Review Reports - Assessing Species Delimitation in <i>Entamoeba</i> (Amoebozoa: Endamoebidae) Using the Small Subunit rRNA Gene: Its Application to the <i>Entamoeba polecki</i> Complex

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Esteban-Sánchez and Ponce-Gordo analyzed the SSU rRNA gene sequences from different members of the genus Entamoeba to differentiate and categorize them. This task is important as morphological characteristics of their trophozoites and cysts are used, as well as molecular methods. Even though different authors have used the SSU rRNA gene sequences to analyze and classify members of this genus, in this manuscript, the authors used a greater number of sequences and included a larger number of species. Additionally, authors include SSU rRNA secondary structures to identify important nucleotide changes in different helices that could be used as Barcodes in the SSU-rDNA sequence for the differentiation between species. Overall, the manuscript is well written and structured. I have several comments that could be addressed to comments that could be addressed to improve the manuscript.

A discussion about the potential pathogenic role of E. dispar, as several reports suggest a role in human disease.
The authors could discuss why E. histolytica in their partitioning analysis cluster with E. nutalli rather than E. dispar, as in other publications using SSU rRNA, E. histolytica and E. dispar cluster together, indicating a closer evolutionary relationship.
The authors may consider the inclusion of a unified figure of the partitioning analysis of all the species included in their analysis, compared to a phylogenetic analysis, which could demonstrate the robustness of their study by comparing the methods and the relationships among Entamoeba species.

Author Response

RESPONSES TO REVIEWER 1

We sincerely thank the reviewer for the careful reading of the manuscript and for the positive and constructive assessment. We particularly appreciate the recognition of the scope of the dataset and the integrative approach adopted in this study. The comments provided have been very helpful in improving the clarity and presentation of the manuscript.

COMMENT 1. A discussion about the potential pathogenic role of E. dispar, as several reports suggest a role in human disease.

RESPONSE 1. We thank the reviewer for this suggestion and fully agree that the potential pathogenic role of E. dispar has been discussed in several studies (e.g., Dolabella et al 2012, doi 10.1016/S1665-2681(19)31494-2; Graffeo et al. 2014, doi 10.1155/2014/498058; da Silva et al. 2021, doi 10.1016/j.actatropica.2021.106114; Mergen et al. 2024, doi 10.1093/ofid/ofae658). This is indeed an important and actively debated aspect of the biology of this species.

However, the aim of the present study is not to address pathogenicity, but rather to evaluate criteria for species identification under an evolutionary species concept using an integrative taxonomic framework, with particular emphasis on the role of genetic data when other diagnostic characters are limited or ambiguous. As such, a discussion of pathogenicity would fall outside the scope of the manuscript and would not directly contribute to its main objectives. For this reason, despite its objective importance, we think it is not relevant for this work and it is not necessary to expand on this topic in the revised version.

COMMENT 2. The authors could discuss why E. histolytica in their partitioning analysis cluster with E. nutalli rather than E. dispar, as in other publications using SSU rRNA, E. histolytica and E. dispar cluster together, indicating a closer evolutionary relationship.

RESPONSE 2. We appreciate this comment, which allows us to clarify the evolutionary relationships among these closely related taxa. Entamoeba histolytica, E. nuttalli, and E. dispar form a well-established phylogenetic group, but multiple studies indicate that E. histolytica is more closely related to E. nuttalli than to E. dispar.

In the SSU rRNA gene, E. histolytica and E. nuttalli differ by only five nucleotide positions (sequence similarity ~99.7%), whereas E. histolytica and E. dispar differ by 26 positions (sequence similarity ~98.6%). Accordingly, phylogenetic analyses consistently recover E. dispar as a sister lineage to a clade comprising E. histolytica and E. nuttalli when the three species are included. This topology has been reported in several studies (see, for example, Clark and Stensvold 2015, doi 10.1007/978-4-431-55200-0_2; Jacob et al. 2016, doi 10.1111/jeu.12249; Al-Habsi et al. 2017, doi 10.1016/j.vetpar.2017.01.013; Elsheikha et al. 2018, doi 10.1016/j.pt.2017.12.008; Jinatham et al. 2019, doi 10.1017/S0031182019000775) and similar relationships have been observed using additional genetic markers (for example, see Feng et al. 2013, doi 10.1007/s00436-013-3299-1). Therefore, the clustering observed in our partitioning analyses is fully consistent with previously published phylogenetic evidence.

