A DNA mini‐barcode for land plants

Small portions of the barcode region – mini‐barcodes – may be used in place of full‐length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini‐barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini‐barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30 472)]. PCR amplification for all mini‐barcodes, as estimated by validated electronic simulation, was successful for 90.2–99.8% of species. Overall Sanger sequence quality for mini‐barcodes was very low – the best mini‐barcode tested produced sequences of adequate quality (B₂₀ ≥ 0.5) for 74.5% of samples. The majority of mini‐barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini‐barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini‐barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini‐barcode D (F52/R193).     

Estimating belowground plant abundance with DNA metabarcoding

Most work on plant community ecology has been performed above ground, neglecting the processes that occur in the soil. DNA metabarcoding, in which multiple species are computationally identified in bulk samples, can help to overcome the logistical limitations involved in sampling plant communities belowground. However, a major limitation of this methodology is the quantification of species’ abundances based on the percentage of sequences assigned to each taxon. Using root tissues of five dominant species in a semi‐arid Mediterranean shrubland (Bupleurum fruticescens, Helianthemum cinereum, Linum suffruticosum, Stipa pennata and Thymus vulgaris), we built pairwise mixtures of relative abundance (20%, 50% and 80% biomass), and implemented two methods (linear model fits and correction indices) to improve estimates of root biomass. We validated both methods with multispecies mixtures that simulate field‐collected samples. For all species, we found a positive and highly significant relationship between the percentage of sequences and biomass in the mixtures (R² = .44–.66), but the equations for each species (slope and intercept) differed among them, and two species were consistently over‐ and under‐estimated. The correction indices greatly improved the estimates of biomass percentage for all five species in the multispecies mixtures, and reduced the overall error from 17% to 6%. Our results show that, through the use of post‐sequencing quantification methods on mock communities, DNA metabarcoding can be effectively used to determine not only species’ presence but also their relative abundance in field samples of root mixtures. Importantly, knowledge of these aspects will allow us to study key, yet poorly understood, belowground processes.     

Prey diversity of Polistes rothneyi koreanus in different landscapes using DNA barcoding

For the present study, we investigated the prey of Polistes rothneyi koreanus, which is the most common social wasp in South Korea, and the relationship between prey diversity and vegetation cover around their nests. Prey was collected over two 6‐hr sampling from seven nests between July and mid‐August 2015 in Daegu, Gyeongsan and Gunwi in South Korea. To analyse the prey spectrum, we identified species using DNA barcodes; to analyse vegetation cover, we used the normalized difference vegetation index in a 200 m radius around the nests. A total of 338 prey samples were collected, and eight orders, 24 families, and 65 species were identified, demonstrating a much broader prey spectrum than those previously recorded for Polistes spp. Lepidoptera were the most prevalent, with 158 samples and 47 species. Nest 7, located in a rural area, had the highest numbers of samples and species per worker (5.2 and 1.9, respectively). Lepidoptera accounted for over half of the prey spectrum for all nests, and the families Noctuidae and Geometridae accounted for 60% of all Lepidoptera. Tenodera sinensis (Mantodea) and Gabala argentata (Noctuidae) were the most ubiquitous species, collected at five locations. Six species and some genera of prey are designated as pests in South Korea, indicating that P. r. koreanus also has a beneficial role in pest control. A higher vegetation cover was associated with significantly higher prey species diversity (R² = .4597, p < .1) and abundance (R² = .5986, p < .05), indicating that vegetation cover is an important factor for maintaining colonies. Therefore, the recent increase in green spaces in South Korean cities is probably a major contributor to the increased density of social wasps.     

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia‐recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH‐psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH‐psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes.     

