Deep‐branching Novel Lineages and High Diversity of Haptophytes in the Skagerrak (Norway) Uncovered by 454 Pyrosequencing

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high‐throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano‐ and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta‐specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep‐branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity.     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

High‐throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long‐read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500‐bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high‐quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny‐aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity‐based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well‐resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.     

dinoref: A curated dinoflagellate (Dinophyceae) reference database for the 18S rRNA gene

Dinoflagellates are a heterogeneous group of protists present in all aquatic ecosystems where they occupy various ecological niches. They play a major role as primary producers, but many species are mixotrophic or heterotrophic. Environmental metabarcoding based on high‐throughput sequencing is increasingly applied to assess diversity and abundance of planktonic organisms, and reference databases are definitely needed to taxonomically assign the huge number of sequences. We provide an updated 18S rRNA reference database of dinoflagellates: dinoref . Sequences were downloaded from genbank and filtered based on stringent quality criteria. All sequences were taxonomically curated, classified taking into account classical morphotaxonomic studies and molecular phylogenies, and linked to a series of metadata. dinoref includes 1,671 sequences representing 149 genera and 422 species. The taxonomic assignation of 468 sequences was revised. The largest number of sequences belongs to Gonyaulacales and Suessiales that include toxic and symbiotic species. dinoref provides an opportunity to test the level of taxonomic resolution of different 18S barcode markers based on a large number of sequences and species. As an example, when only the V4 region is considered, 374 of the 422 species included in dinoref can still be unambiguously identified. Clustering the V4 sequences at 98% similarity, a threshold that is commonly applied in metabarcoding studies, resulted in a considerable underestimation of species diversity.     

Seasonal diversity and dynamics of haptophytes in the Skagerrak,Norway, explored by high‐throughput sequencing

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high‐throughput sequencing. From outer Oslofjorden, S Norway, nano‐ and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta‐specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA‐sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP‐3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in‐depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.     

18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species

The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired‐end (PE; 2 × 150 bp) technology. To critically evaluate the method′s performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual‐based diet analysis methods. The high sensitivity and semi‐quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR‐based results. This molecular approach provides an alternative cost and time effective tool for food‐web analysis.     

Fungal disease and temperature alter skin microbiome structure in an experimental salamander system

Pathogens compete with host microbiomes for space and resources. Their shared environment impacts pathogen–microbiome–host interactions, which can lead to variation in disease outcome. The skin microbiome of red‐backed salamanders (Plethodon cinereus) can reduce infection by the pathogen Batrachochytrium dendrobatidis (Bd) at moderate infection loads, with high species richness and high abundance of competitors as putative mechanisms. However, it is unclear if the skin microbiome can reduce epizootic Bd loads across temperatures. We conducted a laboratory experiment to quantify skin microbiome and host responses (P. cinereus: n = 87) to Bd at mimicked epizootic loads across temperatures (13, 17 and 21°C). We quantified skin microbiomes using 16S rRNA gene metabarcoding and identified operational taxonomic units (OTUs) taxonomically similar to culturable bacteria known to kill Bd (anti‐Bd OTUs). Prior to pathogen exposure, temperature changed the microbiome (OTU richness decreased by 12% and the abundance of anti‐Bd OTUs increased by 18% per degree increase in temperature), but these changes were not predictive of disease outcome. After exposure, Bd changed the microbiome (OTU richness decreased by 0.1% and the abundance of anti‐Bd OTUs increased by 0.2% per 1% increase in Bd load) and caused high host mortality across temperatures (35/45: 78%). Temperature indirectly impacted microbiome change and mortality through its direct effect on pathogen load. We did not find support for the microbiome impacting Bd load or host survival. Our research reveals complex host, pathogen, microbiome and environmental interactions to demonstrate that during epizootic events the microbiome will be unlikely to reduce pathogen invasion, even for putatively Bd‐resistant species.     

Intracellular Diversity of the V4 and V9 Regions of the 18S rRNA in Marine Protists (Radiolarians) Assessed by High-Throughput Sequencing

