A comprehensive genus-level phylogeny and biogeographical history of the Lythraceae based on whole plastome sequences.

BACKGROUND AND AIMS: The Lythraceae are a mainly subtropical to tropical family of the order Myrtales with 28 currently accepted genera and approximately 600 species. There is currently no well-supported phylogenetic and biogeographical hypothesis of the Lythraceae incorporating all currently accepted genera, which we sought to provide. METHODS: Plastomes of representative species of 18 distinct Lythraceae genera were sequenced and annotated. Together with existing sequences, plastomes of all 28 currently accepted genera in the Lythraceae were brought together for the first time. The plastomes were aligned and a Bayesian phylogenetic hypothesis was produced. We then conducted a time-calibrated Bayesian analysis and a biogeographical analysis. KEY RESULTS: Plastome-based Bayesian and maximum-likelihood phylogenetic trees are generally congruent with recent nuclear phylogenomic data and resolve two deeply branching major clades in the Lythraceae. One major clade concentrates shrubby and arboreal South American and African genera that inhabit seasonally dry environments, with larger, often winged seeds, adapted to dispersal by the wind. The second major clade concentrates North American, Asian, African and several near-cosmopolitan herbaceous, shrubby and arboreal genera, often inhabiting humid or aquatic environments, with smaller seeds possessing structures that facilitate dispersal by water. CONCLUSIONS: We hypothesize that the Lythraceae dispersed early in the Late Cretaceous from South American to North American continents, with subsequent expansion in the Late Cretaceous of a North American lineage through Laurasia to Africa via a boreotropical route. Two later expansions of South American clades to Africa in the Palaeocene and Eocene, respectively, are also hypothesized. Transoceanic dispersal in the family is possibly facilitated by adaptations to aquatic environments that are common to many extant genera of the Lythraceae, where long-distance dispersal and vicariance may be invoked to explain several remarkable disjunct distributions in Lythraceae clades.

Diversity of anamorphic Cordyceps (formerly Isaria) isolated from Brazilian agricultural sites.

A total of 53 anamorphic strains of Brazilian Cordyceps species currently maintained in a government-owned culture collection, were reassessed for diversity and species identity using multi-loci-based phylogenetic methods. The strains used in this study were originally obtained from soil samples or were isolated from insects of the orders Hemiptera, Lepidoptera, Coleoptera and Diptera, mostly from agricultural sites. A Bayesian phylogenetic tree was constructed based on a concatenation of five loci (ITS, LSU, RPB1, RPB2 and TEF). In a few cases of ambiguity, morphological traits were also considered for species delimitations. Considerable variability within the set of strains was detected and six Cordyceps species were identified: C. amoenerosea, C. fumosorosea, C. javanica, C. tenuipes and, for the first time, C. brevistroma and C. spegazzinii are reported in Brazil. Four other taxonomically equivocal groups, closely related to other known taxa (C. amoenerosea, C. cateniannulata, C. polyarthra and C. spegazzinii), were also recognized, although further studies will be required to confirm their identifications or their descriptions as new species. Cordyceps javanica was the most common species in our dataset, originally isolated from soil and several different insect orders, and includes 17 strains from the whitefly, Bemisia tabaci. Interestingly, strains previously identified as C. fumosorosea based on morphology and growth characteristics, were shown to be C. javanica, including the active ingredients of some commercial mycoinsecticides. Cordyceps farinosa, usually mentioned in the literature as occurring in Brazil, was not found in our study. Since most strains were from insect crop pests, further studies with hosts from non-agricultural settings or from environmental samples would be advisable for a deeper understanding of the occurrence of anamorphic Cordyceps in Brazil.

Phylogenomic analysis points to a South American origin of Manihot and illuminates the primary gene pool of cassava.

