Diversity of Aphanizomenonflos-aquae (Cyanobacterium) Populations along a Baltic Sea Salinity Gradient

Colony-forming cyanobacteria of the genus Aphanizomenon form massive blooms in the brackish water of the Baltic Sea during the warmest summer months. There have been recent suggestions claiming that the Baltic Sea Aphanizomenon species may be different from Aphanizomenon flos-aquae found in lakes. In this study, we examined variability in the morphology and 16S-23S rRNA internal transcribed spacer (ITS) sequences of A. flos-aquae populations along a salinity gradient from a string of lakes to a fjord-like extension of the Baltic Sea to the open Baltic Sea. Morphological differences among the populations were negligible. We found that the Baltic Sea was dominated (25 out of 27 sequences) by one ITS1-S (shorter band of ITS 1 [ITS1]) genotype, which also was found in the lakes. The lake populations of A. flos-aquae tended to be genetically more diverse than the Baltic Sea populations. Since the lake ITS1-S genotypes of A. flos-aquae are continuously introduced to the Baltic Sea via inflowing waters, it seems that only one ITS1 genotype is able to persist in the Baltic Sea populations. The results suggest that one of the ITS1-S genotypes found in the lakes is better adapted to the conditions of the Baltic Sea and that natural selection removes most of the lake genotypes from the Baltic Sea A. flos-aquae populations.     

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.     

Alexandrium diversaporum sp. nov., a new non-saxitoxin producing species: Phylogeny,morphology and sxtA genes

Species of the PST producing planktonic marine dinoflagellate genus Alexandrium have been intensively scrutinised, and it is therefore surprising that new taxa can still be found. Here we report a new species, Alexandrium diversaporum nov. sp., isolated from spherical cysts found at two sites in Tasmania, Australia. This species differs in its morphology from all previously reported Alexandrium species, possessing a unique combination of morphological features: the presence of 2 size classes of thecal pores on the cell surface, a medium cell size, the size and shape of the 6″, 1′, 2⁗ and Sp plates, the lack of a ventral pore, a lack of anterior and posterior connecting pores, and a lack of chain formation. We determined the relationship of the two strains to other species of Alexandrium based on an alignment of concatenated SSU-ITS1, 5.8S, ITS2 and partial LSU ribosomal RNA sequences, and found A. diversaporum to be a sister group to Alexandrium leei with high support. A. leei shares several morphological features, including the relative size and shapes of the 6″, 1′, 2⁗ and Sp plates and the fact that some strains of A. leei have two size classes of thecal pores. We examined A. diversaporum strains for saxitoxin production and found them to be non-toxic. The species lacked sequences for the domain A4 of sxtA, as has been previously found for non-saxitoxin producing species of Alexandrium.     

Yarrowia porcina sp. nov. and Yarrowia bubula f.a. sp. nov., two yeast species from meat and river sediment

Eleven yeast strains representing two hitherto undescribed species were isolated from different kinds of meat samples in Hungary and one from the sediment of a tropical freshwater river in Southeastern Brazil. The analysis of the sequences of their large subunit rRNA gene D1/D2 domain and the internal transcribed spacer (ITS) regions placed the two new species in the Yarrowia clade. Some of the seven strains representing the first new species can mate and give rise to asci and form ascospores embedded in capsular material, which qualifies it as the third teleomorph species of the Yarrowia clade. The name Yarrowia porcina sp. nov. (type strain: NCAIM Y.02100^T = CBS 12935^T = NRRL Y-63669^T, allotype strain UFMG-RD131^A = CBS 12932^A) is proposed for this new yeast species, which, based on physiological characters, is indistinguishable from Yarrowia lipolytica and some other species of the genus. Considerable intraspecific variability was detected among the sequences of the large subunit rRNA gene D1/D2 domains of the seven strains. The variability among the D1/D2 sequences exceeded the divergence observed among the ITS sequences and in some cases more than 1 % substitution among the D1/D2 sequences was detected. The conspecificity of these strains was supported by the low (0–3 substitutions) sequence divergence among their ITS sequences, the result of a parsimony network analysis utilizing the concatenated ITS and D1/D2 sequences and also by the fingerprint patterns generated by microsatellite primed PCR. No ascospore formation was observed in the group of the other five strains representing the second new species. These strains shared identical D1/D2 and ITS sequences. Yarrowia bubula f.a., sp. nov. (type strain: NCAIM Y.01998^T = CBS 12934^T = NRRL Y-63668^T) is proposed to accommodate these strains.     

