Structural rearrangements and selection promote phenotypic evolution in Anolis lizards

Abstract

The genomic characteristics of adaptively radiated groups could contribute to their high species number and ecological disparity, by increasing their evolutionary potential. Here, we explored the genomic features of Anolis lizards, focusing on three species with unique phenotypes: A. auratus , one of the species with the longest tail; A. frenatus , one of the largest species; and A. carolinensis , one of the species that inhabits the coldest environments. We assembled and annotated two new chromosome-level reference genomes for A. auratus and A. frenatus , and compared them with the available genomes of A. carolinensis and A. sagrei . We evaluated the presence of structural rearrangements, quantified the density of repeat elements, and identified signatures of positive selection in coding and regulatory regions. We detected substantial rearrangements in scaffolds 1, 2 and 3 of A. frenatus different from the other species, in which the rearrangement breakpoints corresponded to hotspots of developmental genes. Further, we detected an accumulation of repeats around key developmental genes in anoles and phrynosomatid outgroups. Finally, we detected signatures of positive selection on coding sequences and regulatory regions of genes relevant to development and physiology that could affect the unique phenotypes of the analyzed species. Our results suggest that anoles have genomic features associated with genes that affect organismal morphology and physiology. This could provide a genomic substrate that promoted phenotypic disparity in anoles, and contributed to their ability to adaptively radiate.

Author Summary
Adaptive radiations are often characterized by high species richness and phenotypic differentiation. Besides the ecological context, the genetic features of organisms could also contribute to their ability to diversify. Anolis lizards are an adaptively radiated group that shows high phenotypic disparity in morphology and physiology. In this study, we explored the genome of four species within the Anolis radiation with distinctive phenotypes. We generated a high-quality chromosome-level reference genome for A. auratus and A. frenatus , and compared them with A. carolinensis and A. sagrei . We detected major structural rearrangements in A. frenatus , a high density of repeat elements around key developmental genes, and signatures of natural selection associated with genes functionally relevant for the analyzed species. Hence, the genomic characteristics of anoles were associated with their unique phenotypic diversity. We highlight the potential relevance of genomic features to influence the ability of groups of organisms to radiate adaptively.

Full text

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Structural rearrangements and selection promote phenotypic evolution in Anolis 1
lizards 2
Raúl Araya-Donoso1, Sarah M. Baty1, Jaime E. Johnson1, Eris Lasku1, Jody M. Taft2, 3
Rebecca E. Fisher1, Jonathan Losos3, Greer A. Dolby4, Kenro Kusumi1, Anthony J. 4
Geneva5* 5
1: School of Life Sciences, Arizona State University, Tempe, AZ 85281 USA 6
2: School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, 7
Johannesburg, South Africa 8
3: Department of Biology, Washington University, Saint Louis, MO, USA 9
4: Department of Biology, University of Alabama at Birmingham, AB, USA 10
5: Department of Biology & Center for Computational and Integrative Biology, Rutgers 11
University–Camden, Camden, NJ 08103 USA 12
*: Correspondence to anthony.geneva@rutgers.edu 13
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Short title: Structural variation and selection in Anolis 15
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Abstract 22
The genomic characteristics of adaptively radiated groups could contribute to their high 23
species number and ecological disparity, by increasing their evolutionary potential. Here, 24
we explored the genomic features of Anolis lizards, focusing on three species with unique 25
phenotypes: A. auratus, one of the species with the longest tail; A. frenatus, one of the 26
largest species; and A. carolinensis, one of the species that inhabits the coldest 27
environments. We assembled and annotated two new chromosome-level reference genomes 28
for A. auratus and A. frenatus, and compared them with the available genomes of A. 29
carolinensis and A. sagrei. We evaluated the presence of structural rearrangements, 30
quantified the density of repeat elements, and identified signatures of positive selection in 31
coding and regulatory regions. We detected substantial rearrangements in scaffolds 1, 2 and 32
3 of A. frenatus different from the other species, in which the rearrangement breakpoints 33
corresponded to hotspots of developmental genes. Further, we detected an accumulation of 34
repeats around key developmental genes in anoles and phrynosomatid outgroups. Finally, 35
we detected signatures of positive selection on coding sequences and regulatory regions of 36
genes relevant to development and physiology that could affect the unique phenotypes of 37
the analyzed species. Our results suggest that anoles have genomic features associated with 38
genes that affect organismal morphology and physiology. This could provide a genomic 39
substrate that promoted phenotypic disparity in anoles, and contributed to their ability to 40
adaptively radiate. 41
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Author Summary (150-200 words) 44
Adaptive radiations are often characterized by high species richness and phenotypic 45
differentiation. Besides the ecological context, the genetic features of organisms could also 46
contribute to their ability to diversify. Anolis lizards are an adaptively radiated group that 47
shows high phenotypic disparity in morphology and physiology. In this study, we explored 48
the genome of four species within the Anolis radiation with distinctive phenotypes. We 49
generated a high-quality chromosome-level reference genome for A. auratus and A. 50
frenatus, and compared them with A. carolinensis and A. sagrei. We detected major 51
structural rearrangements in A. frenatus, a high density of repeat elements around key 52
developmental genes, and signatures of natural selection associated with genes functionally 53
relevant for the analyzed species. Hence, the genomic characteristics of anoles were 54
associated with their unique phenotypic diversity. We highlight the potential relevance of 55
genomic features to influence the ability of groups of organisms to radiate adaptively. 56
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Introduction 65
Adaptively radiated groups of organisms are natural experiments in which the relative roles 66
of ecological and genomic features on speciation and phenotypic differentiation can be 67
