Recent advances on phylogenomics of gymnosperms and a new classification

Living gymnosperms comprise four major groups:cycads,Ginkgo,conifers,and gnetophytes.Relationships among/within these lineages have not been fully resolved.Next generation sequencing has made available a large number of sequences,including both plastomes and single-copy nuclear genes,for reconstruction of solid phylogenetic trees.Recent advances in gymnosperm phylogenomic studies have updated our knowledge of gymnosperm systematics.Here,we review major advances of gymnosperm phylogeny over the past 10 years and propose an updated classification of extant gymnosperms.This new classification includes three classes(Cycadopsida,Ginkgoopsida,and Pinopsida),five subclasses(Cycadidae,Ginkgoidae,Cupressidae,Pinidae,and Gnetidae),eight orders(Cycadales,Ginkgoales,Araucariales,Cupressales,Pinales,Ephedrales,Gnetales,and Welwitschiales),13 families,and 86 genera.We also described six new tribes including Acmopyleae Y.Yang,Austrocedreae Y.Yang,Chamaecyparideae Y.Yang,Microcachrydeae Y.Yang,Papuacedreae Y.Yang,and Prumnopityeae Y.Yang,and made 27 new combinations in the genus Sabina.

Plastid phylogenomics and biogeography of the medicinal plant lineage Hyoscyameae(Solanaceae)

The cosmopolitan family Solanaceae,which originated and first diversified in South America,is economically important.The tribe Hyoscyameae is one of the three clades in Solanaceae that occurs outside of the New World;Hyoscyameae genera are distributed mainly in Europe and Asia,and have centers of species diversity in the Qinghai-Tibet Plateau and adjacent regions.Although many phylogenetic studies have focused on Solanaceae,the phylogenetic relationships within the tribe Hyoscyameae and its biogeographic history remain obscure.In this study,we reconstructed the phylogeny of Hyoscyameae based on whole chloroplast genome data,and estimated lineage divergence times according to the newly reported fruit fossil from the Eocene Patagonia,Physalis infinemundi,the earliest known fossil of Solanaceae.We reconstructed a robust phylogeny of Hyoscyameae that reveals the berry fruit-type Atropa is sister to the six capsule-bearing genera(Hyoscyameae sensu stricto),Atropanthe is sister to the clade(Scopolia,Physochlaina,Przewalskia),and together they are sister to the robustly supported AnisoduseHyoscyamus clade.The stem age of Hyoscyameae was inferred to be in the Eocene(47.11 Ma,95%HPD:36.75e57.86 Ma),and the crown ages of Hyoscyameae sensu stricto were estimated as the early Miocene(22.52 Ma,95%HPD:15.19e30.53 Ma),which shows a close correlation with the rapid uplift of the Qinghai-Tibet Plateau at the Paleogene/Neogene boundary.Our results provide insights into the phylogenetic relationships and the history of the biogeographic diversification of the tribe Hyoscyameae,as well as plant diversification on the Qinghai-Tibet Plateau.

Mitochondrial phylogenomics provides insights into the phylogeny and evolution of spiders(Arthropoda:Araneae)

Spiders are among the most varied terrestrial predators,with highly diverse morphology,ecology,and behavior.Morphological and molecular data have greatly contributed to advances in the phylogeny and evolutionary dynamics of spiders.Here,we performed comprehensive mitochondrial phylogenomics analysis on 78 mitochondrial genomes(mitogenomes)representing 29 families;of these,23 species from eight families were newly generated.Mesothelae retained the same gene arrangement as the arthropod ancestor(Limulus polyphemus),while Opisthothelae showed extensive rearrangement,with 12 rearrangement types in transfer RNAs(tRNAs)and control region.Most spider tRNAs were extremely truncated and lacked typical dihydrouridine or TΨC arms,showing high tRNA structural diversity;in particular,trnS1 exhibited anticodon diversity across the phylogeny.The evolutionary rates of mitochondrial genes were potentially associated with gene rearrangement or truncated tRNAs.Both mitogenomic sequences and rearrangements possessed phylogenetic characteristics,providing a robust backbone for spider phylogeny,as previously reported.The monophyly of suborder,infraorder,retrolateral tibial apophysis clade,and families(except for Pisauridae)was separately supported,and high-level relationships were resolved as(Mesothelae,(Mygalomorphae,(Entelegynae,(Synspermiata,Hypochilidae)))).The phylogenetic positions of several families were also resolved(e.g.,Eresidae,Oecobiidae and Titanoecidae).Two reconstructions of ancestral web type obtained almost identical results,indicating that the common ancestor of spiders likely foraged using a silk-lined burrow.This study,the largest mitochondrial phylogenomics analysis of spiders to date,highlights the usefulness of mitogenomic data not only for providing efficient phylogenetic signals for spider phylogeny,but also for characterizing trait diversification in spider evolution.