Metabarcoding is a powerful tool for exploring microbial diversity in the environment, but its accurate interpretation is impeded by diverse technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra-individual polymorphism) that remain poorly understood. To help interpret environmental metabarcoding datasets, we investigated the intracellular diversity of the V4 and V9 regions of the 18S rRNA gene from Acantharia and Nassellaria (radiolarians) using 454 pyrosequencing. Individual cells of radiolarians were isolated, and PCRs were performed with generalist primers to amplify the V4 and V9 regions. Different denoising procedures were employed to filter the pyrosequenced raw amplicons (Acacia, AmpliconNoise, Linkage method). For each of the six isolated cells, an average of 541 V4 and 562 V9 amplicons assigned to radiolarians were obtained, from which one numerically dominant sequence and several minor variants were found. At the 97% identity, a diversity metrics commonly used in environmental surveys, up to 5 distinct OTUs were detected in a single cell. However, most amplicons grouped within a single OTU whereas other OTUs contained very few amplicons. Different analytical methods provided evidence that most minor variants forming different OTUs correspond to PCR and sequencing artifacts. Duplicate PCR and sequencing from the same DNA extract of a single cell had only 9 to 16% of unique amplicons in common, and alignment visualization of V4 and V9 amplicons showed that most minor variants contained substitutions in highly-conserved regions. We conclude that intracellular variability of the 18S rRNA in radiolarians is very limited despite its multi-copy nature and the existence of multiple nuclei in these protists. Our study recommends some technical guidelines to conservatively discard artificial amplicons from metabarcoding datasets, and thus properly assess the diversity and richness of protists in the environment.     

A Comparison of Methods for Clustering 16S rRNA Sequences into OTUs

Recent studies of 16S rRNA sequences through next-generation sequencing have revolutionized our understanding of the microbial community composition and structure. One common approach in using these data to explore the genetic diversity in a microbial community is to cluster the 16S rRNA sequences into Operational Taxonomic Units (OTUs) based on sequence similarities. The inferred OTUs can then be used to estimate species, diversity, composition, and richness. Although a number of methods have been developed and commonly used to cluster the sequences into OTUs, relatively little guidance is available on their relative performance and the choice of key parameters for each method. In this study, we conducted a comprehensive evaluation of ten existing OTU inference methods. We found that the appropriate dissimilarity value for defining distinct OTUs is not only related with a specific method but also related with the sample complexity. For data sets with low complexity, all the algorithms need a higher dissimilarity threshold to define OTUs. Some methods, such as, CROP and SLP, are more robust to the specific choice of the threshold than other methods, especially for shorter reads. For high-complexity data sets, hierarchical cluster methods need a more strict dissimilarity threshold to define OTUs because the commonly used dissimilarity threshold of 3% often leads to an under-estimation of the number of OTUs. In general, hierarchical clustering methods perform better at lower dissimilarity thresholds. Our results show that sequence abundance plays an important role in OTU inference. We conclude that care is needed to choose both a threshold for dissimilarity and abundance for OTU inference.     

Impact of sequence processing and taxonomic classification approaches on eukaryotic community structure from environmental samples with emphasis on diatoms

Next‐generation sequencing is a common method for analysing microbial community diversity and composition. Configuring an appropriate sequence processing strategy within the variety of tools and methods is a nontrivial task and can considerably influence the resulting community characteristics. We analysed the V4 region of 18S rRNA gene sequences of marine samples by 454‐pyrosequencing. Along this process, we generated several data sets with QIIME, mothur, and a custom‐made pipeline based on DNAStar and the phylogenetic tree‐based PhyloAssigner. For all processing strategies, default parameter settings and punctual variations were used. Our results revealed strong differences in total number of operational taxonomic units (OTUs), indicating that sequence preprocessing and clustering had a major impact on protist diversity estimates. However, diversity estimates of the abundant biosphere (abundance of ≥1%) were reproducible for all conducted processing pipeline versions. A qualitative comparison of diatom genera emphasized strong differences between the pipelines in which phylogenetic placement of sequences came closest to light microscopy‐based diatom identification. We conclude that diversity studies using different sequence processing strategies are comparable if the focus is on higher taxonomic levels, and if abundance thresholds are used to filter out OTUs of the rare biosphere.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont‐rich aphid genus

The bacterial communities inhabiting arthropods are generally dominated by a few endosymbionts that play an important role in the ecology of their hosts. Rather than comparing bacterial species richness across samples, ecological studies on arthropod endosymbionts often seek to identify the main bacterial strains associated with each specimen studied. The filtering out of contaminants from the results and the accurate taxonomic assignment of sequences are therefore crucial in arthropod microbiome studies. We aimed here to validate an Illumina 16S rRNA gene sequencing protocol and analytical pipeline for investigating endosymbiotic bacteria associated with aphids. Using replicate DNA samples from 12 species (Aphididae: Lachninae, Cinara) and several controls, we removed individual sequences not meeting a minimum threshold number of reads in each sample and carried out taxonomic assignment for the remaining sequences. With this approach, we show that (i) contaminants accounted for a negligible proportion of the bacteria identified in our samples; (ii) the taxonomic composition of our samples and the relative abundance of reads assigned to a taxon were very similar across PCR and DNA replicates for each aphid sample; in particular, bacterial DNA concentration had no impact on the results. Furthermore, by analysing the distribution of unique sequences across samples rather than aggregating them into operational taxonomic units (OTUs), we gained insight into the specificity of endosymbionts for their hosts. Our results confirm that Serratia symbiotica is often present in Cinara species, in addition to the primary symbiont, Buchnera aphidicola. Furthermore, our findings reveal new symbiotic associations with Erwinia‐ and Sodalis‐related bacteria. We conclude with suggestions for generating and analysing 16S rRNA gene sequences for arthropod‐endosymbiont studies.     

