Heptageniidae (Insecta, Ephemeroptera) of Thailand

Abstract Nine genera and twenty-two species of heptageniid mayflies from Thailand are defined in this present work as well as one suggested further subgenus, Compsoneuria (Siamoneuria) kovaci (species “incertae sedis”) including some particular characters. Taxonomic remarks, diagnoses, line drawings of key characters, distribution, habitat and biological data, and a larval key to the genera and species are provided. The chorionic eggs of eight genera and eight species were observed and shown using a scanning electron microscope.


Introduction
Heptageniidae is a family of mayflies with around 509 described species and distributed mainly in the Holoarctic, Oriental, and Afrotropical regions (Barber-James et al. 2008). Wang and McCafferty (2004: Part I) "analyzed the generic relationships and presented a phylogenetic classification of the family" while Webb and McCafferty (2008: Part II) defined the genera providing an illustrated key. Following Webb and McCafferty (2008), only 16 genera but more than 150 species of Heptageniidae can be found in the Oriental region (Soldán, 2001).
Heptageniid mayflies are one of the most abundant and common components of benthic communities in Thai running waters. The larvae inhabit slow to fast flowing streams where they occur on the surface of rocks, logs, vegetation, and leaves. Many heptageniid species have been used as indicators of anthropogenic disturbance because they are relatively intolerant of pollution change and as sensitive indicators of organic pollution (Hilsenhoff 1988) and metal pollution (Courtney and Clements 1998, Deacon et al. 2001, Clements 2004, Clark and Clements 2006. Furthermore, individuals of this family were test subjects of toxicity and drift behavior studies (Diamond et al. 1992, Céréghino et al. 2004, Stitt et al. 2006. Heptageniidae have been recorded from Thailand by Polhemus and Polhemus (1988), Braasch (1990), Sites et al. (2001), Sangpradub et al. (2002), Wang and McCafferty (2004), Braasch (2006a), , and recently by Boonsoong (2009, 2010). However, taxonomic revision of the family Heptageniidae in Thailand is urgently needed, because the study of life stages is still in its infancy. This is mainly due to problems of identification, unsettled generic questions, and the lack of use of modern genetic methods to construct a phylogeny of the family Heptageniidae from Southeast Asia. In this paper, we provide a larval key to known genera and species of Thai heptageniid mayflies, with particular emphasis on the problems of identification of several species. Taxonomic remarks, diagnoses, line drawings of key characters, distribution, and habitat and biological data are provided. In addition, the egg chorions of eight Thai heptageniid species were observed. All of the egg specimens used in this study were obtained from mature larvae and adults. The material was first preserved in alcohol and then critical-point dried using carbon dioxide and finally placed on holders and coated with gold. The oological observations of eight heptageniid species were made with a JEOL JSM-5600LV scanning electron microscope. The terminology provided by Koss and Edmunds (1974) is used in this paper.
In the following key and text, abbreviations are as follows: alt (altitude), asl (above sea level), μS/cm (microsiemens per centimeter), comb. Lamellae of gills 1 greatly extended beneath the abdomen (Fig. 1C-D Abdominal terga 2-9 each with long, acute median spine on posterior margin (Fig. 1F) Braasch -Abdominal terga 2-9 each without acute median spine on posterior margin (Fig. 1G) Gill-pairs 1 meet or overlap ventrally to form a friction disc ( Fig. 2A)  Gills 7 slender and pointed (Fig. 4A); robust setae on inner surface of hindtarsi pectinate (Fig. 4B)  Remarks. Epeorus is widely distributed in the Holarctic, northern portion of the Neotropical, Palaearctic, and Oriental regions (Kluge 2004). In tropical Southeast Asia, species of this genus have been reported and described by Braasch (1990Braasch ( , 2011, Braasch and Boonsoong (2010), Soldán (1979, 1984c), and . Nguyen and Bae (2004a) provided larval descriptions and a key to six species of Vietnamese Epeorus ; this was the first comprehensive taxonomic study of the larvae of Epeorus from tropical Southeast Asia. The first record of Epeorus in Thailand is of E. aculeatus Braasch (1990) from Doi Inthanon National Park, Chiang Mai province. Recently, the first Thai imago of E. aculeatus was described by  from Chiang Mai province. Originally, E. unicornutus was recorded from Himalayas (Braasch 2006b) although it had been already collected in Thailand December 1987/1998 from the river Nam Lang, Soppong / Pangmapa, Mae Hong Son province (Braasch, unpublished). Currently, seven species of Epeorus are known from Thailand (Braasch and Boonsoong 2010 Eggs. Egg chorion of E. khayengensis very smooth without any peculiar structure (Fig. 5A), 2-3 micropyles visible in the equatorial area (Fig. 5B).
