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.

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.

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).

Distribution and habitat preference of Ephemeroptera and Trichoptera in subtropical mountain streams: implications for monitoring and conservation.

The assessment of biotic-habitat relationships provides key information to predict biotic responses to perturbations and important tools for river management and monitoring. This study aimed to assess the spatial distribution of Ephemeroptera and Trichoptera in mountain streams of central Argentina. We evaluated the effect of seasonality and identified the variables conditioning the abundance of the assemblages and the habitat with the highest taxonomic richness. Sampling was carried out in four streams (Carcarañá River basin) during high and low water periods. Three lotic habitats were sampled: riffles, coarse substrate runs, and fine substrate runs; and physicochemical and habitat variables were measured. We found differences in assemblage composition, among the 25 genera of Ephemeroptera and Trichoptera, at the habitat level. The most important variables affecting distribution patterns were substrate and flow type, but macroalgae and organic matter (twigs, leaves, and detritus) were also important predictors. Riffles maintained the highest richness but when considering only the Ephemeroptera taxa, fine substrate runs emerged also as an important habitat for these taxa. Our study provided valuable ecological information related to habitat preference of taxa with a key role in stream functioning and of great usefulness for the monitoring of lotic systems.

Clarifying the phylogenetic relationships and taxonomy of Stenonema, Stenacron and Maccaffertium, three common eastern North American mayfly genera.

Stenonema, Stenacron, and Maccaffertium are three closely related genera of mayflies (Ephemeroptera:Heptageniidae) commonly found across North America. Due to their primarily aquatic life history and sensitivity to aquatic pollutants, these mayflies are often used as water quality indicators. However, there is little morphological variation within these genera, leading to difficulties in identification and rampant taxonomic confusion, limiting their utility as bioindicators. In an attempt to resolve the phylogenetic relationships of Stenonema, Stenacron, and Maccaffertium, and to clarify their higher-level classifications, we sequenced regions of two mitochondrial genes (cytochrome oxidase subunit 1 (cox1) and 16S ribosomal RNA (rrnl)) and two nuclear genes (Wingless (Wg) and histone H3) from 60 individuals representing most of the described species in these genera and included data from representatives of three heptageniid genera (Kageronia, Macdunnoa and Pseudiron) proposed in previous studies to be closely related to our focal taxa as well as two more distantly related heptageniid genera (Epeorus and Heptagenia) to root the phylogenies. Maximum likelihood and Bayesian analysis were conducted on single-gene and concatenated multi-gene data sets and species tree methods were utilized to resolve relationships. These analyses resolved Stenacron as a monophyletic group sister to a clade comprising Macdunnoa, Maccaffertium and Stenonema. Maccaffertium was found to be paraphyletic, with Stenonema femoratum resolved within Maccaffertium as sister to M. mexicanum. Many relationships remained unresolved or varied across analyses, making revision of the classification based on phylogenetic considerations challenging. To minimize confusion while naming clades and acknowledging uncertainty in our phylogenetic conclusions, we redefine Stenonema to include Maccaffertium and propose three subgenera-Stenonema, Maccaffertium and Lewisa- for key well-supported clades.

Cryptic lineages of a common alpine mayfly show strong life-history divergence.

Understanding ecological divergence of morphologically similar but genetically distinct species - previously considered as a single morphospecies - is of key importance in evolutionary ecology and conservation biology. Despite their morphological similarity, cryptic species may have evolved distinct adaptations. If such ecological divergence is unaccounted for, any predictions about their responses to environmental change and biodiversity loss may be biased. We used spatio-temporally replicated field surveys of larval cohort structure and population genetic analyses (using nuclear microsatellite markers) to test for life-history divergence between two cryptic lineages of the alpine mayfly Baetis alpinus in the Swiss Alps. We found that the more widespread and abundant cryptic lineage represents a 'generalist' with at least two cohorts per year, whereas the less abundant lineage is restricted to higher elevations and represents a 'specialist' with a single cohort per year. Importantly, our results indicate partial temporal segregation in reproductive periods between these lineages, potentially facilitating local coexistence and reproductive isolation. Taken together, our findings emphasize the need for a taxonomic revision: widespread and apparently generalist morphospecies can hide cryptic lineages with much narrower ecological niches and distribution ranges.

