpinioideae. CYC2 genes were more frequently duplicated in Papilionoideae than in other legumes. We propose that the diversification patterns of both CYC1 and CYC2 genes are not related to the floral symmetry in non-papilionoid Fabales groups, however, gene duplication and functional divergence of CYC2 are essential for the floral zygomorphy of Papilionoideae. This is the first systematic analysis of the CYC-like genes in Fabales and could form the basis for further study of molecular mechanisms controlling floral symmetry in non-model plants of Fabales.

Global transcriptome and gene co-expression network analyses on the development of distyly in Primula oreodoxa.

Distyly is a genetically controlled flower polymorphism that has intrigued both botanists and evolutionary biologists ever since Darwin's time. Despite extensive reports on the pollination and evolution of distylous systems, the genetic basis and mechanism of molecular regulation remain unclear. In the present study, comparative transcriptome profiling was conducted in primrose (Primula oreodoxa), the prime research model for heterostyly. Thirty-six transcriptomes were sequenced for styles at different stages and corolla tube in the three morphs of P. oreodoxa. Large numbers of differentially expressed genes (DEGs) were detected in the transcriptomes of styles across different morphs. Several transcription factors (TFs) and phytohormone metabolism-related genes were highlighted in S-morphs. A growing number of genes showed differential expression patterns along with the development of styles, suggesting that the genetic control of distyly may be more complicated than ever expected. Analysis of co-expression networks and module-trait relationships identified modules significantly associated with style development. CYP734A50, a key S-locus gene whose products degrade brassinosteroids, was co-expressed with many genes in the module and showed significant negative association with style length. In addition, crucial TFs involved in phytohormone signaling pathways were found to be connected with CYP734A50 in the co-expression module. Our global transcriptomic analysis has identified DEGs that are potentially involved in regulation of style length in P. oreodoxa, and may shed light on the evolution and broad biological processes of heterostyly.

Genetics of distyly and homostyly in a self-compatible Primula.

The transition from outcrossing to selfing through the breakdown of distyly to homostyly has occurred repeatedly among families of flowering plants. Homostyles can originate by major gene changes at the S-locus linkage group, or by unlinked polygenic modifiers. Here, we investigate the inheritance of distyly and homostyly in Primula oreodoxa, a subalpine herb endemic to Sichuan, China. Controlled self- and cross-pollinations confirmed that P. oreodoxa unlike most heterostylous species is fully self-compatible. Segregation patterns indicated that the inheritance of distyly is governed by a single Mendelian locus with the short-styled morph carrying at least one dominant S-allele (S-) and long-styled plants homozygous recessive (ss). Crossing data were consistent with a model in which homostyly results from genetic changes at the distylous linkage group, with the homostylous allele (Sh) dominant to the long-styled allele (s), but recessive to the short-styled allele (S). Progeny tests of open-pollinated seed families revealed high rates of intermorph mating in the L-morph but considerable selfing and possibly intramorph mating in the S-morph and in homostyles. S-morph plants homozygous at the S-locus (SS) occurred in several populations but may experience viability selection. The crossing data from distylous and homostylous plants are consistent with either recombination at the S-locus governing distyly, or mutation at gene(s) controlling sex-organ height; both models predict the same patterns of segregation. Recent studies on the molecular genetics of distyly in Primula demonstrating the hemizygous nature of genes at the S-locus make it more likely that homostyles have resulted from mutation rather than recombination.

A symbiotic balancing act: arbuscular mycorrhizal specificity and specialist fungus gnat pollination in the mycoheterotrophic genus Thismia (Thismiaceae).