assessed (1–3). In general, ecological factors and the emergence of ecological opportunity 68
are known to play an important role in determining the ability of a group of organisms to 69
radiate adaptively (4,5). On the other hand, genetic mechanisms could also influence the 70
ability of organisms within radiations to diversify and generate extensive phenotypic 71
variation because clades with greater evolutionary potential could be more likely to radiate 72
adaptively (1,6). Multiple genetic mechanisms could contribute to increased genetic and 73
phenotypic diversity such as chromosome-level structural rearrangements, small-scale 74
structural variation, the dynamics of transposable elements, mutation rates, recombination 75
rates, and the genomic landscape of selection on regulatory elements and/or coding regions 76
(6–10). 77
The relevance of the genomic substrate for highly speciose or adaptively radiated 78
groups of organisms has been discussed before. For example, African lake cichlids show 79
ancient genetic polymorphisms, structural rearrangements, high divergence in regulatory 80
sequences, insertion of transposable elements within regulatory elements, and novel 81
miRNAs (6,8,11). Darwin’s finches also exhibit evidence of ancient polymorphisms, and 82
selection on large-effect loci associated with beak morphology located in genomic islands 83
of low recombination (9,12). Heliconius butterflies present increased genomic variation by 84
hybridization and/or introgression processes, high variability in regulatory regions, genome 85
expansion events caused by an increase in repeat elements, and structural rearrangements 86
(13–16). Therefore, groups that radiate could have more labile genomes that allow for 87
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greater phenotypic diversification. A current challenge is to determine the relative 88
importance of each of these genomic factors, and whether different radiations present 89
similar genomic features that aided diversification or whether different radiations have 90
occurred through different genomic mechanisms. 91
Anolis lizards are an ideal group to assess the relevance of genetic mechanisms for 92
generating and promoting phenotypic diversity. This genus is described as an adaptive 93
radiation with ~400 species distributed in the tropical Americas (17,18). Anolis are 94
considered a model system for evolutionary biology studies because they present extensive 95
phenotypic variation across multiple niche axes. A remarkable characteristic of Anolis 96
evolution is the repeated occurrence of intra-island radiation and morphological 97
differentiation associated with microhabitat use patterns (19–21). Besides morphology, 98
anoles have diversified in behavior, physiology, and sexual dimorphism (22–24). In this 99
context, anoles present a wide range of phenotypic variation compared to other taxa and 100
this diversity may be promoted by ecological and genetic mechanisms. 101
Within the Anolis radiation, some species have distinctive phenotypes that could be 102
adaptive to their niches (Fig. 1A). For example, A. frenatus is one of the largest anole 103
species (Fig. 1B), which may reduce its predation risk and enable a wider dietary breadth, 104
potentially including other anole lizards as prey (25); A. auratus inhabits grasslands and 105
perches on narrow branches and features an extremely long tail (Fig. 1C) that may provide 106
better balance when walking and jumping along narrow surfaces (26,27); and A. 107
carolinensis is one of the species with highest cold tolerance (Fig. 1D), enabling its 108
colonization towards higher latitudes and survival during cold seasons (28). Different types 109
of genomic variation, particularly within coding regions, may control such traits. For 110
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instance, longer tails could be produced by modifications of the number and/or size of the 111
caudal vertebrae, controlled by molecular pathways involved in the axial skeleton 112
development (29–31). A larger body size could be controlled by insulin growth factor or 113
growth hormone pathways (32–34), while cold adaptation could be related to genes 114
regulating oxygen consumption and/or blood circulation (28,35). 115
116
Figure 1. Anolis phylogenetic relationships (A) and genus-wide phenotypic variation in 117
snout-vent length (SVL; B), tail length (TL; C), and thermal climatic niche (D), 118
highlighting the species included in this study (Phylogenetic and morphological data from 119
Poe et al., (2017); temperature data obtained from WorldClim 2 (37) for all species). 120
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121
Some genomic features could be associated with the high ecological disparity 122
described in these Anolis species. Tollis et al. (2018) compared short-read genome 123
assemblies of five species (including A. carolinensis, A. auratus and A. frenatus), and 124
detected high mutation rates in anoles compared to other vertebrates, and signatures of 125
natural selection on genes associated with limb and brain development and hormonal 126
regulation. In some Cuban anole species, an accumulation of gene duplications has been 127
reported (39), and genomic regions undergoing accelerated evolution have been identified 128
in association with thermal biology (40). Furthermore, Anolis genetic diversity could have 129
been fueled by ancient hybridization and introgression processes (41,42). Chromosomal 130
rearrangements could also be relevant because multiple events of chromosome gains and 131
losses have been described within Anolis (43,44), and chromosome fission and fusions have 132
been proposed to determine the evolution of the Anolis X chromosome (45,46). Finally, the 133
dynamics of repeat elements could be relevant because transposable elements can impact 134
the genome by modifying gene regulation patterns, causing mutations, or promoting 135
genome rearrangements (7). A high density of transposable elements within the hoxB and 136
hoxC gene clusters, key regulators of morphological development, has been reported in 137
Anolis (47,48). Nonetheless, genome-wide patterns associated with repeat density and 138
chromosome-level structural rearrangements remain to be explored with a genomic 139
approach, because the analysis of those features requires highly contiguous genome 140
assemblies. 141
Here, we explored the genomic features of species with disparate phenotypes within 142
the adaptively radiated Anolis group. We generated chromosome-level reference genomes 143
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for two Anolis species and found evidence for major structural rearrangements, described a 144
unique pattern of repeat density through the genome, and identified genes under positive 145
