Burrowing ability as a key trait in the establishment of infaunal bivalve populations following competitive release on an extensive intertidal sandflat

Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

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Burrowing ability as a key trait in the establishment of infaunal bivalve populations following competitive release on an extensive intertidal sandflat Seiji Takeuchi a,⁎, Fumihiko Yamada b,1, Hajime Shirozu b, Satoshi Ohashi c, Akio Tamaki a a b c

Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki 852-8521, Japan Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan

a r t i c l e

i n f o

Article history: Received 30 September 2014 Received in revised form 11 January 2015 Accepted 18 January 2015 Available online xxxx Keywords: Tidal flat Venerid clams Burrowing behavior Light/dark conditions Sediment instability Winter mortality

a b s t r a c t Previous research has shown that on an extensive intertidal sandflat in Ariake Sound, Kyushu, Japan, the populations of two powerful bioturbating shrimp species, Upogebia major and Nihonotrypaea japonica, formerly predominated over the high to mid-tide zone but considerably declined almost to the extirpation during 2004 to 2008. This event was followed by higher recruitment of three infaunal clam species, Ruditapes philippinarum, Meretrix lusoria and Mactra veneriformis, than before, most probably owing to competitive release from the shrimp. Thereafter, Me. lusoria and Ma. veneriformis were successful in establishing their adult populations in the newly vacated habitat, but R. philippinarum failed. The surface sediment is more easily eroded by hydrodynamic forcing there than in the low-tide zone where the R. philippinarum population persisted. In the present study, the examination of spatiotemporal size-frequency variations in the clam species detected that their success/failure was primarily caused by the survival of juveniles in the first winter after recruitment. The assessment of potential sediment erodibility through time suggested that the seabed of the high to mid-tide zone was subject to more intense physical disturbance in winter than in the other seasons, especially during the nighttime. This was attributable to seasonally varying (1) prevailing wind directions, (2) daily nighttime length and (3) emergence/submergence durations per tide according to the seasonal change in diurnal tidalheight inequality. We hypothesized that some high burrowing ability of juvenile clams is required for their persistence in the unstable sediment, which was tested by laboratory and field experiments. The burrowing performance of juveniles was higher in Me. lusoria and Ma. veneriformis than in R. philippinarum and was reduced under dark against light conditions in all species. For those juveniles occurring in the high to mid-tide zone, vulnerability to sediment erosion would increase at nights in the wintertime through a combined effect of the stronger wave action and the extension of both nighttime length and nighttime duration of submergence with very shallow waters. With forced repetitive re-burrowing, R. philippinarum reduced burrowing performance, whereas the other two species either enhanced or maintained it. Thus the interspecific difference in the resistivity of juvenile clams to unstable sediments can explain their overwintering success/failure in the high to mid-tide zone. The present findings provide a new perspective for the mechanism of winter mortality of infaunal bivalves inhabiting tidal flats in temperate to boreal regions. © 2015 Elsevier B.V. All rights reserved.

1. Introduction When interference competition is removed from shallow water softsediment benthic community, some inferior competitors may succeed in enlarging local population size by increasing individual density and/or expanding distribution. A number of field experiments have revealed that some inferiors achieved recolonization by adults and/or recruitment ⁎ Corresponding author at: Faculty of Fisheries, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan. Tel.: +81 95 819 2818; fax: +81 95 819 2799. E-mail address: [email protected] (S. Takeuchi). 1 Deceased.

http://dx.doi.org/10.1016/j.jembe.2015.01.011 0022-0981/© 2015 Elsevier B.V. All rights reserved.

at plots from which superiors had been removed (Ólafsson, 1989; Peterson, 1977; Pillay et al., 2007; Posey et al., 1991; Van Colen et al., 2013; Volkenborn and Reise, 2006). Biotic factors such as predation and parasitism (Dumbauld et al., 2011; Flach and Tamaki, 2001; Takeuchi et al., 2013; Thrush et al., 1991) and abiotic factors such as extremely low or high water temperature, freshwater discharge and hydrodynamic forcing (Levin, 1984; Möller, 1986; Nakano et al., 2012; Urabe et al., 2013; Yeo and Risk, 1979) remove dominant species populations from the seabed. The removal could occur on such a large scale that an entire tidal flat is covered (Dumbauld et al., 2011; Flach and Tamaki, 2001; Levin, 1984; Takeuchi et al., 2013; Urabe et al., 2013; Yeo and Risk, 1979). The resultant competitive release would trigger

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S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

the subsequent changes in whole benthic community structure (Flach and Tamaki, 2001; Takeuchi et al., 2013). The response to competitive release is different among inferior species (Levin, 1984; Takeuchi et al., 2013; Thrush et al., 1991; Van Colen et al., 2013; Volkenborn and Reise, 2006). For instance, Volkenborn and Reise (2006) demonstrated that after the removal of an ecosystem engineer, the lugworm, Arenicola marina, from 400-m2 plots on an extensive intertidal sandflat, only one of the two inferior competitors, the deposit-feeding polychaete, Nereis diversicolor, succeeded in the recolonization there, due to its higher tolerance to the emergent anoxic and sulfide sediments. For their success in the distribution expansion following competitive release, inferior competitors should possess not only competence for rapid recolonization and recruitment (Levin, 1984; Thrush et al., 1991) but also physiological and behavioral traits for the adaptation to the newly-vacated habitat conditions (Brock, 1979; Lu and Wu, 2006; Volkenborn and Reise, 2006). Infaunal bivalves are one major taxonomic group in soft-sediment benthic community. Their burrowing ability is a key trait in determining post-(larval) settlement survival. Bivalves in the sediment column can be expelled onto the surface by abrupt sediment erosion induced by intense hydrodynamic forces (Brock, 1979; de Montaudouin, 1997; Lundquist et al., 2004; Sakurai and Seto, 1998; St-Onge and Miron, 2007). Juveniles are more vulnerable to sediment erosion than adults due to limited burrowing depths (Lundquist et al., 2004; St-Onge and Miron, 2007). In general, by re-burrowing quickly, bivalves once exposed onto the sediment surface could avoid lethal risks associated with epi-benthic predation (Gribben and Wright, 2014; Hiddink et al., 2002; Kurihara, 2003), dispersal by waves (Kakino, 2000; Norkko et al., 2001; Toba et al., 2011), and desiccation (Kurihara, 2003). Infaunal bivalves residing in unstable sediment habitats are able to burrow more quickly than those in stable sediment habitats (Alexander et al., 1993; Breum, 1970; Brock, 1979; Stanley, 1970). On an extensive intertidal sandflat in Ariake Sound, western Kyushu, Japan, both competitive release in the infaunal macrobenthic community and the subsequent distribution expansion of some inferior species populations occurred during 2004 to 2008 (Takeuchi et al., 2013). The benthic community mainly consisted of the upogebiid shrimp, Upogebia major (De Haan), the callianassid shrimp, Nihonotrypaea japonica (Ortmann), and the three venerid clams, Ruditapes philippinarum (Adams & Reeve), Mactra veneriformis Deshayes (Reeve) and Meretrix lusoria (Röding) (Nakamura et al., 2010; Nakano et al., 2012; Takeuchi et al., 2013; Tamaki et al., 2008). All species utilize phytoplankton as a major food resource (Yokoyama et al., 2005a,b). The two shrimp species inhibited the larval recruitment of the three clam species most probably through their bioturbation associated with construction and maintenance of burrows (Flach and Tamaki, 2001; Takeuchi et al., 2013; Tamaki et al., 2008). Therefore, competition for food and space would exist among the five species, with the three clams being inferior competitors (Takeuchi et al., 2013; Tamaki et al., 2008). The two shrimp populations predominated over the high to mid-tide zone of the sandflat, but they declined drastically during 2004 to 2008 (Takeuchi et al., 2013; the definition of the tide zones is given therein). We suspected that the declines were caused by hydrodynamic disturbance from a typhoon (Takeuchi et al., 2013) and by intense predation pressure from a dasyatid stingray (Takeuchi and Tamaki, 2014; Takeuchi et al., 2013). Following these events, the three clam species were recruited in a newly vacated space; nevertheless, R. philippinarum could not establish the adult population there, whereas Ma. veneriformis and Me. lusoria did (Takeuchi et al., 2013). Mechanisms for the success/failure in the population establishment remain to be examined. The sediment of the high to mid-tide zone in which the competitive release occurred is more unstable than that of the low-tide zone dominated by R. philippinarum (Yamada et al., 2010). Thus, we hypothesized that some high burrowing ability is required for these clams to survive in the high to mid-tide zone. If so, by comparing the burrowing performance among juveniles of the three clam species, we would be able to present a mechanism for the

