The use of morphometric characteristics in the ...

Biologia 66/3: 425—428, 2011 Section Botany DOI: 10.2478/s11756-011-0034-1

The use of morphometric characteristics in the identification of two morphologically similar charophytes: Chara globularis and Chara virgata ´1 Andrzej Pukacz1, Mariusz Pelechaty2 & Sne˙zana Petrovic 1

Collegium Polonicum, Adam Mickiewicz University Pozna´ n – Europa-Universit¨ at Viadrina, Frankfurt-Oder, Ko´sciuszki 1, PL-69–100 Slubice, Poland; e-mail: [email protected] 2 Department of Hydrobiology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, PL-61–614 Pozna´ n, Poland; e-mail: [email protected]

Abstract: The study reports results of morphometric measurements of two morphologically similar charophytes, Chara globularis Thuill. and Chara virgata K¨ utz. . Although both species differ in ecological spectra, they can co-occur in the same lakes. Morphological differences given in identification keys can be problematic in the field study. Therefore, additional features were tested and suggested as possibly useful, supplementary differences. 10 individuals of both species growing under similar conditions (littoral of a meromictic, mesotrophic lake, mid-Western Poland) were collected in the vegetation season 2005. Statistical tests evidenced that out of 9 features 6 differed the studied species. Axis diameter, the length of internodes and the length of branchlets revealed the most significant differences. Chara virgata is smaller than Chara globularis even in similar or common habitats. The proposed branchlet length: internode length ratio, higher for Chara virgata than for Chara globularis, can be applied as a useful measure for the species differentiation, particularly under conditions where both charophytes co-occur. Key words: Chara virgata; Chara globularis; charophytes; identification; lake environment; morphological features

Introduction Chara globularis Thuill. and Chara virgata K¨ utz. are charophyte species (Chraceae family) whose morphological features usually develop similarly (e.g. Migula 1897; D˛ambska 1964; Krause 1997). It also concerns the development pattern of reproduction organs. Due to the oospore dimensions both species are joined in a common species Chara globularis group (Haas 1994). Since the above-mentioned morphological similarities were emphasized by different authors (e.g. Migula 1897; Groves & Bullock-Webster 1924; Wood & Imahori 1964; D˛ambska 1964; Krause 1997; Cirujano et al. 2007), Chara virgata can be treated as a variety or form of Chara globularis. Although, recent molecular studies have proved their genetic differences with stipulodes to be the most important for the species identification (Mannschreck 2003). These findings are consistent with the Karczmarz’s (1973) opinion according to which Chara globularis and Chara virgata develop differently enough to be distinguished unequivocally as separate species. Apart from stipulodes other morphological differences are given in identification keys (e.g. D˛ambska 1964; Gollerbach & Krasavina 1983; Krause 1997; Blindow et al. 2007; Pelechaty & Pukacz 2008a). No spine-cells develop in Chara globularis, whereas in C. virgata they are present and papilliform. Addition-

ally, the type of cortex tells both species from each other: triple in both species but isostichous in Chara globularis and tylacanthous, with clearly differentiated rows in Chara virgata. Despite their generally dissimilar habitat requirements, the species may co-occur in the same aquatic ecosystems and even build common charophyte meadows under the same habitat conditions (Pelechaty et al. 2004a and references therein). Since then, the need for a comparative analysis of other features, possibly more useful in the field, emerges for further studies. Therefore, the aim of the paper is to compare the length of internodes, axis diameter, number of branchlets and side-branches in every node, length of branchlets, number of branchlet segments, number of ecorticate branchlet segments and number of cells of last ecorticate segment of Chara globularis and C. virgata specimens occurring in the same lake ecosystem. Additionally, the ratio between the length of branchlets and internodes was determined. Material and methods In the study presented 10 individuals of Chara virgata and Chara globularis were analyzed. All the individuals of both Chara species were collected in the vegetation season 2005 from the phytolittoral of a meromictic and mesotrophic lake (Lake Lubi´ nskie, mid-Western Poland, Pelechaty et al. 2004b) and fixed with the formaldehyde solution. For both

