Reports of Baetidae (Ephemeroptera) species from Tafna Basin, Algeria and biogeographic affinities revealed by DNA barcoding.

BACKGROUND: The Mediterranean basin is known to be the cradle of many endemic species. Within mayflies (Insecta, Ephemeroptera), North African species belonging to the family Baetidae remain poorly known and, traditionally, affinities to European fauna were proposed. Recent studies, based on molecular reconstructions, showed closer relationships to Mediterranean islands fauna. NEW INFORMATION: Baetidae were sampled from North-West Algerian wadis (Tafna basin) and involved in COI barcoding reconstructions. Seven species were identified. The subgenus Rhodobaetis is represented by Baetis atlanticus known previously from Macaronesian islands, Europe and Morocco and the Maghrebian endemic Baetis sinespinosus. Specimens, previously identified as Cloeon cf. dipterum, correspond to Cloeon peregrinator and, until now, only reported from Macaronesia. Besides the confirmation of endemicity of some species, such as Procloen stagnicola and B. sinespinosus, our molecular study showed quite original results for relationships between European, insular and Algerian species. Baetis maurus stood out as a North African endemic sister clade to an Iberian clade. Furthermore, we found clear interspecific distances between Algerian and European clades for A. cf. sinaica and B. cf. pavidus, suggesting the presence of cryptic species in Algeria. However, additional studies are needed, as, for the moment, no clear morphological characters were found to separate the different clades and support them as valid species. Nadhira Benhadji, Michel Sartori, Karima Abdellaoui Hassaine, Jean-Luc Gattolliat.

Introduction

The family has a cosmopolitan distribution and represents a quarter of the diversity worldwide both at generic and specific levels (Barber-James et al. 2008, Gattolliat and Nieto 2009, Sartori and Brittain 2015). The genera Leach, 1815 and Leach, 1815 have the largest distribution amongst the family and encompass, respectively, 152 and 74 species (Sartori and Brittain 2015, Jacobus et al. 2019). In Europe, was originally divided into eleven species groups (Müller-Liebenau 1969), of which some are now considered as valid genera ( Waltz and McCafferty, 1994; Novikova and Kluge, 1987; Novikova and Kluge, 1987) or subgenera ( Thomas and Dia, 1999; Jacob, 2003) (Jacob 2003, Novikova and Kluge 1987, Thomas and Dia 2000, Waltz et al. 1994).

The subgenus (corresponding to the group) presently encompasses 43 species, some of them being amongst the most common and abundant mayflies. While some species are widely distributed (e.g. (Pictet, 1843), Soldán and Godunko, 2006), others present a presumably restricted distribution, such as endemic to a single Canary Island ( Gattolliat and Sartori, 2018; Gattolliat and Sartori, 2018; Gattolliat and Sartori, 2018) or known from a restricted area ( Soldán, Godunko and Thomas, 2005 or Soldán and Thomas, 1983 in Algeria) (Gattolliat et al. 2018b, Soldán et al. 2005). Previous molecular studies, based on COI, revealed a high number of independent lineages, which may correspond to cryptic undescribed species (Bisconti et al. 2018, Bisconti et al. 2016, Gattolliat et al. 2015, Gattolliat et al. 2018b, Sroka 2012, Williams et al. 2006).

The species delimitation within the genus Leach, 1815 (sensu Kluge 2016b) is also highly problematic. Most of the reports of the widely-distributed (Linnaeus, 1761) or Stephens, 1835 must be considered with caution as several independent lineages are hidden behind these concepts (Rutschmann et al. 2014, Rutschmann et al. 2017).

Thomas (1998) proposed the first preliminary checklist of mayflies from North Africa, including 25 species. Eleven species are endemic to North Africa: Eaton, 1899; ; Soldán and Thomas, 1983; (Soldán and Thomas, 1983); (Soldán and Thomas, 1983); Soldán and Thomas, 1983; (Soldán and Thomas, 1985); Thomas, 1986; (Thomas and Sartori, 1992); Thomas, 1994 and . Eleven species are originally described from Algeria, ten of them between 1983 and 1986 (Soldán and Thomas 1983a, Soldán and Thomas 1983b, Soldán and Thomas 1985) and no new taxa have been described since 2005 (Soldán et al. 2005). Ten Central European species were reported from Maghreb, but all these identifications should be taken with caution. Remaining species present West Mediterranean distribution including North Africa and Iberian Peninsula ( Thomas, Boumaiza and Soldán, 1983; Kimmins, 1938) or Italian peninsula and South of France ( Grandi, 1949).

