Peer review of: American mastodon mitochondrial genomes suggest multiple dispersal events in response (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Karpinski et al present an analysis of Mastodon phylogeography through time based on 33 new, 
complete mitogenomes. 

All experiments and analyses are conducted thoroughly, and I have little doubts about the data itself. 
Below are a few points that I feel could be improved, though. 

Major points: 
1. Most conclusions presented in this manuscript rely heavily on the ages estimated for the undated 
samples using molecular dating. The estimates produce broad 95% HPD intervals, which is not 
surprising, but which makes their interpretation somewhat speculative. The authors make a case for 
considering the reasonably tight peaks of the HPD distribution rather than the width of the complete 
95% HPD intervals, and while this is a bit arbitrary, I feel it is a fair point to make. However, when so 
much relies on the molecular estimates, I would like to see how well this molecular age estimation 
actually performs with this specific dataset. This can for example be tested through “leave-one-out” 
cross validation of dated samples. I.e, take the dataset as is, but in each iteration, don’t just estimate 
the age of the undated samples, but also the age of one sample for which we know the age. Repeat 
for all dated samples, and check how often the radiocarbon age of these samples 
a) falls within the 95% HPD estimate for the respective sample, and 
b) within the arbitrarily defined “peak” of the HPD distribution, and 
c) how often the radiocarbon age is within the same MIS as the molecular estimate. 
From my experience, this normally works reasonably well, and it really helps to lend some confidence 
to the results. 

2. I find myself slightly skeptical as to how much can be said about the age distribution of clade A 
from two samples. The results are consistent with an earlier migration of clade A to Beringia compared 
to clade Y but with so few samples, this interpretation is standing on very shaky legs and is perhaps 
given a bit too much weight in the discussion and conclusion. And at least with relaxed clock settings, 
there is a significant overlap between the age of clade A and clade Y samples. Perhaps the above cross 
validation test will provide further support, though. 

Minor points: 
Page 6.: When mentioning the 5 major clades (line 87), it would be good to briefly list them, as Fig 1. 
shows 6 clades and it only becomes clear later that clade M is not stable. 

Fig 1 and Fig A2: I am not quite sure what in Figure 1 is expanded compared to Fig A2, as per Figure 
1 legend. They look similar to me. However, it is noticeable that Fig A2 only shows two haplotypes in 
Alberta, while Fig 1 shows three, 

It would be good to indicate in Figure 1 which samples were radiocarbon dated or had a stratigraphic 
date. I can somewhat guess by their names, but it would be easier to have - for example - an asterisk 
by the respective name if there is a date for that sample. 

Michael Knapp 

Reviewer #2 (Remarks to the Author): 

As I am not a geneticist, this review will not focus on the technical aspects of the DNA extraction and 

genomic analysis. As a researcher primarily focused on Pleistocene megafauna and animal 
communities, my review comes from the perspective of a consumer of the genetic literature. 

This work is of substantial value as the first large scale mitochondrial study of extinct North American 
mastodons, with analysis of 33 new mitogenomes. It addresses an existing model of Pleistocene 
mastodon distributions in North America: that populations repeatedly expanded northward into high 
latitudes regional during interglacial periods and were subsequently extirpated in these regions with 
the return to glacial conditions. 

The identification of six previously unknown lineages of mastodons, including two distinct clades in 
eastern Beringia, is the most significant contribution of this paper. These data add to our growing 
knowledge of Pleistocene megafaunal demographics and responses to climate. As the authors correctly 
point out, the only phylogeographic studies of Pleistocene megafauna prior to this study were from 
bison, mammoth, horse, reindeer, and muskox—species adapted to either grassland or steppe-tundra 
environments. This study is one of the first to address megafaunal species adapted to forest or 
woodland habitats. As such there is a broad audience (including geneticists, paleontologists, 
ecologists, and archaeologists) that would be interested in these results. However, difficulties with 
precisely dating many of the samples from northern regions (either directly, using techniques such as 
radiocarbon or luminescence, or with their own attempts at molecular dating) limit definitive 
conclusions about mastodon responses to cyclical glacial-interglacial climate intervals. This manuscript 
has much to contribute to the discipline but requires some relatively minor reworking before 
publication. 

