Friends without benefits: Extensive cytotype sympatry and polyploid persistence in an African geophyte.

PREMISE: Polyploidy is a major factor in plant adaptation and speciation. Multiple mechanisms contribute to autopolyploid frequency within populations, but uncertainties remain regarding mechanisms that facilitate polyploid establishment and persistence. Here we aimed to document and predict cytotype distributions of Oxalis obliquifolia Steud. ex A. Rich. across Gauteng, South Africa, and test for evidence of possible mechanisms, including morphological, phenological, and reproductive traits, that may potentially facilitate polyploid persistence. METHODS: Over 320 O. obliquifolia plants from 25 sites were cytotyped using flow cytometry, and DNA ploidy was confirmed using meiotic chromosome squashes. Cytotypes were mapped and correlations with abiotic variables assessed using ordinations. To assess morphological and phenological associations with cytotype, we grew multiple cytotypes in a common garden, measured phenotypic traits and compared them using linear models and discriminant analyses. Intercytotype reproductive isolation was assessed using crossing experiments, and AMOVAs based on ITS DNA sequences tested for cytogeographic structure. RESULTS: Six cytotypes were identified, and most sites had multiple cytotypes. Abiotic variables were not predictive of cytotype distribution. A clear gigas effect was present. Differences in flower size and phenology suggested pollinator interactions could play a role in polyploid persistence. Intercytotype crosses produced seed at low frequency. DNA data suggested diploids and polyploids were largely reproductively isolated in situ, and polyploidization events were not frequent enough to explain high cytotype sympatry. CONCLUSIONS: Diploids and polyploids are behaving as separate species, despite little observable niche differentiation and non-zero potential intercytotype seed set. Tests on biotic interactions and intercytotype F1 fitness may provide insights into diploid and polyploid coexistence.

Inconsistent expression of the gigas effect in polyploid Oxalis.

PREMISE: It is well-known that whole genome duplication (WGD) has played a significant role in the evolution of plants. The best-known phenotypic effect of WGD is the gigas effect, or the enlargement of polyploid plant traits. WGD is often linked with increased weediness, which could be a result of fitness advantages conferred by the gigas effect. As a result, the gigas effect could potentially explain polyploid persistence and abundance. We test whether a gigas effect is present in the polyploid-rich geophyte Oxalis, at both organ and cellular scales. METHODS: We measured traits in conspecific diploid and polyploid accessions of 24 species across the genus. In addition, we measured the same and additional traits in 20 populations of the weedy and highly ploidy-variable species Oxalis purpurea L., including measures of clonality and selfing as a proxy for weediness. Ploidy level was determined using flow cytometry. RESULTS: We found substantial variation and no consistent ploidy-related size difference, both between and within species, and across traits. Oxalis purpurea polyploids did, however, produce significantly more underground biomass and more bulbils than diploids, consistent with a potential role of WGD in the weediness of this species. CONCLUSIONS: Our results suggest a more nuanced role for the gigas effect, at least in Oxalis. It may be temporary, short-lived, and inconsistently expressed and retained on evolutionary time scales, but in the short term can contribute to lineage success via increased vegetative reproduction.

Symbiotic yeasts from the mycangium, larval gut and woody substrate of an African stag beetle Xiphodontus antilope (Coleoptera: Lucanidae).

Female stag beetles (Lucanidae) possess internal mycangia to maintain microbial cultures. Yeasts from these mycangia may help with larval nutrition in nutrient poor woody substrates, but only a few Lucanidae taxa have been studied and all reports originate from Europe and Asia. We identify the first mycangial yeasts of a South African endemic Lucanidae beetle, Xiphodontus antilope, using nuclear ribosomal RNA and ITS DNA sequence data. In addition we identified yeasts from the larval gut, fecal matter, frass and woody substrate surrounding larvae and pupae. The mycangium of X. antilope was confined to females and is structurally similar to all other Lucanidae. Unlike most Lucanidae that seemingly associate with single species of yeast, or whose mycangia contain yeast monocultures, three yeast species were commonly isolated from X. antilope. Scheffersomyces coipomoensis was the most numerically dominant species on most substrates and in most individuals, but a second, undescribed, Scheffersomyces species was present in high numbers. A third species, also undescribed and unrelated to Scheffersomyces, was recovered from all mycangia but could not be detected in the larval gut, fecal matter, frass or woody substrates. We confirm a close association of Scheffersomyces yeasts with Lucanidae globally, but other taxa may also be involved. We show that the predominant mycangial yeasts also form the predominant yeasts within the larval gut and the woody substrates around the larvae and pupae. This combined external and internal colonization by the same yeasts may provide enhanced opportunities for nutrient acquisition, but this needs validation in future studies.

