Phylogenetic position and plastid genome structure of Vietorchis, a mycoheterotrophic genus of Orchidaceae (subtribe Orchidinae) endemic to Vietnam.

The orchid genus Vietorchis comprises three species, all discovered in the 21 century. Each of these species is achlorophyllous, mycoheterotrophic and is known to be endemic to Vietnam. The type species of the genus, V. aurea, occurs in a single location in northern Vietnam within a lowland limestone karstic area. Vietorchis furcata and V. proboscidea, in contrast, are confined to mountains of southern Vietnam, far away from any limestone formations. Taxonomic placement of Vietorchis remained uncertain for the reason of inconclusive morphological affinities. At the same time, the genus has never been included into molecular phylogenetic studies. We investigate the phylogenetic relationships of two species of Vietorchis (V. aurea and V. furcata) based on three DNA datasets: (1) a dataset comprising two nuclear regions, (2) a dataset comprising two plastid regions, and (3) a dataset employing data on the entire plastid genomes. Our phylogenetic reconstructions support the placement of Vietorchis into the subtribe Orchidinae (tribe Orchideae, subfamily Orchidoideae). This leads to a conclusion that the previously highlighted similarities in the rhizome morphology between Vietorchis and certain mycoheterotrophic genera of the subfamilies Epidendroideae and Vanilloideae are examples of a convergence. Vietorchis is deeply nested within Orchidinae, and therefore the subtribe Vietorchidinae is to be treated as a synonym of Orchidinae. In the obtained phylogenetic reconstructions, Vietorchis is sister to the photosynthetic genus Sirindhornia. Sirindhornia is restricted to limestone mountains, which allows to speculate that association with limestone karst is plesiomorphic for Vietorchis. Flower morphology is concordant with the molecular data in placing Vietorchis into Orchidinae and strongly supports the assignment of the genus to one of the two major clades within this subtribe. Within this clade, however, Vietorchis shows no close structural similarity with any of its genera; in particular, the proximity between Vietorchis and Sirindhornia has never been proposed. Finally, we assembled the plastid genome of V. furcata, which is 65969 bp long and contains 45 unique genes, being one of the most reduced plastomes in the subfamily Orchidoideae. The plastome of Vietorchis lacks any rearrangements in comparison with the closest studied autotrophic species, and possesses substantially contracted inverted repeats. No signs of positive selection acting on the protein-coding plastid sequences were detected.

The Lotus angustissumus Group (Fabaceae): Can Phylogenetic Patterns Be Accommodated by a Taxonomic Concept?

The Lotus angustissimus group represents an example of obvious contradictions between the molecular and morphological data. This group includes from six to eight mostly annual species of Lotus section Lotus, with the center of species diversity in the Mediterranean. We performed a phylogenetic analysis of the genus Lotus with an expanded representation of all known species of the L. angustissimus group using both nuclear (nrITS) and a set of plastid DNA markers and compared the results with traditional taxonomy of this group. Our results demonstrated a non-monophyletic nature of the study group. In addition, the nrITS and plastid dataset tree topologies were incongruent with each other in both Bayesian and ML analyses. We revealed very complex phylogenetic relationships among the species of the group. Important results obtained in this study are: (1) genetic and geographical differentiation in the latitudinal direction (between L. angustissimus and L. praetermissus) and in the longitudinal direction among closely related accessions identified as L. castellanus, L. lourdes-santiagoi and L. palustris; (2) close genetic links between the Anatolian endemic L. macrotrichus and L. praetermissus from Eastern Europe and Central Asia; and (3) the hybrid nature of L. subbiflorus with L. parviflorus as its presumed male parent species. These results are discussed in the context of morphology, biogeography and taxonomy.

When Morphology and Biogeography Approximate Nuclear ITS but Conflict with Plastid Phylogeny: Phylogeography of the Lotus dorycnium Species Complex (Leguminosae).

