A new species of Alainites is described from northern of Morocco Alainites albai sp. nov. It can be separated from the other west Palearctic species by the gill number, the spination of the distal margin of tergites, the leg setation, and the paraproct shape and spination. This species is widespread in the study area but never abundant. It prefers small to medium streams with slow flow, and does not seem to be very sensitive to pollution and water logging activities.

COI, endemics, Maghreb, mayflies, Rif, systematics, West Palaearctic

The mayfly genus Alainites Waltz & McCafferty, 1994 was established to encompass species previously included in the muticus species group of the genus Baetis Leach, 1815 (Waltz et al. 1994). Currently, this genus encompasses 22 species (Barber-James et al. 2013; Kaltenbach and Gattolliat 2021; Phlai-ngam et al. 2022; Yanai et al. 2022). Also, the distribution of Alainites is nearly limited at the Palaearctic and Oriental regions; so far, the highest diversity is found in the West Palaearctic region with eight species. Except Alainites muticus (Linnaeus, 1758) which has a wide distribution in Europe (Bauernfeind and Soldán 2012) and has also been recently reported from western Asia (Armenia, Hrivniak et al. 2018) and Iran (Bojková et al. 2018), this genus has a high endemism rate in the Mediterranean basin. During the last decades, seven species with mostly restricted distributions have been described. These are A. oukaimeden (Thomas & Sartori, 1992) from the Moroccan High Atlas (Thomas and Gagneur 1994; Abdaoui et al. 2010; Zuedzang Abessolo et al. 2021), A. sadati Thomas, 1994 distributed from West Algeria to North Tunisia (Thomas and Gagneur 1994; Zrelli et al. 2012; Benhadji et al. 2020), A. navasi (Müller-Liebenau, 1974) located in the Iberian Peninsula (Müller-Liebenau 1974; Alba-Tercedor 1981, 1982; Puig 1983), A. kars (Thomas & Kazanci, 1989) found in Turkey (Kazanci and Thomas 1989; Novikova and Kluge 1994; Kazanci 2001; Kluge and Novikova 2014) and Armenia (Hrivniak et al. 2018; Sroka et al. 2021), A. albinatii (Sartori & Thomas 1989) mentioned from Corsica (Sartori and Thomas 1989; Gattolliat et al. 2015; Tenchini et al. 2018), A. bengunn Yanai & Gattolliat, 2022 recorded from the sister island of Sardinia (Yanai et al. 2022), and A. gasithi Yanai & Gattolliat, 2022 found recently in Israel (Yanai et al. 2022).

The distinctive taxonomical characters of this genus at the nymphal stage were mentioned by Zrelli et al. (2012) and Sroka et al. (2021) and include a laterally compressed body, closely positioned antennae, an apical prolongation on the paraproct, and a reduced mandibular right prostheca (Müller-Liebenau 1969, 1974; Waltz et al. 1994; Fujitani et al. 2003). West Palearctic species are characterized by the presence of six pairs of gills, except for A. muticus, which has seven. Some larvae of Alainites discovered in the material collected from northern Morocco were different from the Moroccan endemic A. oukaimeden and from A. muticus. It represents the second West Palearctic species with seven pairs of gills. The specimens of this species were previously considered to belong to A. muticus (Dakki 1987; El Bazi et al. 2017; Khadri et al. 2017; Mabrouki et al. 2017; El Alami et al. 2022b). However, detailed morphological and genetic approaches proved that these specimens of Alainites noticeably differed from both the European and Asiatic lineages (Sroka 2012).

In the present study, we describe a new species of Alainites based on nymphs from the Rifean Mountains of Morocco. In addition, distinctive characters of western Palearctic Alainites species are provided. The holotype and part of the paratypes of the new species are housed in the Muséum cantonal des sciences naturelles, Lausanne, Switzerland (MZL); other paratypes are deposited in Laboratory of Ecology, Systematics, Conservation of Biodiversity Tetouan, Morocco (LESCB).

The larvae of Alainites albai sp. nov. has a wide distribution and a wide altitudinal range in the Rif (Fig. 1). The sampling was performed by LESCB team between 1997 and 2022. They were subsequently preserved in 70% or 95% ethanol for description and DNA extraction. 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), using an Olympus SZM100 stereomicroscope.

Figure 1. 

The sampling site localization of Alainites albai sp. nov., in the Rif domain.

