Tetrigidae of Ethiopia: First Species Delimitation via DNA Barcoding and Description of Three New Species^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a valuable contribution to the knowledge of African Orthoptera, particularly the Tetrigidae of Ethiopia. The combination of morphological and molecular evidence, including DNA barcoding, provides a solid foundation for the discovery and description of three new species and several new country records. The results are robust and highlight the importance of integrative taxonomy in understudied faunas. While I find the study  of interest, I have a few comments, mainly concerning the introduction and some of the figures, which I believe could be improved to enhance the clarity of the manuscript.

Introduction – Clarity of Concepts: “However, morphological studies alone often lack the power to detect cryptic species and to evaluate phenotypic plasticity, leading to difficulties in developing consistent concepts of taxonomy and systematics (Hebert et al. 2003, Williams et al. 2006, Moczek 2010).”  The statement “difficulties in developing consistent concepts of taxonomy and systematics” is unclear. Could the authors clarify what specific concepts they are referring to? Are they discussing taxon concepts (i.e., differing interpretations of taxonomic boundaries), species concepts (e.g., morphological vs. biological vs. phylogenetic), or broader systematic frameworks? A more precise explanation would help readers understand the implications of relying solely on morphology.

 

Introduction , Terminology Clarification: ”Hence, barcode data always should be enriched with morphological information to understand complex faunas”  The phrase “complex faunas” is vague in this context. Do the authors mean taxonomically complex groups? Clarifying this would help ensure that the statement is correctly understood, especially in relation to the use of barcode and morphological data.

 

Introduction Clarity  needed:  “Further, DNA barcoding is strongly relying on well curated databases, which are largely not in place…” The phrase “largely not in place” is unclear. Do the authors mean that comprehensive, high-quality barcode reference libraries are missing or incomplete, particularly for biodiverse regions? Please rephrase for clarity.

Introduction Citation needed: “The database lists eight species for northeast tropical Africa, whereas more than 200 species are known for Africa”. This statement needs a citation

 

Figure 1. Distribution and sampling sites of Tetrigidae in Ethiopia.  The current maps are very difficult to interpret due to the lack of conventional cartographic elements. I strongly recommend redrawing all maps using standard and easily recognizable layers, uch as clear geopolitical boundaries, rivers, elevation (if relevant), and labeled key regions. Consider using a base map that improves geographic orientation and enhances clarity for an international audience. Including a legend, north arrow, and scale bar is also essential for proper interpretation.

Figure 9. The quality of the tree has to be improved. Names are very difficult to read.

 

 

Author Response

Dear reviewer, We value the effort and recommendations you provided for our manuscript. We employed the American Journal Experts/AJE language editing for clarity in the research paper, and we incorporated other suggested comments by other reviewers too. Additionally, the quality of the figures and tables has been enhanced as you recommended.

 

Introduction

 

Reviewer #1: Introduction – Clarity of Concepts: “However, morphological studies alone often lack the power to detect cryptic species and to evaluate phenotypic plasticity, leading to difficulties in developing consistent concepts of taxonomy and systematics (Hebert et al. 2003, Williams et al. 2006, Moczek 2010).” The statement “difficulties in developing consistent concepts of taxonomy and systematics” is unclear. Could the authors clarify what specific concepts they are referring to? Are they discussing taxon concepts (i.e., differing interpretations of taxonomic boundaries), species concepts (e.g., morphological vs. biological vs. phylogenetic), or broader systematic frameworks? A more precise explanation would help readers understand the implications of relying solely on morphology.

Response: We attempted to address the inconsistency in precise species identification, especially in cryptic species or species complexes that depend exclusively on morphological diagnosis, like in the case of convergent evolution, which makes classical taxonomy unable to address such issues. Therefore, using integrative taxonomy will resolve such appealing problems in various organisms. Upon careful review, the term “concepts” appears vague and somewhat misleading; we should consider removing it.

Reviewer #1: Terminology clarification: ”Hence, barcode data always should be enriched with morphological information to understand complex faunas”  The phrase “complex faunas” is vague in this context. Do the authors mean taxonomically complex groups? Clarifying this would help ensure that the statement is correctly understood, especially in relation to the use of barcode and morphological data.

Response: Thanks, really, replacing it with “taxonomically complex groups” pronounces well and refines the clarity. We replaced it.

 

Reviewer #1: Clarity needed:“Further, DNA barcoding is strongly relying on well curated databases, which are largely not in place…” The phrase “largely not in place” is unclear. Do the authors mean that comprehensive, high-quality barcode reference libraries are missing or incomplete, particularly for biodiverse regions? Please rephrase for clarity.

Response: We rephrased as commented.

