Functional relationship between woody plants and insect communities in response to Bursaphelenchus xylophilus infestation in the Three Gorges Reservoir region

Abstract To study the effect of the invasion of Bursaphelenchus xylophilus on the functional relationship between woody plants and insect communities, the populations of tree species and insect communities were investigative in the Masson pine forests with different infestation durations of B. xylophilus. In this study, the number of Pinus massoniana began to decrease sharply, whereas the total number of other tree species in the arboreal layer increased gradually with the infestation duration of B. xylophilus. The principal component analysis ordination biplot shows that there was a significant change in the spatial distribution of woody plant species in different Masson pine forest stands. Additionally, a total of 7,188 insect specimens were obtained. The insect population showed an upward trend in stand types with the increase of pine wilt disease infection periods, which demonstrated that the insect community had been significantly affected by the invasion of B. xylophilus. However, the insect diversity indexes were not significantly different among Masson pine forest stands. The structure of insect functional groups changed from herbivorous (He) > omnivorous (Om) > predatory (Pr) > parasitic (Pa) > detritivorous (De) in the control stand to He > Pa > Om, De > Pr after B. xylophilus infestation in the forests. The results showed that the populations of He, Pa, and De increased after the invasion of B. xylophilus, but the populations of Pr decreased. Moreover, the redundancy analysis ordination biplots reflected the complicated functional relationship between woody plant communities and insects after the invasion of B. xylophilus. The present study provides insights into the changes in the community structure of woody plants and insects, as well as the functional relationship between woody plant communities and insect communities after invasion of B. xylophilus.


| INTRODUC TI ON
With increase in the frequency of international trade, some invasive alien species are directly or indirectly introduced into new forest ecosystems. As an external disturbance factor, they inevitably affect the productivity, nutrient cycle, hydrological system, species diversity, and information transmission of the new ecosystem (Ding et al., 2008;Lovett et al., 2006;Westphal et al., 2008). The plant community structure and composition of the forest ecosystem can be significantly changed, and the succession rate of the community can be accelerated after the infestation of invasive alien species (Castello et al., 1995;Gao et al., 2015;Spiegel & Leege, 2013).
Consequently, the species composition and population structure of the animals, insects, and microorganisms that depend on the plant community may also undergo considerable changes (Karban, 2011;Li et al., 2016;Visakorpi et al., 2019).
As one of the most dangerous and destructive forest noxious organism, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle (Nematoda: Aphelenchoididae) causes the pine wilt disease (PWD) and has a strong destructive effect on pine forest ecosystems in China, Japan, Korean, Spain, and Portugal (Abelleira et al., 2011;Futai, 2013;Sousa et al., 2011;Zhao, 2008). The pine sawyer beetle, Monochamus alternatus, is the most efficient insect vector for spreading B. xylophilus in China and other East Asia counties (Li et al., 2020;Mamiya & Enda, 1972;Zhao, 2008). The average number of B. xylophilus carried by M. alternatus is 18,000 per beetle, and the maximum number is 289,000 (Zhao, 2008). In China, B. xylophilus was first discovered in 40 black pines in September 1982 at Sun Yat-Sen's Mausoleum in Nanjing (Cheng et al., 1983;Wan et al., 2005).
Since then, the degree of damage inflicted by B. xylophilus is a continuous large-scale occurrence, which has caused huge economic and ecological losses around China Wan et al., 2005;Zhao, 2008). By August 2020, PWD had spread to 18 provinces and composition and structure (June et al., 2006;Siemann, 1998). As an important environmental indicator, insect species are usually linked to plant communities and are widespread in forest ecosystems (Cédric et al., 2013;Siemann, 1998;Vandewalle et al., 2010).
Woody plant species provide much of the habitat and resource base for insects (Brown et al., 2001;Trotter et al., 2008). A greater diversity of plants provides a greater diversity of resources for insects; consequently, the increase in plant species richness also leads to an increase in insect species richness (Haddad et al., 2001;Knops et al., 1999). Furthermore, a higher diversity of herbivorous insects can provide more food sources for insect predators and parasitoids (Knops et al., 1999).
For the dominant tree species with a large biogeographic distribution, changes in the communities and population structure of this species are likely to affect many other tree or animal species at large landscape scales (Trotter et al., 2008). Masson pine is an important pine tree species in the Three Gorges Reservoir area, which is widely distributed from the bank of the Yangtze River to the top of the mountain (Gao et al., 2015). In this area, the main coniferous forest type was the Masson pine pure forest before the

| Study sites
This study was carried out in Yiling District (latitude 30°32′-31°28′N, longitude 110°51′-111°39′E), which is located in the eastern part of the Three Gorges Reservoir region and contained the demarcation point of the upper and middle reaches of the Yangtze River (Gao et al., 2019). In this area, PWD was first  (Table 1).
Additionally, each Masson pine stand type had three repeated stands, and there were three permanent 15 m × 15 m Masson pine plots in each stand.

