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Ultrastructure and 18S rRNA gene sequence of a small Heterotrophic flagellate Siluania monomastiga gen. et sp. nov. (bicosoecida)
Europ. J. Protisto !. 34 , 4 15--425 (1998) December 7, 1998
European Journal o f
PROTISTOLOGY
Ultrastructure and 18S rRNA Gene Sequence of a Small Heterotrophic Flagellate Siluania monomastiga gen. et sp. nov. (Bicosoecida) Serguei A. Karpov ':". Ralf Kersanach-> " and David M. Williams 2 1Biological
Research Institute of 51. Petersburg State University, Oranienbaumskoye sch. 2, 19890451. Petersburg, Russia -Bot any Department, Cromwell Road, Natural History Museum, London SW7 5BD, UK
Summary
Introduction
Siluania monomastiga gen . et sp. no v. - one of the sm allest fre e-living heterotrophic eukaryotes (1.5-3.0 pm length) h as been investigated using SEM and TEM, and by gene sequence analysis of the small su bu n it rRNA. The flagellate is co vered by only the pla smalemma. The single flagellum h as a unilateral array of tubular ma stigonemes; the flagellar basal body is only composed of doublets of microtubules. One or two additional very short basal bodies may also be present, com posed of single microtubules, of amorphous electron dense material. There is a well developed cyto stome with permanent pharynx, supported by rootlet microtubules and a fibrillar sheet. One mitochondrion wit h tubular cristae contains a spherical body in the centre. D eveloping mastigonemes are located within the perinuclear space. The Golgi apparatus consists of a single diet yo some. The rootlet system of the flagellum is represented by three microtubular bands, numbered as rl, r3 and r4 in accordance with the protocol accepted for biosoecids, and by the fibrillar rootlet having two strands. The cytoskeleton structure and organelle dispos ition of S.monomastiga are similar to those of bicosoecids, particularly Cafeteria roenbergensis. Molecular data confirm this view. The presence of a well developed permanent cytostome/cytopharyn x complex distinguishes S. monomastiga from other bicosoecids, A new famil y Siluan iaceae (accordin g to IBCN) and Siluaniida e (according to IZCN) is erected for this species.
Th e smallest eukaryo tes are comparable in their dim ensio ns' to bact eria. Many intracellular parasitic euk aryot es (Microsp oridia, Apicomplexa) measur e 1-3 pm, but among free-living protoz oan s and algae tiny vegetative cells are relatively rare, more often than not being green algae for example: Micro m onas p usilla [17J, O steococcus tauri [5], members of the Pedinophyceae [2 OJ and Py cnococcaceae [9J. A mo ng th e coloure d chry sophytes th ere are only few examples: Monochrysis parva [29J and memb ers of the Plagophyceae [2]. It might be expected that the y wo uld be extr emely rare amon g free-living heterotrophic flagellates, since if the y have to digest bacteria then they should be larger than the 'foo d' . To the present th ere are only four species of heterotrophic flagellates th at have been described with a cell length around 2-3 pm: th e uniflagellate colourless chrysomonad Oikomonas obliqua [15J, an incertae sedis heterotrophic protist with two flagella Kiitoksia ystaua [33], and the bico soecids Pseudobodo minuta and P. minima [26]. Th e investiga tion of small cell structure has been remarkably p roductive yielding new classes [2, 20], and families [9] of algae. It is likely th at much new information will be gained after studying other unu sual tiny colourless flagellates; this will have significance for taxonomic and phylogenetic research. In spring of 1996, a small unusual heterotrophic flagellate (U H F) was isolated from a fresh water sample collected under the ice of Ryb insk reservoir. It look ed like a prokaryote in respect of its dimension , and just th e presence of one relatively thick flagellum gave us the possibil ity to defin e it as an eukaryote. Th erefore we had no ideas abo ut its taxon omic position before investiga tion of the inte rna l
Key wor ds: Siluania monomastiga; Heterotrophic flagellate; Ultrastructure; SSU rRNA seq uen ce; Bicosoecids, Siluaniaceae, Siluaniidae.
"c orresponding aut ho r ':-"- Present address : N AOS Marine Laboratory, Smith son ian Tropical Research In stitute, APO AA 34002-0948, USA © 1998 by GustavFischer Verlag
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S. A. Karpov, R. Kersanach and D. M. Williams
structure. The ultrastructure and small subunit (18S) rRNA gene sequence of this organism have been investigated and the results are presented in this paper.
Material and Methods A culture of the UHF originates from a fresh water sample collected under the ice of Rybinsk reservoir in April 1996.To obtain a mass culture with a high density of cells it was adapted to a medium consisting of Pratt's solution and cereal infusion with a ratio of 5:1 respectively [14]. Electron microscopy: For scanning electron microscopy, cells were fixed with 1.3% glutaraldehyde in 0.07 M phosphate buffer for 2 h at 4 "C. After dehydration, critical point drying and sputter coating with gold/palladium, the cells were investigated using a SEM JEM-1200 (jeol), For whole mounts, a suspension of cells was placed on the grid, shadowed with gold/palladium and observed using a TEM JEM1200 (jeol). For transmission electron microscopy, cells were fixed with 0.5% osmium tetroxide in 0.05 M phosphate buffer pH 7.2 for 10 minutes on ice, then fixed with 1.3% glutaraldehyde in 0.07 M phosphate buffer for 2 h at 4 °C in the dark. After postfixation with 1% osmium tetroxide in the same buffer for 1 h at 4°C, a pellet was dehydrated in an alcohol series and embedded in Epon resin. Cells were sectioned using a diamond knife on an "Ultracut" (Reichert) ultramicrotome. After staining with uranyl acetate and lead citrate, serial sections were investigated using aJEM-1200 (jeol) electron microscope. DNA extraction: DNA was prepared as described elsewhere [28]. After the last ethanol precipitation, the DNA was resuspended in deionized water and reprecipitated by first adding 1/5 Vol of 4 M NaCI, and then 1 Vol of 13% PEG8000. The precipitation was incubated for 20 minutes on ice and centrifuged for 15 minutes at 14000 rpm at 4°C in a fixed angle rotor (Hermie, model Z323K, rotor, 220.87). The supernatant was carefully removed; the pellet was rinsed with 500 pl of 70% ethanol, and centrifuged for another 2 to 3 minutes as described before. The supernatant was carefully discarded and the pellet was air dried for approximately 20 minutes. DNA was resuspended to a suitable concentration in molecular biology grade water (BDH UK). PCR reactions: PCR reactions amplified aproximately 1,800 bp of the 185 rDNA using primers described elsewhere [6].The reactions were performed using the following profile; 94°C 1 min, 50°C 30 sec, 72 °C 2 min for 10 cycles, then 92 °C 30 sec, 50°C 30 sec, 72 °C 2:30 min for 20 cycles and an extension at 72 °C for 5 min. In each reaction 10-50 ng of PEG precipitated-DNA were added to 10 mM Tris-HCI pH 8.3, 25 mM KCI, 2 mM MgCI 2, 250 mM of dNTPs, 20 pmol of each primer, and 0.5-1 Unit of Ampli-Taq (Perkin-Elmer) to a final volume of 50 microlitres. Sequencing reactions: The 18S rDNA from UHF PCR amplification was cloned using the pGEM-t Easy Kit (Promega). Clones were initially sequenced for the V3 and V4 region of the SSU to verify the sequence consistency among different clones from this reaction. Sequencing reactions were performed using the ABI-Prism Dye Terminator cycle sequencing Kit following manufacturers instructions and run either on ABI 373 or 377 automated sequencers. Among the positively identified clones we chose one and sequenced the whole length with a set of primers described elsewhere [6].
Phylogenetic inference: All small subunit ribosomal rDNA sequences used for the phylogenetic analysis were obtained from GenBank. A total of 1527 bp of unambiguously aligned positions from 47 sequences were used in this study. A distance matrix was created using the DNADIST algorithm [7]. The Jukes/Cantor correction was employed to compute evolutionary distances. This matrix was used to infer a phylogenetic tree using the neighbor-joining algorithm [27J of PHYLIP [7J. Bootstrap resampling (100 replicates) was undertaken to estimate the robustness of internal branches [7J.The same alignment was used as a matrix of unordered character state data as in the DNA-type option of PAUP [31]. All nucleotide characters were weighted equally in the character matrix, and alignment gaps were considered as missing data. Variable characters were subjected to cladistic analysis under the parsimony criterion with PAUP computer package. The most parsimonious cladogram was sought by random sequential addition of taxa in the heuristic search option. The TBR algorithm was used to search for the most-parsimonious cladogram. Unrooted cladograms were calculated, and the ingroup taxa subsequently were rooted with reference to the outgroup. Bootstrap resampling (200 replicates) was also completed for parsimony analysis [7]. A maximum likelihood analysis was performed using the quartet puzzling method [30]. The Hasegawa/Kishino/ Yano substitution model was used [10]. The tree reconstruction method employed was the neighbor-joining algorithm [7J. For quartet puzzling support values, 1000 puzzling steps were performed.