Genetic diversity and gene flow decline with elevation in montane mayflies.

Montane environments around the globe are biodiversity 'hotspots' and important reservoirs of genetic diversity. Montane species are also typically more vulnerable to environmental change than their low-elevation counterparts due to restricted ranges and dispersal limitations. Here we focus on two abundant congeneric mayflies (Baetis bicaudatus and B. tricaudatus) from montane streams over an elevation gradient spanning 1400 m. Using single-nucleotide polymorphism genotypes, we measured population diversity and vulnerability in these two species by: (i) describing genetic diversity and population structure across elevation gradients to identify mechanisms underlying diversification; (ii) performing spatially explicit landscape analyses to identify environmental drivers of differentiation; and (iii) identifying outlier loci hypothesized to underlie adaptive divergence. Differences in the extent of population structure in these species were evident depending upon their position along the elevation gradient. Heterozygosity, effective population sizes and gene flow all declined with increasing elevation, resulting in substantial population structure in the higher elevation species (B. bicaudatus). At lower elevations, populations of both species are more genetically similar, indicating ongoing gene flow. Isolation by distance was detected at lower elevations only, whereas landscape barriers better predicted genetic distance at higher elevations. At higher elevations, dispersal was restricted due to landscape effects, resulting in greater population isolation. Our results demonstrate differentiation over small spatial scales along an elevation gradient, and highlight the importance of preserving genetic diversity in more isolated high-elevation populations.

Distribution and population genetic variation of cryptic species of the Alpine mayfly Baetis alpinus (Ephemeroptera: Baetidae) in the Central Alps.

BACKGROUND: Many species contain evolutionarily distinct groups that are genetically highly differentiated but morphologically difficult to distinguish (i.e., cryptic species). The presence of cryptic species poses significant challenges for the accurate assessment of biodiversity and, if unrecognized, may lead to erroneous inferences in many fields of biological research and conservation. RESULTS: We tested for cryptic genetic variation within the broadly distributed alpine mayfly Baetis alpinus across several major European drainages in the central Alps. Bayesian clustering and multivariate analyses of nuclear microsatellite loci, combined with phylogenetic analyses of mitochondrial DNA, were used to assess population genetic structure and diversity. We identified two genetically highly differentiated lineages (A and B) that had no obvious differences in regional distribution patterns, and occurred in local sympatry. Furthermore, the two lineages differed in relative abundance, overall levels of genetic diversity as well as patterns of population structure: lineage A was abundant, widely distributed and had a higher level of genetic variation, whereas lineage B was less abundant, more prevalent in spring-fed tributaries than glacier-fed streams and restricted to high elevations. Subsequent morphological analyses revealed that traits previously acknowledged as intraspecific variation of B. alpinus in fact segregated these two lineages. CONCLUSIONS: Taken together, our findings indicate that even common and apparently ecologically well-studied species may consist of reproductively isolated units, with distinct evolutionary histories and likely different ecology and evolutionary potential. These findings emphasize the need to investigate hidden diversity even in well-known species to allow for appropriate assessment of biological diversity and conservation measures.

[Temperature and daily emergence of seven genera of Ephemeroptera (Insecta) in a cloud forest stream of tropical Andes]. / Temperatura y emergencia diaria de siete géneros de Ephemeroptera (Insecta) en un río de la Selva Nublada de los Andes Tropicales.

