First record of a size-trimorphic population among euglyphid testate amoebae Текст научной статьи по специальности «Биологические науки»

Protistology 10 (4), 138-147 (2016)

Protistology

First record of a size-trimorphic population among euglyphid testate amoebae

Stefan Luketa

University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Novi Sad, Serbia

| Submitted December 9, 2016 | Accepted December 27, 2016 |

Summary

Morphology and morphometry of moss-dwelling population of Trinema penardi Thomas et Chardez, 1958 from the Island of Rab (Croatia) were investigated based on 196 specimens. Morphometric analysis showed that this population comprises three size-classes: small (shell length 58-61 ^m, shell width 33-34 ^m, aperture width 15-16 ^m), medium (shell length 66-87 ^m, shell width 36-51 ^m, aperture width 17-24 ^m), and large (shell length 87-105 ^m, shell width 49-64 ^m, aperture width 23-29 ^m). Specimens with shell length between 30 and 65 ^m (corresponding to small-size class) were reported earlier, whereas medium and large specimens were described for the first time in the present study. These data show that population of T. penardi from the Island of Rab can be considered as an example of very variable population characterized by discontinuous polymorphism.

Key words: biometry, morphometry, polymorphism, protists, taxonomy, testate amoebae

Introduction

Testate amoebae are free-living predominantly asexual protists characterized by great morphological variability. Luftenegger with co-authors (1988) explained significant intraspecific differences of testate amoebae as results ofpresence of"eco-races" or "geographic variations". Bobrov and Mazei (2004) established three main types ofsize variability in testate amoebae populations: (a) monomorphic populations, (b) polymorphic populations with correlated characters, and (c) populations with high phenotypic plasticity, non-correlated characters and the elements of discreteness. Polymorphism is simultaneous presence of several phenotypes in a population or interrupted diversity of forms

on the common genetic base. In the cases of continuous polymorphism, it is not possible to distinguish two or more clearly defined size classes within a population. This phenomenon is present in several testate amoebae species: Trinema compla-natum (Schonborn and Peschke, 1988; Schonborn, 1992), Euglypha rotunda (Schonborn and Peschke, 1988), Corythionellaminima, C.pontica(Goleman-sky and Todorov, 2006) and Nebela golemanskyi (Todorov, 2010; Longinebelagolemanskyi according to Kosakyan et al., 2016). To the best of my knowledge, only two examples of size discontinuous polymorphism were described among testate amoebae: Centropyxis plagiostoma (Foissner and Korganova, 1995) and Apodera vas (Zapata and Fernández, 2008).

doi:10.21685/1680-0826-2016-10-4-3 © 2016 The Author(s)

Protistology © 2016 Protozoological Society Affiliated with RAS

Euglyphid testate amoebae have a worldwide distribution and they are common in the same habitats as the arcellinid testate amoebae. Their shells are composed of secreted siliceous plates that vary in shape, size and arrangement from one genus to another and from one species to another (Meisterfeld, 2002). The family Trinematidae is a well-studied group of euglyphid testate amoebae characterized by bilateral symmetry and an eccentric, often invaginated aperture. Taxonomy at the species level within this family is based on different types of characters within different genera. Specific situation is within the genus Trinema because here morphometric data are very important for species delimitation. For example, T. lineare, T. enchelys and T. grandis have very similar morphology, but shell length is the key character for distinguishing these species.

During my visit to the Island of Rab (Croatia), I collected moss samples and found shells of euglyphid testate amoeba that morphologically corresponded to T. penardi Thomas et Chardez, 1958. However, only several shells morphometrically corresponded to this species. Other shells were longer and I concluded that the studied population was size-polymorphic. In the present paper I report morphology and morphometric analysis of this specific population.

Material and methods

The material for the present study was extracted from epigenous mosses collected in the forest Dundo on 10 July 2013. This forest is located in the Kalifront Peninsula (44°46'29.8"N, 14°42'45.7"E, ca. 50 m a.s.l.), the Island of Rab, Croatia. The Kalifront Peninsula is characterized by karst relief and forests with holm oak (Quercus ilex) as the dominant species. Morphological characters and morphometric variables were studied using a light microscope Zeiss Axio Imager A1. Images were captured using an AxioCam MRc5 (Zeiss) digital color camera. Measurements were conducted in the program AxioVision 4.9.1. The following measurements were taken for the 196 shells: shell length, shell width, aperture width, aperture length and rim width (Fig. 1). The following descriptive statistics were calculated: extreme values (minimum and maximum), median, arithmetic mean, standard error of the arithmetic mean, standard deviation, coefficient ofvariation in %, skewness and kurtosis.

