Evolutionary origins and diversification of proteobacterial mutualists

Mutualistic bacteria infect most eukaryotic species in nearly every biome. Nonetheless, two dilemmas remain unresolved about bacterial–eukaryote mutualisms: how do mutualist phenotypes originate in bacterial lineages and to what degree do mutualists traits drive or hinder bacterial diversification? Here, we reconstructed the phylogeny of the hyperdiverse phylum Proteobacteria to investigate the origins and evolutionary diversification of mutualistic bacterial phenotypes. Our ancestral state reconstructions (ASRs) inferred a range of 34–39 independent origins of mutualist phenotypes in Proteobacteria, revealing the surprising frequency with which host-beneficial traits have evolved in this phylum. We found proteobacterial mutualists to be more often derived from parasitic than from free-living ancestors, consistent with the untested paradigm that bacterial mutualists most often evolve from pathogens. Strikingly, we inferred that mutualists exhibit a negative net diversification rate (speciation minus extinction), which suggests that mutualism evolves primarily via transitions from other states rather than diversification within mutualist taxa. Moreover, our ASRs infer that proteobacterial mutualist lineages exhibit a paucity of reversals to parasitism or to free-living status. This evolutionary conservatism of mutualism is contrary to long-standing theory, which predicts that selection should often favour mutants in microbial mutualist populations that exploit or abandon more slowly evolving eukaryotic hosts.     

Symposium on Ciliates in Memory of Denis Lynn

Denis Lynn (1947–2018) was an outstanding protistologist, applying multiple techniques and data sources and thus pioneering an integrative approach in order to investigate ciliate biology. For example, he recognized the importance of the ultrastructure for inferring ciliate phylogeny, based on which he developed his widely accepted classification scheme for the phylum Ciliophora. In this paper, recent findings regarding the evolution and systematics of both peritrichs and the mainly marine planktonic oligotrichean spirotrichs are discussed and compared with the concepts and hypotheses formulated by Denis Lynn. Additionally, the state of knowledge concerning the diversity of ciliates in bromeliad phytotelmata and amitosis in ciliates is reviewed.     

Molecular analysis of nematode diversity and the evolution of parasitism.

A thorough and coherent classification of the phylum Nematoda is essential if the evolution of countless phenotypes is to be understood. Here, Mark Dorris, Paul De Ley and Mark Blaxter discuss how the application of molecular phylogenetics is helping to resolve some of the inconsistencies in morphological classification and phylogeny by establishing relationships between free-living and parasitic groups, showing possible patterns underlying the origins of parasitism and placing key nematode species in an evolutionary context for comparative study.     

Molecular evolution of ciliates (Ciliophora) and some related groups of protozoans

The review summarizes current evidence, including the findings related to molecular phylogeny of ciliates (type Ciliophora) and some related groups of protozoans. Based on comparison of the sequences of genes encoding various ribosomal RNAs (rRNAs), the phylogenetic relationships in seven out of eight known classes of ciliates are discussed. The events related to early branching of the eukaryotic tree are briefly presented. The evolutionary history of amitochondrial protists ids considered with regard to reductionistic evolution and archeozoic hypothesis. The phylogenetic relationships among ciliates and sister groups of apicomplexans and dinoflagellates are considered.     

The dauer hypothesis and the evolution of parasitism: 20 years on and still going strong

How any complex trait has evolved is a fascinating question, yet the evolution of parasitism among the nematodes is arguably one of the most arresting. How did free-living nematodes cross that seemingly insurmountable evolutionary chasm between soil dwelling and survival inside another organism? Which of the many finely honed responses to the varied and harsh environments of free-living nematodes provided the material upon which natural selection could act? Although several complementary theories explain this phenomenon, I will focus on the dauer hypothesis. The dauer hypothesis posits that the arrested third-stage dauer larvae of free-living nematodes such as Caenorhabditis elegans are, due to their many physiological similarities with infective third-stage larvae of parasitic nematodes, a pre-adaptation to parasitism. If so, then a logical extension of this hypothesis is that the molecular pathways which control entry into and recovery from dauer formation by free-living nematodes in response to environmental cues have been co-opted to control the processes of infective larval arrest and activation in parasitic nematodes. The molecular machinery that controls dauer entry and exit is present in a wide range of parasitic nematodes. However, the developmental outputs of the different pathways are both conserved and divergent, not only between populations of C. elegans or between C. elegans and parasitic nematodes but also between different species of parasitic nematodes. Thus the picture that emerges is more nuanced than originally predicted and may provide insights into the evolution of such an interesting and complex trait.     

