A machine-learning-based alternative to phylogenetic bootstrap.

MOTIVATION: Currently used methods for estimating branch support in phylogenetic analyses often rely on the classic Felsenstein's bootstrap, parametric tests, or their approximations. As these branch support scores are widely used in phylogenetic analyses, having accurate, fast, and interpretable scores is of high importance. RESULTS: Here, we employed a data-driven approach to estimate branch support values with a probabilistic interpretation. To this end, we simulated thousands of realistic phylogenetic trees and the corresponding multiple sequence alignments. Each of the obtained alignments was used to infer the phylogeny using state-of-the-art phylogenetic inference software, which was then compared to the true tree. Using these extensive data, we trained machine-learning algorithms to estimate branch support values for each bipartition within the maximum-likelihood trees obtained by each software. Our results demonstrate that our model provides fast and more accurate probability-based branch support values than commonly used procedures. We demonstrate the applicability of our approach on empirical datasets. AVAILABILITY AND IMPLEMENTATION: The data supporting this work are available in the Figshare repository at https://doi.org/10.6084/m9.figshare.25050554.v1, and the underlying code is accessible via GitHub at https://github.com/noaeker/bootstrap_repo.

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.

GenomeFLTR: filtering reads made easy.

In the last decade, advances in sequencing technology have led to an exponential increase in genomic data. These new data have dramatically changed our understanding of the evolution and function of genes and genomes. Despite improvements in sequencing technologies, identifying contaminated reads remains a complex task for many research groups. Here, we introduce GenomeFLTR, a new web server to filter contaminated reads. Reads are compared against existing sequence databases from various representative organisms to detect potential contaminants. The main features implemented in GenomeFLTR are: (i) automated updating of the relevant databases; (ii) fast comparison of each read against the database; (iii) the ability to create user-specified databases; (iv) a user-friendly interactive dashboard to investigate the origin and frequency of the contaminations; (v) the generation of a contamination-free file. Availability: https://genomefltr.tau.ac.il/.

Octocorals in the Gulf of Aqaba exhibit high photosymbiont fidelity.

Symbiotic associations, widespread in terrestrial and marine ecosystems, are of considerable ecological importance. Many tropical coral species are holobionts, formed by the obligate association between a cnidarian host and endosymbiotic dinoflagellates of the family Symbiodiniaceae. The latter are abundant on coral reefs from very shallow water down to the upper mesophotic zone (30-70 m). The research on scleractinians has revealed that the photosymbiont lineages present in the cnidarian host play an important role in the coral's ability to thrive under different environmental conditions, such as light regime and temperature. However, little is known regarding octocoral photosymbionts, and in particular regarding those found deeper than 30 m. Here, we used ribosomal (ITS2) and chloroplast (23S) markers to uncover, for the first time, the dominant Symbiodiniaceae taxa present in 19 mesophotic octocoral species (30-70 m depth) from the Gulf of Aqaba/Eilat (northern Red Sea). In addition, using high-throughput sequencing of the ITS2 region we characterized both the dominant and the rare Symbiodiniaceae lineages found in several species across depth. The phylogenetic analyses of both markers were in agreement and revealed that most of the studied mesophotic octocorals host the genus Cladocopium. Litophyton spp. and Klyxum utinomii were exceptions, as they harbored Symbiodinium and Durusdinium photosymbionts, respectively. While the dominant algal lineage of each coral species did not vary across depth, the endosymbiont community structure significantly differed between host species, as well as between different depths for some host species. The findings from this study contribute to the growing global-catalogue of Cnidaria-Symbiodiniaceae associations. Unravelling the Symbiodiniaceae composition in octocoral holobionts across environmental gradients, depth in particular, may enable a better understanding of how specialized those associations are, and to what extent coral holobionts are able to modify their photosymbionts.

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.

Myxozoan infection in thinlip mullet Chelon ramada (Mugiliformes: Mugilidae) in the Sea of Galilee.

