A new genus from Madagascar with strongly enlarged labium (Ephemeroptera, Baetidae)

A new genus of Baetidae, Megalabiops gen. nov., and a new species, M. madagasikara sp. nov., are described from Madagascar based on nymphs. The new genus is characterized by having a strongly enlarged mentum; pedicelli with many long, stout, pointed setae; a brush of dense, short setae between prostheca and mola of both mandibles; an apically pointed maxillary palp with a stout seta at the tip; and a labium with many long, simple setae ventrally on glossae. The patellotibial suture is absent on the fore tibia and present on middle and hind tibiae. The claw is strongly elongated with two rows of denticles. The imago remains unknown and the relationships with other African genera of Baetidae are tentative. Despite being easily identifiable, only two nymphs were found in two highly sampled localities in Madagascar.


Introduction
With nearly one third of all mayfly species worldwide (ca. 1,100 species in 114 genera), the family Baetidae has the highest species diversity among mayflies (updated from Sartori and Brittain 2015;Jacobus et al. 2019). Their distribution is cosmopolitan, excluding only Antarctica, New Zealand, and New Caledonia. Investigations of the molecular phylogeny of the order Ephemeroptera revealed the relatively basal position

Materials and methods
The specimens were collected in 2001 and 2003 from two different locations in the same area of Madagascar. Specimens were preserved in 70%-80% ethanol. Nymphs were dissected in Cellosolve (2-Ethoxyethanol) with subsequent mounting on slides in Euparal medium, using an Olympus SZX7 stereomicroscope.
The DNA of one specimen 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).
The terminology used in the manuscript follows Hubbard (1995) and Kluge (2004).
The new species is established based on morphological characters.

Results
Megalabiops gen. nov. http://zoobank.org/871DA221-4FDB-4CF6-B81B-88E6C35527AB Figures 1-7 Type species. Megalabiops madagasikara gen. et sp. nov., by present designation. Diagnosis. Nymph. This new genus is distinguished by the combination of the following characters: A) body rather short and stocky (Fig. 1); B) base of antennae close to each other, with rather broad carina between them (Fig. 2a); C) pedicellus with many long, stout, pointed setae (Fig. 3a); D) labrum rather small, lateral margins straight, distal margin largely V-shaped; dorsal surface with one long, submedian seta and submarginal arc of setae restricted to a few simple setae (Fig. 3b); E) both mandibles with brush of medium, fine setae between prostheca and mola (Fig. 3c, f ); F) hypopharynx with large, medial tuft of stout setae (Fig. 4a); G) maxillary palp 2-segmented (Fig. 4b); H) labium with strongly enlarged mentum ( Fig. 2b-d); glossae basally broad, narrowing toward apex, shorter than paraglossae; inner ventrolateral   margin of glossae with a row of long, simple setae, close to ventrolateral margin with a row of very long, simple setae, apically with some very robust, long, curved setae; stout paraglossae, slightly curved inward, apex with three rows of stout, pectinate setae; labial palps segment II without distolateral protuberance, segment III slightly pentagonal (Fig. 4d, e); I) forelegs with a row of long, spine-like setae on dorsal margin of femur; femoral patch absent on all legs; patellotibial suture absent on foreleg and present on middle and hind legs; claw strongly elongated, pointed, with two rows of numerous, minute denticles (Fig. 5a, b); J) posterior margin of abdominal tergites with triangular spines, lateral margins without spines (Fig. 6a).
Imagines. Unknown. Etymology. Megalabiops is an arbitrary combination of letters with allusion to the Greek words mega and iops, and the Latin word labium. Megalab-is with reference to the strongly enlarged labium and -iops with reference to the Baetidae which look and move like small fishes. The gender is feminine.
Description. Nymph. (Figs 1-7). Body. Short and stocky (Figs 1, 2). Head. Antenna (Figs 2a,3a). Antennae bases close to each other, with broad carina between them. Pedicellus with many long, stout, pointed setae; scapus bare. Labrum (Fig. 3b). Rather small, lateral margins straight and distal margin largely V-shaped, wider than long; on dorsal surface with one submedian, long, simple seta and submarginal arc of setae restricted to few simple setae; on ventral margin anterolaterally and medially with a row of long, fine setae.
