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\begin{document}

\title{Assisted dispersal and reproductive success in an ant species with
matchmaking}



\author[1]{Mathilde Vidal}%
\author[1]{Jürgen Heinze}%
\affil[1]{University of Regensburg}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Workers of the ant Cardiocondyla elegans drop female sexuals into the
nest entrance of other colonies to promote outbreeding with unrelated,
wingless males. Corroborating results from previous years we document
that carrier and carried female sexuals are typically related and that
the transfer initially occurs mostly from their joint natal colonies to
unrelated colonies. Female sexuals mate multiply with up to seven
genetically distinguishable males. Contrary to our expectation, the
colony growth rate of multiple-mated and outbred female sexuals was
lower than that of inbred or single-mated females, leading to the
question of why female sexuals mate multiply at all. Despite the obvious
costs, multiple mating might be a way for female sexuals to ``pay rent''
for hibernation in an alien nest. We argue that in addition to evading
inbreeding depression from regular sibling mating over many generations,
assisted dispersal might also be a strategy for minimizing the risk of
losing all reproductive investment when nests are flooded in winter.%
\end{abstract}%



\sloppy


\textbf{Assisted dispersal and reproductive success in an ant species
with matchmaking}

Mathilde Vidal \selectlanguage{romanian}ǀ J\selectlanguage{ngerman}ürgen Heinze

LS Zoologie / Evolutionsbiologie, Universität Regensburg,
Universitätsstraße 31, 93040 Regensburg, Germany

\textbf{Abstract}

Workers of the ant \emph{Cardiocondyla elegans} drop female sexuals into
the nest entrance of other colonies to promote outbreeding with
unrelated, wingless males. Corroborating results from previous years we
document that carrier and carried female sexuals are typically related
and that the transfer initially occurs mostly from their joint natal
colonies to unrelated colonies. Female sexuals mate multiply with up to
seven genetically distinguishable males. Contrary to our expectation,
the colony growth rate of multiple-mated and outbred female sexuals was
lower than that of inbred or single-mated females, leading to the
question of why female sexuals mate multiply at all. Despite the obvious
costs, multiple mating might be a way for female sexuals to ``pay rent''
for hibernation in an alien nest. We argue that in addition to evading
inbreeding depression from regular sibling mating over many generations,
assisted dispersal might also be a strategy for minimizing the risk of
losing all reproductive investment when nests are flooded in winter.

\textbf{KEYWORDS} Formicidae, Hymenoptera, population structure, mating
behavior, outbreeding, polyandry



\selectlanguage{romanian}\textbf{1 ǀ Introduction}

In many animals, offspring dispersal is critical to avoid local resource
competition and the risk of inbreeding. Given that dispersal is often
dangerous and young individuals have limited knowledge about the
specific conditions in their surroundings, parents may assist their
young during dispersal. For example, poison frogs may transfer tadpoles
from land to water (Pa\selectlanguage{polish}š\selectlanguage{english}ukonis et al., 2019), wolf spiders may carry
spiderlings (Bonte et al., 2007), and mother bats teach their offspring
to navigate and find suitable habitats (Goldshtein et al., 2022).
Similarly, in several species of ants, workers accompany and even carry
young queens during the founding of new colonies away from their natal
nests (e.g., M\selectlanguage{ngerman}öglich \& Hölldobler, 1974; Fernández-Escudero et al.,
2001; Peeters \& Aron, 2017).