interspecific difference in their population establishment in that zone, following the competitive release. Generally in Japanese waters including our study area, the northwesterly monsoon in winter generates strong wave action, which disturbs the substratum of the north-facing shore (Kakino, 2000; Tamaki, 1987; Yamada and Kobayashi, 2004). This perturbation can expel infaunal bivalves dwelling in the sediment onto the surface, and the exposed individuals must burrow repeatedly to cope with frequent sediment shifting. Repetitive burrowing action of bivalves might result in declining their burrowing performance due to excessive energy consumption (Keino et al., 2005), eventually losing burrowing reactions (Calvez and Guillou, 1998; Keino et al., 2005; Zwarts and Wanink, 1991). Moreover, nighttime length in the middle to high-latitude regions exceeds at least half a day in winter. The nighttime-length extension might lead to the reduction in bivalve burrowing performance, since light is known to stimulate infaunal bivalves to initiate re-burrowing (Ansell et al., 1998; Bonnard et al., 2009; Brock, 1979). Further difficulty for those bivalves inhabiting the middle to higher shore could arise from the seasonal change in the diurnal tidal-height inequality in semi-diurnal tidal regimes. Specifically in the present region, the lowest tide times occur in the daytime during spring to summer and in the nighttime during autumn to winter. Thus, the middle to higher-shore inhabitants might be more subject to very shallow waters around the lowest tide times in the winter nighttime due to strong wind-induced wave surges, experiencing the higher degrees of sediment erosion. So far there is no study relating the seasonal shift in the combined effect of wind, light/ dark and tidal conditions to the population dynamics of infaunal bivalves in tidal flats, especially concerning the cause for their winter mortality. The objective of the present study was to reveal one mechanism that can decide success/failure in the population establishment of the three dominant clam species in a zone with competitive release on the above-mentioned intertidal sandflat. Special attention was paid to winter mortality of juvenile clams. We hypothesized that the burrowing ability of juveniles of the successful species (Me. lusoria and Ma. veneriformis) was higher than that of the failed species (R. philippinarum). To test this hypothesis, we examined the following five items: (1) spatiotemporal size-frequency variations in the clam populations; (2) seasonal variations in hydrodynamic forcing, nighttime length and nighttime emergence/submergence durations of the sandflat per tide and the resultant variation in sediment erodibility, with daily day/night demarcations; (3) juvenile clam burrowing performance under natural conditions; effects of (4) light conditions and (5) reiteration of forced surface exposures on juvenile clam burrowing performance. Finally, we attempted to relate the observed distribution pattern of the three clam species on the sandflat to their juvenile burrowing abilities. 2. Materials and methods 2.1. Study area The intertidal flat for study is located in Ariake Sound, mid-western Kyushu, Japan, being sandwiched between Shirakawa and Tsuboigawa Rivers (32° 47′ N, 130° 36′ E; Fig. 1). The whole area and the maximum distance from the uppermost shore to the mean low water spring tide level (MLWS) are ca. 4.15 km2 and 2.7 km, respectively. The mean tidal ranges are 3.9 m at spring tide and 2.0 m at neap tide (mesotidal regime), with a semidiurnal tidal cycle (tidal gauge station located nearby Kumamoto Port; shown in Fig. 1B, gray-filled square point). The tidal flat can largely be divided into two parts (northern muddy part and southern sandy part), demarcated by the 1.1-km long dike constructed along Tsuboigawa River mouth. Of these, the present study was conducted on the latter part (=3.39 km2; hereafter, Shirakawa sandflat). During late autumn to early spring, the Shirakawa sandflat is subject to northwesterly wind-driven wave action and the resultant intense

S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

A

11

B

C

Fig. 1. (A,B) Study area located in the central part of Ariake Sound, western Kyushu, Japan. The meteorological observatory at Kumamoto Port is shown as a gray filled square point in Fig. 1B. (C) Shirakawa sandflat between Shirakawa and Tsuboigawa Rivers, facing northwest. The dashed lines with MLWS and MLWN indicate mean low water level at spring tide and neap tide, respectively. The water-pressure gauge was installed at Stn 1009 m (white star point). The access road is a concrete road for approaching, by vehicles, the fishery grounds of Ruditapes philippinarum on the lower shore. The sampling for clams and sediments was conducted along the transect.

sediment erosion (Yamada and Kobayashi, 2004; Yamada et al., 2007). In the other seasons, the sandflat receives minimal waves due to the predominant southwesterly winds. The surface sediments (top 1.5-cm layer) are most frequently moderately well-sorted medium sand, with median grain diameter ranging from 0.02 to 0.43 mm (S. Takeuchi et al., unpublished data). The sediment of the high to mid-tide zone is unstable (the definition for zones is given in the last paragraph), whereas that of the low-tide zone is more stable (Yamada et al., 2010). In addition, the latter sediment contains much more shell fragments (A. Tamaki et al., unpublished data) that can be used by R. philippinarum juveniles as a base for their byssus thread attachment (de Montaudouin, 1997). There are fishery grounds of R. philippinarum in the low-tide zone (Takeuchi et al., 2013; Tamaki et al., 2008). A concrete road, which was constructed in 1984 for access by vehicles transporting harvested clams, runs across the middle upper part of the sandflat from the uppermost shore toward seaward (4-m wide, 935-m long; indicated in Fig. 1C, “access road”). It was observed that the mussel, Musculista senhousia, had generated huge mats over a substantial part of the lowtide zone in September 2009 and May 2010 (see Section 3.1). The presence of well-developed mats could alter the surrounding sandy sediments into muddy ones, making a serious impact on fishery grounds of R. philippinarum. A 2129-m long transect for field survey was set on the Shirakawa sandflat from the uppermost shore toward seaward (Fig. 1C). Twentyeight stations were established on the transect, 80 m apart between two successive ones except between the uppermost-shore station and the next seaward one (49 m apart), where highly muddy sediments prevent us from moving through. Hereafter, the station X m away from the uppermost shoreline is expressed as Stn X m. According to the definition in Takeuchi et al. (2013), the transect was divided into three parts based on the elevation profile (Yamada and

Kobayashi, 2004): high-tide zone (Stns 0 m–369 m), mid-tide zone (Stns 449 m–1329 m) and low-tide zone (Stns 1409 m–2129 m). 2.2. Dominant bivalves The three venerid clam species, Ruditapes philippinarum, Meretrix lusoria and Mactra veneriformis, are dominant members of the benthic community on the Shirakawa sandflat, and their adults burrow into the sediment down to 7-cm depth (A. Tamaki et al., unpublished data). The gamete spawning periods and the minimum shell lengths for sexual maturation of these species on the sandflat are October–July and 11.3 mm for R. philippinarum (T. Nakano et al., unpublished data), May–September and 19.0 mm for Me. lusoria (Nakamura et al., 2010), and May–early July and late July–September, and 16.7 mm for Ma. veneriformis (Nakano et al., 2012). It was estimated that newlyrecruited clams [their smallest shell length (SL) defined operationally as 1 mm] occurred approximately 1–3 months after each gamete spawning event (A. Tamaki et al., unpublished data). According to the definition in Takeuchi et al. (2013), clams were categorized into three groups based on shell length: juveniles with SL ≤ 10 mm at the nonreproductive stage; adults with SL N 20 mm at the reproductive stage; and sub-adults with 10 mm b SL ≤ 20 mm at the intermediate stage. 2.3. Spatiotemporal variation in the size structure of clams To investigate the spatiotemporal variation in the body size structure of the three dominant clam species on the Shirakawa sandflat, sampling was conducted along the transect during spring low tides on 20 May, 17 June, 19 July 2008, 24 May, 24 June, 16 September 2009, and 28 May 2010 [samplings in June 2008, and May and June 2009 targeted only the high to mid-tide zone (= Stns 0 m–1329 m)]. At

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each station, two sediment samples were collected with a 25-cm × 25cm quadrat frame forced into 7-cm depth, and those retained on a 1mm mesh sieve were fixed with 10% neutralized seawater formalin. In the laboratory, all target clams were sorted and enumerated. Clam species identification was based on shell shape, ornamentation and color (Takeuchi et al., 2013). Individual density at each station was calculated by averaging the values obtained from the two samples and converted into the density per square meter. Shell length was measured to the nearest 0.01 mm (for details, see Takeuchi et al., 2013), and the length rounded to the one decimal place was used in the subsequent analysis. The temporal change in the clam spatial distributions was detected by examining the shell-length frequency distributions for respective stations over time. In particular, the following two indices were noted: (1) distribution-range length, defined as the distance (m) between the two locations at which, starting from Stn 0 m seaward, the proportional cumulative clam numbers reached 5% and 95% of the total numbers collected through the transect (linear interpolation was made between two actual stations); and (2) central habitat location, defined as the distance (m) from Stn 0 m to the location at which the above cumulative value reached 50%. These indices were calculated from the results on the sampling occasions when all stations on the transect were visited. 2.4. Mud content of surface sediments To investigate spatial variation in the mud content of surface sediments on the Shirakawa sandflat, we collected one sediment sample at each station along the transect on 16 September 2009, using a 10cm × 10-cm quadrat frame forced into 1.5-cm depth, simultaneously with the sampling for clams. The sediment grain-size composition was determined using a laser diffraction particle-size analyzer (SALD-3100, Shimadzu, Co.) or a vibratory sieve shaker with a sieve mesh-size series of 4.0, 2.8, 2.0, 1.0, 0.5, 0.25, 0.125 and 0.063 mm (AS200, Retsch, Co., Ltd.). It is impossible for the former method to analyze the sediment sample containing coarse particles N 3 mm in diameter; the latter method was applied to such cases, in which were involved desalting, drying at 75 °C for a day, removing organisms and biogenic components such as large shell fragments, sieving the sediment, and measuring each fractional sediment weight to the nearest 0.01 g. According to Wentworth (1922), “mud” and “mud content” were defined as the fine particles b 0.063 mm in diameter and its proportion to total weight (%), respectively. The mud content was not obtained directly from the measurement with the laser diffraction particle-size analyzer, which outputs proportional cumulative volumes at given particle-size classes, and thus we estimated the value using liner interpolation between the 0.057- and 0.071-mm particle-size classes. 2.5. Seasonal variation in abiotic environmental factors Seasonal variation in several abiotic factors on the Shirakawa sandflat that were considered relevant to clams' burrowing performance was examined: (1) daily nighttime/daytime length; (2) wind direction and speed; (3) water depth and fluid velocity; (4) duration of emergence/submergence per tide; and (5) potential erodibility of the sediment. Daily nighttime/daytime length was calculated as an alternate period between sunset and sunrise, using data for this location available from National Astronomical Observatory of Japan (http:// www.nao.ac.jp/). Wind direction and speed were recorded hourly at the meteorological observatory located at Kumamoto Port (Fig. 1B) by the Kumamoto Prefecture Government (http://cyber.pref.kumamoto. jp/bousai/). Water depth and fluid velocity at Stn 1009 m (mid-tide zone) were recorded continuously at a rate of 0.2 s during May 2008 to December 2009, using a water-pressure gauge (Wave Hunter 99, IO Technic Co. Ltd.; Fig. 1C, star point). On the same location and occasion, Yamada et al. (2009) also measured concentration of suspended sediment and salinity to estimate suspended sediment and water fluxes,

