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Research Article
Another new species of the genus Habrophlebia Eaton, 1881 (Ephemeroptera, Leptophlebiidae) from the Maghreb
expand article infoMajida El Alami‡, Laurent Vuataz§|, Sara El Yaagoubi‡, Michel Sartori§|
‡ Université Abdelmalek Essaâdi, Tétouan, Morocco
§ Muséum Cantonal des Sciences Naturelles, Département de Zoologie, Lausanne, Switzerland
| University of Lausanne (UNIL), Lausanne, Switzerland

Corresponding author: Majida El Alami ( elalami.majida@gmail.com )

Corresponding author: Michel Sartori ( michel.sartori@vd.ch )

Academic editor: Eduardo Dominguez
© 2023 Majida El Alami, Laurent Vuataz, Sara El Yaagoubi, Michel Sartori.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: El Alami M, Vuataz L, El Yaagoubi S, Sartori M (2023) Another new species of the genus Habrophlebia Eaton, 1881 (Ephemeroptera, Leptophlebiidae) from the Maghreb. ZooKeys 1186: 47-70. https://doi.org/10.3897/zookeys.1186.112796
ZooBank: urn:lsid:zoobank.org:pub:E4831B39-D370-4B42-9439-CA8B3E4E2930
Open Access

Abstract

A new species belonging to the genus Habrophlebia Eaton, 1881 is described at the nymphal stage from the Rif Mountains of Morocco. This species presents unique features, such as the chorionic arrangement of the egg and the ornamentation of the posterior margin of abdominal tergites. It is compared to all west European Habrophlebia species and a table with discriminating characters is given. A phylogenetic reconstruction based on COI sequences fully supports the hypothesis of a new species in the Rif Mountains, with possible further distribution in southern Spain.

Key words

COI, Habrophlebia dakkii sp. nov., mayflies, Morocco, Rif Mountains, Spain, systematics, West Palaearctic

Introduction

Affiliated to the subfamily Habrophlebiinae (Leptophlebiidae), Habrophlebia Eaton, 1881 is a Holarctic genus, represented by a single species in the Nearctic (Peters 1979) and eight in the West Palearctic (Jacob and Sartori 1984; Alba-Tercedor 2000; Bauernfeind and Soldán 2012; Barber-James et al. 2013; Benhadji et al. 2018; Kechemir et al. 2020). So far, five species of this genus have been reported from North Africa: Habrophlebia vaillantorum Thomas, 1986 (Thomas et al. 1999) found in the Moroccan High Atlas; H. consiglioi Biancheri, 1959, described from Sardinia and recently reported from Tunisia (Zrelli et al. 2011, 2016); two species discovered in northern Algeria, H. hassainae Benhadji & Sartori, 2018 and H. djurdjurensis Kechemir, Sartori & Lounaci, 2020; the presence of H. fusca (Curtis, 1834) in the Maghreb (Dakki and El Agbani 1983; Giudicelli and Dakki 1984; Dakki 1987; El Agbani et al. 1992; Lounaci et al. 2000) remains doubtful (Thomas 1998; Thomas et al. 1999) and unconfirmed (El Alami 2002; Benhadji et al. 2018; Kechemir et al. 2020; El Alami et al. 2022a). In fact, in northern Morocco, a review of the ephemeropteran collection from the Rif Mountains, whose specimens come from various streams, revealed the absence of the species in this biogeographical area (El Alami et al. 2022a).

With the exception of H. fusca, H. lauta McLachlan, 1884 and H. eldae Jacob & Sartori, 1984, the majority of species have a very restricted distribution. Habrophlebia antoninoi Alba-Tercedor, 2000, is a southern Iberian endemic, H. vaillantorum is a Moroccan High Atlas endemic, H. hassainae and H. djurdjurensis are Algerian endemics and H. consiglioi was collected only in Sardinia and Tunisia.

The isolation of populations in West Palearctic rivers and streams due to geographical barriers has favored speciation within the genus and contributed to an increase in the endemism rate within its biogeographical zone (Dakki 1987; Cheylan 1990; El Alami et al. 2022a).

The Rif is the most northerly mountain range in Morocco. It is characterized by a number of features that give its aquatic fauna a certain originality (Bennas 2002; El Alami 2002; Blondel et al. 2010; Hajji 2013; Errochdi et al. 2014; El Bazi et al. 2017; Khadri et al. 2017). Although the climate is typically Mediterranean, the western part is subject to Atlantic disturbances and is much wetter than the eastern part. In addition, the geological history of the Rif presents two distinctive features: the dominance of schistose or marly soils, with a limestone formation that extends mainly to the west (Thauvin 1971a, b; El Gharbaoui 1981), and the generally steep relief resulting in valleys that are often deep. The connections between the Rif and the Iberian Peninsula over their geological history have enabled a fauna exchange between the African and European continents and enriched the region’s populations (Bonada et al. 2009; Jaskuła 2015; Poulakakis et al. 2015; Mabrouki et al. 2017; Múrria et al. 2017; Bennas et al. 2018; Slimani 2018; Taybi et al. 2020; El Alami et al. 2022a).

Ongoing research on mayflies from northern Morocco has revealed that the Habrophlebia specimens are not related to either H. vaillantorum or H. fusca. They belong in fact to a new species that has been cited as Habrophlebia sp. in numerous works on the Rif (El Alami 2002; El Bazi et al. 2017; Khadri et al. 2017; Guellaf et al. 2021; El Alami et al. 2022a).

The main objective of the present study is to describe this species based on Mrs El Alami’s collection and from material freshly collected by Ms El Yaagoubi. Morphological and molecular data (COI sequences) were combined to separate the nymphs of the Rif populations from other western Palearctic species. We also provide preliminary information on its distribution and ecological preferences.

Materials and methods

Samplings were performed by the LESCB team between 1997 and 2023 (Fig. 1). They were subsequently preserved in 70% or 95% ethanol for description and DNA extraction.

Figure 1. 

The sampling site localization of Habrophlebia dakkii sp. nov. in the Rif domain.

