Previously, the euthyplociid mayfly from Thailand was reported as Polyplocia spp. without designation to any nominal species. In this study, the genus Polyplocia Lestage, 1921 in Thailand was reviewed. Two species are recognized: Polyplocia orientalis Nguyen & Bae, 2003 and P. nebulosa Gonçalves & Peters, 2016. This is the first report of P. nebulosa in Continental Asia. Cytochrome c oxidase subunit I (COI) data and illustrations based on nymphal characters were used to confirm two different species in Thailand. Additional morphological characteristics of the nymph and eggs of the two species from Thailand are also shown in detail. The taxonomic status of Polyplocia in the Oriental region is reviewed. Keys to known nymphal and imaginal stages are provided and the distribution of Polyplocia in the Oriental region is discussed.

Burrowing mayfly, Continental Asia, distribution, habitat, Insular Asia, taxonomy

Lestage (1921) established the genus Polyplocia, and Polyplocia vitalisi Lestage, 1921 from Tonkin, Vietnam, was considered the type species based on one male imago. Ulmer (1939) described two new species P. campylociella Ulmer, 1939 and P. crassinervis Ulmer, 1939 based on a northern Borneo (now Sabah, Malaysia) subimago. Demoulin (1952) revised the imago characters of Polyplocia as having a transverse pronotum, forewing with MA fork at same level as Rs fork, and at least one intercalary vein in the cubital field with its base connected to CuP, forceps with only one segment and three caudal filaments on the abdomen (Gonçalves and Peters 2016) and provided new figures of P. crassinervis and P. vitalisi. Demoulin (1953) later described the female imago, also from Sarawak, and synonymized the species as P. campylociella (= P. crassinervis) based on wing venation. He later classified a potential nymph of Polyplocia for the first time from West Borneo (now Indonesia) as P. ?crassinervis, despite the synonymy, and identified the nymphal characters: long tusks with truncated apex, antennae almost the same length as the tusks, foretibiae with short apical projection, foretarsi with apical projection, and gills I with two lamellae (Demoulin 1966). Thirty-seven years later, Nguyen and Bae (2003a) described a new species, P. orientalis Nguyen & Bae, 2003, based on nymphs from Dak Lak, Vietnam and stated that this nymph could be associated with P. vitalisi. Gonçalves and Peters (2016) discovered a new species, P. nebulosa Gonçalves & Peters, 2016, based on male and female imagos from Malaysia’s Sabah State, and provisional nymphs assigned to this species are also described. A description of the structure of the chorion was also provided and recommended as a useful tool for recognising species (Gonçalves and Peters 2016). Lastly, Zheng et al. (2023) reported the first nymph-imago association for the genus in P. orientalis from China and expanded the distribution of this species in the Oriental region. Molecular evidence based on cytochrome c oxidase subunit I (COI) revealed an intraspecific distance of 10% between the Vietnamese and Chinese specimens.

To date, the genus Polyplocia is composed of four valid species: P. vitalisi reported only in an adult stage, P. campylociella reported in adult stage with possible nymphal stage, P. orientalis reported in both stages and P. nebulosa reported in adult stage with a possible nymphal stage. The distribution of this genus encompasses the Oriental Realm (Lestage 1921; Ulmer 1939; Demoulin 1966; Nguyen and Bae 2003a; Gonçalves and Peters 2016; Zheng et al. 2023). Polyplocia spp. nymphs from Thailand were mentioned by Kluge (2004) and Gonçalves and Peters (2016). These nymphs may be related to P. orientalis or belong to another species, as they did not possess paired black anterolateral marks on abdominal sterna. Conversely, the genitalia of the male nymphs of Polyplocia spp. from Thailand also had T-shaped penes, as do the nymphs of P. nebulosa (Gonçalves and Peters 2016). In the present study, two species of the genus Polyplocia based on nymphs from Thailand are recorded using morphological and molecular approaches. Some additional descriptions on the nymphal and chorion structures are mentioned. Keys to known species in both stages are provided. The distribution of this genus in Thailand is studied and information on the biological aspects of Polyplocia is provided.

The specimens were collected by hand-picking from headwater streams in the northern and the western parts of Thailand. (Table 1, GPS map versatile navigator (Garmin eTrex 10)). The specimens were preserved in 100% ethanol for molecular and morphological studies. The mature nymphs were reared using earthenware pots connected to an oxygen pump until emergence of winged stages. Measurements (given in mm) and photographs were taken using a NIKON SMZ800 stereoscopic microscope. For scanning electron microscopy (SEM), eggs were dried in a critical point drier (CPD7501) and coated with gold (Sputter Coater SC7620). The SEM photographs were obtained with a FEI Quanta 450 SEM. Final plates were prepared with Adobe Photoshop CC 2022.

