Taxonomy, Distribution and Life Cycle of the Maghrebian Endemic Rhithrogena sartorii (Ephemeroptera: Heptageniidae) in Algeria

: Despite being recorded in Algeria since the nineteenth century, the genus Rhithrogena has never been the object of a taxonomical study and no identiﬁed species is known from this country. Investigations of the relict mountain streams of El Kala, north-eastern Algeria, have led to the discovery of a Rhithrogena population. Morphological and molecular analyses identiﬁed the species as the Maghrebian endemic Rhithrogena sartorii , so far known only from neighboring Tunisia. We report on the species’ distribution, status, and life cycle and discuss its potential role as a bioindicator in environmental monitoring.


Introduction
Rhithrogena, a member of the subfamily Rhithrogeniinae (Heptageniidae), is a Holarctic genus with numerous species in the Palearctic region, occupying mainly cold, fast-flowing, and well-oxygenated headwaters [1,2]. Furthermore, isolated populations of Rhithrogena in mountainous rivers and streams display high levels of endemism and are often on the IUCN Red List [3].
Despite its ecological importance, the taxonomic status of many Rhithrogena species remains a challenge, even in Europe, where taxonomic studies of mayflies are relatively well advanced [4,5]. Based on various nymphal and adult characters, species are lumped into "species groups" [6,7]. However, this grouping remains controversial, marred by cryptic diversity and taxonomic oversplitting [4,5].
The first record of Rhithrogena in North Africa occurred at the edge of the Sahara, when, on 19 March 1895, Eaton [8] collected an immature male at Biskra, Algeria. Eaton went on to speculate that the species might have flown south from the deep canyons descending from the Aures Mountains. This specimen and others encountered in various localities across the Maghreb remained unidentified for several decades until the almost synchronous descriptions from Morocco of a series of new species: Rh. ourika (High Atlas: 1500 and 2600 m) [9], Rh. ayadi (Middle Atlas: 2150 m) [10], Rh. giudicelliorum (High Atlas: 2800 m) [11], and Rh. ryszardi (Middle Atlas: 1260 m) [12].
Subsequently, Vitte [13] described an additional species, Rh. mariae, from the Moroccan Rif. In particular, Rh. mariae differed from other North African Rhithrogena species by Diversity 2021, 13, 547 2 of 13 occurring at a much lower altitude (160 m). Finally, two decades later, Zrelli et al. [14] described a new Rhithrogena species, Rh. sartorii from Tunisia, which gives a total of six known Rhithrogena species in North Africa. Most of the described Maghrebian Rhithrogena species are only known as imagos, whereas Rh. mariae is known at the nymphal and adult stages, and Rh. sartorii at the nymphal and subimaginal stages.
As part of a long-term limnological survey of Algeria, we collected mayfly nymphs from various regions of the country [15], and, in this study, we report the discovery of Rh. sartorii in the relict mountain streams of El Kala, the first record for Algeria, and provide information on its distribution. Because knowledge of immature stages, voltinism, and larval growth patterns provide insights into basic life-history traits and is essential to developing and implementing appropriate conservation strategies [16], we also identified the last three nymphal stages and inferred the species' life cycle.

Study Area
The Algero-Tunisian border is flanked on its northern part by a mountain range known as Kroumiria, where the Kebir-East River emanates. The watershed of the Kebir-East River is second in size only to the Seybouse River in north-eastern Algeria. Further north, the Oued el Eurg basin drains the hills sandwiched between Kroumiria and the Mediterranean Sea ( Figure 1). The climate is typically Mediterranean, with an alternating hot, dry period (May-October) and a rainy season (November-April). Subsequently, Vitte [13] described an additional species, Rh. mariae, from the Moroccan Rif. In particular, Rh. mariae differed from other North African Rhithrogena species by occurring at a much lower altitude (160 m). Finally, two decades later, Zrelli et al. [14] described a new Rhithrogena species, Rh. sartorii from Tunisia, which gives a total of six known Rhithrogena species in North Africa. Most of the described Maghrebian Rhithrogena species are only known as imagos, whereas Rh. mariae is known at the nymphal and adult stages, and Rh. sartorii at the nymphal and subimaginal stages.
As part of a long-term limnological survey of Algeria, we collected mayfly nymphs from various regions of the country [15], and, in this study, we report the discovery of Rh. sartorii in the relict mountain streams of El Kala, the first record for Algeria, and provide information on its distribution. Because knowledge of immature stages, voltinism, and larval growth patterns provide insights into basic life-history traits and is essential to developing and implementing appropriate conservation strategies [16], we also identified the last three nymphal stages and inferred the species' life cycle.