selection that were associated with the unique traits from four species representing 146
divergent phenotypes. These genomic features could fuel genetic diversity and hence, 147
promote the high diversification and phenotypic disparity found within Anolis. 148
149
Results 150
Chromosome level genome assemblies and annotation for A. auratus and A. frenatus 151
We generated Hi-C chromosome-level genome assemblies for two Anolis species (Table 1). 152
Both type specimens were adult females from Panama (Table S1). The resulting assemblies 153
were highly contiguous (N50: A. auratus 281.8 Mbp and A. frenatus 342.7 Mbp) and 154
complete (BUSCO eukaryotic completeness of 93.07% for A. auratus and 86.14 % for A. 155
frenatus). Both species show a similar pattern of repetitive element composition (Fig. S1), 156
which corresponds to roughly 50% of the genome. However, A. auratus shows a recent 157
accumulation of LINEs. We generated genome annotations for both species via the 158
MAKER pipeline (49) using a combination of new RNA sequencing data, and the 159
proteomes of previously sequenced species (see Methods for details). For A. auratus we 160
identified 19,879 genes with an average length of 19,877 bp (Table S2), and 88.2% of all 161
eukaryote BUSCO genes present in the annotation (either complete or fragmented), 162
whereas for A. frenatus 19,643 genes were identified with an average length of 18,033 bp 163
(Table S2) and 76.1% eukaryotic BUSCO genes present. For subsequent analyses, our 164
newly annotated genomes were compared against the chromosome-level reference genomes 165
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of A. carolinensis (AnoCar2.0, (50); and DNAzoo Hi-C Assembly, (51,52)) and A. sagrei 166
(AnoSag2.1, (45), along with the phrynosomatid lizards Urosaurus nigricaudus (53) and 167
Phrynosoma platyrhinos (54). 168
169
Table 1. Genome assembly and annotation statistics for the four analyzed Anolis species. 170
Species
A. auratus
A. frenatus
A. carolinensis
A. sagrei
Genome version
RUC_Aaur_2
RUC_Afre_2
AnoCar2.0
AnoSag2.1
Assembly length
(Gbp)
1.77
1.85
1.79
1.66
N50 (Mbp)
281.8
342.7
150.6
253.6
L50 (n°)
3
3
5
4
Eukaryote
BUSCO
Assembly (%)
C + F: 93.07
C + F: 86.14
C + F: 94.5%
C + F: 100%
% Repeats
48.53
51.27
33
46.3
N° genes
19,879
19,643
21,555
20,033
Average gene
length (bp)
19,877
18,033
32,969
45,059
Eukaryote
BUSCO
Annotation (%)
C + F: 88.2
C + F: 76.1
C + F: 94.5%
C + F: 99.7%
Reference
This study
This study
Alföldi et al.
2011
Geneva et al.
2022
171
Chromosome-level structural rearrangements 172
We performed in silico chromosome painting to assess the synteny conservation among our 173
four Anolis species and U. nigricaudus and P. platyrhinos, using A. sagrei as a reference. 174
Overall, there is high synteny conservation for the main scaffolds or macrochromosomes 175
among those species (Fig. 2A). Interestingly, scaffolds 1, 2, and 3 contain substantial 176
structural rearrangements that are unique to A. frenatus (Fig. 2A, Fig. 2B). The Hi-C data 177
for A. frenatus shows higher contact density within scaffolds and very little interaction 178
between scaffolds 1, 2 and 3 (Fig. 2C, Fig. S2). This observation suggests that the observed 179
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rearrangements are not a sequencing or scaffolding artifact, but rather supports genuine 180
structural differences in this species relative to other Iguanian taxa. To establish a link 181
between the structural rearrangements and their potential functional implications, we 182
identified the genes located within 1 Mbp to the rearrangement breakpoints in scaffolds 1, 2 183
and 3 between A. sagrei and A. frenatus (Table S3). We conducted an enrichment analysis 184
on the list of genes co-located to the breakpoints with g:Profiler (55) which showed 185
significant enrichment of biological processes such as "cellular differentiation", 186
"developmental process" and "pigment granule transport" (Table S4). Further, we 187
quantified the density of genes associated with developmental GO terms along scaffolds 1, 188
2 and 3 of A. frenatus, and we detected that the chromosomal breaks were located in 189
hotspots of genes with developmental functions (Fig. 2D). Among the genes contiguous to 190
the rearrangement breakpoints (Table S3) we identified axin2, a regulator of the Wnt/b-191
catenin and TGF-b pathways that determines chondrocyte maturation and axial skeletal 192
development (56); bmp2, a growth factor determinant for bone development through the 193
BMP-Smad pathway (57); ddit3, transcription factor that influences myogenesis by 194
regulating the GH-IGF1 pathway (58); and twist2, a transcription factor relevant for bone 195
formation and myogenesis (59). 196
Scaffold 7 in A. sagrei has previously been hypothesized to be the X chromosome 197
and the result of a series of autosomal fusions (45,46,60). A. auratus and A. sagrei belong 198
to the Norops clade of Anolis (36) and we found a high degree of synteny conservation 199
between scaffold 7 of these two species, whereas in the species outside of the Norops clade 200
it corresponded to a series of smaller scaffolds (Fig. 2A, Fig. S3). To further explore 201
scaffold 7 evolution within anoles we compared this chromosome against another recently 202
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published Norops clade high-quality genome, A. apletophallus (61), which also revealed 203
high synteny conservation with both A. sagrei and A. auratus (Fig. S3). 204
205
Figure 2. Chromosome-level structural variation across Anolis. A. Synteny between A. 206
sagrei and other anole (A. auratus, A. carolinensis, A. frenatus) and lizard (U. nigricaudus, 207
P. platyrhinos) species for the largest scaffolds representing the chromosomes of each 208
species. B. Synteny between scaffolds 1, 2, and 3 of A. sagrei and A. frenatus showing 209
substantial rearrangements. C. Hi-C density contact matrix for A. frenatus. D. Density of 210
genes associated with developmental GO terms along scaffolds 1, 2 and 3 in A. frenatus. 211
Background colors indicate the homology to A. sagrei scaffolds for different chromosomal 212
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regions and vertical lines indicate the chromosomal breakpoints. Rearrangement 213
breakpoints are within hotspots of developmental genes. 214
215
Repeat density is associated with key developmental genes in Anolis and other pleurodonts 216
We estimated the density of repeats in 500 kb windows throughout the first 6 scaffolds of 217
A. frenatus, A. auratus, A. sagrei, U. nigricaudus and P. platyrhinos based on the repeat 218
element annotation of their genomes. We selected the densest repeat regions corresponding 219
to the top 5% of repeat density and identified the genes present in those regions from our 220
