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\begin{document}

\title{MALE-BIASED DISPERSAL IN A FEMALE-DISPERSED SYMBIOSIS}



\author[1]{Alix Matthews}%
\author[2]{Katrin Kellner}%
\author[2]{Jon Seal}%
\affil[1]{Arkansas State University}%
\affil[2]{University of Texas at Tyler}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
For nearly all organisms, dispersal is a fundamental life history trait
that can shape their ecology and evolution. Variation in dispersal
capabilities within a species exists and can influence population
genetic structure and ecological interactions. In fungus-gardening
(attine) ants, co-dispersal of ants and mutualistic fungi is crucial to
the success of this obligate symbiosis. Female-biased dispersal (and
gene flow) may be favored in attines because virgin queens carry the
responsibility of dispersing the fungi, but a paucity of research has
made this conclusion difficult. Here, we investigate dispersal of the
fungus-gardening ant Trachymyrmex septentrionalis using a combination of
maternally- (mitochondrial DNA) and biparentally-inherited
(microsatellites) markers. We found three distinct, spatially isolated
mitochondrial DNA haplotypes. Two were found in the Florida panhandle
and the other was found in the Florida peninsula. In contrast,
biparental markers illustrated significant gene flow across this region
and minimal spatial structure. The differential patterns uncovered from
mitochondrial DNA and microsatellite markers suggest that most
long-distance ant dispersal is male-biased and that females (and
concomitantly the fungus) have more limited dispersal capabilities.
Consequently, the limited female dispersal is likely an important
bottleneck for the fungal symbiont. This bottleneck could slow fungal
genetic diversification, which has significant implications for both ant
hosts and fungal symbionts regarding population genetics, species
distributions, adaptive responses to environmental change, and
coevolutionary patterns.%
\end{abstract}%



\sloppy




\textbf{INTRODUCTION}

Dispersal is a ubiquitous and fundamental life history trait that plays
a role in the evolutionary ecology of organisms. Dispersal can shape
population genetic differentiation, overall species distributions, and
how species may respond or adapt to environmental change (Clobert,
Baguette, Benton, \& Bullock, 2012; Ellis et al., 2015). For species
with low dispersal capabilities, historical, ecological, or
anthropogenic barriers to movement can influence their population
genetic structure over certain scales (Do\selectlanguage{ngerman}ña et al., 2019; Haye et al.,
2014; Manel, Schwartz, Luikart, \& Taberlet, 2003; Storfer, Murphy,
Spear, Holderegger, \& Waits, 2010). For example, barriers may slow gene
flow between some populations, increase reproductive and genetic
isolation, and ultimately result in genetically divergent populations
(Clark, Brown, Stechert, \& Zamudio, 2010; DiBlasi et al., 2018;
Slatkin, 1987).

Within species, dispersal may be biased towards certain individuals,
which has consequences for a species' genetic diversity and population
genetic structure. For example, in some species, female birds tend to
disperse farther than males, while male mammals tend to disperse farther
than females (sex-biased dispersal) (Greenwood, 1980; Trochet et al.,
2016). While females form the core of societies in ants (and all social
Hymenoptera), considerable variation exists among the dispersal
abilities of males and reproductive females (Bourke \& Franks, 1995;
Cronin, Molet, Doums, Monnin, \& Peeters, 2013; Hakala, Seppä, \&
Helanterä, 2019; Helms, 2018; Jacobs \& Heinze, 2019; Keller, Peeters,
\& Beldade, 2014). For example, some ant species have wingless
individuals (females or males) that can move across scales of just a few
meters, or winged individuals that vary in their ability or tendency to
disperse long distances (over hundreds of meters). Typically, dispersal
pattern and colony founding mode determine intraspecific population
genetic patterns. For example, in species where queens do not fly, males
may fly and be responsible for most long-distance dispersal (Berghoff,
Kronauer, Edwards, \& Franks, 2008; Hakala et al., 2019). In other
species, wingless males and winged females may experience inbreeding due
to a lack of dispersal (Jacobs \& Heinze, 2019). In species with winged
males and females, both sexes may contribute to long-distance dispersal,
gene flow, and near panmictic population structure (Johansson, Seppä,
Helanterä, Trontti, \& Sundström, 2018). On the other hand, some species
with volant sexuals nevertheless show signatures of male-biased
long-distance dispersal (Holzer, Keller, \& Chapuisat, 2009), especially
in species with relatively large and fat-laden queens that produce their
first offspring via metabolic stores (claustral founding) (Helms, 2018).

