﻿First contribution to Labiobaetis Novikova & Kluge in Cambodia (Ephemeroptera, Baetidae), with description of two new species

﻿Abstract Material collected in 2018 in Cambodia gives us first insights into the diversity of Labiobaetis Novikova & Kluge, 1987 in this country. No species has been reported so far. We identified two new species using a combination of morphology and genetic distance (COI, Kimura 2-parameter). They are described and illustrated based on their larvae. A key to all Labiobaetis species of continental Southeast Asia is provided. The interspecific K2P distance between the two new species is 20–21%, the intraspecific distance of one of them is 1%. The total number of Labiobaetis species worldwide is augmented to 156.


Introduction
The genus Labiobaetis Kluge, 1987 (Novikova andKluge 1987) is one of the richest genera of mayflies with 154 previously described species (Barber-James et al. 2013;Sivaruban et al. 2022). The distribution of Labiobaetis is nearly worldwide, exept for the Neotropical realm, New Zealand, and New Caledonia; its main diversity is found in Southeast Asia (Kaltenbach and Gattolliat 2019 and New Guinea Gattolliat 2018, 2021;. The history and concept of the genus Labiobaetis were recently summarized in detail (Shi and Tong 2014;Kaltenbach and Gattolliat 2018). Together with Pseudopannota Waltz & McCafferty, 1987, it belongs to the tribe Labiobaetini, established by Kluge and Novikova (2016) based on a unique combination of imaginal and larval characters. Labiobaetis is part of Baetidae, the family with the highest species diversity among mayflies, comprising over 1160 species in 118 genera (Sartori and Brittain 2015;Jacobus et al. 2019;updated), which is approximately one-third of all mayfly species worldwide.
In the past years, the diversity of Labiobaetis in Southeast Asia was intensely studied with focus on the archipelagos of Indonesia (including the whole of Borneo) and the Philippines (Kaltenbach and Gattolliat 2019). Many new species were described based on morphological and molecular evidence. This contribution will shift our focus to continental Southeast Asia, starting with a first contribution to the knowledge of Labiobaetis in Cambodia. Further studies of the genus in the region are in preparation.
Cambodia is located in the southern part of the Indochinese Peninsula in Southeast Asia, bordering Laos in the northwest, Thailand in the north and the east, and Vietnam in the south and the west, and with a long coastline along the Gulf of Thailand in the west. It is geographically characterized by large central wetlands around Tonle Sap Lake, and by the upper reaches of the Mekong River delta towards Vietnam, surrounded by uplands and low mountains. Cambodia's rich biodiversity is based on its seasonal tropical rainforests.
So far, the specific diversity of Labiobaetis and of Baetidae in general in Cambodia was unknown, despite a first study on mayflies including the first general report of the genus in the country (Chhorn et al. 2020). Some work was done in the neighbouring Vietnam, including a key for the identification of Ephemeroptera (Soldán 1991;Mekong River Commission 2006), and several studies on Baetidae were recently done in the neighbouring Thailand (e.g. Kluge and Suttinun 2020;Suttinun et al. 2020Suttinun et al. , 2021Suttinun et al. , 2022. Intensive exchange between these faunas is likely, as there are only rather low mountain chains with large corridors inbetween, and no other barriers between them. In China, an important study on Labiobaetis was done by Shi and Tong (2014). In the present study, we describe two new species of Labiobaetis from Cambodia based on larval stage.

Materials and methods
Materials used in the study were obtained as part of the Cambodia Entomology Initiative aquatic insect ecological study expeditions (Freitag et al. 2018;Chhorn et al. 2020). The specimens were preserved in 96% ethanol.
Dissection of larvae was done in Cellosolve (2-Ethoxyethanol) with subsequent mounting on slides with Euparal liquid, using an Olympus SZX7 stereomicroscope.
The DNA of part of the 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 2020 for details). Sequencing was done with Sanger's method (Sanger et al. 1977). The genetic variability between specimens was estimated using Kimura-2-parameter distances (K2P;Kimura 1980), calculated with the program MEGA 7 (Kumar et al. 2016;http://www.megasoftware.net).
GenBank accession numbers are given in the sections of examined material. Drawings were made with an Olympus BX43 microscope. To facilitate the determination of species and the comparison of important structures, we partly use a combination of dorsal and ventral aspects in one drawing. Explanations are given in   fig. 1).
Photographs of larvae were taken using a Canon EOS 6D camera and processed with the programs Adobe Photoshop Lightroom (http://www.adobe.com) and Helicon Focus v. 5.3 (http://www.heliconsoft.com). Photographs were subsequently enhanced with Adobe Photoshop Elements 13.

