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Applications of molecular phylogenetic approaches have uncovered evidence of hybridization across numerous clades of life, yet the environmental factors responsible for driving opportunities for hybridization remain obscure. Verbal models implicating geographic range shifts that brought species together during the Pleistocene have often been invoked, but quantitative tests using paleoclimatic data are needed to validate these models. Here, we produce a phylogeny for Heuchereae, a clade of 15 genera and 83 species in Saxifragaceae, with complete sampling of recognized species, using 277 nuclear loci and nearly complete chloroplast genomes. We then employ an improved framework with a coalescent simulation approach to test and confirm previous hybridization hypotheses and identify one new intergeneric hybridization event. Focusing on the North American distribution of Heuchereae, we introduce and implement a newly developed approach to reconstruct potential past distributions for ancestral lineages across all species in the clade and across a paleoclimatic record extending from the late Pliocene. Time calibration based on both nuclear and chloroplast trees recovers a mid- to late-Pleistocene date for most inferred hybridization events, a timeframe concomitant with repeated geographic range restriction into overlapping refugia. Our results indicate an important role for past episodes of climate change, and the contrasting responses of species with differing ecological strategies, in generating novel patterns of range contact among plant communities and therefore new opportunities for hybridization. The new ancestral niche method flexibly models the shape of niche while incorporating diverse sources of uncertainty and will be an important addition to the current comparative methods toolkit. [Ancestral niche reconstruction; hybridization; paleoclimate; pleistocene.]

Background and AimsThe genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach.MethodsWe first compared the performance of phylogenetic analysis based on the plastid matK and trnL–F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species.Key ResultsPhylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0–84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan.ConclusionsThe core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.

Blast is the most devastating disease of rice worldwide. The use of resistance genes is the most effective measure to control the disease. Better understanding of the mechanisms of pathogen infection and plant resistance is important for developing blast resistant cultivars. This chapter reviews the gene-for-gene interactions mediated by pathogen avirulence (AVR) genes and plant resistance (R) genes and shares our experiences of whole genome sequencing (WGS)-based approaches to isolate blast AVR genes and rice R-genes. The chapter also provides a brief summary of current knowledge on interactions of the proteins coded by the isolated blast AVR genes and rice R-genes.

Despite its potential effectiveness for outcrossing, few examples of pollination via mushroom mimicry have been reported. This may be because the conditions under which the strategy can evolve are limited and/or because demonstrating it is challenging. Arisaema is a plant genus that has been suggested to adopt mushroom mimicry for pollination, although no compelling evidence for this has yet been demonstrated. Here, we report that Arisaema sikokianum utilizes mostly a single genus of obligate mycophagous flies (Mycodrosophila) as pollinators, and that the insect community dominated by Mycodrosophila is strikingly similar to those found on some species of wood-decaying fungi. Comparative chemical analyses of Arisaema spp. and various mushrooms further revealed that only A. sikokianum emits a set of volatile compounds shared with some mushroom species utilized by Mycodrosophila. Meanwhile, other closely related and often sympatric Arisaema species do not possess such typical traits of mushroom mimicry or attract Mycodrosophila, thereby likely achieving substantial reproductive isolation from A. sikokianum. Our finding indicates that mushroom mimicry is an exceptional and derived state in the genus Arisaema, thus providing an unprecedented opportunity to study the mechanisms underlying the coordinated acquisition of mimicry traits that occurred during a recent speciation event.

Due to the recent advances in DNA sequencing technologies, whole genome sequencing (WGS)-based approaches are now accelerating the pace of our research toward a better understanding of host-pathogen interactions. Using WGS-based methods, we have isolated three avirulence ( AVR ) genes: AVR-Pia , AVR-Pii and AVR-Pik from Magnaporthe oryzae and two cognate rice resistance ( R -) genes: Pia and Pii . We briefly review our current understanding of the interactions between AVR-Pia and Pia , AVR-Pik and Pik , and AVR-Pii and Pii .