The fungus-gardening (attine) ants are common ants in low to mid
latitudes of the western hemisphere (Branstetter et al., 2017; Nygaard
et al., 2016; Seal \& Tschinkel, 2006). The ants depend on their fungal
cultivar as their main nutritional source, and the fungus depends on the
ants for propagation, survival, and dispersal. Virgin queens (gynes) are
solely responsible for dispersal of the mutualistic fungi (vertical
transmission) by storing it in an infrabuccal pocket prior to the
nuptial flight (Huber, 1905; Mueller, 2002; Mueller, Schultz, Currie,
Adams, \& Malloch, 2001). Thus, symbiotic fungal expansion and
diversification is expected to be largely dependent on dispersal
capabilities of the female ants. As a result, female-biased dispersal
(or at least not male-biased dispersal) may be favored in attine ants;
strict male-biased dispersal would conflict with the dispersal interests
of the fungus (Mueller, 2002) by limiting the expansion and
diversification of the fungal symbiont. However, any conflicts could be
resolved by males and gynes having similar dispersal abilities.
Additionally, gynes of most attines appear to be energetically cheap to
produce and most do not found claustrally (Fernández-Marín, Zimmerman,
\& Wcislo, 2004; Seal, 2009; Seal \& Tschinkel, 2007b); thus, attines
might not be under selection to produce large, heavy gynes that are
generally dispersal-limited because of the energetic demands of flight
(Helms, 2018). Consequently, the relatively lighter attine gynes may fly
just as far as males, thus increasing expansion capabilities of the
co-dispersed fungus.

Surprisingly, there have been very few studies that examine sex-biased
dispersal and gene flow in fungus-gardening ants, even though the
molecular markers necessary to do so have been developed. For instance,
microsatellite markers have been used to examine polyandry, polygyny,
and population structure (Bekkevold, Frydenberg, \& Boomsma, 1999;
Fjerdingstad \& Boomsma, 2000; Helmkampf, Gadau, \& Feldhaar, 2008;
Kellner et al., 2013; Matthews, Rowan, Stone, Kellner, \& Seal, 2020;
Murakami, Higashi, \& Windsor, 2000; Rabeling et al., 2013; Rabeling et
al., 2011; Rabeling et al., 2014) and mitochondrial DNA (mtDNA) markers
have been used to examine phylogeographic structure (i.e., female
dispersal patterns) in several attine species (Cardoso, Cristiano,
Tavares, Schubart, \& Heinze, 2015; Seal, Thiebaud, \& Mueller, 2015;
Solomon, Bacci, Martins, Vinha, \& Mueller, 2008). While at least one
study reported comparable dispersal abilities in both male and female
\emph{Atta colombica} , only a small portion of its known range was
surveyed (Helmkampf et al., 2008). Indeed,\emph{Atta} species are the
only attines that exhibit claustral founding (Fernández-Marín \& Wcislo,
2005; Fernández-Marín et al., 2004; Hölldobler \& Wilson, 2011; Huber,
1905); it therefore remains possible that the large females (among the
largest individual ants in the world) are not capable of flying as far
as males.

In this study, we employ mtDNA markers and diploid microsatellite
markers that were developed using whole genome sequencing (Matthews et
al., 2020) to examine population structure, gene flow, and signatures of
sex-biased dispersal in a fungus-gardening ant species,
\emph{Trachymyrmex septentrionalis}(McCook, 1881) . \emph{T.
septentrionalis} is distributed from Texas to Florida to Long Island,
New York, with a center of genetic diversity found in northern Florida
(Rabeling, Cover, Johnson, \& Mueller, 2007; Seal et al., 2015; Senula
et al., 2019). Prior studies using mtDNA have shown significant
phylogeographic structure across its range. For example, haplotypes are
represented by phylogroups east and west of the Mississippi River, with
western populations consisting of one clade whereas those found in the
east are more diverse, consisting of at least three clades (Mikheyev,
Vo, \& Mueller, 2008; Seal et al., 2015). A preliminary analysis of
nuclear microsatellites supports this overall pattern -- higher
diversity in populations east of the Mississippi River relative to the
west (Matthews et al., 2020).\emph{T. septentrionalis} is one of the
most common and abundant ant species in the longleaf pine forests of
Florida whose populations appear to respond rapidly to annual variations
in climate (especially rainfall) and local conditions (e.g., relief) and
often move significant amounts of soil in the process (Seal \&
Tschinkel, 2006, 2008, 2010; Tschinkel \& Seal, 2016). Therefore,
understanding the population genetics of this species will help us
understand the dispersal biology of an ecologically important symbiosis.

