Incongruence between Nuclear and Chloroplast DNA Phylogenies in <i>Pedicularis</i> Section <i>Cyathophora</i> (Orobanchaceae)

<div><p><i>Pedicularis</i> section <i>Cyathophora</i> 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 <i>Pedicularis</i>, i.e. toothless, toothed, beaked and long-tubed corollas. In this study, we aim to reconstruct a comprehensive phylogeny of section <i>Cyathophora</i>, and compare phylogenetic incongruence between nuclear and chloroplast datasets. Sixty-seven accessions belonging to section <i>Cyathophora</i> and 9 species for other <i>Pedicularis</i> were sampled, and one nuclear gene (nrITS) and four chloroplast genes (<i>matK</i>, <i>rbcL</i>, <i>trnH-psbA</i> and <i>trnL-F</i>) 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 <i>P</i><i>. cyathophylloides</i> (E4 and E5) may show introgression from an ancestor of <i>P</i><i>. cyathophylla</i>. Neither the nrITS dataset nor the concatenated chloroplast dataset were able to adequately resolve relationships among species in the series <i>Reges</i>; 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 <i>Cyathophora</i>, and the chloroplast phylogeny reveals that the long corolla tube with beaked galea is derived from the short one.</p> </div

Geographical information for studied samples of <i>Pedicularis</i> section <i>Cyathophora</i>.

<p>Detailed collection of sample code is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074828#pone.0074828.s001" target="_blank">Table S1</a>. The black, green and light blue dash lines separates sample of <i>P</i>. <i>rex</i> subsp. <i>rex</i>, <i>P</i><i>. thamnophila</i> and the unknown taxon in accordance with the nrITS phylogeny, respectively; and the blue, red and orange dash lines separates samples of <i>P</i><i>. cyathophylla</i>, <i>P</i><i>. cyathophylloides</i> and <i>P</i><i>. superba</i> in accordance with the chloroplast phylogeny, respectively.</p

Four general corolla types in <i>Pedicularis</i> section <i>Cyathophora</i> (Redrawn from Tsoong [14]).

<p>(A) short tubular corolla with a beakless and toothless galea (<i>P</i><i>. cyathophylloides</i>); (B) short tubular corolla with a toothed galea (P. <i>rex</i> subsp. <i>rex</i>); (C) short tubular corolla with a beaked galea (<i>P</i><i>. superba</i>); and (D) long tubular corolla with a beaked galea (<i>P</i><i>. cyathophylla</i>).</p

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

<div><p>Morphological identification of <i>Pedicularis</i> depends on floral characters. However, some important characters may be lost during the process of pressing the specimen. <i>Pedicularis delavayi</i> was described from northwestern Yunnan, and widely adopted as a variety of <i>P</i>. <i>siphonantha</i>. Unfortunately, the name “<i>P</i>. <i>siphonantha</i> var. <i>delavayi</i>’ incorrectly referred to <i>P</i>. <i>milliana</i> (a new species described in this study) or <i>P</i>. <i>tenuituba</i> in some herbarium specimens and publications. Moreover, phylogenetic relationships among <i>P</i>. <i>delavayi</i>, <i>P</i>. <i>siphonantha</i> and its allies (<i>P</i>. <i>milliana</i> and <i>P</i>. <i>tenuituba</i>) were not fully resolved. In this study, we sampled 76 individuals representing 56 taxa. Of them, 10 taxa were from <i>P</i>. <i>siphonantha</i> lineage, and 11 individuals of <i>P</i>. <i>delavayi</i> represented 9 populations. These species were named as <i>P</i>. <i>siphonantha</i> 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 <i>P</i>. <i>siphonantha</i> group was polyphyletic: <i>P</i>. <i>delavayi</i> was sister to <i>P</i>. <i>obliquigaleata</i> in clade A; and the remaining species of <i>P</i>. <i>siphonantha</i> group were monophyletic in clade B, named as <i>P</i>. <i>siphonantha</i> lineage. In the <i>P</i>. <i>siphonantha</i> lineage, <i>P</i>. <i>milliana</i>, <i>P</i>. <i>siphonantha</i>, and <i>P</i>. <i>tenuituba</i> were well supported as monophyletic, and <i>P</i>. <i>dolichosiphon</i> was sister to <i>P</i>. <i>leptosiphon</i>. Morphologically, <i>P</i>. <i>delavayi</i> differs from species of the <i>P</i>. <i>siphonantha</i> 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 <i>P</i>. <i>delavayi</i> as an independent species and describe <i>P</i>. <i>milliana</i> as new species. In addition, <i>P</i>. <i>neolatituba</i> was proposed to reduce as a new synonymy of <i>P</i>. <i>delavayi</i>.</p></div

Field photos of <i>P</i>. <i>delavayi</i> Franch. ex Maxim., <i>P</i>. <i>milliana</i> W. B. Yu, D. Z. Li & H. Wang and <i>P</i>. <i>siphonantha</i> D. Don.

