The Primula edelbergii S-locus is an example of a jumping supergene.

Research on supergenes, non-recombining genomic regions housing tightly linked genes that control complex phenotypes, has recently gained prominence in genomics. Heterostyly, a floral heteromorphism promoting outcrossing in several angiosperm families, is controlled by the S-locus supergene. The S-locus has been studied primarily in closely related Primula species and, more recently, in other groups that independently evolved heterostyly. However, it remains unknown whether genetic architecture and composition of the S-locus are maintained among species that share a common origin of heterostyly and subsequently diverged across larger time scales. To address this research gap, we present a chromosome-scale genome assembly of Primula edelbergii, a species that shares the same origin of heterostyly with Primula veris (whose S-locus has been characterized) but diverged from it 18 million years ago. Comparative genomic analyses between these two species allowed us to show, for the first time, that the S-locus can 'jump' (i.e. translocate) between chromosomes maintaining its function in controlling heterostyly. Additionally, we found that four S-locus genes were conserved but reshuffled within the supergene, seemingly without affecting their expression, thus we could not detect changes explaining the lack of self-incompatibility in P. edelbergii. Furthermore, we confirmed that the S-locus is not undergoing genetic degeneration. Finally, we investigated P. edelbergii evolutionary history within Ericales in terms of whole genome duplications and transposable element accumulation. In summary, our work provides a valuable resource for comparative analyses aimed at investigating the genetics of heterostyly and the pivotal role of supergenes in shaping the evolution of complex phenotypes.

The complete chloroplast genome sequences of five pinnate-leaved Primula species and phylogenetic analyses.

The six pinnate-leaved species are a very particular group in the genus Primula. In the present paper, we sequenced, assembled and annotated the chloroplast genomes of five of them (P. cicutarrifolia, P. hubeiensis, P. jiugongshanensis, P. merrilliana, P. ranunculoides). The five chloroplast genomes ranged from ~ 150 to 152 kb, containing 113 genes (four ribosomal RNA genes, 29 tRNA genes and 80 protein-coding genes). The six pinnate-leaved species exhibited synteny of gene order and possessed similar IR boundary regions in chloroplast genomes. The gene accD was pseudogenized in P. filchnerae. In the chloroplast genomes of the six pinnate-leaved Primula species, SSRs, repeating sequences and divergence hotspots were identified; ycf1 and trnH-psbA were the most variable markers among CDSs and noncoding sequences, respectively. Phylogenetic analyses showed that the six Primula species were separated into two distant clades: one was formed by P. filchnerae and P. sinensis and the other clade was consisting of two subclades, one formed by P. hubeiensis and P. ranunculoides, the other by P. merrilliana, P. cicutarrifolia and P. jiugongshanensis. P. hubeiensis was closely related with P. ranunculoides and therefore it should be placed into Sect. Ranunculoides. P. cicutarrifolia did not group first with P. ranunculoides but with P. merrilliana, although the former two were once united in one species, our results supported the separation of P. ranunculoides from P. cicutarrifolia as one distinct species.

Primula vulgaris (primrose) genome assembly, annotation and gene expression, with comparative genomics on the heterostyly supergene.

Primula vulgaris (primrose) exhibits heterostyly: plants produce self-incompatible pin- or thrum-form flowers, with anthers and stigma at reciprocal heights. Darwin concluded that this arrangement promotes insect-mediated cross-pollination; later studies revealed control by a cluster of genes, or supergene, known as the S (Style length) locus. The P. vulgaris S locus is absent from pin plants and hemizygous in thrum plants (thrum-specific); mutation of S locus genes produces self-fertile homostyle flowers with anthers and stigma at equal heights. Here, we present a 411 Mb P. vulgaris genome assembly of a homozygous inbred long homostyle, representing ~87% of the genome. We annotate over 24,000 P. vulgaris genes, and reveal more genes up-regulated in thrum than pin flowers. We show reduced genomic read coverage across the S locus in other Primula species, including P. veris, where we define the conserved structure and expression of the S locus genes in thrum. Further analysis reveals the S locus has elevated repeat content (64%) compared to the wider genome (37%). Our studies suggest conservation of S locus genetic architecture in Primula, and provide a platform for identification and evolutionary analysis of the S locus and downstream targets that regulate heterostyly in diverse heterostylous species.

Morphological and Molecular Phylogenetic Data Reveal a New Species of Primula (Primulaceae) from Hunan, China.

