Genetic Differentiation of the Mitochondrial Cytochrome Oxidase c Subunit I Gene in Genus Paramecium (Protista,Ciliophora)

Background

The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses.

Methodology/Principal findings

We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure.

Conclusions

Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.     

The Effectiveness of Three Regions in Mitochondrial Genome for Aphid DNA Barcoding: A Case in Lachininae

Background

The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding.

Methodology/Principal Findings

Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in “best match” and 90.8% in “best close match”) and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of “tag barcodes” is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the “barcoding overlap” can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the “best close match” technique.

Conclusions/Significance

A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of “tag barcodes” can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.     

Molecular Differentiation of the African Yellow Fever Vector Aedes bromeliae (Diptera: Culicidae) from Its Sympatric Non-vector Sister Species,Aedes lilii

Introduction

Yellow fever continues to be a problem in sub-Saharan Africa with repeated epidemics occurring. The mosquito Aedes bromeliae is a major vector of yellow fever, but it cannot be readily differentiated from its non-vector zoophilic sister species Ae. lilii using morphological characters. Genetic differences have been reported between anthropophilic Ae. bromeliae and zoophilic Ae. lilii and between forest and domestic populations. However, due to the application of different molecular markers and non-overlapping populations employed in previous studies, interpretation of species delimitation is unclear.

Methodology/Principle Findings

DNA sequences were generated from specimens of Ae. simpsoni s.l. from the Republic of Benin, Tanzania and Uganda for two nuclear genes apolipophorin 2 (apoLp2) and cytochrome p450 (CYPJ92), the ribosomal internal transcribed spacer region (ITS) and the mitochondrial cytochrome c oxidase (COI) barcoding region. Nuclear genes apoLp2 and CYPJ92 were unable to differentiate between species Ae. bromeliae and Ae. lilii due to ancestral lineage sorting, while ITS sequence data provided clear topological separation on a phylogeny. The standard COI barcoding region was shown to be subject to species introgression and unable to clearly distinguish the two taxa. Here we present a reliable direct PCR-based method for differentiation of the vector species Ae. bromeliae from its isomorphic, sympatric and non-biomedically important sister taxon, Ae. lilii, based on the ITS region. Using molecular species verification, we describe novel immature habitats for Ae. lilii and report both sympatric and allopatric populations. Whereas only Ae. lilii is found in the Republic of Benin and only Ae. bromeliae in Tanzania, both species are sympatric in Uganda.

Conclusions/Significance

Our accurate identification method will allow informed distribution and detailed ecological studies that will facilitate assessment of arboviral disease risk and development of future targeted vector control.     

Selection and viability after ingestion of vegetative cells, resting spores and resting cells of the marine diatom, Chaetoceros pseudocurvisetus, by two copepods

Feeding response of two copepods Neocalanus flemingeri and Calanus sinicus on cultures of three life-forms; vegetative cells, resting spores and resting cells, of Chaetoceros pseudocurvisetus was investigated. N. flemingeri fed heavily on the vegetative cells but scarcely responded to feed on the resting spores. C. sinicus showed significantly higher filtering rate on the vegetative cells and resting cells than on the resting spores. Survival of the three life-forms of C. pseudocurvisetus after gut passage of the copepods was also studied. The resting spores could germinate from fecal pellets of both N. flemingeri and C. sinicus; however, both the vegetative cells and the resting cells could not survive ingestion by the copepods. These results suggest that resting spore forming diatoms, such as C. pseudocurvisetus form spores which have a low nutritional value and during gut passage are largely indigestible due to the heavily silicified frustules and thus minimize the effects of grazing by copepods.     

Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima,Bolivia, Assayed with qPCR and 12S Cloning

Background

In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.

Methodology/Principal Findings

We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors).

Conclusions/Significance

We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.     

Cryptic speciation in Brazilian Epiperipatus (Onychophora: Peripatidae) reveals an underestimated diversity among the peripatid velvet worms

Background

Taxonomical studies of the neotropical Peripatidae (Onychophora, velvet worms) have proven difficult, due to intraspecific variation and uniformity of morphological characters across this onychophoran subgroup. We therefore used molecular approaches, in addition to morphological methods, to explore the diversity of Epiperipatus from the Minas Gerais State of Brazil.

