Oligoneuriopsis villosus Bojkova , Godunko, & Staniczek

Sroka, Pavel, Bojkova, Jindriska, Godunko, Roman J., Soldan, Tomas, Namin, Javid Imanpour, Nejat, Farshad, Abdoli, Ashgar & Staniczek, Arnold H., 2019, New Oligoneuriidae (Insecta, Ephemeroptera) from Iran, ZooKeys 872, pp. 101-126 : 101

Oligoneuriopsis villosus Bojkova, Godunko, & Staniczek sp. nov. Figures 5 , 6 , 7 , 8 , Table 2

Etymology.

The name of the new species originates from Latin, meaning hairy, and refers to the dense setation along outer margins of femora and tibiae.

Type material.

Holotype: Male larva, IRAN, Khuzestan Province, right tributary of Marun Rud River, W of Bagh Malek, 31°31.23'N, 49°49.31'E (locality no. 93), 585 m a.s.l., 29.04.2017, leg. Bojková J., Soldán T. & Imanpour Namin J. The holotype is deposited in IECA.

Paratypes: 64 larvae, same locality as holotype, deposited in IECA. Eight larvae, IRAN, Khuzestan Province, Balarud River (right tributary of Dez River), N of Andimeshk, 32°35.29'N, 48°17.32'E (locality no. 80), 230 m a.s.l., 26.04.2017, leg. Bojková J., Soldán T., & Imanpour Namin J., deposited in IECA.

70 larvae, IRAN, Hormozgan Province, Shamil River, Shamil, 27°29.66'N, 56°52.25'E, 63 m a.s.l., 30.04.2017, leg. Staniczek A.H., Godunko R.J., Pallmann M., & Nejat F. (20 larvae deposited in IECA, 20 larvae deposited in SNHM, 20 larvae deposited in SMNS under accessory numbers SMNS_EPH_7555_B_1 and SMNS_EPH_7555_B_2, including DNA voucher specimens with inventory numbers specified in Table 1, and 10 larvae deposited in MMTT_DOE).

110 larvae, IRAN, Hormozgan Province, Roudan River, 5 km N of Dehbarez, 27°28.46'N, 57°15.28'E, 217 m a.s.l., 30.04.2017, leg. Staniczek A.H., Godunko R.J., Pallmann M., & Nejat F. (20 larvae deposited in IECA, 70 larvae deposited in SNHM, and 20 larvae deposited in SMNS under inventory numbers SMNS_EPH_7550_B_1 and SMNS_EPH_7550_V_1-5, including DNA voucher specimens with inventory numbers specified in Table 1).

Localities and biology.

Larvae were collected in two rivers at the southern slopes of Zagros Mountains (right tributary of Marun Rud River and Balarud River) and two other localities (Shamil River and Roudan River) in Hormozgan Province at the western edge of Makran, a semi-desert coastal strip, which stretches along the Gulf of Oman ( Fig. 9 ). Extensive land use is present in most parts of the drainage area, mainly for agricultural and residential matters in all localities. In Marun Rud River basin, the agricultural use is more intense than in other localities and there are several urban settlements in the drainage area of this river.

The species was generally found in shallow river sections (up to 50 cm) with a riverbed composed of coarse and fine gravel, sometimes in combination with cobbles. The localities with the occurrence of this species represented rather small rivers; however, the stream width varied from 4 to 40 m, depending on the river discharge rates at the end of March ( Fig. 10 B–D ).

Larvae were predominantly distributed in river sections with accelerated current and with a minimum amount of alluvial silt. Large larvae (more than 1 cm) were found along stony margins of riffles, while small and medium-sized larvae (up to 1 cm) dwelt anywhere in the riffles.