Labium 4d,e). Mentum strongly enlarged (long, very thick). Glossae basally broad, narrowing toward apex, shorter than paraglossae; inner ventrolateral margin with row of long, simple setae and close to inner margin with row of very long, simple setae; apically with some very robust, long, curved setae. Paraglossae broad, slightly curved inward, with convex outer margin; apically with three rows of long, stout, pectinate setae. Labial palps long, segment II without distolateral protuberance, segment III almost pentagonal, distal margin straight, lateral margin curved.
Caudal filaments (Fig. 1). Inner margin of cerci with 3 to more than 15 long, thin primary swimming setae per segment, increasing in number distally; paracercus bilaterally with 4 to more than 13 long, thin primary swimming setae per segment, increasing in number distally.
Left mandible (Fig. 3f-h). Incisor and kinetodontium partially cleft. Incisor with two denticles, outer denticle blade-like; kinetodontium with three denticles. Prostheca robust, apically denticulate and with comb-shape structure. Margin between prostheca and mola straight; with brush of medium setae between prostheca and mola, length of setae ca. 1/4 of prostheca length; and with minute denticles toward subtriangular process. Subtriangular process long and slender, above level of area between prostheca and mola. Tuft of setae at apex of mola absent.
Both mandibles with lateral margins convex. Basal half laterally with short, fine, simple setae.
Hypopharynx and superlinguae (Fig. 4a). Lingua longer than wide and longer than superlinguae, with prominent central protuberance; with large medial tuft of stout setae. Superlinguae distally rounded, lateral margins rounded; long, fine, simple setae along distal margin.
Maxilla (Fig. 4b, c). Galea-lacinia ventrally with two simple, apical setae under canines. Medially with one bifurcate seta and four medium, simple setae. Maxillary palp ca. 1.4× as long as length of galea-lacinia; palp segment II 1.4× length of segment I; short, fine, simple setae scattered over surface of segment II; apex pointed and with short, stout seta at tip.
Labium (Figs 2b-d, 4d, e). Inner margin of glossa with marginal row of long, simple setae, on ventral surface row of very long, simple setae close to margin; apex with four long, very robust setae, curved toward inner side, and two equally long, spine-like, pectinate setae; outer margin bare. Paraglossa with outer margin and apex convex and inner margin concave, curved inward; ventrally with three rows of long, robust, pectinate setae in apical area and two short, fine, simple setae in anteromedial area; dorsally with row of four long, spine-like setae near inner margin. Mentum strongly enlarged (extended and thickened), especially on ventral side; laterally with medium, fine, simple setae. Labial palp very long, with segment I 0.9× length of segments II and III combined. Segment I ventrally with short, fine, simple setae. Segment II elongated, conical, without protuberance; ventral surface with short, fine, simple setae; dorsally with three spine-like setae near distal margin. Segment III slightly pentagonal; length subequal to width; ventrally covered with short, spine-like setae and short, fine, simple setae.
Thorax. Hind protoptera well developed. Foreleg (Fig. 5a, b). Ratio of foreleg segments 1.8: 1.0: 1.1: 0.6. Femur. Rather broad, length 2.7× maximum width; dorsal margin with ca. nine long, spine-like setae plus ca. five long, spine-like setae close to margin in basal part, length of setae ca. 0.3× maximum width of femur; on apex three long, spine-like setae and some short, fine, simple setae; many short to medium, stout setae along ventral margin; femoral patch absent. Tibia. Stocky, slightly shorter than tarsus; dorsal margin with row of short, fine, simple setae; ventral margin with row of medium, curved, spine-like setae; patellotibial suture absent; connection to tarsus tight. Tarsus. Dorsal margin with row of scarce, short, fine, simple setae; ventral margin with row of short to medium, curved, spine-like setae. Claw strongly elongated, distally pointed, with two rows of numerous minute denticles; denticles decreasing in size distally; subapical setae absent.
