A new Cinygmula McDunnough, 1933 species with distinct imaginal frontal fold from eastern Chinese Himalaya (Ephemeroptera: Heptageniidae).

In the family Heptageniidae (Ephemeroptera), Cinygmula hutchinsoni (Traver, 1939) from central Asia is the only species known to possess pointed claws and a distinct frontal fold. Here, a second similar species with the same combination of characters is described. Cinygmula longissima sp. nov. is found in the eastern Himalaya of China, shows stouter imaginal penes, darker wingbases, and narrower nymphal gills than the previous one. This discovery indicates that this species of Heptageniidae are not limited to central Asia but live in high-altitude alpine habitats.

Life history and mitochondrial genomes of Salassinae and Agliinae (Insecta, Lepidoptera): New insights into the loss of cocooning behaviour and phylogeny of Saturniidae.

The subfamilies Salassinae and Agliinae are two monogeneric groups of the family Saturniidae. They were regarded as the non-cocooning saturniids in Asia. Since very little information on their life history and mitogenome has been reported, their origin and evolution are still poorly understood. In this study, nature-imitated rearing is used to record the life history of two Aglia and five Salassa species. In addition, four complete mitogenomes are presented, which are the first ones of these two subfamilies. The results show that both Salassinae and Agliinae have lost their cocooning. Moreover, the phylogenetic analysis demonstrates that the subfamily Saturniinae is not monophyletic due to the inclusion of Agliinae and Salassinae.

The Exact Morphology of the Species Ephemera pieli Navás, 1934 from Eastern China (Ephemeroptera: Ephemeridae).

The original description and outline figure of the mayfly Ephemera pieli Navás, 1934, were based on male subimagoes, and its morphology and taxonomic status have not been confirmed since then. However, recently collected materials from eastern China, including all stages of this mayfly, reveal several unique characteristics that indicate E. pieli is a valid species in the subgenus Ephemera (Ephemera). These characteristics include forewings with numerous dark dots, hindwings or hind wingpads with one or two distinct median dots, penes covered mostly by the subgenital plate, and the apex of the penis bluntly extended. A neotype is designated.

Report of intersex individuals from a southeastern Chinese Choroterpes facialis (Gillies, 1951) population (Ephemeroptera: Leptophlebiidae).

Previously there were only three cases of mayfly gynandromorphism reported from Asia, occurring in the families Baetidae and Heptageniidae. Here, we report two intersex individuals of Choroterpes facialis (Gillies, 1951) (Ephemeroptera: Leptophlebiidae) from southeastern China. They have similar external morphologies (each having two different eyes, two shortened penes, and female sternum IX) but with one being predominately male and the other being predominately female (one with eggs in the abdomen, but the other apparently with sperm). We believe this to be the first report of a feminized male individual. This phenomenon implies their intersexuality is caused by some similar reasons, such as temperature or parasitism. Remarkably, two intersex specimens found among 1,736 normal individuals shows that gynandromorphism does occur rarely, and only six normal males in the sampling suggest the species C. facialis is partially parthenogenetic at least.

The imaginal and nymphal morphology of Gilliesia pulchra Zhou, 2004 and its contribution to understanding generic phylogeny (Ephemeroptera: Leptophlebiidae).

The nymphs of the leptophlebiid mayfly Gilliesia pulchra Zhou, 2004 have not been reported before, which inhibits deeper understanding of the taxonomy and phylogenetic position of this species and its genus. In 2022, we reared nymphs from southwestern China to the adult stage. All stages are described and photographed here. Nymphs of this species have single deeply-forked gills, somewhat elongated maxillary palpi and two setal tufts on the lingua. Its imaginal characteristics, including wings and penes, are quite similar to G. hindustanica (Gillies, 1951), the type species of the genus. Comparatively, the third species of the genus, G. ratchaburiensis Boonsoong & Sartori, 2015, seems less similar, both in adults and nymphs. Generally, features of G. pulchra nymphs link the two genera Gilliesia Peters & Edmunds, 1970 and Habrophlebiodes Ulmer, 1919 together, while features of the nymphs of G. ratchaburiensis bridge the genera Gilliesia and Dipterophlebiodes Demoulin, 1954.