Water quality at collection sites in Khuzestan Province was good, with pH 8.0-8.4, conductivity 620-760 µS.cm-1, salinity 0.3-0.4 ‰, and temperature 21-29 °C (conductivity and salinity measured on two localities only - the tributary of Marun Rud River and Balarud River). At collection sites in Hormozgan Province, abiotic factors were as following: conductivity 1520-1646 µS.cm, salinity 0.8 ‰, and temperature 32-34 °C.

Emergence of the species can be expected between June and July, as most of larvae occurring in all the localities were small and/or middle-sized at the time of collection. Mayfly taxa found in the same localities included: Baetis s. str. ( Baetidae ), Labiobaetis sp. ( Baetidae ), Nigrobaetis sp. ( Baetidae ), Rhithrogena sp. ( Heptageniidae ), Electrogena sp. ( Heptageniidae ), Choroterpes sp. ( Leptophlebiidae ), Caenis sp. ( Caenidae ), and Prosopistoma sp. ( Prosopistomatidae ).

Diagnosis.

According to the combination of following diagnostic characters, O. villosus sp. nov. can be distinguished from all other representatives of the genus Oligoneuriopsis worldwide:

• head widest across posterolateral corners ( Fig. 5D, F );

• row of long setae along all length of middle- and hind femora and tibiae ( Fig. 6A, D, E, H, I );

• posteromedian projections on abdominal terga absent ( Fig. 5A );

• colouration of abdominal terga dark brown with two pale dots medially ( Fig. 5A );

• setae on surface of terga and gills elongated and bluntly pointed ( Figs 7F, H, J , 8D );

• posterolateral projections of abdominal segments diverging from body axis ( Fig. 7A, B );

• posteromedial setae on sterna III–IV very long and dense, some more than 20 × longer than wide ( Fig. 8 A–C );

• first gill plate markedly smaller than the remaining pairs ( Fig. 7F, G );

• setae on inner distal margin of gill plates II–VII long ( Fig. 7 H–K );

• setae on ventral surface of gill plates near inner distal margin present ( Fig. 7I, K );

• paracercus fully developed ( Fig. 5A, B );

• caudal filaments dark in proximal half, distinct dark band in the middle missing ( Fig. 5A, B ).

Description.

Larva. Mature larvae: body length 13-16 mm (female), 11-12 mm (male), length of cerci approximately 0.4 –0.5× body length, paracercus slightly shorter.

Colouration ( Fig. 5A, B ). Head, thorax and abdomen greyish dark brown with distinctive light (yellowish) ornamentation. When fixed in ethanol, general colouration paler, brown. Legs yellowish with distinctive brown ornamentation forming bands. This colouration apparent only in larger larvae, more than 1 cm long. Smaller larvae uniformly pale, yellowish.

Head. Greyish dark brown, foremargin of head darker, occipital area with yellowish ornamentation forming four longitudinal stripes merging behind ocellar part of head. Antennae yellowish brown proximally, brown distally. Head width/length 1: 1.2 ( Fig. 5 C–F ). Eyes black, slightly exceeding contour of head in dorsal view in males; distance between eyes 2.2 × narrower than eye width in males and 1.5 × wider than eye width in females ( Fig. 5D, F ). Foremargin of head dark, bordered by dense fringe of long bristle-like setae. Labium ventrally with numerous flattened setae of different length not arranged in rows along proximal margin of paraglossae ( Fig. 5 H–J ). Similar setae also situated sparsely along inner margin of paraglossae ( Fig. 5H ). Dorsal side of first segment of labial palp with group of 15-25 thin setae distally ( Fig. 5G ).

