Are stoneworts (Characeae) clonal plants?

Aquatic Botany 100 (2012) 25–34

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Are stoneworts (Characeae) clonal plants? ˛ ∗ , Emilia Rekowska Katarzyna Bociag Department of Plant Ecology, University of Gda´ nsk, Al. Legionów 9, PL 80-441 Gda´ nsk, Poland

a r t i c l e

i n f o

Article history: Received 11 March 2011 Received in revised form 23 February 2012 Accepted 6 March 2012 Available online 16 March 2012 Keywords: Chara Characeae Clonality Architecture of individuals Growth strategy

a b s t r a c t Two questions have been posed: whether the concept of clonality may be applied to charophytes (Characeae) and whether species of the genus Chara differ in the features connected with clonal life strategy. The following criteria of clonality were established: 1/an iterative growth form characterised by a horizontal (above- or underground) axis and vertical (orthotropic) above-ground axes in the architecture of an individual, and 2/the ability of the upright axes to function independently and to regenerate the thallus. We determined the architecture of individuals as well as the ability of the vertical thallus fragments to continue growing and developing after separation from the parent thallus in Chara aspera, Chara globularis, Chara rudis and Chara tomentosa. It was found that both single-axis individuals and those made up of several (mostly 2–3) upright axes (branches) growing from the main horizontal axis occur in the populations of each of these species. The orthotropic axis of the thallus represents a module, a recurring structural unit. The modules, having been separated from the parent thalli, extend to form new vertical branches. However, the strategies of the species discussed in this paper vary in correspondence with differences in habitat. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Clonal plants are characterised by an iterative (recurring) structure. Their individuals consist of similar structural units (modules, ramets). The following basic features differentiate clonal plants from aclonal ones: 1/the iterative growth form and 2/the potential capability of the structural units forming them (modules, ramets) to function completely independently (Harper, 1977, 1985; Harper and White, 1974; Van Groenendael and De Kroon, 1990; De Kroon and Van Groenendael, 1997). Only recently have algologists begun to examine the concept of clonality (Santelices, 1992; Santelices et al., 1995). Studies on clonality of macroalgae concern marine brown, red and green algae (op. cit., Santelices, 2001, 2004; Collado-Vides, 2002). In these works, species growing by multiple repetition of similar units (modules), which survive and function when they become separated from the parent form, are regarded as clonal. Unlike them, aclonal species exhibit primarily vertical growth, and their branches are mostly incapable of survival, functioning or reproduction when separated from the parent form. Inspired by works concerning marine algae written by Santelices (2001, 2004) and Collado-Vides (2002), we have decided to survey clonality as a type of life strategy in stoneworts (Characeae), a group of freshwater macroalgae in the division Chlorophyta.

∗ Corresponding author. Tel.: +48 583412016; fax: +48 583412016. ˛ E-mail address: [email protected] (K. Bociag). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2012.03.007

Stoneworts are thallophytes (Krause, 1997; Lee, 1999), but are more similar to vascular plants in terms of architecture than other macroalgae. Their thallus consists of an upright or prostrate, often branched axis divided into nodes and internodes and forming branchlets and generative organs. Some species also have holdfasts which attach their thalli to the substrate. The prostrate parts of the stipe itself also anchor the plant. Stoneworts reproduce sexually and vegetatively (at least some species). Vegetative propagation occurs through bulbils (Krause, 1997), and thallus fragments (Combroux et al., 2001; Fernández-Aláez et al., 2002; Havens et al., ´ ˛ 2011). and Bociag, 2004; De Winton et al., 2004; Skurzynski The aim of this paper is to check whether a selected charophyte species meet the clonality criteria defined in literature. The application of the clonality of stoneworts could improve our interpretation of observed patterns and test existing and new hypotheses, just as in marine macroalgae (Santelices, 2004; Collado-Vides, 2002) and submerged vascular plants (e.g. Idestam-Almquist and Kautsky, 1995; Vermaat and Verhagen, 1996; Marbà and Duarte, 1998; Wolfer and Straile, 2004; Sintes et al., 2005; Wolfer et al., 2006). Clonality as a life strategy is beneficial in spatially heterogenous environments (Hutchings and Wijesinghe, 1997; Marbà and Duarte, 1998), in undisturbed habitats (Klimes et al., 1997), but also in extremely disturbed areas (Fahrig et al., 1994). Clonal submerged angiosperms and clonal marinae macroalgae occur both at strongly disturbed sites, such as shallow shore zones of seas and oceans, and in more deeply located habitats, undisturbed as they are, but with limited light availability, on different substrates. Under other environmental conditions different features related to clonality are

˛ E. Rekowska / Aquatic Botany 100 (2012) 25–34 K. Bociag,

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Table 1 Lake location and depth range, where the individuals were collected. * Populations from which individuals were collected for the experiment. Lake

