Where did the Central European populations of Ornatoraphidia flavilabris (Costa) come from? (Neuropterida, Raphidioptera, Raphidiidae)

Ornatoraphidia flavilabris (Costa, 1851) is one of 15 snakefly species occurring in southern parts of Central Europe. It is a polycentric 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 uncovered which could serve to differentiate specimens from either distribution center. However, differences were detected in cox1, 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 Europe, 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 Apennine 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.

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. Aspöck 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 flavilabris (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 (i.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 limited 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 Apennine Peninsula? To clarify these questions, we repeated classical morphological examinations and comparisons -as performed years ago, and in particular, complemented this with a phylogeographic study.

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. Tissue was removed from legs of adult specimens preserved in ethanol (96%).
DNA extraction, PCR and sequencing DNA was isolated with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), strictly following the manufacturer's instructions. DNA was stored in a final volume of 100 μl in elution buffer at -20 °C.
Sanger sequencing was performed with a Thermo Fisher Scientific SeqStudio (Thermo Fisher Scientific, MA, USA). Sequences were obtained from both DNA strands and consensus sequences were generated in GenDoc 2.7.0. Obtained 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 (Larkin et al. 2007) and manually edited with GeneDoc 2.7.0 (Nicholas 1997) for further analysis. The three included genes (cox1, cox3 and 28S) were analyzed independently. Alignments of cox1 and cox3 both showed a length of 658 bp without any insertions or deletions. Translation to amino acid sequences for both genes (cox1 and cox3) showed intact reading frames for all included sequences. 28S sequences, which are variable in length, were prealigned with ClustalX. Sequence gaps and ambiguously aligned sections were checked and edited manually with GeneDoc. This manual alignment approach was compared to a computer 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 supplementary files (Suppl. material 2-4).

Pairwise distances
Based on the best fit evolutionary model selection, pairwise Tamura-3-parameter + G (gamma distributed) distances 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 belonging 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.

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 differences do not exist, and thus all populations were assigned to one species (H. Aspöck et al. 1991Aspöck et al. , 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 answer the question whether the Central European populations (at least those of Lower Austria) can be traced back to migrations from the Apennine Peninsula or from the Balkan Peninsula. After alignment, cox1 sequences showed 96 (96/658; 14.6%) variable positions, of which 81 were parsimony informative. Amino acid sequences of all six O. flavilabris specimens showed no differences. The Cox3 sequences 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 cox1 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 sequences analyzed ranged from 0.0% to 15.2% with a mean distance 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% (Ofla1) 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 sequences ranged from 0.0% to 13.3% with a mean distance of 6.0%. Pairwise distances between the O. flavilabris 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% Ofla1 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-parameter 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 pairwise 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% (Ofla1) and 1.5% (Ofla2) ( Table 3). Pairwise distances of O. flavilabris to other included Raphidioptera species were comparably higher and ranged from 2.6% to 4.7% (Suppl. material 7).

Phylogenetic analysis
All sequences included in pairwise sequence calculations were used for the maximum likelihood analysis. Xanthostigma xanthostigma and Puncha ratzeburgi belonging to the Puncha clade were used as outgroups with one exception. For cox1, only X. xanthostigma was used as an outgroup, since no other sequence of the Puncha clade was available from GenBank.
The cox1 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 monophyletic group that was well supported by high bootstrap values. Together they represent the sister group of a O. flavilabris lineage from Greece (Fig. 4A).
The cox3 tree showed three O. flavilabris lineages (Austria, Italy and Greece) with high bootstrap support. Again, the lineages from Austria and Italy formed a sister group, and together they were the sister group of the lineage from Greece. In addition, the lineage from Italy 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 T. amara were only supported by comparably low bootstrap values (Fig. 4B). Also in a recent paper on the phylogeny of Neuropterida based on the transcriptomic 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 subdivided 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 cox1 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, T. amara and P. microstigma belonged to clade 2, unlike tree cox3, however, again the bootstrap support was low (Fig. 4C).

Discussion
Phylogeographic studies have not previously been carried out in Raphidioptera -but for one exception: the Mediterranean Raphidia (R.) mediterranea H. Aspöck, U. Aspöck & Rausch, 1977. Surprisingly in 2013, this species was found in the yard of an old farmhouse in Upper Austria at an altitude of 800 m thriving at an extraordinarily high population density . The larvae developed in the straw of the thatched roof of the farmhouse ). This Central European record is distinctly isolated from the distribution of R. (R.) mediterranea in Italy, the Balkan 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 morphological comparison of both sexes from various populations of the Balkan Peninsula and Apennine Peninsula to specimens from Upper Austria revealed no charac- Table 3. Pairwise Tamura-3-parameter distances (%) of Ornatoraphidia flavilabris based on all three included genes (cox1/cox3/28S).  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 center (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 anthropogenic 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 question arises whether Central Europe was colonized via the Balkan or the Apennine Peninsula. O. flavilabris has been biogeographically characterized as a polycentric Balkanopontomediterranean-Adriatomediterranean faunal element (H. Aspöck et al. 1991), which means that populations 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 colonization pathway was clarified by phylogenomics involving an analysis of three genes (cox1, cox3 and 28S). The study clearly showed that: 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 Peninsula 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 considerable differences between the populations from Greece and those from Italy and Central Europe. Thus, the question arises whether both population groups represent one species. 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, and thus would justify the description of a new taxon (species) or re-installment of a species previously regarded as a synonym (Ronkay and Huemer 2018). Regarding Ornatoraphidia flavilabris the conditions are quite different. We have not found any constant morphological characters which would allow a safe differentiation. Interestingly, the high observed genetic pairwise distances of Italian and Austrian O. flavilabris mitochondrial gene sequences (cox1 and cox3) to both Greek specimens were only marginally lower than those to other included Raphidioptera species, thus, pointing towards the molecular identification of a new (cryptic) species. However, mitochondrial genes might not optimally present taxonomic or phylogenetic relationships among Raphidioptera due to sequence saturation as observed by Haring et al. (2011). While intraspecific 28S genetic distances generally supported the results obtained for cox1 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 Raphidioptera species. Thus, the 28S ribosomal RNA gene possibly reflects the taxonomic status of O. flavilabris more accurately 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 genomic differences is in our opinion -at least in Neuropterida -unjustified. Therefore, we continue to regard all populations 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 morphological 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 refugial center and is of no relevance for the migration of the species to extramediterranean parts of Europe.
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. Dušan Devetak (University of Maribor, Slovenia), and Prof. Dr. Alexi Popov (National Museum of Natural History, Sofia, Bulgaria) for thoroughly reviewing and improving the manuscript and to Mag. Dr. Susanne Randolf (Natural History Museum Vienna), Subject Editor, for carefully supervising the review process. We gratefully acknowledge the Museum für Naturkunde Berlin for waiving the authors' fees.