Molecular phylogeny and inflorescence evolution of Prunus (Rosaceae) based on RAD-seq and genome skimming analyses.

Prunus is an economically important genus widely distributed in the temperate Northern Hemisphere. Previous studies on the genus using a variety of loci yielded conflicting phylogenetic hypotheses. Here, we generated nuclear reduced representation sequencing data and plastid genomes for 36 Prunus individuals and two outgroups. Both nuclear and plastome data recovered a well-resolved phylogeny. The species were divided into three main clades corresponding to their inflorescence types, - the racemose group, the solitary-flower group and the corymbose group - with the latter two sister to one another. Prunus was inferred to have diversified initially in the Late Cretaceous around 67.32 million years ago. The diversification of the three major clades began between the Paleocene and Miocene, suggesting that paleoclimatic events were an important driving force for Prunus diversification. Ancestral state reconstructions revealed that the most recent common ancestor of Prunus had racemose inflorescences, and the solitary-flower and corymb inflorescence types were derived by reduction of flower number and suppression of the rachis, respectively. We also tested the hybrid origin hypothesis of the racemose group proposed in previous studies. Prunus has undergone extensive hybridization events, although it is difficult to identify conclusively specific instances of hybridization when using SNP data, especially deep in the phylogeny. Our study provides well-resolved nuclear and plastid phylogenies of Prunus, reveals substantial cytonuclear discord at shallow scales, and sheds new light on inflorescence evolution in this economically important lineage.

The complete chloroplast genome sequence of Zygophyllum kansuense Y. X. Liou (Zygophyllaceae).

Zygophyllum kansuense Y. X. Liou is a localized species and endemic to China. In this study, the complete chloroplast genome of Z. kansuense was sequenced and described. The length of complete chloroplast genome was 105,383 base pairs (bp) for Z. kansuense, the genome with 33.8% GC content, containing a large single-copy (LSC) of 79,594 bp, a small single-copy (SSC) of 17,061 bp, and a pair of inverted repeats (IRs) of 4,364 bp. The genome contains 112 genes, including 75 protein-coding genes, 33 tRNA genes, and four rRNA genes. The phylogenetic analysis indicated that Z. kansuense and others of Zygophyllum clustered into one clade with a high support value.

Tension zone trapped by exogenous cline: Analysis of a narrow hybrid zone between two parapatric Oxytropis species (Fabaceae).

Hybrid zones have been widely highlighted for their interest in understanding evolutionary processes. It is generally accepted that hybrid zones can be maintained in a balance between dispersal and selection. However, the selective forces can either be endogenous (i.e., genetic incompatibilities between parental taxa) or exogenous (i.e., parental taxa are adapted to different environments). In this study, we evaluated these alternatives and determined the maintenance of a narrow hybrid zone between parapatric distributed Oxytropis diversifolia and O. leptophylla in Nei Mongol, China. For 507 individuals sampled from two populations in the hybrid zone, 12 O. diversifolia populations and five O. leptophylla populations, we measured leaf-morphological characteristics, quantified genetic structure using 11 nuclear microsatellite loci and five chloroplast DNA intergenic regions, collected micro- and macrohabitat data, and conducted geographical cline analysis. We found that the two species differed in leaf morphology, and putative hybrids showed either intermediacy or a bias to O. diversifolia. Parental taxa formed two genetically distinct clusters, while populations in the hybrid zone consisted of both parental forms and various admixed individuals, exhibiting a bimodal pattern. The hybrid zone was coupled to ecological transitions of both microhabitat (i.e., the slope) and macroclimatic conditions. However, the genetic clines were significantly narrower than the environmental cline. Our results indicate that endogenous selection can be primarily responsible for maintaining the hybrid zone, while local adaptation accounts for the position of the zone. We further suggest the probable outcome of hybridization could be introgression.

Comparative Chloroplast Genomics and Phylogenetic Analysis of Zygophyllum (Zygophyllaceae) of China.