In the ant \emph{Cardiocondyla elegans,} workers may carry their female
sexual sisters over several meters and dump them into the nest entrance
of another colony. We have argued that this peculiar behavior
constitutes a type of assisted dispersal and mate choice, allowing young
queens to outbreed with several unrelated males away from their natal
nests (Vidal et al., 2021). The ant genus \emph{Cardiocondyla} is
characterized by an ancestral male polyphenism with winged disperser
males and wingless ``ergatoid'' males, which locally mate with female
sexuals eclosing in their natal nests. Colonies of most
tropical\emph{Cardiocondyla} are relatively small, with only a few dozen
workers and one or several queens. Individual wingless males are
therefore capable of monopolizing mating with all female sexuals by
aggressively excluding other males with their shear- or sickle-shaped
mandibles. In contrast, colonies of Palearctic species are typically
much larger, with several hundred workers and obligatorily a single,
multiply mated queen. Furthermore, female sexuals are produced not
year-round as in the tropics but seasonally. Because of this, wingless
males are no longer capable of individually securing copulations with
all female sexuals and indeed have evolved mutual tolerance (Heinze,
2017). As mating occurs in the natal nest, inbreeding coefficients
determined from microsatellite genotypes are relatively high.
Nevertheless, data suggest that 40 to 80 \% of matings involve unrelated
sexuals (Schrempf et al., 2005; Lenoir et al., 2007; Schrempf, 2014;
Vidal et al., 2021), and observations in the field revealed that in
\emph{C. elegans} these are facilitated by ``royal matchmaking'' by the
workers, which transfer female sexuals between unrelated nests.

Here, we follow up on our earlier studies and provide new data about
queen carrying in the field, the relatedness among carrier and carried
ants, and the growth rate of colonies started by individual queens after
mating only with brothers, one or multiple unrelated males. We expected
that outbreeding and multiple mating are reflected in a better
performance of colonies, i.e., faster colony growth.

\textbf{2} \selectlanguage{romanian}ǀ \textbf{Material and Methods}

\textbf{2.1 ǀ Study species and collection}

\emph{C. elegans} Emery 1869 is a thermophilic ant that builds its nests
in the soil in barely vegetated, sandy patches, e.g., along rivers in
Iberia, France, and Italy. Populations in the Eastern Mediterranean are
now considered to represent a separate species, \emph{C.
dalmatica}Soudek 1925 (Seifert, 2018). From 2014 to 2017 we studied
several populations of \emph{C. elegans} along the rivers Gardon and
Rhone in Southern France. In 2018 and 2019 we concentrated our study on
one site south of Remoulins on Gardon (N 43\selectlanguage{ngerman}° 55\selectlanguage{english}' 43.9\selectlanguage{english}", E 4deg 34\selectlanguage{english}' 5.1\selectlanguage{english}"),
where we repeated our investigation in August 2020 to corroborate
earlier results and retrieve additional detail about the peculiar
phenomenon. For genetic analyses, we also used samples collected at the
same site in August 2019.

The locations of nests were identified by following foragers back to the
nest entrance. Samples of workers were stored in EtOH for the
determination of nestmate relatedness by genotyping the individuals. By
observing nest entrances and following individual pairs of carrying
workers and carried female sexuals, over several minutes, we attempted
to identify the direction of the transfer. For genetic analyses, we
collected the ants with an aspirator immediately before the female
sexual was placed into the entrance of the recipient colony. In
addition, we collected pairs of unknown origin and destination and
either stored them in 100\% EtOH for the determination of genetic
relatedness or kept them alive for later experiments.

