Nothobranchius balamaensis (Cyprinodontiformes: Nothobranchiidae), a new species of annual killifish from northern Mozambique.

A new seasonal killifish of the genus Nothobranchius is described from the Montepuez River system in northern Mozambique. The new species, Nothobranchius balamaensis Bragana Chakona, is differentiated from congeners by its characteristic colour pattern and molecular data further support its taxonomic distinctiveness. Phylogenetic results based on two mitochondrial and three nuclear genes confirms N. balamaensis is closely related to N. kirki and N. wattersi; all three belonging to the Coastal-Inland Clade. The new species is most similar in colour pattern to N. kirki sharing the characteristic of a deep red-orange colouration in the basal, proximal, and medial zones of the caudal and anal fins that grades to orange in the distal zone. This is a key feature that distinguishes these two species from all other Nothobranchius. The main distinguishing features between the new species and N. kirki is the presence of a light blue to white band or series of irregular markings in the proximal zone of the anal fin in N. kirki, versus the absence of such a colour pattern element in N. balamaensis, as well as differences in the dorsal fin pattern. When compared to all population groups of N. wattersi, the colour pattern of N. balamaensis is distinctive. Nothobranchius balamaensis is a relatively slender member of the genus, a characteristic that clearly distinguishes it from both N. kirki and N. wattersi. Nothobranchius balamaensis is currently only known from a few specimens from the type locality.

Genomic fingerprints of palaeogeographic history: The tempo and mode of rift tectonics across tropical Africa has shaped the diversification of the killifish genus Nothobranchius (Teleostei: Cyprinodontiformes).

This paper reports a phylogeny of the African killifishes (Genus Nothobranchius, Order Cyprinodontiformes) informed by five genetic markers (three nuclear, two mitochondrial) of 80 taxa (seven undescribed and 73 of the 92 recognized species). These short-lived annual fishes occupy seasonally wet habitats in central and eastern Africa, and their distribution coincides largely with the East African Rift System (EARS). The fossil dates of sister clades used to constrain a chronometric tree of all sampled Nothobranchius recovered the origin of the genus at ~13.27 Mya. It was followed by the radiations of six principal clades through the Neogene. An ancestral area estimation tested competing biogeographical hypotheses to constrain the ancestral origin of the genus to the Nilo-Sudan Ecoregion, which seeded a mid-Miocene dispersal event into the Coastal ecoregion, followed closely (~10 Mya) by dispersals southward across the Mozambique coastal plain into the Limpopo Ecoregion. Extending westwards across the Tanzanian plateau, a pulse of radiations through the Pliocene were associated with dispersals and fragmentation of wetlands across the Kalahari and Uganda Ecoregions. We interpret this congruence of drainage rearrangements with dispersals and cladogenic events of Nothobranchius to reflect congruent responses to recurrent uplift and rifting. The coevolution of these freshwater fishes and wetlands is attributed to ultimate control by tectonics, as the EARS extended southwards during the Neogene. Geobiological consilience of the combined evidence supports a tectonic hypothesis for the evolution of Nothobranchius.

Phylogenetic relationships amongst the African genera of subtribe Orchidinae s.l. (Orchidaceae; Orchideae): Implications for subtribal and generic delimitations.

Phylogenetic relationships within the Orchideae sensu Pridgeon et al, remain one of the biggest unresolved issues in our understanding of the taxonomy of the orchids. Members of the Orchideae are numerous and widespread in Africa but remain poorly represented in phylogenetic research. In this study we included a broad sampling of African taxa for which we sequenced three plastid (rbcl, matK and trnL + trnL-F) and two nuclear regions (ITS and 18S). We used 368 sequences representing 278 species and 49 genera to infer relationships using the Bayesian Inference and Maximum Likelihood method. Our results show strong support for three clades, two of which almost entirely match the historical circumscription of Orchidinae and Habenariinae, and the third, Bartholininae, sister to the former two, includes the genera Holothrix and Bartholina. Stenoglottis should be assigned to Orchidinae and not to Habenariinae. Several genera such as Habenaria, Cynorkis and Benthamia are shown to be para- or polyphyletic: Bonatea, Centrostigma, Platycoryne and Roeperocharis are all embedded in Habenaria; Physoceras, Arnottia and part of Benthamia are embedded in Cynorkis. We propose a subdivision of Orchideae sensu lato into nine subtribes, but refrain from making generic re-arrangements until more extensive or more in-depth studies have been done.

Leaps and bounds: geographical and ecological distance constrained the colonisation of the Afrotemperate by Erica.

