The Mediterranean snakefly Raphidia mediterranea H. Aspöck, U. Aspöck & Rausch, 1977 – known from many parts of the Balkan Peninsula, several Aegean islands, southern parts of Italy, northwest of Anatolia and a few localities in Eastern Europe, yet not recorded in Central Europe – was surprisingly found with an astoundingly high population density on bushes in the yard of an old farmhouse at a comparatively high altitude (800 m) in Upper Austria, north of the Danube River, in 2013. This spectacular phenomenon was again observed in the following years (2014, 2015, 2016, 2017), and in 2016 the suspicion that the larvae develop in the straw of the thatched roof of the farmhouse could be confirmed by findings of larvae, pupae, and exuviae.It is most likely, that the occurrence of this Raphidia species in Austria is to be traced back to a human-caused introduction at some point in time. It remains, however, unknown when and specifically how this event might have occurred.

Morphologically no substantial differences were found between specimens from Greece, Italy and Upper Austria. In addition, the genetic uniformity (using 3 genes: cox1, cox3, and 28S) of the populations was verified. This supports an earlier hypothesis that the occurrence of the species, as well as that of R. mediterranea in Italy, Anatolia and perhaps elsewhere, may be related to importation of goods involving wood or soil.

A molecular genetic analysis of several Raphidia species confirmed the present morphology-based concept of their systematic position.

The means of dispersal of Raphidioptera are largely unknown. We do not know of any other similar cases of anthropogenic dispersal of a snakefly, but it cannot be excluded that human activities may have played a greater role in the dispersal of Raphidioptera than previously assumed. Phylogenomic studies would therefore be promising to solve some of these questions.

Raphidia mediterranea , man-made dispersal, neozoon, Raphidia , phylogeny

By the 1960s, it was revealed that the Balkan Peninsula was a distribution (and evolution) centre of the order Raphidioptera with an incredibly high number of species (H. Aspöck and U. Aspöck 1965). In the course of the following years, extensive field studies were carried out in various parts of the Balkan Peninsula (H. Aspöck 1987, H. Aspöck et al. 1989, H. Rausch and R. Rausch 2004). These investigations led to the discovery of numerous new species and among them a snakefly species, which – despite slight differences – was morphologically so similar to Raphidia ophiopsis Linnaeus, 1758, that we hesitated to separate it from R. ophiopsis. However, after discovery of unusually large populations of this taxon in various parts of Greece and in biotopes (e.g. in coastal areas with maquis vegetation) ecologically entirely different from those of R. ophiopsis, which is associated with coniferous trees, we decided to describe it as a subspecies of Raphidia ophiopsis: R. ophiopsismediterranea (H. Aspöck et al. 1977). In the meantime, the taxon was surprisingly found in Italy (Apulia) and later in northwest Anatolia. In our monograph (H. Aspöck et al. 1991) we argued that the disjunct distribution could hardly be explained by natural dispersal and we therefore considered that human activities might have been a significant cause of the amplification of the distribution of R. o. mediterranea. One of our arguments was that R. o. mediterranea occurs on the eastern coast of the Apennine Peninsula around Brindisi, a region known for its intensive ship traffic with Greece, which dates back to antiquity. Subsequently, entomologists from Italy found R. o. mediterranea in western parts of the Apennine Peninsula, and from this they concluded that R. o. mediterranea had not been introduced from Greece to Italy by human activities, but that its occurrence in Italy was due to natural dispersal (Letardi 2002, Letardi and Pantaleoni 1996, Pantaleoni 2005). Meanwhile, the taxon was unexpectedly found in Romania (Kis 1984) and Hungary (Sziráki 1993a, b, 2010). Both latter authors studied the taxon carefully and arrived at the conclusion that R. mediterranea is a good species and not a subspecies of R. ophiopsis. Aside from the known and corroborated morphological differences, an important argument for the status of a separate species was the sympatry of both taxa in Romania and Hungary. The arguments of Kis (1984) and Sziráki (1993a) were convincing and accepted by us (H. Aspöck and U. Aspöck 2007, 2013, 2014). Finally, in 2013 R. mediterranea was found in the yard and on the outer walls of an old farmhouse, now representing an open-air museum, at a considerably high altitude (800 m) in Upper Austria (Rausch et al. 2016). It was an absolute surprise to find this Mediterranean snakefly in a comparatively cold region of Austria (Figs 1, 2). Moreover, R. mediterranea occurs there in an extremely high population density. It was suspected that the larvae develop within the straw covering the roof (Rausch et al. 2016), and this could recently be confirmed (Gruppe et al. 2017) (Figs 3, 4). Thus, the question arose: How has R. mediterranea achieved the establishment of a stable population in a locality in Central Europe, which offers unfavourable climatic conditions compared to many other parts of Austria (Fig. 5)? To better evaluate the phylogeographic scenarios of this species, i.e. natural expansion of the distribution range vs. human mediated dispersal, we performed molecular genetic analyses of specimens from Austria, Greece and Italy. The specimens analysed genetically were compared morphologically with specimens from many localities covering the currently known distribution. Moreover, specimens of R. ophiopsis from Upper Austria and other parts of Central Europe were included to corroborate the morphological differences between the two taxa.

