A contribution to the genus Afronurus Lestage, 1924 in China (Ephemeroptera: Heptageniidae, Ecdyonurinae)

The genus Afronurus has several very common mayfl y species in China and they are widely distributed in this country. Some of them are quite similar to each other in both imaginal and nymphal stages. However, these species have not been systematically compared and reviewed so far. In this study, six species are recognized. All nymphs of them share the following characters: gills V–VI with additional arrow-like accessory lobes, branched dentisetae, two rows of bristles and setae on hindtibiae and spotted abdominal terga. The males have divergent penes and clearly expressed titillators. The nymphs of the new species A. drepanophyllus sp. nov. have sickle-like gills I, spotted and striped body color, and males have unique genitalia. The nymphal stages of A. furcatus and A. hunanensis, which are associated and described for the fi rst time, have similar body color to A. drepanophyllus sp. nov., but their pale dots on the head capsules and the shape of the hypopharynx are different. Keys to males and nymphs of the six species are provided.

In China, 18 species under the name of Afronurus or Cinygmina have been reported You 1987;Zhang & Cai 1991;Kang & Yang 1994;Zhou & Zheng 2003;Braasch & Jacobus 2011;Sartori 2014). However, only 4 species were described at both nymphal and imaginal stages, and all original descriptions are very brief and simple. For instance, You (1987) described six Afronurus species from Tibet in brief: A. kouandengensis You, 1987, A. linzhiensis You, 1987, A. yadongensis You, 1987, A. xiasimaensis You, 1987, A. yadongxiasimaensis You, 1987and A. xizangensis You, 1987. However, most of the types are subimagoes, and their exact status demands more research and material. Kang & Yang (1994) described four species from Taiwan based on nymphs. In contrast, Braasch & Jacobus (2011) reported two new species from Hong Kong based on adults. Sartori (2014) transferred another Hong Kong species, Compsoneuria taipokauensis Tong & Dudgeon, 2003, into Afronurus. All these species need to be clarified using more associated specimens. Zhou & Zheng (2003) compared some species of Cinygmina from this country, but most species were known from imaginal stages at that time. In order to know the exact condition of this genus in China, we checked all related materials in our collection. As the first report of a serial study, here we compare and clarify the most common species on the Chinese mainland, leaving those species from the Chinese Hong Kong, Taiwan and Tibet areas for later reports.
As a result, six species are presented in this study, including a new one. They are: A. furcatus (Zhou & Zheng, 2003), A. hunanensis (Zhang & Cai, 1991), A. obliquistriatus , A. rubromaculatus , A. yixingensis  and A. drepanophyllus sp. nov. The eggs, nymphs and males of them are described and photographed. The nymphs of six described Afronurus species have the typical characters within Afronurus specified by Kluge (2004) and Webb & McCafferty (2008): anterior margin of head slightly thickened, mid-and hindtibiae with two rows of setae, maxillae with scattered setae on ventral surface and branched dentisetae. The male imagoes have titillators, which has been inferred by Braasch & Jacobus (2011) and considered as the main character of the Afronurus assamensis group (= Cinygmina) in Asia. In addition, the egg structures have small KCTs at the poles and large KCTs located equatorially, which is consistent with the theories of Belfiore et al. (2003), Kluge (2004) and Webb & McCafferty (2008).

Material and methods
The nymphs were collected by hand net while adults were attracted by lights. Some adults were reared indoors 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 stereo microscope. Some small structures, like mouthparts, claws and gills, were observed and photographed with a microscope camera.
All specimens used in this study are deposited in the Mayfly collection, College of Life Sciences, Nanjing Normal University (NNU).

Taxonomy
Compared to other Asian congeners in the genus, this new species appears to be closely related to the Thailand species A. namnaoensis Braasch & Boonsoong, 2010 in both nymphal and imaginal stages, e.g., the structures of mouthparts, legs and the shape of genitalia. However, the nymph of A. drepanophyllus sp. nov. has larger body size (6.0-8.0 mm); head capsule has 2-5 pale dots on anterior margin and 4-5 pale dots on posterior margin, respectively; abdominal terga yellowish brown and with marked pale dots on terga I-IX; gill I sickle-like; caudal filaments slightly longer than body. In contrast, the nymph of A. namnaoensis has smaller body size (4.5-6.1 mm); head capsule without distinct dot; terga yellowish and with light markings on terga VII-IX; gill I leaf-like; caudal filaments 2× length of body. In male imago, A. drepanophyllus sp. nov. has larger body size (8.0-10.0 mm); abdominal terga I-IX with brown longitudinal stripe and a pair of small pale dots medially; styliger plate with projections laterally; forceps with segment III subequal in length to segment IV and combined segments III and IV about half of segment II. Comparatively, A. namnaoensis has smaller body size (6.2-6.9 mm); terga I-IX with median stripe only; lateral margins of styliger plate slightly expanded; forceps with segment IV half of segment III in length and combined segments III and IV ⅓× length of segment II.

