Loss of Genetic Diversity at an MHC Locus in the Endangered Tokyo Bitterling Tanakia tanago (Teleostei: Cyprinidae) Author(s): Hitoshi Kubota and Katsutoshi Watanabe Source: Zoological Science, 30(12):1092-1101. 2013. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zsj.30.1092 URL: http://www.bioone.org/doi/full/10.2108/zsj.30.1092

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ZOOLOGICAL SCIENCE 30: 1092–1101 (2013)

¤ 2013 Zoological Society of Japan

Loss of Genetic Diversity at an MHC Locus in the Endangered Tokyo Bitterling Tanakia tanago (Teleostei: Cyprinidae) Hitoshi Kubota1* and Katsutoshi Watanabe2 1

Tochigi Prefectural Fisheries Experimental Station, Sarado, Ohtawara, Tochigi 324-0404, Japan 2 Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo, Kyoto 606-8502, Japan

Genetic diversity at a major histocompatibility complex (MHC) class II B gene was examined for two wild and three captive populations of the endangered Tokyo bitterling Tanakia tanago. A specific primer set was first developed to amplify the MHC II B exon 2 locus. Using single strand conformation polymorphism (SSCP) and sequencing analysis, 16 DAB3 alleles were detected with 56 nucleotide substitutions in the 276-bp region. In the putative antigen-binding sites of exon 2, the rate of nonsynonymous substitutions was significantly higher than that of synonymous substitutions (dN/dS = 2.79), indicating positive selection on the retention of polymorphism. The population from the Handa Natural Habitat Conservation Area and that from the Tone River system exhibited low variation (one and three alleles, respectively), whereas the captive population that originated from a mix of three distinct populations had the highest amounts of variation (14 alleles). The levels of heterozygosity at the MHC varied considerably among populations and showed significant correlations with those at putative neutral microsatellite markers, suggesting that genetic drift following a bottleneck has affected MHC variability in some populations. Comparisons between endangered and non-endangered fish species in previous reports and the present results indicate that the number of MHC alleles per population is on average 70% lower in endangered species than non-endangered species. Considering the functional consequence of this locus, attention should be paid to captive and wild endangered fish populations in terms of further loss of MHC alleles. Key words: complex

adaptive variation, captive population, conservation, genetic drift, major histocompatibility

INTRODUCTION The major histocompatibility complex (MHC) is critical to infectious disease resistance (e.g., Hill et al., 1991; Hedrick and Kim, 2000), and its loci exhibit extremely high polymorphism, which is attributed to balancing selection (e.g., Hughes, 1991; Hedrick et al., 2001; Garrigan and Hedrick, 2003). Variation at MHC loci is of particular interest in the conservation of endangered species (e.g., Frankham et al., 2002; Sommer, 2005; Radwan et al., 2009; Ellison et al., 2012); it is thought to be crucial for long-term survival of populations associated with pathogen resistance (e.g., Arkush et al., 2002; Miller et al., 2004; Croisetière et al., 2008) and it plays a role in ecological aspects such as kin recognition and mate choice with resultant inbreeding avoidance (e.g., Olsén et al., 1998; Landry et al., 2001; Reusch et al., 2001; O’Farrel et al., 2012a). Given the evident importance of MHC to adaptive genetic variation, some authorities have proposed that maintenance of MHC variation should be incorporated in the design of conservation strategies for endangered species (Hughes, 1991; Hedrick et al., 2000; * Corresponding author. Tel. : +81-287-98-2888; Fax : +81-287-98-2885; E-mail: [email protected] doi:10.2108/zsj.30.1092

Siddle et al., 2007). Freshwater fish are one of the most threatened groups, resulting in over 60% of species evaluated being threatened (IUCN, 2012). Despite this, variation in MHC genes has only been studied in a subset of endangered fish including Gila topminnow Poeciliopsis occidentalis occidentalis (Hedrick and Parker, 1998; Hedrick et al., 2001), Gila trout Oncorhynchus gilae (Peters and Turner, 2008), brown trout Salmo trutta (Campos et al., 2006; O’Farrell et al., 2012b), Kirikuchi charr Salvelinus leucomaenis (Sato et al., 2010) and Gizani Ladigesocypris ghigii (Moutou et al., 2011). There remains a need for further study of patterns of loss or retention of variation in MHC genes in endangered fishes. The Tokyo bitterling Tanakia tanago (Cyprinidae) is one of the most threatened freshwater fishes in Japan (Nakamura, 1969; Arai, 2003). This species, as well as other bitterling species, has an unusual spawning symbiosis with freshwater mussels in that they deposit their eggs in the gills of living freshwater mussels (mainly Unionidae) (see review by Smith et al., 2004). Although this species was previously distributed throughout the Kanto Plain in the middle of Honshu Island, most of their habitat has been destroyed by human activities over the past few decades, such as urbanization, improvement of paddy fields, and the resultant habitat loss of freshwater mussels. Consequently, wild populations can presently be found only in a few localities in Tochigi and

