Spermatozoa and spermiogenesis of Holocnemus pluchei (Scopoli, 1763) (Pholcidae, Araneae)

Until now, the knowledge on pholcid spermatozoa is based on two species, Pholcus phalangioides and, incompletely, Holocnemus pluchei. To complete this knowledge and to reveal more potential phylogenetic characters, we have investigated sperm ultrastructure and spermiogenesis of H. pluchei. We found that the sperm cells of this species are clearly different from those of P. phalangioides with respect to: (1) the lack of specialization in the cylindrical acrosomal vacuole; (2) a nuclear canal which is located in the periphery and not in the center of the nucleus; (3) a more prominent postcentriolar elongation of the nucleus; (4) the presence of "inner microtubules" in the implantation fossa in early and mid-spermatids; (5) the absence of a helical band of nuclear material; (6) the proximal centriole which is not prolonged; (7) the types of secretion in the seminal fluid (only two types in H. pluchei). Similarities in the spermatozoa of both species concern: (1) a large implantation fossa which contains large amounts of glycogen in mature spermatozoa; (2) absence of a centriolar adjunct; (3) an axonemal basis located in the posterior part of the implantation fossa; (4) the formation of the so-called cleistospermia in the vas deferens. Our results strongly support systematic relationships within Pholcidae placing these two species in different subgroups.     

Simian virus 40 DNA replication in nuclear monolayers.

Simian virus 40 DNA replication has been studied in nuclear monolayers prepared by treatment of monolayers of BSC-1 monkey kidney cells with Nonidet P-40. These nuclear monolayers incorporated [3H]TTP into two types of viral replicative intermediates that sediment as 25-26S and 22-23S species, respectively, in neutral sucrose gradients. The 22-23S species behaves, in dye buoyant density equilibrium gradients, as a late replicative intermediate. Examination of both species in alkaline sucrose gradients revealed the presence of two types of newly synthesized strands: (i) 4-7S strands and (ii) full-length, or nearly full-length, 10-16S strands. At low TTP concentrations (less than 0.5 muM), the two size classes were found in approximately equal amounts. However, at 10 to 50 muM TTP, the proportion of the longer strands increased, with a corresponding decrease in the relative amount of the 4-7S species. Thus, the joining of small, Okazaki-like fragments to the growing chain appears to require a much higher concentration of TTP than the synthesis of the fragments themselves. Replicating simian virus 40 DNA synthesized in the nuclear monolayers is is associated with "M bands", as previously demonstrated for replicating simian virus 40 DNA in cultured whole cells.     

Detection of non-pylori Helicobacter species in "Helicobacter heilmannii"-infected humans

BACKGROUND: A small proportion of patients suffering from chronic active gastritis are diagnosed with gastric Helicobacter species other than Helicobacter pylori. Circumstantial evidence has suggested that these bacteria, also referred to as "Helicobacter heilmannii"-like organisms (HHLO), may be transmitted through animals. The isolation of a Helicobacter bizzozeronii strain from a human patient confirmed this hypothesis. It was the aim of the present study to assess the presence of animal Helicobacter species and H. pylori in humans infected with HHLO, as diagnosed by histology. METHODS: Paraffin-embedded gastric biopsy specimens of 108 HHLO-infected patients (42 women and 66 men) from three clinical centers were screened for the presence of animal gastric Helicobacter species by polymerase chain reaction (PCR), using assays targeting the 16S rDNA region of the three known canine and feline helicobacters (H. bizzozeronii, H. salomonis and H. felis), "Candidatus H. suis", and "Candidatus H. bovis". In addition, the presence of H. pylori was evaluated by multiplex PCR analysis. RESULTS: In 63.4% of the stomachs (64/101) classification of the Helicobacter infection into the above mentioned groups was achieved. Non-pylori Helicobacter species commonly colonizing the stomachs of cats and dogs were found in 48.5% (49/101) of the patients. Fourteen (13.9%) samples tested positive for "Candidatus H. suis", and "Candidatus H. bovis" was demonstrated in 1 (0.9%) patient. The presence of H. pylori was established in 13 patients (12.9%). Eleven stomachs (10.9%) were infected with at least two different Helicobacter species. CONCLUSIONS: This study identifies animal Helicobacter species in the stomach of a large series of HHLO-infected patients, which may have clinical implications in a subset of patients with gastric disease.     

