Discussion
Fig
wasps are a group of special insects that live in the
enclosed
syconia of fig trees.
In
the long evolutionary history, they have maintained a close ecological
relationship with the fig fruits.
Related
to the shelter and relatively safe living environment provided by fig
fruits, the evolution of the fig wasps not only shows an adaption to the
environment within fig syconia, their adaption to the large-scale
geo-climatic environment may also be closely related to the life in the
fig syconia.
This
study focused on TEs in the genomes and tried to explore the
evolutionary traces of the fig wasps in response to the changes of the
microenvironment of fig syconia and the changes in the geo-climatic
macroenvironment.
We first compared the differences in TEs between six pollinator and five
non-pollinator species. The results displayed that in non-pollinators,
not only the TE types were significantly more than those of pollinators,
the total lengths and genome contents of TEs were also significantly
higher than those of
pollinators.
This is inseparable from the relatively complicated lifestyles and
living environment of the non-pollinators.
Compared
with the pollinators,
the
non-pollinators can usually
live
on several host fig species, with various parasitic strategies and
complex feeding habits, which requires a stronger ability to
detect
the hosts and
foods
(Borges 2015). Besides, their longer time living at the external surface
of syconia
for
mating and
oviposition
is
fraught with the risks of
predation
by ants and diseases infection (Ranganathan, Ghara et al. 2010), and
thus also requires a stronger ability to avoid natural enemies and
resist diseases.
It
has been reported that TEs can become active in response to stress
(Horvath, Merenciano et al. 2017), and the active state is the
prerequisite
for TE bursts (Belyayev 2014). In our research results, taking the TEs
of the Gypsy family as an example, their expansion is widespread in fig
wasp
species,
so the contents of TEs in the genomes of both pollinators and
non-pollinators have increased significantly. However, the patterns of
burst of the Gypsy in both groups are quite different, with the degree
of expansion higher in non-pollinators, so there is a significant
difference in the TE contents in the genomes of both groups.
In
addition, the more TE contents in non-pollinators may also be because
that winged male of non-pollinators can fly out of the fig fruits where
they were born in and thus mate outside, which will reduce the
probability of inbreeding (Greeff, Jansen van Vuuren et al. 2009, Cook,
Reuter et al. 2017) and provide more abundant resources for the increase
of TE types; this is also consistent with the results that all
non-pollinators had their own unique TE families.
On
the contrary, in pollinator species, the stricter
host
specificity, single and
sufficient
food sources, stable and relatively hidden habitats, and the higher
degree of inbreeding may be associated with insufficient motivation for
expansion of TEs and thus the lack of TEs in the genomes (Machado,
Robbins et al. 2005, Greeff, Jansen van Vuuren et al. 2009, Cruaud,
Ronsted et al. 2012).
This
is not only reflected in the fact that many pollinators did not have
species-specific TEs, but also that some TE families were absent in some
or all of the pollinator species. For example, hAT-hATm and
insect-specific Crypton-I only existed in the genomes of the
non-pollinator species.
In
order to more clearly reflect the differences in the
composition
of TEs in evolutionary history, the insertion time distribution of TEs
were drawn.
Comparing
the burst patterns of both groups of fig pollinators and
non-pollinators, we found that the burst peaks of non-pollinators were
more complex.
For
example, the burst peaks of the pollinator onF.benjaminapresented
a single concentrated “n” shape, while the burst peaks of three
non-pollinators on the same host fig species presented a complex and
extensive
“m” shapes.
This
implied that both groups of fig wasps experienced different environment
changes in the evolutionary history or responded differently to the same
environmental changes.
It
has been reported that the emergence of fig wasps is
distinctly
seasonal. The pollinators can leave more offsprings in the rainy season,
and non-pollinators produce more offsprings in the dry season (Wang,
Yang et al. 2005, Dong, Peng et al. 2020).
On
the contrary, there is a strong competition between non-pollinators and
pollinators, and the numbers of both groups show a significant
negative
correlation. Therefore, non-pollinators have a higher reproduction rate
in the hot and rainy season (Weiblen 2002, Zhang and Yang 2009).
This
suggests that both groups of fig wasps have different response to the
same environmental changes (Wang, Compton et al. 2015).
Our study detected that almost all the fig wasps had significant TE
burst events in the time range of 32-34 Mya. This period coincided with
the special Eocene-Oligocene (E/O) transisiton period of
~33.7 Mya’s, which was considered to have been an
important stage of the global climate transition in geological history.
Due to the action of the Oi-1glaciation,
the global temperature dropped significantly. With the Antarctic ice
sheet growing rapidly,
the
climate became colder and drier, causing different degrees of extinction
of organisms
and
the decline of broad leaf forests (Zachos, Pagani et al. 2001, DeConto
and Pollard 2003).
The
major burst peaks in non-pollinators and
the
minor burst peaks in pollinators in this period indicated that both
groups were all sensitive to the climate during this period,
but
the non-pollinators retained more TEs than the pollinators. These ice
sheets persisted until the late Oligocene (26 to 27 Mya), when a warming
current reduced the extent of Antarctic ice.
