Antarctic lakes have generally simple periphyton communities when compared with those of lower latitudes. To date, assessment of microbial diversity in Antarctica has relied heavily on traditional direct observation and cultivation methods. In this study, sterilized cotton baits were left submerged for two years in two lakes on King George Island and Deception Island, South Shetland Islands (Maritime Antarctic), followed by assessment of diversity by metabarcoding using high-throughput sequencing. DNA sequences of 44 taxa belonging to four kingdoms and seven phyla were found. Thirty-six taxa were detected in Hennequin Lake on King George Island and 20 taxa were detected in Soto Lake on Deception Island. However, no significant difference in species composition was detected between the two assemblages (Shannon index). Our data suggest that metabarcoding provides a suitable method for the assessment of periphyton biodiversity in oligotrophic Antarctic lakes.

To date, there are very few sequence data for Cyperaceae from mainland Southeast Asia. The aim of the present study was to contribute nuclear ribosomal internal transcribed spacer (ITS) sequences of selected species of Cambodian Cyperaceae to the overall phylogeny of the family. We generated ITS sequences of 38 accessions representing 26 species from Cambodia and used these sequences for phylogenetic analysis together with similar sequences from the National Center for Biotechnology Information GenBank. Our results corroborate recent phylogenetic work in the family and largely confirm established tribal relationships. The backbone of the phylogenetic tree of species-rich genera that have undergone rapid radiations is often weakly resolved (e.g. in Fimbristylis and in the C4 clade of Cyperus). Cryptic variation was revealed in the taxonomically difficult group of Fimbristylis dichotoma, with samples of this taxon appearing in two distinct clades within Fimbristylis. Further addition of geographically spread accessions of taxa will improve our understanding of the complex biogeographical history of the genera in the family. Eleocharis koyamae Tremetsb. & D.A.Simpson is proposed as a new name for E. macrorrhiza T. Koyama.

Molecular techniques are important tools for microbiological studies in different habitats, and the internal transcribed spacer (ITS) has been proved to be useful for analyzing fungal diversity. The aim of this study was to use the ITS region to generate ruminal yeast profile and to identify ruminal yeast. DNA from ruminal digesta was extracted to amplify the ribosomal ITS region. The profile from the PCR products was visualized and the excised bands from the profile were identified as the genera Millerozyma, Pichia, Rhizomucor and Hyphopichia. Overall, the ITS resulted to be a simple, fast and sensitive approach that allowed profiling and identification of ruminal yeast that have not been previously described (Millerozyma and Hyphopichia) in the rumen microbial community.

We evaluated the systematic relationship of the two sections of Raphanus (Brassicaceae) on the basis of morphological and ITS data. We obtained strong support that Raphanus is a polyphyletic group embedded in the Oleracea lineage of the tribe Brassiceae. Section Raphanis, which includes Raphanus raphanistrum, R. pugioniformis and R. sativus, is a strongly supported monophyletic lineage. Section Hesperidopsis is embedded in a different lineage together with Brassica deflexa and B. aucheri. We propose to reinstate the genus Quidproquo in place of Raphanus sect. Hesperidopsis as reflected by both morphological data and ITS phylogeny.

Seven ancient seeds, about 1600 years old, were found during an archaeological excavation in Asar Island which is located in south-western Turkey. These seeds were subjected to germination, in vitro callus induction and molecular characterization experiments to test the viability and plant origin of the seeds. Six of the seven seeds had viable seed components (such as cotyledons) and produced callus tissue in Murashige and Skoog medium supplemented with 2 mg l− 1 6-benzylamino purine (BAP), 0.2 mg l− 1 1-naphthaleneacetic acid (NAA), 20 g l− 1 sucrose, 2 mg l− 1 glycine and 7 g l− 1 agar, but the calli from these seeds failed to yield adventitious shoots. DNA samples from callus tissues produced by ancient seeds in vitro were of good quality. The internal transcribed spacer (ITS) region in nuclear DNA (nDNA) of ancient seeds was amplified successfully. The sequences from amplified ITS DNA products of six ancient seeds indicated that their ITS sequences matched those of Anagyris foetida after subjecting them to BLAST searches in international sequence databases (NCBI). A. foetida is a relict species endemic to the Mediterranean region and used as a herbal medicine. We believe that seed characteristics such as the very hard, extremely smooth and shiny testa, toxic anagyrine alkaloid content and their storage in a pot further improved the longevity of these ancient seeds.

