Intraspecific and interspecific variation in the second internal transcribed spacer sequence for Metastrongylus (Nematoda: Metastrongyloidea) detected by high resolution PCR-linked restriction fragment length polymorphism

Metastrongylus species are important parasites of free-range pigs and wild boar, but little is known about the genetic make-up of natural populations. This study was undertaken to examine sequence variation in internal transcribed spacer 2 of ribosomal DNA within and among three species of Metastrongylus using PCR-linked restriction fragment length polymorphism analysis. In contrast to many other species of bursate nematodes, significant intraspecific variation was detected in restriction fragment length polymorphism profiles among individual worms. In spite of this, it was possible to identify the three species by their distinctive restriction profiles. The findings suggest that the internal transcribed spacer 2 region should be useful for analysing population variation within Metastrongylus species.     

Inonotus taiwanensis sp. nov. (Basidiomycota) from Taiwan

Inonotus taiwanensis (Hymenochaetales) is described as a new species collected from southern Taiwan, and all specimens grew on the trunk of Trema tomentosa. This new species is characterized by having resupinate, effuse-reflexed to pileate basidiocarps, bright yellow context, long setal hyphae in context and trama, setae in hymenium and trama, and fairly small basidiospores. Maximum likelihood and Bayesian inference phylogenies inferred from both sequences of 28S and internal transcribed spacer (ITS) region of rDNA indicated that I. taiwanensis forms a distinct clade within Inonotus, and the species is sister to I. tricolor. The comparison of morphological differences among I. taiwanensis and some closely related Inonotus spp. is presented.     

Single step molecular characterization of morphologically similar black truffle species

Species-specific internal ITS primers that amplify polymerase chain reaction (PCR) products of different lengths were selected to distinguish the morphologically similar ectomycorrhizal fungi T. melanosporum, T. brumale and T. indicum by aligning their internal transcribed spacer sequences and taking into account any incidence of intraspecific variability. In multiplex PCR experiments, the species-specific primers yielded the expected amplicons on template DNA isolated from the above mentioned species, while there was no amplification in PCR reactions carried out on fungal DNA from competing truffle species and host plants.     

DNA barcoding of brown Parmeliae (Parmeliaceae) species: a molecular approach for accurate specimen identification,emphasizing species in Greenland

Warming of Arctic and alpine regions has a substantial impact on high-altitude/-latitude ecosystems. Shifting biomes due to climate change may lead to adjustments in species distributions and potential extinctions. Therefore, detailed monitoring is requisite to assess biologically meaningful shifts in community composition and species distributions. Some Arctic-alpine lichens have been shown to be particularly sensitive to climatic shifts associated with global change. However, accurate identification of lichenized fungal species remains challenging and may limit the effective use of lichens in climate change research. Given the inherent difficulties in accurate identification of lichenized fungi and the potential value of efficient identifications for bio-monitoring research, we investigated the utility of DNA barcode identification of the 13 brown Parmeliae (Ascomycota) species occurring in Greenland. For these species, we assessed monophyly and genetic distances using the nuclear ribosomal internal transcribed spacer region (ITS), the standard DNA barcode for fungi. We also compared intraspecific distance values to a proposed intra-interspecific threshold value for Parmeliaceae to identify nominal taxa potentially masking previously unrecognized diversity. Our results indicated that the 13 brown Parmeliae species occurring in Greenland can be successfully discriminated using the ITS region. All phenotypically circumscribed species were recovered as well-supported, monophyletic clades. Furthermore, our data supported a barcode gap among congeners for all brown Parmeliae species investigated here. However, high intraspecific genetic distances suggest the potential for previously unrecognized species-lineages in at least five species: Melanelia agnata, M. hepatizon, Montanelia disjuncta, M. panniformis, and M. tominii. Our research facilitates effective, long-term bio-monitoring of climate change in Greenland using lichens by providing accurate molecular identification of brown Parmeliae specimens.     

Typing Tuber melanosporum and Chinese black truffle species by molecular markers

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.     

