A new species of Alainites (Ephemeroptera, Baetidae) from Thailand

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.


Introduction
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. 1994McCafferty 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;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).
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. 2018Suttinun et al. , 2020Suttinun et al. , 2021Suttinun et al. , 2022Phlai-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. 2021Suttinun et al. , 2022Phlaingam 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).

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 (http://www.adobe.com). Final plates were prepared and processed with Adobe Photoshop (http:// www.adobe.com). The distribution map was generated with the SimpleMappr software (https://simplemappr.net).
Genetics 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 (GGTCAACAAATCATA-AAGATATTGG) and HCO 2198 (TAAACTTCAGGGT-GACCAAAAAATCA) (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 (http://www.ncbi.nlm.nih.gov/) and new Alainites sequences (this work) were also added in Gen-Bank to update the nucleotides database (Table 2).  (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.
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.
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 spinelike setae; lateral margin with scale bases, bare and not shagreened, femoral villopore absent. 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.
Diagnosis. Alainites siamensis sp. nov. is closely related to A. lingulatus, A. laetficus 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).
Etymology. 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).

Discussion
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. aculatus Tong & Dudgeon, 2000, A. laetificus Müller-Liebenau, 1984, A. pascalae Gattolliat, 2011 (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.

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)

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   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 , 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. laetficus. 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.
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*