Another new species of the genus Habrophlebia Eaton, 1881 (Ephemeroptera, Leptophlebiidae) from the Maghreb Author El Alami, Majida https://orcid.org/0000-0003-2664-646X Universite Abdelmalek Essaadi, Faculte des Sciences, Departement de Biologie, Laboratoire Ecologie, Systematique et Conservation de la Biodiversite (LESB), Unite de Recherche Labellisee CNRST N ° 18. B. P. 2121. Tetouan 93002, Morocco elalami.majida@gmail.com Author Vuataz, Laurent https://orcid.org/0000-0001-9193-8683 Museum Cantonal des Sciences Naturelles, Departement de Zoologie, Palais de Rumine, Place Riponne 6, CH- 1005, Lausanne, Switzerland & University of Lausanne (UNIL), Department of Ecology and Evolution, CH- 1015 Lausanne, Switzerland Author El Yaagoubi, Sara https://orcid.org/0000-0003-1860-6433 Universite Abdelmalek Essaadi, Faculte des Sciences, Departement de Biologie, Laboratoire Ecologie, Systematique et Conservation de la Biodiversite (LESB), Unite de Recherche Labellisee CNRST N ° 18. B. P. 2121. Tetouan 93002, Morocco Author Sartori, Michel https://orcid.org/0000-0003-3397-3397 Museum Cantonal des Sciences Naturelles, Departement de Zoologie, Palais de Rumine, Place Riponne 6, CH- 1005, Lausanne, Switzerland & University of Lausanne (UNIL), Department of Ecology and Evolution, CH- 1015 Lausanne, Switzerland michel.sartori@vd.ch text ZooKeys 2023 2023-12-11 1186 47 70 http://dx.doi.org/10.3897/zookeys.1186.112796 journal article http://dx.doi.org/10.3897/zookeys.1186.112796 1313-2970-1186-47 E4831B39D3704B429439CA8B3E4E2930 7A3F0EFDAEB95D1DB738A19E091BC4F4 Habrophlebia dakkii El Alami, Sartori & Vuataz sp. nov. Figs 2 , 3 , 4 , 5 , 6 , 7 , 8 Habrophlebia sp. in El Alami 2002 ; Khadri et al. 2017 ; El Bazi et al. 2017 . Habrophlebia sp.1 in El Alami et al. 2022a . Material examined. Holotype : one nymph in ethanol (GBIFCH01133087), Morocco , Chefchaouen Province , S 4 Oued Amazithen , Loc. El Ouesteyine ; 35°17.299'N , 4°55.267'W ; alt. 483 m ; 2.IX.2021 ; S. El Yaagoubi leg.; MZL . Paratypes . Morocco , Chefchaouen Province , same data as holotype; 9 nymphs in ethanol (GBIFCH01133086); 1 nymph on slide (GBIFCH01144258-DNA); MZL; same locality as holotype ; 19.IX.2014 ; Khadri leg.; 2 nymphs in ethanol (GBIFCH01133083); MZL • Chefchaouen Province , S 7 Oued Harakat , Loc. Mezine village ; 35°6.133'N , 5°20.767'W ; alt. 740 m ; 31.III.2021 ; S. El Yaagoubi leg.; 6 nymphs in ethanol (GBIFCH01133085); 1 nymph on slide (GBIFCH01144257-DNA); same locality, 29.II.2020 ; 1 nymph on slide (GBIFCH00970950); MZL • Chefchaouen Province , S 5 Oued Sidi Yahia Aarab, Loc. Sidi Yahia Aarab; 35°17.179'N , 4°53.625'W ; alt. 347 m ; 18.VI.2014 ; M. El Alami leg.; 2 nymphs in ethanol (GBIFCH01133079); MZL; 4 nymphs in ethanol; 1 nymph on slide; LESCB; same locality, 27.XI.2021 ; S. El Yaagoubi leg.; 1 nymph in ethanol (GBIFCH01133080); 1 nymph on slide (GBIFCH01144259-DNA); MZL • Chefchaouen Province , S 6 Oued Jnane Nich, Loc. Jnane Nich; 35°16.1856'N , 4°52.128'W ; alt. 215 m ; 12.VIII.2021 ; S. El Yaagoubi leg.; 3 nymphs in ethanol; LESCB • Chefchaouen Province , S 8 Oued Ima sunna, Loc. Tanaqoub; 35°5.533'N , 5°23.604'W ; alt. 684 m ; 31.III.2021 ; S. El Yaagoubi leg.; 2 nymphs in ethanol (GBIFCH01133084); 1 nymph on slide (GBIFCH00970948-DNA); MZL; 1 nymph in ethanol; 1 nymph on slide; LESCB • Chefchaouen Province ; S1 Oued Kelaa , Loc. Akchour; 35°14.333'N , 05°10.144'W ; alt. 460 m ; 17.IV.2008 ; M. El Alami leg.; 3 nymphs in ethanol (GBIFCH01133088); MZL; 13 