Labiobaetis Novikova & Kluge in Borneo (Ephemeroptera, Baetidae)

Abstract Material collected between 2000 and 2014 on the island Borneo, including the Indonesian province of Kalimantan, the Malaysian province of Sabah and Brunei Darussalam, substantially increased our knowledge of Labiobaetis on this island. The total number of Labiobaetis species in Borneo increased to five, as only one species, L. borneoensis (Müller-Liebenau, 1984), was previously reported. Three new species were identified by morphology and partly by using genetic distance (COI, Kimura 2-parameter). They are described and illustrated based on their larvae (Labiobaetis bakerae sp. nov., L. penan sp. nov. and L. dayakorum sp. nov.); in one case, the imago is described as well. New reports of L. borneoensis are presented and the imago of this species is described for the first time. Labiobaetis moriharai (Müller-Liebenau, 1984), originally described from mainland Malaysia (Province Selangor), is reported from Borneo for the first time. The interspecific K2P distances in Borneo are between 19% and 25%, the intraspecific distances are usually between 0% and 1%. The total number of Labiobaetis species worldwide is augmented to 126.


Introduction
The family Baetidae has the highest species diversity among mayflies, comprising 1,070 species in 110 genera Brittain 2015, Jacobus et al. 2019), which is approx. one quarter of all mayfly species worldwide (Gattolliat and Nieto 2009, Jacobus et al. Following combination of characters: A) dorsal surface of labrum with submarginal arc of clavate, apically smooth setae; B) labial palp segment II with large, lobed or thumblike distomedial protuberance, outer margin of protuberance predominantly concave (L. sumigarensis with hook-like modification of the protuberance); C) left mandible without setae at apex of mola, with minute denticles between prostheca and mola; D) six pairs of gills; E) hindwing pads absent; F) distolateral process at scape poorly developed or absent; G) colour of larvae dorsally uniform brown.
Colouration. Head, thorax and abdomen dorsally brown; head and thorax with bright median, dorsal suture. Head, thorax and abdomen ventrally light brown; femur ecru, with brown dorsal margin and brown ventrodistomedial spot, tibia and tarsus brown, caudal filaments ecru.
Antenna (Fig. 2g) with scape and pedicel subcylindrical, with poorly developed distolateral process at scape. Labrum (Fig. 1a). Rectangular, length 0.6× maximum width. Distal margin with medial emargination and a small process. Dorsally with medium, fine, simple setae scattered over surface; submarginal arc of setae composed of 13-15 long, clavate setae. Ventrally with marginal row of setae composed of anterolateral long, feathered setae and medial long, bifid setae; ventral surface with four short, spine-like setae near lateral and anterolateral margin.
Right mandible (Fig. 1b, c). Incisors fused. Outer and inner sets of denticles with 4 + 3 denticles and one minute intermediate denticle. Inner margin of innermost denticle with a row of thin setae. Prostheca robust, apically and distolaterally denticulate. Margin between prostheca and mola straight, with minute denticles. Tuft of setae at apex of mola present.
Left mandible (Fig. 1d, e). Incisors fused. Outer and inner sets of denticles with 4 + 3 denticles and one minute intermediate denticle. Prostheca robust, apically with small denticles and comb-shaped structure. Margin between prostheca and mola 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.
Maxilla (Fig. 1g). Galea-lacinia with two simple, robust apical setae under crown. 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 bipectinate, spine-like seta and 3-4 medium, simple setae. Maxillary palp 1.5× as long as length of galea-lacinia; 2-segmented; palp segment II 1.5× length of segment I; setae on maxillary palp fine and simple, scattered over surface of segments I and II; apex of last segment rounded, with excavation at inner distolateral margin.
