Title Pairwise Comparison of Orthologous Olfactory Receptor Genes Between Two Sympatric Sibling Sea Kraits of the Genus Laticaud

Home , Laticauda frontalis

Pairwise comparison of orthologous olfactory receptor genes Title between two sympatric sibling sea kraits of the genus Laticauda in Vanuatu.

Kishida, Takushi; Hayano, Azusa; Inoue-Murayama, Miho; Author(s) Hikida, Tsutomu

Citation Zoological science (2013), 30(6): 425-431

Issue Date 2013-06

URL http://hdl.handle.net/2433/189751

Type Journal Article

Textversion publisher

Kyoto University Pairwise Comparison of Orthologous Olfactory Receptor Genes Between Two Sympatric Sibling Sea Kraits of the Genus Laticauda in Vanuatu Author(s): Takushi Kishida, Azusa Hayano, Miho Inoue-Murayama and Tsutomu Hikida Source: Zoological Science, 30(6):425-431. 2013. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zsj.30.425 URL: http://www.bioone.org/doi/full/10.2108/zsj.30.425

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. ZOOLOGICAL SCIENCE 30: 425–431 (2013) ¤ 2013 Zoological Society of Japan

Pairwise Comparison of Orthologous Olfactory Receptor Genes Between Two Sympatric Sibling Sea Kraits of the Genus Laticauda in Vanuatu

Takushi Kishida1*, Azusa Hayano2, Miho Inoue-Murayama3, and Tsutomu Hikida4

1Primate Research Institute, Kyoto University, Kanrin, Inuyama, Aichi 484-8506, Japan 2Kyoto University Museum, Yoshida-Honmachi, Sakyo, Kyoto 606-8501, Japan 3Wildlife Research Center, Kyoto University, Tanaka Sekiden-cho, Sakyo, Kyoto 606-8203, Japan 4Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo, Kyoto 606-8502, Japan

Olfaction-based reproductive isolation is widely observed in animals, but little is known about the genetic basis of such isolation mechanisms. Two species of sibling amphibious sea snakes, Laticauda colubrina and L. frontalis live in Vanuatu sympatrically and syntopically, but no natural hybrids have been reported. Adult females of both taxa possess distinctive lipids in the skin, and male L. frontalis distinguishes conspecific females based on olfactory cues. To shed light on the molecular basis of the evolution of olfaction-based isolation mechanisms, olfactory receptor (OR) gene repertoires of both taxa were identified using pyrosequencing-based technology, and orthologous OR gene sets were identified. Few species-specific gene duplications or species-specific gene losses were found. However, the nonsynonymous-to-synonymous substitution rate ratio was relatively higher between orthologous OR genes of L. frontalis and L. colubrina, indicating that L. frontalis and L. colubrina have evolved to possess different olfactory senses. We suggest that L. frontalis and L. colubrina have evolved allopatrically, and this may be a byproduct of the allopatric evolution, and that this dissimilarity may function as a premating isolation barrier, since L. frontalis has returned to the ancestral range (Vanuatu).

Key words: colubrina complex, Laticauda frontalis, olfaction, sea snake, speciation, yellow-lipped sea krait

the tropical Pacific Ocean (Heatwole et al., 2005). From INTRODUCTION 1983 to 1996, extensive field research on sea snakes was Sensory-based reproductive isolation is widely observed conducted in the western Pacific under the leadership of in animals, and is thought to play an important role as an Drs. N. Tamiya (Tohoku Univ.) and T. Tamiya (Sophia isolation barrier between sympatric sibling species. There Univ.), leading to the discovery that two syntopic sibling spe- have been many studies focusing on the molecular basis of cies are included in the populations of the yellow-lipped sea the evolution of such isolation mechanisms, especially focus- krait in Vanuatu (details on this research are described by ing on vision-based isolation mechanisms (e.g., Seehausen Cogger et al. (1987) and Shine et al. (2002)). One species et al., 2008). However, despite the indications that odor- is L. colubrina, and the other, named L. frontalis (de Vis, based reproductive isolation is likely to be important in many 1905), is available (Cogger et al., 1987). The two species metazoan taxa including vertebrates (Coyne and Orr, 2004), are morphologically nearly identical, except that L. colubrina little is known about the molecular basis of the evolution of grows larger than L. frontalis (Fig. 1, Cogger and Heatwole, isolation barriers based on pheromones and other chemicals 2006). They exist sympatrically and syntopically in Vanuatu, , which is due in part to the fact that, unlike in the case of but no natural hybrids have been reported, despite the fact photoreceptors (opsins), there are too many chemosensory that these two species breed at the same time (Shine et al., receptors (CRs) to be investigated in detail. 2002). Shine et al. (2002) showed that adult females of both Sea snakes of the genus Laticauda (Reptilia; Squamata; taxa possess distinctive lipids in the skin, and L. frontalis Serpentes; Elapidae) are a group of monophyletic amphibi- males distinguish conspecific females through their olfactory ous snakes, including the yellow-lipped sea krait Laticauda systems by tongue-flicking on the skin of females. Interest- colubrina. This species spends half of its lifetime on land ingly, L. colubrina males cannot distinguish conspecific (Shetty and Shine, 2002) and is widely distributed around females using olfactory cues, leading Shine et al. (2002) to speculate that male snakes prefer courting larger females, * Corresponding author. Tel. : +81-568-63-0580; meaning that L. colubrina males would be unlikely to court Fax : +81-568-62-9557; L. frontalis-sized (small) females even in the absence of E-mail: [email protected] pheromonal barriers. Based on these studies, L. frontalis Supplemental material for this article is available online. was formally elevated to full species status in 2006 (Cogger doi:10.2108/zsj.30.425 and Heatwole, 2006). In contrast to L. colubrina, the distri- 426 T. Kishida et al.

