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Gigantic Genomes of Salamanders Indicate Body Temperature, not Genome Size, is the Driver of Global Methylation and 5-Methylcytosine Deamination in Vertebrates

Methylation of cytosines at CpG dinucleotide sites silences transposable elements (TEs), sequences that replicate and move throughout genomes. TE abundance drives differences in genome size, but TE silencing variation across genomes of different sizes remains largely unexplored. Salamanders include most of the largest C-values — 9 to 120 Gb. We measured CpG methylation levels in salamanders with genomes ranging from 2N = ~58 Gb to 4N = ~116 Gb. We compared these levels to results from endo- and ectothermic vertebrates with more typical genomes. Salamander methylation levels are ~90%, higher than all endotherms. However, salamander methylation does not differ from the other ectotherms, despite a ~100-fold difference in nuclear DNA content. Because methylation affects the nucleotide compositional landscape through 5-methylcytosine deamination to thymine, we quantified salamander CpG dinucleotide levels and compared them to other vertebrates. Salamanders have comparable CpG levels to other ectotherms, and ectotherm levels are higher than endotherms. These data show no shift in global methylation at the base of salamanders, despite a dramatic increase in TE load and genome size. This result is reconcilable with previous studies by considering endothermy and ectothermy, which may be more important drivers of methylation in vertebrates than genome size.


35
Genomes are composed of sequences with radically different effects on organismal 36 survival and reproduction. Essential genes are required to sustain life, and many are 37 constitutively transcribed in all tissues. In contrast, transposable elements (TEs) ¾ sequences 38 capable of proliferation and movement throughout genomes ¾ are mutagenic, and their 39 transcriptional silencing is required to maintain germline integrity and reproduction (Bourque et 40 al. 2018). Transcription or silencing of different DNA sequences is facilitated by conformational 41 changes to chromatin, which is achieved through epigenetic modifications to both DNA and 42 histones (Venkatesh and Workman 2015). The suppression of TEs relies on transcriptional 43 silencing through methylation of cytosines (5mC) that are adjacent to guanines (i.e. CpG 44 dinucleotide sites) (Bird 2002;Law and Jacobsen 2010;Fedoroff 2012;Venkatesh and Workman 45 2015;Deniz et al. 2019). TEs are a major determinant of overall genome size, which varies > 46 65,000-fold across eukaryotes (Pellicer et al. 2010). How TE suppression via methylation at CpG 47 sites scales with increased TE load and genome size remains incompletely understood (Wolffe 48 1998;Jones and Wolffe 1999). 49 There have been few attempts to explore global methylation at the upper limits of 50 genome size. In Picea abies, the Norway Spruce ¾ which has an unusally large diploid genome 51 size of 20 Gb ¾ the percentage of methylated CpG sites is higher than that found in other 52 monocots and eudicots with smaller genome sizes. CHG sites (where H is any nucleotide that is 53 not a guanine) show the same pattern, whereas methylated CHH sites do not (Ausin et al. 2016). 54 5mC at CpG and CHG are both associated with TE silencing in plants, while 5mC at CHH sites 55 is not, instead forming a barrier between heterochromatin and other genes (Kenchanmane Raju et 56 al. 2019). This single data point suggests that, as genome size increases, the percentage of 57 relationship between genomic gigantism and global methylation in animals remains untested, as 59 does the relationship in any taxon with a genome size above 20 Gb. 60 Global methylation levels can also affect the nucleotide compositional landscape because 61 methylation causes an increase in the rate of specific mutations. Methylated cytosines at CpG 62 sites are predisposed to undergo transition mutations from C to T via deamination (Ehrlich and 63 Wang 1981). Recently, Zhou et al. (2020) demonstrated that the proportion of CpG sites 64 decreases as genome size increases across animal species with genome sizes ranging from 89 Mb 65 to 4 Gb, which was interpreted to reflect deamination-driven transition mutations occurring at 66 higher frequency as TE loads and associated methylation levels increase. Whether this 67 relationship holds for animals with gigantic genome sizes remains unknown. 68 The salamander clade is an appropriate model system for studying the epigenetic factors 69 that control ¾ or fail to control ¾ TE activity in gigantic genomes because it includes diploid 70 genomes that range from 9 to 120 Gb (Šímová and Herben 2012;Sun et al. 2012;Sclavi and 71 Herrick 2019;Gregory 2020;Decena-Segarra et al. 2020). These gigantic and variably-sized 72 genomes reflect the accumulation of extreme levels of TEs, associated with a shift in the 73 dynamics of genome size evolution at the base of the clade ~200 mya (Batistoni et al. 1995;Sun 74 and Mueller 2014;Liedtke et al. 2018). Additionally, there are several polyploid salamander 75 species, providing a different mechanistic path to high levels of nuclear DNA by combining 76 large genomes with increased chromosome copy number (Bogart et al. 2007(Bogart et al. , 2009. 77 Here, we quantify methylation levels across salamanders that encompass their upper 78 range of genome sizes, and we compare these values to those of other vertebrates with more 79 typically sized genomes. We include both endotherms (i.e. birds and mammals) and ectotherms 80 (i.e. fish, other amphibians, reptiles) because body temperature is associated with differences in 81 global methylation (Jabbari et al. 1997). Endotherms, as well as ectotherms inhabiting warm 82 environments, have higher rates of deamination of 5mC nucleotides, resulting in lower global 83 methylation levels and fewer CpG dinucleotides genome-wide (Sved and Bird 1990;Jabbari et 84 al. 1997;Varriale and Bernardi 2006;Bernardi 2007;Bucciarelli et al. 2009). We use these data 85 to test whether methylation levels and genome-wide CpG dinucleotide levels are driven by 86 genome size and/or mode of body temperature regulation. In previous studies, endothermy was 87 confounded with large genome size. The integration of the data from salamanders ¾ which 88 combine huge genome sizes with ectothermy ¾ allows us to decouple genome size from 89 metabolic heat production, revealing that genome size alone lacks explanatory power for 90 methylation and CpG dinucleotide levels across vertebrates. 91 92

