Genetic consequences of Last Glacial-Holocene changes in snowfall regime in Arnica mallotopus populations: A plant confined to heavy-snow areas of Japan.

PREMISE: Snow is an important environmental factor affecting plant distribution. Past changes in snowfall regimes may have controlled the demographies of snow-dependent plants. However, our knowledge of changes in the distribution and demographies of such plants is limited because of the lack of fossil records. METHODS: Population genetic and landscape genetic analyses were used to investigate the response of population dynamics of Arnica mallotopus (Asteraceae)-a plant confined to heavy-snow areas of Japan-to changes in snowfall regimes from the Last Glacial Period to the Holocene. RESULTS: The population genetic analysis suggested that the four geographic lineages diverged during the Last Glacial Period. The interaction between reduced snowfall and lower temperatures during this period likely triggered population isolation in separate refugia. Subpopulation differentiation in the northern group was lower than in the southern group. Our ecological niche model predicted that the current distribution was patchy in the southern region; that is, the populations were isolated by topologically flat and climatically unsuitable lowlands. The landscape genetic analysis suggested that areas with little snowfall acted as barriers to the Holocene expansion of species distribution and continued limiting gene flow between local populations. CONCLUSIONS: These findings indicate that postglacial population responses vary among regions and are controlled by environmental and geographic factors. Thus, changes in snowfall regime played a major role in shaping the distribution and genetic structure of the snow-dependent plant.

The absence of bumblebees on an oceanic island blurs the species boundary of two closely related orchids.

Oceanic islands offer valuable natural laboratories for studying evolution. The Izu Islands, with their recent geological origin, provide an exceptional opportunity to explore the initial evolution on oceanic islands. Another noteworthy aspect is the absence of bumblebee species on most Izu Islands. We used ecological, morphological, and molecular data to investigate the impact of bumblebee absence on the evolution of two closely related orchid species, Goodyera henryi and Goodyera similis, focusing on Kozu Island, the Izu Islands. Our investigation revealed that while G. henryi exclusively relies on a bumblebee species for pollination on the mainland, G. similis is pollinated by scoliid wasps on both the mainland and the island. Intriguingly, all specimens initially categorized as G. henryi on Kozu Island are hybrids of G. henryi and G. similis, leading to the absence of pure G. henryi distribution on the island. These hybrids are pollinated by the scoliid wasp species that also pollinates G. similis on the island. The absence of bumblebees might result in sporadic and inefficient pollination of G. henryi by scoliid wasps, consequently promoting hybrid proliferation on the island. Our findings suggest that the absence of bumblebees can blur plant species boundaries.

Host-Shift Speciation Proceeded with Gene Flow in Algae Covering Shells.

AbstractHost shifts represent the advancement of a novel niche and often lead to speciation in symbionts. However, its mechanisms are not well understood. Here, we focused on the alga Pseudocladophora conchopheria growing on the shells of intertidal snails. Previous surveys have shown that the alga has host specificity-only attaching to the shell of Lunella correensis-but we discovered that the alga attaches to the shells of multiple sympatric snails. A genome-wide single-nucleotide polymorphism analysis (MIG-seq) was performed to determine whether host-associated speciation occurred in the algae. As a result, there was no gene flow or limited gene flow among the algae from different hosts, and some algae were genetically differentiated among hosts. In addition, the demographic estimate revealed that speciation with gene flow occurred between the algae from different hosts. Therefore, these results support the idea that host-shift speciation gradually proceeded with gene flow in the algae, providing insight into the early evolution of host shifts.

Molecular phylogeny and taxonomy of Hosta (Asparagaceae) on Shikoku Island, Japan, including five new species, one new subspecies, and two new status assignments.

