A new species of Cincticostella (Ephemeroptera, Ephemerellidae) from China

Cincticostella tornata, a new species of Ephemerellidae, is described from China. It belongs to the insolta-species group and is closely related to C. femorata. It is hitherto only known from its type locality in central China, more than 1000 km northern to the known distribution of C. femorata. In addition, the COI barcode of the new species is provided.

Cincticostella femorata (Tshernova, 1972) was originally described from Vietnam based on a single larva. It was assigned to Asiatella Tshernova, 1972, but was transferred to Cincticostella as addendum in the same article (Tshernova 1972). This species appears highly de-rived within the insolta-group as the mid and hind femora are greatly flattened with strong chalazae on dorsal and sometimes ventral margins, and the prothorax is much broader than in related species. A closely related species, Cincticostella boja (Allen, 1975) was subsequently established for larvae from Thailand, described and figured but unnamed by Gose (1969). The description was just translated from Gose (1969), and no material was examined by Allen (1975). He considered that C. boja was significantly different from other species and especially from C. femorata by the quadrangular head, the presence of paired submedian tubercles on tergites II to X and the well-developed posterolateral projections on segments VII to IX (Allen 1975). Jacobus et al. (2005) examined five larvae from three different Thai localities. They compared the main morphological characters used by Allen (1975), as well as the number of denticles on claws and the relative density of hair-like setae on the caudal filaments and concluded that all specimens present important but intraspecific variations. They consequently considered C. boja as a junior synonym of C. femorata (Jacobus et al. 2005). Comparisons of original illustrations and descriptions of the two species as well as more recent illustrations of specimens attributed to C. femorata from China (Xie et al. 2009) and Thailand (Martynov et al. 2019) challenged this synonymy. The species is now considered as occurring in Vietnam, Thailand and China (Martynov et al. 2019;Xie et al. 2009).
We had the opportunity to examine material recently collected in China by two of us (DM and WL). Despite important similarities with C. femorata, part of the characters did not match the original and subsequent descriptions of the species. We therefore consider that this material belongs to a new species. The description of this new species may help to better understand the intra-and interspecific variability within C. femorata; in the future C. femorata may appear as a complex of closely related species.

Collecting
The specimens were collected with small sized aquatic net, during a field trip primarily focused on collecting Plecoptera. They were fixed in 75% ethanol on the field. Holotype and paratypes are kept in the MZL (Museum of Zoology, Lausanne, Switzerland), one paratype in the HNHM (Hungarian Natural History Museum, Budapest, Hungary), one paratype in HIST (Henan Institute of Science and Technology) as indicated in the text.

Morphological examination
Аll dissected specimens were entirely mounted on slides in Euparal medium. Drawings and pictures of body parts were made using an Olympus BX51 stereoscopic microscope with a camera lucida or a digital camera Olympus SC50. Extended depth of focus images were obtained using the software Stream Basic 1.9.4. Pictures of the whole larval body were taken with the Visionary LK system (Dun., Inc., USA). Pictures and drawings were subsequently enhanced with Adobe Photoshop CC2015. Genetics DNA of part of the specimens was extracted using non-destructive methods allowing subsequent morphological analysis (see Vuataz et al. 2011 for details). We amplified a 658 bp fragment of the mitochondrial gene cytochrome oxidase subunit 1 (COI) using the primers LCO 1490 (GGTCAACAAATCATAAAGATATTGG) and HCO 2198 (TAAACTTCAGGGTGACCAAAAAAT-CA) (Folmer et al. 1994). The polymerase chain reaction was conducted with an initial denaturation temperature of 98 °C for 30 sec followed by a total of 37 cycles with denaturation temperature of 98 °C for 10 sec, an annealing temperature of 50 °C for 30 sec and an extension at 72 °C for 30 sec, final extension at 72 °C for 2 min. Sequencing was done with Sanger's method (Vuataz et al. 2011). The genetic variability between specimens was estimated using Kimura-2-parameter distances (K2P), calculated with the program MEGA 7 (Kumar et al. 2016). Description. Larva: Body length 8.7-10.0 mm; caudal filaments length 8.0-9.0 mm. Body yellowish-brown.

