Labiobaetis Novikova & Kluge in West Africa (Ephemeroptera, Baetidae), with description of a new species

Material collected between 1984 and 1988 in Guinea and Mali and between 2003 and 2008 in the Ivory Coast substantially increased our knowledge of Labiobaetis Novikova & Kluge in West Africa. We identified eight different species using morphological characters. One species, L. ediai sp. nov., is new to science; it is described and illustrated, based on its nymphs. The status of L. boussoulius (Gillies, 1993) is discussed and the divergent morphology of L. elouardi (Gillies, 1993) is compared to other species of Labiobaetis. A key to the nymphs of all West African species is provided and the distribution of Labiobaetis species in the Afrotropical realm is discussed.


Introduction
The family Baetidae has the highest species diversity amongst mayflies, comprising ca. 1,100 species in 114 genera (updated from Sartori and Brittain 2015, Jacobus et al. 2019, Cruz et al. 2020, which is approximately one third of all mayfly species worldwide. They have a cosmopolitan distribution, except for New Zealand (Gattolliat and Nieto 2009). Investigations of the molecular phylogeny of the Order Ephemeroptera revealed the relatively primitive status of the family (Ogden and Whiting 2005;Ogden et al. 2009;Ogden et al. 2019).
The genus Labiobaetis Novikova & Kluge, 1987, is one of the richest genera of mayflies with 145 previously described species (Barber-James et al. 2013;Kaltenbach et al. 2020 and citations therein;Kaltenbach and Gattolliat 2021). The distribution of Labiobaetis is nearly worldwide, except for the Neotropical realm, New Zealand, New Caledonia and some remote islands. The history and concept of the genus Labiobaetis were recently summarised in detail (Shi and Tong 2014;Kaltenbach and Gattolliat 2018).
Recently, integrative taxonomy was applied on collections from the highly diverse regions of Southeast Asia and New Guinea, but also from Africa and many species were described and named , 2020aKaltenbach et al. 2020). The present contribution will focus on West Africa, which forms its own freshwater ecoregion as one of the five sub-regions of the Afrotropical realm (Barber-James and Gattolliat 2012: fig. 1).
The status of Afrotropical mayfly research was depicted by Barber-James and Gattolliat (2012). The history of taxonomic studies of Labiobaetis in the Afrotropical realm was briefly summarised in Kaltenbach and Gattolliat (2021). West Africa has a long history of mayfly research in general and many collections and studies were undertaken in that region. Between 1974 and1989, the French ORSTOM (Office de la recherche scientifique et technique outre-mer), presently the IRD (Institut de Recherche pour le Développement), conducted field research in West Africa as part of an important onchocerciasis control programme (Lévêque et al. 2003). Aquatic macroinvertebrates were collected in about 100 localities, mainly in Guinea, Ivory Coast and Mali. Most localities were sampled regularly over a period of several years. As the systematics of aquatic insects was still poorly-known at the time, important alpha taxonomic research was conducted and several articles were published, including descriptions of new species and genera. The majority of mayflies known from West Africa were described, based on material collected during this programme (e.g. Elouard and Forge 1978;Gillies 1980Gillies , 1989Gillies , 1993Gillies , 1997Elouard 1986a, b;Elouard and Gillies 1989;Elouard et al. 1990; Gillies and Elouard 1990;Elouard and Hideux 1991;Gillies 1993a, b, 1994;Gattolliat 2006;Gattolliat and Sartori 2006;Kaltenbach and Gattolliat 2020b), including three species of Labiobaetis (Gillies 1993). The present study is partly based on nymphs collected during the ORSTOM programme between 1984 and 1988 in Guinea, Ivory Coast and Mali. Another part of the material came from ecological studies done in the Ivory Coast 2003-2008(Edia et al. 2015 (Fig. 1). Edia et al. (2015: tables 1, 2, fig. 1) also described the environmental characteristics and physical conditions of some of the river systems and a part of the sampling sites in the Ivory Coast (Rivers Ehania, Soumié, Eholié and Noé).
So far, only four species of Labiobaetis are known from West Africa: L. boussoulius (Gillies, 1993), L. elouardi (Gillies, 1993), L. gambiae (Gillies, 1993) and L. piscis McCafferty, 1997 (Gillies 1993;Arimoro and Muller 2010). Here, we report four additional species from different West African countries, one of which is described and illustrated as a new species, based on nymphs (L. glaucus (Agnew, 1961), L. latus (Agnew, 1961), L. vinosus (Barnard, 1932) and L. ediai sp. nov.).The status of L. boussoulius is discussed below; the morphological particularities of L. elouardi are depicted and compared to other species in other realms. The total number of Labiobaetis species worldwide has now been increased to 146.

