The Real Characters of Heptagenia ngi Hsu (1936) from China Representing a New Genus (Ephemeroptera: Heptageniidae) ^†

Abstract

A very common and early named mayfly species from China, Heptagenia ngi Hsu, 1936, has no exact descriptions of its imago nor nymph, so its generic status is not clear. Based on detailed re-descriptions and pictures of all its stages from fresh materials, it is transferred into a new genus Maculogenia Zhou, gen. nov. Its nymph has conspicuous pale-dark colored body, slightly concave posterior margin of head, scattered simple setae on ventral maxillae, spines and very fine hair-like setae on caudal filaments. Similarly, its imago also has remarkable dark stripes and dots on the pale body, small median titillators and short first segment of foretarsi, apically expanded penes without any spines. It is similar to the genera Electrogena, Thamnodontus, and Parafronurus but they can be differentiated easily by above selected key structures.

1. Introduction

Hsu named the species Heptagenia ngi based on a male imago from Hong Kong, China [1], without providing the reason of generic arrangement. Besides the original brief description, this species and its imaginal characteristics were cited and mentioned by Gui, Hubbard and You and Gui [2,3,4], its nymphs were photographed and described briefly by Zhou et al. [5]. Further, because this species has a wide distribution and remarkable colorful body and penes, which was shown clearly by Hsu, it has been identified and included in almost all local fauna books, such as Su and Zhou [6]. However, so far neither exact morphology nor detailed structure has been added to it. Worse, its types have been lost (Prof. Hong Gui, personal communication). Therefore, the real condition of this species needs more work and association to confirm.

In the past 20 years, the generic concepts of the Heptageniidae have been updated greatly [7,8,9] and some Chinese heptageniids have been clarified accordingly [10,11,12,13]. The species Heptagenia ngi Hsu, 1936 on the contrary, is still in the original genus because it lacks more details and nymph–adult association.

In recent years, we have collected and reared many nymphal and imaginal materials of the species H. ngi at multiple locations on the Chinese mainland. In addition, we have also sequenced its mitochondrial genome and associated nymphs to its imagoes. However, we cannot include them in any genus of the family Heptageniidae. Therefore, a new one is established here for it. Its exact morphology is presented and described below.

2. Materials and Methods

The nymphs were collected using a hand net, while the adults were attracted using lights at the same place. Some of the adults were reared from mature nymphs. All materials were stored in ethanol (more than 80%).

All specimens were photographed with a digital camera (Single Lens Reflex) and examined under a stereomicroscope. Some small structures, such as mouthparts, claws, and gills were observed and photographed with a microscope camera.

Eggs were dissected from female imagoes. All SEM (scanning electronic microscope) samples were prepared with a standard protocol: fixed in 4% glutaraldehyde for 5–8 h, rinsed with PBS (physiological saline) 2–3 times (10–15 min each), dehydrated in concentration gradient acetone (30%, 50%, 70%, 80%, 90%, 100%, 10 to 15 min each), and coated with gold film in a vacuum.

The DNA extraction and amplification followed the process by Zheng and Zhou (2021) [14]. The COI sequence of this species was uploaded to GenBank, and its GenBank accession number is listed in

Table 1. GenBank Accession Numbers of the COI sequences used in this research.

Species	GenBank Accession Number
Maculogenia ngi	OP825112.1
Ecdyonurus fractus	LC377313.1

. The sequences were aligned with the software ClustalW.

All specimens used in this study were deposited in the Mayfly Collection, College of Life Sciences, Nanjing Normal University (NNU).

3. Taxonomy

Class Insecta Linnaeus, 1758

Order Ephemeroptera Hyatt and Arms, 1890

Family Heptageniidae Needham, 1901

Maculogenia Zhou, gen. nov.

Nymph: head capsule with slightly thickened anterior margin, dorsal surface with whitish spots, posterior margin slightly concave (Figure 1A,D); ventral surface of maxillae with scattered simple setae, distal dentisetae divided into two branches, proximal dentisetae with fringed margin (Figure 2F and Figure 3A,B); supracoxal sclerites indistinct and round (Figure 1B); mid- and hindtibiae with two rows of spine-like setae on the inner margin and dorsal margin respectively, a row of hair-like setae on the outer margin (Figure 4D); gills I–VII well developed, broad gills I with concave inner margin, gills VII leaf-like, without ventral thread-like lamellae (Figure 4F–I); caudal filaments with spine-like setae and very tiny hair-like setae on articulations (Figure 1D).

