Baetidae (Insecta: Ephemeroptera) of Aurès Mountains (Algeria): A New Species of the Baetis alpinus Species Group, with Notes on Baetis Laech, 1815 Biogeography within Maghreb

Simple Summary The riverine ecosystem of the Aurès Mountains in Algeria is situated in the southern part of the Mediterranean basin. This unique region is an important centre of speciation, with a distinct number of regional endemics—some of which are still undescribed or very poorly studied. Given the importance of biomonitoring the ecological status of surface waters in this region, as well as the need to study and protect the diversity of aquatic fauna, information on the taxonomic composition and status of regional species is essential. Our attention was focused on the mayflies of the genus Baetis Leach, 1815 (Ephemeroptera: Baetidae), a typical element of the river macrozoobenthos within the Aurès Mountains. A new species of Baetis (Baetis) dihyae sp. nov. is described for larvae and attributed to the Baetis alpinus species group using morphological and molecular evidence. The affinities of Baetis (Baetis) dihyae sp. nov. to the closely related Palearctic species Baetis (B.) alpinus (Pictet, 1843); the western Alps endemic Baetis (B.) nubecularis Eaton, 1898; and B. (B.) pasquetorum Righetti & Thomas, 2002 (reported only from a few localities in southern France) are discussed in detail. The record and further description of a new species is the first contribution to the knowledge of the relatively diverse mayfly family Baetidae, distributed in the Aurès Mountains. Establishing a representative level of diversity of the macroinvertebrate fauna of the region is closely linked to both field studies and analyses of the main pathways of invasion of its individual elements. Using an extensive data set on the geology and historical biogeography of different faunal groups as well as analysing the diversity of the Baetis alpinus species-group representatives in Western Europe and the Maghreb, we examined in this contribution its colonisation pathways in North Africa. Abstract A new species, Baetis (Baetis) dihyae sp. nov., belonging to the Baetis alpinus species group, is described and illustrated based on larval material collected in the Aurès Mountains (northeastern Algeria) in 2020–2021. This new species is closely related to three European species, e.g., Baetis (B.) alpinus (Pictet, 1843); B. (B.) nubecularis Eaton, 1898; and B. (B.) pasquetorum Righetti & Thomas, 2002 by the combination of the following characteristics: (i) more than one short, stout bristle at the tip of segment II of the maxillary palp and (ii) a well-developed paracercus. However, the new species clearly differs from all congeners of the Baetis alpinus species group primarily by the (a) structure of mouthparts—with 14–18 long submarginal setae arranged in a single irregular row on the dorsal surface of the labrum; 2–6 short, stout bristles at the tip of segment II of the maxillary palp; and segment II of the labial palp without a considerably developed apico-internal lobe); (b) setation of abdominal terga, with a few triangular-shaped scales sparsely scattered near the posterior margin only; and (c) a well-developed paracercus, comprised of more than 50 segments. Primary data on the biology and distribution of this new species are provided, and molecular affinities are verified by the analysis of COI (barcode) sequences. Detailed notes on the distribution of mayfly species belonging to the Baetis alpinus species group common in Western Europe and the western part of North Africa are presented. The historical movement of Baetis representatives between Europe, North West Africa, and subsequently Algeria, with the land bridges ‘Strait of Gibraltar’ and ‘Strait of Sicily’ as colonization routes, is discussed in detail and identified in the present study as the Western Algeria colonization path and Eastern Algeria colonization path, respectively.


Introduction
Baetis Leach, 1815 is the most diverse genus of Order Ephemeroptera in the West Palaearctic [1].Müller-Liebenau [2] arranged European representatives of this genus into eleven species groups supported later by Jacob [3].One such species group is the Holarctic Baetis alpinus group which is closely related to the B. pavidus Grandi, 1949 and B. lutheri species groups [4].
The Baetis alpinus species group is morphologically distinct, inter alia by a dorsoventrally flattened larva, with a shortened abdomen bearing dark spots and reduced paracercus (less than two-thirds of the cerci), reflecting the rheophilic character of this group.Moreover, the tarsal claws possess a pair of fine bristles subapically as defined by Bauernfeind and Soldán [4].Recently, Godunko et al. [5] reported the distinguishing characters for several European species of the Baetis alpinus group and proposed a new set of features useful for the species delimitation.
In the West Palearctic, Baetis alpinus (Pictet, 1843) is the most widespread group representative within the European mountain ranges [4], where it presents two lineages that radiate from the Alps to the Carpathians and to the Apennines and the Iberic peninsula [6,7].On the other hand, in the Mediterranean islands, the occurrence of B. alpinus endemics is confirmed, and several are described, e.g., B. cyrneus Thomas & Gazagnes, 1984 from Corsica and B. cypronyx Godunko, Soldán & Staniczek, 2017 from Cyprus [5,8].
In northern Africa (namely in the Maghreb), populations of B. maurus Kimmins, 1938 heterospecific to the Iberian populations have been recorded from the Tafna river basin, whilst the species B. berberus Thomas, 1986, probably endemic to Morocco, was described from the High Atlas (Morocco), and B. punicus Thomas, Boumaїza & Soldán, 1983 was reported from the Maghreb area and a single locality in Spain [4,9].There are also populations of some unspecified taxa attributed to "Baetis gr.alpinus" reported from Morocco [10] and the eastern part of Algeria [11,12], which have never been thoroughly taxonomically investigated.
Because of growing interest, the North African region is currently experiencing a plethora of new Ephemeroptera species discoveries-of a distinctly endemic nature [10][11][12][13][14][15][16][17][18][19].Hitherto, the most remarkable results have concerned the fauna of Algeria, the largest country of the Maghreb.In the Aurès region in northeastern Algeria, the first studies on the fauna of the Baetidae began at the same time as the first investigation of mayflies [20][21][22][23].
Due to lack of consistent investigation and the fragmentation of information about mayfly fauna of the Northern and Southern Aurès Mountains (including the Batna and Biskra Provinces) (see, e.g., Dambri et al. [19], Soldán and Thomas [24], Bebba et al. [25]), only several species from this area were reported in the comprehensive checklist of North Africa by Thomas [26]: with 14 reported mayfly taxa, 9 of them are representatives of the family Baetidae.Later, Bebba et al. [25] additionally reported Choroterpes atlas Soldán & Thomas, 1983 (Leptophlebiidae), and recently Dambri et al. [19]  In this contribution, we aim to broaden the knowledge of this spectacular species group within the Maghreb region on the occasion of the description of Baetis (Baetis) dihyae sp.nov.from several valleys in the Aurès Mountains (Algeria).Additional objectives of this contribution are to discuss the differential diagnosis of a new species from other closely related representatives of the Baetis alpinus species group based on both morphological and molecular data.Furthermore, we provide the first biogeographical analysis of this species group in the Maghreb region, with investigation of possible colonization pathways in the context of known paleoclimatic and geologic events.

