Behningiidae and Potamanthidae (Insecta, Ephemeroptera) in Thailand

Abstract The aim of this study is to review the family Behningiidae and Potamanthidae in Thailand. Two genera and three species of Behningiidae are recognised: Protobehningiamerga Peters & Gillies, 1991, Behningiabaei McCafferty & Jacobus, 2006, and Behningianujiangensis Zhou & Bisset, 2019, which is newly reported from Thailand. The egg structure of B.nujiangensis is described for the first time using scanning electron microscopy. The larva of P.merga is redescribed and its distribution is expended northward with a new record from Chiang Mai province. Two genera and five species of Potamanthidae are identified: Potamanthusformosus Eaton, 1892, Rhoenanthusmagnificus Ulmer, 1920 (new record for Thailand), Rhoenanthusobscurus Navás, 1922, Rhoenanthusdistafurcus Bae & McCafferty, 1991, and Rhoenanthusspeciosus Eaton, 1881. Our morphological evidence is supported by COI data for the family Potamanthidae. Diagnostic characters, distributions, and keys are presented for the larvae of all known species of Thai behningiid and potamanthid mayflies.


Introduction
The Behningiidae is a small mayfly family represented by three extant genera (Behningia Lestage, 1930, Dolania Edmunds & Traver, 1959, and Protobehningia Tshernova, 1960 and one fossil genus (Archaeobehningia Tshernova, 1977) (Hubbard 1994). Members of the Behningiidae are known as tuskless burrowing mayflies for their sanddwelling behaviour (Miller et al. 2018). To date, seven species have been described, and three of them have been documented in the Oriental region (two species from Thailand and one species from China) (Zhou et al. 2019). Behningia baei McCafferty & Jacobus, 2006 and Protobehningia merga Peters & Gillies, 1991 have been described from Thailand and to date have only been reported from the type localities (Peters and Gillies 1991;Parnrong et al. 2002;McCafferty and Jacobus 2006). Only larval exuviae of P. merga are known (Peters and Gillies 1991).
In this study, we review the species of Behningiidae and Potamanthidae in Thailand, and we provide the first records of B. nujiangensis Zhou &Bisset, 2019 andR. magnificus Ulmer, 1920. We also redescribe the larva of P. merga, and we present the first description of the egg structure of B. nujiangensis. A distribution map of Thai behningiid and potamanthid mayflies is also provided.

Materials and methods
The specimens were collected from streams and rivers in Thailand and were preserved in absolute ethanol. Measurements (in mm) and photographs were taken using a Nikon SMZ800 and ZEISS Stemi 305 stereoscopic microscope. For scanning electron microscopy (SEM), specimens (head, legs, labrum, labium, labial palp, glossa, paraglossa, and eggs) were dried in a critical point dryer (CPD7501) and coated with gold (Sputter Coater SC7620). The specimens were observed and photographed with an FEI Quanta 450 SEM. The final plates were prepared with Adobe Photoshop CC 2020. The material is deposited in the collection of the Zoological Museum at Kasetsart University in Bangkok, Thailand (ZMKU). The distribution map was constructed using the Simple Mapper website (http://www.simplemappr. net) and GPS coordinates.

Molecular methods
Each specimen was dissected for DNA extraction using a genomic DNA purification kit (NucleoSpin, Macherey-Nagel, Germany) following the manufacturer's protocol. A fragment of the mitochondrial cytochrome oxidase I (COI) was amplified using the primers LCO1490 and HCO2198 (Folmer et al. 1994). The polymerase chain reaction (PCR) conditions and procedure were as described by Gattolliat et al. (2015). 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). The Kimura-2-parameter distances were calculated using the MEGA X program (Kumar et al. 2018). A phylogenetic tree was analysed by the maximum likelihood (ML) method and the Tamura 3-parameter protocol was performed with MEGA X using the likelihood-ratchet method with 1,000 bootstrap replicates. Nucleotide sequences obtained in this study have been deposited in the GenBank database (Table 1) can be distinguished from other Behningia species based on the following characteristics: i) labrum deeply emarginate in a V or U shape at anteromedian margin, ii) labial palp I without concavity on outer margin, iii) labial palp II less than 50% length of labial palp III, iv) tarsus of foreleg as long as tibia and v) coxa of hind leg less than 60% as long as femur.
Remark. The larvae of B. baei were originally described by McCafferty and Jacobus (2006) and collected from Phitsanulok province (Thailand). In this study, we attempted to collect specimens at the type locality (Klong Nam Kub, Ban Khok Phakwan), but no specimens were found during our fieldwork. However, the habitat of the type locality of B. baei is suitable for behningiid larvae, consisting of wadeable, widely flooded rivers with fine sandy bottoms and braided channels (Fig. 7). Diagnosis. The larvae of Behningia nujiangensis Zhou & Bisset, 2019 can be separated from those of other Behningia species based on the following characteristics: i) labrum shallowly emarginate at anteromedian margin ( Fig. 2A), ii) molar areas of mandible with a small apical spine (Fig. 2B), iii) galea-lacinia of maxilla elongated and slender (Fig. 2C), iv) labial palp 3-segmented, segment II as long as segment III (Fig. 2D), v) tarsus of forelegs about 40% the length of tibia (Fig. 3A, B), vi) middle and hind legs with coxa as long as femur (Fig. 3C, D).

