Taxonomic and Ecological Implications of Shell Morphology of Three Testaceans (Protozoa: Rhizopoda) in Russia and Canada

Arch. Protistenkd. 145 (1995): 119-126

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Taxonomic and Ecological Implications of Shell Morphology of Three Testaceans (Protozoa: Rhizopoda) in Russia and Canada ANATOLY

A. BOBROV1), SERGEI B. YAZVENK02,3) & BARRY G. WARNER 2)

') Department of Soil Science, Moscow State University, Moscow, Russia;

2) Department of Geography, University of Waterloo, Waterloo, Ontario, Canada; 3) Department of Higher Plants, Faculty of Biology, Moscow State University, Moscow, Russia Summary: Morphological comparisons of shells of three testaceans, Trigonopyxis arcula, Nebela militaris, and Hyalosphenia papilio were made between a population in Russia and in Canada. Morphological differences were noted in the shells of T. arcula sensu lato, while no major differences in shell morphology were found in the latter two species. We suggest that the degree of variability, found in the two geographically isolated populations of T. arcula sensu lato indicates shell polymorphism. Shell polymorphism may complicate traditional taxonomic treatments which depend solely on morphological features of the shell.

Key Words: Testate amoebae; Sphagnum mires; Shell morphology; Taxonomy; Ecology; Russia, Canada.

Introduction The taxonomy of testate amoebae uses morphological features of their shells as a basis for differentiating species. There is great morphological diversity shown by material used in shell construction, size, shape, ornamentation, surface patterns, and features of the pseudostome which are, therefore, used as aids in taxonomy. As part of a much larger research project aimed at understanding the distribution and ecology of the northern peatland testacean fauna, we had an opportunity to compare material from similar ecological settings in two contrasting geographic regions, namely from Russia and Canada. The value of comparing morphological features of the shells from different populations is demonstrated in the paper by LDFTENEGGER et al. (1988) who examined several populations in the region of Austria. The present paper is one of the first detailed intercontinental comparisons. We have selected three of the dominant and taxonomically remote taxa in the two populations under investigation, Trigonopyxis arcula, Nebela militaris, and Hyalosphenia papilio. Further-

more, in attempting to differentiate species based on current taxonomic treatment, we found it to be difficult to do so in the case of T. arcula sensu lato. This paper reports these comparisons and discusses the implications of our findings.

Study Sites The testaceans come from similar types of Sphagnumdominated mires in continental regions of Russia and Canada. The details of the study sites are as follows.

Ust'Bakchar, Tomsk region, West Siberia, Russia

(Sr34' N; 83°6' E): The site is an open forested boreal mire 360 ha in size, dominated by Pinus sylvestris L. with some Picea abies (L.) KARST. This sample was collected from an open treed part of the mire with scattered Pinus sylvestris where hummock and hollow microtopography of Sphagnum is well-developed. Important vascular plants include Chamaedaphne caly-

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culata (less than 1% coverage), Ledum palustre «1 %), Oxycoccus quadripetalus « 1%) and Eriophorum vaginatum (5% coverage). A sample of Sphagnum capillifolium var. capillifolium (90% coverage) and S. magellanicum (5% coverage) was taken midway on the side of a

Table 1. List of the taxa identified in the Siberian and Ontario populations. Values refer to relative proportions of the total.

hummock for testacean analysis. 979 wetland, Experimental Lakes Area, Ontario, Canada (49 0 38' N; 93 0 43' W): This wetland is developed in a small basin, about 9 ha, of which pond occupies 3 ha, with steep granite walls surrounding it except at its inflow and outflow. The site is an open Sphagnum mire sensu JEGLUM et al. (1974) with scattered Larix laricina (DuRol) K. KOCH. The testacean sample was taken from an open area of the fen three meters landward from the central body of open water. The sample was taken in a small depression with Sphagnum angustifolium. S. capillifolium var. capillifolium and S. magellanicum as the dominant species.

