A note on the laboratory culture of the benthic foraminifer Cornuloculina balkwilli (MacFadyen)

Background: Genetic studies of the Foraminifera provide valuable insights into marine speciation and biogeography, yet the discovery of vitally needed new genetic markers for this important group is being severely limited by an extreme lack of genetic data. The establishment of a laboratory culture from a single, asexually reproducing foraminifer, will be essential to provide enough pooled genetic material from these unicellular organisms, to facilitate full genome sequencing and genetic marker discovery, using next-generation sequencing techniques. Findings: The aim of this study was to develop a simple and inexpensive method of culturing benthic foraminifera, via asexual reproduction, in a controlled laboratory environment. Individual specimens of the benthic foraminfer Cornuloculina balkwilli (MacFadyen) were placed in 7 cm plastic beakers, containing 50 ml natural seawater, filtered to 0.2 μm, and kept at 23°C, with a 12-hour light/dark cycle, and fed weekly on a mixed algal diet of Dunaliella tertiolecta and Phaeodactylum tricornutum. Asexually derived cultures were successfully established from 4 specimens of Cornuloculina balkwilli, originally added to the culture vessels as immature specimens. Many thousands of individuals were present after 6 months. Conclusions: The method presented here demonstrates that only basic laboratory equipment is required to establish and maintain a thriving culture of the benthic foraminfer, C. balkwilli, from a single asexually reproducing specimen, providing an excellent source of genetic material for use in next generation sequencing. The method is easily reproducible and will greatly aid in the discovery of critically needed new genetic markers in the Foraminifera. It also highlights C. balkwilli as a good candidate species for use in the field of environmental micropaleontology

Biogeography and phylogenetics of the planktonic foraminifera

The planktonic foraminifera are a highly abundant and diverse group of marine pelagic protists that are ubiquitously distributed throughout the worlds’ oceans. These unicellular eukaryotes are encased in a calcareous (CaCO3) shell or ‘test’, the morphology of which is used to identify individual ‘morphospecies’. The foraminifera have an exceptional fossil record, spanning over 180 million years, and as microfossils provide a highly successful paleoproxy for dating sedimentary rocks and archiving past climate. Molecular studies, using the small subunit (SSU) ribosomal (r) RNA gene are used here to investigate the biogeographical distributions and phylogenetic relationships of the planktonic foraminifera. Biogeographical surveys of two markedly different areas of the global ocean, the tropical Arabian Sea, and the transitional/sub-polar North Atlantic Ocean, revealed significant genotypic variation within the planktonic foraminifera, with some genetic types being sequenced here for the first time. The foraminiferal genotypes displayed non-random geographical distributions, suggestive of distinct ecologies, giving insight into the possible mechanisms of diversification in these marine organisms. The ecological segregation of genetically divergent but morphologically cryptic genetic types could, however, have serious repercussions on their use as paleoproxies of past climate change. Phylogenetic analyses of the foraminifera based firstly on a partial ~1,000 bp terminal 3´ fragment of the SSU rRNA gene, and secondly on the ~3,000 bp almost complete gene supported the hypothesis of the polyphyletic origins of the planktonic foraminifera, which appear to be derived from up to 5 separate benthic ancestral lineages. The almost complete gene is sequenced here in the planktonic taxa for the first time, though amplification was problematic. In a first step to addressing a pressing need for new genetic markers to support data gained from the SSU rRNA gene, a culture system was established for the benthic foraminifera, in order to provide a reliable source of DNA for EST library construction or full genome sequencing. Finally, to overcome difficulties associated with the PCR amplification of the foraminifera, a new lysis buffer and DNA extraction procedure was developed. A highly successful buffer was created, allowing high quality DNA to be extracted from foraminiferal specimens, whilst leaving the delicate calcitic shell intact for morphological reference

