A comprehensive DNA barcoding reference database for Plecoptera of Switzerland.

DNA barcoding is an essential tool in modern biodiversity sciences. Despite considerable work to barcode the tree of life, many groups, including insects, remain partially or totally unreferenced, preventing barcoding from reaching its full potential. Aquatic insects, especially the three orders Ephemeroptera, Plecoptera, and Trichoptera (EPT), are key freshwater quality indicators worldwide. Among them, Plecoptera (stoneflies), which are among the most sensitive aquatic insects to habitat modification, play a central role in river monitoring surveys. Here, we present an update of the Plecoptera reference database for (meta)barcoding in Switzerland, now covering all 118 species known from this country. Fresh specimens, mostly from rare or localized species, were collected, and 151 new CO1 barcodes were generated. These were merged with the 422 previously published sequences, resulting in a dataset of 573 barcoded specimens. Our CO1 dataset was delimited in 115 CO1 clusters based on a priori morphological identifications, of which 17% are newly reported for Switzerland, and 4% are newly reported globally. Among the 115 CO1 clusters, 85% showed complete congruence with morphology. Distance-based analysis indicated local barcoding gaps in 97% of the CO1 clusters. This study significantly improves the Swiss reference database for stoneflies, enhancing future species identification accuracy and biodiversity monitoring. Additionally, this work reveals cryptic diversity and incongruence between morphology and barcodes, both presenting valuable opportunities for future integrative taxonomic studies. Voucher specimens, DNA extractions and reference barcodes are available for future developments, including metabarcoding and environmental DNA surveys.

Another new species of the genus Habrophlebia Eaton, 1881 (Ephemeroptera, Leptophlebiidae) from the Maghreb.

A new species belonging to the genus Habrophlebia Eaton, 1881 is described at the nymphal stage from the Rif Mountains of Morocco. This species presents unique features, such as the chorionic arrangement of the egg and the ornamentation of the posterior margin of abdominal tergites. It is compared to all west European Habrophlebia species and a table with discriminating characters is given. A phylogenetic reconstruction based on COI sequences fully supports the hypothesis of a new species in the Rif Mountains, with possible further distribution in southern Spain.

A new species of the genus Alainites Waltz & McCafferty, 1994 (Ephemeroptera, Baetidae) from the north of Morocco.

A new species of Alainites is described from northern of Morocco Alainitesalbaisp. nov. It can be separated from the other west Palearctic species by the gill number, the spination of the distal margin of tergites, the leg setation, and the paraproct shape and spination. This species is widespread in the study area but never abundant. It prefers small to medium streams with slow flow, and does not seem to be very sensitive to pollution and water logging activities.

A new species of the Baetisfuscatus group (Ephemeroptera, Baetidae) from Morocco.

Baetisrifensissp. nov. is the first representative of the Baetisfuscatus group to be described from the Maghreb. It was collected from streams in the Rif region of northern Morocco. All species of the B.fuscatus group are morphologically very similar, with slight differences in colour. Thus, in addition to morphological description, species delimitation based on genetic evidence was carried out. The new species was compared with other members of the B.fuscatus group from the Palaearctic region.

Ecdyonurusaurasius sp. nov. (Insecta, Ephemeroptera, Heptageniidae, Ecdyonurinae), a new micro-endemic mayfly species from Aurès Mountains (north-eastern Algeria).

Ecdyonurusaurasius sp. nov., a micro-endemic species reported from several streams within the Aurès Mountains (north-eastern Algeria), is described and illustrated at nymphal, subimaginal and imaginal stages of both sexes. Critical morphological diagnostic characters distinguishing the new species are presented, together with molecular affinities as well as notes on the biology and distribution of the species.

A new species of the genus Prosopistoma Latreille, 1833 (Ephemeroptera, Prosopistomatidae) from Morocco.

We describe a new species of Prosopistoma collected in the High Atlas Mountains of Morocco. Prosopistomamaroccanum sp. nov. appears to be morphologically more similar to the European highly endangered P.pennigerum (Müller, 1785) than to the other Maghrebian species, P.alaini Bojkova & Soldán, 2015. A gene tree including the few available barcode sequences of Palearctic Prosopistoma specimens is provided. Possible affinities with West African species are also discussed.

