Evidence of hybridization between Taenia saginata and Taenia asiatica

There has long been a debate as to the specific status of the cestode Taenia asiatica, with some people regarding it as a distinct species and some preferring to recognize it as a strain of Taenia saginata. The balance of current opinion seems to be that T. asiatica is a distinct species. In this study we performed an allelic analysis to explore the possibility of gene exchange between these closely related taxa. In total, 38 taeniid tapeworms were collected from humans living in many localities including Kanchanaburi Province, Thailand where the two species are sympatric. A mitochondrial DNA (mtDNA)-based multiplex PCR tentatively identified those parasites as T. asiatica (n = 20) and T. saginata (n = 18). Phylogenetic analyses of a mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and two nuclear loci, for elongation factor-1 alpha (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp), assigned all except two individual parasites to the species indicated by multiplex PCR. The two exceptional individuals, from Kanchanaburi Province, showed a discrepancy between the mtDNA and nuclear DNA phylogenies. In spite of their possession of sequences typical of the T. saginata cox1 gene, both were homozygous at the elp locus for one of the alleles found in T. asiatica. At the ef1 locus, one individual was homozygous for the allele found at high frequency in T. asiatica while the other was homozygous for the major allele in T. saginata. These findings are evidence of occasional hybridization between the two species, although the possibility of retention of ancestral polymorphism cannot be excluded.     

Population genetic structure of Penaeus merguiensis in Thailand based on nuclear DNA variation

Polymorphisms of nuclear DNA markers were used to study the genetic variation and differentiation among populations of Penaeus merguiensis in Thailand. The data consisted of three nuclear loci analysed for 163 individuals from five populations collected from the Gulf of Thailand (Trad, Surat Thani and Songkhla) and the Andaman Sea (Satun and Trang). The multilocus F_ST estimated at three nuclear loci revealed great and highly significant differentiation between the Gulf of Thailand and Andaman Sea (F_ST=0.203, P<0.001), mostly due to the polymorphism of locus PvAmy. In addition, significant population differentiation was also found within the Gulf of Thailand. These results were compared to that of a previous mitochondrial DNA survey spanning the same geographical range where two divergent mitochondrial clades were reported. The present study brings support to the fact that the existence of these two clades is not due to a mixture of cryptic species but reflects their phylogeographic origin. The strong genetic structure of P. merguiensis on each side of the Thailand peninsula for both mitochondrial and nuclear genes could thus be linked to the phylogeographic divide between Indian Ocean and Pacific forms on the west and east sides respectively.     

Genetic and morphological diversity of Trisetacus species (Eriophyoidea: Phytoptidae) associated with coniferous trees in Poland: phylogeny,barcoding, host and habitat specialization

Eriophyoid species belonging to the genus Trisetacus are economically important as pests of conifers. A narrow host specialization to conifers and some unique morphological characteristics have made these mites interesting subjects for scientific inquiry. In this study, we assessed morphological and genetic variation of seven Trisetacus species originating from six coniferous hosts in Poland by morphometric analysis and molecular sequencing of the mitochondrial cytochrome oxidase subunit I gene and the nuclear D2 region of 28S rDNA. The results confirmed the monophyly of the genus Trisetacus as well as the monophyly of five of the seven species studied. Both DNA sequences were effective in discriminating between six of the seven species tested. Host-dependent genetic and morphological variation in T. silvestris and T. relocatus, and habitat-dependent genetic and morphological variation in T. juniperinus were detected, suggesting the existence of races or even distinct species within these Trisetacus taxa. This is the first molecular phylogenetic analysis of the Trisetacus species. The findings presented here will stimulate further investigations on the evolutionary relationships of Trisetacus as well as the entire Phytoptidae family.     

Drosophila Melanogaster, Drosophila Simulans: so Similar yet so Different

During the last two decades, the two cosmopolitan species Drosophila melanogaster and Drosophila simulans have been compared with regard to numerous characteristics, ranging from their geographic distribution and ecology to their DNA polymorphism. Various traits have been compared, including morphology, physiology, sexual behavior, allozymes and other proteins, chromosomal inversions, mitochondrial and nuclear DNA, transposable elements, wolbachia etc. Such comparisons reveal similarities and differences between the two species, depending on the trait considered. In most cases, the between-population variability of D. simulans is lower than that of D. melanogaster, but the two species exhibit similar levels of within-population variability. One of the main exceptions is the nucleotide polymorphism of several nuclear regions. Although several hypotheses have been proposed to explain these observations, the evolutionary dynamics of these two species are far from being understood. How have two species sharing a common ancestor in the recent past accumulated so many differences? A brief history of comparisons of the two species, from the first in 1919 by A.H. Sturtevant, and a summary of the hypotheses proposed to explain the similarities and the differences between these species are presented and discussed.     