annotations (Table S5). Within repeat-rich regions in all the analyzed species we detected 221
some developmental genes (Fig. 3) such as the hoxB, hoxC, and hoxD gene clusters, key 222
determinants of the vertebrate body plan (30); notch4, a member of the NOTCH receptors 223
family that are crucial for development (62); and fgf11, member of the fibroblast growth 224
factor (FGF) family which are involved in development and morphogenesis (63). An 225
enrichment analysis was conducted on the lists of genes located within these high repeat-226
density regions for each species with g:Profiler. In general, genes associated with 227
regulatory and developmental biological processes were overrepresented in the high repeat-228
density regions for all species (Table S6; Fig. S4). 229
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230
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Figure 3. Number of repeat elements in 500 kb windows throughout scaffolds 1, 2 and 6 in 232
the analyzed pleurodont species. A higher density of repeats is found close to key 233
developmental genes in the four Anolis and the outgroups. 234
235
Genes under natural selection and divergence in regulatory regions 236
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We identified genes under positive selection among the four Anolis species by calculating 237
the pairwise ratio between non-synonymous to synonymous substitutions (dN/dS) between 238
each species pair (Table S7). We retained genes with dN/dS > 1 overlapping in at least 2 239
out of 3 comparisons for each species (Fig. S5). For A. frenatus 16 genes overlapped 240
including mtpn, a muscle growth factor that shows similar effects to igf1 (64,65); and 241
pdzk1ip1 that regulates and inhibits transforming growth factor (TGF-b) and bone 242
morphogenic protein (BMP) signaling (66). For A. auratus 12 genes overlapped including 243
ramp2 which regulates angiogenesis, cardiovascular development, and influences bone 244
formation (67,68); and dcdc1, associated with bone mineral density (69) and bone 245
degradation in humans (70). In A. carolinensis we detected 6 overlapping genes including 246
lep, a gene relevant to lipid metabolism and energetic balance, and that has thermogenic 247
effects on skeletal muscle (71–73); clps, involved in lipid digestion (74); and stard6, 248
associated with the intracellular transport of sterol and other lipids (75). Anolis sagrei 249
presented 8 overlapping genes including ppdpf1, associated with cell proliferation in 250
multiple types of cancer (76); and s100a1, that can regulate cell growth and proliferation
251
(77). A gene enrichment analysis was run with g:Profiler for each species (Table S8). 252
Among the overrepresented GO terms for A. carolinensis we detected “lipid catabolic 253
process” and “digestion”, for A. auratus “positive regulation of developmental processes”, 254
for A. frenatus “regulation of muscle organ development”, and for A. sagrei “regulation of 255
polarized epithelial cell differentiation”. 256
To identify diverged regulatory regions, we identified genes with the top 1% of 257
divergence in their putative promoter regions (1,000 bp upstream of the transcription start 258
site, (78) for each species pair (Table S9). We retained genes that overlapped in at least 2 259
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out of 3 species comparisons. Within the overlapping genes identified for A. frenatus we 260
found wnt4, key ligand of Wnt/b-catenin signaling that controls development and cell 261
differentiation (79); traf4, an important regulator of embryogenesis and bone development
262
(80); hspg2, which influences skeletal and cardiovascular development (81); and errfi1, that 263
affects cell growth by regulating EGFR signaling (82). In A. carolinensis we detected genes 264
associated with lipid metabolism like plin3, lpin1, ncoa1 (83–85). For A. auratus we found 265
cib2, associated with mechanoelectrical transduction in auditory cells (86). In A. sagrei we 266
found rab3d involved in bone resorption (87); and optn, a gene associated with 267
autoimmune and neurodegenerative disorders (88). 268
We combined these genes with high divergence in the regulatory regions with the 269
genes previously identified as being under positive selection via analysis of dN/dS ratio to 270
generate our candidate gene set. We then used STRING v11 (89) to estimate gene 271
interaction networks for genes in our combined candidate set to obtain an integrative view 272
of evolutionary processes that spanned both regulatory and protein divergence (Fig. 4C). 273
Some genes with dN/dS > 1 were embedded within gene interaction networks of genes with 274
high divergence in regulatory regions (Fig. 4C, Fig. S6). For example, in A. carolinensis 275
several genes in the gene interaction network have functions associated with gene 276
regulation, lipid metabolism, and mitochondria (Fig. 4C). The positively selected lep gene 277
constitutes a central node in the gene interaction network and interacts with other elements 278
of similar function that present high divergence in the regulatory regions. 279
280
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281
Figure 4. Climatic niche characterization across the native distribution range of the four 282
studied anole species. A. Minimum temperature of the coldest month B. Temperature 283
seasonality. A. carolinensis is the species inhabiting the coldest and more thermally 284
seasonal environments. C. Gene interaction network for the genes under natural selection 285
and genes with high divergence on the promoter region for A. carolinensis. Line thickness 286
represents the number of multiple evidence supporting the interaction between two genes. 287
288
Association with phenotypic traits 289
We characterized the realized climatic niche across the native distribution for the four 290
Anolis species and detected that they have different climatic niches (Fig. S7). Among them, 291
A. carolinensis occupies the coldest (Fig. 4A) and most thermally seasonal (Fig. 4B) 292
environments. This is in accordance with the genes under selection and regulatory 293
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divergence mostly associated with biological functions that could influence cold tolerance 294
such as lipid metabolism, mitochondria, and the circulatory system (Fig. 4C). 295
The morphology of the four focal species was also analyzed (Fig. S8). Anolis 296
frenatus is distinct in its larger body size (Fig. 5A). The genes mtpn and pdzk1ip1 were 297
under selection in A. frenatus with respect to the other three species and could influence its 298
larger body size (Fig. 5C). In contrast, A. auratus is characterized by its unique tail 299
elongation (Fig. 5B). We explored the morphology of the caudal vertebrae, and we found 300