Location

Białe

54◦ 22 N 18◦ 11 E 54◦ 5 N 17◦ 51 E 54◦ 14 N 17◦ 58 E 54◦ 17 N 18◦ 2 E 53◦ 53 N 18◦ 11 E 54◦ 14 N 17◦ 32 E 54◦ 1 N 17◦ 19 E 53◦ 58 N 17◦ 16 E 54◦ 2 N 17◦ 21 E 53◦ 55 N 17◦ 3 E 54◦ 4 N 17◦ 52 E 53◦ 47 N 17◦ 35 E Median Mean ± SD Min–Max

Depth [m] Chara aspera

Płocice Wielkie ´ Górne Radunskie ´ Radunskie Dolne Gardliczno ˙ ´ Zuromi nskie Piaszno Długie Borzyszkowskie Dymno Sominko Ostrowite General

C. globularis

C. rudis

C. tomentosa

1.2–5.0* 1.5–3.5

0.2–2.0

0.5–3.0 0.5–3.0 0.05–0.5

4.5

0.7–0.8 0.2–0.5

0.7–3.5*

1.0–6.0*

0.3–0.5

0.3–3.5

2.0–6.0

0.7–5.0

0.3–1.0

0.5–3.0

0.2–0.5 0.3–0.5

0.2–0.5*

0.7–5.0 0.4 0.38 ± 0.17 0.05–0.8

favourable. A large number of modules forming an individual and short spacers between them are beneficial features of clonal architecture, according to the foraging theory, in nutrient-rich habitats (Hutchings and De Kroon, 1994), but also in wave-exposed ones (Collado-Vides, 2002). Vegetative propagation and occupation of space by producing modules is a strategy which proves especially worthwhile in shaded conditions, where the light deficiency limit the colonization rate of the new spaces by generative propagation (Santamaria, 2002). We would like to check if the stonewort species selected for the study, occurring in different habitats of the littoral in temperatezone lakes, employ the clonal life strategy. We expect that they differ in their structural and functional features connected with this strategy: number of modules (orthotropic axes) which form thalli and their fragments’ ability to regenerate depending on the differences in habitats, which can be seen as an adaptation to different environmental conditions. 2. Methods

1.4 1.75 ± 1.35 0.1–5.0

1.0 1.97 ± 1.84 0.3–6.0

1.5 1.88 ± 1.47 0.2–6.0

They are relatively common in northern Europe, including Poland (Krause, 1997; Hutorowicz, 2006; Pełechaty and Pukacz, 2008). Chara aspera occurs on all continents. It grows to a maximum depth of 1 (1.5) m, predominantly occupying shallow habitats disturbed by wave activity. It is a dioecious perennial, but in shallow areas its thallus dies back for the winter (Wood and Imahori, 1965; Krause, 1997; Nielsen, 2003). Chara globularis is widely spread around the world and occupies both shallow and deep habitats in various types of water bodies. It is a monoecious species. Depending on weather conditions and depth, this plant can be annual or overwinter in the form of green thalli (Wood and Imahori, 1965; Krause, 1997; ˙ 2003; Pełechaty and Pukacz, 2008). Chara rudis is Sinkeviciène, mainly found in northern Europe, being less common in Asia, most often in lakes and ponds at a depth of 2–3 m (7 m at most). It is a monoecious perennial (Wood and Imahori, 1965; Krause, 1997). Chara tomentosa occurs throughout the world. It is predominantly found in lakes, and less frequently in other types of water bodies, to a depth of 7 m. It is a dioecious perennial plant which overwinters as a whole thallus or disintegrates into a large number of fragments (Krause, 1997; Torn et al., 2003).

2.1. Assumptions Based on works by Harper and White (1974), Harper (1985) and De Kroon and Van Groenendael (1997), the following criteria of clonality were adopted: (1) the iterative growth form in the architecture of an individual characterised by the presence of a horizontal underground or above-ground main axis and upright orthotropic above-ground axes (branches), and (2) the ability of the upright axes to function independently and regenerate the thallus. The growth form of selected stonewort species was determined on the basis of individuals’ architecture, whereas the ability of vertical axes to function independently and regenerate the thallus was checked by experimental means. 2.2. Subject of study Four stonewort species were selected for the study: Chara aspera Willd., C. globularis Thuill., C. rudis A. Braun and C. tomentosa L.

