Dietary Variation Is Driven by Landscape Heterogeneity in an Insular Omnivorous Endemic Lizard, Revealed by DNA Metabarcoding

Round 1

Reviewer 1 Report

General comments

In this work, Rato and colleagues use DNA metabarcoding and next generation sequencing to characterise the diet of Teira dugesii within its indigenous range on the Madeira archipelago. They found evidence for strong tendencies towards herbivory in this insectivorous species, with males having larger and heavier body sizes consuming the most plant matter. In addition, due to the heterogenous landscape of Madeira archipelago, source population had a strong influence on diet composition.

I really enjoyed reading this manuscript. The methodology was particularly sound and very clearly described. The introduction clearly introduced the topic and described previous literature. The discussion was similarly excellent, placing the results nicely into context with previous work. The only addition I would suggest is perhaps how your results may influence management of this species if it is required at all – whether the species has a threat status or is even overabundant on the archipelago. Alternatively, a discussion of the importance of your results, even if it may simply be extending our knowledge of herbivory in island lizards. Some minor grammar corrections and requests for clarification below.

Detailed comments

Title: The title is a little bit convoluted. Suggest bringing the subject forward to something like this: “Dietary variation is driven by landscape heterogeneity in an omnivorous endemic lizard”

L26: Are plants considered prey? Perhaps change this to diet item occurrences? Check throughout.

L75-76: Change to “they can afford to spend time eating and digesting plants”.

L81: increase gut passage time?

L83-84: Remove the line break here.

L90: Change to “a wide array of habitats” or similar.

L102-103: Change to T. dugesii for consistency with above.

L103-104: Remove line break and combine with above paragraph as still the same idea.

L107: Change to “relationship between herbivory and size”.

L110-112: But you did mention some previous next generation sequencing above? Was that done for this species or another?

L112-113: Number these references for consistency.

L232: Define OTU.

L319: Change this to “varied’. Check tense throughout the results.

L364: Change this to “PerMANOVA” – the acronym was already defined above.

Figure 3b: Add the unit of measurement to x-axis label – e.g., Weight (g).

Figure 6: Make the legends larger, they are too small to read at present.

L559: Bite force?

Supplementary Materials: Include informative figure legends for Figures S1-3.

Author Response

Reviewer #1

 

General comments

In this work, Rato and colleagues use DNA metabarcoding and next generation sequencing to characterise the diet of Teira dugesii within its indigenous range on the Madeira archipelago. They found evidence for strong tendencies towards herbivory in this insectivorous species, with males having larger and heavier body sizes consuming the most plant matter. In addition, due to the heterogenous landscape of Madeira archipelago, source population had a strong influence on diet composition.

I really enjoyed reading this manuscript. The methodology was particularly sound and very clearly described. The introduction clearly introduced the topic and described previous literature. The discussion was similarly excellent, placing the results nicely into context with previous work. The only addition I would suggest is perhaps how your results may influence management of this species if it is required at all – whether the species has a threat status or is even overabundant on the archipelago. Alternatively, a discussion of the importance of your results, even if it may simply be extending our knowledge of herbivory in island lizards. Some minor grammar corrections and requests for clarification below.

R: We thank and appreciate the positive feedback. According to IUCN, Teira dugesii is listed as Least Concern, and with a stable population trend and this category will remain in the updated, which is currently being elaborated. The only significant threat would be the introduction of alien competitors or predators. Beyond preventing them, no other management measure for this species is required. Therefore, the results obtained here support and extend our knowledge on herbivory in insular lizard species.

 

Detailed comments

Title: The title is a little bit convoluted. Suggest bringing the subject forward to something like this: “Dietary variation is driven by landscape heterogeneity in an omnivorous endemic lizard”

R: We think that providing the source of the diet data, informs on the quality of the evidence. Hence, and at the same time following the reviewer’s suggestion the title was changed to “Dietary variation is driven by landscape heterogeneity in an insular omnivorous endemic lizard revealed by DNA metabarcoding”.

 

L26: Are plants considered prey? Perhaps change this to diet item occurrences? Check throughout.

R: Corrected, as suggested.

 

L75-76: Change to “they can afford to spend time eating and digesting plants”.

R: Corrected, as suggested.

 

L81: increase gut passage time?

R: Correct. “time” was added to the end of the sentence.

 

L83-84: Remove the line break here.

R: Line break removed, as suggested.

 

L90: Change to “a wide array of habitats” or similar.

R: Corrected, as suggested.

 

L102-103: Change to T. dugesii for consistency with above.

R: We understand the reviewer point of view but whenever a genus name appears in the beginning of a sentence, it must be written in full. Hence, we will keep it as it is.

 

L103-104: Remove line break and combine with above paragraph as still the same idea.

R: Line break removed, as suggested.

 

L107: Change to “relationship between herbivory and size”.