Hind leg (Fig. 5c). Ratio of hind leg segments 1.8: 1.0: 1.0: 0.6. Femur. Length 4× maximum width. Dorsal margin with ca. six long, spine-like setae plus ca. three long, spine-like setae close to margin; many short to medium, stout setae along ventral margin; femoral patch absent. Tibia. Dorsal margin almost bare; ventral margin with a row of short, spine-like setae. Patellotibial suture present on basal 1/3 area. Tarsus. Dorsal margin bare; ventral margin with a row of short, spine-like setae. Claw as foreleg.
Abdomen. Tergites (Fig. 6a). Posterior margin of tergites with triangular spines, longer than wide. Gills (Fig. 6c). Gills on segments I-VII. Costal and anal margins with small denticles intercalating short, fine, simple setae. Tracheae extending from main trunk to inner and outer margins. Gill I as long as 1/3 length of segment II; gill IV as long as length of segments V, VI and 1/3 VII combined; gill VII reaching end of segment X.  (Fig. 6b). Posterior margin slightly expanded; with ca. 36 stout, marginal spines. Surface scattered with U-shaped scale bases, some fine, simple setae and micropores. Cercotractor with small, marginal spines.

Discussion
Megalabiops gen. nov. clearly belongs to the family Baetidae based on the pisciform body shape (Fig. 1); the nymphal Y-shaped frontal suture reaching ventrally to lateral ocelli (Fig. 5d); the labrum with distinctly expressed median incision (Fig. 3b); the shape of the right and left prostheca (Fig. 3d, g); the shape of the glossae (basally widened, most part narrow) (Fig. 4d); and the anterior outer projection of the femur apex, which is directed toward the inner side of the femur (Fig. 5a, c) (Wang and McCafferty 1996;Kluge 2004). The new genus can be assigned to the Protopatellata Kluge & Novikova, 2011 according to the rank free system of Kluge (Kluge 1997;Kluge and Novikova 2011), based on the absence of a patellotibial suture in the foreleg and its presence in middle and hind legs of the nymph.
African genera of Protopatellata were previously assigned either to the Centroptiloides complex (Lugo-Ortiz and McCafferty 1998a) corresponding to Centroptiloides/ g2 in Kluge's rank free system, or to the Bugilliesia complex (Lugo-Ortiz & McCafferty 1996;Gattolliat 2006;Kluge 2012) corresponding to the Rhithrocloeoninae or Rhithrocloeon/fg1 in Kluge's rank free system. The Centroptiloides complex also includes highly adapted taxa such as the carnivorous genera Nesoptiloides Demoulin, 1973or Barnumus McCafferty & Lugo-Ortiz, 1998. Most genera of this complex have the right incisor and kinetodontium only partially fused, patch of setae between prostheca and mola of the right mandible and the claws with two rows of denticles increasing in size progressively (Lugo-Ortiz and McCafferty 1998a). The Bugilliesia complex is mainly defined at the imaginal stage by the highly modified 2-segmented forceps (Lugo-Ortiz and McCafferty 1996;Kluge 2012).
The following characters differentiate Megalabiops gen. nov. from all other genera of Protopatellata and even of Baetidae in general: antennae with many long, slightly lanceolate setae on pedicelli (Fig. 3a); labium with mentum strongly enlarged (extended and thickened, especially on ventral side), laterally with fine, simple setae (Figs 2b-d, 4d); glossae ventrally with one row of very long setae close to margin and one row of long setae on margin (Fig. 4d).
