Epeorus (Belovius) L-nigrum Matsumura, 1931 (Figs 10–14)

[ Japanese name "Matsumura-hirata-kagerou"]

Epeorus L-nigrum: Matsumura 1931: p. 1477 (male imago) [JAPAN: Hokkaido]; Ishiwata et al. 2018: p. 126, pl. 15, figs 1–4, figs 46-1–46-4, 47-10 (male imago, male subimago, nymph) [JAPAN]; Ishiwata & Fujitani 2018: p. 33, pl. 22, fig. 4 (egg) [JAPAN].

Epeorus l-nigrus: Ishiwata 2001b: p. 186, fig. 10F–J (male imago) [JAPAN: Chiba]; Ishiwata 2002: p. 23 (male imago) [JAPAN: Kanagawa]; Ishiwata & Takemon 2005b: p. 105, figs 46-1–46-4, 47-10 (male imago) [JAPAN]; Inada 2007: p. 111, fig. 1 (male imago) [JAPAN: Hyogo]; Maruyama 2016: p. 173, figs 1-171g, 1-178, 1-275, 1-276, 1-277, 1-278 (male imago, female imago, male subimago, female subimago) [JAPAN: Nara], mis-spelling.

Epeorus latifolium (non Epeorus latifolium Uéno, 1928): Horasawa 1929: p. 253, figs 1–3 (male imago, male subimago, nymph) [JAPAN: Nagano], misidentification (Ishiwata 2001a); Matsumura 1933: p. 96, pl. 21, fig. 8 (female imago) [JAPAN: Hokkaido], misidentification; Imanishi 1934: p. 389, mis-synonymy.