MHC Polymorphism in Tanakia tanago

Chiba regions (Mochizuki, 1997; Nakamura, 1998; Arai, 2003). About 10 local T. tanago populations (wild or captive) are known to exist (Mochizuki, 1997; Arai, 2003). However, reductions in genetic diversity within populations have been detected for almost all of them in terms of both mtDNA and nuclear DNA (AFLP and microsatellites) (Kubota and Watanabe, 2003; Kubota et al., 2008, 2010). Thus, concerns are increasing over the loss of genetic diversity through inbreeding in this species, which may hamper future attempts to reintroduce it to wild habitats. The reduction in genetic diversity at these putatively neutral markers suggests the need to examine whether MHC is similarly affected and how might this inform conservation efforts in remnant T. tanago populations. For this purpose, the present study first developed an experimental system for genotyping the MHC class II B gene, one of the most polymorphic loci of the fish MHC genes (Wegner, 2008). In the MHC class II B of the common carp, Cyprinus carpio, multiple loci were identified, i.e. Cyca-YB (Hashimoto et al., 1990), Cyca-DAB1, Cyca-DAB2 (Ono et al., 1993) and Cyca-DAB3, Cyca-DAB4 (van Erp et al., 1996). Of these, two pairs of loci, Cyca-DAB1 and Cyca-DAB2, and Cyca-DAB3 and Cyca-DAB4, were reported as linked but independent loci originated from gene duplication (van Erp et al., 1996). For other cyprinid species, DAB1 and DAB3 genes have been studied, and examined for their variability (Dixon et al., 1996; Graser et al., 1996; Ottová et al., 2005, 2007; Agbali et al., 2010; Seifertová et al., 2011). Both the DAB1 and DAB3 genes have been reported to exhibit high polymorphism and include positively selected amino acid residues notably in exon 2 (Dixon et al., 1996; Ottová et al., 2005; Seifertová et al., 2011). In the present study, we genotyped the DAB3 gene of T. tanago, and its variation was assessed for wild and captive samples of the species. The levels of the MHC gene in T. tanago populations were then compared with those from other endangered and nonendangered fish species, and implications for future conservation were discussed. MATERIALS AND METHODS Studied samples A total of 148 Tanakia tanago samples used in this study were a subset of the samples used by Kubota et al. (2010), covering two wild populations and three captive populations. The wild populations were collected from the Handa Natural Habitat Conservation Area in Tochigi Prefecture in 1993 (‘Handa wild’, n = 25) and from an anonymous location of the Tone River system in Tochigi Prefecture in 2006 (‘Tone wild’, n = 24). Three captive populations (‘Handa’, ‘Tone’, and ‘Admixed’) were maintained at the Tochigi Prefectural Fisheries Experimental Station. The Handa captive population was founded with 200 wild fish in 1992. The Tone captive population was founded with eight wild fish in 1994. The Admixed captive population originated in 1972 from a mix of three geographically distinct populations, which consisted of fish reared in the National Science Museum, Tokyo (origin uncertain), and wild fish from two populations in Sano and Ohtawara, Tochigi Prefecture (both extinct in the wild) (Nakamura, 1998). Specimens from each captive population were sampled from one or 2 year-classes (1998 year-class of Handa; 1999 and 2000 year-classes of Tone and Admixed) with 19 to 20 individuals. Pelvic-fin tissue samples were taken noninvasively from live fish under the permission of the Japanese National Agency for Cultural Affairs and the Ministry of the Environment and preserved in 99.5% ethanol. For the Handa wild popula-

1093

tion, caudal-fin tissue samples from whole samples stored at –25°C were obtained from Osaka Kyoiku University and transferred to 99.5% ethanol prior to this study. Sequencing of flanking regions of MHC class II B exon 2 for primer design Total genomic DNA was isolated from the fin clips using GenElute Mammalian Genomic DNA Kits (Sigma-Aldrich, St. Louis, MO). The flanking regions of exon 2 in MHC class II B were of interest to facilitate the design of specific primers. A region from intron 1 to exon 4 (ca. 1.5 kbp expected from zebrafish, Danio rerio; Sultmann et al., 1994) was amplified for a subset of the samples analysed (62 of 148 individuals) using primers Tu385 (5′-TGC TGT CGA RCA TTT ACT GGA AC-3′), which was reported for zebrafish by Figueroa (see Graser et al., 1996), and TaMHe4R (5′-TAT GAT GAT TCC CAG CAC CA-3′), newly designed in the exon 4 region of zebrafish (based on Brre-DAB allele; GenBank Accession no U08870, Sultmann et al., 1994). Primers newly designed in this study were developed using Primer3 (Rozen and Skaletsky, 2000). PCR amplification was conducted in a 10 μl reaction volume containing 20–100 ng DNA template, 0.5 units Taq polymerase (TaKaRa EX Taq, TaKaRa, Shiga, Japan), 1× Taq polymerase buffer, 0.25 mM each dNTP and 0.5 μM each primer. PCR profiles started with 2 min at 94°C, followed by 30 cycles of 94°C denaturation for 1 min, 50°C annealing for 1 min and 72°C extension for 2 min. Amplified double-stranded PCR products were purified using ExoSAP-IT (GE Healthcare Bio-Sciences, Piscataway, NJ) and sequenced directly using the BigDye™ Terminator Kit ver. 3.1 (Life Technologies, Carlsbad, CA) on an automated DNA sequencer (Genetic Analyser 310, Life Technologies). Sequences of exon 2 and flanking regions, including intron 1 (ca. 290 bp), were analysed only for homozygous individuals (39 out of 148) using Sequencing Analysis ver. 3.7 (Life Technologies). SSCP and sequencing analyses of MHC class II B exon 2 Based on the flanking sequences determined for exon 2, a new primer set, TaMHe2F (5′-TGT TTC AGC TGA TGG ATA TTA TGA A-3′) and TaMHe2R (5′-GAG TCC TGC AGA TCT CAC CTG-3′), was designed to amplify the region (276-bp) encompassing the whole exon 2 sequence. The forward primer anneals to the end of the intron 1 (7 bp) and the start of the exon 2 (18 bp), and reverse primer anneals to the end of the exon 2 (3 bp) and the start of the intron 2 (18 bp). PCR products with different sequences were first identified using single-strand conformation polymorphism (SSCP) analysis (Orita et al., 1989). PCR products for SSCP analysis were amplified using the reverse primer (TaMHe2R) end-labelled with a fluorescent dye (6-FAM). PCR amplification was conducted in a 10 μl reaction volume containing 20–100 ng DNA template, 0.5 units Taq polymerase (TaKaRa EX Taq), 1× Taq polymerase buffer, 0.25 mM each dNTP and 0.15 μM each primer. PCR profiles started with 1 min at 94°C, 35 cycles of 94°C for 15 sec, 50°C for 15 sec and 72°C for 30 sec followed by 72°C for 7 min. Prior to electrophoresis, 0.4 μl of PCR product was mixed with 0.3 μl of internal size standard (ROX400HD, Life Technologies) and 12 μl of deionised formamide. The loading product was heat-denatured for 2 min at 95°C and then kept at room temperature for more than 30 min for conformational change of single-strand DNA (ssDNA), following the manufacturer’s instructions for the Genetic Analyser 310. Capillary electrophoresis was conducted on an automated DNA sequencer (Genetic Analyser 310) at 30°C using the polymer 3% GeneScan Polymer (Life Technologies) with 10% glycerol in 1× Tris-borate-EDTA (TBE) and the buffer 10% glycerol in 1× TBE, and SSCP patterns were analysed by the software GeneScan ver. 3.7 (Life Technologies). PCR products from homozygotes were directly sequenced using the BigDye™ Terminator Kit with nonlabelled reverse primer.

