The global genetic diversity of planktonic foraminifera reveals the structure of cryptic speciation in plankton.

The nature and extent of diversity in the plankton has fascinated scientists for over a century. Initially, the discovery of many new species in the remarkably uniform and unstructured pelagic environment appeared to challenge the concept of ecological niches. Later, it became obvious that only a fraction of plankton diversity had been formally described, because plankton assemblages are dominated by understudied eukaryotic lineages with small size that lack clearly distinguishable morphological features. The high diversity of the plankton has been confirmed by comprehensive metabarcoding surveys, but interpretation of the underlying molecular taxonomies is hindered by insufficient integration of genetic diversity with morphological taxonomy and ecological observations. Here we use planktonic foraminifera as a study model and reveal the full extent of their genetic diversity and investigate geographical and ecological patterns in their distribution. To this end, we assembled a global data set of ~7600 ribosomal DNA sequences obtained from morphologically characterised individual foraminifera, established a robust molecular taxonomic framework for the observed diversity, and used it to query a global metabarcoding data set covering ~1700 samples with ~2.48 billion reads. This allowed us to extract and assign 1 million reads, enabling characterisation of the structure of the genetic diversity of the group across ~1100 oceanic stations worldwide. Our sampling revealed the existence of, at most, 94 distinct molecular operational taxonomic units (MOTUs) at a level of divergence indicative of biological species. The genetic diversity only doubles the number of formally described species identified by morphological features. Furthermore, we observed that the allocation of genetic diversity to morphospecies is uneven. Only 16 morphospecies disguise evolutionarily significant genetic diversity, and the proportion of morphospecies that show genetic diversity increases poleward. Finally, we observe that MOTUs have a narrower geographic distribution than morphospecies and that in some cases the MOTUs belonging to the same morphospecies (cryptic species) have different environmental preferences. Overall, our analysis reveals that even in the light of global genetic sampling, planktonic foraminifera diversity is modest and finite. However, the extent and structure of the cryptic diversity reveals that genetic diversification is decoupled from morphological diversification, hinting at different mechanisms acting at different levels of divergence.

Macroevolutionary patterns in intragenomic rDNA variability among planktonic foraminifera.

Ribosomal intragenomic variability in prokaryotes and eukaryotes is a genomic feature commonly studied for its inflationary impact on molecular diversity assessments. However, the evolutionary mechanisms and distribution of this phenomenon within a microbial group are rarely explored. Here, we investigate the intragenomic variability in 33 species of planktonic foraminifera, calcifying marine protists, by inspecting 2,403 partial SSU sequences obtained from single-cell clone libraries. Our analyses show that polymorphisms are common among planktonic foraminifera species, but the number of polymorphic sites significantly differs among clades. With our molecular simulations, we could assess that most of these mutations are located in paired regions that do not affect the secondary structure of the SSU fragment. Finally, by mapping the number of polymorphic sites on the phylogeny of the clades, we were able to discuss the evolution and potential sources of intragenomic variability in planktonic foraminifera, linking this trait to the distinctive nuclear and genomic dynamics of this microbial group.

Genetic and morphological divergence in the warm-water planktonic foraminifera genus Globigerinoides.

The planktonic foraminifera genus Globigerinoides provides a prime example of a species-rich genus in which genetic and morphological divergence are uncorrelated. To shed light on the evolutionary processes that lead to the present-day diversity of Globigerinoides, we investigated the genetic, ecological and morphological divergence of its constituent species. We assembled a global collection of single-cell barcode sequences and show that the genus consists of eight distinct genetic types organized in five extant morphospecies. Based on morphological evidence, we reassign the species Globoturborotalita tenella to Globigerinoides and amend Globigerinoides ruber by formally proposing two new subspecies, G. ruber albus n.subsp. and G. ruber ruber in order to express their subspecies level distinction and to replace the informal G. ruber "white" and G. ruber "pink", respectively. The genetic types within G. ruber and Globigerinoides elongatus show a combination of endemism and coexistence, with little evidence for ecological differentiation. CT-scanning and ontogeny analysis reveal that the diagnostic differences in adult morphologies could be explained by alterations of the ontogenetic trajectories towards final (reproductive) size. This indicates that heterochrony may have caused the observed decoupling between genetic and morphological diversification within the genus. We find little evidence for environmental forcing of either the genetic or the morphological diversification, which allude to biotic interactions such as symbiosis, as the driver of speciation in Globigerinoides.

