Improving phylogenetic resolution of the Lamiales using the complete plastome sequences of six Penstemon species.

The North American endemic genus Penstemon (Mitchell) has a recent geologic origin of ca. 3.6 million years ago (MYA) during the Pliocene/Pleistocene transition and has undergone a rapid adaptive evolutionary radiation with ca. 285 species of perennial forbs and sub-shrubs. Penstemon is divided into six subgenera occupying all North American habitats including the Arctic tundra, Central American tropical forests, alpine meadows, arid deserts, and temperate grasslands. Due to the rapid rate of diversification and speciation, previous phylogenetic studies using individual and concatenated chloroplast sequences have failed to resolve many polytomic clades. We investigated the efficacy of utilizing the plastid genomes (plastomes) of 29 species in the Lamiales order, including five newly sequenced Penstemon plastomes, for analyzing phylogenetic relationships and resolving problematic clades. We compared whole-plastome based phylogenies to phylogenies based on individual gene sequences (matK, ndhF, psaA, psbA, rbcL, rpoC2, and rps2) and concatenated sequences. We also We found that our whole-plastome based phylogeny had higher nodal support than all other phylogenies, which suggests that it provides greater accuracy in describing the hierarchal relationships among taxa as compared to other methods. We found that the genus Penstemon forms a monophyletic clade sister to, but separate from, the Old World taxa of the Plantaginaceae family included in our study. Our whole-plastome based phylogeny also supports the rearrangement of the Scrophulariaceae family and improves resolution of major clades and genera of the Lamiales.

Pollinator divergence and pollination isolation between hybrids with different floral color and morphology in two sympatric Penstemon species.

Differential visitation of pollinators due to divergent floral traits can lead to reproductive isolation via assortative pollen flow, which may ultimately be a driving force in plant speciation, particularly in areas of overlap. We evaluate the effects of pollinator behavioral responses to variation of intraspecific floral color and nectar rewards, on reproductive isolation between two hybrid flower color morphs (fuchsia and blue) and their parental species Penstemon roseus and P. gentianoides with a mixed-pollination system. We show that pollinators (bumblebees and hummingbirds) exhibit different behavioral responses to fuchsia and blue morphs, which could result from differential attraction or deterrence. In addition to differences in color (spectral reflectance), we found that plants with fuchsia flowers produced more and larger flowers, produced more nectar and were more visited by pollinators than those with blue flowers. These differences influenced the foraging behavior and effectiveness as pollinators of both bumblebees and hummingbirds, which contributed to reproductive isolation between the two hybrid flower color morphs and parental species. This study demonstrates how differentiation of pollination traits promotes the formation of hybrid zones leading to pollinator shifts and reproductive isolation. While phenotypic traits of fuchsia and red flowers might encourage more efficient hummingbird pollination in a mixed-pollination system, the costs of bumblebee pollination on plant reproduction could be the drivers for the repeated shifts from bumblebee- to hummingbird-mediated pollination.

Developing molecular tools and insights into the Penstemon genome using genomic reduction and next-generation sequencing.

BACKGROUND: Penstemon's unique phenotypic diversity, hardiness, and drought-tolerance give it great potential for the xeric landscaping industry. Molecular markers will accelerate the breeding and domestication of drought tolerant Penstemon cultivars by, creating genetic maps, and clarifying of phylogenetic relationships. Our objectives were to identify and validate interspecific molecular markers from four diverse Penstemon species in order to gain specific insights into the Penstemon genome. RESULTS: We used a 454 pyrosequencing and GR-RSC (genome reduction using restriction site conservation) to identify homologous loci across four Penstemon species (P. cyananthus, P. davidsonii, P. dissectus, and P. fruticosus) representing three diverse subgenera with considerable genome size variation. From these genomic data, we identified 133 unique interspecific markers containing SSRs and INDELs of which 51 produced viable PCR-based markers. These markers produced simple banding patterns in 90% of the species × marker interactions (~84% were polymorphic). Twelve of the markers were tested across 93, mostly xeric, Penstemon taxa (72 species), of which ~98% produced reproducible marker data. Additionally, we identified an average of one SNP per 2,890 bp per species and one per 97 bp between any two apparent homologous sequences from the four source species. We selected 192 homologous sequences, meeting stringent parameters, to create SNP markers. Of these, 75 demonstrated repeatable polymorphic marker functionality across the four sequence source species. Finally, sequence analysis indicated that repetitive elements were approximately 70% more prevalent in the P. cyananthus genome, the largest genome in the study, than in the smallest genome surveyed (P. dissectus). CONCLUSIONS: We demonstrated the utility of GR-RSC to identify homologous loci across related Penstemon taxa. Though PCR primer regions were conserved across a broadly sampled survey of Penstemon species (93 taxa), DNA sequence within these amplicons (12 SSR/INDEL markers) was highly diverse. With the continued decline in next-generation sequencing costs, it will soon be feasible to use genomic reduction techniques to simultaneously sequence thousands of homologous loci across dozens of Penstemon species. Such efforts will greatly facilitate our understanding of the phylogenetic structure within this important drought tolerant genus. In the interim, this study identified thousands of SNPs and over 50 SSRs/INDELs which should provide a foundation for future Penstemon phylogenetic studies and breeding efforts.

A survey of Penstemon's genome size.

Penstemon is the largest genus in North America with more than 270 reported species. However, little is known about its genome size. This information may be useful in developing hybrids for landscape use and for gaining insight into its current taxonomy. Using flow cytometry, we estimated the genome size of approximately 40% of the genus (115 accessions from 105 different species). Genome sizes for both reported and probable diploids range from P. dissectus 2C = 0.94 pg (1C = 462 Mbp) to P. pachyphyllus var. mucronatus 2C = 1.88 pg (1C = 919 Mbp), and the polyploids range from P. attenuatus var. attenuatus 2C = 2.35 pg (1C = 1148 Mbp) to P. digitalis 2C = 6.45 pg (1C = 3152 Mbp). Chromosome counts were done for ten previously published and four previously unreported Penstemon species (P. dissectus, P. navajoa, P. caespitosus var. desertipicti, and P. ramaleyi). These counts were compiled with all previously published chromosome data and compared with the flow cytometry results. Ploidy within this study ranged from diploid to dodecaploid. These data were compared and contrasted with the current taxonomy of Penstemon and previously published internal transcribed spacer and chloroplast DNA phylogenetic work. Based on genome size and previous studies, reassigning P. montanus to the subgenus Penstemon and P. personatus to the subgenus Dasanthera, would better reflect the phylogeny of the genus. Furthermore, our data concur with previous studies suggesting that the subgenus Habroanthus be included in the subgenus Penstemon. The DNA content of subgenus Penstemon exhibits high plasticity and spans a sixfold increase from the smallest to the largest genome (P. linarioides subsp. sileri and P. digitalis, respectively). Our study found flow cytometry to be useful in species identification and verification.