Sacculogenesis of Buddenbrockia plumatellae (Myxozoa) within the invertebrate host Plumatella repens (Bryozoa) with comments on the evolutionary relationships of the Myxozoa

Members of the phylum Myxozoa are obligate parasites, primarily of aquatic organisms. Their phylogeny has remained problematic, with studies placing them within either the Bilateria or Cnidaria. The discovery that the enigmatic Buddenbrockia plumatellae is a myxozoan that possesses distinct bilaterian features appeared to have finally resolved the debate. B. plumatellae is described as a triploblastic 'worm-like' organism, within which typical myxozoan malacospores form. Using EM we examined the early development of the B. plumatellae 'worms' within the bryozoan host Plumatella repens. The initial development involved numerous unicellular, amoeboid pre-saccular stages that were present within the basal lamina of the host's body wall. These stages migrate immediately beneath the peritoneum where a significant host tissue reaction occurs. The stages aggregate, initiating the formation of a 'worm'. The base of a developing 'worm' forms a pseudosyncytium which resolves into an ectoderm surrounding a mesendoderm. The pseudosyncytium is directly anchored into neighbouring host cells via masses of striated fibres. The replication of the ectodermal and mesendodermal cells extends the developing 'worm' into the coelom of the host. The mesendoderm resolves to form a mesoderm and an endoderm. Myogenesis appears to be initiated from the anchored end of the 'worm' and develops along the mesoderm. The aggregation and differentiation of amoeboid pre-saccular stages to initiate the 'worm' draws analogies to the sacculogenesis observed for Tetracapsuloides bryosalmonae, B. plumatellae's sister taxon within the class Malacosporea. The development of a multicellular, spore forming organism, from single cells does not correlate to any bilaterian or cnidarian species. Current phylogenies indicate the Myxozoa are basal bilaterians along with the Acoela and Mesozoa. Comparison with these other basal groups may help to resolve the placement of Myxozoa within the tree of life.     

Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics

Views on myxosporean phylogeny and systematics have recently undergone substantial changes resulting from analyses of SSU rDNA. Here, we further investigate the evolutionary trends within myxosporean lineages by using 35 new sequences of the LSU rDNA. We show a good agreement between the two rRNA genes and confirm the main phylogenetic split between the freshwater and marine lineages. The informative superiority of the LSU data is shown by an increase of the resolution, nodal supports and tree indexes in the LSU rDNA and combined analyses. We determine the most suitable part of LSU for the myxosporean phylogeny by comparing informative content in various regions of the LSU sequences. Based on this comparison, we propose the D5–3′-end part of the LSU rRNA gene as the most informative region which provides in concatenation with the complete SSU a well resolved and robust tree. To allow for simple amplification of the marker, we design specific primer set for this part of LSU rDNA.     

Presidential address: rediscovering parasites using molecular tools--towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium

Molecular biology has provided parasitologists with a fantastic variety of techniques that have had a major impact on research into parasites and parasitism. Molecular tools have revealed the extent and nature of genetic diversity in parasites and this information has made a significant contribution to studies on the population genetics and evolutionary biology of parasites. Similarly, epidemiology has benefited enormously from the application of molecular tools in terms of studying parasite life cycles and transmission, and in the development of specific and sensitive methods for diagnosis and surveillance. However, the theme I wish to develop in this paper is concerned with the contribution molecular tools have made to parasite taxonomy and systematics, and in particular, the fact that in many cases molecular tools are validating the proposals made many years ago by taxonomists and biologists which were discounted or not fully accepted at the time. To do this I have chosen four examples (Echinococcus, Entamoeba, Giardia, Cryptosporidium) where recent research involving molecular characterisation has confirmed observations made many years ago and has resulted in a need to revise the taxonomy of different groups of parasites.     

Conflicting molecular phylogenies of European long-eared bats (Plecotus) can be explained by cryptic diversity

Conflicting phylogenetic signals of two data sets that analyse different portions of the same molecule are unexpected and require an explanation. In the present paper we test whether (i) differential evolution of two mitochondrial genes or (ii) cryptic diversity can better explain conflicting results of two recently published molecular phylogenies on the same set of species of long-eared bats (genus Plecotus). We sequenced 1714bp of three mitochondrial regions (16S, ND1, and D-loop) of 35 Plecotus populations from 10 European countries. A likelihood ratio test revealed congruent phylogenetic signals of the three data partitions. Our phylogenetic analyses demonstrated that the existence of a previously undetected Plecotus lineage caused the incongruities of previous studies. This lineage is differentiated on the species level and lives in sympatry with its sister lineage, Plecotus auritus, in Switzerland and Northern Italy. A molecular clock indicates that all European Plecotus species are of mid or late Pliocene origin. Plecotus indet. was previously described as an intergrade between P. auritus and Plecotus austriacus since it shares morphological characters with both. It is currently known from elevations above 800 m a.s.l. in the Alps, the Dinarian Alps and the Pindos mountains in Greece. Since we could demonstrate that incongruities of two molecular analyses simply arose from the mis-identification of one lineage, we conclude that molecular phylogenetic analyses do not free systematists from a thorough inclusion of morphological and ecological data.     

