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Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: Two New Amitochondrial Heterolobosean Amoebae from Anoxic Environments
Protist, Vol. 154, 281–290, July 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/protist Published online 30 July 2003
Protist
ORIGINAL PAPER
Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: Two New Amitochondrial Heterolobosean Amoebae from Anoxic Environments Charles J. O’Kelly a, Jeffrey D. Silbermanb, Linda A. Amaral Zettler c, Thomas A. Nerad d, and Mitchell L. Sogin c,1 a
Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575, USA Department of Microbiology, Immunology, Molecular Genetics, and Institute of Geophysics and Planetary Physics and Department of Microbiology and Immunology, University of California at Los Angeles, Los Angeles, CA 90095, USA c The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02543, USA d Protistology Department, American Type Culture Collection, Manassas, Virginia 20110-2209, USA b
Submitted January 29, 2003; Accepted April 22, 2003 Monitoring Editor: Michael Melkonian
Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.
Introduction For the most part, the class Heterolobosea (Page 1991; Page and Blanton 1985; Patterson et al. 2000) contains amoebae, amoeboflagellates, and 1
Corresponding author; fax 1508 457 4727 e-mail [email protected]
flagellates that have mitochondria and are, consequently, aerobes. To date, only three non-aerobic species are known by modern methods to belong to the class, all of which have been described within the last two decades. These species are Psalteriomonas lanterna Broers et al. 1990; an amoeboflagellate (Broers et al. 1990), P. vulgaris Broers et al. 1434-4610/03/154/02-281 $ 15.00/0
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1993 (= Lyromonas vulgaris (Broers et al. 1993) Cavalier-Smith 1993), a flagellate (Broers et al. 1993; Cavalier-Smith 1993), and Vahlkampfia anaerobica Smirnov and Fenchel 1996; an amoeba (Smirnov and Fenchel 1996). All of these protists lack mitochondria but have organelles interpreted as hydrogenosomes. Psalteriomonas lanterna occupies a unique phylogenetic position within the Heterolobosea, according to analyses of its nuclear small-subunit (SSU) ribosomal RNA gene sequence (Weekers et al. 1997). Neither of the other anaerobic species has been examined at the molecular level. In the course of investigations on the protistan biota of anaerobic sediments, two anaerobes have been found that are intolerant of aerobic conditions. These amoebae have morphological similarities to other Heterolobosea but have unusual nucleolar morphology. They can be assigned to no known species on the basis of morphological or molecular sequence data. Therefore they are described here as Monopylocystis visvesvarai n. gen., n. sp., and Sawyeria marylandensis n. gen., n. sp. In a previous study (Silberman et al. 2002), the SSU rRNA gene sequence of Sawyeria marylandensis formed a robust clade with Psalteriomonas lanterna in phylogenetic analyses. Here, the SSU rRNA gene from Monopylocystis visvesvarai is added to the analysis. It was found that all three nonaerobes form a single clade within the predominantly aerobic heterolobosean lineage.
Results Morphology of Monopylocystis visvesvarai Monopylocystis visvesvarai produced gymnamoebae and cysts in culture. Locomotive amoebae were of the “limax” type, with an anterior, eruptive hyaline front (Figs 1, 2). They were 17–30 µm long (mean 22 ± 3.7 µm, n = 25) and 6.0–10 µm (mean 8.0 ± 1.4 µm, n = 25) wide. The mean length-width ratio was 2.7. They were “naked” (Fig. 3), lacking tests, scales, or detectable glycocalyx. Non-locomotive cells when attached were circular in outline, and when floating were essentially spherical. No rayed floating forms were observed. The amoebae were active only under anaerobic conditions, ceasing activity and losing cell integrity on exposure to oxygen-containing media. The cells were uninucleate (Figs 1–2). The nuclei had an irregularly polygonal outline (Figs 1, 2, 4). Electron-dense material, interpreted as nucleolar, was appressed to the nuclear envelope (Fig. 4). No other nucleolar material was present. Rough endo-
plasmic reticulum was usually associated with the nucleus, but did not form a complete investment (Fig. 4). Nuclear division was not observed. No Golgi bodies or microbodies were seen. Mitochondria were not observed, but spherical to bacilliform, membrane-bound bodies with uniformly dense contents were scattered throughout the cytoplasm (Figs 4, 5). Food vacuoles contained bacteria (Fig. 2). Cysts were spherical, with a single prominent pore (Fig. 6). They measured 7.5 –13.5 µm (mean 11 ± 1.4 µm, n = 25) in diameter. The cyst wall was thin, unornamented, and unmineralized (Fig. 7). The pore was a protruding aperture in the wall, occluded by a plug of fibrillar material interspersed with dark blocks (Fig. 7).
Morphology of Sawyeria marylandensis Sawyeria marylandensis produced only gymnamoebae in culture. Locomotive amoebae were of the “limax” type, with an anterior, eruptive hyaline front (Figs 8, 9). They were 25–41 µm (mean 31.4 ± 3.2 µm, n = 29) long and 7–12 µm (mean 8.1 ± 1.0 µm, n = 29) wide. The length-width ratio was 3.9. Non-locomotive cells when attached were essentially circular in outline (Fig. 10), and when floating were essentially spherical. No rayed floating forms were observed. The amoebae were “naked” (Fig. 11), lacking tests, scales, or detectable glycocalyx. They were active only under anaerobic conditions, ceasing activity and losing cell integrity on exposure to oxygen-containing media. The cells were uninucleate (Figs 8–10, 12). The nuclei were spherical to slightly ellipsoidal (Figs 8–10) and had either one (Fig. 10) or two (Figs 8–9, 12) peripheral nucleoli that were associated with polar lobes of the nucleus and appressed to the nuclear envelope (Fig. 12). Mean nuclear diameter was 4–5 µm. Rough endoplasmic reticulum usually formed a complete investment around the interphase nucleus (Fig. 12). Dividing nuclei exhibited a cruciform pattern consisting of persistent nucleoli lateral to the chromosomal plate; the nuclear envelope was closed (Fig. 13). No Golgi bodies or microbodies were seen. Mitochondria were not observed, but bacilliform, membrane-bound bodies with marbled contents were scattered throughout the cytoplasm (Figs 11, 14). Food vacuoles contained bacteria (Fig. 12).
