A time-calibrated multi-gene phylogeny of the diatom genus Pinnularia

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Abstract

Pinnularia is an ecologically important and species-rich genus of freshwater diatoms (Bacillariophyceae) showing considerable variation in frustule morphology. Interspecific evolutionary relationships were inferred for 36 Pinnularia taxa using a five-locus dataset. A range of fossil taxa, including newly discovered Middle Eocene forms of Pinnularia, was used to calibrate a relaxed molecular clock analysis and investigate temporal aspects of the genus’ diversification. The multi-gene approach resulted in a well-resolved phylogeny of three major clades and several subclades that were frequently, but not universally, delimited by valve morphology. The genus Caloneis was not recovered as monophyletic, confirming that, as currently delimited, this genus is not evolutionarily meaningful and should be merged with Pinnularia. The Pinnularia–Caloneis complex is estimated to have diverged between the Upper Cretaceous and the early Eocene, implying a ghost range of at least 10 million year (Ma) in the fossil record.

Highlights

► A well-resolved 5-gene phylogeny of the diatom genus Pinnularia is presented. ► Three large clades were detected, containing morphologically recognizable subclades. ► Divergences were placed in time using a fossil-calibrated relaxed molecular clock. ► We report the earliest known fossil of Pinnularia from the Lutetian (>40 Ma). ► The origin of Pinnularia is estimated at 75–50 Ma, implying a ghost range of >10 Ma.

Introduction

Diatoms are an extremely diverse group of unicellular algae that are uniquely characterized by a siliceous cell wall (the frustule) consisting of two valves and a number of girdle bands (Round et al., 1990) and a diplontic life cycle involving gradual size reduction during vegetative divisions and rapid size restitution, usually through sexual reproduction (Chepurnov et al., 2004). In the so-called pennate diatoms the valve pattern is organized bilaterally around the longitudinal axis, and in most cases the valve is elongate. Raphid pennate diatoms possess a pair of longitudinal slits along the apical axis (the raphe system) (Fig. 1), from which extracellular polymeric substances are exuded and used in locomotion and for adhesion to the substratum (Round et al., 1990). The raphe is considered a derived character state that distinguishes the raphid diatoms from the more ancestral araphid pennate forms that lack this structure and from the oldest known forms, the radially organized “centric” taxa (Sims et al., 2006). Based on fossil remains, the araphid pennates first appeared in the Upper Cretaceous (ca. 75 Ma; Chambers, 1966, Hajós and Stradner, 1975), and the raphe-bearing forms soon thereafter, around 70.6–55.8 Ma (Chacon-Baca et al., 2002, Pantocsek, 1889, Singh et al., 2006, Witt, 1886). Monophyly of pennate diatoms as a whole, as well as the raphid pennates, has been documented using SSU rDNA and rbcL sequences (e.g. Kooistra et al., 2003, Sorhannus, 2004, Sorhannus, 2007). Since their origin, raphid pennate diatoms have diversified enormously and account for the majority of the over 200,000 extant species estimated to exist (Mann and Droop, 1996), indicating the evolutionary advantages conferred by the raphe (Sims et al., 2006).

Despite the diversity and ecological success of raphid pennate diatoms, relatively few detailed molecular phylogenetic reconstructions exist. Phylogenies applied at genus to ordinal levels have yielded partly unsupported taxon relationships (e.g. Bruder and Medlin, 2008, Trobajo et al., 2009), in part due to very limited taxon sampling and/or the use of a limited number of genetic markers (Mann and Evans, 2007). In addition, molecular phylogenies of individual genera have focused largely on the identification of cryptic diversity rather than the elucidation of evolutionary relationships between lineages (e.g. Beszteri et al., 2007, Evans et al., 2008, Lundholm et al., 2006). There are a few explicit time-calibrated phylogenies at the ordinal level (e.g. Medlin et al., 1996), the diatoms as a whole (e.g. Kooistra and Medlin, 1996, Medlin et al., 1997a, Sorhannus, 2007), the wider heterokont group (e.g. Brown and Sorhannus, 2010, Medlin et al., 1997b), or even eukaryotes (e.g. Berney and Pawlowski, 2006), but there are none at the generic level; furthermore, few analyses formalize the evolutionary associations between the timing of lineage splitting and ecological, morphological, physiological and/or reproductive strategies, life cycles and geographical distributions (but see Casteleyn et al., 2010). Furthermore, despite important recent micropaleontological discoveries, some of which confirm that particular genera of raphid diatoms are older than previously suspected (Siver and Wolfe, 2007, Siver et al., 2010), numerous gaps remain in the fossil record. As a result, the overall course of evolution in raphid pennate diatoms is not known and the relationships between many groups remain uncertain (Mann and Evans, 2007).

