Allopolyploidy enhances survival advantages for urban environments in the native plant genus Commelina.

BACKGROUND AND AIMS: Urbanization-induced environmental changes affect the geographical distribution of natural plant species. This study focused on how polyploidization, a dynamic genome change, influences the survival and distribution of Commelina communis L. (Cc) and its subspecies, C. communis f. ciliata (Masam.) Murata (Ccfc) which have different chromosome numbers (e.g. Cc: 2n = 88, Ccfc: 2n = 46). The aim is to investigate polyploidization effects on natural plant distribution in urban environments. METHODS: The geographical distribution across urban-rural gradients was investigated at a total of 218 sites in Japan. Stomata size and density were measured and compared between Cc and Ccfc. Flow cytometry determined genome size and polyploidy. Chromosome karyotyping was performed using genomic in situ hybridization (GISH) method. KEY RESULTS: Urban areas were exclusively dominated by Cc, while Cc and Ccfc coexisted in rural areas. Cc had larger and fewer stomata and more than twice the genome size than Ccfc. GISH results indicated that Cc possesses Ccfc and another unknown genome, suggesting allopolyploidy. CONCLUSIONS: Our results show that the ploidy difference affects the geographical distribution, the stomata traits, and genome size between two distinct taxa in the genus Commelina, C. communis as a neo-tetraploid and C. communis f. ciliata, the diploid. Cc is an allopolyploid, therefore, not only polyploidy but also an additional genome with new sets of genes and alleles contributes to Cc having enhance survival potentials in urban environments compared to Ccfc. This is the first investigation to clarify the distribution difference related to urban environments, the difference in stomata traits and genome size, and to conduct chromosome composition in Commelina species.

Does a coexisting congener of a mixed mating species affect the genetic structure and selfing rate via reproductive interference?

Reproductive interference is defined as an interspecific interaction that reduces fitness via mating processes. Although its ecological and evolutionary consequences have attracted much attention, how reproductive interference affects the population genetic structures of interacting species is still unclear. In flowering plants, recent studies found that self-pollination can mitigate the negative effects of reproductive interference. Selfing-biased seed production is expected to increase population-level inbreeding and the selfing rate, and limits gene flow via pollinator outcrossing among populations. We examined the population genetics of the mixed-mating annual herb Commelina communis f. ciliata, focusing on reproductive interference by the sympatric competing congener C. communis using microsatellite markers. First, we found that almost all C. c. f. ciliata populations had relatively high inbreeding coefficients. Then, comparing sympatric and allopatric populations, we found evidence that reproductive interference from a competing congener increased the inbreeding coefficient and selfing rate. Allopatric populations exhibit varied selfing rates while almost all sympatric populations exhibit extremely high selfing rates, suggesting that population selfing rates were also influenced by unexamined factors, such as pollinator limitation. Besides, our findings revealed that reproductive interference from a competing congener did not limit gene flow among populations. We present the first report on how reproductive interference affects the genetic aspects of populations. Our results suggested that the high selfing rate of C. c. f. ciliata promotes its sympatric distribution with C. communis, even in the presence of reproductive interference, although it is not clear whether reproductive interference directly causes the high selfing rate.

Dominance of non-wetland-dependent pollinators in a plant community in a small natural wetland in Shimane, Japan.

Many wetland plants rely on insects for pollination. However, studies examining pollinator communities in wetlands remain limited. Some studies conducted in large wetlands (> 10 ha) have suggested that wetland-dependent flies, which spend their larval stage in aquatic and semi-aquatic habitats, dominate as pollinators. However, smaller wetlands surrounded by secondary forests are more prevalent in Japan, in which pollinators from the surrounding environment might be important. Additionally, information regarding floral traits that attract specific pollinator groups in wetland communities is scarce. Therefore, this study aimed to understand the characteristics of insect pollinators in a small natural wetland (2.5 ha) in Japan. We examined the major pollinator groups visiting 34 plant species and explored the relationship between the flower visitation frequency of each pollinator group and floral traits. Overall, flies were the most dominant pollinators (42%), followed by bees and wasps (33%). Cluster analysis indicated that fly-dominated plants were the most abundant among 14 of the 34 target plant species. However, 85% of the hoverflies, the most abundant flies, and 82% of the bees were non-wetland-dependent species, suggesting that these terrestrial species likely originated from the surrounding environment. Therefore, pollinators from the surrounding environment would be important in small natural wetlands. Flies tend to visit open and white/yellow flowers, whereas bees tended to visit tube-shaped flowers, as in forest and grassland ecosystems. The dominance of flies in small wetlands would be due to the dominance of flowers preferred by flies (e.g., yellow/white flowers) rather than because of their larval habitats.

