Sequence variability at three MHC loci of finless porpoises (Neophocaena phocaenoides)

Immunogenetics (2007) 59:581–592 DOI 10.1007/s00251-007-0223-9

ORIGINAL PAPER

Sequence variability at three MHC loci of finless porpoises (Neophocaena phocaenoides) Shixia Xu & Peng Sun & Kaiya Zhou & Guang Yang

Received: 3 November 2006 / Accepted: 31 March 2007 / Published online: 8 May 2007 # Springer-Verlag 2007

Abstract Major histocompatibility complex (MHC) class II DQB and DRA genes and class I gene of finless porpoises (Neophocaena phocaenoides) were investigated by singlestrand conformation polymorphism and sequence analysis. The DRA, DQB, and MHC-I loci each contained 5, 14, and 34 unique sequences, respectively, and considerable sequence variation was found at the MHC-I and DQB loci. Gene duplication was manifested as three to five distinct sequences at each of the DQB and MHC-I loci from some individuals, and these sequences at each of the two loci separately clustered into four groups (cluster A, B, C, and D) based on the phylogenetic trees. Phylogenetic reconstruction revealed a trans-species pattern of evolution. Relatively high rates of non-synonymous (dN) vs synonymous (dS) substitution in the peptide-binding region (PBR) suggested balancing selection for maintaining polymorphisms at the MHC-I and DQB loci. In contrast, one single locus with little sequence variation was detected in the DRA gene, and no non-synonymous substitutions in the PBR indicated no balancing selection on this gene. Keywords Finless porpoises . MHC . Genetic variation . Balancing selection

Introduction The major histocompatibility complex (MHC) is an important component of the vertebrate immune system S. Xu : P. Sun : K. Zhou : G. Yang (*) Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, 122 Ninghai Road, Nanjing 210097, China e-mail: [email protected]

where it determines which antigens trigger an immune response (Hughes and Yeager 1998). The MHC family includes two major subfamilies, class I and class II genes (Klein 1986). MHC class I genes are expressed on the surface of all nucleated somatic cells. They play an essential role in the immune defense against intracellular pathogens by binding endogenously derived peptides from proteins (mainly viral) in the cytoplasm (Klein 1986). In contrast, MHC class II genes are predominantly involved in monitoring the extracellular environment by presenting peptides mainly derived from parasites to the T cells (Klein 1986; Dengjel et al. 2005). The ability of both classes I and II genes to face various pathogens is believed to be mainly associated with sequence variation among MHC alleles in the peptide-binding region or PBR (Ohta 1998). Variation within the PBR suggests that there has been evolutionary pressure for organisms to combat a wide range of immunological challenges (Abbott et al. 2006). MHC variability reflects evolutionarily relevant and adaptive processes within and among populations and is very suitable to investigate a wide range of open questions in evolutionary ecology and conservation. Most mammals have a high degree of genetic variation at MHC class I and II loci, which is supposed to be an adaptation resulting from the large number of pathogens encountered by natural populations (Klein and Takahata 1990; Hill et al. 1991). The variability of MHC genes is an indicator for the parasite and pathogen resistance, which in turn may influence the long-term survival probability of populations (Paterson et al. 1998; Hedrick et al. 2001a, b; Langefors et al. 2001; Schad et al. 2005). This is particularly important for aquatic species whose chemical and microbial environment is constantly affected by anthropogenic encroachment, and thus, increasing their risk of exposure to novel pathogens (De Swart et al. 1996; Harvell