Diagnosis. The larva of E. khayengensis (Fig. 9D) can be distinguished from that of other congeners by abdominal terga 2-9 without long acute median spine on posterior margin, but bearing long hair-like setae.
Habitat and biology. The larva of E. khayengensis inhabits tropical headwater streams approximately 210 m in alt. The streams range between 6-7 m in width and 10-11 cm in depth. The water temperature ranges between 22-25°C, pH between 6.35-7.15, total dissolved solids are between 27-34 mg/L, and conductivity is between 41-53 μS/cm. The larvae are found in eroded areas of streams where moderately flowing over cobble and sandy bottom.
Remarks. Braasch and Boonsoong (2010)  Diagnosis. The larva of E. unicornutus can be distinguished from that of other congeners by the combination of the following characters: abdominal terga 2-9 each with single, prominent, acute median spine and with a row of short spines on posterior margin, tergum 10 with short spines and hair-like setae on posterior margin.
Habitat and biology. Larvae of E. unicornutus are found in headwater streams shaded under tree canopies in mountainous areas (alt 330 m) where the streams are 10-12 m wide and 10-15 cm in depth. The water temperature ranges between 22-23°C, pH between 7.0-7.2, total dissolved solids range between 18-20 mg/L, and conductivity between 28-30 μS/cm. The larvae are found underneath stones in fast flowing reaches of the streams. The coarse mineral substrate consists of boulder (60%), cobble (30%), gravel and coarse sand (10%), and abundant fallen leaves. E. unicornutus is found under minimally disturbed conditions of Nam Thob streams, Loei province.
Remarks. Braasch (2006b) described this species based on larval specimens from River Indravati near Dalaghat 1,200 m, Nepal, Himalayas. In this study, this species were found from northern and northeastern parts of Thailand. This species is different from other known species of the genus Epeorus by abdominal terga 2-9 each with single, prominent, and acute median spine.

Distribution. Khun Kon Waterfall (Chiang Rai province).
Diagnosis. The larva of E. martinus can be distinguished by the following characteristics: pairs of moderately long acute submedian spines on the abdominal terga 1-9, large but narrow gill 1 forming a sucking disc and gill 7 being unfolded.
Habitat and biology. E. martinus larvae are found in mountain streams with a moderate current, at an elevation of 300-2800 m (Nguyen and Bae 2004a).
Remarks. Webb and McCafferty (2008) did not recognize the subgenera of Epeorus (e.g. Belovius, Iron). However, we hold on the validity of subgenus Belovius and Iron within genus Epeorus (Boonsoong and Braasch, 2010). Therefore, we propose Iron martinus Braasch & Soldán, 1984  Diagnosis. In contrast to the larva of E. longitibius with foretibiae 1.2 × length of forefemora, E. thailandensis has foretibia of equal length; forefemur of new species is with a small femoral spot, E. longitibius without such one; the gills 2-6 are quadrangular and gill 7 has a fold in E. thailandensis, but in E. longitibius gills 2-6 are elongated and gill 7 unfolded.
Habitat and biology. The larvae of E. thailandensis live presumably in high mountain streams with high oxygen concentrations and a faster current where the substrate is mostly stony at an elevation of 2000 m.
