Taxonomy of Micronesian monitors (Reptilia: Squamata: Varanus): endemic status of new species argues for caution in pursuing eradication plans

In the light of recent phylogenetic studies, we re-assess the taxonomy and biogeography of the Varanus populations distributed in the Micronesian islands of Palau, the Western Carolines and the Marianas. Whether these populations are of natural origin or human introductions has long been contentious, but no study has fully resolved that question. Here, we present molecular and morphological evidence that monitor lizards of the Varanus indicus Group reached both Palau and the Mariana Islands sometime in the late Pleistocene and subsequently differentiated into two separate species endemic to each geographical region. One species is confined to the Mariana Islands, and for these populations, we revalidate the name V. tsukamotoi Kishida, 1929. The other species has a disjunct distribution in Palau, the Western Carolines and Sarigan Island in the Northern Marianas and is herein described as V. bennetti sp. nov. Both species are most closely allied to each other, V. lirungensis and V. rainerguentheri, suggesting that colonization of Micronesia took place from the Moluccas. We discuss the biogeographic distributions of both species in the light of the likely colonization mechanism and previous arguments for human introduction, and we argue that bounties for Palauan populations are ill-advised and plans for eradication of some other populations must first demonstrate that they are, in fact, introduced and not native.

Line 157-161 -I think a map with the locations of the samples used in this study would be very helpful.
Line 212-216 -See the comments above on the sample size and LDA. This may not matter because you have a more general problem regarding the sample size, but following the results of the LDA, it would is always good to follow up with cross-validation analysis and report classification error rates.
Line 253 -It seems to me that you need to mention here that you need to revalidate the name and better justify why you would need a redescription.
Line 252-255 -I am a bit confused here. Did you use any individuals from Saipan in the morphological analysis? How many?
Line 278-295 -Ideally you should have all these species from V. indicus group in your LDA, that way it would be much easier to identify potential overlap in the morphological characters. Also, I got a bit confused here because in lines 273-277 you compare with other species in V. indicus group and in the following section you compare with other species of Eupreipiosaurus. From my reading (Line 79) I was assuming these were the same thing. How are they different? What are exactly the species that belong to the V. indicus group?
Line 389-411 -The same comments as above applies to this section.
Line 477 -The term 'species delimitation' here may be misleading. This term refers to a more specific set of protocols and analysis that was not performed by the authors.
Line 502-510 -What are the contributions of your study regarding this discussion?

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments) Comments to the Author(s) I found this paper to be very clear and logical. The methods were well explained as were the analysis. The figures and tables were all very helpful. It was easy to read and well written. I found the (re)descriptions of the two species to be very clear and the diagnoses to be well supported. In the early 1990's when I first saw/collected the indicus on PNG, then Palau, and Guam it was clear they all looked different and I assumed were cryptic taxa. This is very great to finally see this work completed and done so carefully. Also, the indicus I saw on Kosrae in eastern FSM looked strikingly different from these other forms. Clearly the systematics of that set of eastern FSM populations is the next steps as outlined in their paper. Yap is interesting and lacks several taxa such as Cryptoblepharus that occur in the Marianas and Palau. The Varanus there could easily be introduced. Yap really doesn't share any close relationship to the Palau fauna based on our unpublished genetics of other taxa there such as Emoia atrocostata.
Overall I found the paper to be very clean and without much need of revision. Three small notes: 1. Line 59 spelling "revalidate" 2. References need a lot of clean-up 3. Figure 2, not sure how to interpret placement of rainerguentheri (ZFMK 85404) and Varanus sp. (AMSR 121569) Shortland as the same haplotype? That seems weird and not explanation given? This contrasts with placement on Figure 3.