Statistical analysis was conducted using the programs PAST 2.17c and STATISTICA 13.0.

Results

Description of the species

Shell is transparent, colorless, hyaline-ovoid in broad lateral view, in narrow lateral view pointed towards an aperture. Shell surface on the fundus is covered with bigger circular plates of various sizes with smaller oval plates between them, while anterior region is covered only with oval plates. Oval aperture is located in the invagination of a forward ventral part; the aperture border is surrounded by fine dents. Aperture rim is the region bordered by the anterior margin of the shell and one distinct curved line running from aperture to the side of shell. Due to presence of this line, the shell is relatively clearly divided into two parts: anterior (apertural) region and fundus. In the population it is possible to distinguish three size classes, but they do not differ morphologically (Fig. 2) and are thus described together.

Morphometry

Figure 3 shows scatter plot analysis of the correlation between shell length and shell width, indicating presence of three size classes (small, medium and large) without intermediate specimens. Morphometric characters of 196 specimens from the Island of Rab were measured and the results are given in Table 1. Only four specimens are included in small class, while medium and large classes include higher number of specimens. Therefore, only the detailed analyses of the medium and large classes are presented.

Medium class. Coefficients of variation were moderately low for all measured characters (from 4.80% to 9.31%). For basic characters, minimal variability was observed for shell length (4.80%), while the maximal variation coefficient was observed for rim width (8.39%). For ratio characters, minimal variability was observed for aperture width/shell width ratio (5.98%), while the maximal variation coefficient was observed for aperture length/shell width ratio (9.31%).

The most frequent shell length (74 ^m) was registered in 19 specimens (Fig. 4A); the most frequent shell width (43 ^m) was registered in 39 specimens (Fig. 4B), and the most frequent aperture

Fig. 1. Shell outline of Trinema penardi and position of the measured axes. A — shell length; B — shell width; C — aperture width; D — aperture length; E — rim width.

width (21 ^m) was registered in 42 specimens (Fig. 4C). Histogram analysis revealed nearly the same regularity with respect to shell length, shell width and aperture width distribution. All measured specimens had shell length between 66 and 87 ^m. In this case, 64.47% of all specimens had shell width of 73-78 ^m, whereas only 14.47% were smaller than 73 ^m and only 21.05% were larger than 78 ^m. The histograms for shell width and aperture width show similar regularity in size distribution. For example, all measured specimens had shell width between 36 and 51 ^m. In this case, 79.61% of all the specimens had shell width of 41-45 ^m, whereas only 9.21% were narrower than 41 ^m and only 11.18% were wider than 45 ^m. Also, aperture widths ranged from 17 to 24 ^m, but 78.29% of all measured specimens had aperture width of 19-21 ^m, whereas only 21.71% had an aperture narrower than 19 ^m and wider than 21 ^m.

The negative values of skewness for aperture length, rim width and aperture width/shell width ratio suggest an asymmetrical distribution with

a long tail toward lower values. The asymmetry of aperture length (-0.081) and aperture width/ shell width ratio (-0.093) was low, while moderate negative value was observed for rim width (-0.440). High negative values of skewness were not observed for any characters. Aperture width, aperture length/ shell width ratio and aperture length/aperture width ratio displayed low positive skewness values (0.012-0.150), while moderate positive values (0.261-0.343) were observed for shell length, shell width and aperture width/shell length ratio; high positive values (0.539-0.802) were observed for shell width/shell length ratio and aperture length/ shell length ratio. Three characters (aperture width/shell length ratio, aperture width/shell width ratio and aperture length/aperture width ratio) displayed negative kurtosis values (between -0.372 and -0.544), meaning that they were characterized by flatter distribution than a standard Gaussian distribution. This information indicates that the average size group has a higher dispersion. Other variables were found to have positive kurtosis values, indicating a distribution which is sharper than a standard Gaussian distribution. Low positive values (0.009-0.183) were observed for shell length, aperture width, aperture length and aperture length/ shell width ratio. A moderate positive value (0.474) was observed for rim width, while the high positive values (1.155-3.803) were observed for shell width, shell width/shell length ratio and aperture length/ shell length ratio.

