Insights Into The Phylogenetic Position And Endoparasitic Adaptation Mechanism Of Myxozoans

Myxozoans are endoparasitic animals exhibiting complex life cycles that,in most of the known cases,involves an intermediate host,usually a fish,but in rare cases amphibians,reptiles,birds,and mammals.Some species can lead to major disease outbreaks and heavy losses in aquaculture and wild fisheries.Recently,although considerable knowledge has been gained about taxonomy,phylogeny,and life-cycle,questions persist about the origin and adaptive evolution of myxozoans.For nearly three decades,the affiliation of myxozoans has gone through a transition from protozoans to metazoans,and then from bilaterians to cnidarians.However,the precise phylogenetic position within Cnidaria remains uncertain.Moreover,there are over 2,600 species of myxozoans currently recognized,inhabiting diverse habitats.However,the drivers and molecular mechanisms underlying the adaptation of myxozoans to endoparasitism remains unclear.As one of the oldest and most simplified metazoan parasite,resolving the origin and adaptive evolution of myxozoans is important for understanding important characteristics,evolutionary patterns,and mechanisms of metazoans.With the development of omic technology,more and more cnidarian genomes and transcriptomes are published.Thus,the research on myxozoan origin and adaptive evolution analysis now has better technical and data support.In this study,we used phylogenomic,comparative genomic,comparative proteomic,and adaptive evolution analyses to determine the phylogenetic status,divergence time,adaptive contribution of nematocyst,genome evolution,and the molecular mechanism of adaptive evolution.The main results are as follows:1.Phylogenetic analyses of myxozoansBy extensively sampling the major taxa of Myxozoa,free-living cnidarians and other metazoans,we established the most comprehensive metazoan and cnidarian phylogenetic matrix so far.For metazoan matrix,we built on a subset previously published dataset of 1,719 genes,expanding the taxon sampling for myxozoans.The final curated data matrix contained 232 genes(57,930 aa positions)from 103 taxa,with31.1%missing data overall.For cnidarian matrix,our data sets include 76 diverse metazoan taxa and 2 choanoflagellate outgroups.Our primary data set consists of 146orthologous groups,51,598 aa positions,and 24.8%missing data.We estimated both concatenated trees by using two maximum likelihood(ML)methods(RAx ML and IQ-TREE)and a Bayesian approach(Phylo Bayes).Approximately unbiased(AU)tests were conducted to evaluate 17 alternative topologies.Finally,the fast-site removal method was applied to the above two matrices to detect whether fast-site has misled the phylogenetic relationships.Our phylogeny strongly supported the relationships of((Myxozoa+Polypodium)+Medusozoa).The AU-test results showed that most alternative topologies of Myxozoa+Polypodium were rejected significantly.Although the topology Myxozoa+Ctenophore cannot be rejected,the P-Value is low(0.0561)and so is the Bootstrap RELL(0.0000).So,we concluded that this topology was not optimal.The topology and its support values were more resistant to fast-site removal until well over half the data set was removed.The Medusozoa-sistered placement and cluster of Myxozoa+Polypodium were consistently and robustly supported.This study has produced more stable and accurate results by overcoming the evolutionary saturation,long-branch attraction,and other deficiencies in previous molecular phylogenetic analyses.2.Divergence time of myxozoansThe divergence times of the major cnidarian lineages were calculated across the best ML tree using Penalized likelihood(PL)implemented in r8s.Four calibration points were used:the node of crown cnidarians was constrained with maximum age of741 Mya,the minimum age of Medusozoa was set to 570 Mya,the minimum age of Hexacorallia was set to 540 Mya,the rise of hydrozoa was fixed at 500 Mya.We showed that the last common ancestor of cnidarians occurred during the Tonian period approximately 736 Mya.The Medusozoa was estimated to have originated 626.6 Mya.Our calculation of the most recent common ancestor(MRCA)of all myxozoans is dated back to the late Cambrian(492.6 Mya).Currently,the earliest evidence for parasitic relationships has been found in Cambrian.Thus,our analyses revealed that myxozoans are the oldest-known metazoan parasites.Besides,we now have evidence that Myxozoa+Polypodium originated during a period when ancestors to all their currently recognized hosts were also present.We propose that the more convincing scenarios are that fish,or their precursors served as first hosts,or that parasitism was acquired independently in lineages leading to Polypodium and Myxozoa.3.Establishment of a method for isolation intact and clean nematocysts and shell valvesThe nematocyst of myxozoans is essential for