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Keywords:

  • body shape;
  • ecological speciation;
  • food resource;
  • habitat choice;
  • habitat segregation;
  • migration;
  • phenotypic divergence;
  • predation;
  • salinity;
  • species pair

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

When two closely related species migrate to divergent spawning sites, divergent use of spawning habitats can directly reduce heterospecific mating. Furthermore, adaptations to divergent spawning habitats can promote speciation as a by-product of ecological divergence. Here, we investigated habitat isolation and ecological divergence between two anadromous forms of threespine stickleback (Gasterosteus aculeatus), the Japan Sea and Pacific Ocean forms. In several coastal regions of eastern Hokkaido, Japan, these forms migrate to the same watershed to spawn. Our field surveys in a single watershed revealed that segregation of distinct spawning sites between the two forms was maintained within the watershed across multiple years. These spawning sites diverged in salinity and predator composition. Morphological and physiological divergence between the forms also occurs in the direction predicted by ecological differences between the spawning sites. Our data indicate that migration into divergent spawning habitats can be an important mechanism contributing to speciation and phenotypic divergence in anadromous fishes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

When closely related species migrate to distinct spawning sites, mating between individuals from different species is prevented, thereby contributing to prezygotic isolation (Irwin & Irwin, 2005). In addition, differential adaptations for divergent spawning migration or divergent habitat use can result in reproductive isolation as a by-product of ecological divergence (Rice, 1987; Schluter, 2000; Rundle & Nosil, 2005; Funk et al., 2006). For example, ecological selection against migrants and intermediate hybrids can contribute to reproductive isolation (Schluter, 2000; Nosil et al., 2005; Rundle & Nosil, 2005). When traits important for ecological adaptation act as or are linked to mating traits, divergent natural selection on ecological traits can simultaneously promote the evolution of sexual isolation between ecologically divergent forms (Schluter, 2000; Gavrilets, 2004). Because easy-to-detect signals differ between divergent environments, ecological divergence can also drive the divergence in communication systems, including sensory systems and/or mating signals, which can promote speciation (Endler, 1992; Boughman, 2001, 2002; Seehausen et al., 2008). Therefore, it is indispensable to understand the ecological divergence between closely related species that use divergent spawning sites.

Anadromy is common in temperate fishes (Gross et al., 1988); anadromous fishes migrate to rivers or estuaries for spawning, and their juveniles return to the sea for growth. Although ecological divergence and reproductive isolation between anadromous and freshwater-resident forms has been extensively investigated across diverse taxa (Schluter, 2000; Hendry & Stearns, 2003; Quinn, 2005), phenotypic divergence and reproductive isolation between anadromous species pairs has been less studied (for inter-population variation in anadromous fishes, see Schaffer & Elson, 1975; Kinnison et al., 2001). In this study, we characterized ecological divergence and habitat isolation between two closely related anadromous species of threespine stickleback that differentially use the lower and upper reaches within a watershed to spawn.

The ecological transition from the lower to the upper reaches of a coastal river involves many abiotic and biotic shifts that could influence the evolution of diverse aquatic organisms (e.g. Hagen, 1967; Beheregaray & Sunnucks, 2001; Fuller et al., 2007; McCairns & Bernatchez, 2008). For example, differences in salinity between habitats (i.e. higher salinity in the lower reaches and lower salinity in the upper reaches) may lead to divergence in osmoregulation between species inhabiting the different habitats (Foote et al., 1992; Fuller et al., 2007). A difference in the ability to tolerate different salinities could then restrict species to distinct habitats and thus contribute to reproductive isolation. In addition, because of the difference between freshwater and marine ecological communities, the species inhabiting the upper and lower reaches may become specialized for divergent food resources and/or divergent anti-predation strategies (Wootton, 1998).

The threespine stickleback (Gasterosteus aculeatus) species complex contains a variety of morphs that are often reproductively isolated from other morphs (i.e. species). Thus, the threespine stickleback species complex has provided a great model system for elucidation of the ecological mechanisms underlying phenotypic evolution and speciation (Schluter, 2000; McKinnon & Rundle, 2002; Hendry et al., 2009). Extensive studies have been conducted on Canadian stickleback morphs that have diverged along several ecological gradients (e.g. anadromy vs. residency, benthic vs. limnetic, lake vs. stream, and high predation vs. low predation) (Bell & Foster, 1994; Schluter, 2000; McKinnon & Rundle, 2002; McKinnon et al., 2004; Reimchen & Nosil, 2006; Berner et al., 2009; Hendry et al., 2009). These studies have revealed that divergent natural selection plays a substantial role in phenotypic divergence and speciation. For example, adaptation to divergent food resources and divergent predation regimes can cause phenotypic evolution of many morphological traits, such as body shape, body size, foraging apparatus and armour (for reviews, see McPhail, 1994; Reimchen, 1994; Schluter, 2000; McKinnon & Rundle, 2002; Hendry et al., 2009).

