Phylogeny of Alisma (Alismataceae) revisited: implications for polyploid evolution and species delimitation.

Alisma L. is a genus of aquatic and wetland plants belonging to family Alismataceae. At present, it is thought to contain ten species. Variation in ploidy level is known in the genus, with diploids, tetraploids and hexaploids recorded. Previous molecular phylogenetic studies of Alisma have generated a robust backbone that reveals important aspects of the evolutionary history of this cosmopolitan genus, yet questions remain unresolved about the formation of the polyploid taxa and the taxonomy of one particularly challenging, widely distributed species complex. Here we directly sequenced, or cloned and sequenced, nuclear DNA (nrITS and phyA) and chloroplast DNA (matK, ndhF, psbA-trnH and rbcL) of multiple samples of six putative species and two varieties, and conducted molecular phylogenetic analyses. Alisma canaliculatum and its two varieties known in East Asia and A. rariflorum endemic to Japan possess closely related but heterogeneous genomes, strongly indicating that the two species were generated from two diploid progenitors, and are possibly siblings of one another. This evolutionary event may have occurred in Japan. Alisma canaliculatum var. canaliculatum is segregated into two types, each of which are geographically slightly differentiated in Japan. We reconstructed a single phylogeny based on the multi-locus data using Homologizer and then applied species delimitation analysis (STACEY). This allowed us to discern A. orientale as apparently endemic to the Southeast Asian Massif and distinct from the widespread A. plantago-aquatica. The former species was most likely formed through parapatric speciation at the southern edge of the distribution of the latter.

Molecular and Cytogenetic Analysis of rDNA Evolution in Crepis Sensu Lato.

Although Crepis was the first model plant group in which chromosomal changes were considered to play an important role in speciation, their chromosome structure and evolution have been barely investigated using molecular cytogenetic methods. The aim of the study was to provide a better understanding of the patterns and directions of Crepis chromosome evolution, using comparative analyses of rDNA loci number and localisation. The chromosome base number and chromosomal organisation of 5S and 35S rDNA loci were analysed in the phylogenetic background for 39 species of Crepis, which represent the evolutionary lineages of Crepis sensu stricto and Lagoseris, including Lapsana communis. The phylogenetic relationships among all the species were inferred from nrITS and newly obtained 5S rDNA NTS sequences. Despite high variations in rDNA loci chromosomal organisation, most species had a chromosome with both rDNA loci within the same (usually short) chromosomal arm. The comparative analyses revealed several independent rDNA loci number gains and loci repositioning that accompanied diversification and speciation in Crepis. Some of the changes in rDNA loci patterns were reconstructed for the same evolutionary lineages as descending dysploidy.

Exposing the illegal trade in cycad species (Cycadophyta: Encephalartos) at two traditional medicine markets in South Africa using DNA barcoding.

Species in the cycad genus Encephalartos are listed in CITES Appendix I and as Threatened or Protected Species in terms of South Africa's National Environmental Management: Biodiversity Act (NEM:BA) of 2004. Despite regulations, illegal plant harvesting for medicinal trade has continued in South Africa and resulted in declines in cycad populations and even complete loss of sub-populations. Encephalartos is traded at traditional medicine markets in South Africa in the form of bark strips and stem sections; thus, determining the species traded presents a major challenge due to a lack of characteristic plant parts. Here, a case study is presented on the use of DNA barcoding to identify cycads sold at the Faraday and Warwick traditional medicine markets in Johannesburg and Durban, respectively. Market samples were sequenced for the core DNA barcodes (rbcLa and matK) as well as two additional regions: nrITS and trnH-psbA. The barcoding database for cycads at the University of Johannesburg was utilized to assign query samples to known species. Three approaches were followed: tree-based, similarity-based, and character-based (BRONX) methods. Market samples identified were Encephalartos ferox (Near Threatened), Encephalartos lebomboensis (Endangered), Encephalartos natalensis (Near Threatened), Encephalartos senticosus (Vulnerable), and Encephalartos villosus (Least Concern). Results from this study are crucial for making appropriate assessments and decisions on how to manage these markets.

Phylogeny and species delimitations in European Dicranum (Dicranaceae, Bryophyta) inferred from nuclear and plastid DNA.