Phylogenetic Analyses Figure 15 presents the optimal maximum likelihood tree from a SSU rRNA gene data set containing selected Heterolobosea and non-heterolobsean eu-
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Figures 1–7. Light and electron micrographs of Monopylocystis visvesvarai. 1. Locomotive amoeba with anteriorly erumpent hyaloplasmic bulge. Direction of motion is towards the right of the image. Nucleus with angular outline and without a central nucleolus. Scale bar = 5 µm. 2. Locomotive amoeba with laterally erumpent hyaloplasmic bulge and numerous food vacuoles. Direction of motion is towards the right of the image. Scale bar = 5 µm. 3. Cell membrane of locomotive amoeba. No surface structures are evident. Scale bar = 100 nm. 4. Nucleus and associated organelles in the locomotive amoeba. Nucleus with angular outline and parietal encircling nucleolar material. Rough endoplasmic reticulum cisternae are in the vicinity of the nucleus but do not envelop it. Hydrogenosome-like organelles and food vacuoles are present. Scale bar = 500 nm. 5. Detail of hydrogenosomelike organelle. Scale bar = 250 nm. 6. Cyst with single protruding pore. Scale bar = 5 µm. 7. Detail of cyst, with nonmineralized cyst wall and protruding pore with plug. Scale bar = 1.0 µm. Abbreviations for Figure 1–14: b, bacteria; cp, chromosomal plate of mitotic spindle; fv, food vacuole; h, hydrogenosome-like organelle; n, nucleus; no, nucleolus or nucleolar material; p, cyst pore; t, trailing end of amoeba.
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karyotes, with bootstrap values greater than 50% indicated at the nodes. The heteroloboseans, inclusive of Monopylocystis visvesvarai and Sawyeria marylandensis form a monophyletic assemblage supported with 100% bootstrap values (maximum likelihood, distance and parsimony). Figure 16 shows an unrooted maximum likelihood phylogenetic tree, generated from a fine-scale dataset containing only sequences from Heterolobosea, with bootstrap values from distance, parsimony and maximum likelihood analyses. All phylogenetic inferences from this dataset yielded the same tree topology. The analyses demonstrated a sister relationship between Psalteriomonas lanterna and Sawyeria marylandensis. Furthermore, Psalteriomonas lanterna plus Sawyeria marylandensis along with Monopylocystis visvesvarai formed a robust clade with bootstrap values of 100% for all methods tested. Sawyeria marylandensis, Monopylocystis visvesvarai and Psalteriomonas lanterna formed part of a larger clade that included Paravahlkampfia ustiana and Neovahlkampfia damariscottae with good bootstrap support (89/91/83).
Discussion The two amoebae described here are assigned to the Heterolobosea on the basis of the limax form of their locomotive amoebae, their position in phylogenetic trees based on nuclear small-subunit ribosomal RNA gene sequences, and, as seen in Sawyeriamarylandensis, the closed, cruciform mitosis. They are distinct from other Heterolobosea in several ways. Both Monopylocystis visvesvarai and Sawyeria marylandensis are intolerant of normal aerobic environmental conditions. However, as the methods used here probably do not remove all oxygen from
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the culture medium, it is more likely that these protists are microaerophiles (Biagini and Bernard 2000), tolerating and possibly requiring trace amounts of oxygen, rather than strict anaerobes. Both species lack mitochondria with detectable cristae, but have organelles that are similar to mitochondria in shape and size, as do the other known heterolobosean anaerobes, the Psalteriomonas species (Broers et al. 1990, 1993) and Vahlkampfia anaerobica Smirnov and Fenchel (Smirnov and Fenchel 1996). The precise structural features of these organelles, particularly the matrix (which may appear homogeneous or variously heterogeneous) and the number of bounding membranes, are difficult to discern with accuracy, since these features are susceptible to, and vary with, conditions of fixation (Smirnov and Fenchel 1996). The organelles of Psalteriomonas are considered to be hydrogenosomes on the basis of their hydrogenase activity (Broers et al. 1993; Brul et al. 1994). Pending physiological investigations, the analogous organelles of Monopylocystis visvesvarai and Sawyeria marylandensis (as well as those of Vahlkampfia anaerobica) are likewise interpreted as hydrogenosomes. Both Monopylocystis visvesvarai and Sawyeria marylandensis have unusual nucleolar morphologies. Almost all described Heterolobosea have one centrally-located vesicular nucleolus in the interphase nucleus. However, Vahlkampfia anaerobica (Smirnov and Fenchel 1996), and the marine aerobe Heteramoeba clara (Carey and Page 1985), have their nucleolar material distributed along the nuclear periphery, and in the freshwater aerobe Stachyamoeba lipophora (Page 1975, 1987) the nucleolus is present as one, two or three parietal masses. The nucleolar organization of Monopylocystis visvesvarai is similar to that of Heteramoeba clara and Vahlkampfia anaerobica. The latter species is a marine anaerobe; its locomotive amoebae are simi-
Figures 8–14. Light and electron micrographs of Sawyeria marylandensis. 8. Locomotive amoebae in straight-line motion, direction towards the right of the image. The trailing end of locomotive amoebae is frequently slightly bulbous, as here. Nucleus with two nucleoli. Scale bar = 5 µm. 9. Locomotive amoeba in the process of changing direction; former direction towards the top left corner of the image, new direction towards the upper right corner. Bacteria are sometimes associated with the trailing end of the amoeba, as here. Nucleus with two nucleoli. Scale bar = 5 µm. 10. Quiescent amoeba, flattened under coverslip pressure. Nucleus with one nucleolus. Hydrogenosome-like organelles, normally bacilliform, here appear rounded. Scale bar = 5 µm. 11. Cell membrane of locomotive amoeba. No surface structures are evident. A cross-sectional profile of a hydrogenosome-like organelle is present. Scale bar = 250 nm. 12. Nucleus with two parietal nucleoli and apparently complete investment of rough endoplasmic reticulum. Food vacuoles contain bacteria. Scale bar = 500 nm. 13. Nuclear division. The nuclear envelope is closed. Persistent peripheral nucleoli lie alongside the chromosomal plate. Cell fixed and stained according to the Kernichrot procedure. Scale bar = 2 µm. 14. Group of hydrogenosome-like organelles. Scale bar = 500 nm.
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Figure 15. The optimal maximum likelihood tree based on SSU rRNA gene sequence comparisons. Bootstrap values greater than 50% (1000 replicates) for maximum likelihood, ml-distance, and parsimony (respectively) are indicated at the nodes. An asterisk (*) indicates bootstrap values below 50%. The scale bar represents evolutionary distance for the number of changes per site.
lar in dimensions to those of Monopylocystis visvesvarai. The nuclear outline of Vahlkampfia anaerobica is, however, less angular than that of Monopylocystis visvesvarai, and the nucleus in the latter species lacks the close investment of rough endoplasmic reticulum that is present in the former. Also, the uroidal structures and rayed floating forms present in Vahlkampfia anaerobica have not been observed in Monopylocystis visvesvarai, and no cyst stage has been observed in Vahlkampfia anaerobica.
