Chasmogamy and entomophily in Burmannia disticha (Burmanniaceae).

Burmannia shows a set of floral traits that suggest elaborate mechanisms of animal-mediated pollen transfer. These include flower coloration, septal nectaries and a long and narrow floral chamber. The stamens are synorganized with the common style restricting the entrance to the floral chamber, sometimes forming a gynostegium. Contrary to this apparent zoophilous floral syndrome, several species of Burmannia were reported to perform self-pollination via cleistogamy. Understanding of reproductive systems in Burmannia is complicated by scarcity of available results of direct observations on pollination process. Here we present data on pollination biology of B. disticha obtained during field investigations in Vietnam followed by laboratory analyses of ecologically important floral traits and the captured flower visitors. We found that the anthetic perianth is open, i.e. the flower is chasmogamous. The flowers are visited by various Diptera, Hymenoptera, Lepidoptera and Orthoptera. Of them, the bumblebees (Bombus burmensis), a bee (Coelioxys sp.) and some lepidopterans were revealed to carry pollen of B. disticha. Based on the amount of carried pollen, insect behavior during the visits and general knowledge on biology of these insect taxa, we concluded that the bumblebees act as the principal pollinators of B. disticha, whereas the lepidopterans are considered as its possible pollinators. We compared the lengths of proboscises of the captured insects to the depth of the floral chamber, and found that only the bumblebees and lepidopterans should be able to reach the nectar. Finally, we estimated the pollen-ovule ratio of B. disticha as 6.84, which is comparable to the ratio known in autogamous angiosperms. Based on its flower organization and pollination mechanism, we consider B. disticha an entomophilous and predominantly xenogamous species. Its gynostegium is likely an adaptation for pollen transfer by insects with long proboscises. At the same time, earlier investigations together with pollen-ovule ratio indicate that B. disticha possesses a labile pollination strategy, and autogamy sometimes occurs. Since Burmannia is one of the few angiosperm genera that comprise both mycoheterotrophic (achlorophyllous) and autotrophic (green) species, our study provides important evidence for reconstructions of ecological and morphological evolutionary pathways in relation to the mode of organic nutrition.

Structure and Development of Flowers and Inflorescences in Burmannia (Burmanniaceae, Dioscoreales).

Species of the genus Burmannia possess distinctive and highly elaborated flowers with prominent floral tubes that often bear large longitudinal wings. Complicated floral structure of Burmannia hampers understanding its floral evolutionary morphology and biology of the genus. In addition, information on structural features believed to be taxonomically important is lacking for some species. Here we provide an investigation of flowers and inflorescences of Burmannia based on a comprehensive sampling that included eight species with various lifestyles (autotrophic, partially mycoheterotrophic and mycoheterotrophic). We describe the diversity of inflorescence architecture in the genus: a basic (most likely, ancestral) inflorescence type is a thyrsoid comprising two cincinni, which is transformed into a botryoid in some species via reduction of the lateral cymes to single flowers. Burmannia oblonga differs from all the other studied species in having an adaxial (vs. transversal) floral prophyll. For the first time, we describe in detail early floral development in Burmannia. We report presence of the inner tepal lobes in B. oblonga, a species with reportedly absent inner tepals; the growth of the inner tepal lobes is arrested after the middle stage of floral development of this species, and therefore they are undetectable in a mature flower. Floral vasculature in Burmannia varies to reflect the variation of the size of the inner tepal lobes; in B. oblonga with the most reduced inner tepals their vascular supply is completely lost. The gynoecium consists of synascidiate, symplicate, and asymplicate zones. The symplicate zone is secondarily trilocular (except for its distal portion in some of the species) without visible traces of postgenital fusion, which prevented earlier researchers to correctly identify the zones within a definitive ovary. The placentas occupy the entire symplicate zone and a short distal portion of the synascidiate zone. Finally, we revealed an unexpected diversity of stamen-style interactions in Burmannia. In all species studied, the stamens are tightly arranged around the common style to occlude the flower entrance. However, in some species the stamens are free from the common style, whereas in the others the stamen connectives are postgenitally fused with the common style, which results in formation of a gynostegium.