The genus Manihot, with around 120 known species, is native to a wide range of habitats and regions in the tropical and subtropical Americas. Its high species richness and recent diversification only c. 6 million years ago have significantly complicated previous phylogenetic analyses. Several basic elements of Manihot evolutionary history therefore remain unresolved. Here, we conduct a comprehensive phylogenomic analysis of Manihot, focusing on exhaustive sampling of South American taxa. We find that two recently described species from northeast Brazil's Atlantic Forest were the earliest to diverge, strongly suggesting a South American common ancestor of Manihot. Ancestral state reconstruction indicates early Manihot diversification in dry forests, with numerous independent episodes of new habitat colonization, including into savannas and rainforests within South America. We identify the closest wild relatives to Manihot esculenta, including the crop cassava, and we quantify extensive wild introgression into the cassava gene pool from at least five wild species, including Manihot glaziovii, a species used widely in breeding programs. Finally, we show that this wild-to-crop introgression substantially shapes the mutation load in cassava. Our findings provide a detailed case study for neotropical evolutionary history in a diverse and widespread group, and a robust phylogenomic framework for future Manihot and cassava research.

Misleading fruits: The non-monophyly of Pseudopiptadenia and Pityrocarpa supports generic re-circumscriptions and a new genus within mimosoid legumes.

Generic delimitation in Piptadenia and allies (mimosoid legumes) has been in a state of flux, particularly caused by over-reliance on fruit and seed morphology to segregate species out of Piptadenia into the genera Parapiptadenia, Pityrocarpa and Pseudopiptadenia. Although supporting their segregation from Piptadenia, previous phylogenetic analyses suggested that some of these segregated genera are not monophyletic. Here, we test the monophyly of Parapiptadenia, Pityrocarpa and Pseudopiptadenia with dense taxon sampling across these genera, including the type species of each genus. Our analysis recovers Parapitadenia as monophyletic, but places Pseudopiptadenia species in two distinct lineages, one of which includes all three species of Pityrocarpa. Given that the type species of both Pseudopiptadenia and Pityrocarpa are nested in the same clade, we subsume Pseudopiptadenia under the older name Pityrocarpa. The remaining Pseudopiptadenia species are assigned to the new genus Marlimorimia. Alongside high molecular phylogenetic support, recognition of Parapiptadenia, Pityrocarpa and Marlimorimia as distinct genera is also supported by combinations of morphological traits, several of which were previously overlooked.

Mosaic genome evolution and phylogenetics of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and virulence of seven new isolates from the Brazilian states of Minas Gerais and Mato Grosso.

In a comparative analysis of genome sequences from isolates of the baculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from Brazil and Guatemala, we identified a subset of isolates possessing chimeric genomes. We identified six distinct phylogenetically incongruous regions (PIRs) dispersed in the genomes, of between 279 and 3345 bp in length. The individual PIRs possessed high sequence similarity among the affected ChinNPV isolates but varied in coverage in some instances. The donor for four of the PIRs implicated in horizontal gene transfer (HGT) was identified as Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), an alphabaculovirus closely related to ChinNPV, or another unknown but closely related virus. BLAST searches of the other two PIRs returned only ChinNPV sequences, but HGT from an unknown donor baculovirus cannot be excluded. Although Chrysodeixis includens and Trichoplusia ni are frequently co-collected from soybean fields in Brazil, pathogenicity data suggest that natural coinfection of C. includens larvae with ChinNPV and TnSNPV is probably uncommon. Additionally, since the chimeric ChinNPV genomes with tracts of TnSNPV sequence were restricted to a single monophyletic lineage of closely related isolates, a model of progressive restoration of the native DNA sequence by recombination with ChinNPV possessing a fully or partially non-chimeric genome is reasonable. However, multiple independent HGT from TnSNPV to ChinNPV during the evolution of these isolates cannot be excluded. Mortality data suggest that the ChinNPV isolates with chimeric genomes are not significantly different in pathogenicity towards C. includens when compared to most other ChinNPV isolates. Exclusion of the PIRs prior to phylogenetic analysis had a large impact on the topology of part of the maximum-likelihood tree, revealing a homogenous clade of three isolates (IB, IC and ID) from Paraná state in Brazil collected in 2006, together with an isolate from Guatemala collected in 1972 (IA), comprising the lineage uniquely affected by HGT from TnSNPV. The other 10 Brazilian ChinNPV isolates from Paraná, Mato Grosso, and Minas Gerais states showed higher variability, where only three isolates from Paraná state formed a monophyletic group correlating with geographical origin.

