The function of wing bullae in mayflies (Insecta: Ephemeroptera) reveals new insights into the early evolution of Pterygota.

BACKGROUND: Mayflies are basal winged insects of crucial importance for the understanding of the early evolution of Pterygota. Unlike all other insects, they have two successive winged stages, the subimago and the imago. Their forewings feature so-called bullae, which are desclerotized spots in the anterior main veins. Up to now, they have been considered to play a major role in wing bending during flight. RESULTS: We investigated bullae by multiple methods to reveal their structure and arrangement and to gain new information on the evolution of insect flight. Bullae are mostly present in the anterior negative wing veins, disrupting the otherwise rigid veins. High-speed videography reveals that mayfly wings do not bend during flight. Likewise, different arrangements of bullae in different species do not correlate with different modes of flying. Observations on the moulting of subimagines unravel that they are essential for wing bending during the extraction of the imaginal wing from the subimaginal cuticle. Bullae define predetermined bending lines, which, together with a highly flexible wing membrane enriched with resilin, permit wing bending during subimaginal moulting. Bullae are only absent in those species that remain in the subimaginal stage or that use modified modes of moulting. Bullae are also visible in fossil mayflies and can be traced back to stemgroup mayflies of the Early Permian, the 270 million years old Protereismatidae, which most probably had bullae in both fore- and hind wings. CONCLUSIONS: Bullae in mayfly wings do not play a role in flight as previously thought, but are crucial for wing bending during subimaginal moulting. Thus, the presence of bullae is a reliable morphological marker for a subimaginal life stage, confirming the existence of the subimago already in Permian Protereismatidae. A thorough search for bullae in fossils of other pterygote lineages may reveal wheather they also had subimagines and at what point in evolution this life stage was lost. In mayflies, however, the subimago may have been retained due to selective advantages in connection with the transition from aquatic to terrestrial life or due to morphological requirements for a specialized mating flight.

A new Gondwanan mayfly family from the Lower Cretaceous Crato Formation, Brazil (Ephemeroptera: Siphlonuroidea: Astraeopteridae fam. nov.).

The adult holotype of the fossil mayfly Astraeoptera cretacica Brandão et al. 2021 from the Cretaceous Crato Formation, Brazil, is reviewed and attributed to a new family Astraeopteridae fam. nov. Based on alate specimens, we also describe further new representatives of Astraeopteridae fam. nov., namely Astraeoptera vitrea sp. nov. and Astraeoptera oligovenata sp. nov., as well as the new genus and species Eosophobia acuta gen. et sp. nov. A subsequent character analysis of the new material suggests systematic affinities of Astraeopteridae fam. nov. with those extant families of Siphlonuroidea distributed in the Southern Hemisphere. These newly described fossil Siphlonuroidea from the Cretaceous of Brazil thus add to the biogeography and systematics of mayflies.

South African Lagerstätte reveals middle Permian Gondwanan lakeshore ecosystem in exquisite detail.

Continental ecosystems of the middle Permian Period (273-259 million years ago) are poorly understood. In South Africa, the vertebrate fossil record is well documented for this time interval, but the plants and insects are virtually unknown, and are rare globally. This scarcity of data has hampered studies of the evolution and diversification of life, and has precluded detailed reconstructions and analyses of ecosystems of this critical period in Earth's history. Here we introduce a new locality in the southern Karoo Basin that is producing exceptionally well-preserved and abundant fossils of novel freshwater and terrestrial insects, arachnids, and plants. Within a robust regional geochronological, geological and biostratigraphic context, this Konservat- and Konzentrat-Lagerstätte offers a unique opportunity for the study and reconstruction of a southern Gondwanan deltaic ecosystem that thrived 266-268 million years ago, and will serve as a high-resolution ecological baseline towards a better understanding of Permian extinction events.

Cenozoic origins of the genus Calliarcys (Insecta, Ephemeroptera) revealed by Micro-CT, with DNA barcode gap analysis of Leptophlebiinae and Habrophlebiinae.