Methodology/Principal Findings

Our analyses revealed three new species. While Epiperipatus diadenoproctus sp. nov. can be distinguished from E. adenocryptus sp. nov. and E. paurognostus sp. nov. based on morphology and specific nucleotide positions in the mitochondrial cytochrome c oxidase subunit I (COI) and small ribosomal subunit RNA gene sequences (12S rRNA), anatomical differences between the two latter species are not evident. However, our phylogenetic analyses of molecular data suggest that they are cryptic species, with high Bayesian posterior probabilities and bootstrap and Bremer support values for each species clade. The sister group relationship of E. adenocryptus sp. nov. and E. paurognostus sp. nov. in our analyses correlates with the remarkable morphological similarity of these two species. To assess the species status of the new species, we performed a statistical parsimony network analysis based on 582 base pairs of the COI gene in our specimens, with the connection probability set to 95%. Our findings revealed no connections between groups of haplotypes, which have been recognized as allopatric lineages in our phylogenetic analyses, thus supporting our suggestion that they are separate species.

Conclusions/Significance

Our findings suggest high cryptic species diversity and endemism among the neotropical Peripatidae and demonstrate that the combination of morphological and molecular approaches is helpful for clarifying the taxonomy and species diversity of this apparently large and diverse onychophoran group.     

DNA Barcoding Reveals Cryptic Diversity within Commercially Exploited Indo-Malay Carangidae (Teleosteii: Perciformes)

Background

DNA barcodes, typically focusing on the cytochrome oxidase I gene (COI) in many animals, have been used widely as a species-identification tool. The ability of DNA barcoding to distinguish species from a range of taxa and to reveal cryptic species has been well documented. Despite the wealth of DNA barcode data for fish from many temperate regions, there are relatively few available from the Southeast Asian region. Here, we target the marine fish Family Carangidae, one of the most commercially-important families from the Indo-Malay Archipelago (IMA), to produce an initial reference DNA barcode library.

Methodology/Principal Findings

Here, a 652 bp region of COI was sequenced for 723 individuals from 36 putative species of Family Carangidae distributed within IMA waters. Within the newly-generated dataset, three described species exhibited conspecific divergences up to ten times greater (4.32–4.82%) than mean estimates (0.24–0.39%), indicating a discrepancy with assigned morphological taxonomic identification, and the existence of cryptic species. Variability of the mitochondrial DNA COI region was compared within and among species to evaluate the COI region''s suitability for species identification. The trend in range of mean K2P distances observed was generally in accordance with expectations based on taxonomic hierarchy: 0% to 4.82% between individuals within species, 0% to 16.4% between species within genera, and 8.64% to 25.39% between genera within families. The average Kimura 2-parameter (K2P) distance between individuals, between species within genera, and between genera within family were 0.37%, 10.53% and 16.56%, respectively. All described species formed monophyletic clusters in the Neighbour-joining phylogenetic tree, although three species representing complexes of six potential cryptic species were detected in Indo-Malay Carangidae; Atule mate, Selar crumenophthalmus and Seriolina nigrofasciata.

Conclusion/Significance

This study confirms that COI is an effective tool for species identification of Carangidae from the IMA. There were moderate levels of cryptic diversity among putative species within the central IMA. However, to explain the hypothesis of species richness in the IMA, it is necessary to sample the whole family across their broad geographic range. Such insights are helpful not only to document mechanisms driving diversification and recruitment in Carangidae, but also to provide a scientific framework for management strategies and conservation of commercially-important fisheries resources.     

Molecular Identification of Birds: Performance of Distance-Based DNA Barcoding in Three Genes to Delimit Parapatric Species

Background

DNA barcoding based on the mitochondrial cytochrome oxidase subunit I gene (cox1 or COI) has been successful in species identification across a wide array of taxa but in some cases failed to delimit the species boundaries of closely allied allopatric species or of hybridising sister species.

Methodology/Principal Findings

In this study we extend the sample size of prior studies in birds for cox1 (2776 sequences, 756 species) and target especially species that are known to occur parapatrically, and/or are known to hybridise, on a Holarctic scale. In order to obtain a larger set of taxa (altogether 2719 species), we include also DNA sequences of two other mitochondrial genes: cytochrome b (cob) (4614 sequences, 2087 species) and 16S (708 sequences, 498 species). Our results confirm the existence of a wide gap between intra- and interspecies divergences for both cox1 and cob, and indicate that distance-based DNA barcoding provides sufficient information to identify and delineate bird species in 98% of all possible pairwise comparisons. This DNA barcoding gap was not statistically influenced by the number of individuals sequenced per species. However, most of the hybridising parapatric species pairs have average divergences intermediate between intraspecific and interspecific distances for both cox1 and cob.

