The authors have declared that no competing interests exist.
Conceived and designed the experiments: PJK VVA TGS APM. Performed the experiments: DVT JJ KM APM. Analyzed the data: JJ KM APM DT. Contributed reagents/materials/analysis tools: TGS APM VVA PJK. Wrote the paper: DVT JJ APM PJK.
The evolutionary and ecological importance of predatory flagellates are too often overlooked. This is not only a gap in our understanding of microbial diversity, but also impacts how we interpret their better-studied relatives. A prime example of these problems is found in the alveolates. All well-studied species belong to three large clades (apicomplexans, dinoflagellates, and ciliates), but the predatory colponemid flagellates are also alveolates that are rare in nature and seldom cultured, but potentially important to our understanding of alveolate evolution. Recently we reported the first cultivation and molecular analysis of several colponemid-like organisms representing two novel clades in molecular trees. Here we provide ultrastructural analysis and formal species descriptions for both new species,
Alveolates comprise a great portion of protist diversity and include many medically and ecologically important species, such as the malaria parasite (
The colponemids, comprising the single described genus
Despite this evolutionary promise, we known little of colponemids because of their apparently rarity in nature and the difficulty in culturing them. Colponemids remained one of the last alveolate groups for which molecular data was completely missing until recently, and only five colponemid species have been ever described, of which only two have been investigated at the ultrastructural level
New species were identified from samples collected in three distant localities: southern Vietnam wetlands (1), saline lake sediments of Peru seashore (2), and soil and permafrost of Russia (3). Vietnamese samples came from a lake and pool belonging to the Bau Sau wetland complex located in the Cát Tiên National Park, Dong Nai Province, S.R. Vietnam. Cát Tiên National Park has an area of approximately 720 km2 and is located approximately 150 km to the north of Ho Chi Minh City, in the south of Vietnam. The park includes a large area of lowland evergreen tropical and deciduous forest, bamboo woodlands, wetlands and seasonally flooded grasslands, and a small proportion of farmland. The territory is subjected to the tropical monsoon climate with two distinct seasons: a rainy season from April to November and a dry season from December to March. The mean annual rainfall is 2450 mm. The temperature amplitude is very low, varying from 24 to 29°C, and the mean annual temperature is 25.4°C
Peruvian samples came from sediments of the saline lake Supay (76°14′44.38″ W, 14°0′5.18 N″), Pisco Province, Ica Department, Peru. This coastal desert area is subjected to the hot arid climate. The sample was collected at 20 cm depth, (Salinity 35‰, Temp. about 25°C) and contained mainly organic detritus. One clone of the predatory flagellate Colp-5 was isolated.
Russian samples came from soil from the Vorontsovskaya cavernae system, Caucasus and from the permafrost material near Kolyma River, Chukotka, Russia as described previously
Field studies in Vietnam were conducted under permits issued by the administration of Cát Tiên National Park, Vietnam, and authorized by Russian-Vietnam Tropical Centre, Coastal Branch (Nha Trang, Vietnam). No specific permits were required for the described field studies in Russia and Peru. The field studies did not involve endangered or protected species.
The sediment samples, including water, were placed in 50-ml flasks and transported to the laboratory within 5 days. 10 ml sample volumes (water with sediments particles) were analyzed in glass Petri dishes. Species diversity was studied directly after sample arrival and after enrichment with a suspension of
Clonal cultures were isolated from single cells using a micromanipulator fitted with a glass micropipette
Freshwater culture of kinetoplastid
Marine culture of kinetoplastid
Light microscopy observations were conducted using the Carl Zeiss AxioScope A.1 and Biolam-I (Russia) microscopes equipped with DIC and phase contrast water immersion objectives (63x and 70x). The microscopes were equipped with the analog video camera AVT HORN MC-1009/S connected to the Panasonic NV-HS 850 video recorder. Images were acquired using the VHS and S-VHS technology and subsequently digitalized. Short video sequences were obtained in order to facilitate cell identification and describe cell movement, reproduction and feeding behavior. Cells were centrifuged (5000×
Cells grown in clonal laboratory cultures were harvested following peak abundance after eating most of the prey. Cells were collected by centrifugation (10000×
Sequences of Colp-14 and Colp-62 were deposited in GenBank under accession numbers KJ598080 and KJ598081.
