Dtsch. Entomol. Z. 68 (2) 2021, 249-259 | DOI 10.3897/dez.68.70814 yee BERLIN Where did the Central European populations of Ornatoraphidia flavilabris (Costa) come from? (Neuropterida, Raphidioptera, Raphidiidae) Horst Aspéck?, Ulrike Aspéck*, Julia Walochnik’, Edwin Kniha? 1 Medical University Vienna (MUW), Institute of Specific Prophylaxis and Tropical Medicine, Kinderspitalgasse 15, A-1090 Vienna, Austria 2 Natural History Museum, 2"4 Zoological Department, Burgring 7, A-1010 Vienna, Austria 3 University of Vienna, Department of Evolutionary Biology, Althanstrape 14, A-1090 Vienna, Austria http://zoobank. org/20935557-2F F 2-49F 6-BA61-266323E22F77 Corresponding author: Edwin Kniha (edwin.kniha@meduniwien.ac.at) Academic editor: Susanne Randolf # Received 29 June 2021 # Accepted 5 August 2021 Published 30 August 2021 Abstract Ornatoraphidia flavilabris (Costa, 1851) 1s one of 15 snakefly species occurring in southern parts of Central Europe. It 1s a polycen- tric Mediterranean faunal element with refugia in the Apennine Peninsula and the Balkan Peninsula. Two phylogeographic questions are dealt with in this paper: (1) Is it possible to differentiate, morphologically or genetically, the Balkanic populations from the Italian? (2) Did the species reach Central Europe from the Balkan or Apennine Peninsula? These questions were investigated using morphological and molecular biological methods. No morphological characters were un- covered which could serve to differentiate specimens from either distribution center. However, differences were detected in cox/, cox3 and 28S genes which allow for a reliable differentiation. Central European populations were largely identical with populations from Italy, but distinctly different from specimens from Greece. This could lead one to assume that the species migrated from Italy to Central Eu- rope, although colonization from the southeast would appear easier due to more favorable orographic conditions. This discrepancy may be explained by the apparent absence of O. flavilabris from the large central part of the Balkan Peninsula, so that a gap exists between the southern and northern areas inhabited by O. flavilabris. Moreover, the species does not occur in eastern parts of Europe. Thus it would be more probable to assume that the occurrence of the species in the northwest Balkan Peninsula can be traced to migrations from the Apen- nine Peninsula to areas north and northeast of the Adriatic Sea, where O. flavilabris may have colonized the southeast of Central Europe. A migration of Adriatomediterranean faunal elements from the northwest Balkan Peninsula to Central Europe might be of more significance than previously assumed. Key Words Apennine Peninsula, Balkan Peninsula, Mediterranean refugial centers, phylogeography, snakeflies Introduction Raphidioptera (snakeflies) is the smallest order of ho- lometabolous insects. So far, around 250 valid described species are known world-wide: ca. 210 species of Raphi- diidae and >40 species of Inocelliidae. The distribution of snakeflies is confined to the temperate zone of the Hol- arctic, with hotspots of high biodiversity in the Mediter- ranean region, Central Asia, Southeast Asia and southern North America. Europe harbors 82 species, and 57 of these are confined to the Mediterranean region (H. As- pock et al. 1991, 2019, U. Aspock and H. Aspéck 2009, H. Aspock and U. Aspock 2013). Compared to the high number of snakefly species in the Mediterranean parts of Europe, Central Europe har- bors only a moderate Raphidioptera fauna. It comprises Copyright Horst Aspock etal. 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. 