Cincticostella tornata
Head: Covered with thin setae. One pair of small, rounded protuberances between eyes. Genae quadrangular, well developed, angles rounded ( Fig. 1). Antennae with scapus short, pedicellus as broad as long, flagellum of about 25 segments, first segment as long as next three segments.
Mouthparts: Labrum wide, apicolateral angle rounded; apicomedial emargination shallow, dorsal surface densely covered with long, hair-like setae and long stout flattened setae scattered over surface ( Fig. 2A); ventral surface with numerous, long, stout, hair-like setae laterally orientated; apical margin with numerous feathered setae and very thin hair-like setae (  three teeth, two outer teeth stout, broad, apically rounded and one small, blunt tooth; inner incisor with one main stout, apically pointed tooth and one inner vestigial tooth; stout rectangular prostheca with bunch of hair-like setae on inner side (Fig. 2E). Right mandible: outer incisor composed of two spoon-shaped teeth, outer tooth longer than inner; inner incisor composed of two apically pointed teeth, orientated perpendicularly to outer incisor; prostheca consisting of numerous hair-like setae (Fig. 2F); eight hair-like setae on margin below mola (Fig. 2D). Maxilla: apex with transformed ventral canine typical of genus (Kluge 2004); two dentisetae with serrated inner margin; apex of maxilla surface with numerous, long, stout, hairlike setae, some with serrated inner margin; inner margin of galealacinia with 3-4 rows of long, stout, simple setae; 10-12 feathered, stout setae roughly arranged in a row near base of galealacinia surface (Fig. 2G, H). Maxillary palp: three segmented; segments partially fused; segment I slightly longer than half of segments II and III combined; segment III short, pointed, with few thin setae on apex (Fig. 2G, H). Labium with glossae semicircular, almost two times longer than broad; ventral surface of glossae covered with long, stout, simple setae; dorsal surface with an arc of long simple setae parallel to outer margin; glossae protruding over apices of paraglossae; paraglossae semicircular, dorsal surface covered with numerous long stout simple setae, ventral surface with flame-like setae. Ventral surface of mentum and submentum mostly covered with flame-like setae. Labial palp three-segmented; segments I and II equal in length, covered with long, hairlike setae; segment II crescent shaped, outer margin with spine-like long setae and long hair-like setae, inner margin covered with numerous long thin setae; segment III conical, two times longer than broad at base, apex covered with short thin setae (Fig. 2I). Hypopharynx: surface of lingua covered with short setae, most abundant in apical part; apices of superlinguae rounded with numerous hairlike setae (Fig. 2J).
Thorax: dorsal surface covered with scattered small thin setae. Pronotum: broad projection anterolaterally enclosing head, margin rounded except concave apically (black arrow tip on Fig. 3A); projections covered with long hair-like setae; laterally with short flame-like setae. Mesonotum: broad, large, rounded anterolateral projection, covered with long hair-like setae; laterally with short flame-like setae; small pair of triangular protuberances between forewing pads (white arrow tips on Fig. 3A). Forelegs: moderately expanded; length ratio of femur : tibia : tarsus = 2.9 : 2.4 : 1. Forefemora: dorsal surfaces cover with long hair-like setae; dorsal margin slightly concave, with dense setae and with only three blunt chalazae near distal angle; ventral margin with dense setae without serration; apex with one ventral and one dorsal well-developed extension (Fig. 4A). Foretibiae completely covered with long hair-like setae and short stout setae, apex of ventral margin produced and covered with a bunch of dense, long, thin setae. Foretarsi covered with long hair-like setae, ventral margin with stouter longer feathered setae. Foreclaws hooked with a row of four triangular teeth, two proximal teeth closer to each others (Fig. 4D). Mid legs: greatly expanded; length ratio of femur : tibia : tarsus = 3 : 2.7 : 1. Mid femora: dorsal surface cover with hair-like setae; ventral margin with dense setae and four serrated chalazae in proximal half of femora, ending with a well-developed triangular pointed process; dorsal margin with dense setae and eight chalazae progressively larger (Fig. 4B). Mid tibiae similar to foretibiae except setae less abundant and short flame-like setae on dorsal margin and apically. Mid tarsi: ventral margin with setae not feathered. Mid claws similar to foreclaws. Hind legs: greatly expanded; length ratio of femur : tibia : tarsus = 4.2 : 3.2 : 1; hind femora similar to mid femora except chalazae of ventral and dorsal margins: ventral margin with three chalazae medially; dorsal margin with 12 chalazae along margin and progressively larger (Fig.  4C). Hind tibiae and hind tarsi similar to mid tibiae and mid tarsi, respectively. Hind claws similar to foreclaws.
Abdomen: surface of tergites covered with hair-like setae and few stout flame-like setae, absent laterally on tergites IV to IX; distal margin smooth without spines. Tergites I-X with a pair of tubercles, minute on tergites I-III and X (Fig. 4E); highly developed on tergites IV-IX (Fig. 3B): arched, acute tubercles covered with medium setae (Fig. 3C). Sternites densely covered with medium setae; distal margin smooth without spines. Triangular, slender distolateral expansions present on abdominal segments I to IX, highly developed on segments IV to IX, covered with dense flat medium and hair-like setae. Gills present on segments III to VII (Fig. 3E-I); upper lamellae dorsally densely covered with flame-like setae, upper lamellae of gills III to V subrectangular, of gills VI roughly paddle-like, of gills VII suboval and reduced; lower lamellae bifurcate with marginal processes, bilobed on gills III to V, simple on gills VI and VII. Paracercus subequal in length to cerci; cerci and paracercus densely covered with hair-like setae, apex of each segment with very long, flattened setae (Fig. 3D).
Adults. Unknown. Etymology. The Latin word "tornata" means rounded, in reference to the genae and anterolateral projection of mesonotum.
Diagnosis. Cincticostella tornata sp. nov. differs from closely related species of the insolta-group by the following combination of characters: 1) glossae protruded beyond apices of paraglossae; 2) genae extended with round apex; 3) anterolateral projection of pronotum pointed with a concave margin; 4) mesothorax with broad, rounded proximolateral expansion; 5) number and degree of development of chalazae on ventral and dorsal margins of mid and hind femora; 6) degree of development of apicoventral projection on ventral margin of fore-, mid and hind femora; 7) apicolateral projection of tergite IX well developed exceeding 1/3 of tergite X.
Distribution. The new species is only known from the type locality located in the Qinling mountains (Shaanxi province, close to Hanzhong city). This mountain range is considered as a natural boundary between South and North China and between the Palaearctic and Oriental realms (Fig. 5). A huge diversity of plant and animals is considered as endemic from this area, among others a subspecies of the giant panda (Ailuropoda melanoleuca qinlingensis) and the golden snub-nosed monkey  (Rhinopithecus roxellana). At our present state of knowledge, it is not possible to know if Cincticostella tornata sp. nov. is endemic to this mountain range or is more widely distributed through China.
Habitat. The larvae were found in a small forest brook of relatively slow current. Maximum width of the brook is about 1.5 meter; depth varies between 10 and 30 centimetres. The substrate is mostly stony and sandy, mixed with silty patches and a moderate amount of debris. Accompanying fauna included two Holarctic genera of mayflies, Baetis (Baetidae) and Ameletus (Ameletidae), as well as the stoneflies Rhopalopsole basinigra Yang & Yang, 1995, Spaeronemoura grandicauda (Wu, 1973, Nemoura sp., and two species recently described from the same area, namely Amphinemura albicauda Li, Mo, Dong, Yang & Murányi, 2018 and Amphinemura dingoidea Li, Mo, Dong, Yang & Murányi, 2018(Li et al. 2018. Molecular results. A sequence of 658 bp, corresponding to the usual fragment of the COI gene, was obtained. The sequence was deposited in GenBank (Accession number: MT254050). It was compared to other known sequences found in databases such as GenBank and BOLD. The closest sequence corresponds to a North America Ephemerellidae (Drunella walker (Eaton, 1884)) with less than 83% of identity. Around twenty sequences were found with a percentage of identity comprising between 80 and 83, most of them belonging to the families Ephemerellidae and Caenidae. K2P distance was also calculated with a sequence of Cincticostella gosei from Thailand (collected and sequenced by the first author, GenBank accession number: MN186574.1). The distance between the two species was estimated to 21.6% (meaning only 78.4% of identity).