BACKGROUND: The coincidence of long distance dispersal (LDD) and biome shift is assumed to be the result of a multifaceted interplay between geographical distance and ecological suitability of source and sink areas. Here, we test the influence of these factors on the dispersal history of the flowering plant genus Erica (Ericaceae) across the Afrotemperate. We quantify similarity of Erica climate niches per biogeographic area using direct observations of species, and test various colonisation scenarios while estimating ancestral areas for the Erica clade using parametric biogeographic model testing. RESULTS: We infer that the overall dispersal history of Erica across the Afrotemperate is the result of infrequent colonisation limited by geographic proximity and niche similarity. However, the Drakensberg Mountains represent a colonisation sink, rather than acting as a "stepping stone" between more distant and ecologically dissimilar Cape and Tropical African regions. Strikingly, the most dramatic examples of species radiations in Erica were the result of single unique dispersals over longer distances between ecologically dissimilar areas, contradicting the rule of phylogenetic biome conservatism. CONCLUSIONS: These results highlight the roles of geographical and ecological distance in limiting LDD, but also the importance of rare biome shifts, in which a unique dispersal event fuels evolutionary radiation.

Floral Color, Anthocyanin Synthesis Gene Expression and Control in Cape Erica Species.

Introduction: The Cape Floristic Region (CFR) is a biodiversity hotspot, recognized globally for its unusually high levels of endemism. The origins of this biodiversity are a long-standing topic of research. The largest "Cape clade," Erica, radiated dramatically in the CFR, its ca. 690 species arising within 10-15 Ma. Notable between- and within-species flower color variation in Erica may have contributed to the origins of species diversity through its impact on pollinator efficiency and specificity. Methods: We investigate the expression and function of the genes of the anthocyanin biosynthesis pathway that controls floral color in 12 Erica species groups using RT-qPCR and UPLC-MS/MS. Results: Shifts from ancestral pink- or red- to white- and/or yellow flowers were associated with independent losses of single pathway gene expression, abrogation of the entire pathway due to loss of the expression of a transcription factor or loss of function mutations in pathway genes. Discussion: Striking floral color shifts are prevalent amongst the numerous species of Cape Erica. These results show independent origins of a palette of mutations leading to such shifts, revealing the diverse genetic basis for potentially rapid evolution of a speciation-relevant trait.

Targeted NGS for species level phylogenomics: "made to measure" or "one size fits all"?

Targeted high-throughput sequencing using hybrid-enrichment offers a promising source of data for inferring multiple, meaningfully resolved, independent gene trees suitable to address challenging phylogenetic problems in species complexes and rapid radiations. The targets in question can either be adopted directly from more or less universal tools, or custom made for particular clades at considerably greater effort. We applied custom made scripts to select sets of homologous sequence markers from transcriptome and WGS data for use in the flowering plant genus Erica (Ericaceae). We compared the resulting targets to those that would be selected both using different available tools (Hyb-Seq; MarkerMiner), and when optimising for broader clades of more distantly related taxa (Ericales; eudicots). Approaches comparing more divergent genomes (including MarkerMiner, irrespective of input data) delivered fewer and shorter potential markers than those targeted for Erica. The latter may nevertheless be effective for sequence capture across the wider family Ericaceae. We tested the targets delivered by our scripts by obtaining an empirical dataset. The resulting sequence variation was lower than that of standard nuclear ribosomal markers (that in Erica fail to deliver a well resolved gene tree), confirming the importance of maximising the lengths of individual markers. We conclude that rather than searching for "one size fits all" universal markers, we should improve and make more accessible the tools necessary for developing "made to measure" ones.

Evolution of leaf anatomy in arid environments - A case study in southern African Tetraena and Roepera (Zygophyllaceae).

The dry biomes of southern Africa (Desert, Nama Karoo and Succulent Karoo) are home to a rich and diverse xerophytic flora. This flora includes two morphologically diverse clades of Zygophyllaceae, Tetraena and Roepera (Zygophylloideae), which inhabit some of the most arid habitats in the region. Using a plastid phylogeny of Zygophylloideae we assess whether the evolution of putatively adaptive traits (leaf shape, vasculature, mode of water storage and photosynthetic type: C3 versus C4) coincides with the successful colonisation of environments with different drought regimes within southern Africa. Our results show general niche conservatism within arid habitats in Tetraena, but niche shifts from arid to more mesic biomes with longer and/or cooler growing season (Fynbos and Thicket) in Roepera. However, these distinct broad-scale biogeographical patterns are not reflected in leaf anatomy, which seems to vary at more local scales. We observed considerable variability and multiple convergences to similar leaf anatomies in both genera, including shifts between "all cell succulence" leaf types and leaf types with distinct chlorenchyma and hydrenchyma. Our survey of C4 photosynthesis in the Zygophylloideae showed that the C4 pathway is restricted to Tetraena simplex, which also having an annual life history and a widespread distribution, is rather atypical for this group.