| Field surveys
From July to August 2012, the woody plants in the arboreal layer were investigated in each of the 45 plots. For each plot, the basic environmental factors, including elevation, slope, and canopy density, were recorded. The woody plants with a diameter at breast height (DBH) ≥ 2.5 cm were investigated using the method of "Tally" which includes species name, height, DBH, and crown.

| Insect sampling and specimen identification
For each plot, insect sampling was conducted once every 7 days from June to August in 2013 and 2014. Insect specimens were collected by means of "sweep net sampling," "Malaise trap sampling," and "window trap sampling." For the "sweep net sampling" method, a muslin insect swept net was used for sweeping in each corner of the 45 plots, for a total of more than 200 times net was swept for each plot. For the "Malaise trap sampling," a Malaise trap was placed in the center of the 45 plots and was mainly used to trap small insects. For this method, the collected insect specimens could be directly stored in 75% alcohol, which ensured the necessary quality for subsequent molecular identification. Additionally, five window traps (29.5 cm × 19.0 cm × 0.2 cm) were placed at the center and the four end points according to the "five-point sampling" method. The window traps were suspended at a height of approximately 3 m, and the collection device was filled with 75% alcohol in order to preserve the collected insect species.
According to relevant professional books and references, insect individuals were identified as exactly as possible to genus and species. The sampled insect specimens were divided into five groups: herbivorous insects (He), predatory insects (Pr), parasitic insects (Pa), omnivorous insects (Om), and detritivorous insects (De). Moreover, the dominant insect group was the one whose individual number accounted for more than 10% of the total collected insects, common groups accounted for 1.0%-10% of the total number, and the rare groups accounted for less than 1% of the total number (Wang & Wang, 2010).

| Data analysis
One-way ANOVA and Fisher's least significant difference (LSD) test with an alpha value of p <.05 were used to compare the changes in

| Changes in woody plant community structure
The total number of P. massoniana in the healthy ecosystem (ST1) was higher than that in the infected stands (Table 3). With the increase in the infestation duration of PWD, the number of P. massoniana decreased sharply, and the statistical difference between different stand types reached a significant level (F = 6.99, p < .01).
The total number of other tree species in the arboreal layer (except P. massoniana) increased slowly with the increase in the degree of PWD infestation. Additionally, the difference in the total number of the remaining species, ST4 and ST5, reached a significant level when compared with the healthy Masson pine ecosystem (p < .05).
The PCA result and ordination biplot of woody plant species in different Masson pine forest stands are shown in Table 4 and

TA B L E 3
The number of stems for all tree species measured in five Masson pine forest sites infected for different periods by pine wood nematode

| Insect community structure
In this study, we collected and identified 7,188 individual insects, representing 510 species from 15 orders and 152 families (Table 5).
There were significant differences in the levels of order, family, and individual among the collected insects. Overall, the dominant insect communities were Hemiptera, Coleoptera, Hymenoptera, Diptera, and Lepidoptera, which made up more than 10% of the total at the levels of family, species, and individuals. Other insect communities accounted for a relatively small proportion of the total insect population.
There was an upward trend in the insect population at the levels of order, family, species, and individual among different stand types (Table 6). However, there was little variation in insect community composition at the order, family, and species levels among different stand types, and the difference between them was not significant.
Conversely, the individuals were significantly different among different stand types (F = 204.20, p < .01), and the difference between ST1-2 and ST3-5 was significant. As revealed by Figure 3, the insect diversity indexes were not significantly different among five Masson pine forest stands infected by B. xylophilus. Compared with the control stands, the S and H were slightly decreased in ST2 and then showed an increasing trend over the course of PWD infection (Figure 3a and Figure 3b). The changes of H′, D 2 , E, and J were fluctuated, but no significant change trends were observed among different stand types.

| Insect functional groups
The structure of insect functional groups had changed over the course of B. xylophilus infestation (

| Functional relationship between woody plants and insects
For a more intuitive understanding of the complicated functional relationship between woody plant community and insect functional groups, a redundancy analysis was conducted using 16 woody plant species with an importance value of over 1, and five insect functional groups with the numbers of species and individuals. As shown by the RDA results in Table 10, a total of 66.9% canonical eigenvalues in insect functional groups can be explained through ordination by the selected woody plant species.
The RDA ordination biplot with woody plant species and insect functional groups along the first two axes is presented in Figure 6.
As revealed by the ordination biplot, there is a gradient in plots along axis 1, from ST1 very positive to ST5 negative; however, they are