Remarks. Boonsoong and Braasch (2010) misidentified this species as Iron longitibius Nguyen & Bae, 2004 (questionable). In this study, we have re-checked them and found , then we re-identified it as a new species, Epeorus (Iron) thailandensis. The new species is different from the larva of Iron longitibius by characters within length of foretibia, femoral spot, and gill shape of gills 2-7. In this observation, we propose as the new species. Braasch, 1990 http://species-id.net/wiki/Epeorus_aculeatus Diagnosis. The larva of E. aculeatus can be distinguished from that of other congeners by abdominal terga 2-9 ( Fig. 1A) each bearing a pair of long, acute, submedian spines on its posterior margin, and with a median dark brown spot on femoral surface.

Epeorus (Epeorus) aculeatus
Habitat and biology. The larva of E. aculeatus occurs in headwater streams between 600-740 m alt in Thailand and high mountain streams between 1,400-2,800 m in Vietnam (Nguyen and Bae 2004a). The larvae are mostly found under rocks in fast flowing reaches of the streams. The substrate consists of mixed sand/gravel and larger stones such as boulders and cobble.
Remarks. Braasch (1990) described this species based on larval specimens. Then,  described the male imago of E. aculeatus based on reared Thai specimens from Doi Suthep NP, Chiangmai province. This species is different from other known species of the genus Epeorus by each bearing a pair of long, acute, submedian spines on its posterior margin.
Diagnosis. The larva of E. bifurcatus can be distinguished from that of its congeners by pairs of small submedian dorsal tubercles on tergites with acute spines and gill 1 larger than gill 3. In the Figs 7-8 of Nguyen and Bae (2004a) some characters (the femoral spot, the rim of hairs of the hind margin of head and pronotum) are not described. Furthermore, according to our material, the head of the type species is smoothly trapezoid without markings in anterior median half, however, the head is transversely ellipsoid with a broad dark band between its front and hind margins.
Habitat and biology. Nguyen and Bae (2004a) noted that the larva of E. bifurcatus occur in mountain streams ranging between 200-600 m. They were found on the underside of stones in fast flowing sections of the streams.
Remarks. Braasch and Soldán (1979) described this species based on larval specimens and original descriptions are written in Germany. Then, Nguyen and Bae (2004a)  Diagnosis. This species resembles E. bifurcatus however, the larva of E. inthanonensis has paired tubercles on the terga which are more rounded than in E. bifurcatus; they lack spines and gill 1 is smaller than gills 2-6.
Habitat and biology. Braasch and Boonsoong (2010) noted that the larva of E. inthanonensis was found in small mountain streams ranging between 800-2000 m.
Remarks. This species was described by Braasch and Boonsoong (2010). This species resembles E. bifurcatus however, the larva has paired tubercles on the terga which are more rounded than in E. bifurcatus.

Remarks.
The genus Rhithrogena is the most diverse in the Holarctic, with numerous species also in the Palaearctic Asia. However, it seems to be under-represented in Southeast Asia. Braasch and Boonsoong (2009) described R. siamensis Braasch & Boonsoong, 2009 from northern Thailand, this species also occurs in the northeastern and western parts of Thailand. Only two further species of the genus can be found in this area: Rhithrogena parva Ulmer, 1939 from Taiwan (Ulmer 1912) and Rhithrogena diehliana Braasch & Soldán, 1986 from Sumatra (Braasch and Soldán 1986c). Eggs. General shape ovoid. One of the poles terminates with large knob-terminated coiled threads (KCTs) (Fig. 5C), the whole chorion is covered with uniform granules and scattered with small loose KCTs, with a large micropyle on the equatorial plane (Fig. 5D).
Diagnosis. The larva of R. siamensis resembles Rhithrogena (Tumungula) unica Zhou and Peters, 2004 but differs in mouthparts structure and gill 1, with R. siamensis being pointed-crenulate and having longer plica which are more bluntly rounded, whereas that of R. (T.) unica has a few angular crenulations and a shorter, more strongly rounded plica.
Habitat and biology. Rhithrogena siamensis larvae cling to rock surfaces in medium-to fast-flowing water. Collections over most of the year revealed that the flight season at altitudes of 600 m was mainly during March/April, just before the beginning of the monsoon rains in May.