The nucleolar organization of Sawyeria marylandensis is similar to that of Stachyamoeba lipophora. However, the nucleoli of Sawyeria marylandensis have a smooth outline and are consistently found opposite each other in the nucleus, while those of Stachyamoeba lipophora have a more irregular outline and are not consistently placed opposite each other in the nucleus. Also, the nucleoli of Sawyeria marylandensis persist through mitosis, while those of Stachyamoeba lipophora disintegrate (Page
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Figure 16. The phylogenetic tree of heterolobosean SSU rRNA gene sequences inferred from maximum likelihood analysis with the corresponding distance, parsimony, and maximum likelihood bootstrap values (only values greater than 50% are indicated). The two new heterolobosean genera are highlighted in bold. The evolutionary distance for the number of changes per site is represented by the scale bar.
1975). The observation that some Sawyeria marylandensis nuclei have a single nucleolus suggests that nucleolar division in this species occurs early in interphase. In other Heterolobosea, nucleoli either divide or disintegrate during mitotic prophase (Page and Blanton 1985). Monopylocystis visvesvarai is the only anaerobic heterolobosean protist known to have a cyst. The
single protruding plugged pore represents a pore type not previously recorded for the Heterolobosea. The ribosomal RNA gene sequences of Monopylocystis visvesvarai and Sawyeria marylandensis are closely related. Along with Psalteriomonas lanterna they form a clade that includes all of the non-aerobic Heterolobosea. The clade probably also includes Vahlkampfia anaerobica, which may represent a
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second species of Monopylocystis; it is already known that the genus Vahlkampfia, as defined by morphological criteria (Page 1991; Patterson et al. 2000), is polyphyletic (Brown and De Jonckheere 1999). The most parsimonious interpretation of this result is that, within the heteroloboseans, all hydrogenosome-bearing microaerophiles evolved from a single ancestor that gave rise to species with divergent morphological features. On the other hand, anaerobiosis and hydrogenosomes clearly evolved several times among eukaryotes (Embley et al. 1997; Hackstein and Vogels 1997), including several times within the Ciliophora (Embley et al. 1995). Further taxon sampling within the Heterolobosea may reveal other clades that include both aerobic and non-aerobic (microaerophilic or strictly anaerobic) species.
Diagnoses Monopylocystis gen. nov. Uninucleate, bacterivorous, anaerobic or microaerophilic heterolobosean amoebae. Locomotive cells advancing by typical hemispherical hyaloplasmic bulges. Nucleus more or less angular in outline; nucleolar material peripheral, not central, distributed in a thin ring near the nuclear membrane. Golgi body and mitochondria absent, hydrogenosome-like organelles present. Cysts possessing a single pore plugged with gelatinous material. Differentiated from other nonaerobic heterolobosean amoebae by the angular outline of the nucleus, the peripheral distribution of nucleolar material, and the monoporate cyst. Monopylocystis visvesvarai sp. nov. Marine amoeba. Locomotive amoebae 15–30 µm long and 7.5–12.5 µm wide. Cyst with an unmineralized and unornamented wall, diameter 7.5–13.5 µm. Flagellated stages not observed. Differentiated from Vahlkampfia anaerobica Smirnov and Fenchel 1996, by the absence of distinctive uroidal structures and rayed floating forms, and by the angular nuclear outline. Type locality: Sippewissett salt marsh, Falmouth, Massachusetts, USA. Syntypes: Cryopreserved living material, conserved at the American Type Culture Collection (ATCC) as strain 50576. Resin-embedded cells derived from strain 50576 conserved at the Natural History Museum, Smithsonian Institution. Etymology: Monopylocystis, Greek “one plug cyst”, referring to the single plug in the cyst wall; visvesvarai, honoring Dr. Govinda Visvesvara, a leader in free-living amoeba biology.
Sawyeria gen. nov. Uninucleate, bacterivorous, anaerobic or microaerophilic heterolobosean amoebae. Locomotive cells advancing by typical hemispherical hyaloplasmic bulges. Nucleus with one or two parietal nucleoli, often distributed as two approximately equally sized pieces apparently at opposite poles of the nucleus. Golgi body and mitochondria absent, hydrogenosome-like organelles present. Differentiated from other nonaerobic Heterolobosea by the presence of discrete parietal nucleoli. Sawyeria marylandensis sp. nov. Freshwater amoeba. Locomotive amoebae 25–41 µm long and 7–12 µm wide. Nuclear diameter 4–5 µm. Cyst and flagellated stages not observed. Type locality: Iron rich creek emptying into the Potomac River at Great Falls, Maryland, USA. Syntypes: Cryopreserved living material, conserved at the American Type Culture Collection (ATCC) as strain 50653. Resin-embedded cells derived from strain 50653 conserved at the Natural History Museum, Smithsonian Institution. Etymology: Sawyeria, honoring Dr. Thomas K. Sawyer, an internationally recognized expert in the taxonomy of amoebae; marylandensis, referring to the State of Maryland, United States of America, in which the amoeba was isolated.