Prevalence of the trichomycete fungus Harpella melusinae (Harpellales: Harpellaceae) in larval black flies (Diptera: Simuliidae) across a heterogeneous environment

A total of 2063 mid- to late-instar larval black flies were collected from 64 stream sites in South Carolina and screened for the presence of the trichomycete fungus Harpella melusinae. Sixteen of 18 host species were colonized by H. melusinae on at least one occasion. Prevalence of H. melusinae in larvae of Simulium tuberosum cytospecies "A" was highest in acidic streams with low conductivity, whereas H. melusinae colonized larvae of Simulium verecundum most frequently in slower-moving streams. Ecological conditions, therefore, can serve as predictors of the prevalence of H. melusinae. Prevalence in host larvae was significantly lower in the Piedmont ecoregion than in the Mountain ecoregion. Prevalence did not differ in the host species S. verecundum across ecoregions, suggesting that different prevalences among host species might indicate some host preference. The prevalence of H. melusinae differed significantly between two univoltine host species (Simulium venustum and Prosimulium magnum) at the same site but not between two multivoltine host species (S. tuberosum cytospecies "FG" and S. tuberosum cytospecies "CDE"), suggesting that host life history could be important in determining fungal prevalence.     

Replacement of nucleosomal histones by histone H1-like proteins during spermiogenesis in Cnidaria: Evolutionary implications

We have analyzed the chromosomal protein composition of the sperm from several species belonging to three different classes (Hydrozoa, Scyphozoa, Anthozoa) of the phylum Cnidaria. In every instance, the sperm nuclear basic proteins (SNBPs) were found to consist of one to two major protein fractions that belong to the histone H1 family, as can be deduced from their amino acid composition and solubility in dilute perchloric acid, and the presence of a trypsin-resistant core. In those species where mature spawned sperm could be obtained, we were able to show that these proteins completely replace the somatic histones from the stem cells that are present at the onset of spermatogenesis. The presence of a highly specialized histone H1 molecule in the sperm of this phylum provides support for the idea that the protamine-like proteins (PL) from higher groups in the phylogenetic tree (and possibly protamines as well) may all have evolved from a primitive histone H1 ancestor.     

Ubiquity of the St chloroplast genome in St-containing Triticeae polyploids.

Interspecific hybridization occurs between Tritceae species in the grass family (Poaceae) giving rise to allopolyploid species. To examine bias in cytoplasmic DNA inheritance in these hybridizations, the sequence of the 3' end of the chloroplast ndhF gene was compared among 29 allopolyploid Triticeae species containing the St nuclear genome in combination with the H, I, Ns, P, W, Y, and Xm nuclear genomes. These ndhF sequences were also compared with those from diploid or allotetraploid Triticeae species having the H, I, Ns, P, W, St, and Xm genomes. The cpDNA sequences were highly similar among diploid, allotetraploid, allohexaploid, and allooctoploid Triticeae accessions containing the St nuclear genome, with 0-6-nucleotide (nt) substitutions (0-0.8%) occurring between pairs of species. Neighbor-joining analysis of the sequences showed that the ndhF DNA sequences from species containing the St nuclear genome formed a strongly supported clade. The data indicated a strong preference for cpDNA inheritance from the St nuclear genome-containing parent in hybridizations between Triticeae species. This preference was independent of the presence of the H, I, Ns, P, W, and Xm nuclear genomes, the geographic distribution of the species, and the mode of reproduction. The data suggests that hybridizations having the St-containing parent as the female may be more successful.     

Molecular demographic history of the annual sunflowers Helianthus annuus and H. petiolaris--large effective population sizes and rates of long-term gene flow.

Hybridization between distinct species may lead to introgression of genes across species boundaries, and this pattern can potentially persist for extended periods as long as selection at some loci or genomic regions prevents thorough mixing of gene pools. However, very few reliable estimates of long-term levels of effective migration are available between hybridizing species throughout their history. Accurate estimates of divergence dates and levels of gene flow require data from multiple unlinked loci as well as an analytical framework that can distinguish between lineage sorting and gene flow and incorporate the effects of demographic changes within each species. Here we use sequence data from 18 anonymous nuclear loci in two broadly sympatric sunflower species, Helianthus annuus and H. petiolaris, analyzed within an "isolation with migration" framework to make genome-wide estimates of the ages of these two species, long-term rates of gene flow between them, and effective population sizes and historical patterns of population growth. Our results indicate that H. annuus and H. petiolaris are approximately one million years old and have exchanged genes at a surprisingly high rate (long-term N(ef)m estimates of approximately 0.5 in each direction), with somewhat higher rates of introgression from H. annuus into H. petiolaris than vice versa. In addition, each species has undergone dramatic population expansion since divergence, and both species have among the highest levels of genetic diversity reported for flowering plants. Our results provide the most comprehensive estimate to date of long-term patterns of gene flow and historical demography in a nonmodel plant system, and they indicate that species integrity can be maintained even in the face of extensive gene flow over a prolonged period.     