Japan has 16 native species of the genus Hosta Tratt. (Asparagaceae). A recent study on Hosta based on field surveys and molecular phylogenetic analyses resulted in the discovery of six unknown taxa in Kochi Prefecture, Shikoku Island, southwestern Japan. We aimed to identify these unknown taxa. Therefore, we constructed a finely resolved phylogeny for 320 Hosta samples collected from the Honshu, Shikoku, and Kyushu Islands using multiplex inter-simple sequence repeat genotyping by sequencing (MIG-seq). Based on this phylogenetic analysis and related morphological observations, we describe five new species, H.longipedicellatasp. nov., H.minazukiflorasp. nov., H.polyneuronoidessp. nov., H.samukazemontanasp. nov., and H.takiminazukiflorasp. nov. and one new subspecies, H.takiminazukiflorasubsp.grandissubsp. nov. In addition, we propose two new status assignments, H.tardivasubsp.densinerviacomb. and stat. nov. and H.scabrinerviastat. nov. We also propose classifying H.kikutiivar.tosana as a species, H.tosana. Further studies that combine MIG-seq with careful morphological observations are needed for Hosta plants on all Japanese islands, which may result in the discovery of even more undescribed species.

Phylogeographical evidence for historical long-distance dispersal in the flightless stick insect Ramulus mikado.

Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect Ramulus mikado across Japan, based on a multifaceted molecular approach [cytochrome oxidase subunit I (COI) haplotypes, nuclear simple sequence repeat markers and genome-wide single nucleotide polymorphisms]. Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.

Spiranthes hachijoensis (Orchidaceae), a new species within the S. sinensis species complex in Japan, based on morphological, phylogenetic, and ecological evidence.

The systematics of the Old World Spiranthes sinensis (Pers.) Ames species complex (Orchidaceae) has been complicated by its wide distribution and morphological variations. Within the species complex, S. australis Lindl. has been generally accepted as the only Spiranthes Rich. species distributed on the Japanese mainland. The present study provides morphological, phylogenetic, and ecological evidence for the recognition of S. hachijoensis Suetsugu as a new species of the S. sinensis species complex on the Japanese mainland. Spiranthes hachijoensis is morphologically similar to S. hongkongensis S.Y. Hu & Barretto and S. nivea T.P. Lin & W.M. Lin, sharing a degenerated rostellum, pollinia without a viscidium, and distinctly trilobed stigma. However, the taxon can be morphologically distinguished from S. hongkongensis by its glabrous rachis, ovaries, and sepals, and from S. nivea by its papillate labellum disc, larger papillate basal labellum callosities, and glabrous rachis, ovaries, and sepals. The autogamy and flowering phenology (i.e., earlier flowering) of S. hachijoensis are most likely responsible for premating isolation from the sympatric S. australis. A MIG-seq-based high-throughput molecular analysis indicated that the genetic difference between S. hachijoensis and its putative sister species S. sinensis is comparable to, or even greater than, the genetic difference between pairs of other species within the S. sinensis species complex. Our multifaceted approach strongly supports the recognition of S. hachijoensis as a morphologically, phenologically, phylogenetically, and ecologically distinct species.

Monotropastrum kirishimense (Ericaceae), a new mycoheterotrophic plant from Japan based on multifaceted evidence.

Due to their reduced morphology, non-photosynthetic plants have been one of the most challenging groups to delimit to species level. The mycoheterotrophic genus Monotropastrum, with the monotypic species M. humile, has been a particularly taxonomically challenging group, owing to its highly reduced vegetative and root morphology. Using integrative species delimitation, we have focused on Japanese Monotropastrum, with a special focus on an unknown taxon with rosy pink petals and sepals. We investigated its flowering phenology, morphology, molecular identity, and associated fungi. Detailed morphological investigation has indicated that it can be distinguished from M. humile by its rosy pink tepals and sepals that are generally more numerous, elliptic, and constantly appressed to the petals throughout its flowering period, and by its obscure root balls that are unified with the surrounding soil, with root tips that hardly protrude. Based on genome-wide single-nucleotide polymorphisms, molecular data has provided clear genetic differentiation between this unknown taxon and M. humile. Monotropastrum humile and this taxon are associated with different Russula lineages, even when they are sympatric. Based on this multifaceted evidence, we describe this unknown taxon as the new species M. kirishimense. Assortative mating resulting from phenological differences has likely contributed to the persistent sympatry between these two species, with distinct mycorrhizal specificity.