Materials examined. Type specimens: Syntypes: (an identification label by Imanishi, 1933, " Epeorus latifolium Uéno, Syn. Epeorus L-nigrum Matsumura, Det. K. Imanishi 1933"). 1 male imago, " 9 /VIII 1903, Ishiyama"; 1 male imago, "Sapporo, Matsumura; 6 /VII 1915 ", " Epeorus L-nigrum M. det. Matsumura"; 1 male imago and 1 female imago, "Sapporo, Matsumura; 28 /VII 1915 "; 1 male imago (Fig. 33), "Sapporo, Matsumura; 28 /IX 1921 ", "Type Matsumura; Epeorus l-nigrum M" (red label); 1 male imago, "Sapporo, Matsumura; ĭDz (means "garden light"), 10 /IX. 1931 "; 2 male imagines, "Maruyama [in Kanji], 6/7 (year not listed)"; 1 male imago, "Sapporo, Matsumura; 25 / VI ??8 (year unreadable)"; 1 male imago, "Sapproro, Matsumura; 21 /?? 20 (unreadable)"; 1 female imago, "Sapporo, Matsumura" (collection date not listed). Other specimens: JAPAN, HOKKAIDO, ISHIKARI: [3.] Sapporo-shi, Minami-ku, Jozankei, Toyohira-gawa Riv., upstream of confluence of mainstream and Ekiteisawa-gawa stream, 514 m a.s.l. (42°51'18.1" N, 141°08'59.0"E), 1 mature female nymph, 18.X.2003, K. Saito; [4.] Sapporo-shi, Minami-ku, Jozankei, Usubetsu-gawa Stream (a tributary of Toyohira-gawa Riv.), 472 m a.s.l. (42°54'33.5" N, 141°07'28.9"E),1 mature female nymph, 08.IX.2002, K. Saito; [7.] Sapporo-shi, Minami-ku, Jozankei, Otarunai-gawa Stream (a tributary of Toyohira-gawa Riv.), 394 m a.s.l. (43°01'56.1" N, 141°07'42.9"E), 3 mature male nymphs and 1 mature female nymph, 11.IX.2007, K. Saito; [11.] Sapporo-shi, Minami-ku, Toyotaki, Toyohira-gawa Riv., mainstream, 188 m a.s.l. (42°57'38.4" N, 141°13'50.9"E), 1 mature male nymph, 14. VI.2005, K. Saito; ditto, 1 mature male nymph, 13.VIII.2005, K. Saito; ditto, 3 male imagines, 15. VI.2020, T. Takayanagi; [12.] Sapporo-shi, Minami-ku, Makomanai, Toyohira-gawa Riv., mainstream, 82 m a.s.l. (42°59'02.5" N, 141°20'23.5"E), 1 mature female nymph, 12.IV.2019, T. Takayanagi: [13.] Sapporo-shi, Minami-ku, Makomanai, Toyohira-gawa Riv., mainstream, 68 m a.s.l. (42°59'58.5" N, 141°20'26.0"E), 5 mature male nymphs and 6 mature female nymphs, 26. VI.2006, K. Saito; ditto, 2 mature male nymphs and 3 mature female nymphs, 02.IX.2006, K. Saito; [14.] Sapporo-shi, Minami-ku, Minami 30-jo, Nishi 8-chome, Yamahana-gawa Stream (a tributary of Toyohira-gawa Riv.), 51 m a.s.l. (43°01'09.3" N, 141°20'56.1"E), 1 male imago, 23. VI.2018, F. Matsui; [15.] Sapporo-shi, Chuo-ku, Minami 13-jo, Nishi 1-chome, Toyohira-gawa Riv., mainstream, 28 m a.s.l. (43°02'35.3" N, 141°21'31.7"E), 4 mature male nymphs and 1 mature female nymph, 16. V.2020, T. Takayanagi; ditto, 1 mature male nymph and 1 mature female nymph, 19.viii.2020, T. Takayanagi; ditto, 6 mature male nymphs and 1 mature female nymph, 25.IX.2020, T. Takayanagi; ditto, 1 mature female nymph, 24.VII.2021, T. Takayanagi; ditto, 2 mature female nymphs, 08.VIII.2021, T. Takayanagi; [16.] Sapporo-shi, Chuo-ku, Kita 1-jo, Higashi 19-chome, Toyohira-gawa Riv., mainstream, 15 m a.s.l. (43°04'04.8" N, 141°23'13.2"E), 11 mature male nymphs and 15 mature female nymphs, 26. VI.2006, K. Saito; ditto, 3 mature male nymphs and 9 mature female nymphs, 02.IX.2006, K. Saito; ditto, 1 mature male nymph, 25.X.2006, K. Saito; ditto, 1 male imago, 26.VII.2018, T. Takayanagi; ditto, 2 male imagines, 1 mature male nymph and 2 mature female nymphs, 21.VIII.2021, T. Takayanagi; [17.] Sapporo-shi, Kita-ku, Kita 11-jo, Nishi 1-chome, Sosei-gawa Riv. (a tributary of Fushiko-gawa Riv.), 15 m a.s.l. (43°04'30.9" N, 141°21'10.9"E), 2 male imagines and 1 female imago, 23. VI.2021, T. Takayanagi; ditto, 1 female imago, 02.VII.2021, T. Takayanagi; ditto, 1 female imago, 12.VII.2021; [21.] Chitose-shi, Rankoshi, Chitose-gawa Riv., mainstream, 31 m a.s.l. (42°48'34.1" N, 141°34'28.6"E), 1 male imago, 06.VII.2020, T. Ito; [22.] Chitose-shi, Shincho, Chitose-gawa Riv., mainstream, 13 m a.s.l. (42°49'07.7" N, 141°38'25.8"E), 1 male imago and 3 female imagines, 05.IX.2017, N. Kuhara; HIYAMA (outside of the study area): Assabu-cho, Shirooka, Sasamodosawa-gawa Riv., 76 m a.s.l. (41°49'59.0" N, 140°19'57.6"E), 1 male imago, 08.VII.2019, T. Takayanagi.

Nymph (mature, in ethanol) (Figs 10–12).

Body length 9.8–11.0 mm (male), 9.4–9.8 mm (female). Cercus length ca. body length x 1.2.

Head. Shape ellipsoid. Color yellowish-brown to reddish-brown with pale markings. Compound eyes dark-gray to black. Ocelli dark-gray. Antennae brown. Anterior margin densely covered with hair-like setae extending to lateral margins. Dorsal surface of head covered with fine hair-like setae. Sparse longer fine hair-like setae located posterior to eyes (Figs 11a, b). Labrum: anterior and lateral margins evenly convex, anterior margin concave medially. Ventral surface with long antero-lateral bristles, row of short setae and brush of median fine hair-like setae on each side. Dorsal surface with four long bristles medially and two long bristles near each side of antero-lateral margin, and scattered variable-length setae (Fig. 12a). Mandibles: each outer incisor with three apical teeth and serrated margins, inner incisor with two apical teeth (right mandible) or four apical teeth (left mandible) and outer margin of right inner incisor serrated sharply, right inner incisor slender and almost straight. Tuft of long setae at base of inner incisor of right mandible, tuft of long setae and one brachy-pulmose seta on the base of inner incisor of left mandible (Figs 12b, c). Maxillae: one long, feather-like seta at base of apical tooth complex, some specimens with two setae (Fig. 28b). Hypopharynx: superlinguae distally widened, lingua shape subquadrate. Labium: labial palps two segmented, outer margin of proximal one with sparse, thick setae. Distal segment with sparse, hair-like setae on outer margin and dense, brush-like setae on distal 1/3 area. Glossae and paraglossae with long, dense setae.