2.3. Methods of biometric analyses In order to evaluate the features of the stonewort architecture defined as clonality criteria (presence of a horizontal main axis and upright orthotropic above-ground axes, see above), 100 mature individuals were collected from each species, 20 from each of the five populations in lakes of the Pomeranian Lakeland (NW Poland; Table 1). The individuals were obtained in the middle of the growing season (July, August) from different depths and patches with variable density and species composition to cover the widest possible spectrum of phenotypic diversity. Plants were collected by a diver ensuring that the thallus was not broken. After having been taken out of the water, the individuals were placed on white plexiglass in order to analyse their architectural features, such as the presence or lack of the horizontal, non-green thallus part which is at least partially submerged in the sediment, number of orthotropic upright

˛ E. Rekowska / Aquatic Botany 100 (2012) 25–34 K. Bociag,

axes and presence or lack of holdfasts. In addition, the height of the main (longest) thallus axis was measured, regarding it as the measure of the height of an individual. Then each individual was photographed for archiving purposes, dried at 80 ◦ C for 8 h and weighed. The calculations were performed using STATISTICA 7.0 PL and EXCEL 8.0. The significance of the differences between species was verified by the Kruskal–Wallis test. The Mann–Whitney U test with Bonferroni correction was used as a post hoc test (Łomnicki, 2000). 2.4. Methods for the evaluation of habitat conditions Sediment and water samples were taken (from above the plant patch) at the plant collection site in all 5 lakes, and light, thermal and oxygen conditions were determined. The physical and chemical water and sediment properties were measured with standard methods suggested by Eaton et al. (2005). In the water, directly above the plant patch, the following features were determined: light intensity (PAR; LI-COR, LI-250 Light Meter), temperature and oxygen saturation (OXI 96 Oximeter with an EOT 196 electrode). Other parameters measured were: water and sediment pH (320 SET 1 pH-meter with a combined BlueLine 24 pH electrode), water and sediment electrolytic conductivity (LF 96 Conductometer with a TETRA-CON 96 electrode), dissolved organic carbon (DOC) in the water (measuring the absorbance of samples in an Aquaquant Spectrophotometer for  = 330 nm), total nitrogen concentration in the water (Merck Nitrogen (total) Cell Test, measuring absorbance in an Aquaquant Spectrophotometer for  = 340 nm), total phosphorus concentration in the water (after microwave mineralisation with sulphuric and nitric acid, Spectroquant Phosphat-Test, measuring absorbance in an Aquaquant Spectrophotometer for  = 880 nm), calcium concentration in the water (by a complexometric versenate method) and sediment (by the same method, after prior extraction of calcium ions from the sediment using hydrochloric acid (1:1), redox potential in the sediment (320 SET 1 pH-meter with a platinum BlueLine 32 Rx electrode), sediment water content (by weighing water loss after drying for 48 h to constant weight) and organic matter content in the sediment (by weighing sediment loss after ignition for 5 h to constant weight at 550 ◦ C). The statistical analysis was based on arithmetic means, standard deviations, medians and ranges. The significance of the differences between the environmental conditions of species occurrence was verified by the Kruskal–Wallis test. The Mann–Whitney U test was used as a post hoc test (Łomnicki, 2000). A multivariate analysis was also performed using the program CANOCO 4.5. Data for the analyses were standardised in Canoco and not transformed. Detrended correspondence analysis (DCA) showed that water and sediment properties were linear in structure and represented only part of a Gaussian distribution (eigenvalues < 2 SD), thereby calling for the use of principal component (PCA) analyses and redundancy analyses (RDA). Statistical significance was tested using Monte Carlo permutation tests (Ter Braak and Smilauer, 2002). 2.5. Experimental methods A laboratory experiment was performed to check whether the species under study meet the functional criterion of clonality (the ability of the upright axes to function independently and regenerate the thallus, see above) and to determine the related differences between the species. Four-node (3-internode) sections from the apical parts of the orthotropic branches (the apical node and side branches had been cut off) were used in the experiment. The fragments were obtained from individuals in one population (Table 1), one fragment from each randomly collected individual. They were exposed in 215 dm3 tanks for a period of 12 weeks. 50 fragments of C. aspera, 75 of C. rudis, 100 of C. globularis and 100 of

27

C. tomentosa were used. The experiment was conducted for each species in 5 replications (5 × 10, 5 × 15, 5 × 20, 5 × 20 fragments, respectively, randomised block). In the experiment, we used tap water and sterile, autoclaved substrate from the parent population area. The experiment was conducted under long-day conditions (D16 h:N8 h) using Osram L 36W fluorescent tubes. The mean value of light intensity falling on the exposed fragments was 46 ␮mol m−2 s−1 . Water temperature ranged from 19 to 20 ◦ C. After 12 weeks all the fragments were taken out. For each species the proportion of fragments producing new (offspring) axes was determined, calculated from the ratio between the number of fragments giving rise to new axes and number of fragments used in the experiment. Their regenerative potential, defined as the number of new axes produced by a fragment (number of new axes produced during the experiment in relation to the number of thallus sections that resumed growth), was also determined.