Labium (Fig. 1h). Glossa basally broad, narrowing toward apex; shorter than paraglossa; inner margin with five spine-like setae increasing in length distally; apex with two long and one medium, robust, pectinate setae; outer margin with five long, spinelike setae; ventral surface with fine, simple, scattered setae. Paraglossa sub-rectangular, curved inward; apex rounded; with three rows of long, robust, distally pectinate setae in apical area and two or three medium, simple setae in anteromedial area; dorsally with a row of three long, spine-like setae near inner margin. Labial palp with segment I 0.8× length of segments II and III combined. Segment I with fine, simple setae along margins. Segment II with broad, thumb-like distomedial protuberance; distomedial protuberance 0.6× width of base of segment III; inner and outer margins with short, fine, simple setae; dorsally with one or two long, spine-like seta near outer margin. Segment III slightly pentagonal; apex rounded; length 1.1× width; ventrally covered with short, spine-like, simple setae and short, fine, simple setae.
Hindwing pads absent. Foreleg (Fig. 2a, b, c). Ratio of foreleg segments 1.3:1.0:0.6:0.2. Femur. Length ca. 3× maximum width. Dorsal margin with a row of 8-11 curved, spine-like setae; length of setae 0.29× maximum width of femur. Apex rounded; with one pair of curved, spinelike setae, one or a few short stout setae and some fine, simple setae. Stout, lanceolate setae scattered along the ventral margin; femoral patch absent. Tibia. Dorsal margin with a row of stout, apically rounded setae, apically one longer, apically rounded seta. Ventral margin with a row of curved, spine-like setae, on apex a few stout, spine-like, partly bipectinate setae and a tuft of fine, simple setae. Anterior surface scattered with stout, lanceolate setae. Patellotibial suture present on basal 1/3 area. Tarsus. Dorsal margin almost bare. Ventral margin with a row of curved, spine-like setae. Tarsal claw with one row of 9-11 denticles; distally pointed; with two stripes; subapical setae absent.
Terga (Fig. 2d). Surface with rows of U-shaped scale bases and scattered fine, simple, setae. Posterior margin of tergum IV with triangular spines, wider than long.
Gills (Fig. 2e). Present on segments II -VII. Margin with small denticles intercalating fine simple setae. Tracheae partly extending from main trunk towards outer and inner margins. Gill IV as long as length of segments V and 1/3 VI combined. Gill VII as long as length of segments VIII and 1/4 IX combined.
Etymology. Dedicated to Dr. Kate Baker (University of Exeter, UK), who collected the specimens in Brunei.
Distribution. Brunei (Fig. 15c). Biological aspects. The specimens were collected in pools of small lowland forest streams at an altitude of 100 m (Fig. 14).

Description. Larva
Colouration. Head, thorax, and abdomen dorsally brown; head and thorax with bright, median, dorsal suture. Head, thorax, and abdomen ventrally light brown, legs light brown, caudal filaments light brown.
Antenna ( Fig. 4i) with scape and pedicel subcylindrical, without distolateral process at scape. Labrum (Fig. 3a). Rectangular, length 0.6× maximum width. Distal margin with medial emargination and a small process. Dorsally with medium, fine, simple setae scattered over surface; submarginal arc of setae composed of 18-22 long, clavate setae. Ventrally with marginal row of setae composed of lateral and anterolateral long, feathered setae and medial long, bifid setae; ventral surface with five short, spine-like setae near lateral and anterolateral margin.
Right mandible (Fig. 3b, c). Incisors fused. Outer and inner sets of denticles with 4 + 3 denticles and one minute intermediate denticle. Inner margin of innermost denticle with a row of thin setae. Prostheca robust, apically denticulate. Margin between prostheca and mola slightly convex, with a few minute setae. Tuft of setae at apex of mola present.
Left mandible (Fig. 3d, e). Incisors fused. Outer and inner sets of denticles with 4 + 3 denticles and one minute intermediate denticle. Prostheca robust, apically with small denticles and comb-shaped structure. Margin between prostheca and mola 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 (Fig. 3f ). Lingua approx. as long as superlingua. Lingua approx. as broad as long; medial tuft of stout setae well developed; distal half not expanded. Superlingua rounded; lateral margin rounded; fine, long, simple setae along distal margin.
