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Accurate species identification of the mosquitoes in the genus Anopheles is of crucial importance to implement malaria control measures and monitor their effectiveness. We use a previously developed amplicon panel (ANOSPP) that retrieves sequence data from multiple short nuclear loci for any species in the genus. Species assignment is based on comparison of samples to a reference index using k-mer distance. Here, we provide a protocol to generate version controlled updates of the reference index and present its latest release, NNv2, which contains 91 species, compared to 56 species represented in its predecessor NNv1. With the updated reference index, we are able to assign samples to species level that previously could not be assigned. We discuss what happens if a species is not represented in the reference index and how this can be addressed in a future update. To demonstrate the increased power of NNv2, we showcase the assignments of 1789 wild-caught mosquitoes from Madagascar and demonstrate that we can detect within species population structure from the amplicon sequencing data. [ABSTRACT FROM AUTHOR]

Specimen identification at the species level is a critical challenge for understanding community structure and conserving biodiversity. The use of mitochondrial DNA barcodes, in addition to morphology, has proven to be a useful tool for earthworm identification, but it has also raised difficulties. Thus, approaches to delineating molecular operational taxonomic units (MOTUs) from cytochrome c oxidase subunit 1 (COI) or 16S rDNA sequence data often reveal more mitochondrial lineages than morphospecies, raising the question of whether these MOTUs should be used as taxonomic entities in community structure studies. Here, we used a newly acquired dataset of 576 COI barcodes of earthworms from Metropolitan France that were clustered in 36 MOTUS, corresponding to 21 morphospecies. We also incorporated data gathered from the literature to investigate this question. In order to match our MOTUs with already-described mitochondrial lineages, we downloaded reference sequences from the GenBank and BOLD platforms. In light of the difficulties encountered in recovering these sequences, we recommend that any new mitochondrial lineage described in a study be named consistently with previous works. Next, we analyzed the biological, ecological, and molecular data available in the literature on the different mitochondrial lineages that matched our MOTUs in order to determine if there was a consensus for species delimitation. Although the study specimens mainly belong to the Lumbricidae, which is one of the most studied families of earthworms, the data are often missing to determine if the MOTUs correspond to different species. Pending revision of the taxonomy, MOTUs for which mitochondrial divergence has been confirmed by morphological differences should be considered distinct taxonomic entities in community structure studies. In the absence of morphological differences and pending more data, we propose to distinguish for these analyses, within sexually reproducing morphospecies, the MOTUs for which mitochondrial divergence has been confirmed by multilocus nuclear data, while in the case of reproduction by parthenogenesis, ecological differences between MOTUs are necessary to consider them as different taxonomic entities. [ABSTRACT FROM AUTHOR]

Accurate species identification of the mosquitoes in the genus Anopheles is of crucial importance to implement malaria control measures and monitor their effectiveness. We use a previously developed amplicon panel (ANOSPP) that retrieves sequence data from multiple short nuclear loci for any species in the genus. Species assignment is based on comparison of samples to a reference index using k-mer distance. Here, we provide a protocol to generate version controlled updates of the reference index and present its latest release, NNv2, which contains 91 species, compared to 56 species represented in its predecessor NNv1. With the updated reference index, we are able to assign samples to species level that previously could not be assigned. We discuss what happens if a species is not represented in the reference index and how this can be addressed in a future update. To demonstrate the increased power of NNv2, we showcase the assignments of 1789 wild-caught mosquitoes from Madagascar and demonstrate that we can detect within species population structure from the amplicon sequencing data.