and chimpanzee branches (Go and Niimura, 2008) and that this may reflect the fact that these two species speciated allopatrically (Webster, 2009) and have evolved in different environmental niches (Adipietro et al., 2012). At present, in silico screening of whole-genome sequences is the best and indeed the only way to obtain nearly complete OR gene repertoires, but no genomic databases are available for Laticauda sea snakes, or even for elapid snakes. Dehara et al. (2012) suggested that a large number of OR genes can be obtained for a species without any genome databases using pyrosequencing-based OR gene identification methods. In this study, we sampled three Laticauda species, L. colubrina, L. frontalis and L. laticaudata (as an outgroup) in Vanuatu, identified their OR genes using pyrosequencing-based technology, and compared the OR gene repertoires in L. frontalis and L. colubrina in order to investigate how olfactory abilities differentiated between these two species after their genetic split. MATERIALS AND METHODS

Fig. 1. Laticauda colubrina (upper) and L. frontalis (lower) sam- Sampling sea snake specimens pled in Vanuatu. A male L. colubrina and a male L. frontalis were sampled at Ngioriki Islet, Paonangisu village, Vanuatu (17°30′S, 168°25′E). bution of L. frontalis is limited to Vanuatu (Cogger and These two taxa were discriminated in the field by the lateral head Heatwole, 2006; Lane and Shine, 2011). There is one more patterns, as Cogger and Heatwole (2006) suggested. This discrim- ination was confirmed by sequencing mitochondrial genes in the Laticauda species, L. laticaudata, distributed in Vanuatu, laboratory (data not shown). However, no L. laticaudata were found this species is morphologically and phylogenetically not so on the islet. Therefore, a male L. laticaudata was sampled at the closely related to the other two species. The phylogenetic southeast coast of the island of Efate (17°49′S, 168°26′E) at night. relationships of these three Laticauda species were described in Lane and Shine (2011). Sequencing OR genes Studies of how such olfaction-based isolation mecha- The procedures for DNA extraction and OR gene amplification nisms have been achieved, and how L. frontalis and L. of the three Laticauda species followed Kishida and Hikida (2010) colubrina speciated, are of potentially great interest. Shine with OR5B and OR3B primers (Ben-Arie et al., 1994) and et al. (2002) discussed the possibility that L. frontalis arose AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen). The in sympatry with L. colubrina in Vanuatu. In contrast, Lane amplicons are expected to contain part of the open reading frames of OR genes between TM2 and TM7, which are approximately 650 and Shine (2011) suggest that allopatric speciation bp in length without primers. The amplicons were purified using a occurred, in which L. frontalis originated in New Caledonia PCR purification kit (QIAGEN), and then tagged using a GS and re-invaded the ancestral (L. colubrina) range approxi- Titanium Rapid Library MID Adaptors Kit (Roche). The OR amplicon mately 180,000 years ago. Following the view of Lane and libraries of L. frontalis, L. colubina and L. laticaudata were tagged Shine (2011), a hypothesis has been suggested that olfac- with MID6, MID7, and MID8, respectively. Equal amounts of the tion-based isolation mechanisms are achieved through sen- amplicon libraries of L. frontalis and L. colubrina were mixed and sory drive (Endler, 1992; Boughman, 2002), which predicts sequenced together on a GS Junior sequencer (Roche). The ampl- that adaptation of signaling (i.e., lipid chemicals) and sen- icon library of L. laticaudata was also sequenced on a GS Junior sory (i.e., CRs) systems of allopatric populations to different sequencer together with an equal amount of an amplicon library, environments may cause premating isolation upon second- which was not analyzed in this study. Raw reads data obtained using the GS Junior sequencer have been deposited to the DDBJ ary contact of these populations. Sequence Read Archive (DRA) under accession numbers Olfaction is an important sensory modality for animals to DRA000729–DRA000731. perceive surrounding odors, such as environmental odor- We then prepared OR gene amplicons of L. frontalis and L. ants and conspecific pheromones. Seven trans-membrane colubrina once more, and identified OR genes following procedures (7TM) G-protein coupled receptors are known to function as used in a previous study (Kishida and Hikida, 2010) with an CRs across vertebrate species (Nei et al., 2008). Especially ABI3130 capillary sequencer (Applied Biosystems). Approximately notably among such CRs, olfactory receptors (ORs) are 100 colonies were sequenced for each species. present in all vertebrates and are considered to play the pri- mary role in olfaction in amniotes (Nei et al., 2008). The rep- Identification of OR genes ertoire of OR genes varies greatly among amniote species, Reads obtained from the GS Junior sequencer were divided into the three species on the basis of the MID tags, and then assembled and the ecological niche that an animal inhabits is directly into contigs using GS De Novo Assembler ver. 2.5p1 (Roche) with associated with the OR repertoire of the species (Niimura, the following settings; expected depth: 0, minimum read length: 45, 2009; Hayden et al., 2010). In fact, it has been reported that minimum overlap length: 50, minimum overlap identity: 99, heterozy- OR gene repertoires are quite different between the two gotic mode, other parameters: default. Each contig was searched closely-related species, humans and chimpanzees, mainly against the entire mouse protein database, which was retrieved from because of species-specific gene losses in both the human the NCBI website (http://www.ncbi.nlm.nih.gov/protein?term=%22 OR Genes in Syntopic Sibling Sea Snakes 427

