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.

Development of nuclear and chloroplast polymorphic microsatellites for Crossostephium chinense (Asteraceae).

BACKGROUND: Crossostephium chinense is a traditional Chinese medicinal herb and it is often cultivated as an ornamental plant. Previous studies on this species mainly focused on its chemical composition and it was rarely represented in genetic studies, and thus genomic resources remain scarce. METHODS AND RESULTS: Both chloroplast and nuclear polymorphic microsatellites of C. chinense were screened from genome skimming data of two individuals. 64 and 63 cpSSR markers were identified from two chloroplast genomes of C. chinense. A total of 133 polymorphic nSSRs were developed. Ten nSSRs were randomly selected to test their transferability across 35 individuals from three populations of C. chinense, and 20 individuals each of Artemisia stolonifera and A. argyi. Cross-amplifications were successfully done for C. chinense and were partially amplified for both Artemisia species. The number of alleles varied from two to nine. The observed heterozygosity and expected heterozygosity per locus ranged from 0.000 to 0.286 and from 0.029 to 0.755, respectively. CONCLUSIONS: In this study, we developed polymorphic cpSSRs and nSSRs markers for C. chinense based on genome skimming sequencing. These genomic resources will be valuable for population genetics and conservation studies in C. chinense and Artemisia.

The complete chloroplast genome of Papaver setigerum and comparative analyses in Papaveraceae.

Papaver setigerum is an annual herb that is closely related to the opium poppy, P. somniferum. Genetic resources for P. setigerum are scarce. In the present study, we assembled the complete chloroplast (cp) genome of P. setigerum based on genome skimming data, and we conducted comparative cp genome analyses to study the evolutionary pattern in Papaveraceae. The cp genome of P. setigerum is 152,862 bp in length with a typical quadripartite structure. Comparative analyses revealed no gene rearrangement in the Papaveraceae family, although differences were evident in genome size, gene losses, as well as inverted repeats (IR) region expansion and contraction. The rps15 gene has been lost from the genomes of Meconopsis racemosa, Coreanomecon hylomeconoides, P. orientale, P. somniferum, and P. setigerum, and the ycf15 gene is found only in C. hylomeconoides. Moreover, 13 cpDNA markers, including psbA-trnH, rps16-trnQ, trnS-trnG, trnC-petN, trnE-trnT, trnL-trnF, trnF-ndhJ, petA-psbJ, ndhF-rpl32, rpl32-trnL, ccsA-ndhD, ndhE-ndhG, and rps15-ycf1, were identified with relatively high levels of variation within Papaver, which will be useful for species identification in this genus. Among those markers, psbA-trnH is the best one to distinguish P. somniferum and P. setigerum.

Chloroplast genome analyses and genomic resource development for epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae), using genome skimming data.

BACKGROUND: Epilithic sister genera Oresitrophe and Mukdenia (Saxifragaceae) have an epilithic habitat (rocky slopes) and a parapatric distribution in East Asia, which makes them an ideal model for a more comprehensive understanding of the demographic and divergence history and the influence of climate changes in East Asia. However, the genetic background and resources for these two genera are scarce. RESULTS: The complete chloroplast (cp) genomes of two Oresitrophe rupifraga and one Mukdenia rossii individuals were reconstructed and comparative analyses were conducted to examine the evolutionary pattern of chloroplast genomes in Saxifragaceae. The cp genomes ranged from 156,738 bp to 156,960 bp in length and had a typical quadripartite structure with a conserved genome arrangement. Comparative analysis revealed the intron of rpl2 has been lost in Heuchera parviflora, Tiarella polyphylla, M. rossii and O. rupifraga but presents in the reference genome of Penthorum chinense. Seven cp hotspot regions (trnH-psbA, trnR-atpA, atpI-rps2, rps2-rpoC2, petN-psbM, rps4-trnT and rpl33-rps18) were identified between Oresitrophe and Mukdenia, while four hotspots (trnQ-psbK, trnR-atpA, trnS-psbZ and rpl33-rps18) were identified within Oresitrophe. In addition, 24 polymorphic cpSSR loci were found between Oresitrophe and Mukdenia. Most importantly, we successfully developed 126 intergeneric polymorphic gSSR markers between Oresitrophe and Mukdenia, as well as 452 intrageneric ones within Oresitrophe. Twelve randomly selected intergeneric gSSRs have shown that these two genera exhibit a significant genetic structure. CONCLUSIONS: In this study, we conducted genome skimming for Oresitrophe rupifraga and Mukdenia rossii. Using these data, we were able to not only assemble their complete chloroplast genomes, but also develop abundant genetic resources (cp hotspots, cpSSRs, polymorphic gSSRs). The genomic patterns and genetic resources presented here will contribute to further studies on population genetics, phylogeny and conservation biology in Saxifragaceae.