Imago: pale body with dark stripes and dots (Figure 5, Figure 6 and Figure 7D); mesothoracic furcasternum parallel (Figure 7B); first segment of foretibiae of male ca. 1/7× second one (Figure 7C); mid- and hindtarsi less than 1/2× tibiae (Figure 7K,L); crossveins of wings normal, those of female wings with brown cloud (Figure 7F–I); styliger plate concave, apical segment of forceps slightly shorter than previous one; penes almost fused totally, with a pair of median titillators but without any other spines (Figure 8).

Egg: whole surface covered with KCTs (Figure 9).

Diagnosis

The new genus established here, Maculogenia Zhou, gen. nov., has parallel mesothoracic furcasternum in adults and scattered setae on maxillae in nymphs. These characters show that this genus is a member of the subfamily Ecdyonurinae [7,8].

Its nymphs do not have any spines or ridges on their abdomen (in contrast to Notacanthurus), posterolateral angles of abdominal tergites are not enlarged (in contrast to Thalerosphyrus), hypopharynx has round apex (in contrast to Asionurus), nymphal head is almost round (in contrast to Atopopus), pronotum have no distinct posterolateral lobes (in contrast to Ecdyonurus), maxillae have simple hair-like setae on ventral surface (in contrast to most genera of the same subfamily, such as Compsoneuria, Compsoneuriella, and Ecdyogymnurus), supracoxal spurs are blunt (in contrast to Compsoneuriella, Thalerosphyrus), gill I broad, gill VII leaf-like (in contrast to Regulaneuria, Asionurus, and Afronurus), caudal filaments have spine-like setae and few tiny hair-like setae (in contrast to Nixe, Parafronurus, and Rhithrogeniella) [15]. Generally, the nymphs of the new genus are similar to those of the genera Electrogena and Thamnodontus but with an outstanding color pattern.

Although the nymphs of the new genus have no distinct uniqueness except for distinct black/white body and head capsule, the males can be identified easily based on several traits. (1) The first segment of the male foretarsus is only 1/7× of the second one. On the contrary, Afronurus and Parafronurus males have subequal segments I and II, just like the genus Compsoneuriella. (2) Penes of male do not have any spines except titillators. Most genera of the subfamily Ecdyonurinae have various of spines on their penes, like Compsoneuria, Compsoneuriella, Ecdyogymnurus, and Thamnodontus. (3) Two penes fused mostly and apex extended laterally. The penes of genera Regulaneuria, Thalerosphyrus, Electrogena, and Parafronurus are also fused but they do not expand. The penes of Afronurus, which only have titillators as well, are divided deeply and their titillators are usually large and distinct. (4) The mid- and hindtarsi of the new genus are less than 1/2× tibiae, shorter than most other genera, especially Regulaneuria and Thalerosphyrus. (5) Cephalic capsule extends slightly forwards (in contrast to the genera Electrogena and Thamnodontus whose heads have distinct extensions). (6) Crossveins of wings are not reduced (in contrast to Compsoneuriia and Regulaneuria). (7) Their body has various dark stripes and dots. The species in the common genera Regulaneuria, Afronurus, Thalerosphyrus, and Parafronurus are always yellowish to yellow with black or reddish markings.

In general, this new genus is close to Electrogena and Thamnodontus in nymph, but their adults are different because they lack spines on the expanded penes, and their head does not extend either. On the other side, its adult is somehow similar to Parafronurus while their nymphal caudal filaments are not alike because the latter genus have distinct hair-like setae on articulations. Upon its two independent dentiseate and imaginal characteristics, this genus is a member of the tribe Compsonuriini (Rhithrogeniella, Compsoneuria, Compsoneuriella, and Notonurus) sensu Sartori [15]. Except for the setal pattern of maxillae, its nymph is also similar to this tribe.

Species included: Maculogenia ngi (Hsu, 1936) comb. nov.

Distribution: China (southern half).

Maculogenia ngi (Hsu, 1936) comb. nov.

Heptagenia ngi Hsu, 1936: 235. Type: male imago, from Hong Kong, China.

Heptagenia ngi: Hubbard, 1986: 250; Gui, 1985: 84; You and Gui, 1995: 41 (male); Su and Zhou, 1998: 28; Zhou et al., 2015: 127 (nymph, male).

Materials examined

Neotype: 1 male imago, Sixinfamuchang, Wuyi Mountain Natural Reserves, Fujian Province, 320 m, 27°36′19″ N, 117°47′0″ E, 2021-VII-6, De-Wen Gong.

