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Using standard methods, we studied the morphology and 18S rDNA sequence of some peritrich ciliates from tank bromeliads of Costa Rica, Jamaica, and Ecuador. The new genus Orborhabdostyla differs from Rhabdostyla by the discoidal macronucleus. Two species from the literature and a new species from Ecuadoran tank bromeliads are combined with the new genus: O. previpes (Claparède and Lachmann, 1857) nov. comb., O. kahli (Nenninger, 1948) nov. comb., and O. bromelicola nov. spec. Orborhabdostyla bromelicola is a slender species with stalk-like narrowed posterior half and operculariid/epistylidid oral apparatus. An epistylidid relationship is also suggested by the gene sequence. Vorticella gracilis, described by Dujardin (1841) from French freshwater, belongs to the V. convallaria complex but differs by the yellowish colour and the number of silverlines. The classification as a distinct species is supported by the 18S rDNA, which differs nearly 10% from that of V. convallaria s. str. Based on the new data, especially the very stable yellowish colour, we neotypify V. gracilis with the Austrian population studied by Foissner (1979). Vorticella gracilis forms a strongly supported phyloclade together with V. campanula, V. fusca and V. convallaria, while Vorticellides astyliformis and Vorticella microstoma branch in a separate, fully-supported clade that includes Astylozoon and Opisthonecta. The new genus Vorticellides comprises five small (usually < 60 μm), barrel-shaped species with two epistomial membranes: V. aquadulcis (Stokes, 1887) nov. comb., V. astyliformis (Foissner, 1981) nov. comb., V. platysoma (Stokes, 1887) nov. comb., V. infusionum (Dujardin, 1841) nov. comb., and V. (Spinivorticellides) echini (King, 1931) nov. comb. Two of these species are redescribed in the present study: V. astyliformis and V. aquadulcis, which is neotypified with a Costa Rican population. Pseudovorticella bromelicola nov. spec. differs from the congeners by the location of the two contractile vacuoles and the number of silverlines.
The peritrichs are classified into two assemblages (Jankowski 2007, Lynn 2008): the free-living Sessilida and the parasitic Mobilida with their characteristic adhesive disk. The Sessilida, which includes the species described here, comprises about 105 (Lynn 2008) to 140 (Jankowski 2007) genera, showing the great diversity of the group. Although a comprehensive recent review is not available, these genera comprise at least 800 described species. Many of the taxa are epibionts on a great variety of aquatic and semiterrestrial metazoans (Matthes 1982). For instance, some 80 peritrich species and about 10 suctorians and chonotrichs have been reported from freshwater gammarids globally (Schödel 1987). Very likely, the undescribed epibiontic peritrich diversity is much greater than the described one because detailed investigations are rare outside Europe. Likewise, the marine ecosystems are poorly explored, providing a continuous flow of new species (Song and Wang 1999, Sun et al. 2007). Although being comparatively well investigated, new genera and species are still discovered in freshwaters (Ji and Kusuoka 2009, Norf and Foissner 2009). Further, some interesting habitats escaped the attention, for instance, soil and the little water bodies of tank bromeliads (Foissner et al. 2002, 2003). These habitats contain a considerable number of new species, some of which are described in the present study.
Several peritrich genera contain more than 50 nominal species, for instance, Vorticella and Epistylis. A first split of the former into Vorticella (silverlines in transverse rings) and Pseudovorticella (silverline pattern reticulate) was performed by Foissner and Schiffmann (1974), and has been widely accepted (Warren 1987, Song et al. 2009). In the present paper, we propose a second split, using the number of epistomial membranes as a main distinguishing feature. Both splits are supported by 18S rDNA sequences, in which Vorticella appears non-monophyletic (Martin-Cereceda et al. 2007, Li et al. 2008; present paper, Fig. 25).
Peritrichs attracted comparatively many protistologists but reliable species descriptions became available only in the seventies of the past century. We emphasize not only the need of silver preparations and morphometry but also a detailed photographic documentation of the live cells because most species have a characteristic shape difficult to grasp by line drawings. However, the great success of Kahl's monographs is partially based on his outstanding ability to recognize the representative body shape and to show it by “simple” line drawings (Foissner and Wenzel 2004).
The geographic origin of the material is provided in the individual species descriptions. Most samples were from tank bromeliads and were collected and sent to Salzburg by colleagues (see Acknowledgements).
In the Salzburg laboratory, the samples were screened for the species present. To study the peritrichs, which were attached to mud particles in low numbers, we used the following method: The tank water and the mud were sieved through a 500 μm net to remove crustaceans, insect larvae, and large rotifers. Then, the sample was transferred into a Petri dish and enriched with some partially crushed wheat grains to stimulate growth of the natural bacterial community. Concomitantly, coverslips were put on the sample (water) surface, where the peritrichs attached and developed considerable abundances within a few days. With this simple method, sufficient material was obtained for live observations and preparations. Further, such material was used to start a pure culture of Vorticella gracilis. This species was cultivated over half a year in Eau de Volvic (French table water) enriched with some crushed wheat grains. Specimens attached to the bottom of the Petri dish, bacterial flocks, and the coverslips on the culture surface.
Morphological and presentation methods followed Foissner (1991) and Foissner et al. (2002). Briefly, living cells were studied using a high-power oil immersion objective and differential interference contrast. Preparations were performed as described in Foissner (1991). Counts and measurements on silvered specimens were conducted at a magnification of × 1000. In vivo measurements were performed at magnifications of × 100–1000. Illustrations of live specimens were based on free-hand sketches and micrographs, while those of prepared cells were made with a drawing device. Terminology is according to Corliss (1979) and Lynn (2008).