Remarks. Only one species of Rhithrogena was identified in our study as being distributed thoughout Thailand. They live in a rapid current of stream.  . Only one species, so far, is found in northern and northeastern Thai streams.   Kluge, 2004: 173. (comb.) Larva. Braasch and Soldán 1988: 119-124, Figs 1-13. Adult. Unknown. Eggs. Unknown. Distribution. Doi Suthep, Chiang Mai province; Loei province; Soppong, Mae Hong Son province.

Genus
Diagnosis. Trichogenia maxillaris can be differentiated from congeners by the following combination of characteristics: gill lamellae 2-7, long, narrow and sharply pointed; base of outer canines of mandibles without dense lateral brush of setae; supracoxal sclerites short. The other Trichogenia species have gill lamellae 5-7 rounded with a pointed apex, base of outer canines of mandibles with dense lateral brush of setae, and supracoxal sclerites long and pointed.
Habitat and biology. Trichogenia maxillaris larvae occur in small mountain streams. To date, only a few Trichogenia specimens have been collected in Thai streams. This species appears to be a sensitive indicator because larvae were found exclusively in forest stream areas.
Eggs. Chorionic pattern of geometrically arranged small KCTs covering the entire egg surface (Fig. 6A) and interspersed among crenulated granules, folded surface of the chorion, many microgranules densely scattered all over the surface of the chorion, large micropyles on equatorial plane (Fig. 6B).
Distribution. Mae Hong Son province, Mae Chaem district, Doi Inthanon NP, Doi Suthep NP (Chiang Mai province) Diagnosis. Larvae of N. baei are easily identified by having a dorsal median abdominal ridge on tergites 1-9 and denticles on the claws. Larvae of the Indian species Notocanthurus edentatus Braasch, 1986 have no dorsal ridges on the abdomen (Braasch 1986); however, all Himalayan species of Notacanthurus key out by the absence of denticles on the claws. Its prospective penis (Fig. 14, Braasch 1986) is quite unlike the bilobed penes of the Himalayan Nothacanthurus (Figs 4-8, Braasch 1986)" and is expected to be a simple, not bilobed, penis in the male imago.
Habitat and biology. Nothacanthurus baei larvae inhabit small streams and brooks. Larvae were usually found together with those of Asionurus species.
Distribution. Widely distributed in many parts of Thai streams. Diagnosis. The combination of having a distinctly thickened anterior margin of the head capsule, long posterolateral spines on the abdomen, acutely pointed supracoxal spurs, and well-developed lamellae on gills 1 will distinguish Thalerosphyrus from other Ecdyonurinae genera. Within Thalerosphyrus is a 'sinuosus' group of species with larvae having the above-mentioned combination, having in both sexes of adults "costal and subcostal fields with two indistict umbra-brown spots, the first at the beginning of pterostigmatic region, the second directly to the wing tip" (Navás 1933) and a 'determinatus' group, having larvae with short posterolateral spines; however, both sexes of adults have umbra-brown tinged costal and subcostal fields on forewings. In general, the species differentiation in the genus (T. 'sinuosus' group) is unsatisfying and requires verification. The larva of T. sinuosus can be differentiated by the combination of the following characters: gill 3 of rounded shape is much less wide than that of T. vietnamensis (Dang, 1967) Braasch & Boonsoong, 2010. and while in T. flowersi (Venkataraman and Sivaramakrishnan, 1987) the inner side of gill 1 has a straight margin, that of T. vietnamensis (=T. sinuosus?) is slightly concave.
Habitat and biology. T. sinuosus larvae (Fig. 9F) are one of the most widespread mayfly species in Thailand. The larvae are found underneath stones in slow-flowing reaches of streams (water velocity approximately 3-7 cm/sec, water depth ranges between 7-17 cm). The larvae cling to submerged boulders and cobbles. Because they feed by grazing on diatoms, algae and detritus on stream rocks, they prefer rocky substrates in fairly clear to silty sediments.
