Naoyuki Nakahama

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.

Abstract 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.

Abstract Population outbreaks of insects provide an excellent opportunity to elucidate their evolutionary history. In this study, we uncovered the phylogenetic relationships of Dicraeus spp. in Japan and compared the population genetic structure of two bamboo-feeding Dicraeus spp., Dicraeus phyllostachyus and Dicraeus nartshukae, by leveraging a recent nationwide mass flowering event of several bamboo species in Japan. We performed analysis using mitochondrial COI gene sequencing and genome-wide single-nucleotide polymorphisms detected by multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq). We found genetic similarity in COI haplotypes over a large geographical range in the two Dicraeus spp., but, for both species, no concordance was detected between the collected host plants and genetic structures. In the genome-wide single-nucleotide polymorphism data, no clear genetic clusters were detected for D. phyllostachyus populations, whereas D. nartshukae populations were divided into two groups, in the north and south of Japan. Demographic analysis indicated that D. phyllostachyus had greatly expanded its population recently compared with D. nartshukae. These contrasting patterns between the two species might reflect the differences in the demographic history between the species. Our study suggested that the mass flowering event might cascade to the genetic structure of the florivorous insects through trophic interactions.

The use of genetically closely related populations as reintroduction sources is crucial in restoration programmes for endangered species because genetically distant populations may cause genetic disturbances. Aporia hippia (Lepidoptera; Pieridae) is an endangered subalpine grassland butterfly in Japan and its distribution area is restricted to the Asama, Yatsugatake and Akaishi Mountains in central Honshu Island. In the Yatsugatake Mountains, its population is probably already extinct, and reintroduction from the other sites is now under consideration. Here, we estimated the genetic diversity and spatial genetic structure of A. hippia in Japan to identify candidate source populations for reintroduction in extinct populations via multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) and mitochondrial DNA sequencing methods. The MIG-seq method indicated the presence of very few genetic differences among the populations. The mitochondrial DNA sequencing method identified two haplotypes: haplotype A in the Asama, Yatsugatake and northern Akaishi Mountains, and haplotype B in the central and southern Akaishi Mountains. The two haplotypes exhibited only a one base pair substitution between them. Implications for insect conservation: Based on these results, we suggest the existence of two conservation units: the first group consisting of populations in the Asama, Yatsugatake, and northern Akaishi Mountains, and the second group consisting of those in the central and southern Akaishi Mountains. According to this genetic proximity, however, all populations of A. hippia in Japan can also be defined as one conservation unit group, given the risk of inbreeding depression or difficulty of adaptation in different environments.

<title>Abstract</title> The spatial genetic structures of insects are reflected in their habitats. Coastal insects tend to have less genetic differentiation than subterranean insects. However, the general pattern of population structure of coastal species residing in subterranean habitats is unknown. Thalassoduvalius masidai (Coleoptera: Carabidae: Trechinae), endemic to Japan, is a coastal and subterranean beetle. Here, we hypothesized that T. masidai populations disperse among regions by ocean currents, floods and tsunamis, despite their habitat being underground. To test this hypothesis, we performed a phylogeographical analysis of T. masidai using single nucleotide polymorphism data from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) and the mitochondrial COI gene. The phylogenetic network and tree and STRUCTURE analyses indicated that T. masidai populations were divided into three groups: the western Seto Inland Sea–Izu Peninsula coasts, the eastern Seto Inland Sea–Kii Peninsula coasts and the Japan Sea coasts. The genetic diversity of T. masidai was higher in sites with lower longitude and latitude. These results suggest that T. masidai migrated and dispersed from the south-western area of Japan by ocean currents, despite its underground habitat. The genetic analysis data from the present study would also be helpful for the conservation and taxonomic revision of this species.