Maxilla (Fig. 3g, h). Galea-lacinia with two simple, robust apical setae under crown. 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 bipectinate, spine-like seta and three medium, simple setae. Maxillary palp 1.4× as long as length of galea-lacinia; 2-segmented. Palp segment II 1.4× length of segment I. Setae on maxillary palp fine and simple, scattered over surface of segments I and II. Apex of last segment rounded, with strong excavation at inner distolateral margin.
Labium (Fig. 3i). Glossa basally broad, narrowing toward apex; shorter than paraglossa; inner margin with five spine-like setae increasing in length distally; apex with two long and one medium, robust, pectinate setae; outer margin with four long, spinelike setae; ventral surface with short, fine, simple and short, spine-like setae. Paraglossa sub-rectangular, curved inward; apex rounded; with three rows of long, robust, distally pectinate setae in apical area and three medium, simple setae in anteromedial area; dorsally with a row of three long, spine-like 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 broad, thumb-like distomedial protuberance; distomedial protuberance 1.0× width of base of segment III; inner and outer margin with short, fine, simple setae; dorsally with two long, spine-like, simple setae near outer margin. Segment III oblong; apex rounded; length 1.4× width; ventrally covered with short to medium, spine-like, simple setae and short, fine, simple setae.
Foreleg ( Fig. 4a-d). Ratio of foreleg segments 1.1:1.0:0.4:0.1. Femur. Length ca. 3× maximum width. Dorsal margin with a row of 15-19 curved, spine-like, apically rounded setae and many long, fine, simple setae and partly a few stout setae near margin; length of setae 0.28× maximum width of femur. Apex rounded; with one pair of curved, spine-like setae and some short, stout setae. Many stout, lanceolate setae scattered along ventral margin; femoral patch poorly developed. Tibia. Dorsal margin with a row of stout, lanceolate, apically rounded setae and fine, simple setae; on apex one larger, lanceolate, apically rounded seta. Ventral margin with a row of curved, spinelike setae, on apex one bipectinate, spine-like seta and a tuft of long, fine, simple setae. Anterior surface scattered with stout, lanceolate setae. Patellotibial suture present on basal 1/3 area. Tarsus. Dorsal margin with a row of small, stout setae and fine, simple setae. Ventral margin with a row of curved, spine-like setae. Tarsal claw with one row of 9-11 denticles; distally pointed; with three stripes; subapical setae absent.
Terga (Fig. 4e, f ). Surface with rows of U-shaped scale bases. Posterior margin of tergum IV with triangular or rounded spines, wider than long.
Gills (Fig. 4g). Present on segments II -VII. Margin with small denticles intercalating both short and medium, fine, simple setae. Tracheae extending from main trunk to inner and outer margins. Gill IV as long as length of segments V and 1/3 VI combined. Gill VII as long as length of segment VIII.
Forewing (Fig. 12a). Pterostigma with three cross-veins, distal one bifurcated and reaching subcostal vein, in the middle a short one not reaching subcostal vein and the proximal one reaching subcostal vein; double intercalary veins generally shorter than distance between corresponding main veins at wing margin.
Hindwing absent. Genitalia (Fig. 13a). Basal segment of gonostylus (unistyliger) with inner margin apically only slightly expanded; segments I and II almost completely fused; constriction at base of segment II; segment III ovoid. Styliger plate between unistyligers poorly developed, distal margin straight.
Etymology. Dedicated to the indigenous Penan people of Borneo. Distribution. Indonesia: Kalimantan, Brunei, Malaysia: Sabah (Fig. 15d). Biological aspects. The specimens were collected in small, shallow forest streams at altitudes from 100 m to 1,450 m, partly in leaf packs.
Ontogenetic association. With genetics, one male imago shares an identical COI sequence with two larvae from the same location (K2P 0%; Table 3).

Labiobaetis operosus group of species (Kaltenbach and Gattolliat 2019)
Following combination of characters: A) dorsal surface of labrum with submarginal arc of feathered setae; B) labial palp segment II with thumb-like or lobed distomedial protuberance; C) seven pairs of gills; D) hindwing pads well developed; E) distolateral process at scape well developed.
