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Morphology and phylogeny of Vannella croatica n. sp. (Amoebozoa, Discosea, Vannellida)
Accepted Manuscript Title: Morphology and phylogeny of Vannella croatica n. sp. (Amoebozoa, Discosea, Vannellida) Author: Alexey V. Smirnov Natalya Bondarenko A. Glotova Elena Nassonova PII: DOI: Reference:
S0932-4739(15)00097-8 http://dx.doi.org/doi:10.1016/j.ejop.2015.11.002 EJOP 25399
To appear in: Received date: Revised date: Accepted date:
27-7-2015 16-10-2015 2-11-2015
Please cite this article as: Smirnov, A.V., Bondarenko, N., Glotova, A., Nassonova, E.,Morphology and phylogeny of Vannella croatican. sp. (Amoebozoa, Discosea, Vannellida), European Journal of Protistology (2015), http://dx.doi.org/10.1016/j.ejop.2015.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Morphology and phylogeny of Vannella croatica n. sp. (Amoebozoa,
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Discosea, Vannellida)
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Alexey V. Smirnova, *, Natalya Bondarenkoa, A. Glotovaa, Elena Nassonovaa,b
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State University, Universitetskaja nab. 7/9, 199034 St. Petersburg, Russia
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bLaboratory
ave. 4, 194064 St. Petersburg, Russia
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of Cytology of Unicellular Organisms, Institute of Cytology RAS, Tikhoretsky
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Department of Invertebrate Zoology, Faculty of Biology and Soil Sciences, St. Petersburg
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*Corresponding author at : Department of Invertebrate Zoology, Faculty of
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Biology and Soil Sciences, Saint Petersburg State University, Universitetskaya nab. 7/9,
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199034, Saint Petersburg, Russia.
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E-mail address: [email protected] (A.V. Smirnov)
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Abstract We isolated and described a new species of freshwater vannellid amoeba from Krka natural reserve in Croatia ─ Vannella croatica n. sp. This species has certain
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morphological differences from all known vannellids and differs at the level of SSU
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sequence. It resembles in size and morphology Vannella lata; to facilitate direct
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comparison we publish images of the type strain of V. lata CCAP 1589/12 strain (type
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strain, which is now lost) taken in 1999. Vannela croatica feeds on bacteria and can be
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easily grown in large amount in relatively pure culture and thus is suitable for
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molecular and biochemical studies requiring large amounts of material.
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Introduction
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The genus Vannella belongs to the family Vannellidae, order Vannellida, subclass Flabellinia of the class Discosea (Smirnov et al. 2011). It unifies flattened
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amoeba of fan-shaped morphotype with a large frontal area of hyaloplasm (Smirnov
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and Goodkov 1999; Smirnov and Brown 2004). Amoebae of the genus Vannella are
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widely distributed and are among the most common organisms in environmental
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samples, however species diversity within this genus requires further attention.
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Vannellid amoebae have a low number of morphological characters appropriate for
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morphospecies distinction (Page 1976; 1983; 1988; 1991). As a result, the number of
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species, which can be identified at the morphological level is low; in fact, only isolates
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possessing remarkable distinctive characters can be reliably identified or described as
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a new species (Smirnov 2001; 2002). Application of molecular methods allows
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researchers to differentiate vannellid amoebae more precisely and discover several
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new genera of smallest vannellid amoeba best recognised at the molecular level
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(Peglar et al. 2003; Smirnov et al. 2007; Kudryavtsev 2014). It is now clear that
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molecular data are essential to identify or to describe a new species of vannellids;
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remarkable species possessing clear differentiating characters are very rare, and with
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the growth of the number of described species boundaries at the morphological level
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are getting less reliable (Smirnov 2001; Smirnov et al. 2007; Nassonova et al 2010;
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Kudryavtsev 2014).
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In the present paper we provide morphological and molecular data on a new
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species of Vannella – V. croatica n. sp. This species feeds on bacteria and can be easily
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grown in large amount in relatively pure culture and thus is suitable for molecular and
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biochemical studies requiring large amounts of material. It resembles in size and
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morphology Vannella lata; to facilitate direct comparison we publish images of the type
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strain of V. lata CCAP 1589/12.
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Material and Methods
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Samples containing Vannella strain were collected from a freshwater pond in
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Krka natural reserve (Lozovac, Croatia, grid reference 43.801981, 15.965470). Ca. 35
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ml of water containing the top layer of bottom sediments was sampled with a sterile
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Falcon tube. For enrichment cultivation, ca. 500 mg of the sampled material were
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placed in 90 mm Petri dishes filled with artificial medium made by adding vacuum-
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dried wheat grass powder (Weizengras, Sanatur GmbH, D-78224 Singen) to PJ medium
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(Prescott and James 1955) to the final weight concentration of 0.05%. After 7-10 days
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of incubation samples were examined using a Nikon TMF100 inverted phase-contrast
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microscope; detected cells were transferred to fresh medium using tapering Pasteur
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pipette. Strains were cloned, subcloned twice and further cultured in 60 mm Petri
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dishes using the same medium. Clone labelled KRKA 29.9.7.6.1 was selected for further
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studies; it was further subcloned into three clones used in the present work.
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Living trophozoites and cysts were observed, measured and photographed using
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a Nikon TMF100, Leica DM2500 and Leica DMI3000 microscopes equipped with phase
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contrast and DIC optics. Trophozoites were measured on the plastic surface of the Petri
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dish using a 40x objective lens; nuclei were measured on the glass surface of the object
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slides using 100x objective, amoebae were not touched with the coverslip during
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measurements. In order to obtain SSU sequences single cells of amoebae were isolated
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using tapering Pasteur pipettes, washed twice in Millipore 0.2 µm sterilized culture Page 4 of 23
6 medium (freshly made pipette used each time), collected with minimal possible
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amount (1-2 µl) of medium and placed in 200 µl PCR tubes. Tubes were subsequently
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exposed to several rapid freezing-defreezing cycles (4-5 cycles from -180C to room
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temperature), ready PCR mixture was topped on the tube content to the final volume of
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50 µl. Thermal cycle parameters were the following: initial denaturation 10 min. at
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95°C, followed by 40 cycles of 30 s at 94°C, 60 s at 50°C and 120 s at 72°C and
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completed by 10 min at 72°C for final extension. Amplicons were purified using
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Cleanup Mini Purification Kit (Eurogene) and sequenced directly using ABI-PRISM Big
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Dye Terminator Cycle Sequencing Kit.
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Obtained sequences were manually aligned with representative Amoebozoa-wide
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alignment (224 sequences representing all major clades plus outgroups) and the local
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alignment containing all named species of vannellid amoebae and an outgroup (54
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sequences) using SeaView 4.0 (Gouy et al. 2010). The phylogenetic analysis was performed
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using maximum likelihood method as implemented in PhyML program (Guindon and
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Gascuel 2003) with GTR + G + I model suggested by Modeltest program (Posada and
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Crandall 1998); 1302 sites were selected for analysis with the general amoebozoan
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alignment and 1680 sites for the analysis in the local vannellid alignment; the number of
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invariant sites, alpha parameter and tree topology were optimized by PhyML. Bayesian
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analysis was performed using MrBayes 3.1.2, GTR model with gamma correction for
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intersite rate variation (8 categories) and the covarion model. Trees were run as two
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separate chains (default heating parameters) for 5 million generations, by which time they
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had ceased converging (final average standard deviation of the split frequencies was less
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than 0.01). The quality of chains was estimated using Tracer 1.6 package (Rambaut et al.
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2014); basing on the estimates by Tracer, the first 27% of generations were discarded for
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burn-in. Sequence identity matrix was calculated using BioEdit 7.0.9 (Hall 1999).