Mutations in Glucan, Water Dikinase Affect Starch Degradation and Gametophore Development in the Moss Physcomitrella patens.

The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.

Nitrogen-fixing bacteria and Oxalis - evidence for a vertically inherited bacterial symbiosis.

BACKGROUND: Plant-endophyte symbioses often revolve around nitrogen metabolism, and involve varying degrees of intimacy. Although evidence for vertical inheritance of nitrogen-fixing endophytic bacteria is increasing, it is confined mostly to crop plants, and to date no such system has been reported for geophytes. METHODS: Bacterial endophytes associated with Oxalis, the most species-rich geophytic genus form the Cape Flora in southern Africa was studied. Culturable endophytes were isolated from surface-sterilized vegetative and reproductive plant organs for six host species at three locations. Colonies of microbes on various artificial media were morphotyped, enumerated and identified using sequence data. Filter exclusion experiments were conducted to determine if endophytes were vertically transmitted to seeds, determine if mucilage plays a role to actively attract microbes from the soil and to assess microbial richness isolated from the mucilage of Oxalis seedlings. Fluorescent microscopy was implemented in order to visualize endophytic bacteria in cryo-sectioned seeds. RESULTS: Evidence for a novel, vertically transmitted symbiosis was reported. Communities of nitrogen-fixing and plant growth-promoting Bacillus endophytes were found to associate with selected Oxalis hosts from nitrogen-deficient environments of the Cape. Bacillus endophytes were ubiquitous and diverse across species and plant bodies, and were prominent in seeds. Three common nitrogen-fixing Bacillus have known oxalotrophic properties and appear to be housed inside specialised cavities (containing oxalates) within the plant body and seeds. CONCLUSIONS: The discovery of vertical transmission and potential benefits to both host and endophyte suggest a particularly tight mutualism in the Oxalis-endophyte system. This discovery suggests unexpected ways in which geophytes might avoid nitrogen deficiency, and suggest that such symbioses are more common than previously expected.

Oxalis seeds from the Cape Flora have a spectrum of germination strategies.

PREMISE: Seed germination strategy has profound ecological and evolutionary consequences, with transitions between germination strategies receiving renewed recent attention. Oxalis from the Cape Flora, South Africa, has seeds with two contrasting germination strategies: orthodox and recalcitrant. The morphological gulf between these strategies (and potential intermediate morphologies) has been poorly quantified, with questions regarding their ecological function and evolution. We reconsidered this binary classification, emphasizing potential intermediate states. METHODS: Seed physiological traits were used to assign strategies to 64 Oxalis species. We tested for morphological/phenological signal corresponding to defined strategies with cluster, principal component, K-means clustering and discriminant analyses. RESULTS: We showed that an intermediate germination strategy does exist among Cape Oxalis, with two possible morphological groups within each strategy. These could reflect a continuum of germination states, where an ancestral orthodox strategy evolved toward a maximally recalcitrant peak, with a mosaic of intermediate states reflected in extant taxa. CONCLUSIONS: Environmental factors may affect germination strategy and distribution throughout the Cape because recalcitrant and intermediate species are confined to the winter rainfall region. They occupy specialized niches and may face adverse impacts under predicted climate change (hotter and drier winters), meriting focused future conservation.

Repression of Sex4 and Like Sex Four2 Orthologs in Potato Increases Tuber Starch Bound Phosphate With Concomitant Alterations in Starch Physical Properties.

To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.

Two new Sporothrix species from Protea flower heads in South African Grassland and Savanna.

The inflorescences and infructescences of African Protea trees provide habitat for a large diversity of Sporothrix species. Here we describe two additional members, Sporothrix nsini sp. nov. and Sporothrix smangaliso sp. nov., that are associated with the infructescences of various Protea species from grasslands and savannas in the KwaZulu-Natal, North-West, Gauteng and Mpumalanga provinces of South Africa. Their description raises the number of described Protea-associated Sporothrix species to twelve. S. smangaliso sp. nov. is distantly related to other Protea-associated species and, in phylogenies using multiple markers (ITS, beta-tubulin and calmodulin), groups with taxa such as Sporothrix bragantina from Brazil and Sporothrix curviconia from the Ivory Coast. S. nsini sp. nov. resolved as sister to a clade containing four other Protea-associated species within the Sporothrix stenoceras complex. S. nsini sp. nov. was collected from within the same infructescences of Protea caffra that also contained the closely related S. africana and S. protearum. This highlights the need to study and understand the factors that influence host selection and speciation of Sporothrix in this atypical niche.

