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Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals
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Giulia I. M. Pasquesi1,
Richard H. Adams1,
Daren C. Card ORCID: orcid.org/0000-0002-1629-57261,
Drew R. Schield1,
Andrew B. Corbin1,
Blair W. Perry1,
Jacobo Reyes-Velasco1,2,
Robert P. Ruggiero2,
Michael W. Vandewege3,
Jonathan A. Shortt4 &
Todd A. Castoe1
Genome evolution
Mobile elements
Broad paradigms of vertebrate genomic repeat element evolution have been largely shaped by analyses of mammalian and avian genomes. Here, based on analyses of genomes sequenced from over 60 squamate reptiles (lizards and snakes), we show that patterns of genomic repeat landscape evolution in squamates challenge such paradigms. Despite low variance in genome size, squamate genomes exhibit surprisingly high variation among species in abundance (ca. 25–73% of the genome) and composition of identifiable repeat elements. We also demonstrate that snake genomes have experienced microsatellite seeding by transposable elements at a scale unparalleled among eukaryotes, leading to some snake genomes containing the highest microsatellite content of any known eukaryote. Our analyses of transposable element evolution across squamates also suggest that lineage-specific variation in mechanisms of transposable element activity and silencing, rather than variation in species-specific demography, may play a dominant role in driving variation in repeat element landscapes across squamate phylogeny.
Transposable elements (TEs) and other repetitive sequences represent a major fraction of vertebrate genomes—in most mammals, repeat elements comprise 28–58% of the genome1,2, and may comprise more than two-thirds of the human genome3. Several decades of genome research has led to the prevailing view that genome size and genome repeat content are tightly linked, such that shifts in genomic repeat content are expected to result in proportional shifts in vertebrate genome sizes4,5,6. Recently, this correlation has come into question in favor of alternative hypotheses, such as the "accordion" model of co-variation between genomic DNA gained by repeat element expansion and genomic DNA lost through deletion7. It has also been demonstrated that the relationship between genome size and repeat content may vary between vertebrate lineages4,5,8, with some lineages adhering more or less to a particular model or pattern4,6,7,9, underscoring the value of comparative analyses across diverse lineages.
Within vertebrates, our understanding of genome and repeat element evolution is largely biased towards mammals and archosaurian reptiles (mainly birds). The emerging pattern from studies of these groups is that large differences in the repeat element landscape exist among major amniote vertebrate lineages, yet fairly little variation in repeat content and diversity are observed within major amniote groups. For example, estimates based on de novo annotation of TEs in mammal and bird species suggest 1.7-fold and 2.2-fold variation in TE content across species for each group, respectively1,7. Although squamate reptiles (lizards and snakes) represent a major portion of the amniote tree with over 10,000 species spanning more than 200 million years of evolution10, variation in genomic repeat content across squamate reptiles has remained poorly studied. From the few studies to date, genome size appears to be highly conserved in squamate reptiles11, yet the little that we know about repeat element variation suggests that squamate reptile genomes vary greatly in repeat element content12,13.
Motivated to assess whether squamate reptile genomic repeat element landscapes adhere to patterns observed in birds and mammals, we analyzed genomic repeat landscapes across 66 squamate species using low-coverage random whole-genome shotgun sample sequencing data12,13 and draft genome assemblies. We find that squamate reptile genomes indeed challenge the paradigm that genome size and repeat content are tightly linked, and the view that major differences in repeat element content occur only between lineages of amniotes. In addition to contributions from TEs, snake genome repeat content variation is further increased by the largest known instance of microsatellite seeding by long interspersed nuclear elements (LINEs) observed in any living organism. We also find evidence that multiple independent horizontal transfer events and highly idiosyncratic patterns of TE proliferation across squamates have further contributed to extreme variation in genome repeat content in this lineage. We further tested a demographic explanation for variation in repeat content, whereby fluctuations in the effective population size (Ne) of species impact the efficacy of selection against repetitive element insertion14. We find no evidence that Ne explains the distribution and variation in characteristics of the repeat landscape in squamate reptiles, which indicates instead that variation in molecular mechanisms of TE proliferation, silencing, removal, and truncation may underlie the extreme repeat variation observed across squamates. Collectively, our findings challenge existing views related to repeat element and genome size co-evolution, and provide new evidence for unappreciated variation in genomic repeat content within and among major amniote lineages.
Comparison of sampled and assembled genome data
Our analyses of genomic repeat content were based on the assemblies of 12 squamate genomes (including 1 new and 11 published assemblies), and low-coverage, unassembled genomic shotgun read datasets obtained from 54 squamate species (Supplementary Data 1; Castoe et al.13). Previous studies have shown that genomic repeat content estimated from unassembled shotgun genomic datasets are similar to estimates derived from assembled genomes12,13. We confirmed this by comparing repeat annotations from assembled and unassembled genome data from the same species (Supplementary Fig. 1), and also confirmed that repeat estimates derived from unassembled genomic shotgun datasets are effectively independent of the amount of sequence data obtained (Supplementary Fig. 1).
Genome size and repeat content in major amniote groups
Squamate reptile genomes challenge the commonly accepted paradigm that genome size and repeat content are tightly linked4,5,6, and also challenge the prevailing view that large variation in repeat content tends to be characteristic of major clades, rather than highly dynamic within clades1 (Fig. 1). For example, mammalian genome sizes tend to be more highly variable (2.2–6.0 Gbp11; Supplementary Data 2) in comparison with squamate and bird genomes, yet genomic TE estimates demonstrate only moderate levels of clade-specific variation (33.4–56.3%, mean = 44.5%; Fig. 1a, Supplementary Data 3, and Supplementary Note 1). In contrast, birds have smaller genomes and higher conservation of genome sizes (1.0–2.1 Gbp11; Supplementary Data 2), with relatively low levels of TE content (4.6–10.4%, mean = 7.8%, with the only notable exception being the downy woodpecker with an extremely high genomic TE content of 22.5%, which we excluded as an outlier from analyses here; Fig. 1b, Supplementary Data 3, and Supplementary Note 1).
Genomic transposable element (TE) abundance and genome size variation in mammals, birds, and squamate reptiles. Branches on the time-calibrated consensus phylogeny are colored according to the estimated rate of genomic TE evolution. Violin plots show distributions of flow cytometry-based genome size estimates for major groups of a mammals, b birds, and c squamate reptiles, and the associated heat maps reflect the total genomic TE content (%) for each taxon. For squamate reptiles, additional heat maps show percent genomic repeat element content, percent genomic GC content, and percentages of major components contributing to the overall repeat element landscape
With highly conserved genome sizes (1.3–2.8 Gbp) yet extensive variation in genomic content of readily detectable TEs (23.7–56.3%, mean = 41.8%; Fig. 1c), we find that squamate reptiles do not adhere to either of these trends. The relatively high degree of variation in genomic repeat content across remarkably short evolutionary time scales in squamates presents the greatest contrast with birds and mammals. Unlike the clade-specific pattern observed in mammals, the genomic repeat content variation of squamate reptiles exhibits a high degree of variation even between species within the same genus (e.g., within the genera Ophisaurus (44.8–48.9%), Coniophanes (59.4–73%), and Crotalus (35.3–47.3%); Fig. 1c, Supplementary Figs. 2 and 3, and Supplementary Data 4). Across the 66 squamate species sampled, total genomic repeat element content varied from 24.4% to 73.0% (3-fold variation; Fig. 1c). Collectively, our analyses highlight the remarkable finding that the comparatively small genomes of squamates, similar to those of birds, can contain large and highly variable amounts of repeat elements, exceeding the range reported for mammals.
Genomic TE composition across squamate reptiles
The content and evolutionary dynamics of TEs in squamate genomes are unique in many ways when compared to that of mammals and birds, yet squamate genomes also share several key features with both lineages. All three groups have TE landscapes largely dominated by non-long-terminal repeat (non-LTR) retrotransposons. However, unlike mammalian genomes in which L1 LINEs and associated short interspersed nuclear elements (SINEs) are the most dominant and active elements3,15, squamate genomes tend to contain three similarly abundant and active LINE families (CR1, BovB, and L2 LINEs; Fig. 1, Supplementary Fig. 2, and Supplementary Data 4). While CR1 LINEs are ubiquitous across amniote genomes, CR1s are particularly abundant and recently active in squamate genomes (5.1%, compared to ~3.5% in birds and <1% in mammals1), as they tend to be in other non-avian reptiles (i.e., ~10% in crocodilians16). In addition to non-LTR elements, DNA elements are also highly variable and particularly abundant in multiple divergent squamate lineages (Fig. 1). For example, Tc1-Mariner elements have experienced a 2.4-fold expansion in colubroid snakes compared to lizards (mean genomic abundance = 4.23% in colubroid snakes and 1.7% in lizards; Fig. 1, Supplementary Fig. 2, and Supplementary Data 4). The most striking contrast between squamate vs. bird and mammal genomes is that squamate genomes contain an unusually broad diversity of types, subtypes, and families of TEs that appear simultaneously active12,16,17,18,19 (see also below and Supplementary Fig. 3), whereas genomes of mammals and birds tend to have a very small number of active elements (e.g., L1 LINEs and Alu SINEs in mammals, and endogenous retroviruses (ERVs) in birds6,15,20,21).
Guanine-cytosine (GC) content is known to play an important role in genome and repeat element evolution22,23,24,25,26. We found evidence of significant relationships between GC content and total TE content, as well as GC and total microsatellite (or simple sequence repeat; SSR) content, in lizards and colubroid snakes (Supplementary Fig. 4). In contrast, we found no correlation between genomic GC content and any aspect of the genomic repeat element landscape in non-colubroid snake genomes (Supplementary Fig. 4). Consistent with previous studies13, our analyses highlight the surprisingly variable nature of GC content across squamate genomes, which tends to be higher in lizards than in snakes, yet highest in the colubroid snake Coniophanes fissidens (GC = 47.8%; Fig. 1c). These findings are also broadly consistent with previously reported shifts in GC isochore structure in squamate genomes17,26, including the absence of isochore structure in lizard species, and intermediate structure in snakes that appears to represent isochore reacquisition after isochore loss in a squamate ancestor13.