To extract genomic DNA for 18S rDNA phylogenies, about 10 specimens of each species were picked with a micropipette and transferred into 180 μl ATL buffer (Qiagen) and 20 μl Proteinase K (20 mg/ml). Subsequently, the genomic DNA was extracted using the protocol for cultured animal cells of the DNEasy Tissue Kit (Qiagen, Hildesheim, Germany). The 18S rDNA was amplified using the universal eukaryotic primers EukA and EukB (Medlin et al. 1988). The amplification reaction contained 10–20 ng of DNA template, 2.5 U HotStar Taq DNA polymerase (Qiagen) in the manufacturer-provided reaction buffer, 1.5 mM MgCl2, 200 μM of dNTP, and 0.5 μM of each oligonucleotide primer. The final volume was adjusted to 50 μl with sterile distilled water. The PCR protocol for 18S rDNA gene amplification consisted of an initial hot start incubation of 15 min. at 95°C followed by 30 identical amplification cycles (i.e., denaturing at 95°C for 45 s, annealing at 55°C for 1 min., and extension at 72°C for 2.5 min.), and a final extension at 72°C for 7 min. Negative control reactions included Escherichia coli DNA as a template. The resulting PCR products were cleaned with the PCR MinElute Kit (Qiagen) and cloned into a vector using the TA-Cloning kit (Invitrogen, Carlsbad, CA). Plasmids were isolated with Qiaprep Spin Miniprep Kit (Qiagen) from overnight cultures and PCR-reamplified using M13F and M13R primers to screen for inserts of the expected size (about 1.8 kb in case of the SSu-rDNA fragment). Three clones were sequenced bidirectionally (M13 sequence primers) with the Big Dye terminator kit (Applied Biosystems, Foster City, CA) on an ABI 3730 automated sequencer.
For a first assessment of the approximate phylogenetic placement of O. bromelicola, V. astyliformis and V. gracilis, their 18S rDNA sequences were aligned to all 18S rDNA sequences of peritrich ciliates available in GenBank. As an outgroup, we chose a representative each from the orders comprising the class Oligohymenophorea (for a review, see Lynn 2008). Alignments were constructed, using ClustalX (Thompson et al. 1997), and were manually refined in MacClade (Maddison and Maddison 2003), according to conserved regions. Distance, Maximum Likelihood and Bayesian analyses showed a monophyly of the sessilid and mobilid peritrichs, in accordance with the analyses of Gong et al. (2006). Trees with support values are available from the authors.
We then performed a second phylogenetic analysis with a taxon sampling restricted to all 18S rDNA sequences of sessilid peritrichs available from GenBank and the oligohymenophorean Urocentrum turbo as outgroup taxon. The rationale behind this approach is that an analysis with less taxa, which are comparatively closely related to each other, allows the use of a higher number of unmasked, unambiguously aligned nucleotide characters, possibly resulting in a better resolved and supported phylogeny. The respective alignment included 1571 positions. We applied the program Modeltest (Posada and Crandall 1998) to choose the model of DNA substitution that best fitted our data sets. The model suggested by the AIC (Akaike Information Criterion) was GTR + I + G with the proportion of invariable sites (I) being 0.3739 and the gamma distribution shape parameter (G) being 0.5571. Neighbour joining evolutionary distance (NJ) analyses under maximum likelihood criteria were carried out in PAUP* v4.0b8 (Swofford 2002). Bayesian inference trees were obtained by using Mr. Bayes (Ronquist and Huelsenbeck 2003). For the Bayesian trees we ran two simultaneous, completely independent analyses starting from different random trees. This analysis also employed GTR + I + G as the DNA substitution model with the gamma distribution shape parameter, the proportion of invariable sites, base frequencies, and a rate matrix for the substitution model as assessed by Mr. Bayes. Metropolis coupling with three “heated” chains and one “cold” chain was employed to improve the Markov Chain Monte Carlo sampling of the target distribution. We ran 10,000,000 generations and sampled every 10,000th generation, resulting in 1001 samples from the posterior probability distribution. The relative stability of tree topologies was assessed, using 1000 bootstrap replicates and posterior probabilities of 751 Bayesian trees (25% burnin). Heuristic searches for bootstrap analyses employed stepwise addition, starting trees with simple addition of sequences and TBR branch-swapping. Maximum-likelihood bootstrapping analyses were carried out with 1000 replicates using RAxML with the setting as described in Stamatakis et al. (2008). ML and BI analyses were conducted online on the CIPRES Portal V 1.15 (http://www.phylo.org) The GenBank accession numbers of sequences obtained in this study are as follows: O. bromelicola: GQ872428; V. astyliformis: GQ872427; V. gracilis: GQ872429. All individual data sets are available from the Stoeck laboratory.
Solitary Epistylididae (?) with discoidal to ellipsoidal macronucleus and transverse-striate silverline pattern.
Orborhabdostyla bromelicola nov. spec.
Composite of the epistylidid genus Rhabdostyla and the Latin noun orbis (circle), meaning a Rhabdostyla-like ciliate with globular nucleus. Feminine gender according to Aescht (2001).
In addition to the type, two species may be referred to the new genus, viz., Orborhabdostyla brevipes (Claparède and Lachmann, 1857) nov. comb. (basionym: Epistylis brevipes Claparède and Lachmann, 1857) and Orborhabdostyla kahli (Nenninger, 1948) nov. comb. (basionym: Rhabdostyla kahli Nenninger, 1948) nov. comb. Nenninger (1948) established this species for the Rhabdostyla sp. described by Kahl (1935) because she found several populations matching Kahl's description. For details, see description and comparison of O. bromelicola.
The new genus matches Rhabdostyla Kent, 1881, except for the shape of the macronucleus: horseshoe-like in Rhabdostyla, discoidal or globular in Orborhabdostyla. The same split has been performed by Lust (1950) in the genus Opercularia, referring species with discoidal or globular macronucleus to a new genus, Orbopercularia. This split has been widely accepted, for instance, by Corliss (1979) and Lynn (2008). Thus, we split Rhabdostyla, too. See Foissner (1979) and Foissner et al. (1999) for modern descriptions of Rhabdostyla species.