Recently, molecular reconstructions involving were conducted for different projects, in particular for the origin of Macaronesian and Corsican mayflies fauna (Gattolliat et al. 2015, Gattolliat et al. 2018b, Rutschmann et al. 2014, Rutschmann et al. 2017). Despite being not directly focused on North Africa, they included specimens from Tunisia, Algeria and Morocco. These preliminary results for North Africa underlined important links between North African and Macaronesian faunas (in the case of and ). The discovery in Tunisia of a species of , assumed as endemic to Sardinia (Zrelli et al. 2011), also confirmed possible connections between Italy and Maghreb, mainly during crucial geological events, such as the Messinian Salinity Crisis (Gattolliat et al. 2015).

North African species of and Wuillot and Gillies, 1993 have most probably an Afrotropical origin as they are mainly diversified in this area. A dozenspecies of are described in Afrotropics, while the genus is only represented in the Palearctic by a single Maghrebian species () and one in the Arabian Peninsula, Gattolliat and Sartori, 2008 (Gattolliat and Sartori 2008, Kluge 2016a). Despite also being present in Central Europe, is mostly diversified in tropical areas, as proven by its high diversity in Afrotropics (Gattolliat 2001, Lugo-Ortiz and McCafferty 1997) and South East Asia (Kaltenbach and Gattolliat 2018, Kaltenbach and Gattolliat 2019, Kaltenbach and Gattolliat 2020). While a part of the Afrotropical species present a very restricted distribution (Gattolliat 2001, Lugo-Ortiz and McCafferty 1997), a recent molecular reconstruction proved that specimens from Comoro Islands, South Africa and Arabian Peninsula form a monophyletic clade corresponding to (Agnew, 1961) (Gattolliat et al. 2018a).

The present study is the first molecular analysis for Algerian mayflies using the cytochrome oxidase subunit I (COI) region for species delimitation. The main aims are to clarify the status of the different species of present in North West Algeria, especially for Central European species assumed to occur in Maghreb. We also want to clarify the species delimitation in some groups with potential cryptic species and significantdifficulties to identify, based on morphological characters only. Finally, we want to understand the affinities between Maghrebian and neighbouring fauna.

Materials and methods

Sampling

We investigated twelve sampling sites, all located in the Tafna basin in North-West Algeria (Fig. 1); a detailed description of this area is presented in Benhadji et al. (2019). The specimens used for the molecular study are listed in Table 1. They were collected by using a Surber net between April and October 2016, then preserved in 99% ethanol and stored at cold and stable temperature (4°C). A total of 52 larvae were identified at the generic or specific level, based on morphological characters (Benhadji et al. 2019), including spp. (26 specimens), (13 specimens), (four specimens), (one specimen), (four specimens) and (three specimens) (Table 1). Specimens and DNA extractions are housed in the collections of the Museum of Zoology, Lausanne, Switzerland.

Figure 1.

Location of the sampling sites (Tafna basin, Algeria).

Table 1.

List of COI sequenced specimens with Genbank accession number. For sites code, see Benhadji et al. (2019). Each specimen is identified by alphanumeric codes. The two first letters indicate the taxonomic group (RB: ; BP: ; BM: ; AC: ; CO: ; PC: ). ALG indicates the country (i.e. Algeria). The following letter (A to H) with number (1 to 8) indicate the position of the well on the PCR plate. An “F” is added at the end of some codes when only the forward amplification was successful.

