Effect of poly(acrylic acid) molecular mass and end-group functionality on calcium oxalate crystal morphology and growth

A number of series of poly(acrylic acids) (PAA) of differing end-groups and molecular mass were used to study the inhibition of calcium oxalate crystallization. The effects of the end-group on crystal speciation and morphology were significant and dramatic, with hexyl-isobutyrate end groups giving preferential formation of calcium oxalate dihydrate (COD) rather than the more stable calcium oxalate monohydrate (COM), while both more hydrophobic end-groups and less-hydrophobic end groups led predominantly to formation of the least thermodynamically stable form of calcium oxalate, calcium oxalate trihydrate. Conversely, molecular mass had little impact on calcium oxalate speciation or crystal morphology. It is probable that the observed effects are related to the rate of desorption of the PAA moiety from the crystal (lite) surfaces and that the results point to a major role for end-group as well as molecular mass in controlling desorption rate.

An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed.

Petrology and geochemistry of the Quaternary Caldera-forming, Phonolitic Granadilla Eruption, Tenerife (Canary Islands)

The Granadilla eruption at 600 ka was one of the largest phonolitic explosive eruptions from the Las Cañadas volcano on Tenerife, producing a classical plinian eruptive sequence of a widespread pumice fall deposit overlain by an ignimbrite. The eruption resulted in a major phase of caldera collapse that probably destroyed the shallow-level magma chamber system. Granadilla pumices contain a diverse phenocryst assemblage of alkali feldspar + biotite + sodian diopside to aegirine–augite + titanomagnetite + ilmenite + nosean/haüyne + titanite + apatite; alkali feldspar is the dominant phenocryst and biotite is the main ferromagnesian phase. Kaersutite and partially resorbed plagioclase (oligoclase to sodic andesine) are present in some eruptive units, particularly in pumice erupted during the early plinian phase, and in the Granadilla ignimbrite at the top of the sequence. Associated with the kaersutite and plagioclase are small clots of microlitic plagioclase and kaersutite interpreted as quenched blebs of tephriphonolitic magma within the phonolite pumice. The Granadilla Member has previously been recognized as an example of reverse-then-normal compositional zonation, where the zonation is primarily expressed in terms of substantial variations in trace element abundances with limited major element variation (cryptic zonation). Evidence for cryptic zonation is also provided by the chemistry of the phenocryst phases, and corresponding changes in intensive parameters (e.g. T, f O2, f  H2O). Geothermometry estimates indicate that the main body of phonolite magma had a temperature gradient from 860 °C to ∼790 °C, with hotter magma (≥900 °C) tapped at the onset and terminal phases of the eruption. The reverse-then-normal chemical and thermal zonation reflects the initial tapping of a partially hybridized magma (mixing of phonolite and tephriphonolite), followed by the more sequential tapping of a zoned and relatively large body of highly evolved phonolite at a new vent and during the main plinian phase. This suggests that the different magma types within the main holding chamber could have been laterally juxtaposed, as well as in a density-stratified arrangement. Correlations between the presence of mixed phenocryst populations (i.e. presence of plagioclase and kaersutite) and coarser pumice fall layers suggest that increased eruption vigour led to the tapping of hybridized and/or less evolved magma probably from greater depths in the chamber. New oxygen isotope data for glass and mineral separates preclude syn-eruptive interaction between the vesiculating magma and hydrothermal fluids as the cause of the Sr isotope disequilibrium identified previously for the deposit. Enrichment in radiogenic Sr in the pumice glass has more likely been due to low-temperature exchange with meteoric water that was enriched in 87Sr by sea spray, which may be a common process affecting porous and glassy pyroclastic deposits on oceanic islands.

