A new species and a replacement name in Cynanchum (Apocynaceae, Asclepiadeae) from China.

Cynanchumpingtaoi S.Jin Zeng, G.D.Tang & Miao Liao, sp. nov. (Apocynaceae) from Yunnan Province, China, is described and illustrated based on morphological and molecular evidence. Its deeply cordate to reniform leaves and campanulate, large flowers show that it is a member of former Raphistemma Wall., which has been included in Cynanchum L.. It is different from all former Raphistemma species by the broadly ovate corolla lobes, purple-red corolla and connivent corona tip slightly exceeding the corolla throat. Meanwhile, Cynanchumlonghushanense G.D.Tang & Miao Liao, nom. nov. is proposed as replacement name for Raphistemmabrevipedunculatum Y.Wan, which was considered a synonym of Cynanchumhooperianum (Blume) Liede & Khanum but is here reinstated as a distinct species because of significant morphological differences.

Nuclear phylogenomics of Asteraceae with increased sampling provides new insights into convergent morphological and molecular evolution.

Convergent morphological evolution is widespread in flowering plants, and understanding this phenomenon relies on well-resolved phylogenies. Nuclear phylogenetic reconstruction using transcriptome datasets has been successful in various angiosperm groups, but it is limited to taxa with available fresh materials. Asteraceae, which are one of the two largest angiosperm families and are important for both ecosystems and human livelihood, show multiple examples of convergent evolution. Nuclear Asteraceae phylogenies have resolved relationships among most subfamilies and many tribes, but many phylogenetic and evolutionary questions regarding subtribes and genera remain, owing to limited sampling. Here, we increased the sampling for Asteraceae phylogenetic reconstruction using transcriptomes and genome-skimming datasets and produced nuclear phylogenetic trees with 706 species representing two-thirds of recognized subtribes. Ancestral character reconstruction supports multiple convergent evolutionary events in Asteraceae, with gains and losses of bilateral floral symmetry correlated with diversification of some subfamilies and smaller groups, respectively. Presence of the calyx-related pappus may have been especially important for the success of some subtribes and genera. Molecular evolutionary analyses support the likely contribution of duplications of MADS-box and TCP floral regulatory genes to innovations in floral morphology, including capitulum inflorescences and bilaterally symmetric flowers, potentially promoting the diversification of Asteraceae. Subsequent divergences and reductions in CYC2 gene expression are related to the gain and loss of zygomorphic flowers. This phylogenomic work with greater taxon sampling through inclusion of genome-skimming datasets reveals the feasibility of expanded evolutionary analyses using DNA samples for understanding convergent evolution.

A taxonomic revision of Cynanchumthesioides (Apocynaceae) with two new synonyms.

Cynanchumthesioides, a species widely distributed in north-eastern Asia, is revised to include two new synonyms: Vincetoxicumsibiricumf.linearifolium, described from Shandong, China in 1877, but long neglected and Cynanchumgobicum, previously believed to be endemic to Mongolia. Typification for C.thesioides and all its synonyms is given, including lectotypification of V.sibiricumvar.australe and V.sibiricumf.linearifolium. An updated description, three figures showing the diverse habitats, habits and variation in morphological characters, and a general distribution map are also provided.

Nuclear phylogeny and insights into whole-genome duplications and reproductive development of Solanaceae plants.

Solanaceae, the nightshade family, have ∼2700 species, including the important crops potato and tomato, ornamentals, and medicinal plants. Several sequenced Solanaceae genomes show evidence for whole-genome duplication (WGD), providing an excellent opportunity to investigate WGD and its impacts. Here, we generated 93 transcriptomes/genomes and combined them with 87 public datasets, for a total of 180 Solanaceae species representing all four subfamilies and 14 of 15 tribes. Nearly 1700 nuclear genes from these transcriptomic/genomic datasets were used to reconstruct a highly resolved Solanaceae phylogenetic tree with six major clades. The Solanaceae tree supports four previously recognized subfamilies (Goetzeioideae, Cestroideae, Nicotianoideae, and Solanoideae) and the designation of three other subfamilies (Schizanthoideae, Schwenckioideae, and Petunioideae), with the placement of several previously unassigned genera. We placed a Solanaceae-specific whole-genome triplication (WGT1) at ∼81 million years ago (mya), before the divergence of Schizanthoideae from other Solanaceae subfamilies at ∼73 mya. In addition, we detected two gene duplication bursts (GDBs) supporting proposed WGD events and four other GDBs. An investigation of the evolutionary histories of homologs of carpel and fruit developmental genes in 14 gene (sub)families revealed that 21 gene clades have retained gene duplicates. These were likely generated by the Solanaceae WGT1 and may have promoted fleshy fruit development. This study presents a well-resolved Solanaceae phylogeny and a new perspective on retained gene duplicates and carpel/fruit development, providing an improved understanding of Solanaceae evolution.