In this study, we investigated ecological divergence between a Japanese sympatric species pair of threespine stickleback. In contrast to other sympatric stickleback species pairs (McPhail, 1994; McKinnon & Rundle, 2002; Hendry et al., 2009), which contain at least one freshwater-resident population, this Japanese sympatric pair is composed of two divergent anadromous forms of threespine sticklebacks, the Japan Sea and Pacific Ocean forms (Higuchi & Goto, 1996). These two forms are thought to have diverged when the Sea of Japan was geographically isolated from the Pacific Ocean about two million years ago (Higuchi & Goto, 1996; Kitano et al., 2007b). After the last glacial period, the Japan Sea and Pacific Ocean forms were brought into secondary contact. In several coastal regions of eastern Hokkaido, Japan, both forms migrate to the same river or lake to spawn (Higuchi & Goto, 1996; Kume et al., 2005; Kitano et al., 2009). However, even in sympatry, these two forms are reproductively isolated, suggesting that these two forms are incipient species (Higuchi & Goto, 1996; Kitano et al., 2007b, 2009). One of the main isolating barriers is divergence in breeding habitat (habitat isolation): the Japan Sea form usually migrates only to the lower reaches to breed, whereas the Pacific Ocean form usually migrates up the river and breeds in the upper reaches (Kume et al., 2005; Kume, 2007; Kitano et al., 2009). Over 90% of total reproductive isolation in an entire watershed can be explained by habitat isolation (see Supplementary Figure 3 in Kitano et al., 2009). Recently, we found that the two forms have different sex chromosome systems, and traits important for reproductive isolation between the forms in a sympatric site (male mating signals and hybrid male sterility) map to the sex chromosomes (Kitano et al., 2009). However, the genetic basis for habitat isolation is currently unknown.

Previous studies have also revealed morphological divergence between the Japan Sea and Pacific Ocean forms: the Japan Sea form is smaller, has shorter caudal lateral plates and has more numerous gill rakers (Higuchi & Goto, 1996; Kitano et al., 2007b). However, a lack of ecological information about their respective breeding sites makes it difficult to know whether these morphological changes are adaptive. Here, we investigated the divergence in food items and body shape between the Japanese anadromous species pair and divergence in predator composition and salinity between their spawning sites to understand the ecological basis for habitat isolation and phenotypic divergence between two migratory forms of threespine stickleback. Previously, we found a sympatric site, where several hybrid juveniles were caught (Kitano et al., 2009). However, we do not know what determines whether the fish migrate to the native spawning site or the sympatric site. Recently, it is reported that stray fishes that migrate to non-native spawning sites morphologically resemble the fish of the non-native sites (Lin et al., 2008; Bolnick et al., 2009). Therefore, we compared the genetics and morphology of fish breeding in the sympatric site and the native spawning sites.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Fish sampling

This study was conducted in the Bekanbeushi River (43˚4′N, 144˚52′E; Fig. 1) of eastern Hokkaido, Japan, during the breeding season, from June to July (Kume et al., 2005; Kume, 2010), in 2005–2008. Specimens were collected at three sites (Fig. 1): St.1 (northern part of Lake Akkeshi located at ca. 1 km from the river mouth); St.2 (mid-stream of the Bekanbeushi River located at ca. 5.5 km from the river mouth); and St.3 (upstream of this river located at ca. 7 km from the river mouth), using a haul seine and ten minnow traps in 2005–2008. All specimens were identified as either the Japan Sea form or the Pacific Ocean form and as either female or male based on their morphological characteristics (see Higuchi & Goto, 1996; Kitano et al., 2007a, b). Our visual classification of each form was confirmed by genetic analyses of subsets of the fish collected in this study (Kitano et al., 2007b).

image

Figure 1.  Sampling sites (filled circles) of the Japan Sea and Pacific Ocean forms of Gasterosteus aculeatus in the Bekanbeushi River, Hokkaido, Japan. (a) Hokkaido, (b) Akkeshi-Hamanaka region of eastern Hokkaido, (c) the Bekanbeushi River.

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Characterization of breeding sites

Salinity was measured at the middle depth of each site during both low tides and full tides using a salinity metre (nearest 1 psu) four times in 2005–2006. We compared salinity among three sites by Kruskal–Wallis test.

