A New Species of Tachymenoides (Serpentes: Dipsadidae: Tachymenini) from Peru with Comments on the Taxonomic Status of Galvarinus tarmensis (Walker, 1945)^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present manuscript aims to describe a new species of a poorly known genus of snake endemic to Peru, Tachymenoides goodallae (tribe Tachymenini). This is the third species described for the genus; the second species was described in 2023, 127 years after the first description, Tachymenoides affinis (Boulenger, 1896). 

 The authors present a detailed description of the new species and the environment where it occurs, based on morphology and genetic data, also providing comparisons with other species of the genus and related taxa.

The strongest point of the manuscript is to present a phylogenetic analysis, attesting the validity of the new species.

 It is a very important contribution, once it helps to describe the diversity of a group with significant information deficiency, and with a complex and intricated taxonomical history, as it occurs with other genera within Tachymenini. 

 My comments and suggestions intent to improve the quality of the manuscript, so that the information and characters are described as clear as possible, to avoid future taxonomic issues, and to prevent confusion with species diagnosis in an already problematic group.

General concept comments

I have some concerns about the distinctiveness of the new species in relation to Galvarinus tarmensis, and, most importantly, about the insufficiency of data of the species hemipenis.

 Already on the abstract (line 12), the authors stated that the new species is “phylogenetically distinct from Galvarinus tarmensis”; however, G. tarmensis is not included in their phylogenetic analyses, therefore that affirmation cannot be made.

 Regarding the morphological characters, the authors mention the number of dorsals (19/17/15) as an important diagnostic character for T. goodallae, including that as a distinction from the genus Galvarinus (Lines 345-346). However, as seen in table A2, there is a considerable variation, which could weaken the species diagnosis in relation to Galvarinus; unless properly explored and discussed. I recommend that the authors present this variation as the percentage of specimens with 19/19/15, 19/18/15 or 19/17/15. Also, it will be helpful to include a table with the main characters (Ventrals, dorsals, subcaudals, etc) for the three species of Tachymenoides. Likewise, I suggest that the authors present, in their results or discussion section, a comparison focused on the differentiation of the new species in relation to other species of Tachymenini that occur in Peru, informing if they are (or could be) sympatric or not.

The head morphometry could be a good indicative of species/genus differentiation, as it was briefly stated in Figure 12. Although the authors mentioned that the generic affiliation of Galvarinus tarmensis will be discussed in a future publication and, since this species is officially known only by its holotype, it would be important to detail what are the characteristics that are being considered as diagnostic for G. tarmensis; and to explain why the authors are considering the specimen MUSM 38469 (represented in Figure 12) as G. tarmensis.

 Regarding the hemipenis, there is an overall deficiency on its description, with some important points that should be addressed concerning hemipenial terminology.

Some hemipenial characters are mentioned as diagnostic, but there is no image of the fully everted organ. Since there are hemipenes available (among 17 males analyzed), and once there are very few complete hemipenial descriptions published for Tachymenini species, and only one for Tachymenoides, I think it is essential to adequately illustrate the new species hemipenis. I recommend including photographs of the prepared hemipenis of the holotype, or, if not possible, to at least prepare and illustrate the hemipenis of one of the 17 males available (Figure 7 should be redone); preferably more than one, since some degree of variation can occur. Additionally, I disagree with the following diagnostic hemipenial character: in line 314 the authors affirm that “hemipenes are densely covered with large calyces”. The calyces are not present on every portion of the hemipenis, they concentrate on the hemipenial body and on some portions of the capitulum. Again, a photograph or representation of the fully everted hemipenis would facilitate structure visualization, and would allow to highlight these diagnostic features, such as the large calyces on the hemipenial body.

 Some of the hemipenial structures described in the “Description of the holotype” section (lines 367-370) appear to be preparation bias, especially considering that the description is being based on a not fully everted organ. The authors should be very careful with that, to avoid further confusion with species diagnosis, as it is a recurrent problem within the tribe Tachymenini. The description should be based on a prepared, fully everted hemipenis.

 The authors mention that T. affinis and T. goodallae both have hemipenes with large calyces on the asulcate and lateral side of the capitulum (Line 327). However, the calyces are different for each species (in T. goodallae it seems that the calyces are wider and deeper), and the larger calyces are present on the hemipenial body of the asulcate side, not the capitulum. When the authors mention T. affinis hemipenis, what specimen are they referring to? Is it a hemipenis prepared by the authors or is it a specimen from the literature? Please indicate that in the text.

 Additional and specific comments are detailed below, indicated by the line of the text to which they refer to.

 Abstract. The abstract is incomplete, the authors could include a few lines with an overall discussion (main conclusion) in this section.

 Introduction. The introduction is also incomplete and not well-structure. There should be a brief introduction of the tribe, especially regarding closely related genera and species of Tachymenini occurring in Peru, also underlining the underestimated diversity and taxonomical problems within the group (some missing references on Tachymenoides could be added).

 Discussion. I suggest that the authors review their discussion section. There is some information that should be addressed as results, and there should be a more detailed discussion on the diversity and taxonomy of Tachymenoides and other related Tachymenini.

