Bayesian tip-dated phylogeny and biogeography of Cissampelideae (Menispermaceae): Mitigating the effects of homoplastic morphological characters.

The integration of morphological and molecular data is essential to understand the affinities of fossil taxa and spatio-temporal evolutionary processes of organisms. However, homoplastic morphological characters can mislead the placement of fossil taxa and impact downstream analyses. Here, we provide an example of how to mitigate effectively the effect of morphological homoplasy on the placement of fossil taxa and biogeographic inferences of Cissampelideae. We assembled three data types, morphological data only, morphological data with a molecular scaffold and combined morphological and molecular data. By removing high-level homoplastic morphological data or reweighting the morphological characters, we conducted 15 parsimony, 12 undated Bayesian and four dated Bayesian analyses. Our results show that the 14 selected Cissampelideae fossil taxa are placed poorly when based only on morphological data, but the addition of molecular scaffold and combination of morphological and molecular data greatly improve the resolution of fossil nodes. We raise the monotypic Stephania subg. Botryodiscia to generic status and discover that three fossils previously assigned to Stephania should be members of Diploclisia. The Bayesian tip-dated tree recovered by removing homoplastic morphological characters with a Rescaled Consistency Index <0.25 has the highest stratigraphic fit and consequently generates more reasonable biogeographic reconstruction for Cissampelideae. Cissampelideae began to diversify in Asia in the latest Cretaceous and subsequently dispersed to South America around the Cretaceous-Palaeogene boundary. Two dispersal events from Asia to Africa occurred in the Early Eocene and the Late Eocene-Late Oligocene, respectively. These findings provide guidelines and practical methods for mitigating the effects of homoplastic morphological characters on fossil placements and Bayesian tip-dating, as well as insights into the past tropical floristic exchanges among different continents.

Comparative plastome analysis of the sister genera Ceratocephala and Myosurus (Ranunculaceae) reveals signals of adaptive evolution to arid and aquatic environments.

BACKGROUND: Expansion and contraction of inverted repeats can cause considerable variation of plastid genomes (plastomes) in angiosperms. However, little is known about whether structural variations of plastomes are associated with adaptation to or occupancy of new environments. Moreover, adaptive evolution of angiosperm plastid genes remains poorly understood. Here, we sequenced the complete plastomes for four species of xerophytic Ceratocephala and hydrophytic Myosurus, as well as Ficaria verna. By an integration of phylogenomic, comparative genomic, and selection pressure analyses, we investigated evolutionary patterns of plastomes in Ranunculeae and their relationships with adaptation to dry and aquatic habitats. RESULTS: Owing to the significant contraction of the boundary of IRA/LSC towards the IRA, plastome sizes and IR lengths of Myosurus and Ceratocephala are smaller within Ranunculeae. Compared to other Ranunculeae, the Myosurus plastome lost clpP and rps16, one copy of rpl2 and rpl23, and one intron of rpoC1 and rpl16, and the Ceratocephala plastome added an infA gene and lost one copy of rpl2 and two introns of clpP. A total of 11 plastid genes (14%) showed positive selection, two genes common to Myosurus and Ceratocephala, seven in Ceratocephala only, and two in Myosurus only. Four genes showed strong signals of episodic positive selection. The rps7 gene of Ceratocephala and the rpl32 and ycf4 genes of Myosurus showed an increase in the rate of variation close to 3.3 Ma. CONCLUSIONS: The plastomic structure variations as well as the positive selection of two plastid genes might be related to the colonization of new environments by the common ancestor of Ceratocephala and Myosurus. The seven and two genes under positive selection might be related to the adaptation to dry and aquatic habitats in Ceratocephala and Myosurus, respectively. Moreover, intensified aridity and frequent sea-level fluctuations, as well as global cooling, might have favored an increased rate of change in some genes at about 3.3 Ma, associated with adaptation to dry and aquatic environments, respectively. These findings suggest that changing environments might have influenced structural variations of plastomes and fixed new mutations arising on some plastid genes owing to adaptation to specific habitats.