A new species of Primulaceae, Primula undulifolia, is described from the hilly area of Hunan province in south-central China. Its morphology and distributional range suggest that it is allied to P. kwangtungensis, both adapted to subtropical climate, having contiguous distribution and similar habitat, growing on shady and moist cliffs. Petioles, scapes and pedicels of them are densely covered with rusty multicellular hairs, but the new species can be easily distinguished by its smaller flowers and narrowly oblong leaves with undulate margins. Molecular phylogenetic analysis based on four DNA markers (ITS, matK, trnL-F and rps16) confirmed the new species as an independent lineage and constitutes a main clade together with P. kwangtungensis, P. kweichouensis, P. wangii and P. hunanensis of Primula sect. Carolinella.

Sequence capture using RAD probes clarifies phylogenetic relationships and species boundaries in Primula sect. Auricula.

Species-rich evolutionary radiations are a common feature of mountain floras worldwide. However, the frequent lack of phylogenetic resolution in species-rich alpine plant groups hampers progress towards clarifying the causes of diversification in mountains. In this study, we use the largest plant group endemic to the European Alpine system, Primula sect. Auricula, as a model system. We employ a newly developed next-generation-sequencing protocol, involving sequence capture with RAD probes, and map reads to the reference genome of Primula veris to obtain DNA matrices with thousands of SNPs. We use these data-rich matrices to infer phylogenetic relationships in Primula sect. Auricula and examine species delimitations in two taxonomically difficult subgroups: the clades formed by the close relatives of P. auricula and P. pedemontana, respectively. Our molecular dataset enables us to resolve most phylogenetic relationships in the group with strong support, and in particular to infer four well-supported clades within sect. Auricula. Our results support existing species delimitations for P. auricula, P. lutea, and P. subpyrenaica, while they suggest that the group formed by P. pedemontana and close relatives might need taxonomic revision. Finally, we discuss preliminary implications of these findings on the biogeographic history of Primula sect. Auricula.

Phylogeny and biogeography of Primula sect. Armerina: implications for plant evolution under climate change and the uplift of the Qinghai-Tibet Plateau.

BACKGROUND: The historical orogenesis and associated climatic changes of mountain areas have been suggested to partly account for the occurrence of high levels of biodiversity and endemism. However, their effects on dispersal, differentiation and evolution of many groups of plants are still unknown. In this study, we examined the detailed diversification history of Primula sect. Armerina, and used biogeographic analysis and macro-evolutionary modeling to investigate a series of different questions concerning the evolution of the geographical and ecological distribution of the species in this section. RESULTS: We sequenced five chloroplast and one nuclear genes for species of Primula sect. Armerina. Neither chloroplast nor nuclear trees support the monophyly of the section. The major incongruences between the two trees occur among closely related species and may be explained by hybridization. Our dating analyses based on the chloroplast dataset suggest that this section began to diverge from its relatives around 3.55 million years ago, largely coinciding with the last major uplift of the Qinghai-Tibet Plateau (QTP). Biogeographic analysis supports the origin of the section in the Himalayan Mountains and dispersal from the Himalayas to Northeastern QTP, Western QTP and Hengduan Mountains. Furthermore, evolutionary models of ecological niches show that the two P. fasciculata clades have significantly different climatic niche optima and rates of niche evolution, indicating niche evolution under climatic changes and further providing evidence for explaining their biogeographic patterns. CONCLUSION: Our results support the hypothesis that geologic and climatic events play important roles in driving biological diversification of organisms in the QTP area. The Pliocene uplift of the QTP and following climatic changes most likely promoted both the inter- and intraspecific divergence of Primula sect. Armerina. This study also illustrates how niche evolution under climatic changes influences biogeographic patterns.

DNA barcoding evaluation and its taxonomic implications in the species-rich genus Primula L. in China.

The genus Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM) in China as a result of rapid radiation. In order to overcome the difficulty of morphological classification of this genus, we surveyed three plastid regions (rbcL, matK, and trnH-psbA) and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula species across 18 sections, to assess their discriminatory power as barcodes. We found that ITS alone or combined with plastid regions showed the best discrimination across different infrageneric ranks and at species level. We suggest rbcL + matK + ITS as the first choice at present to barcode Primula plants. Although the present barcoding combination performed poorly in many closely related species of Primula, it still provided many new insights into current Primula taxonomy, such as the underlying presence of cryptic species, and several potential improper taxonomic treatments. DNA barcoding is one useful technique in the integrative taxonomy of the genus Primula, but it still requires further efforts to improve its effectiveness in some taxonomically challenging groups.

The draft genome of Primula veris yields insights into the molecular basis of heterostyly.

