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Accurate species delimitation is critical for biodiversity conservation. Integrative taxonomy has been advocated for a long time, yet tools allowing true integration of genetic and phenotypic data have been developed quite recently and applied to few models, especially in plants. In this study, we investigated species boundaries within a group of twelve Pseudophrys taxa from France by analyzing genetic, morphometric and chemical (i.e., floral scents) data in a Bayesian framework using the program integrated Bayesian Phylogenetics and Phylogeography (iBPP). We found that these twelve taxa were merged into four species when only genetic data were used, while most formally described species were recognized as such when only phenotypic (either morphometric or chemical) data were used. The result of the iBPP analysis performed on both genetic and phenotypic data supports the proposal to merge Ophrys bilunulata and O. marmorata on the one hand, and O. funerea and O. zonata on the other hand. Our results show that phenotypic data are particularly informative in the section Pseudophrys and that their integration in a model-based method significantly improves the accuracy of species delimitation. We are convinced that the integrative taxonomic approach proposed in this study holds great promise to conduct taxonomic revisions in other orchid groups.

Environmental DNA is mainly not only used at the interspecific level, to quantify species diversity in ecosystems, but can also be used to quantify intraspecific genetic variability, thus avoiding the need to sample individual tissue. However, errors in the amplification and sequencing of eDNA samples can blur this intraspecific signal and strongly over-estimate genetic diversity. Existing bioinformatics pipelines therefore need to be tested to evaluate whether reliable levels of intraspecific genetic variability can be derived from eDNA samples. Here, we compare the ability of twelve metabarcoding pipelines to detect intraspecific genetic variability combining five programs. All pipelines have common pre-processing steps, a processing data step using programs among obiclean; DADA2; SWARM; and LULU. An additional chimera removal step is also investigated based on two programs (VSEARCH or DADA2). The case study was the natural intraspecific variation within Mullus surmuletus in experimental settings. We developed specific primers for this species, located on the mitochondrial D-loop fragment (barcode MS-DL06). Thirty-nine individuals were collected from the Mediterranean Sea, placed into four aquariums, and their DNA was sequenced on this marker to build an intraspecific reference database. After filtering the aquarium water, DNA was extracted, amplified, and sequenced using the primer pair developed. We then quantified the number of true haplotypes returned by each pipeline and its capacity to eliminate most of the erroneous sequences. We show that the program DADA2 with a two-parent chimeric sequence removal step is the best tool to estimate intraspecific diversity from eDNA. Furthermore, our approach was also able to detect true M. surmuletus haplotypes in two eDNA samples collected in the Mediterranean Sea. We conclude that the combination of an appropriate intrapopulation barcode and a denoising pipeline like DADA2 with a chimeric sequence removal step is promising to make population-level inference using environmental DNA possible., Environmental DNA, 4 (3), ISSN:2637-4943

Supplementary Figures, Tables and Methods for the paper "Environmental DNA metabarcoding reveals and unpacks a biodiversity conservation paradox in Mediterranean marine reserves."

International audience; A series of phylogeographic studies in the Maghreb identified a repeated pattern of deep genetic divergence between an eastern (Tunisia) and western (Morocco) lineage for several taxa but lack of sampling in Algeria made it difficult to know if the range limits between the eastern and western lineages were shared among taxa or not. To address this question, we designed a comparative phylogeographic study using 8 reptile and 3 amphibian species with wide distribution in the Maghreb as models. We selected species where previous studies had identified an East-West phylogeographic divide and collected sampled in Algeria to 1) examine whether the simple East-West divergence pattern still holds after filling the sampling gap in Algeria or if more complex diversity patterns emerge; 2) if the E-W pattern still holds, test whether the limits between the E and W clades are shared between species, suggesting that common historical process caused the E-W divergences; 3) if E-W limits are shared between species, use information on the age of the divergence to identify possible geological or climatic events that could have triggered these E-W differentiations. We found that the E-W pattern was generally maintained after additional sampling in Algeria and identified two common disjunction areas, one around the Algeria-Morocco border, the other one in Kabylia (central Algeria), suggesting that common historical mechanisms caused the E-W divergences in the Maghreb. Our estimates for the times to most common recent ancestors to the E and W clades span a wide range between the Messinian salinity crisis and the Plio-Pleistocene limit (except for one older split), suggesting different origins for the initial divergences and subsequent preservation of the E and W lineages in common climatic refugia in the west and the east of the Maghreb.

Phylogenetic trees of cyt b , ND2 and cyt b + ND2. Bayesian phylogenetic trees of ospreys constructed from: a) the cytochrome b (209 sequences, 1103 nucleotides) showing the four supported clades as well as the geographic origin of the samples; b) the ND2 (39 sequences, 1078 nucleotides) and (c) the concatenated genes (cyt b + ND2; 38 sequences, 2037 nucleotides). In all the trees, four species belonging to the Accipitridae family were used as outgroups. Branch lengths are proportional to the number of substitutions per site and \\ means that branches leading to outgroups have been reduced. For supported clades, bayesian posterior probabilities and ML bootstrap are indicated at nodes, respectively. (DOC 182 kb)

Taxon sampling. Detailed list of the 209 Osprey samples indicating: sample lab code, subspecies according to morphology classification, country of collection, locality, sample type (tp = toepad; wb = wet blood; db = dry blood; ft = feather; fs = fasta sequence), codes, gene bank number accession for cyt b and ND2) and name of the institution and/or collector (with affiliation). (DOC 370 kb)