A preliminary review of Isonychia Eaton, 1871 from Chinese mainland with a re-description of I.kiangsinensis Hsu, 1936 (Insecta, Ephemeroptera, Isonychiidae).

Previously, seven species of the genus Isonychia Eaton, 1871 were reported in China, but they have never been systematically reviewed. After examining our collections from the Chinese mainland, six species and one additional subspecies have been recognized, compared, and photographed. Among them, I.kiangsinensis is redescribed in all stages and a neotype is designated. Its males have triangular penes and nymphs have three dark pigments on each gill. A synonym of I.guixiensisWu et al., 1992 (I.sinensisWu et al., 1992) is confirmed. The males of this species have nearly cylindrical penes and clear abdominal markings. Finally, two species and one subspecies are recorded for the first time in China: I.ussuricasibiricaTiunova et al., 2004, I.ussuricaussurica Bajkova, 1970 and I.vshivkovaevshivkovaeTiunova et al., 2004. Together with the I.ignota (Walker, 1853), I.sexpetalaTiunova et al., 2004, I.formosana (Ulmer, 1912) and possible I.japonica (Ulmer, 1920), they show the rich diversity of the genus Isonychia in China.

A new Neoleptophlebia Kluge, 1997 species from eastern China (Ephemeroptera: Leptophlebiidae).

The genus Neoleptophlebia Kluge, 1997 includes five Asian species. Three of them were reported from northeastern Asia and two were found from Chinese Taiwan Island, leaving a huge geographic gap on the Chinese mainland. Here we find a new one, which is named N. uncinata Zhou sp. nov., from Nanjing municipality, eastern China. Via field collecting and indoor rearing, all life stages were obtained, and its nymphs are found living in small creeks (with a width less than 1 m) and shallow waters (with a depth less than 30 cm). Diagnostically, the imago of this new species has larger lateral penial appendages than its congeners, and its nymph has subequal broadened segments II and III of maxillary and labial palpi. Biogeographically, this species bridges two northern and southern groups of the genus.

A contribution to knowledge of Procloeon Bengtsson, 1915 from the Chinese mainland (Ephemeroptera: Baetidae) with description of a new species.

The mayflies of the genus Procloeon Bengtsson, 1915 are common insects in China. However, partially because of its complicated taxonomic history and numerous species, this genus has never been revised locally in China. Here, we review all reported and related species from the Chinese mainland and confirmed three species of Procloeon occurred namely, Procloeon pennulatum (Eaton, 1870) (= Centroptilum henanensis Zhou et al., 1997, syn. n.) distributed in northern China, P. chinensis (She et al., 1995) (comb. nov.) from southern China and P. strumiferum sp. n. found in eastern China. The imagoes of the new species lack a costal projection of the hindwings but the gills IV of the nymphs have double lamellae. In contrast, both the former species have costal process in the hindwings and double lamellae of gills IVI. The three species can all be differentiated by imaginal genitalia and nymphal color. This revision clarifies all historical species in the genus Procloeon and Centroptilum from the Chinese mainland and provides a base for further research.

A Review of the Genus Serratella Edmunds, 1959 in China with Description of a New Species (Ephemeroptera: Ephemerellidae).

