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.

Extensive Copy Number Variation Explains Genome Size Variation in the Unicellular Zygnematophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

Genome sizes are known to vary within and among closely related species, but the knowledge about genomic factors contributing to the variation and their impacts on gene functions is limited to only a small number of species. This study identified a more than 2-fold heritable genome size variation among the unicellular Zygnematophycean alga, Closterium peracerosum-strigosum-littorale (C. psl.) complex, based on short-read sequencing analysis of 22 natural strains and F1 segregation analysis. Six de novo assembled genomes revealed that genome size variation is largely attributable to genome-wide copy number variation (CNV) among strains rather than mating type-linked genomic regions or specific repeat sequences such as rDNA. Notably, about 30% of genes showed CNV even between strains that can mate with each other. Transcriptome and gene ontology analysis demonstrated that CNV is distributed nonrandomly in terms of gene functions, such that CNV was more often observed in the gene set with stage-specific expression. Furthermore, in about 30% of these genes with CNV, the expression level does not increase proportionally with the gene copy number, suggesting presence of dosage compensation, which was overrepresented in genes involved in basic biological functions, such as translation. Nonrandom patterns in gene duplications and corresponding expression changes in terms of gene functions may contribute to maintaining the high level of CNV associated with extensive genome size variation in the C. psl. complex, despite its possible detrimental effects.

Flora of herbaceous and arboreous plants in Komaba Campus of the University of Tokyo, Japan.

BACKGROUND: Recent studies revealed that green spaces in urban areas are critical for conservation of native biodiversity and that assessment of the present flora of green spaces in urban areas is critical for protection of the native biodiversity. The Komaba Campus of the University of Tokyo embraces a relevant green area, located in a highly urbanised area in Tokyo Metropolis (35.66 N 139.68 E, Japan). The total area of this Campus is 25.4 ha, from which, 4.5 ha are covered by vegetation. Although intense urbanisation can be observed around the Campus, new insect species had been reported for the Campus area, suggesting that the biodiversity on the Campus still demands some attention. Differently from fauna surveys, no flora survey has been done for more than 30 years on the Campus. In this study, we have extensively surveyed the plants diversity on the Komaba Campus of the University of Tokyo, aiming for an update of the plants list on this green urban area in Tokyo. NEW INFORMATION: The survey covered all herbaceous and arboreous plants growing wild on the Campus. Garden plants were excluded in this survey because these plants were supposed to be cultivated. The final dataset contained, in total, 324 taxa, from which 234 were herbaceous plants and 90 were arboreous plants. The top three taxa are as follows: Poaceae (38 taxa), Asteraceae (34 taxa) and Rosaceae (14 taxa), respectively. This is the first update to the Flora of the Komaba Campus of the University of Tokyo in 30 years and represent an important contribution to conservation of native species in the Tokyo metropolis.

Development and characterization of nuclear microsatellite markers to reveal the neutral demographic background of flower color polymorphism in Geranium thunbergii (Geraniaceae).

Nuclear microsatellite markers were developed for Geranium thunbergii, an herbaceous plant characterized by petal color polymorphism. Utilizing RNA sequencing data obtained by next-generation sequencing techniques, we developed and characterized 19 polymorphic microsatellite markers with two to 12 alleles in the nuclear genome. These markers will be used to reveal the genetic structure and demographic history of G. thunbergii in the Japanese archipelago, which will elucidate the genetic background of flower color polymorphism among populations.

Phylogeographic and demographic modeling analyses of the multiple origins of the rheophytic goldenrod Solidago yokusaiana Makino.