The genus Zygophyllum comprises over 150 species within the plant family Zygophyllaceae. These species predominantly grow in arid and semiarid areas, and about 20 occur in northwestern China. In this study, we sampled 24 individuals of Zygophyllum representing 15 species and sequenced their complete chloroplast (cp) genomes. For comparison, we also sequenced cp genomes of two species of Peganum from China representing the closely allied family, Nitrariaceae. The 24 cp genomes of Zygophyllum were smaller and ranged in size from 104,221 to 106,286 bp, each containing a large single-copy (LSC) region (79,245-80,439 bp), a small single-copy (SSC) region (16,285-17,146 bp), and a pair of inverted repeat (IR) regions (3,792-4,466 bp). These cp genomes contained 111-112 genes each, including 74-75 protein-coding genes (PCGs), four ribosomal RNA genes, and 33 transfer RNA genes, and all cp genomes showed similar gene order, content, and structure. The cp genomes of Zygophyllum appeared to lose some genes such as ndh genes and rRNA genes, of which four rRNA genes were in the SSC region, not in the IR regions. However, the SC and IR regions had greater similarity within Zygophyllum than between the genus and Peganum. We detected nine highly variable intergenic spacers: matK-trnQ, psaC-rps15, psbZ-trnG, rps7-trnL, rps15-trnN, trnE-trnT, trnL-rpl32, trnQ-psbK, and trnS-trnG. Additionally, we identified 156 simple sequence repeat (cpSSR) markers shared among the genomes of the 24 Zygophyllum samples and seven cpSSRs that were unique to the species of Zygophyllum. These markers may be useful in future studies on genetic diversity and relationships of Zygophyllum and closely related taxa. Using the sequenced cp genomes, we reconstructed a phylogeny that strongly supported the division of Chinese Zygophyllum into herbaceous and shrubby clades. We utilized our phylogenetic results along with prior morphological studies to address several remaining taxonomic questions within Zygophyllum. Specifically, we found that Zygophyllum kaschgaricum is included within Zygophyllum xanthoxylon supporting the present treatment of the former genus Sarcozygium as a subgenus within Zygophyllum. Our results provide a foundation for future research on the genetic resources of Zygophyllum.

On the Species Delimitation of the Maddenia Group of Prunus (Rosaceae): Evidence From Plastome and Nuclear Sequences and Morphology.

The recognition, identification, and differentiation of closely related plant species present significant and notorious challenges to taxonomists. The Maddenia group of Prunus, which comprises four to seven species, is an example of a group in which species delimitation and phylogenetic reconstruction have been difficult, due to the lack of clear morphological distinctions, limited sampling, and low informativeness of molecular evidence. Thus, the precise number of species in the group and the relationships among them remain unclear. Here, we used genome skimming to generate the DNA sequence data for 22 samples, including 17 Maddenia individuals and five outgroups in Amygdaloideae of Rosaceae, from which we assembled the plastome and 446 single-copy nuclear (SCN) genes for each sample. The phylogenetic relationships of the Maddenia group were then reconstructed using both concatenated and coalescent-based methods. We also identified eight highly variable regions and detected simple sequence repeats (SSRs) and repeat sequences in the Maddenia species plastomes. The phylogenetic analysis based on the complete plastomes strongly supported three main subclades in the Maddenia group of Prunus, while five subclades were recognized based on the nuclear tree. The phylogenetic network analysis detected six hybridization events. Integrating the nuclear and morphological evidence, we proposed to recognize five species within the Maddenia group, i.e., Prunus fujianensis, P. himalayana, P. gongshanensis, P. hypoleuca, and P. hypoxantha. Within this group, the first three species are well-supported, while the gene flow occurring throughout the Maddenia group seems to be especially frequent between P. hypoleuca and P. hypoxantha, eroding the barrier between them. The phylogenetic trees based on eight concatenated hypervariable regions had a similar topology with the complete plastomes, showing their potential as molecular markers and effective barcodes for further phylogeographic studies on Maddenia.

Why More Leaflets? The Role of Natural Selection in Shaping the Spatial Pattern of Leaf-Shape Variation in Oxytropis diversifolia (Fabaceae) and Two Close Relatives.

