Phenotypic Plasticity of Wall Ultrastructure in the Green Alga Pediastrum S.L

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 PEDIASTRUM S.L. 75 P. tetras P.

· + · · · + · + + + + · + + · + ·

subgranulatum cf. P.

· · · · · · · · · · · · · · · · ·

simplex var. simplex P.

· · · · · · · · · + · · · · · · ·

echinulatum var. simpex P.

· · · · · · · · · + · · · · · · · P. orientale P.

· · · · · · · · · + · · · + + + · P. kawraiskyi P.

· · · · · · · · · · · · + + + + +

integrum var. integrum P.

· · · · · · · · · · · · · · · · ·

rugulosum var. duplex P.

· · · · · · · · · + · · · + · + ·

gracillimum var. duplex P.

· · · · · · · · · + · · · · · · ·

duplex var.

taxa duplex P.

· · · · · · + · · · · · · · + · ·

asperum var. duplex P.

· · · · · · · · · · · + · · · · ·

pseudoglabrum var. boryanum P.

· · · · · · · · · + · · + + + · +

perforatum var. boryanum P.

+ + + + + · · · · · · · + · + · +

Pediastrum longicorne var. boryanum P.

· · · · · · · + · · · · + · + · ·

forcipatum var. boryanum P.

· · · · · · · + + + + · + + + · +

cornutum var. boryanum P.

· · · · · · · · · · · + · · · · ·

brevicorne var. boryanum P.

· · · · · · · · · · · · · · · · +

boryanum var. boryanum P.

· + · · · · · + + + + · + + + + +

biradiatum var. biradiatum P.

· · · · · · · · · · · · · · · · ·

angulosum var. angulosum P.

· · · · · · · · · · · · · · · · · P. alternans P. · · · · · · · · · · · · + + + · + Date of sampling 12.08.2009 12.08.2009 03.07.2008 02.07.2008 04.07.2008 02.07.2008 01.07.2008 01.07.2008 29.07.2009 28.07.2009 28.07.2009 27.07.2009 27.07.2009 27.07.2009 05.07.2008, 11.07.2008 11.07.2008, 11.07.2008, 06.07.2008 06.07.2008 Trophy eutrophic eutrophic eutrophic mesotrophic eutrophic mesotrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic metry of water depth (m) 600, 8.0 bodies: area Morpho (ha), maximum 12.2, 13 21.0, 6.5 2 118.3, 73 20.5, 8.8 228.2, 38 3.9, 14 3.4, – 5 3.6, 2.5 11.9, 11.9, 0.8 25.0, 1.5 2.13, – 650, 3.2 7 140, 6.3 2 470, 2.6 1 750, 2.8 2 240, 3.9 Latitude (N)/ Longitude (E) 54°01 ′ /23°01 54°05 ′ /23°04 54°05 ′ /23°05 54°05 ′ /23°01 54°06 ′ /23°05 54°07 ′ /23°04 53°27 ′ /14°32 53°27 ′ /14°42 53°31 ′ /14°27 53°33 ′ /14°39 53°34 ′ /14°38 53°49 ′ /14°58 54°45 ′ /17°35 54°41 ′ /17°21 54°38 ′ /17°10 54°21 ′ /16°15 54°16 ′ /16°06 Locality ca 1.5 km E of Święta ca 1 km NE of Święta

Jezioro Okrągłe lake (SE shore) Jezioro Leszczewek lake in Stary Folwark Wigry Wigry lake / Zatoka Zadworze bay Jezioro Czarne lake near Krzywe Jezioro Pietry lake (SW shore) Jezioro Gałęziste lake near hiking trail Jezioro Rusałka lake in Szczecin Jezioro Dąbie lake in Czarna Łąka Jezioro Bartoszewo lake near Bartoszewo Pond Pond Pond in Golczewo Jezioro Sarbsko lake in Nowęcin Jezioro Łebsko lake in Kluki Jezioro Gardno lake in Gardna Wielka Jezioro Bukowo lake in Dąbkowice Jezioro Jamno lake in Unieście Waterbody no. Waterbody 9 8 7 6 5 4 3 2 1 11 17 16 14 13 12 10 15a Table 1 . Characteristics Table of 49 waterbodies, together with the occurrence of 21 studied taxa in alphabetical order. Data on morphometry from and Lenarczyk trophy (2014), except of for all no. waterbodies 49 cited cited from Lenarczyk and Saługa (2013). samples Water from asterisked localities were maintained live for culturing.

