Potential contribution of floral thermogenesis to cold adaptation, distribution pattern, and population structure of thermogenic and non/slightly thermogenic Symplocarpus species.

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.

Plastome-based backbone phylogeny of East Asian Phedimus (Subgenus Aizoon: Crassulaceae), with special emphasis on Korean endemics.

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 complete chloroplast genome sequence of Thalictrum aquilegiifolium var. sibiricum (Ranunculaceae).

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.

The complete chloroplast genome sequence of Anaphalis margaritacea var. yedoensis (Asteraceae) and phylogenetic relationships within Gnaphalieae.

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.

Construction of three-dimensional weather radar data from volcanic eruption clouds.

Analysis tools of three-dimensional weather radar data (ANT3D) was originally developed at the National Research Institute for Earth Science and Disaster prevention (NIED) to retrieve three-dimensional (3D) precipitation and wind fields for convective storms. In 2013, Kagoshima University significantly revised ANT3D for analyses of volcanic eruption clouds, mainly to improve the temporal and spatial interpolation of radar data and estimation of the advection vector, which is required for temporal interpolation. Detailed information pertaining to these algorithms is listed as additional information in this paper.•Procedures necessary for the construction of three-dimensional (3D) volcanic cloud weather radar data are described.•An algorithm based on temporal and elevation angle interpolation methods was used to create 3D constant altitude plan position indicator (3D CAPPI) data with high temporal and spatial resolution.•Two programs (ANT3D_GUI and the CAPPI viewer) are provided for readers interested in analyzing volcanic eruption cloud radar data.

Genome-Wide Single Nucleotide Polymorphism Analysis Elucidates the Evolution of Prunus takesimensis in Ulleung Island: The Genetic Consequences of Anagenetic Speciation.

Of the two major speciation modes of endemic plants on oceanic islands, cladogenesis and anagenesis, the latter has been recently emphasized as an effective mechanism for increasing plant diversity in isolated, ecologically homogeneous insular settings. As the only flowering cherry occurring on Ulleung Island in the East Sea (concurrently known as Sea of Japan), Prunus takesimensis Nakai has been presumed to be derived through anagenetic speciation on the island. Based on morphological similarities, P. sargentii Rehder distributed in adjacent continental areas and islands has been suggested as a purported continental progenitor. However, the overall genetic complexity and resultant non-monophyly of closely related flowering cherries have hindered the determination of their phylogenetic relationships as well as the establishment of concrete continental progenitors and insular derivative relationships. Based on extensive sampling of wild flowering cherries, including P. takesimensis and P. sargentii from Ulleung Island and its adjacent areas, the current study revealed the origin and evolution of P. takesimensis using multiple molecular markers. The results of phylogenetic reconstruction and population genetic structure analyses based on single nucleotide polymorphisms detected by multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) and complementary cpDNA haplotypes provided evidence for (1) the monophyly of P. takesimensis; (2) clear genetic differentiation between P. takesimensis (insular derivative) and P. sargentii (continental progenitor); (3) uncertain geographic origin of P. takesimensis, but highly likely via single colonization from the source population of P. sargentii in the Korean Peninsula; (4) no significant reduction in genetic diversity in anagenetically derived insular species, i.e., P. takesimensis, compared to its continental progenitor P. sargentii; (5) no strong population genetic structuring or geographical patterns in the insular derivative species; and (6) MIG-seq method as an effective tool to elucidate the complex evolutionary history of plant groups.

Species cohesion of an extremophyte (Carex angustisquama, Cyperaceae) in solfatara fields maintained under interspecific natural hybridization.

BACKGROUND AND AIMS: Hybridization is the main driver of plant diversification, and gene flow via hybridization has multifaceted effects on plant evolution. Carex angustisquama is an extremophyte that grows on soils heavily acidified by volcanism. Despite its habitat distinct from that of other species, this species is known to form interspecific hybrids, implying interspecific gene flow. It is crucial to verify the extent and direction of interspecific gene flow between C. angustisquama and closely related species to understand the evolutionary process of an extremophyte in solfatara fields. METHODS: In this study, expressed sequence tag-simple sequence repeat markers were utilized to infer the extent and direction of interspecific gene flow between C. angustisquama and closely related species. KEY RESULTS: Bayesian clustering and simulation analyses revealed that all individuals of the three hybrid species were classified into the first hybrid generation or first backcross to C. angustisquama; therefore, current interspecific gene flow is limited. Moreover, in the Bayesian inference of historical gene flow based on multispecies samples, the model that assumed no interspecific gene flow was the most strongly supported across all species pairs, including phylogenetically close but ecologically distinctive species pairs. CONCLUSIONS: Our results revealed that interspecific gene flow between C. angustisquama and its related species has been limited both currently and historically. Moreover, our results of Bayesian inference of historical gene flow indicated that extrinsic, rather than intrinsic, factors probably act as isolating barriers between Carex species, with hybrid breakdown via microhabitat segregation being the probable potential barrier. Overall, our findings provide insights into the evolutionary process of an extremophyte in solfatara fields and offer an important example of the mechanisms of diversification of the speciose genus Carex.