It is fascinating and somewhat counter-intuitive that mastodons from northern latitudes (even in 
restricted geographic areas such as eastern Beringia and Alberta) contained specimens from distantly 
related clades. Genetic studies such as this are so valuable in part because they can identify complex 
ecological and evolutionary histories. That said, I would like a deeper discussion of these results in the 
methods and conclusion sections. How do these results—especially the ancient timing of splits in some 
of these clades, well before onset of glacial conditions –fit our larger understanding of mastodon 
origins and evolutionary history? They also diverge from my limited knowledge of the phylogenies of 
North American woolly mammoths and bison, which are arguably the best studied Pleistocene 
megafauna. On page 8, for example, the authors mention that prior work on bison (Shapiro et al, 
Heintzman et al) show a north-south separation of clades related to cyclical climate change. However, 
the changes in bison occurred within the last 100,000 years and are closely tied to clear MIS stages. 
The data presented here suggest much critical differentiation occurred prior to the beginning of the 
Pleistocene. It would be very useful to talk more about this larger patterning. For example, clade M 
contains a late surviving mastodon (30 kya) in Mexico with a lineage split from other north America 
mastodons over 6 mya ago. I would also like to hear more about clade G. Frankly, these are the 
samples with the best temporal control and probably represent a southern population which 
presumably would not undergo periodic extirpation. Because I would assume more genetic continuity 
in southern populations, it would be interesting to know more about this clade. 

The section on mastodon age estimates is the most problematic, not because the median age 
estimates are unbelievable, but because the level of imprecision represented in the 95% HPD is very 
broad. For example, Clade Y’s median age estimates of ~150,000 years have 95% HPD values ranging 
between 50,000-420,000 yrs. As shown in Figure 3b, even under a stricter clock model the best range 
is ~50,000-226,000 yrs. This span covers two complete glacial-interglacial cycles! Results from the 
Clade A analysis are even worse (95% HPD 50,000 – 685,000). Limited available data and the dating 
of individual specimens (rather than tree nodes) understandably increase imprecision. Given that level 
of uncertainty, however, I find it hard to accept some associations of these clades with specific MIS 
stages. The study results appear to be over-interpreted. One way to increase reader confidence would 
be to present the result of this analysis on clade G. These specimens are independently dated with 
radiocarbon dating, so it would be very interested to see how the age estimates from the molecular 
analysis compare to the independent dates. 

Minor issues 
• Not sure Table 1 contributes much. Lisiecki and Raymo’s MIS stage descriptions are broadly 
available. As noted earlier, the temporal precision of the current analysis is not great and so such fine-
scale focus on MIS stages may be problematic. 
• Check the reference column in supplementary Table b. There appear to be some missing or 
incomplete references. 

Matthew Hill 
University of Iowa 

Reviewer #3 (Remarks to the Author): 

The paper by Karpinski et al. greatly expands the genetic dataset currently available for the American 
mastodon. Using robust phylogenetic and molecular dating methodology, the authors find multiple 
occupations of Eastern Beringia by this species, suggesting that the mastodon’s evolutionary history 
was defined by a dynamics of range expansions and contractions. 
I find the paper methodologically sound and clearly written. The conclusions are of interest to the 
community of researchers studying ancient population dynamics and phylogeography. I appreciate 
that the authors performed analyses to ensure quality of the generated data and that they do not 
overinterpret data. On the other hand, I am missing the link between the rather matter-of-factly 
description of results and their potential to address the general issues laid out in the beginning and at 
the end of the paper. I think that the paper would be improved by narrowing its scope and by a more 
careful discussion on the meaning and impact of the results. 

Major comments: 

I think that the generalization of the Abstract and Introduction is not matched by the interpretations 
and extrapolations in the Discussion and Conclusions. Although this study certainly improves our 
understanding of the Pleistocene megafauna range dynamics, I doubt that the spatial and temporal 
detail of the study is sufficient for it to markedly contribute to addressing issues like “how global 
climate change may affect the ecology and distribution of extant species” as indicated in the Abstract. 
I would suggest that the authors narrow down the focus of the paper, so that the areas in which it 
contributes to our understanding of population dynamics are clearer. 