20 male imagoes, West of Mingling Mountain, Yixing City, Jiangsu Province, 1995-V-2, Chao-Dong Zhu; 3 male imagoes, 1 male subimago, 10 female imagoes, 5 female subimagoes, 20 nymphs, Yanzhangxiang, Longquan, Lishui City, Zhejiang Province, 360 m, 28°7′2″ N, 119°1′51″ E, 2021-VII-9, De-Wen Gong; 2 male imagoes, 2 male subimagoes, 5 female imagoes, 3 female subimagoes, 7 nymphs, Sixinfamuchang, Wuyi Mountain Natural Reserves, Fujian Province, 320 m, 27°36′19″ N, 117°47′0″ E, 2021-VII-6, De-Wen Gong; 2 male imagoes, 8 female imagoes, 6 nymphs, Tianmen Mountain, Wuyuan City, JiangXi Province, 2015-VIII-8-12, Yan-Juan Luo, Xiao-Fei Luo; 1 male imago, Fanjing Mountain, Guizhou Province, 2005-IX-21, Zhi-Jie Wang; 4 male imagoes, 6 female imagoes, Tiantangzhai, Luotian City, Hubei Province, 720 m, 31°5′44.7″ N, 115°44′17.52″ E, Shuang Qiu; 4 male imagoes, 2 male subimagoes, 3 female imagoes, 1 female subimago (all imagoes were reared from nymphs), 30 nymphs Jianfeng Town, Ledong County, Hainan Province, 89 m, 2022-VI-28-30, De-Wen Gong, Man-Qing Ding, Xin-He Qiang; 12 male imagoes, 4 female imagoes, Ruyang Management station, Nanling, Guangdong Province, 1890 m, 2020-IX-8-10, Zhen-Xing Ma; 4 male imagoes, 2 female imagoes, Hydropower Station, Huaping Reserve, Guangxi Province, 2020-VIII-20-21, Zhen-Xing Ma; 11 male imagoes, 5 female imagoes, 6 nymphs, Shunfei Hotel, Shunhuang Mountain Ruyang, Hunan Province, 2020-VIII-26-28 Zhen-Xing Ma.

Nymph (in alcohol, Figure 1, Figure 2, Figure 3 and Figure 4): body length 7.0–9.0 mm, caudal filaments 12.0–15.0 mm, body general yellowish brown to dark brown, with various pale and dark dots and stripes (Figure 1A). Head width 2.5–3.0 mm, antennae 2.0–2.5 mm, smooth; anterior margin of capsule slightly thickened, anterior and lateral margins convex and posterior margin slightly concave (Figure 1A,D); dorsal surface of head capsule with 20 pale dots: anterior margin with 6 large pale dots, 5 on the sub-anterior margin, 5 between the base of antennae, and 4 on the posterior margin between the compound eyes (Figure 1D).

Mouthparts: labrum ca. 1/3× width of head, both dorsal and ventral surfaces with dense setae but those on the dorsal surface much longer; ventral surface with median groove (Figure 2A). Both left and right mandibles with long and dense setae on outer margins, prostheca with 8–12 fimbriate bristles (Figure 2C,D,G,H); outer incisor of left mandible with serrated margin and one larger terminal denticle; inner incisor divided into 3 blunt denticles (Figure 2C,G); outer incisor of right mandible with 2 apical terminal denticles; inner one with 2 sharp denticles (Figure 2D,H). Hypopharynx: apex of superlinguae strongly curved and extended into round lobe-like structure, a row of long hair-like setae on outer margin from base to apex; lingua bell-like, with tuft of setae on apex (Figure 2B). Maxillae with simple hair-like setae on ventral surface (Figure 3B), a row of 16–19 comb-shaped setae on the crown of galea-lacinia (Figure 2F), both two distal dentisetae entire and simple, proximal dentiseta bifid (Figure 3A,C); maxillary palpi 3-segmented, first segment with sparse bristles on outer margin, second segment with obviously long and dense setae on outer margin, apex with dense setae brush (Figure 2F). Labium: glossae round lobe-like, inner parts expanded slightly with tuft of long setae; paraglossae expanded into oval lobes, with dense setae and bristles on dorsal surface and anterior margin; labial palpi broad, 2-segmented (Figure 2E).