DNA sequences have been widely used for taxonomy, inferring phylogenetic relationships and identifying species boundaries. Several specific methods to define species delimitations based on molecular phylogenies have appeared recently, with the generalized mixed Yule coalescent (GMYC) method being most popular. However, only few studies on land plants have been published so far and GMYC analyses of bryophytes are missing. Dicranum is a large genus of mosses whose (morpho-)species are partly ill-defined and frequently confused. To infer molecular species delimitations, we reconstructed phylogenetic trees based on five chloroplast markers and nuclear ribosomal ITS sequences from 27 out of 30 species occurring in Europe. We applied the species delimitation methods GMYC and Poisson tree processes (PTP) in order to compare their discriminatory power with species boundaries inferred from the molecular phylogenetic reconstructions and with the morphological species concept. Phylogenetic circumscriptions were congruent with the morphological concept for 19 species, while eight species were molecularly not well delimited, mostly forming closely related species pairs. The automated species delimitation methods achieved similar results but tended to overestimate the number of potential species and exposed several incongruences between the morphological concept and inference from molecular phylogenetic reconstructions. It is concluded that GMYC and PTP methods potentially provide a useful and objective way of delimiting bryophyte species, but studies on further bryophyte data sets are necessary to infer whether incongruences might ensue from evolutionary processes and to test the suitability of these approaches.

Reticulate evolution in North American black-fruited hawthorns (Crataegus section Douglasia; Rosaceae): evidence from nuclear ITS2 and plastid sequences.

BACKGROUND AND AIMS: The taxonomic complexity of Crataegus (hawthorn; Rosaceae, Maleae), especially in North America, has been attributed by some to hybridization in combination with gametophytic apomixis and polyploidization, whereas others have considered the roles of hybridization and apomixis to be minimal. Study of the chemical composition and therapeutic value of hawthorn extracts requires reproducible differentiation of entities that may be difficult to distinguish by morphology alone. This study sought to address this by using the nuclear ribosomal spacer region ITS2 as a supplementary DNA barcode; however, a lack of success prompted an investigation to discover why this locus gave unsatisfactory results. METHODS: ITS2 was extensively cloned so as to document inter- and intraindividual variation in this locus, using hawthorns of western North America where the genus Crataegus is represented by only two widely divergent groups, the red-fruited section Coccineae and the black-fruited section Douglasia. Additional sequence data from selected loci on the plastid genome were obtained to enhance further the interpretation of the ITS2 results. KEY RESULTS: In the ITS2 gene tree, ribotypes from western North American hawthorns are found in two clades. Ribotypes from diploid members of section Douglasia occur in one clade (with representatives of the east-Asian section Sanguineae). The other clade comprises those from diploid and polyploid members of section Coccineae. Both clades contribute ribotypes to polyploid Douglasia. Data from four plastid-derived intergenic spacers demonstrate the maternal parentage of these allopolyploids. CONCLUSIONS: Repeated hybridization between species of section Douglasia and western North American members of section Coccineae involving the fertilization of unreduced female gametes explains the observed distribution of ribotypes and accounts for the phenetic intermediacy of many members of section Douglasia.

Molecular phylogeny and evolutionary history of the Eurasiatic orchid genus Himantoglossum s.l. (Orchidaceae).

BACKGROUND AND AIMS: Lizard orchids of the genus Himantoglossum include many of Eurasia's most spectacular orchids, producing substantial spikes of showy flowers. However, until recently the genus had received only limited, and entirely traditional, systematic study. The aim of the current work was to provide a more robust molecular phylogeny in order to better understand the evolutionary relationships among species of particular conservation concern. METHODS: All putative species of Himantoglossum s.l. were sampled across its geographical range. A large subsample of the 153 populations studied contributed to an initial survey of nuclear ribosomal internal transcribed spacer (nrITS) ribotypes. Smaller subsets were then sequenced for four plastid regions and the first intron of the low-copy-number nuclear gene LEAFY. Rooted using Steveniella as outgroup, phylogenetic trees were generated using parsimony and Bayesian methods from each of the three datasets, supplemented with a ribotype network. KEY RESULTS: The resulting trees collectively determined the order of branching of the early divergent taxa as Himantoglossum comperianum > H. robertianum group > H. formosum, events that also involved significant morphological divergence. Relaxed molecular clock dating suggested that these divergences preceded the Pleistocene glaciations (the origin of the H. robertianum group may have coincided with the Messinian salinity crisis) and occurred in Asia Minor and/or the Caucasus. Among more controversial taxa of the H. hircinum-jankae clade, which are only subtly morphologically divergent, topological resolution was poorer and topological incongruence between datasets was consequently greater. CONCLUSIONS: Plastid sequence divergence is broadly consistent with prior, morphologically circumscribed taxa and indicates a division between H. hircinum-adriaticum to the west of the Carpathians and H. jankae-caprinum (plus local endemics) to the east, a distinction also suggested by nrITS ribotypes. LEAFY phylogenies are less congruent with prior taxonomic arrangements and include one likely example of paralogy. Himantoglossum metlesicsianum fully merits its IUCN Endangered status. Potentially significant genetic variation was detected within Steveniella satyrioides, H. robertianum and H. hircinum. However, confident circumscription of the more derived species of Himantoglossum s.s., including local endemics of hybrid origin, must await future morphometric and population-genetic analyses.