Definition of groups and description of their characters
Labiobaetis operosus group (L. brao sp. nov.) and sumigarensis group (L. kui sp. nov.) were defined and characterized in Kaltenbach and Gattolliat (2019) and .
Colouration (Fig. 5a, b). Head, thorax and abdomen dorsally grey-brown, with pattern as in Figure 6a. Abdominal tergits I and X brighter. Fore protoptera light greybrown with dark striation. Head ventrally brownish, thorax and abdomen ventrally light grey-brown. Legs ecru to light brown, femur with grey-brown distomedial spot, apex and dorsal margin grey-brown. Caudalii grey-brown.
Antenna ( Fig. 2h) with scape and pedicel subcylindrical, with well-developed distolateral process at scape. Labrum (Fig. 1a). Subrectangular, length 0.65× 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 9-11 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 three short, spine-like setae near lateral and anterolateral margin.
Right mandible (Fig. 1b,c). Incisor and kinetodontium fused. Incisor with five denticles; kinetodontium with three denticles, inner margin of innermost denticle with a row of thin setae. Prostheca robust, apically denticulate. Margin between prostheca and mola slightly convex, with few minute denticles. Tuft of setae at apex of mola present.
Left mandible (Fig. 1d, 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 slightly convex, with minute denticles. 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. 1g). 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 spine-like seta and three long, simple setae. Maxillary palp 1.3× as long as length of galea-lacinia; 2-segmented; palp segment II approximately as long as segment I; setae on maxillary palp fine, simple, scattered over surface of segments I and II; apex of last segment rounded, with excavation at inner distolateral margin. Labium (Fig. 1i,j). Glossa basally broad, narrowing toward apex; shorter than paraglossa; inner margin with 8-10 spine-like setae, distalmost seta much longer than other setae; apex with three medium and one short, robust setae; outer margin with ca 6 spine-like setae; ventral surface with fine, simple, scattered setae. Paraglossa subrectangular, 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 four long, spine-like, simple setae near inner margin. Labial palp with segment I 0.8× length of segments II and III combined. Segment I ventrally with short, fine, simple setae. Segment II with broad, extended, thumb-like distomedial protuberance; distomedial protuberance 0.7× width of base of segment III; ventral surface with short, fine, simple setae; dorsally with a row of three long, spine-like setae near outer margin. Segment III rather oblong, apically truncate; length 1.1× width; ventrally covered with short, spine-like, simple setae and short, fine, simple setae.
Hind protoptera well developed. Foreleg (Fig. 2a, b). Ratio of foreleg segments 1.4:1.0:0.6:0.2. Femur. Length ca 3× maximum width. Dorsal margin with 11-25 curved, short, spine-like setae, often one seta additionally near margin in basal area; length of setae 0.14× maximum width of femur. Apex rounded, with a spine-like seta and some short, stout setae. Many stout, lanceolate setae scattered along ventral margin; femoral patch absent. Tibia. Dorsal margin with row of short, stout, apically rounded setae, and some fine, simple setae; many more stout, apically rounded setae along dorsal margin; on apex one seta of same type. Ventral margin with row of short, curved, spine-like setae, on apex some longer setae and a tuft of fine, simple setae. Anterior surface with row of stout, lanceolate setae near ventral margin. Patellatibial suture present on basal 1/3 area. Tarsus. Dorsal margin with row of short, stout setae and some fine, simple setae. Ventral margin with row of curved, spine-like setae. Claw with one row of 7-10 denticles; distally pointed; with ca four stripes; subapical setae absent.
Middle and hind legs. As foreleg, but with reduced or rudimentary femoral patch on middle femur, and reduced or well developed on hind femur. Terga (Fig. 2c). Surface with irregular rows of U-shaped scale bases. Posterior margin of tergum IV with spines varying between mostly triangular to mostly rounded, wider than long.
Tergalii (Fig. 2e). Present on segments I-VII. Margins with small denticles intercalating fine simple setae. Tracheae extending from main trunk to inner and outer margins. Tergalius I ca 2/3 length of segment II. Tergalius IV as long as length of segments V and 1/2 VI combined. Tergalius VII as long as length of segment VIII. Paraproct (Fig. 2f, g). Distally not expanded, with ca 34 stout, marginal spines, and additional row of minute spines along inner, proximal margin. Surface scattered with U-shaped scale bases and fine, simple setae. Cercotractor with numerous small, marginal spines.