Lipid profile of Helicobacter spp.: presence of cholesteryl glucoside as a characteristic feature.

The lipid and fatty acid profiles of eight Helicobacter spp. (H. nemestrinae, H. acinonyx, H. canis, Helicobacter sp. strain CLO-3, "H. rappini" [Flexispira rappini], H. pametensis, Helicobacter sp. strain Bird-B, and Helicobacter sp. strain Bird-C) and the fatty acid profiles of five additional species (H. pylori, H. felis, H. muridarum, H. mustelae, and H. fennelliae) were analyzed and compared. A heterologous fatty acid profile was observed among the Helicobacter spp., and on that basis the species could be divided into two groups. Group A had 19-carbon cyclopropane fatty acid (19:0cyc) and tetradecanoic acid (14:0) as the major fatty acids, and group B characteristically lacked the 19:0cyc and had hexadecanoic acid (16:0) and octadecenoic (18:1) acids as the major fatty acids. The species of group A are primarily gastric colonizers, and those of group B are primarily intestinal colonizers. Seven of the eight species studied showed the unusual and characteristic presence of cholesteryl glucosides (CGs), and most of these seven showed a very large amount (9.7 to 27.4% of the weight of total extractable lipid). The types of CGs and their distribution in different species were as follows: cholesteryl-6-O-acyl-alpha-D-glucopyranoside (cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside in H. nemestrinae and mainly cholesteryl-6-O-dodecanoyl-alpha-D-glucopyranoside in "H. rappini"), cholesteryl-alpha-D-glucopyranoside (H. nemestrinae, H. acinonyx, H. canis, Helicobacter sp. strain CLO-3, and "H. rappini"), and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside (H. nemestrinae, H. acinonyx, H. canis, and Helicobacter sp. strain CLO-3). Besides this, we could also detect cholesteryl acyl glucoside in H. acinonyx, cholesteryl glucoside in Helicobacter sp. strains Bird-B and -C, and cholesteryl phosphatidyl glucoside in "H. rappini" and Helicobacter sp. strain Bird-C. A selective accumulation of free cholesterol was observed in the neutral lipid fractions. On the basis of the detection of CGs in 11 of the 13 species studied so far, the presence of CGs appears to be a characteristic feature of the genus Helicobacter. In view of this and also because of a simple and rapid detection method described herein, the CGs can be used as a valuable chemotaxonomic marker.     

MOLECULAR DEMOGRAPHIC HISTORY OF THE ANNUAL SUNFLOWERS HELIANTHUS ANNUUS AND H. PETIOLARIS—LARGE EFFECTIVE POPULATION SIZES AND RATES OF LONG-TERM GENE FLOW

Hybridization between distinct species may lead to introgression of genes across species boundaries, and this pattern can potentially persist for extended periods as long as selection at some loci or genomic regions prevents thorough mixing of gene pools. However, very few reliable estimates of long-term levels of effective migration are available between hybridizing species throughout their history. Accurate estimates of divergence dates and levels of gene flow require data from multiple unlinked loci as well as an analytical framework that can distinguish between lineage sorting and gene flow and incorporate the effects of demographic changes within each species. Here we use sequence data from 18 anonymous nuclear loci in two broadly sympatric sunflower species, Helianthus annuus and H. petiolaris , analyzed within an "isolation with migration" framework to make genome-wide estimates of the ages of these two species, long-term rates of gene flow between them, and effective population sizes and historical patterns of population growth. Our results indicate that H. annuus and H. petiolaris are approximately one million years old and have exchanged genes at a surprisingly high rate (long-term N_efm estimates of approximately 0.5 in each direction), with somewhat higher rates of introgression from H. annuus into H. petiolaris than vice versa. In addition, each species has undergone dramatic population expansion since divergence, and both species have among the highest levels of genetic diversity reported for flowering plants. Our results provide the most comprehensive estimate to date of long-term patterns of gene flow and historical demography in a nonmodel plant system, and they indicate that species integrity can be maintained even in the face of extensive gene flow over a prolonged period.     