16S rRNA gene metabarcoding and TEM reveals different ecological strategies within the genus Neogloboquadrina (planktonic foraminifer).

Uncovering the complexities of trophic and metabolic interactions among microorganisms is essential for the understanding of marine biogeochemical cycling and modelling climate-driven ecosystem shifts. High-throughput DNA sequencing methods provide valuable tools for examining these complex interactions, although this remains challenging, as many microorganisms are difficult to isolate, identify and culture. We use two species of planktonic foraminifera from the climatically susceptible, palaeoceanographically important genus Neogloboquadrina, as ideal test microorganisms for the application of 16S rRNA gene metabarcoding. Neogloboquadrina dutertrei and Neogloboquadrina incompta were collected from the California Current and subjected to either 16S rRNA gene metabarcoding, fluorescence microscopy, or transmission electron microscopy (TEM) to investigate their species-specific trophic interactions and potential symbiotic associations. 53-99% of 16S rRNA gene sequences recovered from two specimens of N. dutertrei were assigned to a single operational taxonomic unit (OTU) from a chloroplast of the phylum Stramenopile. TEM observations confirmed the presence of numerous intact coccoid algae within the host cell, consistent with algal symbionts. Based on sequence data and observed ultrastructure, we taxonomically assign the putative algal symbionts to Pelagophyceae and not Chrysophyceae, as previously reported in this species. In addition, our data shows that N. dutertrei feeds on protists within particulate organic matter (POM), but not on bacteria as a major food source. In total contrast, of OTUs recovered from three N. incompta specimens, 83-95% were assigned to bacterial classes Alteromonadales and Vibrionales of the order Gammaproteobacteria. TEM demonstrates that these bacteria are a food source, not putative symbionts. Contrary to the current view that non-spinose foraminifera are predominantly herbivorous, neither N. dutertrei nor N. incompta contained significant numbers of phytoplankton OTUs. We present an alternative view of their trophic interactions and discuss these results within the context of modelling global planktonic foraminiferal abundances in response to high-latitude climate change.

A New Integrated Approach to Taxonomy: The Fusion of Molecular and Morphological Systematics with Type Material in Benthic Foraminifera.

A robust and consistent taxonomy underpins the use of fossil material in palaeoenvironmental research and long-term assessment of biodiversity. This study presents a new integrated taxonomic protocol for benthic foraminifera by unequivocally reconciling the traditional taxonomic name to a specific genetic type. To implement this protocol, a fragment of the small subunit ribosomal RNA (SSU rRNA) gene is used in combination with 16 quantitative morphometric variables to fully characterise the benthic foraminiferal species concept of Elphidium williamsoni Haynes, 1973. A combination of live contemporary topotypic specimens, original type specimens and specimens of genetic outliers were utilised in this study. Through a series of multivariate statistical tests we illustrate that genetically characterised topotype specimens are morphologically congruent with both the holotype and paratype specimens of E. williamsoni Haynes, 1973. We present the first clear link between morphologically characterised type material and the unique SSU rRNA genetic type of E. williamsoni. This example provides a standard framework for the benthic foraminifera which bridges the current discontinuity between molecular and morphological lines of evidence, allowing integration with the traditional Linnaean roots of nomenclature to offer a new prospect for taxonomic stability.

Nomenclature for the Nameless: A Proposal for an Integrative Molecular Taxonomy of Cryptic Diversity Exemplified by Planktonic Foraminifera.