Nuclear DNA replicates during zygote development in Arabidopsis and Torenia fournieri

The progression of the cell cycle is continuous in most cells, but gametes (sperm and egg cells) exhibit an arrest of the cell cycle to await fertilization to form a zygote, which then continues through the subsequent phases to complete cell division. The phase in which gametes of flowering plants arrest has been a matter of debate, since different phases have been reported for the gametes of different species. In this study, we reassessed the phase of cell-cycle arrest in the gametes of two species, Arabidopsis (Arabidopsis thaliana) and Torenia fournieri. We first showed that 4’, 6-diamidino-2-phenylindole staining was not feasible to detect changes in gametic nuclear DNA in T. fournieri. Next, using 5-ethynyl-2’-deoxyuridine (EdU) staining that detects DNA replication by labeling the EdU absorbed by deoxyribonucleic acid, we found that the replication of nuclear DNA did not occur during gamete development but during zygote development, revealing that the gametes of these species have a haploid nuclear DNA content before fertilization. We thus propose that gametes in the G1 phase participate in the fertilization event in Arabidopsis and T. fournieri.

The replication of nuclear DNA does not occur during gamete development but during zygote development.     

Speciation slowing down in widespread and long-living tree taxa: insights from the tropical timber tree genus Milicia (Moraceae)

The long generation time and large effective size of widespread forest tree species can result in slow evolutionary rate and incomplete lineage sorting, complicating species delimitation. We addressed this issue with the African timber tree genus Milicia that comprises two morphologically similar and often confounded species: M. excelsa, widespread from West to East Africa, and M. regia, endemic to West Africa. We combined information from nuclear microsatellites (nSSRs), nuclear and plastid DNA sequences, and morphological systematics to identify significant evolutionary units and infer their evolutionary and biogeographical history. We detected five geographically coherent genetic clusters using nSSRs and three levels of genetic differentiation. First, one West African cluster matched perfectly with the morphospecies M. regia that formed a monophyletic clade at both DNA sequences. Second, a West African M. excelsa cluster formed a monophyletic group at plastid DNA and was more related to M. regia than to Central African M. excelsa, but shared many haplotypes with the latter at nuclear DNA. Third, three Central African clusters appeared little differentiated and shared most of their haplotypes. Although gene tree paraphyly could suggest a single species in Milicia following the phylogenetic species concept, the existence of mutual haplotypic exclusivity and nonadmixed genetic clusters in the contact area of the two taxa indicate strong reproductive isolation and, thus, two species following the biological species concept. Molecular dating of the first divergence events showed that speciation in Milicia is ancient (Tertiary), indicating that long-living tree taxa exhibiting genetic speciation may remain similar morphologically.     

DNA Fingerprinting of Pearls to Determine Their Origins

We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in the internal transcribed spacer (ITS) region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry.     

A new species of the genus Trematosphaeria from China

A new species of the genus Trematosphaeria was isolated and identified from the soil in Jiuzhaigou Nature Reserve, southwestern China. The new taxon was studied based on morphological characters and phylogenetic analyses of three nuclear loci, i.e., the internal transcribed spacer rDNA operon (ITS), and large and small subunit nuclear ribosomal DNA (nuc28S rDNA, nuc18S rDNA). The analyzed Trematosphaeria species were clustered in several clades within the Pleosporales and the genus was shown to be polyphyletic. The new species is introduced as Trematosphaeria terricola, and it is characterized by short-papillate ascomata with suborbicular ostiolum, 3-septate, cinnamon-brown, fusiform ascospores with narrowly rounded ends, verruculose at maturity. Trematosphaeria terricola is compared with morphologically similar species T. confusa, T. mangrovis, and T. pertusa, which differ from the former species in the number of septa, and the shape and size of the ascospores with or without a sheath. This is the first report of a Trematosphaeria isolate from alpine soil in China.     

Colour polymorphism in common primrose (Primula vulgaris Huds.): many colours–many species?