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All specimens that were suspected to be SSCP falsehomozygotes by direct sequencing were subjected to TA cloning using the pGEM-T Easy Vector System (Promega, Madison, WI). Insertions were sequenced for 8 to 32 clones to identify both allele sequences. All sequences were aligned using CLUSTAL W (Thompson et al., 1994) and manually adjusted where necessary. Relevant homologous sequences were searched for in GenBank using BLAST programme. The nomenclature of exon 2 sequences obtained followed that of Klein et al. (1990), i.e., Tata-DAB.

search. The rates of synonymous (dS) and nonsynonymous (dN) substitutions were calculated using MEGA with the method of Nei and Gojobori (1986) and applying the Jukes and Cantor (1969) correction. The putative antigen-binding sites (ABS) were identified through comparison with the human HLA class II B sequence (Brown et al., 1993), and the ratio of the excess of dN relative to dS (dN > dS) for putative ABS and non-ABS was tested using a Z-test (Nei and Kumar, 2000). MHC variation was compared with previous genetic diversity data (Ho and He) for six microsatellite loci with an additional population (1999 year-class of Tone) (Kubota et al., 2010). Further, published studies concerning MHC class II B variation in fish populations were assessed and the level of the variation (the number of allele and heterozygosity) was compared between nonendangered and endangered species, including Tokyo bitterling.

Data analysis Genetic variation was assessed for the alleles of the exon 2 region. Observed and expected heterozygosities (Ho and He) were calculated, and deviations from Hardy–Weinberg equilibrium were tested using GENEPOP 3.4 (Raymond and Rousset, 1995). A neighbor-joining tree was constructed in MEGA version 4 (Tamura et al., 2007) using the genetic distance of Jukes and Cantor (1969). This included outgroup DAB sequences from related species that were found to be highly similar to those of T. tanago in the BLAST

RESULTS Detection and characterisation of the MHC class II B locus Direct sequence analysis from the intron 1 to exon 4

Table 1. Polymorphic nucleotide positions from 566 bp (intron1: 290 bp; exon 2: 276 bp) sequences of the MHC class II B gene of Tanakia tanago.

18

21

22

23

25

27

28

29

59

70

71

75

76

77

78

81

82

83

86

97

101

105

106

110

112

G G . .

T .

. . . . . . T T

. . * * . . . .

T . . . . . . . . . . . G G G G

A . . . . . . G G G G G . G G G

C G . . . . . . . . . . . . G . G . G . G . A T T T T T T T T T

T . . . . . . . . . . . C C C C

T . . . . . C . . . . . A A A A

T . . C C C G . . . . . C . C C

T . . . . . . . . . . . G G G G

C . . . . . . . . . . . T T T T

A . . . . . . . . . . . T T T T

T . . . . . . . . . . . G G G G

C . . . . . . . . . . . G G G G

A T T . T . . . . T . . T T T T

G . . . . . . . . . T T T T T T

T . . . . . . . . . . . A A A A

T . . . . . . . . . . . A A A A

A . . . . T T T T . . . . . . .

T . . . . A A A A . . . . . . .

A . . . . G G G G . . . C C C C

T . . . . . C C C . . . . . . .

