DNA extraction and genotyping
DNA was isolated from the sediment by a standard phenol/chloroform
protocol, and then treated with cetyltrimethylammonium bromide (CTAB) to
remove PCR inhibitors as previously described (Ausubel, 1987; Blanton et
al., 2011). For genotyping, duplicate 2 µL of DNA samples per primer
pair was used to PCR amplify 15 polymorphic microsatellite loci with a
SeqStudio-3200 Genetic Analyzer (Thermo Fisher, Carlsbad, CA, USA).
Allele peaks were identified and measured using Peak Scanner software
version 2.0 online workstation (Thermo Fisher, Carlsbad, CA, USA), and
the data were transferred to an Excel template for processing. Data
trimming and organization were automated by custom designed macros.
Allele peaks not conforming to the stepwise mutation model and peaks
<100 pixels in height were excluded from analysis. Allele
frequencies were calculated based on the ratio between each allele
peak´s height and the sum of allele peak heights for each microsatellite
locus (Barbosa et al., 2016). Microsatellite loci and samples with less
than 50% genotyping success were excluded from analyses (Table S1).
Final analyses were performed for 51 infrapopulations (parasites
aggregated within one host) and 15 loci (smms2, smms13, smms16, smms3,
smms17, smms18, smms21, smda23, sm13-478, 1f8a, 29e6a, smu31768,
lg3_sc36b, sc23b, smd28 (Kovach et al., 2021).