Distribution and diversity of members of the bacterial phylum Fibrobacteres in environments where cellulose degradation occurs

The Fibrobacteres phylum contains two described species, Fibrobacter succinogenes and Fibrobacter intestinalis, both of which are prolific degraders of cellulosic plant biomass in the herbivore gut. However, recent 16S rRNA gene sequencing studies have identified novel Fibrobacteres in landfill sites, freshwater lakes and the termite hindgut, suggesting that members of the Fibrobacteres occupy a broader ecological range than previously appreciated. In this study, the ecology and diversity of Fibrobacteres was evaluated in 64 samples from contrasting environments where cellulose degradation occurred. Fibrobacters were detected in 23 of the 64 samples using Fibrobacter genus-specific 16S rRNA gene PCR, which provided their first targeted detection in marine and estuarine sediments, cryoconite from Arctic glaciers, as well as a broader range of environmental samples. To determine the phylogenetic diversity of the Fibrobacteres phylum, Fibrobacter-specific 16S rRNA gene clone libraries derived from 17 samples were sequenced (384 clones) and compared with all available Fibrobacteres sequences in the Ribosomal Database Project repository. Phylogenetic analysis revealed 63 lineages of Fibrobacteres (95% OTUs), with many representing as yet unclassified species. Of these, 24 OTUs were exclusively comprised of fibrobacters derived from environmental (non-gut) samples, 17 were exclusive to the mammalian gut, 15 to the termite hindgut, and 7 comprised both environmental and mammalian strains, thus establishing Fibrobacter spp. as indigenous members of microbial communities beyond the gut ecosystem. The data highlighted significant taxonomic and ecological diversity within the Fibrobacteres, a phylum circumscribed by potent cellulolytic activity, suggesting considerable functional importance in the conversion of lignocellulosic biomass in the biosphere.     

Genetic diversity of rhizobia nodulating lentil (Lens culinaris) in Bangladesh

In order to determine the bacterial diversity and the identity of rhizobia nodulating lentil in Bangladesh, we performed a phylogenetic analysis of housekeeping genes (16S rRNA, recA, atpD and glnII) and nodulation genes (nodC, nodD and nodA) of 36 bacterial isolates from 25 localities across the country. Maximum likelihood (ML) and Bayesian analyses based on 16S rRNA sequences showed that most of the isolates (30 out of 36) were related to Rhizobium etli and Rhizobium leguminosarum. Only these thirty isolates were able to re-nodulate lentil under laboratory conditions. The protein-coding housekeeping genes of the lentil nodulating isolates showed 89.1-94.8% genetic similarity to the corresponding genes of R. etli and R. leguminosarum. The same analyses showed that they split into three distinct phylogenetic clades. The distinctness of these clades from closely related species was also supported by high resolution ERIC-PCR fingerprinting and phenotypic characteristics such as temperature tolerance, growth on acid-alkaline media (pH 5.5-10.0) and antibiotic sensitivity. Our phylogenetic analyses based on three nodulation genes (nodA, nodC and nodD) and cross-inoculation assays confirmed that the nodulation genes are related to those of R. leguminosarum biovar viciae, but clustered in a distinct group supported by high bootstrap values. Thus, our multi-locus phylogenetic analysis, DNA fingerprinting and phenotypic characterizations suggest that at least three different clades are responsible for lentil nodulation in Bangladesh. These clades differ from the R. etli-R. leguminosarum group and may correspond to novel species in the genus Rhizobium.     

