Phylogenetic relationships of Salix (Salicaceae) based on rbcL sequence data

Nucleotide sequences of the chloroplast-encoded rbcL gene were used to examine phylogenetic relationships of the genus Salix together with other allied genera of the family Salicaceae. Phylogenetic analyses of rbcL sequences strongly suggest the monophyly of three commonly recognized genera (Chosenia, Salix, and Toisusu). Two monophyletic groups are recognized within the larger monophyletic group. They do not correspond with any infrageneric taxa proposed so far. With regard to character evolution, it is thought that the reduction of stamen number from more than two stamens to two might occur in at least three lineages and that fused bud scales evolved several times and/or the reverse evolution occurred from fused to free. Some types of pollen surfaces are considered to have evolved independently.     

Phylogenetic relationships of Oxytropis chankaensis Jurtz. and Oxytropis oxyphylla (Pall.) DC. (Fabaceae) inferred from the data of sequencing of the ITS region of the nuclear ribosomal DNA operon and intergenic spacers of the chloroplast genome

The autotetraploid Oxytropis chankaensis Jurtz. is the only representative of the section Baicalia (subgenus Oxytropis, Fabaceae) in Primorskii Krai, and its range is restricted to the sandy belt along the western coast of the Khanka Lake. The type species of the section Baicalia is O. oxyphylla (Pall.) DC., and O. chankaensis is regarded by some authors as its synonym. Analysis of the ITS sequences showed that O. chankaensis is phylogenetically closely related to the species O. oxyphylla and O. racemosa Turcz. from the section Baicalia and also to the species O. anertii Nakai., O. ciliata Turcz., and O. inschanica H. C. Fu & S. H. Cheng from the section Xerobia, but this analysis failed to resolve the phylogenetic relationships of the species within the genus Oxytropis. The analysis of the trnH-psbA, trnL-trnF, trnS-trnG, and petG-trnP regions of cpDNA revealed molecular differences between O. chankaensis and O. oxyphylla that are indicative of considerable divergence of their chloroplast genomes. The ancestral lineages of these species diverged nearly 2 million years ago, which confirms the taxonomic validity of O. chankaensis. Taking into account the ecological specificity of O. chankaensis and the present-day distribution range of O. oxyphylla, it can be assumed that they descended from one ancestral maternal lineage that gave rise to the species of the section Baicalia.     

Phylogenetic Relationships of American Willows (Salix L., Salicaceae)

Salix L. is the largest genus in the family Salicaceae (450 species). Several classifications have been published, but taxonomic subdivision has been under continuous revision. Our goal is to establish the phylogenetic structure of the genus using molecular data on all American willows, using three DNA markers. This complete phylogeny of American willows allows us to propose a biogeographic framework for the evolution of the genus. Material was obtained for the 122 native and introduced willow species of America. Sequences were obtained from the ITS (ribosomal nuclear DNA) and two plastid regions, matK and rbcL. Phylogenetic analyses (parsimony, maximum likelihood, Bayesian inference) were performed on the data. Geographic distribution was mapped onto the tree. The species tree provides strong support for a division of the genus into two subgenera, Salix and Vetrix. Subgenus Salix comprises temperate species from the Americas and Asia, and their disjunction may result from Tertiary events. Subgenus Vetrix is composed of boreo-arctic species of the Northern Hemisphere and their radiation may coincide with the Quaternary glaciations. Sixteen species have ambiguous positions; genetic diversity is lower in subg. Vetrix. A molecular phylogeny of all species of American willows has been inferred. It needs to be tested and further resolved using other molecular data. Nonetheless, the genus clearly has two clades that have distinct biogeographic patterns.     

