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Motomura H.,Tsukuba Botanical Garden | Selosse M.-A.,CNRS Center of Evolutionary and Functional Ecology | Martos F.,CNRS Center of Evolutionary and Functional Ecology | Martos F.,University of Reunion Island | And 2 more authors.
Annals of Botany | Year: 2010

Background and Aims: Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous- achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background. Methods: To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural 13C and 15N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites. Key Results: 13C and 15N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. 13C and 15N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30-50 of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy). Conclusions: It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family. © The Author 2010. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

Shefferson R.P.,University of Georgia | Cowden C.C.,University of Georgia | McCormick M.K.,Smithsonian Environmental Research Center | Yukawa T.,Tsukuba Botanical Garden | And 2 more authors.
Molecular Ecology | Year: 2010

Host breadth is often assumed to have no evolutionary significance in broad interactions because of the lack of cophylogenetic patterns between interacting species. Nonetheless, the breadth and suite of hosts utilized by one species may have adaptive value, particularly if it underlies a common ecological niche among hosts. Here, we present a preliminary assessment of the evolution of mycorrhizal specificity in 12 closely related orchid species (genera Goodyera and Hetaeria) using DNA-based methods. We mapped specificity onto a plant phylogeny that we estimated to infer the evolutionary history of the mycorrhiza from the plant perspective, and hypothesized that phylogeny would explain a significant portion of the variance in specificity of plants on their host fungi. Sampled plants overwhelmingly associated with genus Ceratobasidium, but also occasionally with some ascomycetes. Ancestral mycorrhizal specificity was narrow in the orchids, and broadened rarely as Goodyera speciated. Statistical tests of phylogenetic inertia suggested some support for specificity varying with increasing phylogenetic distance, though only when the phylogenetic distance between suites of fungi interacting with each plant taxon were taken into account. These patterns suggest a role for phylogenetic conservatism in maintaining suits of fungal hosts among plants. We stress the evolutionary importance of host breadth in these organisms, and suggest that even generalists are likely to be constrained evolutionarily to maintaining associations with their symbionts. © 2010 Blackwell Publishing Ltd.

Shirakawa M.,Kyoto University | Ueda H.,Kyoto University | Shimada T.,Kyoto University | Koumoto Y.,Kyoto University | And 6 more authors.
Plant Journal | Year: 2010

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. © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

Godo T.,Botanic Gardens of Toyama | Komori M.,Botanic Gardens of Toyama | Nakaoki E.,Toyama Prefectural University | Yukawa T.,Tsukuba Botanical Garden | Miyoshi K.,Akita Prefectural University
In Vitro Cellular and Developmental Biology - Plant | Year: 2010

The effects of culture conditions on the asymbiotic germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial cool-climate orchid, were examined. Specifically, conditions such as illumination, temperature, and the addition of plant growth regulators to the medium were studied. Mature seeds were harvested from plants that had been collected in Toyama Prefecture, Japan, and maintained at the Botanic Gardens of Toyama. Solidified "New Dogashima" medium was used as the basal medium, and it was supplemented with 6-benzyladenopurine (BA) or α-naphthalene acetic acid (NAA). White light at 40 μmol m-2 s-1, with a 16-h photoperiod, inhibited the germination of seeds by 53-80%, as compared to dark controls in genotypes examined. The optimal temperature for the germination of seeds in darkness was 20°C and the germination frequency reached 60%, whereas it was only 28% at 25°C. While both NAA and BA stimulated germination, BA was more effective than NAA. After storage for 18 mo at 5°C, seeds incubated on medium that contained 0. 2 mg l-1 BA germinated at a frequency of 36%, which was twice that of seeds grown without any plant growth regulators. The frequency of subsequent germination decreased during storage of seeds at 5°C for approximately 2 yr, dropping from 61% to 13%. The protocorms obtained in this study were developed to plantlets readily after transferring to fresh 1/2 MS medium without any plant growth regulators. They were successfully acclimatized in green house after two to three subcultures in vitro. The significant role of a reproducible protocol for the germination of mature seeds is discussed in terms of the ex situ conservation of endangered orchid species. © 2010 The Society for In Vitro Biology.