The Argentine ant Linepithema humile Mayr, native to South America, has been unintentionally introduced worldwide. Although L. humile usually forms an extremely large supercolony all sharing the same haplotype among members in its introduced ranges, four haplotypes (LH1, LH2, LH3, LH4) with different genetic structures have been discovered in Kobe Port (Port-Island and Maya Wharf), Japan. In 2019, a new L. humile population was discovered, the first since the initial invasion in 2006. Therefore, we identified the haplotypes among these newly invaded L. humile populations via mtDNA analysis and then compared the distributions among the haplotypes during these two time periods (2006 versus 2019). The haplotypes of all newly introduced L. humile populations corresponded to only the haplotypes previously detected in Kobe Port. Although the distribution ranges of haplotypes LH1 and LH4 changed little in Maya Wharf over approximately 15 years, major distribution expansions of LH2 and LH3 were found on Port-Island. Given the low natural dispersion ability of L. humile, it is likely that the range expansions of LH2 and LH3 on Port-Island were caused by the extensive transportation of construction materials within Kobe Port. In addition to strengthening the quarantine at existing trading ports, greater attention should be paid to the introduction of biological invaders in conjunction with the transportation of construction materials when new ports are developed and maintained.

Genetic disturbance is propagated by artificial reinforcement or reintroduction of organisms from populations whose genetic structure is sufficiently different from the original populations. Many cultivars were produced from wild plant species, and fears exist that genetic disturbance of wild populations occurred by the human migration of cultivars. In this study, we detected genetic disturbance in Pecteilis radiata [= Habenaria radiata] (Orchidaceae) in the Hyogo Prefecture of Japan using multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq). Also, we estimated the risk of the spread of genetically disturbed individuals based on the results of spatial genetic structure and gene flow. In five out of 33 examined sites, we detected genetically disturbed individuals that possessed the same genetic cluster as cultivar samples. No spatial genetic structure was found among the sites within a 0.64 km radius, which indicated that pollen or seeds of P. radiata freely migrated among these populations. Consequently, we demonstrated that a risk exists of spreading genetic disturbances by gene flow within a radius of 0.64 km from the site where genetic disturbance was found. Raised public awareness of the risk of genetic disturbance would be necessary to prevent additional spread of genetic disturbances by human activities.

Crepidiastrum grandicollum is a critically endangered insular endemic herb found only on two oceanic islands of the Bonin Islands in Japan, namely Chichijima Island and Anijima Island. It is explicitly threatened by herbivory pressure from introduced animals. In 2009, a conservation program for C. grandicollum was begun to ensure its future persistence. To provide further information for conservation planning, we investigated the genetic diversity of C. grandicollum using 13 novel microsatellite markers in 55 individuals from four wild populations and an ex situ living collection. Two closely related woody species were also included for interspecific comparison: seven individuals of Crepidiastrum ameristophyllum and 13 of Crepidiastrum linguifolium. The 13 markers were applicable to all three species and identified 129 alleles in total. We found a clear genetic differentiation between C. grandicollum from Anijima Island and Chichijima Island. Crepidiastrum grandicollum also had low expected heterozygosity and allelic richness in populations compared to the two closely related species. Reconstructed divergence history suggested that differentiation between the islands had occurred several thousand generations ago. We suggest separate conservation units for C. grandicollum on Anijima Island and Chichijima Island given the clear (and putatively historical) genetic differentiation, which may result in speciation in the future.

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.

<title>Abstract</title> An environmental DNA (eDNA) detection method confirms the presence of the subterranean amphipod genus PseudocrangonyxAkatsuka &amp; Komai, 1922 in the city of Kyoto, Honshu, Japan for the first time in more than 90 years. Phylogenetic trees using partial sequences of the 16S rRNA gene of eDNA samples from Kyoto and specimens collected from various locations in Japan and Korea highlight that two genetically distinct taxonomic units inhabit Kyoto’s subterranean environments. Results reveal that the ranges of distinct phylogroups of Pseudocrangonyx widely overlap with each other around Lake Biwa in central western Honshu. The eDNA results increase our understanding of the species richness and complicated evolutionary history of these stygobitic amphipods.

