Plastid genome evolution of a monophyletic group in the subtribe Lauriineae (Laureae,Lauraceae)

Litsea, a non-monophyletic group of the tribe Laureae (Lauraceae), plays important roles in the tropical and subtropical forests of Asia, Australia, Central and North America, and the islands of the Pacific. However, intergeneric relationships between Litsea and Laurus, Lindera, Parasassafras and Sinosassafras of the tribe Laureae remain unresolved. In this study, we present phylogenetic analyses of seven newly sequenced Litsea plastomes, together with 47 Laureae plastomes obtained from public databases, representing six genera of the Laureae. Our results highlight two highly supported monophyletic groups of Litsea taxa. One is composed of 16 Litsea taxa and two Lindera taxa. The 18 plastomes of these taxa were further compared for their gene structure, codon usage, contraction and expansion of inverted repeats, sequence repeats, divergence hotspots, and gene evolution. The complete plastome size of newly sequenced taxa varied between 152,377 bp (Litsea auriculata) and 154,117 bp (Litsea pierrei). Seven of the 16 Litsea plastomes have a pair of insertions in the IRa (trnL-trnH) and IRb (ycf2) regions. The 18 plastomes of Litsea and Lindera taxa exhibit similar gene features, codon usage, oligonucleotide repeats, and inverted repeat dynamics. The codons with the highest frequency among these taxa favored A/T endings and each of these plastomes had nine divergence hotspots, which are located in the same regions. We also identified six protein coding genes (accD, ndhJ, rbcL, rpoC2, ycf1 and ycf2) under positive selection in Litsea; these genes may play important roles in adaptation of Litsea species to various environments.     

Phylogeny of <Emphasis Type="Italic">Litsea</Emphasis> and related genera (Laureae-Lauraceae) based on analysis of <Emphasis Type="Italic">rpb2</Emphasis> gene sequences

The relationship between Litsea and related genera is currently unclear. Previous molecular studies on these taxa using cpDNA and nrITS were unable to produce well-resolved phylogenetic trees. In this study, we explored the potential of the rpb2 gene as a source of molecular information to better resolve the phylogenetic analysis. Although rpb2 was believed to be a single-copy gene, our cloning results showed that most species examined possessed several copies of these sequences. However, the genetic distance among copies from any one species was low, and these copies always formed monophyletic groups in our molecular trees. Our phylogenetic analyses of rpb2 data resulted in better resolved tree topologies compared to those based on cpDNA or nrITS data. Our results show that monophyly of the genus Litsea is supported only for section Litsea. As a genus, Litsea was shown to be polyphyletic. The genera Actinodaphne and Neolitsea were resolved as monophyletic groups in all analyses. They were also shown to be sisters and closer to the genus Lindera than to the genus Litsea. Our results also revealed that the genus Lindera is not a monophyletic group.     

Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed.     

Insights from the docking analysis of biologically active compounds from plant Litsea Genus as potential COX-2 inhibitors

Litsea spp of Laural family are traditionally used as herbal medicine for treating inflammation including gastroenterologia, oedema and rheumatic arthritis. Therefore, it is of interest to investigate and understand the molecular principles for such actions. Here, we have illustrated the binding of thirteen Litsea derived biologically active compounds against the inflammation associated target COX (cyclo-oxygenase) -2 enzymes. We compared the binding information of these compounds with a selected number of already known COX-2 inhibitors. The comparison reflected that some of these compounds such as linderol, catechin, 6''-hydroxy-2'',3'',4'' - trimethoxy-chalcone and litseaone have better or equivalent binding features compared to already known inhibitory compounds namely celecoxib, acetylsalicylic acid, rofecoxib. Therefore, all these small compounds reported from plant Litsea spp were found to possess potential medicinal values with anti-inflammatory properties.     

Evaluation of FT-IR spectroscopy as a tool to quantify bacteria in binary mixed cultures

Fourier-transform infrared (FT-IR) spectroscopy is known as a high-resolution method for the rapid identification of pure cultures of microorganisms. Here, we evaluated FT-IR as a method for the quantification of bacterial populations in binary mixed cultures consisting of Pseudomonas putida and Rhodococcus ruber. A calibration procedure based on Principal Component Regression was developed for estimating the ratio of the bacterial species. Data for method calibration were gained from pure cultures and artificially assembled communities of known ratios of the two member populations. Moreover, to account for physiological variability, FT-IR measurements were performed with organisms sampled at different growth phases. Measurements and data analyses were subsequently applied to growing mixed cultures revealing that growth of R. ruber was almost completely suppressed in co-culture with P. putida. Population ratios obtained by fatty acid analysis as an independent reference method were in high agreement with the FT-IR derived ratios.     