Trichoderma from Brazilian garlic and onion crop soils and description of two new species: Trichoderma azevedoi and Trichoderma peberdyi.

Fifty four Trichoderma strains were isolated from soil samples collected from garlic and onion crops in eight different sites in Brazil and were identified using phylogenetic analysis based on combined ITS region, tef1-α, cal, act and rpb2 sequences. The genetic variability of the recovered Trichoderma species was analysed by AFLP and their phenotypic variability determined using MALDI-TOF. The strain clusters from both typing techniques coincided with the taxonomic determinations made from phylogenetic analysis. The phylogenetic analysis showed the occurrence of Trichoderma asperellum, Trichoderma asperelloides, Trichoderma afroharzianum, Trichoderma hamatum, Trichoderma lentiforme, Trichoderma koningiopsis, Trichoderma longibrachiatum and Trichoderma erinaceum, in the soil samples. We also identified and describe two new Trichoderma species, both in the harzianum clade of section Pachybasium, which we have named Trichoderma azevedoi sp. nov. and Trichoderma peberdyi sp. nov. The examined strains of both T. azevedoi (three strains) and T. peberdyi (12 strains) display significant genotypic and phenotypic variability, but form monophyletic clades with strong bootstrap and posterior probability support and are morphologically distinct from their respective most closely related species.

Complete Genome Sequences of Seven New Chrysodeixis includens Nucleopolyhedrovirus Isolates from Minas Gerais and Mato Grosso States in Brazil.

We report the complete genomic sequences of seven viral isolates from the soybean looper (Chrysodeixis includens) from midwestern and southeastern Brazil. The genomes range from 138,760 to 139,637 bp in length with a G+C content of 39.2% and 140 open reading frames (ORFs).

Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

DNA Barcoding for the Identification of Phyllanthus Taxa Used Medicinally in Brazil.

Plants of the genus Phyllanthus, principally Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus niruri, and Phyllanthus tenellus, are used in Brazilian folk medicine to treat kidney stones as well as other ailments, where the latter two species are listed in the Brazilian Pharmacopeia as quebra-pedra (stone-breaker). However, only P. niruri has been shown to be effective in a clinical setting. Nuclear ribosomal internal transcribed spacer (ITS1 - 5.8S rRNA-ITS2), internal transcribed spacer 2, and chloroplasts rbcL, matK, psbA-trnH, trnL, and trnL-trnF were screened for their potential as DNA barcodes for the identification of 48 Phyllanthus taxa in Brazilian medicinal plant germplasm banks and in "living pharmacies". The markers were also tested for their ability to validate four commercial herbal teas labelled as quebra-pedra. Using the criterion of high clade posterior probability in Bayesian phylogenetic analysis, the internal transcribed spacer, internal transcribed spacer 2, and chloroplast matK, psbA-trnH, trnL, and trnL-trnF markers all reliably differentiated the four Phyllanthus species, with the internal transcribed spacer and matK possessing the additional advantage that the genus is well represented for these markers in the Genbank database. However, in the case of rbcL, posterior probability for some clades was low and while P. amarus and P. tenellus formed monophyletic groups, P. niruri and P. urinaria accessions could not be reliably distinguished with this marker. Packaged dried quebra-pedra herb from three Brazilian commercial suppliers comprised P. tenellus, but one sample was also found to be mixed with alfalfa (Medicago sativa). An herb marketed as quebra-pedra from a fourth supplier was found to be composed of a mixture of Desmodium barbatum and P. niruri.

