A new species of Baetidae, Alainites siamensis sp. nov., is described from Thailand. This new species is closely related to Alainites lingulatus Tong & Dudgeon, 2000, Alainites laetificus (Kang & Yang, 1994) and Alainites yixiani (Gui & Lu, 1999). Species delimitation based on morphological and molecular (mitochondrial COI sequences) evidence is provided. The discovery of this species confirms the wide distribution of Alainites Waltz & McCafferty, 1994 in Southeast Asia.

distribution, diversity, mayflies, Southeast Asia, systematic

The small minnow mayflies (Ephemeroptera: Baetidae) are one of the most common and widespread mayfly family. This family comprises ca. 1,070 species assigned to 110 genera (Sartori and Brittain 2015; Jacobus et al. 2019; Kaltenbach et al. 2020).

The European species of Baetis s.l. Leach, 1815 were firstly classified and divided into eleven species groups by Müller-Liebenau (1969). The muticus species group was subsequently raised to the generic level under Alainites Waltz & McCafferty, 1994 (Waltz et al. 1994; McCafferty and Waltz 1997; Zrelli et al. 2012; Yanai et al. 2022). Nigrobaetis Novikova & Kluge, 1987 was erected for the niger species group (Waltz et al. 1994). Finally, Takobia Novikova & Kluge, 1987 was established and encompassed a single species originally described as Centroptilum maxillare Braasch & Soldán 1983.

Alainites, Nigrobaetis and Takobia were considered as belonging to the Indobaetis complex (Kluge and Novikova 2014). The status of the three genera was widely debated in various studies (Müller-Liebenau 1969; Novikova and Kluge 1987, 1994; Waltz et al. 1994; Waltz and McCafferty 1997; Kluge and Novikova 2014). However, the revisions were not based on examination of type material and detailed descriptions of the type species were missing (see details in Sroka et al. 2021; Yanai et al. 2022). In the last comprehensive studies (Sroka et al. 2021; Yanai et al. 2022), it was highly encouraged to continue to consider Alainites, Nigrobaetis and Takobia as valid genera and wait for a global phylogeny based on molecular and morphological evidence before proposing a definitive classification.

The genus Alainites originally encompassed nine species (Waltz et al. 1994); at that time the diagnostic characters of the larval stage were as follows: i) paraproct with an elongated prolongation; ii) prostheca of the right mandible bifid, reduced to two bristle-like feathered appendages; iii) absence of villopore; and iv) body laterally compressed. At the imaginal stages: i) hindwings, when present, with three longitudinal veins, the second being bifurcated; and ii) segment III of the male forceps spherical to slightly elongated and curved (Waltz et al. 1994; Zrelli et al. 2012; Yanai et al. 2022).

In the Oriental realm, seven species were assigned to Alainites. Part of these species were originally attributed to Baetis, including Baetis laetificus Müller-Liebenau, 1984, Baetis (Acerbaetis) clivosus Kang & Yang, 1994 and Baetis (Acerbaetis) yehi Kang & Yang, 1994. Afterward, these three Baetis species were transferred to Alainites by Waltz et al. (1994). The Chinese species, Alainites yixiani (Gui & Lu, 1999) was also first attributed to Baetis (Gui and Lu 1999). Two new species of Alainites from Hong Kong, Alainites acutulus Tong & Dudgeon, 2000 and Alainites lingulatus Tong & Dudgeon, 2000, were subsequently described (Tong and Dudgeon 2000). The last described species of Alainites in South East Asia, Alainites pascalae Gattolliat, 2011, was reported from Borneo (Gattolliat 2011).

The knowledge of the diversity of Baetidae in Thailand has notably increased recently. Thanks to two large scale surveys of the Baetidae in Thailand project many taxa have been continuously discovered during the last decade (Tungpairojwong and Bae 2015; Phlai-ngam and Tungpairojwong 2018; Suttinun et al. 2018, 2020, 2021, 2022; Phlai-ngam et al. 2022; Tunjpairojwong et al. 2022). Many new taxa have been reported, some of them remain undescribed. Presently, ten genera and 14 species of Baetidae are recorded from this area (Phlai-ngam 2017; Suttinun 2021; Suttinun et al. 2021, 2022; Boonsoong 2022; Phlai-ngam et al. 2022; Tungpairojwong et al. 2022). Based on the remaining under prospected areas and the still unstudied but potentially diversified genera (Baetis, Labiobaetis Novikova & Kluge, 1987, Nigrobaetis), the species diversity of Thai Baetidae will continue to increase rapidly.

Herein, we provide the description and illustration of larval stage of a new species of Alainites from Thailand. The morphological comparison of this new species to related species, provides diagnostic key characters. The species delimitation is also supported by molecular evidence (mitochondrial COI sequences). Additionally, a key to species of the larvae of Alainites from Southeast Asia is also provided.

The new described mayfly species from Thailand, Alainites siamensis sp. nov., is assigned to the genus Alainites Waltz and McCafferty, 1994 based on the following characters: frons with a medial ridge between antenna, absence of distal lobe on antennal scape, prostheca of the right mandible bifid, absence of villopore, paraproct with prolongation (Waltz et al.1994; Fujitani et al. 2003; Zrelli et al. 2012; Yanai et al. 2022). This new species presents similarities with the type species of the genus, Alainites muticus (Linnaeus, 1758); both species possess seven pairs of gills and well-developed paraproct prolongation. The two species can be separated by inconspicuous characters such as the number of setae on dorsal margin of tibia, setation of distal margin and prolongation of paraproct. Alainites muticus is not reported from Southeast Asia and from the Oriental realm in general; the closest report seems to be in Korea (Bae and Park 1998).