Field observations were conducted to examine whether piscivious birds were pecking on the water surface at each site. From St. 2 to St. 3, the birds were visually observed. From the river mouth to St. 2, a web camera was connected to a telescope, and the real time images were transferred to Akkeshi Waterfowl Observation Center (AWOC) and observed by a researcher. The presence of potential predatory fish, such as white-spotted charr (Salvelinus leucomaenis leucomaenis), Sakhalin taimen (Hucho perryi) and Sakhalin river sculpin (Cottus amblystomopsis), was investigated by recording the fish collected in seine nets and traps (see in fish sampling) and by direct observations while scuba diving (30 min.) one time in each year (2005 and 2006) at each site, as well as the results of a previous survey (Kume, 2008). In previous works conducted in different Japanese rivers, it is described that sticklebacks were actually found in stomachs of heron, salmon and sculpin (Nakano, 1992; Goto, 2003).

Stomach content analysis

We compared the stomachs of formalin-fixed sticklebacks collected at native and sympatric sites in 2008. Although we collected sticklebacks from St. 2, all Japan Sea sticklebacks (= 3) at St. 2 had empty stomach and were excluded from analysis. Prey items in the stomachs of one Japan Sea stickleback at St. 1 and two Pacific Ocean sticklebacks at St. 3 were unidentifiable because of digestion, so they were also excluded from analysis. Three Japan Sea sticklebacks and five Pacific Ocean sticklebacks (= 1 at St. 2 and = 4 at St. 3) had empty stomachs and were also excluded from analysis. In total, each prey items found in 15 Japan Sea sticklebacks and 33 Pacific Ocean sticklebacks (= 13 at St. 2, = 20 at St. 3) were counted and weighed to the nearest 0.001 mg. Prey items were classified into nine categories; Mysidae, Amphipoda, Copepoda, Palaemonidae, juvenile fish (stickleback, goby, and unknowns), Nereididae, stickleback eggs (both threespine and ninespine sticklebacks, but largely the former), Chironomus larvae and others.

To clarify whether food resource divergence occurred, stomach contents were compared between the two forms. The per cent index of relative importance (%IRIi) was calculated to characterize diets of the two forms (Pinkas et al., 1971), which combines total weight (%) and frequency of occurrence (%) of each item.

  • image
  • image

where Fi is the frequency of occurrence of item i (%), Wi is the relative weight of item i (% of total) and Ni is the relative number of item i (% of total). We compared dietary components (plankton prey vs. benthic prey) among the two forms of each site by chi-square test.

%IRIi calculations are useful in providing a general description of species diet (Hyslop, 1980). However, these values can be misleading, as they do not measure variability in the diet among species. For this reason, a percentage similarity index (PSI) was used as a measure of interspecific diet variability (Schoener, 1970).

  • image

where Pij is the %IRI of item i in the diet of species j and Pik is the %IRI of item i in the diet of species k. A mean intra-specific general overlap was obtained from all overlap combinations, which permits evaluation of interspecific diet variability (e.g. the degree of diet similarity among species). This index ranges between 0 (no diet overlap, high variability) and 100 (complete overlap, no variability).

Body shape analysis

For body shape analysis, digital pictures of anesthetized fish were taken from the left side of 23 adult Pacific Ocean males, 35 adult Pacific Ocean females, 52 adult Japan Sea males and 24 adult Japan Sea females. Fourteen landmarks were chosen to capture the overall body shape and digitized with tpsDig2 (Rohlf, 2006a; Fig. 2a): (i) anterior tip of upper lip, (ii) anterior base of the first dorsal spine, (iii) anterior base of the second dorsal spine, (iv) anterior base of the third dorsal spine, (v) dorsal base of caudal fin, (vi) caudal end of caudal keel, (vii) ventral base of caudal fin, (viii) base of anal spine, (ix) anterior base of pelvic spine, (x) ventral border of operculum, (xi) posterior edge of anglular, (xii) centre of eye, (xiii) upper end of pectoral fin base and (xiv) lower end of pectoral fin base. All images were aligned, rotated and scaled by the orthogonal least squares Procrustes method with tpsRegr (Zelditch et al., 2004; Rohlf, 2005). From the superimposed landmark configurations, uniform and nonuniform shape components were calculated with tpsRegr. For the full set of uniform and nonuniform components, two-way mancova were run with morph and sex as fixed factors and centroid size as a covariate.

image

Figure 2.  (a) Fourteen landmarks used for the analysis. (b) Body shape change in males (upper) and females (lower). The base of each arrow indicates the mean position of the landmarks in the Pacific Ocean morph. Arrows indicate the direction and magnitude of change in landmark position from the mean position in the Pacific Ocean morph to the mean position in the Japan Sea morph. To increase visibility, the length of each arrow was multiplied by two. The grey areas indicate the head and caudal areas.

