The Taxonomy of the Genus Entamoeba (Archamoebea: Endamoebidae): A Historical and Nomenclatural Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The extensive tables are valuable and constitute a major strength of the manuscript. However, the criteria used for inclusion, synonymy, and taxonomic status assignment are not always made explicit. A brief methodological clarification in the Introduction or Discussion would improve transparency and help readers better understand the authors’ decision-making framework.

The manuscript occasionally uses strongly evaluative terms (e.g., “unjustified”, “incorrect”). While these assessments may be defensible, such wording could be perceived as overly polemical for a review article. Consider adopting more neutral formulations to maintain an objective tone.

Sections reviewing individual species and historical descriptions are extremely detailed and informative, but intermediate synthesis is sometimes lacking. The key conceptual messages may be obscured by the volume of historical material. Brief summary statements at the end of major subsections would improve readability and reinforce the overarching arguments of the review.

P4: The author list contains inappropriate or unintended characters and should be carefully checked and corrected.

British and American English are used inconsistently throughout the manuscript (e.g., “recognised”). Please standardize spelling consistently across the text.

Author Response

See the attached pdf file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, this is a high-quality, authoritative, and carefully researched review that demonstrates exceptional command of the historical and nomenclatural literature on Entamoeba. The manuscript provides a valuable synthesis that clarifies long-standing taxonomic confusion and will serve as an important reference for both parasitologists and taxonomists. Aside from a limited number of conceptual and factual inconsistencies, the review is scientifically sound and suitable for publication after minor revision.

Detailed comments are as follows.

Lines 25–26: The abstract states that “general principles are proposed as a pragmatic synthesis to guide future taxonomic work on Entamoeba,” but none of these principles are summarized or even briefly indicated within the abstract itself. This creates a mismatch between what the abstract claims to deliver and what it actually contains, leaving the reader without a clear sense of the study’s original contribution beyond being a narrative review. To address this, the authors should add one short sentence (or modify lines 25–26) explicitly naming or briefly characterizing the proposed principles (e.g. integration of molecular phylogeny with host association, prioritization of genetic evidence over morphology alone, or explicit nomenclatural criteria). This would align the abstract with good scholarly practice and substantiate its concluding claim.

Lines 32–34: The statement that “several free-living species” belong to Entamoeba represents both a factual inaccuracy and an internal inconsistency. This contradicts established taxonomy and the authors’ own later discussion (e.g. lines 287–295), where species such as E. moshkovskii are correctly treated as primarily free-living or facultatively parasitic, but not representative of a group of several free-living Entamoeba species. At present, Entamoeba is overwhelmingly regarded as a genus of obligate or facultative parasite-associated amoebae, with truly free-living taxa placed in other archamoebid lineages. The authors should revise lines 32–34 to clarify that Entamoeba includes species with parasitic, commensal, or facultatively free-living stages, rather than asserting the existence of multiple free-living species within the genus.

Lines 598–601: The authors state that the recognition of Entamoeba moshkovskii as a distinct, free-living species “has never been questioned.” This claim is factually incorrect and overly categorical. The taxonomic status, ecological niche, and pathogenic potential of E. moshkovskii have been repeatedly debated, particularly regarding whether it is primarily free-living or intestinal with environmental persistence, and whether it can be pathogenic to humans. This controversy is even acknowledged later by the authors themselves (lines 615–618), creating an internal inconsistency. The problem can be resolved by softening the wording in lines 598–601 (e.g. replacing “has never been questioned” with “has generally been accepted, although its ecology and pathogenic potential remain debated”).

Lines 417–428: The authors present a strongly normative conclusion that the reinstatement of Entamoebidae by Cavalier-Smith was “incorrect,” and that Endamoebidae Calkins, 1926 “should be regarded as the correct and valid family-group name.” While the historical analysis is detailed and informative, the language exceeds what is justified for a review article, as Entamoebidae remains accepted by several contemporary taxonomic authorities and databases, and no formal ruling by the ICZN is cited. This should be corrected by reframing the conclusion as an interpretation rather than a definitive resolution (e.g. “can be argued to be incorrect” or “raises serious nomenclatural concerns”), or by explicitly stating that a formal ICZN Opinion would be required to settle the issue conclusively.

