Aquatic adaptation and the evolution of smell and taste in whales
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While olfaction is one of the most important senses in most terrestrial mammals, it is absent in modern toothed whales (Odontoceti, Cetacea). Furthermore, behavioral evidence suggests that gustation is very limited. In contrast, their aquatic sistergroup, baleen whales (Mysticeti) retain small but functional olfactory organs, and nothing is known about their gustation. It is difficult to investigate mysticete chemosensory abilities because experiments in a controlled setting are impossible.
Here, we use the functional regionalization of the olfactory bulb (OB) to identify the loss of specific olfactory functions in mysticetes. We provide the whole-genome sequence of a mysticete and show that mysticetes lack the dorsal domain of the OB, an area known to induce innate avoidance behavior against odors of predators and spoiled foods. Genomic and fossil data suggest that mysticetes lost the dorsal domain of the OB before the Odontoceti-Mysticeti split. Furthermore, we found that all modern cetaceans are revealed to have lost the functional taste receptors.
These results strongly indicate that profound changes in the chemosensory capabilities had occurred in the cetacean lineage during the period when ancestral whales migrated from land to water.
KeywordsAntarctic minke whale genome Archaeoceti Cetacea Chemoreception Olfactory bulb
- D domain
Guanine nucleotide binding protein, alpha transducing 3
G-protein coupled receptor
Million years ago
NAD(P)H dehydrogenase quinone 1
Olfactory cell adhesion molecule
Olfactory medium-chain acyl-CoA synthetase
Olfactory sensory neuron
Trace amine-associated receptor
Taste receptor type 1
Taste receptor type 2
- V domain
Vomeronasal receptor type 1
Vomeronasal receptor type 2
Terrestrial mammals usually have a well-developed sense of smell that can detect various odors using four kinds of G-protein coupled receptors (GPCRs) encoded by different multigene families to each other: olfactory receptors (ORs), trace amine-associated receptors (TAARs) and two types of vomeronasal receptors (V1Rs and V2Rs) . But this sense was greatly reduced in the ancestors of modern cetaceans . Modern cetaceans lack a large number of OR genes [3-5], and odontocetes lost the nervous system structures that mediate olfaction, such as the olfactory tract, olfactory bulb (OB) and cranial nerve I . In addition to the four olfactory GPCRs, two GPCR families are involved in mammalian gustation: TAS1R (taste receptor type 1, the sweet and umami taste receptor) and TAS2R (taste receptor type 2, the bitter taste receptor) . Most of the taste receptor genes have also been lost from dolphin genomes [7,8], though behavioral tests indicate that dolphins can detect several kinds of flavorants . In contrast, mysticetes retained these anatomical structures, although they are small , and it has been suggested that mysticetes use olfaction in foraging . Like those of terrestrial mammals, mysticetes’ olfactory nerves are concentrated in their nasal cavities , and their nasal passages remain filled with air when they dive and keep water out, indicating that mysticetes can smell in air but not underwater. Unfortunately, no mysticete species are kept in laboratories or aquariums, meaning that experiments in a controlled setting are impossible, and thus it is still a mystery how mysticetes use olfaction for their fully aquatic life. Regarding taste, most of their taste receptors have been lost [12,13], but it is not clear whether the remaining receptors are still functional or not.
Olfaction has been studied in laboratory mammals: olfactory sensory neurons (OSNs) are located in the olfactory epithelium of the nasal cavity and each OSN expresses only one chemosensory receptor gene . The axons of the OSNs that express the same receptors converge to a set of glomeruli in the OB that are in a distinct topographic region of the OB . Thus, odorous information received in the olfactory epithelium is converted to topographical maps of activated glomeruli of the OB. The glomerular layer of the OB can be divided into two non-overlapping areas, a dorsal domain (D domain) and a ventral domain (V domain) based on the expression patterns of several domain-specific marker genes [16,17]. D domain-ablated mice (ΔD mice) fail to show innate avoidance behavior against predator odors and spoiled smells .
We previously studied the anatomy and histology of the OB in a single mysticete (bowhead whale Balaena mysticetus) . Olfactory nerves enter the OB from the ventral side in these mysticetes, and connect to glomeruli located on the ventral side. However, unlike OBs in most other mammals, dorsal OSN axons and glomeruli are absent or nearly absent. This distribution of glomeruli resembles that of ΔD mice, and this led us to hypothesize that mysticetes lack the D domain of the OB.