Tanaka N.,Tsukuba Botanical Garden | Demise T.,Chiba University | Ishii M.,Chiba Prefectural Fisheries Research Center | Shoji Y.,Chiba Prefectural Fisheries Research Center | Nakaoka M.,Hokkaido University
Marine Biology | Year: 2011

Massive losses of eelgrass Zostera marina beds in Japan have occurred over the past 100 years. Toward their restoration, transplantation of eelgrass has been attempted in some areas, including Tokyo Bay. This study examined population genetic structures and gene flow in eelgrass in Tokyo Bay to establish guidelines for conducting restoration. Genotypes of a total of 360 individuals from 12 beds were determined using five microsatellite markers. The eelgrass beds in inner bay had above-average genetic diversity. A neighbor-joining tree based on FST values among beds revealed that a strong gene flow had occurred among six beds in the inner bay. Genetic assignment testing of drifting shoots indicated that those with seeds migrate in both directions between the inner and outer bay. We suggested that the restoration of eelgrass in the innermost part of Tokyo Bay, where natural habitats have been lost, should be conducted using the inner bay beds. © 2011 Springer-Verlag.

Ogura-Tsujita Y.,Tsukuba Botanical Garden | Ogura-Tsujita Y.,Japan Women's University | Yokoyama J.,Yamagata University | Miyoshi K.,Chiba University | Yukawa T.,Tsukuba Botanical Garden
American Journal of Botany | Year: 2012

Premise of the study: Mycoheterotrophic plants, which completely depend upon mycorrhizal fungi for their nutrient supply, have unusual associations with fungal partners. The processes involved in shifts in fungal associations during cladogenesis of plant partners from autotrophy to mycoheterotrophy have not been demonstrated using a robust phylogenetic framework.Methods: Consequences of a mycorrhizal shift were examined in Cymbidium (Orchidaceae) using achlorophyllous and sister chlorophyllous species. Fungal associates of the two achlorophyllous mycoheterotrophs (C. macrorhizon and C. aberrans), their close relatives, the chlorophyllous mixotrophs (C. goeringii and C. lancifolium) and an outgroup, the chlorophyllous autotroph C. dayanum, were identified by internal transcribed spacers of the nuclear ribosomal DNA sequences.Key results: Molecular identification of mycorrhizal fungi revealed: (1) the outgroup autotroph is predominantly dependent on saprobic Tulasnellaceae, (2) the mixotrophs associate with the Tulasnellaceae and ectomycorrhizal groups including the Sebacinales, Russulaceae, Thelephoraceae and Clavulinaceae, and (3) the two mycoheterotrophs are mostly specialized with ectomycorrhizal Sebacinales.Conclusion: Fungal partners in Cymbidium have shifted from saprobic to ectomycorrhizal fungi via a phase of coexistence of both nutritional types of fungi. These three phases correspond to the evolution from autotrophy to mycoheterotrophy via mixotrophy in Cymbidium We demonstrate that shifts in mycorrhizal fungi correlate with the evolution of nutritional modes in plants. Furthermore, gradual shifts in fungal partners through a phase of coexistence of different types of mycobionts may play a crucial role in the evolution of mycoheterotrophic plants. © 2012 Botanical Society of America.