Species in the genus Serratella Edmunds, 1959 from China have never been compared and photographed systematically. Six valid Chinese Serratella species are recognized and revised in this paper. Among them, the imagos of S. brevicauda Jacobus et al., 2009 are unknown; the nymph of this species has a stout, strong body, with remarkably short caudal filaments and maxillary palpi. In contrast, only the imago stage of Serratella fusongensis (Su and You, 1988) (=Serratella longipennis Zhou et al., 1997, syn. nov.) is known; it has relatively long penes with small dorsal projections. The nymphs of S. setigera Bajkova, 1967 have small abdominal tergal spines but distinct, stout, blunt bristles on the spines, and the apexes of the male penes are round and shallowly divided. The fourth species, S. acutiformis sp. nov., which was collected from Western China, has sharp penial apexes (imagos) and large abdominal spines (nymphs). Unlike the former four species, S. ignita (Poda, 1761) and S. zapekinae Bajkova, 1967 has sub-quadrate penes without prominent dorsal projections. The nymph of S. ignita has lateral hair-like setae on the caudal filaments, while the nymph of S. zapekinae lacks such setae but has pairs of tubercles on the head and pronotum. Some characters used in the generic delineation of the genera Ephemerella Walsh, 1862 and Serratella, such as nymphal maxillary palpi and hair-like setae on caudal filaments as well as features of the imaginal penes and forelegs, are varied in Chinese species. However, all species in this paper have bifurcate ventral lamellae of gill VI. Our work highlights a need for further comparative systematic study of the genera Serratella, Ephemerella, and another related genus Torleya Lestage, 1917.

Descriptions and Barcoding of Five New Chinese Deuterophlebia Species Revealing This Genus in Both Holarctic and Oriental Realms (Diptera: Deuterophlebiidae).

The monotypic family Deuterophlebiidae of China was recorded twice previously from far northwest upon adults, the most parts of this country have not been investigated, leaving a huge blank of knowledge on their morphology, diversity, biology, or distribution. After deliberated collecting and rearing in recent years, we obtained more than one thousand specimens of Deuterophlebiidae, they are classified into five new species herein: Deuterophlebia sinensis sp. nov., D. yunnanensis sp. nov., D. wuyiensis sp. nov., D. acutirhina sp. nov. and D. alata sp. nov. Detailed descriptions and photographs of gathered life stages are given for these new species. Adults of them can be identified by chaetotaxy and length ratio of flagellomeres and legs, microtrichia on postgena and shape of their clypeus, pupae can be recognized by thoracic spines and abdominal chitin bands, and larvae can be separated by setae on thorax and abdomen. Genetic distances between species are 0.086-0.175 based on their COI genes. This contribution represents the first database of the enigmatic Deuterophlebiidae from China and shows a new distribution pattern of Deuterophlebia. In addition, the discovery throws some light on the origin and biogeography of the genus and family.

Comparative Mitogenome Analyses of Subgenera and Species Groups in Epeorus (Ephemeroptera: Heptageniidae).

Epeorus Eaton, 1881 is a diverse mayfly genus in Heptageniidae comprising more than 100 species which are further divided into nine subgenera and several species groups. However, the classification and the phylogenetic relationships among them are still uncertain. Here, 15 complete mitochondrial genomes of Epeorus were sequenced and compared together with six available ones of same genus in the NCBI database. Based on morphological classification, the 21 mitogenomes were classified into six subgenera (Proepeorus, Epeorus s.str., Belovius, Iron, Caucasiron and Siniron) and four species groups (G1, G2, montanus and longimanus). Among all analyzed mitogenomes, the gene rearrangement of trnI-trnM-trnQ-NCR-ND2 was first found occurring in three species of group G1, whereas the gene block trnI-trnM-trnQ-trnM-ND2 was observed in all other mitogenomes of Epeorus. Furthermore, the genetic composition and codon usage of species in group G1 were also significantly different from all other Epeorus species, except group longimanus. The intergenic spacer between trnA and trnR, which has the stem-loop secondary structure, occurred in all 21 mitogenomes, and the sequences of stems and loops were conserved within species groups. Furthermore, the phylogenetic analyses strongly support the monophyly of all species groups, although three of six recognized subgenera Proepeorus, Belovius, and Iron, were shown as the non-monophyletic groups.

A detailed comparison of two species in the genus Potamanthus Pictet, 1843 from China (Ephemeroptera, Potamanthidae).