Sampling, Type Series, Comparative Material
The larval specimens of B. dihyae sp.nov.were collected with a water net between June 2020 and October 2021 in the Yabous and Inoughissen sites within the Aurès Mountains (Figure 1).All larvae were preserved in 95% ethanol and stored at 4 • C for long-term preservation.The type material of B. dihyae sp.nov is organised into the following collections: holotype (male larva), 1 paratype (1 female larva), and 4 paratypes (4 female larvae) mounted on slides using Faure liquid and 3 paratypes (3 male larvae) and 2 paratypes (1 female and 1 male larvae) used for SEM are in the collection of R.J. Godunko at the Institute of Entomology, Biology Centre of the Czech Academy of Sciences (IE-BC CAS); 1 paratype of a female larva is slide mounted and in the collection of N. Benhadji at the Institute of Technology and Life Sciences, National Research Institute, Szczecin branch, Poland (ITP-PIB); and 14 paratypes (5 male and 9 female larvae) are stored in the collection of B.M. Dambri at the Functional Ecology and Environmental Laboratory, University Batna 2, Algeria (UB2).Type series of Baetis (Baetis) dihyae sp.nov.HOLOTYPE: mature male larva, Algeria, Wilaya de Khenchela, wadi Ekehal, Yabous region, Yabous site, 35 •  Paratypes: 1 mature female larva (the same date and site as holotype), 3 mature male larva (same site as holotype, 28.X.2021), 5 female and 1 male larvae (same site as holotype, 06.VII.2020),Dambri B.M. leg.(IE-BC CAS); 1 female larva mounted on slide, Algeria, Wilaya de Batna, wadi Abiod, Ichmoul region, Inoughissen site, 35

Morphological Study
Line drawings were made using a Zeiss Axioplan microscope with an Olympus BX41 camera lucida.Material was observed with the binocular microscope Leica M205 C. Photographs of larvae were taken using a Leica Z16 APO macroscope and processed with Leica Application Suite™ Version 3.1.8to obtain combined photographs with enlarged depth of field.Photographs were subsequently enhanced with Adobe Photoshop™ CS3.
Two specimens used for SEM investigation were dissected and dehydrated through a stepwise immersion in ethanol and then dried by critical point drying and then mounted on SEM stubs and coated with gold by sputtering using Polaron PS 100.Observations were taken on the scanning electron microscope Jeol JSM 7401F at 4 kV in the Laboratory of Electron Microscopy, Institute of Parasitology, Biology Centre, CAS ( České Budějovice, Czech Republic).

Terminology
In this contribution, the terminology and corresponding acronyms used follow those proposed by Godunko et al. [27] (for the representatives of the subgenus Rhodobaetis Jacob, 2003); Godunko et al. [5] (for the species from Baetis alpinus species group); Godunko et al. [28] (for the species of the genus Nigrobaetis Novikova & Kluge, 1987) are used to describe the body setation (e.g., to characterize the types of stout setae and scales).Initially classification evolved based on the classification and terminology proposed by e.g., Gaino and Rebora [29,30] as a part of SEM studies of the representatives of the genus Baetis Leach, 1815, where several acronyms were proposed (e.g., FT for designation of flat-tipped sensillum, B for sensillum basiconicum, and Hr for hair-like setae).

Molecular Analysis
Three specimens B. dihyae sp.nov.have been successfully barcoded (Table 1).DNA extractions are conserved in the Institute of Biology of the University of Szczecin, Poland.We isolated DNA using DNA Isolation Kit: GeneMATRIX Tissue DNA Purification Kit (EURX) (cat.no.E3550); for PCR, we used PCR Polyerase: DreamTaq™ Hot Start Green PCR Master Mix (Thermofischer Scientific, Waltham, MA, USA) (cat.no.K9021) to the mitochondrial cytochrome oxidase I gene (COI) 658 bp fragments with LCO1490 and HCO2198 primers [31].PCR cycles were performed according to Benhadji et al. [9].We verified PCR success by electrophoresis using the ladder MassRuler DNA Ladder Mix, ready-to-use (Thermofischer Scientific) (cat.no.SM0403), and finally, we purified PCR products using Kit: GeneMATRIX PCR/DNA Clean-Up Purification Kit (EURX) (cat.no.E3520).After purification, we sent samples for sequencing to Macrogen (5 uL of starter + 5 µL of DNA sample) (Address: Meibergdreef 57 1105 BA, Amsterdam, The Netherlands.website link: https://order.macrogen-europe.com/#(accessed on 28 January 2022).We edited, trimmed, and assembled forward and reverse sequence reads using Geneious 10.2.6 [32], with sequence alignments available in the Supplementary Materials: Alignment S1 Using Molecular Evolutionary Genetics Analysis Version 11 (MEGA11); we aligned sequences of each taxon with analogue genus or species selected from Genbank or BoldSystem database [33,34].To perform the biomolecular reconstruction tree, we aligned our sequences with sequences from B. alpinus [6,[35][36][37][38][39][40][41][42][43], B. cyrneus [41,42], B. maurus [9,38] haplogroups and B. melanonyx [6,42,43] as an outgroup.We also performed the reconstruction in MEGA11, selecting the Tamura-Nei model to produce our tree with neighbor-joining analysis, with 1000 normal bootstrap replicates.The GenBank accession numbers for the 3 new sequences (three specimens of a new species) used in the molecular study are given in Table 1; the nomenclature for the gene sequences follows Chakrabarty et al. [44].  2 and 3.  Etymology.The species name "dihyae" is derived from the name of the Amazigh queen Dihya, a historical character who was known as a symbol of people who refuse to be subjugated and are willing to fight to retain their freedom.
Diagnosis (based on larvae).The new species can be easily distinguished from all other representatives of the Baetis alpinus species group by the combination of following diagnostic characters: (i) head with frontal suture widely U-shaped; (ii) tip of segment II of the maxillary palp with up to six short stout setae; (iii) surface of terga: anterior margin with small groups of sparsely scattered tongue-shaped scales (SC-tg); (iv) submarginal irregular row of small spines on the posterior margin of terga III-VIII; (v) paraproct with occasional inconspicuous spine on anterior inner margin of plate; surface of the paraproct plate with occasional 1-3 stout submarginal setae of different size (STSbp); (vi) well-developed paracercus (more than 50 segments of paracercus comprise app.1/2 of cerci length).
Additionally, typical for Baetis alpinus species group, tongue-shaped scales [SC-tg] 10.65−13.20 µm in length were recognized (Table 2; see also Godunko et al. [5]).The body surface is covered also with medium to small stout setae [STS], as well as sensillae of several types (see Table 2).2-4).General colour yellowish grey to dirty yellow; head and thorax dark yellowish-brown to brown.Head light brown; genae and clypeus yellow to yellowish-brown; frons and vertex darker light brown to brown (Figure 4a,b).Two marked brown spots between base of antenna, ocellus, and eye.Facetted surface of larval turbinate eyes brown.Antennae light brown, flagellum paler than scape and pedicel.Pronotum dirty yellow to brown; pair of oblique brownish bands; mesonotum yellowish brown to brown; paired longitudinal brown bands run centrally along initial adult sutures and several spots of same colour centrally and laterally; metanotum brown, unclear brownish smudge centrally (Figures 2a and 3a).Light brown to brown pleurites on lateral sides of thorax (Figures 2b and 3b).Ventral side of thorax paler than dorsal side; sterna yellow to yellowish-brown.Legs pale.Femora and tibia yellowish brown to light brown; apex of tarsi and tarsal claws intensively brown (Figures 2b and 3b).Abdominal terga (Figures 2a,b and 3a,b) yellowish brown to brown.Oblique narrow brown spots anteriorly on tergum I, clearly visible on paler background; similar oblique spots on tergum II; combination of short oblique spots anteriorly, and two elongated spots centrally, on terga III and VI (occasionally V); color pattern on terga VI-IX is represented by a combination of triangular shaped spots anteriorly and two nearly rounded spots centrally; two elongated dark smudges centrally on tergum X. Abdominal sterna yellow to yellowish-brown, paler than terga; a pair of unclear smudges centrally on sterna II-VII (VIII).Cerci and paracercus pale, yellow to dirty yellow and yellowish-brown, and slightly darker distally.

