The adult fluke Stomylotrema vicarium (Stomylotrematidae, Microphalloidea) was described for the first time in Theristicus caerulescens in 1901, but the complete life cycle has remained unknown to date. Here, we found a stomylotrematid trematode in the digestive gland of the endemic apple snail Pomacea americanista. The digestive gland's tubuloacini were compressed by the trematode larvae placed on connective tissues and haemocoel spaces. Non-virgulate, stylet-bearing cercariae showed three pairs of penetration glands with a body, oral sucker and stylet morphometrically similar to those of stylet-bearing, unencysted young metacercariae of S. vicarium found in the aquatic coleopteran Megadytes glaucus, and at a lesser extent with cercariae of S. gratiosus found in the apple snail Pomacea maculata. The larvae molecular phylogeny was inferred using the markers rRNA 28S and ITS1, being these sequences grouped with the sequences of S. vicarium obtained from adult flukes. Together, these findings indicate that the life cycle of S. vicarium begins in P. americanista, thus supporting the hypothesis that the ampullariid snails act as a first intermediate host.

An important step towards developing a successful integrated pest management (IPM) programme for the control of banana pseudostem weevil, Odoiporus longicollis (Olivier), a serious pest of banana in India, is the study of the population structure of the pest. In the present study, the genetic variation among 30 individual weevils of O.longicollis collected from six different locations was assessed by analysing the primary nucleotide sequences of the rDNA ITS1 and ITS2 regions. AMOVA, Mantel test, and the maximum likelihood trees of the haplotypes failed to reveal any phylogeographic structuring, which was confirmed by haplotype analysis and genetic differentiation estimates. The results indicate that the locations are not differentiated, i.e. there is gene flow between the locations. The star-shaped networks revealed a signature of demographic expansion that was confirmed by the different demographic tests. These results provide important information, which is essential for the development of suitable strategies for the control of this banana pest, as well as management of its resistance to insecticides.

Posthodiplostomum minimum utilizes a three-host life cycle with multiple developmental stages. The metacercarial stage, commonly known as ‘white grub’, infects the visceral organs of many freshwater fishes and was historically considered a host generalist due to its limited morphological variation among a wide range of hosts. In this study, infection data and molecular techniques were used to evaluate the host and tissue specificity of Posthodiplostomum metacercariae in centrarchid fishes. Eleven centrarchid species from three genera were collected from the Illinois portion of the Ohio River drainage and necropsied. Posthodiplostomum infection levels differed significantly by host age, host genera and infection locality. Three Posthodiplostomum spp. were identified by DNA sequencing, two of which were relatively common within centrarchid hosts. Both common species were host specialists at the genus level, with one species restricted to Micropterus hosts and the other preferentially infecting Lepomis. Host specificity is likely dictated by physiological compatibility and deviations from Lepomis host specificity may be related to host hybridization. Posthodiplostomum species also differed in their utilization of host tissues. Neither common species displayed strong genetic structure over the scale of this study, likely due to their utilization of bird definitive hosts.

Asynchronous flowering was noted in a recently discovered infestation of siam weed in north Queensland. This may indicate some genetic diversity in the infestation, increasing concerns about the origin of the infestation. Internal transcribed spacer 1 (ITS1) sequence data were obtained for siam weed individuals from north Queensland, Indonesia, Thailand, South Africa, Ivory Coast, Brazil, Colombia, and the U.S. The ITS1 region is 258 base pairs long, and the populations that flower at different times in north Queensland differ by four base substitutions. The genotype common in north Queensland is also reported throughout the native and introduced ranges. The other genotype is reported only in north Queensland and southern Brazil. These data, in conjunction with prior investigations into possible origins, indicate that Brazil is the most likely source of the infestation in Australia.