Fish pathogens near the Arctic Circle: molecular,morphological and ecological evidence for unexpected diversity of Diplostomum (Digenea: diplostomidae) in Iceland

Host–parasite systems at high latitudes are promising model systems for detecting and predicting the impact of accelerated environmental change. A major challenge is the lack of baselines for the diversity and distribution of parasites in Arctic wildlife, especially in the freshwater environment. Here we present the first known estimates of the species diversity and host associations of Diplostomum spp. in sub-Arctic freshwater ecosystems of the Palaearctic. Our analyses integrating different analytical approaches, phylogenies based on mitochondrial and nuclear DNA, estimates of genetic divergence, character-based barcoding, morphological examination, precise detection of microhabitat specialisation and host use, led to the discovery of one described and five putative new species that complete their life-cycles within a fairly narrow geographic area in Iceland. This increases the species richness of Diplostomum in Iceland by 200% and raises the number of molecularly characterised species from the Palaearctic to 17 species. Our results suggest that the diversity of Diplostomum spp. is underestimated globally in the high latitude ecosystems and call for a cautionary approach to pathogen identification in developing the much needed baselines of pathogen diversity that may help detect effects of climate change in the freshwater environment of the sub-Arctic.     

Host associations and evolutionary relationships of avian blood parasites from West Africa

The host specificity of blood parasites recovered from a survey of 527 birds in Cameroon and Gabon was examined at several levels within an evolutionary framework. Unique mitochondrial lineages of Haemoproteus were recovered from an average of 1.3 host species (maximum = 3) and 1.2 host families (maximum = 3) while lineages of Plasmodium were recovered from an average of 2.5 species (maximum = 27) and 1.6 families (maximum = 9). Averaged within genera, lineages of both Plasmodium and Haemoproteus were constrained in their host distribution relative to random expectations. However, while several individual lineages within both genera exhibited significant host constraint, host breadth varied widely among related lineages, particularly within the genus Plasmodium. Several lineages of Plasmodium exhibited extreme generalist host-parasitism strategies while other lineages appeared to have been constrained to certain host families over recent evolutionary history. Sequence data from two nuclear genes recovered from a limited sample of Plasmodium parasites indicated that, at the resolution of this study, inferences regarding host breadth were unlikely to be grossly affected by the use of parasite mitochondrial lineages as a proxy for biological species. The use of divergent host-parasitism strategies among closely related parasite lineages suggests that host range is a relatively labile character. Since host specificity may also influence parasite virulence, these results argue for considering the impact of haematozoa on avian hosts on a lineage-specific basis.     

Phytochemistry and molecular systematics of Triaenophora rupestris and Oreosolen wattii (Scrophulariaceae)

The relationships between the genera Triaenophora, Oreosolen and Rehmannia were investigated. All three genera were previously included in tribe Veroniceae which was part of Scrophulariaceae but which is now included in Plantaginaceae. With regard to the content of iridoid glucosides, Triaenophora rupestris and the much-investigated Rehmannia were almost identical in containing catalpol, ajugol and 6-feruloylajugol. Oreosolen wattii was rather different in having compounds typical for the tribe Scrophularieae (Scrophulariaceae), namely aucubin, harpagide, harpagoside as well as two diesters of rhamnopyranosylcatalpol, one of which, here named oreosolenoside, had not previously been described in the literature. These results are consistent with recent analyses based on DNA sequencing and a phylogenetic tree illustrating the taxonomic relationships is presented.     

Skin peptide and cDNA profiling of Australian anurans: genus and species identification and evolutionary trends

Host defense peptides of 35 species of Australian frogs from the hylids Cyclorana and Litoria, and the myobatrachids Crinia, Limnodynastes and Uperoleia have been identified. The biological activities of the majority of these peptides have been determined and include hormones, neuropeptides, opioids, immunomodulators, membrane active peptides [including antimicrobial, anticancer, antiviral (enveloped viruses like HIV and Herpes) and antifungal peptides], neuronal nitric oxide synthase inhibitors, pheromones and individual peptides with other specific activities. The host defense peptide skin profile can be diagnostic at both the species and higher taxonomic levels; for example, species of Crinia, Litoria and Uperoleia each produce quite different types of peptides. Species of Cyclorana and Limnodynastes are more difficult to characterize by skin peptides alone: species of both genera produce similar peptides with no apparent activity. The skin peptide profiles of frogs from the genera Crinia, Litoria and Uperoleia may be used together with morphological and cognate methods, to differentiate between sub-species and even different population clusters of the same species. Nucleotide sequencing of cDNAs of precursors (pre-pro peptides) of bioactive peptides from the skin glands of various species of the genus Litoria show that the majority of these peptides originated from a single ancestor gene before the break away of Australia from Gondwana. The exceptions are the caerulein neuropeptides {e.g. caerulein [pEQDY(SO₃H)TGWMDF(NH₂)]} which have a different origin to that of other Litoria peptides. Disulfide containing peptides from skin glands of species of Crinia show a different evolutionary route to peptides from species of Litoria.     