Comparison of morphological,DNA barcoding,and metabarcoding characterizations of freshwater nematode communities

Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single‐specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification.     

Toxic HAB species from the Sea of Okhotsk detected by a metagenetic approach,seasonality and environmental drivers

During recent decades, the distribution of harmful algal bloom (HAB) species has expanded worldwide together with the increase of blooms and toxicity events. In this study, the presence of toxic HAB species in the Sea of Okhotsk was investigated based on metagenetic data collected during 6 years of weekly monitoring. Operational taxonomic units (OTUs) associated with the toxic HAB species were detected based on amplifying 18S V7-V9 and 28S D1 rRNA gene regions. In total, 43 unique OTUs associated with toxic HAB species were revealed, with 26 of those previously not reported from the Sea of Okhotsk. More OTUs belonging to dinoflagellates were detected by 18S, whereas a similar number of OTUs associated with dinoflagellates and diatoms were detected by targeting the 28S region. Species belonging to genera Alexandrium, Karenia and Karlodinium were mainly associated with OTUs under Dinophyceae, whereas Bacillariophyceae was represented by the species belonging to genus Pseudo-nitzschia. From the detected OTUs, 22 showed a clear seasonal pattern with the majority of those appearing during summer-autumn. For Alexandrium pacificum, Aureococcus anophagefferens, and Pseudo-nitzschia pungens, the seasonal pattern was detected based on both rRNA regions. Additionally, 14 OTUs were detected during all seasons and two OTUs appeared sporadically. OTUs associated with the toxic species had low relative read abundances, which together with other factors such as similar and variable morphology as well as usage of fixatives, may explain why those species have previously not been detected by light microscopy. Environmental parameters, especially water temperature, significantly (<0.05) influenced the variability in OTU relative abundances and displayed significant (<0.05) correlations with the unique OTUs. The results of this study demonstrate the usefulness of the metagenetic approach for phytoplankton monitoring, which is especially relevant for detecting toxic HAB species.     

Structural alignment of 18S and 28S rDNA sequences provides insights into phylogeny of Phytophaga (Coleoptera: Curculionoidea and Chrysomeloidea)

We performed a comparative study of partial rDNA sequences from a variety of Coleoptera taxa to construct an annotated alignment based on secondary structure information, which in turn, provides improved rRNA structure models useful for phylogenetic reconstruction. Subsequent phylogenetic analysis was performed to test monophyly and interfamilial relationships of the megadiverse plant feeding beetle group known as ‘Phytophaga’ (Curculionoidea and Chrysomeloidea), as well as to discover their closest relatives among the Cucujiformia. Parsimony and Bayesian analyses were performed based on the structural alignment of segments of 18S rRNA (variable regions V4‐V5, V7‐V9) and 28S rRNA (expansion segment D2). A total of 104 terminal taxa of Coleoptera were included: 96 species of Cucujiformia beetles, representing the families and most ‘subfamilies’ of weevils and chrysomeloids (Phytophaga), as well as several families of Cleroidea, Tenebrionoidea and Cucujoidea, and eight outgroups from three other polyphagan series: Scarabaeiformia, Elateriformia and Bostrichiformia. The results from the different methods of analysis agree — recovering the monophyly of the ‘Phytophaga’, including Curculionoidea and Chrysomeloidea as sister groups. The curculionoid and chrysomeloid phylogeny recovered from the aligned 18S and 28S rDNA segments, which is independent of morphological data, is in agreement with recent hypotheses or concepts based on morphological evidence, particularly with respect to familial relationships. Our results provide clues about the evolutionary origin of the phytophagan beetles within the megaclade Cucujiformia, suggesting that the sister group of ‘Curculionoidea + Chrysomeloidea’ is a clade of the ‘Cucujoidea’, represented in this study by species in Boganiidae, Erotylidae, Nitidulidae, Cucujidae and Silvanidae. The Coccinellidae and Endomychidae are not grouped with the latter, and the remaining terminal taxa are nested in Tenebrionoidea and Cleroidea. We propose that the combination of structurally aligned ribosomal RNA gene regions 18S (V4‐V5, V7‐V9) and 28S (D2) are useful in testing monophyly and resolving relationships among beetle superfamilies and families.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.