Arcella catinus PENARD A. vulgaris EHRENBERG Arcella sp. Bullinularia indica (PENARD) Centropyxis aculeata (EHRENBERG) C. aerophila DEFLANDRE v. sphagnicola DEFLANDRE C. ecornis (EHRENBERG) C. laevigata PENARD C. orbicularis DEFLANDRE C. sylvatica (DEFLANDRE) THOMAS Centropyxis sp. Cyclopyxis arcelloides (LEIDY) C. eurystoma (DEFLANDRE) Trigonopyxis arcula (LEIDY) T. arcula PENARD var. major CHARDEZ T. minuta SCHONBORN Heleopera rosea PENARD H. sphagni (LEIDY) H. sylvatica (PENARD) Hyalosphenia elegans LEIDY H. papilio LEIDY H. subflava CASH & HOPKINSON Nebela bohemica TARANEK N. collaris (EHRENBERG) N. militaris PENARD N. minor PENARD N. parvula CASH N. tincta (LEIDY) Phryganella acropodia (HERTWIG & LESSER) Assulina muscorum GREEFF A. seminulum (EHRENBERG) Euglypha compressa CARTER E. compressa CARTER f. glabra WAILES E. filifera (PENARD) E. laevis (EHRENBERG) E. pseudociliata CHARDEZ E. recurvispina BONNET E. rotunda WAILES E. strigosa (EHRENBERG) E. strigosa (EHRENBERG) f. glabra WAILES Euglypha sp. Placocista spinosa (CARTER) Corythion delamarei BONNET & THOMAS C. dubium TARANEK Trinema complanatum PENARD T. complanatum PENARD v. inaequalis

Methods Collections of living Sphagnum mosses were gathered in the field. Fresh moss samples were washed with water on a tea sieve (0.75 mm diameter). The washed concentrate was left in a beaker overnight before decanting off excess water. Water mounts were made of the concentrate containing testaceans. All measurements and photographs were taken under 400X magnification.

Results and Interpretation A total of 51 species and varieties of testaceans were identified from the two samples (Table 1): Trigonopyxis arcula sensu lato (may include T. minuta and T. arcula var. major), Hyalosphenia papilio, and Nebela militaris were the most important taxa found in both samples. Therefore, it was decided to examine in more detail morphological features of the shells of these three taxa. Fifty shells of each of the three species were measured from either population.

Trigonopyxis arcula sensu lato LEIDY 1879 The shell is circular, and occasionally broadly elliptic in apertural view (Figs. 1, 2a-d). It is hemispherical in the lateral view. The pseudostome is positioned on the apertural (ventral) side and has a thickened lip around the periphery. Its shape varies from being triangular, threelobed, irregular to nearly circular (Fig. 3). The edge of the pseudostome can be wavy to somewhat serrated. The colour of the shell can be yellowish-gold to dark brown, often becoming darker as the shell size increa-

Taxon

Siberia Ontario 4.4 0.3 0.0

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11.9 0.3 7.8 12.1 0.0

0.4 0.4 0.7 0.4 0.0 10.6 0.0 3.6 0.4 0.0 0.0 0.7 21.5 0.7 1.5 0.0 21.2 0.4 8.0 5.1 0.4

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DECLOITRE T. lineare PENARD 0.3 T. lineare PENARD v. truncatum CHARDEZ 0.0 Dijj7.ugiella oviformis 0.0 (PENARD) BONNET & THOMAS Pseudodijj7.ugia gracilis SCHLUMBERGER 0.0 v. terricola BONNET & THOMAS Amphitrema flavum (ARCHER) 6.6

0.4 0.4 0.4 0.7 12.0

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Shell Morphology of Three Testaceans

b Fig. 1. Sketch of Trigonopyxis arcula sensu lato showing the typical shape with xenosomes scattered over the shell. This specimen has a typical trilobed pseudostome. a - apertural (ventral) view; b -lateral view. Scale bar: 50 flm. Table 2. Results of the Kolmogorov-Smirnov test for significance of differences in characters of shell size and pseudostome diameter. Species

Character

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Number of shells

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Size

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50 50 50 50

Siberia Ontario Siberia Ontario Siberia Ontario Siberia Ontario Siberia Ontario Siberia Ontario

