Phylogenetic Markers Research Articles

Characterization of the complete mitochondrial genome of Ixodes granulatus (Ixodidae) and its phylogenetic implications.

Ticks are deemed to be second only to mosquitoes as the most common vector of human infectious diseases worldwide that give rise to human and animal diseases and economic losses to livestock production. Our understanding of the phylogenetic analysis between tick lineages has been restricted by the phylogenetic markers of individual genes. Genomic data research could help advance our understanding of phylogenetic analysis and molecular evolution. Mitochondrial genomic DNA facilitated the phylogenetic analysis of eukaryotes containing ticks. In this study, we sequenced and assembled the circular complete mitogenome information of Ixodes granulatus. The 14,540-bp mitogenome consists of 37 genes, including 13 protein-coding genes (PCGs), two genes for ribosomal RNA (rRNAs), and 22 genes for transfer RNA (tRNAs), and the origin of the L-strand replication region. The directions of the coding strand and component genes in the non-Australasian Ixodes mitochondrial genome were similar to those found in most other Australasian Ixodes, except for the loss of a lengthy control region. The phylogenetic tree based on maximum likelihood (ML) and Bayesian inference (BI) computational algorithms showed that I. granulatus exhibits a close relationship with I. hexagonus and I. ricinus. To our knowledge, this is the first study exploring the complete mitogenome for the species I. granulatus. Our results provide new insights for further research on the evolution, population genetics, systematics, and molecular ecology of ticks.

Structural and information-theoretic complexity measures of brain networks: Evolutionary aspects and implications

The evolutionary lineage of neuronal phenotype is notably complex even within a limited number of species. One of the approaches resides in the realm of complex network theory. The theory reduces the connectomic data into a hallmarked set of few parameters, some of which might be correlated with a suitably chosen phylogenetic marker. In this first-of-its-kind attempt, interspecific variations of two structural complexity measures (i.e., clustering coefficient and centrality) along with two independent information-theoretic measures (i.e., von Neumann entropy and multifractality) are investigated to decipher any hidden evolutionary signature considering four mammalian connectomes (i.e., felis catus, mus musculus, macaca mulatta, and homo sapiens). All network complexity measures partially corroborate with the phylogenetic order. Nevertheless, monotonicity of the measures with the chosen phylogenetic marker of genome size has been majorly violated because of the mus musculus data point. On the other hand, von Neumann entropy was found to exhibit an allometric scaling behavior with the community structure of all connectomes (p<0.0001, and R2>0.95). The respective scaling exponent was noted to be monotonic with the genome size. Singularities of the real connectomes were also investigated upon carrying out a similar analysis in three equivalent synthetic network models.

Elevational Gradients Impose Dispersal Limitation on Streptomyces.

Dispersal governs microbial biogeography, but the rates and mechanisms of dispersal remain poorly characterized for most microbial taxa. Dispersal limitation is driven by limits on dissemination and establishment, respectively. Elevation gradients create striking patterns of biogeography because they produce steep environmental gradients at small spatial scales, and these gradients offer a powerful tool to examine mechanisms of dispersal limitation. We focus on Streptomyces, a bacterial genus common to soil, by using a taxon-specific phylogenetic marker, the RNA polymerase-encoding rpoB gene. By targeting Streptomyces, we assess dispersal limitation at finer phylogenetic resolution than is possible using whole community analyses. We characterized Streptomyces diversity at local spatial scales (100 to 3,000 m) in two temperate forest sites located in the Adirondacks region of New York State: Woods Lake (<100 m elevation change), and Whiteface Mountain (>1,000 m elevation change). Beta diversity varied considerably at both locations, indicative of dispersal limitation acting at local spatial scales, but beta diversity was significantly higher at Whiteface Mountain. Beta diversity varied across elevation at Whiteface Mountain, being lowest at the mountain’s base. We show that Streptomyces taxa exhibit elevational preferences, and these preferences are phylogenetically conserved. These results indicate that habitat preferences influence Streptomyces biogeography and suggest that barriers to establishment structure Streptomyces communities at higher elevations. These data illustrate that Streptomyces biogeography is governed by dispersal limitation resulting from a complex mixture of stochastic and deterministic processes.

Reevaluation of the Phylogenetic Diversity and Global Distribution of the Genus "Candidatus Accumulibacter".