Russula brunneoaurantiaca, a novel taxon of Russula subg. Crassotunicata from West Bengal, India, with morpho-molecular analysis and scanning electron microscopy.

This paper describes a new Russula species, R. brunneoaurantiaca, from India with morphological and molecular sequence (nrITS) data, field pictures of basidiocarps, and comparisons with close relatives. Russula brunneoaurantiaca has a brownish orange pileus with a mucilaginous surface, sub-decurrent lamellae that are white to pale orange, a white stipe that turns yellowish brown to brown when bruised, a strong, unpleasant smell, globose to subglobose basidiospores (5.0-9.0 5.0-7.8 m) with an inamyloid suprahilar spot and ornamentation of small isolated conical warts, fusiform hymenial cystidia on gill sides (62.5-82 × 7.5-12.5 µm) and lageniform to sub-lageniform cystidia with filiform apex near the gill edge (80-113 × 7.5-10 µm), fusiform to spindle-shaped pileocystidia, and habitat in association with Castanopsis sp. A complete morphological description, photographs, and molecular sequence-based phylogenetic trees demarcating the position of the novel taxon are provided. RESEARCH HIGHLIGHTS: Scanning electron microscopy (SEM) and subsequent DNA analysis revealed a new species of the genus Russula. SEM analysis is an additional technique to describe the size and shape of its basidiospores as well as their ornamentation. The diagnostic characteristics, habit, habitat, and similarities to related species are given.

Morpho-molecular analyses and scanning electron microscopy reveal two new species of genus Lactarius from the northwestern Himalaya, India.

One of the largest subgenera of the genus Lactarius, Lactarius subg. Russularia is abundant in the forests of the Himalayas. This article proposes Lactarius crassostipes sp. nov. and Lactarius flavocorpus sp. nov. under the L. subgenus Russularia. It includes comprehensive macro- and micro-morphological descriptions, scanning electron microscopy (SEM) micrographs, drawings, and comparisons with related species. The taxa's novelty is validated using phylogenetic analyses based on nrITS. RESEARCH HIGHLIGHTS: SEM and subsequent nrITS-based molecular phylogeny revealed two novel species (L. crassostipes and L. flavocorpus) of the genus Lactarius subg. Russularia. To further characterize the size, form, and ornamentation of these novel species' basidiospores, SEM analysis is an additional tool. In L. crassostipes "crasso" means thick and "stipes" indicates stipe. In contrast to L. flavocorpus, where "flavocorpus" refers to the golden yellow fruiting body, the name alludes to its unique thick stipe. L. crassostipes sp. nov. and L. flavocorpus sp. nov have been collected from the coniferous and broad-leaved forests of Union Territory of Jammu and Kashmir, India, respectively. The diagnostic characteristics, habit, habitat, and similarities to related species are given.

Molecular evidence provides new insights into the evolutionary origin of an ancient traditional Chinese medicine, the domesticated "Baizhi".

