Stenophylax espanioli Schmid 1957

Ruiz-García, Antonio, Zamora-Muñoz, Carmen, Garzón, Andrés & Ferreras-Romero, Manuel, 2018, Description of the last instar larva of Stenophylax espanioli Schmid 1957 (Trichoptera: Limnephilidae) from southern Iberian Peninsula with the barcode of the species and synoptic key for identification of the known Stenophylax larvae from the Iberian Peninsula, Zootaxa 4388 (2), pp. 292-300 : 293-298

Fifth instar larva of Stenophylax espanioli Schmid 1957

Material examined. A total of 44 larvae and 1 pharate male were analysed for this study, including 12 larvae and 1 pharate male collected in Fuente del Boyar, Guadalete Basin, 796 m asl, UTM: 30STF8370; 13 larvae from Alfarnate, Guaro Basin, 851 m asl, UTM: 30S3840; 3 larvae from Alazores, Guadalhorce Basin, 971 m asl, UTM: 30S3840; 1 larva from Fuenfría, Guadalquivir Basin, 971 m asl (leg. Joaquín Márquez-Rodríguez); and 15 larvae from Riogazas, Guadalquivir Basin, 1142m asl, UTM: 30S3841, all in the southern Iberian Peninsula. The samples were taken by hand along a stream section. The conspecificity of larvae and adults was confirmed by study of larval sclerites within the case of the mature pupa with perfectly formed male genitalia and by DNA analysis. Additionally, five larvae were reared in the laboratory for comparison. In the description and synoptic key, the chaetotaxy follows those of Williams and Wiggins (1981) and Wallace et al. (2003).

In addition to morphological analysis, DNA extraction and amplification of one fragment of the “barcode region” of the mitochondrial cytochrome oxidase c subunit I (mtCOI) using primers LCO-1490 and HCO-2198 ( Folmer et al. 1994) were performed. Sequences were edited using the program Chromas Lite, and consensus was obtained using SeqTrace software. Sequences were deposited in GenBank under Accession Nos: MF149119 ; MF149120 . A genetic distances matrix was created from the Barcode of Life Database ( BOLD), and uncorrected p-distances were calculated in Mega 6.3 (Tamura et al. 2013).

Description. Biometry and color. Body length of final instar larva ranging from 21 to 23.5 mm, body width from 3.8 to 4.3 mm, head width from 1.90 to 2.20 mm (n = 12). Sclerotized structures reddish brown in colour, abdominal segments whitish.

Head. Head capsule ( Fig. 1 ) uniformly coloured, darker on mesal sides of frontoclypeal sutures posterior of central constriction, and beside coronal suture. Muscle spots clearly visible and arranged on parietal and dorsal areas, larger and darker near coronal suture; arranged in triangle in posterior third of frontoclypeus. Frontoclypeus subtriangular, with strong central constriction ( Fig. 2 ); head with spinules on parietalia above, below, and behind each eye and extending through areas of frontoclypeus ( Figs. 2, 3 , circles); antennae situated halfway between eye and anterior head margin, short, each consisting of 1 short cylindrical base and 1 short flagellum ( Fig. 3 , red arrow); anterior edge of labrum notched with tuft of setae on each side and 4 short and thick curved setae ( Fig. 5 ); mandibles black ( Figs. 1, 4–6 ), usually right mandible with five and left with four teeth along anterior edge, these teeth of different sizes, ridges in central concavity, and with two external basal setae of different sizes; ventral apotome ( Fig. 6 ) lighter than genae, mesal postgenal suture very short, approximately 25% as long as apotome.

Thorax. Pronotum reddish brown with dark brown muscle attachment spots ( Fig. 7 ); its posterior and lateral margins thickened and darkly striped ( Fig. 8 ); pronotal transverse groove behind anterior third distinct; muscle attachment spots at posterior third arranged as inverse V; dense fringe of short, yellowish setae present along its anterior border, long black setae on rest of sclerite near anterior margin and posterior third; prosternal horn present ( Fig. 8 ). Mesonotum ( Fig. 7 ) composed of two subrectangular sclerites with black sclerotized bands on posterior and posterolateral edges; setae located along anterior edge near median suture (sa 1), anterolateral edge (sa 3), and in posterior third (sa 2). Metanotum ( Fig. 7 ) partially covered by 3 pairs of distinct and small sclerites: anteromedian sclerites (sa 1) narrow and elliptical, their maximum transverse length distinctly smaller than their medial separation; large groups of setae present between pair of posteromedian sclerites (sa 2) and between lateral (sa 3) and posteromedian sclerites on each side; lateral sclerites (sa 3) each with setae only on anterior half ( Figs. 7 , 14 ). Legs reddish brown, row of minute spines present along ventral edge of each femur of every leg ( Fig. 9 ). Forelegs ( Figs. 9–10 ) shorter than others and with broader femora, ventral edges of forefemora each with two strong pale spines, additional setae on posterior faces of forefemora ( Fig. 9 , black arrows), forefemora each with 2 dorsoproximal and 3–5 dorsodistal setae. Meso- and metathoracic femora ( Figs. 9–10 ) each with 2 ventral-edge black setae, 1 dorsoproximal and 5–6 dorsodistal setae; additional setae on anterior and posterior faces of meso- and metathoracic femora, all additional setae situated in central and ventral third of these femora. Tarsal claws of all legs well developed, curved, each with one little basal spine.