76 POLISH BOTANICAL JOURNAL 61(1). 2016 P. tetras P.

+ + + + · · · · + + · + · · · · + + + + + + + + + · + · + · · + +

subgranulatum cf. P.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · + · · ·

simplex var. simplex P.

+ + · + · · · · · · · + · · · · + · · · + · · · · · · · + · · · +

echinulatum var. simpex P.

· · · · + · · · · · · · · · · + + · + · + · + · + · + · · · · · + P. orientale P.

· · · · · · · · · · · · · · · · · · + · + · · · + · · · · · · · · P. kawraiskyi P.

· · · · · · · · · · · · + · · · · · · · · · · · · · · · · · · · ·

integrum var. integrum P.

· · · · · · · · · · · · · · · · · · · · · + · · · · · · · · · · ·

rugulosum var. duplex P.

· + + · · · · · · · · · · · · · + + + · · + · · + · · · · · · · ·

gracillimum var. duplex P.

· · · · · · · · · · + · · · + · · · · · · · · · + · · · · · · · ·

duplex var.

taxa duplex P.

+ + · · · · · + · + · · · · · · · · · · · · · · · · · · · · · + +

asperum var. duplex P.

· + + · · · · · · · + · · · · · + · + · · + · · · · · · + · · · ·

pseudoglabrum var. boryanum P.

· + · + · · · · · · · · + · · · + · · · · + · · · · · · · · · · +

perforatum var. boryanum P.

· · + · · · · · · · · · · · · · · · · + · · + · · · · · · · · · ·

Pediastrum longicorne var. boryanum P.

· + + · · · · · · · + · · · + · · + · + · · + · · · · + · + + · ·

forcipatum var. boryanum P.

· · · + · · + · · · · + + · · · + + + · · + · + · · + · + · · · +

cornutum var. boryanum P.

· · · · · · · · · · + · · · · · · · · · · · · · + · · · · · · · ·

brevicorne var. boryanum P.

+ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

boryanum var. boryanum P.

+ + + + + + + · · + · + · + + + + + + · + + + + · · · · + · + · +

biradiatum var. biradiatum P.

· · · · · · · · · · · · · · · · + + + · · · · + · · · · + · · · ·

angulosum var. angulosum P.

· · · · · · · · · · + · · · · · · · · · · · + · + + · · · · · · · P. alternans P. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Date of sampling 24.07.2008 24.07.2008 24.07.2008 24.07.2008 09.07.2008 22.07.2008 01.09.2008 09.07.2008 01.09.2008 16.06.2008 22.08.2008 17.08.2008 22.08.2008 07.06.2008 22.08.2008 07.06.2008 20.08.2008 10.06.2008 20.08.2008 09.06.2008 21.08.2008 21.08.2008 09.06.2008 21.08.2008 09.06.2008 09.06.2008 21.08.2008 03.07.2008 21.08.2008 25.08.2011 27.06.2008 03.06.2008 11.08.2008 - - - - - Trophy mesotrophic mesotrophic mesotrophic eutrophic meso/eutro phic meso/eutro phic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic meso/eutro phic eutrophic eutrophic eutrophic meso/eutro phic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic eutrophic humoeutro phic eutrophic oligo/ oligo/ mesotrophic mesotrophic eutrophic oligo/ eutrophic eutrophic eutrophic metry of water depth (m) bodies: area Morpho (ha), maximum 3.2, – 1.6, – 1.6, – 0.2, – 307.1, 5.4 0.5, – 0.2, – 148.1, 25.7 0.13, – 0.3, – 26.7, 9.4 2.5, – 66.7, 4.4 0.4, – 20.5, 7.1 1.3, – 54, – 1.9, – 77, – 152.5, 40.5 15, – 1.7, – 2.5, – 25, – 6.6, – 20.0, 7.8 31.7/ 18.2 2 118.3, 73 15, – 0.3, 1.4 0.01, – 0.08, – 5.7, – /19°50 ′ Latitude (N)/ Longitude (E) 51°56 ′ /19°28 51°56 ′ /19°26 51°55 ′ /19°23 51°58 ′ /19°26 52°22 ′ /18°19 50°02 ′ 50°00 ′ /19°44 50°10 ′ /19°48 52°18 ′ /18°16 50°12 ′ /19°48 53°02 ′ /19°00 51°28 ′ /22°57 53°05 ′ /18°49 51°28 ′ /22°56 53°03 ′ /18°33 51°29 ′ /22°55 53°02 ′ /18°30 51°32 ′ /23°06 53°43 ′ /17°32 51°32 ′ /23°05 53°55 ′ /17°51 51°32 ′ /23°03 51°32 ′ /23°02 53°56 ′ /17°49 51°32 ′ /23°02 53°57 ′ /17°48 53°57 ′ /17°47 51°32 ′ /23°01 54°01 ′ /23°03 51°33 ′ /23°01 49°13 ′ /20°00 50°04 ′ /19°58 50°04 ′ /19°57 Locality . Continued