Genetic diversity among endangered Uruguayan populations of Butia Becc. species based on ISSR

The genus Butia Becc. (Arecaceae) comprises 18 species distributed exclusively in South America, four of them in Uruguay: Butia lallemantii, B. paraguayensis, B. yatay and B. capitata. All species show serious regeneration problems which jeopardise their continuity in the near future. Butia lallemantii and B. paraguayensis are in the most critical situation, with restricted distributions (fragmented and reduced by cattle grazing and forestation) and populations with few individuals. Taxonomy in the genus is complex, and species delimitation remains unclear. The aim of this study was to examine the genetic variability of natural populations of B. paraguayensis, B. lallemantii and B. yatay, based on inter?Csimple sequence repeat markers, in view of long term conservation plans and to assist the taxonomic resolution of these closely related species. Twenty individuals from each population were sampled including four populations of B. lallemantii, three of B. yatay and the only B. paraguayensis population reported for Uruguay. Syagrus romanzoffiana was used as outgroup. Five primers were selected based on amplification profiles. Relationships among species were evaluated by constructing dendrograms and principal coordinates analysis. Genetic distance analyses indicate the existence of low variability among Butia species. Variability within populations was high, possibly due to gene flow, past hybridisation or life history traits. This variability provides great potential for recovery. Results do not allow us to suggest changes to the present taxonomic status of these species. We propose management recommendations involving restoration of recruitment and augmentation via stocks from within populations.     

Molecular identification and phylogeny of an aquatic moss species in Antarctic lakes

Due to the morphological variability, the identification of moss species can be difficult when the plant grows in submerged environments. The taxonomic status of an aquatic moss found in lakes of the Sôya Coast region, East Antarctica, had been controversial, and then, it was investigated by molecular phylogenetic and haplotype network analysis of two chloroplast regions (rps4 and trnL-F) and/or the nuclear ribosomal ITS region. Based on the results of the analyses, the moss was assigned to the genus Leptobryum and determined to be conspecific with Leptobryum wilsonii (Mitt.) Broth. described from South America. Almost no genetic variation was observed between all samples from Antarctic lakes and some samples of L. wilsonii from Chile. Molecular and geohistorical evidence suggests that immigration of L. wilsonii into Antarctic lakes took place during the Holocene via long-distance dispersal from South America. This study gives a clear example of the widespread assumption that most of the Antarctic moss species are post-glacial immigrants.     

Population Structure and Distribution Patterns of the Sibling Mosquito Species Culex pipiens and Culex torrentium (Diptera: Culicidae) Reveal Different Evolutionary Paths

Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species'' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima''s D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (F_ST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (Cx. pipiens [88.38%]; Cx. torrentium [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species'' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.     

An Examination of Morphometric Variations in a Neotropical Toad Population (Proceratophrys cristiceps,Amphibia, Anura,Cycloramphidae)

The species Proceratophrys cristiceps belongs to the genus Proceratophrys within the family Cycloramphidae. These amphibians are found exclusively in South America in the morphoclimatic domain of the semi-arid depression zones in northeastern Brazil known as the Caatinga. We examined intrapopulational variation using univariate and multivariate statistics with traditional and geometric morphometrics, which supported the existence of two morphotypes of this species. Our results indicated significant degrees of variation in skeletal characteristics between some natural populations of this species. Careful analyses of variability levels are fundamental to avoid taxonomic errors, principally in populations that demonstrate characteristics intimately associated with their area of occurrence, as is the case of Proceratophrys cristiceps.     

High diversity of Lauraceae from the Oligocene of Ningming,South China

Lauraceae is among the largest and floristically most important woody plant families in subtropical and tropical zones. However, the Cenozoic fossil records of Lauraceae are mostly from mid-latitude regions. Here we described eight lauraceous species from the Oligocene of Ningming, Guangxi, South China on the basis of leaf compressions with cuticular structure, providing new evidence for high diversity of Lauraceae in the low latitude region of the northern hemisphere during the late Paleogene. The extant genera to which the Ningming Oligocene fossils are possibly closely related include Cinnamomum, Neolitsea, Litsea, Alseodaphne, Laurus, and Beilschmiedia. All of these extant genera except Laurus still exist in Guangxi today, whereas Laurus is currently distributed in the Mediterranean and adjacent regions. A cladistic analysis based on leaf architectural and leaf cuticular characters for Paleogene Lauraceae species in the world indicated a possible relationship between European and East Asian Paleogene species.     

