Genome copy number predicts extreme evolutionary rate variation in plant mitochondrial DNA.

Nuclear and organellar genomes can evolve at vastly different rates despite occupying the same cell. In most bilaterian animals, mitochondrial DNA (mtDNA) evolves faster than nuclear DNA, whereas this trend is generally reversed in plants. However, in some exceptional angiosperm clades, mtDNA substitution rates have increased up to 5,000-fold compared with closely related lineages. The mechanisms responsible for this acceleration are generally unknown. Because plants rely on homologous recombination to repair mtDNA damage, we hypothesized that mtDNA copy numbers may predict evolutionary rates, as lower copy numbers may provide fewer templates for such repair mechanisms. In support of this hypothesis, we found that copy number explains 47% of the variation in synonymous substitution rates of mtDNA across 60 diverse seed plant species representing ~300 million years of evolution. Copy number was also negatively correlated with mitogenome size, which may be a cause or consequence of mutation rate variation. Both relationships were unique to mtDNA and not observed in plastid DNA. These results suggest that homologous recombinational repair plays a role in driving mtDNA substitution rates in plants and may explain variation in mtDNA evolution more broadly across eukaryotes. Our findings also contribute to broader questions about the relationships between mutation rates, genome size, selection efficiency, and the drift-barrier hypothesis.

Comparative plastome analysis of Arundinelleae (Poaceae, Panicoideae), with implications for phylogenetic relationships and plastome evolution.

BACKGROUND: Arundinelleae is a small tribe within the Poaceae (grass family) possessing a widespread distribution that includes Asia, the Americas, and Africa. Several species of Arundinelleae are used as natural forage, feed, and raw materials for paper. The tribe is taxonomically cumbersome due to a paucity of clear diagnostic morphological characters. There has been scant genetic and genomic research conducted for this group, and as a result the phylogenetic relationships and species boundaries within Arundinelleae are poorly understood. RESULTS: We compared and analyzed 11 plastomes of Arundinelleae, of which seven plastomes were newly sequenced. The plastomes range from 139,629 base pairs (bp) (Garnotia tenella) to 140,943 bp (Arundinella barbinodis), with a standard four-part structure. The average GC content was 38.39%, but varied in different regions of the plastome. In all, 110 genes were annotated, comprising 76 protein-coding genes, 30 tRNA genes, and four rRNA genes. Furthermore, 539 simple sequence repeats, 519 long repeats, and 10 hyper-variable regions were identified from the 11 plastomes of Arundinelleae. A phylogenetic reconstruction of Panicoideae based on 98 plastomes demonstrated the monophyly of Arundinella and Garnotia, but the circumscription of Arundinelleae remains unresolved. CONCLUSION: Complete chloroplast genome sequences can improve phylogenetic resolution relative to single marker approaches, particularly within taxonomically challenging groups. All phylogenetic analyses strongly support the monophyly of Arundinella and Garnotia, respectively, but the monophylly of Arundinelleae was not well supported. The intergeneric phylogenetic relationships within Arundinelleae require clarification, indicating that more data is necessary to resolve generic boundaries and evaluate the monophyly of Arundinelleae. A comprehensive taxonomic revision for the tribe is necessary. In addition, the identified hyper-variable regions could function as molecular markers for clarifying phylogenetic relationships and potentially as barcoding markers for species identification in the future.

Phylogenetic analysis and divergence time estimation of Lycium species in China based on the chloroplast genomes.