Mus%20musculus%22%5Bporgn%3A__txid10090%5D) using the excluding gaps, between the query sequence and the hit sequences FASTY3.5 program (Pearson et al., 1997). A contig was discarded were calculated. When the nucleotide identity was > 98.5% (frontalis– if its best-hit protein was not an OR, and was replaced by its com- colubrina) or was > 96% (frontalis–laticaudata), and that the plementary base sequence if the coding direction of its best-hit OR sequences were similar to each other over the entire length, they protein was ‘reverse.’ were assumed to be orthologous. These cutoff values were chosen In order to assemble these contigs into OR sequences easily, because, to our knowledge from experiments, nucleotide identities Sanger sequencing-based conspecific sequences were added to of orthologous genomic DNA sequences for L. frontalis-L. colubrina the contigs. The sequences obtained by ABI3130 sequencer were and L. frontalis-L. laticaudata comparisons are generally > 98.5% identified as several OR sequences following the methods of and > 96%, respectively. Then, by using L. colubrina sequences as Kishida and Hikida (2010). Sanger sequencing-based OR sequences queries, we conducted the same procedures and confirmed that we thus obtained are available in the DDBJ/EMBL/GenBank databases obtained the same results. under the following accession numbers; AB754502–AB754548. In the case of L. laticaudata, previously reported Sanger sequencing- OR sequences included in the squamate-specific ORs based OR sequences were retrieved from GenBank under the fol- We first inferred a phylogenetic tree using all Laticauda OR lowing accession numbers (AB524695–AB524714). These Sanger sequences included in the squamate-specific ORs (Fig. 2B). Whole- sequencing-based sequences were mixed with the pyrosequencing- genome sequenced python OR sequences, identified by Dehara et based conspecific contigs, and aligned using the L-INS-i program in al. (2012), were also included. In this phylogenetic tree, eight sets the MAFFT package (Katoh et al., 2005) with manual adjustments. of putative orthologous trios (frontalis–colubrina–laticaudata) and Primer regions and low quality regions (scores < 32 in the sequence three sets of duos (frontalis–colubrina, laticaudata orthologous quality file) were cut off from the sequences. Two sequences that gene not found) were found with bootstrap values > 90%. In all shared > 99.5% similarity with > 50 bp overlaps were considered to cases, we confirmed that the nucleotide identity was > 98.5% encode the same OR sequence and were merged. Finally, the OR between L. frontalis and L. colubrina, and > 96% between L. gene repertoires of these three species were mixed together, and frontalis and L. laticaudata, and that the sequences were similar to aligned using the L-INS-i program with manual adjustments. When each other over the entire length. we found a clearly orthologous gene pair of two species without species-specific duplications and we found two or more fragment Sequence analyses sequences from the third species that strongly resembled the We analyzed each ortholog gene set of Laticauda OR sequences of the two species and did not share overlapping aligned sequences separately. The numbers of nonsynonymous substitu- regions, we merged these fragments into a single sequence and tions (Nd) and synonymous substitutions (Sd) in each branch, as filled gaps with ‘n (base unknown)’. The assembled OR sequences shown in Fig. 3A, were calculated by the method of Nei and Gojobori of L. frontalis, L. colubrina and L. laticaudata are available as (1986) based on the ancestral nucleotide sequences inferred by the Supporting Data. Bayesian method (Yang et al., 1995). Numbers of nonsynonymous sites (N) and synonymous sites (S) were