VTAM: A robust pipeline for validating metabarcoding data using controls.

To obtain accurate estimates for biodiversity and ecological studies, metabarcoding studies should be carefully designed to minimize both false positive (FP) and false negative (FN) occurrences. Internal controls (mock samples and negative controls), replicates, and overlapping markers allow controlling metabarcoding errors but current metabarcoding software packages do not explicitly integrate these additional experimental data to optimize filtering. We have developed the metabarcoding analysis software VTAM, which uses explicitly these elements of the experimental design to find optimal parameter settings that minimize FP and FN occurrences. VTAM showed similar sensitivity, but a higher precision compared to two other pipelines using three datasets and two different markers (COI, 16S). The stringent filtering procedure implemented in VTAM aims to produce robust metabarcoding data to obtain accurate ecological estimates and represents an important step towards a non-arbitrary and standardized validation of metabarcoding data for conducting ecological studies. VTAM is implemented in Python and available from: https://github.com/aitgon/vtam. The VTAM benchmark code is available from: https://github.com/aitgon/vtam_benchmark.

DNA metabarcoding suggests adaptive seasonal variation of individual trophic traits in a critically endangered fish.

Dietary studies are critical for understanding foraging strategies and have important applications in conservation and habitat management. We applied a robust metabarcoding protocol to characterize the diet of the critically endangered freshwater fish Zingel asper (the Rhone streber). We conducted modelling and simulation analyses to identify and characterize some of the drivers of individual trophic trait variation in this species. We found that population density and ontogeny had minor effects on the trophic niche of Z. asper. Instead, our results suggest that the majority of trophic niche variation was driven by seasonal variation in ecological opportunity. The total trophic niche width of Z. asper seasonally expanded to include a broader range of prey. Furthermore, null model simulations revealed that the increase of between-individual variation in autumn indicates that Z. asper become more opportunistic relative to summer and spring, rather than being associated with a seasonal specialization of individuals. Overall, our results suggest an adaptive variation of individual trophic traits in Z. asper: the species mainly consumes a few ephemeropteran taxa (Baetis fuscatus and Ecdyonurus) but seems to be capable of adapting its foraging strategy to maintain its body condition. This study illustrates how metabarcoding data obtained from faeces can be validated and combined with individual-based modelling and simulation approaches to explore inter- and intrapopulational individual trophic traits variation and to test hypotheses in the conventional analytic framework of trophic ecology.

Connectivity and population structure of albacore tuna across southeast Atlantic and southwest Indian Oceans inferred from multidisciplinary methodology.

Albacore tuna (Thunnus alalunga) is an important target of tuna fisheries in the Atlantic and Indian Oceans. The commercial catch of albacore is the highest globally among all temperate tuna species, contributing around 6% in weight to global tuna catches over the last decade. The accurate assessment and management of this heavily exploited resource requires a robust understanding of the species' biology and of the pattern of connectivity among oceanic regions, yet Indian Ocean albacore population dynamics remain poorly understood and its level of connectivity with the Atlantic Ocean population is uncertain. We analysed morphometrics and genetics of albacore (n = 1,874) in the southwest Indian (SWIO) and southeast Atlantic (SEAO) Oceans to investigate the connectivity and population structure. Furthermore, we examined the species' dispersal potential by modelling particle drift through major oceanographic features. Males appear larger than females, except in South African waters, yet the length-weight relationship only showed significant male-female difference in one region (east of Madagascar and Reunion waters). The present study produced a genetic differentiation between the southeast Atlantic and southwest Indian Oceans, supporting their demographic independence. The particle drift models suggested dispersal potential of early life stages from SWIO to SEAO and adult or sub-adult migration from SEAO to SWIO.

One-locus-several-primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies.

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets' complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the "one-locus-several-primer-sets" (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey-predator interactions.

A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies.

The main objective of this work was to develop and validate a robust and reliable "from-benchtop-to-desktop" metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to faecal DNA samples of an invertebrate-eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross-contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.

When Anthropogenic River Disturbance Decreases Hybridisation between Non-Native and Endemic Cyprinids and Drives an Ecomorphological Displacement towards Juvenile State in Both Species.