Etymology. The new species is dedicated to the indigenous Brao people from northeastern Cambodia.
Colouration (Fig. 5c, d). Head, thorax, and abdomen dorsally uniform brown. Head, thorax, and abdomen ventrally light brown. Legs light brown; femur with a brown medial spot, darker on ventral margin, dorsal margin and apex brown. Caudalii light brown.
Antenna (Fig. 4g) with scape and pedicel subcylindrical, without distolateral process at scape. Labrum (Fig. 3a, b). Rectangular, length 0.7× 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 16-18 long, clavate setae. Ventrally with marginal row of setae composed of anterolateral long, feathered setae and medial long, bifid setae; ventral surface with ca three short, spine-like setae near lateral and anterolateral margin.
Right mandible (Fig. 3c, d). Incisor and kinetodontium fused. Incisor with five denticles; kinetodontium with three denticles, inner margin of innermost denticle with a row of thin setae. Prostheca robust, apically denticulate. Margin between prostheca and mola slightly convex. Tuft of setae at apex of mola present.
Left mandible (Fig. 3e, f ). Incisor and kinetodontium fused. Incisor with five denticles; kinetodontium with three denticles. Prostheca robust, apically with small denticles and comb-shaped structure. Margin between prostheca and mola straight, with minute denticles towards subtriangular process. 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. 3h). 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. Medially with one pectinate, spine-like seta and three or four medium, simple setae. Maxillary palp 1.2× as long as length of galea-lacinia; 2-segmented; palp segment II 1.2× length of segment I; setae on maxillary palp fine, simple, scattered over surface of segments I and II; apex of last segment rounded, with excavation at inner distolateral margin. Labium (Fig. 3i). Glossa basally broad, narrowing toward apex; shorter than paraglossa; inner margin with ca six spine-like setae increasing in length distally; apex with two long and one medium, robust, pectinate setae; outer margin with ca four spinelike setae; ventral surface with fine, simple, scattered setae. Paraglossa subrectangular, curved inward; apex rounded; with three rows of long, robust, distally pectinate setae in apical area and a row of 2-4 medium, simple setae in anteromedial area; dorsally with a row of four or five long, spine-like, simple setae near inner margin. Labial palp with segment I 0.8× length of segments II and III combined. Segment I ventrally with short, fine, simple setae. Segment II with extended, slightly hooked, thumb-like distomedial protuberance; distomedial protuberance 0.7× width of base of segment III; ventral surface with short, fine, simple setae; dorsally with two long, spine-like, simple setae near outer margin. Segment III slightly pentagonal; apex rounded, inner apical margin slightly concave; length subequal to width; ventrally covered with short, spine-like, simple setae and short, fine, simple setae.
Hind protoptera absent. Foreleg (Fig. 4a-c). Ratio of foreleg segments 1.3:1.0:0.4:0.2. Femur. Length ca 4× maximum width. Dorsal margin with 10-15 long, curved, spine-like setae; length of setae 0.23× maximum width of femur. Apex rounded, with a pair of long, curved, spine-like setae and some short, stout setae. Many stout, lanceolate setae scattered along ventral margin; femoral patch absent. Tibia. Dorsal margin with row of short, stout, apically rounded setae, on apex one longer, spine-like seta. Ventral margin with row of short, curved, spine-like setae, on apex some longer setae and a tuft of fine, simple setae. Anterior surface scattered with stout, lanceolate setae near ventral margin. Patellatibial suture present on basal 1/3 area. Tarsus. Dorsal margin with some fine, simple setae. Ventral margin with row of curved, spine-like setae. Claw with one row of nine or ten denticles; distally pointed; with ca. five stripes; subapical setae absent. Terga (Fig. 4d). Surface with irregular rows of U-shaped scale bases and scattered fine, simple setae. Posterior margin of tergum IV with triangular spines, wider than long.
Tergalii (Fig. 4e). Present on segments II-VII. Margins with small denticles intercalating fine simple setae. Tracheae extending from main trunk to inner and outer margins. Tergalius IV as long as length of segments V and 1/3 VI combined. Tergalius VII as long as length of segment VIII. Paraproct (Fig. 4f ). Distally slightly expanded, with 33-38 stout, marginal spines. Surface scattered with U-shaped scale bases, fine, simple setae and micropores. Cercotractor with numerous small, marginal spines.
Etymology. The new species is dedicated to the indigenous Kui people from northeastern Cambodia.

