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Most stream bioassessment and biodiversity surveys are currently based on morphological identification of communities. However, DNA metabarcoding is emerging as a fast and cost-effective alternative for species identification. We compared both methods in a survey of benthic macroinvertebrate communities across 36 stream sites in northern Finland. We identified 291 taxa of which 62% were identified only by DNA metabarcoding. DNA metabarcoding produced extensive species level inventories for groups (Oligochaeta, Chironomidae, Simuliidae, Limoniidae and Limnephilidae), for which morphological identification was not feasible due to the high level of expertise needed. Metabarcoding also provided more insightful taxonomic information on the occurrence of three red-listed vulnerable or data deficient species, the discovery of two likely cryptic and potentially new species to Finland and species information of insect genera at an early larval stage that could not be separated morphologically. However, it systematically failed to reliably detect the occurrence of gastropods that were easily identified morphologically. The impact of mining on community structure could only be shown using DNA metabarcoding data which suggests that the finer taxonomic detail can improve detection of subtle impacts. Both methods generally exhibited similar strength of community-environment relationships, but DNA metabarcoding showed better performance with presence/absence data than with relative DNA sequence abundances. Our results suggest that DNA metabarcoding holds a promise for future anthropogenic impact assessments, although, in our case, the performance did not improve much from the morphological species identification. The key advantage of DNA metabarcoding lies in efficient biodiversity surveys, taxonomical studies and applications in conservation biology.

The Malaise trap has gained popularity for assessing diverse terrestrial arthropod communities because it collects large samples with modest effort. A number of factors that influence collection efficiency, placement being one of them. For instance, when designing larger biotic surveys using arrays of Malaise traps we need to know the optimal distance between individual traps that maximises observable species richness and community composition. We examined the influence of spacing between Malaise traps by metabarcoding samples from two field experiments at a site in Waterloo, Ontario, Canada. For one experiment, we used two trap pairs deployed at weekly increasing distance (3m increments from 3 to 27 m). The second experiment involved a total of 10 traps set up in a row at 3m distance intervals for three consecutive weeks.Results show that community similarity of samples decreases over distance between traps. The amount of species shared between trap pairs shows drops considerably at about 15m trap-to-trap distance. This change can be observed across all major taxonomic groups and for two different habitat types (grassland and forest). Large numbers of OTUs found only once within samples cause rather large dissimilarity between distance pairs even at close proximity. This could be caused by a large number of transient species from adjacent habitat which arrive at the trap through passive transport, as well as capture of rare taxa, which end up in different traps by chance.

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5′ terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5′ region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

Summary Ericaceae (the heather family) is a large and diverse group of plants that forms elaborate symbiotic relationships with mycorrhizal fungi, and includes several nonphotosynthetic lineages. Using an extensive sample of fully mycoheterotrophic (MH) species, we explored inter- and intraspecific variation as well as selective constraints acting on the plastomes of these unusual plants. The plastomes of seven MH genera were analysed in a phylogenetic context with two geographically disparate individuals sequenced for Allotropa, Monotropa, and Pityopus. The plastomes of nonphotosynthetic Ericaceae are highly reduced in size (c. 33–41 kbp) and content, having lost all photosynthesis-related genes, and are reduced to encoding housekeeping genes as well as a protease subunit (clpP)-like and acetyl-CoA carboxylase subunit D (accD)-like open reading frames. Despite an increase in the rate of their nucleotide substitutions, the remaining protein-coding genes are typically under purifying selection in full MHs. We also identified ribosomal proteins under relaxed or neutral selection. These plastomes also exhibit striking structural rearrangements. Intraspecific variation within MH Ericaceae ranges from a few differences (Allotropa) to extensive population divergences (Monotropa, Hypopitys), which indicates that cryptic speciation may be occurring in several lineages. The pattern of gene loss within fully MH Ericaceae plastomes suggests an advanced state of degradation.

DNA metabarcoding is an attractive approach for monitoring biodiversity. However, it is subject to biases that often impede detection of all species in a sample. In particular, the proportion of sequences recovered from each species depends on its biomass, mitome copy number, and primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 BINs, a species proxy. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body partitions (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicon generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, other variables did. As expected, the best recovery was obtained from the Single Leg treatment, but the Bulk Abdomen produced a more uniform read abundance than the Bulk Leg or Composite Leg samples. Primer choice also influenced species recovery. Our results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to obtain reproducible results and increase its cost-effectiveness.

The Lennoaceae, a small monophyletic plant family of root parasites endemic to the Americas, are one of the last remaining independently evolved lineages of parasitic angiosperms lacking a published plastome. In this study, we present the assembled and annotated plastomes of two species spanning the crown node of Lennoaceae, Lennoa madreporoides and Pholisma arenarium, as well as their close autotrophic relative from the sister family Ehretiaceae, Tiquilia plicata. We find that the plastomes of L. madreporoides and P. arenarium are similar in size and gene content, and substantially reduced compared to T. plicata, consistent with trends seen in other holoparasitic lineages. In particular, most plastid genes involved in photosynthesis function have been lost, whereas housekeeping genes (ribosomal protein-coding genes, rRNAs, and tRNAs) are retained. One notable exception is the persistence of a rbcL open reading frame in P. arenarium but not L. madreporoides suggesting a nonphotosynthetic function for this gene. Of the retained coding genes, dN/dS ratios indicate that some remain under purifying selection, whereas others show relaxed selection. Overall, this study supports the mounting evidence for convergent plastome evolution in flowering plants following the shift to heterotrophy.

