Complete Plastid Genomes of Nine Species of Ranunculeae (Ranunculaceae) and Their Phylogenetic Inferences.

The tribe Ranunculeae, Ranunculaceae, comprising 19 genera widely distributed all over the world. Although a large number of Sanger sequencing-based molecular phylogenetic studies have been published, very few studies have been performed on using genomic data to infer phylogenetic relationships within Ranunculeae. In this study, the complete plastid genomes of nine species (eleven samples) from Ceratocephala, Halerpestes, and Ranunculus were de novo assembled using a next-generation sequencing method. Previously published plastomes of Oxygraphis and other related genera of the family were downloaded from GenBank for comparative analysis. The complete plastome of each Ranunculeae species has 112 genes in total, including 78 protein-coding genes, 30 transfer RNA genes, and four ribosomal RNA genes. The plastome structure of Ranunculeae samples is conserved in gene order and arrangement. There are no inverted repeat (IR) region expansions and only one IR contraction was found in the tested samples. This study also compared plastome sequences across all the samples in gene collinearity, codon usage, RNA editing sites, nucleotide variability, simple sequence repeats, and positive selection sites. Phylogeny of the available Ranunculeae species was inferred by the plastome data using maximum-likelihood and Bayesian inference methods, and data partitioning strategies were tested. The phylogenetic relationships were better resolved compared to previous studies based on Sanger sequencing methods, showing the potential value of the plastome data in inferring the phylogeny of the tribe.

Phylogeny and Historical Biogeography of the East Asian Clematis Group, Sect. Tubulosae, Inferred from Phylogenomic Data.

The evolutionary history of Clematis section Tubulosae, an East Asian endemic lineage, has not been comprehensively studied. In this study, we reconstruct the phylogeny of this section with a complete sampling using a phylogenomic approach. The genome skimming method was applied to obtain the complete plastome sequence, the nuclear ribosomal DNA (nrDNA), and the nuclear SNPs data for phylogenetic reconstruction. Using a Bayesian molecular clock approach and ancestral range reconstruction, we reconstruct biogeographical history and discuss the biotic and abiotic factors that may have shaped the distribution patterns of the section. Both nuclear datasets better resolved the phylogeny of the sect. Tubulosae than the plastome sequence. Sect. Tubulosae was resolved as a monophyletic group sister to a clade mainly containing species from the sect. Clematis and sect. Aspidanthera. Within sect. Tubulosae, two major clades were resolved by both nuclear datasets. Two continental taxa, C. heracleifolia and C. tubulosa var. ichangensis, formed one clade. One continental taxon, C. tubulosa, and all the other species from Taiwan island, the Korean peninsula, and the Japanese archipelago formed the other clade. Molecular dating results showed that sect. Tubulosae diverged from its sister clade in the Pliocene, and all the current species diversified during the Pleistocene. Our biogeographical reconstruction suggested that sect. Tubulosae evolved and began species diversification, most likely in mainland China, then dispersed to the Korean peninsula, and then expanded its range through the Japanese archipelago to Taiwan island. Island species diversity may arise through allopatric speciation by vicariance events following the range fragmentation triggered by the climatic oscillation and sea level change during the Pleistocene epoch. Our results highlight the importance of climatic oscillation during the Pleistocene to the spatial-temporal diversification patterns of the sect. Tubulosae.

A phylotranscriptome study using silica gel-dried leaf tissues produces an updated robust phylogeny of Ranunculaceae.

The utility of transcriptome data in plant phylogenetics has gained popularity in recent years. However, because RNA degrades much more easily than DNA, the logistics of obtaining fresh tissues has become a major limiting factor for widely applying this method. Here, we used Ranunculaceae to test whether silica-dried plant tissues could be used for RNA extraction and subsequent phylogenomic studies. We sequenced 27 transcriptomes, 21 from silica gel-dried (SD-samples) and six from liquid nitrogen-preserved (LN-samples) leaf tissues, and downloaded 27 additional transcriptomes from GenBank. Our results showed that although the LN-samples produced slightly better reads than the SD-samples, there were no significant differences in RNA quality and quantity, assembled contig lengths and numbers, and BUSCO comparisons between two treatments. Using these data, we conducted phylogenomic analyses, including concatenated- and coalescent-based phylogenetic reconstruction, molecular dating, coalescent simulation, phylogenetic network estimation, and whole genome duplication (WGD) inference. The resulting phylogeny was consistent with previous studies with higher resolution and statistical support. The 11 core Ranunculaceae tribes grouped into two chromosome type clades (T- and R-types), with high support. Discordance among gene trees is likely due to hybridization and introgression, ancient genetic polymorphism and incomplete lineage sorting. Our results strongly support one ancient hybridization event within the R-type clade and three WGD events in Ranunculales. Evolution of the three Ranunculaceae chromosome types is likely not directly related to WGD events. By clearly resolving the Ranunculaceae phylogeny, we demonstrated that SD-samples can be used for RNA-seq and phylotranscriptomic studies of angiosperms.

Corrigendum: Comparative Analysis of Complete Chloroplast Genomes of 13 Species in Epilobium, Circaea, and Chamaenerion and Insights into Phylogenetic Relationships of Onagraceae.

[This corrects the article DOI: 10.3389/fgene.2021.730495.].

Comparative Analysis of Complete Chloroplast Genomes of 13 Species in Epilobium, Circaea, and Chamaenerion and Insights Into Phylogenetic Relationships of Onagraceae.

