Teresa Valor
email: teresa.valor@ctfc.cat

Elena Ormeño

Pere Casals

Temporal effects of prescribed burning on terpene production in Mediterranean pines

Keywords: conifers, fire ecology, Pinus halepensis, Pinus nigra, Pinus sylvestris, plant volatiles, prescribed fire, secondary metabolism

Prescribed burning is used to reduce fuel hazard but underburning can damage standing trees. The effect of burning on needle terpene storage, a proxy for secondary metabolism, in fire-damaged pines is poorly understood despite the protection terpenes confer against biotic and abiotic stressors. We investigated variation in needle terpene storage after burning in three Mediterranean pine species featuring different adaptations to fire regimes. In two pure-stands of Pinus halepensis Mill. and two mixed-stands of Pinus sylvestris L. and Pinus nigra ssp. salzmanni (Dunal) Franco, we compared 24 h and 1 year post-burning concentrations with pre-burning concentrations in 20 trees per species, and evaluated the relative contribution of tree fire severity and physiological condition (δ 13 C and N concentration) on temporal terpene dynamics (for mono-sesqui-and diterpenes). Twenty-four hours post-burning, monoterpene concentrations were slightly higher in P. halepensis than at pre-burning, while values were similar in P. sylvestris. Differently, in the more fire-resistant P. nigra monoterpene concentrations were lower at 24 h, compared with pre-burning. One year post-burning, concentrations were always lower compared with pre-or 24 h post-burning, regardless of the terpene group. Mono-and sesquiterpene variations were negatively related to pre-burning δ 13 C, while diterpene variations were associated with fire-induced changes in needle δ 13 C and N concentration. At both post-burning times, mono-and diterpene concentrations increased significantly with crown scorch volume in all species. Differences in post-burning terpene contents as a function of the pine species' sensitivity to fire suggest that terpenic metabolites could have adaptive importance in fire-prone ecosystems in terms of flammability or defence against biotic agents post-burning. One year postburning, our results suggest that in a context of fire-induced resource availability, pines likely prioritize primary rather than secondary metabolism. Overall, this study contributes to the assessment of the direct and indirect effects of fire on pine terpene storage, providing valuable information about their vulnerability to biotic and abiotic stressors throughout time.

Introduction

Prescribed burning (PB) is the planned use of fire under mild weather conditions to meet defined management objectives [START_REF] Wade | A guide for prescribed fire in southern forests[END_REF]). Prescribed burning is executed mostly for fire risk reduction, but also for forest management, restoring habitats or improving grazing. Generally, prescribed burns are low intensity fires, but certain management objectives require a higher burning intensity to effectively achieve specific goals, such as significantly removing understory or slash. In this case, PB can partially damage trees and affect their vitality in the shortterm. Some studies have analysed the effects of PB on postburning growth [START_REF] Battipaglia | The effects of prescribed burning on Pinus halepensis Mill. as revealed by dendrochronological and isotopic analyses[END_REF][START_REF] Valor | Assessing the impact of prescribed burning on the growth of European pines[END_REF] and tree vitality (see Woolley et al. 2012 for review). Less attention has been dedicated to understanding the effect of PB on secondary metabolites produced by pines [START_REF] Lavoir | Does prescribed burning affect leaf secondary metabolites in pine stands?[END_REF], despite the protection they confer against biotic and abiotic stressors, and their potential to increase plant flammability (Ormeño et al. 2009, Loreto and[START_REF] Loreto | Abiotic stresses and induced BVOCs[END_REF].

