Ecological analyses
In order to evaluate how exhaustively the zooplankton communities of
each station were sampled, rarefaction curves of the detected ASVs were
generated using the QIIME2 diversity alpha-rarefaction plugin,
implementing the median frequency of the reads as the sequencing depth.
Additionally, the number of obtained ASVs from each station was compared
against the non-parametric species richness estimator Chao1
(Chao, 1984;
Supplementary Information Fig. S2). The rarefied number of reads was
used as an abundance proxy to estimate the Shannon diversity index and
structural changes in the zooplankton community among spatial and
temporal scales.
Differences in taxonomic structure among cruises were evaluated at the
order level with bar-plots based on only the most abundant ASVs (cutoff
at > 1% frequency) and at the ASV level by principal
coordinate analysis (PCoA) based on Bray Curtis, Jaccard, and weighted
and unweighted phylogenetic Unifrac distance metrics. The results of the
PCoA were visualized with EMPeror
(Vázquez-Baeza,
Pirrung, Gonzalez, & Knight, 2013). Statistical differences were
evaluated by a permutational multivariate analysis of variance
(PERMANOVA) and were based on 4999 permutations usingbeta-group-significance plugin of QIIME2 v.2019.7
(Anderson, 2001).
Also, shared and/or exclusive non-rarefied ASVs among the same stations
from each cruise (n = 19 and 21 for 18S and COI, respectively) were
visualized using Venn diagrams created with the open web-software
InteractiVenn (http://www.interactivenn.net/
Heberle,
Meirelles, da Silva, Telles, & Minghim, 2015). Significant differences
in the zooplankton diversity index were evaluated with a one-way
analysis of variance (ANOVA) conducted in Statistica (StatSoft Inc.,
Tulsa, USA) with statistical significance evaluated at α = 0.05 (p
< 0.05). Finally, to determine which ASVs were primarily
contributing to the observed spatial-temporal dissimilarities, a SIMPER
analysis based on rarefied ASV abundance was implemented in PRIMER+P v.6
(K. R. Clarke &
Warwick, 2001).