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “
(a, c) general view, short ventral groove, large food and contractile vacuoles; b) bean-shaped cell, nucleus, large food vacuole; d) ovoid cell with wide ventral groove; e) feeding of a starved cell with small anterior rostrum; (f, g, i) general view, large food vacuole and nucleus; h) feeding on the prey. a.f – anterior flagellum, c.v – contractile vacuole, f.v – food vacuole, gr – longitudinal groove, n – nucleus, p.f – posterior flagellum, pr – prey. Scale: 10 µm for all figures.
c.v – contractile vacuole, p.f – posterior flagellum, f.v – food vacuole. Scales: 10 µm for all figures.
Cells of Colp-7 and Colp-7a strains swim rapidly in a spiral or zigzag, usually near the bottom of Petri dishes. The organism is an obligate predator; it takes up smaller flagellates and quickly perishes in the absence of prey. Feeding on bacteria was not observed. After an initial contact with prey, the
c.v – contractile vacuole, a.f – anterior flagellum, p.f – posterior flagellum, f.v – food vacuole. Scales: 10 µm for all figures.
Cells of the
Ultrastructure of
a) Electron micrograph of the cell. Anterior and posterior flagella (af and pf) end with narrowing tips – acronemes (an). The arrows point to the short fold of proximal part of the pf. b) The transversal section at the level of the nucleus (n). In some places the cell contains the alveoli (al) beneath the plasmalemma. Small cytosolic vesicles contain the rudiments of the mastigonemes (mn). Golgi apparatus (Ga) is situated close to the nucleus. Discharged toxicysts (tc) are found outside of the cell. The band of 5+1 microbules (bm) accompanies the pf. Food vacuole (fv) contains remnants of a prey cell. The neighboring cell (nc) is seen. c) The cross section of the pf. The thin fold (fl) is visible. d) Flagella are arranged mutually at an angle of 45 degrees. Right and left bands of microtubules (rbm and lbm) and fibrous band (fb) emerge from the bases of the pf which runs inside the short ventral groove (vg). The secondary microtubules (sm) run near af. The axosoma (as) is visible above the transverse plate (tp). e) The pf inside vg. sm originate from the microtubular band (bm). The conspicuous as is located above the transverse plate (tp) of the flagellum. Mastigonemes (mn) cover the pf. f) The short amorphous rhizoplast (rp) extends from the kinetosome of the posterior flagellum (kpf) towards the nucleus (n). G. Kinetosome area. Three microtubules (rbm) and fibrous bands (fb) lie close to the kpf. vg is armored by the left band of microtubules (lbm). Kinetosome of anterior flagellum (kaf) contains an axosome (as). Single secondary microtubules (sm) are seen. h) pf passes in the vg and bears thin mastigonemes (mn). as is visible in the transitional zone of the flagellum. Scales: 0.5 µm in (c–h); 1 µm in b); 5 µm in a).
a) The appearance of secondary microtubules (sm) from the band of microtubules (bm) going near the proximal end of the kinetosome of the flagellum. Mastigonemes (mn) are included inside the vesicles. Axosome (as) of the flagellum resembles a muff. (b, c) The arrangement of the right band of microtubules (rbm) and left band of microtubules (lbm) lying parallel to the posterior flagellum (pf). The single secondary microtubule (sm) is visible. The band of the microtubules surrounds the kinetosome of posterior flagellum (kpf). d) The nucleus (n) contains a conspicuous compact nucleolus. The extensions of the perinuclear space have mastigonemes (mn). Golgi apparatus (Ga) lies close to the nucleus. e) The mitochondria (mt) contain tubular cristae in cross section. Mastigonemes (mn) are formed inside the perinuclear space. f) Osmiophilis granules represent storage compounds (rs). (g, h, i) The structure of the toxicysts. The longitudinal and transversal sections of mature toxicysts g) and h), empty discharged toxicyst i). Scales: 0.5 µm in (a–c), (h–i); 1 µm in d), e), f).
Both flagella have an ordinary structure (9+2) in section (
The ventral groove starts from the posterior flagellar pocket and extends backwards (
The vesicular nucleus has a central nucleolus represented by an electronic dense material (
Recently, we showed that colponemids fell into two distinct groups in the phylogeny of alveolates: Colp-5 branched at the base of the Myzozoa (apicomplexans, dinoflagellates, and their close relatives), while Colp-7 and Colp-7a branched deeper, and were sister to the only described strain with molecular data,
RAxML phylogenies of eukaryotes based on the 18S rDNA dataset (a), and the 18S+28S rDNA dataset (b). RAxML rapid bootstrap, PhyML aLRT and MrBayes posterior probability branch supports are shown at branches (>50/>0.8/>0.9 are shown as significant; dashes indicate insignificant support). Black dots indicate complete support (100/1/1). Numbers of sequences in collapsed clades are shown in brackets.
