Research Article
Research Article
A new species of Alainites (Ephemeroptera, Baetidae) from Thailand
expand article infoSirikamon Phlai-ngam, Nisarat Tungpairojwong, Jean-Luc Gattolliat§|
‡ Khon Kaen University, Khon Kaen, Thailand
§ Museum of Zoology, Lausanne, Switzerland
| University of Lausanne, Lausanne, Switzerland
Open Access


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.

Key Words

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.

Materials and methods

Collecting samples

Larval specimens were collected by using a hand net or picked manually and sorted from the debris and sediments by using D-framed dip net method. This new species was collected for the first time during a survey of aquatic macroinvertebrates in Phetchabun Province and the survey of aquatic macroinvertebrates project of Kanchanaburi Province (Thailand). The additional specimens were collected during the survey of baetid mayflies in Tak and Kamphaengphet Provinces (Table 1). They were sampled in headwater streams. The specimens were preserved in 95% ethanol. The examined material is deposited in the Collection of Aquatic Insect of Department of Biology at Khon Kaen University in Khon Kaen, Thailand (KKU-AIC) and in the Museum of Zoology in Lausanne, Switzerland (MZL).

Table 1.

GPS coordinates of locations of examined specimens.

Species Provinces GPS coordinates Altitudes (m a.s.l.)
A. siamensis sp. nov. Phetchabun 16°44'27.92"N, 101°34'46.52"E 832
Tak 17°04'52.68"N, 98°45'16.73"E 405
17°01'44.35"N, 98°30'24.47"E 719
Kamphaengphet 17°02'34.94"N, 98°58'39.83"E 154
Kanchanaburi 14°45'08.00"N, 98°48'40.00"E 660

Morphological examination

Part of the specimens were dissected and mounted on microscope slides fixed in Euparal, as specified in the material examined sections below. Ethanol-preserved specimens were studied under a Leica M205 stereomicroscope; microscope slides were drawn from a camera lucida on an Olympus BX51 compound microscope and were subsequently scanned for illustration with the Procreate application (iOS application). Photographs of larvae were taken with a Canon EOS 6D camera and edited with Adobe Lightroom ( Final plates were prepared and processed with Adobe Photoshop ( The distribution map was generated with the SimpleMappr software (


DNA of part of the specimens was extracted using non-destructive methods allowing subsequent morphological analysis (see details in Vuataz et al. 2011). The specimens were amplified for a 658 bp fragment of the mitochondrial gene cytochrome oxidase subunit 1 (COI) using the primers LCO 1490 (GGTCAACAAATCATAAAGATATTGG) and HCO 2198 (TAAACTTCAGGGTGACCAAAAAATCA) (Folmer et al. 1994).

The polymerase chain reaction (PCR) was conducted with an initial denaturation temperature of 94 °C for 5 min followed by a total of 35 cycles with denaturation temperature of 94 °C for 30 sec, an annealing temperature of 48 °C for 40 sec and an extension at 72 °C for 1 min, final extension at 72 °C for 5 min. The sequencing was based the Sanger’s method as developed in Vuataz et al. (2011). Sequences editing and ClustalW alignment were provided. Genetic variability between specimens was calculated using Kimura-2-parameter distances (K2P) model. The molecular reconstruction was analyzed by a maximum likelihood (ML). The best evolution model obtained was Tamura-Nei (TN93+G+I) as the most appropriate for reconstruction based on the lowest AICc and BIC scores, with 100 runs and 1000 bootstrap replicates. All genetic analytical methods were performed by MEGA-X (Kumar et al. 2018). Additional Alainites sequences were obtained from GenBank ( and new Alainites sequences (this work) were also added in GenBank to update the nucleotides database (Table 2).

Table 2.

Sequenced specimens of Alainites (bold text showing new sequences).