Negative legacy effects of past forest use on native plant diversity in semi-natural grasslands on ski slopes.

Over the past century, grassland and forest ecosystems globally have been heavily influenced by land-use changes driven by diverse socioeconomic activities. Ski resorts are a modern land-use type associated with biodiversity loss in mountain ecosystems worldwide. Below the treeline, by contrast, some ski slopes have been shown to provide suboptimal semi-natural habitats for native grassland plants and animals, depending on specific construction and management practices. We compared environmental factors and grassland vegetation between two types of ski slopes in central Japan with different land-use histories: slopes constructed on old pastures (pasture slopes) and slopes constructed by clearing secondary forests or larch plantations established on abandoned pastures during the 1940s-1990s (forest slopes). We examined the effects of land-use history and machine grading as well as other environmental factors on ski slope vegetation, including total species richness and the richness of native, endangered, and exotic plants, using a total of 108 plots of 2 m × 10 m. Compared to pasture slopes, forest slopes exhibited significantly lower richness of native grassland species, including endangered species. Forest slopes were more graded than pasture slopes, resulting in lower native and higher exotic grassland species richness. A significantly lower duration of direct sunlight on forest slopes than on pasture slopes likely decreased endangered species richness. The lower species richness on forest slopes may be also caused by seed dispersal limitations. Our findings demonstrate that ski slopes have good potential to support numerous native grassland plant species, including endangered species, but this potential is significantly and negatively affected by forest use history and concomitant environmental changes. The conservation of semi-natural conditions on pasture slopes as habitats for native grassland species can be promoted through the maintenance of annual mowing practices, avoidance of machine grading, and wider ski courses.

Local environment perceived in daily life and urban green and blue space visits: Uncovering key factors for different age groups to access ecosystem services.

Inequality in access to green and blue spaces is a major issue in the field of environmental justice. Various factors significantly influence visitations to these spaces, including residential, commuting, and shopping areas. However, studies have mainly focused on residential environments. Analyzing the coupling of environmental and socioeconomic factors, including age, income, and childhood experience, and examining the direct and indirect effects of these factors for different age groups are needed to better understand the detailed context of inequalities in access to ecosystem services. We conducted a questionnaire survey and analysis to address these needs in an urban setting. The results demonstrate that several land use categories (i.e., agricultural lands in residential areas, grasslands near shopping areas, and deciduous forests near commuting destinations) positively correlated with visitation frequency, suggesting the lack thereof may be the cause of access inequalities. In particular, the perimeter per unit area of some land cover types, which shows the complexity of the form of each land cover category, was found to be a significant factor. We identified inequality in nature experience to be the key factor correlated with inequality in nature visits among the young age group. For the middle-aged and old age groups, nature-relatedness was positively correlated with visitation frequency. Proper design of the local environment, such as through land use planning, can be an effective measure for all age groups. For instance, long-bordered green areas in the Monsoon Asian region are an attractive traditional landscape, with high accessibility and a comfortable thermal environment. The key factors for each age group should be considered in spatial design and inclusive information-sharing to reduce inequality in access to ecosystem services.

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.

Does sexual dimorphism exist in flowering phenology traits in anemophilous dioecious species? A test with Rumex acetosa.