Line 15 and 16. “ventrals 139‒157, subcaudals 52‒67 in, ..” It seems there is a few words missing here.

Line 17 and 1. I would refrain to use the affirmation that the presence of uncalcified eggs indicate viviparity. That is not always necessarily true. The presence of embryos would be a better indicative of viviparity and, as it seems in Figure 11, it looks like there are embryos present. The authors should highlight that in the text and in the figure.

Line 34. The maps (Figure 1 and 2) should appear after the species description (after the new name appears for the first time in the text).

Line 115 – Figure 2. There is no explanation of Figure 2 along the text, and no mention of how the figure was made. What was the software/model used for species distribution? I recommend that the authors further explain and discuss the figure along the text, or just remove it.

Line 208. Question: In the materials and methods section the authors inform that only the gene 12S was sequenced for both their samples; but in line 208 (Figure 3), the authors state that the phylogenetic analysis was based on concatenated 12S and cytb. Did the authors include ctyb for their samples? How that missing data affected the analysis? That information should be included in the manuscript. 

Perhaps, if cytb sequences are not available for T. goodallae samples, the phylogeny should be based exclusively on 12S sequences.

Line 332. What is this first row of calyces? Is it on the capitulum or the hemipenial body? Proximally or distally? I recommend that the authors illustrate this character. 

Line 339. I consider the conditions of elliptical and vertical pupils to be the same. The “vertical” condition (as seen in Figure 4a of the paper describing Tachymenoides harrisonfordi – Lehr et al 2023) appears to be the elliptical condition contracted (highly illuminated environment). I would not consider that as a diagnostic character between T. harrisonfordi and T. goodallae.

Line 342. I did not understand the character “ventrolateral stripes… posterior to jaws”. Please rephrase or illustrate.

Line 352. Please rephrase or further discuss the sentence “usual complement of head scales present (Figure 6)”

Line 355. Remove the word “two”.

Line 364 and 365. It is not possible to visualize the hemipenis description in Figure 7. See comments on the hempenial morphology above.

Line 365. First row of what?

Line 398 and 399. How did you measure the hemipenis if it is not completely everted? If it was measured on the hemipenis from Figure 7, it cannot be considered.

Lines 401 to 419. Please review the paragraph “Coloration of holotype in life”. A few sentences are confusing (line 404 and 418).

Line 420 and 421. Please rewrite this sentence.

Line 425. Is it possible that males being larger and having more ventral and subcaudals is a sampling artifact? The N of males is considerably bigger than the number of females. I think it would be interesting to discuss that in the manuscript.

Line 448. Please refer the “dark brown lateral stripe” as the postocular stripe.

Line 451. It is not possible to see the yellowish tan ringlet in the specimens from Figure 8. That feature seems to be very common in other taxa, like species of Thamnodynastes and Dryophylax. I recommend reviewing this character.

Lines 602 to 608 should be in the results section, not discussion; or at least it should be rephrased.

Line 634 - Figure 12. Please correct figure legend: the letters for the second specimen are wrong: it should be “Galvarinus tarmensis (MUSM 38469 from Junín, D, E, F,…”

Line 674 (Table A2). Please indicate the meaning of all abbreviations (TaL is not indicated).

Author Response

We thank you very much for your detailed comments and suggestions that helped us improve our manuscript. In bold are our responses.

The present manuscript aims to describe a new species of a poorly known genus of snake endemic to Peru, Tachymenoides goodallae (tribe Tachymenini). This is the third species described for the genus; the second species was described in 2023, 127 years after the first description, Tachymenoides affinis (Boulenger, 1896). 

The authors present a detailed description of the new species and the environment where it occurs, based on morphology and genetic data, also providing comparisons with other species of the genus and related taxa.

The strongest point of the manuscript is to present a phylogenetic analysis, attesting the validity of the new species.

It is a very important contribution, once it helps to describe the diversity of a group with significant information deficiency, and with a complex and intricated taxonomical history, as it occurs with other genera within Tachymenini. 

My comments and suggestions intent to improve the quality of the manuscript, so that the information and characters are described as clear as possible, to avoid future taxonomic issues, and to prevent confusion with species diagnosis in an already problematic group.

General concept comments

I have some concerns about the distinctiveness of the new species in relation to Galvarinus tarmensis, and, most importantly, about the insufficiency of data of the species hemipenis.

Response: We agree that this was a weak point of our manuscript. To fix that, we have included molecular data of two specimens of G. tarmensis collected in Junin in our phylogeny to strengthen our species description and support the distinctiveness of our new species with this taxon. Therefore, we changed the title of the manuscript and we included as coauthors Jack W. Sites Jr. and Claudia Torres who worked on a separate manuscript about the taxonomic status of G. tarmensis. Our phylogeny reveals that G. tarmensis is different from our new species and is part of Tachymenis. Consequently, we move G. tarmensis back to Tachymenis. We have a single photo of one live specimen that we include as Fig. 4.