Sampling Design, Tissue Dissection, and DNA Extraction for Analysis of Global 94
Methylation 95 Our first goal was to test for variation in methylation levels across tissues in salamanders. 96 For this analysis, we focused on the model salamander Ambystoma mexicanum, widely used for 97 studies of development and regeneration (Tank et al. 1976;Seifert et al. 2012;Keinath et al. 98 2015; Sessions and Wake 2020). Additionally, it is one of the few species of salamander to have 99 its entire genome assembled and annotated (Keinath et al. 2015;Nowoshilow et al. 2018). We 100 analyzed brain, heart, lung, liver, intestine, muscle, skin, and testes or ovaries from three adult 101 males and three adult females. 102 genomes. For this analysis, we used muscle tissue as the single representative tissue, and we 104 chose species that span the upper range of diploid genome sizes and ploidy in salamanders. The 105 diploid species were Plethodon cinereus and Necturus beyeri, as well as A. mexicanum. 106 Plethodon cinereus has a range of published genome sizes with an average of ~22 Gb (Gregory 107 2020), although our measurements indicate a genome size of 29.3 Gb (Itgen et al. 2021). The 108 genome size of Necturus beyeri is unknown, but size estimates for congeners are 120 Gb (N. 109 lewisi and N. punctatus) and ~85 Gb (N. maculosus) (Gregory 2020). Ambystoma mexicanum has 110 a genome size of 32.15 Gb (Keinath et al. 2015). The polyploid taxa were triploid and tetraploid 111 unisexual biotypes formed naturally via kleptogenesis (Bogart et al. 2007)

including one haploid 112
A. laterale genome (~29 Gb) and either two or three A. jeffersonianum genomes (haploid = ~29 113 Gb; ~87-116 Gb total). We also included the diploid A. jeffersonianum. Three individuals were 114 sampled from each species/biotype (Table 1). 115 Our third goal was to compare methylation levels in salamanders with those from 116 vertebrates with more typical genome sizes. For this analysis, we combined our new data with 117 previously published methylation levels of brain and liver tissue from fish (Carassius auratus, 118

Luminometric Methylation Assay and Data Standardization 146
To measure DNA methylation levels, we used the luminometric methylation assay 147 (LUMA), a form of pyrosequencing that targets CpG dinucleotides capable of methylation 148 (Hopkinton, MA, USA) using a PyroMark MD system from Qiagen. Duplicate LUMA runs were 150 done for each DNA sample. All assays included four Lambda DNA standards with methylation 151 percentages of 0, 50, 60, and 100% as internal controls. To account for any differences across 152 assay runs, we calibrated against the internal controls using inverse regression calibration (Ott 153 and Longnecker, 2015). Random effects were added per subject and per combination of subject 154 and tissue type to limit batch effects and account for pseudoreplication of duplicate runs. 155