PREMISE: Sexual dimorphism in flowering phenology traits may have evolved under sexual selection and vector-mediated selection. The conspicuous sexual dimorphism and sex-specific selection pressures in flowering phenology traits have been investigated mainly in entomophilous dioecious plants, whereas little is known about this in anemophilous plants. METHODS: We examined sexual dimorphism in flowering onset, flowering peak, flowering duration, maximum proportion of open flowers per inflorescence branch, maximum proportion of newly opening flowers on a given date per branch, and longevity of individual flowers in natural Rumex acetosa populations. Correlations between flowering phenology traits and the degree of flowering overlap with the opposite sex were examined. We also tested whether the overlap of female flowering with male flowering enhanced seed set in female plants. RESULTS: Little sexual dimorphism was observed in flowering onset, peak, duration, and maximum proportion of newly opening flowers. Females had greater floral longevity and greater maximum proportion of open flowers than males. Flowering overlap with the opposite sex significantly increased with the maximum proportion of newly opening flowers and decreased with temporal deviation in the flowering peak in both sexes. Females with greater flowering overlap with males set more seeds in two of the three study populations. CONCLUSIONS: In wind-pollinated R. acetosa, little sexual dimorphism in phenological traits may have evolved to achieve synchronous flowering with the opposite sex. Our results suggest that, in angiosperms, not only common selection but also anemophily-specific selection may shape little sexual dimorphism in R. acetosa, unlike in entomophilous plants.

Historical changes in grassland area determined the demography of semi-natural grassland butterflies in Japan.

Semi-natural grassland areas expanded worldwide several thousand years ago following an increase in anthropogenic activities. However, semi-natural grassland habitat areas have been declining in recent decades due to changes in landuse, which have caused a loss of grassland biodiversity. Reconstructing historical and recent demographic changes in semi-natural grassland species will help clarify the factors affecting their population decline. Here we quantified past and recent demographic histories of Melitaea ambigua (Lepidoptera; Nymphalidae), an endangered grassland butterfly species in Japan. We examined changes in demography over the past 10,000 years based on 1378 bp of mitochondrial COI gene. We then examined changes in its genetic diversity and structure during the last 30 years using nine microsatellite DNA markers. The effective population size of M. ambigua increased about 3000-6000 years ago. In contrast, the genetic diversity and effective population sizes of many populations significantly declined from the 1980s to 2010s, which is consistent with a recent decline in the species population size. Our data suggest that the M. ambigua demography can be traced to changes in area covered by semi-natural grasslands throughout the Holocene.

Low functional diversity promotes niche changes in natural island pollinator communities.

Functional diversity loss among pollinators has rapidly progressed across the globe and is expected to influence plant-pollinator interactions in natural communities. Although recent findings suggest that the disappearance of a certain pollinator functional group may cause niche expansions and/or shifts in other groups, no study has examined this prediction in natural communities with high plant and pollinator diversities. By comparing coastal pollination networks on continental and oceanic islands, we examined how community-level flower visit patterns are influenced by the relative biomass of long-tongued pollinators (RBLP). We found that RBLP significantly correlated with pollinator functional diversity and was lower in oceanic than in continental islands. Pollinator niches shifted with decreasing RBLP, such that diverse species with various proboscis lengths, especially short-tongued species, increasingly visited long-tubed flowers. However, we found no conspicuous negative impacts of low RBLP and the consequent niche shifts on pollinator visit frequencies to flowers in oceanic island communities. Notably, fruit set significantly decreased as RBLP decreased in a study plant species. These results suggest that niche shifts by other functional groups can generally compensate for a decline in long-tongued pollinators in natural communities, but there may be negative impacts on plant reproduction.

Sexual dimorphism in floral longevity and flowering synchrony in relation to pollination and mating success in three dioecious Ilex species.

