Gottschelia (Gottscheliaceae, Marchantiophyta) in Indochina.

Gottschelia, collected for the first time in Indochina, inspired an attempt to review the genus phylogeny to identify a more precise position of Indochinese plants. The genetic distance between African and Asian populations of G. schizopleura sensu lato was confirmed. The two groups should be treated as different species. A new combination, G. microphylla comb. nov., has been proposed for Asian plants. Aside from molecular genetics, distinguishing this species from the presumable strictly African G. schizopleura is also possible by morphological characteristics, as well as by its distribution. At the same time, at least three groups are distinguished among Asian haplotypes of G. microphylla, each of which can be interpreted as a species or, at least, subspecies. A morphological description, intravital photographs of the general habitat, and details of the morphological structures are provided. The position of Gottschelia in the phylogenetic schema of Jungermanniales does not allow us to attribute it to any of the known families and forces us to describe a new family, Gottscheliaceae, which is phylogenetically somewhat related to the Chaetophyllopsidaceae re-evaluated here and very different from Gottscheliaceae morphologically.

New insight into the taxonomy of Cephaloziellaceae (Marchantiophyta): the family of the smallest higher plants on Earth.

An analysis of the phylogeny of Cephaloziellaceae was carried out based on trees constructed for previously and newly obtained sequences of five genes: nuclear ITS1-2 and chloroplast trnL-F, trnG, rbcL, and psbA. Phylogenetic trees inferred from different genes are congruent for the main details; however, the position of several taxa is variable. As a result, a new phylogenetic system of the family was proposed. The narrow genus concept seems to be more appropriate for the family. Cephaloziella spinicaulis is segregated into the new genus Douiniella, the generic status for Prionolobus and Metacephalozia is confirmed, and the dubious generic status of Kymatocalyx is substantiated. The generic independence of Cylindrocolea from Cephaloziella s. str. is confirmed. The small amount of data hinders the description of two more genera from Cephaloziella s.l.

The Position of Lophozia dubia (Lophoziaceae, Marchantiophyta) in the Phylogenetic System of Lophozia and the Distribution of Lophozia in Southeast Eurasia, Extending to Indonesia.

Lophozia pallida, the commonly used name for a rare and little-known Sino-Himalayan species, was found to be a synonym of Lophozia dubia, a forgotten and previously misinterpreted species known in Indonesia. A comparative study of herbarium materials and our collections made it possible to 'extend' the distribution of Lophozia s. str. southward to Indonesia. The description of oil bodies from the species is provided for the first time. The position of the species in the Lophozia phylogenetic system demonstrates its clear differences from the morphologically similar Lophozia guttulata and its phylogenetic relationship with the Japanese-Korean Lophozia koreana.

Integrative Taxonomy Reveals Hidden Diversity in the Aloina catillum Complex (Pottiaceae, Bryophyta).

Aloina catillum is a variable moss typical of xerophytic environments in the Neotropics, characterized against other closely allied Aloina species with well-differentiated leaf border by its setae twisted to the left throughout. In order to clarify its variability and its relationships with the allied species with differentiated leaf border A. brevirostris, A. obliquifolia, and A. rigida, we performed an integrative study including sequence data from four markers (nuclear ITS, plastid atpB-rbcL, trnG, trnL-F), morphometry, and species assembling by automatic partitioning (ASAP) algorithm. Our data suggest that A. catillum consists of at least three species: A. calceolifolia (an earlier name for A. catillum), and two species described here as a new, A. bracteata sp. nov. and A. limbata sp. nov. This latter species includes the specimens previously identified as A. obliquifolia from South America. Additionally, some morphological and molecular variability was also detected in A. limbata, but was not consistent enough to be recognized taxonomically. The study supports the presence of A. brevirostris in the Neotropics and A. rigida is tentatively excluded from South America. Full descriptions of the A. catillum s.l. species and a diagnostic key to this complex in South America are provided.

Genetic evaluation and germplasm identification analysis on ITS2, trnL-F, and psbA-trnH of alfalfa varieties germplasm resources.

In this study, genetic diversity and germplasm identification of 28 alfalfa germplasm cultivars materials were evaluated by analyzing their internal transcribed spacer 2 (ITS2), trnL-F, and psbA-trnH sequences to provide the innovative reference of alfalfa varieties genetic diversity and identify research. The results showed that the fragment average length of ITS2, trnL-F, and psbA-trnH sorting sequences were 455.7 bp, 230.3 bp, and 345.6 bp, respectively. The ITS2 sequence was too conservative to reflect the individual differences between intercultivars and intracultivars in the preliminary experiment. Furthermore, trnL-F and psbA-trnH sequence differences were relatively small between intercultivars but significant between intracultivars. Alfalfa cultivars were divided into four groups by sequence similarity clustering. Alfalfa cultivars trnL-F and psbA-trnH sequences have apparent differences, showing that chloroplast conservative sequences were independent evolution. Compared with trnL-F and psbA-trnH sequences of alfalfa cultivars, psbA-trnH sequence has abundant variation sites and can better reflect the differences between cultivars than the trnL-F sequence. Therefore, the psbA-trnH sequence can identify different alfalfa cultivars and establish the DNA sequence fingerprint.

