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bioRxiv preprint doi: https://doi.org/10.1101/556035; this version posted February 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Title: The receptor D5 shows signs of independent erosion in Toothed and 2 Baleen whales

3 Luís Q. Alves 1,2, Juliana Alves 1,2, Rodrigo Ribeiro 1,2, Raquel Ruivo 1,*, and L. Filipe C. 4 Castro 1,2,* 5 1 CIIMAR/CIMAR—Interdisciplinary Centre of Marine and Environmental Research, 6 University of Porto, 4450-208 Matosinhos, Portugal 7 2 FCUP—Faculty of Sciences, Department of Biology, University of Porto, 4169-007 Porto, 8 Portugal 9 * Correspondence: [email protected] (R.R.); [email protected] (L.F.C.C.) 10 11 Abstract: To compare gene loci considering a phylogenetic framework is a promising approach 12 to uncover the genetic basis of human diseases. Imbalance of dopaminergic systems is suspected 13 to underlie some emerging neurological disorders. The physiological functions of dopamine are 14 transduced via G--coupled receptors, including DRD5 which displays a relatively higher 15 affinity towards dopamine. Importantly, DRD5 knockout mice are hypertense, a condition 16 emerging from an increase in sympathetic tone. We investigated the evolution of DRD5, a high 17 affinity receptor for dopamine, in mammals. Surprisingly, among 124 investigated mammalian 18 genomes, we found that Cetacea lineages (Mysticeti and Odontoceti) have independently lost this 19 gene, as well as the burrowing Chrysochloris asiatica (Cape golden mole). We suggest that DRD5 20 inactivation parallels hypoxia-induced adaptations, such as peripheral vasoconstriction required 21 for deep-diving in Cetacea, in accordance with the convergent evolution of vasoconstrictor 22 in hypoxia-exposed animals. Our findings indicate that Cetacea are natural knockouts for DRD5 23 and might offer valuable insights into the mechanisms of some forms of vasoconstriction 24 responses and in humans.

25 Keywords: dopamine; gene loss; cetacea

26 1. Introduction 27 Dopamine is a neurotransmitter essential for brain function, regulating various physiological 28 processes including locomotion, cognition, and neuroendocrine functions [1,2]. Dopamine 29 molecular actions are transduced via a specific group of G-protein coupled receptors entailing 30 two major classes: DRD1-like and DRD2-like receptors [3,4]. While DRD1-like receptors 31 stimulate cAMP production postsynaptically, DRD2-like receptors inhibit cAMP production both 32 pre and postsynaptically [3]. The genomic structure of the underlying genes is also distinct, with 33 DRD1-like receptors yielding single exon coding regions [3]. DRD1-2 receptor classes have 34 diversified in vertebrate evolution most likely as a result of genome duplications [4]. Interestingly, 35 and antagonist site conservation suggests evolutionary stasis of dopaminergic 36 pathways [4]. Among DRD1-like receptors, the DRD5 subtype displays distinctive features, 37 namely a relatively higher affinity towards dopamine, a putative agonist-independent activity and 38 low level, yet widespread, brain expression [3,5-7]. Nonetheless, the DRD5 seems to display 39 distinct regional and cellular distribution patterns in the brain, when compared to the DRD1 and 40 DRD2 subtypes, with protein enrichment detected in the cerebral cortex, and basal 41 ganglia [7,8]. Peripheral expression has also been found in the adrenals [9], [10] and 42 gastrointestinal tract [11]. Despite the association with [12], attention- 43 deficit/hyperactive disorder [13] and substance abuse [14], gene targeting studies revealed that 44 DRD5 knock-out mice develop hypertension, showing increased from 3 months of 45 age [2]. This hypertensive phenotype appears to result from a defect, 46 leading to an increase in sympathetic tone and, consequently, vasoconstriction [2]. Besides 47 neuronal impairment, DRD5 disruption was also suggested to counter-regulate and modulate the 48 expression of the prohypertensive Angiotensin II Type 1 Receptor (AT1R), involved in renal salt 49 balance, blood pressure and vasoconstriction [15-17].