Mayflies (Ephemeroptera) are among the oldest pterygote insects, with the earliest fossils dating back to the Late Carboniferous. Within mayflies, Leptophlebiidae are a highly diverse and widespread group, with approximately 140 genera and 640 species. Whereas taxonomy, systematics, and phylogeny of extant Leptophlebiidae are in the focus of extensive studies, little is known about leptophlebiid fossil taxa. Because fossil remains of Ephemeroptera in sedimentary rocks are relatively rare, inclusions of mayflies in amber are a unique source of information on their evolution and diversity in the past. Leptophlebiidae found in Cenozoic resins mostly belong to the subfamilies Leptophlebiinae (in Eocene Baltic amber) and Atalophlebiinae (in Miocene Dominican and Mexican ambers). In the present contribution, we confirm the first finding of the genus Calliarcys from Eocene Baltic amber by using Micro-CT, which allowed confirming its generic placement by visualizing diagnostic key characters otherwise hidden by a cloud of turbidity. Additionally, we present first molecular data on the extant species Calliarcys humilis Eaton, 1881 from the Iberian Peninsula and the barcode gap analysis for Leptophlebiinae and Habrophlebiinae.

Evolution of flexible biting in hyperdiverse parasitoid wasps.

One key event in insect evolution was the development of mandibles with two joints, which allowed powerful biting but restricted their movement to a single degree of freedom. These mandibles define the Dicondylia, which constitute over 99% of all extant insect species. It was common doctrine that the dicondylic articulation of chewing mandibles remained unaltered for more than 400 million years. We report highly modified mandibles overcoming the restrictions of a single degree of freedom and hypothesize their major role in insect diversification. These mandibles are defining features of parasitoid chalcid wasps, one of the most species-rich lineages of insects. The shift from powerful chewing to precise cutting likely facilitated adaptations to parasitize hosts hidden in hard substrates, which pose challenges to the emerging wasps. We reveal a crucial step in insect evolution and highlight the importance of comprehensive studies even of putatively well-known systems.

First fossil record of the mayfly family Vietnamellidae (Insecta, Ephemeroptera) from Burmese Amber confirms its Oriental origin and gives new insights into its evolution.

The small, monophyletic mayfly family Vietnamellidae Allen, 1984 has so far only been known from a few extant species of the genus Vietnamella Tshernova, 1972, which are all distributed in the Oriental Realm (Vietnam, Thailand, China, and India). Herein we report the first fossil record of Vietnamellidae based on a male and female imago from Mid-Cretaceous Burmese amber. We establish the new genus Burmella gen. nov. to accommodate these two new Mesozoic specimens. Their attribution to Vietnamellidae is supported by the rounded shape of the hind wings with arched outer margin, the course of thoracic sutures, and characteristics of venation, especially of MP and Cu of the forewings and associated intercalary veins of the cubital field. At the same time, Burmella gen. nov. clearly differs from Vietnamella by a diminished number of longitudinal and cross veins in the hind wings, and by the different shape of male genitalia. This first fossil record of Vietnamellidae supports an age of at least 100 Ma for this taxon.

Diversity and distribution of Epeorus (Caucasiron) (Ephemeroptera, Heptageniidae) in Iran, with descriptions of three new species.

Combining morphological and molecular data in an integrative approach, three new mayfly species of Epeorus (Caucasiron) are described. These include Epeorus (Caucasiron) alborzicus Hrivniak & Sroka, sp. nov. and Epeorus (Caucasiron) shargi Hrivniak & Sroka, sp. nov. from northern Iran, and Epeorus (Caucasiron) zagrosicus Hrivniak & Sroka, sp. nov. from central Iran. They are unambiguously delimited using both distance-based and likelihood-based approaches in the analyses of barcode COI sequences. Each new species is compared with other species of the subgenus and morphological diagnostic characters are provided. Based on extensive sampling of streams throughout the country, the distribution and habitat preferences of all Caucasiron species in Iran are assessed. Altogether, there are now six species recorded, among them also E. (C.) nigripilosus Sinitshenkova, 1976 is reported for the first time in Iran. Five species are distributed in the Alborz Mts. in northern Iran, one species was found in the Zagros Mts. in central Iran.

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.

Revision of fossil Metretopodidae (Insecta, Ephemeroptera) in Baltic amber - Part 4: Description of two new species of Siphloplecton Clemens, 1915, with notes on the new S. jaegeri species group and with key to fossil male adults of Siphloplecton.

The Siphloplecton jaegeri species group is established here for three extinct species, namely for the earlier described Siphloplecton jaegeri Demoulin, 1968, and for two new species from Eocene Baltic amber, Siphloplecton landolti sp. nov. and Siphloplecton studemannae sp. nov. Based on the well-preserved specimens of these species, a diagnosis is provided for the newly established species group. Representatives of the S. jaegeri species group are characterized by the presence of large, medially contiguous eyes, stout pointed setae along the outer margin of the foretibia, three intercalaries in the cubital field of the forewing, and elongated penis lobes, which are apically triangular or rounded, medially contiguous, and with a V-shaped cleft apically. Further new specimens of the S. jaegeri species group are documented that cannot be attributed to species level due to their poor preservation. Finally, a key to male adults of fossil species of Siphloplecton is given.