24-Mar-2020
Dear Dr Weijola, The editors assigned to your paper ("Newly recognized endemism in Micronesian monitors (Reptilia: Squamata: Varanus) argues for caution in pursuing eradication plans") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. I thank the two expert reviewers for their constructive comments, and invite the authors to address/incorporate these comments in a revision.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s)

Review
Weijola and collaborators produced a taxonomical study of monitor lizards in Micronesia. The study describes two new species based on morphological and molecular evidence. There are a lot of issues that I think the authors need to consider before moving forward with publication. Below, I detail my criticisms in the hope to help the authors to revise their manuscript.
Major comments 1. The title suggests that a major component of the paper will be the implications of the taxonomic decisions in the conservation and management of invasive species. However, the introduction of the paper does not contextualize this topic and most of the paper reads like a typical taxonomical study. The issue appears only at the end of the discussion. The authors need to decide what is the emphasis they will carry more on the paper and adjust the manuscript accordingly.
2. Seems to me that this work overlaps a lot with the author's previous work (Weijola et al 2019). I think the authors need to be more detailed and upfront on what are the novel contributions of this study and what were conclusions from the previous study.
3. I am not convinced that the sampling and analysis of the morphological data is bringing robust conclusions about the differences among species (more details below) 4. The molecular evidence shows that the newly proposed species are monophyletic. However, the clade that contains V. bennetti, V. tsukamotoi and V. lirungensis is also monophyletic. Therefore, one could argue that they are all one species. In my opinion, the author's analysis did not show conclusively otherwise. Also, the authors need a statement highlighting that their results are coming from a very limited genetic dataset, of only mitochondrial DNA. It is well reported that results can change dramatically with the addition of more DNA sequences.

Minor comments
Line 70-72 -This is a bit confusing to me. The authors refer to the region as a hotspot of diversity but state that the diversity level for reptiles is lower when compared to other hotspots. The justification used by the authors would be because of the smaller area and isolation of islands. This seems plausible if the authors were justifying the overall diversity and not only reptiles.
Line 73 -Perhaps the lower diversity level of reptiles in the region compared to other hotspots is because there are many more reptiles species to be described than for other groups (e.g. mammals and birds).
Line 77 -What exactly are you referring by "here" in this sentence? It was not clear to me.
Line 78 -What do you mean by 'interesting biogeographic puzzles". This sentence is a bit vague.
Line 79 -How many species are currently recognized on the V. indicus group?
Line 92 -Could the fossil belong to a different group of Varanus? In that scenario, Varanus indicus could still be a recent introduction.
Line 132 -Why that subset of characters?
Line 131-135 -I must say that the sample sizes here for most species are very low for an LDA. The results of this analysis cannot be reliable.
Line 157-161 -I think a map with the locations of the samples used in this study would be very helpful.
Line 212-216 -See the comments above on the sample size and LDA. This may not matter because you have a more general problem regarding the sample size, but following the results of the LDA, it would is always good to follow up with cross-validation analysis and report classification error rates.
Line 253 -It seems to me that you need to mention here that you need to revalidate the name and better justify why you would need a redescription.
Line 252-255 -I am a bit confused here. Did you use any individuals from Saipan in the morphological analysis? How many?
Line 278-295 -Ideally you should have all these species from V. indicus group in your LDA, that way it would be much easier to identify potential overlap in the morphological characters. Also, I got a bit confused here because in lines 273-277 you compare with other species in V. indicus group and in the following section you compare with other species of Eupreipiosaurus. From my reading (Line 79) I was assuming these were the same thing. How are they different? What are exactly the species that belong to the V. indicus group?
Line 389-411 -The same comments as above applies to this section.
Line 477 -The term 'species delimitation' here may be misleading. This term refers to a more specific set of protocols and analysis that was not performed by the authors.
Line 502-510 -What are the contributions of your study regarding this discussion? I found this paper to be very clear and logical. The methods were well explained as were the analysis. The figures and tables were all very helpful. It was easy to read and well written. I found the (re)descriptions of the two species to be very clear and the diagnoses to be well supported. In the early 1990's when I first saw/collected the indicus on PNG, then Palau, and Guam it was clear they all looked different and I assumed were cryptic taxa. This is very great to finally see this work completed and done so carefully. Also, the indicus I saw on Kosrae in eastern FSM looked strikingly different from these other forms. Clearly the systematics of that set of eastern FSM populations is the next steps as outlined in their paper.
Yap is interesting and lacks several taxa such as Cryptoblepharus that occur in the Marianas and Palau. The Varanus there could easily be introduced. Yap really doesn't share any close relationship to the Palau fauna based on our unpublished genetics of other taxa there such as Emoia atrocostata.
Overall I found the paper to be very clean and without much need of revision. Three small notes: 1. Line 59 spelling "revalidate" 2. References need a lot of clean-up 3. Figure 2, not sure how to interpret placement of rainerguentheri (ZFMK 85404) and Varanus sp. (AMSR 121569) Shortland as the same haplotype? That seems weird and not explanation given? This contrasts with placement on Figure 3.

