﻿A preliminary review of Isonychia Eaton, 1871 from Chinese mainland with a re-description of I.kiangsinensis Hsu, 1936 (Insecta, Ephemeroptera, Isonychiidae)

﻿Abstract Previously, seven species of the genus Isonychia Eaton, 1871 were reported in China, but they have never been systematically reviewed. After examining our collections from the Chinese mainland, six species and one additional subspecies have been recognized, compared, and photographed. Among them, I.kiangsinensis is redescribed in all stages and a neotype is designated. Its males have triangular penes and nymphs have three dark pigments on each gill. A synonym of I.guixiensisWu et al., 1992 (I.sinensisWu et al., 1992) is confirmed. The males of this species have nearly cylindrical penes and clear abdominal markings. Finally, two species and one subspecies are recorded for the first time in China: I.ussuricasibiricaTiunova et al., 2004, I.ussuricaussurica Bajkova, 1970 and I.vshivkovaevshivkovaeTiunova et al., 2004. Together with the I.ignota (Walker, 1853), I.sexpetalaTiunova et al., 2004, I.formosana (Ulmer, 1912) and possible I.japonica (Ulmer, 1920), they show the rich diversity of the genus Isonychia in China.


Introduction
The mayflies of the genus Isonychia are relatively large and common aquatic insects.They can be found in almost all of our sampling sites in China and are among the earliest reported species of Chinese Ephemeroptera.For example, I. formosana (Ulmer, 1912) was named based on materials from Taiwan province.However, since then, although a series of researchers have reported and described six additional Isonychia species from Chinese mainland (Ulmer 1925;Hsu 1936;She and You 1988;Wu et al. 1992;Tiunova et al. 2004), no comprehensive review has been conducted.Hsu (1936) named the species I. kiangsinensis based on male imagos and subimagos.Fifty years later, You and Su (1987) described its nymphal stage.However, their original descriptions were extremely brief, their drawings were not accurate, and the types of this species were lost, making the exact morphology and taxonomic status of this species unclear.ZooKeys 1178: 115-141 (2023), DOI: 10.3897/zookeys.1178.104619 Xin-He Qiang & Chang-Fa Zhou: The genus Isonychia of China Similarly, three other species of Chinese Isonychia have been reported based on imagoes only.She and You (1988) described I. hainanensis from Hainan province, southern China.Wu et al. (1992) named two additional species (I.sinensis Wu et al., 1992and I. guixiensis Wu et al., 1992) from Jiangxi province, central China.All of these species need to be compared in detail and their exact characters need to be illustrated.
Furthermore, we believe that there is some confusion in the historical studies on Chinese Isonychia.For instance, 1) the penes in the original picture of I. guixiensis are much smaller than those of others and look like the membranous processes of other congeners.2) Tiunova et al. (2004) synonymized the species I. hainanensis She & You, 1988 with I. ignota (Walker, 1853) without checking any Chinese materials or types.3) Ulmer (1925) reported I. japonica (Ulmer, 1920) (as Chirotonetes japonicus) from China, but Tiunova et al. (2004) confirmed that this species is found in Japan only and doubted the report of Ulmer (1925).
To solve these issues mentioned, it is necessary to systematically revise Chinese Isonychia specimens, especially those from northeastern China.In recent years, we have thoroughly checked our mayfly collections of the family Isonychiidae, focusing on the known species.As a preliminary result, we confirmed six species and one additional subspecies, including three new records, while leaving several possible new species and I. formosana for future studies.The main taxonomic characters of these species are photographed and presented to benefit future works.