Species Locality GenBank Accession Number (GenSeq Nomenclature)
A. talasi Kyrgyzstan MZ983799.1; MZ983800.1
A. sadati Algeria ON072439
A. gasithi Israel ON072440
A. kars Armenia MZ983797.1; MZ983798.1
A. bengunn Sardinia HG934996.1; HG934997.1
A. albinatii Corsica HG934994.1
A. yixiani China GU479735.1
A. muticus Spain SR10E08
Russia SR13E10
A. siamensis sp. nov. (PHET1) Phetchabun, Thailand OP903356
A. siamensis sp. nov. (PHET2) Phetchabun, Thailand OP903357
A. siamensis sp. nov. (PHET3) Phetchabun, Thailand OP903358
A. siamensis sp. nov. (TAK1) Tak, Thailand OP903355
A. siamensis sp. nov. (TAK2) Tak, Thailand OP903359
A. siamensis sp. nov. (KAMP) Kamphaengphet, Thailand OP903360


Alainites siamensis sp. nov.


Holotype: Thailand • Larva; Phetchabun Province, Nam Nao National Park, Yakruae stream; Alt. 832 m. 16°44'27.92"N, 101°34'46.52"E; 7 Mar. 2022; Coll. S. Phlai-ngam and V. Vannachak; 1L on slide GBIFCH00763744 [MZL].

Paratypes: Thailand •20 larvae; same data as holotype; 3L on slides GBIFCH00763745–GBIFCH00763747, [MZL]; 10L in alcohol GBIFCH00763748 [MZL]; 7L in alcohol [KKU-AIC].

Other material

Thailand • 70 larvae; Tak Province, Mae Ra Mad District; Alt. 405 m. 17°04'52.68"N, 098°45'16.76"E; 12 Feb. 2022; Coll. K. Koomput and P. Dapsibhai; 1L on slides GBIFCH00763749 [MZL]; 8L in alcohol GBIFCH00763750 [MZL]; 10L in alcohol [KKU-AIC]. Tak Province, Mae Sod District, small stream near the road (unnamed stream); Alt. 719 m. 17°01'44.35"N, 098°30'24.47"E; 8 Oct. 2020; coll. S. Phlai-ngam; 1L on slide [KKU-AIC]; 14L in alcohol [KKU-AIC]; 1L in alcohol GBIFCH 00673245 [MZL]. Kanchanaburi Province, Thong Pha Phumi District, small stream near the Mining Dr. Phol Kleepbuathe; Alt. 660 m. 14°45'08.00"N, 98°48'40.00"E; 16 Nov. 2022; Coll. S. Phlai-ngam; 1L on slide GBIFCH00763751 [MZL]; 7L in alcohol GBIFCH00763752 [MZL], 8L in alcohol [KKU-AIC]. Phetchabun Province, Nam Nao National Park, Yakruae stream; Alt. 832 m. 16°44'27.92"N, 101°34'46.52"E; 23 Nov. 2020; Coll. S. Phlai-ngam; 15L in alcohol [KKU-AIC]. Kamphaengphet Province, Klong Lan District, Pong Nam Ron, Alt. 154 m. 17°02'34.94"N, 98°58'39.83"E; 12 Feb. 2022; Coll. K. Koomput and P. Dapsibhai; 4L in alcohol [KKU-AIC].


Coloration (Figs 1, 2). General coloration medium brown. Head uniformly medium brown, slightly darker between ocelli and at insertion of antennae. Turbinate eyes in male larva dark orange. Thorax medium brown with indistinct pattern. Pronotum slightly paler than mesonotum and metanotum. Thoracic sternites mostly pale brown. Abdominal tergites medium brown, slightly darker in middle area, distal margin with darker transverse band. Abdominal sternites light brown. Cerci and median caudal filament light brown without bands or pattern.

Figure 1. 

Alainites siamensis sp. nov., female larva (middle instar larva) habitus: A Dorsal view; B Lateral view; C Ventral view. Scale bar: 1 mm.

Figure 2. 

Alainites siamensis sp. nov., male larva (middle instar larva) habitus: A Dorsal view; B Lateral view; C Ventral view. Scale bar: 1 mm.

Body. Maximum length 4.2 mm. Median caudal filament ca. 2/3 of cerci.

Head. Capsule medium brown. Antennae medium brown, filiform.

Mouthparts. Labrum (Fig. 3A): dorsal surface with one central seta and an arc of setae reduced to two lateral setae; about twenty fine stout setae scattered over surface. Ventral surface with five small acute setae near lateral margin. Distal margin fringed with two kinds of setae: long lateral setae strongly feather-like and medially with a shorter row of slightly feather-like setae.

Figure 3. 

