Helen's twins in the Balkans: discovery of two new Paraptychoptera Tonnoir, 1919 species closely related to P. helena Peus, 1958, with systematic revision of the " lacustris " group (Diptera, Ptychopteridae) Author Keresztes, Lujza https://orcid.org/0000-0002-4609-6496 Hungarian Department of Biology and Ecology, Center of Systems Biology, Biodiversity and Bioresources, Advanced Hydrobiology and Biomonitoring Lab, Babes-Bolyai University, 400006 Cluj-Napoca, Romania keresztes2012@gmail.com Author Kappert, Juergen Forsthaus 1, D- 363 94, Sinntal, Germany Author Henning, Maria Hungarian Department of Biology and Ecology, Center of Systems Biology, Biodiversity and Bioresources, Advanced Hydrobiology and Biomonitoring Lab, Babes-Bolyai University, 400006 Cluj-Napoca, Romania Author Toeroek, Edina https://orcid.org/0000-0001-5982-7078 " Lenduelet " Landscape and Conservation Ecology, Institute of Ecology and Botany, Centre for Ecological Research, 2163 Vacratot, Hungary text ZooKeys 2021 2021-11-17 1071 63 81 http://dx.doi.org/10.3897/zookeys.1071.58598 journal article http://dx.doi.org/10.3897/zookeys.1071.58598 1313-2970-1071-63 95BBE51DAD55577AA658388C7DD71744 Ptychoptera (Paraptychoptera) pollux Keresztes & Toeroek sp. nov. Figure 5 Type material. Holotype . male, North Macedonia: Mavrovo, Novo Selo, sweeping the vegetation near a small outflow from Mavrovo Lake, 29. vi, 2017, 990 m, leg. E. Toeroek , 41.721355°N , 20.830103°E . Institutional id for specimen is DCFBG-PT-0003. Diagnosis . Paraptychoptera pollux sp. nov. is known only from a single male collected near a small overflow of the Mavrovo Lake with muddy shore invaded by rich vegetation (Fig. 6 ). The male general habitus and wing venation with spots are highly similar to P. helena and P. castor sp. nov., but the wing spots tend to be reduced, mostly the basal spot and distally band which is divided into two distinct patches. Further differences are in male epandrium: The less developed finger-like subapical process is unique to P. helena , P. castor sp. nov., and P. pollux sp. nov. (Figs 2d , 4e , 5d ). However, in P. pollux sp. nov., this process is much shorter than in P. castor , but comparably longer than in P. helena , with blunt apex and divergent from the basal thorn. However, there is an important difference in the basal thorn orientation. In P. pollux , the basal thorn is oriented upward, while in P. helena the thorn is curved downward. Hypopygium shape with its rounded and slightly inflated apex is the second most distinctive character of P. pollux sp. nov., which differentiates it from both P. castor and P. helena , as well as from other Paraptychoptera species (Figs 2c , 4d , 5c ). Paramere lateral arms of P. pollux are similar to P. castor sp. nov. and P. helena , but are shorter and gradually widened at tip and rounded (Fig. 5h ). The rest of the characters, such the gonocoxite and gonostylus complex, hypandrium and the aedeagus are highly similar to P. helena (Fig. 4g, i ). Figure 5. Ptychoptera pollux sp. nov. a flagellum of antennae b right wing c epandrium, dorsal d subapical lobe of epandrium, ventral e gonocoxite and gonostylus complex, dorsal f gonostylus anterior and medial lobules, caudal g hypandrium, caudal h paramere, ventral i aedeagal complex, dorsal. Figure 6. Habitat of Ptychoptera pollux sp. nov., north Macedonia, Novo Selo village, Mavrovo lake outflow. Description . Medium-sized species, highly similar to its sibling species, P. castor sp. nov. and P. helena . Body length 7.9 mm, wing length 8.5 mm. Head and thorax similar to P. castor . Antennae with 15 segments. Scape elongate, cylindrical, yellowish brown, pedicel globular, pale brown, flagellar segments uniformly dark brown (Fig. 5a ). First flagellar segment shorter than the following two segments together, the others successively shorter and thinner. Each flagellar segment with several long straight black setae and a dense pelt of short dark hairs. Head shining black. Eye large, finely faceted, bare; no ocelli. Clypeus large, elongate, rectangular, flattened, labrum pale brownish. Labellum large, yellowish, maxillary palpus very long with a whip-like fifth segment pale yellow. Thorax dorsally brownish black with metallic blue shining, narrow pronotum, base of postnotum and large parts of episternum, epimeron and metapleuron pale brownish. Coxae orange, legs yellowish, apex of femur, narrow base and apex of tibia, tarsal segments brownish. Wing with three transverse bands of well-developed confluent dark spots in the anterior part of base, middle and distal part of otherwise clear or pale yellowish membrane. Basal spot of the wing more reduced. Distal band interrupted close to ventral edge. Isolated dark spots are present at distal end of Sc and R3 (Fig. 5b ). Wing membrane with macrotrichia. Halter and prehalter yellow. First abdominal tergite blackish to dark brown with a metallic blue shining, narrow yellow stripe close to distal end, well developed yellow band in tergite 2 with a black spot in the middle. Tergite 3 black, covered with yellow setae, the remaining tergites brownish black. Genitalia pale brown. Narrow sternites pale brown at base, becoming yellowish towards the auxiliary sexual organ on segment III. Sternites 4-7 medially reduced to a narrow band with a deep notch in the middle at proximal margins. Auxiliary sexual organ highly similar to P. castor sp. nov. and P. helena . Male terminalia. Epandrium with distinct collar, deeply and widely emarginated behind, hypoproct long tongue-like and furry (Fig. 5c ). Hypoproct lobe widened at apex, rounded, with a shallow notch in the middle. Epandrium lobe long, slightly widened apically. Subapical process of epandrium with a digitiform ventral projection with basal thorn and curved upward, longer, than the blunt digitiform process (Fig. 5c, d ). Apex of the digitiform process with long macrotrichia. Gonocoxite