Plastome phylogenomics and morphological traits analyses provide new insights into the phylogenetic position, species delimitation and speciation of Triplostegia (Caprifoliaceae).

BACKGROUND: The genus Triplostegia contains two recognized species, T. glandulifera and T. grandiflora, but its phylogenetic position and species delimitation remain controversial. In this study, we assembled plastid genomes and nuclear ribosomal DNA (nrDNA) cistrons sampled from 22 wild Triplostegia individuals, each from a separate population, and examined these with 11 recently published Triplostegia plastomes. Morphological traits were measured from herbarium specimens and wild material, and ecological niche models were constructed. RESULTS: Triplostegia is a monophyletic genus within the subfamily Dipsacoideae comprising three monophyletic species, T. glandulifera, T. grandiflora, and an unrecognized species Triplostegia sp. A, which occupies much higher altitude than the other two. The new species had previously been misidentified as T. glandulifera, but differs in taproot, leaf, and other characters. Triplotegia is an old genus, with stem age 39.96 Ma, and within it T. glandulifera diverged 7.94 Ma. Triplostegia grandiflora and sp. A diverged 1.05 Ma, perhaps in response to Quaternary climate fluctuations. Niche overlap between Triplostegia species was positively correlated with their phylogenetic relatedness. CONCLUSIONS: Our results provide new insights into the species delimitation of Triplostegia, and indicate that a taxonomic revision of Triplostegia is needed. We also identified that either rpoB-trnC or ycf1 could serve as a DNA barcode for Triplostegia.

Target sequence capture data shed light on the deeper evolutionary relationships of subgenus Chamaecerasus in Lonicera (Caprifoliaceae).

The genus Lonicera L. is widely distributed in the north temperate zone and is well-known for its high species richness and morphological diversity. Previous studies have suggested that many sections of Lonicera are not monophyletic and phylogenetic relationships within the genus are still poorly resolved. In this study, we sampled 37 accessions of Lonicera, covering four sections of subgenus Chamaecerasus plus six outgroup taxa, to recover the main clades of Lonicera based on sequences of nuclear loci generated by target enrichment and cpDNA from genome skimming. We found extensive cytonuclear discordance across the subgenus. Both nuclear and plastid phylogenetic analyses supported subgenus Chamaecerasus sister to subgenus Lonicera. Within subgenus Chamaecerasus, sections Isika and Nintooa were each polyphyletic. Based on the nuclear and chloroplast phylogenies, we propose to merge Lonicera korolkowii into section Coeloxylosteum and Lonicera caerulea into section Nintooa. In addition, Lonicera is estimated to have originated in the mid Oligocene (26.45 Ma). The stem age of section Nintooa was estimated to be 17.09 Ma (95% HPD: 13.30-24.45). The stem age of subgenus Lonicera was estimated to be 16.35 Ma (95% HPD: 14.12-23.66). Ancestral area reconstruction analyses indicate that subgenus Chamaecerasus originated in East Asia and Central Asia. In addition, sections Coeloxylosteum and Nintooa originated in East Asia, with subsequent dispersals into other areas. The aridification of the Asian interior likely promoted the rapid radiation of sections Coeloxylosteum and Nintooa within this region. Moreover, our biogeographic analysis fully supports the Bering and the North Atlantic Land Bridge hypotheses for the intercontinental migrations in the Northern Hemisphere. Overall, this study provides new insights into the taxonomically complex lineages of subgenus Chamaecerasus and the process of speciation.

Complete Chloroplast Genome Sequence of Triosteum sinuatum, Insights into Comparative Chloroplast Genomics, Divergence Time Estimation and Phylogenetic Relationships among Dipsacales.

Triosteum himalayanum, Triosteum pinnatifidum (Triosteum L., Caprifoliaceae, Dipsacales) are widely distributed in China while Triosteum sinuatum mainly occurrs in northeast China. Few reports have been determined on the genus Triosteum. In the present research, we sequenced 2 chloroplast genomes of Triosteum and analyzed 18 chloroplast genomes, trying to explore the sequence variations and phylogeny of genus Triosteum in the order Dipsacales. The chloroplast genomes of the genus Triosteum ranged from 154,579 bp to 157,178 bp, consisting of 132 genes (86 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes). Comparative analyses and phylogenetic analysis supported the division of Dipsacales into two clades, Adoxaceae and six other families. Among the six families, a clade of Valerianaceae+Dipsacaceae was recovered as a sister to a clade of Morinaceae+Linnaeaceae. A closer relationship of T. himalayanum and T. pinnatifidum among three species was revealed. Our research supported that Loniceraferdinandi and Triosteum was closely related. Zabelia had a closer relationship with Linnaea borealis and Dipelta than Morinaceae. The divergence between T. sinuatum and two other species in Triosteum was dated to 13.4 mya.