Traditionally, rhabdostylids are classified in the Epistylididae. However, the myoneme system and the more or less stalked peristomial disc suggest an operculariid relationship (Foissner 1981). In contrast to the epistylids (Foissner and Schubert 1977), the peristomial disc is not connected to the oral bulge myonemes which, in O. bromelicola, produce three thin, short branches connecting the oral bulge to some ventral body myonemes (Figs (Figs10,10, ,19).19). Vorticellids have, like epistylids, well developed oral bulge myonemes, which are neither connected to the peristomial disc nor the body myonemes (Foissner 1977).
Orborhabdostyla bromelicola branches in a well-supported (NJ/BI/ML – 99/100/79) clade together with Epistylis chrysemydis, E. urceolata, and E. wenrichi (Fig. 25). Thus, the 18S rDNA gene suggests an epistylidid rather than an operculariid affiliation. Even though the support for this relation is very strong, phylogenetic analyses cannot exclude an opercularid relation, as both, operculariids and orborhabdostylids are heavily undersampled and only represented by a single sequence each. Also, it is unfortunate that to date no representative of Rhabdostyla has been sequenced in order to evaluate the phylogenetic relation of Orborhabdostyla and Rhabdostyla.
Size about 65 × 15 μm in field specimens, while 50 × 15 μm in laboratory cultures. Narrowly conical with stalk usually < 10 μm long and 2 μm wide. Contractile vacuole and cytoproct on dorsal wall of vestibulum slightly posterior of oral bulge. On average 63 silverlines from oral end to anlage of aboral ciliary wreath and 16 silverlines from anlage to scopula. Peniculus 2 shortened posteriorly, peniculus 3 composed of three minute kineties. Freshwater, possibly restricted to tank bromeliads and not colonizing any host.
Tanks of Guzmania musaica from Ecuador, province Esmeraldas, underneath Alto Tambo, 270 m above sea level, N01°02′13.7″, W78°37′13.7″. The molecular investigation was performed on a population from Jamaica.
Three slides each with silver nitrate and protargol-impregnated specimens have been deposited in the Biology Centre of the Museum of Upper Austria, Linz (LI). The holotypes and other relevant specimens were marked by black ink circles on the coverslip.
The diagnosis and description are based on field and cultivated specimens, as described in the method section. Size and shape were studied in both, while protargol and silver nitrate data were obtained only from cultivated cells.
Size 50–75 × 13–18 μm in vivo, on average 65 × 15 μm in field specimens, while 40–70 × 13–20 μm, on average 50 × 15 μm in cultivated cells with high coefficient of variation (18%); average length: width ratio thus conspicuously different, viz., 4.3:1 vs. 3.5:1, with extremes of 2.4:1 to 5.2:1 (Table 1). Shape thus conical to narrowly conical, more or less asymmetrical, anterior end transverse to slightly obliquely truncate, posterior third frequently stalk-like narrowed (Figs 1, 6, 11–18, 26, 27, 35, 36). Highly contractile, especially in posterior third, i.e., from anlage of aboral ciliary wreath to scopula. Fully contracted field specimens about 25 μm long with anterior end rounded or slightly projecting (Figs 2, 9, ,19,19, ,41);41); when contracting, conspicuous transverse folds appear in posterior half and the anterior body section folds more or less over the contracted part, giving cells an ellipsoidal to globular shape with a deep indentation containing part of the strongly contracted posterior third (Figs 6–9, 30–32). Distinct folds usually absent from protargol-impregnated specimens (Figs (Figs10,10, ,19),19), i.e., rarely cells appear as shown in Figure 2. Macronucleus usually in anterior dorsal body half near cell's periphery; rarely ventral or lateral; usually ellipsoidal, rarely globular; flattened by about 50% and curved like a hollow hand, one margin frequently folded inwards; on average 10 × 7 μm in protargol preparations, while about 13–15 μm long in vivo; nucleoli minute and numerous. Micronucleus usually close to macronucleus, rarely far away, about 3 × 2 μm in size (Figs 1, 6, 10, ,20,20, 27, 29, 30, 33–35, 38, 39–42; Table 1). Contractile vacuole and cytoproct on dorsal wall of vestibulum slightly to distinctly posterior of oral bulge (Figs 1, 6, 12, 14, 26, 28, 36). Cytoplasm finely granulated, cell thus transparent; granules slightly concentrated in region of anlage of aboral ciliary wreath. Some food vacuoles mainly in posterior body half, 4–6 μm across, contain only few bacteria (Figs (Figs1,1, 26, 27).
Cortex smooth between oral opening and anlage of aboral ciliary wreath, while rugged between anlage and scopula due to distinct transverse ridges (Fig. 1). Pellicular pores mainly within ridges (silverlines), conspicuous because up to 1 μm across, but loosely arranged, i.e., only about 20 pores/100 μm2 (Figs 43, 44; Table 1). Specimens strongly contract, especially in posterior third, when air-dried for silver nitrate impregnation; silverlines thus difficult to count in that region, as indicated by the comparably high coefficient of variation, average possibly slightly higher than shown in Table 1. Silverline pattern basically narrowly striate (average distance between silverlines < 1 μm), except in posterior third, where it is widely striate (> 1 μm, see above). Pattern without peculiarities, some rings bifurcating or ending blindly (Figs (Figs4343--45).45). On average 63 silverlines between oral opening and anlage of aboral ciliary wreath and 16 from there to scopula (Table 1).
Stalk not branched in over 200 specimens analysed; minute, i.e., 1–3 μm long in field specimens, while 3–12 × 1.6–2.5 μm in protargol-prepared cultivated cells, 20 μm long in one field specimen; attached to organic and inorganic debris with adhesive disc up to 5 μm across (Figs 1, 2, 11–18, 29, 30, 35; Table 1). Myoneme system opercularid (see above). Body system comprising 10–15 thick, partially branched strands extending from scopula to anterior body end, where about half become disc retractors attaching to dorsal half of peristomial disc. Oral bulge myonemes inconspicuous, form very thin layer near inner margin of bulge, producing three short branches connected to the ventral body myonemes (Figs 3, 4, 10, 19, 20, ,37).37). No myonemes recognizable in peristomial disc. Another system, composed of fine, rather widely spaced fibres extends whole body length in some protargol-impregnated specimens (Fig. 37).
