A New Species of Vietnamella Tshernova 1972 (Ephemeroptera: Vietnamellidae) from Thailand

Simple Summary The monogeneric family Vietnamellidae is endemic in the Oriental region and found only in the Indomalayan biogeographic realm. Seven nominal species have previously been described, but three are considered to be junior synonyms. However, some provisional species designations have been established. In this study we described formally Vietnamella sp. C as a new species, V. nanensis sp. n., based on the specimens used for initial DNA data and our expanded set of materials from Nan Province, Thailand. Based on morphology, the larva of the new species can be distinguished from those of other congeners. Additional mitochondrial cytochrome oxidase subunit I gene data for the new species are also provided. A key to larvae of all known species in the genus is provided. Abstract The larva, male subimago, female imago, and eggs of V. nanensis sp. n. are described based on specimens from Mae Hong Son and Nan provinces, Thailand. The female subimago is described based on a photograph of a specimen reared to the imago stage. The species previously was distinguished only by DNA barcode data and designated as Vietnamella sp. C. Based on morphology, the larva of the new species can be distinguished with the following combination of characteristics: (i) pattern of serration on the ventral margin of the forefemur, (ii) posterolateral margins of abdominal terga with pairs of acute tubercles, especially terga VI and VII, (iii) a well-developed pair of median ridge projections on tergum X, (iv) the second segment of the maxillary palp being about 1.3× the length of the third segment, and (v) females containing eggs with prominent protuberances on the chorionic surface. A key to larvae of all known species in the genus is provided.

From among the three provisional species, Vietnamella sp. C was only known from a few mitochondrial cytochrome c oxidase subunit I (COI) sequences that had been retrieved by Auychinda et al. [26] from the Barcode of Life Data System (BOLD). No previous attempt had been made to place the larval specimens from which the DNA had been obtained to a species, due to the poor state of systematics of the group (see, e.g., [25,29]). In light of the findings of Auychinda et al. [26], these specimens were investigated again, and new material was discovered, including the male subimago, female subimago, and egg stages. The present study aims to name and describe formally Vietnamella sp. C as a new species based on the specimens used for initial DNA data and our expanded set of materials from Nan Province, Thailand. Additional COI data for the new species are also provided, and these data were analyzed using Bayesian inference methods.

Sampling and Morphological Observations
The newly acquired specimens used for description and photography were collected from Nan Province in Northern Thailand. Larvae were collected from cobble in moderate to fast-flowing areas. Mature larvae were reared using earthenware pots connected to an air supply ( Figure 8C) until emergence of winged stages. The specimens that have been used to obtain initial COI data were retrieved from L.M.J.'s research collection. The chorionic structure was investigated by drying the eggs, coating them with gold, and observing them with a FEI Quanta 450 Scanning Electron Microscope (SEM). The external structures were prepared on permanent slides using Euparal ® as a medium and observed by light microscopy. Final plates were prepared with Adobe Photoshop ® CC 2020. Holotype and paratype specimens of the new species are deposited in the collections of the Zoological Museum at Kasetsart University in Bangkok, Thailand [ZMKU] and the Museum of Zoology in Lausanne, Switzerland [MZL]. L.M.J.'s materials are currently stored in trays with other specimens used for DNA barcoding, and these units will be deposited in the Purdue University Entomological Research Collection, West Lafayette, Indiana, United States of America [PERC]. Our species hypotheses are based on the convergence of the morphological species concept and the phylogenetic species concept.

Molecular Analysis
The COI sequence of Vietnamella nanensis sp. n. was newly amplified from the Wa River, Nan Province, Thailand. The 658 bp sequence was included in the Bayesian tree reconstruction with the other vietnamellid mayflies from GenBank and the BOLD system. The preserved specimens were dissected (thorax) for DNA extraction. Total DNA was extracted using a genomic DNA purification kit (NucleoSpin, Macherey-Nagel, Germany) following the manufacturer's protocol. The COI amplification was performed using LCO1490 and HCO2198 [30]. The polymerase chain reaction (PCR) conditions and procedure were performed as described previously [26]. Purification and sequencing were conducted by Macrogen, Inc. (Seoul, South Korea). The molecular protocols and Bayesian tree construction methods were as previously reported by Auychinda et al. [26]. Nucleotide sequences obtained in this study were deposited in the GenBank database. Other Vietnamella sequences were also obtained from the BOLD system and GenBank, and Teloganella umbrata Ulmer was used as an outgroup (a specimen of Narathiwat Province, Thailand, was amplified and sequenced by the authors); details are presented in Table 1. The Kimura 2-parameter (K2P) genetic distance was analyzed to confirm species delimitation.

