Polish Botanical Journal 61(1): 73–88, 2016 DOI: 10.1515/pbj-2016-0005 PHENOTYPIC PLASTICITY OF WALL ULTRASTRUCTURE IN THE GREEN ALGA PEDIASTRUM S.L. (CHLOROPHYTA, SPHAEROPLEALES) Joanna Lenarczyk1 & Konrad Wołowski Abstract. This study examined wall ultrastructure variability in the microscopic green alga Pediastrum s.l. Its value as a diagnostic character is discussed. Field and cultured material of 21 taxa were compared using light and scanning electron microscopy. Nine ultrastructural elements occurring on the surface of Pediastrum are documented with LM and SEM micrographs. The highest number of taxa showed reticulate ornamentation composed of a trigonal mesh and granules situated on its corners. The paper considers the use of wall ultrastructure to reconcile traditional and modern taxonomical systems with regard to Pediastrum varieties, and addresses the phylogenetic relationships between strains representing different varieties. Key words: cell wall patterns, green algae, Pediastrum, scanning electron microscopy, taxonomy, ultrastructure Joanna Lenarczyk & Konrad Wołowski, Department of Phycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, 31-512 Kraków, Poland; e-mail: [email protected] Introduction Pediastrum Meyen (Chlorophyceae, Sphaerople- types. That work examined 11 species from field, ales) is currently recognized as containing 46 taxa cultured and herbarium material, originating mainly belonging to 27 species (Buchheim et al. 2005; from Europe (e.g., Germany, France, Sweden) and Lenarczyk 2014). Although the genus itself is cos- also from other continents including Asia and mopolitan, only a few species such as Pediastrum North and South America. Parra concluded that boryanum (Turpin) Menegh. and P. duplex Meyen cell wall ornamentation is an important character occur worldwide (Komárek & Jankovská 2001). for distinguishing taxa on the infraspecific level. Other species such as P. privum (Printz) E. Hege- This opinion was supported by Wu (1987), who wald (Kowalska & Wołowski 2010a; Lang et al. revised taxa from Taiwan and documented them 2012) are less common. Taxa of the genus form with SEM micrographs. However, in a study of distinctive flat aggregations of cells (coenobia) granule density on the cell wall surface in cultured as a disc-like structure usually composed of 16, P. boryanum, Nielsen (2000) concluded that those 32 or 64 individual cells. Species and infraspe- structures alone cannot be used as characters for cific taxa are distinguished mainly by the shape diagnosing infraspecific taxa. Wall ultrastructure in of marginal cells, including their lobes and pro- Pediastrum has also been analyzed in taxonomic cesses, incisions between the lobes, and wall work by Couté and Tell (1979), Hegewald and surface ornamentation. The latter character usu- Yamagishi (1994), An et al. (1999), Hegewald ally requires observation by scanning electron and Jeon (2000), McManus and Lewis (2011) microscopy. and others. A broad study of wall structure in Pediastrum Wall ultrastructure has been treated in studies was published by Parra (1979), who introduced of the formation of the coenobium and cell wall terminology for ultrastructure and ornamentation (Gawlik & Millington 1969; Millington & Gawlik 1970; Marchant 1974; Millington et al. 1981), 1 Corresponding author and more recently in research on phylogenetic 74 POLISH BOTANICAL JOURNAL 61(1). 2016 relationships (Buchheim et al. 2005; McManus electron microscopy (SEM) with critical-point drying. & Lewis 2005, 2011; Jena et al. 2014). Additionally, material from six localities (asterisked in The aim of the present study was to determine Table 1) was kept alive for culturing. the variability of wall ultrastructure in Pediastrum Fourteen monoclonal cultures were started with single Pediastrum coenobia transferred with a Narishige taxa observed in field and cultured material from MM 188 micromanipulator into sterile liquid medium various waterbodies in Poland, and to consider (Table 2). The media used were Chu’s medium (Parra these findings in the context of a practical taxo- 1979), a mixture of Chu’s and Knop’s media (Starmach nomical identification system. 1963), and L-S2T2, for which a recipe was available at the website of the ACOI Coimbra Collection of Material and methods Algae [http://acoi.ci.uc.pt/]. The strains were cultured in a Bolarus G-18-2 phytotron at ca 22°C under fluo- Inland water samples containing Pediastrum were col- rescent tubes in the Phycological Laboratory of the W. lected in summer 2008 and 2009 from 48 water bodies Szafer Institute of Botany, Polish Academy of Sciences, in lowland and upland regions of Poland, and in summer Kraków. 