Biodiversity Data Journal 7: e46663 CD doi: 10.3897/BDJ.7.e46663 open access Research Article Salix transect of Europe: additional leaf beetle (Chrysomelidae) records and insights from chrysomelid DNA barcoding Roy Canty, Enrico Ruzzier®!, Quentin C Cronk’, Diana M Percy! + Natural History Museum, Cromwell Road, SW7 5BD, London, United Kingdom § Universtita degli Studi di Padova, Legnaro (Padova), Italy | Natural History Museum, London, United Kingdom q University of British Columbia, Vancouver, Canada Corresponding author: Diana M Percy (diana.percy@ubc.ca) Academic editor: Flavia Rodrigues Fernandes Received: 17 Sep 2019 | Accepted: 27 Oct 2019 | Published: 04 Nov 2019 Citation: Canty R, Ruzzier E, Cronk QC, Percy DM (2019) Salix transect of Europe: additional leaf beetle (Chrysomelidae) records and insights from chrysomelid DNA barcoding. . Biodiversity Data Journal 7: e46663. https://doi.org/10.3897/BDJ.7.e46663 Abstract Occurrence patterns of chrysomelid beetles (Coleoptera: Chrysomelidae), associated with willow (Salix spp.) at 42 sites across Europe, have previously been described. The sites form a transect from Greece (lat. 38.8 °N) to arctic Norway (lat. 69.7 °N). This paper reports additional records and the results of DNA sequencing in certain genera. Examination of further collections from the transect has added 13 species in the genera Aphthona, Chrysomela, Cryptocephalus, Epitrix, Galerucella (2 spp.), Gonioctena, Phyllotreta (2 spp.), Pachybrachis (3 spp.) and Syneta. We also report the sequencing of the DNA regions cytochrome oxidase 1 (CO1) and cytochrome B (cytB) for a number of samples in the genera Plagiodera, Chrysomela, Gonioctena, Phratora, Galerucella and Crepidodera. The cytB sequences are the first available for some of these taxa. The DNA barcoding largely confirmed previous identifications but allowed a small number of re- assignments between related species. Most notably, however, it was evident that the southernmost material (Greece and Bulgaria) of specimens, previously treated as Crepidodera aurata sens. lat., belonged to a distinctive molecular cluster. Morphological re- examination revealed these to be C. nigricoxis Allard, 1878. This is an example of how © Canty R 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. 2 Canty R et al morphotaxonomy and DNA barcoding can work iteratively to refine identification. Our sequences for C. nigricoxis appear to be the first available for this taxon. Finally, there is little geographic structure evident, even in widely dispersed species. Keywords Salicophagy, salicivorous insects, Salicaceae, Chrysomelidae, DNA barcoding, Europe, megatransect Introduction Since early pleas were made for the routine incorporation of a molecular component to taxonomy (“DNA barcoding”) (Hebert et al. 2003a, Hebert et al. 2003b, Tautz et al. 2003), a large amount of literature has accrued and a very large number of sequences backed by voucher specimens have been deposited in standard databases. It is now well established that, in many animal groups, sequencing mitochondrial cytochrome c oxidase subunit 1 (COI) provides a straightforward way of gaining taxonomic insight. Early concerns about molecular methods being somehow antagonistic to morphological taxonomy have given way to acceptance that molecular and morphological taxonomy are complementary, reciprocally illuminating and iterative processes. As part of a study of lowland willow communities sampled from south to north across Europe, we have previously investigated the occurrence and abundance patterns of chrysomelid beetles (Coleoptera: Chrysomelidae) associated with Salix species (Canty et al. 2016). In this study, large numbers of individual beetles were