Genetic variability in Fomes fomentarius reconfirmed by translation elongation factor 1-α DNA sequences and 25S LSU rRNA sequences

The existence of two cryptic species within strains of the wood-decaying fungus Fomes fomentarius was revealed recently based on the internal transcribed spacer (ITS) sequence variability. In this study for the first time the sequences of another molecular markers, partial translation elongation factor 1-α (efa) region and partial 25S large subunit ribosomal RNA gene were obtained and used to evaluate genetic variability of F. fomentarius. Congruent phylogeny was observed for all three markers used confirming the presence of two cryptic species within F. fomentarius. Surprisingly, ITS sequence variability within F. fomentarius was significantly lower compared to the variability of efa sequences (0.023 versus 0.036 nucleotide substitutions per site) questioning the discriminatory power of ITS sequences for fungal species identification.     

Additional data for a new Theileria sp. from China based on the sequences of ribosomal RNA internal transcribed spacers

Theileria sinensis was recently isolated and named as an independent Theileria species that infects cattle in China. To date, this parasite has been described based on its morphology, transmission and molecular studies, indicating that it should be classified as a distinct species. To test the validity of this taxon, the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were cloned and sequenced from three T. sinensis isolates. The complete ITS sequences were compared with those of other Theileria sp. available in GenBank. Phylogenetic analyses based on sequence data for the complete ITS sequences indicate that T. sinensis lies in a distinct clade that is separate from that of T. buffeli/orientalis and T. annulata. Sequence comparisons indicate that different T. sinensis isolates possess unique sizes of ITS1 and ITS2 as well as species-specific nucleotide sequences. This analysis provides new molecular data to support the classification of T. sinensis as a distinct species from other known Theileria spp. based on ITS sequences.     

A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding

Medicinal plants of the Panax genus belonging to Araliaceae family are well-known, rare plants used as tonics in traditional Chinese medicine and have been described in the Chinese Pharmacopoeia. Because of the high price and the huge human demand, these commercial products often contain adulterants. In this study, 377 sequences from four species were analyzed. Single nucleotide polymorphisms (SNPs) were detected and patterns of intragenomic variation in internal transcribed spacer 2 (ITS2) from the four Panax species were studied. Intraspecific variations were analyzed based on three typical DNA barcodings (ITS2, matK and psbA-trnH). Results from this study revealed that intraspecific genetic distances in Panax ginseng and Panax quinquefolius were quite low (0–0.002) and the multi-copy ITS2 could be considered a single locus in the genomes of these two species. Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Considering the mixed powder of P. ginseng and P. quinquefolius, double peaks could be clearly examined at SNP positions and the height of the peaks could indicate the mixed ratio roughly. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species and the mixed powder P. ginseng and P. quinquefolius.     

Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.     

Morphological and molecular characterization of the strawberry black leaf spot pathogen referred to as the strawberry pathotype of Alternaria alternata

The remaining unclarified taxon among the seven known pathotypes of host-selective toxin (HST)-producing Alternaria alternata, namely, the strawberry pathotype (the strawberry black leaf spot pathogen), is taxonomically revised and re-described herein. According to our morphological observations, reference isolates of strawberry and Japanese pear pathotypes, which are toxic to leaves of Japanese pear ‘Nijisseiki’, have conidia that are formed in chains of 3–13, usually without lateral branches, after 7?d incubation on potato-carrot agar. The mean size of the conidia is 27–31?×?11–13?μm. Morphological characteristics of the examined isolates are identical to those of A. gaisen rather than A. alternata. A phylogenetic tree obtained by analysis of a combined dataset of ITS, gapdh, rpb2, tef1, Alt a 1, and endoPG sequences also strongly supports both pathotypes as one species, A. gaisen. We re-describe the fungus as A. gaisen Nagano ex Bokura and propose two formae speciales of the species, A. gaisen f. sp. fragariae producing AF-toxin and f. sp. pyri producing AK-toxin. The epitype specimen and ex-epitype culture of A. gaisen are newly designated.     

Molecular identification of four Panonychus species (Acari: Tetranychidae) in Korea,including new records of P. caglei and P. mori

Four species of the genus Panonychus are identified in Korea including two previously known species P. citri (McGregor) and P. ulmi (Koch) and two newly identified species P. mori Yokoyama and P. caglei Mellot. Morphological diagnostics were observed among the four species in the color of dorsal tubercles and in the shape of male aedeagus. The dorsal tubercles of P. citri, P. ulmi, P. mori and P. caglei are red, white, whitish red and light red, respectively. The aedeagi of male adults are diagnostic in sigmoid‐shaped at the distal end and its shape and length was different among four species. Molecular comparison of the internal transcribed spacer 2 (ITS2) sequence of nuclear DNA and cytochrome‐c oxidase subunit I (COI) nucleotide sequence of mitochondrial DNA among the four species showed divergences 8–12% and 9–12%, respectively. Molecular analysis of the ITS2 and COI sequences revealed their divergences were slightly different among four species. In addition, species‐specific primer sets were designed at the base on ITS2 sequences to precisely diagnose these four species at the molecular level.     

Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area,China

In this paper, species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area, Jiangsu Province of China were investigated during 2010–2011. Based on the sequences analysis of nuclear-encoded ITS (including 5.8S rDNA regions) and 5S rDNA spacer regions, dominant species of both attached and free-floating Ulva samples were identified as Ulva compressa, Ulva linza, Ulva prolifera and Ulva flexuosa. Phylogenetic tree based on sequences of ITS and 5S rDNA spacer regions for attached and free-floating Ulva species was constructed, respectively. Species compositions of the Ulva population attached on aquaculture rafts varied with seasons, and U. prolifera was only found on aquaculture rafts in March 2011 during the 2010–2011 Porphyra yezoensis cultivation season, which had the same sequences of ITS and 5S rDNA spacer regions as that of the dominant species bloomed in the Yellow Sea of China in 2008. Dominant species of the free-floating Ulva population at the early stage of the green tide were U. compressa, U. flexuosa, and U. linza. Free-floating U. prolifera appeared in the middle of May, 2011. ITS sequence similarity rates of U. compressa and U. flexuosa between the attached and free-floating species were 100%. And ITS and 5S rDNA spacer sequences of the attached and the free-floating U. prolifera population also showed no differences. Further study showed that there were two types of free-floating U. prolifera population (Type 5S-A and Type 5S-B) based on 5S rDNA spacer sequences. The present study would provide some useful information for clarifying the outbreak mechanism of green tides occurred in the Yellow Sea, China.     

Intra-individual internal transcribed spacer 1 (ITS1) and ITS2 ribosomal sequence variation linked with multiple rDNA loci: A case of triploid Atractolytocestus huronensis, the monozoic cestode of common carp

Complete sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) and karyological characters of the monozoic (unsegmented) tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia were analysed, revealing considerable intra-genomic variability and triploidy in all analysed specimens. Analysis of 20 sequences of each ITS1 and ITS2 spacer yielded eight and 10 different sequence types, respectively. In individual tapeworms, two to four ITS1 and three to four ITS2 sequence types were found. Divergent intra-genomic ITS copies were mostly induced by nucleotide substitutions and different numbers of short repetitive motifs within the sequence. In addition, triploidy was found to be a common feature of A. huronensis. The karyotype of Slovakian A. huronensis possesses three sets of chromosomes (3n = 24, n = 4m + 3st + 1 minute chromosome), similar to the previously described triploidy in conspecific tapeworms from North America. Fluorescent in situ hybridisation (FISH) with a ssrDNA probe revealed two distinct rDNA clusters for each homologue of the triplet number 2. To date, A. huronensis is the only cestode species in which intra-individual ITS sequence variants were found in parallel with its triploid nature and multiple rDNA loci. Some of these molecular and genetic features were observed in several other species of basal or nearly basal tapeworms of the orders Caryophyllidea and Diphyllobothriidea, which indicates that the phenomena may be characteristic for evolutionarily lower tapeworms and deserve more attention in future studies.     

Intermediate Hosts of Angiostrongylus cantonensis in Tenerife,Spain

The nematode Angiostrongylus cantonensis is the causative agent of human angiostrongyliasis, the main clinical manifestation of which is eosinophilic meningitis. Although this parasite has been found recently in its definitive rat host in Tenerife (Canary Islands, Spain), showing a widespread distribution over the north-east part of the island, there are no available data regarding which snail and/or slug species are acting as intermediate hosts on this island. Consequently, the objective of this work was to determine the possible role of three mollusc species, Plutonia lamarckii, Cornu aspersum and Theba pisana, as intermediate hosts of A. cantonensis in Tenerife. Between 2011 and 2014, 233 molluscs were collected from five biotopes where rats had been found previously to harbor either adult worms or antibodies against A. cantonensis, and the identification was carried out on the basis of morphological features and a LAMP technique. The prevalence of A. cantonensis larvae in the mollusc samples, based on morphological identification, was 19.3%, whereas 59 out of the 98 individuals (60.2%) analyzed by LAMP were positive. Positive results were obtained for the three mollusc species analyzed and two of the positive samples, both obtained from P. lamarckii, were confirmed as positive by 18S rRNA and ITS1 PCR. Sequence analysis of 18S rRNA PCR products showed 100% similarity with previously published A. cantonensis sequences. These results may be relevant from a public health point of view, since all the biotopes from which the samples were obtained were in inhabited areas or areas with human activity, but it is also important from the perspective of a possible transmission to other accidental hosts, such as dogs and horses, animals that are present in some of the areas analyzed.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