\selectlanguage{romanian}\textbf{2.2 ǀ Genetic analyses}

We determined the genotypes of workers and female sexuals at seven
microsatellite loci as described before (CE2-3A, CE2-4A, CE2-4E, CE2-5D,
CE2-12D, Card 8, and Cobs 13; e.g., Lenoir et al. 2007, Vidal et al.
2021). In short, DNA was extracted from whole ants using the CTAB method
(modified from Sambrook \& Russell, 2001) and diluted in 30\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}l of TE
buffer. Polymerase Chain Reactions (PCRs) were performed in a 20 \selectlanguage{greek}μ\selectlanguage{english}l
reaction volume using 1 \selectlanguage{greek}μ\selectlanguage{english}l of DNA with 19 \selectlanguage{greek}μ\selectlanguage{english}l of master-mix (7 \selectlanguage{greek}μ\selectlanguage{english}l H2O,
10 \selectlanguage{greek}μ\selectlanguage{english}l GoTaq, and 1 \selectlanguage{greek}μ\selectlanguage{english}l of each forward and reverse primers). Samples were
amplified following Lenoir et al. (2005) with an initial denaturation
step at 94 \selectlanguage{ngerman}°C for 3\selectlanguage{english} min, 40 cycles at 94\selectlanguage{english} degC for 45\selectlanguage{english} s, with an annealing
temperature according to the primer for 45\selectlanguage{english} s (supplementary table 2.1),
followed by a step of 72\selectlanguage{english} degC for 45\selectlanguage{english} s, and a final extension step at
72\selectlanguage{english} degC for 7\selectlanguage{english} min. Allele sizes were determined by GeneScan(r) 3.1 software
(Applied Biosystems) and GeneScan(r) 500 TAMRA dye as a size standard.
Relatedness among nestmates and pairwise relatedness between female
sexuals and their carriers were calculated for samples from the two
different years separately following Queller and Goodnight (1989) using
the \emph{related} package (Pew et al., 2015) on R software-4.0.3 and
Relatedness v4.2 (Queller \& Goodnight, 1994). Fixation coefficients
(F\textsubscript{ST}) were estimated using GenAlEx v6.51b2 (Peakall \&
Smouse, 2006). The frequency of sibling mating \selectlanguage{greek}α \selectlanguage{english}was calculated from
F\textsubscript{ST} = \selectlanguage{greek}α/\selectlanguage{english}(4-3\selectlanguage{greek}α\selectlanguage{english}) (Pamilo, 1985).

\selectlanguage{romanian}\textbf{2.3 ǀ Colony maintenance}

Individual life pairs of a carrying worker and a carried female sexual
were kept in 1.5 ml Eppendorf tubes with moist cotton and fed with
cookie crumbs. After transfer to the laboratory, we added to each of 43
female sexuals one to three unrelated males from laboratory stock
colonies (one male to 16, two males to 13, and three males to 14 female
sexuals). Additional 112 female sexuals were left without the chance to
mate with additional males. One day later, the males were removed and
the female sexuals were individually placed into 94mm x 16mm Petri
dishes divided by a separator into two equal halves. One half of the
dish was filled with plaster with small tunnels and two or three small
chambers serving as the nest, the other served as a foraging arena, into
which we added a droplet of honey, dead \emph{Drosophila} or chopped up
cockroaches twice per week. To allow the ants to move from the nest to
the foraging arena we made a 5mm wide hole in the plastic separator
using a soldering iron. The nesting chambers were covered by red plastic
foil. To more closely mimic the architecture of natural nests the Petri
Dishes were kept vertically by attaching them to a plaster base.
Experimental nests were kept in incubators under near-natural artificial
conditions matching those found at the collecting site (i.e., from June
to September at summer temperatures of 17\selectlanguage{ngerman}°C -- 34°C, slowly changing by
a weekly temperature decrease of up to 4°C to 5°C -- 13°C in December to
March). Offspring production of isolated female sexuals was recorded
every week for a total of 40 weeks. In addition, we observed offspring
production from similarly housed lab-reared female sexuals, which had
had only access to brothers. These latter colonies were kept alive only
for three months after the hibernation. Note here that we use ``female
sexuals'' even for individuals that have mated and began laying eggs and
which would conventionally be referred to as ``queens.'' However, as
many data refer to both mated, fertile and virgin, non-fertile
individuals, using both terms would have made the text difficult to
read.

In addition to the population genetics software mentioned above,
statistical analyses were done with R 4.0.3 (R Core Team 2020) and
Statistica 6.0 (StatSoft, Tulsa, OK).

