The genome of the king protea, Protea cynaroides.

The king protea (Protea cynaroides), an early-diverging eudicot, is the most iconic species from the Megadiverse Cape Floristic Region, and the national flower of South Africa. Perhaps best known for its iconic flower head, Protea is a key genus for the South African horticulture industry and cut-flower market. Ecologically, the genus and the family Proteaceae are important models for radiation and adaptation, particularly to soils with limited phosphorus bio-availability. Here, we present a high-quality chromosome-scale assembly of the P. cynaroides genome as the first representative of the fynbos biome. We reveal an ancestral whole-genome duplication event that occurred in the Proteaceae around the late Cretaceous that preceded the divergence of all crown groups within the family and its extant diversity in all Southern continents. The relatively stable genome structure of P. cynaroides is invaluable for comparative studies and for unveiling paleopolyploidy in other groups, such as the distantly related sister group Ranunculales. Comparative genomics in sequenced genomes of the Proteales shows loss of key arbuscular mycorrhizal symbiosis genes likely ancestral to the family, and possibly the order. The P. cynaroides genome empowers new research in plant diversification, horticulture and adaptation, particularly to nutrient-poor soils.

Hybridisation and chloroplast capture between distinct Themeda triandra lineages in Australia.

Ecotypes are distinct populations within a species that are adapted to specific environmental conditions. Understanding how these ecotypes become established, and how they interact when reunited, is fundamental to elucidating how ecological adaptations are maintained. This study focuses on Themeda triandra, a dominant grassland species across Asia, Africa and Australia. It is the most widespread plant in Australia, where it has distinct ecotypes that are usually restricted to either wetter and cooler coastal regions or the drier and hotter interior. We generate a reference genome for T. triandra and use whole genome sequencing for over 80 Themeda accessions to reconstruct the evolutionary history of T. triandra and related taxa. Organelle phylogenies confirm that Australia was colonized by T. triandra twice, with the division between ecotypes predating their arrival in Australia. The nuclear genome provides evidence of differences in the dominant ploidal level and gene-flow among the ecotypes. In northern Queensland there appears to be a hybrid zone between ecotypes with admixed nuclear genomes and shared chloroplast haplotypes. Conversely, in the cracking claypans of Western Australia, there is cytonuclear discordance with individuals possessing the coastal chloroplast and interior clade nuclear genome. This chloroplast capture is potentially a result of adaptive introgression, with selection detected in the rpoC2 gene which is associated with water use efficiency. The reason that T. triandra is the most widespread plant in Australia appears to be a result of distinct ecotypic genetic variation and genome duplication, with the importance of each depending on the geographic scale considered.

Coastal dunefields maintain pre-Holocene genetic structure in a rocky shore red alga.

Most intertidal algae have limited dispersal potential, and areas that lack hard substratum suitable for attachment are thus expected to isolate regional populations from each other. Here, we used nuclear and mitochondrial genetic data to compare genetic structure in two co-distributed intertidal red algae with different dispersal potential along the South African coastline. Gelidium pristoides is divided into a south-eastern and a south-western evolutionary lineage separated by extensive, continuous sandy shoreline habitat adjacent to coastal dunefields. In contrast, Hypnea spicifera is genetically homogeneous throughout its range. In G. pristoides, the genetic breaks are associated with contemporary coastal dunefields. The age of the divergence event suggests that this may reflect the effect of older dispersal barriers, and that genetic structure was subsequently maintained by the formation of contemporary coastal dunefields.

Evolution of foraging behaviour: Deep intra-generic genetic divergence between territorial and non-territorial southern African patellid limpets.

Southern Africa is a biodiversity hotspot of patellid limpets, with three genera (Helcion, Cymbula and Scutellastra) identified and described in the region. Scutellastra is the most diverse and most frequently studied of these and, along with Cymbula, includes species with territorial and non-territorial foraging behaviours. We used three mitochondrial markers (12S rRNA, 16S rRNA and COI) and one nuclear marker (ATPSß intron) to assess evolutionary relationships among species of Cymbula and Scutellastra with these two foraging behaviours and to identify which foraging mode is the more ancient. Maximum Likelihood and Bayesian Inference phylogenetic analyses revealed that the species sharing a foraging type are monophyletic in both genera. Territoriality is a derived character, as the clades with this foraging type are nested within a tree that otherwise comprises non-territorial taxa. These include Helcion, which was recovered as sister to the Cymbula/Scutellastra clade, and the next basal genus, Patella, which is ancestral to all southern African patellogastropods. Deep genetic divergence between the two foraging traits reflects strong adaptive effects of resource partitioning in the evolution of southern African patellid limpets.