Oral apparatus epistylidid, however, with rather distinctly stalked peristomial disc resembling operculariids (Figs 1, 6, 11–18, 26–28, 36, 40; Table 1). Peristomial bulge of ordinary distinctness, upper margin very rarely crenelated. Peristomial disc slightly projecting from body proper, average diameter about 10 μm in vivo, inconspicuously to distinctly convex, never umbilicate; obliquely retracted into cell in contracted specimens. Cytopharynx spacious, extends obliquely to near mid-body. Ciliature as typical for peritrichs. Cilia about 15μm long, adoral ciliary spiral performs a 360° turn each around peristomial disc and in cytopharynx; peniculus 2 shortened posteriorly ending between peniculi 1 and 3; peniculus 3 composed of three minute, fan-like spread kineties. Epistomial membrane about halfway between begin of adoral ciliary spiral and vestibular opening, composed of three to five basal bodies (Figs (Figs10,10, ,5050).
Anlage of aboral ciliary wreath in vivo recognizable as slight convexity at beginning of posterior body third (Figs 1, 6, 7, 27, 35); often difficult to recognize in Klein-Foissner silver nitrate preparations, while composed of deeply impregnated, narrowly spaced, oblique dikinetids (?) in protargol slides (Figs 3, 10, 37, 38, 48–50); in Klein-Foissner preparations consists of two narrowly spaced silverlines associated with body silverlines, one anteriorly and another posteriorly, producing a more or less prominent four line pattern (Figs (Figs44,44, ,45).45). Swarmers cylindroid, about 40 μm long, swim very fast, not studied in detail.
As yet found at type locality, as described above, and in a tank bromeliad from Jamaica. Few specimens occurred in the field samples; however, O. bromelicola reproduced rapidly in a culture with tank water containing some squashed wheat grains to stimulate bacterial growth. In field attached to inorganic and organic debris, including insect remnants; did not colonize various dipteran larvae present in the sample.
Of the Rhabdostyla species reviewed in Kahl (1935) and Stiller (1971), only two are similar to O. bromelicola: O. kahli (Nenninger, 1948) and O. brevipes (Claparède and Lachmann, 1857). Orborhabdostyla kahli (Fig. 24) differs from O. bromelicola by the location of the contractile vacuole (ventral vs. dorsal), body shape (cylindroidal vs. conical), and the habitat (epizooic on Lumbriculus vs. attached to dead material). Orborhabdostyla brevipes (Fig. 21) is possibly very similar to O. bromelicola, but so incompletely described that any identification is arbitrary. Claparède and Lachmann (1857) figured only a contracted specimen because the “extended state is like that of Epistylis plicatilis.” Orborhabdostyla brevipes is 80–90 μm long and has been discovered attached to larvae of dipterans in a pond in Berlin; the stalk is minute but broad according to the description and figure of Claparède and Lachmann (1857). Assuming a length of 60 μm of the contracted specimen illustrated by Claparède and Lachmann (1857), the stalk is about 5 μm wide. Taken together, there are three features which distinguish O. brevipes and O. bromelicola: body size (80–90 μm vs. 65 × 15 μm), stalk width (about 5 μm vs. 2 μm), and habitat (epizooic vs. attached to dead matter; our species did not colonize the dipteran larvae present in the field sample!). Synonymy of the populations cannot be excluded until a European population has been characterized with modern methods. However, considering the special habitat (tank bromeliads) and the much narrower stalk, O. bromelicola is likely distinct from O. brevipes. Rhabdostyla brevipes, as described by Penard (1922), is a different species classified by Kahl (1935) as R. brevipes Penard, 1922 (Figs 22, 23).
Great numbers of this species developed in a culture of tank water from a Costa Rican bromeliad. Thus, we could study it in detail and make various preparations to supplement the redescription by Foissner (1979), which is based on life observations and silver nitrate preparations of a population from the Austrian Central Alps (Fig. 51). The life and silver nitrate data from the Costa Rican specimens match Foissner's redescription perfectly (Table 2). Thus, only main characteristics and some additional observations will be mentioned.
Vorticella gracilis is bell-shaped but the size (60–85 × 25–45 μm) and the length: width ratio are highly variable, as shown by Figs 56–65 and the morphometric data (Table 2): = 2.6:1, M = 2.3, SD = 0.6, CV = 24.8, Min = 1.6, Max = 3.7, n = 21. One of the most important features is the yellowish colour, which was stable for the cultivation period, i.e., for over half a year in ordinary Petri dish cultures set up with Eau de Volvic (French Table water) and some crushed wheat grains. These cultures also contained V. astyliformis, which remained colourless. In this connection, it must be emphasized that the re-evaluation of the original notes on the Austrian specimens mention a “yellowish cytoplasm,” not only “yellowish cytoplasmic granules,” as stated in the redescription by Foissner (1979). The macronucleus is J-shaped and its long middle portion extends in the ventral body half, as in the Austrian specimens (Figs (Figs51,51, ,66,66, ,72).72). The protargol preparations show that V. gracilis has an oral ciliature of the convallaria-type, i.e., the adoral ciliary spiral performs about 1.3 turns around the peristomial disc, and peniculus 3 is composed of 3 ciliary rows, of which row 1 is distinctly shortened proximally (Figs 54, 55, ,7272--76);76); further, there is an epistomial membrane (Figs (Figs55,55, ,72).72). For ordinary details, see Figures Figures52,52, 67–71, 77–79.