estimated by the maxi- Identification of orthologous gene sets mum likelihood method (Goldman and Yang, 1994). These calcula- We modified the methods of Go and Niimura (2008) to identify tions were carried out using the CODEML program in the PAML4.4 orthologous OR gene sets. As we suggested previously, the elapid package (Yang, 2007). In cases in which no L. laticaudata OR was snakes OR repertoires include a subfamily named squamate-spe- found as an orthologous sequence, Nd and Sd between the L. frontalis cific ORs, which seems to have diverged rapidly (Kishida and and L. colubrina OR sequence were counted based on the method Hikida, 2010). Among this subfamily, for example, L. frontalis OR of Nei and Gojobori (1986). A sequence was judged to be a pesu- LfrOR25 and L. colubrina OR LcoOR26, which are similar to each dogene if a termination codon and/or frame shift was found in its other in overall bases and are considered to be orthologous, are open reading frame. However, if these frame shifts were caused similar to LfrOR28 and LcoOR29 in the front region of their where three or more repetitions of the same base and the sequence sequences, but are similar to LfrOR26 and LcoOR28 in the rear had not been confirmed by Sanger sequencing, we did not judge region, and are not similar to LfrOR28 and LcoOR29. The LfrOR25 the sequence to be a pseudogene, as such regions tend to be mis- and the LcoOR26 sequences have been confirmed both by the read using the Roche 454 sequencing system. A test of the homo- pyrosequencing-based method and by a Sanger sequencing-based geneity of nonsynonymous/synonymous change ratios (Kishida and method, and it is unlikely that similar artificial chimeras were gener- Thewissen, 2012) was applied to examine whether the number of ated independently in L. frontalis and L. colubrina OR amplicons, nonsynonymous substitutions in particular branches could be con- suggesting that some kinds of gene conversion and/or crossover sidered homogeneous in comparison with that in a compared would be one of the mechanisms that generates this subfamily. This branch. means that simple sequence-similarity based analyses would not be applicable to the OR sequences within this subfamily. Therefore, we RESULTS divided L. frontalis OR sequences into two groups based on the phylogenetic tree shown in Fig. 2A: OR sequences that are not Orthologous relationships of OR genes between L. included in the squamate-specific ORs and OR sequences that are frontalis and L. colubrina included in the squamate-specific ORs. It should be noted that, in Sixty-two, 59, and 60 OR sequences were obtained both groups, the orthologous gene sets were consequently identi- from L. frontalis, L. colubrina and L. laticaudata, respec- fied under the same criteria. tively. The sequence diversity of these ORs is shown in Fig. 2A. Among these sequences, we found 39 sets of ortholo- OR sequences which are not included in the squamate-specific gous trios (frontalis–colubrina–laticaudata) and nine sets of ORs duos (frontalis–colubrina). All orthologous relationships of By using L. frontalis sequences as queries, we conducted OR genes are listed in Supplementary Table S1 online. No BlastN searches (Altschul et al., 1997) against all Laticauda sequences obtained in this study, with the cutoff of e-value < 1.0 × L. frontalis-specific OR gene duplications were found, but 10–10. For each result, the query sequence and the hit sequences one putative L. colubrina-specific OR gene duplication was (excluding the query sequence itself) were aligned using L-INS-i found; L. frontalis LfrOR55 was similar to both L. colubrina with manual inspection, and the nucleotide sequence identities, LcoOR55 (nucleotide identity 99.67%) and LcoOR53 (nucle- 428 T. Kishida et al.