ABSTRACT“Candidatus Accumulibacter” was the first microorganism identified as a polyphosphate-accumulating organism (PAO) important for phosphorus removal from wastewater. Members of this genus are diverse, and the current phylogeny and taxonomic framework appear complicated, with most publicly available genomes classified as “Candidatus Accumulibacter phosphatis,” despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer-scale differentiation into different “types” and “clades”; nevertheless, taxonomic assignments remain inconsistent across studies. Therefore, a comprehensive reevaluation is needed to establish a common understanding of this genus, in terms of both naming and basic conserved physiological traits. Here, we provide this reassessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive data sets. We identified 15 novel species, along with “Candidatus Accumulibacter phosphatis,” “Candidatus Accumulibacter delftensis,” and “Candidatus Accumulibacter aalborgensis.” To compare the species in situ, we designed new species-specific fluorescence in situ hybridization (FISH) probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, “Ca. Accumulibacter” species were widespread in wastewater treatment plants (WWTPs) with phosphorus removal, indicating process design as a major driver for their abundance. Genome mining for PAO-related pathways and FISH-Raman microspectroscopy confirmed the potential for PAO metabolism in all “Ca. Accumulibacter” species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal.IMPORTANCE “Candidatus Accumulibacter” is the most studied PAO, with a primary role in biological nutrient removal. However, the species-level taxonomy of this lineage is convoluted due to the use of different phylogenetic markers or genome sequencing approaches. Here, we redefined the phylogeny of these organisms, proposing a comprehensive approach which could be used to address the classification of other diverse and uncultivated lineages. Using genome-resolved phylogeny, compared to phylogeny based on the 16S rRNA gene and other phylogenetic markers, we obtained a higher-resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of genes and pathways of interest, validated in situ by application of a new set of FISH probes and Raman microspectroscopy, provided additional high-resolution metabolic insights into these organisms.

Phylogenetic Marker Selection and Protein Sequence Analysis of the ORF5 Gene Product of Grapevine Virus A.

Grapevine virus A (GVA), the type species of the Vitivirus genus, is one of the causal agents of the Kober stem grooving disease of the rugose wood complex and one of the most frequently detected viruses in grapevine. There is little information on GVA gene(s) marker useful for phylogenetic analysis. To this aim, a total of 403 leaf samples were collected from vineyards of East and West Azarbaijan provinces in the Northwestern provinces of Iran during 2014–2016 and tested by DAS-ELISA and RT-PCR using ORF5-specific primers. GVA was detected in 56 symptomatic samples, corresponding to 14% of infection, while it was not detected in asymptomatic samples. The ORF5 (p10) protein sequence of eight Iranian isolates was compared to other vitiviruses, showing that the most conserved region resides in the N-terminus, carrying an arginine-rich motif followed by a zinc-finger motif. Next, to define a robust phylogenetic marker representative of the whole genome sequence suitable for phylogenetic and evolutionary studies, phylogenetic trees based on the full genome sequences of all the available GVA isolates and on individual genomic regions were constructed and compared. ORF1, which encodes the RNA-dependent RNA polymerase, was found to be the best phylogenetic marker for GVA classification and evolution studies. These results can be used for further research on phylogenetic analyses, evolution history, epidemiology, and etiology of rugose wood complex, and to identify control measures against GVA and other vitiviruses.

Comparative analysis of the complete plastid genomes in Prunus subgenus Cerasus (Rosaceae): Molecular structures and phylogenetic relationships.