Characterization of the chloroplast genome of Lonicera ruprechtiana Regel and comparison with other selected species of Caprifoliaceae.

Lonicera ruprechtiana Regel is widely used as a greening tree in China and also displays excellent pharmacological activities. The phylogenetic relationship between L. ruprechtiana and other members of Caprifoliaceae remains unclear. In this study, the complete cp genome of L. ruprechtiana was identified using high-throughput Illumina pair-end sequencing data. The circular cp genome was 154,611 bp long and has a large single-copy region of 88,182 bp and a small single-copy region of 18,713 bp, with the two parts separated by two inverted repeat (IR) regions (23,858 bp each). A total of 131 genes were annotated, including 8 ribosomal RNAs, 39 transfer RNAs, and 84 protein-coding genes (PCGs). In addition, 49 repeat sequences and 55 simple sequence repeat loci of 18 types were also detected. Codon usage analysis demonstrated that the Leu codon is preferential for the A/U ending. Maximum-likelihood phylogenetic analysis using 22 Caprifoliaceae species revealed that L. ruprechtiana was closely related to Lonicera insularis. Comparison of IR regions revealed that the cp genome of L. ruprechtiana was largely conserved with that of congeneric species. Moreover, synonymous (Ks) and non-synonymous (Ka) substitution rate analysis showed that most genes were under purifying selection pressure; ycf3, and some genes associated with subunits of NADH dehydrogenase, subunits of the cytochrome b/f complex, and subunits of the photosystem had been subjected to strong purifying selection pressure (Ka/Ks < 0.1). This study provides useful genetic information for future study of L. ruprechtiana evolution.

Linnaea borealis L. var. borealis-In Vitro Cultures and Phytochemical Screening as a Dual Strategy for Its Ex Situ Conservation and a Source of Bioactive Compounds of the Rare Species.

Linnaea borealis L. (Twinflower)-a dwarf shrub in the Linnaeeae tribe of Caprifoliaceae family-is distributed across the Northern Hemisphere. By means of this study, a reliable protocol for efficient micropropagation of uniform L. borealis L. var. borealis plantlets has been provided for the first time; callus culture was also established. Different initial explants, types of cultures, media systems, and plant growth regulators in Murashige and Skoog (MS) media were tested. Agitated shoot cultures in the liquid media turned out to be the best system for the production of sustainable plant biomass. After stabilization of the callus lines, the highest growth index (c.a. 526%) was gained for callus maintained on MS enriched with picloram. TLC and UHPLC-HESI-HRMS analysis confirmed the presence of phenolic acids and flavonoids, and for the first time, the presence of iridoids and triterpenoid saponins in this species. Multiplication of L. borealis shoot culture provides renewable raw material, allowing for the assessment of the phytochemical profile, and, in the future, for the quantitative analyses and the studies of the biological activity of extracts, fractions, or isolated compounds. This is the first report on in vitro cultures of traditionally used L. borealis rare taxon and its biosynthetic potential.

Lineage-Specific Variation in IR Boundary Shift Events, Inversions, and Substitution Rates among Caprifoliaceae s.l. (Dipsacales) Plastomes.

Caprifoliaceae s.l. plastid genomes (plastomes) show that one inversion and two inverted repeat boundary shifts occurred in the common ancestor of this family, after which the plastomes are generally conserved. This study reports plastome sequences of five additional species, Fedia cornucopiae, Valeriana fauriei, and Valerianella locusta from the subfamily Valerianoideae, as well as Dipsacus japonicus and Scabiosa comosa from the subfamily Dipsacoideae. Combined with the published plastomes, these plastomes provide new insights into the structural evolution of plastomes within the family. Moreover, the three plastomes from the subfamily Valerianoideae exhibited accelerated nucleotide substitution rates, particularly at synonymous sites, across the family. The patterns of accD sequence divergence in the family are dynamic with structural changes, including interruption of the conserved domain and increases in nonsynonymous substitution rates. In particular, the Valeriana accD gene harbors a large insertion of amino acid repeat (AAR) motifs, and intraspecific polymorphism with a variable number of AARs in the Valeriana accD gene was detected. We found a correlation between intron losses and increased ratios of nonsynonymous to synonymous substitution rates in the clpP gene with intensified positive selection. In addition, two Dipsacoideae plastomes revealed the loss of the plastid-encoded rps15, and a potential functional gene transfer to the nucleus was confirmed.