Already on the abstract (line 12), the authors stated that the new species is “phylogenetically distinct from Galvarinus tarmensis”; however, G. tarmensis is not included in their phylogenetic analyses, therefore that affirmation cannot be made.

Response: See our above comment, this sentence now makes sense.

Regarding the morphological characters, the authors mention the number of dorsals (19/17/15) as an important diagnostic character for T. goodallae, including that as a distinction from the genus Galvarinus (Lines 345-346). However, as seen in table A2, there is a considerable variation, which could weaken the species diagnosis in relation to Galvarinus; unless properly explored and discussed. I recommend that the authors present this variation as the percentage of specimens with 19/19/15, 19/18/15 or 19/17/15. Also, it will be helpful to include a table with the main characters (Ventrals, dorsals, subcaudals, etc) for the three species of Tachymenoides. Likewise, I suggest that the authors present, in their results or discussion section, a comparison focused on the differentiation of the new species in relation to other species of Tachymenini that occur in Peru, informing if they are (or could be) sympatric or not.

Response: We provided % for all scale variations for T. goodallae sp. nov. to the comparisons, and we overall improved the comparisons, and modified the discussion.

We added to comparisons: The dorsal scales in T. goodallae sp. nov. are mostly in 19/17/15 rows (61.9%, n = 13), followed by 19/19/15 (19.0%, n = 4), 19/18/15 (14.3%, n = 3), and 19/17/14 (4.8%, n = 1); see Table A2. Trevine et al. (2022) mention the dorsal scale row variation in Galvarinus as 19/19/15 (79%, n = 93), 19/19/17 (13%, n = 14), and one specimen in T. tarmensis as 19/19/13.

The head morphometry could be a good indicative of species/genus differentiation, as it was briefly stated in Figure 12. Although the authors mentioned that the generic affiliation of Galvarinus tarmensis will be discussed in a future publication and, since this species is officially known only by its holotype, it would be important to detail what are the characteristics that are being considered as diagnostic for G. tarmensis; and to explain why the authors are considering the specimen MUSM 38469 (represented in Figure 12) as G. tarmensis.

Response: We compared both of our T. tarmensis specimens with the holotype of T. tarmensis and came to the conclusion that our species identification is correct. For the specimen AMNH 117734, referred to as tarmensis by Trevine et al. (2022), we obtained photos from the AMNH, and we confirmed the species identification as T. tarmensis.

Regarding the hemipenis, there is an overall deficiency on its description, with some important points that should be addressed concerning hemipenial terminology.

Response: We agree that the single available hemipenis of the holotype is not fully everted, and our photos and description are not as good as it was done by Trevine et al. (2022), who used the fully everted hemipenis of MUSM 31152 (now our holotype) and described it in detail in Appendix S3 but without images. Unfortunately, this hemipenis disappeared from the collection. We find the description by Trevine et al. (2022) accurate, refer to that in the holotype description, and use our images of the right hemipenis as reference.

We added to the holotype description: Trevine et al. (2022) provided in their Appendix S3 a description of the right hemipenis of MUSM 31152 but did not provide a photograph): “The hemipenis is faintly bilobed with a short capitulum covered in papillated calyces on the lobes and pronounced spinulate calyces on the base of capitulum and distal portion of hemipenial body, extending onto half of the body on the asulcate side. Five rows of body spines that progressively get larger towards the base of the body, and first row with 9 spines encircling the body. Sulcus spermaticus divided at the base of capitulum; subtle constriction between body and base of the hemipenis, base of the organ covered with spines (smaller than those on the body)”. The right hemipenis, not fully everted, is pictured in Fig. 7 as a reference.

Some hemipenial characters are mentioned as diagnostic, but there is no image of the fully everted organ. Since there are hemipenes available (among 17 males analyzed), and once there are very few complete hemipenial descriptions published for Tachymenini species, and only one for Tachymenoides, I think it is essential to adequately illustrate the new species hemipenis. I recommend including photographs of the prepared hemipenis of the holotype, or, if not possible, to at least prepare and illustrate the hemipenis of one of the 17 males available (Figure 7 should be redone); preferably more than one, since some degree of variation can occur. Additionally, I disagree with the following diagnostic hemipenial character: in line 314 the authors affirm that “hemipenes are densely covered with large calyces”. The calyces are not present on every portion of the hemipenis, they concentrate on the hemipenial body and on some portions of the capitulum. Again, a photograph or representation of the fully everted hemipenis would facilitate structure visualization, and would allow to highlight these diagnostic features, such as the large calyces on the hemipenial body.

Response: We deleted our hemipenis description and quote the hemipenis description provided by Trevine et al. (2022) instead. See above.

Some of the hemipenial structures described in the “Description of the holotype” section (lines 367-370) appear to be preparation bias, especially considering that the description is being based on a not fully everted organ. The authors should be very careful with that, to avoid further confusion with species diagnosis, as it is a recurrent problem within the tribe Tachymenini. The description should be based on a prepared, fully everted hemipenis.

Response: We agree. Therefore, we deleted our hemipenis description and quoted the more accurate hemipenis description provided by Trevine et al. (2022) instead.