Chemical Composition of Methanol Extracts from Leaves and Flowers of Anemonopsis macrophylla (Ranunculaceae).

Anemonopsis Siebold et Zucc. is an unstudied single-species genus belonging to the tribe Cimicifugeae (Ranunculaceae). The only species of this genus-Anemonopsis macrophylla Siebold and Zucc.-is endemic to Japan. There are no data on its chemical composition. This work is the first to determine (with liquid chromatography-high-resolution mass spectrometry, LC-HRMS) the chemical composition of methanol extracts of leaves and flowers of A. macrophylla. More than 100 compounds were identified. In this plant, the classes of substances are coumarins (13 compounds), furocoumarins (3), furochromones (2), phenolic acids (21), flavonoids (27), and fatty acids and their derivatives (15 compounds). Isoferulic acid (detected in extracts from this plant) brings this species closer to plants of the genus Cimicifuga, one of the few genera containing this acid and ferulic acid at the same time. Isoferulic acid is regarded as a reference component of a quality indicator of Cimicifuga raw materials. The determined profiles of substances are identical between the leaf and flower methanol extracts. Differences in levels of some identified substances were revealed between the leaf and flower extracts of A. macrophylla; these differences may have a substantial impact on the manifestation of the biological and pharmacological effects of the extracts in question.

The Genus Eranthis: Prospects of Research on Its Phytochemistry, Pharmacology, and Biotechnology.

This review summarizes information about the chemical composition and beneficial properties of species of the genus Eranthis Salisb. from the world's flora. To date, seven out of ~14 species found in Asia and parts of Europe have been studied to various degrees. Here, data are presented on the diversity of sets of chromones, furochromones, triterpene saponins, coumarins, and other classes of secondary metabolites of Eranthis species according to the literature. For new compounds-isolated from Eranthis for the first time-structural formulas are also provided. Among the new compounds, chromones and coumarins predominate, as do triterpene saponins of the olean and cycloartane series and lectin. The results of pharmacological studies are presented showing anti-inflammatory, antioxidant, antiviral, and other types of biological activities found in extracts, in their fractions, and in individual compounds of the aboveground and underground organs and parts of Eranthis species. Despite the limited geographic range of Eranthis plants, it is possible to search for active substances, develop methods for biological and chemical synthesis of the isolated substances, and create a finished therapeutic substance based on them. In addition, it is feasible to obtain the desired standardized pure materials from Eranthis species grown in vitro.

Corrigendum: Organization, phylogenetic marker exploitation, and gene evolution in the plastome of Thalictrum (Ranunculaceae).

[This corrects the article DOI: 10.3389/fpls.2022.897843.].

The synergy of abiotic and biotic factors correlated with diversification of Fumarioideae (Papaveraceae) in the Cenozoic.

Rapid diversification of a group is often associated with exploiting an ecological opportunity and/or the evolution of a key innovation. However, how the interplay of such abiotic and biotic factors correlates with organismal diversification has been rarely documented in empirical studies, especially for organisms inhabiting drylands. Fumarioideae is the largest subfamily in Papaveraceae and is mainly distributed in temperate regions of the Northern Hemisphere. Here, we used one nuclear (ITS) and six plastid (rbcL, atpB, matK, rps16, trnL-F, and trnG) DNA sequences to investigate the spatio-temporal patterns of diversification and potential related factors of this subfamily. We first present the most comprehensive phylogenetic analysis of Fumarioideae to date. The results of our integrated molecular dating and biogeographic analyses indicate that the most recent common ancestor of Fumarioideae started to diversify in Asia during the Upper Cretaceous, and then dispersed multiple times out of Asia in the Cenozoic. In particular, we discover two independent dispersal events from Eurasia to East Africa in the late Miocene, suggesting that the Arabian Peninsula might be an important exchange corridor between Eurasia and East Africa in the late Miocene. Within the Fumarioideae, increased speciation rates were detected in two groups, Corydalis and Fumariinae. Corydalis first experienced a burst of diversification in its crown group at âˆ¼ 42 Ma, and further accelerated diversification from the mid-Miocene onwards. During these two periods, Corydalis had evolved diverse life history types, which could have facilitated the colonization of diverse habitats originating from extensive orogenesis in the Northern Hemisphere as well as Asian interior desertification. Fumariinae underwent a burst of diversification at âˆ¼ 15 Ma, which temporally coincides with the increasing aridification in central Eurasia, but is markedly posterior to the shifts in habitat (from moist to arid) and in life history (from perennial to annual) and to range expansion from Asia to Europe, suggesting that Fumariinae species may have been pre-adapted to invade European arid habitats by the acquisition of annual life history. Our study provides an empirical case that documents the importance of pre-adaptation on organismal diversification in drylands and highlights the significant roles of the synergy of abiotic and biotic factors in promoting plant diversification.

