Research Article |
Corresponding author: Roman J. Godunko ( godunko@seznam.cz ) Academic editor: Lyndall Pereira-da-Conceicoa
© 2019 Pavel Sroka, Jindřiška Bojková, Roman J. Godunko, Tomáš Soldán, Javid Imanpour Namin, Farshad Nejat, Ashgar Abdoli, Arnold H. Staniczek.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Sroka P, Bojková J, Godunko RJ, Soldán T, Namin JI, Nejat F, Abdoli A, Staniczek AH (2019) New Oligoneuriidae (Insecta, Ephemeroptera) from Iran. ZooKeys 872: 101-126. https://doi.org/10.3897/zookeys.872.36098
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Two new species of the mayfly family Oligoneuriidae are described based on larval specimens recently collected in Iran. The first new species, Oligoneuriella tuberculata Godunko & Staniczek, sp. nov., can be distinguished from all its congeners by the presence of pronounced protuberances posteromedially on abdominal terga, highly reduced paracercus, large lamella of gill I, and setation on hind margin of middle and hind femora confined to their basal halves. The second species, Oligoneuriopsis villosus Bojková, Godunko, & Staniczek, sp. nov., remarkably belongs to a mostly Afrotropical genus. The new species clearly differs from all its congeners in the shape of setae on the surface of gills and terga, pattern of body colouration, and the shape of posterolateral projections of abdominal segments. Except for the species description, the generic diagnosis of Oligoneuriopsis Crass, 1947 is briefly discussed. COI barcode sequences of both new species are provided and molecular species delimitation is tested using distance-based and likelihood-based approaches, with both new species unambiguously recognised as separate lineages. The analysis of COI also corroborates the respective affinities of both new species, estimated based on morphology. The two new species of Oligoneuriidae described herein highlight the importance of the Middle East as a centre of diversity of this mayfly family within the Palaearctic.
Barcoding, mayflies, Middle East, Oligoneuriella, Oligoneuriopsis, taxonomy, new species
The mayfly fauna of Iran is still largely unexplored, although considerable progress has been achieved recently.
Oligoneuriella occurs in the Palaearctic and comprises 14 species (
Oligoneuriopsis, the second genus, which we found in Iran, is probably closely related to Oligoneuriella (
As a part of our study, we include new COI barcode sequences of O. tskhomelidzei and the two new species. We analyse these sequences to test the delimitation of the newly proposed species and compare them with all Oligoneuriopsis and Oligoneuriella species, for which COI sequences are available, namely O. bicaudata, O. pallida, O. rhenana, O. skhounate, and two unidentified species from Iraq and China.
Material used for this study was collected by J. Bojková, R.J. Godunko, J. Imanpour Namin, F. Nejat, M. Pallmann, T. Soldán, and A.H. Staniczek during an investigation of different freshwater habitats in Iran. Samples were obtained by kick sampling and specimens were preserved in 96% EtOH. Environmental variables (pH, conductivity, salinity and temperature) were measured using a HACH sensION 5 portable waterproof conductivity meter and HACH Pocket Pro+ Multi 2. Some specimens were dissected and mounted on slides with HydroMatrix (MicroTech Lab, Graz, Austria) to allow detailed microscopic observations. Drawings were made using a stereomicroscope Olympus SZX7 and a microscope Olympus BX41, both equipped with a drawing attachment. Serial habitus photographs were made with a Leica DMC5400 digital camera on a Leica Z16 APO Macroscope using Leica Application Suite Version 3.1.8 and Helicon Focus Pro to obtain stacked photographs with extended depth of field.
For scanning electron microscopy (SEM), eggs were dissected from female last instar larvae and also mouthparts, legs, and gills were dissected. All parts were subsequently dehydrated through a stepwise immersion in ethanol, dried by critical point drying (Leica EM CPD300), and mounted on SEM stubs. The mounted material was coated with a 5 nm Au/Pd layer (Leica EM ACE200) and subsequently examined and photographed with a Zeiss EVO LS 15 scanning electron microscope. All photographs were subsequently sharpened and adjusted in contrast and tonality in Adobe PhotoshopTM CS6.