Photographs and details of structures of two Potamanthus species, P.huoshanensis Wu, 1987 and P.luteus (Linnaeus, 1767), are presented for the first time. Here, based upon Chinese specimens of those species, all external structures are illustrated digitally and compared. The results and photos clearly show that the adults of the two species are different in wing color and genitalia shape, and that their nymphs have different mandibular tusks and forelegs. Specifically, P.luteus has a more colorful body and wings, longer penes and nymphal mandibular tusks but shorter foretarsi than those of P.huoshanensis. This comparison not only confirms the differences between these two similar species, but also supports the updated generic delineations of Potamanthus and Potamanthodes.

Systematics of Potamanthodes nanchangi Hsu, 1936 (Ephemeroptera: Potamanthidae).

The enigmatic mayfly Potamanthodes nanchangi Hsu, 1936 was briefly described based on a male. Currently, this species has an unclear taxonomic position and no supporting type specimen, consequently its recognition, differentiation and classification is vague. In this study, by using fresh specimens from type locality in Jiangxi province at central China, this species is redescribed, photographed, and a neotype is designated. This species evidently belongs to the genus Potamanthodes Ulmer, 1920 by its short mandibular tusks (shorter than head), presence of setae row on forefemora of nymphs, slender and deeply bifurcated V-shaped penes, common base of MP2 and CuA of forewings. Significant morphological characters defining this species are relatively long penial tips, greatly bent R1 of hindwings and small compound eyes of the males, large mandibular tusks and long forelegs of the nymphs. Therefore, the P. nanchangi is indeed a valid and apomorphic species in the genus Potamanthodes.

A Review of the Genus Cloeon from Chinese Mainland (Ephemeroptera: Baetidae).

The widely distributed and species diverse genus Cloeon Leach, 1815 has never been reviewed in China, although nine species names have been reported from this country. After checking types of two species (C. viridulum and C. apicatum) and newly collected materials from more than 50 sites, four species are recognized, compared, and photographed in this research. Among them, the species C. bicolor Kimmins, 1947 is first recorded in China, and its nymphs are described for the first time. The distribution in China of two other species (C. dipterum (Linnaeus, 1761) and C. harveyi (Kimmins, 1947)) are also confirmed. The two species established by Navás (C.apicatum Navás, 1933 = C. navasi Bruggen, 1957, C. pielinum Navás, 1933, syn. nov.) and the species C. micki named by Tong and Dudgeon in 2021 are synonymized with C. viridulum Navás, 1931-the fourth species in this study. Furthermore, the C. virens Klapálek, 1905 from the Chinese mainland, found by Ulmer in 1925, is also regarded as the last species. Among the four species, the C. dipterum and C. harveyi seem closer because of the similar female color patterns of their stigma and ventral abdomen, while the species C. viridulum and C. bicolor are more alike because they have neither pigmented stigma nor stripes on abdominal sterna.

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.

The First Described Nymphs and Detailed Imagoes of the Species Thalerosphyrus cingulatus Navás Revealing a New Mayfly Genus from Eastern China (Ephemeroptera: Heptageniidae, Ecdyonurinae).

The nymph and detailed imaginal morphology of the Chinese mayfly Thalerosphyrus cingulatus have not been reported since it was named by Navás in 1933. Here based on newly collected nymphal and associated imaginal materials of this species from eastern China, we find both nymphs and adults have several extraordinary characters. In adults, forewings have less crossveins in costal and subcostal sections, others crossveins arranged into five regular rows; tibiae and tarsi of midlegs and hindlegs subequal in length; male compound eyes widely separated and penes simple and fused. The nymphs have greatly extended lateral pronotum, round supracoxal spurs and extended dorsal lamellae of gills, especially those of the gills VII; maxillae have two independent distal dentisetae. These combined characters represent a new generic taxon. Therefore, Regulaneuria Zhou, gen. nov. is established here to include the species Regulaneuria cingulata (Navás, 1933) comb. nov. The forewing venation of the species R. cingulata is unique in the family Heptageniidae but similar to some counterparts of Leptophlebiidae and Baetidae.

Three mitochondrial genomes of early-winged insects (Ephemeroptera: Baetidae and Leptophlebiidae).

