Christopher P. Randle

Inferring phylogeny at low taxonomic levels: utility of rapidly evolving cpDNA and nuclear ITS loci

Plant molecular systematic studies of closely related taxa have relied heavily on sequence data from nuclear ITS and cpDNA. Positive attributes of using ITS sequence data include the rapid rate of evolution compared to most plastid loci and availability of universal primers for amplification and sequencing. On the other hand, ITS sequence data may not adequately track organismal phylogeny if concerted evolution and high rDNA array copy number do not permit identification of orthologous copies. Shaw et al. (American Journal of Botany 92: 142-166) recently identified nine plastid regions that appear to provide more potentially informative characters than many other plastid loci. In the present study, sequences of these loci and ITS were obtained for six taxonomic groups in which phylogenetic relationships have been difficult to establish using other data. The relative utility of these regions was compared by assessing the number of parsimony informative characters, character congruence, resolution of inferred trees, clade support, and accuracy. No single locus emerged as the best in all lineages for any of these measures of utility. Results further indicated that in preliminary studies, sampling strategy should include at least four exemplar taxa. The importance of sampling data from independent distributions is also discussed.

Relationships Within and Among Species of the Holoparasitic Genus Hyobanche (Orobanchaceae) Inferred from ISSR Banding Patterns and Nucleotide Sequences

Abstract The systematic relationships of four species in the South African holoparasitic genus Hyobanche were examined using inter-simple sequence repeat (ISSR) banding patterns and nucleotide sequence data from nuclear ribosomal ITS regions and the plastid gene rbcL. Ordination of ISSR data revealed discrete groups, although H. glabrata and H. rubra were indistinguishable. Phylogeny reconstructions based on ISSR and ITS data were congruent and depicted relationships where H. atropurpurea and H. sanguinea form a clade with H. glabrata and H. rubra as their sister group. In contrast, the rbcL tree revealed a topology where H. atropurpurea was sister to a clade that included the other three species. The combination of molecular, morphological, and biogeographical data suggest two alternative hypotheses: (1) that floral evolution has progressed from galeate hirsute corolla tubes to straight glabrous tubes as represented by H. sanguinea, H. glabrata (intermediate morphology), and H. rubra, respectively; or (2) the intermediate floral morphology and distribution of H. glabrata reveal a hybrid origin of this species with H. sanguinea and H. rubra as progenitors. Communicating Editor: Paul Wilson

Phylogeny, taxonomic affinities, and biogeography of Penstemon (Plantaginaceae) based on ITS and cpDNA sequence data

The large and diverse genus Penstemon (ca. 271 species) is endemic to North America and has been divided into six subgenera primarily based on anther dehiscence patterns. Species of Penstemon are known to be pollinated by a variety of insects (hymenopterans, lepidopterans, dipterans) and hummingbirds. Nucleotide sequence data from ITS and two noncoding regions of chloroplast DNA were used to reconstruct the phylogeny of Penstemon. Trees generated from nuclear and chloroplast DNA sequences are incongruent, which is probably the result of hybridization, and not fully resolved, which is likely due to a rapid evolutionary radiation. Penstemon represents a recent continental radiation where speciation has resulted primarily from evolutionary adaptations to ecological niches such as pollinator specialization. The results from these analyses show that the current circumscription of subgenera and sections needs revision to reflect more closely the evolutionary relationships of species. Specifically, species in subgenera Saccanthera, Habroanthus, and Penstemon are polyphyletic. These results also confirm the independent origin of hummingbird floral morphology in 10 clades.