Plastid phylogenomic insights into the evolution of the Caprifoliaceae s.l. (Dipsacales).

The family Caprifoliaceae s.l. is an asterid angiosperm clade of ca. 960 species, most of which are distributed in temperate regions of the northern hemisphere. Recent studies show that the family comprises seven major clades: Linnaeoideae, Zabelia, Morinoideae, Dipsacoideae, Valerianoideae, Caprifolioideae, and Diervilloideae. However, its phylogeny at the subfamily or genus level remains controversial, and the backbone relationships among subfamilies are incompletely resolved. In this study, we utilized complete plastome sequencing to resolve the relationships among the subfamilies of the Caprifoliaceae s.l. and clarify several long-standing controversies. We generated and analyzed plastomes of 48 accessions of Caprifoliaceae s.l., representing 44 species, six subfamilies and one genus. Combined with available Caprifoliaceae s.l. plastomes on GenBank and 12 outgroups, we analyzed a final dataset of 68 accessions. Genome structure was strongly conserved in general, although the boundaries between the Inverted Repeat were found to have contracted across Caprifoliaceae s.l. to exclude rpl2, rps19, and ycf1, all or parts of which are typically present in the IR of most angiosperms. The ndhF gene was found to have been inverted in all plastomes of Adoxaceae. Phylogenomic analyses of 68 complete plastomes yielded a highly supported topology that strongly supported the monophyly of Zabelia and its sister relationship to Morinoideae. Moreover, a clade of Valerianoideae + Dipsacoideae was recovered as sister to a clade of Linnaeoideae + Zabelia + Morinoideae clade, and Heptacodium was sister to remaining Caprifolioideae. The Diervilloideae and Caprifolioideae were successively sister to all other Caprifoliaceae s.l. Major lineages of Caprifoliaceae s.l. were estimated to have diverged from the Upper Cretaceous to the Eocene (50-100 Ma), whereas within-genus diversification was dated to the Oligocene and later, concomitant with global cooling and drying. Our results demonstrate the power of plastid phylogenomics in improving estimates of phylogeny among genera and subfamilies, and provide new insights into plastome evolution across Caprifoliaceae s.l.

AFLP markers show low levels of clonal propagation and high genotypic diversity in the rare, southernmost populations of Linnaea borealis L. (Caprifoliaceae) in the Western Alps.

In plants, clonal propagation is a common reproductive strategy in parallel to sexual reproduction. It has both advantages and drawbacks, and the potential complete loss of sexual reproduction causes serious conservation concerns, especially because population maintenance then only relies on adult survival and low genetic diversity leads to decreased adaptive potential. We investigated the rare, southernmost populations of the mostly circumboreal twinflower Linnaea borealis, located in the Western Alps. Based on 105 AFLP markers and 118 leaf samples, including replicates, we estimated the genetic similarity threshold above which samples belong to a single clone. Although the species is known for extensive clonal propagation, we observed high genotypic diversity within the seven studied populations and almost all samples were genetically distinct. Nevertheless, some clonal samples were detected in two populations, separated by up to 180 m. We found a strong genetic differentiation among populations (overall Fst = 0.38), which was congruent with the previously documented high plastid diversity in the region. We therefore hypothesize that Alpine populations are relicts of the Quaternary glacial periods, when the species probably survived at these lower latitudes before colonizing Northern Europe. Regarding conservation, our results suggest that most extant plants result from sexual reproduction and that populations are not highly threatened. Nevertheless, since clones can be very long-lived and almost no seedlings were observed in recent years, events of sexual reproduction may be ancient. The current reproductive dynamics should therefore be studied to estimate e.g. pollinators activity, proportions of flowering plants, and seed set.

Ploidy-altered phenotype interacts with local environment and may enhance polyploid establishment in Knautia serpentinicola (Caprifoliaceae).