To complement our morphological investigations, we sequenced a 658 bp fragment of the mitochondrial gene cytochrome oxidase subunit 1 (COI hereafter) for seven specimens of Alainites albai sp. nov., following the non-destructive DNA extraction procedure described in Vuataz et al. 2011. The Polymerase Chain Reaction (PCR), purification and sequencing steps were conducted according to the methodology described in El Alami et al 2022a. Forward and reverse sequencing reads were assembled and edited in CodonCode Aligner v. 10.0.2 (Codon-Code Corporation, Dedham, MA), and aligned using MAFFT (Katoh et al. 2019) with default settings as implemented in Jalview v. 2.11.2.6 (Waterhouse et al. 2009). The number of parsimony-informative sites of the alignment was calculated in Mega v. 10.2.4 (Kumar et al. 2018; Stecher et al. 2020).

To augment our molecular dataset, we initially downloaded all COI sequences associated with Alainites available on the GenBank database (identified among species of the genus Takobia Novikova & Kluge, 1987) as of 2 June 2023, resulting in a total of 64 records. Additionally, we obtained the sequences associated with Alainites/Takobia accessible on the BOLDSYSTEMS data portal as of the same date and selectively retained only those that were not shared with GenBank, yielding an additional set of 5 sequences. We then manually excluded GenBank/BOLD sequences obtained from specimens collected outside the western Palearctic region. This selection process was conducted after confirming that the COI sequences of Alainites albai sp. nov. were clearly distinct from the removed sequences (data not shown). A total of 58 sequences remained for further analyses, comprising seven newly generated sequences (Table 1), 46 sequences from GenBank (four from Kjærstad et al. 2012; six from Sroka 2012; two from Cardoni et al. 2015; ten from Gattolliat et al. 2015; one from Corse et al. 2017; seven from Morinière et al. 2017; five from Tenchini et al. 2018; one from Behrens-Chapuis et al. 2021; two from Sroka et al. 2021; two from Roslin et al. 2022; five from Yanai et al 2022; one unpublished iBOL data release) and five from BOLD (DTNHM1449-21, TDAAT323-19, VMCOL019-20–VMCOL021-20). Two additional newly sequenced specimens, namely one Nigrobaetis numidicus (Soldán & Thomas, 1983) from Morocco and one Nigrobaetis rhithralis (Soldán & Thomas, 1983) from Algeria, were included in the study as outgroups (Table 1).

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 4.3.0 (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 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 (HKY+Γ+I) was selected based on the second-order Akaike information criterion (AICc; Hurvich and Tsai 1989) implemented in JmodelTest v. 2.1.10 (Darriba et al. 2012) with three 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 v. 1.10.4 (Suchard et al. 2018) on the CIPRES Science Gateway v. 3.3 (Miller et al. 2010). The input BEAST file was generated in BEAUTi v. 1.10.4 (Suchard et al. 2018), incorporating the selected evolutionary model and partition scheme described above and enforcing a monophyletic constraint on the ingroup (the genus Alainites). 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 60 million generations, logging parameters every 1000 generations. Convergence of the MCMC runs was visually verified in Tracer v. 1.7.2 (Rambaut et al. 2018). The log and tree files from the independent runs were combined using LogCombiner v. 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 v. 1.10.4 (Suchard et al. 2018) with default settings. Visualization and editing of the tree were conducted in iTOL v. 6.7.5 (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) to estimate the most probable number of MOTUs based on our COI alignment. We calculated genetic distances using the Kimura 2-parameter substitution model (Kimura 1980) and selected the species delimitation hypothesis associated to the highest barcode gap width (W) among the two partitions sharing the same best asap-score. The GMYC model, which requires a time-calibrated ultrametric tree as input, implements a maximum likelihood 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 analysis 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 v. 4.6 (Chesters 2013). MCMC chains were run here for a total of 20 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. We conducted mPTP using the web service available at https://mptp.h-its.org, using the BEAST COI gene trees (full dataset) as input (see above).

The discovery of A. albai sp. nov. has increased the biodiversity of Morocco with a new endemic species of mayfly, highlighting the remarkable biodiversity and species richness of Ephemeroptera in the region. The recent identification of two new species, Prosopistoma maroccanum (El Alami et al., 2022a) and Centroptilum alamiae (Gattolliat et al., 2023), has significantly increased the proportion of Ephemeroptera endemism in Morocco to more than 33% (El Alami et al. 2022b). With the potential for further increases in the future, it is crucial to prioritize conservation and protection measures, particularly in sites hosting high species richness and endemic species.

We would like to thank Prof. Boudjéma Samraoui for providing us with the Nigrobaetis rhithralis specimen he collected in Algeria.