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Online Supporting Information for Scordato and Kardish, Prevalence and turnover
in avian malaria
Figure S1: STRUCTURE plots based on microsatellite markers for k = 2. In P.
trochiloides (A), India is a separate genetic cluster from Kyrgyzstan and Siberia. In P.
humei, Siberia is a more distinct genetic cluster than India and Kyrgyzstan. Pairwise FST
values are given below the plots. All pairwise FST values are significant at p < 0.05
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Appendix S1
Microsatellite marker development for P. trochiloides
Microsatellites were developed from samples collected in Indian birds.
Approximately 3 ug of DNA were extracted using Qiagen DNEasy blood and tissue kits.
Isolated DNA was used to construct a genomic library enriched for DNA fragments
containing microsatellite repeats, as described in Price et al 2008. Primers were designed
for 10 loci, of which eight produced reliable genotypes.
We typed 141 P. trochioides used in this study at eight loci. Primers were
fluorescently labeled with FAM, HEX, or NED and run in multiplex. Samples were run
in 10l reactions with 1 l genomic DNA, 5 l HotStarTaq Plus Mastermix (Qiagen),
and 1 M of each primer. The thermocycler profile consisted of an initial denaturing step
at 95˚C for 5 minutes, followed by 35 cycles at 94˚C for 20 seconds, 20 seconds at the
appropriate annealing temperature, and 45 seconds at 72˚C, followed by an elongation of
10 minutes at 72˚C. Primers 9-16 were run at an annealing temperature of 65, 17-24 at an
annealing temperature of 68. Genotyping was conducted at the University of Chicago
Cancer Research Center.
Variability estimates and exclusion probabilities for each locus were determined
in Arlequin. After genotyping all three populations, it was found that two loci (Ptroch13
and Ptroch17) were not in Hardy-Weinberg equilibrium and were dropped from
subsequent analysis. Primer sequences are given in Table S1.
Detection of parasite infections
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To detect Haemoproteus and Plasmodium parasite infections, we used nested
PCR to amplify a 462 base pair portion of the cytochrome b gene from malaria
mitochondrial DNA (modified from Waldenström et al. 2004). Samples were run in 10l
reactions with 1 l genomic DNA, 5 l HotStarTaq Plus Mastermix (Qiagen), and 1 M
of each of the HAEMNF (5’-CATATATTAAGAGAATTATGGAG-3’) and HAEMNR2
(5’-AGAGGTGTAGCATATCTATCTAC-3’) primers. Samples were denatured at 95˚C
for 5 minutes, followed by 35 cycles at 94˚C for 30 seconds, 54˚C for 45 seconds, and
72˚C for 45 seconds, followed by an elongation of 10 minutes at 72˚C. We then added
1l PCR product to 5l HotStarTaq Plus Mastermix (Qiagen) and 1M of the primers
HAEMF (5’-ATGGTGCTTTCGATATATGCATG-3’) and HAEMR2 (5’GCATTATCTGGATGTGATAATGGT-3’). The second set of reactions were denatured
at 95˚C for 5 minutes followed by 35 cycles at 94˚C for 30 seconds, 58˚C for 45 seconds,
and 72˚C for 45 seconds, followed by an elongation of 10 minutes at 72˚C. We optimized
our PCR temperature protocol using positive controls provided by S. Bensch (pers.
comm.). We screened PCR products for presence of parasite DNA on 1.5% agarose gels;
positive samples produced a band of between 400 and 500 base pairs, whereas negative
samples produced no band. Positive samples were sequenced directly from the 5’-end
with the primer HAEMF on an ABI 3730XL DNA Analyzer (Applied Biosystems) at the
University of Chicago Cancer Research Center.
Phylogenetic and population genetic analysis
To assess phylogenetic relationships among malarial strains, parasite mtDNA
sequences were edited and aligned in CodonCode Aligner (CodonCode Corporation). All
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sequences were visually inspected for double peaks, which we considered indicative of a
mixed infection (n = 5). These sequences were not included in phylogenetic analysis. We
performed a BLAST search on all unique malaria haplotypes to identify previously
sequenced strains. Sequences for all unique strains are deposited in GenBank (Accession
nos. KJ396623-KJ396639). The phylogeny was assembled in MrBayes v. 3.1.2 (Ronquist
and Huelsenbeck 2003) using the General Time Reversible model with gamma
distributed rate variation among sites (GTR+G) as suggested by jModelTest 0.1.1
(Posada 2008). Two simultaneous runs were conducted with a sampling frequency of
every 250 generations over three million generations. We discarded 30% of the samples
as burn-in period and used Plasmodium falciparum (GenBank Accession no. AF069609)
as an outgroup. Trees were visualized in FigTree v. 1.3.1
(http://tree.bio.ed.ac.uk/software/figtree/). A minimum spanning tree constructed in
Arlequin v. 3.5 (Excoffier, Laval & Schneider 2005) was drawn by hand.
We analyzed host genetic structure by calculating pairwise FST in microsatellite
frequencies between adult birds in the three populations of P. humei and P. trochiloides
in Arlequin. We then assessed levels of genetic clustering among the three populations
using the Bayesian likelihood method implemented in STRUCTURE (Pritchard,
Stephens & Donnelly 2000), which calculates the posterior probability of individuals
belonging to one of k populations. We used an admixture model with correlated allele
frequencies, set lambda = 1, and allowed alpha to be determined from the data (Falush,
Stephens & Pritchard 2003). For each value of k between 1 and 5 we ran 5 simulations,
using a one million generation burn-in and two million generations to calculate posterior
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probabilities. We selected the most likely number of populations by calculating ∆K
(Evanno, Regnaut & Goudet 2005) with Structure Harvester (Earl and vonHoldt 2012).
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Table S1: Primer sequences and diagnostic information for eight new microsatellite loci
in P. trochiloides. Boldfaced primers were not in Hardy-Weinberg equilibrium in all
populations and were dropped from population genetic analyses.
Supplemental References
Earl, D.A. & vonHoldt, B. M. (2012) STRUCTURE HARVESTER: a website and
program for visualizing STRUCTURE output and implementing the Evanno method.
Conservation Genetics Resources, 4, 359–361.
Evanno, G., Regnaut, S. & Goudet, J. (2005) Detecting the number of clusters of
individuals using the software STRUCTURE: a simulation study. Molecular
Ecology, 14, 2611–2620.
Falush, D., Stephens, M. & Pritchard, J.K. (2003) Inference of population structure using
multilocus genotype data: linked loci and correlated allele frequencies. Genetics, 164,
1567–1587.
Posada, D. (2008) jModelTest: phylogenetic model averaging. Molecular biology and
evolution, 25, 1253–1256.
Price, T.D., Yeh, P.J. & Harr, B. (2008) Phenotypic plasticity and the evolution of
a socially selected trait following colonization of a novel environment.
American Naturalist, 172, S49-S62.
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Waldenström, J., Bensch, S., Hasselquist, D. & Östman, Ö. (2004) A new nested
polymerase chain reaction method very efficient in detecting Plasmodium and
Haemoproteus infections from avian blood. Journal of Parasitology, 90, 191–194.