Detailed DNA barcoding of mayflies in a small European country proved how far we are from having comprehensive barcode reference libraries.

Mayflies (Ephemeroptera) are among the crucial water and habitat quality bioindicators. However, despite their intensive long-term use in various studies, more reliable mayfly DNA barcode data have been produced in a negligible number of countries, and only ~40% of European species had been barcoded with less than 50% of families covered. Despite being carried out in a small area, our study presents the second-most species-rich DNA reference library of mayflies from Europe and the first comprehensive view from an important biodiversity hotspot such as the Western Carpathians. Within 1153 sequences, 76 morphologically determined species were recorded and added to the Barcode of Life Data System (BOLD) database. All obtained sequences were assigned to 97 BINs, 11 of which were unique and three represented species never barcoded before. Sequences of 16 species with high intraspecific variability were divided into 40 BINs, confirming the presence of cryptic lineages. Due to the low interspecific divergence and the non-existing barcoding gap, sequences of six species were assigned to three shared BINs. Delimitation analyses resulted in 79 and 107 putative species respectively. Bayesian and maximum-likelihood phylogenies confirmed the monophyly of almost all species and complexes of cryptic taxa and proved that DNA barcoding distinguishes almost all studied mayfly species. We have shown that it is still sufficient to thoroughly investigate the fauna of a small but geographically important area to enrich global databases greatly. In particular, the insights gained here transcend the local context and may have broader implications for advancing barcoding efforts.

Who is Andesiops peruvianus (Ulmer, 1920) (Ephemeroptera: Baetidae)? New insight from the type basin using morphological and molecular analyses.

Andesiops peruvianus (Ulmer, 1920) is a small minnow mayfly with a distribution throughout the Andes Mountains, mainly in lotic environments. In recent years, the taxonomic status of the species has been shifting, even finding out molecular and morphological evidence to consider it as a cryptic species. In this work, we collected seven specimens of A. peruvianus from the Lake Titicaca in Peru, the type locality and perform molecular and morphological analyses to test their relationship with specimens from other regions. The species delimitation analysis revealed the existence of five MOTUs for specimens identified as A. peruvianus along the Andes Mountains, while the Lake Titicaca specimens formed a single MOTU with high interspecific distance. In this MOTU were found specimens with different types of abdominal gills, from translucent to markedly tracheated supporting the hypothesis of phenotypic plasticity at the abdominal gill level. Also, an extension in the range of tarsal claw denticles, from 10-12 to 10-14, were observed, higher than what were found in other regions. Our results support A. peruvianus as a species complex and reveal A. peruvianus from Titicaca as a single MOTU. Future morphological revisions of topotype specimens as well as from other Andean regions are required to strengthen the diagnosis of informative characteristics at both the larval and adult stage to elucidate the real status of the species.

The taxonomy and molecular phylogeny of two epibiotic colonial peritrich ciliates (Ciliophora, Peritrichia).

Two colonial sessilid peritrichs, Epistylis qingdaoensis n. sp. and Carchesium cyclopidarum Nenninger, 1948, were isolated from a marine crustacean and a freshwater mayfly, respectively. Morphological characters for each species were revealed by in vivo observations and silver staining methods. Epistylis qingdaoensis n. sp. is characterized by the apperance of the colony which is up to 250 µm high and usually contains fewer than eight zooids, the single-layered peristomial lip, the conspicuously conical peristomial disc, and the structure of infundibular polykinety 3 which comprises three isometric ciliary rows. Carchesium cyclopidarum is recognized by the colony height of about 600 µm, the small zooid size of 35-50 × 20-30 µm, and the low number of silverlines. Phylogenetic analyses based on small subunit ribosomal DNA sequences were performed to reveal their evolutionary relationships. Surprisingly, neither species clustered with its congeners. Epistylis qingdaoensis n. sp. nested within a clade of Zoothamnium species that was sister to a clade comprising core Epistylis species and several Zoothamnium species. Carchesium cyclopidarum nested within a clade comprising solitary peritrichs and was distantly related to the type species of Carchesium, C. polypinum. These findings imply for the first time that Carchesium is non-monophyletic.

