The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge.

Sergey Feranchuk; Natalia Belkova; Lubov Chernogor; Ulyana Potapova; Sergei Belikov

doi:10.12688/f1000research.15841.1

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Research Article

The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge.

[version 1; peer review: 2 approved with reservations]

Sergey Feranchuk ^1,2, Natalia Belkova^1,3, Lubov Chernogor¹, Ulyana Potapova¹, Sergei Belikov¹

Sergey Feranchuk ^1,2, Natalia Belkova^1,3, [...] Lubov Chernogor¹, Ulyana Potapova¹, Sergei Belikov¹

PUBLISHED 04 Sep 2018

Author details Author details

¹ Limological institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk, 664033, Russian Federation
² Department of Informatics, National Research Technical University, Irkutsk, 664074, Russian Federation
³ Scientific Centre for Family Health and Human Reproduction Problems, Irkutsk, 664033, Russian Federation

Sergey Feranchuk
Roles: Methodology, Software, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Natalia Belkova
Roles: Conceptualization, Data Curation, Investigation, Resources, Writing – Review & Editing

Lubov Chernogor
Roles: Investigation, Resources, Writing – Review & Editing

Ulyana Potapova
Roles: Validation, Visualization, Writing – Review & Editing

Sergei Belikov
Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Review & Editing

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REVIEWER STATUS

Abstract

Background: The study of ecosystems of the great lakes is important as observations can be extended to ecosystems of larger scale. The ecological crisis of Lake Baikal needs investigations to discover the molecular mechanisms involved in the crisis. The disease of Baikal sponges is one of the processes resulting in the degradation of the littoral zone of the lake.
Methods: The chloroplast genome fragment for the algae endosymbiont of Baikal sponge was assembled from metagenomic sequencing data. The distributions of polymorphic sites were obtained for the genome fragment, separately for samples from healthy sponge, diseased sponge and dead sponge tissues.
Results: The comparative analysis of chloroplast genome sequences suggests that the symbiotic algae from Baikal sponge is close to Choricystis genus of unicellular algae. Also, the distributions of polymorphic sites allowed detection of the signs of extensive mutations in the chloroplasts isolated from the diseased sponge tissues.
Conclusions: The study demonstrate the particular case of evolution at the molecular level due to the conditions of a severe crisis of a whole ecosystem in Lake Baikal. The detection of adaptive mutations in the chloroplast genome is an important feature which could represent the behavior of an ecosystem in the event of a severe crisis.

Keywords

Chlorophyta, Lake Baikal, Chloroplas Genome, Genetic Polymorphism, Mutation Rate

Corresponding author: Sergey Feranchuk

Competing interests: No competing interests were disclosed.

Grant information: This study was supported by the Ministry of Education and Science of the Russian Federation by Government contract project no. 0345-2015-0002, “Molecular Ecology and Evolution of Living Systems of Central Asia in Terms of Fishes, Sponges, and the Microbial Flora Associated with Them” [VI.50.1.4], and the Russian Foundation for Basic Research [16-04-00065, 16-54-150007, 18-04-00224].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2018 Feranchuk S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Feranchuk S, Belkova N, Chernogor L et al. The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge. [version 1; peer review: 2 approved with reservations]. F1000Research 2018, 7:1405 (https://doi.org/10.12688/f1000research.15841.1) First published: 04 Sep 2018, 7:1405 (https://doi.org/10.12688/f1000research.15841.1) Latest published: 14 May 2020, 7:1405 (https://doi.org/10.12688/f1000research.15841.2)

Introduction

Lake Baikal, located in Southeastern Siberia, is the largest by volume and the oldest great lake on the planet, and several signs of ecological crisis in Lake Baikal have been observed since 2010–2011 [Bormotov, 2012; Khanaev et al., 2018; Kravtsova et al., 2014; Timoshkin et al., 2016]. One of these signs is severe disease and death of sponges which is now observed in almost all parts of the lake. The symptoms of the disease begin with the appearance of pink and brown spots on the surface of the sponge and terminate with complete destruction of the sponge tissues. The cause of the disease is still unclear.

Endemic freshwater Baikal sponges (Demospongiae, Lubomirskiidae) dominate their biomass among the benthic organisms of the littoral at depths from 3 to 25 m covering 47% of the available surfaces [Pile et al., 1997]. In healthy condition sponges have a green color, mainly explained by the presence of a photosynthetic symbiont, an intracellular coccoid green algae. This algae belongs to the Cholorophyta division, and is close in taxonomy to the Choricystis genus [Chernogor et al., 2013]. It is natural to assume, that the photosynthetic symbiont is the source of feeding for sponge cells. And, the change of color of sponge tissue could indicate that the chloroplasts of the symbiont are damaged in the early stages of the disease. So, precise study of this algae symbiont could be of critical importance in investigating the cause and consequences of sponge disease.

