Introduction
One of the most enduring challenges for community ecology is how species
diversity maintains in natural communities (Sutherland et al. 2013).
Many mechanisms have been proposed to explain species coexistence and
community assembly, such as negative density dependence (NDD),
environmental filtering, competitive exclusion, and facilitation
(Chesson & Peter 2000, Webb et al. 2006, Adler et al. 2007,
HilleRisLambers et al. 2012, Fichtner et al. 2017, Adler et al. 2018).
Among these various species coexistence mechanisms, NDD is a widely
accepted explanation for the maintenance of forest biodiversity where
seedlings suffer negative effects from host specialized natural enemies
(pathogen and pest) accumulated by local conspecific or phylogenetically
related neighbors (Janzen 1970, Connell 1971, Webb et al. 2006,
Gripenberg et al. 2014, Zambrano et al. 2017, Forrister et al. 2019,
Song et al. 2021). Multiple lineages of natural enemies, such as fungal,
bacterial and viral pathogens together with herbivorous mammals and
insects, may contribute to NDD (Hazelwood et al. 2021, Song & Corlett
2021). The contributions of fungal pathogens and herbivores to NDD have
been documented in many studies, while the contributions of bacterial
pathogens and plant virus were seldom evaluated (Bagchi et al. 2014,
Gripenberg et al. 2014, Hazelwood et al. 2021, Song & Corlett 2021).
Moreover, most previous researches focused mainly on single type of
natural enemy, and studies simultaneously demonstrating the contribution
of multiple natural enemies are still lacking (Hazelwood et al. 2021).
A recently developed community functional genomics strategy may help us
to take an insight into the intrinsic mechanism of NDD from the plant
side and consider multiple natural enemies simultaneously. Plants can
develop multiple molecular responses to microbial pathogens and
herbivores (Wiesner-Hanks & Nelson 2016, Aljbory & Chen 2018,
Ramirez-Prado et al. 2018). The genomic variations involved in these
defense responses of plant species may be related to plant-pathogen or
plant-pest co-evolutionary history, reflect susceptibility of plants to
diseases and pests, and thereby determine growth and survival of
individual plant species as well as the whole plant community (Żmieńko
et al. 2014, Dolatabadian et al. 2017). The community functional
genomics strategy seeks to quantify the similarity of non-model species
via transcriptomic data using Gene Ontology (GO) annotation of
functional genes (Swenson 2012, Swenson et al. 2017). This strategy can
provide information of hundreds of defense related genes and also has
the potential to provide insights into how different lineages of natural
enemies affect plant community structure separately and simultaneously.
Copy number variations (CNVs) and phylogenetic similarity of homologous
genes are two principal methods in community functional genomics to
describe the gene expressional similarity of plant species in response
to biotic and abiotic environments (Han et al. 2017, Zambrano et al.
2017). CNV is defined as copy numbers of homologous genes or size of
gene family (Liu 2014). CNV has been discovered for decades (Bridges
1936, Ohno 1970), which is resulting from gene duplication, gain or
loss, and recently recognized as a common type of genomic variations
(Żmieńko et al. 2014, Dolatabadian et al. 2017). Gene duplication may
induce functional changes of genes such as subfunctionalization,
pseudogenization, and neofunctionalization under rapid evolutionary
processes, or keep their identical function by purifying selection or
concerted evolution (Zhang 2003, Magadum et al 2013). Moreover, the
functional conservation of genes with high sequence similarity, can be
even across distinct phylogenetic lineages (Kater et al 2006, Liu et al
2014). For example, the expression of Arabidopsis ortholog
AtWRKY40 in barley also compromised basal resistance to powdery mildew,
indicating functional conservation of related proteins across dicots and
monocots (Liu et al 2014). The function-conserved gene copies can be
easily detected based on their high levels of sequence similarity
(Magadum et al 2013), and therefore CNVs based on sequence similarity
may act as a useful tool in researches on molecular functional diversity
of species and natural communities. CNVs seem likely to be involved in
many biological processes and can be associated with some important
phenotypic variations (Manolio et al. 2009, Bai et al. 2016). Based on a
functional genetics research on rice, Bai et al. (2016) suggested that
CNVs might be involved in plant resistance to insects. CNVs may also
differ among species and has been implicated in ecological adaptation.
Besides model species, we can also identify CNVs from non-model species
and it had been used in several evolutionary and ecological studies on
microbes (Schirrmeisteret al. 2012, Roller et al. 2016, Nunan et al.
2020), butterflies (Seixas et al. 2021), polar bears (Rinker et al.
2019), and plant species (Zhang et al. 2019, Han et al. 2017). For
example, Han et al. (2017) used CNVs of light-related GOs to test how
differences of tree species in light utilization strategies affect
seedling regeneration in a subtropical forest. The CNVs extracted from
transcriptomic data, defined as functional CNVs, could be useful in
functional ecological researches because functional CNVs could reflect
not only the evolutionary history of species but also the ecological
responses to biotic and abiotic environments.
There are also debates on how CNVs are involved in defense responses and
plant performances (Katju & Bergthorsson 2013, Żmieńko et al. 2014,
Dolatabadian et al. 2017). It is expected that high copy number of
defense genes is advantageous for plants due to the enhanced resistance
against natural enemies, while there might be a trade-off between the
expression of defense genes and plant performance and the high cost in
transcription and translation will in turn limit the copy number of
resistance genes (Lin et al. 2013). Moreover, the copy number of defense
genes might also be the co-evolutionary consequence with exposure chance
to natural enemies, which may reflect pathogen profiles or herbivore
range of host plants (Zhai et al. 2011).
In this study, we extracted CNVs of homologous genes annotated as
defense response to four lineages of natural enemies (fungus, bacterium,
insect and virus). We used partial linear regression analysis to reveal
the effect of gene copy number on seedling survival at species- and
community- level. And we also used generalized linear mixed-effects
models (GLMM) to assess how the neighborhood dissimilarities in
functional CNVs impact seedling survival. For these aims, we
hypothesized that the high copy number of defense response to natural
enemies is associated with high seedling survival (Hypothesis I),
because increased gene copy number may enhance biotic resistance. As the
functional CNVs of defense responses to natural enemies may be strongly
linked to their pathogen profiles or herbivore range, we also
hypothesized that seedlings surround by adult and seedling neighbors
with high dissimilarity in defense-related CNVs should share fewer
natural enemies and therefore have a high survival possibility
(Hypothesis II).