Unparalleled microsatellite abundance in squamate genomes
Our analyses revealed that some squamate genomes contain astonishingly high levels of SSRs, and that genomic SSR content in some snake species is the highest of any previously studied vertebrate (e.g., 14% according to RepeatMasker estimates in Coniophanes fissidens, Supplementary Data 4 and 5, and Supplementary Fig. 5). While previous studies have suggested that the highest variation in SSR content tends to exist among major vertebrate lineages27, with fish, squamate reptiles, and mammalian genomes having similarly high genomic content12,13,17,28, our results provide new evidence that the highest variation known in genomic SSR content exists within lineages—squamates and snakes, specifically. We found up to 10.9-fold variation in the genomic density of SSR loci (262–2845 loci/Mbp) and 16.6-fold variation in SSR-occupied bases per Mbp (4.08–67.94 Kbp/Mbp) among squamates overall, with non-colubroid snakes tending to have the lowest genomic SSR abundance, and colubroid snakes having the highest (Supplementary Data 5, Fig. 2, and Supplementary Figs. 5 and 6). This extreme variation in the genomic SSR content of squamate reptiles exceeds the previous high benchmark set by fish genomes (8.2-fold loci/Mbp and 18.0-fold bp/Mbp variation), and dwarfs that of mammals (5.8-fold loci/Mbp and 5.4 bp/Mbp) and bird genomes (1.8-fold loci/Mbp and 2.8 bp/Mbp)12,13,17,28.
Microsatellite seeding by transposable elements (TEs) in squamate reptiles. a Branches on the time-calibrated consensus phylogeny are colored according to estimated rates of genomic CR1-L3 LINE evolution. Heat maps show the total genomic content (%) of LINE retrotransposon types involved in microsatellite seeding. Associated bar plots represent the total (left), 5mer (middle), and AATAG (right) microsatellite bp/Mbp density frequencies for each genome sampled. Red lines to the right of the bar plots highlight pronounced seeding of 5mer and AATAG microsatellites in colubroid snakes. b The ratio between TE mapping at the 5′ tail of AATAG microsatellite loci (AATAG-adjacent) and TE content averaged over five independent, randomly simulated genomic backgrounds for each class of TEs (SINEs; CR1-L3, Rex, CR1-L2 and BovB LINEs; LTRs; and DNA transposons). Ratios are plotted on a log scale to highlight enriched elements flanking AATAG loci (ratio >1) in contrast to elements more abundant in the genomic background (ratio <1). c Histogram shows joint and conditional probabilities of associations between AATAG loci and CR1-L3 and Rex. Genomic joint probabilities are shown in orange and light blue for CR1-L3 and Rex, respectively. AATAG-adjacent conditional probabilities are shown in red and dark blue for CR1-L3 and Rex, respectively
Largest instance of microsatellite seeding among vertebrates
A peculiar feature of SSR evolutionary dynamics in squamate genomes is the significant shifts in 4mer and 5mer abundances across the squamate tree, including extreme expansion of specific 4mer and 5mer SSRs motifs in colubroid snake genomes (Kruskal–Wallis test p value <0.001, Supplementary Fig. 6 and Supplementary Data 6). Two specific SSR sequence motifs, ATAG and AATAG, account for most of the microsatellite expansion in colubroid snakes, representing a 7.4-fold increase in ATAG (bp/Mbp) and an 87.7-fold increase in AATAG (bp/Mbp) compared to the averages of other squamate genomes (Supplementary Figs. 7 and 8). The extremely high genomic representation of these two similar SSR sequence motifs in snake genomes suggests a motif-specific mechanism has driven their expansion. Previous studies12,13 have suggested that LINE retrotransposons that contain microsatellites on their 3′ end in snakes might lead to SSR genomic expansion through a process called "microsatellite seeding."
To test the hypothesis that microsatellite seeding is responsible for the expansion of particular SSR sequence motifs, we surveyed the regions adjacent to the two most highly expanded SSR motifs (AATAG and ATAG) in eight complete reptile genome assemblies. Consistent with the expectations of microsatellite seeding, we found strong statistical support that CR1-L3 LINEs tend to be immediately adjacent to AATAG loci in colubroid genomes (Fisher's exact test p value <2.2e−16), as well as strong statistical enrichment of AATAG loci at the 3′ end tail of Rex LINEs (p value <2.2e−16) in all squamate genomes sampled, suggesting that both CR1/CR1-L3 and Rex LINEs contribute to microsatellite seeding in squamate genomes (Fig. 2b and Supplementary Data 7). In contrast to elements adjacent to AATAG repeats, we found no evidence of enrichment in adjacency for any particular TE for the second most expanded SSR motif (ATAG) compared to randomly sampled genomic regions; this suggests that the expansion of this motif is not directly driven by microsatellite seeding, although its similarity to AATAG suggests that it might be indirectly related. To further identify the specific LINE element that is responsible for microsatellite seeding of AATAG SSR loci, we calculated the conditional probability of TE-SSR co-occurrence in a genome-wide context compared to the AATAG-adjacent context. Conditional probabilities of AATAG loci and CR1-like LINEs genomic co-occurrence are noticeably different only for CR1-L3 LINEs between colubroid snakes and other squamates (Fig. 2c), and are only barely detectable for Rex LINEs. Additionally, CR1 LINEs are a major contributor to the genomic TE landscape of squamates (particularly colubroid snakes), whereas Rex elements represent a very small fraction. Taken together, our data indicate that microsatellite seeding may be a common ancestral feature of multiple families of squamate LINEs, yet the high activity and expansion of CR1-L3 LINEs has driven associated AATAG loci to extremely high frequencies in colubroid snakes, leading to an astounding 74.73-fold genomic AATAG loci/Mbp increase in this lineage, and the highest levels of genomic SSR content among vertebrates. The ramifications of such extreme levels of homologous SSRs in colubroid snakes, in terms of genome function and evolution, remains uninvestigated. A potential role in mediating increased ectopic recombination leading to gene duplication has been suggested by previous studies that have identified an enrichment of these repeats surrounding tandemly duplicated venom genes in snakes12,29,30. Collectively, these findings imply the exciting possibility that LINE-SSR hybrid elements may have played key roles in the evolution of prominent phenotypes in snakes (i.e., venom evolution).
Multiple independent TE horizontal transfer events
Evidence for the horizontal transfer of BovB LINEs has been identified by previous studies12,31,32,33,34, and our analysis of squamate genomes provides new insight into the complexities of BovB horizontal transfer. Our phylogenetic reconstruction of BovB LINEs, including samples from our squamate genomes and other sequences from GenBank35, highlights multiple horizontal transfer events, and supports ectoparasite-mediated transfers of BovB LINEs into and out of squamate reptile genomes (Fig. 3 and Supplementary Fig. 9a, Supplementary Data 9). We found BovB LINE sequences from squamate species clustering with other groups of metazoans in all branches of our phylogenetic tree, consistent with multiple horizontal transfer events of BovB from lizards to mammals and to other squamates, and from snakes to mammals and other squamates. Previous studies found support for virus-mediated transfer of TEs36, and suggested ectoparasites as potential transmission vectors34,37,38,39,40. Our analyses support the horizontal transfer of BovB from one reptile tick species (Amblyomma limbatum) to colubroid snakes (Supplementary Fig. 9a), and provide the first ever evidence for ectoparasite-mediated transfer from squamate genomes in the case of the reptile tick Bothriocroton hydrosauri. Samples containing BovB elements sequenced from this tick species are deeply nested among lizard-derived BovB sequences, yet are unique in containing a large internal deletion (1691 nt) relative to all other lizard-derived BovB sequences in this clade. Collectively, our analyses of BovB LINE evolution showcase a dynamic history of horizontal transfer that encompasses essentially all forms of the process of transfer into and out of squamate genomes, implicating the role of ectoparasites in both directions of the transfer process.
Evidence for ectoparasite-mediated horizontal transfer of BovB LINEs in squamate reptile genomes. A summarized Bayesian phylogenetic tree of full-length BovB LINE sequences for 87 metazoan species, including two reptile ticks. Branches have been collapsed and colored to represent major clades. Posterior probabilities are shown only at nodes that had posterior support <0.99
Testing explanations of variation in genomic TE abundance
Multiple studies have suggested that purifying selection acting against TE insertions may manifest in correlations between Ne and features of the genomic TE landscape. This prevailing demographic explanation for variation in repeat content has been invoked to describe patterns of genome complexity and evolution across the tree of life, and predicts that lineages with higher Ne should undergo more effective purifying selection and thus lower genomic accumulation of mutationally hazardous DNA41,42. Indeed, previous population (within-species) and phylogenetic (among-species) studies have provided rationale and empirical evidence that TE insertion rates, fixation rates, and abundance may be correlated with Ne14,42,43,44,45. Relative insert length has also been linked to population size at the population level by an ectopic recombination model in which element length is correlated with the strength of selection14,18,43,46,47,48.