BACKGROUND AND AIMS: Mycorrhizal associations in mycoheterotrophic plants are generally more specialized than in autotrophs. Mycoheterotrophs typically bear small, inconspicuous flowers that often self-pollinate to maximize seed set, although some have structurally complex flowers indicative of xenogamy. A trade-off has previously been proposed between specialization in these above- and below-ground symbioses, although empirical data are lacking. METHODS: We used next-generation DNA sequencing to compare the mycorrhizal communities from the roots of a mycoheterotrophic species, Thismia tentaculata (Thismiaceae), and its neighbouring autotrophs. We furthermore conducted detailed assessments of floral phenology and pollination ecology, and performed artificial pollination experiments to determine the breeding system. KEY RESULTS: Thismia tentaculata maintains a symbiotic association with a single arbuscular mycorrhizal Rhizophagus species. The flowers are pollinated by a single species of fungus gnats (Corynoptera, Sciaridae), which are attracted by the yellow pigments and are temporarily restrained within the perianth chamber before departing via apertures between the anthers. The plants are self-compatible but predominantly xenogamous. CONCLUSIONS: Our findings demonstrate that T. tentaculata maintains highly specialized associations with pollinators and mycorrhizal fungi, both of which are widely distributed. We suggest that specialization in multiple symbiotic interactions is possible in mycoheterotrophs if redundant selective pressures are not exerted to further restrict an already constrained suite of life-history traits.

The largest early-diverging angiosperm family is mostly pollinated by ovipositing insects and so are most surviving lineages of early angiosperms.

Insect pollination in basal angiosperms is assumed to mostly involve 'generalized' insects looking for food, but direct observations of ANITA grade (283 species) pollinators are sparse. We present new data for numerous Schisandraceae, the largest ANITA family, from fieldwork, nocturnal filming, electron microscopy, barcoding and molecular clocks to infer pollinator/plant interactions over multiple years at sites throughout China to test the extent of pollinator specificity. Schisandraceae are pollinated by nocturnal gall midges that lay eggs in the flowers and whose larvae then feed on floral exudates. At least three Schisandraceae have shifted to beetle pollination. Pollination by a single midge species predominates, but one species was pollinated by different species at three locations and one by two at the same location. Based on molecular clocks, gall midges and Schisandraceae may have interacted since at least the Early Miocene. Combining these findings with a review of all published ANITA pollination data shows that ovipositing flies are the most common pollinators of living representatives of the ANITA grade. Compared to food reward-based pollination, oviposition-based systems are less wasteful of plant gametes because (i) none are eaten and (ii) female insects with herbivorous larvae reliably visit conspecific flowers.

Evolution of sexual systems and growth habit in Mussaenda (Rubiaceae): Insights into the evolutionary pathways of dioecy.

Dioecy is a rare sexual system that is thought to represent an "evolutionary dead end". While many studies have addressed the evolution of dioecy and/or its relationship with the evolution of the woody habit, few have explored the relationship between dioecy and climbing habit, and their effects on diversification rates. Here, we study the evolution of sexual systems and growth habit in Mussaenda (Rubiaceae) using a robust phylogeny of the genus based on eight plastid regions and a broad sampling of taxa (92 of the 132 species were sampled). A time-calibrated tree was constructed to estimate diversification rates in different clades and its correlates with focal characters. More specifically, we assess evolutionary correlations between dioecy and climbing habit and their respective influences on diversification rates. Ancestral character state reconstructions revealed that distyly is the most likely ancestral state in Mussaenda. Distyly has subsequently given rise to dioecy, short-styled floral monomorphism, and long-styled floral monomorphism. Dioecy has evolved independently at least four times from distyly, and has reversed to homostylous hermaphroditism at least twice, which does not support the "evolutionary dead end" hypothesis. A significant correlation between the evolution of dioecy and climbing growth form was found in Mussaenda. It is possible that a strong association between high net diversification rates and dioecy may exist in Mussaenda, but no association was found with climbing habit.

A Novel, Enigmatic Basal Leafflower Moth Lineage Pollinating a Derived Leafflower Host Illustrates the Dynamics of Host Shifts, Partner Replacement, and Apparent Coadaptation in Intimate Mutualisms.