The quantity and composition of terpenes produced against a stressor can be constrained by the plant's physiological status [START_REF] Sampedro | Costs of constitutive and herbivore-induced chemical defences in pine trees emerge only under low nutrient availability[END_REF]) and genetics [START_REF] Pausas | Secondary compounds enhance flammability in a Mediterranean plant[END_REF], but also by the nature and severity of the stress, and the species affected. The main secondary metabolites biosynthesized in conifers are terpenes and phenols [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology and ethnobotany[END_REF]. In Pinus species, oleoresin is a mixture of terpenes including monoterpenes (volatile metabolites), sesquiterpenes (metabolites with intermediate volatility) and diterpenes (semi-volatile compounds), which are stored in resin ducts of woody and needle tissues [START_REF] Phillips | Resin-based defenses in conifers[END_REF]. Upon stress, plants follow a constitutive or induced strategy to defend themselves from a stressor. Although most Pinus spp. favour the production of constitutive terpenes under stress conditions, they can also synthesize new induced defences [START_REF] Phillips | Resin-based defenses in conifers[END_REF]. The induction timing may be different depending on the chemical groups of terpenes, type of stress, and the species or tissue attacked [START_REF] Lewinsohn | Defense mechanisms of conifers differences in constitutive and wound-induced monoterpene biosynthesis among species[END_REF][START_REF] Achotegui-Castells | Strong induction of minor terpenes in Italian Cypress, Cupressus sempervirens, in response to infection by the fungus Seiridium cardinale[END_REF].

Direct effects of fire such as rising temperatures or heatinduced needle damage can alter terpene production. Increases in air and leaf temperature trigger the emission of volatile terpenes [START_REF] Alessio | Direct and indirect impacts of fire on isoprenoid emissions from Mediterranean vegetation[END_REF]) but their synthesis can also be stimulated if the optimal temperature of enzymes is not exceeded [START_REF] Loreto | Abiotic stresses and induced BVOCs[END_REF]. Benefits of such stimulation include thermoprotection against heat, since terpene volatiles neutralize the oxidation pressure encountered by chloroplasts under thermal stress [START_REF] Vickers | Isoprene synthesis protects transgenic tobacco plants from oxidative stress[END_REF]. As the emission of volatile terpenes in several Mediterranean pines cease 24 h after fire [START_REF] Alessio | Direct and indirect impacts of fire on isoprenoid emissions from Mediterranean vegetation[END_REF] or wounding [START_REF] Pasqua | The role of isoprenoid accumulation and oxidation in sealing wounded needles of Mediterranean pines[END_REF], we hypothesized that the accumulation of monoterpenes will be higher 24 h post-burning, than before PB.

Indirect effects of fire can affect terpene concentrations by means of increasing resource availability [START_REF] Certini | Effects of fire on properties of forest soils: a review[END_REF]. In turn, terpene variations induced by fire could change needle flammability [START_REF] Ormeño | The relationship between terpenes and flammability of leaf litter[END_REF]) and susceptibility to insects [START_REF] Hood | Low-severity fire increases tree defense against bark beetle attacks[END_REF]. The 'growth differentiation balance hypothesis' (GDBH) (Herms andMattson 1992, Stamp 2003) predicts that under poor water and nutrient availabilities, growth is more limited than photosynthesis. Since carbon assimilation is maintained, the excess of carbohydrates favours the synthesis of carbon-based secondary metabolites. On the contrary, when resource availability is high, the growth of plants is not expected to be limited and plants allocate a greater proportion of assimilates to growth rather than to defence traits (Herms andMattson 1992, Stamp 2003). Accordingly, a short-term response following PB should be an increasing demand on the plant for chemical defence if trees are damaged, but with time, if trees heal, increased fertilization and reduced water competition induced by PB [START_REF] Feeney | Influence of thinning and burning restoration treatments on presettlement ponderosa pines at the Gus Pearson Natural Area[END_REF]) could favour carbon allocation to growth rather than chemical defences. Time-course terpene responses of the direct and indirect effects of PB could differ between tree species depending on their fire resistance strategies. In this study, we used pines with contrasting tolerance to surface fires: Pinus halepensis, a fire sensitive species, Pinus sylvestris, moderately fire-resistant and the fire-resister Pinus nigra, which is supposed to be less vulnerable to fire tissue damage due to its pyro-resistant traits (e.g. thicker bark, higher crown base height) [START_REF] Fernandes | Fire resistance of European pines[END_REF]. In agreement with these strategies, we previously found that radial growth was reduced the year of PB in the most firesensitive species and unaffected in P. nigra, while 1 year postburning, growth was augmented in P. nigra and P. halepensis, and reduced in P. sylvestris [START_REF] Valor | Assessing the impact of prescribed burning on the growth of European pines[END_REF]. In consequence, we hypothesized that 1 year post-burning, the concentration of terpenes would be, as a whole, lower than before PB, if fire induces a decrease in nutrient and water competition; this reduction would be lower on damaged trees and in pines defined as having lower fire resistance (e.g., P. halepensis and P. sylvestris).

