Complete chloroplast genomes and phylogenetic relationships of Pedicularis chinensis and Pedicularis kansuensis.

The complete cp genomes of Pedicularis chinensis (GenBank accession number: OQ587614) and Pedicularis kansuensis (GenBank accession number: OQ587613) were sequenced, assembled, and annotated. Their chloroplast (cp) genome lengths were 146,452 bp, and 146,852 bp, respectively; 120 and 116 genes were identified, comprising 75 and 72 protein-coding genes (PCGs), 37 and 36 transfer RNA (tRNA) genes, and 8 and 8 ribosomal RNA (rRNA) genes, for P. chinensis and P. kansuensis, respectively. A simple sequence repeat (SSR) analysis revealed that the repetitive sequences were mainly composed of mononucleotide repeats (A/T motif) and dinucleotide repeats (AT/TA motif). Comparative genomics identified several variant genes (rpl22, rps19, rpl12, ycf1, trnH, psbA, and ndhH) and variant regions (trnS-GGA, trnV-UAC, ndhJ-trnV, ycf4-cemA, ndhE-nhdG, and rpl32-trnL) with a high Pi, indicating the potential to serve as deoxyribo nucleic acid (DNA) barcodes for Pedicularis species identification. The results show that the cp genomes of P. chinensis and P. kansuensis were the same as those of other plants in Pedicularis, with different degrees of AT preference for codons. Large differences in the number of SSRs and the expansion of the inverted repeat (IR) region showed strong variability and interspecific differentiation between these two species and other species represented in the genus Pedicularis. A phylogenetic analysis showed that P. kansuensis had the closest relationship with P. oliveriana, and P. chinensis had the closest relationship with P. aschistorhyncha. These results will facilitate the study of the phylogenetic classification and interspecific evolution of Pedicularis plants.

Chromosome-level genome assembly of Pedicularis kansuensis illuminates genome evolution of facultative parasitic plant.

Parasitic plants have a heterotrophic lifestyle, in which they withdraw all or part of their nutrients from their host through the haustorium. Despite the release of many draft genomes of parasitic plants, the genome evolution related to the parasitism feature of facultative parasites remains largely unknown. In this study, we present a high-quality chromosomal-level genome assembly for the facultative parasite Pedicularis kansuensis (Orobanchaceae), which invades both legume and grass host species in degraded grasslands on the Qinghai-Tibet Plateau. This species has the largest genome size compared with other parasitic species, and expansions of long terminal repeat retrotransposons accounting for 62.37% of the assembly greatly contributed to the genome size expansion of this species. A total of 42,782 genes were annotated, and the patterns of gene loss in P. kansuensis differed from other parasitic species. We also found many mobile mRNAs between P. kansuensis and one of its host species, but these mobile mRNAs could not compensate for the functional losses of missing genes in P. kansuensis. In addition, we identified nine horizontal gene transfer (HGT) events from rosids and monocots, as well as one single-gene duplication events from HGT genes, which differ distinctly from that of other parasitic species. Furthermore, we found evidence for HGT through transferring genomic fragments from phylogenetically remote host species. Taken together, these findings provide genomic insights into the evolution of facultative parasites and broaden our understanding of the diversified genome evolution in parasitic plants and the molecular mechanisms of plant parasitism.

Plastid NDH Pseudogenization and Gene Loss in a Recently Derived Lineage from the Largest Hemiparasitic Plant Genus Pedicularis (Orobanchaceae).

The plastid genome (plastome) is highly conserved in both gene order and content and has a lower mutation rate than the nuclear genome. However, the plastome is more variable in heterotrophic plants. To date, most such studies have investigated just a few species or only holoheterotrophic groups, and few have examined plastome evolution in recently derived lineages at an early stage of transition from autotrophy to heterotrophy. In this study, we investigated the evolutionary dynamics of plastomes in the monophyletic and recently derived Pedicularis sect. Cyathophora (Orobanchaceae). We obtained 22 new plastomes, 13 from the six recognized species of section Cyathophora, six from hemiparasitic relatives and three from autotrophic relatives. Comparative analyses of gene content, plastome structure and selection pressure showed dramatic differences among species in section Cyathophora and in Pedicularis as a whole. In comparison with autotrophic relatives and other Pedicularis spp., we found that the inverted repeat (IR) region in section Cyathophora had expansions to the small single-copy region, with a large expansion event and two independent contraction events. Moreover, NA(D)H dehydrogenase, accD and ccsA have lost function multiple times, with the function of accD being replaced by nuclear copies of an accD-like gene in Pedicularis spp. The ccsA and ndhG genes may have evolved under selection in association with IR expansion/contraction events. This study is the first to report high plastome variation in a recently derived lineage of hemiparasitic plants and therefore provides evidence for plastome evolution in the transition from autotrophy to heterotrophy.

