D Izquierdo

J M Guerra-García

Distribution patterns of the peracarid crustaceans associated with the alga Corallina elongata along the intertidal rocky shores of the Iberian Peninsula

Keywords: Peracarida, Iberian Peninsula, Intertidal, Corallina elongata, biogeography

Spatial patterns of intertidal peracarids associated to the alga Corallina elongata, were studied along the whole Iberian Peninsula. A total of 28215 specimens were collected, comprising 78 different species (57 amphipods, 16 isopods, 4 tanaids and 1 cumacean), most of them with Atlantic-Mediterranean distribution (60%) and only 9% of Mediterranean endemics. Gammarids were dominant in abundance and number of species, representing more than 70% of the total peracarids. The most common species collected during the present study were the caprellid Caprella penantis, the gammarids Hyale schmidti, Hyale stebbingi, Jassa cf. falcata and Stenothoe monoculoides, the isopod Ischyromene lacazei and the tanaid Tanais dulongii. Caprellids and tanaidaceans presented their highest populations in the stations of the Strait of Gibraltar whereas isopods were more abundant in Atlantic stations. Univariate analyses did not reflected differences in number of species, abundance and Shannon-Wiener diversity between Mediterranean and Atlantic. However, cluster analyses and Whittaker index, as measure of ß-diversity, showed a different species composition between Mediterranean and Atlantic and a replacement of species along the coast, especially at the Strait of Gibraltar. The turnover mainly affected to species of the same genera, probably related with sympatric speciation. CCA and BIO-ENV analyses showed high correlations between environmental measures (especially conductivity) and peracarid distribution.

Mediterranean species tolerated higher values of conductivity and temperature, while Atlantic species were associated to stations with higher oxygen concentrations.

Introduction

Understanding assemblages of organisms is based on the quantitative description of patterns of distribution and abundance of species [START_REF] Andrew | Sampling and the description of spatial pattern in marine ecology[END_REF]Mapstone 1987, Underwood et al. 2000), which has become one of the main challenges that studies on biogeography have to face nowadays [START_REF] Pereira | Biogeographic patterns of intertidal macroinvertebrates and their association with macroalgae distribution along the Portuguese coast[END_REF]. The borders in an ecological level are often attributed to climatic conditions [START_REF] Repasky | and the northern distribution of wintering birds[END_REF][START_REF] Wardell-Johnson | Biogeographic barriers in a subdued landscape: the distribution of the Geocrina rosea (Anura: Myobatrachidae) complex in southwestern Australia[END_REF] or antagonistic relationships of competition and predation [START_REF] Hersteinsson | Interspecific competition and the geographical distribution of red and artic foxes (Vulpes vulpes and Alopex lagopus)[END_REF]. Likewise, food selection and habitat complexity may also play an important role in distribution and abundance of marine organisms [START_REF] Duffy | Food and shelter as determinants of food choice by a herbivorous marine amphipod[END_REF][START_REF] Edgar | The influence of seagrass structure on the distribution and abundance of mobile epifauna: pattern and process in a Western Australia Amphibolis bed[END_REF].

Crustaceans exhibit one of the highest ranges of morphological diversity (Martin and Davis 2001). And of these, it is believed that approximately 40 percent of all belongs to the group Peracarida [START_REF] Kaestner | Invertebrate zoology[END_REF]. Most peracarids are bottom-dwelling animals, either infaunal or epifaunal; they have a wide variety of feeding habits, i.e. surface deposit feeding, filter feeding, carnivory, omnivory and even foraminiferivory [START_REF] Gudmundsson | Are foraminifers (Protozoa) important food for small isopods (Crustacea) in the deep-sea? Deep sea[END_REF]De Broyer et al. 2004;[START_REF] Riisgard | Surface deposit feeding versus filter feeding in the amphipod Corophium volutator[END_REF][START_REF] Krapp | Sympagic occurrence of eusirid and lysianassoid amphipods under Antarctic pack ice[END_REF][START_REF] Guerra-García | What do Caprellids (Crustacea: Amphipoda) feed on?[END_REF] and changes in food supply may influence the distribution of the species and the diversity patterns in the benthic environment. Furthermore, peracarids are being studied as bioindicators, since they have shown to be good indicators of environmental changes (Conradi andLópez-González 2001Guerra-García and[START_REF] Guerra-García | The Spatial Distribution of Caprellidea (Crustacea: Amphipoda): A Stress Bioindicator in Ceuta (Nort Africa, Gibraltar Area)[END_REF][START_REF] Ohji | Differences in the acute toxicities of tributylin between the Caprellidea and the Gammaridea (Crustacea: Amphipoda)[END_REF][START_REF] Guerra-García | Crustacean assemblages and sediment pollution in an exceptional case study: a harbour with two opposing entrances[END_REF][START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF]Guerra-García et al. 2009a).

Given that this group lacks pelagic larvae and their capacities for long-distance movement are limited in adults, the studies on biogeography focused on this group have been numerous in recent years (e.g. [START_REF] Chavanich | Rocky intertidal zonation of Gammaridean Amphipods in Long island sound[END_REF][START_REF] Thiel | The zoogeography of algae-associated peracarids along the Pacific coast of Chile[END_REF][START_REF] Castellanos | Isópodos marinos (Crustacea: Isopoda) de las islas Chafarinas (Mediterráneo occidental)[END_REF][START_REF] Broyer | Biodiversity patterns in the Southern Ocean: lessons from Crustacea, 8th SCAR International Biology Symposium[END_REF][START_REF] Chiesa | Species richness and faunistic affinities of the Gammaridea and Corophiidea (Amphipoda) from shallow waters of southern Tierra del Fuego, Argentina: preliminary results[END_REF][START_REF] Winfield | Updated checklist and identification of areas of endemism of benthic amphipods (Caprellidea and Gammaridea) from offshore habitats in the SW Gulf of Mexico[END_REF][START_REF] Myers | The biogeography of Indo-West Pacific tropical amphipods with particular reference to Australia[END_REF]. Nevertheless, research has not been so intensive in the Iberian Peninsula and the knowledge in this field is still fragmentary [START_REF] Bellan-Santini | Faunistics and zoogeography. In: Ruffo S (ed) The Amphipoda of the Mediterranean[END_REF]Guerra-García et al. 2009c,d) and mainly focused on the Strait of Gibraltar [START_REF] Conradi | Relationships between environmental variables and the abundance of peracarid fauna in Algeciras Bay (Southern Iberian Peninsula)[END_REF][START_REF] Castelló | Isopod fauna, excluding Epicaridea, from the Strait of Gibraltar and nearby areas (Southern Iberian Peninsula)[END_REF][START_REF] Guerra-García | The Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa, with the description of three species of Caprella, a key to the species of Caprella, and biogeographical discussion[END_REF].

However, there has been a slight increase on the interest of this region during the last decade (e.g. Ballesteros 1998;[START_REF] Cruz | Spatial distribution of peracarids in the intertidal zone of the Ria Formosa (Portugal)[END_REF][START_REF] Pereira | Biogeographic patterns of intertidal macroinvertebrates and their association with macroalgae distribution along the Portuguese coast[END_REF]Guerra-García et al. in press). The Iberian Peninsula is located in the southwest of Europe and is the westernmost of the three major southern European peninsulas. Its coasts are washed by the Mediterranean on the eastern side and Atlantic on the northern and western side, converging at the Strait of Gibraltar.

Corallina elongata (J. Ellis & Solander, 1786) (hereafter C. elongata) is a macroalga belonging to the family Corallinaceae (Rhodophyta). This species is widely distributed, occurring not only all along Mediterranean [START_REF] Flores-Moya | Contribución a la corología de las macroalgas marinas bentónicas del litoral Malagueño[END_REF][START_REF] Conde | Check-list of Andalusia (S. Spain) seaweeds. III. Rhodophyceae[END_REF][START_REF] Bárbara | Seaweeds of the Ría de A Coruña (NW Iberian Peninsula, Spain)[END_REF][START_REF] Babbini | Recensement de Corallinacées de la Mer Méditerranée et considérations phytogéographiques[END_REF] and Atlantic (Guiry 1977;[START_REF] Neto | Checklist of the benthic marine macroalgae of the Azores[END_REF][START_REF] Araujo | Checklist of benthic marine algae and cyanobacteria of northern Portugal[END_REF], where it has been extensively reported, but also in the Pacific [START_REF] Abbott | Marine red algae of the Hawaiian Islands[END_REF][START_REF] Lee | Marine algae of Jeju[END_REF]. It is a whitish-pink to reddish-lilac calcified algae, with articulated fronds and fish-bone-like arrangement. Its axis is compressed and repeatedly pinnate from discoid base. Coralline algae can be considered as ecosystem engineers, since they can modify the environment by providing shelter from desiccation stress, wave action and predation, enriching biodiversity within its structure [START_REF] Jones | Organisms as ecosystem engineers[END_REF][START_REF] Daleo | Negative effects of an autogenic ecosystem engineer: interactions between coralline turf and an ephemeral green alga[END_REF]. In extreme environments, intertidal coralline algal turfs ameliorate hard physical conditions, allowing the presence of many fauna that otherwise would not dwell at this level [START_REF] Bertness | The community structure of western Atlantic Patagonian rocky shores[END_REF]. Corallina elongata was selected in this study since it is one of the dominant macroalgae along the intertidal ecosystems of the whole Iberian Peninsula [START_REF] Pérez-Cirera | Principales tipos de vegetación bentónica y su zonación en el litoral comprendido entre las rías de Camariñas y de Corme y Lage (Costa de Camelle, La Coruña)[END_REF]Rodriguez-Prieto et al. 1997;[START_REF] Guerra-García | Assessing a quick monitoring method using rocky intertidal communities as a bioindicador: a multivariate approach in Algeciras Bay[END_REF]) and it was present in all sampling stations selected for the present work. Studies on biogeography of macrofauna associated with Corallina worldwide are scarce [START_REF] Dommasnes | On the fauna of Corallina officinalis L. in Western Norway[END_REF][START_REF] Fernández | Estudios estructurales del horizonte de Corallina elongata Ellis y Sol. en el sistema intermareal de Punta Carnero (Algeciras, Cádiz)[END_REF][START_REF] Bitar | Contribution to qualitative and quantitative studies of the macrozoobenthos from a community of Corallina mediterranea (Areschoug) established on the Moroccan Atlantic coast (Temara area)[END_REF][START_REF] Ballesteros | Composición y estructura de la comunidad infralitoral de Corallina elongata Ellis y Solander, 1786, de la Costa Brava (Mediterráneo occidental)[END_REF][START_REF] Kelaher | Spatial patterns of diverse macrofaunal assemblages in coralline turf and their associations with environmental variables[END_REF][START_REF] Kelaher | Influence of physical characteristics of coralline turf on associated macrofaunal assemblages[END_REF]Kelaher and Castill 2002;[START_REF] Kelaher | Experimental transplantations of coralline turf to demonstrate causes of differences in macrofauna at different tidal heights[END_REF][START_REF] Bertness | The community structure of western Atlantic Patagonian rocky shores[END_REF][START_REF] Bussell | Patterns in the invertebrate assemblage associated with Corallina officinalis in tide pools[END_REF][START_REF] Liuzzi | Macrofaunal assemblages associated with coralline turf: species turnover and changes in structure at different spatial scales[END_REF]Guerra-García et al. 2009b), especially in the Iberian Peninsula.