Back to Gondwanaland: can ancient vicariance explain (some) Indian Ocean disjunct plant distributions?

Oceans, or other wide expanses of inhospitable environment, interrupt present day distributions of many plant groups. Using molecular dating techniques, generally incorporating fossil evidence, we can estimate when such distributions originated. Numerous dating analyses have recently precipitated a paradigm shift in the general explanations for the phenomenon, away from older geological causes, such as continental drift, in favour of more recent, long-distance dispersal (LDD). For example, the 'Gondwanan vicariance' scenario has been dismissed in various studies of Indian Ocean disjunct distributions. We used the gentian tribe Exaceae to reassess this scenario using molecular dating with minimum (fossil), maximum (geological), secondary (from wider analyses) and hypothesis-driven age constraints. Our results indicate that ancient vicariance cannot be ruled out as an explanation for the early origins of Exaceae across Africa, Madagascar and the Indian subcontinent unless a strong assumption is made about the maximum age of Gentianales. However, both the Gondwanan scenario and the available evidence suggest that there were also several, more recent, intercontinental dispersals during the diversification of the group.

The identification of oppA gene homologues as part of the oligopeptide transport system in mycoplasmas.

The lack of an annotated oppA gene as part of many oligopeptide permease (opp) operons has questioned the necessity of the oligopeptide-binding domain (OppA) as a part of the Opp transport system in mycoplasmas. This study investigated the occurrence of an oppA gene as part of the oppBCDF operon in 42 mycoplasma genomes. Except for hemoplasma, all mycoplasmas were found to possess one or more copies of the oppBCDF operon and with the help of similarity searches their oppA genes could be identified. Phylogenetic analysis of the combined OppABCDF amino acid sequences allowed them to be grouped into three types. Each type has a unique set of conserved motifs, which are likely to reflect substrate preference and adaption strategies. Our approach allowed the identification of oppA gene homologues for all mycoplasma opp operons and thereby provides a method for re-evaluating the current annotation of oppA genes in mycoplasma genomes.

An approach to identify putative hybrids in the 'coalescent stochasticity zone', as exemplified in the African plant genus Streptocarpus (Gesneriaceae).

The inference of phylogenetic relationships is often complicated by differing evolutionary histories of independently-inherited markers. The causes of the resulting gene tree incongruence can be challenging to identify, often relying on coalescent simulations dependent on unverifiable assumptions. We investigated alternative techniques using the South African rosulate species of Streptocarpus as a study group. Two independent gene trees - from the nuclear ITS region and from three concatenated plastid regions (trnL-F, rpl20-rps12 and trnC-D) - displayed widespread, strongly supported incongruence. We investigated the causes by detecting genetic exchange across morphological borders using morphological optimizations and genetic exchange across species boundaries using the genealogical sorting index. Incongruence between gene trees was associated with ancestral shifts in growth form (in four species) but not in pollination syndrome, suggesting introgression limited by reproductive barriers. Genealogical sorting index calculations showed polyphyly of two additional species, while individuals of all others were significantly associated. In one case the association was stronger according to the internal transcribed spacer data than according to the plastid data, which, given the smaller effective population size of the plastid, may also indicate introgression. These approaches offer alternative ways to identify potential hybridization events where incomplete lineage sorting cannot be rejected using simulations.

Phylogeography and molecular evolution of potato virus Y.

Potato virus Y (PVY) is an important plant pathogen, whose host range includes economically important crops such as potato, tobacco, tomato, and pepper. PVY presents three main strains (PVY(O), PVY(N) and PVY(C)) and several recombinant forms. PVY has a worldwide distribution, yet the mechanisms that promote and maintain its population structure and genetic diversity are still unclear. In this study, we used a pool of 77 complete PVY genomes from isolates collected worldwide. After removing the effect of recombination in our data set, we used bayesian techniques to study the influence of geography and host species in both PVY population structure and dynamics. We have also performed selection and covariation analyses to identify evolutionarily relevant amino acid residues. Our results show that both geographic and host-driven adaptations explain PVY diversification. Furthermore, purifying selection is the main force driving PVY evolution, although some indications of positive selection accounted for the diversification of the different strains. Interestingly, the analysis of P3N-PIPO, a recently described gene in potyviruses, seems to show a variable length among the isolates analyzed, and this variability is explained, in part, by host-driven adaptation.

