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.

Discrimination of conspecifics from heterospecifics in a hybrid zone: Behavioral and chemical cues in ants.

Species and nestmate recognition in social insects occurs mostly through cuticular hydrocarbons acting as chemical cues. These compounds generate a colony-specific odor profile depending on genetic and environmental factors. Species and nestmate recognition results in specific behavioral responses, regulating the level of aggression toward other individuals during an interaction. Although species discrimination and recognition cues have been poorly studied in the context of interspecific hybridization, such systems offer an opportunity to further investigate the influence of heritable and environmental factors on recognition. We explored the strength of discrimination in a hybrid zone between two ant species-Tetramorium immigrans and T. caespitum-by comparing cuticular hydrocarbon profiles and measuring intra- and interspecific worker aggression in both areas of sympatry and areas of allopatry among species. Species cuticular hydrocarbon profiles were well-differentiated and interspecific aggression was high, revealing highly discriminating species recognition cues. Hybrids' cuticular hydrocarbon profiles consisted of a mixture of the parental bouquets, but also exhibited hybrid-specific patterns. Behavioral assays showed that T. immigrans is as aggressive toward hybrids as toward heterospecifics. Finally, aggression between heterospecific workers was lower when interacting individuals came from areas of sympatry among species than from areas of allopatry. Taken as a whole, these findings paint a particularly complex picture of the recognition system in T. immigrans, T. caespitum, and their hybrids, and highlight that hybrid zones afford a still underexplored opportunity for investigating recognition mechanisms and discrimination between species.

Exceptional fossil assemblages confirm the existence of complex Early Triassic ecosystems during the early Spathian.

The mass extinction characterizing the Permian/Triassic boundary (PTB; ~ 252 Ma) corresponds to a major faunal shift between the Palaeozoic and the Modern evolutionary fauna. The temporal, spatial, environmental, and ecological dynamics of the associated biotic recovery remain highly debated, partly due to the scarce, or poorly-known, Early Triassic fossil record. Recently, an exceptionally complex ecosystem dated from immediately after the Smithian/Spathian boundary (~ 3 myr after the PTB) was reported: the Paris Biota (Idaho, USA). However, the spatiotemporal representativeness of this unique assemblage remained questionable as it was hitherto only reported from a single site. Here we describe three new exceptionally diverse assemblages of the same age as the Paris Biota, and a fourth younger one. They are located in Idaho and Nevada, and are taxonomic subsets of the Paris Biota. We show that the latter covered a region-wide area and persisted at least partially throughout the Spathian. The presence of a well-established marine fauna such as the Paris Biota, as soon as the early Spathian, indicates that the post-PTB biotic recovery and the installation of complex ecosystems probably took place earlier than often assumed, at least at a regional scale.

Multiple mating in the context of interspecific hybridization between two Tetramorium ant species.

In eusocial Hymenoptera, haplodiploidy and polyandry may facilitate selection for hybridization. Interspecific hybridization is widespread in ants and can lead to hybrid inviability as well as the formation of new species through hybrid speciation. However, in ants, polyandry is uncommon. By analyzing microsatellite markers on 15 ant workers per colony, we show that the mating system of 28 pure colonies of Tetramorium immigrans, 15 pure colonies of Tetramorium caespitum, and 27 hybrid colonies is a monogyne/polyandrous mating system, with a higher mating rate in T. caespitum (mean = 2.4 males vs. 1.7 in T. immigrans). Hybrid queens, but no hybrid fathers, were deduced from workers' genotypes, in accordance with Haldane's rule extended to haplodiploid organisms, which states that the haploid sex should more often be sterile or inviable. In five colonies, hybridization and multiple mating allowed the simultaneous production of both hybrid and nonhybrid offspring. Although rare, these situations hinted at asymmetrical, larger contributions of T. immigrans vs. T. caespitum males to offspring production. Together, these findings point toward a complex and dynamic mating system in T. immigrans and T. caespitum, and contribute to better understand interspecific hybridization mechanisms and their consequences on genetic and taxonomic diversity. The study of polyandry within a hybrid zone is unprecedented and opens new opportunities to better understand interspecific hybridization mechanisms and their short- to long-term consequences.

Using museum pelt collections to generate pollen prints from high-risk regions: A new palynological forensic strategy for geolocation.

