Species delimitation of tea plants (Camellia sect. Thea) based on super-barcodes.

BACKGROUND: The era of high throughput sequencing offers new paths to identifying species boundaries that are complementary to traditional morphology-based delimitations. De novo species delimitation using traditional or DNA super-barcodes serve as efficient approaches to recognizing putative species (molecular operational taxonomic units, MOTUs). Tea plants (Camellia sect. Thea) form a group of morphologically similar species with significant economic value, providing the raw material for tea, which is the most popular nonalcoholic caffeine-containing beverage in the world. Taxonomic challenges have arisen from vague species boundaries in this group. RESULTS: Based on the most comprehensive sampling of C. sect. Thea by far (165 individuals of 39 morphospecies), we applied three de novo species delimitation methods (ASAP, PTP, and mPTP) using plastome data to provide an independent evaluation of morphology-based species boundaries in tea plants. Comparing MOTU partitions with morphospecies, we particularly tested the congruence of MOTUs resulting from different methods. We recognized 28 consensus MOTUs within C. sect. Thea, while tentatively suggesting that 11 morphospecies be discarded. Ten of the 28 consensus MOTUs were uncovered as morphospecies complexes in need of further study integrating other evidence. Our results also showed a strong imbalance among the analyzed MOTUs in terms of the number of molecular diagnostic characters. CONCLUSION: This study serves as a solid step forward for recognizing the underlying species boundaries of tea plants, providing a needed evidence-based framework for the utilization and conservation of this economically important plant group.

Using scanning electron microscopy and molecular data to discover a new species from old herbarium collections: The case of Phlomoideshenryi (Lamiaceae, Lamioideae).

Phlomoides is one of the largest genera of Lamiaceae with approximately 150-170 species distributed mainly in Eurasia. In this study, we describe and illustrate a new species, P.henryi, which was previously misidentified as P.bracteosa, from Yunnan Province, southwest China. Molecular phylogenetic analyses revealed that P.henryi is found within a clade in which most species lack basal leaves. In this clade, the new species is morphologically distinct from P.rotata in having an obvious stem and, from the rest, by having transparent to white trichomes inside the upper corolla lip. In addition, micro-features of trichomes on the calyx and leaf epidermis can differentiate the new species from other species grouped in the same clade and a key, based on trichome morphology for these species, is provided. The findings demonstrate that the use of scanning electron microscopy can reveal inconspicuous morphological affinities amongst morphologically similar species and play an important role in the taxonomic study of the genus Phlomoides.

Isodonxiaoluzhiensis (Lamiaceae, Nepetoideae), a new species from Yunnan, southwest China.

Isodonxiaoluzhiensis, a new species of the tribe Ocimeae in family Lamiaceae, is described and illustrated. The new species is known only from the type locality, Xiaoluzhi village in Luzhijang dry-hot valley of Yimen County, central Yunnan, southwest China. It is characterized by having a procumbent habit, gracile stems and branches, relatively small leaves and flowers, and the phenology of flowering in winter. The morphological comparisons with its putative closest relatives (I.adenanthus and I.hsiwenii) are also presented.

Comparative analyses of Linderniaceae plastomes, with implications for its phylogeny and evolution.

Introduction: The recently established Linderniaceae, separated from the traditionally defined Scrophulariaceae, is a taxonomically complicated family. Although previous phylogenetic studies based on a few short DNA markers have made great contributions to the taxonomy of Linderniaceae, limited sampling and low resolution of the phylogenetic tree have failed to resolve controversies between some generic circumscriptions. The plastid genome exhibits a powerful ability to solve phylogenetic relationships ranging from shallow to deep taxonomic levels. To date, no plastid phylogenomic studies have been carried out in Linderniaceae. Methods: In this study, we newly sequenced 26 plastid genomes of Linderniaceae, including eight genera and 25 species, to explore the phylogenetic relationships and genome evolution of the family through plastid phylogenomic and comparative genomic analyses. Results: The plastid genome size of Linderniaceae ranged from 152,386 bp to 154,402 bp, exhibiting a typical quartile structure. All plastomes encoded 114 unique genes, comprising 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. The inverted repeat regions were more conserved compared with the single-copy regions. A total of 1803 microsatellites and 1909 long sequence repeats were identified, and five hypervariable regions (petN-psbM, rps16-trnQ, rpl32-trnL, rpl32, and ycf1) were screened out. Most protein-coding genes were relatively conserved, with only the ycf2 gene found under positive selection in a few species. Phylogenomic analyses confirmed that Linderniaceae was a distinctive lineage and revealed that the presently circumscribed Vandellia and Torenia were non-monophyletic. Discussion: Comparative analyses showed the Linderniaceae plastomes were highly conservative in terms of structure, gene order, and gene content. Combining morphological and molecular evidence, we supported the newly established Yamazakia separating from Vandellia and the monotypic Picria as a separate genus. These findings provide further evidence to recognize the phylogenetic relationships among Linderniaceae and new insights into the evolution of the plastid genomes.