The main aim of this study was to demarcate the distribution and abundance patterns of benthic peracarids associated with the alga C. elongata in the intertidal zone along the whole coast of the Iberian Peninsula, and explore the relationship with some environmental variables such as oxygen concentration, turbidity, temperature, pH and conductivity

Material and methods

The present study encompassed the whole coasts of the Iberian Peninsula (Spain and Portugal) (Fig. 1). Nineteen stations were selected in order to cover the greatest range of natural environmental conditions (Figure 1). We chose relatively undisturbed enclaves with low human pressure to avoid the effect of anthropogenic influence on the natural biogeographical and ecological patterns of species.

Sampling was conducted in summer 2008 (June, July and August). The following environmental parameters were measured "in situ" at each sampling site: water temperature, pH, conductivity, dissolved oxygen and turbidity. Temperature and oxygen concentration were measured with an oxymeter CRISON OXI 45; pH and conductivity were measured using a conductivimeter-pHmeter CRISON MM40, and finally turbidity was measured in nephelometric turbidy units (ntu) using a turbidimeter WTW 335 IR. The seaweed C. elongata was selected as substrate and three replicates (quadrats 20 x 20cm) were sampled in each station. Corallina elongata was distributed along the low intertidal level and sublittoral in most of stations and the intertidal belt was selected for this study. The surface was scrapped and the seaweed and associated fauna were collected. The samples were fixed in ethanol 80%, brought to laboratory and sieved using a mesh size of 0.5 mm. Peracarid crustaceans were sorted and identified to species level. Volume of C. elongata of each replicate was estimated as the difference between the initial and final volume when placed into a graduated cylinder with a fixed amount of water (see [START_REF] Pereira | Biogeographic patterns of intertidal macroinvertebrates and their association with macroalgae distribution along the Portuguese coast[END_REF]. Dry weight was also measured (after 24 hours at 70ºC). To estimate the cover of C. elongata in each locality, we used five random quadrats of 50 x 50 cm subdivided with thick fishing line into 25 square units of 10 x 10 cm. The presence/absence of the species was recorded for each unit and data were expressed as cover percentage (mean ± SD of the 5 replicates).

Species richness (S), abundance (N), Shannon-Wiener diversity (H') (Shannon and Weaver 1963) and Pielou's evenness (J) (Pielou 1966) were measured for each station. Possible differences in these parameters between Mediterranean and Atlantic stations were tested by one-way ANOVA, after verifying normality using the Kolmogorov-Smirnov test and the homogeneity of variances using the Levene test. To explore the species replacement along the Atlantic and Mediterranean gradients, the Whittaker index [START_REF] Whittaker | Evolution and meaurement of species diversity[END_REF]) was calculated as measure of ß-diversity. The affinities among stations based on peracarid data were measured through cluster analysis using UPGMA method, based on the Bray-Curtis similarity index. The relationships between environmental measures and peracarid assemblages were studied by Canonical Correspondence Analysis (CCA) and BIO-ENV. Multivariate analyses were carried out using the PRIMER package [START_REF] Clarke | Primer (Plymouth Routines in Multivariate Ecological Research) v5: User Manual /Tutorial[END_REF]

Results

Abiotic data and abundance patterns of C. elongata

Regarding the measures taken for the environmental parameters, it was observed that the Mediterranean stations showed higher values of temperature and conductivity, as well as lower values of oxygen concentration and turbidity than the stations along the Atlantic coast (Fig. 2). Oppositely, pH remained rather unalterable amongst the surveyed sites, with an average of 8.16, ranging from 8 (station 6) to 8.33 (station 10).

Furthermore it was also seen that temperature was negatively correlated with oxygen (r=-0.69, p<0.01) and turbidity (r=-0.54, p<0.05) and positively with conductivity (r=0.73, p<0.01). Station 6 showed abnormal characteristics when compared with the rest of the stations, with the maximum value of turbidity and minimum value of conductivity and temperature.

When focusing on the abundance patterns of C. elongata, the data obtained showed a negative correlation between cover of the seaweed and seawater temperature (r=-0.491, p<0.05); thus, cover of C. elongata on Mediterranean stations was in general below 30%, while cover on the Atlantic stations was closer to 50% (Fig. 3). Biomass, however, did not show significant difference between the Mediterranean and Atlantic, ranging approximately from 500 to 1500 g/m 2 . Therefore, although Mediterranean intertidal was characterised by lower cover percentages of C. elongata, the patches in which the alga was present showed a similar biomass to Atlantic stations. Furthermore, values of volume of C. elongata were significantly correlated with dry weight (r=0.95, p<0.001, n=19).

Peracarid assemblages

A total of 28215 specimens were sorted and examined, out of which more than 87% were crustaceans of the superorder Peracarida, followed in importance by molluscs and annelids. In this study 78 different species of peracarids were identified: 57 species of amphipods, 16 isopods, 4 tanaids and 1 cumacean (Table 1). Gammarids were the dominant group in the majority of stations, representing more than 70% of the total peracarids and 61% of the total macrofauna on average, except on stations 7 (Playa Azul) and 11 (Tarifa) where isopods and caprellids were respectively the dominant groups. The most common species collected during the present study were the caprellid Caprella penantis, the gammarids Hyale schmidtii, Hyale stebbingi, Jassa cf. falcata, Parajassa pelagica, and Stenothoe monoculoides, the isopod Ischyromene lacazei and the tanaid Tanais dulongii. Gammarids did not show any particular preference for either Atlantic or Mediterranean, being equally distributed along the whole Peninsula, whereas caprellids and tanaidaceans presented their highest populations in the stations of the Strait of Gibraltar, and isopods were more abundant in Atlantic stations (Fig. 4).

In connection with the descriptive parameters, the number of species per station ranged from 15 (station 9) to 38 (station 8) (Fig. 5). The highest abundances were measured in the Atlantic stations 2, 6 and 8. Anyway, no significant differences of species richness, abundance and Shannon diversity were found between Mediterranean and Atlantic. Only the Pielou index was significantly higher in the Mediterranean (F=5.3, p<0.05). The Whittaker index, measured between contiguous stations, showed higher values around the Strait of Gibraltar, indicating that the highest replacement of species corresponded to this area (Fig. 6) When examining the cluster based on Bray-Curtis similarity, two main groups can be differentiated. One group included stations 1-8 (Atlantic) and the other one clustered stations 9-19 (Mediterranean and Strait of Gibraltar together) (Fig. 7). Figure 8 and table 2 show the results of the Canonical Correspondence Analysis (CCA). First axis, which absorbed 17.9% of total variance, was significantly correlated with temperature, conductivity, turbidity and oxygen, whereas the second axis mainly correlated with the algal cover. Species such as the gammarids Ampelisca unidentata, Elasmopus vachoni, Ampithoe riedli, Jassa ocia or Elasmopus rapax, isopods such Gnathia sp, or tanaids such as Synapseudes shinoi were associated with stations characterised by higher temperature and conductivity. Caprella penantis, Idotea species, Gammaropsis maculata, Gammarellus homari, Hyale pontica among others were found in oxygenate waters with high levels of turbidity. The highest correlation in the BIO-ENV was measured for conductivity (r=0.55, p<0.05) as unique variable, which was the best to explain peracarid distribution. All the remaining combinations of variables showed lower correlation values.

The classification of species in geographical distribution groups (Fig. 9, Table 1) shows that most species have an Atlantic-Mediterranean distribution (60%). Only the gammarids Gammarellus homari and Peltocoxa damnoniensis, and the isopod Idotea granulosa are restricted to the Atlantic. In connection with the endemic species, only the species Ampithoe riedli, Jassa cadetta, Lembos spiniventris, Stenothoe dollfusi, Cumella limicola, and Dynamene torelliae were Mediterranean endemisms.

Discussion

The peracarid crustaceans constitute the dominant group of epiphytic macrofauna in the intertidal region (Guerra-García et al. in press) and are useful as bioindicators (e.g. [START_REF] Conradi | The amphipod community as a bioindicator in Algeciras Bay (Southern Iberian Peninsula) based on a spatiotemporal distribution[END_REF][START_REF] Sánchez-Moyano | The arthropod community, especially crustacean, as a bioindicator in Algeciras Bay (Southern Spain) based on a spatial distribution[END_REF][START_REF] Guerra-García | The Spatial Distribution of Caprellidea (Crustacea: Amphipoda): A Stress Bioindicator in Ceuta (Nort Africa, Gibraltar Area)[END_REF][START_REF] Ohji | Differences in the acute toxicities of tributylin between the Caprellidea and the Gammaridea (Crustacea: Amphipoda)[END_REF][START_REF] Dauvin | Polychaete/amphipod ratio revisited[END_REF]. On the other hand, C. elongata is one of the most important algae in the intertidal ecosystems (Pérez-Cicera and Maldonado 1982;[START_REF] Guerra-García | Assessing a quick monitoring method using rocky intertidal communities as a bioindicador: a multivariate approach in Algeciras Bay[END_REF]. Consequently, the study of the peracarids associated to C. elongata in the Iberian Peninsula is especially relevant.