Alainites siamensis sp. nov., larval morphology: A Labrum (right: dorsal; left: ventral); B left mandible; C left incisor; D left prostheca; E Right mandible; F Right incisor; G Right prostheca; H Maxilla; I Apex of galea-lacinia; J Labium (right: dorsal; left: ventral); K Hypopharynx.

Left mandible (Fig. 3B–D): inner and outer incisors almost fused, formed by seven blunt denticles (Fig. 3C); prostheca with eleven small pointed denticles apically, (Fig. 3D); edge between prostheca and mola only slightly crenelate near mola; mola with an enlarged triangular pointed tooth, fine setae present apically; proximal part with scattered fine setae, not shagreened (Fig. 3B). Right mandible (Fig. 3E–G): inner and outer incisors almost fused, formed by eight blunt denticles (Fig. 3F), prostheca bifid, both filaments feathered, outer filament half length inner filament (Fig. 3G); edge between prostheca and mola crenelate; proximal part with scattered fine setae, not shagreened (Fig. 3E). Maxilla (Fig. 3H–I): galea-lacinia with 3 enlarged acute teeth on apex (Fig. 3I), 2 rows of long dense basal setae on galea-lacinia; maxillary palpus (Fig. 3H) 2-segmented with scattered small hair-like setae; segment II rounded at apex. Labium (Fig. 3J): glossa slightly shorter than paraglossa; a row of stout setae along outer, inner and apical margins, subapical setal tuft present; paraglossae with 3 rows of simple setae along apical margin; labial palpus 3-segmented with scattered fine setae; segment I slightly shorter than segments II and III combined; segment III subrectangular covered with long robust setae mostly in distal half, short pointed setae mostly along outer margin and abundant fine setae. Hypopharynx with rounded lingua and superlinguae, covered with abundant hair-like setae subapically (Fig. 3K).

Thorax. Hindwing pads well developed.

Foreleg (Fig. 4A). Femur: dorsal margin with a regular row of eight to eleven long robust setae and two subapical setae; ventral margin with abundant short spine-like setae; lateral margin with scale bases, bare and not shagreened, femoral villopore absent.

Figure 4. 

Alainites siamensis sp. nov., larval morphology: A Foreleg; B Fore-tibia; C Mid-tibia; D Hind-tibia; E Tarsal claw; F Abdominal tergite IV; G Posterior marginal spines of tergite IV.

Tibia (Fig. 4B): dorsal margin of fore-tibia with less than five strong setae (usually with 3–4 setae), short fine setae roughly arranged in row; ventral margin with abundant short stout setae ending with patch of long stout feathered setae; lateral margin with abundant scale bases, tibiopatellar suture with short spine-like setae. Tarsus: dorsal margin with few fine setae, ventral margin with abundant pointed setae only slightly increasing in length toward apex, lateral margin with abundant scale bases and a few fine setae. Tarsal claws (Fig. 4E) with one row of 7–9 denticles, increasing in size distally, subapical setae absent. Midleg and hindleg. Similar to foreleg; except ventral margin of femur of hindleg with reduced number of short spine-like setae, generally one or two setae; dorsal margin of mid- tibia (Fig. 4C) and hind-tibia (Fig. 4D) with more than five strong setae (usually more than nine).

Abdomen. Posterior margin of tergite I smooth; posterior margin of tergite II with a few triangular spines mainly in the middle area; posterior margin of tergites III–IX with triangular spines along central portion, absent laterally (Fig. 4F–G). Surface of all tergites not shagreened, with numerous scale bases and few setae. Sternites I–VIII similar to tergites except distal margin smooth, sternite IX distal margin with triangular spines. Gills (Fig. 5A–D) on segments I–VII, with well visible main tracheation but reduced ramification; gill I smallest (Fig. 5A), length of gill I equal to half of tergite II; gill IV (Fig. 5B) and gill V larger than others, length of gill IV equal to tergite V plus half of tergite VI; length of gill V equal to tergite VI plus half of tergite VII; length of gill VII (Fig. 5D) equal to tergite VIII plus half of tergite IX. Surface smooth without any setae or pores, margin serrated with fine simple setae (Fig. 5C). Paraproct (Fig. 5E–F) covered with scale bases and micropores on surface; distal margin with well-developed prolongation; lateral margins of prolongation with numerous small spines, surface without spines (Fig. 5F); distal margin inner to prolongation with 5 huge spines, distal margin outer to prolongation with numerous medium spines (around 20–30 spines); cercotractor with scale bases, distal margin with about 14–16 medium spines (Fig. 5E). Cerci with fine swimming setae along inner margin. Median filament with swimming setae on both margins.