Visualizing weather radar data from volcanic eruption clouds.

This paper is submitted to accompany the article "Analyses of three-dimensional weather radar data from volcanic eruption clouds" [1]; it describes three-dimensional (3D) visualizations of the Sakurajima volcanic eruption clouds and the weather radar data used for analyses, as well as their availability and downloading procedures. The radar data were acquired by an operational X-band weather radar located approximately 11 km south of the Showa vent of Sakurajima in Kagoshima, Japan. The original raw radar data are available from the "XRAIN Precipitation Original Data search and Download System", which is hosted on the website "Data Integration and Analysis System (DIAS)". Animated images of the radar data shown here, which provide a visual explanation of the temporal evolution and the inner structure of volcanic eruption clouds, were created using the program "Analysis Tools of Three-dimensional Weather Radar Data (AN3D)". The detailed methods of ANT3D are provided in the co-submitted article "Construction of three-dimensional weather radar data from volcanic eruption clouds" [2].

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.

Origin of the critically endangered endemic species Scrophularia takesimensis (Scrophulariaceae) on Ulleung Island, Korea: implications for conservation.

Scrophularia takesimensis is a critically endangered endemic species of Ulleung Island, Korea. A previous molecular phylogenetic study based on nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences with very limited sampling suggested that it is most closely related to the clade comprising S. alata and S. grayanoides. To determine the origin of S. takesimensis, we sampled a total of 171 accessions including S. takesimensis (9 populations and 63 individuals) and two closely related species, S. alata (11 populations and 68 individuals) and S. grayanoides (5 populations and 40 individuals) from eastern Asia and sequenced ITS and two chloroplast DNA (cpDNA) non-coding regions. Previously sequenced representative species of Scrophularia (109 taxa for ITS and 80 taxa for cpDNA) were combined with our data set and analyzed. While the global scale ITS phylogenetic tree suggests monophyly for each of the three eastern Asian species, S. takesimensis appears to be more closely related (albeit weakly) to a clade containing eastern North American/Caribbean species than to either S. alata or S. grayanoides. By contrast, the global scale cpDNA phylogenetic tree demonstrates that the eastern North America/Caribbean clade is sister to a clade comprising the three eastern Asian species. In addition, the monophyletic S. takesimensis is deeply embedded within paraphyletic S. alata, sharing its most recent common ancestor with populations from Japan/Sakhalin. Two divergent, geographically structured cp haplotype groups within S. takesimensis suggest at least two independent introductions from different source areas. A new and accurate chromosome number of S. takesimensis (2n = 94) is reported and some conservation strategies are discussed.

Incorporating differences between genetic diversity of trees and herbaceous plants in conservation strategies.

Reviews that summarize the genetic diversity of plant species in relation to their life history and ecological traits show that forest trees have more genetic diversity at population and species levels than annuals or herbaceous perennials. In addition, among-population genetic differentiation is significantly lower in trees than in most herbaceous perennials and annuals. Possible reasons for these differences between trees and herbaceous perennials and annuals have not been discussed critically. Several traits, such as high rates of outcrossing, long-distance pollen and seed dispersal, large effective population sizes (Ne ), arborescent stature, low population density, longevity, overlapping generations, and occurrence in late successional communities, may make trees less sensitive to genetic bottlenecks and more resistant to habitat fragmentation or climate change. We recommend that guidelines for genetic conservation strategies be designed differently for tree species versus other types of plant species. Because most tree species fit an LH scenario (low [L] genetic differentiation and high [H] genetic diversity), tree seeds could be sourced from a few populations distributed across the species' range. For the in situ conservation of trees, translocation is a viable option to increase Ne . In contrast, rare herbaceous understory species are frequently HL (high differentiation and low diversity) species. Under the HL scenario, seeds should be taken from many populations with high genetic diversity. In situ conservation efforts for herbaceous plants should focus on protecting habitats because the typically small populations of these species are vulnerable to the loss of genetic diversity. The robust allozyme genetic diversity databases could be used to develop conservation strategies for species lacking genetic information. As a case study of reforestation with several tree species in denuded areas on the Korean Peninsula, we recommend the selection of local genotypes as suitable sources to prevent adverse effects and to insure the successful restoration in the long term.