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lets and side-branches in every stem node. Then, for every of five whorls of branchlets the: length of banchlets, number of branchlet segments (including ecorticate branchlet segments), number of ecorticate branchlet segments, number of ecorticate cells of branchlet were determined. These all measurements were carried out using stereo microscope and the 0.2 millimetre grid. Additionally, for every individual the branchlet length: internode length ratio was determined. For statistical analyses Statistica 8.1 software was applied. The empirical distribution of most of the measured parameters was not consistent with the theoretical normal one, so the non-parametric statistical test was performed. For proper statistical calculations the individual values of each parameter were averaged and expressed with two decimal places accuracy. For the multidimensional PCA analysis (Canoco for Windows 4.5) all data were transformed using logarithmic transformation (ln(1 + x)).

Results

Fig. 1. Basic morphometric characteristics analyzed in the study presented (example – top whorls of Chara globularis). a – Internode length (of the stem); b – axis diameter; c – branchlet length; d – ecorticate branchlet segments; e – branchlet segment.

species the individuals were collected from the same depth: 1.5–2 m. Undamaged specimens with at least six whorls of branchlets, counting from the apex, were selected. The first, not fully developed whorl of branchlets was passed over and the next five were analyzed. The first stage of the study was to measure the length of internodium, diameter of axis (measured in the middle of internodium) of each individual analyzed (basic features taken under consideration are presented in Fig. 1). For the measurements stereo microscope Zeiss Stemi DV4 with photo set Canon PowerShot A640 was used. To achieve best precision Axio Vision Rel. 4.6 software was applied for the microscopic measurements. The next stage was to determine the number of branch-

To evidence morphological differences between studied charophytes, Mann-Whitney U- test was applied. The results showed that from amongst the morphometric characteristics three did not differentiate between the studied charophytes: the number of branchlets per axis node, number of branchlet segments and number of ecorticate branchlet segments (in each case p > 0.05, Table 1). For the rest of characteristics the differences between Chara virgata and Chara globularis were statistically sound (p < 0.05). Considering the values of Mann-Whitney U-test it may be concluded that the differentiation between both charophytes was to a greatest extent reflected by: axis diameter, internode length and branchlet length (Table 1). The great importance of differences (p < 0.001) in case of the two latter characteristics was also reflected in the branchlet length: internode length ratio, significantly higher for Chara virgata than for Chara globularis. The above-evidenced morphometric differences between Chara virgata and Chara globularis were confirmed by the Principal Component Analysis (Fig. 2). On the assessed diagram both charophytes were spread into two groups, characterized by different or even opposite values of morphometric characteristics. However,

Table 1. Statistical characteristics of morphological features of Chara virgata and Chara globularis, for each species (n = 10). Chara virgata

Internode length [mm] Axis diameter [mm] No. of branchlets No. of side-branches Branchlet length [mm] No. of branchlet segments No. of ecorticate branchlet segments No. of cells of last ecorticate segment Branchlet lenght: internode lenght ratio

Chara globularis

Statistical significance

Min.

Max.

Mean

SD

Min.

Max.

Mean

SD

M-W U-test

p

1.75 0.19 6.00 0.00 5.90 7.00 1.00 1.00 0.90

10.76 0.33 9.00 2.00 20.81 10.25 2.00 1.75 7.69

6.25 0.26 7.22 0.34 15.03 9.29 1.14 1.30 2.66

1.84 0.05 0.58 0.52 3.06 0.51 0.21 0.19 1.15

5.00 0.30 6.00 0.00 8.79 9.00 1.00 1.00 0.63

33.00 0.63 8.00 2.00 39.53 9.88 1.57 2.00 2.86

18.05 0.46 7.20 0.78 21.63 9.38 1.17 1.40 1.34

7.80 0.07 0.53 0.46 7.84 0.20 0.15 0.19 0.49

0.00 0.00 47.50 9.00 0.00 50.00 43.50 21.00 0.00

0.000011 0.000011 0.853428 0.001050 0.000011 1.000000 0.630529 0.028806 0.000011

Abbreviations: Min., minimum; Max., maximum; SD, standard deviation; M-W, Mann-Whitney test; differences statistically significant at p < 0.05.

