3.4 Species tree reconstruction and species delimitation analysis
Both ML and BI phylogenetic analyses and the species tree inferred by SNAPP, all of them based on the SNPs dataset, revealed five major well supported clades (Figure 3A and C). The first and second clades, both feeding on Amaranthaceae and/or Portulacaceae, appear closely related, the first encompassing H. pungens sensu stricto (clade Ar-A) and, the second, mealybugs from northeastern Brazil, Puerto Rico and United States (Florida) (clade BrPRUS-AP), respectively. The third and fourth clades comprised cactus feeding mealybugs: the third from Argentina, Paraguay and Australia (clade ArPaAu-C) and the fourth from southern Brazil and Puerto Rico (clade BrPR-C). The fifth clade was an independent branch formed by Amaranthaceae feeding mealybugs collected in southeastern Brazil (clade Br-A) (Figure 3A and C).
The phylogenetic tree obtained with the mtDNA dataset showed contrasting results with respect to nuclear SNPs (Figure 3B). Four major clades were observed in ML and BI trees, one encompassing H. pungens sensu stricto with populations from northeastern Brazil, Puerto Rico and the United States (Florida) feeding on Amaranthaceae and/or Portulacaceae (clade ArBrPRUS-AP). The clade including cactus feeding mealybugs from southeast Brazil and Puerto Rico (clade BrPR-C) appears as the sister clade of ArBrPRUS-AP. The third clade included populations from Argentina, Paraguay and Australia feeding on Cactaceae (clade ArPaAu-C) that appeared close to Amaranthaceae feeding mealybugs from southeastern Brazil (clade Br-A) (Figure 3B).
Species delimitation analysis based on the BFD method using SNPs data supported a model in which H. pungens was a complex of five species (Model 1, Table S5), supporting the picture depicted by the phylogenetic tree and species tree produced with SNPs data (Figure 3A and C). This means that the clade which included H. pungens sensu stricto (Ar-A) and the clade comprising mealybugs from northeastern Brazil, Puerto Rico and the United States (Florida) feeding on Amaranthaceae and/or Portulacaceae (BrPRUS-AP), as well as the clade of Amaranthaceae feeders from southeastern Brazil (Br-A) may be considered as three separate species. Moreover, the latter appeared as the sister clade of the two clades of cactus feeders, one from Argentina, Paraguay and Australia (clade ArPaAu-C) and the other from southeastern Brazil and Puerto Rico (clade BrPR-C).
GYMC and bPTP, the single locus species delimitation methods based on mtDNA, recovered six (GYMC) and 10 (bTPT) groups (Figure S1). In both cases, GYMC and bPTP delimited four of the five groups identified with SNPs data. These single locus methods did not consider the fifth group that consisted of H. pungens sensu stricto and the population from northeastern Brazil, Puerto Rico and the United States (Florida) on Amaranthaceae and/or Portulacaceae. Both methods delimited three (GYMC) and four (bPTP) additional groups in the clade conformed by mealybugs from Argentina, Paraguay, and Australia feeding on Cactaceae. Two additional splits were considered by bPTP, the first in H. pungens sensu stricto and the second one in mealybugs from southeastern Brazil on Amaranthaceae (Figure S1).