Using our phylogenetic-scale dataset, we tested if features of TE landscapes (i.e., genomic abundance, estimated age of activity, and degree of truncation for BovB and CR1-L3 LINEs) showed evidence of a correlation with estimates of Ne consistent with a demographic model of TE landscape evolution. We first tested for a relationship between Ne and TE landscape characteristics using the median values of Ne estimates derived from pairwise sequentially Markovian coalescent (PSMC) analyses49 for eight published squamate genomes (Fig. 4b–d and Supplementary Fig. 10). With this dataset, we found no evidence supporting a correlation between Ne and CR1-L3 and BovB length or genomic repeat element abundance (Fig. 4c, d and Supplementary Fig. 10c–e). Notably, we found that species with similar Ne estimates (Fig. 4b) showed different levels of truncation and of TE genomic abundance, and that even within a species TE truncation and abundance were poorly correlated (Fig. 4a, c, d and Supplementary Figs. 10 and 11). Second, to further test for correlations between Ne and element abundance or truncation using an approach that is independent of inferences of generation time and mutation rates, and independent of potential biases associated with coalescence-based estimates of Ne (i.e., population substructure, migration, selection)49,50,51,52,53,54,55, we used adult body mass as a proxy for Ne for all species included in our study (as in ref. 56; Supplementary Data 8)57. This approach has the added benefit of leveraging the much larger sample size of our entire dataset (compared to our PSMC analyses using eight complete genomes). Similar to our PSMC-based analyses, we compared body mass to CR1-L3 and BovB genomic abundance, their degree of truncation, and total genomic repeat element and TE abundances. Consistent with our PSMC-based analyses, we failed to find a correlation between body mass and truncation (Fig. 4e and Supplementary Fig. 12b) that would support a demographic model of TE landscape evolution; the only correlative trend that we did find was a correlative trend that is opposite of that predicted by the demographic model between Ne and genomic repeat element abundance instead (i.e., higher Ne was positively correlated with TE abundance; Supplementary Fig. 12d). Finally, to test more generally for evidence that selection acts on TE length at the phylogenetic scale, we tested for a link between TE truncation and TE age18,48,58 using median pairwise divergence of TE copies from their subfamily consensus, π, as a proxy for age for CR1-L3 and BovB families, and found no correlation (Fig. 4f, Supplementary Fig. 13, and detailed in Supplementary Figs. 14–16). While we acknowledge the complexity of testing links between two highly dynamic evolutionary processes (e.g., Ne and TE abundance), and the limitations of methods used to make inferences about these processes (i.e., Ne estimation), all of our analyses fail to provide support for Ne as a strong determinant of variation in the composition and characteristics of the repeat element landscape at the phylogenetic level across squamate reptiles. Although our analyses cannot fully reject a demographic hypothesis that a relationship between Ne and TE characteristics exists (i.e., we can only fail to reject a lack of relationship), the apparently poor explanatory power of the demographic hypothesis in predicting squamate TE activity and abundance suggests that perhaps other factors, such as variation in molecular mechanisms of TE proliferation, silencing, and removal, may better explain the majority of variation in TE abundance at the phylogenetic level in squamates.
Relationships between truncation, effective population size, body mass, and divergence estimates for CR1-L3 and BovB LINE retrotransposons among squamates. a Branches on the time-calibrated consensus phylogenies are colored according to the calculated 3′:5′ read depth coverage ratio for CR1-L3 (left) and for BovB (right) LINEs. Heat maps show the genomic content of CR1-L3 LINEs, total repeats, and BovB LINE retrotransposons represented as percentages of the total genome. For each major clade, violin plots show the density distributions of divergence estimates (pairwise π) for all CR1-L3 and BovB elements compared to the species-specific consensus sequence. b Variation in effective population size (Ne) over time for five snake species scaled by generation time and mutation rate ("g" and "u" on the x-axis). c Relationship between Ne and truncation of CR1-L3 (top) and of BovB (bottom) LINEs for eight squamate species. d Relationship between total genomic abundance of CR1-L3 (top) and BovB (bottom) LINEs and Ne. e Relationship between adult body mass and degree of truncation across 66 squamate species for CR1-L3 (top) and BovB (bottom) LINEs. f Relationship between age (median pairwise π) and truncation for CR1-L3 (top) and BovB (bottom). Summary statistics from phylogenetically independent contrasts (PIC) are shown as insets for each plot in c–f. Statistical analyses were performed after log transformation of truncation values in plots e and f
This broad glimpse into the diversity of repeat structure and composition of squamate reptile genomes suggests that this lineage possesses particularly distinct and often extreme repeat landscape characteristics compared to other amniotes. Our results provide evidence for surprisingly high variation in the content and composition of genomic repeat elements across squamate lineages, including 3-fold variation in the identifiable genomic repeat element content. We also discovered that some snake genomes have experienced microsatellite expansion at unprecedented scales through the process of microsatellite seeding by specific LINEs, leading to genomic microsatellite abundances that are the highest of any known vertebrate genome. Despite such extreme variation in genomic repeat element content, genome size across squamates is remarkably conserved (~0.2-fold variation), challenging the prevailing view that genomic repeat abundance and genome size tend to tightly co-evolve4. These findings provide some of the strongest evidence for a dynamic equilibrium or an "accordion" model, in which genomic DNA gain through TE expansion may be approximately balanced by genomic DNA loss through deletion7,58,59. Overall, these results highlight extreme shifts in the structure of squamate reptile genomes, and further beg the question of whether particular aspects of squamate genome function and evolution are also more unique and variable compared to other vertebrates. These findings argue that squamates may represent a particularly powerful model system for testing hypotheses about genome structure, function, and evolution, and their interactions.
Many previous studies focused on population-level dynamics of TE evolution have shown that differences in Ne and the efficacy of purifying selection acting against TE proliferation have played a major role in structuring the repeat landscape of many eukaryote genomes9,18,46,47,48,58,60,61,62,63. Even in squamate species (e.g., Anolis lizards), variation in Nes has been linked to TE insertion length and fixation probability18,62,63. Our phylogenetic-scale analyses across squamate species, however, recovered no clear evidence linking genomic repeat abundance or activity with Ne estimates in squamates. Although coalescent-based estimates of Ne can be biased by a number of model violations (i.e., population substructure, selection), we also failed to find a significant relationship between genomic repeat characteristics and body mass—a known correlate of Ne. Population size is, however, likely to have influenced other aspects of genome evolution, such as fixation of deletions, that could contribute to the maintenance of nearly constant genome size in squamates.
Our results together with those from previous studies suggest that different evolutionary forces may dominate different evolutionary scales, and that while demographic processes (and purifying selection) may dominate population-level trends in TE evolution, phylogenetic-scale patterns in TE landscapes may be more strongly determined by other processes. Evidence for extreme variation in transcriptional levels of TE-derived transcripts across squamates12, together with evidence from this study of lineage-specific swings in repeat element proliferation, suggest that molecular mechanisms related to TE regulation may be particularly relevant at the phylogenetic scale in squamates. Squamates may, therefore, represent a valuable system for studying the impacts of variation in molecular mechanisms of TE control, such PIWI-interacting RNA dynamics and efficacy, epigenetic silencing of TEs, lineage-specific TE activity, DNA repair mechanisms, and post-insertion 5′ removal of TEs. Further studies are needed to address the question of whether variation in molecular mechanisms of TE silencing and activity, as well as DNA repair, explain variation in squamate genomic TE content, and would provide fascinating insight into the factors that shape genomic repeat landscape variation.
Taxon sampling and library preparation
DNA extraction of 52 squamate samples (total = 45 species) was performed using a phenol–chloroform–isoamyl alcohol (PCI) extraction protocol. Random shotgun genome libraries were prepared by fragmenting DNA samples to an average length of 300–600 bp using a M220 Covaris Ultrasonicator. The NEBNext Illumina DNA Library Prep Kit (New England Biolabs) was used following the manufacturer's protocol to perform fragment-end repair, poly-A tailing, adapter ligation, and library amplification. After library preparation, fragments were size-selected using a BluePippin (Sage Science) for a length of 350–450 bp. Pooled multiplexed libraries were sequenced on an Illumina MiSeq with 300 bp paired-end reads. Paired reads were merged based on sequence overlap and were adapter and quality trimmed using CLC genomics workbench 9.0.1 64. Roche 454 shotgun sequencing data of nine snake species from previous studies12,13 and draft genome assemblies of 12 additional squamate species (Supplementary Data 1) were also included. Our final sampling included a total of 66 different squamate species. For each species, mitochondrial reads were filtered out in CLC genomics workbench 9.0.1 using the complete mitochondrial genome of the most closely related species available on GenBank35. Reads that mapped to the reference were used to assemble species-specific mitochondrial genomes. Reads that did not map to the reference (i.e., nuclear reads) were used for downstream repeat element annotation and analyses.
SSR identification and analysis
We used Pal_finder v.0.02.0365 (Palfinder hereafter) to identify microsatellites. Default Parfinder parameters were used to identify perfect dinucleotide (2mer), trinucleotide (3mer), and tetranucleotide (4mer) that were tandemly repeated for a total length of at least 12 bp. Perfect pentanucleotide (5mer) and hexanucleotide (6mer) tandemly repeated motifs were annotated only if longer than 15 bp. Loci/Mbp and bp/Mbp frequencies were calculated for all microsatellite motifs, length classes (2–6mers), and total content, and summarized per genome and major taxonomic group. Tests for multiple evolutionary rates of microsatellite abundance across lineages, ancestral state reconstruction of genomic microsatellite frequencies, and quantification of microsatellite landscape differentiation among species were performed using the R packages Phytools v.0.4–6066 and APE v.3.367. For the multiple evolutionary rate analysis of microsatellite (and TE) abundance, we conducted censored rate tests using Phytools with 1000 simulations (to compute p values) on 100 randomly sampled posterior trees using the restricted maximum likelihood technique to obtain unbiased estimates of the evolutionary rate parameter (σ)28. We used the time-calibrated phylogeny and the pic function in R (provided by the APE package) to compute phylogenetic independent contrasts for tests of clade-specific differences in genomic microsatellite content. We performed the nonparametric Kruskal–Wallis H test in R after the data rejected normality (Shapiro–Wilks test; p values <0.05 before and after log transformation) and homogeneity of variances (Bartlett's test; p values <0.05 before and after log transformation). Between lineages variation was tested using a post hoc Dunn test for multiple comparisons using the Benjamini–Hochberg correction method in R (Supplementary Data 6).
TE identification and analysis
Squamate genomic repeat elements were annotated according to homology-based and de novo identification approaches. Because repeat element annotation can be highly dependent on the repeat library used, we built large multi-species (clade-specific) repeat libraries that we used to annotate repeats for all members of a clade. To build these clade-specific libraries, we first performed de novo repeat element annotation on each species (except where already published) using RepeatModeler v.1.0.968, followed by further repeat classification in CENSOR69. Second, we built clade-specific de novo repeat element libraries, one for all lizard species (33 species de novo reference library) and one for all snake species (de novo TE libraries for 21 species were combined, and merged with the reference library generated by Castoe et al.13). Each clade-specific library was then filtered to avoid redundancy of highly similar elements. We tested whether using a single squamate-specific library for all species would change the inferred relative TE content and overall amount of repeat identified; we found no detectable difference between the results of the two masking protocols (Supplementary Fig. 17), and therefore decided to use the two clade-specific libraries in order to reduce masking time by reducing the overall library size. Additional classification of unknown (unclassified) elements was achieved by comparing these unclassified elements to all elements that were classified using BLAST70. Additionally, we generated squamate-specific BovB and CR1-L3 LINEs reference sequence libraries for all 66 species included (additional information regarding library generation are provided in the following paragraph).