Acilius kishii Nakane, 1963 (Coleoptera: Dytiscidae) is an endangered diving beetle species distributed in only one location, Lake Yashaga-Ike, Honshu Island, Japan. Acilius japonicus, which is related to A. kishii, is distributed widely in northern Honshu Island and Hokkaido Island in Japan. In this study, we identified 14 microsatellite loci for A. kishii and A. japonicus, including both polymorphic and monomorphic loci, using the next-generation sequencing method. We observed that 5 and 10 loci showed polymorphisms in 31 and 32 individuals of A. kishii and A. japonicus, respectively. The observed and expected heterozygosities were 0.00-1.00 and 0.00-0.74, respectively. These microsatellite loci could be useful for future conservation genetic studies, including monitoring of genetic diversity and extinction risk of A. kishii.

<p>Kirigamine Plateau, where is a habitat for various threatened plants and animals, is one of the most popular natural tourist sites in Japan. In recent years, however, tourism flowers (e.g., Hemerocallis middendorffii) have suffered severe browsing damage by sika deer, and therefore several deer fences have been installed. In this study, to assess the conservation effects of deer fences on species diversity of subalpine semi-natural grasslands, plant and insect surveys with UAV image analysis were conducted in semi-natural grasslands of Kirigamine Plateau. The numbers of flowering plant species, flowers and flowering threatened plant species were higher inside fences than those outside the fences. The numbers of butterflies and bumblebees were also higher inside than outside the fences. UAV image analysis visualized the conservation effects of the deer fences over a wider area. Our results highlight that the installation of deer fences is indispensable for the conservation of both tourism resources and threatened species.</p>

© 2020, Genetics Society of Japan. All rights reserved. Table 1 on p. 135 should be replaced with the corrected Table 1 shown bellow. (Table presented).

To protect endangered and commercially important orchid species, it is important to monitor and accurately identify orchid pests. Japanagromyza tokunagai (Sasakawa) larvae are known to infest the flower buds, stems, and ovaries of various Japanese orchid species. Given that J. tokunagai can infest diverse orchid hosts, J. tokunagai has the potential to be an economically important orchid pest in greenhouses. However, damage by J. tokunagai has previously only been reported in outdoor habitats. Here we revealed that the dipteran larvae infesting the flower buds of Phalaenopsis aphrodite Rchb.f. (Orchidaceae) cultivated in a greenhouse are J. tokunagai based on DNA barcoding, using COI sequences. Infested flower buds remained closed and eventually dropped to the ground, making the P. aphrodite plants commercially worthless. Phalaenopsis aphrodite is one of the most economically important ornamental orchids. Therefore, J. tokunagai is a potential threat to orchid production and we recommend taking precautions to prevent its spread.

Natural hybridization often occurs between insular and widespread species, and investigating these hybridization patterns is important in improving our understanding of the evolution of insular plant species. To this end, we studied the floral morphology and genetic structure of the widespread species Calanthe discolor, the insular closely related species C. izu-insularis and their putative natural hybrids on Honshu Island (mainland) and the Izu Islands (oceanic islands), Japan, using microsatellite markers. Natural hybridization probably occurred on all the investigated Izu Islands in Japan, although C. izu-insularis was morphologically and genetically different from C. discolor. Bumblebees and other relatively large bees are not distributed in the Izu Islands and therefore natural hybridization would have occurred via pollination by relatively small solitary bees that are pollinators of both Calanthe spp. We also showed that in the islands more remote from the mainland, the proportions of insular C. izu-insularis increased and the floral morphologies of both species changed. The results suggested that insular C. izu-insularis evolved on the Izu Islands and the widespread C. discolor adapted to the poor pollinator fauna on oceanic islands.

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 micro satellite 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 inter-species genetic structure and the mixed mating strategy of Commelina species via paternity analysis.

Linepithema humile is one of the most damaging invasive species worldwide. Although chemical control strategies have proven effective for L. humile, the susceptibility of these invasive ants to the insecticide fipronil differs markedly among genetically different supercolonies. In Japan, five mitochondrial L. humile haplotypes were identified from eleven prefectural regions and cities as of 2010. In 2012, a new population was found in Okayama Prefecture. Here, we analyzed mitochondrial DNA from L. humile workers from the Okayama population to better understand the genetic structure of ants in Japan and develop effective control strategies. According to COI-COII and cytochrome b gene sequences, the L. humile Okayama population haplotype was consistent with the 'Japanese main' supercolony-the most invasive supercolony worldwide. Hence, we believe that the Okayama population (Japanese main supercolony) can be easily eradicated because of its early invasion stage, relatively limited distribution range and high sensitivity to fipronil.