Introduction: "Baizhi" is a famous herbal medicine in China, and it includes four landraces named as 'Hangbaizhi', 'Chuanbaizhi', 'Qibaizhi', and 'Yubaizhi'. Long-term artificial selection had caused serious degradation of these germplasms. Determining the wild progenitor of the landraces would be benefit for their breed improvements. Previous studies have suggested Angelica dahurica var. dahurica, A. dahurica var. formosana, or A. porphyrocaulis as potential candidates, but the conclusion remains uncertain, and their phylogenetic relationships are still in controversy. Methods: In this study, the genetic variation and phylogenetic analyses of these species and four landraces were conducted on the basis of both the nrITS and plastome datasets. Results: Genetic variation analysis showed that all 8 population of four landraces shared only one ITS haplotype, meanwhile extremely low variation occurred within 6 population at plastid genome level. Both datasets supported the four landraces might be originated from a single wild germplasm. Phylogenetic analyses with both datasets revealed largely consistent topology using Bayesian inference and Maximum likelihood methods. Samples of the four landraces and all wild A. dahurica var. dahurica formed a highly supported monophyletic clade, and then sister to the monophyly clade comprised by samples of A. porphyrocaulis, while four landraces were clustered into one clade, which further clustered with a mixed branches of A. porphyrocaulis and A. dahurica var. dahurica to form sister branches for plastid genomes. Furthermore, the monophyletic A. dahurica var. formosana was far distant from the A. dahurica var. dahurica-"Baizhi" clade in Angelica phylogeny. Such inferences was also supported by the evolutionary patterns of nrITS haplotype network and K2P genetic distances. The outcomes indicated A. dahurica var. dahurica is most likely the original plant of "Baizhi". Discussion: Considering of phylogenetic inference and evolutionary history, the species-level status of A. dahurica var. formosana should be accepted, and the taxonomic level and phylgenetic position of A. porphyrocaulis should be further confirmed. This study preliminarily determined the wild progenitor of "Baizhi" and clarified the phylogenetic relationships among A. dahurica var. dahurica, A. dahurica var. formosana and A. porphyrocaulis, which will provide scientific guidance for wild resources protections and improvement of "Baizhi".

 Mazusjiangshiense (Mazaceae), a new species from China: evidence from morphological and molecular analyses.

Utilising both morphological and molecular analyses, this study unveils Mazusjiangshiensesp. nov., a novel addition to the Mazaceae family, discovered in Shaowu County, Fujian Province, China. The comprehensive description and illustrations provided here are a result of a meticulous exploration of its morphological features. While bearing a resemblance to M.gracilis, this new-found species is distinguished by three distinct characteristics: its stems are delicately soft, its leaves possess a membranous quality and the ovary is notably villous at the apex. Integration of molecular evidence, derived from the nuclear ribosomal DNA (nrITS) and three plastid DNA sequences (rps16, rbcL and trnL-trnF), unequivocally supports the classification of M.jiangshiense as a distinct species. Notably, the molecular analysis positions it as a sister species to M.spicatus, underscoring the phylogenetic relationships within the genus Mazus. Our research not only introduces M.jiangshiense as a novel taxonomic entity, but also provides a nuanced understanding of its morphological differences and molecular affinities, enriching our comprehension of the diversity and evolutionary relationships of Mazaceae.

Molecular phylogeny of tribe Stachydeae (Lamiaceae subfamily Lamioideae).

Although tribe Stachydeae (Lamiaceae) is considered monophyletic, relationships within the tribe are still poorly understood. The complexity of Stachydeae includes paraphyletic genera, considerable morphological plasticity, a range of ploidy levels, and presumably frequent natural hybridization. We performed parsimony and Bayesian phylogenetic analyses of nuclear (ribosomal ITS) and plastid (trnL intron, trnL-trnF spacer, rps16 intron) DNA sequence data from a taxonomically and geographically broad sampling of the tribe to identify major evolutionary lineages and to test taxonomic hypotheses within this largest of all lamioid tribes. We included 143 accessions corresponding to 121 species, representing both Old and New World species, and all 12 recognized genera of tribe Stachydeae. Both nuclear and plastid data corroborate monophyly of the tribe, with Melittis as sister to all remaining Stachydeae. For the latter well-supported clade, we suggest the phylogenetic name Eurystachys. Within Eurystachys, although monophyly is supported by both nuclear and plastid data for several named and unnamed groups, the majority of recognized taxa appear to be para- or polyphyletic. The taxon compositions of most subclades are congruent between the plastid and nuclear tree topologies, whereas their relative phylogenetic placements are often not. This level of plastid-nuclear incongruence suggests considerable impact of hybridization in the evolution of Stachydeae.

Does One Size Fit All? Variations in the DNA Barcode Gaps of Macrofungal Genera.

