John R Dolan
email: dolan@obs-vlfr.fr

Eun Jin Yang
email: ejyang@kopri.re.kr

Tae Sung-Ho Kang
email: shkang@kopri.re.kr

Siek Rhee
email: rhee@kopri.re.kr

Tae Siek Rhee

Declines in both redundant and trace species characterize the latitudinal diversity gradient in tintinnid ciliates

Keywords: biodiversity, ciliata, rare species, species packing, tintinnida High Latitude Assemblages

Introduction

A very wide variety of taxa, both multicellular and microbial, show a latitudinal diversity gradient [START_REF] Gaston | Biodiversity: an introduction[END_REF]. Typically, species richness peaks in the tropics, often at about 20°, dips slightly at the equator and decreases markedly with latitude both north and south. Tintinnid ciliates of the marine microzooplankton literally provide a textbook example of the pattern [START_REF] Gaston | Biodiversity: an introduction[END_REF]. Slight variations of the pattern characterize other protist taxa of the marine plankton such as foraminifera [START_REF] Rutherford | Environmental controls on the geographic distribution of zooplankton diversity[END_REF] and Ceratium species of the phytoplankton [START_REF] Dolan | The legacy of the last cruise of the Carnegie: a lesson in the value of dusty old taxonomic monographs[END_REF]. The latitudinal diversity gradient is the oldest known biogeographic pattern and the mechanisms generating it have long been debated with dozens of theories proposed, none of which have found wide acceptance [START_REF] Willig | Latitudinal gradients of biodiversity: patterns, process, scale and synthesis[END_REF]. A universal explanation of the latitudinal diversity gradient has been termed 'The Holy Grail of Ecology' [START_REF] Adams | The Holy Grail of ecology: latitudinal gradients[END_REF]. Thus, while there is little agreement as to why the gradient exists, most do acknowledge that species richness often declines with latitude [START_REF] Adams | The Holy Grail of ecology: latitudinal gradients[END_REF]. The structure of species assemblages quite likely often differs with species richness but exactly how is difficult to predict. Declines in species richness can be reflected in various ways with different consequences concerning the ecological characteristics of a species assemblage.

Fewer species can represent a reduction in the overall variety or range of species within an assemblage. Less variety among species can translate into a lower capacity to respond to changes in resource compostion or predation pressure. For example, a smaller range of consumer species could have a lower capacity to exploit changes in the size or qualities of available prey items. Alternatively, lower species richness may represent only fewer forms present in very low concentrations, outside their usual habitat, members of the "accidental biosphere" [START_REF] Weisse | Ciliates and the rare biospher-community ecology and population dynamics[END_REF]). These species likely have low actual or potential ecological impact. Besides a decline in variety, lower species richness may translate into fewer presumptive redundant species (i.e., species of similar ecological characteristics) able to replace dominant species subject to a high specific mortality. Theoretically, the presence of redundant species should increase the resiliency of an assemblage, meaning its capacity to resist perturbation and survive changes in conditions [START_REF] Naeem | Species redundancy and ecosystem reliability[END_REF].

Recent studies have re-emphasized the tremendous diversity of protists in the marine plankton (de [START_REF] Vargas | Eukaryotic plankton diversity in the sunlit ocean[END_REF]. The phylogenetic diversity and extreme ecological complexity of protistan assemblages makes assessing how assemblages are structured a daunting task (e.g., [START_REF] Lima-Mendez | Determinants of community structure in the global plankton interactome[END_REF]. Among planktonic protists, tintinnids are an example of a phylogenetically and ecologically coherent group and so represent a taxon in which study of the structure of assemblages are considerably simplified. Tintinnid ciliates also represent an ideal group to examine with regard to the question of how the structure of species assemblages varies with latitude. This is because not only do they show a typical latitudinal diversity gradient but also the population structure of temperate, sub-tropical and tropical populations are well known and species of similar ecology share similar morphologies allowing identification of ecologically redundant species. Tintinnid ciliates are characterized by the possession of a shell or lorica whose architecture and dimensions form the basis of classic taxonomic schemes (Figure 1).

2013

). Critically, LOD, analogous to gape-size, is related to both the size of the food items ingested and maximum growth rate. The largest prey item ingested is about 0.5 the LOD in longest dimension and a given species feeds most efficiently on prey about 0.25 of its LOD in size. LOD is negatively related to maximum growth rate. This is because LOD is positively related to the volume of the cell occupying the lorica [START_REF] Dolan | Morphology and ecology in tintinnid ciliates of the marine plankton: correlates of lorica dimensions[END_REF]) and tintinnids follow the common inverse relationship between cell size and maximum specific growth rate (Montagnes 2013). Tintinnid species with a similar LOD, or mouth size, are usually similar in terms of both preferred prey size and maximum growth rate; here these similarities are taken as indicating ecological redundancy.

The structure of tintinnid populations, in terms of both species abundance distributions and distributions of species grouped in size-classes, has been characterized for the species-rich assemblages of tropical, sub-tropical and temperate systems [START_REF] Dolan | The neutral community structure of planktonic herbivores, tintinnid ciliates of the microzooplankton, across the SE Pacific Ocean[END_REF]2013). Typically in these systems, dozens of species can be found in material from 10-20 l [START_REF] Dolan | Repeated sampling reveals differential variability in measures of species richness and community composition in planktonic protists[END_REF]. In general, species abundance distributions are log-series or log-normal distributions while grouping forms by size-classes rather than species yields a geometric distribution [START_REF] Dolan | The neutral community structure of planktonic herbivores, tintinnid ciliates of the microzooplankton, across the SE Pacific Ocean[END_REF]2013). Assemblages are overall structured by "mouth size".

Typically there are 4 to 10 abundant species and these are of mouth sizes distinct from one another. The other species, of markedly less abundance, can be divided into two groups. The first is composed of those with mouth sizes similar to one of the dominant species; these are ecologically redundant forms. The second group is formed of species in LOD size-classes distinct from those of the dominants and these species appear to be occasional, ephemeral, or rare species [START_REF] Dolan | Dynamics of core and occasional species in the marine plankton: tintinnid ciliates of the N.W. Mediterranean Sea[END_REF]) and are generally present in very low concentrations (Dolan et al. 2013). However, it is important to note that the categories of ʻredundant' and trace or ʻrare' are not exclusive. If a species found in trace concentrations is in a size-class with other species, then it is both redundant and rare while if alone in a size-class then it is not redundant but rare with ecological characteristics distinct from all the other species.

Tropical, subtropical, and temperate assemblages are characterised by high species richness reflecting both considerable species redundancy as well as the presence of species with morphologies distinct from abundant forms but found in low concentrations. In this study we address the question of how sub-arctic and arctic communities with lower species richness are structured compared to temperate communities.