Mayflies (Ephemeroptera) are a semi-aquatic insect order with comparatively few genomic data available despite their phylogenetic position at the root of the winged-insects and possession of ancestral traits. Here, we provide three mitochondrial genomes (mtgenomes) from representatives of the two most species-rich families, Baetis rutilocylindratus and Cloeon dipterum (Baetidae), and Habrophlebiodes zijinensis (Leptophlebiidae). All mtgenomes had a complete set of 13 protein-coding genes and a conserved orientation except for two inverted tRNAs in H. zijinensis. Phylogenetic reconstructions using 21 mayfly mtgenomes and representatives of seven additional orders recovered both Baetidae and Leptophlebiidae as well supported monophyletic clades, with Ephemeroptera as the sister-taxon to all other winged insects (i.e. Odonata and Neoptera).

The Imaginal Characters of Neoephemera projecta Showing Its Plesiomorphic Position and a New Genus Status in the Family (Ephemeroptera: Neoephemeridae).

The newly collected imaginal materials of the species Neoephemera projecta Zhou and Zheng, 2001 from Southwestern China, which is linking the other genera of the family Neoephemeridae, are described in detail. Nymphs are also photographed for the first time. The morphology of this species shows some characters of the other genera in Neoephemeridae and several autapomorphies. However, most characters can be seen as plesiomorphies of the family. Specifically, the dorsal-oriented fimbriate gills, projected frons and slim labial palpi in nymphs plus large reddishly pigmented wings, many crossveins, 4-segmented forceps with a relatively long basal segment, fused penes and unforked anal vein show that this species is closer to the taxon Fossoriae rather than to the previously considered Potamanthidae. To reflect its primitive position, a new genus, Pulchephemera Zhou gen. n., is established for this species, Pulchephemera projecta comb. n. Its eggs and observed biology are also described.

Comparative Mitogenomic Analysis of Heptageniid Mayflies (Insecta: Ephemeroptera): Conserved Intergenic Spacer and tRNA Gene Duplication.

Large intergenic spacers and tRNA gene duplications have been reported in several insect groups, although little is known about mitogenomes of mayflies. Here, we determined complete mitogenomes of ten heptageniid species and systemically analyzed their mitogenomic features. Both a conserved intergenic spacer (IGS) and trnM duplication were detected in those mitogenomes. The IGS, which was observed in heptageniids, could be further folded into a stable stem-loop structure. The tRNA gene duplication was found in almost all analyzed mitogenomes, and a unique gene block trnI-trnM-trnQ-trnM-ND2 was also discovered. Our analysis demonstrates that the heptageniid gene arrangement pattern can be explained by the tandem duplication-random loss (TDRL) model. Phylogenetic analyses using both Bayesian inference (BI) and maximum likelihood (ML) methods based on the nucleotide and amino acid sequence data recovered the genus Epeorus as monophyletic with strong support. Our results provide a better understanding of mitogenomic evolution in Heptageniidae, as well as novel molecular markers for species identification of mayflies.

The genus Rhoenanthus Eaton, 1881 in China with the redescription of R. hunanensis You amp; Gui, 1995 (Ephemeroptera: Potamanthidae).

The potamanthid mayfly genus Rhoenanthus Eaton, which was not investigated systematically before in China, is revised herein. Six species in the subgenus Potamanthindus are recognized. Among them, R. sapa, R. obscurus and R. coreanus are new records for this country, and the presence in China of the species R. magnificus and R. hunanensis is confirmed, the nymphal stage of the latter species being described for the first time. Phylogenetically and evolutionarily, R. sapa, which has shorter maxillary and labial palpi, foretibiae and less setaceous mouthparts and forelegs, is close to the plesiomorphic genus Potamanthus. At the same time, R. youi, R. obscurus and R. coreanus, which have larger tusks and spines, longer and more setaceous foretibiae, maxillary and labial palpi, are somewhat similar to the other subgenus Rhoenanthus (Rhoenanthus). The Chinese Rhoenanthus (Potamanthindus) species indicate that the genus Rhoenanthus is closer to Anthopotamus rather than Potamanthus, both of them bearing elongated mandibular tusks.