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Results Diagnosis Systematic position according to Smirnov et al. (2011)
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Phylum Amoebozoa Lühe 1913 sensu Cavalier-Smith, 1998
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Subphylum Lobosa Carpenter, 1861 sensu Cavalier-Smith, 2009
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Class Discosea Cavalier-Smith 2004 sensu Smirnov et al., 2011
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Subclass Flabellinia Smirnov et al., 2005 sensu Smirnov et al., 2011
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Family Vannellidae Bovee, 1979
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Vannella croatica n. sp.
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Order Vannellida Smirnov et al., 2005
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Diagnosis: Locomotive form semi-circular or fan-shaped, often with pronounced tail. The
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length of the locomotive form 17-55 μm (average 32-37 μm, depending on clone); breadth
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17-53 μm (average 31-39 μm); L/B ratio 0.54 – 1.86 (average 0.97 – 1.02). Nucleus
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vesicular, rounded or ovoid, with compact rounded or ovoid central nucleolus. The size of
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the nucleus in maximal dimension is 3,5 – 4,8 μm (average 4,8 μm), that of nucleolus 2,2-
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3,5 μm (average 2,8 μm). Floating form of radial type, with up to 15 tapering pseudopodia
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of different length.
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Type location: freshwater pond in Krka natural reserve (Lozovac, Croatia, grid reference
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43.801981 N, 15.96547 E)
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Type material: live strain and DNA sample deposited with core facility centre “Culture
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Collection of Microorganisms” of Saint-Petersburg State University under the reference
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name Vannella croatica KRKA 29.9.7.6.1. SSU sequence obtained from the type strain
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deposited with GenBank under the number KT345693.
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Etymology: named after the country of origin – Croatia
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8 Differential diagnosis: Resembles Vannella lata Page, 1988 but differs by having
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considerably higher length/breadth ratio (average of 1.0 in V. croatica compared to 0.6 in
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V. lata) and pronounced tendency to produce “tail” in locomotion. Has higher number of
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pseudopodia in floating form than V. lata. Differs from the type strain of V. lata by SSU
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sequence. Resembles V. miroides Bovee, 1965, but contains no crystals, which, according to
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Bovee (1965) were present in all strains of V. miroides. Vannella croatica also differs from
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this species by having a less regularly shaped floating form.
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Morphological description
Three clones obtained after subcloning of initial one were studied and found to be
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morphologically identical except slight size differences. Locomotive cells observed on the
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plastic surface using inverted phase contrast optics were semi-circular or fan-shaped, with
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pronounced area of the frontal hyaloplasm (Figs 1-2). Cell morphology on the glass surface
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of the object slide did not differ much from that observed in cultures (Figs 3-14). The
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frontal hyaloplasm occupied from 1/2 to 2/3 of the total cell area (Figs 3-10) and often had
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pronounced lateral intrusions (Figs 3,6,9-10,12). The posterior part of the cell was straight
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or semi-circular (Figs 3, 5-10). Often the posterior part of amoeba adhered to the substrate
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while the cell continued to progress forward, hence the cell formed a pronounced tail (Figs
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4,11). The end of this tail later detached and the entire tail was retracted (see plate S1
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showing subsequent stages of the process). The formation of the tail was more frequent in
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amoebae moving on the clear plastic surface rather than in those moving over the bacterial
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biofilm. Sometimes frontal hyaloplasm was slightly clefted, with pronounced depression in
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the frontal area (Fig. 9). Moving cells often formed depressions on the ventral surface of the
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hyaloplasm (Fig. 13) and waves on its dorsal surface (Fig. 14). In some cells a prominent
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longitudinal ridge was formed for a short time. The ridge was never stable for more than
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several seconds. In older cultures cells often formed lobes on the dorsal surface of the
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hyaline area, sometimes 3-6 lobes were simultaneously seen. Morphometric data are
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provided in the Table 1.
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Cells in non-directed movement often had two prominent areas of the hyaloplasm. Slowly, chaotically moving cells often became elongate and were surrounded with the
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hyaline margin from all sides. Stationary cells usually were rounded and had peripheral
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hyaline margin of variable width.
The floating form was of radial type, with 3-15 long, tapering pseudopodia (Figs. 15-
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19). Developed floating forms had rounded, nearly spherical central mass of the cytoplasm
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and 9-15 pseudopodia of different length; the most of them much exceeded the diameter of
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the central cytoplasmic mass (Figs 15-16). Early-stage floating forms had 2-4 conical
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pseudopodia, sometimes with a pronounced conical hyaline base (Fig. 17). In many floating
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forms spiral coiling of the distal part of pseudopodia was observed (Fig. 18). Sometimes all
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pseudopodia of the floating form were curved in one direction, suggestive of a rotating cell
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(Fig. 19).
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compact rounded central nucleolus (Figs 3-4). The size of the nucleus in maximal
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dimension was 3,5 – 4,8 μm (average 4,8 μm), that of the nucleolus - 2,2-3,5 μm (average
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2,8 μm). The single contractile vacuole is usually located in the posterior part of the cell
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(Figs 3, 5, 9, 12), sometimes – right in the tail. The cytoplasm was filled with numerous
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granules 1-2 μm in size and contained numerous optically empty vacuoles. Crystals were
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never seen; few refractive inclusions (like those visible in Figs 3-4 and 9) were occasional
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pieces of external material or tiny plastic fragments phagocytized or temporarily located
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under the cell. In our cultures amoebae did not form cysts.
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Molecular phylogeny The Amoebozoa-wide ML phylogenetic tree (not shown) clearly indicated that
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the present isolate belongs to the genus Vannella. More restricted tree containing all
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named sequences of vannellid amoebae (Fig. 20) shows with high support that the
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present strain forms a separate clade with some other species of the genus Vannella;
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the closest relatives among named species are V. lata CCAP 1589/12 (type strain);
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among others are isolate ATCC 30945 named as V. miroides and an isolate named V.
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miroides strain 9i. The nearest neighbours of this clade are V. epipetala, and V. placida
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CCAP 1565/2. Another neighbour is the strain ATCC 50745 misidentified as V.
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plurinucleolus (see Smirnov et al. 2007). The overall configuration of the tree
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corresponds to the data published earlier (Smirnov et al. 2007), except that with the
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addition of V.oblongata sequence, two strains of V. plurinucleolus (CCAP1565/7 and
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1565/11) are not neighbours anymore. This is congruent with the results of the
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manual analysis of the alignment; these two sequences have considerable difference in
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the positions 1499-1528 (counted in the sequence of V. plurinucleolus CCAP 1565/7
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strain GB number EF051186) and a number of indels differing them in other regions.
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Genera Ripella, Clydonella and Lingulamoeba form highly supported clades each and
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group together; Paravannella minima with medium support appears to be a basal
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group to this clade, not to entire Vannellida.
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Discussion
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The present isolate with no doubts belongs to the genus Vannella Bovee 1965
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both by morphological and by molecular evidences. Its recognisable locomotive and
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floating forms are characteristic for this amoebae genus; molecular phylogeny places
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this isolate deep inside the genus Vannella. Page 9 of 23
11 Among named species of the genus Vannella the present strain most resembles
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the species V. lata Page, 1988. This relatively briefly described and illustrated species
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has similar general appearance and comparable size range. Page (1988) provides the
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following data for V. lata: breadth 24-46 μm (average 33 μm). Length is not provided
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in this description. The indicated breadth of the cell is close to that of the present strain
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and is comparable with dimensions of clones 2 and 3 (Table 1). Clone 1 is generally
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larger. However, Page (1988 p. 74) indicates that in V. lata “breadth is always greater
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than length, never less” and provides L/B ratio 0.5 - 1.0 with average 0.6. This is not
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the case for our strain, where many cells are elongate and possess a pronounced tail;
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L/B ratio may reach 1.82 with average 0.97 – 1.02. The floating form of V. lata is poorly
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illustrated by Page (1988, p. 75 fig. 29J); he noted that the floating form in his species
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usually has 8 tapering pseudopodia, which may be 3 times longer than the diameter of
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the central cytoplasmic mass. The image published by Page (1988) is very different
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from our strain; however it looks like in this case Page did not observe very well-
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developed floating forms. His illustration does not show any radiating tapering
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pseudopodia. In our strain the number of pseudopodia in the developed floating form
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was often 12-15. Immature floating form in our strain (Fig. 17) does not resemble the
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form illustrated in Page (1988). Page (1991 p. 87 fig. 33h) provided another image of
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the floating form of V. lata, which appears to be properly developed and has tapering
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pseudopodia, hence shorter than those in our strain. Page never mentioned possible
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spiral coiling of pseudopods in floating form, which is rather common in our strain.