Pond near Biała Pond in Dzierżązna Pond in Ciosny Pond in Kotowice Jezioro Pątnowskie lake near Pątnów Oxbow lake near Tyniec Pond in Wołowice Pond in centre of Zelków Jezioro Ślesińskie lake in Ślesin Pond in Czajowice Pond in Rudaw Jezioro Maśluchowskie lake (SW shore) Pond 1.3 km NE of Młyniec Pierwszy Jezioro Uścimowiec lake (SE shore) Pond in Barbarka Jezioro Głębokie lake in Głębokie Peat pit 400 m N of Błotka Anielski Pond near Sosnowica Pond in Karolewo Pond Hetman (S shore) Jezioro Wielewskie Jezioro Wielewskie lake in Wiele Pond Giewont I (NW shore) Pond 300 m W of Libiszów Jezioro Małe Zmarłe lake (N shore) Pond Kościuszko (N shore) Jezioro Zmarłe lake (W shore) Jezioro Kły lake (E shore) Jezioro Bialskie lake (E shore) Wigry/ Wigry/ Zatoka Słupiańska bay Pond Plany (S shore) *Wyżni Czerwony *Wyżni Stawek lake in the Mts Tatra *Concrete-lined pond in Botanic Garden of Jagiellonian University in Kraków Main pond in Botanic Garden of Jagiellonian University in Kraków Waterbody no. Waterbody 32 31 30 29 28 46 45 44 27 43 26 42 25 41 24 40 23 39 22 38 21 37 36 20 35 19 18 34 33 49 48 47 15b Table 1 . Table

J. LENARCZYK & K. WOŁOWSKI: PHENOTYPIC PLASTICITY OF PEDIASTRUM S.L. 77 P. tetras P.

+ + + + · · · · + + · + · · · · + + + + + + + + + · + · + · · + +

subgranulatum cf. P.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · + · · · simplex var. simplex P.

+ + · + · · · · · · · + · · · · + · · · + · · · · · · · + · · · +

echinulatum var. simpex P.

· · · · + · · · · · · · · · · + + · + · + · + · + · + · · · · · + P. orientale P.

· · · · · · · · · · · · · · · · · · + · + · · · + · · · · · · · · P. kawraiskyi P.

· · · · · · · · · · · · + · · · · · · · · · · · · · · · · · · · · integrum var. integrum P.

· · · · · · · · · · · · · · · · · · · · · + · · · · · · · · · · ·

rugulosum var. duplex P.

· + + · · · · · · · · · · · · · + + + · · + · · + · · · · · · · · gracillimum var. duplex P.

· · · · · · · · · · + · · · + · · · · · · · · · + · · · · · · · ·

duplex var. taxa duplex P.

+ + · · · · · + · + · · · · · · · · · · · · · · · · · · · · · + + asperum var. duplex P.

· + + · · · · · · · + · · · · · + · + · · + · · · · · · + · · · · pseudoglabrum var. boryanum P.

· + · + · · · · · · · · + · · · + · · · · + · · · · · · · · · · + perforatum var. boryanum P.

· · + · · · · · · · · · · · · · · · · + · · + · · · · · · · · · ·

Pediastrum longicorne var. boryanum P.

· + + · · · · · · · + · · · + · · + · + · · + · · · · + · + + · · forcipatum var. boryanum P.

· · · + · · + · · · · + + · · · + + + · · + · + · · + · + · · · + cornutum var. boryanum P.

· · · · · · · · · · + · · · · · · · · · · · · · + · · · · · · · ·

brevicorne var. boryanum P.

+ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · boryanum var. boryanum P.

+ + + + + + + · · + · + · + + + + + + · + + + + · · · · + · + · +

biradiatum var. biradiatum P.

· · · · · · · · · · · · · · · · + + + · · · · + · · · · + · · · · angulosum var. angulosum P.

Table 2. Alphabetical list of cultured Pediastrum strains.

