Phylogenetic Markers Research Articles

Structure-Based GC Investigation Sheds New Light on ITS2 Evolution in Corydalis Species.

Guanine and cytosine (GC) content is a fundamental component of genetic diversity and essential for phylogenetic analyses. However, the GC content of the ribosomal internal transcribed spacer 2 (ITS2) remains unknown, despite the fact that ITS2 is a widely used phylogenetic marker. Here, the ITS2 was high-throughput sequenced from 29 Corydalis species, and their GC contents were comparatively investigated in the context of ITS2's characteristic secondary structure and concerted evolution. Our results showed that the GC contents of ITS2 were 131% higher than those of their adjacent 5.8S regions, suggesting that ITS2 underwent GC-biased evolution. These GCs were distributed in a heterogeneous manner in the ITS2 secondary structure, with the paired regions being 130% larger than the unpaired regions, indicating that GC is chosen for thermodynamic stability. In addition, species with homogeneous ITS2 sequences were always GC-rich, supporting GC-biased gene conversion (gBGC), which occurred with ITS2's concerted evolution. The RNA substitution model inferred also showed a GC preference among base pair transformations, which again supports gBGC. Overall, structurally based GC investigation reveals that ITS2 evolves under structural stability and gBGC selection, significantly increasing its GC content.

Genomic features, antimicrobial susceptibility, and epidemiological insights into Burkholderia cenocepacia clonal complex 31 isolates from bloodstream infections in India.

Burkholderia cepacia complex (Bcc) clonal complex (CC) 31, the predominant lineage causing devastating outbreaks globally, has been a growing concern of infections in non-cystic fibrosis (NCF) patients in India. B. cenocepacia is very challenging to treat owing to its virulence determinants and antibiotic resistance. Improving the management of these infections requires a better knowledge of their resistance patterns and mechanisms. Whole-genome sequences of 35 CC31 isolates obtained from patient samples, were analyzed against available 210 CC31 genomes in the NCBI database to glean details of resistance, virulence, mobile elements, and phylogenetic markers to study genomic diversity and evolution of CC31 lineage in India. Genomic analysis revealed that 35 isolates belonging to CC31 were categorized into 11 sequence types (ST), of which five STs were reported exclusively from India. Phylogenetic analysis classified 245 CC31 isolates into eight distinct clades (I-VIII) and unveiled that NCF isolates are evolving independently from the global cystic fibrosis (CF) isolates forming a distinct clade. The detection rate of seven classes of antibiotic-related genes in 35 isolates was 35 (100%) for tetracyclines, aminoglycosides, and fluoroquinolones; 26 (74.2%) for sulphonamides and phenicols; 7 (20%) for beta-lactamases; and 1 (2.8%) for trimethoprim resistance genes. Additionally, 3 (8.5%) NCF isolates were resistant to disinfecting agents and antiseptics. Antimicrobial susceptibility testing revealed that majority of NCF isolates were resistant to chloramphenicol (77%) and levofloxacin (34%). NCF isolates have a comparable number of virulence genes to CF isolates. A well-studied pathogenicity island of B. cenocepacia, GI11 is present in ST628 and ST709 isolates from the Indian Bcc population. In contrast, genomic island GI15 (highly similar to the island found in B. pseudomallei strain EY1) is exclusively reported in ST839 and ST824 isolates from two different locations in India. Horizontal acquisition of lytic phage ST79 of pathogenic B. pseudomallei is demonstrated in ST628 isolates Bcc1463, Bcc29163, and BccR4654 amongst CC31 lineage. The study reveals a high diversity of CC31 lineages among B. cenocepacia isolates from India. The extensive information from this study will facilitate the development of rapid diagnostic and novel therapeutic approaches to manage B. cenocepacia infections.