Evolution of 101 Apocynaceae plastomes and phylogenetic implications.

Apocynaceae are one of the ten species-richest angiosperm families. However, the backbone phylogeny of the family is yet less well supported, and the evolution of plastome structure has not been thoroughly studied for the whole family. Herein, a total of 101 complete plastomes including 35 newly sequenced, 24 reassembled from public raw data and the rest from the NCBI GenBank database, representing 26 of 27 tribes of Apocynaceae, were used for comparative plastome analysis. Phylogenetic analyses were conducted using a combined plastid data matrix of 77 protein-coding genes from 162 taxa, encompassing all tribes and 41 of 49 subtribes of Apocynaceae. Plastome lengths ranged from 150,897 bp in Apocynum venetum to 178,616 bp in Hoya exilis. Six types of boundaries between the inverted repeat (IR) regions and single copy (SC) regions were identified. Different sizes of IR expansion were found in three lineages, including Alyxieae, Ceropegieae and Marsdenieae, suggesting multiple expansion events of the IRs over the SC regions in Apocynaceae. The IR regions of Marsdenieae evolved in two ways: expansion towards the large single copy (LSC) region in Lygisma + Stephanotis + Ruehssia + Gymnema (Cosmopolitan clade), and expansion towards both LSC and small single copy (SSC) region in Dischidia-Hoya alliance and Marsdenia (Asia-Pacific clade). Six coding genes and five non-coding regions were identified as highly variable, including accD, ccsA-ndhD, clpP, matK, ndhF, ndhG-ndhI, trnG(GCC)-trnfM(CAU), trnH(GUG)-psbA, trnY(GUA)-trnE(UUC), ycf1, and ycf2. Maximum likelihood and Bayesian phylogenetic analyses resulted in nearly identical tree topologies and produced a well-resolved backbone comprising 15 consecutive dichotomies that subdivided Apocynaceae into 15 clades. The subfamily Periplocoideae were embedded in the Apocynoid grade and were sister to the Echiteae-Odontadenieae-Mesechiteae clade with high support values. Three tribes (Melodineae, Vinceae, and Willughbeieae), the subtribe Amphineuriinae, and four genera (Beaumontia, Ceropegia, Hoya, and Stephanotis) were not resolved as monophyletic. Our work sheds light on the backbone phylogenetic relationships in the family Apocynaceae and offers insights into the evolution of Apocynaceae plastomes using the most densely sampled plastome dataset to date.

Comparative and phylogenetic analyses of six Kenya Polystachya (Orchidaceae) species based on the complete chloroplast genome sequences.

BACKGROUND: Polystachya Hook. is a large pantropical orchid genus (c. 240 species) distributed in Africa, southern Asia and the Americas, with the center of diversity in Africa. Previous studies on species of this genus have not obtained the complete chloroplast genomes, structures and variations. Additionally, the phylogenetic position of the genus in the Orchidaceae is still controversial and uncertain. Therefore, in this study, we sequenced the complete plastomes of six Kenya Polystachya species based on genome skimming, subjected them to comparative genomic analysis, and reconstructed the phylogenetic relationships with other Orchidaceae species. RESULTS: The results exhibited that the chloroplast genomes had a typical quadripartite structure with conserved genome arrangement and moderate divergence. The plastomes of the six Polystachya species ranged from 145,484 bp to 149,274 bp in length and had an almost similar GC content of 36.9-37.0%. Gene annotation revealed 106-109 single-copy genes. In addition, 19 genes are duplicated in the inverted regions, and 16 genes each possessd one or more introns. Although no large structural variations were observed among the Polystachya plastomes, about 1 kb inversion was found in Polystachya modesta and all 11 ndh genes in the Polystachya plastomes were lost or pseudogenized. Comparative analysis of the overall sequence identity among six complete chloroplast genomes confirmed that for both coding and non-coding regions in Polystachya, SC regions exhibit higher sequence variation than IRs. Furthermore, there were various amplifications in the IR regions among the six Polystachya species. Most of the protein-coding genes of these species had a high degree of codon preference. We screened out SSRs and found seven relatively highly variable loci. Moreover, 13 genes were discovered with significant positive selection. Phylogenetic analysis showed that the six Polystachya species formed a monophyletic clade and were more closely related to the tribe Vandeae. Phylogenetic relationships of the family Orchidaceae inferred from the 85 chloroplast genome sequences were generally consistent with previous studies and robust. CONCLUSIONS: Our study is the initial report of the complete chloroplast genomes of the six Polystachya species, elucidates the structural characteristics of the chloroplast genome of Polystachya, and filters out highly variable sequences that can contribute to the development of DNA markers for use in the study of genetic variability and evolutionary studies in Polystachya. In addition, the phylogenetic results strongly support that the genus of Polystachya is a part of the tribe Vandeae.