Materials and methods
The specimens were collected between 1984 and 1988 in Guinea and Mali by Jean-Marc Elouard (France) during the French ORSTOM programme for the control of onchocerciasis and between 2003 and 2008 in the Ivory Coast by Edia Oi Edia (Université Nangui Abrogoua, Abidjan, Ivory Coast), partly together with one of the authors (JLG). The nymphs were preserved in 70%-96% ethanol. The dissection of nymphs was done in Cellosolve  with subsequent mounting on slides with Euparal liquid, using an Olympus SZX7 stereomicroscope. The DNA of some specimens was extracted using non-destructive methods allowing subsequent morphological analysis (see Vuataz et al. 2011 for details). We amplified a 658 bp fragment of the mitochondrial gene cytochrome oxidase subunit 1 (COI) using the primers LCO 1490 and HCO 2198 (Folmer et al. 1994, see Kaltenbach and Gattolliat 2020a for details). Sequencing was done with Sanger's method (Sanger et al. 1977).
Drawings were made using an Olympus BX43 microscope. To facilitate the determination of the new species and the comparison of important structures with other species, we partly used a combination of dorsal and ventral aspects in one drawing (see Kaltenbach et al. 2020: fig. 1).
Photographs of nymphs were taken using a Canon EOS 6D camera and the Visionary Digital Passport imaging system (http://www.duninc.com) and processed with Adobe Photoshop Lightroom (http://www.adobe.com) and Helicon Focus version 5.3 (http://www.heliconsoft.com). Photographs were subsequently enhanced with Adobe Photoshop Elements 13.
The terminology follows Hubbard (1995) and Kluge (2004). The form of the description follows the one used for other recent descriptions of Labiobaetis, as in Kaltenbach et al. 2020. Abbreviations: NHM The Natural History Museum (London, Great Britain); MZL Musée de Zoologie Lausanne (Switzerland).

Results
List of Labiobaetis species from West Africa
Antenna ( Fig. 6f ) with scape and pedicel subcylindrical, without distolateral process at scape. Labrum (Fig. 7a). Subrectangular, length 0.6× maximum width. Distal margin with medial emargination and small process. Dorsally with long, fine, simple setae scattered over surface; submarginal arc of setae composed of ca. 13 long, feathered  setae. Ventrally with marginal row of setae composed of lateral and anterolateral long, feathered setae and medial long, bifid, pectinate setae; ventral surface with ca. four short, spine-like setae near lateral and anterolateral margin.
Right mandible (Fig. 7b, c). Incisor and kinetodontium fused. Incisor with five denticles; kinetodontium with three denticles, inner margin of innermost denticle with row of thin setae. Prostheca robust, apically denticulate. Margin between prostheca and mola slightly convex, with minute denticles. Tuft of setae at apex of mola present. Left mandible (Fig. 7d,e). Incisor and kinetodontium fused. Incisor with four denticles; kinetodontium with three denticles. Prostheca robust, apically with small denticles and comb-shaped structure. Margin between prostheca and mola almost straight, with minute denticles towards subtriangular process. Subtriangular process long and slender, above level of area between prostheca and mola. Denticles of mola apically constricted. Tuft of setae at apex of mola absent.
Both mandibles with lateral margins almost straight. Basal half with fine, simple setae scattered over dorsal surface.
Hypopharynx and superlinguae (Fig. 7f ). Lingua slightly longer than superlinguae. Lingua longer than broad; medial tuft of stout setae well developed; distal half laterally slightly expanded. Superlinguae distally straight; lateral margins rounded; fine, long, simple setae along distal margin. Maxilla (Fig. 7g). Galea-lacinia ventrally with two simple, apical setae under canines. Inner dorsal row of setae with three denti-setae, distal denti-seta tooth-like, middle and proximal denti-setae slender, bifid and pectinate. Medially with one pectinate, spine-like seta and three or four simple setae. Maxillary palp longer than length of galea-lacinia; 2-segmented; palp segment II 1.5× length of segment I; setae on maxillary palp fine, simple, scattered over surface of segments I and II; apex of last segment rounded, with slight excavation at inner distolateral margin. Labium (Fig. 7h). Glossa basally broad, narrowing towards apex; shorter than paraglossa; inner margin with four or five spine-like setae, distalmost seta much longer than other setae; apex with two long and one medium, robust, pectinate setae; outer margin with four or five spine-like setae increasing in length distally; ventral surface with fine, simple, scattered setae. Paraglossa sub-rectangular, curved inwards; apex rounded; with three rows of long, robust, distally pectinate setae in apical area and one or two short, simple setae in anteromedial area; dorsally with row of three long, spine-like, simple setae near inner margin. Labial palp with segment I 0.7× length of segments II and III combined. Segment I ventrally with short, fine, simple setae. Segment II with rather narrow,   thumb-like distomedial protuberance; distomedial protuberance 0.5× width of base of segment III; ventral surface with short, fine, simple setae; dorsally with four spine-like setae near outer margin. Segment III slightly pentagonal; apex slightly pointed; length 1.1× width; ventrally covered with short, spine-like, simple setae and short, fine, simple setae.
Terga (Fig. 6c). Surface with irregular rows of U-shaped scale bases and scattered fine, simple setae and micropores. Posterior margin of tergum IV with triangular spines, wider than long. Gills (Fig. 6d). Present on segments II-VII. Margin with small denticles intercalating fine simple setae. Tracheae extending from main trunk to inner and outer margins. Gill IV as long as length of segments V, VI and half VII combined; gill VII as long as length of segments VIII, IX and half X combined.
Etymology. Dedicated to the collector of the specimens, Dr. Edia Oi Edia (Université Nangui Abrogoua, Abidjan, Ivory Coast), in recognition of his contribution to the knowledge of aquatic insects from the Ivory Coast.