Thorax: pronotum slightly extended, subequal to head in width (Figure 1A). Supracoxal spurs round (Figure 1B). Legs yellowish brown, femora with 3–4 dark bands dorsally; tibiae pale with basal and median brown bands; tarsi yellowish brown to brown (Figure 4A–C). Femora of all legs with a row of long setae on outer margin, dorsal surface and inner margin with scattered acute bristles. Foretibiae 0.9× of femora in length, outer margin with very tiny setae at base (Figure 4A), inner margin with row of bristles; tarsi ca. 1/3× of tibiae, with sparse setae on surface. Midlegs similar to forelegs except tibiae 0.83× length of femora, with row of fine setae on the outer margin (Figure 4B). Hindleg tibiae 0.71× of femora in length, outer margin of femora with a row of shorter tiny setae, outer submargin of dorsal surface of tibiae with 9–11 acute bristles and tiny setae, outer margin with row of bristles (Figure 4C,D). Claws of all legs with four to five subapical denticles (Figure 4E).

Abdomen: terga I–VI yellowish brown to dark brown, with four pale dots, those on terga I and IV–V are usually larger, making them paler than other terga (some individuals faded, making them look similar to a pair of dark brown longitudinal stripes sub-medially); terga VII–IX pale or light brown, with brown dots on the anterior, posterior, and lateral margins; tergum X pale to light brown with two brown dots and median longitudinal stripe, posterior and lateral margins brown (Figure 1A,C); posterolateral angles of terga III–VIII extended slightly into small acute projections (Figure 1C). Gill I banana-shaped with outer margin expanded (Figure 4F); gills II–IV broad, with lateral margins great expanded (Figure 4G); gill V–VI leaf-like, slightly asymmetric; gill VII narrower than others, with lamellae only (Figure 4I). Base of caudal filaments pale, other parts pale to brown and with brown articulations, whorled spines and extremely fine and sparse hair-like setae present on articulations (Figure 1D).

Male imago (in alcohol, Figure 6A, Figure 7A–D,H,I and Figure 8): body length 7.0–9.0 mm, forewing 9.0–10.0 mm, hindwing 2.5–3.0 mm, cerci 16.0–20.0 mm. Compound eyes contiguous, upper portion grey to pale, lower portion light blue (Figure 5A, Figure 6A and Figure 7A,B). Body brown, with remarkable dots and stripes.

Thorax: mesonotum with apparent transverse suture, lateral parapsidal sutures slightly bent, jointing posterior transverse suture (Figure 7A); mesothoracic furcasternum parallel (Figure 7B). Three nota of thorax with dark brown dots and stripes (Figure 5, Figure 6 and Figure 7A). Femora of all legs brown, with four dark brown bands; tibiae pale but with brown base and apex; tarsi pale to yellowish (Figure 7C). Forelegs: length ratio of femora: tibiae: tarsi = 2.0: 2.2: 3.6, tarsal segments from basal to apical = 0.2: 1.4: 1.0: 0.8: 0.4, segment I reduced in length, about 1/7× of segment II (Figure 7C). Midlegs: length ratio of femora: tibiae: tarsi = 2.2: 1.8: 0.8, tarsal segments arranged in decreasing order as 5, 1, 2, 3, 4. Hindlegs: length ratio of femora: tibiae: tarsi = 2.0: 1.8: 0.5, tarsal segments similar to midlegs. Forewing transparent, stigmatic area semi-hyaline; Rs and MP forked equivalently, MA forked over ½ distance from base to margin (Figure 7H). Hindwing hyaline, with costal projection at base, MP forked in middle, and MA forked much more apically than MP (Figure 7I).

Abdomen: each abdominal tergum has four pale dots; those of terga IV, V, VII are larger, making them almost pale with dark dots or stripes (Figure 7D). Genitalia: styliger plate with median concave (Figure 8). Segment IV of forceps slightly shorter than segment III; the combined length of segments III–IV is less than half of segment II (Figure 8A). Penes almost fused totally, apex extended laterally and with obliquely truncate, ventral surface with remarkable dark spermatic duct, base stem of penes sclerotized laterally; median spine-like titillators reduced (Figure 8). Cerci pale with tiny setae on surface and dark brown articulations.