R: Changed, as suggested.

 

L110-112: But you did mention some previous next generation sequencing above? Was that done for this species or another?

R: Yes, there is the study from Neves, et al. (2022) on introduced T. dugesii from Azores. However, they were mainly interested in detecting sea bird prey items to assess the impact of T. dugesii on bird colonies. They found plants, and invertebrates as well, but no further results are provided regarding richness or composition of the diet.

 

L112-113: Number these references for consistency.

R: Corrected, as suggested.

 

L232: Define OTU.

R: Defined, as suggested.

 

L319: Change this to “varied’. Check tense throughout the results.

R: Corrected, as suggested and tense was checked across the results for consistency.

 

L364: Change this to “PerMANOVA” – the acronym was already defined above.

R: Changed, as suggested.

 

Figure 3b: Add the unit of measurement to x-axis label – e.g., Weight (g).

R: Added, as suggested.

 

Figure 6: Make the legends larger, they are too small to read at present.

R: Size of the legends increased.

 

 

L559: Bite force?

R: Yes, it was a mistake. We meant “bite force”. Corrected, accordingly.

 

Supplementary Materials: Include informative figure legends for Figures S1-3.

R: These were provided in the Supplementary Materials section, after the Conclusions, following the journal’s format.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript titled “Landscape heterogeneity in an oceanic island translated into dietary variation revealed by metabarcoding in an omnivorous endemic lizard”  by Rato et al. analyzes the diet of the Teira dugesii on the Madeira island in different environmental contexts, using DNA metabarcoding, a more reliable and objective approach than the morphological methods for animal diet identification but not without bias, 

 

The manuscript is on the whole well written and organized. Also figures are properly prepared.

 

Here are some of my suggestions:

 

 

Line 70: The authors could broaden the panorama of adaptations also considering whole insular conditions (eg. https:// doi.org/10.1371/journal.pone.0224607)

 

Lines 72-73: This also through adaptations to the gut microbiota level (e.g., https://doi.org/10.1038/s41598-022-16955-0; https://doi.org/10.1242/jeb.138370)

 

Line 112: Carretero, 2004; 112 Carretero et al., 2001 should be reported with the same format of all the references. Maybe this is an error of the automatic system for references elaboration.

 

Line 113-114: The authors should report here that the molecular methods for 8https://doi.org/10.1111/mec.15903), although they are preferable to morphological methods.

 

Line 156: Did the authors estimated the age of the individuals to ensure they were all adults? The size can be conditioned by the achievement of sexual maturity.

 

What happened to the captured individuals? They have been released? It would be better to specify it.

 

Line 187-188: Could the authors specify whether each sample was amplified separately with each of the two primer pairs or whether the plant and animal primers were used together to amplify the same sample.

 

Line 221: On what basis did the authors set the threshold at 26?

 

Line 317: Could the authors report some statistics on the sequencing data? (e.g. number of raw reads obtained, number of trimmed reads, number of items that did not find a taxonomic assignment, etc).

 

Line 319: The authors could also report the standard deviation since these are average values.

 

The authors have produced a useful map in Fig.1 which describes the different vegetation typologies on the island. It would be better to provide a brief but better description of possible animal prey for lizards on the island as well.

 

Lines 375-376: Didn’t the sequencing yield any sequences attributable to Teira dugesii? The authors use universal primers for vertebrates and fail to use a blocking primer for the Teira dugesii DNA, so it was expected that a certain amount of emerged sequences would be assigned to the analyzed species.

 

Figure 6 caption and in the main text: Authors could prefer other term that “prey” when they refers to plant items.

 

Line 449: Maybe “de” is a type error. Did the author intend “the”?

 

Line 488: “and”?

 

Lines 510-511: The authors could report some examples of items recognized with their metabarcoding analysis that escaped from the morphological analysis because it was probably soft bodied items. This would give further support to the molecular method over the morphological one.

 

Lines 563 - 579: There are many concepts repeated elsewhere in the discussion. The authors could try to integrate this part better into the discussion.

Author Response

Reviewer #2

 

This manuscript titled “Landscape heterogeneity in an oceanic island translated into dietary variation revealed by metabarcoding in an omnivorous endemic lizard”  by Rato et al. analyzes the diet of the Teira dugesii on the Madeira island in different environmental contexts, using DNA metabarcoding, a more reliable and objective approach than the morphological methods for animal diet identification but not without bias, 

 

The manuscript is on the whole well written and organized. Also figures are properly prepared.

R: Thank you for the positive feedback.

 

Here are some of my suggestions:

 

Line 70: The authors could broaden the panorama of adaptations also considering whole insular conditions (eg. https:// doi.org/10.1371/journal.pone.0224607)

R: A new sentence has been added referring to the several changes observed in insular taxa in comparison to their mainland ancestors, citing the reference given as example.

