4.1 Hypogeococcus pungens species complex and host plant
use
The specimens used to describe H. pungens were originally
collected on an Amaranthaceae host (Granara de Willink, 1981). Further
collections extended its host range to the Cactaceae, Apocynaceae and
Portulacaceae families (Ben-Dov, 1994; Claps & de Haro, 2001;
Zimmermann, Pérez Sandi Cuen, Mandujano, & Golubov, 2010), suggesting
that H. pungens was a polyphagous species. However, our
population genomics approach pointed to a clear separation between the
mealybugs associated with Amaranthaceae or Cactaceae, and others that
indiscriminately use Amaranthaceae and Portulacaceae species as hosts.
These results confirmed that H. pungens was not a polyphagous
species, but a species complex composed of cryptic species associated
with different host plants as feeding resources. Population genomics,
clustering and species delimitation analyses based on genome wide SNPs
and the mitochondrial gene COI datasets revealed deep genetic
divergence among populations formerly considered as part of H.
pungens.
These results agreed with the previous studies that revealed a deep
genetic divergence, asymmetrical prezygotic and postzygotic reproductive
isolation between the mealybug populations associated with different
hosts from Argentina, and differential preference and performance on
alternative hosts (Poveda-Martínez et al., 2019; Aguirre et al., 2016).
The extension of our sampling effort to a wider geographic area
throughout the native and non-native ranges, along with the use of a
substantially large number of nuclear markers, allowed the confirmation
of the results reported for the Argentine Hypogeococcus mealybugs
(Poveda-Martínez et al., 2019) and new insights of the evolutionary
history of H. pungens sensu lato . In effect, our present results
supported the hypothesis that genetic differentiation throughout the
entire range in South America has mainly been driven by divergent
selection imposed by alternate host plants rather than by isolation
through distance. In fact, Mantel correlation and multiple regression
analyses of genetic distance matrices on geographic and host plant
distance matrices revealed that the latter was a better predictor of
genetic divergence among populations (Table 2).
In this context, our results pointed to host plant use as an important
driver of cryptic divergence in a priori presumed polyphagous
insects. Cryptic divergence has been recurrently observed in the form of
host-associated species that, very likely, diverged as the result of
adaptation to alternate host plants with little morphological divergence
in several insect species (Matsubayashi, Kahono, & Katakura, 2011;
Bagley, Sousa, Niemiller, & Linnen, 2017; Forbes et al., 2017; Zhang,
Bass, Fernández, & Sharanowski, 2018; Driscoe et al., 2019).
Genetic divergence between the cactus feeding mealybugs native to
Argentina and Paraguay, and those introduced to Australia from Argentina
(ArPaAu-C) and cactus feeding mealybugs from southeastern Brazil
(BrPr-C) exceeded that which would be expected for conspecific
populations, as indicated by our species delimitation analyses. In this
context, it is worth mentioning that besides the large geographic
distance separating populations of these clades and the fact that both
feed on Cactaceae, these two putative species fed on cactus that belong
to different genera. Mealybugs of the ArAuPa-C clade fed on species of
the genera Cleistocactus and Harrisia , whereas BrPr-C
thrived on species of other genera like Cereus for native
Brazilian mealybugs, and various genera for non-native Puerto Rican pest
mealybugs (Table S1). It may be argued that divergent selection on
alternate hosts and allopatry appeared as the more likely drivers of
divergence. As a matter of fact, there is evidence that divergent
selection caused by alternate hosts acting on ancestral fragmented
populations is sufficient to produce genetic divergence and incidental
speciation (Nosil et al., 2012; Duque-Gamboa et al., 2018; Doellman et
al., 2019).
Our results also elucidated a certain degree of genetic heterogeneity
within the ArAuPa-C clade, which included cactus feeders sampled in
Argentina, Paraguay, and those introduced in Australia for biological
control of cactus weeds. Both mtDNA and nuclear SNPs revealed a certain
degree of genetic structuring within this clade. Several well
differentiated mtDNA haplotypes were recorded at different locations of
this widespread clade, likely as the result of geographic isolation
(Figure 2C). Also, the phylogenetic tree based on nuclear SNPs showed
internal sub-clades concordant with the results depicted in theCOI network. An evaluation of the external morphology of
specimens collected on cacti in the same localities sampled for the
genomic survey in Argentina revealed subtle morphological variation
(Lucía Claps, University of Tucumán, Argentina, personal communication),
suggesting that genetic heterogeneity in this clade may be indicative of
incipient speciation rather than within species heterogeneity. These
results also helped to end the debate of the controversial origin of the
mealybugs used in the biological control program in Australia (and in
South Africa) (Tomley & McFadyen, 1987; Williams, 1973; Hamon, 1984).