G G G . . . . C C . . . . . . . . . . . . . . . . . . . C C . . . . . . C T . C T . C T . C T .

T . . . . . . . . . . . A A A A

G . . . . . . . . . . . A A A A

T . . . . . . . . . . . A A A A

. . . . . . A A

102

281

140

Tata-DAB*01 Tata-DAB*02 Tata-DAB*03 Tata-DAB*04 Tata-DAB*05 Tata-DAB*06 Tata-DAB*07 Tata-DAB*08 Tata-DAB*09 Tata-DAB*10 Tata-DAB*11 Tata-DAB*12 Tata-DAB*13 Tata-DAB*14 Tata-DAB*15 Tata-DAB*16

exon2

201

intron 1 Position

133

149

150

151

156

162

183

192

193

194

195

197

204

205

206

213

214

225

226

227

234

246

249

250

251

273

203

123

202

120

exon2

T . . . . . . . . . . . A A A A

G . . C . C . . . . . . . . . .

T . . . . . . . . . . . A A A A

G G A C . . A C . . A C . . . . . . . . A C A C . . . . . . . .

A . . . . . . . . . . . T T T T

G . . . . . . . . . . . C C C C

T C . C . C . . . . C C . . . .

G . . . . . . . . . . . A . . A

T . . . . A A A A . . A . A . .

A T . T . T T T T . T T . T . .

T C . C . C C C C . C C . C . .

C A . A . A A A A . A A . A . .

G A G C A . . . . . . G A . . . . . . . A . . A A . . . A . . . A . . . . . . G A . . G A . . G . . . . A G C . . . . . . . . .

A . . . . . . . . . . . T T T T

G . . . . A . . . . . . . . . .

C G T G G G . . . T G T . G . .

A C C C C C . . . C C C . C . .

T . . . . . * . . . . . . . . .

A T . T T T * . . . . T T T T T

C . . . . . * . . . . . . . . .

C . T . . . . . . . . . T T T .

A . . . . C C . . . C C C C . .

C T T T T G T . T T G G T T T .

T G C G C C C . G G C C C C C .

T G . G . . . . G G . . . . . .

T . . . . . . . . . . . G G G G

Accession number AB745676 AB745677 AB745678 AB745679 AB745680 AB745681 AB745682 AB745683 AB745684 AB745685 AB745686 AB745687 AB745688 AB745689 AB745690 AB745691

Dots indicates the same state as in the first taxon. Non-synonymous substitution sites are indicated by Italic typeset. Numbers of positions refer to Brachydanio rerio MHC class II DAB gene sequence (Sultmann et al., 1994). *Indels.

MHC Polymorphism in Tanakia tanago Table 2.

1095

Amino acid sequences translated from nucleotide sequences of exon 2 in MHC class II B gene of Tanakia tanago.

Tata-DAB*01 Tata-DAB*02 Tata-DAB*03 Tata-DAB*04 Tata-DAB*05 Tata-DAB*06 Tata-DAB*07 Tata-DAB*08 Tata-DAB*09 Tata-DAB*10 Tata-DAB*11 Tata-DAB*12 Tata-DAB*13 Tata-DAB*14 Tata-DAB*15 Tata-DAB*16

D . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . .

Y . . . . . . . . . . . . . . .

Y . . . . . . . . . . . . . . .

+ E . . . . . . . . . . . . . . .

Y . . . . . . . . . . . D D D D

+ T . . . . . . G G G G D I V V V

M . . . . . . . . . . . T T T T

+ F . . . S S L . . . . . T M T T

E . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . .

V . . . . . . . . . . . . . . .

Y . . . . . . . . . . . . . . .

S . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . .

S . . . . . . . . . . . . . . .

D . . . . . . . . . . . . . . .

Y . . . . . . . . . . . . . . .

S . . . . . . . . . . . . . . .

D . . . . . . . . . . . . . . .

M . . . . . . . . . . . . . . .

V . . . . . . . . . . . . . . .

Y . . . . . . . . . . . L L L L

L . . . . . . . . . . . . . . .

+ Q L L . L . . . . L H H V V V V

S . . . . . . . . . . . T T T T

+ Y . . . . L L L L . . . N N N N

S . . . . . . . . . . . . . . .

+ F . . . . . . . . . . . . . . .

N . . . . . . . . . . . . . . .

K . . . . . . . . . . . . . . .

V . . . . . A A A . . . . . . .

V . . . . . . . . . . . . . . .

+ D . H . . . . . . H . . Y Y Y Y

+ V . L . . . . . . L . . E E E E

Q . . . . . . . . . . . . . . .

F . . . . . . . . . . . Y Y Y Y

N . . . . . . . . . . . . . . .

S . . . . . . . . . . . . . . .

S . . . . . . . . . . . T T T T

+ V G K F V . . . . . . . . . . L . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y . . . . Y . . . . Y . . . . Y .

Tata-DAB*01 Tata-DAB*02 Tata-DAB*03 Tata-DAB*04 Tata-DAB*05 Tata-DAB*06 Tata-DAB*07 Tata-DAB*08 Tata-DAB*09 Tata-DAB*10 Tata-DAB*11 Tata-DAB*12 Tata-DAB*13 Tata-DAB*14 Tata-DAB*15 Tata-DAB*16

G . . . . . . . . . . . . . . .

V . . . . . . . . . . . L L L L

+ K . . . . . . . . . . . . . . .