The authors mention that T. affinis and T. goodallae both have hemipenes with large calyces on the asulcate and lateral side of the capitulum (Line 327). However, the calyces are different for each species (in T. goodallae it seems that the calyces are wider and deeper), and the larger calyces are present on the hemipenial body of the asulcate side, not the capitulum. When the authors mention T. affinis hemipenis, what specimen are they referring to? Is it a hemipenis prepared by the authors or is it a specimen from the literature? Please indicate that in the text.

 Response: We have corrected as follows our comparisons: Tachymenoides goodallae sp. nov. and T. affinis are of similar size (maximum SVL 433 mm vs. 436 mm in T. affinis, [1]), and have hemipenes with large calyces restricted to the hemipenial body on the asulcate side. However, both species can be distinguished as follows: Tachymenoides goodallae sp. nov. has dorsal scale rows mostly in 19/17/15 (61.9%, n = 13) series (17/17/15 in T. affinis, 81.3%, n = 13, [1]), hemipenial calyces wider (narrower, Fig. S11 in [1] for CORBIDI 11791),

Additional and specific comments are detailed below, indicated by the line of the text to which they refer to.

Abstract. The abstract is incomplete, the authors could include a few lines with an overall discussion (main conclusion) in this section.

Response: we have revised the abstract regarding the changed manuscript title, additional research goals, and additional results.

Introduction. The introduction is also incomplete and not well-structure. There should be a brief introduction of the tribe, especially regarding closely related genera and species of Tachymenini occurring in Peru, also underlining the underestimated diversity and taxonomical problems within the group (some missing references on Tachymenoides could be added).

Response: We have expanded the introduction and included a short taxonomic history of Tachymenini and a newly added goal to resolve the taxonomic status of G. tarmensis.

Discussion. I suggest that the authors review their discussion section. There is some information that should be addressed as results, and there should be a more detailed discussion on the diversity and taxonomy of Tachymenoides and other related Tachymenini.

Response: We have improved the discussion accordingly.

Line 15 and 16. “ventrals 139‒157, subcaudals 52‒67 in, ..” It seems there is a few words missing here.

Response: nothing missing, we corrected and delete “in”.

Line 17 and 1. I would refrain to use the affirmation that the presence of uncalcified eggs indicate viviparity. That is not always necessarily true. The presence of embryos would be a better indicative of viviparity and, as it seems in Figure 11, it looks like there are embryos present. The authors should highlight that in the text and in the figure.

Response: We agree it was poorly worded. We have pointed out the presence of embryos in the figure and text.

Line 34. The maps (Figure 1 and 2) should appear after the species description (after the new name appears for the first time in the text).

Response: We do not follow. Because we mention both figures in the introduction, we keep both following the introduction.

Line 115 – Figure 2. There is no explanation of Figure 2 along the text, and no mention of how the figure was made. What was the software/model used for species distribution? I recommend that the authors further explain and discuss the figure along the text, or just remove it.

Response: We mentioned in Materials and methods hat ArcGis was used to design Figs 1 and 2 but we added more details to read “The maps were designed with ArcGIS (ESRI 2019. ArcGIS Desktop 10.8 version 10.7.0. Redlands, CA: Environmental Systems Research Institute) by J. C. Cusi”.

Line 208. Question: In the materials and methods section the authors inform that only the gene 12S was sequenced for both their samples; but in line 208 (Figure 3), the authors state that the phylogenetic analysis was based on concatenated 12S and cytb. Did the authors include ctyb for their samples? How that missing data affected the analysis? That information should be included in the manuscript. 

Perhaps, if cytb sequences are not available for T. goodallae samples, the phylogeny should be based exclusively on 12S sequences.

Response:  cytb sequences are not available, but c-mos sequences were included and terminals representing the new species have high support values and nested within Tachymenoides. We corrected the text and legend.

Line 332. What is this first row of calyces? Is it on the capitulum or the hemipenial body? Proximally or distally? I recommend that the authors illustrate this character. 

Response: On the base of the capitulum. We point out this character with a green arrow in Fig. 7C.

Line 339. I consider the conditions of elliptical and vertical pupils to be the same. The “vertical” condition (as seen in Figure 4a of the paper describing Tachymenoides harrisonfordi – Lehr et al 2023) appears to be the elliptical condition contracted (highly illuminated environment). I would not consider that as a diagnostic character between T. harrisonfordi and T. goodallae.

Response: Agreed. We deleted the shape of the pupil as diagnostic characters.

Line 342. I did not understand the character “ventrolateral stripes… posterior to jaws”. Please rephrase or illustrate.

Response: we have rephrased this sentence: the ventral coloration is predominately black with two ventrolateral stripes “extending from the angel of jaws caudally for 60 mm in length”.

Line 352. Please rephrase or further discuss the sentence “usual complement of head scales present (Figure 6)”

Response: we added and explained: usual complement of head scales present “consisting of one rostral, two internasals, three prefrontals, one frontal, one supraocular on each side of head, and two parietals“ (Figure 6)

Line 355. Remove the word “two”.

Response: Corrected, deleted “two”.

