Four lineages can be identified in the A. viridifloracomplex
Individuals from twenty populations of the A. viridiflora complex
were sampled for resequencing. Additionally, to ensure that the species
complex with various phenotypes could be regarded as a monophyletic
group, we also sampled other sympatric wild columbine species with theA. viridiflora complex. Whole-genome sequencing of 80 individuals
from nine species was performed, and after filtering, we obtained
1,064,089 high-quality biallelic single nucleotide polymorphisms (SNPs).
We constructed the phylogenetic relationships among the Aquilegiaspecies through the ML and NJ methods based on nuclear genome SNPs. Both
topologies indicated that A. viridiflora , A. kamelinii andA. hebeica shared an MRCA with strong support and relationships
among species were close to each other (Figure S4). Therefore, 672,439
high-quality biallelic SNPs in the 20 populations of the A.
viridiflora complex were used to assess their evolution. Functional
annotations indicated that 55.604% of SNPs were located upstream and
downstream, while 17.454% were in intronic regions, and 7.07% were in
exonic regions of genes. The ML tree inferred from the above SNPS
indicated that the individuals of the A. viridiflora complex were
divided into four lineages (NE, EL, CN and NW): NE comprised A.
kamelinii and the individuals of A. viridiflora distribution in
northeastern China, EL comprised the individuals of A.
viridiflora and A. hebeica distribution in East Shandong South
Liaoning area, the individuals of A. hebeica distribution in
North China belonged to a single lineage (EL), and the individuals ofA. viridiflora distribution in northwestern China belonged to a
single lineage (NW). In this case, the A. viridiflora complex
showed a paraphyletic pattern, that is, NE and EL formed a sister clade,
and the other two lineages, CN and NW, were closely related (Figure 1C).
This revealed a different evolutionary history from the clusters based
on phenotypes.
The population genetic structure of the A. viridiflora complex
indicated that ancestral clustering at K = 4 was optimal
according to the cross-validation error rate (Figure S5). The result of
ancestral inference was obviously consistent with the geographical
distribution of the 20 populations and the phylogenetic relationships
detailed above. Individuals of SZ, LT, YM, XW, HL, HH and HD populations
in the contact regions of lineages showed multiple ancestral
compositions (Figure 1A and 1B), which might reflect recent gene flow
between these lineages. From the PCA plot, the first principal component
(PC1) and second principal component (PC2) explained 6.61% and 4.04%
of the observed variation, respectively. It also showed four distinct
lineages among the 20 populations, while individuals with multiple
ancestral compositions occupied an intermediate space in distinct
lineages (Figure S2B). The neighbor-net phylogenetic network depicted
these patterns by grouping the differential of NE and EL, while CN and
NW were not clearly differentiated, at the same time, it was also proven
that the above individuals had a mixed genetic background (Figure 2A).
In addition, we also detected a significant signal of hybridization in
the above populations at the individual level, in which P1 and P2 did
not belong to a group with hybrids (Table S2). Unsurprisingly, the
neighbor-net phylogenetic network based on 190 polymorphic sites in the
chloroplast genome also showed a little difference from that based on
genome polymorphisms resulting from hybridization and backcrossing of
hybrid lineages but still showed a paraphyletic pattern (Figure S6A).
Taken together, there are four lineages across the sampled A.
viridiflora complex through chloroplast and genome polymorphisms.
To understand the diversity patterns, the nucleotide diversity (π) of
the NE, EL, CN and NW lineages was calculated throughout the genome for
each 50 kb with a 10 kb step-size. Among the four lineages, lineage NW
showed the highest nucleotide diversity, and lineage EL showed the
lowest nucleotide polymorphism (Figure 2B). Based on chloroplast genome
polymorphisms, we detected 27 haplotypes among the 66 A.
viridiflora complex individuals. The haplotype diversity (hd) and
nucleotide diversity (π) for all individuals were 0.971 and 0.00022,
respectively. Among the four lineages, NW had the highest haplotype
diversity and nucleotide diversity (Table S3). Moreover, the haplotypes
in NW were in this network elsewhere, while the haplotypes in the other
groups were limited in the haplotype network (Figure S6B, Table S4).