Y H . H . H . . . . H H . . . .

A . . . . . . . . . . . . . . .

E . . . . . . . . . . . . . . .

+ N . . . . . . . . . . . . . . .

+ F . . . . . . . . . . . . . . .

N . . . . . . . . . . . . . . .

K . . . . . . . . . . . . . . .

D . . . . . . . . . . . N . . N

Q . . . . . . . . . . . . . . .

+ A . . . . . . . . . . . . . . .

Y F . F . I I I I . F I . I . .

+ L I . I . I I I I . I I . I . .

Q . . . . . . . . . . . . . . .

+ Q . . . . . . . . . . . . R . .

+ Q . E . . K . . . E E E L L L L

K . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . .

+ Q A S A A A . . . S A S . A . .

V . . . . . . . . . . . . . . .

D . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . .

+ Y F . F F F * . . . . F F F F F

C . . . . . . . . . . . . . . .

R . . . . . . . . . . . . . . .

+ H . Y . . . . . . . . . Y Y Y .

+ N . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . .

Q . . . . . . . . . . . . . . .

+ I . . . . L L . . . L L L L . .

+ L W S W S A S . W W A A S S S .

D . . . . . . . . . . . . . . .

+ S . . . . . . . . . . . . . . .

+ A . . . . . . . . . . . . . . .

V . . . . . . . . . . . . . . .

R . . . . . . . . . . . . . . .

D . . . . . . . . . . . . . . .

K . . . . . . . . . . . . . . .

S 91 . . . . . . . . . . . A A A A

G . . . . . . . . . . . . . . .

Y . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . .

E . . . . . . . . . . . . . . .

E 50 Q . Q . Q . . . . Q Q V V V V

The amino acid residues are given in the international single-letter code. Amino acid translations were started from the third positions of the nucleotide sequences obtained. *, gap site; +, the residues of the putative antigen-binding sites (Brown et al., 1993).

regions for 62 specimens revealed nucleotide sequences of 290 bp (or 289 bp with a deletion) for the partial intron 1276 bp (or 273 bp with deletions) for the whole exon 2 and 78 bp for the partial intron 2 (Table 1), including 10 exon 2 alleles (Tata-DAB*01, Tata-DAB*02, Tata-DAB*05–*12). Of the sequences detected, three (1.0%) and 33 (12.0%) variable sites were found in intron 1 and exon 2, respectively, indicating extremely higher sequence variability for exon 2. PCR using the primers TaMHe2F and TaMHe2R, designed for amplification of the whole exon 2, successfully amplified ca. 300-bp fragments with the exception of 1 (Tone 2000 year-class) of 148 individuals (99.3%). SSCP analysis of the amplified fragments distinguished 13 exon 2 alleles among the 147 T. tanago. Cloning and sequencing analysis then confirmed the presence of a total of 16 alleles (Tata-DAB*01–*16; GenBank/EMBL/DDBJ accession numbers: AB745676–AB745691) (Table 1). These included two sets of alleles that were not distinguished only by SSCP; i.e. Tata-DAB*11 and *14 and alleles Tata-DAB*04, *06 and *09. However, no individuals were homozygous for these indistinguishable SSCP alleles in the present samples. No more than two alleles were observed per individual, indicating that our primer set amplified only a single locus in this species. No stop codons existed within the exon 2

Table 3. The estimates of nonsynonymous and synonymous substitutions for the putative antigen (ABS) and nonantigen binding sites (Non-ABS). Region

N

dN

dS

dN/dS

P

ABS Non-ABS Total

24 67 91

0.269 ± 0.076 0.045 ± 0.013 0.096 ± 0.020

0.114 ± 0.043 0.067 ± 0.029 0.078 ± 0.024

2.36 0.67 1.23

0.003 ns ns

N, the number of codons; P, levels of significance for dN > dS.

sequences. The 276-bp exon 2 sequences exhibited high polymorphism with 56 (20.3%) nucleotide sites including a deletion of 1 codon. The 56 polymorphic sites included 49 (87.5%) nonsynonymous substitutions. Pairwise sequence divergence among alleles ranged from 1.1% (3 substitutions) to 14.9% (41) (Table 1). In the translated amino acid sequences, 29 of the 91 amino acid positions (31.9%) were variable (Table 2). Of the full set of 91 amino acid positions, 15 of 24 ABS codons (62.5%) were nonsynonymous substitutions, while 14 of 67 non-ABS codons (20.9%) were nonsynonymous. The rate of nonsynonymous substitutions (dN) in the ABS was significantly higher than that of synonymous substitutions (dS) (dN/dS = 2.36, z = 2.828, P = 0.003; Table 3).