Phymactis papillosa is a rocky shore sea anemone that is commonly found in the Pacific Ocean, from the Gulf of California to Tierra del Fuego, and in the Mar del Plata region, Argentina. The genus Phymactis is closely related to Bunodosoma and, due to character plasticity, a number of misidentifications have occurred. Therefore, the presence of P. papillosa in Argentina has been doubted but the matter had not been investigated in detail. Here we analyse P. papillosa specimens from Argentina and compare them, using molecular and morphological markers, to specimens from the species' type locality. In a phylogenetic analysis using 19 allozyme markers and ribosomal internal transcribed spacers sequences of different sea anemone genera, including all West Atlantic Bunodosoma species, we have found that the specimens from Argentina were genetically divergent from P. papillosa from Chile and closely related to West Atlantic Bunodosoma species. The genetic and morphological analyses indicate that those specimens belong to a new species of the genus Bunodosoma, described here as B. zamponii sp. nov.

Neospora caninum and N. caninum-like organisms are cyst-forming coccidian parasites known to cause neuromuscular disorders in dogs and abortion in cattle. In this article we report on the use of the polymerase chain reaction (PCR) for the detection of DNA from N. caninum. After determining the sequence of the internal transcribed spacer 1 (ITSl) of N. caninum and Toxoplasma gondii, and part of the sequences for & species of Sarcocystis, we designed a primer set for the amplification of a 279-base-pair fragment of ITSl from N. caninum. The PCR system made possible the specific detection of 5 N. caninum organisms and no amplification was observed from any of the other cyst-forming coccidia tested, including the closely related T. gondii. Furthermore, we were also able to demonstrate the presence of N. caninum in brain and lung tissue samples from experimentally infected mice. Our data also link the 5-8S rRNA gene for T. gondii and N. caninum to the 16S-like rRNA gene, within the rDNA unit.

The fine-scale genetic variation and population structure of lichen-forming fungi is little known and sampling strategies are rarely recommended or discussed. I tested if the sample sizes of molecular data sets used in recent population studies of Xanthoria parietina revealed all haplotypes potentially present and, accordingly, quantified how many haplotypes were potentially missing in the samples. Data sets were concatenated from two geographical regions in Scandinavia and investigated if the sampling reached saturation at two levels: 1) individual-based using rarefaction curves and 2) population-based using species accumulation curves. At both levels, the matrices of two molecular markers (IGS and ITS) were analysed separately. The molecular markers show similar and parallel patterns in all analyses. Rarefaction analyses did not reveal different patterns for populations in different habitats, i.e., bark and rock. Species accumulation curves estimated with the Chao 1 richness estimator indicated that 23% of the IGS and 8% of the ITS haplotypes were not detected. Corresponding figures from an abundance-based coverage estimator (ACE) were 37% and 18%. Pilot studies are recommended to determine appropriate sample sizes for genetic-based population studies of lichen-forming fungi.

The taxonomic history of members of the 37-collar-spine group within the genus Echinostoma has been very confused. We obtained DNA sequence data from the nuclear rDNA ITS1, 5·8S and ITS2 of 7 nominal species belonging to this group, Echinostoma trivolvis (Cort, 1914), E. revolution (Frölich, 1802), E. caproni Richard, 1964, E. liei Jeyarasasingam et al. 1972, E. paraensei Lie & Basch, 1967, two African isolates, E. sp.I and E. sp.II, and of one 28-collar-spined echinostome, E. hortense (Asada, 1926). Five of the eight species were clearly distinguishable using ITS data. Sequences from the remaining three taxa, E. caproni, E. sp.II and E. liei were identical to one another and the group containing these taxa was distant from other 37-collar-spine species on a phylogenetic tree. E. trivolvis and E. paraensei form a second, but less distinct group within the 37-collar-spine group. The resolution obtained using DNA sequencing will assist in the current reclassification of the group. It also provides a model for future work on sibling species.