Inferring spatial patterns and drivers of population divergence of Neolitsea sericea (Lauraceae), based on molecular phylogeography and landscape genomics.

The relative roles of geography, climate and ecology in driving population divergence and (incipient) speciation has so far been largely neglected in studies addressing the evolution of East Asia's island flora. Here, we employed chloroplast and ribosomal DNA sequences and restriction site-associated DNA sequencing (RADseq) loci to investigate the phylogeography and drivers of population divergence of Neolitsea sericea. These data sets support the subdivision of N. sericea populations into the Southern and Northern lineages across the 'Tokara gap'. Two distinct sublineages were further identified for the Northern lineage of N. sericea from the RADseq data. RADseq was also used along with approximate Bayesian computation to show that the current distribution and differentiation of N. sericea populations resulted from a combination of relatively ancient migration and successive vicariant events that likely occurred during the mid to late Pleistocene. Landscape genomic analyses showed that, apart from geographic barriers, barrier, potentially local adaptation to different climatic conditions appears to be one of the major drivers for lineage diversification of N. sericea.

Rhizosphere microbial community assembly and association networks strongly differ based on vegetation type at a local environment scale.

Introduction: Rhizosphere microbes perform critical functions for their hosts, and their structure is strongly influenced by vegetation type. Although studies on the effects of vegetation on rhizosphere microbial community structure have been conducted at large and global environment scales, studies at local environment scales would eliminate numerous external factors such as climate and soil type, while highlighting the potential influence of local vegetation type. Methods: Here, we compared rhizosphere microbial communities using 54 samples under three vegetation types (herb, shrubs, and arbors, with bulk soil as the control) at the campus of Henan University. 16S rRNA and ITS amplicons were sequenced using Illumina high throughput sequencing. Results and Discussion: Rhizosphere bacterial and fungal community structures were influenced considerably by vegetation type. Bacterial alpha diversity under herbs was significantly different from that under arbors and shrubs. The abundance of phyla such as Actinobacteria was extremely higher in bulk soil than in the rhizosphere soils. Herb rhizosphere harbored more unique species than other vegetation type soils. Furthermore, bacterial community assembly in bulk soil was more dominated by deterministic process, whereas the rhizosphere bacterial community assembly was dominated by stochasticity and the construction of fungal communities was all dominated by deterministic processes. In addition, rhizosphere microbial networks were less complex than bulk soil networks, and their keystone species differed based on vegetation type. Notably, bacterial community dissimilarities were strongly correlated with plant phylogenetic distance. Exploring rhizosphere microbial community patterns under different vegetation types could enhance our understanding of the role of rhizosphere microbes in ecosystem function and service provision, as well as basic information that could facilitate plant and microbial diversity conservation at the local environment scale.

Phylogenomic and syntenic data demonstrate complex evolutionary processes in early radiation of the rosids.