Materials and methods
The species concept and delimitation are based on morphology, and species differentiation mainly follows the work of Tiunova et al. (2004).
The nymphs were collected by hand net, and the adults were attracted by lights.Some of the adults were reared indoors from mature nymphs (mature nymphs were put into a plastic tray with some water from the creeks they lived.The whole system was covered with a mosquito net and oxygen was supplied with a small fish pump).All materials were stored in ethanol (higher than 80%).
All specimens were examined under a stereo microscope (Mingmei Photoelectric, MZ81, Guangzhou, China) and photographed with a digital camera (Single Lens Reflex, Guangzhou, China).Some digital photos of whole nymphs and adults were taken by Sony a7R (Interchangeable Lens Digital Camera).Some small structures such as mouthparts, claws, and penes, were observed and photographed under a microscope camera (Nikon Eclipse 50i, Tokyo, Japan).Eggs were dissected from female imagos, fixed in 4% glutaraldehyde for 5-8 hours to preserve their structure, dehydrated using a series of ethanol solutions (30%, 50%, 70%, 90%, and 100%, 10-15 min each), critically dried to prevent artifacts, mounted onto stubs, coated with gold film in a vacuum, and photographed with a scanning electron microscope (Apreo 2S, Thermo Fisher Scientific Company, Massachusetts, USA).The distribution map is downloaded from the website (http://bzdt.ch.mnr.gov.cn/).All specimens used in this study are deposited in the Mayfly Collection, College of Life Sciences, Nanjing Normal University (NNU), China.
Diagnosis.The male of I. guixiensis has transparent wings (Fig. 1A, B), dark brown forefemora, foretibiae, apical half of tarsal segment I-IV and tarsal segment V (Fig. 1D).Its abdominal terga are brown with a pale median line and dark longitudinal submedian stripes (Fig. 1E).The penes are nearly cylindrical with pointed apicolateral angles, and membranous processes beneath penes are clear (Fig. 1F-H).The females are similar to the males, with concave sternum IX (Fig. 1C).
This species and I. kiangsinensis can be found sympatrically in Jiangxi province, central China.They can be differentiated by the following characters: (1) I. guixiensis is smaller than I. kiangsinensis; (2) the body of I. guixiensis is brown to dark brown (Fig. 1E) while the body of I. kiangsinensis is usually pink to reddish; (3) I. guixiensis has distinct brown markings on abdomen (Fig. 1E) while the I. kiangsinensis has none.(4) their penes are dissimilar: those of I. kiangsinensis are triangular but penes of I. guixiensis are nearly cylindrical with pointed apicolateral angles (Fig. 1G).
Remarks.Wu et al. (1992) named two species, I. guixiensis and I. sinensis, in a single paper, with the penes of I. guixiensis appearing remarkably small.After checking all types of those two species, we found no difference between them, and their abdominal markings are absolutely less distinct than those in the original figures of Wu et al. (1992), and the synonymy of two species is confirmed here.Because the name of I. guixiensis is before I. sinensis in the same paper, the former is retained as the valid name.
Diagnosis.The nymph of this species can be identified by its body with a pale midline from head to abdominal terga VII, tergum X with dark posterior half, and its gills with spines along whole margins (Fig. 2A, B).The male imago is characterized by its wings without any markable painting or pigments, MP of hindwing forked more apically than MA, and two forking points are distinct (Fig. 2C, D), almost total dark forelegs, especially its foretarsi (apical segment usually darker) (Fig. 2G), penes with clear membranous processes ventrally, and apical margin of penis distinctly convex (Fig. 2H-J).The body color is almost brownish to dark brown, with a clear median, longitudinal, pale line on terga I-X, anterior margins of each tergum pale (Fig. 2F).Female imago has a similar color pattern of body and foreleg to the male.Veins of wings are reddish brown to dark, very clear.Posterior margin of sternum IX is shallowly concave (Fig. 2E).
Generally, the males of this species can be identified by their uniform dark forelegs and shorter segment II of gonostyli.The nymphs can be classified by their gills (color and spine pattern) and color pattern of their abdomen.
Remarks.This species was first recorded in China as I. hainanensis by She and You (1988).However, Tiunova et al. (2004) synonymized it with the species I. ignota.We checked the holotype of the former species and found only very delicate and slight differences between them, like color and shape of penes, but we believe those differences are caused by long-term storage in alcohol of the holotype.In April 2023, we also visited some localities of the types in Hainan Island, China, and collected many specimens of the genus Isonychia.However, we did not find any of I. hainanensis.Therefore, we think this synonym is correct.Our record shows the species I. ignota has a wide distribution, from western Europe to southern China.
Four characters can be used to separate nymphs of I. kiangsinensis from other species: (1) abdominal terga I-X with clear pale median longitudinal line (Fig. 7A, C), (2) each gill lobe with two to three spots, the largest one in the middle dark purple, (3) gill lobes VI and VII with spines on apical margins (Fig. 10F, G). ( 4) Tergum X pale in anterior 1/2, brown in posterior 1/2 (Fig. 10J).Compared to known nymphs of Asian Isonychia species, gills of I. kiangsinensis usually have three markings, a large median purple one and two small brown ones.This character is somewhat unique.Generally, this species is larger, more reddish than others, and is distributed south of Yangtze River, China.