To test this hypothesis, we applied a whole-genome shotgun strategy and de Bruijn graph-based algorithms to sequence and assemble the Antarctic minke whale (Balaenoptera bonaerensis, Mysticeti) genome, and compared it to a dolphin (an odontocete) and a cow (an artiodactyl, the cetacean sister group). In addition, we investigated fossils to understand the evolution of whale OB from the morphological aspects. Genetic evidences about mysticete gustation are also examined based on the genome assembly.
Materials and methods
Genome sequencing and assembly
Muscle tissue of Antarctic minke whale was purchased from a fish market in Japan, and the genomic DNA was extracted following the protocol of our previous work . A paired-end sequencing library with average insert size of 330 bp was constructed and sequenced on an Illumina HiSeq2000 sequencer, and then assembled into scaffolds using PLATANUS assembler  ver. 1.2.1. Details about genome sequencing and de novo assembly are described in Additional file 1 §1. The Antarctic minke whale genome assembly thus obtained was named KUjira_1.0.
Cow (Bos taurus, Artiodactyla) genome assembly (UMD_3.1 assembly)  were downloaded from the GenBank FTP site (ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Bos_taurus/Bos_taurus_UMD_3.1/). Bottlenose dolphin (Tursiops truncatus, Odontoceti) genome assembly (Ttru_1.4 assembly)  were also downloaded from the GenBank FTP site (ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Tursiops_truncatus/Ttru_1.4/).
Olfaction-related genes in the cow genome
The loci of the OMACS, NQO1 and OCAM genes in the cow UMD_3.1 genome assembly follow NCBI reference sequence (RefSeq) annotations. The gene ID of each gene is as follows: OMACS, 100299006; NQO1, 519632; OCAM, 535613. We confirmed the RefSeq annotations by comparing translated amino acid sequences with those of other mammals. The amino acid sequences of 15 mouse TAARs (TAAR1 (GenBank accession no. NP_444435.1), TAAR2 (NP_001007267.1), TAAR3 (NP_001008429.1), TAAR4 (NP_001008499.1), TAAR5 (NP_001009574.1), TAAR6 (NP_001010828.1), TAAR7a (NP_001010829.1), TAAR7b (NP_001010827.1), TAAR7d (NP_001010838.1), TAAR7e (NP_001010835.1), TAAR7f (NP_001010839.1), TAAR8a (NP_001010830.1), TAAR8b (NP_001010837.1), TAAR8c (NP_001010840.1), TAAR9 (NP_001010831.1)) and six human TAARs (TAAR2-1 (NP_001028252.1), TAAR2-2 (NP_055441.2), TAAR5 (NP_003958.2), TAAR6 (NP_778237.1), TAAR8 (NP_444508.1), TAAR9 (NP_778227.3)) were used as queries and TAAR sequences were searched against the cow genome assembly using TBLASTN program ver. 2.2.25  with e-value cutoff of <1e-20 and without filtering query sequences. All overlapping sequences of hits with the same orientations were merged. The sequences thus obtained were searched against the mouse protein database (downloaded from the following URL on 14/Oct/2011: http://www.ncbi.nlm.nih.gov/protein/?term=%22Mus+musculus%22%5Bporgn%3A__txid10090%5D) using FASTY program ver. 35.04  and the sequence was discarded if its best hit was not a TAAR gene. Then we aligned all the remaining sequences using the L-INS-i program in the MAFFT package ver. 6.240 [23,24] and looked for the initiation and termination codons. If we could not find initiation and/or termination codons in a sequence, we extended the sequence in the 5’ and/or 3’ direction to find them. If a sequence was interrupted by premature stop codon(s) and/or frame shift(s), or if it lacked one or more trans-membrane (TM) regions completely, the sequence was judged to be a functionless pseudogene. As a result, 17 intact TAAR genes and 14 pseudoegenes were found. The classification of intact cow TAAR genes into TAAR1-9 follows the phylogenetic tree shown in Additional file 1 §3. Deduced amino acid sequences of 142 class I and 828 class II intact OR genes were retrieved from Niimura and Nei .