A Phylogenetic and Biogeographic Analysis of the Cheloneae (Scrophulariaceae) Based on ITS and matK Sequence Data

Abstract A phylogenetic study was conducted on current members (Chelone, Chionophila, Keckiella, Nothochelone, Pennellianthus, Penstemon, and Tetranema) and former members (Collinsia, Freylinia, Halleria, Russelia, Scrophularia, Tonella) of tribe Cheloneae to test for monophyly, examine biogeographic patterns, and assess relationships of Cheloneae to genera previously excluded from the tribe. Nucleotide sequence data from ITS and matK used in separate and combined analyses yielded congruent trees. Our results provide evidence that Collinsia and Tonella should be transferred from Collinsieae to Cheloneae and that Russelieae should be expanded to include Tetranema. Our results also indicate a New World origin for Cheloneae with dispersal of the Asian component over Beringia. North American Cheloneae may have originated in the Klamath Region of the western United States with expansion to the Rocky Mountain/Columbia Plateau region followed by diversification of several genera. We propose a post-Tertiary evolutionary radiation of the large North American genus Penstemon. Communicating Editor: James F. Smith

The evolution and expression of rbcL in holoparasitic sister-genera Harveya and Hyobanche (Orobanchaceae)

The evolution of holoparasitism decreases the adaptive value of genes maintaining the photosynthetic apparatus. These may become pseudogenes through insertion or deletion events resulting in frameshift mutations, or by the evolution of premature stop codons. The holoparasitic sister genera Harveya and Hyobanche have undergone alternate pathways of evolution and expression at the plastid locus rbcL. An open reading frame in all but a single species of Harveya is maintained by purifying selection and is expressed. However, the function of Rubisco in this putative holoparasite is unknown. Conversely, Hyobanche has undergone rbcL pseudogene formation, and comparison of synonymous and nonsynonymous rates of evolution indicates that selection has not played a role in its evolution. This is complicated by the following findings: multiple pseudogene copies of rbcL exist in tissues of Hyobanche, rbcL transcripts also encode pseudogenes, and the large subunit is present in some tissues of Hyobanche. We hypothesize that the rbcL operon is in a state of degradation as may be expected in a holoparasite and is not endogenously expressed. Rather, the large subunit may be taken up from the host plants, and accumulate in tissues as a result of transpiration.

The conflation of ignorance and knowledge in the inference of clade posteriors

The objective Bayesian approach relies on the construction of prior distributions that reflect ignorance. When topologies are considered equally probable a priori, clades cannot be. Shifting justifications have been offered for the use of uniform topological priors in Bayesian inference. These include: (i) topological priors do not inappropriately influence Bayesian inference when they are uniform; (ii) although clade priors are not uniform, their undesirable influence is negated by the likelihood function, even when data sets are small; and (iii) the influence of nonuniform clade priors is an appropriate reflection of knowledge. The first two justifications have been addressed previously: the first is false, and the second was found to be questionable. The third and most recent justification is inconsistent with the first two, and with the objective Bayesian philosophy itself. Thus, there has been no coherent justification for the use of nonflat clade priors in Bayesian phylogenetics. We discuss several solutions: (i) Bayesian inference can be abandoned in favour of other methods of phylogenetic inference; (ii) the objective Bayesian philosophy can be abandoned in favour of a subjective interpretation; (iii) the topology with the greatest posterior probability, which is also the tree of greatest marginal likelihood, can be accepted as optimal, with clade support estimated using other means; or (iv) a Bayes factor, which accounts for differences in priors among competing hypotheses, can be used to assess the weight of evidence in support of clades.
©The Willi Hennig Society 2009

Isolation and characterization of polymorphic microsatellite loci for the three-toed box turtle (Terrapene carolina triunguis) and cross-amplification in other Terrapene species.

We isolated and characterized eight polymorphic microsatellite loci for a Texas population of three‐toed box turtle, Terrapene carolina triunguis, using a refined hybridization capture procedure. All eight primer pairs amplified successfully at all loci in seven Texas ornate box turtles (T. ornata ornata). Due to the decline and conservation concerns of North American box turtles, these isolated microsatellites may be a most valuable tool for evaluating baseline population genetic structure for threatened box turtle populations.

Phylogenetics and diversification of Cotyledon (Crassulaceae) inferred from nuclear and chloroplast DNA sequence data.