An explicit test of Pleistocene survival in peripheral versus nunatak refugia in two high mountain plant species.

Pleistocene climate fluctuations had profound influence on the biogeographical history of many biota. As large areas in high mountain ranges were covered by glaciers, biota were forced either to peripheral refugia (and possibly beyond to lowland refugia) or to interior refugia (nunataks). However, nunatak survival remains controversial as it relies solely on correlative genetic evidence. Here, we test hypotheses of glacial survival using two high alpine plant species (the insect-pollinated Pedicularis asplenifolia and wind-pollinated Carex fuliginosa) in the European Alps. Employing the iDDC (integrative Distributional, Demographic and Coalescent) approach, which couples species distribution modelling, spatial and temporal demographic simulation and Approximate Bayesian Computation, we explicitly test three hypotheses of glacial survival: (a) peripheral survival only, (b) nunatak survival only and (c) peripheral plus nunatak survival. In P. asplenifolia the peripheral plus nunatak survival hypothesis was supported by Bayes factors (BF> 100), whereas in C. fuliginosa the peripheral survival only hypothesis, although best supported, could not be unambiguously distinguished from the peripheral plus nunatak survival hypothesis (BF = 5.58). These results are consistent with current habitat preferences (P. asplenifolia extends to higher elevations) and the potential for genetic swamping (i.e., replacement of local genotypes via hybridization with immigrating genotypes [expected to be higher in the wind-pollinated C. fuliginosa]). Although the persistence of plants on nunataks during glacial periods has been debated and studied over decades, this is one of the first studies to explicitly test the hypothesis instead of solely using correlative evidence.

Nectar supplementation changes pollinator behaviour and pollination mode in Pedicularis dichotoma: implications for evolutionary transitions.

Backgrounds and Aims: Gain or loss of floral nectar, an innovation in floral traits, has occurred in diverse lineages of flowering plants, but the causes of reverse transitions (gain of nectar) remain unclear. Phylogenetic studies show multiple gains and losses of floral nectar in the species-rich genus Pedicularis. Here we explore how experimental addition of nectar to a supposedly nectarless species, P. dichotoma, influences pollinator foraging behaviour. Methods: The liquid (nectar) at the base of the corolla tube in P. dichotoma was investigated during anthesis. Sugar components were measured by high-performance liquid chromatography. To understand evolutionary transitions of nectar, artificial nectar was added to corolla tubes and the reactions of bumble-bee pollinators to extra nectar were examined. Key Results: A quarter of unmanipulated P. dichotoma plants contained measurable nectar, with 0.01-0.49 µL per flower and sugar concentrations ranging from 4 to 39 %. The liquid surrounding the ovaries in the corolla tubes was sucrose-dominant nectar, as in two sympatric nectariferous Pedicularis species. Bumble-bees collected only pollen from control (unmanipulated) flowers of P. dichotoma, adopting a sternotribic pollination mode, but switched to foraging for nectar in manipulated (nectar-supplemented) flowers, adopting a nototribic pollination mode as in nectariferous species. This altered foraging behaviour did not place pollen on the ventral side of the bees, and sternotribic pollination also decreased. Conclusion: Our study is the first to quantify variation in nectar production in a supposedly 'nectarless' Pedicularis species. Flower manipulations by adding nectar suggested that gain (or loss) of nectar would quickly result in an adaptive behavioural shift in the pollinator, producing a new location for pollen deposition and stigma contact without a shift to other pollinators. Frequent gains of nectar in Pedicularis species would be beneficial by enhancing pollinator attraction in unpredictable pollination environments.

Horizontal Gene Transfer in Five Parasite Plant Species in Orobanchaceae.