COMMENT 3. The authors may consider the inclusion of a unified figure of the partitioning analysis of all the species included in their analysis, compared to a phylogenetic analysis, which could demonstrate the robustness of their study by comparing the methods and the relationships among Entamoeba species.

RESPONSE 3. We thank the reviewer for this constructive and well-founded suggestion. In general, integrating species partitioning results with phylogenetic analyses can indeed be informative and is often useful for exploring the consistency between different analytical approaches. However, considering the specific design and objectives of the present study, we believe that such an approach would not be methodologically appropriate in this case.

Species partitioning using ASAP was intentionally performed separately on two phylogenetically coherent groups: (i) the closely related E. histolytica, E. nuttalli and E. dispar, and (ii) the group comprising E. polecki, E. chattoni and E. struthionis, which are phylogenetically related to each other. Both groups are deeply divergent in phylogenetic analysis, as it can be observed in the literature cited in response 2.

This strategy (each group analyzed in separate) was adopted to avoid mixing markedly different evolutionary scales, which would result in separations driven by deep divergences rather than informing fine-scale species partitioning. Because ASAP is a distance-based, non-phylogenetic method designed to explore genetic discontinuities within comparable divergence ranges, a unified phylogenetic analysis including all taxa would be dominated by deep splits between these groups and would not provide additional insight into the partitioning hypotheses addressed in this study. To avoid potential confusion, we have clarified this rationale in the revised manuscript; we have added the following text to material and methods section, lines 291-294:

ASAP analyses were conducted separately within the E. histolytica-like clade and within E. polecki s.l., in order to avoid mixing distinct evolutionary scales, which could result in partitions driven by deep phylogenetic divergences rather than providing informative fine-scale species partitioning.

In addition, we introduced a minimal textual clarification in the Discussion (line 836) to explicitly link the exploratory divergence analyses with the subsequent validation framework:

Once divergence analyses (AMOVA, ASAP) suggest that sequences may represent distinct lineages, validation using non–distance-based criteria is required.

This minor addition improves clarity and prevents potential misinterpretation of the role of these analyses without altering the scope or conclusions of the Discussion.

Once again, we sincerely thank the reviewer for the careful evaluation of the manuscript and for the constructive and insightful comments, which have helped to improve the clarity and overall quality of the work.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The presented manuscript is a substantial study of the molecular genetic variability within the genus Entamoeba that represents an interesting attempt to reconsider the criteria for species delimitation in this important group. Entamoeba is important from the fundamental and practical poins as a group of medical and veterinary relevance. Yet, its species are difficult to identify morphologically and using physiological and ecological criteria (e.g. host specificity). Molecular analysis currently remains the only practical approach. The authors performed an extensive molecular analysis of published previously SSU rRNA sequences of Entamoeba to elaborate improved species boundaries and develop new tools for species identification. The proposed approach is well-justified and may be useful also for other presumably asexual lineages of amoebae poor in morphological characters. I therefore recommend that the paper is published, however, I have several major and minor comments and points to clarify to be considered by the authors (listed below).

Major.

I am wondering, why the authors did not include the sequences not identified to named species in their analysis. I would start from retrieving the named sequences, filtering the dataset, performing preliminary analysis, followed by adding the set of unnamed sequences (i.e. Entamoeba) and performing the second iteration of analysis by clarifying the assignment of “sp.” sequences. In my opinion, this might in some cases enrich the analysis with data and clarify the identity of some unidentified sequences, that could be an additional contribution to the quality of the database.
While discussing the ribosomal RNA variation, the authors might wish to discuss also the option of intragenomic variation. Although considered to be subject of concerted evolution, the multicopy rRNA cluster often demonstrates intragenomic variability in multiple clades of eukaryotes which is a common point now. In my opinion, the authors should consider this option in the Discussion too.
I am a bit skeptical about the secondary structure models presented by the authors (Fig. 3-4). In particular, they are very different from the conventional eukaryotic models developed by Wuyts e al. (2000, 2001 – Nucleic Acids Res. 28: 4698-4708; 29: 5017-5028). In particular, there is a standard set and numbering of helices (1-50) and the structure of the region V4 (expansion segment E23) implies the presence of two pseudoknots. I do not see these features in the models presented by the authors that looks strange.
The nomenclatural problems and type material: there is an on-going discussion on the possibility of establishing total DNA samples as type material in case if other kinds of type material are either not preservable or useless (e.g. if they do not retain the necessary characters and cannot be subject for subsequent reinvestigation with other methods). In my opinion, the authors should reconsider this part of the Discussion (see also comments below).