LfrOR54 LlaOR56 LcoOR52 Pm132 Pm133 otide identity 98.63%). In Pm178_TPm053 Pm188_T Pm014 LfrOR57 (A) LcoOR56 Pm109 this case, we determined LfrOR55 LcoOR53 LfrOR56 LcoOR54 LlaOR57 LcoOR55 Pm124 LfrOR58 that LcoOR53 was not LcoOR57 LlaOR58 Pm208_T Pm265_T Pm101 Pm045 LfrOR51 LcoOR49 orthologous to LfrOR55, LlaOR54 Pm048 LfrOR52 LcoOR50 LlaOR55 Pm205_T LfrOR47 because when LcoOR55 LlaOR50 LcoOR46 Pm007 LfrOR48 LlaOR51 Pm006 LfrOR49 LcoOR47 was used as a query, LlaOR52 Pm253_T LfrOR50 LlaOR53 LcoOR48 Pm004 Pm226_T Pm279_T LfrOR55 was hit prior to Pm076 LfrOR6 LcoOR6 Pm049 LlaOR6 Pm047 LfrOR53 LcoOR53, and because LcoOR51 Pm209_T LfrOR46 LcoOR45 LlaOR49 Pm012 LfrOR43 LcoOR44 detailed phylogenetic anal- Pm203_T Pm231_T LfrOR14 LfrOR44 LfrOR45 Pm125 LfrOR42 LcoOR43 yses indicated the following LlaOR48 Pm123 99 LcoOR18 LfrOR40 LcoOR41 (B) LlaOR46 Pm057 Pm136 Pm058 phylogenetic relationships: Pm131 Pm013 LlaOR20 LfrOR41 LcoOR42 LlaOR47 Pm126 LfrOR59 LcoOR58 ((LfrOR55, LcoOR55), LfrOR60 LlaOR19 Pm169_T LfrOR61 LlaOR59 Pm071 Pm120 Pm038 LcoOR53) (data not Pm039 LfrOR1 LfrOR15 LcoOR1 100 Pm157 Pm040 Pm149 Pm150 Pm118 shown). In addition, we Pm070 LcoOR19 Pm121 Pm041 Pm148 Pm119 Pm160_P Pm072 Pm164_P found that L. frontalis OR Pm173_T LfrOR17 Pm245_T Pm176_T Pm116 Pm198_T Pm161_P 100 Pm154_P LfrOR4 was a pseudogene LfrOR5 100 LcoOR21 LcoOR5 LlaOR5 LfrOR62 LlaOR60 LcoOR59 Pm236_T Pm024 because of a frame shift, LlaOR34 LfrOR18 Pm022 Pm220_T Pm194_T Pm061 LfrOR29 LcoOR31 but LcoOR4, an L. LlaOR33 LlaOR22 Pm216_T Pm190_T Pm187_T Pm268_T Pm037 Pm113 LfrOR30 LfrOR16 colubrina gene ortholo- LcoOR32 Pm278_T Pm200_T Pm255_T Pm192_T Pm254_T Pm211_T Pm170_T LcoOR20 gous to LfrOR4, was intact. Pm165_P Pm111 97 Pm177_T Pm117 Pm272_T LfrOR31 LlaOR35 LfrOR19 However, in the other 47 Pm029 Pm110 100 Pm218_T Pm221_T Pm244_T Pm246_T Pm027 Pm273_T LlaOR23 orthologous sets, no puta- Pm224_T Pm140 Pm193_T Pm144 Pm147 LfrOR3 LcoOR3 Pm264 T tive species-specific gene LlaOR2 Pm028 Pm105 Pm108 Pm106 LfrOR2 LcoOR2 LlaOR1 LfrOR12 duplications or species- Pm010 Pm064 Pm036 Pm146 Pm069 Pm171_T 100 Pm023 LlaOR16 specific pseudogenization Pm085 100 Pm139 Pm185_T Pm104 Pm142 Pm066 Pm223_T Pm115 LcoOR16 mutations could be found. Pm030 Pm222_T Pm250_T Pm252_T Pm277_T Pm090 Pm266_T 98 LlaOR17 Pm075 We found 11 L. frontalis Pm042 Pm077 Pm078 Pm259_T Pm046 Pm256_T LfrOR13 Pm258_T Pm248_T 100 OR pseudogenes among Pm059 Pm097 Pm234_T Pm073 Pm084 Pm079 LcoOR17 Pm212_T Pm270_T LfrOR11 LlaOR14 LcoOR15 LlaOR15 Pm005 LcoOR22 Pm074 LfrOR12 LlaOR16 LcoOR16 LfrOR13 LcoOR17 LlaOR17 Pm264_T LfrOR20 LfrOR14 100 LcoOR18 LlaOR18 LfrOR15 LcoOR19 LlaOR19 LlaOR20 LlaOR24 LfrOR16 LcoOR20 LlaOR21 LcoOR21LfrOR17 LfrOR18 Fig. 2. A neighbor-joining tree LlaOR22 LfrOR19 100 100 LfrOR21 LlaOR23 LfrOR20 LcoOR22 LlaOR24 LfrOR21 of OR genes identified in this LcoOR23 LlaOR25 LcoOR23 LfrOR22 LcoOR24 LlaOR26 LfrOR23 study. L. frontalis ORs are indi- LcoOR25 LlaOR27 LfrOR24 LlaOR25 LfrOR25 100 LcoOR26 LcoOR27 LlaOR29 LlaOR28 cated by red font; L. colubrina, LlaOR30 LfrOR26 LcoOR28 LlaOR26 LfrOR27 LlaOR31 LlaOR32 LfrOR28 green; L. laticaudata, blue. LcoOR29 LcoOR30 Pm008 LfrOR22 Pm134 Pm122 97 100 LlaOR13 Genome-determined python LcoOR13 Pm183_T Pm184_T Pm186_T Pm001 96 LcoOR24 Pm032 LlaOR9 OR genes (indicated by black Pm267_T LlaOR8 Pm182_T Pm240_T Pm251_T LlaOR27 Pm026 Pm080 font), taken from Dehara et al. LfrOR10 LcoOR12 LlaOR11 Pm087 Pm162_P LfrOR9 LfrOR23 LlaOR10 100 (2012), were added to the LcoOR9 LcoOR10 LcoOR11 Pm129 Pm152 LcoOR14 LcoOR25 LlaOR12 tree. (A) The FASTA3.5 pro- Pm153_P LfrOR8 LcoOR8 Pm112 Pm145 Pm130 100 LcoOR27 Pm056 gram (Pearson and Lipman, Pm044 Pm159_P Pm102 Pm230_T Pm051 