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et al. 1999). The studies of MHC variation in marine mammals have, to date, yielded mixed conclusions. Earlier studies found low level of MHC genetic diversity in fin whales (Balaenoptera physalus) and Sei whales (B. borealis; Trowsdale et al. 1989). These results were attributed to a comparatively weak pathogenic pressure in marine habitats. In contrast, sequence analysis of beluga whales (Delphinapterus leucas) MHC-II loci (including DQA, DQB, and DRB) revealed low but measurable polymorphism and non-synonymous substitution rates consistent with selection (Murray et al. 1995; Murray and White 1998). However, recent sequence analysis has revealed high level of MHC diversity in the California sea lion (Zalophus californianus; Bowen et al. 2006), southern elephant seal (Mirounga leonina; Hoelzel et al. 1999), and baiji (Lipotes vexillifer; Yang et al. 2005), etc. The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetaceans widely distributed in the coast waters of the Indo-Pacific Oceans (Reeves et al. 1997). Population subdivisions have been documented in its range especially of Chinese (Gao and Zhou 1995) and Japanese waters (Shirakihara et al. 1994; Yoshida et al. 1995). Of different populations identified for this species, the Yangtze River population is exclusive due to its unique and limited distribution in the middle and lower reaches of the Yangtze River, its small and rapidly declining population size and highly endangered status, and its special adaptation to freshwater environment. Population genetic analyses with the neutral markers such as mitochondrial control region and microsatellites have found low genetic diversity in this species (Yoshida et al. 2001; Yang et al. 2002a, 2003; Xia et al. 2005; Zheng et al. 2005). However, no systematic information on sequence variation in adaptive markers is currently available for the finless porpoise. In this study, sequence variability at exon 2 of one MHC class I gene and two class II genes (DRA and DQB) including part of the putative PBR were investigated using samples of finless porpoises collected mainly in Chinese waters. It is expected to have an in-depth understanding on the behavior of these molecules, especially sequence variability possibly caused by selection pressure. Findings from this study will provide basic information for studying the MHC immunogenetics at a population level and especially to identify genetic basis for its adaptation to freshwater of the Yangtze finless porpoise.

Materials and methods Samples A total of 195 tissue samples (muscle, skeleton, and blood) were collected over a period of more than 20 years from 20

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locations in the coast of China and the middle and lower reaches of the Yangtze River (Table 1, Fig. 1). These samples were assigned to different populations, i.e., the Yangtze River population, the Yellow Sea population, and the South China Sea population, according to the discriminant features established by Gao and Zhou (1995). Voucher specimens are preserved in the Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, China. DNA isolation and PCR The extraction of total genomic DNA from muscles and skeletons followed the procedures described by Sambrook and Russell (2001). Blood sample DNA was extracted using the DNeasy tissue kit (Qiagen, USA) following the manufacturer’s instruction. The exon 2 fragments for MHCI, DRA, and DQB were amplified using three primer sets as shown in Table 2. The primers used to amplify DRA gene were designed against a conserved region among sheep (Ovis aries, GenBank accession, M73983), cattle (Bos taurus, M30120), horse (Equus caballus, L47174), and human (Homo sapiens, M60334; Sena et al. 2003). Polymerase chain reactions (PCRs) were carried out in a Table 1 Samples of finless porpoises analyzed in the present study Populations/sampling sites

Yangtze River population (64) Anhui Province Tongling Wuhu Shanghai City Chongming Island Jiangsu Province Nanjing Jiangpu Zhangjiagang Liuhe Zhenjiang Yixing Jiangxi Province Hukou Yellow Sea population (55) Bohai Sea Jiangsu Province Lusi Rudong Ganyu Liaoning Province Dalian Xingcheng Zhejiang Province Hangzhou Zhoushan South China Sea population (76) Fujian Province Dongshan Pingtan Guangxi Province Beihai

Sample size

Sample symbols (see Fig. 1)

27 1 3 19 8 1 2 1 1 1

A1 A2 S J1 J2 J3 J4 J5 J6 JX

6 33 6 3 2 2 1 2

B JS1 JS2 JS3 L1 L2 Z1 Z2

59 16 1

F1 F2 G

The numerical number in the parentheses refers to the total number of samples in each population.

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Fig. 1 a Schematic map showing the distribution of finless porpoises sampled in this study; b geographical distribution of finless porpoises sampled in this study. See Table 1 for sample symbols

total volume of 50 μl containing 2.5 mM MgCl2, 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 0.2 mM each dNTP, 0.4 μM each primer, 1.0 unit Ex-Taq DNA polymerase (Takara, Japan), and 10–100 ng DNA template. The PCR cycling scheme included an initial denaturation of 5 min at 94°C followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR products were purified using Wizard PCR Preps DNA purification kit (Promega, USA) according to the manufacturer’s instruction. SSCP, cloning, and sequencing All the samples were firstly screened for consistent polymorphism at exon 2 of the MHC-I, DRA, and DQB loci using single-strand conformation polymorphism (SSCP). The selected samples were then characterized at the genetic level by DNA sequence comparison. For SSCP

