Jacek Radwan Institute of Environmental Sciences UJ Institute of Nature Conservation PAS

1. Major histocompatibility complex (MHC) genes: maintenance of diversity and implications for conservation Jacek Radwan Institute of Environmental Sciences UJ Institute of Nature Conservation PAS http://www.eko.uj.edu.pl/radwan

2. Diversity at Major Histocompatibility Complex (MHC) genes • MHC proteins bind antigens of intracellular (MHCI) or extracellular (MHCII) parasites with high specificity • MHC region contains the most polymorphic genes in vertebrates • Polymorphism is concentrated at sites interacting with antigens (ABS) From Janeway 2001

3. Why are MHC genes so polymorphic? heterozygote advantage: production of two different MHC proteins enables presentation of more antigens to the lymphocytes frequency-dependent selection: selection on fast evolving parasites increases frequency of forms that escape presentation by the most common MHC genotypes sexual selection: MHC-dissimilar individuals preferred as mates

4. Fingerprints of historical positive selection are commonly detected in MHC sequences From Janeway 2001 Amino-acid sequences of 2nd exon of MHC-DRB in spotted suslik. Asterisks denote putative ABS (red points in the diagram, based on human MHC; Brown et al. 1993)

5. Detecting selection on protein-coding sequences • Due to degeneration of genetic code nucleotide substitutions can be synonymous and non-synonymous ds, rate of synonymous substitutions/synonymous site, dn, rate of non-synonymous substitutions/non-synonymous site

6. Positive past selection on MHC DRB in spotted suslik

7. voles from 4 different lines derived from the same population; males from different lines than females • typed at class II DRB locus; similar – shared >50% SSCP bands; dissimilar - none

9. Fst=0.04 (12 km) Fst=0.09 Fst=0.07 (14.5 km) Warsaw

10. NGS enables typing of complex, multilocus MHC systems (Babik et al. 2009) From Axter and Sommer 2007; left: from Wegner 2009)

11. From Mardis 2008

12. 74 alleles detected, 58 potentially functional included into further analyses Only 8 most common alleles included into models predicting parasite load

14. GxE coupled with moderate population structure – another mechanism protecting MHC polymoprhism?

15. Why are MHC genes so polymorphic? heterozygote advantage: production of two different MHC proteins enables presentation of more antigens to the lymphocytes frequency-dependent selection: selection on fast evolving parasites increases frequency of forms that escape presentation by the most common MHC genotypes sexual selection: MHC-dissimilar individuals preferred as mates GxE

16. MHC often multiplicated • Within-population cnv • What limits individual MHC diversity (given potential for wider binding range)? Number of DRB seqences/individual bank vole

17. Optimal MHC diversity mediated by a tradeoff associated with possessing large number of alleles (Nowak et al. 1992): + allows presentation of wider array of anigens - decreases a TCR repertoire during negative selection From Woelfing et al. 2009

19. Does balancing selection maintain MHC variation is small populations? www.mysciencebox.org/ island fox (Urocyon littoralis):some MHC variationmaintained despite depletion of neutral variation (Aguillar et al. 2004)

21. Changes in spotted suslik population numbers over last 50 years in SE Poland 1954 143 populations (Surdacki 1963) 1961 81 populations (Surdacki 1963) 1985 32 populations (Męczyński 1991) 2003 7 populations (Męczyński 2004)

22. Spotted suslik habitat

23. Spotted suslik range

24. Spotted suslik range R=2.3 R=4.5

25. Sampling sites

26. Methods: sampling

27. Methods: sampling

28. Methods: sampling

29. Methods: Genetic markers PCR amplification of exon II of MHC DRB gene (201 bp) Alleles separated with SSCP on Elchrom gels, reamplification from bands and sequencing Population genotyping: SSCP in ABI3130 All new alleles confirmed by sequencing from two independent PCR 11 microsatellite loci (Gondek et al. 2006, Hanslik i Kruckenhauser 2000, May et al. 1997). Alleles separated in ABI310

30. Results: diversity at MHC and microsatellites correlated

31. Results: no evidence for recent selection on MHC • Population differentiation (measured by FST) should be lower for loci under balancing selection, and higher under diversifying selection • 95% limits obtained from computer simulations with Fdist (Beaumont & Nichols 1996) diversifying DRB balancing

32. MHC-DRB in S. suslicus: conclusions MHC DRB historically under positive selection No evidence for recent selection MHC diversity correlated with neutral diversity Selection on MHC too weak to resist drift?

