A.M. Soeter , F.A.A. Bakker, M. Velthuis, R.A. Verweij, M.H.S. Kraak & J.A.J. Breeuwer. Adaptation in polluted environments has been considered as evolution in action.This study focused on the effects of metal contamination on chironomid community composition and on genetic adaptation in Chironomus riparius. Using CO-1 sequencing we demonstrated that chironomid community composition was impoverished due to metal pollution. Combining the population genetic structure of C. riparius and copper sensitivity of the F1 larvae we concluded that metal tolerance in C. riparius is most likely due to genetic adaptation.

1. The selective environment: shifts in community composition and the mechanism of tolerance A.M. Soeter F.A.A. Bakker, M. Velthuis, R.A. Verweij, L. Hoitinga, M. Marinkovic, M.H.S. Kraak & J.A.J. Breeuwer

6. Copper sensitivity Differential sensitivity No metal No Panmixis Genetic Adaptation Metal Selection Population genetics Panmixis Equal sensitivity Original situation No metal Metal Phenotypic Plasticity Original situation Hypotheses – mechanism of tolerance

7. Material and Methods - Sampling

8. Methods – Larvae selection

13. Results – Population genetic structure Genetic differentiation P2 P1 R2 Lab 0.381* 0.354* 0.088* 0.333* 7 km 2 km 200 km + 20 years 0.145* 9 km

15. Results – Copper toxicity tests F1 from polluted site less sensitive than lab 100 75 50 25

17. Acknowledgements Supervision Michiel Kraak Hans Breeuwer Ecotoxicology Marino Marinkovic Marie Manguette Genetics Betsie Voetdijk Ling Dong Peter Kuperus Chemical analysis Leo Hoitinga Ton van Wijk Rudo Verweij

18. Chemical analysis Ba Cu Cd Cl - Ni Zn Width Temp. Conductivity pH Velocity µg/L µg/L mg/L µg/L µg/L µg/L M °C µ/cm pH km/h MTR 230.0 2.0 2.0 200.0 5.1 40.0 P1 31-03-09 49.0 1273.0 3.0 36.8 95.0 207.0 1.5 9.4 685.0 6.63 3.57 P2 31-03-09 15.0 15.0 <2.0 1917.3 53.0 44.0 1.8 16.1 8760.0 6.78 5.38 R1 31-03-09 34.0 4.0 <2.0 18.7 3.0 23.0 3.1 11.4 402.0 6.21 2.60 R2 31-03-09 49.0 41.0 <2.0 41.7 12.0 61.0 6.3 11.6 420.0 6.47 2.55 R3 01-04-09 23.0 5.0 <2.0 39.6 <3.0 21.0 3.6 8.1 404.0 6.99 1.90

19. Community composition P1 P2 R1 R2 R3 Number of species found 4 4 8 4 6 Species restricted to polluted sites: Cric. silvestris X Cric. sp . III X Chir. sp. C X Cric. sp. VII X Species occurring at reference and polluted sites: Chir. thummi (sp.) X X X Chir. sp. D X X Cric. sp. I X X X Species restricted to reference sites: Chir. sp. A X Chir. sp. B X Cric. sp. V X Unkn own sp. 1 X Chir. luridus X X Unknown sp. 2 X X X Cric. sp. VI X X Cryptochir. sp. X Cric. sp. II X Cric. sp. IV X

20. Jaccards index P1 P2 R1 R2 R3 P1 - P2 0.5 - R1 0.1 0.1 - R2 0.5 0.5 0.29 - R3 0.125 0 0.25 0.4 -

21. Hardy-Weinberg N Msc2 Msc4 Locus4 Locus6 Ho He p Ho He p Ho He p Ho He P P1 8 0.38 0.79 0.01 0.00 0.43 0.00 0.75 0.92 0.19 0.25 0.77 0.00 P2 13 0.69 0.83 0.12 0.31 0.58 0.01 0.77 0.85 0.42 0.46 0.82 0.08 R2 6 0.67 0.79 0.29 0.67 0.62 1.00 0.17 0.44 0.09 0.67 0.77 0.53 R5 26 0.19 0.50 0.00 0.35 0.43 0.03 0.38 0.40 1.00 0.69 0.62 0.96

22. Population genetic structure P1 P2 R2 P2 0.029 R2 0.145** 0.088** R5 0.354** 0.333** 0.381**