Decision letter (RSOS-200092.R1)
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Review
Weijola and collaborators produced a taxonomical study of monitor lizards in Micronesia. The study describes two new species based on morphological and molecular evidence. There are a lot of issues that I think the authors need to consider before moving forward with publication. Below, I detail my criticisms in the hope to help the authors to revise their manuscript.
Major comments 1. The title suggests that a major component of the paper will be the implications of the taxonomic decisions in the conservation and management of invasive species. However, the introduction of the paper does not contextualize this topic and most of the paper reads like a typical taxonomical study. The issue appears only at the end of the discussion. The authors need to decide what is the emphasis they will carry more on the paper and adjust the manuscript accordingly.
Reply: We have added additional data on this topic to the Introduction that makes the context clearer (lines 99-103, 130-132), and we have altered the title to make it clearer that the conservation implications are immediately important to address for these species but that the paper must necessarily be focused on taxonomy before the conservation matters can logically be addressed.
2. Seems to me that this work overlaps a lot with the author's previous work (Weijola et al 2019). I think the authors need to be more detailed and upfront on what are the novel contributions of this study and what were conclusions from the previous study. Weijola et al. (2019). Further, the present ms. includes a detailed morphological comparison of diagnostic differences among the Micronesian species and their closest relatives; such analysis was completely absent from Weijola et al. (2019). We make these points clearer in the last two paragraphs of the Introduction.

Reply: Yes, there is necessary overlap with the analyses and results of Weijola et al. (2019), but that work was focused purely on the molecular phylogenetics and biogeography of the whole subgenus Euprepiosaurus. In contrast, this current manuscript is a taxonomic and biogeographic revision of the subset of Varanus populations in Micronesia and can best be regarded as a taxonomic extension to that work. This ms. adds to the analyses of Weijola et al. (2019) two taxa absent from that work that are critical for understanding the evolution of the Micronesian species because they are both immediately basal to the Micronesian species. This fortifies and extends upon conclusions about evolutionary relationships and biogeography of the Micronesian species briefly mentioned in
3. I am not convinced that the sampling and analysis of the morphological data is bringing robust conclusions about the differences among species (more details below). Fig  1a) tail/body proportions., and coloration differences. These mensural, meristic, and color-pattern features are-alone or in combination-diagnostic for species across the V. indicus Group. This same combination of features is diagnostic of our two species, consonant with the same conclusions derived from the molecular data, and of the same magnitude and general type as seen between most other recognized species within the V. indicus group. The sample sizes are actually quite good for Varanus species (especially for V. bennetti and V. tsukamotoi), and we have included as many specimens as was practically possible. We respond to this expressed concern in more detail below as the reviewer makes more specific comments.