Results The flagellate is planktonic and almost never glides on a surface. Its short flagellum appears thick and rigid and moves slowly without waving, beating in just one plane. The extreme positions of the flagellum is illustrated in Fig. 1 and 2. In spite of the slow movement of the flagellum, it can swim rather quickly, turning its body 'head-aver-heels'. Some cells can loose their flagellum and settle to the bottom. They can change their body shape from pear- or egg-shape to a more rounded or oval appearance. They do form cysts but the cyst wall is not visible under the light microscope therefore the cysts are not distinguishable from vegetative cells which have lost their flagellum. Cell body dimensions vary from 1.5-3.0 pm, with an average length of 2.2 pm. The flagellum is always of the same length as the body. The details of the outer mor-
Abbreviations to the figures: a = anterior basal body, mo = cytostome, ph = cytopharynx, d = dictyosome of Golgi apparatus, dv = digestive vacuole, f = flagellar base, fm = fibrillar material, fr = fibrillar rootlet, g = spherical granule in the centre of mitochondrion, m = mitochondrion, n = nucleus, nf = non flagellar basal body, p = posterior basal body, r1 = dorsal microtubular rootlet, r3 = cytostomal rootlet, r4 - ventral microtubular rootlet, vp = virus-like particles. D = dorsal, L = left, R = right, V = ventral side of the cell.
A new heterotrophic flagellate Siluania monomastiga
417
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11 Fig. 1-11. Peculiarities of external and internal structure of Siluania monomastiga. 1-2: SEM micrographs: general view of the cell with flagellum being at the extreme positions of its beating. The cytostome (big arrowhead) at the apical end of the cell and a small flagellar pocket (small arrowhead) are visible, Scale bar for Fig. 1,2,4,5: 1 ]Jm, 3: SEM micrograph showing a unilateral disposition of tubular mastigonems. Arrowhead shows that some of them are curved to another side of flagellum, being over glued to its surface. Terminal filament is not visible. Scale bar: 0.5 ]Jm . 4-5: TM micrograph (whole mount preparation) showing (4) two lateral mastigonemes (other detached) each with a terminal filament (arrowhead), and (5) - apical mastigoneme at the tip of flagellum. 6-8: consecutive longitudinal sections through the basal body and cytostome ordered from dorsal to ventral side of the cell. Small arrowheads in Fig. 8 show a spiral fiber in flagellar transition zone, big arrowhead notes the virus-like particles in the perinuclear space. 9-10: two selected serial cross sections of cytostome, showing its outer (9) and inner (10) parts. Arrowhead shows a fibrillar sheet around the pharynx. 11: virus-like particles (vp) in cytoplasm, and also in dictyosome (d) and perinuclear space (arrowheads). Scale bar for Fig. 6-11: 0.2 ]Jm.
418
S. A. Karpov, R. Kersanach and D. M . Williams
phology of the flagellate are visible onl y by scanning electron microscopy.
Ultrastructure The flagellate has a pear-shaped or oval bod y with one flagellum (Fig. 1, 2) which emerges from a pit or small flagellar pocket located laterally near the anterior end . There is a cytostome at the apical position (Fig. 1-3) leading into th e pharynx. A single flagellum is covered by unilateral tubular mastigon emes, which appear tripartite (Fig. 3-4). Using SEM micrographs the 'carrot'-like base and shaft are visible (Fig. 3) and a terminal filament of mastigonemes can be distinguished on whole mount preparations (Fig. 4, 5). A single mastigoneme at the tip of the flagellum has a particularly lon g terminal filament (Fig. 5). The sur face of the cell is smooth and is only covered by the plasmalemma (Fig. 1-3, 6-8 ). A cytostome structure is well developed and co mprises an outer part supported with microtubules, and th e inner part consisting of a long narrow tube (or pharynx) located in th e peripheral cytoplasm of th e cell, and supp ort ed by thin fibrillar material (Fig. 6-10). The microtubules surrounding the outer part of the cytostome originate from th e flagellar basal bod y (Fig. 8) and are formed by a rootlet of 4 microtubules arr an ged in 1+3 (Fig. 8, 10, 11). Thi s rootlet (r3) passes firstl y to the right side! of the cell, then close to the mit ochondrion (Fig. 11, 12), and anteriorly forms a loop to support an outer part of cytostome, passing back to the po sterior part of the cell (Fig. 6-9). It winds back just beneath the plasmalemma, forming a ridge sometimes visible on the cell surface (Fig. 2), passing to the posterior part of the cell which is oft en increased by additional microtubules. There are two more microtubular rootlets consisting of 1-2 microtubules each (Fig. 6-7, 12-19). The left one (r l ) consisting of 2 microtubules, originates from the I The interpretation of rootlet disposition and organelleabsolute orientation is based on clockwise twisting of basal body microtubules when seeing from base-to-tip of the flagellum (Fig. 15-17, and explanation to those serial sections in the legends).
dors al side of the flagellar basal bod y (Fig. 6, 16) and passes to the left (Fig. 19). It turns to the ventral side of th e cell and passes just und ern eath the plasmalemma suppo rting the left rid ge of flagellar pocket (Fig. 14-16). The vent ral rootlet (r4) con sists of 1 microtubule, whi ch passes in th e same direction and in p aral lel to th e flagellar basal body (Fig. 13-1 7). When it reach es the ventral surface of the cell, it turns in a posterior direction (Fig. 12). There is one short fibrillar rootlet composed of two parallel th inly striated bands which ori ginate from th e p roximal end of the basal body and passes to the right between mitochondrion and nucleus (Fig. 18). The mitochondrion resides clos e to th e nucleus, hence th ere is a cavity or groove in it where the fibrillar rootlet p asses (Fig. 18). Almost all ro otlets are associated with flagellar basal bodies, which con sists of doublets (not triplets) of microtubules (Fig. 14-17). Initially, it was assumed th at there was a single basal bod y in the cell and that thi s species is represented by true unifl agellate cells. H owe ver, closer examination of serial sections revealed o ne, and sometimes two, small stru ctu res wh ich can be referred to as vestigal (or rudimentary) non-flagellar basal bodies (Fig. 17, 20). They are located anteriorly and po steriorly with respect to the flagellar basal body and in an orthogonal po sition to it (Fig. 17). They are very short, not mor e th an 50 nm , the anterior one has often a smaller diameter. It con sists of singlets of microtubules as seen in cross section (F ig. 20). The posterior basal body is often represented by just an electron den se spot beneath the flagellar basal bod y. Cells were also found without addition al basal bodies. It should be noted that the r4 rootlet is more ob viously associated with the p osterior basal body (Fig. 17). It can show that the latter should be a permanent structure for interphase cells, but is extremely reduced in this species. The anterior basal bod y does not possess any ro otlets, therefore it can be referr ed to as a product of basal body replication pri or to cell division. The general scheme of the rootlet system is presented in Fig. 23. The flagellar transition zone is relatively simple (Fig . 8, 19). There is a slightly curved transverse plate at th e level of the cell surface. The central microtubules of th e axoneme terminate in a small depression at the centre of
Fig. 12-20. Structure of flagellar apparatus in Siluania monomastiga. 12:longitudinal section in dorso-ventral direction, showing organelle disposition and microtubular rootlets r3 and r4. Scale bar: 0.2 pm. 13-18: consecutive sections of flagellar apparatus (one section between 13 and 14 is missed). Viewis from base-to-tip of flagellum (according to the clockwise twisting of doublets in flagellar basal body (f) in Fig. 15-17). Cell is oriented by anterior end to the right and by posterior end to the left hand. R4 is presented by 1 microtubule. 16:big arrowhead shows r rootlet, small arrowhead - rl. 17: disposition of anterior (a) and posterior (p) basal bodies. Arrowhead shows r3. 18: open arrows show 2 strands of fibrillar rootlet passing in the cavity of mitochondrion (rn). 19: transition zone structure and disposition of main organelles. Arrowheads show a spiral fiber. 20: transverse section of anterior basal body (ab), composed by singlets of microtubules (arrows), fr = fibrill ar rootlet of 2 strands; r1, r3 and r4 = microtubular rootlets. Scale bar in Fig. 13 is 0.2 pm for Fig. 13-20.
A new heterotrophic flagellate Si/uania monomastiga
419
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S. A. Karpov, R. Kersanach and D. M. Williams
this plate (Fig. 19). Two thin fibers are located just beneath the transverse plate (Fig. 8, 19). These fibers occur close to the peripheral doublets of the axoneme and can be classified as spiral fibers or rings [1, 13]. The nucleus is always located on the left of the flagellar basal body with the mitochondrion on the right, ventral and slightly forward (Fig. 8, 19). The nucleus is classified as being vesicular with well developed heterochromatin. It contains the profiles of tubular mastigonemes in the perinuclear space (Fig. 22). A large cisterna of ER with mature tubular mastigonemes can also be found close to the nucleus which is usual for many chrysophytes. It is not possible to define whether the mastigonemes are bipartite or tripartite in those cisternae. The mitochondrion is always located between nucleus and cytopharynx and closely associated with r3 microtubular rootlet (Fig. 8, 10, 11, 13-17, 19). It has true tubular cristae and a spherical electron dense amorphous inclusion or granule in the centre of the mitochondrion (Fig. 8, 11, 12, 19). A dictyosome of the Golgi apparatus is located ventrally to the nucleus (Fig. 11, 12). There is a large food vacuole at the end of cytopharynx (Fig. 21). Dark droplets in this vacuole could be digestive enzymes. Some profiles of this large vacuole, which occasionally are visible in the sections, could be referred to as microbodies. No microbodies or other vesicles bounded with two membranes were found. Some vesicles can be found nearby the flagellar pocket (Fig. 14,15), which may be part of contractile vacuole spongiome; some small vesicles are produced by the dictyosome. The remainder of the cell does not contain any vesicles or endoplasmic reticulum (Fig. 21). Sometimes the droplets which are similar to lipid ones, are visible in the cytoplasm (Fig. 22). The cytoplasm of many cells is filled with small particles of equal diameter (about 25 nm) (Fig. 11, 22). They are similar to virus-like particles. They are likely to be synthesized in the perinuclear space (Fig. 8, 11) and the Golgi apparatus (Fig. 11). In some cells these virus-like particles fill the main part of the cell (Fig. 22), and may be responsible for cell death.