3. Results 3.1. Environmental conditions The examined stonewort populations were found within the epilimnion, to a depth of 6 m in the littoral of the studied lakes. The water in their habitats was warm and well-oxygenated in the summer. However, the habitats of individual species varied in both water and sediment properties (Tables 2 and 3, Fig. 1). The populations of C. aspera occurred only in the shallow littoral zone (0.05–0.8 m). The habitat of this species was accordingly welllit but also disturbed by wave activity (Table 2). The habitat of this species differed from the others especially in terms of properties of the substrate, which was alkaline, but mineral (sandy), calciumpoor and weakly hydrated. Sediment water conductivity was very low, whilst its redox potential relatively high (Table 3). The populations of C. globularis occupied a considerably larger area from a depth of 0.1–5.0 m. Consequently, the relative PAR value had a wide range, and the mean value was significantly lower than in the case of C. aspera. The water contained less calcium than in the other habitats, but it was the richest in phosphorus (Table 2). The substrate was nearly neutral and always reduced, whilst being the richest in calcium and ions dissolved in the sediment water (Table 3). In the more shallow sections of the littoral zone, the sediment was mineral, whereas deeper it was organic. Its water content varied along the depth gradient. C. rudis covered a depth range between 0.3 and 6 m. Thus, the relative PAR intensity varied, just as in the case of C. globularis. The water was characterised by the highest conductivity and amount of calcium ions. However, it was phosphorus-poor (Table 2). The water content of the sediment as well as mineral matter and calcium content were varied (Table 3). The populations of C. tomentosa also covered a vast area, from 0.2 to 6 m of depth. The water was alkaline, calcium-rich and poor in phosphorus (Table 2). The substrate had the highest pH and was very varied in terms of conductivity, calcium content, hydration and mineral matter content (Table 3). The environmental conditions in the populations of the studied species differed in both water and sediment properties (Tables 2 and 3). This was also shown in the conducted PCA analysis (Fig. 1). The first two axes explained 99.9% of the total variation of environmental features (eigenvalues 0.969 and 0.03). Habitats of the species under study differed above all in terms of light and substrate properties. C. aspera occurred in well-lit places, on a mineral and poorly hydrated substrate deficient in mineral salts, which is typical of the shallow littoral. The remaining species occupied sites with worse light conditions and a more hydrated sediment rich

˛ E. Rekowska / Aquatic Botany 100 (2012) 25–34 K. Bociag,

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Table 2 Physico-chemical features of water; presented in the following sequence: number of samples, median, mean ± standard deviation, range. Feature

Chara aspera

C. globularis

C. rudis

C. tomentosa

K-W

U M-W P < 0.001 asp

PAR

pH

Conductivity [␮S cm−1 ]

Ca2+ [mg dm−3 ]

−3

DOC [mg C dm

−3

Ptot [mg dm

]

−3

Ntot [mg dm

]

]

20 55.17 55.1 ± 18.1 33.5–96.0

23 27.35 30.3 ± 22.5 5.2–91.9

16 33.63 39.4 ± 18.8 9.8–73.6

26 31.28 32.1 ± 17.9 8.1–73.6

P < 0.001

6 7.83 – 7.63–8.87

15 8.08 – 7.83–8.63

12 7.87 – 7.7–8.43

17 7.98 – 7.68–8.39

P < 0.005

6 178 144.7 ± 51.5 76.8–186.6

13 186,6 193.7 ± 51.1 118.2–267

12 186,6 194.9 ± 19.9 164.0–225.0

17 176 169.5 ± 20.8 103.8–200.0

P < 0.001

5 31.58 27.5 ± 8.0 16.8–36.9

14 27.35 32.0 ± 6.1 20.6–41.6

12 33.63 37.2 ± 5.2 31.6–47.3

16 31.28 32.8 ± 6.8 24.7–47.3

P < 0.001

5 1.81 1.9 ± 1.0 0.7–3.4

14 1.56 1.5 ± 0.6 0.5–2.5

11 1.13 1.9 ± 1.4 0.7–4.6

15 1.76 1.9 ± 0.9 0.9–3.4

5 0.41 0.86 ± 0.20 0.07–0.58

13 0.51 0.56 ± 0.16 0.37–0.98

10 0.29 0.33 ± 0.29 0.02–1.22

16 0.41 0.33 ± 0.19 0.02–0.77

5 0.43 1.30 ± 1.40 0.23–3.94

16 0.62 0.76 ± 0.49 0.23–2.25

11 0.46 0.60 ± 0.33 0.19–1.11

16 0.43 0.82 ± 0.72 0.19–2.17

P < 0.001

asp glob rud tom asp glob rud tom asp glob rud tom asp glob rud tom

** ** **

rud

tom

**

** –

** – –

– –

–

** ** – –

– –

– **

** ** –

– ** –

–

**

** –

– –

– – **

**

–

** –

**

– – –

–

asp glob rud tom

– – –

asp glob rud tom

** – –

asp glob rud tom

glob

–

– –

– –

–

**

– –

– **

– ** –

–

– – – –

– – –

– –

– –

– – –

–

Table 3 Physico-chemical features of sediment; presented in the following sequence: number of samples, median, mean ± standard deviation, range. Feature