Cuticular coloration (Figures
Hypodermal coloration.Hypoderm without contrasting markings.Head.Solitary B and Hr setae and occasional FT setae on clypeus sparsely scattered across lateral side of frons.Frontal suture widely U-shaped (Figure 9d).Antennae distinctly longer than 1/2 of body length.Scape and pedicel with FT and Hr, and more abundant B setae located mainly along distal margin of each segment; few Ca setae along distal margin of pedicel; no peculiar cuticular corrugation (which is present in some representatives of the Baetis alpinus species group and in the closely related B. lutheri and B. pavidus species groups as defined by Bauernfeind and Soldán [4]).Distal margin of flagellar segments composed by large spines rounded apically, alternating with FT and occasional TR and B (Figure 9a-d, Table 2).Outer mandibular incisor group relatively broad, rounded at apex, moderately separated from inner incisor group by a shallow incision; inner incisor group not prominent, bearing 3-4 small teeth; right and left prostheca nearly of same size, asymmetrical, bearing 8-10 apical teeth (Figure 5d,e).
Maxillary palp two-segmented; segment 1 larger than segment 2; segment 2 with almost parallel outer and inner margins, asymmetrical only apically; tip of segment 2 with a small conical protuberance bearing 2-6 stout setae; surface of both segments with B setae (same type of setae is described as uniporous sensillum basiconicum sensu Gaino and Rebora [30]: 449, Figures 19−21)), densely scattered on distal part of segment 2. Three enlarged, acute teeth situated apically on maxillary galea-lacinia, accompanied by one row of long setae (Figures 6a,b, 9f and 10a-c).
Hypopharynx as in Figure 5c, with prominent central lobe in lingua and marked apical hump on superlinguae.
Labium with relatively slender glossae and paraglossae; glossae slightly shorter than paraglossae; up to 14 stout bristles along inner margin and up to 6 stout setae on outer margin of glossae; 10-14 long bristles grouped into two rows on tip of paraglossae; 6-10 long bristles along outer margin and 4-5 medium sized setae on ventral side of paraglossae.Segment 2 of labial palp 1.20-1.35×longer than segment 3, covered with sparse B and Hr setae and without considerably developed apico-internal lobe; segment 3 nearly symmetrical and evenly rounded apically, slightly broader than long (length/width ratio 0.95-1.10);18-25 slender, pointed, stout setae on dorsal apical surface of segment 3, accompanied by long Hr and slightly shorter B setae; quotient q 1.00-1.08(Figures 6d-i and 10d).Thorax.Surface of pronotum with a few B and occasional FT and Hr setae.Sternal protuberances on meso-and metathorax visible, pointed apically, yellowish-brown to brown.Outer margin of femora with 2-4 rows of long, tiny bristles proximally arranged in 1-2 rows, and one row of shorter bristles distally; long marginal bristles pointed and bluntly pointed apically, alternating with submarginal STS-p and STS-bp setae, elongated Hr and occasional B setae; inner margin with sparse row of small STS-bp setae; surface of femora with STSs-bp and STSs-ov setae and group of tiny setae (Hr and more abundant FT).Outer and inner margins of tibiae with small STS-p and STS-bp setae, alternating with Hr and B; stout setae more abundant along outer margin; surface of tibia with sparse small STS-bp and STSs-ov setae, more abundant along patella-tibial suture.Tarsi with 7-11 middle to elongated STS-p setae along inner margin, and several small to middle sized STS-p and STS-bp setae on outer margin; surface and both margins of tarsi covered with more abundant tiny Hr setae, occasional FT, and small STS-bp setae.Tarsal claws relatively short, moderately hooked; 8-11 teeth arranged in a single row along claw; two subapical hair-like setae (Figures 7a-c and 12a-d).Abdomen.Posterior margin of terga with broad triangular spines of different sizes, bluntly pointed or occasionally pointed apically, and occasionally a few small spines subapically; broader, nearly symmetrical spines along posterior margin of terga III-VIII (IX); marginal broad spines alternating with 1-3 tiny FT and isolated Hr setae.Surface of terga with a few moderately elongated, tongue-shaped [SC-tg] scales, mostly concentrated centrally near posterior margin of segments; solitary scale sockets concentrated also posteriocentrally, mainly on terga IV-VIII; solitary Hr and more abundant FT setae stretched over whole surface of terga I-X.Posterior margin and surface of sterna without spines, stout setae or scales, with B and Hr setae only (Figure 11a-d).
Paraproct plate as in Figure 8c, relatively short, cercotractor not elongated; no prominent spines along inner margin of paraproct (only a few weakly separated small spines were poorly visualized); solitary FT and Hr along of inner margin; same type of setae sparsely scattered on paraproct surface distally; inner margin of cercotractor with inconspicuous/subtle range of bluntly pointed spines spread centrally, a few FT and Hr setae (Figure 8c).
Tracheal gills white to light brown, with slightly pink center; no elongate gill plates; gills moderately asymmetrical, blunt apically, inner margin broadly rounded; gills II-VI asymmetrical; gills I and VII less asymmetrical; serrated margins of gills relatively well marked, with tiny Hr setae inserted at small, articulated bases; FT and Hr setae scattered on dorsal surface of gills; tracheation poorly visible (Figure 8a,b and Figure 11e,f).Morphological characters to distinguish B. dihyae sp.nov.from other representatives of the Baetis alpinus species group, especially from closely related species, are given according to Müller-Liebenau [2], Thomas et al. [46], Thomas and Dia [47,48], Peru and Thomas [49], Jacob [3], Kluge and Novikova [50], Bauernfeind and Soldán [4], and Sroka et al. [45] and are summarized in Table 3.
Male and female adults.Unknown.Distribution and biology.Baetis dihyae sp.nov. is known so far only from two localities of Aurès streams (Figure 1).The species is probably a micro-endemic of the High Mountain ranges of this region.Based on present data, the new species seems to be rare, or localized to several water sources only, since it has not yet been collected from other localities, despite present records on mayfly fauna of the Aurès Mountains.