Behningia nujiangensis
Egg (dissected from mature larva). Length 1.62-1.73 mm, width 1.09-1.26 mm (n = 13); oval (Fig. 4A); with massive amounts of fibrous adhesive material localised at the polar and equatorial regions of the egg (Fig. 4B); chorion densely and finely punctutated, with a weakly developed pentagonal reticulation, circular in shape and convex in the middle (Fig. 4C); funnelform micropyle in the centre of circular accumulations of adhesive material only at the equatorial zone (Fig. 4D).
Remark. The larvae of B. nujiangensis were originally described by Zhou et al. (2019) and collected from China (Nujiang river, Yunnan province, upper Salween river). In Thailand, the samples were collected from the Tard Luang waterfall (fine sandy habitat) in 2011, and specimens were deposited but only identified to the genus level (Dr. Akekawat Vitheepradit, Department of Entomology, Kasetsart University). In this study, we re-examined and identified the specimens. We attempted to collect specimens from the same microhabitat near the Tard Luang waterfall; however, unfortunately, no specimens were found. Head. Anterior margin not projecting, front with densely short goldish setae standing out on the head (Fig. 1B, 5A). Black eyes on dorsolateral margin; ocelli almost white, inner margin of ocelli black in front of compound eye. Antennae at lateral margin of head. Labium extending the entire anterior margin of head, with long setae; labial palp 3-segmented, surface of labial palp covered with rows of long blunt setae, base of second palp segment with the longest setae, first segment longer than other segments (Fig. 5B), glossae and paraglossae with numerous (>20) setae (Fig. 5C). Left mandible and right mandible strong and dentated, mostly similar to P. asiatica. Maxillary palpi 3-segmented, maxilla base extending, apex narrow with terminal tooth.
Abdomen. Similar in colour to head and thorax, abdominal segments elongated and convex, with short straight setae at lateral margin, lateral margin of abdominal segment I-IX with flat projections spine-like in shape. Gill present on segment I-VII, plumose shape, first gill filament single (Fig. 5D); gills II-VII double, upper branch of each gill shorter than lower one. Three caudal filaments fringed with short pale setae, length of median filament as long as lateral filaments. Diagnosis. The larvae of Protobehningia merga Peters & Gillies 1991 can be distinguished from those of P. asiatica based on the following characteristics: i) glossae and paraglossae with more than 20 setae on the ventral surface, ii) maxillary palp segment II 2/3 the length of segment I, each maxillary palp segment completely divided, but segments II and III indistinct (Peters and Gillies 1991).
Distribution. Kanchanaburi and Chiang Mai provinces. Biological aspects. In general, the larvae of behningiid mayflies are rarely collected. In this study, the larvae (middle instar) were found in October (turbidity from flooding, Fig. 8A) and November in a river in Chaing Mai province, whereas Peters and Gillies (1991) found the exuviae and imago during December in Kanchanaburi province (western Thailand). The specimens were collected from the Mae Chaem river, which is submontane and bordered by farmland and residential areas (Fig. 8A). The substrates were covered with fine-and coarse-grained sand (Fig. 8C). The larvae of P. merga were collected using an aquatic net in a fine sandy habitat, where the depth of the sandy bottom was more than 50 cm and near the littoral zone (Fig. 8B, D). The larvae were usually found together with those of the oligoneuriid mayfly, Chromarcys magnifica Navás, 1932 and the gomphid dragonfly, Paragomphus capricornis Förster, 1914.
Remarks. Only two species of Protobehningia are known in the world: Protobehningia asiatica Tshernova & Bajkova 1960 and Protobehningia merga Peters &Gillies 1991. Peters andGillies (1991) used larval exuviae of P. merga for comparison with P. asiatica, but they did not give a more detailed description of the larval stage. The labium structures of our specimens are similar to those of the larval exuviae described by Peters and Gillies (1991). Our new record also expands the geographic distribution of P. merga to northern Thailand. Tarsi of forelegs not fused to tibiae (Fig. 3A); tibiae of hind legs reduced (Fig. 3D)  Tarsi of forelegs fused to tibiae (Fig. 6B); tibiae of hind legs not reduced (Fig. 6D); glossae and paraglossae with more than 20 long stout setae on the ventral surface (Fig. 5C) (Fig. 11A), ii) a subapical cluster of setae on the foretibia, iii) short mandibular tusk (0.10-0.23× length of the head) (Fig. 10A), and iv) relatively small body length.
Distribution. Chanthaburi, Kanchanaburi, Nan, and Chiang Rai provinces. Remark. The adult of P. formosus was described by Eaton (1892) based on materials from Myanmar. Imanishi (1940) described the species Potamanthus kamonis based on imaginal and larval materials from Japan, and P. kamonis was synonymized with P. formosus by Uéno (1969). Potamanthus formosus is widely distributed in East Asia and Southeast Asia (China, Japan, South Korea, Malaysia, Vietnam, Myanmar, and Thailand). In the present study, the specimens were found in eastern, western, and northern Thailand, so P. formosus is the most widespread potamanthid in Thailand (Fig. 14).     Diagnosis. The larvae of Rhoenanthus magnificus (Fig.12) can be distinguished from those of other Rhoenanthus (Potamanthindus) species based on the following characteristics: i) large body size (18-21 mm), ii) mandibular tusks arched inward about 33-34° (angle measurement as shown in Fig. 10D), iii) length of the mandibular tusks ca 1.4× length of head, and iv) length of the foretibiae ca 1.5× length of the forefemora and about 2.9× length of the foretarsi (Fig. 11B) (Nguyen and Bae 2004).