The most relevant aspect of this study lies on the faunistic contribution: seventy eight species of peracarids have been identified during the present study, most of them widely distributed along the Atlantic and Mediterranean, and patterns of abundance along the whole Iberian Peninsula are also included (see Table 1). The percentages of Mediterranean endemics were very low (9%). These results agree with Guerra-García et al. (2009b) which studied the peracarids associated to C. elongata in the Strait of Gibraltar and found 40 species in the 25 selected stations. In the present study we found 42 species in the 3 stations located in the Strait of Gibraltar (see stations 10, 11 y 12 in Fig. 1) with similar biogeographical distribution. The percentage of Mediterranean endemics is very low (3-5% for the Strait and 9% if we consider the whole Iberian Peninsula) when compared to other studies (see [START_REF] Bellan-Santini | Faunistics and zoogeography. In: Ruffo S (ed) The Amphipoda of the Mediterranean[END_REF][START_REF] Conradi | The benthic Gammaridea (Crustacea, Amphipoda) fauna of Algeciras Bay (Strait of Gibraltar): distributional ecology and some biogeographical considerations[END_REF][START_REF] Guerra-García | The Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa, with the description of three species of Caprella, a key to the species of Caprella, and biogeographical discussion[END_REF]. The low number of Mediterranean endemics found for peracarids associated to C. elongata (see also Guerra-García et al. 2009b) contrast with the 37% of endemic amphipods reported by Bellan-Santini and Ruffo (1998), even higher than the 26.6% calculated by [START_REF] Fredj | État des connaissances sur la faune marine méditerranéenne[END_REF] for all the Mediterranean fauna as a whole. [START_REF] Conradi | The benthic Gammaridea (Crustacea, Amphipoda) fauna of Algeciras Bay (Strait of Gibraltar): distributional ecology and some biogeographical considerations[END_REF] also reported a high endemic benthic Gammaridea fauna (18.3% endemics) from Algeciras Bay (Iberian side of the Strait of Gibraltar). Guerra-García et al. (2009b) explained this low endemicity on the basis of the distribution of C. elongata, which is an unspecific substrate widely distributed along the Atlantic and Mediterranean intertidal ecosystems. The peracarid fauna associated to this alga seems to have a low endemic component but a high percentage of lessepsian species (group IV) due to colonization in progress by Indo-Pacific elements via the Suez Canal (Por 1971).

Multivariate analyses based on peracarid abundance clearly showed two groups of stations (Mediterranean versus Atlantic) with stations of the Strait of Gibraltar clustered together with Mediterranean stations. This pattern was also obtained for the caprellid community associated to the seaweed Stypocaulon scoparium (see Guerra-García et al. 2009d) so the present study also reflects that the fauna of the Strait of Gibraltar seems to be more similar to the Mediterranean than to the Atlantic. In any case, along the Iberian Peninsula, the Strait is the area with higher rates of species replacement or turnover, as measured by the Whittaker index in the present study.

Among the physicochemical parameters, hydrodinamism and sedimentation rate are reported in literature as two of the most relevant factors to structure marine communities [START_REF] Moore | Particulate matter in the sublittoral zone of an exposed coast and its ecological significance with special reference to the fauna inhabiting kelp holdfasts[END_REF][START_REF] Dodds | Community interactions between the filamentous alga Cladophora glomerata (L.) Kuetzing, its epiphytes, and epiphyte grazers[END_REF], Gibbons 1998), together with temperature, conductivity, availability of food, predation and habitat structure (Duffy and Hay 2000;[START_REF] Dauby | Trophic diversity within the eastern Weddell Sea amphipod community[END_REF]). In the present study, according to the CCA and BIO-ENV, conductivity was the variable which better explained peracarid distribution along the Iberian Peninsula. Mediterranean is characterised by higher conductivity and temperature and lower values of dissolved oxygen and turbidity. Tanaids showed preferences for Mediterranean stations while isopods were more abundant in Atlantic stations. Amphipods did not show a clear general pattern as a group, since different species showed distinct distribution pattern. The unusual conditions measured in station 6 (low conductivity and oxygen and high turbidity) could be related to rivers influence discharging fresh water. These conditions seem to be excellent for some species, such as Parajassa pelagica. On the other hand, species of the genus Hyale were the most abundant in the present study. In fact, the genus Hyale is well adapted to life among algae of the higher littoral levels [START_REF] Tararam | Vertical distribution of amphipods living on algae of brazilian intertidal rocky shores[END_REF][START_REF] Baldinger | The occurrence of amphipods and other peracarid crustaceans in the rocky littoral zone of Bermuda[END_REF].

In the present study, although there were no significant differences in the number of species and Shannon diversity between Atlantic and Mediterranean stations, some species are substituted by others along the Atlantic-Mediterranean axis. Most of the substitution events affect species of the same genera, probably related to sympatric speciation. Some examples are the substitution of the species Jassa falcata (Atlantic) by J. cadetta (Mediterranean), Apherusa jurinei (Atlantic) by A. mediterranea (Mediterranean), Ampithoe helleri (Atlantic) by A. riedli (Mediterranean) or Stenothoe monoculoides (Atlantic) by S. tergestina (Mediterranean). Nevertheless, there are other factors that could also affect, such as competition or facilitation among species. For example, [START_REF] Viejo | Interactions between mesograzers inhabiting Fucus vesiculosus in northern Spain[END_REF] showed that the feeding activity of the isopod Dynamene bidentata was beneficial for the amphipod Hyale nilsonii, by providing suitable feeding surface on Fucus vesiculosus. A similar interaction seems to occur also in Tarifa Island between H. perieri, D. bidentata and the alga F. spiralis (Guerra-García et al. unpublished data). On the other hand, the higher Pielou index measured in the Mediterranean is probably due to high abundance values of some species in Atlantic stations such as Caprella penantis, Jassa falcata and Parajassa pelagica.

During the last decade, changes in the species distribution in marine ecosystems due to the climatic change are increasing [START_REF] Francour | Are the Mediterranean waters becoming warmer? Information from biological indicators[END_REF][START_REF] Walther | Ecological responses to recent climate change[END_REF][START_REF] Macdonald | Recent climate change in the Arctic and its impact on contaminant pathways and interpretation of temporal trend data[END_REF][START_REF] Harley | The impacts of climate change in coastal marine systems[END_REF][START_REF] Barber | The changing climate of the Arctic[END_REF]. Further studies are necessary to explore if observed patterns of the peracarid species are strictly consequence of natural factors or are already affected by the climatic change.

Figure legends

Figure 1. Study area showing the sampling station. Ogella (1), Oyambre (2), Cetarea (3), Baleo (4), Cabo Silleiro (5), Labruge (6), Playa Azul (7), Vale dos Homens (8), Castelo (9), Bolonia (10), Isla de Tarifa (11), Torreguadiaro (12), Cerro Gordo-Herradura (13), Cabo de Gata ( 14), Cala del tío Ximo (15), Benicassim-Oropesa ( 16), Torrent del Pi (17), Cala de Sant Françesc (18), L'Estartit (19).

  and the PC-ORD programme (McCune and Mefford 1997), whereas for univariate analyses the SPSS programme was used. Species were classified in geographical distribution groups (see also López de la Cuadra and García-Gómez 1994; Conradi and López-González 1999; Guerra-García et al. 2009b). For the present study, five groups were considered: I (Endemic Mediterranean), II (Atlantic and Mediterranean), III (Atlantic, absent in Mediterranean) IV (Atlantic, Mediterranean and Indo-Pacific) and V (Cosmopolitan)

Figure 2 .

 2 Figure 2. Abiotic variables measured in each station (Mean ± SD).

Figure 3 .

 3 Figure 3. Cover percentage and biomass of C. elongata (Mean ± SD) in each station.

Figure 4 .

 4 Figure 4. Abundance patterns of the peracarids associated with C. elongata.

Figure 5 .

 5 Figure 5. Descriptive parameters of the peracarid community measured in each station. Number of species is measured per station (considering the three replicates). Abundance, Shannon diversity and Pielou evenness are mean ± SD of the three replicates

Figure 7 .

 7 Figure 7. Cluster analysis based on the peracarid composition of each station.

Figure 8 .

 8 Figure 8. Graph representation of the stations and caprellid species with respect to the first two axes of the Canonical Correspondence Analysis (CCA).

Figure 9 .

 9 Figure 9. Percentage of species in the zoogeographical groups considered. I (Endemic Mediterranean), II (Atlantic and Mediterranean), III (Atlantic, absent in Mediterranean) IV (Atlantic, Mediterranean and Indo-Pacific) and V (Cosmopolitan).

Table 2 .

 2 Summary of the results of the CCA analysis. *p<0.05, **p<0.01, ***p<0.001. See also Fig. 8.

	Axis 1	Axis 2	Axis 3

Acknowledgements. Financial support of this work was provided by the Ministerio de Educación y Ciencia (Project CGL2007-60044/BOS) co-financed by FEDER funds, and by the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (Project P07-RNM-02524). Special thanks to J. Corzo, M.P. Cabezas and E. Baeza-Rojano for their collaboration during the sampling programme and to J.E. Sánchez-Moyano for his assistance in species identification.


Celine Denais

Carolyn L Dent

Laura Southgate

Jacqueline Hoyle

Dimitra Dafou

Professor Richard Charles Trembath
email: richard.trembath@kcl.ac.uk

David Rajiv

Machado

Rajiv D Machado

a protein integral to extracellular matrix and Golgi organisation and is associated with protein secretion pathways critical in bone development

Keywords: Dymeclin, Skeletal dysplasia, secretion, chondrogenesis, Golgi Dymeclin, Skeletal dysplasia, secretion, chondrogenesis, Golgi

    Dymeclin, the gene underlying Dyggve-Melchior-Clausen syndrome, encodes a protein integral to extracellular matrix and Golgi organisation and is associated with protein secretion pathways critical in bone development.