Remarks. Only one species of Thalerosphyrus (T. sinuosus) was identified from Thailand and this species widely distributed in Thai streams. The larva and adults of this species were adequately described by Braasch and Soldán (1984b) and Navás (1933). Remarks. The heptageniid mayfly genus Compsoneuria was reviewed by . Eleven species were revised from Afro-tropical and Oriental regions. Compsoneuria larvae were found abundantly in lowland rivers on mainland Southeast Asia. There are three species of Compsoneuria mayflies reported from Thailand (Braasch 1990;Braasch 2006a;Braasch and Boonsoong 2010). Compsoneuria larvae occur among submerged vascular plants or roots. Ulmer, 1939 http://species-id.net/wiki/Compsoneuria_thienemanni Figs 3I-J, 7A-B, 9C
Distribution. Mae Hong Son province, Chiang Mai province, Trat province. Diagnosis. Compsoneuria thienemanni is recognised by having gills 2-6 without emarginations and gill 7 leaf-like and pointed apically; pale dots and marks on abdomen as shown in Fig. 3I.
Habitat and biology. Compsoneuria thienemanni larvae (Fig. 9C) are found abundantly in the large rivers at lower altitudes where it is encountered clinging to floating submerged water plants. A few male specimens of C. thienemanni were found in the mountainous region of Mae Hong Son province.
Remarks. The larva and adults of this species were described by Ulmer (1939) and Braasch and Soldán (1986b). The larvae of this species are found abundantly in the low-gradient streams and large rivers in Thailand.
Habitat and biology. Both C. thienemanni and C. langensis larvae are mainly found attached to floating water plants or thick pads of green algae.
Remarks. Braasch and Boonsoong (2010) described this species from Thailand based on larval and imaginal specimens. The larva of this species is different from other known species by gills 2-6 without emarginations, gill 7 lanceolate and rounded apically.

Remarks.
A brief comment on the appropriate placement of C. (S.) kovaci in Braasch and Boonsoong (2010) is required. Larval diagnosis of Compsoneuria (Siamoneuria) is in contradiction to that of Compsoneuria, namely in lacking the combination of long, sharply pointed supracoxal spurs, black spotting on the head capsule and femora, and narrow, apically pointed glossae. Siamoneuria cannot belong in the genus Compsoneuria but probably deserves its own status; it appears to be a morphospecies whose characters are not in accordance with other known genera. For a final definition, important missing details of mouthparts and eggs should be included. For now, we see it as a species as "INCERTAE SEDIS" (Braasch and Boonsoong 2010 Distribution. Nam Lang river, Soppong, Mae Hong Son province, Diagnosis. Larva is conspicuous due to emarginations on gills 2-6, gill 7 being narrowly lanceolate, the head without markings, and the body with paired paramedian spots on tergites 5-8, large median spot on 9, and three distantly arranged small spots at anterior margin of tergite 10.
Habitat and biology. The habitat of the single larva found was submerged roots of a tree standing at the river bank.

Diagnosis.
A. primus differs from A. ulmeri (Braasch and Soldán 1986a) by shorter and more pointed wings of hypopharynx and gill 7 with bulging anterior portion and acutely shaped apically. In A. ulmeri the wings of hypopharynx are longer and have rounded ends whereas gill 7 is narrow and long and hardly extended anterioriorly.  Braasch, 1990 G lamella of gills 7 of A. rainulfiana Braasch, 1990. Habitat and biology. A. primus larvae (Fig. 9B) are often the most abundant in small mountain streams of Thailand. Larvae live beneath rocks and debris. They were found together mostly with those of Notacanthurus baei. Larval habitat preference is similar to that of larvae of N. baei.

Rhithrogeniella ornata
Diagnosis. Male of A. namnaoensis is separated from Vietnamese A. cervina in lacking a median penial cone, by the less deeply notched lobal apex, and titillators curved laterally and in their more medial position. Vietnamese A. dama presents a terminal apex of the penis slightly notched at the inner angles, whereas A. namnaoensis is recognized by somewhat elevated corners on both sides of the apices. Larvae are recognizable by an unmarked forehead, pointed and weakly curved gill 1 and smoothly rounded triangular gill 5 with crosswise projection. Two Vietnamese species, Afronurus meo and A. mnong (Nguyen and Bae, 2003) have gills lacking these projections (Figs 8-10, Figs 18-20, Nguyen and Bae 2003).