Specimen identification at the species level is a critical challenge for understanding community structure and conserving biodiversity. The use of mitochondrial DNA barcodes, in addition to morphology, has proven to be a useful tool for earthworm identification, but it has also raised difficulties. Thus, approaches to delineating molecular operational taxonomic units (MOTUs) from cytochrome c oxidase subunit 1 (COI) or 16S rDNA sequence data often reveal more mitochondrial lineages than morphospecies, raising the question of whether these MOTUs should be used as taxonomic entities in community structure studies. Here, we used a newly acquired dataset of 576 COI barcodes of earthworms from Metropolitan France that were clustered in 36 MOTUS, corresponding to 21 morphospecies. We also incorporated data gathered from the literature to investigate this question. In order to match our MOTUs with already-described mitochondrial lineages, we downloaded reference sequences from the GenBank and BOLD platforms. In light of the difficulties encountered in recovering these sequences, we recommend that any new mitochondrial lineage described in a study be named consistently with previous works. Next, we analyzed the biological, ecological, and molecular data available in the literature on the different mitochondrial lineages that matched our MOTUs in order to determine if there was a consensus for species delimitation. Although the study specimens mainly belong to the Lumbricidae, which is one of the most studied families of earthworms, the data are often missing to determine if the MOTUs correspond to different species. Pending revision of the taxonomy, MOTUs for which mitochondrial divergence has been confirmed by morphological differences should be considered distinct taxonomic entities in community structure studies. In the absence of morphological differences and pending more data, we propose to distinguish for these analyses, within sexually reproducing morphospecies, the MOTUs for which mitochondrial divergence has been confirmed by multilocus nuclear data, while in the case of reproduction by parthenogenesis, ecological differences between MOTUs are necessary to consider them as different taxonomic entities.

The ANOSPP amplicon panel is a genus-wide targeted sequencing panel to facilitate large-scale monitoring of Anopheles species diversity. Combining information from the 62 nuclear amplicons present in the ANOSPP panel allows for a more senstive and specific species assignment than single gene (e.g. COI) barcoding, which is desirable in the light of permeable species boundaries. Here, we present NNoVAE, a method using Nearest Neighbours (NN) and Variational Autoencoders (VAE), which we apply to k-mers resulting from the ANOSPP amplicon sequences in order to hierarchically assign species identity. The NN step assigns a sample to a species-group by comparing the k-mers arising from each haplotype’s amplicon sequence to a reference database. The VAE step is required to distinguish between closely related species, and also has sufficient resolution to reveal population structure within species. In tests on independent samples with over 80% amplicon coverage, NNoVAE correctly classifies to species level 98% of samples within the An. gambiae complex and 89% of samples outside the complex. We apply NNoVAE to over two thousand new samples from Burkina Faso and Gabon, identifying unexpected species in Gabon. NNoVAE presents an approach that may be of value to other targeted sequencing panels, and is a method that will be used to survey Anopheles species diversity and Plasmodium transmission patterns through space and time on a large scale, with plans to analyse half a million mosquitoes in the next five years.

Confocal scanning laser microscopy (CSLM) is becoming a powerful tool for palynological studies. CSLM allows palynomorph image sectioning, internal and surface structures visualization, and 3D reconstruction at a higher resolution than standard light microscopy without extra processing. CSLM images are suitable for several image analysis techniques that could help improve the accuracy and reproducibility of taxa identification. Here, using the palm subtribe Mauritiinae (Arecaceae: Calamoideae: Lepidocaryeae) as a model group, we identify modern and fossil pollen grains using CSLM images coupled with ImageJ/Fiji 1.54f plugins and machine learning statistical analyses. Modern taxa pollen grains including Lepidocaryum tenue Mart. , Mauritia flexuosa L.f., Mauritiella armata (Mart.) Burret and Mauritiella aculeata (Kunth) Burret were obtained from Smithsonian Tropical Research Institute (STRI) pollen collection or herbarium exsiccates. Fossil pollen of Grimsdalea magnaclavata Germeraad et al. 1968 , and Mauritiidites franciscoi (van der Hammen) van der Hammen & Garcia de Mutis 1966, both from Miocene, and Mauritia pollen type from Holocene were obtained from STRI collection. We measured nine shape and exine quantitative parameters, and one qualitative parameter (pollen aperture). Pollen volume was the most important variable (28.270 mean decrease accuracy), followed by pollen aperture (15.003), Skewness (13.466), and spine density (10.246). The machine learning analysis, which included CART and Random Forests, correctly identified both fossil and extant grains. CSLM and the quantitative analysis of morphological traits are a new frontier in palynological studies. • We developed a framework for fossil and modern pollen grain identification. • We used confocal laser scanning microscopy images coupled with open-source image processor software, and machine learning analysis. • Pollen's shape and surface structure showed higher accuracy in machine learning classification than light microscopy morphological traits. • Our framework opens new perspective for automate and digital pollen identification in any plant group. • Our analysis pipeline will become powerful tools to unravel the true potential of palynology. [ABSTRACT FROM AUTHOR]