Molecular Study
The neighbor-joining tree analysis helped in delineating six haplogroups of six B. alpinus species group species' sequences (Figures 14 and 15, Table 4).Three B. dihyae sp.nov.sequences formed a strongly supported monophyletic haplogroup (with a 100% bootstrap).This haplogroup is also supported (bootstrap = 77%) as the single-branched sister clade of one specimen's sequence of B. punicus species from Tunisia.On the other hand, the interspecific genetic distance between B. dihyae sp.nov.and B. punicus haplogroups is quite high (12%), and the latter also morphologically discriminated from its congener above.The B. dihyae sp.nov.haplogroup is highly divergent genetically from all the remaining haplogroups (from 19-25%).

Affinities
Formerly, Thomas and Dia [47] identified a new subgenus, Patites Thomas & Dia, 1999, for two species included in the Baetis alpinus species group sensu Müller-Liebenau [2], namely B. melanonyx (Pictet, 1843) known throughout Europe, B. baroukianus Thomas & Dia, 1984 described from Lebanon, and related species.Afterwards, Godunko et al. [5], whilst describing a new species B. cypronyx from Cyprus, indicated that it is closely related to B. melanonyx and B. baroukianus.However, as a precaution, the attribution of B. cypronyx to the subgenus Patites was not considered by these authors, as this group lacks global revision (J-L.Gattolliat, personal communication), and only 13 species were reassigned to this subgenus by N.J.Kluge (see web resource Ephemeroptera of the World [53]).Most recently, Ephemeroptera of the World [53] implemented the subgenus Patites for all West Palearctic species, and it was also used by Gattolliat et al. [54] for the identification key of Maghrebian Baetidae larvae.Moreover, this key also aligned with distinguishing characters proposed by Thomas and Dia [48], namely the rectangular labrum with one row of fringed distal margin bristles; fused, triangular-shaped mandibular canines; pointed spines on the posterior margin of tergum IV; and a reduced paracercus.
Bauernfeind and Soldán [4] and Godunko et al. [5] avoided this taxonomic concept and adopted the Baetis alpinus species group, one of the groups proposed by Müller-Liebenau [2] and endorsed by Jacob [3].It was again shown that the delimitation of the species of the Baetis alpinus species group can be rather difficult and unclear because there are high levels of clinal variability of taxa included, combined with the disjunctive area of many species (for more details see Godunko et al. [5]).
Additionally, it should be noted that according to the larval characters proposed earlier for the establishment of Patites, B. dihyae sp.nov.undoubtedly does not belong to this subgenus, being a representative of a different and more numerous species group-Baetis gr.alpinus.Furthermore, almost all the features proposed for Patites are not attributed only to this subgenus as they are shared by other representatives of the Baetis alpinus species group:  3), it is more or less rounded and symmetrical as in, e.g., B. alpinus; (v) Posterior margin of abdominal terga: a row of strong spines is characteristic of all congeners of the Baetis alpinus species group sensu Müller-Liebenau [2] and is listed in Bauernfeind and Soldán Together with B. alpinus, B. nubecularis, and B. pasquetorum, B. dihyae sp.nov.occupies an independent position within the Baetis alpinus species group due to features in the (i) structure of the tip of segment 2 of the maxillary palp, which bears more than one stout setae, and (ii) a well-developed paracercus as long as approximately 0.5 of the cerci length (see Table 3).While B. nicolae Thomas & Gazagner, 1983 and B. punicus Thomas, Boumaiza & Soldán, 1983 are both characterized by apical setation of the maxillary palps with one to four (rarely a fifth) setae of different sizes, the paracercus is always diminished to a few segments in these species, in contrast to B. dihyae sp.nov.
Baetis punicus seems to be the closest genetically to B. dihyae sp.nov., with well supported sister-clade haplogroups in the COI tree; yet, with significant genetic distance, these two species share indeed few features: scape and pedicel without stout setae, surface of tegites covered by semi-lunar impressions, and well-developed gills with gill IV and gill V lengths equal to the length of two terga joined together (Thomas et al. [46]).However, they are clearly different by the following characteristics: the shape of the labrum and mandibles, as B. punicus labrum and mandibles (fused with inner inciser group), are similar to those of B. alpinus (Table 3); the surface of terga as B. dihyae sp.nov.bears some tongueshaped setae SC-tg; the length and number of segments of the paracercus are excessively longer in B. dihyae sp.nov.and of up to 60 segments, while it is shorter in B. punicus, possessing only up to 25 segments.
Thus, biomolecular investigations of more populations of B. dihyae sp.nov.and B. punicus are strongly needed to explore this monophyly as the DNA barcoding can show some limitations in species haplogroup delineation (e.g., Funk and Omland [55]; Duran et al. [56]), especially within small sample sizes sequences.Some clear differences between the new species and taxa listed above are recognized in the structure of the mouthparts.The labrum of a new species is rectangular-shaped; however, the lateral posterior margins are more convergent than in the closely related congeners discussed above.
The shape of labial segments 2 and 3 of B. dihyae sp.nov.slightly differs from B. alpinus, B. nubecularis, and B. pasquetorum.Nevertheless, the new species, together with B. alpinus, is characterized by the presence of numerous stout setae situated dorsally on the tip of labial segment 3 (Table 3).Some minor differences, especially in contrast to B. nubecularis and B. pasquetorum, can be observed in the number of long submarginal and short marginal setations of the labrum and the shape of prostheca setation.
Well-defined differences between the species discussed can be found in the shape and other characteristics of the body setation.In contrast to B. nubecularis and B. pasquetorum, B. dihyae sp.nov.possesses weakly developed setation of the abdominal terga surface, with few tongue-shaped scales grouped mainly along the anterior margin of segments.The same type of scales is scattered on the whole surface of abdominal terga in B. alpinus [2,57] and absent in B. nubecularis and B. pasquetorum.The same is recognized in the case of the scale sockets, which are present in the new species and B. alpinus only.Additionally, in contrast to all these species, B. dihyae sp.nov.can be separated by the presence of an irregular submarginal row of small setae, well recognizable on abdominal terga III-VIII.Finally, the paraproct plate of B. dihyae sp.nov.characteristically lacks stout setae in contrast to other species that have a few bluntly pointed submarginal setae; the inner margin of the paraproct in B. dihyae sp.nov.occasionally bears one incospicuous spine, markedly contrasting with developed setation consisting of 4-15 spines in B. alpinus, B. nubecularis, and B. pasquetorum (Table 3).

