Assessing the Impact of Biodiversity (Species Evenness) on The Trophic Position of an Invasive Species (Apple Snails) in Native and Non-Native Habitats Using Stable Isotopes.

Invasive apple snails negatively impact non-native habitats and also human well-being. Here the trophic position of Pomacea canaliculata in native habitats (Maldonado, Uruguay) and non-native habitats (Hangzhou, China and Hawaii, USA) are compared to explain their differential impacts therein. Detritus samples and tissue samples from apple snails were collected in all sites. Apple snail trophic levels were calculated as the difference between the mean δ 15 N values of detritus samples and corresponding apple snail tissue samples, divided by the mean δ 15 N fractionation for nitrogen per trophic level in freshwater habitats. The mean δ 15 N values of detritus in sites served as a baseline (i.e.: zero trophic level), allowing for direct comparisons. Linear regression analysis established correlation between species evenness and apple snail trophic level (R 2 = 0.8602); in line with a Pearson's product-moment correlation value (-0.83) and 95% Con�dence Interval (-0.87, -0.77). Normal quartile plots indicated two normally distributed subsets of apple snail trophic level data: (1) a biodiverse subset containing the Uruguayan and Chinese Lake Sites and (2) the homogenized Hawaiian and Chinese Creek Sites. A precipice value for species

Introduced species are transported outside of their native range by humans and once established, in nonnative habitats, are likely to become invasive pests that result in economic or environmental harm (Pimentel et al., 2005); adversely impacting agriculture and/or natural ecosystems, see Pimentel et al., 2005. Invasive species often led to homogenization, a process which alters habitats by anthropogenic forces. Through homogenization native species are extirpated and replaced by non-native species (McKinney & Lockwood, 1999;Trentanovi et al., 2013;Gámez-Virués et al., 2015). This process (homogenization) generally increases the similarity of species and reduces biotic resistance (Davis, 2003). Invasive species directly and indirectly impact trophic interactions in non-native communities (Simon & Townsend, 2003), via the establishment of new trophic interactions and/or the alteration or eradicating of previously existing ones; thereby reducing the overall complexity and/or stability of nonnative habitats (Jackson et al., 2017 Yusa et al., 2006). Generally, invasive apple snails lack adverse agricultural and/or ecological impacts in native habitats (Francis, 2012;pg. 215). Despite the importance of apple snails in native habitats Pomacea canaliculata is counted amongst 100 of the world's worst invasive species (Lowe et al., 2000) and is a major agricultural pest of aquatic staple crops; such as rice, taro plant, and watercress (Cowie, 2002;Levin, 2006;Gilal et al., 2016). Pomacea canaliculata also shifts non-native freshwater habitats to alternate eutrophic states (Scheffer et al., 2001, Carlsson, Brönmark, & Hansson, 2004; adversely impacting production and environmental heterogeneity, as bottom-up controls act altering community structure, thereby diminishing biodiversity in these newly homogenized freshwater habitats (Carlsson, Brönmark, & Hansson, 2004;Fickbohm & Zhu, 2006). Why apple snails differentially impact native versus non-native habitats is not well understood. Comparisons of the trophic position of Pomacea canaliculata between native and non-native habitats could reveal changes in the trophic position as a key factor in uencing their adverse impacts as invasive species.