250 altogether 15 species: 12 Raphidiidae and 3 Inocelliidae. Six of these species have postglacially expanded their distribution originating from extramediterranean refugial centers and 9 species from Mediterranean refugia. The ranges of most snakeflies in the Mediterranean have not undergone significant expansion but are confined to one of the three peninsulas (Balkan, Apennine, Iberian), and in many cases to small parts only (H. Aspock et al. 2001). Few species have shown considerable expansivity and moved northwards after the last glacial period and colonized Central Europe. Among these are two species which occur on two peninsulas: Ornatoraphidia fla- vilabris (Costa, 1851) and Venustoraphidia nigricollis (Albarda, 1891). Of these, only O. flavilabris (Figs 1, 2) has been recorded from various parts of the Balkan Peninsula, most parts of the Apennine Peninsula (1.e., the southernmost parts to the north) and, moreover, the southern parts of Central Europe (Fig. 3). Since almost all Mediterranean snakeflies are restricted to mostly lim- ited areas on either or both peninsulas, two questions arise. First, do the populations of O. flavilabris belong to a single species? And secondly, where were the Central European populations derived from, the Balkan or Apen- nine Peninsula? To clarify these questions, we repeated classical morphological examinations and comparisons — as performed years ago, and in particular, comple- mented this with a phylogeographic study. Material and methods Morphological examinations Altogether about 75 specimens from 10 localities of the Balkan Peninsula, from 10 localities of the Apennine Peninsula, and from 5 localities in Central Europe were selected for investigation and comparison of characters related to the head, thorax, wings, legs and genitalia of males and females. Molecular analysis Six Ornatoraphidia flavilabris specimens originating from three countries, namely Austria, Italy and Greece (Table 1) were investigated by molecular methods. Tis- sue was removed from legs of adult specimens preserved in ethanol (96%). Table 1. Specimens of Ornatoraphidia flavilabris used in the molecular analysis. voucher _—_ geographic origin (altitude m a.s.1.) coordinates Oflal Greece, Simos (800 m) 38°31.78'N, 21°49.91'E Ofla2 Greece, Parnon Mountain (958 m) 37°06.56'N, 22°43.77'E Ofla3 Italy, Calabria, Aspromonte (1740 m) 38°09.39'N, 15°55.98'E Ofla4 Italy, Calabria, Sila Grande (1298 m) 39°23.61'N, 16°36.43'E Ofla5 Austria, Lower Austria, Eichkogel (358 m) 48°03.75'N, 16°17.55'E Ofla6 Austria, Lower Austria, Gaming (430m) 47°56.57'N, 15°06.59'E dez.pensoft.net Horst Aspock et al.: Central European O. flavilabris Mapping of O. flavilabris distribution Coordinates or locations of previously published O. fla- vilabris records (Navas 1929, H. Aspock and U. Aspéck 1971, Joost 1973, Kofler 1977, Holzel et al. 1980, Devetak 1984, Gepp 1986, H. Aspock et al. 1991, Szira- ki et al. 1992, Popov 1993, 1997, 2000, Letardi 1994, 2003, 2018, 2021, Letardi and Pantaleoni 1996, Panta- leoni and Letardi 1998, Letardi and Migliaccio 2002, Letardi and Biscaccianti 2007, Badano 2008, Aistleitner and Gruppe 2009, Klokoéovnik et al. 2010, Letardi et al. 2010, Badano and Letardi 2010, Tiller 2013, 2015, Sziraki 2014, Devetak et al. 2015, Devetak and Rausch 2016, Hiermann et al. 2018, Letardi and Scalercio 2018) as well as personal unpublished records (Suppl. materi- al 1) were georeferenced into a distribution map using Quantum GIS 3.4.11 (QGIS Development Team 2019) (Fig. 3). DNA extraction, PCR and sequencing DNA was isolated with a Ql[Aamp DNA Mini Kit (Qia- gen, Hilden, Germany), strictly following the manufac- turer’s instructions. DNA was stored in a final volume of 100 ul in elution buffer at -20 °C. PCR amplification of fragments of three different gene fragments, namely cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 3 (cox3) and 28S rRNA gene (28S), was performed with an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany) con- taining 10 x Reaction Buffer B, 2.5 mM MgCl, 1.6 mM dNTPs, 1 uM primers, 1.25 units DNA polymerase and 1-5 pl DNA. Sterile H,O was added to a final volume of 50 ul. For negative controls microbial DNA free water (Qiagen, Hilden, Germany) was added instead of tem- plate DNA. For cox! a 658 bp fragment was amplified using the primers LCO1490 (5’-GGT CAA ATC ATA AAG ATA TTG G-3’) and HCO2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) published by Folmer et al. (1994) for cox3 also a 658 bp fragment was ampli- fied using the primers Raph-cox3fw (5’-TAG TCC ATG ACC HTT AAC AGG-3’) and