Evolution of myxozoan mitochondrial genomes: insights from myxobolids.

BACKGROUND: Myxozoa is a class of cnidarian parasites that encompasses over 2,400 species. Phylogenetic relationships among myxozoans remain highly debated, owing to both a lack of informative morphological characters and a shortage of molecular markers. Mitochondrial (mt) genomes are a common marker in phylogeny and biogeography. However, only five complete myxozoan mt genomes have been sequenced: four belonging to two closely related genera, Enteromyxum and Kudoa, and one from the genus Myxobolus. Interestingly, while cytochrome oxidase genes could be identified in Enteromyxum and Kudoa, no such genes were found in Myxobolus squamalis, and another member of the Myxobolidae (Henneguya salminicola) was found to have lost its entire mt genome. To evaluate the utility of mt genomes to reconstruct myxozoan relationships and to understand if the loss of cytochrome oxidase genes is a characteristic of myxobolids, we sequenced the mt genome of five myxozoans (Myxobolus wulii, M. honghuensis, M. shantungensis, Thelohanellus kitauei and, Sphaeromyxa zaharoni) using Illumina and Oxford Nanopore platforms. RESULTS: Unlike Enteromyxum, which possesses a partitioned mt genome, the five mt genomes were encoded on single circular chromosomes. An mt plasmid was found in M. wulii, as described previously in Kudoa iwatai. In all new myxozoan genomes, five protein-coding genes (cob, cox1, cox2, nad1, and nad5) and two rRNAs (rnl and rns) were recognized, but no tRNA. We found that Myxobolus and Thelohanellus species shared unidentified reading frames, supporting the view that these mt open reading frames are functional. Our phylogenetic reconstructions based on the five conserved mt genes agree with previously published trees based on the 18S rRNA gene. CONCLUSIONS: Our results suggest that the loss of cytochrome oxidase genes is not a characteristic of all myxobolids, the ancestral myxozoan mt genome was likely encoded on a single circular chromosome, and mt plasmids exist in a few lineages. Our findings indicate that myxozoan mt sequences are poor markers for reconstructing myxozoan phylogenetic relationships because of their fast-evolutionary rates and the abundance of repeated elements, which complicates assembly.

The Phylogenetic Position of the Enigmatic, Polypodium hydriforme (Cnidaria, Polypodiozoa): Insights from Mitochondrial Genomes.

Polypodium hydriforme is an enigmatic parasite that belongs to the phylum Cnidaria. Its taxonomic position has been debated: whereas it was previously suggested to be part of Medusozoa, recent phylogenomic analyses based on nuclear genes support the view that P. hydriforme and Myxozoa form a clade called Endocnidozoa. Medusozoans have linear mitochondrial (mt) chromosomes, whereas myxozoans, as most metazoan species, have circular chromosomes. In this work, we determined the structure of the mt genome of P. hydriforme, using Illumina and Oxford Nanopore Technologies reads, and showed that it is circular. This suggests that P. hydriforme is not nested within Medusozoa, as this would entail linearization followed by recirculation. Instead, our results support the view that P. hydriforme is a sister clade to Myxozoa, and mt linearization in the lineage leading to medusozoans occurred after the divergence of Myxozoa + P. hydriforme. Detailed analyses of the assembled P. hydriforme mt genome show that: (1) it is encoded on a single circular chromosome with an estimated size of ∼93,000 base pairs, making it one of the largest metazoan mt genomes; (2) around 78% of the genome encompasses a noncoding region composed of several repeat types; (3) similar to Myxozoa, no mt tRNAs were identified; (4) the codon TGA is a stop codon and does not encode for tryptophan as in other cnidarians; (5) similar to myxozoan mt genomes, it is extremely fast evolving.

Genomic structural plasticity of rodent-associated Bartonella in nature.

Rodent-associated Bartonella species have shown a remarkable genetic diversity and pathogenic potential. To further explore the extent of the natural intraspecific genomic variation and its potential role as an evolutionary driver, we focused on a single genetically diverse Bartonella species, Bartonella krasnovii, which circulates among gerbils and their associated fleas. Twenty genomes from 16 different B. krasnovii genotypes were fully characterized through a genome sequencing assay (using short and long read sequencing), pulse field gel electrophoresis (PFGE), and PCR validation. Genomic analyses were performed in comparison to the B. krasnovii strain OE 1-1. While, single nucleotide polymorphism represented only a 0.3% of the genome variation, structural diversity was identified in these genomes, with an average of 51 ± 24 structural variation (SV) events per genome. Interestingly, a large proportion of the SVs (>40%) was associated with prophages. Further analyses revealed that most of the SVs, and prophage insertions were found at the chromosome replication termination site (ter), suggesting this site as a plastic zone of the B. krasnovii chromosome. Accordingly, six genomes were found to be unbalanced, and essential genes near the ter showed a shift between the leading and lagging strands, revealing the SV effect on these genomes. In summary, our findings demonstrate the extensive genomic diversity harbored by wild B. krasnovii strains and suggests that its diversification is initially promoted by structural changes, probably driven by phages. These events may constantly feed the system with novel genotypes that ultimately lead to inter- and intraspecies competition and adaptation.

Resolving the Microalgal Gene Landscape at the Strain Level: a Novel Hybrid Transcriptome of Emiliania huxleyi CCMP3266.