Line 364 and 365. It is not possible to visualize the hemipenis description in Figure 7. See comments on the hempenial morphology above.

Response: We have addressed the hemipenis description in our above comments.

Line 365. First row of what?

Response: We have deleted and updated our hemipenis description.

Line 398 and 399. How did you measure the hemipenis if it is not completely everted? If it was measured on the hemipenis from Figure 7, it cannot be considered.

Response: Agreed. We deleted the measurements.

Lines 401 to 419. Please review the paragraph “Coloration of holotype in life”. A few sentences are confusing (line 404 and 418).

Response: We agree and have rephrased several sentences to make the content easier to understand.

Line 420 and 421. Please rewrite this sentence.

Response: The ventral surfaces are pale gray and dark gray mottled, with the three narrow longitudinal stripes discernible but slightly faded out. We added red arrows to Figure 6 to indicate the ventral stripes.

Line 425. Is it possible that males being larger and having more ventral and subcaudals is a sampling artifact? The N of males is considerably bigger than the number of females. I think it would be interesting to discuss that in the manuscript.

Response: We added to our discussion: “Our species description is based on 17 males and 4 females, and conclusions regarding the larger size of males (total length 348‒559 mm, n = 17) compared to females (total length 291‒322 mm, n = 4) could be a potential sampling artifact.”

Line 448. Please refer the “dark brown lateral stripe” as the postocular stripe.

Response: we corrected.

Line 451. It is not possible to see the yellowish tan ringlet in the specimens from Figure 8. That feature seems to be very common in other taxa, like species of Thamnodynastes and Dryophylax. I recommend reviewing this character.

Response: We added, “All specimens have a brown iris with a distinct yellowish tan ringlet “(difficult to see in the small images in Figure 8).

Lines 602 to 608 should be in the results section, not discussion; or at least it should be rephrased.

Response: We agree. We have moved all comparisons from the discussion to comparative diagnosis.

Line 634 - Figure 12. Please correct figure legend: the letters for the second specimen are wrong: it should be “Galvarinus tarmensis (MUSM 38469 from Junín, D, E, F,…”

Response: we have corrected the figure legend: Tachymenis tarmensis (MUSM 38469 from Junín, D, E, F,

Line 674 (Table A2). Please indicate the meaning of all abbreviations (TaL is not indicated).

Response: Corrected, we added to the Table A2 legend “TaL is tail length”.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editors,

The framework of this article is complete, but there are several areas that require improvement. Specifically:

1. The phylogenetic relationships of the species were studied using a relatively short 12S sequence, which results in low support for the findings. It is recommended to include cytb sequences and reconstruct the phylogenetic relationships.
   Otherwise, you will need more morphological data to prove that the species is new, especially to find important distinguishing features from other species in the genus.
2. More evidence should be added to support the conclusion that it was viviparous.
3. There needs to be thorough discussion to clarify the relationship between Tachymenis tarmensis and the new species.
4. Additional detailed modifications are provided in the uploaded review manuscript.

Sincerely,

Comments for author File: Comments.pdf

Author Response

We thank you very much for your comments and suggestions, which have helped us improve our manuscript. Below are our responses in bold.

The framework of this article is complete, but there are several areas that require improvement. Specifically:

1. The phylogenetic relationships of the species were studied using a relatively short 12S sequence, which results in low support for the findings. It is recommended to include cytb sequences and reconstruct the phylogenetic relationships.
   Otherwise, you will need more morphological data to prove that the species is new, especially to find important distinguishing features from other species in the genus.

Response: we have added c-mos and included tissues for T. tarmensis to improve our phylogeny and strengthen our findings.

1. More evidence should be added to support the conclusion that it was viviparous.

Response: we paid closer attention to the eggs of two T. goodallae specimens and observed embryos. The new species is viviparous. We deleted the photos of eggs shown previously in Figure 10D because it is misleading and confusing.

1. There needs to be thorough discussion to clarify the relationship between Tachymenis tarmensis and the new species.

Response: With the inclusion of sequences for T. tarmensis and the change of the title of our manuscript, we have clarified the relationships between both taxa.

1. Additional detailed modifications are provided in the uploaded review manuscript.

Response: we address the comments below.

L13: Galvarinus tarmensis” appears abruptly in the abstract. Please provide a brief introduction to its relationship with the new species.

Response: We have modified our introduction and summarized the recent revision of Tachimenini and added the year of description for T. tarmensis.

L17: While there is some basis for this inference, it would be more robust with additional evidence, such as observations of other individuals or support from the literature

Response: We included tissues for T. tarmensis and the only photograph of a life specimen.

L84: The blue circles are not distinct enough; please modify it.

Response: we increased the contrast.

L125: cytb is needed in this article.

Response: not available, but we included c-mos resulting in an improved phylogenetic tree.

L127: Double-stranded PCR

Response: we corrected.

L128: 12S rRNA?

Response: We have corrected the entire method section.

L131: clearly?

Response: corrected: We replaced “cleanly” with “clearly”

L141: The shorter length of the 12S sequences further underscores the need for critical specimens' cytb sequences to be analyzed in this study.