Large class. Coefficients of variation were moderately low for all measured characters and ranged from 4.54% to 10.69%. For basic characters, minimal variability was observed for shell length (4.54%), while the maximal variation coefficient was observed for aperture length (9.54%). For ratio characters, minimal variability was observed for aperture width/shell width (5.43%), while the maximal variation coefficient was observed for aperture length/aperture width (10.69%).

The most frequent shell length (93 and 98 ^m) was registered in 6 specimens (Fig. 4D); the most frequent shell width (58 ^m) was registered in 8 specimens (Fig. 4E), and the most frequent aperture width (26 and 27 ^m) was registered in 9 specimens (Fig. 4F). Analysis of the size frequency distribution of shell length, shell width and aperture width indicates that this morphometric class possesses the continuous polymorphism. All measured specimens had shell length between 87 and 105 ^m. In this case, 60% of all specimens had shell width of 93-

Fig. 2. Light micrographs of Trinema penardi from the Island of Rab (Croatia) — broad lateral view of different specimens showing general shell shape and outline. A — small specimens; B — medium specimens; C — large specimens. Scale bar: 20 ^m.

Table 1. Morphometric characterization of Trinema penardi from the Island of Rab (Croatia) based on 196 specimens (small class 4 specimens, medium class 152 specimens, large class 40 specimens). Measurements are in |jm.

Characters Classes Min Max M x SE SD CV Sk Ku

Shell length small 58 61 60 59.75 - - - - -

medium 66 87 76 75.97 0.30 3.64 4.80 0.261 0.183

large 87 105 97.5 96.55 0.69 4.38 4.54 -0.051 -0.690

Shell width small 33 34 34 33.75 - - - - -

medium 36 51 43 42.95 0.17 2.11 4.91 0.340 1.155

large 49 64 57 57.13 0.49 3.07 5.38 0.027 0.575

Aperture width small 15 16 15 15.25 - - - - -

medium 17 24 20 20.32 0.11 1.30 6.42 0.012 0.121

large 23 29 26 26.15 0.27 1.72 6.57 -0.021 -0.626

Aperture length small 10 12 11.5 11.25 - - - - -

medium 12 19 15 15.26 0.10 1.27 8.31 -0.081 0.009

large 15 23 19 19.25 0.29 1.84 9.54 -0.154 -0.247

Rim width small 7 7 7 7 - - - - -

medium 6 9 8 7.76 0.05 0.65 8.39 -0.440 0.474

large 8 11 10 9.83 0.13 0.84 8.59 -0.189 -0.597

Shell width/shell length small 0.54 0.59 0.57 0.57 - - - - -

medium 0.48 0.71 0.56 0.57 0.00 0.03 6.16 0.539 1.301

large 0.50 0.67 0.59 0.59 0.01 0.04 6.16 -0.386 0.250

Aperture width/shell length small 0.25 0.28 0.25 0.26 - - - - -

medium 0.23 0.32 0.27 0.27 0.00 0.02 7.50 0.343 -0.372

large 0.23 0.31 0.31 0.27 0.00 0.02 6.93 -0.240 -0.186

Aperture length/shell length small 0.17 0.20 0.19 0.19 - - - - -

medium 0.16 0.29 0.20 0.20 0.00 0.02 8.78 0.802 3.803

large 0.17 0.24 0.20 0.20 0.00 0.02 8.64 0.433 -0.320

Aperture width/shell width small 0.44 0.47 0.45 0.45 - - - - -

medium 0.40 0.54 0.47 0.47 0.00 0.03 5.98 -0.093 -0.419

large 0.39 0.50 0.46 0.46 0.00 0.02 5.43 -0.780 0.373

Aperture length/shell width small 0.29 0.36 0.34 0.33 - - - - -

medium 0.27 0.45 0.35 0.36 0.00 0.03 9.31 0.150 0.014

large 0.28 0.40 0.35 0.34 0.01 0.03 9.94 -0.058 -1.006

Aperture length/aperture width small 0.67 0.80 0.74 0.74 - - - - -

medium 0.62 0.90 0.75 0.75 0.01 0.07 8.81 0.015 -0.544

large 0.59 0.96 0.75 0.74 0.01 0.08 10.69 0.381 0.745

Abbreviations: Min and Max - minimum and maximum values, M - median, x - arithmetic mean, SE - standard error of the arithmetic mean, SD - standard deviation, CV - coefficient of variation in %, Sk - skewness, Ku - kurtosis.

