Phylogenomics reveals a new 'megagroup' including most photosynthetic eukaryotes

Advances in molecular phylogeny of eukaryotes have suggested a tree composed of a small number of supergroups. Phylogenomics recently established the relationships between some of these large assemblages, yet the deepest nodes are still unresolved. Here, we investigate early evolution among the major eukaryotic supergroups using the broadest multigene dataset to date (65 species, 135 genes). Our analyses provide strong support for the clustering of plants, chromalveolates, rhizarians, haptophytes and cryptomonads, thus linking nearly all photosynthetic lineages and raising the question of a possible unique origin of plastids. At its deepest level, the tree of eukaryotes now receives strong support for two monophyletic megagroups comprising most of the eukaryotic diversity.     

Phylogenetic placement of diverse amoebae inferred from multigene analyses and assessment of clade stability within 'Amoebozoa' upon removal of varying rate classes of SSU-rDNA

Placing amoeboid lineages on the eukaryotic tree of life is difficult due to the paucity of comparable morphological characters and the limited molecular data available for many groups. This situation has led to the lumping of distantly related lineages into large inclusive groups, such as Sarcodina, that do not reflect evolutionary relationships. Previous analyses of molecular markers with limited taxon sampling reveal members of Sarcodina are scattered in five of the six proposed supergroups. We have used multigene analyses to place seven diverse amoeboid lineages-two Nolandella spp., Rhizamoeba sp., Pessonella sp., Arcella hemisphaerica, Arachnula sp. and Trichosphaerium sp.-on the eukaryotic tree of life. Bayesian analysis of the concatenated data of the four genes sequenced (SSU-rDNA, actin, alpha-tubulin and beta-tubulin), including diverse representatives of eukaryotes, indicates that all seven taxa group within the 'Amoebozoa' supergroup. We further performed separate analyses of the well-sampled SSU-rDNA and actin genes using Bayesian and Maximum Likelihood analyses to assess the positions of our newly characterized taxa. In the case of SSU-rDNA, we performed extensive analyses with removal of the fastest rates classes to evaluate the stability and resolution of various taxonomic hypotheses within 'Amoebozoa'. Five of our seven amoeboid lineages fall within well-supported clades that are corroborated by morphology. In contrast, the positions of Arachnula sp. and Trichosphaerium sp. in the SSU-rDNA gene trees are unstable and vary by analyses. Placement of these taxa will require additional data from slowly evolving genes combined with taxon-rich phylogenetic analyses. Finally, the analyses without the fastest rate classes demonstrate that SSU-rDNA has a limited signal for deep relationships within the 'Amoebozoa'.     

Nitrile hydratase genes are present in multiple eukaryotic supergroups

Background

Nitrile hydratases are enzymes involved in the conversion of nitrile-containing compounds into ammonia and organic acids. Although they are widespread in prokaryotes, nitrile hydratases have only been reported in two eukaryotes: the choanoflagellate Monosiga brevicollis and the stramenopile Aureococcus anophagefferens. The nitrile hydratase gene in M. brevicollis was believed to have arisen by lateral gene transfer from a prokaryote, and is a fusion of beta and alpha nitrile hydratase subunits. Only the alpha subunit has been reported in A. anophagefferens.

Methodology/Principal Findings

Here we report the detection of nitrile hydratase genes in five eukaryotic supergroups: opisthokonts, amoebozoa, archaeplastids, CCTH and SAR. Beta-alpha subunit fusion genes are found in the choanoflagellates, ichthyosporeans, apusozoans, haptophytes, rhizarians and stramenopiles, and potentially also in the amoebozoans. An individual alpha subunit is found in a dinoflagellate and an individual beta subunit is found in a haptophyte. Phylogenetic analyses recover a clade of eukaryotic-type nitrile hydratases in the Opisthokonta, Amoebozoa, SAR and CCTH; this is supported by analyses of introns and gene architecture. Two nitrile hydratase sequences from an animal and a plant resolve in the prokaryotic nitrile hydratase clade.