Some of the most vexing problems of deep level relationship that remain in angiosperms involve the superrosids. The superrosid clade contains a quarter of all angiosperm species, with 18 orders in three subclades (Vitales, Saxifragales and core rosids) exhibiting remarkable morphological and ecological diversity. To help resolve deep-level relationships, we constructed a high-quality chromosome-level genome assembly for Tiarella polyphylla (Saxifragaceae) thus providing broader genomic representation of Saxifragales. Whole genome microsynteny analysis of superrosids showed that Saxifragales shared more synteny clusters with core rosids than Vitales, further supporting Saxifragales as more closely related with core rosids. To resolve the ordinal phylogeny of superrosids, we screened 122 single copy nuclear genes from genomes of 36 species, representing all 18 superrosid orders. Vitales were recovered as sister to all other superrosids (Saxifragales + core rosids). Our data suggest dramatic differences in relationships compared to earlier studies within core rosids. Fabids should be restricted to the nitrogen-fixing clade, while Picramniales, the Celastrales-Malpighiales (CM) clade, Huerteales, Oxalidales, Sapindales, Malvales and Brassicales formed an "expanded" malvid clade. The Celastrales-Oxalidales-Malpighiales (COM) clade (sensu APG IV) was not monophyletic. Crossosomatales, Geraniales, Myrtales and Zygophyllales did not belong to either of our well-supported malvids or fabids. There is strong discordance between nuclear and plastid phylogenetic hypotheses for superrosid relationships; we show that this is best explained by a combination of incomplete lineage sorting and ancient reticulation.

Phylogenomics and integrative taxonomy reveal two new species of Amana (Liliaceae).

Until now the genus Amana (Liliaceae), known as 'East Asian tulips', has contained just seven species. In this study, a phylogenomic and integrative taxonomic approach was used to reveal two new species, Amana nanyueensis from Central China and A. tianmuensis from East China. A. nanyueensis resembles Amana edulis in possessing a densely villous-woolly bulb tunic and two opposite bracts, but differs in its leaves and anthers. Amana tianmuensis resembles Amana erythronioides in possessing three verticillate bracts and yellow anthers, but differs in aspects of its leaves and bulbs. These four species are clearly separated from each other in principal components analysis based on morphology. Phylogenomic analyses based on plastid CDS further support the species delimitation of A. nanyueensis and A. tianmuensis and suggests they are closely related to A. edulis. Cytological analysis shows that A. nanyueensis and A. tianmuensis are both diploid (2n = 2x = 24), different from A. edulis, which is either diploid (northern populations) or tetraploid (southern populations, 2n = 4x = 48). The pollen morphology of A. nanyueensis is similar to other Amana species (single-groove germination aperture), but A. tianmuensis is quite different because of the presence of a sulcus membrane, which creates the illusion of double grooves. Ecological niche modelling also revealed a niche differentiation between A. edulis, A. nanyueensis and A. tianmuensis.

Characterization of the complete chloroplast genome of Peltoboykinia tellimoides (Saxifragaceae).

Peltoboykinia tellimoides, which is distributed in Japan and China, is the type species of Peltoboykinia. In this study, we sequenced and assembled the complete chloroplast (cp) genome of P. tellimoides and reconstructed the phylogeny of Saxifragaceae based on the whole cp genome sequences. The cp genome of P. tellimoides was 156,274 bp in length, comprising a pair of inverted repeat regions (25,099 bp) separated by a large single copy region (88,109 bp) and a small single copy region (17,967 bp). The genome encoded 112 unique genes consisting of 78 different protein-coding genes, 30 transfer RNA and four ribosomal RNA genes, with 16 duplicated genes in the inverted repeats. Phylogenetic analysis indicated that P. tellimoides together with three Chrysosplenium species formed a high support clade, which was sister to Micranthes melanocentra.

Complete chloroplast genome of Plantago asiatica and its phylogenetic position in Plantaginaceae.

Plantago asiatica, an herbaceous perennial species of Plantaginaceae, has been used as a traditional herbal medicine plant in China. In this study, the complete chloroplast (cp) genome of P. asiatica was sequenced and assembled using genome skimming data. The cp genome was 165,045 bp in length including the large single-copy (LSC, 82,964 bp) and small single-copy (SSC, 4,633 bp) regions separated by two copies of inverted region (IR, 38,724 bp). The cp genome encoded 113 unique genes, consisting of 79 protein-coding genes, 30 tRNA genes, and four rRNA genes, additionally with 27 duplicated genes in the IR regions. Phylogenetic analysis indicated that the representative species from Plantago was monophyletic and they were divided into four subgenera. P. asiatica belongs to the subgenus Plantago and was sister to P. rigida with high bootstrap value support.