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In 1999 Alexey Smirnov studied the type strain of V. lata CCAP 1589/12
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deposited by Page and held in CCAP. Illustration made from this strain show cells
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generally corresponding to Page’s description (Figs 21-28). In all cells breadth of the
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cell was equal or exceeded length, sometimes – considerably. The size range seems to
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be slightly wider than provided by Page, according to the measurements made in 1999, Page 10 of 23
12 the largest cells had breadth up to 60 μm (Fig. 26) while the smallest ones were just 24
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μm in maximal dimension (Fig. 21) The floating form (Fig. 28) that was observed was
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better developed that that illustrated in Page (1988) and show tapering pseudopodia,
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hence never coiling like in our strain. It was similar to the floating form shown by Page
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(1991 p. 87 fig. 33h). The number of pseudopodia in the floating form in our
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observations never exceeded six; this is congruent with the image by Page (1991). The
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type strain of V. lata CCAP 1589/12 has been lost, so the only data left (besides Page’s
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description and photographs) are images in Figs 21-28 done by Smirnov from this
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strain, a fragment of SSU sequence done from this strain by Sims et al. (2002, GB
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number AF464917) and complete SSU sequence of this strain done by Smirnov et al.
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(2007, GB number EF051201).
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The SSU sequence of our strain shows much in common with those of V. lata but
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differs in a number of nucleotide positions and it has certain structural differences. The
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sequence identity level between V. lata and the present strain is 0.946. This is a
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relatively low distance but it is comparable with the distance between other named
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species of Vannella, e.g. - between V. simplex and V. persistens – two species rather
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different morphologically with the sequence identity 0.95 (Smirnov and Brown 2004b;
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Smirnov et al. 2007) or that between V. oblongata and V. plurinucleolus CCAP 1565/11,
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which reaches the value of 0.99 (Tabs S1-S2). Taking into consideration morphological
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differences and similar but not identical sequences, we conclude that the present strain
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is a new species, related to V. lata but independent from it. We name it Vannella
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croatica n. sp.
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This species also resembles in morphology Vannella miroides Bovee 1965.
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Locomotive form of our species with flat posterior margin (e.g. in Fig. 6) much
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resembles drawing by Bovee (1965). However, V. miroides appears to be generally
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smaller (greatest dimension 25-35 μm while the present strain reaches 55 μm in Page 11 of 23
13 maximal dimension with average dimensions reaching 39 μm. According to Bovee
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(1965), all strains of V. miroides have bipyramidal crystals in the cytoplasm. This
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feature theoretically may be physiological, but it is noted by Bovee (1965) for several
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independent strains of this species and seems to be regularly appearing. Thus any
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species of appropriate dimension with crystals would more legitimately be called V.
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miroides rather than the present one. Floating form of our species is radial but cannot
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be called “very regular” as it is noted for V. miroides (Bovee 1965). Above analysis
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shows that we cannot associate our strain with V. miroides. However, we must note
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that it resembles Bovee’s description and two isolates named as V. miroides (ATCC
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30945 and strain 9i) group in the same clade. The first one is the nearest neighbour of
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our strain in the tree; it was sequenced by Peglar et al. (2003), while the latter one is a
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side result of a study by Miller et al. (2010). There are no morphological data on either
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of these strains. This evidences that there is a group of vannellids resembling V. lata
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and V. croatica; by description the species V. miroides resembles strains of this group
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but the presence of crystals mentioned in the original description by Bovee (1965)
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require attention; formally only Vannella possessing crystals could be named as V.
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miroides. So far we cannot conclude that there is any reliably re-isolated strain of V.
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miroides.
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A remarkable point is that sequences of two different strains of Vannella
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plurinucleolus CCAP 1565/11 and 1565/7 do not group together in the present tree, as
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they did in Smirnov et al. (2007) paper. This corresponds to the conclusions done from
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manual sequence analysis. Sequence divergence between these two strains is 0.97,
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which is higher than the divergence between V.oblongata and V.plurinucleolus CCAP
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1565/11, which reaches 0.99 (Tab.S2). Both strains were deposited by Frederick Page
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in 1972 at the time of description of V. plurinucleolus (Page 1974), they had the same
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origin (West Mersea, Essex, England) and Page believed them to be co-specific. Page 12 of 23
14 Sequence differences between these strains noted in the positions 1499-1528 are
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considerable; given that the sequence of one of these strains (1565/7) is a result of
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direct sequencing and that of 1565/11 strain is a molecular clone we can suggest that
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this is a case of intraspecific polymorphism of SSU gene mentioned for vannellids
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(Smirnov et al. 2007). This question requires further study.
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The analysis of the GenBank records related to vannellid amoebae clearly shows
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that there should be lots of yet non-described species of Vannella, Ripella and, perhaps,
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other vannellid genera. Within the vannellid amoebae clade, there are more sequences
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belonging to unnamed strains and environmental samples than to presently named
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species. There are likely many more groups of vannellid amoebae that are hardly
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distinguishable morphologically, but are clearly separated at the molecular level. The
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recent establishment of the vannellid genera Ripella (Smirnov et al. 2007) and
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Paravannella (Kudryavtsev 2014) together with the findings reported here on Vannella
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croatica suggest that there may be other presently undescribed amoebae in this
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groups.
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Finally, because Smirnov et al. (2007, 2011) proposed submersion of
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Platyamoeba species within the genus Vannella, the taxa established by Moran et al.
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(2007) are herewith formally transferred to Vannella, as follows: Vannella oblongata
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(Moran et Anderson, 2007) comb. nov. and Vannella contorta (Moran et Anderson
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2007) comb. nov. The authorship retained as given in the original publication,
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containing more authors in the title. Diagnoses and description: Moran et al. (2007).
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Acknowledgements Supported with the Russian Science Foundation 14-14-00474 grant (morphological and molecular studies) and President grant for young PhDs МК-4853.2015.4 to NB
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(alignment and data analysis). This study utilized equipment of the Core Facilities Centres
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“Culture Collection of Microorganisms”, “Development of Molecular and Cell Technologies”
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and “Computing Centre SPbU” of Saint Petersburg State University. Our thanks to Bland
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Finlay and Susan Brown for making the CCAP amoeba collection available for examination by
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Alexey Smirnov in 1999.
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References
3 4
Bovee, E.C., 1965. An emendation of the amoeba genus Flabellula and a descriprtion of Vannella gen. nov. Trans. Am. Microsc. Soc. 84, 217-227.
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5
Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView Version 4: A multiplatform graphical user
7
interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27,
8
221-224.
11 12 13
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phylogenies by maximum likelihood. Syst. Biol. 52, 696-704.
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Guindon, S., Gascuel, O., 2003. A simple, fast and accurate algorithm to estimate large
Hall, T. A., 1999., BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98.
