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.

Evolutionary radiation of the Eurasian Pinus species under pervasive gene flow.

Evolutionary radiation, a pivotal aspect of macroevolution, offers valuable insights into evolutionary processes. The genus Pinus is the largest genus in conifers with c . 90% of the extant species emerged in the Miocene, which signifies a case of rapid diversification. Despite this remarkable history, our understanding of the mechanisms driving radiation within this expansive genus has remained limited. Using exome capture sequencing and a fossil-calibrated phylogeny, we investigated the divergence history, niche diversification, and introgression among 13 closely related Eurasian species spanning climate zones from the tropics to the boreal Arctic. We detected complex introgression among lineages in subsection Pinus at all stages of the phylogeny. Despite this widespread gene exchange, each species maintained its genetic identity and showed clear niche differentiation. Demographic analysis unveiled distinct population histories among these species, which further influenced the nucleotide diversity and efficacy of purifying and positive selection in each species. Our findings suggest that radiation in the Eurasian pines was likely fueled by interspecific recombination and further reinforced by their adaptation to distinct environments. Our study highlights the constraints and opportunities for evolutionary change, and the expectations of future adaptation in response to environmental changes in different lineages.

Evolution of secondary metabolites, morphological structures and associated gene expression patterns in galls induced by four closely related aphid species on a host plant species.

Gall-forming insects induce various types of galls on their host plants by altering gene expression in host plant organs, and recent studies have been conducted for gene expression in galls. However, the evolutionary trajectories of gene expression patterns and the resulting phenotypes have not yet been studied using multiple related species. We investigated the speciation and the diversification process of galls induced by four closely related aphid species (Hormaphidini) on a host plant species (Hamamelis japonica) by examining the phylogenetic congruence between the geographical divergences of aphids and the host plant, and by comparing their gene expression patterns and resulting phenotypes. Phylogenetic analysis of aphids and the host plant showed that geographical isolation among host plant populations has interrupted gene flow in aphids and accelerated the speciation process. The concentration of phenolics and the complexity of the internal structure of galls were correlated with the expression levels of genes for the biosynthesis of phenolics and morphogenesis respectively. These results suggest that the expression levels of genes for the biosynthesis of phenolics and morphogenesis have evolutionarily increased in galls accelerated by the speciation process of aphids due to the distribution change of the host plant, leading to the related phenotypic evolution. Our study showed the evolutionary process of phenotypic traits in galls in the wild from both gene expression and actual phenotype levels.

The phylogenetic reconstruction of the Neotropical cycad genus Ceratozamia (Zamiaceae) reveals disparate patterns of niche evolution.

The cycad genus Ceratozamia comprises 40 species from Mexico, Guatemala, Belize, and Honduras, where cycads occur throughout climatically varied montane habitats. Ceratozamia has the potential to reveal the history and processes of species diversification across diverse Neotropical habitats in this region. However, the species relationships within Ceratozamia and the ecological trends during its evolution remain unclear. Here, we aimed to clarify the phylogenetic relationships, the timing of clade and species divergences, and the niche evolution throughout the phylogenetic history of Ceratozamia. Genome-wide DNA sequences were obtained with MIG-seq, and multiple data-filtering steps were used to optimize the dataset used to construct an ultrametric species tree. Divergence times among branches and ancestral niches were estimated. The niche variation among species was evaluated, summarized into two principal components, and their ancestral states were reconstructed to test whether niche shifts among branches can be explained by random processes, under a Brownian Motion model. Ceratozamia comprises three main clades, and most species relationships within the clades were resolved. Ceratozamia has diversified since the Oligocene, with major branching events occurring during the Miocene. This timing is consistent with fossil evidence, the timing estimated for other Neotropical plant groups, and the major geological events that shaped the topographic and climatic variation in Mexico. Patterns of niche evolution in the genus do not accord with the Brownian Motion model. Rather, non-random evolution with shifts towards more seasonal environments at high latitudes, or shifts towards humid or dry environments at low latitudes explain the diversification of Ceratozamia. We present a comprehensive phylogenetic reconstruction for Ceratozamia and identify for the first time the environmental factors involved in clade and species diversification within the genus. This study alleviates the controversies regarding the species relationships in the genus and provides the first evidence that latitude-associated environmental factors may influence processes of niche evolution in cycads.