Biogeographic diversification of Actaea (Ranunculaceae): Insights into the historical assembly of deciduous broad-leaved forests in the Northern Hemisphere.

The deciduous broad-leaved forests (DBLFs) cover large temperate and subtropical high-altitude regions in the Northern Hemisphere. They are home to rich biodiversity, especially to numerous endemic and relict species. However, we know little about how this vegetation in the Northern Hemisphere has developed through time. Here, we used Actaea (Ranunculaceae), an herbaceous genus almost exclusively growing in the understory of the Northern Hemisphere DBLFs, to shed light on the historical assembly of this biome in the Northern Hemisphere. We present a complete species-level phylogenetic analysis of Actaea based on five plastid and nuclear loci. Using the phylogenetic framework, we estimated divergence times, ancestral ranges, and diversification rates. Phylogenetic analyses strongly support Actaea as monophyletic. Sections Podocarpae and Oligocarpae compose a clade, sister to all other Actaea. The sister relationship between sections Chloranthae and Souliea is strongly supported. Section Dichanthera is not monophyletic unless section Cimicifuga is included. Actaea originated in East Asia, likely the Qinghai-Tibet Plateau, in the late Paleocene (c. 57 Ma), and subsequently dispersed into North America in the middle Eocene (c. 43 Ma) via the Thulean bridge. Actaea reached Europe twice, Japan twice, and Taiwan once, and all these five colonization events occurred in the late Miocene-early Pliocene, a period when sea level dropped. Actaea began to diversify at c. 43 Ma. The section-level diversification took place at c. 27-37 Ma and the species-level diversification experienced accelerations twice, which occurred at c. 15 Ma and c. 5 Ma, respectively. Our findings suggest that the Northern Hemisphere DBLFs might have risen in the middle Eocene and further diversified in the late Eocene-Oligocene, middle Miocene and early Pliocene, in association with climatic deterioration during these four periods.

Individual Differences in Growth and in Accumulation of Secondary Metabolites in Rhodiola rosea Cultivated in Western Siberia.

In this study, growth parameters of underground parts and concentrations of phenylpropanoids, phenylethanoids, flavonoids, hydroxybenzoic acids, and catechins in aqueous-ethanol extracts of 6-year-old cultivated plants of Rhodiola rosea (propagated in vitro) of Altai Mountain origin were analyzed, and differences in chemical composition among plant specimens and between plant parts (rhizome and root) were evaluated. High-performance liquid chromatography detected 13 phenolic compounds. Roots contained 1.28 times higher phenylethanoids levels (1273.72 mg/100 g) than rhizomes did. Overall, the concentration of phenylethanoids in underground organs was not high and ranged from 21.36 to 103.00 mg/100 g. High variation among R. rosea individual plants was noted both in growth characteristics and in levels of secondary metabolites under our cultivation conditions. It was found that concentrations of phenylpropanoids, phenylethanoids, and catechins significantly depend on the plant part analyzed (p ≤ 0.05). Specimen No. 4 is characterized by the highest concentration of rosavins (1230.99 mg/plant) and the lowest concentration of cinnamyl alcohol (62.87 mg/plant). Despite the wide range of values, all 10 tested specimens (underground part) met the minimum requirements of the United States Pharmacopeia (2015) for rosavins (0.3%) and of the Russia State Pharmacopoeia (2015) for the average level of rosavins (roots): (1%).

