Yu Matsuki

The Kuroshio Current influences genetic diversity and population genetic structure of a tropical seagrass, Enhalus acoroides

Information on genetic diversity and differentiation of seagrass populations is essential for the conservation of coastal ecosystems. However, little is known about the seagrasses in the Indo‐West Pacific Ocean, where the worlds highest diversity of seagrasses occurs. The influence of sea currents on these populations is also unknown. We estimated the genetic diversity and population genetic structure and identified reproductive features in Enhalus acoroides populations from the Yaeyama Islands, Hainan Island and the Philippines. The Philippines are situated at the centre of the E. acoroides range, Yaeyama and Hainan are peripheral populations, and the Yaeyama population is at the northern limit of the species range. The powerful Kuroshio Current flows from the Philippines to Yaeyama. Genetic analyses using nine microsatellite markers indicated that reproduction of E. acoroides is mostly sexual. Clonal diversity does not decrease in northern populations, although genetic diversity does. However, the genetic diversity of the Yaeyama populations is greater than that of the Hainan populations. Significant genetic differentiation among most populations was evident; however, the Yaeyama and north‐east Philippines populations were genetically similar, despite being separated by ~1100 km. An assignment test suggested that recruitment occurs from the north‐east Philippines to Yaeyama. The strong current in this region is probably responsible for the extant genetic diversity and recruitment patterns.

Genetic diversity and structure of the tropical seagrass Cymodocea serrulata spanning its central diversity hotspot and range edge

Abstract Persistence of populations at their distributional ranges relies on local population dynamics and the fitness of species with low dispersal potential. We analyzed the population genetic diversity and structure of a tropical seagrass species, Cymodocea serrulata, at 34 sites spanning Philippine (diversity hotspot) and Ryukyu Islands (northern limit of distribution) populations using microsatellite (SSR) markers. Seagrass populations in the diversity hotspot are hypothesized to contain higher genetic diversity and clonal richness than those resulting from expansion or geographic range limits. We tested this hypothesis by comparing the genetic diversity, genetic structure and clonal richness of C. serrulata populations in the Philippines and Ryukyu Islands. C. serrulata populations showed decreased genetic diversity and clonal richness at their northern limit. Clonal reproduction predominated at the northern limit, while sexual reproduction prevailed in the diversity hotspot. Decreased genetic diversity and clonal richness at the northern limit may be the consequence of drift resulting from founder effect, reduced habitat, sea surface temperature and low gene flow and/or natural selection across life stages, wherein clonal reproduction confers greater environmental fitness. Analysis of molecular variance (AMOVA) and the fixation index, FST, showed significant genetic differentiation within and among geographic populations. STRUCTURE analysis revealed that the Ryukyu Islands populations were mosaics of genets from the eastern Philippines, likely carried by the Kuroshio Current.

Development of microsatellite markers in a tropical seagrass Syringodium isoetifolium (Cymodoceaceae)

Nine microsatellite markers of a tropical seagrass species, Syringodium isoetifolium, were developed to investigate genetic diversity and genetic connectivity. The number of alleles detected per locus ranged from two to 16, and the observed and expected heterozygosities ranged from 0.00 to 1.00 and 0.12 to 0.91, respectively. The markers described here are sufficiently polymorphic and informative to investigate the genetic diversity, genetic connectivity and genetic structure of S. isoetifolium.

Development of novel microsatellite markers in a tropical seagrass, Enhalus acoroides

Using the dual-suppression polymerase chain reaction technique, 12 novel polymorphic nuclear microsatellite DNA markers were isolated from the tropical seagrass Enhalus acoroides. The isolated markers provided the polymorphisms of 2–13 alleles per locus in 3 populations located in Japan, China, and the Philippines. The values of the expected heterozygosity fell in the following ranges: 0.000–0.693 (Japan), 0.000–0.623 (China), and 0.000–0.765 (Philippines). Compared to the Philippines population (1 monomorphic locus), a relatively higher proportion of the SSR loci did not show polymorphism in the marginal habitats of E. acoroides (5 loci in Japan; 5 loci in China). These markers may be useful for population and conservation genetics of E. acoroides.

Isolation and characterization of novel microsatellite markers for Cymodocea serrulata (Cymodoceaceae), a seagrass distributed widely in the Indo-Pacific region

Cymodocea serrulata is a tropical seagrass species distributed widely in the Indo-Pacific region. We developed 16 novel microsatellite (simple sequence repeat) markers for C. serrulata using next-generation sequencing for use in genetic studies. The applicability of these markers was attested by genotyping of 40 individuals collected from a natural population in the Philippines. Of the 16 loci, 15 showed polymorphism. For the 15 polymorphic markers, the number of alleles per locus ranged from two to seven, and the observed and expected heterozygosities ranged from 0.131–1.000 and 0.124–0.788, respectively. These markers are useful tools for elucidating genetic diversity, connectivity, and structure in this foundational coastal species.

