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Adaptation in a Peripheral Habitat: Intraspecific Variation in OsmoregulatoryTraits in an Estuarine Hermit Crab (Pagurus minutus)Author(s): Kenji Yoshino, Tsunenori Koga, Koichi Taniguchi and Rei TasakaSource: Journal of Shellfish Research, 33(1):53-59. 2014.Published By: National Shellfisheries AssociationDOI: http://dx.doi.org/10.2983/035.033.0107URL: http://www.bioone.org/doi/full/10.2983/035.033.0107

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ADAPTATION IN A PERIPHERAL HABITAT: INTRASPECIFIC VARIATION

IN OSMOREGULATORY TRAITS IN AN ESTUARINE HERMIT CRAB

(PAGURUS MINUTUS)

KENJI YOSHINO,1* TSUNENORI KOGA,2 KOICHI TANIGUCHI2 AND REI TASAKA2

1Institute of Lowland and Marine Research, Saga University, Saga 840-8502, Japan; 2Facultyof Education, Wakayama University, Wakayama 640-8510, Japan

ABSTRACT We investigated osmoregulatory ability of Pagurus minutus from three sites on the Waka River estuary (upper

reach, river mouth, and middle). The sites have different salinity regimes and we tested whether crabs were adapted to their site

locally or whether they showed phenotypic plasticity. The upper reach had a low salinity (;16) during low tide, unlike the other

two sites (;28–31). The exposure of crabs from each site to seven different salinity levels (0–52.5) showed that P. minutus is

a hyper-hypo-osmoregulator at all sites and is able to survive in 8.75–52.5 salinity levels for 24 h. Crabs from the upper reach,

however, maintained greater hemolymph osmolarity in diluted media, and most of them were able to survive in even 0 salinity

medium for 24 h. Crabs from the other two sites were not able to tolerate this condition and all died. Another experiment,

involving a 1-wk acclimation to 5 decreasing salinitymedia (35 to 0), showed that the survival was about 1.3 times greater for crabs

from the upper reach than from the other two sites. Because the crab density of the upper reach was significantly less than that of

the other two sites, the upper reach is a marginal habitat established by asymmetrical larval dispersal from central saline habitats.

Nevertheless, no crab from the saline sites survived exposure to the salinity of zero medium. These results suggest that the high

osmoregulatory ability of crabs from the upper reach of the estuary does not represent a local adaptation; crabs acquire it after

dispersal through developmental plasticity.

KEY WORDS: developmental plasticity, local adaptation, osmoregulation, phenotypic plasticity, Pagurus minutus

INTRODUCTION

Many marine invertebrate species have planktonic larvalstages with high dispersal potential that contributes greatly totheir range expansion. Range expansion often starts from

adaptation in peripheral conditions of the central habitat.Environmental regimes of peripheral habitats generally differfrom that of the central habitat, so species are challenged in theperipheral environments and may not always achieve range

expansion (Kawecki 2008, Marshall et al. 2010). Adaptation toperipheral habitats can be a precursor of parapatric speciation(e.g., Turelli et al. 2001, Berner et al. 2009, Bolnick et al. 2009).

Understanding how organisms adapt to peripheral conditions isa starting point for understanding niche evolution and contem-porary species diversity in nature.

One fundamental process of adaptation to peripheral hab-itats is strong purifying selection in peripheral populations. Theadaptive divergence during the initial phase may or may not be

involved with genetic differentiation, depending on the relativestrength of selection and the gene flow from the core habitat(reviewed in Sanford and Kelly (2011)). Divergence itself canfacilitate the evolution of reproductive barriers (Berner et al.

2009). Local adaptation may therefore be established afterrange expansion by the evolution of preferences for natalhabitat types or reduced dispersal restricting gene flow between

habitats, because individuals in foreign habitats would havelower fitness (reviewed in Nosil et al. (2005), Rundle and Nosil(2005), and Marshall et al. (2010)). If there is a genetic

correlation between habitat preferences and alleles adaptivein each habitat type (e.g., Bolnick et al. 2009), however, localadaptation could be established through the initial phase ofdivergence.

Another process of adaptation to peripheral habitats isphenotypic plasticity, an expression of different phenotypesby a single genotype. Phenotypic adaptation by plasticity does

not involve genetic modification of the phenotype, so localadaptation by genetic differentiation does not occur. However,recent evidence suggests plasticity itself is determined geneti-

cally, and phenotypic adaptation by developmental plasticityplays a significant role in rapid divergence and speciation(reviewed in Pfennig et al. (2010) and Moczek et al. (2011)).

