Metabolomic Profiling of Antioxidant Compounds in Five Vachellia Species.

The genus Vachellia, previously known as Acacia, belongs to the family Fabaceae, subfamily Leguminosae, which are flowering plants, commonly known as thorn trees. They are traditionally used medicinally in various countries including South Africa for the treatment of ailments such as fever, sore throat, Tuberculosis, convulsions and as sedatives. The aim of this study was to determine biochemical variations in five Vachellia species and correlate their metabolite profiles to antioxidant activity using a chemometric approach. The antioxidant activity of five Vachellia aqueous-methanolic extracts were analyzed using three methods: 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) analysis and the ferric reducing antioxidant power (FRAP) assay by means of serial dilution and bioautography with the thin-layer chromatography (TLC) method. Amongst the Vachellia extracts tested, V. karroo, V. kosiensis and V. xanthophloea demonstrated the highest DPPH, ABTS+ and FRAP inhibitory activity. The antioxidant activities of DPPH were higher than those obtained by ABTS+, although these values varied among the Vachellia species. Proton nuclear magnetic resonance (1H NMR), coupled with multivariate statistical modeling tools such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were performed to profile metabolites responsible for the observed activity. The OPLS-DA categorized the five Vachellia species, separating them into two groups, with V. karroo, V. kosiensis and V. xanthophloea demonstrating significantly higher radical scavenging activity than V. tortilis and V. sieberiana, which clustered together to form another group with lower radical scavenging activity. Annotation of metabolites was carried out using the ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS), and it tentatively identified 23 metabolites of significance, including epigallocatechin (m/z = 305.0659), methyl gallate (m/z = 183.0294) and quercetin (m/z = 301.0358), amongst others. These results elucidated the metabolites that separated the Vachellia species from each other and demonstrated their possible free radical scavenging activities.

Comprehensive chemical characterization and biological evaluation of two Acacia species: A. nilotica and A. ataxacantha.

The genus Acacia (Family Leguminosae) is composed of several medicinal plants used for treating miscellaneous diseases. Amid the important members of this genus, A. nilotica and A. ataxacantha are widely employed for their tremendous healing properties. Hence, this present work aimed to determine the total phenolic and flavonoid contents and investigate the antioxidant, antiproliferative, anti-enzyme and antimicrobial potentials of methanolic and water extracts of leaves and stem bark of A. nilotica and A. ataxacantha obtained by maceration and ultrasonication. The total phenolic and flavonoid contents were obtained in the range of 33.35-116.60 mg GAE/g and 0.26-49.90 mg RE/g, respectively, with the methanolic leaf extracts of both species showing the highest contents. Moreover, the methanolic extracts were observed to display higher antioxidant potentials in almost all antioxidant assays performed compared to the water extracts (ABTS: 52.66-943.81 mg TE/g, DPPH: 8.51-493.90 mg TE/g, CUPRAC: 106.39-1193.75 mg TE/g; FRAP: 31.38-416.21 mg TE/g, and phosphomolybdenum: 0.90-4.17 mM TE/g). However, the water extracts were seen to be better metal chelators than the methanolic extracts (8.47-36.85 mg EDTAE/g). Additionally, all extracts were found to exhibit anti-tyrosinase (30.79-74.80 mg KAE/g) and anti-amylase (0.10-1.10 mM ACAE/g) properties. With the exception of a few extracts, glucosidase and acetylcholinesterase inhibitions (1.69-2.12 mg ACAE/g and 0.42-2.61 mg GALAE/g, respectively) were also demonstrated. While the methanolic extracts of both species showed antimicrobial potency against all the 18 tested microorganisms (gram positive, gram negative, and fungi), the water extracts were effective only against the gram positive bacteria. The extracts were also found to exhibit antiproliferative effects on SH-SY5Y human neuroblastoma cells, with the methanolic extracts showing higher cytotoxic potential than the water extracts. Therefore, this study showed these species to be good sources of antioxidants, enzyme inhibitors, antimicrobials and antiproliferative agents, which could be of great interest for their applications as natural bioactive ingredients in the development of pharmaceuticals and nutraceuticals.