Cultivation shapes genetic novelty in a globally important invader.

Acacia saligna is a species complex that has become invasive in a number of countries worldwide where it has caused substantial environmental and economic impacts. Understanding genetic and other factors contributing to its success may allow managers to limit future invasions of closely related species. We used three molecular markers to compare the introduced range (South Africa) to the native range (Western Australia). Nuclear markers showed that invasive populations are divergent from native populations and most closely related to a cultivated population in Western Australia. We also found incongruence between nuclear and chloroplast data that, together with the long history of cultivation of the species, suggest that introgressive hybridization (coupled with chloroplast capture) may have occurred within A. saligna. While we could not definitively prove introgression, the genetic distance between cultivated and native A. saligna populations was comparable to known interspecific divergences among other Acacia species. Therefore, cultivation, multiple large-scale introductions and possibly introgressive hybridization have rapidly given rise to the divergent genetic entity present in South Africa. This may explain the known global variation in invasiveness and inaccuracy of native bioclimatic models in predicting potential distributions.

A densely sampled ITS phylogeny of the Cape flagship genus Erica L. suggests numerous shifts in floral macro-morphology.

Erica L. is the largest of the 'Cape' clades that together comprise around half of the disproportionately high species richness of the Cape Floristic Region (CFR) of South Africa. Around 840 species of Erica are currently recognised, C.680 of which are found in the CFR, the rest distributed across the rest of Southern Africa, the highlands of Tropical Africa and Madagascar, and Europe. Erica is taxonomically well documented, but very little is known about species-level relationships. We present the first densely sampled phylogenetic analysis of Erica, using nuclear ribosomal DNA sequences (internal transcribed spacers; ITS) of c. 45% of the species from across the full geographic range of the genus, both Calluna and Daboecia (Ericeae; monotypic genera and putative sister groups of Erica), and further Ericoideae outgroups. Our results show both morphological and geographic coherence of some clades, but numerous shifts in floral macro-morphology as represented by variation in individual morphological characters and pollination syndromes. European Ericeae is a paraphyletic grade subtending a monophyletic African/Malagasy Erica. Given the limited resolution of this single gene tree, more data are needed for further conclusions. Clades are identified that will serve as an effective guide for targeted sampling from multiple linkage groups.

Extinction risk and diversification are linked in a plant biodiversity hotspot.

It is widely recognized that we are entering an extinction event on a scale approaching the mass extinctions seen in the fossil record. Present-day rates of extinction are estimated to be several orders of magnitude greater than background rates and are projected to increase further if current trends continue. In vertebrates, species traits, such as body size, fecundity, and geographic range, are important predictors of vulnerability. Although plants are the basis for life on Earth, our knowledge of plant extinctions and vulnerabilities is lagging. Here, we disentangle the underlying drivers of extinction risk in plants, focusing on the Cape of South Africa, a global biodiversity hotspot. By comparing Red List data for the British and South African floras, we demonstrate that the taxonomic distribution of extinction risk differs significantly between regions, inconsistent with a simple, trait-based model of extinction. Using a comprehensive phylogenetic tree for the Cape, we reveal a phylogenetic signal in the distribution of plant extinction risks but show that the most threatened species cluster within short branches at the tips of the phylogeny--opposite to trends in mammals. From analyzing the distribution of threatened species across 11 exemplar clades, we suggest that mode of speciation best explains the unusual phylogenetic structure of extinction risks in plants of the Cape. Our results demonstrate that explanations for elevated extinction risk in plants of the Cape flora differ dramatically from those recognized for vertebrates. In the Cape, extinction risk is higher for young and fast-evolving plant lineages and cannot be explained by correlations with simple biological traits. Critically, we find that the most vulnerable plant species are nonetheless marching towards extinction at a more rapid pace but, surprisingly, independently from anthropogenic effects. Our results have important implications for conservation priorities and cast doubts on the utility of current Red List criteria for plants in regions such as the Cape, where speciation has been rapid, if our aim is to maximize the preservation of the tree-of-life.

Consistent phenological shifts in the making of a biodiversity hotspot: the Cape flora.

