Introduction
Comparative genomics provides a framework for identifying the molecular
mechanisms underlying unique organismal adaptations, in their endless
forms. To date, comparative genomic approaches have revealed the
mechanisms underlying terrestrial adaptations in mudskipper fish (You et
al., 2014), heat tolerance in coral (Bay, Rose, Logan, & Palumbi,
2017), cold stress tolerance in Draba (Nowak et al., 2020), and
extreme longevity in naked mole rats (X. Zhou et al., 2020). In most
cases the search for molecular adaptations has focused on orthologous
single-copy genes, but gene loss and duplication can also be adaptive
and are critical to understanding of how phenotypic adaptations evolve.
Analyses based on highly contiguous genome assemblies have uncovered
gene expansions likely associated with production of urushiol and
anthocyanins in mango (P. Wang et al., 2020), the earliest events of
gene duplication in cytoskeletal and membrane-trafficking families in
eukaryotic cellular evolution (Vosseberg et al., 2020), pseudogenization
in genes associated with testicular descent in afrotherian mammals
(Sharma, Lehmann, Stuckas, Funke, & Hiller, 2018), gene losses
associated with diving–related adaptations in cetaceans (Huelsmann et
al., 2019), and losses associated with physiological and metabolic
adaptations in fruit bats (Sharma, Hecker, Roscito, Foerster, Langer &
Hiller, 2018). Given the importance of gene family evolution, multiple
large-scale genome sequencing consortia such as the Earth BioGenome
Project (Lewin et al., 2018), the Vertebrate Genomes Project (Rhie et
al., 2020), and Bat1K (Teeling et al., 2018) aim to generate
high-quality genome assemblies for species spanning entire clades and
even the entire phylogenetic ‘Tree of Life’, thereby enabling greater
confidence in analyses of gene loss and gene family evolution.
Gene family expansions and contractions are influenced by selection,
including from biological factors such as pathogens. Host-pathogen
interactions are shaped by reciprocal selection, an evolutionary arms
race which has forced hosts to evolve complex immune defense mechanisms
(Papkou et al., 2019; Sironi, Cagliani, Forni, & Clerici, 2015).
Vertebrates have two types of immune response: innate immunity, which is
non-specific and acts as a first line of defense; and adaptive immunity,
which is highly specific and generates immune memory (Delves, Martin,
Burton, & Roitt, 2017; Janeway & Travers 2001.). Several
immune-related gene families that have experienced substantial
evolutionary changes during mammal evolution. While many important
facets of the immune system are conserved, immune gene families have
high rates of evolution whether measured via substitution rate ratios or
birth–death turnover (Bernatchez & Landry, 2003; Goebel et al., 2017;
Minias, Pikus, Whittingham, & Dunn, 2019; Santos et al., 2016; Shultz
& Sackton, 2019; Van Oosterhout, 2009). This is especially true of the
Major Histocompatibility Complex (MHC), which is responsible for
generating cell surface proteins that play essential functions in the
adaptive immune system (Janeway & Travers 2001).
This combination of highly conserved, and highly variable components of
the immune system, is particularly intriguing among bats. Among mammals,
bat diversity is second only to that of rodents, and encompasses over
1,400 species that occupy a broad diversity of ecological niches on six
continents (Fenton & Simmons, 2015; Nogueira et al., 2018). The success
of bats is likely related to a suite of adaptations unique both to the
clade as a whole and to various subclades within the Order Chiroptera.
The most obvious of these is powered flight, allowing bats to occupy a
unique aerial niche not utilized by any other mammal. While this unique
niche limits body size, within that constraint bats have been
exceptionally successful and have diversified in ways unparalleled among
other mammals. For example, bats evolved virtually every mammalian
dietary strategy (e.g., frugivory, carnivory, nectarivory, piscivory)
and have done so in a relatively short evolutionary time frame (Dumont
et al., 2012). Another less obvious but likely more interesting
adaptation is the exceptional longevity and increased health span (the
period of life during which an organism is in generally good health)
exhibited by many bat species given their body size. Many species such
as the Bechstein’s bat (Myotis bechstein) the little brown bat,
Brandt’s bat (Myotis brandtii ), greater mouse-eared bat
(Myotis myotis ) and greater horseshoe bat (Rhinolophus
ferrumequinum ) have unexpectedly long health spans, living 30 - 40
years (Fleischer, Gampe, Scheuerlein & Kerth, 2017; Foley et al., 2018;
Podlustsky, Khritankov, Ovodov & Austad, 2005; Seim et al., 2013;
Wilkinson & Adams, 2019). Such longevity defies the expectation that
large species are longer-lived than small species; despite constrained
body size, bats live longer than other mammals of similar size (Austad
& Fischer, 1991; Healy et al., 2014). Bat longevity and health span may
be influenced by their exposure to extrinsic mortality factors. Powered,
mostly nocturnal flight may lower bats’ exposure to some sources of
extrinsic mortality, including predation (Healy et al., 2014). However,
bats’ propensity to roost in large colonies also increases their
exposure to another source of mortality – infections (Brook & Dobson,
2015; H. Han et al., 2015).Thus, to achieve such longevity and decreased
senescence, long–lived bat populations must overcome the burden of
infectious diseases.
