1urn:lsid:arphahub.com:pub:F9B2E808-C883-5F47-B276-6D62129E4FF4urn:lsid:zoobank.org:pub:245B00E9-BFE5-4B4F-B76E-15C30BA74C02Biodiversity Data JournalBDJ1314-28361314-2828Pensoft Publishers10.3897/BDJ.4.e10003100035945Research ArticleSalix transect of Europe: variation in ploidy and genome size in willow-associated common nettle, Urticadioica L. sens. lat., from Greece to arctic NorwayCronkQuentinquentin.cronk@ubc.ca‡HidalgoOriane§PellicerJaume§PercyDiana|LeitchIlia J.§University of British Columbia, Vancouver, CanadaUniversity of British ColumbiaVancouverCanadaRoyal Botanic Gardens, Kew, United KingdomRoyal Botanic GardensKewUnited KingdomNatural History Museum, London, United KingdomNatural History MuseumLondonUnited Kingdom
2016270920164e10003FF95E640-0516-FFFA-5525-FFC6FFB78E1C1555622507201620092016Quentin Cronk, Oriane Hidalgo, Jaume Pellicer, Diana Percy, Ilia J. LeitchThis is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background
The common stinging nettle, Urticadioica L. sensu lato, is an invertebrate "superhost", its clonal patches maintaining large populations of insects and molluscs. It is extremely widespread in Europe and highly variable, and two ploidy levels (diploid and tetraploid) are known. However, geographical patterns in cytotype variation require further study.
New information
We assembled a collection of nettles in conjunction with a transect of Europe from the Aegean to Arctic Norway (primarily conducted to examine the diversity of Salix and Salix-associated insects). Using flow cytometry to measure genome size, our sample of 29 plants reveals 5 diploids and 24 tetraploids. Two diploids were found in SE Europe (Bulgaria and Romania) and three diploids in S. Finland. More detailed cytotype surveys in these regions are suggested. The tetraploid genome size (2C value) varied between accessions from 2.36 to 2.59 pg. The diploids varied from 1.31 to 1.35 pg per 2C nucleus, equivalent to a haploid genome size of c. 650 Mbp. Within the tetraploids, we find that the most northerly samples (from N. Finland and arctic Norway) have a generally higher genome size. This is possibly indicative of a distinct population in this region.
megatransectgenome sizecytotype variationUrticaVariation on a theme: evolutionary-developmental insights into the Asteraceae flower head657918501100000780European Commissionhttp://doi.org/10.13039/501100000780Introduction
During a recent study of willow (Salix spp.) stands on a latitudinal transect across Europe (Cronk et al. 2015) the opportunity arose to sample individuals of UrticadioicaL.ssp.dioica (the common stinging nettle) that frequently co-occurs with willow in riparian habitats (see under Materials and Methods for further details). Urticadioica is one of the most remarkable plants of Europe. First it possesses a defense, stinging hairs, which are a small marvel of biochemistry and biomechanics. These are highly effective against vertebrate herbivores (Levin 1973, Pollard and Briggs 1984, Pullin and Gilbert 1989, Tuberville et al. 1996). The cell walls of the trichome tip are silicified and brittle (Haberlandt 1914, Barber and Shone 1966, Thurston and Lersten 1969, Thurston 1974, Sowers and Thurston 1979) and break off (like the tip of a glass ampoule) on the slightest mechanical stimulation. The fluid released is a potent and complex mixture of toxins including histamine, oxalic acid and tartaric acid (Emmelin and Feldberg 1949, Fu et al. 2006, Taskila et al. 2000).
Secondly it has an extraordinary biogeographical range, occurring in every corner of Europe, from the shores of the Mediterranean to the Arctic Ocean and from the winter-cold central European plain to the rainswept coasts of western Ireland. Few plants have the ability to grow in such a wide range of climatic conditions. Over this range it is largely native, having spread along its natural habitat of rich alluvial river floodplains. However, it has also become an aggressive ruderal, taking advantage of human disturbance to complete its conquest of Europe through accidental introduction by humans.
Thirdly it is an invertebrate “super-host”. Throughout Europe it provides the food plant for large numbers of specialist and generalist insects, notably in the Lepidoptera, Coleoptera and Hemiptera (Davis 1973, Davis 1975, Davis 1983, Davis 1989, Perrin 1975).
Fourthly, it has exceptional mineral nutrition, being highly phosphate demanding. It ceases growth if phosphate is limiting and responds luxuriantly if phosphate is added, whereas in contrast plants adapted to poor soil scarcely respond to additional phosphate (Pigott 1971, Pigott and Taylor 1964, Taylor 2009). It is not only an indicator of high available phosphate, but it is also a general mineral accumulator, having high concentrations of calcium, nitrogen and phosphorus in its tissues (Müllerová et al. 2014). This may go some way to explaining its attractiveness to herbivorous invertebrates.