Prunus subgenus Cerasus (cherry) is an economically important group that distributed in temperate regions of the northern hemisphere. However, shared interspecific morphological traits and variability across taxa of Cerasus are among the impediments to taxonomic efforts to correctly delimit taxa. This is further complicated by a lack of genetic information on these taxa, with no focused genomic or phylogenetic studies being done on Cerasus. In this study, we conducted comparative analysis on the complete plastid genomes (plastomes) of 20 Cerasus species to gain a greater understanding of the attributes of the plastome of these taxa while helping resolve their phylogenetic placement in Prunus sensu lato and interspecific relationships within the subgenus. Our results displayed that (1) the plastomes of the 20 Cerasus species studied exhibited a typical quadripartite structure with conversed genome arrangement, structure, and moderate divergence. (2) The average size of complete plastomes for the Cerasus taxa studied was 157,861 bp, ranging from 157,458 to 158,024 bp. A total of 134 genes were annotated, including 86 protein-coding genes, 40 tRNAs, and 8 rRNAs across all species. In simple sequence repeat analysis, we found Cerasus had a comparable number of dispersed and tandem repeats to those identified in other angiosperm taxa, with only P. pseudocerasus found to contain trinucleotide repeats. Nucleotide diversity analysis revealed that the trnG-GCC gene and rpl32-trnL region had the highest Pi value showing potential as phylogenetic markers. (3) Two phylogenetic trees of the plastomes verified the monophyletic relationship of Cerasus and provided a more resolved species-level phylogeny. Our study provides detailed plastome information for exploring the phylogeny of subg. Cerasus taxa. We identified various types of repeats and nucleotide diversity hotspots, which can be a reference for species identification and reconstruction of phylogenetic relationships.

Le séquençage à haut débit dans le diagnostic microbiologique

AbstractThe applications of next-generation sequencing (NGS) in health have recently become more widespread, at first in research studies and then in clinical microbiology. The use of NGS has mainly been developed in bacteriology and mycology, especially based on phylogenetic markers (hypervariable regions of rDNA 16S, 18S, or 28S) which allow by metataxonomy, to describe the physiopathological variations of microbiota. By non-targeted metagenomics approaches, the comparison of whole genome NGS data to international databases allows the detection of genomic antimicrobial resistance which is often correlated with the resistance phenotype. Similarly, different strategies to study virulence factors have been developed to compare the genomes of clinical isolates to reference strains and thus to analyze genomic islands. NGS has also shown its efficiency in determining an inter-individual transmission cluster, to identify the source patient or to improve collective management, to search for the etiological agent of an infection.Even though structures such as the Cofrac have taken NGS into account, the lack of standardization limits the routine development of these approaches, due to the need for bioinformatics.

Evolutionary relationships of anglerfishes (Lophiiformes) reconstructed using ultraconserved elements

The macroevolutionary consequences of evolving in the deep-sea remain poorly understood and are compounded by the fact that convergent adaptations for living in this environment makes elucidating phylogenetic relationships difficult. Lophiiform anglerfishes exhibit extreme habitat and predatory specializations, including the use of a fin-spine system as a luring device and unique reproductive strategies where parasitic males attach and fuse to females. Despite their notoriety for these odd characteristics, evolutionary relationships among these fishes remain unclear. We sought to clarify the evolutionary history of Lophiiformes using data from 1000 ultraconserved elements and phylogenomic inference methods with particular interest paid to the Ceratioidei (deep-sea anglerfishes) and Antennarioidei (frogfishes and handfishes). At the suborder level, we recovered similar topologies in separate phylogenomic analyses: The Lophioidei (monkfishes) are the sister group to the rest of the Lophiiformes, Ogcocephaloidei (batfishes) and Antennarioidei (frogfishes) form a sister group, and Chaunacioidei (coffinfishes) and Ceratioidei (deep-sea anglerfishes) form a clade. The relationships we recover within the ceratioids disagree with most previous phylogenetic investigations, which used legacy phylogenetic markers or morphology. We recovered non-monophyletic relationships in the Antennarioidei and proposed three new families based on molecular and morphological evidence: Histiophrynidae, Rhycheridae, and Tathicarpidae. Antennariidae was re-evaluated to include what was known as Antennariinae, but not Histiophryninae. Non-bifurcating signal in splits network analysis indicated reticulations among and within suborders, supporting the complicated history of the Lophiiformes previously found with morphological data. Although we resolve relationships within Antennarioidei, Ceratioidei relationships remain somewhat unclear without better taxonomic sampling.

Reunion of Australasian Possums by Shared SINE Insertions.