The use of pollen as a forensic tool for geolocation is a well-established practice worldwide in cases ranging from the provenance of drugs and other illicit materials to tracking the travel of individuals in criminal investigations. Here we propose a novel approach to generation of pollen databases that uses pollen vacuumed from mammal pelts collected historically from international areas that are now deemed too high risk to visit. We present the results of a study we conducted using mammal pelts collected from Mexico. This new investigative technique is important because, although it would seem that the ubiquitous and geo-specific nature of pollen would make pollen analysis among the most promising forensic tools for law enforcement and intelligence agencies, it is not the case. The process is notoriously slow because pollen identification is a tedious task requiring trained specialists (palynologists) who are few in number worldwide, and the reference materials necessary for geolocation usually are rare or absent, especially from regions of the world that are no longer safe to visit because of war or threat of terrorism. Current forensic palynological work is carried out by a few highly trained palynologists who require accurate databases of pollen distribution, especially from sensitive areas, to do their jobs accurately and efficiently. Our project shows the suitability of using the untapped museum pelt resources to support homeland security programs. This first palynological study using museum pelts yielded 133 different pollen and spore types, including 8 moss or fern families, 12 gymnosperm genera and 112 angiosperm species. We show that the palynological print from each region is statistically different with some important clustering, demonstrating the potential to use this technique for geolocation.

Unexpected Early Triassic marine ecosystem and the rise of the Modern evolutionary fauna.

In the wake of the end-Permian mass extinction, the Early Triassic (~251.9 to 247 million years ago) is portrayed as an environmentally unstable interval characterized by several biotic crises and heavily depauperate marine benthic ecosystems. We describe a new fossil assemblage-the Paris Biota-from the earliest Spathian (middle Olenekian, ~250.6 million years ago) of the Bear Lake area, southeastern Idaho, USA. This highly diversified assemblage documents a remarkably complex marine ecosystem including at least seven phyla and 20 distinct metazoan orders, along with algae. Most unexpectedly, it combines early Paleozoic and middle Mesozoic taxa previously unknown from the Triassic strata, among which are primitive Cambrian-Ordovician leptomitid sponges (a 200-million year Lazarus taxon) and gladius-bearing coleoid cephalopods, a poorly documented group before the Jurassic (~50 million years after the Early Triassic). Additionally, the crinoid and ophiuroid specimens show derived anatomical characters that were thought to have evolved much later. Unlike previous works that suggested a sluggish postcrisis recovery and a low diversity for the Early Triassic benthic organisms, the unexpected composition of this exceptional assemblage points toward an early and rapid post-Permian diversification for these clades. Overall, it illustrates a phylogenetically diverse, functionally complex, and trophically multileveled marine ecosystem, from primary producers up to top predators and potential scavengers. Hence, the Paris Biota highlights the key evolutionary position of Early Triassic fossil ecosystems in the transition from the Paleozoic to the Modern marine evolutionary fauna at the dawn of the Mesozoic era.

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.

SSU rDNA divergence in planktonic foraminifera: molecular taxonomy and biogeographic implications.

The use of planktonic foraminifera in paleoceanography requires taxonomic consistency and precise assessment of the species biogeography. Yet, ribosomal small subunit (SSUr) DNA analyses have revealed that most of the modern morpho-species of planktonic foraminifera are composed of a complex of several distinct genetic types that may correspond to cryptic or pseudo-cryptic species. These genetic types are usually delimitated using partial sequences located at the 3'end of the SSUrDNA, but typically based on empirical delimitation. Here, we first use patristic genetic distances calculated within and among genetic types of the most common morpho-species to show that intra-type and inter-type genetic distances within morpho-species may significantly overlap, suggesting that genetic types have been sometimes inconsistently defined. We further apply two quantitative and independent methods, ABGD (Automatic Barcode Gap Detection) and GMYC (General Mixed Yule Coalescent) to a dataset of published and newly obtained partial SSU rDNA for a more objective assessment of the species status of these genetic types. Results of these complementary approaches are highly congruent and lead to a molecular taxonomy that ranks 49 genetic types of planktonic foraminifera as genuine (pseudo)cryptic species. Our results advocate for a standardized sequencing procedure allowing homogenous delimitations of (pseudo)cryptic species. On the ground of this revised taxonomic framework, we finally provide an integrative taxonomy synthesizing geographic, ecological and morphological differentiations that can occur among the genuine (pseudo)cryptic species. Due to molecular, environmental or morphological data scarcities, many aspects of our proposed integrative taxonomy are not yet fully resolved. On the other hand, our study opens up the potential for a correct interpretation of environmental sequence datasets.

Missing data estimation in morphometrics: how much is too much?

Fossil-based estimates of diversity and evolutionary dynamics mainly rely on the study of morphological variation. Unfortunately, organism remains are often altered by post-mortem taphonomic processes such as weathering or distortion. Such a loss of information often prevents quantitative multivariate description and statistically-controlled comparisons of extinct species based on morphometric data. A common way to deal with missing data involves imputation methods that directly fill the missing cases with model estimates. Over the last years, several empirically-determined thresholds for the maximum acceptable proportion of missing values have been proposed in the literature, whereas other studies showed that this limit actually depends on various properties of the study data set and of the selected imputation method, and is by no way generalizable. We evaluate the relative performances of seven multiple imputation (MI) techniques through a simulation-based analysis under three distinct patterns of missing data distribution. Overall, Fully Conditional Specification and Expectation-Maximization algorithms provide the best compromises between imputation accuracy and coverage probability. MI techniques appear remarkably robust to the violation of basic assumptions such as the occurrence of taxonomically or anatomically biased patterns of missing data distribution, making differences in simulation results between the three patterns of missing data distribution much smaller than differences between the individual MI techniques. Based on these results, rather than proposing a new (set of) threshold value(s), we develop an approach combining the use of MIs with procrustean superimposition of principal component analysis results, in order to directly visualize the effect of individual missing data imputation on an ordinated space. We provide an R function for users to implement the proposed procedure.