Nearly all the strains (Colp-7, 7a, 14 and 62, as well as
The single remaining strain (Colp-5, from Peru) was not specifically related to
The colponemids are bi-flagellar protists that inhabit both marine and freshwater habitats, and can also be found in the soil
The ultrastructural features of
The new species of
Species | Cell size, µm | Cell shape | Comparative length ofanterior flagellum inrelation to body length | Comparative length ofposterior flagellum inrelation to body length | Ventral groove | Anteriorrostrum | Contractilevacuole | Nucleusposition |
10.5–14.5×5.0–6.5(up to 9.0 forsatiated cells) | elongated-oval, not flattened;satiated cells bean-shapedor ovoid | 2 times shorter | 1.5 times longer | short, weaklypronounced | present | present atthe anteriorend | cell center or closerto the anterior end | |
8–12×2.5–4.5 | oval, flattened | about the cell length | 2 times longer | short, well-markedat starvingspecimens | present | present atthe anteriorend | anterior or centralcell part | |
8.5–14×4.0–6.5 | elongated-oval, not flattened | about the cell length | 2 times longer | short, pronounced | absent | absent | anterior cell part | |
17–30×8–15 | ovoid or bean-shaped,not flattened | about the cell length | 1.5 times longer | Long, pronounced | present | present atthe anteriorend | anterior cell part | |
15×13–14 | wide-oval, flattened | slightly shorter than cell | 2 times longer | long, deep, withwide curvilinearmargins | absent | present atthe anteriorend | no data | |
9–15 | elliptical with widely roundedends, flattened | 1.5–2 times longer | 3–4 times longer | long, well-visible | absent | no data | slightly below thecell center | |
9.5–13.5 | elongated-oval or egg-shaped,not flattened | about the cell length | 2 times longer | Absent | present | absent | anterior cell part |
The new genus described here,
The
We observed rapid feeding behavior of
The phylogenetic positions of
Taxonomic diagnoses of the newly described
Assignment. Eukaryota; Alveolata; Colponemidia.
urn:lsid:zoobank.org:act:EBDBD217-D233-4F2C-A621-00513F1B397B.
Type strain: Colp-7a; sediments of the shallow pool, Bau Sau wetland complex, Cát Tiên National park, Dong Nai Province, S.R. Vietnam. The Colp-7a clone is stored in the collection of live protozoan cultures at IBIW RAS.
Type Figure: 2a.
Description. Cells elongated-oval, rigid, not flattened, 10.5–14.5 µm long and 5.0–6.5 µm wide with a small anterior rostrum and rounded posterior end. Anterior end of the cell usually wider than posterior one. Ventral groove is comparatively short and weakly pronounced. Anterior flagellum about half the cell length and makes flapping movements and often curves to dorsal cell surface. Posterior flagellum about 1.5 times the cell length and sometimes undulates in the longitudinal ventral groove. Large contractile vacuole located at the anterior end. Spherical nucleus situated in the center of the cell or slightly closer to the anterior end. Cells swim rapidly, spiral or zigzag. Organism is an obligate predator; it takes up smaller flagellates (e.g.
Comparison. From all other species of
Type locality. Sediments contained organic detritus, plant debris and filamentous algae of the shallow pools and lakes of the Bau Sau wetland complex, Cát Tiên National Park, Dong Nai Province, S.R. Vietnam.
Type sequence. Partial small subunit ribosomal RNA gene of
Etimology. The species name means “Vietnam-dwelling”.
