Eigner Peter 1999: The Complete Paper

Ciliophora, Hypotrichida, Divisional Morphogenesis

European Journal of Protistology

Europ. J. Protistol. 35, 34--48 (1999)

Running title: EIGNER---UPDATE OF THE NATURAL SYSTEM HYPOTRICHIDA

Comparison of Divisional Morphogenesis in Four Morphologically Different Clones of the Genus Gonostomum and Update of the Natural Hypotrich System (Ciliophora, Hypotrichida)

Peter Eigner

peter.eigner@aon.at

Summary

Divisional morphogenesis of a clone of the multimacronucleate Gonostomum kuehnelti Foissner, 1987 (clone I) with four transverse cirri and of three clones of Gonostomum affine (Stein, 1859) Sterki, 1878 with two macronuclear segments are described and compared using protargol impregnation. The three clones of G. affine differ in having two (clone II), four (clone III) and six (clone IV) transverse cirri, whereas all clones have the complete lack of ventral cirri between proximal membranelles and transverse cirri in common. Early morphogenetic processes show that all four clones develop the family characters of the Oxytrichidae Ehrenberg, 1838: "neokinetal 3" anlagen and long primary primordia. Only clone I shows a significant morphogenetic difference by generating proter's first (left) frontal cirrus by the oral primordium. Clones II to IV generate this cirrus de novo. Biometry of middle dividers and specimens in interphase reveals an invariable cirral pattern generated from six ventral anlagen in the clone I, whereas six or seven ventral anlagen, one or two left marginal rows and two to four macronuclear segments develop in the clones II - IV. All four clones are, however, stable in the lack of ventral cirri and, surprisingly, the clones II - IV are also stable (not overlapping) in their different number of transverse cirri. Nevertheless, morphologic differences in the clones II - IV are considered to be probably polymorphic states of G. affine.
Recently published morphogenetic data are analyzed, shown in schematized computer drawings and added to the natural hypotrich system: Lamtostyla edaphoni is transferred to the genus Amphisiella Gourret and Roeser, 1888 within the family Oxytrichidae because of neokinetal 3 anlagen and long primary primordia development. Onychodromopsis flexilis is also assigned to the Oxytrichidae because of neokinetal 3 and long primary primordia, whereas Sterkiella histriomuscorum generates "neokinetal 1" anlagen and long primary primordia are absent and therefore it is assigned to the family Parakahliellidae. A computer generated tree is presented using the programs MacClade and PAUP and consisting of 42 morphogenetically investigated hypotrich species with 6 characters in 15 states. For the homonym Clara the genus name Tetmemena nom. nov. is suggested.

Key Words: Hypotrichida - Divisional morphogenesis - Phylogeny - Gonostomum - Natural system.

 

Introduction

Divisional morphogenesis has been described for Gonostomum strenua (Engelmann, 1862) Sterki, 1878 [33, 38] and G. affine (Hemberger 1982, Dissertation Univ. Bonn, Germany). The present study describes divisional morphogenesis in G. kuehnelti and G. affine, for the latter new morphogenetic data has been found. Old and new data show the genus Gonostomum and its three species G. kuehnelti, G. affine and G. strenua as a well defined and consistent morphologic and morphogenetic group which develops neokinetal 3 anlagen together with long primary primordia in the left ventral field. Thus, they belong to the family Oxytrichidae sensu [15] (Fig. 33).
Sterki separated Stein's Oxytricha affinis and Engelmann's O. strenua from the genus Oxytricha and united them in the genus Gonostomum [19, 40, 41]. He characterized the new genus as different from Oxytricha by the position and shape of the peristome and the different cirral patterns (Figs. 26 - 29). After Sterki such position and shape of the peristome have also been found in other genera as well as in another family: Orthoamphisiella franzi, originally described as Gonostomum franzi, belongs to the Orthoamphisiellidae, because it develops the two rightmost ventral rows by within anlagen [2, 14, 15, 22]. Urosoma macrostyla also has a similar shape and position of the peristome, but has a different ventral cirral pattern; moreover, morphogenesis reveals, in contrast to Gonostomum, dorsomarginal kineties and therefore U. macrostyla belongs to another genus [23]. The peristome in the genera Trachelostyla and Wallackia is similarly shaped and positioned as in Gonostomum [21, 30].
More new genera and species with a morphology similar to Gonostomum have been established or redescribed and several of them were later synonymized with G. affine [4, 9, 10, 11, 12, 24, 28, 39]. The morphologic variability of G. affine and a detailed history of the genus Gonostomum have been published [22, 32]. A family Gonostomatidae was established as well [36].
Two studies dealing with the phylogeny of hypotrich families by using interphase and morphogenetic data were published recently. In the earlier study a set of selected oxytrichid genera was used in order to describe the family Oxytrichidae [5]. In the second study all available detailed descriptions of morphology and morphogenesis were used to describe the families Oxytrichidae, Parakahliellidae and Orthoamphisiellidae. In this study the method of schematized computer drawings was introduced [15]. Short descriptions of methods and results of the two studies are given in the Discussion of the present study and differences are pointed out. Since the appearance of these two studies new descriptions of divisional morphogenesis in Lamtostyla edaphoni, Onychodromopsis flexilis and Sterkiella histriomuscorum have been published. These new descriptions are analyzed by using the methods of [15] and added to the natural system.