We sequenced genomes of five parasite species in family Orobanchaceae to explore the evolutionary role of horizontal gene transfer in plants. Orobanche minor and Aeginetia indica are obligate parasites with no photosynthetic activity, whereas the other three (Pedicularis keiskei, Phtheirospermum japonicum, and Melampyrum roseum) are facultative parasites. By using reference genome sequences and/or transcriptomes of 14 species from Fabaceae and Poaceae, their major host families, we detected 106 horizontally transferred genes (HGT genes), only in the genomes of the two obligate parasites (22 and 84 for Oro. minor and Ae. indica, respectively), whereas none in the three facultative parasites. The HGT genes, respectively, account for roughly 0.1% and 0.2% of the coding genes in the two species. We found that almost all HGT genes retained introns at the same locations as their homologs in potential host species, indicating a crucial role of DNA-mediated gene transfer, rather than mRNA mediated retro transfer. Furthermore, some of the HGT genes might have transferred simultaneously because they located very closely in the host reference genome, indicating that the length of transferred DNA could exceed 100 kb. We confirmed that almost all introns are spliced in the current genome of the parasite species, and that about half HGT genes do not have any missense mutations or frameshift-causing indels, suggesting that some HGT genes may be still functional. Evolutionary analyses revealed that the nonsynonymous-synonymous substitution ratio is on average elevated on the lineage leading to HGT genes, due to either relaxation of selection or positive selection.

Phylogenetic approaches resolve taxonomical confusion in Pedicularis (Orobanchaceae): Reinstatement of Pedicularis delavayi and discovering a new species Pedicularis milliana.

Morphological identification of Pedicularis depends on floral characters. However, some important characters may be lost during the process of pressing the specimen. Pedicularis delavayi was described from northwestern Yunnan, and widely adopted as a variety of P. siphonantha. Unfortunately, the name "P. siphonantha var. delavayi' incorrectly referred to P. milliana (a new species described in this study) or P. tenuituba in some herbarium specimens and publications. Moreover, phylogenetic relationships among P. delavayi, P. siphonantha and its allies (P. milliana and P. tenuituba) were not fully resolved. In this study, we sampled 76 individuals representing 56 taxa. Of them, 10 taxa were from P. siphonantha lineage, and 11 individuals of P. delavayi represented 9 populations. These species were named as P. siphonantha group on the basis of morphological similarity. Nuclear ribosomal internal transcribed spacer (nrITS) and four chloroplast genes/regions were used for phylogenetic analyses. Phylogenetic analyses showed that the P. siphonantha group was polyphyletic: P. delavayi was sister to P. obliquigaleata in clade A; and the remaining species of P. siphonantha group were monophyletic in clade B, named as P. siphonantha lineage. In the P. siphonantha lineage, P. milliana, P. siphonantha, and P. tenuituba were well supported as monophyletic, and P. dolichosiphon was sister to P. leptosiphon. Morphologically, P. delavayi differs from species of the P. siphonantha lineage in having a long petiole (~ 50 mm) and pedicel (~ 40 mm), a ridged corolla tube, and a folded lower-lip of the corolla. Therefore, both morphological characters and phylogenetic evidence strongly supported to reinstate P. delavayi as an independent species and describe P. milliana as new species. In addition, P. neolatituba was proposed to reduce as a new synonymy of P. delavayi.

Resolving interspecific relationships within evolutionarily young lineages using RNA-seq data: An example from Pedicularis section Cyathophora (Orobanchaceae).

Phylogenomics has shown great potential in resolving evolutionary relationships at different taxonomical levels. However, it remains controversial whether all orthologous genes under different selective pressures can be concatenated for phylogenomic reconstruction. Here we used sect. Cyathophora of Pedicularis, one of the most species-rich genera of angiosperms in the alpine and arctic regions of the Northern Hemisphere, as a model to investigate the efficiency of RNA-seq in resolving relationships of closely related congeneric species. Flower transcriptomes were sequenced for all species of sect. Cyathophora and two outgroup species. Forty-one highly conserved single-/low-copy nuclear genes and 1553 orthologous groups (OGs) were identified and concatenated into five datasets based on gene copy numbers and Ka/Ks values to reconstruct the phylogeny of section Cyathophora. We also tested how many genes minimally can resolve the interspecific relationships, and reconstructed the evolutionary history of some floral characters in sect. Cyathophora. The results showed that the five different datasets consistently resolved the interspecific relationships of sect. Cyathophora, and the interspecific relationships can be robustly reconstructed with maximal support when ⩾20 single-/low-copy nuclear genes or 25 OGs are used. Our study suggests that all OGs under different selective pressures can be concatenated for phylogenomic reconstruction, and provides a successful and efficient use of RNA-seq in reconstructing interspecific relationships of a non-model plant group with recent radiations.