Leaf shape exhibits tremendous diversity in angiosperms. It has long been argued that leaf shape can affect major physiological and ecological properties of plants and thus is likely to be adaptive, but the evolutionary evidence is still scarce. Oxytropis diversifolia (Fabaceae) is polymorphic for leaf shape (1 leaflet, 1-3 leaflets, and 3 leaflets) and exhibits clinal variation in steppes of Nei Mongol, China. With two close relatives predominantly fixed for one phenotype as comparison (Oxytropis neimonggolica with 1 leaflet and Oxytropis leptophylla with 5-13 leaflets), we used a comprehensive cline-fitting approach to assess the role of natural selection in shaping the spatial pattern of leaf-shape variation in this system. For 551 individuals sampled from 22 populations, we quantified leaf-morphological differentiation, evaluated patterns of neutral genetic variation using five chloroplast DNA intergenic regions and 11 nuclear microsatellite loci, and performed microhabitat and macroclimatic-association analyses. We found that 1-leaflet proportions in O. diversifolia populations significantly increased from west to east, and three phenotypes also differed in leaflet-blade size. However, compared with the other two species, populations of O. diversifolia showed little neutral genetic differentiation, and no population structure was detected at either marker. We further revealed that the leaf-shape cline could largely be explained by three macroclimatic variables, with leaflet number decreasing and leaflet-blade size increasing with annual precipitation and showing the reverse trends with temperature seasonality and isothermality. Our results suggest that spatially varying abiotic environmental factors contribute to shape the leaf-shape cline in O. diversifolia, while the interspecific pattern may be due to both local adaptation and historical events.

Chloroplast genomes elucidate diversity, phylogeny, and taxonomy of Pulsatilla (Ranunculaceae).

Pulsatilla (Ranunculaceae) consists of about 40 species, and many of them have horticultural and/or medicinal value. However, it is difficult to recognize and identify wild Pulsatilla species. Universal molecular markers have been used to identify these species, but insufficient phylogenetic signal was available. Here, we compared the complete chloroplast genomes of seven Pulsatilla species. The chloroplast genomes of Pulsatilla were very similar and their length ranges from 161,501 to 162,669 bp. Eight highly variable regions and potential sources of molecular markers such as simple sequence repeats, large repeat sequences, and single nucleotide polymorphisms were identified, which are valuable for studies of infra- and inter-specific genetic diversity. The SNP number differentiating any two Pulsatilla chloroplast genomes ranged from 112 to 1214, and provided sufficient data for species delimitation. Phylogenetic trees based on different data sets were consistent with one another, with the IR, SSC regions and the barcode combination rbcL + matK + trnH-psbA produced slightly different results. Phylogenetic relationships within Pulsatilla were certainly resolved using the complete cp genome sequences. Overall, this study provides plentiful chloroplast genomic resources, which will be helpful to identify members of this taxonomically challenging group in further investigation.

Efficient Identification of Pulsatilla (Ranunculaceae) Using DNA Barcodes and Micro-Morphological Characters.

Pulsatilla (Ranunculaceae) comprises about 40 species, many of which have horticultural and/or medicinal importance. However, the recognition and identification of wild Pulsatilla species is difficult due to the presence of complex morphological characters. DNA barcoding is a powerful molecular tool capable of rapidly and accurately distinguishing between species. Here, we assessed the effectiveness of four commonly used DNA barcoding loci-rbcL (R), trnH-psbA ( T ), matK (M), and ITS (I)-to identify species of Pulsatilla from a comprehensive sampling group. Among the four barcoding single loci, the nuclear ITS marker showed the highest interspecific distances and the highest rate of correct identification. Among the eleven combinations, the chloroplast multi-locus R+T and R+M+T combinations were found to have the best species discrimination rate, followed by R+M. Overall, we propose that the R+M+T combination and the ITS marker on its own are, respectively, the best multi- and single-locus barcodes for discriminating among species of Pulsatilla. The phylogenetic analysis was able to distinguish species of Pulsatilla to the subgenus level, but the analysis also showed relatively low species resolution. This may be caused by incomplete lineage sorting and/or hybridization events in the evolutionary history of the genus, or by the resolution limit of the candidate barcodes. We also investigated the leaf epidermis of eight representative species using scanning electronic microscopy. The resulting micro-morphological characters were valuable for identification of related species. Using additional genome fragments, or even whole chloroplast genomes combined with micro-morphological data may permit even higher resolution of species in Pulsatilla.

[A landscape of transcriptome analysis of three sclerotia growth stages in Polyporus umbellatus].