Phylogeny and biogeography of the northern temperate genus Dracocephalum s.l. (Lamiaceae).

The northern temperate genus Dracocephalum consists of approximately 70 species mainly distributed in the steppe-desert biomes of Central and West Asia and the alpine region of the Qinghai-Tibetan Plateau (QTP). Previous work has shown that Dracocephalum is not monophyletic and might include Hyssopus and Lallemantia. This study attempts to clarify the phylogenetic relationships, diversification patterns, and the biogeographical history of the three genera (defined as Dracocephalum s.l.). Based on a sampling of 66 taxa comprising more than 80% from extant species of Dracocephalum s.l., morphological, phylogenetic (maximum parsimony, likelihood, and Bayesian inference based on nuclear ITS and ETS, plastid rpl32-trnL, trnL-trnF, ycf1, and ycf1-rps15, and two low-copy nuclear markers AT3G09060 and AT1G09680), molecular dating, diversification, and ancestral range estimation analyses were carried out. Our results demonstrate that both Hyssopus and Lallemantia are embedded within Dracocephalum and nine well-supported clades can be recognized within Dracocephalum s.l. Analyses of divergence times suggest that the genus experienced an early rapid radiation during the middle to late Miocene with major lineages diversifying within a relatively narrow timescale. Ancestral area reconstruction analyses indicate that Dracocephalum s.l. originated in Central and West Asia and southern Siberia, and dispersed from Central and West Asia into the QTP and adjacent areas twice independently during the Pliocene. The aridification of the Asian interior possibly promoted the rapid radiation of Dracocephalum within this region, and the uplift of the QTP appears to have triggered the dispersal and recent rapid diversification of the genus in the QTP and adjacent regions. Combining molecular phylogenetic and morphological evidence, a revised infrageneric classification of Dracocephalum s.l. is proposed, which recognizes nine sections within the genus.

Leaf epidermal micromorphology in Aspidistra (Asparagaceae): diversity and taxonomic significance.

Micromorphological characters of leaf epidermis were investigated in 69 species of Aspidistra using scanning electron microscopy. Sculpture of epidermis varies from smooth to verrucose and rugose in the genus. The abaxial epidermis of some species bears papillae, whereas the adaxial surface uniformly lacks the papillae. Sculpture type of epidermis and density of papillae are generally found to be stable characters at a species level. The infraspecific variation of epidermis sculpture, where present, ranges from smooth to verrucose or from verrucose to rugose. Micromorphological characters of leaf epidermis are shown to have potential taxonomic significance in Aspidistra; in combination with the type of shoot structure, they allow to subdivide the species into 13 groups. The groups are largely incongruent with floral morphological traits. An identification key to the studied species of Aspidistra based on vegetative characters (gross leaf and shoot morphology and characters of leaf epidermis) is presented.

Genomic comparison of non-photosynthetic plants from the family Balanophoraceae with their photosynthetic relatives.

The plant family Balanophoraceae consists entirely of species that have lost the ability to photosynthesize. Instead, they obtain nutrients by parasitizing other plants. Recent studies have revealed that plastid genomes of Balanophoraceae exhibit a number of interesting features, one of the most prominent of those being a highly elevated AT content of nearly 90%. Additionally, the nucleotide substitution rate in the plastid genomes of Balanophoraceae is an order of magnitude greater than that of their photosynthetic relatives without signs of relaxed selection. Currently, there are no definitive explanations for these features. Given these unusual features, we hypothesised that the nuclear genomes of Balanophoraceae may also provide valuable information in regard to understanding the evolution of non-photosynthetic plants. To gain insight into these genomes, in the present study we analysed the transcriptomes of two Balanophoraceae species (Rhopalocnemis phalloides and Balanophora fungosa) and compared them to the transcriptomes of their close photosynthetic relatives (Daenikera sp., Dendropemon caribaeus, and Malania oleifera). Our analysis revealed that the AT content of the nuclear genes of Balanophoraceae did not markedly differ from that of the photosynthetic relatives. The nucleotide substitution rate in the genes of Balanophoraceae is, for an unknown reason, several-fold larger than in the genes of photosynthetic Santalales; however, the negative selection in Balanophoraceae is likely stronger. We observed an extensive loss of photosynthesis-related genes in the Balanophoraceae family members. Additionally, we did not observe transcripts of several genes whose products function in plastid genome repair. This implies their loss or very low expression, which may explain the increased nucleotide substitution rate and AT content of the plastid genomes.