Genetic connectivity among osprey populations and consequences for conservation: philopatry versus dispersal as key factors

Genetic variability and population structure in osprey were studied using DNA microsatellite markers. Special emphasis was placed on the subspecies living in the Afro-Palearctic (Pandion haliaetus haliaetus). For comparative purposes, American osprey subspecies (P. h. carolinensis, P. h. ridgway) and Indo/Australian subspecies (P. h. cristatus) were included in this analysis. Twenty DNA microsatellite loci were analysed across a total of 200 individuals. Cluster analysis of genetic distances generally grouped populations of osprey in accordance with their subspecific designation and with previous results from mtDNA analysis. Ospreys from America and Australia were clearly separated from P. h. haliaetus suggesting a more ancient isolation which prevented recent gene flow across these groups. Within P. h. haliaetus, significant genetic differentiation was found between populations in northern and southern Europe, suggesting that the Afro-Palearctic group is structured into two interconnected entities (Mediterranean and continental Europe). Population structuring was supported by an assignment test and by analysis of allele-sharing among individuals. At the Mediterranean scale, no significant differences of allelic information were found between populations. Behaviours such as dispersal, migration and philopatry seem to have played simultaneously and in contrary directions in shaping the genetic structure and diversity of populations. Our results provide essential information for reconstructing gene flow and genetic variability among osprey populations at different scales, which call for caution in the proactive management and conservation of the species, namely in the Mediterranean area.     

Phylogeographic history of the woodwardioid ferns,including species from the Himalayas

The woodwardioid ferns are well-represented in the Northern Hemisphere, where they are disjunctly distributed throughout the warm temperate and subtropical regions of North America, Europe, and Asia. To infer the biogeographic history of the woodwardioid ferns, the phylogeny of Woodwardia was estimated using rbcL and rps4 sequences from divergent distribution regions including the Himalayas. Phylogenetic results support Woodwardia as a monophyletic group with Woodwardia areolatae and W. virginica as basal, these two species from eastern North America diverged early, which are sister clades to the remaining species from America, Europe, and Asia. Based on analyses of the fossil records of these species for divergence times, Woodwardia species were estimated to have diverged initially in the Paleogene of North America. After its New World origin, a greater diversification and expansion of Woodwardia occurred in eastern Eurasia, with the European arrival of Woodwardia radicans during the Middle Miocene. Compared to earlier reports, a migration back into North America via the Bering land bridge is consistent with these data.     

Conservation study of an endangered stingless bee (Melipona capixaba—Hymenoptera: Apidae) with restricted distribution in Brazil

Melipona capixaba, popularly known as “uruçu preta”, is a stingless bee restricted to the mountainous Atlantic Rainforest areas of Espírito Santo State, Brazil. Due to the endemism and small population size, this species discovered in 1994 is now considered “vulnerable to extinction”. Using ISSR, PCR–RFLP and microsatellites markers, we studied the genetic variability and structure of M. capixaba from 88 colonies collected throughout the distribution area of the species within Espírito Santo State. The microsatellite, ISSR and mitochondrial haplotype analyses showed that M. capixaba has low genetic variability compared to other insect species. The molecular analyses also indicated a high genetic similarity among the M. capixaba samples, with no clear pattern of structuring. The analyses of molecular variance results indicated that most of the total genetic variation in M. capixaba was explained by the genetic diversity within local populations. Results suggest that the analyzed samples could be treated as a single population for preservation purposes. Thus, given its endemism, local adaptation and low number of natural colonies, efforts for the conservation of M. capixaba should focus on preservation and increasing the number of colonies in the wild, so that M. capixaba can support constant captures and the effects of habitat deforestation in Espírito Santo State.     

Unraveling the evolutionary scenario of the hobo element in populations of Drosophila melanogaster and D. simulans in South America using the TPE repeats as markers

Transposable elements (TEs) are nucleotide sequences found in most studied genomes. These elements are highly diversified and have a large variation in nucleotide structure and mechanisms of transposition. hobo is a member of class II, belonging to hAT superfamily, described inDrosophila melanogaster, and it presents in its Open Reading Frame, a repetitive region encoding the amino acids threonine-proline-glutamic acid (TPE), which shows variability in the number of repeats in some regions of the world. Due to this variability some evolutionary scenarios of the hobo element are discussed, such as the scenario of the invasion of hobo element in populations ofD. melanogaster. In the present study, we investigated 22 DNA sequences of D. melanogaster and seven sequences ofD. simulans, both from South America, to check the number of repetitions of TPE, in order to clarify the evolutionary scenario of thehobo element in these populations. Our results showed a monomorphism in populations of both species in South America, with only three TPE repeats. Hence, we discuss and propose an evolutionary scenario of the invasion of the hobo element in populations of D. melanogaster and D. simulans.     