cox3-rev (5’-ACA TCA ACA AAA TGT CAA TAT CA-3’) published by Haring and U. Aspoéck (2004). The final 28S sequence was ob- tained by the amplification of overlapping fragments ob- tained by the primer pairs Raph28S-1+ (5’-CAG GGG TAA ACC TGA GAA A-3’)/Raph28S-4- (5’-AGC GCC AGT TCT GCT TAC C-3’) and Raph28S83+ (5’-AGC TTT GGG TAC TTT CAG GA-3’)/Raph28S6- (5’-GGA ATA GGA ACC GGA TTC CC-3’) published by Har- ing et al. (2011). Amplification of all three genes was carried out using the following conditions: 95 °C for 15 min, followed by 35 cycles of 95 °C for 1 min (denatur- ation), 52 °C for 1:30 min (annealing) and 72 °C for 2 min (elongation), followed by a final extension of 72 °C for 10 min. Dtsch. Entomol. Z. 68 (2) 2021, 249-259 Pee seme k PS Se ss Figure 1. Ornatoraphidia flavilabris, male. Austria, Eichkogel, 48°03.75'N, 16°17.55'E, 358 m, 10 May 2021, H. & U. Aspock leg., now in coll. NHMW. (Photo: H. Bruckner, NHMW). Length of forewing: 8.4 mm. Figure 2. Ornatoraphidia flavilabris, female. Greece, Phokis, S Pendayi, 38°34.95'N, 22°03.45'E, 960 m, 31 May 2008, H. & U. Aspock leg., now in coll. NHMW. (Photo: P. Sehnal, NHMW). Length of forewing: 10.5 mm. dez.pensoft.net 252 | Ornatoraphidia flavilabris @ record @ record and analyzed in this study Figure 3. Distribution map of Ornatoraphidia flavilabris. The PCR products were subjected to electrophoresis in 2% agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotium, Inc., CA, USA). For further sequencing, bands were analyzed with a Gel DocTM XR+ Imager (Bio- Rad Laboratories, Inc., CA, USA), cut out from the gel and purified with the IllustraTM GFXTM PCR DNA and Gel Purification Kit (GE Healthcare, Buckinghamshire, UK). Sanger sequencing was performed with a Thermo Fisher Scientific SeqStudio (Thermo Fisher Scientific, MA, USA). Sequences were obtained from both DNA strands and con- sensus sequences were generated in GenDoc 2.7.0. Ob- tained sequences were stored in GenBank (MZ313518.1— MZ313535.1) and compared to available sequences using the Basic Local Alignment Search Tool (BLAST) (https:// blast.ncbi.nlm.nih.gov/Blast.cgi) in GenBank. DNA sequence analysis Obtained sequences were aligned with ClustalX 2.1 (Lar- kin et al. 2007) and manually edited with GeneDoc 2.7.0 (Nicholas 1997) for further analysis. The three included genes (cox/, cox3 and 28S) were analyzed independently. Alignments of cox/ and cox3 both showed a length of 658 bp without any insertions or deletions. Translation to ami- no acid sequences for both genes (cox/ and cox3) showed intact reading frames for all included sequences. 28S se- quences, which are variable in length, were prealigned with ClustalX. Sequence gaps and ambiguously aligned sections were checked and edited manually with GeneDoc. dez.pensoft.net Horst Aspock et al.: Central European O. flavilabris This manual alignment approach was compared to a com- puter approach by Haring et al. (2011) and was considered appropriate for this study. After removal of these sections the final alignment resulted in a length of 1602 bp. Final sequence alignments of all three genes are given in supple- mentary files (Suppl. material 2-4). Pairwise distances Based on the best fit evolutionary model selection, pair- wise Tamura-3-parameter + G (gamma distributed) dis- tances were calculated individually for all three genes in MEGAX (Kumar et al. 2018). Therefore, available sequences of Raphidioptera species were downloaded from GenBank. Available sequences of species belong- ing to the “Phaeostigma clade” proposed by Haring et al. (2011), to which also the genus Ornatoraphidia belongs, were chosen as well as sequences of species belonging to the “Puncha clade” which were used as outgroups for tree calculation (Table 2). Phylogenetic analysis To infer the phylogenetic position of the analyzed O. flavilabris samples, maximum likelihood (ML) analysis using the Tamura 3-parameter + G + I model with four discrete gamma values was applied. Nodal support was evaluated by 1000 bootstrap replicates. Dtsch. Entomol. Z. 68 (2) 2021, 249-259 Table 2. Raphidioptera species included in the phylogenetic analysis. 