Biogeography and phylogenetics of the planktonic foraminifera

The planktonic foraminifera are a highly abundant and diverse group of marine pelagic protists that are ubiquitously distributed throughout the worlds’ oceans. These unicellular eukaryotes are encased in a calcareous (CaCO3) shell or ‘test’, the morphology of which is used to identify individual ‘morphospecies’. The foraminifera have an exceptional fossil record, spanning over 180 million years, and as microfossils provide a highly successful paleoproxy for dating sedimentary rocks and archiving past climate. Molecular studies, using the small subunit (SSU) ribosomal (r) RNA gene are used here to investigate the biogeographical distributions and phylogenetic relationships of the planktonic foraminifera. Biogeographical surveys of two markedly different areas of the global ocean, the tropical Arabian Sea, and the transitional/sub-polar North Atlantic Ocean, revealed significant genotypic variation within the planktonic foraminifera, with some genetic types being sequenced here for the first time. The foraminiferal genotypes displayed non-random geographical distributions, suggestive of distinct ecologies, giving insight into the possible mechanisms of diversification in these marine organisms. The ecological segregation of genetically divergent but morphologically cryptic genetic types could, however, have serious repercussions on their use as paleoproxies of past climate change. Phylogenetic analyses of the foraminifera based firstly on a partial ~1,000 bp terminal 3´ fragment of the SSU rRNA gene, and secondly on the ~3,000 bp almost complete gene supported the hypothesis of the polyphyletic origins of the planktonic foraminifera, which appear to be derived from up to 5 separate benthic ancestral lineages. The almost complete gene is sequenced here in the planktonic taxa for the first time, though amplification was problematic. In a first step to addressing a pressing need for new genetic markers to support data gained from the SSU rRNA gene, a culture system was established for the benthic foraminifera, in order to provide a reliable source of DNA for EST library construction or full genome sequencing. Finally, to overcome difficulties associated with the PCR amplification of the foraminifera, a new lysis buffer and DNA extraction procedure was developed. A highly successful buffer was created, allowing high quality DNA to be extracted from foraminiferal specimens, whilst leaving the delicate calcitic shell intact for morphological reference

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

Population differentiation at a regional scale in spadefoot toads: contributions of distance and divergent selective environments

The causes of population differentiation can provide insight into the origins of early barriers to gene flow. Two key drivers of population differentiation are geographic distance and local adaptation to divergent selective environments. When reproductive isolation arises because some populations of a species are under selection to avoid hybridization while others are not, population differentiation and even speciation can result. Spadefoot toad populations Spea multiplicata that are sympatric with a congener have undergone reinforcement. This reinforcement has resulted not only in increased reproductive isolation from the congener, but also in the evolution of reproductive isolation from nearby and distant conspecific allopatric populations. We used multiple approaches to evaluate the contributions of geographic distance and divergent selective environments to population structure across this regional scale in S. multiplicata, based on genotypes from six nuclear microsatellite markers. We compared groups of populations varying in both geographic location and in the presence of a congener. Hierarchical F-statistics and results from cluster analyses and discriminant analyses of principal components all indicate that geographic distance is the stronger contributor to genetic differentiation among S. multiplicata populations at a regional scale. However, we found evidence that adaptation to divergent selective environments also contributes to population structure. Our findings highlight how variation in the balance of evolutionary forces acting across a species’ range can lead to variation in the relative contributions of geographic distance and local adaptation to population differentiation across different spatial scales

PFR²: a curated database of planktonic Foraminifera18S ribosomal DNA as a resource for studies of plankton ecology, biogeography, and evolution

International audiencePlanktonic 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 PFR2, 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 PFR2 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

Geochemical imprints of genotypic variants of <i>Globigerina bulloides</i> in the Arabian Sea

Planktonic foraminifera record oceanic conditions in their shell geochemistry. Many palaeoenvironmental studies have used fossil planktonic foraminifera to constrain past seawater properties by defining species based on their shell morphology. Recent genetic studies, however, have identified ecologically distinct genotypes within traditionally recognized morphospecies, signaling potential repercussions for palaeoclimate reconstructions. Here we demonstrate how the presence of Globigerina bulloides cryptic genotypes in the Arabian Sea may influence geochemical signals of living and fossil assemblages of these morphospecies. We have identified two distinct genotypes of G. bulloides with either cool water (type-II) or warm water (type-I) temperature preferences in the Western Arabian Sea. We accompany these genetic studies with analyses of Mg/Ca and stable oxygen isotope (δ18O) compositions of individual G. bulloides shells. Both Mg/Ca and δ18O values display bimodal distribution patterns. The distribution of Mg/Ca values cannot be simply explained by seawater parameters, and we attribute it to genotype-specific biological controls on the shell geochemistry. The wide range of δ18O values in the fossil assemblage also suggests that similar controls likely influence this proxy in addition to environmental parameters. However, the magnitude of this effect on the δ18O signals is not clear from our data set, and further work is needed to clarify this. We also discuss current evidence of potential genotype-specific geochemical signals in published data on G. bulloides geochemistry and other planktonic foraminiferal species. We conclude that significant caution should be taken when utilizing G. bulloides geochemistry for paleoclimate reconstruction in the regions with upwelling activity or oceanographic fronts