Gale S.W.,Kochi Prefectural Makino Botanical Garden | Maeda A.,Kochi Prefectural Makino Botanical Garden | Chen C.-I.,Taitung District Agricultural Research and Extension Station | Yukawa T.,Tsukuba Botanical Garden
Journal of Plant Research | Year: 2010

An understanding of the extent to which reproductive strategy and seed dispersal lead to the structuring of genetic diversity in space is required when planning measures towards the conservation of endangered plant species. In this study, genetic structure in the endangered terrestrial orchid Nervilia nipponica was investigated using amplified fragment length polymorphisms following extensive sampling throughout the species' range in Japan and intensive sampling at a single population. Limited diversity was found within the species as a whole, but significant structuring was detected between populations. One genotype was common to two widely separated sites, possibly indicative of long-range dispersal. Significant structure was also detected at the intensively sampled site, as a result of the presence of two distinct putative clones. These findings are consistent with observations of the species' ability to set seed autogamously and propagate vegetatively. Given the strong colonising capability inferred for the species, attention should focus on identifying and securing habitat conditions conducive to seed germination and seedling establishment in the development of a conservation strategy. As presently circumscribed, N. nipponica is shown to comprise two polyphyletic taxa, both endemic to Japan, and both distinct from N. taiwaniana, a species that some authors have considered conspecific. © 2010 The Botanical Society of Japan and Springer.

Okuyama Y.,Tsukuba Botanical Garden | Tanabe A.S.,University of Tsukuba | Kato M.,Kyoto University
Molecular Biology and Evolution | Year: 2012

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 nuclearencoded, 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. © The Author 2011.

Ito Y.,University of Tokyo | Ohi-Toma T.,University of Tokyo | Murata J.,University of Tokyo | Tanaka N.,Tsukuba Botanical Garden
American Journal of Botany | Year: 2010

Premise of the study: The monogeneric family Ruppiaceae is found primarily in brackish water and is widely distributed on all continents, many islands, and from subartic to tropical zones. Ruppia taxonomy has been confusing because of its simplified morphology yet high phenotypic plasticity and the existence of polyploidy and putative hybrids. This study addresses the current classification of species in the genus, the origin of putative hybrids and polyploids, and the distribution of Ruppia species. Methods: Separate molecular phylogenetic analyses using plastid DNA and nuclear-encoded PHYB data sets were performed after chromosome observations. Key results: The resultant trees were largely congruent between genomes, but were incongruent in two respects: the first incongruence may be caused by long outgroup branches and their effect on ingroup rooting, and the second is caused by the existence of heterogeneous PHYB sequences for several accessions that may reflect several independent hybridization events. Several morphological species recognized in previous taxonomic revisions appear paraphyletic in plastid DNA and PHYB trees. Conclusions: Given the molecular phylogenies, and considering chromosome number and morphology, three species and one species complex comprising six lineages were discerned. A putative allotriploid, an allotetraploid, and a lineage of hybrid origin were identified within the species complex, and a hybrid was found outside the species complex, and their respective putative parental taxa were inferred. With respect to biogeography, a remarkably discontinuous distribution was identified in two cases, for which bird-mediated seed dispersal may be a reasonable explanation. © 2010 Botanical Society of America.

Ito Y.,University of Tokyo | Tanaka N.,Tsukuba Botanical Garden
Telopea | Year: 2010

The tropical seagrass genus, Halodule, is distributed in warm to tropical areas throughout the world. We performed separate molecular phylogenetic analyses of Halodule based on both plastid and nuclear DNA sequences, followed by haplotype analysis, focusing on plants in the western Pacific area. One western tropical Atlantic species, H. wrightii s.l., and two western Pacific species, H. pinifolia and H. uninervis, were recognised and characterised by leaf morphology. Because samples from the western tropical Atlantic and the western Pacific were nested with each other, allopatric differentiation into both areas may have occurred during the early stages of the evolution of the genus. A hybrid of Halodule was detected at two nearby localities in Okinawa, Japan, together with its co-occurring maternal and paternal species, H. uninervis and H. pinifolia, respectively. Haplotype analysis provided evidence that the hybrid resulted from a single hybridisation event, followed by clonal dispersal to its present localities. © 2011 Royal Botanic Gardens and Domain Trust.

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