Leafflower plant/leafflower moth brood pollination mutualisms are widespread in the Paleotropics. Leafflower moths pollinate leafflower plants, but their larvae consume a subset of the hosts' seeds. These interactions are highly phylogenetically constrained: six clades of leafflower plants are each associated with a unique clade of leafflower moths (Epicephala). Here, we report a previously unrecognized basal seventh pollinating Epicephala lineage-associated with the highly derived leafflower clade Glochidion-in Asia. Epicephala lanceolaria is a pollinator and seed predator of Glochidion lanceolarium. Phylogenetic inference indicates that the ancestor of E. lanceolaria most likely shifted onto the ancestor of G. lanceolarium and displaced the ancestral allospecific Epicephala pollinator in at least some host populations. The unusual and apparently coadapted aspects of the G. lanceolarium/E. lanceolaria reproductive cycles suggest that plant-pollinator coevolution may have played a role in this displacement and provide insights into the dynamics of host shifts and trait coevolution in this specialized mutualism.

Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly.

Background and Aims: The outbreeding floral polymorphism heterostyly frequently breaks down, resulting in the evolution of self-fertilization as a result of homostyle formation. Here, the loss of floral polymorphism in distylous Primula oreodoxa, a sub-alpine species restricted to western Sichuan, China, was examined by investigating the ecological correlates and genetic consequences of mating system transitions. Several key questions were addressed. (1) What are the frequencies, geographical distribution and reproductive characteristics of floral morphs in distylous and homostylous populations? (2) Does increased elevation influence pollinator service and the likelihood of inbreeding in populations? (3) How often has homostyly originated and what are the consequences of the breakdown of distyly for the amounts and distribution of genetic diversity in populations? Methods: Fourteen populations throughout the range of P. oreodoxa were sampled, and morph frequencies and floral characteristics were recorded. Polymorphism at microsatellite loci and chloroplast DNA (cpDNA) variation were used to quantify population genetic structure and genetic relationships among populations. Controlled pollinations and studies of pollen tube growth and fertility were conducted to determine the compatibility status of populations and their facility for autonomous self-pollination. Finally, visitation rates of long- and short-tongued pollinators to distylous and homostylous populations at different elevations were compared to determine if increased elevation was associated with deterioration in pollinator service. Key Results: In contrast to most heterostylous species, both distylous and homostylous morphs of P. oreodoxa are highly self-compatible, but only homostyles have the facility for autonomous self-pollination. Homostyles set significantly more fruit and seeds following open pollination than the distylous morphs. Visitation by long-tongued pollinators was significantly lower in homostylous populations, and overall rates of insect visitation decreased with elevation. Genetic diversity was significantly lower in homostylous populations, with evidence of increased inbreeding at higher elevation. Patterns of cpDNA variation were consistent with multiple transitions from distyly to homostyly and limited gene flow among populations. Conclusions: The results of this study support the hypothesis that the multiple loss of floral polymorphism in distylous P. oreodoxa is associated with unsatisfactory pollinator service, with homostyles benefiting from reproductive assurance as a result of autonomous self-pollination.

Reproductive isolation between sympatric sister species, Mussaenda kwangtungensis and M. pubescens var. alba.

Reproductive isolation defines the biological species concept and plays a key role in the formation and maintenance of species. The relative contributions of different isolating stages has been suggested to be closely associated with phylogenetic relatedness. Few studies have focused on the relative contributions of pre- versus post-zygotic mechanisms, and even fewer have been conducted under strict phylogenetic frameworks. Pre- and post-zygotic reproductive isolation stages have been investigated in the sister species Mussaenda kwangtungensis and M. pubescens var. alba. The two species have partly overlapping distribution ranges and flowering times, while the principal pollinators differed strikingly for them, demonstrating strong pre-zygotic isolations. Natural hybrids were detected by simple sequence repeat markers and their maternal parents were identified based on chloroplast gene sequences. Five out of 81 individuals were suggested to be hybrids that fall into the categories F2, BC1, and BC2 by the NewHybrids analysis. Interspecific crossings resulted in significantly reduced fruit set and seed germination rates. Phylogenetic analysis revealed short Kimura-2-parameter distance between M. kwangtungensis and M. pubescens var. alba. These findings strongly supported the hypothesis that for species with a closer phylogenetic relationship, pre-zygotic isolation plays an important part in limiting gene exchange in sympatric areas.

Phylogenetic position of Guihaiothamnus (Rubiaceae): its evolutionary and ecological implications.