The uniqueness of bats extends to the immune repertoire. Early in the
age of whole-genome analyses, it was clear that inflammation-related
gene families had expanded or contracted, and certain single–copy genes
associated with immunity and cell repair had experienced selection in
bats (G. Zhang et al., 2013). There is still debate as to whether bats
harbor a disproportionately large number of viruses (Moratelli &
Calisher, 2015; Olival et al., 2017), but there is no doubt that several
recent viral intrusions into our own species ultimately originated from
bat hosts (Drexler et al., 2012; Goldstein et al., 2018; Hu et al.,
2017; Memish, Perlman, Van Kerkhove, & Zumla, 2020; Towner et al.,
2007). This likely includes the current SARS-CoV-2 pandemic (Boni et
al., 2020; Lau et al., 2020). Bats appear to have the ability to
tolerate these viruses with few health impacts, hence recent studies
have focused on bat comparative genomics (Jebb et al., 2020) and its
emphasis on viral response (reviewed in: Gorbunova, Seluanov, &
Kennedy, 2020; Hayman, 2019). Although little is known from this
perspective, there is a growing body of functional analyses showing that
bats are unusual among mammals in how they deal with viruses (Ahn et
al., 2019; A. Banerjee et al., 2020; Miller et al., 2016; Schountz,
Baker, Butler, & Munster, 2017; Xie et al., 2018).
The ‘inflammosome’ is typically highly conserved across mammals, but
bats exhibit a reduced inflammatory response that may be tied to their
ability to cope with viral infection while experiencing minimal impact
(Pavlovich et al., 2018). For example, the PYHIN gene family, namely,
appears to have been almost completely lost in bats (Ahn, Cui, Irving,
& Wang, 2016; G. Zhang et al., 2013) while at least one PYHIN gene can
be found in all other eutherians examined. Similarly, in bats, the
inflammatory function of interferons (G. Zhang et al., 2013) appears
distinct and the APOBEC3 repertoire, which is associated with anti-viral
response, is expanded (Jebb et al., 2020; Hayward et al., 2018). All of
these functional patterns suggest an overall dampened inflammatory
reaction despite a robust immune response to viruses whose origins may
lie in the gene repertoires available to bats (A. Banerjee, Rapin,
Bollinger, & Misra, 2017; A. Banerjee et al., 2020).
Gene family evolution also likely plays a role in the unique dietary
ecology of bats. Several studies have found a variety of mechanisms
influencing dietary adaptation. For example, convergent amino acid
substitutions in several lineages of frugivorous bats have occurred
independently (Gutiérrez-Guerrero et al., 2020; Shen, Han, Zhang,
Rossiter, & Zhang, 2012; Teeling et al., 2018; K. Wang et al., 2020),
and are associated with the shift to a high-sugar diet. Another strategy
has been to repurpose a given gene to accommodate such dietary shifts.
(Shen, Han, Jones, Rossiter, & Zhang, 2013). With the exception of
olfactory receptors (Hayden et al., 2014; Hughes et al., 2018;
Tsagkogeorga, Müller, Dessimoz, & Rossiter, 2017), the roles of gene
loss and gain in shaping dietary evolution of bats have not been
comprehensively explored.
Here we investigate bat gene family evolution related to immunity,
metabolism, and dietary adaptations, using the most extensive genomic
sampling within bats to date. Despite variability in quality of
assemblies, the ecological diversity of lineages for which assemblies
are available allows, for the first time, an investigation of gene
family evolution across 10 families, two suborders, and a complete
coverage of the entire range of diets. We find two major patterns.
First, system-wide gene losses related to inflammatory response and
selection on genes associated with antiviral immunity appear to have
influenced bat lineages. This suggests that bats— compared to other
mammals such as cow, dog, horse, pig, mouse and human— have evolved
complex, complementary adaptations across multiple functional pathways
to simultaneously reduce inflammatory response while maintaining strong
antiviral defenses, potentially underlying their suspected tolerance of
viruses. Second, the move from the ancestral arthropod diet to
high-sugar nectar and fruit-based diets is associated with t
lineage-specific gene family expansions in metabolic gene families.