An approach to determining anthocyanin synthesis enzyme gene expression in an evolutionary context: an example from Erica plukenetii.

BACKGROUND AND AIMS: Floral colour in angiosperms can be controlled by variations in the expression of the genes of the anthocyanin pathway. Floral colour shifts influence pollinator specificity. Multiple shifts in floral colour occurred in the diversification of the genus Erica (Ericaceae), from plesiomorphic pink to, for example, red or white flowers. Variation in anthocyanin gene expression and its effects on floral colour in the red-, pink- and white-flowered Erica plukenetii species complex was investigated. METHODS: Next generation sequencing, reverse transcriptase PCR and real-time reverse transcriptase quantitative PCR were used to quantify anthocyanin gene expression. KEY RESULTS: Non-homologous mutations causing loss of expression of single genes were found, indicating that the cause was likely to be mutations in transcription factor binding sites upstream of the 5'-untranslated region of the genes, and this was confirmed by sequencing. CONCLUSIONS: Independent evolution and subsequent loss of expression of anthocyanin genes may have influenced diversification in the E. plukenetii species complex. The approach developed here should find more general application in studies on the role of floral colour shifts in diversification.

The biodiversity hotspot as evolutionary hot-bed: spectacular radiation of Erica in the Cape Floristic Region.

BACKGROUND: The disproportionate species richness of the world's biodiversity hotspots could be explained by low extinction (the evolutionary "museum") and/or high speciation (the "hot-bed") models. We test these models using the largest of the species rich plant groups that characterise the botanically diverse Cape Floristic Region (CFR): the genus Erica L. We generate a novel phylogenetic hypothesis informed by nuclear and plastid DNA sequences of c. 60 % of the c. 800 Erica species (of which 690 are endemic to the CFR), and use this to estimate clade ages (using RELTIME; BEAST), net diversification rates (GEIGER), and shifts in rates of diversification in different areas (BAMM; MuSSE). RESULTS: The diversity of Erica species in the CFR is the result of a single radiation within the last c. 15 million years. Compared to ancestral lineages in the Palearctic, the rate of speciation accelerated across Africa and Madagascar, with a further burst of speciation within the CFR that also exceeds the net diversification rates of other Cape clades. CONCLUSIONS: Erica exemplifies the "hotbed" model of assemblage through recent speciation, implying that with the advent of the modern Cape a multitude of new niches opened and were successively occupied through local species diversification.

Testing reticulate versus coalescent origins of Erica lusitanica using a species phylogeny of the northern heathers (Ericeae, Ericaceae).

Whilst most of the immense species richness of heathers (Calluna, Daboecia and Erica: Ericeae; Ericaceae) is endemic to Africa, particularly the Cape Floristic Region, the oldest lineages are found in the Northern Hemisphere. We present phylogenetic hypotheses for the major clades of Ericeae represented by multiple accessions of all northern Erica species and placeholder taxa for the large nested African/Madagascan clade. We identified consistent, strongly supported conflict between gene trees inferred from ITS and chloroplast DNA sequences with regard to the position of Erica lusitanica. We used coalescent simulations to test whether this conflict could be explained by coalescent stochasticity, as opposed to reticulation (e.g. hybridisation), given estimates of clade ages, generation time and effective population sizes (Ne). A standard approach, comparing overall differences between real and simulated trees, could not clearly reject coalescence. However, additional simulations showed that at the (higher) Ne necessary to explain conflict in E. lusitanica, further topological conflict would also be expected. Ancient hybridisation between ancestors of northern species is therefore a plausible scenario to explain the origin of E. lusitanica, and its morphological similarities to E. arborea. Assuming either process influences the results of species tree and further evolutionary inference. The coalescence scenario is equivocal with regard the standing hypothesis of stepping stone dispersal of Erica from Europe into Africa; whereas reticulate evolution in E. lusitanica would imply that the colonisation of Tropical East Africa by E. arborea instead occurred independently of dispersals within the rest of the African/Madagascan clade.

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5'UTR.

Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5' ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5'UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.