Phylogenetic relationships and intraspecific diversity of a North Patagonian Fescue: evidence of differentiation and interspecific introgression at peripheral populations

Peripheral sites usually offer suboptimal conditions for species with wide distributions, where expression of phenotypic variability and potential interspecific hybridization might be enhanced. The Patagonian steppe, the largest and southernmost dryland ecosystem in South America, is characterized by natural rangelands dominated by grasses. Festuca pallescens is a keystone species with a wide distribution in Patagonia over diverse environments reaching the extreme arid zones in the Somuncura plateau, a biogeographical island. Our aim is to study the phylogenetic relationships among Festuca pallescens populations as well as between this species and the sympatric F. argentina in North Patagonia. We analysed fourteen populations along a west-east transect of about 500 km in North Patagonia with three types of molecular markers: ITS, chloroplast trnL-F and eight nuclear microsatellites. Bayesian inferences, maximum parsimony and maximum likelihood analyses with trnL-F and ITS showed that F. pallescens is related to the Patagonian clade within the Festuca phylogeny. However, the easternmost populations of F. pallescens at Somuncura plateau were highly differentiated from the other populations and clustered with F. argentina (a sympatric species of the Asian-American clade). Principal coordinates analyses and Bayesian clustering performed with nuclear microsatellites as well as morphoanatomical traits, showed an intermediate position of one of these easternmost populations with respect to the two species, suggesting admixture. The high genetic variability observed in these peripheral populations highlight their relevance for conservation and might be indicating the existence of evolutionary processes triggering events of speciation in the Patagonian fescues.     

Insight into genetic relation and diversity of cultivated and semi-domesticated under-utilized Crotalaria species gained using start codon targeted (SCoT) markers

To augment conventional crop improvement approaches in cultivated sunnhemp (Crotalaria juncea L.) and other under-utilized Crotalaria species, genetic diversity of 94 genotypes from seven Crotalaria species was studied using 20 Start Codon Targeted (SCoT) markers. High allele number (1.32), polymorphism information content (0.37) and resolving power (6.59) established SCoT as a reliable marker system for genetic analysis in Crotalaria. All the species except Crotalaria retusa L. exhibited high number of SCoT amplicons. Analysis of molecular variance revealed significant variability between (24.0%) the species as well as within species (76.0%). A cluster analysis identified distinct groups corresponding to the seven species and also identified sub-groups within the species. The sunnhemp cultivars were distant from the landraces, suggesting the need of population improvement using distantly related genotypes. Species relationship identified Crotalaria pallida Aiton to be a close relative of C. juncea. The results of principal coordinate analysis were comparable to that of cluster analysis, revealing high genetic variability in sunnhemp and other semi-domesticated Crotalaria species. The study further suggests some measure for conservation of genetic resources and genetic improvement of these species based on the results of diversity analysis.     

A New Fishfly Species (Megaloptera: Corydalidae: Neohermes Banks) Discovered from North America by a Systematic Revision,with Phylogenetic and Biogeographic Implications

The taxonomy of Megaloptera from the Nearctic region is fairly well known and their faunal diversity has been largely surveyed, even in relatively remote regions. However, the evolutionary history of Nearctic Megaloptera is still poorly known with phylogenetic and biogeographic studies lacking. In this paper, we report a new fishfly species of the endemic North American genus Neohermes Banks, 1908, increasing the total number known of species to six. This new species (Neohermes inexpectatus sp. nov.) is currently known to occur only in California (USA) and is apparently confined to the Northern Coastal Range. The new species resembles the three Neohermes species from eastern North America based on the relatively small body size and the presence of female gonostyli 9. However, our phylogenetic analysis using adult morphological data recovered the new species as the sister species to the remaining Neohermes, which includes two species from western North America and three from eastern North America. According to the present interspecific phylogeny of Neohermes, with reconstructed ancestral areas, the initial divergence within the genus was found to take place in western North America, with a subsequent eastward dispersal. This likely lead to the modern distribution of Neohermes in eastern North America with the closure of the Mid-Continental Seaway, which separated western and eastern North America in the Mid-Late Cretaceous (100–80 MYA) and finally disappeared at the end of the Cretaceous (70 MYA). The uplift of the Cordilleran System probably accounted for the divergence between the eastern and two western Neohermes species.     

