BEAST chronogram for the combined data and hypothesized biogeographic history of Sarraceniaceae.

<p>(A) Mean divergence times estimates are shown at the nodes of the cladogram. 95% posterior probability distribution shown with thick blue lines. Ancestral areas reconstructions from LAGRANGE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039291#pone.0039291-Drummond1" target="_blank">[70]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039291#pone.0039291-Swofford1" target="_blank">[71]</a> shown in boxes near nodes. SA = South America; ENA = Eastern North America; WNA = Western North America; SAf = South Africa; and As = Asia. (B) We hypothesize that Sarraceniaceae originated in the Middle Eocene, perhaps in South America, and achieved its widespread distribution in North and South America by the Late Eocene. An early migration of Sarraceniaceae out of South America during the Eocene may have been facilitated via land connections in the proto-Caribbean. This connection would likely have been unavailable for direct overland migration by the mid-Oligocene, which is consistent with the early Oligocene disjunction of northern (<i>Sarracenia</i>, <i>Darlingtonia</i>) and southern (<i>Heliamphora</i>) members of Sarraceniacace. An East (<i>Sarracenia</i>+<i>Heliamphora</i>)/West (<i>Darlingtonia</i>) disjunction occurred in the very latest Oligocene, and may have been attributable to broad scale cooling and aridification during the late Oligocene.</p

Geographic distribution of Sarraceniaceae.

<p><i>Darlingtonia</i> (A) is restricted to western North America, <i>Sarracenia</i> (B) is widespread in Eastern North America, and <i>Heliamphora</i> (C) occurs in northern South America <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039291#pone.0039291-Juniper1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039291#pone.0039291-Schnell1" target="_blank">[27]</a>. Photographs by the authors.</p

Maximum likelihood phylogeny of Sarraceniaceae based on plastid, nuclear, and mitochondrial data combined.

<p><i>Sarracenia purpurea</i> var. <i>montana</i> was excluded from this analysis (see text). ML bootstrap percentages >65 and Bayesian posterior probabilities >0.85 are indicated at the nodes, respectively. Scale bar shows nucleotide substitutions per site.</p

Structures of the indole-diterpene biosynthesis loci (<i>IDT/LTM</i>) in sequenced genomes.

<p><i>IDT/LTM</i> genes are indicated by single letters, whereby <i>Q = idtQ</i> or <i>ltmQ</i> (in <i>E. festucae</i>), and so forth. Tracks from top to bottom of each map represent the following: genes, repeats, MITEs, and graphs of AT (red) and GC (blue) contents. Each gene is represented by a filled arrow indicating its direction of transcription. Closed circles indicate telomeres, and distances from the telomere on the <i>E. festucae</i> map are indicated in kilobasepairs (kb). Cyan bars representing repeat sequences are labeled with names or numbers to indicate relationships between repeats in the different species. Vertical bars beneath the repeat maps indicate MITEs. Genes for the first fully cyclized intermediate, paspaline, are indicated in blue, those for subsequent chemical decorations are shown in red, and <i>idt/ltmS</i>, with undetermined function, is in purple. Identifiable genes flanking the clusters are indicated in gray, and unfilled arrows indicate pseudogenes. The major pathway end-product for each strain is listed at the right of its map, abbreviated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, and in bold for those confirmed in this study.</p

Phylogenies of <i>rpbA</i> from sequenced isolates and other Clavicipitaceae.

