3.2 Genomic diversity and differentiation in the H.
pungens species complex
Both SNPs and mtDNA datasets revealed
intermediate levels of genetic diversity but great levels of
differentiation among populations. Overall nuclear and mitochondrial
genetic diversity were high across the native range and low in the
non-native range (Table 1). Based on SNPs data, total mean expected
heterozygosity (He= 0.061) was three times greater than observed
heterozygosity (mean Ho= 0.029), probably as a consequence of
differentiation among populations (see below). Expected heterozygosity
varied greatly among populations ranging from 0.267 in the native range
(mean He= 0.102) to as low as 0.003 in the non-native area (mean He=
0.061). Allele richness was 20% higher in the native range (mean Ar =
1.240) than in recently invaded areas (mean Ar = 1.020), and no private
alleles were detected.
The analysis of pairwise Fst
values estimated for the SNPs dataset, considering the a prioridefined populations, sampling area, and host plant family, showed great
global genetic differentiation (Gst= 0.908). In the native range, Fst
estimates ranged from 0.602 for the comparison between BRC (Brazilian
mealybugs feeding on Cactaceae) and BRA (Brazilian mealybugs feeding on
Amaranthaceae and Portulacaceae) to 0.827 in the comparison between ARA
(Argentina mealybugs feeding on Amaranthaceae) and ARC (Argentina,
Paraguay and Australia mealybugs feeding on Cactaceae). Genetic
differentiation (Fst) in the non-native area varied from values as low
as 0.078 between PRA (Puerto Rican mealybugs feeding on Amaranthaceae or
Portulacaceae) and USA (United States mealybugs feeding on
Amaranthaceae) to as high as 0.969 between PRA and PRC (the Puerto Rican
cactus pest) (Table S3).
Clustering analyses also revealed strong population structuring in theH. pungens species complex, not necessarily associated with site
location. Indeed, sNMF and DAPC analyses suggested that the H.
pungens species complex comprises five ancestral populations / clusters
(K) based on cross-entropy and BIC values (Figure 2A and B,
respectively). The first cluster corresponds to what was previously
called H. pungens sensu stricto (Ar-A) (Poveda-MartÃnez et
al., 2019) that included Argentina (Ar) mealybugs feeding on
Amaranthaceae (A). The second cluster included Cactaceae (C) feeding
mealybugs native from Argentina, Paraguay (Pa), and mealybugs collected
in Australia (Au) (ArPaAu-C). The third cluster included populations of
Cactaceae feeding mealybugs from southern Brazil (Br) and the Puerto
Rican (PR) cactus pest (BrPR-C), whereas the fourth cluster encompassed
mealybugs feeding on Amaranthaceae and/or Portulacaceae (AP) species
from northeastern Brazil along with populations from Puerto Rico and the
United States (US) (BrPRUS-AP). The last cluster included mealybugs
collected on Amaranthaceae in southeastern Brazil (Br-A). Admixture
coefficients suggested no admixture between clusters and strong
population genetic structure. Pairwise Fst values considering the five
clusters described above revealed strong genetic differentiation, Fst
estimates ranged from 0.567 in the comparison between Ar-A and BrPRUS-AP
to 0.945 in the comparison between BrPRUS-AP and Br-A (Table S4).
Concerning the mtDNA dataset (Table
1), a total of 27 haplotypes were found in native and non-native ranges.
All haplotypes were present in the native range, 18 haplotypes were
detected in mealybug samples collected on Amaranthaceae and/or
Portulacaceae hosts and 9 on Cactaceae hosts (mean Hd= 0.744). As
expected, the number of haplotypes was substantially lower in non-native
sites, where only three haplotypes were found (mean Hd= 0.174), two in
samples collected on Amaranthaceae and/or Portulacaceae hosts and only
one on Cactaceae hosts.
Four haplogroups, separated by at
least two substitutions, can be recognized based on mtDNA data, each one
characterized by the type of host plant (Amaranthaceae and/or
Portulacaceae, or Cactaceae) and geographic origin (Figure 2C). The
first haplogroup (H1), which contained the most frequent haplotype
(haplotype MT138927) and has a star-like shape, is shared by
Amaranthaceae-feeding mealybugs collected in Argentina, and specimens
from Brazil, Puerto Rico and United States (Florida) feeding on
Amaranthaceae and/or Portulacaceae. The second haplogroup (H2), which
includes two haplotypes, both from mealybugs using Cactaceae as host
plants, one (haplotype MT138924) is shared by the mealybugs from
southeastern Brazil, Puerto Rico and United States (California), and the
other (haplotype MT138923) only found in southeastern Brazil. The
remaining two haplogroups are highly differentiated from H1 and H2. H3
included Cactaceae-feeding mealybugs collected in Argentina, Australia
and Paraguay, and H4 Amaranthaceae-feeding mealybugs from southeastern
Brazil.