A model of bulb evolution in the eudicot genus Oxalis (Oxalidaceae).

The origins and monophyly of the bulbous habit in the eudicot genus Oxalis are uncertain, but key character state transitions in the evolution of true bulbs are currently thought to be reflected in extant pseudobulbous and other geophytic taxa. We test the relationships between the two major groups of bulbous Oxalis taxa, namely the southern African lineage which is centered in the speciose Cape Floristic Region (CFR), and the New World section Ionoxalis, by including the rhizomatous geophyte Oxalis acetosella, the caudiciform stem succulent Oxalis articulata, and the rhizomiform pseudobulbous Oxalis triangularis, in combined phylogenetic analyses of nrITS and trnL-F sequence data. We optimize several key bulbous characters in ancestral state reconstructions on produced phylogenies. Results of our analyses indicate that the evolution of bulbous characters in the genus is more complex than previously thought. Although the two major bulb types are homologous, the rhizomiform pseudobulbous habit arises from within true bulbs, and in most reconstructions the caudiciform stem succulent O. articulata is inferred to have secondarily lost several distinctive bulbous characters. O. acetosella is not as closely related to the bulbous lineage as previously thought. More sampling from other key taxa are needed before the order in which key bulbous characters were acquired can be verified. We discuss these results in terms of the taxonomic and ecological implications for the CFR Oxalis taxa.

Phylogenetic relationships, character evolution and biogeography of southern African members of Zygophyllum (Zygophyllaceae) based on three plastid regions.

The plastid coding rbcL and non-coding trnLF regions of 53 of 55 southern African Zygophyllum species were sequenced and used to evaluate the phylogenetic relationships within the southern African representatives of the genus. Published sequences of the same gene regions of Australian, Asian and North African Zygophyllum species were included to assess the relationships of the species from these regions to the southern African species. The addition of Z. stapffii from Namibia, found to be conspecific with Z. orbiculatum from Angola, lead to a greatly resolved tree. The molecular results were largely congruent with a recent sectional classification of the southern African species and supported their subdivision into subgenera Agrophyllum and Zygophyllum. Reconstruction of the character evolution of capsule dehiscence, seed attachment and seed mucilage showed that these characters allowed a division of southern African species into the two subgenera but that this could not be applied to species occurring elsewhere. Other morphological characters were found to vary and unique character combinations, rather than unique characters, were found to be of systematic value in sectional delimitation. The study suggests that repeated radiations from the horn of Africa to southern Africa and Asia and back lead to the present distribution of the taxa in the subfamily Zygophylloideae. Although this study supports some of the recent taxonomic changes in the group, the unresolved relationships between the proposed genera Tetraena and Roepera and those retained as Zygophyllum species suggest that changes to the taxonomy may have been premature.

Identification of three novel mycoplasma species from ostriches in South Africa.

Mycoplasmas have been implicated in certain clinical syndromes in ostriches and are associated with upper respiratory tract infections. As these infections result in production losses, they are of considerable economic importance to the South African ostrich industry. Although poultry mycoplasmas have been shown to infect ostriches, the existence of unique ostrich-specific mycoplasmas has been suggested. In this study, mycoplasmas were isolated from ostriches in the Klein Karoo, Central Karoo and Garden Route areas of the Western and Northern Cape Provinces of South Africa and identified using 16S rRNA gene sequencing. These sequences indicated that ostriches in these areas carry three unique mycoplasmas and were not infected with chicken mycoplasmas. Phylogenetic analysis of the 16S rRNA sequences of the three isolated ostrich mycoplasmas showed them to be quite divergent and to fall into two distinct phylogenetic groupings. Unique sequences within the 16S rRNA gene of the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of mycoplasma infections in ostriches. Chickens kept in close proximity to infected ostriches were not infected with these ostrich mycoplasmas.

Refugia, dispersal and divergence in a forest archipelago: a study of Streptocarpus in eastern South Africa.