Functional divergence of CYP76AKs shapes the chemodiversity of abietane-type diterpenoids in genus Salvia.

The genus Salvia L. (Lamiaceae) comprises myriad distinct medicinal herbs, with terpenoids as one of their major active chemical groups. Abietane-type diterpenoids (ATDs), such as tanshinones and carnosic acids, are specific to Salvia and exhibit taxonomic chemical diversity among lineages. To elucidate how ATD chemical diversity evolved, we carried out large-scale metabolic and phylogenetic analyses of 71 Salvia species, combined with enzyme function, ancestral sequence and chemical trait reconstruction, and comparative genomics experiments. This integrated approach showed that the lineage-wide ATD diversities in Salvia were induced by differences in the oxidation of the terpenoid skeleton at C-20, which was caused by the functional divergence of the cytochrome P450 subfamily CYP76AK. These findings present a unique pattern of chemical diversity in plants that was shaped by the loss of enzyme activity and associated catalytic pathways.

East Asian-North American disjunctions and phylogenetic relationships within subtribe Nepetinae (Lamiaceae).

Biogeographic disjunctions, including intercontinental disjunctions, are frequent across plant lineages and have been of considerable interest to biologists for centuries. Their study has been reinvigorated by molecular dating and associated comparative methods. One of the "classic" disjunction patterns is that between Eastern Asia and North America. It has been speculated that this pattern is the result of vicariance following the sundering of a widespread Acrto-Teritary flora. Subtribe Nepetinae in the mint family (Lamiaceae) is noteworthy because it contains three genera with this disjunction pattern: Agastache, Dracocephalum, and Meehania. These disjunctions are ostensibly the result of three separate events, allowing for concurrent testing of the tempo, origin, and type of each biogeographic event. Using four plastid and four nuclear markers, we estimated divergence times and analyzed the historical biogeography of Nepetinae, including comprehensive sampling of all major clades for the first time. We recover a well-supported and largely congruent phylogeny of Nepetinae between genomic compartments, although several cases of cyto-nuclear discordance are evident. We demonstrate that the three disjunctions are pseudo-congruent, with unidirectional movement from East Asia at slightly staggered times during the late Miocene and early Pliocene. With the possible exception of Meehania, we find that vicariance is likely the underlying driver of these disjunctions. The biogeographic history of Meehania in North America may be best explained by long-distance dispersal, but a more complete picture awaits deeper sampling of the nuclear genome and more advanced biogeographical models.

Seed morphology of Hypericum (Hypericaceae) in China and its taxonomic significance.

The seed morphology of 40 taxa within the genus Hypericum (Hypericaceae) from China, representing 9 sections of the genus, was examined using both Light and Scanning Electron Microscopy to evaluate the taxonomic relevance of macro- and micro-morphological features. Details articulating variation in seed size, color, shape, appendages, and seed coat ornamentation are described, illustrated, and compared, and their taxonomic importance is discussed. Seeds were generally brown in color and cylindric-ellipsoid to prolonged cylindric in shape. Seed size displayed wide variation, ranging from 0.37-1.91 mm in length and 0.12-0.75 mm in width. Seed appendages were observed as a characteristic morphological feature. Seed surface ornamentation has high phenotypic plasticity, and four types (reticulate, foveolate, papillose, and ribbed) can be recognized. In general, seed color and shape have limited taxonomic significance. However, some other features represent informative characters that can be used efficiently in distinguishing the studied taxa at the section and/or species levels. The findings illustrate that considerable taxonomic knowledge can be obtained by investigating the seed features of Hypericum, and the use of Scanning Electron Microscopy can reveal inconspicuous morphological affinities among species and play a role in taxonomic and systematic studies of the genus Hypericum. RESEARCH HIGHLIGHTS: Macro- and micro-morphological features of seeds of 40 Hypericum taxa from China were examined using Light and Scanning Electron Microscopy, providing the first broad study regarding seed morphology for Hypericum from China. Details and variations of seed size, shape, color, surface ornamentation, and appendages are fully presented. Seed features and their variation have important taxonomic significance at the section and/or species levels within Hypericum.

Plastome sequences fail to resolve shallow level relationships within the rapidly radiated genus Isodon (Lamiaceae).