On Mesopithecus habitat: Insights from late Miocene fossil vertebrate localities of Bulgaria.

The aim of this study is to describe the environments where the cercopithecid Mesopithecus was found during latest Miocene in Europe. For this purpose, we investigate the paleoecology of the herbivorous ungulate mesofauna of three very rich late Miocene fossil localities from southwestern Bulgaria: Hadjidimovo, Kalimantsi and Strumyani. While Mesopithecus has been found in the two first localities, no primate remains have yet been identified in Strumyani. Comparison between localities with and without primates using the herbivore mesofauna allows the cross-corroboration of paleoenvironmental conditions where this primate did and did not live. A multi-parameter statistical approach involving 117 equid and 345 bovid fossil dental and postcranial (phalanges, metapodia, astragali) remains from these three localities provides species to generic-level diet and locomotor habit information in order to characterize the environment in which Mesopithecus evolved. The analysis of dental mesowear indicates that the bovids were mainly mixed feeders, while coeval equids were more engaged in grazing. Meanwhile, postcranial remains show that the ungulate species from Hadjidimovo and Kalimantsi evolved in dry environments with a continuum of habitats ranging from slightly wooded areas to relatively open landscapes, whereas the Mesopithecus-free Strumyani locality was in comparison reflecting a rather contrasted mosaic of environments with predominant open and some more closed and wet areas. Environments in which Mesopithecus is known during the late Miocene were not contrasted landscapes combining open grassy areas and dense forested patches, but instead rather restricted to slightly wooded and homogeneous landscapes including a developed grassy herbaceous layer.

Worldwide genotyping in the planktonic foraminifer Globoconella inflata: implications for life history and paleoceanography.

The planktonic foraminiferal morpho-species Globoconella inflata is widely used as a stratigraphic and paleoceanographic index. While G. inflata was until now regarded as a single species, we show that it rather constitutes a complex of two pseudo-cryptic species. Our study is based on SSU and ITS rDNA sequence analyses and genotyping of 497 individuals collected at 49 oceanic stations covering the worldwide range of the morpho-species. Phylogenetic analyses unveil the presence of two divergent genotypes. Type I inhabits transitional and subtropical waters of both hemispheres, while Type II is restricted to the Antarctic subpolar waters. The two genetic species exhibit a strictly allopatric distribution on each side of the Antarctic Subpolar Front. On the other hand, sediment data show that G. inflata was restricted to transitional and subtropical environments since the early Pliocene, and expanded its geographic range to southern subpolar waters ∼700 kyrs ago, during marine isotopic stage 17. This datum may correspond to a peripatric speciation event that led to the partition of an ancestral genotype into two distinct evolutionary units. Biometric measurements performed on individual G. inflata from plankton tows north and south of the Antarctic Subpolar Front indicate that Types I and II display slight but significant differences in shell morphology. These morphological differences may allow recognition of the G. inflata pseudo-cryptic species back into the fossil record, which in turn may contribute to monitor past movements of the Antarctic Subpolar Front during the middle and late Pleistocene.

Biodiversity is not (and never has been) a bed of roses!

Over the last decades, the critical study of fossil diversity has led to significant advances in the knowledge of global macroevolutionary patterns of biodiversity. The deep-time history of life on Earth results from background originations and extinctions defining a steady-state, nonstationary equilibrium occasionally perturbed by biotic crises and "explosive" diversifications. More recently, a macroecological approach to the large-scale distribution of extant biodiversity offered new, stimulating perspectives on old theoretical questions and current practical problems in conservation biology. However, time and space are practically distinct, but functionally related dimensions of ecological systems. This calls for a spatially-integrated study of biodiversity dynamics at an evolutionary timescale. Indeed, the biosphere is a complex adaptive system whose study cannot be arbitrarily reduced to any single spatial- and/or temporal-scale level of resolution without a loss of content. From such an integrated perspective, a simple fact emerges: in a physically heterogeneous and ever-changing world, spatiotemporal variations in biodiversity are the rule-not the exception.

Can dental microwear textures record inter-individual dietary variations?