The nuclear ribosomal internal transcribed spacer (nrITS) region has been widely used in fungal diversity studies. Environmental metabarcoding has increased the importance of the fungal DNA barcode in documenting fungal diversity and distribution. The DNA barcode gap is seen as the difference between intra- and inter-specific pairwise distances in a DNA barcode. The current understanding of the barcode gap in macrofungi is limited, inhibiting the development of best practices in applying the nrITS region toward research on fungal diversity. This study examined the barcode gap using 5146 sequences representing 717 species of macrofungi from eleven genera, eight orders and two phyla in datasets assembled by taxonomic experts. Intra- and inter-specific pairwise distances were measured from sequence and phylogenetic data. The results demonstrate that barcode gaps are influenced by differences in intra- and inter-specific variance in pairwise distances. In terms of DNA barcode behavior, variance is greater in the ITS1 than ITS2, and variance is greater in both relative to the combined nrITS region. Due to the difference in variance, the barcode gaps in the ITS2 region are greater than in the ITS1. Additionally, the taxonomic approach of "splitting" taxa into numerous taxonomic units produces greater barcode gaps when compared to "lumping". The results show variability in the barcode gaps between fungal taxa, demonstrating a need to understand the accuracy of DNA barcoding in quantifying species richness. For taxonomic studies, variability in nrITS sequence data supports the application of multiple molecular markers to corroborate the taxonomic and systematic delineation of species.

A new species of Sedum (Crassulaceae) from Mount Danxia in Guangdong, China.

Sedumjinglanii, a new species of Crassulaceae from Mount Danxia in Guangdong, China, is described and illustrated. Phylogenetic analysis based on the internal transcribed spacer (ITS) region of nrDNA suggests that the new species belongs to S.sect.Sedum sensu Fu and Ohba (2001) in the "Flora of China", and is sister to a clade comprising S.alfredi and S.emarginatum with high support values (SH-aLRT = 84, UFBS = 95) but is distantly related to S.baileyi. The new species is morphologically similar to S.alfredi but it can be distinguished from the latter in its opposite leaves (vs. alternate leaves), its usually wider leaves (0.4-1.2 cm vs. 0.2-0.6 cm), its usually shorter petals (3.4-4.5 mm vs. 4-6 mm), its shorter nectar scales (0.4-0.5 mm vs. 0.5-1 mm), its shorter carpels (1.5-2.6 mm vs. 4-5 mm), and its shorter styles (0.6-0.9 mm vs. 1-2 mm). The new species can be easily distinguished from S.emarginatum which both have opposite leaves by its short, erect or ascending rhizome (vs. long and prostrate rhizome in the latter), shorter petals (3.4-4.5 mm vs. 6-8 mm) and shorter carpels (1.5-2.6 mm vs. 4-5 mm). It can also be easily distinguished from S.baileyi by its short, erect or ascending rhizome (vs. long and prostrate rhizome) and its shorter style (0.6-0.9 mm vs. 1-1.5 mm).

Applying a modified metabarcoding approach for the sequencing of macrofungal specimens from fungarium collections.

Premise: Fungaria are an underutilized resource for understanding fungal biodiversity. The effort and cost of producing DNA barcode sequence data for large numbers of fungal specimens can be prohibitive. This study applies a modified metabarcoding approach that provides a labor-efficient and cost-effective solution for sequencing the fungal DNA barcodes of hundreds of specimens at once. Methods: We applied a two-step PCR approach using nested, barcoded primers to sequence the fungal nrITS2 region of 766 macrofungal specimens using the Illumina platform. The specimens represent a broad taxonomic sampling of the Dikarya. Of these, 382 Lactarius specimens were analyzed to identify molecular operational taxonomic units (MOTUs) using a phylogenetic approach. The raw sequences were trimmed, filtered, assessed, and analyzed using the DADA2 amplicon de-noising toolkit and Biopython. The sequences were compared to the NCBI and UNITE databases and Sanger nrITS sequences from the same specimens. Results: The taxonomic identities derived from the nrITS2 sequence data were >90% accurate across all specimens sampled. A phylogenetic analysis of the Lactarius sequences identified 20 MOTUs. Discussion: The results demonstrate the capacity of these methods to produce nrITS2 sequences from large numbers of fungarium specimens. This provides an opportunity to more effectively use fungarium collections to advance fungal diversity identification and documentation.

Observations on Pluteaceae in Vietnam: Four New Species and New Records of Pluteus.

Eighteen specimens of Pluteus collected from the tropical forests of Vietnam were studied using morphological and molecular approaches. Pluteus podospilloides, P. semibulbosus, P. chrysaegis and P. septocystidiatus are registered as additional or new records for Vietnam. Four species (P. conformis, P. lucidus, P. subroseus, and P. ornatus) are proposed as new to science, and several other collections (Pluteus sp. 1, P. aff. septocystidiatus, P. aff. pauperculus and P. cf. velutinus) are given an inconclusive taxonomic status for now. The taxonomic positions of all specimens were confirmed using DNA data (nrITS and tef1). Descriptions of the macro- and microscopic features of the studied collections with a discussion of similar taxa are given.

