Masayuki Maki's research while affiliated with Tohoku University and other places

Interspecific hybridization between the short corolla tube species Isodon inflexus and the long corolla tube species I . longitubus was analyzed using genetic and morphometric markers. Bayesian clustering analysis using microsatellites revealed that plants in the contact zone consisted of two I . inflexus individuals, 33 I . longitubus individuals, and 13 hybrid individuals (F 2 -like and backcrosses to both I . inflexus and I . longitubus ). Using the sequences of the psbA-trnH inter genic spacer in chloroplast DNA, three haplotypes were found among pure I . inflexus , while four haplotypes were found among pure I . longitubus . Most hybrid individuals had a haplotype found only in I . inflexus , suggesting that the initial F 1 might have been formed by hybridization with I . inflexus as the maternal parent, and that in later hybrid formation, hybrids or I . inflexus tended to serve as the maternal parent. Although strong prezygotic isolation mechanisms contribute to preventing hybridization between the species, human habitat disturbance might have created the contact zone. Although mature hybrids in the contact zone contained equal proportions of backcrosses to both I . inflexus and I . longitubus , seedlings comprised more individuals originating from backcrosses to I . longitubus . The dominance of backcrosses to I . longitubus was probably caused by the quantitative difference between the parental species in the contact zone. In the contact zone, signs of contemporary hybridization in the nuclear genome might have been diluted by repeated backcrossing. The present study could illustrate the process of unidirectional introgression leading to chloroplast capture, a phenomenon frequently observed in Isodon species in Japan.

The genus Symplocarpus in basal Araceae includes both thermogenic and non/slightly thermogenic species that prefer cold environments. If floral thermogenesis of Symplocarpus contributes to cold adaptation, it would be expected that thermogenic species have a larger habitat than non/slightly thermogenic species during an ice age, leading to increased genetic diversity in the current population. To address this question, potential distribution in past environment predicted by ecological niche modeling (ENM), genetic diversity, and population structure of chloroplast and genome-wide single nucleotide polymorphisms were compared between thermogenic Symplocarpus renifolius and non/slightly thermogenic Symplocarpus nipponicus. ENM revealed that the distribution of S. nipponicus decreased, whereas that of S. renifolius expanded in the Last Glacial Maximum. Phylogeographic analyses have shown that the population structures of the two species were genetically segmented and that the genetic diversity of S. renifolius was higher than that of S. nipponicus. The phylogenetic relationship between chloroplast and nuclear DNA is topologically different in the two species, which may be due to the asymmetric gene flow ubiquitously observed in plants. The results of this study imply that floral thermogenesis of Symplocarpus contributes to expanding the distribution during an ice age, resulting in increased genetic diversity due to cold adaptation.

Although the monophyly of Phedimus has been strongly demonstrated, the species relationships among approximately 20 species of Phedimus have been difficult to determine because of the uniformity of their floral characteristics and extreme variation of their vegetative characters, often accompanied by high polyploid and aneuploid series and diverse habitats. In this study, we assembled 15 complete chloroplast genomes of Phedimus species from East Asia and generated a plastome-based backbone phylogeny of the subgenus Aizoon. As a proxy for nuclear phylogeny, we reconstructed the nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) phylogeny independently. The 15 plastomes of subg. Aizoon were highly conserved in structure and organization; hence, the complete plastome phylogeny fully resolved the species relationships with strong support. We found that P. aizoon and P. kamtschaticus were polyphyletic and morphologically distinct or ambiguous species, and they most likely evolved from the two species complex. The crown age of subg. Aizoon was estimated to be 27 Ma, suggesting its origin to be in the late Oligocene; however, the major lineages were diversified during the Miocene. The two Korean endemics, P. takesimensis and P. zokuriensis, were inferred to have originated recently during the Pleistocene, whereas the other endemic, P. latiovalifolium, originated in the late Miocene. Several mutation hotspots and seven positively selected chloroplast genes were identified in the subg. Aizoon.

The evolutionary transition from outcrossing in heterostylous morphs to selfing in homostylous morphs has occurred in many plant lineages. Homostylous variants with a low degree of herkogamy may increase the reproductive advantage of autonomous self‐pollination when there is little inbreeding depression and/or low pollinator visitation. Oxalis corniculata , a self‐compatible perennial herb derived from a tristylous ancestor, is reported to have two floral morphs. Homostyled plants are broadly distributed in Japan, whereas long‐styled plants are reported to occur only in coastal areas. We examined the hypothesis that a reproductive advantage in avoiding inbreeding depression and/or frequent pollinator visits enables long‐styled plants to live in some areas. Studies on floral variation revealed that the degree of herkogamy varied widely and continuously within and among populations. Homostyled and long‐styled morphs were functionally defined based on the ability of autonomous self‐pollination. Homostyled plants were widely distributed, whereas long‐styled plants were found in and around the two coastal populations and relatively inland populations. Pollination experiments provided no evidence of inbreeding depression in either homostyled or long‐styled plants. Pollinator visitation frequency was higher in the two coastal populations than in the other populations. Thus, reproductive disadvantage in long‐styled plants lacking autonomous self‐pollination has reduced in the two coastal populations, and long‐styled plants may be maintained due to a competitive advantage of large seed size. In contrast, we recorded no comparable frequencies of pollinator visitation in other populations containing long‐styled plants, indicating that there are other factors contributing to the maintenance of long‐styled plants in these areas.