Genetic Structure of the Liriope muscari Polyploid Complex and the Possibility of Its Genetic Disturbance in Japan.

Anthropogenic activities, such as the movement of plants through greening, can result in genetic disturbance that can interfere with local adaptation in wild populations. Although research is underway to prevent genetic disturbance associated with greening, genetic disturbance of intraspecific polyploidy, which is estimated to be present in 24% of vascular plants, has not been well studied. Liriope muscari is a polyploid complex with known diploid (2n = 36), tetraploid (2n = 72), and hexaploid (2n = 108) forms. The plants of this species tolerate dry and hot conditions and are therefore frequently used for greening and gardening. However, the distribution of this polyploid in Japan, its genetic structure, and genetic disturbance are not known. In this study, we investigated the polyploidy distribution and genetic structure in naturally distributed L. muscari in Japan using chloroplast DNA (cpDNA) haplotypes and nuclear DNA (nDNA). Commercially produced individuals were also studied and compared with natural populations to assess any genetic disturbance of the ploidy complex in this species. Chromosome counts, cpDNA, and nDNA results showed three genetically and cytologically distinct groups in Japan: first, a tetraploid group in mainland Japan; second, a hexaploid group in the Ryukyu Islands; and third, a diploid and tetraploid group in the Ryukyu Islands. Significant isolation by distance was also detected within the three groups (p = 0.001). Genetic disturbance due to greening and gardening should be avoided among the three groups. Genetic disturbance can be reduced by using individuals derived from natural populations that are close to the sites used for greening and gardening. For commercially produced individuals, genetic disturbance is unlikely in the Kanto region, an area of high usage, while genetic disturbance is thought possible in the Ryukyu Islands.

Norway spruce postglacial recolonization of Fennoscandia.

Contrasting theories exist regarding how Norway spruce (Picea abies) recolonized Fennoscandia after the last glaciation and both early Holocene establishments from western microrefugia and late Holocene colonization from the east have been postulated. Here, we show that Norway spruce was present in southern Fennoscandia as early as 14.7 ± 0.1 cal. kyr BP and that the millennia-old clonal spruce trees present today in central Sweden likely arrived with an early Holocene migration from the east. Our findings are based on ancient sedimentary DNA from multiple European sites (N = 15) combined with nuclear and mitochondrial DNA analysis of ancient clonal (N = 135) and contemporary spruce forest trees (N = 129) from central Sweden. Our other findings imply that Norway spruce was present shortly after deglaciation at the margins of the Scandinavian Ice Sheet, and support previously disputed finds of pollen in southern Sweden claiming spruce establishment during the Lateglacial.

Intraspecific independent evolution of floral spur length in response to local flower visitor size in Japanese Aquilegia in different mountain regions.

Geographic differences in floral traits may reflect geographic differences in effective pollinator assemblages. Independent local adaptation to pollinator assemblages in multiple regions would be expected to cause parallel floral trait evolution, although sufficient evidence for this is still lacking. Knowing the intraspecific evolutionary history of floral traits will reveal events that occur in the early stages of trait diversification. In this study, we investigated the relationship between flower spur length and pollinator size in 16 populations of Aquilegia buergeriana var. buergeriana distributed in four mountain regions in the Japanese Alps. We also examined the genetic relationship between yellow- and red-flowered individuals, to see if color differences caused genetic differentiation by pollinator isolation. Genetic relationships among 16 populations were analyzed based on genome-wide single-nucleotide polymorphisms. Even among populations within the same mountain region, pollinator size varied widely, and the average spur length of A. buergeriana var. buergeriana in each population was strongly related to the average visitor size of that population. Genetic relatedness between populations was not related to the similarity of spur length between populations; rather, it was related to the geographic proximity of populations in each mountain region. Our results indicate that spur length in each population evolved independently of the population genetic structure but in parallel in response to local flower visitor size in different mountain regions. Further, yellow- and red-flowered individuals of A. buergeriana var. buergeriana were not genetically differentiated. Unlike other Aquilegia species in Europe and America visited by hummingbirds and hawkmoths, the Japanese Aquilegia species is consistently visited by bumblebees. As a result, genetic isolation by flower color may not have occurred.