Specific comments: 

Line 9: “large-scale phylogeographic study” - In light of previous publications on Proboscideans like 
Enk et al. 2016 and Chang et al. 2017, which contained 67 and 54 new mitogenomes, respectively, 
the term ‘large-scale’ becomes questionable and I think that it would be better to use something more 
relevant like ‘continent-wide’. 

Lines 17-19: Can the authors explain how? I think that they should either include more on this in the 
conclusions or the abstract should be phrased in a less generalized way. 

Line 43: I would rephrase the sentence to “most Pleistocene phylogeographic studies on North 
America have focused on megafauna adapted to…” in order to not neglect the numerous studies 
focused e.g. on phylogenetics of Eurasian megafauna. 

Lines 48-50: I’m not quite convinced about this statement. Doesn’t climate-change pressure affect 
both warm-adapted and cold-adapted species in the same extant, just in different or opposite ways? 

Line 78: Even though it comes later in the methods, it would be good to mention here at least a very 

brief information about the samples, for example, where they come from (field-collected or museum 
specimen). 

Line 87: Also, the listing of phylogenetic results comes abruptly. I know the methods are thoroughly 
discussed later, but it would be helpful to at least mention that a number of different extraction 
methods, library build methods, and phylogenetic inference methods were used. Especially, 
understanding what phylogenetic methods were used is important since these are directly compared in 
the main text and in the figure. 

Line 112-122: I find the diverse genetic background of the Alberta samples, especially compared to 
the other samples, one of the most interesting results of the study. And although it is appropriate that 
the authors don’t overinterpret these results because of the small sample size and uncertainty of the 
sample ages, I think it would be interesting to see more information on previously published work 
discussing the role of this region and whether anything is known about mastodon refugia in North 
America in general. I would like to know when was the Ice-Free corridor open and maybe also a bit 
more detail about the bison study by Heintzman et al. 2016 paper mentioned in the text, because 
there might be parallels to this study. Also, including a reference to Fig. 2 in this paragraph would be 
useful, I only noticed that it relates to it later. 

Lines 146-177: I think that the section on age estimates and Fig. 3 could use a more thorough 
discussion. The main message of the paper is basically reduced to one sentence (lines 162 to 164). 

Lines 178-184: I don’t have a strong opinion about this, but the skyline plot could be moved to the 
supplementary since it doesn’t deliver any important information. 

Line 205: “represents a 16.5-fold increase in available molecular data for this species” - quantifying 
the contribution of this dataset seems redundant in the time period of big data being generated at an 
increasing speed. 

Lines 251-259: At first reading, it wasn’t clear to me what the authors meant and I think it would help 
to describe more clearly that a wide spectrum of extraction and library-build protocols was used and 
also to explain the reasoning. 

Table 1: 
I think this could go to the supplementary material since the glacial and interglacial cycles are already 
shown in Fig. 2A. 

Fig. 1: 
Would it be possible to use other colour than two shades of blue? My printer turned the dark blue on 
the tree, but not in the legend, into purple and it created confusion. 
The clade Y star is not immediately visible if one doesn’t look at the supplementary. 
“Circles indicate locations” instead of “Large circles….” – As far as I can see, there is only one size of 
circles. 

Fig. 2: 
I find the depiction in Fig. 2F funny, but also more appropriate for a blog rather than a scientific 
publication. Maybe the authors could instead add another detail showing the progressing glacial 
conditions and absence of mastodons in eastern Beringia. 

Fig. 3: 
I think for consistency, the timeline of Fig. 2 and Fig. 3 should have the same direction. 
I didn’t quite understand which samples are shown. Why do the A and B figures list different samples 
in the legend? 
For visualization purposes, the colours could correspond to the colours of clades in Fig. 2, with the text 

boxes indicating clades highlighted in the same colour. The individual-colour figure could be then 
moved to supplementary. 

Supplementary: 
I think it would be very useful to include the reasoning behind using such a wide variety of laboratory 
methods and how these were piped into a protocol. It’s not immediately obvious if the extracts were 
pooled or which library build method was used on which extract.