Whole genome duplication is a key process in plant evolution and has direct phenotypic consequences. However, it remains unclear whether ploidy-related phenotypic changes can significantly alter the fitness of polyploids in nature and thus contribute to establishment of new polyploid mutants in diploid populations. We addressed this question using a unique natural system encompassing a diploid and its sympatric locally established autotetraploid derivative. By setting a common garden experiment with two manipulated environmental factors (presence/absence of serpentine substrate and competition), we tested whether these two locally important factors differently shape the phenotypic response of the two ploidy levels. Tetraploids attained significantly higher values of both above- and below-ground biomass, and root : shoot ratio compared to their diploid progenitors. Tetraploid superiority in vegetative fitness indicators was most prominent when they were cultivated together with a competitor in nutrient-rich nonserpentine substrate. We show that even genetically very closely related diploids and tetraploids can respond differently to key environmental factors. Provided there are sufficient nutrients, tetraploids can be more successful in tolerating interspecific competition than their diploid progenitors. Such superior performance might have provided an adaptive advantage for the newly established tetraploid promoting colonisation of new (micro-)habitats, which was indeed observed at the natural site.

Combining complete chloroplast genome sequences with target loci data and morphology to resolve species limits in Triplostegia (Caprifoliaceae).

Species represent the most basic unit of taxonomy. As such, species delimitation represents a crucial issue for biodiversity conservation. Taxonomic practices were revolutionized in the last three decades due to the increasing availability of molecular phylogenetic data. The genus Triplostegia (Caprifoliaceae) traditionally consists of two species, T. glandulifera and T. grandiflora, distinguishable mainly based on quantitative morphological features. In this study, we sequenced nine chloroplast loci (i.e., accD, psbK-psbI, rbcL-accD, rpoB-trnC, rps16-trnQ, trnE-trnT, trnF-ndhJ, trnH-psbA, trnS-trnG) and one nuclear locus (ITS) of 16 individuals of Triplostegia representing the entire distribution range of both species recognized. Furthermore, we also obtained whole chloroplast sequences for 11 of the 16 individuals for which silica gel-dried leaves were available. Our phylogenetic analyses integrating chloroplast genome sequences and multiple loci data revealed that Triplostegia includes four main clades that largely match geography. Neither T. grandiflora nor T. glandulifera was recovered as monophyletic and no diagnosable differences in leaf, flower, and pollen traits were detected between the two species, indicating the need for a revised species circumscription within Triplostegia. Our study highlights the importance of combining data from different sources while defining species limits.

The complete chloroplast genome of Kolkwitzia amabilis (Caprifoliaceae), an endangered horticultural plant in China.

The complete chloroplast genome of Kolkwitzia amabilis (Caprifoliaceae) is first presented in the current study. The cp genome of K. amabilis was 156 875 bp in length and composed of two short inverted repeat (IRa and IRb) regions of 23 946 bp which were separated by a small single copy (SSC) region of 18 846 bp and a large single copy (LSC) region of 90 137 bp. The genome encoded 130 genes contained 81 coding genes, 39 tRNA genes, and 8 rRNA genes. The overall AT content of K. amabilis is 61.6% and the corresponding values of the SSC, LSC and IR regions are 66.8%, 64.1%, and 57.3%, respectively. The phylogenetic analysis based on the maximum parsimony tree revealed that K. amabilis was closely related to Lonicera japonica.

Heteroploid Knautia drymeia includes K. gussonei and cannot be separated into diagnosable subspecies.

PREMISE OF THE STUDY: Knautia drymeia is a morphologically variable, diploid and tetraploid temperate forest understory species distributed in southeastern Europe and adjacent areas. The species is an excellent system to explore the influence of polypoidy on taxonomic delineations, the role of hybridization among genetically distant populations in polyploid evolution, and the impact of glacial refugia on the evolution of polyploids. METHODS: Amplified fragment length polymorphism fingerprinting and multivariate analyses of morphological characters were performed on 57 populations spanning the distribution area of K. drymeia. K-means clustering, comparison of in-silico tetraploids and observed tetraploids, and a phylogeographic analysis using relaxed random walks were used to explore the genetic structure within the diploids, to infer the origin of the tetraploids and to reconstruct range expansion through time. Further, we contrasted the morphology and genetic groups with current taxonomy and evaluated the status of the tetraploid Apennine endemic K. gussonei and the intraspecific taxa of K. drymeia. KEY RESULTS: The genetic structure was strongly geographically correlated and yielded four genetic groups; K. gussonei was inseparable from K. drymeia. Distributions of diploid lineages are suggestive of glacial refugia in the northwesternmost and southeastern Balkan Peninsula. Polyploids originated at least two times, as autopolyploids and probably additionally also as allopolyploids. Morphological divergence corresponded with neither genetic groups nor current taxonomy. CONCLUSIONS: Genetic and morphometric data confirmed neither divergence of K. gussonei nor recognition of subspecies within K. drymeia. We therefore propose treating K. drymeia as a morphologically and genetically variable species without infraspecific taxa.