We characterized communities along a latitudinal gradient of decreasing species richness. We examined large-scale or metapopulation characteristics in terms of species and in terms of 'ecotypes', defined here as species of similar feeding ecology and maximum growth rate based on LOD or mouth size. In 2012 we sampled populations of the East Sea/Sea of Japan, the North Pacific Ocean, the Bering Sea and the Chukchi Sea, and the High Arctic along a transect of over 43° of latitude and 5000 km (Figure 2). Notably the year 2012 was a year of record low sea ice extent permitting open water sampling of plankton in the High Arctic, to our knowledge for the first time. For each system 2 to 6 stations were sampled providing at least 2000 ciliate cells representing each assemblage. The parameters estimated for each of the 5 populations were species richness, number of trace species (found as a single cell), number of mouth size-classes, proportion of size-classes with multiple occupants (containing more than one species), and the number of apparently redundant species. For each assemblage the pattern of species abundance distribution was compared with modeled abundance curves constructed using parameters of the particular assemblage for three common models of community organization: geometric, log-normal and log-series. Substituting size-class of oral diameter for 'species', we also determined size-class abundance distributions. Plankton net tows were performed to assess microplankton community composition.

Methods

Sampling & Sample Analysis

For chlorophyll a determinations, water samples of 0.3-1 l were obtained from 7-9 discrete depths between the surface and 100 m depth. Water samples were filtered through a 0.7 µm Whatman glass fiber filter (GF/F) and chlorophyll concentrations determined onboard using a Turner Designs Trilogy model fluorometer calibrated using commercial chlorophyll a standards.

Net tows were made with a 20µm mesh plankton net of 0.45 m diameter towed from 100 m depth to the surface, except at some shallow stations sampled from 50 or 30 m depth. A portion of the net tow material was fixed with Lugol's (2% final concentration) for direct microscopic examination. In the laboratory, multiple 1 -2 ml aliquots of net tow material were diluted and examined in 3 ml settling chambers using an inverted microscope equipped with DIC optics. The entire surface of the settling chamber was examined at 200x total magnification. Tintinnid identifications were made based on lorica morphology and following Kofoid & Campbell (1929, 1939) and [START_REF] Hada | The fauna of Akkeshi Bay IV. The pelagic ciliata[END_REF]. We adopted a conservative approach, distinguishing all forms corresponding to species currently recognized as valid. Species names, occurrences and LOD diameters assigned are given in the supplementary data file.

As previously described and justified [START_REF] Dolan | Comparing taxonomic and morphological biodiversity of tintinnids (planktonic ciliates) of New Caledonia[END_REF]2007[START_REF] Adams | The Holy Grail of ecology: latitudinal gradients[END_REF]2013), species of similar ecology in terms of preferred prey size and maximum growth rate were grouped based on (LOD). Each species was assigned the average dimensions reported in Kofoid & Campbell (1929, 1939) and [START_REF] Hada | The fauna of Akkeshi Bay IV. The pelagic ciliata[END_REF]. Size-class diameters were binned over 4 µm intervals beginning with the overall smallest diameter (11 µm) and continuing to the largest diameter encountered in a given sample.

Total sample volumes examined varied, depending on the concentrations of organisms, and the dilutions employed varied according to concentrations of phytoplankton. However, multiple aliquots were examined until material from at least 100 liters filtered by the net was analyzed for each station. Nominal concentrations of organisms (given in the supplementary file) were calculated based on the volume filtered by the net (calculated from net surface area and depth of the strata sampled) and the volume of net tow material examined. It should be noted that the concentrations reported here are the average values per liter for the portion of the water column sampled, for most stations the top 100 m.

Data Analysis

Tintinnid assemblages were characterized by pooling data by system: East Sea/Sea of Japan stations 1-2; North Pacific stations 3-8; Bering Sea stations 9-12, Chukchi Sea stations A-F, and the High Arctic Sea stations G-H. The parameters estimated for each of the 5 populations were species richness, number of trace species (found as a single cell), number of mouth size-classes, proportion of sizeclasses containing more than one species (multiple occupants), and the number of apparently redundant species. We used simple linear correlation analysis to examine relationships across all stations between species richness and latitude, water temperature, organismal concentrations and chlorophyll a concentrations.

For each assemblage we examined the patterns of both species abundance distribution and the abundance distributions of LOD size-classes. We first made logrank abundance curves by calculating relative abundance for each species and ranking species from highest to lowest and plotting ln(relative abundance) vs. rank.

Then, we constructed hypothetical log-rank abundance curves that could fit the data by using parameters of the particular assemblage. We produced curves for three common models of community organization: geometric series, log-series, and lognormal, as in several previous studies [START_REF] Claessens | A paradox of the ciliates? High ciliate diversity in a resource-poor environment[END_REF][START_REF] Doherty | Ciliate diversity and distribution across an environmental and depth gradient in Long Island Sound, USA[END_REF][START_REF] Dolan | The neutral community structure of planktonic herbivores, tintinnid ciliates of the microzooplankton, across the SE Pacific Ocean[END_REF][START_REF] Dolan | Dynamics of core and occasional species in the marine plankton: tintinnid ciliates of the N.W. Mediterranean Sea[END_REF]2013;[START_REF] Dolan | Repeated sampling reveals differential variability in measures of species richness and community composition in planktonic protists[END_REF][START_REF] Raybaud | Similar patterns of community organization characterize distinct groups of different trophic levels in the plankton of the NW Mediterranean Sea[END_REF].

The observed rank abundance distributions were compared to the hypothetical models using a Bayesian approach: an Akaike goodness of fit calculation (19). Using this approach, an Akaike Information Criterion (AIC) was determined as the natural logarithm of the mean (sum divided by S) of squared deviations between observed and predicted ln(relative abundance) for all ranked S species plus an additional term to correct for the number of estimated parameters, k (1 for geometric series and 2 each for log-series and log-normal distributions): (S + k)/(S-k-2). The lower the calculated AIC value, the better the fit. A difference of 1 in AIC corresponds roughly to a 1.5 evidence ratio; we considered that a minimum difference of 1.0 between AIC values was required to represent a significantly different fit following [START_REF] Burnham | Model selection and multi-model inference: a practical information-theoretic approach[END_REF].

Results & Discussion

The expected latitudinal decline of species richness was evident. In the East Sea/Sea of Japan species richness was much higher than in the higher latitude systems. The East Sea/Sea of Japan also had higher cell and chlorophyll a concentrations.

However, plotting individual station data showed the decline in species richness across the 5 systems to be more closely relatable to sea surface temperature rather than latitude, and independent of the bulk characteristics of tintinnid cell or chlorophyll concentrations (Figure 3).

