A reassessment of the phylogeny and circumscription of Zaluzianskya (Scrophulariaceae).

The genus Zaluzianskya (Scrophulariaceae s.s.) encompasses a diversity of floral and ecological traits. However, this diversity, as described by the current taxonomic circumscription of Zaluzianskya, is an underestimate. We present molecular data suggesting that this genus requires expansion via incorporation of species from other genera and recognition of unnamed cryptic species. This study advances prior molecular phylogenies of the southern African genus through the addition of DNA regions and 51 populations that had not previously been sampled in a published phylogeny. A total of 82 species of Zaluzianskya and related genera are included, adding 48 to those previously sampled. Results are presented from analyses of five DNA regions, including nuclear ITS and four rapidly evolving chloroplast regions (trnL-trnF, rpl16, rps16, and trnS-trnfM). Our primary finding is that the genus Phyllopodium is polyphyletic as currently circumscribed, with some species placed within Zaluzianskya and others grouping with Polycarena, indicating the need for further phylogenetic work on these genera. Preliminary support for the incorporation of Reyemia into Zaluzianskya is reinforced here by the first molecular analysis to include both species of Reyemia and a strong sampling of species across Zaluzianskya and major clades of tribe Limoselleae. The two disjunct, tropical African species of Zaluzianskya are also confirmed as members of this genus. Finally, a broad sampling of 21 populations of Z. microsiphon establishes their phylogenetic division into two to five separate lineages. Hybridization, coevolution, and cryptic speciation may each play a role in the evolution of Z. microsiphon. Further resolution within a clade comprising sections Nycterinia and Macrocalyx is needed to better understand their relationships.

Multilocus phylogenetic inference in subfamily Chlorogaloideae and related genera of Agavaceae - informing questions in taxonomy at multiple ranks.

A series of taxonomic questions at the subfamilial, generic, and intrageneric levels have remained within subfamily Chlorogaloideae s.s. (comprising Camassia, Chlorogalum, Hastingsia, and Schoenolirion) and relatives in Agavaceae. We present the first phylogenetic hypotheses focused on Chlorogaloideae that are based on multiple independent loci and include a wide sampling of outgroups across Agavaceae. In addition to chloroplast regions ndhF and trnL-trnF, we used nrDNA ITS for phylogenetic inference. Incomplete concerted evolution of the latter is indicated by intra-individual site polymorphisms for nearly half of the individuals. Comparisons of four coding and analysis methods for these characters indicate that the region remains phylogenetically informative. Our results confirm that Chlorogaloideae s.s. is not monophyletic, due to the close relationship of Schoenolirion with Hesperaloe and Hesperoyucca, as well as the likely sister relationship between Hesperocallis and core Chlorogaloideae (Camassia, Chlorogalum, and Hastingsia). Chlorogalum is also not monophyletic, being divided with strong support into vespertine and diurnal clades. This study produced the first phylogenetic hypotheses across Hesperaloe, allowing initial tests of several taxonomic disagreements within this genus. Our results reveal the lack of cohesion of H. funifera, indicating that H. funifera ssp. funifera may be more closely related to H. campanulata than to H. funifera ssp. chiangii (=H. chiangii). With potential gene flow between many members of Hesperaloe and a possible hybrid origin for H. campanulata, the genetic relationships within this genus appear complex. Further population-level investigation of many of the taxa in Chlorogaloideae s.l. would benefit our understanding of the evolution and taxonomy of these groups; Camassia and Hastingsia are the current focus of ongoing study.

Origin and diversification of the Greater Cape flora: ancient species repository, hot-bed of recent radiation, or both?

Like island-endemic taxa, whose origins are expected to postdate the appearance of the islands on which they occur, biome-endemic taxa should be younger than the biomes to which they are endemic. Accordingly, the ages of biome-endemic lineages may offer insights into biome history. In this study, we used the ages of multiple lineages to explore the origin and diversification of two southern African biomes whose remarkable floristic richness and endemism has identified them as global biodiversity hotspots (succulent karoo and fynbos). We used parsimony optimization to identify succulent karoo- and fynbos-endemic lineages across 17 groups of plants, for which dated phylogenies had been inferred using a relaxed Bayesian (BEAST) approach. All succulent karoo-endemic lineages were less than 17.5 My old, the majority being younger than 10 My. This is largely consistent with suggestions that this biome is the product of recent radiation, probably triggered by climatic deterioration since the late Miocene. In contrast, fynbos-endemic lineages showed a broader age distribution, with some lineages originating in the Oligocene, but most being more recent. Also, in groups having both succulent karoo- and fynbos-endemic lineages, there was a tendency for the latter to be older. These patterns reflect the greater antiquity of fynbos, but also indicate considerable recent speciation, probably through a combination of climatically-induced refugium fragmentation and adaptive radiation.

Nei's to Bayes': comparing computational methods and genetic markers to estimate patterns of genetic variation in Tolpis (Asteraceae).

Accurate determination of patterns of genetic variation provides a powerful inferential tool for studies of evolution and conservation. For more than 30 years, enzyme electrophoresis was the preferred method for elucidating these patterns. As a result, evolutionary geneticists have acquired considerable understanding of the relationship between patterns of allozyme variation and aspects of evolutionary process. Myriad molecular markers and statistical analyses have since emerged, enabling improved estimates of patterns of genetic diversity. With these advances, there is a need to evaluate results obtained with different markers and analytical methods. We present a comparative study of gene statistic estimates (F(ST), G(ST), F(IS), H(S), and H(T)) calculated from an intersimple sequence repeat (ISSR) and an allozyme data set derived from the same populations using both standard and Bayesian statistical approaches. Significant differences were found between estimates, owing to the effects of marker and analysis type. Most notably, F(ST) estimates for codominant data differ between Bayesian and standard approaches. Levels of statistical significance are greatly affected by methodology and, in some cases, are not associated with similar levels of biological significance. Our results suggest that caution should be used in equating or comparing results obtained using different markers and/or methods of analysis.

