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Paramecium genus: biodiversity, some morphological features and the key to the main morphospecies discrimination
Sergei I. Fokin
Department of Invertebrate Zoology, St. Petersburg State University, Russia
Summary
Biodiversity of the genus Paramecium and its worldwide distribution are discussed. A simple and reliable way of species discrimination based on variation in the morphology of micronuclei and contractile vacuoles is presented.
Key words: contractile vacuole, distribution, key to discrimination, micronuclei, morphospecies, Paramecium
Introduction
Knowledge about distribution and composition of the Paramecium genus has evolved during over two and a half centuries. More than 40 descriptions of representatives were published in the literature for these very well-known ciliates (see: Muller, 1786; Wenrich, 1928; Wichterman, 1986; Fokin and Chivilev, 1999, 2000; Fokin et al., 2004). However, the number of valid morphospecies might be not as high as that, since some of the descriptions (Dumas, 1929; Kahl, 1930; Sramek-Husek, 1954; Baumeister, 1969) were not made properly and cannot be accepted (Fokin, 2002). It has been proposed recently that, in an extension of Jankowski’s earlier suggestion (Jankowski, 1969), the genus Paramecium should be subdivided into four subgenera: Chloroparamecium, Fokin et al., 2004, Helianter, Jankowski 1969, Cypriostomum, Jankowski 1969, Fokin et al., 2004 and Paramecium Jankowski 1969, on the basis of morphological, morphometric, biological and molecular differences
(Fokin et al., 2004). So far we can record 17 morphospecies in the genus. There are: P. bursaria Focke, 1836, P. putrinum Claparede and Lachman, 1858, P. duboscqui Chatton and Brachon, 1933, P. calkinsi Woodruff, 1921, P. polycaryum Woodruff and Spencer, 1923, P. nephridiatum Gelei, 1925, P. woodruffi Wenrich, 1928, P. pseudotrichium Dragesco, 1970, P. caudatum Ehrenberg, 1833, complex of P. aurelia [15 species] (Sonneborn, 1975; Aufderheide et al., 1983), P. multimicronucleatum Powers and Mitchel, 1910, P. jenningsi Diller and Earl, 1958, P. wichtermani Mohammed and Nashed, 1968—1969, P. africanum Dragesco, 1970, P. schewiakoffi Fokin et al., 2001, P. jankowskii Dragesco, 1972 and P. ugandae Dragesco, 1972. The last two, however, should be reinvestigated as they do not share one of the features of the genus, the characteristic structure of quadrulus.
Geographical distribution reported for some of Paramecium spp. does not support the theory that all ciliates are cosmopolitan. On the other hand, this result convinced us that the genus Paramecium
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could be larger than the current representation as many territories still have never been checked by professional ciliate taxonomists (Foissner, 1999; Fokin et al., 2004). Generally, it is more likely to detect new species in remote and therefore rarely investigated areas (South America, Africa, tropical Asia or, on the opposite, Arctic and Antarctic areas). Until recently this point of view was supported by paramecia findings: P. jenningsi — India (Diller and Earl, 1958), P. africanum, P. ugandae, P. jankowskii, P. preudotirichium and P. wichtermani — Africa (Dragesco, 1970; 1972; Mohammed and Nashed, 1968/1969), P. schewiakoffi — China (Fokin et al., 2004).
In Europe the last new valid Paramecium spp. were described 77 years ago from brackish water
— P. duboscqui (Chatton and Brachon, 1933) and 85 years ago from fresh water — P. nephridiatum (Gelei, 1925, 1938). Of course, the ciliate fauna should be investigated in Europe much better than in the rest ofthe world, but a number of species were described from another parts of the Globe: North America — P. polycarium, P. woodruffi, P. calkinsi, P. multimicronucleatum; Africa — P. africanum, P. ugandae, P. jankowskii, P. preudotirichium and P. wichtermani; Asia — P. jenningsi and P. schewiakoffi. Till a few years ago we did not expect any more new European Paramecium morphospecies as we considered it is very unlikely that taxonomists would have missed such obvious ciliates as paramecia (Fokin et al., 2004). However, morphological and molecular analysis of certain paramecia cultures isolated after sampling in Hungary, Germany and Norway revealed, correspondingly, two cryptic Paramecium spp. and one apparently new morphospecies (Fokin et al., 2006).