After successful colonization of peripheral habitats by plastic-ity, the induced character can be fixed by genetic assimilation(West-Eberhard 2003, Crispo 2007). As a result, phenotypic

adaptation by selection and plasticity both involve some geneticdifferentiation. It is, therefore, not easy to discriminate whichprocess (plasticity or purifying selection) was the main contrib-utor to the contemporary patterns of adaptation in natural

systems.One way to overcome this difficulty is to observe divergence

occurring along a gradient of environmental conditions, and

estuaries are an ideal site for this. Because of tides, an estuaryincludes spatially heterogeneous habitats formed by the gradi-ent of salinity, temperature, and/or substrates. Because the

spatial scale of the gradient is often smaller than the larvaldispersal potential of many invertebrates, they are alwayschallenged by differing environmental regimes. Hence, studies

on how these organisms adapt to such environmental regimescould provide an opportunity for exploring not only the processof adaptive divergence of organisms to estuarine habitatsfrom ancestral environments, but also the incipient driver of

the divergence.Among environmental gradients, salinity is a primary factor

determining the distribution of estuarine organisms. In fact,

salinity becomes a barrier restricting the penetration of speciesfrom freshwater and marine to estuarine environments

*Corresponding author: E-mail: c1894@cc.saga-u.ac.jp

DOI: 10.2983/035.033.0107

Journal of Shellfish Research, Vol. 33, No. 1, 53–59, 2014.

53

(reviewed in McLusky and Elliott (2007)). Moreover, closelyrelated species often occupy different habitats, depending on

their osmoregulatory ability (e.g., McLusky & Elliott 2007,Thurman et al. 2010). All estuarine animals are said to beoriginated from either freshwater or marine dwellers (McLusky& Elliott 2007). However little is known of the detailed di-

versification process.In the Waka River estuary, Wakayama Prefecture, Japan,

the inflow of seawater is greater than that of freshwater

(Wakayama Research Organization of Marine Environment2006), and by continuous mixing of the seawater and freshwa-ter, a salinity gradient is formed along an approximately 1-km

stretch from the upper reach to the river mouth. During lowtide, when the effect of freshwater discharge is high, the salinityof the upper reach decreases to ;15 from ;30 during high tide,when the effect of offshore water is high (Wakayama Research

Organization of Marine Environment 2006). Hermit crabs area primary macrobenthos species and several hermit crab speciesare distributed in this estuary, but only Pagurus minutus is

distributed widely from the upper reach to the mouth of theestuary (see Results) throughout the year (T. Koga, pers. obs.).Aquatic hermit crabs are, fundamentally, marine dwellers, and

no freshwater species has been detected to the best of ourknowledge. Hence, the adaptation by P. minutus to estuarineenvironments could be an ideal model system for understanding

the adaptive divergence ofmarine ancestors to estuarine habitats.In marine invertebrates, there are many examples of adap-

tive divergence resulting from both selection (reviewed inSanford and Kelly (2011)) and phenotypic plasticity (e.g.,

Marchinko 2003, Smith 2004, Vaughn 2007, Yoshino et al.2013). There are generally trade-offs involved in the adaptationto local conditions (Angilletta et al. 2003, Hereford 2009). The

wide distribution of Pagurus minutus may represent the aggre-gation of locally adapted demes, each of which has a differingrange of salinity tolerance limit. Alternatively, P. minutus may

acquire a high osmoregulatory ability by phenotypic plasticity,and this euryhalinity enables this species to exploit successfullya wide range of habitat from the ancestral saline environmenttoward the brackish zone. In this study, we evaluated which

hypothesis is most plausible by comparing osmoregulationpatterns, salinity tolerance, and abundance of P. minutus fromthree sites with differing salinity in the Waka River estuary.

