First record of the adult of Cymbalcloeon confirming its position in Baetidae (Insecta, Ephemeroptera)

Biodiversity Data Journal 13: e178464 doi: 10.3897/BDJ.13.e178464 Taxonomy & Inventories First record of the adult of Cymbalcloeon confirming its position in Baetidae (Insecta, Ephemeroptera) Ningning Wang , Di Wang , Jean-Luc Gattolliat , Chanaporn Suttinun , Changfa Zhou ‡ Nanjing Normal University, Nanjing, China § Naturéum, Lausanne, Switzerland | Lausanne University, Lausanne, Switzerland ¶ Chiang Mai University, Chiang Mai, Thailand Corresponding author: Changfa Zhou ([email protected]) Academic editor: Zhi-Teng Chen Received: 18 Nov 2025 | Accepted: 09 Dec 2025 | Published: 22 Dec 2025 Citation: Wang N, Wang D, Gattolliat J-L, Suttinun C, Zhou C (2025) First record of the adult of Cymbalcloeon confirming its position in Baetidae (Insecta, Ephemeroptera). Biodiversity Data Journal 13: e178464. https://doi.org/10.3897/BDJ.13.e178464 Abstract Background The nymphs of Cymbalcloeon sartorii Suttinun et al., 2020 present several unusual characters such as having only three pairs of ventral gills. The imaginal stage of this recently described genus remains unknown. New information In Spring 2025, tens of nymphs as well as morphologically and molecularly associated adults of this species were collected in southwestern China. This represents the first recorded occurrence of the genus in China, and indeed, the first report outside Thailand. The male imagos have tiny bodies, clear median longitudinal ridge on mesonotum, one pair of wings without any marginal intercalary veins, relatively long apical segment of styliger, and distinct penis cover. The morphology of this species shows it is close to ‡ ‡ §,| ¶ ‡ © Wang N et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Baetopus Keffermüller, 1960 in the subfamily Cloeoninae. However, it lost some key structures, like marginal intercalaries, hindwings and most gills. Keywords China, morphology, origin, phylogeny, systematics Introduction The genus Cymbalcloeon and the type-species Cymbalcloeon sartorii Suttinun et al., 2020 were described by Suttinun et al. (2020) based on nymphal specimens collected in Thailand. The nymphs are characterized by three pairs of ventral gills on abdominal segments V–VII, suggesting that Cymbalcloeon represents a highly derived clade within the family Baetidae. In addition to the unique gill structure, the nymphs of C. sartorii exhibit distinctive apomorphies. All three pairs of legs with tibio-patellar sutures and the mandibles lacking setae between the molar and incisor regions are key characters that define distinct lineages within Baetidae, according to Kluge (1997). Other notable features include two-segmented maxillary and labial palpi, the absence of spines along the lateral margins of the terga, the presence of two rows of teeth on the claws, and clearly divided compound eyes. In this context, the discovery and description of the adult stage of the genus are of great significance for understanding its phylogenetic placement. Various attempts were proposed to classify the Baetidae; the family was divided into two (Kazlauskas 1972), subsequently in three (Gillies 1991) and finally in five subfamilies (Kluge 1997) (including a fossil subfamily). However, this kind of classification was questioned (Gattolliat et al. 2008, Nieto 2010, Cruz et al. 2020), and the key character of their divisions (single or double marginal intercalary veins) was already doubted by Riek (1973) and Morihara and Edmunds (1980) as well as most of the subsequent authors. Recent discoveries of additional genera provided valuable insights into the origin and evolution of the Baetidae (Gillies 1990, Kluge and Suttinun 2020); however, genetic analyses and character-based studies are essential to fully resolve the global phylogeny of this highly diverse family. Riek (1973), Landa and Soldán (1985) and Kluge (1997), Kluge (2022), placed the family Siphlaenigmatidae or subfamily Siphlaenigmatinae next to true Baetidae. However, the family Siphlaenigmatidae does not have marginal intercalaries or turban eyes, but it has distinct penes and its forceps have two apical segments instead of one as in Baetidae. Molecular phylogeny confirms the basal position of Baetidae within the order