Luc Lambs

Mohamed Labiod

Climate change and water availability in north-west Algeria: investigation by stable water isotopes and dendrochronology Water International

Keywords: water resources, riparian vegetation, Tafna basin, karst, Algeria

Since the 1970s, rainfall has declined along the North African coast, while the demographic pressure has increased. Supplementing the rainfall data and water level of the Béni Bahdel dam, water isotopic signature and tree ring analyses were used to better understand the effects of climate change (lower rainfall, higher summer temperature) and the water circulation in the Tafna River basin in north-west Algeria. Changes are recommended in water storage and afforestation policies and irrigation techniques.

Introduction

The Tafna River, the closest stream in north-west Algeria to the border with Morocco, takes its source around the Tlemcen Mountains (1400 m). The Tafna basin, with a surface area of 7250 km 2 [START_REF] Dahmani | Traitement des eaux du bassin hydrographique de la Tafna[END_REF], is located in a sub-humid to semi-arid region, with rainfall ranging from 300 to 700 mm/year. Many of the river's tributaries are intermittent streams. Two areas can be distinguished: the higher south composed of chalk plateau and wetter condition due to the altitude, and the north consisting of drier lowland. For the water needs of Oran city, a dam was first built on the Tafna River around 1940, just on the edge of the upper plateau. The excess water was to be used to irrigate the lower lands. At that time, the mean rainfall value was 560 mm/year, so the dam could regulate a volume of 74 million m 3 /year. But since then, the mean rainfall dropped to 450 mm during 1940-1974, and after the dry period of the 1970s, even further, to 342 mm for 1975[START_REF] Adjim | Impact de la sécheresse de 1974-2002 sur les apports du barrage de Béni Bahdel (Nord-Ouest Algérie)[END_REF][START_REF] Adjim | Impact de la sécheresse de 1974-2002 sur les apports du barrage de Béni Bahdel (Nord-Ouest Algérie)[END_REF].

Population growth in Tlemcen and Maghnia has presented two types of problem. The first concerns water supply for population, industry and irrigation. All surface water and shallow groundwater, already scarce, are being fully exploited, and more recently there has also been deep pumping. The second impact on rivers is pollution, which has reached all the lowest river courses. Effects become more obvious during low flow, and some rivers become only fed by wastewaters [START_REF] Gagneur | Twenty years research on the Tafna ecosystem, an Algerian temporary stream[END_REF].

Also, especially in the lower part of the Tafna River basin, since the 1970s many wells and small rivers have become dry, and significant dying of poplars (Populus alba L.) has been observed. At first, the tree loss was attributed to an increase in insect infestation, but *Corresponding author. Email: lambs@cict.fr a recent study [START_REF] Labiod | Devenir du peuplier blanc (Populus alba L.) dans le nord-ouest Algérien. Diagnostic sanitaire de quelques peuplements sur la région de Tlemcen[END_REF]) reveals that the original cause of this degradation in popular health was the lack of water.

Here with the help of the stable water isotope, we try to understand the water circulation along the Tafna basin, its storage within the karst system, and how it affects the riparian vegetation, important for the water bioremediation. We also investigate an older forest to gain insight on the climate change effect on vegetation growth.

Materials and methods

Water stable isotopes

In order to determine the water origin, the tributaries and springs of the Tafna River as well as different groundwaters were sampled, put in capped vials (10 mL for water) and sent to Iso-Analytical Ltd, Sandbach, UK. Oxygen-18 analysis of the samples was performed in triplicate by equilibration with carbon dioxide in septum-capped containers. Deuterium analysis of the samples was performed in triplicate by equilibration with hydrogen gas catalysed by platinum in septum-capped containers. The head spaces of the containers were subsequently measured on a continuous flow isotope ratio mass spectrometer system (Europa Scientific . The equipment was calibrated by using two water standards that are traceable to the primary reference standards V-SMOW2 (Vienna-Standard Mean Ocean Water) and V-SLAP2 (Vienna-Standard Light Antarctic Precipitation) distributed by the IAEA, Vienna. A third traceable water standard was analysed alongside the samples to check the accuracy of the data.

The results are expressed in per mil on the V-SMOW/SLAP scale, for oxygen:

d 18 O V-SMOW2 (‰) = ((18O / 16Osample) / (18O / 16Ostandard) -1) * 1000;
and for deuterium:

d 2 H V-SMOW2 (‰) = (( 2 H / 1 H sample ) / ( 2 H / 1 H standard ) -1) * 1000.

Dendrochronology

Different trees were cored over the Tafna basin, from the riparian white poplar (Populus alba) to older conifers (Juniperius oxycedrus, Pinus halepensis) with an increment borer of 5 mm wide by Suunto, Finland. When possible, some trees were cut to provide whole disks to confirm wood core results. The woods were than sanded and the tree ring widths were read with the help of a LINTAB by RINNTECH, Germany. The aim was to determine the change of tree annual growth over the time.

Results

Figure 1 gives the general morphology of the Tafna with its numerous tributaries originating around the Tlemcen Mountains. The first stable isotope sampling in 2006 [START_REF] Lambs | Etude hydrologique et dendrochronologique du haut bassin de la Tafna, NW Algérie. 4ième Journées de la Société Française des Isotopes Stables[END_REF]) on 18 O compared the variation between the upper basin river and sources, and the sources around the city of Tlemcen. The more negative values (i.e. more depleted in heavy isotopes) showed water coming from a colder area, in general at a higher altitude.