Phylogenetic relationships of Oxytropis chankaensis Jurtz. and Oxytropis oxyphylla (Pall.) DC. (Fabaceae) inferred from the data of sequencing of the ITS region of the nuclear ribosomal DNA operon and intergenic spacers of the chloroplast genome

The autotetraploid Oxytropis chankaensis Jurtz. is the only representative of the section Baicalia (subgenus Oxytropis, Fabaceae) in Primorskii Krai, and its range is restricted to the sandy belt along the western coast of the Khanka Lake. The type species of the section Baicalia is O. oxyphylla (Pall.) DC., and O. chankaensis is regarded by some authors as its synonym. Analysis of the ITS sequences showed that O. chankaensis is phylogenetically closely related to the species O. oxyphylla and O. racemosa Turcz. from the section Baicalia and also to the species O. anertii Nakai., O. ciliata Turcz., and O. inschanica H.C. Fu & S.H. Cheng from the section Xerobia, but this analysis failed to resolve the phylogenetic relationships of the species within the genus Oxytropis. The analysis of the trnH-psbA, trnL-trnF, trnS-trnG, and petG-trnP regions of cpDNA revealed molecular differences between O. chankaensis and O. oxyphylla that are indicative of considerable divergence of their chloroplast genomes. The ancestral lineages of these species diverged nearly 2 million years ago, which confirms the taxonomic validity of O. chankaensis. Taking into account the ecological specificity of O. chankaensis and the present-day distribution range of O. oxyphylla, it can be assumed that they descended from one ancestral maternal lineage that gave rise to the species of the section Baicalia.     

RAD sequencing resolved phylogenetic relationships in European shrub willows (Salix L. subg. Chamaetia and subg. Vetrix) and revealed multiple evolution of dwarf shrubs

The large and diverse genus Salix L. is of particular interest for decades of biological research. However, despite the morphological plasticity, the reconstruction of phylogenetic relationships was so far hampered by the lack of informative molecular markers. Infrageneric classification based on morphology separates dwarf shrubs (subg. Chamaetia) and taller shrubs (subg. Vetrix), while previous phylogenetic studies placed species of these two subgenera just in one largely unresolved clade. Here we want to test the utility of genomic RAD sequencing markers for resolving relationships at different levels of divergence in Salix. Based on a sampling of 15 European species representing 13 sections of the two subgenera, we used five different RAD sequencing datasets generated by Ipyrad to conduct phylogenetic analyses. Additionally we reconstructed the evolution of growth form and analyzed the genetic composition of the whole clade. The results showed fully resolved trees in both ML and BI analysis with high statistical support. The two subgenera Chamaetia and Vetrix were recognized as nonmonophyletic, which suggests that they should be merged. Within the Vetrix/Chamaetia clade, a division into three major subclades could be observed. All species were confirmed to be monophyletic. Based on our data, arctic‐alpine dwarf shrubs evolved four times independently. The structure analysis showed five mainly uniform genetic clusters which are congruent in sister relationships observed in the phylogenies. Our study confirmed RAD sequencing as a useful genomic tool for the reconstruction of relationships on different taxonomic levels in the genus Salix.     

Evolution of the chloroplast genome and polymorphic ITS regions in Allium subg. Melanocrommyum.

Relationships based on PCR-RFLPs of non-coding regions of cpDNA indicate that some of the largest subgenera of the genus Allium and five of the largest sections of the Central Asian subg. Melanocrommyum are artificial. Internested synapomorphic mutations without homoplasy were found only in the chloroplast genomes of plants of subg. Melanocrommyum that occur in the border region of Tajikistan, Uzbekistan, Afghanistan, and Kyrgyzstan. Eighteen of 49 plants surveyed were polymorphic for their ITS regions. Even plants that had identical chloroplast genomes were polymorphic for nuclear ribosomal regions. These individuals had markedly different frequencies of ITS variants that were detected with various restriction enzymes. The geographic partitioning of chloroplast haplotypes and the fact that the ITS variants could not be ordered hierarchically can readily be envisioned to result from gene flow. Processes such as concerted evolution and parallel morphological evolution may also be partly responsible for the disconcordance of mutations in the chloroplast and nuclear genome. However, the chimeric nature of the nuclear ribosomal regions indicates that concerted evolution is not the dominating process in Allium subg. Melanocrommyum.     