Intraspecific and interspecific variation in the second internal transcribed spacer (ITS-2) sequence for Metastrongylus (Nematoda: Metastrongyloidea) detected by high resolution PCR-RFLP

Metastrongylus species are important parasites of free-range pigs and wild boar, but little is known about the genetic make-up of natural populations. This study was undertaken to examine sequence variation in internal transcribed spacer 2 of ribosomal DNA within and among three species of Metastrongylus using PCR-linked restriction fragment length polymorphism analysis. In contrast to many other species of bursate nematodes, significant intraspecific variation was detected in restriction fragment length polymorphism profiles among individual worms. In spite of this, it was possible to identify the three species by their distinctive restriction profiles. The findings suggest that the internal transcribed spacer 2 region should be useful for analysing population variation within Metastrongylus species.     

Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region

Phylogenetic relationships of 6 species in the trematode subfamily Haplorchiinae were analyzed using small and large subunit of ribosomal DNA genes (18S rDNA and 28S rDNA) and internal transcribed spacer subunit II (ITS2) region as molecular markers. Maximum Likelihood and Bayesian inference analyses of combined rDNAs and ITS2 indicated a close relationship between the genera Haplorchis and Procerovum, while these two genera were distinct from Stellantchasmus falcatus. These phylogenetic relationships were consistent with the number of testes but not with the characters of the modification of the seminal vesicle or of the ventral sucker. Although three Haplorchis spp. were, together with Procerovum, in the same cluster, their mutual topology was incongruent between rDNA and ITS2 trees. Phylogenetic analyses using other molecular markers with more species are necessary to work out solid phylogenetic relationships among the species in this subfamily.     

Genetic divergence within the monotypic tree genus <Emphasis Type="Italic">Platycarya</Emphasis> (Juglandaceae) and its implications for species’ past dynamics in subtropical China

Subtropical East Asia harbours a large plant diversity that is often attributed to allopatric speciation in this topographically complex region characterized by a relative climate stability. Here, we use observations of Platycarya, a widespread subtropical Asian tree genus, to explore the consequences of past climate stability on species’ evolutionary history in subtropical China. This genus has a controversial taxonomy: while it is now prevailingly treated as monotypic, two species have been originally described, Platycarya strobilacea and P. longipes. Previous information from species distribution models, fossil pollen data and genetic data based on chloroplast DNA (cpDNA) were integrated with newly obtained genetic data from the two putative species. We used both cpDNA (psbA-trnH and trnL-F intergenic spacers, including a partial trnL gene sequence) and nuclear markers. The latter included sequences of the internal transcribed spacer region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA and random genomic single nucleotide polymorphisms. Using these nuclear genetic markers, we found interspecific genetic divergence fitting with the ‘two species’ scenario and geographically structured intraspecific variation. Using cpDNA markers, we also found geographically structured intraspecific variation. Despite deep inter- and intraspecific genetic divergence, we detected genetic admixture in southwest China. Overall, our findings of genetic divergence within Platycarya support the hypothesis of allopatric speciation. However, episodes of population interconnection were identified, at least in southwest China, suggesting that the genus has had a dynamic population history.     

Characterization of Trichodesmium spp. by Genetic Techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus Trichodesmium. The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of Katagnymene. Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

Molecular identification of Aphidius transcaspicus (Hymenoptera: Braconidae: Aphidiinae), a closely associated species of Aphidius colemani,in Korea

Aphidius colemani (Braconidae: Aphidiinae) is an endoparasitoid and one of the most commercially used parasitoids for the control of various pest aphids. However, species identification of A. colemani is difficult because of the high degree of morphological similarity with closely associated species, such as A. transcaspicus and A. platensis. Recently, we identified a large-scale emergence of parasitoids from mummies of Rhopalosiphum maidis on barley banker plants in Yecheon County, Korea. The morphological characteristics of the forewing, petiole, and antenna indicated that our specimen is A. transcaspicus. The partial nucleotide sequence analysis of the mitochondrial cytochrome oxidase subunit I (COI) gene showed that it is 100% identical to Japanese specimens and 1.69% variable to Greek specimens. Analysis of the phylogenetic tree, haplotypes, and amino acid sequence of the COI gene, as well as the internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA region, indicated that A. transcaspicus is closer to A. platensis than A. colemani, and Korean and Japanese populations were mostly diversified groups at the intraspecific level. This study provides important information to elucidate the genetic variation in A. transcaspicus and the relationships between closely related species within the A. colemani group.     