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Kudryavtsev, A., 2014. Paravannella minima n. g. n. sp. (Discosea, Vannellidae) and distinction of the genera in the vannellid amoebae. Europ. J. Protistol. 50, 258-269.
15
Miller, J.A., Carmichael, A., Ramírez, M.J., Spagna, J.C., Haddad, C.R., Rezác, M., Johannesen, J.,
17 18 19 20 21
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Král, J., Wang, X.P., Griswold, C.E., 2010. Phylogeny of entelegyne spiders: affinities of
Ac ce p
16
d
14
the family Penestomidae (NEW RANK), generic phylogeny of Eresidae, and asymmetric rates of change in spinning organ evolution (Araneae, Araneoidea, Entelegynae). Mol. Phylogenet. Evol. 55, 786-804.
Moran, D.M., Anderson, O.R., Dennett, M.R., Caron, D.A., Gast, R.J., 2007. A description of seven Antarctic marine gymnamoebae including a new subspecies, two new species
22
and a new genus: Neoparamoeba aestuarina antarctica n. subsp., Platyamoeba
23
oblongata n. sp., Platyamoeba contorta n. sp. and Vermistella antarctica n. gen. n. sp. J.
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Eukaryot. Microbiol., 54, 169-183.
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Nassonova, E., Smirnov, A., Fahrni, J., Pawlowski, J., 2010. Barcoding Amoebae: Comparison
2
of SSU, ITS and COI Genes as Tools for Molecular Identification of Naked Lobose
3
Amoebae. Protist 161, 102-115.
6 7
664.
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Page, F.C., 1974. Some marine Platyamoeba of East Anglia. J. Mar. Biol. Assoc. UK 54, 651-
Page, F.C., 1976. An Illustrated Key to Freshwater and Soil Amoebae. Freshwater Biol Assoc., Ambleside.
cr
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Page, F.C., 1983. Marine Gymnamoebae. Institute Terrestrial Ecology, Cambridge.
9
Page, F. C., 1988. A New Key to Freshwater and Soil Gymnamoebae. Freshwater Biological
12
an
11
Association, Ambleside.
Page, F.C., 1991. Nackte Rizopoda. In Nackte Rhizopoda und Heliozoea (Protozoenfauna Band 2). Gustav Fischer Verlag, Stuttgart, New York, pp 3-187.
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Peglar, M. T., Amaral Zettler, L. A., Anderson, O. R., Nerad, T. A., Gillevet, P. M., Mullen, T. E.,
14
Frasca, S., Silberman, J. D., O'Kelly, C. J., Sogin, M. L., 2003. Two new small‐subunit
15
ribosomal RNA gene lineages within the subclass Gymnamoebia. J. Eukar. Microbiol. 50,
16
224-232.
18 19 20 21 22
te
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d
13
Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818.
Prescott, D.M., James, T.W., 1955. Culturing of Amoeba proteus on Tetrahymena. Exp. Cell Res. 8, 256-258.
Rambaut, A., Suchard, M.A., Xie, D., Drummond, A.J., 2014. Tracer v1. 6. Available at: http://beast. bio. ed. ac. uk/Tracer.
23
Sims, G. P., Aitken, R., Rogerson, A., 2002. Identification and phylogenetic analysis of
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morphologically similar naked amoebae using small subunits ribosomal RNA. J.
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Eukaryot. Microbiol. 49, 478-484.
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Smirnov, A. V., Goodkov, A. V., 1999. An illustrated list of basic morphotypes of Gymnamoebia (Rhizopoda, Lobosea). Protistology 1, 20-29. Smirnov, A.V., 2001. Vannella ebro n. sp. (Lobosea, Gymnamoebia), isolated from cyanobacterial mats in Spain. Europ. J. Protistol. 37, 147-153. Smirnov, A.V., 2002. Re-description of Vannella mira (Gymnamoebia, Vannellidae) - an
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often mentioned but poorly known amoeba species. Protistology 2, 178-185.
7
Smirnov, A.V., Brown, S., 2004. Guide to the study and identification of soil amoebae.
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Smirnov, A. V., Nassonova, E. S., Chao, E., Cavalier-Smith, T., 2007. Phylogeny, evolution, and taxonomy of vannellid amoebae. Protist 158, 295-324.
an
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Protistology 3, 148-190.
Smirnov, A. V., Chao, E., Nassonova, E. S., Cavalier-Smith, T., 2011. A revised classification of naked lobose amoebae (Amoebozoa: Lobosa). Protist 162, 545-570.
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19 1 2
Legends to the figures:
3 Figures 1-19. Light microscopy of Vannella croatica. 1-2 – amoebae in culture, on the
5
plastic surface of the Petri dish. Inverted phase contrast. 3-14 – variation in the locomotive
6
forms on the glass surface of the object slide. Arrows indicate nucleus in 3-4; depression on
7
the ventral surface of the hyaloplasm in 13 and waves on its dorsal surface in 14. 15-19 –
8
variation in the floating forms. Scale bar is 20 μm throughout.
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Figure 20. Phylogenetic tree based on SSU rDNA gene. Support values at each node
11
indicated as PP/BS value. Black dots on nodes indicate 1.00/100 support level. Indications
12
in nodes without strong support (<50% BS and < 0.60 PP omitted). All branches are drawn
13
to scale. For convenience all strains are named according to the genera where they belong,
14
independently on the initial genus designation in GenBank (e.g. strains deposited as
15
Platyamoeba are renamed as Vannella). A number of Tubulinea sequences are used as
16
outgroup.
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Figures 21-28. Light microscopy of Vannella lata CCAP 1 589/12 (videoprints done from
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the tape in 1999). DIC on the glass surface of the object slide. 21-27 – variation in the
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locomotive form. 28 - floating form. Scale bar is 20 μm throughout.
21 22
Figures S1-S9. Subsequent stages of the formation and retraction of “tail” in Vannella
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croatica. Image S5 shows the most typical appearance of the “tailed” amoeba. Scale bar is
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20 μm throughout.
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Page 18 of 23
20 1
Table 1. Size data (µm) for length (L) and breadth (B) of Vannella croatica. Three
2
clones originating from the initial strain KRKA 29.9.7.6.1 were independently
3
measured on the plastic surface of the Petri dish using an inverted microscope
L/B average
1.48
1.02
53
39
17
39
29
17
41
19
39
32
19
41
31
M
6
30
cr
27
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0.71
37
L/B min
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0.97
55
5
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7 8 9
1.43
B average
(n=51)
0.67
24
an
Clone 3
0.98
B max
(n=71)
1.86
B min
Clone 2
0.54
L average
(n=94)
L max
Clone 1
L min
clone
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Page 19 of 23
Table
Table 1. Size data (µm) for length (L) and breadth (B) of Vannella croatica. Three clones originating from the initial strain KRKA 29.9.7.6.1 were independently measured on
L/B min
L/B max
L/B average
1.43
0.97
0.71
1.48
1.02
53
17
39
29
17
41
30
19
39
32
19
41
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27
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37
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B average
0.67
55
31
Ac ce pt e
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M
(n=51)
0.98
24
an
Clone 3
1.86
B max
(n=71)
0.54
B min
Clone 2
39
L average
(n=94)
L max
Clone 1
L min
clone
the plastic surface of the Petri dish using an inverted microscope
Page 20 of 23
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Figure
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Figure
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S0932-4739(15)00097-8 http://dx.doi.org/doi:10.1016/j.ejop.2015.11.002 EJOP 25399
To appear in: Received date: Revised date: Accepted date:
27-7-2015 16-10-2015 2-11-2015
Please cite this article as: Smirnov, A.V., Bondarenko, N., Glotova, A., Nassonova, E.,Morphology and phylogeny of Vannella croatican. sp. (Amoebozoa, Discosea, Vannellida), European Journal of Protistology (2015), http://dx.doi.org/10.1016/j.ejop.2015.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
2 1
Morphology and phylogeny of Vannella croatica n. sp. (Amoebozoa,
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Discosea, Vannellida)
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Alexey V. Smirnova, *, Natalya Bondarenkoa, A. Glotovaa, Elena Nassonovaa,b
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State University, Universitetskaja nab. 7/9, 199034 St. Petersburg, Russia
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bLaboratory
ave. 4, 194064 St. Petersburg, Russia
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of Cytology of Unicellular Organisms, Institute of Cytology RAS, Tikhoretsky
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Department of Invertebrate Zoology, Faculty of Biology and Soil Sciences, St. Petersburg
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_________________________
*Corresponding author at : Department of Invertebrate Zoology, Faculty of
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Biology and Soil Sciences, Saint Petersburg State University, Universitetskaya nab. 7/9,
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199034, Saint Petersburg, Russia.