Phylogenetic relationships amongHomo sapiens and related species based on restriction site variations in rDNA spacers

A rapid method, using 12 restriction enzymes, was employed to analyze variations in ribosomal DNA (rDNA) spacers in a study of phylogenetic relationships betweenHomo sapiens and related species. We mapped restriction sites in the external and internal spacer regions and compared the arrangements of sites. The estimated sequence divergence betweenHomo sapiens andPan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus, Hylobates lar, H. agilis, andMacaca fuscata was 2.7, 2.3, 3.8, 7.3, 6.8, 7.8, and 14.1%, respectively. The genetic relationships inferred from these distances generally correspond to those inferred from analyses of other molecular markers in the literature. The divergence betweenH. lar andH. agilis and betweenH. lar andH. syndactylus was 0.34 and 2.4%, respectively.This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and also by the Cooperative Research Program of the Primate Research Institute, Kyoto University.     

Restriction‐site associated DNA sequencing data reveal a radiation of willow species (Salix L., Salicaceae) in the Hengduan Mountains and adjacent areas

The Hengduan Mountains (HDM) in China are an important hotspot of plant diversity and endemism, and are considered to be a secondary diversification center for the woody plant genus Salix L. (Salicaceae). Here we aimed to reconstruct the spatiotemporal evolution of the Salix Chamaetia‐Vetrix clade in the HDM and to test for the occurrence of a local radiation. We inferred phylogenetic relationships based on more than 34 000 restriction‐site associated DNA loci from 27 species. Phylogenetic analyses recovered a well‐resolved tree topology with two major clades, the Eurasian clade and the HDM clade, with a divergence time of ca. 23.9 Ma. Species in the HDM clade originated in the northern part of the range and adjacent areas, and then dispersed into the southern HDM, westwards to the Himalayas and eastwards to the Qinling Mountains. Niche modelling analyses reveal that range contractions occurred in the northern areas during the last glacial maximum, while southward expansions resulted in range overlaps. Reconstructions of character evolution related to plant height, inflorescence, and flower morphology suggest that adaptations to altitudinal distribution contributed to the diversification of the HDM willows. Our data support the occurrence of a radiation in the HDM within the Salix Chamaetia‐Vetrix clade. Dispersal within the mountain system, and to adjacent regions, in addition to survival in glacial refugia shaped the biogeographical history of the clade, while adaptations of the HDM willows along an altitudinal gradient could be important ecological factors explaining the high species diversity of Salix in this area.     

Phylogenetic relationships of Ruteae (Rutaceae): new evidence from the chloroplast genome and comparisons with non-molecular data

Phylogenetic analyses of three cpDNA markers (matK, rpl16, and trnL–trnF) were performed to evaluate previous treatments of Ruteae based on morphology and phytochemistry that contradicted each other, especially regarding the taxonomic status of Haplophyllum and Dictamnus. Trees derived from morphological, phytochemical, and molecular datasets of Ruteae were then compared to look for possible patterns of agreement among them. Furthermore, non-molecular characters were mapped on the molecular phylogeny to identify uniquely derived states and patterns of homoplasy in the morphological and phytochemical datasets. The phylogenetic analyses determined that Haplophyllum and Ruta form reciprocally exclusive monophyletic groups and that Dictamnus is not closely related to the other genera of Ruteae. The different types of datasets were partly incongruent with each other. The discordant phylogenetic patterns between the phytochemical and molecular trees might be best explained in terms of convergence in secondary chemical compounds. Finally, only a few non-molecular synapomorphies provided support for the clades of the molecular tree, while most of the morphological characters traditionally used for taxonomic purposes were found to be homoplasious. Within the context of the phylogenetic relationships supported by molecular data, Ruta, the type genus for the family, can only be diagnosed by using a combination of plesiomorphic, homoplasious, and autapomorphic morphological character states.     

Phylogenetic relationships ofPseudopanax species (Araliaceae) inferred from parsimony analysis of rDNA sequence data and morphology

Sequence data from internal transcribed spacer (ITS) regions of rDNA and data from morphology, cytology and wood anatomy are used to study phylogenetic relationships inPseudopanax. The molecular and non-molecular data are analysed as independent data sets and in combination using parsimony. Results supported the conclusion that the genusPseudopanax is polyphyletic.Pseudopanax species emerge in two major monophyletic groups. The Anomalus group containsPseudopanax anomalus, P. edgerleyi, andP. simplex; these species share a common ancestor withCheirodendron trigynum and more distantly withPseudopanax gunnii. The second major group consists of two smaller groups: the Arboreus group, includingPseudopanax arboreus, P. colensoi, P. kermadecensis, P. laetus, andP. macintyrei, and the Crassifolius/Discolor group, includingP. chathamicus, P. crassifolius, P. discolor, P. ferox, P. gilliesii, P. lessonii, andP. linearis. Meryta species are close relatives of thePseudopanax Arboreus and Crassifolius/Discolor groups.     