For correct identification of even such ‘‘simple’’ ciliates as members of the Paramecium genus, protozoologists must have an adequate general taxonomic knowledge and practice dealing with a particular group of ciliates. The stories of P. duboscqui and P. nephridiatum, which, though widely distributed in Europe, were not recognized there for a long time after their first description (Fokin et al., 1999a, b), just because by some features they reminded another species, clearly show that investigators have to pay considerable attention to the right identification procedure.
In the article I would like to discuss biodiversity of Paramecium spp., their frequency (distribution) in different parts of the world as well as a simple way to discriminate them. For the last matter the variation in morphology of the micronucleus (Mi) and contractile vacuole (CV) of Paramecium spp. will be considered first of all.
Material and methods
Sampling sites, culturing and morphological
ANALYSIS
For the current analysis all ofmy morphological materials connected with Paramecium spp. were used. They came from my own sampling made (1990-2009) in different parts of Europe (former USSR, Poland, Italy, Hungary, Greece, Spain, France, Germany, Denmark, Austria), Asia (former USSR, China, India, Japan), North America (USA, Canada) and some samples provided by colleagues and friends in Europe (Norway), Asia (Israel, Saudi Arabia, Vietnam, Thailand), Australia, Africa (South Africa, Kenya, Tunisia), North America (USA) and South America (Brazil). Alltogether, it totals about 5000 samples. The majority of Paramecium spp. isolated were kept as clone cultures in the own living culture collection and investigated while alive with the help ofa special immobilization device (Skovorodkin, 1990) as well as on fixed material stained by Feulgen reaction and impregnated with silver nitrate. All culturing and stained procedures were described previously (see: Fokin et al., 2004). I have successfully kept in my collection the following morphospecies: P. bursaria, P. putrinum, P. duboscqui, P. calkinsi, P. woodruffi, P. nephridiatum, P. aurelia, P. jenningsi, P. schewiakoffi, P. multimicronucleatum, P. caudatum and Paramecium sp. newly found in Norway, which still has no binominal name. For each species the main morphological characteristics were investigated in 3-6 clones of different origin (if possible) and at least 25-30 cells of each line were used.
Results
Members of the genus Paramecium look very typical and distinctive (Fig. 1) and may be characterized as follows. They are mostly elongate ciliates of cigar or slipper shape (50-300 ^m) with dorsal and ventral surface, a distinct oral groove or depression running from anterior left to middle right of the body on the ventral surface (Wichterman, 1986). The oral groove leads into the vestibulum, which terminates in an opening, the buccal overture, leading into a buccal cavity. Buccal overture is located around the middle ofthe ventral surface (in most Paramecium spp.), or could be shifted forward from the cell’s equator, considerably (P. bursaria, P. putrinum and P. polycaryum) or just a little (P.
Fig. 1. The main morphospecies of Paramecium: a — P. multimicronucleatum, b — P. caudatum, c — P. jenningsi, d — P. schewiakoffi, e — Paramecium sp. (Norway), f — P. woodruffi, g — P. aurelia, h — P. nephridiatum, i — P. bursaria, j — P. calkinsi, k — P. duboscqui, l — P. putrinum, m — P. polycaryum. Abbreviations: Ma — macronucleus, Mi — micronucleus, b — buccal cavity, c — cytoproct. Scale bar = 100 ^m.
schewiakoffi and Paramecium sp. from Norway). A buccal cavity has special buccal ciliature including peniculi (2 sets of ciliary structures each consisting of four converging rows), quadrulus (again four ciliaty rows, but located in some distance from each other) and endoral kinety (membrane), a zigzag row of cilia. This peculiar buccal ciliature is very stable in structure, but can be larger or smaller for different Paramecium spp. It gave the name to the whole subclass Peniculia (Struder-Kypke et al., 2000a, 2000b; Lynn, 2008).
By the general shape of the cell, Paramecium representatives can be divided into two groups: “aurelia”, with a cigar-shaped body and “bursaria”, with the body more like a slipper with obliquely truncated anterior end (Fig. 1). At least two species, however, deviated from those general images — P. pseudotrichium (more ellipsoidal) and the one, newly found in Norway (somehow combining cigar and slipper shape) (Fokin et al., 2006). The same division of the genus members fits for location of slit-like cytoproct on the ventral cell side. All “bursaria”-like species have it close to the posterior end of the cell, while in all the “aurelia”-like members of the genus this structure is situated at some distance from the posterior end (Fokin and Chivilev, 1999, 2000; Fig. 1).