Pictures of nymphal 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 and Helicon Focus ver. 5.3.

Nymphal dissection was performed in Cellosolve or in 10% KOH, and specimens were mounted on slides with Euparal medium, or the dissected parts of the nymphs were mounted directly in Hoyer’s liquid (Alba-Tercedor 1988).

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. Alternatively, pictures were taken using an Olympus CX41 microscope.

Molecular analysis

To complement our morphological investigations, we sequenced a 658 bp fragment of the mitochondrial gene cytochrome oxidase subunit 1 (COI hereafter) for specimens of the new species and other Habrophlebia species collected in the Maghreb. For this, the DNA extraction method described in Vuataz et al. (2011) was used to ensure non-destructive extraction. The Polymerase Chain Reaction (PCR), purification and sequencing steps followed the method outlined in El Alami et al. (2022b). Forward and reverse sequencing reads were assembled and edited in CodonCode Aligner ver. 10.0.2 (Codon-Code Corporation, Dedham, MA). To augment our molecular dataset, we initially downloaded all COI sequences associated with Habrophlebia available on the GenBank database as of 27 August 2023, totaling 69 records. Additionally, we obtained Habrophlebia sequences from the BOLDSYSTEMS data portal on the same date and selectively retained only those not shared with GenBank, yielding an additional set of six sequences. We then manually excluded GenBank/BOLD sequences obtained from specimens collected outside the western Palearctic region or shorter than 250 bp. This selection process was conducted after confirming that the COI sequences of the new species were clearly distinct from the removed sequences (data not shown). We also included sequences from the Freshwater Diversity Identification for Europe (FREDIE) project (unpublished; https://wp.fredie.eu/). A total of 63 sequences remained for further analyses, comprising 18 newly generated sequences (Table 1), 38 sequences from GenBank [five from Cardoni et al. (2015); ten from Gattolliat et al. (2015); 15 from Morinière et al. (2017); three from Behrens-Chapuis et al. (2021); five unpublished International Barcode of Life (iBOL) data releases], five from BOLD (BGMAY026-10, BGMAY092-11, BGMAY446-11, DTNHM444-21, TRSKA4318-20), and two from FREDIE: MO008a_SR4H12 (Morocco, Rif, wadi Farda at Imizzar) and ES035a_SR4E11 (Spain, Cordilleras Béticas, Barranco de los Madroñales near Otivar). Two sequences from specimens of Habroleptoides Schönemund, 1929 were downloaded from GenBank and included in the dataset as outgroups. All sequences were aligned using MAFFT (Katoh et al. 2019) with default settings as implemented in Jalview ver. 2.11.2.7 (Waterhouse et al. 2009). The number of parsimony-informative sites of the alignment was calculated in Mega ver. 10.2.4 (Kumar et al. 2018; Stecher et al. 2020).

Table 1.
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Newly sequenced specimens (nymphs) for the present study, with collection information, GenBank accession numbers and nomenclature details.

Specimen catalogue nb Species Country Stage Locality GPS coordinates Date GenBank ID GenSeq Nomenclature
GBIFCH01144259 Habrophlebia dakkii sp. nov. Morocco Nymph Sidi Yahia Aarab 35°17.179'N, 4°53.625'W 27.xi.2021 OR570530 genseq-2 COI
GBIFCH01144258 Habrophlebia dakkii sp. nov. Morocco Nymph El Ouesteyine 35°17.299'N, 4°55.267'W 1.ix.2021 OR570531 genseq-2 COI
GBIFCH01144262 Habrophlebia dakkii sp. nov. Morocco Nymph Beni idder 35°22.102'N, 5°32.283'W 16.vii.2021 OR570532 genseq-2 COI
GBIFCH00970948 Habrophlebia dakkii sp. nov. Morocco Nymph Tanaqoub 35°5.533'N, 5°23.604'W 31.iii.2021 OR570533 genseq-2 COI
GBIFCH01144257 Habrophlebia dakkii sp. nov. Morocco Nymph Mezine village 35°6.133'N, 5°20.767'W 31.iii.2021 OR570534 genseq-2 COI
GBIFCH00970944 Habrophlebia dakkii sp. nov. Morocco Nymph Jbel Laalam 35°23.387'N, 5°29.953'W 20.iv.2021 OR570535 genseq-2 COI
GBIFCH00970947 Habrophlebia dakkii sp. nov. Morocco Nymph Souk El Had 35°1.283'N, 5°25.300'W 11.iv.2021 OR570536 genseq-2 COI
GBIFCH00970945 Habrophlebia dakkii sp. nov. Morocco Nymph Tzroute 35°16.583'N, 5°31.883'W 2.v.2021 OR570537 genseq-2 COI
GBIFCH01144261 Habrophlebia dakkii sp. nov. Morocco Nymph Tzroute 35°16.583'N, 5°31.883'W 2.v.2021 OR570538 genseq-2 COI
GBIFCH00970946 Habrophlebia dakkii sp. nov. Morocco Nymph Hammadesh 35°22.033'N, 5°32.033'W 20.iv.2021 OR570539 genseq-2 COI
GBIFCH00970949 Habrophlebia sp. 2 Morocco Nymph Afeska 35°10.184'N, 5°13.105'W 2.iv.2021 OR570540 genseq-4 COI
79JJ30_B07 Habrophlebia hassainae Algeria Nymph El Ourit 34°51'57’’N, 1°15'54’’W 1.i.2016 OR570541 genseq-4 COI
79JJ30_G06 Habrophlebia hassainae Algeria Nymph El Ourit 34°51'57"N, 1°15'54"W 5.ii.2016 OR570542 genseq-4 COI
GBIFCH00673196 Habrophlebia djurdjurensis Algeria Nymph Tirourda 36°29.431'N, 4°21.693'E 9.vii.2019 OR570543 genseq-4 COI
GBIFCH00673194 Habrophlebia djurdjurensis Algeria Nymph Echemlili 36°28.267'N, 3°59.84'E 25.v.2018 OR570544 genseq-4 COI
GBIFCH00673195 Habrophlebia djurdjurensis Algeria Nymph Echemlili 36°28.267'N, 3°59.84'E 25.v.2018 OR570545 genseq-4 COI
GBIFCH00673199 Habrophlebia djurdjurensis Algeria Nymph Ouadhias 36°29.279'N, 4°07.362'E 9.vii.2019 OR570546 genseq-4 COI
GBIFCH01211557 Habrophlebia djurdjurensis Algeria Nymph Tala Rana Selloum 2 36°26.902'N, 4°18.820'E 28.iv.2021 OR570547 genseq-4 COI