The dominant species in each assemblage (shown in Supplemental Figure 1) were small-mouthed species, Proplectella expolita, Condonellopsis frigida, Acanthostomella norvegica except in the North Pacific where the relatively largemouthed Ptychocylis obtusa dominated. In each assemblage, the dominant species accounted for 45-86% of the cells encountered (Table 2). Overall, of the 31 species found most (25) were not widely distributed but rather found in only in 1 or 2 of the systems sampled. In contrast, 4 species were found in all 5 populations sampled. These widespread forms were 4 of the 6 species found in the High Arctic Sea. Thus, the few species found in the High Arctic are mostly the widely distributed forms (for details distributions of each species distributions see the supplementary data file "species data").

Characteristics of the populations in the East Sea/Sea of Japan differed considerably from all of the high latitude assemblages (Table 1). Overall species richness was similar to that reported for stations from the California Current system in the Eastern Pacific at about the same latitude (Dolan et al. 2013). The 25 species of the East Sea/Sea of Japan assemblage were distributed in 11 size-classes, most of which were occupied by more than 1 species. Thus, a large portion of the species pool was composed of ʻecological redundants', species occupying a size-class along with 1 or more other species. The size class containing the largest number of species was that of the dominant species, Proplectella expolita, which was accompanied by three other species. The 5 rare or ʻtrace' species (those found as a single individual) were redundants with one exception. Thus only 1 rare form was distinct from all other species in terms of mouth size.

Compared to the East Sea/Sea of Japan, the North Pacific assemblage was composed of fewer species and these were distributed in fewer size-classes (Table 1). Half the size-classes contained redundants as they were occupied by more than 1 species. The dominant species, the large-mouthed Pytchocylis obtusa, was alone in its size-class. None of the trace species formed a distinct size-class, all appeared to be redundants. The Bering Sea assemblage resembled that of the North Pacific in species richness. The assemblage formed 9 size classes with the dominant species, Codonellopsis frigida, sharing a size-class with 2 redundant species. Two other redundant species shared the size class of the second most abundant species Parafavella parumdentata. The 2 trace species were alone in their size-class.

Low species richness characterised both Arctic assemblages (Table 1). In the Chukchi Sea the 8 species found were distributed in 7 size-classes with only the sizeclass of the dominant species, Acanthostomella norvegica, containing a redundant species. The single trace species was alone in its size-class. The High Arctic population, dominated by Ptychocylis obtusa, was singular in having neither rare species nor redundant species; all 6 species found were in distinct size-classes. The latitudinal decline in species richness we found from the East Sea/Sea of Japan to the High Arctic represented declines in numbers of redundant and trace species.

Notably while there were many fewer species in the high latitudes, the total overall size range of LODs found in the assemblages, 11 to 75 µm, was invariant across all the assemblages.

The data reported here are from a summer time sampling as is the case for most studies of high latitude plankton. In lower latitude coastal systems seasonal changes in species richness of tintinnid assemblages are well documented and summer is usually a period of low diversity (reviewed in [START_REF] Dolan | Diversity and distributions of tintinnids[END_REF]. Few data exist concerning seasonality in open water systems, especially in arctic and subarctic waters due to the technical difficulty of sampling in periods other than summer. The gradient we found may be more prominent in other seasons, for example when the ice cover is maximal in the Chukchi Sea and High Arctic. Also worth noting is that rather than harboring cryptic species, some species of the high latitude assemblages have long been suspected to be polymorphic, having variable loricas, which perhaps have been designated wrongly as distinct species.

Among these suspected polymorphs are Parafavella spp (Burkovsky 1973;[START_REF] Davis | Variations of the lorica in the genus Parafavella (Protozoa: Tintinnida) in northern Norway waters[END_REF]) of which 5 are reported here (see supplemental data file). The phenomenon of cryptic species in tintinnids (i.e., [START_REF] Santoferrara | Discrimination of closely related species in tintinnid ciliates: New insights on crypticity and polymorphism in the Genus Helicostomella[END_REF] may be less common compared to polymorphism as there are species known to display different lorica morphologies, but of the same LOD, previously described as different species and/or genera (i.e., Bachy et al. 2012;[START_REF] Laval-Peuto | Sexual reproduction in Favella ehrenbergii (Ciliophora, Tintinnina). Taxonomical implications[END_REF]Kim et al. 2014). At present it is not clear which phenomenon is more common. Among tintinnids and foraminfera cryptic species are generally segregated either temporally or spatially (e.g. [START_REF] Vargas | Molecular evidence of cryptic speciation in planktonic foraminifers and their relation to oceanic provinces[END_REF][START_REF] Xu | Species boundaries in tintinnid ciliates: A case study -Morphometric variabilty, molecular characterisation, and temporal distribution of Helicostomella species (Cciliophora, Tintinnina)[END_REF][START_REF] Santoferrara | Discrimination of closely related species in tintinnid ciliates: New insights on crypticity and polymorphism in the Genus Helicostomella[END_REF] whereas in polymorphic species the different morphotypes occur together (e.g. [START_REF] Dolan | Cyttarocylis ampulla, a polymorphic ciliate of the marine plankton[END_REF]2013;[START_REF] Dolan | Planktonic protists: little bugs pose big problems for biodiversity assessments[END_REF].

The most likely candidates for crypticism are the widespread species, those found from the East Sea/Sea of Japan to the high Arctic such as Ptychocylis obtusa and Acanthostomella norvegica. These species inhabit waters ranging in temperature from 20° to -2° C. However whether or not, for example, an East Sea/Sea of Japan population of a "species" is genetically distinct from that in the High Arctic would not change species inventories or distributions for the geographically distinct assemblages. Given that cryptic forms of tintinnids appear to be segregated either temporally, i.e., seasonally (Xu et al 2015) or spatially i.e., distinct water masses [START_REF] Santoferrara | Discrimination of closely related species in tintinnid ciliates: New insights on crypticity and polymorphism in the Genus Helicostomella[END_REF], they are unlikely to represent hidden diversity within an assemblage. Hence, in comparing assemblages from different systems the true problematic phenomenon is polymorphism that may inflate apparent species richness of an assemblage. If the 5 Parafavella species distinguished here (all found in low numbers: 1-5 cells per assemblage, see supplemental data file) are revealed to be a single species, the species richness of the assemblages of the East Sea/Sea of Japan, North Pacific and Bering Sea may be slightly lower then the numbers reported here.