Evolutionary history of the Arctic flora.

The Arctic tundra is a relatively young and new type of biome and is especially sensitive to the impacts of global warming. However, little is known about how the Arctic flora was shaped over time. Here we investigate the origin and evolutionary dynamics of the Arctic flora by sampling 32 angiosperm clades that together encompass 3626 species. We show that dispersal into the Arctic and in situ diversification within the Arctic have similar trends through time, initiating at approximately 10-9 Ma, increasing sharply around 2.6 Ma, and peaking around 1.0-0.7 Ma. Additionally, we discover the existence of a long-term dispersal corridor between the Arctic and western North America. Our results suggest that the initiation and diversification of the Arctic flora might have been jointly driven by progressive landscape and climate changes and sea-level fluctuations since the early Late Miocene. These findings have important conservation implications given rapidly changing climate conditions in the Arctic.

Phylogenomics and morphological evolution of the mega-diverse genus Artemisia (Asteraceae: Anthemideae): implications for its circumscription and infrageneric taxonomy.

BACKGROUND AND AIMS: Artemisia is a mega-diverse genus consisting of ~400 species. Despite its medicinal importance and ecological significance, a well-resolved phylogeny for global Artemisia, a natural generic delimitation and infrageneric taxonomy remain missing, owing to the obstructions from limited taxon sampling and insufficient information on DNA markers. Its morphological characters, such as capitulum, life form and leaf, show marked variations and are widely used in its infrageneric taxonomy. However, their evolution within Artemisia is poorly understood. Here, we aimed to reconstruct a well-resolved phylogeny for global Artemisia via a phylogenomic approach, to infer the evolutionary patterns of its key morphological characters and to update its circumscription and infrageneric taxonomy. METHODS: We sampled 228 species (258 samples) of Artemisia and its allies from both fresh and herbarium collections, covering all the subgenera and its main geographical areas, and conducted a phylogenomic analysis based on nuclear single nucleotide polymorphisms (SNPs) obtained from genome skimming data. Based on the phylogenetic framework, we inferred the possible evolutionary patterns of six key morphological characters widely used in its previous taxonomy. KEY RESULTS: The genus Kaschgaria was revealed to be nested in Artemisia with strong support. A well-resolved phylogeny of Artemisia consisting of eight highly supported clades was recovered, two of which were identified for the first time. Most of the previously recognized subgenera were not supported as monophyletic. Evolutionary inferences based on the six morphological characters showed that different states of these characters originated independently more than once. CONCLUSIONS: The circumscription of Artemisia is enlarged to include the genus Kaschgaria. The morphological characters traditionally used for the infrageneric taxonomy of Artemisia do not match the new phylogenetic tree. They experienced a more complex evolutionary history than previously thought. We propose a revised infrageneric taxonomy of the newly circumscribed Artemisia, with eight recognized subgenera to accommodate the new results.

Integrative Taxonomy of Nitraria (Nitrariaceae), Description of the New Enigmatic Species and Key to All Currently Known Species.

A new species, Nitraria iliensis sp. nov., is described from the Ili basin, Almaty region, Kazakhstan. It belongs to section Nitraria ser. Sibiricae and is morphologically similar to N. sibirica Pall. An integrative taxonomic approach based on molecular, biochemical and morphological analyses, along with palynological data, was used to delimit this new species. The studied species of the genus are illustrated, and photographs of authentic specimens of the new species, as well as a distribution map of the new species and segregate taxa, are provided. Morphological characters were investigated, more important traits for identification were found, and a new key to distinguish between all species of the genus was prepared.