Systematics and biogeography of the Gondwanan Orthocladiinae (Diptera: Chironomidae)

Restrictions to effective dispersal and gene flow caused by the fragmentation of ancient supercontinents are considered to have driven diversification and speciation on disjunct landmasses globally. Investigating the role that these processes have played in the development of diversity within and among taxa is crucial to understanding the origins and evolution of regional biotas. Within the chironomid (non-biting midge) subfamily Orthocladiinae (Diptera: Chironomidae), a group of genera that are distributed across the austral continents (Australia, New Zealand, South America) have been proposed to represent a relict Gondwanan clade. We used a molecular approach to resolve relationships among taxa with the aim to determine the relative roles that vicariance and dispersal may have played in the evolution of this group. Continental biotas did not form monophyletic groups, in accordance with expectations given existing morphological evidence. Patterns of phylogenetic relationships among taxa did not accord with expected patterns based on the geological sequence of break-up of the Gondwanan supercontinent. Likewise, divergence time estimates, particularly for New Zealand taxa, largely post-dated continental fragmentation and implied instead that several transoceanic dispersal events may have occurred post-vicariance. Passive dispersal of gravid female chironomid adults is the most likely mechanism for transoceanic movement, potentially facilitated by West Wind Drift or anti-cyclone fronts. Estimated timings of divergence among Australian and South American Botryocladius, on the other hand, were congruent with the proposed ages of separation of the two continents from Antarctica. Taken together, these data suggest that a complex relationship between both vicariance and dispersal may explain the evolution of this group. The sampling regime we implemented here was the most intensive yet performed for austral members of the Orthocladiinae and unsurprisingly revealed several novel taxa that will require formal description.

Evolutionary biology of Gondwanan non-biting midges (Diptera: Chironomidae)

The potential restriction to effective dispersal and gene flow caused by habitat fragmentation can apply to multiple levels of evolutionary scale; from the fragmentation of ancient supercontinents driving diversification and speciation on disjunct landmasses, to the isolation of proximate populations as a result of their inability to cross intervening unsuitable habitat. Investigating the role of habitat fragmentation in driving diversity within and among taxa can thus include inferences of phylogenetic relationships among taxa, assessments of intraspecific phylogeographic structure and analyses of gene flow among neighbouring populations. The proposed Gondwanan clade within the chironomid (non-biting midge) subfamily Orthocladiinae (Diptera: Chironomidae) represents a model system for investigating the role that population fragmentation and isolation has played at different evolutionary scales. A pilot study by Krosch et al (2009) indentified several highly divergent lineages restricted to ancient rainforest refugia and limited gene flow among proximate sites within a refuge for one member of this clade, Echinocladius martini Cranston. This study provided a framework for investigating the evolutionary history of this taxon and its relatives more thoroughly. Populations of E. martini were sampled in the Paluma bioregion of northeast Queensland to investigate patterns of fine-scale within- and among-stream dispersal and gene flow within a refuge more rigorously. Data was incorporated from Krosch et al (2009) and additional sites were sampled up- and downstream of the original sites. Analyses of genetic structure revealed strong natal site fidelity and high genetic structure among geographically proximate streams. Little evidence was found for regular headwater exchange among upstream sites, but there was distinct evidence for rare adult flight among sites on separate stream reaches. Overall, however, the distribution of shared haplotypes implied that both larval and adult dispersal was largely limited to the natal stream channel. Patterns of regional phylogeographic structure were examined in two related austral orthoclad taxa – Naonella forsythi Boothroyd from New Zealand and Ferringtonia patagonica Sæther and Andersen from southern South America – to provide a comparison with patterns revealed in their close relative E. martini. Both taxa inhabit tectonically active areas of the southern hemisphere that have also experienced several glaciation events throughout the Plio-Pleistocene that are thought to have affected population structure dramatically in many taxa. Four highly divergent lineages estimated to have diverged since the late Miocene were revealed in each taxon, mirroring patterns in E. martini; however, there was no evidence for local geographical endemism, implying substantial range expansion post-diversification. The differences in pattern evident among the three related taxa were suggested to have been influenced by variation in the responses of closed forest habitat to climatic fluctuations during interglacial periods across the three landmasses. Phylogeographic structure in E. martini was resolved at a continental scale by expanding upon the sampling design of Krosch et al (2009) to encompass populations in southeast Queensland, New South Wales and Victoria. Patterns of phylogeographic structure were consistent with expectations and several previously unrecognised lineages were revealed from central- and southern Australia that were geographically endemic to closed forest refugia. Estimated divergence times were congruent with the timing of Plio-Pleistocene rainforest contractions across the east coast of Australia. This suggested that dispersal and gene flow of E. martini among isolated refugia was highly restricted and that this taxon was susceptible to the impacts of habitat change. Broader phylogenetic relationships among taxa considered to be members of this Gondwanan orthoclad group were resolved in order to test expected patterns of evolutionary affinities across the austral continents. The inferred phylogeny and estimated divergence times did not accord with expected patterns based on the geological sequence of break-up of the Gondwanan supercontinent and implied instead several transoceanic dispersal events post-vicariance. Difficulties in appropriate taxonomic sampling and accurate calibration of molecular phylogenies notwithstanding, the sampling regime implemented in the current study has been the most intensive yet performed for austral members of the Orthocladiinae and unsurprisingly has revealed both novel taxa and phylogenetic relationships within and among described genera. Several novel associations between life stages are made here for both described and previously unknown taxa. Investigating evolutionary relationships within and among members of this clade of proposed Gondwanan orthoclad taxa has demonstrated that a complex interaction between historical population fragmentation and dispersal at several levels of evolutionary scale has been important in driving diversification in this group. While interruptions to migration, colonisation and gene flow driven by population fragmentation have clearly contributed to the development and maintenance of much of the diversity present in this group, long-distance dispersal has also played a role in influencing diversification of continental biotas and facilitating gene flow among disjunct populations.