Repeat element analyses were performed in RepeatMasker v.4.0.671 with default parameter settings. To maximize element identification, we used a custom bash script to specify the order of the four libraries used as references for the masking process: (i) BovB-L3 LINEs library, (ii) Tetrapoda RepBase library (version 20.11, 07 August 201572), (iii) classified elements from the clade-specific library for either snakes or lizards, and (iv) unknown elements from the clade-specific library. We used the BovB-L3 LINEs library first to control for limited sampling and low-quality reference sequences of squamate reptile BovB and L3 LINEs in the tetrapoda library. RepeatMasker output files were post-processed using a custom-modified implementation of the ProcessRepeat script included in the RepeatMasker package. Specifically, we modified the output to include additional summary information in the .tab output file for TE subfamilies that are important and/or frequent in squamate reptiles (e.g., CR1-L3, L2, and Rex). Also, because the provided ProcessRepeat script still reflects old and outdated classification schemes of TEs (e.g., Penelope elements are inappropriately classified as LINEs), we made other modifications to the ProcessRepeat script to correct for such errors according to the classification reported by Chalopin et al6.
Comparing sampled and assembled genomes
We tested whether genomic repeat content estimated from unassembled shotgun genomic datasets were similar to estimates derived from fully assembled genomes. We compared RepeatMasker estimates of total TE genomic abundance between assembled genomes and unassembled shotgun genomic datasets for the same species (Python molurus, Boa constrictor, Thamnophis sirtalis, and Deinagkistrodon acutus) or for two closely related species belonging to the same genus (Gekko gecko vs. Gekko japonicus and Ophisaurus attenuatus vs. Ophisaurus gracilis). We also tested for potential biases due to unequal genomic sampling in the shotgun datasets. We extracted at random subsamples of 3, 5, 8, 10, 30, 50, 100, and 250 Mbp from unassembled genomic shotgun datasets of four species (Python molurus, Gekko gecko, Ophisaurus attenuatus, and Pantherophis emoryi), and compared RepeatMasker estimates of total TE genomic abundance for each. Read extraction was performed using the subsample_fasta.py script from the QIIME pipeline73. Finally, we compared RepeatMasker estimates of total TE genomic abundance in relation to the amount of sequence data obtained for all Illumina and 454 genomic shotgun datasets to test for biases related to sequencing technology, and for biases related to the amount of sequence data collected per individual, vs. estimates of total TE genomic abundance.
CR1 and BovB LINEs phylogenetic and evolutionary analyses
Species-specific consensus sequences for both CR1-L3 and BovB LINE retrotransposons were generated in CLC genomic workbench 9.0.1 using default parameters, a linear gap cost, and the global alignment setting. Nuclear reads for each species were mapped to the consensus sequence of the LINE consensus sequence from the most closely related species available, which was used as initial reference (e.g., both CR1-L3 and BovB reference sequences for the Burmese python were generated by Castoe et al.13 and used as reference for building the consensus for the Mexican burrowing python). The first consensus generated was then used as a new reference for further rounds of re-mapping of nuclear reads until no additional mapping reads were recovered. Consensus sequences were determined by simple majority rule consensus, removing regions with coverage <10x after the second mapping iteration, and <20x in the final mapping. Consensus sequences were aligned in ClustalW74 with a gap open penalty of 50, and alignments were manually adjusted prior to downstream analyses (Supplementary Data 10). To the CR1 consensus sequences generated from our 66 squamate species, we added CR1-L3 and CR1-L2 vertebrate consensus sequences available in RepBase, for a total of 155 sequences (Supplementary Data 10). Squamate BovB consensus sequences we generated from our 66 squamates were combined with other metazoan consensus sequences available in RepBase, for a total of 87 sequences (Supplementary Data 9). Bayesian phylogenetic tree reconstruction analyses of squamate CR1 and BovB LINEs were performed in BEAST275. Two independent analyses were run for 200 million generations each, following the Yule model of speciation and a relaxed log-normal clock model; MCMC chains were sampled every 1000 generations. The program Tracer v.1.676 was used to confirm that the MCMC chains had reached convergence. We conservatively discarded the first 25% of collected MCMC generations as burn-in, based on evidence that the likelihood and parameter values reached stationarity after approximately 15% of the sampling process.
CR1 and BovB LINEs coverage and age analyses
For each species, the species-specific CR1-L3 and BovB consensus sequence was used as a reference to estimate read coverage using the BWA mem alignment tool77, and the BEDTools2 (version 2.26.0) coverage tool78. Coverage counts were normalized by the total number of reads aligned to the full-length reference sequence. Read coverage was estimated for: (i) each 10 bp sliding window, (ii) for the first and second half of the reference sequence, and (iii) for each third of the reference.
We used pairwise sequence divergence from the consensus (pairwise π) as a proxy to infer age and relative element level of activity through time. Pairwise distances values for each element and species were estimated following a custom pipeline starting from BWA alignments. An R79 custom script built on the pegas80 and stringr packages was used to calculate pairwise π estimates using multi-fasta pairwise alignments of reads to the reference. Because we expected multiple TE subfamilies to exist, sequence divergence was estimated by excluding sites that define different CR1 and BovB subfamilies. For each species, we calculated the relative nucleotide frequency for each position in the multiple sequence alignment, and then calculated the mode of the frequency distribution (bins of 0.01) of the most frequent nucleotide at each position. Sites for which the most frequent nucleotide was in a bin more than three bins away from the mode were discarded as defining a separate subfamily.
Time-calibrated phylogeny of 66 squamate reptiles
We estimated a time-calibrated phylogeny for the 66 squamate species in our study and an additional eight outgroup vertebrates. We downloaded and parsed 12 mitochondrial-encoded protein-coding genes for each species with a mitochondrial genome sequence available on GenBank. The same genes were parsed from our de novo assembled mitochondrial genomes after genes were annotated for these using MITOS81. We aligned the 12 protein coding genes encoded on the mitochondrial heavy strand using MUSCLE v.3.8.2182 and concatenated the sequences into an alignment that we used for divergence dating (10,479 bp). Prior to divergence dating, we estimated the best-fit partitioning scheme and associated models of nucleotide substitution using Bayesian information criterion and the heuristic search algorithm provided in PartitionFinder v.1.1.183. We provided a starting partitioning scheme that defined 36 partitions (splitting codon positions for each of the 12 genes), and PartitionFinder identified the best-fit partitioning scheme comprising a single partition for each codon position (three total) and a GTR+I+G model for each partition. We estimated divergence times using BEAST v.2.3.484 with a calibrated Yule model of speciation and a log-normal relaxed clock model. We constrained the topology to that provided from previous studies of the squamate phylogeny and diversification85,86; we also constrained divergence times of a total of seven nodes using fossil calibrations also provided in previous studies. Calibration points and associated prior distributions are given in Supplementary Table 1. Two independent MCMC runs were conducted for 100 million generations each, with MCMC chain sampling every 10,000 generations. We assessed convergence to the posterior based on likelihood and parameter stationarity (effective sample size >200 for all parameters) using the program Tracer. We discarded the first 10% of generations as burn-in, based on the likelihood and parameter values exhibiting stationarity before 10% of sampling was completed.
AATAG microsatellite seeding by TE analyses
We performed adjacency analyses of AATAG and ATAG SSR loci on high-quality assembled genomes for seven snake species, and used the green anole lizard as an outgroup. To increase specificity, genomes were first masked only for simple repeats. We extracted coordinates of annotated AATAG and ATAG SSR loci from the .out RepeatMasker output files, and used these coordinates to extract target regions 400 bp upstream and downstream of each microsatellite locus. We then performed a second run of RepeatMasker to mask only TEs located in the extracted target regions that flank AATAG and ATAG loci. Following this strategy, we were able to annotate TEs located in close proximity to SSR loci, and to differentiate TEs that harbor microsatellite-like regions in their reference sequences. The composition of TEs physically associated with SSR loci regions was then compared to the average of five independent randomly generated genomic backgrounds matching in sample size the corresponding microsatellite landscape. For each species, genomic background reads were generated by using the random tool in the BEDTools2 v.2.26.0 package, in which we specified the number of sequences to be extracted and that their length was to match the SSR-adjacent genomic subsample. The generation of random bed files was performed independently five times per species, the TE composition was averaged across these five genomic backgrounds, and then compared to SSR loci adjacent regions. Fisher's one-tailed exact tests were performed to evaluate the enrichment of TE families in SSR loci regions (at α = 0.01). Finally, to identify the specific element types involved in microsatellite seeding, we estimated genomic and SSR-adjacent conditional probabilities of TE-SSR co-occurrences. We estimated the conditional probability of sampling an AATAG SSR with an adjacent CR1 LINE present within 400 bp, and compared this to the estimated joint probability of sampling an AATAG SSR locus and a CR1 LINE using the genome-wide frequencies. We also calculated the conditional and joint probabilities for Rex LINEs, and compared those to the conditional and joint probabilities of CR1 LINEs, respectively.
Effective population size (N e) estimation
Whole genomic Illumina paired-end reads for eight squamate reptiles species were first preprocessed for quality using Trimmomatic87. Clean paired and unpaired reads were aligned to their respective reference genome assemblies using BWA v.0.7.12, and single nucleotide polymorphisms were called with SAMtools (v.0.1.18) mpileup88. We applied the PSMC49 using a generation time of 3 years across all eight species (which represents the average of generation time approximations available from the literature; Supplementary Table 2) after verifying that the application of a single generation time yielded results consistent with estimates of average Ne produced by the application of generation times within the range reported in the literature. Multiple studies have provided evidence of relatively similar mutation rates across lineages of squamates13,89. Therefore, in our PSMC analyses we used the generalized squamate mutation rate reported in Green et al.89 of 2.4 × 109 /year/site (as estimated from 4-fold degenerate sites between anole and python). To test the robustness of inferred population size estimates, we conducted 100 bootstrap replicate analyses by splitting the scaffolds into smaller segments and randomly sampling the segments with replacement. Default outputs of the psmc_plot.pl script were used to graphically summarize Ne changes over time estimations per each bootstrapped sample (Supplementary Fig. 10b).
