Geosmithia species in southeastern USA and their affinity to beetle vectors and tree hosts

The fungal genus Geosmithia is best known due to one species, G. morbida, which is vectored by the walnut twig beetle (WTB) and contributes to Thousand Cankers Disease (TCD) on walnut and wingnut trees. However, the genus is globally very diverse and abundant, and dominates a ubiquitous but understudied niche – the twig-infesting, phloem-feeding bark beetle mycobiome. The Geosmithia community in North America is only now beginning to be described. Very limited information is available for the South East, despite the region's potential to be a Geosmithia diversity hotspot. To survey the Geosmithia community in the subtropical USA, to assess their beetle and tree associations, and to test for the presence of G. morbida, we systematically deployed branch sections of nine tree species, including three Juglandaceae, in North Florida. We recovered 55 Geosmithia isolates from 195 beetle specimens from 45 exposed branch units. Neither G. morbida nor its beetle vector were detected. We identified 14 Geosmithia species; those in the G. pallida species complex were the most prevalent. Four undescribed phylogenetic species were recovered, indicating that the Geosmithia diversity in North America remains under-documented. Analysis of the association of Geosmithia with beetles and trees suggested that most species are generalists, five display preference for certain tree species, and none is specific to any beetle species.     

Vector Affinity and Diversity of Geosmithia Fungi Living on Subcortical Insects Inhabiting Pinaceae Species in Central and Northeastern Europe

Fungi from the genus Geosmithia (Ascomycota: Hypocreales) are associated with bark beetles (Coleoptera: Scolytinae), though little is known about ecology, diversity, and distribution of these fungi across beetle and its host tree species. This study surveyed the diversity, distribution and vector affinity of Geosmithia isolated from subcortical insects that colonized trees from the family Pinaceae in Central and Northeastern Europe. Twelve Geosmithia species were isolated from 85 plant samples associated with 23 subcortical insect species (including 14 bark beetle species). Geosmithia community composition was similar across different localities and vector species; although the fungal communities associated with insects that colonized Pinus differed from that colonizing other tree species (Abies, Larix, and Picea). Ten Geosmithia species from four independent phylogenetic lineages were not reported previously from vectors feeding on other plant families and seem to be restricted to the vectors from Pinaceae only. We conclude that presence of such substrate specificity suggests a long and stable association between Geosmithia and bark beetles.     

Adaptive traits of bark and ambrosia beetle-associated fungi

A phenotype is the expression of interactions between species genotype and environment. We quantified the contributions of ecological and phylogenetic associations to phenotypic variation in Geosmithia fungi. Geosmithia are symbiotic beetle-associated saprotrophs with a range of life histories and host specificities, including obligate nutritional beetle mutualists (ambrosia fungi) and phytopathogens. We hypothesized that: (1) species phenotypes are better explained by their ecology than by their phylogenetic relationships; (2) niche specialization was accompanied by enzymatic capability losses; and (3) ambrosia Geosmithia species have higher nutritional quality and antibiotic capabilities than species with facultative symbioses. Our results confirmed that long-term co-evolved specialists have reduced metabolic breadth in comparison to generalists. Phytopathogenic G. morbida produces unique enzyme suites with affinity to ligno-cellulose. Mycelia of ambrosia fungi contain large amounts of oleic fatty acid with nutritive and possibly allelopathic function. Overall, our results indicate that Geosmithia ecology have greater effect on species phenotype than their phylogenetic relationships.     

Application of flow cytometry for exploring the evolution of Geosmithia fungi living in association with bark beetles: the role of conidial DNA content

Geosmithia belongs among fungi living in symbiosis with phloem-feeding bark beetles. Several species have altered their ecology to that of obligatory symbiosis with ambrosia beetles, which has led to a shift in their phenotype and caused formation of large spherical conidia. In this study, we pose the following questions; (1) Is the conidial DNA content of Geosmithia correlated with conidial volume?; (2) Is the DNA content of Geosmithia related to the degree of mutual dependence between Geosmithia and their vector? There was a positive and strong correlation between conidial DNA content and conidial volume in Geosmithia. Also species more narrowly associated with the vector tend to have a larger conidial DNA content and volume than less narrowly associated species. Ambrosia fungi achieved the biggest conidial DNA content and volume compared to other species. We suppose that polyploidisation occurred during the evolution of ambrosia species in the genus Geosmithia.     