Rhoenanthus (Rhoenanthus) distafurcus Bae & McCafferty, 1991
Diagnosis. The larvae of Rhoennanthus distafurcus can be distinguished from those of other Rhoenanthus (Rhoenanthus) species based on the following characteristics: i) subapical spine of the mandibular tusk well developed laterally (Fig. 10E), with a simple, short spine, ii) 16-20 medial rounded setae on mandibular tusk (Fig. 10E) iii) length of the mandibular tusks ca 1.7-1.9× length of head, iv) length of foretibiae ca 1.19-1.25× length of the forefemora and about 2.5-2.8× length of the foretarsi, (v) leg with colour marking as in Fig. 11D, and vi) lack of bipectinated setae on the mandible (Soldan and Puthz 2000).

Rhoenanthus (Rhoenanthus) speciosus Eaton, 1881
Diagnosis. The larvae of Rhoenanthus speciosus can be distinguished from those of other Rhoenanthus (Rhoenanthus) species based on the following characteristics: i) lateral subapical spine of the mandibular tusk present (Fig. 10F), ii) absence of medial rounded setae on the mandibular tusk, iii) length of the mandibular tusks ca 1.7-2.3× length of the head, iv) length of the foretibiae ca 1.2-1.23× length of the forefemora and about 2.72-2.82× length of the foretarsi, v) leg with colour marking as in Fig. 11E, and vi) 4 or 5 bipectinated lateral setae on the mandibles (Bae and McCafferty 1991).
Remark. The larvae of Rhoenanthus speciosus were reported by Bae and McCafferty (1991) based on specimens from Indonesia and Malaysia. Parnrong et al. (2002) reported this species from Songkhla province (southern Thailand). In the present study, we found the larva of this species in the nearby Narathiwat province. The distribution of R. speciosus seems to be restricted to the south of the Isthmus of Kra, as was found for another mayfly species, Prosopistoma wouterae (Boonsoong & Sartori, 2019). These findings constitute the northern limit of the known distribution of this species.
Key to genera and species of Potamanthidae in Thailand (adapted from Bae and McCafferty 1991;Nguyen and Bae 2006) 1 Mandibular tusks subequal to, or longer than 1/2 length of head (Fig. 10C Mandibular tusks with large lateral subapical spine with 16-20 medial rounded setae (Fig. 10E)