Investigations of biodiversity, biogeography, and ecological processes rely on the identification of "species" as biologically significant, natural units of evolution. In this context, morphotaxonomy only provides an adequate level of resolution if reproductive isolation matches morphological divergence. In many groups of organisms, morphologically defined species often disguise considerable genetic diversity, which may be indicative of the existence of cryptic species. The diversity hidden by morphological species can be disentangled through genetic surveys, which also provide access to data on the ecological distribution of genetically circumscribed units. These units can be identified by unique DNA sequence motifs and allow studies of evolutionary and ecological processes at different levels of divergence. However, the nomenclature of genetically circumscribed units within morphological species is not regulated and lacks stability. This represents a major obstacle to efforts to synthesize and communicate data on genetic diversity for multiple stakeholders. We have been confronted with such an obstacle in our work on planktonic foraminifera, where the stakeholder community is particularly diverse, involving geochemists, paleoceanographers, paleontologists, and biologists, and the lack of stable nomenclature beyond the level of formal morphospecies prevents effective transfer of knowledge. To circumvent this problem, we have designed a stable, reproducible, and flexible nomenclature system for genetically circumscribed units, analogous to the principles of a formal nomenclature system. Our system is based on the definition of unique DNA sequence motifs collocated within an individual, their typification (in analogy with holotypes), utilization of their hierarchical phylogenetic structure to define levels of divergence below that of the morphospecies, and a set of nomenclature rules assuring stability. The resulting molecular operational taxonomic units remain outside the domain of current nomenclature codes, but are linked to formal morphospecies as regulated by the codes. Subsequently, we show how this system can be applied to classify genetically defined units using the SSU rDNA marker in planktonic foraminifera and we highlight its potential use for other groups of organisms where similarly high levels of connectivity between molecular and formal taxonomies can be achieved.

PFR²: a curated database of planktonic foraminifera 18S ribosomal DNA as a resource for studies of plankton ecology, biogeography and evolution.

Planktonic foraminifera (Rhizaria) are ubiquitous marine pelagic protists producing calcareous shells with conspicuous morphology. They play an important role in the marine carbon cycle, and their exceptional fossil record serves as the basis for biochronostratigraphy and past climate reconstructions. A major worldwide sampling effort over the last two decades has resulted in the establishment of multiple large collections of cryopreserved individual planktonic foraminifera samples. Thousands of 18S rDNA partial sequences have been generated, representing all major known morphological taxa across their worldwide oceanic range. This comprehensive data coverage provides an opportunity to assess patterns of molecular ecology and evolution in a holistic way for an entire group of planktonic protists. We combined all available published and unpublished genetic data to build PFR(2), the Planktonic foraminifera Ribosomal Reference database. The first version of the database includes 3322 reference 18S rDNA sequences belonging to 32 of the 47 known morphospecies of extant planktonic foraminifera, collected from 460 oceanic stations. All sequences have been rigorously taxonomically curated using a six-rank annotation system fully resolved to the morphological species level and linked to a series of metadata. The PFR(2) website, available at http://pfr2.sb-roscoff.fr, allows downloading the entire database or specific sections, as well as the identification of new planktonic foraminiferal sequences. Its novel, fully documented curation process integrates advances in morphological and molecular taxonomy. It allows for an increase in its taxonomic resolution and assures that integrity is maintained by including a complete contingency tracking of annotations and assuring that the annotations remain internally consistent.

Phylogeography of the tropical planktonic foraminifera lineage globigerinella reveals isolation inconsistent with passive dispersal by ocean currents.

Morphologically defined species of marine plankton often harbor a considerable level of cryptic diversity. Since many morphospecies show cosmopolitan distribution, an understanding of biogeographic and evolutionary processes at the level of genetic diversity requires global sampling. We use a database of 387 single-specimen sequences of the SSU rDNA of the planktonic foraminifera Globigerinella as a model to assess the biogeographic and phylogenetic distributions of cryptic diversity in marine microplankton on a global scale. Our data confirm the existence of multiple, well isolated genetic lineages. An analysis of their abundance and distribution indicates that our sampling is likely to approximate the actual total diversity. Unexpectedly, we observe an uneven allocation of cryptic diversity among the phylogenetic lineages. We show that this pattern is neither an artifact of sampling intensity nor a function of lineage age. Instead, we argue that it reflects an ongoing speciation process in one of the three major lineages. Surprisingly, four of the six genetic types in the hyperdiverse lineage are biogeographically restricted to the Indopacific. Their mutual co-occurrence and their hierarchical phylogenetic structure provide no evidence for an origin through sudden habitat fragmentation and their limitation to the Indopacific challenges the view of a global gene flow within the warm-water provinces. This phenomenon shows that passive dispersal is not sufficient to describe the distribution of plankton diversity. Rather, these organisms show differentiated distribution patterns shaped by species interactions and reflecting phylogenetic contingency with unique histories of diversification rates.