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Morphometric characteristics of Chara globularis and Chara virgata

Axis 2 (λ2 = 0.057)

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Fig. 2. PCA output for morphological features analyzed in Chara virgata (circles 1–10) and Chara globularis (filled circles 11–20).

in this case branchlet length have not so strong effect on distinguishing the measured individuals. Moreover, the number of branchlet segments, which is not statistically sound according to Mann-Whitney U- test, is one of the most distinguishing features on the PCA diagram. Construing the PCA diagram and measurements given in Table 1 it may be stated, that in the studied lake Chara virgata is characterized by visibly smaller internodes, branchlets and the diameter of axis as compared to Chara globularis. Side-branches also differentiated both species: usually the lack of side-branches or only one side-branch developed, respectively. Discussion Based on published data, Chara virgata and Chara globularis can be considered species of different or even opposite ecological requirements (Pelechaty et al. 2004a and references therein). Due to own observations and available data sources Karczmarz (1973 with literature summary) concluded that the former species is restricted to oligo- and mesotrophic Lobelia lakes and, so, it occurs more frequently in the area of the Baltic Sea lake districts as compared to other regions. The latter charophyte, Chara globularis, has wide ecological amplitude and is common in a various types of eutrophic waters. Nowadays, it is agreed upon that Chara virgata can occur in lakes with higher phosphorus concentrations, comparable to those given for Chara globularis. According to Blindow’s (1992) studies in Scanian lakes (southern Sweden), phosphate and total phosphorus ranges for both species revealed similar minimum values. In fact, maximum values were much higher for Chara globularis. Along with other small species of charophtes (shoot diameter < 1.0 mm, Blindow 1992), Chara vir-

gata can occur in eutrophic lakes, in which the species is restricted to shallow areas, where light is not a limiting factor, although in one of ultra-oligotrophic lakes charophyte meadows dominated by this species were reported from the depths reaching 100 m (Beauchamp et al. 1992). In shallow eutrophicated waters Chara virgata can grow densely, forming even compact communities. All the above suggests that one can meet both species in the same lakes, at least over the low and intermediate phosphorus concentrations. They can even co-occur and share the same habitats (Pelechaty et al. 2004a). In western Poland, where this study was performed, both species belong to the most common charophytes (Pelechaty & Pukacz 2008b), although by the end of the 20th century Chara virgata had not been reported from this region (Pelechaty et al. 2004a and references therein). This charophyte is considered to be well distributed also in other parts of Poland (G˛abka M., Sugier P., Urbaniak J., pers. communications). Despite the Karczmarz’s opinion (1973) that morphological differences between the two species are distinct, possible difficulties, which can be met when identifying collected material, emerge from numerous works cited in the introductory chapter. It particularly can be a problem in the field when one has to differentiate between both species. The measurements undertaken in this work are consistent with the general conclusion that Chara globularis is bigger then Chara virgata. Importantly, this became true also when both species co-occur under similar conditions where habitat-related morphological variability can be excluded. This study provides evidence for a set of additional morphological differences, which supplement the basic species differences concerning type of cortex, development of spine-cells and stipulodes and can be helpful in the species identification.