Table 2 PCR primers used to amplify three MHC loci in the present study

analysis, 1 μl of the purified PCR product was mixed with 9 μl of loading dye (95% v/v formamide, 20 mM ethylenediamine tetraacetic acid, 0.05% w/v Bromophenol Blue, 0.05% w/v xylene cyanol). After denaturing at 95°C for 10 min and cooling on ice for 5 min, 5 μl of the mixture was loaded into a 10% polyacrylamide gel (38:1, acrylamide/bisacrylamide). Electrophoresis was performed in 1× TBE buffer at 150 V for 16–20 h at room temperature. After completion of the run, SSCP bands were visualized by silver staining procedures. To avoid categorizing PCR artifacts as a new allele based on the SSCP bands, the PCR products were rearranged and separated again on the gel according to assessed similarities. In this study, each sample was analyzed at least twice following the same procedure. For new samples, all known alleles were run as references on each SSCP gel. PCR products showing the same SSCP pattern in the replicates were cloned into the pMD-18T vectors using the TA cloning kit (Takara). For each locus, five to six randomly chosen PCR products were cloned from each SSCP genotype. Four to six clones were picked for each cloned PCR product and sequenced in the forward and/or reverse directions. The sequence reaction was using the BigDye terminator cycle sequencing ready reaction kit (ABI). Automated DNA sequence analysis was performed on an ABI 3730 automated genetic analyzer. Data analysis Sequence variability Statistical analysis of nucleotide and amino acid sequences was computed with MEGA version 3.1 (Kumar et al. 2004). Pairwise nucleotide and amino acid distances among sequences of each locus were respectively estimated by the Kimura 2-parameter model (or K2P) and the Poisson correction method. The average rate of non-synonymous (dN) and synonymous (dS) substitutions were calculated separately for the overall domain, PBR, and non-PBR according to Nei–Gojobori method (Nei and Gojobori 1986), using the Jukes–Cantor correction for multiple

Locus

Size of amplifications (bp)

Primer sequences

Reference

DRA

189

This study

DQB

172

MHC-I

147

5′-AATCATGTGATCATCCAAGCTGAGTTC-3′ 5′-TGTTTGGGGTGTTGTTGGAGCG-3′ 5′-CTGGTAGTTGTGTCTGCACAC-3′ 5′-CATGTGCTACTTCACCAACGG-3′ 5′-TACGTGGMCGACACGSAGTTC-3′ 5′-CTCGCTCTGGTTGTAGTAGCS-3′

Murray et al. (1995) Flores-Ramirez et al. (2000)

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substitutions. The standard errors were obtained by 1,000 bootstrap replicates. The statistical significance of the differences between these rates was tested with a Z test at the 5% level (Kumar et al. 2004). PBR and non-PBR sequences were differentiated, assuming homology predicated against the human MHC molecules (Bjorkman et al. 1987; Brown et al. 1993). In addition, based on tree topology (see below), finless porpoises DQB and MHC-I sequences fell into four clusters. Analyses were also made on each cluster separately. Phylogenetic analysis Median joining network analyses were performed with the software package NETWORK version 4.1 (Bandelt et al. 1999). A phylogenetic tree for each locus using the neighbor-joining method was constructed from K2P nucleotide distances in MEGA3.1 (Kumar et al. 2004). It was used to estimate the genetic relationships among sequences of finless porpoises and with sequences from some other mammalian species. Bootstrap confidence intervals were obtained from 1,000 replicates.

Results Number of unique sequences at three MHC loci DRA A total of six unique sequences were detected for the DRA gene from all the samples. All individuals tested had no more than two sequences, suggesting that the DRA primers used in this study amplified a single locus. Allele NephDRA*06 had a stop codon in the middle part of the sequence, representing most likely a pseudogene, and it was excluded from further analyses. The remaining allele sequences have been submitted to GenBank with accession numbers DQ843609–DQ843613. Of all the alleles identified, Neph-DRA*01 had the highest frequency, while the other alleles only appeared once or twice. DQB Fourteen unique sequences were detected in this study, and they were labeled from Neph-DQB*01 to Neph-DQB*14 (GenBank accession nos. DQ843614–DQ843623, and EF056477–EF056480). Five of these sequences, i.e., NephDQB*06, 07, 08, 09, and 10, have previously been reported from porpoises in Japanese waters (Hayashi et al. 2003). In the 14 sequences, five were found population-specific, i.e., Neph-DQB*02, Neph-DQB*13, and Neph-DQB*11, 12, 14 appeared in the South China Sea population, Yellow Sea population, and Yangtze River population, respectively,