\selectlanguage{romanian}\textbf{3 ǀ Results}

\textbf{3.1 ǀ Population and colony genetic analyses}

Nestmate relatedness and inbreeding coefficients were determined from 42
workers from six colonies collected in 2019 and 113 workers from 16
colonies collected in 2020. Mean relatedness per colony ranged from 0.18
to 0.66 in 2019 and 0.13 to 0.69 in 2020. Across all studied colonies,
the mean relatedness was 0.49 \selectlanguage{ngerman}± SE 0.08 in 2019 and 0.39 ± 0.04 in 2020,
which is in the range of earlier estimates (0.34 ± 0.05, Vidal et al.,
2021). The same applies to the fixation coefficient (2019: 0.43 ± 0.10,
2020: 0.28 ± 0.03, 2018: 0.30 ± 0.03).

As colonies are obligatorily monogynous, i.e., have only one fertile
queen, the lower relatedness suggests multiple mating, which is also
evident from the number of genotypes found among nestmates. The mean
effective mating frequency of queens calculated from nestmate
relatedness following Tarpy and Nielsen (2002) is 2 and 3.5 in our
samples from 2019 and 2020, respectively. The actual mating frequencies
of individual queens that had been collected when carried in the field,
estimated from the genotypes of offspring reared in the laboratory,
revealed a large variation from one to seven mating partners per queen
(mean 4.3 ± SD 1.6, \emph{n} = 6). Though adding unrelated males to
female sexuals in the laboratory resulted in additional mating as
reflected in worker genotypes, mean mating frequencies did not differ
(mean 3.3 ± 2.3, \emph{n} = 12; \emph{t} -test, \emph{t} =
0.950,\emph{p} = 0.346).

The relatedness between carrier and carried female sexual was 0.39 ±
0.03 in 2019 (\emph{n} = 91) and 0.23 ± 0.08 in 2020 (\emph{n} = 8),
i.e., in the range of colony-wise estimates for relatedness but, in
contrast to our earlier study (Vidal et al., 2021), significantly lower
than the estimated mean relatedness in these years (\emph{t} -test,
2019:\emph{t} = 2.90, \emph{p} = 0.005; 2020: \emph{t} = 2.86, \emph{p}
= 0.009). The relatedness of female sexuals to source colonies was 0.29
± 0.14 (\emph{n} = 3) in 2019 and 0.21 ± 0.06 (\emph{n} = 8) in 2020,
the respective values for recipient colonies were 0.03 ± 0.21 and -0.04
± 0.07. Relatedness to the source colony was not different from nestmate
relatedness in 2019 (\emph{t} = 1.98, \emph{p} = 0.09) but significantly
lower in 2020 (\emph{t} = 3.81, \emph{p} = 0.001). Relatedness to the
target colony did not differ from zero (single-sample \emph{t} -test,
2019: \emph{t} = 0.25, \emph{p} = 0.83; 2020: \emph{t} = 1.62, \emph{p}=
0.15).

The large number of pairs of carriers and female sexuals collected in
2019 allowed us to estimate how pairwise relatedness varied with time.
The probability of carrier and female sexual being less closely related
than 0.25 (the value for halfsisters) decreased from 0.17 in the first
phase of our field collection (Aug 14 -- 16, \emph{n} = 6) to 0.43 in
the seventh and last phase (Aug 30, \emph{n} = 14; Spearman rank
correlation, \emph{r\textsubscript{S}} = 0.81, \emph{p} = 0.035).

\selectlanguage{romanian}\textbf{3.2 ǀ Colony growth rate and female mating frequency}

A considerable percentage of female sexuals, which had been transferred
to the laboratory and kept in isolation, died within the first four
weeks of the experiment (40\% of 43 female sexuals with and 26\% of 112
female sexuals without temporary access to males in the lab). Of the
surviving individuals with access to males, 23 (88\%) produced at least
one worker compared to 13 (16\%) of those without. Female sexuals, which
succeeded in producing offspring over the 530 days of the experiment
(\emph{n} = 36), lived significantly longer than those that did not
(\emph{n} = 80; log-rank test, test statistic = -7.57,
\emph{p}\textless{} 0.0001, Figure 1). Dissection of four female sexuals
that died or did not produce any workers revealed an empty spermatheca,
suggesting that also other unproductive female sexuals had been unmated.