Besides species richness, differences between the East Sea/Sea of Japan assemblage and higher latitude populations were also evident in the structures of the assemblages. The patterns of species abundance distributions as well the abundance distributions of size-classes differed among the assemblages (Table 2 and Figure 4). Species abundance distributions of all the high latitude populations were best fit by a geometric distribution in contrast to the log-series distribution of the East Sea/Sea ofJapan. Long-tailed log or log-series distributions, with large numbers of relatively rare species, are commonly observed for most natural assemblages [START_REF] Mcgill | Species abundance distributions: moving beyond single prediction theories to integration within an ecological framework[END_REF]). Long-tailed distributions are typical of abundance curves of planktonic protists determined using molecular techniques (e.g., [START_REF] Bachy | Accuracy of protist diversity assessments: morphology compared to cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study[END_REF][START_REF] Brown | Microbial community structure in the North Pacific Ocean[END_REF][START_REF] Edgcomb | Protistan microbial observatory in the Cariaco Basin, Caribbean. 1. Pyrosequencing vs Sanger insights into species richness[END_REF][START_REF] Orsi | Effect of oxygen minimum zone formation on communities of marine protist[END_REF]. Notably, the pattern can be partially the result of problems arising from sequencing errors and differential sequencing of different taxa, issues which require attention (e.g. [START_REF] Vargas | Eukaryotic plankton diversity in the sunlit ocean[END_REF].

The species abundance distribution of the East Sea/Sea of Japan assemblage was best fit by a log model. Log distributions, either log-normal or log-series are associated with a multitude of factors governing relative species abundance in the case of the log-normal, or immigration and extinction from a metapopulation in the case of log-series (i.e, [START_REF] Hubbell | The unified neutral theory of biodiversity and biogeography[END_REF]. Substituting size-classes for species, similar differences were evident as the log-normal distribution of the East Sea/Sea of Japan population contrasted with the mostly geometric distributions of the high latitude assemblages (Table 3). A geometric distribution represents the result of a priority exploitation of resources by individual species in a community and is classically associated with 'immature' or pioneer communities limited by a single resource such as space (e.g., [START_REF] Whittaker | Evolution and measurement of species diversity[END_REF]. This distribution is also thought to characterize assemblages of low species richness or severe environments [START_REF] Wilson | Methods for fitting dominance/diversity curves[END_REF]. In Antarctic waters, a geometric species abundance distribution was found to describe the species abundance distribution of the entire planktonic ciliate community [START_REF] Wickham | Ciliate distributions and forcing factors in the Amundsen and Bellinghausen Seas (Antarctic)[END_REF]. In tintinnid assemblages, the geometric distribution of LOD size-class abundance is most simply attributable to availability of prey concentration and size given the close relationship between LOD size and prey exploited by tintinnids [START_REF] Dolan | Morphology and ecology in tintinnid ciliates of the marine plankton: correlates of lorica dimensions[END_REF]).

The species found in trace concentrations, just 1 individual in 100 liters, can be considered ʻrare'. The phenomenon of rare species has received a good deal of attention (e.g. [START_REF] Gaston | Rarity[END_REF]). Rare species have become a focus in biodiversity conservation (e.g. [START_REF] Lyons | Rare species and ecosystem functioning[END_REF]. In microbial ecology, the results from highthroughput sequencing commonly suggest the existence of very large numbers of apparently rare species, present in low concentrations (e.g., [START_REF] Dunthorn | Ciliates and the rare biosphere: a review[END_REF].

Rare species, although low in abundance, may have important roles if they possess key functional traits different from abundant species (Mouillot et al. 2013). In the protistan "rare biosphere", rare species may be of importance because they can become abundant if conditions change favoring different species with certain characteristics [START_REF] Caron | Hypotheses on the role of the protistan rare biosphere in a changing world[END_REF]. Admittedly, rare species can also be of minor ecological importance, as in members of the "accidental rare biosphere" [START_REF] Weisse | Ciliates and the rare biospher-community ecology and population dynamics[END_REF]. Regardless of the nature of the rare tintinnid species we found, their numbers decreased markedly with latitude (Figure 5).

Using our non-exclusive categories, a species found as a single cell is defined as rare but if it is in the same LOD size class as another species, it is also a redundant. However, the majority of redundant species were not rare but relatively abundant, as they were found in far greater than trace concentrations. Redundants were most numerous in the size classes of the dominant species and can likely replace a dominant species, for example, one subjected to a high rate of specific mortality from a parasite or predator (i.e., [START_REF] Coats | Parasites of tintinnids[END_REF]. A species can change roles from a minor to a major role. In temperate and sub-tropical assemblages of tintinnids, a species can be dominant in one population and a redundant in another (Dolan et al. 2013). In the Bering Sea Codonellopsis frigida was the dominant form while in Chukchi Sea is a redundant species. It has been argued that the existence of redundant species should increase the capability of an assemblage to exploit changes in conditions [START_REF] Naeem | Species redundancy and ecosystem reliability[END_REF]. Like rare species, we found redundant species declined in relative importance with latitude (Figure 5).

Conclusion

We found the latitudinal decline in species richness of tintinnid ciliates corresponded to fewer rare species, fewer presumed redundant species, and fewer size-classes but without a reduction in the total range of size-classes present. Overall, declines were in the numbers of species accompanying the dominant forms with dominant forms remaining more or less the same set of species. The declines were then in presumed redundant species and numbers of rare species. Both categories declined with latitude to zero values in the High Arctic assemblage (Table 1 and Figure 5). The decline in species richness with latitude in the Northern hemisphere represents declines in the variety of forms in an assemblage and species redundancy.

Consequently there appears to be a latitudinal gradient in the capability of assemblages to adapt to environmental changes in at least with regard to sizespectra of available prey items or the loss of a particular dominant species. The latitudinal diversity gradient in another group of planktonic protists, foraminifera, appears closely related to temperature over a wide variety of time scales [START_REF] Yasuhara | Latitudinal 540 species diversity gradient of marine zooplankton for the last three million years[END_REF], suggesting temperature plays a preponderant role in determining species richness. If changes in species distributions occur with distributions shifted northward, Arctic assemblages may become slightly more species rich and increases in species richness would likely increase the numbers of redundant species.

Theoretically, as redundancy among species increases ecosystem stability [START_REF] Naeem | Species redundancy and ecosystem reliability[END_REF], global climate change may increase the stability of high latitude ecosystems.

However, the relatively small species pools of the northern high latitude assemblages suggest a low capacity to adapt to changing conditions.

About 1 ,

 1 200 species are in the literature (Agatha & Strüder-Kypke 2012); virtually all are restricted to the marine plankton. The diameter of the mouth-end of the lorica, lorica oral diameter (LOD) is a conservative taxonomic character (Laval-Peuto & Brownlee 1986). It does not change with development; newly divided cells form a new lorica with the same oral diameter as the fully developed organism (Agatha et al.

  Data and samples were collected from onboard the Korean Research Icebreaker Araon from July to September in 2012. Data presented here are from 22 stations distributed between the Japan Sea and 82 °N as shown in Figure 1. Exact locations and the Korean Polar Research Station Designations are given the supplementary data file. A Niskin bottle rosette equipped with CTD probes was used to obtain temperature profile data and discrete depth samples for chlorophyll determinations.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Locations sampled between late July and early September in 2012 ranging

Figure 3 .

 3 Figure 3. Relationship of species richness of the tintinnid assemblages to: latitude,

Figure 4 .

