Electronic supplementary material
Appendix 1 – Methods: Unique primer tags
M13f1 atcgtcgGGAAACAGCTATGACCATG
M13f2 atcgtgcGGAAACAGCTATGACCATG
M13f3 atcgctgGGAAACAGCTATGACCATG
M13f4 atcgcgtGGAAACAGCTATGACCATG
M13f5 atcggtcGGAAACAGCTATGACCATG
M13f6 atcggctGGAAACAGCTATGACCATG
M13f7 atctgcgGGAAACAGCTATGACCATG
M13f8 atctggcGGAAACAGCTATGACCATG
M13f9 atctcggGGAAACAGCTATGACCATG
M13f10 atccgtgGGAAACAGCTATGACCATG
M13f11 atccggtGGAAACAGCTATGACCATG
M13f12 atcctggGGAAACAGCTATGACCATG
M13f13 atgctcgGGAAACAGCTATGACCATG
M13f14 atgctgcGGAAACAGCTATGACCATG
M13f15 atgcctgGGAAACAGCTATGACCATG
M13f16 atgccgtGGAAACAGCTATGACCATG
M13f17 atgcgtcGGAAACAGCTATGACCATG
M13f18 atgcgctGGAAACAGCTATGACCATG
M13f19 atgtccgGGAAACAGCTATGACCATG
M13f20 atgtcgcGGAAACAGCTATGACCATG
M13f21 atgtgccGGAAACAGCTATGACCATG
M13f22 atggctcGGAAACAGCTATGACCATG
M13f23 atggcctGGAAACAGCTATGACCATG
M13f24 atggtccGGAAACAGCTATGACCATG
M13f25 attcgcgGGAAACAGCTATGACCATG
M13f26 attcggcGGAAACAGCTATGACCATG
M13f27 attccggGGAAACAGCTATGACCATG
M13f28 attgccgGGAAACAGCTATGACCATG
M13f29 attgcgcGGAAACAGCTATGACCATG
M13f30 attggccGGAAACAGCTATGACCATG
M13f31 actgtcgGGAAACAGCTATGACCATG
M13f32 actgtgcGGAAACAGCTATGACCATG
M13f33 actgctgGGAAACAGCTATGACCATG
M13f34 actgcgtGGAAACAGCTATGACCATG
M13f35 actggtcGGAAACAGCTATGACCATG
M13f36 actggctGGAAACAGCTATGACCATG
M13f37 acttgcgGGAAACAGCTATGACCATG
M13f38 acttggcGGAAACAGCTATGACCATG
M13f39 acttcggGGAAACAGCTATGACCATG
M13f40 actcgtgGGAAACAGCTATGACCATG
M13f41 actcggtGGAAACAGCTATGACCATG
M13f42 actctggGGAAACAGCTATGACCATG
M13f43 acgttcgGGAAACAGCTATGACCATG
M13f44 acgttgcGGAAACAGCTATGACCATG
M13f45 acgtctgGGAAACAGCTATGACCATG
M13f46 acgtcgtGGAAACAGCTATGACCATG
M13f47 acgtgtcGGAAACAGCTATGACCATG
M13f48 acgtgctGGAAACAGCTATGACCATG
M13f49 acgcttgGGAAACAGCTATGACCATG
M13f50 acgctgtGGAAACAGCTATGACCATG
Appendix 2 – Results: 454 sequencing artefact removal
A total of 98,081 reads (sequences) conformed to our initial quality criteria; with complete forward
and reverse unique identifying tags and no N calls. These represented 6,465 unique DNA sequences.
True alleles were separated from sequencing artefacts by two criteria. Firstly a sequence had to be
present in products from at least two PCRs and represented by at least three or more reads in at least
one of the PCRs. This criterion is conservative as it automatically excludes alleles present in a single
individual regardless of the number of reads. However, this is necessary to exclude artefacts produced
during PCR, which is a standard procedure in MHC studies (Babik 2010). This criterion reduced the
data set to 75,819 reads of 392 unique sequences. The next step was to remove unique sequences
below the 3% threshold for mean frequency per individual (Babik et al. 2009), probably representing
artefacts resulting from the sequencing process. This reduced the data to 63,888 reads of 54 “true”
alleles. Samples with less than 50 reads were not considered as a low number of sequences may
induce incomplete genotyping (Babik et al. 2009; Galan et al. 2010), representing 18 of the 183
individuals. Three of the 54 alleles showed possessed stop codons within the sequences and were
removed from further analyses as they were deemed potentially pseudogenes.
Babik, W. 2010 Methods for MHC genotyping in non-model vertebrates. Molecular Ecology
Resources 10, 237-251.