Shine: A novel strategy to extract specific, sensitive and well-conserved biomarkers from massive microbial genomic datasets

BackgroundConcentrations of the pathogenic microorganisms’ DNA in biological samples are typically low. Therefore, DNA diagnostics of common infections are costly, rarely accurate, and challenging. Limited by failing to cover updated epidemic testing samples, computational services are difficult to implement in clinical applications without complex customized settings. Furthermore, the combined biomarkers used to maintain high conservation may not be cost effective and could cause several experimental errors in many clinical settings. Given the limitations of recent developed technology, 16S rRNA is too conserved to distinguish closely related species, and mosaic plasmids are not effective as well because of their uneven distribution across prokaryotic taxa.ResultsHere, we provide a computational strategy, Shine, that allows extraction of specific, sensitive and well-conserved biomarkers from massive microbial genomic datasets. Distinguished with simple concatenations with blast-based filtering, our method involves a de novo genome alignment-based pipeline to explore the original and specific repetitive biomarkers in the defined population. It can cover all members to detect newly discovered multicopy conserved species-specific or even subspecies-specific target probes and primer sets. The method has been successfully applied to a number of clinical projects and has the overwhelming advantages of automated detection of all pathogenic microorganisms without the limitations of genome annotation and incompletely assembled motifs. Using on our pipeline, users may select different configuration parameters depending on the purpose of the project for routine clinical detection practices on the website https://bioinfo.liferiver.com.cn with easy registration.ConclusionsThe proposed strategy is suitable for identifying shared phylogenetic markers while featuring low rates of false positive or false negative. This technology is suitable for the automatic design of minimal and efficient PCR primers and other types of detection probes.

First Report of Fusarium commune Associated with a Root Rot of Grapevine in China.

During last decade, species belonging to Fusarium, Rosellinia, Armillaria and Dactylonectria were confirmed as phytopathogens causing grapevine root diseases (Highet and Nair 1995; Teixeira et al. 1995; Calamit et al. 2021; Ye et al. 2021). From 2020 to 2021, grapevine decline was observed in several vineyards in Beijing region, China. Leaves turned yellow with brown necrotic patches and roots were poorly developed, which was suggesting that a root disease was affecting the vines. The disease incidence was up to 10-15% of the vineyard for sample collection. Symptomatic root samples (cv. 'Red Globe') were collected and tissue fragments were excised at the margin of the symptomatic tissue in order to isolate the potential pathogen. The surface was sterilized using 1.5% sodium hypochlorite for 3 min, followed by 70% ethanol for 30 sec, and rinsed three times with sterile distilled water (Ye et al. 2020). Tissues were dried and placed onto potato dextrose agar (PDA) plates, followed by incubation at 25°C under dark conditions for 3 d. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. Next, single spores were transferred onto new PDA plates and incubated at 25°C for 7 d. Eight isolates numbered with JZB3110172 to JZB3110179 were obtained and their culture characters were identical, and the re-isolation percentage was 100%. Colonies were white to orange, with abundant fluffy aerial mycelium. Macroconidia were fusiform with a slightly curved apical cell and a foot-shaped basal cell, and measured 16.2-43.2 μm × 2.7-4.9 μm (n=50); microconidia were cylindrical, straight to slightly curved, 5.1-13 × 2.1-3.9 μm (n=50). Morphological characters of the isolates resembled to Fusarium commune (Skovgaard et al. 2003). For phylogenetic analysis, genomic DNA of the eight isolates was extracted with a DNA extraction kit (DNeasy plant Mini Kit). PCR amplifications of two phylogenetic markers (EF-1α and RPB2) were performed using the primers EF-1/EF-2 (Geiser et al. 2004) and RPB2-5F2/RPB2-7cR (Liu et al. 1995), respectively. The sequences were deposited in GenBank ON457645 to ON457660. Comparison of base pairs on Maximum likelihood (ML) phylogenetic analysis was conducted using the RAxML-HPC2 tool on XSEDE on the CIPRES Science Gateway platform (http://www.phylo.org/). The sequences of EF-1α and RPB2 of the eight isolates showed 99 to 100% similarity to the reference isolates of F. commune. In the phylogenetic tree, the isolates from this study clustered with the representative strains of F. commune (NRRL 52764, NRRL 28387 and NRRL 52744). Based on morphological characters and the phylogenetic results, all of isolates were identified as F. commune. Koch's postulates were conducted on healthy, 3-month-old grapevine 'Marselan'. Plant roots were trimmed with sterile scissors and then soaked in a spore suspension (1.0 × 106 spores mL-1) or sterile water (as the control) for 30 min. The inoculated grapevines were transplanted into pots and kept in the greenhouse at 25°C. After 14 days, all the inoculated plants developed necrosis and turned yellow. No symptoms were observed on the control. Koch's postulates were fulfilled by re-isolating the fungus from necrotic root tissues. The isolates obtained from the artificially infected tissue were identified again as F. commune based on morphological and molecular analyses. Overall, this is the first report of F. commune associated with a grapevine root rot globally, which lays a foundation for further study and developing disease control methods.

