Characterisation of Microbiome Diversity Unveils Substantial Microbial Variation in Mangrove Soils from Coastal Regions of Malaysia
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The mangrove ecosystems are highly productive and of great ecological importance found in tropical and subtropical coasts, including Malaysia. The microbial communities in the mangrove sediments play an indispensable role in maintaining homeostasis and supporting biodiversity. However, mangroves are facing various threats due to increasing anthropogenic activities that trigger deterioration culminating in irreversible damage to the coastal ecosystem. Thus, it is important to monitor the microbial community to improve our understanding of the impact of anthropogenic pressure on reshaping these ecosystems. This study examines the microbial community diversity in mangrove sediments of southern peninsular Malaysia. High throughput MinION sequencing of the 16S rRNA gene was performed to compare the soil microbiome diversity in 34 samples from eight different mangroves representing Sungai Sedili Kecil and Sungai Sedili Besar that flows into the South China Sea on the east; Sungai Pulai, Sungai Melayu, Sungai Danga, Sungai Skudai and Sungai Johor that joins Straits of Johor in the south; as well as Pulau Kukup from Straits of Malacca on the west. The metagenomic classification performed with 16S rRNA showed 101 taxa comprising 18 phyla. Sequence mean prevalence and total abundance analysis showed Proteobacteria (27-66%), Firmicutes (11-25%), Planctomycetes (3-50%), and Bacteroidetes (5-9%) as the relatively common phyla in all regions. Alpha diversity indices revealed significantly higher richness in samples from mangroves in the estuaries of the South China Sea. Richness indices varied significantly across the straits of Johor, specifically Sungai Johor and Sungai Melayu. Further, Shannon index showed a significant difference in diversity between Sungai Melayu and Sungai Pulai. Higher abundance of Betaproteobacteria, Epsilonproteobacteria and Planctomycete suggest a shift in the microbial community structure. This study stands as the first comprehensive analysis of microbial communities in these mangroves, serving as a valuable reference for future monitoring and conservation endeavours to safeguard these vital ecosystems.
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Comments to Author
The manuscript entitled Characterisation of Microbiome Diversity Unveils Substantial Microbial Variation in Mangrove Soil Sediments from Coastal Regions of Malaysia by Hebbar et al. analyses 16S amplicons using MinION technology to describe the bacterial community composition in sediment samples collected in different areas with very interesting model ecosystems such as mangroves in Malaysian territories with varying anthropogenic impacts. Physical and chemical parameters were also collected and correlated with such findings. The report correctly provides information to access the source raw data in public databases, the methodology is clearly described, the figures represent the result of the analyses described and discussed and the text is written in a logical, clear and concise manner. Main …
Comments to Author
The manuscript entitled Characterisation of Microbiome Diversity Unveils Substantial Microbial Variation in Mangrove Soil Sediments from Coastal Regions of Malaysia by Hebbar et al. analyses 16S amplicons using MinION technology to describe the bacterial community composition in sediment samples collected in different areas with very interesting model ecosystems such as mangroves in Malaysian territories with varying anthropogenic impacts. Physical and chemical parameters were also collected and correlated with such findings. The report correctly provides information to access the source raw data in public databases, the methodology is clearly described, the figures represent the result of the analyses described and discussed and the text is written in a logical, clear and concise manner. Main comments: While the authors are using a method that is commercially available and standardized, it is important that they also discuss and analyze the limitations inherent to this approach, and the authors shall mention them as important factors for the results obtained. 1. It is well established that using primers for the complete amplification of 16S rRNA genes is at least one order of magnitude less efficient, requiring 10X more initial target gene copies to generate a product, which is even more critical when having a mix of competing targets of a bacterial community. https://link.springer.com/article/10.1186/1471-2180-12-56 https://link.springer.com/article/10.1186/s12866-019-1446-2 https://link.springer.com/article/10.1186/s40168-015-0087-4 2. The determination of chimeric sequences is critical in this experimental approach. This must be considered and discussed. When amplifying a complete 16S rRNA gene vs short hypervariable regions alone, the chances to have chimeras are increased. These sequence amplicons are resulting from incomplete extension products between cycles of overlapping PCR from two completely different bacterial taxonomic origins that are annealing in the highly conserved regions contained in the amplicon. https://link.springer.com/article/10.1007/BF02539153 https://www.microbiologyresearch.org/content/journal/micro/10.1099/13500872-142-5-1107 https://academic.oup.com/nar/article/47/18/e103/5527971. 3. I consider that adding a supplementary table with the expanded source data of the final filtered representative sequence, taxonomical classification and frequency per sample used for table 1 and figures 3, 4 and 5 could be useful for the readers. 4. I suggest moving the figure 1 as a supplementary figure. 