Biogeographical Considerations 4.2.1. Overview of Baetis alpinus Species Group in the West Palearctic
Primarily, the Baetis alpinus species group, altogether, is known to encompass cold stenothermic elements [4] and is somewhat restricted to higher altitudes and mountainous streams (200-2600 m a.s.l) ([4,35,39,58-61].They occur in humid to sub-arid climates in intermittent or permanent streams in the West Palaearctic realm, from the Alpine streams in Europe down to the Atlas Mountain ranges in the Maghreb; they are also reported from the Mediterranean islands, e.g., Sardinia [62] and Cyprus [5].Amongst the ten countries of the Balkan Peninsula, B. alpinus is spatially dominant, and B. melanonyx is reported from Slovenia, Albania, and Bulgaria only.The questionable record of B. melanonyx from North Macedonia in Memeti et al. [63] needs to be confirmed.Latitudinally, they are distributed from ca. 50-55 • northern latitude to the Mediterranean and North Africa (the southern limit is at 35 • 16 42 N, 6 • 32 34 E at 1670 m a.s.l. in the Inoughissen site, Wilaya de Batna, Algeria, for B. dihyae sp.nov.).Their eastern limit is in the Levant [4] and the western one is in the Ibero-Moroccan region (Figure 16).Considering only the Baetis alpinus species group in North Africa, records exist from Morocco and Northeastern Algeria [10][11][12] and in this present study.However, several studies containing molecular analyses of studied taxa have unveiled the rich diversity of the Baetis alpinus species group and nominal species B. alpinus, with several cryptic or sibling-species isolated in the West Palearctic [6,7,[9][10][11][12]38,54,64].Thus, the reliable number of taxa of mentioned species group within the Mediterranean region and North Africa should be markedly rich and diverse.
In the Maghreb, recent studies on the taxonomy and distribution of Ephemeroptera, supported by molecular analyses, argue that the Maghrebian B. alpinus represents a cryptic complex of the species in Morocco (morphological studies still not published), where specimens have been shown to be genetically distant from Iberian populations [10] and of the putative endemic in Algeria [11,12,54].These populations occur 20-1500 m a.s.l. in northern Morocco [10].In northeastern Algeria, they appear at the highest site reported in Samraoui et al. [11] (see site O. Nil, 864 m a.s.l), when in the Kebir-East, material was collected at ca. 1200 m a.s.l. in the mountains of Mount Ghora, Kroumiria [12], and have reached peaks of 1420 m in the Yabous site and 1670 m in the Inoughissen site (see present study).
In the Kebir-East, which is the second largest catchment in northeast Algeria, the populations of B. alpinus species group are widespread together with B. atlanticus Soldán & Godunko, 2006 and B. cf.maurus [12].However, they are rare in the Seybouse catchment (the first and largest catchment in northeast Algeria) [11] and are accompanied by the larvae of B. maurus and rarely with taxa belonging to the subgenus Rhodobaetis.
The Baetis alpinus species group has apparently never been collected from the Tafna basin (northwest Algeria), although it has been investigated.Only B. maurus is found in the Tafna basin, along with quite abundant representatives of Rhodobaetis, namely B. sinespinosus and the scarce B. atlanticus [9, [65][66][67].The absence of B. cf.alpinus from this river-basin is quite surprising, as the Tafna watershed is juxtaposed with the Oriental Moroccan region, where it is reported [10].On the other hand, Baetis cf.alpinus elements are also believed to occur in central Algeria (Kabylie), but no published data are available.This region is one of the most diversified in Algeria [24,26], possibly correlated to the high altitude streams and profuse water flow.
Farther East in Tunisia, Maghrebian representatives of B. cf.alpinus have never been recorded, and only B. punicus from the same species group was recorded [54,68].

