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

﻿Abstract Ecdyonurusaurasiussp. 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.

In Africa, only three Ecdyonurinae genera are present: Ecdyonurus is restricted to North Africa, whereas Afronurus and Notonurus Crass, 1947 are found in the Afrotropical region (Webb and McCafferty 2008;Vuataz et al. 2013). Bauernfeind and Soldán (2012) proposed to split the West Palearctic species of the genus Ecdyonurus into two subgenera: Ecdyonurus (25 species) and Helvetoraeticus Bauernfeind & Soldán, 2012 (15 species), according to the arrangement of setae on the superlingua, the number of bristles on the ventral side of the labrum and the number of comb-shaped bristles on the maxilla in nymphs, as well as the shape of the apical sclerite of the male genitalia.
Currently, four taxa of this genus are reported from North Africa (Thomas 1998). Two of them are well-known species with a clear status: Ecdyonurus rothschildi Navás, 1929 and Ecdyonurus ifranensis Vitte & Thomas, 1988, whereas one remains doubtful: Ecdyonurus venosus var. constantinicus Lestage, 1925, and the presence of Ecdyonurus venosus (Fabricius, 1793) mentioned by Gauthier (1928) is still unconfirmed. All of them belong to the subgenus Ecdyonurus. Navás (1929) described Ecdyonurus rothschildi from an oasis in Biskra Province, north-eastern Algeria, based on a male imago. The species was redescribed by Thomas and Dakki (1979) which gave a detailed account of the adult morphology and related it to the E. aurantiacus (Burmeister, 1839) species group. Later, Soldán and Gagneur (1985), proposed the first description of the nymph and an identification key to separate E. rothschildi, E. dispar (Curtis, 1834) and E. aurantiacus nymphs. The species is now known from all Maghreb countries and is one of the most widespread species (Boumaiza and Thomas 1995;Zrelli et al. 2016;Bouhala et al. 2020). Vitte and Thomas (1988) described Ecdyonurus ifranensis at nymphal and adult stages from the Middle Atlas; the species has later been found in other areas of Morocco (El Alami et al. 2022).
The present study aims to examine Ecdyonurus populations from the Aurès region (Algeria). We collected and reared fresh material at all stages. After critical observations and comparison with other Ecdyonurus species, we have clearly distinguished a new Algerian endemic species.

Materials and methods
The material was collected by the first author between February 2020 and November 2021 from six localities from the Aurès region; the sampling sites are located in the Belezma National Park (BNP) and the Western Aurès Massif (Fig. 1). The region is characterized by a semi-arid climate with cold winters and very hot and dry summers. Sampling was performed using a standard benthic net using the kick-sampling method. Imagos and subimagos were obtained by rearing mature nymphs from the Charchar, Yabous and Berbaga sites. All specimens were preserved in 96% ethanol in the field and stored in the laboratory at 4 °C.
The physical and chemical parameters of the water was measured in situ for each sampling site using a multi-probe. The following variables were measured: average water depth, bed width, current velocity with a FLOWATCH flowmeter; conductivity, water temperature and pH using an Adwa AD32 tester and a HANNA HI1271 pH electrode; while dissolved oxygen was recorded using a Lutron PDO-519 Dissolved Oxygen Meter.

Morphological analysis
Morphological characteristics for the description of the new species were used according to Hrivniak et al. (2018). Pictures of habitus were made using a Canon EOS 6D camera and the Visionary Digital Passport imaging system (formerly available and distributed by Dun Inc., Virginia), and processed with Adobe Photoshop Lightroom ver. 4.4. and Helicon Focus ver. 5.3. Four nymphs were dissected in Cellosolve (2-Ethoxyethanol) with subsequent embedding in Euparal medium and mounting on slides. Microscopic pictures were taken using an Olympus BX51 microscope coupled with an Olympus SC50 camera; pictures were enhanced with the stacking software Olympus Stream Basic ver. 2.3.2. and Adobe Photoshop ver. 21.2.2.