Does polyploidy facilitate long-distance dispersal?

BACKGROUND AND AIMS: The ability of plant lineages to reach all continents contributes substantially to their evolutionary success. This is exemplified by the Poaceae, one of the most successful angiosperm families, in which most higher taxa (tribes, subfamilies) have global distributions. Due to the old age of the ocean basins relative to the major angiosperm radiations, this is only possible by means of long-distance dispersal (LDD), yet the attributes of lineages with successful LDD remain obscure. Polyploid species are over-represented in invasive floras and in the previously glaciated Arctic regions, and often have wider ecological tolerances than diploids; thus polyploidy is a candidate attribute of successful LDD. METHODS: The link between polyploidy and LDD was explored in the globally distributed grass subfamily Danthonioideae. An almost completely sampled and well-resolved species-level phylogeny of the danthonioids was used, and the available cytological information was assembled. The cytological evolution in the clade was inferred using maximum likelihood (ML) as implemented in ChromEvol. The biogeographical evolution in the clade was reconstructed using ML and Bayesian approaches. KEY RESULTS: Numerous increases in ploidy level are demonstrated. A Late Miocene-Pliocene cycle of polyploidy is associated with LDD, and in two cases (the Australian Rytidosperma and the American Danthonia) led to secondary polyploidy. While it is demonstrated that successful LDD is more likely in polyploid than in diploid lineages, a link between polyploidization events and LDD is not demonstrated. CONCLUSIONS: The results suggest that polyploids are more successful at LDD than diploids, and that the frequent polyploidy in the grasses might have facilitated the extensive dispersal among continents in the family, thus contributing to their evolutionary success.

Description of a new montane freshwater crab (Arthropoda, Malacostraca, Decapoda, Potamonautidae) from the Eastern Cape, South Africa.

A new species of freshwater crab, Potamonautesamatholesp. nov., is described from the Winterberg-Amathole mountain range in the Eastern Cape Province, South Africa. Morphologically, P.amathole Peer & Gouws, sp. nov. most closely resembles P.tuerkayi but can be distinguished by key morphological characters including the variation in the shape of the subterminal segment of gonopod 2 between both species. Genetically, P.amathole Peer & Gouws, sp. nov. is placed within the clade of small-bodied, mountain-dwelling crabs including P.parvispina, P.parvicorpus, P.brincki, P.tuerkayi, P.baziya, and P.depressus. The new species is found in slow-moving mountain streams and pools at high altitudes. The continued discovery and description of new freshwater crab species reinforces the need for ongoing research, especially in under-sampled regions.

Cryptic variation in an ecological indicator organism: mitochondrial and nuclear DNA sequence data confirm distinct lineages of Baetis harrisoni Barnard (Ephemeroptera: Baetidae) in southern Africa.

