Microbial eukaryotes in the suboxic chemosynthetic ecosystem of Movile Cave, Romania

Abstract

Summary Movile Cave is a small system of partially inundated galleries in limestone settings close to the Black Sea in Southeast Romania. Isolated from the surface for 6 million years, its sulfidic, methane and ammonia‐rich waters harbour unique chemosynthetic prokaryotic communities that include sulphur and ammonium‐metabolizing chemolithotrophs, methanogens, methanotrophs and methylotrophs. The cave also harbours cave‐dwelling invertebrates and fungi, but the diversity of other microbial eukaryotes remained completely unknown. Here, we apply an 18S rRNA gene‐based metabarcoding approach to study the composition of protist communities in floating microbial mats and plankton from a well‐preserved oxygen‐depleted cave chamber. Our results reveal a wide protist diversity with, as dominant groups, ciliates (Alveolata), Stramenopiles, especially bicosoecids, and jakobids (Excavata). Ciliate sequences dominated both, microbial mats and plankton, followed by either Stramenopiles or excavates. Stramenopiles were more prominent in microbial mats, whereas jakobids dominated the plankton fraction of the oxygen‐depleted water column. Mats cultured in the laboratory were enriched in Cercozoa. Consistent with local low oxygen levels, Movile Cave protists are most likely anaerobic or microaerophilic. Several newly detected OTU clades were very divergent from cultured species or environmental sequences in databases and represent phylogenetic novelty, notably within jakobids. Movile Cave protists likely cover a variety of ecological roles in this ecosystem including predation, parasitism, saprotrophy and possibly diverse prokaryote‐protist syntrophies.

Alveolates (dinoflagellates, ciliates and apicomplexans) and Rhizarians are the most common microbial eukaryotes in temperate Appalachian karst caves

“…We also used the PR2 reference database to make taxonomic assignments and 13.2% could be identified to the genus level with 99% certainty, 17.4% with 95% certainty, or 20.4% at 90% certainty. This is similar to Reboul et al (2019) who found that few of their OTUs matched curated/cultured 18S microeukaryotic rDNA sequences.…”

“…Our yields using periphytometers were comparable to another 18S rDNA metabarcoding survey from Movile cave, Romania (Reboul et al ., 2019) which reported microeukaryotes from hypoxic air pockets and noted that they had higher yields from ‘fresh’ samples, meaning microbial mats whose DNA was extracted very soon after collection as opposed to mats that had been cultured for 2 months. In our study, the fact that the glass slides were immersed in DNA extraction buffer immediately after removal from the periphytometer in the cave may have also contributed to the higher ASV yield than those from filtered water.…”

“…When systematic microscopic screens of protists have been conducted, numerous uncharacterized species were found (Coppellotti and Guidolin, 2003; Bastian et al ., 2009; Sigala‐Regalado et al ., 2011; Baković et al ., 2019). Metabarcoding has recently been used in some eukaryotic surveys, including cave walls in a freshwater algae biodiversity survey of the Hawaiian archipelago (Sherwood et al ., 2014), surveys of green algae and diatoms in show cave Lampenflora (Pfendler et al ., 2018; Burgoyne et al ., 2021), soils from the Manao‐Pee Cave in Thailand (Wisechart et al ., 2019), and a survey of microbial mats growing in low oxygen airbells in Movile Cave Romania (Reboul et al ., 2019). Microeukaryotes are essential components of every food web (Corliss, 2002) as they link bacteria and small metazoans so describing them is important for a full understanding of the underground food web.…”

Molecular detection of a novel perkinsid associated with the deep‐sea clam Phreagena okutanii

“…Compared with such animals and prokaryotes, the microbial eukaryotes (protists) in chemosynthetic ecosystems have not been well investigated. Nevertheless, environmental DNA (metabarcoding) surveys of seawater and sediments collected from hydrothermal vents and seeps using the eukaryotic small subunit ribosomal RNA (SSU rRNA) gene as a marker have revealed that a broad range of protists occurs in these environments (Edgcomb et al, 2002; López‐García et al, 2003, 2007; Pasulka et al, 2016; Reboul et al, 2019; Sauvadet et al, 2010; Takishita et al, 2005, 2006, 2007, 2010). Remarkably, a significant fraction of the SSU rRNA gene sequences retrieved through these environmental DNA surveys was closely related to a wide variety of eukaryotic parasite groups, such as Apicomplexa, Perkinsozoa, Syndiniales, Ichthyosporea, and Kinetoplastida.…”

Microbial eukaryote assemblages and potential novel diversity in four tropical East African Great Lakes

“…653environments(Reboul et al 2018). "Novel" OTUs closer to Prostomatea affiliated to sequences from 654 lake sediment (KP059214) and anoxic waters (GU819524).…”