Macroevolutionary patterns in intragenomic rDNA variability among planktonic foraminifera

Introduction

Ribosomal genes (rDNA) play a central role in cellular molecular processes and are present in multiple copies throughout eukaryotic and prokaryotic genomes (Weider et al., 2005; Espejo & Plaza, 2018). The occurrence of multiple copies in the genome is tied to the high demand for ribosomes, the organelles into which the expressed rRNA assembles, making rDNA the most transcribed genome locus (Neidhart, Ingraham & Schaechter, 1990; Kobayashi, 2014). Although high levels of transcription are well-known mutagenic sources (Kim & Jinks-Robertson, 2012), the rDNA gene sequences exhibit a striking homogeneity within a genome. This uniformity is maintained through evolutionary time within a species, resulting from the molecular process of concerted evolution (Elder & Turner, 1995; Espejo & Plaza, 2018), but divergence becomes evident when rDNA sequences from different species are compared. The process preventing divergence among the multiple gene copies within a single genome is gene homogenization, where related genes within a species undergo genetic exchange (Dover, 1982) and the molecular mechanisms that induce it (gene conversion, gene amplification and unequal crossing-over) have been identified in both eukaryotes and prokaryotes (Dover, 1982; Liao, 2000). However, multiple factors can affect the homogenization of ribosomal genes which interfere with concerted evolution, resulting in intragenomic variability appearing among rDNA copies within an organism. This phenomenon is particularly common in microbial organisms, where rDNA intragenomic variability has been linked to the presence of a particularly large number of gene copies per cell (Gong et al., 2013), the displacement of rDNA gene copies among different nuclei (Lanfranco, Delpero & Bonfante, 1999), the presence of non-coding rDNA copies, i.e., pseudogenes (Gribble & Anderson, 2007), and inter-specific hybridization (Boucher et al., 2004; Pillet, Fontaine & Pawlowski, 2012).

Since ribosomal genes are widely used markers for metabarcoding surveys, research on rDNA intragenomic variability has been focused on its biasing effects on microbial diversity or phylogenetic assessments (Thornhill, Lajeunesse & Santos, 2007; Sun et al., 2013; Zhao et al., 2019; Hassler et al., 2022; Sandin, Romac & Not, 2022). Despite the growing literature quantifying intragenomic variability in microbial species, knowledge of how this trait evolves across taxa is remarkably incomplete. Here we use the microbial group of the foraminifera as a case study since their intragenomic variability is widespread (Weber & Pawlowski, 2014). We particularly focus on the planktonic species. This group has a known evolutionary history that can be reconstructed from the fossil record (Aze et al., 2011) and their modest taxonomic diversity with about 50 extant species (Brummer & Kucera, 2022) is structured into three clades, the Spinose, the Non-Spinose and the Microperforate. Most importantly, because of extensive barcoding efforts, the diversity in the planktonic foraminifera group has a near-complete taxonomic coverage of the 3′ end of the SSU rDNA gene, with highly replicated sampling among the taxa (Morard et al., 2015). This comprehensive coverage enables us to systematically evaluate intragenomic variability throughout the group and assess the patterns and macroevolutionary mechanisms responsible for generating such variability. To this end, we expanded the resolution and scope of the existing data by amplifying long sequences using clone libraries, resulting in a collection of 2,403 partial SSU sequences. The data now encompasses five hypervariable regions (or helices) from 33 species of planktonic foraminifera distributed across the major clades of the group. We used these sequences to (i) assess the incidence of intragenomic polymorphism in each species, (ii) examine its effects on the secondary structure of the rDNA molecule, and (iii) explore the evolutionary patterns of the observed incidence and degree of sequence variability in the context of planktonic foraminifera evolutionary history as reconstructed from the fossil record.