The First Two Complete Mitochondrial Genomes of Neoephemeridae (Ephemeroptera): Comparative Analysis and Phylogenetic Implication for Furcatergalia.

Mayflies of the family Neoephemeridae are widespread in the Holarctic and Oriental regions, and its phylogenetic position is still unstable in the group Furcatergalia (mayflies with fringed gills). In the present study, we determined the complete mitogenomes of two species, namely Potamanthellus edmundsi and Pulchephemera projecta, of this family. The lengths of two mitogenomes were 15,274 bp and 16,031 bp with an A + T content of 73.38% and 73.07%, respectively. Two neoephemerid mitogenomes had a similar gene size, base composition, and codon usage of protein-coding genes (PCGs), and the sequenced gene arrangements were consistent with the putative ancestral insect mitogenomes as understood today. The most variable gene of Furcatergalia mitogenomes was ND2, while the most conserved gene was COI. Meanwhile, the analysis of selection pressures showed that ND6 and ATP8 exhibited a relaxed purifying selection, and COI was under the strongest purifying selection. Phylogenetic trees reconstructed based on two concatenated nucleotide datasets using both maximum likelihood (ML) and Bayesian inference (BI) estimations yielded robust identical topologies. These results corroborated the monophyly of seven studied families and supported the family Leptophlebiidae as being of the basal lineage of Furcatergalia. Additionally, the sister-group relationship of Caenidae and Neoephemeridae was well supported. Methodologically, our present study provides a general reference for future phylogenetic studies of Ephemeroptera at the mitogenome level.

Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera).

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.

The complete mitochondrial genome of Choroterpes (Euthralus) yixingensis (Ephemeroptera: Leptophlebiidae) and its mitochondrial protein-coding gene expression under imidacloprid stress.

As one of the most common benthic invertebrates in freshwater, mayflies are very sensitive to changes in water quality and have high requirements for the water environment to allow their nymphs to successfully live and grow. Neonicotinoids, such as imidacloprid, can enter fresh water and pollute the aquatic environment. The present study had two goals: (1) investigate imidacloprid effects on mayfly larvae Choroterpes (Euthralus) yixingensis, and (2) contribute to the phylogenetic status of Ephemeroptera that has always been controversial. Nymphs were collected from Jinhua, China and exposed to different concentrations imidacloprid (5, 10, 20, and 40 µg/L) in the laboratory. Survival of C. yixingensis nymphs decreased as a function of time and imidacloprid concentration with only ~ 55% survival after 72 h exposure to 40 µg/L imidacloprid. After culture under 40 µg/L imidacloprid for 24 h, the steady state transcript levels of mitochondrial COX3, ND4 and ND4L genes were reduced to just 0.07 ± 0.11, 0.30 ± 0.16, and 0.28 ± 0.13 as compared with respective control values (P < 0.01). Steady state transcript levels of ND4 and ND4L were also significantly reduced in a dose-dependent manner (P < 0.05), suggesting that the steady state transcript pattern of these genes in mayfly nymphs can change in response to different levels of environmental contamination. Hence, the mitochondrial protein-coding genes of mayflies could potentially be developed as biomarkers for water ecotoxicity monitoring in the future. In addition, we used the mitochondrial genome sequence of C. yixingensis for an assessment of the phylogenetic tree of Ephemeroptera. The monophyly of Leptophlebiidae was supported and showed that Leptophlebiidae was a sister group to the clade (Baetidae + Caenidae).

Complete mitochondrial genomes of Epeorus carinatus and E. dayongensis (Ephemeroptera: Heptageniidae): Genomic comparison and phylogenetic inference.

The current research on Ephemeroptera is mainly based on its morphology, since only small numbers of mitogenomes have been reported. In this study, the mitogenomes of Epeorus carinatus (15,338 bp) and E. dayongensis (15,609 bp) were sequenced, annotated and compared to genome data from congeners. Both mitogenomes had 23 tRNA genes including standard 22 and one extra tRNAMet. The duplicated tRNAMet gene had been found in other heptageniid species except Paegniodes cupulatus, suggesting it could be used as a molecular synapomorphy for partial Heptageniidae. The phylogenetic analyses based on Bayesian Inference (BI) and Maximum Likelihood (ML) showed that Heptageniidae was monophyletic and the relationships among known Epeorus species were ((E. carinatus + E. herklotsi) + (E. dayongensis + E. sp. 1)), which implied the focal species E. carinatus and E. dayongensis should be grouped into different subgenera.