Development of a high-level gene expression system for the production of bioplastics in sugarcane

Despite various approaches, the production of biodegradable plastics such as polyhydroxybutyrate (PHB) in transgenic plants has met with limited success due largely to low expression levels. Even in the few instances where high levels of protein expression have been reported, the transgenic plants have been stunted indicating PHB is phytotoxic (Poirier 2002). This PhD describes the application of a novel virus-based gene expression technology, termed InPAct („In Plant Activation.), for the production of PHB in tobacco and sugarcane. InPAct is based on the rolling circle replication mechanism by which circular ssDNA viruses replicate and provides a system for controlled, high-level gene expression. Based on these features, InPAct was thought to represent an ideal system to enable the controlled, high-level expression of the three phb genes (phbA, phbB and phbC) required for PHB production in sugarcane at a preferred stage of plant growth. A Tobacco yellow dwarf virus (TbYDV)-based InPAct-phbA vector, as well as linear vectors constitutively expressing phbB and phbC were constructed and different combinations were used to transform tobacco leaf discs. A total of four, eight, three and three phenotypically normal tobacco lines were generated from discs transformed with InPAct-phbA, InPAct-phbA + p1300-TaBV P-phbB/phbC- 35S T, p1300-35S P-phbA-NOS T + p1300-TaBV P-phbB/phbC-35S T and InPAct-GUS, respectively. To determine whether the InPAct cassette could be activated in the presence of the TbYDV Rep, leaf samples from the eight InPActphbA + p1300-TaBV P-phbB/phbC-35S T plants were agroinfiltrated with p1300- TbYDV-Rep/RepA. Three days later, successful activation was indicated by the detection of episomes using both PCR and Southern analysis. Leaf discs from the eight InPAct-phbA + p1300-TaBV P-phbB/phbC-35S T transgenic plant lines were agroinfiltrated with p1300-TbYDV-Rep/RepA and leaf tissue was collected ten days post-infiltration and examined for the presence of PHB granules. Confocal microscopy and TEM revealed the presence of typical PHB granules in five of the eight lines, thus demonstrating the functionality of InPActbased PHB production in tobacco. However, analysis of leaf extracts by HPLC failed to detect the presence of PHB suggesting only very low level expression levels. Subsequent molecular analysis of three lines revealed low levels of correctly processed mRNA from the catalase intron contained within the InPAct cassette and also the presence of cryptic splice sites within the intron. In an attempt to increase expression levels, new InPAct-phb cassettes were generated in which the castorbean catalase intron was replaced with a synthetic intron (syntron). Further, in an attempt to both increase and better control Rep/RepA-mediated activation of InPAct cassettes, Rep/RepA expression was placed under the control of a stably integrated alc switch. Leaf discs from a transgenic tobacco line (Alc ML) containing 35S P-AlcR-AlcA P-Rep/RepA were supertransformed with InPAct-phbAsyn or InPAct-GUSsyn using Agrobacterium and three plants (lines) were regenerated for each construct. Analysis of the RNA processing of the InPAct-phbAsyn cassette revealed highly efficient and correct splicing of the syntron, thus supporting its inclusion within the InPAct system. To determine the efficiency of the alc switch to activate InPAct, leaf material from the three Alc ML + InPAct-phbAsyn lines was either agroinfiltrated with 35S P-Rep/RepA or treated with ethanol. Unexpectedly, episomes were detected not only in the infiltrated and ethanol treated samples, but also in non-treated samples. Subsequent analysis of transgenic Alc ML + InPAct-GUS lines, confirmed that the alc switch was leaky in tissue culture. Although this was shown to be reversible once plants were removed from the tissue culture environment, it made the regeneration of Alc ML + InPAct-phbsyn plant lines extremely difficult, due to unintentional Rep expression and therefore high levels of phb expression and phytotoxic PHB production. Two Alc ML + InPAct-phbAsyn + p1300-TaBV P-phbB/phbC-35S T transgenic lines were able to be regenerated, and these were acclimatised, alcohol-treated and analysed. Although episome formation was detected as late as 21 days post activation, no PHB was detected in the leaves of any plants using either microscopy or HPLC, suggesting the presence of a corrupt InPAct-phbA cassette in both lines. The final component of this thesis involved the application of both the alc switch and the InPAct systems to sugarcane in an attempt to produce PHB. Initial experiments using transgenic Alc ML + InPAct-GUS lines indicated that the alc system was not functional in sugarcane under the conditions tested. The functionality of the InPAct system, independent of the alc gene switch, was subsequently examined by bombarding the 35S Rep/RepA cassette into leaf and immature leaf whorl cells derived from InPAct-GUS transgenic sugarcane plants. No GUS expression was observed in leaf tissue, whereas weak and irregular GUS expression was observed in immature leaf whorl tissue derived from two InPAct- GUS lines and two InPAct-GUS + 35S P-AlcR-AlcA P-GUS lines. The most plausible reason to explain the inconsistent and low levels of GUS expression in leaf whorls is a combination of low numbers of sugarcane cells in the DNA replication-conducive S-phase and the irregular and random nature of sugarcane cells bombarded with Rep/RepA. This study details the first report to develop a TbYDV-based InPAct system under control of the alc switch to produce PHB in tobacco and sugarcane. Despite the inability to detect quantifiable levels of PHB levels in either tobacco or sugarcane, the findings of this study should nevertheless assist in the further development of both the InPAct system and the alc system, particularly for sugarcane and ultimately lead to an ethanol-inducible InPAct gene expression system for the production of bioplastics and other proteins of commercial value in plants.