The phylogenetic significance of leaf anatomical traits of southern African Oxalis.

BACKGROUND: The southern African Oxalis radiation is extremely morphologically variable. Despite recent progress in the phylogenetics of the genus, there are few morphological synapomorphies supporting DNA-based clades. Leaflet anatomy can provide an understudied and potentially valuable source of information on the evolutionary history and systematics of this lineage. Fifty-nine leaflet anatomical traits of 109 southern African Oxalis species were assessed in search of phylogenetically significant characters that delineate clades. RESULTS: A combination of 6 leaflet anatomical traits (stomatal position, adaxial epidermal cells, abaxial epidermal cells, mesophyll, sheath around vascular tissue, degree of leaflet conduplication) clearly support various clades defined by previous DNA-based phylogenetic work. Other, mostly continuous leaflet anatomical traits were highly variable and showed less phylogenetic pattern. CONCLUSIONS: Major and unexpected findings include the transition from ancestral hypostomatic leaflets to adaxially-located stomata in the vast majority of southern African Oxalis, the loss of semi-swollen AB epidermal cells and the gain of swollen adaxial and abaxial epidermal cells in selected clades, and multiple changes from ancestral bifacial mesophyll to isobilateral or homogenous mesophyll types. The information gathered in this study will aid in the taxonomic revision of this speciose member of the Greater Cape Floristic Region and provide a basis for future hypotheses regarding its radiation.

Species-rich and polyploid-poor: Insights into the evolutionary role of whole-genome duplication from the Cape flora biodiversity hotspot.

PREMISE OF THE STUDY: Whole-genome duplication (WGD) in angiosperms has been hypothesized to be advantageous in unstable environments and/or to increase diversification rates, leading to radiations. Under the first hypothesis, floras in stable environments are predicted to have lower proportions of polyploids than highly, recently disturbed floras, whereas species-rich floras would be expected to have higher than expected proportions of polyploids under the second. The South African Cape flora is used to discriminate between these two hypotheses because it features a hyperdiverse flora predominantly generated by a limited number of radiations (Cape clades), against a backdrop of climatic and geological stability. METHODS: We compiled all known chromosome counts for species in 21 clades present in the Cape (1653 species, including 24 Cape clades), inferred ploidy levels for these species by inspection or derived from the primary literature, and compared Cape to non-Cape ploidy levels in these clades (17,520 species) using G tests. KEY RESULTS: The Cape flora has anomalously low proportions of polyploids compared with global levels. This pattern is consistently observed across nearly half the clades and across global latitudinal gradients, although individual lineages seem to be following different paths to low levels of WGD and to differing degrees. CONCLUSIONS: This pattern shows that the diversity of the Cape flora is the outcome of primarily diploid radiations and supports the hypothesis that WGD may be rare in stable environments.

Wounds on Rapanea melanophloeos provide habitat for a large diversity of Ophiostomatales including four new species.

Rapanea melanophloeos, an important canopy tree in Afromontane forests, is commonly utilised for medicinal bark harvesting. Wounds created from these activities provide entrance for many fungi, including arthropod-associated members of the Ophiostomatales and Microascales (ophiostomatoid fungi). In this study we assessed the diversity of wound-associated Ophiostomatales on storm-damaged R. melanophloeos trees in the Afromontane forests of South Africa. Five species were identified based on micro-morphological and molecular phylogenetic analyses. These included Ophiostoma stenoceras and four newly described taxa Sporothrix itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov. and O. noisomeae sp. nov. Four of these are members of the S. schenckii-O. stenoceras complex (O. stenoceras, S. itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov.) while O. noisomeae groups basal in the Ophiostomatales alongside the S. lignivora complex and Graphilbum. In addition to other taxa known from this host, the present study shows that there is a rich, yet still poorly explored, diversity of Ophiostomatales associated with R. melanophloeos in Afromontane forests. More taxa are likely to be discovered with increased research effort. These must be assessed in terms of pathogenicity towards this ecologically and economically important tree.

Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae).

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb-Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb-Seq in phylogenetics.