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Because there was significant sexual dimorphism in body shape (see Results), we next analysed the males and females separately. From the aligned coordinates, the head area (area enclosed by Landmarks 1, 2, 10 and 11; Fig. 2b) and the caudal area (area enclosed by Landmarks 4, 5, 6, 7 and 8; Fig. 2b) were calculated for each sex with tps Utility (Rohlf, 2006b). The area data were ln-transformed and then subjected to ancova with morph as a fixed factor and centroid size as a covariate. Because none of the interactions between morph and centroid size were significant for any area data, the interaction term was excluded from the final model. To visualize body shape divergence between the Japan Sea and Pacific Ocean morphs, tpsRegr and tpsSplin were used (Rohlf, 2004). Fish caught in St. 1 and St. 3 were used. Fish collected in 2005–2008 were used. No significant effect of the sampling year was found for body shape (manova; F1,128 = 1.593, = 0.057). Therefore, we excluded the sampling year from the final model, although analysis including the sampling year as a covariate did not change our conclusion.

Analysis of the fish in the sympatric site

Previously, we genotyped 307 adult fish collected in 2006 (= 108 for St. 1; = 122 for St. 2; = 77 for St. 3) with 13 microsatellite loci (Supplementary Table 4 in Kitano et al., 2009). Here, these data were used to investigate whether there was genetic differentiation between fish that migrated to the sympatric site (St. 2) and fish that migrated to their native spawning sites (St. 1 and St. 3). First, we used a Kruskal–Wallis test to compare the probability of ancestry calculated by STRUCTURE (Fig. 2b in Kitano et al., 2009; = 2) between sites for each form. We also conducted principal coordinate analysis based on the genetic distance matrix (Supplementary Fig. 2b in Kitano et al., 2009) and compared the first principal coordinate between sites for each form by Kruskal–Wallis test.

Standard length (SL) was measured with a vernier calliper from ethanol-fixed fish collected in 2006, which were also used for the genetic analyses. Deformation of the body shape in ethanol-fixed fish precludes the use of geometric morphometrics. Therefore, we measured body depth (BD) and head length (HL) to compare body shape between fish that migrated to the sympatric site (St. 2) and fish that migrated to their native spawning sites (St. 1 and St. 3). All morphological data were ln-transformed and analysed with anova for ln-SL and ancova with ln-SL as a covariate for ln-BD and ln-HL.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Habitat isolation

Divergence in breeding habitat was observed in all years of our field survey (2005–2008). Virtually, all sticklebacks breeding in St. 1 were of the Japan Sea form, whereas nearly all fish in St. 3 were of the Pacific Ocean form. In St. 2, both the Japan Sea and Pacific Ocean forms were found breeding (Fig. 3). These three breeding habitats were significantly different in salinity (Kruskal–Wallis test, < 0.05; Table 1); the average salinity of St. 1 (27.5 psu) was higher than that of St. 2 (5.6  psu), which was then higher than that of St. 3 (3.8 psu).

image

Figure 3.  Frequency distributions of mature Japan Sea (open area) and Pacific Ocean (shaded area) fishes in the Bekanbeushi River, Hokkaido, Japan, in 2005–2008. n indicates sample size.

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Table 1.   Salinity at each site of the Bekanbeushi River, Hokkaido, Japan in 2005–2006.
LocationSalinity (psu)
AverageMinimumMaximum
St. 127.52233
St. 25.6010
St. 33.8010

The occurrence of predatory birds and fishes also differed between these sites (Tables 2 and 3). A larger number of grey heron (Ardea cinerea), black-tailed gull (Larus crassirostris), smew (Mergus albellus), common merganser (M. merganser), red-breasted merganser (M. serrator) and little grebe (Tachybaptus ruficollis) were observed at Lake Akkeshi (St. 1), which is a major feeding habitat for A. cinerea (Sawara et al., 1994). In contrast, only a small number of common kingfisher (Alcedo atthis) and no other birds were found at St. 3 (Table 2). Five species of piscivious fish were observed at St. 1 (Table 3). However, no predatory fishes were found at St. 3 during our field survey, although some nonpiscivous fishes, such as Japanese dace (Tribolodon hakonensis), Sakhalin lake minnow (Phoxinus percnurus sachalinensis) and freshwater type of ninespine stickleback (Pungitius sp.) were observed. Thus, the avian and fish fauna differ between the breeding habitats.

