José Fahrni

How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys

BackgroundOver the past few years, the use of molecular techniques to detect cultivation-independent, eukaryotic diversity has proven to be a powerful approach. Based on small-subunit ribosomal RNA (SSU rRNA) gene analyses, these studies have revealed the existence of an unexpected variety of new phylotypes. Some of them represent novel diversity in known eukaryotic groups, mainly stramenopiles and alveolates. Others do not seem to be related to any molecularly described lineage, and have been proposed to represent novel eukaryotic kingdoms. In order to review the evolutionary importance of this novel high-level eukaryotic diversity critically, and to test the potential technical and analytical pitfalls and limitations of eukaryotic environmental DNA surveys (EES), we analysed 484 environmental SSU rRNA gene sequences, including 81 new sequences from sediments of the small river, the Seymaz (Geneva, Switzerland).ResultsBased on a detailed screening of an exhaustive alignment of eukaryotic SSU rRNA gene sequences and the phylogenetic re-analysis of previously published environmental sequences using Bayesian methods, our results suggest that the number of novel higher-level taxa revealed by previously published EES was overestimated. Three main sources of errors are responsible for this situation: (1) the presence of undetected chimeric sequences; (2) the misplacement of several fast-evolving sequences; and (3) the incomplete sampling of described, but yet unsequenced eukaryotes. Additionally, EES give a biased view of the diversity present in a given biotope because of the difficult amplification of SSU rRNA genes in some taxonomic groups.ConclusionsEnvironmental DNA surveys undoubtedly contribute to reveal many novel eukaryotic lineages, but there is no clear evidence for a spectacular increase of the diversity at the kingdom level. After re-analysis of previously published data, we found only five candidate lineages of possible novel high-level eukaryotic taxa, two of which comprise several phylotypes that were found independently in different studies. To ascertain their taxonomic status, however, the organisms themselves have now to be identified.

The evolution of early Foraminifera

Fossil Foraminifera appear in the Early Cambrian, at about the same time as the first skeletonized metazoans. However, due to the inadequate preservation of early unilocular (single-chambered) foraminiferal tests and difficulties in their identification, the evolution of early foraminifers is poorly understood. By using molecular data from a wide range of extant naked and testate unilocular species, we demonstrate that a large radiation of nonfossilized unilocular Foraminifera preceded the diversification of multilocular lineages during the Carboniferous. Within this radiation, similar test morphologies and wall types developed several times independently. Our findings indicate that the early Foraminifera were an important component of Neoproterozoic protistan community, whose ecological complexity was probably much higher than has been generally accepted.

Bipolar gene flow in deep-sea benthic foraminifera.

Despite its often featureless appearance, the deep‐ocean floor includes some of the most diverse habitats on Earth. However, the accurate assessment of global deep‐sea diversity is impeded by a paucity of data on the geographical ranges of bottom‐dwelling species, particularly at the genetic level. Here, we present molecular evidence for exceptionally wide distribution of benthic foraminifera, which constitute the major part of deep‐sea meiofauna. Our analyses of nuclear ribosomal RNA genes revealed high genetic similarity between Arctic and Antarctic populations of three common deep‐sea foraminiferal species (Epistominella exigua, Cibicides wuellerstorfi and Oridorsalis umbonatus), separated by distances of up to 17 000 km. Our results contrast with the substantial level of cryptic diversity usually revealed by molecular studies, of shallow‐water benthic and planktonic marine organisms. The very broad ranges of the deep‐sea foraminifera that we examined support the hypothesis of global distribution of small eukaryotes and suggest that deep‐sea biodiversity may be more modest at global scales than present estimates suggest.

Molecular data reveal high taxonomic diversity of allogromiid Foraminifera in Explorers Cove (McMurdo Sound, Antarctica)

Abstract Allogromiids are organic-walled or agglutinated, single-chambered Foraminifera, common in deep-sea and polar benthic communities. The simple forms and paucity of distinctive features make allogromiid identification difficult by traditional means. Molecular phylogenetic methods offer alternative tools for species identification and are used here to investigate allogromiid diversity. We obtained 135 partial small-subunit ribosomal DNA sequences of allogromiids collected in Explorers Cove, McMurdo Sound, Antarctica. In contrast to the 27 morphotypes identified, phylogenetic analysis revealed 49 molecular types (considered separate species) that differ by more than 5% of sequence divergence. The 49 genetic types form 28 molecular supra-groups that differ by more than 20% and probably represent distinct genera or families. Large genetic distances separating the molecular types indicate unexpectedly high taxonomic diversity. Comparison of our data with sequences of non-Antarctic allogromiids suggests that Explorers Cove species might be endemic and only distantly related to comparable northern hemisphere fauna.

PHYLOGENY OF ALLOGROMIID FORAMINIFERA INFERRED FROM SSU rRNA GENE SEQUENCES

Allogromiids are classically defined as a group of monothalamous, soft-walled foraminiferans. Recent morphological, cytological, and molecular studies, however, challenge this view, showing that the soft-walled allogromiids are closely related to naked athalamids and unilocular agglutinated foraminiferans. To establish the phylogenetic relationships among these three groups we obtained partial small-subunit ribosomal DNA sequences of 50 species and undetermined morphotypes, and compared them to other foraminiferal taxa. Phylogenetic analyses of our data show that allogromiids, athalamids and astrorhizids comprise an assemblage of 13 lineages branching together at the base of the foraminiferal tree. Among these lineages, two are represented by a single species and four comprise similar genera, while the remaining seven are heterogeneous groups composed of several species having different types of wall structure and different test morphologies. All lineages are relatively well supported, yet the relationships among them are not resolved. In view of our data, we propose to revise the definition of allogromiids to include all naked and testate unilocular granuloreticuloseans that diverged early in the evolution of Foraminifera.