As one of the largest genera of Lamiaceae and of great medicinal importance, Isodon is also phylogenetically and taxonomically recalcitrant largely ascribed to its recent rapid radiation in the Hengduan Mountains. Previous molecular phylogenetic studies using limited loci have only successfully resolved the backbone topology of the genus, but the interspecific relationships suffered from low resolution, especially within the largest clade (Clade IV) which comprises over 80% species. In this study, we attempted to further elucidate the phylogenetic relationships within Isodon especially Clade IV using plastome sequences with a broad taxon sampling of ca. 80% species of the genus. To reduce systematic errors, twelve different plastome data sets (coding and non-coding regions with ambiguously aligned regions and saturated loci removed or not) were employed to reconstruct phylogeny using maximum likelihood and Bayesian inference. Our results revealed largely congruent topologies of the 12 data sets and recovered major lineages of Isodon consistent with previous studies, but several incongruences are also found among these data sets and among single plastid loci. Most of the shallow nodes within Clade IV were resolved with high support but extremely short branch lengths in plastid trees, and showed tremendous conflicts with the nrDNA tree, morphology and geographic distribution. These incongruences may largely result from stochasticity (due to insufficient phylogenetic signal) and hybridization and plastid capture. Therefore, the uniparental-inherited plastome sequences are insufficient to disentangle relationships within a genus which has undergone recent rapid diversification. Our findings highlight a need for additional data from nuclear genome to resolve the relationships within Clade IV and more focused studies to assess the influences of multiple processes in the evolutionary history of Isodon. Nevertheless, the morphology of the shape and surface sculpture/indumentum of nutlets is of systematic importance that they can distinguish the four major clades of Isodon.

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.

Paraphlomishsiwenii (Lamiaceae), a new species from the limestone area of Guangxi, China.

The indumentum of nutlets is shown to be of phylogenetic importance in previous molecular phylogenetic studies of Paraphlomis, a genus of Lamiaceae with approximately 30 species distributed mainly in southern China and Southeast Asia. Nearly half the species of Paraphlomis are known from limestone areas. In this study, we described and illustrated a new species, P.hsiwenii, from the karst mountain forests in Guangxi Zhuang Autonomous Region, China. Our molecular phylogenetic analyses revealed that P.hsiwenii is recovered in a clade consisting of species with hairy nutlets. The new species is morphologically most similar to P.pagantha from the same clade, but they differ in the morphology of lamina bases, length of pedicels and calyces, as well as the morphology of upper corolla lips.

Characterization of the complete chloroplast genome of Salvia trijuga Diels (Lamiaceae).

The complete cp genome of Salvia trijuga Diels was 151,345 bp in length, including a large single-copy region (LSC) of 82,577 bp, a small single-copy region (SSC) of 17,584 bp and a pair of inverted repeats (IRs) of 25,592 bp. The genome contained 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content of this genome was 37.9%, with the corresponding values of LSC, SSC and IR regions being 36.0%, 31.7% and 43.1%, respectively. Further, the phylogenomic analysis strongly supported the sister relationship of Salvia trijuga and Salvia plebeia R. Br.

Six New 3,5-Dimethylcoumarins from Chelonopsis praecox, Chelonopsis odontochila and Chelonopsis pseudobracteata.

Ten 3,5-dimethylcoumarins (1-6 and 8‒11) involving six new ones (1-6), together with a known 3-methylcoumarin (7), were isolated from the aerial parts of three Chelonopsis plants, C. praecox, C. odontochila, and C. pseudobracteata. The structures of the new compounds were determined by extensive HRESIMS, 1D and 2D NMR spectroscopic analyses. According to the substitution at C-5, these coumarins were classified into 5-methyl, 5-hydroxymethyl, 5-formyl, and 5-nor types. All the isolates were assayed for their inhibition on α-glucosidase, protein tyrosine phosphatase 1B, and T-cell protein tyrosine phosphatase in vitro.

A preliminary phylogenetic study of Paraphlomis (Lamiaceae) based on molecular and morphological evidence.

Paraphlomis is a genus of Lamiaceae with about 30 species distributed mainly in subtropical China. In this study, we carried out the first molecular phylogenetic analyses to elucidate the relationships within the genus based on two nuclear and four plastid DNA regions. Our results, which recovered a species of Matsumurella within Paraphlomis, indicate that the genus is not monophyletic. The two sections and most of the series previously described within the genus are also shown to be polyphyletic. Combining with morphological evidence, our study indicates that nutlet morphology rather than calyx morphology is of phylogenetic value for the infrageneric classification of Paraphlomis. Moreover, P. jiangyongensis, a new species from southern China, is here described, and P . coronata, formerly treated as a variety of P . javanica, is here resurrected as a distinct species within the genus.