Methods Isolation, cultivation and cryopreservation: Monopylocystis visvesvarai n. sp. was isolated from a sediment sample taken from Sippewissett Salt Marsh in Falmouth, MA. The amoebae were cultivated xenically in ATCC medium 1525 (Nerad 1993) bacterized with Klebsiella pneumoniae subsp. pneumoniae ATCC 700831. Sawyeria marylandensis n. sp. was isolated from a water sample taken from an iron rich creek emptying into the Potomac River at Great Falls, MD. The amoebae were cultivated xenically in ATCC medium 802 (Nerad 1993) bacterized with Klebsiella pneumoniae subsp. pneumoniae ATCC 700831. Both amoebae were maintained in 16 × 125 mm screwcapped test tubes containing 13 ml of medium. Caps were screwed on tightly and the tubes were incubated at 25 C on a 15 degree horizontal slant. Continuous cultures were maintained by transferring 1 ml of growing amoebae to fresh media on a weekly basis. Both strains were cryopreserved using procedures described by Nerad and Daggett (1992). Light microscopy: Light microscopical observations, on live and Kernichrot stained (Page 1975)
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cells, were made with Zeiss Axioskop compound microscopes outfitted for bright field, phase contrast and differential interference contrast optics. Measurements of live cells were made using a calibrated ocular micrometer. Images were captured electronically with either an Optonics DEI-470 CCD Camera or a Sony DKC-CM30 Digital Still Camera. Transmission electron microscopy: Cells were fixed, serially sectioned, stained and examined using procedures slightly modified from those described in detail elsewhere (O’Kelly 1997; O’Kelly and Patterson 1996). Briefly, exponential-phase cells were fixed in a cacodylate-buffered glutaraldehyde-osmium cocktail for 15 min at room temperature (ca. 20 °C). Fixed cells were filtered onto cellulose acetate/nitrate wafers, washed with distilled water, enrobed in agar, dehydrated in a graded acetone series and embedded in Spurr resin. Blocks were trimmed by hand, serially sectioned with a diamond knife, mounted on naked 600-mesh grids or formvarcoated slots, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 902 transmission electron microscopes operating at 80 kV. DNA isolation, amplification and sequencing: Genomic DNA from Monopylocystis was isolated, and the SSU rRNA gene amplified and sequenced as described elsewhere (Silberman et al. 2002). Phylogenetic analyses: The SSU rRNA gene sequence from Monopylocystis visvesvarai (GenBank accession number AF011463) was aligned with all available heterolobosean sequences in GenBank. Conserved SSU rRNA secondary structure motifs assisted in the alignment of gene sequences. A subset of these heterolobosean sequences and selected representative sequences from non-heterolobosean eukaryotes were chosen for phylogenetic analyses to root the heteroloboseans. This data set included only the most completely determined, published sequences for each of the major heterolobosean genera. A second subset, including only heterolobosean taxa in order to maximize the number of homologous positions available, was chose for a finer-scale analysis. Phylogenetic trees were inferred using maximum likelihood, distance and parsimony criteria. All analyses employed PAUP* version 4.0b10 (Swofford 1998). The rooted data set consisted of 19 taxa and 1,130 aligned positions while the fine-scale data set consisted of 14 taxa and 1,176 aligned positions. To decide on the evolutionary model that best fit the data, likelihood ratio tests (LRT) were performed with Modeltest v. 3.0.6 (Posada and Crandall 1998). The General Time Re-
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versible model incorporating a correction for site-tosite rate heterogeneity plus an estimate of invariable sites (GTR + Γ + I) was chosen as the simplest model that best fit the data for the rooted data set while the Tamura Nei model incorporating a correction for site-to-site rate heterogeneity (TrN + Γ) was chosen for the fine-scale data set (see Swofford et al. (1996) for a more detailed explanation of the various models of evolution). These models of nucleotide substitution were utilized in maximum likelihood and maximum likelihood distance (ml-distance) tree reconstruction. Parsimony analyses were run with gaps treated as missing data, and starting trees obtained using stepwise-addition, random sequence addition, and 10 random addition subreplicates, and the tree-bisection-reconnection branchswapping algorithm. The confidence of topological elements in phylogenies was assessed using 1000 bootstrap resamplings of the rooted data set and 100 resamplings of the fine-scale data set. Internet data dissemination: Updated summaries on the morphology, taxonomy and biology of Sawyeria and Monopylocystis are available from the Protist Image Data Web site (http://megasun.bch. umontreal.ca/protists/excavates/).
Acknowledgements Supported in part by grants from the National Science Foundation (TAN), the Office of Naval Research (CJO), NASA Astrobiology Institute, Vettlesen Foundation (LAZ, JDS, MLS).
References Biagini GA, Bernard C (2000) Primitive anaerobic protozoa: a false concept? Microbiology 146: 1019–1020 Broers CAM, Stumm CK, Vogels GD, Brugerolle G (1990) Psalteriomonas lanterna gen. nov., sp. nov., a free-living amoeboflagellate isolated from freshwater anaerobic sediments. Europ J Protistol 25: 369–380 Broers CAM, Meijers HHM, Symens JC, Stumm CK, Vogels GD, Brugerolle G (1993) Symbiotic association of Psalteriomonas vulgaris n. spec. with Methanobacterium formicicum. Europ J Protistol 29: 98–105 Brown S, De Jonckheere JF (1999) A reevaluation of the amoeba genus Vahlkampfia based on SSU rDNA sequences. Europ J Protistol 35: 49–54 Brul S, Veltman RH, Lombardo MC, Vogels GD (1994) Molecular cloning of hydrogenosomal ferredoxin cDNA from the anaerobic amoeboflagellate Psalteriomonas lanterna. Biochim Biophys Acta 1183: 544–546
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Carey PG, Page FC (1985) A light and electron microscopic study of the marine amoeboflagellate Heteramoeba clara. Arch Protistenkd 130: 313–328
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Embley TM, Horner DS, Hirt RP (1997) Anaerobic eukaryotic evolution: hydrogenosomes as biochemically modified mitochondria? Trends Ecol Evol 12: 437–441 Embley TM, Finlay BJ, Dyal PL, Hirt RP, Wilkinson M, Williams AG (1995) Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates. Proc R Soc Lond B Biol Sci 262: 87–93 Hackstein JHP, Vogels GD (1997) Endosymbiotic interactions in anaerobic protozoa. Antonie Van Leeuwenhoek 71: 151–158 Nerad TA, Daggett P-M (1992) Cryopreservation of Diplomonads other than Giardia. In Lee JJ, Soldo AT (eds) Protocols in Protozoology, vol. 1. Society of Protozoologists, Lawrence, Kansas, pp A70.1–A70.2 O’Kelly CJ (1997) Ultrastructural features of Reclinomonas americana Flavin and Nerad, 1993 (Histionidae): trophic cells, zoospores, and cysts. Europ J Protistol 33: 337–348 O’Kelly CJ, Patterson DJ (1996) The flagellar apparatus of Cafeteria roenbergensis Fenchel and Patterson, 1988 (Bicosoecales = Bicosoecida). Europ J Protistol 32: 216–226 Page FC (1975) A new family of amoebae with fine pseudopodia. Zool J Linn Soc 56: 73–89 Page FC (1987) Transfer of Stachyamoeba lipophora to the class Heterolobosea. Arch Protistenkd 133: 191–197
