Ann Willyard

Use of simultaneous analyses to guide fossil-based calibrations of Pinaceae phylogeny

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 divergence‐time 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.

Reticulate evolution and incomplete lineage sorting among the ponderosa pines.

Interspecific gene flow via hybridization may play a major role in evolution by creating reticulate rather than hierarchical lineages in plant species. Occasional diploid pine hybrids indicate the potential for introgression, but reticulation is hard to detect because ancestral polymorphism is still shared across many groups of pine species. Nucleotide sequences for 53 accessions from 17 species in subsection Ponderosae (Pinus) provide evidence for reticulate evolution. Two discordant patterns among independent low-copy nuclear gene trees and a chloroplast haplotype are better explained by introgression than incomplete lineage sorting or other causes of incongruence. Conflicting resolution of three monophyletic Pinus coulteri accessions is best explained by ancient introgression followed by a genetic bottleneck. More recent hybridization transferred a chloroplast from P. jeffreyi to a sympatric P. washoensis individual. We conclude that incomplete lineage sorting could account for other examples of non-monophyly, and caution against any analysis based on single-accession or single-locus sampling in Pinus.

Evolutionary relationships among Pinus (Pinaceae) subsections inferred from multiple low-copy nuclear loci

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.

Interspecific phylogenetic analysis enhances intraspecific phylogeographical inference: a case study in Pinus lambertiana

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.

Pinus ponderosa: A checkered past obscured four species

PREMISE OF THE STUDY Molecular genetic evidence can help delineate taxa in species complexes that lack diagnostic morphological characters. Pinus ponderosa (Pinaceae; subsection Ponderosae) is recognized as a problematic taxon: plastid phylogenies of exemplars were paraphyletic, and mitochondrial phylogeography suggested at least four subdivisions of P. ponderosa. These patterns have not been examined in the context of other Ponderosae species. We hypothesized that putative intraspecific subdivisions might each represent a separate taxon. METHODS We genotyped six highly variable plastid simple sequence repeats in 1903 individuals from 88 populations of P. ponderosa and related Ponderosae (P. arizonica, P. engelmannii, and P. jeffreyi). We used multilocus haplotype networks and discriminant analysis of principal components to test clustering of individuals into genetically and geographically meaningful taxonomic units. KEY RESULTS There are at least four distinct plastid clusters within P. ponderosa that roughly correspond to the geographic distribution of mitochondrial haplotypes. Some geographic regions have intermixed plastid lineages, and some mitochondrial and plastid boundaries do not coincide. Based on relative distances to other species of Ponderosae, these clusters diagnose four distinct taxa. CONCLUSIONS Newly revealed geographic boundaries of four distinct taxa (P. benthamiana, P. brachyptera, P. scopulorum, and a narrowed concept of P. ponderosa) do not correspond completely with taxonomies. Further research is needed to understand their morphological and nuclear genetic makeup, but we suggest that resurrecting originally published species names would more appropriately reflect the taxonomy of this checkered classification than their current treatment as varieties of P. ponderosa.

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.

A Set of Plastid Loci for Use in Multiplex Fragment Length Genotyping for Intraspecific Variation in Pinus (Pinaceae)

Premise of the study: Recently released Pinus plastome sequences support characterization of 15 plastid simple sequence repeat (cpSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. Methods: Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compared interspecific variation between cpSSRs and nucleotide sequences of ycf1 and tested intraspecific variation for cpSSRs using 911 samples in the P. ponderosa species complex. Results: The cpSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions, and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. Discussion: The protocol allows efficient genotyping of many samples. The cpSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for cpSSR loci in a plastome alignment for the target group.

Incorporating fossils into the Pinaceae tree of life.

PREMISE OF THE STUDY Pinaceae have a rich but enigmatic early fossil record, much of which is represented by permineralized seed cones. Our incomplete knowledge of morphology and anatomy in living and extinct species poses an important barrier to understanding their phylogenetic relationships and timing of diversification. METHODS We expanded a morphology matrix to 46 fossil and 31 extant Pinaceae species, mainly adding characters from stem and leaf anatomy and seed cones. Using parsimony and Bayesian inference, we compared phylogenetic relationships for extant taxa with and without fossils from the morphology matrix combined with an alignment of plastid gene sequences. KEY RESULTS Combined analysis of morphological and molecular characters resulted in a phylogeny of extant Pinaceae that was robust at all nodes except those relating to the interrelationships of Pinus, Picea, and Cathaya and the position of Cedrus. Simultaneous analysis of all fossil and extant species did not result in changes in the relationships among the extant species but did greatly reduce branch support. We found that the placement of most fossils was sensitive to the method of phylogenetic reconstruction when analyzing them singly with the extant species. CONCLUSIONS A robust phylogenetic hypothesis for the main lineages of Pinaceae is emerging. Most Early Cretaceous fossils are stem or crown lineages of Pinus, but close relationships also were found between fossils and several other extant genera. The phylogenetic position of fossils broadly supports the existence of extant genera in the Lower Cretaceous.