The xanthorioid taxa Xanthomendoza borealis and Xanthoria mawsonii are revised using morphological, anatomical, secondary chemical, and molecular characters. We conclude that most specimens determined as X. mawsonii from the Antarctic are conspecific with specimens labelled X. borealis from the Northern Hemisphere. Some specimens named X. mawsonii from the Antarctic are wrongly determined specimens of Xanthoria candelaria (L.) Th. Fr, including the holotype of Xanthoria mawsonii C. W. Dodge. Accordingly, Xanthoria mawsonii C. W. Dodge is a synonym to Xanthoria candelaria (L.) Th. Fr. The only available correct name for the bipolar species, which comprises material previously determined as X. borealis and X. mawsonii, and is distinct from the more widespread taxon X. candelaria, is Xanthomendoza borealis (R. Sant. & Poelt) Søchting, Kärnefelt & S. Kondratyuk.

Lepraria granulata Slavíková, a new species from Eastern and Central Europe, is described. According to its morphology, ITS sequence and ecology, it belongs to the L. neglecta core group. Lepraria granulata is characterized by producing atranorin and two, so far unidentified, fatty acids (‘granulata unknown 1 & 2’) and phylogenetic analyses show that it is clearly distinguished from previously known species. Two additional taxa, provisionally named Lepraria sp. G and Lepraria sp. BG, are also discussed. According to their morphology and ITS sequences, they also belong to the L. neglecta group. Lepraria sp. G is sister to L. granulata whereas Lepraria sp. BG is basal and well separated from all taxa so far known in the group. Their taxonomic status is not yet entirely clear.

Hydro-distilled essential oils from fruits, aerial parts (without fruits) and roots of Pimpinellaspecies native to Turkey and their phylogenetic relationships to one another were examined. Phytochemical investigation of the essential oils of 19 species resulted in isolation of 16 phenylpropanoids, four sesquiterpenes and two azulene-type norsesquiterpenes. The structures of the isolated compounds were determined primarily from 1D- and 2D-NMR experiments as well as liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry. Phylogenetic relationships among 26 species were evaluated using ITS 1, ITS 4 nuclear rDNA and psbA-trnH cpDNA sequences. In this study, significance and occurrence of phenylpropanoids, azulenes and geijerenes are discussed from a phylogenetic, chemical and biosynthetic perspective. The distribution of different classes of compounds and their putative associations with one another as per current knowledge of their biosynthetic pathways indicates that this information, in conjunction with the phylogeny, could provide valuable information regarding the presence and perhaps evolution of the different classes of compounds. Analysis of the phenylpropanoid components indicates that (E)-anethole is an obligatory intermediate of this pathway. The various Pimpinellaspecies differ primarily in their ability to acylate anethole, suggesting that while the pathway leading to anethole is common to this genus, species differ in their enzymatic machinery leading to acylate. The relationship between azulenes and geijerenes is not as intuitive, but all Pimpinellaspecies analysed in this study have the biochemical machinery required to synthesize these chemical classes.