Comparative Analysis of Plastid Genomes in the Non-photosynthetic Genus Thismia Reveals Ongoing Gene Set Reduction.

Heterotrophic plants provide intriguing examples of reductive evolution. This is especially evident in the reduction of their plastid genomes, which can potentially proceed toward complete genome loss. Several milestones at the beginning of this path of degradation have been described; however, little is known about the latest stages of plastome reduction. Here we analyze a diversity of plastid genomes in a set of closely related non-photosynthetic plants. We demonstrate how a gradual loss of genes shapes the miniaturized plastomes of these plants. The subject of our study, the genus Thismia, represents the mycoheterotrophic monocot family Thismiaceae, a group that may have experienced a very ancient (60-80 mya) transition to heterotrophy. In all 18 species examined, the plastome is reduced to 14-18 kb and is highly AT-biased. The most complete observed gene set includes accD, seven ribosomal protein genes, three rRNA, and two tRNA genes. Different clades of Thismia have undergone further gene loss (complete absence or pseudogenization) compared to this set: in particular, we report two independent losses of rps2 and rps18.

Flower structure and development in Vietnamese Sciaphila (Triuridaceae: Pandanales): refined knowledge of the morphological misfit family and implications for taxonomy.

The monocot family Triuridaceae is a morphological misfit with respect to several traits of floral morphology, including the uniformly apocarpous polymerous gynoecium and the famous inside-out flowers of Lacandonia. Although Triuridaceae are crucially important for understanding the floral evolution of Pandanales and angiosperms in general, significant knowledge gaps exist which hamper adequate morphological analysis of flowers in this family. The scarcity of morphological data is also reflected in numerous taxonomic inconsistencies. Here we provide a comprehensive study of reproductive organs of four species of Sciaphila occurring in Vietnam (S. arfakiana, S. densiflora, S. nana and S. stellata) including the first investigation of early floral development and gynoecium phyllotaxis. Our observations are mainly based on SEM images. We confirm the perianth (studied in male flowers) to be two-whorled and report a rare sequence of initiation of perianth parts: the outer tepals show a late congenital fusion, as their free lobes appear before the common perianth tube, whereas the inner tepals show an early congenital fusion, with their free lobes initiating on the tube rim. We interpret the stamen appendages as basal adaxial outgrowths of the stamen filaments. We discuss the number of thecae and locules in anthers of Sciaphila, and conclude that 3- and 4-, but not 2-locular anthers are characteristic of this genus. We describe the carpels as consisting of both ascidiate and plicate zones, the former being extremely short and completely obscured by anthesis. The single ovule is attached in the cross-zone. The style is non-plicate. We analyze gynoecium phyllotaxis by estimating its contact parastichies, and by investigating the number and arrangement of the outermost carpels. The carpel arrangement in a given gynoecium is a result of the balance between whorled and irregular (but not spiral) phyllotaxis. We recognize the following figures of gynoecium merism: 6, 9, 10, 10.5, 11 and 12, with the prevalence of those divisible by three. We discuss our results in the light of general diversity of floral structure of monocots. Our data allow to clarify several issues in taxonomy of Asian Sciaphila and indicate directions of further studies. We report a significant range extension for S. densiflora, which is newly recorded for the flora of Vietnam. We describe for the first time staminodes in female flowers of this species. We reveal two distinct morphs of S. nana in Vietnam. We highlight the need of a thorough revision of S. secundiflora species complex in order to verify the species boundaries and, in particular, to test the identity of the Vietnamese S. stellata.

Rhopalocnemis phalloides has one of the most reduced and mutated plastid genomes known.