This
warm climate lasted until the mid-Miocene (~15 Mya), and
reached
its peak in the late mid-Miocene climate optimum (17 to 15 Mya).
Thereafter, the East Antarctic Ice Sheet(EAIS)began to expand
~ 14 million years ago, and
the
global climate underwent a major reorganization (Groeneveld, Henderiks
et al. 2017).
The
major burst peaks of pollinators were located in the time range of 13-28
Mya, which coincided with the above-mentioned high temperature phase,
and this implied that pollinators might be more sensitive to high
temperature climate changes.
The
early Pliocene was marked by a subtle warming trend until the onset of
Northern
Hemisohere Glaciation (~3.2 Mya), the gradual formation
of Northern Hemisphere Ice Sheet, and present of bipolar state of the
global ice sheet (Tiedemann, Sarnthein et al. 1994, Zachos, Pagani et
al. 2001). During this period, the obvious recent TE bursts in
non-pollinators (0-2 Mya) further demonstrated the sensitivity of this
group to low-temperature climates. To figure out whether the temporal
overlap between the TEs bursts of the fig wasps and the geological
climate changes is widespread, we also investigated the TE burst events
of five species that are not related to fig trees but are widespread and
live in a relatively open
environment,
and found that the major burst peaks of these species were distributed
in 0-3
Mya.
These comparable results indicate that
the
fig wasps
that
are confined to the fig trees in tropical and subtropical regions have
retained more traces of TE bursts related to
geo-climate
changes than the free-living species that are more widespread.
In
order to further clarify the relationship between the TE bursts of fig
wasps and the geo-climate changes, for all the 11 species of fig wasps,
we analyzed the function of genes near TEs
that
were inserted in the four periods representing the growth or decay of
the major continental ice sheet of the Cenozoic. The results showed that
the functions of these genes were mainly involved in the pathways such
as rhythm, signal transduction, and neural activity.
Among
them, the Circadian entrainment pathway (map 04713) was enriched in many
species in all periods. Especially at 31-35 Mya, when the common burst
peaks occurred, this pathway was significantly enriched in up to nine
fig wasps.
Circadian
entrainment is the process
to
activate the internal biological clock of the organism by
recurring
exogenous signals, so that the organism’s endocrine and behavioral
rhythms will be synchronized to environmental cues (Johnson, Elliott et
al. 2003).
There
are interesting and open questions whether the periodic climate changes
will
have a direct impact on the fig wasps, or they will exert selective
pressure on the fig wasps through their influence on the fig trees.
Generally, climate changes have great impacts on plants because they
will change the flowering cycle (Ehrlen and Valdes 2020).
The
year-round phenological variations of fig trees living in the tropics
are generally limited, with fig production featuring intra-individual
flowering synchrony and inter-individual asynchrony throughout the year
(Bronstein, Gouyon et al. 1990).
However,
for the higher latitude regions and the edges of the species’
distribution range, the synchronization of flowering synchrony, and the
productions of wasps and seeds production display seasonal fluctuations,
which are accompanied by a reduction in reproductive success (Chen,
Zhang et al. 2018). Therefore, considering that both parts of the
symbiosis system of the fig and fig wasps have different responses to
the geographic climate differences, it is not difficult to speculate
that, in the evolutionary history of the symbiosis system, the changes
in the flowering periods induced by the rapid paleoclimate changes would
exert
great evolutionary pressure on the fig wasps, because they need to keep
pace with the flowering cycle of the fig trees. In this adaptive
process, the adaptation of the Circadian entrainment genes may play a
pivotal
role. In other words,
periodic
climatic changes can have a huge impact on plants, which in turn can
affect the evolution of symbiotic organisms closely related to plants.
Finally, by analyzing the correlation between the distributions of TEs
and CRMs in the genomes of the fig wasps, we speculated a potential
molecular mechanism for the adaptive evolution of these fig wasps
through the action of TEs.
There
are three main ways for TEs to regulate host genes. One is that newly
inserted or deleted TEs may change the regulation or coding environment
at a specific location, thereby affecting gene expression.
For
example, the adaptive “industrial melanism” phenotype ofBiston
betularia has been evolved through
the
intronic insertion of a DNA transposon, increasing transcript abundance
of the cortex gene during early wing development (van’t Hof,
Campagne et al. 2016).
Second,
TE insertions might also lead to modifications at the
epigenetic
level, which can affect the expression of neighbouring genes. For
example, inArabidopsis ,
gene
expressions are negatively correlated with the density of methylated TEs
(Hollister and Gaut 2009). Third and the last, TEs could be evolved into
regulatory sequences which might affect the downstream gene expression
(Oliver and Greene 2011).
In
the view of the positive role and universality of TEs as regulatory
sequences, we analyzed the relationship between TEs and CRMs based on
homology prediction results, and we found that many TEs in the genomes
may have evolved into functional CRM sequences. The genes near those CRM
sequences that overlapped with TEs were mainly the genes enriched in
pathways related to environmental information processing such as
Circadian entrainment.
This
suggested that in these fig wasp species, some TEs might have evolved
into CRMs to affect nearby gene expression to achieve gene environmental
adaptability.