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E-mail address: [email protected] (A.V. Smirnov)
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Page 1 of 23
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Abstract We isolated and described a new species of freshwater vannellid amoeba from Krka natural reserve in Croatia ─ Vannella croatica n. sp. This species has certain
5
morphological differences from all known vannellids and differs at the level of SSU
6
sequence. It resembles in size and morphology Vannella lata; to facilitate direct
7
comparison we publish images of the type strain of V. lata CCAP 1589/12 strain (type
8
strain, which is now lost) taken in 1999. Vannela croatica feeds on bacteria and can be
9
easily grown in large amount in relatively pure culture and thus is suitable for
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molecular and biochemical studies requiring large amounts of material.
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4 1 2
Introduction
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The genus Vannella belongs to the family Vannellidae, order Vannellida, subclass Flabellinia of the class Discosea (Smirnov et al. 2011). It unifies flattened
6
amoeba of fan-shaped morphotype with a large frontal area of hyaloplasm (Smirnov
7
and Goodkov 1999; Smirnov and Brown 2004). Amoebae of the genus Vannella are
8
widely distributed and are among the most common organisms in environmental
9
samples, however species diversity within this genus requires further attention.
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Vannellid amoebae have a low number of morphological characters appropriate for
11
morphospecies distinction (Page 1976; 1983; 1988; 1991). As a result, the number of
12
species, which can be identified at the morphological level is low; in fact, only isolates
13
possessing remarkable distinctive characters can be reliably identified or described as
14
a new species (Smirnov 2001; 2002). Application of molecular methods allows
15
researchers to differentiate vannellid amoebae more precisely and discover several
16
new genera of smallest vannellid amoeba best recognised at the molecular level
17
(Peglar et al. 2003; Smirnov et al. 2007; Kudryavtsev 2014). It is now clear that
18
molecular data are essential to identify or to describe a new species of vannellids;
19
remarkable species possessing clear differentiating characters are very rare, and with
20
the growth of the number of described species boundaries at the morphological level
21
are getting less reliable (Smirnov 2001; Smirnov et al. 2007; Nassonova et al 2010;
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Kudryavtsev 2014).
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In the present paper we provide morphological and molecular data on a new
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species of Vannella – V. croatica n. sp. This species feeds on bacteria and can be easily
25
grown in large amount in relatively pure culture and thus is suitable for molecular and
Page 3 of 23
5 1
biochemical studies requiring large amounts of material. It resembles in size and
2
morphology Vannella lata; to facilitate direct comparison we publish images of the type
3
strain of V. lata CCAP 1589/12.
4
Material and Methods
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Samples containing Vannella strain were collected from a freshwater pond in
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Krka natural reserve (Lozovac, Croatia, grid reference 43.801981, 15.965470). Ca. 35
10
ml of water containing the top layer of bottom sediments was sampled with a sterile
11
Falcon tube. For enrichment cultivation, ca. 500 mg of the sampled material were
12
placed in 90 mm Petri dishes filled with artificial medium made by adding vacuum-
13
dried wheat grass powder (Weizengras, Sanatur GmbH, D-78224 Singen) to PJ medium
14
(Prescott and James 1955) to the final weight concentration of 0.05%. After 7-10 days
15
of incubation samples were examined using a Nikon TMF100 inverted phase-contrast
16
microscope; detected cells were transferred to fresh medium using tapering Pasteur
17
pipette. Strains were cloned, subcloned twice and further cultured in 60 mm Petri
18
dishes using the same medium. Clone labelled KRKA 29.9.7.6.1 was selected for further
19
studies; it was further subcloned into three clones used in the present work.
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Living trophozoites and cysts were observed, measured and photographed using
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a Nikon TMF100, Leica DM2500 and Leica DMI3000 microscopes equipped with phase
22
contrast and DIC optics. Trophozoites were measured on the plastic surface of the Petri
23
dish using a 40x objective lens; nuclei were measured on the glass surface of the object
24
slides using 100x objective, amoebae were not touched with the coverslip during
25
measurements. In order to obtain SSU sequences single cells of amoebae were isolated
26
using tapering Pasteur pipettes, washed twice in Millipore 0.2 µm sterilized culture Page 4 of 23
6 medium (freshly made pipette used each time), collected with minimal possible
2
amount (1-2 µl) of medium and placed in 200 µl PCR tubes. Tubes were subsequently
3
exposed to several rapid freezing-defreezing cycles (4-5 cycles from -180C to room
4
temperature), ready PCR mixture was topped on the tube content to the final volume of
5
50 µl. Thermal cycle parameters were the following: initial denaturation 10 min. at
6
95°C, followed by 40 cycles of 30 s at 94°C, 60 s at 50°C and 120 s at 72°C and
7
completed by 10 min at 72°C for final extension. Amplicons were purified using
8
Cleanup Mini Purification Kit (Eurogene) and sequenced directly using ABI-PRISM Big
9
Dye Terminator Cycle Sequencing Kit.
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Obtained sequences were manually aligned with representative Amoebozoa-wide
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alignment (224 sequences representing all major clades plus outgroups) and the local
12
alignment containing all named species of vannellid amoebae and an outgroup (54
13
sequences) using SeaView 4.0 (Gouy et al. 2010). The phylogenetic analysis was performed
14
using maximum likelihood method as implemented in PhyML program (Guindon and
15
Gascuel 2003) with GTR + G + I model suggested by Modeltest program (Posada and
16
Crandall 1998); 1302 sites were selected for analysis with the general amoebozoan
17
alignment and 1680 sites for the analysis in the local vannellid alignment; the number of
18
invariant sites, alpha parameter and tree topology were optimized by PhyML. Bayesian
19
analysis was performed using MrBayes 3.1.2, GTR model with gamma correction for
20
intersite rate variation (8 categories) and the covarion model. Trees were run as two
21
separate chains (default heating parameters) for 5 million generations, by which time they
22
had ceased converging (final average standard deviation of the split frequencies was less
23
than 0.01). The quality of chains was estimated using Tracer 1.6 package (Rambaut et al.
24
2014); basing on the estimates by Tracer, the first 27% of generations were discarded for
25
burn-in. Sequence identity matrix was calculated using BioEdit 7.0.9 (Hall 1999).
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3 4 5
Results Diagnosis Systematic position according to Smirnov et al. (2011)
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Phylum Amoebozoa Lühe 1913 sensu Cavalier-Smith, 1998
7
Subphylum Lobosa Carpenter, 1861 sensu Cavalier-Smith, 2009
8
Class Discosea Cavalier-Smith 2004 sensu Smirnov et al., 2011
9
Subclass Flabellinia Smirnov et al., 2005 sensu Smirnov et al., 2011
11
Family Vannellidae Bovee, 1979
12
Vannella croatica n. sp.
