Molecular systematics

The advent of molecular approaches in foraminiferal systematics over the past 20 years has provided new insights in elucidating species relationships and resolving taxonomic uncertainty. Phylogenetic analysis is a powerful approach that provides new lines of taxonomic evidence for species identifications and assessments of biodiversity. These new insights are crucial, as historically species relationships were inferred from differences in the test morphology (as discussed in Section 1.3.1). The molecular approach uses genetic divergence to infer species concepts and evolution (Bowser et al., 2006). Molecular studies have also helped to resolve the phylogenetic position of foraminifera in relation to other distant taxonomic groups, e.g. eukaryotic and prokaryotic protists (Pawlowski et al., 1997; Pawlowski and Holzmann 2002; Pawlowski and Burki, 2009). Moreover, these new lines of taxonomic evidence have also revealed the occurrence of foraminifera in freshwater and terrestrial environments (Holzmann et al., 2003; Lejzerowicz et al., 2010).

Ribosomal RNA genes (rRNA) are the most frequently used genes to resolve phylogenetic relationships between and within taxonomic groups (e.g. Pawlowski and Holzmann, 2002; Schweizer et al., 2008). Although a limited number of other proteins including actin (Fahrni et al., 1997; Flakowski et al., 2005), RNA polymerase (RPB1) (Longet et al., 2003; Flakowski et al., 2005, 2006), ubiquitin (Archibald et al., 2003) and tubulin (Tuschiya, 2003; Habura et al., 2005) have been successfully amplified to elucidate taxonomic relationships. Nevertheless, the ribosomal genes are most commonly employed in molecular systematics, as they possess the advantage of being abundant with several hundreds of copies in each cell, thus providing the opportunity to amplify the rRNA from a single foraminiferal specimen (Bowser et al., 2006). Whilst the small subunit (SSU) rRNA is the most commonly used gene within molecular

systematics (Pawlowski et al., 2013), sequences of the large ribosomal subunit (LSU) have also been used to great effect to elucidate the taxonomic positions of a number of benthic foraminiferal species(e.g. Hayward et al., 2004). The SSU rRNA is considered the optimal genetic marker in phylogenetic approaches, as it exhibits more divergence between species than the LSU rRNA (Bowser et al., 2006).

1.6.1

Cryptic diversity

The new lines of taxonomic evidence provided by molecular systematics have brought into question the criteria by which foraminiferal species are classified, as this approach has revealed previously unrecognised genetic diversity within many of the classical morphospecies concepts. Cryptic species can be defined as “Two or more species that have been classified as a single nominal species and are superficially morphologically indistinguishable” (Bickford, 2007, p.149). Thus, cryptic species are thought to be the product of the inability of classic morphology to resolve species divergence at the evolutionary level (Amato et al., 2007). Cryptic species are a common phenomenon within plantkic foraminifera. For example, out of 26 morphospecies concepts of planktic foraminifera, 66 genetically distinct species were identified (Darling et al., 1999; de Vargas, 1999; Darling et al., 2000, Darling et al., 2004, Darling et al., 2007; Pawlowski and Lecroq, 2010; as reviewed in Darling and Wade, 2008). In contrast, cryptic speciation is seemingly less prevalent within benthic foraminifera and to date cryptic species have only been identified within a limited number of taxa. Cryptic species were found within Ammonia

(Pawlowski et al., 1995; Holzmann et al., 1996; Holzmann and Pawlowski 1997, 2000; Hayward et al., 2004; Pawlowski et al., 2008; Schweizer et al., 2008; Schweizer et al., 2011),

Planoglabratella (Tsuchiya et al., 2000; 2003), Chilostomella (Grimm et al., 2007) and in some monothalous foraminifera (Gooday et al., 2004; Gooday and Pawlowski, 2004; Pawlowski and Holzmann, 2008). The new lines of taxonomic evidence have also revealed the presence of ‘reverse’ cryptic diversity, whereby there is high morphological plasticity but low genetic diversity (Schweizer et al., 2005; André et al., 2012).

The potential misidentification of species using classical morphology based taxonomy has significant ramifications for the interpretations of past, current and future estimates of biodiversity and the understanding of foraminiferal biogeographical distributions. For example, hidden species richness affects the interpretation of ecosystem properties including its stability, identification of the key (foundation) species and trophic levels (Bickford et al., 2007). In addition, the emergence of previously unrecognised cryptic species, could lead to the redefinition of species ecological ranges and preferences. This can have significant implications

for palaeoenvironmental reconstructions, as these investigations are underpinned by an understanding of the biogeography and ecological preferences of extant species e.g. species- specific geochemical calibrations (Murray, 2006). The clarification of cryptic species with molecular systematics within planktic foraminifera has identified the potential to reduce errors in palaeoenvironmental reconstructions (Malgrem et al., 2001; Kucera and Darling, 2002; Darling and Wade, 2008). This highlights the importance of resolving taxonomic relationships within benthic foraminiferal taxonomy in order to improve and constrain climate models. Whilst the advent of molecular systematics has helped to resolve taxonomic relationships within foraminiferal taxonomy, it is important to highlight that this approach is not without its own set of limitations. For example, the robustness of genetic delineations is dependent upon access to large numbers of sequences to establish divergence and to contextualise the delineations (Bowser, 2006). A recent plea by Pawlowski and Holzmann (2014) highlighted that the main limiting factor in elucidating taxonomic relationships using a molecular approach, is that there is currently a dearth of molecular data and there is insufficient sampling at both intraspecific and interspecific levels. The robustness of the genetic delineations produced can also be affected by the choice of genetic out-group (Pawlowski et al., 1997) and potential contamination problems (Pawlowski and Holzmann, 2002). Uncertainties can also arise when rRNA of variable lengths is used to construct phylogenetic trees. This variability in the sequence length arises when only a fragment of the rRNA is successfully amplified, which makes aligning the sequences difficult (Pawlowski et al., 1997; Pawlowski and Holzmann, 2002; Foissner and Hawksworth, 2009; Tsuchiya et al., 2009; Groussin et al,. 2011).

Moreover, to date fossil specimens can only be robustly delineated based upon their test morphology. Although there have been significant advancements in recent years with the extraction of aDNA from fossil specimens (Pawlowska et al., 2014), which has provided a new avenue for assessing and understanding genetic diversity. However, this molecular approach is not currently applicable in everyday taxonomic situations and uncertainty remains about how these new lines of taxonomic evidence can be reconciled with classical morphospecies concepts and nomenclature. Hence, morphology remains invaluable for species delineation, though the need to re-examine classical morphospecies concepts and boundaries in light of new taxonomic (molecular) evidence is recognised.