<p>A, <i>P</i>. <i>siphonantha</i>. B, <i>P</i>. <i>milliana</i>. C-I, <i>P</i>. <i>delavayi</i>: C, G-I, from Daxue Mtn.; D from Hong Mtn.; E from Yulong Mtn.; F from Wuxu Lake. A spreading middle lobe of the corolla lower-lip with emargination indicated by an arrow in A and B; a folded middle lobe of the corolla lower-lip with emargination indicated by an arrow in D-G; a ridged corolla tube indicated by an arrow in G and H; an inflated calyx tube in the middle upper parts indicated by an arrow in H; black seeds indicated by an arrow in I. A and B were taken by Z.-K. Wu; E by H.-D. Li; C, D, and F-I by W.-B. Yu.</p

Sequence characteristics of nrITS and four plastid DNA regions.

<p>Sequence characteristics of nrITS and four plastid DNA regions.</p

Phylogeny of the <i>Pedicularis siphonantha</i> group inferred from Bayesian Inference (BI) and Maximum Likelihood (ML) methods using the combination of nuclear ribosomal internal transcribed spacer (nrITS) and four plastid (<i>matK</i>, <i>rbcL trnH-psbA</i> and <i>trnL-F</i>) datasets.

<p>Topology shows the majority rule consensus of the BI tree. BI posterior probability (PP) ≥ 0.50 and ML bootstrap support (BS) ≥ 50 were annotated on the branch. PP ≥ 0.95 and/or BS ≥ 70 were drawn with thicker and black lines.</p

Phylogenetic reassessment of tribe Anemoneae (Ranunculaceae): Non-monophyly of <i>Anemone</i> s.l. revealed by plastid datasets

<div><p>Morphological and molecular evidence strongly supported the monophyly of tribe Anemoneae DC.; however, phylogenetic relationships among genera of this tribe have still not been fully resolved. In this study, we sampled 120 specimens representing 82 taxa of tribe Anemoneae. One nuclear ribosomal internal transcribed spacer (nrITS) and six plastid markers (<i>atpB-rbcL</i>, <i>matK</i>, <i>psbA-trnQ</i>, <i>rpoB-trnC</i>, <i>rbcL</i> and <i>rps16</i>) were amplified and sequenced. Both Maximum likelihood and Bayesian inference methods were used to reconstruct phylogenies for this tribe. Individual datasets supported all traditional genera as monophyletic, except <i>Anemone</i> and <i>Clematis</i> that were polyphyletic and paraphyletic, respectively, and revealed that the seven single-gene datasets can be split into two groups, i.e. nrITS + <i>atpB-rbcL</i> and the remaining five plastid markers. The combined nrITS + <i>atpB-rbcL</i> dataset recovered monophyly of subtribes Anemoninae (i.e. <i>Anemone</i> s.l.) and Clematidinae (including <i>Anemoclema</i>), respectively. However, the concatenated plastid dataset showed that one group of subtribes Anemoninae (<i>Hepatica</i> and <i>Anemone</i> spp. from subgenus <i>Anemonidium</i>) close to the clade <i>Clematis</i> s.l. + <i>Anemoclema</i>. Our results strongly supported a close relationship between <i>Anemoclema</i> and <i>Clematis</i> s.l., which included <i>Archiclematis</i> and <i>Naravelia</i>. Non-monophyly of <i>Anemone</i> s.l. using the plastid dataset indicates to revise as two genera, new <i>Anemone</i> s.l. (including <i>Pulsatilla</i>, <i>Barneoudia</i>, <i>Oreithales</i> and <i>Knowltonia</i>), <i>Hepatica</i> (corresponding to <i>Anemone</i> subgenus <i>Anemonidium</i>).</p></div

Phylogenetic relationships within tribe Anemoneae based on the combination of nrITS and <i>atpB-rbcL</i> datasets.

<p>The topology is that of the majority rule consensus of BI tree. Bootstrap values of ML are presented under branches, and posterior probability of BI above branches. Topological incongruence between ML and BI trees is indicated by colored nodes/branches, and topology of BI tree shows by dash lines with posterior probability in square bracket under branches.</p

Phylogenetic relationships within tribe Anemoneae the combination of six-plastid-gene dataset.

<p>The six plastid genes are <i>atpB-rbcL</i>, <i>matK</i>, <i>psbA-trnQ</i>, <i>rbcL</i>, <i>rpoB-trnC</i>, and <i>rps16</i>. The topology is that of the majority rule consensus of ML tree. Bootstrap values of ML are presented above branches, and posterior probability of BI under branches. Topological incongruence between ML and BI trees is indicated by colored nodes/branches, and topology of BI tree shows by dash lines with posterior probability in square bracket under branches.</p