Nymph (in alcohol, Figs 7-10): body length 13.0-17.0mm, cerci 8.0-10.0mm, terminal filament 5.0-6.5 mm; body brown to dark amber, with a clear pale median longitudinal line; legs and tail with pale and dark bands, gills with purple markings (Fig. 7A-D).Head: genae brown, lateral 1/3 clypeus brown but median 1/3 pale; scape and pedicel of antennae darker than others, antennae smooth.Length of antennae ~ 3× width of head.Frontal carina pale, area between three ocelli brown, midline of head pale, area between median ocellus and compound eyes pale too.Frontal carina between two antennae, very sharp.Vertex smooth and convex.Dark base of ocelli clear (Fig. 7E, G).Mouthparts: dorsal surface and anterior margin of labrum with long hair-like setae, dorsal surface with additional bristles, ventral surface with two oblique lines of hair-like setae; anterior margin almost straight, lateral margins slightly convex (Fig. 8A).Left mandible: apex of both inner and outer incisor divided into three denticles, outer incisor slightly thinner than inner one; prostheca composed with a distinct spur and a tuft of spines (Fig. 8B).Right mandible: outer and inner incisor divided into two denticles, prostheca composed with a tuft of spines, a line of hair-like setae on mesal margin near molar (Fig. 8C).Hypopharynx: lingua nearly circular, with hair-like setae on ventral apex; superlinguae with straight lateral margins, apex with hair-like setae too (Fig. 8D).Maxillary palpi yellowish brown, length ratio of segments I and II = 1.0: 2.0; apical segment covered with dense hair-like setae and slightly broader than basal one.Two gill tufts between maxilla and labium (Fig. 8E).Galea-lacinia of maxilla with two apical canines, mesal margin with a row of hair-like setae, one distinct spine among them.Crown of maxilla and outer 1/2 surface of both sides with hair-like setae, an additional row of short hair-like setae near canine on ventral surface (Fig. 8F).Glossae and paraglossae of labium heart-shaped, the latter broader than the  former, surface of them with dense hair-like setae, those on margins longer.Labial palpi darker, basal segment: apical segment = 1.0: 2.0, outer margin with long hair-like setae, inner 1/2 surface of it with brush-like setae (Fig. 8G).
Thorax: color brown, with a median pale line and several irregular pale dots or markings on dorsal surface.Mesosternum and metasternum with a projection respectively directed posteriorly, the latter one broader and shorter (Fig. 7F).Gill tuft on forecoxa with pale body but gray filaments.Femora of foreleg with three pale bands on base, middle and apex respectively, tibiae with two pale bands on base and apex, apical 1/2 of tarsi pale, claw pale but with golden apex (Fig. 9A).Inner margin of foreleg with long hair-like setae but outer margin with bristles; apical spine of tibiae ~ 3/4 tarsi, it slightly bent.Claw with five or six denticles (Fig. 9B).Femora: tibiae: tarsi of foreleg = 1.0: 1.0: 0.6.Color pattern of midleg similar to foreleg, both outer and inner margins with spine-like setae only.Claw of midleg with eight or nine denticles (Fig. 9C, D).Femora: tibiae: tarsi of midleg = 1.0: 0.6: 0.4.Color and setae pattern of hind leg similar to midleg (Fig. 9E).Ventral cleft of hind femora usually with four spines (Fig. 9F).Femora: tibiae: tarsi of hind leg = 1.0: 0.5: 0.3.
Abdomen: brown, with a pale median line on terga I-VI, an additional pair of pale median oval dots beside line.Posterolateral angles of terga I-VII extended into blunt lobe while those of terga VIII and IX extended into spines (Fig. 7A-D).Gills I-VII similar in color and structure but larger progressively from anterior to posterior except dorsal gill lobes I-V without apical spines.Each gill lobe with three sclerotized ribs, two along margins and one nearly on middle of dorsal surface.Each gill with three purple dots, a bigger median one, a small apical one and the smallest anterolateral one.Front margin of each gill lobe slightly straighter than hind margin; ventral gill filament tuft with purple median 1/2, other parts pale (Fig. 10A-G).Sternite IX with distinct semi-circular shape cleft in both sexes (Fig. 10H, I).Anterior 1/2 of tergum X pale, posterior 1/2 brown (Fig. 10J).
Remarks.The holotype and paratypes of this species were lost.Both the newly found specimens and original descriptions of Hsu (1936) show this is a valid species; therefore, a male from Jiangxi Province, China (same province as the types) is designated here as its neotype.
The nymphs of I. kiangsinensis have posterolateral projections on terga VIII and IX (Fig. 7A-D), which was missing in the description of You and Su (1987).However, Vasanth et al. (2019) and Muthukatturaja et al. (2021) reported wrongly that I. kiangsinensis has projections on terga I-IX.
Diagnosis.The male of this species is smaller than most congeners but similar to I. ussurica.It has no pigments on wings, MP of hindwings forked more apically than MA, but two forking points are close (Fig. 12A, B), segment II of forceps with straight inner margin, penes nearly cylindrical with convex distal margins and membranous processes with pigmented or sclerotized tip (Fig. 12C-E).
The males of I. ivani Tiunova et Gorovaya, 2010 and I. sexpetala Tiunova et al., 2004 are alike because they have similar cylindrical penes (Fig. 12D) and the basal 1/2 of their foretarsi is usually pale (Fig. 1D), but their membranous processes beneath penes are different.The processes of I. sexpetala have a rounded apex (Fig. 12E) while those of I. ivani are tapered.The nymphs of these two species can be separated by their color pattern of abdominal terga and gills: (1) the pale median longitudinal line of I. sexpetala is shorter than that of I. ivani (Tiunova et al. 2004;Tiunova and Gorovaya 2010), (2) gills of I. ivani have apical small dark dots while those of I. sexpetala are transparent.
Description.See Tiunova et al. (2004).Distribution.China (Heilongjiang province); Russia (Tiunova et al. 2004).Diagnosis.The nymph of this species is smaller than others, abdomen without a clear pale line, tergum X with dark posterior half, and free margin of gills decorated with spines (Fig. 13A).The male of this subspecies is the same as I. ussurica ussurica (see below) except its forewing has no clear band (Fig. 13B-F).
Remarks.The size of brownish transverse band on forewing of our Chinese material seems smaller than that described by Bajkova (1970) and Tiunova et al. (2004) on Russian specimens (Fig. 14A).We regard this difference as a population variation.Tiunova et al. (2004) stated clearly that two subspecies are distributed in different parts of Russia: I. u. sibirica in the western part such as Siberia and Mongolia while I. u. ussurica is found in the eastern part, the Russian Far East.Consistently, our collections of these two subspecies from Heilongjiang province show that the distribution of I. u. sibirica is more western than that of I. u. ussurica.

