Floral pigments and their perception by avian pollinators in three Chilean Puya species.

The Chilean Puya species, Puya coerulea var. violacea and P. chilensis bear blue and pale-yellow flowers, respectively, while P. alpestris considered to be their hybrid-derived species has unique turquoise flowers. In this study, the chemical basis underlying the different coloration of the three Puya species was explored. We first isolated and identified three anthocyanins: delphinidin 3,3',5'-tri-O-glucoside, delphinidin 3,3'-di-O-glucoside and delphinidin 3-O-glucoside; seven flavonols: quercetin 3-O-rutinoside-3'-O-glucoside, quercetin 3,3'-di-O-glucoside, quercetin 3-O-rutinoside, isorhamnetin 3-O-rutinoside, myricetin 3,3',5'-tri-O-glucoside, myricetin 3,3'-di-O-glucoside and laricitrin 3,5'-di-O-glucoside; and six flavones: luteolin 4'-O-glucoside, apigenin 4'-O-glucoside, tricetin 4'-O-glucoside, tricetin 3',5'-di-O-glucoside, tricetin 3'-O-glucoside and selagin 5'-O-glucoside, which is a previously undescribed flavone, from their petals. We also compared compositions of floral flavonoid and their aglycone among these species, which suggested that the turquoise species P. alpestris has an essentially intermediate composition between the blue and pale-yellow species. The vacuolar pH was relatively higher in the turquoise (pH 6.2) and pale-yellow (pH 6.2) flower species, while that of blue flower species was usual (pH 5.2). The flower color was reconstructed in vitro using isolated anthocyanin, flavonol and flavone at neutral and acidic pH, and its color was analyzed by reflectance spectra and the visual modeling of their avian pollinators. The modeling demonstrated that the higher pH of the turquoise and pale-yellow species enhances the chromatic contrast and spectral purity. The precise regulation of flower color by flavonoid composition and vacuolar pH may be adapted to the visual perception of their avian pollinator vision.

Mycobiont identity and light conditions affect belowground morphology and physiology of a mixotrophic orchid Cremastra variabilis (Orchidaceae).

We have investigated whether mycobiont identity and environmental conditions affect morphology and physiology of the chlorophyllous orchid: Cremastra variabilis. This species grows in a broad range of environmental conditions and associates with saprotrophic rhizoctonias including Tulasnellaceae and saprotrophic non-rhizoctonian fungi from the family Psathyrellaceae. We cultured the orchid from seeds under aseptic culture conditions and subsequently inoculated the individuals with either a Tulasnellaceae or a Psathyrellaceae isolate. We observed underground organ development of the inoculated C. variabilis plants and estimated their nutritional dependency on fungi using stable isotope abundance. Coralloid rhizome development was observed in all individuals inoculated with the Psathyrellaceae isolate, and 1-5 shoots per seedling grew from the tip of the coralloid rhizome. In contrast, individuals associated with the Tulasnellaceae isolate did not develop coralloid rhizomes, and only one shoot emerged per plantlet. In darkness, δ13C enrichment was significantly higher with both fungal isolates, whereas Î´15N values were only significantly higher in plants associated with the Psathyrellaceae isolate. We conclude that C. variabilis changes its nutritional dependency on fungal symbionts depending on light availability and secondly that the identity of fungal symbiont influences the morphology of underground organs.

Mycorrhizal specificity differences in epiphytic habitat: three epiphytic orchids harbor distinct ecological and physiological specificity.