Systematic Placement of the Enigmatic Southeast Asian Genus Paralamium and an Updated Phylogeny of Tribe Pogostemoneae (Lamiaceae Subfamily Lamioideae).

Paralamium (Lamiaceae) is a monotypic genus within the subfamily Lamioideae and has a sporadic distribution in subtropical mountains of southeast Asia. Although recent studies have greatly improved our understanding of generic relationships within Lamioideae, the second most species-rich subfamily of Lamiaceae, the systematic position of Paralamium within the subfamily remains unclear. In this study, we investigate the phylogenetic placement of the genus using three datasets: (1) a 69,276 bp plastome alignment of Lamiaceae; (2) a five chloroplast DNA region dataset of tribe Pogostemoneae, and (3) a nuclear ribosomal internal transcribed spacer region dataset of Pogostemoneae. These analyses demonstrate that Paralamium is a member of Pogostemoneae and sister to the monotypic genus Craniotome. In addition, generic-level phylogenetic relationships within Pogostemoneae are also discussed, and a dichotomous key for genera within Pogostemoneae is provided.

Molecular and morphological evidence for a new species of Isodon (Lamiaceae) from southern China.

Isodon brevipedunculatus, a new species from southern China, is described and illustrated. The phylogenetic position of the new species within the genus was analyzed based on two nuclear ribosomal DNA regions and an ingroup sampling of about 80% of Asian species of Isodon. The results show that I. brevipedunculatus is recovered in a clade that consists of species mainly with glandular mericarps and that are distributed in the Sino-Japanese region. Combining molecular and geographical evidence, our study reveals that I. brevipedunculatus is most closely related to Isodon amethystoides and Isodon bifidocalyx, but differs from the former in lamina shape, number of flowers per cyme, and peduncle length, and from the latter in lamina indumentum, calyx morphology, and corolla length.

An updated tribal classification of Lamiaceae based on plastome phylogenomics.

BACKGROUND: A robust molecular phylogeny is fundamental for developing a stable classification and providing a solid framework to understand patterns of diversification, historical biogeography, and character evolution. As the sixth largest angiosperm family, Lamiaceae, or the mint family, consitutes a major source of aromatic oil, wood, ornamentals, and culinary and medicinal herbs, making it an exceptionally important group ecologically, ethnobotanically, and floristically. The lack of a reliable phylogenetic framework for this family has thus far hindered broad-scale biogeographic studies and our comprehension of diversification. Although significant progress has been made towards clarifying Lamiaceae relationships during the past three decades, the resolution of a phylogenetic backbone at the tribal level has remained one of the greatest challenges due to limited availability of genetic data. RESULTS: We performed phylogenetic analyses of Lamiaceae to infer relationships at the tribal level using 79 protein-coding plastid genes from 175 accessions representing 170 taxa, 79 genera, and all 12 subfamilies. Both maximum likelihood and Bayesian analyses yielded a more robust phylogenetic hypothesis relative to previous studies and supported the monophyly of all 12 subfamilies, and a classification for 22 tribes, three of which are newly recognized in this study. As a consequence, we propose an updated phylogenetically informed tribal classification for Lamiaceae that is supplemented with a detailed summary of taxonomic history, generic and species diversity, morphology, synapomorphies, and distribution for each subfamily and tribe. CONCLUSIONS: Increased taxon sampling conjoined with phylogenetic analyses based on plastome sequences has provided robust support at both deep and shallow nodes and offers new insights into the phylogenetic relationships among tribes and subfamilies of Lamiaceae. This robust phylogenetic backbone of Lamiaceae will serve as a framework for future studies on mint classification, biogeography, character evolution, and diversification.

Rediscovery of Mazus lanceifolius reveals a new genus and a new species in Mazaceae.

Mazus lanceifolius (Mazaceae) is a perennial herb with opposite leaves and endemic to central China that has not been collected for 130 years. Rediscovery of this enigmatic species in the wild allows for determination of its phylogenetic position within Mazaceae. Phylogenetic reconstruction of Mazaceae based on DNA sequences from four plastid markers (matK, rbcL, rps16 and trnL-trnF) and nuclear ribosome ITS consistently showed that Mazus was not monophyletic. Mazus lanceifolius is in the most basal clade within Mazaceae, as sister to the remaining species of three recognized genera Dodartia, Lancea and Mazus. These results support the separation of M. lanceifolius from Mazus as a new genus, which was established here as Puchiumazus Bo Li, D.G. Zhang & C.L. Xiang. Meanwhile, a collection from Shennongjia Forestry District of Hubei Province, China, misidentified as "M. lanceifolius" in previous molecular study, is here revealed to represent an undescribed species of Mazus, i.e., M. fruticosus Bo Li, D.G. Zhang & C.L. Xiang, sp. nov. Morphologically, Puchiumazus is clearly distinct from the other three genera by having quadrangular to somewhat ribbed stems, and obviously opposite leaves. In addition, we provide a taxonomic key to the four genera of Mazaceae.