Ephemeroptera but challenges its previously proposed sister relationship with Siphlaenigmatidae (Ogden et al. 2009, Ogden et al. 2019). Identifying the most basal genera within Baetidae is crucial for understanding its evolutionary relationships with other families and for bridging existing phylogenetic gaps. 2Wang N et al In March 2025, we conducted a mayfly collection in Yunnan Province, southwestern China. During this survey, we collected two Cymbalcloeon nymphs along with several imagos that were tentatively associated with them. To obtain additional material for molecular confirmation of the association between stages, we returned to the site in May 2025. The molecular analysis confirms the conspecificity of the nymphs and adults. This allows us to report, for the first time, the adult stage of Cymbalcloeon, providing new insights into the origin and evolutionary history of the family Baetidae. This also represents the first occurrence of the genus in China, and the first report outside of Thailand. Materials and methods Specimen collection and image processing The nymphs were collected by hand net in a small stream, the imagoes and subimagoes were attracted by light-traps. All materials were stored in ethanol (more than 80%). All specimens used in this study are deposited in the Mayfly Collection, College of Life Sciences, Nanjing Normal University (NNU). Most structures are observed directly in ethanol (about 80%). Two genitalia are dissected from male and dissolved in NaOH (10%) for 10 minutes at 40°C. Digital photos were taken by Sony a7R (Interchangeable Lens Digital Camera), and some of them were taken under Nikon Eclipse 50i (Microscope) and Mshot MZ81 (Stereomicroscope). Final plates were prepared with Adobe Photoshop 2022. Eggs were dissected from the female subimago. All SEM samples were dehydrated in graded acetone solutions (70%, 80%, 90%, 2×98% and 2x100%) for 10 min each. Subsequently, the specimens were immersed in 1-1.5 mL HMDS (1,1,1,3,3,3 hexamethyldisilazane; Merck-Suchardt, Darmstadt) in 20 mL glass vials. After a soak of 30 minutes, approximately 90% of the HMDS was removed and the vials were immediately transferred to a desiccator. The bottom of the desiccator was covered by silica gel beads (Merck-Suchardt, Darmstadt）and the desiccator itself was vacuumized. The remaining HMDS was allowed to evaporate overnight under anhydrous conditions. Afterwards, the specimens were sputter coated with gold (BIO-RAD SC510, München) for 80 seconds. The samples were photographed by a Scanning Electron Microscope (Apreo 2S, Thermo Fisher Scientific Company, Waltham, MA, USA). COI sequence Total DNA was extracted from one nymph and two male subimagos of C. sartorii using Animal Genomic DNA Kit (TsingKe Biotech Co., Beijing, China). The mitochondrial gene cytochrome c oxidase subunit I fragment was PCR-amplified using the Premix Taq (Takara Bio Inc., Beijing, China), PCR conditions included initial denaturation at 94 °C for 5 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and First record of the adult of Cymbalcloeon confirming its position in Baetidae ... 3 extension at 72 °C for 40 s, with a final extension at 72 °C for 10 min. Universal COI primers were referenced from Zheng and Zhou (2021). Sequences were aligned using Muscle, and the K2P genetic distances within genus were calculated in MEGA11. The two obtained sequences have been uploaded to GenBank (Table 1). CO1 sequences were compared against reference sequences in the NCBI GenBank and BOLD (Barcode of Life Data System) databases to assess similarity and potential species-level matches. Species GenBank accession number Remarks C. sartorii PV800332 China (Yunnan), Nymph C. sartorii PV800333 China (Yunnan), Male subimago Taxon treatment Cymbalcloeon (Sartorii) sartorii Suttinun et al. 2020 Materials a. scientificName: C. sartorii; country: China; stateProvince: Yunnan; county: Mengla; locality: Man-Nan-Xing, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China; verbatimElevation: 530m; verbatimCoordinates: 21°53.00′N 101°17.00′E; decimalLatitude: 21.8921; decimalLongitude: 101.288; eventDate: 2025-03-18/20; individualCount: 2; lifeStage: nymph; recordedBy: De-Wen Gong, XuHong-Yi Zheng, Ning-Ning Wang, Yu-Xian Sun; occurrenceID: E6970792-99D4-5D80-9234-FAF9B25FC7CD