High-quality carnivoran genomes from roadkill samples enable comparative species delineation in aardwolf and bat-eared fox
Curation statements for this article:-
Curated by eLife
Summary: Collectively, we liked a lot about your paper and we would accordingly like to encourage its continued evolution. However, we felt that the approximately equal balance at present between the roadkill genomics assembly pipeline and the phylogenetic and genetic diversity results was not justified, and we requested a shift accordingly as described below. Second, we require several analytical updates to the manuscript to ensure robustness of the main genetic diversity results.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
In a context of ongoing biodiversity erosion, obtaining genomic resources from wildlife is essential for conservation. The thousands of yearly mammalian roadkill provide a useful source material for genomic surveys. To illustrate the potential of this underexploited resource, we used roadkill samples to study the genomic diversity of the bat-eared fox ( Otocyon megalotis ) and the aardwolf ( Proteles cristatus ), both having subspecies with similar disjunct distributions in Eastern and Southern Africa. First, we obtained reference genomes with high contiguity and gene completeness by combining Nanopore long reads and Illumina short reads. Then, we showed that the two subspecies of aardwolf might warrant species status ( P. cristatus and P. septentrionalis ) by comparing their genome-wide genetic differentiation to pairs of well-defined species across Carnivora with a new Genetic Differentiation index (GDI) based on only a few resequenced individuals. Finally, we obtained a genome-scale Carnivora phylogeny including the new aardwolf species.
Article activity feed
-
-
Reviewer #3:
The manuscript "High-quality carnivore genomes from roadkill samples enable species delimitation in aardwolf and bat-eared fox" is mostly well written and demonstrates an interesting and useful method for sequencing genomes from low-quality samples. They also provide a comprehensive overview of the state of genomics across the Carnivora clade, with some improved species/subspecies designations. I think the work is of broad interest. The analyses are mostly clear and I think a few additional analyses and small improvements could be made prior to publication, but otherwise have no issues.
The additional analyses/clarification I would recommend regards the Genetic differentiation estimate: This is a really interesting statistic! For some of the species you have multiple individuals it seems? Can you explain this a little more …
Reviewer #3:
The manuscript "High-quality carnivore genomes from roadkill samples enable species delimitation in aardwolf and bat-eared fox" is mostly well written and demonstrates an interesting and useful method for sequencing genomes from low-quality samples. They also provide a comprehensive overview of the state of genomics across the Carnivora clade, with some improved species/subspecies designations. I think the work is of broad interest. The analyses are mostly clear and I think a few additional analyses and small improvements could be made prior to publication, but otherwise have no issues.
The additional analyses/clarification I would recommend regards the Genetic differentiation estimate: This is a really interesting statistic! For some of the species you have multiple individuals it seems? Can you explain this a little more in the text. I am just not entirely convinced that the statistic is robust, but I think it would be with a few more analyses. My concern is primarily due to having only two individuals in some of your comparisons, because of population structure/relatedness the random regions you sample could have correlated histories. I think this could be addressed by varying window sizes and replicates across comparisons where you have multiple individuals for both the intraspecific and interspecific calculations.
-
Reviewer #2:
This manuscript from Allio is an interesting mix of approach demonstration (population genomic sampling via roadkill) and application (demographic analyses, questions about taxonomic status, and phylogenomics). There are some valuable results from the application component of the paper. In particular, I appreciate the comparative approach for studying patterns of intra- vs. inter-species genetic diversity. However, there is some rework to fully normalize those comparisons, that I feel is required.
I would suggest the authors be more immediately forthcoming about the sizes of their samples, and perhaps consider changes to the introductory text to avoid giving any mis-impressions to readers about what data are ultimately presented in the manuscript. I had envisioned more of a landscape genetics-level sample and analyses, …
Reviewer #2:
This manuscript from Allio is an interesting mix of approach demonstration (population genomic sampling via roadkill) and application (demographic analyses, questions about taxonomic status, and phylogenomics). There are some valuable results from the application component of the paper. In particular, I appreciate the comparative approach for studying patterns of intra- vs. inter-species genetic diversity. However, there is some rework to fully normalize those comparisons, that I feel is required.
I would suggest the authors be more immediately forthcoming about the sizes of their samples, and perhaps consider changes to the introductory text to avoid giving any mis-impressions to readers about what data are ultimately presented in the manuscript. I had envisioned more of a landscape genetics-level sample and analyses, rather than n=3 individuals per each of the two species. Furthermore, while I think the reporting of the genome assembly qualities is important from confirmatory and quality control perspectives, and while presenting the new assemblies, in my view this shouldn't be set up to be a surprising result. These are very high-quality DNA samples, so we expect to be able to achieve DNA assembly qualities to whatever the invested level using current best-practices data generation and analytical methods.
