Multi-locus phylogenetics, lineage sorting, and reticulation in Pinus subsection Australes.

PREMISE OF THE STUDY: Both incomplete lineage sorting and reticulation have been proposed as causes of phylogenetic incongruence. Disentangling these factors may be most difficult in long-lived, wind-pollinated plants with large population sizes and weak reproductive barriers. METHODS: We used solution hybridization for targeted enrichment and massive parallel sequencing to characterize low-copy-number nuclear genes and high-copy-number plastomes (Hyb-Seq) in 74 individuals of Pinus subsection Australes, a group of ~30 New World pine species of exceptional ecological and economic importance. We inferred relationships using methods that account for both incomplete lineage sorting and reticulation. KEY RESULTS: Concatenation- and coalescent-based trees inferred from nuclear genes mainly agreed with one another, but they contradicted the plastid DNA tree in recovering the Attenuatae (the California closed-cone pines) and Oocarpae (the egg-cone pines of Mexico and Central America) as monophyletic and the Australes sensu stricto (the southern yellow pines) as paraphyletic to the Oocarpae. The plastid tree featured some relationships that were discordant with morphological and geographic evidence and species limits. Incorporating gene flow into the coalescent analyses better fit the data, but evidence supporting the hypothesis that hybridization explains the non-monophyly of the Attenuatae in the plastid tree was equivocal. CONCLUSIONS: Our analyses document cytonuclear discordance in Pinus subsection Australes. We attribute this discordance to ancient and recent introgression and present a phylogenetic hypothesis in which mostly hierarchical relationships are overlain by gene flow.

Phylogenetic relationships and species delimitation in pinus section trifoliae inferrred from plastid DNA.

Recent diversification followed by secondary contact and hybridization may explain complex patterns of intra- and interspecific morphological and genetic variation in the North American hard pines (Pinus section Trifoliae), a group of approximately 49 tree species distributed in North and Central America and the Caribbean islands. We concatenated five plastid DNA markers for an average of 3.9 individuals per putative species and assessed the suitability of the five regions as DNA bar codes for species identification, species delimitation, and phylogenetic reconstruction. The ycf1 gene accounted for the greatest proportion of the alignment (46.9%), the greatest proportion of variable sites (74.9%), and the most unique sequences (75 haplotypes). Phylogenetic analysis recovered clades corresponding to subsections Australes, Contortae, and Ponderosae. Sequences for 23 of the 49 species were monophyletic and sequences for another 9 species were paraphyletic. Morphologically similar species within subsections usually grouped together, but there were exceptions consistent with incomplete lineage sorting or introgression. Bayesian relaxed molecular clock analyses indicated that all three subsections diversified relatively recently during the Miocene. The general mixed Yule-coalescent method gave a mixed model estimate of only 22 or 23 evolutionary entities for the plastid sequences, which corresponds to less than half the 49 species recognized based on morphological species assignments. Including more unique haplotypes per species may result in higher estimates, but low mutation rates, recent diversification, and large effective population sizes may limit the effectiveness of this method to detect evolutionary entities.