Mentha (Nepetoideae, Lamiaceae) is a taxonomically complex genus consisting of 29 species and 11 different hybrids including a number of economically important species among them medical plants used for more than 250 years. Because of its high incidence of polyploidy, diverse morphology, high degree of chromosome number variations and interspecies hybridization, Mentha is considered to be a taxonomical challenge. In this paper, we describe cloning and sequencing of internal transcribed spacers (ITS) 1 and 2 of three mint species: Mentha aquatic L., Mentha longifolia L. (Huds) and Mentha piperita L., collected from natural habitats of Bulgaria. The applicability of ITS1 and ITS2 for molecular fingerprinting and further comparative studies, including the newly obtained ITS1 and ITS2 sequences, was tested in phylogenetic analysis of all eight Mentha species, found in Bulgarian flora.

Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

Host specificity is a fundamental component of a parasite's life history. However, accurate assessments of host specificity, and the factors influencing it, can be obscured by parasite cryptic species complexes. We surveyed two congeneric species of intertidal snail intermediate hosts, Zeacumantus subcarinatus and Zeacumantus lutulentus, throughout New Zealand to identify the number of genetically distinct echinostome trematodes infecting them and determine the levels of snail host specificity among echinostomes. Two major echinostome clades were identified: a clade consisting of an unidentified species of the subfamily Himasthlinae and a clade consisting of five species of the genus Acanthoparyphium. All five Acanthoparyphium species were only found in a single snail species, four in Z. subcarinatus and one in Z. lutulentus. In contrast, the Himasthlinae gen. sp. was found in both hosts, but was more prevalent in Z. lutulentus (97 infections) than Z. subcarinatus (10 infections). At least two of the Acanthoparyphium spp. and the Himasthlinae gen. sp. are widespread throughout New Zealand, and can therefore encounter both snail species. Our results suggest that host specificity is determined by host–parasite incompatibilities, not geographic separation, and that it can evolve in different ways in closely related parasite lineages.

Laelaps giganteus and Laelaps muricola (Mesostigmata; Laelapidae) are widespread and locally abundant host generalists on small mammals in southern Africa. The large host range and complex life history of these ectoparasites may allude to possible intraspecific cryptic diversity in these taxa. To assess genetic and morphological diversity in L. giganteus and L. muricola, we sampled 228 rodents at eight localities in South Africa. This sample included nine previously recorded host species and on these, L. muricola was only recorded from Mastomys natalensis and Micaelamys namaquensis while L. giganteus was found on Rhabdomys dilectus and Lemniscomys rosalia. Phylogenetic analyses of partial mtDNA cytochrome oxidase subunit I (COI) and nuclear ITS1 data strongly supported the recognition of L. giganteus and L. muricola, a scenario partly supported by the Tropomyosin intron. Strong support for evolutionary distinct lineages within L. giganteus is found: L. giganteus lineage 1 is confined to R. dilectus and L. giganteus lineage 2 is confined to L. rosalia. These host specific monophyletic lineages were also separated by 9·84% mtDNA sequence divergence and 3·44% nuclear DNA sequence divergence. Since quantitative morphometric analyses were not congruent with these findings, these two lineages more than likely represent cryptic species.

Oligonychus punicae and Oligonychus perseae (Acari: Tetranychidae) are the most important mite species affecting avocado orchards in Mexico. Here we used nucleotide sequence data from segments of the nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes to assess the phylogenetic relationships between both sympatric mite species and, using only ITS sequence data, examine genetic variation and population structure in both species, to test the hypothesis that, although both species co-occur, their genetic population structures are different in both Michoacan state (main producer) and Mexico state. Phylogenetic analysis showed a clear separation between both species using ITS and COI sequence information. Haplotype network analysis done on 24 samples of O. punicae revealed low genetic diversity with only three haplotypes found but a significant geographical population structure confirmed by analysis of molecular variance (AMOVA) and Kimura-2-parameter (K2P) analyses. In addition, a Mantel test revealed that geographical isolation was a factor responsible for the genetic differentiation. In contrast, analyses of 22 samples of O. perseae revealed high genetic diversity with 15 haplotypes found but no geographical structure confirmed by the AMOVA, K2P and Mantel test analyses. We have suggested that geographical separation is one of the most important factors driving genetic variation, but that it affected each species differently. The role of the ecology of these species on our results, and the importance of our findings in the development of monitoring and control strategies are discussed.

