Original PaperIncomplete Reproductive Isolation Between Genetically Distinct Sympatric Clades of the Pennate Model Diatom Seminavis robusta
Introduction
Over a relatively short period of evolutionary time ( <240 Ma), diatoms have diversified into an estimated 100.000 extant species or more, making them the most species-rich group of microalgae (Mann and Vanormelingen 2013). These estimates are mainly based on the increased use of molecular tools for species distinction, which have revealed that diatom biodiversity had been severely underestimated by traditional morphology-based approaches (Behnke et al., 2004, Godhe and Rynearson, 2017, Mann, 1999). It is now generally accepted that (semi)cryptic diversity, i.e. the existence of genetically distinct diatom species with identical or similar morphologies, is common, with many (semi)cryptic species living in sympatry (Beszteri et al., 2005, Lefebvre et al., 2017, Pinseel et al., 2017, Quijano-Scheggia et al., 2009, Vanelslander et al., 2009, Vanormelingen et al., 2008).
However, the big question is not so much what species are, but what evolutionary processes drive their divergence and keep them distinct (Shapiro et al. 2016). Darwin postulated that species arise by natural selection and competition, keeping them in separate ecological niches (Darwin 1859). Mayr formulated the biological species concept (BSC), in which reproductive isolation is responsible for the maintenance of species as distinct genetic entities. Under the original formulation of the BSC by Ernst Mayr (Mayr 1942), species are defined as “genetically cohesive groups of populations that are reproductively isolated from other such groups”. Although the BSC has become widely embraced in the last decades, critics highlight that it does not give a clear indication of how strong reproductive isolation has to be for two population to qualify as ‘good species’ (Leliaert et al. 2014). For example, studies of genetic structures of plant and animal populations show that gene flow is often substantial between populations that are generally recognized as distinct species (Arnold, 1997, De Queiroz, 2007, Mallet, 2005). An estimated 1-10% of all animal species and up to 25% of plant species can hybridize with at least one other species in their natural environment (Mallet, 2005, Schwenk et al., 2008). These observations urged a broadening of the reproductive isolation criterion for species status to include populations that show “substantial but not necessarily complete” reproductive isolation (Coyne and Orr, 2004, De Queiroz, 2007). Hybrid zones, natural habitats where differentiated populations come into contact and are able to hybridize, were recognized early on as natural laboratories for ecology and evolutionary biology (Hewitt 1988). Since then, hybridization and incomplete reproductive isolation have been well-studied in various macro-organisms such as fish (Harrison et al. 2017), birds (Gill 2014) and land plants (Christe et al. 2016). These increased efforts to identify mechanisms causing reproductive isolation resulted in a renewed interest in the role of ecological and/or sexual selection for the emergence and maintenance of reproductive barriers (Faria et al. 2014).
Despite the enormous species richness of diatoms, little is known about the mechanisms driving diatom speciation, especially sympatric speciation. Consistent with the BSC, a key challenge is to examine the mechanisms and extent of reproductive isolation between populations (Coyne and Orr 2004). Moreover, reproductive isolation is often considered as the hallmark for diatom speciation because of the obligate nature of sexual reproduction in most diatom life cycles, which is related to the physical constraints imposed by their silica cell wall (Chepurnov et al. 2004). Combined with data on genetic divergence, characterization of (incomplete) reproductive isolation may provide insights into diatom speciation mechanisms (Edmands, 2002, Vanormelingen et al., 2008). For this reason, the issue of reproductive isolation in closely related diatom species complexes has gained considerable attention in the last decade (Adams et al., 2009, Amato et al., 2007, Casteleyn et al., 2009, Casteleyn et al., 2010, Chen and Rynearson, 2016, Godhe et al., 2014, Harnstrom et al., 2011, Vanormelingen et al., 2008). However, experimental evidence for incomplete reproductive isolation and widespread hybridization between genetically distinct diatom populations is limited.
Seminavis robusta D.B.Danielidis & D.G.Mann was presented by Chepurnov et al. (2008) as a model system to study life cycle regulation and sexual reproduction in raphid pennate diatoms. Like most raphid pennates, S. robusta has a heterothallic mating system, with two mating types (MT+ and MT−) that form pairs once their cell size drops below the sexual size threshold (SST) of 50 μm. The initial cell size is then restored by gametogenesis, gamete fusion and subsequent auxosporulation. This type of reproductive behavior is considered representative for pennates, the most species-rich group of diatoms (Chepurnov et al. 2008). The status of S. robusta as a model species for sexual reproduction in pennates was further reinforced by the characterization of the chemical signaling pathway involved in mate finding (Gillard et al., 2013, Moeys et al., 2016) and the first description of the genetic basis of sex determination in a diatom (Vanstechelman et al. 2013).
Here, we have explored the genetic and morphological diversity in a set of natural S. robusta isolates collected from different locations in Belgium and the Netherlands. We describe three genetically distinct populations that occur in sympatry. Despite their phylogenetic distinctness, we demonstrate widespread potential for gene flow between these populations.
Section snippets
S. robusta Consists of Three Distinct rbcL Clades
A total of 125 monoclonal S. robusta cultures were established from natural populations originating from the brackish Veerse Meer (VM) and Lake Grevelingen (GM) in the Netherlands (73 and 27 strains, respectively) and from the Spuikom (KOM) coastal lagoon in Belgium (25 strains) (Fig. 1, Table 1). We first sequenced the nuclear encoded ITS1-5.8S-ITS2 (ITS), the hypervariable domains D1-D3 of the 28S rDNA (LSU) and the plastid-encoded RuBisCo (rbcL) gene for a set of 49 strains (32 VM, 9 GM and
Discussion
Phylogenetic and morphometric analyses reveal that natural populations of S. robusta comprise three sympatrically occurring rbcL clades, one of which (clade III) is also morphologically differentiated from the other two. Whereas earlier studies on S. robusta focused on the mechanisms involved in cell and life cycle control (Gillard et al., 2008, Gillard et al., 2013, Moeys et al., 2016, Vanstechelman et al., 2013), more systematic sampling of natural populations revealed a previously undetected
Methods
Sampling area, strain collection and culturing: An initial set of 15 S. robusta strains was isolated in 2012 from a natural sample taken at the VM3 site in the Veerse Meer (the Netherlands). These strains were labeled as PONTON and HASCH instead of VM3, but originate from the same location. Mating type designation of these strains was done by crossing these isolates with S. robusta strains of known mating type (Gillard 2009). As described below, these PONTON and HASCH strains were used as a
Conflicts of Interest
None.
Acknowledgements
The authors thank Sofie D’Hondt (Ghent University) for her help with PCR amplifications and sequence editing and the three anonymous reviewers for their useful comments on the manuscript. This research work was supported financially by the Fund for Scientific Research – Flanders (FWO-Flanders, Belgium, grant number G0D6114N).
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- 1
Corresponding author;
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Present address: Natuurpunt, Coxiestraat 11, 2800 Mechelen, Belgium
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Present address: Department of Marine Sciences, Gothenburg University, Box 461, 40530 Gothenburg, Sweden