BACKGROUND: Baetis harrisoni Barnard is a mayfly frequently encountered in river studies across Africa, but the external morphological features used for identifying nymphs have been observed to vary subtly between different geographic locations. It has been associated with a wide range of ecological conditions, including pH extremes of pH 2.9-10.0 in polluted waters. We present a molecular study of the genetic variation within B. harrisoni across 21 rivers in its distribution range in southern Africa. RESULTS: Four gene regions were examined, two mitochondrial (cytochrome c oxidase subunit I [COI] and small subunit ribosomal 16S rDNA [16S]) and two nuclear (elongation factor 1 alpha [EF1α] and phosphoenolpyruvate carboxykinase [PEPCK]). Bayesian and parsimony approaches to phylogeny reconstruction resulted in five well-supported major lineages, which were confirmed using a general mixed Yule-coalescent (GMYC) model. Results from the EF1α gene were significantly incongruent with both mitochondrial and nuclear (PEPCK) results, possibly due to incomplete lineage sorting of the EF1α gene. Mean between-clade distance estimated using the COI and PEPCK data was found to be an order of magnitude greater than the within-clade distance and comparable to that previously reported for other recognised Baetis species. Analysis of the Isolation by Distance (IBD) between all samples showed a small but significant effect of IBD. Within each lineage the contribution of IBD was minimal. Tentative dating analyses using an uncorrelated log-normal relaxed clock and two published estimates of COI mutation rates suggest that diversification within the group occurred throughout the Pliocene and mid-Miocene (~2.4-11.5 mya). CONCLUSIONS: The distinct lineages of B. harrisoni correspond to categorical environmental variation, with two lineages comprising samples from streams that flow through acidic Table Mountain Sandstone and three lineages with samples from neutral-to-alkaline streams found within eastern South Africa, Malawi and Zambia. The results of this study suggest that B. harrisoni as it is currently recognised is not a single species with a wide geographic range and pH-tolerance, but may comprise up to five species under the phylogenetic species concept, each with limited pH-tolerances, and that the B. harrisoni species group is thus in need of taxonomic review.

Mitochondrial DNA paradox: sex-specific genetic structure in a marine mussel--despite maternal inheritance and passive dispersal.

BACKGROUND: When genetic structure is identified using mitochondrial DNA (mtDNA), but no structure is identified using biparentally-inherited nuclear DNA, the discordance is often attributed to differences in dispersal potential between the sexes. RESULTS: We sampled the intertidal rocky shore mussel Perna perna in a South African bay and along the nearby open coast, and sequenced maternally-inherited mtDNA (there is no evidence for paternally-inherited mtDNA in this species) and a biparentally-inherited marker. By treating males and females as different populations, we identified significant genetic structure on the basis of mtDNA data in the females only. CONCLUSIONS: This is the first study to report sex-specific differences in genetic structure based on matrilineally-inherited mtDNA in a passively dispersing species that lacks social structure or sexual dimorphism. The observed pattern most likely stems from females being more vulnerable to selection in habitats from which they did not originate, which also manifests itself in a male-biased sex ratio. Our results have three important implications for the interpretation of population genetic data. First, even when mtDNA is inherited exclusively in the female line, it also contains information about males. For that reason, using it to identify sex-specific differences in genetic structure by contrasting it with biparentally-inherited markers is problematic. Second, the fact that sex-specific differences were found in a passively dispersing species in which sex-biased dispersal is unlikely highlights the fact that significant genetic structure is not necessarily a function of low dispersal potential or physical barriers. Third, even though mtDNA is typically used to study historical demographic processes, it also contains information about contemporary processes. Higher survival rates of males in non-native habitats can erase the genetic structure present in their mothers within a single generation.

Interventions for recurrent corneal erosions.

