Trichosetodes Ulmer is a genus of Trichoptera in the family Leptoceridae, which can be identified by the crescent shape of the abdominal segment IX from the lateral view, finger-like preanal appendages and a tuft of long hairs anterodorsally on abdominal segment IX (Schmid 1987; Gibon 1991; Malicky 2006a; Malicky and Graf 2020). Fifty-five species have been described worldwide. However, while Trichosetodes spp. has been mainly found from Oriental regions, three and thirteen species have been reported from the East Palearctic and Afrotropical regions, respectively (Malicky 2010; Mey and De Moor 2019; Kimura and Kuranishi 2020; Laudee et al. 2020; Malicky and Graf 2020; Morse 2023). In Asia, T. atisudhara Schmid, 1987, T. compositus Martynov, 1936, and T. pandrosus Malicky, 2006 were reported from Nepal (Malicky 2006b), and T. japonicus Tsuda, 1942 from Japan and the Korean Peninsula (Kimura and Kuranishi 2020; Park and Kong 2020). Yang et al. (2016) reported that seven species of Trichosetodes, namely T. bicornis Yang & Morse, 2000, T. falcatus Yang & Morse, 2000, T. insularis Schmid, 1987, T. lasiophyllus Yang & Morse, 1989, T. phylloideus Yang & Morse, 2000, T. rhamphodes Yang & Morse, 2000 and T. serrayus Yang & Morse, 2000 were found from several parts of China. In India, thirteen species of Trichosetodes have been recorded from several areas (Saini et al. 2001). In Southeast Asia, Malicky (2010) reported that 12 species of Trichosetodes were collected from Thailand, Laos, Peninsular Malaysia, Sumatra (Indonesia) and Java (Indonesia). Oláh (2013) described two new species of Trichosetodes, namely T. harmas Oláh, 2013 and T. sotet Oláh, 2013 from Vietnam. In Myanmar, Malicky and Laudee (2017) described a new species of Trichosetodes, T. asphor Malicky & Laudee, 2017, which was found from Taninthayi Division. Recently, T. kampongspeuensis Malicky & Kong, 2020 in Laudee et al. (2020) from Cambodia was described.

Presented herein is a report encompassing selected findings derived from an extensive investigation into the caddisfly biodiversity within the Mekong River and its network of tributaries. This study led to the collection and subsequent description of three distinct Trichosetodes species originating from the Katieng Waterfall, situated within the confines of Cambodia’s Ratanakiri Province.

Male adult caddisfly specimens were collected overnight with a UV pan light trap (12V, 10W) near Katieng Waterfall and its stream, in Ratanakiri Province, Cambodia. The adult Trichoptera specimens were collected and preserved in 70% ethanol. Adult male genitalia of the new species were excised and macerated in 10% KOH at 60 °C for 30–60 min. The male genitalia of the new species were drawn with pencil while using a compound microscope with a drawing tube, and then final vector-graphic illustrations were prepared from the pencil templates with Adobe Illustrator 2023 software.

The holotypes and some paratypes of the new species were stored in 70% ethanol and were deposited at 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, Lunz am See, Austria (CHM), the National Museum, Prague, Czech Republic (NMPC) and the Clemson University Arthropod Collection, Clemson, South Carolina, USA (CUAC). Terminology of structure of genitalia follows Yang and Morse (2000).

The DNA was extracted from the ethanol-preserved tissue of the specimens, and purified using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The region of mitochondrial DNA (mtDNA) coding 16S rRNA region was amplified by a polymerase chain reaction (PCR) using the primer set 16S rRNA: 5’- TRA CYG TRC AAA GGT AGC -3’ and 5’- CCG GTY TRA ACT CAR ATC ATG T -3’ (Takenaka et al. 2023). Regarding each reaction, 1.0 μL of 10× Ex Taq buffer, 0.8 μL dNTP mixture (included 25 mM MgCl₂), 0.05 μL of 5 U/μL Ex Taq polymerase (TAKARA, Shiga), 0.25 μL of each primer and 2.0 μL of extracted DNA for in total 10 μL were applied. The PCR protocol was: 94 °C for 2 min; 35× (94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min); and 72 °C for 3 min. The PCR products were purified using ExoSAP-IT Express (Thermo Fisher Scientific K.K., Tokyo, Japan). Purified DNA fragments were sequenced directly by an automated method using a BigDye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an automated DNA Sequencer (ABI 3130 or 3130xl DNA Analyzer; Perkin Elmer/Applied Biosystems).