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428 These particularly are: axis diameter, length of internodes and length of branchlets. In a consequence, morphometric differences between both charophytes considered here were clearly expressed in the proposed branchlet length: internode length ratio. For Chara virgata the values of this ratio are significantly higher than for Chara globularis (Table 1, Fig. 2). Thus, this simple ratio can reflect that at Chara virgata individuals the length of branchlets visibly surpass the length of internodes, whereas for Chara globularis the differences are much smaller. This difference was also mentioned by D˛ambska (1964) in her identification key. In the authors’ opinion, this ratio is worth being applied as useful measure for the species differentiation, particularly under conditions where both charophytes co-occur. References Beauchamp D.A., Allen B.C., Richards R.C., Wurtsbaugh W.A. & Goldman G.R. 1992. Lake trout spawning in Lake Tahoe: egg incubation in deep water macrophyte beds. North Amer. J. Fish. Manag. 12: 442–449. Blindow I. 1992. Decline of charophytes during eutrophication; a comparison to angiosperms. Freshwater Biol. 28: 9–14. Blindow I., Krause W., Ljungstrand E. & Koistinen M. 2007. Best¨ amningsnyckel f¨ or kransalger i Sverige. Svensk Bot. Tidskr. 101:165–220. Uppsala, Cirujano S., Cambra J., Sánchez Castillo P.M., Meco A. & Flor Arnau N. 2007. Flora ibérica. Angas continentales. Carofitós (Characeae). Real Jardín Botánico, Madrid, 130 pp. D˛ambska I. 1964. Charophyta – ramienice. Pa´ nstwowe Wydawnictwo Naukowe, Warszawa, 126 pp. Gollerbach M.M. & Krasavina L.K. 1983. Charovyje vodorosli – Charophyta. Opredelitel presnovodnych vodoroslej CCCP. Nauka Leningradskoje Otdelenije, Leningrad, 190 pp.

Groves J. & Bullock-Webster G.R. 1924. The British charophyta, Vol. II, Charae. The Ray Society, London, 129 pp. Haas J. N. 1994. First identification key for charophyte oospores from central Europe. Eur. J. Phycol. 29: 227–235. Karczmarz K.1973. On the ecological requirements of Chara delicatula Agardh. Annales Universitatis Mariae Curie-Sklodowska Lublin – Polonia 28(11): 117–123. KrauseW. 1997. Charales (Charophycae). S¨ usswasserflora von Mitteleuropa, Band 18, Gustav Fischer, Jena, 202 pp. Mannschreck B. 2003. Genetische und morphologische Differenzierung ausgewaehlter Arten der Gattung Chara. PhD thesis. Shaker Verlag. Migula W. 1897. Die Characeen Deutschlands, Oesterreichs und der Schweiz; Unter Ber¨ ucksichtigung aller Arten Europas. In: Rabenhorst L. (ed.), Kryptogamenflora von Deutschland, Oesterreich und der Schweiz, 2. Aufl. Vol. 5. Verlag Eduard Kummer, Leipzig, 765 pp. Pelechaty M. & Pukacz A. 2008a. Klucz do oznaczanie gatunków ramienic (Characeae) w rzekach i jeziorach. Inspekcja Ochro´ ´ ny Srodowiska. Biblioteka Monitoringu Srodowiska, Warszawa, 80 pp. Pelechaty M. & Pukacz A. 2008b. The state of preservation of charophytes (Characeae) in the waters of the Ziemia Lubuska region (mid-western Poland). Oceanol. Hydrobiol. Studies 37(Suppl. 1): 91–97. Pelechaty M., Pukacz A. & Pelechata A. 2004a. Co-occurrence of two stoneworts of reverse ecological spectra in the same lake ecosystem. Habitat requirements ofChara delicatula Agardh and Chara globularis Thuillier in the context of bioindication. Polish J. Environ. Studies 13(5): 551–556. Pelechaty M., Pukacz A. & Pelechata A. 2004b. Diversity of micro- and macrophyte communities in the context of the habitat conditions of a meromictic lake on Lubuskie Lakeland. Limnol. Rev. 4: 209–214. Wood R.D. & Imahori K. 1964. A revision of the Characeae. J. Cramer, Weinheim, 904 pp.


Received December 28, 2009 Accepted August 27, 2010