 

Reviewer #1: Citation needed: “The database lists eight species for northeast tropical Africa, whereas more than 200 species are known for Africa”. This statement needs a citation

Response: Citation inserted as suggested.

 

Materials and methods

Reviewer #1: Figure 1. Distribution and sampling sites of Tetrigidae in Ethiopia.The current maps are very difficult to interpret due to the lack of conventional cartographic elements. I strongly recommend redrawing all maps using standard and easily recognizable layers, such as clear geopolitical boundaries, rivers, elevation (if relevant), and labeled key regions. Consider using a base map that improves geographic orientation and enhances clarity for an international audience. Including a legend, north arrow, and scale bar is also essential for proper interpretation.

Response: All the maps are redrawn as recommended.

Reviewer #1: Figure 9. The quality of the tree has to be improved. Names are very difficult to read.

Response: The quality of the phylogenetic tree and the species names are now improved.

 

regards,

author

Reviewer 2 Report

Comments and Suggestions for Authors

Materials and Methods

Sampling: please give the full name for EBI.

Phylogenetic analyses: please explain in which cases the manual correction of sequences was performed, and the criteria behind the decision to correct sequences.

Results

I cannot pronounce myself on the classic taxonomy because it is outside my expertise. However, I find that the photos in Figures 2, 3, 8 have insufficient quality/resolution. Please provide high quality/resolution photos.

DNA barcoding: please quantify the intra and interspecific divergence between sequences as maximum p-distance. Groups of sequences may form clusters but the condition of low intragroup genetic distance may not be met; in that case, your species inference may not be correct.

“Moreover, the phylogenetic tree also indicates clearly three monophyletic tribes (Tetrigini, Criotettigini and Xerophyllini).” – please highlight the tribes on the tree.

Figure 9: please italicize species names and improve the resolution of the tree.

Discussion

DMNA barcoding in Orthoptera is often difficult, as you mentioned in the Introduction. Please describe and discuss any difficulties that you may have faced with regards to PCR success including no amplification and multiple bands, sequencing success including ambiguous nucleotide positions, etc. In my experience, it is very difficult to obtain 100% success rate in Orthoptera using the Folmer primers.

Author Response

Reviewer #2

Dear reviewer, We value the effort and recommendations you provided for the improvement of our manuscript. I agree with the comments and suggestions you provided. The quality of the figures and tables has been enhanced as you recommended. Additionally, we employed the American Journal Experts/AJE language editing for clarity in the research paper; the certificate of language polishing is also provided.

 

Materials and Methods

Reviewer #2: Sampling: please give the full name for EBI.

Response: The full name for EBI was provided as Ethiopian Biodiversity Institute (EBI).

Reviewer #2: Phylogenetic analyses: please explain in which cases the manual correction of sequences was performed, and the criteria behind the decision to correct sequences.

Response: As we know, automated sequencing can sometimes produce incorrect/ambiguous base calls or even gaps or frameshift errors. In such cases, manual inspection could allow for correction of these errors by examining the underlying data, such as chromatograms, and making informed adjustments just after careful manual inspection of the sequence data. Then, we used Geneious Prime to align the sequences so that the software highlighted the discrepancies, and some areas needed manual correction using the tools interface in the same software. But, in our case, only base gaps have been removed manually, and one frameshift error sequence has corrected the later decision to come to edition after aligning with the reference sequences.

 

Results

Reviewer #2: I cannot pronounce myself on the classic taxonomy because it is outside my expertise. However, I find that the photos in Figures 2, 3, 8 have insufficient quality/resolution. Please provide high quality/resolution photos.

Response: Thanks!. Figures with low resolutions have been replaced with high resolution figures.

Reviewer #2: DNA barcoding: please quantify the intra and interspecific divergence between sequences as maximum p-distance. Groups of sequences may form clusters but the condition of low intragroup genetic distance may not be met; in that case, your species inference may not be correct.

Response: Thanks, really. These are also very important comments that we need to consider. We have included the intra and interspecific divergence between the sequences. The results are included as a supplementary table. Moreover, we also included three species delimitation methods to prove that these clustering would be due to intragenic distance.

Reviewer #2: “Moreover, the phylogenetic tree also indicates clearly three monophyletic tribes (Tetrigini, Criotettigini and Xerophyllini).” – please highlight the tribes on the tree.

Response: This is overlooked, thanks. Just the tribes were highlighted with different colors in the phylogenetic tree.

 

Reviewer #2:  Figure 9: please italicize species names and improve the resolution of the tree.

Response: All the species are italicized, and the figure was improved to a higher resolution.

 

Discussion

Reviewer #2: DMNA barcoding in Orthoptera is often difficult, as you mentioned in the Introduction. Please describe and discuss any difficulties that you may have faced with regards to PCR success including no amplification and multiple bands, sequencing success including ambiguous nucleotide positions, etc. In my experience, it is very difficult to obtain 100% success rate in Orthoptera using the Folmer primers.

