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Marine Environmental Research xxx (2015) 1e9

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Marine Environmental Research

journal homepage: www.elsevier .com/locate/marenvrev

Response of top shell assemblages to cyclogenesis disturbances. A casestudy in the Bay of Biscay

M. Mu~noz-Colmenero a, G.-J. Jeunen a, Y.J. Borrell a, J.L. Martinez b, P. Turrero c,E. Garcia-Vazquez a, *

a Departamento de Biología Funcional, Universidad de Oviedo, Spainb Servicios Científico-T�ecnicos, Universidad de Oviedo, Spainc Universidad Nacional de Educaci�on a Distancia, Campus de Gij�on, Spain

a r t i c l e i n f o

Article history:Received 11 November 2014Received in revised form15 June 2015Accepted 18 June 2015Available online xxx

Keywords:Cyclone eventsGenetic diversityGibbula umbilicalisMetapopulationPhorcus lineatusSize selectionTemporal variability

* Corresponding author. Departamento de Biología Fn, 33006-Oviedo, Spain.

E-mail address: egv@uniovi.es (E. Garcia-Vazquez)

http://dx.doi.org/10.1016/j.marenvres.2015.06.0120141-1136/© 2015 Elsevier Ltd. All rights reserved.

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a b s t r a c t

Cyclones and other climate disturbances profoundly affect coastal ecosystems, promoting changes in thebenthic communities that require time, sometimes even years, for a complete recovery. In this study wehave analysed the morphological and genetic changes occurred in top shell (Gibbula umbilicalis andPhorcus lineatus) assemblages from the Bay of Biscay following explosive cyclogenesis events in 2014.Comparison with previous samples at short (three years before the cyclogenesis) and long (UpperPleistocene) temporal scales served to better evaluate the extent of change induced by these distur-bances in a more global dimension. A significant increase in mean size after the cyclogenesis was foundfor the two species, suggesting selective sweeping of small individuals weakly adhered to substrata. Lossof haplotype variants at the cytochrome oxidase subunit I gene suggests a population bottleneck,although it was not intense enough to produce significant changes in haplotype frequencies. The highpopulation connectivity and metapopulation structuring of the two species in the area likely help thepopulations to recover from disturbances. At a wider temporal scale, cyclogenesis effects seemed tocompensate the apparent decreasing trends in size for P. lineatus occurred after the PleistoceneeHolocene transition. Considering disturbance regimes for population baselines is recommended whenthe long-term effects of climate and anthropogenic pressures are evaluated.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Marine communities are heavily impacted by different envi-ronmental factors, both anthropogenic and natural. Among thelatter, “natural disasters” such as tropical storms or hurricanes,despite their relatively infrequent occurrence, can affect thebenthic marine biota, from continental shelves to deep and abyssalbottoms (Harris, 2014). Natural disturbances are an importantecological process for benthic ecosystems andmight be a key factorcontrolling the spatial distribution of many species in the marineenvironment, with mud and sand bottom communities recoveringfrom disturbances faster than gravel and reef benthos (Harris, 2012,2014).

Although big storms are known to cause profoundmodifications

uncional, C/ Juli�an Clavería s/

.

lmenero, M., et al., Responsesearch (2015), http://dx.doi.o

in nearshore communities (e.g. Morton, 1988), their effect on localbenthic communities is not well known yet, at least in the Bay ofBiscay, mainly due to a lack of spatio-temporal baselines for modeltaxa and species assemblages; this hampers impact assessments inthe zone (e.g. Juanes et al., 2007; Puente et al., 2009). In the lastyears cyclogenesis and storm events are increasing in the region e

some are derived from subtropical processes (Liberato et al., 2013)and may be due to global climate change that promotes instabilityin temperate areas (e.g. Lozano et al., 2004). Storminess is expectedto increase the vulnerability of coastal zones in these regions, withmore intense erosion and subsequent changes in biodiversity(Lozano et al., 2004).

In this study we focused on the effect of the cyclogenesis thataffected the Bay of Biscay in the 2013e2014 winter (cyclone Dirk;see for example http://alert.air-worldwide.com/EventSummary.aspx?e¼727&tp¼31&c¼1, last accessed June 2015), taking the topshells Gibbula umbilicalis (da Costa 1778) and Phorcus lineatus (daCosta 1778; previously Osilinus lineatus) as model species. We have

of top shell assemblages to cyclogenesis disturbances. A case study inrg/10.1016/j.marenvres.2015.06.012

