Full details of the sites and their selection have been given previously (Cronk et al. 2015). Briefly the route from Greece to Arctic Norway was driven in 2015, stopping approximately every 100km to locate and sample a stand of willows (Salix spp.) (Table 1).

Willow associated beetles were collected at every site. A sweep net was used with an attempt to sample from all the taxa of willows present at a site. Willows commonest at a site were sampled more. Sampling duration was approximately 1 hour per site. An attempt was made to separate collections from each species of willow, but as field identification of willows is often difficult and complicated by hybridization this was not always possible. For the purposes of this paper all samples at a site are pooled. The willows at each site and voucher herbarium specimens are given elsewhere (Cronk et al. 2015). Beetle samples were immediately transferred, in the field, into tubes containing 70% alcohol. Alcohol preserved material was then kept at ambient temperature and transferred to the NHM (London) for subsequent sorting. As collecting efficiency may be influenced by environmental conditions the time of day, relative humidity (rH) and temperature (t°C) were also recorded for each site (Table 2; Fig. 1). Relative humidity and temperature were recorded using a Hyelec MS6508 thermohygrometer.

Figure 1.  

Collecting conditions (temperature and relative humidity) at the sites (data plotted from Table 2). In this graph lines of constant absolute humidity (AH; g/m³) and vapour pressure deficit (VPD; kPa) are plotted as dashed lines. VPD is a measure of the drying power of the air. Circles (red line) mark collection localities 1-20 (April 2015) while the triangles (blue line) mark sites 21-41 (June 2015). Note that the environmental conditions during collection are very similar between Central Europe (sites 18-20) in April and Arctic Europe (sites 30-41) in June.

Specimens from each locality were sorted into broad morphospecies, identified and counted. Identifications were made by RC. Most morphospecies likely correspond to biological species. The following works and resources were consulted for the identification of taxa: Hubble (2012); Borowiec (2013); Warchałowski (2003); Warchałowski (2010); Lompe (2002); Watford Coleoptera Group (2016); and the species list of Volf et al. (2015). Some of the most abundant species (Crepidodera aurata Marsham, 1802, C. fulvicornis (Fabricius, 1792), C. plutus (Latreille, 1804), Phratora vitellinae (Linnaeus, 1758), Galerucella lineola (Fabricius, 1781), Plagiodera versicolora (Laicharting, 1781), Chrysomela vigintipunctata Scopoli, 1763 and Gonioctena pallida (Linnaeus, 1758)) were subsampled (one to three individuals per sample from 6 samples per species) from selected localities for imaging and measurement. Measurements were performed using a Zeiss Stemi DV4 dissecting scope and a Minitool miniature measuring scale with a 5mm range calibrated to 0.1mm. Colours were determined by matching to the standard RHS colour chart (RHS 2001). Colour codes were translated to colour names using standard practice (UPOV 2013). Photographs were taken using a Canon EOS 700D, viewing through a Leica MZ12.5 stereomicroscope. Photos were taken via a Dell computer using the Canon EOS 700D Utility Remote Live View programme to take several photos of each specimen at different focus distances. These photos were then combined together to form a fully focused image using the focus stacking software Helicon Focus (version 5.3).

The inter-site latitudinal variation in occurrence of the eight commonest species (Table 4) was examined using Canonical Principal Components Analysis (Redundancy Analysis), with latitude as the explanatory variable. The beetle matrix of counts of individuals (Table 4) was square root transformed to normalise. The beetle matrix was used as the response matrix. Redundancy analysis was performed using the Java package Ginkgo in the software suite B-VegAna (Font Castell 2006).

The list of species encountered is given in Table 3. The sites along the transect yielded 34 morphospecies of willow-associated chrysomelid. The most widespread and abundant of these was C. aurata, which occured at 27 out of 41 sites and >267 individuals were captured in our samples. In all, eight morphospecies were common, occurring at eight or more sites and in considerable abundance (Table 3). The remaining 26 morphospecies were comparatively sparsely distributed with 11 being found at a single site only. The eight common species contributed 1164 counted individuals in our samples. The remaining 26 morphospecies contributed only a further 128 counted individuals (Table 3). Most of the species are known willow-feeders. However, some species taken from willow are commonly recorded as feeding exclusively on other types of plant (Böhme 2001): Donacia aquatica (Carex spp.), D. simplex (Sparganium spp.) and Chrysolina graminis (Asteraceae). Nevertheless they are included here as willow-associated, and examples of beetles that may be taken as by-catch when sampling willows. Site descriptions (Cronk et al. 2015) for sites 29 and 38, where Donacia is present, indicate their suitability (as wetland sites) for these species.

Chrysomelids were rare in Greece and were absent from two Greek sites sampled (2 & 5). However, they were generally abundant at all other sites (from Bulgaria to Norway) (Table 4). The mean number of captured and counted individuals at Greek sites was 2.2 with a range of 0-7. For the remaining sites the mean was 35.6 (range 5-90) (Table 4).