in which data were recorded every second for 20 s at an interval of 10 min. In accord with that data acquisition, 20-s averaged water depth and fluid velocity calculated every 10 min were used in the subsequent analysis to retain consistency between previous and present studies. The water-pressure gauge was retrieved every month for battery exchanges, bringing about a few-day missing data (the duration with no data from 24 February 2009 to 27 March was exceptionally long). The sensor for water pressure was positioned 1.4 cm above the sandflat surface. The measured pressure value was converted into the free surface elevation using linear wave theory. The duration of emergence per tide at the station with the water-pressure gauge was calculated based on the definition of emergence as b 30 cm in water level (detailed descriptions for this threshold are given in the Results, Section 3.2), in which the day/night demarcation was made according to the daily nighttime/daytime length data above. During late autumn to winter, the sandflat is subject to strong northerly-wind induced wave surges that push very shallow waters toward the upper shore around the lowest tide times. As an example of duration covered with such thin water layers, the submergence duration for the 30 cm ≤ water level b 50 cm was calculated, with the day/night demarcation. The fluid velocity at 10 cm above the seabed was recorded with the electromagnetic current meter of the water-pressure gauge. The absolute value of 20-s averaged fluid velocity is expressed as Ū. This timeaveraged values are regarded as the tidal components, but actually include the effects of wind, as the 20-s duration is not too long relative to the (2-year) average wave period of 3 s on the Shirakawa sandflat (Yamada et al., 2009). In theory, the critical water depth-averaged speed (hereafter, Ūcr) designates the fluid velocity with which a few sediment grains begin to move (Soulsby, 1997). The frequency (= number of occurrences) with Ū ≥ Ūcr was counted for every daytime and nighttime, which was defined as potential erodibility of sediment (hereafter, PES). Here, Ūcr was calculated using the following equations (Soulsby, 1997):
1 1 h 7 U cr ¼ 7 ½gðsg−1Þd50 f ðD Þ2 ; for D N 0:1; with d50 0:30 þ 0:055½1− expð−0:020D Þ and 1 þ 1:2D  1 g ðsg−1Þ 3 d50 ; D ¼ v2 f ðD Þ ¼

where h, D⁎, g, ν, sg and d50 represent water depth (m), dimensionless sediment grain diameter, gravitational acceleration (= 9.81 m s− 2), kinematic viscosity of water (=1.0 × 10−6 m2 s−1), ratio of densities of grain and water (= specific gravity of grain) and median grain diameter of sediment (m), respectively. The Ūcr was calculated for the following three sediment types, each composed of the grains with an identical diameter and specific gravity; d50 and sg with (1) 0.0003 m and 2.65 [median grain diameter of the Shirakawa sandflat sediment and specific gravity typical of medium to very coarse sand grains (Soulsby, 1997)]; (2) 0.002 m and 2.65 (very coarse sand); and (3) 0.0058 m and 1.40 (R. philippinarum juvenile-equivalent dimensions; Kakino, 2006), respectively. 2.6. Burrowing ability 2.6.1. Definition for burrowing behaviors To relate the spatiotemporal variation in the size structure of the dominant clam populations on the Shirakawa sandflat to their burrowing abilities, we recorded the burrowing performance of juvenile clams. The typical burrowing behavior is divided into the following four steps: (i) lying on the sediment surface; (ii) starting to insert its foot into the sediment; (iii) attaining firm anchorage and raising its shell vertically; and (iv) burrowing into the sediment, repeating downward motion until complete burial. In this study, the periods during (i) to

S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

(iii) and during (iii) to (iv) were defined as “pre-burrowing time” and “burrowing time”, respectively. These times were measured from the digital video images that were taken by camcorders, using the software TrakAxPC ver. 2.0.108 (http://www.trakax.com/software) to the nearest 0.01 s. The values rounded to the one decimal place were used in the subsequent analysis. To compare burrowing performance among species, burrowing rate index (Stanley, 1970; hereafter, BRI), where burrowing time was normalized to body mass, was used, because burrowing time is known to increase with shell length or weight (Bonnard et al., 2009; Selin, 1999; Stanley, 1970; see Section 3.3.1). BRI is defined as: BRI ¼

p ffiffiffiffiffiffiffiffiffiffiffi 3 Mass  100; BT

where Mass and BT are blotted wet body weight including shell mass (g) and burrowing time (s), respectively. The relationship between pre-burrowing time and body mass is generally unknown, and raw values for the former were adopted. 2.6.2. Field experiment To determine burrowing performance under natural conditions, we conducted field experiments for the burrowing performance of juvenile clams with shell length about 6 mm at Stns 529 m–769 m on the Shirakawa sandflat during daytime spring low tides on 17 June and 19 July 2008, and 24 June 2009. The summary on shell length and number of specimens of each species used in the experiment (n = 33–84) is given in Table 1. Mean pore-water temperature at 1-cm sediment depth during each field experiment was: 31.2 °C on 17 June 2008; 33.5 °C on 19 July 2008; and 27.9 °C on 24 June 2009. We suspected that the difference in seawater temperature between summer and winter by approximately 12 °C on the Shirakawa sandflat (Nakano et al., 2012) might influence both pre-burrowing and burrowing times (Selin, 1999). In winter, however, juveniles of R. philippinarum are

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available only from the low-tide zone which is exposed mostly at night (see Section 3.2); in summer, juveniles of all three species are abundant in the high-tide zone including the above-mentioned stations, which is accessible in the daytime. The weather was fine or cloudy during the experimental periods. Burrowing behaviors were recorded for juveniles placed in depressions where thin seawater remained (ca. 3cm deep), with fine sand [0.24-mm median grain diameter, 1.23 σI (sorting coefficient: Folk and Ward, 1957), and 16.0% mud content]. To avoid the fatigue of specimens due to heating associated with exposure to sunlight for a prolonged time, those collected fresh from the sediment were used (within ca. 15 min from collection). Burrowing behavior was recorded using a digital camcorder (DCR-TRV50, Sony, Co.). The camcorder was fixed above the sediment surface, with the camera lens and the seabed approximately 30 cm apart; we could obtain a sufficient image resolution to detect juvenile burrowing motions. To prevent halation due to reflected lights from the water surface, the monitoring area was shaded with a coarse-mesh black sheet. As a rule, specimens up to nine individuals were gently placed with soft tweezers on the sediment in a 3 × 3 arrangement, with three clams for each species per column. Just prior to the placement on the sediment surface, the clams were photographed on a 1-mm grid water-resistant graph paper in their same arrangement, enabling later shell-length measurement using imageJ 1.46r (http://rsbweb.nih.gov/ij/). Each video recording was started before the arrangement of specimens within the monitoring area and continued for ca. 5 min. For calculation of BRI, body mass was estimated from shell length using nonlinear regression curves (power-law models) for each species, as conducted in Takeuchi et al. (2013, fig. 5), for which both variables were measured from the clams collected at the time of field population surveys (Section 2.3; shell-length range: 3.3–10.0 mm for R. phillipinarum; 2.1–9.8 mm for Me. lusoria; and 2.7–9.9 mm for Ma. veneriformis). The following equations were obtained: Wet massR.p. = 3.662 × 10−4SL2.614 (n: number of clams = 68, R2 = 0.97, p b 0.001); Wet massMe.l. = 2.184 × 10−4SL3.042 (n = 67, R2 =

Table 1 Summary on specimens of three clam species in the field and laboratory experiments testing for burrowing behavior: n (total number of clams tested); percentage proportion of clams used for analysis to the total number tested; and mean shell length ± SD for all clams tested. BRI: burrowing rate index. For Laboratory experiment I, the subscripts at D (dark) and L (light) indicate serial numbers. Experiment

Species

n

Proportion

Field (June 2008)