Material and Methods

The four clones were collected as raw material in 1992 from top soil layers in the village of Schroetten, Deutsch Goritz, Austria and dried for several days. Clone I (Gonostomum kuehnelti) was found in a forest next to the village, clone II (G. affine) on a compost heap and clone III (G. affine) and IV (G. affine) in a rainwater channel underneath a roof. The four raw cultures were set up in petri dishes with local spring water. After a few days a single individual from each of the four raw cultures was isolated. The clones were fed with baker's yeast and maintained at room temperature. Staining was performed according to [26].
Drawings were made with the help of a camera lucida, a scanner and a Power Macintosh computer using a drawing program. To illustrate the changes during morphogenesis old cirri are depicted by contour and new cirri are shaded black.
Terminology is according to [13, 14, 15]. Statistical procedures are according to [37]. Symbols used in the computer drawings are according to [15].
The phylogenetic analysis was carried out using a Power Macintosh computer and the computer programs MacClade 3.04 in order to edit files, explore trees and analyze character evolution upon them [31] and PAUP 3.1[42] in order to search automatically for parsimonious trees. Heuristic search was performed, because the data set was too large to perform exact methods (Fig. 33). The characters A - D are identical with the characters used in [15] to define the three families and therefore weighted 2; the two remaining characters are weighted 1. Search settings: all characters are of the type "ordered". Branches having maximum length zero collapsed to yield polytomies. MULPARS option in effect. One tree held at each step during stepwise addition (addition sequence: "as is"). Branch swapping algorithm: tree bisection-reconnection (TBR).

Results

The very similar and rather simple processes during divisional morphogenesis of both species, Gonostomum kuehnelti and G. affine, can be followed in detail in all decisive stages of morphogenesis in the Figs. 1 - 25 and their legends . Morphologic and morphogenetic differences and similarities are described in Additional description and comparison. The improved diagnoses contain the substantial morphologic and morphogenetic characters. The morphogenetic characters are included to discriminate morphologically similar and closely related taxa.

Gonostomum kuehnelti Foissner, 1987 (clone I; Figs. 1 - 11, 33; Tabs. 1, 2)

Improved diagnosis: 14 macronuclear nodules on average [24]. Six ventral anlagen. Nearly invariable ventral cirral pattern. Ventral cirri between proximal membranelles and transverse cirri absent. Four transverse cirri, usually forming a typical square. Six ventral anlagen develop in each the proter and opisthe by six long primary primordia. The primordia develop in the frontal field right of the adoral zone of membranelles and later split horizontally for proter and opisthe. The long primary primordia are formed first by migrating basal bodies originating from the oral primordium and then also by basal bodies from disaggregating cirri of the frontal field. Anlage 1 generates for the proter one frontal cirrus, for the opisthe one frontal cirrus and the endoral and paroral membrane. Anlage 2 generates in both filial products one frontal and one buccal cirrus; anlage 3 one frontal and one cirrus in the frontal field; each of anlagen 4 - 6 generates two cirri in the frontal field, anlage 5 and 6 additionally generate two transverse cirri each. The anterior two cirri of anlage 6 migrate anteriorly (frontoterminal cirri).