Nuclear phylogenomics of angiosperms and insights into their relationships and evolution

Angiosperms(flowering plants) are by far the most diverse land plant group with over 300,000 species. The sudden appearance of diverse angiosperms in the fossil record was referred to by Darwin as the “abominable mystery,”hence contributing to the heightened interest in angiosperm evolution. Angiosperms display wide ranges of morphological, physiological,and ecological characters, some of which have probably influenced their species richness. The evolutionary analyses of these characteristics help to address questions of angiosperm diversification and require well resolved phylogeny. Following the great successes of phylogenetic analyses using plastid sequences,dozens to thousands of nuclear genes from next-generation sequencing have been used in angiosperm phylogenomic analyses, providing well resolved phylogenies and new insights into the evolution of angiosperms. In this review we focus on recent nuclear phylogenomic analyses of large angiosperm clades, orders, families,and subdivisions of some families and provide a summarized Nuclear Phylogenetic Tree of Angiosperm Families. The newly established nuclear phylogenetic relationships are highlighted and compared with previous phylogenetic results. The sequenced genomes of Amborella,Nymphaea, Chloranthus, Ceratophyllum, and species of monocots, Magnoliids, and basal eudicots, have facilitated the phylogenomics of relationships among five major angiosperms clades. All but one of the 64 angiosperm orders were included in nuclear phylogenomics with well resolved relationships except the placements of several orders. Most families have been included with robust and highly supported placements, especially for relationships within several large and important orders and families.Additionally, we examine the divergence time estimation and biogeographic analyses of angiosperm on the basis of the nuclear phylogenomic frameworks and discuss the differences compared with previous analyses. Furthermore,we discuss the implications of nuclear phylogenomic analyses on ancestral reconstruction of morphological, physiological, and ecological characters of angiosperm groups, limitations of current nuclear phylogenomic studies, and the taxa that require future attention.

Deep genome skimming reveals the hybrid origin of Pseudosasa gracilis (Poaceae: Bambusoideae)

Pseudosasa gracilis(Poaceae:Bambusoideae)is a temperate woody bamboo species endemic to Southcentral China with a narrow distribution.Previous phylogenetic studies revealed an unexpected,isolated phylogenetic position of Ps.gracilis.Here we conducted phylogenomic analysis by sampling populations of Ps.gracilis and its sympatric species Ps.nanunica and Sinosasa polytricha reflecting different genomic signals,by deep genome skimming.Integrating molecular evidence from chloroplast genes and genome-wide SNPs,we deciphered the phylogenetic relationships of Ps.gracilis.Both plastid and nuclear data indicate that Ps.gracilis is more closely related to Sinosasa,which is discordant with the taxonomic treatment.To further explore this molecular-morphological conflict,we screened 411“perfect-copy”syntenic genes to reconstruct phylogenies using both the concatenation and coalescent methods.We observed extensive discordance between gene trees and the putative species tree.A significant hybridization event was detected based on 411 genes from the D subgenome,showing Ps.gracilis was a hybrid descendant between Sinosasa longiligulata and Ps.nanunica,with 63.56%and 36.44%inheritance probabilities of each parent.Moreover,introgression events were detected in the C subgenome between Ps.gracilis and S.polytricha in the same distribution region.Our findings suggest that sympatric hybridization and introgression play a crucial role in the origin of Ps.gracilis.By providing an empirical example of bamboo of hybrid origin using comprehensive analyses based on genomic data from different inheritance systems and morphological characters,our study represents a step forward in understanding of reticulate evolution of bamboos.