Long-Distance Dispersal after the Last Glacial Maximum (LGM) Led to the Disjunctive Distribution of Pedicularis kansuensis (Orobanchaceae) between the Qinghai-Tibetan Plateau and Tianshan Region.

Quaternary climate fluctuations have profoundly affected the current distribution patterns and genetic structures of many plant and animal species in the Qinghai-Tibetan Plateau (QTP) and adjacent mountain ranges, e.g. Tianshan (TSR), Altay, etc. In this greater area disjunct distributions are prominent but have nevertheless received little attention with respect to the historical processes involved. Here, we focus on Pedicularis kansuensis to test whether the current QTP and TSR disjunction is the result of a recent Holocene range expansion involving dispersal across arid land bridge(s) or a Pleistocene range fragmentation involving persistence in refugia. Two chloroplast DNA spacers were sequenced for 319 individuals from 34 populations covering the entire distribution range of this species in China. We found a total of 17 haplotypes of which all occurred in the QTP, and only five in the TSR. Overall genetic diversity was high (HT = 0.882, HS = 0.559) and higher in the QTP than in the TSR. Genetic differentiation among regions and populations was relatively low (GST = 0.366) and little evidence for a phylogeographic pattern emerged. The divergence times for the four main lineages could be dated to the early Pleistocene. Surprisingly, the two ubiquitous haplotypes diverged just before or around the Last Glacial Maximum (LGM) and were found in different phylogenetic lineages. The Species Distribution Model suggested a disappearance of P. kansuensis from the TSR during the LGM in contrast to a relatively constant potential distribution in the QTP. We conclude that P. kansuensis colonized the TSR after the LGM. The improbable long-distance dispersal by wind or water across arid land seed flow may well have had birds or men as vector.

Geographic consistency and variation in conflicting selection generated by pollinators and seed predators.

BACKGROUNDS AND AIMS: Floral traits that attract pollinators may also attract seed predators, which, in turn, may generate conflicting natural selection on such traits. Although such selection trade-offs are expected to vary geographically, few studies have investigated selection mediated by pollinators and seed predators across a geographic mosaic of environments and floral variation. METHODS: Floral traits were investigated in 14 populations of the bumble-bee-pollinated herb, Pedicularis rex, in which tubular flowers are subtended by cupular bracts holding rain water. To study potentially conflicting selection on floral traits generated by pollinators and florivores, stigmatic pollen loads, initial seed set, pre-dispersal seed predation and final viable seed production were measured in 12-14 populations in the field. KEY RESULTS: Generalized Linear Model (GLM) analyses indicated that the pollen load on stigmas was positively related to the exsertion of the corolla beyond the cupular bracts and size of the lower corolla lip, but so too was the rate of seed predation, creating conflicting selection on both floral traits. A geographic mosaic of selection mediated by seed predators, but not pollinators, was indicated by significant variation in levels of seed predation and the inclusion of two-, three- and four-way interaction terms between population and seed predation in the best model [lowest corrected Akaike Information Criterion (AICc)] explaining final seed production. CONCLUSIONS: These results indicate opposing selection in operation: pollinators generated selection for greater floral exsertion beyond the bracts, but seed predators generated selection for reduced exsertion above the protective pools of water, although the strength of the latter varied across populations.

Towards a comprehensive phylogeny of the large temperate genus Pedicularis (Orobanchaceae), with an emphasis on species from the Himalaya-Hengduan Mountains.