The genome sequence of Condylorrhiza vestigialis NPV, a novel baculovirus for the control of the Alamo moth on Populus spp. in Brazil.

Condylorrhiza vestigialis (Lepidoptera: Cambridae), commonly known as the Brazilian poplar moth or Alamo moth, is a serious defoliating pest of poplar, a crop of great economic importance for the production of wood, fiber, biofuel and other biomaterials as well as its significant ecological and environmental value. The complete genome sequence of a new alphabaculovirus isolated from C. vestigialis was determined and analyzed. Condylorrhiza vestigialis nucleopolyhedrovirus (CoveNPV) has a circular double-stranded DNA genome of 125,767bp with a GC content of 42.9%. One hundred and thirty-eight putative open reading frames were identified and annotated in the CoveNPV genome, including 38 core genes and 9 bros. Four homologous regions (hrs), a feature common to most baculoviruses, and 19 perfect and imperfect direct repeats (drs) were found. Phylogenetic analysis confirmed that CoveNPV is a Group I Alphabaculovirus and is most closely related to Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Choristoneura fumiferana DEF multiple nucleopolyhedrovirus CfDEFMNPV. The gp37 gene was not detected in the CoveNPV genome, although this gene is found in many NPVs. Two other common NPV genes, chitinase (v-chiA) and cathepsin (v-cath), that are responsible for host insect liquefaction and melanization, were also absent, where phylogenetic analysis suggests that the loss these genes occurred in the common ancestor of AgMNPV, CfDEFMNPV and CoveNPV, with subsequent reacquisition of these genes by CfDEFMNPV. The molecular biology and genetics of CoveNPV was formerly very little known and our expectation is that the findings presented here should accelerate research on this baculovirus, which will facilitate the use of CoveNPV in integrated pest management programs in Poplar crops.

Complete Genome Sequences of Six Chrysodeixis includens Nucleopolyhedrovirus Isolates from Brazil and Guatemala.

The baculovirus, Chrysodeixis (formerly Pseudoplusia) includens nucleopolyhedrovirus (ChinNPV), is a new Alphabaculovirus pathogenic to Chrysodeixis includens Here, we report the complete genome sequences of six ChinNPV isolates. The availability of these genome sequences will provide information on ChinNPV molecular genetics, promoting understanding of its pathogenicity, diversity, and evolution.

The genome sequence of Pseudoplusia includens single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses.

BACKGROUND: Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated. RESULTS: The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX - Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins. CONCLUSIONS: PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.

Arachis batizocoi: a study of its relationship to cultivated peanut (A. hypogaea) and its potential for introgression of wild genes into the peanut crop using induced allotetraploids.

BACKGROUND AND AIMS: Arachis batizocoi is a wild relative of cultivated peanut (A. hypogaea), an allotetraploid with an AABB genome. Arachis batizocoi was once considered the ancestral donor of the peanut B genome, but cytogenetics and DNA phylogenies have indicated a new genome classification, 'K'. These observations seem inconsistent with genetic studies and breeding that have shown that A. batizocoi can behave as a B genome. METHODS: The genetic behaviour, genome composition and phylogenetic position of A. batizocoi were studied using controlled hybridizations, induced tetraploidy, whole-genome in situ fluorescent hybridization (GISH) and molecular phylogenetics. KEY RESULTS: Sterile diploid hybrids containing AK genomes were obtained using A. batizocoi and the A genome species A. duranensis, A. stenosperma, A. correntina or A. villosa. From these, three types of AAKK allotetraploids were obtained, each in multiple independent polyploidy events. Induced allotetraploids were vigorous and fertile, and were hybridized to A. hypogaea to produce F1 hybrids. Even with the same parental combination, fertility of these F1 hybrids varied greatly, suggesting the influence of stochastic genetic or epigenetic events. Interestingly, hybrids with A. hypogaea ssp. hypogaea were significantly more fertile than those with the subspecies fastigiata. GISH in cultivated × induced allotetraploids hybrids (harbouring AABK genomes) and a molecular phylogeny using 16 intron sequences showed that the K genome is distinct, but more closely related to the B than to the A genome. CONCLUSIONS: The K genome of A. batizocoi is more related to B than to the A genome, but is distinct. As such, when incorporated in an induced allotetraploid (AAKK) it can behave as a B genome in crosses with peanut. However, the fertility of hybrids and their progeny depends upon the compatibility of the A genome interactions. The genetic distinctness of A. batizocoi makes it an important source of allelic diversity in itself, especially in crosses involving A. hypogaea ssp. hypogaea.