Fig. 3. Scatter plot shows the correlation between shell length and shell width of 196 specimens of Trinema penardi from the Island of Rab, Croatia. Legend: ▲ — small specimens, • — medium specimens, ■ — large specimens.

99 ^m, whereas only 17.5% were smaller than 93 ^m and only 22.5% were larger than 99 ^m. The histograms for shell width and aperture width show similar regularity in the distribution. All measured specimens had shell width between 49 and 64 ^m. In this case, 65% ofall the specimens had shell width of 55-58 ^m, whereas only 12.5% were narrower than 55 ^m and only 22.5% were wider than 58 ^m. Also, aperture width ranged from 23 to 29 ^m, but 62.5% of all measured specimens had an aperture width of 25-27 ^m, whereas only 37.5% had an aperture narrower than 25 ^m and wider than 27 ^m.

The positive value of skewness for shell width, aperture length/shell length ratio and aperture length/aperture width ratio suggests an asymmetrical distribution with a long tail toward higher values. The asymmetry of shell width was low, with skewness value of 0.027. High positive values of skewness were observed for aperture length/aperture width ratio (0.381) and aperture length/shell length (0.433). All other variables were characterized by negative values of skewness (between -0.021 and -0.780). Four characters (shell width, shell width/ shell length ratio, aperture width/shell width ratio and aperture length/aperture width ratio) displayed

positive kurtosis values (between 0.250 and 0.745), indicating a distribution which is sharper than a standard Gaussian distribution. All other variables were characterized by negative values of kurtosis (between -0.186 and -1.006), meaning that they were characterized by flatter distribution than a standard Gaussian distribution.

Discussion

In the literature, there are some examples of size-polymorphic testate amoebae species. Todorov (2010) described Nebela golemanskyi and noted that this species is the size-polymorphic one. However, histograms that show the size frequency distribution were not provided. Golemansky and Todorov (2006) based on the analysis of size frequency distribution concluded that Corythionella minima Golemansky, 1970 and C. pontica Golemansky, 1970 are the size-polymorphic species. They noted that C. minima were characterized by increasing size range (shell length 44-60 ^m, shell width 18-27 ^m) and by reduced main size-class in favor of subsidiary classes. In addition, arithmetic means of shell

Fig. 4. Histograms created based on measured specimens of Trinema penardi from the Island of Rab, Croatia. A-C — size frequency distribution of shell length, shell width and aperture width of 152 medium specimens; D-F — size frequency distribution of shell length, shell width and aperture width of 40 large specimens.

length and shell width do not agree with the main size classes and testify to the polymorphism of the species. These authors concluded that C. pontica is a size-polymorphic species by the shell length and a size-monomorphic species by the shell width. Also, the curve of the size classes shows one well-expressed main size class (100-102 pm) and another, not well-expressed subsidiary size class (90-92 pm). All of these cases are characterized by continuous polymorphism because it is not possible to distinguish two or more clearly defined size classes. Golemansky and Todorov (2004) presented morphometric study of eight marine interstitial testate amoebae. Few of these species showed similar size frequency of the shell length and/or shell width as C. minima and C. pontica, but the authors interpreted these cases as size-monomorphic species. This example shows that category of continuous polymorphism is very subjective. It is possible that cases of continuous polymorphic populations are examples oflow action of disruptive selection.

Foissner and Korganova (1995) based on morphometric characterization of Centropyxis pla-giostoma Bonnet et Thomas, 1955 concluded that this species is size-polymorphic. They distinguished two size classes: small (shell length 50-70 pm) and large (shell length 80-120 pm). Zapata and Fernández (2008) described the morphometric characteristics of Apodera vas (Certes, 1889) Loeblich et Tappan, 1961 from two peatlands in southern Chile. This analysis indicates that A. vas is size-polymorphic species with two to three size classes in each population. These size classes are well defined because intermediate specimens are not present. The cases of C. plagiostoma and A. vas are examples of discontinuous polymorphism because it is possible to distinguish two or three clearly defined size classes without intermediate specimens.