Patterson DJ, Rogerson A, Vørs N (2000) Class Heterolobosea. In Lee JJ, Leedale GF, Bradbury P (eds) Illustrated Guide to the Protozoa, 2nd ed. Society of Protozoologists, Lawrence, Kansas, pp 1104–1111 Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818 Silberman JD, Simpson AGB, Kulda J, Cepicka I, Hampl V, Johnson PJ, Roger AJ (2002) Retortamonad flagellates are closely related to diplomonads – Implications for the history of mitochondrial function in eukaryote evolution. Mol Biol Evol 19: 777–786 Smirnov AV, Fenchel T (1996) Vahlkampfia anaerobica n. sp. and Vannella peregrinia n. sp. (Rhizopoda): anaerobic amoebae from a marine sediment. Arch Protistenkd 147: 189–198 Swofford DL (1998) PAUP*. Phylogenetic analyses using parsimony (*and other methods). Sinauer Associates, Sunderland, Massachusetts Swofford DL, Olsen GJ, Waddell PJ, Hillis DM (1996) Phylogenetic Inference. In Hillis DM, Moritz C, Mable BK (eds) Molecular Systematics. Sinauer Associates, Sunderland, Massachusetts, pp 407–514 Weekers PHH, Kleyn J, Vogels GD (1997) Phylogenetic position of Psalteriomonas lanterna deduced from the SSU rDNA sequence. J Euk Microbiol 44: 467–470
Protist
ORIGINAL PAPER
Monopylocystis visvesvarai n. gen., n. sp. and Sawyeria marylandensis n. gen., n. sp.: Two New Amitochondrial Heterolobosean Amoebae from Anoxic Environments Charles J. O’Kelly a, Jeffrey D. Silbermanb, Linda A. Amaral Zettler c, Thomas A. Nerad d, and Mitchell L. Sogin c,1 a
Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575, USA Department of Microbiology, Immunology, Molecular Genetics, and Institute of Geophysics and Planetary Physics and Department of Microbiology and Immunology, University of California at Los Angeles, Los Angeles, CA 90095, USA c The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02543, USA d Protistology Department, American Type Culture Collection, Manassas, Virginia 20110-2209, USA b
Submitted January 29, 2003; Accepted April 22, 2003 Monitoring Editor: Michael Melkonian
Two new species of heterolobosean amoebae from anoxic environments, Monopylocystis visvesvarai and Sawyeria marylandensis, are described on the basis of light microscopy, electron microscopy, and their phylogenetic affiliation based on analyses of nuclear small-subunit ribosomal RNA gene sequences. Both species lack mitochondria but have organelles provisionally interpreted as hydrogenosomes, and neither can tolerate aerobic conditions. As their conditions of culture do not exclude all oxygen, they may be microaerophiles rather than strict anaerobes. Both species have unusual nucleolar morphologies. Monopylocystis visvesvarai, from a marine sediment, has nucleolar material distributed around the nuclear periphery. It is the first non-aerobic heterolobosean protist for which a cyst is known; the cyst is unmineralized and unornamented except for a single, raised, plugged pore. Sawyeria marylandensis, from an iron-rich freshwater stream, has nucleolar material distributed in one or two parietal masses, which persist during mitosis. In phylogenetic analyses of small-subunit rRNA gene sequences, Monopylocystis visvesvarai, Sawyeria marylandensis and Psalteriomonas lanterna converge to form a single clade of non-aerobic (anaerobic/microaerophilic) heteroloboseans.
Introduction For the most part, the class Heterolobosea (Page 1991; Page and Blanton 1985; Patterson et al. 2000) contains amoebae, amoeboflagellates, and 1
Corresponding author; fax 1508 457 4727 e-mail [email protected]
flagellates that have mitochondria and are, consequently, aerobes. To date, only three non-aerobic species are known by modern methods to belong to the class, all of which have been described within the last two decades. These species are Psalteriomonas lanterna Broers et al. 1990; an amoeboflagellate (Broers et al. 1990), P. vulgaris Broers et al. 1434-4610/03/154/02-281 $ 15.00/0
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1993 (= Lyromonas vulgaris (Broers et al. 1993) Cavalier-Smith 1993), a flagellate (Broers et al. 1993; Cavalier-Smith 1993), and Vahlkampfia anaerobica Smirnov and Fenchel 1996; an amoeba (Smirnov and Fenchel 1996). All of these protists lack mitochondria but have organelles interpreted as hydrogenosomes. Psalteriomonas lanterna occupies a unique phylogenetic position within the Heterolobosea, according to analyses of its nuclear small-subunit (SSU) ribosomal RNA gene sequence (Weekers et al. 1997). Neither of the other anaerobic species has been examined at the molecular level. In the course of investigations on the protistan biota of anaerobic sediments, two anaerobes have been found that are intolerant of aerobic conditions. These amoebae have morphological similarities to other Heterolobosea but have unusual nucleolar morphology. They can be assigned to no known species on the basis of morphological or molecular sequence data. Therefore they are described here as Monopylocystis visvesvarai n. gen., n. sp., and Sawyeria marylandensis n. gen., n. sp. In a previous study (Silberman et al. 2002), the SSU rRNA gene sequence of Sawyeria marylandensis formed a robust clade with Psalteriomonas lanterna in phylogenetic analyses. Here, the SSU rRNA gene from Monopylocystis visvesvarai is added to the analysis. It was found that all three nonaerobes form a single clade within the predominantly aerobic heterolobosean lineage.
Results Morphology of Monopylocystis visvesvarai Monopylocystis visvesvarai produced gymnamoebae and cysts in culture. Locomotive amoebae were of the “limax” type, with an anterior, eruptive hyaline front (Figs 1, 2). They were 17–30 µm long (mean 22 ± 3.7 µm, n = 25) and 6.0–10 µm (mean 8.0 ± 1.4 µm, n = 25) wide. The mean length-width ratio was 2.7. They were “naked” (Fig. 3), lacking tests, scales, or detectable glycocalyx. Non-locomotive cells when attached were circular in outline, and when floating were essentially spherical. No rayed floating forms were observed. The amoebae were active only under anaerobic conditions, ceasing activity and losing cell integrity on exposure to oxygen-containing media. The cells were uninucleate (Figs 1–2). The nuclei had an irregularly polygonal outline (Figs 1, 2, 4). Electron-dense material, interpreted as nucleolar, was appressed to the nuclear envelope (Fig. 4). No other nucleolar material was present. Rough endo-
plasmic reticulum was usually associated with the nucleus, but did not form a complete investment (Fig. 4). Nuclear division was not observed. No Golgi bodies or microbodies were seen. Mitochondria were not observed, but spherical to bacilliform, membrane-bound bodies with uniformly dense contents were scattered throughout the cytoplasm (Figs 4, 5). Food vacuoles contained bacteria (Fig. 2). Cysts were spherical, with a single prominent pore (Fig. 6). They measured 7.5 –13.5 µm (mean 11 ± 1.4 µm, n = 25) in diameter. The cyst wall was thin, unornamented, and unmineralized (Fig. 7). The pore was a protruding aperture in the wall, occluded by a plug of fibrillar material interspersed with dark blocks (Fig. 7).