processed and it was impossible with available resources to perform large numbers of genitalia dissections. For this reason, a broad morphospecies concept was used, identifying to species largely using external morphology. We have now been able to test some of these morphospecies assignments using DNA barcoding. This paper reports the new insights that this offers. We also take the opportunity to report additional chrysomelid records from the transect following examination of additional collections. Material and methods Collecting methods Chrysomelid beetles were collected from willows (Salix spp.) by the authors ER and DP at all sites, as previously described (Canty et al. 2016). Details of the sites and the method of their selection have been given in previous papers (Cronk et al. 2015; Canty et al. 2016). The sample sites formed a megatransect from Greece to arctic Norway (Table 1). All collections are deposited in the Natural History Museum, London (BMNH). Table 1. Basic site details. See Cronk et al. (2015) for further details. SITE# 1 oO ON DO oO FF WwW DN Salix transect of Europe: additional leaf beetle (Chrysomelidae) records ... Country Greece Greece Greece Greece Greece Bulgaria Bulgaria Bulgaria Bulgaria Romania Romania Romania Romania Hungary Hungary Hungary Poland Poland Poland Poland Poland Poland Poland Poland Lithuania Lithuania Latvia Latvia Estonia Estonia Finland Finland Finland Lat N 38.80007 38.902 39.306694 40.032685 41.113317 41.412468 42.165622 42.923989 43.739343 44.260343 44.961981 45.510676 46.518504 46.700744 47.665648 48.374291 49.463447 50.470234 50.673994 51.775039 51.775039 52.69398 53.55483 54.06943 54.92583 55.79557 56.71141 57.74963 58.42257 59.40289 60.27299 61.09965 62.04962 Long E 22.4629 22.31015 22.528323 22.175437 23.273893 23.318609 22.998141 23.810563 23.966755 23.786781 23.190337 22.737225 21.512839 21.31268 21.261768 20.725264 21.697255 22.238372 21.823391 21.1971 21.1971 21.8529 22.30299 23.11745 23.7742 24.56678 24.25162 24.4023 24.44063 24.93577 24.65843 25.6282 26.12369 Alt (m) 37 33 177 534 31 90 392 339 35 81 172 556 102 94 91 148 385 157 141 101 101 128 137 174 Date of collection 21-iv-2015 21-iv-2015 22-iv-2015 22-iv-2015 23-iv-2015 23-iv-2015 24-iv-2015 24-iv-2015 24-iv-2015 25-iv-2015 25-iv-2015 26-iv-2015 26-iv-2015 27-iv-2015 27-iv-2015 28-iv-2015 28-iv-2015 29-iv-2015 29-iv-2015 30-iv-2015 11-vi-2015 12-vi-2015 12-vi-2015 13-vi-2015 13-vi-2015 13-vi-2015 14-vi-2015 14-vi-2015 15-vi-2015 15-vi-2015 16-vi-2015 16-vi-2015 17-vi-2015 4 Canty R et al SITE# Country Lat N Long E Alt (m) Date of collection 33 Finland 63.01589 25.80457 139 17-vi-2015 34 Finland 64.05074 25.52664 91 17-vi-2015 35 Finland 64.61287 25.53805 58 18-vi-2015 36 Finland 65.32835 25.29175 1 18-vi-2015 37 Finland 66.24947 23.8945 51 19-vi-2015 38 Finland 67.21253 24.12629 160 19-vi-2015 39 Finland 67.91183 23.63411 233 19-vi-2015 40 Norway 68.8138 23.26658 374 20-vi-2015 4 Norway 69.72487 23.40581 289 20-vi-2015 42 Norway 70.65234 23.66583 67 21-vi-2015 Specimen examination and analysis Morphological procedures followed those used in Canty et al. (2016). A selected subset of specimens was chosen for sequencing (Table 2). These included specimens deemed to be potentially problematic in the original identifications and samples from widespread and variable species. DNA was extracted from material preserved in 90% ethanol. Sequences of mitochondrial cytochrome oxidase subunit 1 (COl) and cytochrome B (cytB) were obtained following protocols for DNA extraction, polymerase chain reaction (PCR) and sequencing described in Percy et al. (2018) with additional primers used for COI (_CO1490 and HCO2198; Folmer et al. 1994). As numerous COI sequences are available