Biodiversity Data Journal : Research Article
Print
Research Article
Salix transect of Europe: additional leaf beetle (Chrysomelidae) records and insights from chrysomelid DNA barcoding
expand article infoRoy Canty, Enrico Ruzzier§,|, Quentin C Cronk, Diana M Percy|
‡ Natural History Museum, Cromwell Road, SW7 5BD, London, United Kingdom
§ Universtità degli Studi di Padova, Legnaro (Padova), Italy
| Natural History Museum, London, United Kingdom
¶ University of British Columbia, Vancouver, Canada
Open Access

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 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).

Basic site details. See Cronk et al. (2015) for further details.

SITE#

Country

Lat N

Long E

Alt (m)

Date of collection

1

Greece

38.80007

22.4629

37

21-iv-2015

2

Greece

38.902

22.31015

33

21-iv-2015

3

Greece

39.306694

22.528323

177

22-iv-2015

4

Greece

40.032685

22.175437

534

22-iv-2015

5

Greece

41.113317

23.273893

31

23-iv-2015

6

Bulgaria

41.412468

23.318609

90

23-iv-2015

7

Bulgaria

42.165622

22.998141

392

24-iv-2015

8

Bulgaria

42.923989

23.810563

339

24-iv-2015

9

Bulgaria

43.739343

23.966755

35

24-iv-2015

10

Romania

44.260343

23.786781

81

25-iv-2015

11

Romania

44.961981

23.190337

172

25-iv-2015

12

Romania

45.510676

22.737225

556

26-iv-2015

13

Romania

46.518504

21.512839

102

26-iv-2015

14

Hungary

46.700744

21.31268

94

27-iv-2015

15

Hungary

47.665648

21.261768

91

27-iv-2015

16

Hungary

48.374291

20.725264

148

28-iv-2015

17

Poland

49.463447

21.697255

385

28-iv-2015

18

Poland

50.470234

22.238372

157

29-iv-2015

19

Poland

50.673994

21.823391

141

29-iv-2015

20

Poland

51.775039

21.1971

101

30-iv-2015

20a

Poland

51.775039

21.1971

101

11-vi-2015

21

Poland

52.69398

21.8529

96

12-vi-2015

22

Poland

53.55483

22.30299

128

12-vi-2015

23

Poland

54.06943

23.11745

137

13-vi-2015

24

Lithuania

54.92583

23.7742

28

13-vi-2015

25

Lithuania

55.79557

24.56678

62

13-vi-2015

26

Latvia

56.71141

24.25162

23

14-vi-2015

27

Latvia

57.74963

24.4023

7

14-vi-2015

28

Estonia

58.42257

24.44063

18

15-vi-2015

29

Estonia

59.40289

24.93577

48

15-vi-2015

30

Finland

60.27299

24.65843

33

16-vi-2015

31

Finland

61.09965

25.6282

84

16-vi-2015

32

Finland

62.04962

26.12369

174

17-vi-2015

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

41

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 (COI) 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 (LCO1490 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).

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

COI

cytB

Chrysomela vigintipunctata

correct

4

MN629768

MN629838

Chrysomela vigintipunctata

correct

7

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

Crepidodera aurata

Crepidodera nigricoxis

4

MN629764

MN629834

Crepidodera aurata

Crepidodera nigricoxis

4

MN629765

MN629835

Crepidodera aurata

Crepidodera nigricoxis

4

MN629773

MN629843

Crepidodera aurata

Crepidodera nigricoxis

7

MN629761

MN629831

Crepidodera aurata

Crepidodera nigricoxis

7

MN629766

MN629836

Crepidodera aurata

Crepidodera nigricoxis

7

MN629767

MN629837

Crepidodera aurata

correct

7

MN629759

MN629829

Crepidodera aurata

correct

8

MN629749

MN629819

Crepidodera aurata

correct

8

MN629750

MN629820

Crepidodera aurata

correct

8

MN629751

MN629821

Crepidodera aurata

correct

8

MN629752

MN629822

Crepidodera aurata

correct

8

MN629753

MN629823

Crepidodera aurata

correct

8

MN629754

MN629824

Crepidodera aurata

correct

8

MN629755

MN629825

Crepidodera aurata

correct

8

MN629756

MN629826

Crepidodera aurata

correct

8

MN629757

MN629827

Crepidodera aurata

correct

8

MN629758

MN629828

Crepidodera aurata

correct

11

MN629774

MN629844

Crepidodera aurata

correct

18

MN629775

MN629845

Crepidodera aurata

correct

25

MN629776

MN629846

Crepidodera aurata

Crepidodera fulvicornis

33

/

MN629847

Crepidodera aurata

Crepidodera fulvicornis

39

MN629777

MN629848

Crepidodera fulvicornis

correct

16

MN629778

/

Crepidodera fulvicornis (a)

correct

23

MN629779

/

Crepidodera fulvicornis (b)

correct

23

MN629780

MN629849

Crepidodera fulvicornis (c)

correct

23

MN629781

MN629850

Crepidodera fulvicornis

correct

27

MN629782

MN629851

Crepidodera fulvicornis

correct

31

MN629783

MN629852

Crepidodera fulvicornis

correct

35

MN629784

MN629853

Crepidodera fulvicornis

correct

39

MN629785

MN629854

Crepidodera plutus

correct

6

MN629748

MN629818

Crepidodera plutus

correct

9

MN629786

MN629855

Crepidodera plutus

correct

11

MN629787

MN629856

Crepidodera plutus

correct

13

MN629788

MN629857

Crepidodera plutus

correct

14

MN629789

MN629858

Crepidodera plutus

correct

19

MN629790

MN629859

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

7

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

41

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

33

MN629816

MN629885

Plagiodera versicolora

correct

39

MN629817

MN629886

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, JN087422, KU188452, KM443640, KJ961764, KM443492

Crepidodera aurata

KJ966066, KJ962544, KF654801, KF656415, KF654798, KJ963892, KM450642, KM445873, KM448484, KM445803

Crepidodera aureola

KF655591, KF655792, KF655954, KF652694, KF652646

Crepidodera browni

KR487413, KR481606, KR490696

Crepidodera fulvicornis

KF656356, KM448864, KF656033, KF656133, KF656534, KF656533, KF655283, KJ963238, 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, KM439994

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, KF656252, 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 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.

Figure 1.  

DNA analysis (NJ tree) using COI and cytB sequences generated in this study. Node support shown only for nodes ≥ 90% bootstrap support.

Figure 2.  

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).

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. Gavrilović and Ćurčić (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).

Figure 3.  

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 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 (SDF13010) 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

References