Chara aspera

C. globularis

C. rudis

C. tomentosa

K-W

pH

5 7.16 – 6.56–7.52

19 7.04 – 6.33–7.52

14 7.16 – 6.72–7.52

21 7.26 – 6.73–8.57

P < 0.001 asp glob rud tom

– – –

5 118.0 133.3 ± 67.9 64.0–250.0

17 392.0 383.4 ± 144.4 83.7–562.0

14 321.0 324.6 ± 134.2 84.0–590.0

21 248.5 268.8 ± 146.9 76.7–590.0

P < 0.001 asp glob rud tom

** ** **

5 −158.0 −119.2 ± 133.1 −279 to 51

17 −279.0 −250.2 ± 66.9 −333 to (−85)

14 −278.0 −250.2 ± 66.9 −327 to (−88)

21 −247.5 −180.7 ± 132.5 −298 to 108

P < 0.001 asp glob rud tom

** ** –

5 0.82 0.7 ± 0.1 0.5–0.8

16 23.70 77.2 ± 99.4 0.8–228.8

14 5.82 47.9 ± 71.6 2.5–240.2

15 13.82 59.0 ± 79.2 0.8–242.4

P < 0.001 asp glob rud tom

** ** **

5 14,01 16.7 ± 7.4 8.6–29.0

19 75,44 72.0 ± 26.4 16.3–99.9

14 72,65 65.0 ± 26.1 14.0–99.9

21 64,35 59.4 ± 28.2 9.9–99.9

P < 0.001 asp glob rud tom

5 99.18 98.3 ± 1.9 94.6–99.7

19 86.97 76.7 ± 22.4 6.8–99.4

14 91.37 83.7 ± 24.6 10.4–98.7

21 91.17 76.1 ± 32.1 1.29–99.6

P < 0.001 asp glob rud tom

U M-W **P < 0.001 asp

Conductivity [␮S cm−1 ]

Redox [mV]

Ca

2+

−1

[mg g DW

]

Water content [%]

Mineral matter [%]

glob

rud

tom

–

– –

– ** –

– ** ** – ** ** – – ** – – **

** ** **

– – **

** ** **

– –

– ** –

** ** –

– ** –

– – –

– ** –

** – –

– ** –

** – –

– ** – –

** – –

˛ E. Rekowska / Aquatic Botany 100 (2012) 25–34 K. Bociag,

29

Fig. 1. Ordination diagram of environmental conditions of species plotted along the 1st and 2nd ordination axes. Abbreviations: W – water, S – sediment, cond. – conductivity, wat. cont. – water content, min. mat. – mineral matter.

in calcium ions, which is characteristic of deeper sections of the littoral. The habitats of C. rudis and C. tomentosa were similar. C. globularis occurred on a substrate with the highest water content and concentration of dissolved salts (Fig. 1). 3.2. Architecture of individuals C. aspera individuals were small, light and made up of 1–9 orthotropic axes connected with a short horizontal main axis usually equipped with holdfasts. The thallus had a modular structure, i.e. the basic, recurring unit was the orthotropic axis. In the sample population, 50% of individuals had single-axis thalli, and 50%, multiaxis ones. Among the latter, 2–3-axis individuals represented the majority, and the more complex individuals (5–9 axes) were less numerous (about 10%; Fig. 2, Table 4). C. globularis individuals were of varied size. The height of the main vertical axis ranged from 24 to 515 mm, and weight of an individual from 0.003 to 0.999 g DW. The thallus had a modular structure and one or more axes (1–11; Fig. 2, Table 4). The connecting plagiotropic part generally did not have holdfasts. The fraction of single-axis individuals represented 65%, whilst that of multiaxis ones was smaller (45%), with 2–3-axis individuals being in the majority (Fig. 2). C. rudis individuals were higher and heavier than the stoneworts described above. The main vertical axis of an individual was 272.9 ± 158.0 mm high, and the individual weighed 0.41 ± 0.33 g DW. The ranges of these features were fairly wide (Table 4). Just as in C. aspera and C. globularis, individuals had a modular structure with one or more axes (1–7) and most often were devoid of holdfasts (Fig. 2). Single-axis individuals represented slightly more than a half of the sample population (52%). The majority of multi-axis ones had 2 axes (Fig. 2, Table 4). C. tomentosa individuals were high and heavy. Their thalli could be as high as 1.5 m and weigh up to 2.5 g DW (Table 4). Most of the individuals had a single axis (69% of the population) without