Molecular analysis
Five specimens belonging to the new species as well as five specimens of Ecdyonurus rothschildi were used for DNA extraction to get a 658 bp fragment of the mitochondrial cytochrome oxidase I gene (COI) (see Table 1). DNA extraction, PCR amplification, sequencing and alignment construction were performed according to Benhadji et al. (2020) or Martynov et al. (2022). One sequence of E. rothschildi was retrieved from GenBank, as well as two sequences of E. aurantiacus and two of E. dispar. Three Electrogena sequences were chosen as the outgroup. We estimated the evolutionary divergence within and between our new species and the other Ecdyonurus species using the COI genetic distances. Both pairwise distance between all sequences and mean distance between and within species were calculated in MegaX (Kumar et al. 2018;Stecher et al. 2020) under the Kimura 2-parameter (K80) substitution model (Kimura 1980). We then applied the recently developed species delimitation method ASAP (Assemble Species by Automatic Partitioning; Puillandre et al. 2021) to our COI data set using the graphical web-interface available at https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html. This distance-based method is similar to the popular ABGD (Automatic Barcode Gap Discovery; Puillandre et al. 2012) approach but has the advantage of providing a score (i.e. asap-score) that indicates the most likely species delimitation. Pairwise genetic distances were computed under the K80 model, and all other settings were set to default. Because ASAP outputs produced two partitions with equal asap-scores, we favored the partition with the smallest p-value.
Finally, we conducted a Bayesian inference gene tree reconstruction in MrBayes ver. 3.2.7a (Ronquist et al. 2012), using the best evolutionary model (GTR + Γ + I) selected in JModelTest ver. 2.1.10 (Darriba et al. 2012) following the second-order Akaike information criterion (AICc). We used five substitution scheme and six gamma categories, with all other parameters set to default. To accommodate different substitution rates among COI codon positions, we analyzed our data set in two partitions, one with first and second codon positions and one with third positions (1 + 2, 3). Two independent analyses of four MCMC chains run for one million generations with trees sampled every 1000 generations were implemented, and 100 000 generations were discarded as a burnin after visually verifying run stationarity and convergence in Tracer ver. 1.7.2 (Rambaut et al. 2018). The consensus tree was visualized and edited in iTOL 6 (Letunic and Bork 2021).
Material is deposited in the following institutions:

IB-US
Institute of Biology, University of Szczecin, Poland;