Taxonomically Urticadioica is part of a complex of closely related taxa and subtaxa (Grosse-Veldmann and Weigend 2015), which includes U.dioicasubsp.subinermis (R. Uechtr.) Hand & Buttler, U.dioicasubsp.sondenii (Simmons) Hyl. and U.dioicasubsp.pubescens (Ledeb.) Domin (Table 1). In addition there are a number of related European perennial nettles that are sometimes confused with Urticadioica, although they are distinctive. These include: Urticagracilis Aiton (the American stinging nettle), Urticakioviensis Rogow. and Urticamembranacea Poir. (Table 1; nomenclature follows Euro+Med PlantBase 2006). Most of these taxa are diploid (Table 1) (typically 2n=26) except for U.dioicasubsp.dioica (common nettle), which is reported as largely, but not completely, tetraploid (Table 1).
Two types of cytological diversity have been found in Urticadioicasubsp.dioica. One is the reported difference in tetraploid chromosome number between 2n=48 and 2n=52 (Skalińska et al. 1974). Such a discrepancy could be due to miscounts, but the repeated reports of both numbers leads to a suspicion that both numbers do exist in nature.
There is also the difference in ploidy level. The possibility must be entertained that counts for Urticadioica of 2n=26 (diploid) refer to one of the infraspecifc taxa and not to U.dioicasubsp.dioica. However there are numerous counts that are candidates for genuine diploid U.dioicasubsp.dioica. For instance Kolnik and Goliašová (in Mráz 2006), reported a chromosome count of 2n=26 for Urticadioica from Závod, Slovakia. Because of the problematic taxonomy of this group it is very important that herbarium voucher specimens are collected in conjunction with any study.
Genome size estimates have also been made for Urticadioica (see Bennett and Leitch 2012 and additional data not yet incorporated into the database), and these results (Table 2) are also indicative of cytotype diversity. Nevertheless, the same cautionary taxonomic considerations apply as well as technological issues arising from the estimation of genome size (e.g. Doležel et al. 2007, Greilhuber et al. 2007, Pellicer and Leitch 2014).
Materials and Methods
Context of study
Urticadioica samples (Table 3) were collected during a survey of willow habitats in a latitudinal transect across Europe: the Salix transect of Europe (Cronk et al. 2015). The aim of this was to survey variation in Urtica and one of its constant herbivores, the Urtica psyllid, Triozaurticae, which were co-sampled. Information on Trioza will be the given in separate papers. Herbarium and living Urtica samples were collected. The aim of the current work was to investigate the extent of ploidy level and genome diversity within the resultant Urtica collection.
Site selection and sampling
Full details of the sites (mainly riverine alluvial habitats), and their selection are given in Cronk et al. (2015). The sites are summarized in Table 3. In all, 42 Salix sites were chosen between Athens (Greece) and Hammerfest (Norway) (Fig. 1). Of these 33 (and one supplementary site) had Urticadioica present and a herbarium voucher specimen was collected from each of these sites (and in addition one specimen of U.membranacea from Greece). Herbarium voucher specimens are deposited in the herbarium of the Natural History Museum, London (BM). Living specimens were also collected for cultivation in London (Queen Mary University of London), for future experimental work. One living specimen was collected from each site (two from site 27). Of the living specimens collected, 27 survived into cultivation and could be used for flow cytometry (see results). The living specimens were grown in London in a 'common garden' (rooftop plant growth facility at Queen Mary University of London, Lat. 51.5234. Long. -0.0423).
Flow cytometry and buffers
Ploidy level (diploid vs tetraploid) was assayed using flow cytometry (as described in Hanson 2005), using a Partec CyFlow flow cytometer with Petroselinumcrispum (parsley) 'Champion Moss Curled' 2C=4.50 pg (Obermayer 2002) as calibration standard. A range of different flow cytometry buffers were tested (including the Galbraith buffer, the general purpose buffer and the LB01 buffer, Pellicer and Leitch 2014). However, only the ‘CyStain PI Absolute P kit’ buffer (Sysmex UK) gave acceptable flow histograms with CVs routinely less than 3%, so it was chosen for estimating the ploidy level of the 29 Urtica specimens.