Although first posited to be of a single origin, the two superfamilies of phalangeriform marsupial possums (Phalangeroidea: brushtail possums and cuscuses and Petauroidea: possums and gliders) have long been considered, based on multiple sequencing studies, to have evolved from two separate origins. However, previous data from these sequence analyses suggested a variety of conflicting trees. Therefore, we reinvestigated these relationships by screening n}{}sim200,000 orthologous short interspersed element (SINE) loci across the newly available whole-genome sequences of phalangeriform species and their relatives. Compared to sequence data, SINE presence/absence patterns are evolutionarily almost neutral molecular markers of the phylogenetic history of species. Their random and highly complex genomic insertion ensures their virtually homoplasy-free nature and enables one to compare hundreds of shared unique orthologous events to determine the true species tree. Here, we identify 106 highly reliable phylogenetic SINE markers whose presence/absence patterns within multiple Australasian possum genomes unexpectedly provide the first significant evidence for the reunification of Australasian possums into one monophyletic group. Together, our findings indicate that nucleotide homoplasy and ancestral incomplete lineage sorting have most likely driven the conflicting signal distributions seen in previous sequence-based studies. [Ancestral incomplete lineage sorting; possum genomes; possum monophyly; retrophylogenomics; SINE presence/absence.]

Looking through the Lens of the Ribosome Biogenesis Evolutionary History: Possible Implications for Archaeal Phylogeny and Eukaryogenesis.

Our understanding of microbial diversity and its evolutionary relationships has increased substantially over the last decade. Such an understanding has been greatly fueled by culture-independent metagenomics analyses. However, the outcome of some of these studies and their biological and evolutionary implications, such as the origin of the eukaryotic lineage from the recently discovered archaeal Asgard superphylum, is debated. The sequences of the ribosomal constituents are amongst the most used phylogenetic markers. However, the functional consequences underlying the analysed sequence diversity and their putative evolutionary implications are essentially not taken into consideration. Here, we propose to exploit additional functional hallmarks of ribosome biogenesis to help disentangle competing evolutionary hypotheses. Using selected examples, such as the multiple origins of halophily in archaea or the evolutionary relationship between the Asgard archaea and Eukaryotes, we illustrate and discuss how function-aware phylogenetic framework can contribute to refining our understanding of archaeal phylogeny and the origin of eukaryotic cells.

An estimate of the deepest branches of the tree of life from ancient vertically evolving genes.

Core gene phylogenies provide a window into early evolution, but different gene sets and analytical methods have yielded substantially different views of the tree of life. Trees inferred from a small set of universal core genes have typically supported a long branch separating the archaeal and bacterial domains. By contrast, recent analyses of a broader set of non-ribosomal genes have suggested that Archaea may be less divergent from Bacteria, and that estimates of inter-domain distance are inflated due to accelerated evolution of ribosomal proteins along the inter-domain branch. Resolving this debate is key to determining the diversity of the archaeal and bacterial domains, the shape of the tree of life, and our understanding of the early course of cellular evolution. Here, we investigate the evolutionary history of the marker genes key to the debate. We show that estimates of a reduced Archaea-Bacteria (AB) branch length result from inter-domain gene transfers and hidden paralogy in the expanded marker gene set. By contrast, analysis of a broad range of manually curated marker gene datasets from an evenly sampled set of 700 Archaea and Bacteria reveals that current methods likely underestimate the AB branch length due to substitutional saturation and poor model fit; that the best-performing phylogenetic markers tend to support longer inter-domain branch lengths; and that the AB branch lengths of ribosomal and non-ribosomal marker genes are statistically indistinguishable. Furthermore, our phylogeny inferred from the 27 highest-ranked marker genes recovers a clade of DPANN at the base of the Archaea and places the bacterial Candidate Phyla Radiation (CPR) within Bacteria as the sister group to the Chloroflexota.

Comparative Analysis of the Complete Chloroplast Genomes of Nine Paphiopedilum Species.

Paphiopedilum is known as “lady’s or Venus” slipper orchids due to its prominent shoe-shaped labellum, with high ornamental value. Phylogenetic relationships among some species in Paphiopedilum genus cannot be effectively determined by morphological features alone or through the analysis of nuclear or chloroplast DNA fragments. In order to provide aid in understanding the evolutionary and phylogenetic relationship in Paphiopedilum at chloroplast (cp) genome-scale level, the complete cp genomes of six Paphiopedilum species were newly sequenced in this study, and three other published cp genome sequences of Paphiopedilum were included in the comparative analyses. The cp genomes of the six Paphiopedilum species ranged from 154,908 bp (P. hirsutissimum) to 161,300 bp (P. victoria-mariae) in size, all constituting four-part annular structures. Analyses of the nucleotide substitutions, insertions/deletions, and simple sequence repeats in the cp genomes were conducted. Ten highly variable regions that could serve as potential DNA barcodes or phylogenetic markers for this diverse genus were identified. Sequence variations in the non-coding regions were greater than that in the conserved protein-coding regions, as well as in the large single copy (LSC) and small single copy (SSC) regions than in the inverted repeat (IR) regions. Phylogenetic analysis revealed that all Paphiopedilum species clustered in one monophyletic clade in the Cypripedioideae subfamily and then subdivided into seven smaller branches corresponding to different subgenus or sections of the genus, with high bootstrap supports, indicate that cp genome sequencing can be an effective means in resolving the complex relationship in Paphiopedilum.