All sequences obtained have been submitted to the DNA data bank of Japan (DDBJ database) (GenBank accession numbers: Trichosetodes carmelae sp. nov., C761851; T. ratanakiriensis sp. nov., LC761852; T. kampongspeuensis, LC761853). Regarding the outgroup, we included the DNA sequence data of Setodes brevicaudatus Yang & Morse, 1989 (GenBank accession numbers: OL678050 and NC069285). Sequence alignment and editing were performed using MEGA v.7.0.26 (Kumar et al. 2016) and CLC Workbench software (CLC bio, Aarhus, Denmark). All sequence data were aligned using MAFFT v.7.222 (Katoh and Standley 2013). Phylogenetic analyses based on the mtDNA 16S rRNA (434 bp) were performed by the Neighbor-Joining (NJ) method using MEGA v.7.0.26 (Kumar et al. 2016) with 1000 bootstrap cycles. Genetic distances (p-distance) between the species were calculated using MEGA v.7.0.26 (Kumar et al. 2016).

Alongside the previously known T. kampongspeuensis, there are now a total of four species of Trichosetodes recorded in Cambodia. The three new species are known from a single locality, and such restricted distribution could be attributed to limited regional data availability, thus indicating the need for a comprehensive survey of aquatic invertebrate diversity in the Mekong River basin. Additionally, considering the known distribution of 19 Trichosetodes species in Southeast Asia (Fig. 7; Malicky 2010; Oláh 2013; Malicky and Laudee 2017; Morse 2023), such restricted distribution could also be attributed to potentially high endemism of this particular genus in the region (Laudee et al. 2022).

Trichosetodes carmelae sp. nov., T. katiengensis sp. nov. and T. ratanakiriensis sp. nov. were collected from a waterfall with cover by montane evergreen rainforest in eastern Cambodia. According to the habitat characteristics where they were collected, the three new species of Trichosetodes are potentially rhithral species that live in waterfalls and small streams where the substrate is dominated by bedrock, boulders and sand (Fig. 6). Moreover, all four species of Trichosetodes recorded from Cambodia were collected from waterfalls (Laudee et al. 2020). Furthermore, T. asphor was also collected from a fast-flowing stream (Malicky and Laudee 2017). However, larvae of T. japonicus which is widely distributed in East Asia including Honshu and Fukuoka Prefectures (Japan), Korean Peninsula and Far East of Russia were mainly found from middle to lower sections of rivers with slow current (Kawai and Tanida 2018). The larvae and pupae of T. imperfectus Ulmer, 1951 from Sumatra, Indonesia were described by Ulmer (1955). Thus, as our collections are based on light trapping and larval stages are still unknown, we cannot exclude the possibility that T. carmelae, T. katiengensis and T. ratanakiriensis inhabit a wide range of habitats, such as larger river sections, lakes and reservoirs like some other Asian species (Kawai and Tanida 2018).

The molecular analysis, centered on the mtDNA 16S rRNA region, consistently mirrored the morphological distinctions observed among the newly identified species. While T. ratanakiriensis sp. nov. and T. kampongspeuensis share similarities in male genitalia characteristics, they exhibit distinct genital morphologies in contrast to T. carmelae sp. nov. Concerning the genetic distance calculated from the mtDNA 16S rRNA region, T. ratanakiriensis sp. nov. and T. kampongspeuensis demonstrated remarkable proximity, with a mere 1.4% divergence. On the contrary, the male genitalia traits of T. carmelae sp. nov. markedly differed from those of T. ratanakiriensis sp. nov. and T. kampongspeuensis, with molecular analysis indicating genetic distances exceeding 6.3%. Our results imply that the mtDNA16S rRNA gene fragment used to infer genetic divergence in Leptoceridae studied proved to be a good tool for supplementing taxonomy and diversity studies of Trichoptera (Takenaka et al. 2023).