Conclusions/Significance

DNA barcoding, if used as a tool for species discovery, would thus fail to identify hybridising parapatric species pairs. However, most of them can probably still assigned to known species by character-based approaches, although development of complementary nuclear markers will be necessary to account for mitochondrial introgression in hybridising species.     

On the value of nuclear and mitochondrial gene sequences for reconstructing the phylogeny of vanilloid orchids (Vanilloideae, Orchidaceae)

Background and Aims

Most molecular phylogenetic studies of Orchidaceae have relied heavily on DNA sequences from the plastid genome. Nuclear and mitochondrial loci have only been superficially examined for their systematic value. Since 40% of the genera within Vanilloideae are achlorophyllous mycoheterotrophs, this is an ideal group of orchids in which to evaluate non-plastid gene sequences.

Methods

Phylogenetic reconstructions for Vanilloideae were produced using independent and combined data from the nuclear 18S, 5·8S and 26S rDNA genes and the mitochondrial atpA gene and nad1b-c intron.

Key Results

These new data indicate placements for genera such as Lecanorchis and Galeola, for which plastid gene sequences have been mostly unavailable. Nuclear and mitochondrial parsimony jackknife trees are congruent with each other and previously published trees based solely on plastid data. Because of high rates of sequence divergence among vanilloid orchids, even the short 5·8S rDNA gene provides impressive levels of resolution and support.

Conclusions

Orchid systematists are encouraged to sequence nuclear and mitochondrial gene regions along with the growing number of plastid loci available.Key words: 26S rDNA, 18S rDNA, 5·8S rDNA, atpA, nad1, orchids, plastid, Vanilla, vanilloid orchids, Vanilloideae     

Genetic variation and relationships of seven sturgeon species and ten interspecific hybrids

Background

Sturgeon cultivation is important for both industry and aquaculture in China. To date, more than 17 species or strains have been farmed for fillets and caviar production. Crossbreeding among different sturgeon species is frequent and the F2 hybrids are fertile. However, large-scale farming can have negative impacts on wild populations i.e. escape of exotic sturgeons and must be taken into consideration. Escape of exotic sturgeons can cause severe ecological problems, including threatening native sturgeon species once the exotic varieties become established or hybridize with native individuals. However, little is known about their genetic resources and variation.

Methods

Genetic diversity and introgression of seven sturgeon species were analyzed using mitochondrial DNA cytochrome oxidase subunit I (COI) and nine microsatellite markers. This study included 189 individuals from seven sturgeon species and 277 individuals from ten lineages of F2 hybrid strains.

Results

MtDNA COI sequences (632 bp long) were generated from 91 individuals across the 17 sturgeon strains and produced 23 different haplotypes. Haplotype diversity was high (h = 0.915 ± 0.015) and nucleotide diversity was low (π = 0.03680 ± 0.00153) in the seven sturgeon species and ten interspecific hybrids. Phylogenetic analyses resulted in almost identical tree topologies, and different haplotype structures were mainly related with sturgeons of different female parents. Analysis of molecular variance revealed that 81.73% of the genetic variance was due to matrilineal differences, while 9.40% resulted from strain variation. Pairwise Fst values obtained with POLYSAT software, were high among strains and ranged from 0.031 to 0.164. Admixture analysis assigned seven distinct groups and ten genotypes of admixed clusters composed of hybrid strains using STRUCTURE when assuming K = 7.

Conclusions

The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions. These relationships were also supported by the results from the microsatellite analysis and contributed to unambiguously identify seven sturgeon species and ten F2 hybrid strains from sturgeon farms in China. Moreover, we found that introgressive hybridization is pervasive, exists in both purebred and hybrid sturgeons, and may reflect widespread mismanagement in sturgeon breeding in China.     

Anatomy and taxonomic status of the chasmosaurine ceratopsid Nedoceratops hatcheri from the upper Cretaceous Lance Formation of Wyoming, U.S.A

Background

The validity of Nedoceratops hatcheri, a chasmosaurine ceratopsid dinosaur known from a single skull recovered in the Lance Formation of eastern Wyoming, U.S.A., has been debated for over a century. Some have argued that the taxon is an aberrant Triceratops, and most recently it was proposed that N. hatcheri represents an intermediate ontogenetic stage between “young adult” and “old adult” forms of a single taxon previously split into Triceratops and Torosaurus.