Primula vulgaris exhibits flower colour polymorphism in the eastern part of its range, especially pronounced on the NE coast of the Black Sea. This polymorphism in the Caucasian populations has been taxonomically described and some segregated species are listed as rare and endangered. We used sequence variation in two chloroplast noncoding regions (trnL–trnF and rpll32–trnL) and the complete nuclear internal transcribed spacer (ITS) of ribosomal DNA region to investigate correspondence between flower colour and geographical distribution of both nuclear and chloroplast haplotypes. It appears that variability in these DNA regions does not correlate with flower colour, being, however, clearly structured geographically. We used nested clade analysis to explore this geographical structure. It seems that the territory of the Colchis refugium on the E coast of the Black Sea contains both the highest flower colour and haplotype diversities. The results suggest that common primroses colonized the NE coast of the Black Sea from this refugium, spreading along the coast westward. At the same time, the analysis of ITS haplotypes indicates that P. vulgaris colonized the Crimea from NW Anatolia. This makes it clear that no segregated species can be recognized within flower colour polymorphic P. vulgaris in the Caucasus region. However, its phylogeography needs further detailed study on a broader scale.     

Chromosomal polymorphism in two species of Hypancistrus (Siluriformes: Loricariidae): an integrative approach for understanding their biodiversity

Structural chromosome changes are widely described in different vertebrate groups and generate genetic, phenotypic and behavioral diversity. During the evolution of loricariids, several rearrangements (fissions, fusions, inversions) seem to have occurred. Hypancistrus, tribe Ancistrini, are highly demanded for fishkeeping around the world. In this tribe, the diploid chromosome number 2n = 52 is considered a synapomorphy, and paracentric-type inversions appear to be involved in the chromosomal evolution of the tribe. The present study investigated the karyotypes of H. zebra and H. cf. debilittera using cytogenetic, classical and molecular tools, as well as DNA barcoding. Data reveal that, although diploid number in both species corroborates the proposed synapomorphy for the tribe, there is a complex karyotype dynamics, reflected in the intense chromosomal polymorphism, resulting from rearrangements involving ribosomal regions (5S and 18S rDNA), which are suggested to be paracentric inversions. Besides, DNA barcode confirms reciprocal monophyletism between the species, validating the existence of two species, only. This scenario, coupled with genomic instability caused by exogenous sequences such as Rex-3 retrotransposons and the species’ sedentary lifestyle, which helps the fast polymorphism fixation, may reflect different phenotypic color patterns in natural populations, as observed in H. cf. debilittera.     

A Morphological and Molecular Perspective of Trichoderma viride: Is It One or Two Species?

Trichoderma (Ascomycetes, Hypocreales) strains that have warted conidia are traditionally identified as T. viride, the type species of Trichoderma. However, two morphologically distinct types of conidial warts (I and II) have been found. Because each type corresponds to a unique mitochondrial DNA pattern, it has been questioned whether T. viride comprises more than one species. Combined molecular data (sequences of the internal transcribed spacer 1 [ITS-1] and ITS-2 regions and of part of the 28S rRNA gene along with results of restriction fragment length polymorphism analysis of the endochitinase gene and PCR fingerprinting), morphology, physiology, and colony characteristics distinguish type I and type II as different species. Type I corresponds to “true” T. viride, the anamorph of Hypocrea rufa. Type II represents a new species, T. asperellum, which is, in terms of molecular characteristics, close to the neotype of T. hamatum.     

Molecular evolution of the leptin exon 3 in some species of the family Canidae

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.     

Phylogeny of firs (genus <Emphasis Type="Italic">Abies</Emphasis>, Pinaceae) based on multilocus nuclear markers (AFLP)

To study the phylogenetic relationships, evolutionary history, and molecular systematics of firs (genus Abies), the phylogenetic reconstruction, based on nuclear multilocus markers—amplified fragment length polymorphism (AFLP)—was conducted. Using seven combinations of selective primers, 84 samples of 39 taxa were genotyped for 553 polymorphic AFLP loci. A comparison with our earlier chloroplast and mitochondrial phylogenies of the genus (in 2014) shows that the nuclear phylogeny generally is more congruent to the chloroplast tree. Most of the clades resolved by the chloroplast phylogeny were supported also in the AFLP tree. Employing the nuclear DNA-based tree, we revealed the presence of new groups and the differences in the topology of several clades. AFLP confirmed the monophyly of Asian species of section Balsamea and their sister position in relation to the American group of species of this section. As shown by the tree of chloroplast DNA, Asian species of section Balsamea do not form a monophyletic group, but belong to the clade comprising the majority of Asian species. Phylogenetically mitochondrial DNA data to a large extent are not congruent to the nuclear and chloroplast DNA trees, and are more in line with geographical distribution of species. Conflicts between nuclear and cytoplasmic phylogeny were analyzed. Taking them into account, we consider the hypothesis of a hybrid origin of particular groups of firs, including ancient hybridization in section Balsamea. A comparison of molecular data with traditional taxonomy of the genus is discussed.     