BACKGROUND: Recurrent corneal erosion is a common cause of disabling ocular symptoms and predisposes the cornea to infection. It may follow corneal trauma. Measures to prevent the development of recurrent corneal erosion following corneal trauma have not been firmly established. Once recurrent corneal erosion develops simple medical therapy (standard treatment) may lead to resolution of the episode. However, some patients continue to suffer when such therapy fails and once resolved further episodes of recurrent erosion may occur. A number of treatment and prophylactic options are then available but there is no agreement as to the best option. OBJECTIVES: To assess the effectiveness and safety of prophylactic and treatment regimens for recurrent corneal erosion. SEARCH METHODS: We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2012, Issue 6), MEDLINE (January 1946 to June 2012), EMBASE (January 1980 to June 2012), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to June 2012), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 13 June 2012. We also contacted researchers in the field. SELECTION CRITERIA: We included randomised and quasi-randomised trials that compared a prophylactic or treatment regimen with another prophylaxis/treatment or no prophylaxis/treatment for patients with recurrent corneal erosion. DATA COLLECTION AND ANALYSIS: Two authors independently extracted data and assessed trial quality. We contacted study authors for additional information. MAIN RESULTS: Seven randomised and one quasi-randomised controlled trial were included in the review. The trials were heterogenous and of poor quality. Safety data presented were incomplete. For the treatment of recurrent corneal erosion, a single-centre trial in the UK with 30 participants showed that oral tetracycline 250 mg twice daily for 12 weeks or topical prednisolone 0.5% four times daily for one week, or both, in addition to standard treatment, accelerated healing rates and improved symptoms. A single-centre trial in Sweden with 56 participants showed that excimer laser ablation in addition to mechanical debridement may reduce the number of erosions and improve symptoms. Furthermore, in a single-centre trial in Germany with 100 participants, transepithelial technique for excimer laser ablation had the same efficacy as the traditional subepithelial excimer laser technique but caused less pain. In a small study of 24 participants in UK, therapeutic contact lens wear was inferior to lubricant drops and ointment in abolishing the symptoms of recurrent corneal erosion and had a high complication rate, although the contact lenses used were the older generation with low oxygen permeability. A recent study in Hong Kong with 48 participants found diamond burr polishing to reduce episodes of recurrent corneal erosion. For prophylaxis of further episodes of recurrent corneal erosion, there was no difference in the occurrence of objective signs of recurrent erosion between hypertonic saline ointment versus tetracycline ointment or lubricating ointment in a small Japanese study with 26 participants. Also, in a single-centre study in the UK with 117 participants, there was no difference in symptom improvement between hypertonic saline versus paraffin ointment when used for prophylaxis. In a UK study with 42 participants, lubricating ointment at night in addition to standard treatment to prevent recurrence following traumatic corneal abrasion (erosion) caused by fingernail injury led to increased symptoms of recurrent corneal erosion compared to standard therapy alone. AUTHORS' CONCLUSIONS: Well-designed, masked, randomised controlled trials using standardised methods are needed to establish the benefits of new and existing prophylactic and treatment regimes for recurrent corneal erosion. International consensus is also needed to progress research efforts towards evaluation of the major effective treatments for recurrent corneal erosions.

Contrasted patterns of hyperdiversification in Mediterranean hotspots.

Dating the Tree of Life has now become central to relating patterns of biodiversity to key processes in Earth history such as plate tectonics and climate change. Regions with a Mediterranean climate have long been noted for their exceptional species richness and high endemism. How and when these biota assembled can only be answered with a good understanding of the sequence of divergence times for each of their components. A critical aspect of dating by using molecular sequence divergence is the incorporation of multiple suitable age constraints. Here, we show that only rigorous phylogenetic analysis of fossil taxa can lead to solid calibration and, in turn, stable age estimates, regardless of which of 3 relaxed clock-dating methods is used. We find that Proteaceae, a model plant group for the Mediterranean hotspots of the Southern Hemisphere with a very rich pollen fossil record, diversified under higher rates in the Cape Floristic Region and Southwest Australia than in any other area of their total distribution. Our results highlight key differences between Mediterranean hotspots and indicate that Southwest Australian biota are the most phylogenetically diverse but include numerous lineages with low diversification rates.

A Comparative Study of the Anatomy of Leaf Domatia in Gardenia thunbergia Thunb., Rothmannia capensis Thunb., and Rothmannia globosa (Hochst.) Keay (Rubiaceae).

Many dicotyledonous plants produce structures called leaf domatia. Approximately 28% of 290 families have species with leaf domatia. These structures are abundant within the Rubiaceae and Vitaceae. 26% and 16% out of 206 representative species cited in literature from 48 plant families belong to the Rubiaceae and Vitaceae respectively. Leaf domatia are usually associated with mites and often mediate mutualistic relationships with predacious mites. These structures are pockets found in the underside of the leaf, where the secondary vein axils meet the major vein. In the present study, we examine the anatomical structures of leaf domatia from three plant species (Gardenia thunbergia Thunb., Rothmannia capensis Thunb., Rothmannia globosa (Hochst.) Keay) from the Rubiaceae family in order to find out if their internal tissues differ. These plants were sectioned and viewed under a Light Microscope in order to document their internal anatomy. A Transmission Electron Microscope was used to search for the presence of cuticular folds in their epidermis, which are thought to assist plant to communicate with mites. Results from this study suggested that the main features of domatial anatomy are the presence of an extra layer of tissue in the lower epidermis, a cuticle, cuticular folds, trichomes and the presence of an invagination. Cuticular folds were present inside the domatia but were not restricted to the domatial lamina. Thus, we conclude that these structures do not assist plant in plant-mite communication. This study provides a better understating of the anatomy of leaf domatia of the Rubiaceae.