The effect of cohabitation of Tubifex tubifex (Oligochaeta: Tubificidae) populations on infections to Myxobolus cerebralis (Myxozoa: Myxobolidae)

The competitive interactions between susceptible and resistant Tubifex tubifex (Oligochaeta: Tubificidae) exposed to Myxobolus cerebralis (Myxozoa: Myxobolidae) infections were investigated in two laboratory trials. Competition was assessed by the total parasite production over the course of the trials in mixed and pure cultures of M. cerebralis exposed worms, and by the genetic analyses of worms from the control and experimental groups at the beginning and end of the experiments. Mixed cultures of resistant and susceptible worms showed a 70% reduction in production of parasites released when compared with pure cultures of susceptible worms. In studies with laboratory and field-collected oligochaetes the mixed cultures at the end of the cohabitation experiments were dominated by resistant Tubifex from lineage V (HB strain) this strain of Tubifex has a competitive advantage over worms from other lineages. The results of this study suggest that certain species of Tubifex may be dead-end hosts to M. cerebralis by absorbing or inactivating the parasite and may also show greater survival compared to susceptible oligochaetes in certain whirling disease enzootic habitats.     

Evolutionary history of frogfishes (Teleostei: Lophiiformes: Antennariidae): a molecular approach

Fishes of the family Antennariidae (order Lophiiformes) are primarily shallow-water benthic forms found in nearly all tropical and subtropical oceans and seas of the world, with some taxa extending into temperate waters. Despite an earlier attempt based on morphology, no previous hypothesis of intergeneric relationships of the Antennariidae exists. To resolve phylogenetic relationships within the Antennariidae, and to test the validity of species groups within Antennarius, DNA sequences from the mitochondrial 16S and cytochrome oxidase c subunit 1 (COI) genes, and nuclear recombination activating gene 2 (RAG2), for 25 described and four undescribed antennariid species, representing 10 of 12 known genera and one undescribed genus, were unambiguously aligned and analyzed using Bayesian and maximum likelihood methods. The markers were partitioned and analyzed for substitution saturation and only the third codon position of COI (COI-3) was found to have reached saturation. However, analysis of both datasets, one with the saturated data and one without, differed only slightly. All molecular analyses recovered two major clades, one comprised of Fowlerichthys, Antennarius, Histrio, and Antennatus; and another containing Rhycherus, Antennariidae gen. et sp. nov., Kuiterichthys, Phyllophryne, Echinophryne, Tathicarpus, Lophiocharon, and Histiophryne. Evidence is presented to illustrate a correlation between phylogeny, geographic distribution, and reproductive life history. The results of these analyses provide the first hypothesis of evolutionary relationships within the Antennariidae.     

Genetic diversity of Garra rufa Heckel, 1843 (Teleostei: Cyprinidae) in Anatolia

In the present study, mitochondrial DNA polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and nuclear DNA inter-simple sequence repeat (ISSR) assays were used to assess the phylogenetic and phylogeographic relationships among Garra rufa samples from Anatolia. The complete mtDNA NADH 3/4 dehydrogenase (ND-3/4) gene amplified by PCR was digested with eight restriction enzymes. These enzymes produced 20 composite haplotypes for G. rufa populations. All the mtDNA haplotypes detected were highly diverged from each other and each lineage had a unique genetic profile. The evaluation of mtDNA PCR-RFLP data coupled with geological history of Anatolia indicated a deep genetic divergence among the mtDNA haplotypes of G. rufa populations from drainages of the Mediterranean and Persian Gulf, suggesting an early isolation of Tigris-Euphrates with Orontes river and other rivers draining into the Mediterranean Sea. In general, data from both mtDNA and nDNA were congruent.     

Morphological diversity of first instar larvae in Miltogramma subgenus Pediasiomyia (Diptera: Sarcophagidae, Miltogramminae)

The morphology of first instar larvae of three species of Miltogramma Meigen subgenus Pediasiomyia Rohdendorf is described using SEM and light microscope techniques. Miltogramma chrysochlamys (Rohdendorf), Miltogramma margiana (Rohdendorf) and Miltogramma przhevalskyi (Rohdendorf) share with other satellite flies the presence of an elongated sensillum basiconicum of the maxillary palpus and numerous longitudinal cuticular ridges on the integument of all segments; and they share with other Miltogramma spp. a maxillary palpus situated on a more or less raised base. While the first instar M. margiana is very similar to first instars of Miltogramma (sensu stricto), first instars of M. chrysochlamys and M. przhevalskyi share several features unique among Miltogramminae: antennal dome situated on a flat or indistinct basal ring, antennal dome flattened, border between pseudocephalon and first thoracic segment with a pair of fleshy processes, and basal ring with trichoid sensillum. First instars of M. chrysochlamys and M. przhevalskyi are unique in having both sb1 and sb2 of the sensilla basiconica of the maxillary palpus elongated, and M. przhevalskyi has an unpaired, median proleg on most abdominal segments.     

