Asuka 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.

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.

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

A Comparative Study of the Fecal Characters of Japanese Serow (Capricornis crispus) and Sika Deer (Cervus nippon)

Abstract. The fecal pellets and fecal pellet group characters of two ungulates, the Japanese serow and sika deer, were compared in Tokushima, Japan, where the species occur sympatrically. Species identification based on 82 fecal DNA samples revealed that 28 and 54 fecal pellet groups were from Japanese serow and sika deer, respectively. The mean values of three fecal pellet groups characters, the maximum diameter, the proportion of adherence of fecal pellets (maximum number of adhering pellets/total pellet number), and pellet number, were significantly different between the two species. These values reflect the differences in their defecation behaviors, and piled fecal pellet groups of Japanese serow can be clearly distinguished from those of sika deer. A stepwise discriminant analysis selected only one fecal pellet group character, the proportion of adherence of fecal pellets, and provided the highest correct classification rate (82.1% for Japanese serow and 88.9% for sika deer). The proportion of adherence of fecal pellets is an important parameter for discriminating between Japanese serow and sika deer on field surveys.

Development of Microsatellite Markers for Isodon longitubus (Lamiaceae)

Premise of the study: Microsatellite markers were developed for Isodon longitubus to study the natural hybridization of the species and its congeners. Methods and Results: A total of 10 primer sets were developed for I. longitubus. From the initial screening, all of 10 loci were polymorphic with five to 19 alleles per locus in the Mt. Ishizuchi population, whereas nine loci were polymorphic with two to 12 alleles per loci in the Toon population. Although one locus was monomorphic at one population, the observed and expected heterozygosity values estimated from 34 I. longitubus samples ranged from 0.273 to 1.000 and from 0.483 to 0.918, respectively. Six primer sets could amplify all three species examined in this study (I. inflexus, I. japonicus, and I. shikokianus). Conclusions: The 10 microsatellite markers developed here will be useful in analyzing the population genetic structure of I. longitubus and in studying the natural hybridization between Isodon species.

Species, sex, and individual identification of Japanese serow ( Capricornis crispus ) and sika deer ( Cervus nippon ) in sympatric region based on the fecal DNA samples

We describe non-invasive molecular techniques for species, sex, and individual identification using fecal of the sympatric species, Japanese serow (Capricornis crispus) and sika deer (Cervus nippon), in Japan. From screening 19 microsatellites using 52 tissue samples of the two species, we selected 14 and 10 highly polymorphic loci for Japanese serow and sika deer, respectively, of which five loci could be amplified across the species. First, species identification using two cross-species-amplifiable loci and sex identification based on a partial amelogenin gene sequence were conducted on 194 fecal samples from Eastern Shikoku, Japan. PCR amplification of 65 fecal samples for new and relatively new fecal pellets identified 35 Japanese serow and 30 sika deer. In Japanese serow, 14 individuals (7 males and 7 females) were identified using 11 of 14 microsatellites. In sika deer, 19 individuals (11 males and 8 females) were identified using 9 of 10 microsatellites. This non-invasive genetic technique using fecal samples should be useful to study the distribution, gene flow, sex ratio, and population size of sympatric Japanese serow and sika deer.

Evidence of pre-introduction hybridization of Formosan sika deer (Cervus nippon taiouanus) on Okinoshima, Wakayama Prefecture, Japan, based on mitochondrial and nuclear DNA sequences

To examine the origin of the Formosan sika deer (Cervus nippon taiouanus) introduced to the island of Okinoshima (Wakayama Prefecture, Japan), sixteen tissue samples from Okinoshima population were analyzed to determine sequence variation in maternal mitochondrial DNA (mtDNA) cytochrome b and nuclear DNA α-lactalbumin (αLAlb). Three cytochrome b haplotypes were found in the Okinoshima population, two Formosan sika deer haplotypes and one Formosan sambar (C. unicolor swinhoei) haplotype. Three nuclear DNA αLAlb sequences, i.e. Formosan sika deer, red deer (C. elaphus), and additional one unknown sequence were detected in the population. Cytonuclear disequilibrium was observed in five individuals, indicating that the Okinoshima population have occurrence of hybrid among the three deer species. The deer introduced on Okinoshima might have originated from a private deer-breeding facility in Taiwan. To avoid native Japanese sika deer (e.g. C. n. centralis) genetic introgression, the Okinoshima population should be isolated from native Japanese sika deer populations.

Indirect estimation of recent sika deer (Cervus nippon) migration in Tsurugi Quasi-National Park, Shikoku, Japan

Abstract. To provide information for the management of sika deer in Tsurugi Quasi-National Park, Shikoku, Japan, we analyzed population genetic structure and migration patterns in the national park and surrounding areas based on from six to seven microsatellite loci using tissue and fecal samples. Bayesian clustering divided the deer into two populations, the Tokushima population and the Kochi population. Twenty-four of 28 samples on Mt. Tsurugi were assigned the Kochi population and the spatial boundary between the two populations was found in the foothills of the Tsurugi mountain range. These results suggest that the invasion of sika deer into Tsurugi Quasi-National Park may have started from the foothills of the Tsurugi mountain range by expansion of the Kochi population. Therefore, proper management of Kochi population is crucial in preventing further migration of sika deer into Tsurugi Quasi-National Park.