Coalescent approaches for estimating Ne and changes in Ne over time (like PSMC) have several intrinsic limitations. Importantly, they rely on explicit assumptions of a single population coalescent model (without subdivision, gene flow, or selection) to estimate the time since the most recent common ancestor of alleles at each locus, as well as an assumed generation time and substitution rate. Population structure has been identified as one major factor that can bias PSMC-based estimates of Ne50,52,90,91. For example, the inferred trend in Ne variation of a structured population can portrait either a bottleneck or an expansion in population size whether the alleles were sampled from the same subpopulation or from different subpopulations, respectively51. Episodes of natural selection can also bias estimates of Ne obtained using PSMC, as selection can manipulate the rate of coalescence at specific loci that are directly or indirectly linked to targets of selection54,55. Given the nature of our data, we are not able to assess the presence and extent of population substructure or selection, and therefore cannot exclude that our PSMC estimates are immune to such biases. Additionally, PSMC has low power at recovering rapid changes in Ne, which may be incorrectly estimated to have occurred over a longer period of time, and cannot recover recent nor very ancient changes in Ne (e.g., younger than ~10 kyBP and older than ~3 myBP for humans)49,51. Thus, we suggest caution when interpreting our PSMC estimates of Ne and Ne changes through time. However, we found low variance across bootstrapped Ne estimates once the most recent and most ancient time points were removed, and patterns of expansion and contraction of Ne are consistent with alternations of glacial and interglacial periods during the middle Miocene climate transition, the Pliocene and the Pleistocene92. In an attempt to reduce potential biases associated with PSMC estimates of recent and ancient changes in Ne, median Ne values were calculated after removing the first and the last time points from each sample. We replicated each analysis (see below) after applying different filtering schemes to the standard PSMC outputs (e.g., removal of 10 and 25% of time point data, and inclusion of only time points between 20 kyBP and 10 myBP). Since all tests provided the same conclusions, we report only analyses performed using median Ne values that were calculated according to the original filtering scheme. Additionally, we replicated all of our analyses using adult body mass as a proxy for Ne56 to avoid potential biases associated with our coalescence-based methods of Ne estimation (i.e., Fig. 4e). For each of the 66 squamate species, we obtained adult body mass measurements from the literature57 which were used to further test for a demographic explanation for variation in repeat content alongside coalescent-based estimates of Ne.
Testing demographic explanations of repeat content variation
We performed linear regression analyses to test for correlations between Ne and LINE truncation, Ne and genomic abundance of BovB and CR1-L3 LINEs, truncation and genomic abundance of repeats, and between truncation and estimates of ages of repeat element activity. We used the pic function in APE and the time-calibrated phylogeny to compute phylogenetic independent contrasts to be used for all linear regressions. These analyses were conducted for both the coalescent-based estimates of Ne and adult body mass as a proxy for Ne. Since truncation values violated assumptions of normality and homogeneity of variance (Shapiro–Wilks test; p values <0.05 and Bartlett's test; p values <0.05), we performed statistical analyses on log-transformed values (Shapiro–Wilks test; p values >0.05 and Bartlett's test; p values >0.05).
New raw, unassembled shotgun sequencing data and new assembled genome data have been deposited at NCBI under the following accessions:PRJNA413172 and PRJNA413201. The authors declare that all data and scripts used in this study are available via public databases or available from the corresponding author upon request.
Smit, A. F. A., Hubley R. & Green P. RepeatMasker Genomic Datasets. http://www.repeatmasker.org/genomicDatasets/RMGenomicDatasets.html. Accessed August 2017.
Platt, R. N., Vandewege, M. W. & Ray, D. A. Mammalian transposable elements and their impacts on genome evolution. Chromosome Res. 26, 25–43 (2018).
de Koning, A. P. J., Gu, W. J., Castoe, T. A., Batzer, M. A. & Pollock, D. D. Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet. 7, doi: 10.1371/journal.pgen.1002384 (2011).
Elliott, T. A. & Gregory, T. R. What's in a genome? The C-value enigma and the evolution of eukaryotic genome content. Philos. Trans. R. Soc. Lond. Ser. B 370, 20140331 (2015).
Canapa, A., Barucca, M., Biscotti, M. A. & Forconi, M. & Olmo, E. Transposons, genome size, and evolutionary insights in animals. Cytogenet. Genome Res. 147, 217–239 (2016).
Chalopin, D., Naville, M., Plard, F., Galiana, D. & Volff, J. N. Comparative analysis of transposable elements highlights mobilome diversity and evolution in vertebrates. Genome Biol. Evol. 7, 567–580 (2015).
Kapusta, A., Suh, A. & Feschotte, C. Dynamics of genome size evolution in birds and mammals. Proc. Natl. Acad. Sci. USA 114, E1460–E1469 (2017).
Agren, J. A. & Wright, S. I. Co-evolution between transposable elements and their hosts: a major factor in genome size evolution? Chromosome Res. 19, 777–786 (2011).
Blass, E., Bell, M. & Boissinot, S. Accumulation and rapid decay of non-LTR retrotransposons in the genome of the three-spine stickleback. Genome Biol. Evol. 4, 687–702 (2012).
Murphy, W. J., Pringle, T. H., Crider, T. A., Springer, M. S. & Miller, W. Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res. 17, 413–421 (2007).
Gregory T. R. Animal genome size database. http://www.genomesize.com/. Accessed August 2017.
Castoe, T. A. et al. Discovery of highly divergent repeat landscapes in snake genomes using high-throughput sequencing. Genome Biol. Evol. 3, 641–653 (2011).
Castoe, T. A. et al. The Burmese python genome reveals the molecular basis for extreme adaptation in snakes. Proc. Natl. Acad. Sci. USA 110, 20645–20650 (2013).
Lynch, M. & Conery, J. S. The origins of genome complexity. Science 302, 1401–1404 (2003).
Huang, C. R. L., Burns, K. H., & Boeke, J. D. Active transposition in genomes. Annu. Rev. Genet. 46, 651–675 (2012).
Suh, A. et al. Multiple lineages of ancient CR1 retroposons shaped the early genome evolution of amniotes. Genome Biol. Evol. 7, 205–217 (2015).
Alfoldi, J. et al. The genome of the green anole lizard and a comparative analysis with birds and mammals. Nature 477, 587–591 (2011).
Tollis, M. & Boissinot, S. Lizards and LINEs: selection and demography affect the fate of L1 retrotransposons in the genome of the green anole (Anolis carolinensis). Genome Biol. Evol. 5, 1754–1768 (2013).
Yin, W. et al. Evolutionary trajectories of snake genes and genomes revealed by comparative analyses of five-pacer viper. Nat. Commun. 7, 13107 (2016).
Brouha, B. et al. Hot L1s account for the bulk of retrotransposition in the human population. Proc. Natl. Acad. Sci. USA 100, 5280–5285 (2003).
Zhang, G. J. et al. Comparative genomics reveals insights into avian genome evolution and adaptation. Science 346, 1311–1320 (2014).
Boissinot, S., Entezam, A. & Furano, A. V. Selection against deleterious LINE-1-containing loci in the human lineage. Mol. Biol. Evol. 18, 926–935 (2001).
Hellen, E. H. & Brookfield, J. F. Alu elements in primates are preferentially lost from areas of high GC content. PeerJ 1, e78 (2013).
Fryxell, K. J. & Moon, W. J. CpG mutation rates in the human genome are highly dependent on local GC content. Mol. Biol. Evol. 22, 650–658 (2005).
Rizzon, C., Marais, G., Gouy, M. & Biemont, C. Recombination rate and the distribution of transposable elements in the Drosophila melanogaster genome. Genome Res. 12, 400–407 (2002).
Georges, A. et al. High-coverage sequencing and annotated assembly of the genome of the Australian dragon lizard Pogona vitticeps. Gigascience 4, 45 (2015).
Neff, B. D. & Gross, M. R. Microsatellite evolution in vertebrates: inference from AC dinucleotide repeats. Evolution 55, 1717–1733 (2001).
Adams, R. H. et al. Microsatellite landscape evolutionary dynamics across 450 million years of vertebrate genome evolution. Genome 59, 295–310 (2016).
Dowell, N. L. et al. The deep origin and recent loss of venom toxin genes in rattlesnakes. Curr. Biol. 26, 2434–2445 (2016).
Ikeda, N. et al. Unique structural characteristics and evolution of a cluster of venom phospholipase A2 isozyme genes of Protobothrops flavoviridis snake. Gene 461, 15–25 (2010).
Kordis, D. & Gubensek, F. Bov-B long interspersed repeated DNA (LINE) sequences are present in Vipera ammodytes phospholipase A2 genes and in genomes of Viperidae snakes. Eur. J. Biochem. 246, 772–779 (1997).
Kordis, D. & Gubensek, F. The Bov-B lines found in Vipera ammodytes toxic PLA2 genes are widespread in snake genomes. Toxicon 36, 1585–1590 (1998).
Kordis, D. & Gubensek, F. Unusual horizontal transfer of a long interspersed nuclear element between distant vertebrate classes. Proc. Natl. Acad. Sci. USA 95, 10704–10709 (1998).
Walsh, A. M., Kortschak, R. D., Gardner, M. G., Bertozzi, T. & Adelson, D. L. Widespread horizontal transfer of retrotransposons. Proc. Natl. Acad. Sci. USA 110, 1012–1016 (2013).
Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Sayers, E. W. GenBank. Nucleic Acids Res. 44, D67–D72 (2016).
Piskurek, O. & Okada, N. Poxviruses as possible vectors for horizontal transfer of retroposons from reptiles to mammals. Proc. Natl. Acad. Sci. USA 104, 12046–12051 (2007).
Novick, P., Smith, J., Ray, D. & Boissinot, S. Independent and parallel lateral transfer of DNA transposons in tetrapod genomes. Gene 449, 85–94 (2010).
Silva, J. C., Loreto, E. L. & Clark, J. B. Factors that affect the horizontal transfer of transposable elements. Curr. Issues Mol. Biol. 6, 57–71 (2004).
Gilbert, C., Schaack, S., Pace, J. K. II, Brindley, P. J. & Feschotte, C. A role for host–parasite interactions in the horizontal transfer of transposons across phyla. Nature 464, 1347–1350 (2010).
Gilbert, C. et al. Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes. Proc. R. Soc. Ser. B 281, 20141122 (2014).
Charlesworth, B. Effective population size and patterns of molecular evolution and variation. Nat. Rev. Genet. 10, 195–205 (2009).