Molecular data All clones were identified as belonging to the same clade in a neighbor-joining analysis. We sequenced the whole length for one of the clones comprising a total of
Fig. 21-22. Peculiarities of internal structure of Siluania monomastiga.
21: location of cytopharynx (ph) and a 'stomach' or digestive vacuole (dv) at the distal end of cytopharingeal tube. 22: virus-like particles (vp) in cytoplasm and tubular mastigonemes (arrowheads) in perinuclear space. Scale bar for both is 0.2 Jlm.
A new heterotrophic flagellate Siluania monomastiga
421
Fig. 23. A scheme of rootl et system organization in Siluania
monomastiga.
A: path ways of rootlets in connection with cytophary nx and flagellar basal body. A view from the anterior end of the cell and slightly from the left. B: composition of microtubular rootl ets. The fibrillar roo tlets are not shown. A view is from the ventr al side of the cell.
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1840 bp. The sequence is depo sited in GenBank under th e Accession No Banklt204816 AF072883. Results of the distance par simony (Fig. 24) analysis are shown. Although there is no clear branching order among the stramenopile groups in any of the analysis done in this work the ampl ified and sequenced PCR product from UHF could be identified as more closely related to Cafeteria roenbergensis (Bicosoecida) th an to any other stramenopile taxon . Thi s relationship is supported by high bootstrap value (100/ 94% ) in all three analyses in this work.
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P. minuta would be more than 3 p rn, as the flagella of smaller organisms would appear relatively thick. This is tru e for our UHF but was not mentioned for P.minuta, therefore, we concur with Larsen and Patterson that the flagellated species P. minuta [26] should be transferred to the genus Cafeteria [16]. In conclusion, at the light microscopi cal level the UHF cannot be ascribed to any kno wn species or genus. To accommodate this taxon we have erected a new genus and species Siluania m onomastiga.
Siluania gen.nov. Discussion This flagellate has been firstly compa red with other small protistan flagellates such as Monochrysis parva [29], Oikomonas obliqua [15], Micromonas pusilla [23] , Kiitoksia ystava [33] and bicosoecids Pseudobodo minuta and P. minima [26]. Among these only Micromonas pusilla has been studied usin g TEM [17, 23], but it is a tru e green flagellate; Kiitoksia ystava is an unplaced heterotrophic protist with two distin ctive flagella [33]; Oikomonas obliqua has a stalk and a flagellum which is lon ger than the cell body; and th e dimensions of the cells are more than 3 pm [15]. The uniflagellate chrysom onad Monochrysis parva [29] has similar dimensions, a short flagellum wh ich emer ges subapically, and was found in fresh water pond s. H owever, no eyespot or contractile vacuole was noticed [29]. On the other hand, M. parva is clearly distin guished from the flagellate under consideration by having a 'brown chromatophore', obovoid body (the anteri or end is broader than th e po sterior one), and its motion is fast and uniform [29]. Th e bicosoecid s Pseudobodo minuta and P.minima should have the same dimensions as the species studied herein -1.5-2.5 x 1-1.5 pm and 2 x 1 urn, respectively [26]. However, these organisms have two flagella and P. minuta was recentl y transferred to the genus Cafeteria minuta [16]. Larsen and Patterson [16] suggested that the dimensions of
Unifl agellate holozoic colourless chrysophyte, flagellum insert ed subapicall y, cytos to me located apically. Type species: Siluania m onomastiga sp.no v. Etymology: The name of the genus is in honour of the Russian St. Siluan of Mount Atho s. Siluania monomastiga sp.nov. (Fig. 1-22, 24) Planktonic freshw ater un iflagellate measuring 1.5- 3.0 pm with one flagellum equal in size to the body; cell egg- or pear-shaped with rounded posterior end, flagellum emerging from small pit; moves by somersaulting. Occurs in cold fresh waters. Type locality: The Rybinsk reservoir (Russia), collected under ice in April 1996 by N .A.Zhgarev. Holotype: Fig. 1-5, 12-20,23. Etymology: 'rnonomasti ga' is Greek for 'uniflagellated'. Concerning the ultra stru ctural features, S. mon omastiga belongs to th e chromophyte or "stramenopiles" series as it has tubular cristae in its mitochondrion and tripartite tubular mastigonemes [25]. S. mon om astiga is not comp arable to the zoosporic fungi that belong to the stramenopiles (Oomycetes and H yphochytridiom ycetes) as it has a simple life cycle consisting of only a un iflagellate vegetative stage and cyst. Pelagom onas calceolata has similar dimensions (1.5- 3 prn) and two transition sp iral fibers between two tranverse plates [2], but differs from S. monomastiga in
422
S. A. Karpov, R. Kersanach and D. M. Williams Pelagornonas calceolata Aureococcus anophageffrrens
100/100
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Sarcinochrysis marina
Pelagophyceae
Pulvinariasp. CCMP292 Pteridomonas danica Apedinella radians
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' - - - - - Rhizochromulina cJ. marina
Alariaesculenia Costana cosllllll
100/100
81/76
Phaeophyceae/ Xanthophyceae
Scyt06iplwnlornentaria
Edocarpussilicul06US
Fig. 24. Distance tree derived from 1527 unambiguous aligned positions of 47 Stramenopile/Haptophyte small subunit ribosomal DNA. Values at internal branches indicate the distance/parsimony bootstrap support (% of 1001200 replicates, values >50% presented).
}
' - - - - - Tribonema aequale
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Helerosigmaakashiwo*
Unclassified Stramenopile Raphidophyceae
Chromulina chromophila
45/50
Hibbtrrdiamagna Chrysonephele paluslris
100/100
}
Chrysophyoeae
' - - - - Ochromonas danicJZ ' - - - - - Oikornonas mutabilis
) Oikomonadaceae
100/100 Nannochloropsisgrannulalll 100/100
Nannochloropsisoculalll
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Eustigmatophyceae
Nannochloropsis salina 96/75
100/93 100/100 55/53 100/100 55/Unidentified Guinea Pig faeceo isolate U26177
} Blastocystida
Blaslocystis sp. ....-----1100/100
Proteromonas laarlile )
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90/75
100/100
Blastocyslis hominis
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. - - - - - - - Cafe~ roenbergensis 100/100
Proteromonadida
) Bicosoecida
UHF . - - - - - - - - - - - - - - Thrauslochytrium kinnei 64/95
lAbyrinlhuloides ha]iotidis Ulkenia profunda
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} Labyrinthulida
Prqmnesium paleliferum P527 100/100
Prymnesium cala1hiferum
79/76
EmilianahUXl!leyi Phaecyslisgloo06a
77/73
Haptophyta
Relicul06phaere socialis
' - - - - - - PllDltroa salina
* Synonym - Olisthodiscus luieus 0.1
A new heterotrophic f lagellate Siluania monomastiga
many essentia l respects: P. calceolata has a lon ger flagellum with paraxial plates, a thin theca coveri ng the plasmalem ma, a chloroplast of chrysophyte type, ext rusomes, and it is a true un iflagellate with no rootlets and cytostome [2]. Bicosoecids and pseud odendromonads are the tw o groups of heterotrophic chro rnophy tes w hich S. m onomastiga is mor e similar to th an any other proti sts. Th e ultra structure of five species of bicosoecids has been investigated before: Bicosoeca plan ctonica [3], B. k epneri and B. lacustris [19], B. maris [22] and Cafeteria roenbergensis [8, 24]. In the first EM works a general structure of the cell and th e feeding app aratu s structure have been sho wn. In common , all bicosoecids are similar to each other in thos e respects. Just the paraxial rod has been found in the posterior flagellum of B. k epneri and B. lacustris [19], and the number of axoneme microtubules is redu ced to 4-5 in the distal part of the posteri or flagellum of B. planctonica [3]. Parti cular attention to th e cytos keleto n has been paid to B. m aris and C. roenberge nsis, whi ch is most essential for comp arison with S. m onomastiga. Our species is mo re similar to C. roenbergensis than to B. m aris. Results fro m molecular analyses (Fig. 24) suggest th e same conclusion . Therefore it is reasonable to compare th ese two alori cate species with each other in more detail. The biflagellate C. roenbergensis repr esents one of the smallest (4- 6 prn) known bicos oecids. The tiny S. monomastiga has on e flagellum but with two additio nal basal bod ies. One of th e latter (the ant erior one) look s too small to be considere d a permanent basal body, and has no roo tlets. Thus, its existence could be connected with th e early stage of kinetid dupli cation during cell division . Perhaps, because of its smaller cell size, there is no recurrent flagellum in S. monomastiga, and cons equently, no rootlets. O nly the ventral microtu bular rootlet (r4) of S.monomastiga can be classified as a rootlet of th e seco nd basal bod y. It is possible th at it could be conside red an addition al microtubule of r3, which correspond s to th at termed "abc" in C. roenbergensis [24]. Th e flagellar base of S. mon om astiga correspo nds to the flagellar base of th e ant erior flagellum in C. roenbergensis. Both species have a 2-st rand left microtubular rootl et (r l ), broad right or cyto stom al microtubular rootlet (r3) with the outer part con sisting of one micr otubule in S. monomastiga which may correspond to that termed "x" in C. roenbergensis [24]. There is also a fibrillar material associ ated with r3 in S. monomastiga but it is mu ch shorter than that in C. roenb ergensis. Th e cytostomal roo tlet (r3) in both species has " L-shaped" form in cross section near its origin, and is connected to the mit ochondrion. Both species also have short fibr illar ro otl ets. However, in S. m onom astiga the fibrillar ro otl et is more closely co nnected with the mitochondrion and both parts pass in one direction. In C. roen -
423
bergensis th e two part s of th is rootlet pass at right angles to each other near to the nuclea r surface. Th e flagellar bases in both species are represented by doublets of peripheral microtubules which can be connected with small cell size. The rootl et system of S. monom astiga differs from that of C. roenbe rgensis in th e follow ing respects: rl has no seco ndary microtubules, the cytos to rnal ro otlet (r3) has an S-shaped form , as th e cytos to me is locat ed anteriorl y to th e flagellum; th e fibrillar roo tlet passes from the proximal end of flagellar basal bod y in S. m onomastiga but in C. roenbergensis it is co nnected with the lateral side in the middle of posterior basal bod y; the fibrillar co nnectives aro und the flagellar base are almost absent in S. monomastiga. Other minor differences between th e rootlet systems have been noted above. S. m on om astiga differs from C. roenbergensis in oth er respects. S.monomastiga has no core inside the basal bodi es, no striated brid ges betw een basal bodies, no microb odi es, no extruso rnes; th ere is a spiral fiber in the flagellar transition zone , and th ere is an electr on den se mater ial in the mitochondrion . Th e primary difference is th at S. m onom astiga has a well develop ed cytostome/cytopharyn x complex. To compare S. monomastiga with other bicosoecids, it is relevant to note th at the transitional spiral fibr es or rings are similar to that structure in B. maris [22J but in the latt er it is locat ed above the tr ansverse plat e. T he spiral fib er located abov e the transverse plate has been found in another repr esent ative of th e bicosoecids Pseudobodo tremulans (in preparation ). Other com mon char acters are restricted by the cyto skeletal str ucture . Parti cularl y essenti al is the L- shaped form of r3 in cro ss sectio , and its association with fibrillar material. These tw o characters have been already found in fou r bicosoecids: B. maris [22], C. roenb ergensis [24], S. monomastiga (present paper) and P. tremulans (in preparation ). Th e most important d ifference of bicosoecids from S. monom astiga is an absence of perm anent cytosto mal struc tures. In th is respect S. monom astiga is more similar to th e pseudodendro mo nads. Pseud od endrom onads have a well develop ed cytophary nx supporte d by broad cyt ostomal microtubular ro otl et, at least one lateral narr ow microtubular rootlet, and a small striated fibrillar rootlet originatin g from th e proximal end of the flagellar base [11, 12, 18]. Unfortunately, pseudodendromonads have not been investi gated in the same detail as C. roenbergensis and S. m onom astiga, therefore a co mparison with th e abso lute orientatio n of cytoskel etal elements is not yet pos sible. In other respects S. m onom astiga differs from pseud od endromonads. The latte r have bod y and flagellar scales and no mastigonemes, there is no spiral fibre in flagellar transition zone and the cell symmetry is different.