The reaction of tadpoles of the common toad (Bufo bufo L.) to chemical signals from individuals of their own and other species

In behavioral experiments, toad tadpoles-recipients chose between two parts of test aquarium with chemical signals of donor tadpoles of the same or different (Rana esculenta) species. The youngest studied tadpoles (43-45 stages) preferred the part of the aquarium with chemical cues from sibs as compared to non-sibs. No reliable difference in "non-sibs: water" system was observed. In "tadpoles of the same species: tadpoles of the other species" system, recipient tadpoles chose "their" part of the aquarium. Hence, toad tadpoles revealed a trend to aggregate with their sibs and avoid R. esculenta toads in the presence of certain chemical cues and in the absence of visual signals.     

Species identification and genetic structure of threatened seahorses in Gran Canaria Island (Spain) using mitochondrial and microsatellite markers

A non-invasive DNA analysis of seahorse populations was carried out after extensive underwater surveys in Gran Canaria Island (Spain). In this geographical area, the presence of two species, Hippocampus hippocampus and H. guttulatus, has been previously reported. Sequencing of 16S ribosomal DNA (16S rDNA) was used for specific identification of live seahorses sampled in situ, as a previous step to evaluate genetic structure based on ten microsatellite markers. Phylogenetic analyses revealed the presence of a single species, H. hippocampus, in the seahorse communities found at Gran Canaria. No evidences of H. guttulatus or interspecific hybrids were found based on 16S rDNA and microsatellite data. The nuclear markers revealed low genetic diversity and lack of population structure across populations of Gran Canaria Island, with evidence of small population sizes. This study provides critical information to support conservation strategies of Gran Canaria seahorses.     

Introgression and selection shaped the evolutionary history of sympatric sister‐species of coral reef fishes (genus: Haemulon)

Closely related marine species with large overlapping ranges provide opportunities to study mechanisms of speciation, particularly when there is evidence of gene flow between such lineages. Here, we focus on a case of hybridization between the sympatric sister‐species Haemulon maculicauda and H. flaviguttatum, using Sanger sequencing of mitochondrial and nuclear loci, as well as 2422 single nucleotide polymorphisms (SNPs) obtained via restriction site‐associated DNA sequencing (RADSeq). Mitochondrial markers revealed a shared haplotype for COI and low divergence for CytB and CR between the sister‐species. On the other hand, complete lineage sorting was observed at the nuclear loci and most of the SNPs. Under neutral expectations, the smaller effective population size of mtDNA should lead to fixation of mutations faster than nDNA. Thus, these results suggest that hybridization in the recent past (0.174–0.263 Ma) led to introgression of the mtDNA, with little effect on the nuclear genome. Analyses of the SNP data revealed 28 loci potentially under divergent selection between the two species. The combination of mtDNA introgression and limited nuclear DNA introgression provides a mechanism for the evolution of independent lineages despite recurrent hybridization events. This study adds to the growing body of research that exemplifies how genetic divergence can be maintained in the presence of gene flow between closely related species.     

Relationships of new sibling species of Paramecium jenningsi based on sequences of the histone H4 gene fragment

Paramecium jenningsi Diller & Earl, 1958 belongs to the "aurelia" subgroup of the genus, together with Paramecium caudatum, Paramecium multimicronucleatum, Paramecium schewiakoffi and species of the Paramecium aurelia complex. The original assumption that the morphospecies P. jenningsi was a single genetic species was questioned because a comparison of genome analyses suggested the possibility that this morphospecies contained two sibling species. To refine understanding of relationships between the strains of P. jenningsi, a molecular phylogenetic analysis was conducted using H4 gene sequences. Some polymorphic sites were found among the compared sequences, and specific patterns of single nucleotide polymorphism (SNP) markers characterize two groups of strains of P. jenningsi. Phylogenetic trees constructed by different methods identified two clearly different groups (from Japan and mainland Asia) whatever the method used. The sequences of the H4 gene analyzed in the present study are closely related, and provide a good subject for phylogenetic analysis. The presence of two isolated groups of strains in the P. jenningsi group can reveal the evolutionary relationship between them; it confirms the presence of two sibling species among the known strains of P. jenningsi, and the close relationships between them and species of the P. aurelia complex.     

European <Emphasis Type="Italic">Hodophilus</Emphasis> (Clavariaceae,Agaricales) species with yellow stipe

Phylogenetic reconstruction of Hodophilus species with a yellow colour on the stipe based on nrITS, nrLSU and rpb2 sequences revealed six European species. All these species correspond to the widely accepted European concept of a single species Hodophilus micaceus. Four of these species are described and illustrated. H. micaceus and H. phaeoxanthus are recognised as two separate species and H. albofloccipes as a synonym of the latter. Two species, H. anatinus and H. cambriensis, are described as new. Possible endemism of H. micaceus and H. cambriensis to the British Isles is discussed. All analysed North American samples represent different species to those found in Europe. The North American species Hygrophorus rugulosus is combined in the genus Hodophilus. The preliminary key uses position and development of the yellow colour during maturation as the most important distinguishing character. The presence of the yellow colour is discussed as a possible synapomorphic character.     