Figure 5. 

Alainites siamensis sp. nov., larval morphology: A Abdominal gill I; B Abdominal gill IV; C Gill margin; D Abdominal gill VII; E Paraproct; F Paraproct prolongation.


Alainites siamensis sp. nov. is closely related to A. lingulatus, A. laetificus and A. yixiani; these species are distributed in the Southeast Asia. The new species can be separated from the other species by the following combination of characters: i) the shape of labial palp, ii) the setation on tibia, iii) the number of denticles of the claws, iv) the degree of development and spination of paraproct prolongation, v) the number of spines on distal margin of paraproct, vi) number of pairs of gills (Table 4).

Ecological notes

The larvae of Alainites siamensis sp. nov. mainly live among aquatic plants and roots of riparian plants in slow to moderately flowing streams (Fig. 6). The species seems to be rather widely distributed in the northwestern and northern Thailand, but is rare and not abundant (Fig. 7).

Figure 6. 

The sampling sites and habitats of Alainites siamensis sp. nov. larvae: A–B Yakruae stream, Nam Nao National Park, Phetchabun Province (type locality); C–D Stream in Mae Ra Mad District, Tak Province; E–F Stream near the Mining Dr. Phol Kleepbuathe, Thong Pha Phum District, Kanchanaburi Province.

Figure 7. 

Distribution map of Alainites siamensis sp. nov. larvae: Closed red triangle shows the type locality, opened red triangle shown other localities.


The specific epithet of this species, siam (noun), refers to the old name of Thailand and to the known distribution of the species.

Molecular result

Sequences of 658 bp, corresponding to a fragment of the COI gene, were obtained from six specimens collected in three localities; the new sequences are deposited in GenBank under accession numbers specified in Table 2. The K2P analysis for genetic distances analysis shows that the three populations of Alainites siamensis sp. nov. have very low intraspecific variation (0% to < 2%). The interspecific distances between A. siamensis sp. nov. and other Alainites species show distances between 17% and 28% (Table 3). In the COI reconstruction, the six larval specimens of Alainites from Thailand were grouped together into the same clade and clearly separated from all other sequenced species of Alainites (Fig. 9).

Table 3.

Genetic distances (COI) between sequenced specimens, using the Kimura 2-parameter.

1 2 3 4 5 6 7 8 9
1 A. siamensis sp. nov. 0.00–0.02
2 A. yixiani 0.20 -
3 A. albinatii 0.21 0.21 -
4 A. kars 0.24 0.20 0.20 0.00
5 A. talasi 0.21 0.24 0.28 0.25 0.00
6 A. sadati 0.22 0.24 0.23 0.23 0.25 -
7 A. bengunn 0.26 0.26 0.20 0.25 0.27 0.24 0.01
8 A. gasithi 0.22 0.23 0.23 0.19 0.24 0.24 0.22 0.00
9 A. muticus 0.22 0.23 0.17 0.22 0.28 0.24 0.20 0.23 0.02
Table 4.

Comparison of larval morphological characters of Alainites siamensis sp. nov. with the closely related Southeast Asian species with seven pairs of gills. Character states based on the original descriptions of individual species.

