Two new species of Ripella (Amoebozoa, Vannellida) and unusual intragenomic variability in the SSU rRNA gene of this genus

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Abstract

Two new species, Ripella decalvata and R. tribonemae (Amoebozoa, Vannellida), are described and the diversity of known strains assigned to the genus analyzed. Ripella spp. are closely similar to each other in the light microscopic characters and sequences of small-subunit (SSU) ribosomal RNA gene, but differences in the cell coat structure and cytochrome oxidase (COI) gene sequences are more prominent. SSU rRNA in R. platypodia CCAP1589/2, R. decalvata and R. tribonemae demonstrates an unusual pattern of intragenomic variation. Sequencing of multiple molecular clones of this gene produced numerous sequence variants in a number of specific sites. These sites were usually terminal parts of several variable helices in all studied strains. Analysis of all known Ripella strains shows that SSU rRNA sites differing between strains of different origin are mainly restricted to these areas of the gene. There are only two sites, which differ between strains, but not within genomes. This intragenomic variability of the SSU rRNA gene, seemingly characteristic of all Ripella spp., was never reported to be so extensive in Amoebozoa. The data obtained show another example of complex organization of rRNA gene cluster in protists and emphasize caution needed when interpreting the metagenomic data based on this marker.

Introduction

The genus Ripella was first established by Smirnov et al. (2007) within the family Vannellidae, however its first member was described more than a century ago. Initially, the species was named Amoeba platypodia (Gläser 1912). It was later transferred into a newly created genus Vannella (Bovee 1965), however, this point of view was not unanimously supported, as Page (1968) first included this species in the genus Flabellula, and only later (Page 1976) started to consider it to be a member of Vannella. This amoeba was considered to be typical yet small freshwater Vannella due to its glycocalyx represented by the pentagonal glycostyles and simple filaments (Page and Blakey 1979), a typical star-shaped floating form with tapering pseudopodia and the change of direction of locomotion by formation of the two temporary parts of the frontal hyaline area moving in opposite directions (summarized in Page 1983). The molecular investigations of the family Vannellidae demonstrated that Vannella platypodia formed a long, deeply branching clade within the Vannellidae together with several unnamed isolates, and a separate genus Ripella was established to accommodate it (Smirnov et al. 2007). Unfortunately, too few members of this genus have been morphologically characterized until now. In spite of the presence of 5 unnamed isolates within a clade corresponding to Ripella (Dyková et al., 1996, Dyková et al., 2005, Dyková et al., 2010, Dyková and Kostka, 2013), the genus has been remaining formally monotypic until now, and its diagnosis was mainly based on the characters of R. platypodia. Therefore, it contains very few differentiating morphological details, and outlines the group most reliably based on small-subunit ribosomal RNA gene sequence characteristics. In particular, it has been shown that members of the genus have an unusually short SSU rRNA gene with a number of deletions (Smirnov et al. 2007).

The purpose of this paper is to describe two new Ripella species and analyze unusual pattern of variability in their small-subunit (SSU) ribosomal RNA gene, which seems to be characteristic for the whole genus.

Section snippets

Amoebae isolation, culturing and microscopy.

Three strains of Ripella were investigated during this study: (1) R. platypodia CCAP 1589/2 originally isolated by F.C. Page (Page 1968); (2) R. decalvata n. sp. Negev 3-2c, isolated from a sample of bottom sediment picked from the freshwater habitat in Ein Yorke’am (Negev desert, Israel; N30.938562 E35.04087) in 2011; (3) R. tribonemae n. sp. SK1 isolated from the culture of a green alga Tribonema sp. kindly provided by Prof. S. Karpov (St-Petersburg State University). The latter strain was

Light microscopic morphology of the living amoebae

Both species demonstrated very similar morphology when studied with the light microscope, we therefore provide a common description paying special attention to the differences in details between the two strains. During locomotion amoebae adopted a fan-shaped morphotype (Smirnov and Brown 2004), and were rounded, fan-shaped or elongated triangular in above view. The anterior margin of the locomotive forms was always arc-shaped, while the lateral and posterior margins could be convex or straight.

Identification of the studied species

An assignment of the new strains to the genus Ripella is fairly easy, as they all form a single, robust clade in the molecular phylogenetic tree of Amoebozoa based on the SSU rRNA gene. This clade includes also previously sequenced R. platypodia and a number of unnamed strains previously studied and illustrated by Dyková et al., 1996, Dyková et al., 2005, Dyková et al., 2010, and Dyková and Kostka (2013). SSU rRNA gene of the studied amoebae shared features characteristic of the genus Ripella,

Taxonomic appendix

Amoebozoa: Discosea: Flabellinia: Vannellida: Vannellidae Bovee, 1970.

Genus Ripella Smirnov et al., 2007. Emended diagnosis: Fan-shaped, spatulate, or discoid amoebae, usually small and rapidly moving, without any ridges or waves on the dorsal surface and always with smooth outlines. 18S rRNA gene markedly shorter than in Vannella spp. and most other Amoebozoa due to deletions in the regions V2 causing shortening of helices 8/e1-10 and absence of helix 10/e1, and V4 causing absence of helices

Acknowledgments

We are grateful to Prof. Sergei Karpov (Saint-Petersburg State University) for providing sample containing Ripella tribonemae and Prof. Jan Pawlowski (University of Geneva) for collecting the sample containing R. decalvata. R. platypodia CCAP 1589/2 was kindly provided by Undine Achilles-Day (Culture Collection of Algae and Protozoa, UK). This study utilized the equipment of the Core Facility Centres “Culture Collection of Microorganisms” and “Development of Molecular and Cell Technologies” of

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