Conclusions/Significance

The evidence presented here demonstrates that nitrile hydratase genes are present in multiple eukaryotic supergroups, suggesting that a subunit fusion gene was present in the last common ancestor of all eukaryotes. The absence of nitrile hydratase from several sequenced species indicates that subunits were lost in multiple eukaryotic taxa. The presence of nitrile hydratases in many other eukaryotic groups is unresolved due to insufficient data and taxon sampling. The retention and expression of the gene in distantly related eukaryotic species suggests that it plays an important metabolic role. The novel family of eukaryotic nitrile hydratases presented in this paper represents a promising candidate for research into their molecular biology and possible biotechnological applications.     

Evaluating support for the current classification of eukaryotic diversity

Perspectives on the classification of eukaryotic diversity have changed rapidly in recent years, as the four eukaryotic groups within the five-kingdom classification--plants, animals, fungi, and protists--have been transformed through numerous permutations into the current system of six "supergroups." The intent of the supergroup classification system is to unite microbial and macroscopic eukaryotes based on phylogenetic inference. This supergroup approach is increasing in popularity in the literature and is appearing in introductory biology textbooks. We evaluate the stability and support for the current six-supergroup classification of eukaryotes based on molecular genealogies. We assess three aspects of each supergroup: (1) the stability of its taxonomy, (2) the support for monophyly (single evolutionary origin) in molecular analyses targeting a supergroup, and (3) the support for monophyly when a supergroup is included as an out-group in phylogenetic studies targeting other taxa. Our analysis demonstrates that supergroup taxonomies are unstable and that support for groups varies tremendously, indicating that the current classification scheme of eukaryotes is likely premature. We highlight several trends contributing to the instability and discuss the requirements for establishing robust clades within the eukaryotic tree of life.     

EEF2 analysis challenges the monophyly of Archaeplastida and Chromalveolata

Background

Classification of eukaryotes provides a fundamental phylogenetic framework for ecological, medical, and industrial research. In recent years eukaryotes have been classified into six major supergroups: Amoebozoa, Archaeplastida, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. According to this supergroup classification, Archaeplastida and Chromalveolata each arose from a single plastid-generating endosymbiotic event involving a cyanobacterium (Archaeplastida) or red alga (Chromalveolata). Although the plastids within members of the Archaeplastida and Chromalveolata share some features, no nucleocytoplasmic synapomorphies supporting these supergroups are currently known.

Methodology/Principal Findings

This study was designed to test the validity of the Archaeplastida and Chromalveolata through the analysis of nucleus-encoded eukaryotic translation elongation factor 2 (EEF2) and cytosolic heat-shock protein of 70 kDa (HSP70) sequences generated from the glaucophyte Cyanophora paradoxa, the cryptophytes Goniomonas truncata and Guillardia theta, the katablepharid Leucocryptos marina, the rhizarian Thaumatomonas sp. and the green alga Mesostigma viride. The HSP70 phylogeny was largely unresolved except for certain well-established groups. In contrast, EEF2 phylogeny recovered many well-established eukaryotic groups and, most interestingly, revealed a well-supported clade composed of cryptophytes, katablepharids, haptophytes, rhodophytes, and Viridiplantae (green algae and land plants). This clade is further supported by the presence of a two amino acid signature within EEF2, which appears to have arisen from amino acid replacement before the common origin of these eukaryotic groups.

Conclusions/Significance

Our EEF2 analysis strongly refutes the monophyly of the Archaeplastida and the Chromalveolata, adding to a growing body of evidence that limits the utility of these supergroups. In view of EEF2 phylogeny and other morphological evidence, we discuss the possibility of an alternative eukaryotic supergroup.     

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

Background  

Over 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).     