253 Taxon GenBank accession coxl cox3 28S Dichrostigma Navas, 1909 Dichrostigma flavipes (Stein, 1863) KJ592551.1 HM543286. 1 HM543378. 1 Ornatoraphidia H. Aspick & U. Aspick, 1968 Ornatoraphidia flavilabris (Costa, 1855) Austria (Ofla5) MZ313522.1 MZ313528.1 MZ313534.1 Austria (Ofla6) MZ313523.1 MZ313529.1 MZ313535.1 Greece (Oflal) MZ313518.1 MZ313524.1 MZ313530.1 Greece (Ofla2) MZ313519.1 MZ313525.1 MZ313531.1 Italy (Ofla3) MZ313520.1 MZ313526.1 MZ313532.1 Italy (Ofla4) MZ313521.1 MZ313527.1 MZ313533.1 Italy - HM543306.1 HM543373.1 Italy - HM543307.1 - Italy = HM543308.1 - Parvoraphidia H. Aspick & U. Aspick, 1968 Parvoraphidia microstigma (Stein, 1863) 7 HM543319.1 HM543366. | Phaeostigma Navas, 1909 Phaeostigma notata (Fabricius, 1781) KJ592465.1 = = Puncha Navas, 1915 Puncha ratzeburgi (Brauer, 1876) = HM543321.1 HM543358.1 Subilla Navas, 1916 Subilla confinis (Stephens, 1836) = HM543325.1 HM543375.1 Turcoraphidia H. Aspick & U. Aspick, 1968 Turcoraphidia amara H. Aspock & U. Aspéck, 1964 = HM543328.1 HM543372. 1 Xanthostigma Navas, 1909 Xanthostigma xanthostigma (Schummel, 1832) KJ592580.1 HM543337.1 HM543360. 1 Results Morphological examinations Specimens of O. flavilabris from Greece had already been carefully studied and compared with specimens from Italy and Central Europe by us in the late 1960s, and again in the following decades. These examinations consistently led to the conclusion that constant differenc- es do not exist, and thus all populations were assigned to one species (H. Aspock et al. 1991, 2001). In the course of the present study we again thoroughly examined many specimens from many localities and could only confirm what we had already concluded half a century ago. Thus, on a morphological basis it is impossible to an- swer the question whether the Central European popula- tions (at least those of Lower Austria) can be traced back to migrations from the Apennine Peninsula or from the Balkan Peninsula. Molecular analysis of O. flavilabris specimens Sequences of all six included O. flavilabris specimens were successfully amplified by PCR and sequenced. The sequence length was 658 bp for all cox/ (GenBank ac- cession: MZ313518.1—MZ313523.1) and cox3 (GenBank accession: MZ313524.1— MZ313529.1) sequences. The 28S sequences of specimens originating from Greece had lengths of 1741 bp (Oflal, GenBank: MZ313530.1) and 1749 bp (Ofla2, GenBank: MZ313531.1), those from Austria had lengths of 1763 bp (Ofla5, Gen- Bank: MZ313534.1) and 1765 bp (Ofla6, GenBank: MZ313535.1) and those from Italy had a length of 1763 bp (Ofla3, GenBank: MZ313532.1; Ofla4, GenBank: MZ313533.1). After alignment, cox] sequences showed 96 (96/658; 14.6%) variable positions, of which 81 were parsimo- ny informative. Amino acid sequences of all six O. fla- vilabris specimens showed no differences. The Cox3 se- quences showed 91 (91/658; 13.8%) variable positions, of which 71 were parsimony informative, which resulted in nine differences at amino acid sequence level. The 28S sequences showed 25 (25/1602; 1.6%) variable positions, of which 18 were parsimony informative. Pairwise distance analysis In total, nine cox] sequences of Raphidioptera with a length of 658 bp were analyzed (Table 2). The overall mean pairwise Tamura-3-parameter distance was 16.0%. Pairwise distances between the six O. flavilabris sequenc- es analyzed ranged from 0.0% to 15.2% with a mean dis- tance of 8.4%. While genetic distances between the O. flavilabris specimens (Ofla5, Ofla6) from Austria and the specimens from Italy were 2.8% (Ofla3) and 3.0% (Ofla4) respectively, genetic distances to the specimens from Greece were considerably higher, being 12.9% (Oflal ) and 13.8% (Ofla2), respectively (Table 3). Pairwise distances of O. flavilabris to other included Raphidioptera species ranged from 15.6% to 19.4% (Suppl. material 5). Altogether, 15 cox3 sequences of Raphidioptera with a length of 658 bp were included in the analysis (Table 2). The overall Tamura-3-parameter distance was 12.8%. Pairwise distances between nine analyzed O. flavilabris dez.pensoft.net 254 Horst Aspock et al.: Central European O. flavilabris Table 3. Pairwise Tamura-3-parameter distances (%) of Ornatoraphidia flavilabris based on all three included genes (cox 1/cox3/28S). 