Table 2.   Avian fauna in the Bekanbeushi River, Hokkaido, Japan, during spring-summer in 2005–2008.
SpeciesLake Akkeshi*Down- to mid-stream†Upstream‡
200520062007200820052006200720082005200620072008
  1. Check mark indicates bird observed (see text).

  2. *St. 1 and river mouth were included.

  3. †From river mouth to St. 2.

  4. ‡From St. 2 to St. 3.

Ardea cinerea    
Ardea intermedia    
Ardea alba    
Egretta garzetta    
Larus crassirostris        
Mergus albellus    
Mergus merganser    
Mergus serrator    
Tachybaptus ruficollis    
Grus japonensis    
Alcedo atthis        
Table 3.   Fish fauna in the Bekanbeushi River, Hokkaido, Japan, during spring-summer in 2005–2008.
SpeciesLake Akkeshi*Down- to mid-stream†Upstream‡
RI§2005200620072008RI§2005200620072008RI§2005200620072008
  1. Check mark indicates fish observed (see text).

  2. *St. 1and river mouth were included.

  3. †From river mouth to St. 2.

  4. ‡From St. 2 to St. 3.

  5. §Reference information (RI) from fish collected in 2000–2003 by Kume (2008).

Hucho perryi             
Salvelinus leucomaenis leucomaenis       
Cottus amblystomopsis              
Cottus nozawae              
Opisthocentrus ocellatus          
Zoarces elongatus          
Myoxocephalus stelleri          

Dietary differences

Stomach content analysis of the 2008 samples revealed that stomach contents among the two forms at each site partly overlapped (PSI = 61.78 for St. 1 vs. St. 2, 51.53 for St. 1 vs. St. 3 and 45.74 for St. 2 vs. St. 3). Both forms mainly preyed on zooplankton (%IRI = 96.18 for the Japan Sea form, and 85.53 at St. 2 and 94.09 at St. 3 for the Pacific Ocean form), such as Mysidae and Amphipoda (Table 4). However, some differences in food resource were found between them. The stomach contents of the Japan Sea form consisted almost exclusively of zooplankton, whereas that of the Pacific Ocean form contained not only zooplankton but also benthic prey (%IRI = 14.47 at St. 2 and 5.92 at St. 3), such as Nereididae and stickleback eggs (Table 4): Chi-square test comparing the frequencies of zooplankton in stomach contents between the Japan Sea form at St. 1 and the Pacific Ocean form at St. 2, χ32 = 36.50, < 0.001; Chi-square test comparing the frequencies of zooplankton in stomach contents between the Japan Sea form at St. 1 and the Pacific Ocean form at St. 3, χ32 = 54.27, < 0.001; Chi-square test comparing the frequencies of zooplankton in stomach contents between the Pacific Ocean form at St. 2 and the Pacific Ocean form at St. 3, χ22 = 69.20, < 0.001; Chi-square test comparing the frequencies of benthic prey in stomach contents between the Pacific Ocean form at St. 2 and the Pacific Ocean form at St. 3, χ12 = 1.976, = 0.160.

Table 4.   Dietary compositions and indexes of relative significance (%IRI) of the Japan Sea and Pacific Ocean forms in each breeding habitat from the Bekanbeushi River, Hokkaido, Japan, in 2008. Prey items were classified into nine categories (see text).
Prey itemsJapan Sea formPacific Ocean formPacific Ocean form
(St. 1; = 15)(St. 2; = 12)(St. 3; = 20)
%F%N%W%IRI%F%N%W%IRI%F%N%W%IRI
Zooplankton
 Mysidae86.6747.9832.5160.7992.3162.6352.8583.8860.0033.3327.5438.36
 Amphipoda40.0025.8811.9213.1730.771.802.290.9975.0015.7953.5254.59
 Copepoda6.671.080.110.0700000000
 Palaemonidae000015.380.774.650.6610.001.179.651.14
 Juvenile fish53.3324.2623.3922.1500000000
Benthic invertebrate
 Nereididae000015.381.5528.943.695.000.581.470.11
 Chironomus larvae000000005.002.920.440.18
 Stickleback egg000030.7733.511.2810.7810.0046.207.385.63
Others13.330.8132.083.8200000000

Divergence in body shape

Because ecologically divergent sympatric species often diverge in body shape, we investigated body shape divergence between the sympatric Japan Sea and Pacific Ocean morphs. Significant divergence was found in body shape between morphs as well as between sexes (two-way mancova with a centroid size as a covariate; morph, F24,106 = 13.10, < 0.001; sex, F24,106 = 20.65, < 0.001; interaction between morph and sex, F24,106 = 1.56, = 0.07; centroid size, F24,106 = 2.56, < 0.001). Males tended to have larger heads than females (Fig. 2), which is consistent with patterns of sexual dimorphism in threespine stickleback reported in previous studies (e.g. Kitano et al., 2007a; Aguirre et al., 2008; Spoljaric & Reimchen, 2008).