We explored the genetic diversity and population genetic structure of an invasive alien species, the raccoon Procyon lotor, which was introduced into the Shikoku Island of Japan, using sequences of the mitochondrial control region and 10 polymorphic microsatellite loci. In 80 individuals examined, we detected two genetically divergent haplotypes, indicating that two maternal lineages had been introduced into Shikoku Island. Population clustering analysis suggested the nonexistence of genetic structure in the area. The relatedness network showed that the raccoon population in Shikoku Island consisted of closely related individuals. The genetic diversity of the Shikoku raccoon population was lower than that in the native range. These results and a prior report indicate that the Shikoku raccoon population is likely to have expanded from small numbers of founders originating from an initial invasive population. Raccoon captures outside Kagawa are decreasing, although past expansions into Tokushima and Ehime suggest that there are no geographical barriers among these areas. Therefore, action should be taken to prevent any re-expansion of the raccoon population in Kagawa. Intensive monitoring and the elimination of dispersers from Kagawa, as well as effort to eradicate potential source populations for expansion in Kagawa, are needed.

Thalictrum aquilegiifolium (Ranunculaceae) is widely distributed in the Eurasian Continent and Japan and comprises some intraspecific taxa. We report here the complete chloroplast genome of T. aquilegiifolium var. sibiricum. The plastome of T. aquilegiifolium var. sibiricum is 156,074 bp in length, containing large (85,457 bp) and small (17,642 bp) single-copy regions which are separated by a pair of inverted repeats (26,487 bp each). The genome consists of 119 genes, including 88 protein-coding, four ribosomal RNA genes, and 27 transfer RNA genes. Our phylogenetic analysis revealed that Thalictrum species formed a highly supported clade, indicating that these species are monophyletic.

Anaphalis margaritacea var. yedoensis is a perennial herb adapted to the severe environment of pebbled river banks, where it is frequently found. In this study, we determined the complete chloroplast genome of A. margaritacea var. yedoensis and uncovered its phylogenetic relationships with other members of Gnaphalieae. The total chloroplast genome size of A. margaritaceae var. yedoensis is 153,231 bp, with a large single-copy region (LSC) of 84,981 bp, a small single-copy region (SSC) of 18,481 bp and a pair of inverted repeat (IR) regions of 24,885 bp. A total of 136 genes were annotated, including 39 tRNA genes, 8 rRNA genes, and 89 protein-coding genes. Phylogenetic analysis showed that A. margaritacea var. yedoensis and another Anaphalis species, A. sinica, do not form a monophyletic group, supporting previous phylogenetic studies using some specific regions of cpDNA that showed the genus Anaphalis is non-monophyletic.

Natural hybridization between the native species Rumex madaio endemic to Japan and the congeneric invasive species R. obtusifolius, which frequently co‐occurs with R. madaio, was examined by molecular identification. At low frequencies, interspecific hybrids between the two species were confirmed using restriction fragment length polymorphism analyses of the internal transcribed spacer region of ribosomal DNA. Whereas these two species can hybridize because their flowering periods overlapped to some extent, the extremely low fertility of R. madaio caused by unknown genetic factors seemed to prevent extensive hybridization between these species. Population genetic analyses based on single nucleotide polymorphisms showed that there was a distinct population genetic structure in R. madaio, suggesting that local populations should be treated separately for genetic conservation of the species.

Adaptation to local pollination regimes and secondary contact of allopatrically differentiated populations with respect to pollination ecology may result in geographical variation in floral traits. We examined the contributions of these two processes in Isodon shikokianus , which showed remarkable geographical variation in corolla tube length in western Japan. Corolla tube length varied among 17 study populations located within a relatively narrow area, and covaried with altitude and distribution of two bumblebee pollinators with different tongue lengths: the longer corolla was found at lower altitudes where the long‐tongued pollinator was more abundant, and vice versa. Additionally, bumblebee species preferentially visited flowers that fit their tongue lengths. Population genetic analysis based on 11 microsatellite loci revealed that populations with long and short corolla tubes constituted genetically distinct groups. Migration rates were low between the groups, but high within each group. These results indicate that two genetically differentiated groups made secondary contact and hybridized, and gene flow between the groups was limited. Thus, the geographical variation in corolla tube length in I. shikokianus may be a result of past allopatric differentiation and subsequent secondary contact of populations with different corolla tube lengths. The variation in corolla tube length within a narrow area may be maintained by selection owing to the altitudinally structured pollinator assemblages. Altitudinal differences in relative abundance of two pollinators and their assortative visitation with respect to corolla tube length may contribute to reproductive isolation between the two groups.