Etymology
The Latin specific name 'drepanophyllus' means 'sickle'. It indicates the nymphs having sickle-like gills I.
Coloration. Body yellowish-brown to brown. Head. Head capsule subquadrangular, anterior margin slightly thickened and with 2-5 small pale dots (some immature individuals less than 5 dots), posterior margin slightly concave; additional 4-5 pale dots located between compound eyes (Fig. 1A). Labrum expanded laterally, with slightly rounded apices; anterior margin with shallow median groove, long and dense setae on dorsal and ventral surfaces, those on dorsal surface longer and denser than others ( Fig. 2A-B); an additional row of short bristles on ventral anterior margin (Fig. 2B). Both mandibles covered with numerous setae on outer margins; prostheca with 3-5 fimbriate bristles ( Fig. 2D-E, H-I); outer incisor of left mandible with serrated margin and one larger terminal denticle; inner incisor shorter than outer incisor and with trifid apex (Fig. 2D, H); outer incisor of right mandible serrated and with 2 apical terminal denticles; inner one divided into 2 sharp denticles ( Fig. 2E, I). Hypopharynx: apex of superlinguae strongly curved and extended into round lobelike structures, row of long hair-like setae on lateral margins from base to apex; lingua bell-like, subequal to superlinguae in length and with tuft of setae at apex (Fig. 2C). Maxillae with scattered fimbriate setae on ventral surface (Figs 2G, 4D), row of 13-15 comb-shaped setae on crown of galea-lacinia, middle combs with 9-13 teeth, distal dentisetae branched, proximal dentisetae bifid and fringed (Fig. 4A); maxillary palpi 3-segmented, first segment with setae on outer margin and basal half of inner margin; second segment obviously longer than basal one, outer margin with long setae, terminal segment with dense setaceous brush (Fig. 2G). Labium: slightly oblong, inner margins of glossae slightly expanded and with tuft of long setae; paraglossae expanded greatly into palpable lobes, with dense setae and bristles on dorsal and anterior margins; labial palpi broad, 2-segmented; ventral surface and free margin with setae brushes; dorsal surface with setae ( Fig. 2F).
tHorax. Pronotum slightly extended laterally, subequal to head in width ( Fig. 1A-B). Supracoxal spurs rounded. Femora of all legs with long setae on outer margins, dorsal surfaces and inner margins with two kinds of spatulate setae, the shorter setae more abundant than longer ones (Fig. 3J). Foretibia subequal in length to femur, base of outer margin with sparse hair-like setae (Fig. 3A), dorsal surface with rows of fine setae and bristles, inner margin with row of bristles only; foretarsi slightly shorter than ⅓ tibia, outer and inner margins with tiny setae. Midleg similar to foreleg, except tibia 0.7× length of femur, with row of hair-like setae on outer margin (Fig. 3B). Hindleg similar to midleg except tibia 0.65× length of femur, outer margin with rows of dense hair-like setae and bristles, dorsal surface with short and long bristles ( Fig. 3C-D). Claws of all legs with 3-4 subapical denticles (Fig. 3E).
abdoMen. Pale but with a longitudinal light brown stripe medially, terga I-VII with pair of pale dots, posterior margin of each tergum with dark brown stripe (Fig. 5F).
Genitalia. Posterior margin of styliger plate convex, lateral margins with two distinct lobes ( Fig. 5D-E). Forceps of segment III subequal to those of segment IV in length, combined segments III-IV approximately half length of segment II. Penial lobes divergent and with U-shaped cleft between them; apices of penial lobes divided into three parts or apices, the median one much broader than inner and outer lobes. Robust but blunt titillators at base of penes, covered by styliger plate and invisible in ventral view (Fig. 5D-E). Cerci pale with dense tiny setae on surface and brown dots on articulations.