Positive selection in AvrP4 avirulence gene homologues across the genus Melampsora

Pathogen genes involved in interactions with their plant hosts are expected to evolve under positive Darwinian selection or balancing selection. In this study a single copy avirulence gene, AvrP4, in the plant pathogen Melampsora lini, was used to investigate the evolution of such a gene across species. Partial translation elongation factor 1-alpha sequences were obtained to establish phylogenetic relationships among the Melampsora species. We amplified AvrP4 homologues from species pathogenic on hosts from different plant families and orders, across the inferred phylogeny. Translations of the AvrP4 sequences revealed a predicted signal peptide and towards the C-terminus of the protein, six identically spaced cysteines were identified in all sequences. Maximum likelihood analysis of synonymous versus non-synonymous substitution rates indicated that positive selection played a role in the evolution of the gene during the diversification of the genus. Fourteen codons under significant positive selection reside in the C-terminal 28 amino acid region, suggesting that this region interacts with host molecules in most sequenced accessions. Selection pressures on the gene may be either due to the pathogenicity or avirulence function of the gene or both.     

Widespread horizontal transfer of the cerato-ulmin gene between Ophiostoma novo-ulmi and Geosmithia species

Previous work had shown that a sequence homologous to the gene encoding class II hydrophobin cerato-ulmin from the fungus Ophiostoma novo-ulmi, the causal agent of Dutch Elm Disease (DED), was present in a strain of the unrelated species Geosmithia species 5 (Ascomycota: Hypocreales) isolated from Ulmus minor affected by DED. As both fungi occupy the same habitat, even if different ecological niches, the occurrence of horizontal gene transfer was proposed. In the present work we have analysed for the presence of the cerato-ulmin gene 70 Geosmithia strains representing 29 species, isolated from different host plants and geographic locations. The gene was found in 52.1 % of the strains derived from elm trees, while none of those isolated from nonelms possessed it. The expression of the gene in Geosmithia was also assessed by real time PCR in different growth conditions (liquid culture, solid culture, elm sawdust, dual culture with O. novo-ulmi), and was found to be extremely low in all conditions tested. On the basis of these results we propose that the cerato-ulmin gene is not functional in Geosmithia, but can be considered instead a marker of more extensive transfers of genetic material as shown in other fungi.     

Fish growth hormone genes: Divergence of intron sequence in charrs of Salvelinus genus

Sequences of the two large introns (C and D) from two paralogous growth hormone genes, GH1 and GH2, were compared in eight charr species of the Salvelinus genus (Osteichthes, Salmonidae). It was demonstrated that the rates of intron divergence in these two genes were remarkably different. Introns in the GH1 gene appeared to be more conservative, while the rate of intron variations was considerably higher in the GH2 gene. These data suggest that noncoding regions of nuclear genes under the selective pressure. The lower congruence of phylogenetic scheme constructed based on an analysis of the GH1 introns compared to that based on the GH2 data, as well as with the traditional views on the evolution of charr species, also favors the supposition on differences in the forces and directions of the selective pressure on the introns.     