Evolution of mitogenomic gene order in Orthoptera.

Mitochondrial gene order has contributed to the elucidation of evolutionary relationships in several animal groups. It generally has found its application as a phylogenetic marker for deep nodes. Yet, in Orthoptera limited research has been performed on the gene order, although the group represents one of the oldest insect orders. We performed a comprehensive study on mitochondrial genome rearrangements (MTRs) within Orthoptera in the context of mitogenomic sequence-based phylogeny. We used 280 published mitogenome sequences from 256 species, including three outgroup species, to reconstruct a molecular phylogeny. Using a heuristic approach, we assigned MTR scenarios to the edges of the phylogenetic tree and reconstructed ancestral gene orders to identify possible synapomorphies in Orthoptera. We found all types of MTRs in our dataset: inversions, transpositions, inverse transpositions, and tandem-duplication/random loss events (TDRL). Most of the suggested MTRs were in single and unrelated species. Out of five MTRs which were unique in subgroups of Orthoptera, we suggest four of them to be synapomorphies; those were in the infraorder Acrididea, in the tribe Holochlorini, in the subfamily Pseudophyllinae, and in the two families Phalangopsidae and Gryllidae or their common ancestor (leading to the relationship ((Phalangopsidae + Gryllidae) + Trigonidiidae)). However, similar MTRs have been found in distant insect lineages. Our findings suggest convergent evolution of specific mitochondrial gene orders in several species, deviant from the evolution of the mitogenome DNA sequence. As most MTRs were detected at terminal nodes, a phylogenetic inference of deeper nodes based on MTRs is not supported. Hence, the marker does not seem to aid resolving the phylogeny of Orthoptera, but adds further evidence for the complex evolution of the whole group, especially at the genetic and genomic levels. The results indicate a high demand for more research on patterns and underlying mechanisms of MTR events in Orthoptera.

The phylogeny and evolutionary ecology of hoverflies (Diptera: Syrphidae) inferred from mitochondrial genomes

Hoverflies (Diptera: Syrphidae) are a diverse group of pollinators and a major research focus in ecology, but their phylogenetic relationships remain incompletely known. Using a genome skimming approach we generated mitochondrial genomes for 91 species, capturing a wide taxonomic diversity of the family. To reduce the required amount of input DNA and overall cost of the library construction, sequencing and assembly was conducted on mixtures of specimens, which raises the problem of chimera formation of mitogenomes. We present a novel chimera detection test based on gene tree incongruence, but identified only a single mitogenome of chimeric origin. Together with existing data for a final set of 127 taxa, phylogenetic analysis on nucleotide and amino acid sequences using Maximum Likelihood and Bayesian Inference revealed a basal split of Microdontinae from all other syrphids. The remainder consists of several deep clades assigned to the subfamily Eristalinae in the current classification, including a clade comprising the subfamily Syrphinae (plus Pipizinae). These findings call for a re-definition of subfamilies, but basal nodes had insufficient support to fully justify such action. Molecular-clock dating placed the origin of the Syrphidae crown group in the mid-Cretaceous while the Eristalinae-Syrphinae clade likely originated near the K/Pg boundary. Transformation of larval life history characters on the tree suggests that Syrphidae initially had sap feeding larvae, which diversified greatly in diet and habitat association during the Eocene and Oligocene, coinciding with the diversification of angiosperms and the evolution of various insect groups used as larval host, prey, or mimicry models. Mitogenomes proved to be a powerful phylogenetic marker for studies of Syrphidae at subfamily and tribe levels, allowing dense taxon sampling that provided insight into the great ecological diversity and rapid evolution of larval life history traits of the hoverflies.

Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera.

Diatoms are one of the most important phytoplankton groups in the world's oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks.