Lotus dorycnium s.l. is a complex of taxa traditionally regarded as members of Dorycnium. It has a wide Mediterranean range, extending in the north to Central and Eastern Europe, and in the east to the Crimea, the Caucasus, and the Western Caspian region. Molecular phylogenetic data support placement of the L. dorycnium complex in the genus Lotus. The present study investigated the phylogeny, phylogeography and morphological variability of the L. dorycnium complex across its distribution range to reveal the main trends in genetic and morphological differentiation in this group. The results of the morphological analyses demonstrated some degree of differentiation, with L. d. ssp. herbaceus, ssp. gracilis, and ssp. anatolicus more or less well defined, whereas ssp. dorycnium, ssp. germanicus, and ssp. haussknechtii can be hardly distinguished from each other using morphology. Analyses of the L. dorycnium complex based on nrITS revealed a tendency towards a geographic differentiation into Western, Eastern, and Turkish groups. Phylogenetic and phylogeographic analyses of the same set of specimens using concatenated plastid markers trnL-F, rps16, and psbA-trnH demonstrated a low resolution between the L. dorycnium complex and L. hirsutus, as well as among the taxa within the L. dorycnium complex, which can be interpreted as evidence of an incomplete lineage sorting or hybridization. The evolutionary processes responsible for incongruence in phylogenetic signals between plastid and nuclear sequences of the morphologically well-defined species L. dorycnium and L. hirsutus were most likely localized in the Eastern Mediterranean. A possibility of rare gene exchange between the L. dorycnium complex and the group of L. graecus is revealed for the first time.

Two centuries from species discovery to diagnostic characters: molecular and morphological evidence for narrower species limits in the widespread SW Australian Anarthria gracilis complex (Restionaceae s.l./Anarthriaceae, Poales).

BACKGROUND: The extreme southwest of Australia is a biodiversity hotspot region that has a Mediterranean-type climate and numerous endemic plant and animal species, many of which remain to be properly delimited. We refine species limits in Anarthria, a Western Australian endemic genus characterised by the occurrence of the greatest number of plesiomorphic character states in the restiid clade of Poales. In contrast to many other groups of wind-pollinated Australian Poales, Anarthria was traditionally viewed as having well-established species limits. All six currently recognised species, which are conspicuous members of some Western Australian plant communities, were described in the first half of the 19th century. They are traditionally distinguished from each other mainly using quantitative characters. METHODS: We examined extensive existing herbarium specimens and made new collections of Anarthria in nature. Scanning electron microscopy and light microscopy were used to study leaf micromorphology. Molecular diversity of Anarthria was examined using a plastid (trnL-F) and a low-copy nuclear marker (at103). This is the first study of species-level molecular diversity in the restiid clade using a nuclear marker. RESULTS: Material historically classified as Anarthria gracilis R.Br. actually belongs to three distinct species, A. gracilis s.str., A. grandiflora Nees and A. dioica (Steud.) C.I.Fomichev, each of which forms a well-supported clade in phylogenetic analyses. Both segregate species were described in the first half of the 19th century but not recognised as such in subsequent taxonomic accounts. Anarthria dioica was first collected in 1826, then wrongly interpreted as a species of Juncus (Juncaceae) and described as Juncus dioicus. We provide a formal transfer of the name to Anarthria and for the first time report its clear and qualitative diagnostic characters: an extremely short leaf ligule and distinctive pattern of leaf epidermal micromorphology. A long ligule is present in A. gracilis s.str. and A. grandiflora. These species differ from each other in leaf lamina morphology and anatomy and have mostly non-overlapping distribution ranges. The narrower definition of species provides a basis for future phylogeographic analyses in Anarthria. Our study highlights a need for more extensive use of nuclear DNA markers in Restionaceae. The use of the low copy nuclear marker at103 allowed a clade comprising all three ligulate species of Anarthria to be recognised. The ligule character is used here for the first time in the taxonomy of Anarthria and merits special attention in studies of other restiids. In general, our study uncovered a superficially hidden but, in reality, conspicuous diversity in a common group of wind-pollinated plants in the southwest of Western Australia.