Olfaction-related genes in the whale and dolphin genomes
Classification of cetacean OR genes into class I/class II
Genes for the sense of taste
TBLASTN searches with e-value cutoff of <1e-5 and without filtering query sequences were employed to identify TAS1R, TAS2R and GNAT3 genes. The amino acid sequence of cow GNAT3 is retrieved from GenBank (accession no. NP_001103452). Using all amniote GNAT3 sequences annotated in Ensembl database (http://www.ensembl.org/index.html) (release 73) as queries, GNAT3 genes were searched against KUjira_1.0 and Ttru_1.4 assemblies. TAS1R genes were also searched against UMD_3.1, KUjira_1.0 and Ttru_1.4 assemblies using all vertebrate TAS1R sequences annotated in Ensembl database (release 70) as queries. In the case of TAS2Rs, we used all intact Euarchontoglires TAS2Rs identified by Hayakawa et al.  as queries and searched against UMD_3.1, KUjira_1.0 and Ttru_1.4 assemblies. All overlapping sequences of hits with the same orientations were merged. The sequences thus obtained were searched against the human (GRCh37 assembly) [33,34] and the mouse (GRCm38 assembly)  genome assemblies using TBLASTX and the sequence was discarded if its best hit was not a GNAT3/TAS1R/TAS2R gene. Because TAS1Rs and GNAT3 are multi-exon genes, the results of TBLASTX were also utilized for subsequent exon annotations. Exon regions and splicing sites of the GNAT3 and TAS1R genes identified in this study were determined by comparing GNAT3 and TAS1R sequences of cetartiodactyls with that of humans and mice using E-INS-i program in the MAFFT package. A taste receptor gene was considered a pseudogene or truncated gene if the same criteria were met that we followed for odorant receptors.
Primers used for amplifying and sequencing the 6 th exon of GNAT3 gene
Within 6th exon of GNAT3 gene
Within 6th exon of GNAT3 gene
This pair of primers are designed to amplify a 299 bp-length region including the partial amino coding region of the 6th exon of GNAT3 gene
Loss of the D domain in all modern whales
The D domain is defined by the expression of the OMACS gene [16,38], and the unique expression of NQO1 gene is also reported . We found that, in both minke whales and dolphins, the OMACS gene is not functional due to the loss of the 5th, 9th, 10th and 11th exons (Figure 1a). Both cetaceans also lost the functional NQO1 gene due to genomic inversion (Figure 1b). These findings suggest that both OMACS and NQO1 genes turned into pseudogenes before the Odontoceti-Mysticeti split.
The V domain is defined by the expression of the OCAM gene [16,40]. Minke whales have maintained the complete coding region of the OCAM gene. In addition to that, dolphins, even though they are anosmic, also have kept this gene under strict purifying selection (Additional file 1 §2), suggesting that this gene has unknown function besides olfaction.
The molecular basis of olfaction relies on the repertoires of four families of chemosensory receptors: TAAR, OR, V1R and V2R . OSNs expressing TAARs project specifically to the D domain of the OB [41,42], and all of the TAAR genes are located in a single gene cluster with no interspersed genes . We found that minke whales have lost the TAAR5, 6, 7 and 8 genes and dolphins have lost the TAAR2-8 genes. In addition, all of the remaining TAAR genes of whales and dolphins are functionless pseudogenes except for the whale TAAR1 gene (Figure 1c), which is not involved in olfaction . Deletion of TAAR5-8 genes from the minke whale genome was also confirmed by PCR (Additional file 1 §4). This finding suggests that both minke whales and dolphins have lost all the olfactory TAARs.
Mammalian ORs can be classified into two subfamilies, class I and class II, based on sequence similarity . Most OSNs expressing class I ORs project specifically to the D domain , whereas OSNs expressing class II ORs project to both the D and V domains . We found only four intact class I OR genes in the minke whale genome and two in the dolphin genome (Figure 2). Both whales and dolphins have kept two intact class I ORs, OR51E1 and OR51E2 (Additional file 1 §3). The expression of these two ORs is highly restricted to prostate tissues [46,47], indicating that these ORs play roles that are not related to olfaction. Minke whales have kept two more intact class I OR genes, but it is difficult to judge whether these two remaining genes are still functional or not. In any case, all cetaceans underwent a significant loss of olfactory-functional class I ORs in evolution. In contrast, 56 intact class II OR genes were found in the minke whale genome, well below the 828 present in cow , but above the ten in dolphins (Figure 2). This is consistent with our previous findings that V domain of the baleen whale OB is small but functional and that baleen whales have a sense of smell .
All of these findings suggest that mysticetes have lost most of the D domain-specific markers and receptors. We conclude that the mysticete OB lacks a region homologous to the D domain of the mouse OB. Putting the loss of the D domain markers in evolutionary perspective, we hypothesize that whales lost the D domain of the OB during the Eocene epoch, which is the time when whale ancestors migrated from land to water.
