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).