5. While it is common to see reports showing phyla composition, at this higher hierarchy the resolution is not meaningful and the differences observed are already evidenced in the alpha diversity metrics. I think it is not of particular importance for the report and figure 3 is not necessary or only as an accessory to figure 4 or as supplementary material. I think is of pivotal importance from now on, to upgrade to current International Code of Nomenclature of Prokaryotes ICNP bacterial taxonomy after 2022, when major changes in upper taxonomic hierarchies occurred, and had been upgraded in the latest classification reference databases of Silva, RDP or NCBI. LPSN contains the current status of the nomenclature and I suggest this must be updated in the manuscript and figures.6. The reformatting of figure 4 is advised. Particularly to set the same bar width for all samples to have a consistent layout, readable font sizes and to avoid redundant presentation of the same data in different figures and formatting. 7. I think that it would be interesting to see in addition to the alpha diversity analyses, the beta diversity analyses results of the bacterial compositions to define if there is clustering significantly related to the point of sampling or to the activity exerted in the sampled ecosystem. 8. I could find the raw sequence datasets as described in the Bioproject with the raw data, but it contains 34 datasets with names indicating they are also from the places sampled, but here 24 are discussed. The authors may explain the reasons not to include those 10 datasets part of the same BioProject in this report i.e. SRR29978932, SRR29978929, etc. 9. I inferred and analyse the patterns of composition from raw data and using RDP classifier 19 https://pmc.ncbi.nlm.nih.gov/articles/PMC11008197/, and I could find as most abundant genera: Woeseia, Limibacillus, Kofleriaceae, Halofilum, Ferruginivarius, Endothiovibrio, Pelagibius, Luteitalea, Ectothiorhodosinus, Nitrospira, Inmirania, Polyangiaceae, Thermoanaerobaculum, Vicinamibacter, Thioalbus, Sulfuriflexus, Longimicrobium, Geomobilimonas, Sandaracinus, Thiogranum, Mangrovivirga, Anaerohalosphaera and Desulfoglaeba among the most relevant. While some of these genera are described and noted in the report, many in apparently high abundance in those samples are not mentioned, which is probably due to the classification system and reference used. Therefore my advice to upgrade such classification using the OTU/ASV sequence used for counting frequencies to improve the resolution of the descriptions that are possible to infer. 10. Proofread and correct minor typos across the manuscript: such as Chao1 vs Choa1, etc. I hope the authors find this review fair, respectful and useful to improve the manuscript.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Both reviewers have raised important issues that should be addressed to move this manuscript forward. Please also improve formatting and figure layout as suggested by the reviewers.
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Comments to Author
Comments for the Author: The manuscript provide 16S amplicon sequencing based microbiome profiling of 34 soil sediments representing 8 different mangroves with detailed protocols, data processing, and statistical analyses. The authors prioritize data accessibility by sharing raw sequences, promoting open science and facilitating future replication. The results are presented clearly with informative figures and tables. The study makes a valuable contribution to studying mangrove soil microbiome diversity offering new insights, well-supported conclusions, and practical recommendations for future research. The manuscript need to be revised with respect to justification for variable number of samples from different study sites. Shift in microbiome observed to be clarified with respect to factors compared?
Comments to Author
Comments for the Author: The manuscript provide 16S amplicon sequencing based microbiome profiling of 34 soil sediments representing 8 different mangroves with detailed protocols, data processing, and statistical analyses. The authors prioritize data accessibility by sharing raw sequences, promoting open science and facilitating future replication. The results are presented clearly with informative figures and tables. The study makes a valuable contribution to studying mangrove soil microbiome diversity offering new insights, well-supported conclusions, and practical recommendations for future research. The manuscript need to be revised with respect to justification for variable number of samples from different study sites. Shift in microbiome observed to be clarified with respect to factors compared? Specific comments: The authors collected samples at a single time point (between June and September) In the results and discussion, they mentioned a shift in the microbiome. The authors should clarify whether this shift refers to a change in microbiota diversity or composition and specify the factors causing this shift. The authors should also explain the reason for the difference in the number of samples collected from different study sites in their study design. If they are comparing their results to previous studies and referring to this as a shift in the microbiome diversity, they should indicate that it is a difference in the microbiome, not a shift in the microbiome. minor comments: Abstract: L32-33: delete Sequence mean prevalence. Introduction Line 94: "Shift in microbiome" will be used when samples are collected at two different time points (different seasons, before and after pollution, etc.). Please clarify and revise Line 100: deleet and, use "unclassified" organism insteas others Materials and methods Line 123: delete YSI Results Line 224: quality control (instead of QC) Discussion Line 322, 330: Revise the text: this observed differences instead of "shift." Line 369: Differenc or deviation? clarify References 157: Nygaard et al., wrong format The following references are not cited in the text but are lister in the reference list. 445- dhariwal 503_ Mongad 547_ Torres Figures Figure 2 can be moved to the supplementary Figure 5: Abbreviations of three regions (ssc, sj and sm) can be given in legend. Supplementary Figure 2: Give abbreviations of sd, sj, sm, sp and ss
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: The study does not involve human subjects, and ethical approval is not required
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