Phylogenetic Placement and Phylogeography of Large-Flowered Lotus Species (Leguminosae) Formerly Classified in Dorycnium: Evidence of Pre-Pleistocene Differentiation of Western and Eastern Intraspecific Groups.

The Mediterranean region is a center of species and genetic diversity of many plant groups, which served as a source of recolonization of temperate regions of Eurasia in Holocene. We investigate the evolutionary history of species currently classified in Lotus sect. Bonjeanea in the context of the evolution of the genus Lotus as a whole, using phylogenetic, phylogeographic and dating analyses. Of three species of the section, L. rectus and L. hirsutus have wide Mediterranean distribution while L. strictus has a disjunctive range in Bulgaria, Turkey, Armenia, Eastern Kazakhstan, and adjacent parts of Russia and China. We used entire nuclear ribosomal ITS1-5.8S-ITS2 region (nrITS) and a plastid dataset (rps16 and trnL-F) to reconstruct phylogenetic relationships within Lotus with an extended representation of Bonjeanea group. We analyzed the phylogeographic patterns within each species based on the plastid dataset. For divergence time estimation, the nrITS dataset was analyzed. Our results confirmed the non-monophyletic nature of the section Bonjeanea. They indicate that Lotus is likely to have diverged about 15.87 (9.99-19.81) million years ago (Ma), which is much older than an earlier estimate of ca. 5.54 Ma. Estimated divergence ages within L. strictus, L. rectus, and L. hisrutus (6.1, 4.94, and 4.16 Ma, respectively) well predate the onset of the current type of Mediterranean climate. Our data suggest that relatively ancient geological events and/or climatic changes apparently played roles in early diversification of Lotus and its major clades, as well as in formation of phylogeographic patterns, in at least some species.

Complete plastome sequence of Paeonia lactiflora Pall. (Paeoniaceae: Saxifragales).

Paeonia lactiflora has been listed as an Endangered species in Russian Federation. The complete plastome was assembled from Next-Generation Sequencing data. It is 152,747 bp in length. It consists of a pair of Inverted Repeat regions (25,651 bp), separated by a small single copy region of 17,033 bp and a large single copy region of 84,412 bp. The plastome encoded 128 genes, including 83 protein coding genes, 37 tRNA, eight rRNA genes, four pseudogenes, and is characterized by loss of the rpl32 and infA genes. Phylogenetic analysis of Paeoniaceae plastomes revealed that P. lactiflora clustered with Eurasian peonies (section Paeoniae).

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.

Complete Plastid Genome of the Recent Holoparasite Lathraea squamaria Reveals Earliest Stages of Plastome Reduction in Orobanchaceae.

Plants from the family Orobanchaceae are widely used as a model to study different aspects of parasitic lifestyle including host-parasite interactions and physiological and genomic adaptations. Among the latter, the most prominent are those that occurred due to the loss of photosynthesis; they include the reduction of the photosynthesis-related gene set in both nuclear and plastid genomes. In Orobanchaceae, the transition to non-photosynthetic lifestyle occurred several times independently, but only one lineage has been in the focus of evolutionary studies. These studies included analysis of plastid genomes and transcriptomes and allowed the inference of patterns and mechanisms of genome reduction that are thought to be general for parasitic plants. Here we report the plastid genome of Lathraea squamaria, a holoparasitic plant from Orobanchaceae, clade Rhinantheae. We found that in this plant the degree of plastome reduction is the least among non-photosynthetic plants. Like other parasites, Lathraea possess a plastome with elevated absolute rate of nucleotide substitution. The only gene lost is petL, all other genes typical for the plastid genome are present, but some of them-those encoding photosystem components (22 genes), cytochrome b6/f complex proteins (4 genes), plastid-encoded RNA polymerase subunits (2 genes), ribosomal proteins (2 genes), ccsA and cemA-are pseudogenized. Genes for cytochrome b6/f complex and photosystems I and II that do not carry nonsense or frameshift mutations have an increased ratio of non-synonymous to synonymous substitution rates, indicating the relaxation of purifying selection. Our divergence time estimates showed that transition to holoparasitism in Lathraea lineage occurred relatively recently, whereas the holoparasitic lineage Orobancheae is about two times older.