The mean values for the two sites differ by about one unit (see Figure 2), respectively -7.52 ± 0.26 and -6.51 ± 0.25, which show the different rainfall and snow origins. More surprising is the stable value over the season, even for the river plateau, thus confirming the importance of a karstic reservoir, with a buffer system of at least over 6 months. Interesting is the sampling in the Mansourah village, above and south-west of the city of Tlemcen, just on the edge of the plateau, where mineral water is produced. The agricultural well displays a d 18 O isotope value of -6.52, whereas at a deeper drilling (70 m) the mineral water has a higher absolute depleted value of -7.52, as the water in the higher Tafna. In this case a double groundwater should exist, one shallow with local water and a deeper one fed by higher-altitude water.

A second isotope investigation was undertaken in spring 2008, looking on both deuterium and 18 O along the Tafna River course, from the underground source until the sea estuary. The conductivity of the main spring and tributaries is very high since the beginning (around 600 µS), due to the time of residence and chalk soil, and increases downstream with the evaporation, pollution and salinity intrusion. The 18 O value for the lower basin ranges between -3.4 and -5.4. Except for the surface water of the Béni Bahdel dam, the water with the higher salinity (1100-2000 µS) has also lower excess deuterium (2.4-5.8).

All the higher altitude sources of the Tafna River system present similar 18 O values, depleted in heavy isotopes: Tafna itself -7.8, Khemis -7.6, Sebdou -7.2, Isser -7.4, and Chouly -7.4 on the Mouillah and -7.2 on the Morocco side. The worldwide and Atlantic reference for the water isotopic measurement is the Global Meteoric Water Line (GMWL) with a slope of 8 and a deuterium excess of 10 as defined by [START_REF] Craig | Isotopic variations in meteoric waters[END_REF]. But for the Mediterranean Sea, due to the closed basin effect, the deuterium excess is much higher, Figure 1. The Tafna River basin. and for the western part of this sea basin, the Western Mediterranean Meteoric Water Line (WMMWL) value is around 14 [START_REF] Celle | Isotopic typology of the precipitation in the western Mediterranean region at three different time scales[END_REF]. For the higher-altitude Tafna sources and tributaries, the deuterium excess ranges between 10 and 14, i.e. between the GMWL and the WMMWL, showing both the Atlantic and Mediterranean rainfall influence (see Figure 3). After the first dam at Béni Bahdel, the value for the Tafna River becomes less negative: (5, around the same as found farther west in Oued Nechef, which takes its source behind the city of Tlemcen, and also the value taken by the Isser River in its lower part. On the Tafna side after the other low-altitude dams, the isotopic value ranges from -4 to -3. The calculated deuterium excess shows higher evaporation for some places (value between 1.4 and 4.7) as well as for the Sebdou River (5.8) where the pollution and human influence are important as revealed by a high ionic content, with a conductivity of 900 µS.

The calculated slope of 6.2 from the Tafna sources until the downstream water sampling shows an important evaporation, as this water sampling was undertaken between February and April. This kind of sampling along the whole Tafna River is not possible in summer due to the drying of many river sections in the lower part of the basin.

The main riparian tree species is the white poplar. Until recent years, in the upper basin, trees growing very close to the river or close to irrigation canal had a high growth rate, mean value from 0.5 to 1 cm/year, whereas in the lower part a much lower rate was present, impacted by insect attacks. Most of the old trees display a lower growth rate since the reduction of rainfall in the 1970s and 1980s. No tree over 45 years old was found. To get a wider view of the rainfall influence on tree growth over the last decades, we had to focus outside of the riparian trees, i.e. on conifer trees. Over the city of Tlemcen, there is a nice Aleppo pine forest and more isolated trees as "oxycedar juniper" (Juniperus oxycedrus) and "thuya de Berbérie" (Tetraclinis articulata), some of these trees being

Discussion

The Tafna basin was mainly investigated under geologic [START_REF] Collignon | Hydrogéologie appliquée des Monts de Tlemcen (Algérie)[END_REF], limnologic [START_REF] Gagneur | Flash floods and drying up as major disturbances upon benthic communities in North-African wadis[END_REF][START_REF] Gagneur | Twenty years research on the Tafna ecosystem, an Algerian temporary stream[END_REF] and hydrologic [START_REF] Dahmani | Traitement des eaux du bassin hydrographique de la Tafna[END_REF][START_REF] Adjim | Impact de la sécheresse de 1974-2002 sur les apports du barrage de Béni Bahdel (Nord-Ouest Algérie)[END_REF] approaches. With the absence of long meteorological series, the drying of the river and the karstic system, it was difficult to study the whole water circulation. The isotopic techniques allow one to follow the water circulation and determine the water origin even with a karstic system as in the upper basin, and to evaluate the evaporation process in the lower 1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 part. The first 18 O measurement in this area was made by Blavoux in 1984-1986, but seems to be shifted (about 1 unit more negative) with the values obtained later by other authors. This could have happened due to exceptional cold conditions during the sampling period or lower mean temperature values during the rainy seasons. The next and more complete isotopic analyses were published by [START_REF] Azzaz | The use of environmental isotopic and hydrochemical tracers to characterize the functioning of karst systems in the Tlemcen Moutains, northwest Algeria[END_REF], and made in April 1991 on about 30 sources and wells on and around the Tlemcen Mountains. They show for the first time a double influence, that of the Mediterranean and the Atlantic, on the high-altitude rainfall, as seen by the deuterium excess value. We have confirmed this mixed origin for the headwaters of the main tributaries of the Tafna River, for whole of the Tafna basin and also for Morocco as well as the Mouillah tributary. Azzaz et al. also measured the resident time of karstic water, and found only a single source with a deeper reservoir and longer storage (around 25 years). From our own data, the absence of a seasonal effect shows that the water is stored for at least 6 months. The city of Algiers is the only Algerian station for the Global Network of Isotopes in Precipitation (GNIP) by IAEA. Rainfall isotopes data is available for 1998 and 2004. The evaporation slope that we have found over the Tafna basin (6.18) is intermediate between the one of Algiers (slope 4.81) and the closest GNIP station on the west part: Bab Bou Idir (7.34), a higher-altitude station in Morocco. This very low slope found for Algiers magnified the significant evaporation during these years. This possible climate change is also seen in tree growth. The study of [START_REF] Labiod | Devenir du peuplier blanc (Populus alba L.) dans le nord-ouest Algérien. Diagnostic sanitaire de quelques peuplements sur la région de Tlemcen[END_REF] has shown that numerous poplars in the lower basin present lower growth rate and higher insect attacks since the 1980s. The diminution of the riparian vegetation has a negative effect on flood buffering, local water vegetation and water bio-remediation. The agricultural surface land increases and with it the nitrate input, while the natural wetland purification area diminishes. This reduction of tree ring width was also observed in nearby older conifers around the city of Tlemcen. Other dendrochronological and tree disease studies have been recently done on century-old cedars in the Chréa forest a few hundred kilometres more east [START_REF] Sbabdji | Cedar tree growth (Cedrus atlantica Manetti) in Chréa National Park, Algeria, and the influence of defoliation by the pine processionary caterpillar (Thaumetopoea pityocampa Schiff)[END_REF]. In that case also, a growth decrease was observed since the 1970s.