1 2 4 5 6 7 8 9 1 O. flavilabris (Eichkogel, Austria)* - 2 O. flavilabris (Gaming, Austria)* 0.0/0.0/0.0 - 3 O. flavilabris (Simos, Greece)* 12.9/11.5/1.1 12.9/11.5/1.1 4 O. flavilabris (Parnon Mt., Greece)* 13.8/12.8/1.5 13.8/12.8/1.5 2.5/3.0/0.5 - 5 O. flavilabris (Calabria, Italy)* 3.0/2.5/0.3 — 3.0/2.5/0.3. 14.3/12.0/1.2 15.2/13.3/1.3 - 6 O. flavilabris (Calabria, Italy)* 2.8/2.5/0.3 2.8/2.5/0.3. 13.9/12.4/1.1 14.8/13.3/1.2 0.5/0.6/0.1 - 7 O. flavilabris (Calabria, Italy)* -/2.5/0.4 -/2.5/0.4 -/12.0/1.3 -/13.3/1.3 -/0.3/0.2— -/0.3/0.1 - 8 O. flavilabris (Emilia-Romagna, Italy)* -/2,8/- -/2.8/- -/11.0/- -/12.1/- -/1.9/- -/1.9/- -/1.9/- - 9 O. flavilabris (Emilia-Romagna, Italy)* -/3,2/- -/2.1/- -/11.4/- -/12.5/- -/2.2/- -/2.2/- -/2.2/- -/0.3/- - *snecimens molecularly analysed in this study, “specimens from GenBank. sequences ranged from 0.0% to 13.3% with a mean dis- tance of 6.0%. Pairwise distances between the O. fla- vilabris specimens from Austria and Italy ranged from 2.5% to 3.2%. Again, much higher pairwise distances were observed between the specimens from Austria with either of those from Greece (11.5% Oflal and 12.8% Ofla2) (Table 3). Pairwise distances of O. flavilabris to other included Raphidioptera species ranged from 13.0% to 21.4% (Suppl. material 6). Overall, 13 28S sequences of Raphidioptera with a length of 1602 bp were included in the analysis (Table 3). The overall mean pairwise intraspecific Tamura-3-pa- rameter distance was 2.9%. Pairwise distances between seven analyzed O. flavilabris sequences ranged from 0.0% to 1.5% with a mean distance of 0.7%. While pair- wise distances between the specimens from Austria and Italy ranged from 0.3% to 0.4%, distances between the specimens from Austria and Greece were 1.1% (Oflal) and 1.5% (Ofla2) (Table 3). Pairwise distances of O. fla- vilabris to other included Raphidioptera species were comparably higher and ranged from 2.6% to 4.7% (Sup- pl. material 7). Phylogenetic analysis All sequences included in pairwise sequence calculations were used for the maximum likelihood analysis. Xantho- stigma xanthostigma and Puncha ratzeburgi belonging to the Puncha clade were used as outgroups with one ex- ception. For cox/, only X. xanthostigma was used as an outgroup, since no other sequence of the Puncha clade was available from GenBank. The cox/ tree resulted in two major clades. Clade 1 included all O. flavilabris sequences, clade 2 comprised Phaeostigma notata and Dichrostigma flavipes (Fig. 4A). Clade 1 was further divided into an O. flavilabris lineage from Austria and a lineage from Italy forming a mono- phyletic group that was well supported by high boot- strap values. Together they represent the sister group of a O. flavilabris lineage from Greece (Fig. 4A). The cox3 tree showed three O. flavilabris \ineages (Austria, Italy and Greece) with high bootstrap support. Again, the lineages from Austria and Italy formed a sis- ter group, and together they were the sister group of the lineage from Greece. In addition, the lineage from Italy dez.pensoft.net was subdivided into two sublineages originating from the Calabria and one from the Emilia-Romagna regions of Italy (Fig. 4B). Parvoraphidia microstigma arose as the sister species of O. flavilabris and P. microstigma + O. flavilabris formed the sister group of Turcoraphidia amara, all together representing a monophyletic group. However, the phylogenetic positions of P. microstigma and 7? amara were only supported by comparably low bootstrap values (Fig. 4B). Also in a recent paper on the phylogeny of Neuropterida based on the transcriptom- ic dataset (Vasilikopoulos et al. 2020) Ornatoraphidia emerged as sister of Parvoraphidia and both being sister of Turcoraphidia. The 28S tree comprised two major clades (Fig. 4C). Clade 1 included all O. flavilabris sequences and was sub- divided into three O. flavilabris lineages (from Austria, Italy and Greece). Clade 2 comprised all other included members of the Phaeostigma group. As