Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing.

Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.

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.

A New Species of the Genus Pseudocrangonyx (Crustacea: Amphipoda: Pseudocrangonyctidae) from Yonaguni Island, Southwestern Japan, and Historical Biogeographic Insights of Pseudocrangonyctids.

The subterranean amphipod genus Pseudocrangonyx is diverse in Far East Asia, including the Japanese Archipelago. However, Pseudocrangonyx species have not been recorded from the Ryukyu Islands, which extend southwest of the Japanese Archipelago. This study describes a new species of Pseudocrangonyx, Pseudocrangonyx dunan sp. nov., from Yonaguni Island, Ryukyu Islands, Japan. Phylogenetic analyses revealed that P. dunan sp. nov. is a sister species to Pseudocrangonyx sp. 4 from Honshu Island, Japan. In addition, three monophyletic groups were found in Pseudocrangonyx, although the phylogenetic positions of several species remain unknown. Our divergence dating indicates that the differentiation of major lineages of Pseudocrangonyx, which contains species from both the Asian continent and the Japanese Archipelago, is concentrated around 20 MYA. These results suggest that the opening of the Sea of Japan is one of the major factors promoting the speciation of Pseudocrangonyx endemic to the archipelago.

Genetic Diversity of Invasive Spartina alterniflora Loisel. (Poaceae) Introduced Unintentionally Into Japan and Its Invasion Pathway.

Among invasive species, aquatic plants pose serious threats to local biodiversity and ecosystem functions. Spartina alterniflora Loisel. (Poaceae), native to the eastern United States, was introduced unintentionally into Japan (Aichi and Kumamoto Prefectures) at around 2010. This invasive species could easily and rapidly spread to estuarine areas of Japan via vigorous trade and transport, making the prediction of its future invasion necessary. Here, the distribution and structure of the genetic variation of S. alterniflora in Japan were examined using chloroplast DNA (cpDNA) and microsatellite genotyping analyses for clarifying its invasion route and process. According to the cpDNA analysis, S. alterniflora populations in Japan had a single haplotype (haplotype C4) that is the most dominant genotype around the Florida Peninsula, the region of its origin, and is also widely found in the introduced populations in the East Asia. Microsatellite analysis also showed a loss of genetic diversity in Japanese S. alterniflora populations (allelic richness (A R) = 1.20-1.39) compared with that in its native region (A R = 4.58-4.59), suggesting a founder effect on S. alterniflora that might have occurred after invasion of the species into Japan. The principal coordinate analysis and The STRUCTURE analysis indicated that no gene mixing among Japanese local populations (Aichi, northern and southern Kumamoto) was observed, indicating that Spartina invasion occurred independently into these regions. Among the three regions, trading between the ports of northern Kumamoto and the U.S. was obviously lower than trading with China. We concluded that invasive S. alterniflora might have independently invaded Japan at different times through an East Asia route, particularly via China (i.e., secondary introduction). Therefore, it is important to strengthen the quarantine control on the importation of commodities, especially of transport vehicles at potential donor spots (i.e., border control/border biosecurity system), and to share information networks on invasive species between each region/port for minimizing further risks of biological species such as Spartina.

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.

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.

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.

Development of microsatellite markers for the endangered orchid Calanthe izu-insularis (Orchidaceae).

Microsatellite markers were developed for the endangered orchid Calanthe izu-insularis (Orchidaceae). This species is unique to the Izu Islands in Japan. Unfortunately, its population size has decreased because of excessive collection for horticultural purposes. In addition, although natural hybridization between C. izu-insularis and C. discolor var. discolor has been reported, morphological differences between C. izu-insularis and the hybridized individuals remain unclear. Using next-generation sequencing, 11 polymorphic microsatellite markers were developed. All developed markers could amplify C. aristulifera and nine markers could amplify C. d. var. discolor, two other orchid species that are also endangered in Japan. The number of alleles and expected heterozygosity at each locus were 1-6 (mean, 2.35) and 0.00-0.79 (mean, 0.30), respectively. These microsatellite markers will help conservation geneticists in their investigation of the proportion of pure C. izu-insularis individuals in the Izu Islands.

Development of microsatellite markers for the semi-natural grassland herb Veronicastrum japonicum (Plantaginaceae).

PREMISE OF THE STUDY: Veronicastrum japonicum (Plantaginaceae) grows in grasslands on Honshu Island, Japan, and is threatened by habitat loss because of rapid land development over recent decades. For the genetic characterization of the remaining populations, microsatellite markers were developed. METHODS AND RESULTS: Twelve polymorphic microsatellite loci were developed using next-generation sequencing. The number of alleles per locus ranged from two to 24 (mean 7.7), and the expected heterozygosity per locus ranged from 0.35 to 0.94 (mean 0.68). CONCLUSIONS: These markers can be used for genetic studies in conservation, such as the evaluation of genetic diversity and genetic structure.

Development of microsatellite markers for the coastal shrub Scaevola taccada (Goodeniaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the coastal shrub species Scaevola taccada to estimate the population genetic structure, which may reflect different seed dispersal patterns. • METHODS AND RESULTS: Thirteen microsatellite primer sets were developed for S. taccada using 454 pyrosequencing. The primer sets were tested on 64 individuals sampled from two populations in Japan. Fragments were amplified using the primers, with one to 10 alleles per locus, and the expected heterozygosity ranged from 0.00 to 0.85. • CONCLUSIONS: These results indicate the utility of markers in S. taccada for broad estimations of the population genetic structure of this species.