Figure 1. 

Raphidia mediterranea, male, from Pelmberg (Upper Austria). Photo H. Bruckner.

Figure 2. 

Raphidia mediterranea, female, from Pelmberg (Upper Austria). Photo H. Bruckner.

Figure 3. 

Raphidia mediterranea, full-grown larva, from Pelmberg (Upper Austria). Photo H. Bruckner.

Figure 4. 

Raphidia mediterranea, pupa, from Pelmberg (Upper Austria). Photo H. Bruckner.

Figure 5. 

Known distribution of Raphidia mediterranea H.A. & U.A. & Rausch. Source of the map see under Material and methods.

In former studies (H. Aspöck et al. 1991 and unpublished), based on male and female genitalia, populations of Raphidia mediterranea from various localities in Greece, Anatolia, and Italy could not be differentiated from each other. This was confirmed again on the basis of more material, particularly specimens from Pelmberg (Upper Austria) whose morphological characters coincide perfectly with those figured in H. Aspöck et al. (1991). Specimens of the genus Raphidia from other parts of Central Europe (except Raphidia ulrikae) proved to be conspecific with Raphidia ophiopsis.

The DNA sequence analysis revealed that the four specimens of R. mediterranea are identical in cox1 and 28S, while in cox3 a single substitution differentiating Ramed-4 from the other (identical) sequences was found. In general, the variation within 28S was extremely low. Except R. ligurica, which shows distances to the other ingroup taxa of 1.08 and 1.35%, respectively, sequences of all other ingroup species differ with p distances below 1% or are even identical. Between Raphidia and Agulla 28S distances ranged from 6.0 to 6.5%. Concerning the mitochondrial marker sequences, p distances between R. mediterranea and R. ophiopsis (the closest relative) were 5.08% in cox1and 5.62% in cox3, while the other species of Raphidia differed between 8.63–14.83% (cox1) and 7.46–16.94% (cox3) from R. mediterranea. Distances between species in the various gene sequences are illustrated by the NJ trees in Suppl. material 1.

To assess the systematic position of R. mediterranea not only on the basis of morphological characters, we performed a phylogenetic analysis based on three genes (cox1, cox3, 28S). The two mt sequences resulted in trees in which the sister group of R. mediterranea is R. ophiopsis. In most analyses, R. alcoholica is the sister group of those two lineages, followed by R. ariadne; only in the BI tree of cox1 the relationships were unresolved (Suppl. material 1). With respect to the relationships of the other species there is a difference concerning R. ligurica and R. ulrikae depending on the marker sequence and the method applied. In some trees R. ulrikae splits from the most basal node, in others it is R. ligurica. Yet, in all trees this node is poorly supported. The tree based on 28S sequences (Suppl. material 1) is congruent with the mt based trees, yet, due to the low variation within this gene, the amount of phylogenetic information is limited. In a BI tree based on the combined marker genes (Fig. 6) all nodes are highly supported.

Figure 6. 

Phylogenetic tree based on BI analysis of six species of Raphidia (with Agulla adnixa as outgroup). Posterior probability values are indicated at the nodes.

The discovery of an isolated and unusually large population of Raphidia mediterranea – a Mediterranean species which has never been found elsewhere in Central Europe – in a farmhouse in a comparatively climatically unfavourable part of Upper Austria had raised the question concerning the origin of this population. It was assumed that morphological and/or genetic differences would be found, if the species had reached Upper Austria long ago by natural means of expansion of the distribution range. Therefore, specimens of the population from Upper Austria were compared with specimens from Greece and Italy. In the present study, the morphology-based results were clearly confirmed by molecular genetic analyses: The four specimens of R. mediterranea (two from Pelmberg (Austria), one from Greece, one from Italy) had almost identical sequences. It is legitimate to conclude that these populations were not separated long ago. The other species of Raphidia are clearly separated (see Fig. 6).