Among the Southeast Asian species, Alainites siamensis sp. nov. can be identified by the shape of labial palp, the setation of tibia, the number of denticles of the claws, the degree of development and spination of paraproct prolongation, the number of spines on distal margin of paraproct and the number of pairs of gills (Table 4). The presence of seven pairs of gills in Alainites siamensis sp. nov. allow the separation of this species from A. acutulus Tong & Dudgeon, 2000, A. laetificus Müller-Liebenau, 1984, A. pascalae Gattolliat, 2011 and A. yehi (Kang & Yang, 1994) which all possess six pairs of gills.

The new species differs from the other species with seven pairs of gills (A. lingulatus Tong & Dudgeon, 2000, A. clivosus (Chang & Yang, 1994) and A. yixiani (Gui & Lu, 1999)) by presence of less than five strong setae on dorsal margin of foretibia (usually with 3–4 setae), tarsal claws with 7–9 denticles, well-developed paraproct prolongation, in opposition to A. clivosus and A. yixiani which possess more than five strong setae on dorsal margin of foretibia and tarsal claws with more than nine denticles. Alainites clivosus can be distinguished from A. yixiani by the abdominal tergites uniformly medium brown while A. yixiani has tergites IX and X lighter than the remaining tergites.

Alainites siamensis sp. nov. can be distinguished from A. lingulatus by the shape of the prolongation of the paraproct and by claws with 7–9 denticles while A. lingulatus has paraproct with a well-developed tongue-like prolongation and claws with 8–11 denticles (Table 4).

Alainites laetificus and A. pascalae share with the new species the distal margin of tergite IV with spines and prolongation of paraproct well developed with numerous spines. Alainites siamensis can be easily separated from these two species by the number of gills.

The molecular analysis clearly confirms that Alainites siamensis sp. nov. is a valid new species as shown by genetic distances between species ranging from 17% to 28% (Table 3), which are greater than 3% generally considered as the maximum value for intraspecific divergence (Hebert et al. 2003). The intraspecific divergences are rather reduced, generally lower than 2%. The genetic distances were calculated between the eight available species, i.e. A. talasi (Novikova & Kluge, 1994), A. sadati Thomas, 1994, A. gasithi Yanai & Gattolliat, 2022, A. kars (Thomas & Kazanci, 1989), A. bengunn Yanai & Gattolliat, 2022, A. albinatii (Sartori & Tomas, 1989), A. muticus (Linnaeus, 1758), and A. yixiani. However, several sequences are missing for part of the Alainites species, especially for the morphologically closely related species A. lingulatus and A. laetificus. Alainites yixiani is the only sequenced species from Southeast Asia available for calculation.

The morphological comparison between species is often challenging by the short inaccurate original description and the poor quality of the illustrations; this remains true also for recently described species such as A. yixiani. A revision of this species is high request and will be necessary to confirm the characters isolated for species identification. However, the molecular analysis based on COI sequences confirm the distinctness of these two species (K2P distance = 20% between the two clades). A complete molecular data analysis of the Southeast Asian Alainites might take time for integrating all previous species reports from this area. We hope that, in a close future, integrative approach including molecular data, accurate descriptions and geographic distribution will help to securely identify the different species of Alainites in Southeast Asia.

Alainites is widely distributed in Southeast Asia, but the eight species are reported from a a single country (Fig. 8). Amazingly, five of the eight are only reported from islands. The new species, Alainites siamensis sp. nov. is only known from Thailand which represents a new report from an inland country in midway between the previous reports in Southeast Asia.

Figure 8. 

Distribution map of Alainites species in Southeast Asia.

Figure 9. 

Phylogenetic reconstruction of Alainites species based on maximum likelihood analysis of sequences of the mitochondrial COI gene. The reconstruction includes representatives of the new species, Alainites siamensis sp. nov. (Orange background), other Alainites species (Blue and gray background) and Liebebiella vera (Black color) as outgroup.

According to ecological requirement and the presumed dispersal abilities of Alainites, we may expect a wider distribution in Southeast Asia which includes most of inland countries of the area, as well as overseas colonization of some islands in particular in the Philippines, as supported by the presence of the genus in Taiwan, Borneo and Hong Kong (Müller-Liebenau 1984; Kang et al. 1994; Tong and Dudgeon 2000; Gattolliat 2011). As already mentioned, almost all species show restricted distribution and are only known from a single country. This is at least partially due to the lack of data in some areas. We can expect new reports from Cambodia, Laos, Myanmar (Burma), or Vietnam; they will almost certainly increase the distribution of part of the species or allow the discovery of new ones. A global molecular study of the different species will greatly help to understand the mechanism of dispersion and speciation within these taxa.

The research was funded by a Science Achievement Scholarship of Thailand (SAST) and Research and Graduate Studies Khon Kaen University. We are thankful to our colleagues for kind support throughout field trips. We are very grateful to the Graduate School of Khon Kaen University for providing a grant to come to the Museum of Zoology, Lausanne (MZL). We are grateful to Céline Stoffel (MZL) for her support with lab work, and Laurent Vuataz (MZL) for his valuable suggestions. We would like to thank the team of MZL, as well as the Department of Biology, Faculty of Science at Khon Kaen University for their assistance and facility supports. Lastly, the authors are grateful to the reviewers, Pavel Sroka and Zohar Yanai, for their valuable recommendations and comments on the manuscript.

This research has been reviewed and approved by the Institutional Animal Care and Use Committee of Khon Kaen University, based on the Ethic of Animal Experimentation of National Research Council of Thailand (Record No. IACUC-KKU-65/63) for collecting mayfly specimens.