Study Area
The Algero-Tunisian border is flanked on its northern part by a mountain range known as Kroumiria, where the Kebir-East River emanates. The watershed of the Kebir-East River is second in size only to the Seybouse River in north-eastern Algeria. Further north, the Oued el Eurg basin drains the hills sandwiched between Kroumiria and the Mediterranean Sea ( Figure 1). The climate is typically Mediterranean, with an alternating hot, dry period (May-October) and a rainy season (November-April).

Sampling
A set of 24 localities (S1-S24), distributed across both O. Bougous, the main tributary of O. El Kebir, and the O. El Eurg watershed, were sampled monthly from November 2018 to June 2021 [15,17]. Mayfly nymphs were collected using a dipnet (500μm mesh size, 35

Molecular Analyses
To complement morphological examinations, we compared mitochondrial DNA sequences of specimens from this study to Tunisian topotype specimens. Specifically, we generated a 658-bp fragment of the cytochrome c oxidase subunit I (COI) gene from five newly-sequenced specimens (two from Algeria and three from the type locality in Tunisia) using LCO1490 and HCO2198 primers [21]. For all specimens, we followed the nondestructive DNA extraction procedure described in [4]. The DNA was extracted using the BioSprint 96 extraction robot (Qiagen Inc., Hilden, Germany). Polymerase Chain Reaction (PCR) was conducted in a volume of 30 µL, consisting of 9 µL (unknown concentration) of template DNA, 1. . We further included one published COI sequence from [5], also corresponding to a topotype specimen ( Table 1). The sequences were aligned using MAFFT [22] as implemented in Jalview 2.11.1.4 [23]. MEGAX [24,25] was used to visualize the alignment, calculate the number of variable sites, define two groups (one group with the two sequences from Algeria, one group with the four topotype sequences from Tunisia), and calculate K2P [26] mean distances within and between groups. Table 1. Codes and origin of specimens examined in the COI analysis. For each specimen, the GBIF code, the sampling information (country, locality, coordinates, and date of sampling), the GenBank accession number of the COI sequence, and the corresponding publication source are provided. All specimens from Tunisia are from the type locality (topotypes).

Morphometry
Rhithrogena nymphs from two localities, Guitna sup (S7) ( Figure 2) and Guitna inf (S8), were selected for measurements. With one exception (see results), measurements were lumped together after inspection of density plots, and Mann-Whitney U tests did not reveal any differences between the two localities. Body length (BL), head width (HW), and length of the mesonotum + wing pad (mn + wsl) were measured using a Precision Steel Rule to the nearest 0.1 mm. The criteria for instar assignment were BL, HW, mn + wsl, and the ratio (mn + wsl)/HW, hereafter referred to as the "Ratio" [27]. Only the last three instars (F-0, F-1, and F-2) were identified; all other stadia were designated as "smaller nymphs". Instars were determined through graphical plots and statistical analyses. The sex of each nymph in the last two instars was determined according to the presence (male) or absence of genital forceps (gonostyli) on the ventral surface of the ninth abdominal segment. Presence in F-0 nymphs of dark wing pads was assumed as evidence of imminent emergence.

Statistical Analysis
A fast, density-based clustering analysis of BL, HW, mn + wsl, and Ratio was performed using DBSCAN (density-based spatial clustering of applications with noise) [28] to identify the last three instars, F-0, F-1, and F-2. The algorithm attempts to identify the structure in the spatial data set by aggregating objects into similar subgroups [29]. All statistical tests were conducted using R software [30].

Statistical Analysis
A fast, density-based clustering analysis of BL, HW, mn + wsl, and Ratio was performed using DBSCAN (density-based spatial clustering of applications with noise) [28] to identify the last three instars, F-0, F-1, and F-2. The algorithm attempts to identify the structure in the spatial data set by aggregating objects into similar subgroups [29]. All statistical tests were conducted using R software [30].

Distribution and Phenology
During the study period, Rhithrogena sartorii nymphs were recorded between January and June at six localities: Pont Bougous (S1), Zitoun Meftah (S4), Guitna sup (S7), Guitna inf (S8), Nouazi (S10), and Kherrata (S19). Nymphs were found in streams that had a substrate made up of cobbles, stones, and boulders and in microhabitats with a relatively cold, fast-flowing current. The nymphal growth and development occurred in winter and spring with marked differences between years: Both in 2019 and 2021, nymphs were first recorded in March, whereas, in 2020, nymphs were first collected in January. In all years, no nymphs were recorded beyond June (Figure 3).

