Modern metagenomic environmental DNA studies are almost completely reliant on next-generation sequencing, making evaluations of these methods critical. We compare two next-generation sequencing techniques – amplicon and shotgun – on water samples across four of Brazil’s major river floodplain systems (Amazon, Araguaia, Paraná, and Pantanal). Less than 50% of phyla identified via amplicon sequencing were recovered from shotgun sequencing, clearly challenging the dogma that mid-depth shotgun recovers more diversity than amplicon-based approaches. Amplicon sequencing also revealed ~27% more families. Overall the amplicon data were more robust across both biodiversity and community ecology analyses at different taxonomic scales. Our work doubles the sampling size in similar environmental studies, and novelly integrates environmental data (e.g., pH, temperature, nutrients) from each site, revealing divergent correlations depending on which data are used. While myriad variants on NGS techniques and bioinformatic pipelines are available, our results point to core differences that have not been highlighted in any studies to date. Given the low number of taxa identified when coupling shotgun data with clade-based taxonomic algorithms, previous studies that quantified biodiversity using such bioinformatic tools should be viewed cautiously or re-analyzed. Nonetheless, shotgun has complementary advantages that should be weighed when designing projects.

The use of venom in intraspecific aggression is uncommon and venom-transmitting structures specifically used for intraspecific competition are found in few lineages of venomous taxa. Next-generation transcriptome sequencing allows robust characterization of venom diversity and exploration of functionally unique tissues. Using a tissue-specific RNA-seq approach, we investigate the venom composition and gene ontology diversity of acrorhagi, specialized structures used in intraspecific competition, in aggressive and non-aggressive polyps of the aggregating sea anemone Anthopleura elegantissima (Cnidaria: Anthozoa: Hexacorallia: Actiniaria: Actiniidae). Collectively, we generated approximately 450,000 transcripts from acrorhagi of aggressive and non-aggressive polyps. For both transcriptomes we identified 65 candidate sea anemone toxin genes, representing phospholipase A2s, cytolysins, neurotoxins, and acrorhagins. When compared to previously characterized sea anemone toxin assemblages, each transcriptome revealed greater within-species sequence divergence across all toxin types. The transcriptome of the aggressive polyp had a higher abundance of type II voltage gated potassium channel toxins/Kunitz-type protease inhibitors and type II acrorhagins. Using toxin-like proteins from other venomous taxa, we also identified 612 candidate toxin-like transcripts with signaling regions, potentially unidentified secretory toxin-like proteins. Among these, metallopeptidases and cysteine rich (CRISP) candidate transcripts were in high abundance. Furthermore, our gene ontology analyses identified a high prevalence of genes associated with “blood coagulation” and “positive regulation of apoptosis”, as well as “nucleoside: sodium symporter activity” and “ion channel binding”. The resulting assemblage of expressed genes may represent synergistic proteins associated with toxins or proteins related to the morphology and behavior exhibited by the aggressive polyp. We implement a multifaceted approach to investigate the assemblage of expressed genes specifically within acrorhagi, specialized structures used only for intraspecific competition. By combining differential expression, phylogenetic, and gene ontology analyses, we identify several candidate toxins and other potentially important proteins in acrorhagi of A. elegantissima. Although not all of the toxins identified are used in intraspecific competition, our analysis highlights some candidates that may play a vital role in intraspecific competition. Our findings provide a framework for further investigation into components of venom used exclusively for intraspecific competition in acrorhagi-bearing sea anemones and potentially other venomous animals.