Taxa	Sites	Codes	Genbank accession
Baetis sinespinosus	SK1	RB_ALG_A06	MT800078
	SK1	RB_ALG_D07	MT800085
	SK1	RB_ALG_E07	MT800086
	SK1	RB_ALG_F05F	MT800093
	SK1	RB_ALG_G05F	MT800095
	SK1	RB_ALG_H05F	MT800096
	CH0	RB_ALG_D01F	MT800091
	CH1	RB_ALG_B03	MT800080
	CH1	RB_ALG_F02	MT800087
	CH1	RB_ALG_H02	MT800089
	CH1	RB_ALG_A03F	MT800090
	CH1	RB_ALG_G02F	MT800094
	CH4	RB_ALG_B04	MT800081
	CH4	RB_ALG_C04	MT800083
	IOM	RB_ALG_F04	MT800088
	TGB	RB_ALG_C10	MT800084
	TGB	RB_ALG_D10F	MT800092
	TFF	RB_ALG_A10	MT800079
	TFF	RB_ALG_B10	MT800082
Baetis atlanticus	SK1	RB_ALG_C07	MT800053
	CH0	RB_ALG_C01F	MT800059
	KH1	RB_ALG_C08	MT800054
	KH1	RB_ALG_D08	MT800055
	KH1	RB_ALG_E08	MT800056
	KH1	RB_ALG_H08	MT800058
	KH1	RB_ALG_F08	MT800057
	KH1	RB_ALG_G08F	MT800060
Baetis cf. pavidus	SK1	BP_ALG_F07	MT800069
	SK1	BP_ALG_G07	MT800071
	CH1	BP_ALG_B02	MT800062
	CH1	BP_ALG_C02	MT800063
	CH1	BP_ALG_D02	MT800065
	CH1	BP_ALG_E02	MT800067
	CH4	BP_ALG_A02	MT800061
	IOM	BP_ALG_C05	MT800064
	TGA	BP_ALG_D09	MT800066
	TGA	BP_ALG_E09	MT800068
	TFF	BP_ALG_F09	MT800070
	TFF	BP_ALG_G09	MT800072
	TFF	BP_ALG_H09	MT800073
Baetis maurus	CH1	BM_ALG_G01	MT800074
	CH1	BM_ALG_H10	MT800075
	SK1	BM_ALG_D05F	MT800076
	SK1	BM_ALG_E05F	MT800077
Acentrella cf. sinaica	IOM	AC-ALG-A05	MT800052
Cloeon peregrinator	CH0	CO_ALG_B01F	MT800100
	CH1	CO_ALG_C03	MT800097
	CH1	CO_ALG_D03	MT800098
	SK1	CO_ALG_D06	MT800099
Procloeon stagnicola	KH1	PC_ALG_A08	MT800101
	KH1	PC_ALG_B08	MT800102
	KH1	PC_ALG_H07	MT800103

COI gene amplifications

We performed DNA extraction using DNeasy Blood & Tissue kit (QIAGEN) and BioSprint 96 extraction robot (Qiagen) by soaking each specimen in buffer and proteinase K at 56°C for an overnight incubation. The mitochondrial DNA cytochrome oxidase c subunit I gene (COI) was amplified using the primers LCO1490 and HCO2198 (Folmer et al. 1994) with an initial denaturation temperature of 98°C for 30 sec followed by a total of 37 cycles with denaturation temperature of 98°C for 10 sec, an annealing temperature of 50°C for 30 sec and an extension at 72°C for 30 sec, final extension at 72°C for 2 min. We checked if the amplification was successful using agarose gel electrophoresis, then we purified PCR products and prepared bi-directional sequencing using the same primers LCO1490 and HCO2198.

COI gene trees

We corrected and edited forward and reverse sequencing reads using Bioedit, then we assembled each of the two complementary sequences using Codon Code Aligner (demo mode) and obtained sequence alignments (Suppl. materials 4, 5, 6, 7, 8, 9) using Jalview 2.10.1 via Mafft alignment as in Vuataz et al. (2011). We aligned sequences of each taxon with analogue genus or species selected from Genbank database (GenBank 2019) or BoldSystem database (BOLD 2019). For , we added sequences corresponding to the known haplogroups of (Gattolliat et al. 2015, Gattolliat et al. 2018b, Lucentini et al. 2011, Williams et al. 2006). For , we selected all available sequences of and several of . For the remaining taxa, in addition to conspecific sequences, we incorporated outgroups sequences (for example, Eaton, 1870, Bengtsson, 1914 and Lestage, 1924 to reconstruction). To delimit the haplogroups, we used ABGD, Automatic Barcode Gap Discovery (Puillandre et al. 2012).