Phylotranscriptomic insights into Asteraceae diversity, polyploidy, and morphological innovation.

Biodiversity is not evenly distributed among related groups, raising questions about the factors contributing to such disparities. The sunflower family (Asteraceae, >26,000 species) is among the largest and most diverse plant families, but its species diversity is concentrated in a few subfamilies, providing an opportunity to study the factors affecting biodiversity. Phylotranscriptomic analyses here of 244 transcriptomes and genomes produced a phylogeny with strong support for the monophyly of Asteraceae and the monophyly of most subfamilies and tribes. This phylogeny provides a reference for detecting changes in diversification rates and possible factors affecting Asteraceae diversity, which include global climate shifts, whole-genome duplications (WGDs), and morphological evolution. The origin of Asteraceae was estimated at ~83 Mya, with most subfamilies having diverged before the Cretaceous-Paleocene boundary. Phylotranscriptomic analyses supported the existence of 41 WGDs in Asteraceae. Changes to herbaceousness and capitulescence with multiple flower-like capitula, often with distinct florets and scaly pappus/receptacular bracts, are associated with multiple upshifts in diversification rate. WGDs might have contributed to the survival of early Asteraceae by providing new genetic materials to support morphological transitions. The resulting competitive advantage for adapting to different niches would have increased biodiversity in Asteraceae.

Analysis of the Complete Plastomes of 31 Species of Hoya Group: Insights Into Their Comparative Genomics and Phylogenetic Relationships.

Hoya is a genus in Apocynaceae-Asclepiadoideae, known for its showy wax flowers, making it a popular ornamental plant. However, phylogenetic relationships among most Hoya species are not yet fully resolved. In this study, we sequenced 31 plastomes of Hoya group species using genome skimming data and carried out multiple analyses to understand genome variation to resolve the phylogenetic positions of some newly sequenced Chinese endemic species. We also screened possible hotspots, trnT-trnL-trnF, psba-trnH, and trnG-UCC, ndhF, ycf1, matK, rps16, and accD genes that could be used as molecular markers for DNA barcoding and species identification. Using maximum likelihood (ML) and Bayesian Inference (BI), a species phylogeny was constructed. The newly assembled plastomes genomes showed the quasi-tripartite structure characteristic for Hoya and Dischidia with a reduced small single copy (SSC) and extremely enlarged inverted repeats (IR). The lengths ranged from 175,404 bp in Hoya lacunosa to 179,069 bp in H. ariadna. The large single copy (LSC) regions ranged from 80,795 bp (Hoya liangii) to 92,072 bp (Hoya_sp2_ZCF6006). The massively expanded IR regions were relatively conserved in length, with the small single-copy region reduced to a single gene, ndhF. We identified 235 long dispersed repeats (LDRs) and ten highly divergent hotspots in the 31 Hoya plastomes, which can be used as DNA barcodes for species identification. The phylogeny supports Clemensiella as a distinct genus. Hoya ignorata is resolved as a relative to Clade VI species. This study discloses the advantages of using Plastome genome data to study phylogenetic relationships.

Asterid Phylogenomics/Phylotranscriptomics Uncover Morphological Evolutionary Histories and Support Phylogenetic Placement for Numerous Whole-Genome Duplications.

