Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

• The reviewed MS focuses on the hidden diversity of the genus Meranoplus, and more specifically on the members of the diversus species group.
• Overall this is a fantastic paper, well-written and well-presented. It was indeed a treat to read.
• The introduction is well-written and convincingly shows the high diversity within the group. The goal of the study is clearly indicated.
• The results are appropriately interpreted and the conclusions are supported by an ample amount of genetic, morphological, and distributional data.
• The paper is aptly illustrated with a great deal (but not overwhelming) of figures.
• The study points out the need for detailed integrated approaches when unravelling the diversity of a tough and diverse group of ants. In addition, the study provides a clear framework for future research on the group.
• This is a well-thought out, well-executed, and well-presented study that contributes a great deal of novel information to our knowledge of a diverse and challenging group, and to our understanding of global patterns of ant diversity.
• The References are complete and, to my knowledge, include all relevant literature.
• I was only able to find a few minor presentation issues which are outlined in the attached.
• This mostly out of curiosity. Figures 2, 5 and 7 show lack of sequenced specimens from the Cape York Peninsula. Are there any specimens collected from the area, which have not yet been sequenced, or is there a complete lack of data from the area? If the latter, is this a sampling artefact, or is there another reason that may help explain this distribution pattern?

Comments for author File: Comments.pdf

Author Response

Comment 1. Overall this is a fantastic paper, well-written and well-presented. It was indeed a treat to read.

Response1. We are most grateful for this positive feedback

 

Comment 2: I was only able to find a few minor presentation issues which are outlined in the attached.

Response 2: We thanks the reviewer for these editorial amendments, all of which we have now attended to.

 

Comment 3: This mostly out of curiosity. Figures 2, 5 and 7 show lack of sequenced specimens from the Cape York Peninsula. Are there any specimens collected from the area, which have not yet been sequenced, or is there a complete lack of data from the area? If the latter, is this a sampling artefact, or is there another reason that may help explain this distribution pattern?

Response 3: To clarify this, we have now added to the Methods: "The Darwin collection contains no specimens from Queensland for either complex, and as far as we are aware none have ever been collected from there."

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

I have read the manuscript entitled "Unrecognised Ant Megadiversity in the Australian Monsoonal Tropics: The Meranoplus berrimah Schödl and M. snellingi Schödl Complexe"  and I want to congratulate you on your hard work. Overall, the paper is well written and its quality is high. However, I must emphasize some aspects that need clarifications:

1. Can the authors explain why only solely CO1 was used to delimitate species? The are also other methods such as nuclear genes and maybe morphometric to enhance the species delimitation process and may add significantly to the value of the paper.
2. As I have seen from the distribution may there is overlapping. Hybridization is known to occur in ants, is it the case within your genus or other species of the genus Meranoplus? If it is the case, some aspects should be provided.
3. In the supplementary files, information from the tables are missing, things like data of sampling is very important. Is the data recent or is it old data? This may change in some way the results.
4. Also, in order to defend the statement that Australia is very rich in species comparable with tropical areas, at least a species (or even more) would have been important to be described in this paper, if the data is available,

 

All the best

Comments for author File: Comments.pdf

Author Response

Comment 1: Overall, the paper is well written and its quality is high.

Response 1: We thank the reviewer for this positive feedback.

Comment 2: Can the authors explain why only solely CO1 was used to delimitate species? The are also other methods such as nuclear genes and maybe morphometric to enhance the species delimitation process and may add significantly to the value of the paper.

Response 2: CO1 is the most widely-used gene for species delimitation in ants, and is especially reliable when integrated with morphological variation, as we have done. Morphological variation in these complexes are primarily qualitative rather than quantitative, and there do not readily lend themselves to formal morphometric analysis. We note that the Schodl's formal taxonomic revision of the Meranoplus diversus group does not include morphometric analysis.  However, in the Results we have now added extra information on morphological differentiation: "In sp. B2 the anterolateral angles of the dorsal shield are square, and in B3 nearly so, rather than acute as in all other species of the complex (Figure 4). In spp. B2 and B4 the posterior margin of the dorsal shield is conspicuously concave medially, compared with only weakly so in spp. B1 and B4 (Figure 4)."

 Comment 3: As I have seen from the distribution may there is overlapping. Hybridization is known to occur in ants, is it the case within your genus or other species of the genus Meranoplus? If it is the case, some aspects should be provided.

Response 3: There is no know hybridization among Australian species of Meranoplus.

Comment 4: In the supplementary files, information from the tables are missing, things like data of sampling is very important. Is the data recent or is it old data? This may change in some way the results.

Response 4: We acknowledge that sampling dates are not included in Table S1. However, we do not see how such information relevant to our analysis of diversity within the complexes, and so we have not included it.

Comment 5:  Also, in order to defend the statement that Australia is very rich in species comparable with tropical areas, at least a species (or even more) would have been important to be described in this paper, if the data is available,

Response 5: Formal taxonomic revision with species descriptions is beyond the scope of this paper, which is about species diversity. Formal descriptions of species would not change our findings.