UNLABELLED: • PREMISE OF THE STUDY: Sexual floral dimorphism may have evolved under pollinator-mediated natural selection by which female and male functions are differently influenced. We hypothesized that mating success is differently influenced by display size between sexes, under which sexual dimorphism in flowering schedule has evolved in dioecious species.• METHODS: We examined sexual dimorphism in the total number of flowers per shoot, maximum daily display size, longevity of individual flowers, and flowering synchrony (maximum proportion of open flowers) in three dioecious Ilex species (I. pedunculosa, I. serrata, and I. crenata) whose female flowers are not smaller than male flowers. We compared pollinator response to natural variation of daily display size between sexes. We also examined the effect of display size on female and male success (fruit set and pollen deposition and removal).• KEY RESULTS: In the three species, male shoots produced significantly more flowers than female shoots did. Although female flowers lasted longer and opened more synchronously than male flowers, maximum daily display size was larger on male shoots than on female shoots. Fruit set was significantly pollen-limited in the field in all species. Pollen deposition and/or fruit set increased with female display size, whereas pollen removal decreased or was approximately constant with male display size in the three species.• CONCLUSIONS: We suggest that sexual dimorphism in floral longevity and flowering synchrony might enhance both female and male success in relation to the display size-mating success (pollinator attraction) association irrespective of flower size dimorphism.

Low flower-size variation in bilaterally symmetrical flowers: Support for the pollination precision hypothesis.

PREMISE OF THE STUDY: The evolutionary shift from radial to bilateral symmetry in flowers is generally associated with the evolution of low flower-size variation. This phenomenon supports the hypothesis that the lower size variation in bilateral flowers can be attributed to low pollinator diversity. In this study, we propose two other hypotheses to explain low flower-size variation in bilateral symmetrical flowers. To test the three hypotheses, we examined the relative importance of pollinator diversity, composition, and bilateral symmetry itself as selective forces on low flower-size variation. METHODS: We examined pollinator diversity and composition and flower-size variation for 36 species in a seminatural ecosystem with high bee richness and frequent lepidopteran visitation. KEY RESULTS: Bilateral flowers were more frequently visited than radial flowers by larger bees, but functional-group diversity of the pollinators did not differ between symmetry types. Although bilateral flowers had significantly lower flower-size variation than radial flowers, flower-size variation did not vary with pollinator diversity and composition but was instead related to bilateral symmetry. CONCLUSIONS: Our results suggest that the lower size variation in bilateral flowers might have evolved under selection favoring the control of pollinator behavior on flowers to enhance the accurate placement of pollen on the body of the pollinator, independent of pollinator type. Because of the limited research on this issue, future work should be conducted in various types of plant-pollinator communities worldwide to further clarify the issue.

Does urbanization promote floral diversification? Implications from changes in herkogamy with pollinator availability in an urban-rural area.

Although land-use changes such as urbanization have dramatically altered plant-pollinator interactions, little is known about their effects on pollen limitation and floral traits. In this study, we examined pollinator visit frequency, reproductive success, and floral trait measurements in 12 populations of the annual andromonoecious Commelina communis in an urban-rural area. Pollinator and mate availability decreased significantly with developed land area around the study site. Most urbanized populations suffered from significant pollinator-limited male and/or female reproductive success. High fruit set in urbanized populations may suggest the presence of high reproductive assurance by selfing. The stigma height and degree of herkogamy significantly decreased with increased pollinator limitation. Petal length, anther height, and/or the pollen:ovule ratio tended to be low in pollinator- and mate-limited urban populations. One urban population with high pollinator availability had flowers with higher herkogamy and stigma height compared to rural populations. These results suggest that urbanization may provide diverse selective forces that could affect the phenotypic variation in floral traits.

Habitat diversification associated with urban development has a little effect on genetic structure in the annual native plant Commelina communis in an East Asian megacity.

Urban development greatly alters the natural and semi-natural habitats of native plants. Urbanisation results in a range of diverse habitats including remnant agricultural lands, urban parks, and roadside habitats. This habitat diversity often promotes trait divergence within urban areas. However, the mechanisms by which diverse urban habitats influence the population genetic structure of individual plant species remain poorly understood. We investigated the effects of urbanisation on genetic diversity and structure within 24 Commelina communis populations across diverse habitat types (rural agricultural land, urban agricultural land, urban park land, and urban roadsides) within the Kyoto-Osaka-Kobe megacity in Japan. We conducted multiplexed inter-simple sequence repeat genotyping to compare genetic diversity among populations in different habitats. We also examined the correlation between Nei's genetic distance and geographic and environmental distances and performed principal coordinate analysis (PCoA) to evaluate genetic differentiation among urban habitats. There were no significant differences in genetic diversity indices between urban and rural populations and among urban habitat types. Although we detected no isolation-by-distance structure in population pairs of the same habitat type and in those of different habitats, the difference in surrounding landscape facilitated genetic differentiation not only between urban and rural habitats but also between different urban habitats. PCoA revealed no clear genetic differentiation among rural and urban habitat populations. Our findings indicate that the establishment of diverse habitat types through urbanisation has no and little impact on genetic diversity and structure, respectively, in C. communis, likely due to its high selfing rate and ability to adapt to urban conditions.