Molecular analysis
The COI ingroup data set was 100% complete (no missing data) and included 25% of parsimony informative sites. The COI gene tree grouped the five sequences of Ecdyonurus aurasius sp. nov. into a well-supported monophyletic clade, and was supported as a distinct species in the ASAP analysis (Fig. 2). The K80 mean genetic distance within the five Ecdyonurus aurasius sp. nov. COI sequences was 0.14%. As expected, all other included species were also recovered as distinct species with high node supports. The K80 mean genetic distance between Ecdyonurus aurasius sp. nov. and the other three species of Ecdyonurus ranged from 7.6% (mean distance to E. rothschildi) to 20.1% (mean distance to E. aurantiacus), with a minimum distance of 7.1% between GBIFCH01119302 / GBIFCH00673192 and EC-CH0 sequences. Description. Male imago Size: body length: 9.0-9.8 mm; forewing length 9.1-10.9 mm; cerci broken. General body color distinctly brown to reddish-brown (Fig. 3A).
Head. Light brown, clypeal plate with blackish maculations; eyes grayish blue separated by a distance equal to the diameter of the frontal ocellus; a brownish lateral stripe present at one third of the ventral side; ocelli apically whitish-yellow, dark brown basally; antennae with scapus medium brown, flagellum grayish brown.
Abdomen. General color brown to rusty tawny. Terga light tawny to rusty tawny. Tergum I dark brown, terga II-VII reddish-brown with two median pairs of light markings, proximal pair elongated and slightly divergent, distal pair subparallel to body axis (Fig. 4A). Segments II-VIII with rusty-brown lateral stripes stretching from anterior to posterior margin of the segment (Fig. 3A) and connected dorso-posteriorly (Fig. 4A); terga VII-X slightly darker that other ones; tergum X reddish-brown, yellowish-brown posteriorly. Abdominal sterna yellowish to light brown, with two pairs of light markings, the proximal pair elongated, and divergent, distal pair rounded (Fig. 4B). Sterna VIII-IX darker. Nervous ganglia well visible and tinted with purple on sterna II-VII. Cerci brown, with joints of segments blackish.
Genitalia. Styliger plate medium brown, lighter in the middle, strongly convex, with two small bumps near gonostyli base; first segment of gonostyli dark brown, second and third lighter (Fig. 4D). Penis lobes yellowish-brown to brown moderately expanded laterally, outer margin rather quadratic (Fig. 4D, E). Basal and lateral sclerites brown, darker than apical sclerite (Fig. 4E). Lateral sclerite rather quadratic slightly larger on inner side; apical sclerite with few medium sized teeth on inner margin (Fig. 5A); basal sclerite outer margin smooth, without teeth. Titillators straight, yellowish-brown, darker on outer margin, with two spines on the dorsal face.   Female imago. Size: body length: 9.9-13.3 mm; forewings length: 10.5-12.9 mm; cerci length: 17.9-21.3 mm. General color of body similar to that in male imago, markedly paler. Head. yellowish-brown; eyes grayish. Thorax. Prothorax yellowish gray to brown. Mesothorax dorsally pale, yellow to yellowish-brown, basisternum and furcasterum medium brown. Abdomen. Terga yellowish laterally and tawny to rusty tawny dorsally. Terga I-VIII with central longitudinal rusty tawny parallel bands and lateral stripes (Fig. 3B). Abdominal sterna yellowish to light brown, especially VIII-IX, segments I-VII generally with two central light short strokes; nervous ganglia strongly tinted with purple on sterna II-VII. Subgenital plate large, whitish and angular, reaching two third of sternum VIII length; subanal plate acutely rounded (Fig. 4C). Cerci brown, with joints blackish.
Male subimago. Size: body length: 9.8-10.5 mm; forewings length: 10.5-11.4 mm; cerci length: 13.3-26.9 mm. Head brown to reddish-brown. Eyes grayish blue. Ocelli as in male imago. Antennae yellowish, brown basally, same than in male imago. Fore legs darker than middle and hind ones. Fore femora intensively brown distally. Middle and hind legs uniformly yellowish gray to yellow. Wings dark gray. Abdominal terga similar to male imago. Sterna slightly lighter than terga. Protuberances of styliger plate well marked, slightly yellowish, gonostyli intensively brown, yellow to whitish-yellow apically. Typical shape of penis already well apparent. Cerci brown.
Mature nymph. Size: body length: up to 7.12 mm for male and 9.6 mm for female; cerci slightly longer than body length. General body color yellowish-brown with pale yellowish markings.
Head. Mean width/length ratio 1.4-1.6, yellowish-brown to brown, with two central light spots near fore margin, and two whitish stripes along the dehiscence line (Fig. 6A). Eyes blackish grey; ocelli whitish grey, antennae with scape and pedicel medium brown; flagellum yellowish-brown.
Thorax. Pronotum. Mean width/length ratio 4.2-5.0, yellowish-brown to brown; lateral projections ca as long as the length of the pronotum; with lateral margin regularly convex, and tip slightly pointed (Fig. 6A). Mesonotum medium brown with yellowish markings.
Abdomen. Terga brownish gray; on terga II-VIII two centrally elongated yellowish spots increasing in size posteriorly and fused on tergum IX; tergum X uniformly medium brown (Fig. 6A). Abdominal sterna yellowish white, nervous ganglia tinted with purple. Posterior margin of terga with large pointed marginal teeth alternating with medium and short ones, and several rows of microdenticles above the margin (Fig. 8C). Posterolateral projections short, weakly sclerotized, reaching from slightly above 1/7 to 1/5 of the length of the following segment (Fig. 6C). Gills grayish brown with distinct brown and developed tracheation; gill I tongueshaped, gills II-VII leaf-shaped, asymmetrical, gills III-IV slightly longer than wide ( Fig. 8D-J). Cerci and paracercus yellowish-brown; each segment with a row of pointed stout setae.