Lynch, M. & Walsh, B. The Origins of Genome Architecture (Sinauer Associates, Sunderland, MA, 2007).
Petrov, D. A., Aminetzach, Y. T., Davis, J. C., Bensasson, D. & Hirsh, A. E. Size matters: non-LTR retrotransposable elements and ectopic recombination in Drosophila. Mol. Biol. Evol. 20, 880–892 (2003).
Le Rouzic, A., Boutin, T. S. & Capy, P. Long-term evolution of transposable elements. Proc. Natl. Acad. Sci. USA 104, 19375–19380 (2007).
Article ADS PubMed Google Scholar
Blumenstiel, J. P., Chen, X., He, M. M. & Bergman, C. M. An age-of-allele test of neutrality for transposable element insertions. Genetics 196, 523–538 (2014).
Song, M. Z. & Boissinot, S. Selection against LINE-1 retrotransposons results principally from their ability to mediate ectopic recombination. Gene 390, 206–213 (2007).
Petrov, D. A., Fiston-Lavier, A. S., Lipatov, M., Lenkov, K. & Gonzalez, J. Population genomics of transposable elements in Drosophila melanogaster. Mol. Biol. Evol. 28, 1633–1644 (2011).
Barron, M. G., Fiston-Lavier, A. S., Petrov, D. A. & Gonzalez, J. Population genomics of transposable elements in Drosophila. Annu. Rev. Genet. 48, 561–581 (2014).
Li, H. & Durbin, R. Inference of human population history from individual whole-genome sequences. Nature 475, 493–U484 (2011).
Nielsen, R. & Beaumont, M. A. Statistical inferences in phylogeography. Mol. Ecol. 18, 1034–1047 (2009).
Mazet, O., Rodriguez, W., Grusea, S., Boitard, S. & Chikhi, L. On the importance of being structured: instantaneous coalescence rates and human evolution—lessons for ancestral population size inference? Heredity 116, 362–371 (2016).
Nadachowska-Brzyska, K., Burri, R., Smeds, L. & Ellegren, H. PSMC analysis of effective population sizes in molecular ecology and its application to black-and-white Ficedula flycatchers. Mol. Ecol. 25, 1058–1072 (2016).
Orozco-TerWengel, P. The devil is in the details: the effect of population structure on demographic inference. Heredity 116, 349–350 (2016).
Schrider, D. R., Shanku, A. G. & Kern, A. D. Effects of linked selective sweeps on demographic inference and model selection. Genetics 204, 1207–1223 (2016).
Adams, R. H., Schield, D. R., Card, D. C. & Castoe T. A. Assessing the impacts of positive selection on coalescent-based species tree estimation and species delimitation. Syst. Biol. https://doi.org/10.1093/sysbio/syy034 (2018).
Figuet, E. et al. Life history traits, protein evolution, and the nearly neutral theory in amniotes. Mol. Biol. Evol. 33, 1517–1527 (2016).
Feldman, A., Sabath, N., Pyron, R. A., Mayrose, I. & Meiri, S. Body sizes and diversification rates of lizards, snakes, amphisbaenians and the tuatara. Glob. Ecol. Biogeogr. 25, 187–197 (2016).
Neafsey, D. E., Blumenstiel, J. P. & Hartl, D. L. Different regulatory mechanisms underlie similar transposable element profiles in pufferfish and fruitflies. Mol. Biol. Evol. 21, 2310–2318 (2004).
Petrov, D. A. Mutational equilibrium model of genome size evolution. Theor. Popul. Biol. 61, 531–544 (2002).
Charlesworth, B., Sniegowski, P. & Stephan, W. The evolutionary dynamics of repetitive DNA in Eukaryotes. Nature 371, 215–220 (1994).
Le Rouzic, A., Payen, T. & Hua-Van, A. Reconstructing the evolutionary history of transposable elements. Genome Biol. Evol. 5, 77–86 (2013).
Ruggiero, R. P., Bourgeois, Y. & Boissinot, S. LINE insertion polymorphisms are abundant but at low frequencies across populations of Anolis carolinensis. Front. Genet. 8, 44 (2017).
Xue, A. T., Ruggiero, R. P., Hickerson, M. J. & Boissinot, S. Differential effect of selection against line retrotransposons among vertebrates inferred from whole-genome data and demographic modeling. Genome Biol. Evol. 10, 1265–1281 (2018).
CLC Genomics Workbench 9.0.1. https://www.qiagenbioinformatics.com/.
Castoe, T. A. et al. Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence. Mol. Ecol. Resour. 10, 341–347 (2010).
Revell, L. J. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, 217–223 (2012).
Paradis, E., Claude, J. & Strimmer, K. APE: Analyses of Phylogenetics and Evolution in R language. Bioinformatics 20, 289–290 (2004).
Smit, A. F. A. & Hubley, R. RepeatModeler Open-1.0.9. http://www.repeatmasker.org/RepeatModeler/ (2008–2017).
Kohany, O., Gentles, A. J., Hankus, L. & Jurka, J. Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor. BMC Bioinform. 7, 474 (2006).
Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9 (2008).
Smit, A. F. A., Hubley, R. & Green, P. RepeatMasker Open-4.0.2. http://www.repeatmasker.or (2013–2015).
Bao, W., Kojima, K. K. & Kohany, O. Repbase update, a database of repetitive elements in eukaryotic genomes. Mob. DNA 6, 11 (2015).
Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).
Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).
Bouckaert, R. et al. BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput. Biol. 10, e1003537 (2014).
Rambaut, A. & Drummond, A. J. Tracer v.1. 4. http://beast.bio.ed.ac.uk/Tracer (2007).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Team R. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria, 2013).
Paradis, E. pegas: an R package for population genetics with an integrated-modular approach. Bioinformatics 26, 419–420 (2010).
Bernt, M. et al. MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313–319 (2013).
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).
Lanfear, R., Calcott, B., Ho, S. Y. & Guindon, S. Partitionfinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol. Biol. Evol. 29, 1695–1701 (2012).
Drummond, A. J. & Rambaut, A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214 (2007).
Benton, M. J. & Donoghue, P. C. J. Paleontological evidence to date the tree of life. Mol. Biol. Evol. 24, 26–53 (2007).
Pyron, R. A., Burbrink, F. T. & Wiens, J. J. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evol. Biol. 13, 93 (2013).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Green, R. E. et al. Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs. Science 346, 1254449 (2014).
Mazet, O., Rodriguez, W. & Chikhi, L. Demographic inference using genetic data from a single individual: separating population size variation from population structure. Theor. Popul. Biol. 104, 46–58 (2015).
Article PubMed MATH Google Scholar
Boitard, S., Rodriguez, W., Jay, F., Mona, S. & Austerlitz, F. Inferring population size history from large samples of genome-wide molecular data—an approximate Bayesian computation approach. PLoS Genet. 12, e1005877 (2016).
Zachos, J., Pagani, M., Sloan, L., Thomas, E. & Billups, K. Trends, rhythms, and aberrations in global climate 65 Ma to present. Science 292, 686–693 (2001).
Support for this work was provided from startup funds from the University of Texas at Arlington to TAC. We acknowledge the Texas Advanced Computing Center (TACC) for providing access to computational resources.
Department of Biology, University of Texas at Arlington, 501S. Nedderman Drive, Arlington, TX, 76019, USA
Giulia I. M. Pasquesi, Richard H. Adams, Daren C. Card, Drew R. Schield, Andrew B. Corbin, Blair W. Perry, Jacobo Reyes-Velasco & Todd A. Castoe
Department of Biology, New York University Abu Dhabi, Saadiyat Island, United Arab Emirates
Jacobo Reyes-Velasco & Robert P. Ruggiero
Department of Biology, Institute for Genomics and Evolutionary Medicine, Temple University, Philadelphia, PA, 19122, USA
Michael W. Vandewege
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, 80045, USA
Jonathan A. Shortt
Giulia I. M. Pasquesi
Richard H. Adams
Daren C. Card
Drew R. Schield
Andrew B. Corbin
Blair W. Perry
Jacobo Reyes-Velasco
Robert P. Ruggiero
Todd A. Castoe
G.I.M.P. and T.A.C. designed and performed experiments, analyzed results, and wrote the manuscript; R.H.A., D.C.C., A.B.C., and D.R.S. collected data and analyzed results; J.R.-V. collected data; B.W.P., R.P.R., M.W.V., and J.A.S. analyzed results; all authors contributed to editing the final manuscript.
Correspondence to Todd A. Castoe.
Supplementary Data 1
Supplementary Data 10
Pasquesi, G.I.M., Adams, R.H., Card, D.C. et al. Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals. Nat Commun 9, 2774 (2018). https://doi.org/10.1038/s41467-018-05279-1
Accepted: 25 June 2018
Transcriptional dynamics of transposable elements in the type I IFN response in Myotis lucifugus cells
Giulia Irene Maria Pasquesi
Conor J. Kelly
Edward B. Chuong
Mobile DNA (2022)
Chromosome-scale genome assembly of the brown anole (Anolis sagrei), an emerging model species
Anthony J. Geneva
Sungdae Park
Jonathan B. Losos
Population genetic and genomic analyses of Western Massasauga (Sistrurus tergeminus ssp.): implications for subspecies delimitation and conservation
Rian Bylsma
Danielle K. Walkup
J. Andrew DeWoody
Conservation Genetics (2022)
Finding and extending ancient simple sequence repeat-derived regions in the human genome
David D. Pollock
Megaevolutionary dynamics and the timing of evolutionary innovation in reptiles
Tiago R. Simões
Oksana Vernygora
Stephanie E. Pierce


You have to go back in order to not make the same mistakes again.
I had to look my demons straight in the face for this album, I had to deal with many things I had kept hidden inside. This album reflects truthfully where I am.
Lions Den by Jess Vincent is recorded and mixed! We recorded this album in our home studio, over the last year. We are looking for your help to fund the important costly finishing processes for this album!