BACKGROUND: Lycium is an economically and ecologically important genus of shrubs, consisting of approximately 70 species distributed worldwide, 15 of which are located in China. Despite the economic and ecological importance of Lycium, its phylogeny, interspecific relationships, and evolutionary history remain relatively unknown. In this study, we constructed a phylogeny and estimated divergence time based on the chloroplast genomes (CPGs) of 15 species, including subspecies, of the genus Lycium from China. RESULTS: We sequenced and annotated 15 CPGs in this study. Comparative analysis of these genomes from these Lycium species revealed a typical quadripartite structure, with a total sequence length ranging from 154,890 to 155,677 base pairs (bp). The CPGs was highly conserved and moderately differentiated. Through annotation, we identified a total of 128-132 genes. Analysis of the boundaries of inverted repeat (IR) regions showed consistent positioning: the junctions of the IRb/LSC region were located in rps19 in all Lycium species, IRb/SSC between the ycf1 and ndhF genes, and SSC/IRa within the ycf1 gene. Sequence variation in the SSC region exceeded that in the IR region. We did not detect major expansions or contractions in the IR region or rearrangements or insertions in the CPGs of the 15 Lycium species. Comparative analyses revealed five hotspot regions in the CPG: trnR(UCU), atpF-atpH, ycf3-trnS(GGA), trnS(GGA), and trnL-UAG, which could potentially serve as molecular markers. In addition, phylogenetic tree construction based on the CPG indicated that the 15 Lycium species formed a monophyletic group and were divided into two typical subbranches and three minor branches. Molecular dating suggested that Lycium diverged from its sister genus approximately 17.7 million years ago (Mya) and species diversification within the Lycium species of China primarily occurred during the recent Pliocene epoch. CONCLUSION: The divergence time estimation presented in this study will facilitate future research on Lycium, aid in species differentiation, and facilitate diverse investigations into this economically and ecologically important genus.

Comparative phylogenomic study of East Asian endemic genus, Corchoropsis Siebold & Zucc. (Malvaceae s.l.), based on complete plastome sequences.

BACKGROUND: Endemic plants are key to understanding the evolutionary history and enhancing biodiversity within their unique regions, while also offering significant economic potential. The East Asian endemic genus Corchoropsis Siebold & Zucc., classified within the subfamily Dombeyoideae of Malvaceae s.l., comprises three species. RESULTS: This study characterizes the complete plastid genomes (plastomes) of C. crenata var. crenata Siebold & Zucc. and C. crenata var. hupehensis Pamp., which range from 160,093 to 160,724 bp. These genomes contain 78 plastid protein-coding genes, 30 tRNA, and four rRNA, except for one pseudogene, infA. A total of 316 molecular diagnostic characters (MDCs) specific to Corchoropsis were identified. In addition, 91 to 92 simple sequence repeats (SSRs) in C. crenata var. crenata and 75 in C. crenata var. hupehensis were found. Moreover, 49 long repeats were identified in both the Chinese C. crenata var. crenata and C. crenata var. hupehensis, while 52 were found in the South Korean C. crenata var. crenata. Our phylogenetic analyses, based on 78 plastid protein-coding genes, reveal nine subfamilies within the Malvaceae s.l. with high support values and confirm Corchoropsis as a member of Dombeyoideae. Molecular dating suggests that Corchoropsis originated in the Oligocene, and diverged during the Miocene, influenced by the climate shift at the Eocene-Oligocene boundary. CONCLUSIONS: The research explores the evolutionary relationships between nine subfamilies within the Malvaceae s.l. family, specifically identifying the position of the Corchoropsis in the Dombeyoideae. Utilizing plastome sequences and fossil data, the study establishes that Corchoropsis first appeared during the Eocene and experienced further evolutionary divergence during the Miocene, paralleling the evolutionary patterns observed in other East Asian endemic species.

Characterization of Firmiana danxiaensis plastomes and comparative analysis of Firmiana: insight into its phylogeny and evolution.

BACKGROUND: Firmiana danxiaensis is a critically endangered and ecologically important tree currently only found in four locations in Danxia or Karst habitats in northern Guangdong Province, China. The specialized habitat preference makes it an ideal model species for study of adaptive evolution. Meanwhile, the phylogenetic relationships of F. danxiaensis in four locations under two landforms are unclear. Therefore, we sequenced its complete chloroplast (cp.) genomes and conducted comprehensive interspecific and intrageneric plastome studies. RESULTS: The F. danxiaensis plastomes in four locations showed a typical quadripartite and circular structure that ranged from 160,832 to 161,206 bp in size, with 112 unique genes encoded. Comparative genomics showed that the plastomes of F. danxiaensis were relatively conserved with high similarity of genome organization, gene number, GC content and SSRs. While the genomes revealed higher biased codon preferences in Karst habitat than those in Danxia habitats. Eighteen and 11 divergent hotpots were identified at interspecific and intrageneric levels for species identification and further phylogenetic studies. Seven genes (clpP, accD, ccsA, ndhH, rpl20, rpoC2, and rps4) were under positive selection and may be related to adaptation. Phylogenetic analysis revealed that F. danxiaensis is sister to F. major and F. simplex. However, the interspecific relationships are not consistent with the habitat types. CONCLUSIONS: The characteristics and interspecific relationship of F. danxiaensis plastomes provide new insights into further integration of geographical factors, environmental factors, and genetic variations on the genomic study of F. danxiaensis. Together, our study will contribute to the study of species identification, population genetics, and conservation biology of F. danxiaensis.