Orchidaceae has diversified in tree canopies and accounts for 68% of vascular epiphytes. Differences in mycorrhizal communities among epiphytic orchids can reduce species competition for mycorrhizal fungi and contribute to niche partitioning, which may be a crucial driver of the unusual species diversification among orchids. Mycorrhizal specificity-the range of fungi allowing mycorrhizal partnerships-was evaluated by assessment of mycorrhizal communities in the field (ecological specificity) and symbiotic cultures in the laboratory (physiological specificity) for three epiphytic orchids inhabiting Japan. Mycorrhizal communities were assessed with co-existing individuals growing within 10 cm of each other, revealing that ecological specificity varied widely among the three species, ranging from dominance by a single Ceratobasidiaceae fungus to diverse mycobionts across the Ceratobasidiaceae and Tulasnellaceae. In vitro seed germination tests revealed clear differences in physiological specificity among the three orchids, and that the primary mycorrhizal partners contributed to seed germination. In vitro compatibility ranges of three orchids strongly reflect the mycorrhizal community composition of wild populations. This suggests that differences in in situ mycorrhizal communities are not strongly driven by environmental factors, but are primarily due to physiological differences among orchid species. This study shows that the symbiotic strategy among the epiphytic orchid species varies from specialized to generalized association, which may contribute to biotic niche partitioning.

Phylotranscriptomic Analyses of Mycoheterotrophic Monocots Show a Continuum of Convergent Evolutionary Changes in Expressed Nuclear Genes From Three Independent Nonphotosynthetic Lineages.

Mycoheterotrophy is an alternative nutritional strategy whereby plants obtain sugars and other nutrients from soil fungi. Mycoheterotrophy and associated loss of photosynthesis have evolved repeatedly in plants, particularly in monocots. Although reductive evolution of plastomes in mycoheterotrophs is well documented, the dynamics of nuclear genome evolution remains largely unknown. Transcriptome datasets were generated from four mycoheterotrophs in three families (Orchidaceae, Burmanniaceae, Triuridaceae) and related green plants and used for phylogenomic analyses to resolve relationships among the mycoheterotrophs, their relatives, and representatives across the monocots. Phylogenetic trees based on 602 genes were mostly congruent with plastome phylogenies, except for an Asparagales + Liliales clade inferred in the nuclear trees. Reduction and loss of chlorophyll synthesis and photosynthetic gene expression and relaxation of purifying selection on retained genes were progressive, with greater loss in older nonphotosynthetic lineages. One hundred seventy-four of 1375 plant benchmark universally conserved orthologous genes were undetected in any mycoheterotroph transcriptome or the genome of the mycoheterotrophic orchid Gastrodia but were expressed in green relatives, providing evidence for massively convergent gene loss in nonphotosynthetic lineages. We designate this set of deleted or undetected genes Missing in Mycoheterotrophs (MIM). MIM genes encode not only mainly photosynthetic or plastid membrane proteins but also a diverse set of plastid processes, genes of unknown function, mitochondrial, and cellular processes. Transcription of a photosystem II gene (psb29) in all lineages implies a nonphotosynthetic function for this and other genes retained in mycoheterotrophs. Nonphotosynthetic plants enable novel insights into gene function as well as gene expression shifts, gene loss, and convergence in nuclear genomes.

Photosynthesis and leaf structure of F1 hybrids between Cymbidium ensifolium (C3) and C. bicolor subsp. pubescens (CAM).

BACKGROUND AND AIMS: The introduction of crassulacean acid metabolism (CAM) into C3 crops has been considered as a means of improving water-use efficiency. In this study, we investigated photosynthetic and leaf structural traits in F1 hybrids between Cymbidium ensifolium (female C3 parent) and C. bicolor subsp. pubescens (male CAM parent) of the Orchidaceae. METHODS: Seven F1 hybrids produced through artificial pollination and in vitro culture were grown in a greenhouse with the parent plants. Structural, biochemical, and physiological traits involved in CAM in their leaves were investigated. KEY RESULTS: Cymbidium ensifolium accumulated very low levels of malate without diel fluctuation, whereas C. bicolor subsp. pubescens showed nocturnal accumulation and diurnal consumption of malate. The F1s also accumulated malate at night, but much less than C. bicolor subsp. pubescens. This feature was consistent with low nocturnal fixation of atmospheric CO2 in the F1s. δ 13C values of the F1s were intermediate between those of the parents. The leaf thickness was thicker in C. bicolor subsp. pubescens than in C. ensifolium, and those of the F1s were more similar to that of C. ensifolium. This was due to the difference in mesophyll cell size. The chloroplast coverage of mesophyll cell perimeter adjacent to intercellular air spaces of C. bicolor subsp. pubescens was lower than that of C. ensifolium, and those of the F1s were intermediate between them. Interestingly, one F1 had structural and physiological traits more similar to those of C. bicolor subsp. pubescens than the other F1s. Nevertheless, all F1s contained intermediate levels of phosphoenolpyruvate carboxylase but as much pyruvate,Pi dikinase as C. bicolor subsp. pubescens. CONCLUSIONS: CAM traits were intricately inherited in the F1 hybrids, the level of CAM expression varied widely among F1 plants, and the CAM traits examined were not necessarily co-ordinately transmitted to the F1s.