Polyporus umbellatus,a traditional Chinese precious medicine as long been used for eliminating dampness,diuresis and have effect on cancer,getting more and more popularly in China recently. And the developmental metabolic process of the medicinal fungus,P. umbellatus,has been gotten more attention. This study is for the first time to explore the three sclerotial growth stages in P. umbellatus,named " white Polyporus"( initial phase), " grey Polyporus"( developmental phase) and " black Polyporus"( mature phase),by utilizing the de novo transcriptome assembly analysis technology. Finally,we obtained 88. 12 Gb sequence containing85 235 unigenes( ≥200 bp) assembled and 100% were annotated. We identified genes differentially expressed among the three stages of the sclerotia and screened out MFSgst,ERG4/ERG24,WD40,Rho A,CYP450,PKS,GSase and CHS1,which may contribute to the production of medicinal secondary metabolites and the defense mechanism against the environmental stress and biological invasion. We did the qRT-PCR trial to verify our results,which is in line with expectations. Our results are purposed to unearth the molecular mechanism of the accumulation of active constituents in different stages of Polyporus sclerotia which can be applied in the production and protection of Polyporus effectively.

The complete chloroplast genome sequence of Oxytropis bicolor Bunge (Fabaceae).

The first complete chloroplast genome of Oxytropis bicolor Bunge is reported and characterized in this study. The whole chloroplast genome was 122,461 base pairs in length with 110 genes, including 76 protein-coding genes, 30 tRNAs, and 4 rRNAs. In addition, the atpF intron was absent. Maximum-likelihood (ML) phylogenetic analysis indicated that O. bicolor and species of Astragalus were closely related, which is congruent with previous studies.

Isolation and characterization of microsatellite loci from Oxytropis diversifolia (Fabaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for a perennial legume from northern China, Oxytropis diversifolia (Fabaceae), to investigate population genetic structure of this taxon, as well as potential hybridization events with closely related taxa in this genus. METHODS AND RESULTS: One hundred and five primer pairs were designed from Illumina sequence data and screened for suitability. Fifteen of these primer pairs were polymorphic, and these primers amplified tri-, tetra-, and pentanucleotide repeats with 10-56 alleles per locus. Cross-amplification tests in three other Oxytropis species from northern China (O. leptophylla, O. neimonggolica, and O. squammulosa) revealed that all of these loci can be amplified successfully and show polymorphism. CONCLUSIONS: These primer pairs can be used to assess the genetic diversity and population structure in future studies of O. diversifolia, as well as studies of potential hybridization events with closely related taxa in this genus.

Floral morphology and morphogenesis in Camptotheca (Nyssaceae), and its systematic significance.

Background and Aims: Camptotheca is endemic to China and there are limited data about the breeding system and morphogenesis of the flowers. Camptotheca is thought to be related to Nyssa and Davidia in Nyssaceae, which has sometimes been included in Cornaceae. However, molecular phylogenetic studies confirmed the inclusion of Camptotheca in Nyssaceae and its exclusion from Cornaceae. The aim of this study was to reveal developmental features of the inflorescence and flowers in Camptotheca to compare with related taxa in Cornales. Methods: Inflorescences and flowers of Camptotheca acuminata at all developmental stages were collected and studied with a scanning electron microscope and stereo microscope. Key Results: Camptotheca has botryoids which are composed of several capitate floral units (FUs) that are initiated acropetally. On each FU, flowers are grouped in dyads that are initiated acropetally. All floral organs are initiated centripetally. Calyx lobes are restricted to five teeth. The hypanthium, with five toothed calyx lobes, is adnate to the ovary. The five petals are free and valvate. Ten stamens are inserted in two whorls around the central depression, in which the style is immersed. Three carpels are initiated independently but the ovary is syncarpous and unilocular. The ovule is unitegmic and heterotropous. Inflorescences are functionally andromonoecious varying with the position of the FUs on the inflorescence system. Flowers on the upper FU often have robust styles and fully developed ovules. Flowers on the lower FU have undeveloped styles and aborted ovules, and the flowers on the middle FU are transitional. Conclusions: Camptotheca possesses several traits that unify it with Nyssa, Mastixia and Diplopanax. Inflorescence and floral characters support a close relationship with Nyssaceae and Mastixiaceae but a distant relationship with Cornus. Our results corroborate molecular inferences and support a separate family Nyssaceae.

Hedysarum L. (Fabaceae: Hedysareae) Is Not Monophyletic - Evidence from Phylogenetic Analyses Based on Five Nuclear and Five Plastid Sequences.