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.

Fine-scale clonal structure of the lingonberry Vaccinium vitis-idaea under the nurse plant Pinus pumila vegetation in an alpine region, Mt. Norikura.

The nurse effect is a positive interaction in which a nurse plant improves the abiotic environment for another species (beneficiary plant) and facilitates its establishment. The evergreen shrub Vaccinium vitis-idaea (a beneficiary plant) grows mainly under the dwarf shrub Pinus pumila (a nurse plant) in the alpine regions of central Japan. However, whether V. vitis-idaea shrubs under various P. pumila shrubs spread through clonal growth and/or seeds remains unclear. We investigated the clonal structure of V. vitis-idaea under the nurse plant P. pumila in Japanese alpine regions. MIG-seq analysis was conducted to clarify the clonal diversity of V. vitis-idaea in isolated and patchy P. pumila plots on a ridge (PATs), and in a plot covered by dense P. pumila on a slope adjacent to the ridge (MAT) on Mt. Norikura, Japan. We detected 28 multilocus genotypes in 319 ramets of V. vitis-idaea across 11 PATs and MAT. Three genets expanded to more than 10 m in the MAT. Some genets were shared among neighboring PATs or among PATs and MAT. These findings suggest that the clonal growth of V. vitis-idaea plays an important role in the sustainability of populations. The clonal diversity of V. vitis-idaea was positively related with the spatial size of PATs and was higher in MAT than in PATs at a small scale. Therefore, the spatial spread of the nurse plant P. pumila might facilitate the nurse effect on the genetic diversity of beneficiary plants, leading to the sustainability of beneficiary populations.

5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species.

Standard models of plant speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality in wind-pollinated trees with long evolutionary histories is more complex: species evolve from other species through isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Therefore, it can provide unique insights into the dynamic speciation processes of lineages that diversified tens of millions of years ago. Here, we provide the first high-throughput sequencing (HTS) of the 5S intergenic spacers (5S-IGS) for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata - F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4963 unique 5S-IGS variants reflect a complex history of hybrid origins, lineage sorting, mixing via secondary gene flow, and intra-genomic competition between two or more paralogous-homoeologous 5S rDNA lineages. We show that modern species are genetic mosaics and represent a striking case of ongoing reticulate evolution during the past 55 million years.

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.

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.

Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change.

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.

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.

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data.

A group of temperate grassland plant species termed the "Mansen elements" occurs in Japan and is widely distributed in the grasslands of continental East Asia. It has been hypothesized that these species are continental grassland relicts in Japan that stretch back to a colder age, but their migration history has not been elucidated. To assess the migration history of the Mansen elements, we performed phylogeographic analyses of Tephroseris kirilowii, a member of this group, using single-nucleotide polymorphisms (SNPs) obtained from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq). It was estimated that the Japanese populations of T. kirilowii were divided from those of continental East Asia at 25.2 thousand years ago (ka) with 95% highest probability density interval (HPD) of 15.3-40.0 ka and that Japanese clades first diverged at 20.2 ka with 95% HPD of 10.4-30.1 ka. As the climatically suitable range during the last glacial maximum (LGM) estimated using ecological niche modeling (ENM) was limited in Japan and there was a slight genetic differentiation among Japanese populations, a post-glacial expansion of T. kirilowii in the Japanese Archipelago was indicated.

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.

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.

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.

Genetic consequences of plant edaphic specialization to solfatara fields: Phylogenetic and population genetic analysis of Carex angustisquama (Cyperaceae).