already observed in the cox! and cox3 tree, the O. flavilabris lineage from Austria formed the sister group of the lineage from Italy, and together they formed the sister group of the lineage from Greece (Fig. 4C). In the 28S tree, 7’ amara and P. microstigma belonged to clade 2, unlike tree cox3, how- ever, again the bootstrap support was low (Fig. 4C). Discussion Phylogeographic studies have not previously been car- ried out in Raphidioptera — but for one exception: the Mediterranean Raphidia (R.) mediterranea H. Aspock, U. Aspock & Rausch, 1977. Surprisingly in 2013, this spe- cies was found in the yard of an old farmhouse in Upper Austria at an altitude of 800 m thriving at an extraordi- narily high population density (Rausch et al. 2016, H. Aspock et al. 2017). The larvae developed in the straw of the thatched roof of the farmhouse (Gruppe et al. 2017). This Central European record is distinctly isolated from the distribution of R. (R.) mediterranea in Italy, the Bal- kan Peninsula and southeast Europe. We concluded that the species must have colonized the farmhouse in Upper Austria by anthropogenic dispersal. However, where did they come from, the Balkan Peninsula or Italy? A mor- phological comparison of both sexes from various popu- lations of the Balkan Peninsula and Apennine Peninsula to specimens from Upper Austria revealed no charac- Dtsch. Entomol. Z. 68 (2) 2021, 249-259 255 A 98 - MZ313520.1 O. flavilabris (Ofla3) Italy LMZ313521.1 O. flavilabris (Ofla4) 99 I M2Z313522.1 O. flavilabris (Oflas) 9g | M2Z313523.1 O. flavilabris (Ofla6) Austria MZ313518.1 O. flavilabris (Ofla1) 99 L_ 1z313519.1 O. flavilabris (Ofla2) ——— KJ592465.1 Phaeostigma notata | 70 ————— KJ592551.1 Dichrostigma flavipes KJ592580.1 Xanthostigma xanthostigma 0.02 Greece B ee MZ313527.1 O. flavilabris (Ofla4) 631] HM543308.1 O. flavilabris MZ313526.1 O. flavilabris (Ofla3) lr HM543307.1 O. flavilabris | Em.-Rom.. Ital 98. HM543306.1 O. flavilabris eae i MZ313528.1 O. flavilabris (Ofla5) | atta 99° MZ313529.1 O. flavilabris (Ofla6) | »MZ313524.1 O. flavilabris (Ofla‘) | eres | 400 L—— MZ313525.1 O. flavilabris (Ofla2) | HM543319.1 Parvoraphidia microstigma HM543328.1 Turcoraphidia amara —— HM543286.1 Dichrostigma flavipes L————_ M543325.1 Subilla confinis —— HM543321.1 Puncha ratzeburgi 58 I$ A HM543337.1 Xanthostigma xanthostigma Calabria, Italy 89 98 C g2 rf MZ313532.1 O. flavilabris (Ofla3) MZ313533.1 O. flavilabris (Ofla4) HM543373.1 O. flavilabris | )§Mz313534.1 O. flavilabris (OflaS) | Austria 96 IMZ313535.1 O. flavilabris (Ofla6) MZ313530.1 O. flavilabris (Ofla1) | Greece 400 L— MZ313531.1 O. flavilabris (Ofla2) | ——— HM543372.1 Turcoraphidia amara HM543366.1 Parvoraphidia microstigma tly a 62 |__ --——— HM543378.1 Dichrostigma flavipes 61 L___________ }}]543375.1 Subilla confinis HM543358.1 Puncha ratzeburgi 99 |_________ }543360.1 Xanthostigma xanthostigma Italy 0.01 Figure 4. Maximum likelihood trees based on cox/ (A.), cox3 (B.), and 28S (C.) sequences of Raphidioptera species. Puncha rat- zeburgi (except cox/ tree) and Xanthostigma xanthostigma were used as outgroups. Vertical colored bars represent Ornatoraphidia flavilabris sequences originating from Austria (green), Italy (red), and Greece (blue). Em.-Rom.=Emilia-Romagna, Italy. Bootstrap values >50% are shown. dez.pensoft.net 256 teristic differences. Thus a molecular study was carried out which showed homogeneity among all populations. These results led to the conclusion that the dispersal of R. (R.) mediterranea occurred from a primary refugial cen- ter (perhaps somewhere in the south of Greece) in recent times by means of human activity. The phylogeography of Ornatoraphidia flavilabris is quite different. Since there are numerous records of this species in Central Europe, the likelihood of anthropogen- ic dispersal is out of the question. However, a post-glacial natural expansion from Mediterranean refugial centers could have led to the present distribution. If so, the ques- tion arises whether Central Europe was colonized via the Balkan or the Apennine Peninsula. O. flavilabris has been biogeographically characterized as a polycentric Balka- nopontomediterranean-Adriatomediterranean faunal ele- ment (H. Aspock et al. 1991), which means that popula- tions of the species were at least present during the last glacial period (about 100,000 to 10,000 years BP) in the Balkan as well as the Apennine Peninsula. The coloniza- tion pathway was clarified by phylogenomics involving an analysis of three genes (cox/, cox3 and 28S). The study clearly showed that: 1. Greek populations (at least from two far distant lo- calities) form a monophyletic group. 