Figs. 1 - 10. Divisional morphogenesis in Gonostomum kuehnelti Foissner, 1987 (clone I; ventral side Figs. 1, 3 - 7, 9; dorsal side Figs. 2, 8, 10). Also see Additional description and comparison of G. kuehnelti and G. affine. White arrows indicate a basal body proliferation and the forming of a field (oral primordium). Small black arrows indicate the position of the paroral membrane (Fig. 1), macronuclear nodules with replication bands (Fig. 2) and tree-like distribution of basal bodies in the frontal field originating from oral primordium (Fig. 3). The buccal cirrus disaggregates (Fig. 3, posterior end of anlage/row 2). Forming of anlage 1 by two basal bodies above the paroral membrane (Fig. 4, anterior arrow) and basal bodies originating from the oral primordium and crossing the endoral membrane (Fig. 4, posterior arrow). These basal bodies proliferate and produce one long primary primordium (Fig. 5), which later condenses to proter and opisthe's left frontal cirrus (Fig. 6) and opisthe's undulating membranes. The cirri in the frontal field disaggregate successively and their basal bodies together with basal bodies originating from the oral primordium form the long primary primordia; the basal bodies from disaggregating cirri contribute only to the anlagen with the same number (e. g. all cirri of the old row 4 contribute only to the new anlage 4). Some old cirri (all from the old row 6), however, do not contribute to the forming of new cirri, and are resorbed during cytokinesis. The long primary primordia five and six form the large "V" of the neokinetal 3 anlagen development which later splits transversely (as the other primordia do) and generate the two rightmost ventral anlagen/rows for proter and opisthe (Figs. 4 - 6, cp. Figs. 14, 16). Figure 6 shows a specimen lying on the right lateral side, therefore the paroral is to the right of the endoral; similarly, the old and new adoral membranelles are positioned like in reorganizers, but the split anlagen and the two new frontal cirri prove it to be a divider. The number of ventral cirri and their migration - as well as the usual division of the marginal rows - are recognizable (Figs. 7, 9). The nuclear apparatus, dorsal kineties and caudal cirri are generated in the usual way (Figs. 8, 10). Numbers (1 - 6) and lines indicate anlagen and cirral rows. Scale bars = 10 µm.

Fig. 11. Divisional morphogenesis in Gonostomum kuehnelti (clone I). The interphase infraciliature and its morphogenesis depicted by symbols is shown in the computer drawing on the left side and for reasons of comparison the ventral cirral pattern of a stained specimen is shown on the right side. Symbols are explained in detail in [15]. In brief, numbers and lines indicate cirral rows/anlagen and are identical in both images. Boxes indicate anlagen and are positioned where the anlagen start to develop: the anterior boxes indicate anlagen for proter, the posterior boxes for opisthe. Except for row 5, which has only one box with the number three, which indicates the neokinetal 3 (N3) anlagen development [15]. The N3 anlage generates the new anlagen 5 and 6 (in italics) for proter and opisthe. Left of the N3 symbol are the anlagen symbols (boxes) for opisthe's anlagen 1 - 4 and the symbol for the oral primordium (grey right angle). In the computer drawing the long primary primordia are indicated by the lines protruding posteriorly (in this species anlagen/rows 1 - 6). Rectangular symbols for cirri indicate used (active) cirri (e. g. row 4), round symbols for cirri indicate unused cirri (cirri which are not used to generate new cirri, e. g. row 6). Right of the right marginal row (row 7) are three dorsal kineties with their anlagen and caudal cirri. L1 = left marginal row. RM = right marginal row. Numbers (1 - 7) and lines indicate anlagen and cirral rows.

Gonostomum affine (Stein, 1859) Sterki, 1878 (clones II - IV; Figs. 12 - 28, 33; Tabs. 1, 2)

Improved diagnosis: Usually two macronuclear nodules. 6 - 7 ventral anlagen. Ventral cirri between proximal membranelles and transverse cirri absent. One to eight transverse cirri. Specimens with four transverse cirri form the same typical square as in G. kuehnelti. Anlagen 1 for proter and opisthe develop by separate primary primordia, the one for proter develops de novo and the one for the opisthe from the oral primordium. The remaining 5 - 6 ventral anlagen develop by long primary primordia in the frontal field right of the adoral zone of membranelles and later split horizontally for proter and opisthe. The long primary primordia are formed first by migrating basal bodies originating from the oral primordium and then also by basal bodies from disaggregating cirri of the frontal field. The anlagen 1 - 6 or 1 - 7 generate 11 - 14 cirri in the frontal field (frontoterminal cirri included) and the transverse cirri. Opisthe's anlage 1 also generates the undulating membranes for opisthe. The anterior two to three cirri of the rightmost ventral anlage 6 or 7 migrate anteriorly (frontoterminal cirri). The transverse cirri originate from the rightmost ventral anlage and also from anlagen left of it.