Reviewer 3 Report

Comments and Suggestions for Authors

The paper of Andersen et al. addresses cryptic diversity within two ant species complexes in Northern Australia. While I find these findings intriguing, I contend that the authors’ conclusions regarding the existence of distinct species are far from conclusive.

First of all, mitochondrial differences alone cannot substantiate reproductive isolation or divergence in the nuclear gene pool. The authors assert that «We followed a species concept based on reproductive isolation and evolutionary independence as evidenced by morphological differentiation between sister (i.e. most closely related) CO1»; however, their methodology provides no evidence for reproductive isolation.

Of course, demonstrating reproductive isolation or nuclear genomic divergence in poorly studied organisms is challenging, leading researchers to frequently use mitochondrial distances as a proxy. However, this part is not adequately described in the paper.What particular distance type was calculated, p-distance, K2P, or some other? And what were the distances? This omission hinders rigorous evaluation of their claims.

Furthermore, evidence for cryptic species based solely on mtDNA data requires clear separation of clades in phylogenetic reconstructions. Examination of the cox1 tree (Fig. 3), however, reveals that branches designated as cryptic species lack bootstrap support; in several instances, they do not even form discrete clades. Consequently, most of the proposed ‘species’ fail to meet currently accepted criteria for species delimitation.

Therefore, I recommend that the authors (1) temper their conclusions with more tentative language and (2) include tables detailing cox1 genetic distances between the proposed ‘species’ and their closest relatives.

Minor comments:

L. 70. «specimens attributable M. berrimah» - attributable to M. berrimah.

L. 88. «DNA sequences were checked and edited in MEGA [11]»  - version?

L. 89 «translated into (invertebrate) proteins» - translated using the invertebrate mitochondrial codon table.

L. 93. «which we also sequenced through BOLD» -what does it mean, sequenced through BOLD?

L. 97 «intraspecific CO1 distance is typically 1–3% [14]» - typical for ants, other groups have widely different distance values.

Author Response

Comment 1: First of all, mitochondrial differences alone cannot substantiate reproductive isolation or divergence in the nuclear gene pool. The authors assert that «We followed a species concept based on reproductive isolation and evolutionary independence as evidenced by morphological differentiation between sister (i.e. most closely related) CO1»; however, their methodology provides no evidence for reproductive isolation.

Response 1: We have used CO1 divergence in association with morphological variation to indicate reproductive isolation, not divergence in the nuclear gene pool, which is not the same thing. Our approach is routinely used in studies of ant diversity.

Comment 2: What particular distance type was calculated, p-distance, K2P, or some other? And what were the distances? This omission hinders rigorous evaluation of their claims.

Response 2: In IQ-TREE v3, genetic distance is calculated from branch lengths as part of the maximum likelihood optimization process. Distance is indicated in our CO1 tree (Figure 3)

 

Comment 3: Furthermore, evidence for cryptic species based solely on mtDNA data requires clear separation of clades in phylogenetic reconstructions. Examination of the cox1 tree (Fig. 3), however, reveals that branches designated as cryptic species lack bootstrap support; in several instances, they do not even form discrete clades. Consequently, most of the proposed ‘species’ fail to meet currently accepted criteria for species delimitation.

Response 3: We acknowledge that bootstrap values are sometimes low, but our species delimitations are not based on CO1 clustering alone - the species that we recognise are also morphologically and geographically coherent.  All our recognised species form discrete CO1 clades - the reviewer appears to be referring to the PTP/bPTP analyses, rather than to the integrated analysis we have used for species recognition.

Comment 4: Minor editorial comments

Response 4: Thank-you for picking these up - we have attended to all of these.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for your reply. I agree with the comments and I am happy with the improvements. However, I must insist on the data from the collection. Is it new or old data. I ask for this, because it is known that old data may alter the quality and quantity of the DNA which is extracted (see https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049710)

Best wishes!

Author Response

Comment: I must insist on the data from the collection. Is it new or old data.

Response: We have now added the year of collection in Supplementary Table S1. We have also added "Collections were made between 1986 and 2023." to the Methods. 

Reviewer 3 Report

Comments and Suggestions for Authors

I have read the revised version of the manuscript. The authors have addressed some of my previous suggestions, which I appreciate. However, I do not agree with the statement that "CO1 divergence in association with morphological variation indicates reproductive isolation"  and I still believe a table with pairwise p-distances should be added. Such a table would allow readers to better assess the genetic differentiation between the proposed species, especially given that the tree does not show these species forming reciprocally monophyletic clades.

Author Response

Comment: I still believe a table with pairwise p-distances should be added. Such a table would allow readers to better assess the genetic differentiation between the proposed species, especially given that the tree does not show these species forming reciprocally monophyletic clades.

Response: We respectfully disagree that such a Table is necessary. Our recognised species actually do occur as reciprocally monophyletic clades in our CO1 tree (Fig. 3), where distance is indicated. More-precise distance data would have no bearing on species delimitation. We note that our similar publications that have previously been published in the journal do not include such data.