Ciliate SSU-rDNA reference alignments and trees for phylogenetic placements of metabarcoding data File S1: Description of the relationships within the main ciliate lineages in the reference trees Ľubomír Rajter, Micah Dunthorn KAYORELICTEA encompasses around 170 morphospecies belonging to 6 families (Cryptopharyngidae, Geleiidae, Kentrophoridae, Loxodidae, Trachelocercidae, and Wilbertomorphidae) (Lynn 2008). Those families represent the main molecular clades in recent SSU-rRNA karyorelictean phylogenetic studies (e.g., Campello-Nunes et al., 2015; Xu et al., 2015, 2013a, 2013b; Yan et al., 2016). The only exception is Cryptopharyngidae with an unknown evolutionary position, as molecular data for this family are missing. This class was monophyletic in the unconstrained tree with high statistical support. We also recognized all five main karyorelictean clades/families with the consistent topology in all our trees. HETEROTRICHEA is a diverse class consisting of ten monophyletic families (Shazib et al. 2014). Each of this family represents well-supported distinct clade in heterotrichean SSU- rRNA phylogeny (Schmidt et al. 2007, Fernandes et al. 2016, Chen et al. 2020). In our trees were all the heterotrichean families also revealed monophyletic. Specifically, the family Peritromidae branched off first in all our trees. Climacostomidae was a sister to Spirostomidae in all trees except the constrained masked tree, where Climacostomidae was a sister to Condylostomatidae. The next branched off a lineage that united four smaller heterotrichean families (Fabreidae, Folliculinidae, Gruberiidae, and Maristentoridae) with two crown species-rich families Blepharismidae and Stentoridae. This topology was also revealed by Shazib et al. (2014) based on multiple genes phylogenetic analyses. SPIROTRICHEA is one of the most diverse Ciliophora class containing three main clades – Euplotia, Hypotrichia, Oligotrichea. Relationships between these three clades had rather paraphyletic character in all our trees. Specifically, the clade Euplotia contained Hypotrichia+Oligotrichea and Hypotrichia contained monophyletic Oligotrichea. This topology is also common in other spirotrichean phylogenetic studies (Kim et al. 2005, Lyu et 1 Ciliate SSU-rDNA reference alignments and trees for phylogenetic placements of metabarcoding data al. 2018, Song et al. 2015). Inconsistent is only the clade Euplotia which is mostly monophyletic (Gao et al 2016), but becomes paraphyletic if sequences from Diophrys genera are included (Gao et al. 2014, this study). Nevertheless, the nodes defining the relationships between these main clades had only low statistical support in the unconstrained tree. Only well supported deep-nodes were for the Oligotrichean’s monophyly (96 bootstrap value) and monophyly of the whole Spirotrichea class (88 bootstrap value). Except for these three core spirotrichean clades, we also included in Spirotrichea two other groups – Licnophoridae and Kitrichidae – following the Adl et al. (2019) system. The affinity of these groups to the class Spirotrichea is still discussed. In all our constrained trees, Licnophoridae and Kitrichidae were monophyletic and branched off first within sprirotricheans, although they were forced to be within the class. In the unconstrained tree, Kitrichidae was in the vicinity of the class Spirotrichea, but Licnophoridae branched outside of Spirotrichea as the earliest branch of the whole SAL lineage. ARMOPHOREA (incl. Cariacotrichea, Muranotrichea and Parablepharismea). This class encompassed four main lineages – Caenomorphidae, Odontostomatida, Metopida, and Clevelandellida – based on the Adl et al. (2019) system. As in our trees are also in the close vicinity of Armophorea clades Cariacotrichea, Muranotrichea, and Parablepharismea, we discuss those here as well. Cariacotrichea always grouped with Parablepharismea and Muranotrichea, creating a sister clade to Armophorea in all our trees. This grouping was also inferred in Rotterová et al. (2020), where the authors described these two new classes: Muranotrichea and Parablepharismea. Within the class Armophorea, Caenomorphidae branched off first in constrained unmasked alignment or they represented a sister group to the Parablepharismidae+Muranotrichidae+Cariacotrichea clade in our masked and V4 unmasked alignments. In the unconstrained tree, Caenomorphids were sister to Litostomatea but only with low statistical support. But this is not unusual as caenomorphids branch outside of the Armophorea in all recent molecular studies (Campello-Nunes et al. 2020, Fernandes et al. 2018 and reference therein, Rotterová et al. 2020), so their definitive phylogenetic home has not been resolved yet. The phylogenetic position of Odontostomatida is stable in all our trees, always