text:
All 75 included individuals were Chinese Han ethnicity and were diagnosed as ANSCLC with EGFR wild type
Posted intrpp
Posted by By kentlandsinitiative December 13, 2021
All 75 included individuals were Chinese Han ethnicity and were diagnosed as ANSCLC with EGFR wild type. studies reported 6-Mercaptopurine Monohydrate that the 5-year survival of non-small cell lung cancer (NSCLC) for all stages is approximately 15%.[4,5] However, the majority of them are diagnosed with advanced or metastatic disease.[6] Unfortunately, the relative 5-year survival rate among such kind of population is only 4%.[7,8] Targeted therapies have been reported to prolong survival for NSCLC,[9C13] especially for epidermal growth factor receptor (EGFR)-mutations by tyrosine kinase inhibitors (TKIs).[14,15] However, these kinds of therapies are not eligible for about 80% patients with NSCLC.[16] Although many tumors are reported not sensitive to single-agent intervention, combined treatment that targets multiple pathways may help to enhance clinical endpoint. It is reported that the signaling pathways of EGFR and cyclooxygenase-2 (COX-2) presents a novel mechanism of EGFR TKI therapy resistance for NSCLC treatment.[17,18] Additionally, it is responsible for tumor proliferation, invasion, and angiogenesis.[19] Thus, combined EGFR and COX-2 may help to potentiate responses for NSCLC. Although several studies have explored the efficacy of erlotinib combined celecoxib (EC) for the treatment of advanced non-small cell lung cancer (ANSCLC), no study specifically investigated the efficacy and safety of EC for ANSCLC patients with EGFR wild type alone.[20C22] Thus, in the present study, we analyzed the efficacy and toxicity of EC in patients with ANSCLC and EGFR wild type only among the Chinese Han population. 2.?Patients and methods 2.1. Ethic approval This study has been approved by the Ethical Committee of the First Affiliated Hospital of Jiamusi University. All patients provided the informed written consent. It was operated 6-Mercaptopurine Monohydrate at First Affiliated Hospital of Jiamusi University from May 2012 to June 2015. 2.2. Patients Seventy-five patients with the confirmed diagnosis of stage IIIB and IV ANSCLC and tumor tissue were available for mutation analysis. All patients were ANSCLC with EGFR wild type.[23] The status of Eastern Cooperative Oncology Group was 0 or 1.[24] Patients had normal functions of hematology, kidney, and liver. Cases were excluded if the subjects were pregnancy or breastfeeding or previous received EGFR 6-Mercaptopurine Monohydrate or COX-2 inhibitor, gastrointestinal ulceration, bleeding or perforation, and severe psychological disorders that affected the treatments. 2.3. Treatment schedule Patients often orally taken erlotinib 150?mg once daily, and celecoxib 200?mg trice daily for a total of 600? mg daily within 30-day cycle. All patients were given the above medication for up to 12 months. Then, patients kept on taking erlotinib orally until the disease progression or unacceptable toxicity achieved. 2.4. Outcome measurements The outcome measurements included progression-free survival (PFS), overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), progress disease (PD), and disease control rate (DCR). Moreover, toxicity was also documented, which was assessed based on the common terminology criteria for adverse events (V3.0).[25] 2.5. Statistical analysis In this study, all included patients were monitored and recorded daily for the treatment-related toxicity. The tumor size measurement was performed by using the standard of response evaluation criteria in solid tumors (RECIST) 1.1.[26] CR was defined as the total tumor disappearance. PFS was set as the time to receive the study medication to disease progression based on the RECIST. OS was calculated at the beginning of study medication applied to the date of death with any reasons. PFS and OS were evaluated by the KaplanCMeier method. All data were analyzed by using SPSS software 18.0 (IBM Corp, Armonk, NY). 3.?Results The characteristics of all included patients are presented in Table ?Table1.1. The mean age was 66.3 years old. All 75 included patients were Chinese Han ethnicity and were Rabbit Polyclonal to EIF3K diagnosed as ANSCLC with EGFR wild type. Of them, 35 (46.7%) subjects were males. As 6-Mercaptopurine Monohydrate for histology, 16 (21.3%) patients were squamous cell carcinoma, and 59 (78.7%) patients were adenocarcinoma. As for stage, 11 (14.7%) patients were IIIB, and 64 (85.3%) patients were IV. Table 1 Characteristics of included patients. Open in a separate window The CR, PR, SD, PD, and DCR of 2-year follow-up were 4.0% (3/75), 6.7% (5/75), 42.6% (32/75), 46.7% (35/75), and 53.3% (52/75), respectively (Table.
Proc Natl Acad Sci U S A 107:10178C10183
Character

redpajama_set_name: RedPajamaCommonCrawl


Andreas Johnson and Mason Marchment each scored twice while netminder Garret Sparks made 29 saves at the Marlies defeated the Texas Stars 6-1 in Game 7 to win their first Calder Cup and the first men's professional hockey title for the city of Toronot since 1967.
Colorado defeated the Florida Everblades in seven games to repeat as Kelly Cup champions in their final season before moving to the AHL next season. The Eagles rallied from a 2-1 deficit in the third period as Gabriel Verpaelst netted the game winner with 2:29 left.
The Wolves defeated the Port Huron Prowlers 3 games to 1 to win the Commissioner's Cup. This was their second FHL title having won it previously in 2015. Gavin Yates led the Wolves with six goals in the playoffs and was awarded the league's MVP.
The Havoc became the first fourth-seeded team to win the SPHL President's Cup beating the Peoria Rivermen 2 games to 1. Sy Nutkevtich, who scored a goal in each playoff game against Peoria, had 13 points (5 goals, 8 assists) in the postseason and was named the MVP. This was the Havoc's second league title. They last won it in 2010.
The Riveters knocked off the defending Isobel Cup champs with a 1-0 win over the Buffalo Beauts. Katie Fitzgerald was the game's MVP as she held Buffalo scoreless. Alexa Gruschow got the game's only goal in the first period for the Riveters. This was their first Isobel Cup title.
The Mudbugs came from behind to beat the Minot Minotauros 2-1 to win the Robertson Cup. Cole Quisenberry tipped home the game winner early in the third period for the Mudbugs. This was the Mudbugs' first title in the NAHL. The franchise had previously won titles in 1999, 2000, 2001 and 2011 in the Western Professional Hockey League (WPHL) and Central Hockey League (CHL).
The Force won their first title in team history winning the Clark Cup over the Youngstown Phantoms. The Force got a late goal from Ty Farmer and an empty net goal from Mark Senden wo win 4-2 in Game 4 and clinch the series 3 games to 1. It took 10 years, 667 games and 63 playoff games for their first title.
The Broncos captured the Memorial Cup for the first time in 25 years. Beck Malenstyn put in an empty net goal to shut the door on the Everett Silvertips for a 3-0 win in Game 6. It wasn't an easy path for the Broncos as it took them seven games to eliminate the Regina Pats in the first round and another seven games to advance past Moose Jaw in the second round. In the Eastern Conference final, they defeated Lethbridge 4 games to 2.
American Hockey League – The San Jose Barracuda and Grand Rapids Griffins are the first two teams to advance to the Calder Cup semifinals. San Jose eliminated San Diego four games to one and Grand Rapids made it by defeated Chicago by the same margin. On the other side of the bracket, the Hershey Bears lead the Providence Bruins three games to two and a chance to wrap it up at home tonight. The Syracuse Crunch and Toronto Marlies are tied at three games apiece with the deciding game to be played tomorrow night in Syracuse.
ECHL – The South Carolina Stingrays kept the Manchester Monarchs from taking a 3-0 lead in the series with a 5-3 win on home ice to cut Manchester's lead to 2-1 in the semifinals. The Toledo Walleye dropped their first home game of the playoffs on Saturday, losing to the Colorado Eagles 4-3. Their semifinal series is now tied at 1-1.
Federal Hockey League – Season completed. Danville Dashers are the champions.
Southern Professional Hockey League – Season completed. Macon Mayhem are the champions.
North American Hockey League – The Lone Star Brahmas won the Robertson Cup National Championship Game with a 3-0 win over the Aston Rebels. Max Prawdzik stopped all 17 shots that came his way and Drake Glover brought home Tournament MVP honors as the NAHL South Division's Lone Star Brahmas won their first-ever NAHL Robertson Cup National Championship on Sunday afternoon at the Essentia Health Duluth Heritage Sports Center.
United States Hockey League – The Sioux City Musketeers and Chicago Steel are tied 1-1 in the Clark Cup Final. Following Sioux City's 5-4 overtime victory in Game 1 and Chicago's 5-1 triumph in Game 2, the Steel will host the Musketeers in Game 3 of the 2017 Clark Cup Final at Fox Valley Ice Arena on Friday night.
Western Hockey League – For the first time in 40 years, the Seattle Thunderbirds are the league champions. History was made Sunday evening at the Brandt Centre in Regina as forward Alexander True lifted the Seattle Thunderbirds to an overtime win in Game 6 of the 2017 WHL Championship Series. The Danish forward scored 12:36 into the first overtime period, completing an electrifying comeback by the visitors. Co-captain Mathew Barzal was named WHL Playoffs MVP after registering 25 points (7G-18A) in 16 games.
Brampton Beast 1, Reading Royals 1.


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Finding Hope Ness
Ontario Power Generation still favors DGR nuclear waste option
February 6, 2020 February 12, 2020 / findinghopeness
A concept image of Ontario Power Generation's DGR for the permanent storage of low and intermediate-radioactive waste at the Bruce Nuclear Site
What are they doing now at Ontario Power Generation (OPG) headquarters in the wake of the recent vote of Saugeen Ojibway Nation (SON) members who voted NO to OPG's plan to bury nuclear waste deep underground at the Bruce Nuclear site?
Are they asking each other, where did we go wrong? Because they did, go wrong.
Are they adding up the huge waste of time, energy and many millions of dollars at public expense in the past two decades? That includes a lengthy and expensive series of public hearings conducted by a joint federal review panel. The hearings were an important part of a Government of Canada approval process that was unexpectedly pre-empted by a side deal commitment OPG made with the SON in 2013 not to build the proposed deep geological repository without their support. That "amounted to a requirement of SON consent," OPG says.
The joint federal review panel went ahead with the hearings that same year, and had to schedule more to meet the public demand to be heard. In 2015 the panel presented its report to the former Conservative government of Prime Minister Stephen Harper. But there was an election coming and the Harper government decided to delay making a final decision, leading to the issuing of a permit for construction by the Canadian Nuclear Safety Commission.