Terrestrial invasion of pomatiopsid gastropods in the heavy-snow region of the Japanese Archipelago

Background  

Gastropod mollusks are one of the most successful animals that have diversified in the fully terrestrial habitat. They have evolved terrestrial taxa in more than nine lineages, most of which originated during the Paleozoic or Mesozoic. The rissooidean gastropod family Pomatiopsidae is one of the few groups that have evolved fully terrestrial taxa during the late Cenozoic. The pomatiopsine diversity is particularly high in the Japanese Archipelago and the terrestrial taxa occur only in this region. In this study, we conducted thorough samplings of Japanese pomatiopsid species and performed molecular phylogenetic analyses to explore the patterns of diversification and terrestrial invasion.     

Phylogenomic analysis supports the monophyly of cryptophytes and haptophytes and the association of rhizaria with chromalveolates

Here we use phylogenomics with expressed sequence tag (EST) data from the ecologically important coccolithophore-forming alga Emiliania huxleyi and the plastid-lacking cryptophyte Goniomonas cf. pacifica to establish their phylogenetic positions in the eukaryotic tree. Haptophytes and cryptophytes are members of the putative eukaryotic supergroup Chromalveolata (chromists [cryptophytes, haptophytes, stramenopiles] and alveolates [apicomplexans, ciliates, and dinoflagellates]). The chromalveolates are postulated to be monophyletic on the basis of plastid pigmentation in photosynthetic members, plastid gene and genome relationships, nuclear "host" phylogenies of some chromalveolate lineages, unique gene duplication and replacements shared by these taxa, and the evolutionary history of components of the plastid import and translocation systems. However the phylogenetic position of cryptophytes and haptophytes and the monophyly of chromalveolates as a whole remain to be substantiated. Here we assess chromalveolate monophyly using a multigene dataset of nuclear genes that includes members of all 6 eukaryotic supergroups. An automated phylogenomics pipeline followed by targeted database searches was used to assemble a 16-protein dataset (6,735 aa) from 46 taxa for tree inference. Maximum likelihood and Bayesian analyses of these data support the monophyly of haptophytes and cryptophytes. This relationship is consistent with a gene replacement via horizontal gene transfer of plastid-encoded rpl36 that is uniquely shared by these taxa. The haptophytes + cryptophytes are sister to a clade that includes all other chromalveolates and, surprisingly, two members of the Rhizaria, Reticulomyxa filosa and Bigelowiella natans. The association of the two Rhizaria with chromalveolates is supported by the approximately unbiased (AU)-test and when the fastest evolving amino acid sites are removed from the 16-protein alignment.     

Developmental expression of the alpha-skeletal actin gene

Background  

Actin is a cytoskeletal protein which exerts a broad range of functions in almost all eukaryotic cells. In higher vertebrates, six primary actin isoforms can be distinguished: alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. Expression of these actin isoforms during vertebrate development is highly regulated in a temporal and tissue-specific manner, but the mechanisms and the specific differences are currently not well understood. All members of the actin multigene family are highly conserved, suggesting that there is a high selective pressure on these proteins.     

Annelid phylogeny and the status of Sipuncula and Echiura

Background  

Annelida comprises an ancient and ecologically important animal phylum with over 16,500 described species and members are the dominant macrofauna of the deep sea. Traditionally, two major groups are distinguished: Clitellata (including earthworms, leeches) and "Polychaeta" (mostly marine worms). Recent analyses of molecular data suggest that Annelida may include other taxa once considered separate phyla (i.e., Echiura, and Sipuncula) and that Clitellata are derived annelids, thus rendering "Polychaeta" paraphyletic; however, this contradicts classification schemes of annelids developed from recent analyses of morphological characters. Given that deep-level evolutionary relationships of Annelida are poorly understood, we have analyzed comprehensive datasets based on nuclear and mitochondrial genes, and have applied rigorous testing of alternative hypotheses so that we can move towards the robust reconstruction of annelid history needed to interpret animal body plan evolution.     