holdfasts. Multi-axis individuals (up to 10 axes) were in the minority (31%), most of them having 2 axes (Fig. 2, Table 4). To sum up, the studied species differed in a statistically significant way with regard to the size of individuals (their weight and height of the vertical axis) except for C. rudis and C. tomentosa, which were similar in weight (Table 4). In terms of thallus structure, however, all of them were similar (Fig. 2, Table 4). They were all modular in structure and could have one or more axes. There were statistically significant differences between C. aspera and C. tomentosa (Table 4). C. aspera had the highest percentage of multiaxis individuals (50%), whilst C. tomentosa, the lowest (31%). In all the species, multi-axes individuals generally had two to three axes, and only few were more complex. The distribution of the number of axes forming a thallus in all the species was similar (skewed rightward, Fig. 2). The performed RDA analysis (Fig. 3) shows the relationships between the architectural features of stoneworts and environmental conditions. It indicates that the length of orthotropic axes and their number predominantly depended on the availability of PAR and substrate specificity (water and mineral matter content). The axis length increased with decreasing light availability, and the substrate contained more water and less mineral matter as depth increased. However, axis number was positively correlated with PAR intensity and mineral matter content in the sediment, and negatively with its water content. This means that multi-axis individuals occurred above all in the shallow, well-lit and mineral littoral. Individuals’ weight in turn depended primarily on the water properties (calcium concentration and conductivity), and weight variation was not related to the other plant features. 3.3. Vegetative growth In C. aspera, only about 35% of fragments of vertical branches formed new axes, whilst the others died back. The regeneration potential of this plant was 1.29 ± 0.59, which means that an

30

˛ E. Rekowska / Aquatic Botany 100 (2012) 25–34 K. Bociag,

Fig. 2. Architecture of single- (A) and multi-axis (B) individuals and their proportions in the population.

average fragment that resumed growth produced one axis and some produced a second (Table 5). The majority of the fragments of C. globularis continued growth after separation from the parent individual. Their regenerative potential was high. Almost 60% of the thallus sections used in the experiment formed new axes. Each of them produced 3 axes on average (2.81 ± 0.92; Table 5). Almost all the thallus sections of C. rudis – 93% – used in the experiment formed new axes. The regeneration potential of this species’ fragments was 2.07 ± 0.89, that is each fragment produced two new axes on average (Table 5). In the case of C. tomentosa, 80% of the thallus sections formed new axes (proportion of fragments producing new axes was

0.8 ± 0.08). However, their regeneration potential was not as high as that of C. globularis and rudis, and each thallus fragment produced one new axis on average (1.39 ± 0.68; Table 5). All in all, the proportion of fragments producing new axes varied for all studied species, however the differences between C. aspera and C. globularis and C. rudis and C. tomentosa were not statistically important. The regeneration potential that is the number of new axes produced by them differed significantly in the four studied species (Table 5). In C. aspera, only 30% of the fragments resumed growth, but their regeneration potential was the lowest. C. globularis was characterised by moderate ability of the fragments to grow, even though their regeneration potential was the highest. As for C. rudis, almost all the thallus fragments began growing,

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Table 4 Structural features of individuals. Feature

Height of main axes [mm]

Weight of individuals [g DW]

Number of orthotropic axes

Presence of holdfastsa

Chara aspera

C. globularis

C. rudis

C. tomentosa

K-W

39.0 45.2 ± 25.1 13.8–152.0

122.1 178.0 ± 136.4 23.9–514.6

223.2 272.9 ± 158.0 29.2–848.1

273.0 416.8 ± 308.6 63.9–1478.7

P < 0.05

0.0122 0.0187 ± 0.0194 0.0016–0.133

0.049 0.084 ± 0.118 0.003–0.999

0.335 0.409 ± 0.329 0.024–1.675

0.282 0.361 ± 0.3402 0.0344–2.422

P < 0.001

1.5 2.28 ± 1.82 1–9

1.0 2.0 ± 1.96 1–11

1.0 1.91 ± 1.40 1–7

1.0 1.58 ± 1.28 1–10

P < 0.001

71

36

36

18

U M-W **P < 0.001 asp

glob

rud

tom

asp glob rud tom

** ** **

**

** **

** ** **

asp glob rud tom

** ** **

asp glob rud tom

– – **

** ** ** ** ** – – –

** ** ** – – –

** ** – ** – –

–

N samples = 100 for each species; presented: median, mean ± standard deviation, range. Abbreviations: asp – Chara aspera, glob – C. globularis, rud – C. rudis, tom – C. tomentosa. a % of individuals in the population.

and their regeneration potential was moderate. In C. tomentosa, most of the fragments gave new axes. However, they had a low regeneration potential (Table 5). 4. Discussion and conclusions 4.1. Clonality of stoneworts in the light of the applied criteria The studied species differed in terms of size, but the general model of their architecture was similar (Fig. 2, Table 4). In each of

them, some of the individuals had one axis, whilst some consisted of a few (mostly 2–3) upright ones (branches) growing from one horizontal axis, which could be equipped with holdfasts. Such an architectural model is mentioned by Santelices (2004) and ColladoVides (2002) as one of several found in marine clonal macroalgae. A recurring unit, or a module according to Harper et al. (1986), is each orthotropic thallus axis that can regenerate. Such a structure is analogous to the one that is common in stoloniferous clonal vascular plants (Harper et al., 1986). The upright axes are equivalent to above-ground shoots, and the horizontal axis to stolons or

Fig. 3. Redundancy analysis (RDA) triplot showing the individual split plot and correlation between the number of orthotropic axes, height of main axes and weight of individuals and environmental variables.

