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, 8 (7), e70501

Phylogenetic Relationships and Species Delimitation in Pinus Section Trifoliae Inferrred From Plastid DNA


Phylogenetic Relationships and Species Delimitation in Pinus Section Trifoliae Inferrred From Plastid DNA

Sergio Hernández-León et al. PLoS One.


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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Geographic distribution of individual samples of Pinus section Trifoliae.
Three of the 191 individuals are of unknown provenance.
Figure 2
Figure 2. Variation of the plastid markers in Pinus section Trifoliae.
Above: The proportion of variable sites for each of the five markers. Below: the total number of variable sites per codon position for the three protein coding regions.
Figure 3
Figure 3. Maximum likelihood tree.
Bootstrap values greater than 50% are shown at branches. A. Pinus subsection Australes. Images from top to bottom are of P . lumholtzii , P . herrerae , P . oocarpa , P . chihuahuana , and P . attenuata . B. Pinus subsections Ponderosae and Contortae. Images from top to bottom are P . scopulorum , P . douglasiana , P . pseudostrobus var. apulcensis , P . coulteri , and P . contorta . The branch leading to the outgroups has been truncated.
Figure 4
Figure 4. Chronogram for Pinus section Trifoliae using three calibration points and lineage-through-time plot.
Chronogram for section Trifoliae. The outgroup has been removed. The two secondary calibration points inferred from a previous relaxed molecular clock are indicated with an asterisk (*), and a fossil calibration point attributed to the California closed-cone pines is indicated with a double asterisk (**). The corresponding lineage-through-time plot is given on the upper left. The transition from speciation to coalescence branching processes gave nonsignificant test results.

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Grant support

This study was supported by the Dirección General de Asuntos del Personal Académico-Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica of the Universidad Nacional Autónoma de México (DGAPA-PAPIIT; IN228209), the United Nations Development Programme, the National Commission for Knowledge and Use of Biodiversity (CONABIO; GE021), and the National Council on Science and Technology (CONACYT; The Barcodes of Life Thematic Network). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.