METHODS

Salinity Condition of the Study Sites

Sampling was conducted during low tide at three stations,separated by approximately 1 km, in the Waka River estuary(Fig. 1). Station A is located in the upper reach of the estuary,

station C is at the river mouth, and station B is intermediatebetween stations A and C. The reproductive season of Pagurusminutus is from winter to early spring, when rainfall is low and

water temperature is expected to be approximately 8–15�C (T.Koga, pers. obs.). As in other hermit crabs, males guard femalesfrom rival males for several days before copulation (e.g.,

Yoshino et al. 2004, Koga et al. 2010). Females spawn soonafter copulation and attach the fertilized eggs to their pleopods.Larvae of P. minutus reared in a laboratory at 14–19�C spend

about 27 days as plankton (Hong 1981) until they settle. As thetemperature decreases, the length of the larval period increases(Oba & Goshima 2004). Larvae from all the sites can spreadthroughout the study area during the planktonic phase.

To investigate the salinity gradient of the three stations, wesampled surface water from each station within 1–2 h duringlow tide, when the effect of freshwater discharge is high, and

measured the salinity with a salinity meter (S-10E; ATAGOCo. Ltd.). We carried out 13 samplings sets between July 2006and January 2008. The Waka River estuary is well mixed; the

difference between surface and bottom salinity is relativelynotable. Benthic organisms in the upper reach experience waterthat varies from brackish to marine with the tide. In fact, thesurface and bottomwater salinity of the site approximately 200m

downriver from station A fluctuates from;15 at low tide to ;30at high tide (Wakayama Research Organization of MarineEnvironment 2006). Thus, the salinity of surface water during

low tide represents the most extreme low salinity conditions thecrabs would experience in this habitat.

Distribution of Hermit Crab Species

Hermit crab samples were collected from three stationsduring low tide on August 4, 5, and 18, 2005 (Fig. 1). We casta circle, 32 cm in diameter, in a random direction 5 times at each

station and collected all the hermit crabs, live gastropods, andempty shells in the circle. The number of hermit crabs of each

Figure 1. Locations of sampling stations in the Waka River estuary.

YOSHINO ET AL.54

species was counted, and the shell species were recorded. Shellsize was measured with calipers to the nearest 0.1 mm. Shell

length was measured for Batillaria spp. and Cerithidea cingu-lata, and shell width was measured for Umbonium moniferum.

Tolerance to Low Salinity

From the samples, we selected and kept Pagurus minutus ofsimilar body size; the remaining crabs were released in theiroriginal site. We used P. minutus collected in 2006 from stations

A, B, and C. Hermit crabs in their shells were exposed for 24 h toartificial seawater of seven different salinities. Crabs wereassigned so they did not differ in size for each treatment to

ensure the effect of shell species was similar among treatmentsand among stations. Before testing, hermit crabs and shells werewiped with a paper towel to remove the internal shell water. Thismay not remove internal shell water completely.However, the effect

should be the same among treatments and among stations, andhence would have no serious impact on the interpretation of theresults. Then, 2–5 crabs were placed in a case (height, 1403 1103

40 mm) with 50 mL test water and were shaken gently to allow thewater to penetrate the shells. The test salinities were set at 0, 8.75,17.5, 26.25, 35, 43.75, and 52.5. These salinities corresponded to 0,

267.5, 535, 802.5, 1070, 1337.5, and 1605 on the milliosmolar scale,respectively. Approximately 30–75 crabs were used for eachtreatment. The number of surviving crabs was counted after 24 h.

A similar test was performed for a longer term. In thisexperiment, we exposed 63 crabs from each of the 3 stations to 5test salinities over 5 consecutive wk to determine their lowsalinity tolerance. Hermit crabs were first placed in water with

a salinity of 35 for 1 wk and then were exposed to a lowersalinity at 1-wk intervals, from 26.25, 17.5, 8.75, to 0. The testwater was renewed every 12 h, and dead crabs during the

interval were removed to reduce the stress of water degrada-tion by the dead crabs on the other live crabs. The number ofsurviving crabs was counted each week. All experiments were

conducted at room temperature (25�C).