Identification of the species is a crucial step in many ecological studies. Sometimes, this could become a challenge, because of animal elusive behavior, low density population or for sympatric species leaving similar signs that are impossible to discriminate based only on their morphology. Here, we set up non-invasive molecular method to discern between the Italian hare (Lepus corsicanus) and the European hare (Lepus europaeus) using High Resolution Melting assay on fecal DNA, for the first time on these species. The Italian hare is endemic of the Central-Southern Italy and Sicily and it is classified vulnerable by the International Union for Conservation of Nature. Our procedure could be a useful tool to help conservation and management strategies, mainly in areas where the Italian hare and the European hare live in sympatry. The 9.5% of the peninsular range distribution of the European hare is took up by the range of the Italian hare. Our workflow allows sure species discrimination, rapidly and inexpensively (in one day at least 36 samples could be processed at costs of about 259 euros, including both DNA extractions and HRM run), also when large numbers of samples have to be processed. Moreover, our method could be widely applicable to other Lepus and/or mammalian species with similar concerns, by small adjustments to the protocol and its further validation, focusing on primes and corresponding HRM annealing temperature. [ABSTRACT FROM AUTHOR]

Many faunal assemblages across southwest Asia contain the remains of multiple wild equid species, which may reflect individual prehistoric human populations' use of different hunting and/or landscape exploitation strategies. Accurate equid species assignments are therefore important. This paper tests the extent to which zooarchaeologists agree on equid species assignments made using commonly used zooarchaeological dental identification criteria. Seven zooarchaeologists individually use published criteria to assign species to equid teeth from Neolithic Çatalhöyük in central Anatolia, then use Fleiss' kappa to measure our reliability of agreement. We assess our degrees of agreement for species assignments made using scanned images versus actual specimens and for mandibular teeth versus maxillary teeth. Having failed to achieve significant agreement, we conclude that zooarchaeologists should be cautious about species assignments made using these methods. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

• Key message : We show that joint multivariate analyses of leaf morphological characters and molecular-genetic markers improve the taxonomic assignment in hybridizing European white oaks. However, model-based approaches using genetic data alone represent straightforward alternatives to laborious, detailed morphological assessments. • Context : In European white oaks, species delimitation is debated because of large overlap of morphological characteristics likely due to hybridization. • Aims : We tested whether joint multivariate analyses of leaf morphology and molecular markers improve the identification of three oak species ( Quercus petraea, Quercus pubescens, Quercus robur) compared to approaches using morphological or genetic variables only. • Methods : We assessed 13 leaf morphological characters and applied eight nuclear microsatellite markers in almost 1400 trees of 71 oak populations across Switzerland. We performed two multivariate approaches with three variable sets (morphology, genetics, combined) and assessed their performance in separating the taxa. We also compared the taxon assignment to a model-based clustering approach ( Structure) based on genetic data alone. • Results : A joint use of morphological and genetic variables led to an improved taxon assignment. Whereas Q. robur could clearly be separated from the two other taxa, there was a certain overlap between Q. petraea and Q. pubescens. The Structure clustering led to the same taxon assignment in 85 % of the individuals. • Conclusion : It is important to consider both morphological and genetic properties in morphologically similar and hybridizing species. However, it might be more efficient to concentrate only on genetic markers than on time-consuming morphological assessments. [ABSTRACT FROM AUTHOR]