 4 Figure 4. Observed abundance distributions of tintinnid assemblages: species

Figure 5 .

 5 Figure 5. The relationships between the average latitude sampled for each of the 5

ACKNOWLEDGEMENTS

The comments and suggestions of Dave Montagnes, 3 reviewers and the editor on previous versions led to significant improvement in the manuscript.

Supplementary information is available at The ISME Journal's website.

trace species encountered (circles). Significant (p < 0.01) negative linear 568 relationships with latitude are evident for both the % of species pool as redundants (n = 5, r = 0.984) and the number of trace species (n = 5, r = 0.987).

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Charline Ladroue

David Hoogewijs

Sophie Gad

Romain Carcenac

Federica Storti

Michel Barrois

Anne-Paule Gimenez-Roqueplo

Michel M Leporrier

Nicole Casadevall

Olivier Hermine

Christophe Marzac

Aurélie Bruno Varet

Betty Gardie
email: betty.gardie@inserm.fr

Jean-Jacques Kiladjian

André Baruchel

Fadi Fakhoury

Brigitte Bressac-De Paillerets

Jean Feunteun

Nathalie Mazure

Jacques Pouysségur

Roland H Wenger

Stéphane Richard

Distinct deregulation of the hypoxia inducible factor by PHD2 mutants identified in

Keywords: hypoxia inducible factor, hypoxia-inducible transcription factor, PHD2, erythrocytosis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background

Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma.

Design and Methods

Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively.

Results

This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category.

Introduction

Secondary erythrocytosis is due to external factors such as increased production of erythropoietin, the origin of which is variable and may result from germline mutations in genes encoding factors involved in the oxygen-sensing pathway. The primary cellular component implicated in oxygen homeostasis is the hypoxia-inducible transcription factor (HIF). HIF operates as a heterodimer composed of a constitutively expressed beta subunit, also known as aryl hydrocarbon receptor nuclear translocator, and an alpha subunit (1α, 2α or 3α) that is tightly regulated by oxygen via post-translational modification. The prolyl-4-hydroxylase domain (PHD) enzymes hydroxylate proline residues located in the oxygen-dependent degradation (ODD) domain of HIF-α. This hydroxylation allows the binding of the von Hipple-Lindau protein (pVHL), the substrate recognition subunit of an E3 ubiquitin ligase complex that induces ubiquitination and subsequent degradation of HIF-α by the proteasome. [START_REF] Kaelin | The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer[END_REF][START_REF] Pouyssegur | Hypoxia signalling in cancer and approaches to enforce tumour regression[END_REF] In the absence of oxygen, HIF-α is stabilized, heterodimerizes with HIF-1b and induces expression of hundreds of genes involved in cell survival, angiogenesis, erythropoiesis and cell proliferation. [START_REF] Pouyssegur | Hypoxia signalling in cancer and approaches to enforce tumour regression[END_REF][START_REF] Wenger | Integration of oxygen signaling at the consensus HRE[END_REF] There is some restricted target gene specificity depending on the HIF-α subunit of the HIF-α/b heterodimeric transcription factor. For example, renal and hepatic erythropoietin is regulated by the HIF-2α subunit in vivo. [START_REF] Rankin | Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin in vivo[END_REF][START_REF] Rosenberger | Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys[END_REF][START_REF] Warnecke | Differentiating the functional role of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2alpha target gene in Hep3B and Kelly cells[END_REF][START_REF] Wenger | Regulated oxygen sensing by protein hydroxylation in renal erythropoietin-producing cells[END_REF] Germline mutations in genes involved in the HIF pathway have been reported in association with syndromes that predispose patients to both neoplasms and/or congenital secondary erythrocytosis. [START_REF] Richard | Von Hippel-Lindau disease[END_REF] The most frequent mutations involve the VHL tumor suppressor gene. Heterozygous germline mutations in this gene are responsible for von Hippel-Lindau (VHL) disease, an autosomal dominant condition predisposing to multiple tumors including central nervous system and retinal hemangioblastomas, clear-cell renal cell carcinoma, pheochromocytomas and pancreatic endocrine tumors. [START_REF] Richard | Von Hippel-Lindau disease[END_REF] Established correlations between genotype and phenotype predict the risk of paraganglioma/pheochromocytoma, with VHL deletions or truncating mutations being associated with a low risk (VHL type 1) and VHL missense mutations being associated with a high risk (VHL type 2). [START_REF] Maher | Phenotypic expression in von Hippel-Lindau disease: correlations with germline VHL gene mutations[END_REF][START_REF] Nordstrom-O'brien | Genetic analysis of von Hippel-Lindau disease[END_REF] In addition, a homozygous 598C>T (R200W) VHL germline mutation has been shown to account for Chuvash congenital polycythemia, an autosomal recessive disease, endemic in the Chuvash Autonomous Republic of the Russian Federation. [START_REF] Ang | Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia[END_REF] Homozygous carriers of the R200W-VHL germline mutation do not develop tumors but instead have Chuvash congenital polycythemia due to high levels of erythropoietin. [START_REF] Ang | Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia[END_REF] The lack of tumor development in this disorder is due to a weak defect of the mutation in terms of its HIF-α regulation (leading to delayed ubiquitination) because of its localization outside pVHL functional domains. [START_REF] Ang | Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia[END_REF] Other homozygous and compound heterozygous polycythemia-associated VHL mutations have also been reported. [START_REF] Nordstrom-O'brien | Genetic analysis of von Hippel-Lindau disease[END_REF] Functional studies of some of these mutants have shown a weak to undetectable defect of HIF-1α regulation (unpublished data).

Recently, a similar phenotype of high erythropoietinassociated polycythemia without associated tumors has been reported in carriers of heterozygous germline mutations in the PHD2 and HIF-2A genes, [START_REF] Al-Sheikh | Disturbance in the HIF-1alpha pathway associated with erythrocytosis: further evidences brought by frameshift and nonsense mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene[END_REF][START_REF] Gale | Autosomal dominant erythrocytosis and pulmonary arterial hypertension associated with an activating HIF2 alpha mutation[END_REF][START_REF] Martini | A novel heterozygous HIF2AM535I mutation reinforces the role of oxygen sensing pathway disturbances in the pathogenesis of familial erythrocytosis[END_REF][START_REF] Percy | Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis[END_REF][START_REF] Percy | A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove[END_REF][START_REF] Percy | A gain-offunction mutation in the HIF2A gene in familial erythrocytosis[END_REF][START_REF] Percy | A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis[END_REF][START_REF] Van Wijk | Erythrocytosis associated with a novel missense mutation in the HIF2A gene[END_REF] with the exception of one patient carrying a H374R-PHD2 mutation. [START_REF] Ladroue | PHD2 mutation and congenital erythrocytosis with paraganglioma[END_REF] This particular patient simultaneously developed congenital secondary erythrocytosis and recurrent paraganglioma, a tumor originating from neural crest cells similar to pheochromocytoma but with an extra-adrenal localization. [START_REF] Ladroue | PHD2 mutation and congenital erythrocytosis with paraganglioma[END_REF] The analysis of the tumor showed a loss of heterozygosity including the wild-type PHD2 allele, suggesting a potential tumor suppressor role of PHD2.