Ecological partitioning and diversity in tropical planktonic foraminifera.

BACKGROUND: Ecological processes are increasingly being viewed as an important mode of diversification in the marine environment, where the high dispersal potential of pelagic organisms, and a lack of absolute barriers to gene flow may limit the occurrence of allopatric speciation through vicariance. Here we focus on the potential role of ecological partitioning in the diversification of a widely distributed group of marine protists, the planktonic foraminifera. Sampling was conducted in the tropical Arabian Sea, during the southwest (summer) monsoon, when pronounced environmental conditions result in a strong disparity in temperature, salinity and productivity between distinct northern and southern water masses. RESULTS: We uncovered extensive genetic diversity within the Arabian Sea planktonic foraminifera, identifying 13 morphospecies, represented by 20 distinct SSU rRNA genetic types. Several morphospecies/genetic types displayed non-random biogeographical distributions, partitioning between the northern and southern water masses, giving a strong indication of independent ecological adaptations. CONCLUSIONS: We propose sea-surface primary productivity as the main factor driving the geographical segregation of Arabian Sea planktonic foraminifera, during the SW monsoon, with variations in symbiotic associations possibly playing a role in the specific ecological adaptations observed. Our findings suggest that ecological partitioning could be contributing to the high levels of 'cryptic' genetic diversity observed within the planktonic foraminifera, and support the view that ecological processes may play a key role in the diversification of marine pelagic organisms.

Surviving mass extinction by bridging the benthic/planktic divide.

Evolution of planktic organisms from benthic ancestors is commonly thought to represent unidirectional expansion into new ecological domains, possibly only once per clade. For foraminifera, this evolutionary expansion occurred in the Early-Middle Jurassic, and all living and extinct planktic foraminifera have been placed within 1 clade, the Suborder Globigerinina. The subsequent radiation of planktic foraminifera in the Jurassic and Cretaceous resulted in highly diverse assemblages, which suffered mass extinction at the end of the Cretaceous, leaving an impoverished assemblage dominated by microperforate triserial and biserial forms. The few survivor species radiated to form diverse assemblages once again in the Cenozoic. There have, however, long been doubts regarding the monophyletic origin of planktic foraminifera. We present surprising but conclusive genetic evidence that the Recent biserial planktic Streptochilus globigerus belongs to the same biological species as the benthic Bolivina variabilis, and geochemical evidence that this ecologically flexible species actively grows within the open-ocean surface waters, thus occupying both planktic and benthic domains. Such a lifestyle (tychopelagic) had not been recognized as adapted by foraminifera. Tychopelagic are endowed with great ecological advantage, enabling rapid recolonization of the extinction-susceptible pelagic domain from the benthos. We argue that the existence of such forms must be considered in resolving foraminiferal phylogeny.

Global molecular phylogeography reveals persistent Arctic circumpolar isolation in a marine planktonic protist.