This species was described by Dujardin (1841) only in the Figure section: “Longeur 55 μm. Dans l'eau de marais conservée pendant long temps. Elle n'est pas décrite dans le texte” (Fig. 53). Foissner (1979) provided a solid redescription and based the identification on the rather slender shape, the sole feature recognizable in Dujardin's “description.” Indeed, V. gracilis is usually more slender than vorticellas of the convallaria-type (~ 2.5:1 vs. 1.8:1, see Table 2 and Foissner et al. 1992), but the most distinctive feature is its yellowish colour, which is very stable within and between populations. In contrast, the yellowish “variety” of V. convallaria, i.e., V. citrina, is likely only an “ecovariety” possibly caused by algal food (Foissner et al. 1992). Interestingly, the number of silverlines is also unique, i.e., it does not match well those of various populations of V. convallaria and V. similis (Fig. 71; Table 2): the former has slightly more silverlines from the anterior body end to the anlage of the aboral ciliary wreath (81 vs. 71), the latter has distinctly more silverlines from the anlage of the aboral ciliary wreath to the scopula (31 vs. 19). Thus, we do not have doubts that the populations represent a distinct species. This is supported by the molecular data (Fig. 25), where V. gracilis branches in a well-supported clade (NJ/BI/ML – 90/100/82) containing V. campanula, V. fusca and V. convallaria. Genetically, V. gracilis is very distinct from V. convallaria with a sequence dissimilarity of 9.8%. Even tough, we currently have no verified guidelines how to translate 18S rDNA sequence similarities into taxonomic hierarchies, such a high dissimilarity clearly points to distinct species. Thus far, to our knowledge no ciliate species have been reported with such a high intergeneric 18S rDNA gene divergence (Li et al. 2008)! The closest known 18S rDNA-relative of V. gracilis is V. fusca with 99.14% sequence similarity. Vorticella fusca has been described rather superficially by Precht (1935). It differs from V. gracilis by the habitat (fresh-water vs. marine, colonizing the alga Enteromorpha), the dark-brown colour (vs. yellowish), the distinctly granulated (vs. smooth) stalk myoneme, the location of the contractile vacuole (ventral vs. dorsal), and the structure of peniculus 3 (Song et al 2009).
Considering the very incomplete original description and several similar species, all discussed by Foissner (1979), V. gracilis needs neotypification. Although the data from the Costa Rican population are more detailed than those from the Austrian Central Alps, we suggest using the Austrian population as a neotype because it is from the same biogeographic region as Dujardin's specimens (Palaearctic, Europe). Based on these and the present data, we fix the species diagnostically.
Size about 80 × 30 μm in vivo; narrowly to ordinarily campanulate. Macronucleus J-shaped. Single contractile vacuole at ventral wall of vestibulum. Cytoplasm yellowish. About 70 silverlines from anterior end to anlage of aboral ciliary wreath and about 20 from there to scopula. Kinety 1 of peniculus 3 distinctly shortened proximally.
Meltwater pool near the Fuschertörl (site 23 in Foissner 1980), Grossglockner-Hochalpenstrasse, Salzburg, Austria, N 47° E 12°, about 2400 m above sea-level.
Six slides with silver nitrate-impregnated specimens from a puddle in the Austrian Central Alps (Grossglockner area) have been deposited in the Biology Centre of the Museum of Upper Austria, Linz (LI). Further, we deposited voucher slides from the Costa Rican population, viz., four slides each with silver nitrate and protargol-impregnated cells. The neotypes and other relevant specimens are marked by black ink circles on the coverslip.
Small, barrel-shaped Vorticellidae with two epistomial membranes and transverse-striate silverline pattern. Peniculus 3 usually composed of two ciliary rows.
Vorticella aquadulcis Stokes, 1887.
Composite of the generic name Vorticella and the substantivated adjective ides, referring to the overall similarity with Vorticella. Masculine gender.
In addition to the type, four other species, formerly classified in Vorticella, may be referred to the new genus Vorticellides: V. astyliformis (Foissner, 1981) nov. comb. (briefly redescribed below); V. platysoma (Stokes, 1887) nov. comb., as redescribed by Foissner et al. 1999 (second epistomial membrane not marked but recognizable in Figure 15, p. 489); V. infusionum (Dujardin, 1841) nov. comb. (see below and Figs 113, 114); and V. (Spinivorticellides) echini (King, 1931) nov. comb., as redescribed by Foissner et al. (2002). The terminations of the species names remain unchanged because aquadulcis and platysoma are appositions; echini is a genitive; infusionum is a genitive plural; and astyliformis has the same termination in the feminine and masculine gender (see ICZN 1999).
As there are many small, barrel-shaped Vorticella species (Kahl 1935, Warren 1986, Song et al. 2009), one may expect that several of them will be transferred to Vorticellides on detailed reinvestigation. Possibly, the second epistomial membrane, which is minute and near the anterior (distal) end of the adoral spiral, has been sometimes overlooked or considered as an irregularity of the adoral ciliary spiral.
About 200 nominal Vorticella species have been described (Kahl 1935, Corliss 1979, Warren 1986). Many of them have been considered to be junior synonyms (Warren 1986). However, more recent research resurrected some of the synonyms and added a considerable number of new species (Foissner 1979; Foissner et al. 1992, 1999; Song et al. 2009). Obviously, Vorticella and Vorticella-like peritrichs are much more divers than supposed previously.
In 1974, Foissner and Schiffmann split the genus in Vorticella (with transverse-striate silverline pattern) and Pseudovorticella (with reticulate silverline pattern). This split has been widely accepted and greatly refined the taxonomy of the vorticellids (Warren 1986, 1987; Song et al. 2009); now, the split is supported also by molecular data (Martin-Cereceda et al. 2007, Li et al. 2008; Fig. 25 of the present study).