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whereas the remaining nine sequences were shared by population pairs. None of the sequences contained insertions/deletions (or indels), or stop codons, suggesting that all sequences might come from functional molecules in the genome. More than two DQB sequences were detected in 8 out the 195 individuals examined in this study. Five of the eight porpoises had three unique sequences, two of them had four, and the last one had five, suggesting that at least three copies of the DQB gene existed in finless porpoises. MHC-I Thirty-four unique sequences were identified for the MHCI gene (GenBank accession nos. DQ843624–DQ843657). No indels or stop codons were detected. However, these sequences are possibly active members of at least three loci rather than a single specific locus because three to five distinct sequences were detected from most of the individuals. The detected number of unique sequences was 13 for 134 clones from 22 Yangtze samples, 22 for 100 clones from 20 Yellow Sea samples, and 26 for 133 clones from 26 South China Sea samples, respectively. The five common sequences of Neph-I*01, 02, 03, 04, and I*07 were the most frequent and widespread in the three populations, and 17 sequences were shared only by some population pairs. In addition, 12 population-specific sequences were identified, four (I*06, I*14, I*23, I*32) in the Yangtze River population, five (I*21, I*24, I*25, I*31, I*34) in the South China Sea population, and three (I*12, I*26, I*27) in the Yellow Sea population, respectively.

Network analysis and phylogenetic reconstruction Median joining networks were constructed for exon 2 sequences of the DRA, DQB, and MHC-I loci, respectively (Fig. 2). The relationship among the sequences did not match the geographical distribution of different populations. The NJ tree of DRA exon 2 alleles (Fig. 3a) revealed that the alleles of finless porpoises constituted a distinct clade with a bootstrap value of 100%. However, according to the phylogenetic trees, four groups can be identified respectively for the DQB and MHC-I. When these groupings were considered separately, each individual contained only one or two alleles, which suggested that these groupings represented different loci. In addition, phylogenetic analyses revealed that sequences did not cluster according to species, but were intermixed with other species. For DQB tree, cluster A comprised six finless porpoises diverse sequences, and four of them were identified in this study, i.e., Neph-DQB*06, 08, 10, and 11. Cluster B also consisted of six sequences, including an allele of the vaquita

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Fig. 2 Median joining networks constructed for the exon 2 sequences of the DRA (a), DQB (b), and MHC-I (c). The circles represent unique sequences, with the area proportional to the frequency of the sequences in three populations except for Neph-DQB*a, Neph-DQB*g, and Neph-DQB*h whose frequencies were not available

(Phocoena sinus), i.e., Phsi-DQB*01, which clustered with Neph-DQB*09, receiving 94% bootstrap support (Fig. 3b). The cluster D had only two finless porpoises sequences, Neph-DQB*03 and 14. The cluster was nested within a larger grouping that included the baiji DQB sequences. Interestingly, Neph-DQB*03 and 14 were found to have identical sequences with Live-DQB*16 and 4 from the baiji

reported in Yang et al. (2005). Similar relationship was noted in the phylogenetic reconstruction for MHC-I gene. Cluster A–D each contained 17, 4, 9, 4 distinct Neph-I sequences, respectively. In the cluster C, Neph-I*04 clustered with five alleles of the vaquita, with a bootstrap value of 94%, and Neph-I*11 and Neph-I*17 also formed a clade clustering with alleles of the vaquita (Fig. 3c).