Methodology/Principal Findings

The holotype skull of Nedoceratops hatcheri was reexamined in order to map reconstructed areas and compare the specimen with other ceratopsids. Although squamosal fenestrae are almost certainly not of taxonomic significance, some other features are unique to N. hatcheri. These include a nasal lacking a recognizable horn, nearly vertical postorbital horncores, and relatively small parietal fenestrae. Thus, N. hatcheri is tentatively considered valid, and closely related to Triceratops spp. The holotype of N. hatcheri probably represents an “old adult,” based upon bone surface texture and the shape of the horns and epiossifications on the frill. In this study, Torosaurus is maintained as a genus distinct from Triceratops and Nedoceratops. Synonymy of the three genera as ontogenetic stages of a single taxon would require cranial changes otherwise unknown in ceratopsids, including additions of ossifications to the frill and repeated alternation of bone surface texture between juvenile and adult morphotypes.

Conclusions/Significance

Triceratops, Torosaurus, and likely Nedoceratops, are all distinct taxa, indicating that species richness for chasmosaurine ceratopsids in the Lance Formation just prior to the Cretaceous-Paleocene extinction was roughly equivalent to that earlier in the Cretaceous.     

Major trends in stem anatomy and growth forms in the perianth-bearing Piperales,with special focus on Aristolochia

Background and Aims

The order Piperales has the highest diversity of growth forms among the earliest angiosperm lineages, including trees, shrubs, climbers and herbs. However, within the perianth-bearing Piperales (Asarum, Saruma, Lactoris, Hydnora, Prosopanche, Thottea and Aristolochia), climbing species only occur in the most species-rich genus Aristolochia. This study traces anatomical and morphological traits among these lineages, to detect trends in growth form evolution and developmental processes.

Methods

Transverse stem sections of different developmental stages of representatives of Asarum, Saruma, Lactoris, Hydnora, Thottea and Aristolochia were compared and anatomical traits were linked to growth form evolution. Biomechanical properties of representative climbers were determined in three-point bending tests and are discussed based on the anatomical observations. Growth form evolution of the perianth-bearing Piperales was reconstructed by ancestral character state reconstruction using Mesquite.

Key Results

While species of Asarum and Saruma are exclusively herbaceous, species of the remaining genera show a higher diversity of growth habit and anatomy. This growth form diversity is accompanied by a more complex stem anatomy and appropriate biomechanical properties. The ancestral growth form of the perianth-bearing Piperales is reconstructed with either a shrub-like or herbaceous character state, while the following three backbone nodes in the reconstruction show a shrub-like character state. Accordingly, the climbing habit most probably evolved in the ancestor of Aristolochia.

Conclusions

Since the ancestor of the perianth-bearing Piperales has been reconstructed with a herb- or shrub-like habit, it is proposed that the climbing habit is a derived growth form, which evolved with the diversification of Aristolochia, and might have been a key feature for its diversification. Observed anatomical synapomorphies, such as the perivascular fibres in Lactoris, Thottea and Aristolochia, support the phylogenetic relationship of several lineages within the perianth-bearing Piperales. In addition, the hypothesis that the vegetative organs of the holoparasitic Hydnoraceae are most probably rhizomes is confirmed.     

Ancient DNA Resolves Identity and Phylogeny of New Zealand's Extinct and Living Quail (Coturnix sp.)

Background

The New Zealand quail, Coturnix novaezealandiae, was widespread throughout New Zealand until its rapid extinction in the 1870''s. To date, confusion continues to exist concerning the identity of C. novaezealandiae and its phylogenetic relationship to Coturnix species in neighbouring Australia, two of which, C. ypsilophora and C. pectoralis, were introduced into New Zealand as game birds. The Australian brown quail, C. ypsilophora, was the only species thought to establish with current populations distributed mainly in the northern part of the North Island of New Zealand. Owing to the similarities between C. ypsilophora, C. pectoralis, and C. novaezealandiae, uncertainty has arisen over whether the New Zealand quail is indeed extinct, with suggestions that remnant populations of C. novaezealandiae may have survived on offshore islands.

Methodology/Principal Findings

Using fresh and historical samples of Coturnix sp. from New Zealand and Australia, DNA analysis of selected mitochondrial regions was carried out to determine phylogenetic relationships and species status. Results show that Coturnix sp. specimens from the New Zealand mainland and offshore island Tiritiri Matangi are not the New Zealand quail but are genetically identical to C. ypsilophora from Australia and can be classified as the same species. Furthermore, cytochrome b and COI barcoding analysis of the New Zealand quail and Australia''s C. pectoralis, often confused in museum collections, show that they are indeed separate species that diverged approximately 5 million years ago (mya). Gross morphological analysis of these birds suggests a parallel loss of sustained flight with very little change in other phenotypic characters such as plumage or skeletal structure.