nymphs in ethanol, 1 nymph on slide; LESCB • Chefchaouen Province , S 2 Oued Talambote, Loc. usine electrique ; 35°16.665'N , 5°13.46.171'W; alt. 129 m ; 2.VI.2021 ; S. El Yaagoubi leg.; 20 nymphs in ethanol; LESCB • Chefchaouen Province , S 3 Oued Laou, Loc. Afertane; 35°20.924'N , 5°11.241'W ; alt. 55 m ; 4.VI.2022 ; S. El Yaagoubi leg.; 20 nymphs in ethanol; LESCB . • Tetouan Province , S 9 Oued El Hamma , Loc. Jbel Laalam; 35°23.387'N , 5°29.953'W ; alt. 200 m ; 20.IV.2021 ; S. El Yaagoubi leg.; 6 nymphs in ethanol (GBIFCH01133076);1 nymph on slide (GBIFCH00970944-DNA); MZL; same locality; 20.II.2022 ; S. El Yaagoubi leg.; 9 nymphs in ethanol; 1 nymph on slide; LESCB • Tetouan Province , S 10 Oued Tisgris, Loc. Hammadesh; 35°22.033'N , 5°32.033'W ; alt. 505 m ; 20.IV.2021 ; S. El Yaagoubi leg.; 8 nymphs in ethanol (GBIFCH01133077); 1 nymph on slide (GBIFCH00970946-DNA); MZL; same data; 7 nymphs in ethanol; LESCB • Tetouan Province , S 11 Oued Taida, Loc. Beni idder; 35°22.102'N , 5°32.283'W ; alt. 507 m ; 16.VII.2021 ; S. El Yaagoubi leg.; 7 nymphs in ethanol (GBIFCH01133081); 1 nymph on slide (GBIFCH01144262-DNA); MZL; same locality; 15.V.2017 ; M. El Alami leg.; 4 nymphs in ethanol; LESCB . • Larache Province , S 14 Oued Stah , Loc. Tzroute; 35°16.583'N , 5°31.883'W ; alt. 766 m ; 2.V.2021 ; S. El Yaagoubi leg.; 16 nymphs in ethanol (GBIFCH01133078); 2 nymphs on slide (GBIFCH00970945-DNA, GBIFCH01144261-DNA); MZL; same data; 8 nymphs in ethanol; 2 nymphs on slide; LESCB; same locality; 18.VI.2022 ; S. El Yaagoubi leg.; 5 nymphs in ethanol; 1 nymph on slide; LESCB . • Ouezzane Province , S 13 Oued Qoub , Loc. Souk El Had ; 35°1.283'N , 5°25.300'W ; alt. 143 m ; 11.IV.2021 ; S. El Yaagoubi leg.; 5 nymphs in ethanol (GBIFCH01133082); 1 nymph on slide (GBIFCH00970947-DNA); MZL; same data; 4 nymphs in ethanol; LESCB; same locality; 3.VI.2022 ; S. El Yaagoubi leg.; 2 nymphs in ethanol; LESCB • Ouezzane Province , S 12 Oued Loukkos, Loc. Souk El Had ; 35°1.350'N , 5°25.233'W ; alt. 140 m ; 11.IV.2021 ; S. El Yaagoubi leg.; 2 nymphs in ethanol; LESCB . Description. Nymph. Coloration and dimensions . Body length of final instar, excluding caudal filaments, 5.2 to 6.5 mm for male and 5.5 to 8 mm for female. Cerci as long as body length. General dark brown coloration with light brown to yellowish markings mainly on abdominal terga. The whole cuticle is shagreened. Head . General coloration light brown; paler area between compound eyes and lateral ocelli; between ocelli, a dark brown mark not reaching the clypeus distally, and extending laterally in front of the compound eyes; vertex sutures yellowish, well visible (Fig. 2 ). Upper portion of male eyes reddish-brown. Antenna with pedicel greyish brown, scape and filament yellowish. Figure 2. Habrophlebia dakkii sp. nov., habitus of the nymph A dorsal view B ventral view C lateral view. Scale bar: 1 mm. Labrum rectangular (Fig. 3A, B ), ca 2 x wider than long; dorsal surface covered distally with scattered stout setae, proximally with long and thin setae; anterior margin with a row of stout, long and spatulate setae medially; emargination narrow, U-shaped with six flat denticles (Fig. 3C ); ventral surface with two bunches of stout setae medially. Figure 3. Habrophlebia dakkii sp. nov., nymphal mouthparts A labrum, dorsal view B labrum, ventral view C emargination of the labrum D left mandible E right mandible. Scale bars: 100 µm ( A, B ), 50 µm ( C ), 200 µm ( D, E ). Mandibles similar to other