INTRODUCTION

The osteochondroplasias are a heterogeneous group of skeletal disorders, the result of inherited defects in bone formation, growth and maintenance [START_REF] Ortega | Matrix remodeling during endochondral ossification[END_REF][START_REF] Superti-Furga | Nosology and classification of genetic skeletal disorders: 2006 revision[END_REF]. To date, some 250 distinct clinical phenotypes have been reported with an incidence of 1 in 4000 across European populations [START_REF] Stoll | Birth prevalence rates of skeletal dysplasias[END_REF]]. Dyggve-Melchior-Clausen syndrome (DMC) is part of a sub-group of osteochondroplasias known as the spondyloepimetaphyseal dysplasias (SEMDs) characterised by malformations of the vertebrae, with abnormal ossification of long bone epiphyses and metaphyses. DMC is a progressive SEMD with additional clinical features that include microcephaly, facial dysmorphism and variable mental retardation [START_REF] Dyggve | Morquio-Ullrichs Disease : An Inborn Error of Metabolism?[END_REF][START_REF] Toledo | Dyggve-Melchior-Clausen syndrome: genetic studies and report of affected sibs[END_REF][START_REF] Beighton | Dyggve-Melchior-Clausen syndrome[END_REF]. Classical radiological features of DMC include the appearance of lacy iliac crests, consistent with widespread bone abnormalities of development and growth [START_REF] Spranger | The Dyggve-Melchior-Clausen syndrome[END_REF], driven by deficient chondrocyte organisation and differentiation with columnar structures that contain populations of degenerating cells [START_REF] Horton | Dyggve-Melchior-Clausen syndrome. A histochemical study of the growth plate[END_REF][START_REF] Nakamura | Dyggve-Melchior-Clausen syndrome without mental retardation (Smith-McCort dysplasia): morphological findings in the growth plate of the iliac crest[END_REF].

DMC is a rare recessive disorder predominantly due to loss-of-function mutations in the DYM gene (MIM ID: 607461) located at chromosome 18q12 [START_REF] Cohn | Mental retardation and abnormal skeletal development (Dyggve-Melchior-Clausen dysplasia) due to mutations in a novel, evolutionarily conserved gene[END_REF][START_REF] El Ghouzzi | Mutations in a novel gene Dymeclin (FLJ20071) are responsible for Dyggve-Melchior-Clausen syndrome[END_REF]. Defects in this gene also result in Smith-McCourt dysplasia (SMC), an allelic variant of DMC without mental retardation [START_REF] Ehtesham | Evidence that Smith-McCort dysplasia and Dyggve-Melchior-Clausen dysplasia are allelic disorders that result from mutations in a gene on chromosome 18q12[END_REF][START_REF] Cohn | Mental retardation and abnormal skeletal development (Dyggve-Melchior-Clausen dysplasia) due to mutations in a novel, evolutionarily conserved gene[END_REF]. The ubiquitously expressed DYM transcript encodes Dymeclin, a novel 669 amino acid protein under tight evolutionary conservation, which does not belong to a recognised protein family and has limited characteristics for functional motifs. Bioinformatic analyses predict the presence of an N-myristoylation site, several transmembrane loops and dileucine motifs but, to date, these have not been experimentally validated [START_REF] Cohn | Mental retardation and abnormal skeletal development (Dyggve-Melchior-Clausen dysplasia) due to mutations in a novel, evolutionarily conserved gene[END_REF][START_REF] El Ghouzzi | Mutations in a novel gene Dymeclin (FLJ20071) are responsible for Dyggve-Melchior-Clausen syndrome[END_REF]. In short, the cellular and molecular function of Dymeclin remains unclear, confounding efforts to better elucidate the processes critical in the genesis of DMC and SMC.

In the present study, the cellular functions of Dymeclin have been investigated by a combinatorial approach. This report is the first to delineate, in human patient cell lines characterised for Dymeclin loss-of-function mutations at the genomic and transcript level, the profound consequences for Golgi and extracellular matrix organisation. Using a zebrafish model, we demonstrate prominent head and cartilage DYM expression during early development. By means of two yeast two-hybrid screens of protein-protein interactions, we identified novel partners that provide insight into Dymeclin-associated cellular function, likely to be critical for chondrocyte development and maintenance.

Together, these data reveal Dymeclin driven processes central to bone development pathways, including Golgi organisation, Golgi-coupled protein secretion, and collagen deposition in the extracellular matrix, thereby illuminating the likely molecular mechanisms underlying DMC pathogenesis.

MATERIALS AND METHODS

Cell culture and transfection

Primary human wild-type (WT) fibroblasts were harvested from skin biopsies obtained under ethics committee approval. All three mutant fibroblast lines were obtained from the Coriell Institute (www.coriell.org), (Table 1). Primary lines, HeLa and U2OS cells were cultured in Dulbecco's modified Eagle's medium with Glutamax (DMEM, GIBCO life technologies) supplemented with 10% foetal bovine serum at 37 °C and 1% penicillin/streptomycin. Transient transfections of HeLa and U2OS cells were performed using Lipofectamine 2000 (Invitrogen) and FuGENE (Roche) respectively in accordance with manufacturers' instructions.

DNA, RNA isolation and RT-PCR

DNA and total RNA was isolated from fibroblast cell lines and whole zebrafish embryos using TRI Reagent (Sigma Aldrich) according to manufacturer's instructions. cDNA was generated by priming with oligo dT using the First strand synthesis SuperScript II kit (Invitrogen). Newly produced cDNA was used as a template in subsequent PCR reactions to amplify regions of the DYM transcript (Human: NM_017653.3, Zebrafish: NM_001002739.1). All human and zebrafish genomic and cDNA primers are available on request. Cycling conditions were as standard. Identified mutations in DMC patient samples were numbered at the nucleotide level on the basis that +1 corresponded to the A of the ATG translation initiation codon in the reference sequence.

Quantitative PCR

Quantitative PCR was carried out containing the TaqMan ABI 7700 with the ABI PRISM Detection System using the Hs00214264_m1 assay which spans the DYM exon 8/9 junction (Applied Biosystems). Briefly, 2 µg of total RNA was reverse transcribed and PCR performed according to manufacturer's instructions to a final volume of 20 µl. A

Yeast two-hybrid screen

The two bait constructs comprising Dymeclin amino acids 1-97 and 270-510 were generated by PCR amplification with full-length DYM cDNA as template and cloned into the yeast expression plasmid pGBDU-C1 [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF]. The yeast strain PJ69-4a was sequentially transformed with the bait plasmid and chondrocyte library using the lithium acetate method devised by Agatep et al [START_REF] Agatep | Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol[END_REF]. The screen was performed by transforming yeast containing the bait plasmid, maintained on SD media lacking uracil, with 60 µg of DNA from the human chondrocyte library. Clones capable of interaction with the bait constructs were selected for by plating transformants on SD/-URA/-LEU/-HIS/ media supplemented with 1 mM 3-aminotriazole (3-AT). To estimate the strength of interaction the clones were subsequently streaked on to minimal media also lacking adenine. The bait plasmid was removed by growing the yeast on SD/-LEU supplemented with 1 g/L 5-fluoroorotic acid to select against plasmids encoding the uracil gene. Plasmids from the chondrocyte library were isolated using the Zymoprep kit (ZymoResearch) and transformed into the E. coli strain DH5α. All library plasmid inserts were sequenced using a primer to the Gal4 activation domain and identified using the BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST).

GST pull-down assays

To generate glutathione S-transferase (GST) fusion proteins, PPIB and GOLM1 were cloned in frame to the 5′ tag in the vector pGEX-4T1 (primers available on request). The bacterial strain BL21 was freshly transformed with these constructs and single colonies grown overnight. Expression of the fusion proteins were induced by the addition of 0.4 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h at 37 °C. Bacterial lysis was achieved by sonication and the extracts were incubated with washed Glutathione-Sepharose 4B beads (Amersham-Pharmacia). [ 35 S]methionine labelled full-length Dymeclin was produced by in vitro translation using the TnT T7-coupled transcription reticulocyte lysate system (Promega) following manufacturer's instructions. 20 µl of the translation reaction was added to purified GST fusion protein immobilised on the beads along with 100 mM PMSF and protease inhibitor cocktail (Roche) at a 7 × concentration.

The final volume was adjusted to 750 µl with cold NETN buffer (0.5% Nonidet P-40, 1 mM EDTA, 20 mM Tris-HCl pH 8, 100 mM NaCl) and the mixture rotated for 90 min at 4 °C. After three washes in NETN buffer, proteins were dissociated from the beads by incubation in 2 × laemmli buffer and separated on a 10% SDS-PAGE gel. A 10% volume of the labelled protein used in the GST pull-down was similarly resolved. Gels were stained in Coomassie blue buffer and exposed to autoradiograph film for up to 5 days. Immunoprecipitation experiments were conducted on transiently transfected HeLa or U2OS cells in 10 cm dishes. Cells were lysed in 1 ml lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM CaCl 2 , 1% Nonidet P-40, 5 mM EDTA and protease inhibitor cocktail). Extracts were incubated overnight at 4 °C with either monoclonal antibody against myc (Sigma Aldrich, clone 9E10) or anti-Flag antibody (Sigma Aldrich, F7425).

Immunoprecipitation and immunoblotting

Protein A Sepharose CL 4B beads reconstituted as a 50% slurry (Amersham) were added to the extracts and incubated for 2 h at 4 °C. Following 5 washes in lysis buffer, protein was eluted into 2 × laemmli buffer and resolved by SDS-PAGE. Proteins were transferred onto nitrocellulose and membranes were probed with myc or Flag antibodies diluted 1/500 in blocking buffer (5% milk powder, 0.1% Tween-20 in PBS). Secondary antibodies were HRP conjugated and diluted according to manufacturer's instructions.

Proteins were detected by chemiluminescence using the EZ-ECL kit (GE Healthcare).

Scanning laser confocal microscopy

Prior to transfection, cells were plated on acid treated coverslips contained in 6-well plates. Transfected cells were washed in PBS, fixed and permeabilised with absolute methanol for 10 min at -20 °C. After blocking with PBS/1%BSA for 1 h at room temperature cells were stained with primary antibodies diluted according to manufacturer's instructions (anti-myc, Abcam, 9E10;anti-GFP, Santa Cruz, sc-8334;Giantin, Abcam, 24586;PDI, Stressgen, SPA-891;anti-PPIB, Sigma Aldrich, HPA012720). Anti-mouse Alexa 488 and anti-rabbit Alexa 555 were employed as

Electron microscopy

Cells were plated onto acid-treated glass coverslips (13 mm in diameter) to a density of 0.5 × 10 5 in 24-well CellBIND Cell Culture Plates (Corning). After reaching sufficient confluency, cells were processed for microscopy analysis by washing with 1 × PBS followed by fixation in 2.5% glutaraldehyde in 0.1 M sodium-cacodylate buffer, pH 7.4.

