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Focal species descriptions
Among the Asteraceae represented in our dataset are some of the most
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noxious weeds and invaders in the temperate world, including the six focal invasive
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taxa of our study. The species in the genus Centaurea, including C. solstitialis, C.
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stoebe ssp. micranthos, and C. diffusa, are native to Europe and Asia, and have
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successfully invaded North American rangelands, making Centaurea the most
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abundant noxious weed genus in western North America (Lejeune & Seastedt 2001).
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In fact, Centaurea is one of only 15 plant genera significantly more likely to contain
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invasive species in North America than expected by chance (Kuester et al. 2014).
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Cirsium arvense Scop is native to temperate Eurasia, where it is one of the foremost
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noxious agricultural weeds (Schroeder et al. 1993). It has spread to four other
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continents and has become one of the most prevalent weeds in North America
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(Moore 1975). Species in the genus Ambrosia (ragweeds), including A. artemisiifolia
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and A. trifida, are native to North America and have become abundant colonizers of
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disturbed habitats across temperate North America, Europe and Asia (Bassett &
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Crompton 1975, 1982). Ragweeds are agricultural weeds and their allergenic pollen
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is one of the primary causes of hay fever (Laaidi et al. 2003). Lastly, thirteen
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members of the genus Helianthus (sunflowers), and in particular H. annuus are
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globally introduced or invasive (Chamberlain and Szocs, 2013; EOL, 2014). The high
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levels of gene flow between members of this genus make it a primary concern for
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transgene escape and the evolution of a “super weed” (discussed in Lai et al. 2012).
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Paralog removal
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To remove potential paralogs from our alignments, we used a tree-based
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approach. Briefly, we constructed gene trees for each orthogroup using RAxML
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version 8.0.6 (Stamatakis 2006). Jmodeltest 2.1.4 was run for each gene to guide the
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selection of the nucleotide substitution model (Darriba et al. 2012). To improve the
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resulting gene tree, we implemented TreeFix v1.1.8 (Wu et al. 2013), which uses the
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species tree topology to guide the reconstruction of the gene tree. Given a gene tree,
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TreeFix finds a “statistically equivalent” tree that minimizes a species tree-based
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cost function. Following this we used the program NOTAUG v2.6 (Durand et al.
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2006) and compared the reconciled gene tree with the species tree (see species tree
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reconstruction below) to identify likely paralogs. We removed sequences that were
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identified as paralogs using this method from the alignments. We included at most
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two sequences of the same species if they were in the alignments. Although this
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method may also eliminate genes crossing species boundaries, we preferred this
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conservative approach to avoid excessive numbers of false positives resulting from
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misidentification of orthology.
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Pairwise comparisons of native and introduced transcriptomes
Specifically, we ran CODEML for the protein coding regions in runmode -2,
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with F3X4 codon frequency. Prior to the analysis we eliminated all alignments with
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average percent identity below 50%, as these likely represented misalignments
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(this procedure was conducted for all alignments, such as the branch-site and site-
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specific analyses). We also removed columns with missing data or gaps and only
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retained sequences with at least 150 nucleotides for the coding regions. As low
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divergence leads to uncertain dN/dS ratio estimates, cases where dS was below 0.01
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were excluded. We also discarded orthogroups showing dS or dN > 2, indicating
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saturation of substitutions. Orthogroups with dN/dS > 1 were considered
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candidates for positive selection.
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PAML branch and branch-site models
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As gaps and other alignment errors have been known to generate false
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positives in dN/dS analyses (Fletcher & Yang 2010; Markova-Raina & Petrov 2011),
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each orthogroup and/or site inferred to be under positive selection was visually
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inspected in AliView 1.07 (Larsson 2014), and any cases of potential misalignment
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(e.g. errors associated with indels) were removed from the analysis. Furthermore,
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because nucleotide changes inferred to be under positive selection did not occur in
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all taxa designated as foreground, we performed an additional set of PAML analyses
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on the remaining orthogroups setting as foreground only taxa in which changes of
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interest were present.
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Gene Ontology analysis
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We assigned GO terms to each orthogroup based on the GO A. thaliana
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mappings to the top hits and removed redundant GO terms. To identify which
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biological processes rapidly evolving genes were associated with, we performed a
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GO enrichment analysis using topGO (Alexa et al. 2006). All orthogroups that were
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not significant in tests of positive selection were used as background. Significance
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for each individual GO-identifier was computed with Fisher's exact test. As GO terms
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are non-independent, we used the parent-child method that determines
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overrepresentation of terms in the context of annotations to the term's parents
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(Grossmann et al. 2007). This approach reduces the dependencies between the
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individual terms, and avoids producing false-positives.
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