Stable isotope analysis (SIA) is widely implemented in ecological studies, over a range of scales, to investigate ecosystem function in terms of animal migration patterns (Hobson, 1999; Rubenstein & Hobson, 2004), resource availability (Jackson et al., 1995;Young et al., 2010), and even the dynamics of carbon, nitrogen, and water in ecosystems (Peterson & Fry, 1987). Stable isotope abundances for 15 N/ 14 N (heavy/light isotopes), when compared amongst species in an ecosystem, can be used to de ne their trophic position; in terms of nitrogen isotope ratios (i.e. 15  Apple Snails, other sympatric animal species, and detritus Pomacea canaliculata and other sympatric animal species were collected by hand, using small aquatic nets, or aerial nets. Once collected, specimens were inventoried, frozen, and thawed. Genetic samples from all Pomacea canaliculata and other metazoan animal species were stored in 95% ethanol, while remaining material, to be used for stable isotope analysis, were rinsed with deionized water, weighed, dried at 60°C for a minimum of 24-hours, subsequently re-weighed, the dried mass recorded, and then ground into a homogeneous powder for stable isotope analysis. Detritus was sampled using 42 cm tall plastic cylinders, with a diameter of 15 cm. These cylinders were pressed into the substrate by hand. The contents of the cylinder were pulled from the water and deposited into labeled plastic bags and later ltered by hand, using a three-piece Soil Sieve Set (1", 0.2", and 0.25" mesh diameter) and running deionized water. Detritus was then dried at 60°C for 24-hours, weighed, and subsequently ground into a homogeneous powder for stable isotope analysis. Pomacea canaliculata and other macroinvertebrate stable isotope and corresponding genetic samples, as well as detritus samples, were stored at -20°C, and later transported to Howard University for long-term storage at -80°C. The biodiversity metric of species evenness was calculated as follows: Evenness= 1/ (# of the most abundant species collected/ total # of organisms collected) Evenness values from all collection sites was compared to the trophic level of Pomaceacanaliculata using linear regression, to ascertain if this metric (species evenness) can explain variation in the trophic postion of Pomacea canaliculata between the ve collections sites. Speci cs on the enumeration of animal species catalogued in each collection site, and subsequently used to calculate species evenness as shown above, is provided in Supplemental Data, Table 2 Results Stable Isotope analysis showed that mean δ 15 N values were highest in Kawainui Marsh and decreased in order between the Chinese Creek Site, Lake Sauce, the Chinese Lake site, and nally Lake Dario. The standard deviation of δ 15 N values was also greatest in the Chinese creek site (see summarized data in Table 1, individual data in Supplementary Data Table 1). As this study relies most heavily on δ 15 N values for subsequent analyses, the δ 13 C values are presented only in Supplementary Data Table 1 to provide a complete data set and for potential use in future research. Species evenness was greatest in Lake Dario and decreased in order from Lake Sauce, to the Chinese lake site, Kawainui Marsh, and the Chinese creek site (see Table 1). The calculated trophic level of Pomacea canaliculata was lowest in Lake Dario. Corresponding trophic level data from the remaining sites increased from Lake Sauce, to the Chinese lake site, to Kawainui marsh, and were greatest in the Chinese creek site; where the variation in the trophic level of Pomacea canaliculata was also greatest (see Table 1).
Species evenness from collection sites correlated with the calculated trophic level of Pomacea canaliculata therein. Linear regression showed species evenness explained 68.2% of trophic level variation for Pomacea canaliculata (see Fig. 1). A subsequently performed linear model (using (1) species evenness and (2) calculated trophic level data for Pomacea canaliculata) produced similar R 2 (0.6821) and adjusted R 2 values (0.6802); while a highly signi cant (p < 0.0001) correlation test also produced a Pearson's product moment value of (-0.83, see Table 2).
Calculated trophic level data were checked for normality. Via a normal quantiles plot, across all collection sites, the majority of these data were found to be normally distributed (see Fig. 2). Those trophic level data that did not t a normal distribution were collected from two sites (those with the lowest values of species evenness); Kawainui Marsh and the Chinese creek site.
As a whole, the calculated trophic level data from all sites explained 68% of the variation observed in trophic level variation, but was composed of two sets of normally distributed data. These data which did not t a normal distribution (see Fig. 2) were utilized in one of two subsequent normal quantile plots (Figs. 3 and 4). The rst subset of trophic level data was collected in Lake Sauce, Lake Dario, and the Chinese lake site (see Fig. 3); the second subset of data was collected in the Chinese creek site and in Kawainui Marsh (see Fig. 4).

