Vietnamellidae (Insecta, Ephemeroptera) of Thailand

Abstract The genus Vietnamella Tshernova, 1972 is investigated in detail for the first time in Thailand. As a consequence, four species are recognized, namely Vietnamella maculosasp. nov., Vietnamella thani Tshernova, 1972, Vietnamella sp. B and Vietnamella sp. C. Herein, larvae and eggs of V. maculosasp. nov. are described and reported from Chiang Rai Province. The larva of Vietnamella sp. B from Tak Province is also described, but not named due to insufficient material, and the imaginal stages and eggs of V. thani Tshernova, 1972 are described and presented for the first time. Our morphological evidence is supported with COI data. The phylogeny showed that four different lineages of the genus Vietnamella occur in Thailand, one of them, viz., Vietnamella sp. C, only known from a couple of COI sequences retrieved from the Barcode of Life Data System (BOLD). Diagnoses for all known Oriental species are also presented.


Introduction
The monogeneric family Vietnamellidae was originally established by Tshernova (1972) [type species: Vietnamella thani Tshernova, 1972] based on larval specimens. The status of the family and the taxonomic history of the genus Vietnamella Tsher-nova, 1972 are reviewed by Jacobus et al. (2005) and Hu et al. (2017) respectively. Nowadays, three species have been described from the Oriental region. They are V. ornata (Tshernova, 1972), V. sinensis (Hsu, 1936) [=V. dabieshanensis You & Su, 1987, V. qingyuanensis Zhou & Su, 1995, V. guadunensis Zhou & Su, 1995, and V. thani (Hu et al. 2017). The larval and imaginal stages of V. sinensis have been described, whereas V. thani is only known from the larval stage and V. ornata (Tshernova, 1972) only from the sub-imaginal stage. Recently, Vietnamella sp. A was described from India, based on its larvae which present the unique character of outer projections on the head with serrations (Selvakumar et al. 2018). However, the authors were unable to confirm species identification using current morphological and molecular data because the larval morphology of V. ornata has never been described, and the distribution of Vietnamella sp. A is close to the type locality of V. ornata (Selvakumar et al. 2018).
The genus Vietnamella is endemic in the Oriental region and is distributed in China, Thailand, India and Vietnam (Tshernova 1972;Jacobus et al. 2005;Hu et al. 2017;Selvakumar et al. 2018). In Thailand, Vietnamella has never been reported at the species level. Here, we review the species of Vietnamella in Thailand and describe a new species in the genus and the imaginal stages of V. thani based on reared specimens. A distribution map and mitochondrial COI sequence data are also provided.

Ethics statement
The present study was approved by the ethics committee of Kasetsart University (approval no. ACKU61-SCI-029) for rearing and collecting the mayfly specimens.

Morphological observations
The vietnamellid larvae were collected from fast-flowing areas of streams in northern and western Thailand. The imagoes were reared from mature larvae in the laboratory. Measurements (mm) and photographs were taken using a Nikon SMZ800 stereoscopic microscope and a Canon EOS 6D camera with MP-E 65 mm macro lens. For scanning electron microscopy (SEM), eggs were dried, coated with gold and observed with a FEI Quanta 450 SEM instrument. Final plates were prepared with Adobe Photoshop CC 2017. The specimens are deposited in the collection of the Zoological Museum at Kasetsart University in Bangkok, Thailand (ZMKU) and at the Museum of Zoology in Lausanne, Switzerland (MZL). The distribution map was generated via the Simple Mapper website using GPS coordinates (http://www. simplemappr.net).