Heterostyly is a genetic polymorphism of anther and stigma heights, which are reciprocally placed among morphs. The maintenance of heterostyly has often been ascribed to disassortative mating, but alternative explanations are also possible, which can be examined by analyzing mating patterns. However, parental analysis with genetic markers has only been applied to a limited number of taxa, despite the phylogenetic diversity of heterostylous species. Here, we examined the mating patterns of a distylous primrose, Primula kisoana (Primulaceae), in its natural habitat, using 11 microsatellite markers. We also examined other fitness components. We found no differences in seed production, vegetative growth, or survival between morphs. However, the siring success of thrum fathers was much higher than that of pin fathers; outcrossing occurred unilaterally between thrum fathers and pin mothers, and selfing was more frequent in thrum plants than in pin plants. These findings are consistent with the explanation that distyly in this species is maintained either by overdominance due to previously reported recessive lethal effects of the thrum allele or by effectively dioecious mating if inbreeding depression is strong. We built a mathematical model of the overdominance hypothesis to confirm that it is logically sound. These results warrant further investigations into the possible roles of overdominance and dioecy in the maintenance of heterostyly.

Background and Aims Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions.Methods The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated.Key Results Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented.Conclusions Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades.

Dried specimens of insects are increasingly seen as genetic resources. However, genetic analysis of dried specimens of insects is hampered by the deterioration of the DNA. In this study, we developed methods for preparing dried specimens of insects with well-preserved DNA, mainly for PCR-based genetic analysis. First, we compared the effects of either exposure to ethyl acetate vapour for from 10 min to 6 h or by freezing on the fragmentation of DNA in order to determine optimal length of time needed for killing insects using the above methods. Second, we compared the fragmentation of DNA after preservation by drying or immersion of legs in 99.5% ethanol or 99% propylene glycol in 0.2-ml tubes. We assessed degrees of fragmentation of DNA by determining polymerase chain reaction (PCR) success rates with primers for 313-, 710- and 1555-bp fragments using DNA that was collected immediately, and at one, six and 12 months after preparing the specimens. Differing times taken to kill insects did not affect the fragmentation of DNA. In dried specimens, DNA was seriously fragmented after one month, whereas that in legs prepared by immersion in 99.5% ethanol or 99% propylene glycol contained long fragments of DNA (1555 bp-) after 12 months. Propylene glycol was more suitable for preservation than ethanol, because the latter evaporates. Thus, to preserve insect DNA we suggest inserting the pin on which an insect is impaled into the hinged lid of a 0.2-ml tube containing 99% propylene glycol so that when the lid is closed the legs of the insect are preserved in the solution.

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.

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.

The monitoring of genetic characters and definition of effective conservation units are important for conservation of endangered semi-natural grassland species.Using molecular markers, we elucidated the recent transitions in genetic diversity for the endangered grassland butterfly Melitaea protomedia (Lepidoptera; Nymphalidae) in Japan. First, we examined changes in genetic diversity and structure from the 1980s to the 2010s from archival and contemporary DNA samples using eight microsatellite markers. Second, we estimated the genetic structure based on 1374bp of mitochondrial COI gene from contemporary samples. We also defined the conservation units of M. protomedia based on the above analysis.The genetic diversity of M. protomedia has significantly declined from the 1980s to the 2010s. Although genetic differentiation was very strong among populations in c. 2010, there was only weak genetic structure in c. 1990. In addition, the number of haplotypes based on mitochondrial DNA is now very low due to recent declines.These findings suggest that effective conservation units for critically endangered species should be determined based on historic (i.e. prior to population declines) as well as contemporary genetic diversity and differentiation, because genetic structure may have changed over time. Genetic analysis of archival DNA is useful to obtain historic genetic information.