Thorax. Greyish dark brown, with yellowish spots submedially on prothorax and yellowish spots and longitudinal stripes on mesothorax. Pleural and ventral part of thorax brown. Legs yellowish-brown, with distinct brown ornamentation ( Fig. 5B ). Coxae and trochanters brown. Proximal half of femora brown, distal half yellowish-brown. Tibiae with narrow brown bands distally and proximally, tarsi with narrow brown band distally. Forecoxae distally with numerous long, bristle-like setae and with about 15 long, thin hair-like setae on inner margin. Foretrochanters distally with numerous long, bristle-like setae. Forefemora length 2 –2.5× width; dorsal surface of forefemora covered with scattered flattened setae of various length; dense group of pointed long flattened setae submarginally near filtering setae; outer margin of forefemora bordered by conspicuous fringe of long hair-like setae ( Fig. 6C ). Filtering setae of femora and tibiae brown. Foretibiae dorsally with irregular row of bristle-like setae in distal quarter ( Fig. 6G ); ventrally with 2-3 subapical setae and several setae apically ( Fig. 6F ). Foretarsi distally with groups of pointed setae. Foreclaws heavily sclerotised at apex, with 6-7 denticles. Middle and hind femora and tibiae with dense fringe of long, hair-like setae along entire length of outer margin ( Fig. 6A, D, E, H, I ). Some hair-like setae present on outer margin of tarsi as well ( Fig. 6I ). Fringes of hair-like setae on femora and middle and hind tibiae are apparent also in (pale) early-instar larvae.

Abdomen. Greyish dark brown, with pair of submedian smudged yellowish spots and conspicuous sublateral yellowish pattern ( Fig. 5A ). Ventral part of abdomen greyish dark brown, with conspicuous yellowish pattern ( Fig. 5B ). Posterolateral processes present on abdominal segments II–IX; bent outwards, apices slightly inwards ( Fig. 7A, B ). Terga sparsely covered with flattened setae of various sizes mixed with numerous microtrichia, largest setae elongated and bluntly pointed apically ( Fig. 8D ). Some sterna with densely assembled setae posteromedially (most prominent on sterna III–IV, present also on sternum V). Individual setae distinctly elongated, some more than 20 × longer than wide, see Fig. 8 A–C ). Sternum IX with wide rounded incision posteriorly, equipped with tiny spines.

Gills ( Fig. 7 ). Gill I asymmetric, bluntly pointed apically ( Fig. 7F, G ), markedly smaller and more elongated than following gills, which are oval and almost symmetric ( Fig. 7D, E, H–K ). Size ratio between gill I and IV approximately 0.8: 1 (length) and 0.7: 1 (width). All gill pairs equipped with bundle of whitish filaments slightly shorter than respective gill plate (filaments are slightly longer in gill I). Dorsal surface of gill I covered with elongated setae of various size ( Fig. 7F ). Ventral surface of gill I with submarginal setae only ( Fig. 7G ). Dorsal surface of gills II–VII with sparsely scattered, elongated, and bluntly pointed setae ( Fig. 7D, H, J ). Similar setae occurring also on margins of gill plates. Setae bordering inner part of distal margin relatively long ( Fig. 7 H–K ). Ventral surface of gill plates II–VII with numerous hair-like setae along outer margin of ventral cavity ( Fig. 7E, I, K ). These setae slightly plumose. Second group of minute setae situated near inner distal margin ( Fig. 7I, K ).

Cerci dark brown, lighter towards apex, with inner marginal fringe of fine hair-like setae. Paracercus reaching approx. 2/3 of cerci length, with dense setation laterally ( Fig. 5A, B ).

Egg. Shape of eggs studied deformed due to extraction from mature larvae ( Fig. 8E ). Eggs 250-320 μm long and 190-240 μm wide; chorionic surface covered with circled terminal fibre clusters (diameter 10 μm). Micropyle shallow, circular; sperm guide well apparent ( Fig. 8E ). Terminal fibre clusters finely sculptured, with leaf-like, flat microstructures ( Fig. 8F ).

Imago and subimago. Unknown.

Affinities.