on GenBank, we were able to align our own sequences with previously published ones (Table 3). Aligned sequences were analysed using neighbour-joining (NJ) with uncorrected (p) distances in PAUP* (Swofford 2003). Bootstrap support was obtained using 1000 replicates. Sequences generated as a result of this study are all deposited in GenBank (accession numbers MN629748 - MN629886) (Table 2). Table 2. Samples sequenced in this study, reassignments made, and sequences deposited in GenBank: COI (cytochrome oxidase 1), cytB (cytochrome B). Original species ID Reassignment ID Site col cytB Chrysomela vigintipunctata correct 4 MN629768 MN629838 Chrysomela vigintipunctata correct 4 MN629769 MN629839 Chrysomela vigintipunctata correct 11 MN629770 MN629840 Chrysomela vigintipunctata correct 16 MN629771 MN6298341 Chrysomela vigintipunctata correct 21 MN629772 MN629842 Crepidodera aurata Crepidodera nigricoxis 3 MN629760 MN629830 Crepidodera aurata Crepidodera nigricoxis 4 MN629762 MN629832 Crepidodera aurata Crepidodera nigricoxis 4 MN629763 MN629833 Salix transect of Europe: additional leaf beetle (Chrysomelidae) records ... Original species ID Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera aurata Crepidodera fulvicornis Crepidodera fulvicornis (a) Crepidodera fulvicornis (b) Crepidodera fulvicornis (c) Crepidodera fulvicornis Crepidodera fulvicornis Crepidodera fulvicornis Crepidodera fulvicornis Crepidodera plutus Crepidodera plutus Crepidodera plutus Crepidodera plutus Crepidodera plutus Crepidodera plutus Reassignment ID Crepidodera nigricoxis Crepidodera nigricoxis Crepidodera nigricoxis Crepidodera nigricoxis Crepidodera nigricoxis Crepidodera nigricoxis correct correct correct correct correct correct correct correct correct correct correct correct correct correct Crepidodera fulvicornis Crepidodera fulvicornis correct correct correct correct correct correct correct correct correct correct correct correct correct correct 2 o onnaoanvnoanoanvno vn von wo on nN NN fF HF F col cytB MN629764 MN629834 MN629765 MN629835 MN629773 MN629843 MN629761 MN629831 MN629766 MN629836 MN629767 MN629837 MN629759 MN629829 MN629749 MN629819 MN629750 MN629820 MN629751 MN629821 MN629752 MN629822 MN629753 MN629823 MN629754 MN629824 MN629755 MN629825 MN629756 MN629826 MN629757 MN629827 MN629758 MN629828 MN629774 MN629844 MN629775 MN629845 MN629776 MN629846 / MN629847 MN629777 MN629848 MN629778 / MN629779 / MN629780 MN629849 MN629781 MN629850 MN629782 MN629851 MN629783 MN629852 MN629784 MN629853 MN629785 MN629854 MN629748 MN629818 MN629786 MN629855 MN629787 MN629856 MN629788 MN629857 MN629789 MN629858 MN629790 MN629859 6 Canty R et al Original species ID Reassignment ID Site col cytB Crepidodera plutus correct 21 MN629791 MN629860 Galerucella lineola correct 7 MN629792 MN629861 Galerucella lineola correct 11 MN629793 MN629862 Galerucella lineola correct 19 MN629794 MN629863 Galerucella lineola correct 26 MN629795 MN629864 Galerucella lineola correct 34 MN629796 MN629865 Galerucella lineola correct 39 MN629797 MN629866 Gonioctena pallida correct 32 MN629798 MN629867 Gonioctena pallida correct 34 MN629799 MN629868 Gonioctena pallida correct 35 MN629800 MN629869 Gonioctena pallida correct 37 MN629801 MN629870 Gonioctena pallida correct 39 MN629802 MN629871 Gonioctena pallida correct 41 MN629803 MN629872 Phratora vitellinae Phratora polaris £ MN629804 MN629873 Phratora vitellinae Phratora vulgatissima 15 MN629805 MN629874 Phratora vitellinae Phratora polaris 20 MN629806 MN629875 Phratora vitellinae Phratora polaris 26 MN629807 MN629876 Phratora vitellinae correct 32 MN629808 MN629877 Phratora vitellinae correct 4 MN629809 MN629878 Plagiodera versicolora correct 6 MN629810 MN629879 Plagiodera