BACKGROUND: The flowering plant Primula veris is a common spring blooming perennial that is widely cultivated throughout Europe. This species is an established model system in the study of the genetics, evolution, and ecology of heterostylous floral polymorphisms. Despite the long history of research focused on this and related species, the continued development of this system has been restricted due the absence of genomic and transcriptomic resources. RESULTS: We present here a de novo draft genome assembly of P. veris covering 301.8 Mb, or approximately 63% of the estimated 479.22 Mb genome, with an N50 contig size of 9.5 Kb, an N50 scaffold size of 164 Kb, and containing an estimated 19,507 genes. The results of a RADseq bulk segregant analysis allow for the confident identification of four genome scaffolds that are linked to the P. veris S-locus. RNAseq data from both P. veris and the closely related species P. vulgaris allow for the characterization of 113 candidate heterostyly genes that show significant floral morph-specific differential expression. One candidate gene of particular interest is a duplicated GLOBOSA homolog that may be unique to Primula (PveGLO2), and is completely silenced in L-morph flowers. CONCLUSIONS: The P. veris genome represents the first genome assembled from a heterostylous species, and thus provides an immensely important resource for future studies focused on the evolution and genetic dissection of heterostyly. As the first genome assembled from the Primulaceae, the P. veris genome will also facilitate the expanded application of phylogenomic methods in this diverse family and the eudicots as a whole.

Population expanding with the phalanx model and lineages split by environmental heterogeneity: a case study of Primula obconica in subtropical China.

BACKGROUND: Current and historical events have both affected the current distribution patterns and intraspecific divergence of plants. While numerous studies have focused on the Qinghai-Tibetan Plateau (QTP), the impacts of such events on the flora of subtropical China remain poorly understood. Subtropical China is famous for its highly complex topography and the limited impact from glaciation during the Pleistocene; this may have resulted in a different genetic legacy for species in this region compared to fully glaciated areas. METHODOLOGY/PRINCIPAL FINDINGS: We used plastid and nuclear DNA sequence data and distribution modeling to analyze the divergence patterns and demographic history of Primula obconica Hance, a widespread herbaceous montane species in subtropical China. The phylogenetic analysis revealed two major lineages (lineage A and lineage B), representing a west-east split into the Yunnan and Eastern groups, and the Sichuan and Central groups, respectively. The Eastern and Central groups comprised relatively new derived haplotypes. Nested Clade Analysis and Bayesian Skyline Plot analyses both indicated that P. obconica mainly experienced a gradual expansion of populations. In addition, the simulated distribution of P. obconica during the Last Glacial Maximum was slightly larger than its present-day distribution. CONCLUSION/SIGNIFICANCE: Our results are the first to identify a west-east migration of P. obconica. The gradual expansion pattern and a larger potential distribution range in cold periods detected for P. obconica indicate that the population expansion of this species is consistent with the phalanx model. In addition, the current patterns of genetic differentiation have persisted as a result of the extensive environmental heterogeneity that exists in subtropical China.

Phylogenetic analysis of Primula section Primula reveals rampant non-monophyly among morphologically distinct species.

The type section of Primula (Primulaceae), here considered to include seven species, is phylogenetically quite isolated in its genus. Although its species are popular ornamentals, traditional medicinal plants and model organisms for the study of heterostyly, the section has not yet been studied from a phylogenetic or evolutionary perspective. Using phylogenetic analysis of nuclear ITS and plastid data from all species and subspecies, we find that widespread Primula elatior is genetically heterogeneous and non-monophyletic to most if not all of the other ingroup taxa. The Genealogical Sorting Index (GSI) indicates that the assumption of all currently accepted species being independent lineages is consistent with the data. It is possible that P. elatior in its current circumscription may represents the disjointed remnant of an ancestral species from which the other recognized species diverged. However, based on available data, the alternative possibility of introgression explaining the non-monophyly of this species cannot be excluded. Species trees show P. elatior and P. veris as sister species. Primula vulgaris and P. juliae are closely related, while, in contrast to previous assumptions, P. renifolia does not appear to be a close relative of P. megaseifolia. With the section's isolation from the rest of the genus and very short internal branches, our dataset also presents a case study of the confounding effects of different branch length priors on the Bayesian estimation of resulting branch length estimates. Experimental runs using different priors confirm the problem of resulting estimates varying by orders of magnitude, while topology and relative branch lengths seem unaffected.

Polyploid evolution and Pleistocene glacial cycles: A case study from the alpine primrose Primula marginata (Primulaceae).