Daily emergence of mayflies in Neotropical rivers and their causes have been poorly studied. In temperate zones, this process is better known and attributed to several factors. In this work, we studied the daily emergence of subimagines of several Ephemeroptera genera in La Picón River of a Venezuelan Andean cloud forest and its relation with changes of environmental temperature. Four emergence traps were placed along a reach of 50 m of the stream, each one was examined each two hours in a 24 hr cycle to capture the newly emerged subimagos. This procedure was repeated for eight dates between November-2007 and February-2008 for a total of 32 observations in each sampling hour. The subimagos were reared to adults and identified to genus. The relative density of emergence per trap was calculated for each genus and sampling hour. Water and air temperature were measured each hour during the daily cycle of observation, and the averages of temperature and hour-degrees of air and water were calculated for each hour from the eight dates studied. Seven genera were identified: Leptohyphes Eaton, 1882 and Haplohyphes Allen 1966 (Leptophlebiidae); Prebaetodes Lugo-Ortiz and McCafferty, 1996, Andesiops Lugo-Ortiz and McCafferty, 1999, Baetodes Needham and Murphy, 1924 and Americabaetis Kluge, 1992 (Baetidae); and Thraulodes Ulmer, 1920 (Leptophlebiidae); being the more abundant Leptohyphes (38.4 %) and Thraulodes (20.5 %). The emergence occurred between 11:00 am and 23:00 pm showing the following: a) an emergence initiated during daylight hours by organisms of Leptohyphes, Prebaetodes and Haplohyphes; b) a nocturnal emergence, in Thraulodes, Andesiops, Baetodes and Americabaetis; and c) two peaks: one diurnal produced by Leptohyphes and other nocturnal with predominance of Thraulodes. These results are the first records on the diurnal daily emergence in Andesiops, Prebaetodes, Americabaetis, Haplohyphes, and Leptohyphes, as well as the nocturnal emergence in Thraulodes. It was evidenced that Leptohyphes, with small nymphs (average head width = 1.05 mm) needed to accumulate less hour-degrees to initiate the emergence than those required by Thraulodes whose nymphs are larger (average head width = 2.01 mm). This disparity in the emergence energy requirements must be consequence of differences between the sizes of mature nymphs of both genera; facts which rely on the constancy of sizes shown by these taxa along an altitudinal-thermal gradient and the little daily and seasonal variability of water temperature in La Picón River. In the daily lapse when the emergence occurred, the air and water average temperatures were higher than those registered in the no-emergence lapse; therefore; it is suggested that during the daily lapse, when this process occurs, the environment is thermally favorable for the emergence of subimagos and their survival out of water.

The nymph of Gilliesia Peters & Edmunds, 1970 (Ephemeroptera: Leptophlebiidae), with description of a new species from Thailand.

The new species Gilliesia ratchaburiensis sp. nov. is described based on male and female imagos (some of them reared from nymphal stages), nymphs and eggs collected in western Thailand. The nymph of Gilliesia, which is described for the first time, has bifid gills, a dense patch of setae on the ventral side of the glossae, no posterolateral spines on abdominal segment VIII, maxillary palpi 3-segmented and very reduced maxillary canines. Compared to congeners, the male imagos of the new species have penis lobes more straight and with the apical portion bent laterally but not ventrally, and female abdominal sternum 9 with a U-shaped, deep, median cleft. Phylogenetically, Gilliesia seems to be more similar to Dipterophlebiodes than to Habrophlebiodes and other Leptophlebiinae. The present finding in Thailand expands the distribution of Gilliesia in tropical Southeast Asia.

Effect of land use on mayfly assemblages structure in Neotropical headwater streams.