Degradation of key photosynthetic genes in the critically endangered semi-aquatic flowering plant Saniculiphyllum guangxiense (Saxifragaceae).

BACKGROUND: Plastid gene loss and pseudogenization has been widely documented in parasitic and mycoheterotrophic plants, which have relaxed selective constraints on photosynthetic function. More enigmatic are sporadic reports of pseudogenization and loss of important photosynthesis genes in lineages thought to be fully photosynthetic. Here we report the complete plastid genome of Saniculiphyllum guangxiense, a critically endangered and phylogenetically isolated plant lineage, along with genomic evidence of reduced chloroplast function. We also report 22 additional plastid genomes representing the diversity of its containing clade Saxifragales, characterizing gene content and placing variation in a broader phylogenetic context. RESULTS: We find that the plastid genome of Saniculiphyllum has experienced pseudogenization of five genes of the ndh complex (ndhA, ndhB, ndhD, ndhF, and ndhK), previously reported in flowering plants with an aquatic habit, as well as the surprising pseudogenization of two genes more central to photosynthesis (ccsA and cemA), contrasting with strong phylogenetic conservatism of plastid gene content in all other sampled Saxifragales. These genes participate in photooxidative protection, cytochrome synthesis, and carbon uptake. Nuclear paralogs exist for all seven plastid pseudogenes, yet these are also unlikely to be functional. CONCLUSIONS: Saniculiphyllum appears to represent the greatest degree of plastid gene loss observed to date in any fully photosynthetic lineage, perhaps related to its extreme habitat specialization, yet plastid genome length, structure, and substitution rate are within the variation previously reported for photosynthetic plants. These results highlight the increasingly appreciated dynamism of plastid genomes, otherwise highly conserved across a billion years of green plant evolution, in plants with highly specialized life history traits.

Episodic and guanine-cytosine-biased bursts of intragenomic and interspecific synonymous divergence in Ajugoideae (Lamiaceae) mitogenomes.

Synonymous substitution rates in plant mitochondrial genomes vary by orders of magnitude among species, whereas synonymous rates among genes within a genome are generally consistent. Exceptionally, genes within the Ajuga reptans (Lamiaceae) mitochondrial genome exhibit unprecedented intragenomic heterogeneity in synonymous sequence divergence, but the biological mechanisms underlying this rate variation remain unclear. We tracked the origin and evolutionary trajectory of mitochondrial rate variations by dense sampling in Ajugoideae and found differences in the timing and magnitude of rate acceleration for particular genes. The most divergent genes accelerated earlier, retained a high rate across Ajugoideae, and are generally devoid of RNA editing, whereas moderately diverged genes accelerated later and retained relatively higher RNA editing frequency. The acceleration of mutation rates correlates with increased guanine-cytosine (GC) content, suggesting a key role for GC-biased gene conversion and/or repair after the breakage of ancestral gene clusters.

Leveraging plastomes for comparative analysis and phylogenomic inference within Scutellarioideae (Lamiaceae).

Scutellaria, or skullcaps, are medicinally important herbs in China, India, Japan, and elsewhere. Though Scutellaria is the second largest and one of the more taxonomically challenging genera within Lamiaceae, few molecular systematic studies have been undertaken within the genus; in part due to a paucity of available informative markers. The lack of informative molecular markers for Scutellaria hinders our ability to accurately and robustly reconstruct phylogenetic relationships, which hampers our understanding of the diversity, phylogeny, and evolutionary history of this cosmopolitan genus. Comparative analyses of 15 plastomes, representing 14 species of subfamily Scutellarioideae, indicate that plastomes within Scutellarioideae contain about 151,000 nucleotides, and possess a typical quadripartite structure. In total, 590 simple sequence repeats, 489 longer repeats, and 16 hyper-variable regions were identified from the 15 plastomes. Phylogenetic relationships among the 14 species representing four of the five genera of Scutellarioideae were resolved with high support values, but the current infrageneric classification of Scutellaria was not supported in all analyses. Complete plastome sequences provide better resolution at an interspecific level than using few to several plastid markers in phylogenetic reconstruction. The data presented here will serve as a foundation to facilitate DNA barcoding, species identification, and systematic research within Scutellaria, which is an important medicinal plant resource worldwide.