Osmoregulation

To determine whether the mechanism of osmoregulationdiffers among sites, we measured the osmolarity of hemolymph

of Pagurus minutus from the 3 stations independent of thesurvival experiments. Between 150 and 200 P. minutus weresampled from stations A, B, and C from June 2007 to January

2008. We exposed the crabs to the salinity test for 24 h using thesame method as for the short-term exposure experiment,assuming that 24 h is sufficient for reaching osmotic equilibrium

(based on results published by Young (1979) and Rhodes-Ondiand Turner (2010), which show that it takes approximately 12 hto reach equilibrium). Twelve to 17 crabs from each station wereassigned to each salinity condition. After exposure, the hemo-

lymph of each crab was extracted for measuring osmolarity bycutting a portion of the shield with a razor blade and insertinga hematocrit tube. The extracted hemolymph was pipetted

into a 1.5-mL microtube, diluted with 90 mL distilled water,and preserved in the freezer at –30�C until measurement. Themeasurement of osmotic pressure was conducted using an

osmometer (SemiMicro Osmometer K-7400; KNAUER). Dur-ing the course of the experiment, the seawater was renewed toprevent changes in salinity resulting from evaporation.

Data Analyses

Differences in crab density were analyzed using a generalized

linear model (GLM) with a Poisson error distribution. Multiplecomparisons were performed after type 1 error adjustmentaccording to Holm�s method, when necessary.We also analyzeddifferences in the frequency of occupied shell species using

a GLMwith a Poisson error distribution, pooling the data fromfive replications at each station. Differences in crab survivalrates in the long-term experiment were estimated with Kaplan–

Meier curves, and statistical significance was tested with a log-rank test. Differences in hemolymph osmolarity were tested by2-way or 1-way analysis of variance (ANOVA), after verifying

homogeneity of variance using Levene�s test. In all cases,Levene�s test showed lack of heteroskedasticity, and hence notransformation was performed. Pairwise comparisons were also

performed, if necessary, using Holm�s type 1 error adjustment.We also determined effect size and the 95% confidence interval(CI) for significant results in pairwise comparisons. Effect sizeof pairwise comparisons after ANOVA and GLM was calcu-

lated based on bias-corrected, standardized mean differencemeasure (Hedges� d) using t or z statistics and sample size(Nakagawa & Cuthill 2007). We used a hazard ratio as an effect

size for survival analyses between the pairs. All statisticalanalyses were performed with the freeware R, version 2.8.1 (RDevelopment Core Team 2008).

RESULTS

Salinity Condition of the Study Site

The salinity during low tide at station A was 15.8 ± 2.5(mean ± SD), suggesting the influence of freshwater discharge.The salinities at stations B and C were 28.4 ± 1.9 and 30.9 ± 2.2,

respectively, which are close to that of the open sea. Hence, thesalinity at stations B and C would be relatively stable regardlessof tide, but stationAwould be exposed to brackish water during

low tide.

Distribution Patterns and Occupied Shell Species

In our study area, 3 hermit crab species coexist. The crabPagurus minutus is more numerous than Diogenes nitidimanusand Clibanarius infraspinatus, and the latter 2 species were notpresent at station A (Fig. 2). Although P. minutus inhabited all

stations, its density differed significantly among the 3 stations(PoissonGLM, df¼ 2, chi square¼ 56.6,P < 0.0001; Fig 2). Thedensity of crabs at station A was significantly less than that at

stations B (z ¼ 5.40, P < 0.0001; Hedges� d ± 95% CI, 3.1 ± 2.0)and C (z ¼ 6.51, P < 0.0001; Hedges� d ± 95% CI, 3.7 ± 2.0).These results suggest that among the 3 hermit crab species, only

P. minutus penetrates successfully into the upper reach, and it isa marginal habitat for them.

The hermit crab Pagurus minutus used primarily 3 shell

species: Batillaria sp., Cerithidea cingulata, and Umboniummoniferum. Other shell species used included Hinia festiva,Thais clavigera, and Turbo cornatus. The frequency of occupiedshell species differed significantly between any 2 stations (G test,

allG3 > 16.5, allP < 0.001 < adjusted P¼ 0.016). Because liveU.moniferum are not found at station A, U. moniferum shells arenot available for hermit crabs there. More than 80% of crabs

used Batillaria or Cerithidea shells in similar frequency (i.e.,

OSMOREGULATION OF P. MINUTUS 55

;40% each) at station A. At station B, approximately 60%

crabs used Batillaria shells, and 25% crabs used Cerithidea orUmbonium shells at a similar frequency (i.e., ;12% each). Atstation C, more than 80% of the crabs used Umbonium or

Batillaria shells in similar frequency (;40% each), and fewcrabs used Cerithidea shells. Shell size was similar betweenstations for each shell species (Table 1). Hence, similar-sizecrabs mostly use the same shell species even if habitat origin

differs.