Asterids are one of the most successful angiosperm lineages, exhibiting extensive morphological diversity and including a number of important crops. Despite their biological prominence and value to humans, the deep asterid phylogeny has not been fully resolved, and the evolutionary landscape underlying their radiation remains unknown. To resolve the asterid phylogeny, we sequenced 213 transcriptomes/genomes and combined them with other data sets, representing all accepted orders and nearly all families of asterids. We show fully supported monophyly of asterids, Berberidopsidales as sister to asterids, monophyly of all orders except Icacinales, Aquifoliales, and Bruniales, and monophyly of all families except Icacinaceae and Ehretiaceae. Novel taxon placements benefited from the expanded sampling with living collections from botanical gardens, resolving hitherto uncertain relationships. The remaining ambiguous placements here are likely due to limited sampling and could be addressed in the future with relevant additional taxa. Using our well-resolved phylogeny as reference, divergence time estimates support an Aptian (Early Cretaceous) origin of asterids and the origin of all orders before the Cretaceous-Paleogene boundary. Ancestral state reconstruction at the family level suggests that the asterid ancestor was a woody terrestrial plant with simple leaves, bisexual, and actinomorphic flowers with free petals and free anthers, a superior ovary with a style, and drupaceous fruits. Whole-genome duplication (WGD) analyses provide strong evidence for 33 WGDs in asterids and one in Berberidopsidales, including four suprafamilial and seven familial/subfamilial WGDs. Our results advance the understanding of asterid phylogeny and provide numerous novel evolutionary insights into their diversification and morphological evolution.

Phylogeny and biogeography of Pachygoneae (Menispermaceae), with consideration of the boreotropical flora hypothesis and resurrection of the genera Cebatha and Nephroia.

The tribe Pachygoneae consists of four genera with about 40 species, primarily distributed in tropical and subtropical Asia and America, also in Australasia and Africa. This tribe presents an ideal model to investigate the origin of the tropical and subtropical amphi-Pacific disjunction pattern. More specifically, it allows us to test whether the tropical lineages diverged earlier than the subtropical ones during the fragmentation of the boreotropical flora. In this study, we reconstructed the phylogeny of Pachygoneae using five plastid (rbcL, atpB, matK, ndhF, trnL-F) and one nuclear (26S rDNA) DNA regions. Our results indicate that Pachygoneae is not monophyletic unless Cocculus pendulus and Cocculus balfourii are excluded. We resurrected the genus Cebatha to include these two species and established a new tribe for this genus. Within Pachygoneae, the species of Cocculus are distributed in three different clades, among which two are recognized as two distinct genera, Cocculus s.str. and Nephroia resurrected, and one species is transferred into Pachygone. Our molecular dating and ancestral area reconstruction analyses suggest that Pachygoneae began to diversify in tropical Asia around the early-middle Eocene boundary (c. 48 Ma) and expanded into the New World by c. 44 Ma. In the New World, tropical Hyperbaena originated in the late Eocene (c. 40 Ma), whereas the subtropical Cocculus carolinus and Cocculus diversifolius originated later, in the early Oligocene (c. 32  Ma). These two timings correspond with the two climatic cooling intervals, which suggests that the formation and breakup of the boreotropical floral may have been responsible for the amphi-Pacific disjunct distribution within Pachygoneae. One overland migration event from Asia into Australasia appears to have occurred in the early to late Miocene.

A well-resolved fern nuclear phylogeny reveals the evolution history of numerous transcription factor families.

Ferns account for 80% of nonflowering vascular plant species and are the sister lineage of seed plants. Recent molecular phylogenetics have greatly advanced understanding of fern tree of life, but relationships among some major lineages remain unclear. To better resolve the phylogenetic relationships of ferns, we generated transcriptomes from 125 ferns and two lycophytes, with three additional public datasets, to represent all 11 orders and 85% of families of ferns. Our nuclear phylogeny provides strong supports for the monophyly of all four subclasses and nearly all orders and families, and for relationships among these lineages. The only exception is Gleicheniales, which was highly supported as being paraphyletic with Dipteridaceae sister to a clade with Gleicheniaceae + Hymenophyllales. In addition, new and strongly supported phylogenetic relationships are found for suborders and families in Polypodiales. We provide the first dated fern phylogenomic tree using many nuclear genes from a large majority of families, with an estimate for separation of the ancestors of ferns and seed plants in early Devonian at ∼400 Mya and subsequent gradual divergences of fern orders from ∼380 to 200 Mya. Moreover, the newly obtained fern phylogeny provides a framework for gene family analyses, which indicate that the vast majority of transcription factor families found in seed plants were already present in the common ancestor of extant vascular plants. In addition, fern transcription factor genes show similar duplication patterns to those in seed plants, with some showing stable copy number and others displaying independent expansions in both ferns and seed plants. This study provides a robust phylogenetic and gene family evolution framework, as well as rich molecular resources for understanding the morphological and functional evolution in ferns.