Morphology of Sawyeria marylandensis Sawyeria marylandensis produced only gymnamoebae in culture. Locomotive amoebae were of the “limax” type, with an anterior, eruptive hyaline front (Figs 8, 9). They were 25–41 µm (mean 31.4 ± 3.2 µm, n = 29) long and 7–12 µm (mean 8.1 ± 1.0 µm, n = 29) wide. The length-width ratio was 3.9. Non-locomotive cells when attached were essentially circular in outline (Fig. 10), and when floating were essentially spherical. No rayed floating forms were observed. The amoebae were “naked” (Fig. 11), lacking tests, scales, or detectable glycocalyx. They were active only under anaerobic conditions, ceasing activity and losing cell integrity on exposure to oxygen-containing media. The cells were uninucleate (Figs 8–10, 12). The nuclei were spherical to slightly ellipsoidal (Figs 8–10) and had either one (Fig. 10) or two (Figs 8–9, 12) peripheral nucleoli that were associated with polar lobes of the nucleus and appressed to the nuclear envelope (Fig. 12). Mean nuclear diameter was 4–5 µm. Rough endoplasmic reticulum usually formed a complete investment around the interphase nucleus (Fig. 12). Dividing nuclei exhibited a cruciform pattern consisting of persistent nucleoli lateral to the chromosomal plate; the nuclear envelope was closed (Fig. 13). No Golgi bodies or microbodies were seen. Mitochondria were not observed, but bacilliform, membrane-bound bodies with marbled contents were scattered throughout the cytoplasm (Figs 11, 14). Food vacuoles contained bacteria (Fig. 12).
Phylogenetic Analyses Figure 15 presents the optimal maximum likelihood tree from a SSU rRNA gene data set containing selected Heterolobosea and non-heterolobsean eu-
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Figures 1–7. Light and electron micrographs of Monopylocystis visvesvarai. 1. Locomotive amoeba with anteriorly erumpent hyaloplasmic bulge. Direction of motion is towards the right of the image. Nucleus with angular outline and without a central nucleolus. Scale bar = 5 µm. 2. Locomotive amoeba with laterally erumpent hyaloplasmic bulge and numerous food vacuoles. Direction of motion is towards the right of the image. Scale bar = 5 µm. 3. Cell membrane of locomotive amoeba. No surface structures are evident. Scale bar = 100 nm. 4. Nucleus and associated organelles in the locomotive amoeba. Nucleus with angular outline and parietal encircling nucleolar material. Rough endoplasmic reticulum cisternae are in the vicinity of the nucleus but do not envelop it. Hydrogenosome-like organelles and food vacuoles are present. Scale bar = 500 nm. 5. Detail of hydrogenosomelike organelle. Scale bar = 250 nm. 6. Cyst with single protruding pore. Scale bar = 5 µm. 7. Detail of cyst, with nonmineralized cyst wall and protruding pore with plug. Scale bar = 1.0 µm. Abbreviations for Figure 1–14: b, bacteria; cp, chromosomal plate of mitotic spindle; fv, food vacuole; h, hydrogenosome-like organelle; n, nucleus; no, nucleolus or nucleolar material; p, cyst pore; t, trailing end of amoeba.
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karyotes, with bootstrap values greater than 50% indicated at the nodes. The heteroloboseans, inclusive of Monopylocystis visvesvarai and Sawyeria marylandensis form a monophyletic assemblage supported with 100% bootstrap values (maximum likelihood, distance and parsimony). Figure 16 shows an unrooted maximum likelihood phylogenetic tree, generated from a fine-scale dataset containing only sequences from Heterolobosea, with bootstrap values from distance, parsimony and maximum likelihood analyses. All phylogenetic inferences from this dataset yielded the same tree topology. The analyses demonstrated a sister relationship between Psalteriomonas lanterna and Sawyeria marylandensis. Furthermore, Psalteriomonas lanterna plus Sawyeria marylandensis along with Monopylocystis visvesvarai formed a robust clade with bootstrap values of 100% for all methods tested. Sawyeria marylandensis, Monopylocystis visvesvarai and Psalteriomonas lanterna formed part of a larger clade that included Paravahlkampfia ustiana and Neovahlkampfia damariscottae with good bootstrap support (89/91/83).
Discussion The two amoebae described here are assigned to the Heterolobosea on the basis of the limax form of their locomotive amoebae, their position in phylogenetic trees based on nuclear small-subunit ribosomal RNA gene sequences, and, as seen in Sawyeriamarylandensis, the closed, cruciform mitosis. They are distinct from other Heterolobosea in several ways. Both Monopylocystis visvesvarai and Sawyeria marylandensis are intolerant of normal aerobic environmental conditions. However, as the methods used here probably do not remove all oxygen from
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the culture medium, it is more likely that these protists are microaerophiles (Biagini and Bernard 2000), tolerating and possibly requiring trace amounts of oxygen, rather than strict anaerobes. Both species lack mitochondria with detectable cristae, but have organelles that are similar to mitochondria in shape and size, as do the other known heterolobosean anaerobes, the Psalteriomonas species (Broers et al. 1990, 1993) and Vahlkampfia anaerobica Smirnov and Fenchel (Smirnov and Fenchel 1996). The precise structural features of these organelles, particularly the matrix (which may appear homogeneous or variously heterogeneous) and the number of bounding membranes, are difficult to discern with accuracy, since these features are susceptible to, and vary with, conditions of fixation (Smirnov and Fenchel 1996). The organelles of Psalteriomonas are considered to be hydrogenosomes on the basis of their hydrogenase activity (Broers et al. 1993; Brul et al. 1994). Pending physiological investigations, the analogous organelles of Monopylocystis visvesvarai and Sawyeria marylandensis (as well as those of Vahlkampfia anaerobica) are likewise interpreted as hydrogenosomes. Both Monopylocystis visvesvarai and Sawyeria marylandensis have unusual nucleolar morphologies. Almost all described Heterolobosea have one centrally-located vesicular nucleolus in the interphase nucleus. However, Vahlkampfia anaerobica (Smirnov and Fenchel 1996), and the marine aerobe Heteramoeba clara (Carey and Page 1985), have their nucleolar material distributed along the nuclear periphery, and in the freshwater aerobe Stachyamoeba lipophora (Page 1975, 1987) the nucleolus is present as one, two or three parietal masses. The nucleolar organization of Monopylocystis visvesvarai is similar to that of Heteramoeba clara and Vahlkampfia anaerobica. The latter species is a marine anaerobe; its locomotive amoebae are simi-
Figures 8–14. Light and electron micrographs of Sawyeria marylandensis. 8. Locomotive amoebae in straight-line motion, direction towards the right of the image. The trailing end of locomotive amoebae is frequently slightly bulbous, as here. Nucleus with two nucleoli. Scale bar = 5 µm. 9. Locomotive amoeba in the process of changing direction; former direction towards the top left corner of the image, new direction towards the upper right corner. Bacteria are sometimes associated with the trailing end of the amoeba, as here. Nucleus with two nucleoli. Scale bar = 5 µm. 10. Quiescent amoeba, flattened under coverslip pressure. Nucleus with one nucleolus. Hydrogenosome-like organelles, normally bacilliform, here appear rounded. Scale bar = 5 µm. 11. Cell membrane of locomotive amoeba. No surface structures are evident. A cross-sectional profile of a hydrogenosome-like organelle is present. Scale bar = 250 nm. 12. Nucleus with two parietal nucleoli and apparently complete investment of rough endoplasmic reticulum. Food vacuoles contain bacteria. Scale bar = 500 nm. 13. Nuclear division. The nuclear envelope is closed. Persistent peripheral nucleoli lie alongside the chromosomal plate. Cell fixed and stained according to the Kernichrot procedure. Scale bar = 2 µm. 14. Group of hydrogenosome-like organelles. Scale bar = 500 nm.