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In conclusion, the uniflagellate S. monomastiga cannot be placed among the pseudodendromonads. It is certainly more similar to bicosoecids, particularly in respect of cytoskeletal structure, a fact confirmed by molecular data. Unfortunately, there are little molecular data available for other bicosoecids which may allow a more definitive position for Siluania. The close position of Cafeteria and Siluania in all three molecular analyses allows them to be placed in one order Bicosoecales in spite of some morphological differences. There are two families in the order Bicosoecales (sensu [21])2: Cafeteriaceae, which includes bicosoecids without loricae, and Bicosoecaceae, which includes those with loricae. However, representatives from both families do not have a permanent cytostome. Therefore, although Siluania could be formally placed in the family Cafeteriaceae (as a flagellate without lorica), we cannot do this because of presence of the permanent cytostome structure. On the other hand, it is not correct to establish a new order for this species being based just on that. Siluaniaceae Karpov (present paper) Apochlorotic uniflagellated 'chrysophytes' without a lorica; with unilateral mastigonemes; a transition spiral fiber located under the transverse plate; a permanent cytostome with developed cytopharynx present and supported by a microtubular rootlet. Type genus: Siluania Karpov (present paper) Regardless of whether a new family is erected, the diagnosis of the order Bicosoecales has to be amended as there are not known bicosoecids with a permanent cytostome. Order Bicosoecales (Grasse) emend. Karpov (present paper) 'Chrysophycean' heterotrophic flagellates without plastids, having one or two flagella, with or without a lorica; feeding apparatus represented by lip or by permanent cytostome with cytopharynx; transitional spiral fiber, if present, located above or under transverse plate; the broadest microtubular rootlet connected with cytostome region, often "L-shaped" in cross section and associated with fibrillar material. Sedentary and planktonic, freshwater and marine. According to the International Botanical Code of Nomenclature the order Bicosoecales (Grasse) emend. Karpov (present paper) includes 3 families: We cannot accept the classification of Tom Cavalier-Smith [4], where these families belong to different orders. It seems not necessary to introduce the new order Anoecales, which has incorrect diagnosis (neither Cafeteria, nor any other bicosoecids have sagenogenetosome or ciliary transitional helix-character. Therefore it is proposed in this paper to erect a new family in the order Bicosoecales.
• Siluaniaceae Karpov (present paper) • Siluania Karpou (present paper) Cafeteriaceae Moestrup, 1995 Cafeteria Fenchel & Patterson, 1988, Pseudobodo Griessmann, 1913 Bicosoecaceae Stein, 1878 Bicosoeca Stein, 1878 Accoding to the International Zoological Code of Nomenclature the order Bicosoecida (Grasse) emend. Karpov (present paper) includes 3 families: • Siluaniidae Karpov (present paper) Cafeteriidae Moestrup, 1995 Bicosoecidae Stein, 1878 Acknowledgements: This work was partly supported by the ex-quota visit programme of the Royal Society of London. The authors are also grateful to Dr. B.s.e. Leadbeater as the bulk of this work was undertaken in his laboratory of the School of Biological Sciences of Birmingham University. The molecular work was undertaken with financial assistance from NERC grant awarded to DMW and T. Martin Embley.
References
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3 4 5
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2
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Andersen R. A. (1991): The cytoskeleton of chromophyte algae. Protoplasma 164,143-159. Andersen R. A., Saunders G. A., Paskind M. P. and Sexton J. P. (1993): Ultrastructure and 18S rRNA gene sequence for Pelagomonas calceolata gen. et sp. nov. and the description of a new algal class, the Pelagophyceae classis nov. J. Phycol. 29, 701-715. Belcher J. H. (1975): The fine structure of the loricate colourless flagellate Bicoeca planctonica Kisselew. Arch. Protistenkd. 117, 78-84. Cavalier-Smith T. (1997): Sagenista and Bigyra, two phyla of heterotrophic heterocont chromists. Arch. Protistenkd. 148,253-267. Chretiennot-Dinet M.-J., Courties e., Vaquer A., Nevex J., Claustre H., Lautier J. and Machado M.e. (1995): A new marine picoeucaryote: Osteococcus tauri gen. et sp. nov. (Chlorophyta, Prasinophyceae). Phycologia 34, 285-292. Embley T. M., Finlay B. J., Thomas R. H. and Dyal P. L. (1992): The use of rRNA sequences and fluorescent probes to investigate the phylogenetic positions of the anaerobic ciliate Metopus palaeformis and its archaeobacterial endosymbiont.J. Gen. Microbiol. 138, 1479-1487. Felsenstein J. (1995): PHYLIP - Phylogeny inference package. ( version 3.572), University of Washington, Seattle Fenchel T. and Patterson D. J. (1988): Cafeteria roenbergensis nov. gen., nov. sp., a heterotrophic microflagellate from marine plankton. Mar. Microb. Food Webs, 1988, 3, 9-19. Guillard R. R. L., Keller M. D., O'Kelly e. J. and Floyd G. L. (1991): Pycnococcus provasolii gen. et sp. nov., a coccoid prasinoxanthin-containing phytoplankter from the western North Atlantic and Gulf of Mexico. J. Phycol. 27, 39-47.
A new heterotrophic flagell ate 5iluania mon omastiga
10 H asegawa M., Kish ino H . and Yano K. (1985): D atin g o f the human-ape splitt ing by a mol ecular cloc k of mit ocho nd rial DNA.J. Mo l. Evol, 22, 160-1 74. 11 H ibb erd D. J. (1976): O bserva tio ns on th e ultrastructure of three new species of Cyathobodo Petersen et Hansen (Csa lpinx, Cintricatus, and C simplex) and on the external mo rpho logy of Pseudodendrom onas v lkii Bourrelly. Protistologica 12,249-261. 12 H ibb erd D. J. (1985): O bservatio ns o n the ult rastructure o f new species of Pseudodendrom onas Bourrelly (P. ope rculifera and P. insignis) and Cya tho bodo Peter sen and H an sen (Cpeltatus and Cgemmatus), Pseud odend romo nadida ord. no v. Ar ch . Protistenkd. 129,3-11. 13 Karp ov S. A. and Fokin S. I. (1995): T he str uctu ral di versity of flagellar transitional zon e in heterotrophic flagellates and other protists. In: Karpov, S. A. (ed.): The biology of fr ee-living heterotrophic flagellates, pp. 1038-1052. N auka, St. Petersburg. 14 Karp ov S. A. and Leadbeater B. S. C. (1997): Cell and nuclear d ivision in fre shwater choan oflagellate Mon osiga ova ta. Eu ro p. J. Pr otistol . 33, 323-334. 15 Kent, W. S. (1880- 1882): A manu al of th e Infusoria. 1-3, 9 13 pp. 16 Lar sen]. and Patterson D .]. (1990): Some flagellates (P rotist a) fro m trop ical marine sediments . J. N at. Hist. 24, 80 1- 937. 17 Manton I. and Parke M. (1960): Fu rt her ob servations o n small green flagellates w ith sp ecial reference to po ssibl e relatives of Chromulina p usilla Butcher. J. Mar. BioI. Assoc. U .K. 39,275-298. 18 M ignotJ.-P. (1974a): Etude ultrastructurale d 'un protiste flagelle incol ore: Pseudodendrom onas v lkii Bourrelly . Proti stol ogica 10,394-41 2. 19 Mig no t J. -P. (1974b): Etude uhrastructurale de s Bicoeca, protiste flagell(s. Proti stologica, 10,544-565. 20 Moestrup O. (1991): Fu rth er studies of p resumabl y primitive green algae, including the description of Pedinophyceae class nov. and Resulror gen. nov. J. Ph ycol. 27, 119-133. 21 Mo estrup O. (1995): Current status of chrysophyt e "s plint er groups": synurophytes, pedinellids, silicoflagellates. In: Sangren c., Smol J. P. and Kristiansen J. (cds.): Chrysophyte algae: ecology, ph ylo geny, development, pp. 75- 91. Cambridge Universit y Press, Ca mbridge.