The most distinctive and useful for species’ discrimination, to my mind, are type and number ofthe Mi as well as the structure of CV. In the Paramecium genus we can find all the possible variations of the Mi types and CV structure occurring within all the Ciliophora (Fokin, 1986, 1997, 2001, 2002). Both of the features are very easy to check using living and fixed (stained by Feulgen and impregnated) cells.
Unfortunately, even in such a solid monograph as “The biology of Paramecium”(Wichterman, 1986), only two characteristic types of the nucleus according to the Mi morphology, “caudatum” and “aurelia”, are given. This is far away from the real picture.
There are four main morphological types of the Mi organization (Fig. 1, 2). The first, large “compact” type ofthe Mi can be found in P. bursaria, P.putrinum, P. caudatum. The size ofgenerative nucleus in those cases is, approximately, 6-10 ^m. The single Mi is barrel-like, spherical to ovoid (or in P. bursaria often spindle-like) in shape (Fig. 2). The generative nuclei, in fact, are not equal in those species according to the fine structure and should be recorded as the “compact a” (P. caudatum), “b” (P. bursaria) and “c” (P. putrinum) (Fig. 2 Ia-c) (Fokin, 1997). Very similar to the “compact c” type Mi type was found in the newly discovered species from Norway (Fokin et al., 2006).
Using light microscopy, however, it is possible to discriminate only the Mi type of P. caudatum from the nucleus of other two species. P. caudatum always has the Mi with the so-called “cap”, a space without chromatin at the nuclear pole. In P.putrinum and P. bursaria this peculiar structure is absent. Differences between P. putrinum and P. bursaria micronuclei can be revealed only with the use of electron microscopy. The Mi of P. bursaria has permanent specific lamellar structures connected with chromatin bodies (Lewis, 1975; Fokin, 1997, 2002).
The second type ofthe Mi is the large “chromosomal” nucleus corresponding to characteristic of P. jenningsi and P. schewiakoffi (Fig. 2 II). This type of nucleus is also relatively large (approximately, 5-9
Fig. 2. Morphological types of Paramecium micronuclei. I — “compact” type: P. caudatum (a),
P. bursaria (b), P. putrinum (c); II — “chromosomal” type (P. jenningsi, P. schewiakoffi and, probably, P. wichtermani); III — “endosomal” type (P. duboscqui, P. woodruffi, P. calkinsi and P. nephridiatum); IV — “vesicular” type (P. multimicronucleatum, P. aurelia and P. polyca-ryum). Bar = 10 ^m.
^m) and almost spherical, with a reticular structure. The chromatin mass occupying most of the Mi volume appears as a Feulgen-positive reticulum composed ofnumerous interlaced chromatin ropes. This mass was separated from the nuclear envelope by a distinctive space without chromatin.
Some of the common morphospecies, euryha-line, but mainly brackish water (Smurov and Fokin, 1999) have a relatively small (approximately 1.5-5.0 ^m) “endosomal” type of the Mi — P. woodruffi, P. nephridiatum, P. calkinsias well as P. duboscqui (Fig. 2 III). There are several generative nuclei per cell, usually 2-6. However, in case of P. calkinsi and P. duboscqui, most of the lines investigated had strictly
2 Mi. Generative nuclei in these species are spherical, with only P. duboscqui having lens or spindlelike micronuclei. The chromatin mass occupies in this case the main volume of the nucleus, but is separated from the nuclear envelope by the usual distinctive “clean” space (Fig. 2).
The small “vesicular” type of the Mi (approximately, 1-3.0 ^m) was recorded for P. aurelia complex spp., P. polycaryum and P. multimicronucleatum (Fig. 2 IV). The spherical nuclei have chromatin mass occupying only the periphery of the nuclear volume, and in the center there is a Feulgen-negative “vesicle”. There are always two Mi for all representatives of P. aurelia complex spp.; P. polycaryum has 2-5 Mi (on the average, 3) and P. multimicronucleatum — 3-5 (on the average, 4).