2. Italian populations (at least from two far distant lo- calities) are closely related. 3. The two Greek populations can clearly be differen- tiated from the Italian populations. 4. The two specimens of O. flavilabris from Central Europe (Austria) are identical to each other (with one exception of the 28S rRNA gene sequences) and closely related to the populations from Italy. The conclusion drawn is that postglacial migration to Central Europe had its origin in the Apennine Peninsula. This is astounding since migration from the Balkan Pen- insula would seem to be easier with regard to orography (cf. Schmitt 2020). Nevertheless, O. flavilabris could still have colonized Central Europe from the northwest Balkan Peninsula, if we assume that present-day populations, e.g. in Slovenia, are to be traced back to (possibly postglacial, if not even earlier) migrations from Italy. This assumption is strongly corroborated by the fact that there is a large gap comprising central parts of the Balkan Peninsula between the range of O. flavilabris in the south (Greece, Albania, Macedonia, Bulgaria) and that in the north (Slovenia). Aside from phylogeography, a final taxonomic aspect must be discussed. The molecular results show considera- ble differences between the populations from Greece and those from Italy and Central Europe. Thus, the question arises whether both population groups represent one spe- cies. In the recent past an increasing number of cryptic species has been detected in various groups of arthropods on the basis of molecular differences. However, in these cases, morphological differences could be found that, although slight and often inconspicuous, were constant, dez.pensoft.net Horst Aspock et al.: Central European O. flavilabris and thus would justify the description of a new taxon (species) or re-installment of a species previously regard- ed as a synonym (Ronkay and Huemer 2018). Regard- ing Ornatoraphidia flavilabris the conditions are quite different. We have not found any constant morphological characters which would allow a safe differentiation. In- terestingly, the high observed genetic pairwise distances of Italian and Austrian O. flavilabris mitochondrial gene sequences (coxl and cox3) to both Greek specimens were only marginally lower than those to other included Raphidioptera species, thus, pointing towards the molec- ular identification of a new (cryptic) species. However, mitochondrial genes might not optimally present taxo- nomic or phylogenetic relationships among Raphidiop- tera due to sequence saturation as observed by Haring et al. (2011). While intraspecific 28S genetic distances gen- erally supported the results obtained for cox/ and cox3, all three genes showing low distances between Austrian and Italian specimens and about 3-fold higher distances to Greek specimens, this gene revealed a better separation between O. flavilabris and the other included Raphidiop- tera species. Thus, the 28S ribosomal RNA gene possibly reflects the taxonomic status of O. flavilabris more accu- rately than the two mitochondrial marker genes, thereby justifying our decision of differentiating O. flavilabris into two lineages rather than two (sub)species. A description of new taxa solely on the basis of genom- ic differences is in our opinion — at least in Neuropterida — unjustified. Therefore, we continue to regard all pop- ulations of O. flavilabris as a single — morphologically monotypic — species, which means that there is no reason to differentiate subspecies. Nevertheless, further studies on the genomic and mor- phological characters of