Phylogenetic relationships among cultivated Allium species from restriction enzyme analysis of the chloroplast genome

Summary The genus Allium contains many economically important species, including the bulb onion, chive, garlic, Japanese bunching onion, and leek. Phylogenetic relationships among the cultivated alliums are not well understood, and taxonomic classifications are based on relatively few morphological characters. Chloroplast DNA is highly conserved and useful in determining phylogenetic relationships. The size of the chloroplast genome of Allium cepa was estimated at 140 kb and restriction enzyme sites were mapped for KpnI, PstI, PvuII, SalI, XbaI, and XhoI. Variability at restriction enzyme sites in the chloroplast DNA was studied for at least three accessions of each of six cultivated, old-world Allium species. Of 189 restriction enzyme sites detected with 12 enzymes, 15 mutations were identified and used to estimate phylogenetic relationships. Cladistic analysis based on Wagner and Dollo parsimony resulted in a single, most-parsimonious tree of 16 steps and supported division of the species into sections. Allium species in section Porrum were distinguished from species in sections Cepa and Phyllodolon. Two species in section Rhiziridium, A. schoenoprasum and A. tuberosum, differed by five mutations and were placed in separate lineages. Allium cepa and A. fistulosum shared the loss of a restriction enzyme site and were phylogenetically closer to each other than to A. schoenoprasum. This study demonstrates the usefulness of restriction enzyme site analysis of the chloroplast genome in the elucidation of phylogenetic relationships in Allium.     

Phylogenetic relationships in the orchid genus Serapias L. based on noncoding regions of the chloroplast genome

A molecular phylogenetic analysis was performed on 14 species of the Mediterranean unrewarding orchid genus Serapias using sequences of four noncoding regions of chloroplast DNA. This study has led to a new interpretation of the evolutionary relationships in this genus. The well-defined phylogenetic tree supports a division of taxa into two main clades, each including two minor groups. The molecular relationships found in this study differ from those defined by traditional systematic morphological assessments. By comparing the variation in sequence to variations in floral traits, we propose that the split in the two main lineages reflects an early differentiation of flower size, perhaps due to the shift from allo- to self-pollination. Conversely, the relationships within each minor group do not reflect floral size variation; therefore, we presume that this diversification resulted from genetic drift, local selection forces, and multiple, independent transitions towards self-pollination and polyploidy.     

Phylogenetic relationships among Artemisia species based on nuclear ITS and chloroplast psbA-trnH DNA markers

The taxonomic and phylogenetic relationships within the genus Artemisia s.l. (Asteraceae) are controversial, and it has been considered 1 to 8 different genera. This work re-investigated the phylogenetic relationships in Artemisia using nuclear ribosomal (ITS) and chloroplast psbA-trnH DNA sequences using three sections of Artemisia, Dracunculus, and Serphidium. Three phylogenetic trees were conducted separately on the basis of ITS, psbA-trnH and combined sequences using maximum parsimony. The results showed that the three sections were clearly separated from each other, and that the heterogamous Dracunculus and Artemisia are closely related to each other than either to homogamous Serphidium. This may suggest the taxonomic importance of capitulum morphology in Artemisia s.l. Our data also cast doubt on the use of cytogenetic similarity e.g., basic chromosome number in grouping Serphidium and Artemisia s.s. Furthermore, AMOVA analysis showed a higher level of ITS (55.29%) and combined ITS+cppsbA-trnH (55.63%) variations among sections. This provides further evidence for separation of these three sections and supports the phylogenetic results. The higher ITS nucleotide differences detected in Artemisia (30.4737) compared to very low value in Dracunculus (2.3333) and Serphidium (1.23077) may propose that the Artemisia comprises of several incipient sections. This supports the previous suggestion that Artemisia is a complex group.     