No. Taxon Strain Medium Origin of isolated coenobia (water body no.) 1 P. alternans 01.160708 Chu’s and Knop’s mixture Jezioro Gardno lake in Gardna Wielka (3) 2 P. biradiatum var. biradiatum 02.040908 Chu’s Pond Hetman (southern shore) (38) 3 P. biradiatum var. biradiatum 05.040908 Chu’s Pond Hetman (southern shore) (38) Concrete-lined pond in Botanic Garden of 4 P. boryanum var. brevicorne 06.030608 Chu’s and Knop’s mixture Jagiellonian University in Kraków (48) 5 P. boryanum var. cornutum 19.090508 Chu’s Jezioro Zatorek lake

6 P. boryanum var. longicorne 02.290811a L-S2T2 Wyżni Czerwony Stawek lake (49) Concrete-lined pond in Botanic Garden of 7 P. boryanum var. longicorne 13.280608 Chu’s and Knop’s mixture Jagiellonian University in Kraków (48) Concrete-lined pond in Botanic Garden of 8 P. boryanum var. longicorne 22.280608 Chu’s and Knop’s mixture Jagiellonian University in Kraków (48) 9 P. duplex cf. var. duplex 16.160708 Chu’s and Knop’s mixture Jezioro Jamno lake in Unieście (1) 10 P. duplex var. rugulosum 10.090508 Chu’s Jezioro Zatorek lake 11 P. duplex var. rugulosum 13.090508 Chu’s and Knop’s mixture Jezioro Zatorek lake 12 P. integrum var. integrum 09.170408 Chu’s and Knop’s mixture Jezioro Warnowskie lake 13 P. orientale 12.160708 Chu’s and Knop’s mixture Jezioro Łebsko lake in Kluki (4)

14 P. cf. subgranulatum 19.300112 S2T2 Wyżni Czerwony Stawek Lake (49)

Table 3. Nine structural elements on the surface of coenobia in 21 analyzed Pediastrum taxa, and the states used in statistical analysis. 0 – no, 1 – yes.