Mullets (Mugilidae) are economically important fish in Israel. Two species of mugilids (i.e., the thinlip mullet Chelon ramada and the flathead grey mullet Mugil cephalus) have been stocked in the Sea of Galilee (Lake Kinneret) in order to increase fishermen's income and lake water quality. These catadromous species do not reproduce in the lake, consequently, fingerlings have been introduced every year since 1958. Following a survey of myxozoan infections in the Sea of Galilee, we described Myxobolus pupkoi n. sp. infecting the gill arches, and reported Myxobolus exiguus from visceral peritoneum and gall bladder of C. ramada. The prevalence of infection of both Myxobolus pupkoi n. sp. and M. exiguus were 11.5% (2/23). Our study indicates that the parasites infecting C. ramada belong to a lineage of myxozoans infecting mugilids. This result suggests that the infection took place in the Mediterranean Sea, where the fingerlings were caught, before their introduction into the Sea of Galilee. Since 2018 only farm-raised fingerlings have been introduced. We thus recommend to closely monitor the presence of these parasites in the future to determine if the presence of parasites disappear with the introduction of farm-raised fingerlings.

Two novel myxosporean parasite species of Ceratomyxa Thélohan, 1892 from the banded cusk-eel Raneya brasiliensis (Kaup) (Ophidiiformes: Ophidiidae) off Patagonia, Argentina.

We described two novel myxozoan parasite species Ceratomyxa argentina n. sp. and Ceratomyxa raneyae n. sp. from the gall bladder of Raneya brasiliensis (Kaup) from the Patagonian coast of Argentina. Both species can be distinguished from other ceratomyxids by myxospore and polar capsule (nematocyst) morphology and morphometry, fish host and geographic locality. Phylogenetic reconstruction using ssrDNA gene sequences showed that the two new species are placed in a long-branching ceratomyxid clade which also include Ceratomyxa appendiculata Thélohan, 1892, Ceratomyxa anko Freeman, Yokoyama and Ogawa, 2008, Ceratomyxa pantherini Gunter, Burger and Adlard, 2010 and Pseudoalataspora kovalevae Kalavati, MacKenzie, Collins, Hemmingsen and Brickle, 2013. This study documents additional biodiversity of marine myxozoans in the South Atlantic, a region still largely unexplored for this group of parasitic cnidarians.

And Then There Were Three : Extreme Regeneration Ability of the Solitary Chordate Polycarpa mytiligera.

Extensive regenerative ability is a common trait of animals capable of asexual development. The current study reveals the extraordinary regeneration abilities of the solitary ascidian Polycarpa mytiligera. Dissection of a single individual into separate fragments along two body axes resulted in the complete regeneration of each fragment into an independent, functional individual. The ability of a solitary ascidian, incapable of asexual development, to achieve bidirectional regeneration and fully regenerate all body structures and organs is described here for the first time. Amputation initiated cell proliferation in proximity to the amputation line. Phylogenetic analysis demonstrated the close affinity of P. mytiligera to colonial species. This evolutionary proximity suggests the ability for regeneration as an exaptation feature for colonial lifestyle. P. mytiligera's exceptional regenerative abilities and phylogenetic position highlight its potential to serve as a new comparative system for studies seeking to uncover the evolution of regeneration and coloniality among the chordates.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode.

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

Myxosporea (Myxozoa, Cnidaria) Lack DNA Cytosine Methylation.

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.

Illuminating Genetic Mysteries of the Dead Sea Scrolls.

The discovery of the 2,000-year-old Dead Sea Scrolls had an incomparable impact on the historical understanding of Judaism and Christianity. "Piecing together" scroll fragments is like solving jigsaw puzzles with an unknown number of missing parts. We used the fact that most scrolls are made from animal skins to "fingerprint" pieces based on DNA sequences. Genetic sorting of the scrolls illuminates their textual relationship and historical significance. Disambiguating the contested relationship between Jeremiah fragments supplies evidence that some scrolls were brought to the Qumran caves from elsewhere; significantly, they demonstrate that divergent versions of Jeremiah circulated in parallel throughout Israel (ancient Judea). Similarly, patterns discovered in non-biblical scrolls, particularly the Songs of the Sabbath Sacrifice, suggest that the Qumran scrolls represent the broader cultural milieu of the period. Finally, genetic analysis divorces debated fragments from the Qumran scrolls. Our study demonstrates that interdisciplinary approaches enrich the scholar's toolkit.