To explore and visualize the COI evolutionary divergence, we employed pairwise genetic distances and gene tree approaches. COI pairwise distances were calculated using the dist.dna function from the ape 5.7-1 package (Paradis and Schliep 2019) in R ver. 4.3.1 (R Core Team 2023), selecting the raw model and the pairwise.deletion option, corresponding to uncorrected p-distances (see Srivathsan and Meier 2012) with missing data removed in a pairwise way. Mean, minimum, and maximum distances within and between putative COI species, referred to as Molecular Operational Taxonomic Units (MOTUs) hereafter, were calculated using the ddply function from the plyr ver. 1.8.8 package (Wickham 2011). The assignment of COI sequences to MOTUs was determined based on the results of the species delimitation analyses (as described below). Prior to reconstructing the COI gene tree, the best evolutionary model (GTR+Γ) was selected based on the second-order Akaike information criterion (AICc; Hurvich and Tsai 1989) implemented in JmodelTest ver. 2.1.10 (Darriba et al. 2012) with five substitution schemes, six gamma categories and default values for other parameters. To account for different substitution rates among COI codon positions, we analyzed our data set in two partitions, one with first and second codon positions, and the other with third positions (1 + 2, 3). Bayesian inference analysis was performed using BEAST ver. 1.10.4 (Suchard et al. 2018) on the CIPRES Science Gateway ver. 3.3 (Miller et al. 2010). The input BEAST file was generated in BEAUTi ver. 1.10.4 (Suchard et al. 2018), incorporating the selected evolutionary model and partition scheme described above. A relaxed molecular clock model (uncorrelated lognormal) and a UPGMA starting tree were used, with default settings for other parameters. Two independent Markov chain Monte Carlo (MCMC) analyses were run for 50 million generations, logging parameters every 1000 generations. Convergence of the MCMC runs was visually verified in Tracer ver. 1.7.2 (Rambaut et al. 2018). The log and tree files from the independent runs were combined using LogCombiner ver. 1.10.4 (Suchard et al. 2018), after discarding the initial 10% of trees as burn-in, ensuring that all parameters reached effective sample size values > 200. The maximum clade credibility tree was obtained using TreeAnnotator ver. 1.10.4 (Suchard et al. 2018) with default settings and then visualized and edited in iTOL ver. 6.8 (Letunic and Bork 2021).

Finally, we applied three contrasting single-locus species delimitation methods to our COI dataset: the distance-based ASAP (Assemble Species by Automatic Partitioning; Puillandre et al. 2020), and the tree-based GMYC (General Mixed Yule-Coalescent; Pons et al. 2006; Fujisawa and Barraclough 2013) and mPTP (multi-rate Poisson Tree Processes; Kapli et al. 2017) approaches. ASAP, an improved version of the ABGD (Automatic Barcode Gap Discovery; Puillandre et al. 2012) approach, was employed using the ASAP webserver (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html), computing the genetic distances from our COI alignment under simple p-distances with all other settings set to default. The GMYC model, which requires a time-calibrated ultrametric tree as input, implements a maximum likelihood (ML) approach that defines a threshold separating the branches modelled under speciation events (Yule process) from those described by allele neutral coalescence. The ultrametric tree used as input for GMYC was generated in BEAST, following the same procedure described earlier. However, a reduced dataset was utilized, in which outgroups were excluded and haplotypes were pruned (see Talavera et al. 2013) using Collapsetypes ver. 4.6 (Chesters 2013). MCMC chains were run here for a total of 30 million generations. GMYC was run in R using the SPLITS package 1.0–20 (Ezard et al. 2009). We favored the single-threshold version of the GMYC model because it was shown to outperform the multiple-threshold version (Fujisawa and Barraclough 2013). The mPTP approach, an extension of the PTP method by Zhang et al. (2013), also exploits phylogenetic differences within and between species, but with the advantage of directly using the number of substitutions from a phylogenetic tree, eliminating the need for time calibration. The input ML tree for mPTP was generated in RAxML-NG ver. 1.1.0 (Kozlov et al. 2019), selecting the all-in-one (ML search + bootstrapping) option and MRE-based bootstrap convergence criterion. The best model of evolution and the partition scheme specified above, as well as 50 random and 50 parsimony starting trees, were implemented.

Abbreviations

MZL Muséum Cantonal des Sciences Naturelles, Lausanne (Switzerland);

LESCB Laboratoire d’Ecologie, Systématique et Conservation de la Biodiversité (Morocco).

Results

Systematics

Habrophlebia dakkii El Alami, Sartori & Vuataz, sp. nov.

https://zoobank.org/D4B71565-041B-417A-8575-8348702DCF73
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, S4 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, S7 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, S5 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, S6 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, S8 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 Kelâa, 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, S2 Oued Talambote, Loc. usine électrique; 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, S3 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, S9 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, S10 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, S11 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, S14 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, S13 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, S12 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× 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× 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× longer than the maximum width, segments 2 as long as segment 3, ca 0.7× length of segment 1; segment 3 ca 1.6× 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× 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× 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× 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× length of tibiae.

Hind legs (Fig. 5B) with femora 2.8× 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× 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× 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.

