Dylan O. Burge; Kaila Mugford; Amy P. Hastings; Anurag A. Agrawal

“ The center of diversity for Pachypodium is Madagascar, with 16 endemic species; the remaining five species are restricted to southern Africa (Fig. 1; Table 1) . Most Pachypodium species are narrowly distributed, with specialized ecology ( [1] ; [6] ; [5] ) ; habitats vary from desert to subhumid grassland, although most species are restricted to extremely arid environments (i.e., 8–34 cm annual precipitation; [5] ) . ”

“ We treated gaps as missing data for all phylogenetic analyses.
Topology testing
We used Templeton’s nonparametric test (1983) , as implemented in PAUP* v 4.0 ( [27] ) , to evaluate several key phylogenetic relationships. Templeton’s test compares pairs of topologies, measuring relative statistical support for the trees within a sequence dataset (alignment) . For these tests, we compared the best tree from the original parsimony tree search (see above) to the best tree from a search using a constraint (e.g., African Pachypodium constrained as monophyletic) . ”

“ The genus Pachypodium contains 21 species of succulent, generally spinescent shrubs and trees found in southern Africa and Madagascar. Pachypodium has diversified mostly into arid and semi-arid habitats of Madagascar, and has been cited as an example of a plant group that links the highly diverse arid-adapted floras of Africa and Madagascar. However, a lack of knowledge about phylogenetic relationships within the genus has prevented testing of this and other hypotheses about the group.Methodology/Principal Findings. ”

“ Additional molecular phylogenetic work will be required to obtain better support for basal-branching relationships in Pachypodium, particularly the relationship between African and Malagasy species. This work will likely require the sequencing of additional loci, from both the chloroplast and nuclear genome. Resolution of relationships among species from [6] ’s ( [6] ) section Gymnopus will also require additional work. In Gymnopus, a number of widespread species (e.g., P. densiflorum and P. brevicaule) are non-monophyletic. ”

“ We use DNA sequence data from the nuclear ribosomal ITS and chloroplast trnL-F region for all 21 Pachypodium species to reconstruct evolutionary relationships within the genus. We compare phylogenetic results to previous taxonomic classifications and geography. Results support three infrageneric taxa from the most recent classification of Pachypodium, and suggest that a group of African species (P. namaquanum, P. succulentum and P. bispinosum) may deserve taxonomic recognition as an infrageneric taxon. ”

“ Variable, the number of variable characters in the ingroup, followed by the number of variable characters in the full alignment (in parentheses) ; PIC, the number of parsimony-informative characters in the ingroup, followed by the number of parsimony informative characters in the full alignment (in parentheses) . Following individual alignment of ITS and trnL-F, we endeavored to create a combined alignment. Preliminary analyses showed that for the single Pachypodium sample represented by more than one cloned ITS sequence (P053; Table 1) , the four sequences formed a monophyletic group. ”

“ However, our results do not resolve relationships among major African and Malagasy lineages of the genus.Conclusions/Significance. We present the first molecular phylogenetic analysis of Pachypodium. Our work has revealed five distinct lineages, most of which correspond to groups recognized in past taxonomic classifications. Our work also suggests that there is a complex biogeographic relationship between Pachypodium of Africa and Madagascar. ”

“ Those species that occur in more mesic habitats (up to 200 cm annual precipitation; [5] ) tend to occupy rocky outcrops that are probably edaphically arid.
Geographic distribution of Pachypodium.Inset is sampling of Pachypodium in Madagascar (Appendix 1) . Sampling in Africa not mapped. Data for distributions is approximate, adapted from [7] and [1] . Table 1 "Pachypodium species, sampling, geography, and traits. ”

“ For seeds, up to three excised embryos from a single parent plant were pooled prior to DNA extraction ( [21] ) . DNA was extracted from seeds when silica-dried material for the same plant was not available, or proved recalcitrant to extraction of high quality DNA. All polymerase chain reactions were performed using Qiagen Taq DNA Polymerase. Amplifications were performed using an initial incubation at 94°C for 10 min and 30 cycles of three-step PCR (1 min at 94°C, 30 s at 45°C, and 2 min at 72°C) , followed by final extension at 72°C for 7 min. ”

“ ITS tree is midpoint rooted. Zigzag line indicates that the branch connecting the outgroup to Pachypodium is not shown to scale (see Treefiles S4 and S5) . Maximum parsimony searches based on ITS data alone resulted in 4851 trees of 324 steps (Table 4; Treefile S4B) ; a total of 12 internal nodes had bootstrap (BS) support greater than or equal to 95% (Treefile S4C) . Searches using trnL-F data alone resulted in 8 trees of 71 steps (Table 4; Treefile S5B) ”

“ Bayesian consensus phylograms for individual genetic regions.Left, ITS; right, trnL-F. Numbers above branches are Bayesian posterior probability (PP) from the 50% majority rule consensus tree; thickened branches have PP of 1.0. ”