The evening primrose family, Onagraceae, is a well defined family of the order Myrtales, comprising 22 genera widely distributed from boreal to tropical areas. In this study, we report and characterize the complete chloroplast genome sequences of 13 species in Circaea, Chamaenerion, and Epilobium using a next-generation sequencing method. We also retrieved chloroplast sequences from two other Onagraceae genera to characterize the chloroplast genome of the family. The complete chloroplast genomes of Onagraceae encoded an identical set of 112 genes (with exclusion of duplication), including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The chloroplast genomes are basically conserved in gene arrangement across the family. However, a large segment of inversion was detected in the large single copy region of all the samples of Oenothera subsect. Oenothera. Two kinds of inverted repeat (IR) region expansion were found in Oenothera, Chamaenerion, and Epilobium samples. We also compared chloroplast genomes across the Onagraceae samples in some features, including nucleotide content, codon usage, RNA editing sites, and simple sequence repeats (SSRs). Phylogeny was inferred by the chloroplast genome data using maximum-likelihood (ML) and Bayesian inference methods. The generic relationship of Onagraceae was well resolved by the complete chloroplast genome sequences, showing potential value in inferring phylogeny within the family. Phylogenetic relationship in Oenothera was better resolved than other densely sampled genera, such as Circaea and Epilobium. Chloroplast genomes of Oenothera subsect. Oenothera, which are biparental inheritated, share a syndrome of characteristics that deviate from primitive pattern of the family, including slightly expanded inverted repeat region, intron loss in clpP, and presence of the inversion.

Natural Hybrid Origin of the Controversial "Species" Clematis × pinnata (Ranunculaceae) Based on Multidisciplinary Evidence.

Interspecific hybridization is common and has often been viewed as a driving force of plant diversity. However, it raises taxonomic problems and thus impacts biodiversity estimation and biological conservation. Although previous molecular phylogenetic studies suggested that interspecific hybridization may be rather common in Clematis, and artificial hybridization has been widely applied to produce new Clematis cultivars for nearly two centuries, the issue of natural hybridization of Clematis has never been addressed in detail. In this study, we tested the hybrid origin of a mesophytic and cold-adapted vine species, Clematis pinnata, which is a rare and taxonomically controversial taxon endemic to northern China. Using field investigations, flow cytometry (FCM), phylogenomic analysis, morphological statistics, and niche modeling, we tested hybrid origin and species status of C. pinnata. The FCM results showed that all the tested species were homoploid (2n = 16). Phylonet and HyDe analyses based on transcriptome data showed the hybrid origins of C. × pinnata from either C. brevicaudata × C. heracleifolia or C. brevicaudata × C. tubulosa. The plastome phylogeny depicted that C. × pinnata in different sampling sites originated by different hybridization events. Morphological analysis showed intermediacy of C. × pinnata between its putative parental species in many qualitative and quantitative characters. Niche modeling results suggested that C. × pinnata had not been adapted to a novel ecological niche independent of its putative parents. These findings demonstrated that plants of C. × pinnata did not formed a self-evolved clade and should not be treated as a species. The present study also suggests that interspecific hybridization is a common mechanism in Clematis to generate diversity and variation, and it may play an important role in the evolution and diversification of this genus. Our study implies that morphological diversity caused by natural hybridization may overstate the real species diversity in Clematis.

An updated phylogenetic and biogeographic analysis based on genome skimming data reveals convergent evolution of shrubby habit in Clematis in the Pliocene and Pleistocene.

Convergent evolution, often viewed as the inevitable outcome of natural selection, has received special attention since the time of Darwin. Clematis is well known for its climbing habit, but it has some shrubby species, known as sect. Fruticella s.l. The shrubby Clematis species are distributed in the dry habitats of Central Asia and adjacent areas showing possible convergent evolution. In this study, we assembled the complete plastome and nuclear ribosomal DNA (nrDNA) sequences of 56 Clematis species, representing most sections and covering most of the shrubby species, to reconstruct their evolutionary histories. Using both maximum likelihood and Bayesian methods, the plastome and nrDNA datasets generated similar, but not identical, phylogenetic relationships, which are better resolved than in previous studies. Then, molecular dating, historical range reconstruction, and character optimization analyses were conducted based on this updated phylogenetic framework. All the morphological characters widely used for taxonomy were shown to have evolved multiple times. Molecular dating inferred that Clematis diverged from its sister in the mid Miocene, and all six major clades of Clematis originated during the late Miocene, with a species radiation during the Pliocene to Pleistocene. The results clearly showed that the shrubby habit evolved independently in four lineages of Clematis in Asia. We also revealed that the shrubby lineages have emerged since the very beginning of Pliocene. Asian monsoon variation in the Pliocene and glacial period fluctuation in the Pleistocene may be the driving forces for the origin and diversification of the shrubby Clematis in Central Asia and adjacent dry areas.

The complete chloroplast genome of a distinctive meadow-rue, Thalictrum foeniculaceum (Ranunculaceae).

Thalictrum foeniculaceum is a morphologically distinctive species in the genus with very reduced filiform compound leaves and big pinkish flowers occurring in dry slopes of northern China. Herein, we report the first complete chloroplast genome sequence of T. foeniculaceum. The chloroplast genome sequence was 155,923 bp in length, with large and small single-copy regions (LSC with 85,323 bp and SSC with 17,628 bp in length) separated by two inverted repeat regions (IRs with 26,486 bp). The total GC content was 38.3%. The complete plastome sequence contained 111 genes, including 77 protein-coding, 30 tRNA, and four rRNA genes. The phylogenetic analysis of Thalictrum based on the complete chloroplast genomes available online was also presented in this study.