It has been suggested that plants can recognize their kin neighbours and non-self neighbours, and this recognition can have affect direct and inclusive fitness for plants growing with relatives (Hamilton, 1964). Kin recognition is often expressed through decreasing competition by lower investments to fine roots (Gruntman & Novoplansky, 2004; Dudley & File, 2007; Bhatt & Dudley, 2010; Biedrzycki et al., 2010), producing less leaf area (Ninkovic, 2003) or less leaf overlap in kin groups compared to strangers (Crepy & Casal, 2015), and these changes may increase fitness in sibling groups (Tonsor, 1989; Biernaskie, 2011). However, plants may have trade-offs between their traits (Schlichting, 1986; Casper & Jackson, 1997; Cahill & Casper, 2000; Toledo-Aceves & Swaine, 2008), making it difficult to determine if kin recognition in morphological traits has fitness consequences (File et al., 2012; Till-Bottraud & de Villemereuil, 2016). Some physiological performance traits which can sufficiently reveal plant competitive interactions may also be candidates for kin recognition responses. For example, photosynthesis is likely to be changed if carbon-use efficiency is affected by neighboring plants (McCormick et al., 2006; Lendenmann et al., 2011). The competition between plants could alter the storage of carbohydrates (Benayas et al., 2003) and thus increase communication by higher roots activity (Yoder, 1999; Callaway & Aschehoug, 2000; Islam et al., 2007; Biedrzycki et al., 2010). Studies have found that plants growing together with their clones decreased their root respiration rate compared to plants growing with non-self neighbours (Meier et al., 2013). However, other physiological responses to the relatedness of neighbours remain less unexplored and need more investigation (Till-Bottraud and de Villemereuil, 2016).
In addition to reducing competition, kin recognition could also be revealed through increasing resource use efficiency, especially when the fitness is not available for direct measurements (Cheplick, 1992; James et al., 2005; File et al., 2011, 2012; Meier et al., 2013; Simonsen et al., 2014). Unlike inter-specific plants which access multiple soil resources by consuming different nutrient forms (Mc Kane et al., 2002) or extending roots differently to both horizontal and vertical soil layers (Cahill et al., 2010), kin plants significantly decreased uptake of total nitrogen (N) and NO3−-N than strangers to reduce N competition (Zhang et al., 2015), with improved use efficiency of N and other nutritive elements (Maestre et al., 2009; Zhang et al., 2015; Till-Bottraud & de Villemereuil, 2016; Li et al., 2017, 2018). Because N is a major nutrient limiting plant growth in most terrestrial ecosystems (LeBauer & Treseder, 2008 Ecology) and sensitive to kin interactions (Zhang et al., 2016; Li et al., 2017, 2018), the amounts and forms of available soil N, their uptake and allocation in plant tissues may play a significant role in kin recognition or competition (Cheplick, 1992; Zhang et al., 2015). Therefore, clarifying how kin recognition affects soil N cycling as well as the related functional genes of soil microbes can provide insights into the mechanisms for plant competition or cooperation with relatives. However, it is remains unexplored so far.
Nitrification and denitrification are two critical processes of the soil N cycling, which are mediated by specific functional microbial populations. Nitrification converts ammonia to nitrate via nitrite (Farmaha, 2014; Wrage et al., 2001). The nitrification intensity is thought to be catalyzed primarily by autotrophic ammoniaoxidizing bacteria (AOB) belonging to β- or γ-proteobacteria usually in neutral and alkaline soils (De Boer & Kowalchuk, 2001; Prosser, 1990; Jia & Conrad, 2009; Xia et al., 2011) and by ammonia-oxidizing archaea (AOA) (Konneke et al., 2005; Leininger et al., 2006; Treusch et al., 2005; Venter et al., 2004) especially in acidic soils (Prosser & Nicol, 2012; Yao et al., 2011; Zhang et al., 2012; Gubry-Rangin et al., 2010; Lu et al., 2012). In contrast, denitrification has been considered to be an important pathway for N loss from soil through N2 and N2O in terrestrial ecosystems. It can be predicted by the genetic expression of nirK, nirS, nosZ and etc. (Thamdrup and Dalsgaard, 2002; Dalsgaard et al., 2003; Lam et al., 2009). The absorption and use efficiency of available N by plants under N-limited conditions is closely related with the conversion of nitrification and denitrification involved with related functional microbes. Besides, biological N fixation is also another important pathway of N input through fixing atmospheric N2 to bio-available N, which is achieved by diazotrophs using nitrogenase encoded principally by the genes of nifH (encoding the nitrogenase reductase subunit) (Rösch et al., 2002, Wartiainen et al., 2008, Tu et al., 2016, Fan et al., 2018). Responses of such functional genes related to soil N cycling to kin interactions may provide new insights into mechanism-based replenishment to kin response on N use efficiency (NUE).
The rhizosphere microbes can be stimulated by root exudates and thus accelerate soil carbon and nitrogen turnover, affecting the nutrient acquisition of soil microorganisms and plants (Eisenhauer et al, 2012). Numerous studies show that there is a direct relationship between the exudates of plant root system and the soil enzyme activity, because plant root exudates themselves contain enzymes and certainly have impact on the original soil enzyme activity (Dijkstra et al, 2007). Moreover, 20–60% that carbon fixed by plants through photosynthesis is transported to plant roots, and 40%-70% of which is released into rhizosphere as root exudates (Kuzyakov et al, 2000). Therefore, a large amount of carbon and nitrogen accumulate in rhizosphere soil and become the substrate of microbial activities, which affects the growth and structure of microbial community (Singh et al, 2006) as well as the release and circulation of soil nutrients mediated by soil microbes (Dinkerlaker et al., 1989; Fisk et al., 2015). It was found that the increased carbon-nitrogen ratio of plant root exudates could increase the abundance of soil microorganisms, because higher carbon-nitrogen ratio would promote soil microorganisms to secret more extracellular enzymes to accelerate the decomposition of organic matter and provide carbon and nitrogen for the growth of microorganisms (Zhang et al., 2013). Additionally, root exudates are suggested to be kin signals among plant relatives, but it remains unclear which root exudates participate in kin recognition (Biedrzycki et al., 2010). Therefore, the study of root exudates could not only clarify how the kin recognition signals are passed, but also whether the microbial responses and soil enzyme effect are induced by root exudates response to relatedness.
Kin effects have been reported in some crop species such as Hordeum vulgare (Ninkovic, 2003), Pisum sativum (Meier et al., 2013), Lupinus angustifolius (Milla et al., 2009; 2012), Oryza sativa (Fang et al., 2013), perhaps because these crop species are planted widely in the world and are most likely to have relatives growing together and sensitive to N utilization (Hess and De Kroon, 2007; File et al., 2011). In this experiment, we chose Glycine max to represent monoculture plants and examine how plant responds to kin neighbors through fitness, morphology, physiology and nutritional absorption performance. We test the following hypotheses: 1) G. max plants respond to kin by increasing physiological growth efficiency and fitness; 2) input of plant root exudates (mainly carbon and nitrogen production rate) could be kind of kin signals, and increased of which response amplifies the consequences of growing with kin; and 3) the relatedness of plants in a pot affects functional genes related to the soil N cycling and leads to the differences of NUE between plants growing with kin and strangers. Testing these hypotheses will provide a new perspective on kin interactions and benefit on our understanding of how they evolve (File et al., 2012; Simonsen et al., 2014).