Variations and reduction of plastome are associated with the evolution of parasitism in Convolvulaceae.

Parasitic lifestyle can often relax the constraint on the plastome, leading to gene pseudogenization and loss, and resulting in diverse genomic structures and rampant genome degradation. Although several plastomes of parasitic Cuscuta have  been reported, the evolution of parasitism in the family Convolvulaceae which is linked to structural variations and reduction of plastome has not been well investigated. In this study, we assembled and collected 40 plastid genomes belonging to 23 species representing four subgenera of Cuscuta and ten species of autotrophic Convolvulaceae. Our findings revealed nine types of structural variations and six types of inverted repeat (IR) boundary variations in the plastome of Convolvulaceae spp. These structural variations were associated with the shift of parasitic lifestyle, and IR boundary shift, as well as the abundance of long repeats. Overall, the degradation of Cuscuta plastome proceeded gradually, with one clade exhibiting an accelerated degradation rate. We observed five stages of gene loss in Cuscuta, including NAD(P)H complex → PEP complex → Photosynthesis-related → Ribosomal protein subunits → ATP synthase complex. Based on our results, we speculated that the shift of parasitic lifestyle in early divergent time promoted relaxed selection on plastomes, leading to the accumulation of microvariations, which ultimately resulted in the plastome reduction. This study provides new evidence towards a better understanding of plastomic evolution, variation, and reduction in the genus Cuscuta.

Comparative plastome analysis and taxonomic classification of snow lotus species (Saussurea, Asteraceae) in Central Asia and Southern Siberia.

Four species of Saussurea, namely S. involucrata, S. orgaadayi, S. bogedaensis, and S. dorogostaiskii, are known as the "snow lotus," which are used as traditional medicines in China (Xinjiang), Kyrgyzstan, Mongolia, and Russia (Southern Siberia). These species are threatened globally, because of illegal harvesting and climate change. Furthermore, the taxonomic classification and identification of these threatened species remain unclear owing to limited research. The misidentification of medicinal species can sometimes be harmful to health. Therefore, the phylogenetic and genomic features of these species need to be confirmed. In this study, we sequenced five complete chloroplast genomes and seven nuclear ITS regions of four snow lotus species and other Saussurea species. We further explored their genetic variety, selective pressure at the sequence level, and phylogenetic relationships using the chloroplast genome, nuclear partial DNA sequences, and morphological features. Plastome of the snow lotus species has a conserved structure and gene content similar to most Saussurea species. Two intergenic regions (ndhJ-ndhK and ndhD-psaC) show significantly high diversity among chloroplast regions. Thus, ITS and these markers are suitable for identifying snow lotus species. In addition, we characterized 43 simple sequence repeats that may be useful in future population genetic studies. Analysis of the selection signatures identified three genes (rpoA, ndhB, and ycf2) that underwent positive selection. These genes may play important roles in the adaptation of the snow lotus species to alpine environments. S. dorogostaiskii is close to S. baicalensis and exhibits slightly different adaptation from others. The taxonomic position of the snow lotus species, confirmed by morphological and molecular evidence, is as follows: (i) S. involucrata has been excluded from the Mongolian flora due to misidentification as S. orgaadayi or S. bogedaensis for a long time; (ii) S. dorogostaiskii belongs to section Pycnocephala subgenus Saussurea, whereas other the snow lotus species belong to section Amphilaena subgenus Amphilaena; and (iii) S. krasnoborovii is synonymous of S. dorogostaiskii. This study clarified the speciation and lineage diversification of the snow lotus species in Central Asia and Southern Siberia.