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22 Moestrup o . and Thomsen H . A. (1976): Fin e structural studies on the flagellat e genus Bicoe ca. I. Bicoeca maris with parti cular emp hasis to the flagellar appara tus . Prot istologica 12, 101-120. 23 No rr is R. E. (1980): Prasinophyte s, In: De velopments in Marin e Biology. Vo1.2. Phytoplagellates, pp. 85-145. Elsevier N orth Holland, New York. 24 O' Kelly C. J. and Patterson D . J. (1996): The flagellar apparatu s of Cafe teria roenbergensis Fe nc hel & Patterson, 1988 (Bicos oe cales = Bicosoecida). Europ. J. Pr oti stol . 32, 216-226. 25 Patt erson D .J. (1989): Stramenop iles: ch ro mop hy tes from a protistan perspective. In: Green J. c., Leadbeater B. S. C. and Diver W. L. (eds .): Th e chr omophyte algae: problems and perspectives, pp. 357- 379. Clarendon Press, O xford. 26 Ruinen J. (1938): Notizen tiber Salzflagellaten. II. tiber die Verbreitu ng der Salzfla gellaten. Arch. Protistenkd. 90, 210-258. 27 Saitou N. and Nei M. (1987): The neighbor-joining meth od: A new method for recon stru ctin g phylogenetic tr ees. Mol. BioI. Evol. 4, 406-425. 28 Sambrook J., Fr itsch E. F. and Man iatis T. (1989): Mole cular C loni ng - A lab or atory Manu al. Cold Spring Harbour Labor atory Pre ss. 29 Sku ja, H. (1956): Taxo nom ische und biologischen Studien uber das Ph ytoplank ton Schw ed ische r Binnengewasser, N ova Acta Regiae Societ atis Scienti arurn Upsaliensis, Ser. IV, 16 (3), 1-404. 30 Strimmer K. and von Haeseler A. (1996): Quartet pu zzling: A quartet maximum-likelihood method for recon structing tree top olo gies. Mol. BioI. Evol. 13, 964-969. 31 Swofford D. L. (1993): PAUP - Ph ylogenetic anal ysis using parsim ony. (v.3.1.1), Syn auer. 32 Verhagen F.]. M., Z6lffel M., Bru gerolle G . and P atterson D . ]. (1994): Adriamonas peritocrescens gen. nov., sp. nov., a new free-living soil flagellate (Pro tista, Pseudoden dromonadida, incertae sedis). Europ. J. Protistol. 30, 295-308. 33 Vors, N. (1992): Heterotrophi c amoebae, flagellates and heliozoa from the Tvarminne area, Gulf of Finland, in 1988-1990. Ophelia 36, 1-109.
European Journal o f
PROTISTOLOGY
Ultrastructure and 18S rRNA Gene Sequence of a Small Heterotrophic Flagellate Siluania monomastiga gen. et sp. nov. (Bicosoecida) Serguei A. Karpov ':". Ralf Kersanach-> " and David M. Williams 2 1Biological
Research Institute of 51. Petersburg State University, Oranienbaumskoye sch. 2, 19890451. Petersburg, Russia -Bot any Department, Cromwell Road, Natural History Museum, London SW7 5BD, UK
Summary
Introduction
Siluania monomastiga gen . et sp. no v. - one of the sm allest fre e-living heterotrophic eukaryotes (1.5-3.0 pm length) h as been investigated using SEM and TEM, and by gene sequence analysis of the small su bu n it rRNA. The flagellate is co vered by only the pla smalemma. The single flagellum h as a unilateral array of tubular ma stigonemes; the flagellar basal body is only composed of doublets of microtubules. One or two additional very short basal bodies may also be present, com posed of single microtubules, of amorphous electron dense material. There is a well developed cyto stome with permanent pharynx, supported by rootlet microtubules and a fibrillar sheet. One mitochondrion wit h tubular cristae contains a spherical body in the centre. D eveloping mastigonemes are located within the perinuclear space. The Golgi apparatus consists of a single diet yo some. The rootlet system of the flagellum is represented by three microtubular bands, numbered as rl, r3 and r4 in accordance with the protocol accepted for biosoecids, and by the fibrillar rootlet having two strands. The cytoskeleton structure and organelle dispos ition of S.monomastiga are similar to those of bicosoecids, particularly Cafeteria roenbergensis. Molecular data confirm this view. The presence of a well developed permanent cytostome/cytopharyn x complex distinguishes S. monomastiga from other bicosoecids, A new famil y Siluan iaceae (accordin g to IBCN) and Siluaniida e (according to IZCN) is erected for this species.
Th e smallest eukaryo tes are comparable in their dim ensio ns' to bact eria. Many intracellular parasitic euk aryot es (Microsp oridia, Apicomplexa) measur e 1-3 pm, but among free-living protoz oan s and algae tiny vegetative cells are relatively rare, more often than not being green algae for example: Micro m onas p usilla [17J, O steococcus tauri [5], members of the Pedinophyceae [2 OJ and Py cnococcaceae [9J. A mo ng th e coloure d chry sophytes th ere are only few examples: Monochrysis parva [29J and memb ers of the Plagophyceae [2]. It might be expected that the y wo uld be extr emely rare amon g free-living heterotrophic flagellates, since if the y have to digest bacteria then they should be larger than the 'foo d' . To the present th ere are only four species of heterotrophic flagellates th at have been described with a cell length around 2-3 pm: th e uniflagellate colourless chrysomonad Oikomonas obliqua [15J, an incertae sedis heterotrophic protist with two flagella Kiitoksia ystaua [33], and the bico soecids Pseudobodo minuta and P. minima [26]. Th e investiga tion of small cell structure has been remarkably p roductive yielding new classes [2, 20], and families [9] of algae. It is likely th at much new information will be gained after studying other unu sual tiny colourless flagellates; this will have significance for taxonomic and phylogenetic research. In spring of 1996, a small unusual heterotrophic flagellate (U H F) was isolated from a fresh water sample collected under the ice of Ryb insk reservoir. It look ed like a prokaryote in respect of its dimension , and just th e presence of one relatively thick flagellum gave us the possibil ity to defin e it as an eukaryote. Th erefore we had no ideas abo ut its taxon omic position before investiga tion of the inte rna l
Key wor ds: Siluania monomastiga; Heterotrophic flagellate; Ultrastructure; SSU rRNA seq uen ce; Bicosoecids, Siluaniaceae, Siluaniidae.
"c orresponding aut ho r ':-"- Present address : N AOS Marine Laboratory, Smith son ian Tropical Research In stitute, APO AA 34002-0948, USA © 1998 by GustavFischer Verlag
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structure. The ultrastructure and small subunit (18S) rRNA gene sequence of this organism have been investigated and the results are presented in this paper.
Material and Methods A culture of the UHF originates from a fresh water sample collected under the ice of Rybinsk reservoir in April 1996.To obtain a mass culture with a high density of cells it was adapted to a medium consisting of Pratt's solution and cereal infusion with a ratio of 5:1 respectively [14]. Electron microscopy: For scanning electron microscopy, cells were fixed with 1.3% glutaraldehyde in 0.07 M phosphate buffer for 2 h at 4 "C. After dehydration, critical point drying and sputter coating with gold/palladium, the cells were investigated using a SEM JEM-1200 (jeol), For whole mounts, a suspension of cells was placed on the grid, shadowed with gold/palladium and observed using a TEM JEM1200 (jeol). For transmission electron microscopy, cells were fixed with 0.5% osmium tetroxide in 0.05 M phosphate buffer pH 7.2 for 10 minutes on ice, then fixed with 1.3% glutaraldehyde in 0.07 M phosphate buffer for 2 h at 4 °C in the dark. After postfixation with 1% osmium tetroxide in the same buffer for 1 h at 4°C, a pellet was dehydrated in an alcohol series and embedded in Epon resin. Cells were sectioned using a diamond knife on an "Ultracut" (Reichert) ultramicrotome. After staining with uranyl acetate and lead citrate, serial sections were investigated using aJEM-1200 (jeol) electron microscope. DNA extraction: DNA was prepared as described elsewhere [28]. After the last ethanol precipitation, the DNA was resuspended in deionized water and reprecipitated by first adding 1/5 Vol of 4 M NaCI, and then 1 Vol of 13% PEG8000. The precipitation was incubated for 20 minutes on ice and centrifuged for 15 minutes at 14000 rpm at 4°C in a fixed angle rotor (Hermie, model Z323K, rotor, 220.87). The supernatant was carefully removed; the pellet was rinsed with 500 pl of 70% ethanol, and centrifuged for another 2 to 3 minutes as described before. The supernatant was carefully discarded and the pellet was air dried for approximately 20 minutes. DNA was resuspended to a suitable concentration in molecular biology grade water (BDH UK). PCR reactions: PCR reactions amplified aproximately 1,800 bp of the 185 rDNA using primers described elsewhere [6].The reactions were performed using the following profile; 94°C 1 min, 50°C 30 sec, 72 °C 2 min for 10 cycles, then 92 °C 30 sec, 50°C 30 sec, 72 °C 2:30 min for 20 cycles and an extension at 72 °C for 5 min. In each reaction 10-50 ng of PEG precipitated-DNA were added to 10 mM Tris-HCI pH 8.3, 25 mM KCI, 2 mM MgCI 2, 250 mM of dNTPs, 20 pmol of each primer, and 0.5-1 Unit of Ampli-Taq (Perkin-Elmer) to a final volume of 50 microlitres. Sequencing reactions: The 18S rDNA from UHF PCR amplification was cloned using the pGEM-t Easy Kit (Promega). Clones were initially sequenced for the V3 and V4 region of the SSU to verify the sequence consistency among different clones from this reaction. Sequencing reactions were performed using the ABI-Prism Dye Terminator cycle sequencing Kit following manufacturers instructions and run either on ABI 373 or 377 automated sequencers. Among the positively identified clones we chose one and sequenced the whole length with a set of primers described elsewhere [6].