Abdomen. Lateral fringe ( Figs. 11, 12 ) on each side extending from anterolateral edge of abdominal segment III to end of abdominal segment VIII. Gills each with single-filament in dorsal, dorsolateral, ventrolateral and ventral positions anteriorly and posteriorly. Distribution of filaments on abdominal segments similar to that of S. crossotus (see Ruiz-García & Ferreras-Romero 2007). Abdominal segment I with 1 dorsal and 2 lateral fleshy protuberances (humps; Figs. 13-14 ); dorsal hump ( Fig. 13 ) with continuous transverse group of setae anteriorly, no setae posteriorly; posterodorsal region of each lateral protuberance ( Fig. 14 ) with 2 small sclerites each with central hole but without setae ( Fig. 14 ), region anterior of protuberance without setae; abdominal sternum I ( Figs. 15–16 ) central area without setae ( Fig. 15 ), more evident in some individuals than others ( Fig. 16 ). Posterior third of abdominal dorsum VIII with 15–18 setae ( Fig. 17 ). Abdominal tergum IX with 8 long black setae on posterior edge ( Fig. 18 , black arrows), including 2 A-setae and 2 C-setae of equal length ( Figs. 18 , 19 ) and 1 or 2 posterolateral setae ( Fig. 18 , red arrows); ventrally with 2 pairs of setae of different sizes, outer pair being longer than inner pair ( Fig. 20 , red arrows). Anal claws each with accessory hook on dorsal side ( Fig. 21 , black arrow).

Larval case. Case length 22–25 mm, case width 5.8–6.3 mm; larval case straight and slightly conical, although some cylindrical ( Fig. 22 ), composed of mineral particles of different sizes and posterior half or third consisting of leaf fragments; occasionally case composed entirely of mineral matter. As in other limnephilids, early instars construct cases of plant material. Cases of later instars of mineral material, with plant material often remaining at older, posterior end during transition.

DNA analysis. The analysis of the barcode region of an adult female of S. espanioli and the hitherto unknown larva collected in the same locality (Puerto del Boyar) showed a genetic distance of 0.00% ( Table 1). This value certainly fits well within the intraspecific variability of mtCOI usually observed in caddisflies ( Pauls et al. 2009, 2010; Previsic et al. 2009, 2014; Graf et al. 2015), and the uncorrected p-distances recorded in the mtCOI gene from these two individuals ( Table 1), relative to the other species, are in line with interspecific distances commonly reported in Limnephilidae ( Graf et al. 2005, 2015). Thus, the data enable confident association of the larva and the female of S. espanioli . Furthermore, the association of larvae and adults is based not only on comparisons of sequence from these specimens ( Zhou et al. 2007), but also by morphological study and laboratory breeding. The publication of these data contributes also to collaboration with the global Trichoptera barcode initiative (Zhou et al. 2016), providing DNA sequences from species not previously sequenced.

Diagnosis. According to the key for the identification of Stenophylax larvae of the Iberian Peninsula presented by Sáinz-Bariáin & Zamora-Muñoz (2012), the larva of S. espanioli keys with S. crossotus . However, the larvae of these two species can be distinguished by the following morphological characteristics:

- The larva of S. espanioli is distinctly larger than that of S. crossotus , and with more reddish coloration;

- head spinules cover a large area of the parietalia behind and above the eye in S. crossotus , whereas in S. espanioli they also extend across the frontoclypeus;

- additional setae are present on the posterior faces of the forefemora in S. espanioli , missing in S. crossotus ;

- posterior face setae of mid- and hind femora occur on their ventral third in S. crossotus , posterior face setae of mid- and hind femora occur on their central and ventral third of femora of S. espanioli ;

- the center of the ventral protuberance of abdominal segment I has an area without setae in S. espanioli , but S. crossotus has setae in this area.

Figures

FIGURES 1–6. Stenophylax espanioli Schmid 1957, final instar larva. (1) head capsule, dorsal; (2) head capsule, left dorsolateral, blue circles referring to spinules; (3) head capsule, right lateral, blue circles referring to spinules, red arrow pointing to right antenna; (4) mandibles, dorsal; (5) mandibles and labrum, dorsal; (6) head capsule, ventral.

FIGURES 7–12. Stenophylax espanioli Schmid 1957, final instar larva. (7) thorax, dorsal; (8) prothorax, left lateral; (9) left legs, posterior faces, black arrows pointing to additional setae; (10) left legs, anterior faces; (11) habitus, left lateral; (12) posterior abdominal segments, right lateral.

FIGURES 13–18. Stenophylax espanioli Schmid 1957, final instar larva. (13) abdominal segment I, dorsal; (14) abdominal segments I and II, right lateral; (15–16) abdominal sternum I of two specimens of the same population; (17) abdominal dorsum VIII, dorsal, red arrows pointing to posterolateral setae; (18) abdominal tergum IX, dorsal, black arrows pointing to long A and C setae, red arrows pointing to posterolateral setae.

FIGURES 19–22. Stenophylax espanioli Schmid 1957, final instar larva. (19) abdominal tergum IX, left lateral, black arrows pointingto A andC setae; (20) posterior abdominal segments, ventral, red arrows pointing to setaeof sternum IX; (21) leftanal claw, ventral, black arrow pointing to accessoryhook; (22) larval case.