Resolving the Mortierellaceae phylogeny through synthesis of multi-gene phylogenetics and phylogenomics

Early efforts to classify Mortierellaceae were based on macro-and micromorphology,but sequencing and phylogenetic studies with ribosomal DNA(rDNA)markers have demonstrated conflicting taxonomic groupings and polyphyletic genera.Although some taxonomic confusion in the family has been clarified,rDNA data alone is unable to resolve higher level phylogenetic relationships within Mortierellaceae.In this study,we applied two parallel approaches to resolve the Mortierel-laceae phylogeny:low coverage genome(LCG)sequencing and high-throughput,multiplexed targeted amplicon sequenc-ing to generate sequence data for multi-gene phylogenetics.We then combined our datasets to provide a well-supported genome-based phylogeny having broad sampling depth from the amplicon dataset.Resolving the Mortierellaceae phylogeny into monophyletic genera resulted in 13 genera,7 of which are newly proposed.Low-coverage genome sequencing proved to be a relatively cost-effective means of generating a high-confidence phylogeny.The multi-gene phylogenetics approach enabled much greater sampling depth and breadth than the LCG approach,but has limitations too.We present this work to resolve some of the taxonomic confusion and provide a genus-level framework to empower future studies on Mortierellaceae diversity and evolution.

Phylogenomics of non-model ciliates based on transcriptomic analyses

Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep se- quencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phyloge- nomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epi- plasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.

New insights into intergeneric relationships of Hickeliinae(Poaceae:Bambusoideae) revealed by complete plastid genomes

The Hickeliinae(Poaceae:Bambusoideae) is an ecologically and economically significant subtribe of tropical bamboos restricted to Madagascar,Comoros,Reunion Island,and a small part of continental Africa(Tanzania).Because these bamboos rarely flower,field identification is challenging,and inferring the evolutionary history of Hickeliinae from herbarium specimens is even more so.Molecular phylogenetic work is critical to understanding this group of bamboos.Here,comparative analysis of 22 newly sequenced plastid genomes showed that members of all genera of Hickeliinae share evolutionarily conserved plastome structures.We also determined that Hickeliinae plastome sequences are informative for phylogenetic reconstructions.Phylogenetic analysis showed that all genera of Hickeliinae are monophyletic,except for Nastus,which is paraphyletic and forms two distant clades.The type species of Nastus(Clade Ⅱ) is endemic to Reunion Island and is not closely related to other sampled species of Nastus endemic to Madagascar(Clade Ⅵ).Clade Ⅵ(Malagasy Nastus) is sister to the Sokinochloa+Hitchcockella clade(Clade Ⅴ),and both clades have a clumping habit with short-necked pachymorph rhizomes.The monotypic Decaryochloa is remarkable in having the longest floret in Bambuseae and forms a distinct Clade Ⅳ.Clade Ⅲ,which has the highest generic diversity,consists of Cathariostachys,Perrierbambus,Sirochloa,and Valiha,which are also morphologically diverse.This work provides significant resources for further genetic and phylogenomic studies of Hickeliinae,an understudied subtribe of bamboo.

Comparative genomics of widespread and narrow-range white-bellied rats in the Niviventer niviventer species complex sheds light on invasive rodent success

Widespread species that inhabit diverse environments possess large population sizes and exhibit a high capacity for environmental adaptation,thus enabling range expansion.In contrast,narrow-range species are confined to restricted geographical areas and are ecologically adapted to narrow environmental conditions,thus limiting their ability to expand into novel environments.However,the genomic mechanisms underlying the differentiation between closely related species with varying distribution ranges remain poorly understood.The Niviventer niviventer species complex(NNSC),consisting of highly abundant wild rats in Southeast Asia and China,offers an excellent opportunity to investigate these questions due to the presence of both widespread and narrow-range species that are phylogenetically closely related.In the present study,we combined ecological niche modeling with phylogenetic analysis,which suggested that sister species cannot be both widespread and dominant within the same geographical region.Moreover,by assessing heterozygosity,linkage disequilibrium decay,and Tajima's D analysis,we found that widespread species exhibited higher genetic diversity than narrow-range species.In addition,by exploring the“genomic islands of speciation”,we identified 13 genes in highly divergent regions that were shared by the two widespread species,distinguishing them from their narrow-range counterparts.Functional annotation analysis indicated that these genes are involved in nervous system development and regulation.The adaptive evolution of these genes likely played an important role in the speciation of these widespread species.