Discussion
Within the insolta-group, C. femorata and C. tornata share two presumably derived characters, namely the anterolateral expanded prothorax and the extremely flattened mid and hind femora. The degree of development, the position and the orientation of the tubercles on the abdominal tergites of C. tornata are different from other species of the group. However, these characters must be considered with caution as it was recently demonstrated in a species of Notacanthella Jacobus and Mc-Cafferty, 2008. Two morphotypes were present based on the shapes of the tubercles, but these intraspecific variabilities mainly correspond to different ontogenic stages (earlier stages having more prominent tubercles)  (Auychinda et al. 2020). As already mentioned, C. femorata and C. tornata only differ by minute characters which may be easily overlooked. In particular, when using the keys to identify species of Cincticostella (Allen 1975;Xie et al. 2009), one will arrive to C. femorata without discrepancies. This easily explains why all the larvae with expanded prothorax, flattened femora and developed genae were previously identified as C. femorata. A global revision involving all available material from South East Asia is highly recommended for the species delimitation of C. femorata, including the confirmation or rejection of the synonymy of C. boja with C. femorata as well as the correct attribution of the material from South and Central China to one of these species. This revision should be ideally based on integrative taxonomy, especially by using both morphological and molecular evidences. The comparison of the characters in the closely related species is summarized in Table 1. Noteworthy, the imagos all these taxa remain unknown. The recent discovery and description of imaginal C. fusca revealed that male genitalia (penes and gonopods), tergite pattern and pigmentation of hind wings are re-liable characters to distinguish the different species of Cincticostella (Zhang et al. 2020). We may expect that imaginal characters will confirm the separation and validity of these closely related species. The type-locality of C. tornata is located 1200 km north to those of C. femorata and 2350 km north to those of C. boja (1700 km north to the closest report from Thailand) (Martynov et al. 2019). These long distances may explain the differences observed between taxa and may be the results of vicariant speciation. Xie et al. (2009) indicated that Cincticostella species were frequently collected in the recent years (meaning probably during the first decade of this century). However, they did not indicate where in China C. femorata was found and how widespread the species is. It will be of first interest from a biogeographic point of view to properly identify this material. This will allow the clarification of the distribution of C. femorata in China and may also clarify the distribution of C. tornata. It is also not excluded that collected specimens mentioned by Xie et al. (2009) partly belong to C. tornata or even to a still undescribed species.