Genetics
COI sequences were obtained from both new species (see type material sections). The genetic distance (K2P) between them is 20-21%, 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). A very limited genetic distance of 1% was found between two specimens of L. kui sp. nov., as expected for the same location.

Assignment to Labiobaetis and to species groups
For the assignment of the new species to Labiobaetis we refer to Kluge and Novikova (2014), Müller-Liebenau (1984), and McCafferty and Waltz (1995). Labiobaetis is characterized by a number of characters, some of which are not found in other taxa (Kluge and Novikova 2014): antennal scape sometimes with a distolateral process (Fig. 2h); maxillary palp two segmented with excavation at inner distolateral margin of segment II, excavation may be poorly developed or absent (Figs 1g, 3h); labium with paraglossae widened and glossae diminished; labial palp segment II with distomedial protuberance (Figs 1i, 3i). 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 by Gattolliat (2018, 2019). The morphological groups within Labiobaetis are primarily a working tool but could also serve as a basis for future studies on the generic or subgeneric delimitations and phylogeny of this genus. The inclusion of nuclear gene sequences may prove that some of them are natural groups. The two species in Cambodia belong to different groups, one to the operosus goup and one to the sumigarensis group. The operosus group is mainly characterized by A) labrum dorsally with submarginal arc of feathered setae; B) distolateral process at scape well developed; C) seven pairs of tergalii; D) hind protoptera well developed (see Kaltenbach et al. 2020: 40). The sumigarensis group is mainly characterized by A) labrum dorsally with submarginal arc of clavate setae; B) left mandible without setae at mola apex; C) six pairs of tergalii; D) hind protoptera absent; E) colour dorsally uniform brown (see Kaltenbach et al. 2020: 63).
These groups are widespread and highly diversified in Asia. Species of the operosus group are also known from India, Malaysia, Indonesia, and the Philippines; and species of the sumigarensis group from India, Sri Lanka, Malaysia, Indonesia, Brunei, China, Taiwan, and the Philippines (Müller-Liebenau 1984;Müller-Liebenau and Hubbard 1985;Kang et al. 1994;Shi and Tong 2014;Kubendran et al. 2015;Gattolliat 2019, 2020;Sivaruban et al. 2022). None of these groups are known from New Guinea Gattolliat 2018, 2021;.

Genetic distance
The genetic distances between the two new species of Labiobaetis in the Cambodia (20-21%, K2P) is rather high, which is in line with the genetic distances found in Indonesia (11-24%; Kaltenbach and Gattolliat 2019), Borneo (19-25%; Kaltenbach and Gattolliat 2020), and the Philippines (15-27%; . Ball et al. (2005) reported a mean interspecific, congeneric distance of 18% for mayflies from the United States and Canada.
The number of sampled localities and different habitats is until now very limited and the vast majority of the country was not covered by collection activities so far (Fig. 6b). Therefore, we can expect that the number of Labiobaetis species in Cambodia will substantially increase with further collections.