Premise of the Study The detection of environmental DNA (eDNA) using high-throughput sequencing has rapidly emerged as a method to detect organisms from environmental samples. However, eDNA studies of aquatic biomes have focused on surveillance of animal species with less emphasis on plants. Pondweeds are important bioindicators of freshwater ecosystems, although their diversity is underestimated due to difficulties in morphological identification and monitoring. Methods A protocol was developed to detect pondweeds in water samples using atpB-rbcL and ITS2 markers. The water samples were collected from the Grand River within the rare Charitable Research Reserve, Ontario (RARE). Short fragments were amplified using primers targeting pondweeds, sequenced on an Ion Torrent Personal Genome Machine, and assigned to the taxonomy using a local DNA reference library and GenBank. Results We detected two species earlier documented at the experimental site during ecological surveys (Potamogeton crispus and Stuckenia pectinata) and three species new to the RARE checklist (P. foliosus, S. filiformis, and Zannichellia palustris). Discussion Our targeted approach to track the species composition of pondweeds in freshwater ecosystems revealed underestimation of their diversity. This result suggests that eDNA is an effective tool for monitoring plant diversity in aquatic habitats.

The genus Cuscuta (Convolvulaceae, the morning glory family) is one of the most intensely studied lineages of parasitic plants. Whole plastome sequencing of four Cuscuta species has demonstrated changes to both plastid gene content and structure. The presence of photosynthetic genes under purifying selection indicates that Cuscuta is cryptically photosynthetic. However, the tempo and mode of plastid genome evolution across the diversity of this group (~200 species) remain largely unknown. A comparative investigation of plastid genome content, grounded within a phylogenetic framework, was conducted using a slot-blot Southern hybridization approach. Cuscuta was extensively sampled (~56% of species), including groups previously suggested to possess more altered plastomes compared with other members of this genus. A total of 56 probes derived from all categories of protein-coding genes, typically found within the plastomes of flowering plants, were used. The results indicate that two clades within subgenus Grammica (clades ‘O’ and ‘K’) exhibit substantially more plastid gene loss relative to other members of Cuscuta. All surveyed members of the ‘O’ clade show extensive losses of plastid genes from every category of genes typically found in the plastome, including otherwise highly conserved small and large ribosomal subunits. The extent of plastid gene losses within this clade is similar in magnitude to that observed previously in some non-asterid holoparasites, in which the very presence of a plastome has been questioned. The ‘K’ clade also exhibits considerable loss of plastid genes. Unlike in the ‘O’ clade, in which all species seem to be affected, the losses in clade ‘K’ progress phylogenetically, following a pattern consistent with the Evolutionary Transition Series hypothesis. This clade presents an ideal opportunity to study the reduction of the plastome of parasites ‘in action’. The widespread plastid gene loss in these two clades is hypothesized to be a consequence of the complete loss of photosynthesis. Additionally, taxa that would be the best candidates for entire plastome sequencing are identified in order to investigate further the loss of photosynthesis and reduction of the plastome within Cuscuta.

Background DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. Methods We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. Results All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven–by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. Conclusion Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an interdisciplinary approach taking into account the processes involved in production of herbal supplements, as well as biocomplexity of plant-plant and plant-fungal biological interactions.

Premise of the study: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Methods: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Results: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Discussion: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic ...

Unlike parasitic plants, which are linked to their hosts directly through haustoria, mycoheterotrophic (MHT) plants derive all or part of their water and nutrients from autothrophs via fungal mycorrhizal intermediaries. Ericaceae, the heather family, are a large and diverse group of plants known to form elaborate symbiotic relationships with mycorrhizal fungi. Using PHYA sequence data, we first investigated relationships among mycoheterotrophic Ericaceae and their close autotrophic relatives. Phylogenetic results suggest a minimum of two independent origins of MHT within this family. Additionally, a comparative investigation of plastid genomes (plastomes) grounded within this phylogenetic framework was conducted using a slot-blot Southern hybridization approach. This survey encompassed numerous lineages of Ericaceae with different life histories and trophic levels, including multiple representatives from mixotrophic Pyroleae and fully heterotrophic Monotropeae and Pterosporeae. Fifty-four probes derived from all categories of protein coding genes typically found within the plastomes of flowering plants were used. Our results indicate that the holo-mycoheterotrophic Ericaceae exhibit extensive loss of genes relating to photosynthetic function and expression of the plastome but retain genes with possible functions outside photosynthesis. Mixotrophic taxa tend to retain most genes relating to photosynthetic functions but are varied regarding the plastid ndh gene content. This investigation extends previous inferences that the loss of the NDH complex occurs prior to becoming holo-heterotrophic and it shows that the pattern of gene losses among mycoheterotrophic Ericaceae is similar to that of haustorial parasites. Additionally, we identify the most desirable candidate species for entire plastome sequencing.

The exact phylogenetic position of Gnetales, a small, highly modified group of gymnosperms with an accelerated rate of molecular evolution, is one of the most challenging issues for seed plant systematics. Recent results from entire plastid genome (ptDNA) sequencing revealed the absence of the entire suite of plastid ndh genes in several species of Gnetales and the pine family (Pinaceae) potentially highlighting a major structural feature linking these two groups—concerted loss of all plastid genes for the NADH dehydrogenase complex. However, the precise extent of ndh gene loss in gymnosperms has not been surveyed. Using a slot-blot hybridization method, we probed all 11 ndh genes in 162 species from 70 of 85 gymnosperm genera. We find that all ndh genes are absent across Gnetales and Pinaceae, but not in any other group of gymnosperms. This feature represents either a major synapomorphy for a clade consisting of these two lineages or, less likely, a convergent loss. Our survey substantially extends previous inferences based on more limited sampling and, if the former evolutionary interpretation is correct, it provides additional support for the contentious “gnepine” hypothesis, which places Gnetales as sister to Pinaceae.