versicolora correct 12 MN629811 MN629880 Plagiodera versicolora correct 16 MN629812 MN629881 Plagiodera versicolora (a) correct 20 MN629813 MN629882 Plagiodera versicolora (b) correct 20 MN629814 MN629883 Plagiodera versicolora (c) correct 20 MN629815 MN629884 Plagiodera versicolora correct ce) MN629816 MN629885 Plagiodera versicolora correct 39 MN629817 MN629886 Table 3. GenBank sequences included in the phylogenetic analysis. The sample in bold under Phratora polaris was downloaded from GenBank as P. tibialis. Species (Chrysomelidae) GenBank Accession numbers Chrysomela vigintipunctata AY027624,KM451318, KM443123, JNO87422, KU188452, KM443640, KJ961764, KM4 43492 Crepidodera aurata KJ966066, KJ962544, KF654801, KF656415, KF654798, KJ963892, KM450642, KM4458 73, KM448484, KM445803 Salix transect of Europe: additional leaf beetle (Chrysomelidae) records ... 7 Species (Chrysomelidae) GenBank Accession numbers Crepidodera aureola KF655591, KF655792, KF655954, KF652694, KF652646 Crepidodera browni KR487413, KR481606, KR490696 Crepidodera fulvicornis KF656356, KM448864, KF656033, KF656133, KF656534, KF656533, KF655283, KJ9632 38, KJ964506, KJ962307 Crepidodera heikertingeri KR487651, KT608408, KT608832 Crepidodera plutus KM452345, KM441553 Crepidodera sculpturata KR486405 Crepidodera sp. KM849066, KR490063, KR483107, KR483276, KM845706 Galerucella lineola KJ963510, KF652931, KC336454, KJ966162, KC336452, KF652986, KF652930, KM4399 94 Galerucinae sp. KR485283, KR487847 Gonioctena pallida FJ346952, FJ346941, FJ346950, FJ346944, KJ962854, FJ346935, FJ346934, FJ346975, FJ346931, FJ346859 Phratora atrovirens KJ965539 Phratora frosti KM841607, KM846081, KR119812 Phratora polaris KJ965979, KM449319, KJ963698, KM442534, KM848244, KJ967261 Phratora purpurea KM845219, KR481952, KM845523 Phratora vitellinae KM443624, KJ963556, KJ963944, KM447598, KF656305 Phratora vulgatissima KJ962797, KF656615, KF656399, KM445038, KM442140 Plagiodera versicolora KR480773, KR483766, KM439446, KJ962066, KF656648, KF652968, KF652966, KF656 252, KF656237 Results Taxonomic insights from molecular barcoding We used DNA sequencing to test and, if necessary, refine our morphospecies assignments made previously (Canty et al. 2016). Generally, the barcoding results confirmed the morphospecies assignments and provide well-supported species clusters (Figs 1, 2). However, the Chrysomelidae barcoding analysis revealed that some specimens were incorrectly assigned in Canty et al. (2016) (Table 2; Fig. 2). These were all due to using broad morphospecies concepts for Phratora vitellinae (Linnaeus, 1758) and Crepidodera aurata Marsham, 1802. In Phratora, three specimens assigned to Phratora vitellinae clustered in the barcoding data with sequences identified on GenBank as P. polaris Schneider, 1886; and one specimen assigned to Phratora vitellinae clustered with 8 Canty R et al GenBank sequences of P. vulgatissima (Linnaeus, 1758). In Crepidodera, two specimens assigned to Crepidodera aurata clustered with GenBank sequences, plus our own sequences, for C. fulvicornis Fabricius, 1792. oof L a | Gonioctena pallida 11 tae 21 Chrysomela vigintipunctata 16 4 100 zi} ji 2s] Phratora polaris oft 10°F 32] Phratora vitellinae 98 15 Phratora vulgatissima 90850 100 o. Plagiodera versicolora Crepidodera aurata 99,9 pac 8 92 99) 4 4 3 100) 7 " bnew: 100 Z Crepidodera nigricoxis loo) 4 4 4 100 23 23 33* 16 23 i ic je 100 7 Crepidodera fulvicornis 39 35 39 31 11 14 9 100 S Crepidodera plutus 13 19 7 92g 74 100 . a6 Galerucella lineola — 0.005 substituionsisite Figure 1. doi DNA analysis (NJ tree) using COI and cytB sequences generated in this study. Node support shown only for nodes 2 90% bootstrap support. In addition, we noted that