\textbf{MATERIALS AND METHODS}

\textbf{Study site and field methods}

\emph{Trachymyrmex septentrionalis} colonies were found in Florida, USA.
Florida is located entirely within the North American Coastal Plain,
which is a global biodiversity hotspot owing to its unique geologic
history and diverse ecosystems (Griffith, 1994; Noss, 2012; Noss et al.,
2015). Samples were collected across two primary regions in Florida that
correspond with known biogeographic breaks in the state (e.g., (Seal et
al., 2015)); the panhandle (which included two distinct ranger districts
within the Apalachicola National Forest and a nearby barrier island) and
the peninsula, represented by samples collected in North Central Florida
near Gainesville and in Central Florida near Orlando (Figure 1).

In the panhandle, the Apalachicola National Forest (ANF) is divided into
two ranger districts that occur on either side of the Ochlockonee River:
the Apalachicola Ranger District on the west and the Wakulla Ranger
District on the east. The Apalachicola Ranger District (ARD) is
comprised mainly of pine flatwood forest with high herbaceous cover,
little relief, and a high water table (Abrahamson \& Harnett,
1990).\emph{Trachymyrmex septentrionalis} colonies in pine flatwoods are
restricted to `habitat islands' of somewhat higher elevation (relative
to the water table) and population sizes likely vary temporally as
annual rainfall varies from drought to very wet (Seal \& Tschinkel,
2010). On the other hand, the Wakulla Ranger District (WRD) contains
significant stands of sandhill forest (i.e., remnants of Pleistocene
sand dunes; (Myers, 1990)) that are excessively drained and support many
species associated with deserts (Brown, Stone, \& Carlisle,
1990).\emph{T. septentrionalis} colonies are found throughout the
sandhills, absent only from frequently inundated areas near ephemeral
ponds. Unlike flatwoods populations, those found in sandhills do not
likely fluctuate very much in size, at least with respect to rainfall
(Seal \& Tschinkel, 2006, 2010). Both ranger districts are dominated and
characterized by longleaf pine (\emph{Pinus palustris} ) and sandy
soils. Located just south of the ANF, St. George Island is a sandy
barrier island approximately 6km from the mainland. Despite its low
relief and susceptibility to hurricanes, the island contains several
stands of pine forest on elevated regions (Gornish \& Miller, 2010;
Huffman, Platt, \& Grissino-Mayer, 2004) which harbor several species
typical of sandhills further inland, such as \emph{T. septentrionalis}
and the seed-harvesting ant\emph{Pogonomyrmex badius} . The Florida
peninsula is separated from the panhandle by the Suwanee River, a known
biogeographic barrier to many species (including ants), owing to
different soils and geological history (Brown et al., 1990; Richter et
al., 2014; Strehl \& Gadau, 2004). Thus, the collections were made
across multiple ecological gradients and potential dispersal barriers.

\emph{Trachymyrmex septentrionalis} colonies were identified in the
field by their distinctive and steep crescent-shaped mounds, which are
built up by ants piling excavated chamber soil (Seal \& Tschinkel,
2006). Individual worker ants were collected directly from colonies in
2013 and 2018. All workers were preserved in 95\% ethanol at -20ºC until
DNA extraction. Only one individual worker per colony was used for
sequencing. All relevant specimen information (e.g., collection date,
region/location) can be found in Table S1.

\textbf{Microsatellite genotyping and genetic diversity}

We extracted genomic DNA from 64 individual \emph{T.
septentrionalis}workers using a QIAamp DNA Micro Kit (QIAGEN). We
implemented the M13-tail polymerase chain reaction (PCR) method
(Schuelke, 2000), which involves three primers in the PCR reaction:
forward primers (Matthews et al., 2020) with an M13-tail at the 5' end,
an unlabeled reverse primer, and a universal M13 primer labeled with
6-FAM (6-carboxy-fluorescine) fluorescent dye. These were used to
amplify nine microsatellite loci (Ts21, Ts25, Ts32, Ts33, Ts39, Ts41,
Ts43, Ts46, Ts5) (Matthews et al., 2020).

