Hiroyuki Kurokochi

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 16 microsatellite markers in Eusideroxylon zwageri by next-generation sequencing

The Ulin, Eusideroxylon zwageri, is a useful tropical tree now threatened by excessive logging. We used next-generation sequencing to isolate and characterize 16 polymorphic and codominant microsatellite (simple sequence repeat) markers in 32 E. zwageri trees in east Kalimantan, Indonesia. The number of alleles for each locus ranged from 2 to 19 and observed and expected heterozygosities ranged from 0.13 to 0.91 and from 0.18 to 0.92, respectively. These microsatellites may be useful for genetic studies and conservation of E. zwageri.

Transcriptional responses of a bicarbonate-tolerant monocot, Puccinellia tenuiflora, and a related bicarbonate-sensitive species, Poa annua, to NaHCO3 stress.

Puccinellia tenuiflora is an alkaline salt-tolerant monocot found in saline-alkali soil in China. To identify the genes which are determining the higher tolerance of P. tenuiflora compared to bicarbonate sensitive species, we examined the responses of P. tenuiflora and a related bicarbonate-sensitive Poeae plant, Poa annua, to two days of 20 mM NaHCO3 stress by RNA-seq analysis. We obtained 28 and 38 million reads for P. tenuiflora and P. annua, respectively. For each species, the reads of both unstressed and stressed samples were combined for de novo assembly of contigs. We obtained 77,329 contigs for P. tenuiflora and 115,335 contigs for P. annua. NaHCO3 stress resulted in greater than two-fold absolute expression value changes in 157 of the P. tenuiflora contigs and 1090 of P. annua contigs. Homologs of the genes involved in Fe acquisition, which are important for the survival of plants under alkaline stress, were up-regulated in P. tenuiflora and down-regulated in P. annua. The smaller number of the genes differentially regulated in P. tenuiflora suggests that the genes regulating bicarbonate tolerance are constitutively expressed in P. tenuiflora.

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 18 microsatellite markers in Pieris japonica, a poisonous tree insulated from the browsing pressure of herbivores, using a next-generation sequencer

Pieris japonica is a poisonous tree species that is rarely eaten by herbivorous animals, which could allow it to expand its distribution and change ecosystems. Using a next-generation sequencer, 18 microsatellite markers were isolated from P. japonica and characterized. The number of alleles at each locus ranged from 2 to 11. The observed and expected heterozygosities ranged from 0.13 to 1.00 and 0.32 to 0.88, respectively. These markers will be useful for genetic studies of P. japonica, which will be essential for conservation of its surrounding environment.

Fifteen novel microsatellite markers for two Amphiprion species (Amphiprion frenatus and Amphiprion perideraion) and cross-species amplification

Abstract Anemonefishes are popular model species for research on population connectivity via larval dispersal, and understanding this connectivity is beneficial when designing marine protected areas. We developed 15 microsatellite markers for two anemonefishes, Amphiprion frenatus and Amphiprion perideraion. Of these 15 markers, 10 worked well for both species, while three were specific for A. frenatus and two were specific for A. perideraion. For A. frenatus, the number of alleles at each locus ranged from 4 to 28, and the observed and expected heterozygosities ranged from 0.258 to 0.938 and from 0.285 to 0.953, respectively. For A. perideraion, the respective numbers were 4–14, 0.344–0.969, and 0.412–0.868. These microsatellite markers will be useful for the study of population connectivity in these two species.

Development of 12 microsatellite markers in Euptelea polyandra by a random tailed genome-walking method using Phi29 DNA polymerase

Twelve microsatellite (simple sequence repeat, SSR) markers were isolated from Euptelea polyandra, a Japanese endemic deciduous tree species, based on a random tailed genome-walking method using Phi29 DNA polymerase. There were 2 to 11 alleles per locus, and the observed and expected heterozygosities ranged from 0.05 to 0.90 and from 0.233 to 0.883, respectively.

Development of 13 polymorphic chloroplast DNA markers in Quercus gilva, a regionally endemic species in Japan

Quercus gilva is an evergreen oak species which is native to Japan, continental China and the islands of Taiwan. However, because of historical logging practices and habitat destruction in ancient times, Q. gilva is threatened with extinction in several regions of Japan. In this study, 13 polymorphic markers spanning the whole chloroplast DNA of Q. gilva were developed, using next-generation sequencing technology. These markers could distinguish between two individuals established in close proximity to one another as well as those from geographically-distinct regions. This indicates that the markers will be suitable for detecting the genetic structure and phylogeography of Q. gilva on both a large and a fine scale, potentially providing us with useful information for the conservation of this species.

Local-Level Genetic Diversity and Structure of Matsutake Mushroom (Tricholoma matsutake) Populations in Nagano Prefecture, Japan, Revealed by 15 Microsatellite Markers

The annual yield of matsutake mushrooms (Tricholoma matsutake) has consistently decreased in Japan over the past few decades. We used 15 polymorphic and codominant simple sequence repeat (SSR) markers, developed using next-generation sequencing, to carry out genetic analyses of 10 populations in Nagano, Japan. Using the SSRs, we identified 223 genotypes, none of which was observed in more than one population. The mean expected heterozygosity and standardized allelic richness values were 0.67 and 4.05, respectively. Many alleles appeared in only one of the 10 populations; 34 of these private alleles were detected with a mean number per population of 3.4. The fixation index (FST) and standardized genetic differentiation (G′ST) values were 0.019 and 0.028, respectively. Analysis of molecular variance (AMOVA) showed that the contribution of among population, among genets within a population, and within genets variation to the total variation was 2.91%, 11.62%, and 85.47%, respectively, with genetic differentiation being detected for all sources. Twenty-eight of 45 pairwise FST values were significantly larger than zero, and no pattern of isolation by distance was detected among the 10 populations. Bayesian-based clustering did not show clear differences among populations. These results suggest that reestablishment of a colony would be best accomplished by transplantation within a field; if this is not possible, then transplantation from within several dozen kilometers will cause little damage to the original population genetic structure.

Marine protected area restricts demographic connectivity: Dissimilarity in a marine environment can function as a biological barrier

Abstract The establishment of marine protected areas (MPAs) can often lead to environmental differences between MPAs and fishing zones. To determine the effects on marine dispersal of environmental dissimilarity between an MPA and fishing zone, we examined the abundance and recruitment patterns of two anemonefishes (Amphiprion frenatus and A. perideraion) that inhabit sea anemones in different management zones (i.e., an MPA and two fishing zones) by performing a field survey and a genetic parentage analysis. We found lower levels of abundance per anemone in the MPA compared to the fishing zones for both species (n = 1,525 anemones, p = .032). The parentage analysis also showed that lower numbers of fishes were recruited from the fishing zones and outside of the study area into each anemone in the MPA than into each anemone in the fishing zones (n = 1,525 anemones, p < .017). However, the number of self‐recruit production per female did not differ between the MPA and fishing zones (n = 384 females, p = .516). Because the ocean currents around the study site were unlikely to cause a lower settlement intensity of larvae in the MPA, the ocean circulation was not considered crucial to the observed abundance and recruitment patterns. Instead, stronger top‐down control and/or a lower density of host anemones in the MPA were potential factors for such patterns. Our results highlight the importance of dissimilarity in a marine environment as a factor that affects connectivity.