Although most plant species are photosynthetic, several hundred species have lost the ability to photosynthesize and instead obtain nutrients via various types of heterotrophic feeding. Their plastid genomes markedly differ from the plastid genomes of photosynthetic plants. In this work, we describe the sequenced plastid genome of the heterotrophic plant Rhopalocnemis phalloides, which belongs to the family Balanophoraceae and feeds by parasitizing other plants. The genome is highly reduced (18,622 base pairs vs. approximately 150 kbp in autotrophic plants) and possesses an extraordinarily high AT content, 86.8%, which is inferior only to AT contents of plastid genomes of Balanophora, a genus from the same family. The gene content of this genome is quite typical of heterotrophic plants, with all of the genes related to photosynthesis having been lost. The remaining genes are notably distorted by a high mutation rate and the aforementioned AT content. The high AT content has led to sequence convergence between some of the remaining genes and their homologs from AT-rich plastid genomes of protists. Overall, the plastid genome of R. phalloides is one of the most unusual plastid genomes known.

Checklist to the Elatostema (Urticaceae) of Vietnam including 19 new records, ten new combinations, two new names and four new synonyms.

Elatostema (Urticaceae) comprises several hundred herbaceous species distributed in tropical and subtropical Africa, Asia, Australia and Oceania. The greatest species richness occurs on limestone karst in Southeast Asia. Taxonomic revisions of Elatostema are largely out of date and contradict each other with respect to the delimitation of Elatostema and Pellionia. Most herbaria in SE Asia and worldwide contain significant amounts of unidentified material. As part of a broader revision of Elatostema in SE Asia, we present an updated checklist for Vietnam based on field visits, a review of specimens in herbaria worldwide, a review of type material and nomenclature. We recognize 77 taxa (75 species and two infraspecific taxa) of Elatostema in Vietnam, 23 of which were previously ascribed to Pellionia. Nineteen of these are new records for the country, i.e., E. attenuatoides, E. austrosinense, E. backeri, E. brunneinerve, E. crassiusculum, E. crenatum, E. fengshanense, E. glochidioides, E. malacotrichum, E. nanchuanense, E. oblongifolium, E. obtusum, E. oppositum, E. pergameneum, E. prunifolium, E. pseudolongipes, E. pycnodontum, E. salvinioides and E. xichouense. We place E. baviensis in synonymy of E. platyphyllum, E. colaniae in synonymy of E. myrtillus, P. macroceras in synonymy of E. hookerianum, and P. tetramera in synonymy of E. dissectum for the first time. Fourteen taxa (18% of all the recognized taxa) are endemic to Vietnam, which makes Elatostema one of the richest genera for endemic species in this country; this level of endemism is comparable to levels observed in Orchidaceae. Our checklist suggests that the highest diversity and endemism of Elatostema occurs in northern Vietnam, and that there is the greatest floristic similarity of northern Vietnam to SW China. The relationship among floristic regions is also investigated. We could find no records of Elatostema for 33 out of 63 provincial units of Vietnam, including all the southernmost provinces. We propose that further studies on the diversity of Elatostema in central and southern Vietnam are severely needed.

Staminate flower of Prunus s. l. (Rosaceae) from Eocene Rovno amber (Ukraine).

The late Eocene ambers provide plethora of animal and plant fossils including well-preserved angiosperm flowers from the Baltic amber. The Rovno amber from NW Ukraine resembles in many aspects the Baltic amber; however, only fossilized animals and some bryophytes have yet been studied from the Rovno amber. We provide the first detailed description of an angiosperm flower from Rovno amber. The flower is staminate with conspicuous hypanthium, double pentamerous perianth and whorled androecium of 24 stamens much longer than the petals. Sepals are sparsely pubescent and petals are densely hirsute outside. The fossil shares important features with extant members of Prunus subgen. Padus s. l. (incl. Laurocerasus, Pygeum and Maddenia), especially with its evergreen paleotropical species. It is described here as a new species Prunus hirsutipetala D.D.Sokoloff, Remizowa et Nuraliev. Our study provides the first convincing record of fossil flowers of Rosaceae from Eocene of Europe and the earliest fossil flower of Prunus outside North America. Our record of a plant resembling extant tropical species supports palaeoentomological evidences for warm winters in northwestern Ukraine during the late Eocene, as well as suggesting a more significant role of tropical insects in Rovno amber than inferred from Baltic amber.

