Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

Isolation and characterization of microsatellites from a cicada, Yezoterpnosia nigricosta (Hemiptera: Cicadidae), distributed in subarctic and cool temperate forests.

The cicada Yezoterpnosia nigricosta (Hemiptera: Cicadidae) is distributed in subarctic and cool temperate forests in Japan, China and the Russian Far East. Due to its limited and isolated distribution at higher altitudes in cool temperate forests on the main island of Japan, especially in the central and southern areas, this species is listed as a threatened or near-threatened species on the Red List of 12 prefectures in Japan. Moreover, there are concerns about the impacts of climate change on the species' distribution and population demography. In this study, seventeen microsatellite markers were developed for Y. nigricosta, and marker suitability was evaluated using 32 individuals from two populations in Nagano prefecture (central Japan) and Hokkaido, a northern island of Japan. The number of alleles, expected heterozygosity and fixation index at each locus were 1-15 (mean = 4.294), 0.000-0.914 (mean = 0.519) and -0.225-0.456 (mean = 0.108), respectively. Furthermore, there was moderate genetic differentiation between the two populations (FST = 0.111, F'ST = 0.237). These markers will be useful to evaluate the genetic structure and to infer population demographic history of Y. nigricosta populations, which can contribute to population genetics studies of this species.

Efficient PCR Amplification Protocol of Nuclear Microsatellites for Exuviae-Derived DNA of Cicada, Yezoterpnosia nigricosta.

Although insect exuviae-based genetics is challenging, it can be a valuable method for obtaining reliable DNA resources by non-invasive sampling. This approach is especially effective when the target species is endangered/endemic or when sampling the adult is difficult. One example is cicadas, which during molt leave their exoskeletons on tree trunks, making them easily collectable. While cicada exuviae-derived DNA has previously been employed for mitochondrial DNA sequencing, this study aimed to develop a reliable method for the PCR amplification of nuclear microsatellite loci from cicada exuviae derived DNA for application in molecular ecology, conservation and population genetics. Five different PCR amplification protocols were performed, and the fragment patterns compared with those obtained using DNA extracted from adult individuals. Moreover, the relationship between the freshness of the exuviae and genotyping success was evaluated. TaKaRa LA Taq provided the best performance in the PCR amplification of DNA isolated from cicada exuviae and the electropherogram showed a clear fragment pattern that was equivalent to that obtained from the DNA extracted from the adult individual. This result suggests that cicada exuviae-derived DNA can be amplified by PCR and that multiple independent loci of nuclear DNA microsatellite markers can be easily genotyped. This study demonstrates that fresh cicada exuviae provide high quality DNA, which can be used for microsatellite genotyping. The methods developed in this study are applicable not only for cicada but other insect species for which exuviae are available. Thus, this study can make a significant contribution to insect sciences.

Spatiotemporal distribution of juvenile chum salmon in Otsuchi Bay, Iwate, Japan, inferred from environmental DNA.

To understand the ecology of juvenile chum salmon during early marine life after their downstream migration, we developed a quantitative PCR-based environmental DNA (eDNA) method specific for chum salmon and investigated the spatiotemporal distribution of eDNA in Otsuchi Bay, Iwate, Japan. Indoor aquarium experiments demonstrated the following characteristics of chum salmon eDNA: (1) the eDNA shedding and degradation were time- and water temperature-dependent and the bacterial abundance could contribute to the eDNA decay, (2) fecal discharge may not be the main source of eDNA, and (3) a strong positive Pearson correlation was found between the number of juveniles and the eDNA amounts. As we discovered strong PCR inhibition from the seawater samples of the bay, we optimized the eDNA assay protocol for natural seawater samples by adding a further purification step and modification of PCR mixture. The intensive eDNA analysis in the spring of 2017 and 2018 indicated that juvenile chum salmon initially inhabited in shallow waters in the shorefront area and then spread over the bay from January to June. The eDNA data also pointed out that outmigration of juvenile chum salmon to open ocean temporarily suspended in April, possibly being associated with the dynamics of the Oyashio Current as suggested by a previous observation. The eDNA method thus enables us large-scale and comprehensive surveys without affecting populations to understand the spatiotemporal dynamics of juvenile chum salmon.

Transgenerational seasonal timer for suppression of sexual morph production in the pea aphid, Acyrthosiphon pisum.