Figs. 12 - 19. Shape, macronuclear nodules and contractile vacuole from live cells (Fig. 12) and divisional morphogenesis from protargol slides (Figs. 13 - 19) in Gonostomum affine (Stein, 1859) Sterki, 1878 (clone II; ventral side Figs. 13, 14, 16 - 18; on the right side of Fig. 13 there are two macronuclear nodules and two micronuclei; dorsal side Fig. 15, 19). Morphogenesis in G. kuehnelti and G. affine is very similar. Thus, only deviating processes are described. The new anlage 1 commences development similar to that of G. kuehnelti (cp. arrows in Fig. 4 and in Fig. 14), but in G. affine the two anlagen for proter and opisthe are obviously not connected in any stage, i. e. the anlage 1 in G. affine is not generated by a long primary primordium (see next two stages). The basal bodies which the posterior arrow is pointing to (Fig. 14) are possibly used for generating anlage 2 (and not for anlage 1 as in Fig. 4). Some adoral membranelles in Fig. 14 were deleted to make basal bodies and membranes recognizable. Arrows in Figs. 16, 17 indicate developments for producing anlage 1 for proter (anterior arrows) and opisthe (posterior arrow). Opisthe's anlage 1 probably develops from the basal bodies below the streaks for anlage 2 (Fig. 16). Three dorsal kineties develop for each proter and opisthe (Fig. 19). Caudal cirri are not recognizable yet. Numbers (1 - 6) and lines indicate anlagen and cirral rows. Scale bars = 10 µm.

Fig. 20. Divisional morphogenesis in Gonostomum affine (clone II). The interphase infraciliature and its morphogenesis depicted by symbols is shown in the computer drawing on the left side and for reasons of comparison the ventral cirral pattern of a stained specimen is shown on the right side. Symbols are explained in detail in [15]. Some symbols are explained briefly in the legend to Fig. 11. Morphogenesis in G. affine and G. kuehnelti is similar, thus the description given for Fig. 11 is also valid for Fig. 20. The difference is that anlage 1 develops separately for proter and opisthe in G. affine whereas it develops by long (common) primary primordia in G. kuehnelti. Another difference is that the anlagen/rows 5 and 6 each constantly produce two transverse cirri in G. kuehnelti. In G. affine a total of 1 - 8 transverse cirri is produced (Figs. 21 - 25, Tab. 1, 2). L1 = left marginal row. RM = right marginal row. Numbers (1 - 7) and lines indicate anlagen and cirral rows.

Additional description and comparison of G. kuehnelti and G. affine

In both species the new adoral membranelles develop from the oral primordium, marginal rows and dorsal kineties develop from within anlagen, caudal cirri develop from the posterior end of the dorsal kineties and the macronuclear nodules from a single mass that has been formed shortly before by a fusing of the old macronuclear nodules.
Early morphogenetic processes are very similar in both species, and thus they are shown only once, either in the drawings of morphogenesis of G. kuehnelti (Figs. 1 - 10) or of G. affine (Figs. 13 - 19): (1) basal body proliferation shown for G. kuehnelti (Fig. 1, white arrow) was also observed in G. affine; (2) the distribution of basal bodies shown for G. kuehnelti (Fig. 3, black arrow) was also observed in G. affine; (3) an early streak development for the rightmost ventral anlage shown for G. affine (Fig. 13, arrow) was also found in G. kuehnelti. The paroral membrane (Fig. 1, black arrow) of the proter is obviously unchanged in all stages of morphogenesis. However, some reorganization of the proter's endoral and paroral, especially in G. kuehnelti, cannot be excluded with certainty. In figure 7 both the short anterior paroral on the surface and the long endoral membrane below the surface are optically in one line.
The most conspicuous differences in morphogenetic processes of Gonostomum kuehnelti and G. affine (besides the development of a different number of macronuclear nodules) are in the origin of the anlage 1. In G. kuehnelti it develops from a single, very long primary primordium whereas in G. affine anlagen 1 develop by separate primary primordia.
Special (subpellicular) cortical granules were not observed in the clone I of G. kuehnelti, which is in contrast to the type population [24].
Biometry was carried out for clone I and II using specimens in the middle stages of morphogenesis in order to determine the number of ventral anlagen and their cirri (Tab. 1). All specimens of clone I as well as 17 of 21 investigated dividers of clone II develop six ventral anlagen in each proter and opisthe. Of the remaining four dividers of clone II, in one divider seven anlagen develop in each proter and opisthe, in one divider seven anlagen develop only in proter, and in two dividers seven anlagen only in opisthe. Very few specimens with three and four connected macronuclear nodules were also found in clone II. Most or all macronuclear nodules in the four clones are connected by very thin bridges, although these connections can only be observed in well stained specimens. As usual, more cirri are generated in middle stages of division than later exist in interphase: they disappear again during migration and cytokinesis shortly after they have been generated (cp. Tab. 1 and Tab. 2, clone I).
All four clones completely lack ventral cirri between proximal membranelles and transverse cirri. Clone I with four transverse cirri is nearly 100 % stable in its cirral pattern as it is reported for the raw culture of its type population (Fig. 11; [24]). The clone III has also four transverse cirri and develops compared to clone I a quite similar nearly invariable ventral cirral pattern (Fig. 21, Tabs. 1, 2). However, six of 365 specimens in clone III have a second left marginal row. In these six specimens one left marginal row occurs with the usual length and parallel to it it develops the second row with a variable number of cirri: 2, 3, 3, 8, 8 or 11. Each of the clones II - IV is nearly stable in its different number of transverse cirri ; only one specimen of clone II has four transverse cirri.
Cysts of clone II were observed in different stages to be resorbing cortical structures and fusing their macronuclear nodules. As a result, the cysts became 20 µ in diameter, with one rounded up macronucleus in the center and a smooth surface.

