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-Martínez 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.