Evidence for extensive cryptic speciation in trematodes of butterflyfishes (Chaetodontidae) of the tropical Indo-West Pacific

Molecular data from the cytochrome c oxidase subunit I (cox1) mitochondrial DNA gene and the second internal transcribed spacer (ITS2) nuclear rDNA region were used to test the current morphologically-based taxonomic hypothesis regarding species of Monorchiidae (Hurleytrematoides) from chaetodontid and tetraodontid fishes from six sites in the tropical Indo-West Pacific (TIWP): Heron and Lizard Islands off the Great Barrier Reef (GBR, Australia), Moorea (French Polynesia), New Caledonia, Ningaloo Reef (Australia) and Palau. The 16 morphospecies analysed differed from each other by a minimum of 55 bp (9.1%) over the mitochondrial cox1 and 8 bp (1.6%) over the ITS2 DNA regions. For two species, Hurleytrematoides loi and Hurleytrematoides sasali, specimens from the same host species in sympatry differed at levels comparable to those between pairs of distinct morphospecies for both cox1 and ITS2 sequences. We take this as evidence of the presence of combinations of cryptic species; however, we do not propose new species for these taxa because we lack identified morphological voucher specimens. For seven species, Hurleytrematoides coronatum, Hurleytrematoides deblocki, Hurleytrematoides faliexae, H. loi, Hurleytrematoides morandi, H. sasali and Hurleytrematoides sp. A, samples from some combinations of localities had base pair differences that were equal to or greater than differences between some pairs of distinct morphospecies for one or both cox1 and ITS2 sequences. For three species, H. coronatum, H. loi and H. morandi, one haplotype differed from every other haplotype by more than the morphospecies benchmark. In these cases morphological specimens could not be distinguished by morphology. These data suggest extensive cryptic richness in this genus. For the present we refrain from dividing any of the morphospecies. This is because there is a continuum of levels of intra- and interspecific genetic variation in this system, so that distinguishing the two would be largely arbitrary.     

Natural hybridization between Paragonimus bangkokensis and Paragonimus harinasutai

Paragonimus bangkokensis and Paragonimus harinasutai, which are morphologically distinguishable species, often co-infect in the same crab intermediate hosts. Recent molecular phylogenetic analyses revealed that these two species are genetically close to each other and are considered as the sister species. While we have been studying Paragonimus adult worms obtained from the lungs of a cat experimentally infected with Paragonimus metacercariae which were morphologically identified as P. harinasutai collected from central Viet Nam, one out of 6 adult worms has grouped cuticular spines, which is a feature of P. bangkokensis. By molecular analyses, the CO1 sequence of this specimen was identical with that of P. bangkokensis, but the ITS2 and the D2 region of 28S rDNA sequences showed a two peak pattern. Then, PCR products of the ITS2 and the D2 region of 28S rDNA sequences were ligated to TOPO vector and subcloned to determine the heterozygosity. Two types of sequences were obtained from each ITS2 and D2 region of 28S; one was identical with P. harinasutai and the other with P. bangkokensis. Taking all these morphological and molecular data together, we identified this adult worm as a hybrid specimen of P. bangkokensis and P. harinasutai.     

Two new Pythium species from China based on the morphology and DNA sequence data

In an investigation of Pythium species in lawn grassland of south China, two new species, Pythium breve and P. baisense, were identified based on morphological characteristics and DNA sequence data. These two new species differ morphologically from the other Pythium species by the oogonia encompassed by many antheridia and with 1?C3 antheridia on the wavy and curved stalks. Furthermore, P. baisense with complexly lobed antheridial stalks differs from P. breve with antheridial stalks entwining the hyphae nearby the oogonia with several turns. Results of phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species based on the internal transcribed spacer region (ITS1, 5.8S, ITS2) and cytochrome c oxidase subunit I (COXI) gene sequences using maximum parsimony and Bayesian methods. These two new species are described and illustrated in detail.     

The phylogenetic position of an <Emphasis Type="Italic">Armillaria</Emphasis> species from Amami-Oshima,a subtropical island of Japan,based on elongation factor and ITS sequences

An undetermined Armillaria species was collected on Amami-Oshima, a subtropical island of Japan. The phylogenetic position of the Armillaria sp. was determined using sequences of the elongation factor-1α (EF-1α) gene and the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The phylogenetic analyses based on EF-1α and ITS sequences showed that this species differs from known Japanese taxa of Armillaria. The sequences of this species and A. novae-zelandiae from Southeast Asia were contained in a strongly supported clade, which was adjacent to a well-supported sister clade containing A. novae-zelandiae from Australia and New Zealand.