Colouration. Head, thorax and abdomen dorsally brown; head and thorax with bright median, dorsal suture, abdominal segment X light brown. Head, thorax and abdomen ventrally light brown, legs light brown with a brown spot medially and apically on femur, caudal filaments light brown.
Antenna (Fig. 6g) with scape and pedicel subcylindrical, with well-developed distolateral process at scape. Labrum (Fig. 5a). Rectangular, length 0.7× maximum width. Distal margin with medial emargination and a small process. Dorsally with medium to long, fine, simple setae scattered over surface; submarginal arc of setae composed of 10-12 long, feath- ered setae. Ventrally with marginal row of setae composed of anterolateral long, feathered setae and medial long, bifid setae; ventral surface with five short, spine-like setae near lateral and anterolateral margin.
Right mandible (Fig. 5b, c). Incisors fused. Outer and inner sets of denticles with 4 + 3 denticles and one minute intermediate denticle. Inner margin of innermost denticle with a 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. 5d, e). Incisors fused. Outer and inner sets of denticles with 4 + 3 denticles and one minute intermediate denticle. Prostheca robust, apically with small denticles and comb-shaped structure. Margin between prostheca and mola 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 present.
Both mandibles with lateral margins almost straight. Basal half with fine, simple setae scattered over dorsal surface.
Maxilla (Fig. 5g). Galea-lacinia with two simple, robust apical seta under crown. 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 bipectinate, spine-like seta and four medium, simple setae. Maxillary palp 1.2× as long as length of galea-lacinia; 2-segmented; palp segment II 1.6× length of segment I; setae on maxillary palp fine and simple, scattered over surface of segments I and II; apex of last segment rounded, with excavation at inner distolateral margin.
Labium (Fig. 5h, i). Glossa basally broad, narrowing toward apex; shorter than paraglossa; inner margin with seven or eight spine-like setae increasing in length distally; apex with two long and one medium, robust, pectinate setae; outer margin with five or six long, spine-like setae; ventral surface with short, fine, simple, scattered setae. Paraglossa sub-rectangular, curved inward; apex rounded; with three rows of long, robust, distally pectinate setae in apical area and two medium, simple setae in anteromedial area; dorsally with a row of three long, spine-like setae near inner margin. Labial palp with segment I 0.9× length of segments II and III combined. Segment I ventrally with short, fine, simple setae. Segment II with large, lobed distomedial protuberance; distomedial protuberance 0.7× width of base of segment III; inner and outer margin with short, fine, simple setae; dorsally with two medium, spine-like, simple setae near outer margin. Segment III slightly pentagonal; apex truncate; length 1.1× width; ventrally covered with short, spine-like, simple setae and short, fine, simple setae.
Hindwing pads (Fig. 6h) well developed. Foreleg (Fig. 6a, b). Ratio of foreleg segments 1.1:1.0:0.4:0.2. Femur. Length ca. 4× maximum width. Dorsal margin with a row of 12-14 curved, spine-like setae; length of setae 0.26× maximum width of femur. Apex rounded, with one pair of curved, spine-like setae and some short, stout setae. Many short, stout, lanceolate setae scattered along the ventral margin; femoral patch absent. Tibia. Dorsal margin with a row of short, stout setae, on apex one longer seta, and a row of short, stout setae close to dorsal margin. Ventral margin with a row of curved, spine-like setae, on apex two spine-like seta and a tuft of long, fine, simple setae. Anterior surface scattered with stout, lanceolate setae. Patellotibial suture present on basal 1/3 area. Tarsus. Dorsal margin with a row of short, stout setae. Ventral margin with a row of curved, spine-like setae. Tarsal claw with one row of 9-13 denticles; distally pointed; with four stripes; subapical setae absent.
Terga (Fig. 6c, d). Surface with irregular rows of U-shaped scale bases and scattered fine, simple setae. Posterior margin of tergum IV with rounded or triangular spines, wider than long. Gills (Fig. 6e). Present on segments I -VII. Margin with small denticles intercalating fine simple setae. Tracheae extending from main trunk to inner and outer margins. Gill I as long as length of ½ segment II. Gill IV as long as length of segments V and 1/3 VI combined. Gill VII as long as length of segments VIII and 1/3 IX combined.