Figs. 21, 22. Gonostomum affine (clone III, ventral side Fig. 21; dorsal side Fig. 22). Note that the number and pattern of transverse cirri are as in G. kuehnelti (Fig. 1). L1 = left marginal row. Numbers (1 - 7) and lines indicate anlagen and cirral rows. Scale bars = 10 µm.

Figs. 23 - 25. Gonostomum affine (clone IV, ventral side Fig. 23, 25; dorsal side Fig. 24). Interphase specimens (Figs. 23, 24). The late stage of divisional morphogenesis shows the origin of six transverse cirri (Fig. 25). L1 = left marginal row. Numbers (1 - 7) and lines indicate anlagen and cirral rows. Scale bars = 10 µm.

Figs. 26 - 29. Gonostomum affine (Stein, 1859) Sterki, 1878 (Figs. 26, 28 from [40], Fig. 26 modified. Fig. 27 from [41] modified), G. strenua (Engelmann, 1862) Sterki, 1878 (Fig. 29 from [19]). Figs. 26, 28 show that ventral cirri between proximal membranelles and transverse cirri are absent, whereas the ventral row in Fig. 29 extends below the proximal membranelles. Sterki explains the different position and shape of the oral apparatus in his new genus Gonostomum (Fig. 27, a indicates the left body margin). Such oral apparatus has since been also found in species which do not belong to the genus Gonostomum (see Introduction).

 

Discussion

The three Gonostomum species (Figs. 11, 20, 33)

The genus Gonostomum Sterki, 1878 and its three species are now a morphogenetically and morphologically well defined group within the family Oxytrichidae Ehrenberg, 1838. The position of its three species shown in the cladogram (tree, Fig. 33) is similar to the position of G. strenua (Engelmann, 1862) Sterki, 1878 in the evolutionary line shown in Fig. 3 in [15]. The present study shows G. affine (Fig. 20) and G. kuehnelti (Fig. 11) as having the same morphogenetic pattern in the right ventral field, i. e. the two rightmost ventral anlagen (cirral rows) are generated by the neokinetal 3 anlagen development. Anlagen in the left ventral field develop by within anlagen and by long primary primordia that originate in the posterior (oral primordium) and anterior (disaggregating cirri) part of the body. A previous description of divisional morphogenesis of G. affine shows developments similar to those in the present study (Hemberger. Dissertation, 1982). In contrast to the present study, however, the complete renewal of proter's endoral and paroral membrane is described in the aforementioned dissertation. In the present study such a renewal was not observed (cp. Figs. 13, 14, 16-18).
The most conspicuous discriminating interphase characters of the three species are: G. strenua - two macronuclear nodules, ventral cirri present, 4 - 7 transverse cirri, G. affine - two macronuclear nodules, ventral cirri absent, 1 - 7 transverse cirri, and G. kuehnelti - multimacronucleate, ventral cirri absent, 4 transverse cirri.

Onychodromopsis flexilis Stokes, 1887 (Figs. 30, 33)

The recently published detailed description of divisional morphogenesis in O. flexilis shows the development of neokinetal 3 anlagen in the right and long primary primordia in the left ventral field. Therefore I agree with the authors of [34] that this species belongs to the family Oxytrichidae Ehrenberg, 1838 although the family Oxytrichidae is defined quite differently in the two studies [15, 34]. Its inner right and outer left marginal rows are produced similarly to those of Parakahliella macrostoma (Fig. 24 in [15]). In both species anlagen develop for the "regular" right and left marginal row, shortly after that anlagen develop parallel to them and generate the additional marginal rows.