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.

A specific SNP-based multiplex PCR assay for the simultaneous identification of two biological ingredients for the Chinese patent medicine, Danggui Buxue pill.

Background: An increasing number of Chinese patent medicines (CPM) have been widely used in East Asian and North American countries, and the safety and efficacy of CPM have highly attracted public attention. However, it is difficult to supervise the authenticity of multiple biological ingredients within CPM based on microscopic inspection and physical and chemical detection. The raw materials may have similar characteristics of tissue structures and ergastic substances or similar chemical composition and contents when substitutes and/or adulterants are added. DNA molecular markers have been used to distinguish the biological ingredients within CPM based on conventional PCR assay. However, it was proved to be time- and labor-consuming and reagent-wasting, as multiple PCR amplification strategies were required for identifying the complex species composition within CPM. Here, we took the CPM (Danggui Buxue pill) as an example and aimed to establish a specific SNP-based multiplex PCR assay and simultaneously determine the authenticity of the two biological ingredients (Angelicae Sinensis Radix and Astragali Radix) within this CPM. Methods: We, respectively, designed the species-specific primers based on highly variable nrITS for discriminating Angelicae Sinensis Radix and Astragali Radix from their common substitutes and adulterants. The specificity of the primers was checked through conventional PCR assay and multiplex PCR assay. Furthermore, we used a handcrafted Danggui Buxue pill sample (DGBXP) to optimize annealing temperatures for the primers with multiplex PCR, and the sensitivity was also assessed. Finally, fourteen batches of commercial Danggui Buxue pills were used to verify the stability and practicability of the established multiplex PCR assay. Results: Two pairs of highly species-specific primers for amplifying Angelicae Sinensis Radix and Astragali Radix were screened, and our established multiplex PCR assay showed high specificity and sensitivity (lowest detection concentration: 4.0 × 10-3 ng/µL) at an optimal annealing temperature of 65°C. The method could simultaneously identify both biological ingredients within the Danggui Buxue pill. Conclusion: The specific SNP-based multiplex PCR provided a simple, time-, and labor-saving method for the simultaneous identification of the two biological ingredients within Danggui Buxue pills. This study was expected to provide a novel qualitative quality control strategy for CPM.

Liparismacrosepala (Orchidaceae), a new species from southwest China with its phylogenetic position.

A new orchid species, Liparismacrosepala, is illustrated and described from Yunnan Province, China, based on morphological and molecular analyses. This plant is characterised by the ovoid-fusiform, slightly compressed pseudobulbs with 4 or 5 leaves with slightly crisped margins on their apical half, dorsal sepal heart-shaped, lip with a bituberculate basal callus and a thickened folded lateral lobe on each side, centrally with one cavity with slightly raised margins, the column with a single pair of broadly triangular, obtuse wings. Maximum Likelihood and Bayesian Inference analyses of combined nrITS and plastid matK DNA sequences place this species in section Cestichis.

Mazusdanxiacola (Mazaceae), a distinct new species endemic to Danxia landform in Jiangxi Province, eastern China.

Mazusdanxiacola, a new species endemic to Danxia landform in east Jiangxi Province, eastern China, is described and illustrated. The systematic placement of this new species was confirmed by molecular phylogenetic analyses based on four plastid markers (matK, rbcL, rps16 and trnL-trnF) and nuclear ribosome ITS sequence, and its specific relationships within Mazus were discussed. Morphologically, the new species is clearly different from other Mazus species by having a series of uncommon traits, i.e., annual habit, without stolons and basal leaves, single, erect and unbranched stems, long petiolate leaves abaxially grayish green to silver gray, truncate to broadly cuneate leaf bases, racemes extremely elongated up to 35 cm long, white corolla, and palate densely covered by conspicuous clavate gland-like hairs. The new species is assigned to Critically Endangered (CR) according to the IUCN Red List Categories and Criteria.

Primulalongipilosa (Primulaceae), a new species from Yunnan, China.

Primulalongipilosa from SW Yunnan, China, is described as a species new to science and illustrated. The systematic placement of this new species is also discussed based on an nrITS molecular tree. It is morphologically most similar to P.mollis, but differs from the latter in its racemose inflorescence, green calyx tube, pink to pink rose corolla, stamens at 1/3 length above the base of the corolla tube and applanate globose capsule.