Habrophlebia species (Fig. 3D, E ). Maxilla (Fig. 4 ) stocky, subapical row of 6 or 7 pectinate setae (Fig. 4A ); maxillary palp with three segments, segment 1 and 2 subequal in length, and longer than segment 3; segment 3 triangular a little bit 1.5 x longer than wide at base; all setae on palp segments stout and entire (Fig. 4B ). Figure 4. Habrophlebia dakkii sp. nov., nymphal mouthparts A maxilla B maxillary palp C labium D hypopharynx. Scale bars: 200 µm ( A, C, D ), 100 µm ( B ). Hypopharynx with highly developed superlinguae terminated by a membranous digitation (Fig. 4D ). Labium (Fig. 4C ) with rhomboid glossae, outer margin and apex covered by short, broad setae; paraglossae enlarged laterally, covered with fine and long setae on the dorsal surface; with long, thick setae on the outer margin; labial palp with three segments, inner margin of segment 1 highly dilated near the middle, about 0.8 x longer than the maximum width, segments 2 as long as segment 3, ca 0.7 x length of segment 1; segment 3 ca 1.6 x longer than wide at base, conical shape and the inner margin with one row of longer and robust setae. Thorax . Pro- and mesonotum yellowish to light brown, with greyish brown maculae, on medium and lateral margins (Fig. 2A ). Legs light to medium brown; dorsal surface of femora almost entirely washed with greyish brown macula; tarsi and tibiae generally lighter, except sometimes in mature nymphs. Fore legs (Fig. 5A ) femora elongated, ca 1.2 x longer than wide, upper surface covered with long, entire and pointed setae; fore tibiae subequal in length to femora, outer margin with thin and long setae, inner margin with several rows of long stout and feathered setae especially near apex; tarsi 0.4 x length of tibiae, outer margin with long and thin setae, inner margin with long and pointed feathered setae. Middle legs similar to fore legs, femora ca 2.5 x longer than wide, dorsal surface of femora with more numerous and slightly longer stout and pointed setae; tibiae and femora of subequal length; tarsi 0.5 x length of tibiae. Hind legs (Fig. 5B ) with femora 2.8 x longer than wide, dorsal surface covered with stout, long, pointed and feathered setae (Fig. 5C ); ventral surface with few feathered setae; hind tibiae as long as hind femora, outer margin with scattered stout, pointed setae; inner margin with stout, pointed, scattered setae; tarsi 0.4 x length of tibiae, outer margin with long and thin setae, inner margin with long, stout, pointed setae. Claws (Fig. 5D ) of all legs slightly hooked, with 15 to 18 long, thin, pointed denticles that decrease in size from the apex to the tarsus. Figure 5. Habrophlebia dakkii sp. nov., nymphal legs A fore femur B hind femur C setae on upper face of hind femur D claw. Scale bars: 200 µm ( A, B ); 50 µm ( C ); 100 µm ( D ). Figure 6. Habrophlebia dakkii sp. nov., posterior margin of abdominal tergites IV to IX of the nymph. Scale bar: 100 µm . Abdomen . Grey to dark brownish terga with characteristic light markings (Fig. 2A ). Tergites I-II with a dark brown color, terga III-IX with two sublateral elongated dark brown maculae, joining on the posterior margin, leaving two lateral light areas pronounced on tergites III-VIII; posterior marking sometimes absent on tergites IX-X. Sternite I entirely greyish brown (Fig. 2B ); sternites II-VIII yellowish with lateral, anterior and posterior bands brown, and two parasagittal nervous ganglia greyish browns; sternite IX lighter brown, in male nymphs