Understanding adaptation mechanisms is important in evolutionary biology. Parallel adaptation provides good opportunities to investigate adaptive evolution. To confirm parallel adaptation, it is effective to examine whether the phenotypic similarity has one or multiple origins and to use demographic modeling to consider the gene flow between ecotypes. Solidago yokusaiana is a rheophyte endemic to the Japanese Archipelago that diverged from Solidago virgaurea. This study examined the parallel origins of S. yokusaiana by distinguishing between multiple and single origins and subsequent gene flow. The haplotypes of noncoding chloroplast DNA and genotypes at 14 nuclear simple sequence repeat (nSSR) loci and single-nucleotide polymorphisms (SNPs) revealed by double-digest restriction-associated DNA sequencing (ddRADseq) were used for phylogeographic analysis; the SNPs were also used to model population demographics. Some chloroplast haplotypes were common to S. yokusaiana and its ancestor S. virgaurea. Also, the population genetic structures revealed by nSSR and SNPs did not correspond to the taxonomic species. The demographic modeling supported the multiple origins of S. yokusaiana in at least four districts and rejected a single origin with ongoing gene flow between the two species, implying that S. yokusaiana independently and repeatedly adapted to frequently flooding riversides.

Temperature-related cline in the root mass fraction in East Asian wild radish along the Japanese archipelago.

Wild plants with a wide distribution, including those exposed to a wide variety of environmental conditions, may have variations in key functional traits relevant for agricultural applications. The East Asian wild radish (Raphanus sativus var. raphanistroides) is an appropriate model plant because it is widely distributed and has outstanding sink capacity as well as two cultivars within the species. Multiple common garden trials with 14 populations and three testing sites were conducted across the Japanese archipelago to quantify variations in yield and allocation. Significant inter-population variations and interaction effects with testing sites were detected for the root and shoot mass and the root mass fraction (RMF). While the rank order of the population changed drastically among sites and the variance components of genetic effects were small in yield traits (2.4%-4.7%), RMF displayed a large genetic variance (23.2%) and was consistently higher in the northern populations at all sites. Analyses revealed that the mean temperature of growing season of the seed origin was the most prominent factor explaining variation in RMF, irrespective of the sites. We concluded that the trait of resource allocation had a temperature-related cline and plants in cooler climates could invest more resources into their roots.

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.

Where should wild species be sampled? New method based on isolation-by-distance objectively gives the answer.

Random sampling is an important statistical assumption, but virtually impossible when sampling a wild species as we cannot know where all the individuals exist. While interpopulation or intrataxa sampling methods have been developed, there are currently few intrataxon sampling methods to objectively decide where to sample wild taxa. We suggest a new sampling method which computes appropriate sampling locations from coordinates, assuming geographical autocorrelation of phylogeny within a taxon (isolation-by-distance). The computed locations encompass the highest genetic diversity, providing a genetically representative sample. In addition, it can utilize presence/absence information during sampling to reoptimize sampling scheme. Comparing to the single existing method of the similar purpose, the merits of ours is unnecessity of environmental data resulting in easy application, and is theoretically deduced. We tested this method using published phylogeographical data. The test result was generally encouraging, but the method failed where species showed uniform genetic structure or recent distribution expansion which violate the assumption of geographical autocorrelation of phylogeny. Though simple, our method constructs a methodological and statistical foundation for sampling wild species, and is applicable to revising taxonomic study and conservation biology.

Genetic consequences of being a dwarf: do evolutionary changes in life-history traits influence gene flow patterns in populations of the world's smallest goldenrod?

BACKGROUND AND AIMS: Contrasting life-history traits can evolve through generations of dwarf plant ecotypes, yet such phenotypic changes often involve decreased plant size and reproductive allocation, which can configure seed dispersal patterns and, subsequently, population demography. Therefore, evolutionary transitions to dwarfism can represent good study systems to test the roles of life-history traits in population demography by comparing genetic structure between related but phenotypically divergent ecotypes. METHODS: In this study, we examined an ecotypic taxon pair of the world's smallest goldenrod (stem height 2.6 cm) in alpine habitats and its closely related lowland taxon (30-40 cm) found on Yakushima Island, Japan. Genetic variation in chloroplast DNA sequences, nuclear microsatellites and genome-wide single-nucleotide polymorphisms were used to investigate 197 samples from 16 populations, to infer the population genetic demography and compare local genetic structure of the ecotypes. KEY RESULTS: We found a pronounced level of genetic differentiation among alpine dwarf populations, which were much less geographically isolated than their lowland counterparts. In particular, several neighbouring dwarf populations (located ~500 m apart) harboured completely different sets of chloroplast haplotypes and nuclear genetic clusters. Demographic modelling revealed that the dwarf populations have not exchanged genes at significant levels after population divergence. CONCLUSIONS: These lines of evidence suggest that substantial effects of genetic drift have operated on these dwarf populations. The low-growing stature and reduced fecundity (only 3.1 heads per plant) of the dwarf plants may have reduced gene flow and rare long-distance seed dispersal among habitat patches, although the effects of life-history traits require further evaluation using ecological approaches.

