2
Soininen et al. Shedding new light on the diet of Norwegian lemmings: DNA
metabarcoding of stomach content
3
Appendix 1.
1
4
5
Details of taxonomic adjustments
6
For seed plants, we first verified the taxonomic annotation of sequences based on the
region’s flora (Lid and Lid 2005, Mossberg and Stenberg 2005, Norwegian Biodiversity
Information Centre and GBIF Norway 2012). Several vascular plant genera are represented
only by one species in the study area. We therefore attributed sequences assigned to these
genera to the respective species (e.g. Empetrum nigrum, Geranium sylvaticum). When a
species was identified that is not present in the study area and several possible species could
come in question, the adjustment was done to a less specific level (e.g. Euphrasia tatarica
was assigned to genus Euphrasia). For each identified taxon, we also checked whether the
taxonomic reference library included all closely related taxa possibly present in the area. If
this was the case and when possible, sequences of missing taxa available in EMBL were
compared to the sequences in the taxonomic reference library. If no unambiguous
identification of the retrieved sequences was possible, the identification was moved to a less
specific taxonomic rank (e.g. from species to genus). Furthermore, we moved sequences
assigned to Vaccinium ovalifolium to Vaccinium myrtillus, because the former is not present
in Europe, but the two have almost identical g-h region (accession numbers GQ245635GQ245641 in EMBL). In total, 99.7% and 0.3% of the sequences included in the final seed
plant dataset were identified based on the combined arctic and boreal reference library and
reference sequences from EMBL, respectively.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
31
We did similar verifications for bryophytes, i.e. comparison to regional flora (Hill et al. 2006,
Norwegian Biodiversity Information Centre and GBIF Norway 2012) and reference library
coverage. We changed the taxonomic annotation from species to genera for two taxa. First,
we moved Dicranum flexicaule to genus Dicranum, because its close relative D. fuscenses
was not included in the taxonomic reference library and we could therefore not inarguably
differentiate between these two species. Further, we moved Sphagnum russowii to genus
Sphagnum, as sections are probably the lowest level of true recognition within this genus
(Shaw 2000; Shaw et al. 2010).
32
References
33
34
35
36
37
38
Hill MO, Bell N, Bruggeman-Nannenga MA, Brugues M, Cano MJ, Enroth J, Flatberg KI, Frahm JP,
Gallego MT, Garilleti R, Guerra J, Hedenas L, Holyoak DT, Hyvonen J, Ignatov MS, Lara F,
Mazimpaka V, Munoz J, Söderström L (2006) An annotated checklist of the mosses of Europe
and Macaronesia. Journal of Bryology 28 (198-267)
24
25
26
27
28
29
30
Lid J, Lid DT (2005) Norsk flora, 7th ed. (in Norwegian). Samlaget, Oslo.
39
40
41
42
43
44
45
46
Mossberg B, Stenberg L (2005) Suuri Pohjolan Kasvio (in Finnish).Tammi, Helsinki.
Norwegian Biodiversity Information Centre and GBIF Norway (2012) Species Map Service 1.6
(http://artskart.artsdatabanken.no/). Accessed July 2012.
Shaw AJ (2000) Phylogeny of the Sphagniopsida based on chloroplast and nuclear DNA sequences.
Bryologist 103 (2):277-306
Shaw AJ, Devos N, Cox CJ, Boles SB, Shaw B, Buchanan AM, Cave L, Seppelt R (2010) Peatmoss
(Sphagnum) diversification associated with Miocene Hemisphere climatic cooling? Molecular
Phylogenetics and Evolution 55 (3):1139-1145.
47
48
49
50
Supplementary Table S1 Sequence analysis detailed for each of the three primer pairs used in order of execution. The samples were sequenced
as a part of a batch of 192 samples comprised partly of samples not presented in this study. First part of the sequence analysis was done for the
whole dataset of 192 samples, using software OBITools (available at http://www.grenoble.prabi.fr/trac/OBITools). Thereafter, a new dataset
was composed consisting of lemmings only (focal dataset of each step denoted in the first column).
