Protist, Vol. 170, 121–140, April 2019 http://www.elsevier.de/protis Published online date 13 October 2018 ORIGINAL PAPER Extensive Cryptic Diversity in the Terrestrial Diatom Pinnularia borealis (Bacillariophyceae) a,b,c,1,2 d,2 d d Eveline Pinseel , Jana Kulichová , Vojtechˇ Scharfen , Pavla Urbánková , b,c,3 a,3 Bart Van de Vijver , and Wim Vyverman a Protistology & Aquatic Ecology (PAE), Department of Biology, Faculty of Science, Ghent University, Krijgslaan 281-S8, B–9000 Ghent, Belgium b Research Department, Botanic Garden Meise, Nieuwelaan 38, B–1860 Meise, Belgium c Ecosystem Management Research Group (ECOBE), Department of Biology, Faculty of Science, University of Antwerp, Universiteitsplein 1, B–2610 Wilrijk, Antwerp, Belgium d Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, CZ–12801 Prague 2, Czech Republic Submitted April 30, 2018; Accepted October 2, 2018 Monitoring Editor: Wiebe H. C. F. Kooistra With the increasing application of molecular techniques for diatom species discovery and identification, it is important both from a taxonomic as well as an ecological and applied perspective, to understand in which groups morphological species delimitation is congruent with molecular approaches, or needs reconsideration. Moreover, such studies can improve our understanding of morphological trait evo- lution in this important group of microalgae. In this study, we used morphometric analysis on light microscopy (LM) micrographs in SHERPA, detailed scanning electron microscopy (SEM), and cyto- logical observations in LM to examine 70 clones belonging to eight distinct molecular lineages of the cosmopolitan terrestrial diatom Pinnularia borealis. Due to high within-lineage variation, no con- clusive morphological separation in LM nor SEM could be detected. Morphological stasis due to the “low-morphology” problem or stabilizing selection, as well as parallel/convergent evolution, phenotypic plasticity and structural inheritance are discussed as potential drivers for the observations. Altogether, P. borealis is truly cryptic, in contrast to the majority of other diatom species complexes which turned out to be pseudo-cryptic following detailed morphological analysis. © 2018 Elsevier GmbH. All rights reserved. Key words: Diatoms; LSU rDNA; molecular phylogenies; morphometrics; rbcL; shape. Introduction Diatoms (Bacillariophyta) are highly diverse and, 1 Corresponding author. 2 with an estimated number of species ranging These authors contributed equally and should be considered between 30,000 and 200,000 (Mann 1999; Mann shared first authors 3 These authors contributed equally and should be considered and Vanormelingen 2013), are generally consid- shared last authors ered to be the most species-rich group of algae on e-mail [email protected] (E. Pinseel). https://doi.org/10.1016/j.protis.2018.10.001 1434-4610/© 2018 Elsevier GmbH. All rights reserved. 122 E. Pinseel et al. Earth. Diatoms are widely used in various applied and mosses (Krammer 2000; Krammer and Lange- sciences such as water quality biomonitoring (Kelly Bertalot 1988; Souffreau et al. 2013b), which is 1998) and paleo-ecological reconstructions (Smol well-adapted to living in extreme environments and Stoermer 2010), mainly because of the pos- (Hejduková et al. 2017; Souffreau et al. 2013a; sibility of long-term preservation of their siliceous Stock et al. 2018). Occasionally, P. borealis can cell wall in marine, lake and peat sediments and also be found living in the littoral zones of fresh- the specific ecological preferences of individual water lakes and ponds (Pinseel et al. 2017a; taxa. These applications are based on the assump- Rumrich et al. 2000). Based on morphological tion that individual species can be identified based features of the cell wall discernible in LM, tax- on the morphological features of the cell wall dis- onomists separated about 66 different varieties and cernible in light microscopy (LM). Nevertheless, formae within P. borealis (Kociolek et al. 2018), an increasing number of molecular studies has although most of these considerably overlap in their shown that many, often common, diatom mor- morphological features resulting in an uncertain phospecies harbour distinct molecular variation, taxonomic status. Recent molecular phylogenetic which likely corresponds to species-level differ- analyses based on the nuclear encoded D1–D3 entiation (Beszteri et al. 2007; Kermarrec et al. region of the LSU rDNA (28S) and the plastid 2013; Pinseel et al 2017b; Quijano-Scheggia et al. gene rbcL revealed that P. borealis consists of sev- 2009; Souffreau et al. 2013b; Vanelslander et al. eral distinct lineages that likely separated several 2009; Vanormelingen et al. 2013). The presence millions of years ago (Souffreau