Samples were negatively stained with 1% uranyl acetate in water. Stained sections were examined on a FEI Tecnai T12 BioTWIN transmission electron microscope and images captured on a Gatan BioScan Model 792 MSC SI003 1 camera.

Proliferation assay

Cells to be tested were pelleted and CyQUANT GR dye/lysis buffer was added to each cell pellet. A cell dilution series ranging from 100 to 50 × 10 4 cells was created in a 96well cell culture microplate (Corning) with CyQUANT GR dye/lysis buffer, in final volumes of 200 µl per well. A control 200 µl well with no cells (CyQUANT GR dye/lysis buffer only) was also prepared. The samples were incubated in darkness for 2 to 5 min at room temperature. The fluorescence of each sample was measured with a CytoFluor 2350 fluorescence microplate reader, with 485 nm (±10 nm) excitation and 530 nm (±12.5 nm) emission filters. For each experiment, a standard calibration curve was generated with measured fluorescence values versus cell number, as determined from cell suspensions using a haemocytometer (6-well culture plates of cells were harvested on days 1 to 8).

Apoptosis analysis

Stress-induced apoptosis was quantified and differentiated from necrosis using the Annexin-V-FLUOS direct fluorescence staining kit (Roche) according to the manufacturer's instructions, which allows simultaneous staining of cell surface phosphatidylserine (with Annexin-V-FLUOS) and necrotic cells (with propidium iodide).

Apoptotic cells were measured in mass cultures wherein cells were grown beyond the stage of confluence to achieve stress-induced apoptosis [START_REF] Tannock | Evidence against apoptosis as a major mechanism for reproductive cell death following treatment of cell lines with anti-cancer drugs[END_REF]. 1 × 10 6 cells were then washed with PBS, stained with Annexin V and Propidium Iodide for 15 min at room temperature and analysed using a Becton and Dickinson Aria II flow cytometer. Experiments were performed in triplicate and a student t-test was used to measure significance.

Whole-mount in situ hybridisation

Digoxigenin (DIG) labelled riboprobe was synthesised from plasmids harbouring fulllength zebrafish dymeclin using the DIG RNA labelling kit (Roche). In situ hybridisation was performed as described previously [START_REF] Jowett | Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin-and fluorescein-labelled probes[END_REF]. Briefly, prehybridisation, followed by probe hybridisation at 1/100 dilution was conducted at 68 °C overnight. The embryos were incubated with alkaline phosphatase-conjugated anti-DIG antibody (Roche). The embryos were allowed to develop colour in nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3'-indolyphosphate-p-toluidine salt (NBT/BCIP) (Roche) in developing buffer for 1.5 h. The embryos were photographed with a Nikon FMZ microscope.

RESULTS

Identification and characterisation of DYM mutations in patient cell lines

DNA extracted from the three fibroblast lines (DMC10568, DMC02437 and DMC04997) was screened for DYM mutations by direct sequencing. In DMC10568, a homozygous single base pair deletion was identified within exon 10 (c.1004delT) predicting a frameshift and the incorporation of an aberrant termination codon 65 amino acids downstream (p.L335CfsX65). Homozygous acceptor splice site mutations were detected in DMC02437 and DMC04997 in intron 10 (c.1126-1G>A) and intron 11 (c.1252-1G>A) respectively (Fig. 1A). Sequence analysis of gel excised bands from patient cDNA indicated complete skipping of exon 11 in DMC02437 resulting in an in-frame truncated mRNA (Fig. 1B). Two major splice isoforms were identified in DMC04997, namely an in-frame retention of the terminal 219 bases of intron 11, and a deletion of the first 28 bp of exon 12 leading to a loss of frame (Fig. 1B). The gene defects identified in samples DMC02437 and DMC04997 represent novel mutations. Quantitative RT-PCR confirms the effect of all mutations to be a considerable reduction in levels of the Dymeclin transcript (Fig. 1C).

Dymeclin is localised to the Golgi apparatus and cytoplasm

We sought to establish the distribution of Dymeclin in HeLa cells and the osteosarcoma line U2OS using transiently transfected GFP-tagged constructs, due to the absence of a Giantin, a marker of the cis-Golgi, demonstrated a high degree of co-localisation indicating punctate enrichment of Dymeclin in the Golgi of both cell lines (Fig. 2A). By contrast, no co-localisation was observed with PDI, a protein integral to the endoplasmic reticulum (ER) (Fig. 2B).

Phenotypic profiling of cells harbouring Dymeclin mutations

Visualisation of Golgi and ER structure by confocal microscopy revealed significant differences between WT and mutation-positive fibroblasts. In all three DMC lines, the Golgi was markedly distended and disorganised by comparison to WT cells (Fig. 3A).

These changes to organelle structural conformation are unlikely to be due to impaired cytoskeletal scaffolding as there was no demonstrable evidence of disruption to either the microtubular or actin networks in mutant cells (data not shown). Notably, electron micrographs of fibroblasts from DMC04997 illustrate that by contrast to the WT control, DMC fibroblasts have a significantly lower density of collagen fibrils on the cell surface (Fig. 3B). Measurement of the proliferative capacity of the three mutant cell cultures revealed no significant differences to controls over a period of eight days (Fig. 3C). The apoptotic cell population, measured by FACS analysis, was also unaltered under normal culture conditions (data not shown). However, a significantly higher early apoptotic cell population was observed for the mutants when stressed-induced apoptosis was achieved by growing cells beyond the level of confluence (Fig. 3C). Golgi were still evident upon the introduction of 0.5 µg of exogenous wild-type Dymeclin. However, increasing the amount of transfected Dymeclin to 1 µg effectively reversed abnormal Golgi conformation and positioning in mutant cells to a largely normal phenotype (Fig. 4B). Taken together, these data indicate that the impact of Dymeclin loss on sub-cellular morphology is a striking dysregulation of Golgi structure and impaired deposition of extracellular collagen.

Dymeclin expression during zebrafish embryonic development

The temporal expression of the dym transcript was analysed in zebrafish embryos by RT-PCR at different developmental time points ranging from the 32-cell stage and up to 48 hours post-fertilisation (hpf). Dymeclin was strongly expressed in early embryo development but decreased dramatically by 48 hpf (Fig. 5A). As active zygotic expression is considered to occur at the blastula stage these data signify that dymeclin is highly expressed both maternally and zygotically. To examine the spatio-temporal expression of the gene, whole mount in situ hybridisation in zebrafish was performed at 1, 2, 3 and 5 days post fertilisation (dpf). At all experimental time points studied, dym staining was highly evident in the head region, specifically the fore-, mid-and hindbrain.

To better examine the developing jaw, the eyes were removed after 5 dpf. Dymeclin, at this stage, was highly expressed in cartilaginous structures, in particular, the mandibular arch otherwise referred to as Meckel's cartilages and in ceratobranchial arches (Fig. 5B).

These expression studies suggest a prominent role for dymeclin in brain and cartilage formation at the earliest stages of embryonic development.

Novel Dymeclin interacting partners identified by yeast two-hybrid screens

To uncover Dymeclin-associated pathways of potential importance to bone development and regulation, the Gal4 yeast two-hybrid system was used to screen a human chondrocyte library using two bait constructs in independent experiments. Both baits were confirmed as non-toxic and neither auto-activated the yeast auxotrophic reporter genes. Each screen was conducted under high stringency conditions to select for the strongest and most stable interactions. Of the putative protein partners identified in these assays two were prioritised for further study, namely PPIB and GOLM1, identified as binding the predicted N-terminal myristoylation domain and the conserved central region of Dymeclin respectively. This approach yielded a novel set of protein interactions to those previously described in the literature, yet the present findings complement and expand on the emerging hypothesis that the wider Dymeclin network predominantly comprises proteins with roles in ER-Golgi secretion and export [START_REF] Osipovich | Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic[END_REF]. To substantiate the interaction between Dymeclin and proteins identified in the yeast twohybrid screen, GST pull-down assays were performed. Following incubation with GSTtagged PPIB and GOLM1, 35 S radiolabelled full-length Dymeclin was successfully recovered with each protein but not with GST alone, thereby validating physical interaction (Fig. 6A). To investigate these interactions in a mammalian in vivo setting, myc-tagged WT Dymeclin was used in immunoprecipitation experiments with FLAGtagged PPIB and GOLM1. Subsequent immunoblotting revealed co-precipitation of both proteins with Dymeclin. In control experiments, lysates from cells transfected with myc-Dymeclin and empty FLAG vector did not show the presence of bands pertaining to PPIB and GOLM1 (Fig. 6B). Additionally, the specificity of the interaction with these proteins was indicated by the inability of Dymeclin to bind the randomly chosen mitochondrial protein, SOD2 (data not shown).

In vitro and in vivo

Co-localisation of full-length Dymeclin and PPIB in HeLa cells

The sub-cellular localisation of myc-Dymeclin and PPIB in HeLa cells was examined by indirect immunofluorescence using antibodies against the myc tag and native PPIB.

When expressed alone PPIB was dispersed throughout the cytoplasm while Dymeclin, as previously described, was cytosolic with punctate Golgi staining. However, when coexpressed, PPIB co-localised with Dymeclin and displayed a perinuclear distribution, likely aggregating within Golgi and/or ER structures (Fig. 6C). As GOLM1 has long been established to localise to the same cis-Golgi structures as Dymeclin, these data were not replicated in this study [START_REF] Bachert | Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GP73 produces marker for hepatocellular carcinoma[END_REF].

DISCUSSION

To address the physiological role of Dymeclin and mechanistic basis of DMC disease precipitation and progression, this report utilised a combined approach employing molecular, imaging, proteomic and model system studies.

We utilised a resource of three human DMC fibroblast cell lines to elucidate the effects of Dymeclin mutation on organelle morphology. The cell lines were first characterised at the genomic and transcript level for DYM mutations. The splice junction mutations detected in patient lines resulted in heterogeneous consequences for the transcript; in one case both in-frame intronic retention and exonic skipping resulting in premature truncation. However, quantitative PCR performed on all available lines demonstrated a profound loss of DYM mRNA, thus indicating that insignificant levels of translational product was likely to be generated in our patient series.