R. philippinarum

66

Me. lusoria

67

Ma. veneriformis

63

R. philippinarum

33

Me. lusoria

63

Ma. veneriformis

84

R. philippinarum

76

Me. lusoria

34

Ma. veneriformis

79

R. philippinarum

40

Me. lusoria

24

Ma. veneriformis

40

R. philippinarum

31

Me. lusoria

24

Ma. veneriformis

33

Pre-burrowing time: 73% BRI: 73% Pre-burrowing time: 85% BRI: 85% Pre-burrowing time: 81% BRI: 81% Pre-burrowing time: 73% BRI: 73% Pre-burrowing time: 87% BRI: 87% Pre-burrowing time: 77% BRI: 75% Pre-burrowing time: 87% BRI: 86% Pre-burrowing time: 85% BRI: 79% Pre-burrowing time: 92% BRI: 87% Pre-burrowing time: 70, 78, 95 and 98% BRI: 38, 73, 68 and 90%; both for D1, L1, D2 and L2, respectively Pre-burrowing time: 67, 67, 100 and 100% BRI: 54, 54, 100 and 100%; both for D1, L1, D2 and L2, respectively Pre-burrowing time: 75, 80, 98 and 100% BRI: 70, 80, 93 and 100%; both for D1, L1, D2 and L2, respectively Pre-burrowing time: 68% BRI: 68% Pre-burrowing time: 54% BRI: 54% Pre-burrowing time: 76% BRI: 76%

Field (July 2008)

Field (June 2009)

Laboratory I

Laboratory II

Shell length (mm) 4.4 ± 0.8 3.2 ± 0.9 2.8 ± 0.6 7.3 ± 1.4 6.0 ± 1.1 6.6 ± 1.3 6.9 ± 1.2 6.7 ± 2.2 6.1 ± 0.6 8.7 ± 0.9 10.5 ± 2.1 8.2 ± 0.7 5.0 ± 1.5 4.2 ± 1.3 4.9 ± 1.6

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Fig. 2. Shell-length frequency distributions of three clam species for respective stations along the transect on the Shirakawa sandflat over time during May 2008 to May 2010. The clam individual densities are expressed in logarithmic scale.

S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

0.99, p b 0.001); and Wet massMa.v. = 1.785 × 10−4SL3.073 (n = 66, R2 = 0.99, p b 0.001), where Wet mass (clam species expressed as subscripts) and SL are blotted wet body mass including shell mass (g) and shell length (mm), respectively. The interspecific differences in each of pre-burrowing time and BRI were tested using non-parametric, Kruskal-Wallis test, followed by Steel-Dwass multiple-comparison test. All statistical analyses were performed using “R” (R Core Team, 2012). The mean proportions of unusable clam numbers for analysis to the total number tested due to non-burrowing within each recording time window or obscure images through all experimental occasions were: 23% for R. philippinarum; 15% for Me. lusoria; and 18% for Ma. veneriformis (for details, see Table 1). 2.6.3. Laboratory experiment I The effect of light conditions on burrowing performance of juvenile clams with shell length about 9 mm was examined in the laboratory (laboratory experiment I). The summary on shell length and number of specimens of each species used in the experiment (n = 24–40) is given also in Table 1. Specimens were collected at Stns 609 m–769 m on the Shirakawa sandflat during spring low tide on 13 July 2010 and transported to the laboratory within 9 h. The collected clams were kept in a cool box with seawater when transported. In the laboratory, the animals were transferred to a cylindrical bucket (22-cm diameter and 23-cm height) containing seawater and field-collected sediment and kept there for two days, unfed, to become acclimated to the laboratory conditions. The seawater was aerated and maintained at a mean temperature of ca. 24 °C using the room air conditioner. After the above acclimation, we conducted the experiment I, where each clam was allowed to burrow under the alternate four dark/light (D/L) conditions, 1st dark (hereafter, D1), 1st light (L1), 2nd dark (D2) and 2nd light (L2). Prior to the D1, the clam was acclimated to the dark condition for 2 h. The mean photon flux densities

15

(μmol s− 1 m− 2; measured with Compact-LW, JFE Advantech, Co.) were 0.1 (± 0.0 SD, n: number of data = 120), 15.3 (± 0.4 SD, n = 60), 0.1 (± 0.1 SD, n = 80) and 15.1 (± 0.2 SD, n = 60) for D 1, L 1, D2 and L2, respectively. The D/L conditions were controlled by switching the room light off/on. The photon flux densities (μmol s−1 m−2) under D1 and D2 were similar to the in situ nighttime light conditions, with mean and maximum of 0.1 (±0.3 SD, n = 111) and 2.0 at Stn 769 m on the Shirakawa sandflat measured during 11–25 August 2010, respectively, whereas those densities under L1 and L2 were considerably lower than the in situ daytime values, with mean and maximum of 265.0 (±301.9 SD, n = 104) and 1355.1, respectively (S. Takeuchi et al., unpublished data). Weak lights with photon flux densities b 30 μmol s−1 m−2 that were comparable to those in L1 and L2 were occasionally recorded on the sandflat at a daytime spring high tide on cloudy or rainy days. Specimens were placed in a cylindrical container (18.5-cm diameter and 20-cm height), with 6-cm deep seawater at 24.1 °C (± 0.1 SD, n = 40) and 32.8 salinity over the 8.5-cm thick sediment taken from the sandflat, which were processed as below: passing through a sieve with 2-mm mesh openings and decanting off silt and clay particles, yielding a sediment fraction of 0.3-mm median grain diameter, 0.62 σ1 and 0.3% mud content. The seawater was not aerated during the experiment to avoid agitating the water. The sediment surface of the container was equally divided into four sectors with acrylic plates, each containing one clam for each species (i.e., three species per sector). Burrowing behavior was recorded with a digital camcorder (HDR-XR500V, Sony, Co.), which can capture images under dark conditions by using infrared shooting mode. The camcorder was fixed above to cover the entire sectors. Video recording under each D/L condition was started before the specimens were placed on the sediment surface and continued for ca. 2 h. We made ten sets of containers and camcorders simultaneously. Following the completion of each recording, specimens were acclimated to

A

B

C

Fig. 3. (A,B) Huge mats with sandy mud generated by the mussel, Musculista senhousia, on the lower Shirakawa sandflat (photographed on 28 May 2010). (C) Distribution of mud (silt-clay) content of sediments along the transect on 16 September 2009.

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A

B C

D

E

F

G

H

I

S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

17

the subsequent D/L conditions for 2 h before the start of the next recording, during which they were individually maintained in the sediment in a refrigerator ice-cube tray soaked with gently aerated seawater to retain respective clam identification. Specimens' shell length and blotted wet body weight including shell mass were measured after the experiment. To detect the effect of D/L on burrowing performance (preburrowing time and BRI), we used three generalized linear mixed models (GLMMs) and a null model with random effects of individuals, assuming a gamma distribution and a log-link function: (1) GLMM with fixed effects of D/L and cumulative number of re-burrowing series (CNRB; no. = 1,.., 4); (2) GLMM with fixed effect of D/L; (3) GLMM with fixed effect of CNRB; and (4) null model with no fixed effect. The best-fit model was selected from these models based on Akaike's Information Criterion (AIC; Akaike, 1973). Model construction was made using “R” (R Core Team, 2012). The mean proportions of unusable clam numbers for analysis to the total number tested due to non-burrowing within each recording time window or obscure images through the experiment were: 24% for R. philippinarum; 20% for Me. lusoria; and 13% for Ma. veneriformis (for details, see Table 1).

before the specimen was set on the sediment surface and continued for ca. 2 h. One clam was introduced into the left edge section. The clam that had completed burrowing was immediately retrieved gently with a small stainless spoon. Then the aquarium was slid leftward so that the sediment of the adjacent section came into the camcorder view, on which the clam was gently placed. Specimens' shell length and blotted wet body weight including shell mass were measured after the experiment. To detect the effect of CNRB on re-burrowing performance, we used two models with random effects of individuals, assuming a gamma distribution and a log-link function: (1) GLMM with fixed effect of CNRB; and (2) null model with no fixed effect. The best-fit model was selected based on AIC. The proportions of unusable clam numbers for analysis to the total number tested due to non-burrowing within each recording time window or obscure images through the experiment were: 32% for R. philippinarum; 46% for Me. lusoria; and 24% for Ma. veneriformis (Table 1).