Endangered island endemic plants have vulnerable genomes.

Loss of genetic diversity is known to decrease the fitness of species and is a critical factor that increases extinction risk. However, there is little evidence for higher vulnerability and extinction risk in endangered species based on genomic differences between endangered and non-endangered species. This is true even in the case of functional loci, which are more likely to relate to the fitness of species than neutral loci. Here, we compared the genome-wide genetic diversity, proportion of duplicated genes (PD), and accumulation of deleterious variations of endangered island endemic (EIE) plants from four genera with those of their non-endangered (NE) widespread congeners. We focused on exhaustive sequences of expressed genes obtained by RNA sequencing. Most EIE species exhibited significantly lower genetic diversity and PD than NE species. Additionally, all endangered species accumulated deleterious variations. Our findings provide new insights into the genomic traits of EIE species.

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.

Phylogeographic analysis suggests two origins for the riparian azalea Rhododendron indicum (L.) Sweet.

Ecological speciation is an important factor in the diversification of plants. The distribution of the woody species Rhododendron indicum, which grows along rivers and is able to withstand water flow when rivers flood (i.e. it is a rheophyte), is disjunct, in contrast to the widespread distribution of its relative, Rhododendron kaempferi. This study aimed to elucidate the phylogenetic relationships between R. indicum and R. kaempferi and the evolutionary processes that gave rise to them. The sequences of three non-coding chloroplast DNA regions (total length 1977 bp) were obtained from 21 populations covering the ranges of the two species. In addition, genome-wide SNPs were genotyped from 20 populations using a genotyping by sequencing method. Leaf morphologies were measured for eight representative populations. Two chloroplast DNA haplotypes, which were detected in R. indicum, were shared between the two species. Genome-wide SNPs identified two lineages in R. indicum and these lineages did not constitute a monophyletic group. Each of these two lineages was related to geographically close populations of R. kaempferi. Leaf morphology, which is a characteristic feature in rheophytes, was not differentiated between the two lineages in R. indicum. The morphological similarity between the two heterogeneous lineages may be a result of parallel evolution from R. kaempferi or of introgressive hybridization between the species due to strong selective pressure imposed by flooding.

Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis.

Lineage-specific duplicated genes likely contribute to the phenotypic divergence in closely related species. However, neither the frequency of duplication events nor the degree of selection pressures immediately after gene duplication is clear in the speciation process. Here, using Illumina DNA-sequencing reads from Arabidopsis halleri, which has multiple closely related species with high-quality genome assemblies (A. thaliana and A. lyrata), we succeeded in generating orthologous gene groups in Brassicaceae. The duplication frequency of retained genes in the Arabidopsis lineage was ∼10 times higher than the duplication frequency inferred by comparative genomics of Arabidopsis, poplar, rice and moss (Physcomitrella patens). The difference of duplication frequencies can be explained by a rapid decay of anciently duplicated genes. To examine the degree of selection pressure on genes duplicated in either the A. halleri-lyrata or the A. halleri lineage, we examined positive and purifying selection in the A. halleri-lyrata and A. halleri lineages throughout the ratios of nonsynonymous to synonymous substitution rates (KA/KS). Duplicate genes tended to have a higher proportion of positive selection compared with non-duplicated genes. Interestingly, we found that functional divergence of duplicated genes was accelerated several million years after gene duplication compared with immediately after gene duplication.