In-vitro symbiotic germination of seeds of five mycoheterotrophic Gastrodia orchids with Mycena and Marasmiaceae fungi.

We performed in-vitro germination tests on seeds from five Gastrodia orchids (G. confusa, G. elata var. elata, G. elata var. pallens, G. nipponica, and G. pubilabiata) using one Marasmiaceae and two Mycena isolates. Mycena sp. 1 promoted germination of all five Gastrodia orchids, with root and/or tuber formation observed in G. confusa, G. nipponica, and G. pubilabiata. No additional growth was observed in the other two orchids. Mycena sp. 2 induced G. confusa, G. elata var. elata, and G. nipponica germination, whereas Marasmiaceae sp. 1 induced G. nipponica and G. pubilabiata germination. Phylogenetic analyses indicated that the two Mycena isolates represent distinct lineages within the Mycenaceae. Mycena sp. 1 and Marasmiaceae sp. 1 are closely related to Mycena abramsii and Marasmiellus rhizomorphogenus, respectively. Our results imply that Mycena and marasmioid fungi play important roles in early development in Gastrodia species, and that Mycena fungi in particular may be common mycobionts of Gastrodia species. Root and/or tuber development was observed with four plant-fungus combinations, implying that these associations persist throughout the life cycle, whereas G. elata var. elata may require different associates over time. Our findings will contribute to elucidating the mycorrhizal associations of mycoheterotrophic orchids throughout their life cycle.

Phylogenetic analysis and character evolution of tribe Arethuseae (Orchidaceae) reveal a new genus Mengzia.

Delimitation of the tribe Arethuseae has varied considerably since it was first defined. The relationships within Arethuseae, particularly within the subtribe Arethusinae, remain poorly elucidated. In this study, we reconstructed the phylogeny of Arethuseae, using six plastid markers (matK, ycf1, rbcL rpoc1, rpl32-trnL and trnL-F) from 83 taxa. The ancestral state reconstruction of 11 selected morphological characters was also conducted to identify synapomorphies and assess potential evolutionary transitions. Morphological character comparision between the distinct species Bletilla foliosa and other species are conducted. Our results unequivocally supported the monophyly of Arethuseae, which included highly supported clades and a clear synapomorphy of non-trichome-like lamellae. Furthermore, B. foliosa formed a separate clade in the subtribe Arethusinae, instead of clustering with the other Bletilla species in the subtribe Coelogyninae. The morphological characters comparision further showed that the B. foliosa clade could be distinguished from other genera in Arethuseae by multiple characters, including presence of lateral inflorescence, three lamellae with trichome-like apex and four pollinia. In light of these molecular and morphological evidences, we propose Mengzia as a new genus to accommodate B. foliosa and accordingly provide descriptions of this new genus and combination.

Mycobiont diversity and first evidence of mixotrophy associated with Psathyrellaceae fungi in the chlorophyllous orchid Cremastra variabilis.