Complete plastome genomes of three medicinal heliotropiaceae species: comparative analyses and phylogenetic relationships.

BACKGROUND: Heliotropiaceae is a family of the order Boraginales and has over 450 species. The members of the family Heliotropiaceae have been widely reported to be used in traditional medicine Over time, the classification of Heliotropiaceae has remained uncertain and has moved from family to subfamily, or conversely. RESULTS: In the present study, we sequenced, analyzed, and compared the complete plastomes of Euploca strigosa, Heliotropium arbainense, and Heliotropium longiflorum with the genomes of related taxa. The lengths of the plastomes of E. strigosa, H. arbainense, and H. longiflorum were 155,174 bp, 154,709 bp, and 154,496 bp, respectively. Each plastome consisted of 114 genes: 80 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. The long repeats analysis indicated that reverse, palindromic, complement and forward repeats were all found in the three plastomes. The simple repeats analysis showed that the plastomes of E. strigosa, H. arbainense, and H. longiflorum contained 158, 165, and 151 microsatellites, respectively. The phylogenetic analysis confirmed two major clades in the Boraginales: clade I comprised Boraginaceae, while clade II included Heliotropiaceae, Ehretiaceae, Lennoaceae, and Cordiaceae. Inside the family Heliotropiaceae, E. strigosa is nested within the Heliotropium genus. CONCLUSIONS: This study expands our knowledge of the evolutionary relationships within Heliotropiaceae and offers useful genetic resources.

The complete plastomes of thirteen Libanotis (Apiaceae, Apioideae) plants: comparative and phylogenetic analyses provide insights into the plastome evolution and taxonomy of Libanotis.

BACKGROUND: The genus Libanotis Haller ex Zinn, nom. cons., a contentious member of Apiaceae, encompasses numerous economically and medicinally significant plants, comprising approximately 30 species distributed across Eurasia. Despite many previous taxonomic insights into it, phylogenetic studies of the genus are still lacking. And the establishment of a robust phylogenetic framework remains elusive, impeding advancements and revisions in the taxonomic system for this genus. Plastomes with greater variability in their genetic characteristics hold promise for building a more robust Libanotis phylogeny. RESULTS: During our research, we sequenced, assembled, and annotated complete plastomes for twelve Libanotis species belong to three sections and two closely related taxa. We conducted a comprehensive comparative analysis through totally thirteen Libanotis plastomes for the genus, including an additional plastome that had been published. Our results suggested that Libanotis plastome was highly conserved between different subclades, while the coding regions were more conserved than the non-coding regions, and the IR regions were more conserved than the single copy regions. Nevertheless, eight mutation hotspot regions were identified among plastomes, which can be considered as candidate DNA barcodes for accurate species identification in Libanotis. The phylogenetic analyses generated a robustly framework for Libanotis and revealed that Libanotis was not a monophyletic group and their all three sections were polygenetic. Libanotis schrenkiana was sister to L. sibirica, type species of this genus, but the remainders scattered within Selineae. CONCLUSION: The plastomes of Libanotis exhibited a high degree of conservation and was effective in enhancing the support and resolution of phylogenetic analyses within this genus. Based on evidence from both phylogeny and morphology, we propose the recognition of "Libanotis sensu stricto" and provide taxonomic recommendations for other taxa that previously belonged to Libanotis. In conclusion, our study not only revealed the phylogenetic position and plastid evolution of Libanotis, but also provided new insights into the phylogeny of the family Apiaceae and phylogenetic relationships within the tribe Selineae.

Phylogeny and diversification of genus Sanicula L. (Apiaceae): novel insights from plastid phylogenomic analyses.