Taxon mesh mesh ridges spines punctae granules verrucae wrinkles tetragonal polygonal trigonal mesh trigonal P. alternans 0 0 1 0 1 0 0 0 0 P. angulosum var. angulosum 0 0 0 0 0 1 0 0 0 P. biradiatum var. biradiatum 0 1 0 0 1 0 0 0 0 P. boryanum var. boryanum 0 1 0 0 0 0 1 0 0 P. boryanum var. brevicorne 0 1 0 0 0 0 1 0 0 P. boryanum var. cornutum 0 1 0 0 0 0 1 0 0 P. boryanum var. forcipatum 1 1 0 0 0 0 0 0 0 P. boryanum var. longicorne 0 1 0 0 0 0 1 0 1 P. boryanum var. perforatum 0 1 0 0 0 0 1 0 0 P. boryanum var. pseudoglabrum 1 1 1 0 0 0 0 0 0 P. duplex var. asperum 0 0 0 0 0 1 0 0 0 P. duplex var. duplex 0 0 0 0 0 0 0 0 0 P. duplex var. gracillimum 0 0 0 0 0 0 0 0 0 P. duplex var. rugulosum 0 1 0 0 0 0 0 0 1 P. integrum var. integrum 0 1 0 0 0 0 1 0 1 P. kawraiskyi 1 0 1 0 0 0 0 0 0 P. orientale 0 1 0 0 1 0 0 0 0 P. simplex var. echinulatum 0 0 0 1 0 0 1 1 0 P. simplex var. simplex 0 1 0 0 0 0 1 0 1 P. cf. subgranulatum 0 1 0 0 0 0 1 0 0 P. tetras 0 0 0 0 0 0 0 0 0 J. LENARCZYK & K. WOŁOWSKI: PHENOTYPIC PLASTICITY OF PEDIASTRUM S.L. 79 aldehyde was first rinsed with 10% ethanol (3× 20 min) made the pattern resemble the granulate type (3) and then dehydrated in a series of nine ethanol solutions in P. boryanum var. forcipatum. Besides the ver- increasing from 20% to 100% (20–30 min each) and rucate pattern (type 4) composed of large verrucae finally in 100% acetone (2 × 30 min). The material and punctae, an old coenobium of P. kawraiskyi was placed in micropore capsules, critical-point dried (Fig. 2l) exhibited scabrate-type (2) ornamentation with CO2 in an Anderson apparatus, and mounted on aluminum stubs with double-sided carbon tape. All the composed of tiny verrucae and punctae, making materials were sputter-coated with carbon or gold and the species most similar to P. boryanum var. pseu- viewed using Hitachi S-4700 or JEOL JSM 5410 scan- doglabrum and P. boryanum var. forcipatum in ning electron microscopes at the Institute of Geology the classification diagram (Fig. 1). In contrast to and the Institute of Zoology of the Jagiellonian Uni- P. kawraiskyi, P. alternans (Fig. 5a–c) had only versity, Kraków. one wall pattern, composed of wrinkles and ver- The nine structural elements observed on the surface rucae (type 6b), which was not shared with other of coenobia in 21 analyzed taxa and the states used species. Pediastrum alternans formed one clade in statistical analysis are given in Table 3. The wall with P. biradiatum var. biradiatum (Fig. 4k–l) and patterns of Pediastrum taxa were compared by hierar- chical clustering in Past ver. 2 (Hammer et al. 2001) P. orientale (Fig. 4g–j), because the three taxa were and visualized as a classification dendrogram (Fig. 1). covered with wrinkles (Table 3, Fig. 1). Clustering was based on Jaccard’s coefficient and the The reticulate pattern with a trigonal mesh UPGMA method. Morphological terminology follows and granules (type 7c) was shared by the highest Parra (1979) and Ziembińska-Tworzydło and Kohlman- number of taxa (8), mostly P. boryanum varieties. Adamska (2003). The classification system used here The density and size of granules, as well as the for Pediastrum follows Komárek and Jankovská (2001). shape of the mesh, differed between and within some P. boryanum varieties. The densest granula- Results tion, up to 18–20 granules per cell width, was observed in P. boryanum var. boryanum (Fig. 6b) and Depending on the occurrence or absence of ul- P. boryanum var. brevicorne A. Braun (Fig. 6c), trastructural elements, seven types of wall pat- and the least dense (6–7 granules per cell width) tern were distinguished: (1) smooth, (2) scabrate, in P. boryanum var. boryanum, P. boryanum var. (3) granulate, (4) verrucate, (5) araneose, (6) ru- longicorne Reinsch (Fig. 6d–f) and P. boryanum gulate, and (7) reticulate; they are described in var. perforatum (Racib.) Nitardy (Fig. 6i). Granule Table 4. Rugulate ornamentation comprised two density was a stable feature in P. boryanum var. subtypes based on the elements situated on the longicorne, but SEM revealed differences in the wrinkles; reticulate ornamentation comprised three shape of granules and mesh between cultures from subtypes depending on the shape of mesh and the different localities. The granules were delicate and elements on it. the mesh trigonal in coenobia from lowland waters Most of the patterns were shared by at least two (Fig. 6d, e), but the granules were prominent and taxa. Three ornamentation types were represented rounded, and the mesh polygonal or trigonal, in by single taxa: granulate (3) by P. boryanum var. a strain from Wyżni Czerwony Stawek lake in the forcipatum (Corda) Chodat (Fig. 3a–h), verrucate Tatra Mountains (Fig. 6f). (4) by P. kawraiskyi Schmidle (Figs 2 & 3i–l), Similarly to P. boryanum, P. simplex Meyen and rugulate with verrucae (6b) by P. alternans varieties were also covered with mesh and ad- Nygaard (Fig. 5a–c) (Table 4, Fig. 1). There were ditional elements. Pediastrum simplex var. echi- some irregularities in the granulate wall pattern of nulatum Wittr. was covered with spines disposed P. boryanum var. forcipatum which made the pat- on a trigonal or tetragonal mesh (type 7b; Figs 1 tern resemble the scabrate type (2) in P. boryanum & 5h–k). However, the differences between the var. pseudoglabrum Parra (Fig. 2f–k). The surface spines of this variety and the granules of P. simplex of P. boryanum var. pseudoglabrum was some- var. simplex (Fig. 6l) were so slight in some cells times covered with short rows of granules, which that it was difficult to distinguish the two taxa. 80 POLISH BOTANICAL JOURNAL 61(1). 2016

abc

d e f

g h i

j k l

Fig. 2. Cell wall surface in Pediastrum Meyen: smooth (a–e) and scabrate (f–l). a, c, d – P. duplex Meyen var. duplex, b – P. duplex cf. var. duplex, e – P. tetras (Ehrenb.) Ralfs, f–k – P. boryanum var. pseudoglabrum (Turpin) Menegh., l – P. kawraiskyi Schmidle. Arrows – punctae (f, h, l) or rows of granules (k). Scale bars: a–c – 40 µm; g – 20 µm; d–f, h–l – 5 µm. J. LENARCZYK & K. WOŁOWSKI: PHENOTYPIC PLASTICITY OF PEDIASTRUM S.L. 81