The evolutionary origin of periodical mass‐flowering plants (shortly periodical plants), exhibiting periodical mass flowering and death immediately after flowering, has not been demonstrated. Within the genus Strobilanthes (Acanthaceae), which includes more than 50 periodical species, Strobilanthes flexicaulis on Okinawa Island, Japan, flowers gregariously every 6 years. We investigated the life history of S. flexicaulis in other regions and that of closely related species together with their molecular phylogeny to reveal the evolutionary origin of periodical mass flowering. S. flexicaulis on Taiwan Island was found to be a polycarpic perennial with no mass flowering and, in the Yaeyama Islands, Japan, a monocarpic perennial with no mass flowering. Molecular phylogenetic analyses indicated that a polycarpic perennial was the ancestral state in this whole group including S. flexicaulis and the closely related species. No distinctive genetic differentiation was found in S. flexicaulis among all three life histories (polycarpic perennial, monocarpic perennial, and periodical plant). These results suggest that among S. flexicaulis, the periodical mass flowering on Okinawa Island had evolved from the polycarpic perennial on Taiwan Island via the monocarpic perennial in the Yaeyama Islands. Thus, the evolution of life histories could have taken at the level of local populations within a species.

Copyright by the Ecological Society of America. Okuyama, Y., Okamoto, T., Kjærandsen, J. & Kato, M. (2018). Bryophytes facilitate outcrossing of Mitella by functioning as larval food for pollinating fungus gnats. Ecology, 99(8), 1890-1893. Source at https://doi.org/10.1002/ecy.2364.

Floral scents are among the key signals used by pollinators to navigate to specific flowers. Thus, evolutionary changes in scents should have strong impacts on plant diversification, although scent-mediated plant speciation through pollinator shifts has rarely been demonstrated, despite being likely. To examine whether and how scent-mediated plant speciation may have occurred, we investigated the Asimitellaria plant lineage using multidisciplinary approaches including pollinator observations, chemical analyses of the floral scents, electroantennographic analyses and behavioural bioassays with the pollinators. We also performed phylogenetically independent contrast analyses of the pollinator/floral scent associations. First, we confirmed that the pairs of the sympatric, cross-fertile Asimitellaria species in three study sites consistently attract different pollinators, namely long-tongued and short-tongued fungus gnats. We also found that a stereoisomeric set of floral volatiles, the lilac aldehydes, could be responsible for the pollinator specificity. This is because the compounds consistently elicited responses in the antennae of the long-tongued fungus gnats and had contrasting effects on the two pollinators, that is triggering the nectaring behaviour of long-tongued fungus gnats while repelling short-tongued fungus gnats in a laboratory experiment. Moreover, we discovered that volatile composition repeatedly switched in Asimitellaria between species adapted to long-tongued and short-tongued fungus gnats. Collectively, our results support the idea that recurrent scent-mediated speciation has taken place in the Asimitellaria–fungus gnat system.

► we isolated flavonoids and xanthones from the flowers and leaves of Iris rossii. ► we obtained the chloroplast DNA sequences and determined the phylogenetic tree. ► Ten compounds were isolated and characterized from the flowers and leaves. ► Three C-arabinosylflavones were isolated from Iris for the first time. ► Phylogeny revealed relationships between I. rossii and series Chinenses species.

Attelabid weevils manipulate specific structures of their host plants in a species-specific manner, e.g., cutting a shoot, cutting a leaf, rolling a leaf, or constructing sophisticated wrapped leaf rolls, presumably to secure the survivorship of eggs or larvae. To depict the evolutionary history of maternal plant-manipulation behaviours and larval feeding strategies of the family Attelabidae, molecular phylogenetic analyses were conducted by sequencing the nuclear 18S and 28S ribosomal DNA and the mitochondrial cytochrome oxidase subunit I genes. Our analyses indicated that the attelabid weevils form a monophyletic group, and that maternal plant-cutting behaviour originated in a common ancestor of Attelabidae, but was subsequently lost in several lineages. Monophyly of the subfamily Attelabinae was also recovered with high support, but the subfamily Rhynchitinae was not recovered as monophyletic. By employing maximum-likelihood-based ancestral state reconstructions, larval leaf-blade feeding was inferred to have evolved from boring of cut shoots/petioles. Moreover, maternal leaf-rolling behaviours likely originated independently in the Attelabinae and Byctiscini lineages, and in several Deporaini lineages. As the sophisticated behaviours constructing wrapped leaf rolls of Attelabinae originated only once and has not been lost from the lineage, these complex and innovative behaviours may have contributed to the success and diversification of the lineage.