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Table 4. Observed frequencies of the 16 alleles at the class II B exon 2 locus in the wild and captive Tanakia tanago populations examined. Handa Allele

Tone

Admixed

Wild (1993)

’98 year-class

Wild (2006)

’99 year-class

’00 year-class

’99 year-class

’00 year-class

(n = 25)

(n = 20)

(n = 24)

(n = 20)

(n = 19)

(n = 20)

(n = 20)

Tata-DAB*01

–

–

–

–

–

–

0.05

Tata-DAB*02

–

–

0.29

0.30

0.13

–

–

Tata-DAB*03

–

–

–

–

–

–

0.03

Tata-DAB*04

–

–

–

–

–

0.03

0.03

Tata-DAB*05

–

–

0.38

0.58

0.42

–

–

Tata-DAB*06

–

–

–

–

–

0.08

0.08

Tata-DAB*07

–

–

–

–

–

0.33

0.05

Tata-DAB*08

–

–

–

–

–

0.18

0.30

Tata-DAB*09

–

–

–

–

–

0.08

0.13

Tata-DAB*10

–

–

–

–

–

0.05

–

Tata-DAB*11

–

–

–

–

–

–

0.10

Tata-DAB*12

1.00

1.00

–

–

–

0.20

0.20

Tata-DAB*13

–

–

–

–

–

0.03

0.05

Tata-DAB*14

–

–

–

–

–

0.03

–

Tata-DAB*15

–

–

–

–

–

0.03

–

Tata-DAB*16

–

–

0.33

0.13

0.45

–

–

Evaluation of genetic variability of exon 2 in T. tanago A BLAST search showed that Tata-DAB*01 was most similar to the MHC class II Sqce-DAB3*28 allele in the European chub Squalius cephalus (HQ595146; Seifertová and Šimková, 2011) with a sequence similarity of 92.7% (253/273 bp, E value = 2 × 10–104 in BLAST search) (Fig. 1). All of the 100 top-ranked sequences were of MHC class II DAB genes (both DAB1 and DAB3) of cyprinid species [European chub, common carp, Rio Grande silvery minnow Hybognathus amarus and common bream Abramis brama]. Phylogenetic analysis for the exon 2 sequences of T. tanago and other cyprinids identified two major groups corresponding to DAB1 and DAB3 lineages. All Tata-DAB sequences from T. tanago clustered with all DAB3 sequences of other cyprinids, as well as Hyam-DAB1*D (Rio Grande silvery minnow) and DAB1*04C6 (zebrafish) (Fig. 1). The Tata-DAB sequences from T. tanago made two clusters in the tree (Tata-DAB*01–*12 and *13–*16). Some DAB sequences from other cyprinids were positioned between the two Tata-DAB clusters, representing trans-species polymorphism. The Tone and Admixed populations possessed both Tata-DAB groups’ allele(s) (Table 4). The Handa wild and Handa 1998-captive populations were both monomorphic with the Tata-DAB*12 allele (Table 4). Although all samples of the Tone population (wild, 1999-

Fig. 1. A neighbor-joining tree for the class II B exon 2 alleles of Tanakia tanago and some other cyprinid species, with one Salmo salar allele as outgroup. Numbers on branches are bootstrap values resulting from 1,000 replicates. DNA database accession numbers for other species: rose bitterling (Rooc-DAB1*01–16: GU080071– 87); European chub (Sqce-DAB3*28: HQ595146); common bream (DAB3*04: AJ811677); Rio Grande silvery minnow (Hyam-DAB1*D: JF792469; Hyam-DAB3*S: JF792486); zebrafish (DAB1*04-C6: AY103492); common carp (Cyca-DAB1*01: Z47731; CycaDAB3*0801: JF742726); Atlantic salmon (DAB*0201: AJ438971).

MHC Polymorphism in Tanakia tanago Table 5. Observed and expected heterozygosities at the class II B exon 2 locus and microsatellites (six loci) for Tanakia tanago populations. Population or year-class

Microsatellites†

MHC Ho

He

Ho

He

0.00 0.00

0.00 0.00

0.13 0.13

0.14 0.29

Wild (2006) ’99 year-class‡ ’00 year-class

0.58 0.65 0.47

0.68 0.58 0.62

0.35 0.56 0.55

0.41 0.55 0.60

Admixed ’99 year-class ’00 year-class*

0.60 0.50

0.83 0.85

0.72 0.70

0.76 0.73

Handa Wild (1993) ’98 year-class Tone

*Significant deviation from HWE for MHC, at P < 0.05 level after Bonferroni correction. †Data from Kubota et al. (2010) with ‡ an additional population.

captive and 2000-captive) shared three alleles (alleles TataDAB*02, *05 and *16), a significant difference in allele frequency was observed between the two year-classes of the captive population (exact G test using the Markov chain, P < 0.05). The Admixed captive populations had high amounts of genetic variation as represented by 10 alleles in both 1999 and 2000 year-classes, with significant difference in allele frequency between the year-classes (P < 0.05). The expected heterozygosity value was extremely low in the Handa populations (He = 0.00), moderate in the Tone populations (0.58–0.68) and high in the Admixed captive populations (0.83–0.85) (Table 5). In most of the samples (4/5), Ho exhibited slight deficits from Hardy–Weinberg expectations; in particular, the deficiency was significant for the 2000 year-class of Admixed population after Bonferroni correction at a 5% significance level (P < 0.007). The levels of He at the MHC were higher than the microsatellites (Table 5) with the exception of the monomorphic Handa population and were positively correlated with the average He for the microsatellites (Spearman’s r = 0.847, P < 0.05). DISCUSSION MHC variation and effects of genetic drift In MHC class II B genes (DAB) of cyprinid species, two major functional gene groups (DAB1 and DAB3 groups) have been identified (Ono et al., 1993; Dixon et al., 1996; van Erp et al., 1996; Ottová et al., 2005; Seifertová and Šimková, 2011). The nucleotide sequences from Tanakia tanago determined in this study showed high homology to DAB3 alleles from other cyprinid species and were consequently concluded to be the homologous gene to exon 2 of the MHC class II DAB3. Agbali et al. (2010) reported on the MHC class II DAB1 gene and evaluated its variation for rose bitterling Rhodeus ocellatus. Because the primers used in this study also successfully amplified the putative MHC class II DAB3 gene for some other bitterlings (Kubota et al., unpublished data), both DAB1 and DAB3 are currently potentially available for bitterlings. These will help in progressing conser-