simple, cylindrical. Gonostylus with an anterior lobule divided into a dorsal triangular process, ventral part with a small rostrum (Fig. 5f ). Middle lobe strong sclerotised sickle-like curved process, with long fine hairs at the end (Fig. 5e, f ). Gonostylus apical lobe narrow fleshly process, subequal with secondary lobe (Fig. 5e ). Secondary lobe slightly curved at apex, with strong erect spines. Hypandrium wide, long and elongate crests in the middle, including a narrow slit between them, from which the eversible sac protrudes. The transverse scale at the base of the slit is less developed, with two lateral wings and a triangular process in the middle, membranous and densely pilose. Hypandrium apex terminal division less developed, but distinct as a narrow band with pointed apex (Fig. 5g ). Paramere lateral arms less developed, rounded apically and widened with less-developed setae close to the interior of the apex. Apical processes of paramere well developed, rectangular, with a recurring thorn-like formation (Fig. 5h ). Aedeagal complex highly similar to other Paraptychoptera species. Apex of aedeagus concave, with a depression in the middle, subapical lobe of aedeagus rounded (Fig. 5i ). Female unknown. Etymology . The specific epithet is named after Pollux, the twin brother of Castor in Greek mythology, known together as the Dioscuri, both twin brothers of Helena, because together with P. castor they share close morphological similarity with P. helena and all together they form a distinct monophyletic unit among Paraptychoptera , as was recovered by our cladistic analysis (Fig. 7 ). Figure 7. Single most parsimonious tree (1392 steps) based on 53 morphological characters. Bootstrap (B) values over 50% are noted above the corresponding branches, respectively. Branch support was calculated by bootstrap with 10000 replicates. Character states are shown above branches. Cladistic analyses .The parsimony analyses of the 53 different morphological characters selected in the present study resulted in a single most parsimonious tree (Fig. 7 ). As shown in our parsimony analyses (Fig. 7 .), the eleven different Paraptychoptera species are divided into two major monophyletic clades, with a highly divergent monophyletic unit, including five species, P. agnes , P. lacustris , P. helena , P. castor sp. nov., and P. pollux sp. nov. This monophyletic unit is supported by common morphological features, such as the soft and lobe-like basal scale, hypandrium with a long, but not bilobate narrow ribbon-like process or reduced and the simpler lateral arms of paramere (characters 36, 52). Among this group, a distinct lineage is represented by P. agnes , highly different from all other members of the clade by the presence of a conspicuous epandrial lobe, with a ventral lobe close to the base, and a transverse projection with a comb of long setae, unique only to this species, in addition to the particularly shaped hypoproct and epandrial subapical process, and also by the uniquely shaped secondary lobe of the apical stylus inflated and globulose at apex, besides the details of hypandrium and parameres (characters 8, 12, 13, 23, 30, 37, 47). The present cladistic analyses recovered the " Ptychoptera lacustris " species group as a monophyletic unit, which was also noted by Stubbs (1993) and Zwick and Stary (2003) , but in a restricted sense, containing only four species: P. lacustris , P. helena , P. castor sp. nov. and P. pollux sp. nov. Further, the previously considered P. loncicauda and P. paludosa were excluded and mostly based on a weekly developed auxiliary sexual organ in " Ptychoptera lacustris " group, but well developed in later species, and a weakly developed and reduced subhemispherical membranous basal scale in " Ptychoptera lacustris " group in contrast with the well-developed and chitinous scale in P. longicauda and P. lacustris . There was also the presence of the stripe-like and bifurcate or cross-shaped terminal division of the hypandrium apex in P. longicauda and P. paludosa , in contrast with the weakly developed terminal division of hypandrium apex in " Ptychoptera lacustris " group. This latter was missing in P. lacustris or reduced to a tapering ribbon-like protrusion in P. helena and allies (characters 38, 48). However, within the " Ptychoptera lacustris " group the subapical lobes of epandrium have a conspicuously similar shape in P. castor sp. nov., P. helena and P. pollux sp. nov., while in P. lacustris such a formation is totally absent. Ptychoptera lacustris is also divergent from the three closely-related Balkan species by the presence of a small triangular hypoproct and rounded subspherical basal scale of the hypandrium (characters 21, 39). Ptychoptera helena and the two newly discovered species of the " Ptychoptera lacustris " group are morphologically highly similar, but deeply divergent from all other Paraptychoptera species, having unique epandrial clasper lobes that are slightly divergent toward the tip, and epandrial subapical lobes reduced to a finger-like short projection with a basal chitinous thorn, hypandrium apex terminal division that is highly reduced, finger-like, with pointed apex, lacking laterally directed spines, but thin hairs are sometimes present (only in P. castor such a process is absent). Further, the aedeagus tip has a unique shape, with a small depression in the middle (characters 29, 40, 44, 45). Ptychoptera helena and P. pollux sp. nov. are highly similar, but minor differences are present in the shape of the hypopygium, and the design of the subapical lobe of the epandrium (characters 24, 26). Further, they are distinctly different from P. castor sp. nov. by the presence of a long subapical epandrial lobe (character 19), which is as long as its basal chitinous thorn, as well as the long harpoon-like hypoproct, unique only to this species (character 25).