Differences in gene expression are termed expression level polymorphisms (ELPs). Here, we propose a new ELP class, bimodal ELPs (bELPs), as a criterion to screen for genes that are responsible for natural phenotypic variation and/or that are targeted by balancing selection. bELP genes are characterized by two expression level modes. Genomic scans based on nucleotide sequences are not ideal for identifying genes targeted for selection. A critical concern is that several genes can be present in the selection-targeted regions identified by such scans. This situation indicates the importance of integrating genomic sequence data and other information, such as gene expression data. Comparative transcriptomics is useful for determining evolutionarily and ecologically important polymorphisms. In a genome-wide expression screen of 34 accessions, we identified 344 Arabidopsis thaliana genes exhibiting bELPs. Population genetic analysis revealed that bELP genes had high nucleotide diversities and long linkage disequilibriums. The highest nucleotide diversity (11-fold greater than the genomic mean) was found in the At1g23780 gene, which encodes a putative F-box protein. We observed a clear association between the expression mode and sequence type of the At1g23780 gene. Our results suggest that bELPs will be useful for the screening and functional analysis of genes responsible for phenotypic polymorphisms. Such a "multi-omics" approach has the potential to facilitate the scanning of genes relevant to balanced polymorphisms not only in A. thaliana, but also in other model and non-model organisms.

The reconstruction of an ancient polyploidization history is often challenging, although it is a crucial step in clarifying the mechanisms underlying the contemporary success and diversity of polyploids. Phylogenetic relationships of duplicated gene pairs of polyploids, with respect to their orthologs in related diploids, have been used to address this problem, but they often result in conflicting topologies among different genes. Asimitellaria is an East Asian endemic tetraploid lineage of perennials (genus Mitella; Saxifragaceae) that has diversified in riparian habitats. Phylogenetic analyses of four nuclear-encoded, single-copy (per haploid) genes GBSSI-A, GBSSI-B, GS-II, and PepCK all supported a single allopolyploid origin of Asimitellaria, but they did not lead to a consensus about which diploid lineage gave rise to each of the Asimitellaria subgenomes. To address this issue, we used an integrated approach, whereby the four gene data sets and an additional nuclear ribosomal external transcribed spacer and internal transcribed spacer (including a 5.8S ribosomal DNA) data set were concatenated in all possible combinations, and the most probable data combination was determined together with the phylogenetic inference. This resulted in relatively robust support for the two closely related North American diploid species as the ancestral lineages of the Asimitellaria subgenomes, suggesting ancient intercontinental migration of the diploid or tetraploid lineages and subsequent tetraploid diversification in the Japanese Archipelago. The present approach enabled sorting out the duplicated genes into their original combinations in their preduplication ancestors under a maximum-likelihood framework, and its extension toward genome sequencing data may help in the reconstruction of ancestral, preduplicated, whole-genome structures.

Rice blast caused by an ascomycete fungus Magnaporthe oryzae is the most devastating disease of rice worldwide. Thus understanding of the molecular mechanisms of Magnaporthe-rice interactions is crucial to devise efficient means of disease control. The availability of whole genome sequences of both M. oryzae and rice enables us to analyze their DNA polymorphisms on the genomic scale for the purpose of association genetics as well as for population genomics analysis. Studies on the association between phenotypes-DNA polymorphisms allowed us to isolate three M. oryzae AVR genes, AVR-Pia, AVR-Pii, AVR-Pik/km/kp as well as a rice R-gene, Pia. Population genomics addresses the patterns of DNA polymorphisms to identify the genomic regions under natural selection, a potentially useful tool to isolate the genes involved in plant-pathogen interactions. Recent progress in next-generation sequencing technologies predicts that these approaches would be commonly used to elucidate various host-pathogen interactions.

SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.