Pseudostome Hyalosphenia papilio

Length Width Pseudostome

Nebela militaris

Length Width Pseudostome

ses. The shell is predominantly organic in composition containing scattered large organic and mineral particles (xenosomes) over the shell. These particles are usually more abundant on the dorsal side than on the ventral side. Smaller shells generally lack xenosomes. In order to test the differences between the populations, the Kolmogorov-Smimov test was selected. It is a univariate test that has been shown to be sensitive to any differences between two populations (CAMPBELL 1989). Results of testing suggest that differences between Siberian and Ontario populations in shell size and pseudostome diameter are statistically insignificant (Table 2). However, according to the scatter diagram (Fig. 4),

Standard deviation

P

93.42 85.82 20.78 16.84

27.29 20.38 6.67 5.24

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98.14 103.18 65.62 67.90 27.80 29.56

8.60 7.58 4.70 5.08 1.72 2.89

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70.86 72.26 41.74 39.84 17.50 17.64

4.45 3.50 4.77 3.44 1.41 1.22

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>.05 <.01 (*) >.05 <.001 (**) >.05 <.001 (**) >.05

the two populations do exhibit differences. In the Ontario population shell size and pseudostome diameter are tightly correlated (r = 0.941); the smaller shells have smaller pseudostome diameters and the larger shells have larger pseudostome diameters. On the contrary, in the Siberian population such a relation is virtually lacking (r 0.033). This may indicate that the Ontario population is more homogeneous than the Siberian population. Another important observation is the degree of variability in the shape of the pseudostome in both populations (Fig. 3). The edge of the pseudostome varies from being smooth, slightly wavy, to somewhat serrated.

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Fig. 3. Sketches of the range of shapes in pseudostome of T. arcula sensu lato.

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Fig. 2. Micrographs of Trigonopyxis arcula sensu lato (a-d), Nebela militaris (e-f), and Hyalosphenia papilio (g-h). Scale bar: 20 !Jm.

In broad lateral view, the shell is pyriform or narrowly pyriform with a convex pseudostome (Figs. 2e-f, 5). The pseudostome is surrounded by a narrow organic collar. There is only a slight restriction towards the pseudostome in narrow lateral view. In apertural view, the shell is elliptic in shape with the pseudostome appearing as an elongated slit. A small pore is present on each side of the lower third of the shell. The pores are poorly visible in light field but are obvious under SEM (OGDEN & HEDLEY 1980). The shell is covered by a mixture of oval and circular shell plates (idiosomes) of different sizes. The patterns are quite variable and some distinct types are recognized in our material (Fig. 6). Occasionally, the shells lack idiosomes. Measurements of shell length showed no statistically significant difference between the Siberian and Ontario populations (Table 2), nor were any differences noted in the diameters of the pseudostome, whereas shell width is significantly different. A scatter diagram and results of correspondence analysis (TER BRAAK 1988) indicate, however, that the two populations are essentially similar and each of them is homogeneous (Fig. 7a-b).

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Fig. 4. Scatter diagram of shell diameter vs. pseudostome diameter in Trigonopyxis arcula, the populations from Siberia (.) and Ontario (0).

Discussion This study is one of the first attempts to compare shell morphology in two geographically isolated populations

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Sample size Source

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95-168 93.7-112.5 85-120 38-194

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of similar species in the northern hemisphere. The shells of Trigonopyxis arcula sensu lato showed that the two populations are different in the way the characters are correlated but they are similar in the overall range of variation. The wide range in shell size may relate to the existence of different species or varieties (Table 3). Shells larger than 140 11m have been treated as T. arcula var. major CHARDEZ (CHARDEZ 1960). At the other extreme are the small shells. Shells less than 70 11m have been described as Trigonopyxis minuta (SCHONBORN & PESCHKE 1988). Both of our populations are quite similar in containing significant numbers of shells attributed to T. minuta.