In this study, we have reliably demonstrated in vitro that Dymeclin is indeed highly concentrated in the Golgi apparatus but also present in the cytosol. These observations are in accordance with recent publications and suggest that Dymeclin is a peripheral nonanchored Golgi protein capable of shuttling between cellular compartments [START_REF] Osipovich | Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic[END_REF][START_REF] Dimitrov | The gene responsible for Dyggve-Melchior-Clausen syndrome encodes a novel peripheral membrane protein dynamically associated with the Golgi apparatus[END_REF]. Immunofluorescence analysis of various cellular compartments emphasised the critical importance of Dymeclin to Golgi structure. In all cell lines analysed, Golgi disorganisation was the most prominent pathogenic feature. functionally related to families of structural Golgi proteins including the SNAREs and GRASPs that act in Golgi ribbon formation and/or tethering [Shorter and Warren, 1999;Shorter et al., 1999;[START_REF] Wang | A direct role for GRASP65 as a mitotically regulated Golgi stacking factor[END_REF]. The developing skeleton has been shown to be particularly affected by mutations in genes disrupting protein secretion and Golgi structure. Electron microscopic analysis of patient fibroblasts noted a marked loss of collagen fibrils surrounding the cell [START_REF] Nakamura | Dyggve-Melchior-Clausen syndrome without mental retardation (Smith-McCort dysplasia): morphological findings in the growth plate of the iliac crest[END_REF][START_REF] El Ghouzzi | Mutations in a novel gene Dymeclin (FLJ20071) are responsible for Dyggve-Melchior-Clausen syndrome[END_REF].

Restoration of Golgi structure and juxta-nuclear positioning in patient lines by the overexpression of wild-type protein is a compelling indication that Dymeclin may be

This would suggest impairment in the formation of the extracellular matrix, fundamental for chondrogenesis and bone development.

Patient cells displayed normal rates of proliferation and under standard culture conditions apoptosis also appeared unaffected. However, DMC cells apoptosed at a significantly higher level than WT controls when the process was forced. This finding would suggest that cells lacking Dymeclin are intrinsically more fragile and cell death more likely to occur when the micro-environment is challenged.

By contrast to the mouse and human, embryogenesis in zebrafish can be monitored from the earliest stages as embryos develop outside the mother's body [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF].

We studied temporal dym expression in this developmental system from the pre-blastula stage to full embryo formation (48 hpf). Dym, detected as both a maternal supply and, at the blastula stage, as a zygotic transcript was highly expressed throughout embryogenesis indicating the likely requirement of the protein to embryological developmental processes. Embryonic spatio-temporal expression of dym, examined by whole-mount in situ hybridisation of zebrafish embryos, revealed high levels of the transcript in the Deleted: Electron microscopic analysis of patient fibroblasts confirmed previous observations of an enlarged ER and abnormally high vacuolation but, importantly, we noted a marked loss of collagen fibrils surrounding the cell. Both in vitro and in vivo GOLM1 and PPIB displayed high affinity for Dymeclin. GOLM1, a protein of unclear function, stably localises to the cis-Golgi under steady-state conditions. However, when levels of the serine protease furin are increased, typically by TGF-β ligands, the GOLM1 ectodomain is cleaved and secreted via the trans-Golgi and endosomal network into the cytosol and extracellular space [START_REF] Bachert | Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GP73 produces marker for hepatocellular carcinoma[END_REF]. Under physiological conditions, furin cycles from cell membrane to the trans-Golgi network and is dispersed throughout the cell in stark contrast to Dym deficient mouse cells where furin accrues in the Golgi [START_REF] Nakayama | Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins[END_REF][START_REF] Rockwell | Precursor processing by kex2/furin proteases[END_REF][START_REF] Osipovich | Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic[END_REF].

This abnormal accumulation of furin in DMC cells would result in pathogenically high levels of GOLM1 cleavage and release, albeit with unknown functional consequences. PPIB, also known as cyclophilin B, belongs to a highly conserved protein family associated with protein post-translational modification and is resident in the ER of all cell types [START_REF] Price | Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence[END_REF]. PPIB directly binds procollagen in complex with P3HI and CRTRAP and directs it to the Golgi to promote biosynthesis of the mature protein [START_REF] Price | Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence[END_REF]. Collagen is a key element in generating the osteoid framework required for bone formation [START_REF] Riminucci | Building bone tissue: matrices and scaffolds in physiology and biotechnology[END_REF]. PPIB mutations generating null alleles in mouse and human lead to the condition osteogenesis imperfecta, characterised by bone fragility due to osteoporosis, reduced bone mass and growth defects [START_REF] Choi | Severe osteogenesis imperfecta in cyclophilin Bdeficient mice[END_REF][START_REF] Van Dijk | PPIB mutations cause severe osteogenesis imperfecta[END_REF][START_REF] Barnes | Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding[END_REF]. Of note, collagen accumulates in the ER of mutant mouse cells with entry into Golgi significantly delayed. Although collagen is secreted to the cell surface, fibril formation and morphology is abnormal [START_REF] Choi | Severe osteogenesis imperfecta in cyclophilin Bdeficient mice[END_REF]. As described here and by others Dymeclin plays a central role in multiple protein trafficking pathways opening up the possibility that Dymeclin provides the link between collagen processing by the PPIB complex and vesicular transport [START_REF] Osipovich | Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic[END_REF]. The loss of interaction between Dymeclin and PPIB may be the mechanistic basis underlying the paucity of collagen fibres observed on the surface of DMC cells by ultrastructural analysis. When taken together, these data underscore a prominent physiological role for Dymeclin in stabilising the Golgi network for effective protein secretion and, further, implicate the PPIB pathway interaction as potentially key to the pathogenesis of DMC.

Through the use of genetically characterised human patient fibroblast lines, this study establishes Dymeclin as integral to Golgi and extracellular matrix structure and provides novel insights into the cellular basis of DMC, in particular the impact of mutation on apoptosis. Expanding the network of interacting proteins consolidates a central position for Dymeclin in intracellular protein secretion and, additionally, identifies specific pathways that provide insight into both cartilage/bone formation and the molecular mechanisms underlying DMC. These data would imply that the molecular basis of DMC shares a common aetiology with other skeletal dysplasias, for example, X-linked spondyloepiphyseal dysplasia tarda and achondrogenesis type 1A [START_REF] Gedeon | The molecular basis of X-linked spondyloepiphyseal dysplasia tarda[END_REF][START_REF] Smits | Lethal skeletal dysplasia in mice and humans lacking the golgin GMAP-210[END_REF]. These findings are complemented by zebrafish studies demonstrating early and specific dymeclin expression in neural and cartilage development.

Formatted

  the cDNA template was used as a control. The housekeeping gene, cyclophilin A (Hs99999904_m1) was used as an internal control. Each experiment was performed in triplicate. For data evaluation, resulting Ct values acquired in real-time qPCR for Dymeclin transcripts were normalized to the Ct values of cyclophilin A transcripts to yield the ∆Ct value. Relative expression level (fold change) of Dymeclin in each experimental sample was calculated using 2-∆∆Ct method where ∆Ct = Ct (target gene) -Ct (reference gene) and ∆∆Ct = ∆Ct (treated) -∆Ct (control).

  secondary antibodies. Before examination, coverslips were mounted with Vectashield containing DAPI (Vector labs) to facilitate nuclear staining. Confocal microscopy was Zeiss LSM510 laser scanning microscope (Zeiss, Thornwood, NY) using a Zeiss 63 × 1.4 numerical aperture oil immersion lens.

  Sample fluorescence was measured as above, and growth curves were plotted as fluorescence versus time.

  the native protein. Confocal microscopy revealed the presence of Dymeclin in the cytosol but with marked focal concentrations. Co-staining with

  Due to the marked perturbation of Golgi structure in Dymeclin deficient patient lines, we sought to further interrogate the role of Dymeclin in regulating Golgi morphology. Overexpression of WT Dymeclin in HeLa and U2OS lines resulted in substantive condensation of the Golgi by comparison to cells transfected with GFP alone and neighbouring untransfected cells (Fig.4A). To exclude the possibility that the Golgi compaction was due to transfection based toxicity mock transfections were used as a control. To further corroborate these observations we next over-expressed wild-type Dymeclin in mutant fibroblasts exhibiting grossly distended Golgi structures. Abnormal

  cartilage structures, closely correlating with the key sites of disease in DMC.

  Future

  be aimed at further elucidating the functional relationship between Dymeclin and identified interactors to further interrogate pathways implicated in chondrogenesis and bone development.

Figure 1 .

 1 Figure 1. Genetic analysis of DMC fibroblast cell lines. (A) Chromatograms of genomic mutations identified in the three DMC lines, altered bases are indicated by arrows and base deletion by a triangle. (B) Effect of the splice junction mutations was examined by RT-PCR and gel electrophoresis. The expected PCR products in WT controls and those observed in the mutant samples are marked. Filled arrowheads show the two splice isoforms observed in DMC04997. In each case, aberrant products were gel excised and sequenced to determine the effect of mutations on the transcript. Chromatograms of WT controls are displayed to the left. (C) Levels of DYM transcript measured by quantitative RT-PCR show greatly reduced expression in all mutants by comparison to the WT control fibroblast line. Measurements were standardised against the housekeeping gene cyclophilin A. Identified mutations in DMC patient samples were numbered at the nucleotide level on the basis that +1 corresponded to the A of the ATG translation initiation codon in the reference sequence.

Figure 2 .

 2 Figure 2. Dymeclin localises to the cytosol and Golgi. (A) Focal co-localization is observed with Giantin (red), a component of the Golgi apparatus, 24 hrs post-transfection of GFP-Dymeclin (green). White arrows indicate areas of overlapping fluorescence. (B) By contrast, GFP-Dymeclin (green) displays no evidence for co-localization with the ER marker PDI (red).

Figure 3 .

 3 Figure 3. Morphological consequences of Dymeclin loss-of-function. (A) The Golgi and ER in wild-type (WT) fibroblasts and three patient lines were visualised by Giantin (red)

Figure 4 .

 4 Figure 4. Over-expression of Dymeclin leads to Golgi compaction. (A) Comparative to fibroblasts transfected with empty GFP vector increasing amounts of WT GFP-Dymeclin (0.5µg -2µg) cause condensation of the Golgi, stained with Giantin (red). Golgi position in transfected cells is indicated by the white arrows. (B) WT GFP-Dymeclin transfected into DMC lines leads to progressive restoration of Golgi morphology, indicated by the white arrows, by comparison to neighbouring untransfected cells, where the Golgi is identified by double-headed arrows.

Figure 5 .