The aim of this study was to test the effect of agricultural and forestry land use on the structure of mayfly assemblages in low-order streams. Twenty-nine headwater streams were investigated in the state of São Paulo. We analyzed 15 streams in pristine areas (mixed tropical rainforest, semideciduous forest and dense tropical rainforest), and 14 streams covered with sugarcane, eucalyptus and pasture. Mayfly richness obtained by rarefaction curves was higher in pristine areas (21 genera), especially in mixed and semideciduous forest when compared to land use (9 genera), where values were particularly low in sugarcane plantation (3 genera). The non-metric multidimensional scaling (NMDS) ordination showed clear difference in mayfly assemblages between land uses and pristine areas, supported by analysis of similarity (R=0.67, p=0.001). In partial redundancy analysis (pRDA), the environmental descriptors that best explained differences in assemblage structure were Riparian, Channel and Environmental Inventory (RCE) index score, percentage of fine sediment stream substrate, water pH and land elevation. Our results show that agricultural and forestry land use has a strong negative effect on the structure of mayfly assemblages. These results also support the use of mayflies as environmental indicators, as some genera were sensitive to changes in land use, while others responded to naturally occurring changes in the study area.

A new species of Labiobaetis (Ephemeroptera: Baetidae) from the southern Western Ghats in India, with comments on the taxonomic status of Labiobaetis.

A new species of Labiobaetis Novikova & Kluge, 1987, Labiobaetis soldani sp. nov., is described from the larvae and reared male and female imagoes from Gadana River in the southern Western Ghats in India. Brief ecological notes are appended. The taxonomic status of Labiobaetis is commented on in light of the morphological traits of the larvae and associated imagoes.

A new species of Languidipes Hubbard (Ephemeroptera, Polymitarcyidae) from Borneo.

The genus Languidipes is currently represented by three species distributed in southeastern Asia, India, and Sri Lanka. Languidipes corporaali is the most widely distributed species, and both, male and female imagos, as well as nymphs, are known. In contrast, the other species, L. taprobanes and L. lithophagus, are only known from nymphs. Here, we describe a new species, Languidipes janae sp nov, based on male imagos collected from Borneo, Indonesia. This new species is characterized by the presence of ommation on mesonotum, and penis almost completely divided, with sub-quadrate base and a small outer projection basal to the long and slender distal arms. This constitutes the first record of the genus for Borneo. A cladistic analysis of the subfamily Asthenopodinae supports its taxonomic status.

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.

Automated identification of aquatic insects: A case study using deep learning and computer vision techniques.

Deep learning techniques have recently found application in biodiversity research. Mayflies (Ephemeroptera), stoneflies (Plecoptera) and caddisflies (Trichoptera), often abbreviated as EPT, are frequently used for freshwater biomonitoring due to their large numbers and sensitivity to environmental changes. However, the morphological identification of EPT species is a challenging but fundamental task. Morphological identification of these freshwater insects is therefore not only extremely time-consuming and costly, but also often leads to misjudgments or generates datasets with low taxonomic resolution. Here, we investigated the application of deep learning to increase the efficiency and taxonomic resolution of biomonitoring programs. Our database contains 90 EPT taxa (genus or species level), with the number of images per category ranging from 21 to 300 (16,650 in total). Upon completion of training, a CNN (Convolutional Neural Network) model was created, capable of automatically classifying these taxa into their appropriate taxonomic categories with an accuracy of 98.7 %. Our model achieved a perfect classification rate of 100 % for 68 of the taxa in our dataset. We achieved noteworthy classification accuracy with morphologically closely related taxa within the training data (e.g., species of the genus Baetis, Hydropsyche, Perla). Gradient-weighted Class Activation Mapping (Grad-CAM) visualized the morphological features responsible for the classification of the treated species in the CNN models. Within Ephemeroptera, the head was the most important feature, while the thorax and abdomen were equally important for the classification of Plecoptera taxa. For the order Trichoptera, the head and thorax were almost equally important. Our database is recognized as the most extensive aquatic insect database, notably distinguished by its wealth of included categories (taxa). Our approach can help solve long-standing challenges in biodiversity research and address pressing issues in monitoring programs by saving time in sample identification.

The adults of Camelobaetidiusjanae Dominique & Thomas, 2001 and C. yacutinga Nieto, 2003, with a new synonym for C. leentvaari Demoulin, 1966 (Ephemeroptera: Baetidae).