Interspecific hybridization is one of the major factors leading to phylogenetic incongruence among loci, but the knowledge is still limited about the potential of each locus to introgress between species. By directly sequencing three DNA regions: chloroplast DNAs (matK gene and trnL-F noncoding region), the nuclear ribosomal external transcribed spacer (ETS) region, and internal transcribed spacer (ITS) regions, we construct three phylogenetic trees of Asian species of Mitella (Saxifragaceae), a genus of perennials in which natural hybrids are commonly observed. Within this genus, there is a significant topological conflict between chloroplast and nuclear phylogenies and also between the ETS and the ITS, which can be attributed to frequent hybridization within the lineage. Chloroplast DNAs show the most extensive introgression pattern, ITS regions show a moderate pattern, and the ETS region shows no evidence of introgression. Nonuniform concerted evolution best explains the difference in the introgression patterns between the ETS region and ITS regions, as the sequence heterogeneity of the ITS region within an individual genome is estimated to be twice that of an ETS in this lineage. Significant gene conversion patterns between two hybridizing taxa were observed in contiguous arrays of cloned ETS-ITS sequences, further confirming that only ITS regions have introgressed bidirectionally. The relatively slow concerted evolution in the ITS regions probably allows the coexistence of multiple alleles within a genome, whereas the strong concerted evolution in the ETS region rapidly eliminates heterogeneous alleles derived from other species, resulting in species delimitations highly concordant with those based on morphology. This finding indicates that the use of multiple molecular tools has the potential to reveal detailed organismal evolution processes involving interspecific hybridization, as an individual locus varies greatly in its potential to introgress between species.

The first example of pollination by fungus gnats in the eudicots is reported. The genus Mitella (Saxifragales) is characteristically produces minute, inconspicuous, mostly dull-coloured flowers with linear, sometimes pinnately branched, petals. To understand the function of these characteristic flowers, we studied the pollination biology of four Mitella species with different floral traits and different sexual expression: dioecious M. acerina , gynodioecious M. furusei var. subramosa , and hermaphroditic M. stylosa var. makinoi and M. integripetala. Flower-bagging experiments showed that wind pollination did not occur in the dioecious and gynodioecious species. Two years of observations of flower visitors at six study sites in Japan revealed that the principal pollinators of all four Mitella were specific species of fungus gnats (Mycetophilidae), which landed on the flowers with their long spiny legs settling on the petals. Characteristically, numerous pollen grains were attached to the fungus gnats in specific locations on the body. Although, on average, 1.3‐2.6 fungus gnats visited each inflorescence per day, the fruit set of both bisexual and female flowers exceeded 63%. These results suggest that fungus gnats are highly efficient pollinators of Mitella spp., and that Mitella flowers are morphologically adapted to pollination by fungus gnats. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 144 , 449‐460.

Weevils are one of the dominant taxonomic groups in terrestrial ecosystem, diversifying to more than 60,000 described species. Although the most derived weevil group, Curculionidae, has adapted to and is utilising almost all parts of plant, basal groups show relatively limited larval feeding habits, such as pollen, seed, or fungus-infested wood feeders. Thus, it seems that ancestral larval infesting plant parts of weevils were restricted to N-rich, induced-defenseless, and temporal resources. Among the basal weevils, Attelabidae obtained such resources for their larvae by evolving unique maternal plant-manipulations: attelabid females manipulate specific young plant tissues of their host plants in a species-specific manner, e.g. cutting a shoot or a leaf, rolling a leaf, or constructing sophisticated wrapped leaf rolls, before and after oviposition presumably to secure the survivorship of eggs or larvae. Molecular phylogenetic analyses based on the nuclear 18S and 28S ribosomal DNA and the mitochondrial COI genes indicated that the maternal plant-cutting behaviour originated in a common ancestor of Attelabidae, but was subsequently lost in the several lineages. The monophyly was recovered for the subfamily Attelabinae with high support, but not for the subfamily Rhynchitinae. By employing maximum likelihood-based ancestral state reconstructions, the larval leaf-blade feeding was inferred to have evolved from the boring of cut shoots/petioles. Moreover, the maternal leaf-rolling behaviour might have originated independently in the Attelabinae, Byctiscini, and also in several Deporaini lineages. Since the sophisticated behaviour of Attelabinae, i.e. constructing wrapped leaf rolls, have originated only once and not been lost in the lineage, these complex and innovative behaviours may have contributed to the success of the lineage diversification.