PCRs were performed in a 10 \selectlanguage{greek}μ\selectlanguage{english}L mix containing 1 \selectlanguage{greek}μ\selectlanguage{english}L of 10X PCR buffer
(1.0X; Applied Biosystems), 1 \selectlanguage{greek}μ\selectlanguage{english}L Bioline\selectlanguage{ngerman}® dNTP mix (1 mM each; 0.1 mM as
a proportion of the total), 1.5 \selectlanguage{greek}μ\selectlanguage{english}L of 25 mM MgCl\textsubscript{2} (3.75
mM; Applied Biosystems), 0.5 \selectlanguage{greek}μ\selectlanguage{english}L of 20 \selectlanguage{greek}μ\selectlanguage{english}M BSA (1 \selectlanguage{greek}μ\selectlanguage{english}M; New England
Biolabs), 0.3 \selectlanguage{greek}μ\selectlanguage{english}L of 2 \selectlanguage{greek}μ\selectlanguage{english}M tag labeled primer (0.06 \selectlanguage{greek}μ\selectlanguage{english}M; forward primer
with M13 tag), 0.6 \selectlanguage{greek}μ\selectlanguage{english}L of a 2 \selectlanguage{greek}μ\selectlanguage{english}M universal dye-labeled primer (0.12 \selectlanguage{greek}μ\selectlanguage{english}M;
FAM label with M13 tag), 1 \selectlanguage{greek}μ\selectlanguage{english}L of 2 \selectlanguage{greek}μ\selectlanguage{english}M unlabeled primer (0.2 \selectlanguage{greek}μ\selectlanguage{english}M; reverse
primer), 0.1 \selectlanguage{greek}μ\selectlanguage{english}L of \emph{Taq} polymerase (0.5 U; Applied Biosystems),
and 1 \selectlanguage{greek}μ\selectlanguage{english}L of DNA template. Nuclease-free water was used to make up the
remaining volume. The following thermocycling profile was used on an
Eppendorf Mastercycler: initial denaturation of 4 min at 95\selectlanguage{ngerman}°C, followed
by 25 (or 30) cycles of 30 s at 95°C, 45 s at the primer-specific
annealing temperature found by a temperature gradient program, 45 s at
72°C, then 8 cycles of 30 s at 95°C, 45s at 53°C, and 45 s at 72°C,
followed by a final extension of 5 min at 72°C. Diluted PCR products
were run on an Applied Biosystems 3730 Genetic Analyzer and fragments
were sized with LIZ600 size standard at the University of Texas at
Austin DNA Sequencing Facility in Austin, Texas, USA. We scored alleles
using Geneious v10.2.3 (Kearse et al., 2012).

For each locus, we used GenAlEx v6.5 (Peakall \& Smouse, 2006; Peakall
\& Smouse, 2012) to estimate the number of alleles (\emph{K} ), observed
and expected heterozygosity (H\textsubscript{o} and H\textsubscript{e}),
and the probability of identity (PI; the probability of two independent
samples having the same genotype). We assessed deviations from
Hardy-Weinberg equilibrium (HWE) expectations using GENEPOP v4.2
(Rousset, 2008) across each locus for the overall dataset, as well as
across each locus within each population. GENEPOP was also used to test
for linkage disequilibrium (LD) across all pairs of loci.