the completion of their life-cycle,and serves as an ideal model for investigating the contribution of phenotypes to adaptive evolution.To facilitate subsequent experiments,develop a new,fast and efficient method for myxospore dissection that can be utilized to isolate nematocysts and shell valves from representative species Myxobolus honghuensis,Myxobolus wulii,and Thelohanellus kitauei.This was achieved through spore purification by sucrose density gradient ultracentrifugation,spore disruption by sonication and nematocyst/shell valve isolation by Percoll density gradient ultracentrifugation.For nematocyst isolation,the50%,70%,90%Percoll gradient led to optimal results:nematocysts in an almost pure state(>98%)were collected from the 50%/70%interface and the middle of the 70%layer.After the secondary purification,which repeated the optimal Percoll gradient process,intact,clean and unextruded nematocysts were successfully isolated from the three myxozoans.For shell valve isolation,a final 100%Percoll centrifugation at 6580g for 15 min resulted in the optimal isolation of shell valves,to>98%purity in the upper part of the 100%layer.Repeating the process led to intact and clean shell valves.To further study the nematocysts,we evaluated whether heat,high salinity and various chemicals can trigger the discharge of M.honghuensis spores/nematocysts,and whether heavy metals can affect the discharge response.Our results showed that heat,urea,and ammonia successfully triggered the discharge of most spores and isolated nematocysts,whereas Na Cl,acetic acid,ethanol,sodium bicarbonate and Ca Cl₂ did not.With regard to heavy metals,we showed that a 30 min exposure of spores/nematocysts to Zn SO₄,Ba Cl₂,Fe Cl₃,Mn Cl₂ or Cd Cl₂,dramatically impaired discharge of nematocysts.This new tool will facilitate subsequent analyses and enable a better understanding of the ecological and evolutionary significance of parasitic spores.4.CCPRD:A novel analytical framework for the comprehensive proteomic reference database construction of myxozoansWe propose a framework for constructing a customized comprehensive proteomic reference database(CCPRD)from draft genomes and deep sequencing transcriptomes.Its effectiveness is demonstrated by incorporating the proteomes of nematocysts from Myxobolus honghuensis,Myxobolus wulii,and Thelohanellus kitauei.The performance of the CCPRD was assessed by comparing its MS results with four alternative databases:(1)a six-frame translation of the transcriptomes(trans?6?frame);(2)a six-frame translation of the transcriptome plus genomes(genome+transcriptome?6?frame);(3)a contaminant database made by adding manually selected host and bacteria sequences to the CCPRD(CCPRD?contam);and(4)adding back sequences(both from genomes and transcriptomes)that were removed by the decontamination process to the CCPRD(CCPRD?remove).All of the databases were evaluated with respect to size,the number of identified peptides and proteins,and completeness.Our results show that the database CCPRD?contam identified many more peptides and proteins than the CCPRD,which suggests the existence of contamination in the source omic data and proves the necessity of decontamination.For the database CCPRD?remove,adding back sequences that were removed during the decontamination process did not increase the number of identified peptides and proteins,which suggests that our method is not overdecontaminated.our database significantly outperforms alternative approaches in peptide and protein identification numbers,database size,and completeness.The CCPRD could benefit the proteomic results by detecting 19.1-43.8%more peptides while reducing a maximum 84.6%of database-size.We used the SSRCalc model to estimate the hydrophobicity index of peptides based on their sequences and chemical modifications,which can be linearly mapped to the retention time.Both the linear fitting results of overall and database-specific peptides suggest that our results are valid and CCPRD can improve the overall validity(higher R²).The redundancy removal analyses of database-specific peptide identifications revealed that,even with redundancy removed,the number of CCPRD-specific peptides still outperformed the alternative databases.This suggested that the increasing of identifications in CCPRD is not due to the increasing of replicate identifications.This further demonstrates that CCPRD does improve the performance of the database as a whole,rather than simply increasing the number of false positives or repetitive sequences.This study provides technical guarantees for downstream comparative proteomic analysis and references for other proteome projects involving nonmodel organisms.5.Quantitatively defining the role of nematocyst in cnidarian adaptationIn this study,the role of nematocysts in cnidarians adaptive success was studied by taking advantage of all available nematocyst