[Study on genetic-chemical relation of Pterocephali Herba based on DNA barcode and UFLC].

DNA barcoding technique in combination with UFLC analysis technology was used to evaluate the quality of Tibetan medicine Pterocephalus hookeri from species identification and chemical qualitative and other aspects. Hybrid identification was established by DNA barcoding; UFLC-PDA was adopted to analyse fingerprint of different parts of Pterocephali Herba, and SPSS and Grey relation software were used for data analysis. The result showed that DNA barcoding is an accurate and reliable method in origin identification of Pterocephalus hookeri. The compounds in overground is more than underground by analysis of the different part fingerprint by UFLC. The genetic gene may be involved in the secondary metabolites of iridoid glycosides. Pertinence between gene and chemical component, as a new model established, could be suited for quality evaluation and resources protection.

Cytotype diversity and genome size variation in Knautia (Caprifoliaceae, Dipsacoideae).

BACKGROUND: Polyploidisation is one of the most important mechanisms in the evolution of angiosperms. As in many other genera, formation of polyploids has significantly contributed to diversification and radiation of Knautia (Caprifoliaceae, Dipsacoideae). Comprehensive studies of fine- and broad-scale patterns of ploidy and genome size (GS) variation are, however, still limited to relatively few genera and little is known about the geographic distribution of ploidy levels within these genera. Here, we explore ploidy and GS variation in Knautia based on a near-complete taxonomic and comprehensive geographic sampling. RESULTS: Genome size is a reliable indicator of ploidy level in Knautia, even if monoploid genome downsizing is observed in the polyploid cytotypes. Twenty-four species studied are diploid, 16 tetraploid and two hexaploid, whereas ten species possess two, and two species possess three ploidy levels. Di- and tetraploids are distributed across most of the distribution area of Knautia, while hexaploids were sampled in the Balkan and Iberian Peninsulas and the Alps. CONCLUSIONS: We show that the frequency of polyploidisation is unevenly distributed in Knautia both in a geographic and phylogenetic context. Monoploid GS varies considerably among three evolutionary lineages (sections) of Knautia, but also within sections Trichera and Tricheroides, as well as within some of the species. Although the exact causes of this variation remain elusive, we demonstrate that monoploid GS increases significantly towards the limits of the genus' distribution.

Molecular phylogeny and biogeographic diversification of linnaeoideae (caprifoliaceae s. L.) disjunctly distributed in Eurasia, North America and Mexico.

Linnaeoideae is a small subfamily of erect or creeping shrubs to small trees in Caprifoliaceae that exhibits a wide disjunct distribution in Eurasia, North America and Mexico. Most taxa of the subfamily occur in eastern Asia and Mexico but the monospecific genus Linnaea has a circumboreal to north temperate distribution. In this study, we conducted phylogenetic and biogeographic analyses for Linnaeoideae and its close relatives based on sequences of the nuclear ribosomal ITS and nine plastid (rbcL, trnS-G, matK, trnL-F, ndhA, trnD-psbM, petB-D, trnL-rpl32 and trnH-psbA) markers. Our results support that Linnaeoideae is monophyletic, consisting of four eastern Asian lineages (Abelia, Diabelia, Dipelta and Kolkwitzia), the Mexican Vesalea, and Linnaea. The Mexican Vesalea was formerly placed in Abelia, but it did not form a clade with the eastern Asian Abelia; instead Vesalea and Linnaea are sisters. The divergence time between the eastern Asian lineages and the Mexican Vesalea plus the Linnaea clade was dated to be 50.86 Ma, with a 95% highest posterior density of 42.8 Ma (middle Eocene) to 60.19 Ma (early Paleocene) using the Bayesian relaxed clock estimation. Reconstructed ancestral areas indicated that the common ancestor of Linnaea plus Vesalea may have been widespread in eastern Asia and Mexico or originated in eastern Asia during the Eocene and likely migrated across continents in the Northern Hemisphere via the North Atlantic Land Bridges or the Bering Land Bridge. The Qinling Mountains of eastern Asia are the modern-day center of diversity of Kolkwitzia-Dipelta-Diabelia clade. The Diabeliaclade became highly diversified in Japan and eastern China. Populations of Diabelia serrata in Japan and eastern China were found to be genetically identical in this study, suggesting a recent disjunction across the East China Sea, following the last glacial event.