Afronurus furcatus
Head. Head capsule subquadrate, anterior margin of head with 4 small pale dots and basal part of antennae with 2 pale dots; posterior margin of head slightly concave and with 4 pale dots between the compound eyes (Fig. 7A). Labrum 0.5× width of head, laterally tapering and slightly curved; anterior margin with shallow emargination; an additional row of short bristles on ventral anterior margin (Fig. 8A). Both mandibles covered with numerous setae on outer margins; prostheca with 6-8 fimbriate bristles (Fig. 8C-D, G-H); outer incisor of left mandible with serrated margin and one larger terminal denticle; inner incisor shorter than outer incisor and with 3 blunt denticles (Fig. 8C, G); outer incisor of right mandible serrated and with 2 apical terminal denticles; inner one divided into 2 sharp denticles (Fig. 8D, H). Hypopharynx: apex of superlinguae strongly curved and extended into round lobe-like structures, long hair-like setae up to the lower part of superlinguae; lingua bell-like, subequal to superlinguae in length and with tuft of setae at apex (Fig. 8B). Maxillae with scattered fimbriate setae on ventral surface (Figs 4D, 8F), row of 17-20 comb-shaped setae on crown of galea-lacinia, middle combs with 10-15 teeth, distal dentisetae branched, proximal dentisetae bifid (Fig. 4B); maxillary palpi with setae on outer margin and basal half of inner margin of first segment; segment II longer than the former one and outer margin with long setae, terminal segment with dense setal brush (Fig. 8F). Labium: glossae lobe rounded, inner margin slightly expanded, with tuft of long setae; paraglossae expanded greatly into palpable lobes, with dense setae and bristles on dorsal and free margins; labial palpi 2-segmented, ventral surface and free margin of second segment with setal brush, dorsal surface with scattered golden setae (Fig. 8E).
tHorax. Pronotum slightly expanded laterally, wider than head (Fig. 7A). Supracoxal spurs rounded. Femora of all legs with row of long setae on outer margins, dorsal surfaces with spatulate setae and inner margins with short bristles. Foretibia subequal in length to femur, outer margin with sparse hair-like setae near base, inner margin with row of bristles; foretarsi 0.4× as long as tibiae, outer and inner margins with tiny setae (Fig. 9A). Midleg similar to foreleg except tibia 0.91× of femur in length, outer margin with row of hair-like setae, dorsal surface with rows of hair-like setae and bristles; tarsi approximately ⅓ length of tibia (Fig. 9B). Hindleg tibia 0.8× of femur in length, outer margin and dorsal surface with rows of long setae and bristles, inner margin with row of bristles and very tiny setae (Fig. 9D); tarsi about ¼ length of tibia (Fig. 9C). Claws of all legs with 3-4 subapical denticles (Fig. 9E).

Egg
Oval, chorion mostly smooth and decorated with small KCTs at each pole, larger KCTs located equatorially (Fig. 6B).

Remarks
The male imago of A. furcatus is similar to the one of A. hunanensis due to color pattern of abdominal terga, plate-like titillators and bifurcated apices of penial lobes. However, they can be separated by the following characters: 1) the body color of A. furcatus is bright yellow when it is alive (Fig. 12A) while reddish brown in A. hunanensis (Fig. 12B); 2) A. furcatus has slightly divergent penial lobes, which are connected by a foliated structure (Fig. 13A-B), while penes of A. hunanensis are obviously divergent and with deeply cleft (Fig. 13C-D); 3) the plate-like titillators of A. hunanensis are smaller than in A. furcatus (Fig. 13A-D).

Afronurus hunanensis
Head. Head capsule ellipsoid, anterior margin unicolor, posterior margin slightly concave and with 4 pale dots in the middle of the compound eyes (Fig. 7D). Labrum ca half width of head, lateral margins expanded slightly; free margin nearly straight, both surfaces with setae but those on dorsal surface relatively longer and denser; ventral surface with shallow median groove, an additional row of bristles on ventral anterior margin (Fig. 10A). Both mandibles covered with numerous long setae on outer margins; prostheca with 5-7 fimbriate bristles ( Fig. 10C-D, G-H); outer incisor of left mandible with serrated margin and a large terminal denticle; inner incisor divided into 3 blunt denticles (Fig. 10C, G); outer incisor of right mandible serrated with 2 apical terminal denticles; inner one divided into 2 sharp denticles (Fig. 10D, H). Hypopharynx: apex of superlinguae extended into round lobe-like structures, row of long hair-like setae on lateral margins from base to apex; lingua bell-like, much shorter than superlinguae, apex with tuft of setae (Fig. 10B). Maxillae with fimbriate setae on ventral surface (Figs 4D, 10F), row of 16-18 comb-shaped setae on crown of galea-lacinia, middle combs with 10-13 teeth, distal dentisetae branched, proximal dentisetae bifid and fringed (Fig. 4C); maxillary palpi 3-segmented, basal segment with scattered setae on both outer and inner margins, second segment with long setae on outer margin and terminal segment with setal brush (Fig. 10F). Labium: glossae lobe rounded, inner margins slightly expanded, with tuft of long setae; paraglossae expanded into distinct lobes, with dense setae and bristles on dorsal surface and outer margin; labial palpi 2-segmented, ventral surface and free margin of second segment with setal brush (Fig. 10E).
tHorax. Pronotum slightly extended laterally, subequal in length to head (Fig. 7D). Supracoxal spurs rounded. Femora of all legs with acute and spatulate setae on dorsal surfaces, outer margins with long setae and inner margins with short bristles. Foretibia subequal in length to femur, outer margin with tiny setae at base; foretarsi approximately ⅓ length of tibiae (Fig. 11A). Midleg similar to foreleg, but tibia 0.89× of femur in length, outer margin and dorsal surface with rows of sparse setae (Fig. 11B). Hindleg similar to midleg except tibia 0.7× of femur in length and dorsal surface of tibia with long and short bristles (Fig. 11C-D). Claws of all legs with 4 subapical denticles (Fig. 11E).