BACKGROUND: The genus Sanicula L. is a unique perennial herb that holds important medicinal values. Although the previous studies on Sanicula provided us with a good research basis, its taxonomic system and interspecific relationships have not been satisfactorily resolved, especially for those endemic to China. Moreover, the evolutionary history of this genus also remains inadequately understood. The plastid genomes possessing highly conserved structure and limited evolutionary rate have proved to be an effective tool for studying plant phylogeny and evolution. RESULTS: In the current study, we newly sequenced and assembled fifteen Sanicula complete plastomes. Combined with two previously reported plastomes, we performed comprehensively plastid phylogenomics analyses to gain novel insights into the evolutionary history of this genus. The comparative results indicated that the seventeen plastomes exhibited a high degree of conservation and similarity in terms of their structure, size, GC content, gene order, IR borders, codon bias patterns and SSRs profiles. Such as all of them displayed a typical quadripartite structure, including a large single copy region (LSC: 85,074-86,197 bp), a small single copy region (SSC: 17,047-17,132 bp) separated by a pair of inverted repeat regions (IRs: 26,176-26,334 bp). And the seventeen plastomes had similar IR boundaries and the adjacent genes were identical. The rps19 gene was located at the junction of the LSC/IRa, the IRa/SSC junction region was located between the trnN gene and ndhF gene, the ycf1 gene appeared in the SSC/IRb junction and the IRb/LSC boundary was located between rpl12 gene and trnH gene. Twelve specific mutation hotspots (atpF, cemA, accD, rpl22, rbcL, matK, ycf1, trnH-psbA, ycf4-cemA, rbcL-accD, trnE-trnT and trnG-trnR) were identified that can serve as potential DNA barcodes for species identification within the genus Sanicula. Furthermore, the plastomes data and Internal Transcribed Spacer (ITS) sequences were performed to reconstruct the phylogeny of Sanicula. Although the tree topologies of them were incongruent, both provided strong evidence supporting the monophyly of Saniculoideae and Apioideae. In addition, the sister groups between Saniculoideae and Apioideae were strongly suggested. The Sanicula species involved in this study were clustered into a clade, and the Eryngium species were also clustered together. However, it was clearly observed that the sections of Sanicula involved in the current study were not respectively recovered as monophyletic group. Molecular dating analysis explored that the origin of this genus was occurred during the late Eocene period, approximately 37.84 Ma (95% HPD: 20.33-52.21 Ma) years ago and the diversification of the genus was occurred in early Miocene 18.38 Ma (95% HPD: 10.68-25.28 Ma). CONCLUSION: The plastome-based tree and ITS-based tree generated incongruences, which may be attributed to the event of hybridization/introgression, incomplete lineage sorting (ILS) and chloroplast capture. Our study highlighted the power of plastome data to significantly improve the phylogenetic supports and resolutions, and to efficiently explore the evolutionary history of this genus. Molecular dating analysis explored that the diversification of the genus occurred in the early Miocene, which was largely influenced by the prevalence of the East Asian monsoon and the uplift of the Hengduan Mountains (HDM). In summary, our study provides novel insights into the plastome evolution, phylogenetic relationships, taxonomic framework and evolution of genus Sanicula.

Testing plastomes and nuclear ribosomal DNA sequences as the next-generation DNA barcodes for species identification and phylogenetic analysis in Acer.

BACKGROUND: Acer is a taxonomically intractable and speciose genus that contains over 150 species. It is challenging to distinguish Acer species only by morphological method due to their abundant variations. Plastome and nuclear ribosomal DNA (nrDNA) sequences are recommended as powerful next-generation DNA barcodes for species discrimination. However, their efficacies were still poorly studied. The current study will evaluate the application of plastome and nrDNA in species identification and perform phylogenetic analyses for Acer. RESULT: Based on a collection of 83 individuals representing 55 species (c. 55% of Chinese species) from 13 sections, our barcoding analyses demonstrated that plastomes exhibited the highest (90.47%) species discriminatory power among all plastid DNA markers, such as the standard plastid barcodes matK + rbcL + trnH-psbA (61.90%) and ycf1 (76.19%). And the nrDNA (80.95%) revealed higher species resolution than ITS (71.43%). Acer plastomes show abundant interspecific variations, however, species identification failure may be due to the incomplete lineage sorting (ILS) and chloroplast capture resulting from hybridization. We found that the usage of nrDNA contributed to identifying those species that were unidentified by plastomes, implying its capability to some extent to mitigate the impact of hybridization and ILS on species discrimination. However, combining plastome and nrDNA is not recommended given the cytonuclear conflict caused by potential hybridization. Our phylogenetic analysis covering 19 sections (95% sections of Acer) and 128 species (over 80% species of this genus) revealed pervasive inter- and intra-section cytonuclear discordances, hinting that hybridization has played an important role in the evolution of Acer. CONCLUSION: Plastomes and nrDNA can significantly improve the species resolution in Acer. Our phylogenetic analysis uncovered the scope and depth of cytonuclear conflict in Acer, providing important insights into its evolution.