Comparative methylomics and chromatin accessibility analysis in Osmanthus fragrans uncovers regulation of genic transcription and mechanisms of key floral scent production.

Linalool and ionone are two important aromatic components in sweet osmanthus petals, and the regulatory mechanisms that produce these two components remain unclear. In this study, we employed whole-genome methylation sequencing and ATAC-seq technology to analyze the genomic DNA methylation status and chromatin accessibility of the sweet osmanthus cultivars 'Zaohuang' and 'Chenghong Dangui'. Results showed that the promoter region of TPS2, a key gene in the linalool synthesis pathway, was less methylated in 'Chenghong Dangui' than in 'Zaohuang'. The chromatin was more accessible in 'Chenghong Dangui' than in 'Zaohuang', which resulted in a much stronger expression of this gene in 'Chenghong Dangui' than in 'Zaohuang'. This eventually led to a high quantity of linalool and its oxides in the petals of 'Chenghong Dangui', but there were lower levels present in the petals of 'Zaohuang'. These results suggest that DNA methylation and chromatin accessibility play major roles in linalool synthesis in sweet osmanthus. The methylation level of the promoter region of CCD4, a key gene for ionone synthesis, was higher in 'Zaohuang' than in 'Chenghong Dangui'. The chromatin accessibility was lower in 'Zaohuang' than in 'Chenghong Dangui', although the expression of this gene was significantly higher in 'Zaohuang' than in 'Chenghong Dangui'. ChIP-seq analysis and a series of experiments showed that the differential expression of CCD4 and CCD1 in the two cultivars may predominantly be the result of regulation by ERF2 and other transcription factors. However, a 183-bp deletion involving the CCD4 promoter region in 'Chenghong Dangui' may be the main reason for the low expression of this gene in its petals. This study provides an important theoretical basis for improving selective breeding of key floral fragrance components in sweet osmanthus.

Elsholtziazhongyangii (Lamiaceae), a new species from Sichuan, China.

Elsholtziazhongyangii (Lamiaceae), a new species from Sichuan Province, China, is described and illustrated. The new species is morphologically similar to E.feddeif.feddei, but it can be easily distinguished from E.feddeif.feddei by smaller corolla (3.2-3.5 mm vs. 4.5-5.3 mm), bract indumentum (glabrous, except margin ciliate vs. villous, especially on veins abaxially, glabrous adaxially) and bract stalked (ca. 1.2 mm vs. sessile). Phylogenetic analyses, based on two nuclear ribosomal (ETS, ITS) and five plastid (rbcL, matK, trnL-F, ycf1, ycf1-rps15) regions, confirmed that the new species formed a monophyletic clade with robust support. The new species is currently known from western Sichuan.

The complete chloroplast genome sequence of Zelkova sinica and phylogenetic implications in Rosales.

Zelkova sinica is a popular landscape plant in China because of its wide adaptation, strong disease resistance, large crown and beautiful fall color. Here, we assembled the complete chloroplast (cp) genome of Z. sinica based on genome skimming data. The cp genome is 158,924 bp in length including two copies of inverted region (IR, 26,427 bp) separated by the large single copy (LSC, 87,318 bp) and small single copy (SSC, 18,752 bp) regions. It encodes 111 unique genes, containing 77 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, with 18 duplicated genes in the IR regions. Phylogenetic analysis shows that Z. sinica is sister to Z. schneideriana in Ulmaceae family.

Development of genomic resources for the genus Celtis (Cannabaceae) based on genome skimming data.