The genus Camelobaetidius Demoulin, 1966 has 39 species distributed throughout the Nearctic and Neotropical Regions, being one of the most studied genera of the family Baetidae. Based on material from Brazil, the aim of the present paper is to describe adults of C. janae Dominique & Thomas, 2001 and C. yacutinga Nieto, 2003, and to recognize a new synonym for Camelobaetidius leentvaari Demoulin, 1966 (= C. mantis Traver & Edmunds, 1968 nov. syn.) based on the examination of type material.

New species of Hermanella complex (Ephemeroptera: Leptophlebiidae) from Brazilian Atlantic Forest.

In the present work, based on material from Brazilian Atlantic Forest, four new species of the Hermanella complex are described. The main characteristics that distinguish the new species from its congeners are, in Hermanella amere sp. nov.: (1) subgenital plate yellow, with wide projection near base of forceps; (2) penis lobe with ventral, robust, posterioly directed spine; in Hermanella nigra sp. nov.: (1) subgenital plate brown washed with gray, with wide projection near inner base of forceps; (2) penis lobe with a distomedial membranous projection and ventral, robust, posteriorly directed spine; in Hylister obliquus sp. nov.: (1) subgenital plate yellowish brown, with pointed projection near inner base of forceps; (2) penis lobe with ventral, short, narrow, posteromedially directed spine; in Traverella insolita sp. nov.: (1) subgenital plate strongly projected posteriorly, forming three broad and short projections; (2) penis lobe laterally sinuous and apically rounded, with a ventral, long, narrow spine curved toward the midline of the body. Modified keys of male imagos are provided for the three genera, whereas comments regarding their taxonomy are presented. Additionally, Hermanella mazama (Nascimento, Mariano & Salles 2012 in Lima et al. 2012), comb. nov., is proposed.

A new species of Prosopistoma (Ephemeroptera: Prosopistomatidae) from China with a key to Oriental species.

A new species of the monogeneric family Prosopistomatidae, Prosopistoma ocellatum sp. n., is described and illustrated based on mature larval stages from Guangxi and Hainan, southern China. The new species can be readily distinguished from the other members of Prosopistoma by the following combination of characters: antenna 6-segmented, segment III much longer than segments IV-VI; three bristles at the base of the inner canine; 10-11 pectinate setae on ventral margin of fore tibiae, ventral and basal half surface of all femora with dense scale-like structures, and color pattern of eye-spot on the mesonotum. An update key to the known Oriental species is provided.

Thraulodes bonito sp. nov. (Ephemeroptera: Leptophlebiidae) from Rio de Janeiro State, Southeastern Brazil.

A new species of Thraulodes Ulmer, 1920 is described from Rio de Janeiro. The species resembles the recently published Thraulodespelicanus Mariano and Froehlich, 2011 (in Mariano et al. 2011) due to the length and width of the penes. However, Thraulodes bonito sp. nov. differs from this and all other species of the genus by the wide penes, the triangular shape of the styliger plate with median projection short and apex rounded, the abdominal color pattern and the presence of 1-3 weakly marked cross veins basal to bullae. The new species is described based on male imagos, female subimagos and nymphs.

Paracloeodes caldensis (Ephemeroptera: Baetidae), an atypical new species from the Colombian Andes.

In the present work, an atypical new species of Paracloeodes Day (Ephemeroptera: Baetidae) is described based on nymphs collected in the Caldas Department, Central Cordillera of Colombia. This represents the first report of the genus from Colombia. The main characteristics that distinguish the new species from congeners are: abundant setae on femora, antennae length twice the width of the head capsule, maxilla with palp twice the length of the galea-lacinia, segment II of the labial palp with a rounded projection and 2.5 times the width of segment III, tarsal claws half the length of their respective tarsi and the abdominal color pattern. The concepts of the genera Paracloeodes, Varipes and Rivudiva are discussed in light of the discovery of P. caldensis n. sp.