Incorporación de diferencias de diversidad genética entre árboles y plantas herbáceas en estrategias de conservación Resumen Las revisiones que resumen la diversidad genética de las plantas en relación con sus características ecológicas y biológicas muestran que los árboles forestales tienen más diversidad genética a nivel de población y de especie que las plantas anuales o las perennes herbáceas. Sumado a esto, la diferenciación genética entre poblaciones es significativamente más baja en los árboles que en la mayoría de las perennes herbáceas y las anuales. Hasta la fecha no se han discutido críticamente las posibles explicaciones de estas diferencias entre los árboles y las perennes herbáceas y las plantas anuales. Varias características, como las tasas altas de alogamia, la dispersión a gran distancia de polen y semillas, el gran tamaño de la población efectiva (Ne ), la estatura arbórea, la baja densidad poblacional, la longevidad, el solapamiento de generaciones y la presencia dentro de comunidades sucesionales tardías, pueden generar en los árboles una menor sensibilidad a los cuellos de botella genéticos y una mayor resistencia a la fragmentación del hábitat o al cambio climático. Recomendamos que las directrices para las estrategias de conservación genética estén diseñadas de manera diferente para las especies arbóreas que para otro tipo de plantas. Ya que la mayoría de las especies arbóreas encajan dentro de un escenario LH (baja [L] diferenciación genética y alta [H] diversidad genética), las semillas de los árboles podrían tomarse de unas cuantas poblaciones dispersas a lo largo del área de distribución de la especie. Por lo anterior, para la conservación in situ de los árboles, la translocación es una opción viable para incrementar la Ne . Al contrario de esta situación, las especies herbáceas raras del sotobosque con frecuencia son especies HL (alta diferenciación y baja diversidad). En el escenario HL, las semillas deberían ser recolectadas de muchas poblaciones con diversidad genética alta y los esfuerzos de conservación in situ para las plantas herbáceas deberían enfocarse en la protección del hábitat ya que las poblaciones típicamente pequeñas de estas especies son vulnerables a la pérdida de la diversidad genética. Las robustas bases de datos de diversidad genética aloenzimática podrían usarse para desarrollar estrategias de conservación para las especies que carecen de información genética. Como caso de estudio de reforestación con varias especies arbóreas en áreas deforestadas de la Península de Corea, recomendamos la selección de genotipos locales como fuente adecuada para prevenir los efectos adversos y para asegurar la restauración exitosa a largo plazo.

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.

Comparison of Whole Plastome Sequences between Thermogenic Skunk Cabbage Symplocarpus renifolius and Nonthermogenic S. nipponicus (Orontioideae; Araceae) in East Asia.

Symplocarpus, a skunk cabbage genus, includes two sister groups, which are drastically different in life history traits and thermogenesis, as follows: The nonthermogenic summer flowering S. nipponicus and thermogenic early spring flowering S. renifolius. Although the molecular basis of thermogenesis and complete chloroplast genome (plastome) of thermogenic S. renifolius have been well characterized, very little is known for that of S. nipponicus. We sequenced the complete plastomes of S. nipponicus sampled from Japan and Korea and compared them with that of S. renifolius sampled from Korea. The nonthermogenic S. nipponicus plastomes from Japan and Korea had 158,322 and 158,508 base pairs, respectively, which were slightly shorter than the thermogenic plastome of S. renifolius. No structural or content rearrangements between the species pairs were found. Six highly variable noncoding regions (psbC/trnS, petA/psbJ, trnS/trnG, trnC/petN, ycf4/cemA, and rpl3/rpl22) were identified between S. nipponicus and S. renifolius and 14 hot-spot regions were also identified at the subfamily level. We found a similar total number of SSR (simple sequence repeat) motifs in two accessions of S. nipponicus sampled from Japan and Korea. Phylogenetic analysis supported the basal position of subfamily Orontioideae and the monophyly of genus Symplocarpus, and also revealed an unexpected evolutionary relationship between S. nipponicus and S. renifolius.

Multiple origins and the population genetic structure of Rubus takesimensis (Rosaceae) on Ulleung Island: Implications for the genetic consequences of anagenetic speciation.

To determine the origin and genetic consequences of anagenesis in Rubus takesimensis on Ulleung Island, Korea, we compared the genetic diversity and population structure of R. takesimensis with those of its continental progenitor R. crataegifolius. We broadly sampled a total of 315 accessions in 35 populations and sequenced five noncoding regions of chloroplast DNA. Rubus takesimensis emerged as nonmonophyletic and several geographically diverse continental populations were likely responsible for the origin of R. takesimensis; the majority of R. takesimensis accessions were sisters to the clade containing accessions of R. crataegifolius, primarily from the Korean peninsula, while rare accessions from three populations shared common ancestors with the ones from the southern part of the Korean peninsula, Jeju Island, and Japan. A few accessions from the Chusan population originated independently from the Korean peninsula. Of 129 haplotypes, 81 and 48 were found exclusively in R. crataegifolius and R. takesimensis, respectively. We found unusually high genetic diversity in two regions on Ulleung Island and no geographic population structure. For R. crataegifolius, two major haplotype groups were found; one for the northern mainland Korean peninsula, and the other for the southern Korean peninsula and the Japanese archipelago. Compared with populations of R. crataegifolius sampled from Japan, much higher haplotype diversity was found in populations from the Korean peninsula. The patterns of genetic consequences in R. takesimensis need to be verified for other endemic species based on chloroplast DNA and independent nuclear markers to synthesize emerging patterns of anagenetic speciation on Ulleung Island.

Phylogeographic Analysis and Genetic Structure of an Endemic Sino-Japanese Disjunctive Genus Diabelia (Caprifoliaceae).

The Sino-Japanese Floristic Region (SJFR) is a key area for plant phylogeographical research, due to its very high species diversity and disjunct distributions of a large number of species and genera. At present, the root cause and temporal origin of the discontinuous distribution of many plants in the Sino-Japanese flora are still unclear. Diabelia (Caprifoliaceae; Linnaeoideae) is a genus endemic to Asia, mostly in Japan, but two recent discoveries in China raised questions over the role of the East China Sea (ECS) in these species' disjunctions. Chloroplast DNA sequence data were generated from 402 population samples for two regions (rpl32-trnL, and trnH-psbA) and 11 nuclear microsatellite loci were screened for 549 individuals. Haplotype, population-level structure, combined analyses of ecological niche modeling, and reconstruction of ancestral state in phylogenies were also performed. During the Last Glacial Maximum (LGM) period after the Tertiary, Diabelia was potentially widely distributed in southeastern China, the continental shelf of the East China Sea and Japan (excluding Hokkaido). After LGM, all populations in China have disappeared except those in Zhejiang which may represent a Glacial refuge. Populations of Diabelia in Japan have not experienced significant bottleneck effects, and populations have maintained a relatively stable state. The observed discontinuous distribution of Diabelia species between China and Japan are interpreted as the result of relatively ancient divergence. The phylogenetic tree of chloroplast fragments shows the characteristics of multi-origin evolution (except for D. sanguinea). STRUCTURE analysis of nuclear Simple Sequence Repeat (nSSR) showed that the plants of the Diabelia were divided into five gene pools: D. serrata, D. spathulata, D. sanguinea, D. ionostachya (D. spathulata var. spathulata-Korea), and populations of D. ionostachya var. ionostachya in Yamagata prefecture, northern Japan. Molecular evidence provides new insights of Diabelia into biogeography, a potential glacial refuge, and population-level genetic structure within species. In the process of species differentiation, ECS acts as a corridor for two-way migration of animals and plants between China and Japan during glacial maxima, providing the possibility of secondary contact for discontinuously distributed species between China and Japan, or as a filter (creating isolation) during glacial minima. The influence of the ECS in speciation and biogeography of Diabelia in the Tertiary remains unresolved in this study. Understanding origins, evolutionary histories, and speciation will provide a framework for the conservation and cultivation of Diabelia.