2011 from Wyżni Czerwony Stawek lake in the Tatra Wall patterns were observed in preserved field mate- Mountains (Table 1). Additional water samples from rial (Table 1) and living isolates (Table 2) by both light Jezioro Warnowskie lake and Jezioro Zatorek lake used (LM) and scanning electron microscopy (SEM). LM to initiate some of the Pediastrum cultures were ob- studies employed Nikon OPTIPHOT-2, Jenaval (Carl tained in 2008 (Table 2). The samples were taken with Zeiss) and Nikon Eclipse 600 microscopes. For SEM a plankton net from 1 or 2 localities of each waterbody. the material was prepared and analyzed in two ways. The material from each locality was preserved with The material preserved with formaldehyde and the living 2–4% formaldehyde, except for three samples (3, 5, 8) isolates were first rinsed with distilled water, then placed collected in 2009 (Table 1), which were preserved with on cover glasses and air-dried at ca 35°C. The cover 1–2% glutaraldehyde in cacodylate buffer in order to glasses were affixed to aluminum stubs with double- apply the protocol for preparing samples for scanning sided carbon type. The material preserved with glutar- Similarity 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 P. tetras P. duplex var. gracillimum P. duplex var. duplex 1 P. angulosum var. angulosum P. duplex var. asperum 5 P. boryanum var. forcipatum 3 P. boryanum var. pseudoglabrum P. kawraiskyi 4 2 P. biradiatum var. biradiatum P. orientale 6a P. alternans 6b P. boryanum var. boryanum P. boryanum var. brevicorne P. boryanum var. cornutum P. boryanum var. perforatum 7c P. cf. subgranulatum P. duplex var. rugulosum P. boryanum var. longicorne 7a P. integrum var. integrum P. simplex var. simplex 7c P. simplex var. echinulatum 7b Fig. 1. Similarities between wall patterns in Pediastrum, based on Jaccard’s coefficient and UPGMA clustering. For numbers of types of wall patterns see Table 4. Drawings after Lenarczyk (2014). J. LENARCZYK & K. WOŁOWSKI: PHENOTYPIC PLASTICITY OF PHENOTYPIC PLASTICITY WOŁOWSKI: K. & LENARCZYK J. Table 1. Characteristics of 49 waterbodies, together with the occurrence of 21 studied taxa in alphabetical order. Data on morphometry and trophy of all waterbodies cited from Lenarczyk (2014), except for no. 49 cited from Lenarczyk and Saługa (2013). Water samples from asterisked localities were maintained live for culturing. Pediastrum taxa Morpho metry of water Latitude (N)/ Date of angulosum biradiatum boryanum cornutum forcipatum longicorne perforatum pseudoglabrum Locality bodies: area Trophy brevicorne Longitude (E) sampling integrum simplex asperum duplex gracillimum (ha), maximum var. echinulatum var. rugulosum var. var. var. var. var. var. var. var. var. var. Waterbody no. Waterbody depth (m) var. var. var. var. var. var. var. var. subgranulatum cf. P. tetras P. P. kawraiskyi P. orientale P. simpex P. simplex P. P. alternans P. angulosum P. biradiatum P. boryanum P. boryanum P. boryanum P. boryanum P. boryanum P. boryanum P. boryanum P. duplex P. duplex P. duplex P. duplex P. integrum P. 1 Jezioro Jamno lake in Unieście 54°16′/16°06′ 2 240, 3.9 eutrophic 06.07.2008 + · · + + · + · + + · · · · · + · · · · · 2 Jezioro Bukowo lake in Dąbkowice 54°21′/16°15′ 1 750, 2.8 eutrophic 06.07.2008 · · · + · · · · · · · · · + · + + · · · + 3 Jezioro Gardno lake in Gardna 54°38′/17°10′ 2 470, 2.6 eutrophic 11.07.2008, + · · + · · + + + + · + · · · + + · · · · Wielka 12.08.2009 4 Jezioro Łebsko lake in Kluki 54°41′/17°21′ 7 140, 6.3 eutrophic 11.07.2008 + · · + · · + · · + · · · + · + + · · · + 5 Jezioro Sarbsko lake in Nowęcin 54°45′/17°35′ 650, 3.2 eutrophic 05.07.2008, + · · + · · + + + + · · · · · + · · · · + 12.08.2009 6 Pond in Golczewo 53°49′/14°58′ 2.13, – eutrophic 27.07.2009 · · · · · + · · · · + · · · · · · · · · · PEDIASTRUM 7 Pond ca 1 km NE of Święta 53°34′/14°38′ 25.0, 1.5 eutrophic 27.07.2009 · · · + · · + · · · · · · · · · · · · · + 8 Pond ca 1.5 km E of Święta 53°33′/14°39′ 11.9, 0.8 eutrophic 27.07.2009 · · · + · · + · · + · · + + · · + + + · + 9 Jezioro Bartoszewo lake near 53°31′/14°27′ 3.6, 2.5 eutrophic 28.07.2009 · · · + · · + · · · · · · · · · · · · · + Bartoszewo 10 Jezioro Dąbie lake in Czarna Łąka 53°27′/14°42′ 5 600, 8.0 eutrophic 28.07.2009 · · · + · · + + · · · · · · · · · · · · + S.L. 11 Jezioro Rusałka lake in Szczecin 53°27′/14°32′ 3.4, – eutrophic 29.07.2009 · · · · · · · · · · · + · · · · · · · · · 75 12 Jezioro Gałęziste lake near hiking 54°07′/23°04′ 3.9, 14 mesotrophic 01.07.2008 · · · · · · · · · · · · · · · · · · · · + trail 13 Jezioro Pietry lake (SW shore) 54°06′/23°05′ 228.2, 38 eutrophic 01.07.2008 · · · · · · · · + · · · · · · · · · · · · 14 Jezioro Czarne lake near Krzywe 54°05′/23°01′ 20.5, 8.8 mesotrophic 02.07.2008 · · · · · · · · + · · · · · · · · · · · · 15a Wigry lake / Zatoka Zadworze bay 54°05′/23°05′ 2 118.3, 73 eutrophic