Expression of stress-related genes in zebrawood (Astronium fraxinifolium, Anacardiaceae) seedlings following germination in microgravity.

Seeds of a tropical tree species from Brazil, Astronium fraxinifolium, or zebrawood, were germinated, for the first time in microgravity, aboard the International Space Station for nine days. Following three days of subsequent growth under normal terrestrial gravitational conditions, greater root length and numbers of secondary roots was observed in the microgravity-treated seedlings compared to terrestrially germinated controls. Suppression subtractive hybridization of cDNA and EST analysis were used to detect differential gene expression in the microgravity-treated seedlings in comparison to those initially grown in normal gravity (forward subtraction). Despite their return to, and growth in normal gravity, the subtracted library derived from microgravity-treated seedlings was enriched in known microgravity stress-related ESTs, corresponding to large and small heat shock proteins, 14-3-3-like protein, polyubiquitin, and proteins involved in glutathione metabolism. In contrast, the reverse-subtracted library contained a comparatively greater variety of general metabolism-related ESTs, but was also enriched for peroxidase, possibly indicating the suppression of this protein in the microgravity-treated seedlings. Following continued growth for 30 days, higher concentrations of total chlorophyll were detected in the microgravity-exposed seedlings.

Pseudoplusia includens single nucleopolyhedrovirus: genetic diversity, phylogeny and hypervariability of the pif-2 gene.

The soybean looper (Pseudoplusia includens Walker, 1857) has become a major pest of soybean crops in Brazil. In order to determine the genetic diversity and phylogeny of variants of Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IA to -IG), partial sequences of the genes lef-8, lef-9, pif-2, phr and polh were obtained following degenerate PCR and phylogenetic trees constructed using maximum parsimony and Bayesian methods. The aligned sequences showed polymorphisms among the isolates, where the pif-2 gene was by far the most variable and is predicted to be under positive selection. Furthermore, some of the pif-2 DNA sequence mutations are predicted to result in significant amino acid substitutions, possibly leading to changes in oral infectivity of this baculovirus. Cladistic analysis revealed two closely related monophyletic groups, one containing PsinNPV isolates IB, IC and ID and another containing isolates IA, IE, IF and IG. The phylogeny of PsinSNPV in relation to 56 other baculoviruses was also determined from the concatenated partial LEF-8, LEF-9, PIF-2 and POLH/GRAN deduced amino acid sequences, using maximum-parsimony and Bayesian methods. This analysis clearly places PsinSNPV with the Group II Alphabaculovirus, where PsinSNPV is most closely related to Chrysodeixis chalcites NPV and Trichoplusia ni SNPV.

Morphology and molecular taxonomy of Evlachovaea-like fungi, and the status of this unusual conidial genus.

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.

Occurrence of Metarhizium spp. in Central Brazilian soils.

The biodiversity of entomopathogenic fungi in tropical ecosystems is still little investigated, and the objective of this study was to isolate and identify fungi of the entomopathogenic genus Metarhizium (Hypocreales: Clavicipitaceae) present in undisturbed soils of the Central Brazilian Cerrado. A total of 107 Metarhizium isolates was obtained from soils collected from Cerrado sites in the state of Goiás; gene sequences from 63 of these were obtained and compared. Among these, one was confirmed to be M. anisopliae sensu stricto; 53 were very closely allied to M. anisopliae but require more extensive genetic characterization to determine if they might represent a new taxon in the M. anisopliae species complex. Eight of these Cerrado isolates were referable to M. robertsii, and the remaining isolate is the first South American (and Southern Hemisphere) collection of M. flavoviride var. pemphigi. These findings underline the need for better characterization of the diversity of these widely distributed fungi in Brazil.

