2.2. Reproductive isolation
Mating incompatibility between I. graellsii males and I.
elegans and hybrid females is known in populations from the north-west
hybrid region (Sánchez-Guillén, Wellenreuther, & Cordero-Rivera, 2011),
but not its potential temporal and spatial variation. Thus, the strength
of the reproductive barriers involved in preventing copulation in
north-central was used to test temporal and/or spatial variation.
Similar data from north-west populations are available from an
unpublished study (Arce-Valdés, Ballén-Guapacha, Chávez-Rios &
Sánchez-Guillén, under review).
For reproductive isolation analysis, last instar larvae of approximately
200 I. elegans and 200 I. graellsii individuals, both from
north-central Spain (Mateo, Valbornedo and Villar), were sampled in
June-July of 2016-2017. Larvae were transported to the laboratory and
maintained until adulthood (for details about larval rearing methodology
see Sánchez-Guillén et al., 2005; Van Gossum, Sánchez-Guillén &
Cordero-Rivera, 2003). Mechanical isolation to reach the copulation was
estimated by measuring incompatibility between the male cerci and female
prothorax (males attempt to grasp females) and the incompatibility
between male-female genitalia (both genitalia come into contact), in
heterospecific crosses of I. elegans with I. graellsiifemales, and I. graellsii males with I. elegansfemales. Additionally, hybrids in the laboratory (from crosses betweenI. elegans and I. graellsii ) were used to produce
backcrosses to measure mechanical isolation in both directions (hybrid
males with I. elegans females and I. elegans females with
hybrid females; and hybrid males with I. graellsii females andI. graellsii males with hybrid females).
2.3 Genome-wide SNP markers
2.3.1 DNA extraction and RAD library
preparation
Genomic DNA from head and thorax tissues of 187 individuals from the 20
populations (6-10 individuals per site; Table 1) was extracted with
Qiagen DNeasy Blood & Tissue Kit. Extracted DNA was quantified using
Nanodrop and visually controlled for DNA degradation using a 1% agarose
gel. Five single-digest RAD DNA libraries were processed according to
the protocol implemented in Etter et al. (2011) and modified in Dudaniec
et al. (2018). All samples, plus five sample replicates, were
distributed across the five separately prepared RAD libraries with 40
unique barcodes used per library (sourced from Metabion). Each library
was paired-end sequenced (2*100 bp) on a separate lane of an Illumina
HiSeq 2500 at SNP&SEQ Technology Platform at Uppsala University,
yielding 180 million read pairs per lane (i.e. per library).
2.3.2. Quality checking
and SNP calling
Libraries were processed using pipelines within STACKS v2.2 (Catchen,
Hohenlohe, Bassham, Amores, & Cresko, 2013; Catchen, Amores, Hohenlohe,
Cresko, & Postlethwait, 2011). Raw reads were demultiplexed with
process_radtags and PCR clones were identified and discarded with
clone_filter using default parameters. Sequence reads were aligned to
the I. elegans draft genome assembly (Chauhan et al., 2021) using
BOWTIE2 v.2.3 (mismatch allowance per seed alignment of 1, maximum
mismatch penalty of 6 and minimum of 2, maximum fragment length of 1000
bp and minimum of 100 bp, Langmead & Salzberg, 2012). We used the
ref_map pipeline to detect SNPs using default parameters. Only SNPs
with a minor allele frequency of >0.05 and a maximum
observed heterozygosity of 0.7 were retained. Moreover, the locus had to
occur in 80% of the individuals in a population and in 18 of the 20
populations to be included in the final SNP set. SNP markers were
filtered to include only a single random SNP on each RAD tag to create a
data set without closely linked loci (using the write_single_snp
option in STACKS). Finally, by using the I. elegans reference
genome (Chauhan et al., 2021) SNPs were filtered to include only those
located on autosomal scaffolds.
Exploratory analyses of population structure revealed possible
hybridization in two of the I. graellsii samples from Seyhouse
(Algeria); probably with a third Ischnura species (see
Supplementary Figure S1). These two samples were removed from further
analyses leaving the final total sample size of 185 (Table 1).
2.3.3. Identification of
species-specific loci
Diagnostic species-specific loci are powerful markers for assigning
later generation hybrids and detecting introgressed alleles in
population genetic studies (Hohenlohe, Amish, Catchen, Allendorf, &
Luikart, 2011). To provide a list of species-specific markers,
alternatively fixed SNPs between the parental species from the
allopatric distribution (n=43 I. elegans and n=25 I.
graellsii ) were identified using VCFtools v0.1.16 (Danecek et al.,
2011). SNPs for each of the two allopatric regions that had only one
allele (–max-maf 0) were selected, and then, shared loci
between the two allopatric regions were found using the intersect(
) function of R (R Core Team, 2016). Following that, we applied to
those loci the Hardy-Weinberg test implemented in VCFtools
(–hardy ), and removed loci fixed for the same allele in the
two species (HE=0). The remaining 381 SNPs (out of the
5,702 SNPs) were considered as the species-specific markers set.