1 bioRxiv preprint doi: https://doi.org/10.1101/556035; this version posted February 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

50 Whole genome sequencing has greatly expanded our capacity to comprehend evolutionary 51 history, the role of adaptation or the basis for phenotype differences across the tree of life. Multi- 52 genome comparisons have also been powerful to recognize the molecular basis of human diseases, 53 a field named as phylomedicine [18-21]. Here we investigate the evolution of DRD5 in 54 mammalian species. By analysis 124 genomes covering 16 orders, we show that independent 55 coding debilitating mutations occurred in the ancestors of Mysticeti and Odontoceti (Cetacea). 56 Our findings suggest that these species are natural KOs for this and might 57 offer valuable insights into the mechanisms of some forms of essential hypertension. 58 59 2. Results 60 To examine the annotation tags and distribution of the DRD5 gene across mammals, 119 61 annotated mammalian genomes available at NCBI (National Center of Biotechnology 62 Information), were scrutinized for the presence of DRD5 gene annotation and each respective 63 protein product description screened for the ‘low-quality protein’ (LQ) tag. This examination 64 resulted in 10 species presenting DRD5 annotation tagged as ‘low-quality protein’ producing gene, 65 including Ovis aries (sheep), Phascolarctos cinereus (koala), Bison bison bison (plains bison), 66 Myotis davidii (vesper bat), Ochotona princeps (American pika) and 5 cetacean species. The latter 67 included Lagenorhynchus obliquidens (Pacific white-sided dolphin), Neophocaena 68 asiaeorientalis asiaeorientalis (Yangtze finless porpoise), Delphinapterus leucas (beluga whale), 69 Physeter catodon (sperm whale) and Orcinus orca (killer whale). Each genomic sequence 70 corresponding to the DRD5 LQ annotations was examined and the coding sequence (CDS) 71 manually predicted [22]. Given the prominence of DRD5 LQ annotations in Cetacea we 72 scrutinized other cetacean species with available, but unannotated genomes, Balaenoptera 73 bonaerensis (Antarctic minke whale), Eschrichtius robustus (gray whale), Balaena mysticetus 74 (bowhead whale), Sousa chinensis (Indo-Pacific humpback dolphin), or with annotated genomes 75 lacking DRD5 annotations: Lipotes vexillifer (Yangtze River dolphin) and Balaenoptera 76 acutorostrata scammoni (minke whale) (Figure 1). Additionally, Tursiops truncatus (bottlenose 77 dolphin), presenting a seemingly intact DRD5 gene annotation, without the ‘low-quality protein’ 78 tag, as well as Hippopotamus amphibius (common hippopotamus) predicted DRD5 coding 79 sequence (Figure 1), representing the closest extant lineage of Cetacea, were equally inspected. 80 Other mammals with annotated genome without DRD5 gene annotation were also scrutinised, 81 namely: Microcebus murinus (gray mouse lemur), Jaculus jaculus (lesser Egyptian jerboa), 82 Chrysochloris asiatica (Cape golden mole), Erinaceus europaeus (western European hedgehog), 83 Elephantulus edwardii (Cape elephant shrew) and Condylura cristata (star-nosed mole). In total, 84 124 mammalian species were inspected and an in-depth description regarding analysed species 85 list and genomic sequences accession numbers are available at Supplementary Table 1. The 86 Supplementary Figure 1 presents a multiple translation alignment of the NCBI non-‘low-quality 87 protein’ tagged mammalian DRD5 orthologous sequences. The alignment also includes the 88 predicted H. amphibius DRD5 sequence and excludes T. truncatus DRD5 sequence, afterwards 89 demonstrated as pseudogenized (see below). The examined sequences exhibit a substantial degree 90 of conservation (average pairwise identity of over 80%), with minor variations in expected protein 91 size. The protein sequence conservation is particularly noticeable at the c-terminus (see below).

2 bioRxiv preprint doi: https://doi.org/10.1101/556035; this version posted February 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

92

93 Figure 1. Comparative synteny maps of DRD5 genomic in Cetacea and H. 94 amphibius. Blue squares indicate the presence of DRD5 in genome annotation. Red 95 squares represent the absence of DRD5 annotation in the corresponding genome. H. 96 amphibius genes found in the same genomic scaffold are represented by dark grey 97 squares and underlined with a bar indicating the corresponding accession number. Not 98 found genes due to the end of genomic region are represented by white squares.