˛ E. Rekowska / Aquatic Botany 100 (2012) 25–34 K. Bociag,

32 Table 5 Functional features of the clonality of the species. Feature

Proportion of fragments producing new axes

Regenerative potential of a fragment producing new axes

Chara aspera

C. globularis

C. rudis

C. tomentosa

K-W

0.30 0.34 ± 0.152 0.2-0.6

0.60 0.59 ± 0.055 0.50–0.65

0.93 0.93 ± 0.047 0.87–1.0

0.80 0.80 ± 0.08 0.7–0.9

P < 0.05

1 1.29 ± 0.588 1–3

3 2.81 ± 0.92 1–4

2 2.07 ± 0.89 1–4

1 1.39 ± 0.68 1–4

P < 0.001

U M-W *P < 0.01, **P < 0.001 asp

glob

rud

tom

asp glob rud tom

* *

–

* *

* *

asp glob rud tom

** ** **

* * ** ** **

– ** **

** ** **

**

N samples = 50, 75, 100, 100 respectively for each species; presented: median, mean ± standard deviation, range. Abbreviations: asp – Chara aspera, glob – C. globularis, rud – C. rudis, tom – C. tomentosa.

rhizomes. The performed experiments showed that most fragments of orthotropic axes separated from the parent thalli survived and regenerated thallus by forming new axes/branches (Table 5). Thus, these orthotropic axes may be regarded as modules (ramets) in functional terms as well. The development of stoneworts, which are not differentiated into organs, is not as rigorously determinate as in higher plants. The side axes display indeterminate growth like the main axis and can form in every node (Van den Hoek et al., 1995). Consequently, according to the definition proposed by Collado-Vides (2002) for filamentous marine algae, under which a module is a result of constant division of the apical or any other node cell, not only the vertical axes can be regarded as modules, but also each new branch of the thallus, at least in structural terms. ´ ˛ (2011) and Bociag The results of the experiments by Skurzynski show that in stoneworts even single-node axis fragments are capable of resuming growth and regenerating the thallus. Based on this, it may be assumed that each axis fragment with at least one node is a ramet. However, our field observations indicated that in nature stonewort thalli do not spontaneously fall apart into such short fragments, but divide by separation of upright side axes from the parent thallus in the process of ageing, disintegration and breaking up of ageing horizontal axes. Still, it is known that the disintegration of thalli may be enhanced by external factors, such as hydrodynamic disturbance (Keddy, 1982; Garbey et al., 2006) or the activity of aquatic fauna (De Winton et al., 2002; Lammens et al., 2004). Under such conditions, much smaller fragments break off the parent thallus, especially in species which are strongly encrusted with calcium and therefore fragile. The composition of the vegetative ´ ˛ diaspore bank of Chara rudis recorded in Skurzynski and Bociag’s (2011) work confirms this. It included both single- and multi-node fragments.

4.2. Clonality of stoneworts and environmental conditions The apparent growth strategies of the four individual species tested are slightly different (Table 6). This may be interpreted as an adaptation to different habitats (Tables 2 and 3, Figs. 1 and 3). According to Fischer and van Kleunen (2002), “clonality and clonal life history traits are likely adaptive, but it is not clear to what extent or which features related to clonality constitute adaptations to which environmental factors”. Our multivariate analysis showed that C. aspera differed most from the others with regard to the occupied habitat. It occurred in very shallow and well-lit habitats, which were also strongly disturbed by wave activity. This species was also different in terms of the clonal features of its architecture and ability to regenerate: it had relatively complex, often multiaxis thalli, but the capacity of its thallus fragments for resuming growth after separation was limited in comparison with the other species (Table 6). The formation of short but complex multi-axis thalli anchored to many points of the substrate by holdfasts can be interpreted as adaptive in reducing the likelihood of individuals being pulled out by wave action. The same was the case with marine algae and angiosperms found in tidal areas (Vermaat et al., 1987; Collado-Vides, 2002). The positive correlation between the number of thallus-forming axes and the properties of the shallow littoral was also confirmed by the RDA analysis (Fig. 3). What is more, the habitats of C. aspera are prone to freezing in winter and periodic drying in summer. In such an environment the life span of thallus fragments is limited, so clonal regeneration may be less worthwhile. The production of bulbils buried in the sediment and a large number of oospores is much more profitable, just as in periodically drying water bodies of arid regions of Australia and the Mediterranean (Grillas et al., 1991; Casanova, 1994; Casanova and Brock, 1990, 1996, 1999).