Microalgae are key ecological players with a complex evolutionary history. Genomic diversity, in addition to limited availability of high-quality genomes, challenge studies that aim to elucidate molecular mechanisms underlying microalgal ecophysiology. Here, we present a novel and comprehensive transcriptomic hybrid approach to generate a reference for genetic analyses and resolve the microalgal gene landscape at the strain level. The approach is demonstrated for a strain of the coccolithophore microalga Emiliania huxleyi, which is a species complex with considerable genome variability. The investigated strain is commonly studied as a model for algal-bacterial interactions and was therefore sequenced in the presence of bacteria to elicit the expression of interaction-relevant genes. We applied complementary PacBio Iso-Seq full-length cDNA and poly(A)-independent Illumina total RNA sequencing, which resulted in a de novo-assembled, near-complete hybrid transcriptome. In particular, hybrid sequencing improved the reconstruction of long transcripts and increased the recovery of full-length transcript isoforms. To use the resulting hybrid transcriptome as a reference for genetic analyses, we demonstrate a method that collapses the transcriptome into a genome-like data set, termed "synthetic genome" (sGenome). We used the sGenome as a reference to visually confirm the robustness of the CCMP3266 gene assembly, to conduct differential gene expression analysis, and to characterize novel E. huxleyi genes. The newly identified genes contribute to our understanding of E. huxleyi genome diversification and are predicted to play a role in microbial interactions. Our transcriptomic toolkit can be implemented in various microalgae to facilitate mechanistic studies on microalgal diversity and ecology. IMPORTANCE Microalgae are key players in the ecology and biogeochemistry of our oceans. Efforts to implement genomic and transcriptomic tools in laboratory studies involving microalgae suffer from the lack of published genomes. In the case of coccolithophore microalgae, the problem has long been recognized; the model species Emiliania huxleyi is a species complex with genomes composed of a core and a large variable portion. To study the role of the variable portion in niche adaptation, and specifically in microbial interactions, strain-specific genetic information is required. Here, we present a novel transcriptomic hybrid approach, and generated strain-specific genome-like information. We demonstrate our approach on an E. huxleyi strain that is cocultivated with bacteria. By constructing a "synthetic genome," we generated comprehensive gene annotations that enabled accurate analyses of gene expression patterns. Importantly, we unveiled novel genes in the variable portion of E. huxleyi that play putative roles in microbial interactions.

Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs.

Serotonin (5-HT) neurons, the major components of the raphe nuclei, arise from ventral hindbrain progenitors. Based on anatomical location and axonal projection, 5-HT neurons are coarsely divided into rostral and caudal groups. Here, we propose a novel strategy to generate hindbrain 5-HT neurons from human pluripotent stem cells (hPSCs), which involves the formation of ventral-type neural progenitor cells and stimulation of the hindbrain 5-HT neural development. A caudalizing agent, retinoid acid, was used to direct the cells into the hindbrain cell fate. Approximately 30%-40% of hPSCs successfully developed into 5-HT-expressing neurons using our protocol, with the majority acquiring a caudal rhombomere identity (r5-8). We further modified our monolayer differentiation system to generate 5-HT neuron-enriched hindbrain-like organoids. We also suggest downstream applications of our 5-HT monolayer and organoid cultures to study neuronal response to gut microbiota. Our methodology could become a powerful tool for future studies related to 5-HT neurotransmission.

A cnidarian parasite of salmon (Myxozoa: Henneguya) lacks a mitochondrial genome.

Although aerobic respiration is a hallmark of eukaryotes, a few unicellular lineages, growing in hypoxic environments, have secondarily lost this ability. In the absence of oxygen, the mitochondria of these organisms have lost all or parts of their genomes and evolved into mitochondria-related organelles (MROs). There has been debate regarding the presence of MROs in animals. Using deep sequencing approaches, we discovered that a member of the Cnidaria, the myxozoan Henneguya salminicola, has no mitochondrial genome, and thus has lost the ability to perform aerobic cellular respiration. This indicates that these core eukaryotic features are not ubiquitous among animals. Our analyses suggest that H. salminicola lost not only its mitochondrial genome but also nearly all nuclear genes involved in transcription and replication of the mitochondrial genome. In contrast, we identified many genes that encode proteins involved in other mitochondrial pathways and determined that genes involved in aerobic respiration or mitochondrial DNA replication were either absent or present only as pseudogenes. As a control, we used the same sequencing and annotation methods to show that a closely related myxozoan, Myxobolus squamalis, has a mitochondrial genome. The molecular results are supported by fluorescence micrographs, which show the presence of mitochondrial DNA in M. squamalis, but not in H. salminicola. Our discovery confirms that adaptation to an anaerobic environment is not unique to single-celled eukaryotes, but has also evolved in a multicellular, parasitic animal. Hence, H. salminicola provides an opportunity for understanding the evolutionary transition from an aerobic to an exclusive anaerobic metabolism.

The Multipartite Mitochondrial Genome of Enteromyxum leei (Myxozoa): Eight Fast-Evolving Megacircles.

Myxozoans are a large group of poorly characterized cnidarian parasites. To gain further insight into their evolution, we sequenced the mitochondrial (mt) genome of Enteromyxum leei and reevaluate the mt genome structure of Kudoa iwatai. Although the typical animal mt genome is a compact, 13-25 kb, circular chromosome, the mt genome of E. leei was found to be fragmented into eight circular chromosomes of ∼23 kb, making it the largest described animal mt genome. Each chromosome was found to harbor a large noncoding region (∼15 kb), nearly identical between chromosomes. The protein coding genes show an unusually high rate of sequence evolution and possess little similarity to their cnidarian homologs. Only five protein coding genes could be identified and no tRNA genes. Surprisingly, the mt genome of K. iwatai was also found to be composed of two chromosomes. These observations confirm the remarkable plasticity of myxozoan mt genomes.