The establishment of the generic attribution of O. villosus sp. nov. is not straightforward, although affinities to the genera Oligoneuriella Ulmer, 1924 and Oligoneuriopsis Crass, 1947 are obvious based on the shape of head, legs, and gills (see Edmunds 1961; Bauernfeind and Soldán 2012). However, differences between Oligoneuriella and Oligoneuriopsis are very subtle and the separation of these genera is not clear. The first description of the genus Oligoneuriopsis was published by Crass (1947), with the type species Oligoneuriopsis lawrencei Crass, 1947, mainly based on the adult stage. Its larva was not described in detail, but briefly commented as being identical to larvae described and depicted earlier by Barnard (1940), who assigned them to " Elassoneuria trimeniana ". This opinion is followed by later authors, for example Demoulin (1970). Edmunds (1961) considered Oligoneuriopsis as closely related to Oligoneuriella . In the larval key to genera, the presence of the lamella of gill I in Oligoneuriella and its absence in Oligoneuriopsis was used as the crucial diagnostic character separating the genera ( Edmunds 1961). Nevertheless, Agnew (1980) clearly stated that this character is not valid, since a small lamella is present also in Oligoneuriopsis , including the type species O. lawrencei . Instead, he proposed the arrangement of the setae medially on abdominal sterna being more developed in Oligoneuriopsis than in Oligoneuriella as a reliable diagnostic character. Bauernfeind and Soldán (2012) mentioned more conspicuous, very long setae medially on sterna, lamella of gill I reduced, membranous, and cerci with dark band in the middle as diagnostic for Oligoneuriopsis . In our view, the length of posteromedial setae on sterna probably mostly represents a reliable character, although it varies, and the setae are reported 3 –6× longer than wide in known species of Oligoneuriella ( Sroka et al. 2015) and even more than 10 × longer than wide in Oligoneuriella tuberculata sp. nov., described above. However, in O. villosus sp. nov., some of these setae are more than 20 × longer than wide ( Fig. 8B, C ), which fulfils the criteria for Oligoneuriopsis and roughly also agrees with our observation of setae length in Oligoneuriopsis skhounate from North Africa. We observed that the lamella of gill I in O. skhounate is small-sized compared to other gills, but structurally not different. The same condition applies also to O. villosus sp. nov. ( Fig. 7C ). A clearer morphological definition of Oligoneuriopsis would be possible after a more thorough study of the Afrotropical material, including the type species O. lawrencei , as already pointed out by Bauernfeind and Soldán (2012). Ideally such a study should also include adults, since several adult characters separating Oligoneuriella from Oligoneuriopsis have been published, mostly concerning wing venation and male genitalia ( Bauernfeind and Soldán 2012).

At this time, we consider it justified to assign the new species to Oligoneuriopsis based on following characters shared with O. skhounate (and presumably also with other Afrotropical representatives of Oligoneuriopsis ): (i) setae posteromedially on abdominal sterna very long, some more than 20 × longer than wide; (ii) lamella of gill I significantly smaller than remaining gill pairs; and (iii) row of setae along entire length of posterior margin of femora and outer margin of tibiae on middle and hind legs. The second and third characters are also depicted in the figures provided by Barnard (1940), presumably representing the larva of O. lawrencei , which supports their diagnostic value for the separation of Oligoneuriopsis from Oligoneuriella .

In the genus Oligoneuriopsis , six species have been described up to now ( Bauernfeind and Soldán 2012). Five of them are distributed exclusively in the Afrotropics; only a single species, O. skhounate , occurs also in the Palaearctic (North Africa and the Iberian Peninsula). The occurrence of Afrotropical species in Iran is extremely unlikely (even more when taking into account that most species are known from South Africa only). Nevertheless, all these species can be morphologically distinguished from O. villosus sp. nov. (except O. dobbsi (Eaton, 1912) with unknown larva).