Mitochondrial phylogenomics provides conclusive evidence that the family Ancyrocephalidae is deeply paraphyletic

BackgroundUnresolved taxonomic classification and paraphyly pervade the flatworm class Monogenea: the class itself may be paraphyletic and split into Polyopisthocotylea and Monopisthocotylea; there are some indications that the monopisthocotylean order Dactylogyridea may also be paraphyletic; single-gene markers and some morphological traits indicate that the family Ancyrocephalidae is paraphyletic and intertwined with the family Dactylogyridae.MethodsTo attempt to study the relationships of Ancyrocephalidae and Monopisthocotylea using a phylogenetic marker with high resolution, we sequenced mitochondrial genomes of two fish ectoparasites from the family Dactylogyridae: Dactylogyrus simplex and Dactylogyrus tuba. We conducted phylogenetic analyses using three datasets and three methods. Datasets were ITS1 (nuclear) and nucleotide and amino acid sequences of almost complete mitogenomes of almost all available Monopisthocotylea mitogenomes. Methods were maximum likelihood (IQ-TREE), Bayesian inference (MrBayes) and CAT-GTR (PhyloBayes).ResultsBoth mitogenomes exhibited the ancestral gene order for Neodermata, and both were compact, with few and small intergenic regions and many and large overlaps. Gene sequences were remarkably divergent for nominally congeneric species, with only trnI exhibiting an identity value > 80%. Both mitogenomes had exceptionally low A + T base content and AT skews. We found evidence of pervasive compositional heterogeneity in the dataset and indications that base composition biases cause phylogenetic artefacts. All six mitogenomic analyses produced unique topologies, but all nine analyses produced topologies that rendered Ancyrocephalidae deeply paraphyletic. Mitogenomic data consistently resolved the order Capsalidea as nested within the Dactylogyridea.ConclusionsThe analyses indicate that taxonomic revisions are needed for multiple Polyopisthocotylea lineages, from genera to orders. In combination with previous findings, these results offer conclusive evidence that Ancyrocephalidae is a paraphyletic taxon. The most parsimonious solution to resolve this is to create a catch-all Dactylogyridae sensu lato clade comprising the current Ancyrocephalidae, Ancylodiscoididae, Pseudodactylogyridae and Dactylogyridae families, but the revision needs to be confirmed by another marker with a sufficient resolution.Graphical

The Complete Mitochondrial Genome of Dendrogale murina (Tupaiidae) and Phylogeny of Scandentia.

In this paper, we report the complete mitochondrial genome of the northern smooth-tailed treeshrew Dendrogale murina, which was sequenced for the first time using the Illumina next-generation sequencing (NGS) technology. The total length of the mitochondrial genome is 16,844-16,850 bp and encodes 37 genes, including two ribosomal RNAs (rRNAs) 12S and 16S, 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), and a D-loop in the characteristic arrangement of family Tupaiidae (Mammalia: Scandentia). The overall base composition of the complete mitochondrial DNA is A (33.5%), C (25.5%), G (13.9%), and T (27.1%). Phylogenetic analysis of Scandentia mitochondrial genomes showed a classic pattern, which was revealed previously while using individual phylogenetic markers. The result of the current study is consistent with one based on the latest morphological studies, with the basal position of Ptilocercus and Dendrogale sister to the rest of the Tupaiidae genera. The divergence time of the Dendrogale genus is estimated as Eocene-Oligocene, with the mean value of 35.8 MYA, and the Ptilocercus genus probably separated at about 46.3 MYA. We observe an increase in the age of all nodes within the Scandentia, except for a decrease in the age of separation of Ptilocercus. This result can be explained both by the addition of new mitochondrial genome data in the analysis and the usage of new calibration points from recently published data.

New Mitogenomes of the Polypedilum Generic Complex (Diptera: Chironomidae): Characterization and Phylogenetic Implications.

Mitochondrial genomics, as a useful marker for phylogenetics and systematics of organisms, are important for molecular biology studies. The phylogenetic relationships of the Polypedilum generic complex remains controversial, due to lack taxonomy and molecular information. In this study, we newly sequenced mitogenomes of 14 species of the Polypedilum generic complex. Coupled with three recently published sequences, we analyzed the nucleotide composition, sequence length, and evolutionary rate of this generic complex. The control region showed the highest AT content. The evolution rate of protein coding genes was as follows: ATP8 > ND6 > ND5 > ND3 > ND2 > ND4L > ND4 > COX1 > ND1 > CYTB > APT6 > COX2 > COX3. We reconstructed the phylogenetic relationships among the genera within the Polypedilum generic complex based on 19 mitochondrial genomes (seventeen ingroups and two outgroups), using Bayesian Inference (BI) and Maximum Likelihood (ML) methods for all databases. Phylogenetic analysis of 19 mitochondrial genomes demonstrated that the Endochironomus + Synendotendipes was sister to Phaenopsectra + Sergentia.