Phylogenetic inference: All small subunit ribosomal rDNA sequences used for the phylogenetic analysis were obtained from GenBank. A total of 1527 bp of unambiguously aligned positions from 47 sequences were used in this study. A distance matrix was created using the DNADIST algorithm [7]. The Jukes/Cantor correction was employed to compute evolutionary distances. This matrix was used to infer a phylogenetic tree using the neighbor-joining algorithm [27J of PHYLIP [7J. Bootstrap resampling (100 replicates) was undertaken to estimate the robustness of internal branches [7J.The same alignment was used as a matrix of unordered character state data as in the DNA-type option of PAUP [31]. All nucleotide characters were weighted equally in the character matrix, and alignment gaps were considered as missing data. Variable characters were subjected to cladistic analysis under the parsimony criterion with PAUP computer package. The most parsimonious cladogram was sought by random sequential addition of taxa in the heuristic search option. The TBR algorithm was used to search for the most-parsimonious cladogram. Unrooted cladograms were calculated, and the ingroup taxa subsequently were rooted with reference to the outgroup. Bootstrap resampling (200 replicates) was also completed for parsimony analysis [7]. A maximum likelihood analysis was performed using the quartet puzzling method [30]. The Hasegawa/Kishino/ Yano substitution model was used [10]. The tree reconstruction method employed was the neighbor-joining algorithm [7J. For quartet puzzling support values, 1000 puzzling steps were performed.
Results The flagellate is planktonic and almost never glides on a surface. Its short flagellum appears thick and rigid and moves slowly without waving, beating in just one plane. The extreme positions of the flagellum is illustrated in Fig. 1 and 2. In spite of the slow movement of the flagellum, it can swim rather quickly, turning its body 'head-aver-heels'. Some cells can loose their flagellum and settle to the bottom. They can change their body shape from pear- or egg-shape to a more rounded or oval appearance. They do form cysts but the cyst wall is not visible under the light microscope therefore the cysts are not distinguishable from vegetative cells which have lost their flagellum. Cell body dimensions vary from 1.5-3.0 pm, with an average length of 2.2 pm. The flagellum is always of the same length as the body. The details of the outer mor-
Abbreviations to the figures: a = anterior basal body, mo = cytostome, ph = cytopharynx, d = dictyosome of Golgi apparatus, dv = digestive vacuole, f = flagellar base, fm = fibrillar material, fr = fibrillar rootlet, g = spherical granule in the centre of mitochondrion, m = mitochondrion, n = nucleus, nf = non flagellar basal body, p = posterior basal body, r1 = dorsal microtubular rootlet, r3 = cytostomal rootlet, r4 - ventral microtubular rootlet, vp = virus-like particles. D = dorsal, L = left, R = right, V = ventral side of the cell.
A new heterotrophic flagellate Siluania monomastiga
417
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11 Fig. 1-11. Peculiarities of external and internal structure of Siluania monomastiga. 1-2: SEM micrographs: general view of the cell with flagellum being at the extreme positions of its beating. The cytostome (big arrowhead) at the apical end of the cell and a small flagellar pocket (small arrowhead) are visible, Scale bar for Fig. 1,2,4,5: 1 ]Jm, 3: SEM micrograph showing a unilateral disposition of tubular mastigonems. Arrowhead shows that some of them are curved to another side of flagellum, being over glued to its surface. Terminal filament is not visible. Scale bar: 0.5 ]Jm . 4-5: TM micrograph (whole mount preparation) showing (4) two lateral mastigonemes (other detached) each with a terminal filament (arrowhead), and (5) - apical mastigoneme at the tip of flagellum. 6-8: consecutive longitudinal sections through the basal body and cytostome ordered from dorsal to ventral side of the cell. Small arrowheads in Fig. 8 show a spiral fiber in flagellar transition zone, big arrowhead notes the virus-like particles in the perinuclear space. 9-10: two selected serial cross sections of cytostome, showing its outer (9) and inner (10) parts. Arrowhead shows a fibrillar sheet around the pharynx. 11: virus-like particles (vp) in cytoplasm, and also in dictyosome (d) and perinuclear space (arrowheads). Scale bar for Fig. 6-11: 0.2 ]Jm.
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S. A. Karpov, R. Kersanach and D. M . Williams
phology of the flagellate are visible onl y by scanning electron microscopy.
Ultrastructure The flagellate has a pear-shaped or oval bod y with one flagellum (Fig. 1, 2) which emerges from a pit or small flagellar pocket located laterally near the anterior end . There is a cytostome at the apical position (Fig. 1-3) leading into th e pharynx. A single flagellum is covered by unilateral tubular mastigon emes, which appear tripartite (Fig. 3-4). Using SEM micrographs the 'carrot'-like base and shaft are visible (Fig. 3) and a terminal filament of mastigonemes can be distinguished on whole mount preparations (Fig. 4, 5). A single mastigoneme at the tip of the flagellum has a particularly lon g terminal filament (Fig. 5). The sur face of the cell is smooth and is only covered by the plasmalemma (Fig. 1-3, 6-8 ). A cytostome structure is well developed and co mprises an outer part supported with microtubules, and th e inner part consisting of a long narrow tube (or pharynx) located in th e peripheral cytoplasm of th e cell, and supp ort ed by thin fibrillar material (Fig. 6-10). The microtubules surrounding the outer part of the cytostome originate from th e flagellar basal bod y (Fig. 8) and are formed by a rootlet of 4 microtubules arr an ged in 1+3 (Fig. 8, 10, 11). Thi s rootlet (r3) passes firstl y to the right side! of the cell, then close to the mit ochondrion (Fig. 11, 12), and anteriorly forms a loop to support an outer part of cytostome, passing back to the po sterior part of the cell (Fig. 6-9). It winds back just beneath the plasmalemma, forming a ridge sometimes visible on the cell surface (Fig. 2), passing to the posterior part of the cell which is oft en increased by additional microtubules. There are two more microtubular rootlets consisting of 1-2 microtubules each (Fig. 6-7, 12-19). The left one (r l ) consisting of 2 microtubules, originates from the I The interpretation of rootlet disposition and organelleabsolute orientation is based on clockwise twisting of basal body microtubules when seeing from base-to-tip of the flagellum (Fig. 15-17, and explanation to those serial sections in the legends).
dors al side of the flagellar basal bod y (Fig. 6, 16) and passes to the left (Fig. 19). It turns to the ventral side of th e cell and passes just und ern eath the plasmalemma suppo rting the left rid ge of flagellar pocket (Fig. 14-16). The vent ral rootlet (r4) con sists of 1 microtubule, whi ch passes in th e same direction and in p aral lel to th e flagellar basal body (Fig. 13-1 7). When it reach es the ventral surface of the cell, it turns in a posterior direction (Fig. 12). There is one short fibrillar rootlet composed of two parallel th inly striated bands which ori ginate from th e p roximal end of the basal body and passes to the right between mitochondrion and nucleus (Fig. 18). The mitochondrion resides clos e to th e nucleus, hence th ere is a cavity or groove in it where the fibrillar rootlet p asses (Fig. 18). Almost all ro otlets are associated with flagellar basal bodies, which con sists of doublets (not triplets) of microtubules (Fig. 14-17). Initially, it was assumed th at there was a single basal bod y in the cell and that thi s species is represented by true unifl agellate cells. H owe ver, closer examination of serial sections revealed o ne, and sometimes two, small stru ctu res wh ich can be referred to as vestigal (or rudimentary) non-flagellar basal bodies (Fig. 17, 20). They are located anteriorly and po steriorly with respect to the flagellar basal body and in an orthogonal po sition to it (Fig. 17). They are very short, not mor e th an 50 nm , the anterior one has often a smaller diameter. It con sists of singlets of microtubules as seen in cross section (F ig. 20). The posterior basal body is often represented by just an electron den se spot beneath the flagellar basal bod y. Cells were also found without addition al basal bodies. It should be noted that the r4 rootlet is more ob viously associated with the p osterior basal body (Fig. 17). It can show that the latter should be a permanent structure for interphase cells, but is extremely reduced in this species. The anterior basal bod y does not possess any ro otlets, therefore it can be referr ed to as a product of basal body replication pri or to cell division. The general scheme of the rootlet system is presented in Fig. 23. The flagellar transition zone is relatively simple (Fig . 8, 19). There is a slightly curved transverse plate at th e level of the cell surface. The central microtubules of th e axoneme terminate in a small depression at the centre of
Fig. 12-20. Structure of flagellar apparatus in Siluania monomastiga. 12:longitudinal section in dorso-ventral direction, showing organelle disposition and microtubular rootlets r3 and r4. Scale bar: 0.2 pm. 13-18: consecutive sections of flagellar apparatus (one section between 13 and 14 is missed). Viewis from base-to-tip of flagellum (according to the clockwise twisting of doublets in flagellar basal body (f) in Fig. 15-17). Cell is oriented by anterior end to the right and by posterior end to the left hand. R4 is presented by 1 microtubule. 16:big arrowhead shows r rootlet, small arrowhead - rl. 17: disposition of anterior (a) and posterior (p) basal bodies. Arrowhead shows r3. 18: open arrows show 2 strands of fibrillar rootlet passing in the cavity of mitochondrion (rn). 19: transition zone structure and disposition of main organelles. Arrowheads show a spiral fiber. 20: transverse section of anterior basal body (ab), composed by singlets of microtubules (arrows), fr = fibrill ar rootlet of 2 strands; r1, r3 and r4 = microtubular rootlets. Scale bar in Fig. 13 is 0.2 pm for Fig. 13-20.