Unfortunately, it is quite common that in the first description the authors do not provide the precise information about the Mi structure. According to the pictures from the literature, we can assume that most African species: P. africanum, P. pseudotrichium,
and P. jankowskii have the “endosomal” Mi, but P. ugandae and P. wichtermani have, probably, the “compact” but relatively small Mi (Dragesco, 1970, 1972; Mohammed and Nashed, 1968/1969). Unfortunately, except P. africanum (S. Krenek, personal communication) none of these species were kept in any living culture collections, and I could not check their morphology myself.
Structure of CV in the genus members, as it was already mentioned, can also be of different types (Fokin, 1987) (Fig. 3). These organelles are commonly two in number and are located close to the dorsal surface in the endoplasm, usually directly beneath the cortex. They are situated at a distance of approximately one quarter up to one third of the entire body length from the anterior and posterior ends. CV connects with the outside through pores permanently located in the cortex. There is a slightly conical, very short (except in P. putrinum) canal decorated by helically arranged microtubules (Fig. 3g-i). The number of pores per one CV in a species is a distinctive feature (Fokin, 1987; Fokin et al., 1999a). For example, P. caudatum and P. aurelia complex spp. always have one pore per CV, but P. bursaria and P. nephridiatum, from 2 to 5. The last species reminds very much P. woodruffi and the above-mentioned feature (several pores per CV) helps to discriminate these two species, which have often been confused before (Jankowski, 1969; Fokin, 1986). Sometimes P. multimicronucleatum cell has three CV;
P. ugandae cell usually has 5-7 CV (up to 9), but no species has only one CV, which is a very common situation for the close related genus Frontonia (Andreoli et al., 2007).
In the literature two types of CV within the Paramecium genus are indicated (Wichterman, 1986). In one, called canal-fed type, a number of radial (collecting or nephridial) canals radiate from the vacuole and empty into it. This type is characteristic of all paramecia except P. putrinum. The latter has a vesicle-fed type, in which a number of small vesicles (or vacuoles) lie close to the main vacuole and empty into it. The CV of P. putrinum are located a bit deeper in the endoplasm and connect with the cortex by a rather long convoluted tube (canal) ending within the cortex (Fig. 3f, i). It could be treated as a very long pore. For all the other species the pore structure looks as a very short slightly conical tube mounted in the cortex. In fact, P. duboscqui has a similar organization of CV, but without the long tube, as in this species CV are situated very close to the cortex layer. Almost the same structure of CV (vesicle-fed type without
9
Fig. 3. Morphological types of Paramecium contractile vacuole. a — “canal-fed” type with long canals and one pore (all members of Paramecium subgenus); b — “canal-fed” type with long canals and several pores (P. bursaria); c — “canal-fed” type with short canals and one pore (P. woodruffi, P. calkinsi, P. polycaryum); d - “canal-fed” type with short canals and several pores (P. nephridiatum); e — “collecting vesicles” type with short outlet canal (pore) (P. duboscqui); f — “collecting vesicles” type with long outlet canal (tube) (P. putrinum); g — the scheme of organization for CV types “a-d”; h - the scheme of organization for CV type “e”; i - the scheme of organization for CV type “f”. For the last type, the distribution of microtubules (mt) on the central ampoule’s (ca) surface is unknown. Abbreviations: cc — collecting canals, cv — collecting vesicles, ot — outlet tube, p — pore.
long excretory tube) was discovered recently for Paramecium sp. from Norway (Fokin et al., 2006). Thus, we can assume that three distinctive types of CV organization exist within the genus (Fokin, 1987,2002).
Inside group which has canal-fed type of CV some variation in the number and size of canals could be recorded (Fig. 3 a-d). For P. bursaria there
are 5-7 (more often, 5-6) long canals; P. woodruffi
— 8-16 (more often, 10-12) relatively short ones; P. nephridiatum — 8-15 (more often, 10-11) short ones; P. calkinsi — 8-10 (more often, 9) short; P. polycaryum — 6-8 also short (more often, 7); P. caudatum — 5-8 (more often, 7) long canals; P. aurelia complex — 5-7 (more often, 5-6) long canals; P. multimicronucleatum — 7-9 (more often, 8) long
canals; P. jenningsi — 6-8 (more often, 7) long canals; P. schewiakoff — 5-8 (more often, 7) long canals. According to Dragesco (1972), P. pseudotrichium has only three collecting canals in the CV.