Conclusion

The upper Tafna River is not easy to study since it is formed by a wide karstic and cave system with an underground river up to 3.4 km long. Some water outflow from this system has even been found on the Morocco side. The total underground water of this system is estimated to be tens of billions of cubic metres and thus certainly buffers the seasonal rainfall fluctuation, but also prolongs the lack of water in the following years. This means that the real effect of the rainfall over the head basin will be only visible in the following year. The lower Tafna basin now has four additional dams that increase water evaporation and enhance sediment retention. The pimary aim of these dams was to improve the buffer capacity for the dry year, but now the rainfall is too scarce to fill the dam completely during the wet season. The drier and hotter weather condition over all North Africa and higher water needs will oblige water planners to think again regarding the distribution of water resources and the wastewater recycling. For instance, natural karstic system should be preferred for water storage as against open dams with their high evaporation. Also preserving the remaining forests and planting drought-resistant trees will reduce aridity. For agriculture, drip techniques would reduce water requirements. With the lowering of the groundwater table and lower river discharge, care must to taken to prevent salt intrusion from the sea or the numerous chots (salty lakes).

Figure 2 .

 2 Figure 2. Comparison of the mean water isotopic value (d 18 O) for the upper Tafna basin and the middle Tafna basin, Tlemcen area, during March 2006 (dark grey) and August 2006 (light grey); n represents the number of different water samples.

  old. Figure4reports typical dendrochronological curves versus the annual rainfall. Here also, a decrease in growth could be seen since 1975.

Figure 3 .

 3 Figure 3. Deuterium and 18 O characteristics of the Tafna water during February and April 2008.

Figure 4 .

 4 Figure 4. Comparison of the annual conifer tree growth (mm/year) on the forest above Tlemcen and the annual rainfall (in hundreds of mm/year), this last curve being shifted to avoid overlapping, showing the corresponding decrease since 1975.

Acknowledgements

We are grateful to H. Adjim (Université de Tlemcen) and J. Gagneur (Ecolab, Toulouse) for interesting discussion on the Tafna basin hydrology and to F. Guibal (IMEP, Aix-en-Provence) for advice for the Aleppo pine dendrochronologic approach.


Jérôme Orivel
email: jerome.orivel@ecofog.gf

Luc Lambs

Pierre-Jean G Malé

Céline Leroy

Julien Grangier

Thierry Otto

Angélique Quilichini

Alain Dejean

Joshua J Tewksbury

C Orivel

Leroy

P.-J G Malé

P.-J G Malé

Dynamics of the association between a long-lived understory myrmecophyte and its speciWc associated ants

Keywords: Allomerus decemarticulatus, Hirtella physophora, Lifespan, Mutualism, Myrmecophyte

Myrmecophytic symbioses are widespread in tropical ecosystems and their diversity makes them useful tools for understanding the origin and evolution of mutualisms. Obligate ant-plants, or myrmecophytes, provide a nesting place, and, often, food to a limited number of plant-ant species. In exchange, plant-ants protect their host plants from herbivores, competitors and pathogens, and can provide them with nutrients. Although most studies to date have highlighted a similar global pattern of interactions in these systems, little is known about the temporal structuring and dynamics of most of these associations. In this study we focused on the association between the understory myrmecophyte Hirtella physophora (Chrysobalanaceae) and its obligate ant partner Allomerus decemarticulatus (Myrmicinae). An examination of the life histories and growth rates of both partners demonstrated that this plant species has a much longer lifespan (up to about 350 years) than its associated ant colonies (up to about 21 years). The size of the ant colonies and their reproductive success were strongly limited by the available nesting space provided by the host plants. Moreover, the resident ants positively aVected the vegetative growth of their host plant, but had a negative eVect on its reproduction by reducing the number of Xowers and fruits by more than 50%. Altogether our results are important to understanding the evolutionary dynamics of ant-plant symbioses. The highly specialized interaction between long-lived plants and ants with a shorter lifespan produces an asymmetry in the evolutionary rates of the interaction which, in return, can aVect the degree to which the interests of the two partners converge.