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Differences in the pseudostome are noteworthy. The pseudostome of both T. arcula var. major and T. minuta are described as being serrated, whereas it is smooth in T. arcula (SCHONBORN & PESCHKE 1988). We observed serrated pseudostomes in both large and small shells in our material. The shape of the pseudostome is quite variable (OGDEN & HEDLEY 1980). T. minuta is described as having an irregular pseudostome in contrast to the regular triangle or trilobed pseudostome of T. arcula

Shell Morphology of Three Testaceans

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Fig. 9b. Correspondence analysis of shells in H. papilio, the populations from Siberia (.) and Ontario (0).

(SCHONBORN & PESCHKE 1988). However, all the small shells which we observed had regular triangular pseudostome with slightly wavy edges. We would suggest, based on these observations, that patterns of the edge of the pseudostome are not good characteristics for differentiating species or varieties of Trigonopyxis. The size range given in the taxonomic literature to separate taxa of T. arcula sensu lato requires further consideration. Our data give little-support for recognizing different species within the T. arcula complex. T. minuta and T. arcula var. major may represent morphological variations within this large polymorphic complex. Therefore, it may be more reasonable to treat T. arcula sensu lato as a single polymorphic species until a more comprehensive study is accomplished. The concept of shell polymorphism and major differences in shell morphology within populations and between them has long been known within the Rhizopoda. In Trigonopyxis arcula a significant variability of shell diameter was reported (HOOGENRAAD & DE GROOT 1937, 1952). Recent discussions on the topic have appeared in the literature, namely the work of SCHONBORN (1990, 1992), who concluded that genetic links seem to be an important mechanism for shell polymorphism. It is possible also, that some environmental control associated with microhabitat may be responsible for the variability and distribution of Trigonopyxis arcula sensu lato. Reliance on shell morphology alone in differentiating species should be accepted with some caution. We

believe that scrupulous morphological, ecological and genetic studies should be undertaken on the same material to improve our understanding of the nature of species and infraspecific structure in testaceans. It has long been believed that the Rhizopoda, in general, are cosmopolitan in distribution, or at the very least, comprise a, fauna of the northern hemisphere separate from the southern hemisphere (HOOGENRAAD & DE GROOT 1979). We have found statistically significant differences in some key morphological characters of the three testaceans which we studied. However, such subtle size differences may be insufficient to treat populations as separate taxa. This view differs somewhat from that of SCHONBORN et a1. (1983) who feel that such differences, if statistically significant, do have taxonomic value. With respect to Trigonopyxis arcula (Fig. 4), contrasting interrelations between the shell size and pseudostome diameter have been found in the Siberian and Ontario populations. We can speculate that this fact might indicate greater genetic heterogeneity in the Siberian population. The importance of environmental factors on the distribution of testaceans has been found in our ongoing work on the ecology of testaceans. In comparing Fennoscandian and Canadian faunas, clear ecological dissimilarities in microhabitat have been found for the same apparent species (TOLONEN et a1. 1992; CHARMAN & WARNER 1992). BEYENS et a1. (1990) found similar differences in the Arctic faunas.

126

A. A. BOBROV et aI.

Conclusions 1. Detailed morphological comparison of shell size characteristics of two geographically isolated populations of testaceans revealed statistically significant differences between the Siberian and Ontario populations of Nebela militaris and Hyalosphenia papilio, but not

Trigonopyxis arcula. 2. However, these differences seem insufficient for the taxonomic treatment and may more reflect geographic and ecologic, rather than taxonomic variation. Caution should be exercised in establishing taxa which are based on statistically significant differences in size. 3. Taxonomy based on shell morphology for identifying species and varieties in T. arcula s.l. is probably incomplete, and T. arcula may be better regarded as a single polymorphic complex rather than as two or more distinct species. 4. Although it is believed that testate amoebae are cosmopolitan in distribution, we have found that morphological differences may exist for some species in geographically isolated parts of their geographic range. Acknowledgements: We thank Prof. Jv. GELTZER and Prof. V. TIKHOMIROV for encouraging this study, Dr. D. ALEXEEv for helpful discussion on taxonomy, and Dr. L. INISHEVA for providing the material from Tomsk. This work was supported by the Russian Fund for Fundamental Research and the Natural Sciences and Engineering Research Council of Canada.