Material is deposited in the collections of the Biology Centre CAS, Institute of Entomology, České Budějovice, Czech Republic (
For DNA extraction, one severed leg of each specimen was processed with Qiagen DNeasy Blood & Tissue Kit following the manufacturer’s instructions for tissue samples. The resulting DNA-elute was used in a PCR reaction with the primer pair of LCO1490 and HCO2198 for COI targeting, utilising a Thermo Scientific™ Phire Tissue Direct PCR Master Mix Kit for samples of O. tskhomelidzei and a Qiagen Multiplex PCR Kit for the two new species. The PCR amplification was performed as follows: Initial heat activation at 95 °C for 15 min and 35 cycles of denaturation at 94 °C for 0:30 min. For Thermo Scientific protocol, annealing was performed at 50 °C for 1:30 min and elongation at 68 °C for 0:45 min, for Qiagen protocol, annealing was performed at 56 °C for 1:30 min and elongation at 72 °C for 1:30 min. Both protocols followed a final elongation step at 72 °C for 10 min. The PCR products were enzymatically purified using ExoI/FAP. The purified product was sequenced via the EZ-seq single direct service by Macrogen (Amsterdam, Netherlands). Resulting chromatograms were assembled and final sequences were error checked using Geneious suite version 10.2.3. COI sequences were deposited at Barcode of Life Data Systems (http://www.boldsystems.org) under accession numbers specified in Table
Species | Location | Voucher specimen collection code | Bold Process ID | GenBank accessionnumber | Source |
O. rhenana | Germany | – | GBEPT372-14 | KY261779 | GenBank |
Italy | – | GBA22942-15 | LN734762 | GenBank | |
Italy | – | GBA22943-15 | LN734763 | GenBank | |
France | – | GBMIN65367-17 | MF458765 | GenBank | |
Germany | – | GBEPT1975-14 | KY261297 | GenBank | |
France | – | GBMIN65371-17 | MF458760 | GenBank | |
Germany | – | GBEPT344-14 | KY262278 | GenBank | |
France | – | GBMIN65370-17 | MF458763 | GenBank | |
Germany | – | GBEPT1221-14 | KY261341 | GenBank | |
Germany | – | FBAQU839-10 | KY262106 | GenBank | |
Germany | – | FBAQU1261-12 | KY262260 | GenBank | |
O. pallida | Hungary | – | – | – |
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Hungary | – | GBMIN65365-17 | KU609047 | GenBank | |
O. tskhomelidzei | Iran | SMNS_EPH_7724_V_1 | EPHIR014-19 | – | newly sequenced |
Iran | SMNS_EPH_7596_V_4 | EPHIR012-19 | – | newly sequenced | |
Iran | SMNS_EPH_7596_V_6 | EPHIR013-19 | – | newly sequenced | |
O. bicaudata | Iraq | – | BMIKU058-09 | – | Bold Systems |
Iraq | – | BMIKU056-09 | – | Bold Systems | |
Iraq | – | BMIKU054-09 | – | Bold Systems | |
O. tuberculata sp. nov. | Iran | SMNS_EPH_7574_V_1 | EPHIR003-19 | – | newly sequenced |
Iran | SMNS_EPH_7574_V_2 | EPHIR004-19 | – | newly sequenced | |
Iran | SMNS_EPH_7574_V_4 | EPHIR005-19 | – | newly sequenced | |
Iran | SMNS_EPH_7574_V_5 | EPHIR006-19 | – | newly sequenced | |
Iran | SMNS_EPH_7574_V_6 | EPHIR002-18 | – | newly sequenced | |
Oligoneuriella sp. 1 | Iraq | – | BMIKU059-09 | – | Bold Systems |
Iraq | – | BMIKU040-09 | – | Bold Systems | |
Iraq | – | BMIKU037-09 | – | Bold Systems | |
Iraq | – | BMIKU031-09 | – | Bold Systems | |
Oligoneuriella sp. 2 | China | – | XJDQD857-18 | – | Bold Systems |
China | – | XJDQD856-08 | – | Bold Systems | |
O. skhounate | Spain | – | – | – |
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O. villosus sp. nov. | Iran | SMNS_EPH_7550_V_2 | EPHIR008-19 | – | newly sequenced |
Iran | SMNS_EPH_7555_V_3 | EPHIR011-19 | – | newly sequenced | |
Iran | SMNS_EPH_7555_V_2 | EPHIR010-19 | – | newly sequenced | |
Iran | SMNS_EPH_7550_V_4 | EPHIR009-19 | – | newly sequenced | |
Iran | SMNS_EPH_7550_V_1 | EPHIR007-19 | – | newly sequenced |
