Rhyacophila septentrionis McLachlan 1865

Description of the larva

Colour: Live specimen golden-olive (Hickin 1954), pale ventrally. Specimens preserved in ethanol reddish brown, dorsum dark, ventrally pale; on abdomen, semicircular-triangular pale anterior dorsolateral areas, dorsocentral area of segments with darker circular pale spots, connected caudally with anterior dorsolateral areas through pale lines, forming W-shape. Sclerotized areas of head and thorax dark brown with pale areas, black in some places (Fig 22a).

Head (Figs 14, 22b). Maximum width in posterior third of head, lateral margins almost parallel (tapering slightly towards anterior end, but not constricted at level of eyes, Mackereth 1954, fig 1S; Hickin 1967, fig 40; tapering anteriorly, Edington & Hildrew 1981). Cephalic capsule (Figs 14a, 14b), dorsally and dorsolaterally dark brown, anterior third and areas adjacent to frontoclypeal and anterior coronal sutures and anterior dorsolateral areas paler (dorsal posterior region of head with conspicuous pattern of dark spots on lighter background, Edington & Hildrew 1981, Fig 33, arrow a); ventral side pale, with central and slightly darker U-shape area from middle to posterior end of protogula covered with pale spots (Fig 14c), aboral margins of genae just before union with small black fold (Hickin 1954, fig 4B, 1967, fig 54); in lateral view anterior edge and submentum reddish brown, posterior edge black; oval brown area with dark spots extending laterally from anterior edge of occipital foramen to anterior third of head (Fig 14b). Frontoclypeal apotome (Fig 14d) pale; 2 oval dark brown submesal muscle attachment spots in anterior third between setae #4; dark brown pigmentation in posterior third heart-shaped (crescent shaped, Mackereth 1954, fig 1S; Hickin 1967, fig 40), including pits #4 but not setae #6, with pair of oval lateral and 2–3 circular mesal paler brown muscle attachment spots with dark borders, anterocentral area pale with two oval pale brown spots in anteromesal position (Figs 14a, 22a, 22b). Two-thirds of posterior dorsal area of head brown, marked with darker and paler muscle attachment spots; submesally one pair of dark brown triangular patches with paler spots with dark borders outside posterior edges of frontal sutures; light triangular area on juncture of frontal and coronal sutures, small pale area around pit #9; three pale spots around setae #15, #16, and #19–21, last one connected with posterior pale areas; dark brown dots in posterior area of genae (Mackereth 1954, fig 1S; Hickin 1967, fig 40); dorsal head pits with dark brown borders, posteromesal edge of occipital foramen black.

Mandibles (Fig 15) asymmetrical, as in other rhyacophilids; left one bigger than right one. Subapicomesal blade of left mandible slightly concave, right mandible with small tooth in middle, both with darker apices (Figs 15, 22a). Labrum and maxillolabium as in other Rhyacophila species. Mentum rectangular, its anterior half slightly sclerotized. Labrum with small transparent area in central anterior edge.

Thorax (Figs 16, 22c): Anterior half of pronotum pale or very light coloured, posterior half with three darker areas, one median and two lateral (Edington & Hildrew 1981, fig 35; Hickin 1954, fig 5B, 1967 fig 56), in darker specimens posterior half brown, with mesal area darker. Sinuous posterior margin bordered by black band, posteriorly and posterolaterally light brown submesally; posterior half of pronotum (Fig 16a) dark, with narrow semicircular area extending anterolaterally and small area laterally, lateral area pale in each pronotal plate; central dark region triangular, tapered posteriorly and reaching posterior edge of sclerite, anteriorly truncate and wider than posteriorly, pale brown spots inside triangular region reaching posterior edge; each side of pronotal plate with two or three lines of pale brown spots with dark border of different shapes and parallel to posterior border; pale areas around setae 2–3#, 5# and 6#, four–six brown spots around setae #5; curved row of two or three dark dots extending from setae #5 toward anterolateral corner, two pale brown spots with dark borders anterolateral and posterolateral of seta 6#, circular and elongate respectively (Figs 16b, 22c); with narrow black band in anterolateral angles, extending posterad laterally above lateral margins to posterolateral corners (Fig 16b); anterior margin light brown, with a line of black spots near anterior angle; mesal area of posterior marginal band black (Fig 16b, 22c).

Legs: Similar to those of other Rhyacophila species. Colour pale yellowish, each with tarsus and claw darker, proximal parts of coxa, femur and distal parts of trochanters of fore- and midlegs black or dark brown. Narrow pale dark brown band in proximal dorsolateral area of tibia and distal dorsolateral area of femur of each pair of legs, each inner and outer face of each femur with band of dark brown circular spots. Forelegs each ventrally with spicules, extending from distal half of trochanter, all of femur, tibia and tarsus. Setae black or dark brown, some transparent in ventral area.

Abdomen (Figs 17, 18, 22d, 22e): Similar to other Rhyacophila species. Colour as for meso- and metathoracic segments, in preserved larvae semicircular-triangular pale anterior dorsolateral areas, dorsocentral area of each segment with darker circular pale spots, connected caudally with anterior dorsolateral areas through pale lines, forming W-shape, pale yellow ventrally. Widths of anterior abdominal segments similar, narrower in posterior segments. Lateral abdominal gills in tufts of 25–30 filaments. Dorsal sclerite of segment IX (Figs 17, 22d) light coloured, darker in central area, with continuous black band on anterior edge and two discontinuous black bands on posterior edge, posterior to setae #2; band of anterior edge narrower in center, surrounding anteriorly pit #1; pale spots inside central area, two pale areas laterally. Anal prolegs typical of group, each with long sword process (Figs 18a, 18b, 22e). Anal claws each with three short teeth on ventral edge (Figs 18a, 18c, 22e), basoventral hook black basally and reddish brown apically (Figs 18a, 18b).

Description of the pupa

Biometrics of pupa and cocoon. Male pupa: 10.00 mm (1 specimen); female pupa: 11.60 mm (1 specimen). Cocoon length: 14.40 mm (1 specimen).

Head: Distribution of setae on dorsal head similar to R. fasciata. Mandibles dark brown, left one with two large teeth and right one with three, anterior tooth bigger, with numerous fine teeth on inner blade. Antennae of variable length, reaching posterior edge of segment VII and posterior edge of segment IV in male and female studied, respectively.

Thorax: Tubercles of prothorax each with 6 setae on male and 4 setae on female studied. Hind wing pads reaching posterior edge of abdominal segment III of male and posterior edge of segment IV of female studied.

Abdomen: Paired anterior hook plates pedunculated, almost circular and present from abdominal terga IV to VII (Fig 11), more elongated on segment VII, each with more than 30 spines; also tergum III with pair of small flat hook plates each with 10–15 spines. Paired posterior hook plates sessile on terga III–V, almost circular, each with more than 30 spines.

Description of the imago

Male body length: 7.70 mm (1 specimen) each forewing: 12.20 mm, each hind wing: 10.60 mm; female, length: 8 mm (1 specimen), each forewing: 11.50 mm, each hind wing: 10.30 mm.

Colour: Specimens preserved in ethanol dark brown dorsally, yellowish ventrally, spurs yellowish brown, setae brown, females generally darker than males; small black or dark brown spots dorsally. Forewings dark with small pale spots, pale in preserved specimens, darker in females, hind wings pale; each with dark area on leading edge between subcostal (Sc) and middle space between first radial (R 1) and second radial (R 2) veins in fore- and hind wings, with some darker stripes; each forewing hyaline in circular-oval zone, connecting medial (M) and medio-cubital (m-cu) veins and in distal area of second cubital vein (Cu 2) (Holzenthal et al. 2007); in each hind wing proximal segment of medial veins and connection area (M 1-4), not conspicuous, second cubital (Cu 2) vein not conspicuous either totally or partially.

Male genitalia (Figs 19, 24, 31b, 31c, 34): Apical segment of each inferior appendage (Figs 19A, 24a, 34c) with basal and distal edges diverging, dorsal and ventral edges slightly diverging, posterior edge of 2nd segment of each inferior appendage slightly concave, ventral edge slightly convex, 2.5 times longer that dorsal edge (Figs 19A, 24a). Apicodorsal vertex angular, apicoventral angle rounded, projecting as thick lobe narrowing progressively to apex.

Parameres in ventral view curved posteromesad in apical half (Figs 19 BV p, 24c, 34d). In lateral view (Fig 19 BL) each constricted at short distance beyond base, dilated at middle, with rounded dorsal margin, without spines in midventral margin, few long and thick spines on lateroventral area; midlateral surface covered by fine spicules or setae from middle to ventral edge, absent in middle anteroventral and posterodorsal edges.

Aedeagus (phallicata) in lateral view (Fig 19 CL) with dorsal margin concave and posterior corner of concavity hooked anterad, posterior edge of aedeagus straight, projected posteroventrad in small ventral apex (Fig 19 CL); ventral lobe of aedeagus semicircular (Fig 19 BV vl). Lateroventral lobes of phallus straight, posterior edges strongly convex, apicolateral margins semicircular (Fig 19 CD lvl).

Apicodorsal lobe of segment IX dilated subapicolaterally (Figs 19 DD al, 24b, 31b, 34b), preanal appendages (Fig 19 DD pa, 24b, 31b, 34b) round, almost as long as apicodorsal lobe, covered partially by it, mainly posteriorly; in ventral view apical band (Fig 19 DV ab, 31c) V-shaped, longer than wide, posterior edges rounded, almost parallel, non-sclerotized inside area (Fig 19 DV va) straight, anal sclerites triangular (Fig 19 DV as).

Female genitalia (Figs 20, 26, 32a, 35): In lateral view (Figs 20L, 26L, 32a, 35c), posterodorsal margin of segment VIII valves on each side round, posterolateral margin with small excision in middle, ventral edge convex, dorsal edge with dorsolateral triangular projection (Fig 20L).

In dorsal view (Figs 20D, 26D, 35b), with indentation between segment VIII valves, projections triangular with rounded corners, delimiting oval space (Fig 20D).

In ventral view (Fig 20V, 26V, 35d) segment VIII valves forming two elongate sclerites, distal 2/3 of proximal edges diverging, distal edges straight or slightly concave; intersegmental membrane with pair of deep-dark, slightly elongate sclerites.

Morphological characters diagnosing larvae of R. fasciata and R. septentrionis

Head: In R. fasciata ventral side of head pale, in some specimens one or two rows of low conspicuous pale spots from posterior edge to halfway up the head. In R. septentrionis central area with slightly darker U-shape area from middle to posterior end of protogula, covered with pale spots.

In R. fasciata the pair of dark brown patches outside posterior edge of frontal suture in the shape of open wings, with dark spots inside. In R. septentrionis the pair of dark brown patches more nearly triangular, with paler spots with dark borders inside.

In R. fasciata mandibles dark reddish brown, uniformly coloured, left mandible with inner blade slightly convex. In R. septentrionis mandibles reddish brown, with darker apices, left mandible with inner blade slightly concave.

Thorax: In R. fasciata on posterior half three dark areas, one median and two lateral, two lines of dark spots inside median area, circular spot on anterior half; posterior area with two to three rows of brown spots with dark border in circular-oval shape and parallel to posterior border. In R. septentrionis on posterior half three dark areas (in Hickin 1954) or dark base with darker mesal stripe with two lines of pale spots inside, two pale brown spots with dark borders anterolateral and posteriolaterally to seta 6#, circular and elongate respectively; posterior area with two-three lines of darkly-bordered pale brown spots of different shapes and parallel to posterior border. In R. fasciata anterior margin with black spot near anterolateral angle; mesal area of posterior marginal band of sclerite dark brown or black posteriorly. In R. septentrionis anterior margin with a line of black spots near anterolateral angle, light brown in middle; mesal area of posterior marginal band of each sclerite black posteriorly.

Abdomen: In R. fasciata dorsal sclerite of segment IX uniformly coloured, brown spots in posteromesal area, band of anterior edge with two extensions surrounding pit #1, connected with dark stripes in darker larvae. In R. septentrionis sclerite of segment IX light coloured, mesal area slightly darker, with paler spots inside.

Morphological characters diagnosing pupae of R. fasciata and R. septentrionis

Abdomen: In R. fasciata paired anterior hook plates palmate, pair of small flat anterior plates without spines on tergum III; paired posterior hook plates slightly oblong transversally. In R. septentrionis paired anterior hook plates almost circular, pair of small flat anterior plates on tergum III with 10–15 spines; paired posterior hook plates almost circular.

Morphological characters diagnosing males of R. fasciata and R. septentrionis

Genitalia: In R. fasciata, posterior edge of 2nd segment of each inferior appendage slightly concave dorsally and convex ventrally, ventral edge slightly concave and 2.0 times longer than dorsal edge, apicodorsal vertex slightly angular. In R. septentrionis, posterior edge of 2nd segment of each inferior appendage completely concave, ventral edge slightly convex and 2.5 times longer than dorsal edge, apicodorsal vertex more conspicuously angular.

In R. fasciata parameres in lateral view each slender near base, dilated in middle, with almost parallel dorsal and ventral margins in central area (S-shape), with two rows of long and thick spines on midventral margin, pointing outward; midlateral surface with sparse fine spicules or setae, from middle posteroventral to posterodorsal edge, covering most of apex, absent on middle anteroventral and dorsal edges. In R. septentrionis, parameres each slender near base, dilated in middle, with rounded dorsal margin, without spines in midventral margin, few long and thick spines on lateroventral area; midlateral surface covered by fine spicules or setae, from middle to ventral edge, absent on middle anteroventral and posterodorsal edges.

In R. fasciata posterior edge of aedeagus straight, projected posteroventrad and rounded at posteroventral apex; ventral lobe of aedeagus triangular, lateral edges nearly straight; lateroventral lobes of phallus straight, posterior edges convex, apicolateral margins rounded. In R. septentrionis posterior edge of aedeagus straight, projected posteroventrad in small posteroventral protrusion; ventral lobe of aedeagus semicircular; lateroventral lobes of phallus straight, posterior edges convex, apicolateral margins semicircular.

In R. fasciata apicodorsal lobe of segment IX slightly dilated subapicolaterally, almost round, with small apicomesal excision in some specimens; preanal appendages round, shorter than apicodorsal lobe, almost covered by it; in ventral view, apical band V-shaped and 1.8 times as long as wide, posterior arms widely separated, nonsclerotized area between arms of apical band heart-shaped and with posteromesal excision and posterior edges rounded. In R. septentrionis apicodorsal lobe of segment IX strongly dilated subapicolaterally; preanal appendages round, almost as long as apicodorsal lobe, covered partially by it, mainly posteriorly; in ventral view, apical band V-shaped and 1.2 times as long as wide, posterior arms close together and almost parallel, non-sclerotized area between arms of apical band straight and narrow and without posteromesal excision.

Morphological characters diagnosing females of R. fasciata and R. septentrionis

Genitalia: In R. fasciata in lateral view with dorsal margin of segment VIII 3/4 as long as venter and with prominent dorsolateral projection, posterolateral margin of valve on each side irregular, with 1 or 2 posterior projections, posteroventral projection more conspicuous, ventral margin slightly concave near base and straight or convex distally, in some specimens slightly indented posteriorly. In R. septentrionis in lateral view, with dorsal margin of segment VIII half as long as venter and with prominent dorsolateral projection, posterolateral margin of valve on each side round and with small excision in middle, ventral margin convex.

In R. fasciata in dorsal view with indentation between segment VIII valves nearly enclosed by pair of thick posterolateral projections, with rounded apical and subapical corners, small projection on apical corner of some specimens. In R. septentrionis with indentation between segment VIII valves nearly enclosed by pair of triangular projections, with more-nearly acute apical corners.

In R. fasciata in ventral view segment VIII valves forming two elongate sclerites, basal third of proximal edges parallel, distal edges irregular, straight, diverging; intersegmental membrane with pair of dark ovoid or slightly elongate sclerites. In R. septentrionis in ventral view segment VIII valves forming two elongate sclerites, basal 1/3 of proximal edges converging, distal edges straight or slightly concave, diverging; intersegmental membrane with pair of deep-dark slightly elongate sclerites.

Genetic analysis

Rhyacophila fasciata has a wide area of distribution in northern and central Europe and there is no GeneBank entry from the type locality (Elberfeld, Germany), so we have included some sequences from localities around it (Belgium and Germany). In the first analysis, we used these sequences, together with others from localities that previously have been checked as R. fasciata from Bosnia and Herzegovina, Belgium, Croatia, Czech Republic, Estonia, Finland, Germany, Kosovo, Norway, Poland, Republic of Macedonia, Russia, Slovakia and Sweden, together with one sequence of R. cf. nubila from Russia and three of R. cf. obliterata from Poland and Russia.

In the second analysis, to build the tree, we selected a total of 52 sequences: a maximum of two sequences of R. fasciata per country (25 in total), as well as 3 sequences of R. septentrionis, 3 sequences of R. denticulata, 4 sequences of R. sociata from Spain and France (promoted to species by Valladolid et al. 2018), 3 sequences of R. kykladica (promoted to species by Valladolid et al. 2019), 7 sequences of R. fasciata delici Kučinić & Valladolid (in Valladolid et al. 2020) and 3 sequences of R. fasciata viteceki Valladolid & Kučinić 2020 (in Valladolid et al. 2020), 1 sequence of R. cf. nubila from Russia and 3 of R. cf. obliterata from Poland and Russia (see Table 1 for references). The intra- and interspecific distances were calculated with all the sequences of each species (162 in total), as well as the bootstrap values of main branches in the second tree. The second tree (as a graphic) includes only some selected specimens and the bootstrap values cited.

The first tree (Fig 27) shows that, despite the wide distribution of the specimens, the group is quite homogeneous genetically, with three well-supported branches, one for the R. fasciata specimens (bootstrap value of 98) (Fig 27a), a second for R. septentrionis specimens (bootstrap value of 99) (Fig 27b), and the last for the cf. obliterata specimens (b.v. of 99) (Fig 27d). Intraspecific distances (maximum composite likelihood model) are lower than 1% (0.0041 in the R. fasciata Group, 0.0014 in the R. septentrionis Group and 0.0053 in the R. cf. obliterata Group), while interspecific distances are higher than 1% (R. fasciata-R. septentrionis = 0.0290, R. fasciata-R. cf. obliterata = 0.1040, R. fasciata-R. cf. nubil a = 0.0787, R. septentrionis-R. cf. obliterata = 0.0867, R. septentrionis-R. cf. nubila = 0.0618).

The second tree (Fig 28) shows clear differences between R. fasciata and the other species included, that previously were considered as R. fasciata or subspecies of R. fasciata. Based on the divergence sequences, there are seven well-supported branches, corresponding to R. fasciata (bootstrap value of 99) (Fig 28a), R. f. delici (b.v. of 98) (Fig 28c), R. septentrionis (b.v. of 90) (Fig 28d), R. denticulata (b.v. of 99) (Fig 28e), R. sociata (b.v. of 97) (Fig 28f), R. kykladica (b.v. of 100) (Fig 28g), all clearly different from R. cf. nubila (Fig 28h) and R. cf. obliterata (b.v. of 100) (Fig 28i) branches. Rhyacophila f. viteceki (Fig 28b) appears as an intermediate group between R. f. delici and R. fasciata groups. Intraspecific distances (maximum composite likelihood model) are lower than 1% (Table 2a), being the lowest in R. septentrionis, R. f. viteceki, and R. kykladica (0.0014, 0.0017, and 0.0019, respectively) while interspecific distances are higher than 1% (Table 2b) among all of these species. The lower distances are between R. septentrionis and R. f. viteceki (0.0101) and between R. f. delici and R. f. viteceki (0.0203).