David S. Gernandt

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology

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.

Phylogeny and classification of Pinus

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 terminal-positioned umbo on the ovulate cone scale is diagnostic of subsect. Strobus.

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.

Phylogenetics of Lophodermium from pine

Lophodermium comprises ascomycetous fungi that are both needle-cast pathogens and asymptomatic endophytes on a diversity of plant hosts. It is distinguished from other genera in the family Rhytismataceae by its filiform ascospores and ascocarps that open by a longitudinal slit. Nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were used to infer phylogenetic relationships within Lophodermium. Twenty-nine sequences from approximately 11 species of Lophodermium were analyzed together with eight sequences from isolates thought to represent six other genera of Rhytismataceae: Elytroderma, Lirula, Meloderma, Terriera, Tryblidiopsis and Colpoma. Two putative Meloderma desmazieresii isolates occurred within the Lophodermium clade but separate from one another, one grouped with L. indianum and the other with L. nitens. An isolate of Elytroderma deformans also occurred within the Lophodermium clade but on a solitary branch. The occurrence of these genera within the Lophodermium clade might be due to problems in generic concepts in Rhytismataceae, such as emphasis on spore morphology to delimit genera, to difficulty of isolating Rhytismataceae needle pathogens from material that also is colonized by Lophodermium or to a combination of both factors. We also evaluated the congruence of host distribution and several morphological characters on the ITS phylogeny. Lophodermium species from pine hosts formed a monophyletic sister group to Lophodermium species from more distant hosts from the southern hemisphere, but not to L. piceae from Picea. The ITS topology indicated that Lophodermium does not show strict cospeciation with pines at deeper branches, although several closely related isolates have closely related hosts. Pathogenic species occupy derived positions in the pine clade, suggesting that pathogenicity has evolved from endophytism. A new combination is proposed, Terriera minor (Tehon) P.R. Johnst.

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.

Phylogenetics of Pinus Subsections Cembroides and Nelsoniae Inferred from cpDNA Sequences

Abstract We sequenced chloroplast DNA from the matK, rbcL, and rpl16 regions to infer interrelationships within the pinyon pines (Pinus subsections Cembroides and Nelsoniae). Pinyons, together with subsections Balfourianae, Gerardianae, and Krempfianae have been classified in section Parrya, characterized by a dorsal umbo (raised area) on the ovulate cone scale. All three cpDNA regions support the separation of pinyon pines into subsection Cembroides and a monotypic subsection Nelsoniae and indicate that section Parrya is paraphyletic. We propose restricting section Parrya to the North American clade (subsections Cembroides, Nelsoniae, and Balfourianae) and transferring the Asian subsections Gerardianae and Krempfianae to section Quinquefolius (generally known as section Strobus). The data moderately support a sister relationship between subsections Nelsoniae and Balfourianae, rendering the pinyons paraphyletic. Several monophyletic groups can be identified within subsection Cembroides, including a sister relationship between P. maximartinezii and P. pinceana, which is at variance with morphological cladistic analyses. In general, relationships inferred from cpDNA are less consistent with morphological evidence than with internal transcribed spacer region data, despite paralogy in the latter marker.

Phylogenetic Relationships of Pinus Subsection Ponderosae Inferred from Rapidly Evolving cpDNA Regions

Abstract Pinus subsection Ponderosae includes approximately 17 tree species distributed from western Canada to Nicaragua. We inferred phylogenetic relationships of multiple accessions for all widely recognized species from 3.7 kb of CpDNA sequence (matK, trnD-trnY-trnE spacer, chlN-ycf1 spacer, and ycf1). The sister relationship between subsections Ponderosae and Australes was corroborated with high branch support, and several clades, most with lower branch support, were identified within subsection Ponderosae. Pinus jeffreyi was sister to P. coulteri, P. sabiniana, and P. torreyana. Californian accessions of P. ponderosa and P. washoensis occurred in a clade separate from P. arizonica and P. scopulorum from the southwestern United States. Western Mexican species P. cooperi and P. durangensis had CpDNA sequences identical to one or more accessions of P. arizonica and P. scopulorum, and together these taxa were closely related to clades of P. engelmannii-P. devoniana (Mexico) and P. douglasiana-P. yecorensis-P. maximinoi (western Mexico to Guatemala). A well supported clade of taxa from Mexico and Central America included P. pseudostrobus, P. montezumae, P. hartwegii, P. maximinoi (one of three accessions), P. nubicola, and P. donnell-smithii. Chloroplast DNA sequences were nonmonophyletic for most species, although the degree of support varied.

Pinus nelsonii and a Cladistic Analysis of Pinaceae Ovulate Cone Characters

Abstract The complexity of ovulate cones and their preservation as fossils makes them promising material for reconstructing the evolutionary history of gymnosperms, but phylogenetic analyses of cone morphological characters of Pinaceae have been inconclusive. We describe the ovulate cone anatomy of Pinus nelsonii, a rare and phylogenetically isolated pinyon pine endemic to Mexico, and add the species together with Pinus ponderosa and the fossil Pinus belgica to a recoded and expanded ovulate cone morphology matrix for fossil and extant Pinaceae. The cone anatomy of Pinus nelsonii conforms to previous generic concepts of Pinus. Despite its phylogenetically isolated position among the soft pines (Pinus subgenus Strobus) and thus potential for displaying plesiomorphic features, the cone of Pinus nelsonii is unlike the oldest Pinus fossil cones in possessing enlarged, functionally wingless seeds partially embedded in scale tissue, and in lacking sclerenchyma in the cortex of the axis, in the bract, and in the scale. Cladistic analysis of cone morphology characters recovers several Pityostrobus species in a clade with Pinus. Although the inferred relationships among living species do not coincide in several respects to molecular studies, adding taxa and further exploration of characters promise to clarify relationships.

Plastid DNA Diversity Is Higher in the Island Endemic Guadalupe Cypress than in the Continental Tecate Cypress

Background Callitropsis guadalupensis (Guadalupe cypress) is endemic to Guadalupe Island, Mexico, where it is the dominant species of the only forest. The species has suffered declining numbers following the introduction of goats to the island over 150 years ago. Callitropsis guadalupensis is closely related to Callitropsis forbesii (Tecate cypress), distributed in small isolated populations in mainland Baja California and southern California. The objective of the present study was to compare the genetic diversity of the island endemic to the continental species. Methodology/Principal Findings We measured genetic diversity in Callitropsis guadalupensis (n = 54) from Guadalupe Island and in Callitropsis forbesii (n = 100) from five populations in mainland Baja California. The plastid DNA trnS-trnG spacer and the trnL-trnF region were chosen for characterization. Thirty-four haplotypes were observed, of which six were shared between both species. One of these haplotypes was also shared with three other species, Callitropsis lusitanica, Callitropsis montana, and Callitropsis stephensonii. Haplotype diversity (h) and nucleotide diversity (π) were significantly higher for Callitropsis guadalupensis (h = 0.698, π = 0.00071) than for Callitropsis forbesii (h = 0.337, π = 0.00024). Conclusions/Significance Callitropsis guadalupensis shows no evidence of a founder effect or of a genetic bottleneck, and can be added to a growing list of insular species with higher genetic diversity than their mainland relatives.

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.