1097

vation genetic studies of bitterlings, which include many endangered species (Kitamura, 2008). Although SSCP is an economical, powerful procedure for identifying sequence differences in amplified DNA fragments, our protocol could not distinguish some alleles. Because the electrophoretic mobility of fragments in SSCP is affected by the temperature and concentration of polymers (Kuhn et al., 2005), electrophoresis under different conditions may enable resolution of all alleles without cloning or sequencing analyses. In cyprinid species, the polymorphisms in exon 2 of both DAB1 and DAB3 are reportedly maintained by positive selection (Ottová et al., 2005). Similarly, T. tanago showed a large dN/dS value, especially in ABS (dN/dS = 2.36), and trans-species evolution of alleles, both suggesting that historical positive selection for the retention of adaptive polymorphism has shaped patterns of nucleotide diversity at this locus (Nei, 1987; Hughes and Nei, 1989; Hedrick, 1999; Bernatchez and Landry, 2003). The contemporary balancing selection usually results in an excess of heterozygosity and/ or a more even allelic frequency than under neutrality (reviewed in Bernatchez and Landry, 2003; Garrigan and Hedrick, 2003; Sommer, 2005). However, low levels of variation, correlation with levels of microsatellite variation and heterozygote deficiencies were observed in T. tanago; all of these suggest that variation at this locus would be determined by recent demographic events (i.e., bottleneck), as was the case for many endangered species (Bernatchez and Landry, 2003; Garrigan and Hedrick, 2003; Sommer, 2005; Radwan et al., 2009). In fact, the levels of Ne of endangered fish population, including T. tanago, are often small enough to override an effect of selection (i.e., Ne < 50) (Fiumera et al., 1999; Schwartz and May, 2008; Sato and Harada, 2008; Kubota et al., 2010). Significant differences in allele frequency between the year-classes in the captive populations of T. tanago also suggest strong genetic drift specifically in captivity. The presence of monomorphic populations in the MHC class II B exon 2 gene is a hallmark of endangered species when compared between endangered and non-endangered fish species (Table 6). The average number of alleles per population in the gene was calculated as 3.3 (range: 1–13, n = 49 populations) for endangered species and 10.3 (range: 3–43, n = 113 populations) for non-endangered species, indicating a significant decrease in endangered species (Mann-Whitney U-test, Z = 8.738, P < 0.001); i.e., the allelic variability in endangered fish declines to roughly 30% of that in non-endangered fish. Significant differences in heterozygosity were also observed between endangered (average He = 0.554, n = 37 populations) and non-endangered fish populations (average He = 0.770, n = 107 populations), even after excluding monomorphic populations (Mann-Whitney Utest, Z = 6.235, P < 0.001). Tanakia tanago was one of the species with the lowest level of variability. Many bottlenecked populations that have lost variability in MHC genes have reportedly still persisted (see reviews by Sommer, 2005; Radwan et al., 2009), and little direct evidence exists that population viability suffers from the loss of variability in MHC. However, similar to other vertebrates, MHC genes in fish have been shown to be associated with disease resistance at the population level under experimental conditions (Hedrick et al., 2001; Arkush et al., 2002;

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H. Kubota and K. Watanabe

Table 6. Synopsis of non-model fish population studies from which number of alleles (A), observed heterozygosity (Ho) and expected heterozygosity (He) at MHC class II B exon 2 locus are available.

