Occurrence Of Cryptic Species Research Articles

The burst of satellite DNA in Leptidea wood white butterflies and their putative role in karyotype evolution.

Satellite DNAs (satDNAs) are abundant components of eukaryotic genomes, playing pivotal roles in chromosomal organization, genome stability and evolution. Here, we combined cytogenetic and genomic methods to characterize the satDNAs in the genomes of Leptidea butterflies. Leptidea is characterized by the presence of a high heterochromatin content, large genomes and extensive chromosomal reshuffling as well as the occurrence of cryptic species. We show that, in contrast to other Lepidoptera, satDNAs constitute a considerable proportion of Leptidea genomes, ranging between 4.11%-11.05%. This amplification of satDNAs, together with the hyperactivity of transposable elements, contributes to the substantial genome expansion in Leptidea. Using chromosomal mapping, we show that, particularly LepSat01-100 and LepSat03-167 satDNAs, are preferentially localized in heterochromatin exhibiting variable distribution that could influence in the highly diverse karyotypes within the genus. The satDNAs also exhibit W-chromosome accumulation, suggesting their involvement in sex chromosome evolution. Our results provide insights into the dynamics of satDNAs in Lepidoptera genomes and highlight their role in genome expansion and chromosomal organization, which could influence the speciation process. The high proportion of repetitive DNAs in the genomes of Leptidea underscores the complex evolutionary dynamics revealing the interplay between repetitive DNAs and genomic architecture in the genus.

Molecular identification of the ubiquitous green algae Ulva reveals high biodiversity, crypticity, and invasive species in the Atlantic-Baltic Sea region

Correct species identification is fundamental for assessment and understanding of biodiversity. Erroneous species identification may impede conservation management and may delay detection of invasive species. The ubiquitous green algal genus Ulva is known for its wide environmental tolerance, plastic morphology, occurrence of cryptic species and ambiguous species concepts that hinder clear identification. We used molecular monitoring to assess species diversity and distribution of Ulva along the full Atlantic-Baltic Sea salinity gradient (> 10,000 km). Ulva specimens were collected from Denmark, Finland, Germany, Norway, and Sweden. DNA barcoding analysis of the tufA gene revealed 20 genetic entities in total, of which 11 could be identified to species level (U. californica, U. flexuosa, U. torta, U. linza, U. prolifera, U. fenestrata, U. australis, U. intestinalis, U. compressa, U. gigantea, U. lacinulata). Nine entities (Ulva sp. 1–9; [Ulva capillata]) yielded novel sequence reads that belonged to either unidentified species, species complexes, or singletons. At least 3 of the discovered species (U. australis, U. californica, U. gigantea) are considered non-native and potentially invasive. Furthermore, considerable differences between the observed and the historically estimated species distributions were found. The highest diversity was recorded in the Atlantic and Skagerrak region whereas only two entities of taxonomically accepted species where found north-east the Blekinge coast. Our study shows that the species diversity of Ulva in the study area is diverging from previous reports, and that molecular methods are imperative for species identification in this morphologically plastic genus. Furthermore, the presence of non-native species indicates a necessity for further fine-scale monitoring in specific areas to e.g. mitigate formation of green tides.

DNA Barcoding of Fish Species Diversity in Guizhou, China

Guizhou is an important ecological barrier in the upper reaches of the Yangtze River and the Pearl River basins with abundant fish species. However, fish from these regions are threatened by anthropogenic activities, including overfishing and habitat destruction. Here, we assessed the fish diversity including more than half of the species from the region using DNA barcoding (partial sequence of cytochrome c oxidase subunit I (COI) gene). We obtained 800 mitochondrial COI barcode sequences from 82 genera, 18 families and 8 orders of fishes. The average Kimura two-parameter (K2P) distances within species and genera were 0.35% and 5.44%, respectively. The average interspecific distance was 15.54 times higher than the mean intraspecific distance. Moreover, DNA barcodes revealed 175 operational taxonomic units (OTUs) based on consensus demarcation schemes. Barcoding gaps were detected in 94.81% of morphospecies. Three fish species (Schistura fasciolata, Vanmanenia pingchowensis, and Misgurnus dabryanus) have considerable intraspecific variability, and each was divided into multiple molecular operational taxonomic units (MOTUs) using molecular definition methods (Automatic Barcode Gap Discovery, Refined Single Linkage, General Mixed Yule Coalescent, and Poisson Tree Processes), possibly indicating the occurrence of cryptic species. Altogether, our study reveals the complex diversity of fish species in Guizhou Province, serving as a reference for the conservation and monitoring of fish species in this region.