Seed dispersal limitation causes negative legacy effect on restoration of grassland plant diversity on ski slopes.

Past forest use often has a long-term negative impact on the recovery of the original plant composition of semi-natural grasslands, which is known as a legacy effect. This study investigates the impact of seed dispersal limitations on the restoration of grassland plant diversity on ski slopes with past forest use, highlighting the negative legacy effect on biodiversity recovery. Focusing on ski areas, our research contrasts the vegetation on ski slopes originally created on semi-natural grasslands such as pasture (pasture slopes) and constructed by clearing secondary forests or conifer plantations (forest slopes). We examined species richness and diversity, considering seed dispersal modes, grassland management history, and seed source proximity. We reveal that the proximity to species-rich grassland sources is pivotal for the restoration of native grassland vegetation. Particularly, wind-dispersed species show significant recovery on slopes with sustained management for more than 70 years and those with neighboring species-rich grasslands, suggesting that both the duration of management and the proximity to seed sources are critical for overcoming the legacy effects of past forest use. Meanwhile, gravity-dispersed species failed to recover their richness and diversity regardless of both the duration of management and the proximity to seed source grasslands, which their diversity recovered where seed sources neighbored. Our findings emphasize the importance of considering seed dispersal limitation and management history in the restoration and conservation of grasslands and their biodiversity, particularly in landscapes experiencing past human intervention.

Topography- and management-mediated resource gradients maintain rare and common plant diversity around paddy terraces.

Examining the causes of interspecific differences in susceptibility to bidirectional land-use changes (land abandonment and use-intensification) is important for understanding the mechanisms of global biodiversity loss in agricultural landscapes. We tested the hypothesis that rare (endangered) plant species prefer wet and oligotrophic areas within topography- and management-mediated resource (soil water content, nutrient, and aboveground biomass) gradients, making them more susceptible to both abandonment and use-intensification of agricultural lands. We demonstrated that topography and management practices generated resource gradients in seminatural grasslands around traditional paddy terraces. Terraced topography and management practices produced a soil moisture gradient within levees and a nutrient gradient within paddy terraces. Both total and rare species diversity increased with soil water content. Total species diversity increased in more eutrophied areas with low aboveground biomass, whereas rare species diversity was high under oligotrophic conditions. Rare and common species were differentially distributed along the human-induced nutrient gradient, with rare species preferring wet, nutrient-poor environments in the agricultural landscapes studied. We suggest that conservation efforts should concentrate on wet, nutrient-poor areas within such landscapes, which can be located easily using land-use and topography maps. This strategy would reduce the costs of finding and conserving rare grassland species in a given agricultural landscape.

Inflorescence architecture affects pollinator behaviour and mating success in Spiranthes sinensis (Orchidaceae).

• Despite the wide inflorescence diversity among angiosperms, the effects of inflorescence architecture (three-dimensional flower arrangement) on pollinator behaviour and mating success have not been sufficiently studied in natural plant populations. • Here, we investigated how inflorescence architecture affected inter- and intra-plant pollinator movements and consequent mating success in a field population of Spiranthes sinensis var. amoena (S. sinensis). In this species, the flowers are helically arranged around the stem, and the degree of twisting varies greatly among individuals. The large variation in inflorescence architecture in S. sinensis results from variation in a single structural parameter, the helical angle (the angular distance between neighbour-flower directions). • The numbers of visits per inflorescence and successive probes per visit by leaf-cutting bees decreased with helical angle, indicating that individual flowers of tightly twisted inflorescences received less visitations. As expected from pollinator behaviour, pollinia removal and fruit set of individual flowers decreased with helical angle. Meanwhile, geitonogamy decreased in tightly twisted inflorescences. • Our novel findings demonstrate that natural variation in inflorescence architecture significantly affects pollinator behaviour and reproductive success, suggesting that inflorescence architecture can evolve under pollinator-mediated natural selection in plant populations. We also discuss how diverse inflorescence architectures may have been maintained in S. sinensis populations.