New Oligoneuriidae (Insecta, Ephemeroptera) from Iran.

Two new species of the mayfly family Oligoneuriidae are described based on larval specimens recently collected in Iran. The first new species, Oligoneuriella tuberculata Godunko & Staniczek, sp. nov., can be distinguished from all its congeners by the presence of pronounced protuberances posteromedially on abdominal terga, highly reduced paracercus, large lamella of gill I, and setation on hind margin of middle and hind femora confined to their basal halves. The second species, Oligoneuriopsis villosus Bojková, Godunko, & Staniczek, sp. nov., remarkably belongs to a mostly Afrotropical genus. The new species clearly differs from all its congeners in the shape of setae on the surface of gills and terga, pattern of body colouration, and the shape of posterolateral projections of abdominal segments. Except for the species description, the generic diagnosis of Oligoneuriopsis Crass, 1947 is briefly discussed. COI barcode sequences of both new species are provided and molecular species delimitation is tested using distance-based and likelihood-based approaches, with both new species unambiguously recognised as separate lineages. The analysis of COI also corroborates the respective affinities of both new species, estimated based on morphology. The two new species of Oligoneuriidae described herein highlight the importance of the Middle East as a centre of diversity of this mayfly family within the Palaearctic.

'Rolling' stoneflies (Insecta: Plecoptera) from mid-Cretaceous Burmese amber.

This contribution describes seven new species of fossil stoneflies from Cretaceous Burmese amber, all of which are dedicated to present and past members of the Rolling Stones. Two species-Petroperla mickjaggeri gen. nov. sp. nov. and Lapisperla keithrichardsi gen. nov. sp. nov.-are placed in a new family Petroperlidae within the stemline of Systellognatha. The first Cretaceous larval specimen of Acroneuriinae, Electroneuria ronwoodi gen. nov. sp. nov., is also described along with another four new species that are placed within the Acroneuriinae genus Largusoperla Chen et al., 2018: Largusoperla charliewattsi sp. nov., Largusoperla brianjonesi sp. nov., Largusoperla micktaylori sp. nov., and Largusoperla billwymani sp. nov. Additional specimens of Acroneuriinae are described without formal assignment to new species due to insufficient preservation. Implications for stonefly phylogeny and palaeobiogeography are discussed.

Initial commented checklist of Iranian mayflies, with new area records and description of Procloeon caspicum sp. n. (Insecta, Ephemeroptera, Baetidae).

An initial checklist of mayflies (Ephemeroptera) of Iran is compiled based on critical review of available literature data, complemented with new data from 38 localities of Gilan and Ardabil provinces. At present, altogether only 46 species and 25 genera are known from Iran, 18 species are reported as new to Iran in this study. Some previously published data are critically evaluated and doubtful taxa are excluded from the list. Basic analysis of the distribution and biogeography of recorded species is given. Procloeon (Pseudocentroptilum) caspicum Sroka, sp. n. is described based on mature larva and egg. Critical differential diagnostic characters distinguishing the species from related taxa are discussed in detail.

Baetis (Baetis) cypronyx sp. n., a new species of the Baetis alpinus species-group (Insecta, Ephemeroptera, Baetidae) from Cyprus, with annotated checklist of Baetidae in the Mediterranean islands.

A detailed description of the larvae of Baetis (Baetis) cypronyx sp. n., a representative of the Baetis alpinus species-group within the mayfly family Baetidae, is provided, including a differential diagnosis with regard to closely related species of the group, especially Baetis melanonyx (Pictet, 1843) and Baetis baroukianus Thomas & Dia, 1984. The new species is mainly distinguished by mouthparts (i.e. the shape and setation of labrum, maxillary and labial palps, details of paraglossae and mandibular incisors), setation of legs and abdominal terga, and length of paracercus. All available data on the biology of this putative endemic species of Cyprus are summarized. Annotated distributional data of the 33 species of Baetidae so far recorded from the Mediterranean islands are given, including new records and also including first data from Malta.

Revision of fossil Metretopodidae (Insecta: Ephemeroptera) in Baltic amber-Part 3: Description of two new species of Siphloplecton Clemens, 1915, with notes on the re-discovered lectotype of Siphloplecton macrops (Pictet-Baraban & Hagen, 1856).