Transcriptomic and life history responses of the mayfly Neocloeon triangulifer to chronic diel thermal challenge.

To better understand the effects of transient thermal stress in an aquatic insect, we first identified static temperatures associated with fitness deficits, and then reared larvae from egg hatch to adulthood under diurnally variable regimens including daily forays into deleterious temperatures. We sampled mature larvae at the coolest and warmest portions of their respective regimens for RNA-seq analysis. Few transcripts (28) were differentially expressed when larvae oscillated between favorable temperatures, while 614 transcripts were differentially expressed when experiencing daily transient thermal stress. Transcripts associated with N-glycan processing were downregulated while those associated with lipid catabolism and chitin turnover were significantly upregulated in heat stressed larvae. An across-regimen comparison of differentially expressed transcripts among organisms sampled at comparable temperatures demonstrated that the effects of daily thermal stress persisted even when larvae were sampled at a more optimal temperature (806 differentially expressed transcripts). The chronically stressed population had reduced expression of transcripts related to ATP synthesis, mitochondrial electron chain functions, gluconeogenesis and glycolytic processes while transcripts associated with cell adhesion, synaptic vesicle transport, regulation of membrane potential and lipid biosynthesis increased. Comparisons of constant vs. variable temperatures revealed that the negative consequences of time spent at stressful temperatures were not offset by more time spent at optimal temperatures.

Novel gene rearrangement pattern in the mitochondrial genomes of Torleya mikhaili and Cincticostella fusca (Ephemeroptera: Ephemerellidae).

The mayfly family Ephemerellidae (Insecta: Ephemeroptera) is distributed around the world and has very high species diversity. However, its evolution pattern of mitogenome and phylogenetic relationships within Ephemeroptera remain unclear. In this study, the complete mitochondrial genomes (mitogenomes) of Torleya mikhaili (15,042 bp) and Cincticostella fusca (15,135 bp) were firstly determined and analyzed. Two ephemerellid mitogenomes shared similar gene organization with 37 typical genes as well as a putative control region. Compared with other reported mitogenomes of mayflies, the unique gene order (I'-CR-Q-M) was found in these two mitogenomes. Although the observed rearrangement pattern is novel within ephemeropteran mitogenomes, it could be explained presumably by the mechanisms of tandem duplication-random loss and recombination. The phylogenetic analyses using both Bayesian inference (BI) and maximum likelihood (ML) methods based on four nucleotide datasets placed three ephemerellid species together. Furthermore, the phylogenetic relationships of the three genera were recovered as ((Ephemerella + Cincticostella) + Torleya).

Genomic adaptations to aquatic and aerial life in mayflies and the origin of insect wings.

The evolution of winged insects revolutionized terrestrial ecosystems and led to the largest animal radiation on Earth. However, we still have an incomplete picture of the genomic changes that underlay this diversification. Mayflies, as one of the sister groups of all other winged insects, are key to understanding this radiation. Here, we describe the genome of the mayfly Cloeon dipterum and its gene expression throughout its aquatic and aerial life cycle and specific organs. We discover an expansion of odorant-binding-protein genes, some expressed specifically in breathing gills of aquatic nymphs, suggesting a novel sensory role for this organ. In contrast, flying adults use an enlarged opsin set in a sexually dimorphic manner, with some expressed only in males. Finally, we identify a set of wing-associated genes deeply conserved in the pterygote insects and find transcriptomic similarities between gills and wings, suggesting a common genetic program. Globally, this comprehensive genomic and transcriptomic study uncovers the genetic basis of key evolutionary adaptations in mayflies and winged insects.

Space colonization by branching trachea explains the morphospace of a simple respiratory organ.