BACKGROUND: Dental microwear analyses are commonly used to deduce the diet of extinct mammals. Conventional methods rely on the user identifying features within a 2D image. However, recent interdisciplinary research has lead to the development of an advanced methodology that is free of observer error, based on the automated quantification of 3D surfaces by combining confocal microscopy with scale-sensitive fractal analysis. This method has already proved to be very efficient in detecting dietary differences between species. Focusing on a finer, intra-specific scale of analysis, the aim of this study is to test this method's ability to track such differences between individuals from a single population. METHODOLOGY/PRINCIPAL FINDINGS: For the purposes of this study, the 3D molar microwear of 78 individuals from a well-known population of extant roe deer (Capreolus caprelous) is quantified. Multivariate statistical analyses indicate significant seasonal and sexual differences in individual dental microwear design. These are probably the consequence of seasonal variations in fruit, seed and leaf availability, as well as differences in feeding preference between males and females due to distinct energy requirements during periods of rutting, gestation or giving birth. Nevertheless, further investigations using two-block Partial Least-Squares analysis show no strong relationship between individual stomach contents and microwear texture. This is an expected result, assuming that stomach contents are composed of food items ingested during the last few hours whereas dental microwear texture records the physical properties of items eaten over periods of days or weeks. CONCLUSIONS/SIGNIFICANCE: Microwear 3D scale-sensitive fractal analysis does detect differences in diet ranging from the inter-feeding styles scale to the intra-population between-season and between-sex scales. It is therefore a possible tool, to be used with caution, in the further exploration of the feeding biology and ecology of extinct mammals.

Good genes and good luck: ammonoid diversity and the end-Permian mass extinction.

The end-Permian mass extinction removed more than 80% of marine genera. Ammonoid cephalopods were among the organisms most affected by this crisis. The analysis of a global diversity data set of ammonoid genera covering about 106 million years centered on the Permian-Triassic boundary (PTB) shows that Triassic ammonoids actually reached levels of diversity higher than in the Permian less than 2 million years after the PTB. The data favor a hierarchical rather than logistic model of diversification coupled with a niche incumbency hypothesis. This explosive and nondelayed diversification contrasts with the slow and delayed character of the Triassic biotic recovery as currently illustrated for other, mainly benthic groups such as bivalves and gastropods.

Oxygen isotope fractionation between human phosphate and water revisited.

The oxygen isotope composition of human phosphatic tissues (delta18OP) has great potential for reconstructing climate and population migration, but this technique has not been applied to early human evolution. To facilitate this application we analyzed delta18OP values of modern human teeth collected at 12 sites located at latitudes ranging from 4 degrees N to 70 degrees N together with the corresponding oxygen composition of tap waters (delta18OW) from these areas. In addition, the delta18O of some raw and boiled foods were determined and simple mass balance calculations were performed to investigate the impact of solid food consumption on the oxygen isotope composition of the total ingested water (drinking water+solid food water). The results, along with those from three, smaller published data sets, can be considered as random estimates of a unique delta18OW/delta18OP linear relationship: delta18OW=1.54(+/-0.09)xdelta18OP-33.72(+/-1.51)(R2=0.87: p [H0:R2=0]=2x10(-19)). The delta18O of cooked food is higher than that of the drinking water. As a consequence, in a modern diet the delta18O of ingested water is +1.05 to 1.2 per thousand higher than that of drinking water in the area. In meat-dominated and cereal-free diets, which may have been the diets of some of our early ancestors, the shift is a little higher and the application of the regression equation would slightly overestimate delta18OW in these cases.

High-precision determination of 18O/16O ratios of silver phosphate by EA-pyrolysis-IRMS continuous flow technique.

A high-precision, and rapid on-line method for oxygen isotope analysis of silver phosphate is presented. The technique uses high-temperature elemental analyzer (EA)-pyrolysis interfaced in continuous flow (CF) mode to an isotopic ratio mass spectrometer (IRMS). Calibration curves were generated by synthesizing silver phosphate with a 13 per thousand spread in delta(18)O values. Calibration materials were obtained by reacting dissolved potassium dihydrogen phosphate (KH(2)PO(4)) with water samples of various oxygen isotope compositions at 373 K. Validity of the method was tested by comparing the on-line results with those obtained by classical off-line sample preparation and dual inlet isotope measurement. In addition, silver phosphate precipitates were prepared from a collection of biogenic apatites with known delta(18)O values ranging from 12.8 to 29.9 per thousand (V-SMOW). Reproducibility of +/- 0.2 per thousand was obtained by the EA-Py-CF-IRMS method for sample sizes in the range 400-500 microg. Both natural and synthetic samples are remarkably well correlated with conventional (18)O/(16)O determinations. Silver phosphate is a very stable material and easy to degas and, thus, could be considered as a good candidate to become a reference material for the determination of (18)O/(16)O ratios of phosphate by high-temperature pyrolysis.