Then, the newly elected Liberal government of Prime Minister Justin Trudeau also put off making a final decision and asked OPG for more information. To this day, the decision has not been made. It's not clear what the government will do, now that OPG, an Ontario Crown Corporation, has said it will honour its commitment to SON. But it certainly appears OPG's application for a permit to build the DGR as planned will be withdrawn.
Is OPG prepared to say something like, sorry, we blew it? Because it did, time and time again, as far back as 2004, when in October of that year it signed a hosting agreement with Kincardine and other municipalities near the Bruce Nuclear generating station. The agreement required the municipalities to use their "best efforts" to support the proposed DGR or risk losing millions of dollars worth of payments, "at the sole discretion" of OPG. Thus, did the publicly-owned corporation show the extent it was prepared to go to get what it wanted.
OPG was prepared to offer the SON communities a compensation package reportedly worth $150 million if the result of the recent vote had been YES. But SON members, 1,058 of the 1,232 who voted, were not so easily bought off.
In the immediate aftermath of the vote OPG said it will look for "an alternate solution" for the permanent storage of low and intermediate-level nuclear waste that takes many thousands of years to lose its radioactivity.
If anybody had asked me, before or after the SON membership vote, I would have said the idea of burying nuclear waste anywhere near the shores of Lake Huron, one of The Great Lakes share between Canada and the U.S., was always a bad idea. And that's no matter how many scientific studies were done and concluded it was an okay thing to do. The optics were terrible, and the scope of the adverse public reaction largely predictable; except for the Town of Kincardine and other municipalities bought off by OPG in 2004, and sentiment elsewhere in the Grey-Bruce area where over the years nuclear has become a sacred, economic cow. There were also clear signs, literally, the DGR was bound to become a serious thorn in the side of U.S.-Canada relations if construction had been approved.
A map prepared by Michigan Radio in 2015
A mischievous mind might ask, is this truly the end of OPG's plans to build a deep geological repository somewhere, possibly even elsewhere in the SON territory, farther away than 1.2 kilometres from the shores of a Great Lake?
A closer look at OPG's January 31 media release shows OPG is still committed to the DGR concept, as an integral part of nuclear energy's role as a "vital tool in fighting climate change," as a, wait for it, "non-emitting source of electricity."
Under the heading of "Key Facts on OPG and the DGR project" the media release says "The DGR project, as submitted to the Canadian Nuclear Safety Commission in 2005, would have been built 680 metres below the Bruce site, in strong, dry and impermeable rock that has been isolated from the lake or any groundwater for hundreds of millions of years.
"High-level waste, or used fuel, in Canada is destined for a separate DGR for which a site selection process is underway, managed by a separate company, the Nuclear Waste Management Organization (NWMO)," OPG also notes. It does not mention that the Municipality of South Bruce, near Bruce Nuclear, and in the SON territory, is one of the two communities left on the NWMO's host-site short list.
Nor does, the OPG release say anything about OPG's long-time, senior-partner connection and working relationship with the NWMO.
The "Key Facts" part of the release also says, "The Municipality of Kincardine was a willing hosting community for OPG's DGR since 2005. Four adjacent municipalities also passed resolutions to support the project." It does not say how they were financially obligated to show that support.
Under the heading, "Frequently asked questions." another OPG document poses a question about the role of SON and other indigenous communities may play in developing an alternate solution for the permanent storage of low and intermediate-level nuclear waste:
"Over the years, OPG and SON have been building a relationship based on mutual respect, collaboration and trust. OPG looks forward to continuing this relationship, and committed to remain in dialogue with SON regarding permanent disposal of the waste in its territory," (my italics) the prepared answer says.
Getting back to the NWMO and OPG's connections to it, the Canadian government set up the NWMO in 2002 with a mandate to find a permanent solution for the permanent, long-term storage of the highly radioactive used fuel. At the time it was widely thought the most likely solution would be burial deep in the very hard and very old, igneous rock of the vast Canadian Shield. Indeed, a research centre in the shield area of northern Manitoba had already been set up to look at that idea.
The NWMO's board of directors is composed of representatives from all the owner/operators of nuclear reactors in Canada. Because most of them are in Ontario, OPG is in effect NWMO's senior partner. In 2003 OPG began to raise concerns at the NWMO about the Canadian Shield assumption and suggested there were sedimentary rock formations in southern Ontario that merited further study.
In July 2004 OPG received a report from Martin Mazurek of the Institute of Geological Sciences, University of Bern, Switzerland and shared it with the NWMO. The geo-scientific assessment examined "the suitability of Palaeozoic sedimentary rock occurring beneath southern Ontario to host a Deep Geological Repository (DGR) for used nuclear fuel."
The report concluded there were many good reasons why the shales and limestones beneath southern Ontario "provide a highly suitable environment to host a deep geological repository for spent fuel . . . From a geo-scientific perspective, the chance of success to complete a convincing safety case is substantial," the executive summary said. The body of the report also cited the "Bruce Megablock" as a specific area worthy of further consideration.
Soon after that, the NWMO shut down the Canadian Shield research centre in Manitoba because it was no longer necessary. Clearly, there had been a big turning point in the approach being taken in Canada to the deep-rock burial of nuclear waste.
At the time, I found the OPG-commissioned report amid numerous background materials on the NWMO website. Someone in that organization thought it was important enough to show to a Nuclear Waste Management Regional Forum for southern Ontario aboriginal communities in Toronto in the fall of 2004. Copies of the executive summary were distributed to the 19 First Nation Chiefs and other representatives of aboriginal communities on hand.
The Assembly of First Nations submitted a report about the results of the southern Ontario forum to the NWMO on Dec. 17, 2004. The deep geological repository idea was heavily criticized: "There was strong opposition to the idea that nuclear fuel waste can be 'isolated' from the environment in this way," There was also a lot of suspicion, an "underlying" feeling that the NWMO, the nuclear energy industry, and even the government, already favour that approach, the AFN said.
I don't know for a fact that the Chiefs and perhaps other members of the two SON First Nations at the time were at that meeting, but it seems more than likely.
I don't say that qualifies as fulfillment back then of a "duty to consult" when that was long before the Supreme Court of Canada made that ruling. But I do say OPG knew as early as 2004 that a DGR in southern Ontario was going to be a hard sell in any First Nation traditional territory
The hosting agreement between OPG and Kincardine and other municipalities near Bruce Nuclear had already been signed that same year. As mentioned above, a section of the agreement obligates the municipalities to use their "best efforts" to support the proposed DGR and its approval; otherwise, they would risk seeing the payments cancelled, at "the sole discretion" of OPG. At the time those grants were estimated to total up to $35 million. A timeline for "milestones" in the agreement anticipated 2017 would be a reasonable date to expect a licence for construction of the proposed DGR would be finally approved and issued.
Section 2.2 of hosting agreement says the payments "shall terminate immediately" and any money, including interest, still set aside in special accounts will "immediately be returned … if the DGR can't be advanced or constructed because of events outside of the reasonable or actual control of the parties."
So, it appears those payments, or any continuation of them, are finished, with OPG's decision to honor the results of the SON vote. But OPG has said municipalities would be consulted as part of the development of a new DGR proposal.
It also remains to be seen how the SON vote against the OPG deep-geological project could affect the NWMO`s consideration of South Bruce as a site for its proposed DGR for highly radioactive used, nuclear fuel. South Bruce is, after all, in the SON territory. The only other potential site under consideration is Ignace, in northern Ontario`s Canadian Shield region.
Canada's growing stockpile of used nuclear fuel is currently being storied in above ground facilities regarded as "temporary" at nuclear facilities, including OPG's Western Waste Management Facility at Bruce Nuclear, near the Lake Huron town of Kincardine.
While the two DGR's are supposed to be separate proposals, not surprisingly, they have often been confused in the public mind.
The press release announcing the results of the SON vote described it as an "historic exercising of our Aboriginal and Treaty Rights and free, prior and informed consent in our Territory. The Communities have voted against the DGR."
It said 170 votes were cast in favor of the development, while 1,058 were opposed, and four ballots were spoiled, for a total of 1,232. There were four spoiled ballots.
The release quoted Chief Lester Anoquot of the Chippewas of Saugeen First Nation speaking to the importance of the vote as an "historic milestone and momentous victory for our People."
Chief Anoquot said, "We worked for many years for our right to exercise jurisdiction in our Territory and the free, prior and informed consent of our People to be recognized," he said. "As Anishinaabe, we didn't ask for this waste to be created and stored in our Territory, but it is here. We have a responsibility to our Mother Earth to protect both her and our Lands and Waters. Today, our People have voted against the DGR; tells us that we must work diligently to find a new solution for the waste."
That doesn't like SON is anxious to keep its DGR options open.
OPG isn't going to give up on the DGR concept easily. It has a lot of time, energy, and money — public money — invested in it. At times OPG was so eager to get what it wanted it went too far to manipulate outcomes, for example, in its dealings with municipalities in Bruce Nuclear area. It used its place as senior partner in the NWMO to open the door wide to the deep burial of nuclear waste in southern Ontario's sedimentary rock formations, highly radioactive used fuel, as well as its own low and intermediate level nuclear waste. That concept, with the possibility of two DGRs somewhere near the Bruce Nuclear site, appears to be still in play.
In fairness to OPG, the deep-rock burial idea for the long-term storage of nuclear waste (500 to a million years) has been around for a long time. Atomic Energy Canada Limited (AECL) came up with it in 1988. AECL suggested burial in what it called the 'plutonic rock' of the Canadian Shield.
The Canadian government reacted by setting up an environmental review panel, the 'Seaborn Panel,' to study that concept in more detail, and travel the country to gauge public attitude toward the idea. The panel found a lot of skepticism. It the Seaborn panel 10 years to present its final report. One of its main recommendations was for the government to set up a Nuclear Waste Management Organization. But the panel said it should have an "arm's length" board of directors representing key stakeholders, but not including the owner/operators of nuclear facilities, like OPG. But the Liberal government of former prime Minister Jean Chretien did exactly what the panel said should be avoided.
And the rest is history, I guess one could say.
(As of this writing the SON has not been able to say how many of its more than 4,500 members were eligible to vote, other than to say that number includes members under age 16. As a result, it is not possible to say what percentage of SON members actually voted.)
DGR, Nuclear Waste, Saugeen Ojiway Nation
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