O. flavilabris including other populations will be useful to corroborate the conclusions presented here. Moreover, the search for cryptic species (taxa) in Raphidioptera should be enhanced and may yield surprising results. Conclusion The occurrence of Ornatoraphidia flavilabris in Central Europe can be traced to a postglacial migration of the species from the Apennine Peninsula via the northwest Balkan Peninsula. The distribution of O. flavilabris in the south of the Balkan Peninsula represents an isolated refu- gial center and is of no relevance for the migration of the species to extramediterranean parts of Europe. Acknowledgements Hubert Rausch (Scheibbs, Austria) has kindly provid- ed the specimen of O. flavilabris from Gaming (Lower Austria) and has made available several records of O. fla- vilabris from the Balkan Peninsula. Mag. Harald Bruck- ner and Peter Sehnal (Natural History Museum Vienna) Dtsch. Entomol. Z. 68 (2) 2021, 249-259 provided the photographs of adult O. flavilabris. Dr. Dr. John Plant (Madison, Connecticut, USA) critically read the manuscript and polished the English. Grateful thanks to all these colleagues! Sincere thanks also to Dr. Davide Badano (Sapienza University of Rome, Italy), Prof. Dr. Duan Devetak (University of Maribor, Slovenia), and Prof. Dr. Alexi Popov (National Museum of Natural His- tory, Sofia, Bulgaria) for thoroughly reviewing and im- proving the manuscript and to Mag. Dr. Susanne Randolf (Natural History Museum Vienna), Subject Editor, for carefully supervising the review process. 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Zoosystema 37: 581-594. https://dot.org/10.5252/z2015n4a4 Vasilikopoulos A, Misof B, Meusemann K, Lieberz D, Flouri T, Beutel RG, Niehuis O, Wappler T, Rust J, Peters RS, Donath A, Podsiad- lowski L, Mayer C, Bartel D, Bohm A, Liu S, Kapli P, Greve C, Jep- son JE, Liu X, Zhou X, Aspéck H, Aspéck U (2020) An integrative phylogenomic approach to elucidate the evolutionary history and divergence times of Neuropterida (Insecta: Holometabola). BMC Evolutionary Biology 20: e64. https://doi.org/10.1186/s12862-020- 01631-6 Supplementary material 1 Personal unpublished O. flavilabris records used for the distribution map Authors: Horst Aspéck, Ulrike Aspock, Julia Walochnik, Edwin Kniha Data type: locations Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.70814.suppl1 Supplementary material 2 Multiple sequence alignment based on cytochrome c oxidase subunit 1 (cox1) sequences Authors: Horst Aspéck, Ulrike Aspock, Julia Walochnik, Edwin Kniha Data type: Sequence alignment Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.70814.suppl2 Dtsch. Entomol. Z. 68 (2) 2021, 249-259 Supplementary material 3 Multiple sequence alignment based on cytochrome c oxidase subunit 3 (cox3) sequences Authors: Horst Aspock, Ulrike Aspock, Julia Walochnik, Edwin Kniha Data type: Sequence alignment Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/dez.68.70814.suppl3 Supplementary material 4 Multiple sequence alignment based on 28S rRNA gene (28S) sequences Authors: Horst Aspock, Ulrike Aspéck, Julia Walochnik, Edwin Kniha Data type: Sequence alignment Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.70814.suppl4 Supplementary material 5 Pairwise Tamura-3-parameter distances (“%) based on cytochrome c oxidase subunit 1 (cox1) gene sequences Authors: Horst Aspock, Ulrike Aspéck, Julia Walochnik, Edwin Kniha Data type: Pairwise genetic distances Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/dez.68.70814.suppl5 259 Supplementary material 6 Pairwise Tamura-3-parameter distances (“%) based on cytochrome c oxidase subunit 3 (cox3) gene sequences Authors: Horst Aspock, Ulrike Aspock, Julia Walochnik, Edwin Kniha Data type: Pairwise genetic distances Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.70814.suppl6 Supplementary material 7 Pairwise Tamura-3-parameter distances (%) based on 28S rRNA gene (28S) gene sequences Authors: Horst Aspock, Ulrike Aspéck, Julia Walochnik, Edwin Kniha Data type: Pairwise genetic distances Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/dez.68.70814.suppl7 dez.pensoft.net