The sequencing and comparative analysis of chloroplast DNA is a conventional method for detailed study of planctonic algae [Lemieux et al., 2014; Lemieux et al., 2015]. Normally, chloroplast DNA sequences are determined using cultivated algae and de novo assembly of genomic DNA reads [Twyford & Ness, 2017]. But for uncultured species, the chloroplast genome can be obtained using metagenome sequencing [Worden et al., 2012]. For symbiotic algae from Baikal sponges, this strategy is probably more efficient, and the comparative analysis of samples of healthy and diseased sponges can provide a deeper look into the features of chloroplast genome affected by sponge disease. The presence and properties of polymorphic sites on the chloroplast genome could be an effective way to investigate the variations of genome sequence depending on the disease state of the sponge. The study of the distribution of bacterial strains depending on geographic location [Truong et al., 2017] can be mentioned as a precedent, where gene-batteries typical for gut microbiome were compared using the distribution of polymorphic sites in genome sequences, using metagenome sequencing.

Methods

Sampling and sequencing

Three samples of freshwater sponge Lubomirskia baicalensis were collected from Lake Baikal in the Bol'shiye Koty area (51° 90´ 69 N ´´, 105° 07´ 05 E´´) at a depth of 10 m by scuba divers in June 2016. One sample was obtained from the sponge that was healthy in appearance (exhibiting a green colour), one sample was taken from diseased sponge and one from dead rotten sponge tissues. The collected samples were immediately placed in containers with Baikal water and ice and transported to the lab, maintaining a constant water temperature. For all three samples Illumina pair-end reads were obtained by DNA metagenome sequencing in Novogene Inc. (Illumina PE 150). The extraction and sequencing of RNA in the samples was also performed at Novogene Inc., to represent their metatranscriptome content. This was possible only for healthy and diseased sponge tissues; not enough RNA was extracted from the rotten tissues. The DNA metagenomic reads were processed by a conventional bioinformatics pipeline implemented at Novogene Inc, including the filtering of sequencing errors and the assembly of contigs using SoapDeNovo assembler [Luo et al., 2012]. RNA metatranscriptomic reads were filtered and trimmed using Trimmomatic 0.35 software [Bolger et al., 2014].

Assembly and annotation of the chloroplast genome fragment

The sequence of chloroplast genome Choricystis parasitica (NC_025539) was used as a template for assembly and for comparative analysis because it is the chloroplast genome closest to the available genomes. For the assembly of the targeted genome, the following steps were performed:

1. Scaffolds were obtained by de novo assembly of each DNA metagenomic sample by conventional utilities with the use of the SoapDeNovo assembler
2. Scaffolds were aligned to the reference sequence of chloroplast using Blastn (evalue threshold 1e-40)
3. The paired-end sequence reads from 3 DNA metagenomics libraries and 2 RNA metatransriptomic libraries were aligned to the selected scaffolds using Bowtie2 (v. 2.2.6)
4. The aligned reads from all samples were collected and assembled de novo using the Inchworm assembler from the Trinityrnaseq 2.6.5 package [Grabherr et al., 2011] with the lowest possible tolerance to sequencing errors and the highest possible value of k-mer size (K=31)
5. The contigs obtained after the de novo assembly by Inchworm were compared with the reference genome; this allowed the selection of a single contig of length 55638 with high homology to the reference genome. It was the only contig which could be reliably identified as a fragment of the chloroplast genome.

Open reading frames were identified in the obtained contig, and most of the identified proteins are annotated following the annotations of the reference genome. TrnaSCAN 1.4 software [Lowe & Eddy, 1997] was used for identification of the transport RNA in the putative chloroplast sequence, and the locations of 18S rRNA and 23S rRNA were identified by a direct alignment with reference rRNAs using the Mummer 3.23 package [Kurtz et al., 2004].

Identification of polymorphisms

In order to separate it from traces of sequencing errors, the selection of the polymorphic sites in the genome was implemented following the approach described in [Truong et al., 2017]. Each of the RNA and DNA samples represented as pair-end reads was separately aligned to the assembled fragment of the chloroplast genome using Bowtie2; the alignments were then processed using the Samtools 1.7 software pipeline with conventional settings, and the approach proposed in [Truong, 2017] was used to identify the polymorphic sites.

Describing the algorithm, for each position s on the alignment of the reads against the Ns is defined as the total number of reads covering it, and Ts is defined as the number of reads supporting the most abundant allele. Given the sequencing error rate E, the non-polymorphic null hypothesis was rejected if the probability that the number Ns − Ts of reads coming from the non-dominant allele is <α = 0.05. This is estimated using the probability mass function of a binomial distribution with Ns trials and the successful rate 1 − E. The error rate was set to 0.01 for Illumina sequencing. The bases with quality below 30 were removed and the reads with an average identity to the reference below 99% were ignored before applying the statistical test. Failing to reject the null hypothesis reflects the absence of alternative alleles or inability of distinguishing between low-coverage potential alternative alleles and sequencing noise.

Thus, the number of polymorphic sites could be counted for each gene. Another property of each gene is the number of polymorphic sites where the count of alternative alleles is higher than the count of dominant allele (Ts < Ns - Ts). This property could detect mutations in the sample genotype and phenotype, for each gene.

