*5.4. Statistical Analysis*

We tested for significant compositional differentiation using a hierarchical multivariate approach that is robust to the compositional nature of the data [44]. First, we brought the relative abundance data out of the compositional simplex space using the isometric log-ratio (ilr) transformation, which also avoids the zero-sum constraint of the centered-log ratio (clr) transformations but at the cost of one dimension of the data. The ilr-transformed peak data were then subjected to permutational multivariate analysis of variance based on distances using the adonis function from the vegan R package [45], with all peak dimensions as a response and environment as a predictor. Significance and R<sup>2</sup> were estimated using 10,000 permutations of the raw data. We then visualized the venom phenotype of each snake in multivariate space using robust principal components analysis (PCA) as implemented in the pcaCoDA function from the robCompositions R package [46].

After detection of a significant global association between environment and venom composition, we conducted pairwise posthoc PERMANOVA, as described above, between the venom compositions of all pairs of environments to determine which environments differed significantly. Lastly, we preserved the full dimensionality of the data but removed the data from the simplex using the clr-transformation, to determine which peaks specifically varied across environments. To do this, we use the lm function from R stats, with the clr-transformed abundance of an individual peak as the response and environment of origin as the predictor. We repeated this test for all 26 peaks and used the false-discovery rate (fdr) approach to correct the *p*-values for multiple tests.

For the functional analyses (in vitro and in vivo assays), firstly, the data were evaluated for a normal distribution (Shapiro–Wilk), and then differences among the means were evaluated by one-way ANOVA, followed by a Tukey post-test (for multiple comparisons). Data that did not meet the normality criteria were analyzed using a non-parametric test (Kruskal–Wallis). Results represent the mean and standard deviation or standard error, as

appropriate, and the level of significance was set at *p* ≤ 0.05. Data were analyzed using the GraphPad Prism statistical program (version 7.00 for Windows, GraphPad Software, San Diego, CA, USA).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/toxinstoxins13110814/s1, Figure S1: Chromatographic profiles of *B. atrox* venoms of different habitats from west of Pará State, Brazilian Amazon; Figure S2: Characteristics and localization of areas of snake collection.

**Author Contributions:** Conceptualization, L.F.S. and A.M.M.-d.-S.; formal analysis, L.F.S., M.L.H., R.H.V.M., B.P., H.L.G. and A.M.M.-d.-S.; funding acquisition, A.M.M.-d.-S.; investigation, L.F.S., T.H.M.D.-R., M.M.T.R., R.H.V.M. and B.P.; methodology, L.F.S., M.L.H., T.H.M.D.-R., M.M.T.R., B.P. and H.L.G.; project administration, A.M.M.-d.-S.; resources, H.M.C.; software, M.L.H.; supervision, A.M.M.-d.-S.; writing—original draft, L.F.S.; writing—review and editing, L.F.S., M.L.H., R.H.V.M., H.L.G. and A.M.M.-d.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Coordenação de Aperfeiçoamento de Pessoal nível superior (CAPES 063/2010-Toxinology-AUXPE 1209/2011); Fundação de Amparo à Pesquisa de São Paulo (FAPESP 2016/50127-5; 2014/13124-2; 2017/15170-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 303958/2018-9). Additional support was provided by Fundação de Amparo à Pesquisa do Estado do Amazonas-FAPEAM (PRO-ESTADO) to AMMS. as Manaus Visiting Professor. LFS and THMDR were students in the Science Graduate Program— Toxinology (Instituto Butantan).

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki. The snakes were captured under ICMBio/SISBio license 32098-1 and SISGEN number A78BD88 and CEUAIB 1244/14, and its handling was undertaken according to the guidelines and permits (CEUAIB 1244/14, Instituto Butantan). The procedures involving mice, chickens, and chicks were approved by the Ethics Committee on Animal Use of the Instituto Butantan (Protocol Number: 13710-14).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