Molecular analysis
The collected specimens were fixed in absolute ethanol and preserved under refrigeration for description and DNA extraction. Collection details of the specimens of the three species used for the DNA experiment are shown in Table 1. Part of the specimens was extracted by using non-destructive methods. Total DNA was extracted 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 (658 bp) using the primers LCO1490 (5'-GGT CAA ATC ATA AAG ATA TTG G-3') and HCO2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3'), designed by Folmer et al. (1994). Polymerase chain reaction (PCR) conditions were as follows: a 25 μl final total volume containing 12 μl of PCR Master Mix solution, 1.5 μl (10 μM) of each primer, 5 μl of DNA and 5 μl of sterile water. PCR was performed as follows: 5 minutes at 94 °C, then 30 seconds at 94 °C, 30 seconds at 48 °C and 60 seconds at 72 °C (40 cycles), and a final elongation step at 72 °C for 10 minutes (Gattolliat et al. 2015). Purification and sequencing were conducted by Macrogen, Inc. (South Korea). Sequence alignment and editing were performed using ClustalW. The phylogenetic tree was analysed by Bayesian inference using MrBayes. The evolution model obtained was General Time Reversible Model and Gamma distributed with invariant sites (GTR+G+I). Nucleotide sequences obtained in this study have been deposited in GenBank database. Other analysed mayfly sequences were obtained from the Barcode of Life Data System (BOLD): Vietnamella sp. C (THMAY031-09.COI-5P, THMAY148-12.COI-5P and THMAY149-12. COI-5P); and GenBank: Vietnamella sp. 1 (KM207084.1; KM244655.1) and V. dabieshanensis (HM067837.1). Other Ephemerelloidea COI sequences from GenBank including Dudgeodes palnius (LC057264.1), Teloganella indica (LC057266.1), Teloganopsis deficiens (HQ958649.1) were added to the analysis. Potamanthellus edmundsi (MN186576) was used as an outgroup. Description. Mature larva (in alcohol, Fig. 1A). Body length 11.25 mm without cerci; cerci 4.8 mm; body brown with dark brown markings on thorax and femora.