Geographic Range Expansion and Taxonomic Notes of the Shortfin Neoscopelid Neoscopelus cf. microchir (Myctophiformes: Neoscopelidae) in the North-Eastern Atlantic

One specimen of the shortfin neoscopelid Neoscopelus microchir Matsubara, 1943, has been recorded for the first time on the Porcupine Bank, southwestern Ireland, providing a new northern limit of distribution for the eastern Atlantic. Morphometric and meristic parameters confirm the taxonomic identification. However, DNA barcoding shows deficiencies in current taxonomy and the potential occurrence of cryptic species. On this basis, the specimen is cautiously reported as Neosopelus cf. microchir pending a taxonomic revision of the genus.

Cryptic species in a Vulnerable seabird: shorttailed albatross consists of two species

The occurrence of cryptic species within a threatened taxon is rare, but where they do occur, understanding species boundaries is essential for planning an effective conservation strategy. The short-tailed albatrossPhoebastria albatrusis a Vulnerable seabird that mainly breeds on Torishima and the Senkaku Islands in the western North Pacific. Although it has been tacitly regarded as a single management unit with 2 breeding sites, the species is known to comprise 2 genetically separated populations (Senkaku-type and Torishima-type). However, morphological examination of birds from both populations has not been conducted owing to the difficulty in accessing the Senkaku Islands. In this study, we examined the morphological differences between immigrants from the Senkaku Islands to Torishima (Senkaku-type) and native birds on Torishima (Torishima-type) and found significant differences in morphological characteristics between the 2 bird types. In general, Torishima-type birds were larger than Senkaku-type birds, whereas Senkaku-type birds had relatively longer beaks. Based on the morphological differences found in this study as well as genetic and ecological differences revealed in previous studies, we believe that Senkaku- and Torishima-type birds should be classified as different cryptic species. To the best of our knowledge, this is the first case of cryptic species being identified in a threatened avian species.

Italian odonates in the Pandora's box: A comprehensive DNA barcoding inventory shows taxonomic warnings at the Holarctic scale.

The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA-based monitoring relies on validated reference data sets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658-bp 5' end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the data set to 1,294 DNA barcodes. A multi-approach species delimitation analysis involving two distance (OT and ABGD) and four tree-based (PTP, MPTP, GMYC and bGMYC) methods was used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct molecular operational units, whereas the remaining ones were classified as 'warnings' (i.e. showing a mismatch between morphospecies assignment and DNA-based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped into three categories depending on if they showed low, high or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA-based monitoring studies.

Molecular discrimination of Ancistrus lineages (Siluriformes: Loricariidae) using barcode DNA tool

Although several species of Ancistrus have been described from the Amazon and Paraguay river basins in the states of Amazonas and Mato Grosso, Brazil, the taxonomic status of most specimens from these regions remains doubtful. In the present work, cytogenetic and molecular data were used to discriminate and isolate unexpected Ancistrus lineages from the Amazon and Paraguay basins. For that, it was used DNA barcoding based on mitochondrial Cytochrome Oxidase Subunit I (COI) gene and cytogenic data to perform such molecular discrimination. The analyzed sequences had 669 bp, of which 171 bp were conserved and 491 bp were variable. The Neighbor-joining and Bayesian analysis revealed 21 distinct groups in topology. The genetic distances within each group was 0.4%, 21 times smaller than the mean distance observed among groups, which was 8.4%. These values showed seven distinct lineages of Ancistrus from the studied points of the Amazon basin and eight lineages from the Paraguay basin points. Our results illustrate the efficiency of this technique for the discrimination of the Ancistrus lineages once it indicates the occurrence of cryptic species in these regions, which cannot yet be identified either with just chromosomal or morphological analyzes.