 5 Figure 5. Dymeclin expression in zebrafish embryos. (A) RNA recovered from labelled stages of development was reverse transcribed and used to PCR amplify a 1500 bp fragment of the Dymeclin transcript. Expression is consistently high in early development but decreases by 2 days post-fertilisation. (B) In situ hybridisation reveals intense staining throughout the brain and head region at 1, 2 and 3 dpf. In addition, dymeclin expression becomes detectable in the neuromast sensory organs (n) as highlighted by the

Figure 6 .

 6 Figure 6. Validation and localisation of Dymeclin interacting proteins. (A) GST pulldown assays were employed to assess in vitro binding between Dymeclin and GSTtagged PPIB and GOLM1. The association of both proteins with Dymeclin is indicated by the presence of radio-labelled Dymeclin on the autoradiograph, by contrast Dymeclin is not pulled-down by the GST alone control (left panel). 10% of in vitro translated (IVT) Dymeclin used in the pull-down experiment is shown in the panel to the right. (B) HeLa cells were co-transfected with myc-Dymeclin and FLAG-tagged PPIB and GOLM1. Transfected protein levels were assessed by immunoblotting total lysate with anti-FLAG antibody (left panel). Arrows indicate full-length protein. Immunoprecipitation with antimyc antibody followed by western blot analysis using antibodies against the FLAG tag revealed co-precipitation of PPIB and GOLM1. No protein bands were observed when empty FLAG vector was co-transfected. The asterisk indicates the presence of the immunoglobulin heavy chain. (C) Endogenous PPIB (red) localisation in singly transfected HeLa cells is cytosolic. Co-transfection with GFP-Dymeclin (green) reveals high levels of overlapping perinuclear staining depicted in the merge image (right panel).

  Genetic analysis of DMC fibroblast cell lines. (A) Chromatograms of genomic mutations identified in the three DMC lines, altered bases are indicated by arrows and base deletion by a triangle. (B) Effect of the splice junction mutations was examined by RT-PCR and gel electrophoresis. The expected PCR products in WT controls and those observed in the mutant samples are marked. Filled arrowheads show the two splice isoforms observed in DMC04997. In each case, aberrant products were gel excised and sequenced to determine the effect of mutations on the transcript. Chromatograms of WT controls are displayed to the left. (C) Levels of DYM transcript measured by quantitative RT-PCR show greatly reduced expression in all mutants by comparison to the WT control fibroblast line. Measurements were standardised against the housekeeping gene cyclophilin A. 210x297mm (300 x 300 DPI) Dymeclin localises to the cytosol and Golgi. (A) Focal co-localization is observed with Giantin (red), a component of the Golgi apparatus, 24 hrs post-transfection of GFP-Dymeclin (green). White arrows indicate areas of overlapping fluorescence. (B) By contrast, GFP-Dymeclin (green) displays no evidence for co-localization with the ER marker PDI (red). 210x297mm (300 x 300 DPI) Morphological consequences of Dymeclin loss-of-function. (A) The Golgi and ER in wild-type (WT) fibroblasts and three patient lines were visualised by Giantin (red) and PDI (green) immunofluorescence. Mutant cells display considerably distended Golgi by comparison to WT. (B) Electron micrographs of DMC04497 fibroblasts reveal a depletion of collagen fibres on the cell surface relative to WT cells as indicated by the black arrows. Nuclei are labelled (N). (C) Proliferation assays show no statistical difference in proliferation capacity between mutant and WT fibroblasts over an 8 day period (p=0.6610). FACS analysis of Annexin V stained cells following induced apoptosis demonstrate significantly larger populations of mutant cells undergoing apoptosis. 297x210mm (300 x 300 DPI) of Dymeclin leads to Golgi compaction. (A) Comparative to fibroblasts transfected with empty GFP vector increasing amounts of WT GFP-Dymeclin (0.5µg -2µg) cause condensation of the Golgi, stained with Giantin (red). Golgi position in transfected cells is indicated by the white arrows. (B) WT GFP-Dymeclin transfected into DMC lines leads to progressive restoration of Golgi morphology, indicated by the white arrows, by comparison to neighbouring untransfected cells, where the Golgi is identified by double-headed arrows. 297x210mm (300 x 300 DPI) Dymeclin expression in zebrafish embryos. (A) RNA recovered from labelled stages of development was reverse transcribed and used to PCR amplify a 1500 bp fragment of the Dymeclin transcript. Expression is consistently high in early development but decreases by 2 days post-fertilisation. (B) In situ hybridisation reveals intense staining throughout the brain and head region at 1, 2 and 3 dpf. In addition, dymeclin expression becomes detectable in the neuromast sensory organs (n) as highlighted by the arrows. At 5 dpf, the eyes were removed to facilitate assessment of the cartilaginous structures. The staining becomes more prominent in the cartilage of the head, in particular, ceratohyal (ch), ceratobranchial (cb) and lower jaw (lj) structures. Negative controls using the sense strand of dymeclin are displayed in the far-right panels. 297x210mm (300 x 300 DPI) Validation and localisation of Dymeclin interacting proteins. (A) GST pull-down assays were employed to assess in vitro binding between Dymeclin and GST-tagged PPIB and GOLM1. The association of both proteins with Dymeclin is indicated by the presence of radio-labelled Dymeclin on the autoradiograph, by contrast Dymeclin is not pulled-down by the GST alone control (left panel). 10% of in vitro translated (IVT) Dymeclin used in the pull-down experiment is shown in the panel to the right. (B) HeLa cells were co-transfected with myc-Dymeclin and FLAG-tagged PPIB and GOLM1. Transfected protein levels were assessed by immunoblotting total lysate with anti-FLAG antibody (left panel). Arrows indicate full-length protein.Immunoprecipitation with anti-myc antibody followed by western blot analysis using antibodies against the FLAG tag revealed coprecipitation of PPIB and GOLM1. No protein bands were observed when empty FLAG vector was cotransfected. The asterisk indicates the presence of the immunoglobulin heavy chain. (C) Endogenous PPIB (red) localisation in singly transfected HeLa cells is cytosolic. Co-transfection with (green) reveals high levels of overlapping perinuclear staining depicted in the merge image (right panel). 210x297mm (300 x 300 DPI)

Table 1 . Clinical characteristics of DMC subjects. Page 33 of 32 John Wiley & Sons, Inc. Human Mutation

 1

	TABLES
	Identifier	Gender	Ethnic	Consanguinity	Clinical summary
			origin
	DMC02437 Female	Unknown	Unknown	DMC diagnosis with no
					additional information
	DMC04997 Male	Lebanon	Yes	Mental retardation
					Dwarfism
	DMC10568 Male	Pakistan	Yes	Mental retardation
					Mottled iliac crests
					Dwarfism

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

This work was supported by the British Heart Foundation [RG/08/006/25302 to R.C.T., FS/07/036 to R.D.M., FS/06/051 to C.D.). The authors acknowledge financial support from the Department of Health via the National Institute for Health Research comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust.

CONFLICT OF INTEREST STATEMENT

None to declare.


Jaakko Nevalainen
email: jaakko.nevalainen@uta.fi

Jyrki Möttönen

Hannu Oja

A spatial rank test and corresponding estimators for several samples

Keywords: Kruskal-Wallis test, Multivariate several samples rank test, Spatial Hodges-Lehmann estimator, Spatial rank

In the several samples location problem, it is usually of interest to present estimates of treatment effects along with the test. The spatial Hodges-Lehmann estimators ∆ ij of the differences between treatments i and j are apparent companions to a multivariate Kruskal-Wallis test. However, these estimators generally fail to satisfy the property ∆ ij = ∆ ik + ∆ kj , making them incompatible with each other. In this paper we consider adjusted estimators possessing this property. A simulation study is carried out in order to study their finite sample efficiencies. Limiting distributions and efficiencies are presented as well.

A c c e p t e d m a n u s c r i p t 1 Introduction

Let X = (X 1 , . . . , X c ) be a p × N data matrix consisting of c independent random samples X 1 = (x 11 , . . . , x 1n 1 ), X 2 = (x 21 , . . . , x 2n 2 ), . . .

X c = (x c1 , . . . , x cnc ),
from p-variate continuous distributions F (x -µ 1 ), F (x -µ 2 ), . . ., F (x -µ c ), respectively,

where N = n 1 + . . . + n c . Knowing little of the underlying distribution, we are interested in finding out whether there are differences in location between the samples, and if so, estimating those differences.

First recall the notions of spatial sign and rank. A multivariate extension of univariate sign, the spatial sign of vector x ij , is defined as

S(x ij ) =        x ij -1 x ij , if x ij = 0; 0, if x ij = 0,
where • denotes the Euclidian length. Thus, the spatial sign is a p-variate unit vector.

The empirical spatial centered rank of x ij among the data set X is defined as

R N (x ij ) = 1 N c k=1 n k l=1 S(x ij -x kl ).
This gives a vector inside the unit sphere pointing from the center of the data cloud X approximately to the direction of x ij . Spatial ranks are data dependent, but they converge uniformly in probability to their theoretical values R( The hypotheses of interest are

x ij ) = E [S (x ij -x)], x ∼ F .
H 0 : µ 1 = • • • = µ c versus H 1 : µ 1 , . . . , µ c not all equal.
A classical univariate nonparametric test for the problem is the Kruskal-Wallis test. Write

R i = 1 n i n i j=1 R N (x ij )
for the group average of empirical spatial ranks with respect to the entire sample. Under the null hypothesis, [START_REF] Möttönen | Multivariate spatial sign and rank methods[END_REF][START_REF] Choi | An approach to multivariate rank tests in multivariate analysis of variance[END_REF], where

Q 2 = c i=1 n i R T i B -1 R i D -→ χ 2 p(c-1)
B = ave R N (x ij )R N (x ij ) T
is a consistent estimator (under H 0 ) of the spatial rank covariance matrix

B = E 0 R(x ij )R(x ij ) T .

