Nota Lepi. 43 2020: 265-279 | DOI 10.3897/nl.43.52581 Research Article Phtheochroa carpatiana sp. nov. (Lepidoptera, Tortricidae), the Carpathian representative of the Phtheochroa frigidana species-group ZOLTAN Kovacs!, SANDOR Kovacs’, BOYAN ZLATKOV°, PETER HUEMER* sir. Campul Mare nr. 133, RO-530240 Miercurea Ciuc, Romania; kovkopp@gmail.com 2 str. Oltului nr. 57 bloc 6 sc. A ap. 6, RO-520027 Sfantu Gheorghe, Romania; skovacssandor@gmail.com 3 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Blvd, 1000 Sofia, Bulgaria; bzlatkov@gmail.com 4 Tiroler Landesmuseen Betriebsgesellschaft m.b.H., Sammlungs- und Forschungszentrum, Naturwissenschaftliche Sammlungen, Krajnc-Str. 1, A-6060 Hall in Tirol, Austria; p.huemer@tiroler-landesmuseen.at http://zoobank. org/B377EFE7-6761-411F-901E-80BF 1A4BBC4B Received 26 March 2020; accepted 16 June 2020; published: 28 September 2020 Subject Editor: Maria Heikkila Abstract. Phtheochroa carpatiana sp. nov. is described from the Southern Carpathians (Romania). It is close- ly related to the other five members of the P. frigidana species-group. Adults, male and female genitalia, and the habitat of the new species are described and figured, molecular data and some details of the biology are given. The species inhabits alpine meadows above 2100 m, the highest regions of the Southern Carpathians. Adults are on the wing from the end of June to the beginning of August. Phtheochroa carpatiana demon- strates considerable intraspecific variation in some details of the vesica in the male genitalia. Introduction Phtheochroa frigidana s. \at. is a species-complex in which the true level of diversity is difficult to ascertain using traditional taxonomic methods. Initially, the group was believed to consist of only two species, the mainly western P. frigidana (Guenée, 1845) and the eastern P. drenowskyi (Rebel, 1916), both considered as widely distributed in the alpine zone of different European high mountain systems. This approach, however, led to controversial results when identifying individ- uals from other regions, especially from the Apennines (Trematerra 2003) and the western Balkan Peninsula (Razowski 2009). A recent study by Zlatkov and Huemer (2017), involving material originating from the major- ity of the mountain ranges of Europe, resulted in major reinterpretations of the taxonomy of this group. Combining a new morphological technique, the eversion of the vesica in the male genitalia, along with DNA barcode sequencing of the mitochondrial COI gene resulted in five morpholog- ically and genetically different geographically separated species, one from each mountain range involved in the study: P. cantabriana Zlatkov & Huemer, 2017 from the Cantabrian Mountains, P. frigidana (Guenée, 1845) from the Pyrennees, P. alpinana Zlatkov & Huemer, 2017 from the south-western Alps, P. apenninana Zlatkov & Huemer, 2017 from the Apennines and P. schaw- erdae (Rebel, 1908) (with P. drenowskyi Rebel, 1916 as its junior synonym) from the Dinaric, Rila and Pirin Mountains of the Balkan Peninsula. Additionally, based only on available literature sources, two other neighbouring populations from the Slovenian Alps and the Carpathian Moun- tains are assumed to be conspecific with P. schawerdae. 266 ZOLTAN Kovacs et al.: Phtheochroa carpatiana sp. nov. from the Carpathians A subsequent re-examination of the Carpathian material originally identified as P. drenowskyi (Karisch and Stanescu 2003; Kovacs and Kovacs 2005) raised questions regarding the conspecific- ity of these specimens with P. schawerdae, as small but constant differences were found both in the external morphology and in the male and female genitalia. Further arguments for the description of a new species from the Southern Carpathians were provided by the analysis of DNA barcode se- quences and examination of the everted vesica in the male genitalia, following the same procedures as in the revision by Zlatkov and Huemer (op. cit.). Material and methods Eighty-six specimens were examined that were collected from three of the four major mountain ranges of the Southern Carpathians: the Bucegi, Fagaras and Parang Mountains. The examined ma- terial was dried, pinned and set. The terminology of wing pattern follows Razowski (1970, 2009) and Zlatkov and Huemer (2017). Genitalia preparation techniques followed the methodology of Robinson (1976). Vesicae were dissected and everted under a Carl Zeiss stereomicroscope Stemi 2000c following Zlatkov (2011). The everted vesicae were drawn, photographed and measured following Zlatkov and Huemer (2017) through a Carl Zeiss Jena Amplival compound microscope equipped with a camera lucida and Canon EOS 