Phylogenetic relationships ofMicrosphaera andErysiphe sectionErysiphe (powdery mildews) inferred from the rDNA ITS sequences

The genusMicrosphaera has been considered to be derived from sectionErysiphe of the genusErysiphe by a single event. Cleistothecial appendages are the most distinct difference between the two genera and have an important role for overwintering. To understand the phylogenetic relationship betweenErysiphe sectionErysiphe andMicrosphaera more precisely, phylogenetic trees were constructed using the nucleotide sequences of the rDNA ITS region from 11Erysiphe (sectionErysiphe) and 16Microsphaera taxa. The phylogenetic trees indicated the close relationship between the two genera. However, the generaErysiphe (sectionErysiphe) andMicrosphaera did not group into separate monophyletic lineages; instead, they formed several small clusters that were mixed together. This result suggests that the differentiations of the genera occurred two or more times independently. This also supports the idea that appendage morphology does not always accurately reflect the phylogeny of the powdery mildews because the morphology of appendages may evolve convergently under the selection pressure of their particular biotopes (host plants). Contribution No. 144 from the Laboratory of Plant Pathology, Mie University.     

Leaf epidermal features of Salix species (Salicaceae) and their systematic significance

? Premise of the study: The classification of the genus Salix has historically been intrinsically difficult due to its propensity toward plasticity and high variation in diagnostic morphological characters. We investigated leaf epidermal characteristics, focusing on the stomatal apparatus because it may provide critical insights into the evolution and taxonomy of Salix and its closely related genera. ? Methods: Light microscopy was used to examine the leaf epidermal features in 32 taxa of Salix. ? Key results: Characters such as shape, size, and density of stomatal complexes were very useful in differentiating Salix species. Variation in features of stomatal apparatus in Salix is wider than previously known. Moreover, the type of stomatal complex proved to be very helpful in discriminating Chosenia as members of the genus Salix. ? Conclusions: The results of the present study support the placement of Chosenia within Salix and the combining of subgenera Chamaetia and Vetrix because of similarities in their unique stomatal apparatus.     

Eighteen microsatellite loci in Salix arbutifolia (Salicaceae) and cross-species amplification in Salix and Populus species

Salix arbutifolia is a riparian dioecious tree species that is of conservation concern in Japan because of its highly restricted distribution. Eighteen polymorphic loci of dinucleotide microsatellites were isolated and characterized. Among these, estimates of the expected heterozygosity ranged from 0.350 to 0.879. Cross-species amplification was successful at 9-13 loci among six Salix species and at three loci in one Populus species.     

Phylogenetic position of the coral symbiont Ostreobium (Ulvophyceae) inferred from chloroplast genome data

The green algal genus Ostreobium is an important symbiont of corals, playing roles in reef decalcification and providing photosynthates to the coral during bleaching events. A chloroplast genome of a cultured strain of Ostreobium was available, but low taxon sampling and Ostreobium's early‐branching nature left doubt about its phylogenetic position. Here, we generate and describe chloroplast genomes from four Ostreobium strains as well as Avrainvillea mazei and Neomeris sp., strategically sampled early‐branching lineages in the Bryopsidales and Dasycladales respectively. At 80,584 bp, the chloroplast genome of Ostreobium sp. HV05042 is the most compact yet found in the Ulvophyceae. The Avrainvillea chloroplast genome is ~94 kbp and contains introns in infA and cysT that have nearly complete sequence identity except for an open reading frame (ORF) in infA that is not present in cysT. In line with other bryopsidalean species, it also contains regions with possibly bacteria‐derived ORFs. The Neomeris data did not assemble into a canonical circular chloroplast genome but a large number of contigs containing fragments of chloroplast genes and showing evidence of long introns and intergenic regions, and the Neomeris chloroplast genome size was estimated to exceed 1.87 Mb. Chloroplast phylogenomics and 18S nrDNA data showed strong support for the Ostreobium lineage being sister to the remaining Bryopsidales. There were differences in branch support when outgroups were varied, but the overall support for the placement of Ostreobium was strong. These results permitted us to validate two suborders and introduce a third, the Ostreobineae.