Three new species of caddisflies (Trichoptera, Hydroptilidae, Polycentropodidae, Leptoceridae) from Khon Phapheng Waterfall, the Mekong River, Laos

Abstract The Mekong River is a hotspot area for freshwater biodiversity, but caddisfly diversity is largely understudied. Three new species of caddisflies from three different families are described and illustrated from Khon Phapheng Waterfall, the Mekong River, Laos; Orthotrichiachoengthongi Malicky & Laudee, sp. nov. (Hydroptilidae), Pseudoneureclipsiskhonphaphengensis Malicky & Thamsenanupap, sp. nov. (Polycentropodidae), and Setodeskarrilai Malicky & Laudee, sp. nov.Orthotrichiachoengthongi Malicky & Laudee, sp. nov. can be differentiated from the most similar OrthotrichiatritonMalicky 2008 by the specific shape of segment X which is long and tubular, pointed apically and curved inward then immediately upward in dorsal view. Pseudoneureclipsiskhonphaphengensis Malicky & Thamsenanupap, sp. nov. differs from the similar P.kaineus Malicky & Bunlue in Malicky et al. 2004 by the shape of the inferior appendages that are usually broad, almost circular in lateral view. Setodeskarrilai Malicky & Laudee, sp. nov. is mainly different to S.omphale Malicky & Changthong in Malicky et al. 2004 by the distal part of segment X which has a brush-like process.


Introduction
The Mekong River is one of the main rivers in Southeast Asia, flowing from the Tibetan Plateau through several countries to the South China Sea. A large human population in the Southeast Asian region depends on it as a source for subsistence (Campbell 2009; Mekong River Commission 2020). The ecological system in the Mekong River is also varied as it runs from the Tibetan Plateau through to the South China Sea, and the lower Mekong basin is a hotspot area for aquatic biodiversity (Campbell 2009). At the place where the Mekong River zigzags from the Korat Plateau, which is in the northeastern Thailand, through the Tonle Sab basin, a broad valley to the east of the Khorat Plateau, the river level drops several meters over many waterfalls, such as Li Phi fall and Khon Phapheng waterfall (Campbell 2009).
The aim of this research study was to describe new species of Trichoptera from the falls in the Mekong River, and thus to provide a valuable contribution to the knowledge on South-East Asian caddisfly diversity.

Materials and methods
Adult stage caddisfly specimens were collected with a UV pan light trap (12 V, 10 W) set beside Khon Phapheng waterfall, Mekong River, at night. The caddisfly specimens were preserved in 70% ethanol. For identifying to species level, the male genitalia of the caddisflies were observed under a stereomicroscope. The male genitalia were dissected and the muscle tissues macerated by heating in 10% KOH at 60 °C for 30-60 minutes then soaked in a detergent solution before being transferred back to 70% ethanol. The genitalia of caddisflies were identified to species level after Malicky (2010). For the new species of caddisflies, drawings of the male genitalia were made in pencil using a compound microscope equipped with a drawing tube, and the drawings were used to produce the final vector graphics in Adobe Illustrator software.
Holotypes are preserved and stored in the Princess Maha Chakri Sirindhorn Natural History Museum, Prince of Songkla University, Hat Yai Campus, Hat Yai district, Songkhla province, Thailand (PSUNHM). Some paratypes are deposited in the collection of Hans Malicky (CHM), the Clemson University Arthropod Collection (CUAC), and the National Museum, Prague, Czech Republic (NMPC). Terminology for genitalia structures for different genera follows Wells (1979) and Marshall (1979) for the genus Orthotrichia, Oláh and Johanson 2010 for the genus Pseudoneureclipsis, and Laudee and Malicky 2016 for the genus Setodes.  Diagnosis. The new species is similar to Orthotrichia triton Malicky, 2008 from Borneo in the general shape of the male genitalia. However, it can be differentiated in that the shape of tergum X of Orthotrichia choengthongi Malicky & Laudee, sp. nov. is long and tubular, pointed apically and curved inwards then abruptly upwards in dorsal view; however, is stout basally then curved slightly downward in O. triton. Moreover, the united inferior appendages of the new species are collectively trilobed; but bilobed in O. triton.  1C). Posterolateral processes of tergum IX asymmetrical; left process shorter, bilobed, each lobe pointed apically; right process longer, tubular, bifurcate apically with acute apices in dorsal view (Fig. 1A); in left lateral view, directed caudad, short, pointed apically (Fig. 1B); in right lateral view (Fig. 1C), somewhat triangular, short. Dorsal spine of tergum IX lanceolate, curved upward, pointed apically in dorsal view (Fig. 1A). Tergum X long tubular, pointed apically, curved mesad then abruptly upward in dorsal view (Fig. 1A); shaped nearly as isosceles triangle, pointed apically in right lateral view. United inferior appendages trilobed, asymmetrical, each lobe shaped as isosceles triangle and acute apically, with middle lobe smallest and closer to right lobe in ventral view, left lobe thicker than right one in lateral view (Fig. 1B, C). Pair of tubular processes of inferior appendages long, slender, each with long seta apically ( Fig. 1B-D). Paramere spine long, acute apically in ventral view (Fig. 1D). Phallus, long, enlarged and bilobed apically, with titillator encircling segment 1 time tightly against it near the middle (Fig. 1E).