M. Mu~noz-Colmenero et al. / Marine Environmental Research xxx (2015) 1e92

chosen top shells because we have a baseline of previous geneticand morphological population data (e.g. Turrero et al., 2014), andbecause top shells aremodel species useful for assessing the impactof climate (e.g. Mieszkowska et al., 2006; Hawkins et al., 2009).Rapid alterations in the distributional limits of P. lineatus and G.umbilicalis have been described following climate change in themid-1980s (Mieszkowska, 2009), and top shells are vulnerable towave exposure since their sizes change along with this and otherfactors (e.g. Crothers, 2001; Preston and Roberts, 2007). Followingthis, we expected them to be sensitive to the effects of the cyclo-genesis, which modify different parameters of the environmentsuch as turbidity, nutrients dissolved in the water, intensity of waveaction on the coast, etc. (Harris, 2014). Selection against vulnerablesize classes, too big or too small for proper anchoring to the sub-strate under the influence of strong windstorms, would be ex-pected in more exposed beaches, affecting population sizedistributions and even population genetic diversity if the stormscaused very intense mortality. Although the removal of individualsis not necessarily the only effect of these storms, due to the strengthof these events in recent years we considered it as one of the factorswith most modifying power over these populations. The sequenceof events would be: A storm comes to shore. More vulnerable(small and exposed) individuals are swept out. If many individualsare lost from an exposed population, a bottleneck and subsequentgenetic drift is expected. These effects are expected to be lessintense in less exposed beaches, and therefore differences betweenexposed (open) and sheltered beaches are expected for both sizeand population genetic variation (populations from more exposedbeaches are expected to be less variable).

2. Materials and methods

2.1. Studied area and post-cyclogenesis sampling

The taxonomic nomenclature we use follows that currentlyaccepted in theWorld Register of Marine Species (WoRMS, Boxshallet al., 2014). The top shell species G. umbilicalis (da Costa 1778) andP. lineatus (da Costa 1778) were chosen as models due to the exis-tence of previous observations and genetic data from the region.

The study area was the central coastal part of the Spanish Bay ofBiscay, in the region of Asturias (43�4000000N/42�5305000 Nto �7�1200000W/4�3202000W; see Fig. 1). Rocky beaches were visitedin late March just after the end of the cyclogenesis that occurredduring the previous winter (cyclone Dirk in December 2013 andlater episodes; see for example http://poleshift.ning.com/profiles/blogs/north-atlantic-wave-bombs, last accessed June 2015). Thetype of geological substrate, mainly calcareous, is very similar in allthe considered beaches. None of tem is significantly affected bycontamination. At least partially sheltered rocky shores werepreferred for sampling to minimize wave exposure, although evensheltered beaches were impacted during the cyclogenesis event.This was apparent when sampling the beaches of Andrín andVidiago (Fig. 1), where no gastropods could be found except for afew G. umbilicalis specimens. Sampling of the two consideredspecies was conducted from three affected (more open) beaches(Otur, Verdicio and Tor�o), where sand had been removed and morerocks than usual were exposed, and three less affected (moresheltered) beaches, where sand was still in place and rock exposurehad not changed noticeably (Perlora, La Griega and P�oo).

At each location, samples were obtained at random (i.e. withoutselection for size) from the intertidal transect, covering an area ofapproximately 2000 m2. For reasons of comparable habitat andsampling effort, naked rocks with <10% algae coverage were tar-geted for sampling. Sampling effort was roughly 10% (one indi-vidual collected per ten individuals observed). The samples were

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visually identified based on morphological traits (see for exampleCrothers, 2003) and stored in 96% ethanol until genetic analyseswere performed. The ethanol was changed twice on consecutivedays to improve tissue preservation.

2.2. Prehistoric and contemporary (pre-cyclogenesis) baselines

Details of the baselines can be found in Turrero et al. (2014), andcan be summarised as follows: remains of the species P. lineatus(formerly named Osilinus lineatus) were found in 6 archaeologicalsites (Balmori, Coberizas, Cuetu laMina, La Lloseta, La Riera and TitoBustillo) in the archives of the Archaeological Museum of Asturias,and the maximum widths of 1106 archaeological top shells weremeasured. These were found in strata from three different cultural/technological phases: Solutrean, from ~20000 years ago (20 ka) to~17 ka; Magdalenian, from ~17 ka to ~11.5 ka; and Epipalaeolithic,from ~11.5 ka to 6 ka (roughly coincident with the beginning of theHolocene epoch).

Contemporary shellfish were randomly sampled during winter(December to January) from 2009 to 2011, with average tempera-tures between 9 and 10 �C (comparable to the 10.2 �C average forMarch 2014 in the region; see A.E.MET, 2015 for weather data),from the accessible intertidal level of rocky coast close to thearchaeological sites. This area corresponds to the eastern part of thesampling area of the present study, between La Griega and Tor�o. Atotal of 135 top shells were collected from six areas, visuallyidentified and stored in 96% ethanol for further genetic identifica-tion employing the Barcoding COI gene (Donald et al., 2012).