In this contribution, we provide a complementary description of the re-discovered lectotype of Siphloplecton macrops (Pictet-Baraban & Hagen, 1856). Additionally, complementary descriptions of S. barabani Staniczek & Godunko, 2012 and S. picteti Staniczek & Godunko, 2012 based on new material are given. Two new species, S. sartorii sp. nov. and S. gattolliati sp. nov., are described from male imagines and attributed to two newly defined species groups within Siphloplecton.

New fossil insect order Permopsocida elucidates major radiation and evolution of suction feeding in hemimetabolous insects (Hexapoda: Acercaria).

With nearly 100,000 species, the Acercaria (lice, plant lices, thrips, bugs) including number of economically important species is one of the most successful insect lineages. However, its phylogeny and evolution of mouthparts among other issues remain debatable. Here new methods of preparation permitted the comprehensive anatomical description of insect inclusions from mid-Cretaceous Burmese amber in astonishing detail. These "missing links" fossils, attributed to a new order Permopsocida, provide crucial evidence for reconstructing the phylogenetic relationships in the Acercaria, supporting its monophyly, and questioning the position of Psocodea as sister group of holometabolans in the most recent phylogenomic study. Permopsocida resolves as sister group of Thripida + Hemiptera and represents an evolutionary link documenting the transition from chewing to piercing mouthparts in relation to suction feeding. Identification of gut contents as angiosperm pollen documents an ecological role of Permopsocida as early pollen feeders with relatively unspecialized mouthparts. This group existed for 185 million years, but has never been diverse and was superseded by new pollenivorous pollinators during the Cretaceous co-evolution of insects and flowers. The key innovation of suction feeding with piercing mouthparts is identified as main event that triggered the huge post-Carboniferous radiation of hemipterans, and facilitated the spreading of pathogenic vectors.

Towards an 18S phylogeny of hexapods: accounting for group-specific character covariance in optimized mixed nucleotide/doublet models.

The phylogenetic diversification of Hexapoda is still not fully understood. Morphological and molecular analyses have resulted in partly contradicting hypotheses. In molecular analyses, 18S sequences are the most frequently employed, but it appears that 18S sequences do not contain enough phylogenetic signals to resolve basal relationships of hexapod lineages. Until recently, character interdependence in these data has never been treated seriously, though possibly accounting for the occurrence of biased results. However, software packages are readily available which can incorporate information on character interdependence within a Bayesian approach. Accounting for character covariation derived from a hexapod consensus secondary structure model and applying mixed DNA/RNA substitution models, our Bayesian analysis of 321 hexapod sequences yielded a partly robust tree that depicts many hexapod relationships congruent with morphological considerations. It appears that the application of mixed DNA/RNA models removes many of the anomalies seen in previous studies. We focus on basal hexapod relationships for which unambiguous results are missing. In particular, the strong support for a "Chiastomyaria" clade (Ephemeroptera+Neoptera) obtained in Kjer's [2004. Aligned 18S and insect phylogeny. Syst. Biol. 53, 1-9] study of 18S sequences could not be confirmed by our analysis. The hexapod tree can be rooted with monophyletic Entognatha but not with a clade Ellipura (Collembola+Protura). Compared to previously published contributions, accounting for character interdependence in analyses of rRNA data presents an improvement of phylogenetic resolution. We suggest that an integration of explicit clade-specific rRNA structural refinements is not only possible but an important step in the optimization of substitution models dealing with rRNA data.

A hexapod nuclear SSU rRNA secondary-structure model and catalog of taxon-specific structural variation.

RNA molecules and in particular the nuclear SSU RNA play an important role in molecular systematics. With the advent of increasingly parameterized substitution models in systematic research, the incorporation of secondary-structure information became a realistic option compensating interdependence of character variation. As a prerequisite, consensus structures of eukaryotic SSU RNA molecules have become available through extensive comparative analyses and crystallographic studies. Despite extensive research in hexapod phylogenetics, consensus SSU RNA secondary structures focusing on hexapods have not yet been explored. In this study, we compiled a representative hexapod SSU data set of 261 sequences and inferred a specific consensus SSU secondary-structure model. Our search for conserved structural motives relied on a combined approach of thermodynamic and covariation analyses. The hexapod consensus-structure model deviates from the canonical eukaryotic model in a number of helices. Additionally, in several helices the hexapod sequences did not support a single consensus structure. We provide consensus structures of these sections of single less-inclusive taxa, thus facilitating the adaptation of the consensus hexapod model to less-inclusive phylogenetic questions. The secondary-structure catalog will foster the application of RNA structure models in phylogenetic analyses using the SSU rRNA molecule, and it will improve the realism of substitution models and the reliability of reconstructions based on rRNA sequences.