Dataset
Whole dataset
Final dataset of
lemmings
g-h
Sequences with an error in the primer
Sequences with an error in the tag sequence
Sequences with fewer reads discarded
Unrealistically short sequences removed, threshold length
Potential PCR errors discarded (using OBIcleana), criteria
GenBank database accessed
Software used for sequence annotation
Minimum match with reference sequence
Mean no. sequence reads per sample
Mean no. taxa per sample in final dataset
Sequences assigned to species level
Sequences assigned to genus level
Sequences assigned to family level
c-h
ITS-Fungi
2 errors allowed
Removed
<4
8
50
clustering threshold 10%
16th April 2012
EcoTag (available as part of OBITools)
98%
98%
2405 (range 23-12510)
581 (range 74-1516)
15.4 (range 6-27)
8.9 (range 3-16)
45%
57%
27%
31%
26%
9%
50
90%
44 (range 0-225)
3 (range 0-9)
12%
1%
4%
51
52
53
a=OBIclean
(included in OBITools) identifies progressive changes of one bp, defines clusters which include a maximum threshold proportion of
changed sequences, and keeps the most abundant sequence of the cluster
54
55
56
57
58
59
60
61
62
Supplementary Table S2 Rare plant species and genera recorded in the diets of Norwegian
lemmings (N=40) during a population density peak in northern Norway using DNA
metabarcoding of chloroplast trnL intron. Included are taxa which composed on average <
0.1% of seed plants in diets, determined using primer pair g-h and taxa which composed on
average < 0.1% of mosses in diets, determined using primer pair c-h. See methods for
details. Column “Frequency” refers to the number of individuals from which the taxa in
question was found. Column “Change” shows taxa which identity was changed based on
regional flora; “+” indicates that at least part of the sequences included in the taxon were reassigned to a more specific taxonomic level,”-“ the opposite.
Group
Taxa
Seed plants Andromeda polifolia
Arabis alpina
Bartsia alpina
Caltha palustris
Chamerion angustifolium
Comarum palustre
Dryas octopetala
Geranium sylvaticum
Geum rivale
Lathyrus pratensis
Linnaea borealis
Lotus corniculatus
Melampyrum pratense
Parnassia palustris
Phalaroides arundinacea
Pinus sylvestris
Saussurea alpina
Trientalis europaea
Trollius europaeus
Vaccinium vitis-idaea
Alchemilla
Calamagrostis
Epilobium
Euphrasia
Galium
Larix
Luzula
Plantago
Rhinanthus
Stellaria
Papaver
Bryophytes Hylocomiastrum pyrenaicum
Hylocomium splendens
Frequency
1
1
5
4
1
2
1
5
1
1
1
1
1
1
1
4
5
6
2
4
3
7
1
1
2
2
1
1
1
1
2
1
3
Change
+
+
+
+
+
+
+
+
-
Kiaeria glacialis
Lophozia wenzelii
Pohlia wahlenbergii
Saniona uncinata
Bryum
Sciuro-hypnum
63
1
1
1
1
2
2
64
65
66
67
68
69
Supplementary Table S3 Fungal taxa ingested by Norwegian lemmings (N=40) during a
population density peak in northern Norway, determined with the primer pair ITS5 and
5.8S_fungi on stomach content DNA. Sequences identified to lower taxonomic levels are
included at the higher levels. Frequency: number of individuals in whose stomach content
DNA-sequences of a taxon. Size class indicates to which fungal size class (micromycete/
macromycete) the taxa belong.
Division
Class
Family
Species
Ascomycota
Venturiaceae
Venturia sp.
Venturia atriseda
No rank
Leotiomycetes
Helotiaceae
Gremminella sp.
Thelebolaceae
Herpotrichiellaceae
Lecanoromycetes
Teloschistaceae
Cladophialophora
minutissima
Caloplaca sp.
Caloplaca flavocitrina
Saccharomycetes
Dipodascaceae
Galactomyces
geotrichum
Yarrowia lipolytica
Basidiomycota
Exobasidiomycetes
Agaricomycetes
Tremellomycetes
Exobasidiaceae
Schizophyllaceae
No rank Tremellales
Exobasidium rostrupii
Schizophyllum sp.
Trichonosporales sp.
LM547
no rank
No rank
Leucosporidiales
No rank
Leucosporidium
No rank
No rank Fungi
70
71
Size
class
21
4
3
Dothideomycetes
Eurotoimycetes
Frequency
2
1
1
9
1
1
8
3
1
1
1
4
4
1
3
17
3
2
2
2
14
1
13
4
38
micro
micro
micro
micro
micro
micro
micro
micro
micro
macro
micro
micro