Isonychia vshivkovae vshivkovae Tiunova et al., 2004 (first record from China)
Isonychia (Isonychia) vshivkovae vshivkovae Tiunova et al., 2004: 24 (nymph and adults).Types from Russia.Diagnosis.The nymph of this species can be identified by its gills without spines along apical margins, body with a longitudinal median pale stripe from head to abdominal tergum IX, and tergum X with a pair of pale spots (Fig. 15A).The male imago can be differentiated easily by its outer half pigmented forewings, especially pterostigma, MP of hindwing forked equally as MA (Fig. 15B,  C), relatively long and strong penes, apical half of penes slightly expanded and apical margins almost straight, membranous processes between penes and styligers are absent (Fig. 15E-G), basal half of each foretarsus is pale but apical half dark.Body is uniformly reddish brown to dark brown.Female imago  has a similar color pattern of body and foreleg to male, pterostigma of forewing pigmented only, veins are clear.Posterior margin of sternum IX is shallowly concave (Fig. 15D).
Remarks.Tiunova et al. (2004) divided I. vshivkovae Tiunova et al., 2004 into two subspecies, I. v. vshivkovae and I. v. sinitshenkovae.The former is distributed in the eastern part of Asian Russia and has pigmented forewings.Based upon the pigmented forewings and the collection location, our material is identified as Isonychia vshivkovae vshivkovae.Generally, species differences in both the nymph and adult stages are relatively few.The species can only be separated by tiny structures (the shape of penes and their membranous projections, the shape of forceps, and the spine pattern on the gill plate) or color patterns (stains on wings or pigment on forelegs) (Table 1).