that the long tail in A. auratus is associated with an elongation of the caudal vertebrae 301
rather than an increase in the number of vertebrae when compared to the other species (Fig. 302
5D). The relative length of the trunk vertebrae of A. auratus did not differ from the other 303
species (Fig. S9). Anolis frenatus also features a longer tail and longer caudal vertebrae 304
than A. sagrei and A. carolinensis, but not as long as A. auratus (Fig. 5D). Among the 305
genes under selection in A. auratus we detected ramp2 and dcdc1, which could be 306
associated with the vertebral elongation phenotype (Fig. 5E). 307
308
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309
Figure 5. Morphological variation in distinctive traits of the analyzed species. A. A. 310
frenatus stands out for its large body size. B. A. auratus its characterized by a long tail. C. 311
Selection on the mtpn and pdzk1ip1 genes could influence A. frenatus body size. D. The 312
long tail in A. auratus is caused by an elongation of the caudal vertebrae rather than the 313
addition of more vertebrae. E. Selection on ramp2 and dcdc1 could influence the vertebral 314
elongation in A. auratus. 315
316
Discussion 317
Genomic features can influence speciation and promote phenotypic variation within 318
adaptive radiations (1,90). Here, we explored the genomic features, namely chromosomal 319
rearrangements and repeat element concentration, potentially contributing to diversity and 320
phenotypic disparity within the Anolis radiation. Our results indicate that major structural 321
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rearrangements, high densities of TEs around developmental genes, and signatures of 322
natural selection and divergence on regulatory regions are associated with genes that could 323
affect the unique phenotypes in these Anolis species. 324
325
Major structural rearrangements within Anolis 326
Our synteny analysis detected major chromosomal rearrangements within Anolis. 327
Chromosome fissions and fusions have been previously described as highly relevant in 328
anoles (43,44), but we also identified an important number of translocations, inversions, 329
and deletions among the analyzed species. Chromosome-level structural variations can 330
directly influence speciation by disrupting meiosis in heterozygotes and reducing fertility in 331
hybrids or generating barriers to gene flow (91,92). Moreover, they can modify the gene 332
regulation and recombination patterns (10,93). 333
Chromosomes 1, 2 and 3 presented a substantial rearrangement in A. frenatus (Fig. 334
2B). Hi-C analysis suggests this is a true rearrangement and not a technical artifact given 335
that contact maps show strong within-chromosome interactions and little to no interactions 336
between these chromosomes (Fig. 2C, Fig. S2). This rearrangement was absent in the other 337
anole species and the outgroups, which suggests this chromosomal mutation occurred in 338
this lineage. Anolis frenatus is part of the basal Dactyloa clade of Anolis (Fig. 1A; (36). 339
Chromosomes 1, 2, and 3 are bigger in other Dactyloa anoles when compared to non-340
Dactyloa karyotypes (Table S10), which suggests this mutation could be shared among 341
dactyloans and evolved early during the lineage history. Chromosomal breaks in A. frenatus 342
were located in areas with a high density of genes with developmental functions (Fig. 2D), 343
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including some genes highly relevant to skeletal and muscle development and growth like 344
axin2, bmp2, ddit3, and twist2 (56–59). This suggests adaptive structural rearrangements in 345
A. frenatus (and potentially other Dactyloa) that could potentially have influenced the 346
evolution of body size and morphology. Thus, these major structural rearrangements could 347
have altered the gene regulation patterns of the developmental genes adjacent to them 348
(10,93), and hence enhanced the evolution of phenotypic variability in anoles. 349
Our results also allowed us to explore patterns of sex chromosome evolution across 350
Anolis. Anoles share a single ancestral XY sex chromosome system but have commonly 351
experienced chromosomal fission and fusion, including fusions that involve sex 352
chromosomes (44,94). In A. sagrei the X chromosome (scaffold 7) has been reported as the 353
fusion of chromosomes 9, 12, 13 and 18 from A. carolinensis (45,46,60). Further, 354
Giovannotti et al. (46) described chromosome 7 homology between A. sagrei and A. 355
valencienni, both belonging to the Norops clade of anoles. Our results are consistent with 356
this finding (Fig. 2A; Fig. S3) and expand the homology for the sex chromosome to all 357
three analyzed Norops-clade anoles (A. auratus, A. apletophallus and A. sagrei), with only 358
within-chromosome structural changes such as inversions and deletions differing among 359
these species (Fig. S3). Norops is one of the most diverse clades within Anolis (36) with 360
~200 species. Our findings suggest that the X-autosome fusions detected in Anolis sagrei 361
arose early in the clade and highlight the relevance of sex chromosome evolution for anole 362
diversification (44,94). 363
364
Key developmental genes in repeat-rich regions in Anolis and other pleurodonts. 365
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We quantified the density of repeat elements through the genome for four anoles and two 366
phrynosomatid outgroup species. Repeat elements can be a source of genetic variation 367
because they can modify gene regulation patterns, be a source of mutations, and trigger 368
structural rearrangements (7,95). We found a high density of repeats associated with key 369
developmental genes such as notch4, fgf11, and the hoxB, hoxC and hoxD clusters in Anolis 370
and the outgroups (Fig. 3; (31,62,63)). Furthermore, the enrichment analysis detected that 371
genes located in repeat-rich regions were mostly associated with developmental and 372
regulatory functions (Fig. S4, Table S6). Feiner (47,48) reported that anoles have an 373
accumulation of repeats around the hoxB and hoxC gene clusters when compared against 374
other more distantly related squamates but did not include other pleurodont lizards such as 375
the prhynosomatids U. nigricaudus and P. platyrhinos. Thus, our analysis expands this 376
pattern of repeat element accumulation to other genes that also affect development (Table 377
S5) and indicates that this is not a feature exclusive to Anolis but is also present in other 378
species from the Pleurodonta clade of Iguania. Pleurodont lizards include some of the most 379
diverse vertebrate genera with respect to species number and morphological variation (e.g. 380
Anolis, Liolaemus, Sceloporus; (96,97)). Therefore, the accumulation of repeat elements 381