To reconstruct the trees, we used Mega version 10.0.4; we chose the best evolutionary model using the AICc criteria (Akaike 1974), then we set to run a Maximum Likelihood bootstrap analysis with 1000 normal bootstrap replicates.

Checklists

Checklist of the species of the Tafna basin (North-West Algeria)

Hyatt & Arms, 1890

015A1E71-E333-5221-9814-225FF797BA68

Leach, 1815

3FAF161E-2364-543C-B25B-13A942C6785C

Bengtsson, 1912

70EF0C3E-5A11-5940-9607-A74E9CC6683F

Bogoescu, 1931

EAE1FE9C-ECED-5F31-88AE-F82D05D2F53D

Leach, 1815

ACF7B2E0-1AD0-594A-969D-6DCE74853EB1

Soldán and Godunko 2006

178EF1C3-F5F6-5242-ABFA-9C3920A9B996

Kimmins, 1938

C7233785-EEA5-589D-845D-7CCB25F3FA60

Grandi, 1949

AB6B940D-255C-5B41-AC6D-B030AD61599E

Soldán and Thomas, 1983

89613628-8222-5FEB-8ACD-97F834398E20

Leach, 1815

FA06CCC9-0475-5137-9079-C70775C6EF19

Gattolliat and Sartori, 2008

CA10A785-8255-5D83-A0AE-88209FE89D12

Bengtsson, 1915

CB811D74-9C3B-56F9-9DED-8398387C7667

Soldán and Thomas, 1984

40E16844-D041-5061-B5B0-2083F07193EF

Analysis

Species delimitations

Based on the molecular analysis, seven species were recognised or confirmed in the Tafna basin sites.

Subgenus

We obtained 11 haplogroups of (Suppl. material 1, Table 2) with the 27 Algerian sequences differentiating into two haplogroups (Fig. 2): a) RB_Gp1, a strongly supported monophyletic clade (97% BS) also containing Macaronesian and Iberian sequences from ; b) RB_Gp2, a monophyletic clade with a strong bootstrap support (98%) containing only Algerian sequences of . RB_Gp1 and RB_Gp2 have, respectively, 1.65% and 0.48% of intraspecific distance. RB_Gp1 and RB_Gp2 are poorly supported as a monophyletic clade (25% BS); the two haplogroups present an interspecific distance of 15.2%.

Table 2.

Distances within (in bold) and between haplogroups. RB-Gp1: ; RB-Gp2: Haplogroups with Algerian sequences are underlined.

	RB_Gp1	RB_Gp2	RB_Gp3	RB_Gp4	RB_Gp5	RB_Gp6	RB_Gp7	RB_Gp8	RB_Gp9	RB_Gp10	RB_Gp11
RB_Gp1	0.02
RB_Gp2	0.15	0.004
RB_Gp3	0.16	0.14	n/c
RB_Gp4	0.15	0.14	0.12	0.004
RB_Gp5	0.15	0.14	0.14	0.10	0.01
RB_Gp6	0.17	0.17	0.18	0.18	0.20	n/c
RB_Gp7	0.18	0.16	0.17	0.14	0.16	0.12	n/c
RB_Gp_8	0.20	0.19	0.17	0.17	0.20	0.14	0.12	0.01
RB_Gp_9	0.18	0.16	0.15	0.17	0.20	0.19	0.18	0.21	0.003
RB_Gp10	0.19	0.17	0.17	0.16	0.17	0.19	0.18	0.19	0.10	0.01
RB_Gp11	0.26	0.24	0.23	0.23	0.25	0.22	0.23	0.25	0.23	0.22	0.002

Figure 2.

Maximum Likelihood tree including representative of using TN93 (+G+I) model; only bootstrap supports (BS) higher than 70% are indicated on the corresponding branch.

We obtained two haplogroups of (Fig. 3, Table 3): a highly-supported clade gathering Algerian specimens and two sequences from southern France (BP_Gp1) and a second clade (BP_Gp2) which is composed by from Italy. Both haplogroups are highly supported as sister clades. BP_Gp1 possesses a very low intraspecific distance (0.2%) and high interspecific distance with its sister clade BP_Gp2 (11%).

