ORIGINAL ARTICLE Against all odds: reconstructing the evolutionary history of Scrophularia (Scrophulariaceae) despite high levels of incongruence and reticulate evolution Agnes Scheunert 1 & Günther Heubl 1 Received: 4 August 2016 /Accepted: 30 November 2016 # Gesellschaft für Biologische Systematik 2017 Abstract The figwort genus Scrophularia (Scrophulariaceae), widespread across the temperate zone of the Northern Hemisphere, comprises about 250 species and is a taxonomi- cally challenging lineage displaying large morphological and chromosomal diversity. Scrophularia has never been exam- ined in a large-scale phylogenetic and biogeographic context and represents a useful model for studying evolutionary his- tory in the context of reticulation. A comprehensively sampled phylogeny of Scrophularia was constructed, based on nuclear ribosomal (ITS) and plastid DNA sequences (trnQ-rps16 intergenic spacer, trnL-trnF region) of 147 species, using Bayesian inference and maximum likelihood approaches. Selected individuals were cloned. A combination of coding plastid indels and ITS intra-individual site polymorphisms, and applying Neighbor-Net and consensus network methods for adequate examination of within-dataset uncertainty as well as among-dataset incongruence, was used to disentangle phy- logenetic relationships. Furthermore, divergence time estima- tion and ancestral area reconstruction were performed to infer the biogeographic history of the genus. The analyses reveal significant plastid-nuclear marker incongruence and consider- able amounts of intra-individual nucleotide polymorphism in the ITS dataset. This is due to a combination of processes including reticulation and incomplete lineage sorting, possibly complicated by inter-array heterogeneity and pseudogenization in ITS in the presence of incomplete con- certed evolution. Divergence time estimates indicate that Scrophularia originated during the Miocene in Southwestern Asia, its primary center of diversity. From there, the genus spread to Eastern Asia, the New World, Europe, Northern Africa, and other regions. Hybridization and polyploidy played a key role in the diversification history of Scrophularia, which was shaped by allopatric speciation in mountainous habitats during different climatic periods. Keywords Scrophularia . Incongruence . Reticulate evolution . Intra-individual polymorphism . 2ISP . Allopatric speciation Introduction In recent years, an increasing number of phylogenetic studies in plants, based on molecular sequence information from nu- merous independent loci, have revealed discordance among chloroplast and nuclear gene trees as well as gene trees in general. Although methodological issues in data collection or analysis might be responsible for some of these observa- tions, incongruence may also be due to conflicting genealog- ical histories (e.g., Rokas et al. 2003; van der Niet and Linder 2008). These can be caused by gene duplications or losses, or by incomplete lineage sorting (ILS; Maddison 1997; Degnan and Rosenberg 2009). Furthermore, processes involving retic- ulation, i.e., gene flow among species, have been identified, e.g., horizontal gene transfer, introgression, and homo- or polyploid hybridization. These phenomena are common in plants (Rieseberg and Wendel 1993; Rieseberg et al. 1996; Wendel and Doyle 1998; Mason-Gamer 2004; Richardson and Palmer 2007); reticulation is now even regarded as a Electronic supplementary material The online version of this article (doi:10.1007/s13127-016-0316-0) contains supplementary material, which is available to authorized users. * Agnes Scheunert agnes.scheunert@gmx.net 1 Systematic Botany and Mycology, Department Biology I, and GeoBio-Center LMU, Ludwig-Maximilians-University Munich, Menzinger Strasse 67, 80638 Munich, Germany Org Divers Evol DOI 10.1007/s13127-016-0316-0