Multigene barcoding and phylogeny of selected Engraulidae species

Anchovies (Engraulidae) are one of the ecologically important groups and often difficult to identify due to their small size and overlapping morphological characters. In the present study, reference DNA barcodes were generated for 82 individuals representing 13 species of Engraulidae family using mitochondrial Cytochrome c oxidase subunit I (COI) and 16S rRNA genes. The average genetic distance value of COI gene for conspecific, congeneric and confamilial is 0.25, 20.45 and 22.28%, respectively. Mitochondrial 16S rRNA showed an average divergence value of 0.60, 10.28 and 14.37% for within species, between species and within families, respectively. Comparison of the present study reference barcodes with the reported sequences revealed high frequency of misidentification of species and possible occurrence of cryptic species in this family. Phylogenetic tree reconstructed using different methodologies revealed monophyletic nature of genus Stolephorus and the evolutionary relationship within genus Stolephorus is defined as ([S. insularis: S. tamilensis] S. dubiosus (S. waitei [S. commersonnii: S. indicus])).

Cryptic species in an ancient flowering‐plant lineage (Hydatellaceae, Nymphaeales) revealed by molecular and micromorphological data

The flora of the southwestern Australian biodiversity hotspot is rich in endemic species, many of which remain to be discovered or properly described; estimates of species diversity and levels of endemism should take into account the possible occurrence of cryptic species. Here we explore taxonomic diversity in a Western Australian lineage belonging to the primarily Australian genus Trithuria, the sole genus of Hydatellaceae (Nymphaeales). Recent molecular evidence supports the existence of cryptic species in self‐pollinating members of section Trithuria. We investigate Western Australian plants currently classified as T. australis s.l., a self‐pollinating member of the section Hydatella. Using evidence from microsatellite data (SSRs), an expanded molecular phylogenetic analysis based on four plastid markers, and fruit micromorphology, we suggest that material traditionally classified as T. australis s.l. belongs to at least four species. Two species occur in the northern part of the distribution range of the group (31° S to 33°27′ S), and two in the southern part (33°27′ S to 35° S). Each northern species has distinctive fruit micromorphology not recorded in other members of the genus. The two southern species are well characterized by molecular characters and seem to be allopatric, but lack obvious morphological differences from each other. We describe one of the northern species as T. fitzgeraldii sp. nov. However, clarifying the names of the other three species is currently problematic as T. australis and another available name are based on collections made 117 years ago, from localities distant from any subsequent records of Hydatellaceae. Based on genome size estimations, we also demonstrate two ploidy levels in the T. australis complex. Our study supports the view that species diversity in Hydatellaceae is strongly underestimated.

Ribosomal DNA polymorphisms reveal genetic structure and a phylogeographic pattern in the Burgundy truffle Tuber aestivum Vittad.

ABSTRACTEctomycorrhizal ascomycetes belonging to the genus Tuber produce edible fruiting bodies known as truffles. Tuber aestivum, in particular, is a fungus appreciated worldwide and has a natural distribution throughout Europe. Most of the molecular studies conducted on this species have been focused on the question as to whether or not T. aestivum and the morphologically similar T. uncinatum are conspecific. Conversely, only a handful of studies have assessed the level and distribution of genetic diversity and occurrence of phylogeographic patterns in this species. Here, we analyzed the genetic diversity of T. aestivum over a wide geographic range, performing an extensive sampling of specimens from Turkey, which is novel, to the best of our knowledge. We compared the internal transcribed spacer (ITS) profiles of 45 samples collected in different Turkish areas with those of 144 samples from all over Europe. We identified 63 haplotypes, 32 of which were exclusively present in Turkey. The majority of these haplotyes were also population specific. Haplotype network analysis and statistical tests highlighted the presence of a genetic structure and phylogeographic pattern, with three spatially distinct genetic clusters (northeastern Europe, southern Europe, and Turkey), with Turkey representing a diversity hotspot. Based on these results, we hypothesize the presence of glacial refugia for T. aestivum in Turkey, whereas European populations likely experienced a population bottleneck. The possible occurrence of cryptic species among Turkish T. aestivum samples also emerged. Our results are of practical relevance for the marketing of T. aestivum truffles and mycorrhizal seedlings and the preservation of the biodiversity of this species.

