# Phytoplankton Diversity in the Northern Adriatic Sea: Insights and Inconsistencies from Microscopy and Metabarcoding

## TL;DR

This study compares microscopy and DNA metabarcoding to assess phytoplankton diversity in the northern Adriatic Sea, finding that combining both methods improves accuracy and reliability of biodiversity assessments.

## Contribution

The study introduces a combined approach of microscopy and DNA metabarcoding with correction factors to enhance phytoplankton diversity assessment.

## Key findings

- Metabarcoding detected higher diversity, especially for small and fragile phytoplankton species.
- Microscopy was more effective for identifying larger species like diatoms and coccolithophores.
- Applying correction factors based on genetic material improved consistency between methods.

## Abstract

Phytoplankton plays a fundamental role in marine ecosystems and is widely used to assess environmental change. In this study, light microscopy and DNA metabarcoding approaches were used to investigate phytoplankton communities at three long-term monitoring sites in the northern Adriatic Sea. By combining the two approaches, more than 500 species were recorded. Metabarcoding detected substantially higher diversity, particularly among small and fragile organisms that are difficult to identify under a microscope, whereas microscopy was better for identifying larger species with distinctive morphology. Because species contain different amounts of genetic material, DNA data can overestimate or underestimate their true contribution to the community. Indeed, after applying correction factors that account for these differences, the DNA-based estimates became more consistent with the microscopy results for several phytoplankton groups. The differences among the samples were influenced more by the approach used than by the sampling location. Overall, combining these two approaches provides an accurate and robust picture of phytoplankton communities. In this way, it is possible to improve the reliability of marine biodiversity assessments and to support better environmental monitoring and management in coastal seas.

Phytoplankton is a key component of marine ecosystems and a sensitive indicator of environmental change. In this study, light microscopy (LM) and DNA metabarcoding (18S-V4, 18S-V9, and rbcL) were combined to assess differences in phytoplankton diversity and community structure across three LTER sites in the northern Adriatic Sea, and to evaluate the methodological effects on community assessment. A total of 329 genera and 527 species were recorded by integrating both the approaches. Metabarcoding (MB) revealed increased taxonomic richness than LM, particularly for dinoflagellates and small phytoflagellates, while LM was better for identifying the diatoms and coccolithophores. The rbcL marker improved the taxonomic resolution for the diatoms compared to the 18S regions. The proportion of species shared among the sites increased from 13% with LM to 33–42% with MB, suggesting that MB may effectively reduce the discrepancies observed when relying solely on LM. Cluster analysis performed on species-relative abundances grouped the samples by approaches rather than sites, showing that methodological variability exceeded the ecological differences. The relative abundance patterns differed between methods but became more comparable after applying correction factors based on the 18S rRNA gene copy numbers, particularly for the dinoflagellates. Overall, MB enhances biodiversity assessment and comparability among sites, while LM remains essential for morphological validation and for abundance assessment.