Thorax. Color yellowish-brown to reddish-brown with pale markings dorsally, white ventrally (Fig 10). Pronotum roundly projecting laterally (Fig. 29a). Long hair-like setae on medial line to posterior margin of pronotum. Long hair-like setae on medial line to posterior area of mesonotum. Legs. Each anterior face of femur with dark-brown transverse markings on its proximal, medial, and distal parts and hypodermal black spots on its base and middle (Fig. 11c). Dorsal edge of femur with dense long blade-like setae, ventral edge with sparse short bluntly pointed spines. Femoral surface with diversely shaped setae: oblong, spatulate or semi-circular. Those near ventral edge of each femur tend to be oblong, and those near dorsal edge of each femur tend to be semi-circular. Tibia with two dark-brown markings on base and middle parts and long hair-like setae on dorsal edge. Tarsus darker in proximal area and dorsal edge with long hair-like setae (Fig. 11c). Tarsal claw with 4–5 small denticles.

Abdomen. Terga yellowish-brown to reddish-brown, each tergum III–IX with a pair of dark spots. Sterna white. Surface of each tergum with a cluster of long setae located to each side of midline. Posterior margin of each tergum with long setae and long, sharp denticles which are uniform and arranged in an even line (Figs 12g, 31b). Tergum I–VII each with three postero-lateral projections, ventral one blunt, lateral one long, pointed and bending slightly upward and dorsal one blunt (Fig. 30b). Posterior margin of Sternum IX with deep medial emargination forming sharp tips and covered in hair-like setae (Figs 12d, e). Dorsal surface of cercus with a row of fine setae. Gills. Gills III and IV largest in size, VII smallest. Each gill with variably sized, dark-violet spots on its posterior half, ground color slightly purple (Fig. 11d). Gills II–VII with anal-proximal projections, dark colored (Fig. 11d). Costal margin of gill I with fine hair-like setae. II–VII costal margin with rough surface of spines and distal margin with fine setae. Each gill plate with filaments forming a fan-shape together. Gill VII without longitudinal fold.

Male (in ethanol), redescription (Fig. 13).

Body length 9.6–12.9 mm. Cercus length ca. body length x 2.8. Forewing length 9.3–13.3 mm.

Head. Color white. Frons shape triangular. Compound eyes dark-gray to black, light-blueish-gray when alive. Ocelli grayish-white with dark-gray border (Fig. 13g).

Thorax. Ground color brownish-white to translucent, internal tissues visible. Pronotum with a pair of black line. Mesonotum with thick black markings on adjacent parts of sutures (Fig. 13g). Meso- and metapleuron with black markings (Fig. 13a). Wings. Hyaline, veins white to translucent. Pterostigmatic area cloudy. Forewing with black markings on the base of radius and cubitus to costal brace, junction of costal brace and costa and base of costa (Fig. 13e). Forewing axillary cord color black. Hindwing with black spot on the base of cubitus to the base of radius. Legs. Anterior face of fore femur with black line on its proximal half. Fore tibia with dark spot on its distal tip. Fore tarsus longest with each segment becoming shorter distally and each joint dark. Mid and hind femur with discontinuous black lines. Fourth segment of mid and hind tarsus shortest (Figs 13b–d). Tarsal claws dissimilar, one hooked-shape and the other oblong.

Abdomen. Ground color white to translucent. Posterior margin of each tergum color dark. Segments VIII– X color white, internal organs and tissues visible (Fig. 13a). Posterior margin of styliger plate shape edged and triangular, forceps base with developed projection which has sharp spines. Forceps four-segmented, with short comb-like spines on each inner surface, distal two segments dark in color, fourth segment with sharp setae on its surface. Penis lobes without spines, titillators developed (Fig. 13f). Cercus white.

Female (in ethanol) (Fig. 14).

Body length 10.5–13.1 mm. Cercus length ca. body length x 2.1. Forewing length 12.2–15.1 mm.