Data that effectively demonstrate the benefits of a 3D CAPPI algorithm.

The data presented in this article are related to the research article entitled "Three-dimensional analysis of the initial stage of convective precipitation using an operational X-band polarimetric radar network" [1]. The data presented were obtained using a three-dimensional constant-altitude plan-position-indicator (3D CAPPI), which was generated by a new method proposed by [1]. The data used to create the 3D CAPPI were derived from two X-band polarimetric radar installations in the Kanto region of Japan, Ebina (139.39°E, 35.40°N), and Shin-yokohama (139.60°E, 35.51°N). These data are superior to operational radar data in terms of their temporal and spatial resolution. These high resolution data can indicate a rapidly developing storm, such as localized precipitation. It is particularly important to understand the early stages of storms in terms of numerical and short-term models. These data show the time of appearance, life cycle, and evolution of each cell that constitutes a storm in three-dimensional detail.

Development and characterization of EST-SSR markers for Carex angustisquama (Cyperaceae), an extremophyte in solfatara fields.

PREMISE OF THE STUDY: Expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed for Carex angustisquama (Cyperaceae) to investigate the evolutionary history of this plant that is endemic to solfatara fields in northern Japan. METHODS AND RESULTS: Using RNA-Seq data generated by the Illumina HiSeq 2000, 20 EST-SSR markers were developed. Polymorphisms were assessed in C. angustisquama and the closely related species C. doenitzii and C. podogyna. In C. angustisquama, many loci were monomorphic within populations; the average number of alleles ranged from one to five, and levels of expected heterozygosity ranged from 0.000 to 0.580, while all markers were polymorphic in a population of C. doenitzii. This indicates that low genetic polymorphism of C. angustisquama is likely due to the species' population dynamics, rather than to null alleles at the developed markers. CONCLUSIONS: These markers will be used to assess genetic diversity and structure and to investigate evolutionary history in future studies of C. angustisquama and related species.

Development of microsatellite markers for the wind cave-associated shrub Lonicera alpigena subsp. glehnii (Caprifoliaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the wind cave-associated shrub Lonicera alpigena subsp. glehnii to conduct phylogeographic studies on the species. METHODS AND RESULTS: Based on the sequence data obtained by 454 sequencing, a total of 81 primer pairs were designed and 18 successfully amplified the microsatellite regions. These markers were highly variable (i.e., average number of alleles per locus = 6.2 [range = 2-15]; average expected heterozygosity per locus = 0.489 [range = 0.149-0.729]). Cross-species amplification of the primers was tested in 10 congeneric taxa (L. caerulea var. emphyllocalyx, L. chamissoi, L. chrysantha, L. gracilipes var. glandulosa, L. japonica, L. kurobushiensis, L. morrowii, L. ramosissima, L. sachalinensis, and L. strophiophora), and six to 11 primers amplified the microsatellite markers. CONCLUSIONS: The microsatellite markers developed in this study will be useful for phylogeographic studies and conservation genetics of L. alpigena subsp. glehnii as well as congeneric species.

The giant mycoheterotrophic orchid Erythrorchis altissima is associated mainly with a divergent set of wood-decaying fungi.

The climbing orchid Erythrorchis altissima is the largest mycoheterotroph in the world. Although previous in vitro work suggests that E. altissima has a unique symbiosis with wood-decaying fungi, little is known about how this giant orchid meets its carbon and nutrient demands exclusively via mycorrhizal fungi. In this study, the mycorrhizal fungi of E. altissima were molecularly identified using root samples from 26 individuals. Furthermore, in vitro symbiotic germination with five fungi and stable isotope compositions in five E. altissima at one site were examined. In total, 37 fungal operational taxonomic units (OTUs) belonging to nine orders in Basidiomycota were identified from the orchid roots. Most of the fungal OTUs were wood-decaying fungi, but underground roots had ectomycorrhizal Russula. Two fungal isolates from mycorrhizal roots induced seed germination and subsequent seedling development in vitro. Measurement of carbon and nitrogen stable isotope abundances revealed that E. altissima is a full mycoheterotroph whose carbon originates mainly from wood-decaying fungi. All of the results show that E. altissima is associated with a wide range of wood- and soil-inhabiting fungi, the majority of which are wood-decaying taxa. This generalist association enables E. altissima to access a large carbon pool in woody debris and has been key to the evolution of such a large mycoheterotroph.

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.