Here, we propose a second split of Vorticella, using a rather difficult feature, viz., the number of epistomial membranes (one or two). The epistomial membranes are difficult to recognize because they consist of only few basal bodies, forming minute rows at the anterior (distal) end of the adoral ciliary spiral (epistomial membrane 2) and/or near the level of the vestibular entrance (epistomial membrane 1). Further, the epistomial membranes are ciliated only in the swarmer stage, except of Opisthonecta, where membrane 1 forms a conspicuous tuft in the trophic cells (Foissner 1975). Nothing is known about the function of the epistomial membranes, but the activation in the swarmer stage suggests some role in habitat selection. Likewise, nothing is known on their genesis and whether membrane 1 and 2 are homologous at all.
Most peritrichs have only one epistomial membrane near the anterior end or at the level of the vestibular entrance of the adoral ciliary spiral. However, Foissner et al. (1992) reported two epistomial membranes in a species of the Vorticella infusionum-complex (Fig. 114). Since then, we found two epistomial membranes in some other vorticellas (Foissner et al. 1999, 2002), and here we describe them in detail for Vorticella aquadulcis Stokes, 1887. The five species now collected in Vorticellides have not only two epistomial membranes in common but also a small body size (usually < 60 μm), a barrel-like body shape, and a minute peniculus 3 consisting of only two kineties. However, the most important and unique feature is the two epistomial membranes, one near the anterior (distal) end of the adoral ciliary spiral, the other rather far away from the anterior end, i.e., at level of the vestibular entrance.
The second split is also supported by the molecular data, where the genus Vorticella appears biphyletic (Martin-Cereceda et al. 2007, Li et al. 2008, Fig. 25 in the present paper). One clade contains, inter alia, “typical” campanulate species, viz., Vorticella gracilis and V. convallaria, while the other clade consists of small, barrel-shaped species, such as V. astyliformis and V. microstoma, as well as of several stalkless peritrichs, such as Opisthonecta spp. and Astylozoon enriquesi. The latter lacks any epistomial membrane (Foissner, unpubl.), while Hastatella has two membranes (Foissner, unpubl.).
The identification needs silver impregnation, exept of V. (Spinivorticellides) echini, which has distinct spines on the body surface (for a detailed redescription, see Foissner et al. 2002). Vorticellides infusionum is usually distinctly larger than V. astyliformis, V. platysoma and V. aquadulcis. Vorticellides platysoma and V. aquadulcis are highly similar (Table 5), and thus possible synonymous. Further studies are required on the macronucleus (ellipsoidal to reniform vs. elongate reniform to semicircular) and peniculus 3.
We included in the new genus Vorticellides also the Vorticella microstoma available in GenBank because it was contained in the same clade as Vorticellides astyliformis. As far as we know, the morphological identity of the GenBank species has been not documented. We would be not surprised, if the GenBank species belongs to the Vorticellides infusionum-complex because species of this complex are very frequent and have been often mixed with Vorticella microstoma. See Foissner et al. (1992) how to separate species of the V. microstoma – and V. infusionum-complex.
This species was discovered by Foissner (1981) in soil from the Austrian Central Alps. Later, it was found in soils globally (Foissner 1998). We re-investigated a slide from the type series to obtain more detailed data on the epistomial membrane and peniculus 3. The second population was found at Namibian site 23 (Foissner et al. 2002), and the third population occurred in the tank of a bromeliad from Costa Rica. The slides investigated have been deposited in the Biology Centre of the Museum of Upper Austria, Linz (LI). Relevant specimens have been marked by black ink circles on the coverslip.
The reinvestigation of the type and the Namibian population showed that both have two epistomial membranes in exactly the same position as V. aquadulcis, redescribed below. Thus, we do not provide new figures. The same applies to peniculus 2, which consists of two rows (Foissner 1981) but row 2 is only about half as long as row 1.
The population from a tank bromeliad of Costa Rica is highly similar to the specimens described from Austrian soils by Foissner (1981). Even a sophisticated feature like the number of pellicular pores is quite similar: about 100 pores/100 μm2 (Tables (Tables3,3, ,5).5). There are, however, a few slight differences and additional observations. Size 28–35 × 12–20 μm in vivo, on average 30 × 15 μm with a length:width ratio of 1.7–2.5:1, while the Austrian specimens are 30–50 μm long. Stalk 2–3 μm thick. Specimens attached to debris and bacterial flocs. Cortical striation as distinct as in type population, i.e., crest distance > 1 μm (Figs 80–83).
There are many similar species, most depicted and briefly discussed in Foissner (1981) and Foissner et al. (1992, 2002). Especially, V. costata appears so similar to V. astyliformis that synonymy appears likely. However, there is a massive difference in a little used feature, viz., the number of pellicular pores: about 100/100 μm2 in three populations of V. astyliformis (Foissner 1981, Fig. 35; Foissner et al. 2002, Fig. 378d; present study), while about 16–30 in V. costata and related species (Foissner 1979, Fig. 39; Foissner et al. 1992, Figs 22, 23 of the V. aquadulcis complex). Further, the oral bulge is considerably narrower in V. astyliformis than V. costata, which provides the former with a very characteristic appearance (Figs 80–82): ratio of width of body and peristomial bulge 1.4–2.0:1 vs. 1.2:1, as calculated from the present observations, the micrographs and drawings in the literature cited above and in Sommer (1951), and Foissner et al. (2002, Fig. 378a).
Two populations were studied from a tank bromeliad of Costa Rica and a pond in the botanical garden of the town of Linz, Austria, respectively. Protargol preparations were made only from the Costa Rican population, which grew well in the natural sample enriched with some wheat grains. The specimens attached to organic debris and to coverslips on the water surface (Figs 94–98).
“Body ovate or pyriform, very slightly changeable in shape, less than twice as long as broad, slightly constricted beneath the peristome border, the cuticular surface strongly and conspicuously striate transversely; peristome more than one-half the body-centre in breadth, but not equaling it, the border thickened, not everted; ciliary disc obliquely elevated; pedicle from two to three times as long as the body. Length of body, 35 μm. Hab.–Fresh water; attached to rootlets of Lemna. Solitary, or few together. Contracted body obovate.”