Controls on iron in soils and soil waters of a forested, coastal catchment in subtropical Australia

Soluble organic matter derived from exotic Pinus vegetation forms stronger complexes with iron (Fe) than the soluble organic matter derived from most native Australian species. This has lead to concern about the environmental impacts related to the establishment of extensive exotic Pinus plantations in coastal southeast Queensland, Australia. It has been suggested that the Pinus plantations may enhance the solubility of Fe in soils by increasing the amount of organically complexed Fe. While this remains inconclusive, the environmental impacts of an increased flux of dissolved, organically complexed Fe from soils to the fluvial system and then to sensitive coastal ecosystems are potentially damaging. Previous work investigated a small number of samples, was largely laboratory based and had limited application to field conditions. These assessments lacked field-based studies, including the comparison of the soil water chemistry of sites associated with Pinus vegetation and undisturbed native vegetation. In addition, the main controls on the distribution and mobilisation of Fe in soils of this subtropical coastal region have not been determined. This information is required in order to better understand the relative significance of any Pinus enhanced solubility of Fe. The main aim of this thesis is to determine the controls on Fe distribution and mobilisation in soils and soil waters of a representative coastal catchment in southeast Queensland (Poona Creek catchment, Fraser Coast) and to test the effect of Pinus vegetation on the solubility and speciation of Fe. The thesis is structured around three individual papers. The first paper identifies the main processes responsible for the distribution and mobilisation of labile Fe in the study area and takes a catchment scale approach. Physicochemical attributes of 120 soil samples distributed throughout the catchment are analysed, and a new multivariate data analysis approach (Kohonen’s self organising maps) is used to identify the conditions associated with high labile Fe. The second paper establishes whether Fe nodules play a major role as an iron source in the catchment, by determining the genetic mechanism responsible for their formation. The nodules are a major pool of Fe in much of the region and previous studies have implied that they may be involved in redox-controlled mobilisation and redistribution of Fe. This is achieved by combining a detailed study of a ferric soil profile (morphology, mineralogy and micromorphology) with the distribution of Fe nodules on a catchment scale. The third component of the thesis tests whether the concentration and speciation of Fe in soil solutions from Pinus plantations differs significantly from native vegetation soil solutions. Microlysimeters are employed to collect unaltered, in situ soil water samples. The redox speciation of Fe is determined spectrophotometrically and the interaction between Fe and dissolved organic matter (DOM) is modelled with the Stockholm Humic Model. The thesis provides a better understanding of the controls on the distribution, concentration and speciation of Fe in the soils and soil waters of southeast Queensland. Reductive dissolution is the main mechanism by which mobilisation of Fe occurs in the study area. Labile Fe concentrations are low overall, particularly in the sandy soils of the coastal plain. However, high labile Fe is common in seasonally waterlogged and clay-rich soils which are exposed to fluctuating redox conditions and in organic-rich soils adjacent to streams. Clay-rich soils are most common in the upper parts of the catchment. Fe nodules were shown to have a negligible role in the redistribution of dissolved