In terms of number of morphospecies per site, Greek sites had an average of 1 species (range 0-2) and the other sites an average of 4.7 species (range 2-12) (Table 3). All sites together had an average number of species per site of 4.2.

The commonest species and their site distributions are detailed in Table 4. The species showed clear evidence of geographical patterning (Table 4). Of the eight abundant species, three were very widespread along the transect (C. aurata, P. vitellinae and G. lineola). Two had northern-biased distributions (C. fulvicornis and Gonioctena pallida) and three southern-biased distributions (P. versicolora, C. vigintipunctata and C. plutus).

Redundancy analysis showed that variation in occurrence of chrysomelids (common species) was, as expected, highly correlated with latitude. Latitude was able to explain 23.2% of the total variance in the beetle matrix. When the latitude input order was randomized multiple times, latitude was only able to explain around 2% of the variance by chance alone (mean=2.26%, standard deviation = 0.71). However this correlation with latitude is mainly due to (1) the paucity chrysomelids at the southernmost sites (Greece) and (2) the difference between a distinctly boreal chrysomelid fauna north of Poland contrasting with a rather homogeneous central European fauna from Bulgaria to Poland (sites 6 to 23). When sites 6 to 23 are analyzed separately there is little association with latitude (6.8%) and this is not much better than random (random: 3.66%, SD 1.36).

We noted considerable variation in colour and size of the common beetles from population to population but within populations they tended to be fairly homogeneous. All the common species displayed great chromatic variation (Table 5; Fig. 2; Fig. 3) with the exception of G. lineola. In this species no variation in colour was detectable by the human eye. Species also differed in their size variation: most were quite variable between populations but the three species of Crepidodera were comparatively invariant in size (Table 5).

Figure 2.  

Images of representative examples of common species from different populations. Chrysomela vigintipunctata, Crepidodera aurata, C. fulvicornis, C. plutus. Populations are referred to a map (left). Scale bars = 1 mm.

Figure 3.  

Images of representative examples of common species from different populations. Galerucella lineola, Gonioctena pallida, Phratora vitellinae, Plagiodera versicolora. Populations are referred to a map (left). Scale bars = 1 mm.

The distribution and abundance of chrysomelids does not just vary geographically. These beetles are well known for temporal variation, both phenological (timing of appearance), population build-up during a year and interannual (year to year) variation driven by episodic outbreaks and population control by parasites and predators. The variation between willow stands, and across Europe will reflect both spatial and temporal patterns. Nevertheless, our “snapshot” of variation gives a clear idea of the variation across Europe to be encountered in a particular year. It also provides the possibility for follow-up specifically to quantify temporal variation. Another advantage of collecting along a geographically wide megatransect is that a full picture of morphological variation within a species is gained (as summarized in Table 5). Biogeographical work in central Europe (Schmitt and Rönn 2011) characterized Crepidodera fulvicornis as "widely distributed", while Gonioctena pallida and Phratora vulgatissima were characterized as "southern", on the basis of 63,000 records. The differences in biogeographical pattern reported here could be due to the "snapshot effect" or simply to the different (more easterly) region being examined. Further work will be needed to distinguish these two hypotheses.

It is clear that our sampling reveals a considerable difference between Greece and Bulgaria. This may reflect the comparative rarity of willows in the strongly anthropogenically disturbed and dry Mediterranean climate of Greece, which would deny willow-associated beetles the ready access to this food-plant resource that they have over the rest of Europe. Another possible explanation is that the paucity of Salix-associated chrysomelids in Greece in 2015 is the consequence of phenology or interannual variation (the spring was noted to have been exceptionally warm in Greece in 2015).

Another potential distributional breakpoint we note is around site 23 (northern Poland) which appears to mark a division between the southern-biased common species which end around here (at sites 20-25) and the northern-biased species C. fulvicornis which comes in strongly at site 25 (admittedly with southern outliers to site 11). The other northern-biased species, Gonioctena pallida, does not fit the pattern so well, coming in at site 32 (Finland). However this may be due to our late timing of collection with respect to what is clearly a more cryophilous beetle. Generally, the apparent transition point in northern Poland may reflect a genuine biogeographical shift or may simply reflect the particular circumstances of phenology and collection time.

Although this transect was north-south in orientation, the effect of east-west biogeographical boundaries can be seen in the comparative rarity of P. vulgatissima (3 sites only). This beetle is sometimes stated to be the commonest willow-associated chrysomelid in Europe (as also implied by its Linnaean epithet) so it might appear odd that it was not more abundant in our samples. However it is a species primarily of NW Europe, being particularly abundant in Sweden and Germany westwards to the UK and Norway. Our transect goes through the eastern edge of its range so the comparative rarity in our samples in not surprising.