loss of MHC variability might thus contribute to increased susceptibility to He Reference Species average A (range) Ho such diseases and conseEndangered species quently caused the popula2 (1–3) n.a. 0.00–0.50 Sato et al. (2010) Kirikuchi charra tion decline. A reintroduction Spanish Brown trout 4 (1–13) 0.00–0.74 0.00–0.90 Campos et al. (2006) programme for the captive Gila trout 3.5 (2–5) 0.20–0.80 0.29–0.76 Peters and Turner (2008) population is currently being Gila topminnow 2.4 (1–5) 0.00–0.80 0.00–0.71 Hedrick et al. (2001) planned, but water quality Yaqui topminnow 7.5 (6–8) 0.50–0.75 0.76–0.79 Hedrick et al. (2001) has not improved. Caution Gizani 3.5 (3–4) 0.22–0.46 0.61–0.71 Moutou et al. (2011) must be paid in terms of a Tokyo bitterling 2.2 (1–3) 0.00–0.65 0.00–0.68 this study susceptibility of this population to diseases during the Non-endangered species reintroduction process. b Atlantic salmon 6 (5–7) 0.57–0.92 0.58–0.76 Landry and Bernatchez (2001) The Admixed captive c Atlantic salmon 12.3 (10–16) 0.78–0.88 0.85–0.86 Landry and Bernatchez (2001) population has retained a Atlantic salmonc 10.7 (8.4–12.9)e 0.72–1.00 0.72–0.91 Dionne et al. (2007) large number of alleles (13 8.4 (6–12) n.a. 0.65–0.87 Langefors (2005) Atlantic salmonc of 16 alleles). A recent c Atlantic salmon 16 0.88 0.85 Landry et al. (2001) study showed that this popc,d California steelhead 12.8 (6–19) 0.62–0.96 0.72–0.96 Aguilar and Garza (2006) ulation is an admixture of 3.4 (3–4) 0.22–0.75 0.20–0.73 Kim et al. (1999) Chinook salmonc,d two historically divergent Evans and Neff (2009), Chinook salmonc 9 (8–10) 0.47–0.92 0.45–0.81 groups and retains relatively Evans et al. (2010) high overall genetic diversity Sockeye salmonc 11f 0.54 (mean) 0.55 (mean) Beacham et al. (2004) (Kubota et al., 2010). This Lake trout 43 n.a. n.a. Dorschner et al. (2000) population reproduces most Killifish 10.5 (3–20) n.a. n.a. Cohen (2002) easily among captive popuChinese rose bitteling 17 n.a. n.a. Agbali et al. (2010) lations, and no apparent Guppy 9.3 (4–15) n.a. 0.09–0.86 Fraser et al. (2010) outbreeding depression has Species: aexcluding genetically admixed populations; bland-locked populations; canadromous populabeen observed in captivity d e f tions; partially declining. average A: allelic richness; total number of alleles; n.a. not available. (Kubota et al., unpublished data). This population has Croisetière et al., 2008) or at the individual level under field been deemed to be of poor value for captive breeding and conditions (Wegner et al., 2003; Evans and Neff, 2009; reintroduction because of crossbreeding. However, this popFraser and Neff, 2010). Thus, populations exhibiting low ulation, which has high MHC diversity, should be considered MHC variability will be more vulnerable to novel pathogens. to be an important genetic source for confronting newly In freshwater fish, several exotic infectious disease outemerging diseases. In terms of disease resistance, crossing breaks have occurred in wild populations by disease transthis population with populations that have limited MHC varimission through stockings for recreational fisheries and ation may result in beneficial effects on adaptive potential, pathogen transfer from farmed fish (e.g., whirling disease in i.e. genetic restoration (see Weeks et al., 2011 for review). salmonids, bacterial cold water disease in ayu Plecoglossus Of course, intentional crossing with the Admixed captive altivelis, koi herpesvirus disease in common carp and population should be conducted only when there is evidence enteric septicaemia of catfish; Dobson and Foufopoulos, that a population is clearly suffering from inbreeding depres2001). Prevention of the spread of newly emerging pathosion (Edmands, 2007), since this population is an admixture gens to endangered species’ habitats will be crucial for conof different ESUs (Kubota et al., 2010). servation management of freshwater fish, including T. tanago. In some fish species, MHC-dependent mate preferences have been observed (Landry et al., 2001; Reusch et Implications for conservation al., 2001; reviewed in Bernatchez and Landry, 2003; Agbali Monomorphism at the MHC locus observed in the et al., 2010). Recently, Agbali et al. (2010) demonstrated Handa population of T. tanago, as well as the low levels of experimental evidence that females choose MHC-dissimilar variability at neutral markers (Kubota and Watanabe, 2003; males as mates and that the offspring of dissimilar matings Kubota et al., 2008, 2010), suggests that this population have significantly higher survival rates in the rose bitterling. went through a severe bottleneck in the wild until 1993, In the present study, several rare MHC alleles (≤ 3%) were when the specimens were taken. Although the size of the found to have been retained in one captive population of T. Handa population was roughly estimated at several thoutanago (Admixed population) despite the fact that this popsand individuals in the early 1990s (Nakamura, 1998), the ulation has been bred in captivity for more than 30 years population started to decline in 1996, and no individuals without incorporation of new individuals from other populahave been found in this habitat since 2001 (Oda, 2005). tions. The loss of these rare MHC alleles may have been Eutrophication of the source irrigation pond is thought to be delayed by MHC-disassortative mating preferences because responsible for this extinction (Fukumoto et al., 2008). Eutrofree mate choice might have been possible in this captive phication has been reported to increase parasitism and bacpopulation, which has been reproduced using living freshwaterial infections in fish (Skinner, 1982; Lafferty, 1997); the ter mussels. Undoubtedly, MHC-disassortative mating pref-

MHC Polymorphism in Tanakia tanago

erences would not operate in artificial insemination or in vitro fertilisation. In captive breeding of bitterlings, artificial insemination has recently spread under circumstances in which many mussel species are also threatened with extinction and have become increasingly difficult to use (Maehata, 1997; Negishi, 2008). Despite such constraints, we propose that opportunities for mate preference should be provided as much as possible to maintain various genotypes in MHC by conducting natural breeding. In addition, to establish suitable genetic management for captive breeding programmes, further studies are needed to document how each breeding protocol influences variability in MHC in comparison to neutral loci. ACKNOWLEDGMENTS We are grateful to K. Iguchi for the constructive comments. We also thank M. Amagai for technical advice, N. Sakae for laboratory assistance, and A. Kumagai for providing information on fish diseases. This research was conducted as the Wild Aquatic Biodiversity Conservation Project of the Fisheries Agency of Japan and was conducted with the permission of the Japanese National Agency for Cultural Affairs.

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