Mid-Infrared Spectroscopy as a Rapid Tool to Qualitatively Predict the Effects of Species, Regions and Roasting on the Nutritional Composition of Australian Acacia Seed Species.

In recent times, the popularity of adding value to under-utilized legumes have increased to enhance their use for human consumption. Acacia seed (AS) is an underutilized legume with over 40 edible species found in Australia. The study aimed to qualitatively characterize the chemical composition of 14 common edible AS species from 27 regions in Australia using mid-infrared (MIR) spectroscopy as a rapid tool. Raw and roasted (180 °C, 5, 7, and 9 min) AS flour were analysed using MIR spectroscopy. The wavenumbers (1045 cm-1, 1641 cm-1, and 2852-2926 cm-1) in the MIR spectra show the main components in the AS samples. Principal component analysis (PCA) of the MIR data displayed the clustering of samples according to species and roasting treatment. However, regional differences within the same AS species have less of an effect on the components, as shown in the PCA plot. Statistical analysis of absorbance at specific wavenumbers showed that roasting significantly (p < 0.05) reduced the compositions of some of the AS species. The results provided a foundation for hypothesizing the compositional similarity and/or differences among AS species before and after roasting.

Nutritional, anti-nutritional, antioxidant, physicochemical and functional characterization of Australian acacia seed: effect of species and regions.

BACKGROUND: Acacia seed (AS) is an underutilized legume widely distributed in the world, with majority of the species (>70%) found in Australia. Generally, the seeds are not only rich in protein, dietary fibre and potassium, but also possess anti-nutritional compounds. In recent years, there have been an increase in the cultivation of some Australian acacia species such as Acacia victoriae, Acacia cowleana and Acacia coriacea from different regions. However, there is limited information on the composition, anti-nutrients, antioxidant and functional properties of flour from these widely grown Australian AS species. Thus, the present study aimed to assess the properties of these Australian AS species from different geographical regions. RESULTS: The A. cowleana and A. coriacea were characterized by high protein, fat, potassium and soluble carbohydrate. However, higher starch and fibre contents were present in A. victoriae. Greater amounts of anti-nutrients, total phenolics and flavonoids were found in A. cowleana and A. coriacea seeds, whereas A. victoriae had higher 2,2-diphenyl-1-picrylhydrazyl radical-scavenging capacity. A. victoriae and A. coriacea demonstrated the highest water absorption and solubility index, respectively. However, A. cowleana showed the highest oil absorption index. There was less variation in the composition and properties within species from different regions. CONCLUSION: All samples showed promising nutritional characteristics, although with sufficient diversity to indicate that Australian acacia seeds can be utilized to develop a range of new (functional) food products. Overall, the information obtained will help the food industries with the selection of AS species for food application.

Intraspecific variation in spermoderm pattern of tribe Acacieae (Mimosoideae) using scanning electron microscopy techniques.

Current research carried out in Pakistan is the first report on spermoderm ornamentation of eight species of tribe Acacieae (Mimosoidae) by using scanning electron microscopic techniques representing two genera, Fedherbia and Acacia were examined. Different spermoderm ornamentation were observed, described and discussed for their taxonomic importance. Seeds surfaces of the studied tribe possess novel variations in macro and micro morphology. Great variations were observed in both qualitative and quantitative characters of seeds. Seeds shape was oblong, ovate to elliptical and spermoderm ornamentation was levigate, rugose, polygonal and discoid, colliculate, and papillose type. These variations in the spermoderm ornamentation can be used as an aid in identification and classification of the members of tribe Acacieae.

Contrasting Influences of Geographic Range and Distribution of Populations on Patterns of Genetic Diversity in Two Sympatric Pilbara Acacias.

The influence of geographic range on species persistence has long been of interest and there is a need for a better understanding of the genetic consequences for species with restricted distributions, particularly with the increasing rate of global species extinctions. However, the genetic effects of restricted range are often confounded by the impacts of population distribution. We compared chloroplast and nuclear genetic diversity and differentiation in two acacias, the restricted, patchily distributed Acacia atkinsiana and the widespread, semi-continuously distributed A. ancistrocarpa. Lower intra-population diversity and higher differentiation between populations were seen in A. atkinsiana compared to its widespread congener, A. ancistrocarpa. There was little evidence of geographical influences on population genetic structure in A. ancistrocarpa whereas A. atkinsiana exhibited nuclear genetic structure with isolation by distance, differentiation of near-coastal populations from those in the ranges, and differentiation of peripheral populations from those in the centre of the distribution. These results are consistent with expectations of the effect of geographic range and population distribution on genetic diversity, but indicate that distribution of populations rather than geographic range has influenced the observed genetic structure. The contrasting patterns observed here demonstrate that conservation approaches for species management and ecological restoration need to consider the distribution of populations in geographically restricted species.