2.6.4. Laboratory experiment II The effect of reiteration of forced surface exposures on re-burrowing performance of juvenile clams with shell length about 5 mm was examined also in the laboratory (laboratory experiment II). The summary on shell length and number of specimens of each species used in the experiment (n = 24–33) is given also in Table 1. Specimens were collected at Stns 609 m–769 m on the Shirakawa sandflat during daytime spring low tides on 24 May and 24 June 2009 and transported to the laboratory in a way similar to the laboratory experiment I. The collected clams were kept in a container (54 cm long × 39 cm wide × 31 cm high) with seawater and sediment for the night, after which they were transferred to an aquarium placed inside a laboratory located by the sea and kept there until the end of the experiment [mean water temperature, 21.6 °C (±1.7 SD, n: number of data = 4465); salinity, 33.8 (±0.7 SD, n = 4465)]. The aquarium consisted of two tanks: outer one (215 cm long × 215 cm wide × 50 cm high) and inner one containing clams (100 cm long × 50 cm wide × 45 cm high). Filtered seawater passed through a cartridge filter with 10-μm mesh openings (TOCEL, Advantec,Co.) was continuously introduced into the outer tank. The seawater running through the outer tank was introduced into the inner tank using a pump that operated automatically during 21:00–9:00 every day. To feed the clams, we introduced 1.6 × 105 cells of the microalga, Chaetoceros gracilis, into the inner tank at 9:00. For the experiment, those clams that had been treated above for at least five days were used. Each clam took a series of 15-time forced re-burrowing under light conditions with mean photo flux density of 14.1 μmol s− 1 m− 2 (± 0.1 SD, n = 122). The aquarium for the experiment was a rectangular parallelepiped made of thin acrylic plates, with a 90-cm long × 10-cm high frontal plate and a 4-cm wide side plate. The inside was filled with 4-cm deep seawater with 34.0 salinity and the 4-cm thick sediment prepared by the same method as in the laboratory experiment I (0.3-mm median grain diameter, 0.53 σI and 0.04% mud content). The aquarium was equipped with four underside corner wheels for smooth lateral movement. The frontal plate of the aquarium was equally divided into 15 sections with vertical lines. Water temperature was maintained at a mean of 21.3 °C (± 0.3 SD, n = 284) using the room air conditioner. Burrowing behavior was laterally recorded with a digital camcorder (DCRTRV50, Sony, Co.) fixed on a tripod. Each recording was started

3.1. Spatiotemporal variation in the size structure of clams

3. Results

In the Shirakawa sandflat benthic community, the patterns of survival process in the high to mid-tide zone (= Stns 0 m–1329 m) before and after the winter each year were different among the three dominant clam species (Fig. 2). Although the distribution range of juveniles of R. philippinarum spread over the whole intertidal gradient, that of subadults and adults was mostly limited to the low-tide zone (= Stns 1409 m–2129 m). By contrast, the distribution ranges of all three growth stages of the other two species extended over the whole intertidal zone. The mean distribution-range lengths (± SD, n: number of sampling occasions) in juvenile, sub-adult and adult, and their reduction rate from juvenile to adult stages were: 1478 m (± 88, n = 4), 676 m (± 562, n = 4), 671 m (± 213, n = 4) and 54.6% for R. philippinarum; 1228 m (± 210, n = 4), 971 m (± 133, n = 4), 1162 m (± 268, n = 4) and 5.4% for Me. lusoria; and 1269 m (± 257, n = 4), 903 m (± 311, n = 3), 1091 m (± 311, n = 4) and 14.1% for Ma. veneriformis, respectively. The mean central habitat locations (± SD, n) in juvenile, sub-adult and adult were: 1459 m (± 352, n = 4), 1466 m (± 350, n = 4) and 1610 m (± 242, n = 4) for R. philippinarum; 700 m (± 149, n = 4), 667 m (± 256, n = 4) and 690 m (± 178, n = 4) for Me. lusoria; and 755 m (± 142, n = 4), 800 m (±257, n = 3) and 1061 m (±143, n = 4) for Ma. veneriformis, respectively. Juveniles of R. philippinarum that had been recruited in the high to mid-tide zone were observed until autumn (e.g., 16 September 2009), but most of them disappeared in the next spring. Exceptionally, during September 2009 to May 2010, (sub-)adults of R. philippinarum which were distributed in the low-tide zone almost disappeared (Fig. 2). Their mean individual density (± SD, number of stations = 10) in May 2008 was 130.4 (±177.6) inds m−2, whereas that in May 2010 was 6.4 (±20.2) inds m−2. In the meantime, huge mats generated by the mussel, Musculista senhousia, and accumulated mud were observed also over a middle part of the low-tide zone (Stns 1569 m– 1809 m; Fig. 3). The mud contents at the mussel-invaded stations were remarkably higher (mean 62.6%) than those at the other stations (mean 2.3%). Mean individual density (± SD, number of stations = 4) of juveniles of R. philippinarum at Stns 1569 m–1809 m after the mussel mat formation was much lower (40.0 ± 13.1 inds m− 2) than before (722.0 ± 801.9 inds m−2).

Fig. 4. Seasonal variation in abiotic environmental factors on the Shirakawa sandflat. (A) Percentage proportions of monthly cumulative wind directions (16 divisions) with monthly averaged speeds at Kumamoto Port during 2008 to 2009 (http://cyber.pref.kumamoto.jp/bousai/). (B) Time series of 20-s averaged, absolute fluid velocity, Ū, calculated for a 10-min interval at Stn 1009 m on the Shirakawa sandflat from May 2008 to December 2009, with the large values (N0.75 m s−1) eliminated as noises, as indicated in the overall fluid-velocity frequency distribution (C). Seasonal variation at Stn 1009 m in daily (D) nighttime/daytime-length proportions, (E) emergence duration, (F) submergence duration with a thin water layer (30 cm ≤ water level b 50 cm) and (G–I) potential erodibility of three types of sediments, each with an identical grain-diameter and specific-gravity composition. In (E)–(I), the nighttime and daytime occupancies are demarcated.

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S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

3.2. Seasonal variation in abiotic environmental factors The northwest-facing Shirakawa sandflat is subject to intense wave action in the wintertime. Northerly (northerly–west-northwesterly) winds were predominant in this season (Fig. 4A). For instance, the proportion of these winds to the total in late autumn to winter [November– February (hereafter, collectively termed winter); 44.2%] was about double that in summer (June–August; 24.5%), and the monthly averaged wind speed (from all directions inclusive) in winter (mean ± SD = 5.6 ± 0.5 m s−1, n: number of months through the two years = 8) was also higher than in summer (4.0 ± 0.4 m s−1, n = 6). The maximum of the monthly averaged northerly wind speed observed in February 2008 was 7.4 m s−1. At the water-level monitoring station (Stn 1009 m; Fig. 1C, star point), the fluid velocity sensor recorded noises with relatively large values mainly during the periods with the low water levels less than 30 cm [i.e., Ū N 0.75 m s−1 (= mean + SD for all possible “normal” and “abnormal” measurements inclusive)], accounting for 6% of the total measurements (Fig. 4B,C). The occurrence of these values suggests temporally abrupt changes in very shallow water depths, and thus they were eliminated from the subsequent analysis. Here, the length of any period with water levels b 30 cm is broadly defined as the duration of emergence of the sandflat at Stn 1009 m. Overall, the calculation of Ū for the submergence period was made for 77% of all recorded data. After summer, the low Ū values began to increase in mid-October. The higher values appear to be associated with the stronger northerly winds in winter. The monthly averaged value in winter (mean ± SD = 0.12 ± 0.02 m s−1, n: number of months = 6) was double that in summer (0.07 ± 0.01 m s− 1, n = 6). The maximum value (0.74 m s−1) was recorded in January, 2009. The proportion of daily nighttime length exceeded 50% during 27 September 2008 to 17 March 2009, with the maximum (58.3%) on 21 December 2008 (Fig. 4D). Stn 1009 m emerged mainly during daytime low tides in summer and during nighttime low tides in winter, primarily reflecting the seasonal reversal of diurnal tidal-height inequality patterns (Fig. 4E). Daily averaged emergence duration and the nighttime occupancy in each daily total were: 7.7 h (±1.3 SD, n: number of days = 132) and 30.8% (±8.2 SD) in summer; and 6.8 h (±2.1 SD, n = 90) and 63.5% (±14.7 SD) in winter, respectively. Daily averaged duration of submergence with a thin water layer (30 cm ≤ water level b 50 cm) and the nighttime occupancy in each daily total were: 1.4 h (± 0.6 SD, n = 151) and 49.0% (±15.2 SD) in summer and 4.5 h (±3.5 SD, n = 144) and 57.2% (± 17.2 SD) in winter, respectively (Fig. 4F). Such greater daily submergence duration in winter is attributable to northerly wind-induced wave surges that push thin water layers toward the upper shore, even overcoming ebb tidal current forces (S. Takeuchi et al., personal observation). The greater proportion of the nighttime occupancy in the daily duration of submergence with very shallow waters in winter is attributable to the extension of both nighttime length and nighttime emergence duration. All these physical settings peculiar to winter can cause greater erosion of the surface sediment of the sandflat than in the other seasons. In fact, potential erodibility of sediment (PES) was enhanced sharply in the winter nighttime (Fig. 4G–I). In the case with 0.0003-m sediment median grain diameter and 2.65 specific gravity, the daily integrated PES (including both day and night) was much higher in winter than in summer [mean ± SD per day, 7.05 ± 11.59 (n: number of days = 164) vs. 0.04 ± 0.22 (n = 184); Fig. 4G]. The maximum value (60 per day) was recorded on 3 January 2009, and through this month, the mean nighttime occupancy in each daily PES was 70.8% (± 27.7 SD, n = 25). Similar seasonally changing patterns were observed for the other two sediment types (Fig. 4H,I). 3.3. Burrowing ability 3.3.1. Field experiment The relationship between pre-burrowing time and body mass of clams was different among species. Pre-burrowing time was not

A

B

Fig. 5. Field experiments. (A) Pre-burrowing time and (B) burrowing rate index for three clam species. The box plots display medians (line in boxes), 25th and 75th percentiles (lower- and upper-sides of box), 10th and 90th percentiles (error bars), and outliers (circles). Significant differences detected by Steel-Dwass multiple-comparison test are indicated; NS (non-significant); *** (p b 0.001); ** (0.001 b p b 0.01); * (0.01 b p b 0.05).

