Silent mutation rate estimates for Pinus vary 50-fold, ranging from angiosperm-like to among the slowest reported for plants. These differences either reflect extraordinary genomic processes or inconsistent fossil calibration, and they have important consequences for population and biogeographical inferences. Here we estimate mutation rates from 4 Pinus species that represent the major lineages using 11 nuclear and 4 chloroplast loci. Calibration was tested at the divergence of Pinus subgenera with the oldest leaf fossil from subg. Strobus (Eocene; 45 MYA) or a recently published subg. Strobus wood fossil (Cretaceous; 85 MYA). These calibrations place the origin of Pinus 190-102 MYA and give absolute silent rate estimates of 0.70-1.31x10(-9) and 0.22-0.42x10(-9).site-1.year-1 for the nuclear and chloroplast genomes, respectively. These rates are approximately 4- to 20-fold slower than angiosperms, but unlike many previous estimates, they are more consistent with the high per-generation deleterious mutation rates observed in pines. Chronograms from nuclear and chloroplast genomes show that the divergence of subgenera accounts for about half of the time since Pinus diverged from Picea, with subsequent radiations occurring more recently. By extending the sampling to encompass the phylogenetic diversity of Pinus, we predict that most extant subsections diverged during the Miocene. Moreover, subsect. Australes, Ponderosae, and Contortae, containing over 50 extant species, radiated within a 5 Myr time span starting as recently as 18 MYA. An Eocene divergence of pine subgenera (using leaf fossils) does not conflict with fossil-based estimates of the Pinus-Picea split, but a Cretaceous divergence using wood fossils accommodates Oligocene fossils that may represent modern subsections. Because homoplasy and polarity of character states have not been tested for fossil pine assignments, the choice of fossil and calibration node represents a significant source of uncertainty. Based on several lines of evidence (including agreement with ages inferred using calibrations outside of Pinus), we conclude that the 85 MYA calibration at the divergence of pine subgenera provides a reasonable lower bound and that further refinements in age and mutation rate estimates will require a synthetic examination of pine fossil history.

Uncertainties in the age and phylogenetic position of Pinaceae fossils present significant obstacles to our understanding of the timing of diversification in the family. We demonstrate that simultaneous phylogenetic analyses of chloroplast DNA (matK and rbcL) and nonmolecular characters that include both extant genera and a limited number of fossil taxa provide useful hypotheses for calibrating molecular trees. Root placements varied for Pinaceae, with Bayesian analyses recovering mutually monophyletic subfamilies Pinoideae and Abietoideae and parsimony analyses recovering Abietoideae as paraphyletic by placing the root between Cedrus and the remaining genera. The inferred phylogenetic positions of fossil taxa Pityostrobus bernissartensis as the sister group to Pinus and Pseudolarix erensis as the sister group to extant Pseudolarix were used to guide divergencetime calibrations; these calibrations yielded an Early Cretaceous and an Early Jurassic age for crown-group Pinaceae, respectively. The older age estimates based on Pseudolarix erensis are supported by weaker evidence from the fossil record but are consistent with recent reports of Early Cretaceous leaf fossils that appear to coincide with extant genera. There remains a great need to characterize the anatomy of extant and fossil species and to code additional nonmolecular characters.

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Sequence data from nrITS and cpDNA have failed to fully resolve phylogenetic relationships among Pinus species. Four low-copy nuclear genes, developed from the screening of 73 mapped conifer anchor loci, were sequenced from 12 species representing all subsections. Individual loci do not uniformly support either the nrITS or cpDNA hypotheses and in some cases produce unique topologies. Combined analysis of low-copy nuclear loci produces a well-supported subsectional topology of two subgenera, each divided into two sections, congruent with prior hypotheses of deep divergence in Pinus. The placements of P. nelsonii, P. krempfii, and P. contorta have been of continued systematic interest. Results strongly support the placement of P. nelsonii as sister to the remaining members of sect. Parrya, suggest a moderately well-supported and consistent position of P. krempfii as sister to the remaining members of sect. Quinquefoliae, and are ambiguous about the placement of P. contorta. A successful phylogenetic strategy in Pinus will require many low-copy nuclear loci that include a high proportion of silent sites and derive from independent linkage groups. The locus screening and evaluation strategy presented here can be broadly applied to facilitate the development of phylogenetic markers from the increasing number of available genomic resources.

Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a 1600-km latitudinal range extending from Oregon, USA, to northern Baja California, Mexico. Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast DNA (cpDNA) survey of Pinus subsect. Strobus with comprehensive geographical sampling of P. lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographical differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an additional 72 individuals demarcated a narrow 150-km contact zone in northeastern California. In the contact zone, maternal (megagametophtye) and paternal (embryo) haplotypes were identified in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene resistance, previously determined for 41 seed zones, differ significantly among seed zones that are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that includes Pinus albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058). These results are consistent with a Pleistocene migration of whitebark pine into North America and subsequent chloroplast introgression from whitebark pine to sugar pine. This study demonstrates the importance of placing phylogeographical results in a broader phylogenetic context.

Abstract—Pinus subsection Cembroides comprises approximately 15 taxa distributed from the southwestern United States to south central Mexico. Despite previous phylogenetic studies based on morphology, nuclear ribosomal DNA, and plastid DNA, we still lack a robust phylogenetic hypothesis and clear delimitation for the closely-related species within the group. We studied the evolutionary relationships within subsection Cembroides and explored incomplete lineage sorting and reticulation using low-copy number nuclear genes. Concatenation and multispecies coalescent phylogenies were inferred from samples representing all taxa from subsection Cembroides and outgroups corresponding to the closely-related subsections Balfourianae, Nelsoniae, Gerardianae, and Krempfianae. The concatenation and coalescence-based trees mainly agreed with one another in recovering Pinus subsection Cembroides as monophyletic and in recovering similar relationships among species as in previous plastid DNA-based studies. Phylogenetic position and admixture analysis suggest that P. californiarum should be treated as a separate species from P. monophylla. Furthermore, our results support recognizing P. fallax as a species rather than as an infraspecific taxon of P. monophylla or P. edulis. The ASTRAL-III tree was consistent with the presence of very high levels of ILS in the group of pinyon pines with small cones. Analyses that account for both incomplete lineage sorting and reticulation identify some unexpected hybridization scenarios that were not reported in the literature.

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.