Comparative and phylogenetic analyses of Loranthaceae plastomes provide insights into the evolutionary trajectories of plastome degradation in hemiparasitic plants.

BACKGROUND: The lifestyle transition from autotrophy to heterotrophy often leads to extensive degradation of plastomes in parasitic plants, while the evolutionary trajectories of plastome degradation associated with parasitism in hemiparasitic plants remain poorly understood. In this study, phylogeny-oriented comparative analyses were conducted to investigate whether obligate Loranthaceae stem-parasites experienced higher degrees of plastome degradation than closely related facultative root-parasites and to explore the potential evolutionary events that triggered the 'domino effect' in plastome degradation of hemiparasitic plants. RESULTS: Through phylogeny-oriented comparative analyses, the results indicate that Loranthaceae hemiparasites have undergone varying degrees of plastome degradation as they evolved towards a heterotrophic lifestyle. Compared to closely related facultative root-parasites, all obligate stem-parasites exhibited an elevated degree plastome degradation, characterized by increased downsizing, gene loss, and pseudogenization, thereby providing empirical evidence supporting the theoretical expectation that evolution from facultative parasitism to obligate parasitism may result in a higher degree of plastome degradation in hemiparasites. Along with infra-familial divergence in Loranthaceae, several lineage-specific gene loss/pseudogenization events occurred at deep nodes, whereas further independent gene loss/pseudogenization events were observed in shallow branches. CONCLUSIONS: The findings suggest that in addition to the increasing levels of nutritional reliance on host plants, cladogenesis can be considered as another pivotal evolutionary event triggering the 'domino effect' in plastome degradation of hemiparasitic plants. These findings provide new insights into the evolutionary trajectory of plastome degradation in hemiparasitic plants.

Comparative analysis of chloroplast genomes of Pulsatilla species reveals evolutionary and taxonomic status of newly discovered endangered species Pulsatilla saxatilis.

BACKGROUND: Pulsatilla saxatilis, a new species of the genus Pulsatilla has been discovered. The morphological information of this species has been well described, but its chloroplast genome characteristics and comparison with species of the same genus remain to be reported. RESULTS: Our results showed that the total length of chloroplast (cp.) genome of P. saxatilis is 162,659 bp, with a GC content of 37.5%. The cp. genome contains 134 genes, including 90 known protein-coding genes, 36 tRNA genes, and 8 rRNA genes. P. saxatilis demonstrated similar characteristics to other species of genus Pulsatilla. Herein, we compared cp. genomes of 10 species, including P. saxatilis, and found that the cp. genomes of the genus Pulsatilla are extremely similar, with a length of 162,322-163,851 bp. Furthermore, The SSRs of Pulsatilla ranged from 10 to 22 bp in length. Among the four structural regions of the cp. genome, most long repeats and SSRs were detected in the LSC region, followed by that in the SSC region, and least in IRA/ IRB regions. The most common types of long repeats were forward and palindromic repeats, followed by reverse repeats, and only a few complementary repeats were found in 10 cp. genomes. We also analyzed nucleotide diversity and identified ccsA_ndhD, rps16_trnK-UUU, ccsA, and rbcL, which could be used as potential molecular markers for identification of Pulsatilla species. The results of the phylogenetic tree constructed by connecting the sequences of high variation regions were consistent with those of the cp. gene phylogenetic tree, and the species more closely related to P. saxatilis was identified as the P. campanella. CONCLUSION: It was determined that the closest species to P. saxatilis is P. campanella, which is the same as the conclusion based on pollen grain characteristics, but different from the P. chinensis determined based on morphological characteristics. By revealing information on the chloroplast characteristics, development, and evolution of the cp. genome and the potential molecular markers, this study provides effective molecular data regarding the evolution, genetic diversity, and species identification of the genus Pulsatilla.

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.

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.