Discussion

Until the late 1970s, only two species of Ephemeroptera were known from the Rif, due to the works of Eaton (1899), Navás (1922, 1929, 1935), Lestage (1925) and Kimmins (1938). This fauna corresponded to two Baetidae recorded in the regions of Tétouan and Chefchaouen [Baetis rhodani (Pictet, 1843) and Procloeon cf. concinnum]. Since then, a large number of surveys have been carried out to augment this initial list with an additional 35 species, resulting in a total species richness of 37 taxa, which constitutes more than 68% of Morocco’s Ephemeroptera (Dakki and El Agbani 1983; Giudicelli and Dakki 1984; El Alami and Dakki 1998; El Alami et al. 2000, 2022a; Kaltenbach et al. 2022; Gattolliat et al. 2023; El Alami et al. 2023; El Yaagoubi et al. 2023). These investigations have collected no less than three new species from the Rif, including Habrophlebia dakkii sp. nov.

Morphology characteristics

The main characters used to distinguish the hitherto known species from the new one, are presented in Table 2. At the egg stage, Habrophlebia dakkii sp. nov. presents a unique chorionic arrangement among Habrophlebia species, composed of fine granulations evenly distributed on the whole surface, without any other attachment structures (ribs, polar caps). Habrophlebia dakkii sp. nov. is most similar to H. eldae, especially concerning the abdominal spines on the posterior margin of abdomen, although those of H. eldae are larger than those of H. dakkii. The latter mainly differs from the former by the number of gill filaments. Habrophlebia dakkii can be easily separated from the other Maghrebian species; from H. hassainae by the number of denticles on the claw, by the number of gill filaments, and by the shape of posterior abdominal spines on tergites V; from H. djurdjurensis also by the shape of posterior abdominal spines on tergites V-VIII; and from H. vaillantorum by the number and the size of denticles on the tarsal claw, the number of gill filaments, and by the shape of posterior abdominal spines on tergites V-IX.

Table 2.
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Taxonomic criteria differentiating nymphs and eggs of Western Palaearctic Habrophlebia species: (0) Present study; (1) Thomas et al. 1999; (2) Wagner at al., 2007; (3) Mazzini and Gaino 1985 (H. eldae sub. nom. H. fusca); (4) Zrelli et al. 2011; (5) Alba-Tercedor 2000; (6) Benhadji et al. 2018; (7) Kechemir et al. 2020; (8) Biancheri 1959; (9) Kimmins 1954; (10) Landa 1969; (11) M. Sartori, pers. obs.; (12) Jacob and Sartori 1984; (13) Bauernfeind and Soldán 2012; (14) Thomas and Bouzidi 1986; (15) Righetti 2020; (16) Belfiore and Gaino 1985.

Character H. fusca H. lauta H. eldae H. consiglioi H. antoninoi (5) H. vaillantorum H. hassainae (6) H. djurdjurensis (7) H. dakkii sp.nov. (0)
Egg chorionic structure ribs long, punctuated (1) ribs long, not punctuated (2) ribs long, slightly punctuated (3) ribs barbed, long , punctuated (3,4) ribs forming a reticulated mesh ribs long, not punctuated (1) ribs short, not punctuated ribs long, not punctuated without ribs, entirely covered with small granulations
Position of the costal process of hind wing middle (9) middle (10) middle (11) middle (8) distal middle (1) middle middle middle
Bristles on upper face of hindfemur truncated, entire (12) pointed, entire (12) pointed, fringed (12) pointed, fringed (12) ? pointed, entire (13) pointed, fringed pointed, fringed pointed, fringed
Number of denticles on claws 11–13 (11) 14–16 (11) 14–17 (11, 15) 15–17 (11) ? 13–16 (1, 14) 18–22 15–18 15–18
Size of distal denticles on claws normal (11) normal (11) normal (11) normal (11) ? reduced (1, 14) normal normal normal
Number of filaments on dorsal (costal) and ventral (anal) lamellae of gills II-VI 7–8; 3–4 (13) 5–7; 4–5 (13) 3–7; 2–4(13) 3–6; 1–3 (16) ? 5–6; 4–5 (1, 14) 9–12; 5–8 8–11; 4–7 6–9; 3–4
Shape and size of posterior spines on abdominal segment IX truncated, wider than long (1, 2, 16) triangular, as long as wide at base (2, 16) triangular, 2–3× longer than wide at base (2, 16) triangular, 1.5–2× longer than wide at base (16) ? minute, needle-shaped (1) triangular, 2–3× longer than wide at base lanceolate, 2–3× longer than wide at base triangular, 2–3× longer than wide at base
Shape and size of posterior spines on abdominal segment VIII truncated, wider than long (16) triangular, as long as wide at base (16) triangular, 2–3× longer than wide at base (16) triangular, 1.5–2× longer than wide at base (16) ? ? (probably minute, needle-shaped) triangular, 2–3× longer than wide at base minute, needle-shaped triangular, 2–3× longer than wide at base
Shape and size of posterior spines on abdominal segment VII truncated, wider than long (16) triangular, as long as wide at base (16) triangular, 2–3× longer than wide at base (16) triangular, 1.5–2× longer than wide at base (16) ? minute, needle-shaped (1) triangular, 2× longer than wide at base minute, needle-shaped triangular, 2–3× longer than wide at base
Shape and size of posterior spines on abdominal segment V truncated, wider than long (16) triangular, as long as wide at base (16) triangular, 2–3× longer than wide at base (16) triangular, as long as wide at base (16) ? ? (probably minute or absent) minute, needle-shaped minute, needle-shaped triangular, 2–3× longer than wide at base

Genetic characteristics

While the 11 sequences from the new species group together in a well-supported COI clade (Fig. 9), they are sister to the sequence of H. sp. 1 from Spain (Andalusia) obtained from a young nymph, showing a minimal p-distance of 0.9% between both lineages. Such a genetic distance is typically found within species, as reported in insects in general (e.g., Virgilio et al. 2010) and especially in mayflies (e.g., Ball et al. 2005; Morinière et al. 2017). This observation is further corroborated by the results obtained from the three species delimitation methods. To conclusively determine whether these lineages represent distinct species and potentially establish the new species as a Moroccan endemic or if they constitute a single species with an extended geographic range, additional investigations in the Iberian Peninsula are imperative with new material. A similar situation occurred with the recently described baetid species Baetis rifensis El Yaagoubi, Vuataz & Gattolliat, 2023 from Morocco. Its closest COI sequences were from three specimens sampled in the Iberian Peninsula, but in this case, the minimum p-distance between the Moroccan and Iberian lineages was higher (3.2%; El Yaagoubi et al. 2023).