correlated with body mass for R. philippinarum (r = − 0.04, p N 0.05, n: number of specimens = 138) and Me. lusoria (r = 0.09, p N 0.05, n = 141), whereas a weak positive correlation was detected for Ma. veneriformis (r = 0.29, p b 0.001, n = 189) (plots of data not shown). Because of this interspecific inconsistency, no normalization of preburrowing time to body mass was made, and the raw data for preburrowing time were used. On the other hand, burrowing time was positively correlated with body mass for all clam species (R. philippinarum, r = 0.25, 0.001 b p b 0.01, n = 137; Me. lusoria, r = 0.26, 0.001 b p b 0.01, n = 139; and Ma. veneriformis, r = 0.42, p b 0.001, n = 183) (plots of data not shown), and thus it is appropriate to use BRI for the interspecific comparison. The interspecific order in pre-burrowing time was not consistent through the three experimental occasions. The interspecific differences were significant in June 2008 and June 2009 (Kruskal-Wallis test, p b 0.001; Fig. 5A) and non-significant in July 2008 (p N 0.05). The Steel-Dwass multiple comparison test detected significantly shorter pre-burrowing time for Ma. veneriformis than for the other two species in June 2008 and for R. philippinarum than for Ma.veneriformis in June 2009. The median pre-burrowing times (first–third quartiles) for R. philippinarum, Me. lusoria and Ma. veneriformis through all experiments were 66.0 s (31.7–117.3), 71.0 s (34.3–164.4) and 64.6 s (33.0–120.2), respectively. The interspecific order in BRI was consistent

S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

through the three experimental occasions. The differences were significant on all occasions (Kruskal-Wallis test, p b 0.001; Fig. 5B). Ma. veneriformis was the most rapid burrower (Steel-Dwass multiple comparison tests, 0.001 b p b 0.01), followed by Me. lusoria and R. philippinarum in order. The median BRIs (first–third quartiles) for R. philippinarum, Me. lusoria and Ma. veneriformis were 0.61 (0.44– 0.74), 0.70 (0.51–1.02) and 1.40 (0.80–2.12), respectively. According to the definition in Stanley (1970), R. philippinarum and Me. lusoria can be classified as “moderately rapid” burrowers and Ma. veneriformis as a “moderately rapid or rapid” burrower. 3.3.2. Laboratory experiment I The pre-burrowing time of the three clam species was strongly affected by dark/light (D/L) conditions and weakly by increment in cumulative number of re-burrowing (CNRB; no. = 1,.., 4) (Fig. 6A and Table 2). The generalized linear mixed model (GLMM) with fixed effects of D/L and CNRB was accepted as best-fit for each species (Table 2). Each best-fit model indicates that the pre-burrowing time is shorter under light than under dark conditions, slightly decreasing with CNRB; mean pre-burrowing times under dark (D1 and D2) and light (L1 and L2) were: 355.4 and 51.1 s for R. philippinarum; 233.6 and 67.2 s for Me. lusoria; and 715.0 and 182.5 s for Ma. veneriformis, respectively. The pre-burrowing time exhibited greater intraspecific difference under dark than under light conditions (Fig. 6A); the standard deviations under D1, L1, D2 and L2 were: 591.5, 90.5, 450.3 and 254.6 s for R. philippinarum; 461.7, 236.8, 250.2 and 34.3 s for Me. lusoria; and 1104.0, 495.6, 539.3 and 224.9 s for Ma. veneriformis, respectively. The BRIs of the three clam species were also affected by D/L conditions (Fig. 6B and Table 2). The GLMM with fixed effect of D/L was accepted as best-fit for each species (Table 2). Each best-fit model indicates that BRI is lower under dark than under light (Fig. 6B); mean BRIs under dark and light were: 0.14 and 0.28 for R. philippinarum; 0.47 and 0.66 for Me. lusoria; and 0.76 and 1.40 for Ma. veneriformis, respectively.

A

B

Fig. 6. Laboratory experiment I. (A) Pre-burrowing time and (B) burrowing rate index for three clam species in response to alternate dark/light conditions: D1 [1st dark, with cumulative number of re-burrowing (CNRB) = 1]; L1 (1st light, with CNRB = 2); D2 (2nd dark, with CNRB = 3); and L2 (2nd light, with CNRB = 4). The box plots display medians, 25th and 75th percentiles, 10th and 90th percentiles, and outliers as in Fig. 5.

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The interspecific difference in each of pre-burrowing time and BRI was statistically tested. The differences in pre-burrowing time were significant in all D/L conditions (Kruskal-Wallis test, 0.01 b p b 0.05). In most cases, Ma. veneriformis specimens started burrowing latest among the three clam species; the median pre-burrowing times (first–third quartiles) in D1, L1, D2 and L2 were: 537.2 s (196.8–958.9), 31.2 s (23.0–47.1), 173.0 s (83.5–297.1) and 33.4 s (21.6–61.0) for R. philippinarum; 207.0 s (98.0–448.6), 62.4 s (35.0–102.6), 159.6 s (96.3–343.7) and 47.0 s (28.5–77.5) for Me. lusoria; and 775.9 s (291.3–1580.2), 193.4 s (54.4–523.2), 517.1 s (228.0–657.5) and 90.7 s (34.9–244.7) for Ma. veneriformis, respectively (Fig. 6A). The Steel-Dwass multiple comparison test detected significant differences between: Ma. veneriformis N Me. lusoria in D1 (0.01 b p b 0.05); R. philippinarum b the other two species in L1 (0.01 b p b 0.05); Ma. veneriformis N the other two species in D2 (p b 0.001); and Ma. veneriformis N R. philippinarum in L2 (0.01 b p b 0.05). The differences in BRI were also significant in all cases (Kruskal-Wallis test, 0.01 b p b 0.05). In most cases, R. philippinarum was the slowest burrower among the three clam species; the median BRIs (first–third quartiles) in D1, L1, D2 and L2 were: 0.18 (0.07–0.29), 0.31 (0.24–0.43), 0.17 (0.09–0.22) and 0.30 (0.21–0.40) for R. philippinarum; 0.48 (0.39–0.63), 0.85 (0.40–1.11), 0.52 (0.38–0.70) and 0.70 (0.40–0.99) for Me. lusoria; and 0.43 (0.10–0.96), 1.68 (0.28–2.36), 0.78 (0.40–1.31) and 1.62 (0.66–2.18) for Ma. veneriformis, respectively (Fig. 6B). The Steel-Dwass multiple comparison test detected significant differences between: R. philippinarum b Me. lusoria in D1 (0.001 b p b 0.01); R. philippinarum b the other two species in L1 (0.001 b p b 0.01) and in D2 (p b 0.001); and R. philippinarum b Me. lusoria b Ma. veneriformis in L2 (0.001 b p b 0.01). 3.3.3. Laboratory experiment II The raw data used for the analysis of experimental results are given in Appendix A. The pre-burrowing time was affected by CNRB (no. = 1,.., 15) in different ways among the three clam species (Fig. 7A); with CNRB, the time was prolonged for R. philippinarum and shortened for Me. lusoria and Ma. veneriformis. The GLMM with fixed effect of CNRB was accepted as best-fit for each species (Table 3). Each best-fit model indicates that the mean pre-burrowing times vary with CNRB: from 69.3 (no. 1) to 103.5 (no. 15) s for R. philippinarum, from 98.8 to 28.3 s for Me. lusoria, and from 101.6 to 12.1 s for Ma. veneriformis. The BRIs were also affected by CNRB for R. philippinarum and Me. lusoria but not for Ma. veneriformis (Fig. 7B); with CNRB, BRI decreased for R. philippinarum and increased for Me. lusoria. The GLMM with fixed effect of CNRB was accepted as best-fit for R. philippinarum and Me. lusoria, and the null model was accepted for Ma. veneriformis (Table 3). Each best-fit model indicates that the mean BRIs of the former two species vary with CNRB: from 0.52 (no. 1) to 0.38 (no. 15) for R. philippinarum and from 0.81 to 0.91 for Me. lusoria; it is constant at 1.30 for Ma. veneriformis. The interspecific difference in each of pre-burrowing time and BRI was tested in CNRB no. 1 and CNRB no. 15. The differences in pre-burrowing time were significant in both CNRB numbers (Kruskal-Wallis test, 0.01 b p b 0.05). Ma. veneriformis specimens started burrowing earliest among the three clam species; the median pre-burrowing times (first–third quartiles) in no. 1 and no. 15 were: 45.2 s (23.8–93.5) and 85.6 s (64.0–122.5) for R. philippinarum; 48.6 s (32.8–74.4) and 25.2 s (18.1–37.5) for Me. lusoria; and 18.3 s (7.2–42.1) and 10.2 s (7.0–13.0) for Ma. veneriformis, respectively. The Steel-Dwass multiple comparison test detected significant differences between: Ma. veneriformis b the other two species in no. 1 (0.01 b p b 0.05); and Ma. veneriformis b Me. lusoria b R. philippinarum in no. 15 (p b 0.001). The differences in BRI were also significant in all cases (Kruskal-Wallis test, p b 0.001). R. philippinarum was the slowest burrower among the three clam species; the median BRIs (first–third quartiles) in no. 1 and no. 15 were: 0.39 (0.33–0.59) and 0.36 (0.30–0.51) for R. philippinarum; 0.79 (0.68–1.03) and 1.00