Association of Geosmithia fungi (Ascomycota: Hypocreales) with pine- and spruce-infesting bark beetles in Poland

Bark beetles (Coleoptera, Scolytinae) are known to be associated with fungi, particularly species of the orders Ophiostomatales and Microascales. However, very little is known about other ectosymbionts of phloeophagous bark beetles on Pinaceae. In this study, we examined the Geosmithia species associated with eight bark beetle species infesting Picea abies and Pinus sylvestris branches in Poland. Fungi were isolated from 1 731 samples collected from 14 study sites. We identified a total of 653 isolates that were sorted into nine taxa based on their phenotypic similarity and phylogeny of their ITS-LSU regions of rDNA, β-tubulin, elongation factor 1α and the second-largest subunit of the RNA polymerase II gene. They represented nine species without formal names. There were large quantitative and qualitative differences in the composition of Geosmithia communities between P. sylvestris and P. abies trees. The proportion of samples infested with Geosmithia species suggests that this association is more widespread among bark beetles infesting branches of P. sylvestris. In addition, these beetles were vectors of different Geosmithia species compared with than the beetles that colonize P. abies. In mixed-conifer forests, the Geosmithia communities were more diverse and richer than in pure spruce or pine stands, where the insects Pityogenes chalcographus and Pityophthorus pityographus with low host-specificity play a distributing role for various Geosmithia species. Among eight bark beetle species examined, only P. bidentatus, P. pityographus, P. chalcographus and Polygraphus poligraphus acted as effective vectors for Geosmithia species. The following hypothesis emerges from these studies: changes in the composition of ectosymbionts of pine- and spruce-infesting bark beetles in Central Europe run along a gradient of thickness of the wood substrata preferred by insects.     

Multilocus sequence analysis of the actinobacterial genus Kribbella

Multilocus sequence analysis (MLSA) was used to refine the phylogenetic analysis of the genus Kribbella, which currently contains 17 species with validly-published names. Sequences were obtained for the 16S rRNA, gyrB, rpoB, recA, relA and atpD genes for 16 of the 17 type strains of the genus plus seven non-type strains. A five-gene concatenated sequence of 4099 nt was used to examine the phylogenetic relationships between the species of the genus Kribbella. Using the concatenated sequence of the gyrB-rpoB-recA-relA and atpD genes, most Kribbella type strains can be distinguished by a genetic distance of >0.04. Each single-gene tree had an overall topology similar to that of the concatenated sequence tree. The single-gene relA tree, used here for the first time in MLSA of actinobacteria, had good bootstrap support, comparable to the rpoB and atpD gene trees, which had topologies closest to that of the concatenated sequence tree. This illustrates that relA is a useful addition in MLSA studies of the genus Kribbella. We propose that concatenated gyrB-rpoB-recA-relA-atpD gene sequences be used for examining the phylogenetic relationships within the genus Kribbella and for determining the closest phylogenetic relatives to be used for taxonomic comparisons.     

Whole-gene positive selection, elevated synonymous substitution rates, duplication, and indel evolution of the chloroplast clpP1 gene

Background

Synonymous DNA substitution rates in the plant chloroplast genome are generally relatively slow and lineage dependent. Non-synonymous rates are usually even slower due to purifying selection acting on the genes. Positive selection is expected to speed up non-synonymous substitution rates, whereas synonymous rates are expected to be unaffected. Until recently, positive selection has seldom been observed in chloroplast genes, and large-scale structural rearrangements leading to gene duplications are hitherto supposed to be rare.

Methodology/Principle Findings

We found high substitution rates in the exons of the plastid clpP1 gene in Oenothera (the Evening Primrose family) and three separate lineages in the tribe Sileneae (Caryophyllaceae, the Carnation family). Introns have been lost in some of the lineages, but where present, the intron sequences have substitution rates similar to those found in other introns of their genomes. The elevated substitution rates of clpP1 are associated with statistically significant whole-gene positive selection in three branches of the phylogeny. In two of the lineages we found multiple copies of the gene. Neighboring genes present in the duplicated fragments do not show signs of elevated substitution rates or positive selection. Although non-synonymous substitutions account for most of the increase in substitution rates, synonymous rates are also markedly elevated in some lineages. Whereas plant clpP1 genes experiencing negative (purifying) selection are characterized by having very conserved lengths, genes under positive selection often have large insertions of more or less repetitive amino acid sequence motifs.