certain specimens assigned to Crepidodera aurata formed a distinct molecular cluster, distinct from our own C. aurata sequences and from all others downloaded from GenBank. These specimens were the southernmost specimens of our C. aurata from sites 3 and 4 (Greece) and site 7 (Bulgaria). This prompted a morphological re- examination of these samples, including dissections of genitalia and these specimens were identified with C. nigricoxis Allard, 1878 (Fig. 3; Table 2). The two species are very similar in external morphology and variable (Fig. 3). Nevertheless, the molecular data clearly separates them (Figs 1, 2). Our sequences for C. nigricoxis appear to be the first to be made available for this taxon. Gavrilovié and Curéié (2013) note that C. nigricoxis is found on Salix alba L. Although we did not distinguish willow species at the point of collection, Salix alba was present at all the sites where we recorded C. nigricoxis (Cronk et al. 2015). Salix transect of Europe: additional leaf beetle (Chrysomelidae) records ... i2 Plagiodera versicolora 100-3 (transect: 0.3%) 100 52h Chrysomela vigintipunctata 16, (transect: 0.7%) 3234 , ! 41 Gonioctena pallida fae (transect: 1.2%) 39 37, 26 7 ~ 6 20 | Phratora polarigtransect: 0.5%) 1008] Phratora frosti ?59 Phratora atrovirens 98 Phratora vulgatissima 100 100 100 ($ ] Phratora purpurea le 41 100 32 ] Phratora vitellindéransect: 0.2%) 100 39 19 737° Galerucella lineol&transect: 1.5%) 100 34 J 23 Crepidodera fulvicornis 100] F285 (transect: 1.8%) 1 Crepidodera plutus 100 11 (transect: 0.5%) 100, 100-43 ] Crepidodera browni og 93 Crepidedera sp . . cs |] Crepidodera heikertingeri *O0 Crepidodera sculpturata 100 — , | Crepidodera sp 8, | Crepidodera aurata too] 48 (transect: 1.8%) 100 f in 7 100 | Crepidodera aureola 43 47 | Crepidodera nigricox{éransect: 1.5%) 7 Galerucinae indet — _ 0.005 substitutions/site Figure 2. | doi DNA barcoding analysis using COI sequences generated in this study and from GenBank. Sequences from this study show the site number and those obtained from GenBank are indicated by a black circle (GenBank accessions given in Table 3). Node support shown only for nodes > 90% bootstrap support. Maximum intraspecific divergences are shown (for our transect samples only), estimated using uncorrected (p) distances (see methods). 10 Canty R et al Crepidoderaaurata i 60 GOH 090 Foe Crepidodera nigricoxis ‘c= oC60CaE HG Crepidodera plutus | 6 i - SEN Figure 3. | doi Comparative figure of similar species in the genus Crepidodera Dejean, 1836 species, showing size and colour variation of Crepidodera aurata Marsham, 1802 and C. nigricoxis Allard, 1878, with an example of Crepidodera plutus (Latreille, 1804) for comparison. Site number given for each individual. Scale bars whole insect = 2 mm, aedeagus = 0.5 mm. DNA barcoding clearly distinguishes the species. Finally, our analysis indicates that a specimen from GenBank (KM442534.1: voucher GBOL_Col_FK_7108), identified as Phratora tibialis (Suffrian, 1851), may in fact be P. polaris (Table 3; Fig. 2). Phylogeographic patterns There is little phylogeographic structure evident from the sequence data, even for widely dispersed taxa along the transect. Fig. 2 (COI data) is suggestive of a split in Crepidodera fulvicornis between northern samples (Finland: 31, 35, 39) in one clade and southern samples (Hungary: 16, Poland: 23, Latvia: 27) in the other (e.g. a Zoogeographic boundary around Estonia or the Gulf of Finland), but one sample from Finland (site 33) that only sequenced for cytB (Fig. 1) clusters with the southern clade. The absence of clear phylogeographic patterns in the chrysomelids is similar to our findings for curculionids (Canty et al. in review), but differs