DNA metabarcoding reveals diverse but selective diet of three-spined stickleback in a coastal ecosystem

Transcription

1 DNA metabarcoding reveals diverse but selective diet of three-spined stickleback in a coastal ecosystem Jakubavičiūtė E* 1, Bergström U 2, Haenel Q 3, Bourlat SJ 4, Eklöf J 5 1 Nature Research Centre, Vilnius, Lithuania 2 Department of Aquatic Resources, Institute of Coastal Research, Swedish University of Agricultural Sciences (SLU), Öregrund, Sweden 3 Zoological Institute, University of Basel, Switzerland 4 Department of Marine Sciences, University of Gothenburg, Box 463, Gothenburg, Sweden 5 Department of Ecology, Environment and Plant Sciences, Stockholm University, Sweden Tallinn, 2017

2 Outline Introduction: DNA metabarcoding Stickleback diet diversity Stickleback diet selectivity Comparing the methods: visual stomach content analysis vs DNA metabarcoding 2

3 DNA metabarcoding DNA barcode - any DNA sequence used for identification at any taxonomic level; DNA barcoding - taxon identification using a standardized DNA region; DNA metabarcoding - the use of NGS to identify multiple species in a sample using DNA barcodes; OTU operational taxonomic unit - gene sequence reads, clustered based on sequence similarity. Kress et al.,

4 DNA metabarcoding Biodiversity assesment, trophic interactions, community composition... Environmental DNA (edna) metabarcoding edna in aquatic environments - non-invasive monitoring tool 4

5 Material & Methods 196 three-spined stickleback samples TL=57.7 ±7.6 mm (SD) Visual inspection of stomach contents DNA metabarcoding Zooplankton abundance data in bays Benthos abundance data in bays 5

6 Workflow Fish dissection, visual identification of stomach contents DNA extraction PCR Amplification of 313 bp region in COI (Leray et al., 2013) Universal and blocking primers Taxa1 (OTU)... # of reads Taxa2 (OTU)... # of reads Taxonomic assignement Sequences clustered into OTUs and compared against reference databases Sequencing Illumina MiSeq platform, final dataset of reads 6

7 Data analysis %N bar - proportions of prey in the stomachs based on the number of OTU reads per taxon %F vis and %F bar - frequency of occurrence based on barcoding and visual data Jacobs s index for diet selectivity estimations J=(r p)/(r+p 2pr) r - %OTU reads of certain prey species in stomach (%N bar ) p - proportion of certain prey in the environment (zooplankton/ benthos) J [-1;1] 7

8 Diet diversity of three-spined stickleback 120 taxa revealed by metabarcoding in the stomachs of sticklebacks 15 phyla 27 classes 52 orders 66 families 83 genus 84 species Primary prey 103 taxa 8

9 Proportion of different classes in stomachs (%N bar ) Actinopterygii 2% Bivalvia 1% Gastropoda 1% Polychaeta 1% Malacostraca 3% Ostracoda 10% Branchiopoda 15% Insecta 48% Maxillopoda 19% 9

10 Proportion of different families in stomachs (%N bar ) Gasterosteidae 2% Cyclopidae 2% Temoridae 6% Asellidae 1% Tellinidae 1% Cytherideidae 1% Tachidiidae 13% Chironomidae 55% Podonidae 19% 10

11 Relative abundance, % Nbar Main prey species Dicrotendipes modestus Tanytarsus usmaensis Eurytemora affinis Pleopis polyphemoides Tachidius discipes Chironomus aprilinus Frequency of occurence, %Fbar 11

12 Diet selectivity preferred rejected *P<0.05 **P<

13 % Nbar Fish size and diet Differences in stomach content depending on fish size (Permanova, P=0.044). Diet of the smallest fish (<4.5 cm) group differed from the others 100 n=8 n=158 n= <4,5cm 4,5-6,5 cm >6,5 cm S M L Diptera Harpacticoida Calanoida Cyclopoida Diplostraca Podocopida Amphipoda Isopoda Littorinimorpha Table 1. Anosim results, differences in diet between fish size groups. Medium Large P values ( cm) (>6.5 cm) Small (<4.5cm) 0,0037 0,0062 Large (>6.5 cm) 0,6576 R values Small (<4.5cm) 0,2623 0,2821 Large (>6.5 cm) -0,

14 DNA metabarcoding vs visual inspection Mean taxonomic rank assigned to items within individual stomachs Number of taxa identified per stomach 14

15 Relative abundance, % Nbar Compliance of the two methods: visual vs barcoding R² = Chironomidae Insecta Maxillipoda Branchiopoda 10 Amphipoda Ostracoda Gastropoda Malacostraca Bivalvia 0 0 Annelida Frequency of occurence, % Fvis 15

16 DNA metabarcoding vs visual inspection Visual inspection DNA metabarcoding Mean taxonomic rank Order Genus Mean number of taxa identified per stomach 1.96 ± 1 (SD) 21.7 ± 8.8 (SD) Total number of taxa identified

17 Methodological shortcomings Secondary consumption 103 out of 120 taxa are primary prey? Some taxa detected by visual inspection only (Temora longicornis, Bosminidae, Hydracarina), some may be underestimated (Bivalvia) Quantification primer-template mismatches Species differ in amount of DNA/per gram taxon-specific differences in how well DNA amplifies PCR bias in amplification may result in skewed recovery of sequences 17

18 Take home message In general, the two methods gave consistent results with the same prey taxa dominating. High-throughput DNA sequencing is a promising method for estimating the composition and richness of three-spined stickleback diet, but that some further technical development is needed to efficiently capture all species in the diet. 18

19 Aitäh! Eglė Jakubavičiūtė PhD student Laboratory of Marine Ecology Nature Research Centre, Akademijos str. 2, LT-08412, Lithuania 19