Low Salinity Tolerance of Pagurus minutus

All crabs survived regardless of the collection site after 24-hexposure to a series of test salinities except for 0 salinity media(Table 2). About 98% of Pagurus minutus from station A

survived under 0 salinity conditions, but all individuals fromstations B and C died (chi square ¼ 143.4, df ¼ 2, P < 0.0001).The long-term survival experiment (Fig. 3) also showed signif-

icant differences in the survival curves among collection sites(log-rank test, chi square ¼ 56.4, df ¼ 2, P < 0.0001), and thecrabs from station A showed 1.25 and 1.3 times greater survivalrates than those from station B (chi square ¼ 36.9, df ¼ 1, both

P < 0.0001; hazard ratio, 1.25; 95%CI, 1.16–1.35) and station C(chi square ¼ 47.4, df ¼ 1, P < 0.0001; hazard ratio, 1.30; 95%CI, 1.22–1.34), respectively. No difference was found in survival

between stations B and C (chi square ¼ 0.7, df ¼ 1, P ¼ 0.417).

Osmoregulation

Because we could not obtain osmolarity data in 0 mediumfrom stations B and C, we performed a 3-way ANOVA, takingsite, salinity of external medium, and sex as independentvariables, excluding the 0-salinity medium. There was no 3-

way interaction (F10,118¼ 0.35, P¼ 0.96) and sex-related effects(main effect; F1,118¼ 2.83,P¼ 0.1, sex3site; F2,118¼ 0.057,P¼0.94; sex3medium salinity; F10,118¼ 0.35, P¼ 0.96). However,

the pattern of osmolarity of Pagurus minutus hemolymph after24-h exposure to the test medium differed depending on the siteof origin (site3medium salinity, F5,118 ¼ 2.26, P ¼ 0.019; Fig.

4). Hence, we performed a 1-way ANOVA for each test salinity,pooling both sexes, and found significant differences amongthe 3 sites under conditions of 8.75 salinity (F2,23 ¼ 13.0, P ¼

0.0002) and 26.25 salinity (F2,23 ¼ 6.25, P ¼ 0.007). Pairwisecomparisons showed thatP. minutus from station Amaintaineda greater osmolarity at 8.75 salinity than those from station B

(t15 ¼ –4.10, adjusted P < 0.001; Hedges� d ± 95% CI, –1.9 ±1.23) and station C (t15¼ –4.74, adjusted P < 0.001; Hedges� d ±95% CI, –2.2 ± 1.29). Also, at 26.25 salinity, crabs from station

A maintained significantly and almost significantly greaterosmolarity than crabs from station B (t14 ¼ –3.53, adjustedP ¼ 0.0055; Hedges� d ± 95 %CI ¼ –1.67 ± 1.15) and from

station C (t16 ¼ –2.03, adjusted P ¼ 0.054; Hedges� d ± 95%CI ¼ –0.91 ± 0.98), respectively. However, there were nosignificant differences in osmolarity between stations B and Cin either 8.75 salinity (t16 ¼ –0.66, adjusted P ¼ 0.52) or 26.25

salinity (t16 ¼ –1.69, adjusted P ¼ 0.11) media. These resultsindicate that P. minutus from the 3 habitats are all hyperhypo-osmoregulators, and osmoregulatory ability is greater in crabs

from the upper reach than from the other 2 sites.

DISCUSSION

Although crabs could not cope completely with dilutedsalinity, as indicated by the mortality in the long-term experi-ment, we clearly detected a difference in osmoregulatory ability

among crabs from the 3 habitats. The most clear-cut was theresult of 24-h exposure to the 0-salinity medium, in which 98%of the crabs from the upper reach survived but all crabs from thesaline habitats died. Moreover, Pagurus minutus from the upper

Figure 2. Distribution and abundance of 3 hermit crab species at each

station.

TABLE 1.

The size of shell species frequently occupied by Pagurusminutus.

Shell species

(measured length) Station Mean % SD (mm) n

Batillaria (SH) A 18.2 ± 6.5 13

B 18.6 ± 3.6 61

C 17.6 ± 4.2 54

Cerithidea (SH) A 22.5 ± 1.5 11

B 21.3 ± 2.8 11

C 22.9 ± 2.3 6

Umbonium (SW) A — 0

B 13.7 ± 1.7 14

C 12.9 ± 1.6 43

SH, shell height; SW, shell width.