Discussion
Our results (as illustrated by Fig. 1 and Table 1) indicate that species evenness in native and non-native habitats is a good predictor of the average calculated trophic level of Pomacea canaliculata; explaining more than 68% of the variation observed. What is likely more telling is the realization that these data from ve unique collection sites can be placed into two normally distributed subsets (as shown by normal quartile plots in Figs. 2, 3, and 4).
The trophic levels, depicted in Fig. 1, are estimates based on the detritus stable isotope data. Lake Dario was anthropogenically disturbed by the removal of macrophytes, leading to the alteration of production and the accrual of detrital material in the lake. This may explain why a subset of apple snails was nitrogen poor therein, resulting in a calculated trophic level below the baseline (or zero trophic level) ; as seen in Fig. 1. In the cases of these speci c snails they had less 15 N than the detritus samples average within that speci c site (as listed in Table 1); hence the negative value for their respective trophic level. This anomaly was only evident in Dario; and only in a subset of snails. The high level of species diversity (high species evenness value) and the diminished production, due to anthropogenic in uences within Lake Dario, together may account for this apparition in the resultant trophic levels of a subset of snails in Lake Dario. Figure 2 illustrates that the trophic level data from all ve collection sites, when taken together, diverge from normality and that a subset of those data is statistically distinct. In Fig. 2 the x-axis is a normal quartile scale and the y-axis is a compendium for the trophic level data from all collection sites; the dotted blue lines represent the 95% con dence intervals for a normal distribution of data. It therefore illustrates the desired test for data normality across all collection sites. Figures 3 and 4, respectively, test two distinct subsets of three and two collection sites for normality independently. Again, Figs. 3 and 4, display 95% con dence intervals that illustrate that these two distinct subsets of data do not diverge from normality.
The value of species evenness, our metric of biodiversity within all ve collection sites, decreases from Lake Dario (4.9), to Lake Sauce (4.6), to the Chinese Lake site (3.7), in the rst subset of data. Species evenness continues to decline in the second subset of data, with Kawainui Marsh (2.4), reaching a minimum value in the Chinese creek site (2.2; Table 1). One explanation for this phenomenon may be that there is a tipping point, or threshold value, for species evenness in habitats (whether native or non-native); in-between corresponding values observed in the Chinese Lake site (3.7) and Kawainui marsh (2.4). Once met (e.g.: as species evenness is reduced to and/or below this point) the trophic level of Pomacea canaliculata therein would diverge from normality with corresponding data from: (1) the native habitat of invasive Pomacea canaliculata (Lake Sauce and Lake Dario), where they have no ill ecological impacts, or (2) the less biodiverse, but statistically indistinct (see Fig. 3), Chinese Lake site.
These ve collection sites can therefore be separated into two categories: (1) the more biodiverse and less homogenized subset of statistically indistinct collection sites (Lake Sauce, Lake Dario, and the Chinese Lake site; see Fig. 3); and (2) the less biodiverse and more homogenized collection sites (Kawainui Marsh and the Chinese creek site; see Fig. 4). These data, from all ve collection sites, provide a capable tool for predicting the trophic level occupied by Pomacea canaliculata in native and non-native habitats; and possibly the negative ecological impacts they have in less diverse and more so homogenized habitats over broad geographical distances (see Fig. 1 and Table 2).
This data supports the following prediction: where species evenness values, and therefore biodiversity, are low (e.g.: in the Chinese Creek site and the Kawainui Marsh site) Pomacea canaliculata occupy trophic levels higher than corresponding snails (Pomacea canaliculata) collected in sites where species evenness values, and therefore biodiversity, are high (e.g.: in the Lake Sauce, Lake Dario, and the Chinese Lake site). Hence, the calculated trophic level of this invasive species, Pomacea canaliculata, is inversely tied to species evenness in the native and/or non-native habitats where it is found; but the occupancy of higher trophic levels may be directly tied to the known adverse ecological impacts of invasive apple snails.