Molecular analysis
The partial sequence of the mitochondrial COI gene (658 bp) of P. merga (MW792224) found in Thailand was analysed and compared with the sequence of Dolania americana (BIT011-04) from BOLD. However, there is no available sequence for the genus Behningia. The intergeneric genetic distance between these two genera was 22.39%, as determined by the Kimura 2-parameter (K2P) model. For Potamanthidae, the phylogenetic tree of the ML analysis is shown in Figure 15 and depicts four clearly separated clades delineating four species. No sequence of Rhoenanthus speciosus are included due to unsuccessful DNA extraction. Analysis of the K2P genetic distance to confirm the species delimitation revealed that the intraspecific genetic distances vary between 0.2-5.4%, whereas the interspecific distances are high, ranging from 14-20% (Table 2). The lowest interspecific distance value was found between R. magnificus and R. obscurus (14%), which share close morphological characters.  Figure 15. The COI phylogenetic construction based on the maximum likelihood (ML) analysis of family Potamanthidae in Thailand.

Discussion
The discovery of an additional species of Behningia in Thailand reveals the high diversity of the behningiid mayflies in the country. The presence of B. nujiangensis was confirmed based on morphological evidence according to Zhou et al. (2019) and McCafferty and Jacobus (2006). In the present study, larvae of B. nujiangensis were collected from a stream in Chiang Mai province, whereas Zhou et al. (2019) described the species from the Nujiang river (China, upper Salween river), a short section of the river that flows through northern Thailand (Fig. 9). The habitat of B. nujiangensis is restricted to sandy bottoms in streams or rivers. The larval exuviae and imagoes of P. merga were known only from a river in Kanchanaburi province (western Thailand) by Peters and Gillies (1991). In addition, larvae of P. merga were collected from the Mae Chaem river, Chiang Mai province (northern Thailand). This a second report and a new distribution record for this species. The eggs of the genus Behningia are the largest known among mayflies, with B. nujiangensis reaching more than 1 mm in length. The length of eggs of Behningia lestagei (0.9-1 mm) and Dolania americana (0.7-0.8 mm) were reported by Keffermul-ler (1959) and Koss and Edmunds (1974). The egg structure of B. nujiangensis is similar to that of Behningia lestagei. The position of adhesive material differs between Behningia and Dolania. However, the egg of Protobehningia is still unknown. The presence of R. magnificus in Thailand was confirmed based on the morphological characters proposed by Nguyen and Bae (2004). This species is a new record in Thailand. In this study, the larvae of R. magnificus were collected from streams and rivers, where they were often found at the interface of small stones and finer substrate (sand and gravel) in the slow current streams, as previously reported by Nguyen and Bae (2004). Our results allow us to conclude that five valid species of the family Potamanthidae exist in Thailand, as supported by morphological and molecular analyses.

Conservation issues of the Behningiidae
The larvae of Behningiidae are restricted to fine sandy habitats (Peters and Gillies 1991;McCafferty and Jacobus 2006;Park et al. 2019;Zhou et al. 2019). The habitat of Behningia tshernovae Edmunds & Traver, 1959 in Korea is restricted to fine sand streams, and high-quality water is needed for its survival (Park et al. 2019). Behningia ulmeri is a very rare and extremely endangered European lowland species. In Poland, it may have become extinct as well, and any protective measures there would seem useless (Bauernfeind and Soldán 2012). Among the Thai behningiid mayflies, B. baei was found in the Klong Namkub (Phitsanoluk province) in 2002 and B. nujiangensis in the Tard Luang waterfall (Chiang Mai province) in 2011, whereas Protobehningia merga was found in the Khwae Noi river (Kanchanaburi province) in 1987 and has not been found again in re-samplings. Thai streams and rivers are altered by channel alterations, dam constructions, and sand harvesting. The sandy habitats have gradually decreased in Thailand, and this has threatened the survival of sand-dwelling organisms, including behningiid mayflies. The conservation of fine sandy habitat is, therefore, required to protect this extremely specialized psammophilous fauna.