Phylogenetic position of Protoopalina intestinalis based on SSU rRNA gene sequence

https://doi.org/10.1016/j.ympev.2004.05.009Get rights and content

Abstract

A robust recognition of phylogenetic affinities of Opalinidae—the peculiar multinucleated intestine commensals of frogs—is hindered by the absence of reliable molecular data. Up to now all attempts to sequence opalinid genes failed, as the obtained sequences labeled as Protoopalina intestinalis, Cepedea virguloidea, and Opalina ranarum in GenBank apparently originate from a zygomycete contamination. In this paper, we present the first molecular data for the family Opalinidae—SSU rRNA gene of P. intestinalis. Our phylogenetic analyses undoubtedly show opalinids as a sister group to Proteromonas within the Stramenopila clade, confirming the monophyly of Patterson’s order Slopalinida. The enigmatic genus Blastocystis is resolved with high statistical support as a sister group to Slopalinida. The information contained in the SSU rRNA gene proved insufficient to uncover broader affinities of this group to other groups of Stramenopila. Nevertheless, our analyses clearly demonstrate that Cavalier-Smith’s phylum Bigyra, which comprises Oomycetes and their relatives together with Slopalinida and Blastocystis, is not monophyletic.

Introduction

Opalinids, first observed by Leeuwenhoek in 1683 (Dobell, 1932), are large (up to 2.8 mm), multinucleated, multiciliated protozoa with unusual morphology and ultrastructure. They are very common in the cloacae of frogs and toads. They are also the representatives of last few higher eukaryotic taxa that were not studied with molecular phylogenetic methods because of lack of DNA sequence data. Four genera of family Opalinidae are separated into two subfamilies (Metcalf, 1923). Subfamily Protoopalininae comprises binucleated genera Protoopalina (with cylindrical cells) and Zelleriella (with flattened cells). The second subfamily—Opalininae—comprises multinucleated genera Cepedea (cylindrical cells) and Opalina (flattened cells).

Opalinids resemble ciliates in having multiple flagella and were for a long time considered to be related to them (e.g., Stein, 1860—after Delvinquier and Patterson, 1993; Metcalf, 1923). In the 1950s, the hypothesis of opalinid–ciliate affinity was abandoned and opalinids were deemed to be either an isolated taxon in the phylum Zooflagellata or were treated as a separate phylum: Opalinata (e.g., Corliss, 1955; Grassé, 1952).

The first hypothesis on opalinid affinity based on reliable morphological data was formulated after a detailed ultrastructural study by Patterson (1985). The pattern of subpelicular microtubules and the structure of basal bodies and flagellar transition zone of opalinids strongly resembled those of Karotomorpha bufonis (Brugerolle and Joyon, 1975). Karotomorpha, the commensal in the frog intestine, is a uninucleated flagellate with four flagella. Genus Karotomorpha and related genus Proteromonas belong to the family Proteromonadidae. Patterson established a new order Slopalinida comprising families Proteromonadidae and Opalinidae. He also suggested a relationship between Slopalinida and heterokont algae due to the similarities in their basal body structures and the proposed homology between the somatonemes of Proteromonas and the mastigonemes of heterokont organisms.

The phylogenetic analysis of Silberman et al. (1996) confirmed the assumption that Proteromonas lacertae belongs to the Stramenopila. Quite surprisingly, the study also revealed that the hitherto enigmatic genus Blastocystis, the aciliated multinucleated gut parasite of both vertebrates and invertebrates with a spherical cell and a large central vacuole, is the sister group of Proteromonas.

These results and similarities in the structure of basal bodies and flagellar transition zone led Cavalier-Smith to postulate a new phylum Bigyra in the kingdom Chromista. The phylum Bigyra comprises Slopalinida, Blastocystis, Oomycetes, Hyphochytrea, and Developayella (Cavalier-Smith, 1997, Cavalier-Smith, 1998).

Although there are quite strong morphological indications of opalinid affinities to Stramenopila, we are still unable to confirm the hypotheses of Patterson and Cavalier-Smith on the basis of sequence data. The inclusion of opalinids in Stramenopila is based merely on their similarities in the structure of basal bodies and on the proposed relationship between opalinids and the family Proteromonadidae. The information contained in the SSU rDNA sequence of opalinids would not only clarify the phylogenetic position of the group, but could also elucidate the evolutionary history of the group Stramenopila.

Here we report the sequence of the SSU rRNA gene of Protoopalina intestinalis. We also demonstrate the phylogenetic affinities of opalinids and we examine the proposed monophyly of the order Slopalinida and the class Bigyra.

Section snippets

DNA isolation, SSU rDNA amplification, and sequencing

Opalinids of the species P. intestinalis were isolated with a Pasteur pipette from the cloaca of a fire-bellied toad (Bombina bombina). The isolate contained numerous specimens of Protopalina, diplomonads, bacteria, and a few ciliates. Protoopalina cells were then separated from most of the other eukaryotes and bacteria by filtration through sterile filter paper (pore size ∼10 μm). The filter was rinsed with 10 ml of physiological solution and the material on the upper side of the filter was

Results and discussion

We determined 1751 bp of P. intestinalis SSU rRNA gene. The G-C content is quite low: 34.6%. The sequence contains many unique A-T rich regions especially in those positions that are known to vary among different eukaryotic taxa.

Analysis of the data set containing main eukaryotic groups showed that P. intestinalis belonged to the Stramenopila group (Fig. 2). All methods supported this result with high bootstrap values (ML 93%, MP 87%, and LogDet 98%). The relatively low posterior probability

Acknowledgment

This work was supported by grants J13/98113-B4 and 269/2004/B-BIO/PrF.

References (23)

  • C. Dobell

    Anthony van Leeuwenhoek and his ‘Little Animals’

    (1932)
  • Cited by (0)

    View full text