Molecular phylogeny and taxonomy of the Hydrangeaserrata complex (Hydrangeaceae) in western Japan, including a new subspecies of H.acuminata from Yakushima.

According to the contemporary classification of Hydrangea native to Japan, H.serrata is a polymorphic species including six varieties. We discovered a plant identified as H.serrata, but morphologically distinct from previously known varieties, in Yakushima island where approximately 50 endemic species are known. To determine the relationship of this plant with previously known varieties, we examined morphology and constructed a highly resolved phylogeny of H.serrata and its relatives using three chloroplast genomic regions, rbcL, trnL intron, psbA-trnH, and two nuclear genomic regions, ITS1 and ITS2, and Multiplex ISSR genotyping by sequencing (MIG-seq). Based on these morphological and phylogenetic observations, we describe Hydrangeaacuminatasubsp.yakushimensissubsp. nov. as a newly discovered lineage in Yakushima, Japan and propose Hydrangeaminamitanii stat. nov. and Hydrangeaacuminatasubsp.australisstat. nov. which were previously treated as varieties of H.serrata.

 Hydrangeamarunoi (Hydrangeaceae), a new species from Osumi Peninsula, southern Japan.

Hydrangeamarunoi Tagane & S. Fujii, from the Kimotsuki Mountains in the Ohsumi Peninsula, southern Japan, is described and illustrated. It is morphologically similar to H.alternifolia in having three-petaloid calyx lobes in marginal flowers, but is distinguished by the larger stamen number, and longer styles and seeds. Multiplex ISSR genotyping by sequencing (MIG-seq) demonstrated that the new species is monophyletic and closely related to H.amamiohsimensis and H.moellendorffii rather than H.alternifolia.

Morphological, ecological, and molecular phylogenetic approaches reveal species boundaries and evolutionary history of Goodyeracrassifolia (Orchidaceae, Orchidoideae) and its closely related taxa.

Species delimitation within the genus Goodyera is challenging among closely related species, because of phenotypic plasticity, ecological variation, and hybridization that confound identification methods based solely on morphology. In this study, we investigated the identity of Goodyeracrassifolia H.-J.Suh, S.-W.Seo, S.-H.Oh & T.Yukawa, morphologically similar to Goodyeraschlechtendaliana Rchb.f. This recently described taxon has long been known in Japan as "Oh-miyama-uzura" or "Gakunan" and considered a natural hybrid of G.schlechtendaliana and G.similis Blume (= G.velutina Maxim. ex Regel). Because the natural hybrid between G.schlechtendaliana and G.similis was described as G.×tamnaensis N.S.Lee, K.S.Lee, S.H.Yeau & C.S.Lee before the description of G.crassifolia, the latter might be a synonym of G.×tamnaensis. Consequently, we investigated species boundaries and evolutionary history of G.crassifolia and its closely related taxa based on multifaceted evidence. Consequently, morphological examination enabled us to distinguish G.crassifolia from other closely related species owing to the following characteristics: coriaceous leaf texture, laxly flowered inflorescence, long pedicellate ovary, large and weakly opened flowers, and column with lateral appendages. Ecological investigation indicates that G.crassifolia (2n = 60) is agamospermous, requiring neither pollinators nor autonomous self-pollination for fruit set, whereas G.schlechtendaliana (2n = 30) is neither autogamous nor agamospermous but is obligately pollinator-dependent. MIG-seq-based phylogenetic analysis provided no evidence of recent hybridization between G.crassifolia and its close congeners. Thus, molecular phylogeny reconstructed from MIG-seq data together with morphological, cytological, and ecological analyses support the separation of G.crassifolia as an independent species.