| D ISCUSS I ON
The present study provides new insights into the functional relationship between woody plants and insect communities after the ST4 ST5 Under natural conditions, B. xylophilus infestation will cause serious damage and loss to a healthy pine forest ecosystem, but with the continuous succession and natural recovery of the ecosystem, the species diversity of the plant community will increase significantly, and the pine forest will evolve and develop in a more advanced direction Spiegel & Leege, 2013;Zhao, 2008 Similar results were found in many previous studies, which showed that the invasion of B. xylophilus can lead to the succession of the pure P. massoniana forest ecosystem into a mixed coniferous and broadleaf forest ecosystem or even a broadleaf forest ecosystem (Gao et al., 2015;Shi et al., 2007;Wang et al., 2014). By comparing and analyzing the niche indices of the main woody plant species, the vegetation communities in the woody plant layer did not degenerate in the direction of the shrub layer after the invasion of the Masson pine ecosystem by B. xylophilus. In general, the structure of new forest ecosystems formed by succession tends to be more stable and has a strong ability to resist forest pests and diseases (Hambäck et al., 2000;Humphrey et al., 1999;Jobidon et al., 2004;Li et al., 2012).

Number of individuals
To    (Gao et al., 2013). Additionally, the number of insect species and individuals showed an upward trend in Masson pine forest stand types with increasing PWD infection periods, which demonstrated that the insect community was significantly affected by the invasion of B. xylophilus. Previous studies have indicated that a greater diversity of woody plant species provides much more habitats and resources for insects (Brown et al., 2001;Cédric et al., 2013;Root, 1973;Siemann, 1998;Trotter et al., 2008;Vandewalle et al., 2010). Therefore, through this study, we found that the invasion of B. xylophilus can affect the community structure and composition, change the spatial and structural distribution As a multidimensional property of natural system, biodiversity represents the variety and heterogeneity of organisms and usually quantified by diversity indices (Daly et al., 2018;Morris et al., 2014). Insect diversity is an important part of biodiversity and one of the key indicators to study the structure and function of insect community (Barton & Evans, 2017;Christensen et al., 1996). In this research, we compared insect diversity indexes The structure of insect functional groups can reflect the development and health status of a specific forest ecosystem (Haddad et al., 2001;June et al., 2006;Karban, 2011;Visakorpi et al., 2019). In this study, the magnitude of relative abundance for insect functional groups in the control stand type (ST1) was He > Om > Pr > Pa > De, whereas after infection, the magnitude of relative abundance for insect functional groups was altered (ST2-ST5: He > Pa > Om, De > Pr). This result indicated that the structure of insect functional groups changed over the course of B. xylophilus infestation in the Masson pine forests. As a crucial part of the biological communities in forest ecosystems, the population dynamics and spatial distribution of insect functional groups are significantly restricted by woody plant species (Bezemer et al., 2014;Cédric et al., 2013;Root, 1973;Tchakonté et al., 2015;Vandewalle et al., 2010). Additionally, the outbreak of PWD also has multiple effects on growth rates, canopy structure, and composition of woody plant species, which in turn may alter the structure of insect functional groups ( Li et al., 2012;Root, 1973;Veblen et al., 1991). Moreover, the number of insect species and individuals of herbivorous insects increased with an increase in the invasion duration of B. xylophilus, and the difference of individuals reached a significant level among stand types. In order to control the population of herbivorous insects and enhance degradation of plant and insect residues (Haddad et al., 2001;June et al., 2006;Trotter et al., 2008;Visakorpi et al., 2019), the relative abundance of parasitic insects and detritivorous insects in this study also increased by varying degrees after the invasion of B. xylophilus, with values increasing from 7.86% to 16.69% and from 2.10% to 11.16%, respectively.
As revealed by the RDA ordination graph, the population spatial distribution of insect functional groups was significantly restricted by woody plant species. The correlation between parasitic insects and woody plant species indicated that broad-leaved tree species may provide more resources and ecological niches for parasitic insects. This result is consistent with a previous study showing that the number and diversity index of parasitic insects increased with an increase in the proportion of broad-leaved tree species in a coniferous forest ecosystem in Zhejiang Province (Wang et al., 2014). According to "Resource Concentration Hypothesis," the increase in plant species diversity can provide more habitats and food resources, which may significantly affect the structure of insect functional groups and increase the species and individual numbers of herbivorous and parasitic insects (Gao et al.,2013(Gao et al., , 2018Knops et al., 1999;Root, 1973;Taki et al., 2010). As the population of parasitic and predatory insects continues to increase, the number of herbivores, including M. alternatus, which is the vector of B. xylophilus, may be limited.

| CON CLUS IONS
The

ACK N OWLED G M ENTS
Special thanks go to the anonymous reviewers for their valuable comments and suggestions. We also thank Dewen Song and Kezhou He for their assistance with the collection of data. We used Editage (www.edita ge.com) for English language editing.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.