Changes in marine species abundance can impact ecosystems' stability and sustainability of fisheries. In the eastern Atlantic Ocean, harbour seals (Phoca vitulina) are top predator occupying highest trophic level in coastal marine food webs. Although, the overall European population decline, harbour seals have increased dramatically since the 1990's at the southern limit of their European distribution along the French coast of the English Channel. However, little is known about the feeding habits of the expanding peripheral populations limiting the assessment of the role and the impact of this predator in these coastal ecosystems. Here, we investigated the sex-specific diet of harbour seals during summer in the Bay of Somme, the largest colony in the English Channel. We collected 91 faecal samples from haul-out sites mainly used by harbour seal but also by few grey seals. Molecular methods have been used to discard grey seal samples and differentiate gender. Collectively, the 86 faecal samples of harbour seals contained the remains of 3327 prey belonging to at least 13 fish species, and represented ∼109 kg of consumed fish (of which 85% were soles and plaices). Most of the fish consumed were juvenile and small flatfish (Buglossidium luteum, Microchirus variegatus, Solea vulgaris, Pegusa lascaris, Pleuronectes platessa and Platichthys flesus). Hard-part identification further showed a similar diet between the sexes in terms of primary prey consumed, but a greater diversity of preys in the male diet. The dependence of harbour seals on flatfish has not been reported elsewhere and has significant implications for the sustainability of the important flatfish nursery in the Bay of Somme. Consumption estimation and ecosystem modelling are now required to provide robust assessment of the effect of harbour seal predation on population dynamics of the flatfish nursery, on ecosystem of the Bay of Biscay at-large, and finally on interactions with fisheries. [ABSTRACT FROM AUTHOR]

The freshwater amphipod Gammarus fossarum Koch, in Panzer, 1836 is a locally abundant keystone species mainly occuring in European headwaters but also in larger rivers. Genetic studies in the past 25 years have revealed three cryptic species within nominal G. fossarum (types A, B and C). Assignments of specimens to these types were based on allozyme and 16 S markers. Today, a fragment of the cytochrome c oxidase subunit 1 ( CO1) is primarily used as a genetic marker for species assignments (‘DNA Barcoding’), yet not a single CO1 sequence of G. fossarum is available in the Barcode of Life Database. We analysed new CO1 and 16 S data for German, Hungarian and Croatian G. fossarum specimens and compared them with 16 S and CO1 sequences of G. fossarum from GenBank. Thereby, we close the gap between traditional allozyme- and 16 S-based species assignments and modern CO1 barcoding. Studying genetic variation in 55 specimens from 29 populations, we identified between 11 and 23 novel and genetically distinct clades using distance- and tree-based methods. Our results suggest that G. fossarum comprises several additional, yet unrecognised, species in particular from the Balkan region. Therefore, a taxonomic revision and biogeographic reconsideration of the G. fossarum complex is urgently needed. [ABSTRACT FROM AUTHOR]

Success of species assignment using DNA barcodes has been shown to vary among plant lineages because of a wide range of different factors. In this study, we confirm the theoretical prediction that gene flow influences species assignment with simulations and a literature survey. We show that the genome experiencing the highest gene flow is, in the majority of the cases, the best suited for species delimitation. Our results clearly suggest that, for most angiosperm groups, plastid markers will not be the most appropriate for use as DNA barcodes. We therefore advocate shifting the focus from plastid to nuclear markers to achieve an overall higher success using DNA barcodes. [ABSTRACT FROM AUTHOR]

DNA barcoding is the assignment of individuals to species using standardized mitochondrial sequences. Nuclear data are sometimes added to the mitochondrial data to increase power. A barcoding method for analysing mitochondrial and nuclear data is developed. It is a Bayesian method based on the coalescent model. Then this method is assessed using simulated and real data. It is found that adding nuclear data can reduce the number of ambiguous assignments. Finally, the robustness of coalescent-based barcoding to departures from model assumptions is studied using simulations. This method is found to be robust to past population size variations, to within-species population structures, and to designs that poorly sample populations within species. is available online at . [ABSTRACT FROM AUTHOR]

Sturgeon disappeared from the Rhône River in the mid 70's without certitude about which species it was and about the existence of a sympatry between European sturgeon, Acipenser sturio, and Adriatic sturgeon, A. naccarii, in this watershed. In order to reach a reliable specific determination of this extinct sturgeon population, archaeozoological remains of the Jardin d'Hiver in Arles city, on the Rhône river banks, were genetically analysed, following strict criteria of authentication for the ancient DNA work. The rich collection of Arles sturgeon bone remains stems from human activities between the 6th and the 2nd Century BC. Sequences of 86 bp of the cytochrome b gene were obtained on four bones, from different anatomical parts of the fish and from different archaeological layers. All gave A. sturio diagnostic sequences. This preliminary analysis is an essential first step in the project of sturgeon reintroduction in the Rhône River. Thus, further analyses on a larger sample are necessary to comfort this result and to solve the question of sympatry with A. naccarii. [ABSTRACT FROM AUTHOR]