2.3. Morphometric analysis

The maximum widths of the shells were measured using aVernier Caliper (±0.1 mm). For graphical representation, shellmeasurements were grouped into mean-centred shell width cate-gories every 2.5 mm.

2.4. Genetic analysis

DNA extraction was carried out following Estoup et al.’s (1996)resin-based Chelex protocol. The region of the mitochondrialgene cytochrome oxidase I (COI) was PCR-amplified with AppliedBiosystem‘s Veriti Thermal Cycler from specimen DNA according tothe procedure revised by Geller et al. (2013). The primersjgLCO1490 (TITCIACIAAYCAYAARGAYATTGG) and jgHCO2198(TAIACYTCIGGRTGICCRAARAAYCA) were used. PCR was preparedwith 4 mL 5� PCR buffer, 0.2 mL Taq polymerase (Promega), 2 mMMgCl2, 0.5 mM of each primer, 2 mL of 2.5 mM dNTP and 2 mL ofgenomic DNA in 20 mL reactions. PCR conditions were an initialdenaturing step at 95 �C for 5 min; then 35 cycles of 1 min at 95 �C,1 min at 46 �C, 1.30 min at 72 �C; and a final 7 min at 72 �C. PCRproducts were examined on a 2% agarose gel stained with ethidiumbromide.

The illustra™ ExoStar™ 1-Step GE Healthcare Life Sciences pro-tocol was applied to the PCR products, that were sequencedemploying the BigDye Terminator Cycle Sequencing Kit v3.1 andanalysed on a 3130 xl Genetic Analyzer (Applied Biosystems)Automated Sequencer at the Unit of Genetic Analysis of the Uni-versity of Oviedo.

Sequence chromatograms were edited using Seqman Lasergenev7.0.2. (DNASTAR).

DNA sequences were treated using different programs. FASTAfiles were compiled per species after morphological classificationwas confirmed by comparison of the DNA dataset with referencesequences using the nBLAST program within NCBI (http://blast.st-va.ncbi.nlm.nih.gov/). Alignment of COI sequences per species

of top shell assemblages to cyclogenesis disturbances. A case study inrg/10.1016/j.marenvres.2015.06.012

Fig. 1. Location of the studied region and the sites mentioned in the text. Beaches in clear grey: 1, Otur (affected); 2, Verdicio (affected); 3, Perlora (less affected); 4, La Griega (lessaffected); 5, P�oo de Llanes (less affected); 6, Tor�o (affected); 7, Andrín (not sampled); 8, Vidiago (not sampled). Archaeological sites in white: A, La Lloseta, Tito Bustillo; B, Coberizas;C, Cuetu la Mina, La Riera; D, Balmori.

M. Mu~noz-Colmenero et al. / Marine Environmental Research xxx (2015) 1e9 3

was performed with the ClustalW program within Mega 5.10(Tamura et al., 2013). COI sequences were loaded into DnaSP v 5.10(Librado and Rozas, 2009) to generate a haplotype data file. Thehaplotypes found for each species were submitted to GenBank(http://www.ncbi.nlm.nih.gov/) for availability in a public re-pository. The level of sequence polymorphism (genetic diversity)was measured by different parameters: Nh (number of sequencevariants or haplotypes), Hd (haplotype diversity, that is, the prob-ability of two randomly chosen haplotypes in the sample beingdifferent) and п (nucleotide diversity, or the mean number of dif-ferences between all pairs of haplotypes in the sample). They werecalculated with ARLEQUIN v3.5 software (Excoffier et al., 2005).Haplotype networks were constructed with NETWORK v4.6.1.2(www.fluxus-engineering.com). Different haplotypes are repre-sented by circles connected by lines, where the mutations betweenthem are represented. The diameter of each circle is proportional tothe frequency of the corresponding haplotype.

2.5. Statistical analyses

For the statistical analyses, samples from each chronologicalperiod, and status (affected/less-affected beaches) for post-disturbance samples, were pooled spatially. Dispersal distancesfor Phorcus populations, that is, their advance into new locations,can reach 1 km in less than one decade (Little et al., 2012), and thepotential for population connectivity at this spatial scale has beenconfirmed by little or null regional population differentiation fortop shells (e.g. Keith et al., 2011). Between-population differences ata smaller geographic scale, as is the present case, are therefore notexpected.