Understanding the impact of non-native species on native species is a major challenge in molecular ecology, particularly for genetically compatible fish species. Invasions are generally difficult to study because their effects may be confused with those of environmental or human disturbances. Colonized ecosystems are differently impacted by human activities, resulting in diverse responses and interactions between native and non-native species. We studied the dynamics between two Cyprinids species (invasive Chondrostoma nasus and endemic Parachondrostoma toxostoma) and their hybrids in 16 populations (from allopatric to sympatric situations and from little to highly fragmented areas) corresponding to 2,256 specimens. Each specimen was assigned to a particular species or to a hybrid pool using molecular identification (cytochrome b and 41 microsatellites). We carried out an ecomorphological analysis based on size, age, body shape, and diet (gut vacuity and molecular fecal contents). Our results contradicted our initial assumptions on the pattern of invasion and the rate of introgression. There was no sign of underperformance for the endemic species in areas where hybridisation occurred. In the unfragmented zone, the introduced species was found mostly downstream, with body shapes similar to those in allopatric populations while both species were found to be more insectivorous than the reference populations. However, high level of hybridisation was detected, suggesting interactions between the two species during spawning and/or the existence of hybrid swarm. In the disturbed zone, introgression was less frequent and slender body shape was associated with diatomivorous behaviour, smaller size (juvenile characteristics) and greater gut vacuity. Results suggested that habitat degradation induced similar ecomorphological trait changes in the two species and their hybrids (i.e. a transition towards a pedomorphic state) where the invasive species is more affected than the native species. Therefore, this study reveals a diversity of relationships between two genetically compatible species and emphasizes constraints on the invasion process in disturbed areas.

Non-homogeneous combination of two porous genomes induces complex body shape trajectories in cyprinid hybrids.

INTRODUCTION: Hybridization is a common phenomenon in fish and is considered to be a major source of diversification. Deciphering the remoulding of genomic regions and phenotypes in zones where hybrid specimens occur is of particular interest to elucidate the emergence of evolutionary novelties. This approach is particularly challenging because the first step of hybridization seems to be the most important stage in the emergence of hybrid lineages. However, the signal can be significantly altered after only a few generations. RESULTS: We studied 41 microsatellites and partial cytochrome b gene sequences in 970 specimens belonging to two fish species (Chondrostoma nasus and Parachondrostoma toxostoma) in allopatric/parapatric zones, hybrids between them in a natural sympatric zone: the Ardèche basin. We showed that the genomic architecture in hybrids presented pattern heterogeneity of selection for the different loci. Indeed, the upstream part of the river (Rosières and Labeaume) presented an overdominant fitness of heterozygotes (12.20%) corresponding to a genomic compatibility, and underselection was observed for 4.88%-7.32% of the loci tested indicating a genomic incompatibility. Moreover the upstream station (Rosières) presented a positive selection of invasive C. nasus homozygotes (17.07% to 21.95%) indicating that hybridization may increase the fitness of admixed individuals.We showed that hybrid morphology (body shape based on 21 landmarks) correlated with genomic dilution indicating a species fingerprint. However, we demonstrated that the hybrid morphology was not a linear modification between the two parental species but a trade-off between several correlated traits. CONCLUSIONS: Hybrid specimens present a mosaic of genomic combination, showing regions with genomic compatibility and others with genomic incompatibility between the two species. Positive selection (invasive advantage ranging from 9.76% to 21.95% of the loci) was evidenced in the upstream part of the Ardèche indicating that environmental selection makes a substantial contribution. Although the presence of a dam is known to impose heterogeneous hybrid zones between these two species, we demonstrated in this study that a natural environment can also generate a hybrid zone with a large number (and diversity) of hybrids. The combination of the two genomes in the hybrids results in complex ontogenetic trajectories (with different morphological traits evolving at different rates) that correspond to novel developmental pathways.

Phylogenetic analysis of Thecosomata Blainville, 1824 (holoplanktonic opisthobranchia) using morphological and molecular data.

Thecosomata is a marine zooplankton group, which played an important role in the carbonate cycle in oceans due to their shell composition. So far, there is important discrepancy between the previous morphological-based taxonomies, and subsequently the evolutionary history of Thecosomata. In this study, the remarkable planktonic sampling of TARA Oceans expedition associated with a set of various other missions allowed us to assess the phylogenetic relationships of Thecosomata using morphological and molecular data (28 S and COI genes). The two gene trees showed incongruities (e.g. Hyalocylis, Cavolinia), and high congruence between morphological and 28S trees (e.g. monophyly of Euthecosomata). The monophyly of straight shell species led us to reviving the Orthoconcha, and the split of Limacinidae led us to the revival of Embolus inflata replacing Limacina inflata. The results also jeopardized the Euthecosomata families that are based on plesiomorphic character state as in the case for Creseidae which was not a monophyletic group. Divergence times were also estimated, and suggested that the evolutionary history of Thecosomata was characterized by four major diversifying events. By bringing the knowledge of palaeontology, we propose a new evolutionary scenario for which macro-evolution implying morphological innovations were rhythmed by climatic changes and associated species turn-over that spread from the Eocene to Miocene, and were shaped principally by predation and shell buoyancy.

Improving PCR detection of prey in molecular diet studies: importance of group-specific primer set selection and extraction protocol performances.