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.

Codonoblepharonteae, a New Major Lineage among Orthotrichoideae (Orthotrichaceae, Bryophyta).

Orthotrichoideae aggregates epiphytic mosses widespread throughout temperate regions and high tropical mountains of the world. Recently, important advances have been made in elucidating its phylogenetic relationships and evolutionary patterns. Fourteen genera are currently recognized within the subfamily, which are spread over two main tribes: Orthotricheae, comprising Orthotrichinae and Lewinskyinae, and Zygodonteae. Despite the progress, some groups have received little attention, as is the case of genus Codonoblepharon. Recent studies have suggested that this genus may represent a separate lineage from Zygodonteae, in which it traditionally has been considered. Although, none of the studies were conclusive as they did not include a representative sampling of the Codonoblepharon species. This work aims to evaluate the taxonomic position of Codonoblepharon and its phylogenetic relationships within Orthotrichoideae. For this purpose, we present an updated phylogenetic tree based on four different loci, one belonging to the nuclear genome (ITS2) and the rest to the plastid genome (rps4, trnG and trnL-F). The phylogenetic reconstruction recovers all samples of Codonoblepharon in a monophyletic group, sister to the rest of the subfamily, constituting a lineage independent of the two currently recognized tribes. For this reason, we propose the new tribe Codonoblepharonteae to accommodate Codonoblepharon.

Screening of universal DNA barcodes for identifying grass species of Gramineae.

There is currently international interest in applying DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of four candidate plant DNA barcoding regions [rbcL, matK, trnL-F, and internal transcribed spacer (ITS)] in seven genera of Gramineae including Agropyron, Bromus, Elymus, Elytrigia, Festuca, Leymus, and Lolium. Fourteen accessions were analyzed, and matK and ITS showed the highest species, subspecies, and variety discriminatory power, each resolving 11 accessions. Species discrimination using rbcL and trnL-F was lower, resolving 7 and 8 accessions, respectively. Subspecies and variety discrimination using rbcL and trnL-F could not identify 4 accessions of Agropyron. A technical system can be established using the proposed DNA barcode to rapidly and reliably identify the seven genera of Gramineae. This study serves as a "useful reference" for identifying the genetic diversity of grass germplasm resources. DNA barcoding can be utilized to uncover the relatives of different species within the same family or between different families. It can also be used to determine the related groups of important herbage, turfgrass, and crops and provide crucial background information for discovering excellent genes and improving existing crop varieties.

Circumscription and Phylogenetic Position of Two Propagulose Species of Syntrichia (Pottiaceae, Bryophyta) Reveals Minor Realignments within the Tribe Syntricheae.

In the course of a worldwide revision of the genus Syntrichia, we identified problems in the circumscription of some species of the genus as well as among some allied genera grouped in the tribe Syntricheae. This is the case for the two propagulose Syntrichia amphidiacea and S. gemmascens, closely related to Streptopogon. We analyzed phylogenetic relationships between these species, based on nuclear (ITS) and two plastid (trnL-F and trnG) markers and morphological features. Species delimitation using molecular data was consistent with our preliminary morphological inference. Phylogenetic analyses were performed using maximum likelihood and Bayesian inference methods. Our results placed Syntrichia amphidiacea in the Streptopogon clade. Syntrichia gemmascens is also included in Streptopogon in spite of the discrepancy of the ITS and plastid relationships, which could be evidence of an exchange of genetic material between species in various lineages in the Pottioideae. Streptopogon is maintained as a separate genus on the basis of morphology characters, and we consider the differentiation of laminal papillae and the presence of a stem central strand as new characters in the genus. We accept Sagenotortula as distinct genus sister to Syntrichia. We consider the lack of costal dorsal epidermis and the differentiation of a crescent-shaped costal dorsal stereid band as distinctive generic characters in Syntrichia. Additionally, we include Syntrichia percarnosa as a new synonym for S. breviseta. Three names are lectotypified.

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.

Hemiboeakaiyangensis, a new species of Gesneriaceae endemic to Guizhou, China.