We analysed COI to test the validity of the morphological species concept of both new species. COI of O. tuberculata sp. nov., O. villosus sp. nov., and O. tskhomelidzei were newly sequenced. Remaining sequences used in the analysed dataset were downloaded from GenBank and Bold Systems. Two sequences (one from Oligoneuriopsis skhounate and one from Oligoneuriella pallida) were obtained from
To split a sequences alignment dataset into candidate species, the alignment was analysed using Automatic Barcode Gap Discovery (ABGD) (
Species delimitation was also tested using single-loci coalescence based General Mixed Yule Coalescent model (GMYC) (
An ultrametric COI gene tree was reconstructed under a relaxed molecular clock (uncorrelated lognormal distribution) using BEAST 2.4.8 (
The name of the new species refers to the presence of protuberances posteromedially on terga, which is a character unknown for any other species of Oligoneuriella. A tuberculum is the Latin expression for protuberance.
Holotype
: Male larva, IRAN, Kohgiluyeh and Boyer-Ahmad Province, Kata, Marbor River, 31°10.71'N, 51°15.78'E, 1562 m a.s.l., 04.05.2017, leg. Staniczek A.H., Godunko R.J., Pallmann M. & Nejat, F. The holotype is deposited at
Paratypes
: 165 larvae, same locality as holotype (40 larvae deposited in
Summary of main larval characters of O. villosus sp. nov. and O. tuberculata sp. nov.
O. villosus sp. nov. | O. tuberculata sp. nov. | |
Body length (mm) | 13–16 | >9–12 (fully mature larvae not available) |
Colour pattern | greyish dark brown with distinctive light (yellowish) ornamentation | yellowish-white, light brown to light dirty olivaceous, without marked ornamentation |
Compound eyes of male exceeding / not exceeding head margin laterally | slightly exceeding | unknown (fully mature male larvae not available) |
Basal setae on paraglossae | sparse, slightly elongated, not arranged in rows | sparse, slightly elongated, not arranged in rows |
Setae distally on segment I of labial palps | approx. 15–25, short, hair-like | more than 30, short, hair-like |
Hair-like setae proximally on posterior margin of middle- and hind femora | dense and long, forming fringe along all length of femur | dense and long, forming fringe, reaching 1/3–1/2 of femur length |
Submedial row of spine-like setae dorsally on fore tibiae | irregular row of bristle-like setae in distal quarter | 3–4 setae arranged in irregular row subapically |
Presence of posterolateral projections on abdominal segments | II–IX | (II) III–IX |
Shape of posterolateral projections of last abdominal segments | bent outwards, apices slightly inwards | nearly straight, slightly diverging from body axis |
Posteromedial setae on sterna III-IV | some more than 20× longer than wide | up to 10–15× longer than wide |
Size of first gill plate compared to remaining pairs | markedly smaller | significantly larger |
Setae on inner distal margin of gill plates II–VII | long | slightly elongated |
Setae on ventral surface of gill plates near inner distal margin | present | absent |
Paracercus | fully developed | vestigial |
The single known locality of O. tuberculata sp. nov. in Marbor River is situated in the southeastern part of the Zagros Mountains within the Khersaan River basin (Fig.
Marbor River at the type locality is a small, premontane river, 15–22 m wide, with depths up to 1.5–1.7 m (Fig.
Water quality at Marbor River was good, with conductivity 264 µS.cm, salinity 0.1 ‰, and temperature 15 °C.
The emergence of the species can be expected approximately between July and August, as all larvae collected here in May were small and only half-grown with small wing pads. Together with O. tuberculata sp. nov., only three other mayfly taxa were collected: Baetis (Rhodobaetis) sp. (Baetidae), Rhithrogena sp. (Heptageniidae) and Epeorus sp. (Heptageniidae).