"Nested" cryptic diversity in a widespread marine ecosystem engineer: a challenge for detecting biological invasions.

BACKGROUND: Ecosystem engineers facilitate habitat formation and enhance biodiversity, but when they become invasive, they present a critical threat to native communities because they can drastically alter the receiving habitat. Management of such species thus needs to be a priority, but the poorly resolved taxonomy of many ecosystem engineers represents a major obstacle to correctly identifying them as being either native or introduced. We address this dilemma by studying the sea squirt Pyura stolonifera, an important ecosystem engineer that dominates coastal communities particularly in the southern hemisphere. Using DNA sequence data from four independently evolving loci, we aimed to determine levels of cryptic diversity, the invasive or native status of each regional population, and the most appropriate sampling design for identifying the geographic ranges of each evolutionary unit. RESULTS: Extensive sampling in Africa, Australasia and South America revealed the existence of "nested" levels of cryptic diversity, in which at least five distinct species can be further subdivided into smaller-scale genetic lineages. The ranges of several evolutionary units are limited by well-documented biogeographic disjunctions. Evidence for both cryptic native diversity and the existence of invasive populations allows us to considerably refine our view of the native versus introduced status of the evolutionary units within Pyura stolonifera in the different coastal communities they dominate. CONCLUSIONS: This study illustrates the degree of taxonomic complexity that can exist within widespread species for which there is little taxonomic expertise, and it highlights the challenges involved in distinguishing between indigenous and introduced populations. The fact that multiple genetic lineages can be native to a single geographic region indicates that it is imperative to obtain samples from as many different habitat types and biotic zones as possible when attempting to identify the source region of a putative invader. "Nested" cryptic diversity, and the difficulties in correctly identifying invasive species that arise from it, represent a major challenge for managing biodiversity.

Climate-driven genetic divergence of limpets with different life histories across a southeast African marine biogeographic disjunction: different processes, same outcome.

Genetic divergence among populations of marine broadcast spawners in the absence of past geological barriers presents an intriguing challenge to understanding speciation in the sea. To determine how differences in life history affect genetic divergence and demographic histories across incomplete dispersal barriers, we conducted a comparative phylogeographic study of three intertidal limpets (Siphonaria spp.) represented on either side of a biogeographic disjunction separating tropical and subtropical marine provinces in southeastern Africa. Using a combination of mitochondrial and nuclear sequence data, we identified two distinct evolutionary lineages each in both Siphonaria concinna (a planktonic disperser) and S. nigerrima (a direct developer), and panmixia in a second planktonic disperser, S. capensis. Although phylogeographic breaks were present in two species, how these became established differed depending on their life histories. In the direct developer, lack of gene flow following divergence, and demographic expansion from a small initial size in the species' subtropical population, point to a single colonisation event. In contrast, the evolutionary lineages of the planktonic disperser split into two genetic lineages with much larger initial population sizes and southward gene flow continued at least periodically, indicating that divergence in this species may have been driven by a combination of reduced larval dispersal and divergent selection. These findings help explain why the presence or absence of phylogeographic breaks often appears to be independent of species' dispersal potential.

Molecular systematics of the African snake family Lamprophiidae Fitzinger, 1843 (Serpentes: Elapoidea), with particular focus on the genera Lamprophis Fitzinger 1843 and Mehelya Csiki 1903.