(a–d)
(a–f)
Cell flattened and oval, 8–12 µm long and 2.5–4.5 µm wide with small rostrum and pointed distal part. Flagellar bases situated in anterior part of the cell, subapically. Anterior flagellum about the cell length or slightly longer, often curves to the dorsal cell surface. Posterior flagellum about twice cell length, situated in ventral groove and makes undulating movements there. Large contractile vacuole situated in the anterior part of the cell; median nucleus in the anterior or central cell part. Young specimens with the small rostral anterior end of cell and rounded posterior end (
Cell elongated-oval, 8.5–14 µm long and 4.0–6.5 µm wide. Anterior end of the cell wider than posterior one. Two heterodynamic flagella appear separately from two flagellar pockets. Anterior flagellum about the cell length, posterior one about twice the cell length, situated in short ventral groove and makes undulating movements there. The nucleus is situated anterior, whereas the large food vacuole is posterior. Alveoli are not numerous. The extrusive organelles are of the toxicysts type. Contractile vacuole is absent. Cells multiply by binary longitudinal division. Cysts have not been found. Cells swim in zigzag path near substrate. The organism is an obligate predator and takes up small heterotrophic flagellates (like
Cells are ovoid or bean-shaped, 17–30 µm long and 8–15 µm wide, with well-visible rostrum. Anterior flagellum about as long as the cell, and makes flapping movements; the posterior flagellum is about 1.5 times as long as the cell, and undulates in the longitudinal ventral groove. Large spherical nucleus and contractile vacuole situated in anterior part of cell. Large food vacuole in posterior. Cytoplasm contains light-refracting granules. Amphora-shaped toxicysts about 0.9–1.5 µm long. Cells multiply by longitudinal division. Cysts not found. Inhabit fresh-water, including benthos of ponds, reservoirs, sewage waters as well as soils rich in humus. Swims in a spiral pattern, and after making contact with prey stops and within 1–2 minutes captures it. Paralyzing effects on prey not revealed. Pattern of absorption of food was described by Zhukov and Mylnikov
Cells oval, wide and flattened, with the anterior part wider than posterior one. Deep longitudinal ventral groove, with wide curvilinear margins. Ventral groove narrowed in middle part of cell and widened at the cell ends (especially near the posterior end). Cells are about 15 µm long and 13–14 µm wide. Flagella originating from ventral groove near the anterior end. Anterior flagellum slightly shorter than the cell; posterior flagellum about twice as long as the cell. Median contractile vacuole situated at the anterior end of the cell. Cytoplasm contains light-refracting granules. Cysts not found. Observed rarely in the marine waters of the Rio de Janeiro gulf, Brazil
Cells rigid, flattened, elliptical, with widely rounded ends. Distinctive median longitudinal ventral groove divides the cell into two equal parts. Cell length about 9–15 µm. Subapical anterior flagellum is about 1.5–2.0 times as long as the cell; subapical posterior flagellum is about 3–4 times as long as the cell. Spherical nucleus situated slightly posterior to the cell center, near dorsal side and distinguishable only after fixation. Organism always attaches to the substratum by the distal end of posterior flagellum and jerks constantly backwards and forwards. Data about feeding absent. Cysts not found. Rare species, observed in soils of England
Assignment. Eukaryota; Alveolata; Acavomonidia.
urn:lsid:zoobank.org:act:D0A71F0F-C760-4236-84D3-E937A0A98C14.
Type Figure: 3a.
Description. The cell is biflagellate, not dorsoventrally compressed, elongated-oval shape with a rounded posterior end. Flagella originate near the anterior cell end. The anterior flagellum makes flapping movements, the posterior flagellum is straight and rigid, does not make obvious movements. A large food vacuole is located at the posterior end of the cell. Longitudinal ventral groove is absent. Obligate fast-swimming predator, consumes smaller flagellates.
Comparison. From very similar representatives of the genus
Type species.
Etimology. The genus name means “without cave (ventral groove)”.
urn:lsid:zoobank.org:act:3B010EA2-C5BE-462B-947F-A2488C6C422C.
Type strain: Colp -5; sediments of saline lake Supay, Pisco Province, Ica Department, Peru. The clone Colp-5 was stored in the collection of live protozoan cultures at IBIW RAS, but perished after one month of cultivation.
Type Figure: 3a.
Description. Cell is elongated-oval or egg-shaped, rigid, not flattened, 9.5–13.5 µm long and 6.0–10.0 µm wide with a small anterior rostrum. The anterior flagellum is about the cell length, very thin, frequently curves to dorsal cell surface. The posterior flagellum is about 2 times the cell length, straight and rigid. A spherical nucleus is situated at the anterior end of the cell. A large food vacuole is located at the posterior end of the cell. Cells swim rapidly, rectilinearly or in a spiral. Organism is an obligate predator; it consumes smaller flagellates (e.g.
Comparison. Single representative of the genus, from similar species of
Type locality. Organic detritus of the saline lake Supay, Pisco Province, Ica Department, Peru.