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Fig. 3 Neighbor-joining phylogenetic trees constructed based on nucleotide sequences of three MHC loci from a matrix of Kimura 2-parameter nucleotide distances. Numbers at branch-points represent bootstrap support values, and only bootstrap values ≥50% (1,000 replicates) were shown. a DRA. In addition to the five Neph-DRA alleles identified in this study (in the frame), sequences from other species were also included: water buffalo (Bubalus bubalis, Bula-DRA*01: AF385488; Bula-DRA*02: AF385489), lowland anoa (B. depressicornis, Ande-DRA*01: AF385483; Ande-DRA*02: AF385484), cattle (BoLA-DRA: M30120), sheep (Ovar-DRA: M73983), ass (E. asinus, ELA-DRA*05: L47171; ELA-DRA*JBD3: AJ575296; ELA-DRA*JBD17: AJ575297; ELADRA*JBH45: AJ575298;), horse (ELA-DRA*03: L47172, ELADRA*01: L47174; ELA-DRA*02: M60100; ELA-DRA*JBH11: AJ575295), Grant’s zebra (E. boehmi, ELA-DRA*JBZ185: AJ575299), onager (E. hemionus, ELA-DRA*04: L47173), California sea lion (Zaca-

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DRA*01-Zaca-DRA*06: AY491450-AY491455). b DQB. In addition to the 14 Neph-DQB sequences identified in this study, the following sequences were also included: finless porpoise (Neph-a: AB164212; Neph-g: AB164218; Neph-h: AB164219), Yangtze river dolphin (LiveDQB*4: AY177153; Live-DQB*5: AY177283; Live-DQB*8: AY177286; Live-DQB*11: AY177289; Live-DQB*13: AY177291; Live-DQB*16: AY333383; Live-DQB*28: AY333395; Live-DQB*29: AY333396), vaquita (Phsi-DQB*01: AY170897), narwhal (Momo-DQB*0201: U16991), beluga whale (Dele-DQB*0201: U16989; Dele-DQB*0101: U16986), cattle (BoLA-DQB1*2: U77787; BoLA-DQB1*3: U77788; BoLA-DQB2*1: U77795; BoLA-DQB2*2A: U77796). c MHC-I. In addition to the 34 Neph-I sequences in this study, some sequences of other cetaceans and ungulates were downloaded from GenBank: vaquita (Phsi*01-Phsi*06: AY170890-AY170895)

Immunogenetics (2007) 59:581–592 Fig. 4 Alignment of predicted amino acid sequences of MHC DRA (a), DQB (b), and MHC-I (c) exon 2 from finless porpoises. Dots indicate residues identical to the reference sequences. Putative peptide binding sites (Brown et al. 1993; Bjorkman et al. 1987) are marked with asterisks

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Sequence variability of three MHC loci DRA Sequence polymorphism analysis showed that the overall variation was very low, with 2.1% variability at the nucleotide level (four polymorphic sites of 189 base pairs sequenced) and 4.8% at the amino acid level (three polymorphic sites of 63 amino acid residues; Fig. 4a). On the other hand, the similarity between DRA alleles was extremely high, with each pair containing only a maximum of two different nucleotides. For the deduced amino acid sequences, three of the four polymorphic sites represented non-synonymous substitutions (Fig. 4a), but none was found in the α1 domain which encodes the putative MHC class II PBR (Brown et al. 1993; Stern et al. 1994). DQB Compared with Neph-DRA, DQB had a relatively higher variability of 12.8% (22/172) at the nucleotide level and 24.6% (14/57) at the amino acid level (Fig. 4b). The number of pairwise nucleotide differences between pairs of 14 Neph-DQB sequences ranged from 1 (Neph-DQB*01 vs Neph-DQB*02) to 16 (Neph-DQB*10 vs Neph-DQB*14), and the number for amino acid varied from 0 (NephDQB*06 vs Neph-DQB*11) to 11 (Neph-DQB*10 vs NephDQB*14). For sequences of cluster B, the number of pairwise nucleotide differences between pairs of sequences ranged from one (Neph-DQB*07 vs Neph-DQB*12) to nine (Neph-DQB*04 vs Neph-DQB*09). In contrast, sequences of the cluster A, C, and D showed relatively conservative