Because of the presence of sexual dimorphism, we next investigated male and female body shape separately. The Japan Sea males had a smaller head area but a larger caudal area than the Pacific Ocean males (Fig. 2b; = 23 Pacific Ocean males and 52 Japan Sea males; head area: anova, F1,72 = 21.95, < 0.001; caudal area: anova, F1,72 = 5.17, = 0.026). Similarly, the Japan Sea females had a smaller head area than the Pacific Ocean females (Fig. 2b; = 35 Pacific Ocean females and 24 Japan Sea females, anova, F1,56 = 22.18, < 0.001), although the caudal area did not differ significantly between morphs (F1,56 = 0.53, = 0.470).

Characterization of the fish in the sympatric site

We investigated whether the fish breeding in the sympatric site were genetically or morphologically more ‘hybrid-like’ than the fish breeding in the native spawning sites. Neither the probability of assignment to each form calculated by STRUCTURE nor the first principal coordinate value differed between sites for either form (Kruskal–Wallis test, > 0.05; Table 5), suggesting that the fish breeding in the sympatric site were not genetically more ‘hybrid-like’ than the fish breeding in the native spawning sites. However, we found that the Pacific Ocean males breeding in the sympatric site were smaller than the Pacific Ocean males breeding in the native spawning site (anova, F1, 38 = 10.1, = 0.0029; Table 5), whereas no difference was found in any other body shape traits measured (Table 5).

Table 5.   Genetic and morphological comparison of each form between different breeding sites. For genetic data, probability of assignment to each form calculated by STUCRURE as well as principal coordinate score based on the genetic distance matrix was compared (mean ± SE). For genetic data, males and females did not differ, so they were pooled. All morphological data are shown in mm (mean ± SE). Fifty-nine Japan Sea male caught in St. 1, 49 Japan Sea female caught in St. 2, five Japan Sea male caught in St. 1, four Japan Sea female caught in St. 3, 28 Pacific Ocean male caught in St. 2, 85 Japan Sea female caught in St. 2, 12 Pacific Ocean male caught in St. 3 and 65 Pacific Ocean female caught in St. 3 were used.
 Japan Sea formPacific Ocean form
St. 1St. 2St. 2St. 3
  1. *< 0.1, †= 0.005 (anova).

Assignment0.997 ± 0.000.988 ± 0.010.997 ± 0.000.998 ± 0.00
PCo1−0.410 ± 0.01−0.400 ± 0.0420.241 ± 0.000.244 ± 0.00
Male standard length (SL)63.44 ± 0.3763.14 ± 0.5875.01 ± 0.50†78.07 ± 0.94†
Female SL69.87 ± 0.4468.87 ± 1.1481.13 ± 0.28*82.03 ± 0.39*
Male head length (HL)19.89 ± 0.1419.92 ± 0.1423.98 ± 0.1724.32 ± 0.33
Female HL20.18 ± 0.1319.85 ± 0.3023.92 ± 0.0824.02 ± 0.10
Male body depth (BD)14.24 ± 0.0914.97 ± 0.0418.69 ± 0.1318.87 ± 0.19
Female BD15.04 ± 0.1115.90 ± 0.5519.66 ± 0.0919.76 ± 0.09

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Habitat isolation

Previous field surveys from 2000 through 2003 identified divergent spawning sites between the Japan Sea and Pacific Ocean forms (Kume et al., 2005; Kume, 2007). In this study, our field surveys conducted from 2005–2008 confirmed the presence of habitat isolation between the forms. In a nearby sympatric river, the Charo River, Pacific Ocean fish were mainly found breeding in the upper reaches, whereas Japan Sea fish were mainly found breeding in the lower reaches in 2005 and 2006 (Appendix S1). Thus, the segregation of breeding habitats between these two forms occurs in similar ways in multiple regions of sympatry.