Discussion
Ecdyonurus aurasius sp. nov. belongs to the subgenus Ecdyonurus by the shape of the apical sclerite of male genitalia and the single row of stout setae on the ventral side of the labrum. However, this species presents some intermediate characters between the subgenera Ecdyonurus and Helevetoraeticus; the number of comb-shaped setae on the crown of the galea-lacinia is generally less than 20 in Ecdyonurus s.s., whereas our species exhibits a range from 16 to 22 setae; the setae on the lateral margin of superlingua are supposed to be long, including the tip, whereas in our species, those at the tip are shorter. We can also add the posterolateral projections on the abdomen which are very short, and the nervous ganglia tinted in purple, two characters not frequent in Ecdyonurus s.s. but more common in Helvetoraeticus. Nevertheless, we are confident that our new species belongs to the subgenus Ecdyonurus.
By the shape of the penis lobes and the posterolateral projections of the abdomen, E. aurasius sp. nov. is closely related to E. aurantiacus, E. dispar, E. rothschildi, and E. ifranensis. The first two are considered as Mediterranean faunal elements, expanding to Central Europe or even the British Islands for E. dispar (Bauernfeind and Soldán 2012). The nymph of E. aurasius sp. nov. can be separated from those of E. aurantiacus and E. dispar by the nervous ganglia tinted with purple, and the tongue-shaped gill I,  from E. dispar also by the shape of the stout setae on the dorsal surface of femora (acute and pointed in the latter). The new species presents more affinities with the two other North African endemics but can be distinguished from E. rothschildi by the much longer pronotal projections, the shape of the stout setae on the dorsal surface of femora (pointed in the latter), the shape of the gills (more symmetrical in E. rothschildi) and the shape of the glossae ((inner margin rounded and convex in E. rothschildi). Ecdyonurus aurasius sp. nov. differs from E. ifranensis by the shape of the labrum (less broad in E. ifranensis), the shape of the stout setae on the dorsal surface of femora (pointed in E. ifranensis), and the shape of the glossae similar to E. rothschildi. In males, E. aurasius sp. nov. differs from E. rothschildi, E. dispar and E. aurantiacus by the compound eyes separated and not touching (character not stated in E. ifranensis description), from E. aurantiacus and E. dispar by the posterior margin of the basal sclerite smooth, and from E. ifranensis by the first transversal vein in the costal field surrounded by a dark brown maculation (the same in E. rothschildi), and by the shape of the posterior margin of the basal sclerite rounded (straight in E. ifranensis). It is also worth noting that E. aurasius sp. nov. differs from the two other North African species by the nervous ganglia tinted in purple in female imagos, whereas they are colorless in E. rothschildi and E. ifranensis.

Distribution and biology
Ecdyonurus aurasius sp. nov., as known so far, is restricted to the Aurès region. The species has been recorded from only six localities in the Western Aurès area; most habitats are located in the highest part of the streams, within altitudes ranging from 1010 to 1800 m a.s.l. These sites are represented by small mountain watercourses with gravel substrate (Fig. 9). The average annual water temperature ranges from 5 °С to 18 °С with high concentration of dissolved oxygen (6.5 to 9.35 mg/L). The nymphs were sampled under current velocity ranging from 0.24 to 0.48 m/sec, the average streams width from 60 cm to 1.50 m, with depth from 10 to 35 cm, and pH from 6.8 to 7.2. The highest population density was recorded at the Charchar site (60 individuals/m 2 ) and the lowest one was observed at the Bouailef site (2-5 individuals/m 2 ).
The mature nymphs and subimagos (together with early-instar nymphs) were observed in May/June and another generation observed in September/October, thus suggesting a bivoltine life cycle. The other Ephemeroptera species sporadically occurring in the same sites were Caenis luctuosa (Burmeister, 1839), Baetis chelif Soldan, Godunko & Thomas, 2005 and Baetis sinespinosus Soldán & Thomas, 1983. production. Our sincere thanks to Sonia Zrelli (Bizerte, Tunisia), Mokhtar Benlasri (Marrakech, Morocco) Lina Kechemir (Tizi-Ouzou, Algeria) and Boudjéma Samraoui (Guelma, Algeria) for providing material useful for this study. Céline Stoffel (MZL) is thanked for her dedicated work with the molecular lab. We also express our gratitude to Robert Czerniawski, Director of IB-US for encouraging this collaboration and Tomasz Krepski for providing support and lab material for morphological observations in IB-US laboratory. Comments from and discussion with the two reviewers, Lubos Hrivniak and Ernst Bauernfeind, greatly helped to improve the manuscript. This research was supported by the Algerian Ministère de l'Enseignement Supérieur et de la Recherche Scientifique.