Revealing hidden diversity in gonostomatid ciliates (Ciliophora, Hypotrichia): integrative evidence supports two new species, a new genus, and Wallackiidae fam. nov.

BACKGROUND: Gonostomatidae are among the most common terrestrial hypotrichs and are readily recognized by a distinctive oral apparatus (knee-shaped adoral zone of membranelles; monokinetidal undulating membranes with a widely spaced paroral and densely spaced endoral). A further key diagnostic trait is morphogenesis, in which the proter and opisthe share primary frontal-ventral-transverse (FVT) primordia that later split into secondary primordia. Despite their apparent diversity in soils, comparatively few gonostomatid species have been described, and most lack complete morphogenetic and/or molecular documentation. This insufficient documentation obscures relationships and contributes to taxonomic instability, highlighting the need for integrative taxonomy combining morphology, detailed morphogenesis, and multigene phylogenetics.

RESULTS: From Korean soils we discovered two novel hypotrichs with a gonostomatid oral apparatus. The first is a new gonostomatid species with five FVT cirral rows and one or two transverse cirri. Its morphogenesis is distinctive within Gonostomatidae: the rightmost cirral row arises predominantly from anlage V with a short posterior contribution from anlage IV (i.e., pseudorow). Phylogenetic analyses show marker-dependent placements: the new species clusters with Metagonostomum gonostomoidum in the 18S rDNA tree but with Paragonostomoides xianicum in ITS1-5.8S-ITS2, 28S rDNA, and multi-gene datasets, indicating that the 18S rDNA marker lacks sufficient phylogenetic signal unlike the other markers; integrative evidence supports establishment of Metagonostomoides gen. nov. The second species, Wallackia koreana sp. nov., has a buccal cirral row reduced to two buccal cirri and an adoral zone occupying ~ 62% of body length; it clusters with W. bujoreani and Cladotricha australis, and the three group with Chaetospiridae in ITS1-5.8S rDNA-ITS2 and multi-gene datasets. A comparative review further shows that Wallackia and Neowallackia deviate from core gonostomatids by lacking shared primary primordia (proter and opisthe anlagen form independently), supporting Wallackiidae fam. nov. Re-evaluation of the "gonostomatid-type" oral apparatus across hypotrichs restricts it to Gonostomatidae, Chaetospiridae, and Wallackiidae fam. nov.

CONCLUSION: We document two new soil hypotrich species from Korea, one representing a new gonostomatid genus (Metagonostomoides gen. nov.) and the other a new Wallackia species (W. koreana sp. nov.), and show that their integrative placement helps resolve relationships among hypotrichs with a gonostomatid oral apparatus. The combined morphological, morphogenetic, and multigene phylogenetic evidence supports recognition of Wallackiidae fam. nov. and restricts the gonostomatid-type oral apparatus to Gonostomatidae, Chaetospiridae, and Wallackiidae fam. nov.