iron in the catchment. They are formed by the erosion, colluvial transport and chemical weathering of iron-rich sandstones. The ferric horizons, in which nodules are commonly concentrated, subsequently form through differential biological mixing of the soil. Whereas dissolution/ reprecipitation of the Fe cements is an important component of nodule formation, mobilised Fe reprecipitates locally. Dissolved Fe in the soil waters is almost entirely in the ferrous form. Vegetation type does not affect the concentration and speciation of Fe in soil waters, although Pinus DOM has greater acidic functional group site densities than DOM from native vegetation. Iron concentrations are highest in the high DOM soil waters collected from sandy podosols, where they are controlled by redox potential. Iron concentrations are low in soil solutions from clay and iron oxide rich soils, in spite of similar redox potentials. This is related to stronger sorption to the reactive clay and iron oxide mineral surfaces in these soils, which reduces the amount of DOM available for microbial metabolisation and reductive dissolution of Fe. Modelling suggests that Pinus DOM can significantly increase the amount of truly dissolved ferric iron remaining in solution in oxidising conditions. Thus, inputs of ferrous iron together with Pinus DOM to surface waters may reduce precipitation of hydrous ferric oxides and increase the flux of dissolved iron out of the catchment. Such inputs are most likely from the lower catchment, where podosols planted with Pinus are most widely distributed. Significant outcomes other than the main aims were also achieved. It is shown that mobilisation of Fe in podosols can occur as dissolved Fe(II) rather than as Fe(III)-organic complexes. This has implications for the large body of work which assumes that Fe(II) plays a minor role. Also, the first paper demonstrates that a data analysis approach based on Kohonen’s self organising maps can facilitate the interpretation of complex datasets and can help identify geochemical processes operating on a catchment scale.

Defining the role of phytoene synthase in carotenoid accumulation of high provitamin A bananas

Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.

The Prehistory of Potyviruses: Their Initial Radiation Was during the Dawn of Agriculture

Background: Potyviruses are found world wide, are spread by probing aphids and cause considerable crop damage. Potyvirus is one of the two largest plant virus genera and contains about 15% of all named plant virus species. When and why did the potyviruses become so numerous? Here we answer the first question and discuss the other. Methods and Findings: We have inferred the phylogenies of the partial coat protein gene sequences of about 50 potyviruses, and studied in detail the phylogenies of some using various methods and evolutionary models. Their phylogenies have been calibrated using historical isolation and outbreak events: the plum pox virus epidemic which swept through Europe in the 20th century, incursions of potyviruses into Australia after agriculture was established by European colonists, the likely transport of cowpea aphid-borne mosaic virus in cowpea seed from Africa to the Americas with the 16th century slave trade and the similar transport of papaya ringspot virus from India to the Americas. Conclusions/Significance: Our studies indicate that the partial coat protein genes of potyviruses have an evolutionary rate of about 1.1561024 nucleotide substitutions/site/year, and the initial radiation of the potyviruses occurred only about 6,600 years ago, and hence coincided with the dawn of agriculture. We discuss the ways in which agriculture may have triggered the prehistoric emergence of potyviruses and fostered their speciation.