PHD2 and VHL act in concert to regulate HIF-α. Based on the observation that VHL mutation carriers display different phenotypes depending on the relative capacity of the VHL mutants to regulate HIF, we sought to define the genotypephenotype relationship regarding the capacity of PHD2 mutants to differentially regulate HIF-α. Here we report a functional study comparing five PHD2 variants associated with isolated congenital secondary erythrocytosis with the mutation identified in a patient with recurrent paraganglioma.

Design and Methods

Patients and mutation screening

Thirty-four patients who did not fulfill World Health Organization diagnostic criteria for polycythemia vera were investigated. The local ethics committee of Kremlin Bicetre Hospital approved the study and all patients provided written informed consent. Blood samples were collected from all patients and germline DNA was extracted and analyzed by direct sequencing. [START_REF] Ladroue | PHD2 mutation and congenital erythrocytosis with paraganglioma[END_REF] DNA from the blood of healthy donors of Caucasian origin was used as a control.

Assay of hypoxia inducible factor transcriptional activity

In order to assay HIF transcriptional activity, dual luciferase assays were performed in Hek293T cells as described previously. [START_REF] Ladroue | PHD2 mutation and congenital erythrocytosis with paraganglioma[END_REF] A pGL3promoter vector expressing luciferase under the control of hypoxia response elements [START_REF] Dayan | The oxygen sensor factor-inhibiting hypoxia-inducible factor-1 controls expression of distinct genes through the bifunctional transcriptional character of hypoxia-inducible factor-1alpha[END_REF] was used. Cells were exposed to hypoxic conditions (1% O2) for 4 h before extraction.

Assay of hypoxia inducible factor stability

HeLa cells were transiently co-transfected with increasing amounts (50-200 ng) of an expression vector encoding the HIF-2α ODD domain (amino acids 404-569), fused to yeast Gal4 DNAbinding domain and Herpes simplex VP16-derived transactivation domain, together with the Gal4-response element driven firefly luciferase reporter, pGRE5xE1b (125 ng), and a Renilla luciferase control plasmid (4 ng). [START_REF] Koditz | Oxygendependent ATF-4 stability is mediated by the PHD3 oxygen sensor[END_REF] Twenty-four hours post-transfection, cells were cultured under either normoxic (20% O2) or hypoxic (1% O2) conditions for an additional 16 h, and firefly luciferase activity was determined and normalized to Renilla luciferase activity.

Hydroxylation assay

PHD2 and HIF-ODD proteins (plasmid pcDNA3-HA-Gal4-HIF-1α-ODD was a generous gift from WG Kaelin Jr, Dana-Farber Cancer Institute, Boston, USA) [START_REF] Ohh | Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein[END_REF] were produced in wheat germ extract in vitro using the TnT transcription-translation kit (Promega). The hydroxylation reaction was carried out by mixing PHD2 and HIF-1α-ODD (amino acids 536-652) proteins in a reaction buffer containing co-factors (Fe 2+ , ascorbic acid and 2-oxoglutarate) as described previously. [START_REF] Huang | Sequence determinants in hypoxiainducible factor-1alpha for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3[END_REF] The samples were incubated at 30°C, collected at different time points, and immunoblotted with anti-HA (Tebu) and anti-HIF-1α (Pro 564)-OH (Cell-Signaling Technology) antibodies.

Results

We sequenced the PHD2 gene on germline DNA from a

C. ladroue et al.

series of Caucasian patients with unexplained polycythemia associated with normal or elevated serum erythropoietin levels (Figure 1A). Four novel heterozygous sequence variants were identified in the PHD2 gene [c.G471C, p.Gln157His (Q157H); c.C599A, p.Pro200Gln (P200Q); c.G760C, p.Asp254His (D254H); c.C1192T, p.Arg398X (R398X)] as well as the already described c.G1112A, p.Arg371His (R371H) mutation [START_REF] Percy | A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove[END_REF] (Figures 1A andB). Genetic testing was performed on available parents and relatives of the PHD2 mutation carriers, but no mutation was found except in the mother of the PHD2-R398X carrier who presented characteristics of polycythemia (Figure 1A). She harbors the PHD2 mutation but with a mosaic status as demonstrated by the reduced height of the peak on the sequence chromatogram (Figure 1B). The low proportion of the mutated allele was confirmed by a quantitative allelespecific oligonucleotide method (data not shown).

Concerning the other families, only a few relatives were available for further genetic and clinical investigations but there was no history of familial polycythemia. Briefly, the brother, the sister and the son of patient #2295 and the father of patient #2403 agreed to genetic testing and were not carriers of PHD2 mutation. Other parents had died previously, of known causes in two cases: esophageal cancer for the mother of patient #2403 and colon cancer for the mother of patient #2412. Finally, patient #0424 was an adopted child, without children.

The frequencies of the different variants were evaluated in a control population (Figure 1C). Only the Q157H variant was found and was, therefore, classified as a polymorphism. Analysis of amino acid conservation supports this conclusion, as the Q157 amino acid is not conserved either between species or within the PHD protein family (PHD1 and 3) (Online Supplementary Table S1). In contrast, amino acids P200 and R371 are highly conserved and the D254 amino acid is fully conserved, similar to H374 described in our previous study. [START_REF] Ladroue | PHD2 mutation and congenital erythrocytosis with paraganglioma[END_REF] As far as concerns the location of the amino acids in the protein, D254, like H374, is part of the PHD2 catalytic site. [START_REF] Mcdonough | Cellular oxygen sensing: crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2)[END_REF] The P200 amino acid is located within the nuclear localization signal (NLS) implicated in the shuttling of PHD2 between the cytoplasm and the nucleus which plays a crucial role in HIF regulation. [START_REF] Steinhoff | Cellular oxygen sensing: importins and exportins are mediators of intracellular localisation of prolyl-4-hydroxylases PHD1 and PHD2[END_REF] We tested the shuttling of the P200Q mutation by immunofluorescence but we did not observe any impact of the mutation on the capacity of PHD2 to shuttle (data not shown).

The functional consequences of these mutations on PHD2 activity were first evaluated using a hypoxia reporter assay based on the transcriptional activity of endogenous HIF (accumulated during 4 h of hypoxia). A luciferase reporter gene driven by a hypoxia response element derived from the erythropoietin 3' enhancer was used. Adding wild-type PHD2 led to a dose-dependent suppression of HIF-α-mediated luciferase expression (Figure 2A). As expected, the PHD2 variant with the Q157H polymorphism reduced luciferase activity to the same extent as wild-type PHD2 (Figure 2A). Within the different missense mutations, we observed two types of mutants: one type (including PHD2-P200Q and R371H) did not affect the capacity of PHD2 to regulate HIF-α transcriptional activity, whereas a second type (including PHD2-D254H and H374R) completely abrogated PHD2 activity (Figure 2A).