Selected specimens were dissected for DNA extraction. Total DNA was extracted using a genomic DNA purification kit (NucleoSpin, Macherey-Nagel, Germany), following the manufacturer’s protocol. The COI amplification was performed using LCO1490 and HCO2198 (Folmer et al. 1994). The polymerase chain reaction (PCR) conditions and procedure were as described by Kwanboon et al. (2021). The PCR products were purified using a Gel and PCR Clean-up Kit (NucleoSpin, Macherey-Nagel, Germany) and were sequenced by ATGC Co., Ltd (Thailand). Other analysed Polyplocia orientalis sequences were obtained from GenBank (OP347109; OP962407). Potamanthus formosus Eaton, 1892, retrieved from GenBank (MZ453438), was used as an outgroup. The genetic distances between species were determined using Kimura-2-parameter distances (Kimura 1980), calculated with the MEGA11 program (Tamura et al. 2021). Sequence alignment and editing were performed using ClustalW in MEGA11. A phylogenetic tree was analysed by the maximum likelihood (ML) method and the most appropriate evolutionary model was calculate using the Find Best DNA/Protein Models (ML) option test provided with MEGA11. The Tamura-Nei 93 model and a proportional discrete Gamma distribution (TN93+G) was performed with MEGA11 using the likelihood-ratchet method with 1000 bootstrap replicates. The GenBank accession numbers are given in Table 2, nomenclature of gene sequences follows Chakrabarty et al. (2013). Nucleotide sequences obtained in this study have been deposited in GenBank. The distribution map was generated with the SimpleMappr software (https://simplemappr.net; Shorthouse 2010).

The material was deposited in the collection of the Zoological Museum at Kasetsart University in Bangkok, Thailand (ZMKU) and the Veterinary Anatomy and Pathology Museum, Chiang Mai University, Thailand (VMCMU).

We followed all guidelines of the Animal Ethics Committee of Kasetsart University (approval no. ACKU61-SCI-029) for collecting the mayfly specimens.

Specimens of the Polyplocia from Thailand were studied based on the following nymphal characters defined by Demoulin (1966). Of the nominal species that are newly reported to the country, P. orientalis is distributed in northern Thailand (Chiang Rai and Chiang Mai provinces). Some variations in the morphological characters of the Thai P. orientalis were observed in this study (Table 4): Labrum slightly concave on anterior margin, nearly straight; the mandibular tusks are cylindrical, pointed apically and strongly arched inward (18.6° in curvature), with 7–9 spines on 1/3 of the tusk length distally. The second species, P. nebulosa, is distributed in western Thailand (Kanchanaburi and Phetchaburi provinces). Nymphs of these two species from Thailand can be easily separated by the presence of anterolateral black marks ventrally on P. nebulosa abdomen and their absence in P. orientalis. As we have only a very limited description of P. campylociella nymphs, their differences are not discussed in this study. The combinations of characters used to differentiate nymphs of known species are listed in Table 4.

The egg structure of all Polyplocia species has a similar chorionic surface pattern that forms an irregular mesh with raised ridges; however, we found small differences between the eggs of Thai and Chinese specimens of P. orientalis. The specimen from Thailand is oval-shaped, with two visible micropores, while P. orientalis from China and Vietnam has a barrel-shaped egg with no mention of a micropore (Nguyen and Bae 2003a; Zheng et al. 2023). The egg of the Thai P. orientalis shows different characters than P. nebulosa in terms of shape; the length vs width ratio, and the number of visible micropores. Gonçalves and Peters (2016) mention that the structure of the chorion should be considered as a foundation for future study of the genus. The present study confirmed that the structure of the chorion is useful for recognising the species.

The first molecular study of the genus Polyplocia was conducted by Zheng et al. (2023). The intraspecific distance of P. orientalis from Vietnam and China was mentioned to be as high as 10%. In our study, the maximum intraspecific distance between the Thai P. orientalis was relatively high, at 11%. When we compared the intraspecific distance of specimens from Thailand and Vietnam (10–11%) and Thailand and China (6–7%), the result from the genetic point of view also supported assigning the Polyplocia from northern Thailand to P. orientalis, as the maximum intraspecific distance of all specimens of P. orientalis is 11%. The genetic distances between the P. orientalis from northern Thailand and the sequence of P. nebulosa from western Thailand support the separation of this genus in Thailand into two species due to the high interspecific distance that ranges between 18% and 24%. The genetic distances of all known sequences of P. orientalis (sequences from Vietnam and China were added) and P. nebulosa from western Thailand also confirmed the separation into two species, with an interspecific distance of 18–24%. The result of interspecific distances between P. campylociella from Malaysia and two species from the Continental Asia are relatively high at the maximum 22% for P. orientalis and 23% for P. nebulosa. Kwanboon et al. (2021) also reported a variation in the interspecific distance as high as 14–20% for congeneric burrowing mayflies in Southeast Asia. The ML tree consists of two main clades; the P. nebulosa and P. campylociella clades and a P. orientalis clade (Fig. 9). The P. nebulosa and P. campylociella clades could represent two closely related species since they share some morphological characteristics, such as an abdominal sternum with two blackish marks on the anterolateral edges and wings with dark clouds around the cross veins and margins (Table 4). We recommend that molecular studies (i.e., the COI gene) be included as a basis for future studies of all burrowing mayflies, based on our findings and the studies of other burrowing mayflies.