Gradual vs. abrupt reduction of carpels in syncarpous gynoecia: A case study from Polyscias subg. Arthrophyllum (Araliaceae: Apiales).

PREMISE OF THE STUDY: Revealing the relative roles of gradual and abrupt transformations of morphological characters is an important topic of evolutionary biology. Gynoecia apparently consisting of one carpel have evolved from pluricarpellate syncarpous gynoecia in several angiosperm clades. The process of reduction can involve intermediate stages, with one fertile and one or more sterile carpels (pseudomonomery). The possible origin of monomery directly via an abrupt change of gynoecium merism has been a matter of dispute. We explore the nature of gynoecium reduction in a clade of Araliaceae. METHODS: The anatomy and development of unilocular gynoecia are investigated using light and scanning electron microscopy in two members of Polyscias subg. Arthrophyllum. Gynoecium diversity in the genus is discussed in a phylogenetic framework. KEY RESULTS: Unilocular gynoecia with one fertile ovule have evolved at least four times in Polyscias, including one newly discovered case. The two unilocular taxa investigated are unicarpellate, without any traces of reduced sterile carpels. Carpel orientation is unstable, and the ovary roof and style contain numerous vascular bundles without clearly recognizable dorsals or ventrals. In contrast to pluricarpellate Araliaceae and Apiaceae, the cross zone is apparently oblique in the unicarpellate species. CONCLUSIONS: No support was found for gradual gynoecium reduction via pseudomonomery. The abrupt origin of monomery via direct change of gynoecium merism and the unstable carpel orientation observed are related to the general lability of the flower groundplan in Polyscias. The apparent occurrence of the unusual oblique cross zone in unicarpellate Araliaceae can be explained by developmental constraints.

Exploring the limits for reduction of plastid genomes: a case study of the mycoheterotrophic orchids Epipogium aphyllum and Epipogium roseum.

The question on the patterns and limits of reduction of plastid genomes in nonphotosynthetic plants and the reasons of their conservation is one of the intriguing topics in plant genome evolution. Here, we report sequencing and analysis of plastid genome in nonphotosynthetic orchids Epipogium aphyllum and Epipogium roseum, which, with sizes of 31 and 19 kbp, respectively, represent the smallest plastid genomes characterized by now. Besides drastic reduction, which is expected, we found several unusual features of these "minimal" plastomes: Multiple rearrangements, highly biased nucleotide composition, and unprecedentedly high substitution rate. Only 27 and 29 genes remained intact in the plastomes of E. aphyllum and E. roseum-those encoding ribosomal components, transfer RNAs, and three additional housekeeping genes (infA, clpP, and accD). We found no signs of relaxed selection acting on these genes. We hypothesize that the main reason for retention of plastid genomes in Epipogium is the necessity to translate messenger RNAs (mRNAs) of accD and/or clpP proteins which are essential for cell metabolism. However, these genes are absent in plastomes of several plant species; their absence is compensated by the presence of a functional copy arisen by gene transfer from plastid to the nuclear genome. This suggests that there is no single set of plastid-encoded essential genes, but rather different sets for different species and that the retention of a gene in the plastome depends on the interaction between the nucleus and plastids.

The plastid genome of mycoheterotrophic monocot Petrosavia stellaris exhibits both gene losses and multiple rearrangements.

Plastid genomes of nonphotosynthetic plants represent a perfect model for studying evolution under relaxed selection pressure. However, the information on their sequences is still limited. We sequenced and assembled plastid genome of Petrosavia stellaris, a rare mycoheterotrophic monocot plant. After orchids, Petrosavia represents only the second family of nonphotosynthetic monocots to have its plastid genome examined. Several unusual features were found: retention of the ATP synthase genes and rbcL gene; extensive gene order rearrangement despite a relative lack of repeat sequences; an unusually short inverted repeat region that excludes most of the rDNA operon; and a lack of evidence for accelerated sequence evolution. Plastome of photosynthetic relative of P. stellaris, Japonolirion osense, has standard gene order and does not have the predisposition to inversions. Thus, the rearrangements in the P. stellaris plastome are the most likely associated with transition to heterotrophic way of life.