around developmental genes could be a source of genetic variation that fueled 382
morphological innovation in these pleurodont groups (48). Exploring the potential effects 383
of the repeat accumulation on genetic and phenotypic variation for these lizard groups is 384
key to understanding whether TE dynamics contribute to their evolvability and 385
diversification. However, additional genomes assembled from within pleurodonts and other 386
iguanians are needed to identify specifically when this pattern arose. 387
388
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Selection on coding regions and regulatory divergence associate with unique phenotypes in 389
Anolis 390
For the analyzed species we detected some candidate genes under selection and genes with 391
high divergence in their regulatory regions, potentially associated with their unique 392
phenotypes (Fig. 4, Fig. S6). Anolis carolinensis presented signatures of selection and high 393
regulatory divergence on genes potentially driving cold adaptation. In general, ectotherm 394
adaptation to cold environments involves physiological processes of oxygen consumption 395
and blood circulation (28,98). Among the genes under selection in A. carolinensis, leptin 396
(lep) was a central node in the gene interaction network (Fig. 4). Moreover, other genes 397
associated with lipid metabolism (e.g. clps, stard6, ncoa1, lpin1, plin3) were also identified 398
in our analysis. Lipid metabolism has been proposed as a potential thermal adaptation in 399
ectotherms (99). For instance, it could be an alternative for energy obtention during cold 400
seasons with lower resource availability (100), or it could be associated with changes in cell 401
membrane composition impacting fluidity in colder temperatures (101). Genes associated 402
with lipid metabolism have been identified as undergoing accelerated evolution when 403
comparing Cuban anole species with different thermal biology (40). Furthermore, genes 404
interacting with leptin and involved in lipid metabolism have been identified as under-405
selection in A. cybotes populations inhabiting cold high-elevation environments (102). 406
Therefore, it is possible that changes in lipid metabolism could constitute an adaptation to 407
cold environments in A. carolinensis. We also detected divergence in the regulatory region 408
of genes associated with the circulatory system and mitochondria (Fig. 4). Populations of A. 409
carolinensis inhabiting colder environments show lower oxygen consumption rates, and 410
signatures of selection and changes in the expression of genes associated with the 411
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circulatory system (28,103). Thus, changes in these genes could enhance oxygen intake for 412
low oxygen availability under cold temperatures in A. carolinensis versus other anole 413
species. 414
A. auratus stands out for its long tail. This species is usually found on the grass in 415
dense vegetation patches, and a long tail may provide better balance when walking or 416
jumping across narrow perches (26,27). Body elongation is a convergent phenotype in 417
several reptiles, and most species develop longer bodies through the addition of vertebrae 418
(29). However, the extremely long tail in A. auratus is achieved by elongation of the caudal 419
vertebrae rather than the addition of more segments (Fig. 5D). The longest caudal vertebrae 420
in A. auratus are located distal to the ninth caudal vertebrae (e.g., Ca10-21, Fig. 5D). In 421
anoles, the m. caudofemoralis longus originates from the proximal caudal vertebrae (e.g. 422
Ca2-8 in A. sagrei, (104); Ca2-9 in A. heterodermus, A. tolimensis, and A. valencienni, 423
(104,105); and Ca3-8 in A. carolinensis, (106). This primary hip joint extensor is essential 424
for locomotion and may also assist with lateral flexion of the tail when the hindlimb is 425
fixed (106). Therefore, caudal vertebral elongation in A. auratus is most pronounced in a 426
region of the tail that is less functionally constrained. The pattern of caudal vertebrae 427
elongation in A. auratus is similar to that seen in the tail of arboreal Peromyscus 428
maniculatus (107), the cervical vertebrae of giraffes (108), the trunk of some plethodontid 429
salamanders (109) and some fish species (110). Among the mechanisms that could 430
determine caudal vertebral elongation are genes associated with axial development and 431
determinants of the caudal region such as the hox13 genes, fgf8, or fgfr1 432
(30,31,107,108,111). In our genetic data, we detected signatures of selection in ramp2 and 433
dcdc1, which influence bone development (67,69). Heterozygote knockout mice for ramp2 434
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present skeletal abnormalities such as lower bone density and delayed development of the 435
lumbar vertebrae, producing a similar pattern of vertebral elongation (112). In Peromyscus 436
maniculatus, dcdc1 is located within a locus associated with tail length (107). Thus, the 437
mutations in these genes could contribute to the unique tail phenotype in A. auratus. 438
Finally, A. frenatus is characterized by a large body size and relatively long limbs. 439
In general, vertebrate body size is determined by genes associated with insulin growth 440
factors and growth hormone pathways (32,34,113,114). Our selection analysis identified 441
some candidate genes potentially associated with large body size in A. frenatus. We 442
detected signatures of selection on the mtpn and pdzk1ip1 genes, both involved in muscle 443
development, growth, and morphogenesis (64–66,115,116). Injection of mtpn in mice 444
produces increased body and muscle weights (117). Moreover, among the genes that 445
presented high divergence in regulatory regions for A. frenatus we identified other genes 446
highly relevant for development. For instance, wnt4 can be modulated by the growth 447
hormone (118), and mice with overexpression of wnt4 present dwarfism (119). Further, 448
knockout mice for traf4 show reduced body weight than wildtype mice (120). 449
Overall, the genes under selection and with high divergence in their regulatory 450
regions perform relevant biological functions that could affect the phenotypes of the 451
analyzed species. This indicates that the combination of mechanisms acting at different 452
hierarchical levels can aid in the generation of adaptive phenotypes in anoles. Changes in 453
regulatory regions could provide more evolvability than changes in protein-coding 454
sequences that are in general more constrained to mutations given their biological function 455
(40,121). Therefore, exploring the effects of regulatory sequence divergence and regulatory 456
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RNAs on gene expression and their impacts on species traits is key to understanding how 457