The direction of rotation during swimming has always been used for P. calkinsi discrimination. It is admitted that, when free-swimming, the members of the genus spiral characteristically to the left (anticlockwise) and only P. calkinsi spirals to the right (clockwise). In fact, both P. duboscqui and P. calkinsi spiral to the right, but the former does so always and P. calkinsi, preferably (Fokin et al., 1999b). Those two species were apparently mixed up of the basis of this feature, and identified as P. calkinsi only. I believe, this was one of the reason why P. duboscqui was not found for such a long time after its first description in 1933. Moreover, P. calkinsi, P. nephridiatum, Paramecium sp. (from Norway) and P. woodruffi can easily change the rotation’s direction. The cause of this rotation and possible mechanism to change direction of spiraling was discussed in the literature, as in many cases it is really a characteristic feature for species discrimination (see: Seravin, 1970).
Discussion
According to the results of my investigation, it is possible to propose the new discrimination key to the main representatives ofthe Paramecium genus . The most promising way to the correct discrimination appears to be the use of the combination of the Mi and CV characteristics. Ofcourse, the cell size should be taken into consideration as well, but in most of the species investigated by me, the length variation was rather significant across different populations and even inside clonal cultures (especially for brackish water species): 140-170 (P. nephridiatum -18%), 160-210 (P. woodruffi — 24%), 100-140 (P. calkinsi
— 29%) and 110-150 (P. duboscqui — 27%). For P. aurelia complex this variation has the same range: 120-170 (30%).
Swimming behavior as a discrimination feature works only for P. duboscqui and, partly (as this species can change it), for P. calkinsi, which, however, could be discriminated from majority of other Paramecium spp. by rotation to the right. But at least three more species (P. woodruffi, P. nephridiatum and Paramecium sp. from Norway) can also rotate to the right from time to time as well. In fact, P. duboscqui has very distinctive trajectory of swimming as well which is easily discriminated it from the rest of paramecia (Fokin et al., 1999b).
The number and length of collecting canals
can be used for discrimination as well. However, the first characteristic clearly separated only P. pseudotrichium from the rest of paramecia, while the second, the “aurelia” group from the “bursaria” group.
Key to discrimination of the main species
1 (10) Cells cigar-shaped, rounded anteriorly, pointed or conic posteriorly, the CV always with long radial canals and one pore. During swimming cell rotating to the left (anticlockwise).
2 (5) Single Mi belongs to the “compact a” type,
cell size around 200 ^m................P. caudatum
3 (4) Two Mi belong to the “vesicular” type, cell
size 120-170 ^m.................P. aurelia complex
4 (3) Several Mi ofthe “vesicular” type, cell size
200-250 ^m.................P. multimicronucleatum
5 (2) Another types of the Mi
6 (9) Mi belongs to the “chromosomal” type
7 (8) Single Mi ofthe “chromosomal” type, cell
size around 200 ^m..................P. schewiakoffi
8 (7) Two Mi of the “chromosomal” type, cell
size around 170 ^m.....................P. jenningsi
9 (6) Several Mi of the “endosomal” type, cell
size more that 200 ^m................P. wichtermani
10 (1) Cells shorter and wider, like a slipper, dorsoventrally flattened with obliquely truncated anterior end and broadly rounded posterior end. The CV either with long radial canals and several pores or with short radial canals or without canals (vesicle-fed type). If cell more close to cigar in shape, CV of vesicle-fed type with several pores. Some species can rotate in both direction or to the right (clockwise).
11 (12) CV with long collecting canals and se-
veral pores, single Mi of the “compact b” type. Usually with chlorellae symbionts (“green paramecia”), rarely aposymbiotic...........P. bursaria
12 (11) CV with short collecting canals and single pore, more than two Mi.
13 (18) Several Mi ofthe “endosomal” type, cell size not less than 120 ^m.
14 (15) Cell size 160-180 ^m.......P. woodruffi
15 (14) Cell size less than 160 ^m.
16 (17) CV with short collecting canals and
several pores. Usually 3-4 Mi, swimming rotation to both directions (right and left)...P. nephridiatum
17 (16) CV with single pore, two Mi. Swimming
rotation to both directions, but preferably to the right..................................P. calkinsi
18 (13) Another type of Mi, cell size smaller than 120 ^m.