Mapping CAP-A satellite DNAs by FISH in Sapajus cay paraguay and S. macrocephalus (Platyrrhini, Primates)

Satellite DNAs such as Cap-A sequences are potentially informative taxonomic and phylogenetic markers useful for characterizing primate genomes. They have also been used as cytogenetic markers facilitating species identification in many taxa. The aim of this work is to map Cap-A sequences by FISH (fluorescent in situ hybridization) on two Platyrrhini (Primates) species genomes, Sapajus cay paraguay and S. macrocephalus, in order to study their distribution pattern on chromosomes. The Cap-A probes showed bright signals with almost the same interstitial pattern of distribution in correspondence with C and CMA3 rich regions on six pairs of chromosomes in both Sapajus species. An additional pair was detected on S. macrocephalus. The analysis of the results, compared with previous literature data on other phylogenetically close New World species, shows that Cap-A satellite sequences have a genus-specific pattern, but with slight species-specific patterns that are useful as phylogenetic and taxonomic markers.

A Guide to Gene-Centric Analysis Using TreeSAPP.

Gene-centric analysis is commonly used to chart the structure, function, and activity of microbial communities in natural and engineered environments. A common approach is to create custom ad hoc reference marker gene sets, but these come with the typical disadvantages of inaccuracy and limited utility beyond assigning query sequences taxonomic labels. The Tree-based Sensitive and Accurate Phylogenetic Profiler (TreeSAPP) software package standardizes analysis of phylogenetic and functional marker genes and improves predictive performance using a classification algorithm that leverages information-rich reference packages consisting of a multiple sequence alignment, a profile hidden Markov model, taxonomic lineage information, and a phylogenetic tree. Here, we provide a set of protocols that link the various analysis modules in TreeSAPP into a coherent process that both informs and directs the user experience. This workflow, initiated from a collection of candidate reference sequences, progresses through construction and refinement of a reference package to marker identification and normalized relative abundance calculations for homologous sequences in metagenomic and metatranscriptomic datasets. The alpha subunit of methyl-coenzyme M reductase (McrA) involved in biological methane cycling is presented as a use case given its dual role as a phylogenetic and functional marker gene driving an ecologically relevant process. These protocols fill several gaps in prior TreeSAPP documentation and provide best practices for reference package construction and refinement, including manual curation steps from trusted sources in support of reproducible gene-centric analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Creating reference packages Support Protocol 1: Installing TreeSAPP Support Protocol 2: Annotating traits within a phylogenetic context Basic Protocol 2: Updating reference packages Basic Protocol 3: Calculating relative abundance of genes in metagenomic and metatranscriptomic datasets.

Mining various genomic resources to resolve old alpha-taxonomy questions: A test of the species hypothesis of the Proteocephalus longicollis species complex (Cestoda: Platyhelminthes) from salmonid fishes

High-throughput sequencing strategies became commonly employed to study non-model parasites, but the corresponding genomes and transcriptomes were seldom mined following the original publication. Similar to the data generated with genome skimming techniques based on shallow-depth shotgun genomes, various genomic and transcriptomic resources can be screened for useful molecular phylogenetic markers traditionally characterised with Sanger sequencing. Here, we provide an example of a strategy using reduced-representation genomic as well as transcriptomic data to obtain broad insights into the molecular diversity of the cestode Proteocephalus longicollis, a common parasite of salmonids distributed throughout the Holarctic region. We extract popular mitochondrial and nuclear ribosomal markers from various genomic resources for hundreds of parasite specimens from multiple European whitefish populations and compare those with Proteocephalus representatives from other species of salmonids and various geographical regions. In contrast with the previous morphology-based assessments, molecular phylogeny reveals a high degree of genetic divergence between Proteocephalus isolates from different salmonids, contrastingly low genetic differentiation within the parasite’s populations hosted by the European whitefish (Coregonus lavaretus species complex), and a sister species relationship of Proteocephalus from European whitefish and Proteocephalus percae, a parasite of European perch (Perca fluviatilis). Proteocephalus spp. from North American lake whitefish, brown trout and Arctic charr each formed clearly distinct lineages. These results advance our understanding of the interrelationships of the Proteocephalus-aggregate, a well-recognized clade of Holarctic freshwater fish proteocephalids, and support resurrection of some of the nominal species of Proteocephalus, including Proteocephalus exiguus La Rue, 1911 from North American coregonids and Proteocephalus fallax La Rue, 1911 from European C. lavaretus, reserving Proteocephalus longicollis (Zeder, 1800) exclusively for parasites of Salmo trutta.