Pm262_T Pm217_T LfrOR28 1988) was used to calculate Pm202_T 93 Pm201_T Pm257_T Pm094 Pm197_T Pm158_P Pm219_T LcoOR29 opt scores pairwise between Pm043 Pm092 Pm214_T Pm215_T Pm003 LfrOR7 LlaOR7 all combinations of OR LcoOR7 LlaOR32 Pm232_T Pm228_T Pm180_T Pm249_T Pm276_T 99 100 sequence pairs, and the opt Pm181_T Pm020 LfrOR25 Pm189_T Pm086 Pm210_T Pm016 Pm235_T score matrix thus obtained Pm151 Pm099 Pm195_T LcoOR26 Pm241_T LlaOR3 Pm098 Pm174_T Pm175_T was used as the distance Pm163_P Pm019 LlaOR28 Pm261_T 97 Pm135 Pm179_T Pm269_T matrix. Note that this tree is Pm060 Pm015 Pm137 93 LlaOR30 Pm274_T Pm128 Pm009 Pm167_T unrooted. (B) Details of a sub- Pm050 Pm275_T Pm238_T LcoOR30 Pm082 Pm017 Pm052 Pm002 tree named squamate-specific Pm096 Pm021 Pm081 Pm156_P LfrOR26 Pm033 99 Pm107 Pm166_P ORs (Kishida and Hikida, Pm138 Pm204_T Pm206_T Pm207_T LcoOR28 Pm091Pm213_T Pm067 2010). Distance matrix was Pm062 LfrOR4 LcoOR4 LlaOR4 Pm263_T 98 LfrOR27 Pm095 Pm114 calculated based on Kimura’s Pm089 Pm155_P Pm227_T Pm280_T 0.05 Pm243_T Pm088 95 LlaOR31 Pm271_T 2-parameter method. Boot- Pm103 Pm168_T (substitutions/site) Pm083 Pm011 Pm055 Pm054 Pm018 strap values were obtained by LfrOR32 LfrOR33 LcoOR33 LlaOR36 Pm035 LfrOR34 LcoOR34 500 resamplings, and values > LlaOR38 LlaOR37 LlaOR39LcoOR35 LlaOR40 LlaOR41 LfrOR35 90% are shown. Sets of puta- Pm233_T LfrOR36 LcoOR36 LcoOR37 LlaOR42 Pm191_T tive orthologous OR gene Pm196_T Pm260_T LfrOR37 LcoOR38 LlaOR43 Pm034 LfrOR39 trios/duos, supported with > LcoOR40 LlaOR45 Pm141 Pm242_T Pm143 Pm225_T LfrOR38LcoOR39 90% bootstrap values, are LlaOR44 Pm127 Pm068 Pm247_T Pm172_T Pm093 indicated with brown vertical Pm239_T Pm063 Pm025 Pm100 Pm031 Pm237_T Pm065 bars. Pm199_T Pm229_T OR Genes in Syntopic Sibling Sea Snakes 429 the other 47 sets of ortholo- (A) L. frontalis (B) gous trios/duos, and in all cases, the L. colubrina frontalis branch 30 ortholog of each L. frontalis changed branch OR was also a pseudogene, unknown colubrina branch sharing the same pseudog- 20 changed in enization mutations with its L. colubrina both branches orthologous L. frontalis OR. Note that we analyzed only changed in branch colubrina branch between the TM2 and TM7 10 regions, thus genes that seem to be intact may in fact changed in laticaudata be pseudogenes. For exam- frontalis branch number of orthologous gene sets ple, it is still possible that L. laticaudata both LfrOR4 and LcoOR4 changed not changed are pseudogenes and share identical pseudogenization (C) Numbers and rates of nonsynonymous and synonymous substitutions mutations in the region in each branch N S d d ω between TM1 and TM2. In d d N S any case, these data indi- frontalis branch 19 12 0.0016 0.0025 0.65 cate that, unlike in the case colubrina branch 17 10 0.0014 0.0021 0.69 of humans and chimpanzees laticaudata branch 95 91 0.0081 0.0189 0.43 (Go and Niimura, 2008), L. Nd: number of nonsynonymous substitutions Sd: number of synonymous substitutions frontalis and L. colubrina dN: the number of nonsynonymous substitutions per site Nd/N maintain similar OR gene rep- dS: the number of synonymous substitutions per site Sd/S ertoires to each other. These ω: nonsynonymous to synonymous substitution rate ratio dN/dS 11 sets of pseudogene Fig. 3. (A) A schematic phylogenetic tree and branch names used in this study. Note that this tree is orthologs were excluded from unrooted, and that the root is expected to be located on the laticaudata branch. (B) The numbers of further analyses. orthologous gene sets for which nonsynonymous changes had occurred only in the frontalis branch/only in the colubrina branch/in both branches/branch unknown, as no orthologous OR