Introduction

The evolutionary persistence of mutualisms depends on the alignment of the reproductive interests of the interacting partners. If each partner gains from the association, the costs and beneWts involved in maintaining the interaction can vary in both space and time. Such variability is the primary source of conXicts of interest between the partners-especially if it is associated with a horizontal mode of transmission-and promotes cheating. Hence, the challenge is to identify the mechanisms that maintain evolutionarily stable, interspeciWc mutualisms in spite of the ubiquity of cheating [START_REF] Bronstein | Ecological dynamics of mutualist/antagonist communities[END_REF][START_REF] Herre | The evolution of mutualisms: exploring the paths between conXict and cooperation[END_REF][START_REF] Sachs | The evolution of cooperation[END_REF]). Several factors, such as partner choice (i.e., Wltering), partner Wdelity or spatial structure (i.e., limited dispersal) have been identiWed as fostering the alignment of the mutualists' interests [START_REF] Foster | A general model for the evolution of mutualisms[END_REF][START_REF] Herre | The evolution of mutualisms: exploring the paths between conXict and cooperation[END_REF][START_REF] Szilágyi | The evolution of intermediate castration virulence and ant coexistence in a spatially structured environment[END_REF]. Consequently, both the spatial and temporal dynamics of the interactions are important features in understanding mutualistic associations between species.

Mutualistic ant-plant associations, which are widespread in tropical ecosystems, oVer a suitable model for exploring this issue. Obligate ant-plants, or myrmecophytes, provide a nesting space, and, often, food to a limited number of plant-ant species. In exchange, plant-ants protect their host plants from phytophagous animals, competitors and pathogens, and can provide them with nutrients [START_REF] Beattie | Ant-plant interactions[END_REF][START_REF] Heil | Protective ant-plant interactions as model systems in ecological and evolutionary research[END_REF][START_REF] Solano | Ant-fed plants: comparison between three geophytic myrmecophytes[END_REF]. Ant-plants and plantants share a common interest in growth, but not necessarily in reproduction. For both parties, the reproductive investments of the other are useless to the interaction. Winged sexuals are not involved in protecting the plant foliage and the energy invested by plants in producing Xowers does not increase the nesting space for the ant colony. Consequently, natural selection should favor cheating to maximize each species' own beneWts. Indeed castrating parasites have been identiWed in several antplant mutualisms [START_REF] Edwards | Tolerating castration by hiding Xowers in plain sight[END_REF][START_REF] Gaume | Ant-plant conXicts and a novel case of castration parasitism in a myrmecophyte[END_REF][START_REF] Izzo | Cheating the cheater: domatia loss minimizes the eVects of ant castration in an Amazonian ant-plant[END_REF][START_REF] Stanton | Sterilization and canopy modiWcation of a swollen thorn acacia tree by a plant-ant[END_REF][START_REF] Yu | A castration parasite of an ant-plant mutualism[END_REF]. If castration can clearly reduce or even prevent plant reproductive success, it has, nevertheless, to be balanced against the overall reproductive activity of the plant throughout its lifetime.

In this study we concentrated on a one-to-one mutualism between the Neotropical understory myrmecophyte Hirtella physophora Martius & Zuccharini (Chrysobalanaceae) and its obligate and speciWc ant-partner Allomerus decemarticulatus Mayr (Myrmicinae). Due to the highly specialized nature of the H. physophora-A. decemarticulatus association, we focused on the temporal structuring and dynamics of this association by studying the life histories and growth rates of both partners. Through Weld inventories and using an experimental approach, we investigated the lifespan of both the plants and their associated ant colonies, and the beneWcial or detrimental eVects they can have on the reproductive growth of the other.

Materials and methods

Study site and model

This study was conducted between 2000 and 2009 in the pristine forest situated around the Weld station at Petit Saut, Sinnamary, French Guiana (05°03Ј30.0ЉN, 52°58Ј34.6ЉW).

Hirtella physophora grows strictly in the understory of pristine Amazonian forests and mostly in patches located on the upper slopes of hillsides [START_REF] Solano | Myrmecophytes of the understory of French Guianian rainforests: their distribution and their associated ants[END_REF]. These treelets have long-lived leaves that bear extraXoral nectaries and a pair of pouches at the base of each lamina. The leaf pouches that shelter ant colonies, diVer both morphologically and anatomically from the lamina [START_REF] Leroy | Comparison between the anatomical and morphological structure of leaf blades and foliar domatia in the ant-plant Hirtella physophora (Chrysobalanaceae)[END_REF]. Each H. physophora is almost always associated with A. decemarticulatus (99% of the inhabited plants, the remaining being inhabited by Crematogaster sp. aV. crucis), with a single colony per plant. Moreover, A. decemarticulatus has never been found in association with another myrmecophyte species in the study area [START_REF] Solano | Myrmecophytes of the understory of French Guianian rainforests: their distribution and their associated ants[END_REF]. The A. decemarticulatus workers protect their host plants through their predatory behavior, including by building a gallery-shaped trap along the stems to capture prey [START_REF] Dejean | Arboreal ants build traps to capture prey[END_REF].

Peak Xowering periods in H. physophora in French Guiana occur from December to February and June to August. InXorescences arise at axillary fasciculate racemes and they comprise three to six Xowers [START_REF] Prance | Monograph of the Chrysobalanaceae[END_REF].

Life history and growth rate of H. physophora A total of 1,320 H. physophora individuals were tagged, and their height and trunk diameters were measured. The measurements of trunk diameters were taken with calipers on the lowest part of the base of the trunk, closest to the ground.

The growth rate of H. physophora was estimated by recording changes in the trunk diameters of 36 tagged Hirtella trees over 6 years (2000)(2001)(2002)(2003)(2004)(2005)(2006). We also monitored 257 individuals for 3 years (2003)(2004)(2005)(2006), and recorded the number of trees that were heavily damaged by falling trees or branches plus those that had dried out after the formation of a tree-fall gap in their vicinity.

Radiocarbon dating the trees

Due to the lack of true annual growth rings in many tropical species, the estimation of the age of such plant individuals cannot be made dendrometrically. We therefore used radiocarbon ( 14 C) dating together with estimations of diametrical increments of the trunk to address this question. Four wood samples from H. physophora trees were analyzed along with samples taken from Pinus caribaea Morelet and Avicennia germinans (L.) L. of known ages growing in neighboring areas and that served as references (see Table 1 for details on trunk diameters, wood density and weight of samples). The pine sample was taken from a plantation whose age is known, while the mangrove tree is a natural pioneer species whose age was deduced from aerial photographs.