Head. Color white. Frons shape triangular. Compound eyes dark-gray to black, light-blueish gray when alive.

Thorax. Coloration as in male imago (Fig. 14a). Wings. Veins slightly brown. Basal markings as in male imago (Fig. 14a). Legs. Coloration and markings as in male imago. Fore tarsus not elongated. Fourth tarsal segment of each leg shortest, other segments almost equal length (Figs 14b–d). Tarsal claws dissimilar as in male imago.

Abdomen. White to translucent, greenish eggs visible when alive. Posterior margin of subgenital plate almost straight. Posterior margin of sternum IX with shallow emargination and both tips relatively sharp (Fig. 14e). Cercus white.

Diagnosis and comparison. Nymph. The nymph of E. L-nigrum is quite similar to that of E. latifolium . Among our samples, the body size of E. latifolium is larger than E. L-nigrum from May to June (reaching up to 15 mm vs. 11 mm, respectively). However, in samples collected later in the season, E. latifolium individuals become smaller. The following morphological characteristics can be used to differentiate the nymphs of the two species: 1) the shape of the marginal denticles on the abdominal terga differs, with E. latifolium having short, fine denticles (Figs 7k, 31a) and E. L-nigrum having long, sharp denticles (Figs 12g, 31b); 2) the posterior tip of abdominal sternum IX is rounded in E. latifolium (Figs 7i, j) but pointed in E. L-nigrum (Figs 12d, e); and 3) the outer margin of the left mandible’s inner incisor lacks sharp serrations in E. L-nigrum (Figs 12c, f) but has sharp serrations in E. latifolium (Fig. 7c). However, it appears that characteristics 1) and 2) are reliable diagnostic features earlier in the summer, but become less distinct later in the season (after September). In this study, specimens were differentiated by a combination of these two features. The serration on the mandibles may vary depending on the time elapsed since molting, but observations of the inner cuticle of the incisor suggest that serration is consistently absent in E. L-nigrum (Fig. 12f). This morphological trait may be useful for identifying this species, although it requires dissection and high-magnification observation. The proposed morphological description of E. L-nigrum nymphs is based on specimens identified through molecular analysis. Male imago. Distinguishing between E. L-nigrum and E. latifolium in male imago is straightforward based on their penial structures: E. L-nigrum has titillators, which are absent in E. latifolium . Additionally, E. L-nigrum lacks lateral projections, whereas E. latifolium possesses them. The black markings at the base of the forewing also differ: E. L-nigrum has "L" or "Z"-shaped markings on the costal brace and the base of the radius, while E. latifolium displays spotted markings only on the costal brace.

Remarks. Nymph. The nymph was described for the first time. Male imago. The specimens here identified as E. L-nigrum agree with the original description (Matsumura 1931) and the syntype specimens. Female imago. The female imago was described for the first time. Subgenus attribution. This species can be attributed to the subgenus Belovius by the following features: 1) wide-rounded anal-proximal projection on gills II–VI (Fig. 11d); 2) Male imago with titillators on its penis (a valuable character in Belovius) (Fig. 13f) (Kluge 2004). Tshernova (1981) probably observed this species and established the subgenus Belovius . Further notes are in the discussion section.

This species was originally described based on male imaginal specimens collected in "Sapporo". The description appeared in 6000 Illustrated Insects of Japan-Empire (Matsumura 1931; 1932), a publication noted for numerous taxonomic and nomenclatural issues that have complicated species concepts for many Japanese insects due to its insufficient descriptions (see Uéno 1931c regarding mayflies). Fortunately, we were able to examine syntypes of E. L-nigrum and found no contradictions in morphological characters among the materials examined (Fig. 33). This species was considered a synonym of E. latifolium from Imanishi (1934) onward; however, Ishiwata (2001b) suggested that E. L-nigrum and E. latifolium could be distinguished by the shape of male genitalia, recognizing E. L-nigrum (spelled as " E. l-nigrus ") as a valid species (Ishiwata 2001a). This study supports that view.

Emergence periods and habitat. Fully mature nymphs and imagines were continuously found from June to October, suggesting that this species is multivoltine. Mature nymphs collected in May and June were relatively large (ca., 11 mm), while those obtained from August to October were smaller (ca., 8 mm). This species is most common in the midstream riffles of the Toyohira-gawa River (localities [11.]–[17.]) but is absent from smaller mountain streams (localities [10.], [19.]).

Distribution. Japan (Hokkaido, Honshu, Shikoku, and Kyushu).