The legume family (Fabaceae) exhibits a high level of species diversity and evolutionary success worldwide. Previous phylogenetic studies of the genus Hedysarum L. (Fabaceae: Hedysareae) showed that the nuclear and the plastid topologies might be incongruent, and the systematic position of the Hedysarum sect. Stracheya clade was uncertain. In this study, phylogenetic relationships of Hedysarum were investigated based on the nuclear ITS, ETS, PGDH, SQD1, TRPT and the plastid psbA-trnH, trnC-petN, trnL-trnF, trnS-trnG, petN-psbM sequences. Both nuclear and plastid data support two major lineages in Hedysarum: the Hedysarum s.s. clade and the Sartoria clade. In the nuclear tree, Hedysarum is biphyletic with the Hedysarum s.s. clade sister to the Corethrodendron + Eversmannia + Greuteria + Onobrychis clade (the CEGO clade), whereas the Sartoria clade is sister to the genus Taverniera DC. In the plastid tree, Hedysarum is monophyletic and sister to Taverniera. The incongruent position of the Hedysarum s.s. clade between the nuclear and plastid trees may be best explained by a chloroplast capture hypothesis via introgression. The Hedysarum sect. Stracheya clade is resolved as sister to the H. sect. Hedysarum clade in both nuclear and plastid trees, and our analyses support merging Stracheya into Hedysarum. Based on our new evidence from multiple sequences, Hedysarum is not monophyletic, and its generic delimitation needs to be reconsidered.

Multiple Events of Allopolyploidy in the Evolution of the Racemose Lineages in Prunus (Rosaceae) Based on Integrated Evidence from Nuclear and Plastid Data.

Prunus is an economically important genus well-known for cherries, plums, almonds, and peaches. The genus can be divided into three major groups based on inflorescence structure and ploidy levels: (1) the diploid solitary-flower group (subg. Prunus, Amygdalus and Emplectocladus); (2) the diploid corymbose group (subg. Cerasus); and (3) the polyploid racemose group (subg. Padus, subg. Laurocerasus, and the Maddenia group). The plastid phylogeny suggests three major clades within Prunus: Prunus-Amygdalus-Emplectocladus, Cerasus, and Laurocerasus-Padus-Maddenia, while nuclear ITS trees resolve Laurocerasus-Padus-Maddenia as a paraphyletic group. In this study, we employed sequences of the nuclear loci At103, ITS and s6pdh to explore the origins and evolution of the racemose group. Two copies of the At103 gene were identified in Prunus. One copy is found in Prunus species with solitary and corymbose inflorescences as well as those with racemose inflorescences, while the second copy (II) is present only in taxa with racemose inflorescences. The copy I sequences suggest that all racemose species form a paraphyletic group composed of four clades, each of which is definable by morphology and geography. The tree from the combined At103 and ITS sequences and the tree based on the single gene s6pdh had similar general topologies to the tree based on the copy I sequences of At103, with the combined At103-ITS tree showing stronger support in most clades. The nuclear At103, ITS and s6pdh data in conjunction with the plastid data are consistent with the hypothesis that multiple independent allopolyploidy events contributed to the origins of the racemose group. A widespread species or lineage may have served as the maternal parent for multiple hybridizations involving several paternal lineages. This hypothesis of the complex evolutionary history of the racemose group in Prunus reflects a major step forward in our understanding of diversification of the genus and has important implications for the interpretation of its phylogeny, evolution, and classification.

Polyphyly of the Padus group of Prunus (Rosaceae) and the evolution of biogeographic disjunctions between eastern Asia and eastern North America.

Prunus subgenus Padus is a group with a wide distribution in temperate eastern Asia and eastern North America with one species extending to Europe and one to Central America. Phylogenetic relationships of subgenus Padus were reconstructed using sequences of nuclear ribosomal ITS, and plastid ndhF gene, and rps16 intron and rpl16 intron. Prunus subgenus Padus is shown to be polyphyletic. Taxa of subgenus Padus and subgenus Laurocerasus are highly intermixed in both the ITS and the plastid trees. The results support two disjunctions between eastern North America and Eurasia within the Padus group. One disjunction is between Prunus virginiana of eastern North America and P. padus of Eurasia, estimated to have diverged at 2.99 (95 % HPD 0.59-6.15)-4.1 (95 % HPD 0.63-8.59) mya. The other disjunction is between P. serotina and its Asian relatives. The second disjunction may have occurred earlier than the former one, but the age estimate is difficult due to the unresolved phylogenetic position of the P. serotina complex.