Two lineages of Lemna aequinoctialis (Araceae, Lemnoideae) based on physiology, morphology, and phylogeny.

Lemna aequinoctialis Welw. is a widely spread species that has diverse physiological and molecular properties. Flower characteristics are important factors in deducing taxonomical status; however, owing to the rarity of flowering observations in Lemna, studying them has been a prolonged challenge. In this study, physiological and morphological analyses were conducted by inducing flowering, and molecular analysis was done based on the two chloroplast DNA loci (matK, atpF-atpH intergeneric spacer) of L. aequinoctialis sensu Landolt (1986) from 70 strains found in 70 localities in Japan, Korea, Thailand, and the US. In total, 752 flowering fronds from 13 strains were observed based on axenic conditions. Two different trends in flower organ development-protogyny and adichogamy-were detected in these strains. Their physiological traits were divided into two groups, showing different morphological features based on frond thickness, root cap, and anther sizes. Molecular analysis showed two lineages corresponding to two physiological groups. These were identified as L. aequinoctialis sensu Beppu et al. (1985) and L. aoukikusa Beppu et Murata based on the description of the nomenclature of L. aoukikusa. These were concluded as independent taxa and can be treated as different species. Furthermore, the distribution of L. aoukikusa is not only limited to Japan.

Gene expression shapes the patterns of parallel evolution of herbicide resistance in the agricultural weed Monochoria vaginalis.

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterization and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

Ecotypic divergences of the alpine herb Potentilla matsumurae adapted to fellfield-snowbed habitats across a series of mountain sky islands.

PREMISE: Divergent selection due to environmental heterogeneity can lead to local adaptation. However, the ecological and evolutionary processes of local adaptation that occurs across multiple regions are often unknown. Our previous studies reported on the ecotypic divergence within a local area of variation of Potentilla matsumurae, an alpine herb adapted to the fellfield-snowbed environment. Here we investigated large-scale geographic patterns of ecotypic differentiation in this species to infer local adaptation and selective forces across multiple regions. METHODS: We compiled information on the overall distributions of fellfield and snowbed habitats on the mountains in Japan across the distribution of the species. Next, we conducted common garden experiments to test the adaptive divergence of the fellfield-snowbed plants derived from multiple regions. Finally, we evaluated phylogeographic structures based on cpDNA and allozyme variations and inferred the evolutionary history of ecotype differentiation. RESULTS: The mosaic distribution of the fellfield-snowbed ecotypes across isolated mountaintops constitutes indirect evidence for habitat-specific natural selection. The significant difference in survivorship between the ecotypes observed in a controlled snow environment provides more substantial evidence of local selection. Phylogeographic structures support the hypothesis that ecotypic divergence events from fellfield to snowbed populations occurred independently in at least two distinct regions. CONCLUSIONS: Ecotypic divergence of P. matsumurae has occurred across a series of mountain sky islands. Local selection in snowy environments is a driving force that maintains the divergent ecotypes across multiple mountain regions and can contribute to the diversification of plants in heavy-snow regions.

Evaluation of genetic consequences of stocking on the southern-margin populations of white-spotted charr.

Coldwater-adapted freshwater fishes, especially their populations along warm-range margins, are most vulnerable to the climate oscillations associated with global warming. Stocking is a major strategy for avoiding the extinction of these species. However, while stocking can reverse the decline of isolated populations, it may also result in a loss of genetic diversity in the native local population due to the introgressive replacement of hatchery genes. To plan an adequate strategy for conserving locally adapted populations, the genetic impacts of stocking on native lineages should be evaluated from small river branches to wide-ranging drainage areas. We investigated the population genetic structure of white-spotted charr (Salvelinus leucomaenis) within its southern range (Lake Biwa basin, Japan). By applying genome-wide SNP analysis to the population's genetic structure, we assessed the extent of genetic introgression resulting from stocking. White-spotted charr in the Lake Biwa watershed constitutes a distinctive genetic group, within which apparent genetic differentiation was observed. The hatchery-reared fish line commonly used for supplementation stocking in the catchment was discernable from the native population, enabling us to analyze genetic introgression across the entire drainage area. Admixed individuals resulting from hatchery introgression were observed in most of the stocked sites that showed relatively high heterozygosity and nucleotide diversity. However, their genetic differentiation was much lower than that of native populations. The supplementation history as well as the road availability contributed substantially to the introgression of hatchery genes. Populations with the native genetic structure remained in the upstream regions of the tested rivers. However, their heterozygosity and nucleotide diversity were low when compared with that of the populations with hatchery supplementation. Our results shed light on the genetic impacts of stocking on isolated native populations and suggest that conventional supplementation methods cannot preserve a unique biodiversity in the distribution margin.