The snake family Lamprophiidae Fitzinger (Serpentes: Elapoidea) is a putatively Late Eocene radiation of nocturnal snakes endemic to the African continent. It incorporates many of the most characteristic and prolific of Africa's non-venomous snake species, including the widespread type genus Lamprophis Fitzinger, 1843 (house snakes). We used approximately 2500 bases of mitochondrial and nuclear DNA sequence data from 28 (41%) of the approximately 68 recognised lamprophiid species in nine of the eleven genera to investigate phylogenetic structure in the family and to inform taxonomy at the generic level. Cytochrome b, ND4 and tRNA gene sequences (mitochondrial) and c-mos sequences (nuclear) were analysed using Maximum Likelihood, Bayesian Inference and Maximum Parsimony methods. The genus Mehelya Csiki, 1903 was paraphyletic with respect to Gonionotophis Boulenger, 1893. To address this, the concept of Gonionotophis is expanded to include all current Mehelya species. The genus Lamprophis emerged polyphyletic: the enigmatic Lamprophis swazicus was sister to Hormonotus modestus from West Africa, and not closely related to its nominal congeners. It is moved to a new monotypic genus (Inyoka gen. nov.). The remaining Lamprophis species occur in three early-diverging lineages. (1) Lamprophis virgatus and the widely distributed Lamprophis fuliginosus species complex (which also includes Lamprophis lineatus and Lamprophis olivaceus) formed a clade for which the generic name Boaedon Duméril, Bibron & Duméril, 1854 is resurrected. (2) The water snakes (Lycodonomorphus) were nested within Lamprophis (sensu lato), sister to Lamprophis inornatus. We transfer this species to the genus Lycodonomorphus Fitzinger, 1843. (3) We restrict Lamprophis (sensu strictissimo) to a small clade of four species endemic to southern Africa: the type species of Lamprophis Fitzinger, 1843 (Lamprophis aurora) plus Lamprophis fiskii, Lamprophis fuscus and Lamprophis guttatus.

Phylogenetic relationships of Aristida and relatives (Poaceae, Aristidoideae) based on noncoding chloroplast (trnL-F, rpl16) and nuclear (ITS) DNA sequences.

PREMISE: The cosmopolitan and ecologically important grass subfamily Aristidoideae comprises the widely distributed genus Aristida (250-290 species), Stipagrostis (50 species, with an African-Asian distribution), and Sartidia (five species, Africa and Madagascar). The subfamily includes species with C(3) (Sartidia and a single species of Aristida) and C(4) photosynthetic pathways. Rigorous phylogenetic reconstructions of species relationships are required to explain the biogeographic, physiological, and ecological diversity within this subfamily. METHODS: Chloroplast (trnL-F, rpl16) and nuclear (ITS) DNA sequences were obtained from 198 accessions, and the combined data set was subjected to parsimony, maximum likelihood, and Bayesian inference analyses. Dating analyses calibrated using previously published node ages were conducted to determine the ages of major radiations. RESULTS: The C(3) Sartidia is sister to a monophyletic Stipagrostis, and the (Sartidia, Stipagrostis) clade is sister to Aristida. Within Aristida, the only known C(3) species, A. longifolia, is sister to the remainder of the genus. Infrageneric sections of Aristida were not supported, and there are no synapomorphic morphological characters for the clades retrieved. Within Aristida, monophyletic Australian, African, North American, and South American clades are retrieved. CONCLUSIONS: The subfamily dates back to the late Miocene, with the major lineages present by the Pliocene. With one exception, regional clades of Aristida evolved in the Pliocene. The C(3) photosynthetic pathway is hypothesized to be the pleisomorphic condition for the subfamily, wherein two independent C(4) pathways (each with unique anatomical and genetic features) evolved, one within Aristida and one in Stipagrostis.

Reticulation, data combination, and inferring evolutionary history: an example from Danthonioideae (Poaceae).

We explore the potential impact of conflicting gene trees on inferences of evolutionary history above the species level. When conflict between gene trees is discovered, it is common practice either to analyze the data separately or to combine the data having excluded the conflicting taxa or data partitions for those taxa (which are then recoded as missing). We demonstrate an alternative approach, which involves duplicating conflicting taxa in the matrix, such that each duplicate is represented by one partition only. This allows the combination of all available data in standard phylogenetic analyses, despite reticulations. We show how interpretation of contradictory gene trees can lead to conflicting inferences of both morphological evolution and biogeographic history, using the example of the pampas grasses, Cortaderia. The characteristic morphological syndrome of Cortaderia can be inferred as having arisen multiple times (chloroplast DNA [cpDNA]) or just once (nuclear ribosomal DNA [nrDNA]). The distributions of species of Cortaderia and related genera in Australia/New Guinea, New Zealand, and South America can be explained by few (nrDNA) or several (cpDNA) dispersals between the southern continents. These contradictions can be explained by past hybridization events, which have linked gains of complex morphologies with unrelated chloroplast lineages and have erased evidence of dispersals from the nuclear genome. Given the discrepancies between inferences based on the gene trees individually, we urge the use of approaches such as ours that take multiple gene trees into account.