New species of Ophiostomatales from Scolytinae and Platypodinae beetles in the Cape Floristic Region, including the discovery of the sexual state of Raffaelea.

Olea capensis and Rapanea melanophloeos are important canopy trees in South African Afromontane forests. Dying or recently dead individuals of these trees are often infested by Scolytinae and Platypodinae (Curculionidae) beetles. Fungi were isolated from the surfaces of beetles emerging from wood samples and their galleries. Based on micro-morphological and phylogenetic analyses, four fungal species in the Ophiostomatales were isolated. These were Sporothrix pallida and three taxa here newly described as Sporothrix aemulophila sp. nov., Raffaelea vaginata sp. nov. and Raffaelea rapaneae sp. nov. This study represents the first collection of S. pallida, a species known from many environmental samples from across the world, from Scolytinae beetles. S. aemulophila sp. nov. is an associate of the ambrosia beetle Xyleborinus aemulus. R. rapaneae sp. nov. and R. vaginata sp. nov. were associated with a Lanurgus sp. and Platypodinae beetle, respectively, and represent the first Raffaelea spp. reported from the Cape Floristic Region. Of significance is that R. vaginata produced a sexual state analogous with those of Ophiostoma seticolle and O. deltoideosporum that also grouped in our analyses in Raffaelea s. str., to date considered an asexual genus. The morphology of the ossiform ascospores and anamorphs of the three species corresponded and the generic circumscription of Raffaelea is thus emended to accommodate sexual states. The two known species are provided with new combinations, namely Raffaelea seticollis (R.W. Davidson) Z.W. de Beer and T.A. Duong comb. nov. and Raffaelea deltoideospora (Olchow. and J. Reid) Z.W. de Beer and T.A. Duong comb. nov.

High ploidy diversity and distinct patterns of cytotype distribution in a widespread species of Oxalis in the Greater Cape Floristic Region.

BACKGROUND AND AIMS: Genome duplication is widely acknowledged as a major force in the evolution of angiosperms, although the incidence of polyploidy in different floras may differ dramatically. The Greater Cape Floristic Region of southern Africa is one of the world's biodiversity hotspots and is considered depauperate in polyploids. To test this assumption, ploidy variation was assessed in a widespread member of the largest geophytic genus in the Cape flora: Oxalis obtusa. METHODS: DNA flow cytometry complemented by confirmatory chromosome counts was used to determine ploidy levels in 355 populations of O. obtusa (1014 individuals) across its entire distribution range. Ecological differentiation among cytotypes was tested by comparing sets of vegetation and climatic variables extracted for each locality. KEY RESULTS: Three majority (2x, 4x, 6x) and three minority (3x, 5x, 8x) cytotypes were detected in situ, in addition to a heptaploid individual originating from a botanical garden. While single-cytotype populations predominate, 12 mixed-ploidy populations were also found. The overall pattern of ploidy level distribution is quite complex, but some ecological segregation was observed. Hexaploids are the most common cytotype and prevail in the Fynbos biome. In contrast, tetraploids dominate in the Succulent Karoo biome. Precipitation parameters were identified as the most important climatic variables associated with cytotype distribution. CONCLUSIONS: Although it would be premature to make generalizations regarding the role of genome duplication in the genesis of hyperdiversity of the Cape flora, the substantial and unexpected ploidy diversity in Oxalis obtusa is unparalleled in comparison with any other cytologically known native Cape plant species. The results suggest that ploidy variation in the Greater Cape Floristic Region may be much greater than currently assumed, which, given the documented role of polyploidy in speciation, has direct implications for radiation hypotheses in this biodiversity hotspot.

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.

New primers for single-copy nuclear-encoded chloroplast-expressed glutamine synthetase (ncpGS) in Oxalidaceae.

PREMISE OF THE STUDY: New primers were developed for the nuclear marker glutamine synthetase (ncpGS) in Oxalidaceae. • METHODS AND RESULTS: New forward and reverse primers were designed and tested across a wide range of Oxalidaceae. Selected taxa were sequenced to confirm homology. Potential for phylogenetic study was assessed by comparing sequenced taxa with commonly used nuclear and plastid markers. • CONCLUSIONS: Four out of five Oxalidaceae genera and all tested Oxalis spp. amplified successfully. Sequencing confirmed homology of the amplicon. Parsimony analysis of ncpGS showed that it is a promising candidate for future phylogenetic work in Oxalidaceae.

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.