The Effects of Atrazine, Diuron, Fluazifop-P-butyl, Haloxyfop-P-methyl, and Pendimethalin on Soil Microbial Activity and Diversity

Understanding the impacts of herbicides on soil microbial communities is important, as these organisms mediate a wide range of ecosystem services. Here, we investigated whether the diversity and function of soil microbial communities were significantly influenced by one-off applications of atrazine, diuron, fluazifop-P-butyl, haloxyfop-P-methyl and pendimethalin as pure compounds at their recommended doses over multiple time points (1, 3, 7, 14, 30 and 60 days). Phylogenetic marker gene sequencing revealed that none of the herbicides influenced the numbers of bacterial and archaeal taxa or the evenness of their abundances. Similarly, none of the herbicides influenced the composition of bacterial and archaeal communities, except for diuron, fluazifop-P-methyl and pendimethalin, which were associated with larger relative abundances of a small number of OTUs on day 30 only. Functionally, none of the herbicides significantly influenced fluorescein diacetate hydrolysis (FDA) and beta-glucosidase activities or the induced respiratory responses of soil microbial communities to a range of substrates. These data indicate that the active herbicide ingredients tested may have minimal non-target effects when applied once at their recommended dose. Given their frequent use, it is important to next consider whether these herbicides have more pronounced effects at higher doses and application frequencies.

Exploring Evolutionary Relationships within Neodermata Using Putative Orthologous Groups of Proteins, with Emphasis on Peptidases

The phylogenetic relationships within Neodermata were examined based on putative orthologous groups of proteins (OGPs) from 11 species of Monogenea, Trematoda, and Cestoda. The dataset included OGPs from BUSCO and OMA. Additionally, peptidases were identified and evaluated as phylogenetic markers. Phylogenies were inferred using the maximum likelihood method. A network analysis and a hierarchical grouping analysis of the principal components (HCPC) of orthologous groups of peptidases were performed. The phylogenetic analyses showed the monopisthocotylean monogeneans as the sister-group of cestodes, and the polyopisthocotylean monogeneans as the sister-group of trematodes. However, the sister-group relationship between Monopisthocotylea and Cestoda was not statistically well supported. The network analysis and HCPC also showed a cluster formed by polyopisthocotyleans and trematodes. The present study supports the non-monophyly of Monogenea. An analysis of mutation rates indicated that secreted peptidases and inhibitors, and those with multiple copies, are under positive selection pressure, which could explain the expansion of some families such as C01, C19, I02, and S01. Whilst not definitive, our study presents another point of view in the discussion of the evolution of Neodermata, and we hope that our data drive further discussion and debate on this intriguing topic.

Analysis of Complete Chloroplast Genome: Structure, Phylogenetic Relationships of Galega orientalis and Evolutionary Inference of Galegeae.

Galega orientalis, a leguminous herb in the Fabaceae family, is an ecologically and economically important species widely cultivated for its strong stress resistance and high protein content. However, genomic information of Galega orientalis has not been reported, which limiting its evolutionary analysis. The small genome size makes chloroplast relatively easy to obtain genomic sequence for phylogenetic studies and molecular marker development. Here, the chloroplast genome of Galega orientalis was sequenced and annotated. The results showed that the chloroplast genome of G. orientalis is 125,280 bp in length with GC content of 34.11%. A total of 107 genes were identified, including 74 protein-coding genes, 29 tRNAs and four rRNAs. One inverted repeat (IR) region was lost in the chloroplast genome of G. orientalis. In addition, five genes (rpl22, ycf2, rps16, trnE-UUC and pbf1) were lost compared with the chloroplast genome of its related species G. officinalis. A total of 84 long repeats and 68 simple sequence repeats were detected, which could be used as potential markers in the genetic studies of G. orientalis and related species. We found that the Ka/Ks values of three genes petL, rpl20, and ycf4 were higher than one in the pairwise comparation of G. officinalis and other three Galegeae species (Calophaca sinica, Caragana jubata, Caragana korshinskii), which indicated those three genes were under positive selection. A comparative genomic analysis of 15 Galegeae species showed that most conserved non-coding sequence regions and two genic regions (ycf1 and clpP) were highly divergent, which could be used as DNA barcodes for rapid and accurate species identification. Phylogenetic trees constructed based on the ycf1 and clpP genes confirmed the evolutionary relationships among Galegeae species. In addition, among the 15 Galegeae species analyzed, Galega orientalis had a unique 30-bp intron in the ycf1 gene and Tibetia liangshanensis lacked two introns in the clpP gene, which is contrary to existing conclusion that only Glycyrrhiza species in the IR lacking clade (IRLC) lack two introns. In conclusion, for the first time, the complete chloroplast genome of G. orientalis was determined and annotated, which could provide insights into the unsolved evolutionary relationships within the genus Galegeae.

