Extensive Cryptic Diversity and Ecological Associations Uncovered among Mexican and Global Collections of Naegleria and Vermamoeba Species by 18S Ribosomal DNA, Internal Transcribed Spacer, and Cytochrome Oxidase Subunit I Sequence Analysis

ABSTRACT Free-living amoebae (FLA) are phagocytic protists that play crucial roles in microbial communities as significant microbial grazers. However, our current knowledge of their diversity, ecology, and population genetic structures is marginal due to the shallow and biased sampling of ecosystems and the use of few, poorly resolving molecular markers. Thirty-two FLA were isolated from soil and water samples collected across representative ecosystems of the State of Morelos in Central Mexico, including the drinking water distribution system (DWDS) from the state capital. We classified our isolates as members of Acanthamoeba, Vermamoeba, Naegleria, and Tetramitus by 18S ribosomal DNA (rDNA) sequencing. Vermamoeba isolates were recovered exclusively from the DWDS samples. In contrast, Naegleria strains displayed a broad distribution in soil and water samples across the natural ecosystems. We used a combination of phylogenetic and population genetic analyses of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (COI) sequences from our isolates and a comprehensive set of reference sequences to analyze the currently known diversity of Naegleria spp. Significant associations were uncovered between the most prevalent lineages of Naegleria and Vermamoeba and broad ecological and geographical variables at regional and global levels. The population structure and cryptic diversity within the Naegleria galeacystis-Naegleria americana and Vermamoeba vermiformis species complexes were thoroughly analyzed. Our results prove that the genus Vermamoeba, which was previously thought to consist of only one species, actually encompasses at least seven widely distributed species, as indicated by consistent evidence from Bayesian phylogenetics, two species-delimitation programs, and population genetics analyses. IMPORTANCE Our study sheds new light on the population genetic structure of V. vermiformis and diverse Naegleria species. Using improved molecular markers and advanced analytical approaches, we discovered that N. americana, previously considered a single species, actually contains multiple distinct lineages, as revealed by COI sequencing. These lineages are highly differentiated, with little gene flow between them. Our findings demonstrate that the genus Vermamoeba holds multiple cryptic species, requiring a significant taxonomic revision in light of multilocus sequence analyses. These results advance our understanding of the ecology, molecular systematics, and biogeography of these genera and species complexes at both regional and global scales. This study has significant implications for diagnosing amoebal infections and evaluating health risks associated with FLA in domestic and recreational waters.


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62. You could clarify that you're referring to FLA species/genus (which many may be considered broadly distributed erroneously due to the issues you mention), not FLA as whole group (which is a wide group, broadly distributed).

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The authors thank the reviewer for the constructive criticism, which helped us improve the manuscript. Line numbering corresponds to the Marked-Up_Manuscript.pdf file.
62. This issue was clarified. The sentence was re-structured as follows, providing selected references to sustain the statement: L88-91. FLA species are generally thought to be broadly distributed (16,17). However, poorly resolving molecular markers, combined with a shallow sampling of a few ecosystems, constrain our knowledge of the diversity and ecological features of FLA on local, regional, and global geographical scales (18-20).
68. These are all excellent and relevant references added to the manuscript. However, they are cited in the discussion to avoid excessive citations in the Introduction. For example, these references (73-75) nicely fit the last sentences of the first paragraph of the discussion.
The reference to the COI-metabarcoding paper was cited again at the end of the discussion section on COI markers.

L694-700
This analysis revealed that the analyzed COI segment is significantly more polymorphic than the ITS region and, therefore, better suited for ecological inference and molecular systematics studies. Furthermore, COI markers pave the way for finer-grained association studies between FLA lineages and environmental variables, particularly if coupled with high-throughput sequencing of lineagespecific COI-barcodes generated directly from environmental DNA, as recently reported (75). 92. This problematic sentence was rephrased for clarity as follows: L119-121 Combined phylogenetic and population genetic analyses of the new sequences generated in this study and selected global reference sequences confirmed the identification of the isolates at the species level based on the current taxonomy.
170. The following sentence was added to explicitly provide our biological interpretation of the statistical test:

L200-202
This result indicates that the sampled organisms from both genera have strongly differentiated environmental distributions, which may result from distinct habitat preferences or adaptations. 551. The problematic sentence (last one in the following paragraph, provided for context) was corrected (identifying instead of to identify) and improved (shortened and avoiding passive voice) as follows: L590-598 However, we found that both delimitation methods require some data pre-processing to perform optimally, like collapsing sequences to haplotypes. In addition, mPTP requires at least two haplotypes per ESU, as the shifting point between speciation and coalescent processes cannot be determined for singleton species or units because no coalescence events are available for them in the dataset (46). Therefore, we paid great attention to identifying and excluding singleton units from datasets to be analyzed with mPTP to avoid lumping these lineages with sister units or clades. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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