Tadashi Yamashiro

Species identification based on the faecal DNA samples of the Japanese serow (Capricornis crispus)

The Japanese serow (Capricornis crispus) is endemic to mountain regions in Japan. Due to the difficulty of directly observing Japanese serows, traces such as faeces have been utilized to study their distribution and estimate their population size. However, the recent population increase of sika deer (Cervus nippon) often leads misidentification of sika deer faecal pellets as those of Japanese serows. Therefore, we developed species identification methods to differentiate between the Japanese serow and sika deer based on mtDNA cytchrome b gene sequences, and applied the method to faecal samples collected for a survey program on the Japanese serow. In 67 field-collected faecal samples examined, we could amplify mtDNA at relatively high PCR succession rates (86.6%). Species identification based on PCR–RFLPs using the cytochrome b gene revealed that 41.4% of faecal pellets of sika deer were misidentified as being from Japanese serow. This suggests that the present population size of the Japanese serow may be overestimated. This species identification method eliminates such misidentifications and should become a powerful tool for studying Japanese serow, including their distribution, precise population size, and estimations of density.

Was Jeju Island a glacial refugium for East Asian warm-temperate plants? Insights from the homosporous fern Selliguea hastata (Polypodiaceae)

PREMISE OF THE STUDY We posed two hypotheses for broad scenarios of postglacial recolonization of Korea by the warm-temperate vegetation: (1) that extant Korean populations are derived from a single refugium, or (2) that they are derived from multiple refugia. We chose a homosporous fern typical of East Asian warm-temperate vegetation, Selliguea hastata, to test which of the two scenarios is more likely and to check whether Japan contained putative glacial refugia. METHODS Using 16 allozyme loci, we obtained genotypes of 756 individuals from 20 populations, representative of the whole distribution area in Korea (including Jeju Island), Japan, and Taiwan. We assessed genetic variability within and among populations, Wrights F-statistics, and conducted analysis of molecular variance, model-based Bayesian clustering, and bottleneck tests. KEY RESULTS We found no allozyme variation within populations of S. hastata in mainland Korea, whereas genetic polymorphism was detected for populations from Jeju Island, Japan (in particular a population from southeastern Shikoku), and Taiwan. The levels of inbreeding within populations were high, consistent with the potential of S. hastata for intragametophytic selfing. CONCLUSIONS Data on allelic richness together with Bayesian clustering methods suggest a pattern of postglacial recolonization of mainland Korea from a single refugium, probably located either on Jeju Island or in Japan. Jeju Island should merit the highest priority for conservation biogeography, as it played a role as a Quaternary refugium for arctic-alpine, boreal, temperate as well as warm-temperate plants, as suggested here.

Intraspecific morphological and genetic differentiation in Scrophularia grayana (Scrophulariaceae)

Scrophularia grayana, which is distributed throughout northern Japan and Sakhalin, and its locally endemic variety var. grayanoides, have been examined morphometrically and genetically. Principal-component analysis using a total of 26 morphological characteristics revealed that these taxa are morphologically differentiated, but that the difference is not distinct. These two taxa have the same number of chromosomes in the somatic cells, 2n = 94, suggesting that ploidal level difference is not relevant to their divergence. The distributions of the taxa are adjoining in the north of Japanese mainland Honshu. Nevertheless, principal-coordinate analysis using putative 112 ISSR loci indicated they are genetically very distinct. Many taxon-specific alleles were found, and many of the alleles were fixed in each taxon. This genetic information suggests that a relatively long time has passed since the taxa became differentiated and that gene flow has rarely occurred between them, although morphological similarity has been maintained, probably because of natural selective forces.

A comparative study of the reproductive character and genetic diversity of an autogamous Tylophora matsumurae and its progenitor Tylophora tanakae (Apocynaceae-Asclepiadoideae)

Reproductive character and genetic diversity were investigated for an autogamous species of Tylophora matsumurae endemic to the Ryukyu Islands and its progenitor species T. tanakae. Approximately one-fourth of bagged flowers set fruits in T. matsumurae, although no fruits were obtained from bagged flowers in T. tanakae. In situ pollen tube germination was observed with high frequency in all five populations examined in T. matsumurae. Furthermore, in T. matsumurae, anther sacs were not dehisced even at anthesis. These results suggest the highly autogamous nature of T. matsumurae. No allozymic variation was detected in all seven populations examined in T. matsumurae. The phenogram constructed using the neighbor-joining method based on Neis unbiased genetic distance indicated that T. matsumurae clustered with the Okinoerabu Island population of T. tanakae. The estimated outcrossing rate of four populations of T. tanakae varied from 0.18 to 0.59. It is probable that T. matsumurae had been derived from the predominantly self-pollinating population of T. tanakae, and rapidly enlarged its distribution area.

Utilization on extrafloral nectaries and fruit domatia of Canavalia lineata and C. cathartica (Leguminosae) by ants

Utilization of the extrafloral nectaries (EFNs) and fruits of Canavalia lineata and C. cathartica by ants was investigated at 30 sites in Japan. The fruits of C. lineata and C. cathartica were inhabited by five and eight ant species, respectively. Ant nesting periods and their utilization of EFNs differed between C. lineata and C. cathartica. Canavalia lineata flowers once a year, and periods of EFN-utilization and fruit-nesting by ants do not overlap. The fruit-nesting ants on C. lineata seem to invade the plant from the holes made by moth larvae or breaches made by decay. The ants nesting on the fruits of C. lineata may defend the plant against seed herbivores because they feed on moth larvae. Canavalia cathartica flowers several times over a year, and fruits are found throughout the year; therefore, periods of EFN-utilization and fruit-nesting by ants are overlapped. Canavalia cathartica offers year-round nesting sites and food for ants, and therefore may receive a higher defensive effect from ants than C. lineata.