Brugia malayi is one of the parasitic worms which causes lymphatic filariasis in humans. Its geographical distribution includes a large part of Asia. Despite its wide distribution, very little is known about the genetic variation and molecular epidemiology of this species. In this study, the internal transcribed spacer 1 (ITS1) nucleotide sequences of B. malayi from microfilaria-positive human blood samples in Northeast Borneo Island were determined, and compared with published ITS1 sequences of B. malayi isolated from cats and humans in Thailand. Multiple alignment analysis revealed that B. malayi ITS1 sequences from Northeast Borneo were more similar to each other than to those from Thailand. Phylogenetic trees inferred using Neighbour-Joining and Maximum Parsimony methods showed similar topology, with 2 distinct B. malayi clusters. The first cluster consisted of Northeast Borneo B. malayi isolates, whereas the second consisted of the Thailand isolates. The findings of this study suggest that B. malayi in Borneo Island has diverged significantly from those of mainland Asia, and this has implications for the diagnosis of B. malayi infection across the region using ITS1-based molecular techniques.

Recent studies have shown that some digenean trematodes previously identified as single species due to the lack of distinguishing morphological characteristics actually consist of a number of genetically distinct cryptic species. We obtained mitochondrial 16S and nuclear ITS1 sequences for the redial stages of Acanthoparyphium sp. and Curtuteria australis collected from snails and whelks at various locations around Otago Peninsula, New Zealand. These two echinostomes are well-known host manipulators whose impact extends to the entire intertidal community. Using phylogenetic analyses, we found that Acanthoparyphium sp. is actually composed of at least 4 genetically distinct species, and that a cryptic species of Curtuteria occurs in addition to C. australis. Molecular data obtained for metacercariae dissected from cockle second intermediate hosts matched sequences obtained for Acanthoparyphium sp. A and C. australis rediae, respectively, but no other species. The various cryptic species of both Acanthoparyphium and Curtuteria also showed an extremely localized pattern of distribution: some species were either absent or very rare in Otago Harbour, but reached far higher prevalence in nearby sheltered inlets. This small-scale spatial segregation is unexpected as shorebird definitive hosts can disperse trematode eggs across wide geographical areas, which should result in a homogeneous mixing of the species on small geographical scales. Possible explanations for this spatial segregation of the species include sampling artefacts, local adaptation by first intermediate hosts, environmental conditions, and site fidelity of the definitive hosts.

Accurate identification of aspermic Fasciola forms in Japan remains difficult because of their morphological variations. In order to characterize the forms genetically, nucleotide sequences of ribosomal internal transcribed spacer (ITS1 and ITS2) and mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NDI) genes in 34 liver flukes from 16 prefectures in Japan were analysed. Two major forms represented by Fsp 1 and Fsp 2 had sequences identical to or closely resembling those of F. hepatica and F. gigantica, respectively, in all the 4 DNA markers and were mainly distributed in northern and eastern-western parts of Japan, respectively. Fsp 1 and Fsp 2 would have been introduced into Japan with infected cattle of 2 distinct lineages via the Korean Peninsula and spread through limited parts of Japan (northern and eastern-western parts) together with the movement of each cattle lineage. The Japanese form (Fsp 1/2), which showed heterozygosity in ribosomal DNA and Fsp 2 haplotype in mitochondrial DNA, may have originated in interspecific cross hybridization between paternal F. hepatica and maternal F. gigantica.