Taxonomy
Head. Brown with a pair of occipital tubercles, a single sub-occipital tubercle medially; two projections below eyes; inner pairs of projections small, spine-like and sharp ( Fig. 2A); outer pair large, triangular, cone shaped without any serrated spines (Fig. 4A, D). Labrum, similar to other vietnamellid mayflies, anterior half of dorsal surface and margins with relatively long setae, ventral surface with short setae (Fig.  2B). Labium, glossae width greater than length, glossae and paraglossae with dense setae on surface, setae on dorsal surface and margins longer; labial palpi three segmented, basal segment broader and longer than the second, apical segment very small; palpi with tiny setae (Fig. 2C). Left mandible, slender, a little concave at sub-median area; molar block-like shape with a tuff of short setae below inner molar margin (Fig.  2D). Right mandible, slender, slightly concave at sub-median area; molar block-like shape with a row of setae below inner molar margin (Fig. 2E). Maxillae slender; maxillary palpi three segmented, with tiny setae; length ratio from basal to apical = 1.3:1.2:1 (Fig. 2F). Hypopharynx, lingua square and superlinguae nearly round, with setae on surface ( Fig. 2G) Thorax. Pronotum with small moderately sharp anterolateral projections, and slightly pointed protuberances below the anterolateral projection (Fig. 3A). Forefemur, strongly expanded with serrations or teeth projections on ventral margin (Fig. 3B); transverse ridge serrated with small rounded setae (Fig. 5A, B) and long thin setae near inner dorsal margin. Midfemur without any projection, expanded, dorsal margin convex apically and with a row of hair-like setae (Fig. 3C). Hindfemur without any projection, expanded, longer than midfemur, dorsal margin with a row of hair-like setae (Fig. 3D). All claws similar, strongly hooked with a single small denticle basally (Fig. 3E).
Abdomen. Tergites I-X with a pair of median ridges or tubercles; posterolateral angles of tergites II-X extended into sharp projection; tergite VII with a pair of tubercles ( Fig. 4G) and tergite X with well-developed a pair of tubercles (Fig. 4J); lateral margins of tergite with dense setae. Gill on segments I-VII: gill I finger-like with setae ( Fig. 3F); gills on segments II-VI similar in structure, with dorsal and ventral lamellae, the latter further divided into two clusters, each with several smaller lobes   Diagnosis. The larva of Vietnamella maculosa sp. nov. is most similar to that of V. thani and V. sinensis in the lack of serrations on the outer projection on the head, but it can be separated from V. thani based on the following characteristics: i) the pattern of serration on the ventral margin of the forefemur, ii) the setae on the transverse ridge of the forefemur, iii) a well-developed pair of median ridge projections of tergite X. It can be separated from V. sinensis by the proportion of the maxillary palp on the second segment, which is slightly longer than on the third segment (1.2:1), whereas in V. sinensis it is clearly longer (1.6:1).
Remarks. The larvae of Vietnamella maculosa sp. nov. have a unique colour pattern, with brown and dark brown banding spread over the body and femurs. However, this colour can change in intensity depending on the life stage and environment. Other species, like V. thani, have variation in body colour but do not have this pattern.  Description. Larva (in alcohol, Fig. 1B). Body length 6 mm without cerci; body reddish with dense setae lateral and on margin of head.
Head. Reddish with a sharp pair of occipital tubercles and a single sub-occipital tubercle medially; two projections below eyes: inner pairs of projections small, spinelike and sharp; outer pair large, triangular, cone shaped with five unequal serrated spines (Fig. 4B, E), basal spine the largest. Labrum, similar to V. thani and V. maculosa sp. nov. Labium, glossae width greater than length, glossae and paraglossae with dense setae on surface, labial palpi three segmented, basal segment broader and longer than the second, apical segment small; palpi with tiny setae. Left mandible and right mandible slender, mostly similar to the other Vietnamella species. Maxillae slender, maxillary palpi three segmented with tiny setae, length ratio from basal to apical = 1.3:1:1. Hypopharynx, lingua and superlinguae nearly round with setae on surface.
Thorax. Pronotum with small sharp anterolateral projections, slight protuberances below the anterolateral projections. Forefemur strongly expanded with serrations or teeth projections on ventral margin; transverse ridge serrated with small setae. Midfemur without any projection. Hindfemur without any projection, expanded, longer than midfemur; dorsal margin with a row of hair-like setae. All claws similar, with one small denticle basally.
Abdomen. Tergite I-VI and VIII-X with pair of median ridges or tubercles progressively; tergite VII with a single tubercle (Fig. 4H); posterolateral angles of terga II-IX extended into sharp projection; tergite X with a pair of moderately-developed tubercles (Fig. 4K); lateral margins of tergite with dense setae. Gills on segments III-VI similar in structure with dorsal and ventral lamella, the latter further divided into two clusters, each with several smaller lobes; gill VII small, with two lamellae but ventral lamella divided into three lobes.
Diagnosis. The larva of Vietnamella sp. B can be separated from those of other species based on the following characteristics: i) outer pairs of projections on the head are large and stout, triangular, and cone shaped, with five unequally serrated spines (one large spine + four small spines), and ii) the abdominal tergites II-IX have a pair of projections or tubercles at the posterolateral margin except for tergite VII which has only a single projection (Fig. 4H).
Remarks. The larval description given herein agrees with larvae of other species belonging to the genus Vietnamella, including the presence of a pair of projections on the head, the expanded femur and the forefemur with serrations on the outer dorsal margin. The larva described here has serrated spines on the outer projection of the head that differ from the other valid species of Vietnamella.The outer serrated projection is similar to that of Vietnamella sp. A from India (Selvakuma et al. 2018), but it differs in the number and character of the outer projection spines (four equal serrated spines in Vietnamella sp. A, five unequal serrated spines in Vietnamella sp. B). Although Vietnamella sp. B was not a mature larva, the phylogenetic analysis showed it belonged to a clearly different clade and had a high genetic distance compared to the other species ( Fig. 12; Table 2). Formal description of this species is pending more material.
Distribution. Tak Province. Adult and egg. Unknown.   Diagnosis. The larva of Vietnamella thani can be distinguished from those of other Vietnamella based on the following characteristics: i) outer pairs of projections on the head are long, triangular, and cone shaped without serrated spines; ii) the first and second segments of the maxillary palpi have an equal length ratio; iii) the forefemur has a serrated transverse ridge with spatulate setae (Fig. 5C); and iv) the abdominal tergite X either lacks or has a poorly-developed pair of tubercles (Fig. 4L).
Remarks. The larvae of Vietnamella thani are widely distributed in Thailand. They inhabit fast-flowing streams (Fig. 10A) but have never been reported in southern Thailand. Vietnamella thani have notable outer pairs of projections on head without serrated spines that differ from Vietnamella sp. B. The larvae show colour variations and can be greenish, yellow or brown. The imaginal stages of V. thani have similar characters to those of V. sinensis but lack the longitudinal vein on the stigmatic area of the forewing and they have less crossveins between Sc and RA on the hindwings ( Table 4). The egg structure is covered by a membrane, which leads to unclear sculpturing of the surface, especially in the posterior part of the egg. Thus, the egg from this study showed little difference from the egg structure of V. sinensis.