The Influence of Solid and Liquid Systems In Vitro on the Growth and Biosynthetic Characteristics of Microshoot Culture of Spiraea betulifolia ssp. aemiliana.

The paper focuses on the growth dynamics and biosynthetic characteristics of the microshoot culture of Spiraea betulifolia ssp. aemiliana obtained in vitro in agar-solidified and liquid media. Microshoots cultured in either type of media showed similar growth dynamics. The most active culture growth was observed from day 35 to day 60. A comparative analysis of the contents of flavonoids and phenol carboxylic acids showed a higher level of phenol carboxylic acids (5.3-6.84%) and a stronger 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity (half-maximal inhibitory concentration: 341 µg/mL) in S. betulifolia ssp. aemiliana microshoots grown in the liquid medium compared to the microshoots cultured in the solid medium. The flavonoid content of the cultured microshoot did not depend on the consistency of the medium. High-performance liquid chromatography (HPLC) was employed to study the profile and levels of phenolic compounds in microshoots, intact plants, and ex vitro-acclimated S. betulifolia ssp. aemiliana plants. The concentration of kaempferol glycosides was found to be higher in microshoots (1.33% in the solid medium, 1.06% in the liquid medium) compared to intact plants and ex vitro-acclimated plants. Thus, the microshoots of S. betulifolia ssp. aemiliana cultured in the liquid medium rapidly increase their biomass and are an inexpensive promising source of biologically active antioxidant substances, mainly phenol carboxylic acids and kaempferol glycosides.

Phylogeny and biogeography of Tiliacoreae (Menispermaceae), a tribe restricted to tropical rainforests.

BACKGROUND AND AIMS: Modern tropical rainforests house the highest biodiversity of Earth's terrestrial biomes and are distributed in three low-latitude areas. However, the biogeographical patterns and processes underlying the distribution of biodiversity among these three areas are still poorly known. Here, we used Tiliacoreae, a tribe of pantropical lianas with a high level of regional endemism, to provide new insights into the biogeographical relationships of tropical rainforests among different continents. METHODS: Based on seven plastid and two nuclear DNA regions, we reconstructed a phylogeny for Tiliacoreae with the most comprehensive sampling ever. Within the phylogenetic framework, we then estimated divergence times and investigated the spatiotemporal evolution of the tribe. KEY RESULTS: The monophyletic Tiliacoreae contain three major clades, which correspond to Neotropical, Afrotropical and Indo-Malesian/Australasian areas, respectively. Both Albertisia and Anisocycla are not monophyletic. The most recent common ancestor of Tiliacoreae occurred in Indo-Malesia, the Afrotropics and Neotropics in the early Eocene, then rapidly diverged into three major clades between 48 and 46 Ma. Three dispersals from Indo-Malesia to Australasia were inferred, one in the middle Eocene and two in the late Oligocene-late Miocene, and two dispersals from the Afrotropics to Indo-Malesia occurred in the late Eocene-Oligocene. CONCLUSIONS: The three main clades of Anisocycla correspond to three distinct genera [i.e. Anisocycla sensu stricto and two new genera (Georgesia and Macrophragma)]. Epinetrum is a member of Albertisia. Our findings highlight that sea-level fluctuations and climate changes in the Cenozoic have played important roles in shaping the current distribution and endemism of Tiliacoreae, hence contributing to the knowledge on the historical biogeography of tropical rainforests on a global scale.

LC-HRMS for the Identification of Quercetin and Its Derivatives in Spiraea hypericifolia (Rosaceae) and Anatomical Features of Its Leaves.