After a normalization test, non-parametric tests were performedto compare the effect of the cyclogenesis on shell size at a shortscale (comparing post-disturbance data with the contemporarybaseline, affected eopen beaches- and less affected eshelteredbeaches-, pairwise to check all possible comparisons). We usedManneWhitney tests to compare means and Kolmor-ogoveSmirnov tests to compare the distributions of frequencies. In

Please cite this article in press as: Mu~noz-Colmenero, M., et al., Responsethe Bay of Biscay, Marine Environmental Research (2015), http://dx.doi.o

order to obtain the same information, graphic and non-parametriccomparisons were done between different statuses of exposure(open versus sheltered) and/or periods (Solutrean, Magdalenian,Epipalaeolithic, contemporary baseline in 2009e2011 and post-disturbance in 2014), not taking into account the status of thebeaches from the paleontological samples because we did notknow the beach of origin for each sample. Statistical analyses ofmorphological measures were performed with SPSS Statisticssoftware version 17.0.2 and PAST v3.0 (Hammer et al., 2001). Thesignificance of the differences between locations or chronologicalperiods for species distribution was tested using non-parametriccontingency Chi-Square.

Finally, we compared, by parametric ANOVA two-ways analyses,the effect in P. lineatus (a species for which we had a baseline) withthat in G. umbilicalis, to determine if both species were similarlyaffected or not after the disturbances.

3. Results

3.1. Population genetic diversity

PCR amplification of COI gene provided a total of 146 sequences495-nucleotide long for G. umbilicalis samples collected aftercyclone Dirk, 72 from affected beaches and 74 from less-affectedlocations (Table 1). They were submitted to GenBank, where theyare available with accession numbers KP064694eKP064756. For P.lineatus 63 sequences from less-affected sites and 25 from affectedlocations were obtained, all 524 nucleotide long. Their GenBankaccession numbers are KP064757eKP064788.

For G. umbilicalis only a few sequences (GenBank accessionnumbers JN241973eJN241977) were available before the cyclo-genesis (Table 1). These individuals were highly variable at thisgene with Hd higher than 0.9, and five different haplotypes forseven individuals sampled (Nh/N ¼ 0.71). In contrast, after thedisturbance this ratio was Nh/N ¼ 0.437, with 63 different haplo-types for 144 individuals sampled. The individuals sampled fromless-affected locations exhibited only slightly higher diversity (both

of top shell assemblages to cyclogenesis disturbances. A case study inrg/10.1016/j.marenvres.2015.06.012

Table 1Genetic diversity at the cytochrome oxidase I gene for top shells Gibbula umbilicalis and Phorcus lineatus at six locations sampled after 2014's cyclogenesis, by location, byexposure status (affected or less-affected) and in the whole region, as well as regional data from before the disturbance. N, sample size; Nh, Hd and p: number of haplotypes,haplotype diversity and nucleotide diversity respectively (standard error given in parenthesis).

Location Gibbula umbilicalis Phorcus lineatus

N Nh Hd p N Nh Hd p

Otur 28 14 0.913 (0.030) 0.0055 (0.0036) 21 16 0.962 (0.030) 0.0098 (0.0055)Verdicio 8 5 0.857 (0.108) 0.0058 (0.0039) 2 2 1.000 (0.500) 0.0134 (0.0143)Perlora 42 27 0.963 (0.017) 0.0085 (0.0048) 20 7 0.842 (0.049) 0.0075 (0.0044)La Griega 22 14 0.952 (0.027) 0.0064 (0.0038) 22 14 0.944 (0.029) 0.0097 (0.0055)P�oo 10 8 0.956 (0.059) 0.0075 (0.0047) 21 10 0.881 (0.047) 0.0096 (0.0054)Tor�o 36 27 0.976 (0.015) 0.0079 (0.0045) 2 2 1.000 (0.500) 0.0134 (0.0143)Pre-disturbance 7 5 0.905 (0.103) 0.0077 (0.0050) 24 14 0.938 (0.030) 0.0095 (0.0053)Post-disturbance (whole) 146 63 0.957 (0.008) 0.0072 (0.0041) 88 32 0.924 (0.016) 0.0093 (0.0051)Affected (more open) 72 36 0.948 (0.015) 0.0068 (0.0039) 25 18 0.957 (0.029) 0.0097 (0.0054)Less-affected (sheltered) 74 35 0.959 (0.010) 0.0076 (0.0043) 63 23 0.913 (0.019) 0.0093 (0.0051)

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Hd and p) than those sampled from affected locations (Table 1).The global picture given by the haplotype network (Fig. 2) for

Gibbula, with two main lineages and many singletons, does notindicate a preferential loss of haplotypes from exposed versus less-exposed sites. FST values for pairwise comparisons between pre-and post-disturbance samples were not significant (FST values of0.06622 with P ¼ 0.06306 and 0.03102 with P ¼ 0.13514, forcomparison of pre-cyclone with affected and less-affected post-cyclone samples respectively, both not significant). Differencesbetween affected and less-affected post-cyclone samples were notsignificant (FST ¼ 0.00145 with P ¼ 0.54955).