The anti-inflammatory and antinociceptive effects of proteins extracted from Acacia farnesiana seeds / Efeito antiiflamatório e antinociceptivo de proteínas extraídas de sementes de Acacia farnesiana

ABSTRACT Seeds of Acacia farnesiana are commonly sold in the local markets of northeastern Brazil as a therapeutic agent. The present work aimed to evaluate the anti-inflammatory and analgesic activities of proteins obtained from A. farnesiana seeds. Five different protein fractions (albumin, globulin, prolamin, acidic and basic glutelins) were obtained and investigated for the protein pattern, the presence of hemagglutinating and proteolytic activities. The globulin fraction (GLB) was also evaluated for anti-inflammatory and analgesic activities. Globulins reduced the paw edema induced by carrageenan in a dose-dependent manner, which was accompanied by a reduction of myeloperoxidase activity (p < 0.05). Additionally, GLB reduced the neutrophil peritoneal migration induced by carrageenan. However, GLB was not able to inhibit the edema triggered by dextran. Pre-treatment with globulins reduced the abdominal constrictions induced by acetic acid as well as the paw licking time induced by formalin (69.1% at first phase). However, it did not produce a significant antinociceptive effect in the hot plate test (55-56 °C). Treating the GLB with heat (at 100 °C for 30 min) abolished its anti-edematogenic and hemagglutinating activities. Our results showed that seeds from A. farnesiana are a source of proteins with anti-inflammatory and analgesic properties.

RESUMO Sementes de Acacia farnesiana são comumente vendidas em feiras locais no nordeste do Brasil como agente terapêutico. O presente trabalho objetivou avaliar as atividades antiinflamatória e antinociceptiva de proteínas obtidas de sementes de A. farnesiana. Cinco frações protéicas distintas (albuminas, globulinas, prolaminas, glutelinas ácidas e básicas) foram obtidas e investigadas quanto o perfil de proteínas, presença de atividade hemaglutinante e proteolítica. A fração globulina (GLB) também foi avaliada quanto a presença de atividade antiinflamatória e analgésica. Globulinas reduziram o edema de pata induzido por carragenina de modo dependente da dose que foi acompanhada da redução da atividade da mieloperoxidase (p < 0,05). Em adição, GLB reduziu a migração de neutrófilos para cavidade peritoneal induzida por carragenina. Entretanto, GLB não foi capaz de inibir o edema induzido por dextrana. O pré-tratamento com globulinas reduziu as contorções abdominais induzidas por ácido acético, bem como o tempo de lambedura da pata induzida por formalina (69.1% na primeira fase). Por outro lado, GLB não produziu um efeito antinociceptivo significante no teste de placa quente (55-56 °C). O pré-tratamento de GLB com calor (100 °C por 30 min) aboliu sua atividade anti-edematogênica e hemaglutinante. Nossos resultados mostraram que sementes de A. farnesiana são fonte de proteínas com propriedades antiinflamatórias e analgésicas.

Ground level photosynthetically active radiation dynamics in stands of Acacia mearnsii De Wild.

The objective is to study the dynamics of photosynthetic radiation reaching the soil surface in stands of Acacia mearnsii De Wild and its influence on height growth in stands. This fact gives rise to the formulation of the following hypothesis for this study: "The reduction of the incidence of light inside the stand of black wattle will cause the inflection point in its height growth when this reaches 4 to 5 m in height, i.e. when the stand is between 2 and 3 years of age". The study was conducted in stands in the state of Rio Grande do Sul, Brazil, where diameters at breast height, total height and photosynthetically active radiation available at ground level were measured. The frequency tended to be more intense when the age of the stands increases. It was evident that a reduction of light incidence inside the forest occurred, caused by canopy closure. Consequently, closed canopy propitiated the competition of plants. This has affected the conditions for growth in diameter and height of this species, reason why it becomes possible to conceive the occurrence of an inflection point in the growth of these two variables, confirming the formulated hypothesis.