References BEYENS, L., CHARDEZ, D. & DE BAERE, D. (1990): Ecology of terrestrial testate amoebae from coastal lowlands on Devon Island (NWT, Canadian Arctic). Polar BioI. 10: 431-440. BONNET, L. & THOMAS, R. (1960): Fauna terrestris et d'eau douce des Pyrenees-Orientales. Thecamoebiens du sol. Paris. CHARDEZ, D. (1960): Introduction ill etude des Thecamoebiens du sol. Bull. Inst. agron. et Stat. Rech Gembloux. 28: 118-132. CAMPBELL, R. C. (1989): Statistics for Biologists. 3rd ed. Cambridge. CHARMAN, D. 1. & WARNER, B. G. (1992): Relationship between testate amoebae (Protozoa: Rhizopoda) and microenvironmental parameters on a forested peatland in northeastern Ontario. Can. J. Zool. 70: 2474-2482. HOOGENRAAD, H. R. & DE GROOT, A. A. (1937): Biometri-

sche Untersuchungen an SiiBwasserrhizopoden. Arch. Hydrobiol. 31: 101-132. - - (1952): Thekamobe Moosrhizopoden aus Asien. Arch. Hydrobiol. 47: 263-287. - - (1979): Die geographische Verbreitung der Siisswasser-Rhizopoden. Hydrobiol. Bull. 13: 152-171. JEGLVM, J. K., BOISSONNEAV, A. N. & HAAVISTO, V. F. (1974): Towards a wetland classification for Ontario. Dep. Environment, Canadian Forest Service, Sault Ste. Marie, Ont. Inf. Rep. 0-X-215. LEIDY, J. (1879): Freshwater Rhizopods of North America. In: United States Geological Survey of the Territories. Vol. 12, Washington, 324 pp. LUFrENEGGER, G., PETZ, W., BERGER, H., FOISSNER, W. & ADAM, H. (1988): Morphologic and biometric characterization of twenty-four soil testate amoebae (Protozoa, Rhizopoda). Arch. Protistenkd. 136: 153-189. OGDEN, C. G. & HEDLEY, R. H. (1980): An atlas of freshwater testate amoebae. Oxford. SCHONBORN, W. (1990): Shell polymorphism and habitat structure in Testacea (Rhizopoda). J. Protozool. 37: 62A. - (1992): Adaptive polymorphism in soil-inhabiting testate amoebae (Rhizopoda): Its importance for delimitation and evolution of asexual species. Arch. Protistenkd. 142: 139-155. - & PESCHKE, T. (1988): Biometric studies on species, races, ecophenotypes and individual variations of soilinhabiting Testacea (Protozoa: Rhizopoda), including Trigonopyxis minuta n. sp. and Corythion asperulum n. sp. Arch. Protistenkd. 136: 345-363. - FOISSNER, W. & MEISTERFELD, R. (1983): Licht- und rasterelektronenmikroskopische Untersuchungen zur Schalenmorphologie und Rassenbildung bodenbewohnender Testaceen (Protozoa: Rhizopoden) sowie VorschHige zur biometrischen Charakterisierung von Testaceen-Scha1en. Protistologica 19: 553-566. TER BRAAK, C. 1. F. (1988): CANOCO - a FORTRAN program for canonical community ordination by [partial] [detrended] [canonical] correspondence analysis, principal components analysis and redundancy analysis (version 2.1). Agricultural Mathematics Group, Wageningen. TOLONEN, K., WARNER, B. G. & VASANDER, H. (1992): Ecology of testaceans (Protozoa: Rhizopoda) in mires in southern Finland. I. Autecology. Arch. Protistenkd. 142: 119-138. VOLZ, P. (1929): Studien zur Biologie der bodenbewohnenden Thekamoben. Arch. Protistenkd. 68: 349-408. Accepted: July 15, 1994 Corresponding author: Dr. SERGEI B. YAZVENKO, University of Waterloo, Department of Geography, Isaiah Bowman Building, Waterloo, Ontario, Canada N2L 3Gl.