Large-scale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages

https://doi.org/10.1016/j.ympev.2014.04.020Get rights and content

Highlights

  • New EST data for 11 ciliates.

  • Ciliates are robustly monophyletic.

  • Protocruzia is not a member of Spirotrichea.

  • We place Protocruzia incertae sedis in Ciliophora.

  • Strong association of Spirotrichea, Armophorea and Litostomatea.

Abstract

The Ciliophora is one of the most studied protist lineages because of its important ecological role in the microbial loop. While there is an abundance of molecular data for many ciliate groups, it is commonly limited to the 18S ribosomal RNA locus. There is a paucity of data when it comes to availability of protein-coding genes especially for taxa that do not belong to the class Oligohymenophorea. To address this gap, we have sequenced EST libraries for 11 ciliate species. A supermatrix was constructed for phylogenomic analysis based on 158 genes and 42,158 characters and included 16 ciliates, four dinoflagellates and nine apicomplexans. This is the first multigene-based analysis focusing on the phylum Ciliophora. Our analyses reveal two robust superclades within the Intramacronucleata; one composed of the classes Spirotrichea, Armophorea and Litostomatea (SAL) and another with Colpodea and Oligohymenophorea. Furthermore, we provide corroborative evidence for removing the ambiguous taxon Protocruzia from the class Spirotrichea and placing it as incertae sedis in the phylum Ciliophora.

Introduction

Over the past few years, phylogenomic approaches have been employed to untangle deep relationships among major microbial eukaryotic lineages and place divergent taxa of evolutionary significance (Brown et al., 2012, Brown et al., in press, Burki et al., 2007, Burki et al., 2009, Burki et al., 2012, Hampl et al., 2009, Parfrey et al., 2010, Zhao et al., 2012). Large-scale phylogenomic analyses are now being utilized to resolve questions associated with the shallower nodes of the eukaryotic tree of life (Bachvaroff et al., 2011, Burki et al., 2010). One protistan group where such analyses have never been performed is the phylum Ciliophora. This is mainly because sufficient data exist for only a limited number of taxa (Abernathy et al., 2007, Aury et al., 2006; Ricard et al., 2008, Swart et al., 2013). Consequently, phylogenetic inference has been based largely on the 18S ribosomal RNA; however, a single locus is insufficient to infer robust phylogenetic relationships (Gribaldo and Philippe, 2000). Thus, several evolution and taxonomy related questions remain unresolved.

The ciliate tree is divided in two deep lineages – the Postciliodesmatophora and Intramacronucleata, a split that is supported by both molecular and morphological lines of evidence (Baroin-Tourancheau et al., 1998, Embley et al., 1995, Gao et al., 2010, Hirt et al., 1995, Lynn, 1996, Lynn, 2003). Beyond this deep division, it is generally agreed that there are 11 major ciliate lineages or classes and a twelfth single species clade of Cariacotrichea (Adl et al., 2012, Lynn, 2008, Orsi et al., 2012, Stoeck et al., 2003). Most of these classes are strongly supported by morphology, with the exception of the “riboclasses” – the Armophorea and Plagiopylea, which are identified only by sequences of the 18S rRNA genes, as included taxa lack any morphological synapomorphies (Bernhard et al., 1995, Bernhard et al., 2001, Cameron et al., 2001, Embley et al., 1995, Greenwood et al., 1991, Leipe et al., 1994, Lynn, 2008, Lynn et al., 1999, Lynn and Strüder-Kypke, 2002, Snoeyenbos-West et al., 2004, Stechmann et al., 1998). Nevertheless, the monophyly of the class Spirotrichea has been challenged in several studies, notably those that include Protocruzia spp. While Protocruzia has been formally assigned to the Spirotrichea (Lynn, 2008), its phylogenetic position remains as one of the most ambiguous since it is rarely recovered with the Spirotrichea (Bernhard and Schlegel, 1998, Shin et al., 2000, Song and Wilbert, 1997). Some researchers have suggested that Protocruzia be assigned its own independent lineage status (Li et al., 2010).

The phylogenetic relationships between the classes of the phylum generally are uncertain, although there is a robust clustering of Colpodea, Oligohymenophorea, Nassophorea, Prostomatea, Plagiopylea and Phylopharyngea, named CONthreeP (Adl et al., 2012, Lynn, 2008). The class Spirotrichea is of uncertain affiliation making it an orphan lineage in the ciliate tree, though some studies do recover a moderately supported cluster with the Litostomatea and Armophorea (Da Silva Paiva et al., 2013, Riley and Katz, 2001, Vd’ačný et al., 2010).

To examine whether the relationships between some of the classes differ to those recovered by 18S rRNA phylogenies and to elucidate the phylogenetic position of Protocruzia, we increased both taxon and character sampling by obtaining RNAseq data from 11 ciliate taxa. Some of these taxa belong to ciliate lineages for which only limited data are available, namely the Colpodea, Litostomatea and Heterotrichea. Phylogenomic analyses of 158 genes show maximally supported groupings of Colpodea + Oligohymenophorea and Spirotrichea + Armophorea + Litostomatea. Furthermore, our study illustrates that Protocruzia is not a spirotrich, though it remains unclear, if it is an independent lineage or a member of the Heterotrichea.

Section snippets

Identification, isolation and culturing

Details regarding identification, isolation and culturing of individual organisms are provided in Supplementary Information section, Appendix A.

RNA extraction, cDNA libraries, Illumina sequencing, EST clustering and annotation

Details regarding RNA extraction from individual organisms are provided in Supplementary Information section, Appendix A.

Poly(A)+ RNA was isolated and cDNA libraries with an insert size of ∼200 bp were constructed according to the standard protocol of the National Center for Genome Resources (NCGR, http://ncgr.org). The cDNA libraries were then sequenced

Results and discussion

In the present study, we increased the taxonomic breadth of the ciliate clade by integrating data from 11 ciliates for which EST data were not available previously with five pre-existing genomic datasets. Our taxon sampling covered six major ciliate groups – Heterotrichea, Colpodea, Oligohymenophorea, Litostomatea, Armophorea and Spirotrichea. We assembled a 158-gene dataset containing 42,158 amino acid positions. This is the largest “ciliate-based” phylogenetic dataset assembled to date in

Conclusions

Our aims in this first phylogenomic analysis of major clades of the phylum Ciliophora were to confirm the monophyly of the group, to resolve the phylogenetic position of the cytologically unusual ciliate Protocruzia, and to explore the deeper relationships within the phylum. We have vigorously confirmed the monophyly of the Ciliophora in agreement with ultrastructural, rRNA gene studies and some protein gene sequences. We postulate that Protocruzia is not a spirotrich, but its exact position

Acknowledgments

This research was funded in part by the Gordon and Betty Moore Foundation through Grant #2637 to the National Center for Genome Resources. Samples MMETSP0125-20120918 (Aristerostoma sp.), MMETSP0210-20121227 (Condylostoma magnum), MMETSP0205-20121125 (Euplotes focardii), MMETSP0213-20121227 (Euplotes harpa), MMETSP0209-20121228 (Litonotus sp.), MMETSP0127-20121128 (Platyophrya macrostoma), MMETSP0216-20120918 (Protocruzia adherens), MMETSP0211-20121228 (Pseudokeronopsis riccii),

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