DISCUSSION
The goals of this study were to examine the population structure, gene
flow, and signatures of sex-biased dispersal in a fungus-gardening ant
species across several ecological gradients in Florida, USA. Overall,
our results suggest that most long-distance dispersal is conducted by
males. Specifically, females appear to have more limited dispersal as
the mtDNA haplotypes are strongly geographically clustered (Figure 4).
There were two COI clades found primarily in the Florida panhandle (Ts
Clade I and Ts Clade II) and one that was restricted to the Florida
peninsula (Ts Clade III). There were only three ants collected on the
peninsula that somewhat clustered with panhandle ants: one with Ts Clade
I (however, this specimen was separated from the Ts Clade I by 32
mutation steps) and two with Ts Clade II, separated by 12 mutation
steps. These three ants were notably collected in North Central Florida,
near Gainesville, geographically closer to the panhandle than the
samples collected in Central Florida near Orlando. In contrast to mtDNA
data, biparental microsatellite markers suggest significant gene flow
across Florida (approximately 360km, linear distance between the Central
region in Orlando and St. George Island, Florida) and minimal spatial
structure (Figures 2 and 3). These results indicate considerable
admixture of microsatellite alleles across the range of this study,
which considering the geographic clustering of mtDNA COI sequences,
likely arise from male movement. Together, these results suggest that
males are responsible for most long-distance dispersal while females
(and concomitantly, their co-dispersed fungal symbionts;
(Tesson et al., 2015)) do not disperse
very far. This result was surprising since T. septentrionalisqueens are not relatively large nor especially endowed with fat stores,
which could impact their flying ability
(Helms, 2018;
Seal, 2009;
Seal & Tschinkel, 2007b).
It is not clear how much of a barrier that rivers present to females
since we did find evidence of some trans-river female dispersal;
however, the distance that females seem to move across rivers is much
shorter relative to the distance males appear to disperse. The Suwannee
River may be an important barrier to female dispersal, though not
impenetrable since two peninsular haplotypes (in three individuals) were
found clustered with the two panhandle clades (Figure 4). Conversely,
the Ochlocknee River in the panhandle may not be a significant dispersal
barrier to either sex considering the extensive admixture of
microsatellite alleles (Figure 2) and shared haplotype clades (Ts Clades
I and II; and even identical haplotypes in some cases) in both the ARD
and WRD (Figure 4). The latter finding is surprising considering the
differing ecologies and environments (i.e., frequently flooded flatwoods
in the ARD and dry, xeric sandhills in the WRD). That being said, the
finding of reduced genetic diversity in the ARD relative to the WRD
(Table 2) could suggest recent expansion in the WRD. As a result, on
small scales (10s of kilometers), T. septentrionalis appears to
be a very mobile species, capable of rapid population growth and
extensive dispersal capabilities, but there are limits to their
expansion abilities across larger scales (>100s of
kilometers). Possible explanations for this conclusion could be related
to Pleistocene bottlenecks and then subsequent expansion, and a
subsequent time lag in the expansion of COI haplotypes. Therefore, it
would appear likely that males have a greater dispersal capability than
females. Field studies measuring the variation in flight distance within
and between sexes could further inform our results.
Evidence is currently lacking as to whether male-biased dispersal is the
general rule in the tribe Attini. This is surprising considering how
important female dispersal is for the range expansion and ultimately
evolution of the fungal symbiont (Mueller
et al., 2001). For example, the basal neoattine Mycetophylax
simplex exhibited relatively minor mtDNA (COI) variation across its
range in Brazilian Atlantic Forest
(Cardoso et al., 2015), which suggests
that females are capable of long-distance dispersal. However, as a lower
attine (i.e., an early branching lineage of attini), Mycetophylaxlikely has smaller queens than Trachymyrmex sensu lato ants and
other members of the so-called ‘higher attini’
(Seal, 2009); thus, the energetic cost of
dispersal for Mycetophylax compared to Trachymyrmex sensu
lato could possibly be lower. As another example, Mycocepurus
smithii indicated stronger gene flow and little spatial structure in
populations across the Panamanian isthmus (inferred from
microsatellites) unlike their fungal symbionts that were more spatially
structured, though the study did not also employ mtDNA markers like the
present study (Kellner et al., 2013).
While spatially structured fungal symbionts could point to limited
female dispersal and long-distance male dispersal like we found withT. septentrionalis , males are rare if not absent in M.
smithii , which exhibit thelytokous parthenogenesis in Panama
(Kellner et al., 2013). Thus, M.
smithii female movement (and some level of disruption to vertical fungi
transmission) likely explains the patterns in central Panama. While
lower attines such as Mycetophylax and Mycocepuruscultivate fungi that are likely capable of independent life, fungi grown
by higher attini such as Trachymyrmex and Atta are not
(Schultz & Brady, 2008). Solomon et al.