abc

d e f

g h i

j k l

Fig. 3. Cell wall surface in Pediastrum Meyen: granulate (a–h) and verrucate (i–l). a–h – P. boryanum var. forcipatum (Corda) Chodat, i–l – P. kawraiskyi Schmidle. Arrows – granules (b), punctae (d–f) or deviations of cell wall (g, h). Scale bars: a, i – 40 µm; b, c, j – 20 µm; e, g, h, k – 10 µm; d, f, l – 5 µm. 82 POLISH BOTANICAL JOURNAL 61(1). 2016

a b c

d e f

g h i

j k l

Fig. 4. Cell wall surface in Pediastrum Meyen: araneose (a–f) and rugulate with granules (g–l). a & b – P. angulosum (Ehrenb.) Menegh. var. angulosum, c–f – P. duplex var. asperum (A. Braun) Hansg., g–j – P. orientale (Skuja) Jankovská & Komárek, k & l – P. biradiatum Meyen var. biradiatum. Arrows – granules (i). Scale bars: a, b – 60 µm; c, d – 40 µm, e–h; l – 10 µm; i–k – 5 µm. J. LENARCZYK & K. WOŁOWSKI: PHENOTYPIC PLASTICITY OF PEDIASTRUM S.L. 83

a b c

d e f

g hi

j k l

Fig. 5. Cell wall surface in Pediastrum Meyen: rugulate with verrucae (a–c), reticulate with granules on polygonal mesh (d–g), reticulate with spines on trigonal or tetragonal mesh (h–k), and reticulate with granules on trigonal mesh (l). a–c – P. alternans Nygaard, d–g – P. duplex var. rugulosum Racib., h–k – P. simplex var. echinulatum Wittr., l – P. boryanum (Turpin) Menegh. var. boryanum. Arrows – verrucae (b), granules (g) or spines (j, k). Scale bars: a, d, h, i, l – 40 µm; f, g – 10 µm; b, c, e, j, k – 5 µm. 84 POLISH BOTANICAL JOURNAL 61(1). 2016

a b c

d e f

g h i

j k l

Fig. 6. Wall surface: reticulate with granules on trigonal mesh, in Pediastrum Meyen. a – P. boryanum (Turpin) Menegh. cf. var. boryanum, b – P. boryanum var. boryanum, c – P. boryanum var. brevicorne A. Braun, d–f – P. boryanum var. longicorne Reinsch, g & h – P. boryanum var. cornutum (Racib.) Sulek, i – P. boryanum var. perforatum (Racib.) Nitardy, j – P. cf. subgranulatum (Racib.) Jankovská & Komárek, k – P. integrum Nägeli var. integrum, l – P. simplex Meyen var. simplex. Scale bars: d, g, h, j – 20 µm; e, k, l – 10 µm; a–c, f, i – 5 µm. J. LENARCZYK & K. WOŁOWSKI: PHENOTYPIC PLASTICITY OF PEDIASTRUM S.L. 85