this feature could promote anole phenotypic diversity. 458
459
Conclusions 460
Our analysis of highly contiguous genome assemblies of four anole species allowed us to 461
identify genomic features that could contribute to the extensive phenotypic variation among 462
Anolis species. In Anolis, chromosome-level structural rearrangements could directly 463
generate reproductive isolation, and affect the gene regulation patterns of genes relevant to 464
development and morphological configuration. Further, a high density of repeat elements 465
close to key developmental genes could also contribute to variation in the expression of 466
such genes. Finally, natural selection on few coding sequences but relevant to species traits, 467
in addition to divergence in regulatory regions could also play a role in shaping phenotypic 468
diversity. The interaction between these genomic characteristics and selection pressures 469
potentially enabled the evolution of disparate phenotypes within anoles, but further analysis 470
of a wider sample of high-quality genomes would help to formally test this hypothesis. We 471
highlight that besides ecological opportunity, genomic features can also be extremely 472
relevant for promoting adaptive radiation. 473
474
Methods 475
Sampling and type specimens 476
The A. auratus specimen was collected in Gamboa, Panama, and the A. frenatus specimen 477
in Soberania National Park, Panama (Collecting Permits: SE/A-33-11 and SC/A-21-12, 478
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Autoridad Nacional de Ambiente, ANAM, Republic of Panama; IACUC Protocol: 2011-479
0616-2014-07 Smithsonian Tropical Research Institute). Additional samples of A. 480
carolinensis and A. sagrei obtained from the Sullivan Company (Nashville, TN) and 481
Marcus Cantos Reptiles (Fort Myers, FL) were included for morphological analyses 482
(IACUC Protocol: Arizona State University 19-1053R and 12-1247R). Table S1 shows the 483
number of individuals collected per species and locations used for reference genome 484
assemblies and morphological analyses. Specimens were euthanized by intracoelomic 485
injection of sodium pentobarbital (IACUC Protocols 09-1053R, 12-1274R, and 15-1416R 486
ASU). The type specimens for the A. auratus and A. frenatus reference genomes 487
corresponded to adult females. 488
489
Reference Genomes 490
We generated new reference genomes for A. auratus and A. frenatus. Skeletal muscle from 491
the A. auratus type specimen, and liver and heart from A. frenatus type specimen were sent 492
for DNA extraction and whole genome sequencing. The RUC_Aaur_2 and RUC_Afre_2 493
genomes were sequenced by Dovetail Genomics on an Illumina PE150 platform, de novo 494
assembled with meraculous v2.2.2.5 (122). HiRise v2.1.6-072ca03871cc (123) scaffolding 495
was performed with Chicago and Hi-C chromatic conformation capture libraries. The 496
published genome assemblies and annotations of A. carolinensis (AnoCar2.0, (50); and Hi-497
C assembly from DNAzoo, (51,52)) and A. sagrei (AnoSag2.1, (45)) were included for 498
comparative genomic analyses. Table 1 shows the assembly statistics for the four Anolis 499
genomes. Additionally, we included the reference genome of the phrynosomatids 500
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Phrynosoma platyrhinos (MUOH_PhPlat_1.1, (54)) and Urosaurus nigricaudus 501
(ASU_Uro_nig_1, (53)) for some comparative genomic analyses. 502
A genome annotation was generated for A. auratus and A. frenatus. For each 503
species, repeats were identified on the genome sequences by using RepeatModeler v2.0.1 504
(124), and then repeat elements were soft-masked on the assembly with RepeatMasker 505
v4.1.1 (125). To aid in annotation we generated a de novo transcriptome for each species 506
using tail and ovary/yolk for A. auratus and brain and ovary for A. frenatus. Tissue samples 507
were collected from the same animals used for genome sequencing. Tissues were sent to 508
the Yale Center for Genomic Analyses (YCGA; West Haven, CT) for RNA extraction, 509
cDNA poly-A-enriched Illumina library preparation and sequencing on an Illumina 510
NovaSeq S4 platform using 150-bp paired end reads. Read quality was assessed with 511
FastQC v0.11.7 (126), and reads were trimmed with Trimgalore v0.6.8 (127). Then a de 512
novo transcriptome assembly was generated with Trinity v2.12.0 (128). The generated 513
transcriptomes were used as evidence for each species genome annotation respectively. 514
Multiple iterations of Maker v3.01.03 (49) were run to annotate the genomes. We 515
used the species-specific transcripts, and the protein-coding sequences from A. carolinensis 516
and A. sagrei as evidence. A first round of Maker was run for aligning and mapping 517
transcript and protein evidence. Then, two additional rounds of ab initio gene model 518
prediction using Augustus v3.4.0 (129) and SNAP v2006-07-28 (130) were run. After each 519
round of Maker, the Annotation Edit Distance (AED) was recorded, and annotation 520
completeness was assessed with BUSCO v5.4.2 (131) on the predicted transcripts obtained 521
from Maker, comparing against the eukaryote and sauropsid gene datasets. 522
523
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Chromosome-level structural rearrangements 524
We investigated synteny among the main scaffolds from the four analyzed Anolis species 525
along with the phrynosomatids P. platyrhinos and U. nigricaudus by in silico chromosome 526
painting. For this analysis, we used the high contiguity DNAzoo Hi-C-scaffolded genome 527
assembly of A. carolinensis (51,52). All species were compared against the A. sagrei 528
genome as a reference, because it is the species with the most contiguous and complete 529
genome among our samples (45). The first 14 scaffolds from A. sagrei, representative of its 530
chromosomes, were split in chunks of 100 bp with “faSplit” v438 from the UCSC 531
Bioinformatic Utilities (132). Then, we used blastn v2.10.0 (133) to map each fragment 532
onto the 5 other species’ genome. We retained matches with at least 50 bp length, and that 533
were contiguous in at least 5 matches (54). To further explore the chromosome X evolution 534
within Anolis, we compared chromosome 7 from A. sagrei to the closely related A. 535
apletophallus (61) following the same methodology. 536
537
Hi-C data analysis 538
Link density histograms were generated with Juicer v2.0 (134) by mapping paired reads 539
from the Hi-C libraries for A. auratus and A. frenatus to the finished genome assembly to 540
assess chromatin conformation and to validate our chromosomal rearrangements. Hi-C 541
contact maps were visualized with Juicebox v1.9.8 (52). 542
543
Developmental genes located in A. frenatus scaffolds 1, 2 and 3 rearrangement 544