Response: cytb is not available, but we included c-mos.

L147: Here, the number of male and female specimens used for morphological studies should be introduced.

Response: Accepted: we included the sample size for males and females.

L175: Please clarify this sentence to avoid any misunderstanding. You mean "Photographs of preserved specimens were taken while they were temporarily immersed in ethanol to minimize reflections"?

Response: corrected, we changed to read “The specimens (17 males, 4 females) were fixed…. ”.

L188: The support rate is low; it is recommended to add cytb sequences for concatenated analysis.

Response: cytb is not available, but we included c-mos.

L208: Phylogenetic analysis was based on concatenated 12S rRNA and cytochrome 208 b gene regions.

Response: We have changed the legend of Figure 3 as follows: Maximum likelihood (ML) tree showing relationships of the new species and other Tachymenini genera. Phylogenetic analysis was based on concatenated 12S, cytb, and c-mos gene regions. Bootstrap values are shown on nodes.

L546-548: Please make sure it's ovovivparous or viviparous?

Response: The new species is viviparous.

L585: Full name ‘Tachymenoides ’should be used in this instance, rather than abbreviations.

Response: corrected, we replaced “T.” with “Tachymenoides”.

L587: There is no logical connection between wooden snake and the nomenclature of the species.

Response: We completely modified the discussion.

L607: The number of specimens to be examined should be listed here

Response: the cited information for the specimens was taken from the literature, and the sample size was not presented in this reference. Consequently, we cannot correct it here.

L608-609: The use of personal communications as evidence does not sufficiently support this conclusion.

Response: Agreed, we included new coauthors to our manuscript and now can use tissues and a photos of T. tarmensis.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear editor,

I hereby present my considerations on the revised version of the manuscript: "A new species of Tachymenoides (Serpentes: Dipsadidae: Tachymenini) from Peru" (Manuscript ID taxonomy-3438241).

The authors have thoroughly addressed all corrections and suggestions, significantly improving the quality of the manuscript.

Nonetheless, I still have some reservations regarding the use of cytb in the analysis. Cytochrome-b was used exclusively for outgroup taxa (other Tachymenini), and was not sequenced for Tachymenis tarmensis and Tachymenoides goodallae. Could the missing data be influencing the topology? It would be helpful if the authors could present a version of the tree excluding the cytb sequences to assess how the missing data might impact the results. This tree could be presented as supplementary material, and could be used to improve the discussion.

Additionally, it would be highly beneficial to describe the intraspecific hemipenial morphology for Tachymenoides goodallae, particularly given the lack of comparisons or detailed descriptions for this genus. This information would greatly enhance the manuscript.

Also, I still strongly recommend that Figures containing maps be placed immediately following the first mention of the new species in the text. Furthermore, the quality of some of the images could be improved (contrast and brightness) (Figures 13 and 11). Figure 6 (the separate file) appears to be in lower resolution, please verify if the correct file format has been uploaded.

Additional minor corrections and suggestions are noted as comments or incorporated directly into the attached PDF file.

Comments for author File: Comments.pdf

Author Response

Dear reviewer, we appreciate your time revising our manuscript and are grateful for your suggestions, most of which we have accepted. See below our responses in bold.

The authors have thoroughly addressed all corrections and suggestions, significantly improving the quality of the manuscript.

Nonetheless, I still have some reservations regarding the use of cytb in the analysis. Cytochrome-b was used exclusively for outgroup taxa (other Tachymenini), and was not sequenced for Tachymenis tarmensis and Tachymenoides goodallae. Could the missing data be influencing the topology? It would be helpful if the authors could present a version of the tree excluding the cytb sequences to assess how the missing data might impact the results. This tree could be presented as supplementary material, and could be used to improve the discussion.

Response: We added the tree (Figure S1) and legend below as supplementary material. We comment on this tree without cytb in the Results and Discussion section. Overall, the new species is nested within Tachymenoides with high support as in the tree including cytb, but the position of T. tarmensis within Tachymenis has low support. We expect that addition of cytb sequences in a new phylogenetic analysis will corroborate not only relationships within Tachymenis but also increase support values.

Maximum likelihood (ML) tree showing relationships of the new species and other 
Tachymenini genera. Phylogenetic analysis was based on concatenated 12S and c-mos gene
regions. Bootstrap values are shown on nodes.

Additionally, it would be highly beneficial to describe the intraspecific hemipenial morphology for Tachymenoides goodallae, particularly given the lack of comparisons or detailed descriptions for this genus. This information would greatly enhance the manuscript.

Response: We agree that there is an overall lack of information regarding the hemipenis morphology in Tachimenini, and no males are known for T. tarmensis. However, it is not the scope of our manuscript to solve such problems.

Also, I still strongly recommend that Figures containing maps be placed immediately following the first mention of the new species in the text. Furthermore, the quality of some of the images could be improved (contrast and brightness) (Figures 13 and 11). Figure 6 (the separate file) appears to be in lower resolution, please verify if the correct file format has been uploaded.

Response: We have rearranged the figures and placed the maps next to the results. Furthermore, we increased the contrast and brightness for Figures 6, 11, and 13.