\textbf{Amplification and sequencing of mtDNA}

In order to augment our microsatellite data, we included mtDNA
previously sequenced from Seal et al. (2015; n = 61) and sequenced new
samples directly for this study (n = 26). A total of 55 samples had both
microsatellite and mtDNA sequenced (Table S1). Again, DNA was extracted
from whole individual ants using a QIAamp DNA Micro Kit (QIAGEN), and a
779-bp sequence was obtained from the COI-tRNA Leucine-COII region of
mtDNA. We used the following primers: C1-J2195 (alias CO1-RLR;
5\selectlanguage{english}'-TTGATTTTTTGGTCATCCAGAAGT-3\selectlanguage{english}'); and C2-N-3661 (alias Barbara; 5\selectlanguage{english}'-
CCACAAATTTCTGAACATTGACCA-3\selectlanguage{english}'(Simon et al., 1994)). PCR mixtures and
cycling profiles were identical to Seal et al. (2015). PCR products were
then purified and sequenced at the University of Texas at Austin's DNA
Sequencing Facility on an Applied Biosystems 3730 DNA Analyzer.
Chromatograms were visually checked and resolved in Geneious v10.2.3
(Kearse et al., 2012), and sequences were aligned using MEGA v6.06
(Tamura, Stecher, Peterson, Filipski, \& Kumar, 2013) using the ClustalW
algorithm (Thompson, Higgins, \& Gibson, 1994). New sequences are
deposited into GenBank under accession numbers MN088095-MN088120 (Table
S1). mtDNA is known to have several properties that make evolutionary
conclusions problematic, such as pseudogenes or nuclear insertions
(numts) (Beckenbach, 2009; Cristiano, Cardoso, \& Fernandes-Salomao,
2014; Martins et al., 2007; Toews \& Brelsford, 2012). Therefore, we
examined for stop codons in our sequences (other than at the COI-tRNA
Leucine transitions) (Seal et al., 2015). Sequences were also long and
frequently readable at \textgreater{}800bp. Two primary advantages of
mtDNA sequences are that 1) sequences can be readily obtained and 2) the
problems associated with mtDNA are understood and can be easily
examined, whereas the problems associated with nuclear markers are more
uncertain (Bowen et al., 2014; Moreau, 2009). Moreover, a preliminary
examination of the microsatellite markers of \emph{T.
septentrionalis}used in this study supported one of the main findings of
mtDNA-based studies in this species: pronounced genetic differentiation
across the Mississippi River Valley (Matthews et al., 2020), thus making
it unlikely that the COI sequences analyzed were numts, which usually
lack variation because of purifying selection in nuclear genomes
(Martins et al., 2007). Therefore, mtDNA sequences in this species are
likely robust genetic tools (Matthews et al., 2020; Mikheyev et al.,
2008; Seal et al., 2015).

\textbf{Population genetic analyses}

\emph{Microsatellites}

We measured population genetic structure by Analysis of Molecular
Variance (AMOVA) (Excoffier, Smouse, \& Quattro, 1992) and by pairwise
Fst values between populations in Arlequin v3.5.2.2 (Excoffier \&
Lischer, 2010). Significance was tested using 1,000 permutations. We
generated several subsets to examine signatures of population
differentiation related to the varying ecologies and potential barriers
to dispersal across our collection sites. Specifically, our subsets
were: a) all regions; b) all regions with the island excluded; c) ANF
panhandle regions only (i.e., ARD and WRD); d) peninsular regions only
(i.e., North Central near Gainesville and Central near Orlando); and e)
ANF regions as one collective region and peninsular regions as one
collective region. For each subset, we adjusted the allowed missing data
(from 0.15 to 0.5) in order to include all nine loci in all analyses.

To further assess population genetic structure, we used the Bayesian
clustering algorithm implemented through STRUCTURE v2.3.4 (Pritchard,
Stephens, \& Donnelly, 2000), which uses allele frequency data to assign
individuals into genetic clusters (K). These analyses were executed with
no prior population information and under the admixture model with
correlated allele frequencies. In order to determine K, we assessed K =
1 through K = 10 with default parameters. To explore each K value, we
performed 10 replicates with a burnin value of 50,000 and 500,000 MCMC
iterations. We determined the optimal K value by assessing the maximum
likelihood values and \selectlanguage{greek}Δ\selectlanguage{english}K (Evanno, Regnaut, \& Goudet, 2005) implemented
in Structure Harvester v0.6.94 (Earl \& von Holdt, 2012). We then
averaged the 10 STRUCTURE replicates for the optimal K through the
CLUMPAK pipeline (Kopelman, Mayzel, Jakobsson, Rosenberg, \& Mayrose,
2015) and visualized the results in DISTRUCT v1.1 (Rosenberg, 2004).

We performed Mantel tests to test whether genetic distances correlate
with geographic distance (isolation by distance; IBD). We generated
several subsets to test for IBD. Specifically, we considered the
following: a) all sampling sites across all regions; b) all sampling
sites excluding the island; c) all sampling sites within the ANF; and d)
all sampling sites within the peninsula. For measures of genetic
distances, we used pairwise Fst values obtained from Arlequin and
linearized them as Fst/(1-Fst) (Rousset, 1997). Negative Fst values, if
present, were set to zero. Otherwise, the absolute difference between
values would be inflated, when in actuality they are effectively zero
(i.e., no genetic differentiation). We used Geographic Distance Matrix
Generator v1.2.3 (Ersts, 2013) to generate pairwise geographic distances
(in km) from the GPS coordinates of the sampling sites. We implemented
Mantel tests using the ade4 package (Dray \& Dufour, 2007) in R (Team,
2018) with 9,999 permutations.