Spiraea hypericifolia L. is affiliated with the section Chamaedryon Ser. of the genus Spiraea L. (Rosaceae). Similar to many other Spiraea species, S. hypericifolia most often accumulates flavonols among other flavonoids, in particular quercetin and its derivatives. An ethanol-water extract from the aerial part of S. hypericifolia collected in the vicinity of the Ilyichovo settlement (Krasnoyarsk Krai, Russia) was analyzed by liquid chromatography with high-resolution mass spectrometry. Primary and secondary metabolites were found in the extract; structural interpretation consistent with quercetin and its derivatives was proposed for 10 of them. Major compounds were various glycosides of quercetin containing glucose (four compounds), galactose (one compound), xylose (two compounds), arabinose (one compound), or rutinose (one compound) as a carbohydrate residue. Isorhamnetin and 3-O-methylquercetin-3'-O-ß-D-glucopyranoside were identified among methyl-containing compounds. The latter compound and reynoutrin, rhamnetin-3-O-ß-D-xylopyranosyl-ß-D-glucopyranoside, and quercetin-3-O-(6″-O-malonyl)-ß-D-glucoside have not been previously found in S. hypericifolia. Data on the presence of quercetin and its derivatives in the extract of S. hypericifolia expand the understanding of the possible practical use of this plant. In addition, the microscopic features of S. hypericifolia leaves were studied. The diagnostic features of the leaf blade necessary for the authentication of raw materials were revealed: straight-walled epidermis cells, stomata located on both sides of the leaf blade (amphistomatic type), two types of trichomes, and wrinkled cuticula with nodi. The main anatomical diagnostic features of the leaves of S. hypericifolia were determined, which makes it possible to assess the authenticity of the raw material.

A complete genus-level phylogeny reveals the Cretaceous biogeographic diversification of the poppy family.

Angiosperms, a trigger for the Cretaceous Terrestrial Revolution (KTR), underwent a rapid expansion and occupied all the environments during the Mid-Upper Cretaceous. Yet, Cretaceous biogeographic patterns and processes underlying the distribution of angiosperm diversity in the Northern Hemisphere are still poorly known. Here, we elucidated the biogeographic diversification of the angiosperm family Papaveraceae, an ancient Northern Hemisphere clade characterized by poor dispersal ability and high level of regional endemism. Based on both plastome and multi-locus datasets, we reconstructed a robust time-calibrated phylogeny that includes all currently recognized 45 genera of this family. Within the time-calibrated phylogenetic framework, we conducted 72 biogeographic analyses by testing the sensitivity of uncertainties of area delimitation, maxarea constraints, and the parameters of the model, i.e., j (describing jump-dispersal events) and w (modifying dispersal multiplier matrices), to ancestral range estimations. We also inferred ancestral habitat and ecological niches. Phylogenetic analyses strongly support Papaveraceae as monophyletic. Pteridophylloideae is strongly supported as sister to Hypecoideae-Fumarioideae. Our results indicate that the j parameter and number of predefined areas strongly affect ancestral range estimates, generating questionable ancestral ranges, whereas maxarea constraint and w parameter have no effect and improve model fit. After accounting for these uncertainties, our results indicate that Papaveraceae differentiated in Asian wet forests during the Lower Cretaceous and subsequently occupied the Asian and western North American arid and open areas. Three dispersals from Asia to western North America via the Bering land bridge occurred in the Mid-Upper Cretaceous, largely in agreement with the KTR. Habitat shift and ecological niche divergence resulted in the subsequent disjunctions between Asia and western North America. These findings suggest that the interplay of range expansion and niche divergence-driven vicariance might have shaped Cretaceous biogeographic patterns of angiosperms with Papaveraceae-like ecological requirements and dispersal abilities in the Northern Hemisphere, hence contributing to the knowledge on the geographic expansion of angiosperms during the KTR.

Karyotypes and Physical Mapping of Ribosomal DNA with Oligo-Probes in Eranthis sect. Eranthis (Ranunculaceae).