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Figure 15. The optimal maximum likelihood tree based on SSU rRNA gene sequence comparisons. Bootstrap values greater than 50% (1000 replicates) for maximum likelihood, ml-distance, and parsimony (respectively) are indicated at the nodes. An asterisk (*) indicates bootstrap values below 50%. The scale bar represents evolutionary distance for the number of changes per site.
lar in dimensions to those of Monopylocystis visvesvarai. The nuclear outline of Vahlkampfia anaerobica is, however, less angular than that of Monopylocystis visvesvarai, and the nucleus in the latter species lacks the close investment of rough endoplasmic reticulum that is present in the former. Also, the uroidal structures and rayed floating forms present in Vahlkampfia anaerobica have not been observed in Monopylocystis visvesvarai, and no cyst stage has been observed in Vahlkampfia anaerobica.
The nucleolar organization of Sawyeria marylandensis is similar to that of Stachyamoeba lipophora. However, the nucleoli of Sawyeria marylandensis have a smooth outline and are consistently found opposite each other in the nucleus, while those of Stachyamoeba lipophora have a more irregular outline and are not consistently placed opposite each other in the nucleus. Also, the nucleoli of Sawyeria marylandensis persist through mitosis, while those of Stachyamoeba lipophora disintegrate (Page
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Figure 16. The phylogenetic tree of heterolobosean SSU rRNA gene sequences inferred from maximum likelihood analysis with the corresponding distance, parsimony, and maximum likelihood bootstrap values (only values greater than 50% are indicated). The two new heterolobosean genera are highlighted in bold. The evolutionary distance for the number of changes per site is represented by the scale bar.
1975). The observation that some Sawyeria marylandensis nuclei have a single nucleolus suggests that nucleolar division in this species occurs early in interphase. In other Heterolobosea, nucleoli either divide or disintegrate during mitotic prophase (Page and Blanton 1985). Monopylocystis visvesvarai is the only anaerobic heterolobosean protist known to have a cyst. The
single protruding plugged pore represents a pore type not previously recorded for the Heterolobosea. The ribosomal RNA gene sequences of Monopylocystis visvesvarai and Sawyeria marylandensis are closely related. Along with Psalteriomonas lanterna they form a clade that includes all of the non-aerobic Heterolobosea. The clade probably also includes Vahlkampfia anaerobica, which may represent a
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second species of Monopylocystis; it is already known that the genus Vahlkampfia, as defined by morphological criteria (Page 1991; Patterson et al. 2000), is polyphyletic (Brown and De Jonckheere 1999). The most parsimonious interpretation of this result is that, within the heteroloboseans, all hydrogenosome-bearing microaerophiles evolved from a single ancestor that gave rise to species with divergent morphological features. On the other hand, anaerobiosis and hydrogenosomes clearly evolved several times among eukaryotes (Embley et al. 1997; Hackstein and Vogels 1997), including several times within the Ciliophora (Embley et al. 1995). Further taxon sampling within the Heterolobosea may reveal other clades that include both aerobic and non-aerobic (microaerophilic or strictly anaerobic) species.
Diagnoses Monopylocystis gen. nov. Uninucleate, bacterivorous, anaerobic or microaerophilic heterolobosean amoebae. Locomotive cells advancing by typical hemispherical hyaloplasmic bulges. Nucleus more or less angular in outline; nucleolar material peripheral, not central, distributed in a thin ring near the nuclear membrane. Golgi body and mitochondria absent, hydrogenosome-like organelles present. Cysts possessing a single pore plugged with gelatinous material. Differentiated from other nonaerobic heterolobosean amoebae by the angular outline of the nucleus, the peripheral distribution of nucleolar material, and the monoporate cyst. Monopylocystis visvesvarai sp. nov. Marine amoeba. Locomotive amoebae 15–30 µm long and 7.5–12.5 µm wide. Cyst with an unmineralized and unornamented wall, diameter 7.5–13.5 µm. Flagellated stages not observed. Differentiated from Vahlkampfia anaerobica Smirnov and Fenchel 1996, by the absence of distinctive uroidal structures and rayed floating forms, and by the angular nuclear outline. Type locality: Sippewissett salt marsh, Falmouth, Massachusetts, USA. Syntypes: Cryopreserved living material, conserved at the American Type Culture Collection (ATCC) as strain 50576. Resin-embedded cells derived from strain 50576 conserved at the Natural History Museum, Smithsonian Institution. Etymology: Monopylocystis, Greek “one plug cyst”, referring to the single plug in the cyst wall; visvesvarai, honoring Dr. Govinda Visvesvara, a leader in free-living amoeba biology.
Sawyeria gen. nov. Uninucleate, bacterivorous, anaerobic or microaerophilic heterolobosean amoebae. Locomotive cells advancing by typical hemispherical hyaloplasmic bulges. Nucleus with one or two parietal nucleoli, often distributed as two approximately equally sized pieces apparently at opposite poles of the nucleus. Golgi body and mitochondria absent, hydrogenosome-like organelles present. Differentiated from other nonaerobic Heterolobosea by the presence of discrete parietal nucleoli. Sawyeria marylandensis sp. nov. Freshwater amoeba. Locomotive amoebae 25–41 µm long and 7–12 µm wide. Nuclear diameter 4–5 µm. Cyst and flagellated stages not observed. Type locality: Iron rich creek emptying into the Potomac River at Great Falls, Maryland, USA. Syntypes: Cryopreserved living material, conserved at the American Type Culture Collection (ATCC) as strain 50653. Resin-embedded cells derived from strain 50653 conserved at the Natural History Museum, Smithsonian Institution. Etymology: Sawyeria, honoring Dr. Thomas K. Sawyer, an internationally recognized expert in the taxonomy of amoebae; marylandensis, referring to the State of Maryland, United States of America, in which the amoeba was isolated.