We next studied the PHD2 mutants using a one-hybrid reporter assay based on the capacity of PHD2 to induce HIF-α protein instability. Cells were co-transfected with expression vectors containing the different PHD2 mutants (50 to 200 ng), the oxygen-dependent degradation domain of HIF-2α (HIF-2α-ODD) fused to yeast Gal4 DNA-binding domain and Herpes simplex VP16-derived transactivation domain, together with a reporter vector expressing luciferase under the control of a Gal4-response element. In this assay, luciferase expression reflects the stability of HIF-2α-ODD. A trend similar to that in the previous test was obtained: there was no detectable effect of the P200Q and R371H substitutions on PHD2 activity (which was compa-Deregulation of HIF by PHD2 mutants haematologica | 2012; 97(1) Figure 1. Identification of PHD2 mutations in patients with polycythemia. (A) Table of patients diagnosed with erythrocytosis and a PHD2 variation. Ht: hematocrit; Hb: hemoglobin; RC: number of red cells; EPO: erythropoietin; m: male; f: female; N: normal; NA: not available; *: previously described. [START_REF] Ladroue | PHD2 mutation and congenital erythrocytosis with paraganglioma[END_REF] (B) Sequence chromatogram of PHD2 in the area of the mutated nucleotide. Wild-type DNA was used as reference (Ref) (top) and compared to germline DNA of the patient (bottom). Only one relative of a patient was available for genetic testing (the mother of the PHD2-R398X carrier). She harbors the PHD2 mutation but with a mosaic pattern as demonstrated by the reduced height of the peak on the sequence chromatogram. (C) Frequency of the PHD2 variations in a control population.

A B C

rable to that of the wild-type PHD2) and there was total abolition of the D254H mutant activity (comparable to that of the truncated PHD2 variant R398X) (Figure 2B). Identical results were obtained using a HIF-1α-ODD stability reporter (data not shown). The experiment was repeated with the P200Q-PHD2 mutant in oxygen-regulated erythropoietin-expressing cells (including the human hepatoma cell line Hep3B and renal erythropoietin-producing cells, a new cell model isolated from the tumor-free tissue of a patient with renal carcinoma) but no significant difference was observed (data not shown). [START_REF] Frede | Oxygen-regulated expression of the erythropoietin gene in the human renal cell line REPC[END_REF] We next performed a sensitive in vitro assay in order to test the ability of the PHD2 mutants to hydroxylate HIF-1α in a time-dependent manner. In this assay, in vitro-translated PHD2 proteins were mixed with HIF-1α-ODD in the presence of co-factors necessary for the hydroxylation reaction. The capacity of the different PHD2 mutants to hydroxylate HIF-1α was measured by immunoblotting using an antibody specific for the hydroxylated HIF-1α-ODD (HIF-OH, Figure 3A). The H374R and D254H substitutions totally impaired HIF-1α hydroxylation (Figure 3A). By contrast the R371H mutant behaved like the wild-type PHD2 and the P200Q mutant, although capable of hydroxylating HIF-1α (Figure 3A), showed a reproducible and consistent delay (Figure 3B).

Discussion

Taken together, these results show that PHD2 mutations can be divided into several different classes in terms of their effects on HIF regulation. Genotype/phenotype correlations cannot be established for PHD2 mutations because they are rare events that have been reported in only ten families to date (including those in the present study), in contrast to the 945 families described with a VHL mutation. [START_REF] Nordstrom-O'brien | Genetic analysis of von Hippel-Lindau disease[END_REF] In addition, the parents of the PHD2 mutation carriers reported in the literature were either dead or not available for further genetic and clinical investigations (including one parent who died of esophageal cancer [START_REF] Percy | A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis[END_REF] ). In our study, only one parent was genetically tested and diagnosed as a mosaic carrier which prevents any conclusion regarding the developed phenotype. Nonetheless, regarding the close functional relation between VHL and PHD2 in the regulation of HIF and the implication of the HIF pathway in the genesis of pheochromocytoma, [START_REF] Dahia | A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas[END_REF][START_REF] Eisenhofer | Distinct gene expression profiles in norepinephrine-and epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel-Lindau syndrome[END_REF][START_REF] Favier | The Warburg effect is genetically determined in inherited pheochromocytomas[END_REF][START_REF] Pollard | Expression of HIF-1alpha, HIF-2alpha (EPAS1), and their target genes in paraganglioma and pheochromocytoma with VHL and SDH mutations[END_REF] we can hypothesize similarities between the various types of mutants and raise the question of a possible risk of development of paraganglioma/pheochromocytoma in PHD2 mutation carriers. Subjects with one category of mutantion (P200Q and R371H) display features similar to those with the VHL-R200W mutantion responsible for Chuvash polycythemia without any increased risk of neoplasia. [START_REF] Ang | Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia[END_REF] Like the VHL-R200W mutant, which is located outside functional domains, the PHD2-P200Q and R371H mutations are not located in the catalytic domain of the enzyme and have a moderate impact on HIFα regulation. In addition, the VHL-R200W mutant only induces delayed ubiquitination of HIFα which may be comparable to the delayed hydroxylation of HIFα observed with the PHD2-P200Q mutant. Interestingly, Pro200 is only one residue N-terminal to Cys201 which has been shown to chelate zinc and cadmium ions, providing evidence for the existence of a second metal binding site on PHD2. [START_REF] Mecinovic | Use of mass spectrometry to probe the nucleophilicity of cysteinyl residues of prolyl hydroxylase domain 2[END_REF][START_REF] Mecinovic | ESI-MS studies on prolyl hydroxylase domain 2 reveal a new metal binding site[END_REF] This Cys201 affects PHD2 hydroxylation activity and appears to be implicated in redox signaling in vitro. [START_REF] Nytko | Vitamin C is dispensable for oxygen sensing in vivo[END_REF] The very close location to the functionally important Cys201 residue could be the cause of the delayed hydroxylation of HIF-1α by the PHD2haematologica | 2012; 97(1) P200Q mutant. Intriguingly, concerning the R371H mutant, the previously reported loss-of-function effect of this mutant [START_REF] Percy | A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove[END_REF] could not be confirmed by any of the three tests of our study. The R371H mutation segregates with erythrocytosis in two different families (described herein and by Percy et al. [START_REF] Percy | A novel erythrocytosis-associated PHD2 mutation suggests the location of a HIF binding groove[END_REF] ) and is unequivocally involved in this pathology. We currently cannot explain why, in our hands, the R371H mutation failed to abolish PHD2 catalytic activity. The PHD2 expression vectors were re-sequenced and their functions confirmed by immunoblotting. Parallel experiments with other PHD2 mutants confirmed the validity of our assays. For this category of mutants which have a moderate impact on HIFα regulation, we cannot rule out potential indirect regulation on the oxygen sensing pathway via PHD2-interacting proteins. Indeed, during the past decade a large number of PHD2-interacting proteins have been discovered, including both upstream regulators and downstream targets of PHD2, substantially increasing the complexity of the PHD/HIF oxygen-sensing regulation pathway. [START_REF] Wenger | Regulated oxygen sensing by protein hydroxylation in renal erythropoietin-producing cells[END_REF] Moreover, PHD2 has been reported to have hydroxylation-independent gene regulatory functions. [START_REF] Bordoli | Prolyl-4hydroxylase PHD2-and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis[END_REF][START_REF] Chan | Tumor vasculature is regulated by PHD2-mediated angiogenesis and bone marrow-derived cell recruitment[END_REF][START_REF] Shao | Two distinct roles for EGL-9 in the regulation of HIF-1-mediated gene expression in Caenorhabditis elegans[END_REF] Another category, including the PHD2-R398X mutation and three other PHD2 truncated mutations described previously, [START_REF] Al-Sheikh | Disturbance in the HIF-1alpha pathway associated with erythrocytosis: further evidences brought by frameshift and nonsense mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene[END_REF] can be compared to the VHL truncation mutations (VHL disease type 1) which are not associated with the development of pheochromocytomas. Subject with these first two categories could be considered at low risk of developing paraganglioma/pheochromocytoma.