Oligoneuriopsis lawrencei Crass, 1947 differs in the shape of head, widest anterior to the eyes ( Agnew 1973, fig. 1) contrary to O. villosus sp. nov. with the head widest across posterolateral corners ( Fig. 5D, F ). Furthermore, O. lawrencei possesses very long setae on margin of gill plates ( Agnew 1980, fig. 8). O. jessicae Agnew, 1973 profoundly differs by having posteromedial spine-like projections on abdominal terga ( Agnew 1973, fig. 1), missing in O. villosus sp. nov. ( Fig. 5A ). O. elisabethae Agnew, 1973 differs from O. villosus sp. nov. in colouration. O. elisabethae exhibits a uniform light brown colour, without any indication of an abdominal pattern ( Agnew 1973), whereas O. villosus sp. nov. possesses a distinct pair of submedian, smudged, yellowish spots and a conspicuous, sublateral, yellowish pattern on the dorsal side of abdomen ( Fig. 5A ).

The nearest distributed congeneric species O. skhounate is morphologically similar to O. villosus sp. nov. It even shares the colouration pattern of legs with alternating darker and lighter bands ( Fig. 5B ; Dakki and Giudicelli 1980, figs 28, 29). Nevertheless, several morphological characters allow the separation of these two species: (i) setae on the surface of gills and terga are shorter in O. skhounate , longer and more pointed in O. villosus sp. nov. ( Fig. 8D ); (ii) colouration is different, prominent posteromedian pale spot on terga in O. skhounate ( Dakki and Giudicelli 1980, fig. 20) is missing in O. villosus sp. nov. ( Fig. 5A ). Moreover, the dark band at half length of caudal filaments, depicted for O. skhounate ( Dakki and Giudicelli 1980; fig. 20), is also missing in O. villosus sp. nov. ( Fig. 5A, B ); (iii) in O. skhounate , posterolateral projections of abdominal segments are oriented parallel to the body axis, and lateral margins of individual segments are convex ( Dakki and Giudicelli 1980, fig. 20). In O. villosus sp. nov., posterolateral projections of abdominal segments are diverging from the body axis, with lateral margins straight or slightly concave ( Fig. 7A, B ).

Al-Zubaidi and Al-Kayatt (1986) without further details already reported findings of Oligoneuriopsis sp. from north Iraq. An unidentified species of Oligoneuriopsis was also collected recently from the same area and deposited in the Bold database as Oligoneuriopsis sp. MAA01. This record contains two barcode sequences, which are not publicly accessible, but exhibiting 90.03-90.63% similarity to the haplotypes of O. villosus sp. nov. sequenced in the present study. This level of similarity rather corresponds with interspecific distances found in Oligoneuriidae (see Fig. 11C ). Therefore, we may assume the existence of more Oligoneuriopsis species in the Middle East.

Molecular species delimitation.

The ABGD analysis of the COI distance matrix generated 11 stable groups ( Fig. 11A ). The mean genetic distances within groups generated by ABGD ranged between 0.003 and 0.011 and the mean distances between groups between 0.060 and 0.234; for the histogram of distances see Fig. 11C .

The GMYC model recognised 11 ML entities, with confidence interval 11-11 ( Fig. 11A, B ). We used single threshold GMYC, since multiple threshold is prone to over splitting species that are not evenly sampled throughout its distributional area ( Fujisawa and Barraclough 2013; Hrivniak et al. 2019). The Maximum Likelihood of GMYC model was 197.4186, compared to the likelihood of the null model 181.5407. The likelihood ratio test significantly rejected the null model expecting uniform coalescent branching rates across entire tree (likelihood ratio=31.75579, p=1.271505e-07).

The clusters delimited as distinct species were congruent in both approaches. Both methods of the molecular species delimitation also unambiguously supported the designation of O. tuberculata sp. nov. and O. villosus sp. nov., which were recovered as distinct units in both analytical approaches. Apart from the two new species described herein, previously known species recognised as distinct entities in both ABGD and GMYC included O. pallida , O. bicaudata , O. tskhomelidzei , and O. skhounate . The species identified morphologically as O. rhenana was split into three putative COI species, which indicates that this widely distributed taxon (occurring in most of Europe, see Bauernfeind and Soldán 2012) actually represents a species complex. The putative cryptic species within O. rhenana were left undescribed for the moment, since extensive material was not at our disposal. Within the dataset, a further two unidentified putative species were recognised, one from China and one from Iraq. These clusters possibly represented a further two undescribed species, left unnamed for the time being.