Molecular analysis
The phylogenetic tree of vietnamellid mayflies and the other families of Ephemerelloidea was constructed from 658 bp of COI sequences by Bayesian interference. The results show that Vietnamellidae is clearly separated from the others. Six clades can be recognized within Vietnamellidae with high posterior probability value support for the morphospecies: Vietnamella maculosa sp. nov., Vietnamella sp. B, Vietnamella sp. C, Vietnamella sp. 1, V. dabieshanensis and V. thani (Fig. 12) Moreover, K2P genetic distance was analyzed to confirm species delimitation. The intraspecific genetic distances vary between 0-6.7 % whereas interspecific distances are very high, ranging from 16-31% (Table 2). The phylogenetic tree and K2P value result confirm that there are four different species of Vietnamella in Thailand.

Discussion
Comparison of the larvae of Vietnamella, including V. thani, V. sinensis, Vietnamella sp. A, Vietnamella sp. B and Vietnamella maculosa sp. nov., is presented in Table 3. Vietnamella ornata, which was previously reported from Yunnan, China is not included because its larvae are still unknown (Tshernova 1972). The comparisons showed that a major character, the serration of the outer projection on the head, can distinguish Vietnamella into two groups (serration and non-serration groups).The non-serration group consists of V. thani and Vietnamella maculosa sp. nov., which is most similar to V. sinensis. They have a second segment of the maxillary palp that is longer than the other segments in V. sinensis but is of medium or nearly equal length in V. thani and Vietnamella maculosa sp. nov. The serration group includes Vietnamella sp. A (India) and Vietnamella sp. B (Thailand).  Egg structure of Vietnamella species has a similar pattern of a polar cap covering half of the egg chorion; however, we found little difference between the three species known at that stage: V. maculosa sp. nov., V. thani and V. sinensis. In addition, V. maculosa sp. nov. has a rod-shaped KCT that is different from that in V. thani and V. sinensis which have oval-shaped KCT. The chorionic surface of V. maculosa sp. nov. and V. thani have a protuberance which is smaller than in V. sinensis (Hu et al. 2017, fig. 6). From our results, it appears that the egg structure can be useful for species identification at least in the three Vietnamella species investigated. Considering other ootaxonomic investigations, egg structure of some mayflies can be used for identification at the species level (Sivaramakrishnan and Venkataraman 1987;Ubero-Pascal and Puig 2007).
Our phylogeny indicated the existence of six different species of Vietnamella in the Oriental region, with four of them found in Thailand. Only one species, V. thani, can be found in western Thailand. Surprisingly, three species distributed in northern Thailand are revealed here by molecular analysis. Herein, only V. maculosa can be described as new species. However, we suppose that Vietnamella sp. B and Vietnamella sp. C are putative new species which could be formally described when more material becomes available. Although our molecular phylogenetic result clearly showed species delimitation in Thailand, there are still ambiguities in other areas where COI sequence or morphological data are incomplete. Thus, we infer that the sequences of Vietnamella sp. 1 (KM207084.1; KM244655.1) may belong to V. ornata because their specimens were collected near the type locality (Fig. 13) (Tang et al. 2014). Vietnamella sp. A was recently reported from India and the authors suspect it can represent the unknown larvae of V. ornata (Selvakumar et al. 2018). In addition, V. dabieshanensis (HM067837.1) is Figure 13. Distribution map of Vietnamella in the Oriental region. Each different shape represents a different species. Our specimens are only distributed in Thailand and including the data of Vietnamella sp. C from the BOLD system. Outside of Thailand distribution of Vietnamella is based on previous records including V. ornata (Tshernova, 1972), V. sinensis (Hu et al. 2017), Vietnamella sp. 1 (Tang et al. 2014), Vietnamella sp. A (Selvakumar et al. 2018) and V. thani (Hu et al. 2017 and unpublished data). now considered as a junior synonym of V. sinensis (Hu et al. 2017) and this sequence likely refers to this species.
Although ambiguous classifications of Vietnamellidae still remain, our results allow us to conclude that at least four valid species (V. thani, V. ornata, V. sinensis and V. maculosa sp. nov.) exist, as supported by our morphological and molecular analyses. The findings of this study also extend the species diversity, imaginal description and phylogeny for future considerations of the Vietnamellidae.
for short-term research in Lausanne, Switzerland. The authors would like thank the subject editor, Dr. Changfa Zhou, and Dr. Sivaramakrishnan KG for their comments on the manuscript. Finally, we are thankful to the members at the Zoology Museum of Lausanne, Switzerland for their assistance and the use of their facilities.