Morphological and molecular characterisation of the Popijac's Yellow Sally, Isoperla popijaci sp. nov., a new stenoendemic stonefly species from Croatia (Plecoptera, Perlodidae) Author Hlebec, Dora https://orcid.org/0000-0001-5967-864X Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia dora.hlebec@gmail.com Author Sivec, Ignac Slovenian Museum of Natural History, Presernova 20, 1000 Ljubljana, Slovenia Author Podnar, Martina https://orcid.org/0000-0002-5782-3222 Croatian Natural History Museum, Demetrova 1, 10000 Zagreb, Croatia Author Skejo, Josip https://orcid.org/0000-0002-2554-4499 Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia Author Kucinic, Mladen Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia text ZooKeys 2021 2021-12-16 1078 85 106 http://dx.doi.org/10.3897/zookeys.1078.66382 journal article http://dx.doi.org/10.3897/zookeys.1078.66382 1313-2970-1078-85 359F5F541DFA4A788F7BD8B042F40E1E 4BE607A8C859518DA44B0AFF7768FDC3 Isoperla popijaci Hlebec & Sivec sp. nov. Figures 1A-E , 2A-G , 3A, B , 4A-D Material examined ( 1♂ HT, 10♂♂ PTs, 23♀♀ PTs and 8 larvae PTs): 1♂ HT (96% ethanol) Original label: Croatia , Lika , Krbava field, Krasulja rivulet, karstic source Severova Cave ; 44°40.78'N , 15°37.87'E , 21 June 2019 , I. Sivec leg. (CNHM: CPSH) ; 6♂♂ PTs and 11♀♀ PTs (96% ethanol) same data as for the holotype ; 5 larvae PTs (96% ethanol) 09 April 2015 , I. Sivec leg.; 3 larvae PTs (96% ethanol) 22 February 2021 , D. Hlebec leg.; 1♂ PT and 3♀♀ PT s (96% ethanol) 2 June 2021 , I. Sivec leg.; 3♀♀ PTs (96% ethanol) 18 June 2021 , D. Hlebec leg.; 3♂♂ PTs and 6♀♀ PTs (96% ethanol) karstic source nearby village Mirici , 44°43.14'N , 15°38.09'E , 2 June 2021 , I. Sivec leg. Type material depository. HT (1♂) and 31 PTs (7♂♂+18♀♀+6 larvae) in Zagreb, Croatia (CNHM), Collection of Plecoptera Sivec & Hlebec, under accession number CPSH 1-32; and 10 PTs (3♂♂+5♀♀+2 larvae) in Ljubljana, Slovenia (PMSL). Type locality. Croatia, Lika, Krbava field, Krasulja rivulet, karstic source Severova Cave, 44°40.78'N ; 15°37.87'E ; 640 m a.s.l. Diagnosis. The new species I. popijaci sp. nov. belongs to the I. tripartita species group, with divided medial penial armature into upper and lower coloured portions. It has, however, a specific penial armature on the ventral lobe of the penis, different from all known Isoperla species. The upper medial armature is subdivided, and the lower medial armature is present in two scale spike-like areas. The proximal part has a pair of drop-shaped areas armoured with spines, longer at the tip and shorter at the base. The medial penial armature with a field of shorter spines as in Figure 4C . Only a few irregular spines on the lateral side of the penis in the area of the upper armature of the penis. Description. Macropterous in both sexes, medium-sized species with yellow head and pronotum. Adult. Body length: HT male 18.5 mm; PTs : males 17-19 mm (n = 10), females 16.5-18 mm (n = 23). Forewing length: HT male 12 mm; PTs : males 11-13.5 mm, females 11.5-14 mm. Colouration. General colour uniformly brownish (Figure 1C ), slightly paler ventrally and laterally. Figure 1. Morphology of Isoperla popijaci sp. nov. A head and pronotum in dorsal view (adult male HT) B habitus (larval PT) C habitus (adult male PT) D female terminalia in ventral view (PT) E everted male copulatory organ (HT). Scale bar: 0.5 mm A-E . Head. The central part of the head pale yellowish; darker at the lower part and between ocelli; slightly darker in the frontal and lateral part. M-line and tentorial callosities weakly expressed and inconspicuous. Pale spot positioned centrally between the ocelli, paler in the central distal part of the head. Eyes slightly smaller than the area delimited by the three ocelli. Scape and pedicel dark brown. Palpi uniformly cream coloured. The distal part of the antennae pale and the proximal segments darker (Figures 1A , 2A ). Wings. Wings translucent brownish, venation dark brown. Pronotum. Pronotum yellowish, rectangular with angled edges. Medial and lateral parts of the pronotum pale; central part on both sides slightly darker and with dark brown textured surface (Figures 1A , 2A ). Figure 2. Morphology of Isoperla popijaci sp. nov. A head and pronotum in dorsal view (adult female PT) B terminalia in ventral view (adult female PT) C terminalia in ventral view (adult male HT) D head and pronotum in dorsal view (larval PT) E abdomen in dorsal view and detail of a distal segment of a cercus (larval PT) F right maxilla in dorsal view (larval PT) G penial armature (adult male HT). Scale bars: 1 mm ( A-D ); 0.5 mm ( E-G ). Mesothorax and metathorax. Ventral surface of thorax uniformly brownish; dorsal side slightly darker, lateral part lighter. Mesonotum and metanotum predominantly dark brown. Legs. Femora and tibia brownish, same as body colouration. Tarsi slightly darker than femora and tibia on the dorsal side and pale ventrally. Male abdomen. Mesobasisternum and metabasisternum brown in the middle and darker laterally. Ventral surface of male abdomen uniformly brownish, slightly