A new species of Gesneriaceae from Guizhou, China, Hemiboeakaiyangensis sp. nov., is described and illustrated. We investigated its phylogenetic position and relationships with 13 other species of Hemiboea C.B.Clarke, which present large morphological diversity in the genus, based on molecular analyses of the nuclear ribosomal internal transcribed spacer (ITS) and the chloroplast trnL-F intron-spacer sequences. The molecular phylogenetic analyses revealed that the new species is most closely related to H.ovalifolia. A diagnostic table and discussion of morphological characters are provided to differentiate the new species from H.longisepala, H.flaccida and H.ovalifolia.

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.

Two cryptic species of California mustard within Caulanthus lasiophyllus.

PREMISE: Cryptic species are evolutionarily distinct lineages lacking distinguishing morphological traits. Hidden diversity may be lurking in widespread species whose distributions cross phylogeographic barriers. This study investigates molecular and morphological variation in the widely distributed Caulanthus lasiophyllus (Brassicaceae) in comparison to its closest relatives. METHODS: Fifty-two individuals of C. lasiophyllus from across the species' range were sequenced for the nuclear ribosomal internal transcribed spacer region (ITS) and the chloroplast trnL-F region. A subset of these samples were examined for the chloroplast ndhF gene. All 52 individuals were scored for 13 morphological traits, as well as monthly and annual climate conditions at the collection locality. Morphological and molecular results are compared with the closest relatives-C. anceps and C. flavescens-in the "Guillenia Clade." To test for polyploidy, genome size estimates were made for four populations. RESULTS: Caulanthus lasiophyllus consists of two distinct lineages separated by eight ITS differences-eight times more variation than what distinguishes C. anceps and C. flavescens. Fewer variable sites were detected in trnL-F and ndhF regions, yet these data are consistent with the ITS results. The two lineages of C. lasiophyllus are geographically and climatically distinct; yet morphologically overlapping. Their genome sizes are not consistently different. CONCLUSIONS: Two cryptic species within C. lasiophyllus are distinguished at the molecular, geographic, and climatic scales. They have similar genome sizes and are morphologically broadly overlapping, but an ephemeral basal leaf character may help distinguish the species.

Blepharostoma trichophyllum S.L. (Marchantiophyta): The Complex of Sibling Species and Hybrids.

Blepharostoma trichophyllum was found to be a species collectiva formed by several strongly genetically different species. The taxonomic diversity in the group is the possible result of radiation in early stages; then, these taxa likely survived for a long time in similar environmental conditions, which resulted in stasis. Presently, the existing taxa are similar one to another and may be morphologically distinguished with difficulties. The most taxonomically valuable morphological characteristics include oil bodies and cells in the leaf segment features. The most diverse genotypes (the vast majority of which are treated here as distinct species) were found in amphi-Pacific Asia, which may reflect the evolutionary history of the genus or may be the consequence of more profound sampling in the macro-region in comparison with other parts of the Holarctic.

Two new combinations in Oreocharis (Gesneriaceae) based on morphological, molecular and cytological evidence.

The newly-circumscribed genus Oreocharis is recently enlarged by incorporating ten other genera with high floral diversity. In this study, our morphological, molecular and cytological evidence supports our adding two species from other two different genera (Boeica and Beccarinda) to Oreocharis. The special corolla shape (campanulate or flat-faced) and related short filament of these two new combinations, Oreocharis guileana and O. baolianis, further enrich the diversity of floral characters of the enlarged Oreocharis. Meanwhile, some supplementary and amended descriptions of these two species are made here. Our morphological, molecular and geographical data indicate that O. guileana is related to O. pilosopetiolata to a certain extent. For O. baolianis, however, our current dataset does not allow conclusions on the species relationship within Oreocharis.

First Phylogeny of Bitterbush Family, Picramniaceae (Picramniales).

Picramniaceae is the only member of Picramniales which is sister to the clade (Sapindales (Huerteales (Malvales, Brassicales))) in the rosidsmalvids. Not much is known about most aspects of their ecology, geography, and morphology. The family is restricted to American tropics. Picramniaceae representatives are rich in secondary metabolites; some species are known to be important for pharmaceutical purposes. Traditionally, Picramniaceae was classified as a subfamily of Simaroubaceae, but from 1995 on, it has been segregated containing two genera, Picramnia and Alvaradoa, with the recent addition of a third genus, Nothotalisia, described in 2011. Only a few species of the family have been the subject of DNA-related research, and fewer than half of the species have been included in morphological phylogenetic analyses. It is clear that Picramniaceae remains a largely under-researched plant group. Here we present the first molecular phylogenetic tree of the group, based on both chloroplast and nuclear markers, widely adopted in the plant DNA barcoding. The main findings are: The family and its genera are monophyletic and Picramnia is sister to two other genera; some clades corroborate previous assumptions of relationships made on a morphological or geographical basis, while most parts of the molecular topology suggest high levels of homoplasy in the morphological evolution of Picramnia.