BACKGROUND: Recent studies highlighted the role of Pleistocene climatic cycles in polyploid speciation and of southern Alpine refugia as reservoirs of diversity during glacial maxima. The polyploid Primula marginata, endemic to the southwestern Alps, includes both hexaploid and dodecaploid cytotypes that show no ecological or morphological differences. We used flow cytometry to determine variation and geographic distribution of cytotypes within and between populations and analyses of chloroplast (cp) and nuclear ribosomal (nr) DNA sequences from the Internal Transcribed Spacer (ITS) region to infer the evolutionary history of the two cytotypes and the auto- vs. allopolyploid origin of dodecaploid populations. RESULTS: We did not detect any intermediate cytotypes or variation of ploidy levels within populations. Hexaploids occur in the western and dodecaploids in the eastern part of the distributional range, respectively. The cpDNA and nrDNA topologies are in conflict, for the former supports shared ancestry between P. marginata and P. latifolia, while the latter implies common origins between at least some ITS clones of P. marginata and P. allionii. CONCLUSIONS: Our results suggest an initial episode of chloroplast capture involving ancestral lineages of P. latifolia and P. marginata, followed by polyploidization between P. marginata-like and P. allionii-like lineages in a southern refugium of the Maritime Alps. The higher proportion of ITS polymorphisms in dodecaploid than in hexaploid accessions of P. marginata and higher total nucleotide diversity of ITS clones in dodecaploid vs. hexaploid individuals sequences are congruent with the allopolyploid hypothesis of dodecaploid origin.

QTL analysis of heterostyly in Primula sieboldii and its application for morph identification in wild populations.

BACKGROUND AND AIMS: Primula sieboldii is a perennial clonal herb that is distributed around the Sea of Japan and is endangered in Japan. Its breeding system is characterized by heteromorphic self-incompatibility, and the morph ratio within a population is very important for reproductive success. The aims of this study were to construct a linkage map, map the S locus as a qualitative trait and quantitative trait loci (QTLs) for floral morphological traits related to heterostyly, and predict the morph type in wild populations by using molecular markers for devising a conservation strategy. METHODS: A linkage map was constructed with 126 markers. The QTLs for four floral traits and the S locus were mapped. Using the genotypes of loci that were located near both the S locus and the QTLs with large effects, morphs of 59 wild genets were predicted. KEY RESULTS: The linkage map consisted of 14 linkage groups (LGs). The S locus was mapped to LG 7. Major QTLs for stigma and anther heights were detected in the same region as the S locus. These QTLs exhibited high logarithm of the odds scores and explained a high percentage of the phenotypic variance (>85 %). By analysing these two traits within each morph, additional QTLs for each trait were detected. Using the four loci linked to the S locus, the morphs of 43 genets in three wild populations could be predicted. CONCLUSIONS: This is the first report of a linkage map and QTL analysis for floral morphology related to heterostyly in P. sieboldii. Floral morphologies related to heterostyly are controlled by the S locus in LG 7 and by several QTLs in other LGs. Additionally, this study showed that molecular markers are effective tools for investigating morph ratios in a population containing the non-flowering individuals or during the non-flowering seasons.

Reconsideration for conservation units of wild Primula sieboldii in Japan based on adaptive diversity and molecular genetic diversity.

Primula sieboldii E. Morren is a perennial clonal herb that is widely distributed in Japan, but in danger of extinction in the wild. In a previous study, we revealed the genetic diversity of the species using chloroplast and nuclear DNA and used this information to define conservation units. However, we lacked information on adaptive genetic diversity, which is important for long-term survival and, thus, for the definition of conservation units. In order to identify adaptive traits that showed adaptive differentiation among populations, we studied the genetic variation in six quantitative traits within and among populations for 3 years in a common garden using 110 genets from five natural populations from three regions of Japan. The number of days to bud initiation was adaptive quantitative trait for which the degree of genetic differentiation among populations (QST) was considerably larger than that in eight microsatellite markers (FST). The relationship between this trait and environmental factors revealed that the number of days to bud initiation was negatively correlated, with the mean temperature during the growing period at each habitat. This suggests that adaptive differentiation in the delay before bud initiation was caused by selective pressure resulting from temperature differences among habitats. Our results suggest that based on adaptive diversity and neutral genetic diversity, the Saitama population represents a new conservation unit.

Exudate flavonoids of Primula spp: structural and biogenetic chemodiversity.