Response: Dear, sure that there are many problems while studying with the orthopteran genome for which I have not yet encountered the problems for this study batch. However, much evidence has come to light as I am studying with a lot of grasshoppers from diverse origins of various species, and I encountered many difficulties, as you stated, starting from failure in PCR amplification to full sequence ambiguities, particularly Oedipodinae. However, it is dependent upon the type of primer selection and choice of polymerase; this is even more important even if we selected the right primer, which can be altered by the level of genome size, which is greatly different across taxa. However, for a batch, almost the success rate was 99% with very nice sequence quality; I encountered only one low-quality sequence that might be due to many contributing factors. That was why I did not discuss this in the manuscript.

 

Thanks,

Author

Reviewer 3 Report

Comments and Suggestions for Authors

Please find my comments in the attached file. Additionally, kindly pay closer attention to sentence structure and grammatical issues.

Comments for author File: Comments.pdf

Author Response

Dear reviewer, We value the effort and recommendations you provided for the improvement of our manuscript. I agree with all of the critical comments raised here below and tried to include all the comments and suggestions. We employed the American Journal Experts/AJE language editing for more clarity in the research paper. Additionally, the quality of the figures and tables has been enhanced as you recommended.

 

Reviewer #3: British or American English? (e.g., grey or gray?)

Response: This should have to follow the consistency of using American English as “gray.” The error has been resolved with the language edition, which was polished by American Journal Experts/AJE.

Reviewer #3: Correct spacing between paragraphs.

Response: corrected.

Reviewer #3: Page 3: Table 1 should be presented first, as it is mentioned first.

Response: Sure, table 1 was first mentioned.

Reviewer #3: Figure 1: The alignment of maps needs to be corrected; the scale below overlaps.

Response: The maps were aligned and changed to clearer maps as suggested by the other reviewers too.

Reviewer #3: Figure 1: The use of a dot or cross (⚫ or X) would be more visually appealing. In addition, the colour red makes for difficult viewing.

Response: Definitely, the suggestion is accepted, and all the maps were replaced with better, more informative, and more understandable ones.

Reviewer #3: Figure 1: Consistency in font size within the legend is crucial.

Response: Thanks. I agree that all the fonts are adjusted to consistency.

Reviewer #3: In text, “Figure 1A and B,” however; the legend only mentions “Figure 1.”

Response: Figure 1 is used for the reference within the text as suggested.

Reviewer #3: Headings for figures and tables should be correctly aligned.

Response: Really, all the headings for figures and tables were aligned; however, it might be interrupted by the journal's automatized submission format. I agree that the headings are aligned.

Reviewer #3: Table 1: Text size can be reduced so that column headings read correctly.

Response: The text size of Table 1 and 2 was reduced to fit the table column.

Reviewer #3: Collector names written out consistently, i.e. Tarekegn F. {OR} F. Tarekegn.

Response: I checked the name of the collector repeatedly; I didn't find any inconsistency.

Reviewer #3: ….but we introduce a new measure: the ratio, fastigium width/width of eyes… A comma should follow the term ratio, as well as in the subsequent sentence.

Response: This is resolved following language edition.

Reviewer #3: ….which has the difficulty in that the eyes are implanted obliquely on the head as seen in dorsal view.

Response: resolved following the language edition.

Reviewer #3: Page 7: “dected” change to detect.

Response: changed to detect

Reviewer #3: Page 8: …9° 22’40’’N, 37°25’12’’E, 19. VII. 2023, collector; Tarekegn F. leg. The term ‘collector’ is not consistently being used.

Response: The term ‘collector’ is consistently used.

Reviewer #3: Page 9: Figure 4 images and legend description does not correspond.

Response: The images and legend descriptions corresponded to the following comments.

Reviewer #3: Page 9: Description: Figure descriptions should follow a chronological order.

Response: ordered.

Reviewer #3: Page 10: Table 2: Comparison of Paratettix tanai and P. geminus…. Headings should be understandable when read alone. Therefore, the genus name should be written out fully before being abbreviated. In addition, all scientific names must be italicized.

Response: I changed the table heading to be more independent and easily understandable. The scientific names are italicized.

Reviewer #3: Page 11: Finally, Paratettix ruwenzoricus (Rehn, 1914) is clearly different as…. Consistency is required for author names of species, which should be placed in brackets with a comma separating the name and date.

Response: all the authors' names and years were used consistently as suggested.

Reviewer #3: Figure 5: Legend and images do not correspond.

Response: As commented, the legend and images are corresponded.