According to the combination of following diagnostic characters, O. tuberculata sp. nov. can be distinguished from all other representatives of the genus Oligoneuriella worldwide:
• Body pale, abdominal terga with inconspicuous, pale maculae laterally (Fig.
• eyes of male laterally not exceeding head margin (Fig.
• dorsal side of foretibia with 3–4 setae arranged into irregular row subapically (Fig.
• dense rows of long, hair-like setae on posterior margin of proximal 1/3–1/2 of middle- and hind femora (Fig.
• posterolateral processes of abdominal segments relatively narrow (Fig.
• terga II–IX with unpaired protuberance posteromedially (Fig.
• posteromedial flattened setae on sterna III–IV up to 10–15× longer than wide (Fig.
• first gill plate significantly larger than remaining gill plates, without ridge near outer margin dorsally (Fig.
• sparse setation on dorsal and ventral surfaces of gill plates II–VII (Fig.
• ventral surface of gill plates II–VII with setae sparsely scattered only along outer margin of ventral cavity (Fig.
• paracercus vestigial, approximately 5-segmented.
Oligoneuriella tuberculata sp. nov., larvae A habitus in dorsal view B habitus in ventral view C habitus in lateral view D head of male in lateral view (immature larva) E head of male in dorsal view (immature larva) F head of female in lateral view G head of female in dorsal view H detail of setae on distal part of labial palp segment I in dorsal view I detail of setae on proximal margin of paraglossae in ventral view. Scale bars: 2.0 mm (A–C); 0.5 mm (D–G); 0.1 mm (H); 0.2 mm (I). Same scale bar for D and E. Same scale bar for F and G.
Oligoneuriella tuberculata sp. nov., larvae, legs A forefemur in dorsal view B middle femur in dorsal view C hind femur in dorsal view D middle leg under SEM in dorsal view E micro sculpture of leg cuticula under SEM in dorsal view F apical part of foretibia with subapical setae in ventral view G shape of foretibia with subapical setae in dorsal view. Scale bars: 0.5 mm (A–D, F, G); 0.02 mm (E). Same scale bar for A–C. Same scale bar for F and G.
Oligoneuriella tuberculata sp. nov., larvae, abdomen A abdomen under SEM in dorsal view B detail of tergal protuberances under SEM C abdomen under SEM in ventral view D detail of setae on sternum III under SEM E detail of setae on sterna III and IV F detail of setae on surface of terga G outline of abdomen. H detail of abdomen tip under SEM with bases of cerci and vestigial paracercus. Scale bars: 0.5 mm (A–C, H); 0.2 mm (D–F); 1 mm (G).
Oligoneuriella tuberculata sp. nov., larvae, gills A gill IV under SEM in dorsal view B gill IV under SEM in ventral view C gill plate I in dorsal view D gill plate I in ventral view E gill plate IV in dorsal view F gill plate IV in ventral view G gill plate VII in dorsal view H gill plate VII in ventral view. Scale bars: 0.1 mm (A–H). Same scale bar for C–H.
Larva. Submature larvae: body length 9–12 mm (female), 8–9 mm (male), length of cerci approximately 0.70–0.85× body length, paracercus vestigial, 5-segmented.
Colouration (Fig.
Head. Pale, yellowish-brown to dirty olive, slightly darker centrally; ocellar area brown. Antennae unicoloured, yellow. Head width/length 1 : 1.1–1.3 in submature larvae (Fig.
Thorax. Yellowish-brown to light dirty olive; base of wing pads with diffuse whitish maculation; two unclear whitish spots on mesonotum laterally; indistinct elongated maculae centrally. Pleural and ventral side paler than dorsal. Legs pale, whitish-yellow to yellow or slightly olivaceous, without any distinct ornamentation. Coxae and trochanters of same colour as other leg segments. Surface of legs covered with elongated bristles, spatulate and hair-like setae and small setae with star-like distal end (up to 15 μm long). Forecoxae with at least 30 long bristle-like setae distally and at least 20 long stout setae on inner margin. Foretrochanters with tuft of dense, long bristle-like setae distally. Femora unicoloured, occasionally with small diffuse spot distally. Forefemora length 2.0–2.1× of their width; dorsal surface covered with few flattened setae of various length, arranged in irregular sparse rows near filtering setae centrally; sparse row of stout bristle-like setae extends along 1/3–1/4 of length of outer margin, and 13–17 short bluntly pointed setae in submarginal area (Fig.