Phylogeography of mugwort (Artemisia indica), a native pioneer herb in Japan.

Many phylogeographic studies of various tree species have been conducted to elucidate the locations of refugia and the colonization patterns during the Pleistocene. However, only a few large-scale phylogeographic studies have been conducted on herbaceous plants, especially scarce on herbs that are adapted to disturbance. Artemisia indica is a fast-growing perennial herb found in open habitats. To examine the basic information on the genetic structure of this species, we investigated the chloroplast DNA variation within and among populations across Japan. We detected 26 haplotypes in 604 individuals from 28 Japanese populations. The haplotype A1 had wide geographic distribution, and its close relatives were locally present. Some putative ancestral lineages were found mainly in the Kyushu region. This may be because several lineages migrated from the Eurasian continent to the northern coast in Kyushu via the Korean peninsula during the Pleistocene, and the A1 haplotype expanded northward, whereas others remained in southern areas. Phylogenetic distant haplotypes were present mainly in the Kanto region. Because the geographic distribution pattern of these haplotypes in this region is believed to be unnatural, these haplotypes may be derived from commercial sources for re-vegetation during the last few decades.

Copy Number Variation in Acetolactate Synthase Genes of Thifensulfuron-Methyl Resistant Alopecurus aequalis (Shortawn Foxtail) Accessions in Japan.

Severe infestations of Alopecurus aequalis (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of A. aequalis growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in ALS gene copy numbers among accessions were revealed. Two copies, ALS1 and ALS2, were conserved in all accessions, while some carried two additional copies, ALS3 and ALS4. A single-base deletion in ALS3 and ALS4 further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the ALS gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the ALS1 or ALS2 gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each ALS gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of ALS genes in A. aequalis will aid future analysis of the mechanism of resistance.

Morphological and genetic variations of Potentilla matsumurae (Rosaceae) between fellfield and snowbed populations.

Identifying ecological factors associated with local differentiation of populations is important for understanding microevolutionary processes. Alpine environments offer a unique opportunity to investigate the effects of habitat-specific selective forces and gene flow limitations among populations at a microscale on local adaptation because the heterogeneous snowmelt patterns in alpine ecosystems provide steep environmental changes. We investigated the variation in morphological traits and enzyme loci between fellfield and snowbed populations of Potentilla matsumurae, a common alpine herb with a wide distribution along snowmelt gradients in northern Japan. We found significant differences in morphological traits between fellfield and snowbed habitats in a northern distribution region. These differences were maintained when plants were grown under uniform conditions in a greenhouse. Allozyme variations among 15 populations from geographically separated regions with different historical backgrounds showed that the populations are more genetically differentiated between the fellfield and snowbed habitats within a region than between populations occupying the same habitat type in different regions. These results suggest that variation in snowmelt regimes could be a driving force creating local adaptation and genetic differentiation of alpine plant populations.

Intraspecific variations in seedling emergence and survival of Potentilla matsumurae (Rosaceae) between alpine fellfield and snowbed habitats.

Potentilla matsumurae has a wide distribution from wind-blown fellfields to snowbeds in alpine regions of Japan. The environmental factors influencing seedling establishment differ between the fellfield and snowbed habitats; plants growing in each habitat may therefore have different germination strategies. Using a reciprocal sowing experiment, patterns of seedling emergence and survivorship were examined in both habitat types in the Taisetsu Mountains, Japan. Seeds derived from a fellfield population germinated earlier than did those derived from a snowbed population at both habitats, and the germination of fellfield seeds continued throughout the growing season. The timing of seedling emergence greatly affected subsequent survival at the fellfield. Seedlings that emerged in the first half of the growing season had low survivorship during the first year because of frost and drought damage, but the remaining seedlings had high survivorship during the winter; seedlings that emerged in the latter half of the growing season showed the opposite trend. At the snowbed, seedling survival was high throughout the growing season. Germination experiments in the laboratory highlighted a difference in the sensitivity of seeds from the fellfield and snowbed populations to fluctuating temperatures. These results indicate that intraspecific variation in emergence and survivorship may occur over a small scale in an alpine environment.