darker dorsally. A few proximal segments of cerci pale, with rest dark brown. Penis (everted). Divided into four lobes, with a basal section in everted position. The medial penial armature on the ventral surface of the penis divided into an upper and a lower part, both are coloured (Figures 2C, G , 4A ), upper part rather pale. The upper medial penial armature is further subdivided into left and right arms, elongated, delimited from scales of the lateral lobes (Figure 4C ). Length of the arms is 200-250 µm , width 100-120 µm . Scales of the upper medial penial armature forming a drop-shaped area, spike-like, with longer scales at the tip and shorter ones at the base. Length of the scales 25-37 µm , width 7-9 µm at the base. The lower part of the medial penial armature subdivided, with an irregular upturned V-shaped area and bearing very short spines (Figure 4D ). Length of the areas 220-250 µm , width 100-140 µm . The scales are spike-like, thinner than in the upper medial armature. The ventral lobe hemispherical, covered with hair-like scales, in some places ciliated scales. The medial lobe small with diverse scales. Lateral penial armatures located on the lateral lobes, above the basal section, small and indistinct with only a few spines. Detail of the lateral lobe as in Figure 4B . Female abdomen. All tergites uniformly brownish. Sternites slightly paler brownish. A few basal segments of cerci pale, rest of cerci dark brown. Subgenital plate large and wide reaching near the end of sternite IX (widely concave in the middle) (Figure 2B ). Egg. Chorion light brown, 0.34-0.38 mm long and 0.29-0.33 mm wide (n = 22). Chorion with marked ornamentation of irregular round shape. Follicular cell impressions with finer inner punctations. Hatching line distinct. Micropyles not well recognisable. Collar and anchor missing (Figure 3A, B ). Figure 3. Egg of Isoperla popijaci sp.nov. A whole egg, lateral view B detail of hatching line, lateral view Figure 4. Extruded penis of Isoperla popijaci sp. nov. A male abdomen with extruded penis, ventral view B detail of penial armature on the lateral lobe, dorsal view C scales of the upper medial penial armature, dorsal view D pair of the scales spike-like on the ventral lobe, dorsal view. Larva. Body length of not-completely-mature larva 14-16 mm (n = 8). General colour pale brownish; with darker markings on head and abdomen. Body and legs typically pilose. Swimming hairs present on femora, tibiae and tarsi. Posterior abdominal fringe short and cercal fringe no longer than width of cercal segment. General colour of the head brownish, with a darker transversal mask connecting eyes and ocelli (Figure 2D ). M line indistinct. Eyes well developed. Mouth parts and basal parts of antennae pale coloured; distal part of antennae dark brownish. Lacinia bidentate; inner margin with 4-5 stout setae and a row of short thin setae below subapical tooth. Pronotum rounded; brownish; with indistinct darker pattern centrally and distinctly paler laterally (Figure 2D ). Pronotal setal fringe with short bristles and bearing only a few longer setae at posterior margin. Ventral side of the body and leg pale coloured. Abdominal tergites darker, brown with a pair of relatively large drop-shaped pale spots in the middle of the abdomen (Figure 2E ). Paraprocts and cerci uniformly pale. Setation on distal section of cercal segments with rather uniform setae and single larger dorsal setae. Etymology. The specific name is the genitive singular of the Latinised version of the surname Popijac (Popiacus, -i, m.), given in honour of colleague Dr Aleksandar Popijac and his achievements in field research and knowledge of the Plecoptera fauna in Croatia. Distribution and ecology. The species was collected at the entrance to the Severova Cave, occasional karstic source of the intermittent Krasulja rivulet in Krbava field and two year later (on 2 June 2021) near the karstic source of the same rivulet, near the village of Mirici . The Severova Cave (old name Hrnjakova Cave) is located on the northern edge of the Krbava field (karst field located near settlement Krbavica in the vicinity of the Plitvice Lakes National Park). The temporary Krasulja rivulet is part of the hydrogeological system of the Krbavica River (Figure 5E ). For several months a year, the water runs from the cave and forms the Krasulja rivulet, which flows into the Krbavica River and sinks on the south side of the field. When the discharge of the Krasulja falls below 60 l/sec, the water-flow ceases from Severova Cave ( Malinar and Cepelak 2009 ). The stream does not have a rich stonefly fauna and the species found at this locality, except the newly-described species of Isoperla , are Amphinemura standfussi (Ris, 1902) and Nemoura cinerea (Retzius, 1783). The substrate at the collection site of larvae was mainly composed of larger fractions. Figure 5. Type locality of the Popijac's Yellow Sally, Isoperla popijaci sp. nov.: Severova Cave in Croatia A and B photographs in wet phase C, D photographs in dry phase E map (blue circle indicates type locality).