While it initially appeared that female sexuals, which had been given
temporary contact with two or three males in the lab, produced
considerably more offspring than those with contact with only one male
or without contact (data not shown), paternity analyses of ten offspring
workers per female sexual revealed the opposite pattern. The number of
offspring varied significantly with mating frequency (Kruskal-Wallis
test, \emph{H} \textsubscript{(6,18)} = 14.97, \emph{p} = 0.02) and
surprisingly average productivity declined with mating frequency
(Spearman rank correlation, \emph{r\textsubscript{S}} = -0.816,
\emph{p}\textless{} 0.001, Figure 2). Furthermore, a comparison between
these field-collected and lab-reared female sexuals, which could only
have mated with their brothers, did not reveal a difference in the
number of offspring reared during the first three months after
hibernation (Kruskal-Wallis test, \emph{H} \textsubscript{(19,40)} =
20.80, \emph{p}= 0.348, Figure 2).

\textbf{4 ǀ Discussion}

Our new population and colony genetic data corroborate earlier estimates
(Vidal et al., 2021) that colonies of the ant \emph{Cardiocondyla
elegans} in Southern France typically harbor a single, multiply mated
queen and that a large percentage of mating episodes involve siblings.
Workers promote outbreeding by transferring female sexuals between
nests. We have previously hypothesized that during the mating period in
summer female sexuals are carried repeatedly from nest to nest (Vidal et
al., 2021). While first related workers will carry them away from their
natal nests, later unrelated workers will move them from a first
unrelated recipient colony to another. Our data appear to support this
hypothesis. First, the percentage of carriers and female sexuals that
were less closely related than 0.25 decreased from mid to late August
2019. Second, in contrast to our previous study (Vidal et al., 2021),
the average relatedness between carrier and carried female sexual was
significantly lower than mean nestmate relatedness in both years,
probably because in 2019 and 2020 more sexuals were collected late in
the mating season. Third, genotyping the offspring of carried female
sexuals showed that several of them had mated with six or seven
genetically distinguishable males, which suggests the ``visit'' of more
than one unrelated colony.

A large fraction of field-collected female sexuals died without
producing workers when housed in artificial nests in the laboratory.
Dissections of a small number of corpses, in which the ovaries had not
yet decayed, revealed the absence of sperm in the spermatheca,
suggesting that also other unproductive individuals had been virgins.
Their higher mortality matches the finding that in social insects mating
and reproduction increase life expectancy (e.g., Hartmann \& Heinze,
2003; Schrempf et al., 2005), in contrast to many solitary insects, in
which virgins outlive reproductively active, mated females (e.g.,
Service, 1989; Chapman et al., 1998; Limberger et al., 2021). A positive
association between fecundity and longevity appears to be a trait
associated with the evolution of perennial insect societies (e.g.,
Parker, 2010; Rodrigues \& Flatt, 2016, Jaimes-Ni\selectlanguage{ngerman}ño et al., 2022).

Contrary to our expectation colony growth rates were highest in colonies
with a singly or doubly mated queen, while colonies with queens with
higher mating frequencies were much less productive. Multiple mating has
been shown to increase colony growth rate in the western harvester ant
(\emph{Pogonomyrmex} occidentalis; Wiernasz et al., 2004), increase the
resistance to pathogens in bees and wasps (e.g., Shykoff \&
Schmid-Hempel, 1991; Seeley \& Tarpy, 2007; Saga et al., 2020) and a
more efficient division of labor (e.g., Oldroyd \& Fewell, 2007). As in
our study colony growth rate was monitored only for about one year and
no sexuals were produced during this time we cannot exclude that
multiple mating in \emph{C. elegans} might have a beneficial effect
later in the life of a colony or under more variable environmental
conditions in the field.