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Table 2 Laboratory experiment I. Three generalized linear mixed models (GLMMs) and null model used to detect effects of dark/light (D/L) conditions and cumulative number of re-burrowing (CNRB; no. = 1,.., 4) on pre-burrowing time and burrowing rate index (BRI) for three clam species. The case with no fixed effect is listed as “null”. Akaike's information criterion (AIC) for each model is indicated; Δ AIC means residual from AIC of the best-fit model. Species

Response variable (y)

R. philippinarum Pre-burrowing time

BRI

Me. lusoria

Pre-burrowing time

BRI

Ma. veneriformis Pre-burrowing time

BRI

Fixed effects

Random effect

Model name

Equation

D/L, CNRB

Individuals

GLMM 1

If D/L = “dark”, y = exp(6.1284 − 0.5448 × ln CNRB) 1647.21 If D/L = “light”, y = exp(6.1284 − 1.6464 − 0.5448 ×

D/L

Individuals

GLMM 2

CNRB Null D/L, CNRB

Individuals Individuals Individuals

GLMM 3 Null model GLMM 1

D/L

Individuals

GLMM 2

CNRB Null D/L, CNRB

Individuals Individuals Individuals

GLMM 3 Null model GLMM 1

D/L

Individuals

GLMM 2

CNRB Null D/L, CNRB

Individuals Individuals Individuals

GLMM 3 Null model GLMM 1

D/L

Individuals

GLMM 2

CNRB Null D/L, CNRB

Individuals Individuals Individuals

GLMM 3 Null model GLMM 1

D/L

Individuals

GLMM 2

CNRB Null D/L, CNRB

Individuals Individuals Individuals

GLMM 3 Null model GLMM 1

D/L

Individuals

GLMM 2

CNRB Null

Individuals Individuals

GLMM 3 Null model

(0.73–1.20) for Me. lusoria; and 1.42 (0.82–2.04) and 1.19 (0.95–1.48) for Ma. veneriformis, respectively. The Steel-Dwass multiple comparison test detected significant differences between: R. philippinarum b Me. lusoria b Ma. veneriformis in no. 1 (0.01 b p b 0.05); and R. philippinarum b the other two species in no. 15 (p b 0.001). 4. Discussion In a number of infaunal bivalves, newly-recruited densities were not necessarily reflected on adult numerical or biomass densities depending on post-recruitment survival processes (Beukema et al., 2010; Dethier et al., 2012; Seitz, 2011). On the Shirakawa sandflat, the success/failure in the population establishment of three clam species (R. philippinarum, Me. lusoria and Ma. veneriformis) following the competitive release seemed to be decided by the survival process of juveniles in the first winter after recruitment (Fig. 2). Juveniles of the two successful species (Me. lusoria and Ma. veneriformis) recruited in the above-mentioned vacated habitat overwintered and grew into the (sub-)adult stage, whereas juveniles of the failed species (R. philippinarum) mostly disappeared in the next spring. By contrast, R. philippinarum juveniles recruited in

ln CNRB) If D/L = “dark”, y = exp(5.8332) If D/L = “light”, y = exp(5.8332 − 1.9558) y = exp(6.1870 − 1.2731 × ln CNRB) y = exp(5.5445) If D/L = “dark”, y = exp(−2.04361 + 0.05577 × ln CNRB) If D/L = “light”, y = exp(−2.04361 + 0.70745 +

AIC

1656.73

0.2510 × ln CNRB) If D/L = “dark”, y = exp(−0.2768) If D/L = “light”, y = exp(−0.2768 + 0.6160) y = exp(−0.3531 + 0.4682 × ln CNRB) y = exp(−0.16371)

Accepted

9.51

0

4.22

981.51 998.20 13.53

29.87 46.56 2.00

0

13.08

2011.76 2047.07 316.30

31.05 66.36 0.08

323.95 327.95

Accepted

10.34 10.38 0 Accepted

1993.79

316.22

Accepted

31.68 35.37 0 Accepted

955.86

ln CNRB) If D/L = “light”, y = exp(−0.7437960 + 0.3238736 − 0.0008222 × ln CNRB) 11.53 If D/L = “dark”, y = exp(−0.74446) If D/L = “light”, y = exp(−0.74446 + 0.32356) y = exp(−0.7193 + 0.1503 × ln CNRB) 21.87 y = exp(−0.56786) 21.90 If D/L = “dark”, y = exp(6.8763 − 0.6760 × ln CNRB) 1980.71 If D/L = “light”, y = exp(6.8763 − 0.9937 − 0.6760 × ln CNRB) If D/L = “dark”, y = exp(6.4702) If D/L = “light”, y = exp(6.4702 − 1.2953) y = exp(6.8240 − 1.0656 × ln CNRB) y = exp(6.0585) If D/L = “dark”, y = exp(−0.4825 + 0.2510 × ln CNRB) If D/L = “light”, y = exp(−0.4825 + 0.5283 +

0

Best-fit model

1724.73 77.51 1770.31 123.10 –134.45 1.81

0.05577 × ln CNRB) If D/L = “dark”, y = exp(−1.99653) –136.25 If D/L = “light”, y = exp(−1.99653 + 0.72519) y = exp(−1.8035 + 0.3317 × ln CNRB) –104.58 y = exp(−1.4646) –100.89 If D/L = “dark”, y = exp(5.6648 − 0.4364 × ln CNRB) 951.64 If D/L = “light”, y = exp(5.6648 − 1.0149 − 0.4364 × ln CNRB) If D/L = “dark”, y = exp(5.3469) If D/L = “light”, y = exp(5.3469 − 1.1828) y = exp(5.6495 − 0.8346 × ln CNRB) y = exp(4.939) If D/L = “dark”, y = exp(−0.7437960 − 0.0008222 ×

Δ AIC

0

Accepted

7.73 11.73

the low-tide zone overwintered to reach the (sub-)adult stage. As with some other infaunal bivalves (e.g., Cerastoderma edule, Macoma balthica and Me. lusoria), active migration of juveniles and/or subadults from the high to mid-tide zone to the low-tide zone might explain the decrease in the R. philippinarum population in the former zone (de Montaudouin, 1997; Hiddink et al., 2002; Nakamura, 2013). However, this is unlikely to occur in R. philippinarum, which adopts an anti-dispersal strategy adhering to small hard substrata such as shell fragments and live clam surfaces buried in the sediment (de Montaudouin, 1997; S. Takeuchi et al., personal observation). Thus the success/failure in population persistence in the high to mid-tide zone is most likely to be explained by the site- and species-specific winter mortality. Exceptionally, most of the adult populations of the three clam species in the low-tide zone disappeared during September 2009 to May 2010 (Fig. 2). In the middle part there, huge mats generated by the mussel, Musculista senhousia, had accrued to accumulate mud most probably transported from the Shirakawa River during the preceding rainy season (Yamada and Kobayashi, 2004; Fig. 3). Well-developed mussel beds create anoxic and sulfide conditions (Creese et al., 1997; Crooks, 1998; Ito and

S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

21

Fig. 7. Laboratory experiment II. Curves (solid lines) representing the best-fit models for pre-burrowing time and burrowing rate index for three clam species versus re-burrowing series (based on raw data given in Appendix A). The gray and light gray bands about the solid line indicate 50% and 90% confidence intervals, respectively.

Kajihara, 1981), which have damaged several fishery grounds of R. philippinarum in Japanese waters (Ito and Kajihara, 1981; Sugawara et al., 1961; Uchida, 1965). On the Shirakawa sandflat, the sediments of the high to mid-tide zone are unstable (Yamada et al., 2007), which would be unsuitable for juveniles of R. philippinarum to survive in (Breber, 2002; Ponurovskii, 2000, 2008; Yamada et al., 2007). By contrast, the sediment of the low-tide zone is stable and contains more shell fragments (Yamada et al., 2010), which can be used by R. philippinarum juveniles as a base for their byssus thread attachment to prevent dispersal (de Montaudouin, 1997). Yamada et al. (2007) used a three-dimensional

laser scanner technique to detect a change between two consecutive dates in winter for wave-generated sediment ripples in the low-tide zone (R. philippinarum-dominant area) and the mid-tide zone (nondominant area) on the Shirakawa sandflat. They found that the former surface exhibited a flatter and lower relief and a smaller daily change. Vertical displacement of ca. 10-mm thick sediment could erode juvenile clams with shell lengths ≤ 10 mm. The proportion of ripples with wave heights N 10 mm increased by ca. 6% and ca. 18% per day in the R. philippinarum-dominant area and the non-dominant area, respectively. The habit of attaching to shell fragments by R. philippinarum juveniles would not function efficiently in the high to mid-tide zone, of which

Table 3 Laboratory experiment II. Generalized linear mixed model (GLMM) and null model used to detect effects of re-burrowing cumulative number (CNRB; no. = 1,.., 15) on pre-burrowing time and burrowing rate index (BRI) for three clam species. The case with no fixed effect is listed as “null”. Akaike's information criterion (AIC) for each model is indicated; Δ AIC means residual from AIC of the best-fit model. Species