Also interesting is the close genetic relationship between H. hassainae and H. djurdjurensis; both species are only separated by a minimum genetic distance of 2.1%, which suggests they could belong to the same species. Morphologically however, both species differ by a number of characters (Table 2), among which are the shape and size of spines on the posterior margin of abdominal tergites, the number of gill filaments and the number of denticles on the claw. Such a low COI divergence between species, possibly indicating recent reproductive isolation, has been sporadically documented in animals (Hebert et al. 2003), including mayflies (e.g., Morinière et al. 2017), and appears to be relatively frequent among closely related species pairs of stoneflies (Vuataz pers. obs.)

Our COI analyses have identified a new Habrophlebia lineage in the Rif region, labeled as H. sp. 2 (Fig. 9). Initial morphological examinations suggest it may be a new species, which will be further investigated in a future study. Additionally, we could not assign names to several other COI clades. While H. sp. 3 from Corsica and H. sp. 4 from Sicily were previously identified as H. eldae in Gattolliat et al. (2015) and Tenchini et al. (2018), respectively, the COI distances between those three lineages (see Fig. 9) are more indicative of distinct species (with minimum p-distance of H. eldae–H. sp. 3: 6.7%; H. eldae–H. sp. 4: 8.5%; H. sp. 3–H. sp. 4: 7.7%), as pointed out by Tenchini et al. (2018). Similarly, H. sp. 5 from Bulgaria, presently labeled as H. fusca in GenBank, clearly differs from H. fusca and all other lineages in our dataset. The p-distance to its nearest neighbors, H. eldae and H. lauta (12.2%), strongly supports H. sp. 5 as a distinct, new species. These results emphasize the need for more taxonomic research on this genus in Europe.

Distribution and ecology

This species is widely distributed in the Rif, where it occupies a large range of biotope types, spanning from sea level up to an altitude of 780 m. It also tolerates wide variations in water conductivity (35 to 1112 µS/cm). Habrophlebia dakkii has a clear preference for poorly mineralized headwaters with moderate current velocity. The substrate is characterized by pebbles, gravel, sand and silt covered in some places with algae and submerged macrophytes, which provide excellent refuge for nymphs when the current is stronger.

The same remark was made by Gagneur and Thomas (1988) concerning Habrophlebia hassainae (sub. nom. H. gr. fusca) found in Algeria.

Since this species has been found in localities on the Oued Ouergha, which is the Rif tributary of the Oued Sebou, we believe that its presence in the Haut Sebou (Middle Atlas) and the coastal Meseta is highly probable.

Nymphs of Habrophlebia dakkii sp. nov. can be found all year long, but are most abundant in spring, when temperatures are optimal for their development.

Acknowledgments

We express our gratitude to Céline Stoffel (MZL) for her dedicated work with the molecular lab. Nadhira Benhadji (Tlemcen, Algeria now Szczecin, Poland), Lina Kechemir (Tizi-Ouzou, Algeria), Sarah Labdaoui and Boudjéma Samraoui (Gelma and Annaba, Algeria), Cesc Múrria (Barcelona, Spain), Ossama Khadri (Tetouan, Morocco) are thanked for donation of precious material. Constructive remarks by Pavel Sroka (Ceske Budejovice, Cech Republic) and Nadhira Benhadji helped to improve the manuscript.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

No funding was reported.

Author contributions

Conceptualization: MS, MEA. Data curation: LV, SEY. Formal analysis: LV. Funding acquisition: MS. Investigation: SEY. Methodology: MEA. Project administration: MEA. Software: LV. Validation: MS, MEA. Writing - original draft: MEA. Writing - review and editing: MS, LV, MEA.

Author ORCIDs

Majida El Alami https://orcid.org/0000-0003-2664-646X

Laurent Vuataz https://orcid.org/0000-0001-9193-8683

Sara El Yaagoubi https://orcid.org/0000-0003-1860-6433

Michel Sartori https://orcid.org/0000-0003-3397-3397

Data availability

All of the data that support the findings of this study are available in the main text.