Characters A. clivosus A. lingulatus A. yixiani A. siamensis sp. nov.
Shape of terminal segment of labial palp Sub-rectangular shaped; lateral margin slightly rounded Sub-rectangular shaped; lateral margin almost straight Sub-rectangular shaped; lateral margin slightly rounded Sub-rectangular shaped; lateral margin almost straight
Setation of dorsal margin of foretibia More than five strong setae More than five strong setae More than five strong setae Less than five strong setae (usually 3–4 setae)
Number of denticles of the tarsal claws 11–13 denticles 8–11 denticles 9–11 denticles 7–9 denticles
Abdominal tergites Uniformly medium brown Uniformly medium brown in mature female; tergites I–VI light brown and tergites VII–X brown in mature male larvae Tergites I–VIII uniformly medium brown; IX and X light brown Uniformly medium brown
Distal margin of abdominal tergites Tergite I smooth; tergites II–X with triangular spines along central portion, absent laterally Tergite I smooth, tergites II–X with triangular spines ? Tergite I smooth; tergite II with a few triangular spines mainly in the middle area; tergites III–IX with triangular spines along central portion, absent laterally
The degree of development of paraproct prolongation Moderately developed Well-developed tongue-like Moderately developed Well-developed
Distribution Taiwan Hong Kong China mainland Thailand
Reference Kang et al. (1994) Tong and Dudgeon (2000) Gui and Lu (1999) This work


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.

Key to the larvae of Alainites from Southeast Asia*

1 7 pairs of gills 2
6 pairs of gills 5
2 Paraproct with a tongue-like prolongation, broader apically than medially A. lingulatus
Paraproct with a moderately or well-developed prolongation, not tongue-like 3
3 Abdominal tergites uniformly medium brown 4
Abdominal tergites I–VIII uniformly medium brown; tergites IX and X light brown A. yixiani
4 Tarsal claws with 7–9 denticles A. siamensis sp. nov.
Tarsal claws with 11–13 denticles A. clivosus
5 Distal margin of tergite IV with spines; prolongation of paraproct well developed with numerous spines 6
Distal margin of tergite IV without spines; prolongation of paraproct moderately developed or well-developed tongue-like projection 7
6 More than ten strong setae on the dorsal margin of mid tibia; short spine-like setae between prostheca and mola A. pascalae
Less than five strong setae on the dorsal margin of mid tibia; long spine-like setae between prostheca and mola A. laetificus
7 Paraproct with short, slightly acute prolongation (moderately developed); ventral margin of fore femora with robust, simple setae A. acutulus
Paraproct with well-developed tongue-like prolongation; ventral margin of fore femora without setae A. yehi

* Based on the original description and illustration.


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.