Fig. 2. Haplotype network constructed from Gibbula umbilicalis cytochrome oxidase I genetheir frequency. Black, pre-disturbance samples; white and grey, post-cyclone samples from

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Baseline P. lineatus samples obtained before the cyclogenesis(N ¼ 24), with GenBank accession numbers JN241979eJN241991(formerly named O. lineatus following older taxonomic nomencla-ture), were moderately variable with Hd and p values of 0.938 and0.009 respectively (Table 1), and 14 haplotypes for 24 individuals(Nh/N ¼ 0.58). After the disturbance, a total of 32 haplotypes werefound for a total of 88 samples (Nh/N ¼ 0.36), which is a consid-erable reduction in general diversity. Differences between affectedand less-affected locations for haplotype and nucleotide diversitywere not consistent across sites; two affected localities, Tor�o andVerdicio, have only two sequences. The haplotype network (Fig. 3)

sequences. Each circle represents a haplotype, and their diameters are proportional tomore exposed and less exposed locations respectively.

of top shell assemblages to cyclogenesis disturbances. A case study inrg/10.1016/j.marenvres.2015.06.012

Fig. 3. Haplotype network constructed from Phorcus lineatus cytochrome oxidase I gene sequences. Each circle represents a haplotype, and their diameters are proportional to theirfrequency. Black, pre-disturbance samples; white and grey, post-cyclone samples from affected and less-affected locations respectively.

Table 2Comparison of top shell sizes from different sampling locations in the Bay of Biscayafter 2014's cyclogenesis. A: Mean size (in mm) ± standard error (N) of theconsidered species in the six sampling locations (Otur, Verdicio, Perlora, La Griega,P�oo and Tor�o), listed from west to east. B: Non-parametric analyses performed onPhorcus lineatus data to compare the post-disturbance samples with the pre-disturbance baseline, taking into account the exposure status of the sampling sites(exposed or less exposed, affected and less affected by the cyclogenesisrespectively).

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exhibited a complex shape with different lineages, somewhatsimpler than the network obtained for G. umbilicalis (Fig. 2) butwith the similar feature of showing less shared haplotypes thansingletons. For changes in haplotype frequencies between pre- andpost-cyclone samples, FST value was not significant (FST ¼ 0.0196with P¼ 0.099). As expected, FST between affected and less-affectedpost-cyclone samples was also not significant (FST ¼ 0.0203 withP ¼ 0.946).

Location Status Gibbula umbilicalis Phorcus lineatus

A)Otur Exposed 14.81 ± 0.92 (((40) 14.94 ± 4.23 (((54)Verdicio Exposed 12.59 ± 1.54 (115) 15.54 ± 2.40 (98)Perlora Less-exposed 10.88 ± 1.33 (188) 13.92 ± 3.15 (39)La Griega Less-exposed 12.12 ± 2.55 (27) 12.66 ± 3.67 (110)P�oo Less-exposed 11.86 ± 1.73 (18) 13.13 ± 3.36 (76)Tor�o Exposed 12.95 ± 1.26 (67) 15.00 ± 1.87 (7)All exposed Exposed 13.10 ± 1.59 (222) 15.31 ± 3.12 (159)All less-exposed Less-exposed 11.11 ± 1.61 (233) 13.04 ± 3.49 (225)

Comparisons ManneWhitneytest

KolmorogoveSmirnov test

B)Pre-disturbance vs. post-

disturbanceU ¼ 24,785,P ¼ 0.803

Z ¼ 2.074, P ¼ 0.000

Affected beaches (pre vs. post) U ¼ 5544, P ¼ 0.005 Z ¼ 2.031, P ¼ 0.001Less-affected beaches (pre vs. post) U ¼ 1004.500,

P ¼ 0.000Z ¼ 4.172, P ¼ 0.000

Post-disturbance (affected vs. less-affected)

U ¼ 11,279,P ¼ 0.000

Z ¼ 2.592, P ¼ 0.000

Pre-disturbance (affected vs. less-affected)

U ¼ 165.500,P ¼ 0.000

Z ¼ 4.840, P ¼ 0.000

3.2. Top shell size

A total of 455 G. umbilicalis and 384 P. lineatus specimens weresampled and measured after cyclone Dirk and subsequent episodesaffected the region (Table 2A). A size baseline for P. lineatus from theregion, sampled before 2014, is available (Turrero et al., 2014). Thecomparison of mean size between pre- (contemporary baseline)and post-cyclogenesis samples shows that differences are not sig-nificant (U ManneWhitney ¼ 24,785, P ¼ 0.803), but a comparisonbetween size class frequencies shows their differences are highlysignificant (Z KolmorogoveSmirnov ¼ 2.074, P ¼ 0.000). As awhole, post-disturbance samples exhibited a higher proportion oflarge individuals than pre-disturbance samples (Fig. 4). All thecomparisons between more exposed (open) versus less exposed(sheltered) beaches within periods and between periods weresignificant too, as can be seen on Table 2B. In the pre-cyclogenesissamples a wider variety was found, with extensive differences be-tween open and sheltered beaches. These differences were reducedafter the storms due to a clear decrease of the frequency of smallertop shells in the more sheltered beaches, and a decrease of theintermediate size classes in the more open beaches (Fig. 4),resulting in a greater similarity between the different beaches.