Laboratory-reared queens that were only given the chance to mate with
their brothers produced similar numbers of offspring during the first
three months after hibernation as single- or double-mated queens. This
means that outbreeding does not immediately lead to a higher fitness
than a single round of inbreeding. In honeybees and many other
Hymenoptera with single locus complementary sex determination (sl-CSD)
inbreeding results in the production of sterile or inviable diploid
males (e.g., van Wilgenburg et al., 2006; Darvill et al., 2012).
While\emph{Cardiocondyla} appears to have a different mechanism of sex
determination, in which sister-brother and even mother-son mating does
not result in the production of diploid males (Schrempf et al., 2006;
Schmidt et al., 2013), repeated sib-mating nevertheless results in
decreased lifespan and reproductive success (Schrempf et al., 2006).
That a single round of sib-mating does not immediately result in
inbreeding depression in \emph{C. elegans} does therefore not exclude
any possible negative effects of inbreeding over several generations.

The significant initial costs of multiple mating and the efforts
associated with assisted outbreeding raise the question of why \emph{C.
elegans} has evolved the carrying of female sexuals and polyandry.
Female sexuals may mate ``for convenience'' when resistance to courting
males is too costly, as has been suggested for the polyandrous queens
of\emph{Plagiolepis pygmaea} (Trontti et al., 2007). However, previous
observations of mating behavior in \emph{Cardiocondyla} clearly show
that female sexuals are well capable of defending themselves even
against the most vicious courtship by much larger males (e.g., Heinze et
al., 2021). Instead, female sexuals of \emph{C. elegans} might copulate
repeatedly ``to pay rent'' for hibernation in an alien colony. Colonies
should not tolerate alien queens, which already mated with alien males
and are no longer available for mating with the workers' brothers, as
this would pose the risk of usurpation and queen replacement without
fitness benefits. If the adopted queen mated with the resident males
this might increase the potential reproductive success of the latter and
therefore the inclusive fitness of the workers. Such a hypothesis might
also explain why workers carry alien female sexuals from their nest to
other, unrelated recipient colonies -- doing so they disperse the sperm
of their brothers without risking that their own queen is later
replaced, in a way passing the buck like in the ``Black Peter'' or ``Old
Maid'' card game, in which players try to win by getting rid of an
unmatchable card. In any case, by exporting female sexuals to other
nests, colonies increase the likelihood that their sexual offspring
survive hibernation. Temporary flooding of nests on the banks of Loire
during winter results in high colony mortality (Lenoir et al., 2007) and
assisted dispersal may be a reasonable risk-averse strategy in
unpredictable environments.

ACKNOWLEDGMENTS

The study was supported by DFG (He 1623/42).

CONFLICT OF INTEREST

None declared.

AUTHOR CONTRIBUTIONS

Mathilde Vidal: Investigation and data analysis (lead)

Jürgen Heinze: Conceptualization (lead); investigation and data analysis
(supporting); writing--original

draft (lead)

ETHICAL APPROVAL

The ants were collected with the permit TREL1902817S/132 issued by
Access and Benefit-Sharing Clearing-House (ABSCH 2019). No other permits
were required for the study.

DATA AVAILABILITY STATEMENT

Raw data, individual genotypes, pairwise relatedness estimates and
colony growth data are available at
https://doi.org/10.6084/m9.figshare.19982081.

\textbf{ORCID}

\emph{Mathilde Vidal} https://orcid.org/0000-0003-2178-7052

\emph{Jürgen Heinze} https://orcid.org/0000-0002-9403-5946

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} Survival time of female sexuals of the
ant\emph{Cardiocondyla elegans} from Southern France that produced
worker offspring in the laboratory () or failed to do so (). The symbol
+ indicates censored information. Unproductive female sexuals had a
significantly shorter life expectancy.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} Offspring production by female sexuals of the
ant\emph{Cardiocondyla elegans} that had access only to brothers or in
addition to one to seven unrelated males. Multiple-mated individuals
appeared to produce considerably fewer offspring than inbred or outbred,
single- or double-mated female sexuals

\selectlanguage{english}
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