The Complete Sequence of the Acacia ligulata Chloroplast Genome Reveals a Highly Divergent clpP1 Gene.

Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 174,233 bp in size, comprising inverted repeats of 38,225 bp and single-copy regions of 92,798 bp and 4,985 bp [corrected]. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease complex.

Genetic and genomic diversity studies of Acacia symbionts in Senegal reveal new species of Mesorhizobium with a putative geographical pattern.

Acacia senegal (L) Willd. and Acacia seyal Del. are highly nitrogen-fixing and moderately salt tolerant species. In this study we focused on the genetic and genomic diversity of Acacia mesorhizobia symbionts from diverse origins in Senegal and investigated possible correlations between the genetic diversity of the strains, their soil of origin, and their tolerance to salinity. We first performed a multi-locus sequence analysis on five markers gene fragments on a collection of 47 mesorhizobia strains of A. senegal and A. seyal from 8 localities. Most of the strains (60%) clustered with the M. plurifarium type strain ORS 1032T, while the others form four new clades (MSP1 to MSP4). We sequenced and assembled seven draft genomes: four in the M. plurifarium clade (ORS3356, ORS3365, STM8773 and ORS1032T), one in MSP1 (STM8789), MSP2 (ORS3359) and MSP3 (ORS3324). The average nucleotide identities between these genomes together with the MLSA analysis reveal three new species of Mesorhizobium. A great variability of salt tolerance was found among the strains with a lack of correlation between the genetic diversity of mesorhizobia, their salt tolerance and the soils samples characteristics. A putative geographical pattern of A. senegal symbionts between the dryland north part and the center of Senegal was found, reflecting adaptations to specific local conditions such as the water regime. However, the presence of salt does not seem to be an important structuring factor of Mesorhizobium species.

Accumulation and precipitation of magnesium, calcium, and sulfur in two Acacia (Leguminosae; Mimosoideae) species grown in different substrates proposed for mine-site rehabilitation.

PREMISE OF THE STUDY: Few studies have investigated the effects of substrates on the accumulation and precipitation of magnesium, calcium, and sulfur in plants. Acacia stipuligera and A. robeorum growing in their natural habitats with different substrates show different accumulation and precipitation patterns of these elements. Here, we compared the accumulation and precipitation of magnesium, calcium, and sulfur in A. stipuligera and A. robeorum grown in different substrates proposed for mine-site rehabilitation and expected the differences in substrates to have significant effects on the accumulation and precipitation of these elements in the two species. METHODS: Saplings were grown in sandy topsoil or in a topsoil-siltstone mixture in a glasshouse. Phyllode magnesium, calcium, and sulfur concentrations of 25-wk-old plants were measured. Precipitation of these elements in phyllodes and branchlets was investigated by means of scanning electron microscopy and energy-dispersive x-ray spectroscopy. KEY RESULTS: Phyllode magnesium, calcium, and sulfur concentrations were generally significantly greater in A. robeorum than in A. stipuligera. The two species responded in unique ways to the substrate, with A. stipuligera having similar phyllode magnesium and calcium concentrations in both substrates, but greater sulfur concentration in the topsoil-siltstone mixture, while A. robeorum showed lower phyllode magnesium, calcium, and sulfur concentrations in the topsoil-siltstone mixture. For both substrates, mineral precipitates were observed in both species, with A. robeorum having more mineral precipitates containing magnesium, calcium, and sulfur in its phyllodes than A. stipuligera did. CONCLUSIONS: The accumulation and precipitation patterns of magnesium, calcium, and sulfur are more species-specific than substrate-affected.

Salt- and alkaline-tolerance are linked in Acacia.