The fish that migrated to the mid-stream region (St. 2) to breed may be the main contributors to gene flow between these two forms. If migratory behaviour has a heritable component, hybrids are expected to perform intermediate migratory behaviour (Irwin & Irwin, 2005). However, F1 hybrid adults were very rare in the entire watershed (only one F1 adult was found among 422 adult fish analysed in 2006 and 2007; Kitano et al., 2009), possibly because of the low fitness of F1 hybrids. Therefore, we could not examine whether hybrids migrate to the mid-stream. Importantly, we found that the Pacific Ocean fish breeding in the sympatric site were smaller in body size than the Pacific Ocean males breeding in the native spawning site. Phenotype-dependent habitat choice has been also found in several fishes (Lin et al., 2008; Bolnick et al., 2009): migrants are morphologically similar to the native fish in their new habitats. Thus, phenotype-dependent habitat choice might be important also for the Pacific Ocean stickleback in the Bekanbeushi River.

Ecological divergence

The most conspicuous difference that we found among the three breeding sites was in salinity. Several lines of evidence indicate that the Japan Sea and Pacific Ocean forms have different freshwater tolerance. Japan Sea juveniles suffer from severe thyroid goitre and show higher mortality in fresh water after the age of about 2 months, whereas Pacific Ocean juveniles of a similar age survive these conditions (Hamada, 1975; Honma et al., 1977; Yamada, 2003). Thus, selection of breeding habitat matches the difference in freshwater tolerance of juveniles, although there are no apparent differences in freshwater or saltwater tolerance between adults of the two forms. In St. Lawrence estuary, Canada, four species of stickleback (Gasterosteus aculeatus, G. wheatlandi, Pungitius pungitius and Apeltes quadracus) use divergent breeding habitats, which may be based on difference in salinity preference behaviour (Audet et al., 1985). Because olfaction is an important modality in stickleback perception (McLennan, 2003; Kozak et al., 2009), olfactory signals may also play a role in habitat choice of sticklebacks like in anadromous salmonids (e.g. Quinn, 2005). Further studies on the physiological basis for divergence in habitat choice between the Japanese sympatric pair will help to understand the mechanisms underlying reproductive isolation between anadromous fishes.

Competition for food resources is an important driver of phenotypic evolution (Schluter, 2000). We found some evidence for divergence in food exploitation in 2008; the stomachs of virtually all Japan Sea fish contained only zooplankton, whereas those of the Pacific Ocean fish contained both zooplankton and benthic prey. Japan Sea fish have more gill rakers than Pacific Ocean fish (Kitano et al., 2007b); a higher number of gill rakers is characteristic of planktivores (McPhail, 1994; Schluter, 2000). Thus, these two forms may be specialized for exploiting divergent food resources. However, this difference in stomach content was not found between the two forms in our previous study using 2003 samples; both forms preyed almost exclusively zooplankton and only Japan Sea fish ate threespine stickleback eggs (Kume & Torii, 2009). Thus, feeding habits may change year by year, possibly because of annual changes in the relative abundance of food resources in the breeding sites, suggesting a potential temporal change in the strength of divergent natural selection on foraging traits. However, we could not compare between prey items of both forms in sympatric site. Therefore, further comparative studies on feeding habits are needed to test this prediction.

Predation regime is another important factor that can drive phenotypic evolution and even speciation (Reimchen, 1994; Walker, 1997; Reimchen & Nosil, 2006; Langerhans et al., 2007). We found that the lower reaches have more diverse predatory birds and fishes. Therefore, predation pressure might be higher in the lower reaches than in the upper reaches. Previous work suggests that fish in high-predation habitats have smaller heads and larger caudal regions than fish in low-predation habitats, because this body shape is suitable for high performance escape swimming (Walker, 1997; Langerhans et al., 2007). Consistent with this prediction, Japan Sea males and females have smaller heads, and Japan Sea males have larger caudal regions. Furthermore, our preliminary behavioural studies indicate that the Japan Sea form has a better turning velocity of escape response than the Pacific Ocean form (J. Kitano, unpublished data). Thus, body shape divergence in the Japanese species pair has occurred in the direction predicted by predation regime, although we cannot exclude the possibility that the morphological divergence evolved as adaptations to divergence in marine environments while they are out in the sea between the Sea of Japan and the Pacific Ocean.

The armour plates are also an important component of defence against predators in sticklebacks (Reimchen, 1994). Although armour plate number does not differ between forms (Kitano et al., 2007a), the plates in the caudal regions of the fish are shorter in the Japan Sea form (Kitano et al., 2007b). Because bending of the caudal body trunk is required for burst escape swimming (Bergstrom, 2002), a reduction of plate height only on the caudal region of the fish may be suitable for enhancing the escape performance in the Japan Sea form. Further studies on the anti-predator strategies of these two forms as well as more quantitative analysis (e.g. stomach content analysis of predatory animals) will be required to understand the role of predation in phenotypic divergence between the Japan Sea and Pacific Ocean forms.