The OB communicates with the nasal cavity via the cribriform plate. The cribriform plate fossilizes and its shape can be used to deduce the shape of the OB, thus tracing the reduction of the D domain in evolutionary time. Cetaceans originated around 50 million years ago (MYA) , and their basal family is Pakicetidae . In this family, the orbits are close together near the midline and the OB is located just anterior to the orbit . As a consequence, the OB is very small . However, we investigated the skull of a pakicetid Ichthyolestes and found that a part of the cribriform plate faces dorsally, and is perforated by many small foramina, presumably for cranial nerve I (Figure 3ab). On the other hand, the cribriform plate of remingtonocetids, Eocene whales closer to the divergence of mysticetes and odontocetes, differs from that of pakicetids, in that the OB faces ventral, similar to bowhead whales and there is no indication for dorsally projecting fibers of cranial nerve I (Figure 3c-g). Thus, whereas pakicetids show connections of the cribriform plate on the dorsal side of the OB, these connections are lost in remingtonocetids. In effect, the olfactory anatomy of modern minke whales resembles that of late Eocene whales . This suggests that the D domain was lost during the course of the Eocene, but was present in the earliest cetaceans.
Loss of the vomeronasal olfaction in basal cetaceans
No intact V2R genes exist in the cetaceans and cattle (Figure 2), suggesting that this gene family was lost in the cetartiodactyl lineage before the cow-cetacean split, congruent with a previous report . In contrast, cattle have 40 intact V1R genes whereas mysticetes have only two and odontocetes just one (Figure 2). Absence of the VO, in which V1Rs are expressed [1,53], can be inferred from fossils, and this suggests that the organ was lost around 45 MYA, before the divergence of odontocetes and mysticetes. The vomeronasal ducts of mammals pass through the anterior palatine foramina, and the absence of these foramina implies that the organ is absent. Whereas these foramina are still present in the earliest whales pakicetids , they have been lost in remingtonocetids , and it is likely that the VO was lost at this node of the cladogram.
Loss of the sense of sweet, umami and bitter tastes in whales
Taken together, these results describe the outline of chemosensory evolution in cetaceans during the land to water transition. Cetaceans are derived from artiodactyls with well-developed olfactory and vomeronasal organs [55,56], although their V2Rs were already lost . Amphibious basal cetaceans emerged around 50 MYA, when olfactory organs were reduced, but retained both D and V domains. Around 45 MYA, the cetacean family Remingtonocetidae underwent significant changes in their chemical senses, losing the VO and the D domain of the OB. At this time, V1Rs, OMACS, NQO1, olfactory TAARs as well as most of class I OR genes are speculated to have lost their functions. Remingtonocetus are considered to have been one of the earliest whales that acquired well-established underwater hearing systems , and it is possible that, at this point in evolution, the importance of olfaction as a sense decreased. Basically, these conditions were maintained in modern mysticete whales. It is not obvious that physiological studies using mice can be directly extended to other mammals. However, it is reasonable to assume that the olfactory capability of mysticetes is similar to that of ΔD mice, i.e., that mysticetes lack innate avoidance behavior against predator odors and spoiled smells. Terrestrial animals cannot prey on fully aquatic whales, and whales’ predators, such as sharks and killer whales, cannot be detected by smelling in air. In addition, unlike the nares of other mammals, whales’ nares are not located at the tip of their snout, and whales’ nasal passage is not connected directly to their oral cavity, indicating that it is difficult for whales to rely on olfaction to judge whether something they are about to swallow is edible or not. Further studies will test this assumption. The evolution of taste cannot be traced in mysticete evolution, but gene evidence indicates that all cetaceans lack functional receptors for sweet, bitter and umami flavors. Although some Neogene odontocetes had a large OB chamber and well-developed cribriform plate , modern odontocetes reduced their chemical senses even further, losing the entire OB with further loss of class II OR genes.
This study indicates that all modern cetaceans lack innate avoidance behavior against spoiled smells, and the sense of tastes. This could be one of the reasons why cetaceans often die from ingesting inedible debris , and has implications for whale conservation.
Availability of supporting data
The Antarctic minke whale genome data sets supporting the results of this article are available in the DDBJ/EBI/NCBI databases under the following BioProject ID: PRJDB1465. The 6th exons of the GNAT3 genes amplified and sequenced in this study are available in the DDBJ/EMBL/GenBank databased under the following accession numbers: AB897678, AB897679, AB897680, AB897682 and AB897683.
This work was supported by Excellent Graduate Schools of Biodiversity & Evolution of Kyoto University, Leading Graduate Program of Primatology & Wildlife Science of Kyoto University, Yamada Science Foundation and JSPS KAKENHI (24770075 to TK and 12J04270 to TH). The super computer system of National Institute of Genetics (NIG), Research Organization of Information and Systems (ROIS) was used in part for assembling the whale genome. We are grateful to Rei Kajitani, Osamu Nishimura, Yasuhiro Go and Elizabeth Nakajima for technical advices and helpful comments. Muscle tissue of a hippopotamus was kindly provided by Takeshi Wada, Osaka Museum of Natural History.
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