particularly at the amino acid level, with each pair containing only a maximum of three different amino acids. The dN and dS were calculated for all the sequences and for each cluster (Table 3). The rate of dN in all 14 NephDQB sequences was more than four times higher than that of dS (P=0.071, Z test of positive selection) in the putative PBR, while the rate decreased to two (P=0.148, Z test of positive selection) in the non-PBR (Table 3). An excess of non-synonymous substitutions was also found in the PBR at each cluster of the DQB. MHC-I The variability was 47 of 147 (32.0%) in nucleotide sequences and 25 of 49 (51.0%) in amino acid residues (Fig. 4c). Nucleotide sequence variation between all pairwise comparisons of 34 Neph-I sequences ranged from 1 (Neph-I*02 vs Neph-I *26) to 25 nucleotides (Neph-I *14 vs Neph-I *26), whereas amino acid substitutions ranged from 1 (Neph-I *02 vs Neph-I *26) to 19 (Neph-I *14 vs Neph-I *22). The sequences of each cluster were highly divergent, except for cluster B where there was only two nucleotide difference. For instance, nucleotide sequence variation between pairwise comparisons of cluster A sequences ranged from 1 (Neph-I*09 vs Neph-I *10) to 15 (Neph-I*21 vs Neph-I *22), while amino acid substitutions ranged from 1 (Neph-I*09 vs Neph-I *10) to 12 (Neph-I*21 vs Neph-I *22). In addition, the Z test showed that dN was greater than dS for the MHC-I sequences, except for the cluster B where there was no dN and dS in the PBR (Table 3). The dN/dS ratio was even higher when only putative PBR was included as shown in Table 3.

Table 3 Average rates of non-synonymous substitutions (dN), synonymous substitutions (dS), with standard errors obtained through 1,000 bootstrap replicates in parentheses, and results of the Z test of positive selection Loci

DRA All sequences DQB All sequences Cluster A Cluster B Cluster C Cluster D MHC-I All sequences Cluster A Cluster B Cluster C Cluster D

All domain

PBR

Non-PBR

dN

dS

dN/dS

P

dN

dS

dN/dS

P

dN

dS

dN/dS

P

0.9 (0.5)

0.8 (0.9)

1.1

0.476

0 (0)

3.8 (4.5)

0

1.000

1.2 (0.7)

0 (0)

–

0.038

5.2 1.0 4.1 1.7 2.5

2.0 (0.9) 1.0 (1.0) 0 (0) 0 (0) 4.1 (2.8)

2.6 1.0 – – 0.6

0.037 1.000 0.001 0.036 1.000

10.0 (3.5) 1.7 (1.7) 7.2 (3.0) 4.6 (3.1) 10.9 (9.0)

2.3 (3.0) 0 (0) 0 (0) 0 (0) 0 (0)

4.3 – – – –

0.071 0.156 0.009 0.079 0.111

3.7 1.4 2.6 0.8 2.3

(1.7) (1.0) (1.3) (0.8) (1.7)

1.8 (1.0) 0 (0) 1.4 (1.0) 0 (0) 0 (0)

2.1 – 1.9 – –

0.148 0.075 0.220 0.158 0.073

3.5 (1.4) 1.2 (1.2) 0 (0) 2.6 (1.1) 4.6 (2.7)

3.1 6.1 – 3.1 3.3

0.003 0.001 0.048 0.017 0.012

26.9 (8.0) 22.6 (7.0) 0 (0) 24.3 (7.9) 45.1 (16.4)

1.7 (1.3) 4.2 (4.9) 0 (0) 2.2 (2.1) 6.4 (7.5)

15.8 5.4 0 11.0 7.0

0.002 0.005 – 0.004 0.025

4.8 2.8 1.9 3.9 7.3

(1.5) (1.2) (1.1) (1.6) (2.3)

3.0 (1.4) 0.1 (0.1) 0 (0) 2.9 (1.4) 4.0 (3.0)

1.6 28 – 1.3 1.8

0.188 0.009 0.042 0.307 0.157

(1.5) (0.7) (1.3) (1.0) (1.9)

10.7 (2.5) 7.3 (2.0) 1.4 (0.8) 8.1 (2.2) 15.2 (3.6)

dS and dN values are given as percentages per site.