References

  • Alba-Tercedor J (1988) Ephemeroptera. Bases para un curso práctico de Entomología. Barrientos, J.A. (coord.). Asociación Española de Entomología 6: 359–372.
  • Alba-Tercedor J (2000) Habrophlebia antoninoi sp.n., a new species from Spain, with an account of the European species of Habrophlebia Eaton, 1881 (Ephemeroptera: Leptophlebiidae, Habrophlebiinae). Aquatic Insects 22(1): 1–7. https://doi.org/10.1076/0165-0424(200001)22:1;1-Z;FT001
  • Ball SL, Hebert PD, Burian SK, Webb JM (2005) Biological identifications of mayflies (Ephemeroptera) using DNA barcodes. Journal of the North American Benthological Society 24(3): 508–524. https://doi.org/10.1899/04-142.1
  • Bauernfeind E, Soldán T (2012) The Mayflies of Europe (Ephemeroptera). Apollo Books, Ollerup, 781 pp.
  • Behrens-Chapuis S, Herder F, Geiger MF (2021) Adding DNA barcoding to stream monitoring protocols – What’s the additional value and congruence between morphological and molecular identification approaches? PLoS ONE 16(1): e0244598. https://doi.org/10.1371/journal.pone.0244598
  • Belfiore C, Gaino E (1985) Le specie italiane del genere Habrophlebia Eaton, 1881 (Ephemeroptera, Leptophlebiidae). Bollettino dell’Associazione Romana di Entomologia 39: 11–18. [1984]
  • Benhadji N, Hassaine KA, Sartori M (2018) Habrophlebia hassainae, a new mayfly species (Ephemeroptera: Leptophlebiidae) from North Africa. Zootaxa 4403(3): e557. https://doi.org/10.11646/zootaxa.4403.3.8
  • Bennas N (2002) Coléoptères aquatiques Polyphaga du Rif (Nord du Maroc): Faunistique, Ecologie et Biogéographie. Thèse Doctorat d’Etat. Université Abdelmalek Essaâdi.
  • Bennas N, L’mohdi O, El-Haissoufi M, Charfi F, Ghlala A, El-Alami M (2018) New data on the aquatic insect fauna of Tunisia. Transactions of the American Entomological Society 144(3): 575–592. https://doi.org/10.3157/061.144.0309
  • Biancheri E (1959) Note sugli Efemerotteri italiani. IX. Descrizione di una nuova specie della Sardegna: Habrophlebia consiglioi sp. n. Bollettino della Società Entomologica Italiana 89(3–4): 35–38.
  • Blondel J, Aronson J, Bodiou J-Y, Boeuf G (2010) The Mediterranean Region: Biological Diversity in Space and Time (2e édn.). OUP Oxford, Oxford, 392 pp.
  • Bonada N, Múrria C, Zamora-Muñoz C, El Alami M, Poquet JM, Puntí T, Moreno JL, Bennas N, Alba-Tercedor J, Ribera C, Prat N (2009) Using community and population approaches to understand how contemporary and historical factors have shaped species distribution in river ecosystems. Global Ecology and Biogeography 18(2): 202–213. https://doi.org/10.1111/j.1466-8238.2008.00434.x
  • Cardoni S, Tenchini R, Ficulle I, Piredda R, Simeone M, Belfiore C (2015) DNA barcode assessment of Mediterranean mayflies (Ephemeroptera), benchmark data for a regional reference library for rapid biomonitoring of freshwaters. Biochemical Systematics and Ecology 62: 36–50. https://doi.org/10.1016/j.bse.2015.07.035
  • Cheylan G (1990) Endémisme et spéciation chez les Mammifères méditerranéens. Vie et Milieu 40: 137–143.
  • Dakki M (1987) Ecosystème d’eau courante du haut Sebou (Moyen Atlas); Etudes typologiques et analyses écologiques et biogéographie des principaux peuplements entomologiques. Travaux de l’Institut scientifique (Rabat) Serie Zoologie 42: 1–99.
  • Dakki M, El Agbani M-A (1983) Ephéméroptères d’Afrique du Nord: 3. Eléments pour la connaissance de la faune marocaine. Bulletin de l’Institut Scientifique, Rabat 7: 115–126.
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): e772. https://doi.org/10.1038/nmeth.2109
  • Eaton AE (1899) List of Ephemeridae hitherto observed in Algeria, with localities. Entomologist’s Monthly Magazine 35: 4–5.
  • El Agbani MA, Dakki M, Bournaud M (1992) Etude typologique du Bou Regreg (Maroc) : Les milieux aquatiques et leurs peuplements en macroinvertébrés. Bulletin d’écologie 23: 103–113.
  • El Alami M (2002) Taxonomie, écologie et biogéographie des Ephéméroptères du Rif (Nord du Maroc). Thèse Doctorat d’Etat. Université Abdelmalek Essaâdi.
  • El Alami M, Dakki M (1998) Peuplements d’Ephéméroptères et de Trichoptères de l’Oued Laou (Rif Occidentale, Maroc): distribution longitudinale et biotypologie. Bulletin de l’Institut Scientifique, Rabat 21: 51–70.
  • El Alami M, Dakki M, Errami M, Alba-Tercedor J (2000) Nouvelles données sur les Baetidae du Maroc (Insecta: Ephemeroptera). Zoologica Baetica 11: 105–113.
  • El Alami M, El Yaagoubi S, Gattolliat J-L, Sartori M, Dakki M (2022a) Diversity and Distribution of Mayflies from Morocco (Ephemeroptera, Insecta). Diversity 14(6): e498. https://doi.org/10.3390/d14060498
  • El Alami M, Benlasri M, Sartori M, Vuataz L, Ghamizi M (2022b) A new species of the genus Prosopistoma Latreille, 1833 (Ephemeroptera, Prosopistomatidae) from Morocco. ZooKeys 1117: 203–218. https://doi.org/10.3897/zookeys.1117.83539
  • El Alami M, Vuataz L, El Yaagoubi S, Gattolliat J-L (2023) A new species of the genus Alainites Waltz & McCafferty, 1994 (Ephemeroptera, Baetidae) from the north of Morocco. ZooKeys 1176: 221–241. https://doi.org/10.3897/zookeys.1176.107829
  • El Bazi R, El Alami M, Khadri O, Errochdi S, Slimani M, Bennas N (2017) Projet du parc naturel de Bouhachem (Nord-Ouest du Maroc) II: Ephemeroptera, Plecoptera, Trichoptera. Boletin de la SEA 61: 55–66. [SEA]
  • El Gharbaoui A (1981) La terre et l’homme dans la péninsule tingitane: étude sur l’homme et le milieu naturel dans le Rif occidental. Travaux de l’Institut scientifique. Série géologie et géographie physique, ISSN 0851-299X; 15. Edition de l’Institut Scientifique de Rabat, 439 pp.