Schonborn (1992) described morphological variability of soil-dwelling population of Trinema complanatum Penard, 1890 based on large number of specimens from Germany. The author observed two morphologically clearly delineated morphs: rounded and waisted. In addition, shells of the rounded morph can be divided into two size classes: small (shell length 21.9-31.2 pm) and large (shell length 34.4-50.0 pm). This author noted that the morphs can be maintained by selection; therefore, their shape may have an adaptive significance. Based on experiments with clonal cultures he concluded: "The morphs can be kept relatively constant in clonal cultures. But in the course oftime individuals

occur with features of the other morphs. These, again selected, also remained constant for a certain time. But the morphs tend, to a different degree, to produce other forms. So, the waisted morph only rarely produced rounded shells. It was not possible to select another morph from this morph. Large-rounded forms frequently produced waisted shells. Their progeny were kept in clonal cultures and produced also only few rounded shells. In nature the converse way also may occur: selection of rounded shells from waisted forms. The small-rounded morph is very constant and the large-rounded morph produced only few small-shelled individuals. In the nature it may be probably that the inverted way is also possible. Generally, in Trinema complanatum the shell-size seems to be to select difficulty, because here may occur a non-genetic, modificatoric size-span" (Schonborn, 1992, p. 152).

The discontinuous polymorphism is the result of strong action of disruptive selection. However, experiments with clone cultures conducted by Schonborn (1992) indicated that disruptive selection must not necessarily lead to speciation. In addition, Schonborn and Peschke (1988) concluded that "a visible discontinuous polymorphism is less suitable to produce local races because its genetically fixed size spectrum can be run through quickly and therefore extinguish a local difference". For these reasons, further studies are needed to clarify taxonomic status of three morphologically similar, but morphometrically different species: T. lineare, T. enchelysand T. grandis. Luftenegger et al. (1988) delimited T. lineare and T. enchelys based on shell length (24-41 pm versus 41-52 pm, respectively). In addition, Chattopadhyay and Das (2003) based on many sympatric populations of these species concluded that they are only morphometrically delineated, but with intermediate specimens (shell length 18.78-40.69 pm versus 37.56-81.38 pm, respectively). Third species with similar morphology is T. grandis, but shell length of this species varies from 100 to 125 pm (Mazei and Tsyganov, 2006). It is possible that these three species are size classes of the same size-trimorphic species.

Thomas and Chardez (1958) noted the following measurements for Trinemapenardi: shell length 4255 pm, shell width 20-30 pm and aperture width 1015 pm. There are several notes and brief descriptions of T. penardi available in the later literature. Luftenegger et al. (1988) based on 26 specimens from Austria recorded shell lengths of 30-65 pm, shell widths 16-35 pm, aperture widths 8-16 pm, aperture

heights 5-13 pm and apertural rim widths 5-8 pm. Alekperov and Snegovaya (2000) noted shell lengths 30-50 pm, shell widths 25-30 pm and aperture widths 12-16 pm for a population collected from the Apsheron Peninsula, Azerbaijan. Chattopadhyay and Das (2003) measured morphometric characters of 16 moss-dwelling specimens from Uttarakhand (India) and recorded the following measurements: shell length 43.82-46.95 pm, shell width 28.17-31.30 pm, aperture width 12.52-14.09 pm, aperture height 10.95-14.09 pm and apertural rim width 6.26 pm. All mentioned authors reported similar morphometric values for this species. After summarizing these data, I have concluded that T. penardi is characterized by the following measurements: shell length 30-65 pm, shell width 16-35 pm, aperture width 8-16 pm, aperture height 5-14 pm and aperture rim width 5-8 pm. Measurements of small class from the Island of Rab correspond more or less to these values. To the best ofmy knowledge, medium and large size classes of T. penardi were not described to date. These data show that population of T. penardi from the Island of Rab can be considered as an example of very variable population characterized by discontinuous polymorphism.

Acknowledgments

I am very grateful to Dr. Laszlo Barsi (University of Novi Sad, Serbia) for permission to use the Zeiss Axio Imager A1 light microscope, to the Referee (Y.M.) for the constructive comments and to the Editor for English editing.

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Address for correspondence: Stefan Luketa. Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, 21000 Novi Sad, Serbia; e-mail: stefan.luketa@dbe.uns. ac.rs