Plastid phylogenomics of Robinsonia (Senecioneae; Asteraceae), endemic to the Juan Fernández Islands: insights into structural organization and molecular evolution.

BACKGROUND: The genus Robinsonia DC. (tribe Senecioneae, Asteraceae) endemic to the Juan Fernández Islands in Chile is one of the most conspicuous insular plant groups in the world. Unlike typical herbaceous Asteraceae plants, these plants demonstrate spectacular and unusual rosette tree growth forms as shown by the alpine giant senecios (genus Dendrosenecio, tribe Senecioneae) endemic to the East African mountains. However, monophyly of the genus and phylogenetic relationships among species of Robinsonia as well as their plastome evolution remain elusive. This study aims to explore their phylogeny, species diversification, and molecular evolution based on the complete plastome sequences in the context of adaptive radiation on oceanic islands. RESULTS: The insular Robinsonia plastomes are highly conserved in their structures and organization of contents. Five divergence hotspots as potential chloroplast markers and five positively selected coding genes (accD, ndhF, rpoA, ycf1, and ycf2) are identified. Robinsonia plastomes has an overall nucleotide diversity higher than that of the sky island Dendrosenecio, but much lower than herbaceous Senecio. Phylogenetic analysis demonstrates the monophyly of Robinsonia and identifies two major infrageneric lineages. Both Robinsonia and Dendrosenecio are deeply nested within large genus Senecio. CONCLUSIONS: While plastid genomes of Robinsonia are highly conserved, their sequences strongly demonstrated the monophyly of the genus and inferred robust interspecific relationships, including herbaceous Senecio and woody Dendrosenecio. Different sets of positively selected chloroplast genes, five for Robinsonia and two for Dendrosenecio, may play an important role in the adaptation strategies of these fascinating woody species in insular and continental sky island habitats. Overall phylogenetic positions and sister lineages of Robinsonia and Dendrosenecio require additional study based on broader sampling of Senecio.

Phylogenetic incongruence in an Asiatic species complex of the genus Caryodaphnopsis (Lauraceae).

BACKGROUND: Caryodaphnopsis, a group of tropical trees (ca. 20 spp.) in the family Lauraceae, has an amphi-Pacific disjunct distribution: ten species are distributed in Southeast Asia, while eight species are restricted to tropical rainforests in South America. Previously, phylogenetic analyses using two nuclear markers resolved the relationships among the five species from Latin America. However, the phylogenetic relationships between the species in Asia remain poorly known. RESULTS: Here, we first determined the complete mitochondrial genome (mitogenome), plastome, and the nuclear ribosomal cistron (nrDNA) sequences of C. henryi with lengths of 1,168,029 bp, 154,938 bp, and 6495 bp, respectively. We found 2233 repeats and 368 potential SSRs in the mitogenome of C. henryi and 50 homologous DNA fragments between its mitogenome and plastome. Gene synteny analysis revealed a mass of rearrangements in the mitogenomes of Magnolia biondii, Hernandia nymphaeifolia, and C. henryi and only six conserved clustered genes among them. In order to reconstruct relationships for the ten Caryodaphnopsis species in Asia, we created three datasets: one for the mitogenome (coding genes and ten intergenic regions), another for the plastome (whole genome), and the other for the nuclear ribosomal cistron. All of the 22 Caryodaphnopsis individuals were divided into four, five, and six different clades in the phylogenies based on mitogenome, plastome, and nrDNA datasets, respectively. CONCLUSIONS: The study showed phylogenetic conflicts within and between nuclear and organellar genome data of Caryodaphnopsis species. The sympatric Caryodaphnopsis species in Hekou and Malipo SW China may be related to the incomplete lineage sorting, chloroplast capture, and/or hybridization, which mixed the species as a complex in their evolutionary history.

Repeated migration, interbreeding and bottlenecking shaped the phylogeography of the selfing grass Brachypodium stacei.