Phylogenetic analysis

The chloroplast genome sequences of Picocystis salinarum (NC_024828), Myrmecia israelensis (KM462861), Botryococcus braunii (KM462884), Coccomyxa subellipsoidea (NC_015084), Hydrodictyon reticulatum (NC_034655), Mychonastes jurisii (NC_028579) and Chlorella vulgaris (NC_001865) were used for a reconstruction of the phylogenetic trees for the 16S ribosomal RNA (rrs gene) and the ATP synthase subunit beta (atpB gene). The nucleotide sequences of the selected genes were aligned using Mafft 7.27 software [Katoh & Standley, 2013]. The trees were constructed using the FastMe 2.1.5.1 software [Lefort et al., 2015], with the distance-based neighbor-joining method to select tree topology and Jukes-Cantor measure to calculate the distances between genes.

Analysis was performed using custom scripts in Python 2.7 (see Data and software availability section).

Results

The chloroplast genome of the Choricystis parasitica algae is a circular DNA 94206 base pairs in length. The comparison of open reading frames of the candidate genome fragment from the metagenomic samples with annotated genes of C. parasitica support the statement that this genome fragment of length 55638 is a large part of the chloroplast genome of algae close to the C. parasitica species. Figure 1 illustrates the order of genes in these two related chloroplasts. The comparison of gene sequences shows them to be up to 98% identical in these two species.

Figure 1. The comparison of gene positions for the chloroplast genome of Choricystis parasitica and the fragment of chloroplast genome of symbiotic algae in Lubomirskia baikalensis sponge samples.

Upper track: C.parasitica chloroplast; Lower track: Chloroplast of sponge symbiont. The start position of the C.parasitica chloroplast sequence was changed to fit the location of the fragment shown at the bottom. The rRNA and tRNA locations are shown in brown color for both tracks. Text labels show the locations of several selected genes in both genomes.

The phylogenetic trees for the two selected genes, 16S ribosomal RNA and ATP synthase beta (Figure 2) in general confirm the conventional relations between Cholorophyta algae [Lemieux et al., 2014; Lemieux et al., 2015]. Figure 2 suggests that the symbiotic algae of L. baikalensis sponge is close in taxonomy to the Choricistys genus.

Figure 2. Phylogenetic tree for two chloroplast genes: 16S ribosomal RNA (rrs) and ATP synthase subunit beta (atpB).

Bars located at the node for the studied chloroplast genome represent the relative number of polymorphic positions, in all 5 studied samples, at the 1:1 scale.

The bars on Figure 2 which show the proportion of polymorphic positions in the genes of symbiotic algae in metagenomic samples is comparable in scale with the distances between genera. This observation needs discussion, because a timescale which separates the origins of the close genera in Figure 2 implies a much larger timescale than that which could characterize the separation of the chloroplast strains detected in the metagenome. Partially this can be explained by the RNA editing and similar modifications which lead to the accumulation of polymorphic positions.

A different view of the unexpectedly high proportion of polymorphic sites in the metagenomic samples is illustrated in Figure 3. Here, the proportion of polymorphic sites, and the proportion of polymorphic sites with a low abundance of dominant allele (“mutations”) is shown separately for each DNA and RNA sample. The results of Figure 3 are presented separately for each gene frame, and for a whole set of genes.

Figure 3. The relative proportion of polymorphic sites in a chloroplast genome of sponge symbiont, for the five samples studied.

The results for each of the annotated genes are show in the bottom part. The upper line presents the integrated results for each sample. The proportions of polymorphic sites and the sites with high levels of alternative alleles (“mutations”) are shown as pie charts, relatively to a total number of polymorphic sites in all samples. The proportion of sites which are polymorphic in some other samples, but not in the given sample, are shown in light blue. The legend on the right shows a color scheme used to represent three types of sites. The circle radius represents a total number of sequencing reads aligned to the gene segment and used to identify polymorphic sites. The scale of the circle radii is transformed for better appearance, to compensate for the high variations in the numbers of aligned reads.

The proportion of polymorphic sites in the DNA and RNA metagenomes for the sample of healthy sponge tissues reflects the natural situation, where the quantity of matrix RNA in the chloroplast organelle is in general higher than the quantity of DNA. Here the polymorphic sites in the DNA sequences may arise due to natural heterogeneity of chloroplast genomes, but the dominant strain is clearly identified. The number of polymorphic sites in the RNA sequences is slightly higher than in DNA sequences due to RNA editing and other modifications.

In contrast, in the DNA and RNA samples of the diseased tissue, the quantity of chloroplast DNA is decreased, and the quantity of RNA is decreased even more, reflecting the fact of disease and the low level of chloroplast activity. Importantly, the number of polymorphic sites is sharply higher in the remaining DNA sequences, and the alternative alleles are presented in high proportion. And, for RNA sequences, the dominant allele is present much less than alternative alleles. For the case of dead tissue, where RNA couldn't be extracted, the discussion about the observed number of DNA molecules and the proportion of polymorphic sites is beyond the scope of this study.

The natural assumption about the diseased but alive tissue is that living cells are desperately trying to survive. Adaptation to a changed environment is the one of the best ways to survive. The accumulation of mutations is a straightforward form of adaptation, and this could be confirmed by the results of Figure 3 for the sample of diseased sponge tissue. The rapid increase of mutations in the genome can be observed in the chart for chloroplast DNA, and as it may be suggested from the chart for chloroplast RNA, that the mutations which help survival are fixed in the cells which still continue to develop.