99 2.1. ORF disrupting mutations of DRD5 in Cetacea 100 For cetacean species with annotated genomes, we started by examining the DRD5 gene locus, 101 including neighbouring genes, to verify and elucidate the orthology of the annotated and non- 102 annotated genes and outline the genomic regions to be inspected. All analysed loci were found to 103 be conserved, including in both L. vexillifer and B. a. scammoni, which lacked previous DRD5 104 gene annotations (Figure 1). Subsequent manual annotation of all collected cetacean genomic 105 sequences revealed DRD5 gene erosion across all analysed species, except in B. a. scammoni, for 106 which the DRD5 coding status could not be accessed due to fragmentation of the 5’ end of the

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107 respective genomic region (presence of sequencing gaps (Ns)). In detail, a conserved 2 nucleotide 108 deletion was detected for the full set of Odontoceti species examined, except for P. catodon that 109 presented a premature in the middle of the gene and a single nucleotide insertion near 110 the end of the gene (Figure 2). The deletion alters the reading frame, leading to a drastic change 111 in downstream amino acid composition. Additionally, a premature stop codon shortens the 112 protein’s expected size—31 amino acid shorter than hippopotamus—a characteristic not found in 113 any of the examined coding DRD5 sequences. Additionally, non-conserved mutations were found 114 in L. vexillifer which presented 2 premature stop codons and D. leucas which displayed a single 115 nucleotide deletion near the 5’ end of the gene (Figure 2). D. leucas presumed DRD5 sequence 116 presented another noticeable feature. A massive and abrupt alignment identity decrease was 117 observed when aligned with the Bos taurus (cow) reference against the genomic target region of 118 D. leucas. The alignment identity drop was observed approximately in the middle of the complete 119 alignment length for this species, suggesting that the DRD5 gene sequence is interrupted, and 120 further supporting pseudogenization (Figure 3). Regarding Odontoceti, at least one ORF- 121 abolishing mutation was validated using available genomic Sequence Read Archives (SRAs) 122 experiments for all studied species, excluding S. chinensis and L. vexillifer for which no genomic 123 sequencing runs were available at the NCBI SRA database (Supplementary Material 1). 124

125 126 Figure 2. Schematic representation of the DRD5 gene ORF abolishing annotated 127 mutations regarding Cetacea suborders Odontoceti (light gray) and Mysticeti (dark 128 gray). Thicker bars represent 2 nucleotide insertions (blue) or deletions (orange). 129 Thinner bars represent single nucleotide insertion (blue) or deletion (orange). Premature 130 stop codons are represented by red thick bars. Unknown regions, either resulting from 131 genome poor assembly or coverage (Ns in Balaenoptera acutorostrata scammoni) or 132 due to small genomic scaffold size (Balaenoptera bonaerensis), are represented by 133 black regions. Hippopotamus amphibius presents a complete functional DDR5 gene. 134 Two elucidative boxes represent the conserved mutations found in Odontoceti (2 135 nucleotide deletion) and in Mysticeti (1 nucleotide deletion).

136 Regarding the Mysticeti suborder, a conserved single nucleotide deletion was detected in all 137 species except B. a. scammoni (Figure 2). A non-conserved 2 nucleotide insertion was found also 138 found in B. bonaerensis and an insertion of one nucleotide was detected at E. robustus DRD5 139 sequence (Figure 2). Again, at least one ORF-abolishing mutation was validated using genomic 140 SRA experiments for all analysed species (Supplementary Material 1). Importantly, no conserved 141 mutations were detected between Odontoceti and Mysticeti clades, suggesting that DRD5 142 pseudogenization events occurred independently after the divergence of both lineages (Figure 2). 143 To increase the robustness of our analysis, we further scrutinized the genome of the extant sister 144 clade of the Cetacea, the Hippopotamidae and were able to predict a fully functional CDS for 145 DRD5 in H. amphibius, supporting the loss of DRD5 after Cetacea diversification.