Orthotrichia choengthongi
Etymology. The species epithet honors Asst. Prof. Dr Suchart Choengthong, Prince of Songkla University, Surat Thani Campus. Diagnosis. The male genitalia of P. khonphaphengensis sp. nov. are similar to those of P. kaineus Malicky & Bunlue in Malicky et al. 2004 from Thailand. However, P. khonphaphengensis sp. nov. is strikingly different from P. kaineus and the other species of the genus by the usually broad, almost circular inferior appendages with 1.5 × longer than its width in lateral view, while the inferior appendages of P. kaineus are oval and 3.2 × longer than its width in lateral view. In addition, the phallus of P. khonphaphengensis sp. nov. bearing the hooked phallic sclerite which are lacking in P. kaineus.
Etymology. The species is named for the type locality, Khon Phapheng waterfall.   Malicky et al. 2004 from Thailand. Both species share similar characteristics of five lobes of each inferior appendage. However, the most prominent difference is the structure of segment X, i.e., the distal part of segment X in S. karrilai sp. nov. has a brush-like process, visible in lateral and dorsal views, which is lacking in S. omphale. In addition, the phallus of S. karrilai sp. nov. has a pair of very long, thin, pointed parameres with distal 1/3 bent and twisted meso-upward; whereas the parameres of S. omphale are curved downward.

Setodes karrilai
Description. Length of each male forewing 6.5-7.0 mm (n = 18); color in alcohol of head, thorax, forewings, and abdomen yellow; femora and tibiae brown or dark brown; wings clear and transparent, yellowish with brown veins.
Male genitalia as in Figure 3. Segment IX somewhat triangular with long sinuous edges posteriorly, convex anteriorly in lateral view (Fig. 3B); square in ventral view (Fig. 3C). Segment X long, slender, divided into basal segment and apical segment (Fig. 3A, B); basal segment mostly tubular with shallow and broad excision apically, apical segment slender, tubular with brush of straight hairs at posterior end in dorsal view (Fig. 3A); in lateral view, basal segment tubular, apical segment thicker apically and with apical brush (Fig. 3B). Preanal appendages very small, leaf-like with scattered setae (Fig. 3A, B). Inferior appendages each five-lobed (Fig. 3B); dorsal lobe long, tubular and erect basally then curved caudad, two finger-like mesal lobes tubular and with upper mesal lobe longer than lower mesal lobe, basoventral lobe small and knotlike, ventral lobe very long and sword-like with scattered setae in lateral view (Fig. 3B). Phallus long, slender, bent downwards with pair of very long, thin, and pointed parameres with distal 1/3 bent and twisted meso-upward in lateral view (Fig. 3D).
Etymology. The species epithet honors Assoc. Prof. Dr Seppo Karrila, Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus.

Discussion
Three new species of caddisflies were described from Khon Phapheng waterfall, where the whole Mekong River drops 30 meters over the waterfalls. These species are Orthotrichia choengthongi sp. nov., Pseudoneureclipsis khonphaphengensis sp. nov., and Setodes karrilai sp. nov. The three new species are potamon species that live in large rivers. The substrate is dominated by bedrocks, boulders, and sand ( Fig. 4A-D). There is no previously reported species of Orthotrichia in Laos. However, more than 50 species of the genus have been reported from adjacent countries (Malicky 2010), thus we expect ad-  and Dudgeon 1990 reported that the larvae are often found under rocks in streams. The Orthotrichia spp. in South-East Asia show restricted regional distribution, as only 9 of the 54 species are reported to be widely distributed (Malicky and Chantaramongkol 2007;Malicky 2010).
The genus Pseudoneureclipsis has been previously reported from Laos, i.e., six species were known; thus including P. khonphaphengensis sp. nov. there are now seven species of Pseudoneureclipsis recorded from Laos. In addition to the Laotian species, there are three species (P. romosa, P. usia and P. kaineus) with wider distribution in South-East Asia (Malicky 2010). Pseudoneureclipsis romosa and P. kaineus are potamon species as they were found from the Mekong River (Laudee and Malicky 2017) but Thapanya et al. 2004 reported that Pseudoneureclipsis spp. are found also in mountain streams in Doi Suthep-Pui and Doi Inthanon National Parks, Chiang Mai Province, Thailand. The larvae of the genus Pseudoneureclipsis live on undersides of stones in shallows of fast flowing water (Dudgeon 1999).
In addition to the description of a new species, for Setodes karrilai sp. nov. we give data on its distribution, as it was collected from several provinces in Laos, not only from the Khon Phapheng waterfall. Thus, it is a widespread species in the Mekong River, which is found from central to southern Laos. The genus Setodes is represented by twenty Laotian species, among the more than eighty species that have been reported from South-East Asia (Malicky 2010) and the 285 species reported from the Oriental Biogeographic Region (Morse 2021). The genus Setodes inhabits a variety of habitats, such as waterfalls, small streams, and rivers (Malicky and Chantaramongkol 2006;Laudee and Malicky 2016). In addition, Setodes larvae have been reported to burrow into sand deposits of riffle areas (Merill and Wiggins 1971).