The analysis of COI corroborated the affinity of O. villosus sp. nov. to Oligoneuriopsis . From the taxa included, O. villosus sp. nov. exhibited the highest sequence similarity with O. skhounate from Southern Spain. However, the distance between O. villosus sp. nov. and O. skhounate clearly separated them into two distinct species. Regarding the affinities of O. tuberculata sp. nov., O. bicaudata was recovered as the most closely related species from the ones sequenced, which is in accordance with the morphological character distributions.

Figures

Figure 5. Oligoneuriopsis villosus sp. nov., larvae A habitus in dorsal view B habitus in ventral view C head of male in lateral view D head of male in dorsal view E head of female in lateral view F head of female in dorsal view G detail of setae on distal part of labial palp segment I in dorsal view H-I setae on paraglossae in ventral view J detail of basal portion of paraglossae under SEM in ventral view. Scale bars: 5 mm (A, B); 1 mm (C-F); 0.2 mm (G-J). Same scale bar for C and D. Same scale bar for E and F.

Figure 6. Oligoneuriopsis villosus sp. nov., larvae, legs A hind leg under SEM in dorsal view B micro sculpture of leg cuticula under SEM in dorsal view C forefemur in dorsal view D middle femur in dorsal view E hind femur in dorsal view F apical part of foretibia with subapical setae in ventral view G shape of foretibia with subapical setae in dorsal view H middle tibia and tarsus in dorsal view I hind tibia and tarsus in dorsal view. Scale bars: 1 mm (A, C-E); 0.02 mm (B); 0.5 mm (F-I). Same scale bar for C, D and E. Same scale bar for F and G. Same scale bar for H and I.

Figure 7. Oligoneuriopsis villosus sp. nov., larvae, abdomen and gills A abdomen outline, male B abdomen outline, female C proximal portion of abdomen under SEM in lateral view D gill IV under SEM in dorsal view E gill IV under SEM in ventral view F gill plate I in dorsal view G gill plate I in ventral view H gill plate IV in dorsal view I gill plate IV in ventral view J gill plate VII in dorsal view K gill plate VII in ventral view. Scale bars: 1 mm (A, B); 0.5 mm (C); 0.2 mm (D-K). Same scale bar for F-K.

Figure 8. Oligoneuriopsis villosus sp. nov., larvae and eggs A general view of basal part of abdomen under SEM in ventral view B detail of setae on sternum IV C detail of setae on sternum IV under SEM D detail of setae on surface of terga E egg, general view F detail of egg chorionic structure. Scale bars: 1 mm (A); 0.1 mm (B, D); 0.5 mm (C); 0.05 mm (E); 0.01 mm (F).

Figure 9. Map of Iran with localities of Oligoneuriella tuberculata sp. nov. (star) and Oligoneuriopsis villosus sp. nov. (circles).

Figure 10. Photos of the localities with occurrence of Oligoneuriella tuberculata sp. nov. and Oligoneuriopsis villosus sp. nov. A Marbor River near Kata (type locality of O. tuberculata sp. nov.) B right tributary of Marun Rud River near Bagh Malek (type locality of O. villosus sp. nov.) C Balarud River near Andimeshk (locality of O. villosus sp. nov.) D Shamil River near Shamil (locality of O. villosus sp. nov.).

Figure 11. Results of the molecular species delimitation: A maximum clade credibility COI gene tree. The red branches represent species delimited by GMYC. Columns on the right illustrate groups delimited by both approaches used B lineage through time plot, generated by GMYC. The red vertical line indicates the threshold time between inter- and intraspecific branching C distribution of distances calculated by ABGD.