Plants assimilate 14 C during photosynthesis, and once trapped in the wood, the amount of 14 C decreases according to the exponential law of radioactive decay (T 1/2 = 5,730 years), so that the 14 C content can permit us to date the age of the wood. However, concentrations of atmospheric 14 C doubled between 1950 and 1965 due to the aboveground explosions of atomic bombs; and then declined [START_REF] Nydal | Tracing bomb 14 C in the atmosphere 1962-1980[END_REF]. Consequently, radiocarbon-dating wood formed before or after the detonation of the atomic bombs requires diVerent methods of calculation. For trees formed after 1950, these changes in atmospheric 14 C are directly detectable by measuring radiocarbon. Dating older wood is complicated by the temporal variation in atmospheric 14 C resulting from massive emissions of CO 2 containing million-year-old 14 C that can trigger an overestimation of the age of the wood [START_REF] Nydal | Tracing bomb 14 C in the atmosphere 1962-1980[END_REF][START_REF] Stuiver | High-precision decadal calibration of the radiocarbon time scale, AD 1950-2500 BC[END_REF]. The age of wood samples is usually expressed after calibration as a percentage of modern C (PMC) for periods following the explosion of the bombs, or in the years before the present for trees that date from before the explosion of the bombs (BP years; where the present is arbitrarily deWned as 1950).

We used the Calibomb program (http://calib.qub.ac.uk/ CALIBomb) and the Wellington data set programmed for the Southern Hemisphere to estimate the age of the wood samples displaying a PMC value greater than 100, or wood formed after 1950. For the samples displaying a PMC value less than 100, or wood formed before 1950, we used the Calib 5.02 program (http://calib.qub.ac.uk/calib/calib.html) with the calibration curve shcal04.14c proposed by [START_REF] Mccormac | SHCal04 Southern Hemisphere calibration 0-11.0 cal kyr BP[END_REF].

Evaluating the lifespan of A. decemarticulatus colonies

To evaluate the lifespan of A. decemarticulatus colonies, 193 H. physophora individuals were monitored twice each year between 2002 and 2005 (800 days). Each time, we recorded the presence or absence of an ant colony and its developmental status according to the quality of the galleries interconnecting the leaf pouches. Indeed the building of the gallery-shaped trap by A. decemarticulatus enables us to estimate the development of the colony and its death as the fungus they use in the galleries dies soon after the death of the ant colony. Consequently, the death of a given colony and the recolonization of the plant between two censuses can be easily determined as the trap is built after the colony develops (i.e., the length of the galleries is linked to the increase in the number of inhabited domatia on the plant). We hypothesized that all ages were represented in the ant colonies at the Wrst census and that the death of the colonies was directly related to their age. This permitted us to establish a linear regression of the number of surviving colonies as a function of time, and the rate of colony extinction was used to estimate the half-life and maximum age of the colonies.

Ants' eVect on plant growth and reproduction

To assess both the short-and long-term eVects of the ants on the plants, each month for 16 months we monitored the vegetative and reproductive growth rates of individuals from which the ants were removed (n = 25) as well as control plants (n = 22). Both groups of plants were similar in size expressed as the number of leaves (mean § SE = 17.04 § 3.6 vs. 16.5 § 5.3 for ant-excluded and control plants, respectively, two-sample t-test: t = 0.335, df = 45, P = 0.74). We eliminated the ants by injecting an aqueous solution (2‰) of pyrethrum 25% inside all of the domatia. The rapid knockdown eVect of the pyrethrum immediately killed all of the resident ants, and its photolability prevented any residual eVects on phytophagous insects. As founding queens re-colonized certain plants during the course of the experiment, we re-injected the pyrethrum solution into the domatia occupied by new queens 3 times over the 16 months. We recorded the total number of leaves, the number of new leaves as well as the number of Xoral buds, Xowers and fruits on each plant individual monthly. Control and ant-excluded plants were compared according to either their total vegetative or reproductive investments during the 16-month period. We started the census 2 months after removing the ants because [START_REF] Grangier | Indirect defense in a highly speciWc ant-plant mutualism[END_REF] demonstrated that excluding the ants has no eVect on the rate of herbivory after 40 days.

To investigate the potential castrating virulence of A. decemarticulatus, 23 H. physophora bearing at least two early stage inXorescences were selected. Ant workers were removed from one inXorescence and their access to the Xoral buds was prevented using Tanglefoot; while the other inXorescence was used as a control. The numbers of Xowers as well as the number of fruits produced were counted each week after treatment.

Size and structure of the ant colonies

We collected the ant colonies inhabiting 35 H. physophora, which ranged in size from having 7-96 domatia, and we investigated the relationship between colony size and the number of domatia on the host plant. All of the leaves and stems on each plant individual were cut oV and placed into a plastic bag, and then preserved in 70% ethanol. Each domatium was then dissected and the number of workers, pupae, larvae, queens, and alate males and females was recorded. The number of workers that were in the galleries built under the stems of the plant was also recorded.

Results

Diameter and height distribution of H. physophora trees

The frequency distributions for height and stem diameter of the 1,320 plants measured were in both cases unimodal with a mean diameter of 1.6 § 0.94 cm and a mean height of 1.31 § 1.0 m (Fig. 1; data not shown for height distribution). The structure of the distributions showed that most of the trees were in the middle of the size range for both diameter and height, with a relatively small number of seedlings and a constant decrease toward the tallest individuals.