Guihaiothamnus (Rubiaceae) is an enigmatic, monotypic genus endemic to southwestern China. Its generic status has never been doubted because it is morphologically unique by having rosette habit, showy, long-corolla-tubed flowers, and multi-seeded indehiscent berry-like fruits. The genus has been postulated to be a relict in the broad-leaved forests of China, and to be related to the genus Wendlandia, which was placed in the subfamily Cinchonoideae and recently classified in the tribe Augusteae of the subfamily Dialypetalanthoideae. Using combined evidence from palynology, cytology, and DNA sequences of nuclear ITS and four plastid markers (rps16, trnT-F, ndhF, rbcL), we assessed the phylogenetic position of Guihaiothamnus in Rubiaceae. Our molecular phylogenetic analyses placed the genus deeply nested within Wendlandia. This relationship is corroborated by evidence from palynology and cytology. Using a relaxed molecular clock method based on five fossil records, we dated the stem age of Wendlandia to be 17.46 my and, the split between G. acaulis and related Wendlandia species in southwestern China to be 2.11mya. This young age, coupled with the derived position in Wendlandia, suggests an evolutionary derivation rather than an evolutionary relict of G. acaulis. Its rosette habit and large showy flowers, which are very distinctive from other Wendlandias, are interpreted as a result of recent rapid adaptation to rock and cliff habitats.

Pre- and post-zygotic reproductive isolation between co-occurring Mussaenda pubescens var. alba and M. shikokiana (Rubiaceae).

Reproductive isolation is a fundamental requirement for speciation and includes several sequential stages. Few studies have determined the relative contributions of pre- and post-zygotic reproductive isolation in plants, especially between relative species with clear differentiation in flower form. To investigate the mechanisms responsible for reproductive isolation in sympatric Mussaenda pubescens var. alba and Mussaenda shikokiana (Rubiaceae) in Guangxi Province, China, we made observations of flowering phenology, patterns of insect visitation, and conducted pollination experiments, including artificial hybridization. The two species had overlapping flowering times and were pollinated by overlapping pollinators; however, their relative importance differed significantly with M. pubescens visited more commonly by bees and M. shikokiana more frequently by butterflies. Using vegetative and floral characters and molecular evidence based on nuclear ribosomal internal and external transcribed spacer regions we detected seven naturally occurring hybrids among a sample of approximately 125 individuals. Hybrids were characterized by morphologies that most closely resembled their maternal parents based on chloroplast evidence. Studies of artificially synthesized and natural hybrids demonstrated that hybrid seed had very low germination rates and naturally occurring hybrids exhibited pollen sterility. Post-zygotic reproductive isolating mechanisms play a primary role in limiting gene exchange between co-occurring species and maintaining species integrity in areas of sympatry.

Different mechanisms underlie similar species-area relationships in two tropical archipelagoes.

Despite much research in the field of island biogeography, mechanisms regulating insular diversity remain elusive. Here, we aim to explore mechanisms underlying plant species-area relationships in two tropical archipelagoes in the South China Sea. We found positive plant species-area relationships for both coral and continental archipelagoes. However, our results showed that different mechanisms contributed to similar plant species-area relationships between the two archipelagoes. For coral islands, soil nutrients and spatial distance among communities played major roles in shaping plant community structure and species diversity. By contrast, the direct effect of island area, and to a lesser extent, soil nutrients determined plant species richness on continental islands. Intriguingly, increasing soil nutrients availability (N, P, K) had opposite effects on plant diversity between the two archipelagoes. In summary, the habitat quality effect and dispersal limitation are important for regulating plant diversity on coral islands, whereas the passive sampling effect, and to a lesser extent, the habitat quality effect are important for regulating plant diversity on continental islands. More generally, our findings indicate that island plant species-area relationships are outcomes of the interplay of both niche and neutral processes, but the driving mechanisms behind these relationships depends on the type of islands.

Phylogenetic relationships and biogeography of Asia Callicarpa (Lamiaceae), with consideration of a long-distance dispersal across the Pacific Ocean -insights into divergence modes of pantropical flora.