Substantial differences could not be found in morphological characters, particularly in male and female genitalia, or in the sequences of three genes (cox1, cox3, 28S). This implies that all presently known and examined populations of R. mediterranea originated from a single glacial refugium. This refugium can reasonably be assumed to be in the south of the Balkan Peninsula as a part of the large balkanopontomediterranean refugium (H. Aspöck et al. 1991). From there the species reached other parts of Europe (and Anatolia) not long ago. Natural dispersal from the south of the Balkan Peninsula to isolated areas of the north of the Balkan Peninsula, to southern parts of Italy, to parts of Eastern Europe and particularly parts of Central Europe is highly unlikely. Consequently, an anthropogenic introduction into various regions is highly probable. Raphidia mediterranea is a euryoecious species, whose larvae live mainly in the detritus of roots of bushes, but sometimes also under bark.

In Greece, the species occurs in many regions, in various habitats at altitudes of 10 – 1200 m and often in high population densities. Thus, it might have been occasionally transported to new habitats by ships carrying wood or soil. This could have occurred already in antiquity and throughout the past centuries.

The discovery of the isolated population of R. mediterranea in Upper Austria and the failure to detect any morphological or genetic differences between these vastly distant populations supports our previous hypothesis (Aspöck et al. 1977, 1980, 1991, 2001) of unintentional introduction by human activities. A natural dispersal – per continuitatem or by wind – can convincingly be excluded. How did R. mediterranea come to Upper Austria? We now know definitely that the larvae develop within the straw of the roof (Gruppe et al. 2017). Thus, it is a reasonable assumption that this snakefly was introduced with straw from somewhere on the Balkan Peninsula. The straw presently on the roof is from Austria, but in the past it may have been imported. It is also possible that live adults (theoretically one female would be sufficient) were introduced (e.g. via car, truck or bus) from the Mediterranean region to Pelmberg and subsequently the female laid eggs in the straw. Until now, no studies have been published indicating that larvae develop in straw on roofs. In Central Europe thatched roofs have become rare, but in eastern and southeastern parts of Europe such roofs are still frequent in certain regions. It would be easy and exciting to examine these habitats for snakeflies. It would particularly be interesting to know whether other species of Raphidioptera can also develop in straw of thatched roofs where they would feed on mites, spring-tails, Psocoptera, larvae of beetles and other small arthropods living in the straw.

Concerning the systematic position of R. mediterranea, the phylogenetic analysis based on three genes confirmed our view of the systematics of R. ophiopsis, R. mediterranea, R. alcoholica, R. ariadne, R. ulrikae and R. ligurica (H. Aspöck et al. 1991, 2001). It is of particular interest that R. alcoholica is the sister species of R. ophiopsis + R. mediterranea, thus confirming the close relationship of the three taxa once regarded as subspecies of R. ophiopsis.

We know little about the formation of distribution patterns of Raphidioptera. Snakeflies are generally characterised by low, in many cases extremely low, expansivity, and many species have hardly enlarged their distribution beyond their glacial refugial areas. In Central Europe, 16 species of Raphidioptera (13 Raphidiidae and 3 Inocelliidae) occur, 10 of these are of Mediterranean origin and presumably have reached Central Europe after the last glacial period, i.e. within the past 10,000 years (H. Aspöck 2008, 2010, H. Aspöck et al. 1991, 2001, H. Aspöck and U. Aspöck 2015). At least in Austria, R. mediterranea must be regarded as a human introduced neozoon.

Cordial thanks to Mag. Harald Bruckner (NHMW) for the photographs and for preparing the distribution map. The technical assistance of cand. med. Alexandra Szewczyk, BA, (Vienna) is gratefully acknowledged. We thank Prof. Dr. Sven Bacher (Fribourg) and Prof. Dr. Peter Duelli (Zürich) for their constructive reviews. We are very thankful to DDr. John Plant (Guilford, Connecticut) for polishing the English. We gratefully acknowledge the Museum für Naturkunde Berlin for waiving the author’s fees.