Genetic and morphometric evidence for parallel evolution of the Globigerinella calida morphotype

Molecular genetic investigations of the highly abundant extant planktonic foraminifera plexus Globigerinella siphonifera/Globigerinella calida have recently shown this group to be the genetically most diverse one within planktonic foraminifera, separating it into 12 distinct genetic types belonging to three main genetic lineages. Independently, several morphological or physiological variants have been described within the group, but the correlation between the high genetic diversity and the phenotypic variability remains unclear. In this study, we combine genetic data with morphometric analyses of shell shape and porosity of genotyped individuals of the different genetic lineages. Our morphometric measurements suggest a differentiation of three morphotypes within the plexus, two of which possess the elongated chambers described as a typical trait of G. calida. These two morphotypes with elongated chambers are associated with two distinct genetic lineages. The G. calida morphology therefore appears to have evolved twice in parallel. Unexpectedly, we show that the two morphotypes with elongated chambers can be separated from each other by characters seen in the lateral view of their shells. This implies that the taxonomy of the extant members of the genus Globigerinella should be revised. A comparison with the original descriptions and type specimens of members of the genus shows that two genetic types of one major lineage correspond to G. calida. The second group with elongated chambers is associated with a recently diverged genetic type and we propose to reinstate the name Globigerinella radians for this distinct form. The remaining nine of the 12 genetic types correspond to the G. siphonifera morphology, and in the absence of evidence for morphological differentiation, they form a paraphyletic morpho-taxon. Our results highlight the prevalence of parallelism in the evolution of shell morphology in planktonic foraminifera even at the lowest level of relatedness represented by genetic types

Genetic diversity and ecology of the planktonic foraminifers Globigerina bulloides , Turborotalita quinqueloba and Neogloboquadrina pachyderma off the Oman margin during the late SW Monsoon

The molecular work was funded by an Advanced Fellowship award to K. Darling (UK Natural Environment Research Council (NERC); NER/J/S/2000/00860 and NE/D009707/1). SA received support from a DAAD fellowship (A0998101) and HS was supported by a DFG grant (SCHU 1605/2-1).The tropical waters of the Arabian Sea are among the richest biological areas of the world. The highly complex monsoonal system is particularly challenging for palaeoenvironmental study, which relies heavily upon understanding the modern-day ecology of planktonic foraminiferal assemblages and their geochemical signatures throughout the monsoonal cycle. Major upwelling responders such as G. bulloides, T. quinqueloba and N. pachyderma, typically associated with cooler mid to higher latitude ecosystems, are also found in number in the tropical Arabian Sea. Due to the more usual cooler water affinity of these morphospecies, the oceanographically isolated tropical upwelling ecosystem of the Arabian Sea potentially harbours new ecologically distinct genotypes (ecotypes). Samples were collected off the Oman margin at 15 stations towards the end of the summer monsoon to determine the genetic profiles of these morphospecies in both upwelling and open ocean regimes. Phylogenetic analysis of their small subunit (SSU) rDNA sequences revealed several new genetically distinct ecotypes. Two genetically divergent ecotypes of G. bulloides (Types Ia and IIf) were identified along the cruise track. Type Ia, a member of the G. bulloides warm water lineage, was found in both the upwelling and open ocean regions. The second genotype (IIf), a member of the G. bulloides cool water lineage, was found only in more marginal late upwelling cooler waters. Initial visual assessment of G. bulloides images suggests that it may be morphologically cryptic. Two highly divergent genotypes of T. quinqueloba (Types Ib and IIe) were also identified, which were largely confined to the eastern and northern Arabian Sea. Type IIe is a new member of the T. quinqueloba cool water lineage which points to its potential cool water affinity, but genotyping numbers are too low to confirm a specific association with upwelling. A new highly divergent genotype of N. pachyderma (Type VIII) was also identified at the western and southern stations. Comparison of global upwelling system genotype assemblages currently indicate little regional commonality. This complicates regional palaeoproxy understanding, since geochemical calibrations are known to be species and genotype specific. Detailed studies of the ecology and diversity of genotypes within each system should therefore be carried out to ensure the accuracy of palaeorecord interpretation.Publisher PDFPeer reviewe

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