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We explored which genes were located adjected to the rearrangement among scaffolds 1, 2, 545
and 3 detected between A. frenatus and A. sagrei. For this, we pulled from the A. sagrei 546
annotation the genes located within 1 Mbp from the scaffold breakpoints identified with the 547
synteny analysis. We performed an enrichment analysis on the genes located in these 548
regions with g:Profiler ve111_eg58_p18_30541362 (55) to assess which biological 549
processes were overrepresented in that gene list. Then, we extracted the list of genes 550
present in scaffolds 1, 2, and 3 in A. frenatus to identify if the chromosomal breaks were 551
located in hotspots of genes with developmental function. We identified and extracted all 552
the GO terms included in the list of genes located on each scaffold with g:Profiler, and we 553
retained only the genes matching GO terms that included any of the keywords: 554
“development”, “morpho”, “growth” or “organ”. We then calculated the number of genes 555
with those developmental functions along each chromosome in 500 kbp windows in R 556
v4.1.2 (135) with a custom script. 557
558
Repeat density through the genomes 559
For A. auratus, A. frenatus, A. sagrei, U. nigricaudus and P. platyrhinos we calculated the 560
repeat density for each one of the largest 6 scaffolds. The number of repeats was calculated 561
in 500 kbp windows, and we retained repeats longer than 50 bp and with a score value over 562
10 (47). Then, we selected the 500 kbp windows corresponding to the highest 1% of repeat 563
density per scaffold for each species with a custom script in R, and identified which genes 564
were located in those high repeat density regions using the respective genome annotations. 565
An enrichment analysis was performed to identify the most represented gene ontology 566
(GO) categories on the list of genes situated in high repeat regions for each species with 567
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g:Profiler, and the enriched GO terms were semantically organized and visualized with 568
Revigo v1.8.1 (136). 569
570
Identification of genes under positive selection and regulatory elements with high 571
divergence 572
We looked for potential genes under positive selection among the four Anolis species by 573
calculating the ratio between non-synonymous to synonymous mutations (dN/dS) between 574
orthologs from species pairs with the “orthologr” package (137) in R using the Comeron’s
575
(138) method. To identify genes positively selected in each species, we retained the genes 576
overlapping in at least 2 out of 3 comparisons between the focal and the other three species. 577
Then, an enrichment analysis was performed on that gene list with g:Profiler to identify the 578
most represented GO terms for each species. 579
To assess regulatory regions with high divergence we focused on the 1000 kb 580
upstream of the transcription start, which includes the promoter region (78). We compared 581
orthologs between species pairs previously identified with “orthologr” in R. Each ortholog 582
pair was aligned with mafft v7.520 (139) and the genetic distance between aligned 583
orthologs was estimated with the “bio3d” package (140) in R. We considered the genes 584
with the top 1% of genetic distance as genes with the highest divergence in their regulatory 585
regions between species pairs. For each species we retained the genes overlapping in at 586
least 2 out of 3 comparisons. Finally, we used STRING v12.0 (89) to evaluate gene 587
interactions among the genes under selection and the genes with high regulatory divergence 588
for each species. 589
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590
Climatic niche analyses 591
For each species, occurrence records were obtained from the Global Biodiversity 592
Information Facility (141). Occurrences were deduplicated and manually curated to 593
accurately represent the native distribution of each species. Raster data for the 19 594
bioclimatic variables was obtained from the WorldClim v2 database (37). For each 595
occurrence point, the corresponding values of the 19 bioclimatic variables were obtained. 596
We compared the climatic niche between the four analyzed species. Climatic variation was 597
visualized with a Principal Component Analysis (PCA) in R, and the main variables 598
differentiating species were identified. 599
600
Morphological analyses 601
Additional samples for the four Anolis species were included for morphological analyses. 602
Skeletal data was obtained from osteological preparations following Tollis et al. (2018) 603
modification of amphibian protocols, or from micro-computed tomography (microCT) 604
images collected in a Siemens Inveon micro-CT scanner at the RII Translational 605
Bioimaging Resource at the University of Arizona (Table S1). For skeletal preparations, 606
individuals were photographed with a scale in a stereodissecting microscope (Nikon 607
SMZ800 with Coolpix 995 digital camera), and morphological traits were measured with 608
ImageJ v1.53k (142). For microCT scans, digital images were analyzed and measured with 609
InVesalius v3.1.1 (143). For each species we measured snout–vent length (SVL), axilla–610
groin distance (AGD), forelimb total length (FLL), forelimb autopod length, forelimb 611
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stylopod length, forelimb zeugopod length, hindlimb total length (HLL), hindlimb autopod 612
length, hindlimb stylopod length, hindlimb zeugopod length, head width (HW), head length 613
(HL), head height (HH) and tail length (TL). We also analyzed the osteology of the caudal 614
vertebrae for the four Anolis species. For this, we measured the distance from the distal end 615
of the cotyle to the proximal tip of the condyle on each caudal vertebra. All measurements 616
apart from SVL were standardized by dividing by the distance between the snout to the end 617
of the sacral vertebrae as an approximation to body size. We compared microCT and 618
skeletal preparation measurements with a paired T-test to assess possible bias in the 619
sampling methodology (Table S11). 620
Data Availability 621
All raw read files have been accessioned to the NCBI SRA under BioProject 622
#PRJNA1096315. Final genome assemblies and annotations have been accessioned to the 623
Harvard Dataverse (https://doi.org/10.7910/DVN/F9NDWL). 624
Funding 625
This project was supported by funding from NSF grant DEB-1927194 to AJG and JL and 626
the College of Liberal Arts and Sciences at Arizona State University (ASU) to KK. RAD 627
was supported by the Doctoral scholarship 72200094 (ANID, Chile) and the Peabody 628
Family Memorial Award. Support for EL was provided by the ASU School of Life 629
Sciences Undergraduate Research Program. 630
631
References 632
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