Elsevier

Protist

Volume 163, Issue 3, May 2012, Pages 415-434
Protist

Original Paper
COI Barcoding of Nebelid Testate Amoebae (Amoebozoa: Arcellinida): Extensive Cryptic Diversity and Redefinition of the Hyalospheniidae Schultze

https://doi.org/10.1016/j.protis.2011.10.003Get rights and content

We used Cytochrome Oxidase Subunit 1 (COI) to assess the phylogenetic relationships and taxonomy of Nebela sensu stricto and similar taxa (Nebela group, Arcellinida) in order to clarify the taxonomic validity of morphological characters. The COI data not only successfully separated all studied morphospecies but also revealed the existence of several potential cryptic species. The taxonomic implications of the results are: (1) Genus Nebela is paraphyletic and will need to be split into at least two monophyletic assemblages when taxon sampling is further expanded. (2) Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, and the mixotrophic Hyalosphenia papilio branch within the Nebela group in agreement with the general morphology of their shell and the presence of an organic rim around the aperture (synapomorphy for Hyalospheniidae). We thus synonymise Hyalospheniidae and Nebelidae. Hyalospheniidae takes precedence and now includes Hyalosphenia, Quadrulella (previously in the Lesquereusiidae) and all Nebelidae with the exception of Argynnia and Physochila. Leptochlamys is Arcellinida incertae sedis. We describe a new genus Padaungiella Lara et Todorov and a new species Nebela meisterfeldi n. sp. Heger et Mitchell and revise the taxonomic position (and rank) of several taxa. These results show that the traditional morphology-based taxonomy underestimates the diversity within the Nebela group, and that phylogenetic relationships are best inferred from shell shape rather than from the material used to build the shell.

Introduction

Free-living protists make up a large part of the Earth's biodiversity (Medinger et al. 2010) and are of major ecological importance at the global scale (Adl and Gupta 2006). The vast majority of this diversity is, however, not yet described (Piganeau et al. 2011) and existing descriptions are often imprecise (Adl et al., 2008, Caron, 2009). Yet reliable taxonomy is an essential prerequisite for understanding the ecology, biogeography, and evolution of any group of organisms. Unfortunately, poor taxonomy is one of the curses of the study of free-living protists, leading, for instance, to endless debates about the existence of biogeographical patterns in the distribution of free-living protists (Finlay et al., 2004, Foissner, 2008, Heger et al., 2009, Mitchell and Meisterfeld, 2005).

DNA-based studies often show that traditional taxonomy underestimates diversity of both macroscopic and microscopic organisms (Harper et al., 2009, Hebert et al., 2004a, Hebert et al., 2004b). Due to a lower taxonomic effort and the lack of easily recognized morphological features, the expectation is that the amount of cryptic diversity (i.e. genetic diversity that is not reflected in observable morphological features) in microscopic organisms is very high.

Arcellinid amoebae are a good model for taxonomy and evolutionary studies of free-living protists because of their diversity, abundance and taxonomically diagnostic shell. The distinct ecological requirements of testate amoebae species, including both the arcellinids (Amoebozoa: Arcellinida) and the euglyphids (Rhizaria: Cercozoa: Euglyphida), and the preservation of their shells in peat and sediments make them good bioindicators for palaeoecological studies and environmental monitoring (Charman 2001). In addition, testate amoebae were shown to play important roles in the cycling of carbon, nitrogen and silica in terrestrial ecosystems (Aoki et al., 2007, Schröter et al., 2003, Wilkinson, 2008). However, as for most protists, data on total biodiversity, geographic distribution, morphology, phylogeny and ecology of this group of organisms are still very incomplete and controversial.