Brachypodium stacei is the most ancestral lineage in the genus Brachypodium, a model system for grass functional genomics. B. stacei shows striking and sometimes contradictory biological and evolutionary features, including a high selfing rate yet extensive admixture, an ancient Miocene origin yet with recent evolutionary radiation, and adaptation to different dry climate conditions in its narrow distribution range. Therefore, it constitutes an ideal system to study these life history traits. We studied the phylogeography of 17 native circum-Mediterranean B. stacei populations (39 individuals) using genome-wide RADseq SNP data and complete plastome sequences. Nuclear SNP data revealed the existence of six distinct genetic clusters, low levels of intra-population genetic diversity and high selfing rates, albeit with signatures of admixture. Coalescence-based dating analysis detected a recent split between crown lineages in the Late Quaternary. Plastome sequences showed incongruent evolutionary relationships with those recovered by the nuclear data, suggesting interbreeding and chloroplast capture events between genetically distant populations. Demographic and population dispersal coalescent models identified an ancestral origin of B. stacei in the western-central Mediterranean islands, followed by an early colonization of the Canary Islands and two independent colonization events of the eastern Mediterranean region through long-distance dispersal and bottleneck events as the most likely evolutionary history. Climate niche data identified three arid niches of B. stacei in the southern Mediterranean region. Our findings indicate that the phylogeography of B. stacei populations was shaped by recent radiations, frequent extinctions, long-distance dispersal events, occasional interbreeding, and adaptation to local climates.

Phylogenomic conflict analyses of the plastid and mitochondrial genomes via deep genome skimming highlight their independent evolutionary histories: A case study in the cinquefoil genus Potentilla sensu lato (Potentilleae, Rosaceae).

Phylogenomic conflicts are widespread among genomic data, with most previous studies primarily focusing on nuclear datasets instead of organellar genomes. In this study, we investigate phylogenetic conflict analyses within and between plastid and mitochondrial genomes using Potentilla as a case study. We generated three plastid datasets (coding, noncoding, and all-region) and one mitochondrial dataset (coding regions) to infer phylogenies based on concatenated and multispecies coalescent (MSC) methods. Conflict analyses were then performed using PhyParts and Quartet Sampling (QS). Both plastid and mitochondrial genomes divided the Potentilla into eight highly supported clades, two of which were newly identified in this study. While most organellar loci were uninformative for the majority of nodes (bootstrap value < 70%), PhyParts and QS detected conflicting signals within the two organellar genomes. Regression analyses revealed that conflict signals mainly occurred among shorter loci, whereas longer loci tended to be more concordant with the species tree. In addition, two significant disagreements between the two organellar genomes were detected, likely attributed to hybridization and/or incomplete lineage sorting. Our results demonstrate that mitochondrial genes can fully resolve the phylogenetic relationships among eight major clades of Potentilla and are not always linked with plastome in evolutionary history. Stochastic inferences appear to be the primary source of observed conflicts among the gene trees. We recommend that the loci with short sequence length or containing limited informative sites should be used cautiously in MSC analysis, and suggest the joint application of concatenated and MSC methods for phylogenetic inference using organellar genomes.

Phylogenomics of mulberries (Morus, Moraceae) inferred from plastomes and single copy nuclear genes.

Mulberries (genus Morus), belonging to the order Rosales, family Moraceae, are important woody plants due to their economic values in sericulture, as well as for nutritional benefits and medicinal values. However, the taxonomy and phylogeny of Morus, especially for the Asian species, remains challenging due to its wide geographical distribution, morphological plasticity, and interspecific hybridization. To better understand the evolutionary history of Morus, we combined plastomes and a large-scale nuclear gene analyses to investigate their phylogenetic relationships. We assembled the plastomes and screened 211 single-copy nuclear genes from 13 Morus species and related taxa. The plastomes of Morus species were relatively conserved in terms of genome size, gene content, synteny, IR boundary and codon usage. Using nuclear data, our results elucidated identical topologies based on coalescent and concatenation methods. The genus Morus was supported as monophyletic, with M. notabilis as the first diverging lineage and the two North American Morus species, M. microphylla and M. rubra, as sister to the other Asian species. In the Asian Morus species, interspecific relationships were completely resolved. However, cyto-nuclear discordances and gene tree-species tree conflicts were detected in the phylogenies of Morus, with multiple evidences supporting hybridization/introgression as the main cause of discordances between nuclear and plastid phylogenies, while gene tree-species tree conflicts were mainly caused by ILS.