Forewing (Fig. 12b). Pterostigma with seven cross-veins, only two proximal ones reaching subcostal vein; double intercalary veins shorter than distance between corresponding main veins at wing margin. Genitalia (Fig. 13b). Basal segment of gonostylus (unistyliger) with inner margin apically slightly expanded; segments I and II almost completely fused; constriction at base of segment II; segment III quadrangular. Styliger plate between unistyligers trapezoidal, distal margin slightly concave.
Distribution. Indonesia: Kalimantan, Malaysia: Sabah, Brunei (Fig. 15b). Biological aspects. The specimens were collected at altitudes between 100 m to 300 m, partly on bottom gravel, rock surface or submerged wood in stream run or riffles.

Distribution
The material treated in this study was collected in ca. 20 localities in Borneo, which belong to four different areas, one area in Brunei, two in Sabah (Malaysia), and one in Kalimantan (Indonesia) (Fig. 15). There are still many regions in Borneo as well as in Southeast Asia in general where no sampling of mayflies has yet been done and many species known to date are from a single population only. This implies that the diversity and the distribution must be considered as very preliminary. However, the distribution of the Labiobaetis species seems to be very diverse. Labiobaetis moriharai  has a large distribution (continental and insular) and the other species are endemic to Borneo (Fig. 15). In terms of altitude, the Labiobaetis species of Borneo were found from sea level to mountain areas up to 1,450 m. The GPS coordinates of the locations of examined material are given in Table 2.

Genetics
COI sequences were obtained from two of the three new species (Table 1) as well as from the two other species. In two cases (L. penan sp. nov. and L. borneoensis) a male imago could be associated with larvae: the COI sequences of the two ontogenetic stages were identical. The genetic distances (K2P) between the species in Borneo are between 19% and 25%, and therefore much higher than 3.5%, which is generally considered as a likely maximal value for intraspecific divergence (Hebert et al. 2003, Ball et al. 2005, Zhou et al. 2010 (Table 3). Very limited genetic distances (between 0% and 1%) were found between specimens of the same species, as in L. penan sp. nov., L. borneoensis, and partly in L. bakerae sp. nov. The only exception is L. bakerae sp. nov.: in this species one larva has a distance of 6% from the two others, despite being collected in the same area and having no morphological difference.

Discussion
For the assignment of the new species to Labiobaetis we are referring to Kluge and Novikova (2014), Müller-Liebenau (1984a) and McCafferty and Waltz (1995). Labiobaetis is characterized 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 (Fig. 6g); maxillary palp two segmented with excavation at inner distolateral margin of segment II, excavation may be poorly developed or absent (Kaltenbach and Gattolliat 2019: figs 1o-q); labium with paraglossae widened and glossae diminished; labial palp segment II with distomedial protuberance (Kaltenbach and Gattolliat 2019: fig. 1g-n). All these characters vary and may be secondarily lost (Kluge and Novikova 2014). The concept of Labiobaetis is also based on additional characters, summarized and discussed in Gattolliat (2018, 2019). Two of the three new species (L. bakerae sp. nov., L. penan sp. nov.) belong to the rather large sumigarensis group and the third one (L. dayakorum sp. nov.) to the operosus group (Müller-Liebenau and Hubbard 1985, Kaltenbach and. Labiobaetis bakerae sp. nov. and L. penan sp. nov. can be distinguished by the number of clavate setae forming an arc on the dorsal surface of the labrum (13-15 in L. bakerae sp. nov., ca. 22 in L. penan sp. nov.), the number of setae at the dorsal margin of the femur (8-11 in L. bakerae sp. nov., 15-19 in L. penan sp. nov.) and the presence of split tips of the marginal spines of the paraproct in L. penan sp. nov. (Fig. 4h). Labiobaetis bakerae sp. nov. is morphologically closely related to L. jacobusi Kubendran and Balasubramanian from India and L. geminatus (Müller-Liebenau and Hubbard) from Sri Lanka (Müller-Liebenau and Hubbard 1985, Kubendran et al. 2015. From the first species L. bakerae sp. nov. is different in the shape of the