Distribution and Phenology
During the study period, Rhithrogena sartorii nymphs were recorded between January and June at six localities: Pont Bougous (S1), Zitoun Meftah (S4), Guitna sup (S7), Guitna inf (S8), Nouazi (S10), and Kherrata (S19). Nymphs were found in streams that had a substrate made up of cobbles, stones, and boulders and in microhabitats with a relatively cold, fast-flowing current. The nymphal growth and development occurred in winter and spring with marked differences between years: Both in 2019 and 2021, nymphs were first recorded in March, whereas, in 2020, nymphs were first collected in January. In all years, no nymphs were recorded beyond June (Figure 3).

Taxonomy
Rhithrogena sartorii nymphs are characterized by the following combination of characters: (1) All gills are crenulated ( Figure 4A-C); (2) compared to Rh. insularis ( Figure 5A), the plica of the dorsal face of the first gill is well expressed, clearly triangular, the leading edge somewhat concave ( Figure 5B); (3) the lateral sclerites of the first sternite are slightly turned backward, sometimes perpendicular to the body axis ( Figure 4C); (4) the upper face of femora of all legs has a well-expressed rounded blackish hypodermal macula (Figure 4B); (5) the crown of the galea-lacinia has 9-11 comb-shaped bristles, each with 6-7 teeth ( Figure 5C).

Molecular Analyses
There were no missing data, gaps, or ambiguous sites in the COI alignment, and a total of four variable sites were recorded. The K2P mean distances within groups were 0.25% and 0.15% for Tunisian (topotype) and Algerian sequences, respectively. The K2P mean distance between groups was 0.23% (maximum distance: 0.61%). Two Tunisians and one Algerian specimen shared the same COI haplotype.

Taxonomy
Rhithrogena sartorii nymphs are characterized by the following combination of characters: (1) All gills are crenulated ( Figure 4A-C); (2) compared to Rh. insularis ( Figure 5A), the plica of the dorsal face of the first gill is well expressed, clearly triangular, the leading edge somewhat concave ( Figure 5B); (3) the lateral sclerites of the first sternite are slightly turned backward, sometimes perpendicular to the body axis ( Figure 4C); (4) the upper face of femora of all legs has a well-expressed rounded blackish hypodermal macula ( Figure 4B); (5) the crown of the galea-lacinia has 9-11 comb-shaped bristles, each with 6-7 teeth ( Figure 5C).

Molecular Analyses
There were no missing data, gaps, or ambiguous sites in the COI alignment, and a total of four variable sites were recorded. The K2P mean distances within groups were 0.25% and 0.15% for Tunisian (topotype) and Algerian sequences, respectively. The K2P mean distance between groups was 0.23% (maximum distance: 0.61%). Two Tunisians and one Algerian specimen shared the same COI haplotype.

Morphometry
A total of 524 nymphs were measured. Overall, nymphal body length (BL) ranged from 2.0 to 10.8 mm, while the ranges of head width (HW) and mesonotum length + wing pad length (mn + wsl) were 0.8-3.4 and 0.2-5.2 mm, respectively. Females' BL were Diversity 2021, 13, 547 8 of 13 marginally longer than males' (one-way ANOVA: F 1,164 = 3.6, p = 0.06). All other measured morphometric characters did not differ between the sexes. Assignment of the last three final instars suggested the presence of three clusters corresponding to F-0: Ratio ≥ 1.1, F-1: 1.1 > Ratio ≥ 0.75, F-2: 0.75 > Ratio ≥ 0.5. The rest may be grouped into the category "smaller instars" (Figure 6a). The allometric growth of wing pads (mn + wsl) at the F-0 instar contrasting sharply with the isometric growth of BL and HW (Figure 6a,b) is noteworthy.  (Figure 6a). The allometric growth of wing pads (mn+wsl) at the F-0 instar contrasting sharply with the isometric growth of BL and HW (Figure 6a,b) is noteworthy.  The density-based clustering algorithm, DBSCAN, using the following parameters: eps (maximum radius between two neighbors belonging to the same cluster) = 0.35, and MinPts (minimum number of neighbors required to form a cluster) = 15, and the variables: BL, HW, mn + wsl, and Ratio, confirmed the preliminary visual inspection by assigning 318 nymphs into three clusters corresponding to F-0, F-1, and F-2 (Figure 6b). The rest (206) corresponded to "smaller nymphs" and noise.