Figure 3.

Maximum Likelihood tree including a representative of using the General Time Reversible model (+G+I); only bootstrap supports (BS) higher than 70% are indicated on the corresponding branch.

Table 3.

Distances within (in bold) and between (BP_Gp1-BM_Gp2), (BP_Gp3) and haplogroups (BP_Gp4-BP_Gp7). Haplogroup with Algerian sequences is underlined.

	BP_Gp1	BP_Gp2	BP_Gp3	BP_Gp4	BP_Gp5	BP_Gp6	BP_Gp7
BP_Gp1	0.002
BP_Gp2	0.11	0.003
BP_Gp3	0.21	0.23	0.004
BP_Gp4	0.22	0.24	0.23	n/c
BP_Gp5	0.23	0.25	0.24	0.08	0.006
BP_Gp6	0.23	0.25	0.25	0.15	0.15	0.008
BP_Gp7	0.24	0.24	0.24	0.23	0.22	0.23	n/c

We delineated three haplogroups (Suppl. material 2, Table 4). Our sequences (BM_Gp1) belong to a strongly-supported monophyletic haplogroup identified as (Fig. 4). This haplogroup is highly supported as the sister clade of specimens identified also as (BM_Gp2 and BM_Gp3), but coming from Spain; the distances between these clades, all identified as , are of interspecific level (15-16%).

Table 4.

Distances within (in bold) and between (BM_Gp1-BM_Gp3) and haplogroups (BM_Gp4-BM_Gp7). Haplogroup with Algerian sequences is underlined.

	BM_Gp1	BM_Gp2	BM_Gp3	BM_Gp4	BM_Gp5	BM_Gp6	BM_Gp7
BM_Gp1	0
BM_Gp2	0.16	0.003
BM_Gp3	0.15	0.16	0.007
BM_Gp4	0.24	0.24	0.23	0.043
BM_Gp5	0.26	0.24	0.21	0.23	0.006
BM_Gp6	0.24	0.23	0.22	0.25	0.19	0.06
BM_Gp7	0.25	0.26	0.22	0.25	0.19	0.19	0.04

Figure 4.

Maximum Likelihood tree including a representative of using TN93 (+G+I) model; only bootstrap supports (BS) higher than 70% are indicated on the corresponding branch.

The reconstruction divided sequences into 6 haplogroups (Fig. 5, Table 5). The single sequence from Algeria was recovered as an independent clade (AC_Gp5) distant at least 19% from other clades. AC_Gp6 haplogroup is the sister clade of AC_Gp5; it contains sequences from France and Italy gathered with a very strong BS (100%).

Figure 5.

Maximum Likelihood tree including a representative of spp using the General Time Reversible model (+I); only bootstrap supports (BS) higher than 70% are indicated on the corresponding branch.

Table 5.

Distances within (in bold) and between haplogroups. AC-Gp1: . AC-Gp2: . AC-Gp3: . AC-Gp4: . AC-Gp5: . AC-Gp6: . Haplogroup with Algerian sequence is underlined.

	AC_Gp1	AC_Gp2	AC_Gp3	AC_Gp4	AC_Gp5	AC_Gp6
AC_Gp1	0
AC_Gp2	0.19	0.011
AC_Gp3	0.23	0.21	0
AC_Gp4	0.22	0.22	0.24	0.002
AC_Gp5	0.25	0.25	0.26	0.22	n/c
AC_Gp6	0.26	0.25	0.24	0.25	0.19	0.003

We obtained 11 haplogroups from the reconstruction (Suppl. material 3, Table 6) including six highly supported haplogroups. The clade containing all haplogroups of s.l. is a monophyletic clade with a high BS (99%).

Table 6.

Distances within (in bold) and between haplogroups. CO_Gp1-CO-Gp6: sl. CO_Gp2: Haplogroup with Algerian sequences is underlined.