Energetic costs

It has been suggested that one benefit of spawning in the upper reaches is a lowered risk of predation (Whoriskey & FitzGerald, 1985; Kedney et al., 1987; Wootton, 1998). Our data support this hypothesis. However, by migrating to the upper reaches, fish are also faced with several costs (Wootton, 1998), such as osmoregulation (see Discussion) and migratory swimming (Bernatchez & Dodson, 1987; Quinn, 2005). In the Bekanbeushi River, adults of the Pacific Ocean form migrate for an additional 5.5 km when compared to the Japan Sea form (Kume et al., 2005; Kume, 2007; this study). By reducing migration distance, the Japan Sea form might reduce the energetic costs of migration and the risk of osmotic shock, but this strategy may expose them to higher predation pressures. By contrast, the Pacific Ocean form breeds in a place with less piscivores but migrates for additional distance and is then faced with the need to tolerate osmotic changes.

The difference in body size between these two forms is unlikely to result from their different ages, because previous studies on the otoliths revealed that breeding fish of both forms were at the age of 1 year (Higuchi, 1996). Larger body size is expected to evolve in fish that perform long-distance migration (Bernatchez & Dodson, 1987). In accordance with this prediction, the Pacific Ocean form is larger than the Japan Sea form (Kitano et al., 2007b; Table 5). Interestingly, body size divergence contributes to asymmetric behavioural isolation between these two forms (Kitano et al., 2009). Thus, the different reproductive strategies that underlie habitat isolation between the Japanese sympatric forms may shape phenotypic divergence and reproductive isolation between them.

Reproductive isolation between anadromous fishes

It is now well appreciated that ecological speciation plays important roles in diversification of north temperate fishes (Taylor, 1999; Schluter, 2000; Landry et al., 2007). In this study, we report that ecological divergence can be an important isolating mechanism between anadromous fishes. First, the divergence in spawning sites (ecogeographical isolation) directly reduces the frequency of heterospecific encounters. Second, phenotypic divergence in freshwater tolerance and morphology might have evolved as an adaptation to divergent breeding habitats and/or migratory behaviour and contributed to reproductive isolation between the forms by reducing the fitness of immigrants. Ecological selection against hybrids was suggested in a previous study: the frequency of adult hybrids was lower than that of hybrid juveniles (Kitano et al., 2009). Furthermore, like in many other sympatric stickleback species pairs (McKinnon & Rundle, 2002; McKinnon et al., 2004), body size divergence may be linked not only to sexual isolation (Kitano et al., 2009) but also to ecological divergence in the Japanese species pair. Because ecological divergence can also drive the divergence in communication systems (Endler, 1992; Boughman, 2001, 2002; Seehausen et al., 2008), it will be important to know how the environmental differences between the estuary and the upstream might influence the evolution of sensory systems and mating traits in fishes.

Recently, we demonstrated that hybrid male sterility and male mating signals are important for reproductive isolation between the forms in sympatric site; these traits were mapped to the sex chromosomes (Kitano et al., 2009). Theory predicts that genetic linkage between mating signals and ecological traits promote speciation (Gavrilets, 2004). The Japanese stickleback species pair thus provides an opportunity to test this prediction by directly comparing the genomic loci important for ecological isolation with those for sexual isolation and intrinsic isolation.

Finally, several stickleback species are now threatened with extinction because of human disturbances (Mori, 2003). Our work clearly demonstrates that preservation of ecologically diverse environments in a watershed is essential for conserving incipient anadromous species. Thus, divergence in spawning migration should be given further consideration as an important mechanism contributing to the evolution of reproductive isolation between closely related anadromous fishes.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We wish to express our thanks to T.E. Reimchen, C.L. Peichel and three anonymous reviewers for valuable comments on the manuscript, C. Torii, T. Sato and J. N. Negishi for valuable discussions, S. Awano, N. Suzuki, T. Ueda, Y. Machida, Y. Meguro, and the staffs of AWOC for collecting samples, S. Takeyama for use of facility during our field work, and Y. Kayaba, Y. Harada, C.L. Peichel and M. Kawata for use of laboratory.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Appendix S1. Segregation of breeding habitats between two migratory forms of Gasterosteus aculeatus.

Figure S1. Sampling sites and frequency distributions of two migratory forms of Gasterosteus aculeatus in the Charo River, Hokkaido, Japan, in 2005 and 2006.

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