Cryptic species diversity in the Hypsolebiasmagnificus complex, a clade of endangered seasonal killifishes from the São Francisco River basin, Brazilian Caatinga (Cyprinodontiformes, Aplocheilidae).

A great diversity of animal species adapted to life in the semi-arid Caatinga of northeastern Brazil, including seasonal killifishes, has been reported in the last three decades. More recently, field and molecular data have shown a high occurrence of cryptic species. The killifish group herein analysed, the Hypsolebiasmagnificus species complex, is endemic to the middle and southern portion of the Caatinga, occupying about 120 km along the floodplains of the middle São Francisco River and some adjacent tributaries. Species of this complex are rare and presently considered threatened with extinction, being uniquely found in pools protected by trees and bushes. Single-locus delimitation methods were used to test species limits of populations displaying different colour patterns along the whole distribution of the complex. All analyses consistently supported the three nominal species and two new, herein described: H.gardneri Costa, sp. n., from the floodplains of the middle São Francisco River and H.hamadryades Costa, sp. n., from the Gorotuba River floodplains. The phylogenetic analysis highly supports H.hamadryades as sister to a clade comprising H.gardneri and H.harmonicus. Our field observations suggest that H.hamadryades is a miniature species. This study indicates that the H.magnificus complex comprises cryptic species apparently endemic to small areas and extremely vulnerable to environmental changes, deserving high concern.

Contrasting biological features in morphologically cryptic Mediterranean sponges.

Sponges are key organisms in the marine benthos where they play essential roles in ecological processes such as creating new niches, competition for resources, and organic matter recycling. Despite the increasing number of taxonomical studies, many sponge species remain hidden, whether unnoticed or cryptic. The occurrence of cryptic species may confound ecological studies by underestimating biodiversity. In this study, we monitored photographically growth, fusions, fissions, and survival of two morphologically cryptic species Hemimycale mediterranea Uriz, Garate & Agell, 2017 and H. columella (Bowerbank, 1874). Additionally, we characterized the main environmental factors of the corresponding species habitats, trying to ascertain whether some abiotic factors were correlated with the distribution of these species. Sponge monitoring was performed monthly. Seawater samples were collected the same monitoring days in the vicinity of the target sponges. Results showed contrasting growth and survival patterns for each species: H. mediterranea totally disappeared after larval release while 64% of individuals of H. columella survived the entire two years we monitored. The species also differed in the number of fissions and fusions. These events were evenly distributed throughout the year in the H. mediterranea population but concentrated in cold months in H. columella. No measured environmental factor correlated with H. mediterranea growth rates, while temperature and dissolved organic nitrogen were negatively correlated with H. columella growth rates. The strong differences in depth distribution, survival, growth, fusions, and fissions found between these two cryptic species, highlights the importance of untangling cryptic species before ecological studies are performed in particular when these species share geographical distribution.

Morphological and molecular characterization of Bysmatrum subsalsum (Dinophyceae) from the western Mediterranean Sea reveals the existence of cryptic species.

Bysmatrum subsalsum is a cosmopolitan dinoflagellate species that inhabits marine and transitional habitats. Despite its wide distribution, information on the morphological variability, phylogeny and ecology of B.subsalsum is scarce. In this study, we provide morphological and molecular data on B.subsalsum strains and wild cells from different locations in the Mediterranean Basin. The dynamics of cell abundances and the associated environmental conditions during a field bloom are also described. Genetic sequences of B.subsalsum obtained in this study showed large intraspecific differences, clustering in two well-differentiated clades. Despite a certain degree of variation with respect to cell size, apical pore complex (APC) morphology and size, and cingulum displacement, cells from the two clades showed similar morphological traits. These findings indicated the occurrence of cryptic species. Comparisons of themorphology of our B.subsalsum specimens with the few descriptions available in the literature revealed larger than previously known intraspecific morphological variability. Phylogenetic trees inferred from the concatenated SSU, 5.8S-ITS, and LSU rRNA and the individual 5.8S-ITS regions suggested the inclusion of Bysmatrum in the Peridiniales and a close phylogenetic relationship with Peridinium sensu stricto. However, the low statistical support prevented the assignment of Bysmatrum to a particular family of Peridiniales. Ecological data obtained from a bloom in La Pletera salt marshes (Catalan Coast, Spain) suggested the species reaches high cell abundances at water temperatures >20°C and salinity levels >30. Our results add new information regarding the morphology, phylogeny, and ecology of B.subsalsum.