Babik, W., Taberlet, P., Ejsmond, M. J. & Radwan, J. 2009 New generation sequencers as a tool for
genotyping of highly polymorphic multilocus MHC system. Molecular Ecology Resources 9,
713 - 719.
Galan, M., Guivier, E., Caraux, G., Charbonnel, N. & Cosson, J. F. 2010 A 454 multiplex sequencing
method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies.
BMC Genomics 11, 296.
All samples
(genomic DNA)
Group 1
adapter-A
primerF
Seq.
primerR
A-adapter
Group 2
1st PCR
adapter-T
primerF
Seq.
primerR
T-adapter
Dilute product 1:20
Group 1a
Group 2a
Group 1b
Group 2b
2nd PCR
primer1
primer1
primer1
primer1
primer1
primer1
primer1
primer1
primer2
primer2
primer2
primer2
primer2
primer2
primer2
primer2
Pool A
Pool B
(approx. equimolar quantities)
(approx. equimolar quantities)
tag1
tag1
tag1
tag1
tag1
tag1
tag1
tag1
454 DNA
library
preparation
tag2
tag2
tag2
tag2
tag2
tag2
tag2
tag2
Final Pool
454 sequencing
Separate individuals by unique
primer/adapter/tag combination
= individual 1
= individual 2 etc.
Figure S1. Diagrammatic representation of parallel genotyping, via 454 sequencing, implementing a
minimal number of unique primers.
Figure S2. Alignment of MHC class I exon 3 amino-acid sequences of Kryptolebias marmoratus.
Dots indicate identity with the amino-acid sequence of allele S1-01. Grey highlights indicate sites
under positive selection identified by likelihood analysis.
A
B
C
Figure S3. Results of cluster analysis (Paired linkage a) Pearson’s correlation, b) Euclidean, c) Cosine
distance measure) defining supertypes, derived from physicochemical properties of amino acids at 11
positively selected codon sites. Significant bootstrapping values (n = 1000) indicated.
Table S1. Initial generalized linear models aimed at evaluating the effect of the number of MHC
supertypes on parasite loads. Model selection was then applied for the best fit of the data according to
AICc values.
d.f.
F
P
a) Total parasite load
No. of MHC supertypes (MHC)
Sampling site (S)
Heterozygosity (HL)
Fish length (L)
1
3
1
1
0.002
0.955
0.012
0.003
0.965
0.435
0.914
0.955
MHC2
MHC x S
MHC x HL
MHC x L
S x HL
SxL
S x MHC2
HL x L
HL x MHC2
L x MHC2
1
3
1
1
3
3
3
1
1
1
0.009
0.486
0.005
0.003
0.077
0.713
0.483
0.262
0.002
0.004
0.927
0.697
0.945
0.955
0.972
0.557
0.698
0.615
0.968
0.948
b) Bacterial gill cysts
MHC
S
HL
L
1
3
1
1
1.236
0.143
0.233
0.494
0.281
0.933
0.635
0.491
MHC2
MHC x S
MHC x HL
MHC x L
S x HL
SxL
S x MHC2
HL x L
HL x MHC2
L x MHC2
1
3
1
1
3
3
3
1
1
1
1.203
1.125
1.343
0.743
1.166
1.153
1.043
0.465
1.291
0.694
0.287
0.365
0.262
0.400
0.350
0.355
0.397
0.504
0.271
0.416
c) Trichodinids
MHC
S
HL
L
1
3
1
1
0.007
0.250
0.165
0.016
0.935
0.860
0.690
0.902
MHC2
MHC x S
MHC x HL
1
3
1
0.032 0.861
0.176 0.911
0.169 0.686
MHC x L
S x HL
SxL
S x MHC2
HL x L
HL x MHC2
L x MHC2
1
3
3
3
1
1
1
0.008
0.156
0.174
0.175
0.087
0.202
0.000
0.931
0.924
0.913
0.912
0.772
0.659
0.993
d) Acanthocephalans
MHC
S
HL
L
1
3
1
1
0.116
3.291
0.491
0.304
0.737
0.044
0.493
0.588
MHC2
MHC x S
MHC x HL
MHC x L
S x HL
SxL
S x MHC2
HL x L
HL x MHC2
L x MHC2
1
3
1
1
3
3
3
1
1
1
0.292
1.806
0.610
0.418
0.431
1.521
1.732
0.029
0.281
0.622
0.596
0.182
0.445
0.526
0.733
0.243
0.196
0.867
0.603
0.441
Figure S4. Comparisons of parasite loads at each MHC supertype (mean ± 1 SE). Asterisks indicate
significant differences after Bonferroni corrections for multiple tests.