Mixotrophy (MX, also called partial mycoheterotrophy) in plants is characterized by isotopic abundances that differ from those of autotrophs. Previous studies have evaluated mycoheterotrophy in MX plants associated with fungi of similar ecological characteristics, but little is known about the differences in the relative abundances of 13C and 15N in an orchid species that associates with several different mycobionts species. Since the chlorophyllous orchid Cremastra variabilis Nakai associates with various fungi with different ecologies, we hypothesized that it may change its relative abundances of 13C and 15N depending on the associated mycobionts. We investigated mycobiont diversity in the chlorophyllous orchid C. variabilis together with the relative abundance of 13C and 15N and morphological underground differentiation (presence or absence of a mycorhizome with fungal colonization). Rhizoctonias (Tulasnellaceae, Ceratobasidiaceae, Sebacinales) were detected as the main mycobionts. High differences in δ13C values (- 34.7 to - 27.4 ‰) among individuals were found, in which the individuals associated with specific Psathyrellaceae showed significantly high relative abundance of 13C. In addition, Psathyrellaceae fungi were always detected on individuals with mycorhizomes. In the present study, MX orchid association with non-rhizoctonia saprobic fungi was confirmed, and the influence of mycobionts on morphological development and on relative abundance of 13C and 15N was discovered. Cremastra variabilis may increase opportunities to gain nutrients from diverse partners, in a bet-hedging plasticity that allows colonization of various environmental conditions.

Mycoheterotrophic seedling growth of Gentiana zollingeri, a photosynthetic Gentianaceae plant species, in symbioses with arbuscular mycorrhizal fungi.

We found mycoheterotrophic seedling growth (initial mycoheterotrophy) of Gentiana zollingeri, a spring-flowering photosynthetic species of Gentianaceae family. Small seeds (about 300 µm in length) were buried in a habitat by using seed packets, and development of the subterranean seedlings to form shoots, more than 3 cm in length, was observed in symbiosis with arbuscular mycorrhizal (AM) fungi in the dark (i.e., underground of a field). Hyphal coils and their degenerations were observed in the root cortical cells of the subterranean seedlings as well as those of adult plants. Among the mycobionts identified on the basis of partial small subunit rDNA sequences, it was found that AM fungi of a lineage in Glomeraceae dominantly colonized, and the AM fungi were also dominant in adult individuals of G. zollingeri in three habitats separated one another by 17.2, 34.7, and 49.6 km. Though initial mycoheterotrophy in symbioses with AM fungi has been observed in some pteridophytes, this is the first study to demonstrate this type of symbiosis in a photosynthetic seed plant. The mycoheterotrophy means that an energy distribution occurs through the hyphal bridges of AM fungi among different photosynthetic seed plants, which may be important in constructing plant species diversity in some ecosystems.

Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi.

Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant-fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.

The mycorrhizal community of the epiphytic orchid Thrixspermum japonicum is strongly biased toward a single Ceratobasidiaceae fungus, despite a wide range of fungal partners.

PREMISE: Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. METHODS: We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300 km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. RESULTS: We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. CONCLUSIONS: Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host-tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats.

Molecular phylogenetic study of the tribe Tropidieae (Orchidaceae, Epidendroideae) with taxonomic and evolutionary implications.

The orchid tribe Tropidieae comprises three genera, Tropidia, Corymborkis and Kalimantanorchis. There are three fully mycoheterotrophic species within Tropidieae: Tropidia saprophytica, T. connata and Kalimantanorchis nagamasui. A previous phylogenetic study of K. nagamasui, based only on plastid matK data, placed K. nagamasui outside the clade of Tropidia and Corymborkis without support. In this study, we performed phylogenetic analyses using a nuclear ribosomal DNA spacer (ITS1-5.8S-ITS2), a low-copy nuclear coding gene (Xdh) and a mitochondrial intron (nad1b-c intron) to study the phylogenetic relationships within Tropidieae. We included six photosynthetic and all three fully mycoheterotrophic Tropidieae species. The resulting phylogenetic trees placed these fully mycoheterotrophic species inside the Tropidia clade with high support. In our trees, these three species do not form a monophyletic group together, because the photosynthetic T. graminea is nested amongst them. Our results also suggest that the loss of photosynthetic ability occurred at least twice in Tropidia.

Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves.

Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25 mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30 min at room temperature (20 °C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30 min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information.

New taxa of Rhododendron tschonoskii alliance (Ericaceae) from East Asia.