A study of the relationships of cultivated peanut (Arachis hypogaea) and its most closely related wild species using intron sequences and microsatellite markers.

BACKGROUND AND AIMS: The genus Arachis contains 80 described species. Section Arachis is of particular interest because it includes cultivated peanut, an allotetraploid, and closely related wild species, most of which are diploids. This study aimed to analyse the genetic relationships of multiple accessions of section Arachis species using two complementary methods. Microsatellites allowed the analysis of inter- and intraspecific variability. Intron sequences from single-copy genes allowed phylogenetic analysis including the separation of the allotetraploid genome components. METHODS: Intron sequences and microsatellite markers were used to reconstruct phylogenetic relationships in section Arachis through maximum parsimony and genetic distance analyses. KEY RESULTS: Although high intraspecific variability was evident, there was good support for most species. However, some problems were revealed, notably a probable polyphyletic origin for A. kuhlmannii. The validity of the genome groups was well supported. The F, K and D genomes grouped close to the A genome group. The 2n = 18 species grouped closer to the B genome group. The phylogenetic tree based on the intron data strongly indicated that A. duranensis and A. ipaënsis are the ancestors of A. hypogaea and A. monticola. Intron nucleotide substitutions allowed the ages of divergences of the main genome groups to be estimated at a relatively recent 2·3-2·9 million years ago. This age and the number of species described indicate a much higher speciation rate for section Arachis than for legumes in general. CONCLUSIONS: The analyses revealed relationships between the species and genome groups and showed a generally high level of intraspecific genetic diversity. The improved knowledge of species relationships should facilitate the utilization of wild species for peanut improvement. The estimates of speciation rates in section Arachis are high, but not unprecedented. We suggest these high rates may be linked to the peculiar reproductive biology of Arachis.

Shotgun sequencing for microsatellite identification in Ilex paraguariensis (Aquifoliaceae).

PREMISE OF THE STUDY: Ilex paraguariensis is a native tree species from Brazil, Argentina, and Paraguay that is used in the production of beverages, medicines, and cosmetics. Primers flanking microsatellites were developed to investigate genetic parameters in the species. • METHODS AND RESULTS: Using microsatellites cloned from an I. paraguariensis shotgun genomic library, 25 pairs of primers were designed and synthesized. Levels of polymorphism were evaluated in 24 individuals from two populations. Twenty loci were polymorphic, and an average of 4.8 and 4.5 alleles per locus were detected in the two populations, respectively. The mean observed heterozygosity was lower than the expected heterozygosity (0.54 vs. 0.60), indicating a departure from Hardy-Weinberg equilibrium and suggesting endogamy in both populations. • CONCLUSIONS: The reported set of markers is highly informative and constitutes a powerful tool for the development of genetic characterization studies in I. paraguariensis.

Identification and sequence analysis of the Condylorrhiza vestigialis MNPV p74 gene.

The baculovirus Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV), isolated from C. vestigialis infected larvae in Paraná (Brazil), was identified in our laboratory. A full-length clone was obtained from the CoveMNPV genome, of the gene that encodes the homolog to baculoviral p74, essential for oral infectivity which was then sequenced and characterized. The CoveMNPV p74 gene (GenBank accession number EU919397) contains an ORF of 1935 bp that encodes a deduced protein of 73.61 kDa. The phylogenetic affiliations of the CoveMNPV gene were determined by a heuristic search of 40 aligned baculovirus p74 nucleotide sequences using maximum parsimony (PAUP 4.0b4a). The phylogenetic analysis placed CoveMNPV within lepidopteran nucleopolyhedrovirus (NPV) Group I, Clade A, as being the closest to Choristoneura fumiferana defective NPV.