On the Origin of Baetis Leach, 1815 in North Africa
According to Razeng et al. [69], speciation in mayflies has been associated with fluctuations in sea levels, continental uplift, and glaciation as a gene flow barrier (see also Bisconti et al.,Sekiné et al.,).Indeed, several geological and climatic event developments may have shaped the mosaic pathway of mayfly distribution in the Mediterranean basin and the Maghreb area in particular.The geological pattern of the Mediterranean was mainly modelled by the Alps orogenesis that started in the Cenozoic, from the Oligocene to the late Pliocene [73,74]; it provoked major changes in the adjacent area between Europe and Africa due to extensional tectonics.
There are few palaeozoogeographical investigations involving aquatic insects of Pterygota in the North African or Mediterranean region (for Odonata see [75]).Except for some species, mayflies are commonly considered poor dispersers, exhibiting a reduced gene flow between catchments, thus promoting genetic divergence [69].
However, the opposite has been demonstrated.Namely, the mayfly taxa successfully crossed from islands to continent and vice versa, as shown, e.g., for Madagascar and Africa [76], the Iberian Peninsula and Macaronesian islands [41], and for latter and North Africa [9].Thus, Ephemeroptera unite all the three qualities to test biogeographical patterns recommended by Schmitt [77], especially (i) high dispersal ability, and then, (ii) once established, large stable populations (iii) that exhibit more or less sedentary and nonmigratory behaviour.Generally, the Maghreb populations of the three countries show discrepancies in species richness, with west-east decrease.However, such differences in the abundance and population structure are not a unique situation, and east-west differentiations were also noticed in reptiles [78][79][80] or mammals [81] due to physical barriers such as the Atlas Mountains.
Recently, it has been increasingly argued that during the transit from southern Europe through the Mediterranean islands to North Africa, populations of several species groups within the genus Baetis have acquired distinct new morphological characteristics and molecular identities-adaptations that have raised them to the rank of a distinct new species with microendemic status [9,54].
Concerning the Afrotropical mayflies, a Gondwanan origin is likely.They could not have crossed the Sahara Desert belt, even with the presence of the Nile as a semitranscontinental river, flowing from Lake Victoria (Uganda, Kenya, Tanzania) to the Nile delta (Egypt).This is probably due to the lentic/potamophilic character of the Nile River, which starts from Sudan's hot and desert climate in the Sahel and Sahara of Northeastern Africa-not a habitat favoured by rheophilic or cold stenothermic mayflies.
To understand the migration routes within the region discussed, paleogeography should be considered.Major events occurred mainly between the Late Oligocene to the Quaternary, with the most prominent shifts happening during the Miocene, Pliocene, and Pleistocene series (e.g., Messinian Salinity Crisis (MSC); Quaternary ice age and deglaciations, Alps orogenesis) [73,74].Also, during Quaternary glaciation/deglaciation, the Saharan belt moved vertically back and forth many times, resulting in differentiation of some terrestrial populations of Northwest and Northeast Africa, such as beetles, snails, reptiles, and mammals such as shrews [78,80,81,[95][96][97].
However, the Saharan Belt was not an obstacle for some Pterygota, such as the order Odonata (another group in the Paleoptera), as they are strong fliers compared to Ephemeroptera.Thus, approximately 20 Afrotropical species of Odonata reached North Africa, and even further North to Europe, the Levant, and even the Arabic or Persian Gulf, through the Nile Corridor, which seemingly acted as a highway for most of these species; six of these species were found only in Egypt [75].In the previously cited study, only 2 species (belonging to the Coenagrionidae family) of the 19 species are reported from the Levant (Pseudagrion torridum Selys, 1876) and the Arabic Gulf in West Saudi Arabia (Ceriagrion glabrum (Burmeister, 1839)), and 11 of the 19 species are or were (and now extinct) found in Algeria: it took only a few decades for these species to spread.The Libellulidae Trithemis annulata (Beauvois, 1807) even crossed to the Canary Islands due to recent climate change and was considered by Boudot et al. [75] as an indicator of expansion.
Thus, the humid periods in the Pleistocene or Sahelian Holocene shifted the Sahara belt and so facilitated faunal movement northward, also explaining the presence of relict Afrotropical species in the Maghreb region.Nevertheless, after these periods, these regions most likely represented a barrier to the expansion of Afrotropical taxa of aquatic fauna, as was shown in the case of Odonata [75].Another Afrotropical colonizer is the biting midge Culicoides imicola Kieffer, 1913 (Ceratopogonidae), which formerly had an Afrotropical distribution that might have extended from the West Palearctic into the Mediterranean basin due to climate change in the Late Pleistocene or Early Holocene [98].

Exploration of Possible Colonization Pathways
Through examining the history of fauna, whether of vertebrates or invertebrates, and the recurrent and consistent findings of colonization patterns, the movement of Baetis representatives between Europe, Northwest Africa, and subsequently, Algeria, can be understood, with the land-bridges 'the Strait of Gibraltar' and 'the Strait of Sicily' acting as colonization routes (see Figure 17) as follows:

•
Eastern Algeria colonization path (Strait of Sicily): Schmitt [77] considered the Adriatic-Mediterranean refugial area (continental Italy) to be geographically less diversified and smaller and with generally less complicated paleogeographical processes than the Iberian Peninsula and the Atlantic-Mediterranean region.Even if the Apennines were supposedly connected to Tunisia by Sicily and Malta since the Oligocene (30 Ma) [73,74], this colonization path does not seem to be likely, at least not for all the majority of mayflies as their diversity is seemingly much higher in the western part of the Maghreb than the eastern part, with Ibero-Maghrebian closely related representatives differing morphologically, genetically, or both (e.g., see Gattolliat et al. [54] for some Baetidae).However, Pliocene sea-level changes could have had unanticipated effects on North African freshwater taxa.While the data about aquatic insects are scarce, some information on other groups of aquatic biota were published.For example, Carranza and Wade [99] speculated that ancestral populations of Pleurodeles Michahelles, 1830 (Amphibia: Salamdridae) found themselves isolated in the Edough Peninsula (northeast Algeria) after the division of the Afro-European land-bridge (ca.5.3 Ma ago), and that marine transgression during the Pliocene might have transformed Edough Peninsula into an island, initiating speciation (ca.4.2 Ma ago, during the Upper-middle Pliocene).This speciation resulted in two new species restricted to the 'fossil island' Edough Peninsula, separated by lowland marshes from the rest of the continental land, and one other species inhabiting the rest of Algeria and Tunisia [99].The reduced diversification of mayfly fauna of North Tunisia compared to Algeria and Morocco can be explained the same way, as well as the isolation of the Sardinia vagrant Habrophlebia consiglioi Biancheri, 1959 (Leptophlebiidae) in northern Tunisia [100].The latter probably had a successful colonization between the MSC (6 Ma) and the Pliocene (ca. 5 Ma).
In many palaeozoogeographical studies, the evolution of circum-Mediterranean Arthropoda species was shown to be crafted by paleoclimatic and geologic events in several ways.In a climate model, Trájer et al. [101] implied that the desiccation of the Mediterranean Sea during the Messinian (end of the Miocene) was a turning point for three Mediterranean sandfly species of the genus Phlebotomus (Psychodidae): speciation was rapid, but migration was not.According to the same authors, the intense hot climate and the high salinity of the Mediterranean abyssal plain during this time inhibited Europe-North Africa cross-migration, whether through the Gibraltar or Sicily straits.On the other hand, several former studies qualified the MSC as 'the trans-Mediterranean migration period' of sandfly ancestors, and thus, the Sicilian Strait route should not necessarily be disregarded.Several studies also addressed the possibility of faunal north-south (Italian Peninsula-North Africa) exchanges of land biota and freshwater invertebrates [101][102][103].