Several new accessions of the genus Primula and of the closely related Cortusa matthioli have been studied for their exudate flavonoid profiles. Unsubstituted flavone, 5-hydroxy-, 2'-hydroxyflavone and 5,2'-dihydroxyflavone were found as main components. Several other rather unusual substitution patterns were also found. 8-O-Substituted flavones appear to be accumulated more often than 6-O-substituted products. Chalcones with corresponding substitution patterns were of scattered occurrence, while flavanones could so far not be detected in exudates of Primula species. The distribution of externally accumulated flavonoids, including literature data, is related to current taxonomic views on the infrageneric grouping of Primula and related taxa. Organ-specific accumulation, biosynthetic and chemosystematic aspects are briefly addressed.

Disentangling reticulate evolution in an arctic-alpine polyploid complex.

Although polyploidy plays a fundamental role in plant evolution, the elucidation of polyploid origins is fraught with methodological challenges. For example, allopolyploid species may confound phylogenetic reconstruction because commonly used methods are designed to trace divergent, rather than reticulate patterns. Recently developed techniques of phylogenetic network estimation allow for a more effective identification of incongruence among trees. However, incongruence can also be caused by incomplete lineage sorting, paralogy, concerted evolution, and recombination. Thus, initial hypotheses of hybridization need to be examined via additional sources of evidence, including the partitioning of infraspecific genetic polymorphisms, morphological characteristics, chromosome numbers, crossing experiments, and distributional patterns. Primula sect. Aleuritia subsect. Aleuritia (Aleuritia) represents an ideal case study to examine reticulation because specific hypotheses have been derived from morphology, karyology, interfertility, and distribution to explain the observed variation of ploidy levels, ranging from diploidy to 14-ploidy. Sequences from 5 chloroplast and 1 nuclear ribosomal DNA (nrDNA) markers were analyzed to generate the respective phylogenies and consensus networks. Furthermore, extensive cloning of the nrDNA marker allowed for the identification of shared nucleotides at polymorphic sites, investigation of infraspecific genetic polymorphisms via principal coordinate analyses PCoAs, and detection of recombination between putative progenitor sequences. The results suggest that most surveyed polyploids originated via hybridization and that 2 taxonomic species formed recurrently from different progenitors, findings that are congruent with the expectations of speciation via secondary contact. Overall, the study highlights the importance of using multiple experimental and analytical approaches to disentangle complex patterns of reticulation.

Comparative phytochemical and morphological analyses of three Italian Primula species.

The taxonomy of alpine Primula species has long been in dispute because of high morphologic variability and several hybridisations. In Primula species, the trichome height and the colour of hair-tips are usually indicated as diacritic characters, but in our experience this is not adequate. The present study, focused on Primula auricula, Primula daonensis and Primula hirsuta, therefore proposes the use of other morphologic trichome parameters (size and dimensional ratio of stalk, neck and gland head). Phytochemical investigations about the flavonoid composition (epicuticular and vacuolar) of leaves, as taxonomic markers, have also been performed. We report the isolation and identification of two new flavonol glycosides, isorhamnetin 3-O-(2,6-di-O-beta-D-glucopyranosyl-beta-D-glucopyranoside) (1) and kaempferol 3-O-(2-O-alpha-L-rhamnopyranosyl-6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) (2) and of eight known flavonoids. Size and dimensional ratio of the three trichome elements (stalk, neck and glandular head) are typical for each species analysed. The flavonoid profile well characterise the entities under study. Three different profiles have been obtained with both vacuolar and epicuticular flavonoids. The morphologic and phytochemical markers proposed in this work seem to be parameters which significatively discriminate the species under study.

Evolution of biogeographic patterns, ploidy levels, and breeding systems in a diploid-polyploid species complex of Primula.

Primula sect. Aleuritia subsect. Aleuritia (Aleuritia) includes diploid, self-incompatible heterostyles and polyploid, self-compatible homostyles, the latter generally occurring at higher latitudes than the former. This study develops a phylogenetic hypothesis for Aleuritia to elucidate the interactions between Pleistocene glacial cycles, biogeographic patterns, ploidy levels and breeding systems. Sequences from five chloroplast DNA loci were analyzed with parsimony to reconstruct a phylogeny, haplotype network, and ancestral states for ploidy levels and breeding systems.The results supported the monophyly of Aleuritia and four major biogeographic lineages: an amphi-Pacific, a South American, an amphi-Atlantic and a European/North American lineage. At least four independent switches to homostyly and five to polyploidy were inferred. An Asian ancestor probably gave origin to an amphi-Pacific clade and to a lineage that diversified on the European and American continents. Switches to homostyly occurred exclusively in polyploid lineages, which mainly occupy previously glaciated areas. The higher success of the autogamous polyploid species at recolonizing habitats freed by glacial retreat might be explained in terms of selection for reproductive assurance.