The Piroplasmida Babesia, Cytauxzoon, and Theileria in farm and companion animals: species compilation, molecular phylogeny, and evolutionary insights.

The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz's rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed.

Comparing genetic markers efficiencies for discrimination between two commercially important holothuroids in the Mediterranean Sea, Holothuria polii and Holothuria sanctori.

Sea cucumber (bche-de-mer, Echinodermata: Holothuroidea) is one of the top internationally traded seafood varieties. Besides its direct nutritional benefits, it is continuously used in the traditional medicine in different areas and cultures in the world. This world-wide interest triggered various issues related to stocks declining and risks of species extinction. For these reasons, the current study was designed to provide molecular tools for accurate discrimination between two sea cucumber species that prevail the Mediterranean of these echinoderms in Egypt, that are Holothuria polii and H. sanctori. The power of three gene markers, i.e., 16S rDNA, 28S rDNA, and Histone H3 in achieving accurate DNA-based identification, as well as elucidating clear phylogenetic and genetic diversity differences between those two species was assessed. Among the three genes, 16S rDNA showed the highest potentials as genetic and phylogenetic species discrimination marker. Both 28S rDNA and H3 exhibited the least number of holothuroid reference sequences in the GenBank database. For genetic diversity within each species population, 16S rDNA exhibited the best potentials, followed by H3. 28S rDNA showed no genetic polymorphism at all. Moreover, the collective data of both H3 and 16S rDNA suggested a possible role of asexual reproduction behavior in H. sanctori in the reduction of genetic diversity, as a possible response to overfishing. Hence, the current research can recommend the simultaneous application of both 16S rDNA and H3 as accurate markers for genetic discrimination among H. polii, H. sanctori and other different holothuroid species.

First Report on Mitochondrial Gene Rearrangement in Non-Biting Midges, Revealing a Synapomorphy in Stenochironomus Kieffer (Diptera: Chironomidae).

Simple SummaryGene rearrangement is an additional type of data to support relationships of taxa, with rearrangement synapomorphies identified across multiple orders and at many different taxonomic levels. The concept to use mitochondrial gene rearrangements as phylogenetic markers has been proposed since the mid-1980s, the synapomorphic gene rearrangements have been identified from many lineages. However, mitochondrial gene rearrangement has never been observed in the non-biting midges (Diptera: Chironomidae). Here, seven new mitogenomes of the genus Stenochironomus were sequenced and analyzed. Coupled with published data, phylogenetic analyses were performed within Chironominae. The present study showed that mitogenomes of Stenochironomus are showing a higher A+T bias than other chironomid species. A synapomorphic gene rearrangement that the gene order rearranges from trnI-trnQ-trnM to trnI-trnM-trnQ was identified within Stenochironomus, which is the first instance of mitochondrial gene rearrangement discovered in the Chironomidae. The monophyly of the genus Stenochironomus was strongly supported by mitogenomes. Our study provides new insights into the mitochondrial gene order of Chironomidae, and provides a valuable resource for understanding synapomorphic gene rearrangements.(1) Background: Gene rearrangement of mitochondrial genome, especially those with phylogenetic signals, has long fascinated evolutionary biologists. The synapomorphic gene rearrangements have been identified across multiple orders and at many different taxonomic levels, supporting the monophyletic or systematic relationships of related lineages. However, mitochondrial gene rearrangement has never been observed in the non-biting midges (Diptera: Chironomidae); (2) methods: in this study, the complete mitogenomes of seven Stenochironomus species were sequenced and analyzed for the first time; (3) results: each mitogenome of Stenochironomus contains 37 typical genes and a control region. The whole mitogenomes of Stenochironomus species exhibit a higher A+T bias than other published chironomid species. The gene order rearranges from trnI-trnQ-trnM to trnI-trnM-trnQ in all the seven mitogenomes of Stenochironomus, which might be act as a synapomorphy of the genus, supporting the monophyletic of Stenochironomus species. In addition, another derived gene cluster: trnA-trnG-ND3-trnR exists in Stenochironomus tobaduodecimus. The derived gene orders described above are the first case of mitochondrial gene rearrangement in Chironomidae. Coupled with published data, phylogenetic relationships were reconstructed within Chironominae, and strongly supported the monophyly of Stenochironomus; (4) conclusions: our study provides new insights into the mitochondrial gene order of Chironomidae, and provides a valuable resource for understanding the synapomorphic gene rearrangements.