Branching morphogenesis helps increase the efficiency of gas and liquid transport in many animal organs. Studies in several model organisms have highlighted the molecular and cellular complexity behind branching morphogenesis. To understand this complexity, computational models have been developed with the goal of identifying the "major rules" that globally explain the branching patterns. These models also guide further experimental exploration of the biological processes that execute and maintain these rules. In this paper we introduce the tracheal gills of mayfly (Ephemeroptera) larvae as a model system to study the generation of branched respiratory patterns. First, we describe the gills of the mayfly Cloeon dipterum, and quantitatively characterize the geometry of its branching trachea. We next extend this characterization to those of related species to generate the morphospace of branching patterns. Then, we show how an algorithm based on the "space colonization" concept (SCA) can generate this branching morphospace via growth towards a hypothetical attractor molecule (M). SCA differs from other branch-generating algorithms in that the geometry generated depends to a great extent on its perception of the "external" space available for branching, uses few rules and, importantly, can be easily translated into a realistic "biological patterning algorithm". We identified a gene in the C. dipterum genome (Cd-bnl) that is orthologous to the fibroblast growth factor branchless (bnl), which stimulates growth and branching of embryonic trachea in Drosophila. In C. dipterum, this gene is expressed in the gill margins and areas of finer tracheolar branching from thicker trachea. Thus, Cd-bnl may perform the function of M in our model. Finally, we discuss this general mechanism in the context of other branching pattern-generating algorithms.

The impact of Miocene orogeny for the diversification of Caucasian Epeorus (Caucasiron) mayflies (Ephemeroptera: Heptageniidae).

A common hypothesis for the high biodiversity of mountains is the diversification driven by orogeny creating conditions for rapid in situ speciation of resident lineages. The Caucasus is a young mountain system considered as a biodiversity hotspot; however, the origin and evolution of its diversity remain poorly understood. This study focuses on mayflies of the subgenus Caucasiron, one of the most diversified stenotopic mayflies inhabiting various types of streams throughout the Caucasus. Using the time-calibrated phylogeny based on two mitochondrial (COI, 16S) and three nuclear (EF-1α, wg, 28S) gene fragments, we tested the role of Caucasian orogeny in biogeography, diversification patterns, and altitudinal diversification of Caucasiron mayflies. We found that orogeny promoted the lineage diversification of Caucasiron in the Miocene. The highest diversification rate corresponding with the uplift of mountains was followed by a significant slowdown towards the present suggesting minor influence of Pleistocene climatic oscillations on the speciation. The Caucasiron lineages cluster into three principal clades originating in the Upper Miocene. We found a strong support that one of the three clades diversified via allopatric speciation in the Greater Caucasus isolated in the Parathetys Sea. The other two clades originating most likely outside the Greater Caucasus diversified towards high and low altitude, respectively, indicating possible role of climatic factors and/or passive uplift on their differentiation. Current high Caucasiron diversity in the Greater Caucasus is a result of in situ speciation and later immigration from adjacent mountain ranges after the Parathetys Sea retreat. Our phylogeny supported the monophyly of Rhithrogeninae, Epeorus s.l., Caucasiron, and Iron. Epeorus subgenus Ironopsis was found paraphyletic, with its European representatives more closely related to Epeorus s.str. than to Iron. Therefore, we re-arranged taxa treated within Ironopsis to comply with the phylogeny recovered herein.

Genealogical Position of Japanese Populations of the Globally Distributed Mayfly Cloeon dipterum and Related Species (Ephemeroptera, Baetidae): A Molecular Phylogeographic Analysis.

In the present study, we add genetic data of the mayfly Cloeon dipterum collected from the Japanese Islands to the established molecular phylogenetic knowledge in the mitochondrial COI gene of Cloeon mayflies. Cloeon dipterum is a typical cosmopolitan species that includes six intraspecific haplotype groups. The present phylogenetic analysis revealed that haplotypes of the Japanese C. dipterum constitute a seventh group together with a haplotype from Korea. This East Asian group forms a sister group with previously known European and North American haplotype groups (i.e., the clade CT1 to CT3). The present phylogenetic analysis further revealed the occurrence of two described species (C. dipterum and Cloeon ryogokuensis) and possibly three species (Cloeon sp. 1 to 3) in Japan. Consideration is given to the degree of genetic differentiation, divergence time, and differentiation process among these seven genetic groups.

Direct analysis of vicariance in Neotropical mayflies (Ephemeroptera).