•
Western Algeria colonization path (Strait of Gibraltar): The West Mediterranean area (Atlantic-Mediterranean refugial area sensu Schmitt [77]), including the Iberian Peninsula and Maghreb, is known as the most studied shelter for Southern species during the MSC and the glaciations cycles (see also Pinto-Juma et al. [97]).
The Strait of Gibraltar opening and separation of North Africa (end of the MSC; ca.5.33 Ma), followed by the historically unequalled decrease in sea levels during the Late Glacial Maximum, unveiled continental shelves on both sides of the Mediterranean and new islands [104].
This acted as a "stepping stone" to the flora and fauna from both continents [97].These processes divided the Atlantic-Mediterranean refugial area and enhanced the possibility of radiation by vicariance for various taxa due to isolations [77,97,[105][106][107].
For the mayflies, the West Mediterranean north-south affinities of Baetis suggest a rather western Mediterranean dispersal or migration pathway [9,54].
Based on this suggestion, we could suppose that colonization of North Africa by Baetis ancestors probably began after the Middle Miocene (15 Ma) and not before.Before the Oligocene, there was more or less a stable climate and geological situation in Europe, and so Baetis fauna were not pressured to migrate and find new favourable ecological niches further in the south.
In regard to the first Northwest African refuge (north of Morocco) and the expansion from the western to eastern Maghreb, the movement of fauna may have occurred through the Mauretanic Mediterranean, as suggested by de Lattin [108] based on butterfly studies-considering the fact that the Rif mountains more or less blocked the dispersal of many taxa previously cited.In the case of Central Europe, Monaghan et al. [64] suggested that the fragmentation of alpine streams by lakes or dams creates barriers that hinder the gene flow of B. alpinus and their habitat distribution along the streams and thus isolates populations.Furthermore, Leys et al. [6] highlighted a genetic divergence within cryptic lineages of B. alpinus in European mountain ranges and attributed this to spatial isolation among mountain massifs.Following the scheme of Leys et al. [6], geographic barriers and allopatric speciation induced by stream fragmentations are most likely also the reason behind the presence of several sibling species of the Baetis alpinus species group in the Maghreb, and due to the physical barriers of high mountain ranges, especially in Morocco and Algeria, in these countries the species can be found only in the north central and eastern regions.
The hypothesis discussed above corroborates with Hrivniak et al. [109], which proved that the grouping within Epeorus (Caucasiron) Eaton 1881 species, in the Greater Caucasus range, happened between the Later Miocene and the Pliocene (between 10-5 Ma).Additionally, Poulakakis [110] described the MSC as one of the most pertinent geological events in the biogeography and biodiversity of both terrestrial and aquatic Mediterranean-dwelling fauna.Recently, Trájer et al. [100] proved that the MSC encouraged migration of some other sandfly species of the genus Phlebotomus.
Northwest Africa is typically considered as the southern retreat for Northwest Mediterranean fauna during harsh climate fluctuations (e.g., Glaciations cycles) [97].It is also considered that the land bridges of Gibraltar of Sicily acted as colonization channels during sea-level oscillations, and that the Strait of Gibraltar worked as a path of migration to the south (Maghreb) and of re-migration to the north (Iberian Peninsula) after better climate conditions returned [111,112].
Pinto-Juma et al. [97] demonstrated that Moroccan Cicada (Cicadidae) populations exhibit a greatly diversified haplotype diversity, which infers that they are demographically more stable in the Northwest African refuge than the Iberian populations.This can only confirm the hospitality of this refuge.Regarding mayfly populations of either side of the Rif Mountains, they are highly endemic, certainly due to the long height isolation period of their ancestor during the Holarctic.Finally, the checklist of Thomas [26] suggests that mayfly fauna of Baetidae and Baetis elements in North Central Algeria might be richer that the rest of the country due to high peaks and wadis with permanent and high discharge.

Figure 1 .
Figure 1.Map of sampling sites in the Aurès Mountains (Algeria).

Figure 8 .
Figure 8. Baetis (Baetis) dihyae sp.nov.larva, paratype, gills: (a) gill I-III; (b) gill IV-VII; (c) details of paraproct, ventral view.Roman numbers refer to the respective gill pairs.Without scale bars.Mouthparts.Labrum moderately oblong-shaped, width/length ratio of labrum 1.40-1.45;strong posterior convergence of lateral margins; 1 + 14-18 long submarginal setae on dorsal surface arranged in a single irregular row; 6-12 smaller stout setae laterally on both margins; dorsal surface of labrum covered with densely arranged B and only a few FT and Hr setae grouped posterolaterally.Median incurvation of labrum anterior margin moderately shallow and wide (Figure 5a,b and Figure 9e).Outer mandibular incisor group relatively broad, rounded at apex, moderately separated from inner incisor group by a shallow incision; inner incisor group not prominent, bearing 3-4 small teeth; right and left prostheca nearly of same size, asymmetrical, bearing 8-10 apical teeth (Figure5d,e).Maxillary palp two-segmented; segment 1 larger than segment 2; segment 2 with almost parallel outer and inner margins, asymmetrical only apically; tip of segment 2 with a small conical protuberance bearing 2-6 stout setae; surface of both segments with B setae (same type of setae is described as uniporous sensillum basiconicum sensu Gaino and Rebora [30]: 449, Figures19−21)), densely scattered on distal part of segment 2. Three enlarged, acute teeth situated apically on maxillary galea-lacinia, accompanied by one row of long setae (Figures6a,b, 9f and 10a-c).Hypopharynx as in Figure5c, with prominent central lobe in lingua and marked apical hump on superlinguae.Labium with relatively slender glossae and paraglossae; glossae slightly shorter than paraglossae; up to 14 stout bristles along inner margin and up to 6 stout setae on outer margin of glossae; 10-14 long bristles grouped into two rows on tip of paraglossae; 6-10 long bristles along outer margin and 4-5 medium sized setae on ventral side of paraglossae.Segment 2 of labial palp 1.20-1.35×longer than segment 3, covered with sparse B and Hr setae and without considerably developed apico-internal lobe; segment 3 nearly symmetrical and evenly rounded apically, slightly broader than long (length/width ratio 0.95-1.10);18-25 slender, pointed, stout setae on dorsal apical surface of segment 3, accompanied by long Hr and slightly shorter B setae; quotient q 1.00-1.08(Figures6d-i and 10d).