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.
Thorax. Pronotum with moderately sharp anterolateral projections and slightly pointed protuberances below anterolateral projection. Mesonotum without projections and tubercles. Forefemora strongly expanded with serrations or tooth-like projections on ventral margin ( Figure 3A); transverse ridge serrated with spatulate setae and long, thin setae near inner dorsal margin; dorsal and ventral margins with simple, fine setae. Midfemora moderately expanded, dorsal margin smooth and with row of hair-like setae, ventral margin with small serration apically ( Figure 3B). Hindfemora moderately expanded, more slender than midfemora, dorsal margin smooth, with row of hair-like setae; ventral margin with small serration apically ( Figure 3C). All claws similar, strongly hooked, each with basal protuberance and 4-5 fine sub-apical setae ( Figure 3D).
Abdomen. Terga covered with simple, short setae; Terga I-X each with pair of median ridges or tubercles; posterolateral angles of terga II-X extended into sharp projections with dense simple setae; terga VI-VII each with pair of acute tubercles ( Figures 1C and 3E) and tergum X with well-developed pair of blunt tubercles ( Figure 3F). Gills I finger-like with setae; gills on segments II-VI similar in structure ( Figure 3G-K); gills on segment VII small, with ventral lamella divided into three lobes ( Figure 3L). Caudal filaments with long setae on both lateral side at the middle part.
Head. Eyes with dorsal part yellowish and ventral part pale brown ( Figure 4A-C).
Eggs (dissected from imago, Figure 6). Ovoid, with length approximately 310 µm, width approximately 220 µm; nearly half of egg covered with helmet-shaped polar cap ( Figure 6A). Rod-shaped knob terminated coiled thread (KCT) around egg body; 2 or 3 tagenoform-type micropyles at center, without protuberances in micropyle area ( Figure 6B). Chorionic surface with protuberances, prominent in posterior area ( Figure 6C). Abdomen. Terga covered with simple, short setae; Terga I-X each with pair of median ridges or tubercles; posterolateral angles of terga II-X extended into sharp projections with dense simple setae; terga VI-VII each with pair of acute tubercles (Figures 1C and 3E) and tergum X with well-developed pair of blunt tubercles ( Figure 3F). Gills I finger-like with setae; gills on segments II-VI similar in structure ( Figure 3G-K); gills on segment VII small, with ventral lamella divided into three lobes ( Figure 3L). Caudal filaments with long setae on both lateral side at the middle part.       Female subimago ( Figure 7B). Body length 12-14 mm without cerci. Head. Eyes yellowish.

Thorax. Brownish veins and crossveins brown.
Abdomen. Brownish, terga segment VI-IX with pair of median pale line (similar to male subimago).
Diagnosis. The larva of Vietnamella nanensis sp. n. is most similar to that of V. thani Tshernova, but can be separated from the latter and other Vietnamella species based on the following combination of characteristics: i) pattern of serration on the ventral margin of the forefemur ( Figures 1A,B and 3A), ii) posterolateral margins of the abdominal terga with pairs of acute tubercles, especially in terga VI and VII ( Figures 1A,C and 3E), iii) well-developed pair of median ridge projections of tergum X ( Figure 3F), iv) second segment of the maxillary palp being slightly longer than the third segment (1.3:1), and v) chorionic surface with prominent protuberances (imago stage).
In the alate stages, the anterior margin of forewings is brown and has fewer cross-veins than in the other species. The venation of hind wings shows cross veins between Subcosta (Sc) and Radius sector (RA) similar in number with V. sinensis [31] but more numerous compared to V. thani. The penis of the subimago is most similar to that of V. thani and clearly different from the one of V. ornata (Tshernova) [24].
Remarks. The morphology of the alate stages was described from male subimago and female imago associated with larvae by rearing method. In addition, the female subimago was photographed before its imago emerged ( Figure 7B).
Etymology. The specific epithet is a reference to the province Nan in Thailand, where the type material was collected and reared. Female subimago ( Figure 7B). Body length 12-14 mm without cerci.
Head. Eyes yellowish. Thorax. Brownish veins and crossveins brown. Abdomen. Brownish, terga segment VI-IX with pair of median pale line (similar to male subimago). Diagnosis. The larva of Vietnamella nanensis sp. n. is most similar to that of V. thani Tshernova, but can be separated from the latter and other Vietnamella species based on the following combination of characteristics: (i) pattern of serration on the ventral margin of the forefemur ( Figure 1A,B and Figure 3A), (ii) posterolateral margins of the abdominal terga with pairs of acute tubercles, especially in terga VI and VII ( Figure 1A,C and Figure 3E), (iii) well-developed pair of median ridge projections of tergum X ( Figure 3F), (iv) second segment of the maxillary palp being slightly longer than the third segment (1.3:1), and (v) chorionic surface with prominent protuberances (imago stage). In the alate stages, the anterior margin of forewings is brown and has fewer cross-veins than in the other species. The venation of hind wings shows cross veins between Subcosta (Sc) and Radius sector (RA) similar in number with V. sinensis [31] but more numerous compared to V. thani. The penis of the subimago is most similar to that of V. thani and clearly different from the one of V. ornata (Tshernova) [24]. Remarks. The morphology of the alate stages was described from male subimago and female imago associated with larvae by rearing method. In addition, the female subimago was photographed before its imago emerged ( Figure 7B). Etymology. The specific epithet is a reference to the province Nan in Thailand, where the type material was collected and reared.