 

Lines 72-73: This also through adaptations to the gut microbiota level (e.g., https://doi.org/10.1038/s41598-022-16955-0; https://doi.org/10.1242/jeb.138370)

R: Thank you for the suggested references. These were added to line 82, where we refer on the intestinal helminth communities.

 

Line 112: Carretero, 2004; 112 Carretero et al., 2001 should be reported with the same format of all the references. Maybe this is an error of the automatic system for references elaboration.

R: The Reviewer is correct and the mistaken was corrected.

 

Line 113-114: The authors should report here that the molecular methods for (https://doi.org/10.1111/mec.15903), although they are preferable to morphological methods.

R: Thank you for the suggested reference. This was added to the sentence.

 

Line 156: Did the authors estimated the age of the individuals to ensure they were all adults? The size can be conditioned by the achievement of sexual maturity.

R: All individuals used in this study were adults. In fact, this was missing from the methods section and was now described. The females used here had a SVL between 47.93 and 72.14 mm, and males between 50.27 and 79.96 mm, which are within the size intervals of adults for this species (see Galán and Vicente, 2003).

 

Galán, P., and Vicente, L. (2003). Reproductive characteristics of the insular lacertid Teira dugesii. Herpetological Journal 13(3), 149-154.

 

What happened to the captured individuals? They have been released? It would be better to specify it.

R: Yes, all captured individuals were later released in the same place of capture. This was now added to the text.

 

Line 187-188: Could the authors specify whether each sample was amplified separately with each of the two primer pairs or whether the plant and animal primers were used together to amplify the same sample.

R: The samples were amplified separately for each primer pair. This is now described in the methods. Nevertheless, the description of two different PCR cycles implies that the amplification was done separately.

 

Line 221: On what basis did the authors set the threshold at 26?

R: This threshold was defined following Andújar et al. (2018) and Martins et al. (2022). These references were now added to the text.

 

Andújar, C., Arribas, P., Gray, C., Bruce, C., Woodward, G., Yu, D.W., et al. (2018). Metabarcoding of freshwater invertebrates to detect the effects of a pesticide spill. Molecular Ecology 27(1), 146-166.

 

Martins, B., Silva-Rocha, I., Rocha, R., Mata, V.A., Gonçalves, Y., and Rato, C. (2022). Trophic interactions of an invasive gecko in an endemic-rich oceanic island: insights using DNA metabarcoding. Frontiers in Ecology and Evolution In press.

 

Line 317: Could the authors report some statistics on the sequencing data? (e.g. number of raw reads obtained, number of trimmed reads, number of items that did not find a taxonomic assignment, etc).

R: The Reviewer is correct, and that data is now described in the results.

 

Line 319: The authors could also report the standard deviation since these are average values.

R: Standard deviations are now provided.

 

The authors have produced a useful map in Fig.1 which describes the different vegetation typologies on the island. It would be better to provide a brief but better description of possible animal prey for lizards on the island as well.

R: The Reviewer is correct but unfortunately that information is not available as a geographic shapefile. Moreover, in Madeira Island the endemic taxa of the Animal kingdom alone accounts for 1189 species, which are all potential prey for T. dugesii. That amount of information would be very hard to be displayed in a map or text.

 

Lines 375-376: Didn’t the sequencing yield any sequences attributable to Teira dugesii? The authors use universal primers for vertebrates and fail to use a blocking primer for the Teira dugesii DNA, so it was expected that a certain amount of emerged sequences would be assigned to the analyzed species.

R: The Reviewer is correct and that information is now provided in the first paragraph of the results.

 

Figure 6 caption and in the main text: Authors could prefer other term that “prey” when they refers to plant items.

R: True, see textbook by Krebs that explicitly separates herbivory from predation. The term “prey” was changed to “diet item” when referring to plants.

 

Krebs, C.J. 2014. Ecology: the experimental analysis of distribution and abundance (6th int. ed.). Pearson, Harlow, UK

 

Line 449: Maybe “de” is a type error. Did the author intend “the”?

R: Indeed, this was a typo which is now corrected to “the”.

 

Line 488: “and”?

R: This was another typo. We meant “…, and Porto,…”. Corrected.

 

Lines 510-511: The authors could report some examples of items recognized with their metabarcoding analysis that escaped from the morphological analysis because it was probably soft bodied items. This would give further support to the molecular method over the morphological one.

R: The author is correct, and the best example is the capacity of the molecular methods to assign plants up to the species level (or any taxonomical level, for that matter). This was now added to text.

 

Lines 563 - 579: There are many concepts repeated elsewhere in the discussion. The authors could try to integrate this part better into the discussion.

R: After careful reading, we cannot find any repeated concepts between these text lines and the remaining discussion, and so we have not changed this part of the text.

Author Response File: Author Response.pdf