A new heterotrophic flagellate Si/uania monomastiga
419
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S. A. Karpov, R. Kersanach and D. M. Williams
this plate (Fig. 19). Two thin fibers are located just beneath the transverse plate (Fig. 8, 19). These fibers occur close to the peripheral doublets of the axoneme and can be classified as spiral fibers or rings [1, 13]. The nucleus is always located on the left of the flagellar basal body with the mitochondrion on the right, ventral and slightly forward (Fig. 8, 19). The nucleus is classified as being vesicular with well developed heterochromatin. It contains the profiles of tubular mastigonemes in the perinuclear space (Fig. 22). A large cisterna of ER with mature tubular mastigonemes can also be found close to the nucleus which is usual for many chrysophytes. It is not possible to define whether the mastigonemes are bipartite or tripartite in those cisternae. The mitochondrion is always located between nucleus and cytopharynx and closely associated with r3 microtubular rootlet (Fig. 8, 10, 11, 13-17, 19). It has true tubular cristae and a spherical electron dense amorphous inclusion or granule in the centre of the mitochondrion (Fig. 8, 11, 12, 19). A dictyosome of the Golgi apparatus is located ventrally to the nucleus (Fig. 11, 12). There is a large food vacuole at the end of cytopharynx (Fig. 21). Dark droplets in this vacuole could be digestive enzymes. Some profiles of this large vacuole, which occasionally are visible in the sections, could be referred to as microbodies. No microbodies or other vesicles bounded with two membranes were found. Some vesicles can be found nearby the flagellar pocket (Fig. 14,15), which may be part of contractile vacuole spongiome; some small vesicles are produced by the dictyosome. The remainder of the cell does not contain any vesicles or endoplasmic reticulum (Fig. 21). Sometimes the droplets which are similar to lipid ones, are visible in the cytoplasm (Fig. 22). The cytoplasm of many cells is filled with small particles of equal diameter (about 25 nm) (Fig. 11, 22). They are similar to virus-like particles. They are likely to be synthesized in the perinuclear space (Fig. 8, 11) and the Golgi apparatus (Fig. 11). In some cells these virus-like particles fill the main part of the cell (Fig. 22), and may be responsible for cell death.
Molecular data All clones were identified as belonging to the same clade in a neighbor-joining analysis. We sequenced the whole length for one of the clones comprising a total of
Fig. 21-22. Peculiarities of internal structure of Siluania monomastiga.
21: location of cytopharynx (ph) and a 'stomach' or digestive vacuole (dv) at the distal end of cytopharingeal tube. 22: virus-like particles (vp) in cytoplasm and tubular mastigonemes (arrowheads) in perinuclear space. Scale bar for both is 0.2 Jlm.
A new heterotrophic flagellate Siluania monomastiga
421
Fig. 23. A scheme of rootl et system organization in Siluania
monomastiga.
A: path ways of rootlets in connection with cytophary nx and flagellar basal body. A view from the anterior end of the cell and slightly from the left. B: composition of microtubular rootl ets. The fibrillar roo tlets are not shown. A view is from the ventr al side of the cell.
R
L
A
1840 bp. The sequence is depo sited in GenBank under th e Accession No Banklt204816 AF072883. Results of the distance par simony (Fig. 24) analysis are shown. Although there is no clear branching order among the stramenopile groups in any of the analysis done in this work the ampl ified and sequenced PCR product from UHF could be identified as more closely related to Cafeteria roenbergensis (Bicosoecida) th an to any other stramenopile taxon . Thi s relationship is supported by high bootstrap value (100/ 94% ) in all three analyses in this work.
r3 [l B
r4° 1L-
nf
_
P. minuta would be more than 3 p rn, as the flagella of smaller organisms would appear relatively thick. This is tru e for our UHF but was not mentioned for P.minuta, therefore, we concur with Larsen and Patterson that the flagellated species P. minuta [26] should be transferred to the genus Cafeteria [16]. In conclusion, at the light microscopi cal level the UHF cannot be ascribed to any kno wn species or genus. To accommodate this taxon we have erected a new genus and species Siluania m onomastiga.
Siluania gen.nov. Discussion This flagellate has been firstly compa red with other small protistan flagellates such as Monochrysis parva [29], Oikomonas obliqua [15], Micromonas pusilla [23] , Kiitoksia ystava [33] and bicosoecids Pseudobodo minuta and P. minima [26]. Among these only Micromonas pusilla has been studied usin g TEM [17, 23], but it is a tru e green flagellate; Kiitoksia ystava is an unplaced heterotrophic protist with two distin ctive flagella [33]; Oikomonas obliqua has a stalk and a flagellum which is lon ger than the cell body; and th e dimensions of the cells are more than 3 pm [15]. The uniflagellate chrysom onad Monochrysis parva [29] has similar dimensions, a short flagellum wh ich emer ges subapically, and was found in fresh water pond s. H owever, no eyespot or contractile vacuole was noticed [29]. On the other hand, M. parva is clearly distin guished from the flagellate under consideration by having a 'brown chromatophore', obovoid body (the anteri or end is broader than th e po sterior one), and its motion is fast and uniform [29]. Th e bicosoecid s Pseudobodo minuta and P.minima should have the same dimensions as the species studied herein -1.5-2.5 x 1-1.5 pm and 2 x 1 urn, respectively [26]. However, these organisms have two flagella and P. minuta was recentl y transferred to the genus Cafeteria minuta [16]. Larsen and Patterson [16] suggested that the dimensions of
Unifl agellate holozoic colourless chrysophyte, flagellum insert ed subapicall y, cytos to me located apically. Type species: Siluania m onomastiga sp.no v. Etymology: The name of the genus is in honour of the Russian St. Siluan of Mount Atho s. Siluania monomastiga sp.nov. (Fig. 1-22, 24) Planktonic freshw ater un iflagellate measuring 1.5- 3.0 pm with one flagellum equal in size to the body; cell egg- or pear-shaped with rounded posterior end, flagellum emerging from small pit; moves by somersaulting. Occurs in cold fresh waters. Type locality: The Rybinsk reservoir (Russia), collected under ice in April 1996 by N .A.Zhgarev. Holotype: Fig. 1-5, 12-20,23. Etymology: 'rnonomasti ga' is Greek for 'uniflagellated'. Concerning the ultra stru ctural features, S. mon omastiga belongs to th e chromophyte or "stramenopiles" series as it has tubular cristae in its mitochondrion and tripartite tubular mastigonemes [25]. S. mon om astiga is not comp arable to the zoosporic fungi that belong to the stramenopiles (Oomycetes and H yphochytridiom ycetes) as it has a simple life cycle consisting of only a un iflagellate vegetative stage and cyst. Pelagom onas calceolata has similar dimensions (1.5- 3 prn) and two transition sp iral fibers between two tranverse plates [2], but differs from S. monomastiga in
422
S. A. Karpov, R. Kersanach and D. M. Williams Pelagornonas calceolata Aureococcus anophageffrrens
100/100
}
Sarcinochrysis marina
Pelagophyceae
Pulvinariasp. CCMP292 Pteridomonas danica Apedinella radians
} Dictyochophyceae
' - - - - - Rhizochromulina cJ. marina
Alariaesculenia Costana cosllllll
100/100
81/76
Phaeophyceae/ Xanthophyceae
Scyt06iplwnlornentaria
Edocarpussilicul06US
Fig. 24. Distance tree derived from 1527 unambiguous aligned positions of 47 Stramenopile/Haptophyte small subunit ribosomal DNA. Values at internal branches indicate the distance/parsimony bootstrap support (% of 1001200 replicates, values >50% presented).