  • Errochdi S, El Alami M, Vinçon G, Abdaoui A, Ghamizi M (2014) Contribution to the knowledge of Moroccan and Maghrebin stoneflies (Plecoptera). Zootaxa 3838(1): 46–76. https://doi.org/10.11646/zootaxa.3838.1.2
  • Ezard T, Fujisawa T, Barraclough T (2009) splits: SPecies’ LImits by Threshold Statistics version 1.0-20 from R-Forge. https://rdrr.io/rforge/splits/ [June 8, 2023]
  • Fujisawa T, Barraclough TG (2013) Delimiting species using single-locus data and the Generalized Mixed Yule Coalescent approach: A revised method and evaluation on simulated data sets. Systematic Biology 62(5): 707–724. https://doi.org/10.1093/sysbio/syt033
  • Gagneur J, Thomas AGB (1988) Contribution à la connaissance des Ephéméroptères d’Algérie. I. Répartition et écologie (1ère partie) (Insecta, Ephemeroptera). Bulletin de la Société d’Histoire Naturelle de Toulouse 124: 213–223.
  • Gattolliat J-L, Cavallo E, Vuataz L, Sartori M (2015) DNA barcoding of Corsican mayflies (Ephemeroptera) with implications on biogeography, systematics and biodiversity. Arthropod Systematics & Phylogeny 73(1): 3–18. https://doi.org/10.3897/asp.73.e31813
  • Gattolliat J-L, Samraoui B, Benhadji N, Kechemir L, Zrelli S, El Yaagoubi S, El Alami El Moutaouakil M, Sartori M (2023) Baetidae (Baetidae, Ephemeroptera) in the Maghreb: State of the art, key, and perspectives. ZooKeys 1139: 137–163. https://doi.org/10.3897/zookeys.1139.94586
  • Giudicelli J, Dakki M (1984) Les sources du Moyen Atlas et de Rif (Maroc): Faunistique (description de deux espèces nouvelles de Trichoptères), écologie, intérêt biogéographique. Bijdragen tot de Dierkunde 54(1): 83–100. https://doi.org/10.1163/26660644-05401007
  • Guellaf A, El Alami M, Kassout J, Errochdi S, Khadri O, Kettani K (2021) Diversity and ecology of aquatic insects (Ephemeroptera, Plecoptera and Trichoptera) in the Martil basin (Northwestern Morocco). Community Ecology 22(3): 331–350. https://doi.org/10.1007/s42974-021-00058-3
  • Hajji K (2013) Contribution à la connaissance des Trichoptères (Trichoptera) du Rif (nord du Maroc). Boletin de la Asociacion Espanola de Entomologia, 181–216.
  • Hebert PD, Ratnasingham S, De Waard JR (2003) Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, Series B, Biological Sciences 270: S96–S99. https://doi.org/10.1098/rsbl.2003.0025
  • Jacob U, Sartori M (1984) Die europäischen Arten der Gattung Habrophlebia Eaton (Ephemeroptera, Leptophlebiidae). Entomologische Abhandlungen Staatliches Museum für Tierkunde Dresden 48(5): 45–52.
  • Jaskuła R (2015) The Maghreb – one more important biodiversity hot spot for tiger beetle fauna (Coleoptera, Carabidae, Cicindelinae) in the Mediterranean region. ZooKeys 482: 35–53. https://doi.org/10.3897/zookeys.482.8831
  • Kaltenbach T, Vuataz L, Samraoui B, El Yaagoubi S, El Alami M, Gattolliat J-L (2022) Two new species of Centroptilum Eaton, 1869 from North Africa (Ephemeroptera, Baetidae). ZooKeys 1131: 71–97. https://doi.org/10.3897/zookeys.1131.91017
  • Kapli P, Lutteropp S, Zhang J, Kobert K, Pavlidis P, Stamatakis A, Flouri T (2017) Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo. Bioinformatics 33(11): 1630–1638. https://doi.org/10.1093/bioinformatics/btx025
  • Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20(4): 1160–1166. https://doi.org/10.1093/bib/bbx108
  • Khadri O, El Alami M, El Bazi R, Slimani M (2017) Ephemeroptera’s diversity and ecology in streams of the ultramafic massif of Beni Bousera and in the adjacent non-ultramafic sites (NW, Morocco). Journal of Materials & Environmental Sciences 8: 3508–3523.
  • Kimmins DE (1954) A revised key to the adults of the British species of Ephemeroptera with notes on their ecology. Scientific Publication – Freshwater Biological Association 15: 1–71.
  • Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A (2019) RAxML-NG: A fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35(21): 4453–4455. https://doi.org/10.1093/bioinformatics/btz305
  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution 35(6): 1547–1549. https://doi.org/10.1093/molbev/msy096
  • Landa V (1969) Jepice-Ephemeroptera. Fauna CSSR 18: 1–352.
  • Lestage J (1925) Ephémères, Plécoptères et Trichoptères recueillis en Algérie et liste des espèces connues actuellement de l’Afrique du Nord. Bulletin de la Société d’histoire naturelle d’Afrique du Nord 16: 8–18.
  • Letunic I, Bork P (2021) Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Research 49(W1): W293–W296. https://doi.org/10.1093/nar/gkab301
  • Lounaci A, Brosse S, Thomas A, Lek S (2000) Abundance, diversity and community structure of macroinvertebrates in an Algerian stream: The Sébaou wadi. Annales de Limnologie – International. Annales de Limnologie 36(2): 123–133. https://doi.org/10.1051/limn/2000008
  • Mabrouki Y, Taybi AF, El Alami M, Berrahou A (2017) New and interesting data on distribution and ecology of Mayflies from Eastern Morocco (Ephemeroptera). Journal of Materials & Environmental Sciences 8: 2839–2859.
  • Mazzini M, Gaino E (1985) Fine structure of the egg shells of Habrophlebia fusca (Curtis) and H. consiglioi Biancheri (Ephemeroptera: Leptophlebiidae). International Journal of Insect Morphology & Embryology 14(6): 327–334. https://doi.org/10.1016/0020-7322(85)90013-3
  • Miller M, Pfeiffer WT, Schwartz T (2010) Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees. Proceedings of the Gateway Computing Environments Workshop, 14 Nov 2010, New Orleans, 8 pp. https://doi.org/10.1109/GCE.2010.5676129