Life Cycle
Both in 2019 and 2021, the nymphs first appeared in March, and development proceeded quickly with F-0 nymphs with pigmented wing pads occurring from April to June (Figure 7a-d). In 2020, nymphal development was more protracted, with nymphs collected from January to June, but in all three years, the emergence spanned April to June, coinciding with the drying up of streams in early summer. The size difference between the two sampling sites in May 2020 (BL: log e (Wmann-Whitney) = 5.44, N = 76, p = 2.09 × 10 −6 ; HW:log e (Wmann-Whitney) = 5.20, N = 76, p = 1.19 × 10 −7 ) and the persistence of "small instars" nymphs in June 2020 at Guitna inf (S8) while Guitna sup (S7) dried up is noteworthy. The density-based clustering algorithm, DBSCAN, using the following parameters: eps (maximum radius between two neighbors belonging to the same cluster) = 0.35, and MinPts (minimum number of neighbors required to form a cluster) = 15, and the variables: BL, HW, mn + wsl, and Ratio, confirmed the preliminary visual inspection by assigning 318 nymphs into three clusters corresponding to F-0, F-1, and F-2 (Figure 6b). The rest (206) corresponded to "smaller nymphs" and noise.

Life Cycle
Both in 2019 and 2021, the nymphs first appeared in March, and development proceeded quickly with F-0 nymphs with pigmented wing pads occurring from April to June (Figure 7a-d). In 2020, nymphal development was more protracted, with nymphs collected from January to June, but in all three years, the emergence spanned April to June, coinciding with the drying up of streams in early summer. The size difference between the two sampling sites in May 2020 (BL: loge (Wmann-Whitney) = 5.44, N = 76, p = 2.09 × 10 -6 ; HW:loge (Wmann-Whitney) = 5.20, N = 76, p = 1.19 × 10 -7 ) and the persistence of "small instars" nymphs in June 2020 at Guitna inf (S8) while Guitna sup (S7) dried up is noteworthy.

Distribution
Both in Tunisia [14] and in Algeria, the distribution of Rhithrogena sartorii was restricted to the Kroumiria mountain range. Overall, the species seems confined to the metarhithral and parapotamal river reaches. The lower end of the altitudinal range of the species' habitats (200-650 m), almost matching Rh. mariae, which is able to colonize lower stretches (160 m) in Morocco, is noteworthy [13].

Distribution
Both in Tunisia [14] and in Algeria, the distribution of Rhithrogena sartorii was restricted to the Kroumiria mountain range. Overall, the species seems confined to the metarhithral and parapotamal river reaches. The lower end of the altitudinal range of the species' habitats (200-650 m), almost matching Rh. mariae, which is able to colonize lower stretches (160 m) in Morocco, is noteworthy [13].

Taxonomy
Recently described from Tunisia, Rh. sartorii was thought to belong to the insularisspecies group [14]. However, after preliminary investigations reported in [5], a careful re-examination of these nymphs conducted here confirmed that this species is more related to species of the so-called sowai-group [6]. Specifically, the shape of the plica, which is clearly concave (always less prominent and convex in species of the hybrida-group- Figure 5A), and the lateral sclerites of the first sternite, which can be slightly turned backward (always perpendicular to body axis in hybrida-group). Species of the sowai-group are poorly known; seven species have been described with only one in the nymphal stage [31], although an unnamed species has been described at the nymphal stage from Portugal [32]. All of these species are restricted to the Mediterranean basin. Finally, the lack of true affinities with species of the hybrida-group is demonstrated by the quite isolated position Rh. sartorii occupies in the phylogeny of European species of the genus based on mitochondrial and nuclear markers [5].
With a maximum of 0.61%, the COI K2P distances between sequences from Algeria and topotypes from Tunisia are very low, typically corresponding to intraspecific divergence in previous mayfly barcoding studies (e.g., [33][34][35][36]). Moreover, given that one of our specimens has the same COI haplotype as the two topotype specimens and that the combination of morphological characters fully fits the Rh. sartorii description, we can be confident in our identification. This is not surprising, as both populations are located in the same mountain range (Kroumiria), only c. 30 km distant from each other.