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer–fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

Forests with different flora and vegetation types harbor different assemblages of flower visitors, and plant-pollinator interactions vary among forests. In monsoon-dominated East and Southeast Asia, there is a characteristic gradient in climate along latitude, creating a broad spectrum of forest types with potentially diverse pollinator communities. To detect a geographical pattern of plant-pollinator interactions, we investigated flowering phenology and pollinator assemblages in the least-studied forest type, i.e., tropical monsoon forest, in the Vientiane plain in Laos. Throughout the 5-year study, we observed 171 plant species blooming and detected flower visitors on 145 species. Flowering occurred throughout the year, although the number of flowering plant species peaked at the end of dry season. The dominant canopy trees, including Dipterocarpaceae, bloomed annually, in contrast to the supra-annual general flowering that occurs in Southeast Asian tropical rain forests. Among the 134 native plant species, 68 were pollinated by hymenopterans and others by lepidopterans, beetles, flies, or diverse insects. Among the observed bees, Xylocopa, megachilids, and honeybees mainly contributed to the pollination of canopy trees, whereas long-tongued Amegilla bees pollinated diverse perennials with long corolla tubes. This is the first community-level study of plant-pollinator interactions in an Asian tropical monsoon forest ecosystem.

The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.

SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.

The widespread pattern of parallel flower evolution as an adaptation for particular pollinator agents, known as "pollination syndromes", has long drawn attention from evolutionary biologists. Here, we report parallel evolution of saucer-shaped flowers and an associated unusual pollination system within the lineage Heucherina, a group of saxifragaceous genera. Field observations reveal that 18 of 28 plant species studied are pollinated almost exclusively by fungus gnats (Mycetophilidae). Among the 18 species with a fungus-gnat pollination system, 13 have characteristic saucer-shaped flowers and are pollinated mainly by several unspecialized mycetophilid genera with short mouthparts. We performed phylogenetic analyses using nucleotide sequences of external and internal transcribed spacers of nuclear ribosomal DNA and reconstructed ancestral floral morphologies with an establishment of the model of floral character evolution under a maximum-likelihood framework. Our analysis indicates that there is significant directionality in the evolutionary shifts of floral forms in the Heucherina. The inferred phylogeny further supports four origins of saucer-shaped flowers, which is shared among 14 species that are traditionally classified into the genus Mitella. In addition, our analysis indicates the extensive polyphyly of genus Mitella, as also suggested previously. The results suggest that the flower-visiting fungus gnats have caused convergent selection for the saucer-shaped flower repeatedly evolved within Heucherina.

Background Although DNA sequence analysis is becoming a powerful tool for identifying species, it is not easy to assess whether the observed genetic disparity corresponds to reproductive isolation. Here, we compared the efficiency of biological species identification between nuclear ribosomal and chloroplast DNA sequences, focusing on an Asian endemic perennial lineage of Mitella (Asimitellaria; Saxifragaceae). We performed artificial cross experiments for 43 pairs of ten taxonomic species, and examined their F1 hybrid pollen fertility in vitro as a quantitative measure of postzygotic reproductive isolation. Results A nonlinear, multiple regression analysis indicated that the nuclear ribosomal DNA distances are sufficient to explain the observed pattern of F1 hybrid pollen fertility, and supplementation with chloroplast DNA distance data does not improve the explanatory power. Overall, with the exception of a recently diverged species complex with more than three biological species, nuclear ribosomal DNA sequences successfully circumscribed ten distinct biological species, of which two have not been described (and an additional one has not been regarded as a distinct taxonomic species) to date. Conclusion We propose that nuclear ribosomal DNA sequences contribute to reliable identification of reproductively isolated and cryptic species of Mitella. More comparable studies for other plant groups are needed to generalize our findings to flowering plants.