genes of L. laticaudata Nonsynonymous substitu- were found, and the number of orthologous gene sets for which no nonsynonymous differences were tions within orthologous found between L. frontalis and L. colubrina. (C) Numbers and rates of nonsynonymous and synonymous gene sets substitutions in each branch. All the orthologous gene trios were calculated together (N = 11787.4, S = As shown in Fig. 3, in 4817.6). many cases (28/37 = 76%), nonsynonymous differences are found between L. frontalis OR genes and their L. Table 1. Test of homogeneity of nonsynonymous/synonymous colubrina orthologs, and the actual rate would be even change ratios. higher because we sequenced only the region between TM2 b Nd Sd p value and TM7 of the OR genes. Nonsynonymous substitutions a occurred at nearly equal frequencies in the frontalis and frontalis and colubrina branches 45 27 colubrina branches (Fig. 3B), and the nonsynonymous to laticaudata branches 95 91 0.065* a All orthologous trios and duos were calculated together. synonymous substitution rate ratios were higher in both b branches compared with that in the laticaudata branch (Fig. p value was calculated using Fisher’s exact test (one-tailed). * weakly significant (p < 0.1) 3C). This tendency was weakly significant according to the test of homogeneity of nonsynonymous/synonymous change ratios (P = 0.065, Table 1). Their primer sets seem to be more efficient compared with DISCUSSION ours, but they obtained only approximately 330 bp for each Approximately 60 OR genes were identified for each OR gene, which was too short to conduct further sequence species, and these sequences did not seem to be concen- analyses. In any case, the OR gene repertoires obtained by trated extremely in any specific subtrees (Fig. 2A). Dehara the PCR-based pyrosequencing method were far from com- et al. (2012) reported that 280 OR genes were identified plete, but this method is nonetheless one of the best ways based on the in silico screening of the python draft genome to identify a large number of OR genes without any refer- database. We thus may have obtained approximately 20% ence genome databases and at a reasonable cost. In par- of the total OR gene repertoires, based on the assumption ticular, this method would work efficiently for identifying that pythons and Laticauda sea snakes possess almost orthologous gene sets from multiple species, as the equal numbers of OR genes, though we cannot judge sequences were obtained under the same primer bias for all whether this assumption is valid or not. Dehara et al. (2012) species and thus if a sequence was obtained from a spe- identified 96 OR genes from rat snakes following nearly the cies, its ortholog would also be expected to be sequenced same protocols as used here, but using another primer sets. from the other species. Actually, 62 OR sequences were 430 T. Kishida et al. obtained from L. frontalis in this study, and among 77% (48/ olfactory senses. It is expected that most of the ORs ana- 62) of them, the orthologous genes were obtained from L. lyzed in this study have been evolved to adapt the chemical colubrina. environments surrounding snake habitats. Therefore, if As shown in Results, there were few species-specific these two Laticauda species have evolved sympatrically gene duplications or species-specific gene losses when the and syntopically on land, these two species should maintain OR gene repertoire of L. frontalis was compared with that of similar OR gene repertoires. In contrast, our results can be L. colubrina. This means that, both L. frontalis and L. explained easily if these two species have evolved allopatri- colubrina are expected to have