Systematics of the avian family Alaudidae using multilocus and genomic data

The family Alaudidae,larks,comprises 93-100 species(depending on taxonomy)that are widely distributed across Africa and Eurasia,with single species extending their ranges to North and northernmost South America and Australia.A decade-old molecular phylogeny,comprising~80%of the species,revealed multiple cases of parallel evolution and large variation in rates of morphological evolution,which had misled taxonomists into creating many non-monophyletic genera.Here,we reconstruct the phylogeny of the larks,using a dataset covering one mitochondrial and 16 nuclear loci and comprising all except one of the currently recognised species as well as several recently proposed new species(in total 133 taxa;not all loci available for all species).We provide additional support using genome-wide markers to infer a genus-level phylogeny based on near-complete generic sampling(in total 51 samples of 44 taxa across 40 species).Our results confirm the previous findings of rampant morphological convergence and divergence,and reveal new cases of paraphyletic genera.We propose a new subfamily classification,and also that the genus Mirafra is divided into four genera to produce a more balanced generic classification of the Alaudidae.Our study supports recently proposed species splits as well as some recent lumps,while also questioning some of the latter.This comprehensive phylogeny will form an important basis for future studies,such as comparative studies of lark natural history,ecology,evolution and conservation.

Comparison and phylogeny on mitochondrial genome of marine and freshwater taxa of genus Hildenbrandia(Florideophyceae,Rhodophyta)

Hildenbrandia is an early diverged lineage in Florideophyceae,Rhodophyta.The species diversity of this genus is still unresolved due to the simple morphology and limited molecular information.The mitochondrial genome of freshwater H.jigongshanensis was determined in this study.The freshwater H.jigongshanensis had a larger mitochondrial genome than the marine H.rubra and GC content was higher.Collinear alignment structure was observed between the mitochondrial genomes of H.jigongshanensis and H.rubra,except for one block that was encoded on the complement strand.More introns were found in mitochondrial genome of H.jigongshanensis than in H.rubra,and H.jigongshanensis shares the common feature with Bangiophyceae that two introns were distributed in cox1.Comparison of mitochondrial genome organization suggests that H.jigongshanensis preserves characters that could be hypothetically more similar to the ancestor of Bangiophyceae and Florideophyceae,which differ with previous studies based on chloroplast,and nuclear markers.More mitochondrial genomes and phylogenetic analyses combing nuclear,chloroplast and mitochondrial genomes are needed to clarify this discrepancy.Mitochondrion-based phylogeny in this study resulted in better solution at both the deep and recent derived nodes than single-gene phylogenies.Most protein-coding genes between H.jigongshanensis and H.rubra were identical except atp8,which was present in H.jigongshanensis while absent from H.rubra.This finding follows the trend that high Ka/Ks ratio genes are more frequently lost than low Ka/Ks ratio ones in red algae.

Summary of Laurasiatheria （Mammalia） Phylogeny

Laurasiatheria is one of the richest and most diverse superorders of placental mammals. Because this group had a rapid evolutionary radiation, the phylogenetic relationships among the six orders of Laurasiatheria remain a subject of heated debate and several issues related to its phylogeny remain open. Reconstructing the true phylogenetic relationships of Laurasiatheria is a significant case study in evolutionary biology due to the diversity of this suborder and such research will have significant implications for biodiversity conservation. We review the higher-level （inter-ordinal） phylogenies of Laurasiatheria based on previous cytogenetic, morphological and molecular data, and discuss the controversies of its phylogenetic relationship. This review aims to outline future researches on Laurasiatheria phylogeny and adaptive evolution.

Genomic insights into ruminant evolution: from past to future prospects

Ruminants(Ruminantia) are among the most successful herbivorous mammals, exhibiting wideranging morphological and ecological characteristics(such as headgear and multichambered stomach)and including various key livestock species(e. g.,cattle, buffalo, yak, sheep, and goat). Understanding their evolution is of great significance not only in scientific research but also in applications potential for human society. The rapid growth of genomic resources provides unprecedented opportunities to dissect the evolutionary histories and molecular mechanisms underlying the distinct characteristics of ruminants. Here we summarize our current understanding of the genetic, morphological, and ecological diversity of ruminants and provide prospects for future studies.