Pinnularia Ehrenberg (1843) is one of the most species-rich genera of raphid pennate diatoms, with 2465 taxon names recorded in Algaebase of which 412 are currently accepted (Guiry and Guiry, 2011). The genus occurs globally in freshwater habitats of varying pH and trophic status, and to a lesser extent in moist soils, peatlands, spring seeps and marine coastal environments (Krammer, 2000, Round et al., 1990). Members of Pinnularia and the closely related genus Caloneis Cleve (1894) have linear-lanceolate, blunt-ended or occasionally capitate valves with a central raphe system (Fig. 1) that terminates internally in helictoglossae at the poles (Figs. 1b and 2b, d, f and h). They possess either two plate-like plastids (Fig. 1y and z) or a single H-shaped plastid (Fig. 1a′–c′). Pinnularia is characterized by the presence of a chambered, double-walled valve structure in which the outer surface is ornamented by multiple rows of small pores forming multiseriate striae (detail in Fig. 2c), whereas the inner wall of each chamber or alveolus is perforated by a large transapically elongate aperture (Figs. 1b and 2b, d, f and h) (Round et al., 1990). Caloneis is similar, except that the inner aperture is smaller and often circular, and sometimes there are two apertures per stria. Despite the species richness, morphological diversity and ecological significance of Pinnularia and Caloneis in freshwater and terrestrial ecosystems, the evolutionary relationships among species are poorly known. Bruder et al. (2008) constructed a molecular phylogeny of Pinnularia and Caloneis using 18S, 28S and rbcL genes for 15 species. Whereas some well-supported Pinnularia lineages were recovered and Caloneis was scattered among Pinnularia, overall support for the clades was low, suggesting that more exhaustive sampling, with respect to both taxa and genetic markers, is needed to produce a well-resolved phylogeny.

The temporal aspects of the diversification of Pinnularia are also poorly documented, despite the fact that numerous fossils have been reported for the genus. To date, the oldest known diatoms reliably assigned to Pinnularia originate from the Wagon Gap Formation of Wyoming, USA (Lohman and Andrews, 1968). The age of these sediments is not known precisely because diatom-containing sediment clasts have been redeposited in a carbonate conglomerate, but their age lies between the Late Eocene and Early Oligocene (3532 Ma). Dating from the same period, the Oamaru diatomite deposits also contain two species of Pinnularia (Desikachary and Sreelatha, 1989). From the Early Miocene onwards, diverse morphological types of Pinnularia are reported from freshwater deposits or as freshwater inwash in marine deposits (for Miocene deposits including Pinnularia see for e.g. Hajós, 1986, Héribaud, 1902, Lewis et al., 2008, Li et al., 2010, Ognjanova-Rumenova and Vass, 1998, Pantocsek, 1886, Saint Martin and Saint Martin, 2005, Servant-Vildary et al., 1988, VanLandingham, 1991, Yang et al., 2007). The origin of Pinnularia thus predates the Late Eocene, and the occurrence of several taxa within the Wagon Gap material suggests an even earlier origin for the genus. Thus, despite the well-established fossil record dating from after the diversification of Pinnularia into different morphological types, the earlier fossil record is very scarce and too fragmentary to provide detailed information about the timing of early diversification events. With this in mind, and given that ghost lineages are anticipated to be common in algae in general and diatoms in particular (Brown and Sorhannus, 2010), it is particularly relevant to produce a well-resolved, time-calibrated phylogeny for this genus.

The goal of the present study is to reconstruct a molecular phylogeny for a representative selection of Pinnularia taxa spanning the morphological variability of the genus, and to infer a time-calibrated phylogeny constrained by accurately dated fossil representatives. To achieve this, we sequenced two nuclear markers (18S rDNA, 28S rDNA), two plastid markers (rbcL, psbA) and a mitochondrial marker (cox1) from 36 species of the genus and inferred phylogenies using partitioned models in a likelihood framework. We also present new Pinnularia fossils from the Middle Eocene of Canada, which are included as constraints in the relaxed molecular clock analyses.

Section snippets

Taxon sampling

In a first step, the rbcL and LSU of 139 cultured Pinnularia and Caloneis strains were sequenced as described below and all available additional rbcL and LSU sequences downloaded from GenBank. From a maximum parsimony guide tree produced from these sequences, one strain per species was selected (in a few cases, sequence divergence was so high that we selected two or even more strains) for sequencing of three additional loci (18S rDNA, psbA and cox1). Sequences from additional species that were

Dataset properties

Our 44-taxon, five-locus dataset is 87% filled (192 sequences; Table 1) and includes 4852 sites of which 1012 (21%) are parsimony-informative. The most complete markers are 28S rDNA and rbcL (0 of 44 missing), followed by 18S rDNA (4 sequences or 9% missing), psbA (8 or 18% missing) and cox1 (16 or 36% missing). Missing sequences were due to failed PCR or because they were not available in GenBank (Table 1). Previous studies revealed that taxa with missing sequence data (up to 95% of missing

Relationships within Pinnularia

The current study presents a multi-gene phylogeny of the genus Pinnularia and extends the sampling of Bruder et al. (2008) threefold. Our five-locus analysis yielded a well-supported phylogenetic tree, with Pinnularia and Caloneis resolved in a single monophyletic clade, in accordance with the results of Bruder and Medlin (2008) and Bruder et al. (2008). Our phylogeny shows that Caloneis (as currently defined) is not monophyletic, but rather that its species were recovered in various places

Acknowledgments

We thank Aloisie Poulíčková for providing cultures of Pinnularia borealis and Elie Verleyen for providing live environmental samples. Alex Ball enthusiastically assisted with confocal microscopy. Sequencing was done by Andy Vierstrate. Funding was provided by the Fund for Scientific Research-Flanders (FWO-Flanders, comprising a PhD fellowship to CS, post-doctoral fellowships to HV and PV, and Research Grant 3G/0533/07). Research on fossil diatoms is partially supported by the US National

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    Present address: Laboratory of Aquatic Ecology and Evolutionary Biology, Katholieke Universiteit Leuven, Charles Debériotstraat 32, 3000 Leuven, Belgium.

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