A review of global diversity in avian haemosporidians (Plasmodium and Haemoproteus: Haemosporida): new insights from molecular data

Biogeographic patterns of parasite diversity are useful for determining how host–parasite interactions can influence speciation. However, variation in methodologies and sampling effort can skew diversity estimates. Avian haemosporidians are vector-transmitted blood parasites represented by over 1300 unique genetic lineages spread across over 40 countries. We used a global database of lineage distributions for two avian haemosporidian genera, Plasmodium and Haemoproteus, to test for congruence of diversity among haemosporidians and their avian hosts across 13 geographic regions. We demonstrated that avian haemosporidians exhibit similar diversity patterns to their avian hosts; however, specific patterns differ between genera. Haemoproteus spp. diversity estimates were significantly higher than those of Plasmodium spp. in all areas where the genera co-occurred, apart from the Plasmodium spp.-rich region of South America. The geographic distributions of parasite genera also differed, with Haemoproteus spp. absent from the majority of oceanic regions while Plasmodium spp. were cosmopolitan. These findings suggest fundamental differences in the way avian haemosporidians diverge and colonise new communities. Nevertheless, a review of the literature suggests that accurate estimates of avian haemosporidian diversity patterns are limited by (i) a concentration of sampling towards passerines from Europe and North America, (ii) a frequent failure to include microscopic techniques together with molecular screening and (iii) a paucity of studies investigating distributions across vector hosts.     

Molecular diversity and phylogeny of rhizobia associated with Lablab purpureus (Linn.) grown in Southern China

As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes.     

Molecular phylogenetics and diversification of the genus Sporophila (Aves: Passeriformes)

The evolutionary affinities within and among many groups of nine-primaried oscines remain unresolved. One such group is Sporophila, a large genus of New World tanager-finches. Our study focused particularly on clarifying the relationship between this genus and a closely related one, Oryzoborus, and on examining the phylogenetic affinities of the "capuchinos," a group of 11 Sporophila species that share a similar male plumage coloration pattern. Our phylogenetic analyses, based on 498 bp of mitochondrial DNA sequence, indicated that: (1) Oryzoborus is embedded within a well-supported clade containing all Sporophila species, which strongly suggests that both genera should be merged, (2) the species of capuchinos comprise a monophyletic group, implying that the plumage patterns common to all probably arose only once, and (3) the capuchinos clade is comprised of two sub-clades, one including two species that are distributed in northern South America and the other one containing eight species that are present south of the Amazon River. Mean sequence divergence among the southern capuchinos species was extremely low, suggesting a rapid radiation within the last half-million years that may be related to the high level of sexual selection present in the genus and might have been promoted by marine ingressions and egressions that occurred in some southern coastal regions of South America in the Late Pleistocene.     

The secondary structure of the unusually long 18S ribosomal RNA of the myxozoan Sphaerospora truttae and structural evolutionary trends in the Myxozoa

The nearly complete 18S rRNA sequence of the myxozoan parasite Sphaerospora truttae shows an extraordinary length (2,552bp) in comparison with other myxozoans and with metazoans in general (average 1,800-1,900bp). The sequence shows nucleotide insertions in most variable regions of the 18S rRNA (V2, V4, V5 and V7), with especially large expansion segments in V4 and V7. In the myxozoans, nucleotide insertions and specific secondary structures in these regions of the gene were found to be strongly related to large scale phylogenetic clustering and thus with the invertebrate host type. Whereas expansion segments were generally found to be absent in the malacasporeans and the clade of primary marine myxozoan species, they occur in all taxa of the clade containing freshwater species, where they showed a consistent secondary structure throughout. The longest expansion segments occur in S. truttae, Sphaerospora elegans and Leptotheca ranae, which represent a clade that has emerged after the malacosporeans and before the radiation of all other myxozoan genera. These three species demonstrate structural links to the malacosporeans as well as other unique features. A smaller number of nucleotide insertions in different subhelices and specific secondary structures appear to have evolved independently in two marine genera, i.e. Ceratomyxa and Parvicapsula. The secondary structural elements of V4 and V7 of the myxozoan 18S rRNAs were found to be highly informative and revealed evolutionary trends of various regions of the gene hitherto unknown, since previous analyses have been based on primary sequence data excluding these regions. Furthermore, the unique features of the V4 region in S. truttae allowed for the design of a highly specific PCR assay for this species.