Two Rivers, Two Methods: DNA and Microscopes Reveal Hidden Diatom Diversity in the Euphrates-Tigris

Deep in southern Iraq, where the Euphrates and Tigris rivers wind through one of the Middle East’s most ecologically stressed drainage basins, an international team of researchers has carried out the first combined morphological and DNA metabarcoding survey of epiphytic diatoms in the country. The study, published in Metabarcoding and Metagenomics, examined microscopic algae living on the surfaces of submerged aquatic plants at six sampling sites near the city of Basra, and its findings reveal both the remarkable hidden biodiversity of these waters and the technical challenges of reading it with molecular tools.

The Euphrates-Tigris catchment is one of the major drainage basins of the Middle East, but decades of overexploitation have pushed the water system to its ecological limits. Iraqi waterbodies suffer from salinization driven by water losses caused by upstream dam construction, water policies during the Iraq wars and the intensifying impact of climate change on a region that already experiences minimal rainfall. Against this backdrop, diatoms, a diverse group of microalgae with silicified cell walls, serve as the world’s most widely used bioindicators of water quality, reflecting ecosystem conditions with unique sensitivity. Epiphytic diatoms, which live attached to submerged macrophytes, are particularly interesting because they interact with their living hosts in ways that can shape their own community structure.

Accurate taxonomic identification of diatoms is essential for sound biodiversity evaluation, yet classical identification by light microscopy requires highly specialized taxonomic skills and can produce discrepancies between researchers. Cryptic diversity, species that look identical under the microscope but are genetically distinct, further hinders accurate identification. In regions where literature on the local diatom flora is scarce, researchers often rely on European floras, which may force identifications into existing taxonomic concepts and overlook local variation or unique species. DNA metabarcoding, which enables the concurrent identification of numerous species from environmental samples, has become an increasingly popular alternative, but until now there has been a notable absence of such studies in Iraq.

The research team collected 47 samples during the summer of 2019, the dry season, and the winter of 2020, the rainy season, at six sites across two water bodies: the River Tigris in the city of Al-Qurnah and the River Euphrates in the city of Al-Midaina. Because no stones were available as habitats at the sampling stations, samples were taken separately from 11 different taxa of macrophytes and macroalgae, including common reed, cattail, hornwort, watermilfoil, several pondweed species, green algae of the genus Cladophora, naiad and sea aster. At least five stems and branches of each plant were shaken vigorously with distilled water to dislodge the attached diatoms, and the resulting suspensions were preserved with 70 percent alcohol for transport to the Botanic Garden and Botanical Museum at Freie Universität Berlin, where each sample was split for morphological and molecular analysis.

For the morphological analysis, samples were treated with 35 percent hydrogen peroxide to oxidize organic materials, and the cleaned diatom frustules were mounted in the high refractive index medium Naphrax. Using a Zeiss Axioplan microscope with differential interference contrast and a 100x Plan Apochromat objective, the researchers counted a minimum of 400 valves per sample to document the presence and relative abundance of each taxon, and turned to a Hitachi FE 8010 scanning electron microscope when finer details were needed. This meticulous dual-microscope approach identified 284 infrageneric taxa belonging to 59 genera, a species richness notably higher than in previous Iraqi studies. Remarkably, 46 species were recorded for the first time in the region, and 62 others could not be identified to species level and are possibly new to science, including Prestauroneis furatensis, which was newly described from the epiphytic community of the River Euphrates.

The metabarcoding analysis targeted the V4 region of the 18S rRNA gene, amplified with the DIV4 primer pair and sequenced on an Illumina MiSeq platform with 300-bp paired-end reads. After bioinformatic processing with the DADA2 pipeline, which denoises reads and infers amplicon sequence variants, and quality control with the metabaR package to remove contaminants and tag-jumps, 5,461,214 reads remained from 43 successfully amplified samples. These reads belonged to 2,953 ASVs, of which 1,454, or 49.2 percent, were classified as Bacillariophyta, the diatoms. The molecular approach recovered 54 genera and 108 species, with many ASVs assigned to the same taxa, and in most cases metabarcoding detected more taxa per sample than microscopy.