et al. 2013b). Sev- of (pseudo)cryptic diversity not only hampers the eral of these lineages showed niche-differentiation use of diatom morphologies in applied sciences, regarding optimal growth temperature and upper but also influences our understanding of species temperature limits for growth, altogether suggest- distributions, niche differentiation and diatom evo- ing they represent distinct species (Souffreau et al. lution. However, in recent years, the development 2013b). However, the molecular lineages identified of various tools for quantitative morphometric anal- by Souffreau et al. (2013b) did not show obvi- ysis of diatom valves, such as landmark analysis ous morphological discontinuities in LM preventing (Bookstein 1997) or SHERPA (Kloster et al. 2014, their separation using traditional measurements of 2016), has revealed that several members of these their valve morphologies, such as length, width morphospecies complexes are morphologically dif- and stria densities. Nevertheless, although they ferentiated in a subtle way, often involving small could shed light on the true morphological diver- differences in valve shape (Beszteri et al. 2005a; sity of this species complex, detailed morphometric Kloster et al. 2018; Mann et al. 2004; Poulíckovᡠtechniques were not used, nor was ultrastruc- et al. 2010; Veselá et al. 2009). Some studies ture examined. The possibility to reliably identify found a link between morphological and ecolog- lineages within P. borealis using morphological fea- ical differentiation (Kulichová and Fialová 2016; tures would be valuable in light of recent interests Potapova and Hamilton 2007; Urbánková et al. to use terrestrial diatom species for monitoring of 2016). In other cases, morphometric analyses soil ecological quality (Barragán et al. 2018) or to were used to distinguish or describe species with provide the much desired link between molecu- highly similar morphologies (Fránková et al. 2009; lar studies and original type material for which no Mann et al. 2004; Theriot 1992; Van de Vijver genetic material is available. Furthermore, consid- et al. 2013). Morphometric methods can thus be ering the amount of already available data on P. a powerful tool to study the morphological varia- borealis, this taxon forms an ideal model system to tion in diatoms, and to reveal potential links among study the (morphological) evolution and differentia- morphological, molecular and eco(physio)logical tion of species-level lineages within diatoms. differentiation between and within closely related In this study, monoclonal cultures of P. borealis diatom taxa. This could have important implica- were established, originating from various (sub- tions for (paleo)environmental inferences where )polar, temperate and Mediterranean sites in the morphologically similar and often difficult to identify Northern and Southern hemisphere. First, using diatom taxa are usually lumped together, thereby their sequences of the nuclear encoded D1–D3 ignoring the potential presence of different eco- LSU rDNA (28S) and the plastid gene rbcL, the phy- types. logenetic position of these newly obtained strains One diatom species complex that has gained was assessed in relation to the already existing P. interest over the last years is Pinnularia borealis borealis phylogeny (Pinseel et al. 2017a; Souffreau Ehrenberg. It is a common, cosmopolitan com- et al. 2013b) and additional new lineages within P. ponent of (semi)terrestrial habitats such as soils borealis were delineated. The choice for D1–D3 Cryptic Diversity in Pinnularia borealis 123 28S was motivated by the fact that this marker Results proved to be highly variable in the genus Pinnularia Ehrenberg, highlighting its usefulness to distinguish Taxon Sampling between closely related Pinnularia species (Kollár In total 48 new strains of Pinnularia borealis were et al. 2018). Although rbcL is distinctly less vari- established (Fig. 1, Supplementary Material Table able in the genus Pinnularia (Kollár et al. 2018), S1). The dataset was complemented with cul- it is a useful marker to provide a good resolu- ture material from 22 P. borealis strains already tion at deeper nodes in the phylogeny. Second, published in Souffreau et al. (2013b) (Fig. 1, Sup- morphometric analyses and ultrastructural analy- plementary Material Table S1), resulting in a total of sis in scanning electron microscopy (SEM) were 70 strains of P. borealis to be examined for their mor- performed on all newly obtained strains as well phology in this study. For the phylogenetic analysis as a selected subset of previously studied strains (see below), all published strains were analysed (Souffreau et al. 2013b), to check the hypothesis to put the results in a wider context (Supplemen-