BACKGROUND: Striking interspecific variations in floral traits of the large temperate genus Pedicularis have given rise to controversies concerning infra-generic classifications. To date, phylogenetic relationships within the genus have not been well resolved. The main goal of this study is to construct a backbone phylogeny of Pedicularis, with extensive sampling of species from the Himalaya-Hengduan Mountains. Phylogenetic analyses included 257 species, representing all 13 informal groups and 104 out of 130 series in the classification system of Tsoong, using sequences of the nuclear ribosomal internal transcribed spacer (nrITS) and three plastid regions (matK, rbcL and trnL-F). Bayesian inference and maximum likelihood methods were applied in separate and combined analyses of these datasets. RESULTS: Thirteen major clades are resolved with strong support, although the backbone of the tree is poorly resolved. There is little consensus between the phylogenetic tree and Tsoong's classification of Pedicularis. Only two of the 13 groups (15.4 %), and 19 of the 56 series (33.9 %) with more than one sampled species were found to be strictly monophyletic. Most opposite-/whorled-leaved species fall into a single clade, i.e. clade 1, while alternate leaves species occur in the remaining 12 clades. Excluding the widespread P. verticillata in clade 1, species from Europe and North America fall into clades 6-8. CONCLUSIONS: Our results suggest that combinations of morphological and geographic characters associated with strongly supported clades are needed to elucidate a comprehensive global phylogeny of Pedicularis. Alternate leaves are inferred to be plesiomorphic in Pedicularis, with multiple transitions to opposite/whorled phyllotaxy. Alternate-leaved species show high diversity in plant habit and floral forms. In the Himalaya-Hengduan Mountains, geographical barriers may have facilitated diversification of species with long corolla tubes, and the reproductive advantages of beakless galeas in opposite-/whorled-leaved species may boost speciation at high altitude.

[Predicting the dispersal routes of alpine plant Pedicularis longiflora (Orobanchaceae) based on GIS and species distribution models].

Pedicularis longiflora experienced extensive populations' expansion in Quaternary, but the dispersal corridors were still unclear. According to the distribution patterns of haplotypes based on chloroplast DNA variation, the dispersal routes were predicted using species distribution models (MXENT) and the least-cost path method. Two possible dispersal routes from the southeastern part of Tibetan Plateau (TP) to interior were identified. The populations of East Himalayas-Hengduan Mountains region expanded to the western part of TP along with the Yarlung Zangbo River valley and the lower altitudes of the north slope of Himalayas. The expansion trend was also proved by SDMs based on two historical periods containing the Last Interglacial and the Last Glacial Maximum. In conclusion, identification of dispersal routes is significant to the evolutionary history of alpine plants and the protection of special species in TP.

High mountain origin, phylogenetics, evolution, and niche conservatism of arctic lineages in the hemiparasitic genus Pedicularis (Orobanchaceae).

The origin of the arctic flora covering the northernmost treeless areas is still poorly understood. Arctic plants may have evolved in situ or immigrated from the adjacent ecosystems. Frequently arctic species have disjunctive distributions between the Arctic and high mountain systems of the temperate zone. This pattern may result from long distance dispersal or from glacial plant migrations and extinctions of intermediate populations. The hemiparasitic genus Pedicularis is represented in the Arctic by c. 28 taxa and ranks among the six most species-rich vascular plant genera of this region. In this study, we test the hypothesis that these lineages evolved from predecessors occurring in northern temperate mountain ranges, many of which are current centers of diversity for the genus. We generated a nuclear ribosomal and chloroplast DNA phylogeny including almost all of the arctic taxa and nearly half of the genus as a whole. The arctic taxa of Pedicularis evolved 12-14 times independently and are mostly nested in lineages that otherwise occur in the high mountains of Eurasia and North America. It appears that only three arctic lineages arose from the present-day center of diversity of the genus, in the Hengduan Mountains and Himalayas. Two lineages are probably of lowland origin. Arctic taxa of Pedicularis show considerable niche conservatism with respect to soil moisture and grow predominantly in moist to wet soils. The studied characteristics of ecology, morphology, and chromosome numbers of arctic Pedicularis show a heterogeneous pattern of evolution. The directions of morphological changes among the arctic lineages show opposing trends. Arctic taxa are chiefly diploid, the few tetraploid chromosome numbers of the genus were recorded only for arctic taxa. Five arctic Pedicularis are annuals or biennials, life forms otherwise rare in the Arctic. Other genera of the Orobanchaceae consist also of an elevated number of short-lived species, thus hemiparasitism may favor this life form in the Arctic.

Incongruence between nuclear and chloroplast DNA phylogenies in pedicularis section Cyathophora (Orobanchaceae).