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Compared with the older (prehistoric) samples, post-cyclogenesis top shells were more comparable to our

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Fig. 4. Size classes of Phorcus lineatus before and after the cyclogenesis Dirk in open(above) and sheltered (below) beaches, presented as proportion of each size class incm. Post-disturbance, N ¼ 225; pre-disturbance, N ¼ 135.

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Epipalaeolithic samples than to the samples collected before thecyclogenesis (Fig. 5). The respective size means for Epipalaeolithic,pre- and post-disturbance samples are 14.39, 13.52 and 13.98 mm.The difference in mean size between Epipalaeolithic and post-disturbance 2014 samples is significant for both exposed (meansize 15.31; t ¼ 3.214, P ¼ 0.0014 for samples with equal variance)and less-exposed (mean size 13.04; t ¼ 4.815, P ¼ 2.06 � 10�6 forsamples with unequal variance) sites. These differences are oppo-site in sign because top shells from exposed beaches were biggerthan those found in Epipalaeolithic sites, whereas those from lessexposed locations were smaller. When all post-cyclone samples are

Fig. 5. Size distributions (proportion of each size class in cm) of Phorcus lineatuspopulations inhabiting the studied areas in different chronological moments. Solu-trean, N ¼ 74; Magdalenian, N ¼ 557; Epipalaeolithic, N ¼ 358; pre-disturbance,N ¼ 135; post-disturbance, N ¼ 225.

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considered as a single population the difference in size with Epi-palaeolithic samples is not statistically significant (t ¼ 1.722,P ¼ 0.085 for samples with unequal variance).

Taking into account the two species, top shell samples fromlocations more affected by the cyclogenesis were larger than thosesampled from less-affected beaches, although for the same type ofbeach P. lineatuswere always bigger than G. umbilicalis (Table 2A). Atwo-way ANOVA, performed to discriminate if the two speciesunderwent the same effect after the cyclogenesis, was highly sig-nificant for differences in size between species as well as betweenaffected and less-affected beaches (Table 3). The interaction be-tween both factors was not significant (F ¼ 2.039, P ¼ 0.154,Table 3), indicating that the size difference between samples fromaffected and less-affected beaches within species was similar forthe two species.

The distribution in size classes (Fig. 6) shows the differencesrevealed by the ANOVA. The distribution obtained from samples ofsheltered beaches contains a much higher proportion of top shellsfrom small size classes than that from samples of more open bea-ches, for the two species. It is also evident that G. umbilicalis wassmaller than P. lineatus, and that the shape of the size class distri-bution for open and sheltered beaches was similar within species,with a higher variance in P. lineatus.

3.3. Changes in top shell assemblages

It is important to take into account the spatial component ofspecies assemblages, because the relative abundance of Gastropodspecies varies along the studied coast due to a shift in environ-mental conditions around Cape Pe~nas, in the centre of the region(Mu~noz-Colmenero et al., 2012; Turrero et al., 2012). Consideringonly the locations where data on top shell species compositionwere available before 2014 e that is, the eastern part of the studiedarea (Tor�o, La Griega and P�oo) e, it can be noted that the assem-blage changed (Fig. 7), with a significant increase in G. umbilicalispresence, which was scarce before the cyclogenesis (Chi-square of10.98 for 1 degree of freedom, P ¼ 0.001). This species was clearlydominant in the more exposed beaches in 2014 (Table 1A). More-over, in the two beaches visited in 2014 after the cyclogenesiswhere sampling was discarded (Vidiago and Andrín), only a few G.umbilicalis were found, and no P. lineatus.

4. Discussion

The results obtained in this study reveal alterations in coastaltop shell populations from the Bay of Biscay following exposure tocyclone Dirk. Small individuals were less abundant after thedisturbance for the two model species studied, G. umbilicalis and P.lineatus, and regional genetic diversity (at COI DNA sequences)seems to have been reduced although haplotype frequencies didnot change significantly. These effects are consistent with asweeping effect of the cyclone at both morphological and geneticlevels.

Although there is no simple explanation linking the reduction of

Table 3Two-way ANOVA comparing the mean sizes of the studied top shell populations.Status, open and sheltered beaches; Species, Gibbula umbilicalis and Phorcus lineatus.

