Ciliate diversity in rodrigo de freitas lagoon (Rio de Janeiro, Brazil) from an integrative standpoint

Abstract

The Rodrigo de Freitas Lagoon is a highly eutrophic lacustrine system and has one of the longest histories of exploration and anthropic alteration in Brazil. Despite its relevance, limited studies explored the diversity of micro-eukaryotes in the lagoon. Ciliates (Alveolata, Ciliophora) are overlooked in environmental microbiology, especially in tropical and subtropical ecosystems, resulting in limited knowledge about their diversity and functional relevance in South American habitats, particularly in coastal lagoons. To fill this gap, here we investigated the diversity of ciliates in a brackish coastal lagoon in an urban area of Rio de Janeiro, Brazil, applying and comparing the performance of morphological and metabarcoding approaches. The metabarcoding analysis, based on high-throughput sequencing of the hipervariable region V4 of the 18S rRNA genes detected 37 molecular operational taxonomic units (MOTUs) assigned to Ciliophora, representing only about a half (56.9%) of the diversity detected by microscopy, which counted 65 ciliate morphotypes. The most representative classes in both approaches were Spirotrichea and Oligohymenophorea. The metabarcoding analysis revealed that 35.3% of the ciliate MOTUs had less than 97% similarity to available sequences in the NCBI database, indicating that more than one-third of these MOTUs potentially represents still not represented or undescribed ciliate species in current databases. Our findings indicate that metabarcoding techniques can significantly enhance the comprehension of ciliate diversity in tropical environments, but the scarcity of reference sequences of brackish ciliates in molecular databases represents a challenge to the taxonomic assignment of the MOTUs. This study provides new insights into the diversity of ciliates in a threatened coastal lagoon, revealing a vast array of still unknown and rare ciliate taxonomic units in tropical environments.

Keywords: Brackish water; Brazil, Rio de Janeiro; Ciliophora; Coastal lagoon; Eukaryotes; Metabarcoding; Microscopy.

Fig. 1

Geographical location of the sampling sites in Rodrigo de Freitas lagoon, Rio de Janeiro, Brazil

Fig. 2

Living (A–D,F,H,J,L,N,P,R,T,V,X) and protargol stained (E,G,I,K,M,O,Q,S,U,W) ciliates from RFL under light microscopy. A Caenomorpha sp. B Metopus sp. 1. C Metopus sp. 2. D,E Metopus vestitus (F,G) cf. Muranothrix sp. H,I Condylostoma arenarium. J,K Condylostoma patulum. L,M Condylostoma remanei. N,O Spirostomum teres. P,Q Copemetopus verae. R,S Peritromus kahli. T,U Linostomella vorticella. V,W Corlissina maricaensis. X Parduczia sp. Scale bars: 30 µm (A); 50 µm (D–G,R–U); 100 µm (B,C,H–K,N–Q,V,W); 200 µm (L,M,X)

Fig. 3

Living (A,D,F,H,J,L,N,P,R,T,V,X) and protargol stained (B,C,E,G,I,K,M,O,Q,S,U,W,Y) ciliates from RFL under light microscopy. A,B Tracheloraphis sp. C Trachelocerca sp. D,E Parablepharisma brasiliensis. F,G Parablepharisma granulata. H,I Enchelyodon trepida. J,K Lacrymaria sp. L,M Kentrophyllum verrucosum. N,O Litonotus bergeri. P,Q Litonotus bonnensis. R,S Litonotus uninucleatus. T,U Loxophyllum elegans. V,W Mesodinium rubrum. X,Y Cyclidium sp. Scale bars: 10 µm (V,Y); 25 µm (P–S); 50 µm (D–M,T,U); 100 µm (A–C,N,O)

Fig. 4

Living (A,C,E,G,I,K,M–P,R,T,V) and protargol stained (B,D,F,H,J,L,Q,S,U) ciliate specimens from RFL under light microscopy. A,B Paramecium sp. C,D Frontonia mengi. E,F Frontonia subtropica. G,H Pleuronema coronatum. I,J Cristigera media. K,L Uronema sp. M,N Vorticella sp. O–Q Zoothamnium sp. R,S Dysteria cristata. T,U Dysteria procera. V Protocruzia sp. Scale bars: 20 µm (I–L,R,S,V); 50 µm (A,B,G,H,M,N,Q,T,U); 100 µm (C–F); 200 µm (O,P)

Fig. 5

Living (A,C,D,F,H,I,K,M,O,Q,S) and protargol stained (B,E,G,J,L,N,P,R,T) ciliate specimens from RFL under light microscopy. A,B Plagiopyla sp. C Sonderia sp. D,E Pinacocoleps sp. 1. (F,G Pinacocoleps sp. 2. H Coleps sp. I,J Lagynus elegans. K,L Holophrya coronata. M,N Holophrya sp. O,P Deviata brasiliensis. Q,R Aspidisca steini. S,T Diophrys oligothryx. Scale bars: 15 µm Q,R; 30 µm (F,G,S,T); 50 µm (A–E,H–J,M–P); 250 µm (K,L)

Fig. 6

Living (A,C,E,H,J,L,N,P,R,T,W-X,Z,A2) and protargol stained (B,D,F,G,I,K,M,O,Q,S,U,Y,B2,C2) ciliates from RFL under light microscopy. A,B Euplotes vannus. C,D Euplotes woodruffi. E,F Euplotes sp. 1. G Euplotes sp. 2. H,I Euplotes sp. 3. J,K Holosticha pullaster (L,M) Novistrombidium apsheronicum. N,O Parallelostrombidum sp. P,Q Uronychia setigera. R,S Protogastrostyla pulchra. T,U Psedokeronopsis sp. V Psedokeronopsis flavicans. W Trachelostyla sp. X,Y Urosoma macrostyla. Z Amphisiella sp. (A2–C2) Notoholosticha sp. Scale bars: 25 µm (E,F,J,K); 30 µm (G–I,L,M,P–S); 50 µm (A–D,N,O,T–C2)

Fig. 7

A Morphotype species accumulation curve per sampling event. The blue shading represents the standard deviation. B Venn’s diagram of the number of ciliate MOTUs classified at genus level by VSEARCH (blue), BLAST (red) and SKLEARN (green) algorithms using SILVA as reference database. C, D Stacked barplots of the ciliate MOTUs relative abundances by genus (C) and species (D) retrieved with SKLEARN. Only MOTUs with abundance ≥ 2% were included. The relative abundance of unclassified MOTUs is remarkably high. E Species accumulation curve of ciliate MOTUs richness by sequencing depth

Fig. 8

Maximum likelihood phylogenetic tree of ciliates based on V4 18S-rDNA gene sequences, including the sequences retrieved from RFL (in red font)