Conclusions/Significance

We found positive selection of the clpP1 gene in various plant lineages to correlated with repeated duplication of the clpP1 gene and surrounding regions, repetitive amino acid sequences, and increase in synonymous substitution rates. The present study sheds light on the controversial issue of whether negative or positive selection is to be expected after gene duplications by providing evidence for the latter alternative. The observed increase in synonymous substitution rates in some of the lineages indicates that the detection of positive selection may be obscured under such circumstances. Future studies are required to explore the functional significance of the large inserted repeated amino acid motifs, as well as the possibility that synonymous substitution rates may be affected by positive selection.     

Species-specific molecular signature of Commiphora species of Saudi Arabia inferred from internal transcribed spacer sequences of nuclear ribosomal DNA

The deciduous habit and tendency to produce flowers prior to developing leaves, and a predominantly dioecious system of breeding in the genus Commiphora leads to difficulties in its taxonomic identification at species level. The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups. The present study deals the ITS sequence of nrDNA based molecular genotyping of seven species of the genus Commiphora of Saudi Arabia. The molecular phylogenetic analysis of ITS sequences of nrDNA of Commiphora species distributed in Saudi Arabia reveals the the occurrence of C. madagascariens in Saudi Arabia.     

Molecular phylogenetics and historical biogeography of Rhinolophus bats

The phylogenetic relationships within the horseshoe bats (genus Rhinolophus) are poorly resolved, particularly at deeper levels within the tree. We present a better-resolved phylogenetic hypothesis for 30 rhinolophid species based on parsimony and Bayesian analyses of the mitochondrial cytochrome b gene and three nuclear introns (TG, THY and PRKC1). Strong support was found for the existence of two geographic clades within the monophyletic Rhinolophidae: an African group and an Oriental assemblage. The relaxed Bayesian clock method indicated that the two rhinolophid clades diverged approximately 35 million years ago and results from Dispersal Vicariance (DIVA) analysis suggest that the horseshoe bats arose in Asia and subsequently dispersed into Europe and Africa.     

Bark and ambrosia beetles (Curculionidae: Scolytinae), their phoretic mites (Acari) and associated Geosmithia species (Ascomycota: Hypocreales) from Virgilia trees in South Africa

Bark and ambrosia beetles are ecologically and economically important phloeophagous insects that often have complex symbiotic relationships with fungi and mites. These systems are greatly understudied in Africa. In the present study we identified bark and ambrosia beetles, their phoretic mites and their main fungal associates from native Virgilia trees in the Cape Floristic Region (CFR) of South Africa. In addition, we tested the ability of mites to feed on the associated fungi. Four species of scolytine beetles were collected from various Virgilia hosts and from across the CFR. All were consistently associated with various Geosmithia species, fungi known from phloeophagous beetles in many parts of the world, but not yet reported as Scolytinae associates in South Africa. Four beetle species, a single mite species and five Geosmithia species were recovered. The beetles, Hapalogenius fuscipennis, Cryphalini sp. 1, and Scolytoplatypus fasciatus were associated with a single species of Elattoma phoretic mite that commonly carried spores of Geosmithia species. Liparthrum sp. 1 did not carry phoretic mites. Similar to European studies, Geosmithia associates of beetles from Virgilia were constant over extended geographic ranges, and species that share the same host plant individual had similar Geosmithia communities. Phoretic mites were unable to feed on their Geosmithia associates, but were observed to feed on bark beetle larvae within tunnels. This study forms the first African-centred base for ongoing global studies on the associations between arthropods and Geosmithia species. It strengthens hypotheses that the association between Scolytinae beetles and dry-spored Geosmithia species may be more ubiquitous than commonly recognised.     