Crassulaceae includes approximately 35 genera and 1500 species of leaf and stem succulent flowering plants. The family is nearly cosmopolitan in distribution, but is particularly diverse in southern Africa, where five genera comprising approximately 325 species are found. One of these genera, Cotyledon, includes 10 species that are largely confined to South Africa, where they are commonly found on rocky hillsides, coastal flats, and cliff faces. Species of Cotyledon are characterized by five-parted, pendulous, sympetalous flowers, but the genus is highly diverse in growth form, flower color and size, and leaf morphology. One particularly variable species, C. orbiculata, has been divided into five varieties based on leaf morphology and biogeography; however, the monophyly of this species as well as the relationships among the varieties have not previously been investigated. Parsimony analyses of a combined data set of DNA sequences from chloroplast and nuclear genome provided the first estimate of phylogeny for Cotyledon, and resulted in two minimum-length trees and a fully resolved phylogeny for the genus. Results indicate that C. orbiculata is not monophyletic and suggest the need for additional studies and a revised classification within the genus.

Effects of urbanization on genetic diversity, gene flow, and population structure in the ornate box turtle (Terrapene ornata)

Large-scale declines in North American box turtle (Terrapene spp.) populations have been attributed to habitat fragmentation as a result of urbanization. We compared microsatellite markers and mitochondrial control region sequences of Ornate Box Turtles (Terrapene ornata) in two populations (a natural and an urban habitat) to test two hypotheses. We hypothesized that urban populations of T. ornata experience genetic bottlenecks due to road mortality and habitat fragmentation, and that roadways represent a barrier to gene flow among turtle populations, resulting in increased fragmentation of gene pools. Both populations shared similar allelic diversity and observed heterozygosity, with eight and seven of twelve microsatellite loci exhibiting heterozygote deficiency in the natural and urban populations, respectively. The number of mitochondrial control region haplotypes in the urban population was nearly four times that of the natural population, although only one haplotype occurred in appreciable frequency in both populations. We did not detect conclusive evidence of a recent genetic bottleneck in the urban population, thereby rejecting our first hypothesis. We detected weak differentiation among populations on opposing sides of a large highway, but did not detect any evidence of population structure, thereby rejecting our second hypothesis. This study indicates that a population of T. ornata with moderate road mortality currently has high genetic diversity, moderate inbreeding, and displays some evidence of genetic differentiation, but no conclusive evidence of recent genetic bottlenecks or unique genetic clustering. We suggest this is primarily due to the species long generation time and is a positive aspect of their life-history.

Identification and persistence of Pinus pollen DNA on cotton fabrics: A forensic application.

Advances in plant genomics have had an impact on the field of forensic botany. However, the use of pollen DNA profiling in forensic investigations has yet to be applied. Five volunteers wore a jacket with Pinus echinata pollen-containing cotton swatches for a 14-day period. Pollen decay was evaluated at days 0, 3, 6, 9 and 14 by microscopy. Pollen grains were then transferred to slides using a portable forensic vacuum handle. Ten single grains per swatch were isolated for DNA analysis. DNA was extracted using a high throughput extraction method. A nine-locus short tandem repeat (STR) multiplex system, including previously published primers from Pinus taeda, was developed. DNA was amplified by PCR using fluorescent dyes and analyzed by capillary electrophoresis. Pollen counts from cotton swatches in a 14-day period exhibited an exponential decay from 100% to 17%. The success rate of PCR amplification was 81.2%. Complete and partial STR profiles were generated from 250 pollen grains analyzed (44% and 37%, respectively). Due to the limited amount of DNA, drop-in events were observed (1.87%). However, the rate of contamination with pollen from other pine individuals originating from environmental sources was 4.4%. In conclusion, this study has shown that pollen can be a stable source of forensic DNA evidence, as a proof-of-principle, and that may persist on cotton clothing for at least 14 days of wear. This method can be applied in forensic cases where pollen grains larger than 10 μm (e.g., from herbs or trees) may be transferred to clothing (worn by suspect or victim) by primary contact.