<p>The phylogenetic tree is based on nucleotide alignment for a portion of the RNA polymerase II largest subunit gene, <i>rpbA</i>. This tree is rooted with <i>Fusarium graminearum</i> as the outgroup. Epichloae are indicated in green, <i>Claviceps</i> species are indicated in blue, <i>Periglandula</i> species are indicated in red, and <i>Aciculosporium take</i> is in black. Species for which genomes were sequenced in this study are shown in bold type, and asterisks indicate plant-associated fungi. Alkaloids listed are the major pathway end-products predicted from the genome sequences, abbreviated as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g002" target="_blank">Figure 2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g004" target="_blank">Figure 4</a>. Other abbreviations: (−) = some genes or remnants present, but not predicted to make alkaloids of this class, – = no genes present for this alkaloid class, EA = ergot alkaloids may be produced; IDT = indole-diterpenes may be produced, (ΔR*) = deletion of terminal reductase domain of <i>perA</i>.</p

GC proportions in genic and repeat DNA of sequenced genomes.^a

a<p>Abbreviations: CDS = coding sequence, GC = proportion of sequence that is G or C, non-Rpt-IG = nonrepetitive intergenic DNA, Rpt = repetitive DNA.</p>b<p>Statistics for <i>P. ipomoeae</i> are tentative because the assembly was filtered by selecting only contigs containing tBLASTx matches to genome sequences from the other Clavicipitaceae.</p

Relative repeat contents in specialized metabolite clusters of <i>Epichloë festucae</i>.

<p>Log-ratios of repeat sequences (Rpt) to coding sequences (CDS) are shown in order of increasing proportions of repeats. Open boxes represent clusters that are apparently nonfunctional due to inactivation of signature genes.</p

Alkaloid biosynthesis genes in sequenced isolates.^a

a<p>Abbreviations: <i>Ata = Aciculosporium take</i> MAFF 241224, and other strains are abbreviated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-t004" target="_blank">Table 4</a>, except that <i>Egl</i> = both <i>E. glyceriae</i> strains, E277 and E2772, which had identical sets of alkaloid genes. Symbols are: + = Apparently functional gene, Ψ  = pseudogene, Δ = deleted reductase-encoding domain of <i>perA</i> (<i>perA</i>-Δ<i>R*</i>). Two symbols in a cell indicate two gene copies.</p

Alkaloid profiles of sequenced isolates.^a

a<p>Strains are abbreviated as follow: <i>Cpu</i> = <i>Claviceps purpurea</i> 20.1, <i>Cfu</i> = <i>C. fusiformis</i> PRL 1980, Cpa = <i>C. paspali</i> RRC-1481, <i>Eam</i> = <i>Epichloë amarillans</i> E57, <i>Ebe</i> = <i>E. brachyelytri</i> E4804, <i>Eel</i> = <i>E. elymi</i> E56, <i>Ef</i>1 = <i>E. festucae</i> Fl1, <i>Ef</i>2 = <i>E. festucae</i> E2368, <i>Egl</i> = <i>E. glyceriae</i> E2772, <i>Et</i>8 = <i>E. typhina</i> E8, <i>Et</i>5 = <i>E. typhina</i> E5819, <i>Nga</i> = <i>N. gansuense</i> E7080, <i>Ngi</i> = <i>N. gansuense</i> var. <i>inebrians</i> E818, <i>Nun</i> = <i>N. uncinatum</i> E167, <i>Pip</i> = <i>P. ipomoeae</i> IasaF13. Symbols: + = present, (+) = intermediate inferred to be synthesized because downstream product is present, − = not predicted and not detected, (−) = predicted but not detected, nt = predicted but not tested, ERA = ergotamine, ERB = ergobalansine, ERC = ergocryptine, ERV = ergovaline. Blank cells indicate compounds not predicted from genotype, and not tested.</p>b<p>Identification of IDT-436 and terpendoles E, I, J, K, M, M, and A are tentative because authentic standards are unavailable.</p

Summary of loline alkaloid-biosynthesis pathway.

<p>Arrows indicate one or more steps catalyzed by products of the genes indicated. Arrows and genes in blue indicate steps in synthesis of the first fully cyclized intermediate (NANL). Arrows and genes in red indicate steps in modification of NANL to give the variety of lolines found in the epichloae. Asterisks indicate <i>LOL</i> genes that were newly discovered in the genome sequence of <i>E. festucae</i> E2368.</p