Hammondia heydorni is regarded as a protozoan parasite that uses canids, e.g. dogs and foxes, as definitive hosts, but clinical signs of infection are rare. This study therefore took advantage of the opportunity to study an oocyst population from the faeces of a dog suffering from intermittent bouts of diarrhoea. Oocysts from the naturally infected dog were shown to be H. heydorni by using the polymerase chain reaction combined with DNA sequencing as a diagnostic tool.The nucleotide sequence data reported in this paper are available from GenBank under the following Accession numbers DQ183058, DQ183059 and DQ022687. A comparison of the first internal transcribed spacer (ITS1) sequence of ribosomal DNA obtained with those from other dog and fox oocysts, previously regarded as H. heydorni, showed these oocysts contained identical ITS1 sequences. However, the oocyst DNA from the fox and dog differed by the presence/absence of a 9 bp insertion/deletion within intron 1 of the alpha tubulin gene, and this difference was conserved across a number of different oocyst populations from the 2 species of host. A PCR assay was established that takes advantage of this insertion/deletion and is able to differentiate between the 2 oocyst populations. This study therefore provides evidence that H. heydorni oocysts from dogs and foxes represent 2 distinct genetic lineages that can be differentiated using a PCR, which targets the alpha tubulin locus.

The evolutionary relationships of several, mainly Iberian, Jasione taxa (Campanulaceae) were investigated using molecular data. A parsimony analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA was partially successful in elucidating some interspecific alliances. One of the main clades included three morphologically rather disparate species from S Spain and Morocco which might indicate that these areas were refugia during the last glaciations. The fact that most of the other taxa fell in a large polytomy suggests a recent origin of these species. Amplified fragment length polymorphisms (AFLPs) were analysed for a larger sample set of two taxa from within the ITS polytomy to assess the partition of genetic variation between and within populations. All the British accessions named J. montana var. montana were found to be close to those so-named from Spain. However, individual accessions of J. montana var. montana from Argyll (Scotland), Devon (SW England) and the Shetland Islands proved to be genetically different from each other. This might suggest a low level of genetic diversification from a common progenitor due to a rapid northward migration. It is proposed that J. montana var. bracteosa and J. maritima var. sabularia merit recognition at higher rank. The possibility that J. montana vars gracilis and latifolia are mere repetitive segregants of J. montana var. montana is considered.

Freshwater snails belonging to the genus Biomphalaria are intermediate hosts of the trematode Schistosoma mansoni in the Neotropical region and Africa. In Brazil, one subspecies and ten species of Biomphalaria have been identified: B. glabrata, B. tenagophila, B. straminea, B. occidentalis, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and B.t. guaibensis. However, only the first three species are found naturally infected with S. mansoni. The classical identification of these planorbids is based on comparison of morphological characteristics of the shell and male and female reproductive organs, which is greatly complicated by the extensive intra-specific variation. Several molecular techniques have been used in studies on the identification, genetic structure as well as phylogenetic relationships between these groups of organisms. Using the randomly amplified polymorphic DNAs (RAPD) analysis we demonstrated that B. glabrata exhibits a remarkable degree of intra-specific polymorphism. Thus, the genetics of the snail host may be more important to the epidemiology of schistosomiasis than those of the parasite itself. Using the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) in intra-populational and intra-specific studies we have demonstrated that snails belonging to the B. straminea complex (B. straminea, B. kuhniana and B. intermedia) clearly presented higher heterogeneity. Using the low stringency polymerase chain reaction (LS-PCR) technique we were able to separate B. glabrata from B. tenagophila and B. tenagophila from B. occidentalis. To separate all Brazilian Biomphalaria species we used the restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer region (ITS) of the DNA gene. The method also proved to be efficient for the specific identification of DNA extracted from snail eggs. Recently we have sequenced the ITS2 region for phylogenetic studies of all Biomphalaria snails from Brazil.