Two new species of Centroptilum Eaton, 1869 from North Africa (Ephemeroptera, Baetidae).

Based on recently collected larvae from Algeria and Morocco, the species delimitation within the genus Centroptilum Eaton, 1869 in that region is validated. Two new species are described and illustrated, one from north-eastern Algeria, and one from North Morocco, using an integrated approach with morphological and molecular evidence. A table summarising the morphological differences between the new species and Centroptilumluteolum (Müller, 1776) from Central Europe is provided. Further, molecular evidence for additional undescribed species of Centroptilum in other regions of the West Palearctic is provided and discussed.

Electrogena brulini sp. nov. and E. vipavensis Zurwerra & Tomka, 1986 syn. nov. revealed by integrative taxonomy of E. gridellii (Grandi, 1953) (Ephemeroptera: Heptageniidae).

A new species belonging to the speciose genus Electrogena Zurwerra & Tomka, 1985 is described based on all stages from material collected in Switzerland, northern Italy and Slovenia. Electrogena brulini Wagner sp. nov. is closely related to E. gridellii (Grandi, 1953). A genetic analysis based on the mitochondrial gene CO1, including 9 specimens from 5 populations, reveals a K2P distance of 22% between both species. Our investigations demonstrate that the description of the nymph of E. gridellii sensu Belfiore (1996) refers in fact to both species. Therefore, E. gridellii is redescribed in all stages. The number of bristles on each mandibular prostheca in the nymphs, the shape of titillators in male genitalia and the shape of the subanal plate in female imagines are the main characters allowing discrimination of these two species. This study also shows that in this case it is possible and helpful to use colour patterns to separate two closely related species. The re-examination of the type material of Electrogena vipavensis Zurwerra & Tomka, 1986 described from western Slovenia indicates that this species is a junior subjective synonym of E. gridellii.

Molecular phylogeny and timing of diversification in Alpine Rhithrogena (Ephemeroptera: Heptageniidae).

BACKGROUND: Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown. RESULTS: Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups. CONCLUSIONS: Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects.

Endemism and diversification in freshwater insects of Madagascar revealed by coalescent and phylogenetic analysis of museum and field collections.

The biodiversity and endemism of Madagascar are among the most extraordinary and endangered in the world. This includes the island's freshwater biodiversity, although detailed knowledge of the diversity, endemism, and biogeographic origin of freshwater invertebrates is lacking. The aquatic immature stages of mayflies (Ephemeroptera) are widely used as bio-indicators and form an important component of Malagasy freshwater biodiversity. Many species are thought to be microendemics, restricted to single river basins in forested areas, making them particularly sensitive to habitat reduction and degradation. The Heptageniidae are a globally diverse family of mayflies (>500 species) but remain practically unknown in Madagascar except for two species described in 1996. The standard approach to understanding their diversity, endemism, and origin would require extensive field sampling on several continents and years of taxonomic work followed by phylogenetic analysis. Here we circumvent this using museum collections and freshly collected individuals in a combined approach of DNA taxonomy and phylogeny. The coalescent-based GMYC analysis of DNA barcode data (mitochondrial COI) revealed 14 putative species on Madagascar, 70% of which were microendemics. A phylogenetic analysis that included African and Asian species and data from two mitochondrial and four nuclear loci indicated the Malagasy Heptageniidae are monophyletic and sister to African species. The genus Compsoneuria is shown to be paraphyletic and the genus Notonurus is reinstalled for African and Malagasy species previously placed in Compsoneuria. A molecular clock excluded a Gondwanan vicariance origin and instead favoured a more recent overseas colonization of Madagascar. The observed monophyly and high microendemism highlight their conservation importance and suggest the DNA-based approach can rapidly provide information on the diversity, endemism, and origin of freshwater biodiversity. Our results underline the important role that museum collections can play in molecular studies, especially in critically endangered biodiversity hotspots like Madagascar where entire species or populations may go extinct very quickly.

Toward a DNA taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) using a mixed Yule-coalescent analysis of mitochondrial and nuclear DNA.

Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera) inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC) model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1) marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality) or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe.