Rhyacophila anatolica Ekingen & Valladolid, 2024

Valladolid, Maria, Ekingen, Pinar, Arauzo, Mercedes, Dorda, Beatriz A. & Rey, Isabel, 2024, The Rhyacophila fasciata Species Complex (Trichoptera: Rhyacophilidae) in Turkey with description of a new species, Rhyacophila anatolica Ekingen & Valladolid, sp. nov., based on morphological, genetic, and ecological evidence, Zootaxa 5537 (3), pp. 301-324 : 306-320

Rhyacophila anatolica Ekingen & Valladolid , sp. nov.

Introduction: This species has been found in northern Turkey, mainly in the western and middle part of the Black Sea Region of Anatolia.

Derivatio nominis: Derived from the name of the area ( Anatolia) of Turkey where the new species has been found.

Material examined:

Holotype ♂: TURKEY (ANA 6), Çitdere, Yenice , Karabük, 41.032ºN, 32.436ºE, 1000 m a.s.l., 06/x/2020, leg. P. Ekingen [ RHY–ÖRN7 ( HUTC)]

Paratypes: TURKEY [ HUTC, 38 ♂♂ + 6 ♀♀ + 1 ♂ P + 1 ♀P + 3 L]; 21 ♂♂ + 1 ♀, same locality as holotype, 06/x/2020 (P. Ekingen) ; 5 ♂♂ Çitdere, Yenice , Karabük, 06/x/2020 (P. Ekingen) ; 5 ♂♂ Çitdere, Yenice , Karabük, 11/viii/2020 (P. Ekingen) ; 2 ♂♂ ŞimŞir Stream , Yenice, Karabük, 06/x/2020 (P. Ekingen) ; 1 ♂ + 1 ♀, Yedigöller National Park , Şelale, Bolu, 28/x/2023, (P. Ekingen) ; 3 ♂♂ + 4 ♀♀, Yedigöller National Park , Şelalenin yanındaki kol, Bolu, 28/x/2023, (P. Ekingen) ; 1 ♂, Yedigöller National Park , Derin Göl’e dökülen akarsu, Bolu, 28/x/2023 (P. Ekingen) [ HUTC] ; 1 ♂P + 1 ♀P, ŞimŞir Stream , Yenice, Karabük, 26/10/2019, (P. Ekingen) ; 3 L, ŞimŞir Stream , Yenice, Karabük, 11/viii/2020, (P. Ekingen) .

LARVA

Figs 2–7

Biometrics of last instar larva and prepupa: Length: 14.5–16.5 mm (x̄ = 15.8, SD = 1.09, n = 3). Maximum width of larva at metathorax: 2.3–2.8 mm (x̄ = 2.6, n = 3). Specimens preserved in ethanol yellowish, with dorsum darker.

Head ( Figs 2 , 13a ). Length: 1.7–1.8 mm (x̄ = 1.7, SD = 0.06, n = 3). Width: 1.21–1.24 mm (x̄ = 1.22, SD = 0.02, n = 3). Maximum width at posterior third of head, lateral margins slightly converging anteriorly, round posteriorly. Sclerotized areas of head generally pale with dark areas and spots, dorsal head pits with dark borders. Labrum with small transparent area in middle of anterior edge. Frontoclypeal apotome ( Figs. 2a, 2d ) pale, anterior edge concave; pair of small, transversely oval, pale brown, submesal muscle attachment spots in anterior third between setae S4; dark pigmentation in posterior third U-shaped, with truncate or rounded anterolateral edges on pits P4 but not around setae S6, anteromesal portion of dark pigmentation paler brown, somewhat square, main portion of dark pigmentation with four (two central circular and two lateral transversely oval) pale ring-shape muscle attachment spots ( Figs. 2a, 2d ). Parietals of cephalic capsule ( Figs 2a–2b ), with pair of longitudinal subdorsal bands in posterior two-thirds, these longitudinal subdorsal bands each darker and somewhat triangular, acute anteriorly and without muscle attachment spots, gradually broader posteriorly, and with two lines of 4–6 transversely oval, dark brown muscle attachments spots perpendicular to edges, forming light circular area surrounding juncture of frontal and coronal sutures, including pairs of pits P9 and setae S17, these longitudinal subdorsal bands narrow, light brown posteriorly, converging mesally toward pale yellowish longitudinal stripe along middle portion of coronal suture, with three or four spots aligned along each side of coronal suture, and 6–8 spots next to posterior edge; two pairs of pale spots around bases of setae S15 and S16 ( Figs. 1a , 2a ); laterally with longitudinally rectangular dark band in posterodorsal third, with oval dark spots ( Fig. 2b ); ventrally pale except anteroventral edge, and submentum reddish brown ( Fig. 2c ). Posterior occipital foramen brown with black corners at anteroventral area, dark brown line surrounding remainder of foramen.

Mandibles ( Figs 3D, 3V ) asymmetrical, as in other rhyacophilids; left mandible slightly longer than right one. Left mandible with mesal border of inner blade nearly straight, right mandible with inconspicuous, small tooth in middle, and concave subapically. Maxillolabium and labrum as in other Rhyacophila species.

Thorax ( Figs 4 , 13b ). Anterior half of pronotum pale or very light coloured. Anterior margin straight or slightly convex ( Figs 4a , 13b ), light brown, anterolateral borders strongly angulate ( Fig. 4b ); each side with two or three dark brown spots and black band from setae S22 and S 23 in anterior edge, and narrow black band in lateral edge touching broad posterolateral band ( Fig. 4b ). Posterior half of pronotum ( Fig. 4a ) with three vaguely defined dark regions separated by light zones and by setae S2 and S3 and pit P2 near mid-length of pronotum; (1) central dark region trapezoidal, tapered posteriorly and reaching posterior edge of sclerite, anteriorly truncate near midlength of pronotum, slightly wider than posteriorly, its lateral edges slightly concave or straight, pair of submesal lines of six or seven slightly dark brown, longitudinally oval spots inside this central dark region, reaching posterior edges of region; (2 and 3) pair of lateral dark regions irregular, each with its mesal edge convex, posterior edge convex and parallel to posterior mid-height pale brown border, and anterior edge diagonal between its anterior subdorsal edge and its posterior sublateral edge. Each lateral dark region with four or five brown spots around setae S5, transversely oval spot near its diagonal edge, and two transverse rows of circular or slightly transversely oval brown spots parallel to its posterior border. Posterior margin of pronotum strongly sinuous, with posterior border of each half black, this border at mid-height pale brown anteriorly ( Figs 4a, 4b , 13b ); each posterolateral margin projecting posterad and bordered by black from posterolateral corner, touching anterolateral band.

Legs. Similar to those of other Rhyacophila species. Colour yellowish, tarsi light reddish brown, proximal regions of coxae, trochanters, femora, and tibiae each with narrow black band, small circular black spots in both faces of each femur distally. Forelegs each with anterior and ventral rows of spicules, extending along distal half of trochanter, proximal half of femur, distal half of tibia, and distal half of tarsus. Setae black or dark brown, some transparent ventrally.

Abdomen ( Figs 5–7 , 13c, 13d ). Similar to that of other species of Rhyacophila . Preserved larva reddish brown dorsally, two stripes of anterolateral triangular pale spots, connected anteriorly and posteriorly with pale bar in central area, and two central stripes of oval dark spots ( Fig. 5 ), ventral side pale. Widths of anterior abdominal segments similar, narrower in posterior segments. Lateral abdominal gills in tufts of 20–35 fine filaments. Tergite IX ( Figs 6 , 13c ) yellowish, with black band on anterior edge and two black areas around central setae on posterior edge. Anal prolegs typical of group, each with long sword process ( Figs 7a; 7b , sp; 13d). Anal claws each with three teeth on ventral edge ( Figs 7a, 7c ), its basoventral hook black basally and reddish brown apically ( Figs 7b , bh; 13c).

Diagnosis of larvae

Head ( Figs 13a, 14a ). In R. anatolica , the pair of dark brown patches outside the posterior edge of the frontal suture are more or less triangular in shape, inside each patch two rows of dark spots aligned almost perpendicular to the frontal suture and a row of three or four spots aligned along sides of coronal suture. In R. fasciata the pair of dark brown patches outside the posterior edge of the frontal suture are in the shape of open wings, inside the darker anterior extension of each dark brown patch there is only a row of dark spots aligned parallel to the frontal suture, two groups of spots outside coronal suture ( Valladolid et al. 2021, fig. 4a).

In R. anatolica each side of the head has laterally a rectangular dark band with two lines of darker spots, extending one-third the distance from the posterior edge. In R. fasciata , each side of the head has laterally an oval dark area with darker spots, extending half the distance from the posterior edge ( Valladolid et al. 2021, fig. 4b).

In R. anatolica the left mandible has its inner blade nearly straight, right mandible with inconspicuous tooth. In R. fasciata the left mandible has its inner blade slightly convex, right mandible with small tooth ( Valladolid et al. 2021, figs 5Dl, 5Vl).

Thorax: In R. anatolica the anterior half of the pronotum is pale; each side with two or three dark brown spots, and black band from setae S22 and S 23 in anterior edge, and narrow black band in lateral edge touching broad posterolateral band. In R. fasciata the black band in each anterolateral angle extends posterad to join with the broad, black band of the posterolateral margin ( Valladolid et al. 2021, figs 6a, 6b); there is a conspicuous black circle sublaterally one-third of the distance from the anterior margin. In R. anatolica on the posterior half of the pronotum, the median dark stripe is truncate anteriorly and slightly wider than posteriorly, and with its lateral edges slightly concave or straight, pair of submesal lines of six or seven slightly dark brown, longitudinally oval spots inside this central dark region, reaching posterior edge. The posterior marginal band at mid-height is pale brown and black anteriorly and posteriorly, respectively. In R. fasciata on the posterior half of the pronotum the median dark stripe is convex anteriorly and with its lateral edges slightly convex. The posterior marginal band at mid-height is dark brown anteriorly and posteriorly, and black in the middle area ( Valladolid et al. 2021, fig. 6b).

Abdomen. In R. anatolica tergite IX yellowish, the band of the anterior edge is continuous, black; the central area of the tergite is pale; on the posterior edge a pair of black areas is on the posterior margin behind the bases of the central setae. In R. fasciata tergite IX ( Valladolid et al. 2021, fig. 7) the band of the anterior edge is continuously black in darker larvae, and has a pair of dark brown extensions surrounding the pair of pits P1; the central area of the tergite is uniformly light brown; on the posterior edge the pair of black areas surround the bases of the central setae.

PUPA

Figs 8l, 8r

Biometrics of pupa and cocoon. Pupal length (total): 8.7–12.3 mm (x̄ = 10.2, SD = 1.04, n = 13) [male pupa: 8.7–10.75 mm (x̄ = 9.8, SD = 0.77, n = 8); female pupa: 9.3–12.3 mm (x̄ = 10.9, SD = 1.17, n = 5)]. Cocoon length (total): 11.7–16.0 mm (x̄ = 13.6, SD = 1.14, n = 13) [male cocoon length: 11.7–14.0 mm (x̄ = 13.2, SD = 0.76, n = 8); female cocoon length: 12.0–16.0 mm (x̄ = 14.2, SD = 1.46, n = 5)].

Head. Mandibles reddish brown, with basis pale, left mandible with two preapical teeth, distal tooth larger, and right mandible with three preapical teeth, distal tooth larger, both mandibles with numerous fine teeth on inner blade. Antennae of variable length, reaching posterior edge of abdominal segment IV to posterior edge of segment VIII (from middle of segment VII to posterior edge of segment VIII in males, posterior edge of segment IV to middle of segment VI in females).

Thorax: Tubercles of prothorax each with 5 setae. Hind wing pads variously reaching posterior edge of abdominal segment III to posterior edge of segment IV.

Abdomen: Paired anterior hook plates pedunculate, and present on abdominal terga IV to VII ( Fig. 8 ), each with 5–10 spines (AIV, the smallest plates) and 10–20 spines (AV and AVII). Paired posterior hook plates PIII–PV sessile and circular, plates PIV slightly bigger, slightly transversely oval (length: width = 1: 1.2).

Diagnosis of pupae

Abdomen. In R. anatolica the anterior hook plates AIV are much smaller than the other plates; the paired posterior hook plates are almost circular except for oval PIV (length: width = 1: 1.2). In R. fasciata ( Valladolid et al. 2021, fig. 10), a pair of small flat anterior plates AIII without spines, and the paired anterior hook plates AIV are slightly smaller than the other plates; the paired posterior hook plates PIII–PV are all slightly transversely oval (length: width = 1: 1.3).

ADULT

Figs 10–12

Colour. Specimens preserved in ethanol reddish brown (some specimens yellowish) dorsally, yellowish ventrally, spurs reddish brown, setae brown, females generally darker than males; small black or dark brown spots in dorsal area of abdomen, in lateral view, some specimens with large elongate black spots marking border between dorsal and ventral areas. Forewings ( Fig 10a ) dark, each with small pale spots or uniformly coloured, one or two central transversal dark bands (fascia) in distal area more or less conspicuous, and one central dark spot in proximal area, with pale thick stigma (St) on leading edge between subcostal (Sc) and first radial (R 1) veins, hyaline circular zone in connection area of medial vein (M) and m-cu crossvein; additional crossvein connecting costal (C) and subcostal (Sc) veins in some specimens, crossveins connecting R 1 +R 2 (r) and fifth radial (R 5), and first medial (M 1) veins (r-m) present, distal segment of second anal (A 2) connecting first and third anal (A 1 and A 3) not conspicuous. Hind wings ( Fig. 10b ) pale, each with pale thick stigma on leading edge between subcostal (Sc) and second radial (R2) veins; proximal portion of medial vein (M) not conspicuous, median crossvein m missing; first cubital (Cu 1a-b) thicker; proximal crossvein cu-a connecting second cubital (Cu 2) and first anal (A 1) veins missing.

MALE

Biometrics. Type series: Lengths: Body (distance from front of head to distal edge of segment IX): 7.5–10.2 mm (x̄ = 9.0, SD = 0.67, n = 43), each forewing 9.6–12.5 (x̄ = 11.2, SD = 0.69, n = 43), each hind wing 8.3–10.9 mm (x̄ = 9.7, SD = 0.61, n = 43). Holotype male Lengths: Body, 9.7 mm, each forewing 11.1 mm, each hind wing 9.7 mm.

Genitalia ( Figs 11 , 15 ) as for R. fasciata except as follows: 2nd segment of each inferior appendage ( Figs 11A , 15a ) with basal and distal edges diverging, dorsal and ventral edges diverging, posterior edge straight or slightly convex, ventral edge slightly concave, 2 times longer that dorsal edge; apicodorsal vertex of 2nd segment ~ 120° and round, apicoventral angle ~ 50°, projecting as thick lobe tapering to round apex.

Parameres in ventral view curved posteromesad in distal half ( Figs 11 BV, p; 15c). In lateral view ( Figs 11 BL, 15d) each constricted 1/3 distance beyond base, dilated at middle, with round dorsal and ventral margins, almost parallel; along midventral margin abundant spines; midlateral surface with long spicules or setae of similar size, from midventral to dorsal edges. Aedeagus (phallicata) in lateral view ( Fig. 11 CL) with anterodorsal margin and posterior corner of dorsal concavity hooked anterad, apical edge of aedeagus concave; ventral lobe of aedeagus in ventral view triangular ( Fig. 11 BV, vl), in lateral view vertical edge concave, posteroventral angle broadly convex and obtuse ( Fig. 11 CL). In dorsal view posterior edges of lateroventral lobes of phallus apically convex, apicolateral margins straight or slightly convex, lateral margins diverging anterad ( Fig. 11 CD, lvl).

Apicodorsal lobe of segment IX in dorsal view dilated subapicolaterally, wider than long ( Figs 11 DD, al; 15b), with broad apicomesal excision; preanal appendages ( Fig. 11 DD, pa) rectangular, 2/3 as long as apicodorsal lobe, posterior edges convex. In ventral view, apical band oval ( Fig. 11 DV, ab), much longer than wide, its posterior arms parallel and directed posterad, and round apically, its non-sclerotized mesal area triangular ( Fig. 11 DV, va); anal sclerites triangular, posterior edges straight or slightly convex ( Fig. 11 DV, as).

Diagnosis of males

Genitalia. In R. anatolica the posterior edge of the second segment of each inferior appendage is straight or slightly convex. In R. fasciata , the posterior edge of the second segment of each inferior appendage is slightly concave dorsally, and convex ventrally ( Fig 16a ).

In R. anatolica the posterior edge of the aedeagus is concave in lateral view; the ventral lobe of the aedeagus is subtriangular, in lateral view round, obtuse; posterior edges of the lateroventral lobes of phallus are convex, the apicolateral margins are straight or slightly convex, and the lateral margins are diverging anteriorly. In R. fasciata , the posterior edge of the aedeagus is straight, projected posteroventrad, and rounded at its posteroventral apex; the ventral lobe of the aedeagus is subtriangular and round posteroventrally ( Fig. 16c ), its lateral edges are nearly straight, almost parallel; the lateroventral lobes of the phallus are straight, its posterior edges convex, apicolateral margins rounded.

In R. anatolica the apicodorsal lobe of segment IX in dorsal view is dilated subapicolaterally, wider than long ( Figs 15b ; 11 DD, al), with broad apicomesal excision; the preanal appendages are elongate rectangular, with posterior edges convex; the apical band in ventral view ( Fig. 11 DV, ab) is oval, 1.4 times as long as wide, its posterior arms are parallel and directed posterad; the non-sclerotized area between these arms ( Fig. 11 DV, va) is triangular; the anal sclerites ( Fig. 11 DV, as) are straight or slightly convex apically. In R. fasciata the apicodorsal lobe of segment IX in dorsal view ( Fig. 16b ; Valladolid et al. 2021, fig. 12DD, al) is dilated subapicolaterally, almost round, with a shallow apicomesal excision in some specimens; the preanal appendages ( Valladolid et al. 2021, fig. 12DD, pa) are round, convex laterally; the apical band in ventral view ( Valladolid et al. 2021, fig. 12DV, ab) is 1.2 times as long as wide, its posterior arms widely separate and straight; the non-sclerotized area between these arms ( Valladolid et al. 2021, fig. 12DV, va) is heart-shaped, and with a wider posteromesal excision; the anal sclerites ( Valladolid et al. 2021, fig. 12DV, as) are truncated apically.

In R. anatolica the parameres in lateral view ( Fig. 11 BL) are convex on dorsal and ventral margins, each has two rows of long spines along all its midventral margin; the midlateral surface has spicules or setae, similar in size from middle ventral to dorsal edges. In R. fasciata the parameres in lateral view ( Fig. 16d ; Valladolid et al., 2021, fig 12BL) are convex basally, each has two rows of long, thick spines on the posterior midventral margin, each row with more than three spines pointing laterad; the midlateral surface has sparse fine setae from the middle of the anteroventral edge to the posterodorsal edge, covering most of the apex, but absent in the middle anteroventral and posterodorsal edges.

FEMALE

Biometrics. Body length (distance from front of head to distal edge of segment VIII): 8.5–10.6 mm (x̄ = 9.6, SD = 0.83, n = 6), each forewing 9.5–12.8 mm (x̄ = 11.6, SD = 1.13, n = 6), each hind wing 8.7–11.0 mm (x̄ = 10.2, SD = 0.81, n = 6).

Genitalia ( Figs 12 , 17 ). In lateral view ( Figs 12L , 17L ), posterior margin of segment VIII valves on each side triangular, with dorsolateral projection directed dorsomesad, posterior dorsolateral and posteroventral margins concave, posterolateral projection round. In dorsal view ( Figs 12D , 17D ), with indentation between segment VIII valves nearly enclosed by pair of dorsolateral suboval projections directed dorsomesad, slightly dilated at apices, with rounded apical and subapical corners, delimiting bean-shape space. In ventral view ( Figs 12V , 17V ) segment VIII valves forming two elongate sclerites, concave mesally, and diverging in apical halves, separated anteriorly; intersegmental membrane distally with pair of dark, ovoid or slightly elongate sclerites, converging anteriorly.

Diagnosis of females

Genitalia. In R. anatolica lateral view ( Figs 12L , 17L ) each of the segment VIII lateral valves is triangular, with an apicodorsal projection; the oblique posterodorsal and posteroventral margins are concave, meeting at a round apex. In R. fasciata lateral view ( Fig. 18L ; Valladolid et al. 2021, fig. 13L) each of the segment VIII lateral valves is quadrate with an apicodorsal projection; the upper posterior margin is more nearly vertical, and with an irregular edge having one or two posterior sinuosities ( Valladolid et al. 2021, figs 13La, 13Lb), the posteroventral margin is slightly concave sub-basally, and straight or convex distally, in some specimens slightly indented posteriorly, the upper posterior margin and the posteroventral margin meet at a round, obtuse angle.

In R. anatolica dorsal view ( Figs 12D , 17D ), the indentation between the segment VIII valves is nearly enclosed by pair of suboval apicodorsal projections, slightly dilated at their apices, delimiting a bean-shape membranous space. In R. fasciata dorsal view ( Fig. 18D ; Valladolid et al. 2021, fig. 13Da), the indentation between the segment VIII valves is nearly enclosed by a pair of thick and tapered apicodorsal projections delimiting an oval membranous space; a small projection occurs anteromesally in some specimens.

In R. anatolica ventral view ( Figs 12V , 17V ), the segment VIII valves form two elongate sclerites, each with concave and convex edges in posterior and anterior areas respectively, widely separated basally; the intersegmental membrane has a pair of ovoid elongate sclerites, converging anteriorly. In R. fasciata ventral view ( Fig. 18V ; Valladolid et al. 2021, fig. 13V), the segment VIII valves form two elongate sclerites that are narrowly separated basally, the mesal margins are parallel in their basomesal 1/4, and straight and divergent ~ 47° in their apicomesal 3/4, their lateral margins convex; their intersegmental membrane has a pair of ovoid elongate sclerites separated, that are almost parallel.

Phylogenetic considerations

The combined BI + ML tree ( Fig. 19 ) shows clear differences between R. fasciata and the other species included, that previously were considered as R. fasciata or subspecies of R. fasciata . ML (bootstrap support, BS) and BI (posterior probabilities, PP) phylogenetic analyses recovered similar topologies, supporting two major clades: monophyletic R. kykladica (BS = 86, PP = 1) ( Fig. 19k ), and the rest of species of the “ R. fasciata Species Complex” (BS = 69, PP = 0.97). Inside this last clade, we found six well supported groups, and three paraphyletic groups: (1) paraphyletic R. septentrionis (BS = 69, PP = 0.97) ( Fig. 19b ), (2) paraphyletic R. viteceki (BS = 51, PP = 0.82) ( Fig. 19c ), (3) monophyletic R. loeffleri (BS = 75, PP = 0.98) ( Fig. 19d ), (4) paraphyletic R. macedonica (BS = 76, PP = 0.98) ( Fig. 19e ), and (5) monophyletic R. fasciata (BS = 73, PP = 0.97) ( Fig. 19f ) as sister species. (6) Monophyletic R. delici (BS = 85, PP = 0.99) ( Fig. 19g ), (7) monophyletic R. denticulata (BS = 85, PP = 0.99) ( Fig. 19h ), (8) monophyletic R. sociata (BS = 90, PP = 0.97) ( Fig. 19i ), and (9) monophyletic R. anatolica (BS = 99, PP =0.99) ( Fig. 19j ), as sister species.

Intraspecific distances (maximum composite likelihood model) are lower than 1% ( Table 2a ), being the lowest in R. septentrionis , R. viteceki , and R. kykladica (0.0020, 0.0026, and 0.0030, respectively). Interspecific distances are higher than 1% ( Table 2b ) among all the species of the Complex. The lower distances are between R. septentrionis and R. viteceki (0.0100) and between R. loeffleri and R. viteceki (0.0163).

Discussion

The use of the combination of selected characters in males (e.g., inferior appendages, dorsal lobes, parameres, and aedeagus), and females (shape of the sclerotized lateral valves of segment VIII in dorsal, ventral, and lateral views) has allowed morphological descriptions of new species of the “ R. fasciata Species Complex” ( Valladolid et al. 2019), the resurrection of species considered synonyms of R. fasciata ( Valladolid et al. 2018, 2021), and changes of status from subspecies to species ( Valladolid et al. 2018, 2019, 2022). In the successive studies that we have carried out, our hypothesis has been supported that the morphological differences, which until now have been considered simple intraspecific variations, are actually interspecific differences, since, in those species with a wide distribution, and that coincide in an area with another species of the “ R. fasciata Species Complex”, the differences between them and the specimens with which they share territory are greater than the differences with other specimens of their species that are further away, sometimes hundreds or thousands of kilometers. In this study, these characters show the presence of a new species of this group, Rhyacophila anatolica .

Mitochondrial DNA (mtDNA) sequences can be useful as a first indicator in species delineation, and, combined with careful morphological, behavioural, distributional, and ecological analysis, can help to establish accurate species boundaries ( Bickford et al. 2007; Dasmahapatra & Mallet 2006; Galtier et al. 2009). Most recent studies show the importance of mtDNA in the emergence of new species through mitochondrial-nuclear (mitonuclear) coevolution. Among the mtDNA, the nucleotide sequence of the COI gene has been established as a highly effective DNA barcode for diagnosing the species boundaries of animals ( Bucklin et al. 2011; Dasmahapatra & Mallet 2006; Hebert et al. 2003b; Hill 2016). In most metazoan populations, there is a correspondence between the species designated by COI and species previously described morphologically ( Tavares & Baker 2008; Tavares et al. 2011); furthermore, discovery of barcode gaps often leads to the morphological/behavioural/distributional/ecological recognition of new species upon further study ( Bickford et al. 2007; Hebert et al. 2004; Hill 2016).

The corresponding differences observed at genetic and morphological levels corroborate the hypothesis that R. fasciata and R. anatolica are two different species: The intraspecific distances were always less than 1% divergence, and interspecific distances were higher than 1% ( Table 2 ). This result is similar to what we found in specimens of R. denticulata and R. sociata (Valladolid et. al. 2018), R. kykladica ( Valladolid et al. 2019) , R. delici , and R. viteceki ( Valladolid et al. 2020) , or R. loeffleri ( Valladolid et al. 2023) . Other authors that have analysed the divergence of COI sequences between different species of several taxonomic groups found that more than 98% of the species pairs show divergences greater than 2% (e.g., Hebert et al. 2003b), and that the intraspecific divergences usually are less than 1% ( Avise 2000). We also agree with the premise of Hebert et al. (2003a) that we can find new species by their genetic divergences from known species assemblages. In this case, the species was identified originally as R. fasciata .

In future studies we intend to prepare Cytochrome B gene sequences for these specimens. This gene, used extensively and particularly for vertebrates, has enabled researchers to resolve relationships among closely related taxa (Tauzt et al. 2003) and, combined with the COI findings, could lead us to a better knowledge of this group of Trichoptera in Europe. Another option that we would like to explore is the inclusion of a nuclear gene in the study, which complements the information from the mitochondrial DNA.

Our successive studies of the “ R. fasciata Species Complex” have increased the number of sequences (haplotypes), and provided more information about the historical relationships among the species previously described and possible new species, mainly in those species apparently sharing a recent common ancestor. Based on our morphological and genetic evidence, we therefore propose a new species: Rhyacophila anatolica Ekingen & Valladolid (sp. nov.) from Turkey. All the specimens from this country cited as R. fasciata fasciata by Malicky & Sipahiler (1993), Sipahiler (2007, 2008, 2012, 2013, 2014, 2016), and Sipahiler & Malicky (1987) belong to this new species.

Finally, the descriptions of species included in the “ R. fasciata Species Complex”, together with the addition of a few more descriptions of new species in this group (in preparation) will allow us to prepare a synopsis of the diagnostic characteristics of the Complex in a future publication.

Distribution and ecology

Figure 20 presents the current knowledge of the distribution of R. anatolica sp. nov. in Turkey. The species is found in the western and central Black Sea Region, and east of the Marmara Region, in small forest streams flowing into the Black Sea and the Marmara Sea. The substrate consists of boulder, cobble, pebble, and gravel, with sand in very small amounts, with rich stream bank vegetation, and consequently entrained fallen leaves and twigs. Sampling sites are relatively isolated from human influence, and range from 35 to 1534 m a.s.l. Flight activity occurs usually from late May to November. In Karabük, larvae are found in alkaline waters (pH 7.58–8.65), in streams 2–12 m wide, and with water temperatures from 10.41 to 19.53ºC, during three seasons (spring, summer, and autumn) ( Ekingen 2022).

Figures

FIGURE 1. Setae (S) and sensory pits (P) of the head and thorax of Rhyacophila larvae. 1a, head, dorsal; 1b, prothorax, right sclerite, dorsal (from Williams & Wiggins 1981).

FIGURE 8. Rhyacophila anatolica Ekingen & Valladolid sp. nov., paired abdominal hook plates of male pupa. 8l and 8r, left and right hook plates, respectively. A = anterior hook plates, dorsal, P = posterior hook plates, dorsal; III–VII = abdominal terga III through VII, dorsal. Scale bars: 50 μm.

FIGURE 19. Phylogenetic relationships among the species of the “Rhyacophila fasciata Species Complex” included in this study. The ML tree represents a combination of Bayesian inference and maximum likelihood trees based on COI. Support for each node is represented by the posterior probabilities (PP) resulting from the Bayesian inference analysis and the bootstrap support values (BS) obtained for the maximum likelihood tree (PP/BS, respectively). 19a, Rhyacophila cf. obliterata, outgroup; 19b–19k, R. fasciata Complex: 19b, Rhyacophila septentrionis McLachlan 1865; 19c, R. viteceki Valladolid & Kučinić 2020 (by Valladolid et al. 2020); 19d, R. loeffleri Valladolid & Waringer (by Valladolid et al. 2023); 19e, R. macedonica Karaouzas, Valladolid & Ibrahimi 2022 (by Valladolid et al. 2022); 19f, R. fasciata Hagen 1859; 19g, R. delici Kučinić & Valladolid 2020 (by Valladolid et al. 2020); 19h, R. denticulata McLachlan 1879; 19i, R. sociata Navás 1916; 19j, R. anatolica sp. nov.; 19k, R. kykladica Malicky & Sipahiler 1993. Data for specimens are summarized in Table 1 (R. anatolica) and by Valladolid et al 2024. Scale bar: mean number of nucleotide substitutions per site or nucleotide position on the respective branch.

FIGURE 20. Spatial distribution of Rhyacophila anatolica Ekingen & Valladolid sp. nov. in Turkey. Star: specimens with DNA information. B. Bulgaria, C: Cyprus, Ge: Georgia, Gr: Greece, I: Iraq, R: Russia, S: Syria.

FIGURES 2–7. Larva of Rhyacophila anatolica Ekingen & Valladolid sp. nov. 2a–2d, head: 2a, dorsal; 2b, right lateral; 2c, ventral; 2d, frontoclypeus, dorsal. 3D–3V, mandibles from last instar larval exuviae, left (l) and right (r): 3D, dorsal; 3V, ventral. 4a–4b, prothorax: 4a, dorsal; 4b, left hemisclerite, left lateral. 5, abdominal segment III, dorsal, A = anterior edge. 6, abdominal tergite IX. 7a–7c, details of anal prolegs: 7a, right anal claw, right lateral; 7b, detail of basolateral plate with sword process (sp) and basoventral hook (bh), right lateral; 7c, detail of right claw, right lateral. Scale bars: unlabelled = 1 mm; ● = 0.5 mm.

FIGURES 13–14. Larvae of Rhyacophila anatolica Ekingen & Valladolid sp. nov. and Rhyacophila fasciata Hagen 1859. 13, R. anatolica: 13a, head, dorsal; 13b, prothorax, dorsal; 13c, abdominal tergite IX, dorsal; 13d, left anal proleg, left lateral. 14, R. fasciata: 14a, head, dorsal. 14b, prothorax, dorsal; 14c, abdominal tergite IX, dorsal; 14d, left anal proleg, left lateral. Scale bars: 1 mm.

FIGURES 9–10. Wings of Rhyacophila fasciata Hagen 1859 and Rhyacophila anatolica Ekingen & Valladolid sp. nov. 9a, forewing of R. fasciata, lectotype [Museum of Comparative Zoology of Harvard University, Cambridge, MA, USA (MCZH), with permission]; 9b, forewing of R. fasciata male from Slovakia; 9c, hind wing of R. fasciata male from Slovakia. 10a, forewing of R. anatolica male. 10b, hind wing of R. anatolica male. St = stigma. Major veins: C = costa, Sc = subcosta, R 1 –R = 5 radius, M 1 –M 4 = media, Cu1–Cu2 = cubitus, A1–A3 = anal; m-cu = crossvein between medial and cubital veins, cu-a = crossvein between second cubital and first anal veins (from Holzenthal et al. 2007). Scale bars: 2 mm. 9a: specimen preserved dry, 9b–c, 10a–b: specimens preserved in ethanol.

FIGURES 11–12. Male and female genitalia of Rhyacophila anatolica Ekingen & Valladolid sp. nov., respectively. 11, male: 11A, 2nd segment of left inferior appendage, left lateral; 11BV, 11BL, parameres: 11BV, parameres (p) and ventral lobe of aedeagus (phallicata) (vl), ventral; 11BL, left paramere, left lateral. 11CL, 11CD, aedeagus (phallicata) and lateroventral lobes: 11CL, aedeagus and its left lateroventral lobe, left lateral; 11CD, aedeagus and its lateroventral lobes (lvl), dorsal. 10DD, apicodorsal lobe of segment IX (al) and preanal appendages (pa), dorsal; 11DV, segment X, ventral, ab = apical band, as = anal sclerites, va = non-sclerotized ventral area. 12, female segments VIII–XI: 12L, left lateral; 12D, dorsal; 12V, ventral. Scale bars: unlabelled = 1 mm; ● = 0.5 mm; * = 200 μm.

FIGURES 15–18. Males and females of Rhyacophila anatolica Ekingen & Valladolid sp. nov. and Rhyacophila fasciata Hagen 1859. 15, male of R. anatolica: 15a, 2nd segment of left inferior appendage, left lateral; 15b, apicodorsal lobe of segment IX and preanal appendages, dorsal; 15c, parameres and ventral lobe of aedeagus (phallicata), ventral; 15d, left paramere, left lateral. 16, male of R. fasciata: 16a, 2nd segment of left inferior appendage, left lateral; 16b, apicodorsal lobe of segment IX and preanal appendages, dorsal; 16c, parameres and ventral lobe of aedeagus (phallicata), ventral; 16d, left paramere, left lateral. 17, female segments VIII–XI of R. anatolica: 17L, left lateral; 17D, dorsal; 17V, ventral. 18, female segments VIII–XI of R. fasciata: 18L, left lateral; 18D, dorsal; 18V, ventral. Scale bars: Figures 15a–c, 16a–c = 0.5 mm, Figures 15d, 16d = 200 μm, Figures 17, 18 = 1 mm.