Celtis is a Cannabaceae genus of 60-70 species of trees, or rarely shrubs, commonly known as hackberries. This woody genus consists of very valuable forest plants that provide important wildlife habitat for birds and mammals. Although previous studies have identified its phylogenetic position, interspecific relationships within Celtis remain unclear. In this study, we generated genome skimming data from five Celtis species to analyze phylogenetic relationships within the genus and develop genome resources. The plastomes of Celtis ranged in length from 158,989 bp to 159,082 bp, with a typical angiosperm quadripartite structure, and encoded a total of 132 genes with 20 duplicated in the IRs. Comparative analyses showed that plastome content and structure were relatively conserved. Whole plastomes showed no signs of gene loss, translocations, inversions, or genome rearrangement. Six plastid hotspot regions (trnH-psbA, psbA-trnK, trnG-trnR, psbC-trnS, cemA-petA and rps8-rpl14), 4097 polymorphic nuclear SSRs, as well as 62 low or single-copy gene fragments were identified within Celtis. Moreover, the phylogenetic relationships based on the complete plastome sequences strongly endorse the placement of C. biondii as sister to the ((((C. koraiensis, C. sinensis), C. tetrandra), C. julianae), C. cerasifera) clade. These findings and the genetic resources developed here will be conducive to further studies on the genus Celtis involving phylogeny, population genetics, and conservation biology.

Complete chloroplast genome of Trophis caucana and its phylogenetic implication in Rosales.

Trophis caucana, which belongs to Moraceae, is a tree species lived in a humid climate at low and middle altitudes. The complete chloroplast (cp) genome of T. caucana was sequenced and assembled in this study. The cp genome is 161,445 bp in length with comprising two copies of inverted region (IR, 25,894 bp) separated by the large single copy (LSC, 89,633 bp) and small single copy (SSC, 20,024 bp) regions. It encodes 111 unique genes, consisting of 77 protein-coding genes, 30 tRNA genes, and four rRNA genes, with 19 duplicated genes in the IR regions. Phylogenetic analysis indicates that T. caucana is sister to Antiaris toxicaria in Moraceae family.

The complete chloroplast genome sequence of purple mullein (Verbascum phoeniceum L.).

Verbascum phoeniceum, known as purple mullein or temptress purple, is a species native to Central Europe, Central Asia, and Western China. In the present study, the chloroplast (cp) genome of V. phoeniceum was assembled using genome skimming sequencing. The cp genome of V. phoeniceum is 153,348 bp in length comprising two copies of inverted regions (IR, 25,430 bp) separated by the large single-copy (LSC, 84,601 bp) and small single copy (SSC, 17,887 bp) regions. It encodes 114 unique genes, consisting of 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, with 20 duplicated genes in the IR regions. Phylogenetic analysis indicates that V. phoeniceum exhibits a closer relationship with Scrophularia rather than Buddleja.

The complete chloroplast genomes of seventeen Aegilops tauschii: genome comparative analysis and phylogenetic inference.

The D genome progenitor of bread wheat, Aegilops tauschii Cosson (DD, 2n = 2x = 14), which is naturally distributed in Central Eurasia, ranging from northern Syria and Turkey to western China, is considered a potential genetic resource for improving bread wheat. In this study, the chloroplast (cp) genomes of 17 Ae. tauschii accessions were reconstructed. The cp genome sizes ranged from 135,551 bp to 136,009 bp and contained a typical quadripartite structure of angiosperms. Within these genomes, we identified a total of 124 functional genes, including 82 protein-coding genes, 34 transfer RNA genes and eight ribosomal RNA genes, with 17 duplicated genes in the IRs. Although the comparative analysis revealed that the genomic structure (gene order, gene number and IR/SC boundary regions) is conserved, a few variant loci were detected, predominantly in the non-coding regions (intergenic spacer regions). The phylogenetic relationships determined based on the complete genome sequences were consistent with the hypothesis that Ae. tauschii populations in the Yellow River region of China originated in South Asia not Xinjiang province or Iran, which could contribute to more effective utilization of wild germplasm resources. Furthermore, we confirmed that Ae. tauschii was derived from monophyletic speciation rather than hybrid speciation at the cp genome level. We also identified four variable genomic regions, rpl32-trnL-UAG, ccsA-ndhD, rbcL-psaI and rps18-rpl20, showing high levels of nucleotide polymorphisms, which may accordingly prove useful as cpDNA markers in studying the intraspecific genetic structure and diversity of Ae. tauschii.