Molecular Analysis
The COI analysis revealed three major clades, namely Vietnamella sp. B, V. thani, and a group of Vietnamella species with distribution in Northern Thailand and China. The last major clade was distinguished into four clusters and referred to four different species: V. maculosa Auychinda, Sartori & Boonsoong, V. cf. ornata, V. sinensis, and V. nanensis sp. n. The V. nanensis sp. n. cluster is divided into two branches based on geography including Mae Hon Son and Nan provinces (Figure 9).
The intraspecific genetic distances varied between 0% and 3.4%, whereas interspecific distances were very high, ranging from 16.1% to 31.2% (Table 2).   Vietnamella species with distribution in Northern Thailand and China. The last major clade was distinguished into four clusters and referred to four different species: V. maculosa Auychinda, Sartori & Boonsoong, V. cf. ornata, V. sinensis, and V. nanensis sp. n. The V. nanensis sp. n. cluster is divided into two branches based on geography including Mae Hon Son and Nan provinces (Figure 9). The intraspecific genetic distances varied between 0% and 3.4%, whereas interspecific distances were very high, ranging from 16.1% to 31.2% (Table 2).

Discussion and Conclusions
The currently known larvae of Vietnamella species can be clearly distinguished from one another by their morphology, and their character comparison is shown in Table 3. Vietnamella ornata is not included, as it has been reported only from the subimaginal stage [24]. Vietnamella sp. A from India might represent the larva of V. ornata [27]; more work is needed in this area. Both morphological and molecular evidence shows similar preliminary results for species numbers and delimitation.
We found that the venation of the stigma area of the forewing can be used to separate Vietnamella species into two groups as follows: i) with divided longitudinal vein, including species that have been found only in China (V. sinensis and V. ornata) and ii) without divided longitudinal vein, including three species (V. nanensis sp. n., V. maculosa and V. thani). The MA fork position of forewings of V. nanensis sp. n. shows intraspecific variation, with one specimen having the MA fork situated medially and another having it submedially. The penis of the male subimago of V. nanensis sp. n. is most similar to V. thani, whereas it differs completely from V. ornata. Therefore, the combination of wing and male genitalia characters can be considered for species diagnosis of the adult stages. The details of the adult characters are presented in Table 4.
The chorionic structure can be used for distinguishing between the four species V. sinensis [25], V. maculosa, V. thani [26], and V. nanensis sp. n. The eggs of Vietnamella show a unique body shape and the helmet-like shape of the polar cap. Differences exist in the details of the chorionic surface and the KCT shape (Table 3). Vietnamella nanensis sp. n. shows prominent protuberances on the posterior

Discussion and Conclusions
The currently known larvae of Vietnamella species can be clearly distinguished from one another by their morphology, and their character comparison is shown in Table 3. Vietnamella ornata is not included, as it has been reported only from the subimaginal stage [24]. Vietnamella sp. A from India might represent the larva of V. ornata [27]; more work is needed in this area. Both morphological and molecular evidence shows similar preliminary results for species numbers and delimitation.
We found that the venation of the stigma area of the forewing can be used to separate Vietnamella species into two groups as follows: i) with divided longitudinal vein, including species that have been found only in China (V. sinensis and V. ornata) and ii) without divided longitudinal vein, including three species (V. nanensis sp. n., V. maculosa and V. thani). The MA fork position of forewings of V. nanensis sp. n. shows intraspecific variation, with one specimen having the MA fork situated medially and another having it submedially. The penis of the male subimago of V. nanensis sp. n. is most similar to V. thani, whereas it differs completely from V. ornata. Therefore, the combination of wing and male genitalia characters can be considered for species diagnosis of the adult stages. The details of the adult characters are presented in Table 4.  [25] Definition based on Figure 1A; p. 383. b [27] Definition based on Figure 1; p. 995.  The chorionic structure can be used for distinguishing between the four species V. sinensis [25], V. maculosa, V. thani [26], and V. nanensis sp. n. The eggs of Vietnamella show a unique body shape and the helmet-like shape of the polar cap. Differences exist in the details of the chorionic surface and the KCT shape (Table 3). Vietnamella nanensis sp. n. shows prominent protuberances on the posterior surface, and the egg size is the largest of the four species. The COI reconstruction showed the existence of six species of Vietnamella, in agreement with a previous study [26].