While the morphological identification of prey remains in predators' faeces is the most commonly used method to study trophic interactions, many studies indicate that this method does not detect all consumed prey. Polymerase chain reaction-based methods are increasingly used to detect prey DNA in the predator food bolus and have proven efficient, delivering highly accurate results. When studying complex diet samples, the extraction of total DNA is a critical step, as polymerase chain reaction (PCR) inhibitors may be co-extracted. Another critical step involves a careful selection of suitable group-specific primer sets that should only amplify DNA from the targeted prey taxon. In this study, the food boluses of five Rattus rattus and seven Rattus exulans were analysed using both morphological and molecular methods. We tested a panel of 31 PCR primer pairs targeting bird, invertebrate and plant sequences; four of them were selected to be used as group-specific primer pairs in PCR protocols. The performances of four DNA extraction protocols (QIAamp(®) DNA stool mini kit, DNeasy(®) mericon food kit and two of cetyltrimethylammonium bromide-based methods) were compared using four variables: DNA concentration, A(260) /A(280) absorbance ratio, food compartment analysed (stomach or faecal contents) and total number of prey-specific PCR amplification per sample. Our results clearly indicate that the A(260) /A(280) absorbance ratio, which varies between extraction protocols, is positively correlated to the number of PCR amplifications of each prey taxon. We recommend using the DNeasy(®) mericon food kit (QIAGEN), which yielded results very similar to those achieved with the morphological approach.

A PCR-based method for diet analysis in freshwater organisms using 18S rDNA barcoding on faeces.

The development of DNA barcoding from faeces represents a promising method for animal diet analysis. However, current studies mainly rely on prior knowledge of prey diversity for a specific predator rather than on a range of its potential prey species. Considering that the feeding behaviour of teleosts may evolve with their environment, it could prove difficult to establish an exhaustive listing of their prey. In this article, we extend the DNA barcoding approach to diet analysis to allow the inclusion of a wide taxonomic range of potential prey items. Thirty-four ecological clade-specific primer sets were designed to cover a large proportion of prey species found in European river ecosystems. Selected primers sets were tested on isolated animal, algal or plant tissues and thereafter on fish faeces using nested PCR to increase DNA detection sensitivity. The PCR products were sequenced and analysed to confirm the identity of the taxa and to validate the method. The methodology developed here was applied to a diet analysis of three freshwater cyprinid species that are assumed to have similar feeding behaviour [Chondrostoma toxostoma toxostoma (Vallot 1837), Chondrostoma nasus nasus (Linnaeus, 1758) and Barbus barbus, (Linneaus 1758)]. These three species were sampled in four different hydrographic basins. Principal Component Analysis based on prey proportions identified distinct perilithon grazer and benthophagous behaviours. Furthermore, our results were consistent with the available literature on feeding behaviour in these fish. The simplicity of the PCR-based method and its potential generalization to other freshwater organisms may open new perspectives in food web ecology.

Trade-off between morphological convergence and opportunistic diet behavior in fish hybrid zone.

BACKGROUND: The invasive Chondrostoma nasus nasus has colonized part of the distribution area of the protected endemic species Chondrostoma toxostoma toxostoma. This hybrid zone is a complex system where multiple effects such as inter-species competition, bi-directional introgression, strong environmental pressure and so on are combined. Why do sympatric Chondrostoma fish present a unidirectional change in body shape? Is this the result of inter-species interactions and/or a response to environmental effects or the result of trade-offs? Studies focusing on the understanding of a trade-off between multiple parameters are still rare. Although this has previously been done for Cichlid species flock and for Darwin finches, where mouth or beak morphology were coupled to diet and genetic identification, no similar studies have been done for a fish hybrid zone in a river. We tested the correlation between morphology (body and mouth morphology), diet (stable carbon and nitrogen isotopes) and genomic combinations in different allopatric and sympatric populations for a global data set of 1330 specimens. To separate the species interaction effect from the environmental effect in sympatry, we distinguished two data sets: the first one was obtained from a highly regulated part of the river and the second was obtained from specimens coming from the less regulated part. RESULTS: The distribution of the hybrid combinations was different in the two part of the sympatric zone, whereas all the specimens presented similar overall changes in body shape and in mouth morphology. Sympatric specimens were also characterized by a larger diet behavior variance than reference populations, characteristic of an opportunistic diet. No correlation was established between the body shape (or mouth deformation) and the stable isotope signature. CONCLUSION: The Durance River is an untamed Mediterranean river despite the presence of numerous dams that split the river from upstream to downstream. The sympatric effect on morphology and the large diet behavior range can be explained by a tendency toward an opportunistic behavior of the sympatric specimens. Indeed, the similar response of the two species and their hybrids implied an adaptation that could be defined as an alternative trade-off that underline the importance of epigenetics mechanisms for potential success in a novel environment.