Results
The flow cytometry results are given in Table 3. In all, 24 plants have flow cytometry results consistent with tetraploidy whereas five plants, 7-5 (Bulgaria), 11-4 (Romania), while 31-12, 32-11, 34-6 (southern Finland), have results consistent with diploidy. The identity of these diploid plants was checked and confirmed as U.dioicasens. lat. As diploidy is often associated with stinglessness, information on the presence of stinging hairs was collected after cultivation in a common garden (Queen Mary University of London, QMUL) for one year (Table 4). Information on flowering time in the common garden is also given. Flowering time shows an overall correlation with latitude (generally with late flowering plants coming from Finland and Norway, although there are some exceptions (Table 4). Voucher specimens of both the original specimens and plants after cultivation (in common garden conditions for one year) are deposited at the Natural History Museum, London (BM).
At the tetraploid level, some variation in the estimated genome sizes was detected, with the northern populations tending to have higher 2C-values compared with the more southerly ones (Table 3; Fig. 2). To confirm that this intraspecific variation was genuine rather a technical artefact, leaves from the two individuals showing the largest difference in 2C-value (i.e. 28-10, 37-6) were co-processed. This resulted in two distinct peaks in the flow histogram (Fig. 3), indicative of biologically real difference in C-values at the tetraploid level.
Discussion
The results confirm that the tetraploid is the dominant cytotype in our sample of U.dioica but that diploid plants do occur relatively frequently (at least in SE Europe and S. Finland). A more extensive survey of cytotype variation in Romania and Bulgaria, as well as around the Baltic would be of interest. Ploidy level has been shown to correspond with morphological characters (Geltman 1984; Geltman 1986). The possibility must be therefore be examined that the diploid samples here belong to the diploid taxa Urticadioicassp.pubescens (Ledeb.) Domin (synonym: U.dioicassp.galeopsifolia (Wierzb. ex Opiz) J. Chrtek), U.dioicassp.subinermis (R. Uechtr.) Weigend or Urticadioicassp.sondenii (Simmons) Hyl. These taxa typically lack stinging hairs on the leaves. U.dioicassp.pubescens typically has a pubescence of long non-stinging hairs whereas U.dioicassp.sondenii is glabrous. We are cautious in assigning any of the individuals studied here to those taxa without further study of the populations, which may not be homogeneous. Of the diploids, only 11-4 and 32-11 can be considered stingless. None of the diploids here are glabrous (although many have an indumentum of very short hairs), ruling out U.dioicassp.sondenii. Only 32-11 (with few stinging hairs on leaves and relatively long pubescence) can be considered a reasonable match for U.dioicassp.pubescens. However this plant generally resembles the other diploids, 31-12 and 34-6, which vary in stinging hairs and pubescence. Until the populations from which these plants come can be examined critically we tentatively assign all our samples to the variable U.dioicassp.dioica.
The finding of diploids in SE Europe raises the possibility that the widespread tetraploid form of Urticadioicasubsp.dioica, which has also become a weed, may have originated there, and the diploids may have survived glacial episodes in S. European refugia. The origin of the diploids of S. Finland is as yet unknown, although a phylogeographic analysis might be informative here. Another interesting result is the discovery of intraspecific C-value variation, particularly the generally higher C-values in the far north. This may be indicative of a distinct population of nettles in the north, and again this would benefit from more detailed cytogeographic study.
Acknowledgements
We thank especially Paul Fletcher (Organismal Biology Facilities Manager, School of Biological and Chemical Sciences, Queen Mary University of London) for his expert care of the living Urtica collection described in this study. We also thank Enrico Ruzzier and Gavin Broad (Natural History Museum, London, UK) for assistance with the collection of Urtica. Funding for the fieldwork was partly provided by the Natural History Museum (London, UK) Life Sciences Departmental Investment Fund (SDF13010) to DMP. QCC acknowledges appointments by RBG Kew (as Honorary Research Associate) and by Queen Mary University of London (as Visiting Professor), which greatly facilitated the conduct of this study. OH was supported by the Marie Sklodowska Curie Action Individual Fellowship program (CAPITULA – grant agreement n°657918). Finally, we thank the reviewers (D. Geltman and M. Weigend) for their very helpful comments on the manuscript.
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Map of Urtica sample sites. Squares: diploids; diamonds: tetraploids; red line = route of transect (Lat. = latitude, Long. = longitude).
Scatter plot of genome size (2C-value, pg) values (as given in Table 3), plotted against latitude (Table 3). Only tetraploids (4x) are shown; diploid samples (2x) are not plotted. Note the generally higher genome size of the high latitude samples (see Table 3).