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Additional minor corrections and suggestions are noted as comments or incorporated directly into the attached PDF file.

Response: we have addressed all suggestions/comments marked in the PDF.

L39: This sentence is disconected with the rest of the paragraph.

Response: The new paragraph is an error and we connected this with the following sentence.

L61: “Glavarinus:

Response: we corrected the typo to read Galvarinus.

L182: Why cytb was included in the analysis once it was not sequenced for the new species and Tachymenis tarmensis?

Response: 12S and c-mos were sequenced for new species of Tachymenoides and T. tarmensis about ten years ago, and at that time CAP and JSW did not considered to sequence cytb.

L195/196: BYU DNA Sequencing Center.

Response: we changed to read “..the DNA Sequencing Center (DNASC) at Brigham Young University”.

L247: by J.C. Cusi.

Response: we deleted “by J.C. Cusi”.

L251: I recommend to change the generic abbreviation of Tachymenoides to “Ta.”  to avoid confusion with Tachymenis (T.)

Response: we follow this recommendation throughout the manuscript.

L352 and following: “collected”

Response: we inserted “collected” to all dates.

L435: Was this information collected based on which hemipenis? Did the authors prepared any hemipenes or do they refer to literature data? Please indicate that in the text.

Response: We added to the Methods: Hemipenial morphology follows Dowling and Savage [15] and Zaher [16] “and is based on two specimens (holotype MUSM 31152, paratype MUSM 18580) with (partially) everted hemipenes”.

L447: Figures 6 and 7

Response: we inserted Figures 6 and 7 here.

L454: delete “cloacal scale divided (usually divided)”,

Response: we deleted “cloacal scale divided (usually divided)”,

L465: “angel”

Response: we corrected the typo to read “angle”

L510: Head length of the specimen FMNH 5698?

Response: we added the head length (17.0 mm) to the figure legend.

L514: MUSM 31152.

Response: we added the number and deleted it in the parenthesis.

L517-519: usual complement of head scales present consisting of one rostral, two internasals, two prefrontals, one frontal, one supraocular on each side of the head, and two parietals

Response: We changed to read “head scales consist of one rostral, two internasals, two prefrontals, one frontal, one supraocular on each side of the head, and two parietals      ”

L530/31: insert “the”, delete parenthesis

Response: we corrected.

L567-569: dorsal, delete comma

Response: we corrected.

L573-576: Please rewrite this sentence. “the head has an irregularly shaped dark brown stripe running across the nasal, upper halves of loreal, preocular, between suture of postoculars, diagonally across the lower half of anterior temporal, upper half of 7th supralabial, and 8th supralabial to the corner of the mouth”

Response: We changed to read: “the head has an irregularly shaped dark brown canthal stripe covering the nasal, upper halves of loreal, and preocular; a dark brown postocular stripe extends between the suture of postoculars, diagonally across the lower half of anterior temporal, upper half of 7th supralabial, and 8th supralabial to the corner of the mouth”

L611/612: Is there a photograph of this hemipenis?

Response: we do not have a photograph. We added “partially” to everted hemipenes.

L618: Figure 8E.

Response: we referred to the wrong figure and corrected to Figure 10E.

L758: “sp. nov.”

Response: we corrected to read “Tachymenoides goodallae sp. nov.”

L828: “pholidosis”

Response: we replaced pholidotic with pholidosis.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I hope this message finds you well. I would like to express my appreciation for the significant improvements made in your manuscript; the results presented are now much more convincing.

However, there remain some minor imperfections. It is important to refine these areas to ensure the clarity and impact of your work are maximized.

1、Figure 2. The legend in the figure conflicts with the textual description below, making it difficult for readers to understand. Please revise and simplify to make it clear.

2、Although comparing the genetic distances of a certain gene sequence among species cannot be used as the sole criterion for determining species validity, it does have a certain utility and is a common practice in species classification research. It is recommended that the author include this information to assist readers in making their judgments.

3、In the Abstract, the authors may need to briefly introduce which gene segments were used and what analytical methods were employed.

4、It is preferable to unify the point shapes and colors in Figure 1 and Figure 2. The color of the points for the holotype specimens should ideally correspond to the color representing that particular species.

5、Regarding the 6th point location, Ingenio, are both species present there? Why? Please provide an explanation or possible reasons from the authors.

6、Is there a misidentification of Glavarinus tarmensis in historical specimens? The authors should briefly introduce how the new species mentioned in the text was previously considered to be Galvarinus tarmensis in Trevine et al.'s paper, and suggest adding a 'sensu stricto' description for the revised Thamnodynastes tarmensis.



Sincerely,

Comments on the Quality of English Language

L61 Glavarinus？

Author Response

Dear reviewer, we appreciate your time revising our manuscript and are grateful for your suggestions, most of which we have accepted. See below our responses in bold.

Dear Authors,

I hope this message finds you well. I would like to express my appreciation for the significant improvements made in your manuscript; the results presented are now much more convincing.

However, there remain some minor imperfections. It is important to refine these areas to ensure the clarity and impact of your work are maximized.