Phylogenetic diversity of true morels (Morchella), the main edible non-timber product from native Patagonian forests of Argentina

Morchella species are edible fungi in high demand and therefore command high prices in world markets. Phenotypic-based identification at the species-level remains inadequate because of their complex life cycles, minor differences and plasticity of morphological characteristics between species, and the lack of agreement between scientific and common names. In Patagonia–Argentina, morels are associated with native forests of Austrocedrus chilensis (Cordilleran or Chilean cypress) and Nothofagus antarctica (ñire) and several exotic conifers that were introduced from western North America. Little is known about their taxonomy and phylogenetic relationships with other species in the genus. This work focused on the identification of collections of Morchella from Patagonia and their phylogenetic relationships with other species from the Northern Hemisphere. The comparison was made by analysis of DNA sequences obtained from four loci: the nuclear ribosomal internal transcribed spacer region (ITS) and the partial RNA polymerase I gene (RPB1) for the complete collection; and ITS, RPB1, RNA polymerase II gene (RPB2), and translation elongation factor (EF1-α) for the species-rich Elata Subclade. Analyses of individual and combined data sets revealed that Patagonian morels belong to the Elata Clade and comprised three strongly supported species-level lineages from both Patagonian native forest, and exotic trees introduced from western North America. One lineage was identified as Morchella frustrata phylogenetic species Mel-2, which is known from the USA and Canada. The second lineage, which appeared to be ‘fire-adapted’, was identified as Morchella septimelata phylogenetic species (Mel-7), which is also known from the USA. This species was collected from burned native forests mainly composed of A. chilensis and N. antarctica but also Pseudotsuga menziesii (Mirb.) Blanco, which is native to western North America. The phylogenetic analyses suggested that the third species from Patagonia was nested within the species-rich Elata Subclade and represents a new species-level lineage (informally designated Mel-37) within Elata Clade. The present collections from Patagonia constitute the southernmost latitude from which Morchella has been reported to date. The identification of two Argentine morels as North American taxa is therefore a remarkable biogeographic pattern. In view of the hypothesis that the Elata Clade originated in western North America, we speculate that at least two of the lineages colonized South America from North America via long distance dispersal, migration or, more likely, they were introduced with the exotic tree species that they were collected near.     

Physiological and anatomical trait variability of dominant C4 grasses

Climate variability is a key driver of physiological responses in common grass species in grasslands of North America. Differences in microanatomical traits among coexisting species may influence physiological responses to climate variability over large geographic scales. The goal of this research was to determine leaf-level physiological and microanatomical trait variability among four dominant C₄ grass species across a natural precipitation gradient. Physiological traits were observed to vary significantly across the gradient with greater variability than microanatomical traits. Microanatomical traits were shown to predict physiological responses in A. gerardii and P. virgatum, but the nature of the relationships varied between species. These results illustrate that microanatomical and physiological traits vary across a precipitation gradient, there are clear linkages between microanatomy and physiology in grass species, and this evidence underscores the need for further investigation using phylogenetically diverse assemblages.     

Molecular characterization revealed Fasciola specimens in Ecuador are all Fasciola hepatica,none at all of Fasciola gigantica or parthenogenic Fasciola species

All 225 Fasciola flukes obtained from domestic animals (73 cattle, 7 sheep and 1 pig) of 18 distinct geographic areas in Ecuador-South America, were identified as Fasciola hepatica, based on molecular analyses of nuclear pepck and pold genes, and mitochondrial nad1gene as well as the morphological observation of sperm within the seminal vesicles. Fasciola gigantica and parthenogenic Fasciola forms endemic to Asian countries were not found in this study, although zebu cattle and water buffalos have introduced into South America from Asia; this could be due to the absence of suitable intermediate host snails. The results of pepck analysis using multiplex PCR developed previously showed that 32 of the flukes could not be confirmed as F. hepatica, suggesting that the method is unreliable for the accurate discrimination of F. hepatica, and that pepck gene of the species consists of multiple loci, not a single locus. The results of genetic diversity, phylogenetic, and network analyses based on mitochondrial nad1 sequences suggest that F. hepatica populations in South America, including Ecuador, formed from the ancestral F. hepatica individuals introduced into the continent along with anthropogenic movement of livestock infected with the species.