Pedicelliops Kaltenbach & Gattolliat, 2020, another genus of Protopatellata, also has two rows of setae of different length ventrally on and close to the margin of the glossae and similar stout, curved setae at their apex (Kaltenbach and Gattolliat 2020b: fig. 4i). Further, it also has a carina between the antennae, a similar shape of the mandibles (outer lateral margins convex) and a brush of setae between prostheca and mola of both mandibles (Kaltenbach and Gattolliat 2020b: figs 2d, 4b, d). However, the other mouthparts are dissimilar, the legs are strongly adapted with long setae at the femur and the pedicellus is bilaterally strongly enlarged (Kaltenbach and Gattolliat 2020b: figs 4, 5a, c, 3a). From the illustrations, it appears that also Micksiops bicaudatus (Gillies, 1990) has glossae with shorter and longer setae on ventral side (Gillies 1990: fig. 91), but other mouthparts and legs are dissimilar to Megalabiops gen. nov.
Elongated claws with two rows of numerous denticles increasing in length toward the apex are also typical for species of Bugilliesia Lugo-Ortiz & McCafferty, 1996. However, the legs are generally very slender and there is no tight connection between tibia and tarsus. Additionally, the maxillary palp has a small third segment, the glossae are without rows of long setae, the labial palp segment II has a distomedial protuberance, and the submarginal arc of setae on dorsal surface of the labrum is composed of lanceolate setae ).
The legs of Megalabiops gen. nov. are similar to Delouardus djabala Lugo-Ortiz & McCafferty, 1999: the tibia is slightly shorter than the tarsus, with a tight connection between them; the claws are elongated with two rows of denticles; and the setation is similar. Further, there are some long, simple setae on the pedicellus (but not stout, slightly lanceolate setae as in Megalabiops gen. nov.) and both mandibles have a brush of fine, simple setae between prostheca and mola. The claws of Megalabiops gen. nov. and the tight connection between tibia and tarsus are also similar to Cheleocloeon Wuillot & Gillies, 1993. However, Delouardus and Cheleocloeon are different in other characters from Megalabiops gen. nov., e.g. the labium without long setae on the glossae and the labial palp segment II with a large distomedial protuberance. The sister genera Delouardus and Cheleocloeon belong to the Anteropatellata according to the rank free system of Kluge (Kluge and Novikova 2011), with a patellotibial suture on all legs of the nymph.
A remarkable setation on the antennal base is also present in two Neotropical Anteropatellata: Rivudiva trichobasis Lugo-Ortiz & McCafferty, 1998 has many long, robust, simple setae on scape and pedicellus and Spiritiops silvudus Lugo-Ortiz & Mc-Cafferty, 1998 has many minute, fine, simple setae scattered over surface of scape and pedicellus (Lugo-Ortiz and McCafferty 1998b).
As a conclusion, the relationship of Megalabiops gen. nov. with other genera remains unresolved and knowledge of the imaginal stage, especially of the male genitalia and the number of intercalary veins of the wings is of major importance to confirm the possible relationship with the Centroptiloides complex or Rhithrocloeoninae (sensu Kluge 2012). Indeed, Bugilliesia Lugo-Ortiz & McCafferty, 1996, and related genera belonging to this subfamily, possess a unique character: the loss of the last segment of the gonostylus.
The description of Megalabiops gen. nov. is based on only two nymphs. Remarkably, these nymphs were found in two frequently sampled localities, where different field trips of the ORSTOM and MZL teams (Elouard and Gibon 2001) as well as a monthly survey of this catchment (Oliarinony et al. 2016) were done. Intensive fieldwork and many studies were done in Madagascar by one of the authors (e.g. Gattolliat and Sartori 1998, 2000, 2001Gattolliat 2001Gattolliat , 2002Gattolliat , 2006), and because of its reasonable size and its ease of recognition, we cannot assume that additional material stored in our collection was overlooked. It could mean that either this species is extremely rare, or it occurs in microhabitats, which were only rarely or not at all sampled, and the two specimens were collected by chance.
The number of localities and different habitats sampled in Madagascar are relatively high, but there are still areas where no collection activities have occurred (especially in the Northeast of the island including the Masoala Peninsula). Taking into account the obvious biological richness of this island, it would be prudent to assume that the number of genera and species of Baetidae will continue to increase with further fieldwork and collections in Madagascar.