DNA barcoding herbaceous and woody plant species at a subalpine forest dynamics plot in Southwest China

Although DNA barcoding has been widely used to identify plant species composition in temperate and tropical ecosystems, relatively few studies have used DNA barcodes to document both herbaceous and woody components of forest plot. A total of 201 species (72 woody species and 129 herbaceous species) representing 135 genera distributed across 64 families of seed plants were collected in a 25 ha CForBio subalpine forest dynamics plot. In total, 491 specimens were screened for three DNA regions of the chloroplast genome (rbcL, matK, and trnH‐psbA) as well as the internal transcribed spacers (ITS) of nuclear ribosomal DNA. We quantified species resolution for each barcode separately or in combination using a ML tree‐based method. Amplification and sequencing success were highest for rbcL, followed by trnH‐psbA, which performed better than ITS and matK. The rbcL + ITS barcode had slightly higher species resolution rates (88.60%) compared with rbcL + matK (86.60%) and rbcL + trnH‐psbA (86.01%). The addition of trnH‐psbA or ITS to the rbcL + matK barcode only marginally increased species resolution rates, although in combination the four barcodes had the highest discriminatory power (90.21%). The situations where DNA barcodes did not discriminate among species were typically associated with higher numbers of co‐occurring con‐generic species. In addition, herbaceous species were much better resolved than woody species. Our study represents one of the first applications of DNA barcodes in a subalpine forest dynamics plot and contributes to our understanding of patterns of genetic divergence among woody and herbaceous plant species.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

DNA barcoding commercially important fish species of Turkey

DNA barcoding was used in the identification of 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. A total of 1765 DNA barcodes using a 654‐bp‐long fragment of the mitochondrial cytochrome c oxidase subunit I gene were generated for 89 commercially important freshwater and marine fish species found in Turkish ichthyofauna. These species belong to 70 genera, 40 families and 19 orders from class Actinopterygii, and all were associated with a distinct DNA barcode. Nine and 12 of the COI barcode clusters represent the first species records submitted to the BOLD and GenBank databases, respectively. All COI barcodes (except sequences of first species records) were matched with reference sequences of expected species, according to morphological identification. Average nucleotide frequencies of the data set were calculated as T = 29.7%, C = 28.2%, A = 23.6% and G = 18.6%. Average pairwise genetic distance among individuals were estimated as 0.32%, 9.62%, 17,90% and 22.40% for conspecific, congeneric, confamilial and within order, respectively. Kimura 2‐parameter genetic distance values were found to increase with taxonomic level. For most of the species analysed in our data set, there is a barcoding gap, and an overlap in the barcoding gap exists for only two genera. Neighbour‐joining trees were drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. Results of this study supported DNA barcoding as an efficient molecular tool for a better monitoring, conservation and management of fisheries.     

Egg identification of three economical important fish species using DNA barcoding in comparison to a morphological determination

Accurate identification of fish eggs to species level is a challenging task as many species have similar egg sizes and morphology. The results of egg determination of three economically important fish species are presented by using DNA barcoding in comparison to the classical morphological determination. About 500 fish eggs from the Celtic Sea were collected, morphologically identified and used for DNA analysis. In total, DNA barcodes were successfully obtained from 98% of the investigated eggs, including 167 DNA barcodes from 169 morphologically identified fish eggs of Merluccius merluccius (98.8%), 257 of 262 Scomber scombrus (98.1%), and 47 from 50 Trachurus trachurus (94%). Overall, species identification with DNA barcodes showed a congruence of 96.2% to identification by morphology, whereas 3.8% (n = 18) of the analyzed eggs were morphologically assigned to the wrong species. The highest number of incorrect identified eggs was for S. scombrus (n = 15). Our study highlights the usefulness of DNA barcoding for valid fish egg identification but also indicates the robustness of the classical morphology‐based approach.     

DNA metabarcoding reveals that 200‐μm‐size‐fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes

Microeukaryotic plankton (0.2–200 μm) are critical components of aquatic ecosystems and key players in global ecological processes. High‐throughput sequencing is currently revolutionizing their study on an unprecedented scale. However, it is currently unclear whether we can accurately, effectively and quantitatively depict the microeukaryotic plankton communities using traditional size‐fractionated filtering combined with molecular methods. To address this, we analysed the eukaryotic plankton communities both with, and without, prefiltering with a 200 μm pore‐size sieve –by using SSU rDNA‐based high‐throughput sequencing on 16 samples with three replicates in each sample from two subtropical reservoirs sampled from January to October in 2013. We found that ~25% reads were classified as metazoan in both size groups. The species richness, alpha and beta diversity of plankton community and relative abundance of reads in 99.2% eukaryotic OTUs showed no significant changes after prefiltering with a 200 μm pore‐size sieve. We further found that both >0.2 μm and 0.2–200 μm eukaryotic plankton communities, especially the abundant plankton subcommunities, exhibited very similar, and synchronous, spatiotemporal patterns and processes associated with almost identical environmental drivers. The lack of an effect on community structure from prefiltering suggests that environmental DNA from larger metazoa is introduced into the smaller size class. Therefore, size‐fractionated filtering with 200 μm is insufficient to discriminate between the eukaryotic plankton size groups in metabarcoding approaches. Our results also highlight the importance of sequencing depth, and strict quality filtering of reads, when designing studies to characterize microeukaryotic plankton communities.     