Phylogeographic analysis of the East Asian goldenrod (Solidago virgaurea complex, Asteraceae) reveals hidden ecological diversification with recurrent formation of ecotypes.

Background and Aims: The processes and mechanisms underlying lineage diversification are major topics in evolutionary biology. Eurasian goldenrod species of the Solidago virgaurea complex show remarkable morphological and ecological diversity in the Japanese Archipelago, with ecotypic taxa well adapted to specific environments (climate, edaphic conditions and disturbance regimes). The species complex is a suitable model to investigate the evolutionary processes of actively speciating plant groups, due to its ability to evolve in relation to environmental adaptation and its historical population dynamics. Methods: Two chloroplast markers, 18 nuclear microsatellite markers and ddRAD-sequencing were used to infer population genetic demography of S. virgaurea complex with its related species/genera. Key Results: Our analysis showed that populations in Japan form an evolutionary unit, which was genetically diverged from adjacent continental populations. The phylogenetic structure within the archipelago strongly corresponds to the geography, but interestingly there is no concordance between genetic structure and ecotypic boundaries; neighbouring populations of distinct ecotypes share a genetic background. Conclusions: We propose that the traits specific to the ecotypic entities are maintained by natural selection or are very recently generated and have little effect on the genomes, making genome-wide genetic markers unsuitable for detecting ecotypic differentiation. Furthermore, some sporadically distributed taxa (found as rheophytes and alpine plants) were repeatedly generated from a more widespread taxon in geographically distant areas by means of selection. Overall, this study showed that the goldenrod complex has a high ability to evolve, enabling rapid ecological diversification over a recent timeframe.

Simultaneous evaluation of the effects of geographic, environmental and temporal isolation in ecotypic populations of Solidago virgaurea.

Early stages of ecological speciation can create populations with an ecology and reproduction timing distinct from those of related populations. Landscape genetic models incorporating environmental heterogeneity and population-specific reproductive traits enable the processes of population genetic differentiation to be inferred. We investigated genome-wide genetic variation in ecotypic populations of Solidago virgaurea sensu lato, a herbaceous plant inhabiting a wide range of habitats (woodlands, serpentine barrens and alpine grasslands) and displaying remarkable variation in flowering time. Simultaneous evaluation of environmental factors revealed an overwhelming effect of soil type differences on neutral genetic differentiation, compared with elevational differences. This result probably reflects the abrupt environmental changes generated by geological boundaries, whereas mountain slopes exhibit clinal changes, facilitating gene exchange between neighbouring populations. Temporal isolation was positively associated with genetic differentiation, with some early-flowering serpentine populations having allele frequencies distinct from adjacent nonserpentine populations. Overall, this study highlights the importance of ecological processes and of evolution of flowering time to promote genetic differentiation of S. virgaurea populations in a complex landscape.

Application of a simplified method of chloroplast enrichment to small amounts of tissue for chloroplast genome sequencing.

PREMISE OF THE STUDY: High-throughput sequencing of genomic DNA can recover complete chloroplast genome sequences, but the sequence data are usually dominated by sequences from nuclear/mitochondrial genomes. To overcome this deficiency, a simple enrichment method for chloroplast DNA from small amounts of plant tissue was tested for eight plant species including a gymnosperm and various angiosperms. METHODS: Chloroplasts were enriched using a high-salt isolation buffer without any step gradient procedures, and enriched chloroplast DNA was sequenced by multiplexed high-throughput sequencing. RESULTS: Using this simple method, significant enrichment of chloroplast DNA-derived reads was attained, allowing deep sequencing of chloroplast genomes. As an example, the chloroplast genome of the conifer Callitris sulcata was assembled, from which polymorphic microsatellite loci were isolated successfully. DISCUSSION: This chloroplast enrichment method from small amounts of plant tissue will be particularly useful for studies that use sequencers with relatively small throughput and that cannot use large amounts of tissue (e.g., for endangered species).