The high-latitude planktonic foraminifera have proved to be particularly useful model organisms for the study of global patterns of vicariance and gene flow in the oceans. Such studies demonstrate that gene flow can occur over enormous distances in the pelagic marine environment leading to cosmopolitanism but also that there are ecological and geographical barriers to gene flow producing biogeographic structure. Here, we have undertaken a comprehensive global study of genetic diversity within a marine protist species, the high-latitude planktonic foraminiferan Neogloboquadrina pachyderma. We present extensive new data sets from the North Pacific and Arctic Oceans that, in combination with our earlier data from the North Atlantic and Southern Oceans, allow us to determine the global phylogeography of this species. The new genetic data reveal a pattern of Arctic circumpolar isolation and bipolar asymmetry between the Atlantic and Pacific Oceans. We show that the ancestry of North Pacific N. pachyderma is relatively recent. It lies within the upwelling systems and subpolar waters of the Southern Hemisphere and remarkably not within the neighboring Arctic Ocean. Instead, the Arctic Ocean population forms a genetic continuum with the North Atlantic population, which became isolated from the southern populations much earlier, after the onset of Northern hemisphere glaciation. Data from the planktonic foraminiferal morphospecies Globigerina bulloides is also introduced to highlight the isolation and endemism found within the subpolar North Pacific gyre. These data provide perspective for interpretation and discussion of global gene flow and speciation patterns in the plankton.

Molecular evidence links cryptic diversification in polar planktonic protists to Quaternary climate dynamics.

It is unknown how pelagic marine protists undergo diversification and speciation. Superficially, the open ocean appears homogeneous, with few clear barriers to gene flow, allowing extensive, even global, dispersal. Yet, despite the apparent lack of opportunity for genetic isolation, diversity is prevalent within marine taxa. A lack of candidate isolating mechanisms would seem to favor sympatric over allopatric speciation models to explain the diversity and biogeographic patterns observed in the oceans today. However, the ocean is a dynamic system, and both current and past circulation patterns must be considered in concert to gain a true perspective of gene flow through time. We have derived a comprehensive picture of the mechanisms potentially at play in the high latitudes by combining molecular, biogeographic, fossil, and paleoceanographic data to reconstruct the evolutionary history of the polar planktonic foraminifer Neogloboquadrina pachyderma sinistral. We have discovered extensive genetic diversity within this morphospecies and that its current "extreme" polar affinity did not appear until late in its evolutionary history. The molecular data demonstrate a stepwise progression of diversification starting with the allopatric isolation of Atlantic Arctic and Antarctic populations after the onset of the Northern Hemisphere glaciation. Further diversification occurred only in the Southern Hemisphere and seems to have been linked to glacial-interglacial climate dynamics. Our findings demonstrate the role of Quaternary climate instability in shaping the modern high-latitude plankton. The divergent evolutionary history of N. pachyderma sinistral genotypes implies that paleoceanographic proxies based on this taxon should be calibrated independently.

Cryptic species of planktonic foraminifera: their effect on palaeoceanographic reconstructions.

Shells of planktonic foraminifera recovered from marine sediments provide a multitude of important palaeoproxies. Most of these proxies are based on the assumption that each morphospecies of planktonic foraminifera represents a genetically continuous species with a unique habitat. Recent discovery of hitherto hidden genetic diversity among modern planktonic foraminifera has significant repercussions on palaeoproxies derived from their fossil shells. We have compiled all available data on this genetic diversity. To date, 33 cryptic genetic types were found in 9 out of the 22 sequenced morphospecies of modern planktonic foraminifera. An examination of this database suggests that cryptic genetic diversity may be a prevalent pattern among modern planktonic foraminifera, but that the total number of cryptic genetic types per morphospecies is not large and that most genetic types show a non-random pattern of distribution in the oceans. Using modern distribution data from the Atlantic Ocean as constraints, the relationship between abundances of three genetic types of Globigerina bulloides and sea-surface temperature has been modelled and this model has been applied to a database of species counts in Atlantic coretops (761 samples). Trials with artificial neural networks (ANNs), the modern analogue technique and Imbrie-Kipp transfer functions showed that the splitting of G. bulloides into three genetic types resulted in substantial reduction in the prediction error rate (by 5 to 34%) and that this improvement was by far greatest in ANN trials (on average more than 20%). We conclude that such a large reduction in error rate occurred because the models resonated with a real pattern in the original data. This study indicates that genetic diversity among planktonic foraminifera may become more of a gift than malaise to palaeoproxies. If it becomes possible to distinguish these genetic types in the fossil record, the accuracy of proxies based on planktonic foraminifera will indeed substantially increase.