Molecular Identification of Algal Endosymbionts in Large Miliolid Foraminifera: 2. Dinoflagellates

Abstract Large miliolid foraminifers of the subfamily Soritinae bear symbiotic dinoflagellates morphologically similar to the species of the “Symbiodinium” complex, commonly found in corals and other marine invertebrates. Soritid foraminifers are abundant in coral reefs and it has been proposed that they share their symbionts with other dinoflagellate-bearing reef dwellers. In order to test this hypothesis, we have analysed partial large subunit ribosomal DNA sequences from dinoflagellates symbionts obtained from 28 foraminiferal specimens, and compared them to the corresponding sequences of Symbiodinium-like endosymbionts from various groups of invertebrates. Phylogenetic analysis of our data shows that all soritid symbionts belong to the “Symbiodinium” species complex, within which they form seven different molecular types (Fr1–Fr7). Only one of these types (Fr1) branches within a group of invertebrate symbionts, previously described as type C. The remaining six types form sister groups to coral symbionts previously designed as types B, C, and D. Our data indicate a high genetic diversity and specificity of Symbiodinium-like symbionts in soritids. Except for type C, we have found no evidence for the transmission of symbionts between foraminifers and other symbiont-bearing invertebrates from the same localities. However, exchanges must have occurred frequently between the different species of Soritinae, as suggested by the lack of host specificity and some biogeographical patterns observed in symbiont distribution. Our data suggest that members of the subfamily Soritinae acquired their symbionts at least three times during their history, each acquisition being followed by a rapid diversification and independent radiation of symbionts within the foraminiferal hosts.

Phylogeny of the infraorder Culicomorpha (Diptera: Nematocera) based on 28S RNA gene sequences

Phylogenetic relationships between the families of the infraorder Culicomorpha were investigated by using partial 28S ribosomal RNA gene sequences. All families traditionally placed in this infraorder were investigated and confirmed as clades. On the other hand, some of the morphological relationships between these families were found to be in disagreement with phylogenies based on molecular characters. Our results did not support the generally accepted division of the Culicomorpha into two superfamilies, the Culicoidea (Culicidae + Corethrellidae + Chaoboridae + Dixidae) and the Chironomoidea (Chironomidae + Ceratopogonidae + Simuliidae + Thaumaleidae). Precisely, if the sister‐group relationship between Culicidae, Chaoboridae and Corethrellidae was clearly confirmed, the Dixidae, traditionally considered as closely related to these two families, were not placed close to them on our trees. On the other hand, strong evidence was found for grouping together the Simuliidae and the Thaumaleidae, in spite of the cytological and morphological differences between these two families. The position of the Ceratopogonidae was uncertain, and the Chironomidae appeared as a possible sister group to the rest of Culicomorpha. The phylogenetic positions of the groups characterized by feeding on vertebrate blood or insect haemolymph (the Culicidae, Chaoboridae, Ceratopogonidae and Simuliidae) suggest that haematophagy has appeared at least twice in the evolution of Culicomorpha.

Molecular evidence that Reticulomyxa filosa is a freshwater naked foraminifer.

ABSTRACT Reticulomyxa filosa is a freshwater protist possessing fine granular, branching and anastomosing pseudopodia and therefore traditionally placed in the class Granuloreticulosea, order Athalamida, as a sister group to the order Foraminiferida. Recent studies have revealed remarkable similarities in pseudopodial motility and ultrastructure between R. filosa and foraminifera (e.g. Allogromia laticollaris), prompting us to conduct a molecular phylogenetic analysis of these seemingly disparate organisms. We sequenced the complete small‐subunit of the ribosomal DNA of the cultured strain of R. filosa and compared it to the corresponding sequences of other protists including 12 species of foraminifera. We also sequenced and analyzed the actin coding genes from R. filosa and two species of foraminifera, Allogromia sp. and Ammonia sp. the analysis of both data sets clearly shows that R. filosa branches within the clade of foraminifera, suggesting that R. filosa is in fact a freshwater naked foraminiferan.

Small-Subunit Ribosomal RNA Gene Sequences of Phaeodarea Challenge the Monophyly of Haeckel's Radiolaria

In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups.

ACTIN PHYLOGENY OF FORAMINIFERA

Molecular phylogenies of foraminifera are commonly inferred from the small subunit rRNA (SSU) genes, which can easily be obtained from single cells isolated from environmental samples. The SSU phylogenies, however, are often biased by heterogeneity of substitution rates, and their resolution of higher level relationships is often very low. The sequences of protein-coding genes provide an important alternative source of phylogenetic information, yet their availability from foraminifera has been limited until now. Here, we report the first extensive protein sequence data for foraminifera, which comprises 90 actin sequences for 27 species representing five major foraminiferan groups. Our analysis enables grouping foraminiferan actins into two main paralogs, ACT1 (actin type 1) and ACT2 (actin type 2), and several actin-deviating proteins. Phylogenetic analyses of ACT1 and ACT2 confirm the general structure of foraminiferan phylogenies inferred from SSU rDNA sequences. In particular, actin phylogenies support (1) the paraphyly of monothalamous foraminifera, including the allogromiids, astrorhizids and athalamids; (2) the independent divergence of miliolids and their close relationship to Miliammina ; (3) the monophyly of rotalids; and (4) the rotaliid ancestry of globigerinids. Some foraminiferan taxa can be distinguished in actin sequences by the presence of group-specific introns (rotaliids, allogromiids) or absence of any introns (soritids ACT1).