BACKGROUND: The best documented survival responses of organisms to past climate change on short (glacial-interglacial) timescales are distributional shifts. Despite ample evidence on such timescales for local adaptations of populations at specific sites, the long-term impacts of such changes on evolutionary significant units in response to past climatic change have been little documented. Here we use phylogenies to reconstruct changes in distribution and flowering ecology of the Cape flora--South Africa's biodiversity hotspot--through a period of past (Neogene and Quaternary) changes in the seasonality of rainfall over a timescale of several million years. RESULTS: Forty-three distributional and phenological shifts consistent with past climatic change occur across the flora, and a comparable number of clades underwent adaptive changes in their flowering phenology (9 clades; half of the clades investigated) as underwent distributional shifts (12 clades; two thirds of the clades investigated). Of extant Cape angiosperm species, 14-41% have been contributed by lineages that show distributional shifts consistent with past climate change, yet a similar proportion (14-55%) arose from lineages that shifted flowering phenology. CONCLUSIONS: Adaptive changes in ecology at the scale we uncover in the Cape and consistent with past climatic change have not been documented for other floras. Shifts in climate tolerance appear to have been more important in this flora than is currently appreciated, and lineages that underwent such shifts went on to contribute a high proportion of the flora's extant species diversity. That shifts in phenology, on an evolutionary timescale and on such a scale, have not yet been detected for other floras is likely a result of the method used; shifts in flowering phenology cannot be detected in the fossil record.

Estimating the age of fire in the Cape flora of South Africa from an orchid phylogeny.

Fire may have been a crucial component in the evolution of the Cape flora of South Africa, a region characterized by outstanding levels of species richness and endemism. However, there is, to date, no critical assessment of the age of the modern fire regime in this biome. Here, we exploit the presence of two obligate post-fire flowering clades in the orchid genus Disa, in conjunction with a robust, well-sampled and dated molecular phylogeny, to estimate the age by which fire must have been present. Our results indicate that summer drought (winter rainfall), the fire regime and the fynbos vegetation are several million years older than currently suggested. Summer drought and the fynbos vegetation are estimated to date back to at least the Early Miocene (ca 19.5 Ma). The current fire regime may have been established during a period of global cooling that followed the mid-Miocene Climatic Optimum (ca 15 Ma), which led to the expansion of open habitats and increased aridification. The first appearance of Disa species in the grassland biome, as well as in the subalpine habitat, is in striking agreement with reliable geological and palaeontological evidence of the age of these ecosystems, thus corroborating the efficacy of our methods. These results change our understanding of the historical mechanisms underlying botanical evolution in southern Africa, and confirm the potential of using molecular phylogenies to date events for which other information is lacking or inconclusive.

A rapid and inexpensive method for the direct PCR amplification of DNA from plants.

PREMISE OF THE STUDY: We present a rapid and inexpensive alternative to DNA isolation for polymerase chain reaction (PCR) amplification from plants. • METHODS AND RESULTS: The method involves direct PCR amplification from material macerated in one buffer, followed by dilution and incubation in a second buffer. We describe the procedure and demonstrate its application for nuclear and plastid DNA amplification across a broad range of vascular plants. • CONCLUSIONS: The method is fast, easy to perform, cost-effective, and consequently ideal for large sample numbers. It represents a considerable simplification of present approaches requiring DNA isolation prior to PCR amplification and will be useful in plant systematics and biotechnology, including applications such as DNA barcoding.

Genetic variability within the coat protein gene of Grapevine fanleaf virus isolates from South Africa and the evaluation of RT-PCR, DAS-ELISA and ImmunoStrips as virus diagnostic assays.

Grapevine fanleaf virus (GFLV) is responsible for severe fanleaf degeneration in grapevines of all major wine producing regions of the world, including South Africa. In order to successfully control the spread of the virus, specific and reliable diagnostic assays are necessary. The genetic variability of 12 GFLV isolates recovered from naturally infected grapevine plants in the Western Cape region of South Africa were characterised. These samples were subjected to RNA extraction, RT-PCR analysis and sequencing of the coat protein gene (2CCP). Sequence identities between different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the 2CCP gene sequences showed that the South African isolates form two distinct clades or sub-populations. The specificity and sensitivity of three diagnostic techniques (rapid-direct-one-tube-RT-PCR, DAS-ELISA and ImmunoStrips) for the detection of GFLV were analysed to determine the appropriate diagnostic assay for virus infection. Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for detection. This is the first report on sequence analysis of full-length 2CCP gene cDNA clones of GFLV isolates from South Africa.

Enzyme-linked immunosorbent assay detection of Ralstonia solanacearum in potatoes: the South African experience.

Outbreaks of bacterial wilt caused by Ralstonia solanacearum in South Africa in the 1980s necessitated the development of a sensitive assay for its detection. In this chapter, the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Ralstonia solanacearum including antibody production, the ELISA method itself, potato sample preparation for testing, ELISA result validation and interpretation, and confirmation of infection are discussed. Data showing the drop in the Ralstonia solanacearum infection rate, after testing was implemented, are presented and shows how the use of this method has brought the disease under control in South Africa.

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.