Estimation of Treatment Effects

Denote the difference between treatments i and j by ∆ ij = µ j -µ i . Natural companion estimator ∆ ij to the test is the multivariate two-sample spatial Hodges-Lehmann estimator, that is, the sample spatial median of the n i n j pairwise differences x jk -x il (k = 1, . . . , n j ; l = 1, . . . , n i ). Note that ∆ ij = -∆ ji and ∆ ii = 0. Möttönen and Oja (1995) state but do not prove the following asymptotic result concerning this estimator:

A c c e p t e d m a n u s c r i p t

1. Under general assumptions,

√ N ( ∆ ij -∆ ij ) D -→ N p 0, λ i + λ j λ i λ j A -1 BA -1 .
Here

A = E 1 x jk -x il -∆ ij I p -S(x jk -x il -∆ ij )S(x jk -x il -∆ ij ) T .
To find a covariance matrix estimate for ∆ ij , the A matrix can be estimated by

A = ave 1 x jk -x il -∆ ij I p -S(x jk -x il -∆ ij )S(x jk -x il -∆ ij ) T ,
where the average is taken over all possible pairs (x ij , x kl ) from all the samples. An estimate of the B matrix can be obtained via

B = ave R N (x ij -∆ 1i )R N (x ij -∆ 1i ) T .
For completeness of this paper, a heuristic proof of the limiting normality is presented in the Appendix. [START_REF] Chaudhuri | Multivariate location estimation using extension of R-estimates through U -statistics type approach[END_REF] considers the spatial one-sample Hodges-Lehmann estimator and [START_REF] Hodges | Estimates of location based on rank tests[END_REF] the univariate two-sample estimation problem.

The inconvenience with the above estimators-just like in the univariate case, or when using multivariate marginal ranks-is that the obtained estimates are not generally compatible in the sense that

∆ ij = ∆ ik + ∆ kj .
To overcome this problem, consider competing estimators of treatment effects. An estimator of the difference between the ith and the jth treatment using the kth treatment as a reference is

∆ ij•k = ∆ ik + ∆ kj . ( 1
)
This type of estimator can be useful in a situation where the treatment effect of interest cannot be estimated directly, but only via a third treatment. Note that

∆ ij•i = ∆ ij•j =
A c c e p t e d m a n u s c r i p t

∆ ij = 1 c c k=1 ∆ ij•k (2)
yields a generalization of the univariate estimator proposed by [START_REF] Lehmann | Robust estimation in analysis of variance[END_REF]. The adjusted estimators (1) and ( 2) are consistent only if

n i N → λ i , 0 < λ i < 1, for all i = 1, . . . , c.
Estimators that are consistent under a weaker condition that n i N → λ i and

n j
N → λ j are obtained by weighting the estimators by the relative group size of the reference sample [START_REF] Spjøtvoll | A note on robust estimation in analysis of variance[END_REF]:

∆ij = 1 N c k=1 n k ∆ ij•k (3)
When n 1 = . . . = n c the spatial Spjøtvoll's estimators (3) reduce to the spatial Lehmann's estimators (2).

Theorem 2 Assume that min {n i , n j , n k } → ∞ and that n i N → λ i , n j N → λ j and n k N → λ k , 0 < λ i , λ j , λ k < 1.
Then, under general assumptions,

√ N ∆ ij•k -∆ ij P -→ 0
for all i, j, k.

Corollary 1 Under the assumptions of Theorem 2,

√ N ∆ ij -∆ ij P -→ 0 and √ N ∆ij -∆ ij P -→ 0 for all i, j, k.
Theorem 2 and Corollary 1 imply that the alignment of the estimates (with respect to the other c -2 treatments) does not alter their limiting distributions. However, it is unclear what happens to the efficiency of the adjusted estimators in finite samples, particularly if the sample sizes are widely disparate. This question will be addressed in the next section. Recall that the spatial Hodges-Lehmann estimator is much more efficient than the mean difference vector for heavy-tailed distributions, and nearly as efficient at the normal model (Table 1). As the adjusted estimators share the limiting distribution of the spatial Hodges-Lehmann estimator, their asymptotic relative efficiencies are identical as well.

The finite sample efficiencies of the adjusted estimators

∆

A c c e p t e d m a n u s c r i p t 7

Finally, the spatial Spjøtvoll's estimator ∆12 seems to have the same efficiency as the spatial Hodges-Lehmann estimator ∆ 12 when n 3 is small (Figure 3). As n 3 increases and n 1 , n 2 remain fixed, the estimator tends to ∆ 12•3 . Thus, for large n 3 it can be slightly better than ∆ 12 . The weighting procedure enables the spatial Spjøtvoll's estimator ∆12 to protect itself against efficiency losses due to extreme group allocations in both directions, thus making it a superior estimator. If the sample sizes are approximately the same, it does not make a difference which estimator is used.

Affine Invariant/Equivariant Versions

The test and the estimators based on spatial ranks are orthogonally invariant and equivariant but not affine invariant and equivariant. However, if V is an affine equivariant estimator of shape in the sense that V(HX + g1 T N ) ∝ H VH T , then

• any orthogonally invariant test computed on the transformed data set Y = V -1/2 X is affine invariant [START_REF] Randles | A simpler, affine-invariant, multivariate, distribution-free sign test[END_REF][START_REF] Möttönen | Multivariate nonparametric tests in a randomized complete block design[END_REF], and

• any orthogonally equivariant estimator computed on the transformed data set, and retranformed, V 1/2 ∆ (Y), is affine equivariant. This procedure is widely known as the transformation retransformation technique [START_REF] Chakraborty | On a transformation and re-transformation technique for constructing affine equivariant multivariate median[END_REF]Chakraborty et al., 1998;Chakraborty and Chaudhuri, 1998).

Therefore, a solution is to perform the multivariate spatial rank test on a transformed data set Y, and to retransform the treatment difference estimates obtained from Y back to the original scale by V 1/2 . Here the most natural approach is to use spatial ranks in the estimation of the shape matrix V [START_REF] Oja | Multivariate nonparametric tests[END_REF]. One possibility is to apply

p c i=1   1 n i n i j=1 R n i (y ij )R n i (y ij ) T   = c i=1   1 n i n i j=1 R n i (y ij ) T R n i (y ij )   I p , (4)
where

y ij = V -1/2 x ij and R n i (y ij )
is the spatial rank of y ij among y i1 , . . . , y in i . After standardization by V -1/2 obtained from (4) the spatial ranks appear as if they were from a spherical distribution. For similar definitions of shape matrices based on spatial signs, see earlier work of [START_REF] Tyler | A distribution-free M -estimator of multivariate scatter[END_REF] and [START_REF] Dümbgen | On Tyler's M -functional of scatter in high dimension[END_REF]. [START_REF] Oja | Multivariate nonparametric tests[END_REF] also gave an algorithm for the computation of a shape matrix similar to (4). The algorithm always seems to converge, but the actual proof is missing. S(y j -

x i ) - 1 n n j=1 R(y j ) + 1 m m i=1 R(x i )   P -→ 0. Therefore √ N   ∆ + A -1   1 n n j=1 R(y j ) - 1 m m i=1 R(x i )     P -→ 0
and the result follows.

Proof of Theorem 2. Let x 1 , . . . , x m , y 1 , . . . , y n and z 1 , . . . , z l be three independent random samples from F (x). Let N = m + n + l. By Theorem 1

√ N ∆ xz + A -1 1 l l k=1 R(z k ) - 1 m m i=1 R(x i ) P -→ 0, and
√ N   ∆ zy + A -1   1 n n j=1 R(y j ) - 1 l l k=1 R(z k )     P -→ 0.
But then simply

√ N   ∆ xy•z + A -1   1 n n j=1 R(y j ) - 1 m m i=1 R(x i )     P -→ 0

  12•3 , ∆ 12 and ∆12 relative to the unadjusted estimator ∆ 12 shown in Figures 1, 2 and 3, respectively, are based on simulations from a univariate normal distribution (10000 repetitions) and a bivariate spherical normal distribution (1000 repetitions) for three groups. Efficiencies for spherical distributions in general are likely to be approximately the same. The simulations were conducted in R (R Development Core Team, 2004).The efficiency of the estimator ∆ 12•3 , based on a reference sample of size 1 (n 3 = 1), is approximately 0.7 (p = 1) and 0.8 (p = 2). At this point, the value is merely the observed relative efficiency of the the difference of the (spatial) medians related to the (spatial) Hodges-Lehmann estimator. Adding a few observations to the third group quickly improves the performance of the estimator ∆ 12•3 . At n 1 = n 2 = n 3 , the observed relative efficiency is close to unity (Figure1). Our further simulation studies suggest that, as n 1 = n 2 remain fixed and n 3 increases, the finite sample efficiency increases even beyond 1, but only very slightly.The behavior of the spatial Lehmann's estimator ∆ 12 , being the average of the ∆ 12•k estimators, is very similar (Figure2). It is superior to the estimator ∆ 12•3 , because it is never worse than 90% efficient, and because it reaches the efficiency of the spatial Hodges-Lehmann estimator much faster. The reasons are easy to see: as ∆ 12 = 1 3 (2 ∆ 12 + ∆ 12•3 ), the (spatial) Hodges-Lehmann estimator ∆ 12 receives the most weight in the computation.

  defined by the implicit equation

Figure 1 :

 1 Figure 1: The observed relative efficiency of ∆ 12•3 relative to ∆ 12 .

Figure 2 :Figure 3 :

 23 Figure 2: The observed relative efficiency of ∆ 12 relative to ∆ 12 .

  [START_REF] Hettmansperger | Affine invariant multivariate rank tests for several samples[END_REF] gave a multivariate extension of the Kruskal-Wallis test, identical to the classical test in the univariate case, based on affine invariant ranks. Similar approach can be taken with spatial ranks as outlined next.

Table 1 :

 1 Asymptotic relative efficiency of the spatial Hodges-Lehmann estimator relative to the mean difference vector under multivariate t-distributions[START_REF] Möttönen | On the efficiency of multivariate spatial sign and rank tests[END_REF].

	12

n j N → λ j , 0 < λ i , λ j <

Acknowledgements

The research work of Jaakko Nevalainen and Hannu Oja was partially supported by research grants from the Academy of Finland and by the doctoral program of School of Statistical Information, Inference, and Data Analysis (SIIDA).

Appendix

Proof of Theorem 1. Let x 1 , . . . , x m and y 1 , . . . , y n be two independent random samples from F (x -µ x ) and F (y -µ y ). It is not a restriction to assume that

Suppose that ∆ is √ N -consistent for ∆, where N = m + n. This can be shown in the multivariate case as in [START_REF] Nevalainen | On the multivariate spatial median for clustered data[END_REF]. Write ∆ * = √ N ∆. Then the Taylor