Pollination of Vietnamese Aspidistra xuansonensis (Asparagaceae) by female Cecidomyiidi flies: larvae of pollinator feed on fertile pollen in anthers of anthetic bisexual flowers.

UNLABELLED: • PREMISE OF THE STUDY: Aspidistra is a species-rich, herbaceous monocot genus of tropical Southeast Asia. Most species are recently discovered and apparently endangered, though virtually nothing is known about their biology. Species of the genus are primarily distinguished using flower morphology, which is enormously diverse. However, the pollination process has not been directly observed in the center of diversity of the genus (N Vietnam and S China). Indirect and partly direct data on the only widely cultivated species of the genus (A. elatior) placed it among angiosperms with the most unusual pollination biology, though these data are highly controversial, suggesting pollen transfer by mollusks, crustaceans, flies, or possibly tiny soil invertebrates such as collembolans.• METHODS: Pollination of Aspidistra xuansonensis in the center of diversity of the genus was studied using visual observations and videos and light and scanning electron microscopy investigation of flowers and their pollinators. Pollinators and their larvae were molecularly barcoded.• KEY RESULTS: Aspidistra xuansonensis is pollinated by female cecidomyiid flies (gall midges). They oviposit on anthers, and larvae develop among the pollen mass. Molecular barcoding proved taxonomic identity of the larvae and the flies. The larvae neither damage floral parts nor cause gall formation, but feed on pollen grains by sucking out their content. The larvae move out of the flowers before decomposition starts. Carebara ants steal developing larvae from flowers but do not contribute to pollination.• CONCLUSIONS: More than one kind of myiophily is present in Aspidistra. Brood site pollination was documented for the first time in Aspidistra. The pollination system of A. xuansonensis differs from other kinds of brood site pollination in the exit of the larvae prior to the decomposition of floral parts.

Phylogenetics of Anthyllis (Leguminosae: Papilionoideae: Loteae): Partial incongruence between nuclear and plastid markers, a long branch problem and implications for morphological evolution.

Phylogenetic relationships in the genus Anthyllis (Leguminosae: Papilionoideae: Loteae) were investigated using data from the nuclear ribosomal internal transcribed spacer regions (ITS) and three plastid regions (psbA-trnH intergenic spacer, petB-petD region and rps16 intron). Bayesian and maximum parsimony (MP) analysis of a concatenated plastid dataset recovered well-resolved trees that are topologically similar, with many clades supported by unique indels. MP and Bayesian analyses of the ITS sequence data recovered trees that have several well-supported topological differences, both among analyses, and to trees inferred from the plastid data. The most substantial of these concerns A. vulneraria and A. lemanniana, whose placement in the parsimony analysis of the ITS data appears to be due to a strong long-branch effect. Analysis of the secondary structure of the ITS1 spacer showed a strong bias towards transitions in A. vulneraria and A. lemanniana, many of which were also characteristic of certain outgroup taxa. This may contribute to the conflicting placement of this clade in the MP tree for the ITS data. Additional conflicts between the plastid and ITS trees were more taxonomically focused. These differences may reflect the occurrence of reticulate evolution between closely related species, including a possible hybrid origin for A. hystrix. The patterns of incongruence between the plastid and the ITS data seem to correlate with taxon ranks. All of our phylogenetic analyses supported the monophyly of Anthyllis (incl. Hymenocarpos). Although they are often taxonomically associated with Anthyllis, the genera Dorycnopsis and Tripodion are shown here to be more closely related to other genera of Loteae. We infer up to six major clades in Anthyllis that are morphologically well-characterized, and which could be recognized as sections. Four of these agree with various morphology-based classifications, while the other two are novel. We reconstruct the evolution of several morphological characteristics found only in Anthyllis or tribe Loteae. Some of these characters support major clades, while others show evidence of homoplasy within Anthyllis.