}
' - - - - - Tribonema aequale
1------ Coccoid Slramenopile. 1144 ) L....._ _
}
Helerosigmaakashiwo*
Unclassified Stramenopile Raphidophyceae
Chromulina chromophila
45/50
Hibbtrrdiamagna Chrysonephele paluslris
100/100
}
Chrysophyoeae
' - - - - Ochromonas danicJZ ' - - - - - Oikornonas mutabilis
) Oikomonadaceae
100/100 Nannochloropsisgrannulalll 100/100
Nannochloropsisoculalll
}
Eustigmatophyceae
Nannochloropsis salina 96/75
100/93 100/100 55/53 100/100 55/Unidentified Guinea Pig faeceo isolate U26177
} Blastocystida
Blaslocystis sp. ....-----1100/100
Proteromonas laarlile )
'--
90/75
100/100
Blastocyslis hominis
'--
. - - - - - - - Cafe~ roenbergensis 100/100
Proteromonadida
) Bicosoecida
UHF . - - - - - - - - - - - - - - Thrauslochytrium kinnei 64/95
lAbyrinlhuloides ha]iotidis Ulkenia profunda
.-----':;";"':''::'';;'';---1 ' - - - - - - - L..-
} Labyrinthulida
Prqmnesium paleliferum P527 100/100
Prymnesium cala1hiferum
79/76
EmilianahUXl!leyi Phaecyslisgloo06a
77/73
Haptophyta
Relicul06phaere socialis
' - - - - - - PllDltroa salina
* Synonym - Olisthodiscus luieus 0.1
A new heterotrophic f lagellate Siluania monomastiga
many essentia l respects: P. calceolata has a lon ger flagellum with paraxial plates, a thin theca coveri ng the plasmalem ma, a chloroplast of chrysophyte type, ext rusomes, and it is a true un iflagellate with no rootlets and cytostome [2]. Bicosoecids and pseud odendromonads are the tw o groups of heterotrophic chro rnophy tes w hich S. m onomastiga is mor e similar to th an any other proti sts. Th e ultra structure of five species of bicosoecids has been investigated before: Bicosoeca plan ctonica [3], B. k epneri and B. lacustris [19], B. maris [22] and Cafeteria roenbergensis [8, 24]. In the first EM works a general structure of the cell and th e feeding app aratu s structure have been sho wn. In common , all bicosoecids are similar to each other in thos e respects. Just the paraxial rod has been found in the posterior flagellum of B. k epneri and B. lacustris [19], and the number of axoneme microtubules is redu ced to 4-5 in the distal part of the posteri or flagellum of B. planctonica [3]. Parti cular attention to th e cytos keleto n has been paid to B. m aris and C. roenberge nsis, whi ch is most essential for comp arison with S. m onomastiga. Our species is mo re similar to C. roenbergensis than to B. m aris. Results fro m molecular analyses (Fig. 24) suggest th e same conclusion . Therefore it is reasonable to compare th ese two alori cate species with each other in more detail. The biflagellate C. roenbergensis repr esents one of the smallest (4- 6 prn) known bicos oecids. The tiny S. monomastiga has on e flagellum but with two additio nal basal bod ies. One of th e latter (the ant erior one) look s too small to be considere d a permanent basal body, and has no roo tlets. Thus, its existence could be connected with th e early stage of kinetid dupli cation during cell division . Perhaps, because of its smaller cell size, there is no recurrent flagellum in S. monomastiga, and cons equently, no rootlets. O nly the ventral microtu bular rootlet (r4) of S.monomastiga can be classified as a rootlet of th e seco nd basal bod y. It is possible th at it could be conside red an addition al microtubule of r3, which correspond s to th at termed "abc" in C. roenbergensis [24]. Th e flagellar base of S. mon om astiga correspo nds to the flagellar base of th e ant erior flagellum in C. roenbergensis. Both species have a 2-st rand left microtubular rootl et (r l ), broad right or cyto stom al microtubular rootlet (r3) with the outer part con sisting of one micr otubule in S. monomastiga which may correspond to that termed "x" in C. roenbergensis [24]. There is also a fibrillar material associ ated with r3 in S. monomastiga but it is mu ch shorter than that in C. roenb ergensis. Th e cytostomal roo tlet (r3) in both species has " L-shaped" form in cross section near its origin, and is connected to the mit ochondrion. Both species also have short fibr illar ro otl ets. However, in S. m onom astiga the fibrillar ro otl et is more closely co nnected with the mitochondrion and both parts pass in one direction. In C. roen -
423
bergensis th e two part s of th is rootlet pass at right angles to each other near to the nuclea r surface. Th e flagellar bases in both species are represented by doublets of peripheral microtubules which can be connected with small cell size. The rootl et system of S. monom astiga differs from that of C. roenbe rgensis in th e follow ing respects: rl has no seco ndary microtubules, the cytos to rnal ro otlet (r3) has an S-shaped form , as th e cytos to me is locat ed anteriorl y to th e flagellum; th e fibrillar roo tlet passes from the proximal end of flagellar basal bod y in S. m onomastiga but in C. roenbergensis it is co nnected with the lateral side in the middle of posterior basal bod y; the fibrillar co nnectives aro und the flagellar base are almost absent in S. monomastiga. Other minor differences between th e rootlet systems have been noted above. S. m on om astiga differs from C. roenbergensis in oth er respects. S.monomastiga has no core inside the basal bodi es, no striated brid ges betw een basal bodies, no microb odi es, no extruso rnes; th ere is a spiral fiber in the flagellar transition zone , and th ere is an electr on den se mater ial in the mitochondrion . Th e primary difference is th at S. m onom astiga has a well develop ed cytostome/cytopharyn x complex. To compare S. monomastiga with other bicosoecids, it is relevant to note th at the transitional spiral fibr es or rings are similar to that structure in B. maris [22J but in the latt er it is locat ed above the tr ansverse plat e. T he spiral fib er located abov e the transverse plate has been found in another repr esent ative of th e bicosoecids Pseudobodo tremulans (in preparation ). Other com mon char acters are restricted by the cyto skeletal str ucture . Parti cularl y essenti al is the L- shaped form of r3 in cro ss sectio , and its association with fibrillar material. These tw o characters have been already found in fou r bicosoecids: B. maris [22], C. roenb ergensis [24], S. monomastiga (present paper) and P. tremulans (in preparation ). Th e most important d ifference of bicosoecids from S. monom astiga is an absence of perm anent cytosto mal struc tures. In th is respect S. monom astiga is more similar to th e pseudodendro mo nads. Pseud od endrom onads have a well develop ed cytophary nx supporte d by broad cyt ostomal microtubular ro otl et, at least one lateral narr ow microtubular rootlet, and a small striated fibrillar rootlet originatin g from th e proximal end of the flagellar base [11, 12, 18]. Unfortunately, pseudodendromonads have not been investi gated in the same detail as C. roenbergensis and S. m onom astiga, therefore a co mparison with th e abso lute orientatio n of cytoskel etal elements is not yet pos sible. In other respects S. m onom astiga differs from pseud od endromonads. The latte r have bod y and flagellar scales and no mastigonemes, there is no spiral fibre in flagellar transition zone and the cell symmetry is different.
424
S. A. Karpov, R. Kersanach and D. M. Williams
In conclusion, the uniflagellate S. monomastiga cannot be placed among the pseudodendromonads. It is certainly more similar to bicosoecids, particularly in respect of cytoskeletal structure, a fact confirmed by molecular data. Unfortunately, there are little molecular data available for other bicosoecids which may allow a more definitive position for Siluania. The close position of Cafeteria and Siluania in all three molecular analyses allows them to be placed in one order Bicosoecales in spite of some morphological differences. There are two families in the order Bicosoecales (sensu [21])2: Cafeteriaceae, which includes bicosoecids without loricae, and Bicosoecaceae, which includes those with loricae. However, representatives from both families do not have a permanent cytostome. Therefore, although Siluania could be formally placed in the family Cafeteriaceae (as a flagellate without lorica), we cannot do this because of presence of the permanent cytostome structure. On the other hand, it is not correct to establish a new order for this species being based just on that. Siluaniaceae Karpov (present paper) Apochlorotic uniflagellated 'chrysophytes' without a lorica; with unilateral mastigonemes; a transition spiral fiber located under the transverse plate; a permanent cytostome with developed cytopharynx present and supported by a microtubular rootlet. Type genus: Siluania Karpov (present paper) Regardless of whether a new family is erected, the diagnosis of the order Bicosoecales has to be amended as there are not known bicosoecids with a permanent cytostome. Order Bicosoecales (Grasse) emend. Karpov (present paper) 'Chrysophycean' heterotrophic flagellates without plastids, having one or two flagella, with or without a lorica; feeding apparatus represented by lip or by permanent cytostome with cytopharynx; transitional spiral fiber, if present, located above or under transverse plate; the broadest microtubular rootlet connected with cytostome region, often "L-shaped" in cross section and associated with fibrillar material. Sedentary and planktonic, freshwater and marine. According to the International Botanical Code of Nomenclature the order Bicosoecales (Grasse) emend. Karpov (present paper) includes 3 families: We cannot accept the classification of Tom Cavalier-Smith [4], where these families belong to different orders. It seems not necessary to introduce the new order Anoecales, which has incorrect diagnosis (neither Cafeteria, nor any other bicosoecids have sagenogenetosome or ciliary transitional helix-character. Therefore it is proposed in this paper to erect a new family in the order Bicosoecales.
• Siluaniaceae Karpov (present paper) • Siluania Karpou (present paper) Cafeteriaceae Moestrup, 1995 Cafeteria Fenchel & Patterson, 1988, Pseudobodo Griessmann, 1913 Bicosoecaceae Stein, 1878 Bicosoeca Stein, 1878 Accoding to the International Zoological Code of Nomenclature the order Bicosoecida (Grasse) emend. Karpov (present paper) includes 3 families: • Siluaniidae Karpov (present paper) Cafeteriidae Moestrup, 1995 Bicosoecidae Stein, 1878 Acknowledgements: This work was partly supported by the ex-quota visit programme of the Royal Society of London. The authors are also grateful to Dr. B.s.e. Leadbeater as the bulk of this work was undertaken in his laboratory of the School of Biological Sciences of Birmingham University. The molecular work was undertaken with financial assistance from NERC grant awarded to DMW and T. Martin Embley.
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3 4 5
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A new heterotrophic flagell ate 5iluania mon omastiga
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