	CO_Gp1	CO_Gp2	CO_Gp3	CO_Gp4	CO_Gp5	CO_Gp6	CO_Gp7	CO_Gp8	CO_Gp9	CO_Gp10	CO_Gp11
CO_Gp_1	0.002
CO_Gp_2	0.09	0.002
CO_Gp_3	0.10	0.10	0.002
CO_Gp_4	0.11	0.11	0.07	0.012
CO_Gp_5	0.11	0.11	0.08	0.08	0.021
CO_Gp_6	0.09	0.09	0.1	0.1	0.1	0.002
CO_Gp_7	0.15	0.15	0.17	0.17	0.16	0.16	0.01
CO_Gp_8	0.18	0.19	0.19	0.19	0.20	0.20	0.15	0.00
CO_Gp_9	0.19	0.21	0.2	0.19	0.19	0.20	0.20	0.22	0.013
CO_Gp_10	0.17	0.19	0.17	0.18	0.18	0.19	0.19	0.20	0.11	0.005
CO_Gp_11	0.17	0.19	0.16	0.17	0.17	0.17	0.18	0.18	0.18	0.19	n/c

The CO_Gp2 haplogroup, which includes four sequences from Algeria and sequences of from Madeira (type locality) and Gran Canaria (Fig. 6), is highly supported as a monophyletic haplogroup (100%). This haplogroup has a low intraspecific distance (0.2%) and high interspecific distance with all the other haplogroups of s.l., for instance with CO_Gp1 with which it has the least distance (8.9%). Consequently, the sequences from Algeria and Gran Canaria belong to .

Figure 6.

Maximum Likelihood tree including a representative of spp. using the General Time Reversible model (+G+I); only bootstrap supports (BS) higher than 70% are indicated on the corresponding branch.

In this reconstruction, six haplogroups (Fig. 7, Table 7) were obtained. from Algeria (PC_Gp1) forms a well-supported monophyletic haplogroup. PC_Gp1 has a high interspecific distance from PC_Gp2 (16.2%), which corresponds to the closest European species (Bengsston, 1912) and from the remaining sequences (from 16% to 23.7%).

Figure 7.

Maximum Likelihood tree including a representative of spp using the General Time Reversible model (+G+I); only bootstrap supports (BS) higher than 70% are indicated on the corresponding branch.

Table 7.

Distances within (in bold) and between haplogroups (PC_Gp1-PC_Gp5) and group (PC_Gp6). PC-Gp1: . PC-Gp2 – PC-Gp3: . PC-Gp4: . Haplogroup with Algerian sequences is underlined.

	PC_Gp1	PC_Gp2	PC_Gp3	PC_Gp4	PC_Gp5	PC_Gp6
PC_Gp1	0.006
PC_Gp2	0.16	0.027
PC_Gp3	0.18	0.19	n/c
PC_Gp4	0.19	0.19	0.20	0.016
PC_Gp5	0.19	0.20	0.21	0.20	0.037
PC_Gp6	0.22	0.23	0.24	0.24	0.23	0

Discussion

The different trees we obtained allowed us to better understand the composition of Algerian . Based on our analysis, we can link the Algerian lineages with their sister-groups, calculate the maximum and minimum distances and evaluate which lineages may represent putative species.

Maghrebian endemic species

is a subgenus of and corresponds to the concept of species-group (Jacob 2003, Müller-Liebenau 1969). It is widely distributed in all West Palearctic streams (Gattolliat et al. 2015). Three species of are reported from Algeria, two of them are endemic: and (Bauernfeind and Soldán 2012, Thomas 1998). A representative of s.l. is also reported (Thomas 1998). As noticed by Sroka (2012), it is generally difficult to find morphological characters to support the molecular species delimitation within . In the present case, the absence of a single rudimentary scale on the tip of the maxillary palp and the presence of four rows of setae at the apex of the paraglossae indicate that the clade RB_Gp2 corresponds to is a well-supported monophyletic clade and presents high interspecific distances with all the other European and Mediterranean species. According to our data and reports from literature, this species seems to be endemic to Algeria. However, its presence in nearby countries, such as Tunisia and Morocco, will not be surprising as several populations were provisionally identified as s.l. (Mabrouki et al. 2017, Thomas 1998, Zrelli et al. 2016) and the present study is based on material collected very close to the border of Morocco.