Materials and Methods

Results

The incidence of intragenomic variability varied greatly across the hypervariable regions and the species analysed (Fig. 1). On average, Non-Spinose species had a higher incidence, reaching levels beyond 50% for each of the helices studied. Within this clade, the maximum values were observed in G. eastropacia (100%, 37f), N. pachyderma (75%, 41f), G. conglomerata (100%, 43e and 45e–47f), G. hexagonus (100%, 43e), G. menardii (100%, 45e–47f) and G. truncatulinoides (70%, 49e). Conversely, the incidence was above 50% in only the three Spinose species T. sacculifer (100%, 41f), G. elongatus (70%, 45e–47f) and G. siphonifera (75%, 49e) and absent in G. conglobatus. Intragenomic variability was also prevalent among the Microperforate taxa (Fig. 1). Their average incidence was 53%, ranging between 13% and 100% except for G. minuta, which displayed no polymorphism.

To investigate the extent of the mutations causing the polymorphism, we proceeded to analyse the number of polymorphic sites and tested for differences between clades (Fig. 2). We found that the number of polymorphic sites varied between 1 and 36 base pairs. Remarkably, the number of times the presence of polymorphisms was detected in a specimen (i.e., mutation events) was substantially higher in Non-Spinose species, irrespectively of the number of polymorphic sites ascribable to the specific mutation event (Fig. 2B). In addition, the species D. anfracta and B. variabilis (Other in Fig. 2A), exhibited a distribution centred towards a higher number of polymorphic sites (IQR = 8).

The Kruskal–Wallis test showed that the number of polymorphic sites statistically differed among the clades analysed (p = 0.01) (Fig. 2A). Specifically, the number of polymorphic sites was found to be significantly smaller in the Spinose clade compared to the Microperforate (p = 0.02), and Non-Spinose (p = 0.004) clades and in comparison with the species D. anfracta and B. variabilis (p = 0.01) as revealed by the paired Dunn test (Fig. 2).

To assess the effect of polymorphisms on the SSU secondary structure, we counted the number of mutations located in the Stem or Loop regions of the molecule to obtain the Stem/Loop ratio (SLr) (Figs. 2C and 2D) and tested for differences between clades. The SLr assumed values larger than 0 in 88% of the polymorphisms observed, indicating that a higher number of mutations were located in the stem regions in the vast majority of the specimens. The Kruskal–Wallis test showed that the SLr statistically differed among the clades analysed (p = 0.01) (Fig. 2D). In particular, the paired Dunn test (Fig. 2) indicated that the SLr was significantly higher in the Microperforate clade compared to the Spinose (p = 0.02), and Non-Spinose (p = 0.03) species.

We further investigated the macroevolutionary signal behind intragenomic variability in planktonic foraminifera by conducting the Abouheif’s C_mean phylogenetic test (Abouheif, 1999). We performed the analysis separately for each morphogroup, using the fossil phylogeny for Spinose and Non-Spinose (Aze et al., 2011) and the molecular phylogeny for Microperforate species (Morard et al., 2019). The species-specific traits tested were: the median number of polymorphic sites (Mps), the average number of polymorphic sites (Aps) and the overall incidence of intragenomic variability (Iniv). A significant positive phylogenetic signal was retrieved in the Non-Spinose phylogeny for the Aps (C_mean = 0.28, p = 0.02) and Mps (C_mean = 0.35, p = 0.01) traits.

Finally, the trait with the strongest phylogenetic signal, Mps, was selected for the reconstruction of the ancestral state and plotted on the fossil phylogeny of each morphogroup. The median number of polymorphic sites varied within major clades but was on average higher in Non-Spinose (3.34) and Microperforate (4.73) than in Spinose (1.75). The highest Mps values were observed in the Spinose species T. quinqueloba (Mps = 8), the Non-Spinose species N. pachyderma (Mps = 8), N. incompta (Mps = 11) and the Microperforate species T. iota (Mps = 8.5). Importantly, the analysis suggested that some polymorphic sites (~3 base pairs) were present in the ancestor of the group and have been successively reduced in some lineages, mostly Spinose, but multiplied in others (Fig. 3).

A stark reduction for polymorphic sites was also evident when data from the benthic Rotaliida and planktonic clades were compared (Fig. 4). The boxplot in Fig. 4 shows that the Mps for benthic Rotaliida (Mps = 12) was three times larger than in Microperforates (Mps = 4), four times larger than in Non-Spinose (Mps = 3) and six times larger than in Spinose (Mps = 2) for the 37f, 41f and 43e helices.