Reply: The morphological differences between the proposed species (Varanus bennetti and V. tsukamotoi) are clear and significant, particularly in scalational characters, (see
4. The molecular evidence shows that the newly proposed species are monophyletic. However, the clade that contains V. bennetti, V. tsukamotoi and V. lirungensis is also monophyletic. Therefore, one could argue that they are all one species. In my opinion, the author's analysis did not show conclusively otherwise. Also, the authors need a statement highlighting that their results are coming from a very limited genetic dataset, of only mitochondrial DNA. It is well reported that results can change dramatically with the addition of more DNA sequences. Reply: The reviewer suggests that since V. bennetti, V. lirungensis and V. tsukamotoi group together as a monophyletic clade, they could as well be called just one species. It is true that diagnosing species (i.e., identifying those characters and their geographic distribution that suggests evolutionarily independent lineages) is always a hypothesis and sometimes somewhat subjective. For that reason scientists should always carefully evaluate all available evidence. To make our interpretation as solid as possible we have used an integrative approach using a wide set of characters, basing our species boundaries on concordance between both molecular and morphological evidence.
But the reviewer seems especially swayed by molecular evidence. In this case, our phylogenetic results based on molecular data indicate that our specimens of each species form three separate clades in both parsimony and maximum likelihood analyses. Nodal support values for these three species are high (JF=99/BS=99 for V. bennetti; JF=97/BS=100 for V. lirungensis and JF=94/BS=93 for V. tsukamotoi), and each species is supported by a number of synapomorphic characters (6, 4 and 4 respectively). These results show that the available molecular evidence supports the distinction of these three species.
Since both lines of evidence (molecular and morphological) clearly show that these three evolutionary lineages form cohesive and diagnostic groups that are different from each other, it is well justified to call them separate species. The reviewer suggests that we disregard this evidence and consider these three lineages as populations of a single species, but this approach would not follow good scientific practice nor common sense. Furthermore, there is more to species recognition than just molecules, and we would be curious as to what characters the reviewer would use to diagnose this alternative "species". It would contain nothing but a hodgepodge of morphological features, making it undiagnosable vis-a-vis the remaining members of the V. indicus Group. And it makes no sense at all to postulate that a single, rarely dispersing lineage represents a single species across ca. 1600 km of ocean containing only sparsely distributed small islands. Further, the reviewer's suggestion is that all these populations represent V. lirungensis, yet the populations we describe as new do not fit the diagnosis for that species. And cramming their morphological diversity into a revised so-called "V. lirungensis" would require such an expanded morphological diagnosis of V. lirungensis that it would also require that most or all other species in the V. indicus Group be subsumed in the same "species", bringing us back to the state of taxonomic understanding of several decades ago, to wit: a single wideranging V. indicus. Our dataset includes two mitochondrial genes with a total of 1000bp, this is stated on lines 175-176 and lines 199-202. We see no need to emphasize this again elsewhere in the ms., and we don't see that this is a limitation of our study inasmuch as taxa of the V. indicus Group are closely related (having diverged in only the past 1.3 MY), and nuclear genes would be unlikely to add much useful information to our study, given their slower evolutionary rates.

Minor comments
Line 70-72 -This is a bit confusing to me. The authors refer to the region as a hotspot of diversity but state that the diversity level for reptiles is lower when compared to other hotspots. The justification used by the authors would be because of the smaller area and isolation of islands. This seems plausible if the authors were justifying the overall diversity and not only reptiles.
Reply: In this paragraph we state that the region has been classified as one of the world's biodiversity hotspots by Conservation International and go on to discuss how this relates to reptiles. We find this to be a logical and relevant introduction to a manuscript describing two endemic species from this region. We suspect that the reviewer might not have noted the citation and therefore think that we ourselves have identified the region as a biodiversity hotspot.
Line 73 -Perhaps the lower diversity level of reptiles in the region compared to other hotspots is because there are many more reptiles species to be described than for other groups (e.g. mammals and birds).
Reply: Incorrect. Some of CI's hotspots have high reptile diversity; others have low reptile diversity. In this case, the Polynesia/Micronesia hotspot has low reptile diversity because of the small land areas involved, the remoteness of these islands to source regions, and the simplified habitats available on many of the islands (see lines 70-77). CI's hotspots were defined solely on the basis of endemic plant diversity and degree of human threat to habitats. Hence, whether reptile diversity is high or low in these areas had no bearing on their recognition and is a separate issue than the fact that they are recognized as hotspots.
Line 77 -What exactly are you referring by "here" in this sentence? It was not clear to me.
Reply: We refer to Palau as a continuation to the previous sentence. To avoid confusion we changed "here" to "Palau".
Line 78 -What do you mean by 'interesting biogeographic puzzles". This sentence is a bit vague.