Comparative Analyses of Global Methylation 156
First, we tested for differences in methylation levels among the A. mexicanum tissues 157 using a mixed model ANOVA, with tissue as a fixed effect factor and individual and duplicate 158 runs as random effect factors, followed by a Tukey HSD to test for significance between tissues. 159 Next, we tested for differences in methylation levels among the polyploid unisexual salamander 160 biotypes A. laterale (LJJ) and A. laterale (LJJJ) and the four diploid species A. jeffersonianum 161 (JJ), A. mexicanum, P. cinereus, and N. beyeri using a mixed model ANOVA, with species as a 162 fixed effect factor and individual and duplicate runs as random effect factors. We then performed 163 a Tukey HSD to test for significance between species/biotype. Finally, we tested whether 164 methylation levels vary across vertebrates as a function of salamander vs. non-salamander (i.e. 165 genome size ³ 29 Gb vs. genome size £ 6.4 Gb), ploidy (i.e. diploid vs. polyploid), and body 166 temperature regulation (i.e. endothermy vs. ectothermy). We assigned species to the appropriate 167 subgroup(s) and tested for variation between subgroups using linear regression contrasts. We 168 carried out all analyses in R Studio (Martin 2021;Team 2021) using R packages emmeans 169 (Lenth 2021), parameters (Lüdecke et al. 2020), and lme4 (Bates et al. 2015). We used the 170 ggpubr package (Kassambara 2020) to visualize the results. 171 Our final goal was to test whether enormous genome sizes translate into changes in the 173 nucleotide compositional landscape, as predicted based on the relationships among genome size, 174 transposable element load, methylation level, and C à T deamination mutation inferred across 175 53 animals (47 vertebrates, 6 invertebrates) with more typical genome sizes (89 Mb to 4 Gb) 176 (Zhou et al. 2020). C à T deamination mutations occur preferentially at methylated CpG 177 dinucleotides (Ehrlich and Wang 1981); thus, higher levels of methylation, associated with 178 methylation-based silencing of a greater TE load, are predicted to correlate with fewer CpG 179 dinucleotides genome-wide than are expected based on nucleotide frequencies (Zhou et al. 180 2020). We obtained publicly available genomic sequence data for nine species of salamanders 181 with varying genome sizes: Desmognathus ochrophaeus (~15 Gb; SRA046120.1), Pleurodeles We tested for a relationship between genome size and CpG O/E dinucleotides among the 201 nine salamander species using a linear regression analysis. We also corrected for phylogeny 202 using phylogenetic independent contrasts (PIC) with the tree for the nine focal species 203 subsampled from a comprehensive amphibian phylogeny (Pyron and Wiens, 2011). PIC analyses 204 were carried out using R packages ape (Paradis and Schliep 2019), Geiger (Harmon et al. 2008), 205 nlme (Pinheiro et al. 2020), and phylools (Revell 2012 (Zhou et al. 2020); this study included full 207 genome assemblies from species with genome sizes ranging from 0.36 to 4.78 Gb and showed a 208 negative correlation between genome size and CpG O/E (Zhou et al. 2020). To ensure that the 209 datasets were comparable, we subsampled Illumina reads of 20 species from the Zhou et al. 210 dataset to generate datasets with coverage that approximated the coverage of the salamander 211 datasets, and we calculated CpG O/E from these subsampled datasets. We then compared the 212 CpG O/E values based on full genome assemblies to those we calculated from subsampled short 213 read datasets using linear regression and found a significant positive correlation between the two 214 sets of estimates (p < 0.00001, R = 0.83; subsampled estimates equally likely to be slightly above 215 or below whole-geome estimates), suggesting that comparisons across these different datasets are 216 unlikely to introduce bias due to comparing whole-genome assemblies to unassembled read 217 datasets. 218 219

Methylation Levels Do Not Vary Across Salamander Species 226
Methylation levels are not significantly different between salamander species, despite 227 differences in diploid genome size and ploidy (df = 5, F-value = 1.11, p = 0.4; Figure 2). We 228 note that the two species with the most nuclear DNA ¾ the tetraploid Ambystoma biotype LJJJ 229 and the diploid Necturus beyeri ¾ have the largest intraspecific variances.