Screen shot from the Partec CyFlow flow cytometer showing flow histogram obtained from analysing Urticadioica accessions 28-10 (peak 1, 2C=2.36 pg) and 37-6 (peak 2, 2C=2.59 pg) showing two distinct peaks and hence demonstrating genuine intraspecific variation in genome size between these two tetraploid individuals (28-10 and 37-6: see Table 3). The graph shows the relative fluorescence (indicative of DNA amount) in thousands of cell nuclei. The machine also gives summary statistics for the peaks. Note the very low coefficient of variation (CV%) of 2.48% and 2.89%.
https://binary.pensoft.net/fig/91651
Some nettle taxa reported in Europe with representative chromosome counts. There are very large numbers of counts for Urticadioica and the list below does not aim to be comprehensive. For a full summary see the Chromosome Counts Database, CCDB (Rice et al. 2014).
Name
Notes
Representative chromosome counts
U. dioica L. subsp. dioica
The common stinging nettle
2n=26 (Kolník M. and Goliašová, in Mráz 2006); 2n=48 (Májovský et al. 1987); 2n=48, 52 (Skalińska et al. 1974); 2n=48, 52 (Lippert 2006); 2n=52 (Löve and Kjellqvist 1974); 2n=52 (Corsi et al. 1999)
U.dioicasubsp.subinermis (R. Uechtr.) Weigend
2n=24/26 (Lippert 2006)
U.dioicasubsp.sondenii (Simmons) Hyl.
2n=26 (Geltman 1984)
U.dioicasubsp.pubescens (Ledeb.) Domin
Syn. U.galeopsifolia
2n=26 (Geltman 1984); 2n=26 (McAllister 1999)
U.gracilis Aiton
Syn. U.dioicasubsp.gracilis (Aiton) Selander
2n=26, 52 (Woodland et al. 1982)
U.kioviensis Rogow.
2n=26 (Kolník M. and Goliašová, in Mráz 2006)
U.membranacea Poir.
2n=22 (Corsi et al. 1999)
Previous genome size estimates in Urticadioica s.l.
Estimated using FC:PI with LB01 or MgSO4 buffer and Solanum lycopersicum L. ‘Stupické polní rané’ (2C=1.96 pg) as calibration standard.
Bainard et al. 2011
U.dioica
3.1
52
UK
Estimated using Fe with Senecio vulgaris (PBI population (2C=3.16 pg) as calibration standard.
Mowforth 1986
U.dioica
2.34
n/a
Germany
Estimated using FC:PI with Galbraith buffer. Calibration standard unclear.
Barow and Meister 2003
U.dioica
2.16
n/a
West Balkans, Central Bosnia, Serbia Macedonia
Estimated using FC:PI with Galbraith buffer and Petunia hybrid ‘PxPC6’ (2C=2.85 pg) as calibration standard.
Pustahija et al. 2013
Locations of the Urtica samples collected in April and June 2015, together with estimated genome size (2C-values) and ploidy levels made from the living material (herb. = only herbarium material available).
Sample
Latitude (N)
Longitude (E)
Country
River/ location
2C-value (pg)
Ploidy Level (x)
Living material/Flow cytometry
2-4
38.902
22.31015
Greece
R. Sperchios, near Leianokladi, east of Lamia
-
-
Herb. only
4-4
40.032685
22.175437
Greece
Stream near Kokkinogeia, Thrace
2.46
4
Yes
5-3
41.113317
23.273893
Greece
At R. Struma, near Lithotopos
-
-
Herb. only
6-5
41.412468
23.318609
Bulgaria
R. Struma, near Topolnitsa
2.41
4
Yes
7-5
42.165622
22.998141
Bulgaria
R. Struma, north of Boboshevo
1.35
2
Yes
8-3
42.923989
23.810563
Bulgaria
R. Kalnitza, near Botevgrad
2.46
4
Yes
11-4
44.961981
23.190337
Romania
R. Jiu, north of Rovinari
1.33
2
Yes
12-3
45.510676
22.737225
Romania
Meadow near Paucinesti, Carpathian region
-
-
Herb. only
13-4
46.518504
21.512839
Romania
R. Crisul Alb, at Chisineu-Cris
-
-
Herb. only
14-6
46.700744
21.31268
Hungary
R. Fekete-Koros, near Gyula
2.46/ 2.46
4
Yes (x2)
15-5
47.665648
21.261768
Hungary
Drainage ditches near R. Hortobagy, north-east of Balmazujvaros
2.42
4
Yes
16-7
48.374291
20.725264
Hungary
R. Bodva, south of Szendro
2.43
4
Yes
17-4
49.463447
21.697255
Poland
R. Panna, at Tylawa
-
-
Herb. only
18-4
50.470234
22.238372
Poland
Fields north of Rudnik nad Sanem
2.48
4
Yes
19-7
50.673994
21.823391
Poland
R. Leg, near Gorzyce
-
-
Herb. only
20-6
51.775039
21.1971
Poland
R. Pilica, at Warka
2.42
4
Yes
21-11a
52.69398
21.8529
Poland
R. Bug, near Brok
2.48
4
Yes
22-6
53.55483
22.30299
Poland
Meadow near R. Biebrza at Wasocz, near Szczuczyn
2.44
4
Yes
23-6
54.06943
23.11745
Poland
R. Czarna Hancza, near Sejny on road from Suwalki
2.45
4
Yes
24-11
54.92583
23.7742
Lithuania
Embankment of River at Kaunas
2.40
4
Yes
26-15
56.71141
24.25162
Latvia
Near R. Misa, between Iecava and Kekana
-
-
Herb. only
27-6 & 7
57.74963
24.4023
Latvia
R. Salaca short distance inland from Salacgriva
2.40
4
Yes (27-7)
28-10
58.42257
24.44063
Estonia
Field near Parnu
2.36
4
Yes
29-7
59.40289
24.93577
Estonia
R. Pirita at Lagedi near Tallinn
2.44
4
Yes
30-8
60.27299
24.65843
Finland
Near Lake Bodom, Espoo, Finland
n.d.