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.     

Narrow Genetic Variability in Cicer arietinum L. as Revealed by RFLP Analysis

A subgenomic library constructed from small Pstl restriction fragments (0.4 to 2.0 kb) yielded 83.18% low-copy clones. Using 17 random genomic and 5 heterologous probes in 65 probe-enzyme combinations, Restriction Fragment Length Polymorphism (RFLP) for nuclear DNA was studied in five desi and five kabuli type chickpea cultivars. Only two clones revealed polymorphism in the cultivars tested. No polymorphism in chickpea varieties was detected with four Random Amplified Polymorphic DNA (RAPD) markers studied. However, some degree of polymorphism between C. arietinum and its wild relative C. reticulatum was detected. The RFLP analysis of chloroplast and mitochondrial genomes showed no polymorphism.     

Molecular and morphometric identification of Trypanosoma (Megatrypanum) minasense in blood samples of marmosets (Callithrix: Callithrichidae) from the city of Rio de Janeiro,Brazil.

Callithrix jacchus and C. penicillata marmosets are invasive to the state of Rio de Janeiro, Brazil, threatening the native and vulnerable C. aurita. Both invasive species can be hosts of Trypanosoma cruzi, T. minasense, T. rangeli and T. devei. We aim to investigate the occurrence of trypanosomatids in Callithrix sp. from Jardim Botânico do Rio de Janeiro, located in a central and populous area of the city. Fifteen marmosets were captured. Blood samples were collected for light microscopy and molecular genetics analysis. Parasites morphometric values were evaluated for species identification. DNA was extracted from blood samples by phenol-chloroform method, for partial amplification of the 18S rRNA gene. PCR products were sequenced and aligned using BLAST®. A maximum likelihood phylogenetic tree was constructed to analyze the proximity between the observed sequences. By light microscopy, trypomastigotes were detected in five of the fifteen marmosets. Morphometric measurements and size polymorphism corresponded to those previously described for T. minasense. The DNA sequences of approximately 600 base pairs of the 18S rRNA gene were obtained for three samples with 99% identity with T. minasense sequence, forming a cluster in the phylogenetic tree and corroborating morphometric analysis. Trypanosoma minasense is a highly specific parasite to non-human primates considered as non-pathogenic. There is no evidence of infection in humans and these parasite findings from invasive marmosets do not support additional risks for the native species.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra

Pinus mugo (dwarf mountain pine) is an important component of European mountain ecosystems. However, little is known about the present genetic structure and population differentiation of this species at the DNA level, possibly due to a lack of nuclear microsatellite markers (SSR) developed for Pinus mugo. Therefore in this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. This cross-species amplification approach is much faster and less expensive than isolation and characterization of new microsatellite markers. The transfer rates from the source species to Pinus mugo were moderately low (26%). There were no differences in microsatellite repeat motifs between the source species and Pinus mugo. Nuclear microsatellite markers successfully transferred to Pinus mugo can be applied to various genetic studies on this species, due to the high level of their polymorphism and high value of polymorphic information content.     

Hybrid status of Tibouchina urvilleana Cogn. revealed by ITS sequences of nuclear ribosomal DNA

Tibouchina urvilleana Cogn. is native to southern Brazil and currently cultivated as an important ornamental shrub in frost free areas around the world. Its rapid vegetative growth and sterility suggests that it might be of hybrid origin. In this study, Internal Transcribed Spacer (ITS) of nuclear ribosomal DNA and chloroplast trnL-trnF spacer from T. urvilleana and three other congeneric species were sequenced to test its hybrid status. Cloning sequencing revealed two distinct types of ITS sequences from T. urvilleana, with one type almost identical with Tibouchina aspera. Genetic distance between the two types was much larger than the average interspecific genetic distances calculated from other Tibouchina species. Sequencing of chloroplast trnL-trnF spacer showed that T. urvilleana has identical sequence with T. aspera, but differed from other congeneric species by one nucleotide substitution and two indels. Molecular data demonstrated clearly that T. urvilleana indeed was a hybrid, with T. aspera or closely related species acting as the maternal parent.