Our results showed a high interspecific distance between the Algerian clade and its European sister species ; thus, it confirms the validity of . This latter differs from P. especially by the flat and rounded bristles on the labrum margin; the pointed apex of the gills with an extremely reduced second lamella and also by the lateral margins of the abdominal segments which possess spines from segment V to IX. The species was originally described from Algeria (Soldán and Thomas 1983a) and was subsequently discovered in Tunisia (Boumaiza and Thomas 1995). The report of from Morocco (El Alami et al. 2000) may be a misidentification and may also concern . Identification of material collected by the last author (Jean-Luc Gattolliat) and stored in the MZL collection confirmed the presence of this species in Morocco (unpublished data). This species should be therefore considered as endemic to Maghreb.

is a representative of the species-group. The species is considered as an Atlanto-Mediterranean element (Bauernfeind and Soldán 2012). It was originally described from Morocco (Kimmins 1938), then reported from the Iberian Peninsula (Alba-Tercedor 1982, Müller-Liebenau 1974). Algerian haplotypes present high distances with presumably conspecific specimens from Spain (Murria et al. 2017), as well as with the other Euro-Mediterranean species belonging to the species-group. Our results tend to prove that is, in fact, a Magrebian endemic species (originally described from Morocco) and that at least one sister undescribed species occurs in the Iberian Peninsula. These preliminary results must be confirmed by sequencing additional populations from Spain and Maghreb and by morphological evidence. With the presence of a second rudimentary row of denticles on claws, both Maghrebian and Iberian populations possess a unique morphological character amongst the genus (Thomas et al. 1983).

is a South and Central European species (Bauernfeind and Soldán 2012). It was reported from Tunisia (Boumaiza and Thomas 1995) and then from Algeria (Mebarki et al. 2017). In his checklist of North African mayflies, Thomas (1998) considered the specific identification as possibly incorrect, referring maybe to Alba-Tercedor & El Alami 1999 described from Morocco (Alba-Tercedor and El Alami 1999). The important distances between our unique haplotype and those from France and Italy tend to confirm that Algerian specimens do not belong to . Unfortunately, no sequence is available for We refrain to attribute the specimens from Algeria to , as important distinctive characters do not match between our specimens and the original description. Especially, the Algerian specimens possess long setae along the dorsal margin of the femora (similar to ), while present much shorter ones (Alba-Tercedor and El Alami 1999). There is, therefore, some probability that our specimens represent an undescribed species from North Africa. Additional sequences and close morphological studies are needed to confirm this hypothesis.

Western Mediterranean species

is a Western Mediterranean species and was originally described from Italy and then reported from the Maghreb (Bauernfeind and Soldán 2012, Jacob 2003). In Algeria and Tunisia, it is one of the most common and abundant species at low to middle elevation and is rather tolerant to pollution and low oxygenation (Benhadji et al. 2019, Boumaiza and Thomas 1995). The Algerian haplotypes present low genetic distance with specimens from southern France, confirming the link for this species between North African and South European populations. The sequences from Sicily, Italy (BP_Gp2) were not assigned to with certainty by Tenchini et al. (2018) and may represent an undescribed species close to or, alternatively, may be correctly associated with and the specimens from Algeria and from South of France represent a new species.

Widely-distributed species

was first considered as an endemic species from Madeira (Gattolliat et al. 2008), then was also found on the Canary Islands (Rutschmann et al. 2017). It belongs to the species-group from which it can be separated by minute morphological characters (Gattolliat et al. 2008). Algerian haplotypes present a low genetic distance with Madeiran specimens which prove their conspecificity. This discovery is rather surprising as was first thought to be an insular endemic. Molecular studies showed mayflies are able to colonise islands, even for such taxa with presumably low dispersal capacity (Monaghan et al. 2005, Rutschmann et al. 2014). The origin of the species cannot yet be proven and two scenarios can be proposed: either a colonisation of Macaronesia from Continental Europe, then a speciation process on the islands and a subsequent colonisation of North Africa or, alternatively, a colonisation of North Africa by a European lineage followed by a speciation process and subsequently a colonisation of Macaronesia.