Comparative chloroplast genomics and phylogenetics of Fagopyrum esculentum ssp. ancestrale -a wild ancestor of cultivated buckwheat.

BACKGROUND: Chloroplast genome sequences are extremely informative about species-interrelationships owing to its non-meiotic and often uniparental inheritance over generations. The subject of our study, Fagopyrum esculentum, is a member of the family Polygonaceae belonging to the order Caryophyllales. An uncertainty remains regarding the affinity of Caryophyllales and the asterids that could be due to undersampling of the taxa. With that background, having access to the complete chloroplast genome sequence for Fagopyrum becomes quite pertinent. RESULTS: We report the complete chloroplast genome sequence of a wild ancestor of cultivated buckwheat, Fagopyrum esculentum ssp. ancestrale. The sequence was rapidly determined using a previously described approach that utilized a PCR-based method and employed universal primers, designed on the scaffold of multiple sequence alignment of chloroplast genomes. The gene content and order in buckwheat chloroplast genome is similar to Spinacia oleracea. However, some unique structural differences exist: the presence of an intron in the rpl2 gene, a frameshift mutation in the rpl23 gene and extension of the inverted repeat region to include the ycf1 gene. Phylogenetic analysis of 61 protein-coding gene sequences from 44 complete plastid genomes provided strong support for the sister relationships of Caryophyllales (including Polygonaceae) to asterids. Further, our analysis also provided support for Amborella as sister to all other angiosperms, but interestingly, in the bayesian phylogeny inference based on first two codon positions Amborella united with Nymphaeales. CONCLUSION: Comparative genomics analyses revealed that the Fagopyrum chloroplast genome harbors the characteristic gene content and organization as has been described for several other chloroplast genomes. However, it has some unique structural features distinct from previously reported complete chloroplast genome sequences. Phylogenetic analysis of the dataset, including this new sequence from non-core Caryophyllales supports the sister relationship between Caryophyllales and asterids.

Complete sequence of the duckweed (Lemna minor) chloroplast genome: structural organization and phylogenetic relationships to other angiosperms.

The complete nucleotide sequence of the duckweed (Lemna minor) chloroplast genome (cpDNA) was determined. The cpDNA is a circular molecule of 165,955 bp containing a pair of 31,223-bp inverted repeat regions (IRs), which are separated by small and large single-copy regions of 89,906 and 13,603 bp, respectively. The entire gene pool and relative positions of 112 genes (78 protein-encoding genes, 30 tRNA genes, and 4 rRNA genes) are almost identical to those of Amborella trichopoda cpDNA; the minor difference is the absence of infA and ycf15 genes in the duckweed cpDNA. The inverted repeat is expanded to include ycf1 and rps15 genes; this pattern is unique and does not occur in any other sequenced cpDNA of land plants. As in basal angiosperms and eudicots, but not in other monocots, the borders between IRs and a large single-copy region are located upstream of rps19 and downstream of trnH, so that trnH is not included in IRs. The model of rearrangements of the chloroplast genome during the evolution of monocots is proposed as the result of the comparison of cpDNA structures in duckweed and other monocots. The phylogenetic analyses of 61 protein-coding genes from 38 plastid genome sequences provided strong support for the monophyly of monocots and position of Lemna as the next diverging lineage of monocots after Acorales. Our analyses also provided support for Amborella as a sister to all other angiosperms, but in the bayesian phylogeny inference based on the first two codon positions Amborella united with Nymphaeales.