2.4.1 Assignment to hybrid classes: Hybrid Index (HI) and
Heterozygosity (Het)
We used the R package INTROGRESS v1.2.3 (Gompert & Alex Buerkle, 2010)
to calculate individual introgression coefficients; hybrid index
(HI-values) and individual heterozygosity (HET-values), and used both of
them to classify individuals into different hybrid classes (c.f. Jordan
et al., 2018). INTROGRESS was used with the subset of 381
species-specific SNPs, as the assignment to hybrid classes can be
inexact when using nondiagnostic markers (Buerkle, 2005). INTROGRESS
when using species-specific allele SNPs, calculates the hybrid index as
the proportion of alleles inherited from one species, and the
heterozygosity, as the proportion of alleles that are heterozygous,
ranging from 0 (pure species) to 1 (F1 hybrids) because
of pure species are 100% homozygous, while F1 hybrids
are 100% heterozygous (Gompert & Buerkle, 2010). Thus, the HI-value
gives the proportion of alleles inherited from one species, in this caseI. elegans (e.g. 1.00=100% I. elegans , and 0.00 =100%I. graellsii , alleles), whereas HET-values, which range from 0.00
to 1.00 (0.00=all sites are homozygous, 1.00=all sites are
heterozygous), indicate the timing of the hybridization event. First
generation hybrids (F1 individuals) are expected to be
heterozygous at all species-specific SNPs, while later-generation
hybrids and backcrosses will have a lower heterozygosity levels, and the
HI-values of F1 and F2 individuals will
be close to 0.5, while backcrosses will have a HI-value below (or up to)
0.5 (Fitzpatrick, 2012). However, because of the lack of pure I.
elegans and pure I. graellsii in the three hybrid regions
[only 3 out of the 102 individuals were pure I. elegans (2) or
pure I. graellsii (1)], criteria for F1 and
F2 hybrid classes were relaxed. Thus, we classified
individuals into eight parental and hybrid classes (cf., Milne &
Abbott, 2008; Walsh et al., 2015): (i) pure I. elegans (HI=1.000;
HET≤0.000), (ii) pure I. graellsii (HI=0.000; HET≤0.000), (iii)
introgressed-elegans (HI=0.900-0.999; HET≤0.118), (iv)
introgressed-graellsii (HI=0.001-0.100; HET≤0.118), (v)
backcross-elegans (HI=0.601-0.899; HET=0.118-0.449), (vi)
backcross-graellsii (HI=0.101-0.399; HET=0.118-0.449), (vii)
relaxed F1 hybrids (HI=0.400-0.600; HET≥0.600), and
(viii) relaxed F2 hybrids (HI=0.400-0.600;
HET=0.450-0.599).
To investigate whether the hybrid regions have similar proportions of
hybrid classes, the observed hybrid-class distributions of individuals
from north-central and north-west (north-east was not included in the
statistical analyses due to small sample sizes) were used to estimate
the predicted distribution of individuals in admixture-classes using a
contingency table (assuming random distribution) and then compared to
the observed admixture-class distribution in both hybrid regions.
Z-tests with Yates’s corrections for small sample sizes were used to
test for differences in the proportions of each hybrid class category
between north-west and north-central hybrid regions.
The twelve analyzed populations from the Spanish hybrid zone were
assigned to three qualitative measures that reflects their genotypic
compositions. This classification depends on the frequency distribution
of the different hybrid classes: 1) unimodal hybridization pattern, when
the distribution spans a range of admixture and backcrosses towards both
parental species; 2) bimodal hybridization pattern, when the
distribution is deviated to the two parental or parental-like genotypes,
and few hybrids (F1 and F2 hybrids) are
present (Jiggins & Mallet, 2000); and 3) introgressed hybridization
pattern when the distribution was deviated to one parental-like
genotype.