Forecasting pollination declines through DNA barcoding: the potential contributions of macroecological and macroevolutionary scales of inquiry.

While pollinators are widely acknowledged as important contributors to seed production in plant communities, we do not yet have a good understanding of the importance of pollinator specialists for this ecosystem service. Determination of the prevalence of pollinator specialists is often hindered by the occurrence of cryptic species and the limitations of observational data on pollinator visitation rates, two areas where DNA barcoding of pollinators and pollen can be useful. Further, the demonstrated adequacy of pollen DNA barcoding from historical records offers opportunities to observe the effects of pollinator loss over longer timescales, and phylogenetic approaches can elucidate the historical rates of extinction of specialist lineages. In this Viewpoint article, we review how advances in DNA barcoding and metabarcoding of plants and pollinators have brought important developments to our understanding of specialization in plant-pollinator interactions. We then put forth several lines of inquiry that we feel are especially promising for providing insight on changes in plant-pollinator interactions over space and time. Obtaining estimates of the effects of reductions in specialists will contribute to forecasting the loss of ecosystem services that will accompany the erosion of plant and pollinator diversity.

Parallel evolution evidenced by molecular data in the banded-tetra (Astyanax fasciatus)

Astyanax is well known as a model for developmental biology studies, particularly with regard to Mexico's cave populations. More than 130 species of Astyanax are already known, most of which live in South America. The occurrence of cryptic species and species complexes elucidated by chromosomal and genetic studies demonstrates that the relationship between morphology and molecular evolution is quite complex within this group. In this work, we demonstrate that morphology does not follow the path of vicariant processes observed in Astyanax fasciatus populations, which separated about three million years ago, although molecular data suggests its separation in two species.

Tracing shifts of oceanic fronts using the cryptic diversity of the planktonic foraminifera Globorotalia inflata

AbstractThe use of planktonic foraminifera in paleoceanographic studies relies on the assumption that morphospecies represent biological species with ecological preferences that are stable through time and space. However, genetic surveys unveiled a considerable level of diversity in most morphospecies of planktonic foraminifera. This diversity is significant for paleoceanographic applications because cryptic species were shown to display distinct ecological preferences that could potentially help refine paleoceanographic proxies. Subtle morphological differences between cryptic species of planktonic foraminifera have been reported, but so far, their applicability within paleoceanographic studies remains largely unexplored. Here we show how information on genetic diversity can be transferred to paleoceanography using Globorotalia inflata as a case study. The two cryptic species of G. inflata are separated by the Brazil‐Malvinas Confluence (BMC), a major oceanographic feature in the South Atlantic. Based on this observation, we developed a morphological model of cryptic species detection in core top material. The application of the cryptic species detection model to Holocene samples implies latitudinal oscillations in the position of the confluence that are largely consistent with reconstructions obtained from stable isotope data. We show that the occurrence of cryptic species in G. inflata can be detected in the fossil record and used to trace the migration of the BMC. Since a similar degree of morphological separation as in G. inflata has been reported from other species of planktonic foraminifera, the approach presented in this study can potentially yield a wealth of new paleoceanographical proxies.

New set of nuclear primers for the ribosomal regions in Proseriata (Platyhelminthes)

The order Proseriata (Platyhelminthes) represents one of the most abundant soft-bodied meiofaunal groups. These minute interstitial organisms are characterized by a very simple morphology, which often hides the occurrence of cryptic species. Accordingly, molecular analyses are often needed to provide reliable taxonomic assessment and/or species identification. For these purposes the 18S and 28S rDNA genes have been so far the markers of choice. Here, we present a set of new primers for the sequencing of the whole ribosomal region in Proseriata, which improves the size of the sequenced segment from the current 3100 bp (18S + 28S D1–D6) to about 6300 bp. A broader molecular dataset may be used to achieve deeper insights on the systematics of Proseriata.