1、Figure 2. The legend in the figure conflicts with the textual description below, making it difficult for readers to understand. Please revise and simplify to make it clear.

Response: We have improved both maps (Figures 3 and 4) and made the figure legends easier to read.

• Figure 3. Map of Peru with the distribution of Tachymenoides goodallae nov. and Tachymenis tarmensis. The blue star is the type locality (Chacos) of Ta. goodallae sp. nov., the pink star is the type locality (Tarma) of T. tarmensis. Blue circles (1‒7) are collecting sites of paratypes of Ta. goodallae sp. nov.: 1 = Chacos, type locality (Region Pasco), 2 = Puagmaray (Region Pasco), 3 = road between Auquimarca and Uchuhuerta (Region Pasco), 4 = Auquimarca (Region Pasco), 5 = Huallamayo (Region Pasco), 6 = Ingenio (Region Junín), 7 = Pacchani (Region Puno). Pink triangles (1‒2) are our collecting sites of T. tarmensis: 1: Aco (Region Junín), 2: Pichjapuquio (Region Junín).
• Figure 4. Estimated distributional area based on the seven collecting sites of Tachymenoides goodallae nov. The distance (airline) from the northernmost (2) to the southernmost (7) collecting site is 782.27 km; the estimated distributional area (in green) is 3,6981.23 km². Bold numbers (1, 6; 1, 2) indicate collecting sites whose specimens were used for the phylogeny (Figure 1). The type locality (Tarma, pink star) of T. tarmensis and the collecting site of Ta. goodallae sp. nov. at Ingenio (blue circle 6) overlap; both localities are separated by 3.5 km airline.

2、Although comparing the genetic distances of a certain gene sequence among species cannot be used as the sole criterion for determining species validity, it does have a certain utility and is a common practice in species classification research. It is recommended that the author include this information to assist readers in making their judgments.

Response: We have estimated genetic distances on a 12S maximum likelihood tree using Species Delimitation plugin in Geneious Pro (see Materials and Methods). Genetic distance between the new species and Tachymenoides affinis (0.020) is similar to distance between this species and Ta. harrisonfordi (0.027), but genetic distance within new species is much lower (0.003). We have added this new information to the Results section. 

3、In the Abstract, the authors may need to briefly introduce which gene segments were used and what analytical methods were employed.

Response: Done. We added to the abstract: “A maximum likelihood phylogenetic tree based on two mitochondrial (12S and cyt-b) genes and one nuclear (c-mos) gene shows that” the new species is the sister taxon of T. affinis and distinct from Galvarinus tarmensis, which we transfer back to Tachymenis.

4、It is preferable to unify the point shapes and colors in Figure 1 and Figure 2. The color of the points for the holotype specimens should ideally correspond to the color representing that particular species.

Response: We agree, and have improved both maps (now Figures 3 and 4).

5、Regarding the 6th point location, Ingenio, are both species present there? Why? Please provide an explanation or possible reasons from the authors.

Response: We have added to Figure 3 legend following: The type locality (Tarma, pink star) of T. tarmensis and the collecting site of Ta. goodallae sp. nov. at Ingenio (blue circle 6) overlap; both localities are separated by 3.5 km airline.

6、Is there a misidentification of Glavarinus tarmensis in historical specimens? The authors should briefly introduce how the new species mentioned in the text was previously considered to be Galvarinus tarmensis in Trevine et al.'s paper, and suggest adding a 'sensu stricto' description for the revised Thamnodynastes tarmensis.

Response: We are not aware of historic misidentifications, at least not in the MUSM, except for our holotype, which was incorrectly identified as the first male of G. tarmensis. The other specimen (AMNH from Tarma) was correctly identified as G. tarmensis.

We already have pointed out in our introduction the misidentifications and morphological arguments used by Trevine et al. (2022), but we included now that MUSM 31152 was identified in the collection as Thamnodynastes sp.: “Trevine et al. [1] identified two additional specimens (AMNH 117734 a pregnant female from Tarma, Region Junín, Peru, and MUSM 31152 (identified in the collection as Thamnodynastes sp.) a male from Chacos, Region Pasco, Peru) as G. tarmensis based on “melanic coloration, smaller SVL size, and ventral and subcaudal counts”, and on hemipenal characters that are shared with the other members of Galvarinus such as the enlarged first row of calyces [1]. Trevine et al. [1] described the hemipenis for G. tarmensis based on MUSM 31152.

We added to the: “Furthermore, we show that the male specimen (MUSM 31152) from the Region Pasco, previously identified as G. tarmensis by Trevine et al. [1], represents a new species that is distributed in the regions of Pasco, Junín, and Puno. Twenty additional specimens of our new species were misidentified in the herpetological collection of the Natural History Museum of the San Marcos University (Lima, Peru) as Tachymenis affinis and T. peruviana.”

We refrain from providing a “sensu stricto” description because of the limited specimens we have, no males (= no hemipenis), and just a single photograph in life. Hopefully, future fieldwork will reveal more specimens to fill this knowledge gap.

Author Response File: Author Response.pdf