Three new taxa, Rhododendron sohayakiense Y.Watan. & T.Yukawa (Ericaceae), and its two varieties, var. kiusianum Y.Watan., T.Yukawa & T.Minamitani and var. koreanum Y.Watan. & T.Yukawa are described and illustrated from Japan and South Korea. They can be distinguished from each other and from the other members of the R. tschonoskii alliance, i.e. R. tschonoskii, R. tetramerum, R. trinerve and R. tsusiophyllum, through their combination of leaf size, leaf morphologies including lateral nerves on abaxial leaf surface, corolla morphologies including number of corolla lobes, style length and anther form. Phylogenetic inferences based on chloroplast DNA and genome-wide sequences revealed that each of the three new taxa is monophyletic and they further form a clade. Distributions of the three taxa are also clearly separated from each other and also from the other members of the R. tschonoskii alliance.

A leafless epiphytic orchid, Taeniophyllum glandulosum Blume (Orchidaceae), is specifically associated with the Ceratobasidiaceae family of basidiomycetous fungi.

Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has chloroplast-containing cortical cells and is the sole photosynthetic organ for carbon gain. All orchids are entirely dependent on mycorrhizal fungi for their carbon supply during seed germination, and this mycorrhizal association generally persists in adult plants. However, our knowledge of the mycorrhizal association of leafless epiphytic orchids remains limited, and the contribution of the mycorrhizal association to nutrient acquisition in these orchid species is largely unknown. In this study, the mycorrhizal fungi of a leafless epiphytic orchid, Taeniophyllum glandulosum, were identified molecularly using 68 mature plants and 17 seedlings. In total, 187 fungal internal transcribed spacer sequences were obtained, of which 99% were identified as Ceratobasidiaceae. These sequences were classified into five operational taxonomic units (OTUs) based on 97% sequence similarity. The most frequent sequence was OTU1, which accounted for 91% of all Ceratobasidiaceae sequences, although other phylogenetically distinct Ceratobasidiaceae fungi were detected. These results show that T. glandulosum is specifically associated with a particular group of Ceratobasidiaceae. All mycorrhizal fungi found in T. glandulosum seedlings belonged to OTU1, which was also found in adult plants on the same host tree. The mycorrhizal fungi from 13 host tree species were compared, and T. glandulosum was preferentially associated with OTU1 on 11 tree species. In conclusion, T. glandulosum is specifically associated with Ceratobasidiaceae fungi and this specific association remains throughout the orchid life cycle and is found on divergent host tree species.

Low assimilation efficiency of photorespiratory ammonia in conifer leaves.

Glutamine synthetase (GS) localized in the chloroplasts, GS2, is a key enzyme in the assimilation of ammonia (NH3) produced from the photorespiration pathway in angiosperms, but it is absent from some coniferous species belonging to Pinaceae such as Pinus. We examined whether the absence of GS2 is common in conifers (Pinidae) and also addressed the question of whether assimilation efficiency of photorespiratory NH3 differs between conifers that may potentially lack GS2 and angiosperms. Search of the expressed sequence tag database of Cryptomeria japonica, a conifer in Cupressaceae, and immunoblotting analyses of leaf GS proteins of 13 species from all family members in Pinidae revealed that all tested conifers exhibited only GS1 isoforms. We compared leaf NH3 compensation point (γNH3) and the increments in leaf ammonium content per unit photorespiratory activity (NH3 leakiness), i.e. inverse measures of the assimilation efficiency, between conifers (C. japonica and Pinus densiflora) and angiosperms (Phaseolus vulgaris and two Populus species). Both γNH3 and NH3 leakiness were higher in the two conifers than in the three angiosperms tested. Thus, we concluded that the absence of GS2 is common in conifers, and assimilation efficiency of photorespiratory NH3 is intrinsically lower in conifer leaves than in angiosperm leaves. These results imply that acquisition of GS2 in land plants is an adaptive mechanism for efficient NH3 assimilation under photorespiratory environments.

Phylogenomic inference in extremis: A case study with mycoheterotroph plastomes.