Reviewer #3: Page 12: Diagnosis: Description does not match images. Description: …total broadt of eyes + fastigium…total breadth?, Figures are not being referred to here.

Response: Figures with the corresponding descriptions are referred to.

Reviewer #3: Can Figure 6 not be moved to Figure 2? There is quite a bit of mention of Figure 6 in the text, prior to viewing the diagram.

Response: Because Figure 6 is a reference for both P. tanai and P. geminus, we preferred to refer to it after the description details, not very far. Figure 6 was mentioned two times before the figure but almost on the same page to bind the two new species' descriptions of key characters.

Reviewer #3: Page 14: The main identification characters for the genus Leptacrydium (Chopard 1945) in Ethiopia… Author names placed in brackets.

Response: placed in brackets.

Reviewer #3: Figure 7 heading: D) head and anterior pronotum, lateral view

Response: added.

Reviewer #3: Page 15: Images are not being referenced in the description.

Response: Figures are referred to in the description.

Reviewer #3: Page 16: ….in L. nNaqamteensis (Figure 7A-E), Again, there is mention of a Figure 1B (Distribution records), Even within a single species, they can be variable (e.g., Loxilobus bantu), antennae slender (15 segments including scapus and pedicle)…

Response: corrected and incorporated. However, I was unable to address what for antennae slender (15 segments including scapus and pedicle)… Would you clarify the question, please?

Reviewer #3: Please check consistency for the term ‘alae’ as I have noticed the spelling ‘alea’.

Response: checked, and I used only ‘alea’ consistency.

Reviewer #3: Suggestion: Frontal view can also be referred to as “anterior” view. This will coincide with the term’s “lateral” and “dorsal” view.

Response: Thanks for the suggestions; we used “anterior” view in place of frontal view.

Reviewer #3: In all Figure headings: the term ‘Body’ can be written in lowercase.

Response: the lowercase “body” is used consistently.

Reviewer #3: Page 17: The heading ‘DNA barcoding’ could be written as DNA barcoding analysis or results,  BS needs to be explained before abbreviated, Interestingly, both the Paratettix new species are distinct from each another… Mention the two species you are referring to, Suggestion: indicate the three tribes in Figure 9.

Response: “DNA barcoding” is written as “DNA barcoding results.” BS is defined first.The two new Paratettix species are mentioned.

Reviewer #3: Page 18: Figure 9 heading, the abbreviation for bootstrap is not provided,

Response: provided.

Reviewer #3: In Ethiopia, only a single species had been registered so far, P. meridionalis. (missing comma), This was only clear clarified after a genetical study of Ethiopian specimens, which are now described as Paratettix geminus sp. nov., which a species that is morphologically is very close similar to P. meridionalis. Another species, P. tanai sp. nov., is also new to science and could also likewise be mistaken for P. meridionalis.

Response: With the given comments and with the support of language editions made by the American Journal expert, all the necessary corrections are considered.

Reviewer #3: Page 19: We here Here, we sampled across a wider geographical range and identified the specimens based on both morphological and DNA barcoding data, Awkward sentence. We did not find P. meridionalis, but instead were able to discover two novel species P. tanai sp. nov. and P. geminus sp. nov., which are predominantly found in Ethiopia and both species coexist across varying geographical ranges from east to west and from north to south of Ethiopia. So far, we know nothing, about their distribution in other neighboring countries…

Response: all the necessary corrections are considered.

Reviewer #3: Please check format of “Figure xyz” as page 4 has “Fig.” instead.

Response: checked and corrected.

Reviewer #3: Page 20: Awkward sentences. We record Leptacrydium in Ethiopia for the first time, with one new species, L. naqamteensis sp. nov., currently the as so far only known species of this genus for the country. Leptacryidium naqamteensis sp. nov. also was also found coexisting with the two species of Paratettix described herein. Hence, it may be expected to find this species also in Ethiopia in the future as well, or to assign some of the other Eastern African populations to the newly described species.

Response: All comments are incorporated.

Reviewer #3: References supporting the distribution records of genera are lacking.

Response: Distribution records of the genus is supported with reference.

Reviewer #3: Page 21: Consider rephrasing the following sentences. A comparable approach to identify Tetrigidae species with some problem on the basis of COI gene sequences was used by Hawlitschek et al (2017) who successfully sequenced some species of Tetrigidae; Tetrix kraussi, Paratettix meridionalis, Tetrix depressa and Tetrix bipunctata from Europe. Supported by Backed with the morphological differences, we demonstrate the high value of integrative taxonomy for understudied cryptic species groups. Please reconsider sentence structure and wording for the conclusion.

Response: Comments are incorporated.

 

Thanks,

Author

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have incorporated the prescribed corrections and suggestions. The manuscript is now fit for publication and I have no further comments.