The distribution of aquatic insects has been poorly explored in quantitative analyses aiming at the historical reconstruction of area relationships in the Neotropics. Ephemeroptera is an ancient group, characterized by its low vagility, and of high richness and endemicity in this region. Systematic knowledge of the group has enormously increased in the last decades, achieving a sufficient background to explore biogeographical historical patterns. Our aim is to reconstruct area history in the Neotropics using the rationale of Barrier biogeography (Hovenkamp protocol). We present eleven mayfly phylogenies, representing groups that evolved independently at least from the Jurassic (i.e., not a one-taxon history). With these groups, we conducted independent biogeographical analyses (using Vicariance Inference Program), and extracted the events that repeated in two or more clades. We found fifty-eight TVEs (Traceable Vicariant Events), from which four were found at least twice, thus constituting SVEs (Supported Vicariant Events).

The mitochondrial genomes of palaeopteran insects and insights into the early insect relationships.

Phylogenetic relationships of basal insects remain a matter of discussion. In particular, the relationships among Ephemeroptera, Odonata and Neoptera are the focus of debate. In this study, we used a next-generation sequencing approach to reconstruct new mitochondrial genomes (mitogenomes) from 18 species of basal insects, including six representatives of Ephemeroptera and 11 of Odonata, plus one species belonging to Zygentoma. We then compared the structures of the newly sequenced mitogenomes. A tRNA gene cluster of IMQM was found in three ephemeropteran species, which may serve as a potential synapomorphy for the family Heptageniidae. Combined with published insect mitogenome sequences, we constructed a data matrix with all 37 mitochondrial genes of 85 taxa, which had a sampling concentrating on the palaeopteran lineages. Phylogenetic analyses were performed based on various data coding schemes, using maximum likelihood and Bayesian inferences under different models of sequence evolution. Our results generally recovered Zygentoma as a monophyletic group, which formed a sister group to Pterygota. This confirmed the relatively primitive position of Zygentoma to Ephemeroptera, Odonata and Neoptera. Analyses using site-heterogeneous CAT-GTR model strongly supported the Palaeoptera clade, with the monophyletic Ephemeroptera being sister to the monophyletic Odonata. In addition, a sister group relationship between Palaeoptera and Neoptera was supported by the current mitogenomic data.

Pesticide contamination drives adaptive genetic variation in the endemic mayfly Andesiops torrens within a semi-arid agricultural watershed of Chile.

Agrichemical contamination can provoke evolutionary responses in freshwater populations. It is a particularly relevant issue in semi-arid regions due to the sensitivity of endemic species to pollutants and to interactions with temperature stress. This paper investigates the presence of pesticides in rivers within a semi-arid agricultural watershed of Chile, testing for their effects on population genetic characteristics of the endemic mayfly Andesiops torrens (Insecta, Ephemeroptera). Pesticides were detected in sediment samples in ten out of the 30 sites analyzed throughout the upper part of the Limarí watershed. To study the evolutionary impact of such contamination on A. torrens, we used a genome-wide approach and analyzed 2056 single nucleotide polymorphisms (SNPs) loci in 551 individuals from all sites. Genetic differentiation was weak between populations, suggesting high gene flow across the study area. While we did not find evidence of pesticide effects on genetic diversity nor on population differentiation, the allele frequency of three outlier SNP loci correlated significantly with pesticide occurrence. Interrogation of genomic resources indicates that two of these SNPs are located within functional genes that encode for the low-density lipoprotein receptor-related protein 2 and Dumpy, both potentially involved in insect cuticle resistance processes. Such genomic signatures of local adaptation are indicative of past adverse effects of pesticide exposure on the locally adapted populations. Our results reveal that A. torrens is sensitive to pesticide exposure, but that a high gene flow may confer resilience to contamination. This research supports the contention that A. torrens is an ideal model organism to study evolutionary responses induced by pesticides on non-target, endemic species.

Concordance between molecular biogeography of Dipteromimus tipuliformis and geological history in the local fine scale (Ephemeroptera, Dipteromimidae).