PREMISE OF THE STUDY: Phylogenomic studies employing large numbers of genes, including those based on plastid genomes (plastomes), are becoming common. Nonphotosynthetic plants such as mycoheterotrophs (which rely on root-associated fungi for essential nutrients, including carbon) tend to have highly elevated rates of plastome evolution, substantial genome reduction, or both. Mycoheterotroph plastomes therefore provide excellent test cases for investigating how extreme conditions impact phylogenomic inference. METHODS: We used parsimony and likelihood analysis of protein-coding gene sets from published and newly completed plastomes to infer the phylogenetic placement of taxa from the 10 angiosperm families in which mycoheterotrophy evolved. KEY RESULTS: Despite multiple very long branches that reflect elevated substitution rates, and frequently patchy gene recovery due to genome reduction, inferred phylogenetic placements of most mycoheterotrophic lineages in DNA-based likelihood analyses are both well supported and congruent with other studies. Amino-acid-based likelihood placements are broadly consistent with DNA-based inferences, but extremely rate-elevated taxa can have unexpected placements-albeit with weak support. In contrast, parsimony analysis is strongly misled by long-branch attraction among many distantly related mycoheterotrophic monocots. CONCLUSIONS: Mycoheterotrophic plastomes provide challenging cases for phylogenomic inference, as substitutional rates can be elevated and genome reduction can lead to sparse gene recovery. Nonetheless, diverse likelihood frameworks provide generally well-supported and mutually concordant phylogenetic placements of mycoheterotrophs, consistent with recent phylogenetic studies and angiosperm-wide classifications. Previous predictions of parallel photosynthesis loss within families are supported for Burmanniaceae, Ericaceae, Gentianaceae, and Orchidaceae. Burmanniaceae and Thismiaceae should not be combined as a single family in Dioscoreales.

Phylotranscriptomic analysis and genome evolution of the Cypripedioideae (Orchidaceae).

PREMISE OF THE STUDY: The slipper orchids (Cypripedioideae) are a morphologically distinct subfamily of Orchidaceae. They also have some of the largest genomes in the orchids, which may be due to polyploidy or some other mechanism of genome evolution. We generated 10 transcriptomes and incorporated existing RNA-seq data to infer a multilocus nuclear phylogeny of the Cypripedioideae and to determine whether a whole-genome duplication event (WGD) correlated with the large genome size of this subfamily. Knowing more about timing of ancient polyploidy events can help us understand the evolution of one of the most species-rich plant families. METHODS: Transcriptome data were used to identify low-copy orthologous genes to infer a phylogeny of Orchidaceae and to identify paralogs to place any WGD events on the species tree. KEY RESULTS: Our transcriptome phylogeny confirmed relationships published in previous studies that used fewer markers but incorporated more taxa. We did not find a WGD event at the base of the slipper orchids; however, we did identify one on the Orchidaceae stem lineage. We also confirmed the presence of a previously identified WGD event deeper in the monocot phylogeny. CONCLUSIONS: Although WGD has played a role in the evolution of Orchidaceae, polyploidy does not appear to be responsible for the large genome size of slipper orchids. The conserved set of 775 largely single-copy nuclear genes identified in this study should prove useful in future studies of orchid evolution.

Identification of Two Phenanthrene Derivatives from Australasian Allied Species in Genus Dendrobium.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.

Genetic diversity in the endangered terrestrial orchid Cypripedium japonicum in East Asia: Insights into population history and implications for conservation.

Little is known about levels and patterns of genetic diversity for the entire range of endangered orchids native to China, Korea, and Japan. In this study, we focus on Cypripedium japonicum and suggest three hypotheses: 1) that genetic drift has been a primary evolutionary force; 2) that populations in central and western China harbor higher levels of genetic variation relative to those from eastern China; and 3) that C. japonicum in China maintains the highest genetic variation among the three countries. Using ISSR and SCoT markers, we investigated genetic diversity in 17 populations to test the three hypotheses. As anticipated, we found low levels of genetic diversity at the species level with substantially high degree of genetic divergence, which can be mainly attributed to random genetic drift. Chinese populations harbor the highest within-population genetic variation, which tends to increase from east to west. We also found a close relationship between Korean populations and central/western Chinese populations. Historical rarity coupled with limited gene flow seems to be important factors for shaping genetic diversity and structure of C. japonicum. Our results indicate that the mountain areas in central and western China were likely refugia at the Last Glacial Maximum.