The anisakid nematodes morphologically corresponding with Pseudoterranova decipiens sensu lato (s.l.) (Krabbe, 1878) from different seal or sea lion hosts and geographical origins, previously identified as Pseudoterranova krabbei, P. decipiens (s.s.), P. bulbosa, P. azarasi and P. cattani by multilocus enzyme electrophoresis, were characterized using a DNA approach. Also a population of P. decipiens (s.l.) from Chaenocephalus aceratus, the blackfin icefish, from Antarctica and another from Osmerus eperlanus, the European smelt, from Germany were included in the study. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by PCR from individual nematodes and analysed by single-strand conformation polymorphism (SSCP), followed by selective sequencing. While no variation in single-stranded ITS-1 and ITS-2 profiles was detected among samples representing each of the species or populations (with the exception of slight microheterogeneity), SSCP analysis of the ITS-2 amplicons allowed the unequivocal differentiation of all of the 5 sibling species of P. decipiens (s.l.) examined, which was supported by sequence differences in ITS rDNA. Samples representing the P. decipiens (s.l.) population from O. eperlanus had the same SSCP profile as those of P. decipiens (s.s.), which was supported by a lack of nucleotide difference in the ITS between them, suggesting that the former represented P. decipiens (s.s.). Based on SSCP results and ITS sequence data, P. decipiens (s.l.) from C. aceratus was genetically most distinct with respect to all other members of Pseudoterranova examined, which indicated that it may represent P. decipiens E (based on geographical origin) or a distinct species. These findings and the molecular approach taken should have important implications for studying the life-cycles, transmission patterns, epidemiology and population genetics of these anisakid nematodes, and the diagnosis of their infections.

Maturation of the large subunit rRNAs includes a series of cleavages that result in removal of the internal transcribed spacer (ITS2) that separates mature 5.8S and 25/28S rRNAs. Previous work demonstrated that formation of higher order secondary structure within the assembling pre-ribosomal particle is a prerequisite for accurate and efficient pre-rRNA processing. To date, it is not clear which specific sequences or secondary structures are required for processing. Two alternative secondary structure models exist for Saccharomyces cerevisiae ITS2. Chemical and enzymatic structure probing and phylogenetic comparisons resulted in one structure (Yeh & Lee, J Mol Biol, 1990, 211:699–712) referred to here as the “hairpin model.” More recently, an alternate folded structure was proposed (Joseph et al., Nucleic Acids Res, 1999, 27:4533–4540), called here the “ring model.” We have used a functional genetic assay to examine the potential significance of these predicted structures in processing. Our data indicate that elements of both structural models are important in efficient processing. Mutations that prevent formation of ring-specific structures completely blocked production of mature 25S rRNA, whereas those that primarily disrupt hairpin elements resulted in reduced levels of mature product. Based on these results, we propose a dynamic conformational model for the role of ITS2 in processing: Initial formation of the ring structure may be required for essential, early events in processing complex assembly and may be followed by an induced transition to the hairpin structure that facilitates subsequent processing events. In this model, yeast ITS2 elements may provide in cis certain of the functions proposed for vertebrate U8 snoRNA acting in trans.

Of the 455 known genera in Apiaceae (Umbelliferae), 41% are monotypic and 26% comprise only two or three species each. Similarly, of the 16 genera constituting Scandiceae Spreng. subtribe Scandicinae Tausch, seven (Balansaea Boiss. & Reut., Kozlovia Lipsky, Krasnovia Schischk., Myrrhis Mill., Myrrhoides Fabr., Sphallerocarpus DC. and Todaroa Parl.) are monotypic, and two (Neoconopodium Pimenov & Kljuykov and Tinguarra Parl.) are bitypic. Phylogenetic analysis of the subtribe, using molecular (rDNA ITS), morphological and anatomical data, indicates that the number of genera may be reduced with only three, Todaroa, Sphallerocarpus and Myrrhis, retained as monotypic. Remaining taxa form eight clades that are supported by high bootstrap values and are morphologically distinct. Four of these clades (Anthriscus Pers., Geocaryum Coss., Osmorhiza Raf. and Scandix L.) are equivalent to currently recognized genera. Kozlovia, Krasnovia and Neoconopodium form a well-supported clade that may be recognized as the single genus Kozlovia, and Myrrhoides is grouped with Chaerophyllum L. Tinguarra and Athamanta L. form a monophyletic group that is well supported by analyses of morphology, fruit anatomy, and combined morphological, anatomical and ITS sequence data; however, this group is not maintained in separate analyses of ITS sequences. Similarly, the group formed by Conopodium W. D. J. Koch and Balansaea is monophyletic in morphological, anatomical, and combined analyses, but is not supported by the separate analyses of ITS sequence data. All of these groups are well delimited on the basis of fruit characters that have long been regarded as essential in umbellifer taxonomy. One new combination is proposed: Athamanta montana (Webb ex H. Christ.) Spalik, A. Wojew. & S. R. Downie.