Discussion

As previously shown for benthic foraminifera (Holzmann, Piller & Pawlowski, 1996; Pillet, Fontaine & Pawlowski, 2012; Weber & Pawlowski, 2014; Girard et al., 2022), the occurrence of intragenomic variability is also widespread among planktonic foraminifera (Fig. 1). The incidence of this trait is particularly high among the Non-Spinose and Microperforate species together with the closely related species B. variabilis and D. anfracta. Importantly, our analysis shows that intragenomic polymorphisms are also found on the V9 region in 24 of the species analysed, with incidence values above 60% in G. truncatulinoides and G. siphonifera (Fig. 1). The V9 is a widely used marker for assessing the diversity of microbial plankton in metabarcoding surveys (e.g., de Vargas et al., 2015; Piredda et al., 2017), thus the extensive presence of intragenomic variability in this region calls for a careful interpretation of diversity estimates of planktonic foraminifera community based on this marker.

Admittedly, while providing a valid overview of the occurrence of intragenomic variability in planktonic foraminifera, the incidence is impacted by the different cloning efforts (sampling bias) for the species present in our compiled dataset (Fig. 1). Such disparity is likely the result of earlier observations that showed no intragenomic variability among Spinose lineages (André et al., 2014) and high levels among Non-Spinose and Microperforates (Darling et al., 2006; Morard et al., 2016, 2019), which possibly skewed later cloning effort towards species belonging to these two clades. Another factor contributing to this asymmetric effort is the good quality data resulting from the direct sequencing of Spinose specimens that, thus, do not require cloning for obtaining interpretable sequences.

Conversely, the number of polymorphic sites and SLr are robust measures, independent of the number of specimens analysed. Together, these two parameters can provide a verifiable framework to understand potential sources and mechanisms behind the extent and evolution of intragenomic variability in planktonic foraminifera ribosomal genes.

The first pattern emerging from our analyses is that the occurrence of polymorphic sites on the ribosomal genes is a common trait in every planktonic foraminifera clade (Figs. 1 and 2). A potential explanation for this result is that we included non-coding sequences in our analyses (Gribble & Anderson, 2007). However, our RNA molecule simulations suggest that it is unlikely that the sequences analysed are pseudogenes since most of the polymorphic sites are located on paired (stem) regions (Fig. 2D), indicating that their ribosomal secondary structure is subject to functional constraints (Smit, Widmann & Knight, 2007). Alternatively, the presence of a particularly large number of rDNA copies has also been proposed as a source of intragenomic polymorphisms in ciliates (Gong et al., 2013) and fungi (Simon & Weiss, 2008). A recent investigation using single-cell qPCR has shown that planktonic foraminifera carry between ~300 and 350,000 copies of the SSU genes, a trait shared among species of different clades (Milivojevic et al., 2021) and common also in benthic lineages (Weber & Pawlowski, 2013). Notably, in their study, Milivojevic et al. (2021), show that the number of gene copies in planktonic foraminifera varies between different species and could reflect the ploidy status of the sequenced specimens.

Another genomic feature associated with intragenomic polymorphism is nuclear dualism or heterokaryosis. It has been proposed that, in ciliates, protists characterised by the presence of a diploid micronucleus and a polyploid macronucleus, polymorphic sites could result from rDNA amplification occurring during the development or amitotic divisions of the macronucleus (Wang et al., 2019). Importantly, canonical heterokaryosis has been also reported in several benthic foraminifera species within the order Rotaliida (reviewed in Goetz et al. (2022)), the clade from which modern planktonic lineages diversified from benthic ancestors (Pawlowski, Holzmann & Tyszka, 2013).

Finally, intragenomic polymorphism could be the result of interspecific hybridization as shown for some benthic species (Pillet, Fontaine & Pawlowski, 2012), however, given that no evidence of this phenomenon exists for planktonic foraminifera (Weiner et al., 2014), we can rule it out as a possible explanation of our results.