A comparative karyotype analysis of four species of yellow-flowered Eranthis sect. Eranthis, i.e., E. bulgarica, E. cilicica, E. hyemalis, and E. longistipitata from different areas, has been carried out for the first time. All the studied specimens had somatic chromosome number 2n = 16 with basic chromosome number x = 8. Karyotypes of the investigated plants included five pairs of metacentric chromosomes and three pairs of submetacentric/subtelocentric chromosomes. The chromosome sets of the investigated species differ mainly in the ratio of submetacentric/subtelocentric chromosomes, their relative lengths, and arm ratios. A new oligonucleotide probe was developed and tested to detect 45S rDNA clusters. Using this probe and an oligonucleotide probe to 5S rDNA, 45S and 5S rDNA clusters were localized for the first time on chromosomes of E. cilicica, E. hyemalis, and E. longistipitata. Major 45S rDNA clusters were identified on satellite chromosomes in all the species; in E. cilicica, minor clusters were also identified in the terminal regions of one metacentric chromosome pair. The number and distribution of 5S rDNA clusters is more specific. In E. cilicica, two major clusters were identified in the pericentromeric region of a pair of metacentric chromosomes. Two major clusters in the pericentromeric region of a pair of submetacentric chromosomes and two major clusters in the interstitial region of a pair of metacentric chromosomes were observed in E. longistipitata. E. hyemalis has many clusters of different sizes, localized mainly in the pericentromeric regions. Summarizing new data on the karyotype structure of E. sect. Eranthis and previously obtained data on E. sect. Shibateranthis allowed conclusions to be formed about the clear interspecific karyological differences of the genus Eranthis.

Phylogenomics, plastome degradation and mycoheterotrophy evolution of Neottieae (Orchidaceae), with emphasis on the systematic position and Loess Plateau-Changbai Mountains disjunction of Diplandrorchis.

BACKGROUND: Mycoheterotrophy is a unique survival strategy adapted to dense forests and has attracted biologists' attention for centuries. However, its evolutionary origin and related plastome degradation are poorly understood. The tribe Neottieae contains various nutrition types, i.e., autotrophy, mixotrophy, and mycoheterotrophy. Here, we present a comprehensive phylogenetic analysis of the tribe based on plastome and nuclear ITS data. We inferred the evolutionary shift of nutrition types, constructed the patterns of plastome degradation, and estimated divergence times and ancestral ranges. We also used an integration of molecular dating and ecological niche modeling methods to investigate the disjunction between the Loess Plateau and Changbai Mountains in Diplandrorchis, a mycoheterotrophic genus endemic to China that was included in a molecular phylogenetic study for the first time. RESULTS: Diplandrorchis was imbedded within Neottia and formed a clade with four mycoheterotrophic species. Autotrophy is the ancestral state in Neottieae, mixotrophy independently originated at least five times, and three shifts from mixotrophy to mycoheterotrophy independently occurred. The five mixotrophic lineages possess all plastid genes or lost partial/all ndh genes, whereas each of the three mycoheterotroph lineages has a highly reduced plastome: one lost part of its ndh genes and a few photosynthesis-related genes, and the other two lost almost all ndh, photosynthesis-related, rpo, and atp genes. These three mycoheterotrophic lineages originated at about 26.40 Ma, 25.84 Ma, and 9.22 Ma, respectively. Diplandrorchis had presumably a wide range in the Pliocene and migrated southward in the Pleistocene. CONCLUSIONS: The Pleistocene climatic fluctuations and the resultant migration resulted in the Loess Plateau-Changbai Mountains disjunction of Diplandrorchis. In the evolution of mycoheterotrophic lineages, the loss of plastid-encoded genes and plastome degradation are staged and irreversible, constraining mycoheterotrophs to inhabit understories with low light levels. Accordingly, the rise of local forests might have promoted the origin of conditions in which mycoheterotrophy is advantageous.

Organization, Phylogenetic Marker Exploitation, and Gene Evolution in the Plastome of Thalictrum (Ranunculaceae).