Figure 11 .
Figure 11.Baetis (Baetis) dihyae sp.nov.larva, SEM: (a) details of posterior margin and surface of terga III; (b) details of posterior margin and surface of terga VI, dorsal view; (c) details of posterior margin terga I, centrally; (d) details of surface of terga I, centrally; (e) outer margin of gill VI; (f) details of outer margin of gill VI.Cerci 0.54-0.57×body length.Paracercus consists of more than 50 segments.Posterior margin of cercal and paracercal segments each with a row of broad, triangular spines, mainly blunt and bluntly pointed apically, alternating with B setae, occasionally grouped with 2-3 such types of setae (Figure 12e,f).Morphological characters to distinguish B. dihyae sp.nov.from other representatives of the Baetis alpinus species group, especially from closely related species, are given according to Müller-Liebenau[2], Thomas et al.[46], Thomas and Dia[47,48], Peru and Thomas[49], Jacob[3], Kluge and Novikova[50], Bauernfeind and Soldán[4], and Sroka et al.[45] and are summarized in Table3.Male and female adults.Unknown.Distribution and biology.Baetis dihyae sp.nov. is known so far only from two localities of Aurès streams (Figure1).The species is probably a micro-endemic of the High Mountain

Figure 14 .
Figure 14.COI neighbor-joining tree, using Tamura-Nei model, with collapsed haplogroups of B. alpinus in blue, B. punicus in green, Baetis dihyae sp.nov. in red, B. cyrneus in purple, B. maurus in orange, and B. melanonyx in black.

( i )
Labrum: the rectangle-shaped labrum attributed to Patites is as well observed in new species B. alpinus, B. nubecularis, and B. pasquetorum.The long, submarginal bristles on the dorsal surface of the labrum are disposed in one row (irregular in B. dihyae sp.nov.), and a mean number of bristles ranging between 1 + 14-21 is also a characteristic of whole Baetis gr.alpinus, which is characterized by the presence of 1 + 10-22 to 1 + 18-22 such bristles (e.g., B. alpinus); (ii) Maxillary palp: B. dihyae sp.nov.bears more than one stout seta on the tip of the maxillary palp, unlike the subgenus Patites; (iii) Mandibles: the prominent mandibular incisors are described as triangular and often fused for all representatives of Patites and the Baetis alpinus species group; (iv) Labial palps: segment 3 is listed as stout in Patites; for B. dihyae sp.nov.and closely related species (see Table [4]; (vi) Surface of abdominal terga: in contrast to Patites with numerous scales and their bases densely scattered on the surfaces of terga; B. dihyae sp.nov. is characterized by a small number of such scales along the anterior margin of the segment and their absence (including scale bases) anteriorly and centrally; (vii) Paracercus: this character excludes B. dihyae sp.nov.from the subgenus Patites, which comprises species with a diminished paracercus; B. dihyae sp.nov.has a long paracercus as in, e.g., B. alpinus.

Figure 16 .
Figure 16.Diversity and distribution of the Baetis alpinus species group in the Western Palearctic.Around the Mediterranean basin, several endemic species of the Baetis alpinus species group appear: B. pasquetorum in the Southern Alps of France, B. cyrneus in Corsica, B. cypronyx in Cyprus, and B. maurus with apparently different species populations in Spain and in North Africa [9].Additionally, two other species are known from the Maghrebian area, namely B. berberus Thomas, 1986, considered as a Moroccan endemic, and B. punicus, described from Tunisia and occurring in the whole Maghreb [4].Considering only the Baetis alpinus species group in North Africa, records exist from Morocco and Northeastern Algeria[10][11][12] and in this present study.However, several studies containing molecular analyses of studied taxa have unveiled the rich diversity of the Baetis alpinus species group and nominal species B. alpinus, with several cryptic or sibling-species isolated in the West Palearctic[6,7,[9][10][11][12]38,54,64].Thus, the reliable number of taxa of mentioned species group within the Mediterranean region and North Africa should be markedly rich and diverse.In the Maghreb, recent studies on the taxonomy and distribution of Ephemeroptera, supported by molecular analyses, argue that the Maghrebian B. alpinus represents a cryptic complex of the species in Morocco (morphological studies still not published), where specimens have been shown to be genetically distant from Iberian populations[10] and of the putative endemic in Algeria[11,12,54].These populations occur 20-1500 m a.s.l. in northern Morocco[10].In northeastern Algeria, they appear at the highest site reported in Samraoui et al.[11] (see site O. Nil, 864 m a.s.l), when in the Kebir-East, material was collected at ca. 1200 m a.s.l. in the mountains of Mount Ghora, Kroumiria[12], and have reached peaks of 1420 m in the Yabous site and 1670 m in the Inoughissen site (see present study).In the Kebir-East, which is the second largest catchment in northeast Algeria, the populations of B. alpinus species group are widespread together with B. atlanticusSoldán &

Figure 17 .
Figure 17.Colonization pathways of the Baetis alpinus species group in North Africa (for detailed description, see Section 4.2.3).Green areas represents the connection of land-bridges (strait of Gilbraltar and strait of sicily) to North africa.Black arrows represent the sens of colonization.