Edaphic specialization is one of the main drivers of plant diversification and has multifaceted effects on population dynamics. Carex angustisquama is a sedge plant growing only on heavily acidified soil in solfatara fields, where only extremophytes can survive. Because of the lack of closely related species in similar habitats and its disjunct distribution, the species offers ideal settings to investigate the effects of adaptation to solfatara fields and of historical biogeography on the genetic consequences of plant edaphic specialization to solfatara fields. Here, genome-wide single nucleotide polymorphisms were used to reveal the phylogenetic origin of C. angustisquama, and 16 expressed sequence tag-simple sequence repeat markers were employed to infer population demography of C angustisquama. Molecular phylogenetic analysis strongly indicated that C. angustisquama formed a monophyletic clade with Carex doenitzii, a species growing on nonacidified soil in the sympatric subalpine zone. The result of population genetic analysis showed that C. angustisquama has much lower genetic diversity than the sister species, and notably, all 16 loci were completely homozygous in most individuals of C. angustisquama. Approximate Bayesian computation analysis supported the model that assumed hierarchical declines of population size through its evolutionary sequence. We propose that the edaphic specialist in solfatara fields has newly attained the adaptation to solfatara fields in the process of speciation. Furthermore, we found evidence of a drastic reduction in genetic diversity in C. angustisquama, suggesting that the repeated founder effects associated with edaphic specialization and subsequent population demography lead to the loss of genetic diversity of this extremophyte in solfatara fields.

Role of ancient lakes in genetic and phenotypic diversification of freshwater snails.

Endemic organisms of ancient lakes have been studied as models to understand processes of speciation and adaptive radiation. However, it remains unclear how ancient lakes play roles in genetic and phenotypic diversity of freshwater mollusks. In the present study, we focus on viviparid freshwater snails in the ancient lakes of East and Southeast Asia (Japan and China) to address this question. Using molecular phylogenetic analyses based on mitochondrial (COI, 16S) and nuclear genes (18S, 28S, H3), we show that patterns of species diversification in viviparid lineages. Colonization to ancient lakes occurred independently in China and Japan at least four times, with subsequent diversification into more than two species within each lake group. Morphological analyses of fossil related viviparids suggest parallel phenotypic evolution occurred in the different lakes and ages. Each lake contained a single lineage which was phenotypically diversified relative to those from other sites. Using genome-wide SNPs obtained by MIG-seq, we also examined the genetic structure of three Japanese viviparids, including two endemic species of ancient Lake Biwa. The results suggest that these two species diversified from the population of the third species living in wetlands surrounding the lake. These findings suggest that rapid diversification of lineages and phenotypic divergence can occur in ancient lakes compared to other habitats. Formation of large lakes probably promotes speciation and phenotypic divergence as a result of adaptation into different microhabitats. High numbers of ancient lakes could be a driver of species diversity in Asian viviparid snails.

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.

The phylogeography of the cycad genus Dioon (Zamiaceae) clarifies its Cenozoic expansion and diversification in the Mexican transition zone.

Background and Aims: Biogeographic transition zones are promising areas to study processes of biogeographic evolution and its influence on biological groups. The Mexican transition zone originated due to the overlap of Nearctic and Neotropical biota, which promoted great biological diversification. However, since most previous studies in this area were focused on revealing the phylogeography of Nearctic plants, how historical biogeographic configuration influenced the expansion and diversification of the Neotropical flora remains almost unknown. Using the cycad genus Dioon (Zamiaceae), this study aimed to test whether the biogeographic provinciality of the Mexican transition zone reflects the history of diversification of Neotropical plants. Methods: Two chloroplast DNA (cpDNA) regions were analysed from 101 specimens of 15 Dioon species to reveal the distribution of haplogroups. In addition, genome-wide single nucleotide polymorphisms (SNPs) from 84 specimens were used to test the concordance between phylogenetic clusters and the biogeographic provinces. An ultrametric tree was constructed from the sequences containing SNPs to reconstruct the biogeographic events of vicariance and dispersal of Dioon across the Neotropical biogeographic provinces. Key Results: Four Dioon lineages with strong phylogeographic structures were recognized using both cpDNA and SNP data. The lineages correspond to two clades that originated from a common ancestor in Eastern Mexico. One clade expanded and diversified in South-east Mexico and Central America. Another clade diversified into three lineages that dispersed to North-east, South and North-west Mexico. Each lineage was biogeographically delimitated. Biogeographic provinces might have provided disparate ecological conditions that facilitated speciation in Dioon since the Miocene. Conclusions: The current genetic structure and species diversity of Dioon depict the history of expansion and diversification of the northernmost Neotropical provinces. Past biogeographic connectivities were favoured by elevated topographies, since mountain systems served as corridors for the migration of Dioon and as refugia of tropical communities that diversified during the formation of modern Neotropical forests.