Long-term persisting hybrid swarm and geographic difference in hybridization pattern: genetic consequences of secondary contact between two Vincetoxicum species (Apocynaceae–Asclepiadoideae)

BackgroundDuring glacial periods, glacial advances caused temperate plant extirpation or retreat into localized warmer areas, and subsequent postglacial glacial retreats resulted in range expansions, which facilitated secondary contact of previously allopatric isolated lineages. The evolutionary outcomes of secondary contact, including hybrid zones, dynamic hybrid swarm, and resultant hybrid speciation, depends on the strengths of reproductive barriers that have arisen through epistatic and pleiotropic effects during allopatric isolation. The aim of this study was to demonstrate refugia isolation and subsequent secondary contact between two perennial Asclepioid species and to assess the genetic consequences of the secondary contact. We modeled the range shift of two ecologically distinct Vincetoxicum species using the species distribution model (SDM) and assessed the genetic consequences of secondary contact by combining morphological and genetic approaches. We performed morphometric analysis (592 individuals) and examined 10 nuclear microsatellites (671 individuals) in V. atratum, V. japonicum, and putative hybrid populations.ResultsMultivariate analysis, model-based Bayesian analysis, and non-model-based discriminant analysis of principal components confirmed the hybridization between V. atratum and V. japonicum. High pollen fertility and a lack of linkage disequilibrium suggested that the hybrid populations may be self-sustaining and have persisted since V. atratum and V. japonicum came into contact during the post-glacial period. Moreover, our findings show that the pattern of hybridization between V. atratum and V. japonicum is unidirectional and differs among populations. Geographically-isolated hybrid populations exist as genetically distinct hybrid swarms that consist of V. atratum-like genotypes, V. japonicum-like genotypes, or admixed genotypes. In addition, Bayesian-based clustering analysis and coalescent-based estimates of long-term gene flow showed patterns of introgressive hybridization in three morphologically ‘pure’ V. japonicum populations.ConclusionIn this study, we demonstrated that climatic oscillations during the Quaternary period likely led to species range shift and subsequently secondary contact. Hybrid populations may be self-sustaining and have persisted since V. atratum and V. japonicum came into contact during the post-glacial period. Pattern of hybridization between V. atratum and V. japonicum is unidirectional and differs among populations. We concluded that these differences in the genetic consequences of secondary contact are caused by historical colonization processes and/or natural selection.

Development of microsatellite markers for the endangered grassland perennial herb Vincetoxicum atratum (Apocynaceae–Asclepiadoideae)

Eight microsatellite markers were developed for the endangered grassland perennial herb Vincetoxicum atratum. The number of alleles ranged from 4 to 14, and the expected heterozygosities were from 0.575 to 0.933 in a population of V. atratum. Five of the eight loci did not significantly deviated from the Hardy–Weinberg equilibrium. All eight loci were tested for cross-species amplification in five other species of Vincetoxicum in Japan. These microsatellite loci will be useful for conservation genetics of V. atratum and other species of Vincetoxicum.

Isolation and Characterization of Microsatellite Markers for Canavalia cathartica and C. lineata (Fabaceae)

Premise of the study: Microsatellite markers were developed for the sea-dispersed legume Canavalia cathartica to investigate its genetic diversity and population structure. We also applied these microsatellite markers to the closely related species C. lineata. Methods and Results: Ten primer sets were developed for C. cathartica, and all of the primers were amplified in C. lineata. The number of alleles per locus ranged from two to 13 and one to 10 for C. cathartica and C. lineata, respectively. The expected heterozygosity ranged from 0.375 to 0.870 and from 0.071 to 0.877 for C. cathartica and C. lineata, respectively. Conclusions: The 10 microsatellite markers developed in this study can be used to analyze the population genetic structure of C. cathartica and other related taxa.

Genetic Diversity and Divergence in Populations of the Threatened Grassland Perennial Vincetoxicum atratum (Apocynaceae-Asclepiadoideae) in Japan

We examined the genetic diversity and structure in populations of the endangered grassland herb Vincetoxicum atratum using 11 polymorphic microsatellite loci. Although the populations were small and disconnected, our molecular data indicated that the species maintains relatively high levels of genetic diversity and connectivity among populations. Population clustering analyses detected 2 to 3 clusters and most of the populations of V. atratum comprised admixture of these genetic clusters. These admixtures likely formed during the process of colonizing habitats that had been disturbed by human activities. However, STRUCTURE clustering detected low-admixtures in populations occurring in rocky maritime sites, which may not be suitable for agriculture/rangeland activities. High genetic diversity and population connectivity suggested that loss of the remaining populations by grassland reduction might be an immediate threat for this species. Small grasslands populations managed by local farmers need appropriate conservation practices. Although our results showed genetic diversity and gene flow among populations of V. atratum were high, it is possible that this resulted from the historical continuous distribution of the species. To examine this hypothesis, further periodical monitoring of the genetic diversity and the genetic differentiation for the species is needed for a conservation action of the species.

Geographic Origin and Genetic Structure of Introduced Sika Deer, Kerama Deer (Cervus nippon keramae) on Ryukyus Inferred from Mitochondrial DNA Sequences

1 Environmental Symbiosis Studies, Graduate School of Integrated Arts and Sciences, The University of Tokushima, 1-1 Minamijosanjima, Tokushima 770-8502, Japan 2 Faculty of Human Development, Department of Child Education, Minami Kyushu University, 376-1 Tateno, Miyakonojo, Miyazaki 885-0035, Japan 3 Okinawa International Peace Research, 2-24-2 Nishi, Naha 900-0036, Japan 4 Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Hayama, Kanagawa 240-0193, Japan