Methods Isolation, cultivation and cryopreservation: Monopylocystis visvesvarai n. sp. was isolated from a sediment sample taken from Sippewissett Salt Marsh in Falmouth, MA. The amoebae were cultivated xenically in ATCC medium 1525 (Nerad 1993) bacterized with Klebsiella pneumoniae subsp. pneumoniae ATCC 700831. Sawyeria marylandensis n. sp. was isolated from a water sample taken from an iron rich creek emptying into the Potomac River at Great Falls, MD. The amoebae were cultivated xenically in ATCC medium 802 (Nerad 1993) bacterized with Klebsiella pneumoniae subsp. pneumoniae ATCC 700831. Both amoebae were maintained in 16 × 125 mm screwcapped test tubes containing 13 ml of medium. Caps were screwed on tightly and the tubes were incubated at 25 C on a 15 degree horizontal slant. Continuous cultures were maintained by transferring 1 ml of growing amoebae to fresh media on a weekly basis. Both strains were cryopreserved using procedures described by Nerad and Daggett (1992). Light microscopy: Light microscopical observations, on live and Kernichrot stained (Page 1975)
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cells, were made with Zeiss Axioskop compound microscopes outfitted for bright field, phase contrast and differential interference contrast optics. Measurements of live cells were made using a calibrated ocular micrometer. Images were captured electronically with either an Optonics DEI-470 CCD Camera or a Sony DKC-CM30 Digital Still Camera. Transmission electron microscopy: Cells were fixed, serially sectioned, stained and examined using procedures slightly modified from those described in detail elsewhere (O’Kelly 1997; O’Kelly and Patterson 1996). Briefly, exponential-phase cells were fixed in a cacodylate-buffered glutaraldehyde-osmium cocktail for 15 min at room temperature (ca. 20 °C). Fixed cells were filtered onto cellulose acetate/nitrate wafers, washed with distilled water, enrobed in agar, dehydrated in a graded acetone series and embedded in Spurr resin. Blocks were trimmed by hand, serially sectioned with a diamond knife, mounted on naked 600-mesh grids or formvarcoated slots, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 902 transmission electron microscopes operating at 80 kV. DNA isolation, amplification and sequencing: Genomic DNA from Monopylocystis was isolated, and the SSU rRNA gene amplified and sequenced as described elsewhere (Silberman et al. 2002). Phylogenetic analyses: The SSU rRNA gene sequence from Monopylocystis visvesvarai (GenBank accession number AF011463) was aligned with all available heterolobosean sequences in GenBank. Conserved SSU rRNA secondary structure motifs assisted in the alignment of gene sequences. A subset of these heterolobosean sequences and selected representative sequences from non-heterolobosean eukaryotes were chosen for phylogenetic analyses to root the heteroloboseans. This data set included only the most completely determined, published sequences for each of the major heterolobosean genera. A second subset, including only heterolobosean taxa in order to maximize the number of homologous positions available, was chose for a finer-scale analysis. Phylogenetic trees were inferred using maximum likelihood, distance and parsimony criteria. All analyses employed PAUP* version 4.0b10 (Swofford 1998). The rooted data set consisted of 19 taxa and 1,130 aligned positions while the fine-scale data set consisted of 14 taxa and 1,176 aligned positions. To decide on the evolutionary model that best fit the data, likelihood ratio tests (LRT) were performed with Modeltest v. 3.0.6 (Posada and Crandall 1998). The General Time Re-
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versible model incorporating a correction for site-tosite rate heterogeneity plus an estimate of invariable sites (GTR + Γ + I) was chosen as the simplest model that best fit the data for the rooted data set while the Tamura Nei model incorporating a correction for site-to-site rate heterogeneity (TrN + Γ) was chosen for the fine-scale data set (see Swofford et al. (1996) for a more detailed explanation of the various models of evolution). These models of nucleotide substitution were utilized in maximum likelihood and maximum likelihood distance (ml-distance) tree reconstruction. Parsimony analyses were run with gaps treated as missing data, and starting trees obtained using stepwise-addition, random sequence addition, and 10 random addition subreplicates, and the tree-bisection-reconnection branchswapping algorithm. The confidence of topological elements in phylogenies was assessed using 1000 bootstrap resamplings of the rooted data set and 100 resamplings of the fine-scale data set. Internet data dissemination: Updated summaries on the morphology, taxonomy and biology of Sawyeria and Monopylocystis are available from the Protist Image Data Web site (http://megasun.bch. umontreal.ca/protists/excavates/).
Acknowledgements Supported in part by grants from the National Science Foundation (TAN), the Office of Naval Research (CJO), NASA Astrobiology Institute, Vettlesen Foundation (LAZ, JDS, MLS).
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Patterson DJ, Rogerson A, Vørs N (2000) Class Heterolobosea. In Lee JJ, Leedale GF, Bradbury P (eds) Illustrated Guide to the Protozoa, 2nd ed. Society of Protozoologists, Lawrence, Kansas, pp 1104–1111 Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818 Silberman JD, Simpson AGB, Kulda J, Cepicka I, Hampl V, Johnson PJ, Roger AJ (2002) Retortamonad flagellates are closely related to diplomonads – Implications for the history of mitochondrial function in eukaryote evolution. Mol Biol Evol 19: 777–786 Smirnov AV, Fenchel T (1996) Vahlkampfia anaerobica n. sp. and Vannella peregrinia n. sp. (Rhizopoda): anaerobic amoebae from a marine sediment. Arch Protistenkd 147: 189–198 Swofford DL (1998) PAUP*. Phylogenetic analyses using parsimony (*and other methods). Sinauer Associates, Sunderland, Massachusetts Swofford DL, Olsen GJ, Waddell PJ, Hillis DM (1996) Phylogenetic Inference. In Hillis DM, Moritz C, Mable BK (eds) Molecular Systematics. Sinauer Associates, Sunderland, Massachusetts, pp 407–514 Weekers PHH, Kleyn J, Vogels GD (1997) Phylogenetic position of Psalteriomonas lanterna deduced from the SSU rDNA sequence. J Euk Microbiol 44: 467–470