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146 147 148

149

150

151 Figure 3. B. taurus reference DRD5 gene (single-exon) aligned against the genomic 152 target regions of D. leucas. Lower bar represents the reference exon and the upper bar 153 represents the genomic target region. An alignment identity graph is presented above 154 the alignment. Green represents very high alignment identity values, followed by yellow 155 (mid to high alignment identity values) and finally red, indicating a very low alignment 156 identity value. The hypothetical approximate DRD5 gene real size in D. leucas is marked 157 by the clear abrupt alignment identity drop and is represented by a green shadowed 158 rectangle.

159 2.2. Other mammalian species showing DRD5 LQ tags have a coding gene 160 Our initial analysis revealed the presence of at least one ORF-abolishing mutation in O. aries, 161 P. cinereus, B. bison bison and O. princeps. These are in some cases suggestive of gene 162 inactivation and not sequencing artefacts [18,22]. To investigate whether these DRD5 gene 163 sequences are eroded in these species, we performed a meticulous manual annotation including 164 SRA validation. Our results revealed sequencing reads supporting the functionality of the gene, 165 rebutting each inactivation mutation and suggesting that DRD5 is, in fact, coding in these species 166 (Supplementary Material 2). Regarding M. davidii, the fragmentation of the genomic region (Ns) 167 flanked by upstream and downstream DRD5 neighbouring genes impeded us to infer the DRD5 168 coding status in this species, although, the visible nucleotide aligning regions showed no ORF 169 disruptive mutations, excepting for an 8 nucleotide deletion near the end of the gene, leading to a 170 STOP codon, suggesting that this gene possibly is lightly truncated but still functional in this 171 species. Interestingly, regarding O. princeps mutational SRA validation, two scenarios were 172 observed: approximately 50% of aligned reads supported the absence of a premature stop codon 173 in the DRD5 gene of this species, with the remaining set of aligning reads supporting the presence 174 of a premature stop codon in the same species. This suggest that an allelic pseudogenization event 175 might have occurred in O. princeps. 176 177 2.3. Chrysochloris asiatica presents a non-functional DRD5 gene 178 Next, we examined other mammalian species with annotated genome yet lacking DRD5 gene 179 annotations. Results were inconclusive regarding the coding status of M. murinus, C. cristata, J. 180 jaculus and E. europaeus DRD5, due to the fragmentation (Ns) of the genomic region flanked by 181 the upstream and downstream DRD5 neighbouring genes, and the unavailability of whole genome 182 shotgun contigs, spanning our target gene. For E. edwardii we were able to deduce a fully 183 functional DRD5 coding sequence. Curiously, C. asiatica presented a single nucleotide insertion 184 in the 5’ end of the gene (validated by genomic SRA, see Supplementary Material 3), suggesting 185 that DRD5 is pseudogenized in this species. 186

187 3. Discussion 188 The rise of large-scale genomic sequencing projects has emphasized the role of gene loss as 189 a potent driver of evolutionary change: underlying phenotypic adaptations or neutral regressions 190 in response to specific environmental cues and niches [23-28]. Interestingly, gene loss 191 mechanisms seem pervasive in lineages that endured drastic environmental adaptations in the 192 course of evolution, such as Cetacea, entailing niche-specific physiological and morphological 193 adaptations [25,29-33].