(if not stated otherwise, data are from the Costa Rican neotype population): Size 23–40 × 13–24 μm in vivo, usually about 35 × 20 μm (Table 4), matching Stokes' notion. Shape of Austrian and Costa Rican specimens more variable than in Stokes' population, i.e., usually more or less pyriform or barrel-like, rarely obovate, conical, slenderly conical or globular; never campanulate because oral bulge distinctly narrower than mid-body (Figs 94–98, 110–112; Table 4); contracted specimens globular. Macronucleus in or slightly above mid-body, elongate reniform to semicircular, with conspicuous nucleoli (Figs 85, 86, 90, ,99);99); micronucleus not impregnated with the protargol method used. Contractile vacuole on ventral wall of vestibulum underneath oral bulge (Figs (Figs85,85, 96, 97). Cytoplasm colourless, usually packed with 4–7 μm-sized food vacuoles and some lipid droplets about 1 μm across. Feeds on bacteria, forming rather transparent masses in the food vacuoles (Figs 85, 94--9898).
Cortex as distinctly striate transversely as in Stokes' North American specimens; striation recognizable even at low magnification (~ × 100) because crests comparatively high (~ 1 μm) and about 1.5 μm apart, as measured in vivo and calculated from average body length and number of silverlines (Figs (Figs85,85, 94–98, 110–112; Table 4). Silverline pattern widely striate (average distance between silverlines > 1 μm), without peculiarities, on average 18 silverlines (19 in Austrian specimens) from anterior body end to anlage of aboral ciliary wreath and 6 silverlines from there to scopula, both in Costa Rican and Austrian specimens (Tables (Tables4,4, ,5);5); two silverlines in oral bulge. On average 31 and 25 pellicular pores/100 μm2 in Costa Rican and Austrian specimens, respectively; most pores underneath silverlines in Costa Rican cells, while above in Austrian ones (Figs (Figs85,85, 102, 105, 106, 108, 109; Tables Tables4,4, ,55).
Stalk vorticellid, i.e., with flattened, spirally contracting myoneme; 2–2.5 μm wide and 20–105 μm long, on average 48 μm and thus short, as mentioned by Stokes (1887); attached to debris and coverslips by a leaf-like disc about 3 μm across. Scopular organelles 1–1.5 μm long, in some specimens two rings one upon the other (Figs 85–88, 94–98, 110–112; Table 4). Myoneme system vorticellid and rather loose, i.e., six to eight strands originate in the surroundings of the scopula and extend anteriorly, becoming bi-to quadripartite before attaching to the adoral ciliary spiral (Figs 86, 87, 91, 92, ,99).99). Oral bulge myoneme dense, with short, posteriorly directed extensions, forming a granular ring (Figs (Figs87,87, 100); no myonemes recognizable in peristomial disc.
Oral apparatus typical, except of the two epistomial membranes, structures very small and thus difficult to analyze (Figs 86, 88–90, 93, 99–101, 103, 104, 107; Table 4). Peristomial bulge and peristomial disc about 5 μm high in feeding specimens, vestibulum of ordinary size, extends obliquely to body centre. Peristomial bulge 2–2.5 μm thick, slightly projecting from body proper and thus distinct, considerably narrower than widest site in mid-body (~ 15 μm vs. 19 μm), shows two transverse striae (silverlines). Peristomial disc slightly narrower than peristomial bulge (12 μm vs. 15 μm; Table 4), surface flat to inconspicuously convex, never umbilicate both in Costa Rican and Austrian specimens; slightly obliquely elevated in feeding cells (Figs (Figs85,85, 94–98, 110–112). Adoral ciliary spiral performs slightly more than one turn (~ 400°) around peristomial disc, before plunging into the vestibulum, performing a further turn and splitting into three peniculi (Figs 85, 86, 88–90, 92, 99–101). Peniculus 1 and germinal kinety without peculiarities. Peniculus 2 only about 5 μm long, first row slightly shortened proximally. Peniculus 3 composed of only two ciliary rows: row 1 distinctly longer than row 2, slightly curved or sigmoidal, ends near proximal end of peniculus 1; row 2 distinctly shortened proximally, composed of only three to five basal bodies (Figs (Figs93,93, 99–101, 107). Epistomial membrane 1 at level of vestibular entrance, i.e., rather far away from distal end of adoral ciliary spiral, composed of three to five basal bodies. Epistomial membrane 2 slightly ahead of distal end of adoral ciliary spiral, composed of three basal bodies (Figs 86, 88–90, 92, 103, 104).
Anlage of aboral ciliary wreath hardly recognizable in vivo, in posterior quarter of cell and distinct in silver nitrate preparations, appearing as a granular mass boardered by a silverline each anteriorly and posteriorly (Figs 102, 105); in protargol preparations composed of minute, slightly oblique pairs (?) of basal bodies (Figs 86–88, 90, 91, 99–101, 107); when activated in swarmers, composed of oblique, 2–3 μm long kineties (Figs 106, 108, 109).
The three reliable records available (Stokes 1887 and the two populations described here) indicate that V. aquadulcis is probably widespread in various freshwater habitats, ranging from ponds to bromeliad tanks. Many records of this and similar species were reviewed by Foissner et al. (1992). They conclude that species of the V. aquadulcis-complex prefer rather clean, beta-mesosaprobic habitats, while records from sewage plants are possibly misidentifications.
See key to species and V. astyliformis.
Considering the very incomplete original description and many similar species, most discussed by Warren (1986) and Foissner et al. (1992, 1999), V. aquadulcis needs neotypification. Ideally, the neotype should be from the locus classicus or from nearby (ICZN 2009). However, this regulation has been questioned in the case of microscopic organisms, many of which have been very poorly described and have a large areal (Foissner 2002).
Of the two populations studied, only that from Costa Rica has been fully investigated and will thus be fixed as a neotype, although it is about 3300 km away from the type area, i.e., a pond in the surroundings of the town of Trenton, New Jersey, USA, where Stokes lived and worked. To use the Costa Rican population as a neotype is supported by the high similarity with the North American (Stokes 1887) and the European (Figs (Figs84,84, 108–112; Table 5) populations, indicating a cosmopolitan distribution and wide ecological range (ordinary ponds to tanks of bromeliads).