We describe a scenario of plant speciation across a relict forest archipelago in South Africa involving Pleistocene habitat expansion-contraction cycles, dispersal and adaptation to lower temperatures. This is the first population level study using molecular data in South African forests and has significant implications for conservation efforts in this area. Populations of the mesophytic forest floor herbs Streptocarpus primulifolius sensu lato and Streptocarpus rexii were sampled throughout their range in the naturally fragmented forests of eastern South Africa in order to investigate population genetic and phylogenetic patterns within the species complex, using nuclear microsatellites, nuclear ribosomal ITS (internal transcribed spacer) sequences and chloroplast genome sequences. S. primulifolius harbours high levels of genetic diversity at both the nuclear (mean HE = 0.50) and the chloroplast level (each population fixed for a unique haplotype). This is consistent with populations of these coastal species being Pleistocene relicts. In contrast, populations of S. rexii in cooler habitats at higher altitudes and lower latitudes harbour little or no nuclear genetic diversity (mean HE = 0.09) and most share a common chloroplast haplotype. The split of S. rexii from populations intermediate between the two species (S. cf. primulifolius) occurred between 0 and 0.44 million years ago according to the calibrated ITS phylogeny of the taxa. The low genetic diversity and homogeneity of S. rexii is congruent with this species having reached its current range during the Holocene. We found no evidence of monophyly of any of the taxa in this study, which we consider a consequence of recent evolution in a fragmented habitat.

Phylogenetic relationships in Disa based on non-coding trnL-trnF chloroplast sequences: evidence of numerous repeat regions.

Sequence data from the intron and spacer of the trnL-F chloroplast region elucidate the phylogenetic relationships of the tribe Diseae (Orchidoideae: Orchidaceae). Within Diseae, 41 species of Disa, two of Brownleea, three of Satyrium, and two of Corycium were included, with five species of Habenaria sensu lato (Orchideae) and one epidendroid as outgroups. The sequences revealed substitutions and considerable length variation, due mainly to the presence of repeat motifs. Phylogenetic analysis using parsimony revealed five distinct clades. The branching order of the five weakly supported the paraphyly of Diseae, with the successive divergence of Brownleea, Corycium, Habenaria, Satyrium, and Disa. Within the monophyletic Disa, three main groupings appeared, two strongly supported clades representing sect. Racemosae and sect. Coryphaea and the third grouping containing several clades currently grouped into sections based on morphological phylogenies. Some discrepancies between the molecular phylogeny and the phylogeny based on morphological characters may require reevaluation of some of the morphological characters. The presence of different numbers of repeat motifs, both among different taxa and within taxa, indicates that these characters may be phylogenetically informative at the population level.

Antibody responses to La Sota strain vaccines of Newcastle disease virus in ostriches (Struthio camelus) as detected by enzyme-link ed immunosorbent assay.

Because of the fact that South Africa is a Newcastle disease virus (NDV)-endemic country, major concerns exist that the export of ostrich meat could transmit velogenic strains of this disease. The ability to transmit the virus could be reduced by effective vaccination of South African ostriches. In this study, two vaccination trials were conducted to assess serum antibody production in response to vaccination with La Sota strain NDV vaccines. To this end, a commercially available chicken anti-NDV enzyme-linked immunosorbent assay (ELISA) was modified for the detection of anti-NDV antibodies in ostrich serum. The results obtained with this ELISA were verified by comparison with an indirect ELISA. In the first trial, ostriches were immunized subcutaneously four times with different volumes of an inactivated vaccine and their immune response was determined from 2.5 mo up to the ideal slaughter age of 14 mo. Results indicated that ostriches responded in a dose-dependent manner and gave support for the vaccination schedule currently recommended to South African farmers. In a second trial, immunization by eyedrop with a live La Sota vaccine of 5-wk-old ostriches did not elicit a humoral immune response. The results indicate that it is highly unlikely that ostriches that have been vaccinated according to the recommended vaccination schedule can transmit the virus.

The effect of cytochrome b5 on progesterone metabolism in the ovine adrenal.