(2008) reported mtDNA (COI) clusters in three Atta species across
continental scales, which suggests limited female dispersal, but did not
examine whether males were capable of dispersing longer distances.
Interestingly, ddRADseq (i.e., diploid markers) in Atta texanashowed evidence of spatial structure and isolation by distance across a
north-south gradient in Texas (850km). Though fungal symbionts also
illustrate significant north-south differentiation in this species, the
patterns are not concordant with their host ants
(Mueller, Mikheyev, Solomon, & Cooper,
2011; Smith et al., 2019), which could
indicate independent/differential dispersal patterns of males, females,
and fungal symbionts via unknown mechanisms.
Our results support greater dispersal abilities in male T.
septentrionalis than females. Consequently, this suggests that the
dispersal abilities of the vertically transmitted symbiotic fungus (and
further associated microbial symbionts
(Ishak et al., 2011;
Ronque, Lyra, Migliorini, Bacci, &
Oliveira, 2020)) are likely also limited and thus also exhibits spatial
structure, unless the fungus also has the ability of independent
dispersal as suggested in A. texana(Smith et al., 2019). Limited female and
symbiont co-dispersal could represent a significant bottleneck to fungal
diversification (and associated microbes). Bottlenecks are a common
feature among vertically transmitted symbionts, which generally exhibit
eroded genetic variation and reduced genomes compared to horizontally
exchanged relatives (Bennett, McCutcheon,
MacDonald, Romanovicz, & Moran, 2014;
Douglas, 2010;
Helms, Ijelu, & Haddad, 2019;
Nikoh, Hosokawa, Oshima, Hattori, &
Fukatsu, 2011). Bottlenecks may not only influence population
demographics but also the adaptive abilities of co-dispersed symbionts
under varying environments. Consequently, the overall coevolutionary
patterns and associations observed in the fungus-farming ant symbiosis
may be constrained by limited female dispersal especially in the higher
attini that are characterized by obligate symbionts and large-bodied,
fatter queens.
The approximately 49 ant species in the genus formerly known asTrachymyrmex (now split into 3 genera
(Solomon et al., 2019)) grow
conservatively 4-5 phylotypes of fungi
(Ješovnik et al., 2017;
Luiso, Kellner, Matthews, Mueller, &
Seal, 2020; Solomon et al., 2019). One
possible explanation is that ant host diversification in these derived
lineages has happened at a faster rate than their fungal symbionts
because of limited female ant dispersal. Whether attine ants and their
fungal symbionts have different evolutionary (or expansion) rates is
currently unknown. The most recent genome-level examinations suggested
that attine fungal genomes have lower diversity of metabolic genes
compared to free-living fungi; however, this was based on transcriptomes
(measures of gene expression) as we lack fully annotated attine fungal
genomes because attine fungi are functionally polyploid
(Kooij, Aanen, Schiott, & Boomsma, 2015;
Kooij, Poulsen, Schiøtt, & Boomsma,
2015; Nygaard et al., 2016).
Alternatively, since neither ant nor fungi have to evolve at similar
rates, reduced fungal lineage diversity could be due to higher
evolutionary and subsequent extinction rates among the fungi, such that
fungal diversification may occur more rapidly with ants adopting novel
fungal strains and discarding others as climate and parasite pressure
change the outcome of the interaction
(Mehdiabadi, Hughes, & Mueller, 2006;
Seal & Mueller, 2014;
Seal, Schiøtt, & Mueller, 2014;
Seal & Tschinkel, 2007a). Furthermore,
some phylogenetic analyses suggest that the fungal lineages typically
grown by leaf-cutter ants (i.e., Clade A fungi grown by Atta andAcromyrmex ) (Mueller et al., 2018)
are younger than the ant lineages
(Mikheyev, Mueller, & Abbot, 2010;
Nygaard et al., 2016) which suggests a
recent domestication event. However, analyses of more recent datasets
have called this conclusion into question since some non-leaf-cutting
ants grow Clade A fungi (Mueller et al.,
2017; Mueller et al., 2018;
Schultz et al., 2015), indicating that
the fungi may have been around for as long as the less derivedTrachymyrmex ants. In conclusion, a greater understanding of the
dispersal biology of these species could vastly improve our ability to
understand and ultimately predict how host and symbiont populations
expand and evolve across larger geographic and macroevolutionary scales.