Size about 35 × 20 μm in vivo. Shape highly variable, usually pyriform, obovate or conical. Macronucleus semicircular in transverse axis of mid-body. Single contractile vacuole at ventral wall of vestibulum. Cortex distinctly transverse-striate, with about 19 silverlines from anterior end to anlage of aboral ciliary wreath and an average of 6 silverlines from there to the scopula. Epistomial membrane 1 at level of vestibular entrance, membrane 2 at distal end of adoral ciliary spiral. Peniculus 3 composed of two kineties, with kinety 2 distinctly shortened proximally.
Tank bromeliads from Costa Rica, Central America. Unfortunately, a more exact locality cannot be provided because the collector did not specify the site.
Six slides with protargol-impregnated cells and two slides with silver-nitrate impregnated specimens from the Costa Rican neotype population have been deposited in the Biology Centre of the Museum of Upper Austria, Linz (LI). Further, five slides with silver nitrate-impregnated specimens from Linz, Austria, have been deposited at the same locality. The neo-types and other relevant specimens have been marked by black ink circles on the coverslip.
Size about 65 × 40 μm in vivo; pyriform to campanulate. Macronucleus J-shaped. Two ventral contractile vacuoles. On average 41 silverlines from anterior end to anlage of aboral ciliary wreath and 13 silverlines from there to scopula.
Tanks of Guzmania scherzeriana (Bromeliaceae) in the garden of the tropic station La Gamba, Costa Rica, N 8°42′, W 83°12′, 70 m above sea-level.
Composite of the plant genus Bromelia (Bromeliaceae) and the Latin verb colere (inhabiting), referring to the habitat in which the species was discovered.
Size 60–75 × 30–50 μm in vivo, on average about 65 × 40 μm; shape narrowly pyriform to pyriform and more or less campanulate, length:width ratio 1.3–1.9:1, usually about 1.7:1. Fully contracted specimens globular with projecting peristomial bulge (Figs 115-126; Table 6). Macronucleus J-shaped and extending longitudinally in dorsal half of cell. Micronucleus not observed. Two contractile vacuoles at ventral wall of vestibulum: one in mid-portion, the second near its end. Cytoproct on dorsal wall of vestibulum underneath peristomial bulge (Fig. 115). Cytoplasm colourless, with many food vacuoles 7–9 μm across and containing bacteria. Stalk up to three times body length, i.e., up to 200 μm long and 4–5 μm wide. Stalk myoneme without granules, contracts spirally. Cells attached to debris and bottom of Petri dish (Figs 115, 122–124).
Cortex almost smooth in vivo as alveoli flat and without inclusions (Figs 115, 119–125). Silverline pattern reticulate, typical of genus (Figs 127–129). On average 41 transverse silverlines between anterior end and anlage of aboral ciliary wreath and 13 from there to scopula (Table 6). Anlage of aboral ciliary wreath a slight convexity or concavity at beginning of posterior third of body, comprises three or four narrowly spaced silverlines in Klein-Foissner silver nitrate preparations, particularly discernible at cell margin, otherwise appearing as a thick dark line (Figs 115, 117, 119, 120, 127–129).
Oral apparatus of usual structure. Peristomial bulge about 5 μm thick, slightly narrower to slightly wider than broadest body region. Peristomial disc projecting slightly from peristomial bulge, flattened to slightly concave. Vestibulum and cytopharynx spacious, extending obliquely to dorsal side and mid-body (Figs 115, 119–125).
As yet found only at type locality.
Unfortunately, we lost the protargol slides, and thus the description is incomplete. However, the differences in the pattern of the contractile vacuoles and the number of silverlines are so distinct that Pseudovorticella bromelicola cannot be confused with congeners.
Of the Pseudovorticella species reviewed by Warren (1987) and ourselves, three congeners have two contractile vacuoles and a J-shaped macronucleus: Pseudovorticella foissneri Sun et al., 2007; P. sphagni Foissner and Schiffmann, 1974; and P. monilata (Tatem, 1870) Foissner and Schiffmann, 1974. Pseudovorticella foissneri is a marine species mainly characterized by its extremely flattened, asymmetrical body. Pseudovorticella sphagni, as described by Foissner and Schiffmann (1974) and Foissner (1979), differs from P. bromelicola by the location of the contractile vacuoles (one each on ventral and dorsal vestibular wall, checked in three populations; vs. both ventral) and the number of silverlines from the anterior end to the anlage of the aboral ciliary wreath (25–30 vs. 39–43) and from the anlage to the scopula (8–10 vs. 11–15). Pseudovorticella monilata is possibly most similar to P. bromelicola but differs distinctly in the number of silverlines from the anterior end to the anlage of the aboral ciliary wreath (15–23 vs. 39–43), while the number of silverlines from the anlage to the scopula is similar (9–18 vs. 11–15). These values are important because they are based on five populations of P. monilata (for a review, see Foissner et al. 1992). Further, the cortical alveoli are usually much more prominent in P. monilata than in P. bromelicola (see micrographs in Foissner et al. 1992).
Very recently, Song et al. (2009) described and redescribed 25 Pseudovorticella species from marine habitats in China. None resembles P. bromelicola.
Supported by the Austrian Science Foundation (FWF project P-20360-B17) and the German Science Foundation (DFG, STO-414/2-3). The technical assistance of Mag. Barbara Harl, Mag. Gudrun Fuss, Robert Schörghofer, and Andreas Zankl is greatly acknowledged. Special thanks to Prof. Dr. Till (University of Vienna), Miss Pfandl (limnological station Mondsee), and Mag. Werner Huber (Tropic Station La Gamba, Costa Rica) for collecting samples from Ecuador and Costa Rica, respectively.