Together, these observations can help provide an interpretation for the widespread occurrence of intragenomic polymorphism in planktonic clades (Fig. 1) as well as its presence in the ancestral nodes of the group (Fig. 3): we speculate that this genomic feature could represent an evolutionary relict deriving from benthic ancestors characterised by nuclear dualism and/or disproportionately high rDNA gene copy number.

The high number of polymorphic sites among benthic species shown in Fig. 4 also suggests that intragenomic variability is a shared trait within the order Rotaliida and therefore, potentially present in the benthic ancestors of the planktonic clades. Furthermore, our analysis indicates a substantial reduction in the number of polymorphic sites in the planktonic taxa (Fig. 4). Interestingly, this pattern appears to trace the estimated time of diversification of the planktonic clades from the benthos (Morard et al., 2022). The Spinose clade is the most ancient and on average displays a lower number of polymorphic sites than the more recently diversified Microperforate clade which shows the highest (Fig. 4). This suggests that planktonic clades have gradually reduced their intragenomic variability through their evolution.

We should also note that our explanation potentially implies the presence of heterokaryosis in modern planktonic foraminifera, a hypothesis that would require further investigations employing laboratory techniques like DAPI staining combined with confocal microscopy to be confirmed (e.g., Bellec, Maurer-Alcala & Katz, 2014).

The second pattern emerging from our analyses is that there are significantly fewer polymorphic sites present in the rDNA copies of the Spinose clade compared to those of the Microperforate and Non-Spinose clades. (Fig. 2A). This observation holds across the different magnitudes of mutation events observed, ruling out the possibility that the difference is simply the result of single polymorphic sites potentially ascribable to random errors introduced during the polymerase chain reaction amplification process (Fig. 2B).

The prevalence of intragenomic variability in Non-Spinose and Microperforates suggests the presence in these species of a mechanism that hinders the process of gene homogenization of the rDNA gene copies, amplifying the effects of the elevated gene copy number or nuclear dualism discussed above. The explanation might lie in the different life cycle strategies of some planktonic foraminifera species. Previous studies on ciliates and fungi have shown that in multinucleate organisms, intragenomic rDNA polymorphism is higher when multiple macronuclei or nuclei are present within the same cell or spore (Lanfranco, Delpero & Bonfante, 1999; Zhao et al., 2019). This is because the rDNA gene copies emerging during genome multiplication in different (macro) nuclei undergo gene conversion within the same nucleus but not between all nuclei, originating polymorphic copies within the same specimen (Lanfranco, Delpero & Bonfante, 1999). Indeed, during the asexual generation, foraminifera are multinucleated while sexually reproducing individuals only possess a single nucleus (Goetz et al., 2022). To date, asexual reproduction has been only reported in Microperforate and Non-Spinose species (Davis et al., 2020; Takagi, 2020; Meilland et al., 2022), while Spinose species have only been observed to reproduce via gametogenesis (i.e., sexually) (e.g., Bijma, Erez & Hemleben, 1990). We speculate that the incidence of asexual reproduction in a lineage could drive the elevated number of polymorphic sites that we observe in these two clades. Although the prevalence of multinucleated asexually reproducing planktonic foraminifera needs further investigation, the significant phylogenetic signal of Mps and Aps recorded in the Non-Spinose group suggests that intragenomic polymorphism is a heritable trait, albeit likely due to a secondary correlation with a higher incidence of asexual reproduction within the clade.

Conclusions

In this study, we investigated the occurrence and extent of intragenomic variability in planktonic foraminifera by analysing a compilation of 2,403 sequences from the clone libraries of 33 species. Our analysis shows that intra-individual mutations on SSU genes are common across all studied clades, but are more prevalent in Non-Spinose and Microperforate species. Furthermore, we identified the number of polymorphic sites as an important macroevolutionary trait, possibly reflecting the evolution of the nuclear and genomic architecture in the group. Our findings significantly advance our understanding of the molecular biology of planktonic foraminifera and underline the importance of studying genomic features in a phylogenetic context to gain insights into the evolutionary history of microbes.

Supplemental Information