Sum of squares df Mean square F p (same)

Exposure status: 804.406 1 804.406 123.4 7.87E-27Species: 755.616 1 755.616 115.9 2.13E-25Interaction: 13.2923 1 13.2923 2.039 0.1537Within: 5444.26 835 6.52007Total: 7132.8 838

of top shell assemblages to cyclogenesis disturbances. A case study inrg/10.1016/j.marenvres.2015.06.012

Fig. 6. Size distribution (proportion of each size class in cm) for the populations ofGibbula umbilicalis (Gu, green) and Phorcus lineatus (Pl, blue) inhabiting affected(dashed line) and less-affected (solid line) locations. (For interpretation of the refer-ences to colour in this figure legend, the reader is referred to the web version of thisarticle.)

Fig. 7. Gibbula umbilicalis and Phorcus lineatus assemblages found in the intertidal area of the eastern sampling points (La Griega, P�oo, Tor�o) before and after 2014's cyclogenesis,presented as the proportion of each species. Data for 2014 affected and less-affected beaches are also presented separately.

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small size classes and cyclone Dirk, they do seem to be connectedsince this reduction occurred in both species regardless of theirdifferent species-specific mean size. Top shell juveniles attachrelatively weakly to the substrate (e.g. Hayakawa et al., 2008); itcould be hypothesized that small individuals exhibit less adhe-siveness to the substrate than bigger ones. They would therefore bemore vulnerable to sudden intense windstorms, which wouldinvolve massive movements of sediments, carrying sand and otherparticles that would especially affect smaller, more fragile in-dividuals. However, very small individuals refuged inside rockcrevices and small holes would be more protected against wavesweeping, being more difficult to remove. This would explain theapparent removal of intermediate size classes in the more open

Please cite this article in press as: Mu~noz-Colmenero, M., et al., Responsethe Bay of Biscay, Marine Environmental Research (2015), http://dx.doi.o

locations, which on the other hand are also more exposed to wavesin normal conditions.

The lack of small size classes has been often considered anindication of sporadic settlement events (e.g. Lewis, 1986; Zacherlet al., 2003; Lima et al., 2006). However, the results found in thisstudy suggest an alternative or complementary explanation inareas vulnerable to sediment disturbances, since a lack of small topshells could reflect a recent sweeping due to big storms or cyclones.From our results we can see that these climatic events acted in thedirection of homogenizing the P. lineatus population, removing thesmall individuals from both more exposed and sheltered beachesrefugees (Fig. 4A). The great strength of the waves brought on bythese cyclones cancels the effect of refuge for juveniles of thesespecies that the sheltered beaches provide in normal weatherconditions.

Related to this, some discussion on recruitment in these twospecies is necessary. Reproduction and development of the gonadsfor both species usually happens in the summer months(Mieszkowska et al., 2006, 2007), with the peak depending on thetemperature at the area. The increase in temperature resulting fromglobal warming has led to successful recruitment for these speciesin the North Atlantic (Mieszkowska et al., 2007). Because of this,successful recruitment is expected unless some disturbance mod-ifies the usual conditions. One such disturbance would be thecyclogenesis processes in our study, which would remove juvenilesalready settled and would drag away those that were trying tosettle, preventing them from adhering to the substrate. The autumn

months are usually the months of higher population density, cor-responding to the later stages of settlement in the intertidal(Ahmedou Salem et al., 2014). The heavy storms the coast sufferedwould have impeded the new individuals to thrive. On the otherhand, the post-storm recovery (or the resistance to sweeping) of G.umbilicalis and P. lineatus found in this study was better for theformer, especially in open locations. This is consistent with theinterpretation given above, based on the removal of small but in-termediate size classes, i.e. the small individuals that were notsmaller enough for hiding in crevices and intricate holes. Thiswould be reflected in higher standard deviation for post-than forpre-disturbance samples (very small and big individuals wouldsurvive). Gibbula, which is generally smaller than Phorcus,

of top shell assemblages to cyclogenesis disturbances. A case study inrg/10.1016/j.marenvres.2015.06.012

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recovered much better after the storm (Fig. 6), indicating that theyresisted better the cyclogenesis impact than the other bigger topshell.