Toxicity of the Diatom Genus Pseudo-nitzschia (Bacillariophyceae): Insights from Toxicity Tests and Genetic Screening in the Northern Adriatic Sea.

Diatoms of the genus Pseudo-nitzschia H.Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species—P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle—have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed.

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring.

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

Concerted and Independent Evolution of Control Regions 1 and 2 of Water Monitor Lizards (Varanus salvator macromaculatus) and Different Phylogenetic Informative Markers.

Simple SummaryThe evolutionary patterns and phylogenetic utility of duplicate control regions (CRs) in 72 individuals of Varanus salvator macromaculatus and other varanids have been observed. Divergence of the two CRs from each individual revealed a pattern of independent evolution in CRs of varanid lineage. This study is a first step towards developing new phylogenetic evolutionary models of the varanid lineage, with accurate evolutionary inferences to provide basic insights into the biology of mitogenomes.Duplicate control regions (CRs) have been observed in the mitochondrial genomes (mitogenomes) of most varanids. Duplicate CRs have evolved in either concerted or independent evolution in vertebrates, but whether an evolutionary pattern exists in varanids remains unknown. Therefore, we conducted this study to analyze the evolutionary patterns and phylogenetic utilities of duplicate CRs in 72 individuals of Varanus salvator macromaculatus and other varanids. Sequence analyses and phylogenetic relationships revealed that divergence between orthologous copies from different individuals was lower than in paralogous copies from the same individual, suggesting an independent evolution of the two CRs. Distinct trees and recombination testing derived from CR1 and CR2 suggested that recombination events occurred between CRs during the evolutionary process. A comparison of substitution saturation showed the potential of CR2 as a phylogenetic marker. By contrast, duplicate CRs of the four examined varanids had similar sequences within species, suggesting typical characteristics of concerted evolution. The results provide a better understanding of the molecular evolutionary processes related to the mitogenomes of the varanid lineage.

Comparative genomic analysis of dwarf Vibrio myoviruses defines a conserved gene cluster for successful phage infection.

Tailed bacteriophages have been at the center of attention, not only for their ability to infect and kill pathogenic bacteria but also due to their peculiar and intriguing complex contractile tail structure. Tailed bacteriophages with contractile tails are known to have a Myoviridae morphotype and are members of the order Caudovirales. Large bacteriophages with a genome larger than 150 kbp have been studied for their ability to use multiple infection and lysis strategies to replicate more efficiently. On the other hand, smaller bacteriophages with fewer genes are represented in the GenBank database in greater numbers, and have several genes with unknown function. Isolation and molecular characterization of a newly reported bacteriophage named Athena1 revealed that it is a strongly lytic bacteriophage with a genome size of 39,826 bp. This prompted us to perform a comparative genomic analysis of Vibrio myoviruses with a genome size of no more than 50 kbp. The results revealed a pattern of genomic organization that includes sets of genes responsible for virion morphogenesis, replication/recombination of DNA, and lysis/lysogeny switching. By studying phylogenetic gene markers, we were able to draw conclusions about evolutionary events that shaped the genomic mosaicism of these phages, pinpointing the importance of a conserved organization of the genomic region encoding the baseplate protein for successful infection of Gram-negative bacteria. In addition, we propose the creation of new genera for dwarf Vibrio myoviruses. Comparative genomics of phages infecting aquatic bacteria could provide information that is useful for combating fish pathogens in aquaculture, using novel strategies.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3.

ABSTRACT Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1–3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS–PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1–3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.