Eaton's Rhithrogena
In contrast to the relatively straightforward identification of Rh. sartorii, Eaton's Rhithrogena still remains shrouded in mystery. Unless the specimen is retrieved, we will probably never know which species of Rhithrogena Eaton [8] collected in Algeria, but we can safely rule out many of the known North African species on the basis of their limited distribution and ecology. Indeed, four species (Rh. ourika, Rh. ayadi, Rh. giudicelliorum, and Rh. ryszardi) occupy an altitudinal range between 1260 and 2800 m. Likewise, based on the location, Biskra, where Eaton has recorded the specimen, we can safely assume that the species was able to stand high temperatures in one phase of its life cycle. North African rheophilic Rhithrogena species, such as Rh. mariae, are present at low altitudes, but due to their localized distribution in the Rif, this latter is unlikely to represent a good candidate (but see [13]) for Eaton's Rhithrogena, which once inhabited the edge of the desert.
In addition, based on the flight period (late winter) of Eaton's specimen, we can also exclude that it was Rh. sartorii that emerges in late spring. The delayed nymphal growth and development of Rh. sartorii are suggestive of a univoltine winter/spring life cycle, whereas nymphal development in Eaton's species probably occurred in late autumn and winter, similar to the life cycle of Rh. germanica [37,38]. Based on all these elements and the extensive surveys of mayflies of the Aures Mountains (unpublished), we hypothesize that Eaton's Rhithrogena has probably gone extinct.

Life Cycle
Nymphal development of Rh. sartorii occurred during winter and spring, but there was considerable annual variation (January-March) in the first records of nymphs, probably linked to the close relationship between egg development and water temperature [39,40]. For instance, eggs of the cold stenothermal Rh. loyolaea and Rh. nivata rarely hatch at temperatures above 10 • C, thus restricting the species to cold streams [41,42]. If this was the case for Rh. sartorii, this threshold would limit hatching to winter months. In addition, once hatching is underway, the differential growth rates in small nymphs may be responsible for the extended period of their presence [41].
Rhithrogena sartorii managed a single generation per year, with the nymphal stage spread over winter and spring. According to Clifford's classification [43], the species exhibited a seasonal univoltine cycle (Us-Uw) where the egg stage and part of the nymphal stage overwinters. Nevertheless, the univoltine life cycle of Rh. sartorii is quite unusual, with a winter and vernal growth and a long embryonic diapause during the warm months. It is somewhat distinct from the life cycle of Rh. germanica, a univoltine winter species, which emerges in Central Europe between February and April, undergoes a summer embryonic diapause with eggs hatching once the temperature drops in October [37].
Furthermore, the presence of small nymphs of Rh. sartorii in June may either suggest a protracted egg hatching period or a proclivity for the species to undertake a second generation if environmental conditions are adequate. In all three years, the habitats dried up, and thus, this question deserves further investigation. Although Rhithrogena species are known to be mainly univoltine [43], plasticity in voltinism has been demonstrated, ranging from semivoltinism for Rh. loyalae [44] to partial bivoltinism [45], and even bivoltinism [46] for Rh. semicolorata and Rh. diaphana, respectively.

Conservation
Although species of the genus Rhithrogena may be perceived as less threatened, as their rhithral habitats may be contending with lesser anthropogenic pressures than downstream habitats, they are highly sensitive to various environmental factors [47,48]. In addition, due to historical factors (transboundary region and previous war zone), the El Kala district has been relatively maintained as a hotspot of freshwater biodiversity. However, despite its status as a Man and the Biosphere Reserve, the area is now under severe anthropogenic pressures fueled by a burgeoning population [49,50]. With its restricted distributional range encompassing the Tunisian and Algerian Kroumiria, Rh. sartorii is clearly an endemic species of conservational concern. Moreover, in most sites and during the three-year study, the species was never abundant. Thus, the limited range, low abundance, and narrow ecological niche (rheobiont associated with riffles) make this threatened species and its habitat vulnerable to various natural and anthropogenic stressors (climate change, pollution, land conversion, etc.). Rhithrogena sartorii may act as a useful bioindicator of such scarce habitats and an umbrella species for the conservation of the unique freshwater biodiversity hosted by the Kroumiria mountain range that spans north-eastern Algeria and north-western Tunisia [15,51,52]. Unless urgent steps are taken to lessen human encroachment on its habitats, this imperiled Maghrebian microendemic may rapidly go extinct.

Conclusions
A survey of the highlands of the El Kala region, north-eastern Algeria, has led to the discovery of a species of Rhithrogena that occupied the hyporhithral and parapotamal river reaches. Molecular and morphological analyses identified the species as Rh. sartorii, a Maghrebian microendemic confined to the Kroumiria mountain range and environs on the Algero-Tunisian border. The species exhibited a univoltine life cycle (Us-Uw) with emergence spread between April and June. Rhithrogena sartorii is threatened due to the species' limited range and the mounting anthropogenic pressures (water abstraction, fire, pollution, etc.) in the region.