Finding a correct species assignment for aMetschnikowiastrain: insights from the genome sequencing of strain DBT012

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.

Primer design for the amplification of the ammonium transporter genes from the uncultured haptophyte algal species symbiotic with the marine nitrogen-fixing cyanobacterium UCYN-A1.

The multiple symbiotic partnerships between closely related species of the haptophyte algae Braarudosphaera bigelowii and the nitrogen-fixing cyanobacteria Candidatus Atelocyanobacterium thalassa (UCYN-A) contribute importantly to the nitrogen and carbon cycles in vast areas of the ocean. The diversity of the eukaryotic 18S rDNA phylogenetic gene marker has helped to identify some of these symbiotic haptophyte species, yet we still lack a genetic marker to assess its diversity at a finer scale. One of such genes is the ammonium transporter (amt) gene, which encodes the protein that might be involved in the uptake of ammonium from UCYN-A in these symbiotic haptophytes. Here, we designed three specific PCR primer sets targeting the amt gene of the haptophyte species (A1-Host) symbiotic with the open ocean UCYN-A1 sublineage, and tested them in samples collected from open ocean and near-shore environments. Regardless of the primer pair used at Station ALOHA, which is where UCYN-A1 is the pre-dominant UCYN-A sublineage, the most abundant amt amplicon sequence variant (ASV) was taxonomically classified as A1-Host. In addition, two out of the three PCR primer sets revealed the existence of closely-related divergent haptophyte amt ASVs (>95% nucleotide identity). These divergent amt ASVs had higher relative abundances than the haptophyte typically associated with UCYN-A1 in the Bering Sea, or co-occurred with the previously identified A1-Host in the Coral Sea, suggesting the presence of new diversity of closely-related A1-Hosts in polar and temperate waters. Therefore, our study reveals an overlooked diversity of haptophytes species with distinct biogeographic distributions partnering with UCYN-A, and provides new primers that will help to gain new knowledge of the UCYN-A/haptophyte symbiosis.

The Chromosome Counts Database (CCDB).

For decades, plant biologists have been interested in the determination and documentation of chromosome numbers for extant taxa. This central cytological character has been used as an important phylogenetic marker and as an indicator for major genomic events such as polyploidy and dysploidy. Due to their significance and the relative ease by which chromosome numbers can be obtained, chromosome numbers have been extensively recorded across the plant kingdom and documented in a wide variety of resources. This makes the collection process a wearing task, often leading to partial data retrieval. In 2015, the Chromosome Counts Database (CCDB) was assembled, being an online unified community resource. This database compiles dozens of different chromosome counts sources, of which a significant portion had been unavailable before in a digitized, searchable format. The vast amount of data assembled in CCDB has already enabled a large number of analyses to examine the evolution of different plant hierarchies, as well as the application of various follow-up analyses, such as ploidy-level inference using chromEvol. CCDB ( http://ccdb.tau.ac.il/ ) encourages data sharing among the botanical community and is expected to continue expanding as additional chromosome numbers are recorded.

Molecular detection of some Gram-negative bacterial species using folp gene sequences

In this study, the folp gene encoding DHPS was tested as a possible alternative phylogenetic marker for more closely related Gram-negative bacterial species. In this new method, 854 bp were implemented for classification instead of 1435 mostly used in 16S gene detection. Phylogenetic analysis was performed based on DNA sequences obtained from the GenBank including the most important Gram-negative bacterial species. The 16S rRNA-based tree openly portrayed three distinct clusters where cluster 1