Morphometric study of third-instar larvae from five morphotypes of the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae)

The occurrence of cryptic species among economically important fruit flies strongly affects the development of management tactics for these pests. Tools for studying cryptic species not only facilitate evolutionary and systematic studies, but they also provide support for fruit fly management and quarantine activities. Previous studies have shown that the South American fruit fly, Anastrepha fraterculus, is a complex of cryptic species, but few studies have been performed on the morphology of its immature stages. An analysis of mandible shape and linear morphometric variability was applied to third-instar larvae of five morphotypes of the Anastrepha fraterculus complex: Mexican, Andean, Ecuadorian, Peruvian and Brazilian-1. Outline geometric morphometry was used to study the mouth hook shape and linear morphometry analysis was performed using 24 linear measurements of the body, cephalopharyngeal skeleton, mouth hook and hypopharyngeal sclerite. Different morphotypes were grouped accurately using canonical discriminant analyses of both the geometric and linear morphometry. The shape of the mandible differed among the morphotypes, and the anterior spiracle length, number of tubules of the anterior spiracle, length and height of the mouth hook and length of the cephalopharyngeal skeleton were the most significant variables in the linear morphometric analysis. Third-instar larvae provide useful characters for studies of cryptic species in the Anastrepha fraterculus complex.

DNA Barcoding to Improve the Taxonomy of the Afrotropical Hoverflies (Insecta: Diptera: Syrphidae).

The identification of Afrotropical hoverflies is very difficult because of limited recent taxonomic revisions and the lack of comprehensive identification keys. In order to assist in their identification, and to improve the taxonomy of this group, we constructed a reference dataset of 513 COI barcodes of 90 of the more common nominal species from Ghana, Togo, Benin and Nigeria (W Africa) and added ten publically available COI barcodes from nine nominal Afrotropical species to this (total: 523 COI barcodes; 98 nominal species; 26 genera). The identification accuracy of this dataset was evaluated with three methods (K2P distance-based, Neighbor-Joining (NJ) / Maximum Likelihood (ML) analysis, and using SpeciesIdentifier). Results of the three methods were highly congruent and showed a high identification success. Nine species pairs showed a low (< 0.03) mean interspecific K2P distance that resulted in several incorrect identifications. A high (> 0.03) maximum intraspecific K2P distance was observed in eight species and barcodes of these species not always formed single clusters in the NJ / ML analayses which may indicate the occurrence of cryptic species. Optimal K2P thresholds to differentiate intra- from interspecific K2P divergence were highly different among the three subfamilies (Eristalinae: 0.037, Syrphinae: 0.06, Microdontinae: 0.007–0.02), and among the different general suggesting that optimal thresholds are better defined at the genus level. In addition to providing an alternative identification tool, our study indicates that DNA barcoding improves the taxonomy of Afrotropical hoverflies by selecting (groups of) taxa that deserve further taxonomic study, and by attributing the unknown sex to species for which only one of the sexes is known.

DNA barcode assessment of Mediterranean mayflies (Ephemeroptera), benchmark data for a regional reference library for rapid biomonitoring of freshwaters

Accurate identification of aquatic species is fundamental to freshwater research. In this paper, we targeted Ephemeroptera, a key taxonomic insect group for biomonitoring of water bodies and present an overview on the efficacy of the DNA barcoding approach to document species identity in the Mediterranean region. We sequenced the mitochondrial cytochrome c oxidase (COI) in 39 nominal species. Sample discrimination and species identification were investigated by evaluating haplotype identity and similarity, intra-/interspecific genetic distances, optimal identification of barcoding gap thresholds, estimates of species monophyly and comparative species matches on available reference libraries. The resolving power of the obtained data was discussed in the light of statistical tools such as Spider R-package and Poisson Tree Processes. High levels of species identification were achieved with all the used methodologies, and the occurrence of cryptic species was suggested. We conclude that DNA barcoding is a powerful tool for taxonomic research in Mediterranean mayflies, with great promise to ameliorate biodiversity inventories of freshwater ecosystems and to provide the necessary accuracy for water quality assessment programs. Our results further indicated we need to upgrade the current regional mayfly diversity knowledge. The development of a Mediterranean reference library could integrate this new information system.