1300D digital camera. The female genitalia were drawn in a similar manner after being placed on a cavity slide without compression. The descrip- tion of the phalli follows the same publication (Zlatkov and Huemer op. cit.); terminology of the cornuti follows Anzaldo et al. (2014). Samples of the everted vesicae from all the collecting sites were studied to assess possible geographical variation. More specimens were also examined from the Fagaras Mountains to detect potential intraspecific variability. DNA samples from three specimens were prepared according to the prescribed standards and processed at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) to obtain DNA barcodes following the standard high-throughput protocol described in deWaard et al. (2008). Two of the resulting sequences (629 bp and 558 bp) and a short- er sequence (307 bp) were analysed together with 11 sequences already published by Zlatkov and Huemer (2017) and a sequence of Phtheochroa rugosana (Hubner, 1799) used as an outgroup. Se- quences were submitted to GenBank; further details including complete voucher data and images can be accessed in the public dataset “Phtheochroa frigidana species-group [DS-AALARANE]” https://doi.org/10.5883/DS-AALARANE in the Barcode of Life Data Systems (BOLD; Ratnasin- gham and Hebert 2007). Degrees of intra- and interspecific variation of DNA barcode fragments were calculated under the Kimura 2 parameter model of nucleotide substitution using analytical tools of BOLD systems v. 4.0. (http:// www.boldsystems.org). A Neighbor-joining tree of DNA barcode data of currently sequenced taxa was constructed using MEGA6 (Tamura et al. 2013) under the K2P model for nucleotide substitutions. Photographs of the adults were taken using a Canon EOS—6D camera with a Sigma 105 mm lens and focus stacking method. Photos of the habitats and adults in nature were taken using Sony DSC-H2 and DSC—W830 digital cameras. Institutional abbreviations NMNHS National Museum of Natural History, Sofia, Bulgaria; TLMF Tiroler Landesmuseum Ferdinandeum, Innsbruck, Austria. Nota Lepi. 43: 265-279 267 Results Phtheochroa carpatiana Kovacs, Kovacs, Zlatkov & Huemer, sp. nov. http://zoobank.org/8DE83A9B-E8ED-4BA F-A985-6170FOF99BB3 Figs 1-6, 12 Type material. Holotype. RoMANIA; ° 3’; Carpatii Meridionali, Muntii Fagaras, Caldarea Balea; [45°36’12”N, 24°37’ 18”E ]; 2100-2200 m; 17 Jul. 2019; S. & Z. Kovacs legit & coll. (Miercurea Ciuc) (Fig. 1). Paratypes. 78 33,7 2 Q, all collected by S. & Z. Kovacs and if not otherwise mentioned are deposited in the collection of S. & Z. Kovacs (Figs 2-3). Romanla; * 9; Carpatii Meridionali, M[un]tii Fagaras, Balea; 2100 m; 9 Aug. 1992; genit. prep. no. 468/9/ Kovacs (1998); * 3 33; Carpatii Meridionali, Muntii Fagaras, Varful Laitel; 2300 m; 21 Jul. 2015; * 40 04, 3 29; Carpatii Merid- ionali, Muntii Fagdras, Caldarea Bélea; 2100-2200 m; 14 Jul. 2016; genit. prep. no. 1909/4/ and 1910/9/ Kovacs (2017), genit. prep. no. 1/3.2.2020/¢ and 3/20.1.2020/9/ Zlatkov; [Barcode identification number] TLMF Lep 27414, TLMF Lep 27444: 1 3,1 2 coll. TLMF, 1 9 coll. NMNHS:; « 24 44; same data as for preceding; 17 Jul. 2019; genit. prep. no. 2262/3/ Kovacs (2019), genit. prep. no. 1/20.1.2020/<', 2/3.2.2020/¢ and 3/3.2.2020/3// Zlatkov; 1 4 coll. NMNHS; °3 3d, 2 99: same data as for preceding; 18 Jul. 2019; + 4; Carpatii Meridionali, Muntii Fagaras, Iezerul Caprei; 2350 m; 17 Jul. 2019; °2 43,1 9; Carpatii Meridionali, Muntii Bucegi, V[4r]f[ul]. Caraiman; 2380 m; 27 Jun. 2003; genit. prep. no. 1486/ cat Figure 8. The habitat of Phtheochroa carpatiana sp. nov., Fagaras Mountains, general view, 2100-2500 m, westwards of Iezerul Caprei Peak. Habitat. Phtheochroa carpatiana inhabits the highest mountains in the Romanian Carpathians. The moths can be found in the alpine zone from 2100 to 2380 m above sea level. The substrate is silicate in Fagaras (Fig. 8) and the Parang Mountains (Fig. 9), and conglomerate in the Bucegi (Fig. 10). This species prefers sunny meadows, thickly covered with grasses (Fig. 11). In most of its collecting sites it is found with other Carpathian endemic species such as Dichrorampha carpa- talpina Kovacs & Kovacs, 2019 (Tortricidae) and Catoptria orientellus (Herrich-Schaffer, [1855 ]) (Crambidae). Phenology. Univoltine, adults are on the wing from the end of June to the beginning of August, being highly dependent on general weather conditions. Biology. The early stages and the host-plant are unknown. Adults fly during the day in sunshine or they rest on the upper part of grasses. In cloudy weather they immediately hide deep in the vegetation. The flight of the males is not fast, and only when