The observed signs of extensive mutations in response to severe stress are somewhat controversial when compared to a the widely accepted concept of molecular clocks and a theory of neutral evolution [Kimura, 1968; Margoliash, 1963; Zuckerkandl & Pauling, 1962] where mutations are appear randomly and are independent from the environment. But in several studies the presence of adaptive mutations in response to stress has been detected in certain species, as reviewed in [Rosenberg, 2001; Wright, 2004]. So the present result cannot be treated as completely inadequate, in conditions of severe and unusual crisis of the whole ecosystem.

Discussion

The signs of the large-scale ecological crisis in Lake Baikal are confirmed from many sources, and ecological crises on such a large scale are rare in the documented history of water ecosystems. Sequencing technologies have appeared only in a recent years, and, to the authors best knowledge, no cases of large and sharp changes in ecosystems have been documented using the tools of molecular biology.

The importance of Lake Baikal itself as an ecosystem with an unusual diversity of endemic species, and as a glorious source of pure drinking water, is a subject high above the economic and pragmatic reasons which are usually considered in molecular biology studies. However the present results suggest that the conventional approaches of molecular biology may be insufficient to adequately describe situations of ecological crisis. In particular, the observations of rapid accumulation of mutations in the chloroplast genomes in the diseased tissues could indicate that the concept of molecular clocks is inappropriate in rapidly changing ecosystems.

What’s more, using the tools of molecular biology to study the Baikal ecosystem has another importance; it is a unique chance to accumulate observations about a rapidly changing environment. Great lakes are themselves simplified cases of large-scale marine ecosystems. The presented results, as a part of all Baikal ecosystem studies, could find application, not only in the challenge of minimizing the consequences of the crisis in Baikal, but also in the possible future global challenges caused by sudden changes in ecosystems of any scale.

In particular, the reconstructed genome of the symbiotic algae may improve knowledge about a cause of sponge disease, and indirectly narrow the possible strategies to prevent the spread of destruction in the Baikal ecosystem. The presented description of the genome may be helpful in the evolutionary studies of marine and freshwater Cholorophyta algae.

Data availability

The nucleotide sequence of the chloroplast genome fragment is deposited to Genbank under the accession number: MH591948

Nucleotide sequences have also been deposited with the European Nucleotide Archive (ENA) of the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) under study number: ERP110335.

Software availability

The sequencing reads and source codes of scripts sufficient to reproduce the presented results are available from GitHub: https://github.com/sferanchuk/bsponge_chloroplast

Archived source code at time of publication available at: https://doi.org/10.5281/zenodo.1326765 [Feranchuk, 2018].

(License: CC BY 4.0).

Custom scripts on Python (v 2.7) were used to run the pipeline and present the results. Python libraries pysam (0.14.1), biopython (1.66) and matplotlib (2.2.2) are required to run the scripts.

Grant information

This study was supported by the Ministry of Education and Science of the Russian Federation by Government contract project no. 0345-2015-0002, “Molecular Ecology and Evolution of Living Systems of Central Asia in Terms of Fishes, Sponges, and the Microbial Flora Associated with Them” [VI.50.1.4], and the Russian Foundation for Basic Research [16-04-00065, 16-54-150007, 18-04-00224].

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements

We thank Dr. Colin Brown for valuable help in the work presented in the manuscript; and we thank Dr. Dmitry Kuzmin and Vadim Sharov from the Siberian Federal University for assistance in the data processing.

Faculty Opinions recommended

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Version 2

VERSION 2 PUBLISHED 04 Sep 2018

Author details Author details

Sergey Feranchuk
Roles: Methodology, Software, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Natalia Belkova
Roles: Conceptualization, Data Curation, Investigation, Resources, Writing – Review & Editing

Lubov Chernogor
Roles: Investigation, Resources, Writing – Review & Editing

Ulyana Potapova
Roles: Validation, Visualization, Writing – Review & Editing

Sergei Belikov
Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

Article Versions (2)

version 2

Revised

Published: 14 May 2020, 7:1405

https://doi.org/10.12688/f1000research.15841.2

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Published: 04 Sep 2018, 7:1405

https://doi.org/10.12688/f1000research.15841.1

© 2018 Feranchuk S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Feranchuk S, Belkova N, Chernogor L et al. The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge. [version 1; peer review: 2 approved with reservations]. F1000Research 2018, 7:1405 (https://doi.org/10.12688/f1000research.15841.1)

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Reviewer Report 23 Jan 2019

Roman Kondratov, Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.17291.r42769

The massive presence of sponges is known as playing a leading role in the process of biofiltration of Baikal's water. Recently the cases of sponge disease have been expanded rapidly. The significance of the current study is coming from the attempt to investigate molecular aspects of sponge disease using a systems biology approach. In the present submission the authors provide data on the genome and transcriptome of chloroplasts from symbiotic algae. Metagenomic and metatranscriptomic sequencing data was used for a template-based assembly of the chloroplast genome of algae, and a mapping of sequencing reads to the obtained genome sequence was a subject of interpretation, based on a detection of polymorphic sites. The advantage of the study is the exploration of natural samples, improvement of methods of bioinformatical analysis and provocative ideas. I think that the manuscript deserves to be indexed, but there are concerns which the authors need to address:

What was the number of analyzed biological replicas or independent samples? Do the authors expect the same spectra if another sample(s) of sponge tissues would be processed with the same pipeline? What would be the distribution of variations in polymorphic sites in another series of experiments?
Were the “healthy”, “diseased” and “dead” samples collected from the same spot? How can the authors be sure that they are dealing with the same species? Could it be that what the authors proposed as “adaptive mutations” is in fact a difference between different subspecies?