The complete plastome of Panax stipuleanatus: Comparative and phylogenetic analyses of the genus Panax(Araliaceae)

Panax stipuleanatus(Araliaceae) is an endangered and medicinally important plant endemic to China.However, phylogenetic relationships within the genus Panax have remained unclear. In this study, we sequenced the complete plastome of P. stipuleanatus and included previously reported Panax plastomes to better understand the relationships between species and plastome evolution within the genus Panax.The plastome of P. stipuleanatus is 156,069 base pairs(bp) in length, consisting of a pair of inverted repeats(IRs, each 25,887 bp) that divide the plastome into a large single copy region(LSC, 86,126 bp) and a small single copy region(SSC, 8169 bp). The plastome contains 114 unigenes(80 protein-coding genes,30 tRNA genes, and 4 r RNA genes). Comparative analyses indicated that the plastome gene content and order, as well as the expansion/contraction of the IR regions, are all highly conserved within Panax. No significant positive selection in the plastid protein-coding genes was observed across the eight Panax species, suggesting the Panax plastomes may have undergone a strong purifying selection. Our phylogenomic analyses resulted in a phylogeny with high resolution and supports for Panax. Nine proteincoding genes and 10 non-coding regions presented high sequence divergence, which could be useful for identifying different Panax species.

Plastome characteristics of Cannabaceae

Cannabaceae is an economically important family that includes ten genera and ca. 117 accepted species.To explore the structure and size variation of their plastomes, we sequenced ten plastomes representing all ten genera of Cannabaceae. Each plastome possessed the typical angiosperm quadripartite structure and contained a total of 128 genes. The Inverted Repeat(IR) regions in five plastomes had experienced small expansions(330 e983 bp) into the Large Single-Copy(LSC) region. The plastome of Chaetachme aristata has experienced a 942-bp IR contraction and lost rpl22 and rps19 in its IRs. The substitution rates of rps19 and rpl22 decreased after they shifted from the LSC to IR. A 270-bp inversion was detected in the Parasponia rugosa plastome, which might have been mediated by 18-bp inverted repeats. Repeat sequences, simple sequence repeats, and nucleotide substitution rates varied among these plastomes.Molecular markers with more than 13% variable sites and 5% parsimony-informative sites were identified, which may be useful for further phylogenetic analysis and species identification. Our results show strong support for a sister relationship between Gironniera and Lozanell(BS ? 100). Celtis, CannabisHumulus, Chaetachme-Pteroceltis, and Trema-Parasponia formed a strongly supported clade, and their relationships were well resolved with strong support(BS ? 100). The availability of these ten plastomes provides valuable genetic information for accurately identifying species, clarifying taxonomy and reconstructing the intergeneric phylogeny of Cannabaceae.

Summary of Laurasiatheria(Mammalia)Phylogeny

Laurasiatheria is one of the richest and most diverse superorders of placental mammals.Because this group had a rapid evolutionary radiation,the phylogenetic relationships among the six orders of Laurasiatheria remain a subject of heated debate and several issues related to its phylogeny remain open.Reconstructing the true phylogenetic relationships of Laurasiatheria is a significant case study in evolutionary biology due to the diversity of this suborder and such research will have significant implications for biodiversity conservation.We review the higher-level(inter-ordinal)phylogenies of Laurasiatheria based on previous cytogenetic,morphological and molecular data,and discuss the controversies of its phylogenetic relationship.This review aims to outline future researches on Laurasiatheria phylogeny and adaptive evolution.

Extreme plastid RNA editing may confound phylogenetic reconstruction:A case study of Selaginella(lycophytes)

Cytidine-to-uridine(C-to-U)RNA editing is common in coding regions of organellar genomes throughout land plants.In most cases RNA editing alters translated amino acids or creates new start codons,potentially confounds phylogenetic reconstructions.In this study,we used the spike moss genus Selaginella(lycophytes),which has the highest frequency of RNA editing,as a model to test the effects of extreme RNA editing on phylogenetic reconstruction.We predicted the C-to-U RNA editing sites in coding regions of 18 Selaginella plastomes,and reconstructed the phylogenetic relationships within Selaginella based on three data set pairs consisted of plastome or RNA-edited coding sequences,first and second codon positions,and translated amino acid sequences,respectively.We predicted between 400 and 3100 RNA editing sites of 18 Selaginella plastomes.The numbers of RNA editing sites in plastomes were highly correlated with the GC content of first and second codon positions,but not correlated with the GC content of plastomes as a whole.Contrast phylogenetic analyses showed that there were substantial differences(e.g.,the placement of clade B in Selaginella)between the phylogenies generated by the plastome and RNA-edited data sets.This empirical study provides evidence that extreme C-to-U RNA editing in the coding regions of organellar genomes alters the sequences used for phylogenetic reconstruction,and might even confound phylogenetic reconstruction.Therefore,RNA editing sites should be corrected when plastid or mitochondrial genes are used for phylogenetic studies,particularly in those lineages with abundant organellar RNA editing sites,such as hornworts,quillworts,spike mosses,and some seed plants.