The noticeable increase in the calculated trophic level of Pomacea canaliculata in less biodiverse and/or homogenized habitats, which here are comprised of two non-native habitats (e.g.: Kawainui Marsh and the Chinese creek sites), provides a mechanism by which Pomacea canaliculata responds to reduced interspeci c competition (and/or increased intraspeci c competition during novel biological invasions) for essential resources (e.g.: food and space). Reduced interspeci c competition in less biodiverse (more homogenized) and/or less productive habitats consequentially shifts the trophic position of Pomacea canaliculata. This trophic shift may cause the well-known adverse ecological impacts associated with Pomacea canaliculata that extirpate both aquatic and riparian plants in invaded habitats and facilitate trophic cascades via the bottom-up control of community structure, see Carlsson, Brönmark, & Hansson (2004) and Scheffer et al. (2001). This ultimately contributes to further loss of biodiversity and increasing homogenization therein.

Conclusion
This study has provided data that support the proposed mechanism by which the adverse ecological impacts of Pomacea canaliculata in non-native habitats are explained. These data also provide a means of predicting the susceptibility of non-native habitats to said ill ecological effects. As biodiversity declines within non-native habitats, the arrival of this biological invader leaves open niche space within these habitats which this invasive species can readily ll. What may be more signi cant is that the data illustrate a clear relationship between reduced biodiversity in habitats and the prevalence of adverse ecological impacts from invasive apple snails therein. These adverse ecological impacts differ starkly from the impacts of invasive apple snails in pristine and/or anthropogenically disturbed portions of the native range, as well as in more biodiverse and less homogenized non-native habitats (e.g.: the Chinese Lake site), where the deleterious effects of Pomacea canaliculata are absent or less obvious.
The fact that Pomacea canaliculata can have variable ecological impacts in native versus non-native habitats suggests that this proposed mechanism of trophic shifts may explain changes in invasive apple snail feeding behavior and/or diet between habitats. It appears that the trophic shifting, as de ned by the data presented in this publication, demonstrates that as Pomacea canaliculata move from biodiverse native habitats, to anthropogenically disturbed native habitats, and nally to more and more homogenized (and therefore less biodiverse) non-native habitats may correspond to a shift in their ecological niche, diet, and inherent (natural versus unnatural) ecological role. This is a plausible explanation as to how invasive apple snails transition from being primarily opportunistic generalists, in highly productive and relatively biodiverse native habitats within the Pantanal, to macrophyte and/or riparian plant specialists in more homogenized, less biodiverse and by extension less interspeci cally competitive, non-native habitats.
Stable isotope studies could be used preemptively, at the forefront of biological invasion, to determine habitats' susceptibility to biological invaders and the adverse ecological impacts predicted by the invasion front hypothesis (Iacarella et al., 2015). Conversely, in habitats already suffering these ill ecological effects, stable isotope studies can provide insights into: 1) the changes to habitats' trophic ecology which may facilitate these impacts and 2) determine the likelihood that habitats may be restored to proper function. The trophic level of Pomacea canaliculata, as well as other biological invaders, may be predictable based on differences in biodiversity (species evenness) and stable isotope data. This may provide a powerful tool in the conservation and /or restoration of habitats invasive species endanger and/or inhabit. Future studies would bene t from evidence inferred from the stable isotope data collected within these ve collection sites to examine the speci c contribution of available food resources to the diet of Pomacea canaliculata. In doing so, the observations from this study can be supported, or disproven, by indirectly inferring and de ning the components and proportions available food resources contribute to invasive apple snail (or other biological invaders') diets; in native and/or non-native habitats along a compendium of species evenness (a measure of biodiversity and/or homogeneity) values.

Funding
This work was supported the Frederic Weiss Memorial Award from the Conchologist of America, 2018.

Authors' Contributions
KESII conceived project design and designed methodologies, completed all eldwork, prepared samples, analyzed samples, processed data, analyzed data, wrote and edited all manuscript drafts. CF assisted with project design and methodologies, provided training, provided laboratory support, analyzed samples, assisted with data analysis, and edited all manuscript drafts. FJ assisted with project design and methodologies, assisted with general project management, provided funding and laboratory space, assisted with data analysis, and edited all manuscript drafts.