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.

A new mitochondrial gene order in the banded cusk-eel Raneya brasiliensis (Actinopterygii, Ophidiiformes).

The complete mitochondrial genome of the banded cusk-eel, Raneya brasilensis (Kaup, 1856), was obtained using next-generation sequencing approaches. The genome sequence was 16,881 bp and exhibited a novel gene order for a vertebrate. Specifically, the WANCY and the nd6 - D-loop regions were re-ordered, supporting the hypothesis that these two regions are hotspots for gene rearrangements in Actinopterygii. Phylogenetic reconstructions confirmed that R. brasiliensis is nested within Ophidiiformes. Mitochondrial genomes are required from additional ophidiins to determine whether the gene rearrangements that we observed are specific to the genus Raneya or to the subfamily Ophidiinae.

A genome wide survey reveals multiple nematocyst-specific genes in Myxozoa.

BACKGROUND: Myxozoa represents a diverse group of microscopic endoparasites whose life cycle involves two hosts: a vertebrate (usually a fish) and an invertebrate (usually an annelid worm). Despite lacking nearly all distinguishing animal characteristics, given that each life cycle stage consists of no more than a few cells, molecular phylogenetic studies have revealed that myxozoans belong to the phylum Cnidaria, which includes corals, sea anemones, and jellyfish. Myxozoa, however, do possess a polar capsule; an organelle that is homologous to the stinging structure unique to Cnidaria: the nematocyst. Previous studies have identified in Myxozoa a number of protein-coding genes that are specific to nematocytes (the cells producing nematocysts) and thus restricted to Cnidaria. Determining which other genes are also homologous with the myxozoan polar capsule genes could provide insight into both the conservation and changes that occurred during nematocyst evolution in the transition to endoparasitism. RESULTS: Previous studies have examined the phylogeny of two cnidarian-restricted gene families: minicollagens and nematogalectins. Here we identify and characterize seven additional cnidarian-restricted genes in myxozoan genomes using a phylogenetic approach. Four of the seven had never previously been identified as cnidarian-specific and none have been studied in a phylogenetic context. A majority of the proteins appear to be involved in the structure of the nematocyst capsule and tubule. No venom proteins were identified among the cnidarian-restricted genes shared by myxozoans. CONCLUSIONS: Given the highly divergent forms that comprise Cnidaria, obtaining insight into the processes underlying their ancient diversification remains challenging. In their evolutionary transition to microscopic endoparasites, myxozoans lost nearly all traces of their cnidarian ancestry, with the one prominent exception being their nematocysts (or polar capsules). Thus nematocysts, and the genes that code for their structure, serve as rich sources of information to support the cnidarian origin of Myxozoa.

Extensive mitochondrial gene rearrangements in Ctenophora: insights from benthic Platyctenida.

BACKGROUND: Complete mitochondrial (mt) genomes have been sequenced for thousands of animals and represent a molecule of choice for many evolutionary studies. Nevertheless, some animal groups have remained under-sampled. Ctenophora (comb jellies) is one such example, with only two complete mt sequences determined hitherto for this phylum, which encompasses ca. 150-200 described species. This lack of data derives from the extremely fast mt evolutionary rate in this lineage, complicating primer design and DNA amplification. Indeed, in the two ctenophore mt genomes sequenced to date, i.e. those of Mnemiopsis leidyi (order Lobata) and Pleurobrachia bachei (order Cydippida), both rRNA and protein coding genes exhibit an extraordinary size reduction and have highly derived sequences. Additionally, all tRNAs, and the atp6 and atp8 genes are absent. In order to determine whether these characteristics are shared by other ctenophores, we obtained the complete mt genomes of three benthic ctenophores belonging to the so far unsampled order of Platyctenida: Coeloplana loyai, Coeloplana yulianicorum and Vallicula multiformis. RESULTS: The mt genomes of benthic ctenophores reveal the same peculiarities found in Mnemiopsis and Pleurobrachia, demonstrating that the fast evolutionary rate is a general trait of the ctenophore mt genomes. Our results also indicate that this high evolutionary rate not only affects the nucleotide substitution but also gene rearrangements. Indeed, gene order was highly rearranged among representatives of the different taxonomic orders in which it was close to random, but also quite variable within Platyctenida, in which the genera Coeloplana and Vallicula share only four conserved synteny blocks. However, the two congeneric Coeloplana species display exactly the same gene order. Because of the extreme evolutionary rate, our phylogenetic analyses were unable to resolve the phylogenetic position of ctenophores within metazoans or the relationships among the different Ctenophora orders. Comparative sequence-analyses allowed us to correct the annotation of the Pleurobrachia mt genome, confirming the absence of tRNAs, the presence of both rRNA genes, and the existence of a reassignment of codon TGA from tryptophan to serine for this species. CONCLUSIONS: Since Platyctenida is an early diverging lineage among Ctenophora, our findings suggest that the mt traits described above are ancestral characteristics of this phylum.