Evaluation of sampling effort required to assess pollen species richness on pollinators using rarefaction.

PREMISE: Understanding the flower visitation history of individual pollinators is key in the study of pollination networks, but direct tracking is labor intensive and, more important, does not capture information about the previous interactions of an individual. Therefore, a protocol to detect most of the pollen species on the body surfaces of an individual pollinator could elucidate its flower visitation history. METHODS AND RESULTS: Under a microscope, we observed 6.0-µL droplets from a sample solution (1.0 or 3.0 mL) containing pollen grains collected from individuals of six major pollinator functional groups. To clarify how many droplets need to be observed to detect all pollen species within the solution, we examined up to 10 droplets collected from each individual insect. Sample-based rarefaction curve analyses of the data showed that we could detect ~90% of the pollen species and the plant-pollinator links in the networks by observing six droplets. CONCLUSIONS: The rarefaction curve analysis for pollen-on-pollinator studies is a useful preliminary step for minimizing the time and labor required while maximizing the data on the flower visitation history of each individual pollinator and revealing any hidden flower-pollinator interactions.

Corrigendum: Development of microsatellite markers for the annual andromonoecious herb Commelina communis f. ciliata (Commelinaceae) [Genes Genet. Syst. (2019) 94, p. 133-138].

Table 1 on p. 135 should be replaced with the corrected Table 1 shown bellow.

Flower orientation enhances pollen transfer in bilaterally symmetrical flowers.

Zygomorphic flowers are usually more complex than actinomorphic flowers and are more likely to be visited by specialized pollinators. Complex zygomorphic flowers tend to be oriented horizontally. It is hypothesized that a horizontal flower orientation ensures effective pollen transfer by facilitating pollinator recognition (the recognition-facilitation hypothesis) and/or pollinator landing (the landing-control hypothesis). To examine these two hypotheses, we altered the angle of Commelina communis flowers and examined the efficiency of pollen transfer, as well as the behavior of their visitors. We exposed unmanipulated (horizontal-), upward-, and downward-facing flowers to syrphid flies (mostly Episyrphus balteatus), which are natural visitors to C. communis. The frequency of pollinator approaches and landings, as well as the amount of pollen deposited by E. balteatus, decreased for the downward-facing flowers, supporting both hypotheses. The upward-facing flowers received the same numbers of approaches and landings as the unmanipulated flowers, but experienced more illegitimate landings. In addition, the visitors failed to touch the stigmas or anthers on the upward-facing flowers, leading to reduced pollen export and receipt, and supporting the landing-control hypothesis. Collectively, our data suggested that the horizontal orientation of zygomorphic flowers enhances pollen transfer by both facilitating pollinator recognition and controlling pollinator landing position. These findings suggest that zygomorphic flowers which deviate from a horizontal orientation may have lower fitness because of decreased pollen transfer.

Development of microsatellite markers for the annual andromonoecious herb Commelina communis f. ciliata (Commelinaceae).

Commelina communis f. ciliata (Commelinaceae), a newly distinguished taxon, is an annual andromonoecious herb exhibiting a mixed mating system, the details of which remain unclear. We developed microsatellite markers for use in exploring the evolution of andromonoecy and mixed mating in the species. Fifteen microsatellite loci were developed using next-generation sequencing. The primer sets were used to evaluate 65 C. communis f. ciliata individuals from three populations in Japan; we found 1-13 alleles per locus and the expected heterozygosity ranged from 0.00 to 0.76. The markers are potentially useful to examine intra- and interspecies genetic structure and the mixed mating strategy of Commelina species via paternity analysis.