Efficient Monitoring of Adult and Immature Mosquitoes Through Metabarcoding of Bulk Samples: A Case Study for Non-Model Culicids With Unique Ecologies.

The rapid and economical monitoring of mosquitos is imperative to understanding the dynamics of both disease vectors and nuisance species. In light of technological advances in mosquito sampling and DNA sequencing, health agencies can now utilize the full potential of metabarcoding pipelines for rapid and standardizable surveillance. Here, we describe mosquito spatial and temporal variation, with particular focus on Mansonia Blanchard species, in the Madeira (Rondônia State) and the Ribeira (São Paulo) watersheds, Brazil using metabarcoding of the D2 rDNA marker. Sampling and molecular pipelines were used to evaluate the taxonomic contribution of mosquitos in pools of culicids collected en masse from macrophyte-roots (immatures) and from Mosquito Magnet traps and protected human landings (adults). Results for adult captures are comparable to morphological diagnoses and clarify previously unknown temporal and spatial species turnover. Metabarcoding of immature stages also confirmed the extent of the geographical distribution of some species and each taxon's association with macrophyte species. Given the benefits of metabarcoding, such as taxonomic acuity, high throughput processing, and objectivity, we suggest such techniques should be more fully incorporated into culicid monitoring schemes. The metabarcoding protocol described herein paired with standardized field sampling schemes, when used by mosquito monitoring professionals, offers substantial improvements in terms of practicality, speed and cost.

Culicidae-centric metabarcoding through targeted use of D2 ribosomal DNA primers.

A practical limitation to many metabarcoding initiatives is that sampling methods tend to collect many non-target taxa, which become "amplicon noise" that can saturate Next Generation Sequencing results and lead to both financial and resource inefficiencies. An available molecular tool that can significantly decrease these non-target amplicons and decrease the need for pre-DNA-extraction sorting of bycatch is the design of PCR primers tailored to the taxa under investigation. We assessed whether the D2 extension segment of the 28S ribosomal operon can limit this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude from amplification most other taxa likely to be collected with current sampling apparatuses. We show that, given enough sequencing depth, D2 is an effective marker for the detection of mosquito sequences within mock genomic DNA pools. As few as 3,050 quality-filtered Illumina reads were able to recover all 17 species in a bulk pool containing as little as 0.2% of constituent DNA from single taxa. We also mixed these mosquito DNA pools with high concentrations of non-Culicidae bycatch DNA and show that the component mosquito species are generally still recoverable and faithful to their original relative frequencies. Finally, we show that there is little loss of fidelity in abundance parameters when pools from degraded DNA samples were sequenced using the D2 primers.

Metabarcoding Analyses Enable Differentiation of Both Interspecific Assemblages and Intraspecific Divergence in Habitats With Differing Management Practices.

Spatial and temporal collections provide important data on the distribution and dispersal of species. Regional-scale monitoring invariably involves hundreds of thousands of samples, the identification of which is costly in both time and money. In this respect, metabarcoding is increasingly seen as a viable alternative to traditional morphological identification, as it eliminates the taxonomic bottleneck previously impeding such work. Here, we assess whether terrestrial arthropods collected from 12 pitfall traps in two farms of a coffee (Coffea arabica L.) growing region of Sao Paulo State, Brazil could differentiate the two locations. We sequenced a portion of the cytochrome oxidase 1 region from minimally processed pools of samples and assessed inter- and intraspecific parameters across the two locations. Our sequencing was sufficient to circumscribe the overall diversity, which was characterized by few dominant taxa, principally small Coleoptera species and Collembola. Thirty-four operational taxonomic units were detected and of these, eight were present in significantly different quantities between the two farms. Analysis of community-wide Beta diversity grouped collections based on farm provenance. Moreover, haplotype-based analyses for a species of Xyleborus beetle showed that there is significant population genetic structuring between the two farms, suggesting limited dispersal. We conclude that metabarcoding can provide important management input and, considering the rapidly declining cost of sequencing, suggest that large-scale monitoring is now feasible and can identify both the taxa present as well as contribute information about genetic diversity of focal species.

Concordant phylogeographies of 2 malaria vectors attest to common spatial and demographic histories.

The phylogeography of South American lineages is a topic of heated debate. Although a single process is unlikely to describe entire ecosystems, related species, which incur similar habitat limitations, can inform the history for a subsection of assemblages. We compared the phylogeographic patterns of the cytochrome oxidase I marker from Anopheles triannulatus (N = 72) and previous results for A. darlingi (N = 126) in a broad portion of their South American distributions. Both species share similar population subdivisions, with aggregations northeast of the Amazon River, in southern coastal Brazil and 2 regions in central Brazil. The average capital EF, Cyrillic(ST) between these groups was 0.39 for A. triannulatus. Populations northeast of the Amazon and in southeastern Brazil are generally reciprocally monophyletic to the remaining groups. Based on these initial analyses, we constructed the a priori hypothesis that the Amazon and regions of high declivity pose geographic barriers to dispersal in these taxa. Mantel tests confirmed that these areas block gene flow for more than 1000 km for both species. The efficacy of these impediments was tested using landscape genetics, which could not reject our a priori hypothesis but did reject simpler scenarios. Results form summary statistics and phylogenetics suggest that both lineages originated in central Amazonia (south of the Amazon River) during the late Pleistocene (579 000 years ago) and that they followed the same paths of expansion into their contemporary distributions. These results may have implications for other species sharing similar ecological limitations but probably are not applicable as a general paradigm of Neotropical biogeography.

Habitat fragmentation and biodiversity: testing for the evolutionary effects of refugia.

Concordant areas of endemism among taxa have important implications both for understanding mechanisms of speciation and for framing conservation priorities. Here we discuss the need for careful testing of phylogeographic data for evidence of such concordance, with particular reference to the Indonesian island of Sulawesi. This is because there are good reasons to question whether concordance between taxa is likely to be a common pattern, and because of the serious implications of incorrectly concluding that the biodiversity of a given area can be partitioned in this way.