The effects of salt concentration and H-1 depletion on the digestion of calf thymus chromatin by micrococcal nuclease.

We have removed histone H1 specifically from calf thymus nuclei by low pH treatment, and studied the digestion of such nuclei in comparison with undepleted nuclei. By a number of criteria the nuclei do not appear damaged. The DNA repeat-length in nuclear chromatin is found to be the same (192 +/- 4 bp) in the presence or absence of H1. These experiments demonstrate that the core histone complex of H2A, H2B, H3, and H4 can itself protect DNA sequences as long as 168 bp from nuclease. Our interpretation is that this represents an important structural element in chromatin, carrying two full turns of superhelical DNA. Depending on conditions of digestion this 168 bp fragment may be metastable and is normally rapidly converted by exonucleolytic trimming to the well-known "core-particle" containing 145 bp. Larger stable DNA fragments observed indigestion of H-1 depleted nuclei appear to arise from oligomers assembled from 168 bp cores in close contact exhibiting trimming of 0-20 bp at the ends. Electrophorograms of undepleted nuclear digests reveal oligomer bands in several size classes, each corresponding to one or more combinations of 168 bp particles, H1-protected spacers of about 20 bp length, and particles with ends trimmed to varying degrees.     

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

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

Inter-species variation in unpalatability does not explain polymorphism in a mimetic species

Conspicuous colouration in unpalatable organisms acts as a warning signal of their unprofitability, a phenomenon known as aposematism. The protection conferred by such colouration can lead to evolutionary convergence in warning signals between aposematic species, because sharing warning signals reduces the per capita cost of predator learning. Consequently, most aposematic species display a single colour pattern and participate in a single mimetic community (i.e. mimicry ring) at any given locality. However, some, like the Amazonian butterfly Heliconius numata, are polymorphic and participate in several mimicry rings within the same locality. We tested whether the unexpected polymorphism of H. numata could be due to a weak defence against predators. Poorly defended species participating in a mimicry ring are subject to negative frequency dependent selection, because their presence weakens the protection provided by the shared signal. This could promote polymorphism and participation in multiple mimicry rings. Using wild caught great tits (Parus major), we compared the palatability of H. numata to one of its locally monomorphic co-mimics (Mechanitis polymnia) and to two other locally monomorphic Heliconius species (H. melpomene and H. erato). The tested birds strongly rejected the polymorphic species H. numata, as well as the two other Heliconius species. Unexpectedly, a significantly weaker rejection was found towards M. polymnia, which relies on different toxic compounds to Heliconius. Our study demonstrates that the origin of polymorphic mimicry in H. numata is unlikely to stem from low unpalatability and raises new questions on defence variation within mimetic communities.     

Ex vivo detection of histone H1 modified with advanced glycation end products

A number of oxidative stress agents cause DNA and protein damage, which may compromise genomic integrity. Whereas oxidant-induced DNA damage has been extensively studied, much less is known concerning the occurrence and fate of nuclear protein damage, particularly of proteins involved in the regulation and maintenance of chromatin structure. Protein damage may be caused by the formation of reactive carbonyl species such as glyoxal, which forms after lipid peroxide degradation. It may also result from degradation of early protein glycation adducts and from methylglyoxal, formed in the process of glycolytic intermediate degradation. Major adducts indicative of protein damage include the advanced glycation end product (AGE) carboxymethyllysine (CML) and argpyrimidine protein adducts. Thus, the formation of CML and argpyrimidine protein adducts represents potential biomarkers for nuclear protein damage deriving from a variety of sources. The purpose of this study was to identify and quantify AGE adducts formed in vivo in a nuclear protein, specifically histone H1, using CML and argpyrimidine as biomarkers. Histone H1 was isolated from calf thymus collected immediately after slaughter under conditions designed to minimize AGE formation before isolation. Using antibodies directed against oxidative protein adducts, we identified CML, argpyrimidine, and protein crosslinks present in the freshly isolated histone H1. Detailed mass spectroscopy analysis of histone H1 revealed the presence of two specific lysine residues modified by CML adducts. Our results strongly suggest that glycation of important nuclear protein targets such as histone H1 occurs in vivo and that these oxidative changes may alter chromatin structure, ultimately contributing to chronic changes associated with aging and diseases such as diabetes.