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.

The utility of automated analysis of inter-simple sequence repeat (ISSR) loci for resolving relationships in the Canary Island species of Tolpis (Asteraceae).

Plants of oceanic islands, often remarkably divergent morphologically from continental relatives, are useful models for studying evolution and speciation because evolution is telescoped in time and space. Prior studies revealed little DNA sequence variation within the clade of ca. 10 Canary Island species of Tolpis, which precluded resolving species relationships. The present study assessed the utility of automated analysis of inter-simple sequence repeat (ISSR) loci for resolving relationships within the clade using 264 individuals from 36 populations of all recognized species and three undescribed morphological variants. Similarity (Dice coefficient) and Fitch parsimony were used to generate neighbor-joining (NJ) and strict consensus trees (MP), respectively. All individuals of the morphologically distinct endemic species formed clusters in both trees. There is also support for clusters of two undescribed variants in the NJ tree. Individuals from a morphologically variable complex consisting primarily of two species are not well resolved at population or species levels. The NJ and MP trees are not congruent at deeper levels, including relationships among species. Results are interpreted in terms of the biology of the species, and the utility of automated analysis of ISSR markers for interpreting patterns of evolution of Tolpis in the Canary Islands is discussed.

Allozyme diversity within and divergence among species ofTolpis(Asteraceae-Lactuceae) in the Canary Islands: systematic, evolutionary, and biogeographical implications.

Plants endemic to oceanic islands represent some of the most unusual and rare taxa in the world. Enzyme electrophoresis was used to assess genetic diversity within and divergence among all endemic species of a small genus of plants on the Canary Islands. Our results show that the genus Tolpis is similar to many other island groups in having generally low allozyme divergence among species, with the highest divergence found among four groups of endemics. The two rare and highly localized species T. glabrescens and T. crassiuscula are each divergent from all other species in the Canaries. Tolpis coronopifolia is also divergent at allozyme loci; this is the only endemic species that is a self-compatible annual (or weak biennial). A large, morphologically variable species complex consisting of T. laciniata and T. lagopoda together with several named and unnamed morphological variants shows low allozyme divergence among its elements. The evolution of polyploidy from diploid ancestors in situ in oceanic archipelagos is uncommon, but the tetraploid T. glabrescens is an exception. Allozyme data do not implicate any extant diploid Tolpis species as parents of the polyploid. It is possible that T. glabrescens originated early in the evolution of Tolpis in the Canary Islands and that its parents are now extinct. The nonendemic T. barbata shows no greater divergence from the Canary Island endemics than some endemics exhibit among themselves. Both changes in allele frequencies and unique alleles are responsible for genetic divergence among species of Tolpis.

Life history affects the evolution of reproductive isolation among species of Coreopsis (Asteraceae).

The evolution of reproductive isolation within Coreopsis is investigated by integrating phylogenetic data with estimates of pollen viability of plants from inter- and intraspecific crosses. Three different models that predict F1 fitness are compared. The first uses ITS pairwise distances, the second is based on phylogenetic branch lengths derived from DNA sequences, and the third elaborates on the second model by dividing branch length according to reconstructions of the evolution of life history. This is the first study to use phylogenetic branch-length estimates for predicting levels of reproductive isolation. Estimated branch lengths (model 2) predict hybrid fitness more accurately than simply genetic distance (model 1) but only very slightly. This is probably because the two variables are strongly correlated in Coreopsis. Prediction is substantially improved by allowing evolutionary rates to differ between annual and perennial branches (model 3). A bootstrapping procedure indicates that the life-history effect is statistically significant. The more rapid evolution of reproductive isolation within annual species of Coreopsis may be due to differing mechanisms of reproductive isolation, that is, chromosomal rearrangements rather than genetic incompatibilities.

Hybridization and gene flow between a day- and night-flowering species of Zaluzianskya (Scrophulariaceae s.s., tribe Manuleeae).

Despite apparent ethological isolation based on specialized pollination systems, hybridization between day-flowering Zaluzianskya microsiphon and night-flowering Z. natalensis has been proposed due to intermediate individuals found in sympatric populations of these species. The extent of this putative hybridization was investigated using inter-simple sequence repeat (ISSR) markers and principal components analysis (PCA) of morphological traits. The species are genetically similar, but show some intra- and interspecific variation in band frequencies. Neighbor-joining analyses of the ISSR data demonstrated that although the species largely formed distinct groups, several individuals from the sympatric populations of each species and the "hybrids" clustered together rather than with members of their own species. These results are consistent with hybridization, although they could also indicate historical similarity. Nine loci were present only in individuals of Z. microsiphon, the "hybrids," and sometimes the sympatric individuals of Z. natalensis. In contrast, only one locus showed the reverse pattern. This suggests unidirectional gene flow from Z. microsiphon to Z. natalensis, which is also supported by population-level examinations of four loci. Ordination revealed separate phenotype clusters for each species, with hybrid individuals located in between but often closer to the Z. natalensis cluster. One hypothesis is that hybrids are backcrossing with Z. natalensis, leading to introgression of Z. microsiphon genetic material.