b. scientificName: C. sartorii; country: China; stateProvince: Yunnan; county: Mengla; locality: Man-Nan-Xing, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China; verbatimElevation: 530m; verbatimCoordinates: 21°53.00′N 101°17.00′E; decimalLatitude: 21.8921; decimalLongitude: 101.288; eventDate: 2025-03-18/20; individualCount: 15; sex: male; lifeStage: imago; recordedBy: De-Wen Gong, Xu-Hong-Yi Zheng, Ning-Ning Wang, Yu-Xian Sun; occurrenceID: 86F631A9-6C54-5B4E-A9E5-416B5740C879 c. scientificName: C. sartorii; country: China; stateProvince: Yunnan; county: Mengla; locality: Man-Nan-Xing, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China; verbatimElevation: 530m; verbatimCoordinates: 21°53.00′N 101°17.00′E; decimalLatitude: 21.8921; decimalLongitude: 101.288; eventDate: 2025-03-18/20; individualCount: 2; sex: female; lifeStage: imago; recordedBy: De-Wen Gong, Xu-Hong-Yi Zheng, Ning-Ning Wang, Yu-Xian Sun; occurrenceID: 0092A0E4-8941-5670-A3A5-453F160CED6B d. scientificName: C. sartorii; country: China; stateProvince: Yunnan; county: Mengla; locality: Man-Nan-Xing, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China; verbatimElevation: 530m; verbatimCoordinates: 21°53.00′N 101°17.00′E; decimalLatitude: 21.8921; decimalLongitude: 101.288; eventDate: 2025-03-18/20; individualCount: 20; sex: male; lifeStage: subimago; recordedBy: DeTable 1. The species and their COI sequences used in this research. 4Wang N et al Wen Gong, Xu-Hong-Yi Zheng, Ning-Ning Wang, Yu-Xian Sun; occurrenceID: A60DB51B-8EE6-52E1-B8EA-48183513F517 e. scientificName: C. sartorii; country: China; stateProvince: Yunnan; county: Mengla; locality: Man-Nan-Xing, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China; verbatimElevation: 530m; verbatimCoordinates: 21°53.00′N 101°17.00′E; decimalLatitude: 21.8921; decimalLongitude: 101.288; eventDate: 2025-03-18/20; individualCount: 3; sex: female; lifeStage: subimago; recordedBy: DeWen Gong, Xu-Hong-Yi Zheng, Ning-Ning Wang, Yu-Xian Sun; occurrenceID: 12437BA3-835A-5BA1-AD82-486B5E8225B4 f. scientificName: C. sartorii; country: China; stateProvince: Yunnan; county: Mengla; locality: Man-Nan-Xing, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China; verbatimElevation: 530m; verbatimCoordinates: 21°53.00′N 101°17.00′E; decimalLatitude: 21.8921; decimalLongitude: 101.288; eventDate: 5/10/2025; individualCount: 10; lifeStage: nymph; recordedBy: De-Wen Gong and Di Wang; occurrenceID: 18207030-8E64-52DD-806C-3C35976AA8B3 Description Male imago (in alcohol). Body length 3.4 mm, forewing 3.0 mm, cerci 3.6 mm (Fig. 1A, B, E). Head. Light reddish brown (Fig. 1A, B). Scape and pedicel cylindrical, light brown, flagella light brown to whitish, ca. 6.5x pedicel in length, antennal length equivalent to head width (Fig. 1A, B). Dorsal surface of upper portion of compound eyes orange, lateral surface pale grey (Fig. 1E); upper portion of eyes cylindrical (Fig. 1E); lower Figure 1. Imaginal habitus of C. sartorii. A male (dorsal view); B male (ventral view); C female (dorsal view); D female (ventral view); E male (lateral view); Subimaginal habitus of C. sartorii: F female subimago (lateral view); G male subimago (lateral view).  First record of the adult of Cymbalcloeon confirming its position in Baetidae ... 5 portion black, semi-spherical (Fig. 1E); facets of compound eyes hexagonal (Fig. 2D). Ocelli with brown base but pale apex (Fig. 1A). Thorax. Nota of thorax brown to deep brown but with pale sutures (Fig. 1A) with distinct longitudinal median ridge on mesonotum (Fig. 1E, Fig. 3D). Scutella of mesoand metanota expanded into projections (Fig. 1E). Foreleg pale brown, length ratio of forefemur: tibia: tarsus=1.0: 1.5: 1.7; length ratio of segments of foretarsus=1.0: 7.5: 4.9: 3.0: 1.4; foreleg with two claws, one acute, one blunt (Fig. 2G). Basal half of midfemur brown, other part light brown, tibia and tarsus light brown (Fig. 2H). Length ratio of midfemur: tibia: tarsus=2.5: 3.3: 1.0; length ratio of three free segments of midtarsus =1.3: 1.0: 2.3; one claw acute, one claw blunt (Fig. 2H). Basal half of hindfemur brown, other part light brown (Fig. 2I). Length ratio of hindfemur: tibia: tarsus=2.5: 3.2: 1.0; length ratio of three segments of hindtarsus=1.1: 1.0: 2.0 (Fig. 2I). Claws similar to foreor midlegs (Fig. 2J). Costal and subcostal veins of forewings light brown, other longitudinal veins whitish to light brown (Fig. 2A); crossvein greatly reduced, only 4-5 crossveins between C Figure 2. Imaginal characters of C. sartorii. A forewing of male; B forewing of male subimago; C forewing of male subimago enlarged (arrows indicating the forking points of MA₁ & MA₂ and MP₁ & MP₂); D compound eyes; E genitalia (ventral view); F subgenital plate; G foreleg of male; H midleg of male; I hindleg of male; J claw of hindleg; K genitalia (dorsal view, showing the penial bridge).  