Pedicularis section Cyathophora is a monophyletic group characterized by perfoliate leaf and/or bract bases at each node. This section comprises four series, corresponding to four general corolla types of Pedicularis, i.e. toothless, toothed, beaked and long-tubed corollas. In this study, we aim to reconstruct a comprehensive phylogeny of section Cyathophora, and compare phylogenetic incongruence between nuclear and chloroplast datasets. Sixty-seven accessions belonging to section Cyathophora and 9 species for other Pedicularis were sampled, and one nuclear gene (nrITS) and four chloroplast genes (matK, rbcL, trnH-psbA and trnL-F) were sequenced. Phylogenetic analyses show that the topologies and networks inferred from nrITS and the concatenated chloroplast datasets were incongruent, and the nrITS phylogenies and network agreed with the morphology-based taxonomy to some degree. The chloroplast genome of two Sichuan samples of P. cyathophylloides (E4 and E5) may show introgression from an ancestor of P. cyathophylla. Neither the nrITS dataset nor the concatenated chloroplast dataset were able to adequately resolve relationships among species in the series Reges; this is most likely due to incomplete lineage sorting and/or introgression/hybridization. The nrITS phylogeny indicates the beakless (toothed and toothless) and beaked galeas may have evolved independently within section Cyathophora, and the chloroplast phylogeny reveals that the long corolla tube with beaked galea is derived from the short one.

Extensive population expansion of Pedicularis longiflora (Orobanchaceae) on the Qinghai-Tibetan Plateau and its correlation with the Quaternary climate change.

The Qinghai-Tibetan Plateau (QTP) is thought to be more strongly affected by the Quaternary glaciations than most other regions of the same latitude. It would be of great interest to investigate the population genetic structure of organisms distributed on the platform and its correlation with the Quaternary climatic oscillations. Here we used the chloroplast (cp)DNA trnT-trnF sequence to study genetic variation and phylogeography of Pedicularis longiflora, an alpine herb with extensive distribution on the QTP. Based on a range-wide sampling comprising 41 populations and 910 individuals, we detected 30 cpDNA haplotypes that were divided into five clades by phylogenetic and network analyses and a strong phylogeographical structure. All haplotypes but one in the three basal clades occur exclusively in the southeast QTP, whereas haplotypes in the young clade V occupy almost the whole species range. In particular, the young haplotype H18 occurs in 420 individuals, even at a frequency of 100% in some QTP platform populations and the Altai population. The haplotype distribution pattern, together with molecular clock estimation and mismatch distribution analysis, suggests that the southeast QTP was either a refuge for P. longiflora during the Quaternary climatic change or is the place of origin of the species. The present wide distribution of the species on the QTP platform has resulted from recent population expansions which could be dated back to 120,000-17,000 years ago, a period mostly before the last glacial maximum. The possible relationships among geographic genetic structure, climatic change and species diversification in Pedicularis are also discussed.

Chloroplast DNA phylogeography of Pedicularis ser. Gloriosae (Orobanchaceae) in Japan.

Phylogeographic analyses using chloroplast DNA (cpDNA) variation were performed for Pedicularis ser. Gloriosae (Orobanchaceae). Eighty-one plants of 18 populations of 6 species (P. gloriosa, P. iwatensis, P. nipponica, P. ochiaiana, P. sceptrum-carolinum and P. grandiflora) were analyzed. Fifteen distinct haplotypes were identified based on six cpDNA regions: the intergenic spacer between the trnT and trnL 3'exon, trnL 3'exon-trnF, atpB-rbcL, accD-psaI, the rpl16 intron and the trnK region (including the matK gene). Via phylogenetic analyses of the haplotypes, two continental species, P. sceptrum-carolinum and P. grandiflora, were placed at the most ancestral position in the trees. The former species is widely distributed in the Eurasian continent, and the latter is distributed in Far East Asia. Two robust major cpDNA clades (clades I and II) were revealed in the Japanese archipelago, although the statistical values of monophyly of these clades were weak. Clade I included the haplotypes (A-1, A-2, B-1, B-2 and J) of three species (P. gloriosa, P. iwatensis and P. ochiaiana), and Clade II included seven haplotypes (C-D, E-1, E-2 and F-H) of P. nipponica. These results suggest that this series originated on the Eurasian continent and that subsequently populations at the eastern edge of the continent differentiated into the two Japanese lineages.