Morphological and genetic characterization of Porrocaecum angusticolle (Molin, 1860) (Nematoda: Ascaridomorpha) from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic

Porrocaecum angusticolle is a nematode species mainly parasitic in the birds of Accipitriformes and Strigiformes. However, some aspects of the morphology of P. angusticolle remain insufficiently known. In the present study, the detailed morphology of P. angusticolle was studied using light and, for the first time, scanning electron microscopy, based on newly collected specimens from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic. Some previously unreported morphological features of taxonomic significance were observed. The nuclear and mitochondrial DNA markers, including partial large ribosomal DNA (28S), complete internal transcribed spacer (ITS-1 + 5.8S + ITS-2), cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2) of P. angusticolle were sequenced for molecular identification of this species. There was no intraspecific genetic variation detected in the 28S and ITS regions among different individuals of P. angusticolle, but low level of intraspecific nucleotide divergence was found in the cox1 (0.26–0.78%) and cox2 regions (1.0%). The 28S and cox2 of P. angusticolle were sequenced for the first time. Our molecular evidence supported the validity of both P. angusticolle and P. depressum. The newly obtained genetic data are helpful for further studies of DNA-based taxonomy, population genetics and phylogeny of the genus of Porrocaecum.     

Molecular Evidence Shows Low Species Diversity of Coral-Associated Hydroids in Acropora Corals

A novel symbiosis between scleractinians and hydroids (Zanclea spp.) was recently discovered using taxonomic approaches for hydroid species identification. In this study, we address the question whether this is a species-specific symbiosis or a cosmopolitan association between Zanclea and its coral hosts. Three molecular markers, including mitochondrial 16S and nuclear 28S ribosomal genes, and internal transcribed spacer (ITS), were utilized to examine the existence of Zanclea species from 14 Acropora species and 4 other Acroporidae genera including 142 coral samples collected from reefs in Kenting and the Penghu Islands, Taiwan, Togian Island, Indonesia, and Osprey Reef and Orpheus Island on the Great Barrier Reef, Australia. Molecular phylogenetic analyses of the 16S and 28S genes showed that Acropora-associated Zanclea was monophyletic, but the genus Zanclea was not. Analysis of the ITS, and 16S and 28S genes showed either identical or extremely low genetic diversity (with mean pairwise distances of 0.009 and 0.006 base substitutions per site for the 16S and 28S genes, respectively) among Zanclea spp. collected from diverse Acropora hosts in different geographic locations, suggesting that a cosmopolitan and probably genus-specific association occurs between Zanclea hydroids and their coral hosts.     

Morphological variability and molecular characterisation of Dichelyne (Cucullanellus) pleuronectidis (Yamaguti, 1935) (Ascaridida: Cucullanidae) from the flatfish Pleuronichthys cornutus (Temminck & Schlegel) (Pleuronectiformes: Pleuronectidae) in the East China Sea

Cucullanid nematodes identified morphologically as Dichelyne (Cucullanellus) pleuronectidis (Yamaguti, 1935) were collected from the ridged-eye flounder Pleuronichthys cornutus (Temminck & Schlegel) (Pleuronectiformes: Pleuronectidae) in the East China Sea. Their examination using light microscopy and, for the first time, scanning electron microscopy, revealed several important, but previously unreported morphological features and the presence of remarkable morphological differences in the intestinal caecum and deirids among some individuals. Consequently, specimens of D. pleuronectidis were characterised using molecular methods by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA to test whether the present material with broad range of morphological variability, represents a complex of sibling species or a single species. The results of molecular analyses proved that the differences in the intestinal ceacum and deirids should be considered as intraspecific variation and that the nematode material collected from P. cornutus in the East China Sea represented a single species, D. pleuronectidis. These new morphological and genetic data contributed to an accurate diagnosis of this hitherto insufficiently known nematode and also indicated that a more rigorous study based on morphological and genetic data with broader representation of the Cucullanidae is required to assess whether the traditionally used diagnostic character of absence or presence of intestinal ceacum is of generic importance in distinguishing Dichelyne and Cucullanus.     