On the general genetic diversity and taxonomic questions, from my own experience I know that genetic differentiation metrics are not necessarily precisely comparable between a new study based on genome sequence data and an existing published dataset. Sample size affects false positive and false negative SNP calling error rates and sequence coverage and the variation among samples can also make a difference. Thus, especially since this leads to a key result/conclusion (i.e. that the two subspecies of aardwolf may deserve species status), it isn't sufficient that "similar individual sampling was available" for the carnivoran comparative datasets. The datasets should be equalized with sample number and individual sequence coverage (using downsampling) and then SNPs re-called using the same approach, before making the comparison. From the methods it wasn't clear to me the extent to which this all was done. It does appear that the same number of samples were used, and that the SNP calling approach was likely re-done from the read data (although please be more explicit about this, in the description). However, it doesn't appear that the sequence read data were subsampled for equivalency across the samples, which should be incorporated. Hopefully the results are similar, but there can be big changes that affect interpretation, so a careful approach is required.
The study design and proposed expanded use of roadkill samples in population genomics led me to think of this study, one of my all-time favorites: Brown & Bomberger Brown (2013), Where has all the road kill gone?, Current Biology. For the present study, the question of potential biases in the sample for similar or related reasons is beyond the scope of investigation; this is not relevant for the sample sizes collected and analyses conducted. However, the importance of keeping this possibility in mind should at least be noted given the more expansive promotion of the wider inclusion of roadkill samples in population genomic studies. E.g. could the sample be biased towards individuals with genetically-mediated and/or culturally learned behavioral tolerance of human-disturbed habitats, etc., rather than a truly random sample representative of the overall landscape.
In the methods section, the collection process and permits for the four samples from South Africa are described in detail. (Could you preemptively explain that the IUCN status for these two species is Least Concern, and thus that CITES permits are not required for the international transport of the samples?). However, the same information is not provided for the two East African samples that were included in the study (also, I think that there should not be two separate sampling sections in the manuscript). Please provide these details or expanded explanations.
-
Reviewer #1:
The manuscript by Allio et al. tries to justify that roadkill can be a useful source for genomic sequencing and even genome assembly level data. The authors cover all aspects of using this resource, from a new protocol to extract DNA, through generating a hybrid short- and long-read genome assembly and to various applications, showing that this data can be used in phylogenomic and population genomic analyses. Although I think that the manuscript is useful in highlighting how this resource can be analysed, it covers a lot of different topics and covers them in varying depth, which makes it difficult to follow and understand the real importance of the different sections.
Major comments:
-Overall, this manuscript left me a bit confused about what is the main scope. It covers a lot of different topics from the laboratory-end of …
Reviewer #1:
The manuscript by Allio et al. tries to justify that roadkill can be a useful source for genomic sequencing and even genome assembly level data. The authors cover all aspects of using this resource, from a new protocol to extract DNA, through generating a hybrid short- and long-read genome assembly and to various applications, showing that this data can be used in phylogenomic and population genomic analyses. Although I think that the manuscript is useful in highlighting how this resource can be analysed, it covers a lot of different topics and covers them in varying depth, which makes it difficult to follow and understand the real importance of the different sections.
Major comments:
-Overall, this manuscript left me a bit confused about what is the main scope. It covers a lot of different topics from the laboratory-end of the spectrum, e.g. protocol used to get good DNA out of roadkills and how to assemble these genomes with a hybrid genome assembly, and crossing into a phylogenomic analyses making taxonomic suggestions and an analyses of the complete carnivora group, plus a demographic analyses showing the changes of population size over time.
I was left with an impression that the authors tried to cover a lot of different topics but did not go deep enough in any of those. As a consequence, the results section ends up sounding somewhat shallow, while the discussion takes up a lot of space.
What I would suggest if this manuscript is indeed to serve as a roadmap to roadkill genomics, is to add a figure showing the pipeline and then adjust the structure of the manuscript accordingly. For example, one box in the figure would correspond to one heading in the results/methods, where the DNA analyses is explained - reasoning why a special protocol is needed, what is the main difference to existing methods, how does the yield compare to other methods, etc.
And then the different topics explored in this manuscript could be shown as different examples of the application - taxonomic questions on intra-/inter-species level, higher level taxonomic analyses (of the whole Carnivora), population genomic analyses, etc. Highlighting this as examples of the potential use of the roadkill genomes would make it understandable why this paper is trying to cover aardwolf and bat-eared fox genomics from so many ends.
-Even though showing that roadkill samples can be useful for analysing particular species for which obtaining samples is difficult in other ways, I'm missing a discussion of how difficult it is to obtain roadkill samples and what are the ramifications. Can this approach be generally applied due to legislation reasons, do you need permits, do you find enough roadkill to rely on this source or do you only see it as an opportunistic sampling scheme?
-Genome assembly is not exactly my field of expertise; therefore, I would like the authors to better explain how their hybrid, short- and long-read genome assembly approach is novel. My impression was that such hybrid assemblies are now a rather common and well-established practice. But a lot of space is dedicated to explaining this topic in the introduction and again in the discussion, which to me is something obvious and reads more like a review than a research article. But maybe I'm missing something obvious here, in which case I'd like the authors to make it clearer.
-
Summary: Collectively, we liked a lot about your paper and we would accordingly like to encourage its continued evolution. However, we felt that the approximately equal balance at present between the roadkill genomics assembly pipeline and the phylogenetic and genetic diversity results was not justified, and we requested a shift accordingly as described below. Second, we require several analytical updates to the manuscript to ensure robustness of the main genetic diversity results.
-
