Microsatellite genotyping
As published microsatellite loci for titi monkeys (Martins, 2015;
Mendoza et al., 2015; Menescal, Gonçalves, Silva, Ferrari, & Schneider,
2009) revealed unreliable results for our study species, we established
a new set of 27 di-repeat microsatellite loci that can be universally
applied to New World monkeys (details are described in Supplementary
Methods and Supplementary Tables 3–4). To simplify library preparation
for genotyping by sequencing, we added adapter nucleotide sequences to
the 5’ end of the locus-specific primers.
We amplified all 27 loci in single multiplex PCR reactions using the
Qiagen Multiplex PCR Kit (Qiagen) with a total volume of 25 µL and
containing 12.5 µL 2x Multiplex Master Mix, 1 µL of primer pool (0.2 µM
of each primer), 1 µL of DNA extract (ca. 200 ng total DNA) and 10.5 µL
of RNase-free water. Amplifications were performed with initial
denaturation at 95°C for 15 min, 40 cycles of denaturation at 94°C for
30 sec, annealing at 57 °C for 1.5 min, extension at 72 °C for 1 min and
a final extension at 60°C for 30 min. PCR products were checked on 1.5%
agarose gels together with non-template controls. To prevent false
homozygosity due to allelic dropout, we repeated each multiplex reaction
three times (Barbian et al., 2018). In some samples, the total multiplex
reaction with all 27 loci yielded low number of sequencing reads; in
these cases, we additionally amplified the loci in 3 separate multiplex
reactions with the following primer pools: chr01b–chr07a,
chr08a–chr12a, chr12b–chrXa, as this method usually yielded more
reads (see Supplementary Materials and Methods for details). The
reactions and PCR condition for 3 separate multiplex reactions were the
same as for the total multiplex reaction.
Following amplification, we pooled 5 µL of each multiplex PCR product
(or of each PCR product of 3 separate multiplex reactions), purified the
pooled products with the Monarch PCR & DNA Cleanup Kit (New England
BioLabs) and quantified them using Qubit Fluorometer (Thermo Fisher). To
prepare sequencing libraries, we performed indexing PCRs using Kapa HiFi
Hotstart ReadyMix PCR Kit (Roche) with a total volume of 25 µL
containing 12.5 µL 2x Kapa HiFi Hotstart ReadyMix, 1 µL (0.5 µM) of each
indexing primer (containing individual barcodes) and 100 ng purified PCR
product. Indexing PCRs were done with an initial denaturation step at
98°C for 45 sec, followed by 4 cycles of denaturation at 98°C for 15
sec, annealing at 62°C for 30 sec and extension at 72 °C for 30 sec, and
a final extension step at 72°C for 1 min. Full-length libraries were
purified with the Monarch PCR & DNA Cleanup Kit (New England BioLabs)
and quantified using Qubit Fluorometer (Thermo Fisher). Fragment sizes
and molarities were quantified using Bioanalyzer 2100 (Agilent
Technologies). Libraries were diluted to 10 nM and then pooled and
sequenced using Miseq Reagent Kit v2 with PhiX DNA (Illumina) added on
the MiSeq system (Illumina). Sequencing was performed with 51 forward
and 251 reverse read cycles. Only the reverse reads were used for
further analysis, while forward reads were only used for MiSeq quality
control.
After sequencing, the samples were demultiplexed using MiSeq Reporter
software and then processed using the CHIIMP analysis pipeline (Barbian
et al., 2018). The CHIIMP pipeline calls alleles by first producing
unique sequences with relevant attributes (read counts, sequence
length, etc.) for each MiSeq sequence file, querying the sequences for
potential PCR artifacts, such as stutter sequences, and then removing
all sequences that do not match the locus attributes. All alleles called
by CHIIMP were manually checked to validate the results and to correct
automated allele calling for those loci that contain “wobble”
positions in the primer sequences and are incorrectly processed by
CHIIMP.
To control for possible misidentification of animals in the field, we
genotyped most individuals from 2–3 independent samples. We also used a
PCR-based sexing assay (Di Fiore, 2005) to confirm reported sex (and to
sex young individuals for whom sex could not be identified in the
field). To control for laboratory mistakes, we genotyped each extraction
twice.
Of 27 loci, 9 either consistently failed to amplify in our study animals
(chr06b, chr11f, chr16b) or proved to be monomorphic (chr02a, chr02b,
chr04a, chr10b, chr12a, chr13b) and were excluded from further analysis.
The final set consisted of 18 loci, including 17 autosomal and one
X-linked locus (chrXa) (Supplementary Table 5). All animas were
genotyped at least at 10 loci (16.5 loci on average).
As X-linked loci are haploid in males, we performed all of the following
summary statistics tests using data from both sexes for autosomal loci
and using data only from females (diploid for X-linked loci) for locus
chrXa.
We checked all loci for the presence of null alleles, allelic dropout
and stuttering using Micro-Checker 2.2.5 (Van Oosterhout, Hutchinson,
Wills, & Shipley, 2004). We assessed Hardy–Weinberg equilibrium (HWE)
and calculated observed and expected heterozygosity with PopGenReport
2.2.2 (Adamack & Gruber, 2014). Since the presence of family structure
can cause deviations from HWE and bias population genetic analyses,
especially in monogamous species, we only included adults in this
analysis. The analysis indicated that the population was in HWE. Two
loci, chr01b and chr21a, departed from HWE, likely due to the presence
of relatives in a study group and/or small sample size.
One of these two loci, chr01b, also showed evidence of null alleles.
However, this locus did not show any mismatches for the known
mother/offspring dyads (see below). Consequently, we ran all further
analyses using two sets of data, one with the full set of loci and
another one with locus chr01b excluded. Since the results from these two
sets did not differ substantially, we present all further results only
for the reduced data set.