Indeed, Australian Hypogeococcus mealybugs were genetically close
to the cactus feeding samples collected in northwestern Argentina and
Paraguay, but not, as suggested by other authors, to Hypogeococcus
festerianus Lizer & Trelles, another cactophagous species inhabiting
central-western Argentina, or H. pungens (Julien & Griffiths,
1999; Zimmermann, Pérez, Cuen, Mandujano, & Golubov, 2010; McFadyen,
2012).
Overall, our data provided evidence of two factors that influenced at
least 48% (Table 2) of the genetic divergence between the mealybugs
considered as part of the H. pungens species complex. Host plant
associations seemed to be the primary force influencing genetic
divergence, followed by the limited gene flow induced by isolation by
distance. Still, these results should be interpreted with caution since
other factors could also affect the distribution of genetic variation
that remained unexplained. Local adaptation to different environmental
conditions and/or ecological interactions with natural enemies or
competitors may impose varying selective pressures in different
geographic locations. A recent survey of parasitoids and
hyperparasitoids associated with Hypogeococcus mealybugs in South
America identified Leptomastidea hypogeococci Triapitsyn
(Hymenoptera: Encyrtidae) as a widespread primary parasitoid, able to
attack all members of the H. pungens complex, whereasAnagyrus cachamai Triapitsyn, Logarzo & Aguirre and A.
lapachosus Triapitsyn, Aguirre & Logarzo (also encyrtid primary
parasitoids from Argentina and Paraguay) were only found associated toH. pungens sensu stricto and the ArAuPr-C clade
(Triapitsyn et al., 2018). The influence of such differential ecological
interactions with natural enemies might be affecting patterns of genetic
divergence in this species complex.
Results of phylogenetic and species delimitation analyses were not
entirely congruent. Analysis with mtDNA visualized four major clades
whereas genome wide SNPs allowed to detect five well supported clades
(Figure 3). The main difference being that Hypogeococcusmealybugs from northeastern Brazil, Puerto Rico and United States
(BrPRUS-AP clade) and H. pungens sensu stricto (Ar-A) were
different species according to nuclear SNPs, while these clades appeared
collapsed in the same group with mtDNA data (Figure 3). Such
mito-nuclear inconsistencies have often been reported in insects
(Weigand et al., 2017; Hinojosa et al., 2019). Even though the mtDNA has
been useful to trace the evolutionary history in many species groups
(Hebert, Penton, Burns, Janzen, & Hallwach, 2004; Ball & Armstrong,
2006), incomplete lineage sorting and past hybridization events may
obscure species delimitation based only on mtDNA data, particularly in
recently diverged taxa (Hickerson, Meyer, & Moritz, 2006; Hinojosa et
al., 2019;
Després,
2019). For instance, Moreyra et al., (2019) reported a mitogenomic study
in a cluster of closely related cactophagous Drosophila spp.
inhabiting the southern cone of South America and found that the
evolutionary history inferred by mitogenomes is not completely
concordant with the phylogeny depicted by nuclear genomes (Hurtado,
Almeida, Revale, & Hasson, 2019). The authors proposed that either
incomplete lineage sorting (ILS) and/or introgressive hybridization
could account for the pattern observed. A word of caution is needed
before arriving at definitive conclusions in the present study due to
limitations of the mtDNA dataset, that consisted of only a few hundred
base pairs of the mtDNA gene encoding COI . In contrast, the
evolutionary history depicted by the nuclear genomic dataset may be
considered more reliable since it consisted of more than one thousand
widely distributed SNPs.
Phylogenetic trees based on either nuclear SNPs or mtDNA indicated that
feeding on Amaranthaceae was the more likely ancestral condition.
However, results yielded by mtDNA and SNPs datasets were not completely
congruent concerning the evolution of host plant use; both cactus
feeding species formed a derived monophyletic clade in the tree based on
nuclear SNPs, while cactophagy appeared to have evolved twice in the
tree obtained with mtDNA data. However, to unveil the ancestral host in
these clades, and to evaluate the evolution of host plant use and the
biogeographic history of the genus in the continent, other well
delimited cactophagous species of the genus Hypogeococcus from
South America, such as Hypogeococcus spinosus Ferris and H.
festerianus , should be included in an expanded analysis.