Plastome Evolution in Saxifragaceae and Multiple Plastid Capture Events Involving Heuchera and Tiarella.

Saxifragaceae, a family of over 600 species and approximately 30 genera of herbaceous perennials, is well-known for intergeneric hybridization. Of the main lineages in this family, the Heuchera group represents a valuable model for the analysis of plastid capture and its impact on phylogeny reconstruction. In this study, we investigated plastome evolution across the family, reconstructed the phylogeny of the Heuchera group and examined putative plastid capture between Heuchera and Tiarella. Seven species (11 individuals) representing Tiarella, as well as Mitella and Heuchera, were selected for genome skimming. We assembled the plastomes, and then compared these to six others published for Saxifragaceae; the plastomes were found to be highly similar in overall size, structure, gene order and content. Moreover, ycf15 was lost due to pseudogenization and rpl2 lost its only intron for all the analyzed plastomes. Comparative plastome analysis revealed that size variations of the plastomes are purely ascribed to the length differences of LSC, SSC, and IRs regions. Using nuclear ITS + ETS and the complete plastome, we fully resolved the species relationships of Tiarella, finding that the genus is monophyletic and the Asian species is most closely related to the western North American species. However, the position of the Heuchera species was highly incongruent between nuclear and plastid data. Comparisons of nuclear and plastid phylogenies revealed that multiple plastid capture events have occurred between Heuchera and Tiarella, through putative ancient hybridization. Moreover, we developed numerous molecular markers for Tiarella (e.g., plastid hotspot and polymorphic nuclear SSRs), which will be useful for future studies on the population genetics and phylogeography of this disjunct genus.

The complete chloroplast genome of Butomus umbellatus L. and its phylogenetic position.

Butomus umbellatus, which belongs to Butomaceae, is a plant species typical of littoral communities of river and stream shores. Here, we reported and characterized the complete chloroplast (cp) genome of B. umbellatus and analyzed its phylogenetic position based on the complete cp genome sequences of 10 species within the core Alimatales. The cp genome is 158,107 bp in length, which consists of a large single-copy region (LSC, 88,140 bp； GC content: 34.8%), a small single-copy region (SSC, 19,695 bp; GC content: 30.1%), and a pair of inverted repeat regions (IRs, 25,136 bp; GC content: 42.9%). It harbours 113 unique genes, including 79 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes with an overall GC content of 36.8%. The phylogeny inference showed that Butomaceae and Hydrochariaceae formed a high supported clade, which was sister to Alismataceae. This result was similar to the floral development from petaloid Alismatales, and also similar to the phylogenetic studies of Alismales based on 83 plastid genes, but with much higher bootstrap support.

The complete chloroplast genome of Fagus crenata (subgenus Fagus) and comparison with F. engleriana (subgenus Engleriana).

This study reports the whole chloroplast genome of Fagus crenata (subgenus Fagus), a foundation tree species of Japanese temperate forests. The genome has a total of 158,227 bp containing 111 genes, including 76 protein-coding genes, 31 tRNA genes and 4 ribosomal RNA genes. Comparison with the only other published Fagus chloroplast genome, F. engeleriana (subgenus Engleriana) shows that the genomes are relatively conserved with no inversions or rearrangements observed while the proportion of nucleotide sites differing between the two species was equal to 0.0018. The six most variable regions were, in increasing order of variability, psbK-psbI, trnG-psbfM, rpl32, trnV, ndhI-ndh and ndhD-psaC. These highly variable chloroplast regions in addition to 160 chloroplast microsatellites identified (of which 46 were variable between the two species) will provide useful genetic resources for studies of the inter- and intra-specific genetic structure and diversity of this important northern hemisphere tree genus.