Validation of Hostaalata (Asparagaceae) as a new species and its phylogenetic affinity.

Molecular phylogenetic studies of Hostapulchella (Asparagaceae) and its relatives, which are native to Japan, have been conducted and resulted in a highly resolved phylogeny. Specifically, the relationship of H.pulchella to H.alata Hatusima, nom. nud. is investigated. These data include genome-wide SNPs obtained through conducting multiplexed ISSR genotyping by sequencing (MIG-seq). Based on these phylogenetic results, morphological observations, distribution, and differences in flowering periods of H.alata collections sympatric with H.pulchella, we find the two species closely related, but distinct. As such, we formally describe Hostaalata sp. nov. from the Oita Prefecture of Kyushu island, southwestern Japan.

A strategic sampling design revealed the local genetic structure of cold-water fluvial sculpin: a focus on groundwater-dependent water temperature heterogeneity.

A key piece of information for ecosystem management is the relationship between the environment and population genetic structure. However, it is difficult to clearly quantify the effects of environmental factors on genetic differentiation because of spatial autocorrelation and analytical problems. In this study, we focused on stream ecosystems and the environmental heterogeneity caused by groundwater and constructed a sampling design in which geographic distance and environmental differences are not correlated. Using multiplexed ISSR genotyping by sequencing (MIG-seq) method, a fine-scale population genetics study was conducted in fluvial sculpin Cottus nozawae, for which summer water temperature is the determinant factor in distribution and survival. There was a clear genetic structure in the watershed. Although a significant isolation-by-distance pattern was detected in the watershed, there was no association between genetic differentiation and water temperature. Instead, asymmetric gene flow from relatively low-temperature streams to high-temperature streams was detected, indicating the importance of low-temperature streams and continuous habitats. The groundwater-focused sampling strategy yielded insightful results for conservation.

Evolutionary history of Hemerocallis in Japan inferred from chloroplast and nuclear phylogenies and levels of interspecific gene flow.

The perennial herb genus Hemerocallis (Asphodelaceae) shows four flowering types: diurnal half-day, diurnal one-day, nocturnal half-day, and nocturnal one-day flowering. These flowering types are corresponding to their main pollinators, and probably act as a primary mechanism of reproductive isolation. To examine how the four flowering types diverged, we reconstructed the phylogeny of the Japanese species of Hemerocallis using 1615 loci of nuclear genome-wide SNPs and 2078 bp sequences of four cpDNA regions. We also examined interspecific gene flows among taxa by an Isolation-with-Migration model and a population structure analysis. Our study revealed an inconsistency between chloroplast and nuclear genome phylogenies, which may have resulted from chloroplast capture. Each of the following five clusters is monophyletic and clearly separated on the nuclear genome-wide phylogenetic tree: (I) two nocturnal flowering species with lemon-yellow flowers, H. citrina (half-day flowering) and H. lilioasphodelus (one-day flowering); (II) a diurnal one-day flowering species with yellow-orange flowers, H. middendorffii; (III) a variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. disticha; (IV) another variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. aurantiaca, and a diurnal one-day flowering species with yellow-orange flowers, H. major; (V) a diurnal half-day flowering species with yellow-orange flowers, H. hakuunensis. The five clusters are consistent with traditional phenotype-based taxonomy (cluster I, cluster II, and clusters III-V correspond to Hemerocallis sect. Hemerocallis, Capitatae, and Fulvae, respectively). These findings could indicate that three flowering types (nocturnal flowering, diurnal one-day flowering, and diurnal half-day flowering) diverged in early evolutionary stages of Hemerocallis and subsequently a change from diurnal half-day flowering to diurnal one-day flowering occurred in a lineage of H. major. While genetic differentiation among the five clusters was well maintained, significant gene flow was detected between most pairs of taxa, suggesting that repeated hybridization played a role in the evolution of those taxa.