with genitalia well visible and styliger plate dark brown. Posterolateral expansions only on segments VIII and IX. Posterior margin of tergite IX with well-developed narrow and pointed spines, ca 2-3 x longer than wide at base (Fig. 6 ); shape and size of posterior spines on abdominal segments V to VIII similar, a little bit smaller than those on segment IX; tergites I-IV with barely visible spines (Fig. 6 ). Gills present on segments I-VII; all gills long and large; first gill (Fig. 7A ) with dorsal lamella bearing 3 or 4 filaments, ventral lamella with 2-3 filaments, gills II-VII (Fig. 7B-D ) with 3-4 and 6-9 filaments on the ventral and dorsal lamella respectively. Cerci and paracercus yellowish brown, medium brown in mature nymphs (Fig. 2A-C ). Figure 7. Habrophlebia dakkii sp. nov., nymphal gills A gill I B gill III C gill IV D gill VII. Eggs ovoid, ca 155 µm x 80 µm , without ribs; chorion surface regularly decorated by numerous and small granulations (Fig. 8 ). Figure 8. Habrophlebia dakkii sp. nov., egg A overview of the chorionic structure B detail of the chorion with micropyle. Scale bar: 50 µm . Imago. Unknown. Etymology. The first author dedicates this species to her former mentor, Professor Dakki Mohamed. He contributed significantly to her training and specialization in the hydrobiological study of Moroccan streams. Molecular analysis. The COI data set was> 97% complete and included 26% of parsimony informative sites. Pairwise COI distances across all sequences ranged from 0% to 18.2%. All species delimitation methods were fully congruent in delimiting nine Habrophlebia MOTUs (Fig. 9 ). Among these, the 11 sequences from the new species formed a strongly supported monophyletic clade, closely related to the sequence of H. sp. 1 from Spain (Andalusia) from which it exhibits a minimal p-distance of 0.9%. According to our species delimitation methods, both lineages were grouped into a single MOTU. Similarly, the sequences of H. djurdjurensis and H. hassainae formed two distinct, well-supported, sister clades, that were merged into the same MOTU (Fig. 9 ). The overall mean p-distance within MOTUs was 1.0% (mean range: 0.4%-1.9%), while the overall mean p-distance between MOTUs was 12.9% (mean range: 7.0%-16.9%). The maximum p-distance within MOTUs varied from 1.3% ( H. sp. 4) to 4.2% ( H. djurdjurensis + H. hassainae ). The minimum p-distance between MOTUs ranged from 6.7% ( H. sp. 3- H. eldae ) to 12.3% ( H. fusca - H. lauta ). The maximum p-distance within the new species was 1.5%, whereas it was 1.8% for both H. djurdjurensis and H. hassainae . The minimum p-distance between the new species and H. sp. 1 was 0.9%, and between H. djurdjurensis and H. hassainae was 2.1%. Figure 9. Bayesian (BEAST) maximum clade credibility COI tree of the genus Habrophlebia in the West Palearctic. Branch ends labelled with GBIF or 79JJ30 codes indicate newly sequenced specimens; the other codes correspond to sequences obtained from various sources (see material and methods). Colored vertical boxes indicate species delimitation hypothesis (MOTUs) according to the ASAP, mPTP, and GMYC methods. For each MOTU, the corresponding species names (where available) and the country (-region) of origin is provided, with the newly described species and associated GBIF codes specified in bold. Circles on branches indicate Bayesian posterior probabilities> 0.9. The outgroup branches are presented in grey, along with their corresponding labels and species names.