Parahellenia,a new genus segregated from Hellenia(Costaceae)based on phylogenetic and morphological evidence

Previous studies recognized three major lineages of the family Costaceae:a South American clade,an Asian clade and a Costus clade.However,the genus Hellenia within the Asian clade has been shown to be non-monophyletic and its morphology has not been studied carefully.Therefore,the complete plastid genomes of Hellenia species were obtained and the monophyly of Hellenia was tested through four different datasets in this study.Plastid phylogenomic analyses of Costaceae revealed that Hellenia is strongly supported as paraphyletic.Two major clades are recovered,namely the Hellenia s.s.subclade and the Parahellenia subclade.Phylogenetic analyses based on an enlarged taxon sampling of the Asian clade using a two chloroplast markers dataset(trnK intron and trnL-F spacer)confirmed the paraphyly of Hellenia.Meanwhile,morphological analyses suggested that members of the Parahellenia subclade differ from the remaining Hellenia species in many characters including inflorescences,bracts,stigma,axillary buds,floral tubes and labellum.According to the present molecular and morphological evidence,the latter subclade is recognized as a new genus,Parahellenia.Two new species are described,four new combinations are made,and identification keys are also provided.

Complete genome of Cobetia marina JCM 21022T and phylogenomic analysis of the family Halomonadaceae

Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. ~To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022 ~T using single molecule real-time sequencing technology(SMR^T) and performed comparative genomics and phylogenomics analyses. ~The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 t RNA genes, and 21 r RNA genes. ~The C. marina JCM 21022 ~T genome contained a set of crucial genes involved in surface colonization processes. ~The comparative genome analysis indicated the significant diff erences between C. marina JCM 21022 ~T and Cobetia amphilecti KMM 296(formerly named C. marina KMM 296) resulted from sequence insertions or deletions and chromosomal recombination. Despite these diff erences, pan and core genome analysis showed similar gene functions between the two strains. ~The phylogenomic study of the family Halomonadaceae is reported here for the first time. We found that the relationships were well resolved among every genera tested, including Chromohalobacter, Halomonas, Cobetia, Kushneria, Zymobacter, and Halotalea.

Lycophyte transcriptomes reveal two whole-genome duplications in Lycopodiaceae:Insights into the polyploidization of Phlegmariurus

Lycophytes are an ancient clade of the non-flowering vascular plants with chromosome numbers that vary from tens to hundreds.They are an excellent study system for examining whole-genome duplications(WGDs),or polyploidization,in spore-dispersed vascular plants.However,a lack of genome sequence data limits the reliable detection of very ancient WGDs,small-scale duplications(SSDs),and recent WGDs.Here,we integrated phylogenomic analysis and the distribution of synonymous substitutions per synonymous sites(Ks)of the transcriptomes of 13 species of lycophytes to identify,locate,and date multiple WGDs in the lycophyte family Lycopodiaceae.Additionally,we examined the genus Phlegmariurus for signs of genetic discordance,which can provide valuable insight into the underlying causes of such conflict(e.g.,hybridization,incomplete lineage sorting,or horizontal gene transfer).We found strong evidence that two WGD events occurred along the phylogenetic backbone of Lycopodiaceae,with one occurring in the common ancestor of extant Phlegmariurus(Lycopodiaceae)approximately 22-23 million years ago(Mya)and the other occurring in the common ancestor of Lycopodiaceae around 206-214 Mya.Interestingly,we found significant genetic discordance in the genus Phlegmariurus,indicating that the genus has a complex evolutionary history.This study provides molecular evidence for multiple WGDs in Lycopodiaceae and offers phylogenetic clues to the evolutionary history of Lycopodiaceae.