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Order Vannellida Smirnov et al., 2005
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Diagnosis: Locomotive form semi-circular or fan-shaped, often with pronounced tail. The
14
length of the locomotive form 17-55 μm (average 32-37 μm, depending on clone); breadth
15
17-53 μm (average 31-39 μm); L/B ratio 0.54 – 1.86 (average 0.97 – 1.02). Nucleus
16
vesicular, rounded or ovoid, with compact rounded or ovoid central nucleolus. The size of
17
the nucleus in maximal dimension is 3,5 – 4,8 μm (average 4,8 μm), that of nucleolus 2,2-
18
3,5 μm (average 2,8 μm). Floating form of radial type, with up to 15 tapering pseudopodia
19
of different length.
20 21
Type location: freshwater pond in Krka natural reserve (Lozovac, Croatia, grid reference
22
43.801981 N, 15.96547 E)
23
Type material: live strain and DNA sample deposited with core facility centre “Culture
24
Collection of Microorganisms” of Saint-Petersburg State University under the reference
25
name Vannella croatica KRKA 29.9.7.6.1. SSU sequence obtained from the type strain
26
deposited with GenBank under the number KT345693.
27
Etymology: named after the country of origin – Croatia
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Page 6 of 23
8 Differential diagnosis: Resembles Vannella lata Page, 1988 but differs by having
2
considerably higher length/breadth ratio (average of 1.0 in V. croatica compared to 0.6 in
3
V. lata) and pronounced tendency to produce “tail” in locomotion. Has higher number of
4
pseudopodia in floating form than V. lata. Differs from the type strain of V. lata by SSU
5
sequence. Resembles V. miroides Bovee, 1965, but contains no crystals, which, according to
6
Bovee (1965) were present in all strains of V. miroides. Vannella croatica also differs from
7
this species by having a less regularly shaped floating form.
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Morphological description
Three clones obtained after subcloning of initial one were studied and found to be
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morphologically identical except slight size differences. Locomotive cells observed on the
12
plastic surface using inverted phase contrast optics were semi-circular or fan-shaped, with
13
pronounced area of the frontal hyaloplasm (Figs 1-2). Cell morphology on the glass surface
14
of the object slide did not differ much from that observed in cultures (Figs 3-14). The
15
frontal hyaloplasm occupied from 1/2 to 2/3 of the total cell area (Figs 3-10) and often had
16
pronounced lateral intrusions (Figs 3,6,9-10,12). The posterior part of the cell was straight
17
or semi-circular (Figs 3, 5-10). Often the posterior part of amoeba adhered to the substrate
18
while the cell continued to progress forward, hence the cell formed a pronounced tail (Figs
19
4,11). The end of this tail later detached and the entire tail was retracted (see plate S1
20
showing subsequent stages of the process). The formation of the tail was more frequent in
21
amoebae moving on the clear plastic surface rather than in those moving over the bacterial
22
biofilm. Sometimes frontal hyaloplasm was slightly clefted, with pronounced depression in
23
the frontal area (Fig. 9). Moving cells often formed depressions on the ventral surface of the
24
hyaloplasm (Fig. 13) and waves on its dorsal surface (Fig. 14). In some cells a prominent
25
longitudinal ridge was formed for a short time. The ridge was never stable for more than
26
several seconds. In older cultures cells often formed lobes on the dorsal surface of the
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Page 7 of 23
9 1
hyaline area, sometimes 3-6 lobes were simultaneously seen. Morphometric data are
2
provided in the Table 1.
3
Cells in non-directed movement often had two prominent areas of the hyaloplasm. Slowly, chaotically moving cells often became elongate and were surrounded with the
5
hyaline margin from all sides. Stationary cells usually were rounded and had peripheral
6
hyaline margin of variable width.
The floating form was of radial type, with 3-15 long, tapering pseudopodia (Figs. 15-
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19). Developed floating forms had rounded, nearly spherical central mass of the cytoplasm
9
and 9-15 pseudopodia of different length; the most of them much exceeded the diameter of
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the central cytoplasmic mass (Figs 15-16). Early-stage floating forms had 2-4 conical
11
pseudopodia, sometimes with a pronounced conical hyaline base (Fig. 17). In many floating
12
forms spiral coiling of the distal part of pseudopodia was observed (Fig. 18). Sometimes all
13
pseudopodia of the floating form were curved in one direction, suggestive of a rotating cell
14
(Fig. 19).
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The nucleus was of the vesicular type, rounded or slightly ovoid, with the single
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compact rounded central nucleolus (Figs 3-4). The size of the nucleus in maximal
17
dimension was 3,5 – 4,8 μm (average 4,8 μm), that of the nucleolus - 2,2-3,5 μm (average
18
2,8 μm). The single contractile vacuole is usually located in the posterior part of the cell
19
(Figs 3, 5, 9, 12), sometimes – right in the tail. The cytoplasm was filled with numerous
20
granules 1-2 μm in size and contained numerous optically empty vacuoles. Crystals were
21
never seen; few refractive inclusions (like those visible in Figs 3-4 and 9) were occasional
22
pieces of external material or tiny plastic fragments phagocytized or temporarily located
23
under the cell. In our cultures amoebae did not form cysts.
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Molecular phylogeny The Amoebozoa-wide ML phylogenetic tree (not shown) clearly indicated that
3
the present isolate belongs to the genus Vannella. More restricted tree containing all
4
named sequences of vannellid amoebae (Fig. 20) shows with high support that the
5
present strain forms a separate clade with some other species of the genus Vannella;
6
the closest relatives among named species are V. lata CCAP 1589/12 (type strain);
7
among others are isolate ATCC 30945 named as V. miroides and an isolate named V.
8
miroides strain 9i. The nearest neighbours of this clade are V. epipetala, and V. placida
9
CCAP 1565/2. Another neighbour is the strain ATCC 50745 misidentified as V.
10
plurinucleolus (see Smirnov et al. 2007). The overall configuration of the tree
11
corresponds to the data published earlier (Smirnov et al. 2007), except that with the
12
addition of V.oblongata sequence, two strains of V. plurinucleolus (CCAP1565/7 and
13
1565/11) are not neighbours anymore. This is congruent with the results of the
14
manual analysis of the alignment; these two sequences have considerable difference in
15
the positions 1499-1528 (counted in the sequence of V. plurinucleolus CCAP 1565/7
16
strain GB number EF051186) and a number of indels differing them in other regions.
17
Genera Ripella, Clydonella and Lingulamoeba form highly supported clades each and
18
group together; Paravannella minima with medium support appears to be a basal
19
group to this clade, not to entire Vannellida.
21 22
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Discussion
23 24
The present isolate with no doubts belongs to the genus Vannella Bovee 1965
25
both by morphological and by molecular evidences. Its recognisable locomotive and
26
floating forms are characteristic for this amoebae genus; molecular phylogeny places
27
this isolate deep inside the genus Vannella. Page 9 of 23
11 Among named species of the genus Vannella the present strain most resembles
2
the species V. lata Page, 1988. This relatively briefly described and illustrated species
3
has similar general appearance and comparable size range. Page (1988) provides the
4
following data for V. lata: breadth 24-46 μm (average 33 μm). Length is not provided
5
in this description. The indicated breadth of the cell is close to that of the present strain
6
and is comparable with dimensions of clones 2 and 3 (Table 1). Clone 1 is generally
7
larger. However, Page (1988 p. 74) indicates that in V. lata “breadth is always greater
8
than length, never less” and provides L/B ratio 0.5 - 1.0 with average 0.6. This is not
9
the case for our strain, where many cells are elongate and possess a pronounced tail;
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L/B ratio may reach 1.82 with average 0.97 – 1.02. The floating form of V. lata is poorly
11
illustrated by Page (1988, p. 75 fig. 29J); he noted that the floating form in his species
12
usually has 8 tapering pseudopodia, which may be 3 times longer than the diameter of
13
the central cytoplasmic mass. The image published by Page (1988) is very different
14
from our strain; however it looks like in this case Page did not observe very well-
15
developed floating forms. His illustration does not show any radiating tapering
16
pseudopodia. In our strain the number of pseudopodia in the developed floating form
17
was often 12-15. Immature floating form in our strain (Fig. 17) does not resemble the
18
form illustrated in Page (1988). Page (1991 p. 87 fig. 33h) provided another image of
19
the floating form of V. lata, which appears to be properly developed and has tapering
20
pseudopodia, hence shorter than those in our strain. Page never mentioned possible
21
spiral coiling of pseudopods in floating form, which is rather common in our strain.