TABLE 2.

The number of Pagurus minutus that survived exposure to

salinity media for 24 h.

Site Time (h)

Salinity of external

media

0 8.75 17.5 26.25 35 43.75 52.5

Station A 0 75 45 45 45 45 45 45

24 72 45 45 45 45 45 45

Station B 0 40 50 50 50 50 45 45

24 0 50 50 50 50 45 45

Station C 0 30 45 45 45 45 45 45

24 0 45 45 45 45 45 45

YOSHINO ET AL.56

reach were able to maintain greater osmolarity in diluted mediafor 24 h (i.e., 8.75 salinity and 26.25 salinity) than crabs from theother 2 saline sites. The high osmoregulatory ability of crabs

from the upper reach is not merely ascribed to an artifact fora difference in the initial hemolymph osmolarity of crabs amongsites by the lack of a reasonable acclimation in the experiment.First, osmotic equilibrium is established in about 12 h in hermit

crabs (Shumway 1978, Young 1979, Rhodes-Ondi & Turner2010). Second, the initial osmolarity should be less in the crabsfrom the upper reach than in those from the saline sites, because

hemolymph osmolarity in hypotonic media was regulated lowerthan that in hypertonic media. In the long-term experiment,crabs from the upper reach also showed 1.2–1.3 times greater

survival than those from the saline sites, indicating that crabsfrom the upper reach could maintain an osmotic balance longerthan those from the saline sites. This small increase in survival

would be adaptive enough for the crabs from the upper reachto live there, because it is only during low tide that they areexposed to low salinity. Increased hyperregulatory ability hasalso been reported in the evolution of osmoregulation in the

copepod Eurytemora affinis, which invaded freshwater habitats(Lee et al. 2003, Lee et al. 2012). A significantly lower density inthe upper reach suggests that the upper reach is a peripheral

habitat and that P. minutus there adaptively enhance highosmoregulatory ability in the process of habitat expansiontoward the brackish zone.

Local adaptation would be unlikely to explain the highosmoregulatory ability of the crabs in the upper reach. If localadaptation is the case, a certain fraction of highly tolerant crabsshould exist in the saline sites as a source of larval supply.

However, all crabs died at the 2 saline sites when exposed to 0-salinity medium, in contrast to more than 95% survival fromthe upper reach. It is also difficult to ascribe the absence of

freshwater-tolerant alleles in the saline sites to their rarity. If so,a high larval dispersal from the saline habitats is necessaryfor ensuring a sufficient supply of the rare alleles to and the

successful colonization of the upper reach. Also, strong selec-tion against less tolerant alleles is needed. Otherwise, thefixation of the rare allele could be swamped by high amounts

of the other maladaptive larvae (Lenormand 2002, Bridle &

Vines 2006). However, the selective force does not seem to bestrong enough to remove all the alleles other than tolerantalleles, even if larvae are more susceptible to osmotic stress than

adults (Anger 2003). Rainfall is generally low during thereproductive season of Pagurus minutus in our area, and thesalinity of the upper reach is 12.3 at the lowest. In our

experiment, however, all the crabs survived the 8.75-salinitymedium regardless of habitat origin. Local adaptation seemsunlikely if low-salinity-tolerant alleles are actually rare.

Figure 3. Kaplan–Meier curves ofPagurusminutus from each station during

5 successive wk. Initial sample sizes are 63 individuals in all settings.

Figure 4. Hemolymph osmolarity (mean % 95% confidence intervals) of

Pagurus minutus exposed to the test salinities for 24 h. Dashed lines

indicate isosmotic states to external media. Numbers near plots denote

sample size. Different letters indicate significant differences among

stations at media salinity.

OSMOREGULATION OF P. MINUTUS 57

Some other potential explanations for the absence offreshwater-tolerant crabs in the 2 saline habitats include the

maternal effect (e.g., Marshall 2008) and habitat selection bylarvae (e.g., Keough&Downes 1982, Jenkins 2005). The geneticeffects of the crabs in the 2 saline sites might be masked by thematernal effect, leading to the absence of freshwater-tolerant

crabs there. Alternatively, Pagurus minutus larvae might settleselectively, depending on their tolerance. Hermit crab larvaecan settle selectively in their preferred sites at a small spatial

scale (Oba & Goshima 2004). We suggest that these explana-tions are flawed for the following reasons. If maternal effects areso strong, colonization in the upper reach would be inhibited.