The First Mitochondrial Genomics and Evolution SMBE-Satellite Meeting: A New Scientific Symbiosis.

The central role of the mitochondrion for cellular and organismal metabolism is well known, yet its functional role in evolution has rarely been featured in leading international conferences. Moreover, the contribution of mitochondrial genetics to complex disease phenotypes is particularly important, and although major advances have been made in the field of genomics, mitochondrial genomic data have in many cases been overlooked. Accumulating data and new knowledge support a major contribution of this maternally inherited genome, and its interactions with the nucleus, to both major evolutionary processes and diverse disease phenotypes. These advances encouraged us to assemble the first Mitochondrial Genomics and Evolution (MGE) meeting-an SMBE satellite and Israeli Science foundation international conference (Israel, September 2017). Here, we report the content and outcome of the MGE meeting (https://www.mge2017.com/; last accessed November 5, 2017).

Molecular diversity of benthic ctenophores (Coeloplanidae).

Coeloplanidae, the largest family of benthic ctenophores, comprises 33 species, all described based on traditional morphological characteristics, such as coloration, length, and number of aboral papillae, which are highly variable and can be affected by fixation methods and environmental conditions. Thus, there is a need for reliable genetic markers to complement the morphological identifications at the species level. Here, we analyzed 95 specimens from 11 morphologically distinct species of benthic ctenophores from the Red Sea and Sulu Sea, and tested selected regions of four genetic markers (ITS1, 18S rRNA, 28S rRNA and COI) for their ability to differentiate between species. We show that the barcoding region of the mitochondrial gene, cytochrome oxidase subunit I (COI), is highly variable among species of Coeloplanidae, and effectively discriminates between species in this family. The average Kimura-2-parameter (K2P) distance between species-level clades was 10%, while intraspecific variation was ~30 times lower (0.36%). COI-based phylogeny supported the delineation of four recently described new species from the Red Sea. The other nuclear markers tested were found to be too conserved in order to separate between species. We conclude that COI is a potential molecular barcode for the family Coeloplanidae and suggest to test it in pelagic ctenophores.

Eastern Mediterranean Mobility in the Bronze and Early Iron Ages: Inferences from Ancient DNA of Pigs and Cattle.

The Late Bronze of the Eastern Mediterranean (1550-1150 BCE) was a period of strong commercial relations and great prosperity, which ended in collapse and migration of groups to the Levant. Here we aim at studying the translocation of cattle and pigs during this period. We sequenced the first ancient mitochondrial and Y chromosome DNA of cattle from Greece and Israel and compared the results with morphometric analysis of the metacarpal in cattle. We also increased previous ancient pig DNA datasets from Israel and extracted the first mitochondrial DNA for samples from Greece. We found that pigs underwent a complex translocation history, with links between Anatolia with southeastern Europe in the Bronze Age, and movement from southeastern Europe to the Levant in the Iron I (ca. 1150-950 BCE). Our genetic data did not indicate movement of cattle between the Aegean region and the southern Levant. We detected the earliest evidence for crossbreeding between taurine and zebu cattle in the Iron IIA (ca. 900 BCE). In light of archaeological and historical evidence on Egyptian imperial domination in the region in the Late Bronze Age, we suggest that Egypt attempted to expand dry farming in the region in a period of severe droughts.

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.