Saline or alkaline soils present a strong stress on plants that together may be even more deleterious than alone. Australia's soils are old and contain large, sometimes overlapping, areas of high salt and alkalinity. Acacia and other Australian plant lineages have evolved in this stressful soil environment and present an opportunity to understand the evolution of salt and alkalinity tolerance. We investigate this evolution by predicting the average soil salinity and pH for 503 Acacia species and mapping the response onto a maximum-likelihood phylogeny. We find that salinity and alkalinity tolerance have evolved repeatedly and often together over 25 Ma of the Acacia radiation in Australia. Geographically restricted species are often tolerant of extreme conditions. Distantly related species are sympatric in the most extreme soil environments, suggesting lack of niche saturation. There is strong evidence that many Acacia have distributions affected by salinity and alkalinity and that preference is lineage specific.

Delayed colonisation of Acacia by thrips and the timing of host-conservatism and behavioural specialisation.

BACKGROUND: Repeated colonisation of novel host-plants is believed to be an essential component of the evolutionary success of phytophagous insects. The relative timing between the origin of an insect lineage and the plant clade they eat or reproduce on is important for understanding how host-range expansion can lead to resource specialisation and speciation. Path and stepping-stone sampling are used in a Bayesian approach to test divergence timing between the origin of Acacia and colonisation by thrips. The evolution of host-plant conservatism and ecological specialisation is discussed. RESULTS: Results indicated very strong support for a model describing the origin of the common ancestor of Acacia thrips subsequent to that of Acacia. A current estimate puts the origin of Acacia at approximately 6 million years before the common ancestor of Acacia thrips, and 15 million years before the origin of a gall-inducing clade. The evolution of host conservatism and resource specialisation resulted in a phylogenetically under-dispersed pattern of host-use by several thrips lineages. CONCLUSIONS: Thrips colonised a diversity of Acacia species over a protracted period as Australia experienced aridification. Host conservatism evolved on phenotypically and environmentally suitable host lineages. Ecological specialisation resulted from habitat selection and selection on thrips behavior that promoted primary and secondary host associations. These findings suggest that delayed and repeated colonisation is characterised by cycles of oligo- or poly-phagy. This results in a cumulation of lineages that each evolve host conservatism on different and potentially transient host-related traits, and facilitates both ecological and resource specialisation.

[Seedlings growth and survival of five Acacia (Fabaceae) species that coexists in neotropical semi-arid forests of Argentina, under different light and water availability conditions]. / Crecimiento y supervivencia de plántulas de cinco especies de Acacia (Fabaceae), que coexisten en bosques secos neotropicales de argentina, en distintas condiciones de disponibilidad de luz y agua.

Seedlings growth and survival of five Acacia (Fabaceae) species that coexists in neotropical semi-arid forests of Argentina, under different light and water availability conditions. Seedling establishment is one of the most risky stages of plants, especially in arid and semiarid regions, where low water availability and high solar radiation influence its emergence, development and survival. In seasonally dry xerophytic forests occurring in North-Western Córdoba, central Argentina, five neotropical species of Acacia co-exist: A. aroma, A. caven, A. atramentaria, A. gilliesii and A. praecox. With the aim to evaluate growth variables and survival of these five species seedlings, in response to water stress and different light availability conditions, a greenhouse experiment was undertaken from March to June of 2010. Although small differences were found between species (F = 5.66, p = 0.001), all of them showed high percentages of seedling survival in response to different light and water treatments, suggesting that seedlings would be tolerant to water stress and could be established both in light and shade. On the other hand, although all species showed an increase in growth in light conditions and without water stress, we have found some trends towards a greater growth in the seedlings ofA. aroma, A. caven and A. atramentaria when compared to those of A. praecox and A. gilliessi in most of the variables considered (F = 41.9, p < 0.0001; F = 7.06, p < 0.0001; F = 53.59, p < 0.0001). This pattern was confirmed through a cluster analysis that classified the species in two main groups. These results, together with others already reported, would indicate a regenerative niche differentiation that might be favoring the regional coexistence of these five species in semiarid forests in central Argentina.