Phylogenetic analyses of chloroplast gene (rbcL, matK), intron (rpl16, rps16, rpoC1) and nuclear ribosomal DNA internal transcribed spacer (ITS) sequences and chloroplast DNA restriction sites, with supplementary data from variation in size of the chloroplast genome inverted repeat, have been used to elucidate major clades within Apiaceae (Umbelliferae) subfamily Apioideae Drude. This paper summarizes the results of previously published molecular cladistic analyses and presents a provisional classification of the subfamily based on taxonomic congruence among the data sets. Ten tribes (Aciphylleae M. F. Watson & S. R. Downie, Bupleureae Spreng., Careae Baill., Echinophoreae Benth., Heteromorpheae M. F. Watson & S. R. Downie, Oenantheae Dumort., Pleurospermeae M. F. Watson & S. R. Downie, Pyramidoptereae Boiss., Scandiceae Spreng. and Smyrnieae Spreng.) are erected or confirmed as monophyletic, with Scandiceae comprising subtribes Daucinae Dumort., Scandicinae Tausch and Torilidinae Dumort. Seven additional clades are also recognized but have yet to be treated formally, and at least 23 genera examined to date are of dubious tribal or clade placement. The utility of these different molecular markers for phylogenetic inference in Apioideae is compared based on maximum parsimony analyses of subsets of previously published molecular data sets. Of the six loci sequenced, the ITS region is seen to be evolving most rapidly and rbcL is the most conservative. Intermediate in rate of evolution are matK and the three chloroplast introns; with rpl16 and rps16 evolving slightly faster than matK or rpoC1. The analysis of restriction sites, however, provided 2–4 times more parsimony informative characters than any single DNA locus sequenced, with estimates of divergence just slightly lower than that of the ITS region. The trees obtained from separate analyses of these reduced data sets are consistent with regard to the major clades inferred and the relationships among them. Similar phylogenies are obtained by combining data or combining trees, representing the supermatrix and supertree approaches to phylogenetic analysis, respectively. The inferred relationship among the tribes and informally recognized major clades within Apioideae is presented.

Systematic relationships within the Hawaiian group Tetraplasandra A. Gray, Munroidendron Sherff and Reynoldsia sandwicensis A. Gray are investigated here based on parsimony analysis of nucleotide sequences from the internal transcribed spacer (ITS 1 and 2) regions and the non-transcribed spacer of 5S (5S-NTS) nuclear ribosomal DNA. Results indicate that Tetraplasandra, Munroidendron and Reynoldsia sandwicensis form a single monophyletic group, and that Reynoldsia A. Gray is paraphyletic. Both data sets support combining Reynoldsia sandwicensis with Munroidendron and treating it as distinct from Reynoldsia species outside Hawaii. Both data sets support two major clades: a Munroidendron/Reynoldsia sandwicensis clade and a Tetraplasandra clade, but they are discordant in relation to the monophyly of Tetraplasandra. 5S-NTS results support treating Tetraplasandra, Munroidendron and Reynoldsia sandwicensis as a single genus, whereas ITS results suggest that Tetraplasandra represents a distinct genus from Reynoldsia sandwicensis and Munroidendron. Tetraplasandra gymnocarpa (Hillebr.) Sherff is the only member of the Araliaceae with a completely superior ovary, and in T. kavaiensis (H. Mann) Sherff the ovary is partially superior. Together, they form a well-supported clade within the Hawaiian complex, confirming the hypothesis that in Tetraplasandra superior ovaries evolved in Hawaii from an epigynous ancestor.