Remarks
The nymphs of A. hunanensis and A. yixingensis can be separated into one group due to the anterior margins of head capsules being without any dot. However, the following characters can help differentiate them: 1) lingua of A. hunanensis is much shorter than superlinguae ( Fig. 10B) but in A. yixingensis it is subequal in length (as in Fig. 8B); 2) glossae of A. hunanensis are round (Fig. 10E) while slightly oblong in A. yixingensis (as in Fig. 2F); 3) number of comb-shaped setae on maxillae: 16-18 in A. hunanensis (middle combs with 10-13 teeth) and 12-14 in A. yixingensis (middle combs with 7-10 teeth); 4) gill VII of A. hunanensis is broad (Fig. 11I) but narrow, leaf-like in A. yixingensis (as in Fig. 9F); 5) caudal filaments of A. hunanensis are pale at base and yellowish-brown in other parts (Fig. 7F), whereas caudal filaments of A. yixingensis have dark brown dots on every two or three segments (Fig. 7O).
In male imago, A. hunanensis has plate-like titillators and bifurcated apices of penial lobes (Fig. 13C-D, I-J). In contrast, A. yixingensis has spine-like titillators and a remarkable projection between divergent penial lobes.

Remarks
In China, the nymphs of A. obliquistriatus and A. rubromaculatus are the most difficult to distinguish, especially when they are young or typical stripes are faded. Herein, the following combination of characters are differentiable: 1) lingua is much shorter than superlinguae in A. obliquistriatus (as in Fig. 10B), while that in A. rubromaculatus is subequal in length (as in Fig. 2C); 2) number of comb-shaped setae on maxillae: 11-13 in A. obliquistriatus and 14-16 in A. rubromaculatus; 3) A. obliquistriatus has lateral oblique stripes on terga I-X (Fig. 7H) and A. rubromaculatus has pale dots on terga I-X only (Fig. 7K); 4) posterolateral projections of A. obliquistriatus are moderately developed on segments III-VIII (Fig. 7H) but weakly developed in A. rubromaculatus (as in Fig. 7E); 5) caudal filaments of A. obliquistriatus have brown stripes on every two or three articulations (Fig. 7I), but A. rubromaculatus has dark brown stripes on some articulations (Fig. 7L).
In male imago, the color pattern of abdominal terga and shape of genitalia can be used as valid characters to distinguish them: 1) A. obliquistriatus has unique oblique stripes on abdomen (Fig. 12C), while A. rubromaculatus has reddish longitudinal stripes laterally (Fig. 12D); 2) penial lobes of A. obliquistriatus are slightly divergent (Fig. 13E-F), whereas A. rubromaculatus has a distinct conelike projection between obviously divergent penial lobes (Fig. 13G-H).

Remarks
Among the six valid Chinese Afronurus species, A. yixingensis and A. hunanensis are separated into one group because of the similar color patterns of the head capsule in nymphs. However, as stated above for A. hunanensis, the nymphs can be separated by shape of hypopharynx, glossae, gills VII, number of comb-shaped setae on maxillae and color patterns of caudal filaments. In the male imago, they can easily be differentiated by shape of penes, titillators and projection between penial lobes.  (Zhou & Zheng, 2003) (NNU). C-D. A. hunanensis (Zhang & Cai, 1991) (NNU). E-F. A. obliquistriatus  (NNU). G-H. A. rubromaculatus  (NNU). I-J. A. yixingensis

Ecology
Among the six Chinese Afronurus species studied in this paper, A. obliquistriatus, A. rubromaculatus and A. yixingensis are more common than the others and they are frequently found at the collecting sites. The last instar nymphs of A. obliquistriatus are recorded molting at about 10:00-12:30 AM or 17:00-19:30 PM while the last instar nymphs of A. rubromaculatus and A. yixingensis molted at about 16:00-19:00 PM local time, and these three species are observed molting underwater. The subimagoes persisted for 1-2 days and molted at 12:00-15:00 PM, the whole process lasting about 2-4 minutes. Moreover, in another observed species, A. drepanophyllus sp. nov., the last instar nymph molted at about 17:00-18:00 PM and the male subimago molted at 15:00-16:00 PM, the process lasting about 90 seconds.