labial palp, the longer maxil- lary palp (compared to galea-lacinia) and the shorter medial tuft of the hypopharynx (Fig. 1f-h; Kubendran et al. 2015: figs 44, 47, 48). From the second species, L. bakerae sp. nov. differs by the very poorly developed distolateral scape process (rather well developed in L. geminatus), the shape of the labial palp (distomedial protuberance of seg-ment II more slender and with a clearly concave distal outer margin in L. geminatus), the distinct denticles between prostheca and mola of the left mandible (hardly visible in L. geminatus), the maxillary palp with a pronounced distolateral excavation (less developed in L. geminatus) and the shape of the triangular spines at anterior margin of tergum IV (generally much wider than long; as wide as long in L. geminatus, with pronounced points) (Figs 1b, g, h, 2d, g;Müller-Liebenau and Hubbard 1985: figs 5b, d, e, g, 22). The third new species, L. dayakorum sp. nov., is morphologically close to L. paraoperosus Kaltenbach and Gattolliat from Sumatra, but differentiated in the following characters: thorax and abdomen of L. dayakorum sp. nov. dorsally uniform brown (Fig. 11a) and with a distinct pattern in L. paraoperosus (Fig. 11b), shape of the labial palp (Figs 5h, 7d), denticles of the right mandible (4+1+3 in L. dayakorum sp. nov., 4+3 in L. paraoperosus), and size and shape of the hindwing pads (Figs 6h, 7e).
In general, the genetic distances between the different species of Labiobaetis are rather high in Borneo, between 19% and 25% (K2P , Table 3), which is in line with the genetic distances found in New Guinea (avg. 22%; Kaltenbach and Gattolliat 2018) and Indonesia (11%-24%; Kaltenbach and Gattolliat 2019). Ball et al. (2005) reported a mean interspecific, congeneric distance of 18% for mayflies from the United States and Canada.
The intraspecific distances are mostly very low as expected, ranging from 0% to 1% (K2P). This result is certainly biased as it is based on a limited number of sequenced specimens per species, which were mostly from a single population. But there is one exception, L. bakerae sp. nov., where one specimen has an intraspecific distance of 6% to another specimen of the same population as well as to a specimen of another population. Compared to the usual distances between different Labiobaetis species in that region and because there is no morphological difference, this distance is surprising, but can be still considered as intraspecific. Ball et al. (2005) also reported a case with 6% intraspecific distance in a mayfly in North America and intraspecific K2P distances of more than 3.5% are also not uncommon within Plecoptera (Gill et al. 2015, Gattolliat et al. 2016.
In addition to the five species cited in this paper, we obtained two additional COI sequences with clearly interspecific genetic distance to other specimens with similar morphology. In one case, one specimen is highly similar to L. borneoensis, but with a K2P distance of 16%. In the other case, one specimen is morphologically very close to L. penan sp. nov. and partly damaged, but with a K2P distance of 22%. Because of the limited amount of material and the absence of morphological support, they have to remain species hypotheses for now without further treatment in this paper. Additional material will be necessary to confirm their status in the future. We also have specimens of two additional undescribed species, which have some morphological differences to their closest species. Unfortunately, the material is insufficient or partly damaged and we could not extract DNA. We therefore also refrain to describe them.
The number of sampled localities and different habitats is still very limited and there are large regions, especially in mountainous areas, without any collection activities so far (Fig. 15). In addition, we have four species hypotheses based on genetics only or based on morphological differences without genetics, which may be confirmed as valid species in the future. Therefore, we may assume that the number of Labiobaetis species in Borneo will continue to increase substantially with further collections in the future. Thereby, inter-disciplinary collaborations between ecologists and taxonomists may contribute to the discovery of new species in these remote, tropical regions (Baker et al. 2019).