Ecological variation within traditional diatom morphospecies: diversity of Frustulia rhomboides sensu lato (Bacillariophyceae) in European freshwater habitats JANA VESELA ´ *, PAVLA URBA ´ NKOVA ´ ,KATER ˇ INA C ˇ ERNA ´ AND JIR ˇ I ´ NEUSTUPA Department of Botany, Faculty of Science, Charles University in Prague. Bena ´tska ´ 2, CZ-12801 Prague, Czech Republic VESELA ´ J., URBA ´ NKOVA ´ P., C ˇ ERNA ´ K. AND NEUSTUPA J. 2012. Ecological variation within traditional diatom morphospecies: diversity of Frustulia rhomboides sensu lato (Bacillariophyceae) in European freshwater habitats. Phycologia 51: 552–561. DOI: 10.2216/11-101.1 More than 300 strains of Frustulia rhomboides sensu lato were isolated from populations across Europe to estimate its molecular diversity and geographic distribution in oligotrophic freshwater habitats. Phylogenetic analyses, based on partial large subunit (LSU) rDNA sequences, revealed six lineages that did not fully correspond with the separation of F. rhomboides into traditional varieties. Although four closely related lineages showed substantial overlaps in morphology, the characteristic morphological features could be determined using morphometric techniques. The phylogenetic lineages of F. rhomboides showed different patterns of distribution among regions. Sequence frequencies within samples from different habitats indicated that distribution of the common lineages most likely reflected their different ecological preferences rather than dispersal constraints. KEY WORDS: Biogeography, Cryptic species, Diatoms, Frustulia rhomboides, Geometric morphometrics, LSU rDNA INTRODUCTION A multidisciplinary approach to the discovery and delimita- tion of protist species (e.g. Behnke et al. 2004; Mann et al. 2004; Lowe et al. 2005a; Pro ¨schold & Leliaert 2007; Weisse et al. 2008) has renewed a debate regarding species concepts (Mann 1999, 2010; de Queiroz 2005, 2007; Fenchel & Finlay 2006). Molecular genetic methods, morphometric tech- niques, breeding experiments, and cultivation under con- trolled conditions have revealed hidden diversity within traditional morphologically defined species. However, conflicting evidence regarding species boundaries indicated that rates of morphological and molecular evolution may be decoupled (Philippe et al. 1994; Alverson 2008), and reproductively compatible strains may be isolated by spatial or temporal separation (Behnke et al. 2004; Casteleyn et al. 2008). Furthermore, the molecular markers may not reflect the ecophysiological differentiations of the strains (Fenchel 2005; Lowe et al. 2005b; Weisse 2008), and the measured theoretical niche may be broader than the realized niche (Boenigk et al. 2007; Vanelslander et al. 2009). These phenomena suggest that species discovery and description should be based upon a combination of results acquired by different approaches instead of relying on any single approach (de Quieroz 2007; Mann 2010). Frustulia rhomboides (Ehrenberg) De Toni sensu Krammer & Lange-Bertalot (1986) contains five intraspecific taxa in Europe: F. rhomboides var. rhomboides, F. rhomboides var. saxonica (Rabenhorst) De Toni, F. rhomboides var. crassi- nervia (Brebisson) Ross, F. rhomboides var. amphipleuroides (Grunow) De Toni, and F. rhomboides var. viridula (Brebisson) Cleve. Recent nomenclatural changes raised these morphotypes to the species level, Frustulia krammeri Lange-Bertalot & Metzeltin, Frustulia saxonica Rabenhorst, Frustulia crassinervia (Brebisson) Lange-Bertalot & Krammer, Frustulia amphipleuroides (Grunow) Cleve-Euler, and Frus- tulia erifuga Lange-Bertalot & Krammer (for details, see Lange-Bertalot & Metzeltin 1996; Lange-Bertalot & Jahn 2000; Lange-Bertalot 2001). However, these taxonomic changes and the greater number of micrographs of natural populations from diverse geographic areas have instead complicated the distinctions between F. krammeri (F. rhomboides), F. saxonica, and F. crassinervia (Siver & Baskette 2004). The most commonly reported taxa world- wide are the morphotypes F. saxonica and F. crassinervia (Lange-Bertalot 2001; Siver & Baskette 2004; Beier & Lange-Bertalot 2007); both are characteristic members of phytobenthos in acid, peaty waters (such as Sphagnum bogs), and many ephemeral habitats (Krammer & Lange- Bertalot 1986; Round et al. 1990; Wehr & Sheath 2002). In the present study, diverse European regions and habitats were sampled to obtain monoclonal cultures in order to (1) analyse the phylogenetic structure, (2) assess the qualitative and quantitative morphological variation of the lineages, and (3) describe the distribution patterns of the lineages. MATERIAL AND METHODS Between October 2007 and June 2010, 62 benthic samples were taken from diverse freshwater oligotrophic biotopes (ombrotrophic peat bogs, minerotrophic mires, Sphagnum dominated littoral of lakes, and ephemeral habitats) in nine European regions. Localities in the Czech Republic (CZ), western Ireland (IE), and south-western France (FR) were studied in detail; samples from other localities were incidentally collected, i.e. the Azores Islands (PT), Ger- many (DE), the Netherlands (NL), Slovakia (SK), Slovenia * Corresponding author (vesela6@natur.cuni.cz). Phycologia (2012) Volume 51 (5), 552–561 Published 4 September 2012 552