DOI: 10.5061/dryad.n5tb2rbxp
title: The evolution of the Aristolochia pallida complex (Aristolochiaceae) contradicts traditional taxonomy and reflects large-scale glacial refugia in the Mediterranean

description: The taxonomy of the Mediterranean Aristolochia pallida complex has been
 under debate for several decades with the following species currently
 recognized: A. pallida, A. lutea, A. nardiana, A. microstoma, A.
 merxmuelleri, A. croatica and A. castellana. These taxa are distributed
 from Iberia to Turkey. To reconstruct phylogenetic and biogeographic
 patterns, we employed cpDNA sequence variation using both non-coding
 (intron and spacer) and protein-coding regions (i.e. trnK intron, matK
 gene and trnK-psbA spacer). Our results show that the morphology-based
 traditional taxonomy was not corroborated by our phylogenetic analyses.
 Aristolochia pallida, A. lutea, A. nardiana and A. microstoma were not
 monophyletic. Instead, strong geographic signals were detected. Two major
 clades, one exclusively occurring in Greece and a second one of
 pan-Mediterranean distribution, were found. Several subclades distributed
 in Greece, NW Turkey, Italy, as well as amphi-Adriatic subclades and a
 subgroup of southern France and Spain were revealed. The distribution
 areas of these groups are in close vicinity to hypothesised glacial
 refugia areas in the Mediterranean. According to molecular clock analyses,
 the diversification of this complex started around 3–3.3 my, before the
 onset of glaciation cycles, and the further evolution of and within major
 lineages falls into the Pleistocene. Based on these data, we conclude that
 the Aristolochia pallida alliance survived in different Mediterranean
 refugia rarely with low, but often with a high potential for range
 extension, and a high degree of morphological plasticity.
description: We included all currently
 accepted species of the <em>A. pallida </em>complex in our
 analysis (<em>A. pallida, A. lutea, A. nardiana, A. microstoma, A.
 merxmuelleri, A. croatica </em>and<em> A.
 castellana</em>). In total, 87 accessions of the aforementioned taxa
 covering both the entire distribution range as well as the phenotypic
 plasticity were included. Additional 41 accessions of 33 Aristolochia taxa
 were selected to include the entire diversity of
 <em>Aristolochia</em> section <em>Diplolobus</em>
 (subsection <em>Aristolochia</em> and
 <em>Podanthemum</em>) as well as more distantly related
 outgroups from the remaining <em>Aristolochia</em> lineages.
 The Mediterranean <em>Aristolochia</em> species belong to
 <em>Aristolochia</em> subsection
 <em>Aristolochia</em> whereas
 <em>Aristolochia</em> subsection
 <em>Podanthemum</em> is distributed from Asia to Africa. Both
 taxa belong to <em>Aristolochia</em> subgenus
 <em>Aristolochia</em>. <em>Aristolochia</em>
 subgenus <em>Siphisia</em> is sampled with three accessions
 (<em>A. westlandii, A. salvadorensis, A.
 macrophylla</em>).  Total genomic
 DNA was extracted from herbarium specimens, fresh leaves of cultivated
 plants, and silica-dried leaf material, collected in natural populations,
 respectively.  We used the chloroplast trnK-matK-psbA region
 for phylogenetic reconstructions (i.e. <em>trnK</em> intron,
 <em>matK</em> gene and
 <em>trnK</em>-<em>psbA</em>
 spacer).
 Sequences were aligned using MAFFT v7.388 as
 implemented in GENEIOUS followed by a manual check. Some very variable
 poly A-T regions were excluded from the analysis. This concerns the
 alignment positions 1–64, 377–1143, 1243–1257, 1471–1480, 1532–1566,
 2341–2358, 3363–3387, 3695–3709, 3857–3955, and
 4009–4061.