6Wang N et al and Sc, 3-4 between Sc and R , two between other neighbored longitudinal veins (Fig. 2A). MA and MP detached from MA and MP respectively (Fig. 2A–C). Membrane of forewing transparent (Fig. 2A). Sc, Rs and Rs with bullae; CuP with a back-curved base but most part of it and A straight (Fig. 2A). Abdomen. Segment I, VI-X reddish brown, other segment pale with dark brown stripes on lateral margins of terga; terga I-VI with a pair of submedian reddish dots respectively, but in most cases, these dots indistinct to invisible; terga VII-X with a pair of additional submedian stripes respectively (Fig. 1A, B, E). Cerci pale. Genitalia: Pedestal of gonostylus distinctly wider than gonostylus (forceps) (Fig. 2E, K, Fig. 3B); gonostylus with two segments, basal half of segment I slightly widened; segment II with slightly broadened apex, its length subequal to half segment I (Fig. 2 E, K, Fig. 3B). Subgenital plate with tiny hair-like setae on surface (Fig. 3B, C). Penis cover distinct, nearly quadrate in shape (Fig. 2K). Gonovectes or penial arms smoothly curved (Fig. 2K). Female imago (in alcohol). Body length 2.9 mm, forewing 3.0 mm, cerci broken (Fig. 1 C, D). Similar to male imago but with more reddish pigments, especially thorax and abdomen (Fig. 1C, D). Length ratio of forefemur: tibia: tarsus=1.2: 1.1: 1.0; length ratio of segments of foretarsus=1.0: 2.8: 1.7: 1.7: 2.1 (Fig. 1C, D). Midlegs broken. Hindlegs similar to forelegs in color (Fig. 1C, D). Length ratio of hindfemur: tibia: tarsus=2.0: 3.2: 1.0; length ratio of segments of hindtarsus=1.1: 1.5: 1.0 (Fig. 1C, D). 1 2 2 1 1 1 5 1 Figure 3. SEM photos. A genitalia of male subimago; B genitalia of male imago; C subgenital plate of male imago; D mesonotum of male imago; E tarsus of hindleg; F egg from female subimago.  First record of the adult of Cymbalcloeon confirming its position in Baetidae ... 7 Male subimago: (in alcohol). Body length 3.1 mm, forewing 2.4 mm, cerci 4.4 mm (Fig. 1G). Length ratio of forefemur: tibia: tarsus=1.5: 1.0: 1.7; length ratio of midfemur: tibia: tarsus=1.8: 1.6: 1.0; length ratio of segments of hindtarsus=3.4: 4.1: 1.0 (Fig. 1G). Veins of forewing slightly clearer than male, MA and MP connected to MA and MP with a crossvein respectively (Fig. 2B). Genitalia distinct, gonostyliger and segment II of styliger oriented inwards but segment I of styliger oriented outwards; length of them: segment I > segment II > gonostyliger (Fig. 3A). Female subimago: (in alcohol). Body length 2.7 mm, forewing 2.9 mm, cerci 4.0 mm (Fig. 1F). Length ratio of forefemur: tibia: tarsus=1.5: 1.4: 1.0; Length ratio of midfemur: tibia: tarsus=2.0: 2.0: 1.0; Length ratio of hindfemur: tibia: tarsus=3.5: 3.9: 1.0 (Fig. 1F). Nymph (in alcohol) (see Suttinun et al. 2020 and Figs 4, 5, 6). 2 2 1 1 Figure 4. Nymphal habitus of C. sartorii. A male nymph (dorsal view); B male nymph (ventral view); C female nymph (dorsal view); D female nymph (ventral view).  8Wang N et al The main characteristics including: Body length 2.5 mm, cerci 1.3 mm (Fig. 4A–D). Body color pattern similar to adults, pale body with reddish dots and streaks; thorax distinctly broader and more robust than abdomen (Fig. 4A–D). Mouthparts shown in figure 5, labrum with a very shallowly concave anterior margin (Fig. 5A); mandibles without any setae between mola and incisor, outer margin without setae, prostheca Figure 5. Mouthpart of C. sartorii. A labrum; B left mandible; C right mandible; D maxilla; E labium; F hypopharynx.  Figure 6. Nymphal characters of C. sartorii. A foreleg; B midleg; C hindleg; D Gill V; E Gill VI; F Gill VII.  First record of the adult of Cymbalcloeon confirming its position in Baetidae ... 9