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Discussion Gene duplication and genetic variation of MHC genes in finless porpoises Many mammalian species have one single DRA locus (Chu et al. 1994; Takada et al. 1998), which is further approved by the present study. All individuals examined in this study had no more than two alleles of the DRA gene, strongly suggesting that the DRA primers used in this study had amplified a single locus in finless porpoises. However, some strong evidences of gene duplication were found for the DQB and MHC-I loci. As shown in “Results”, three, four, or five distinct sequences were detected in some individuals at the DQB and MHC-I loci, suggesting that at least three copies of DQB and MHC-I existed in finless porpoises. This was further supported by the phylogenetic trees which revealed that four loci might be identified at the DQB and MHC-I. Gene duplication was corroborated in humpback whales (Megaptera novaeangliae), southern right whales (Eubalaena australis), and gray whales (Eschrichtius robustus; Baker et al. 2006; Flores-Ramirez et al. 2000). These duplications could be analogous or homologous with the cattle, which are also known to have two or three transcribed DQB and MHC-I loci (Ellis and Ballingall 1999; Ellis et al. 1999). Further, although Baker et al. (2006) suggested that DQB duplication in the baleen whale (suborder Mysticeti) and baiji (suborder Odonotoceti), an early divergence of the toothed whales (suborder Odonotoceti; Cassens et al. 2000), is consistent with retention of an ancestral condition shared with the ruminants and loss in the more derived cetaceans such as the beluga, the narwhal (Monodon monoceros included in the family Monodontidae), and the true dolphins (Delphinus delphis included in the family Delphinidae), this was not supported by the finless porpoise (family Phocoenidae) examined in the present study. Despite population decline in recent years, finless porpoises seem to retain considerable MHC genetic variation, which is approved by the big number of unique sequences. A total of five DRA, 14 DQB, and 34 MHC-I unique sequences were identified in 195 finless porpoises examined in the current study. When each cluster was presumed to represent a distinct locus, the number of alleles at each cluster for the DQB and MHC-I genes was greater than or comparable to those of some other marine mammal species/populations. For example, four, five, three, and two DQB alleles was separately included in cluster A, B, C, and D in this study. In contrast, two Mian-DQB alleles have been identified in 110 Northern elephant seals (Mirounga angustrirostris), five Dele-DQB alleles in 233 beluga, one Momo-DQB allele in 12 narwhals (Weber et al. 2004; Murray et al. 1995), and eight Mile-DQB alleles in 109 Southern elephant seals

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(Hoelzel et al. 1999). In addition, high level of sequence variation between sequences also indicates that the MHC genes have significant genetic diversity. Similar to other mammalian species, DRA gene showed very low sequence divergence in finless porpoises, with a low dN/dS ratio (Table 3) and a low sequence variability of 2.1 and 4.8%, respectively, at the nucleotide level and amino acid level. In contrast, a considerable sequence variation was detected for the DQB not only for all the sequences but also for sequences at each putative locus. Nucleotide sequence variation among pairwise comparisons of all Neph-DQB sequences ranged from 0.58 to 9.30%. For sequences of cluster B, the number of pairwise nucleotide differences between pairs of sequences ranged from 1 (Neph-DQB*07 vs Neph-DQB*12) to 9 (NephDQB*04 vs Neph-DQB*09). Considerable sequence variation was also evidenced by the high dN/dS ratio as shown in Table 3. However, of the three genes investigated in this study, MHC-I showed the most extensive variability. Both variation at the nucleotide and amino acid levels and dN/dS ratio at the MHC-I locus were much higher than those at the DQB and DRA loci. Further, all populations had relatively large number of sequences at the MHC-I locus, ranging from 13 (Yangtze River population) to 26 (South China Sea population), with a mean of 20.3. However, the Yangtze River population had relatively fewer unique sequences than the other two populations (13 sequences out of 64 Yangtze samples vs 22 out of 54 Yellow Sea samples and 26 out of 76 South China Sea samples). This suggested that the Yangtze population might have experienced historically genetic bottlenecks when they dispersed into the Yangtze River and adapted to freshwater. Although relatively fewer Neph-I sequences were observed in the Yangtze River population, the divergence between sequences was comparable to the populations in the Yellow Sea or South China Sea. For instance, the mean nucleotide sequence variation among the MHC-I sequences in the Yangtze River population was 10.37±1.90%, 8.58±1.58% in the Yellow Sea population and 8.02±1.56% in the South China Sea population. Such pattern of a low number of MHC sequences, with a high degree of divergence between sequences in the Yangtze finless porpoise, implied that those divergent sequences were very ancient and could predate the population bottleneck. This phenomenon was also found in some other bottlenecked species (e.g., Hedrick et al. 1999; Hoelzel et al. 1999). Balancing selection The MHC encompasses highly polymorphic genes, and variation at these loci is believed to be under some forms of balancing selection (Potts and Wakeland 1993; Apanius et al. 1997; Aguilar et al. 2004) such as pathogen-driven