Type sequence. Partial small subunit ribosomal RNA gene of
Etimology. The species name means “Peru dwelling”.
Previous alveolate classifications tended to treat the colponemids as unknowns. Cavalier-Smith initially placed
Infrakingdom Alveolata Cavalier-Smith 1991 Primarily single-celled eukaryotes with cortical alveolae, ciliary pit or micropore, tubular or ampulliform mitochondria cristae, extrusomes with dense cores. | |||
Phylum Myzozoa Cavalier-Smith 2004 Predominantly haploid, typically uninucleate alveolates with zygotic meiosis; lacking separate macronuclei; ancestrally and typically with two centrioles and cilia only; anterior cilium often with simple hairs. Trichocysts typically with a dense basal rod that is square in cross section and a less dense distal region composed of hollow twisted tubules. When trichocysts are present cortical alveoli are typically inflated and morphologically discrete, often with internal plates; when trichocysts are absent they are typically highly compressed and often fused into an inner membrane complex. Myzocystosis and/or rhoptries and micronemes are very widespread, and possibly even ancestral. | Phylum Acavomonidia nom. nov. Free-living bi-flagellar protists with rigid cell and vesicular nucleus. Predators, capture prey as a whole and lack special structures for food uptake (as opposed to sucking structures in Myzozoa employed during myzocytosys). No reproduction or resting cysts. | Phylum Colponemidia nom. nov. Bi-flagellar cells with three-membrane alveolar pellicle, vesicular nucleus has a central nucleolus, two microtubule bands armour the longitudinal groove. The anterior flagellum carries fine nontubular mastigonemes at its proximal end. The posterior flagellum with the proximal fold, undulates in ventral groove. Amphora-like or bottle-shaped extrusomes related to toxicysts type. Micropores are absent. Predators, capturing prey as a whole by a longitudinal groove uptake. Binary longitudinal cell division. No reproduction or resting cysts. | Phylum Ciliophora Doflein 1901 [Ciliata Perty 1852, Infusoria Bütschli 1887] Cells with nuclear dimorphism, including a typically polygenomic macronucleus and at least one diploid micronucleus; somatic kinetids having a postciliary microtubular ribbon arising from triplet 9, a kinetodesmal fibril or striated rootlet homologue arising near triplets 5–8, and a transverse microtubular ribbon arising in the region of triplets 4–6; sexual reproduction, when present, by conjugation typically with mutual exchange of haploid gametic nuclei that fuse to form the synkaryon or zygotic nucleus. |
Class Acavomonea cl. nov. Order Acavomonida ord. nov. Family Acavomonidae fam. nov. urn:lsid:zoobank.org:act:63976BCD-ED40-46E0-8F16-0133DC0896C7 Diagnosis as for phylum Acavomonidia. | |||
Class Colponemea Cavalier-Smith 1993. Emend Free-living zooflagellates; centrioles diverge at nearly 90° | |||
Type genus |
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Taxonomy of Myzozoa is not discussed here because we believe that systematics of this phylum cannot be currently resolved with certainty and requires clarification using molecular data. | |||
Order Colponemida Cavalier-Smith 1993. Family Colponemidae Cavalier-Smith and Chao 2004 Free-living biciliates with inflated cortical alveoli over all cell surface; cilia subapical; posterior cilium in deep gutter; with toxicysts, but no rhoptries. | |||
Type species |
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Phylum is represented now by the single genus |
Phylum Acavomonidia includes the single genus
We also create the phylum Colponemidia, and move the class Colponemea Cavalier-Smith 1993 and order Colponemida Cavalier-Smith 1993 from Myzozoa to phylum Colponemidia. This classification is strongly supported by molecular phylogenies and by the fact that colponemidians lack apical complex-like structures and do not feed by myzocytosis. However, we retain the order Algovorida Cavalier-Smith 2004 previously classified inside the class Colponemea by Cavalier-Smith and Chao
The authors would like to thank Dr. Dmitry Scherbakov, Dr. Evgeny Gusev, Dr. Vladimir Gusakov for help with sample collection as well as the staff of the Russian-Vietnam Tropical Centre, Coastal Branch (Nha Trang, Vietnam), especially to Nguyen Th? Hai Thanh and Tran Duc Dien for their assistance with trip management and sampling. This field study has been part of the project “Ecolan 3.2.” of the Russian-Vietnam Tropical Centre.