  • Morinière J, Hendrich L, Balke M, Beermann AJ, König T, Hess M, Koch S, Müller R, Leese F, Hebert PDN, Hausmann A, Schubart CD, Haszprunar G (2017) A DNA barcode library for Germany’s mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera). Molecular Ecology Resources 17(6): 1293–1307. https://doi.org/10.1111/1755-0998.12683
  • Múrria C, Bonada N, Vellend M, Zamora‐Muñoz C, Alba‐Tercedor J, Sainz‐Cantero CE, Garrido J, Acosta R, El Alami M, Barquín J, Derka T, Álvarez‐Cabria M, Sáinz‐Bariain M, Filipe AF, Vogler AP (2017) Local environment rather than past climate determines community composition of mountain stream macroinvertebrates across Europe. Molecular Ecology 26(21): 6085–6099. https://doi.org/10.1111/mec.14346
  • Navás SL (1922) Insectos de la excursion de D. Ascensio Cordina à Marruecos. Trabajo del Museo de Ciencias Naturales Barcelona 4: 119–127.
  • Navás SL (1929) Insectes Névroptères et voisins de Barbarie (Septième série). Bulletin de la Société d’histoire naturelle de l’Afrique du Nord 20: 57–60.
  • Navás SL (1935) Insectos de Berberia. Boletín de la Sociedad Entomológica de España, Madrid 18: 45–50. [and 77–100]
  • Peters WL (1979) Taxonomic status and phylogeny of Habrophlebia and Habroleptoides (Leptophlebiidae: Ephemeroptera). In: Pasternak K, Sowa R (Eds) Proceedings of the Second International Conference on Ephemeroptera. Panstwowe Wydawnictwo Naukowe, Warszawa-Krakow, 51–56.
  • Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S, Kamoun S, Sumlin WD, Vogler AP (2006) Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology 55(4): 595–609. https://doi.org/10.1080/10635150600852011
  • Poulakakis N, Kapli P, Lymberakis P, Trichas A, Vardinoyiannis K, Sfenthourakis S, Mylonas M (2015) A review of phylogeographic analyses of animal taxa from the Aegean and surrounding regions. Journal of Zoological Systematics and Evolutionary Research 53(1): 18–32. https://doi.org/10.1111/jzs.12071
  • Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7. Systematic Biology 67(5): 901–904. https://doi.org/10.1093/sysbio/syy032
  • Righetti B (2020) Détermination des larves de Leptophlebiidae de France métropolitaine: un outil pratique pour identifier au genre les exemplaires dépourvus de trachéobranchies (Ephemeroptera). Ephemera 21: 109–122. [2019]
  • Slimani M (2018) Etat écologique et biodiversité des macroinvertébrés aquatiques du Haut Bassin Versant du Loukkos (Nord-Ouest du Maroc) et effet de l’intermittence et des altérations anthropiques. Thèse de Doctorat. Abdelmalek Essaâdi.
  • Stecher G, Tamura K, Kumar S (2020) Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Molecular Biology and Evolution 37(4): 1237–1239. https://doi.org/10.1093/molbev/msz312
  • Suchard MA, Lemey P, Baele G, Ayres DL, Drummond AJ, Rambaut A (2018) Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evolution 4(1): vey016. https://doi.org/10.1093/ve/vey016
  • Talavera G, Dincă V, Vila R (2013) Factors affecting species delimitations with the GMYC model: Insights from a butterfly survey. Methods in Ecology and Evolution 4(12): 1101–1110. https://doi.org/10.1111/2041-210X.12107
  • Taybi AF, Mabrouki Y, Dakki M, Berrahou A, Millán A (2020) Longitudinal distribution of macroinvertebrate in a very wet North African Bas In: Oued Melloulou (Morocco). Annales de Limnologie – International. Annales de Limnologie 56: 1–17. https://doi.org/10.1051/limn/2020016
  • Tenchini R, Cardoni S, Piredda R, Simeone MC, Belfiore C (2018) DNA barcoding and faunistic criteria for a revised taxonomy of Italian Ephemeroptera. The European Zoological Journal 85(1): 253–266. https://doi.org/10.1080/24750263.2018.1480732
  • Thauvin J (1971a) Domaines du Rif et du Maroc Oriental: La Zone Axiale du Rif. Ressources en eau du Maroc. Notes et Mémoire du Service Géologique (Rabat). Notes et mémoire du Service Géologique, Rabat, 43–67.
  • Thauvin J (1971b) Domaines du Rif et du Maroc Oriental: Présentation du Domaine Rifain. Ressources en eau du Maroc. Notes et Mémoire du Service Géologique (Rabat). Notes et mémoire du Service Géologique, Rabat, Maroc, 27–42.
  • Thomas AGB (1998) A provisional checklist of the mayflies of North Africa (Ephemeroptera). Bulletin de la Société d’Histoire Naturelle de Toulouse 134: 13–20.
  • Thomas AGB, Bouzidi A (1986) Trois Ephéméroptères nouveaux du Haut Atlas marocain (Heptageniidae, Baetidae, Leptophlebiidae). Bulletin de la Société d’Histoire Naturelle de Toulouse 122: 7–10.
  • Thomas A, Gaino E, Marie V (1999) Complementary description of Habrophlebia vaillantorum Thomas, 1986, in comparison with H. fusca (Curtis, 1984) (Ephemeroptera, Leptophlebiidae). Ephemera 1: 9–21.
  • Vuataz L, Sartori M, Wagner A, Monaghan MT (2011) Toward a DNA Taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) Using a Mixed Yule-Coalescent Analysis of Mitochondrial and Nuclear DNA. PLoS ONE 6(5): e19728. https://doi.org/10.1371/journal.pone.0019728
  • Wagner A, Lubini V, Vicentini H (2007) Habrophlebia eldae Jacob & Sartori, 1984 (Ephemeroptera: Leptophlebiidae) neu für die Schweizer Fauna. Mitteilungen der schweizerischen entomologischen Gesellschaft 80: 91–98.
  • Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview Version 2–A multiple sequence alignment editor and analysis workbench. Bioinformatics 25(9): 1189–1191. https://doi.org/10.1093/bioinformatics/btp033
  • Zrelli S, Boumaiza M, Bejaoui M, Gattolliat J-L, Sartori M (2011) New reports of mayflies (Insecta: Ephemeroptera) from Tunisia. Revue Suisse de Zoologie 118: 3–10.
  • Zrelli S, Boumaiza M, Bejaoui M, Gattolliat J-L, Sartori M (2016) New data and revision of the Ephemeroptera of Tunisia. Inland Water Biology (Supplement 3): 99–106.
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