We used chloroplast DNA sequences from matK and rbcL to infer the phylogeny for 101 of the approximately 111 species of Pinus (Pinaceae). At the level of subsection and above, the cpDNA tree is congruent with phylogenies based on nuclear DNA with one notable exception: cpDNA sequences from subsect. Contortae are sister to all other North American hard pines rather than occupying a more derived position in the same clade. We used the cpDNA tree plus evidence from nuclear ribosomal DNA and morphology to propose a new classification for the genus. The molecular phylogenies are symmetrical at the deepest branches of the genus, allowing for the delineation of two subgenera, each with two sections that form sister groups. Within sections, clades were slightly asymmetric and sometimes ambiguously resolved. To accomodate ambiguity in some interrelationships, avoid the creation of new ranks, and retain traditional names, we recognised up to three monophyletic subsections per section. Subgenus Pinus (the diploxylon, or hard pines) is divided into the predominantly Eurasian and Mediterranean section Pinus, composed of subsections Pinus and Pinaster, and the strictly North American section Trifoliae, composed of subsections Australes, Ponderosae, and Contortae. Subgenus Strobus (the haploxylon, or soft pines) is divided into the strictly North American section Parrya, composed of subsections Cembroides, Nelsoniae, and Balfourianae, and the Eurasian and North American section Quinquefoliae, composed of subsections Gerardianae, Krempfianae, and Strobus. Mapping of ten morphological and distributional characters indicates that two were diagnostic for infrageneric taxa: the number of vascular bundles per leaf distinguishes subgenus Pinus from subgenus Strobus, and a terminal­positioned umbo on the ovulate cone scale is diagnostic of subsect. Strobus.

Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified ∼120 kb plastomes from eight species (seven Pinus, one Picea) in 35 reactions. Pooled products were ligated to modified adapters that included 3 bp indexing tags and samples were multiplexed at four genomes per lane. Tagged microreads were assembled by de novo and reference-guided assembly methods, using previously published Pinus plastomes as surrogate references. Assemblies for these eight genomes are estimated at 88–94% complete, with an average sequence depth of 55× to 186×. Mononucleotide repeats interrupt contig assembly with increasing repeat length, and we estimate that the limit for their assembly is 16 bp. Comparisons to 37 kb of Sanger sequence show a validated error rate of 0.056%, and conspicuous errors are evident from the assembly process. This efficient sequencing approach yields high-quality draft genomes and should have immediate applicability to genomes with comparable complexity.

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.

How coniferous forests evolved in the Northern Hemisphere remains largely unknown. Unlike most groups of organisms that generally follow a latitudinal diversity gradient, most conifer species in the Northern Hemisphere are distributed in mountainous areas at middle latitudes. It is of great interest to know whether the midlatitude region has been an evolutionary cradle or museum for conifers and how evolutionary and ecological factors have driven their spatiotemporal evolution. Here, we investigated the macroevolution of Pinus, the largest conifer genus and characteristic of northern temperate coniferous forests, based on nearly complete species sampling. Using 1,662 genes from transcriptome sequences, we reconstructed a robust species phylogeny and reestimated divergence times of global pines. We found that ∼90% of extant pine species originated in the Miocene in sharp contrast to the ancient origin of Pinus, indicating a Neogene rediversification. Surprisingly, species at middle latitudes are much older than those at other latitudes. This finding, coupled with net diversification rate analysis, indicates that the midlatitude region has provided an evolutionary museum for global pines. Analyses of 31 environmental variables, together with a comparison of evolutionary rates of niche and phenotypic traits with a net diversification rate, found that topography played a primary role in pine diversification, and the aridity index was decisive for the niche rate shift. Moreover, fire has forced diversification and adaptive evolution of Pinus. Our study highlights the importance of integrating phylogenomic and ecological approaches to address evolution of biological groups at the global scale.