DNA extraction, nSSR genotyping and cpDNA sequencing
DNA extraction . Total genomic DNA was extracted using the MATAB
protocol and chloroform separation with slight modifications following
Mariac et al . . We checked DNA quality and quantity using a
NanoDropTM One spectrophotometer (Thermo
ScientificTM). Extracted DNA was diluted in 50 μL
Tris-EDTA (TE) buffer (1X) pH 8.0 and stored at −20°C.
nSSR genotyping . All individuals were genotyped using 14
polymorphic nuclear nSSR markers, with PCR conditions as described in
Kusuma et al. . As one of the primer pair (Myr 42 locus)
allowed for the amplification of two different genomic regions,
corresponding information was split into two, namely Myr 42a andMyr 42b (see Kusuma et al ., 2020 for details). From this,
we obtained our final dataset of 15 diploid genotypes for each
individual. Genotyping was done on an ABI 3500 XL (Applied Biosystems)
at CIRAD (Montpellier, France) using 1 μL of PCR product, 12 μL of Hi-Di
Formamide (Applied Biosystems) and 0.3 μL GeneScan™ 500 LIZ™ dye Size
Standard (Applied Biosystems). Genotypes were analyzed for each locus
using the automatic procedure implemented in Geneious
Prime® v.2022.2.2 and manually corrected.
cpDNA sequencing . We selected three to five individuals per
subpopulation for genomic library construction (N = 90; see
Supplementary File 1 for details) as well as four additional individuals
of the sister species Myristica argentea (commonly known as the
Papuan nutmeg, from Fakfak, Papua, Indonesia) that has been used as an
outgroup in phylogenetic network analyses (N = 94 in total; see
Supplementary File 1 for details). The protocols for library
preparation, chloroplast enrichment, multiplexing, and sequencing are
described in Mariac et al . . All sequenced individuals were
analyzed as described in Scarcelli et al. . In short, data were
demultiplexed and cleaned using the scripts DEMULTADAPT
(https://github.com/Maillol/demultadapt)
and CUTADAPT v.1.2.1 . Reads were then filtered using a PERL script
(Supplementary File 2). After discarding low quality reads, mapping was
performed with BWA v.0.7.12 using the M. fragrans cpDNA genome as
reference. Unmapped reads were discarded and mapped reads were sorted
using SAMTOOLS v.1.9 . Then VARSCAN v.2.3.7 was used to call Single
Nucleotide Polymorphisms (SNPs). All SNPs were filtered using VCFtools ,
by deleting all the indels, and all non-biallelic SNPs. Then, we
calculated the mean depth per individual, mean depth per SNP, percentage
of missing data per individual, and percentage of missing data per SNP
using VCFtools . We provide the command lines and scripts to prepare
sequence data, mapping and calling of SNPs in Supplementary File 3.
Genetic diversity and population structure of M. fragrans