Gene copy numbers of defense responses affect seedling survival
The result of partial linear regression analysis showed that at species level the copy numbers in defense response to insect have a negative effect on survival rates, and at community level (seedling stations) the copy numbers of defense response to fungus, insect and virus showed a negative effect on seedling survival. These results are inconsistent with some previous studies that showed that resistance was enhanced as gene copy number increased (Slabaugh et al. 2003, Bradeen et al. 2009). However, low gene copy numbers of biotic resistance are also found in some plant species (Lin et al. 2013, Xue et al. 2020), which may help them to gain evolutionary advantages due to a trade-off effect between cost on biotic resistance and growth performance. Although high copy number of defense gene is expected to be advantageous for better resistance against pathogens, the copy number is also limited to balance biological cost including not only energy for transcription and translation but also their toxic effect (Lin et al. 2013). Another potential explanation is that the low gene copy number within the GO in a local community may be due to the low presence of attacks from natural enemies. This hypothesis was supported by a recent genomic research on balsam poplar, which showed that a lower pathogenic pressure resulted in a lower defense gene copy number (Prunier et al. 2019). In the evolutionary process, plant-pathogen and plant-herbivore interactions may influence gene gain-and-loss and gene copy number of plant species (Liu 2014) and the copy number of defense related genes can act as an indicator of the abundance of natural enemies (Zhai et al. 2011). In our study, all tree seedlings within one community are exposed to the same putative natural enemy pool, which means that a species with lower copy number of defense genes may be less attacked by natural enemies and thus has a higher survival rate.
Moreover, this result also indicated that tree seedlings seemed more susceptible to gene copy numbers of defense response to insect rather than to other natural enemies, suggesting that the pressure from insect pests could be a key driver in structuring seeding community.
Conspecific NDD and neighborhood functional CNV dissimilarity in defense affect seedling survival
A significant NDD effect from con-specific seedlings was detected in our study, which agrees with the result of Wang et al. (2020) in the same forest. The significant NDD in the subtropical forest infers a high intra-specific competition because of the increased seedling density in these years after an extreme winter storm (Wang et al. 2020). Additionally, conspecific neighboring adults had no significant effect on seedling survival, which can be explained by storm-induced decrease in tree neighbor density (Man et al. 2011, Wang et al. 2020).
In theory, all the four lineages of natural enemies can mediate negative density dependence (NDD) caused by Janzen–Connell mechanisms (Song et al. 2021). Many studies so far have focused on fungal pathogens rather than other natural enemy types and it is suggested that fungal pathogen is the key diver of NDD (Bell et al. 2006, Liang et al. 2016, Song & Corlett 2021). However, we didn’t detect significant effects of functional CNV dissimilarity on defense to fungus in the present study. There may be two potential reasons for this. Firstly, the transcriptome extracted from the leaves cannot reflect the defense responses to root-associated pathogens, which play a vital role in structuring plant community (Chen et al. 2019, Tedersoo et al. 2020). Secondly, a counteraction effect may occur since both fungal pathogens and mutualistic endophytes exist simultaneously in seedlings (Chen et al. 2019).
Our results also show, for the first time, that pressure from bacterial and viral pathogens may also be a key driver of NDD for seedlings in subtropical forest. Functional CNV dissimilarity to adult neighbors in defense response to bacterium showed a significant and positive effect on seedling survival, which means that seedlings with distinct defense response to bacterial pathogens from adult neighbors have higher probability of survival as predicted by NDD. This pattern is similar to the results of Zambrano et al. (2017) who also found that neighborhood dissimilarity of several defense genes have positive effects on individual survival. Our result suggests that the functional CNV dissimilarity between plants species can well reflect the niche differentiation in response to specialized bacteria. In other words, species with similar defense responses to bacterium may have similar bacterial pathogen profiles and thus seedlings may be killed by bacterial pathogen accumulated by adult neighbors with similar defense responses to bacterium. Moreover, since the defense response to bacterium has significant phylogenetic signal, this result may also explain conspecific and phylogeny related NDD. At last, this result is reasonable because many bacterial pathogens can infect plant leaves and are fatal to seedlings (Xin et al. 2018) and our transcriptomic data from seedling leaves can well reflect the interation between seedlings and phyllosphere bacteria.
The Functional CNV dissimilarity to adult neighbors in defense response to virus also had a significant and positive influence on seedling survival. In other words, seedlings growing near adult neighbor with distinct defense response to virus survive more, similar to the NDD pattern in defense response to bacterium. While viral pathogens infect almost all plants in terrestrial ecosystems (Lefeuvre et al. 2019), plant viruses also have high host specificity. For example, a study on host range of 29 plant virus species indicated that 69% viruses were restricted to a single plant family (García-Arenal & McDonald 2003). Due to the high host-specificity of plant virus, the positive effect of functional CNV dissimilarity in defense response to virus on seedling survival is also reasonable.
Although the NDD mediated by insect herbivores have been well documented in natural forests (Forrister et al. 2019) and manipulative experiment using insecticide (Jia et al. 2020), the contributions of insect herbivores to NDD are still debated (Bagchi et al. 2010, Gripenberg et al. 2014, Song & Corlett 2021). In this study we detected an opposite pattern that functional CNV dissimilarity to seedling neighbors in defense response to insect had a negative effect on seedling survival (Fig. 3), suggesting that seedling survival was higher when surrounded by neighbor seedlings with similar defense response to insect. This result is similar to previous studies showing that the survival of focal species increases in the presence of functionally similar or phylogenetic related neighbors, which could be explained by habitat preference (Godoy et al. 2014, Lebrija-Trejos et al. 2014, Wang et al. 2020). However, it seems unlikely the case in this study, because we found the neighbor seedlings rather than adults with similar anti-insect responses have a significant positive effect on seedling survival, while the habitat preference states that the habitats occupied by closely related adult neighbors might provide suitable resources and enhance seedling survival (Lebrija-Trejos et al. 2014). This indicates that insect-mediated biotic filtering rather than habitat preference may be involved in the mechanism underlying seedling dynamics that only specific seedlings beyond the host range of local insects survive. Another possible reason for this pattern is competitive exclusion driven by competitive ability rather than niche differences (Cavender-Bares et al. 2004, Mayfield & Levine 2010). During the competitive process, species with high competitive ability can displace the others and result in a clustering pattern (Mayfield & Levine 2010). We also found that the gene copy number of defense response to insect is negatively associated with seedling survival. This result, together with negative effect of functional CNV dissimilarity of defense insect on neighbor seedlings, suggests that the copy number of defense response to insect can reflect competitive ability of plant species and result in functional clustering of the seedling community. Taken together, it can be concluded that the competitive exclusion mechanism caused by the fitness difference of defense response to insect should be a key driver of seeding communities.