Crecimiento y supervivencia de plántulas de cinco especies de Acacia (Fabaceae), que coexisten en bosques secos neotropicales de Argentina, en distintas condiciones de disponibilidad de luz y agua / Seedlings growth and survival of five Acacia (Fabaceae) species that coexists in neotropical semiarid forests of Argentina, under different light and water availability conditions

Seedling establishment is one of the most risky stages of plants, especially in arid and semiarid regions, where low water availability and high solar radiation influence its emergence, development and survival. In seasonally dry xerophytic forests occurring in North-Western Córdoba, central Argentina, five neotropical species of Acacia co-exist: A. aroma, A. caven, A. atramentaria, A. gilliesii and A. praecox. With the aim to evaluate growth variables and survival of these five species seedlings, in response to water stress and different light availability conditions, a greenhouse experiment was undertaken from March to June of 2010. Although small differences were found between species (F=5.66, p=0.001), all of them showed high percentages of seedling survival in response to different light and water treatments, suggesting that seedlings would be tolerant to water stress and could be established both in light and shade. On the other hand, although all species showed an increase in growth in light conditions and without water stress, we have found some trends towards a greater growth in the seedlings of A. aroma, A. caven and A. atramentaria when compared to those of A. praecox and A. gilliessi in most of the variables considered (F=41.9, p<0.0001; F=7.06, p<0.0001; F=53.59, p<0.0001). This pattern was confirmed through a cluster analysis that classified the species in two main groups. These results, together with others already reported, would indicate a regenerative niche differentiation that might be favoring the regional coexistence of these five species in semiarid forests in central Argentina.

El establecimiento de la plántula es una de las etapas más riesgosas para las plantas, especialmente en zonas áridas y semiáridas donde la sequía y alta radiación solar influyen sobre su emergencia, desarrollo y supervivencia. Se evaluó en invernadero la supervivencia y variables de crecimiento en plántulas sometidas a estrés hídrico y a distintas condiciones de luz, en cinco especies de Acacia (A. aroma, A. caven, A. atramentaria, A. gilliesii y A. praecox) que coexisten en los bosques xerófilos de Córdoba, Argentina. Aunque se encontraron diferencias entre las especies (F=5.66, p=0.001), todas tuvieron altos porcentajes de supervivencia en las distintas condiciones de luz y agua, sugiriendo que serían tolerantes al estrés hídrico y podrían establecerse bajo luz o sombra. Si bien todas las especies mejoraron el crecimiento con luz y sin estrés hídrico, A. aroma, A. caven y A. atramentaria mostraron una tendencia hacia un mayor crecimiento en la mayoría de las variables consideradas (F=41.9, p<0.0001; F=7.06, p<0.0001; F=53.59, p<0.0001). Estos resultados sumados a otros ya reportados indicarían una diferenciación de nichos regenerativos favoreciendo la coexistencia regional de estas especies en los bosques xerófilos de Córdoba.

DNA barcoding for conservation, seed banking and ecological restoration of Acacia in the Midwest of Western Australia.

We used DNA barcoding to address an important conservation issue in the Midwest of Western Australia, working on Australia's largest genus of flowering plant. We tested whether or not currently recommended plant DNA barcoding regions (matK and rbcL) were able to discriminate Acacia taxa of varying phylogenetic distances, and ultimately identify an ambiguously labelled seed collection from a mine-site restoration project. Although matK successfully identified the unknown seed as the rare and conservation priority listed A. karina, and was able to resolve six of the eleven study species, this region was difficult to amplify and sequence. In contrast, rbcL was straightforward to recover and align, but could not determine the origin of the seed and only resolved 3 of the 11 species. Other chloroplast regions (rpl32-trnL, psbA-trnH, trnL-F and trnK) had mixed success resolving the studied taxa. In general, species were better resolved in multilocus data sets compared to single-locus data sets. We recommend using the formal barcoding regions supplemented with data from other plastid regions, particularly rpl32-trnL, for barcoding in Acacia. Our study demonstrates the novel use of DNA barcoding for seed identification and illustrates the practical potential of DNA barcoding for the growing discipline of restoration ecology.

Comparison of random and SSR-enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization in Acacia harpophylla F. Muell. Ex Benth.

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state-of-the-art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north-eastern Australia, we show that high-throughput shotgun pyrosequencing technology, so-called second-generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large-scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200,908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost-effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.

The evolutionary history and biogeography of Mimosoideae (Leguminosae): an emphasis on African acacias.