proteomes and 111 cnidarian transcriptomes/genomes(including 7 newly generated myxozoan transcriptome and/or genome data sets).We found high variability within and between parasitic and free-living cnidarian nematocyst proteins(NEMs).Besides,the proteomic comparison results are inconsistent with their phylogeny.Thus,these results support the innate plastic adaptation strategies of nematocyst,highlighting its possible significant role in undertaking adaptive evolution.We also suggested that nematocysts probably originated autogenously from very few proteins and are free from precursors.Therefore,their possible contribution to the organismal adaptation is free from considering the influence of external symbionts in downstream analysis.Furthermore,we studies the evolutionary trade-off of core set/nonconcomitant set before and after the major lineage expansion was examined.We found a pattern of"decentralized modification"in which the putative core set has undergone few evolutionary events before and after their expansion to the shared ancestor of cnidarian and most active evolutionary events are in nonconcomitant set.The most plausible explanation for the decentralized modification is that the rise of a nematocyst prototype is followed by a rapid diversification of cnidarians and there is a relatively short time for evolutionary events to occur before nematocysts rapidly expand into various lineages.This is also reasonable support for the key role of nematocyst in cnidarian evolution.Finally,we found all NEMs have experienced approximately 50%more adaptive amino acid changes on average compared to non-NEMs.NEMs with the strongest evidence of adaptation(BUSTED P-values lower than 10^-5)have up to 60%adaptive excess(permutation test P<2.2e^-16,10⁷ iterations).Moreover,the Fisher's exact test on the NEMs against background proteins showed that selective pressure of the background protein was significantly enriched(P-value=0.004658,0.01117,0.007638).These results demonstrate the predominant role of nematocysts in successful cnidarian adaptation and constitute an important basis for studies aimed at quantitatively defining the role of phenotypic novelty in adaptation.6.Mosaic evolution of the Myxobolus honghuensis genomeTo gain insight into the genetic mechanisms of myxozoan adaptation,we report the genome sequencing and analysis of myxozoan Myxobolus honghuensis,an endoparasitic cnidarian infecting the gibel carp.The genome size was estimated,by k-mer analysis using Jellyfish software,to be 206 Mb.The FALCON assembled genome contained 1,118 contigs totaling 161 Mb with a contig N50 size of 1.3 Mb.Adaptive evolution analysis revealed that Myxobolus honghuensis has the largest described myxozoan genome,which is less reduced and less compact,due to gene retention,large intron,transposon insertion,but not polyploidy.Comparative analysis showed neural gene depletion and the presence of the simplest animal immune components.Conversely,genes involved in stress resistance,invasion,energy metabolism,and cellular processes,are intensified by gene expansion and positive selection,as evolutionary adaptations to endoparasitism.M.honghuensis has conserved myogenic components compared with free-living cnidarians,including less reduced Wnt and Hedgehog pathways.Collectively,our study reveals mosaic genome evolution in M.honghuensis,in which different genomic regions reveal different patterns and degrees of gene conservation,divergence,depletion,and enhancement.Far from genomic degeneracy,M.honghuensis genome displays considerable innovation and expansion,chiefly in the form of multi-copy effector gene families and the domains that seem to regulate their expression and diversity.These results suggest that myxozoans are not as genetically simple as previously thought.At least for myxozoans,the transition to parasitism is driven by both genomic simplification and complexification,while the relative contributions of these two evolutionary regimes vary across species.This study has changed and/or expanded our views on myxozoan genome complexity and evolution,and has important implications for an in-depth understanding of parasite evolution,a fundamental issue in this evolutionary biology.In summary,to address the origin of myxozoans,this study constructs the most reliable cnidarian species tree to date based on phylogenomics and estimates the divergence time of myxozoans.On this basis,drivers and evolutionary mechanisms of adaptive evolution in myxozoans were elucidated from both phenotypic(nematocyst)and molecular(M.honghuensis genome)level.The nematocyst was found to be a key phenotype for successful adaptive evolution in cnidarians,including myxozoans.Comparative genomics imply that myxozoan genome evolution reflects the trade-off between genomic simplification(caused by loss of redundant genes)and complexification(caused by increased adaptability).