Keys to
The six Isonychia species and one subspecies of Chinese mainland presented in the present paper show that, except I. ignota which has a wide distribution, the other five species can be divided geographically into southern and northern groups.The southern group includes two species (I.guixiensis and I. kiangsinensis) in south of the Yangtze River, while the northern one has three species and one subspecies (I.sexpetala, I. ussurica sibirica, I. ussurica ussurica and I. vshivkovae vshivkovae) found in northeastern China (Fig. 16).The huge gap in their geographical distribution highlights the need for more collections.
Additionally, Ulmer (1925) reported that I. japonica was found in southern Guangdong (Kuangtung) Province, China.However, Tiunova et al. (2004) clarified that this species is only found in Japan.In our opinion, this species may be found in northeastern China but will not be found in the southern part of  this country.So for now, we exclude this species from Chinese mayfly fauna pending more material from Guangdong Province and further molecular work on the related species.
So far, at least seven Isonychia species and one subspecies have been confirmed as occurring in China (including I. formosana).Other material in our collection suggest that China has more species than are currently known and hosts the most diverse species in Asia.

Figure 1 .
Figure 1.Imaginal structures of I. guixiensis A forewing of male B hindwing of male C subanal plate of female (ventral view) D foreleg of male E abdomen of male (dorsal view) F male genitalia (ventral view) G penes (dorsal view) H membranous processes (ventral view).

Figure 2 .
Figure 2. Imaginal and nymphal structures of I. ignota A nymph (dorsal view) B gill VII C forewing of male D hindwing of male E subanal plate of female (ventral view) F body of male imago (dorsal view) G foreleg of male H male genitalia (ventral view) I penes (dorsal view) J membranous processes (ventral view).

Figure 3 .
Figure 3. Structures of I. kiangsinensis male imago A habitus (lateral view) B head (dorsal view) C thorax (ventral view) D abdomen (dorsal view) E foreleg (arrow indicating the gill relics on forecoxa) F claw of foreleg G midleg H hindleg I claw of midleg.

Figure 4 .
Figure 4. Male structures of I. kiangsinensis A-E male imago F-G male subimago A forewing B hindwing C male genitalia (ventral view) D male genitalia (dorsal view) E penes (ventral view) F habitus (lateral view) G genitalia (ventral view).

Figure 5 .
Figure 5. Female structures of I. kiangsinensis A imago (lateral view) B subimago (lateral view) C head of imago (dorsal view) D head and prothorax of imago (ventral view, arrow indicating the gill relics on forecoxa) E abdomen of imago (dorsal view).

Figure 6 .
Figure 6.Structures of I. kiangsinensis female imago A forewing B hindwing C subanal plate D abdominal segments VII-X (ventral view) E abdominal segments VII-X (lateral view).

Figure 7 .
Figure 7. Nymphal structures of I. kiangsinensis A female nymph (dorsal view) B female nymph (ventral view) C male nymph (dorsal view) D male nymph (ventral view) E head of female F thorax of female (ventral view) G antenna H caudal filaments.

Figure 8 .
Figure 8. Mouthparts of I. kiangsinensis nymph A labrum B left mandible C right mandible D hypopharynx E maxilla F galea-lacinia of maxilla G labium.

Figure 9 .
Figure 9. Nymphal legs of I. kiangsinensis A foreleg B apical spine of foretibiae C midleg D claw of midleg E hind leg F ventral cleft of hind femur (ventral view).

Figure 11 .
Figure 11.Egg of I. kiangsinensis A overall view B surface enlarged C micropyle.

Figure 12 .
Figure 12.Male structures of I. sexpetala A forewing B hindwing C genitalia (ventral view) D penes (dorsal view) E membranous processes (ventral view).

Figure 13 .
Figure 13.Imaginal and nymphal structures of I. ussurica sibirica A nymph (dorsal view) B forewing of male C hindwing of male D male genitalia (ventral view) E penes (dorsal view) F membranous processes (ventral view).

Figure 14 .
Figure 14.Imaginal structures of I. ussurica ussurica A forewing of male B hindwing of male C subanal plate of female (ventral view) D male genitalia (ventral view) E penes (dorsal view) F membranous processes (ventral view).

Figure 15 .
Figure 15.Imaginal and nymphal structure of I. vshivkovae vshivkovae A nymph (dorsal view) B forewing of male C hindwing of male D subanal plate of female E male genitalia (ventral view) F penes (dorsal view) G male genitalia enlarged (ventral view).

Table 1 .
Comparison of Isonychia from Chinese mainland.