Table 6 Features of the growth strategy in the species under study. Feature

Chara aspera

C. globularis

C. rudis

C. tomentosa

Size of individuals Architecture of individuals

Small Single- or multi-axis (ratio in population 1:1) mostly with holdfasts

Medium-sized Mostly single-axis, less often with many axes (ratio in population 2:1) usually without holdfasts

Large Single- or multi-axis (ratio in population 1:1) usually without holdfasts

Large Mostly with one, less often many axes (ratio in population 2:1) rarely with holdfasts

Ability of modules to resume growth

Low (most do not resume growth)

Average (60% resume growth)

High (almost all resume growth)

High (almost all resume growth)

Regeneration potential of modules

Low (fragment mostly forms 1 new axis)

High (3 axes/fragment on average)

Average (2 axes/fragment on average)

Low (1 axis/fragment)

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In the populations of the remaining species under study (C. globularis, C. rudis, C. tomentosa) the fraction of multi-axis individuals was smaller, but the fragments of their axes could resume growth and regenerate the thallus. These species occurred over a wider depth range, up to 5–6 m, and were consequently far less exposed to disturbance. Under such conditions, the complex structure of the thallus was not necessary for the anchorage in the substrate, but clonal regeneration could be a worthwhile strategy. Especially at the deep edge of a stand, where the production of gametangia as well as the recruitment of new individuals from oospores are probably limited (Casanova and Brock, 1999; Asaeda et al., 2007; ´ ˛ 2009), clonal regeneration may enhance Skurzynski and Bociag, persistence of a population. Chara globularis (Table 6) had the highest regeneration capacity. For this widespread species, a high capacity for clonal regeneration may ensure population persistence despite limitations to sexual reproduction. Thus, in compliance with the proposed hypothesis, the differences among the studied stonewort species in their structural and functional features were connected with clonal life strategies. All in all, the stoneworts under study can be regarded as clonal plants. However, according to our hypothesis, the growth strategies of the species included in this work, especially C. aspera and C. globularis, are different, which results from the fact that they occupy different habitats. Taking into account the high phenotypic plasticity of stoneworts, one may expect individuals of one species to develop clonally in different ways depending on the environmental conditions. Within this group, both clonal and aclonal plant species are likely to occur, as well as the intermediate forms possessing an incomplete set of clonal properties (Sachs, 2002; Klimeˇsová and Martinková, 2004). Acknowledgements We wish to express our appreciation to Prof. Józef Szmeja for his comments on the text. We are grateful to Jan Vermaat and anonymous referees for their helpful remarks on the manuscript, to Marek Merdalski and Krzysztof Bana´s for their assistance in the field works and help in statistical analyses and to Emilia Pokojska for translating this text into English. This work was financed by education funds for 2007–2013 as a research project (projects no. N N304 4113 33 and N N304 4116 38 of the Ministry of Science and Higher Education). References Asaeda, T., Rajapakse, L., Sanderson, B., 2007. Morphological and reproductive acclimations to growth of two charophyte species in shallow and deep water. Aquat. Bot. 86, 393–401. Casanova, M.T., 1994. Vegetative and reproductive responses of charophytes to water-level fluctuations in permanent and temporary wetlands in Austalia. Aust. J. Mar. Freshwater Res. 45, 1409–1419. Casanova, M.T., Brock, M.A., 1990. Charophyte germination and establishment from the seed bank of an Australia temporary lake. Aquat. Bot. 36, 247–254. Casanova, M.T., Brock, M.A., 1996. Can oospore germination patterns explain charophyte distribution in permanent and temporary wetlands? Aquat. Bot. 54, 297–312. Casanova, M.T., Brock, M.A., 1999. Life histories of charophytes from permanent and temporary wetlands in Eastern Australia. Aust. J. Bot. 47, 383–397. Collado-Vides, L., 2002. Clonal architecture in marine macroalgae: ecological and evolutionary perspectives. Evol. Ecol. 15, 531–545. Combroux, I., Bornette, G., Willby, N.J., Amoros, C., 2001. Regenerative strategies of aquatic plants in disturbed habitats: the role of the propagule bank. Arch. Hydrobiol. 152, 215–235. De Kroon, H., Van Groenendael, J., 1997. The Ecology and Evolution of Clonal Plants. Backhuys Publishers, Leiden. De Winton, M.D., Casanova, M.T., Clayton, J.S., 2004. Charophyte germination and establishment under low irradiance. Aquat. Bot. 79, 175–187. De Winton, M.D., Taumoepeau, A.T., Clayton, J.S., 2002. Fish effects on charophytes establishment in a shallow, eutrophic New Zealand lake. N. Z. J. Mar. Freshwater Res. 36, 815–823. Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., 2005. Standard Methods for the Examination of Water and Wastewater. Am. Publ. Health Ass., Washington.

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