The comparison between the two methods exposed striking technical discrepancies. At the genus level, 38 genera were retrieved in both datasets, while 21 genera were identified only by morphology, including Amphipleura, Aulacoseira, Caloneis, Prestauroneis and Synedropsis, and 16 only by metabarcoding, including Campylodiscus, Chaetoceros, Eunotia, Fistulifera and Skeletonema. The incomplete taxonomic reference database for the studied sites turned out to be the major limitation of the molecular approach, as many morphologically identified species in genera such as Mastogloia and Achnanthidium are absent from any reference library. Intraspecific genetic variation, multiple rRNA operons within diatom genomes evolving at different rates, and sequencing artifacts can also generate multiple ASVs from a single species, artificially inflating molecular richness. Conversely, some abundant species identified microscopically, such as Navicula recens, Navicula rostellata and Nitzschia clausii, had no corresponding match in the metabarcoding inventories, possibly because of insufficient DNA concentrations in those samples.

Community-level statistics revealed further contrasts. Shannon diversity indices and average taxa richness were mostly higher in the metabarcoding analyses than in the morphological ones, with the highest molecular species count of 345 recorded at one winter Euphrates station and the lowest of 5 at another. Both approaches agreed that taxon richness was lowest at station E3 on the Euphrates and highest at station E1 on the same river. The non-metric multidimensional scaling plots for morphology showed a clear seasonal effect in diatom community composition, separating the dry summer from the rainy winter, and PERMANOVA confirmed statistically significant differences between the two seasons and the two rivers. In contrast, the NMDS plots for the 18SV4 analyses did not show a seasonal effect for the same samples, a discrepancy the authors attribute to gaps in the DNA barcode reference databases, particularly at the species level, which hinder accurate taxonomic assignment.

The ecological picture emerging from the morphological data is consistent with the environmental stress documented in the region. Pennate diatoms dominated the epiphytic assemblages, constituting 93.7 percent of identified species, while centric diatoms accounted for only 6.3 percent. The distribution of diatom assemblages showed a prevalence of freshwater species at 65.5 percent, followed by brackish species at 13.5 percent and marine species at 10 percent, with the remaining 11 percent being euryvalent species tolerant of a broad range of conditions. Several taxa of marine origin, including Tabularia fasciculata, Seminavis strigosa and Surirella striatula, were recorded, likely because the Qurna marsh connects directly with the Shatt Al-Arab River and receives water from the Arabian Gulf during high tides, creating a conduit for marine diatom migration to a region situated around 200 kilometers from the Gulf. Brackish diatoms such as Bacillaria paxillifera and Nitzschia filiformis reflect salinity fluctuations that intensify as less freshwater flows from the rivers and more seawater ingress from the Gulf.

The study demonstrates that combining metabarcoding with classical microscopy increases the detection and identification of diatom species and lays the foundation for understanding diatom biodiversity in this region. Even amid major advances in molecular tools for biodiversity assessment, the role of morphology in species discovery remains central. The authors recommend repeating the analysis with the rbcL marker, which has shown promise in providing higher resolution for diatom identification, and producing unialgal diatom strains from the studied sites to close the gaps in reference libraries. Such investigations are essential for illustrating the importance of sustainable ecosystem management and the conservation of water as a vital resource, both regionally and globally, in a catchment that climate change, overuse of waters and subsequent salinization have brought to the brink of ecological devastation.

Subject of Research: Diatom biodiversity in the Euphrates-Tigris rivers revealed by combined microscopy and DNA metabarcoding

Article Title: Comparison of the biodiversity of epiphytic diatoms in the Euphrates-Tigris rivers using morphological and metabarcoding analyses

Article References: Mohamad, H., Schimani, K., Al-Shaheen, M., Abarca, N., Jahn, R., Al-Handal, A., Kusber, W.-H., & Zimmermann, J. (2024). Comparison of the biodiversity of epiphytic diatoms in the Euphrates-Tigris rivers using morphological and metabarcoding analyses. Metabarcoding and Metagenomics, 8, Article e135082. https://doi.org/10.3897/mbmg.8.135082

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.135082

Keywords: diatoms, DNA metabarcoding, Euphrates, Tigris, biodiversity, bioindicators, water quality, Iraq, 18S rRNA, epiphytic algae, Comparison, epiphytic