Conclusion/Significance

Ancient DNA has proved invaluable for the detailed analysis and identification of extinct and morphologically cryptic taxa such as that of quail and can provide insights into the timing of evolutionary changes that influence morphology.     

Biogeography and evolution of a widespread Central American lizard species complex: Norops humilis, (Squamata: Dactyloidae)

Background

Caribbean anole lizards (Dactyloidae) have frequently been used as models to study questions regarding biogeography and adaptive radiations, but the evolutionary history of Central American anoles (particularly those of the genus Norops) has not been well studied. Previous work has hypothesized a north-to-south dispersal pattern of Central American Norops, but no studies have examined dispersal within any Norops lineages. Here we test two major hypotheses for the dispersal of the N. humilis/quaggulus complex (defined herein, forming a subset within Savage and Guyer’s N. humilis group).

Results

Specimens of the N. humilis group were collected in Central America, from eastern Mexico to the Canal Zone of Panama. Major nodes were dated for comparison to the geologic history of Central America, and ancestral ranges were estimated for the N. humilis/quaggulus complex to test hypothesized dispersal patterns. These lineages displayed a northward dispersal pattern. We also demonstrate that the N. humilis/quaggulus complex consists of a series of highly differentiated mitochondrial lineages, with more conserved nuclear evolution. The paraphyly of the N. humilis species group is confirmed. A spatial analysis of molecular variance suggests that current populations are genetically distinct from one another, with limited mitochondrial gene flow occurring among sites.

Conclusions

The observed south-to-north colonization route within the Norops humilis/quaggulus complex represents the first evidence of a Norops lineage colonizing in a south-to-north pattern, (opposite to the previously held hypothesis for mainland Norops). One previously described taxon (N. quaggulus) was nested within N. humilis, demonstrating the paraphyly of this species; while our analyses also reject the monophyly of the Norops humilis species group (sensu Savage and Guyer), with N. tropidonotus, N. uniformis, and N. marsupialis being distantly related to/highly divergent from the N. humilis/quaggulus complex. Our work sheds light on mainland anole biogeography and past dispersal events, providing a pattern to test against other groups of mainland anoles.

Electronic supplementary material

The online version of this article (doi:10.1186/s12862-015-0391-4) contains supplementary material, which is available to authorized users.     

Investigating the structural impacts of I64T and P311S mutations in APE1-DNA complex: a molecular dynamics approach

Background

Elucidating the molecular dynamic behavior of Protein-DNA complex upon mutation is crucial in current genomics. Molecular dynamics approach reveals the changes on incorporation of variants that dictate the structure and function of Protein-DNA complexes. Deleterious mutations in APE1 protein modify the physicochemical property of amino acids that affect the protein stability and dynamic behavior. Further, these mutations disrupt the binding sites and prohibit the protein to form complexes with its interacting DNA.

Principal Findings

In this study, we developed a rapid and cost-effective method to analyze variants in APE1 gene that are associated with disease susceptibility and evaluated their impacts on APE1-DNA complex dynamic behavior. Initially, two different in silico approaches were used to identify deleterious variants in APE1 gene. Deleterious scores that overlap in these approaches were taken in concern and based on it, two nsSNPs with IDs rs61730854 (I64T) and rs1803120 (P311S) were taken further for structural analysis.

Significance

Different parameters such as RMSD, RMSF, salt bridge, H-bonds and SASA applied in Molecular dynamic study reveals that predicted deleterious variants I64T and P311S alters the structure as well as affect the stability of APE1-DNA interacting functions. This study addresses such new methods for validating functional polymorphisms of human APE1 which is critically involved in causing deficit in repair capacity, which in turn leads to genetic instability and carcinogenesis.     

Rate heterogeneity in six protein-coding genes from the holoparasite Balanophora (Balanophoraceae) and other taxa of Santalales

Background and Aims

The holoparasitic flowering plant Balanophora displays extreme floral reduction and was previously found to have enormous rate acceleration in the nuclear 18S rDNA region. So far, it remains unclear whether non-ribosomal, protein-coding genes of Balanophora also evolve in an accelerated fashion and whether the genes with high substitution rates retain their functionality. To tackle these issues, six different genes were sequenced from two Balanophora species and their rate variation and expression patterns were examined.

