Campanuloideae_Roucela_atpB_matK_petD_rbcL_Spacer_AT11_AT70

FASTA alignment file of all Campanuloideae taxa (including Roucela species) included in the study. Alignment contains atpB, matK, petD, rbcL, atpB-rbcL spacer region, AT11, and AT70 sequences

Phylogeny of Campanuloideae (Campanulaceae) with Emphasis on the Utility of Nuclear Pentatricopeptide Repeat (PPR) Genes

<div><p>Background</p><p>The Campanuloideae (Campanulaceae) are a highly diverse clade of angiosperms found mostly in the Northern Hemisphere, with the highest diversity in temperate areas of the Old World. Chloroplast markers have greatly improved our understanding of this clade but many relationships remain unclear primarily due to low levels of molecular evolution and recent and rapid divergence. Furthermore, focusing solely on maternally inherited markers such as those from the chloroplast genome may obscure processes such as hybridization. In this study we explore the phylogenetic utility of two low-copy nuclear loci from the pentatricopeptide repeat gene family (PPR). Rapidly evolving nuclear loci may provide increased phylogenetic resolution in clades containing recently diverged or closely related taxa. We present results based on both chloroplast and low-copy nuclear loci and discuss the utility of such markers to resolve evolutionary relationships and infer hybridization events within the Campanuloideae clade.</p><p>Results</p><p>The inclusion of low-copy nuclear genes into the analyses provides increased phylogenetic resolution in two species-rich clades containing recently diverged taxa. We also obtain support for the placement of two early diverging lineages (<i>Jasione</i> and <i>Musschia</i>-<i>Gadellia</i> clades) that have previously been unresolved. Furthermore, phylogenetic analyses of PPR loci revealed potential hybridization events for a number of taxa (e.g., <i>Campanula pelviformis</i> and <i>Legousia</i> species). These loci offer greater overall topological support than obtained with plastid DNA alone.</p><p>Conclusion</p><p>This study represents the first inclusion of low-copy nuclear genes for phylogenetic reconstruction in Campanuloideae. The two PPR loci were easy to sequence, required no cloning, and the sequence alignments were straightforward across the entire Campanuloideae clade. Although potentially complicated by incomplete lineage sorting, these markers proved useful for understanding the processes of reticulate evolution and resolving relationships at a wide range of phylogenetic levels. Our results stress the importance of including multiple, independent loci in phylogenetic analyses.</p></div

Plastid Phylogeny.

<p>Best tree from maximum likelihood analysis of combined plastid dataset: <i>atpB</i>, <i>matK</i>, <i>petD</i>, <i>rbcL</i>, and <i>trnL</i>-<i>F</i>. Numbers above branches are bootstrap values >50%. Numbers below branches indicate posterior probabilities >.70 from Bayesian analysis. Letters A-K refer to nodes and clades discussed in the text.</p

Combined Plastid and PPR Phylogeny.

<p>Best tree from maximum likelihood analysis of combined plastid-PPR dataset: <i>atpB</i>, <i>matK</i>, <i>petD</i>, <i>rbcL</i>, <i>trnL</i>-<i>F</i>, <i>PPR11</i>, and <i>PPR70</i>. Numbers above branches are bootstrap values >50%. Numbers below branches indicate posterior probabilities >.70 from Bayesian analysis. Letters refer to nodes and clades discussed in the text. Nodes for which bootstrap values are increased compared to the plastid-only analysis are highlighted in blue.</p

Divergence Time Estimates for Combined Plastid and PPR Tree.

<p>Chronogram from BEAST analysis of the combined plastid-PPR dataset. Scale bar is in millions of years before present. Green star indicates placement of fossil constraint. Green diamonds indicate age constraints obtained from a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094199#pone.0094199-Bell1" target="_blank">[53]</a>. Numbers above branches indicate mean age estimates for clades (in millions of years). Error bars around nodes correspond to 95% highest posterior distributions of divergence times for clades discussed in the text.</p

Comparison of Support for Plastid-only Tree and Combined Plastid-PPR Tree.

<p>Maximum-likelihood trees from the plastid dataset (a) and the combined plastid-PPR dataset (b) with taxon names removed. Branches are shaded relative to BS support with darker branches indicating higher support. Letters correspond to clade/node names in text and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094199#pone-0094199-g001" target="_blank">Figure 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094199#pone-0094199-g003" target="_blank">Figure 3</a>. Support for many clades is increased with the inclusion of PPR loci while other areas of the tree remain poorly supported.</p

Chloroplast and Nuclear Loci Used in this Study.

<p>Chloroplast and Nuclear Loci Used in this Study.</p

Maximum Parsimony Strict consensus tree of <i>Campanula</i> and relatives (D680).

<p>Part of the cladogram showing detailed relationships for outgroup and sister lineages, and clades Cam01, <i>Jasione</i>-<i>Feeria</i>, and Cam02 to Cam04. Values below branches indicate bootstrap support for the sustained clades. Gray boxes indicate the respective outgroup and sister clades; blue boxes refer to “Cam” clades containing at least one accession of <i>Campanula</i> (Cam01 to Cam17; see text). A blue dot indicates the crown node of <i>Campanula</i> s.lat. Pictures are representative specimens for clades Cam01 (<i>Campanula primuliifolia</i>), Cam02 (<i>Campanula exigua</i>), Cam03 (<i>Campanula persicifolia</i>), and Cam04 (<i>Legousia falcata</i>). All photos from Guilhem Mansion.</p

Characteristics of the respective phylogenetic analyses for the three datasets (D088, D101, and D680).

<p>BS = bootstrap, JK = jackknife, pp = posterior probability.</p

Chronogram of <i>Campanula</i> and relatives (D680) inferred from the penalized-likelihood method implemented in r8s, and dated using one fossil constraint (yellow spiral).

<p>The yellow box refers to the time span between the stem and crown node of <i>Campanula</i> s.lat. Clades are represented by triangles proportional in size to the number of included accessions. Gray triangles indicate the respective outgroup and sister clades; blue triangles refer to “Cam” clades containing at least one accession of <i>Campanula</i> (Cam01 to Cam17; see text). White bars represent 95% confidence intervals (CI) for the respective node ages (blue: crow ages; white: stem ages). An asterisk indicates nodes for which CI could not be calculated. Ma = Mega Annuum or Million years; LOBE = Lobelioideae; CYPHI: Cyphioideae; CA-CYA: Campanuloideae-Cyanantheae; CA-WAH: Campanuloideae-Wahlenbergieae.</p