In the mantle cavity of the heterobranch snail Physella acuta, collected from a lake in Slovakia (Central Europe), we identified the peritrich ciliate Trichodina chlorophora harboring endosymbiotic green algae. To elucidate the evolutionary origins of this tripartite consortium, we determined the phylogenetic affiliations of all three partners and conducted a detailed morpho-molecular characterization of the ciliate, a central component of this hyper-symbiotic system. The European population of T. chlorophora closely matched North American populations previously described from physinine snails. The diagnostic features of T. chlorophora include: body diameter of 41–83 μm after dry silver nitrate impregnation; denticle ring 23–39 μm wide, with 23–30 denticles and 9–11 radial pins per denticle; denticles 5.7–7.8 μm long; adoral ciliary spiral performing ~ 1.13 turns (390°–409°) around peristomial disc; and a horseshoe-shaped macronucleus. Phylogenetic analyses revealed that: (1) the host snails are closely related to North American conspecifics, reflecting the human-mediated introduction of this invasive gastropod to Europe; (2) trichodinids colonized aquatic snails multiple times independently from poikilothermic vertebrate hosts, with T. chlorophora clustering with freshwater congeners from frogs, snails, and planarians; and (3) the endosymbiotic green algae comprise two species: Chlorella sp., closely related to endosymbionts of heliozoans and cnidarians, and Jaagichlorella geometrica, which clusters with epiphytic congeners. While the algae exhibit low host specificity, snail-dwelling Trichodina species show high phylogenetic host specificity. The parallel emergence of green algae-bearing trichodinids in physinine and planorbid snails suggests co-evolutionary processes that independently gave rise to interdependent associations among aquatic snails, ciliates, and zoochlorellae.

Astome ciliates live in the digestive tract of a broad spectrum of marine, freshwater, and terricolous annelids. In aquatic lumbriculid and criodrilid oligochaetes collected in Central Europe, we rediscovered three insufficiently known astomes: Hoplitophrya secans, Mesnilella clavata, and Buchneriella criodrili. Their morphology was studied using in vivo observation, protargol, and dry silver nitrate impregnation. Multiple nuclear and mitochondrial molecular markers were used to determine their phylogenetic positions and reconstruct their evolutionary history. According to our phylogenetic analyses: (1) mouthless ciliates isolated from annelids form a robustly supported monophylum within the class Oligohymenophorea, (2) the progenitor of astomes invaded the digestive tract of marine polychaetes during the Paleozoic era, (3) lumbricid earthworms likely served as a source of astomes for criodrilid, almid, and megascolecid earthworms, (4) the ancestral host of the earthworm-dwelling astome clade led an endogeic lifestyle, and (5) there were multiple independent transfers of astomes from endogeic to epigeic and anecic earthworms. These findings support previous views of the annelid phylogeny, suggesting that astomes reside and evolve in tandem with annelids for several hundred million years.