Patterns of nuclear and organelle inheritance among artificial hybrids of the seaweeds Fucus serratus and F. evanescens were detected using single-strand conformation polymorphism (SSCP). Three alleles were identified in the 231 bp rDNA-ITS1 gene (nuclear): two in F. serratus and one in F. evanescens. Alleles differed by 1–2 bp and all hybrids possessed one allele from each parent. Two haplotypes were present in the 288 bp Rubisco spacer (chloroplast), differentiated by a 33 bp indel. Two haplotypes differing by a single nucleotide were found in a 135 bp region of nad11 gene (mitochondrion). Both organelles are maternally inherited, as all hybrids contained the haplotypes of the parent contributing the egg. Although laboratory hybrids among Fucus spp. have been produced previously, this is the first time that both nuclear and cytoplasmic genetic markers have been used to document inheritance patterns. SSCPs analysed on an automated sequencer offer a rapid and powerful approach for identifying suspected hybrids from field samples, as well as a screen for intraspecific and intra-individual variation in DNA regions prior to confirmation of variations by sequencing.

Uncertainties about the identity of type specimens of red algae have frequently led to taxonomic and nomenclatural confusion. A procedure for extracting PCR-amplifiable DNA from formalin-fixed material and herbarium specimens was used to investigate the taxonomic status of several South African Gigartinaceae. We compared nucleotide sequences in the internal transcribed spacer 1 (ITS1) region in type specimens and other historically important collections presently referred to Gigartina bracteata, G. radula and G. stiriata. The following opinions are supported: (1) Fucus bracteatus S.G. Gmelin, Chondrodictyon capense Kützing and Iridaea clathrata Decaisne represent a single species of South African Gigartina (G. bracteata (S.G. Gmelin) Setchell & Gardner) in which the disintegration of tetrasporangial sori results in a reticulate thallus. (2) Mastocarpus polycarpus Kützing, M. incrassatus Kützing and Iridaea lapathifolia Kützing represent a single species of South African Gigartina (G. polycarpa (Kützing) Setchell & Gardner) that has often, but erroneously, been called G. radula (Esper) J. Agardh. (3) Mastocarpus verrucosus Kützing is a later heterotypic synonym of Iridaea papillosa Bory (Sarcothalia papillosa (Bory) Leister) and was based on material that probably came from southern South America rather than from South Africa, the provenance given by Kützing. (4) Fucus stiriatus Turner and Sphaerococcus burmannii C. Agardh represent a single species of South African Sarcothalia (S. stiriata (Turner) Leister).

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5·8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.

Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris; (1) C. muris genotype A; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.

When generating kelp hybrid crosses in vitro, small blades are frequently seen in single-sex cultures of microscopic gametophytes as well as in putative hybrid crosses. We demonstrate a polymerase chain reaction (PCR)-based method that quickly determines the presence/absence of possible parental genomes in the observed blades. ITS1 nrDNA fragments that varied in length in a species-specific way were amplified. Amplification products from possible hybrid blades, apogamic blades and parental gametophytes were compared on agarose gels. Using these methods, we were able to identify a number of apogamic blades (i.e. formed directly from gametophytic cells) as well as a true hybrid sporophyte blade in crosses between Alaria marginata (Alariaceae) and Lessoniopsis littoralis (Lessoniaceae), members of two separate families of the Laminariales. To our knowledge this is the first genotypic confirmation of a hybrid kelp.

The first internal transcribed spacer (ITS1) of the nuclear ribosomal DNA repeat was sequenced for members of the Schistosoma japonicum species complex (S. malayensis, S. mekongi and 2 geographical isolates of S. japonicum). The ITS1 is composed of 3 distinct regions: the 5′ end (23 nucleotides); a tract of approximately 90–140 nucleotides, which occurs up to 7 times in tandem, the number varying even within an individual in all species investigated in this study; the 3′ region (378 nucleotides), which lacks repeats. There is size and sequence variation among copies of the ITS1 repeat within a single individual. The relative abundances of size variants of ITS1 in S. japonicum have been ascertained by hybridizing genomic digests with an ITS1 probe. Multiple repeats and intra-individual variation in numbers and abundance of these is a feature of the Asian schistosomes, but not generally of African schistosomes. Possible reasons for this difference in ITS1 between African and Asian schistosomes are discussed. The ITS1 repeat sequences described for African schistosomes are different to, and cannot be aligned with, those from the Asian species described here, whereas the remainder of the ITS1 can be aligned quite easily.