I will recommend to tone down the conclusions and to discuss possible alternative interpretations. The statement on a presence of adaptive mutations in the chloroplast genome is too strong. It requires confirmation with more independently obtained samples. There is also no evidence that even if these mutations will be confirmed, that they are “true adaptive” mutations and provide the organism with any physiological advantage. Therefore the title is misleading in my opinion.

I suggest also to provide more details of the overall design of the study and on novelty in bioinformatics approaches.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Gene expression, biological rhythms, metabolism

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 14 May 2020

Sergey Feranchuk, Forest Research Institute, Belarus

14 May 2020

Author Response

Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed ... Continue reading Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed results can be doubted. I did consider his remarks in a most full extent.

I respect the experience of Prof. Kondratov who did suggest a presence of several subspecies of algae in a sponge host. In the revised version I explicitly told about possible presence of subspecies in the samples and add an extra chart which shows the distribution of algae subspecies around Baikal. And I did explicitly specify some of most important details of the study, and did remove some extra material from the second edition, trying to keep the format and to put more accents on the main of the declared results.

I did change the declared statement of the manuscript, to propose a proof of a possibility rather than a proof of an observation. In the revised version I tried to provide a more explicit "proof of possibility", that the adaptation can be just one of the many contributions to the observed distribution of polymorphic sites. And I add an additional argumentation why just a possibility of the adaptation is worth to be presented as a scientific result. In short, the situation around Baikal is complicated, and just an idea how this complication can be resolved is worth to be said.
Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed results can be doubted. I did consider his remarks in a most full extent.

I respect the experience of Prof. Kondratov who did suggest a presence of several subspecies of algae in a sponge host. In the revised version I explicitly told about possible presence of subspecies in the samples and add an extra chart which shows the distribution of algae subspecies around Baikal. And I did explicitly specify some of most important details of the study, and did remove some extra material from the second edition, trying to keep the format and to put more accents on the main of the declared results.

I did change the declared statement of the manuscript, to propose a proof of a possibility rather than a proof of an observation. In the revised version I tried to provide a more explicit "proof of possibility", that the adaptation can be just one of the many contributions to the observed distribution of polymorphic sites. And I add an additional argumentation why just a possibility of the adaptation is worth to be presented as a scientific result. In short, the situation around Baikal is complicated, and just an idea how this complication can be resolved is worth to be said.
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Author Response 14 May 2020

Sergey Feranchuk, Forest Research Institute, Belarus

14 May 2020

Author Response

Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed ... Continue reading Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed results can be doubted. I did consider his remarks in a most full extent.

I respect the experience of Prof. Kondratov who did suggest a presence of several subspecies of algae in a sponge host. In the revised version I explicitly told about possible presence of subspecies in the samples and add an extra chart which shows the distribution of algae subspecies around Baikal. And I did explicitly specify some of most important details of the study, and did remove some extra material from the second edition, trying to keep the format and to put more accents on the main of the declared results.

I did change the declared statement of the manuscript, to propose a proof of a possibility rather than a proof of an observation. In the revised version I tried to provide a more explicit "proof of possibility", that the adaptation can be just one of the many contributions to the observed distribution of polymorphic sites. And I add an additional argumentation why just a possibility of the adaptation is worth to be presented as a scientific result. In short, the situation around Baikal is complicated, and just an idea how this complication can be resolved is worth to be said.
Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed results can be doubted. I did consider his remarks in a most full extent.

I respect the experience of Prof. Kondratov who did suggest a presence of several subspecies of algae in a sponge host. In the revised version I explicitly told about possible presence of subspecies in the samples and add an extra chart which shows the distribution of algae subspecies around Baikal. And I did explicitly specify some of most important details of the study, and did remove some extra material from the second edition, trying to keep the format and to put more accents on the main of the declared results.

I did change the declared statement of the manuscript, to propose a proof of a possibility rather than a proof of an observation. In the revised version I tried to provide a more explicit "proof of possibility", that the adaptation can be just one of the many contributions to the observed distribution of polymorphic sites. And I add an additional argumentation why just a possibility of the adaptation is worth to be presented as a scientific result. In short, the situation around Baikal is complicated, and just an idea how this complication can be resolved is worth to be said.
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Reviewer Report 08 Jan 2019

Michael G. Sadovsky, Siberian Federal University, Krasnoyarsk, Russian Federation

Approved with Reservations

https://doi.org/10.5256/f1000research.17291.r42738

Well, on one hand I should say ``Yes’’ to this submission. At least, it completely meets all the up-to-date customs and observances in genomics and molecular biology. On the other hand, the authors rely on a number of (quite complex and apparent) software packages, workbenches and pipelines, and this is the matter of my general scepticism. Actually, we all become the hostages of those software tools; one has to trust software designers and believe the output of the programs is correct, free of mistakes, stably working, etc. Somebody may say ``You, physicists and mathematicians, do use Wolfram’s Mathematica and there is no collapse in math, nor in physics’’. Reciprocally, I would like to draw attention to an Elsevier journal devoted to Microsoft Excel errors solely (McCullough and Heiser, 2007¹).