Many aphid species switch reproductive modes seasonally, with the sexual generations appearing in autumn. Sexual generations are induced by short days. It has been reported that the appearance of sexual morphs is suppressed by a transgenerational factor (a seasonal timer) over several generations after hatching from overwintered eggs. The present study examined whether the seasonal timer measures the number of days from hatching or the number of generations from hatching using the pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae). Effects of temperature and photoperiod on the seasonal timer were also examined by successive rearing. The ability to produce sexual morphs was strongly suppressed in stem mothers (the foundress generation), and gradually recovered over successive generations produced during a few months. The duration for which the seasonal timer could function depended on the number of days from hatching and temperature, but not on photoperiod or the number of generations from hatching. We thus showed in a single study that the seasonal timer of the pea aphid has all the physiological characteristics shown in separate studies in different aphid species.

A new species of Megoura (Hemiptera, Aphididae) from Japan.

A new species of the genus Megoura, M. lathyricola sp. n., was collected from Lathyrus japonicus subsp. japonicus (Leguminosae) in seashore areas of northern and southern Japan. This species is described and illustrated, and a revised key to the identification of the world species of Megoura is presented.

Unique haplotypes in ant-attended aphids and widespread haplotypes in non-attended aphids.

Aphid species within the genus Tuberculatus Mordvilko (Hemiptera: Aphididae) exhibit a variety of interactions with ants, ranging from close associations to non-attendance. A previous study indicated that despite wing possession, ant-attended Tuberculatus species exhibited low dispersal rates compared with non-attended species. This study examined if presence or absence of mutualistic interactions and habitat continuity of host plants affected intraspecific genetic diversity and genetic differentiation in mitochondrial DNA cytochrome oxidase I (COI) sequences. Sympatric ant-attended Tuberculatus quercicola (Matsumura) (Hemiptera: Aphididae) and non-attended Tuberculatus paiki Hille Ris Lambers (Hemiptera: Aphididae) were collected from the daimyo oak Quercus dentata Thunberg (Fagales: Fagaceae) in Japan and examined for haplotype variability. Seventeen haplotypes were identified in 568 T. quercicola individuals representing 23 populations and seven haplotypes in 425 T. paiki representing 19 populations. Haplotype diversity, which indicates the mean number of differences between all pairs of haplotypes in the sample, and nucleotide diversity were higher in T. quercicola than T. paiki. Analysis of molecular variance (AMOVA) showed higher genetic differentiation among populations within groups of T. quercicola (39.8%) than T. paiki (22.6%). The effects of attendant ant species on genetic differentiation in T. quercicola were not distinguishable from geographic factors. Despite low dispersal rates, host plant habitat continuity might facilitate widespread dispersal of a T. quercicola haplotype in Hokkaido. These results suggested that following T. quercicola colonization, gene flow among populations was limited, resulting in genetic drift within populations. However, frequent T. paiki dispersal is clearly evident by low genetic differentiation among populations within groups, resulting in lower haplotype diversity.

Allelic and genotypic diversity in long-term asexual populations of the pea aphid, Acyrthosiphon pisum in comparison with sexual populations.

Many aphid species exhibit geographical variation in the mode of reproduction that ranges from cyclical parthenogenesis with a sexual phase to obligate parthenogenesis (asexual reproduction). Theoretical studies predict that organisms reproducing asexually should maintain higher allelic diversity per locus but lower genotypic diversity than organisms reproducing sexually. To corroborate this hypothesis, we evaluated genotypic and allelic diversities in the sexual and asexual populations of the pea aphid, Acyrthosiphon pisum (Harris). Microsatellite analysis revealed that populations in central Japan are asexual, whereas populations in northern Japan are obligatorily sexual. No mixed populations were detected in our study sites. Phylogenetic analysis using microsatellite data and mitochondrial cytochrome oxidase subunit I (COI) gene sequences revealed a long history of asexuality in central Japan and negated the possibility of the recent origin of the asexual populations from the sexual populations. Asexual populations exhibited much lower genotypic diversity but higher allelic richness per locus than did sexual populations. Asexual populations consisted of a few predominant clones that were considerably differentiated from one another. Sexual populations on alfalfa, an exotic plant in Japan, were most closely related to asexual populations associated with Vicia sativa L. The alfalfa-associated sexual populations harboured one COI haplotype that was included in the haplotype clade of the asexual populations. Available evidence suggests that the sexuality of the alfalfa-associated populations has recently been restored through the northward migration and colonization of alfalfa by V. sativa-associated lineages. Therefore, our results support the theoretical predictions and provide a new perspective on the origin of sexual populations.