22
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In 1999 Alexey Smirnov studied the type strain of V. lata CCAP 1589/12
23
deposited by Page and held in CCAP. Illustration made from this strain show cells
24
generally corresponding to Page’s description (Figs 21-28). In all cells breadth of the
25
cell was equal or exceeded length, sometimes – considerably. The size range seems to
26
be slightly wider than provided by Page, according to the measurements made in 1999, Page 10 of 23
12 the largest cells had breadth up to 60 μm (Fig. 26) while the smallest ones were just 24
2
μm in maximal dimension (Fig. 21) The floating form (Fig. 28) that was observed was
3
better developed that that illustrated in Page (1988) and show tapering pseudopodia,
4
hence never coiling like in our strain. It was similar to the floating form shown by Page
5
(1991 p. 87 fig. 33h). The number of pseudopodia in the floating form in our
6
observations never exceeded six; this is congruent with the image by Page (1991). The
7
type strain of V. lata CCAP 1589/12 has been lost, so the only data left (besides Page’s
8
description and photographs) are images in Figs 21-28 done by Smirnov from this
9
strain, a fragment of SSU sequence done from this strain by Sims et al. (2002, GB
us
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1
number AF464917) and complete SSU sequence of this strain done by Smirnov et al.
11
(2007, GB number EF051201).
an
10
The SSU sequence of our strain shows much in common with those of V. lata but
13
differs in a number of nucleotide positions and it has certain structural differences. The
14
sequence identity level between V. lata and the present strain is 0.946. This is a
15
relatively low distance but it is comparable with the distance between other named
16
species of Vannella, e.g. - between V. simplex and V. persistens – two species rather
17
different morphologically with the sequence identity 0.95 (Smirnov and Brown 2004b;
18
Smirnov et al. 2007) or that between V. oblongata and V. plurinucleolus CCAP 1565/11,
19
which reaches the value of 0.99 (Tabs S1-S2). Taking into consideration morphological
20
differences and similar but not identical sequences, we conclude that the present strain
21
is a new species, related to V. lata but independent from it. We name it Vannella
22
croatica n. sp.
23
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12
This species also resembles in morphology Vannella miroides Bovee 1965.
24
Locomotive form of our species with flat posterior margin (e.g. in Fig. 6) much
25
resembles drawing by Bovee (1965). However, V. miroides appears to be generally
26
smaller (greatest dimension 25-35 μm while the present strain reaches 55 μm in Page 11 of 23
13 maximal dimension with average dimensions reaching 39 μm. According to Bovee
2
(1965), all strains of V. miroides have bipyramidal crystals in the cytoplasm. This
3
feature theoretically may be physiological, but it is noted by Bovee (1965) for several
4
independent strains of this species and seems to be regularly appearing. Thus any
5
species of appropriate dimension with crystals would more legitimately be called V.
6
miroides rather than the present one. Floating form of our species is radial but cannot
7
be called “very regular” as it is noted for V. miroides (Bovee 1965). Above analysis
8
shows that we cannot associate our strain with V. miroides. However, we must note
9
that it resembles Bovee’s description and two isolates named as V. miroides (ATCC
us
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1
30945 and strain 9i) group in the same clade. The first one is the nearest neighbour of
11
our strain in the tree; it was sequenced by Peglar et al. (2003), while the latter one is a
12
side result of a study by Miller et al. (2010). There are no morphological data on either
13
of these strains. This evidences that there is a group of vannellids resembling V. lata
14
and V. croatica; by description the species V. miroides resembles strains of this group
15
but the presence of crystals mentioned in the original description by Bovee (1965)
16
require attention; formally only Vannella possessing crystals could be named as V.
17
miroides. So far we cannot conclude that there is any reliably re-isolated strain of V.
18
miroides.
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19
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A remarkable point is that sequences of two different strains of Vannella
20
plurinucleolus CCAP 1565/11 and 1565/7 do not group together in the present tree, as
21
they did in Smirnov et al. (2007) paper. This corresponds to the conclusions done from
22
manual sequence analysis. Sequence divergence between these two strains is 0.97,
23
which is higher than the divergence between V.oblongata and V.plurinucleolus CCAP
24
1565/11, which reaches 0.99 (Tab.S2). Both strains were deposited by Frederick Page
25
in 1972 at the time of description of V. plurinucleolus (Page 1974), they had the same
26
origin (West Mersea, Essex, England) and Page believed them to be co-specific. Page 12 of 23
14 Sequence differences between these strains noted in the positions 1499-1528 are
2
considerable; given that the sequence of one of these strains (1565/7) is a result of
3
direct sequencing and that of 1565/11 strain is a molecular clone we can suggest that
4
this is a case of intraspecific polymorphism of SSU gene mentioned for vannellids
5
(Smirnov et al. 2007). This question requires further study.
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1
The analysis of the GenBank records related to vannellid amoebae clearly shows
7
that there should be lots of yet non-described species of Vannella, Ripella and, perhaps,
8
other vannellid genera. Within the vannellid amoebae clade, there are more sequences
9
belonging to unnamed strains and environmental samples than to presently named
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6
species. There are likely many more groups of vannellid amoebae that are hardly
11
distinguishable morphologically, but are clearly separated at the molecular level. The
12
recent establishment of the vannellid genera Ripella (Smirnov et al. 2007) and
13
Paravannella (Kudryavtsev 2014) together with the findings reported here on Vannella
14
croatica suggest that there may be other presently undescribed amoebae in this
15
groups.
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Finally, because Smirnov et al. (2007, 2011) proposed submersion of
17
Platyamoeba species within the genus Vannella, the taxa established by Moran et al.
18
(2007) are herewith formally transferred to Vannella, as follows: Vannella oblongata
19
(Moran et Anderson, 2007) comb. nov. and Vannella contorta (Moran et Anderson
20
2007) comb. nov. The authorship retained as given in the original publication,
21
containing more authors in the title. Diagnoses and description: Moran et al. (2007).
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Page 13 of 23
15 1 2 3 4
Acknowledgements Supported with the Russian Science Foundation 14-14-00474 grant (morphological and molecular studies) and President grant for young PhDs МК-4853.2015.4 to NB
6
(alignment and data analysis). This study utilized equipment of the Core Facilities Centres
7
“Culture Collection of Microorganisms”, “Development of Molecular and Cell Technologies”
8
and “Computing Centre SPbU” of Saint Petersburg State University. Our thanks to Bland
9
Finlay and Susan Brown for making the CCAP amoeba collection available for examination by
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Alexey Smirnov in 1999.
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Page 14 of 23
16 1 2
References
3 4
Bovee, E.C., 1965. An emendation of the amoeba genus Flabellula and a descriprtion of Vannella gen. nov. Trans. Am. Microsc. Soc. 84, 217-227.
ip t
5
Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView Version 4: A multiplatform graphical user
7
interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 27,
8
221-224.
11 12 13
us
phylogenies by maximum likelihood. Syst. Biol. 52, 696-704.
an
10
Guindon, S., Gascuel, O., 2003. A simple, fast and accurate algorithm to estimate large
Hall, T. A., 1999., BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98.