I am far from the idea to say that the study is wrongly arranged or badly accomplished; I just want to stress the point that there should be some special efforts done to ensure the results are at least stable. For example, what happens with assembled contigs, if we randomly remove a small part of reads? If a series of runs of an assembler yields (almost) the same contigs set, then the results could be used for further analysis. The problem arises, if one gets a number of sets of contigs with a sounding difference between them. The paper has no answer on that point; a comparative study (like that one presented by the authors) should have some proofs of the absence of artefacts affecting the comparison of fine differences between biological objects involved in the study. Meanwhile, I pretty well understand that such output testing falls beyond the customs and habits of NGS sequenced data treatment and I am in the smallest minority. So, from that point of view the paper completely meets all the custom data treatment procedures and in such capacity should be recommended for indexing.

Another important issue of the paper is that it presents an attempt to tie together ecological (environmental) processes, and some genetic background that may stand behind. Here the word `crisis’ used by the authors makes a point: regularly, ecological crisis is stipulated as a rather fast running process in a community resulting in serious (and inevitable) loss of the greater part of species from the community. Maybe, this word is too strong here: what if the observed infection intrusion is just a regular (while long ranged) periodic event in the community? Nonetheless, the scientific merit of the paper is obvious, the results and conclusions are sounding and up-to-date, and paper should be indexed.

The paper needs major revisions in its English. The paper is written in a version I dare say is Runglish. There are too many lines in the manuscript that look like a literal translation from Russian of (quite boring) scientific Russian-style. I myself can decipher what the authors mean, since my mother language is also Russian. I am absolutely sure that the current version of the paper will fall out of comprehension for the greatest majority of readers who have no active Russian. To begin with, the title must be changed. No signs, at all. The correct version should be like ``Evidences of the adaptive mutations in chloroplast genomes of some algae endosymbionts of Baikal sponge’’.

Same in the Abstract (Background paragraph): instead of “The study of ecosystems of the great lakes is important as observations can be extended to ecosystems of larger scale. The ecological crisis of Lake Baikal needs investigations to discover the molecular mechanisms involved in the crisis. The disease of Baikal sponges is one of the processes resulting in the degradation of the littoral zone of the lake” there should be something like “Monitoring and investigation of the great lakes ecosystem provides a sounding background to forecast the greater scale ecosystem dynamics. Changes in the Baikal lake biota observed nowadays demand deeper investigations of the molecular mechanisms standing behind these former. The endemic Baikal sponge disease may cause a degradation of littoral ecosystem of the lake”. I am far from the idea that my version is the best, but the original one must be rewritten.

Unfortunately, there are many more similar problem lines in the manuscript, so very strong revisions in the English are absolutely necessary.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. McCullough B, Heiser D: On the accuracy of statistical procedures in Microsoft Excel 2007. Computational Statistics & Data Analysis. 2008; 52 (10): 4570-4578 Publisher Full Text

Competing Interests: No competing interests were disclosed.

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Author Response 14 May 2020

Sergey Feranchuk, Forest Research Institute, Belarus

14 May 2020

Author Response

I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.

First of ... Continue reading I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.

First of all, he was right that crises like the crisis on Baikal could anyway happen in the past. The need to survive in the times of severe crises can be encoded in genome. This idea was introduced to the revised version, as an additional support to the hypothesis about adaptive mutations.

To answer the remark about "closeness" and insufficient robustness of the software, I did several other runs of the assembly. I agree with Prof. Sadovsky about "closeness" and over-complication of some software, and this is why I did choose Inchworm assembler in the initial version of the pipeline, as the most lightweight and straightforward of the available assemblers. In additional runs I tried another assemblers. The correctness of the assembled chloroplast sequence was anyway confirmed, and the fact of verification was pointed out in the second revision.

To answer the remark about "Russian" style of language. This question is in part beyond the scope of the discussion. It is unlikely that me who is Russian will speak the same English as a man from England. But Prof. Sadosvky was right that the meaning of the text in the first edition was unclear in many parts. And in the revised version I put much more attention to a choice of words and grammatic constructions, to use only those words, for which I am certain in their meaning. The text can anyway look unusual to one who know in perfect the context of all words in English, but at least I do my best to make the meaning of the text the most clear.
I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.

First of all, he was right that crises like the crisis on Baikal could anyway happen in the past. The need to survive in the times of severe crises can be encoded in genome. This idea was introduced to the revised version, as an additional support to the hypothesis about adaptive mutations.

To answer the remark about "closeness" and insufficient robustness of the software, I did several other runs of the assembly. I agree with Prof. Sadovsky about "closeness" and over-complication of some software, and this is why I did choose Inchworm assembler in the initial version of the pipeline, as the most lightweight and straightforward of the available assemblers. In additional runs I tried another assemblers. The correctness of the assembled chloroplast sequence was anyway confirmed, and the fact of verification was pointed out in the second revision.