The systematics of Mimosoideae has been in a state of flux, which reflects overall poor knowledge of the evolution and biogeography of this group. Preliminary molecular phylogenetic analyses suggest the tribal system of Mimosoideae needs a complete revision. This has led to the use of new generic names for Acacia sensu lato (s.l. hereafter) following the re-typification of Acacia with an Australian type: (i) Acacia sensu stricto (s.s. hereafter), Vachellia, Senegalia, Acaciella and Mariosousa. This study reconstructs the evolutionary history of Mimosoideae, using the most comprehensive sampling to date, with an emphasis on African species. It aims to reconstruct the phylogenetic relationships among the five recognized genera within Acacieae as the precursor to elucidate the paleo-biogeography of mimosoids and their adaptation to open habitats in the Cenozoic. The basal position of Mimoseae lineages with regards to Vachellia and Senegalia+Mariosousa+Acaciella+Ingeae+Acacia s.s. clades is a novel finding. Vachellia (formerly Acacia subgenus Acacia) is found monophyletic. A grade including the remaining Mimoseae lineages is found sister to the Senegalia+Mariosousa+Acaciella+Ingeae+Acacia s.s. clade. The major clades originated in the late Oligocene-early Miocene (∼25mya). The transitions from close to open habitats occurred during the Miocene for at least four mimosoid lineages. These are interpreted as responses to increased seasonality leading to fire climates and drying trends in the Miocene, which allowed the expansion of open habitats, such as savannas, worldwide.

Leaf miner and plant galler species richness on Acacia: relative importance of plant traits and climate.

Diversity patterns of herbivores have been related to climate, host plant traits, host plant distribution and evolutionary relationships individually. However, few studies have assessed the relative contributions of a range of variables to explain these diversity patterns across large geographical and host plant species gradients. Here we assess the relative influence that climate and host plant traits have on endophagous species (leaf miners and plant gallers) diversity across a suite of host species from a genus that is widely distributed and morphologically variable. Forty-six species of Acacia were sampled to encapsulate the diversity of species across four taxonomic sections and a range of habitats along a 950 km climatic gradient: from subtropical forest habitats to semi-arid habitats. Plant traits, climatic variables, leaf miner and plant galler diversity were all quantified on each plant species. In total, 97 leaf mining species and 84 plant galling species were recorded from all host plants. Factors that best explained leaf miner richness across the climatic gradient (using AIC model selection) included specific leaf area (SLA), foliage thickness and mean annual rainfall. The factor that best explained plant galler richness across the climatic gradient was C:N ratio. In terms of the influence of plant and climatic traits on species composition, leaf miner assemblages were best explained by SLA, foliage thickness, mean minimum temperature and mean annual rainfall, whilst plant gall assemblages were explained by C:N ratio, %P, foliage thickness, mean minimum temperature and mean annual rainfall. This work is the first to assess diversity and structure across a broad environmental gradient and a wide range of potential key climatic and plant trait determinants simultaneously. Such methods provide key insights into endophage diversity and provide a solid basis for assessing their responses to a changing climate.

Neotropical mutualism between Acacia and Pseudomyrmex: phylogeny and divergence times.

The interaction between Acacia and Pseudomyrmex is a textbook example of mutualism between ants and plants, nevertheless aspects of its evolutionary biology have not been formally explored. In this paper we analyze primarily the phylogenies of both New World Acacia and of their associated species of ants, and the geographic origin of this mutualism. Until now, there has been no molecular analysis of this relationship in terms of its origin and age. We analyzed three chloroplast markers (matK, psaB-rps14, and trnL-trnF) on a total of 70 taxa of legumes from the subfamily Mimosoideae, and two nuclear regions (long-wavelength rhodopsine and wingless) on a total of 43 taxa of ants from subfamily Pseudomyrmecinae. The monophyly of subgenus Acacia and within the New World lineages that of the myrmecophilous Acacia group was established. In addition, our results supported the monophyly of the genus Pseudomyrmex and of the associated acacia-ants P. ferrugineus group. Using Bayesian methods and calibration data, the estimated divergence times for the groups involved in the mutualism are: 5.44+/-1.93 My for the myrmecophilous acacias and 4.58+/-0.82 My for their associated ant species, implying that their relationship originated in Mesoamerica between the late Miocene to the middle Pliocene, with eventual diversification of both groups in Mexico.