disturbed do they fly fast and hide deep within the vegetation. Females call on the vegetation. They were much more rarely collected than males: when we observed freshly emerged females we counted 40 males and only 4 females. One pair in copula was also observed on low vegetation (Fig. 12). Following the end of the copulation with the first male, a second attracted male immediately started to mate with the female. Phtheochroa carpatiana does not seem to be active by night as none were collected by light traps installed at sites where specimens were observed during the same day. 274 ZOLTAN Kovacs et al.: Phtheochroa carpatiana sp. nov. from the Carpathians = L, a Ss ape Eb — oe ees SS ae : = Figure 9. The habitat of Phtheochroa carpatiana sp. nov., Parang Mountains, general view, 2100-2500 m, westwards of Mohorul Peak. Distribution. Phtheochroa carpatiana seems to be widespread and locally common in the three main mountain ranges in the Southern Carpathians: Bucegi, Fagaras and Parang (Fig. 13). It may also occur in the other parts of the Southern Carpathians, at least in the very similar Retezat Moun- tains, where we made only one unsuccessful attempt to find it. Phtheochroa carpatiana replaces P. drenowskyi in the checklist of the Romanian Lepidoptera (Rakosy and Goia 2007). Etymology. The specific name is a feminine adjective derived from the name of the Carpathi- ans, the mountain range where the new Phtheochroa species was discovered. Discussion The members of the Phtheochroa frigidana species-group are geographically separated and their collecting sites are a guide to their identification. Externally the species are similar, and only the genitalia provide reliable diagnostic characters in this group. In ventral view of the male genitalia the ventral phallic process is straight in P. frigidana, curved to the right at almost 90° in P. schaw- erdae and almost similarly curved (to around 45°) in all other species: it is widest in P. apenninana, slenderer in P. cantabriana, evenly narrowing in P. alpinana, and slightly sinuous in P. carpatiana. The everted vesica has a single dorsal diverticulum in P. frigidana, but in all other species has two distinct diverticula which differ in position: in P. alpinana dorsal and ventral, in P. carpatiana Nota Lepi. 43: 265-279 275 Figure 10. The habitat of Phtheochroa carpatiana sp. nov., Bucegi Mountains, Caraiman Peak, 2380 m, west-facing slope. dorso- and ventrolateral, in P. apenninana lateral, and distal and lateral with the angle between the axes of the right diverticulum and the sclerotized phallus smaller (100—120°) in P. schawerdae and larger (120—140°) in P. cantabriana. The female genitalia are unknown in P. apenninana and P. cantabriana; the ductus bursae is longer in P. frigidana, P. alpinana and P. schawerdae and shorter in P. carpatiana. The small mem- branous diverticulum of the ductus bursae is long and slender in P. schawerdae, shorter and wider in P. alpinana (Z\atkov and Huemer op. cit.), widest in P. carpatiana and has not been figured in P. frigidana. Female genitalia figures referring to the P. frigidana species-group in the literature before Zlatkov and Huemer (op. cit.) are incomplete, as they do not depict the membranous diver- ticulum on the ventral wall of the ductus bursae (Razowski 1961, 1970, 2002, 2009; Karisch and Stanescu 2003; Kovacs and Kovacs 2005). The paucity of available material of most species is the major hindrance to a thorough study of the group. Phtheochroa carpatiana is the only species with a larger type series allowing a Study of variability, which appears to be much higher than expected. The variation of the size of P. carpatiana is the same as in all other related species of the P. frigidana species- group. The variable shape of the transtilla in specimens of the same population has already been mentioned in P. schawerdae by Zlatkov and Huemer (op. cit.) and observed in the case of the 276 ZOLTAN Kovacs et al.: Phtheochroa carpatiana sp. nov. from the Carpathians Figure 11. The habitat of Phtheochroa carpatiana sp. nov., Fagaras Mountains, Caldarea Balea, 2100— 2200 m, west-facing slope. vesica and antrum of P. carpatiana during the present study. The presence of two cornuti of normal size and development on the right diverticulum instead of one, and diverticula having larger diameters in a specimen from the Fagaras Mountains is remarkable. Such a condition has never been found either in any species of the P. frigidana species-group or in other Cochylini groups with two cornuti, each attached to its own diverticulum (P. reisseri (Razowski, 1970), P. unionana (Kennel, 1900), P. procerana (Lederer, 1863), Eugnosta magnificana (Rebel, 1914), E. lathoniana (Hubner, [1800])). In general, P. carpatiana demonstrates variation in some details of the vesica. The lack of detected variation in other species of the P. frigidana species-group may be due to the small number of available males (e.g., only two males are known of P. alpinana). It must be emphasized that the observed variation in P. carpatiana 1s not geographically related. Another representative in the tribe Cochylini, Eugnosta magnificana, also demonstrates remarkable variation of the vesica, mainly in its size (Zlatkov 2018). Since there are no particular studies on variation of the phallus and especially the vesica in the Cochylini or other Tortricidae groups, any further conclusions on the variation of the vesica in P. carpatiana would be speculative. Interspecific DNA barcode divergences to the Nearest Neighbor in the P. frigidana species-group roughly range from 3 % to 4 % (Table 1) though based on low sampling. Where such high levels of barcode variation are found within a series believed to be from a single species, it often reflects Nota Lepi. 43: 265-279 277 Figure 12. Phtheochroa carpatiana sp. nov., pair in copula, Fagaras Mountains, Caldarea Balea, 2100— 2200 m, 14.vi1.2016. Table 1. Barcode gap analysis in the Phtheochroa frigidana species-group. For each species the number of barcoded specimens are given (n), the mean and maximum intra-specific values are compared to the Nearest Neighbor (NN) distance (in %). For singletons N/A is displayed for intra-specific values. Species n Mean Intra-Sp Max Intra-Sp Nearest Species Distance to NN Phtheochroa apenninana — 2 0 0 Phtheochroa schawerdae 3.93 Phtheochroa cantabriana 3 0.2 0.31 Phtheochroa frigidana B.02 Phtheochroa alpinana 1 N/A 0 Phtheochroa carpatiana 3.47 Phtheochroa frigidana 1 N/A 0 Phtheochroa cantabriana 3.62 Phtheochroa schawerdae 4 0.98 1.61 Phtheochroa carpatiana 3.14 Phtheochroa carpatiana 3 0 0 Phtheochroa schawerdae 3.14 overlooked species or at least requires further integrative analysis (Hausmann et al. 2013), but there is no fixed level of divergence that indicates species status (Kekkonen et al. 2015). It is notable that the flight period for P. frigidana mentioned by Razowski (1970, 2002, 2009) is from April to July, which is not reflected by the material in recent studies, as the collecting dates of all six currently known species in the P. frigidana species-group are between the end of June and beginning of August. Further studies are necessary to clarify which one of the species of the P Jrigidana species-group has an earlier flying period. The status of two specimens from the Slovenian Alps, one mentioned as P. frigidana by Lesar et al. (2009) and another deposited in the J. Klimesch collection (Lepiforum), thought to be P schawerdae by Zlatkov and Huemer (op. cit.) 1s uncertain. 278 ZOLTAN Kovacs et al.: Phtheochroa carpatiana sp. nov. from the Carpathians @ Phtheochroa cantabriana Zlatkov & Huemer, 2017 @ Phtheochroa frigidana (Guenée, 1845) Phtheochroa alpinana Zlatkov & Huemer, 2017 | @ Phtheochroa sp. 3 @ Phtheochroa apenninana Zlatkov & Huemer, 2017 @ Phtheochroa schawerdae (Rebel, 1908) @ Phtheochroa carpatiana sp. n. Figure 13. Distribution map of the Phtheochroa frigidana species-group based on examined material (after Zlatkov and Huemer 2017, modified) and literature sources for Phtheochroa sp. from the Slovenian Alps. Map created with SimpleMappr (http://www.simplemappr. net). Acknowledgments We are most grateful to Paul D.N. Hebert and the entire team at the Canadian Centre for DNA Barcoding (CCDB, Guelph, Canada) for carrying out the sequence analyses. PH is particularly indebted to the Promotion of Educational Policies, University and Research Department of the Autonomous Province of Bolzano — South Tyrol for funding the project “Genetische Artabgrenzu- ng ausgewahlter arktoalpiner und boreomontaner Tiere Stidtirols”. Special thanks go to Robert J. Heckford (Plympton, U. K.) for linguistic corrections and improvement of the manuscript. Zoltan Csata (Sfantu Gheo- rghe, Romania) helped with photographing the adults. We are grateful for constructive comments of the editor and reviewers which improved the quality of the manuscript. References Anzaldo SS, Dombroskie J, Brown JW (2014) Morphological variation, taxonomic distribution, and phylo- genetic significance of cornuti in Tortricinae (Lepidoptera: Tortricidae). Proceedings of the Entomological Society of Washington 116 (1): 1-31. https://do1.org/10.4289/0013-8797.116.1.1 deWaard JR, Ivanova NV, Hajibabaei M, Hebert PDN (2008) Assembling DNA barcodes: Analytical Proto- cols. 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