Thalictrum is a phylogenetically and economically important genus in the family Ranunculaceae, but is also regarded as one of the most challengingly difficult in plants for resolving the taxonomical and phylogenetical relationships of constituent taxa within this genus. Here, we sequenced the complete plastid genomes of two Thalictrum species using Illumina sequencing technology via de novo assembly. The two Thalictrum plastomes exhibited circular and typical quadripartite structure that was rather conserved in overall structure and the synteny of gene order. By updating the previously reported plastome annotation of other nine Thalictrum species, we found that the expansion or contraction of the inverted repeat region affect the boundary of the single-copy regions in Thalictrum plastome. We identified eight highly variable noncoding regions-infA-rps8, ccsA-ndhD, trnSUGA-psbZ, trnHGUG-psbA, rpl16-rps3, ndhG-ndhI, ndhD-psaC, and ndhJ-ndhK-that can be further used for molecular identification, phylogenetic, and phylogeographic in different species. Selective pressure and codon usage bias of all the plastid coding genes were also analyzed for the 11 species. Phylogenetic relationships showed Thalictrum is monophyly and divided into two major clades based on 11 Thalictrum plastomes. The availability of these plastomes offers valuable genetic information for accurate identification of species and taxonomy, phylogenetic resolution, and evolutionary studies of Thalictrum, and should assist with exploration and utilization of Thalictrum plants.

Optimization of Biomass Accumulation and Production of Phenolic Compounds in Callus Cultures of Rhodiola rosea L. Using Design of Experiments.

Rhodiola rosea L. is a valuable medicinal plant with adaptogenic, neuroprotective, antitumor, cardioprotective, and antidepressant effects. In this study, design of experiments methodology was employed to analyze and optimize the interacting effects of mineral compounds (concentration of NO3- and the ratio of NH4+ to K+) and two plant growth regulators [total 6-benzylaminopurine (BAP) and α-naphthylacetic acid (NAA) concentration and the ratio of BAP to NAA] on the growth and the production of total phenolic compounds (TPCs) in R. rosea calluses. The overall effect of the model was highly significant (p < 0.0001), indicating that NH4+, K+, NO3-, BAP, and NAA significantly affected growth. The best callus growth (703%) and the highest production of TPCs (75.17 mg/g) were achieved at an NH4+/K+ ratio of 0.33 and BAP/NAA of 0.33, provided that the concentration of plant growth regulators was 30 µM and that of NO3- was ≤40 mM. According to high-performance liquid chromatography analyses of aerial parts (leaves and stems), in vitro seedlings and callus cultures of R. rosea contain no detectable rosarin, rosavin, rosin, and cinnamyl alcohol. This is the first report on the creation of an experiment for the significant improvement of biomass accumulation and TPC production in callus cultures of R. rosea.

Immigration dynamics of tropical and subtropical Southeast Asian limestone karst floras.

Ex situ origins and dispersal of taxa have played important roles in the assembly of island-like biodiversity hotspots. Insular limestone karsts in Southeast Asia are hotspots of biodiversity and endemism, but the immigration processes of their unique floras are still poorly known. Here, we used Gesneriaceae as a proxy to investigate the immigration dynamics of tropical and subtropical Southeast Asian karst floras. We present the most comprehensive phylogenetic analysis of the Old World gesneriads to date based on twelve loci. By estimating divergence times and reconstructing ancestral states (habitat, soil type and range), we found that immigration into subtropical Southeast Asian karst floras first occurred in the Early Miocene, with two peaks in the Early-Middle Miocene and the Pliocene-Early Pleistocene, whereas immigration into tropical Southeast Asian karsts initiated in the Late Eocene, with two peaks in the Late Oligocene and the Late Miocene. We also discover that Southeast Asian karst biodiversity comprises immigrant pre-adapted lineages and descendants from local acid soil ancestors, although niche shift from acid soil to karst in tropical Southeast Asian islands was lacking. This study advances our understanding of the historical assembly of Southeast Asian karst floras.