Evolutionary history of mammalian sucking lice (Phthiraptera: Anoplura)

Background  

Sucking lice (Phthiraptera: Anoplura) are obligate, permanent ectoparasites of eutherian mammals, parasitizing members of 12 of the 29 recognized mammalian orders and approximately 20% of all mammalian species. These host specific, blood-sucking insects are morphologically adapted for life on mammals: they are wingless, dorso-ventrally flattened, possess tibio-tarsal claws for clinging to host hair, and have piercing mouthparts for feeding. Although there are more than 540 described species of Anoplura and despite the potential economical and medical implications of sucking louse infestations, this study represents the first attempt to examine higher-level anopluran relationships using molecular data. In this study, we use molecular data to reconstruct the evolutionary history of 65 sucking louse taxa with phylogenetic analyses and compare the results to findings based on morphological data. We also estimate divergence times among anopluran taxa and compare our results to host (mammal) relationships.     

Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists

Background  

Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).     

Untangling the Phylogeny of Amoeboid Protists1

ABSTRACT. The amoebae and amoeboid protists form a large and diverse assemblage of eukaryotes characterized by various types of pseudopodia. For convenience, the traditional morphology‐based classification grouped them together in a macrotaxon named Sarcodina. Molecular phylogenies contributed to the dismantlement of this assemblage, placing the majority of sarcodinids into two new supergroups: Amoebozoa and Rhizaria. In this review, we describe the taxonomic composition of both supergroups and present their small subunit rDNA‐based phylogeny. We comment on the advantages and weaknesses of these phylogenies and emphasize the necessity of taxon‐rich multigene datasets to resolve phylogenetic relationships within Amoebozoa and Rhizaria. We show the importance of environmental sequencing as a way of increasing taxon sampling in these supergroups. Finally, we highlight the interest of Amoebozoa and Rhizaria for understanding eukaryotic evolution and suggest that resolving their phylogenies will be among the main challenges for future phylogenomic analyses.     

Phylogeny of Annelida (Lophotrochozoa): total-evidence analysis of morphology and six genes

Background  

Annelida is one of the major protostome phyla, whose deep phylogeny is very poorly understood. Recent molecular phylogenies show that Annelida may include groups once considered separate phyla (Pogonophora, Echiurida, and Sipunculida) and that Clitellata are derived polychaetes. SThe "total-evidence" analyses combining morphological and molecular characters have been published for a few annelid taxa. No attempt has yet been made to analyse simultaneously morphological and molecular information concerning the Annelida as a whole.     

Morphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts: <Emphasis Type="Italic">Auranticordis quadriverberis</Emphasis> n. gen. et sp. (Cercozoa)

Background  

Comparative morphological studies and environmental sequencing surveys indicate that marine benthic environments contain a diverse assortment of microorganisms that are just beginning to be explored and characterized. The most conspicuous predatory flagellates in these habitats range from about 20–150 μm in size and fall into three major groups of eukaryotes that are very distantly related to one another: dinoflagellates, euglenids and cercozoans. The Cercozoa is a diverse group of amoeboflagellates that cluster together in molecular phylogenies inferred mainly from ribosomal gene sequences. These molecular phylogenetic studies have demonstrated that several enigmatic taxa, previously treated as Eukaryota insertae sedis, fall within the Cercozoa, and suggest that the actual diversity of this group is largely unknown. Improved knowledge of cercozoan diversity is expected to help resolve major branches in the tree of eukaryotes and demonstrate important cellular innovations for understanding eukaryote evolution.     