Wing shape as a potential discriminator of morphologically similar pest taxa within the Bactrocera dorsalis species complex (Diptera: Tephritidae)

Four morphologically cryptic species of the Bactrocera dorsalis fruit fly complex (B. dorsalis s.s., B. papayae, B. carambolae and B. philippinensis) are serious agricultural pests. As they are difficult to diagnose using traditional taxonomic techniques, we examined the potential for geometric morphometric analysis of wing size and shape to discriminate between them. Fifteen wing landmarks generated size and shape data for 245 specimens for subsequent comparisons among three geographically distinct samples of each species. Intraspecific wing size was significantly different within samples of B. carambolae and B. dorsalis s.s. but not within samples of B. papayae or B. philippinensis. Although B. papayae had the smallest wings (average centroid size=6.002 mm±0.061 SE) and B. dorsalis s.s. the largest (6.349 mm±0.066 SE), interspecific wing size comparisons were generally non-informative and incapable of discriminating species. Contrary to the wing size data, canonical variate analysis based on wing shape data discriminated all species with a relatively high degree of accuracy; individuals were correctly reassigned to their respective species on average 93.27% of the time. A single sample group of B. carambolae from locality 'TN Malaysia' was the only sample to be considerably different from its conspecific groups with regards to both wing size and wing shape. This sample was subsequently deemed to have been originally misidentified and likely represents an undescribed species. We demonstrate that geometric morphometric techniques analysing wing shape represent a promising approach for discriminating between morphologically cryptic taxa of the B. dorsalis species complex.

A toolbox for animal call recognition

Monitoring the natural environment is increasingly important as habit degradation and climate change reduce theworld’s biodiversity.We have developed software tools and applications to assist ecologists with the collection and analysis of acoustic data at large spatial and temporal scales.One of our key objectives is automated animal call recognition, and our approach has three novel attributes. First, we work with raw environmental audio, contaminated by noise and artefacts and containing calls that vary greatly in volume depending on the animal’s proximity to the microphone. Second, initial experimentation suggested that no single recognizer could dealwith the enormous variety of calls. Therefore, we developed a toolbox of generic recognizers to extract invariant features for each call type. Third, many species are cryptic and offer little data with which to train a recognizer. Many popular machine learning methods require large volumes of training and validation data and considerable time and expertise to prepare. Consequently we adopt bootstrap techniques that can be initiated with little data and refined subsequently. In this paper, we describe our recognition tools and present results for real ecological problems.

Polyandry and paternity skew in natural and experimental populations of Drosophila serrata

Many species engage in polyandry, resulting in the potential for sexual selection to continue post-copulation through sperm competition and/or cryptic female choice. The relative importance of pre- vs. post-copulatory processes remains unknown for most species despite this information being fundamental for understanding the evolutionary consequences of sexual selection. The Australian fruit fly Drosophila serrata has become a prominent model system for studying precopulatory sexual selection, such as mating preferences and their influence on the evolution of sexually selected traits. Here, we investigated polyandry and the potential for post-copulatory sexual selection in this species using indirect paternity analysis. We genotyped 21 wild-caught and 19 laboratory-reared mothers and their offspring (a total of 787 flies) at six microsatellite loci and found extensive polyandry, with all broods surveyed having at least two sires. Female remating rates were higher than in other Drosophila surveyed to date and no significant differences were found between laboratory and field populations. Additionally, we found evidence for biased sperm usage in several broods of D. serrata. Paternity skew occurred more frequently in broods from the field population than the laboratory one, suggesting differences between the two environments in the level of post-copulatory sexual selection. Our data suggest that D. serrata represents a promising system for studying the interaction between pre- and post-copulatory sexual selection in driving the evolution of sexually selected phenotypes.