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selection (Hedrick 2002; Penn et al. 2002; McClelland et al. 2003). Three lines of evidences support that balancing selection plays a role in maintaining MHC sequence polymorphism at the MHC-I and DQB loci. Excess of nonsynonymous than synonymous substitutions in the PBR was considered as an indication of balancing selection (Bernatchez and Landry 2003), and this phenomenon was observed in the PBR at each of the two loci and each DQB and MHC-I cluster except for MHC-I cluster B. The findings from the current study suggested that the evolution rate was faster for non-synonymous than synonymous sites, which could be due to the action of balancing selection to favor new variants and increase MHC diversity (Hughes and Nei 1989). Similar pattern has been found in a number of other species, e.g., Spanish ibex (Capra pyrenaica; Amills et al. 2004), Bighorn sheep (O. canadensis; Gutierrez-Espeleta et al. 2001), and Eurasian beaver (Castor fiber; Babik et al. 2005), etc. Second, a high level of divergence among MHC sequences within a population is suggested as another evidence that balancing selection is acting to maintain MHC variation (Richardson and Westerdahl 2003). In this study, considerable divergence was detected especially among sequences of the DQB and MHC-I genes within a population. For example, in the Yangtze River population, sequences were highly divergent with up to 18.4% (27/147) and 36.7% (18/49) at the nucleotide and amino acid levels, respectively. A trans-species polymorphism was manifested at the DQB and MHC-I loci among some sequences of the finless porpoise and vaquita, which could be regarded as the third evidence for balancing selection. As revealed in the phylogenetic trees, some sequences of finless porpoises clustered with those of the vaquita at the presumed locus, e.g., Neph-DQB*09 clustering with Phsi-DQB*01 in DQB cluster B, Neph-I*04 clustering with Phsi-DQB*06 in MHC-I cluster C, etc. In the case of the conserved DRA gene, no nonsynonymous substitutions were identified in the PBR, which may imply no balancing selection on the DRA gene. This is known to be true of other mammals such as human, mouse (Mus musculus), and California sea lion (Pimtanothai et al. 2001; Janitz et al. 1998; Bowen et al. 2004). Trans-species or convergent evolution between the finless porpoise and baiji This study identified that the two unique sequences of the finless porpoise (Neph-DQB*03 and 14) were identical to their counterparts in the baiji (Live-DQB*16 and 4). In addition, identical and homologous sequences were also found at the DRA and MHC-I loci in the two populations (data not shown). The possibility of “PCR artifacts” was excluded because each unique sequence was identified from at least two replicates of PCR products in a large

Immunogenetics (2007) 59:581–592

population. In addition, we have obtained some sequences of mitochondrial control region from the same DNA extractions (Yang et al. 2003, 2007). The finless porpoise is known to be sympatric with the baiji in the Yangtze River. The conservation of MHC sequences between these two species can be the result of trans-species evolution or convergent evolution. Trans-species pattern of evolution is a common occurrence when MHC alleles of two closely related species are compared (Klein 1987), whereas the convergence hypothesis explains the observed similarity as having arisen independently by balancing selection driven by selection pressure (Kriener et al. 2000). Trans-species pattern of evolution appears acceptable to explain the sharing MHC sequences or motif between the finless porpoise and vaquita, as both species belong to the same family Phocoenidae. However, the finless porpoise (especially the Yangtze population) and baiji are highly divergent, although the two species are sympatric in the Yangtze River. The former belongs to the family Phocoenidae of the superfamily Delphinoidea (Rice 1998), and the latter is a member of the family Lipotidae under the superfamily Lipotoidea (De Muizon 1988; Yang et al. 2002b). It is reasonable to infer that the conserved MHC sequences or motifs in both species have resulted from similar selection processes under the same pathogenic pressure in the Yangtze River. Studies using other molecular markers are underway in our laboratory to characterize these features among the selected populations. Acknowledgments This study was supported through the National Natural Science Foundation Commission of China Grants, No.30270212, No.30470253, and No.30670294 and “Qinglan Project” of Jiangsu Province awarded to Dr. Guang Yang. The authors thank Anli Gao, Xin-Rong Xu, Hua Chen, and Qing Chang for collecting samples during the years, and members of the Institute of Genetic Resources, Nanjing Normal University, for their contributions to this paper.

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