Global isolation by distance despite strong regional phylogeography in a small metazoan

Background  

Small vagile eukaryotic organisms, which comprise a large proportion of the Earth's biodiversity, have traditionally been thought to lack the extent of population structuring and geographic speciation observed in larger taxa. Here we investigate the patterns of genetic diversity, amongst populations of the salt lake microscopic metazoan Brachionus plicatilis s. s. (sensu stricto) (Rotifera: Monogononta) on a global scale. We examine the phylogenetic relationships of geographic isolates from four continents using a 603 bp fragment of the mitochondrial COI gene to investigate patterns of phylogeographic subdivision in this species. In addition we investigate the relationship between genetic and geographic distances on a global scale to try and reconcile the paradox between the high vagility of this species and the previously reported patterns of restricted gene flow, even over local spatial scales.     

AIR: A batch-oriented web program package for construction of supermatrices ready for phylogenomic analyses

Background  

Large multigene sequence alignments have over recent years been increasingly employed for phylogenomic reconstruction of the eukaryote tree of life. Such supermatrices of sequence data are preferred over single gene alignments as they contain vastly more information about ancient sequence characteristics, and are thus more suitable for resolving deeply diverging relationships. However, as alignments are expanded, increasingly numbers of sites with misleading phylogenetic information are also added. Therefore, a major goal in phylogenomic analyses is to maximize the ratio of information to noise; this can be achieved by the reduction of fast evolving sites.     

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

Background  

Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.     

Molecular phylogenetics reveal multiple tertiary vicariance origins of the African rain forest trees

Background  

Tropical rain forests are the most diverse terrestrial ecosystems on the planet. How this diversity evolved remains largely unexplained. In Africa, rain forests are situated in two geographically isolated regions: the West-Central Guineo-Congolian region and the coastal and montane regions of East Africa. These regions have strong floristic affinities with each other, suggesting a former connection via an Eocene pan-African rain forest. High levels of endemism observed in both regions have been hypothesized to be the result of either 1) a single break-up followed by a long isolation or 2) multiple fragmentation and reconnection since the Oligocene. To test these hypotheses the evolutionary history of endemic taxa within a rain forest restricted African lineage of the plant family Annonaceae was studied. Molecular phylogenies and divergence dates were estimated using a Bayesian relaxed uncorrelated molecular clock assumption accounting for both calibration and phylogenetic uncertainties.     

Biodiversity,biogeography, and connectivity of polychaetes in the world's largest marine minerals exploration frontier

Aim

The abyssal Clarion-Clipperton Zone (CCZ), Pacific Ocean, is an area of commercial importance owing to the growing interest in mining high-grade polymetallic nodules at the seafloor for battery metals. Research into the spatial patterns of faunal diversity, composition, and population connectivity is needed to better understand the ecological impacts of potential resource extraction. Here, a DNA taxonomy approach is used to investigate regional-scale patterns of taxonomic and phylogenetic alpha and beta diversity, and genetic connectivity, of the dominant macrofaunal group (annelids) across a 6 million km² region of the abyssal seafloor.

Location

The abyssal seafloor (3932–5055 m depth) of the Clarion-Clipperton Zone, equatorial Pacific Ocean.

Methods

We used a combination of new and published barcode data to study 1866 polychaete specimens using molecular species delimitation. Both phylogenetic and taxonomic alpha and beta diversity metrics were used to analyse spatial patterns of biodiversity. Connectivity analyses were based on haplotype distributions for a subset of the studied taxa.

Results

DNA taxonomy identified 291–314 polychaete species from the COI and 16S datasets respectively. Taxonomic and phylogenetic beta diversity between sites were relatively high and mostly explained by lineage turnover. Over half of pairwise comparisons were more phylogenetically distinct than expected based on their taxonomic diversity. Connectivity analyses in abundant, broadly distributed taxa suggest an absence of genetic structuring driven by geographical location.

Main Conclusions

Species diversity in abyssal Pacific polychaetes is high relative to other deep-sea regions. Results suggest that environmental filtering, where the environment selects against certain species, may play a significant role in regulating spatial patterns of biodiversity in the CCZ. A core group of widespread species have diverse haplotypes but are well connected over broad distances. Our data suggest that the high environmental and faunal heterogeneity of the CCZ should be considered in future policy decisions.