Even if tree height and diameter showed a positive correlation, the variance between groups of individuals was not homogeneous [14 groups from 0.1 to 0.3 cm in diameter to >4 cm; Fig. 1; Levene test F (13,1306) = 61.24, P < 0.01], highlighting the large variation observed in tree height as the trees age and, consequently, the amount of damage they can sustain during their lifetime. Indeed, during the 3-yearlong monitoring period, a total of 29 trees out of 257 were aVected in some way, resulting in 4% of the trees aVected each year.

Determining the age of the trees

The 14 C analyses conducted using the Calibomb program for wood with PMC values greater than 100 generally resulted in one single date (Table 1). The Calibomb program estimated the ages of two trees of known ages (P. caribaea and A. germinans) to within 1-2 years of their real ages. The evaluation of the ages of the two of H. physophora tree samples with 1.9-and 2.2-cm-wide trunks corresponded to 48 and 50 years, respectively (Table 1). Thus, the corresponding mean growth rate from seedlings of such trees was about 0.042 cm/year [0.040 (1.9/48) and 0.044 (2.2/50) for the 1.9-and the 2.2-cm-wide trees, respectively]. Note that the diVerences in age and trunk diameter reXect possible variations in environmental conditions for these two individuals, but their growth rate was quite similar.

The 14 C analyses concerning the two largest H. physophora trees (2.9-and 3.2-cm-wide trunks) resulted in PMC values lower than 100, and thus had to be recalibrated using the Calib 5.02 program. This recalibration resulted in three theoretically possible dendro-ages for each sample (H. physophora sample C, 73 § 75 BP, 150 § 30 BP and 244 § 25 BP; H. physophora s a m p l e D , 3 6 § 30 BP, 110 § 30 BP and 240 § 20 BP). This multi-age result originates from the very low rate of decrease in atmospheric 14 C and from the fact that this logarithmic curve is not smooth: it has many small peaks, which can be matched with the measured PMC value. To select the right age, we used the growth rate of adult trees determined in the Weld by measuring the increase in trunk diameters between (mean § SE) 2.078 § 0.755 to 2.181 § 0.776 cm. The mean diVerence (0.103 § 0.070 cm) corresponds to a mean growth of 0.0171 cm/year over this period for H. physophora trees whose trunk diameters were already about 2 cm, so about half from early growth, knowing that these 2-cm-wide trees are around 50 years old. Consequently, a tree with a 3-cm-wide trunk is theoretically approximately 108 years old [50 + (1/0.0171)], permitting us to estimate that H. physophora samples C and D were 150 and 110 BP-years old, respectively (Table 1). These BP values correspond to real ages of about 205 and 165 years, respectively.

To evaluate the age of the individual in our dataset with the widest trunk (6 cm), we based our calculations on the ages of the 14 C-dated trees with trunk diameters of about 3 cm (see above). We thus estimated that this tree was between approximately 340 [165 + (2.8/0.0171)] and 385 [205 + (3.1/0.0171)] years old.

Evaluating the lifespan of A. decemarticulatus colonies

The linear regression of the number of surviving ant colonies as a function of time resulted in r 2 = 0.97, permitting us to estimate a colony half-life (i.e., the point in time at which 50% of the initial number of colonies would have survived) at 10.13 years. As it takes about 1 year for a colony to reach maturity (J.O., personal observation), a better estimate of the half-life of the colonies would be closer, therefore, to 11 years. The estimated maximum age of the colonies is 20.36 years (a minimum of 21 years starting from foundation), and, thus, by far shorter than that of the host H. physophora individuals.

Ants' eVect on plant growth and reproduction

Removing the ants over a long period of time aVected the vegetative growth of the plants, and the H. physophora leaves produced after the ants were removed suVered from herbivory (Fig. 2). Indeed, the plants devoid of ants produced a signiWcantly lower number of new leaves than the control plants (mean number per plant § SE = 17.4 § 7.4 vs. 24.9 § 6.7 for ant-excluded and control plants, respectively; Mann-Whitney U test: U = 134.5, P < 0.01), which resulted in a lower vegetative growth rate over the course of the experiment for plants devoid of ants (Fig. 2; Table 2). However, no signiWcant diVerences in reproductive growth rates could be discerned. Reproductive investment occurred in most of the plants (21/25 vs. 19/22 for ant-excluded and control plants, respectively; Fisher's exact test P = 0.57) and the reproductive eVort of those plants did not diVer between the two groups (Xoral buds, mean number per plant § SE = 21.4 § 13.0 vs. 22.9 § 12.9 for ant-excluded and control plants, respectively; Mann-Whitney U test, U = 40.5, P = 0.29; Xowers, 4.6 § 2.8 vs. 5.6 § 3.1, U = 51.5, P = 0.64). The percentage of plants that produced fruits did not diVer between both groups (5/25 vs. 7/22 for ant-excluded and control plants, respectively; Fisher's exact test P = 0.28). Moreover, and as usually observed in H. physophora, fruit production was very low and the diVerence between plants that produced fruits was not sig-niWcant between the two groups (mean number per plant § SE = 1.4 § 0.5 vs. 2.3 § 1.3 for ant-excluded and control plants, respectively, Mann-Whitney U test: U = 12.5, P =0.42).

Nevertheless, the presence of the ants had a negative eVect on the reproduction of their host plants that was demonstrated when the ants were selectively removed from the inXorescences of their host (Fig. 3). The inXorescences to which the ants had access indeed produced signiWcantly fewer Xowers than the ant-excluded ones (mean percentage of Xowers per inXorescence compared to the initial number of buds § SE = 24.5 § 32.3 vs. 76.9 § 24.8 for control and ant-excluded inXorescences, respectively; Wilcoxon test: W =193, P < 0.0001). This negative eVect was also reXected in the number of plants that produced fruits (12/23 vs. 3/23 for control and ant-excluded inXorescences, respectively; Fisher's exact test P = 0.005) and in the number of fruits produced (mean percentage of fruits per inXorescence compared to the initial number of buds § SE = 2.3 § 1.3 vs. 17.1 § 3.8 for control and ant-excluded inXorescences, respectively; Wilcoxon test: W = 78, P < 0.0025).