n.d.
Yes
31-12
61.09965
25.6282
Finland
Drainage flowing into lake Vesijärvi at Paimela near Lahti
1.33
2
Yes
32-11
62.04962
26.12369
Finland
Lake near Toivakka
1.34
2
Yes
34-6
64.05074
25.52664
Finland
R. Pyhäjoki, at Joutenniva, south of Haapavesi
1.31
2
Yes
35-8
64.61287
25.53805
Finland
Tributary of the R. Siikajoki near Mankila
2.49
4
Yes
37-6
66.24947
23.8945
Finland
Small river between Kainuunkylä and Väystäjä
2.59
4
Yes
38-11
67.21253
24.12629
Finland
Near Vaattojärvi
2.52
4
Yes
39-16
67.91183
23.63411
Finland
River Muonion (Muonionjoki) just south of Muonio
2.51
4
Yes
42-8
70.65234
23.66583
Norway
Jansvannet Lake, Hammerfest
2.54/ 2.53
4
Yes (x2)
SUPPLEMENTARY SITES
i-D-1 & 2
38.1261
22.45348
Greece
[Urticamembranacea]
-
-
Herb. only (fem. & mas.)
ii-D-4
65.32443
25.3153
Finland
Kestilä
2.42
4
Yes
Urtica phenotype in common garden (London). Fl. (flowering) time refers to category of flowering performance in 2016; 1 = early flowering (flowering before 16 May); 2 = mid-June (flowering by 10 June); 3 = late June (21 June); 4 = early July (2 July); 5 = late or not flowering (not flowering by early July). Stinging hairs refers to the typical number of stinging hairs per leaf; 1 = <10; 2 = 10-50; 3 = 50-100; 4 = >100. Numbers are given for: adaxial surface (first number)/abaxial surface (second number).
Accession
Fl. time
Stinging hairs
Notes
4-4
1
3/3
Well-armed.
6-5
2
2/3
Moderately well-armed.
7-5 (diploid)
4
1/3
Tall plant with rather narrow leaves but abundant stinging hairs on undersides of leaves. Non-stinging hairs very short.
8-3
5
1/3
Moderately well-armed.
11-4 (diploid)
3
1/1
Leaves largely stingless except on petiole. Shortly pubescent on veins and stems.
14-6
1
3/4
Well-armed.
15-5
2
1/1
Leaves largely stingless, except on petiole. Shortly pubescent on veins.
16-7
2
3/4
Well-armed.
18-4
2
3.4
Well-armed.
20-6
2
3/4
Well-armed.
21-11
2
2/3
Moderately well-armed.
22-6
3
3/4
Well-armed.
23-6
2
3/4
Well-armed.
24-11
2
1/1
Largely stingless except on petiole and midrib. Pubescent on veins.
27-7
3
3/4
Well-armed.
28-10
3
1/1
Largely stingless except on petiole, pubescent with rather long hairs on veins.
29-7
2
3/4
Well-armed.
30-8
5
2/3
Moderately well-armed.
31-12 (diploid)
3
1/3
Moderately armed below, other pubescence of rather sparse very short hairs.
32-11 (diploid)
3
1/1
Leaves very largely unarmed below, stinging hairs mainly on inflorescence, petiole and stem, otherwise similar to previous, but stems and veins covered with longer non-stinging hairs.
34-6 (diploid)
4
1/3
Moderately well-armed; other pubescence of very short hairs.