Species distribution area is determined by both biotic and abiotic factors. In particular, significant geological events influence the biodiversity and the genetic structures of the organisms inhabiting the area. The establishment of physical barriers (e.g., mountains, rivers), drives species differentiation by their interference with biological dispersal or gene flow. The Japanese Islands have a high degree of biodiversity. This study focused on the Kii Peninsula, which stands out as a region of exhibiting particularly high biodiversity, and also exceptionally high endemism. The Kii Peninsula has experienced active mountain formation ever since the Quaternary period. In this study, we investigate the influence of geological events on the establishment of genetic diversity. We focused on the mayfly, Dipteromimus tipuliformis. Phylogenetic analyses were performed utilizing the mitochondrial DNA 16S rRNA and COI regions, and the nuclear DNA histone H3, PEPCK and 28S rRNA regions. As a result, it was shown that this mayfly exhibits a genetic structure that strongly reflects the geological history of the Kii Peninsula, and detected their dispersal process across the Median Tectonic Line. This is a unique and significant study, in that it clearly shows the relationship between the phylogenetic evolution of this mayfly and the corresponding geological history in surprisingly geographic fine scale.

The First Establishment of "Hand-Pairing" Cross-Breeding Method for the Most Ancestral Wing Acquired Insect Group.

Insects are the most diverse organisms in the world and have been in existence since ca. 480 Ma; given this, they can provide profound insights into evolution. Among them, the order Ephemeroptera is one of the most basal clades of winged insects. This makes Ephemeroptera a significant key taxon in understanding the macro-evolution or the insect groundplan. In the development of biological evolutionary studies of this taxon, it is important to establish a technique for cross-breeding. Furthermore, the establishment of these techniques also makes a great contribution in the fields of micro-evolution. In a non-model taxon, the mayfly, subcultivation in the laboratory has been thus far considered impossible. With the exception of some parthenogenetic strains, it is extremely difficult to mate these insects in artificial environments. In this study, we established a successful artificial mating technique, i.e., a "hand-pairing" based cross-breeding method for mayflies. Furthermore, we also succeeded in clearly verifying by a genotyping method that the offspring reproduced by hand-pairing were in fact derived from the actual male and female which were used for hand-pairing. We established a reproductive experimental technique for hand-pairing of Dipteromimus tipuliformis and verified this technique by means genotyping. This technique could allow the artificial control of fertilization timing, and result in offspring which can be verified as to their status by means of genotyping. This achievement will be extremely important in the future for both the macro- and micro-evolutionary studies of insects.

The origin of the odorant receptor gene family in insects.

The origin of the insect odorant receptor (OR) gene family has been hypothesized to have coincided with the evolution of terrestriality in insects. Missbach et al. (2014) suggested that ORs instead evolved with an ancestral OR co-receptor (Orco) after the origin of terrestriality and the OR/Orco system is an adaptation to winged flight in insects. We investigated genomes of the Collembola, Diplura, Archaeognatha, Zygentoma, Odonata, and Ephemeroptera, and find ORs present in all insect genomes but absent from lineages predating the evolution of insects. Orco is absent only in the ancestrally wingless insect lineage Archaeognatha. Our new genome sequence of the zygentoman firebrat Thermobia domestica reveals a full OR/Orco system. We conclude that ORs evolved before winged flight, perhaps as an adaptation to terrestriality, representing a key evolutionary novelty in the ancestor of all insects, and hence a molecular synapomorphy for the Class Insecta.

Fast and accurate identification of cryptic and sympatric mayfly species of the Baetis rhodani group.

OBJECTIVE: Species of the Baetis rhodani group are among the most widespread mayflies of the Palearctic region. However, frequent occurrence of morphologically cryptic species complicates the identification of sympatric species. Here, we proposed and tested a method for the fast, accurate, and cost-effective assignment of a large number of individuals to their putative species, based on high resolution melting profiles of a standard mitochondrial gene fragment. We tested this method using a system of three recently identified cryptic species inhabiting the Tyrrhenian Islands (western Mediterranean basin). RESULTS: Highly species-specific high resolution melting profiles were obtained, allowing the unequivocal attribution of each individual to the respective species. This assay provides a convenient and easily customizable alternative to traditional barcoding approaches, provided that the mayfly taxa occurring within the geographic area of interest have been previously identified and their high resolution melting profiles assessed.