Assignment to Labiobaetis and affinities
For the assignment of the new species to Labiobaetis, we refer to Kluge and Novikova (2014). Labiobaetis is characterised by a number of derived characters, some of which are not found in other taxa (Kluge and Novikova 2014): antennal scape sometimes with a distolateral process; maxillary palp two segmented with excavation at inner distolateral margin of segment II, excavation may be poorly developed or absent (Fig. 7g); labium with paraglossae widened and glossae diminished; labial palp segment II with distomedial protuberance (Fig. 7h). The concept of Labiobaetis is also based on additional characters, summarised and discussed in Gattolliat (2018, 2019). L. ediai sp. nov. is morphologically related to L. vinosus, sharing the absence of a distolateral process on scape, the absence of hind protoptera and six pairs of gills. The main differences are the narrow distomedial protuberance seen in labial palp segment II (broad in vinosus) (Figs 2h, 7h), the number of spine-like setae at the dorsal margin of the fore femur (7-9 in L. ediai sp. nov.; 8-18 in L. vinosus) and the presence or absence of setae at the apex of the left mola (present in L. vinosus, absent in L. ediai sp. nov.).

Labiobaetis boussoulius and L. glaucus
Labiobaetis boussoulius was described by Gillies (1993), based on imagos, subimagos and nymphs from a single location, the Milo River near Boussoulé in Guinea. The species is very similar to the common and very widespread L. glaucus, which is also present in Guinea. Additionally, L. glaucus is known to have a very variable nymphal morphology . Therefore, we investigated the morphology of L. boussoulius in comparison to L. glaucus: the male genitalia are indistinguishable (Gillies 1993: fig. 3;Gattolliat et al. 2018: fig. 46), both have a remarkable triangular expansion at the inner margin at the apex of segment I and the base of segment II of the gonostyli; the colouration of the male imago seems to be slightly different, according to the descriptions of Gillies 1993: p. 214 andGattolliat et al. 2018: p. 97, but the specimen of L. boussoulius is from Guinea and the one of L. glaucus from Saudi Arabia and the colours may have altered by the long preservation in alcohol before their description; Gillies 1993: p. 214 indicates 0-4 partial cross veins in the pterostigma of the male wing of L. boussoulius (Gillies 1993: fig. 1), but the male holotype has four longer cross veins not reaching the subcostal vein and one shorter cross vein, which is the same as in a male of L. glaucus from Saudi Arabia (GBIFCH00235731, GenBank MH070317); most characters of the nymph are identical (Gillies 1993: figs 4, 6-12;Gattolliat et al. 2018: figs 34-44;Lugo-Ortiz and McCafferty 1997: figs 27-50;Lugo-Ortiz et al. 2000: figs 1-11). We notice that the maxillary palp of L. boussoulius is 2-segmented as usually seen in Labiobaetis and not 3-segmented as shown in Gillies 1993: fig. 5. However, we identified four morphological differences in the nymphs, based on the material at our disposal: the maxillary palp of L. boussoulius has a more pronounced distolateral excavation at segment II than L. glaucus and segment II is longer in comparison to segment I in L. boussoulius (1.8-1.9×) than in L. glaucus (ca. 1.5×) (Fig. 2a-c); the fore femur of L. boussoulius is usually more slender than the one of L. glaucus (length 3.1-4.1× maximum width vs. 2.7-3.2×) (Fig. 3a, b); and L. boussoulius has less spinelike setae at the dorsal margin of the fore femur (9-12) than L. glaucus (13-18). Gattolliat et al. 2018 published a COI barcode of L. boussoulius (GenBank MH070309, GBIFCH00517528), which is different from the one of L. glaucus. However, the specimen is from the Ivory Coast and not from Guinea and no material is available for morphological re-investigation. Therefore, the assignment of this sequence to L. boussoulius remains uncertain. In conclusion, we continue to consider L. boussoulius as a valid species, based on four slight morphological differences of the nymph; but due to the known high variability of L. glaucus, we recommend that its status is confirmed with genetic investigations of fresh material of L. boussoulius from Guinea and L. glaucus from West Africa.