Production of D-alanine from DL-alanine using immobilized cells of Bacillus subtilis HLZ-68.

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce D-alanine from DL-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher L-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on L-alanine consumption were examined. Maximum L-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of DL-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete L-alanine degradation within 60 h, leaving 185 g of D-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. D-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted D-alanine was 99.1 and 99.6%, respectively.

Multiple Polyploidization Events across Asteraceae with Two Nested Events in the Early History Revealed by Nuclear Phylogenomics.

Biodiversity results from multiple evolutionary mechanisms, including genetic variation and natural selection. Whole-genome duplications (WGDs), or polyploidizations, provide opportunities for large-scale genetic modifications. Many evolutionarily successful lineages, including angiosperms and vertebrates, are ancient polyploids, suggesting that WGDs are a driving force in evolution. However, this hypothesis is challenged by the observed lower speciation and higher extinction rates of recently formed polyploids than diploids. Asteraceae includes about 10% of angiosperm species, is thus undoubtedly one of the most successful lineages and paleopolyploidization was suggested early in this family using a small number of datasets. Here, we used genes from 64 new transcriptome datasets and others to reconstruct a robust Asteraceae phylogeny, covering 73 species from 18 tribes in six subfamilies. We estimated their divergence times and further identified multiple potential ancient WGDs within several tribes and shared by the Heliantheae alliance, core Asteraceae (Asteroideae-Mutisioideae), and also with the sister family Calyceraceae. For two of the WGD events, there were subsequent great increases in biodiversity; the older one proceeded the divergence of at least 10 subfamilies within 10 My, with great variation in morphology and physiology, whereas the other was followed by extremely high species richness in the Heliantheae alliance clade. Our results provide different evidence for several WGDs in Asteraceae and reveal distinct association among WGD events, dramatic changes in environment and species radiations, providing a possible scenario for polyploids to overcome the disadvantages of WGDs and to evolve into lineages with high biodiversity.

Inaccessible Biodiversity on Limestone Cliffs: Aster tianmenshanensis (Asteraceae), a New Critically Endangered Species from China.

Aster tianmenshanensis G. J. Zhang & T. G. Gao, a new species of Asteraceae from southern China is described and illustrated based on evidence from morphology, micromorphology and molecular phylogeny. The new species is superficially similar to Aster salwinensis Onno in having rosettes of spatulate leaves and a solitary, terminal capitulum, but it differs by its glabrous leaf margins, unequal disc floret lobes and 1-seriate pappus. The molecular phylogenetic analysis, based on nuclear sequences ITS, ETS and chloroplast sequence trnL-F, showed that the new species was nested within the genus Aster and formed a well supported clade with Aster verticillatus (Reinw.) Brouillet et al. The new species differs from the latter in having unbranched stems, much larger capitula, unequal disc floret lobes, beakless achenes and persistent pappus. In particular, A. tianmenshanensis has very short stigmatic lines, only ca. 0.18 mm long and less than 1/3 of the length of sterile style tip appendages, remarkably different from its congeners. This type of stigmatic line, as far as we know, has not been found in any other species of Aster. The very short stigmatic lines plus the unequal disc floret lobes imply that the new species may have a very specialized pollination system, which may be a consequence of habitat specialization. The new species grows only on the limestone cliffs of Mt. Tianmen, Hunan Province, at the elevation of 1400 m. It could only be accessed when a plank walkway was built across the cliffs for tourists. As it is known only from an area estimated at less than 10 km2 and a walkway passes through this location, its habitat could be easily disturbed. This species should best be treated as Critically Endangered based on the International Union for Conservation of Nature Red List Categories and Criteria B2a.