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194 By comparing 124 mammalian genomes, we document the independent erosion of a 195 dopamine receptor, DRD5, in both Cetacea lineages, Mysticeti and Odontoceti. Dopamine, a 196 neurotransmitter and signalling molecule, is involved in distinct functions both in the central 197 nervous system and peripheral tissues: including movement, feeding, sleep, reward, learning and 198 memory as well as in the regulation of olfaction, hormone pathways, renal functions, immunity, 199 sympathetic regulation and cardiovascular functions, respectively [3]. More specifically, the 200 disruption of DRD5-dependent pathways in rodents was shown to increase blood pressure and 201 sympathetic tone, promoting vasoconstriction, thus yielding a hypertensive phenotype [2,15]. 202 The observed gene loss distribution, and predicted phenotypic outcome, is consistent with 203 the peripheral vasoconstriction mechanism described in Cetacea, suggested to counterbalance 204 deep-diving induced hypoxia [34,35]. In fact, Cetacea have developed a number of physiological 205 adaptations to offset hypoxia: notably, increased blood volume and oxygen-transport protein 206 levels (hemoglobin, neuroglobin and myoglobin), allowing oxygen stores in blood, muscle 207 tissues, and brain, reduced heart rate or bradycardia, apnea and peripheral vasoconstriction [34- 208 36]. Peripheral vasoconstriction allows the regional compartmentalization of blood supplies, 209 reducing blood flow in more hypoxia-tolerant tissues, such as skin, muscle, spleen or kidney, 210 while maintaining arterial blood flow to the central nervous system and heart [36]. Moreover, the 211 deviation of muscle blood supply reduces lactate accumulation [36]. Thus, in Cetacea, DRD5 loss 212 could contribute for the peripheral vasoconstriction requirements of diving. Also, by shifting renal 213 salt balance, DRD5 could also play a role in the maintenance of an adequate blood volume and 214 pressure [15-17,37]. 215 The predicted vasoconstriction phenotype is in agreement with a previous work reporting 216 episodes of adaptive evolution (positive selection) in genes related with hypoxia tolerance in 217 Cetacea, including genes involved in oxygen transport and regulation of vasoconstriction [35]. In 218 addition, by expanding their analysis to other non-aquatic hypoxic environments, such as 219 underground tunnels, they uncovered convergent evolution scenarios in species adapted to diving 220 and burrowing [35]. A similar convergence is observed in the present work. In fact, in the mole 221 C. asiatica, DRD5 was also predicted non-functional. Although this was the single DRD5 loss 222 example found outside Cetacea, one cannot discard the putative contribution of alternative 223 molecular events (i.e. post-translational mechanisms) towards trait loss [24], or event distinct 224 physiological adaptations to overcome oxygen deprivation. 225 Besides diving physiology, DRD5-dependent sympathetic tone alterations could also 226 contribute to the idiosyncratic sleep behavior observed in Cetacea [30,38]. Several physiological 227 adjustments occur during sleep, encompassing thermoregulation, as well as endocrine, immune, 228 pulmonary and cardiovascular functions [39]. In most mammals, sleep states lead to a decrease 229 of the sympathetic tone, inducing vasodilation and decreasing blood pressure [39]. Thus, DRD5 230 loss could prevent sympathetic tone decrease in resting states paralleling the unihemispheric 231 sleeping behavior and long-term vigilance observed in Cetacea. 232 Overall, our findings provide evidence for natural occurring KO for DRD5. Besides 233 highlighting a molecular signature for vasoconstriction and blood pressure regulation in Cetacea, 234 naturally occurring DRD5 KO could also provide useful frameworks to gain insight into 235 hypertension and heart failure-induced peripheral vasoconstriction responses in humans [40,41]. 236 237 4. Materials and Methods 238 4.1. Synteny analysis 239 To build the synteny maps for the DRD5 gene locus in Cetacea and H. amphibius (Figure 1) 240 several annotated Cetacea genome assemblies were inspected and scrutinised using the NCBI 241 browser, namely O. orca (GCF_000331955.2), L. obliquidens (GCF_003676395.1), T. truncatus 242 (GCF_001922835.1), D. leucas (GCF_002288925.1), N. a. asiaeorientalis (GCF_003031525.1), 243 L. vexillifer (GCF_000442215.1), P. catodon (GCF_002837175.1) and B. a. scammoni 244 (GCF_000493695.1). B. taurus (GCF_002263795.1), a fully terrestrial relative of extant 245 cetaceans, was used as reference. Next, (1) if DRD5 was annotated, the following procedure was 246 used: five flanking genes, considering only the ones characterised and tagged as protein coding,