Our focus here is on a group of arcellinid testate amoebae including the “core Nebelas” sensu Lara et al. (2008) and most closely related taxa (i.e. the clade containing Apodera vas Certes, 1889 and Nebela lageniformis Penard, 1890), hereafter referred to as the “Nebela group”. This group contains some of the most remarkable and common species of testate amoebae, including both easily identifiable species, and problematic species-complexes. Members of this group are especially abundant in mosses and forest litter, and more rarely in other biotopes such as freshwater pools, etc. (Meisterfeld, 2002, Todorov, 2002). The classification of this group is based on characters of the test such as composition (proteinaceous or agglutinated), shape of the aperture (circular, oval or curved) and shape of the shell (mostly flask-shaped but more or less elongated and in some cases with appendages, a keel, horns etc.). The classification of the Nebela group has changed considerably over time (Fig. 1), mainly depending on which morphological trait has been considered as phylogenetically most relevant at the different taxonomical levels.

Molecular tools now make it possible to reassess the validity of this taxonomy. However until now only one study has examined the phylogeny of the Nebela group based on molecular methods (SSU rRNA), but with very partial coverage of the described morpho-species (Lara et al. 2008). This study showed that the Nebelidae sensu Meisterfeld (2002) was paraphyletic as Argynnia dentistoma Penard,1890 appeared only distantly related to members of genus Nebela Leidy, 1874. In addition, members of genera Apodera Loeblich and Tappan, 1961, Hyalosphenia Stein, 1859, Nebela and Porosia Jung, 1942, were intermingled in a robust clade informally called “core Nebelas”. However, the species delineations and the phylogenetic relationship between members of the “core Nebelas” remained unclear, partly because of under-sampling and partly because these close-related species could hardly be discriminated on the basis of the less variable SSU rRNA gene. We therefore investigated the species delineations and the phylogenetic relationships within genus Nebela and related taxa based on mitochondrial cytochrome oxydase gene subunit 1 (COI) sequences. This marker is commonly used for DNA barcoding in animals (Hebert et al., 2003a, Hebert et al., 2003b) and has been shown to be well suited for delimiting species of ciliates, dinoflagellates, vannellid amoebae or euglyphid testate amoebae (Barth et al., 2006, Chantangsi et al., 2007, Heger et al., 2010, Lin et al., 2009, Nassonova et al., 2010). Our data confirm the usefulness of COI sequences for taxonomic studies of certain Arcellinida species. In addition our combined molecular and morphological results lead us to propose several nomenclatural changes.

Section snippets

Results

A total of 59 sequences were obtained from 24 morphospecies, most of which were also characterized by scanning electron and light microscopy. The fragment lengths ranged from 308 bp to 668 bp. Only 6 sequences were shorter than 560 bp (Table 1). COI separated efficiently the different morphospecies, including closely related ones and our molecular data suggest the presence of cryptic species within several morphospecies.

The results of our phylogenetic reconstructions are shown in Figure 2.

Discussion

The use of molecular markers offers a way to reassess the validity of taxonomic systems based on morphology and provides new criteria for species discrimination. Molecular taxonomy has revealed the presence of a large cryptic or pseudo-cryptic diversity (Hebert et al., 2004b, Heger et al., 2011a, Kolisko et al., 2010) while molecular phylogeny and phylogenomics have led to major revisions in the classification of most groups of organisms (Baldauf, 2003, Burki et al., 2008). In this study we

Methods

Sampling and species isolation: Testate amoebae were obtained from Sphagnum, other mosses and forest litter collected from different geographical sites (Table 1). They were extracted by sieving and back sieving using appropriate mesh size and isolated individually with a narrow diameter pipette under the dissecting microscope. Cells were rinsed with demineralized water. We characterized the morphology of these distinct “populations” (=individuals of a given morphospecies isolated from one

Acknowledgements

This work was funded by Swiss NSF projects n° 205321-109709 / 1 & 2 to E. Mitchell; Science and Technology Cooperation Program Switzerland - Russia grant IZLR Z3_128338 to E. Mitchell & E. Lara; Swiss NSF Ambizione grant n° PZ00P2_122042 to E. Lara, Swiss NSF PBELP2-122999 to T. Heger; and the National Science and Engineering Research Council of Canada (NSERC 283091-09) to B. Leander. T. Heger and B. Leander are also supported by the Tula Foundation's Centre for Microbial Diversity and

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