Minor.

Title and throughout the manuscript: Entamoebidae is the correct spelling of the family, not Endamoebidae.

Lines 19-21: “we established reference SSU rDNA sequences for those Entamoeba species for which the sequences are currently available” sounds unclear; please, rephrase.

Line 89 and throughout the ms: four-nucleated -> tetranucleate

Line 91 and throughout the ms: one-nucleated -> uninucleate (or mononucleate), etc.

Line 121: please, correct S0SU

Lines 132-143: “differed from their corresponding reference sequence by at least one stretch of five or more contiguous positions” – could you please clarify what you mean by “stretch”? Did you mean five positions one after another each of which varies between species?

Line 291-292: which tool did you use for the generation of consensus sequences?

Lines 324-328: which tools did the authors use to detect chimeras? Please, mention in methods section.

Lines 650-651: DNA sequences do not meet the criteria of types, but total DNA itsef, in my opinion, does. The Code states that “an animal or part of an animal” is eligible as type material. DNA extracted from cells evidently counts as “part of an animal”. I’ve had a discussion in person on this point some time ago with experts who are involved in the protists’ part of ICZN, and they are in favor of the approach that a deposited total DNA sample may indeed serve as type material. The authors may stress this point. You may also consider the following paper in this discussion: https://www.cell.com/trends/genetics/fulltext/S0168-9525(25)00193-3

Line 652: Given my above comment, I would not agree with the statement made here. Please, also not the Recommendation 73G from the ICZN: “Recommendation 73G. Specific reasons for designation of an unpreserved specimen as the name-bearing type. An author should provide detailed reasoning why at least one preserved specimen, whether a complete individual organism or a part of such an individual, was not used as the name-bearing type for the new taxon and why the formal naming of the taxon is needed at a point in time when no preserved name-bearing type will be available.” (https://www.iczn.org/the-code/the-code-online/)

Lines 662-667: In line with the previous comments, I would suggest not just “replacing numerical designations with names”, but establishing the proper species. If deposited DNA samples are available, there should not be problems with type material designation.

Lines 696-698: the problem of variability at the ends (especially if there are single-nucleotide substitutions) may also be caused by the application of different primers by different research groups. Normally, the terminal parts of SSU rRNA molecule are conservative, that allows us to use conserved “universal” primers however, there are several variations of these primers that involve nucleotides in the middle part. If a primer site is included in the sequence submitted to GenBank, it contributes to the variation if different researchers use different primers.

Line 790: nutalli -> nuttalli

In Supplementary File 1: what do colors of the helices indicate?

Author Response

The responses include some figures; please read the pdf file.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I am happy with the changes the authors made to the manuscript and I suggest acceptance after some minor corrections.

Minor comments:

I fully understand and support all the reasons to consider Endamoebidae as a priority over Entamoebidae, yet, this is unusual, as Entamoebidae is much more frequently used now. Probably, it would make sense to add a short comment for those not fully familiar with numenclatural history of these taxa.

Line 22: standartize -> standartized(?)

Lines 19-23: this single long sentence is difficult to read. I would suggest splitting it in two (e.g. To apply these criteria, we assembled a curated set of reference SSU rDNA sequences for those Entamoeba species identified at the species level (bearing valid specific epithets) for which sequence data are currently available, providing a standardize framework for the accurate assignment of published sequences. We determined their SSU rRNA secondary structures using previously published experimental data and in silico analyses.)

Author Response

We would like to thank the reviewer once again for the thoroughness and care with which this manuscript was read. We have followed the reviewer’s comments, corrected the linguistic errors, and added a sentence explaining the rationale for using Endamoebidae as the family name. We have also incorporated a reference to a manuscript that we have recently submitted for publication (a historical review of the taxonomy of the genus Entamoeba) which is currently available as a preprint and provides a detailed discussion supporting the use of this family name.