Phloxine B staining is compatible with high‐throughput DNA barcoding of meiofauna

Modern, integrative biodiversity research requires methods capable of bridging the gap between detailed morphological observations and the scalability of DNA sequencing. Integrative approaches like megabarcoding generate DNA sequences, while morphological functional data and abundance data are retained. For small, transparent, and highly abundant animals like meiofauna, the necessity to sort the specimens from organic matter and sediment forms a major bottleneck, as the use of stains required for enhanced manual or automated specimen detection can inhibit downstream molecular workflows. To address this, we tested the compatibility of phloxine B staining with DNA sequencing to facilitate simultaneous morphological and molecular specimen processing. Meiofauna preserved in ethanol, propylene glycol, or a DMSO/EDTA/saturated NaCl solution (DESS) was incubated with phloxine B for 30min or 24h. Specimens were manually isolated, the V1–V2 region of the 18S rDNA gene was sequenced, and success was quantified across major meiofaunal phyla. Nematodes, copepods, and annelids consistently showed high sequencing success irrespective of the preservative or staining incubation time. Platyhelminths showed lower success, likely due to misidentification or primer limitations rather than dye inhibition. Overall, these findings demonstrate that phloxine B is compatible with downstream DNA amplification and sequencing, enabling efficient integration of morphological and molecular data. The approach offers high potential for broader application in other microscopic taxa, supporting the way to comprehensive, high-throughput biodiversity assessments or ecological monitoring.

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