Genome‐skimming provides accurate quantification for pollen mixtures

Studies on foraging partitioning in pollinators can provide critical information to the understanding of food‐web niche and pollination functions, thus aiding conservation. Metabarcoding based on PCR amplification and high‐throughput sequencing has seen increasing applications in characterizing pollen loads carried by pollinators. However, amplification bias across taxa could lead to unpredictable artefacts in estimation of pollen compositions. We examined the efficacy of a genome‐skimming method based on direct shotgun sequencing in quantifying mixed pollen, using mock samples (five and 14 mocks of flower and bee pollen, respectively). The results demonstrated a high level of repeatability and accuracy in identifying pollen from mixtures of varied species ratios. All pollen species were detected in all mocks, and pollen frequencies estimated from the number of sequence reads of each species were significantly correlated with pollen count proportions (linear model, R² = 86.7%, p = 2.2e?16). For >97% of the mixed taxa, pollen proportion could be quantified by sequencing to the correct order of magnitude, even for species which constituted only 0.2% of the total pollen. In addition, DNA extracted from pollen grains equivalent to those collected from a single honeybee corbicula was sufficient for genome‐skimming. We conclude that genome‐skimming is a feasible approach to identifying and quantifying mixed pollen samples. By providing reliable and sensitive taxon identification and relative abundance, this method is expected to improve our understanding in studies that involve plant–pollinator interactions, such as pollen preference in corbiculate bees, pollen diet analyses and identification of landscape pollen resource use from beehives.     

Complete mitochondrial genome of the black‐tailed hornet,Vespa ducalis (Hymenoptera: Vespidae): Genomic comparisons in Vespoidea

We sequenced the complete mitochondrial genome (mitogenome) of the black‐tailed hornet, Vespa ducalis (Hymenoptera: Vespidae). The genome was 15,779‐bp long and contained typical sets of genes [13 protein‐coding genes (PCGs), 22 tRNAs, and 2 rRNAs]. The V. ducalis A + T‐rich region was 166‐bp long and was the shortest of all sequenced Vespoidea genomes, including Vespa. The genome was highly biased toward A/T nucleotides—80.1 % in the whole genome, 77.8 % in PCGs, 83.4–85.6 % in RNAs, and 92.8 % in the A + T‐rich region. These values are well within the typical range for genes and regions of Vespoidea mitogenomes. Start and stop codons in several Vespa species—including V. ducalis—were diversified, despite these species belonging to the same genus. In comparison with the ancestral mitogenomes, Vespa mitogenomes—including that of V. ducalis—showed substantial gene rearrangement; however, we detected no gene rearrangement among Vespa species. We conducted phylogenetic reconstruction based on concatenated sequences of 13 PCGs and two rRNAs (12,755 bp ) in available species of Vespoidea—21 species in six subfamilies in two families (Vespidae and Formicidae). The Bayesian inference and maximum likelihood (ML) methods revealed that each family formed strong monophyletic groups [Bayesian posterior probability (BPP) = 1; ML, 100 %]. Moreover, V. ducalis and V. mandarinia formed a strong sister group (BPP = 1; ML, 94 %).     

Emperipolesis mediated by CD8+ T cells correlates with biliary epithelia cell injury in primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell‐in‐cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty‐six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early‐stage PBC (stages I and II, n = 39) and late‐stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin‐eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3⁺ and CD8⁺ T cells correlated with the advancement of emperipolesis (R² = 0.318, P < .001; R² = 0.060, P < .05). The cell numbers of TUNEL‐positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R² = 0.236, P < .001; R² = 0.267, P < .001). We conclude that emperipolesis mediated by CD8⁺ T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts.     