Fauna of Cerambycidae (Insecta: Coleoptera) in Komaba Campus of the University of Tokyo, a highly urbanised area in Japan.

Urban green spaces play an important role in maintaining urban biodiversity in the Tokyo Metropolis, Japan. Plant-dependent insect assemblages such as Cerambycidae, in particular, are likely influenced by the existence of green spaces in Tokyo's urbanised environments. This study is the first comprehensive inventory of the cerambycid fauna in the Komaba Campus of the University of Tokyo. A cerambycid assemblage composed of a total of 25 species was recorded within the Komaba Campus site and compared to cerambycid assemblages in nine other green spaces distributed throughout Tokyo. The results indicated that the species number in the campus was similar to that recoded in a similar-sized green space in coastal Tokyo. Fewer cerambycid species were, however, found at the campus site than in larger-sized green spaces within Tokyo. Moreover, species compositions in urbanised areas were markedly different from those in suburbanised parks, mountains and forests within Tokyo.

Gondwanan conifer clones imperilled by bushfire.

Global increases in fire frequency driven by anthropogenic greenhouse emissions and land use change could threaten unique and ancient species by creeping into long-term fire refugia. The perhumid and mountainous western half of Tasmania is a globally important refugium for palaeo-endemic, fire intolerant lineages, especially conifers. Reproductive strategy will be crucial to the resilience of these organisms under warmer, dryer and more fire prone climates. This study analysed clonal versus sexual reproduction in old growth plots dominated by the palaeo-endemic conifer Athrotaxis cupressoides (Cupressaceae), a species that lacks any traits to tolerate frequent landscape fire. Across most of the seven plots the amount of sexually derived individuals was lower than clonally derived with, on average, 60% of all stems belonging to the same multi-locus lineage (MLL) (i.e. were clonal). Some MLLs were large spanning over 10 s of metres and consisted of up to 62 stems. The high mortality after fire and the rarity of sexual regeneration means that the range of this fire-intolerant species is likely to contract under enhanced fire regimes and has a limited capacity to disperse via seed to available fire refugia in the landscape.

Wide distribution range of rhizobial symbionts associated with pantropical sea-dispersed legumes.

To understand the geographic distributions of rhizobia that associated with widely distributed wild legumes, 66 nodules obtained from 41 individuals including three sea-dispersed legumes (Vigna marina, Vigna luteola, and Canavalia rosea) distributed across the tropical and subtropical coastal regions of the world were studied. Partial sequences of 16S rRNA and nodC genes extracted from the nodules showed that only Bradyrhizobium and Sinorhizobium were associated with the pantropical legumes, and some of the symbiont strains were widely distributed over the Pacific. Horizontal gene transfer of nodulation genes were observed within the Bradyrhizobium and Sinorhizobium lineages. BLAST searches in GenBank also identified records of these strains from various legumes across the world, including crop species. However, one of the rhizobial strains was not found in GenBank, which implies the strain may have adapted to the littoral environment. Our results suggested that some rhizobia, which associate with the widespread sea-dispersed legume, distribute across a broad geographic range. By establishing symbiotic relationships with widely distributed rhizobia, the pantropical legumes may also be able to extend their range much further than other legume species.

Development and characterization of SSR markers for Aster savatieri (Asteraceae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) markers were developed for Aster savatieri (Asteraceae) and the serpentine variety A. savatieri var. pygmaeus to re-evaluate their taxonomic status. METHODS AND RESULTS: Using RNA-Seq data, 22 expressed sequence tag (EST)-SSR markers were developed. Polymorphisms were assessed in A. savatieri and in A. savatieri var. pygmaeus. The average number of alleles ranged from four to 15, and expected heterozygosity ranged from 0.417 to 0.870. Transferability was examined in six representative species of Japanese Aster and in Solidago virgaurea subsp. asiatica var. asiatica, a member of the tribe Astereae (Asteraceae); most of the loci were transferable to these examined species. CONCLUSIONS: These markers will be useful for genetic studies of variation in A. savatieri and other Aster species that occur in Japan.