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.

We included all currently accepted species of the A. pallida complex in our analysis (A. pallida, A. lutea, A. nardiana, A. microstoma, A. merxmuelleri, A. croatica and A. castellana). 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 Aristolochia section Diplolobus (subsection Aristolochia and Podanthemum) as well as more distantly related outgroups from the remaining Aristolochia lineages. The Mediterranean Aristolochia species belong to Aristolochia subsection Aristolochia whereas Aristolochia subsection Podanthemum is distributed from Asia to Africa. Both taxa belong to Aristolochia subgenus Aristolochia. Aristolochia subgenus Siphisia is sampled with three accessions (A. westlandii, A. salvadorensis, A. macrophylla). 

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. trnK intron, matK gene and trnK-psbA 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.