DNA barcoding of Gaultheria L. in China (Ericaceae: Vaccinioideae)

Abstract Four DNA barcoding loci, chloroplast loci rbcL, matK, trnH‐psbA, and nuclear locus internal transcribed spacer (ITS), were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P‐distance, Wilcoxon signed rank test, and tree‐based analyses. This study included 186 individuals from 89 populations representing 30 species. For all individuals, single locus markers showed high levels of sequencing universality but were ineffective for species resolvability. Polymerase chain reaction amplification and sequencing were successful for all four loci. Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH‐psbA. A combination of matK and ITS was the most efficient DNA barcode among all studied regions, however, they do not represent an appropriate candidate barcode for Chinese Gaultheria, by which only 11 out of 30 species can be separated. Loci rbcL, matK, and trnH‐psbA, which were recently proposed as universal plant barcodes, have a very poor capacity for species separation for Chinese Gaultheria. DNA barcodes may be reliable tools to identify the evolutionary units of this group, so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Temporal variation in coat colour (genotypes) supports major changes in the Nordic cattle population after Iron Age

Variation in coat colour genotypes of archaeological cattle samples from Finland was studied by sequencing 69 base pairs of the extension locus (melanocortin 1‐receptor, MC1R) targeting both a transition and a deletion defining the three main alleles, such as dominant black (E^D), wild type (E⁺) and recessive red (e). The 69‐bp MC1R sequence was successfully analysed from 23 ancient (1000–1800 AD) samples. All three main alleles and genotype combinations were detected with allele frequencies of 0.26, 0.17 and 0.57 for E^D, E⁺ and e respectively. Recessive red and dominant black alleles were detected in both sexes. According to the best of our knowledge, this is the first ancient DNA study defining all three main MC1R alleles. Observed MC1R alleles are in agreement with calculated phenotype frequencies from historical sources. The division of ancient Finnish cattle population into modern Finnish breeds with settled colours was dated to the 20th century. From the existing genotyped populations in Europe (43 breeds, n = 2360), the closest match to ancient MC1R genotype frequencies was with the Norwegian native multicoloured breeds. In combined published genotype data of ancient (n = 147) and genotypes and phenotypes of modern Nordic cattle (n = 738), MC1R allele frequencies showed temporal changes similar to neutral mitochondrial DNA and Y‐chromosomal haplotypes analysed earlier. All three markers indicate major change in genotypes in Nordic cattle from the Late Iron Age to the Medieval period followed by slower change through the historical periods until the present.     

Phylogenomics and multigene phylogenies decipher two new cryptic marine algae from California,Gelidium gabrielsonii and G. kathyanniae (Gelidiales,Rhodophyta)

Molecular surveys are leading to the discovery of many new cryptic species of marine algae. This is particularly true for red algal intertidal species, which exhibit a high degree of morphological convergence. DNA sequencing of recent collections of Gelidium along the coast of California, USA, identified two morphologically similar entities that differed in DNA sequence from existing species. To characterize the two new species of Gelidium and to determine their evolutionary relationships to other known taxa, phylogenomic, multigene analyses, and morphological observations were performed. Three complete mitogenomes and five plastid genomes were deciphered, including those from the new species candidates and the type materials of two closely related congeners. The mitogenomes contained 45 genes and had similar lengths (24,963–24,964 bp). The plastid genomes contained 232 genes and were roughly similar in size (175,499–177,099 bp). The organellar genomes showed a high level of gene synteny. The two Gelidium species are diminutive, turf‐forming, and superficially resemble several long established species from the Pacific Ocean. The phylogenomic analysis, multigene phylogeny, and morphological evidence confirms the recognition and naming of two new species, describe herein as G. gabrielsonii and G. kathyanniae. On the basis of the monophyly of G. coulteri, G. gabrielsonii, G. galapagense, and G. kathyanniae, we suggest that this lineage likely evolved in California. Organellar genomes provide a powerful tool for discovering cryptic intertidal species and they continue to improve our understanding of the evolutionary biology of red algae and the systematics of the Gelidiales.     

DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya–Hengduan Mountains

The Himalaya–Hengduan Mountains encompass two global biodiversity hotspots with high levels of biodiversity and endemism. This area is one of the diversification centres of the genus Rhododendron, which is recognized as one of the most taxonomically challenging plant taxa due to recent adaptive radiations and rampant hybridization. In this study, four DNA barcodes were evaluated on 531 samples representing 173 species of seven sections of four subgenera in Rhododendron, with a high sampling density from the Himalaya–Hengduan Mountains employing three analytical methods. The varied approaches (nj , pwg and blast ) had different species identification powers with blast performing best. With the pwg analysis, the discrimination rates for single barcodes varied from 12.21% to 25.19% with ITS < rbcL < matK < psbA‐trnH. Combinations of ITS + psbA‐trnH + matK and the four barcodes showed the highest discrimination ability (both 41.98%) among all possible combinations. As a single barcode, psbA‐trnH performed best with a relatively high performance (25.19%). Overall, the three‐marker combination of ITS + psbA‐trnH + matK was found to be the best DNA barcode for identifying Rhododendron species. The relatively low discriminative efficiency of DNA barcoding in this genus (~42%) may possibly be attributable to too low sequence divergences as a result of a long generation time of Rhododendron and complex speciation patterns involving recent radiations and hybridizations. Taking the morphology, distribution range and habitat of the species into account, DNA barcoding provided additional information for species identification and delivered a preliminary assessment of biodiversity for the large genus Rhododendron in the biodiversity hotspots of the Himalaya–Hengduan Mountains.     

Feasibility of monitoring advanced melanoma patients using cell‐free DNA from plasma

To determine the feasibility of liquid biopsy for monitoring of patients with advanced melanoma, cell‐free DNA was extracted from plasma for 25 Stage III/IV patients, most (84.0%) having received previous therapy. DNA concentrations ranged from 0.6 to 390.0 ng/ml (median = 7.8 ng/ml) and were positively correlated with tumor burden as measured by imaging (Spearman rho = 0.5435, p = .0363). Using ultra‐deep sequencing for a 61‐gene panel, one or more mutations were detected in 12 of 25 samples (48.0%), and this proportion did not vary significantly for patients on or off therapy at the time of blood draw (52.9% and 37.5% respectively; p = .673). Sixteen mutations were detected in eight different genes, with the most frequent mutations detected in BRAF, NRAS, and KIT. Allele fractions ranged from 1.1% to 63.2% (median = 29.1%). Among patients with tissue next‐generation sequencing, nine of 11 plasma mutations were also detected in matched tissue, for a concordance of 81.8%.     

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.     

Monitoring cellular C:N ratio in phytoplankton by means of FTIR‐spectroscopy

Statistical growth rate modelling can be applied in a variety of ecological and biotechnological applications. Such models are frequently based on Monod or Droop equations and, especially for the latter, require reliable determination of model input parameters such as C:N quotas. Besides growth rate modelling, a C:N quota quantification can be useful for monitoring and interpretation of physiological acclimation to abiotic and biotic disturbances (e.g., nutrient limitations). However, as high throughput C:N quota determination is difficult to perform, alternatives need to be established. Fourier‐transformed infrared (FTIR) spectroscopy is used to analyze a variety of biochemical, chemical, and physiological parameters in phytoplankton. Hence, a quantification of the C:N quota should also be feasible. Therefore, using FTIR spectroscopy, six phytoplankton species from among different phylogenetic groups have been analyzed to determine the effect of nutrient limitation on C:N quota patterns. The typical species‐specific response to increasing nitrogen limitation was an increase in the C:N quota. Irrespective of this species specificity, we were able to develop a reliable multi‐species C:N quota prediction model based on FTIR spectroscopy using the partial least square regression (PLSR) algorithm. Our data demonstrate that the PLSR approach is more robust in C:N quota quantification (R² = 0.93) than linear correlation of C:N quota versus growth rate (R² ranges from 0.74 to 0.86) or biochemical information based on FTIR spectra (R² ranges from 0.82 to 0.89). This accurate prediction of C:N values may support high throughput measurements in a broad range of future approaches.