maintained the size of their cally, as Lane and Shine (2011) suggested. OR repertoires after their genetic split. The OR repertoire The present work is relatively descriptive about the has been shown to undergo extensive gains and losses of genomic basis of the sense of smell among Laticauda sea genes during vertebrate evolution (Nei et al., 2008), and it snakes living in Vanuatu. It remains unclear whether ORs has long been discussed that the number of OR genes can are involved in conspecific recognition or not, and whether serve as an indicator for assessing the olfactory ability of the such ORs were included in this analysis or not. In this study, animal (e.g., Dehara et al., 2012). In addition, orthologous we simply showed that L. frontalis and L. colubrina have genes are assumed to perform equivalent functions (Go and evolved to maintain similar numbers of OR genes, but that Niimura, 2008; Nehrt et al., 2011). According to these views, they may possess different senses of smell to each other. It the olfactory abilities of both L. frontalis and L. colubrina are is possible that this dissimilarity functions as a premating expected to be nearly equivalent to that of their last common isolation barrier since L. frontalis has returned to the ances- ancestor, and no significant changes have occurred after tral range, but this has not been confirmed yet. Further stud- their genetic isolation. However, as Nei et al. (2008) dis- ies will be required to reveal the evolution of odor-based cussed, even a small number of amino acid changes could isolation mechanisms between L. frontalis and L. colubrina. alter the function of OR genes. Keller et al. (2007) showed ACKNOWLEDGMENTS that only two amino acid changes on a human OR OR7D4 alter the human odor perception drastically. Adipietro et al. We are grateful to all JICA members resident in Vanuatu, (2012) extended this view and showed that even a small especially Yuriko Igarashi, Yusuke Inokuchi, and Akihito Motegi for number of nonsynonymous substitutions can change the supporting our field works in Vanuatu; Henry Maulaiwia, Chief of Paonangisu village for allowing us to study sea snakes at Ngioriki ligand potency and efficiency of ORs dramatically. Our data Islet; Michihisa Toriba, Takashi Hayakawa and Yasuhiro Go for sug- shows that in many cases (76% or more), the amino acid gestive comments; Elizabeth Nakajima for checking the English of sequences were different between L. frontalis and L. colubrina the text. Through the courtesy of Robert Jimmy and Moses Amos, orthologs, suggesting that their olfactory abilities are differ- Vanuatu Fisheries Department, we were permitted to sample and ent from each other. Unexpectedly, nonsynonymous substi- export sea snake specimens under the following permission num- tutions occurred almost equally in both the frontalis and bers; 06795/2009 and 07072/2010. Roche GS Junior sequencer colubrina branches, indicating that both L. frontalis and L. was operated by the Cooperation Research Program of Wildlife colubrina has changed its olfactory sense. This may sug- Research Center of Kyoto University. This work was funded by gest that the surrounding environmental odors have Global COE program (A06) of Kyoto University, and by MEXT changed, even in the same place since the time when their KAKENHI (grant nos. 22770082 and 24770075) to TK. ancestors lived. REFERENCES Most of the OR genes possessed by Laticauda sea Adipietro KA, Mainland JD, Matsunami H (2012) Functional Evolu- snakes are expected to function only on land (Kishida and tion of Mammalian Odorant Receptors. PLoS Genet 8: Hikida, 2010). 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