Analysis of synonymous codon usage patterns of HPRT1 gene across twelve mammalian species

Genetic changes in Hypoxanthine guanine phosphoribosyltransferace (HPRT1) gene can alter the expression of the dopamine neurotransmitter leads to abnormal neuron function, a disease called Lesch-Nyhan syndrome (LNS). Although different studies were conducted on LNS, information on codon usage bias (CUB) of HPRT1 gene is limited. The present study examines the genetic determinants of CUB in HPRT1 gene using twelve mammalian species. In the coding sequence of HPRT1 genes, A/T ending codons was most frequently used. A higher ENC value was observed indicating lower HPRT1 gene expression in the selected mammalian species. Correlation analysis indicates that compositional constraints under mutation pressure can involve in CUB of HPRT1 genes among the selected mammalian species. Relative synonymous codon usage (RSCU) value revealed that the codons such as ACT, AGG, ATT and AGC were over-represented in each of the mammalian species. Result from the analysis of the RSCU indicates that compositional constraint is a key driver for the variation in codon usage. Ratio of nonsynonymous (dN) and synonymous (dS) substitution further suggested that purifying selection occurs among the HPRT1 gene of studied mammals to maintain its protein function under the process of evolution. Our findings report an insight into the codon usage patterns of HPRT1 gene and will be useful for LNS management.     

ASPM and the Evolution of Cerebral Cortical Size in a Community of New World Monkeys

The ASPM (abnormal spindle-like microcephaly associated) gene has been proposed as a major determinant of cerebral cortical size among primates, including humans. Yet the specific functions of ASPM and its connection to human intelligence remain controversial. This debate is limited in part by a taxonomic focus on Old World monkeys and apes. Here we expand the comparative context of ASPM sequence analyses with a study of New World monkeys, a radiation of primates in which enlarged brain size has evolved in parallel in spider monkeys (genus Ateles) and capuchins (genus Cebus). The primate community of Costa Rica is perhaps a model system because it allows for independent pairwise comparisons of smaller- and larger-brained species within two taxonomic families. Accordingly, we analyzed the complete sequence of exon 18 of ASPM in Ateles geoffroyi, Alouatta palliata, Cebus capucinus, and Saimiri oerstedii. As the analysis of multiple species in a genus improves phylogenetic reconstruction, we also analyzed eleven published sequences from other New World monkeys. Our exon-wide, lineage-specific analysis of eleven genera and the ratio of rates of nonsynonymous to synonymous substitutions (d_N/d_S) on ASPM revealed no detectable evidence for positive selection in the lineages leading to Ateles or Cebus, as indicated by d_N/d_S ratios of <1.0 (0.6502 and 0.4268, respectively). Our results suggest that a multitude of interacting genes have driven the evolution of larger brains among primates, with different genes involved in this process in different encephalized lineages, or at least with evidence for positive selection not readily apparent for the same genes in all lineages. The primate community of Costa Rica may serve as a model system for future studies that aim to elucidate the molecular mechanisms underlying cognitive capacity and cortical size.     

RAPD and ISSR analyses of species and populations of the genus Stachys

Molecular analysis of the genome was performed for 14 species of the genus Stachys. RAPD and ISSR analyses of the Stachys genome revealed 574 polymorphic fragments, including genus-and species-specific markers. Based on the patterns, UPGMA and the Jacquard coefficient were used to estimate the genetic distances between Stachys species and populations and to construct dendrograms reflecting the phylogenetic relationships among the Stachys species. Molecular analysis of the Stachys genome refined the phylogenetic positions of some species and revealed synonymous species.     

Phylogeny and taxonomy of the genus Cylindrocladiella

The genus Cylindrocladiella was established to accommodate Cylindrocladium-like fungi that have small, cylindrical conidia and aseptate stipe extensions. Contemporary taxonomic studies of these fungi have relied on morphology and to a lesser extent on DNA sequence comparisons of the internal transcribed spacer regions (ITS 1, 2 and 5.8S gene) of the ribosomal RNA and the ??-tubulin gene regions. In the present study, the identity of several Cylindrocladiella isolates collected over two decades was determined using morphology and phylogenetic inference. A phylogeny constructed for these isolates employing the ??-tubulin, histone H3, ITS, 28S large subunit and translation elongation factor 1-alpha gene regions resulted in the identification of several cryptic species in the genus. In spite of the 18 new Cylindrocladiella species described in this study based on morphological and sequence data, several species complexes remain unresolved.