Development of novel microsatellite markers for Cymodocea rotundata Ehrenberg (Cymodoceaceae), a pioneer seagrass species widely distributed in the Indo-Pacific

Genetic diversity and population genetic structure are key components of seagrass resilience and contribute to an understanding of its conservation and management. We isolated 29 polymorphic microsatellite (SSR) markers from a widely distributed pioneer seagrass, Cymodocea rotundata, by two methods; next generation sequencing and compound SSR marker isolation. Twenty-nine markers had 2–14 alleles per locus, and the observed and expected heterozygosity ranged from 0 to 0.688, and 0.113 to 0.770, respectively. These loci will facilitate investigation of the genetic diversity and population genetic connectivity and structure of C. rotundata.

Development of microsatellite markers for Thalassia hemprichii (Hydrocharitaceae), a widely distributed seagrass in the Indo-Pacific

Nine microsatellite markers of a widely-distributed seagrass, Thalassia hemprichii, were developed to investigate genetic diversity and genetic connectivity. The number of alleles detected per locus ranged from 3 to 26, and the expected and observed heterozygosities ranged from 0.00 to 1.00 and 0.00 to 0.87, respectively. The markers described here are sufficiently polymorphic and informative to investigate the genetic diversity, genetic connectivity and genetic structure of T. hemprichii.

Pollination Efficiencies of Insects Visiting Magnolia obovata, as Determined by Single-Pollen Genotyping

The genetic composition of pollen grains that are transported to flowers affects the reproductive success and fitness of the plants and the genetic structure of the plant population. For example, for plants that suffer inbreeding depression, the pollen from another conspecific individual is essential for effective reproduction. Many researchers have focused on the pollination processes in various plants and have sought to understand relationships between plants and pollinators. Here, we show the latest approach, which evaluates the contribution of flower-visiting insects by direct genotyping of pollen grains. The genotypes of pollen grains adhering to flower beetles, small beetles, and bumblebees that visited flowers of Magnolia obovata were determined directly. The genetic traits of transported pollen differed by insect type. Most of the pollen adhering to small beetles and bumblebees was self-pollen (pollen transported to a different region on the same tree). On the other hand, an average of 70% of pollen grains adhering to flower beetles was transported from other reproductive trees. We also described the patterns of pollen movements by performing a paternity analysis on pollen grains using trees in our study populations as candidate male parents. Although most of the pollen that adhered to small beetles and bumblebees was moved within a short range of distance, pollen grains that adhered to flower beetles tended to travel longer distances. Our results showed that small beetles and bumblebees rarely move between plants, cause geitonogamous pollen flow, and may negatively affect reproduction in M. obovata, which undergoes substantial inbreeding depression in the early life stages. In contrast, flower beetles contribute to the outcrossing of M. obovata, transporting genetically diverse outcross pollen. Our evaluation is consistent with the traditional idea that the flowers of Magnoliaceae have features of a beetle pollination syndrome. The direct genotyping of pollen grains provides powerful evidence for the traditional idea of pollination syndrome and offers new insights, shedding light on the mutualism and coevolution of plants and flower visitors.

Development of 10 novel polymorphic microsatellite markers for the Indo-Pacific horned starfish, Protoreaster nodosus

Ten novel polymorphic nuclear microsatellite simple sequence repeat (SSR) markers were isolated from an Indo-Pacific horned starfish, Protoreaster nodosus. The isolated SSR markers provided polymorphisms of 2-9 alleles per locus in three populations obtained from Indonesia and the Philippines. The expected and observed heterozygosities ranged from 0.049 to 0.691 and from 0.050 to 0.800, respectively. Pairwise FST values among three populations ranged from 0.018 to 0.050 (global FST=0.031). All P. nodosus individuals collected from three populations exhibited different genotypes for the ten identified SSR markers, indicating that P. nodosus reproduces sexually. Then the developed SSR markers will be useful for studying the population and conservation genetics of P. nodosus.

Development of 11 microsatellite markers in Pinus parviflora by the dual-suppression technique and next-generation sequencing

Pinus parviflora Sieb. et Zucc. var. parviflora is a coniferous tree species distributed in mountainous areas of temperate zones from southern Tohoku to the Kyushu area in Japan. Some P. parviflora populations have become small and endangered due to massive death resulting from pine wilt disease, scab canker, and presumably climate change. We developed 11 microsatellite markers for P. parviflora by the dual-suppression technique and next-generation sequencing using 32 individuals of P. parviflora collected from Aokigahara at the foot of Mt. Fuji. The number of alleles for each locus ranged from two to 10. The averages of observed and expected heterozygosities were 0.61 and 0.59, respectively. These markers will become powerful tools for assessing genetic diversity, genetic connectivity, and genetic structure in P. parviflora populations, which will facilitate our understanding and conservation of P. parviflora.