\emph{Mitochondrial DNA}

Using the COI mtDNA alignment, we examined haplotype diversity and
calculated basic genetic polymorphism statistics and across regions and
within regions using DNAsp v6 (Rozas et al., 2017). We then
reconstructed a haplotype network for the COI data (n = 87; combined
samples from Seal et al. (2015) and new sequences directly from this
study) using the TCS method (Clement, Posada, \& Crandall, 2000) in
PopArt v1.7 (Leigh \& Bryant, 2015).

\textbf{RESULTS}

\textbf{Genetic diversity of microsatellite DNA}

The mean number of alleles per locus across nine loci in regions of
Florida was 18.9. The mean observed heterozygosity was 0.688, while the
mean expected heterozygosity was higher at a value of 0.859 (Table 1).
After Bonferroni correction for multiple comparisons across all regions,
four of the nine loci showed deviations from expectations under HWE
(Table 1). However, by region and locus, only four HWE tests were
significant after Bonferroni corrections and no loci deviated in more
than two regions (Table 1). Therefore, we retained all nine loci for
downstream analyses. There were no cases of linkage disequilibrium
detected for any pair of loci (raw genotypic data for microsatellites
are available in Table S2).

\textbf{Population genetic analyses}

\emph{Microsatellites}

Overall, AMOVAs did not reveal significant population differentiation
(overall Fst values were between 0.000 and 0.035 and all associated Fst
p-values were \textgreater{} 0.05). Thus, the amount of genetic
variation attributable to differences between the assigned population
subsets was low and insignificant; most of the genetic differentiation
occurred within and among individuals (Table S3). Alternatively,
according to our STRUCTURE results, there are two distinct genetic
clusters (K = 2) in our dataset, providing evidence for genetic
differentiation across Florida with signatures of gene flow between
clusters. Specifically, the samples from St. George Island were largely
assigned to one cluster. Likewise, the samples from the peninsula region
were largely assigned to another single cluster with minimal admixture.
The samples from the ANF appear to have substantial (nearly 50\%
overall) admixture of alleles associated with the peninsula and St.
George Island clusters (Figure 2).

Finally, based on results of the Mantel tests of the association between
genetic distance and geographic distance, we found weak to little
evidence of IBD across the different subsets (Figure 3). Thus, the
amount of genetic variation explained by geographic distance is low,
suggesting that the genetic differentiation of our samples is continuous
across geography rather than illustrating discrete populations separated
by specific barriers.

\emph{Mitochondrial DNA}

In contrast to the diploid microsatellite markers, mtDNA markers
indicated pronounced population differentiation. There were two distinct
haplotype clades found in the panhandle and one in the peninsula. Three
specimens collected in the North Central peninsula (Gainesville)
appeared to be most similar to those found in the panhandle, however,
even these were genetically unique (Figure 4). Across the combined 87
samples, there were 38 mitochondrial haplotypes identified and were
defined by 71 polymorphic sites, which consisted of 33 singleton
variable sites and 38 parsimony informative sites. Overall haplotype
diversity was high and overall nucleotide diversity was low, with the
same being true within each region (the Central region is an exception
to high haplotype diversity), suggesting a recent population expansion
(Table 2).

\textbf{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
\emph{T. septentrionalis}queens 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), \emph{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 (\textgreater{}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 \emph{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), \emph{Mycetophylax}likely has smaller
queens than \emph{Trachymyrmex} sensu lato ants and other members of the
so-called `higher attini' (Seal, 2009); thus, the energetic cost of
dispersal for \emph{Mycetophylax} compared to \emph{Trachymyrmex} sensu
lato could possibly be lower. As another example, \emph{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 with\emph{T. septentrionalis} , males are rare
if not absent in \emph{M. smithii} , which exhibit thelytokous
parthenogenesis in Panama (Kellner et al., 2013). Thus, \emph{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 \emph{Mycetophylax} and
\emph{Mycocepurus}cultivate fungi that are likely capable of independent
life, fungi grown by higher attini such as \emph{Trachymyrmex} and
\emph{Atta} are not (Schultz \& Brady, 2008). Solomon et al. (2008)
reported mtDNA (COI) clusters in three \emph{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 \emph{Atta
texana}showed 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 \emph{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 \emph{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
as\emph{Trachymyrmex} (now split into 3 genera (Solomon et al., 2019))
grow conservatively 4-5 phylotypes of fungi (Je\selectlanguage{polish}š\selectlanguage{english}ovnik 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, Schi\selectlanguage{ngerman}øtt, \&
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 \emph{Atta}
and\emph{Acromyrmex} ) (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 derived\emph{Trachymyrmex} 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.