A last category could be compared to the VHL missense mutations involved in VHL disease type 2, associated with a high risk of paraganglioma/pheochromocytoma. This category includes the previously described PHD2-H374R mutation identified in a 43-year-old patient with paragan-glioma. A loss of the PHD2 wild-type allele was demonstrated in the patient with this tumor, arguing for a tumor suppressor role of PHD2. [START_REF] Ladroue | PHD2 mutation and congenital erythrocytosis with paraganglioma[END_REF] No PHD2 mutations have been identified in other series of patients affected by pheochromocytomas (73 patients with hereditary paraganglioma and pheochomocytoma syndrome, Gimenez-Roqueplo, Hôpital Européen Georges Pompidou, Paris, unpublished data) and/or renal carcinoma, [START_REF] Astuti | Mutation analysis of HIF prolyl hydroxylases (PHD/EGLN) in individuals with features of phaeochromocytoma and renal cell carcinoma susceptibility[END_REF] but the risk that germline PHD2 mutation carriers have of developing tumors should not be underestimated. Indeed, like His374, Asp254 is highly conserved, and located in the catalytic site of PHD2. [START_REF] Mcdonough | Cellular oxygen sensing: crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2)[END_REF] Moreover, the D254H mutation results in a severe loss of function. Therefore, although no cases of paraganglioma or pheochromocytoma have yet been detected in the D254H-PHD2 mutation carrier, this mutation may be considered as a potential candidate for tumor predisposition.

In conclusion, using three different approaches we demonstrated that distinct PHD2 mutations have differential effects on HIF regulation. We suggest that, by analogy to VHL mutations, carriers of particular PHD2 mutations may be prone to tumor development. These patients would then require screening for tumor prevention and early detection.

Figure 2 .

 2 Figure2. Functional study of PHD2 mutants using luciferase reporter assays. (A) PHD2-dependent regulation of endogenous HIF in an assay based on a hypoxia response element reporter gene. Cells were co-transfected in a 12-well format with various amounts of pcDNA3-HA-PHD2 expression vectors (to enable the expression of the same amount of PHD2 proteins) in addition to pGL3 reporter vectors encoding firefly luciferase under the control of a sensitive hypoxia response element and Renilla luciferase as a control of transfection efficiency. Cells were placed in hypoxic conditions (1% O2) for 4 h in order to accumulate endogenous HIFα before being collected. Results are given in percentage of firefly luciferase activity normalized to Renilla luciferase activity. The amount of HA-PHD2 transfected (PHD2) was quantified by immunoblotting using an anti-HA antibody. (B) The effect of PHD2 effect on HIF-2α protein stability in a one-hybrid reporter assay. Cells were cotransfected in a 6-well format with various amounts of pcDNA3-HA-PHD2 expression vectors (200, 100 and 50 ng), a Gal4-VP16-HIF-2αODD (amino acids 404-569) construct as well as a Gal4 response element-driven firefly luciferase reporter and a Renilla luciferase control plasmid. Twenty-four hours post-transfection cells were incubated for 16 h in normoxic or hypoxic conditions. Results are mean values of three independent experiments performed in triplicate.

Figure 3 .

 3 Figure 3. Functional analysis of PHD2 mutants using an in vitro hydroxylation assay. (A) Immunoblot estimation of HIF-1α-ODD protein hydroxylation in vitro. PHD2 and HIF-1α-ODD proteins were synthesized separately by in vitro transcription-translation reactions. The hydroxylation reaction was then processed by mixing HA-PHD2 and HA-HIF-1α-ODD proteins in a reaction buffer containing PHD2 enzymatic co-factors. The reaction was carried out at 30°C and samples were collected after 0, 10, 30, and 60 min of incubation. For immunoblotting, 10 mL aliquots of the hydroxylation reaction assays were separated by SDS-PAGE, blotted, and incubated with an anti-HA antibody [to quantify PHD2 and total HIF-ODD (HIF)] and anti-hydroxylated HIF-1 antibody [to quantify hydroxylated HIF-ODD (HIF-OH)]. (B) Quantification of hydroxylated HIF-1α. The proportion of hydroxylated HIF-1α was measured and related to total HIF-1α. The 100% value corresponds to the quantity of HIF-1α hydroxylated by the PHD2-WT protein after 1 h. Means were obtained with three independent experiments.

haematologica | 2012; 97[START_REF] Kaelin | The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer[END_REF]

Hummel and Bruno Cassinat for their precious help in recruiting patients and Sylvie Hermouet for scientific discussionsFunding: this work was supported by grants from the "Association pour la Recherche sur le Cancer" (ARC), the French National Cancer Institute ("INCa", PNES Rein, and Réseau National Prédispositions héréditaires au cancer du rein) and the French Ligue Nationale contre le Cancer (Comités du Cher et de l'Indre) as well as by the HypoxiaNet COST Action TD0901. DH is supported by a postdoctoral Marie Curie IEF Fellowship from the European Commission and the Sassella Stiftung. DH and RHW are supported by grants from the State Secretariat of Education and Research (C10.0106) and from the NCCR Kidney.CH, funded by the SNF.

Authorship and Disclosures

The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.

Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.

Deregulation of HIF by PHD2 mutants haematologica | 2012; 97 (1)