from those found in a hemipteran taxon (the nettle psyllid; Psylloidea, Hemiptera) sampled along the transect in which population structure suggests distinct regional clades (Wonglersak et al. 2017). Additional chrysomelid records from the transect Since the publication of Canty et al. (2016), examination of additional material from general collections by DP over the transect has brought to light some further records (all single individuals per site, unless otherwise stated). The additional records are: Aphthona cf. lutescens (Gyllenhal, 1808) (site 22); Chrysomela lapponica Linnaeus, 1758 (site 40 and also in supplementary site ii-I [site details in Cronk et al. 2015]); Cryptocephalus ocellatus Salix transect of Europe: additional leaf beetle (Chrysomelidae) records ... 11 Drapiez, 1819 (site 20a); Epitrix sp. (site 22 - two individuals); Galerucella cf. nymphaeae (Linnaeus, 1758) (site 37); Galerucella cf. sagittariae (Gyllenhal, 1813) (site 38); Gonioctena cf. olivacea (Forster, 1771) (site 39); Phyllotreta cf. vittula (Redtenbacher, 1849) (site 24); Phyllotreta undulata (Kutschera, 1860) (sites 27, 30); Pachybrachis hieroglyphicus Laicharting, 1781 (site 20a); Pachybrachis sp. (site 20); Pachybrachis cf. Salfii Burlini, 1956 (site 31) ; and Syneta sp. (site 35). Some of these are not generally associated with willows and are probably accidental by-catch (e.g. Galerucella nymphaeae and Galerucella sagittariae). These additional records do not materially change the basic data or conclusions of Canty et al. (2016), but bring the total number of species to 47 (not 34). Discussion The barcoding, described here, provides a good example of the value of iterative molecular and morphological processes in taxonomy. In this case, a broad morphospecies concept allowed determination of those species that have the greatest geographic and morphological variation. These could then be targeted for barcoding to determine patterns of molecular variation. In the case of Crepidodera aurata sens. lat. this led to the distinguishing of two divergent molecular clusters. This in turn led to a re-appraisal of the morphology and to the refinement of the concept of C. aurata and the recognition of C. nigricoxis as its apparent replacement (at least in our sampling) in southern Europe (Greece and Balkans). This very small example thus serves to emphasise that morphological and molecular taxonomy, taken together and applied iteratively, are powerful adjuncts. Acknowledgements Funding for the fieldwork was partly provided by the Natural History Museum (London, UK) Life Sciences Departmental Investment Fund (SDF 13010) to DMP. We thank Gavin Broad (NHM) for advice and help in the field. Author contributions RC identified and analysed the beetles, extracted DNA and contributed to the writing of the paper; ER collected the beetles and contributed to the writing of the paper; QC co-wrote the paper and contributed to the analysis and planning of the work; DP contributed to the collection of beetles, co-wrote the paper, analysed the molecular data, planned and directed the work and obtained funding for the study. Conflicts of interest None 12 Canty R et al References . Canty R, Ruzzier E, Cronk Q, Percy D (2016) Salix transect of Europe: patterns in the most abundant chrysomelid beetle (Coleoptera: Chrysomelidae) herbivores of willow from Greece to Arctic Norway. Biodiversity Data Journal 4: e10194. https://doi.org/10.3897/ BDJ.4.e10194 . Cronk Q, Ruzzier E, Belyaeva |, Percy D (2015) Salix transect of Europe: latitudinal patterns in willow diversity from Greece to arctic Norway. Biodiversity Data Journal 3: e6258. https://doi.org/10.3897/bdj.3.e6258 . 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