\textbf{ACKNOWLEDGEMENTS}

The work was supported by a National Science Foundation grant to JNS
(CAREER, IOS-152822) and KK (DEB-1354629). Travel to Florida was also
funded by a UT-Tyler Internal Research Grant to JNS. We thank Walter
Tschinkel for providing logistical support while collecting in North
Florida. Joshua King and Andrea Lucky provided \emph{T.
septentrionalis}specimens from the Orlando and Gainesville, Florida
areas, respectively. This is paper No. 4 of the Ant Symbiosis
Laboratory.



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\textbf{AUTHOR CONTRIBUTIONS}

JNS and KK conceived the study, secured funding and permits, and
collected samples. AEM conducted the molecular work and data analyses
with contributions from JNS and KK. AEM and JNS led manuscript writing,
and all authors edited and approved the final version. The authors
declare no competing interests.

\textbf{DATA ACCESSIBILITY}

COI sequence data corresponding to the samples used in this study are
deposited in GenBank under accession numbers MN088095-MN088120,
KP282952-KP282961, and KP282964- KP283014 and can be found in Table S1.
Microsatellite genotypes can be found in Table S2.

\textbf{TABLES}

\textbf{Table 1.} Details of the nine polymorphic microsatellite loci
analyzed for \emph{Trachymyrmex septentrionalis} ants across Florida.
Superscript \textsuperscript{a} indicates deviation from Hardy-Weinberg
expectations after Bonferroni corrections. Bolded and underlined
p-values indicate deviation after Bonferroni correction11Note.
Abbreviations include: the number of individuals genotyped (N), number
of alleles observed (K), observed heterozygosity per locus for all
regions (H\textsubscript{oL}), expected heterozygosity per locus for all
regions (H\textsubscript{eL}), probability of identity per locus for all
regions (PI\textsubscript{L}), observed heterozygosity per region by
locus (H\textsubscript{o}), Hardy-Weinberg Equilibrium p-values per
region by locus (HWE), St. George Island (SGI), ARD (Apalachicola Ranger
District), and WRD (Wakulla Ranger District)\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllll@{}}
\toprule
& \textbf{All regions} & \textbf{All regions} & \textbf{All regions} &
\textbf{All regions} & \textbf{All regions} & \textbf{SGI} &
\textbf{SGI} & \textbf{ARD} & \textbf{ARD} & \textbf{WRD} & \textbf{WRD}
& \textbf{North Central} & \textbf{North Central} & \textbf{Central} &
\textbf{Central}\tabularnewline
\midrule
\endhead
\textbf{Locus} & \textbf{N} & \textbf{K} & \textbf{Ho\textsubscript{L}}
& \textbf{He\textsubscript{L}} & \textbf{PI\textsubscript{L}} &
\textbf{Ho} & \textbf{HWE} & \textbf{Ho} & \textbf{HWE} & \textbf{Ho} &
\textbf{HWE} & \textbf{Ho} & \textbf{HWE} & \textbf{Ho} &
\textbf{HWE}\tabularnewline
Ts5\textsuperscript{a} & 53 & 12 & 0.585 & 0.827 & 0.051 & 0.750 & 0.771
& 0.654 & 0.006 & 0.538 & 0.005 & 0.500 & 1 & 0.250 &
0.142\tabularnewline
Ts21\textsuperscript{a} & 50 & 11 & 0.66 & 0.823 & 0.048 & 0.000 & 0.009
& 0.852 & 0.014 & 0.700 & 0.409 & 0.167 & 0.011 & 0.667 &
0.599\tabularnewline
Ts25 & 61 & 22 & 0.787 & 0.829 & 0.028 & 0.667 & 1 & 0.778 & 0.039 &
0.769 & 0.586 & 0.818 & 0.163 & 0.857 & 0.571\tabularnewline
Ts32 & 61 & 16 & 0.754 & 0.864 & 0.031 & 0.500 & 0.428 & 0.769 & 0.199 &
0.846 & 0.401 & 0.636 & 0.014 & 0.857 & 0.297\tabularnewline
Ts33 & 63 & 14 & 0.714 & 0.776 & 0.072 & 0.400 & 0.300 & 0.778 & 0.110 &
0.692 & 0.929 & 0.909 & 0.089 & 0.429 & 0.174\tabularnewline
Ts39\textsuperscript{a} & 63 & 22 & 0.492 & 0.919 & 0.012 & 0.667 &
0.413 & 0.519 & \emph{0} & 0.583 & 0.002 & 0.273 & \emph{0} & 0.429 &
0.012\tabularnewline
Ts41 & 60 & 17 & 0.8 & 0.843 & 0.037 & 0.500 & 0.214 & 0.792 & 0.364 &
0.833 & 0.116 & 0.909 & 0.553 & 0.857 & 0.556\tabularnewline
Ts43\textsuperscript{a} & 55 & 34 & 0.6 & 0.937 & 0.007 & 1.000 & 1 &
0.609 & \emph{0} & 0.500 & \emph{0} & 0.636 & 0.008 & 0.333 &
0.003\tabularnewline
Ts46 & 59 & 22 & 0.797 & 0.921 & 0.011 & 0.500 & 0.003 & 0.833 & 0.119 &
0.833 & 0.180 & 0.800 & 0.478 & 0.857 & 0.052\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Population genetic diversity indices
of\emph{Trachymyrmex septentrionalis} ants across Florida, as well as
separated by site with major region indicated in parentheses11Note.
Abbreviations include: sample size (n), number of unique haplotypes (h),
haplotype (hd) and nucleotide (\selectlanguage{greek}π\selectlanguage{english}) diversities with standard deviation
(SD), and the average number of nucleotide differences (k).\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Region} & \textbf{n} & \textbf{h} & \textbf{hd (SD)} & \selectlanguage{greek}\textbf{π
(SD)}\selectlanguage{english} & \textbf{k}\tabularnewline
\midrule
\endhead
All & 87 & 38 & 0.940 (0.013) & 0.010 (0.00097) & 8.1\tabularnewline
Island (Panhandle) & 5 & 3 & 0.700 (0.218) & 0.001 (0.00045) &
1\tabularnewline
Apalachicola Ranger District (Panhandle) & 27 & 9 & 0.795 (0.054) &
0.007 (0.00093) & 5.3\tabularnewline
Wakulla Ranger District (Panhandle) & 30 & 19 & 0.947 (0.026) & 0.007
(0.00052) & 5.7\tabularnewline
North Central (Peninsula) & 11 & 7 & 0.873 (0.089) & 0.018 (0.00583) &
14.2\tabularnewline
Central (Peninsula) & 14 & 3 & 0.275 (0.148) & 0.002 (0.00137) &
1.8\tabularnewline
\bottomrule
\end{longtable}