To answer the remark about "Russian" style of language. This question is in part beyond the scope of the discussion. It is unlikely that me who is Russian will speak the same English as a man from England. But Prof. Sadosvky was right that the meaning of the text in the first edition was unclear in many parts. And in the revised version I put much more attention to a choice of words and grammatic constructions, to use only those words, for which I am certain in their meaning. The text can anyway look unusual to one who know in perfect the context of all words in English, but at least I do my best to make the meaning of the text the most clear.
Competing Interests: No competing interests were disclosed. Close
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Author Response 14 May 2020

Sergey Feranchuk, Forest Research Institute, Belarus

14 May 2020

Author Response

I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.

First of ... Continue reading I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.

First of all, he was right that crises like the crisis on Baikal could anyway happen in the past. The need to survive in the times of severe crises can be encoded in genome. This idea was introduced to the revised version, as an additional support to the hypothesis about adaptive mutations.

To answer the remark about "closeness" and insufficient robustness of the software, I did several other runs of the assembly. I agree with Prof. Sadovsky about "closeness" and over-complication of some software, and this is why I did choose Inchworm assembler in the initial version of the pipeline, as the most lightweight and straightforward of the available assemblers. In additional runs I tried another assemblers. The correctness of the assembled chloroplast sequence was anyway confirmed, and the fact of verification was pointed out in the second revision.

To answer the remark about "Russian" style of language. This question is in part beyond the scope of the discussion. It is unlikely that me who is Russian will speak the same English as a man from England. But Prof. Sadosvky was right that the meaning of the text in the first edition was unclear in many parts. And in the revised version I put much more attention to a choice of words and grammatic constructions, to use only those words, for which I am certain in their meaning. The text can anyway look unusual to one who know in perfect the context of all words in English, but at least I do my best to make the meaning of the text the most clear.
I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.

First of all, he was right that crises like the crisis on Baikal could anyway happen in the past. The need to survive in the times of severe crises can be encoded in genome. This idea was introduced to the revised version, as an additional support to the hypothesis about adaptive mutations.

To answer the remark about "closeness" and insufficient robustness of the software, I did several other runs of the assembly. I agree with Prof. Sadovsky about "closeness" and over-complication of some software, and this is why I did choose Inchworm assembler in the initial version of the pipeline, as the most lightweight and straightforward of the available assemblers. In additional runs I tried another assemblers. The correctness of the assembled chloroplast sequence was anyway confirmed, and the fact of verification was pointed out in the second revision.

To answer the remark about "Russian" style of language. This question is in part beyond the scope of the discussion. It is unlikely that me who is Russian will speak the same English as a man from England. But Prof. Sadosvky was right that the meaning of the text in the first edition was unclear in many parts. And in the revised version I put much more attention to a choice of words and grammatic constructions, to use only those words, for which I am certain in their meaning. The text can anyway look unusual to one who know in perfect the context of all words in English, but at least I do my best to make the meaning of the text the most clear.
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Version 2

VERSION 2 PUBLISHED 04 Sep 2018

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Version 2 (revision) 14 May 20		read	read	read
Version 1 04 Sep 18	read	read

Michael G. Sadovsky, Siberian Federal University, Krasnoyarsk, Russian Federation
Roman Kondratov, Cleveland State University, Cleveland, USA
Ashraf Al-Ashhab, Dead Sea and Arava Science Center, Masada, Israel
OPhir Nave, Jerusalem College of Technology, Jerusalem, Israel

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Reviewer Report

2 Views

23 Nov 2020 | for Version 2

OPhir Nave, Department of Mathematics and Computer Science, Jerusalem College of Technology, Jerusalem, Israel

2 Views Cite this report Responses(0)

Approved

The paper is very interesting and has an important application.

The abstract summarized the main results of the paper.
The authors revised the introduction according to the comments.
The introduction is not complete. The authors must add a relevant list of references to this section.
5 paragraphs of the introduction are without even one reference.
Also the section "Materials and methods" is without an extensive list of references. This section is very important, and the authors need to present the relevant research list.
The results section needs to be revised. Since as it is present in the current version, it is not absolutely clear what the authors achieve from the study.
The analysis of the FIgures must be ended in detail.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Mathematical model and application to engineering and medicine

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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3 Views

16 Nov 2020 | for Version 2

Ashraf Al-Ashhab, Microbial Metagenomics Division, Dead Sea and Arava Science Center, Masada, Israel

3 Views Cite this report Responses(1)

Approved

The authors have based their study on chloroplast polymorphic sites in algae endosymbiont of Baikal sponge of diseased, healthy and dead sponge tissue.

The main concern is that they based all their analysis and conclusions from one sample following each category. The authors also used 150 PE sequencing, while for better assembly and more accurate evaluation of polymorphic sites they could have used a longer amplicon sequencing.
The authors have used PE reads to obtain while in the analysis they were treated and independent unpaired read, why did they do this? At which stage the sequencing errors were checked and quality control used is not clear.
The authors have used a genome of Choricystis parasitica (NC_025539) as a template assembly, and not enough information of why they have chosen this and not others and more than one.
Methods on RNA and DNA extraction, library preparation, and size selection are not listed in the manuscript.
The authors also should indicate some information about contigs length distribution following assembly.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

microbiome

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response

16 Nov 2020

Sergey Feranchuk, Limological institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk, 664033, Russian Federation

Many thanks for the approving.