Labiobaetis elouardi
The morphology of Labiobaetis elouardi is very atypical for this genus: the labrum is rectangular (Fig. 8a), the setae of the submarginal arc of setae on the dorsal surface of the labrum are feathered with a broad middle part, representing a new type of seta, which was not known, so far, in Labiobaetis (Fig. 8h-j), the apex of both mandibles is cleft between incisor and kinetodontium (Fig. 8b, c), the glossae are much shorter than the paraglossae (Fig. 8d) and the paraproct has only two or three strongly devel-oped, marginal spines (Fig. 8f, g). Usually, in Labiobaetis, the labrum is much more subrectangular and laterally rounded, the feathered setae on the dorsal surface of the labrum have a slender middle part (Fig. 8k), incisors and kinetodontium are fused till the apex in both mandibles, the glossae are only slightly shorter than the paraglossae (except in a few species, such as the Malagasy species L. fabulosus Lugo-Ortiz & Mc-Cafferty, 1997) and the paraproct has many and less pronounced marginal spines. The usual type of feathered setae (Fig. 8k) is present in all other Afrotropical species Gattolliat 2001) as well as in the groups operosus and difficilis from Southeast Asia (Kaltenbach and Gattolliat 2019) and in the group orientis from New Guinea .
However, Labiobaetis is generally very variable in many characters and there are other species with similar characters in other regions: species of the groups vallus and sumigarensis, as well as L. moriharai Müller-Liebenau, 1984 andL. paravultuosus Kaltenbach & have a nearly rectangular labrum; an undescribed species from Borneo has similar setae dorsally on the labrum; species of the vallus group, as well as L. bakerae Kaltenbach & Gattolliat, 2020, L. pakpak Kaltenbach & Gattolliat, 2019, L. baganii Kaltenbach, Garces & Gattolliat, 2020, L. delocadoi Kaltenbach, Garces & Gattolliat, 2020, L. giselae Kaltenbach, Garces & Gattolliat, 2020and L. mendozai Kaltenbach, Garces & Gattolliat, 2020, have a slight cleft between the incisor and kinetodontium; and L. paravitilis  and L. fabulosus also have glossae much shorter than paraglossae. We keep L. elouardi in the concept of Labiobaetis, because of the presence of a distolateral process at the scape (Fig. 8e), a slight excavation at the inner distolateral margin of maxillary palp segment II, the large distomedial protuberance at labial palp segment II and the presence of a broad subgenital plate between the gonopods, which are all important characters of the genus. Further, Labiobaetis is known to have very different types of setae (simple, feathered, clavate, dendritic, lanceolate) forming the submarginal arc on the dorsal surface of the labrum (Kaltenbach et al. 2020: fig. 2a-e).

Distribution of Labiobaetis in the Afrotropical realm
In Kaltenbach and Gattolliat 2021, we compared the distribution of Labiobaetis species in the Afrotropical realm to Southeast Asia and New Guinea, where the majority of species live. Apart from Madagascar, where all Labiobaetis species are endemic to the Island (Gattolliat 2001), some Afrotropical species have a wide or even very wide distribution (L. glaucus, L. latus, L. potamoticus and L. vinosus), whereas most species in Southeast Asia and New Guinea are restricted to smaller regions or are endemic to an island. The reason probably lies in the high geographic complexity of the region (islands in Southeast Asia, extreme landscape structure in New Guinea), facilitating allopatric speciation and endemicity (Toussaint et al. 2013(Toussaint et al. , 2014. The huge African continent is, in comparison, geographically less structured, which generally facilitates wider distributions of species. The present study confirms large distribution ranges of some Labiobaetis species in the Afrotropical realm: L. glaucus is additionally present in Guinea, Ivory Coast and Mali and can be considered as Panafrotropical (except Madagascar), the same probably applies for L. vinosus (additionally found in the Ivory Coast) and L. latus (additionally found in Guinea, Ivory Coast and Mali) and also L. piscis (additionally found in Guinea, Ivory Coast and Mali) have a very large distribution range as well. Labiobaetis gambiae seems to have a wider distribution at least in West Africa (known from Gambia, Ivory Coast and Mali) and may have been easily overlooked in other areas due to its close similarity with L. piscis. In contrast, L. boussoulius and L. elouardi seem to be rare and local species, while L. ediai sp. nov. is, so far, only known from the Ivory Coast, but it is not rare.
A high number of localities and different habitats have been sampled in West Africa due to the ORSTOM activities in the past (Lévêque et al. 2003), but there are still areas where no collecting has yet been carried out. Additionally, the distribution of Labiobaetis species in Africa is often much more widespread than in other regions. Therefore, we may expect a few, but not many, more species to be discovered in West Africa with further collections.