M
9
cr
6
Kudryavtsev, A., 2014. Paravannella minima n. g. n. sp. (Discosea, Vannellidae) and distinction of the genera in the vannellid amoebae. Europ. J. Protistol. 50, 258-269.
15
Miller, J.A., Carmichael, A., Ramírez, M.J., Spagna, J.C., Haddad, C.R., Rezác, M., Johannesen, J.,
17 18 19 20 21
te
Král, J., Wang, X.P., Griswold, C.E., 2010. Phylogeny of entelegyne spiders: affinities of
Ac ce p
16
d
14
the family Penestomidae (NEW RANK), generic phylogeny of Eresidae, and asymmetric rates of change in spinning organ evolution (Araneae, Araneoidea, Entelegynae). Mol. Phylogenet. Evol. 55, 786-804.
Moran, D.M., Anderson, O.R., Dennett, M.R., Caron, D.A., Gast, R.J., 2007. A description of seven Antarctic marine gymnamoebae including a new subspecies, two new species
22
and a new genus: Neoparamoeba aestuarina antarctica n. subsp., Platyamoeba
23
oblongata n. sp., Platyamoeba contorta n. sp. and Vermistella antarctica n. gen. n. sp. J.
24
Eukaryot. Microbiol., 54, 169-183.
Page 15 of 23
17 1
Nassonova, E., Smirnov, A., Fahrni, J., Pawlowski, J., 2010. Barcoding Amoebae: Comparison
2
of SSU, ITS and COI Genes as Tools for Molecular Identification of Naked Lobose
3
Amoebae. Protist 161, 102-115.
6 7
664.
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5
Page, F.C., 1974. Some marine Platyamoeba of East Anglia. J. Mar. Biol. Assoc. UK 54, 651-
Page, F.C., 1976. An Illustrated Key to Freshwater and Soil Amoebae. Freshwater Biol Assoc., Ambleside.
cr
4
Page, F.C., 1983. Marine Gymnamoebae. Institute Terrestrial Ecology, Cambridge.
9
Page, F. C., 1988. A New Key to Freshwater and Soil Gymnamoebae. Freshwater Biological
12
an
11
Association, Ambleside.
Page, F.C., 1991. Nackte Rizopoda. In Nackte Rhizopoda und Heliozoea (Protozoenfauna Band 2). Gustav Fischer Verlag, Stuttgart, New York, pp 3-187.
M
10
us
8
Peglar, M. T., Amaral Zettler, L. A., Anderson, O. R., Nerad, T. A., Gillevet, P. M., Mullen, T. E.,
14
Frasca, S., Silberman, J. D., O'Kelly, C. J., Sogin, M. L., 2003. Two new small‐subunit
15
ribosomal RNA gene lineages within the subclass Gymnamoebia. J. Eukar. Microbiol. 50,
16
224-232.
18 19 20 21 22
te
Ac ce p
17
d
13
Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818.
Prescott, D.M., James, T.W., 1955. Culturing of Amoeba proteus on Tetrahymena. Exp. Cell Res. 8, 256-258.
Rambaut, A., Suchard, M.A., Xie, D., Drummond, A.J., 2014. Tracer v1. 6. Available at: http://beast. bio. ed. ac. uk/Tracer.
23
Sims, G. P., Aitken, R., Rogerson, A., 2002. Identification and phylogenetic analysis of
24
morphologically similar naked amoebae using small subunits ribosomal RNA. J.
25
Eukaryot. Microbiol. 49, 478-484.
Page 16 of 23
18
2 3 4 5
Smirnov, A. V., Goodkov, A. V., 1999. An illustrated list of basic morphotypes of Gymnamoebia (Rhizopoda, Lobosea). Protistology 1, 20-29. Smirnov, A.V., 2001. Vannella ebro n. sp. (Lobosea, Gymnamoebia), isolated from cyanobacterial mats in Spain. Europ. J. Protistol. 37, 147-153. Smirnov, A.V., 2002. Re-description of Vannella mira (Gymnamoebia, Vannellidae) - an
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1
often mentioned but poorly known amoeba species. Protistology 2, 178-185.
7
Smirnov, A.V., Brown, S., 2004. Guide to the study and identification of soil amoebae.
11 12
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Smirnov, A. V., Nassonova, E. S., Chao, E., Cavalier-Smith, T., 2007. Phylogeny, evolution, and taxonomy of vannellid amoebae. Protist 158, 295-324.
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Protistology 3, 148-190.
Smirnov, A. V., Chao, E., Nassonova, E. S., Cavalier-Smith, T., 2011. A revised classification of naked lobose amoebae (Amoebozoa: Lobosa). Protist 162, 545-570.
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19 1 2
Legends to the figures:
3 Figures 1-19. Light microscopy of Vannella croatica. 1-2 – amoebae in culture, on the
5
plastic surface of the Petri dish. Inverted phase contrast. 3-14 – variation in the locomotive
6
forms on the glass surface of the object slide. Arrows indicate nucleus in 3-4; depression on
7
the ventral surface of the hyaloplasm in 13 and waves on its dorsal surface in 14. 15-19 –
8
variation in the floating forms. Scale bar is 20 μm throughout.
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4
9
Figure 20. Phylogenetic tree based on SSU rDNA gene. Support values at each node
11
indicated as PP/BS value. Black dots on nodes indicate 1.00/100 support level. Indications
12
in nodes without strong support (<50% BS and < 0.60 PP omitted). All branches are drawn
13
to scale. For convenience all strains are named according to the genera where they belong,
14
independently on the initial genus designation in GenBank (e.g. strains deposited as
15
Platyamoeba are renamed as Vannella). A number of Tubulinea sequences are used as
16
outgroup.
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17
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18
Figures 21-28. Light microscopy of Vannella lata CCAP 1 589/12 (videoprints done from
19
the tape in 1999). DIC on the glass surface of the object slide. 21-27 – variation in the
20
locomotive form. 28 - floating form. Scale bar is 20 μm throughout.
21 22
Figures S1-S9. Subsequent stages of the formation and retraction of “tail” in Vannella
23
croatica. Image S5 shows the most typical appearance of the “tailed” amoeba. Scale bar is
24
20 μm throughout.
25
Page 18 of 23
20 1
Table 1. Size data (µm) for length (L) and breadth (B) of Vannella croatica. Three
2
clones originating from the initial strain KRKA 29.9.7.6.1 were independently
3
measured on the plastic surface of the Petri dish using an inverted microscope
L/B average
1.48
1.02
53
39
17
39
29
17
41
19
39
32
19
41
31
M
6
30
cr
27
ip t L/B max
0.71
37
L/B min
us
0.97
55
5
Ac ce p
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7 8 9
1.43
B average
(n=51)
0.67
24
an
Clone 3
0.98
B max
(n=71)
1.86
B min
Clone 2
0.54
L average
(n=94)
L max
Clone 1
L min
clone
4
Page 19 of 23
Table
Table 1. Size data (µm) for length (L) and breadth (B) of Vannella croatica. Three clones originating from the initial strain KRKA 29.9.7.6.1 were independently measured on
L/B min
L/B max
L/B average
1.43
0.97
0.71
1.48
1.02
53
17
39
29
17
41
30
19
39
32
19
41
cr
27
us
37
ip t
B average
0.67
55
31
Ac ce pt e
d
M
(n=51)
0.98
24
an
Clone 3
1.86
B max
(n=71)
0.54
B min
Clone 2
39
L average
(n=94)
L max
Clone 1
L min
clone
the plastic surface of the Petri dish using an inverted microscope
Page 20 of 23
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Figure
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Figure
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ed
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Figure
Page 23 of 23