19 (20) Several Mi of “vesicular” type, cell size
Table 1. Paramecium species distribution according to the literature and own data.
Species Europe Asia N. America S. America Africa Australia Reference of the first description
P. bursaria + + + + + + Focke, 1836
P. putrinum + + + - - + Claparede and Lachmann, 1858
P. duboscqui + + + + + - Chatton and Brachon, 1933
P. calkinsi + + + - - - Woodruff, 1921
P. woodruffi + + + - - - Wenrich, 1928
P. polycaryum + + + - + - Woodruff and Spencer, 1923
P. caudatum + + + + + + Ehrenberg, 1833
P. aurelia + + + + + + Muller, 1773
P. sonneborni - - + - - - Aufderheide et al., 1983
P. jenningsi + + + - + - Diller and Earl, 1958
P. wichtermani - + - - - - Mohammed and Nashed, 1968/1969
P. multimicronucleatum + + + + + + Power and Mitchell, 1910
P. schewiakoffi - + - - - - Fokin et al., 2001
Paramecium sp. + - - - - - Fokin et al., 2006
Notes: + presence; - either absence or not yet found.
70-110 ^m..............................P. polycaryum
20 (19) Single Mi of “compact c” type, CV
of vesicle-fed type with several pores, cell size 170-190 ^m, swimming rotation in both directions .....................Paramecium sp. (Norway)
21 (24) CV without canals with the only pore.
22 (23) Pore of CV short, slightly conical, two Mi spindle-shaped, swimming rotation to the right
(clockwise), cell size more that 110 ^m..............
.......................................P. duboscqui
23 (22) CV with long convoluted outlet canal
(pore), single Mi of “compact c” type, swimming rotation to the left (anticlockwise), cell size less that 110 ^m.................................P. putrinum
24 (25) Two CV with 7-9 collecting canals, cell size more than 300 ^m, number of Mi usually 7 P. africanum
25 (24) Two CV with 3 collecting canals only,
cell size not more than 100 ^m, two Mi...............
..................................P. pseudotrichium
Other two species described by Dragesco (1972) in Africa, P. ugandae and P. jankowskii, should be rechecked, as the composition of quadrulus reported for them, 5 or 6 ciliary rows, is very unusual for Paramecium spp.
Though paramecia have been reported from many geographic areas, most findings are from well investigated regions, such as Europe and North America (Table 1). Very likely this means that for
the South Hemisphere we simply have no clear information, as a lot of territories there were not checked professionally at all. At present, I can say that P. bursaria, P. caudatum, P. multimicronuclea-tum and P. aurelia complex (as a morphospecies) could be found everywhere (cosmopolitan). P. putrinum as well as P. polycaryum, P. jenningsi, P. woodruffi, P. calkinsi, P. duboscqui and P. nephridiatum could be found relatively often, but not everywhere, as they have some preferences for the climate. On the contrary, P. schewiakoJJi, P. sonneborni and all “African” species have an apparently resticted distribution (are endemic) and are strongly associated with the areas of warm (or somehow specific) climate (Table 1). It also should be taken in consideration that all euryhaline species: P. duboscqui, P. calkinsi, P. nephridiatum and P. woodruffi mainly could be found in brackish water (salinity from 10 to 25%c).
As mentioned above, P. nephridiatum, P. duboscqui were often confused with P. woodruffi and P. calkinsi, respectively (Fokin et al., 1999a, 1999b). Misidentification could also be the case for fresh water Paramecium sp. from Norway (Fokin et al., 2006). This “chimeric” paramecium at first glance looks like P. caudatum (size, general shape, one large Mi), but the type of the Mi is the “compact c” (as in P. putrinum) and the CV structure reminds P. duboscqui (Fokin et al. 2006). This example shows clearly that even in Europe some new ciliates can still
be found. Genetic variation within the Paramecium genus appears to be even more strong than the morphological one (Fokin, 2002; Fokin et al., 2006) and we hope to find a number of “cryptic” species.
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Address for correspondence: Sergei I. Fokin. Department of Invertebrate Zoology, St. Petersburg State University, Universitetskaya emb. 7/9, St.Petersburg, Russia, 199034 Russia; e-mail: [email protected]