Nonadaptive processes governing early stages of polyploid evolution: Insights from a primary contact zone of relict serpentine Knautia arvensis (Caprifoliaceae).

• Premise of the study: Contact zones between polyploids and their diploid progenitors may provide important insights into the mechanisms of sympatric speciation and local adaptation. However, most published studies investigated secondary contact zones where the effects of genome duplication can be confounded by previous independent evolution of currently sympatric cytotypes. We compared genetically close diploid and autotetraploid serpentine cytotypes of Knautia arvensis (Caprifoliaceae) in a primary contact zone and evaluated the role of adaptive and nonadaptive processes for cytotype coexistence.• Methods: DNA flow cytometry was used to determine ploidy distribution at various spatial scales (from across the entire contact zone to microgeographic). Habitat preferences of diploids and polyploids were assessed by comparing vegetation composition of nearby ploidy-uniform sites and by recording plant species immediately surrounding both cytotypes in mixed-ploidy plots.• Key results: Tetraploids considerably outnumbered their diploid progenitors in the contact zone. Both cytotypes were segregated at all investigated spatial scales. This pattern was not driven by ecological shifts, because both diploids and tetraploids inhabited sites with nearly identical vegetation cover. Certain interploidy niche differentiation was indicated only at the smallest spatial scale; ecologically nonadaptive processes were most likely responsible for this difference.• Conclusions: We conclude that a shift in ecological preferences (i.e., the adaptive scenario) is not necessary for the establishment and evolutionary success of autopolyploid derivatives in primary contact zones. Spatial segregation that would support ploidy coexistence can also be achieved by ecologically nonadaptive processes, including the founder effect, limited dispersal ability, intense clonal growth, and triploid block.

Disentangling relationships among the diploid members of the intricate genus Knautia (Caprifoliaceae, Dipsacoideae).

The genus Knautia (Caprifoliaceae, Dipsacoideae) encompasses 40-60 species mainly distributed in western Eurasia, with highest species diversity in the Alps and the Balkan Peninsula. It is traditionally regarded as one of the taxonomically most challenging European genera due to the widespread occurrence of polyploidy, the high incidence of hybridisation and the maintenance of morphologically intermediate forms. A prerequisite for assessing the complex spatiotemporal diversification of a polyploid group is a comprehensive hypothesis of the phylogenetic relationships among its diploid members. To this end, DNA sequence data (nrDNA ITS and plastid petN(ycf6)-psbM) combined with AFLP fingerprinting were performed on 148 diploid populations belonging to 35 taxa. Phylogenies obtained by maximum parsimony and Bayesian analyses were used to test the monophyly of the genus and its three sections Trichera, Tricheroides and Knautia, to provide insights into its evolutionary history and to test previous hypotheses of inter- and intrasectional classification. Both nuclear and chloroplast datasets support the monophyly of Knautia and its three sections, with ambiguous placement of K. cf. degenii. The majority of species belong to the nearly exclusively perennial section Trichera (x=10). Within section Trichera all markers revealed largely unresolved phylogenetic relationships suggesting rapid radiation and recent range expansion. In addition, extensive sharing of plastid haplotypes across taxa and wide geographic ranges of plastid haplotypes and ribotype groups were observed. The molecular data are partly at odds with the traditional informal grouping of taxa within section Trichera. Whereas the traditional groups of K. dinarica, K. drymeia and K. montana can be maintained, the new, smaller and well supported Midzorensis and Pancicii Groups as well as the SW European Group are separated from the heterogeneous traditional K. longifolia group. The former groups of K. arvensis, K. dalmatica, K. fleischmannii and K. velutina are clearly polyphyletic. Their diploid members have to be rearranged into the Xerophytic Group, the Carinthiaca Group, and the Northern and Southern Arvensis Groups. The annual sections Tricheroides (x=10) and Knautia (x=8) with only a few taxa are resolved in the ITS and plastid trees on long branches as early diverging lineages within the genus.