Polyplocia nymphs from Thailand were mentioned by Kluge (2004) as Polyplocia sp. (locality and character are unknown) and by Gonçalves and Peters (2016) as Polyplocia spp. from many localities in northern Thailand. In this study, we collected specimens from one of the localities in which Polyplocia spp. were found by Gonçalves and Peters (2016) (Chiang Rai Province, Doi Luang National Park, Namtok Pu Keang). Therefore, the Polyplocia spp. of Gonçalves and Peters (2016) are proposed to be P. orientalis like the northern Thailand Polyplocia from this study. Although, the Polyplocia sp. of Kluge (2004) is not assigned to either P. nebulosa or P. orientalis, as the data are still lacking.

The distribution of the genus Polyplocia Lestage, 1921 in the Oriental region is shown in Fig. 1. Polyplocia orientalis was reported in Vietnam (Dak Lak, Lam Dong and Thua Thien Hue) and China (Yunnan) and, in this study, in the northern part of Thailand (Chiang Rai, Chiang Mai, Phrae, Phayao and Nan). Therefore, this species has the potential to be assigned as the dominent species of Polyplocia in mainland Asia in the Oriental region. Surprisingly, the discovery of P. nebulosa in Thailand is the first report of this species from Continental Asia, as all other specimens were reported from East Malaysia (Sabah) or Insular Asia. This wide range of distribution of mayflies between Continental and Insular Asia has been reported in many families. For example, Potamanthellus caenoides Ulmer, 1939 (Neoephemeridae) is known as a widespread mayfly in the Oriental region. This species has also been recorded from the Insular Asia, in Indonesia (Sumatra, Java, Bali, Lombok and Flores), Malaysia (Sabah and Sarawak) and the Philippines (Mindanao), and in Continental Asia, in Malaysia (the Malay peninsula), Thailand (Chiang Mai), India (western Ghat) and Vietnam (Dak Lak) (Bae and McCafferty 1998; Nguyen and Bae 2003b; Sartori et al. 2003; Selvakumar et al. 2015; Banazair and Christopher 2019). Such wide distribution is also observed for the burrowing mayfly Rhoenanthus (Rhoenanthus) speciosus Eaton, 1881 of the family Potamanthidae. This species was reported from the Insular Asia, in Indonesia (Sumatra) and East Malaysia (Sabah) and from Continental Asia, in west Malaysia (Pahang) and Thailand (Songkla and Narathiwat) (Bae and McCafferty 1991; Parnrong et al. 2002; Kwanboon et al. 2021). The most diverse family in the Oriental region, Baetidae, also has a widespread species, Labiobaetis moriharai Müller-Liebenau, 1984. This species was recorded in Insular Asia in Brunei (Temborong) and Malaysia (Sabah) and in Continental Asia in Malaysia (Selangor) and Vietnam (Dong Nai) (Kaltenbach and Gattolliat 2020). The distribution of P. campylociella is limited to eastern Malaysia and the distribution of the type species P. vitalisi is limited to the type locality (Tonkin, Vietnam); the nymph remains a mystery.

Our study allows us to conclude that the genus Polyplocia in Thailand is represented by two species, P. orientalis and P. nebulosa, based on a combination of different morphological characters, egg characters and molecular evidence. We have reported the first nominal species records of this family in Thailand and expanded the distribution of these two species. The distribution of P. orientalis in Thailand is limited to the northern parts and mainland Asia, while the distribution of P. nebulosa extends from Insular Asia to Continental Asia, in western Thailand in our study. We expect to find a broader distribution of this genus in Thailand, especially in the southern and the eastern parts, as we gain a more in-depth understanding of the distribution pattern of this genus in the Oriental region.

This research was partially supported by Chiang Mai University, the Faculty of Veterinary Medicine, Chiang Mai University, and the Centre of Excellence on Biodiversity (BDC) Office of Higher Education Commission. We are most grateful to Assistant Professor Dr. Akekawat Vitheepradit (Department of Entomology, Faculty of Agriculture, Kasetsart University) who kindly provide specimens for this study. We are thankful to Dr. Chonlakarn Auychinda (Department of Biology, Faculty of Science, Silpakorn University) for his support with lab work and preparation of the COI barcode. Sarawak Forestry Permission to conduct research on biological resources - Permit SFC.810-4/6/1 (2023) - 023. We are most grateful to our colleagues for assistance during field trips. Further, we would like to thank the Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University and Department of Zoology, the Faculty of Science at Kasetsart University in Bangkok for their assistance and use of their facilities. The authors are also grateful to the reviewers for their valuable recommendations and comments on the manuscript.