Larval habitat selection cannot explain the origin of low-salinity-tolerant alleles that contributed to the colonization inthe upper reach. Both maternal effect and larval habitatselection would not be drivers of incipient divergence in the

current osmoregulatory ability of P. minutus, whereas they mayreinforce divergence after colonization in the upper reach issuccessful.

In contrast, developmental plasticity can explain much moreeasily the absence of freshwater-tolerant crabs in the 2 salinehabitats. Adult salinity tolerance and performance of osmoreg-

ulation can be an integrated output of developmental pathways,each of which is molded by environmental conditions duringontogeny. Developmental plasticity is the irreversible response

to different environmental regimes. Only migrants grown in theupper reach may develop high osmoregulatory ability andtolerance limits. In the grapsid crab Neohelice (formerly Chas-magnathus) granulata, previous exposure of eggs and larvae to

a lower salinity enhanced the hyperosmoregulatory capacity offirst zoea in conditions of decreased salinity (Charmantier et al.2002, Gimenez & Anger 2003). Rearing the copepod Euryte-

mora affinis from eggs in high-salinity conditions enhances itstolerance to high salinity (Lee & Petersen 2003). The larvae ofP. minutus transported to the upper reach may develop high

tolerance while growing to the adult stage.Whether such enhanced ability to accommodate low salinity

reduces tolerance to high salinity is an interesting issue. Suchfunctional trade-offs are actually found in the copepod Eur-

ytemora affinis (Lee et al. 2003) and the shrimpMacrobrachiumamazonicum (Charmantier & Anger 2011). However, this trade-off was not observed in Pagurus minutus in water of our

experimental salinity regimes, because significantly differentosmoregulation abilities were not found in isosmotic andhypersaline regimes (i.e., salinity 35–52.5 media) among crabs

from the 3 populations. Although there is no reason to supposethat crabs tolerant to low salinity would incur greater fitness

costs at the saline sites, where costly regulation is not necessary,there still might be fitness trade-offs if maintenance of high

osmoregulatory ability is costly.Interestingly, Pagurus minutus showed an enhanced toler-

ance limit to salinity stress. These crabs from the upper reachshowed tolerance to freshwater media for at least 24 h. This was

surprising because the average salinity in their habitat is muchgreater (;15.8) than freshwater, even if salinity might decreaseto freshwater levels, depending on the season or daily weather

conditions, such as heavy rainfall. At the genetic level, salinitystressors increase gene expression of heat shock proteins,such as Hsp70 and Hsp90, as well as thermal stress (Feder &

Hofmann 1999), and the same response is found in severalcrustaceans (e.g., Spees et al. 2002, Lee et al. 2011). Heat shockproteins function as molecular chaperones that fix otherenzymes damaged by stressors and contribute in part to stress

tolerance (Feder & Hofmann 1999). According to comparativestudies on thermal tolerance limits in marine gastropod conge-ners, the production of heat shock proteins in the species with

a low thermal tolerance limit reaches a peak at lower temper-atures than in those with a high thermal tolerance limit(Tomanek (2008) and references therein). The same mechanism

might work for tolerance to salinity stress.In summary, Pagurus minutus from the 3 habitats were all

hyper-hypo-osmoregulators, and showed high tolerance and

osmoregulatory ability to a wide range of salinity regimesduring the short term that they would experience in the field.However, P. minutus from the upper reach has an adaptivelyenhanced osmoregulatory ability and tolerance limit to low

salinity compared with individuals from the more saline sites.Although firm conclusions await rigid data on gene flow, a likelyprocess for the contemporary osmotic adaptation is develop-

mental plasticity, which would have allowedP. minutus alone toexploit the brackish zone in the estuary, unlike other hermitcrab species. Developmental plasticity can play a crucial role in

niche evolution and speciation, with subsequent genetic assim-ilation. Contemporary niche differences in closely related speciesin estuaries might also have diverged from osmoregulation-flexible ancestors that colonized a marginal habitat.

ACKNOWLEDGMENTS

We appreciate all the members of the Laboratory of AnimalEcology in the Wakayama University for field sampling. SeijiGoshima gave valuable comments on the earlier draft of this

manuscript. Patricia Backwell kindly corrected the English onthe final version of the manuscript.

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