I agree and confirm that the study has been done with methodological inconsistencies which are mentioned in the concerns. A consistent research should be based on several replicates of sponges in each phase of disease; sequencing should be more fine-tuned, more deep and should make use of long amplicon extraction. The answers are in the reality of scientific business rather than in the science itself so I would like to omit the more detailed explanations.

Answering the more specific concerns:

- Reads were unpaired and mixed after filtering on a template of a similar chloroplast. After this stage, the chances of mis-assembly which are intended to be reduced by a filtering of mate-pairs, were of lesser importance than a need to obtain as long contig as possible, from a filtered pool of reads.

- A close similarity of the target algae species to Choricystis species was observed earlier in vitro [Chernogor 2013] and by 16S rRNA annotation to Silva database.

- In the primary Inchworm assembly, the targeted 55K contig was followed just by "noisy" contigs with lengths not more than 5K b.p., so the distribution of contig lengths was not explicitly indicated.

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Competing Interests

No competing interests were disclosed.

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3 Views

28 May 2020 | for Version 2

Roman Kondratov, Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA

3 Views Cite this report Responses(0)

Approved

The authors addressed my concerns. I am fine with the current version.

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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26 Views

23 Jan 2019 | for Version 1

Roman Kondratov, Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA

26 Views Cite this report Responses(1)

Approved With Reservations

What was the number of analyzed biological replicas or independent samples? Do the authors expect the same spectra if another sample(s) of sponge tissues would be processed with the same pipeline? What would be the distribution of variations in polymorphic sites in another series of experiments?
Were the “healthy”, “diseased” and “dead” samples collected from the same spot? How can the authors be sure that they are dealing with the same species? Could it be that what the authors proposed as “adaptive mutations” is in fact a difference between different subspecies?

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Gene expression, biological rhythms, metabolism

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Responses (1)

Author Response

14 May 2020

Sergey Feranchuk, Forest Research Institute, Belarus

Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed results can be doubted. I did consider his remarks in a most full extent.

I respect the experience of Prof. Kondratov who did suggest a presence of several subspecies of algae in a sponge host. In the revised version I explicitly told about possible presence of subspecies in the samples and add an extra chart which shows the distribution of algae subspecies around Baikal. And I did explicitly specify some of most important details of the study, and did remove some extra material from the second edition, trying to keep the format and to put more accents on the main of the declared results.

I did change the declared statement of the manuscript, to propose a proof of a possibility rather than a proof of an observation. In the revised version I tried to provide a more explicit "proof of possibility", that the adaptation can be just one of the many contributions to the observed distribution of polymorphic sites. And I add an additional argumentation why just a possibility of the adaptation is worth to be presented as a scientific result. In short, the situation around Baikal is complicated, and just an idea how this complication can be resolved is worth to be said.

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

23 Views

08 Jan 2019 | for Version 1

Michael G. Sadovsky, Siberian Federal University, Krasnoyarsk, Russian Federation

23 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. McCullough B, Heiser D: On the accuracy of statistical procedures in Microsoft Excel 2007. Computational Statistics & Data Analysis. 2008; 52 (10): 4570-4578 Publisher Full Text

Competing Interests

No competing interests were disclosed.

Respond to this report

Responses (1)

Author Response

14 May 2020

Sergey Feranchuk, Forest Research Institute, Belarus

I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.

First of all, he was right that crises like the crisis on Baikal could anyway happen in the past. The need to survive in the times of severe crises can be encoded in genome. This idea was introduced to the revised version, as an additional support to the hypothesis about adaptive mutations.

To answer the remark about "closeness" and insufficient robustness of the software, I did several other runs of the assembly. I agree with Prof. Sadovsky about "closeness" and over-complication of some software, and this is why I did choose Inchworm assembler in the initial version of the pipeline, as the most lightweight and straightforward of the available assemblers. In additional runs I tried another assemblers. The correctness of the assembled chloroplast sequence was anyway confirmed, and the fact of verification was pointed out in the second revision.

To answer the remark about "Russian" style of language. This question is in part beyond the scope of the discussion. It is unlikely that me who is Russian will speak the same English as a man from England. But Prof. Sadosvky was right that the meaning of the text in the first edition was unclear in many parts. And in the revised version I put much more attention to a choice of words and grammatic constructions, to use only those words, for which I am certain in their meaning. The text can anyway look unusual to one who know in perfect the context of all words in English, but at least I do my best to make the meaning of the text the most clear.

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge.

Abstract

Keywords

Introduction

Methods

Sampling and sequencing

Assembly and annotation of the chloroplast genome fragment

Identification of polymorphisms

Phylogenetic analysis

Results

Figure 1. The comparison of gene positions for the chloroplast genome of Choricystis parasitica and the fragment of chloroplast genome of symbiotic algae in Lubomirskia baikalensis sponge samples.

Figure 2. Phylogenetic tree for two chloroplast genes: 16S ribosomal RNA (rrs) and ATP synthase subunit beta (atpB).

Figure 3. The relative proportion of polymorphic sites in a chloroplast genome of sponge symbiont, for the five samples studied.

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