Habitat and biology. Larvae of A. namnaoensis (Fig. 9A) are probably the most abundant species on rocks and stones in Nam Lang River and elsewhere in current waters of northern and northeastern Thailand. These mayflies are an important food source for headwater stream fishes (Cyclocheilichthys apogon, Devario regina, Opsarius pulchellus, and Rasbora rasbora).
Habitat and biology. The larvae of A. rainulfiana were found in headwater streams. Remarks. Only larva of A. rainulfiana was reported from northern part of Thailand and was described by Braasch (1990). The adults of A. rainulfiana are unknown.
Diagnosis. This species is unique in the genus because of its abdominal pigmentation: terga pale yellow medially and reddish laterally. The male genitalia have an obvious projection between the two lobes. The larvae of this species are larger and have more pale dots and marks on head and body than those of the other known species (Figs 7, 9, 10, Zhou and Zheng 2003), gill 5 or 6 are provided with a small, thin projection (Fig. 8).
Habitat and biology. Larvae of A. rubromaculata were the only representatives of Afronurus encountered in the large river Mekong in February 2002 along the Thai-Laos border in the utmost north of Thailand. It is regularly found as a resident together with the dominant A. namnaoensis on stones and rocks in Nam Lang River, altitude 600 m (Braasch 2006). This species is also found in Vietnam.
Remarks. The larva and adults of A. rubromaculata were adequately described by You et al. (1981), Wu et al. (1986) and Zhou and Zheng (2003). Only larva of A. rubromaculata was reported in Thailand (Braasch and Boonsoong 2010).
A key to the eggs of known genera and species of Heptageniidae in Thailand 1 KCTs absent (Fig. 5A)  Rounded tubercles are scattered all over the surface of the chorion; micropyle border not strongly thickened (Fig. 6C)  Chorionic surface folded, with many densely scattered crenulated granules; small KCTs covering the entire egg surface (Fig. 6A)  Chorionic surface with many sizes of granular matrix; large KCTs randomly scattered (Fig. 7A)

Conclusions and recommendations
Heptageniidae is the most diverse and abundant mayfly family in Thailand. The total number of Thai Heptageniidae described to date amounts to 9 genera and 22 species. The results presented here show that the Thai heptageniid fauna is dominated by Oriental genera. In addition, three Southeast Asian endemic genera morphospecies (Asionurus, Siamoneuria and Trichogenia) are found in Thailand. The species of the genera Afronurus and Thalerosphyrus are the most abundantly and widely distributed species found in Thai streams. Early studies of Thai heptageniid mayflies provide an important base on which to continue the study of these insects. It is important, now, to investigate the fauna further, which will lead to a better biogeographical understanding, and, at the same time, to begin a study of the biology and ecology, which is still very limited for species recorded in Thailand. Data on the ecology of heptageniids in Asian streams is limited. Dudgeon (1996) gives some preliminary data on life histories of five Hong Kong species, and estimates of their secondary production. Boonsoong (2002) presents some preliminary data on life history and diet of Afronurus species from Nam Nao National Park. These larvae show a non-seasonal multivoltine. Based on gut analyses, larvae are non-selective generalists. They feed mainly on detritus and diatoms and could be categorized as scrapers. Larvae of heptageniid mayflies often occur in reaches of fast-flowing streams where mixed substrates are composed of cobble, pebble, and gravel. In general, the distribution of heptageniid larvae depends upon substrate type and water current. In addition to basic taxonomic research, revision of unclear or poorly defined genera, and association of larval and adult stages by rearing, investigation priorities of the Thai Heptageniidae can be summarized briefly as follows: study of life cycle and ecology of individual species distribution; of heptageniid larvae with respect to different water conditions.