Male subimago (in alcohol): body length 7.0–9.0 mm, cerci 12.0–14.0 mm, forewing 9.0–10.0 mm, and hindwing 2.5–3.0 mm. Body brown with dark dots and markings, color pattern resembles nymph and imago. Forewing semi-hyaline, crossveins pigmented brown to dark brown. Femora: tibiae: tarsi of forelegs = 2.0: 1.8: 2.2, tarsal segments arranged in decreasing order as 2, 3, 4, 5, 1; those of midlegs = 2.2: 1.8: 1.0, tarsal segments arranged in decreasing order as 5, 2, 3, 1. 4; those of hindlegs = 2.5: 1.8: 0.8, tarsal segments arranged in decreasing order as 5, 1, 2, 3, 4. Cerci pale to yellowish, with tiny setae on surface.

Female imago (in alcohol, Figure 6B,C and Figure 7E–G): body length 8.0–10.0 mm, cerci 15.0–17.0 mm, forewing 8.0–10.0 mm, hindwing 2.5–3.0 mm (Figure 5B and Figure 6B,C). Color pattern similar to nymph and imago but generally darker. Wings with conspicuous brown crossveins (Figure 7F,G). Femora: tibiae: tarsi of forelegs = 2.1: 2.2: 2.4, tarsal segments arranged in decreasing order as 2, 3, 4, 5, 1; femora: tibiae: tarsi of midlegs = 2.6: 2.0: 0.8, tarsal segments arranged in decreasing order as 5, 1, 2, 3, 4; femora: tibiae: tarsi of hindlegs = 2.8: 1.8: 0.5, tarsal segments similar to midlegs. Sternum VII extended posteriorly, near the posterior margin of sternum VIII; subanal plate with the convex posterior margin (Figure 7E). Cerci pale with dark brown articulations.

Female subimago (in alcohol): body 8.0–10.0 mm, cerci 12.0–13.0 mm; forewing 8.0–10.0 mm, hindwing 2.5–3.0 mm. Femora: tibiae: tarsi of forelegs = 2.4: 2.2: 1.4, tarsal segments arranged in decreasing order as 2, 3, 5, 1, 4; those of midlegs = 2.8: 2.4: 1.0, tarsal segments in decreasing order as 5, 2, 1, 3, 4; those of hindlegs = 3.0: 2.0: 1.0, tarsal segments similar to midlegs. Color pattern resembles female imago but dull, wings with more pigments around crossveins.

Egg (Figure 9): oval, chorion decorated with irregular small tubercle-like projections (Figure 9A). Small knob-terminated coiled threads (KCTs) densely concentrated on the whole surface, micropyle (M) situated equatorially (Figure 9B).

Distribution: South of China (Anhui, Fujian, Guangxi, Guizhou, Hainan, Henan, Hong Kong, Hunan, Jiangxi, Zhejiang).

Remarks: At the species level, the male of Maculogenia ngi (Hsu, 1936) has no apical spines on penes, forewings are totally hyaline and with pale veins except for dark subcostal brace. The color pattern of the male is also outstanding. Similarly, the female also has a mottled body, and its wings have brown veins, especially the crossveins. The nymph of this species has a narrow labrum and colorful body.

4. Discussion

In China, Kang and Yang named the species Electrogena fracta from Chinese Taiwan province upon its egg structure and nymphal stage [16]. Later, Ishiwata transferred it to the genus Ecdyonurus Eaton, 1868, in the checklist of Japan in 2001 [17,18], whereas Shieh et al. still used the name Electrogena fracta [19]. Judging from the original description and pictures, we assume that this species is another one of the genus Maculogenia Zhou, gen. nov. We also sequenced the COI gene of the Maculogenia ngi (Hsu, 1936). The K2P distance of those two species is 14.1%. The genetic distance between mayfly congeneric species varies greatly, from 6.7% to 32.9% [20], or greater than 0.18 in Baetidae [21]. However, at present, we do not want to transfer it into the genus Maculogenia formally because we do not have any Taiwanese materials to ensure.

Usually, the mayflies show distinct sexual dimorphism of males and females. For instance, in the genus Ephemera (Ephemeridae), the males have more colorful wings and conspicuous markings on their abdomen. In contrast, in the genera Cloeon (Baetidae) and Habrophlebiodes (Leptophlebiidae), the crossveins and wings of females are more distinct and pigmented than those of the males [22]. The adults in Heptageniidae show diverse patterns of sexual dimorphism, as the male can be more beautiful (such as the species Paegniodes cupulatus, Epeorus melli) or similar to each other (such as the species of Afronurus) [23]. The species Maculogenia ngi shows another extreme example. In this case, the crossveins of female wings are pigmented brown cloud while the veins of the male wings are totally pale except for a dark dot on the subcostal brace.