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control

Background

Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism.

Results

We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio.

Conclusions

Knowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.     

Unusually high evolutionary rate of the elongation factor 1 alpha genes from the Ciliophora and its impact on the phylogeny of eukaryotes

The elongation factor 1 alpha (EF-1 alpha) has become widely employed as a phylogenetic marker for studying eukaryotic evolution. However, a disturbing problem, the artifactual polyphyly of ciliates, is always observed. It has been suggested that the addition of new sequences will help to circumvent this problem. Thus, we have determined 15 new ciliate EF-1 alpha sequences, providing for a more comprehensive taxonomic sampling of this phylum. These sequences have been analyzed together with a representation of eukaryotic sequences using distance-, parsimony-, and likelihood-based phylogenetic methods. Such analyses again failed to recover the monophyly of Ciliophora. A study of the substitution rate showed that ciliate EF-1 alpha genes exhibit a high evolutionary rate, produced in part by an increased number of variable positions. This acceleration could be related to alterations of the accessory functions acquired by this protein, likely to those involving interactions with the cytoskeleton, which is very modified in the Ciliophora. The high evolutionary rate of these sequences leads to an artificial basal emergence of some ciliates in the eukaryotic tree by effecting a long-branch attraction artifact that produces an asymmetric topology for the basal region of the tree. The use of a maximum-likelihood phylogenetic method (which is less sensitive to long-branch attraction) and the addition of sequences to break long branches allow retrieval of more symmetric topologies, which suggests that the asymmetric part of the tree is most likely artifactual. Therefore, the sole reliable part of the tree appears to correspond to the apical symmetric region. These kinds of observations suggest that the general eukaryotic evolution might have consisted of a massive radiation followed by an increase in the evolutionary rates of certain groups that emerge artificially as early branches in the asymmetric base of the tree. Ciliates in the case of the EF-1 alpha genes would offer clear evidence for this hypothesis.     

Endosymbiotic bacteria associated with nematodes, ticks and amoebae

Endosymbiosis is a mutualistic, parasitic or commensal symbiosis in which one symbiont is living within the body of another organism. Such symbiotic relationship with free-living amoebae and arthropods has been reported with a large biodiversity of microorganisms, encompassing various bacterial clades and to a lesser extent some fungi and viruses. By contrast, current knowledge on symbionts of nematodes is still mainly restricted to Wolbachia and its interaction with filarial worms that lead to increased pathogenicity of the infected nematode. In this review article, we aim to highlight the main characteristics of symbionts in term of their ecology, host cell interactions, parasitism and co-evolution, in order to stimulate future research in a field that remains largely unexplored despite the availability of modern tools.     

Highly divergent actins from karyorelictean, heterotrich, and litostome ciliates

We have cloned, sequenced, and characterized cDNA of actins from five ciliate species of three different classes of the phylum Ciliophora: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma japonicum, Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Loxodes striatus uses UGA as the stop codon and has numerous in-frame UAA and UAG, which are translated into glutamine. The other four species use UAA as the stop codon and have no in-frame UAG nor UGA. The putative amino acid sequences of the newly determined actin genes were found to be highly divergent as expected from previous findings of other ciliate actins. These sequences were also highly divergent from other ciliate actins, indicating that actin genes are highly diverse even within the phylum Ciliophora. Phylogenetic analysis showed high evolutionary rate of ciliate actins. Our results suggest that the evolutionary rate was accelerated because of the differences in molecular interactions.     