Bauernfeind and Soldán (2012) stated that is a species endemic to Madeira, while Rutschmann et al. (2014) showed later that the species has a much wider distribution. They pointed out its European or North-African (Morocco) origin and suggested a recent colonisation of Madeira. is widely distributed in Atlantic Europe as proven by the recent reports of the species from the United Kingdom (Macadam et al. 2018). This species remains difficult to distinguish morphologically from other species (Soldán and Godunko 2006). The main discriminating character between and the North-African endemic is the number of regular rows of long setae at the apex of the paraglossae (the usual three rows in and four rows in ) and the apex of the maxillary palp (with one typical small apical scale in and without the apical scale in ). The genetic distances amongst Macaronesian, Iberian and Algerian specimens unequivocally confirm the conspecificity of the different populations. The ecological preference of in the Tafna catchment is similar to that of the lowlands of Madeira (Soldán and Godunko 2006): larvae prefer coarse substrate composed of rocks, cobbles or pebbles in low, moderate to fast current velocities; they were less abundant and only present in relatively-preserved sites at higher altitude (CH0; CH1; KH1; SK1; IOM). In comparison, larvae have a much wider ecological range and are more pollution resistant; they are also highly abundant and present in all sampling sites with various substrates and velocities.

Concluding remarks

We summarise the state of the knowledge and the implication of the present study for the fauna of North-West Algeria in Table 8. As we mostly found interspecific distances between Algerian and European lineages, our results generally highlighted and confirmed the high endemism of North African . According to the present knowledge, the endemism may be restricted to Algeria or to the Maghreb, even for species that were supposed to present West Palearctic distribution (Western Europe and North Africa). The link with the Iberian Peninsula is less strong than expected, as no species included in the study is shared only between the two areas. From a genetic point of view, only one case of sister-species was found (). is the only species with a West Mediterranean distribution, as the same species occurs in Algeria and South of France. Finally, our study confirms the presence of Macaronesian and Atlantic species in Maghreb and, therefore, endorses the preliminary results of Rutschmann et al. (2014), Rutschmann et al. (2017).

Table 8.

Distribution of the of the Tafna catchment. Distribution prior to study, based on 1Bauernfeind and Soldán (2012); 2Fauna Europa (Fauna Europaea 2020); 3Macadam et al. (2018).

Operational Taxonomic Units	Implications from this study
Acentrella cf. sinaica	New unnamed species in North Africa
Baetis maurus	Endemic to North Africa1,2; new unnamed species in Iberian Peninsula
Baetis cf. pavidus	North Africa and South of France1,2
Baetis (Rhodobaetis) atlanticus	First report for North Africa; known from Macaronesia and Atlantic Europe1,3
Baetis (Rhodobaetis) sinespinosus	Confirmation of North African endemism1
Cloeon peregrinator	First report for North Africa; known from Macaronesia1
Procloeon stagnicola	Confirmation of North African endemism1

The next steps will be to sequence more specimens from different areas of Algeria and also from Morocco and Tunisia to confirm the monophyly of the different North African clades. The results, especially the validation of the new species hypotheses, need to be confirmed by integrative methods. Only morphological evidence and more mitochondrial or nuclear genes can validate the specific status of these clades. Our study may have implications outside of North Africa, as our results suggest that one or two lineages, previously supposed to belong to , may represent new species in Spain, as well as the presumably non-conspecificity of the French and Italian lineages of .

Complete Maximum Likelihood tree including representative of using TN93 (+G+I) model

Biomolecular tree

File: oo_420313.tif

Complete Maximum Likelihood tree including representative of using TN93 (+G+I) model

Biomolecular tree

File: oo_420317.tif

Complete Maximum Likelihood tree including representative of spp. using General Time Reversible model (+G+I)

Biomolecular tree

File: oo_420319.tif

RB_Sequence alignment

Nucleotide sequences

File: oo_435076.fasta

BP_sequence alignment

Nucleotide sequence

File: oo_435079.fas

BM_sequence alignment

Nucleotide sequences

File: oo_435073.fas

AC_sequence alignment

Nucleotide sequences

File: oo_435075.fas

CO_sequence alignment

Nucleotide sequences

File: oo_435071.fas

PC_sequence alignment

Nucleotide sequences

File: oo_435074.fas