Size and structure of the ant colonies

There was a positive and linear relationship between the size of the ant colony and the number of domatia on the host plant (Fig. 4; r 2 = 0.8356). This relationship accounts for most of the variation in colony size and strongly suggests that for all plant sizes, the amount of available nesting space is an important limiting factor for colony growth. The lack of curvilinearity in the relationship (test of signiWcance for domatia 2 : F = 0.109, P = 0.80) also showed that the amount of available nesting space is probably one of the most important limiting factors in colony development.

The onset of alate production begins early in colony development (e.g., when there are fewer than 300 workers), but most of the alate individuals are produced in colonies containing more than 600 workers. Moreover, the number of alates (brood and adult males and females), and thus the rate of alate production is also strongly correlated with the amount of nesting space oVered by the host plant (Fig. 4; r 2 = 0.7984). The same relationship exists when colony size rather than the number of domatia is taken into account. Then, the amount of available nesting space not only limits colony size, but also its rate of reproduction.

Discussion

Altogether our results highlight some important features that can deeply inXuence the evolutionary dynamics of ant-plant symbioses. As in most ant-plant mutualisms, the myrmecophytism in H. physophora clearly enhances the growth of individuals, but A. decemarticulatus also negatively aVects its host's reproduction by reducing by twothirds the number of Xowers and thus the number of fruits. However, such a negative eVect has to be balanced over the entire lifetime of the plant individuals and it appears that the overall eVect of the ants on their host plant is clearly positive. Indeed, even if the reproductive eVort does not diVer between ant-inhabited plants and plants devoid of ants, the latter nevertheless suVer from herbivory. Thus, over the long term, this can be detrimental to their growth and survival.

Functioning and importance of myrmecophytism Through their predatory behavior, which includes the constant patrolling of their host tree foliage and their induced response to foliar wounds [START_REF] Grangier | Indirect defense in a highly speciWc ant-plant mutualism[END_REF], resident ants protect the foliage of their host plant. This also beneWts the ants as the production of new leaves is the only means by which their nesting space can increase. The limitation in colony growth and reproduction induced by the amount of available nesting space highlights the importance of this limiting factor for the ants [START_REF] Fonseca | Nesting space limits colony size of the plant-ant Pseudomyrmex concolor[END_REF][START_REF] Fonseca | Amazonian ant-plant interactions and the nesting space limitation hypothesis[END_REF]. According to these results, it is in A. decemarticulatus' best interest to favor the vegetative growth of their host plant, like in any protective ant-plant symbioses, not only in terms of the production of more food rewards or domatia, but also to maximize their own reproductive success. Moreover, castration also enables host-plant resources to be reallocated from reproduction to vegetative growth [START_REF] Frederickson | ConXict over reproduction in an ant-plant symbiosis: why Allomerus octoarticulatus ants sterilize Cordia nodosa trees[END_REF][START_REF] Yu | Parasites of mutualisms[END_REF]). Castrating parasites have been identiWed in several systems and especially in all of those involving the Allomerus ant species studied so far [START_REF] Izzo | Cheating the cheater: domatia loss minimizes the eVects of ant castration in an Amazonian ant-plant[END_REF][START_REF] Yu | A castration parasite of an ant-plant mutualism[END_REF]. Allomerus decemarticulatus is no diVerent in this matter than the other Allomerus species. But, A. decemarticulatus destroy about two-thirds of the Xoral buds whereas the other Allomerus species generally destroy 100% of the Xoral buds, reducing their host reproduction to zero (but see [START_REF] Edwards | Tolerating castration by hiding Xowers in plain sight[END_REF]). In the present case, the combination of both the positive eVect of the Allomerus on the vegetative growth of H. physophora and the much longer longevity of the host plants than their speciWc plant-ants are important factors favoring the persistence of the interaction. The loss of ant inhabitation by the plant can be considered as a Wtness valley as the loss of biotic protection would aVect the survival of the plants. In absence of alternative solution for H. physophora, this system could be thus considered as an example of a local adaptive peak.

Beside its myrmecophytism, H. physophora also shares several biological traits with many plant species from the understory that enables its survival in this environment, such as shade tolerance and slow growth, mortality, and recruitment rates [START_REF] Easdale | Tree life histories in a montane subtropical forest: species diVer independently by shade-tolerance, turnover rate and substrate preference[END_REF][START_REF] Gourlet-Fleury | Grouping species for predicting mixed tropical forest dynamics: looking for a strategy[END_REF][START_REF] Nascimento | Demographic and life-history correlates for Amazonian trees[END_REF][START_REF] Vieira | Slow growth rates of Amazonian trees: consequences for carbon cycling[END_REF]). Moreover, like for any plant species in the understory, the impact and consequences of the physical damage caused by debris falling from the canopy are of importance. The frequency of the damage suVered by H. physophora is of the same magnitude as that recorded in other studies, with around 4% of the trees aVected per year (Clark andClark 1989, 1991;[START_REF] Gartner | Breakage and regrowth of Piper species in rain forest understory[END_REF]). However, the ability of this plant species to develop reiterations from its lower part reXects its adaptation to these frequent disturbances.