Large-subunit ribosomal RNA systematics of symbiotic dinoflagellates: morphology does not recapitulate phylogeny

Biochemical, histological, physiological, and genetic evidence indicates that dinoflagellates symbiotic with marine invertebrates are a heterogeneous complex of taxa, representing at least five genera in three orders. Despite a wealth of data regarding morphological, biochemical, and behavioral differences among symbiotic dinoflagellates, knowledge concerning patterns of diversification is limited. I analyzed approximately 900 bp of the 5' end of the large-subunit ribosomal RNA gene from 14 dinoflagellate isolates: six cultured Symbiodinium specimens, two cultured symbiotic Gymnodinium, two algal samples isolated from reef-building corals, an algal sample obtained from cultures of the jellyfish Cassiopea xamachana, and three free-living Gymnodinium isolates. Results show that morphological similarities among the examined symbiotic taxa do not necessarily correspond with molecular phylogeny. The included Symbiodinium taxa represent a paraphyletic assemblage while Gymnodinium is reconstructed as a polyphyletic assemblage. Analysis indicates that all the included symbiotic dinoflagellates descended from a common, symbiotic ancestor (though within the dinoflagellates, symbiosis is a polyphyletic trait). Additionally, two free-living dinoflagellates emerge within the symbiotic clade, suggesting that the symbiotic lifestyle has been lost at least once in this group. It has been hypothesized that rates of evolution within mutualistic endosymbioses should be reduced relative to free-living taxa. However, results indicate that rates of molecular, morphological, biochemical and behavioral change are similar among branches leading to symbiotic and free-living dinoflagellates.     

Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.     

Endophytic fungus-vascular plant-insect interactions

Insect association with fungi has a long history. Theories dealing with the evolution of insect herbivory indicate that insects used microbes including fungi as their principal food materials before flowering plants evolved. Subtlety and the level of intricacy in the interactions between insects and fungi indicate symbiosis as the predominant ecological pattern. The nature of the symbiotic interaction that occurs between two organisms (the insect and the fungus), may be either mutualistic or parasitic, or between these two extremes. However, the triangular relationship involving three organisms, viz., an insect, a fungus, and a vascular plant is a relationship that is more complicated than what can be described as either mutualism or parasitism, and may represent facets of both. Recent research has revealed such a complex relationship in the vertically transmitted type-I endophytes living within agriculturally important grasses and the pestiferous insects that attack them. The intricacy of the association depends on the endophytic fungus-grass association and the insect present. Secondary compounds produced in the endophytic fungus-grass association can provide grasses with resistance to herbivores resulting in mutualistic relationship between the fungus and the plant that has negative consequences for herbivorous insects. The horizontally transmitted nongrass type-II endophytes are far less well studied and as such their ecological roles are not fully understood. This forum article explores the intricacy of dependence in such complex triangular relationships drawing from well-established examples from the fungi that live as endophytes in vascular plants and how they impact on the biology and evolution of free-living as well as concealed (e.g., gall-inducing, gall-inhabiting) insects. Recent developments with the inoculation of strains of type-I fungal endophytes into grasses and their commercialization are discussed, along with the possible roles the endophytic fungi play in the galls induced by the Cecidomyiidae (Diptera).     

ORIGINS,EVOLUTION, AND DIVERSIFICATION OF CLEPTOPARASITIC LINEAGES IN LONG‐TONGUED BEES

The evolution of parasitic behavior may catalyze the exploitation of new ecological niches yet also binds the fate of a parasite to that of its host. It is thus not clear whether evolutionary transitions from free‐living organism to parasite lead to increased or decreased rates of diversification. We explore the evolution of brood parasitism in long‐tongued bees and find decreased rates of diversification in eight of 10 brood parasitic clades. We propose a pathway for the evolution of brood parasitic strategy and find that a strategy in which a closed host nest cell is parasitized and the host offspring is killed by the adult parasite represents an obligate first step in the appearance of a brood parasitic lineage; this ultimately evolves into a strategy in which an open host cell is parasitized and the host offspring is killed by a specialized larval instar. The transition to parasitizing open nest cells expanded the range of potential hosts for brood parasitic bees and played a fundamental role in the patterns of diversification seen in brood parasitic clades. We address the prevalence of brood parasitic lineages in certain families of bees and examine the evolution of brood parasitism in other groups of organisms.     

Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.     

Comparative analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in ciliates (Alveolata,Ciliophora) and evaluation of its suitability as a biodiversity marker

The mitochondrial cytochrome c oxidase subunit 1 (COI) gene of ciliates was first successfully sequenced in species of the genera Tetrahymena and Paramecium (Class Oligohymenophorea). The sequence of the COI gene is extremely divergent from other eukaryotes and includes an insert, which is over 300 nucleotides long. In this study, we designed a primer pair that successfully amplified the COI gene of ciliates from five different classes: Heterotrichea, Spirotrichea, Oligohymenophorea, Nassophorea and Colpodea. These classes represent the diversity of the phylum Ciliophora very well, since they are widely distributed on the ciliate small subunit rRNA tree. The amplified region is approximately 850 nucleotides long and corresponds to the general barcoding region; it also includes the insert region. In this study, 58 new COI sequences from over 38 species in 13 orders are analysed and compared, and distance trees are constructed. While the COI gene shows high divergence within ciliates, the insert region, which is present in all classes, is even more divergent. Genetic distances calculated with and without the insert region remain in the same range at the intraspecific level, but they differ considerably at or above genus level. This suggests that the entire barcoding region is under similar selective constraints and that the evolutionary rate of the ciliate COI is extremely high and shows unequal rate variation. Although many problems still remain regarding standardization of barcoding methods in ciliates, the development of a universal or almost universal primer combination for the Phylum Ciliophora represents important progress. As shown in four examples, the resolution of COI at the intraspecific level is much greater than that of any nuclear genes and shows great potential to (1) identify species based on molecular data if a reliable database exists, and (2) resolve the relationships of closely related ciliate taxa and uncover cryptic species.     

An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

Evolution of parasite life history traits: myths and reality

Parasitism has evolved independently several times in many different animal lineages. Observations made on distantly related parasites have revealed a variety of adaptations to parasitism, including changes in physiology, morphology, and life history traits. These observations have led parasitologists to formulate general rules about the evolution of parasites, rules that define a common evolutionary path presumably followed by all parasitic organisms. Robert Poulin uses recent evidence to question the generality of these rules and to show that parasite evolution may take different roads. The selective pressures acting on parasites are diverse and may guide their evolution in any direction, just as they have shaped a wide variety of free-living organisms.     

车轮虫分类与系统发育研究进展

车轮虫属一大类寄生性纤毛虫原生动物,可不同程度地给宿主造成危害,故对其研究既具有理论意义,又具有经济价值。文中简单回顾了车轮虫的分类研究和系统发育研究的历史。在分类学研究领域方面,全面介绍了目前车轮虫科已发现属的寄生部位和地理分布等;而系统发育研究领域方面,则介绍了车轮虫研究在萌芽期、探索期和发展期这三个时期的进展,包括形态学和分子生物学两方面的研究内容。并对该领域今后应进行的研究提出了建议。     

Cortical ultrastructure of Coleps bicuspis Noland, 1925 and the phylogeny of the class Prostomatea (Ciliophora)

The ultrastructure of Coleps bicuspis Noland, 1925 is described. The ciliate is a typical prostomate: the somatic kinetid is a monokinetid with a postciliary ribbon at triple 9, a kinetodesmal fibril originating near triplets 5, 6, 7 and an apparently radial transverse ribbon at triplet 4. The oral area is circular and has three brosse kineties associated with it. The brosse kineties are composed of dikinetids whose anterior kinetosome bears a tangential transverse ribbon and whose posterior kinetosome bears the fibrillar associates typical of a somatic monokinetid. The oral dikinetids are oriented parallel to the circumference of the oral cavity, which is surrounded by oral papillae and oral ridges. Pairs of nematodesmata, originating from oral dikinetid kinetosomes, are typically triangular in transection. A phylogeny of rhabdophoran ciliates is presented using the mixed parsimony algorithm and is discussed with reference to the systematic revisions of the phylum Ciliophora.