Methods

Sequences including nuclear PI, euAP3, TM6, LFY and RPB2 and mitochondrial matR were determined from two Balanophora spp. and compared with selected hemiparasitic species of Santalales and autotrophic core eudicots. Gene expression was detected for the six protein-coding genes and the expression patterns of the three B-class genes (PI, AP3 and TM6) were further examined across different organs of B. laxiflora using RT-PCR.

Key Results

Balanophora mitochondrial matR is highly accelerated in both nonsynonymous (d_N) and synonymous (d_S) substitution rates, whereas the rate variation of nuclear genes LFY, PI, euAP3, TM6 and RPB2 are less dramatic. Significant d_S increases were detected in Balanophora PI, TM6, RPB2 and d_N accelerations in euAP3. All of the protein-coding genes are expressed in inflorescences, indicative of their functionality. PI is restrictively expressed in tepals, synandria and floral bracts, whereas AP3 and TM6 are widely expressed in both male and female inflorescences.

Conclusions

Despite the observation that rates of sequence evolution are generally higher in Balanophora than in hemiparasitic species of Santalales and autotrophic core eudicots, the five nuclear protein-coding genes are functional and are evolving at a much slower rate than 18S rDNA. The mechanism or mechanisms responsible for rapid sequence evolution and concomitant rate acceleration for 18S rDNA and matR are currently not well understood and require further study in Balanophora and other holoparasites.     

DNA barcode detects high genetic structure within neotropical bird species

Background

Towards lower latitudes the number of recognized species is not only higher, but also phylogeographic subdivision within species is more pronounced. Moreover, new genetically isolated populations are often described in recent phylogenies of Neotropical birds suggesting that the number of species in the region is underestimated. Previous COI barcoding of Argentinean bird species showed more complex patterns of regional divergence in the Neotropical than in the North American avifauna.

Methods and Findings

Here we analyzed 1,431 samples from 561 different species to extend the Neotropical bird barcode survey to lower latitudes, and detected even higher geographic structure within species than reported previously. About 93% (520) of the species were identified correctly from their DNA barcodes. The remaining 41 species were not monophyletic in their COI sequences because they shared barcode sequences with closely related species (N = 21) or contained very divergent clusters suggestive of putative new species embedded within the gene tree (N = 20). Deep intraspecific divergences overlapping with among-species differences were detected in 48 species, often with samples from large geographic areas and several including multiple subspecies. This strong population genetic structure often coincided with breaks between different ecoregions or areas of endemism.

Conclusions

The taxonomic uncertainty associated with the high incidence of non-monophyletic species and discovery of putative species obscures studies of historical patterns of species diversification in the Neotropical region. We showed that COI barcodes are a valuable tool to indicate which taxa would benefit from more extensive taxonomic revisions with multilocus approaches. Moreover, our results support hypotheses that the megadiversity of birds in the region is associated with multiple geographic processes starting well before the Quaternary and extending to more recent geological periods.     

Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae)

Background and Aims

Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data.

Methods

Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals.

Key Results

Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation.

Conclusions

The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile.     

A Validated Methodology for Genetic Identification of Tuna Species (Genus Thunnus)

Background

Tuna species of the genus Thunnus, such as the bluefin tunas, are some of the most important and yet most endangered trade fish in the world. Identification of these species in traded forms, however, may be difficult depending on the presentation of the products, which may hamper conservation efforts on trade control. In this paper, we validated a genetic methodology that can fully distinguish between the eight Thunnus species from any kind of processed tissue.

Methodology

After testing several genetic markers, a complete discrimination of the eight tuna species was achieved using Forensically Informative Nucleotide Sequencing based primarily on the sequence variability of the hypervariable genetic marker mitochondrial DNA control region (mtDNA CR), followed, in some specific cases, by a second validation by a nuclear marker rDNA first internal transcribed spacer (ITS1). This methodology was able to distinguish all tuna species, including those belonging to the subgenus Neothunnus that are very closely related, and in consequence can not be differentiated with other genetic markers of lower variability. This methodology also took into consideration the presence of introgression that has been reported in past studies between T. thynnus, T. orientalis and T. alalunga. Finally, we applied the methodology to cross-check the species identity of 26 processed tuna samples.

Conclusions

Using the combination of two genetic markers, one mitochondrial and another nuclear, allows a full discrimination between all eight tuna species. Unexpectedly, the genetic marker traditionally used for DNA barcoding, cytochrome oxidase 1, could not differentiate all species, thus its use as a genetic marker for tuna species identification is questioned.