Fig. 30. Divisional morphogenesis in Onychodromopsis flexilis Stokes, 1887. The interphase infraciliature and its morphogenesis depicted by symbols is shown in the computer drawing on the left side and for reasons of comparison the ventral cirral pattern of a stained specimen is shown on the right side [34]. Symbols are explained in detail in [15]. Some symbols are explained briefly in the legend to Fig. 11. In the right marginal row 8 (RM) two anlagen similar to neokinetal 1 anlagen are shown to be generating the inner marginal row 7. Similar processes are reported for marginal rows left of the left marginal row (L1). Right of row 8 the development of two dorsomarginal kineties and a split dorsal kinety is shown [15]. Except for the right and left marginal rows O. flexilis develops a morphogenetic pattern very similar to that of Stylonychia mytilus and S. lemnae (cp. Fig. 30 of the present study and Fig. 39 in [15]). L1 = left marginal row. RM = right marginal row. Numbers (1 - 8) and lines indicate anlagen and cirral rows.

Lamtostyla edaphoni Berger and Foissner, 1987 (Figs. 31, 33)

In a recent study divisional morphogenesis of Lamtostyla edaphoni is described and Amphisiella australis is combined with the genus Lamtostyla [34]. The morphologic and morphogenetic comparison shows that Amphisiella australis (and its morphogenesis) is very similar to L. edaphoni (cp. Fig. 31 of the present study and Fig. 18 in [15]; [1, 8, 34, 43]). Most biometric values overlap. Both show the same morphogenetic peculiarity pointed out in [15], i. e. the posterior cirri of the second ventral anlage from the right form a conspicuous second (oral) primordium (large arrow in Fig. 6 in [34]). However, one cirrus left of the amphisiellid cirral row (ACR) constantly occurs in L. edaphoni, whereas for A. australis 2-10 cirri are reported in this position (Tab. 3).
The authors of [34] divide amphisiellids into two groups (Lamtostyla/Amphisiella) based on the apokinetal (Lamtostyla) and parakinetal (Amphisiella) development of the oral primordium. On this basis they combine Amphisiella australis with the genus Lamtostyla and do not recognize a former combination (Amphisiella perisincirra [18]). They give no definition or reference for the character pair apokinetal/parakinetal. Moreover, it has been shown that the earliest stages of morphogenesis may occur in one species in different places [46]. Therefore, the character pair parakinetal/apokinetal development of the oral primordium is obviously of very low or no taxonomic and phylogenetic value within the Euhypotrichina [20]. There can hardly be a division of a group of hypotrichs based on this character pair as done in [34].
The genus Amphisiella Gourret and Roeser, 1888 is a well defined morphologic and morphogenetic group and can be characterized (1) as member of the family Oxytrichidae (neokinetal 3 anlagen development for the two rightmost ventral cirral rows and long primary primordia in the left ventral field present [15]), (2) by an amphisiellid cirral row (which, however, is not as distinct as in other closely related species, cp. Figs. 13 - 20 in [15]), (3) by presence of transverse cirri, and (4) by absence of dorsomarginal kineties and caudal cirri (Figs. 31, 33. Figs. 18 - 20 in [15]. [18]). Therefore, I suggest to combine Lamtostyla edaphoni, which possesses all these characters, with the genus Amphisiella: Amphisiella edaphoni (Berger and Foissner, 1987) nov. comb.
Species assignable to the genus Amphisiella Gourret and Roeser, 1888: A. marioni Gourret and Roeser, 1888; A. perisincirra (Hemberger, 1985) Eigner and Foissner, 1994 (Basionym and former combination: Tachysoma perisincirra, Lamtostyla perisincirra); A. edaphoni (Berger and Foissner, 1987) nov. comb. (Basionym: Lamtostyla edaphoni), Amphisiella australis Blatterer and Foissner, 1988 [1,15, 18]).

Fig. 31. Divisional morphogenesis in Amphisiella edaphoni (Berger and Foissner, 1987) nov. comb. (Basionym: Lamtostyla edaphoni [34]). The interphase infraciliature and its morphogenesis depicted by symbols is shown in the computer drawing on the left side and for reasons of comparison the ventral cirral pattern of a stained specimen is shown on the right side [34]. Symbols are explained in detail in [15]. Some symbols are explained briefly in the legend to Fig. 11. L1 = left marginal row. RM = right marginal row. Numbers (1 - 6) and lines indicate anlagen and cirral rows.