There are about 140 species of Callicarpa L. 1753 (Lamiaceae), with more species richness in tropical to subtropical Asia and the New World. The genus might provide an insight into the amphi-Pacific disjunction pattern of tropical and subtropical vegetation. This study has greatly improved the phylogenetic underpinning for Callicarpa, derived from more inclusive taxonomic samplings, and employing data on both two-nuclear and eight-chloroplast regions. To address time and patterns of diversification in Callicarpa, we conducted divergence time and biogeographic analyses, and inferred shifts in the distribution areas across the phylogenetic clades. Our phylogenetic results show that Callicarpa is monophyletic with respect to the groups considered, and eight well-supported primary clades were discerned in the combined analyses. Our estimates indicated that the crown group of Callicarpa originates around the Late-Eocene (ca. 36.23 Ma) and diversification within most clades is concentrated in the Miocene and continued to the Pleistocene. In addition, our biogeographic analyses suggested that the probable ancestor of the Callicarpa crown clade originated in East Asia and Southeast Asia. Multiple dispersal and vicariance events contributed to the current distribution of the taxa. Furthermore, this genus expanded eastward out of East and Southeast Asia to the New World by long-distance dispersal, which inspired us to better understand the amphi-Pacific disjunct distribution.

Comprehensive transcriptomic profiling reveals complex molecular mechanisms in the regulation of style-length dimorphism in Guettarda speciosa (Rubiaceae), a species with "anomalous" distyly.

Background: The evolution of heterostyly, a genetically controlled floral polymorphism, has been a hotspot of research since the 19th century. In recent years, studies on the molecular mechanism of distyly (the most common form of heterostyly) revealed an evolutionary convergence in genes for brassinosteroids (BR) degradation in different angiosperm groups. This floral polymorphism often exhibits considerable variability that some taxa have significant stylar dimorphism, but anther height differs less. This phenomenon has been termed "anomalous" distyly, which is usually regarded as a transitional stage in evolution. Compared to "typical" distyly, the genetic regulation of "anomalous" distyly is almost unknown, leaving a big gap in our understanding of this special floral adaptation strategy. Methods: Here we performed the first molecular-level study focusing on this floral polymorphism in Guettarda speciosa (Rubiaceae), a tropical tree with "anomalous" distyly. Comprehensive transcriptomic profiling was conducted to examine which genes and metabolic pathways were involved in the genetic control of style dimorphism and if they exhibit similar convergence with "typical" distylous species. Results: "Brassinosteroid homeostasis" and "plant hormone signal transduction" was the most significantly enriched GO term and KEGG pathway in the comparisons between L- and S-morph styles, respectively. Interestingly, homologs of all the reported S-locus genes either showed very similar expressions between L- and S-morph styles or no hits were found in G. speciosa. BKI1, a negative regulator of brassinosteroid signaling directly repressing BRI1 signal transduction, was identified as a potential gene regulating style length, which significantly up-regulated in the styles of S-morph. Discussion: These findings supported the hypothesis that style length in G. speciosa was regulated through a BR-related signaling network in which BKI1 may be one key gene. Our data suggested, in species with "anomalous" distyly, style length was regulated by gene differential expressions, instead of the "hemizygous" S-locus genes in "typical" distylous flowers such as Primula and Gelsemium, representing an "intermediate" stage in the evolution of distyly. Genome-level analysis and functional studies in more species with "typical" and "anomalous" distyly would further decipher this "most complex marriage arrangement" in angiosperms and improve our knowledge of floral evolution.

Development of microsatellite markers from Mussaenda pubescens (Rubiaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed and characterized in Mussaenda pubescens for further study of its levels of genetic diversity and changes in population genetic structure in reproductive character displacement and in shifts of sexual systems. METHODS AND RESULTS: Nineteen microsatellite loci were amplified successfully in M. pubescens, 17 of which were polymorphic. A maximum of eight alleles were detected per locus in 68 individuals at population level. The observed and expected heterozygosities varied from 0 to 1.000 and 0 to 0.882, respectively. CONCLUSIONS: These newly developed microsatellite markers will be useful in further investigations of genetic diversity and gene flow among populations of M. pubescens and its congeneric species.