Molecular variation of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda: Pseudophyllidea) in different fish host species based on ITS rDNA sequences

The molecular variation in Bothriocephalus acheilognathi Yamaguti, 1934 from 11 species of freshwater fish collected in Australia, China, the Czech Republic, England and Hawaii was investigated by determining the nucleotide sequences of the internal transcribed spacer region. The length of the first and second internal transcribed spacer sequences of multiple individuals ranged from 553 to 571 bp and 553 to 615 bp, and the G + C content from 53.1 to 53.5%. The percentage sequence divergence varied between 0 and 0.9% in the ITS1 and 0 and 6.6% in the ITS2, respectively, indicating the occurrence of intraspecific variation. It is demonstrated that the fragment length variation resulted primarily from microsatellite polymorphisms present in the ITS region, especially in the ITS2 region. Phylogenetic analyses revealed that B. acheilognathi examined in this study consisted of three closely related genotypes with certain degrees of host-specificity, and the genotype representing isolates from Cyprinus carpio L. was the most common and diverse form within the species B. acheilognathi.     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.     

Variability of Ribosomal DNA ITS-2 and Its Utility in Detecting Genetic Relatedness of Pearl Oyster

The objective of this study was to detect interspecific and intraspecific genetic variations of the second internal transcribed spacer of ribosomal DNA (ITS-2), and explore the feasibility of using it as a molecular marker phylogenetic analyses and species identification among pearl oysters. ITS-2 sequences of 6 pearl oysters were amplified via polymerase chain reaction. The amplified DNA fragments were about 500 bp, spanning the partial sequences of 5.8S and 28S rRNA genes. The GC contents of all species used in this study were higher than the AT contents. The variations of sequences involved substitutions as well as insertions/deletions and were mainly concentrated in spacer regions. Sequences of about 30-bp in spacer regions showed no variations among 5 Pincatda species. Intraindividual and intraspecific polymorphisms of ITS-2 sequences were detected in some species; the interspecific variability was significantly larger than the variability within species, and the variability at the genus level was higher than that at the species level. Both neighbor-joining and parsimony analyses of ITS-2 sequences revealed the distinguishable species boundary of 6 pearl oysters, and indicated that P. chemnitzi and P. nigra were the closely related species, as were P. maxima and P. margaritifera. The findings revealed that ITS-2 sequences could be an appropriate tool for phylogenetic study of pearl oysters.     

Genetic covariation of the marine fungal symbiont Haloguignardia irritans (Ascomycota, Pezizomycotina) with its algal hosts Cystoseira and Halidrys (Phaeophyceae, Fucales) along the west coast of North America

The fungal endophyte Haloguignardia irritans induces gall formation on the brown algal genera Cystoseira and Halidrys occurring from Oregon to Baja California, Mexico. Here we examine genetic covariation and compare rDNA phylogenies to investigate the coevolutionary histories of H. irritans and its algal hosts. Despite recognition of H. irritans as a single morphological species, internal transcribed spacer rDNA sequences representative of its geographic range are characterized by sequence variation at the intraspecific to intrageneric levels. An assessment of parallel cladogenesis between endophyte and host phylogenies provides evidence for a combination of independent fungal divergence and host jumping, similar to that observed in terrestrial lichens. Our results suggest that reduced gene flow due to geographic isolation is a major contributing factor to more concerted covariation observed at one island site, rather than to differences among algal host species alone. Because geography and its effects on gene flow can create heterogeneous mosaics of coevolution for symbioses in terrestrial environments, our results support the notion that conservation efforts toward the maintenance of genetic diversity in marine environments should likewise consider geographic complexity and its effects on coevolving marine species.     