  • Bae YJ, Park SY (1998) Alainites, Baetis, Labiobaetis and Nigrobaetis (Ephemeroptera: Baetidae) in Korea. The Korean Journal of Systematics Biology 14: 1–12.
  • Boonsoong B (2022) Mayfly larvae in Thailand. Pre-One Part., Ltd., 469 pp.
  • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.
  • Fujitani T, Hirowatari T, Tanida K (2003) Genera and species of Baetidae in Japan: Nigrobaetis, Alainites, Labiobaetis, and Tenuibaetis n. stat. (Ephemeroptera). Limnology 4(3): 121–129.
  • Gattolliat J-L (2011) A new species of Alainites (Ephemeroptera: Baetidae) from Borneo (East Kalimantan, Indonesia). Journal of the Swiss Entomological Society 84: 185–192.
  • Gui H, Lu L (1999) A new species of the genus Baetis from China (Ephemeroptera: Baetidae). Acta Entomologica Sinica 42: 297–299. [in Chinese]
  • Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003) Biological identifications through DNA barcodes. Proceedings of The Royal Society B 270: 313–321.
  • Kaltenbach T, Garces JM, Gattolliat J-L (2020) A new genus of Baetidae (Insecta, Ephemeroptera) from Southeast Asia. European Journal of Taxonomy 612: 1–32.
  • Kang SC, Chang HC, Yang CT (1994) A Revision of the genus Baetis in Taiwan (Ephemeroptera, Baetidae). Journal of Taiwan Museum 47: 9–44.
  • Kluge NJ, Novikova EA (2014) Systematics of Indobaetis Müller-Liebenau & Morihara, 1982, and related implications for some other Baetidae genera (Ephemeroptera). Zootaxa 3835(2): 209–236.
  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms (Version 10.0.2). Molecular Biology and Evolution 35: 1547–1549.
  • Müller-Liebenau I (1969) Revision der europaischen Arten der Gattung Baetis Leach, 1815 (Insecta, Ephemeroptera). Gewasser und Abwasser 48/49: 1–214.
  • Müller-Liebenau I (1984) New genera and species of the family Baetidae from West Malaysia (River Gombak) (Insecta: Ephemeroptera). Spixiana 7: 253–284.
  • Novikova EA, Kluge NJ (1987) Systematics of the genus Baetis (Ephemeroptera, Baetidae), with description of new species from Middle Asia. Vestnik Zoologii 1987: 8–19. [in Russian]
  • Novikova EA, Kluge NJ (1994) Mayflies of the subgenus Nigrobaetis (Ephemeroptera, Baetidae, Baetis). Entomologicheskoe Obozrenie 73: 623–644. [in Russian]
  • Phlai-ngam S (2017) Species diversity of baetid mayflies (Ephemeroptera: Baetidae) in North and Northeast of Thailand. Thesis of Master Degree, Khon Kaen University, Khon Kaen.
  • Phlai-ngam S, Tungpairojwong N (2018) First record of Platybaetis bishopi Müller-Liebenau, 1980 and Baetiella bispinosa (Gose, 1980) (Ephemeroptera: Baetidae) from Thailand. Chiang Mai Journal of Science 45(2): 774–783.
  • Phlai-ngam S, Boonsoong B, Gattolliat J-L, Tungpairojwong N (2022) Megabranchiella gen. nov., a new mayfly genus (Ephemeroptera, Baetidae) from Thailand with description of two new species. ZooKeys 1125: 1–31.
  • Sartori M, Brittain JE (2015) Order Ephemeroptera. In: Thorp J, Rogers DC (Eds) Ecology and General Biology: Thorp and Corvich’s Freshwater Invertebrates. Academic Press, Boston, 873–891.
  • Sroka P, Yanai Z, Palatov D, Gattolliat J-L (2021) Contribution to the knowledge of the genus Takobia Novikova & Kluge, 1987 (Ephemeroptera, Baetidae) in Central Asia. ZooKeys 1071: 127–154.
  • Suttinun C (2021) Systematics of the family Baetidae (Order Ephemeroptera) in Southern and Western of Thailand. Doctoral dissertation, Kasetsart University, Bangkok.
  • Suttinun C, Gattolliat J-L, Boonsoong B (2018) A new species of Platybaetis Müller-Liebenau, 1980 (Ephemeroptera: Baetidae) from Thailand, with description of the imago of Platybaetis bishopi Müller-Liebenau, 1980. Zootaxa 4378(1): 85–97.
  • Suttinun C, Kaltenbach T, Gattolliat J-L, Boonsoong B (2021) A new species and first record of the genus Procerobaetis Kaltenbach & Gattolliat, 2020 (Ephemeroptera, Baetidae) from Thailand. ZooKeys 1023: 13–28.
  • Suttinun C, Gattolliat J-L, Boonsoong B (2022) First report of the genus Tenuibaetis (Ephemeroptera, Baetidae) from Thailand revealing a complex of cryptic species. ZooKeys 1084: 165–182.
  • Tong X, Dudgeon D (2000) Two new species of Alainites (Ephemeroptera: Baetidae) from Hong Kong, China. Pan-Pacific Entomology 76: 115–120.
  • Tungpairojwong N, Phlai-ngam S, Jacobus LM (2022) A new species of Acentrella Bengtsson, 1912 (Ephemeroptera: Baetidae) from Thailand. Zootaxa 5125(4): 351–378.
  • Vuataz L, Sartori M, Wagner A, Monaghan MT (2011) Toward a DNA taxonomy of alpine Rhithrogena (Ephemeroptera: Heptageniidae) using a mixed yule-coalescent analysis of mitochondrial and nuclear DNA. PLoS ONE 6(5): e19728.
  • Waltz RD, McCafferty WP (1997) New generic synonymies in Baetidae (Ephemeroptera). Entomological News 108: 134–140.
  • Waltz RD, McCafferty WP, Thomas A (1994) Systematics of Alainites n. gen., Diphetor, Indobaetis, Nigrobaetis n. stat., and Takobia n. stat. (Ephemeroptera, Baetidae). Bulletin de la Société d’Histoire naturelle de Toulouse 130: 33–36.
  • Zrelli S, Gattolliat J-L, Boumaiza M, Thomas A (2012) First record of Alainites sadati Thomas, 1994 (Ephemeroptera: Baetidae) in Tunisia, description of the larval stage and ecology. Zootaxa 3497(1): 60–68.