Sterkiella histriomuscorum (Figs. 32, 33)

Two descriptions of divisional morphogenesis of S. histriomuscorum exist now, a detailed one in [35] and one in [7] as Histriculus muscorum. The latter was used in [15] to establish a natural system.
Morphogenetic processes in Sterkiella histriomuscorum [35] are nearly the same as those described for Tetmemena vorax (Stokes, 1885) nom. nov. (Stylonychia vorax, Clara vorax) and T. pustulata (Müller, 1786) nom. nov.
(Kerona pustulata, Stylonychia pustulata, Clara pustulata; cp. Fig. 32 of the present study and Fig. 37 in[15]. Tetmemena nom nov. see below). Only the origin of opisthe's anlage 4 is described for S. histriomuscorum as developing by a possible contribution of some basal bodies from opisthe's anlagen 1 - 3 (Fig. 32). The only morphologic difference are the undulating membranes, which are parallel in Tetmemena vorax and T. pustulata [46], whereas they are shown to be parallel only in one late stage of morphogenesis in S. histriomuscorum [35]. In other descriptions of morphogenesis, for instance in Bakuella pampinaria, the membranes are also shown in some specimens to be parallel and in others to be distinctly intersecting [16]. In [5] much emphasis is put on the character pair parallel/intersected membranes, but the position of the body (e. g. lateral or straight) on slides and the staining technique may strongly influence the position and shape of the membranes on slides (cp. Figs. 6, 7). This ambiguity is also shown in a description of Sterkiella histriomuscorum, which in vivo shows rather parallel membranes, yet when stained they are shown as intersecting (Histriculus muscorum in [22]). Thus, at least the species described in [35] as Sterkiella histriomuscorum (membranes in stained specimen usually crossing) is probably Tetmemena vorax or T. pustulata (membranes usually parallel). Similarly, in [47] Sterkiella (Histriculus) is considered to be a synonym of Tetmemena nom. nov. (Stylonychia).

Fig. 32. Divisional morphogenesis in Sterkiella histriomuscorum (formerly Histriculus muscorum). The interphase infraciliature and its morphogenesis depicted by symbols is shown in the computer drawing on the left side and for reasons of comparison the ventral cirral pattern of a stained specimen is shown on the right side [35]. Symbols are explained in detail in [15]. Some symbols are explained briefly in the legend to Fig. 11. The species shown here in Fig. 32 and described in [35] is possibly the same as Tetmemena vorax nom. nov. or T. pustulata nom. nov. for which also a description of morphogenesis exists (cp. the present Fig. 32 and Fig. 37 in [15]). In row 4 the anterior part of a neokinetal 1 (N1) anlagen development [15] is shown (anterior box with the number 1) which generates proter's anlagen 4 - 6 (numbers in italics). The N1 anlage in row 5 generates the opisthe's anlagen 5 and 6 (in italics). The opisthe's anlage 4 is generated by the disaggregating second cirrus of row 4 (within anlage touching this cirrus) and possibly by some basal bodies from the oral primordium (symbolized by the connection to opisthe's anlagen 1 - 3 and the oral primordium). Right of the right marginal row (RM), two dorsomarginal kineties and one split dorsal kinety are indicated [15]. Long primary primordia are absent. L1 = left marginal row. RM = right marginal row. Numbers (1 - 7) and lines indicate anlagen and cirral rows.

The tree (Fig. 33)

Of the six characters used to compute the tree, the first four characters (A - D) are identical with the characters used in [15] to define the families. The Orthoamphisiellidae are regarded as the ancestors of the Oxytrichidae and Parakahliellidae, because the Orthoamphisiellidae generate all their ventral cirri by within anlagen. Two more characters are used to show the clusters within the families (E - F). The number of cirri in the two rightmost ventral rows is obviously reduced from an ancestral to a derived state (E), i. e. the number of cirri in the two rightmost ventral rows are considered to be of great value for the determination of the position of a species or genus in the evolutionary line within its family [15]. For instance, Gonostomum kuehnelti (species number 13) has a maximum of only eight cirri in the two rightmost ventral rows, like a group of derived oxytrichids (14 - 18), whereas G. affine and G. strenua have a maximum of more than 8 cirri (Fig. 11, cirral rows 5, 6; Tabs. 1, 2; [33, 38]). Dorsomarginal kineties (F) are generated in all Parakahliellidae and in a group of Oxytrichidae (14 - 18) which is nearly identical with the group having fewer than 9 cirri in the two rightmost ventral rows.
This tree contains most taxa of the hand generated evolutionary line using Hennig's method [29] in Figs. 2, 3 in [15]. Gonostomum affine (Fig. 20), G. kuehnelti (Fig. 11) and Onychodromopsis flexilis (Fig. 30) are added. Histriculus muscorum is renamed to Sterkiella histriomuscorum (Fig. 32). Deviata abbrevescens is not included in the tree, because it is regarded as an oxytrichid in an ancestral state having characters only similar to other oxytrichids [15]. Likewise Engelmanniella mobilis, Psilotricha succisa and Circinella arenicola are not included because of ambiguous characters or missing data. These three species are considered, however, to be more closely related to the Orthoamphisiellidae than to the other two families, because the cirri on right ventral side are generated by within anlagen [15].
In [15] the genus Stylonychia has been divided and the genus name Clara was suggested for Stylonychia pustulata and S. vorax. Later I was informed by Prof. Dr. Foissner that Clara is preoccupied (homonym). Therefore, Tetmemena nom. nov. is suggested as Tetmemena pustulata (Müller, 1786) nom. nov. and T. vorax (Stokes, 1885) nom. nov. Derivatio nominis: Gr., tetmemene (cut, divided) from temno (cut, divide), because the genus Stylonychia is not monophyletic and had to be divided [15].

