Results
Alpha diversity
The mean Chao1 index across all crosses was 59.7 ranging from 41.7 (PUNT) to 69.1 (YIAL); for Shannon diversity, the mean across all crosses was 4.33, ranging from 3.97 (PUNT) to 4.54 (YIAL, Figure 2) within crosses. Large standard errors in the alpha diversity measures were observed across all crosses in the study for Shannon and Chao1 indices.
Our likelihood ratio tests showed no significant differences due to cross or sire effects using the Chao1 and Shannon indices, but a significant difference was found between replicate pens for the Chao1 index (F = 1.53, P = 0.038; Table 1) and not for Shannon's index. Post-hoc pairwise t-tests analyses showed no statistically significant differences in the means between the pairs of crosses for Chao1 and Shannon indices (Table 1), corroborating the overall analysis results.
Beta diversity
Overall, the first two PCoAs accounted for approximately 37% of all variance in Bray-Curtis distances across all sample pairs, and approximately 22% (PC1 = 15.81%, PC2 = 6.31%) for Jaccard distances. The multidimensional clustering revealed YIAL as an outlier cross, CHILL as intermediate, and the remaining stocks clustering more closely together on the axes (Figure 3).
Using the overall PERMANOVA model, significant cross effects were found in the microbial community structure using Bray-Curtis distances (P = 0.0025, pseudo-F = 1.74) and Jaccard distances (P = 0.00040, pseudo-F = 1.55); however, no sire or replicate pen effects were found on community composition (Table 2). Cross-specific nested-PERMANOVA models showed significant sire effects within CHILL on Bray-Curtis distances (P = 0.028, pseudo-F = 1.48), and within CHILL and NIT on Jaccard distances (P = 0.0017, pseudo-F = 1.35 and P = 0.009, pseudo-F = 1.32, respectively). No significant sire effects were found within other crosses, and no replicate pen effects were found for any cross for Bray-Curtis (Supplementary table 1) and Jaccard distances (Supplementary table 2). PERMDISP showed no significant differences of within-group dispersions among crosses for Bray-Curtis (P = 0.83, pseudo-F = 0.42) and Jaccard distances (P = 0.35, pseudo-F = 0.99). Further, no pairwise PERMDISP differences were found among crosses (Supplementary table 2).
Pairwise PERMANOVA tests using Bray-Curtis distances showed that YIAL was statistically different from all hybrid-crosses except CHILL (P > 0.05); further, CHILL was different than CAP (P = 0.0028); however, all other pairwise PERMANOVA comparisons showed a lack of significant pairwise differences (Table 3). Similarly, PERMANOVA tests using Jaccard distances showed that YIAL was statistically different from all hybrid-crosses except CHILL, with CHILL being statistically different from CAP (P = 0.0028) and BQ (P = 0.040), and other all pairwise PERMANOVA comparisons showing a lack of significant differences (Table 3).
ASV-level analysis
In total, 124 ASVs were found to be common to all crosses, and the largest number of unique ASVs per cross were distinctly found to be in YIAL (429), CHILL (293) and QUIN (206) (Figure 3). Considering the taxonomic families of unique ASVs occurring across the hyrid-crosses at least 10 times or more, YIAL showed the largest number of unique ASVs for 8 families (Sphingomonadaceae; Moraxellaceae; Corynebacteriaceae; Chitinophagaceae; Pseudomonadaceae; Beijerinckiaceae; Paracaedibacteraceae; Pirellulaceae; Caulobacteraceae), with CHILL showing the highest number of unique ASVs for Lactobacillaceae (Figure 4). Furthermore, the biggest number of ASVs found common to all hybrid-crosses were taxonomically classified as Bacillaceae, Burkholderiaceae, Mycoplasmataceae, or Streptococcaceae.
Using multiple (FDR-corrected) Kruskal-Wallis tests implemented in the LefSe algorithm, 17 ASVs showed significant differences among crosses (Figure 5). Multiple non-parametric pairwise comparisons within each ASV showed significant differences in the means for fourteen ASVs between pairs of hybrid-crosses (Figure 6)