34. Should we expect balancing selection to maintain MHC variation is small populations? Simultaneous action of heterozygote advantage and freqency-dependence in small populations leads to higher initial depletoin of MHC polymorphism than drift (Ejsmont and Radwan, 2010, Conservatiom Genetics)

35. MHC alleles able to present a novel antigen unavailable when a new parasite emerges Parasites adapt easier when number of host genotypes limited Hughes (1991): Maintenance of MHC variation should be one of main aims of genetic rescue programs Loss of MHC diversity and conservation

36. Does the decreased MHC diversity threaten survival of bottlenecked species? European bison threatened by posthitis – a disease causing male infertility Only four MHC-DRB alleles (n=172), compared to >100 in cattle ds/dn<1, evidence of historical selection on DRB No association of DRB alleles or their heterozygosity with posthitis or intensity of infection with invasive nematode Asworthius sidemi Radwan et al. 2007. Mol. Ecol.16: 531-540

37. Does the decreased MHC diversity threaten survival of bottlenecked species? European bison threatened by posthitis – a disease causing male infertility Only four MHC-DRB alleles (n=172), compared to >100 in cattle ds/dn<1, evidence of historical selection on DRB No association of DRB alleles or their heterozygosity with posthitis or intensity of infection with invasive nematode Asworthius sidemi MHC unimportant, or alleles conferring resistance lost during bottleneck? Radwan et al. 2007. Mol. Ecol.16: 531-540

38. species with reduced genetic MHC variation appear moresusceptible to infection cheetah – feline peritonitis (O’Brien 1990) Tasmanian devil – transmissible tumor (Siddle et al. 2007) but some heavily bottlenecked populations increase despite depletion of MHC polymorphism beaver (Ellegren et al. 1993, Babik et al. 2005) MHC and conservation

40. DRB in beaver historically under positive selection, yet most populations now monomoprhic

41. MHC and neutral variation in the great crested newt, Triturus cristatus Reasons to study MHC variation in great crested newt: Long-standing inter-population differences in the level of genetic diversity: vast majority of current range of postglacial origin (low mtDNA and allozyme diversity), but refugial populationsstill present Amphibians have been declining worldwide at an unprecedented rate

42. Causes of amphibian decline • Habitat loss and fragmentation • Environmental pollution • UV-B • Invasive species • Climate change • Emerging pathogens:chytrid fungus Batrachochytrium dendrobatidis

43. MHC and neutral variation in the great crested newt, Triturus cristatus: samples PGE 20 PGE: postglacial expansion area, characterised by low mtDNA and allozyme variation REF: refugial area 24 19 37 REF 5 19 5 8

44. MHC in T. cristatus: variation in β chain exon 2 dn/ds= 5.0 at ABS of expressed locus, indicating historical selection Pseudogenes evolved neutrally ? PS II PS III DAB PS I Babik et al. 2009, Mol Ecol, 18: 769-81

45. T. cristatus: MHCII diversity within populations All 43 sequences found in the species are present in REF populations Only six sequences in 63 individuals from PGE populations The same two expressed alleles present in all PGE populations E G P REF PS I - III DAB Babik et al. 2009, Mol Ecol, 18: 769-81

46. MHC variation in T. cristatus – conclusions • Expressed MHC alleles are highly polymorphic in refugial area, most likely due to balancing selection revealed by sequence evolution • Low MHC diversity in northern populations is likely to result of a founder effect during postglacial colonisations • all populations carry the same two DAB alleles • similar pattern occurs for mtDNA, allozymes, microsatellites and MHC pseudogenes • Since postglacial expansion (about 10 000 y ago) no apparent population declines reported

47. Implications for conservation Long-term survival despite low MHC diversity: MHC less important than inbreeding? But: emergent pathogens may make monomorphic populations susceptible (e.g. chytrid fungus) bias in the data: it is easier to document low MHC diversity in species which survied than in extinct ones needed: data on MHC diversity vs parasite load Conservation of MHC diversity and avoidance of inbreeding depression can sometimes be achieved simultaneously (eg.refugial area), but conflicts may occur (captive breeding)

48. Acknowledgements Collaborators: Ola Biedrzycka, Wiesiek Babik, Agnieszka Kloch, Maciek Pabijan, Pim Arntzen, Dan Cogălniceanu, Walter Durka, Jan Wójcik, Agata Kawałko, Anna Bajer, Edward Siński Funding:, Alexander von Humboldt Foundation grant 3-Fokoop-POL/1022634, Ministry of Science and Higher Education grant 0494 ⁄ P04 ⁄ 2005 ⁄, Foundation for Polish Science

49. Examples of endangered species where the effects of parasites on survival or population dynamics have been documented

50. Methods: primer design ? ? Known sequence Unknown primer-binding sequences • Primers located in conservative regions (as determined on the basis of mamalian sequences) of MHCII DRB locus, coding for peptide binding region • Design of suslik-specific primers with vectorette (bubble) PCR