Diversification of CYCLOIDEA expression in the evolution of bilateral flower symmetry in Caprifoliaceae and Lonicera (Dipsacales).

BACKGROUND AND AIMS: The expression of floral symmetry genes is examined in the CYCLOIDEA lineage following duplication, and these are linked to changes in flower morphology. The study focuses on Dipsacales, comparing DipsCYC2 gene expression in Viburnum (radially symmetrical Adoxaceae) to members of early-diverging lineages of the bilaterally symmetrical Caprifoliaceae (Diervilla and Lonicera). METHODS: Floral tissue from six species, which included dorsal, lateral and ventral regions of the corolla, was dissected. RNA was extracted from these tissues and each copy of DipsCYC2 was amplified with reverse transcriptase PCR. KEY RESULTS: Members of DipsCYC2 were expressed across the corolla in the radially symmetrical Viburnum plicatum. A shift to bilaterally symmetrical flowers at the base of the Caprifoliaceae was accompanied by a duplication of the DipsCYC2 gene, resulting in DipsCYC2A and DipsCYC2B, and by loss of expression of both of these copies in the ventral petal. In Lonicera (Caprifolieae), there is a shift from flowers with two dorsally and three ventrally oriented corolla lobes to a clear differentiation of dorsal, lateral and ventral lobes. This shift entailed a decoupling of expression of DipsCYC2A and DipsCYC2B; DipsCYC2B continues to be expressed in the dorsal and lateral lobes, while DipsCYC2A expression is restricted to just the two dorsal lobes. A reversion to more radially symmetrical flowers within Lonicera was accompanied by a re-expansion of expression of both DipsCYC2A and DipsCYC2B. CONCLUSIONS: The transition to bilateral symmetry in Caprifoliaceae involved: (a) duplication of an ancestral DipsCYC2 gene; (b) the loss of expression of both of these copies in the ventral petal; and (c) changes in the zone of expression, with one copy continuing to be expressed across the dorsal and lateral petals, and the other copy becoming restricted in expression to the dorsal corolla lobes.

Ploidy distribution and DNA content variations of Lonicera caerulea (caprifoliaceae) in Japan.

Ploidy level and geographical distribution were investigated in Japanese Lonicera caerulea L. Flow cytometric analysis revealed the presence of DNA diploid and DNA tetraploid plants in Japan. Chromosome observation confirmed that diploid and tetraploid plants showed 2n = 2x = 8 and 2n =4x = 36, respectively. The DNA diploid populations were found only in lowland mires, Betsukai, Bekanbeushi, Kushiro and Kiritappu located in eastern Hokkaido. On the other hand, DNA tetraploid populations were distributed in a wide area of Hokkaido, and mainland of Japan. The habitats of DNA tetraploid plants were lowland to alpine region. The DNA content measurement with flow cytometry revealed significant differences in the relative DNA contents among DNA tetraploid populations. The relative DNA content within DNA tetraploid populations varied 1.157-fold at maximum, and might correlate with altitude indicating that DNA contents were smaller as altitude increases. The wide area of distribution in various environments of DNA tetraploid plants suggested the adaptability of the tetraploid plants. Although diploid and tetraploid populations were found, no triploid was detected, indicating crossing difficulty between diploid and tetraploid as confirmed by crossing experiment.

[DNA barcoding in medicinal plants Caprifoliaceae].

OBJECTIVE: To determine the candidate sequences which can be used as DNA barcode to identify species in Caprifoliaceae family by screening out from four different DNA fragments sequences. METHOD: PCR amplification, sequencing efficiency, differential intra- and interspecific divergences, the DNA barcoding gap and identification efficiency were used to evaluate these loci. RESULT: The ITS2 was used as a candidate sequence of DNA barcode to identify the species in Caprifoliaceae family, whose rate of success in identification in genera level was 100% and in species 96.6%, and psbA-trnH as a complementary barcode to ITS2 for Caprifoliaceae.