Fig. 33. Computer generated strict consensus tree (programs: MacClade and PAUP [31, 42]) consisting of 42 morphogenetically investigated hypotrich species and six characters (A - F) in 15 states (data from [15] and the present study). A: forming of an anlage in the rightmost ventral cirral row during division (1 = present; 0 = absent). B: anlagen development for the rightmost ventral row (0 = "within" in the rightmost ventral row; 1 = "neokinetal" in the second rightmost ventral row). C: which neokinetal development generates the rightmost ventral row (0 = not relevant; 1 = neokinetal 1; 2 = neokinetal 3). D: development of long primary primordia (0 = not relevant; 1 = present; 2 = absent). E: number of cirri in the two rightmost ventral rows (0 = > 30 cirri; 1 = 9 - 30 cirri; 2 = < 9 cirri). F: development of dorsomarginal kineties (0 = absent; 1 = present).

The two natural systems of the family Oxytrichidae

Berger and Foissner [5] selected a set of 13 genera that are usually assigned to the Oxytrichidae. Their investigation shows two autapomorphies for these selected Oxytrichidae: 18 ventral cirri in a certain pattern and the fragmentation of dorsal kineties. Their cladograms divide the Oxytrichidae in two groups: in one group the cirrus V/3 participates in early morphogenetic processes, the other group has three synapomorphies, viz. a rigid body, an oral apparatus of more than 40% of body length, and the lack of cortical granules. All these characters used in [5] are discussed in detail in [15, p. 569].
A decisive difference between the two studies [5, 15] is the selection and number of investigated taxa. Berger and Foissner [5] investigate a selected set of genera with certain characters, in [15] all available detailed descriptions of morphology and morphogenesis were used, i. e. 43 species in 35 genera. This way it was possible to reveal the relationship of all hypotrichs, for instance of the Oxytrichidae that are in a derived state and also those which are in an ancestral state where they have up to 80 ventral cirri. Three morphogenetic patterns on the right ventral side of the cell's body were found [15]. These three patterns, which generate the cirri on the right ventral side, are the "neokinetal 1" (Parakahliellidae), "neokinetal 3" (Oxytrichidae) and "within" anlagen processes (Orthoamphisiellidae). The neokinetal 1 and neokinetal 3 processes are both complex in their development and are accompanied by presence (neokinetal 3) and absence (neokinetal 1) of long primary primordia in other parts of the cell's body. Thus, the two autapomorphies of the Oxytrichidae are the neokinetal 3 and the long primary primordia processes [15]. The two neokinetal processes and their value for reconstructing the phylogeny could only be found with the help of schematized computer drawings as used in [15]. The Orthoamphisiellidae generate the cirri on the right ventral side and also on the left ventral side by usual within anlagen. In the Oxytrichidae and Parakahliellidae cirri on the left ventral side are usually also generated by within anlagen. Since most cirri on the ventral side and cilia on the dorsal side are generated by within anlagen, the neokinetal anlagen are considered to be derived and the Orthoamphisiellidae are considered to be ancestors of Oxytrichidae and Parakahliellidae. The differences in the two studies in the assignment of taxa to the Oxytrichidae are shown by two groups in the cladogram of the present study (Fig. 33): one group (11 - 18) is assigned in both studies to the Oxytrichidae and the other group (34 - 41) is assigned in [5] also to the Oxytrichidae(except for Pattersoniella vitiphila) and in the present study to the Parakahliellidae.

 

Acknowledgements: Supported by The Austrian Science Fund (FWF, Project P11707BIO).

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