Extensive interlineage hybridization in the predominantly clonal Hydrilla verticillata.

PREMISE: The submersed aquatic plant Hydrilla verticillata ("hydrilla") is important ecologically and economically due to its aggressive growth in both indigenous and nonindigenous regions. Substantial morphological variation has been documented in hydrilla, including the existence of monoecious and dioecious "biotypes." Whereas plastid sequence data have been used previously to explore intraspecific diversity, nuclear data have yet to be analyzed in a phylogenetic context. Molecular and morphological analyses were used to evaluate the genetic diversity and phylogenetic relationships of native and introduced populations. METHODS: Nuclear (internal transcribed spacer-ITS; phytoene desaturase-PDS) and plastid (trnL-F) sequence data were evaluated phylogenetically using likelihood and Bayesian methods. Leaf morphologies were compared among clades that were identified in phylogenetic analyses. RESULTS: Data from both ITS and PDS show multiple instances of polymorphic sequences that could be traced to two or more lineages, including both invasive biotypes in the Americas. Leaf morphological data support the distinctness of lineages and provide a metric for distinguishing monoecious and dioecious biotypes in the United States. CONCLUSIONS: Nuclear molecular data indicate far greater genetic diversity than could be estimated using plastid markers. Substantially divergent copies of nuclear genes, found in multiple populations worldwide, likely result from interlineage hybridization. Invasive monoecious and dioecious hydrilla biotypes in the Americas are genetically distinct, with both biotypes resulting from admixture among Eurasian progenitors. Genetic similarity to populations in India and South Korea, respectively, implicates these as likely origins for the dioecious and monoecious biotypes.

Integrating DNA Barcoding and Traditional Taxonomy for the Identification of Dipterocarps in Remnant Lowland Forests of Sumatra.

DNA barcoding has been used as a universal tool for phylogenetic inferences and diversity assessments, especially in poorly studied species and regions. The aim of this study was to contrast morphological taxonomy and DNA barcoding, using the three frequently used markers matK, rbcL, and trnL-F, to assess the efficiency of DNA barcoding in the identification of dipterocarps in Sumatra, Indonesia. The chloroplast gene matK was the most polymorphic among these three markers with an average interspecific genetic distance of 0.020. The results of the molecular data were mostly in agreement with the morphological identification for the clades of Anthoshorea, Hopea, Richetia, Parashorea, and Anisoptera, nonetheless these markers were inefficient to resolve the relationships within the Rubroshorea group. The maximum likelihood and Bayesian inference phylogenies identified Shorea as a paraphyletic genus, Anthoshorea appeared as sister to Hopea, and Richetia was sister to Parashorea. A better discriminatory power among dipterocarp species provided by matK and observed in our study suggests that this marker has a higher evolutionary rate than the other two markers tested. However, a combination of several different barcoding markers is essential for reliable identification of the species at a lower taxonomic level.

Phylogenetic Reconstruction and Divergence Time Estimation of Blumea DC. (Asteraceae: Inuleae) in China Based on nrDNA ITS and cpDNA trnL-F Sequences.

The genus Blumea is one of the most economically important genera of Inuleae (Asteraceae) in China. It is particularly diverse in South China, where 30 species are found, more than half of which are used as herbal medicines or in the chemical industry. However, little is known regarding the phylogenetic relationships and molecular evolution of this genus in China. We used nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) and chloroplast DNA (cpDNA) trnL-F sequences to reconstruct the phylogenetic relationship and estimate the divergence time of Blumea in China. The results indicated that the genus Blumea is monophyletic and it could be divided into two clades that differ with respect to the habitat, morphology, chromosome type, and chemical composition of their members. The divergence time of Blumea was estimated based on the two root times of Asteraceae. The results indicated that the root age of Asteraceae of 76-66 Ma may maintain relatively accurate divergence time estimation for Blumea, and Blumea might had diverged around 49.00-18.43 Ma. This common ancestor had an explosive expansion during the Oligocene and Miocene and two major clades were differentiated during these epochs 29.60 Ma (17.76-45.23 Ma 95% HPD (Highest Posterior Density). Evidence from paleogeography and paleoclimate studies has confirmed that Blumea experienced differentiation and an explosive expansion during the Oligocene and Miocene.

Phenotypic and Genetic Variation of an Interspecific Centaurium Hybrid (Gentianaceae) and Its Parental Species.

Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species.