Morphological and molecular differentiation of two new species of Pseudoacanthocephalus Petrochenko, 1958 (Acanthocephala: Echinorhynchidae) from amphibians and reptiles in the Philippines, with identification key for the genus

The genus Pseudoacanthocephalus Petrochenko, 1958 currently includes 14 species of acanthocephalans parasitic in amphibians and reptiles worldwide. This work describes two new species of Pseudoacanthocephalus from amphibians and reptiles collected in several localities on Luzon Island, Philippines. Pseudoacanthocephalus nickoli n. sp. was found in two species of frogs, Rana luzonensis Boulenger and Rana similis (Günther), and Pseudoacanthocephalus smalesi n. sp. was found in a scincid lizard, Sphenomorphus abdictus Brown & Alcala. Differential diagnoses of the two new species of Pseudoacanthocephalus from their congeners are provided. Comparative analysis of nuclear ribosomal rRNA sequences encompassing the 3′ end of 18S nuclear rDNA gene, internal transcribed spacer region (ITS1+5.8S+ITS2), and 5′ end of the 28S gene strongly corroborated the morphological evidence and demonstrated significant differences between the two new species as well as between these species and closely related species from continental China and Vietnam. No intraspecific sequence variability was detected among different individuals representing each of the examined species. This is the first report of Pseudoacanthocephalus in the Philippines. A key to known species of Pseudoacanthocephalus is provided.     

Genetic variation in native and introduced populations of the 'New Zealand flatworm', Arthurdendyus triangulatus

The internal transcribed spacer regions (ITS1 and ITS2) including the 5.8S region of the ‘New Zealand flatworm’, Arthurdendyus triangulates, are 1004 base pairs in length. Restriction fragment length polymorphism analysis of PCR products (PCR‐RFLP) was conducted on A. triangulates specimens from 45 locations in Northern Ireland, Scotland, England and New Zealand. Seven restriction endonucleases (Alu I, Rsa I, Sau3A I, Cfo I, Nde I, Dde I, and Mbo I) were used to reveal intraspecific variation. Analysis of molecular variance revealed the presence of population genetic substructuring, with most genetic heterogeneity present between populations rather than between individuals or geographical regions. No distinct differences were found between Northern Irish and Scottish populations but phylogenetic analysis supports the hypothesis of multiple introductions from New Zealand. There was no significant relationship between genetic distance and geographic distance, as would be expected for natural spread, indicating that this species is largely anthropochorous, even in parts of New Zealand.     

Pleurotus opuntiae revisited – An insight to the phylogeny of dimitic Pleurotus species with emphasis on the P. djamor complex

The name Pleurotus opuntiae is indiscriminately used for describing mushrooms with white to off-white to white-grey pilei with short or absent stipe and dimitic hyphal system, which grow on plants of the genera Opuntia, Yucca, Agave, Phytolacca etc. However, the outcome of the present study evidences that this name should be reserved for specimens deriving from the Mediterranean area only; an epitype originating from Italy on Opuntia ficus-indica is designated. Pertinent material was sequenced by using the internal transcribed spacer region (ITS) and found to be phylogenetically related to P. djamor from Kenya and Nigeria, while members of the P. djamor complex from other continents were clearly more distant. Results were further corroborated by examining the large subunit of nuclear ribosomal DNA (LSU) and the second subunit of RNA polymerase II (RPB2). The P. djamor complex shows high intraspecific polymorphism evidenced by sequence divergence and genetic distance values, presents a cosmopolitan distribution and also comprises material initially identified as P. flabellatus, P. opuntiae, P. ostreatoroseus, P. parsonsiae and P. salmoneostramineus. An ITS tree including representative specimens from all major Pleurotus species is provided for the first time and ambiguous taxa are discussed in the context of new findings.