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247 from each side of DRD5 gene, were collected. (2) If DRD5 gene annotations were not found, B. 248 taurus DRD5 direct flaking genes were used as reference genes to collect the corresponding 249 flanking genes. In detail, CPEB2 was used as an anchor to search the rightwards DRD5 flanking 250 genes, and OTOP1 to search the leftwards genes. Regarding H. amphibius, the synteny map was 251 built via blast searches against the assembled, fragmented and not annotated genome of the same 252 species, available at NCBI (GCA_002995585.1). B. taurus DRD5 flanking genes were used as 253 reference, and using the discontiguous megablast task from blastn, the best blast hit (the hit with 254 the highest alignment identity and query coverage) was retrieved and the coordinates of alignment 255 in the target genome carefully inspected. H. amphibius synteny map was then built by sorting the 256 genes according to the subject alignment coordinates within genes aligning at the same genomic 257 scaffold. 258 259 4.2. Sequence retrieval and gene annotation 260 To perform manual gene annotation, all genomic regions of DRD5 sequences tagged as LQ 261 were directly collected from NCBI. Regarding cetacean species with annotated genomes but 262 without DRD5 gene annotations (L. vellixifer and B. a. scammoni), the genomic sequence ranging 263 from the upstream to the downstream flanking genes was collected. For non-cetacean mammals, 264 with annotated genomes but without DRD5 annotations two procedures were followed. (1) For 265 each species, two DRD5 direct flanking genes were selected from a reference phylogenetic close 266 species displaying non ‘low-quality protein’ DRD5 annotation (see Supplementary Table 2) and 267 the test species region flanked by the same reference genes was further collected. (2) If the same 268 flanking region exhibited severe fragmentation (Ns), using the same phylogenetic close species 269 (Supplementary Table 2) DRD5 coding sequence as query, blastn searches were conducted against 270 the Whole Shotgun Contigs dataset of the corresponding species via discontiguous megablast 271 task. The genomic sequence corresponding to the blast hit with the highest alignment identity and 272 query coverage amongst the others was retrieved. 273 For cetacean species with no annotated genomes (B. bonaerensis, E. robustus, B. mysticetus, 274 S. chinensis), as well as H. amphibius, genomic sequences were retrieved through blastn searches 275 in the corresponding genome assembly using the B. taurus DRD5 coding sequence as query. The 276 best genomic scaffold considered for each species corresponded to the blast hit with the highest 277 query coverage and identity value amongst total hits. Due to the presence of a fragmented 278 genomic region concerning T. truncatus DRD5 gene annotation, the same blast search procedure 279 was executed for this species, using as target the Whole Genome Shotgun contig dataset of the 280 same species. The gene annotation concerning this species was performed in the retrieved contig 281 corresponding to the blast hit with the highest query coverage and alignment identity value. 282 Collected genomic sequences were further imported into Geneious Prime 2019 283 (www.geneious.com) and the DRD5 gene coding sequences were manually annotated for each 284 species. Briefly, using the built-in map to reference tool with the highest sensibility parameter 285 selected, the reference single-exon DRD5 gene, 3’ and 5’ UTR flanked, was mapped against the 286 corresponding genomic sequence of the in-study species and aligned regions carefully screened 287 for ORF abolishing mutations including frameshift mutations and premature stop codons. For 288 Cetacea and H. amphibius DRD5 gene annotation, B. taurus DRD5 was selected as reference. 289 Regarding non-cetacean mammals DRD5 annotation, different references were chosen according 290 to the phylogenetic relationships between reference and test species (Supplementary Table 2). 291 The identified mutations were next validated using the sequencing run raw data (sequencing 292 reads) retrieved from at least two independents genomic NCBI SRA projects (when available). 293 294 Author Contributions: conceptualization, R.R. and L.F.C.C.; methodology, L.Q.A. and 295 L.F.C.C.; investigation, L.Q.A., R.R. and J.A.; data curation, L.Q.A.; writing—original draft 296 preparation, R.R., L.Q.A. and L.F:C.C.; writing—review and editing, All; supervision, R.R.; 297 project administration, L.F.C.C.; funding acquisition, L.F.C.C..

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298 Funding: This work was supported by Project No. 031342 co-financed by COMPETE 2020, 299 Portugal 2020 and the European Union through the ERDF, and by FCT through national funds.

300 Acknowledgments: We acknowledge the various genome consortiums for sequencing and 301 assembling the genomes.

302 Conflicts of Interest: The authors declare no conflict of interest.

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