\textbf{FIGURES}

\textbf{Figure 1.} Localities of \emph{Trachymyrmex
septentrionalis}specimen sampling sites in this study, color-coded by
major region. The Apalachicola National Forest is highlighted in green.
Major cities are denoted by gray stars and major rivers are labeled.

\textbf{Figure 2.} Bayesian model-based genetic clusters (K = 2)
inferred from STRUCTURE analyses using nine microsatellite loci from
64\emph{Trachymyrmex septentrionalis} ants across Florida. Each vertical
bar represents an individual and each section between black bars
represents major regions, which are labeled above. The two colors
represent the two unique genetic clusters, and the proportion of each
color represents the assignment probability of that individual to each
cluster.

\textbf{Figure 3.} Isolation by distance scatterplots showing the
relationship between genetic differentiation (transformed pairwise Fst
values) and geographic distance (distance between sites in km) as
identified by Mantel tests for \emph{Trachymyrmex septentrionalis} ants
in Florida. The analyses were conducted across (A) all sampling sites
across all regions, (B) all sampling sites excluding SGI, (C) all
sampling sites within the ANF only, and (D) all sampling sites within
the peninsula only. Regression coefficients and p-values are displayed
for each analysis.

\textbf{Figure 4.} A TCS haplotype network for \emph{Trachymyrmex
septentrionalis} constructed with COI mitochondrial DNA sequences. Each
circle represents a unique haplotype and the relative size of circles is
proportional to the number of individuals harboring that haplotype.
Colors correspond to sampling sites and align with Figure 1. Tick marks
on branches indicate the number of mutations between haplotypes. Small
black circles represent predicted intermediate haplotypes. Haplotype
clades (Ts Clades I-III) are circled, highlighted, and labeled.

\textbf{Hosted file}

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