as sister to Armophorea within APM-clade, same as in other molecular studies (e.g., 2 Ciliate SSU-rDNA reference alignments and trees for phylogenetic placements of metabarcoding data Campello-Nunes et al. 2020). It’s worth mentioning that Fernandes et al. (2018) recently proposed odontostomatids as separate class, but we follow the Adl et al. (2018) system where they belong to Armophorea. Metopida contained all its families (Apometopidae, Metopidae, and Tropidoatractidae), but it is paraphyletic as it also encompassed monophyletic Clevelandellida. This result corresponds with other recent molecular studies (e.g., Bourland et al. 2020). LITOSTOMATEA contains two free-living subclasses (Rhynchostomatia and Haptoria) and an endosymbiotic subclass (Trichostomatia). But many litostomatean taxa also have an uncertain phylogenetic position, and intraclass phylogeny is not entirely resolved yet (Vďačný et al. 2014). The phylogenetic position of enigmatic Mesodiniidae is discussed in the incertae sedis part below. In all the trees, branched off the first two long-branched litostomatean taxa: Helicoprorodon and then Chaenea. Lacrymariida and Rhynchostomatia clustered together in all the trees except the V4 unmasked alignment in which Lacrymariida clustered with Homalozoon, and Rhynchostomatia represented a separate clade. But the bootstrap support for Lacrymariida+Rhynchostomatia grouping is low (39) in the unconstrained tree. Didiniida and Pleurostomatida grouped together in all trees except the masked alignment tree in which are both as distinct clades, but the statistical support for sister relationship of these two clades is low (33) in the unconstrained tree. Finally, two haptorian species-rich orders Spathidiida and Haptorida are mixed and contained endosymbiotic subclass Trichostomatia as in all the other phylogenetic studies (e.g., Vďačný et al. 2014). Within Trichostomatia branched off first two entodiniomorphids groups (Spirodiniidae and Buetscheliida) and then Vestibulifera that contained Macropodiniida. The crown trichostomatian groups consisted of the rest of the entodiniomorphids (i.e., Isotrichidae, Blepharocorythina, Polydiniellidae, Spirodiniida, Troglodytellidae, Cycloposthiidae, and Ophyoscolecidae). COLPODEA. All its four main lineages - Platyophryida, Bursariomorphida, Cyrtolophosidida, and Colpodida - were recognized in all constrain trees. The only difference 3 Ciliate SSU-rDNA reference alignments and trees for phylogenetic placements of metabarcoding data was in the topology. Platyophryida branched off first, then Bursariomorphida as a sister to Cyrtolophosidida+Colpodida in both unmasked alignments. On the contrary, Bursariomorphida branched off first, then Platyophryida as a sister to Cyrtolophosidida+Colpodida in masked and V4 unmasked alignments. Though both topology scenarios are plausible (Vďačný and Foissner 2019), platyophryids-branching-first topology has slightly more molecular support (Rajter et al. 2020). OLIGOHYMENOPHOREA. Oligohymenophorea is a diverse class that encompasses six main lineages: Apostomatia, Astomatia, Hymenostomatia, Peniculia, Peritrichia, and Scuticociliatia. Relationships among these main lineages varied based on the masking strategy used. We can also see in the unconstrained trees very low bootstrap support for nodes defining these relationships. The monophyly of Peniculia had full-statistical support but excluding Urocentrum turbo that cluster outside of this clade in all our trees. Peritrichia is bifurcated to its two primary groups – Mobilida and Sessilida – in all our trees. The statistical supports for the monophyly of both groups are high (96, 100) although the support for the monophyly of Peritrichia itself is low (65). This could by the reason that in the masked alignment are Mobilida and Sessilida not grouped together. Astomatia is monophyletic with moderate statistical support (90) although the Haptophrya planarium and Durchoniella brasili may clustered outside (see the trees from masked and V4 unmasked alignments). Apostomatia and Hymenostomatia were always monophyletic with full-statistical support in unconstrained tree. Finally, Scuticociliatia was non-monophyletic and got only low statistical support in the unconstrained tree. But their three main lineages – Loxocephalida, Pleuronematida and Philasterida – were recognized in all our trees and had moderate to full support (91, 95, and 100) in the unconstrained tree. NASSOPHOREA. Nassophorea represents a smaller group compare to other ciliate classes and contains two main lineages – Nassulida and Microthoracida. We forced these two lineages to cluster together as they always represent separate clades in the SSU-rRNA phylogenies. In our masked and V4 unmasked alignments was Nassulida paraphyletic