The production of glucocorticoids and mineralocorticoids in the endoplasmic reticulum (ER) of the mammalian adrenal cortex are, to a great extent, regulated by the relative activities of the steroid 21-hydroxylase (P450c21) and steroid 17 alpha-hydroxylase (P450c17) enzymes. Progesterone can be 17 alpha-hydroxylated to yield 17 alpha-hydroxyprogesterone which, under certain conditions and in certain species, can be further lyased to adrostenedione by the same enzyme. P450c21 can 21-hydroxylate 17 alpha-hydroxyprogesterone to yield cortisol but also converts progesterone to corticosterone. Cytochrome b5 (cyt b5) can also participate in the regulation of adrenal microsomal steroid hydroxylase activities by changing the rates of the P450c21 and P450c17 reactions or by affecting the 17 alpha-hydroxylation:17,20-lyase ratio of progesterone, by P450c17. We investigated the metabolism of progesterone by sheep adrenal microsomes to identify the products of the different steroid hydroxylase activities in the ER and to investigate the influence of cyt b5 on progesterone metabolism using purified ovine cyt b5 and anti-cyt b5. The P450c17-activity in sheep adrenal microsomes is inhibited by the addition of purified cyt b5 while anti-cyt b5 IgG stimulates the 17 alpha-hydroxylation of progesterone. No 17,21-lyase-activity towards progesterone could be detected in sheep adrenal microsomes.

Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells.

In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.

The production of highly specific anti-testosterone antisera using acid-treated bacteria as immunogenic carrier.

A novel and effective procedure for the production of highly specific anti-testosterone antibodies is described. It involved pretreatment of experimental animals with tolerogens followed by immunization with conjugates of testosterone covalently linked to acid-treated Salmonella minnesota R595 bacteria as immunogenic carriers. Antibodies elicited by this procedure showed minimal cross-reactivity towards 5 alpha-dihydrotestosterone and some of them were successfully used in radioimmunoassays for the determination of serum testosterone levels, even without the need for prior extraction.

Human serum amyloid A protein. Behaviour in aqueous and urea-containing solutions and antibody production.

Human serum amyloid A protein (apo-SAA) can be prepared by gel filtration of delipidated acute-phase high-density lipoprotein in the presence of urea. The resultant apo-SAA is soluble (greater than 90% solubility) in a wide range of buffer solutions, with all of the six major isoforms of apo-SAA being equally soluble. In urea-containing solutions the isoforms behave qualitatively differently in various urea concentrations, probably reflecting subtle primary-structure variations. The higher-pI isoforms are only completely unfolded at greater than 7 M-urea. By immunizing with apo-SAA adsorbed to acid-treated bacteria (Salmonella minnesota R595), high-titre antibodies can easily be elicited in rabbits.

Immune carrier properties of acid-treated Salmonella minnesota R595 bacteria. The immune response to TNP-bacterial conjugates in rabbits and mice.

Salmonella minnesota R595 bacteria from which the core region of the lipopolysaccharide on the cell wall had previously been removed by mild acid treatment were trinitrophenylated. Differing amounts of these trinitrophenyl naked bacterial conjugates (TNP-NB), covering a range of epitope densities, were used for immunising mice and rabbits via the intraperitoneal or intravenous routes without adjuvants. It was found that such acid-treated, naked bacteria were effective carriers for the covalently linked hapten, TNP, with an optimum epitope density of 15 micrograms TNP/mg NB. Significant immune responses were obtained with dose levels as low as 50 ng TNP. The possible applications of acid-treated, naked bacteria as universal carriers having inherent adjuvant activity are discussed.

Acid-treated, naked bacteria as immune carriers for protein antigens.

Salmonella minnesota R595 bacteria were stripped of their natural antigenic determinants to yield acid-treated, naked bacteria. The proteins, human apolipoprotein A1 and carcino-embryonic antigen, were adsorbed to naked bacteria and these complexes were used to immunise rabbits. Although the antibody titres obtained were comparable to those achieved using Freund's adjuvant emulsions, much less antigen was needed for immunisation. This technique could be of great value where the amount of protein available for immunisation is very small.