Levels and CpG Dinucleotide Levels Across Vertebrate Genomes 258
Despite spanning a ~100-fold difference in genome size, the methylation levels of 259 ectotherms were not significantly different from one another. Additionally, the methylation 260 levels of polyploids were not significantly different from diploids. In contrast, endotherms had 261 significantly lower levels of methylation compared to ectotherms, with or without the inclusion 262 vertebrates is the maintenance of a high body temperature by the production of metabolic heat, 264 and not the amount of DNA in the nuclear genome. 265 Endothermy and ectothermy have been proposed as the primary determinants of global 266 methylation levels in vertebrates before (Jabbari et al. 1997). Mammals and birds, which have 267 high, endothermically-maintained body temperatures, have lower methylation levels than 268 amphibians and reptiles, which have lower body temperatures. This difference in methylation is 269 paralleled in congeneric species of gobies that live in vastly different temperatures in the Gulf of 270 California¾the species that lives in warmer temperatures has lower levels of DNA methylation 271 (Bucciarelli et al. 2009). Similarly, temperate and tropical fish have lower methylation levels 272 than polar fish (Varriale and Bernardi 2006). In all of these cases, the lower methylation levels 273 found in animals with higher body temperatures likely reflect the higher rates of 5mC 274 deamination of CpG dinucleotides at warmer temperatures, leading to faster loss of methylated 275 cytosines from the genome (Jabbari et al. 1997;Varriale and Bernardi 2006;Bernardi 2007;276 Bucciarelli et al. 2009 predicted by their endothermic body temperature regulation. We re-analyzed the data from Zhou 289 et al, running a linear regression with first genome size as the predictor, followed by 290 ectothermy/endothermy. The R 2 value increased from 0.26 (when using genome size as the 291 predictor) to 0.48 (when using ectothermy vs. endothermy). This suggests that much of the signal 292 within the Zhou et al. dataset comes from the fact that mammals are endothermic and also tend to 293 have larger genomes than fish, reptiles, and frogs¾but it is endothermy itself, rather than 294 genome size, that is driving the CpG O/E pattern (Supplemental Figures 1 and 2). Within the 295 non-salamander ectotherms alone, however, genome size is negatively correlated with CpG O/E 296 based on linear regression analysis (p = 0.009), suggesting a possible relationship between TE 297 load, methylation, and CpG loss in ectotherms with relatively small genomes that warrants 298 further investigation. 299 Given these results, is there a potential mechanistic relationship between endothermy and 300 the slightly increased TE load of mammals? Higher deamination rates would decrease 301 methylation of TE sequences in mammals, which might negatively impact their silencing and 302 allow for greater activity. In addition, higher deamination rates would yield higher transition 303 mutation rates of TE sequences, which in turn could have two outcomes: 1) the production of 304 divergent TE sequences that escape sequence-specific TE silencing, leading to greater TE 305 activity, and/or 2) the production of TE sequences that lose their functional ORFs, rendering 306 them incapable of autonomous transposition and leading to decreased TE activity. There are also 307 groups that do not show an association between endothermy and increased TE load and genome 308 size. Birds ¾ also endothermic ¾ have lower TE loads and smaller genome sizes, and 309 salamanders ¾ ectotherms ¾ have the highest TE loads and largest genomes among tetrapods. 310 In total, TE activity and abundance reflect the interaction of diverse forces, which may or may 311 not include a relationship between methylation, mutation, and TE silencing that contributes to 312 mammals' slightly larger genome sizes relative to non-salamander vertebrates. Methylation levels (percentage methylated cytosines at CpG dinucleotide sites) of DNA extracted from different tissues in Ambystoma mexicanum. Lungs have significantly lower methylation levels compared to some tissues: Brain -Lungs , Heart -Lungs, Gill -Lungs, and Liver -Lungs.

Fig. 2
Methylation levels (percentage of methylated cytosines at CpG dinucleotide sites) of six different species/biotypes of salamanders with different amounts of nuclear DNA. Teal represents diploid species and orange represents polyploid species. There are no significant differences among groups.

Fig. 3
Methylation levels (percentage of methylated cytosines at CpG dinucleotide sites) across vertebrates. Diploid, triploid, or tetraploid genome sizes are listed with species names. Orange dots indicate endothermy and teal dots indicate ectothermy. Blue font indicates salamanders. Ectotherms have higher methylation levels than endotherms (p < 0.001).

Species
Genome Size (Gb)