Discussion may be connected with a taxonomical problem in P. orientale pointed out by Kowalska and Among the 21 taxa investigated, only Pediastrum Wołowski (2010b). Based on the variability of wall alternans (Fig. 5a–c) is characterized by a single surface and cell shape, Kowalska and Wołowski wall pattern – rugulate with verrucae (type 6b) (2010b) suggested that P. orientale is a complex – not shared with other taxa (Table 4, Fig. 1). It taxon composed of several similar species. Further follows that this pattern is a reliable diagnostic studies comparing the morphology and phyloge- character for the species. Kowalska and Wołowski netic relations of its monoclonal strains are needed (2010b) proposed verrucae as a new taxonomical to address the taxonomical problem; until it is re- character for P. alternans, since Nygaard (1949) solved, wall ornamentation cannot be regarded as did not mention it in the original description. a diagnostic character in P. orientale. In our analysis the wall pattern of P. alternans The variability of the reticulate pattern with proved to be most similar to that of P. biradiatum granules disposed regularly on a trigonal mesh var. biradiatum (Fig. 4k–l) and P. orientale (Fig. (type 7c) is so great that this pattern has little di- 4g–j) in the classification dendrogram (Fig. 1). agnostic value for Pediastrum taxa. Pediastrum These three taxa have wrinkles on their surface, boryanum var. boryanum (Fig. 6b), with its wide but with different additional elements: verrucae in range of granule density, is a good example of that P. alternans and granules in the other two. The three variability. Lenarczyk (2015) stated that granule taxa are not morphologically similar (Lenarczyk density within a single P. boryanum var. bory- 2014). No molecular data on P. orientale have been anum strain does not change significantly when published so far, but some of our preliminary data cultured at different nutrient concentrations, but in indicate that P. alternans forms a clade with P. bo- preliminary studies, McManus and Lewis (2005) ryanum strains. This means that the similarities in found that the nutrient status of the culture me- wall ultrastructure in P. alternans, P. biradiatum dium influences phenotypic variability in a strain var. biradiatum and P. orientale could have arisen with P. boryanum-like morphology, including independently in their evolution. granule density. In our present study we found Our description of the wall surface in P. orien- that granule density in P. boryanum var. brevicorne tale differs from those given by other authors. (Fig. 6c) was within the range of P. boryanum var. Komárek and Jankovská (2001) described its or- boryanum (Fig. 6b). According to Komárek and namentation as a mesh with granules, Sulek (1969) Jankovská (2001), these varieties differ from each as rugulate with tiny depressions, and Nygaard other in the length of processes and the depth of (1977) as covered with pores in oblique decussate marginal cell incisions, but Nielsen (2000) stated series. The differences between the descriptions that the variety brevicorne should be rejected and

Table 4. Types of wall surface in Pediastrum taxa noted in the present study.