Evolutionary consequences of the asymmetry in the partners' lifespan Because the plants are colonized early in their development, they will host several successive ant colonies during their lifetime. Neither the ontogenetic successions of diVerent partners nor the secondary polygyny of A. decemarticulatus colonies have been noted in H. physophora, thus requiring the de novo colonization of the plant by founding queens [START_REF] Djiéto-Lordon | Ecology of an improbable association: the pseudomyrmecine plant-ant Tetraponera tessmanni and the myrmecophytic liana Vitex thyrsiXora (Lamiaceae) in Cameroon[END_REF][START_REF] Feldhaar | Maintaining an ant-plant symbiosis: secondary polygyny in the Macaranga triloba-Crematogaster sp. association[END_REF][START_REF] Feldhaar | Patterns of the Crematogaster-Macaranga association: the ant partner makes the diVerence[END_REF][START_REF] Young | Ants on swollen-thorn acacias: species coexistence in a simple system[END_REF]. The temporal succession of colonies induces periods of time during which the plants are not protected by their associated plant-ants and, thus these periods are unfavorable to their growth and survival. However, given that the re-colonization process is rapid and continuous throughout the year, these unfavorable periods of time are generally kept to a minimum. Moreover, the evolutionary persistence of this association is favored by the highly specialized nature of the interaction that ensures the constant re-colonization of the plants by A. decemarticulatus queens [START_REF] Grangier | Mechanisms driving the speciWcity of a myrmecophyte-ant association[END_REF].

Altogether these results demonstrate that the specialized interaction between long-lived plants and ants with a shorter lifespan produces an asymmetry in the generational turnover and the evolutionary rates of the two partners. On the one hand, such an asymmetry could be seen as a destabilizing factor for the association because the populations of the long-lived mutualist can only respond slowly to increases in densities among the shorter-lived partner. Thus, when members of this shorter-lived guild of mutualists compete for access to their host, asymmetries in the generational turnover may increase the intensity of this competition [START_REF] Stanton | Interacting guilds: moving beyond the pairwise perspective on mutualisms[END_REF]. However, in the present case interspeciWc competition for the occupancy of H. physophora individuals is extremely weak [START_REF] Grangier | Mechanisms driving the speciWcity of a myrmecophyte-ant association[END_REF]). Thus, the evolutionary persistence of the H. physophora-A. decemarticulatus association seems possible due to the absence of an alternative partner for the plant combined with the dynamics of the interaction which enables H. physophora to still produce oVspring during their lifetime.

Fig. 1

 1 Fig. 1 Distribution of the trunk base diameters of a population of 1,320 Hirtella physophora. The age of the plants is indicated based on radiocarbon dating Diameter at the base (cm)

Fig. 2

 2 Fig. 2 Vegetative growth expressed as the mean number §SE of leaves in ant-removed (empty circles) and control (Wlled circles) H. physophora plants during a 16-month Weld experiment Number of months since ant exclusion 0 2 4 6 8 1 01 21 41 6

Fig. 3 Fig. 4

 34 Fig. 3 Allomerus decematiculatus' impact on the reproduction of H. physophora expressed as the number of Xowers (a) or fruits (b) produced according to the initial number of buds per inXorescences in ant-excluded or control inXorescences. Error bars above and below the boxes indicate the 90th and 10th percentiles, the ends of the boxes indicate the 25th and 75th percentiles and crosses indicate outliers

Table 1

 1 Sample information, accelerator mass spectrometry 14 C measurements, and minimum AD age (relative to 2005) for the wood samples of Hirtella physophora, Pinus Caribaea and Avicennia germinans

	Sample name	Laboratory	Trunk	Density Sample	13 C	Error	PMC	Error	Minimum	Age	Growth
		code	diameter		weight	(‰)	13 C		PMC	age	14 C BP	rate
			(cm)		(g)							(cm/years)
	P. caribaea	Poz 13575 14.9	0.69	0.65	¡26.1 0.1	118.00 0.39	17	-	0.876
	A. germinans	Poz 13574 14.3	0.66	0.26	¡23.8 0.8	114.30 0.40	14	-	1.021
	H. physophora sample A Poz 13595	1.9	0.95	0.30	¡37.7 0.1	103.05 0.36	48	-	0.040
	H. physophora sample B Poz 13594	2.2	0.95	0.40	¡34.3 0.4	101.44 0.35	50	-	0.044
	H. physophora sample C Poz 13598	2.9	0.93	0.40	¡36.7 1.4	98.12 0.35	-	150 § 30 -
	H. physophora sample D Poz 13592	3.2	0.93	0.24	¡32.8 0.5	98.67 0.34	-	110 § 30 -
	PMC percentage of modern C

Table 2

 2 Repeated-measures ANOVA examining the impact of ant removal from the plant on the number of H. physophora leaves over time

		df	MS	FP value	G-G a
	Treatment	1	947.47	1.531	0.222
	Error	45	618.98
	Time	16	22.10	5.530	<0.001	<0.001
	Time £ treatment	16	19.08	4.775	<0.001	0.001
	Error	720	3.99
	MS mean square
	a Probabilities corrected for sphericity are provided using the Green-
	house-Geisser correction (G-G)

Acknowledgments

We are grateful to T. Goslar from the Poznan Radiocarbon Laboratory, Poland and M. Paterne from the LSCE CNRS-CEA, France, for their help in the determination of the 14 C age, to C. Trontin for her valuable assistance in the lab and to Andrea Dejean for proofreading the manuscript. We would like to thank V. Rico-Gray and two anonymous reviewers for their valuable comments on the manuscript. We would also like to thank the Laboratoire Environnement de Petit Saut for furnishing logistical assistance. Financial support for this study was provided by the Programme Amazonie II (project 2ID) of the French Centre National de la Recherche ScientiWque (CNRS), by the Programme Convergence 2007-2013 Région Guyane (project DEGA) from the European Community, by a research program of the French Agence Nationale de la Recherche (research agreement no. ANR-06-JCJC-0109-01), and by the ESF-EURO-CORES/TECT/BIOCONTRACT program. The experiments comply with the current laws of France.