Comparative analysis of spatial genetic structures in sympatric populations of two riparian plants, Saxifraga acerifolia and Saxifraga fortunei.

PREMISE: The genetic structure between plant populations is facilitated by the spatial population arrangement and limited dispersal of seed and pollen. Saxifraga acerifolia, a local endemic species in Japan, is a habitat specialist that is confined to waterfalls in riparian environments. Its sister species, Saxifraga fortunei, is a generalist that is widely distributed along riverbanks. Here, we examined sympatric populations of the two Saxifraga species to test whether the differences in habitat preference and colonization process influenced regional and local genetic structures. METHODS: To reveal genetic structures, we examined chloroplast microsatellite variations and genome-wide nucleotide polymorphisms obtained by genotyping by sequencing. We also estimated the gene flow among and within populations and performed landscape genetic analyses to evaluate seed and pollen movement and the extent of genetic isolation related to geographic distance and/or habitat differences. RESULTS: We found strong genetic structure in the specialist S. acerifolia, even on a small spatial scale (<1 km part); each population on a different waterfall in one river system had a completely different predominant haplotype. By contrast, the generalist S. fortunei showed no clear genetic differentiation. CONCLUSIONS: Our findings suggest that the level of genetic isolation was increased in S. acerifolia by the spatially fragmented habitat and limited seed and pollen dispersal over waterfalls. Habitat differentiation between the sister taxa could have contributed to the different patterns of gene flow and then shaped the contrasting genetic structures.

A new subspecies of Stellariaalsine (Caryophyllaceae) from Yakushima, Japan.

An unknown taxon of Stellaria was discovered in Yakushima, a Japanese island known to harbor several endemic species. To determine the identity of this taxon, this study employed MIG-seq for the reconstruction of a finely resolved phylogenetic tree of the newly discovered taxon, along with some related species of Stellaria. The results showed that the newly discovered taxon is a relative of S.alsine. Based on this result, Stellariaalsinesubsp.nanasubsp. nov. was published.

UV bullseye contrast of Hemerocallis flowers attracts hawkmoths but not swallowtail butterflies.

The color and patterns of animal-pollinated flowers are known to have effects on pollinator attraction. In this study, the relative importance of flower color and color contrast patterns on pollinator attraction was examined in two pollinator groups, swallowtail butterflies and hawkmoths using two Hemerocallis species; butterfly-pollinated H. fulva and hawkmoth-pollinated H. citrina, having reddish and yellowish flowers in human vision, respectively. Flowers of both species have UV bullseye patterns, composed of UV-absorbing centers and UV-reflecting peripheries, known to function as a typical nectar guide, but UV reflectance was significantly more intense in the peripheries of H. citrina flowers than in those of H. fulva flowers. Comparison based on the visual systems of butterflies and hawkmoths showed that the color contrast of the bullseye pattern in H. citrina was more intense than that in H. fulva. To evaluate the relative importance of flower color and the color contrast of bullseye pattern on pollinator attraction, we performed a series of observations using experimental arrays consisting of Hemerocallis species and their hybrids. As a result, swallowtail butterflies and crepuscular/nocturnal hawkmoths showed contrasting preferences for flower color and patterns: butterflies preferred H. fulva-like colored flower whereas the preference of hawkmoths was affected by the color contrast of the bullseye pattern rather than flower color. Both crepuscular and nocturnal hawkmoths consistently preferred flowers with stronger contrast of the UV bullseye pattern, whereas the preference of hawkmoths for flower color was incoherent. Our finding suggests that hawkmoths can use UV-absorbing/reflecting bullseye patterns for foraging under light-limited environments and that the intensified bullseye contrast of H. citrina evolved as an adaptation to hawkmoths. Our results also showed the difference of visual systems between pollinators, which may have promoted floral divergence.