Regarding genetic diversity, the results of this study are a goodexample of bottleneck effects. Top shells reproduce once a year inmid-summer and live for several years (e.g. Crothers, 2003). Sincepopulation sizes are large, they have long dispersal capacity andspatial connectivity is generally ensured (e.g. Keith et al., 2011;Little et al., 2012), pronounced gene drift and thus genetic differ-ences between consecutive generations are not expected.Removing a part of the population, likely the younger generation/s,would reduce a part of the general diversity because some single-tons would be lost, but the population's genetic pool would notchange substantially. Moreover, replenishment of the lost variationby immigration from neighbouring, less exposed, beaches is ex-pected in a short time thanks to the top shells' dispersal capacity.Top shells would function as metapopulations and genetic diversitylosses in a part of their distribution would be rapidly compensatedby high gene flow. These dynamics would enable top shells tocolonize neighbouring environments; actually, these species andother intertidal gastropods are in expansion under the currentclimate change (e.g. Hellberg et al., 2001; Hawkins et al., 2008,2009; Mu~noz-Colmenero et al., 2012; Rubal et al., 2014).

We have found a significant change in size for both speciesunder study that was dependent on the exposure to the cyclo-genesis, which promoted spatial heterogeneity in the small regionstudied. Moreover, in some locations (Vidiago, Andrín) almost alltop shells were swept away by the cyclogenesis, with only a fewindividuals of one species left. Patchy distributions of a speciesmight therefore be explained from different exposure to distur-bance events. Our results support the inclusion of disturbanceregime concepts with other biophysical variables that define thefundamental niches of marine species for predictive habitatmodelling, as proposed by Harris (2014). Under normal conditions,exposure of the beaches would be an important factor for intertidalpopulations, varying the intensity at which the sea affects them, butunder conditions of hard climatic disturbances, the effect of ho-mogenization of the affected coasts should be taken into account ehere represented by a smaller range of sizes and the loss of geneticvariation.

Instability due to intense disturbances such as cyclogeneseswould produce rapid changes in size in top shell populations,contributing to increase the average size in the long term bysporadically sweeping smaller size classes (when disturbances areproduced, which may vary depending on the particular climaticregime of each region). This has enormous importance forcomparative studies at large temporal scales: if samples from onlyone or a few years are considered as representative of modernpopulations, they may reflect just a transient status that maychange significantly after a single disturbance event. Comparingsamples from one or two undisturbed years with chronologicalperiods that include at least various cyclonic events likely underrepresents the real evolution and status of the studied biota. Forinstance, known periods of global climatic change such as theMediaeval Warm Period or the Little Ice Age (e.g. see Desprat et al.,2003; Eiríksson et al., 2006) are likely to have produced one orseveral such events. The inclusion of samples from years and zonesaffected by disturbances in regional baselines is therefore highlyrecommended.

The results of this study also help to explain, at least partially,the scarcity of G. umbilicalis in archaeological sites of this regionwhere Patella, Phorcus and Littorina were by far the most intenselyexploited Gastropods (e.g. Ortea, 1986; Guti�errez-Zugasti, 2011;Turrero et al., 2014). Ortea (1986) explained the almost totalabsence of this species from Upper Palaeolithic remains due to the

Please cite this article in press as: Mu~noz-Colmenero, M., et al., Responsethe Bay of Biscay, Marine Environmental Research (2015), http://dx.doi.o

difficult extraction of the whole body from the shell with a pointedobject like a pin (the Upper Palaeolithic is represented in our studyby the Solutrean andMagdalenian periods). However, the shells canbe broken as easily as those of the widely consumed Phorcus.Preference for bigger shellfish (selection for size, as seen forexample in Guti�errez-Zugasti, 2011 and Turrero et al., 2012) wouldalso explain the differential Palaeolithic exploitation of these twospecies. Since under the same conditions Gibbula grows smallerthan Phorcus, the latter species would be preferred for harvesting.

5. Conclusions

The significantly bigger size of G. umbilicalis and P. lineatusspecimens from beaches affected by 2014's cyclogenesis in the Bayof Biscay when compared to less-affected sampling points suggeststhe cyclogenesis had a sweeping effect over small individuals. Asignificant increase in the average regional size after the cyclo-genesis compensated the apparent size decline of P. lineatus afterthe Upper Palaeolithic, suggesting that reference baselines shouldtake into account disturbances when evaluating the long-termevolution of intertidal communities. On the other hand, signifi-cant changes in COI haplotype frequencies were not found despitethe reduction of haplotype variation. High connectivity betweenpopulations and a metapopulation structuring of top shells wouldexplain their high capacity for the recovery of population geneticvariability.

Acknowledgements

The authors are grateful to the Museo de Arqueología de Astu-rias for granting permission to study part of their collections, and toan anonymous reviewer for his/her helpful suggestions forimproving data analysis. M. Mu~noz-Colmenero holds a NationalSpanish Grant (reference AP-2010-5211). This study has beensupported by the Regional Government of Asturias (grant numberSV-PA-13-ECOEMP-41; GRUPIN14-093) and the Spanish NationalProject MINECO CGL2013-42415-R. This is a contribution from theMarine Observatory of Asturias (Spain).

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