Intraspecific mitochondrial gene variation can be as low as that of nuclear rRNA.

Background: Mitochondrial DNA (mtDNA) has long been used to date historical demographic events. The idea that it is useful for molecular dating rests on the premise that its evolution is neutral. Even though this idea has long been challenged, the evidence against clock-like evolution of mtDNA is often ignored. Here, we present a particularly clear and simple example to illustrate the implications of violations of the assumption of selective neutrality. Methods: DNA sequences were generated for the mtDNA COI gene and the nuclear 28S rRNA of two closely related rocky shore snails, and species-level variation was compared. To our knowledge, this is the first study to use nuclear rRNA at this taxonomic level, presumably because this marker is assumed to evolve so slowly that it is only suitable for phylogenetics.   Results: Even though high inter-specific divergence reflected the faster evolutionary rate of COI, intraspecific genetic variation was similar for both markers. As a result, estimates of population expansion times based on mismatch distributions differed between the two markers by millions of years. Conclusions: Assuming that 28S evolves effectively clock-like, these findings can be explained by variation-reducing purifying selection in mtDNA at the species level, and an elevated divergence rate caused by diversifying selection between the two species. Although these two selective forces together make mtDNA suitable as a marker for species identifications by means of DNA barcoding because they create a 'barcoding gap', estimates of demographic change based on this marker can be expected to be highly unreliable. Our study contributes to the growing evidence that the utility of mtDNA sequence data beyond DNA barcoding is limited.

Habitat requirements affect genetic variation in three species of mayfly (Ephemeroptera, Baetidae) from South Africa.

This study investigates genetic diversity in three species of Ephemeroptera, one eurytopic and therefore widespread (Afroptilum sudafricanum) and two stenotopic and thus endemic (Demoreptus natalensis and Demoreptus capensis) species, all of which co-occur in the southern Great Escarpment, South Africa. Mitochondrial DNA was analysed to compare the genetic diversity between the habitat generalist and the two habitat specialists. Afroptilum sudafricanum showed no indication of population genetic structure due to geographic location, while both Demoreptus species revealed clear genetic differentiation between geographic localities and catchments, evident from phylogenetic analyses and high FST values from AMOVA. In addition, the phylogenetic analyses indicate some deeper haplotype divergences within A. sudafricanum and Demoreptus that merit taxonomic attention. These results give important insight into evolutionary processes occurring through habitat specialisation and population isolation. Further research and sampling across a wider geographic setting that includes both major mountain blocks of the Escarpment and lowland non-Escarpment sites will allow for refined understanding of biodiversity and associated habitat preferences, and illuminate comparative inferences into gene flow and cryptic speciation.

Using fossils and molecular data to reveal the origins of the Cape proteas (subfamily Proteoideae).

The angiosperm family Proteaceae is a distinct component of the Cape Floristic Region biodiversity hotspot with 330 endemic species. Phylogenetic analyses of subfamily Proteoideae using sequence data from one nuclear and six plastid loci show that most of this diversity is contained in two distinct Cape floral clades. Molecular dating analyses, using Bayesian and penalized likelihood methods and four phylogenetically supported fossil age constraints, reveal contrasting histories for these two clades. The genus Protea belongs to a lineage that may have been in Africa since the Late Cretaceous but began to diversify in the Cape only 5-18 Myr ago. In contrast, the Leucadendrinae clade presumably arrived in the region no earlier than 46 Myr ago by long-distance dispersal from an Australian ancestor and the extant members of this clade began to diversify in the Cape 22-39 Myr ago. These results join a growing number of case studies that challenge the commonly accepted view that most of the Cape flora radiated synchronously in the Late Miocene and Early Pliocene when a Mediterranean climate settled in the region.