Type Wall pattern Description 1 smooth without any concave or convex elements (Fig. 2a–e) 2 scabrate tiny verrucae and punctae, exceptionally short rows of granules (Fig. 2f–l) 3 granulate granules in regular rows and punctae (Fig. 3a–h) 4 verrucate verrucae or verrucae and punctae (Fig. 3i–l) 5 araneose ridges lying irregularly (Fig. 4a–f) 6 rugulate a winkles with granules (Fig. 4g–l) b winkles with verrucae (Fig. 5a–c) 7 reticulate a granules on a polygonal mesh (Fig. 5d–g) b spines on a trigonal or tetragonal mesh (Fig. 5h–k) c granules on a trigonal mesh (Figs 5l, 6a–l) 86 POLISH BOTANICAL JOURNAL 61(1). 2016 included into the nominate variety, as their cell Lewis]; the two strains differed only slightly in shape varies depending on ontogeny and there is their 26S rDNA and rbcL sequences. no difference in granule density. Therefore density We also observed some wall features differing should not be regarded as a diagnostic character from those given in the literature as character- for the variety brevicorne. We also found that the istic for P. simplex var. simplex, P. duplex var. coenobia of P. boryanum var. cornutum (Racib.) rugulosum Racib., P. tetras (Ehrenb.) Ralfs and Sulek (Fig. 6g, h) and P. boryanum var. perfo- P. duplex var. duplex. In P. simplex var. simplex ratum (Fig. 6i) are similar in granule density and (Fig. 6l) the granules were disposed regularly on in coenobium shape and cell shape. This supports a trigonal mesh (type 7c), irregularly on a po- Nielsen’s (2000) doubts about the distinction be- lygonal mesh (type 7a), but not on wrinkles as tween these varieties. stated by Komárek and Jankovská (2001). Ac- Some preliminary data (Lenarczyk & Saługa cording to Parra (1979), trigonal mesh occurs in 2013) indicate that in the phylogenetic tree the younger specimens and then turns into an irregular strain P. boryanum var. longicorne isolated from pattern whose elements are similar to wrinkles mountains (Table 2, no. 6) is not closely related to building Y-shaped connections. Besides the reticu- two other strains of P. boryanum var. longicorne late ornamentation in P. simplex var. simplex, Parra isolated from lowland (Table 2, nos 7 and 8). That (1979) observed fossulate ornamentation in his suggestion is supported by morphological differ- newly described P. simplex var. pseudoglabrum. ences between the lowland and mountain strains In P. duplex var. rugulosum the granules were we observed in the present study. The variety lon- of different sizes (Fig. 5d–g). The bigger ones gicorne is a complex taxon. covered the mesh or wrinkles to which they were The taxonomical position of P. boryanum var. affixed (Fig. 5g). Hindák and Hindáková (2008) pseudoglabrum (Fig. 2i–h) and P. boryanum var. may also have observed coenobia with such gran- forcipatum (Fig. 3a–h) is questionable as well, ules, as they stated that the surface of this taxon is in view of their similar cell shape and deviations covered with granules, not reticulate with granules from the scabrate (type 2) and granulate (type 3) (type 7a). wall patterns. According to Lenarczyk (2014), The single coenobium of P. tetras observed P. boryanum var. pseudoglabrum probably is P. bo- by SEM (Fig. 2e) was smooth with sparse sites ryanum var. forcipatum with not well developed of roughness resembling granules or verrucae. granulate ornamentation. Parra (1979) noted various ornamentation types To distinguish between P. simplex var. simplex in this species: irregularly granulate, reticulate, (Fig. 6l) covered with granules and P. simplex var. and rugulate with granules. Shubert and Wilk- echinulatum (Fig. 5h–k) covered with spines, and Woźniak (2003) found a rugulate coenobium in to identify the range of spine length in the latter a waterbody in Puławy, central Poland. Wu (1987) variety from field material, analysis of these fea- distinguished P. tetras var. tetras with a reticu- tures in monoclonal cultures is necessary. By that late pattern and P. tetras var. tetraodon (Corda) method the length of structural elements, spines Hansg. with a granulate one. McManus and Lewis and granules can be measured in specimens un- (2005) observed a rugulate/reticulate surface in ambiguously representing individual P. simplex different strains of P. tetras from Europe and North genotypes. Parra (1979) and Wu (1987) did not dis- America, and thick wrinkles in another strain from tinguish the variety echinulatum, but determined North America. Komárek and Jankovská (2001) P. simplex coenobia with spines as P. simplex var. and McManus and Lewis (2005) indicated that simplex. McManus and Lewis (2011) genetically strains with P. tetras-like morphology may repre- analyzed single strains of P. simplex var. simplex sent separate taxa. [as Monactinus simplex var. simplex (Meyen) A smooth surface (type 1) was observed in Corda] and P. simplex var. echinulatum [as M. sim- two P. duplex varieties: P. duplex var. duplex plex var. echinulatum (Wittr.) H. McManus & L. A. (Fig. 2a, c, d) and P. duplex var. gracillimum. Due J. LENARCZYK & K. WOŁOWSKI: PHENOTYPIC PLASTICITY OF PEDIASTRUM S.L. 87 to the shared surface type, Sulek (1969) and Parra the full range of wall variability in individual taxa (1979) proposed that the latter variety should be in- throughout their life cycle, and to determine the cluded into the nominate one. However, McManus impact of particular environmental factors more et al. (2011) used molecular and morphometric precisely. methods to describe a new genus Lacunastrum H. McManus and a new combination L. gracil- Acknowledgements. We thank Professor Brian A. Whitton and Michael Jacobs for reviewing the Eng- limum (W. & G. S. West) H. McManus (formerly lish, the anonymous reviewers for helpful remarks and P. duplex var. gracillimum). suggestions on the manuscript, and Professor Lubomira We observed the last type of wall surface – Burchardt for the water samples from Jezioro War- araneose (type 5) – in two varieties from different nowskie lake and Jezioro Zatorek lake used to initiate species: P. angulosum (Ehrenb.) Menegh. var. an- some of the Pediastrum cultures. The study was funded gulosum (Fig. 4a, b) and P. duplex var. asperum in part by the Polish Ministry of Science and Higher (A. Braun) Hansg. (Fig. 4c–f). McManus and Education for 2008–2010 (grant no. N N303 070534) Lewis (2011) reported phylogenetic results based and by the statutory fund of the W. Szafer Institute of on a single strain of P. duplex var. asperum and Botany, Polish Academy of Sciences, including in-house grants for young scientists in 2012 and 2015. seven strains of P. angulosum, which were grouped in two different clades consisting mainly of P. duplex, not assigned names to variety level. Thus, it References seems likely that araneose ornamentation evolved independently in various Pediastrum lineages. An S. S., Hegewald E. & Jeon S. L. 1999. Pediastrum privum In addition to differences in wall patterns be- (Printz) Hegewald new to Korea. Algae 14: 83–85. tween taxa, our SEM study of cultured material Buchheim M., Buchheim J., Carlson T., Braband A., Hep- perle D., Krienitz L., Wolf M. & Hegewald E. 2005. disclosed changes in wall patterns during coeno- Phylogeny of the Hydrodictyaceae (Chlorophyceae): infer- bium growth and aging. In older specimens the ences from rDNA data. J. 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Received 13 August 2015