Abdomen (Fig.
Gills (Fig.
Cerci whitish, unicoloured, with inner marginal fringe of fine, hair-like setae. Paracercus vestigial, 5-segmented (Fig.
Egg, imago, and subimago. Unknown.
Among the Palaearctic genera of Oligoneuriidae, attribution of O. tuberculata sp. nov. to the genus Oligoneuriella is obvious, based on the shape of head, legs, and gills (see
Within Oligoneuriella, the most closely related species to O. tuberculata sp. nov. are O. bicaudata
(i) The first gill plate is markedly larger than gill pairs II–VII, nearly circular and symmetric, without a ridge near the outer margin in O. tuberculata sp. nov. In contrast, O. pectinata has an oval gill I, only slightly larger than the remaining gill pairs, with an indistinct ridge close to the outer margin (see Fig.
(ii) The setation on the surface of gills: in O. tuberculata sp. nov., dorsal and ventral surface of the gill I is equipped with very few flattened setae (Fig.
(iii) All three species can be separated by the shape of abdominal segments. Unpaired posteromedial protuberances on terga are characteristic for O. tuberculata sp. nov. (Fig.
(iv) All three species have a pale body colouration and an inconspicuous colour pattern on the abdominal terga. However, O. pectinata is slightly darker, with a pair of diffuse median spots on terga, whereas O. tuberculata sp. nov. is characterised by the presence of diffuse maculae laterally. Abdomen of O. bicaudata is pale brown, without any distinct pattern (
An analysis of diagnostic characters based on adults is impossible at present, as adults are not known for O. tuberculata sp. nov. and O. bicaudata. The colouration pattern and size of eyes would slightly differ in fully mature larvae from the material described herein.
The name of the new species originates from Latin, meaning hairy, and refers to the dense setation along outer margins of femora and tibiae.
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
Paratypes
: 64 larvae, same locality as holotype, deposited in
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
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
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.
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.
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.
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).
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.
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.
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.).
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).
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.
• row of long setae along all length of middle- and hind femora and tibiae (Fig.
• posteromedian projections on abdominal terga absent (Fig.
• colouration of abdominal terga dark brown with two pale dots medially (Fig.
• setae on surface of terga and gills elongated and bluntly pointed (Figs
• posterolateral projections of abdominal segments diverging from body axis (Fig.
• posteromedial setae on sterna III–IV very long and dense, some more than 20× longer than wide (Fig.
• first gill plate markedly smaller than the remaining pairs (Fig.
• setae on inner distal margin of gill plates II–VII long (Fig.
• setae on ventral surface of gill plates near inner distal margin present (Fig.
• paracercus fully developed (Fig.
• caudal filaments dark in proximal half, distinct dark band in the middle missing (Fig.
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.
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.
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.
Abdomen. Greyish dark brown, with pair of submedian smudged yellowish spots and conspicuous sublateral yellowish pattern (Fig.
Gills (Fig.
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.
Egg.
Shape of eggs studied deformed due to extraction from mature larvae (Fig.
Imago and subimago. Unknown.
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
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
In the genus Oligoneuriopsis, six species have been described up to now (
Oligoneuriopsis lawrencei Crass, 1947 differs in the shape of head, widest anterior to the eyes (
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.
The ABGD analysis of the COI distance matrix generated 11 stable groups (Fig.
The GMYC model recognised 11 ML entities, with confidence interval 11–11 (Fig.
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.
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
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.
We are grateful to M. Sartori (Musée cantonal de zoologie, Lausanne) for consultations concerning the systematic placement of O. villosus sp. nov. and to M. Al-Saffar for information on O. bicaudata. The authors are grateful to Milan Pallmann (