Response variable (y)

Fixed effects

Random effect

Equation

AIC

Δ AIC

Best-fit model

R. philippinarum

Pre-burrowing time

CNRB Null CNRB Null CNRB Null CNRB Null CNRB Null CNRB Null

Individuals Individuals Individuals Individuals Individuals Individuals Individuals Individuals Individuals Individuals Individuals Individuals

y = exp(4.23859 + 0.14819 × ln CNRB) y = exp(4.52012) y = exp(−0.6625 − 0.1092 × ln CNRB) y = exp(−0.86336) y = exp(4.5926 − 0.4609 × ln CNRB) y = exp(3.8013) y = exp(−0.21158 + 0.04327 × ln CNRB) y = exp(−0.13061) y = exp(4.62077 − 0.78604 × ln CNRB) y = exp(3.2905) y = exp(0.28510 − 0.01387 × ln CNRB) y = exp(0.25927)

3306.49 3318.29 –190.57 –180.49 1776.32 1846.80 –46.43 –44.62 3211.36 3318.61 502.98 501.31

0 11.80 0 10.08 0 70.47 0 1.80 0 107.26 1.67 0

Accepted

BRI Me. lusoria

Pre-burrowing time BRI

Ma. veneriformis

Pre-burrowing time BRI

Accepted Accepted Accepted Accepted

Accepted

22

S. Takeuchi et al. / Journal of Experimental Marine Biology and Ecology 466 (2015) 9–23

sediment had lower shell fragment contents (Yamada et al., 2010). There, some high burrowing ability would be required for juvenile clams to maintain themselves in the sediment. It has been reported that high burrowing ability is a critical trait for infaunal bivalves to survive in unstable sediment habitats (Alexander et al., 1993; Breum, 1970; Brock, 1979; Kakino, 2000; Sakurai and Seto, 1998; Stanley, 1970). The laboratory experiments of the present study have clearly demonstrated that BRI of the two successful clam species (Me. lusoria and Ma. veneriformis) was higher than that of R. philippinarum (Figs. 6 and 7). A similar tendency was observed in the field experiment (Fig. 5B). Regarding pre-burrowing time, however, the interspecific order was not consistent (Figs. 5–7). This might be ascribed to a size-dependent pre-burrowing time for Ma. veneriformis specimens used in the experiments; pre-burrowing time was weakly positively correlated with body mass only for this species (Section 3.3.1). A similar tendency was recorded for the clam, Macoma balthica (see Tallqvist, 2001). In the present study, Ma. veneriformis started burrowing earliest among the three clam species on the first field experimental occasion and in laboratory experiment II, in which the specimens' mean shell length was smaller than those in the other cases (Table 1). On the contrary, this species started burrowing latest on the third field experimental occasion and in laboratory experiment I, in which the specimens' mean shell length was greater than those in the other cases. Thus, it is inconclusive now to relate the interspecific order in pre-burrowing time to the field abundance pattern of the three clam species. The nighttime length in the middle to high-latitude regions exceeds at least half a day in winter. This would lead to the reduction in bivalve burrowing performance, as the time to start light-induced re-burrowing behavior is suppressed or prolonged (Ansell et al., 1998; Bonnard et al., 2009; Brock, 1979). In the present study, the burrowing performance of the three clam species was affected by the dark/light (D/L) conditions (Fig. 6). Infaunal bivalves in estuarine and coastal shallow waters possess photoreceptive cells in their mantle and siphon tip (Morton, 2008) and respond to D/L changes by closure/retraction of siphon and valve adduction (Wilkens, 2008) and by emerging on the sediment surface (Ansell et al., 1998; Richardson et al., 1993). Our results revealed that juvenile clams of the three species responded to light stimulations, shortening pre-burrowing time and enhancing BRI. Such behavioral trait of clams will decrease the risk of encountering with epi-benthic predators. Other marine infauna exhibit similar behavioral responses to light conditions [polychaetes (Lee et al., 2004), bivalves (Hiddink et al., 2002), sea cucumbers (Mercier et al., 1999)]. From the viewpoint of winter mortality of the three clam species on the Shirakawa sandflat, their lowered burrowing performance under dark conditions would increase vulnerability to increased sediment erosion caused by a combined seasonal effect of the stronger wave action and the extension of both nighttime length and nighttime duration of submergence with very shallow waters (Fig. 4). On stormy days in winter on the Shirakawa sandflat, clams exposed on the surface must burrow repeatedly to cope with shifting surface sediment. The frequent re-burrowing might result in reducing burrowing performance of clams due to excessive energy consumption (Keino et al., 2005). In fact, the increasing cumulative number of re-burrowing (CNRB) deteriorated the burrowing performance of R. philippinarum (Fig. 7). Similar tendencies for infaunal bivalves were reported by Breum (1970) and Selin (1999), as described as “fatigue” or “tiredness”. Keino et al. (2005) demonstrated for R. philippinarum that glycogen content and condition factor decreased with CNRB. We suspect that the fatigue-like phenomenon observed for the present R. philippinarum could be caused by energy consumption. Moreover, the burrowing depth of this species might become shallower with CNRB, due to decreasing body mass associated with energy consumption (Calvez and Guillou, 1998; Keino et al., 2005; Zwarts and Wanink, 1991), which may make juvenile clams more vulnerable to sediment erosion. The frequent exposures to sediment surface would cause juveniles of R. philippinarum to be plunged into a negative spiral, accelerating subsequent exposures. By

contrast, the two successful species (Me. lusoria and Ma. veneriformis) either enhanced or maintained their burrowing performance with CNRB (Fig. 7). In particular, it is notable that BRI of Me. lusoria was not significantly different from that of Ma. veneriformis in CNRB no. 15 in spite of a significantly lower value for the former species in CNRB no. 1 (Section 3.3.3). These traits would enable the two clam species to resist frequent sediment erosion, especially in the wintertime. Finally, for future research, it must be pointed out that the examination of the following two items is required to further support the present “burrowing ability hypothesis”: (1) how the clam behaviors observed at the present summer high temperatures vary at winter low temperatures and (2) how the difference in submergence duration per tide between high to mid-tide zone and low-tide zone (means of 6.3 to 8.1 h vs. 9.2 h; Yamada et al., 2012, table 1) affect the survival of overwintering clams. The natural expanse of the uppermost Shirakawa sandflat is cut with a concrete wall at the present uppermost shoreline (Fig. 1C), which is ca. 40 cm above the mean sea level and ca. 40 cm below mean high water neap tide level (Yamada et al., 2012). Thus the submergence duration may be a limiting factor for growth but not for survival. In conclusion, burrowing ability can be one key trait for success/ failure in the population establishment of clam species in an area with shifting sediment on intertidal sandflats. The negative effect of the winter environmental conditions on the burrowing performance of juvenile clams can induce mortality in the unstable sediment. Specifically on the Shirakawa sandflat, the difference in burrowing ability between the successful and failed species was one possible critical cause for the determination of their population dynamics and community dominance following the competitive release from the bioturbating shrimp. The present findings provide a new perspective for the mechanism of winter mortality of infaunal bivalves inhabiting tidal flats in temperate to boreal regions. Acknowledgements We thank the staff of Oshima Fisheries Cooperative Associations for allowing sampling on the Shirakawa sandflat, and S. Mandal, Y. Agata, Y. Takahara, T. Nakano, Y. Saitoh, S. Sen-ju, W. Jinno and A. Inomata for assistance with field and/or laboratory works. We also appreciate numerous constructive comments and corrections from the two anonymous reviewers. Regrettably, one of the authors, F. Yamada, passed away while the paper was under review. This paper will be in memory of our friendships for years. This research was partly supported by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research 19310148 to AT. [ST]. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jembe.2015.01.011. References Akaike, H., 1973. Information theory and an extension of the maximum likelihood principle. In: Petrov, B.N., Csáki, F. (Eds.), Proceedings of the Second International Symposium on Information Theory. Akadémiai Kiadó, Budapest, pp. 267–281. Alexander, R.R., Stanton Jr., R.J., Dodd, J.R., 1993. Influence of sediment grain size on the burrowing of bivalves: correlation with distribution and stratigraphic persistence of selected neogene clams. Palaios 8, 289–303. Ansell, A.D., Günther, C.-P., Burrows, M.T., 1998. Partial emergence of the bivalve Donax vittatus in response to abrupt changes in light intensity and before spawning. J. Mar. Biol. Assoc. UK 78, 669–672. Beukema, J.J., Dekker, R., Philippart, C.J.M., 2010. Long-term variability in bivalve recruitment, mortality, and growth and their contribution to fluctuations in food stocks of shellfisheating birds. Mar. Ecol. Prog. Ser. 414, 117–130. Bonnard, M., Roméo, M., Amiard-Triquet, C., 2009. Effects of copper on the burrowing behavior of estuarine and coastal invertebrates, the polychaete Nereis diversicolor and the bivalve Scrobicularia plana. Hum. Ecol. Risk. Assess. 15, 11–26. Breber, P., 2002. Introduction and acclimatisation of the Pacific carpet clam, Tapes philippinarum, to Italian waters. In: Leppäkoski, E., Gollasch, S., Olenin, S. (Eds.), Invasive Aquatic Species of Europe. Kluwer Academic Publishers, Netherlands, pp. 120–126.

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