Lines 889–894: There is a conceptual and taxonomic inconsistency in the treatment of free-living species within the histolytica (tetranucleate) group. Here, Entamoeba moshkovskii, E. ecuadoriensis, and E. marina are grouped together as free-living species while still being implicitly retained within the histolytica group, which is defined earlier (lines 700–707) strictly on the basis of cyst nuclear number and traditionally applied to intestinal species. This creates a conceptual conflict, as cyst morphology in E. ecuadoriensis and E. marina is either poorly documented or not demonstrably comparable to that of classical tetranucleate intestinal species, and their ecological separation from host-associated taxa undermines the usefulness of this grouping. The authors should either state explicitly that inclusion of these taxa in the histolytica group is provisional, or separate free-living species into a distinct subsection where molecular affinity is discussed independently of cyst-based morphology.

Lines 985–989: There is a methodological inconsistency in how molecular evidence is weighed against morphology and host association. An isolate from a laboratory rat is reassigned from E. muris to E. coli ST2 solely on sequence identity, while the alternative interpretation—that E. coli ST2 may represent a rodent-associated lineage or a cryptic taxon within the E. muris complex—is dismissed without justification. This contradicts the authors’ repeated caution elsewhere (e.g. lines 740–745, 781–786) that genetic similarity alone is insufficient for species assignment. This can be corrected by acknowledging both interpretations as plausible and by clearly stating that, in the absence of corroborating morphological or ecological data, such assignments should remain tentative.

Lines 1115–1119: The authors state that by the end of the 20th century E. bovis, E. ovis, E. dilimani, E. bubalus, and E. polecki were “generally accepted as valid species in ungulates,” while later (lines 1141–1148) they argue that genetic evidence supports the synonymization of E. ovis with E. bovis and, by extension, suggests that E. dilimani and E. bubalus may also be synonyms. This creates a major internal inconsistency: species are first presented as taxonomically stable and later implicitly downgraded without a clear transition or synthesis. The authors should revise lines 1115–1119 to clarify that this acceptance was historical and provisional, and add a concise synthesis at the end of Section 7.3 explicitly stating which species they currently regard as valid, which as probable synonyms, and which as unresolved.

Lines 1447–1457: The authors argue that a physically deposited total DNA extract could, “in principle,” qualify as a name-bearing type under Art. 72.5.1 of the Code and present this as a plausible option for Entamoeba taxonomy. This interpretation is highly problematic and overstates what is currently accepted under the ICZN. While Art. 72.5.1 allows parts of animals to serve as name-bearing types, the Code does not explicitly recognize DNA extracts as acceptable type material, and the prevailing consensus is that DNA extracts alone do not meet the requirements for name-bearing types, particularly in unicellular organisms lacking a preserved organismal voucher. Although the authors partly acknowledge these difficulties later (lines 1458–1468), the initial framing is misleading. This section should be revised to state clearly that the acceptability of DNA extracts as name-bearing types is not established under the current Code, remains controversial, and should be treated as a theoretical discussion rather than a viable current practice.

Lines 1551–1560: The authors state that species delimitation in Entamoeba should follow a lineage-based species concept and that any single line of evidence supporting evolutionary independence “may be sufficient” for species delimitation, while simultaneously claiming that this approach is consistent with integrative taxonomy. This represents a major conceptual inconsistency. Integrative taxonomy is explicitly based on the congruence of multiple independent lines of evidence, particularly in taxa with cryptic diversity and limited morphology such as Entamoeba. Presenting single-criterion delimitation as fully compatible with integrative taxonomy conflates species hypothesis generation with formal species recognition and risks legitimizing taxonomic decisions based on minimal evidence. This can be fixed by clearly distinguishing between provisional species hypotheses and formal species delimitation, and by emphasizing that corroboration from multiple independent data sources should remain the standard wherever feasible.

Author Response

See the attached pdf file.

Author Response File: Author Response.pdf