The objectives of this study were to evaluate the effects of relatively high-intensity PB (enough to remove understory and ladder fuels) on mono-, sesqui-and diterpene storage in Pinus spp., comparing 24 h and 1 year post-burning concentrations with pre-burning concentrations. We modelled the relative change of terpene concentrations at two sampling times: (i) 24 h post-burning, as a function of fire severity and pre-burning physiological condition and (ii) 1 year post-burning, as a function of fire severity and PB-induced changes in pine physiological conditions. Additionally, we aimed to identify the most representative terpenes of each sampling time since burning.

Materials and methods

The study was established in three sites situated in the NE Iberian Peninsula (Catalonia, Spain): two plots in mixed-stands of P. nigra ssp. salzmanni (Dunal) Franco and P. sylvestris L. at Miravé and Lloreda localities, situated in the foothills of the Pyrenees; and two other plots in a pure-stand of P. halepensis Mill. at El Perelló locality, in the Southern part of Catalonia. The P. halepensis stand is located in areas of dry Mediterranean climate while the mixed-stands of P. nigra and P. sylvestris are situated in temperate cold sub-Mediterranean climate with milder summers and colder winters (Table 1). In the sub-Mediterranean sites, soils are developed from calcareous colluviums (0.5-1 m deep) and thus classified as Calcaric cambisols (FAO 2006); in the Mediterranean site, they are developed from limestones (0.4-0.5 m deep) and classified as Leptic Regosol (FAO 2006). The understory is dominated by Buxus sempervirens L. and Viburnum lantana L., in the P. nigra and P. sylvestris mixedstands, and by Pistacia lentiscus L. and Quercus coccifera L. in the P. halepensis stand.

Experimental design: tree selection and prescribed burning

A total of four plots (30 × 30 m) were set up: one in each of the mixed-stand of P. nigra and P. sylvestris, and two in the pure P. halepensis stand. Each plot was burnt in spring 2013 (Table 2). Prescribed burns were conducted by the Forest Actions Support Group (GRAF) of the Autonomous Government (Generalitat de Catalunya) using a strip headfire ignition pattern. Prescribed burning aimed to decrease fuel hazard by reducing surface and ladder fuel loads. Between 90% and 100% of the surface fuel load was consumed in all plots. Needle terpene concentration, fire features and tree physiological condition were studied in 9/10 dominant or co-dominant pines per species in each plot. Each tree was sampled on three occasions for analysing terpene concentration: 24 h before PB (pre-burning), 24 h and 1 year after PB (24 h post-burning and 1 year post-burning, respectively). δ 13 C and N concentrations of 1-year-old needles were also analysed as a proxy of physiological condition in pre-burning and 1 year post-burning samples.

Before PB, selected trees were identified with a metal tag. Their diameter at breast height (DBH), total height and height to live crown base were measured. During fires, the fire residence time (minutes) above 60 °C and the maximum temperature at the base of the trunk were measured for the selected trees with K-thermocouples (4 mm) connected to dataloggers (Testo 175), packed with a fireproof blanket and buried into the soil. Temperatures were recorded every 10 s. The maximum temperatures registered at the soil surface occurred in the P. nigra and P. sylvestris plots, while the highest residence time above 60 °C was recorded in the P. halepensis plots (Table 2). One week after PB, the crown volume scorched was visually estimated to the nearest 5% as an indicator of fire severity. Foliage scorch was defined as a change in needle colour resulting from direct foliage ignition or indirect heating [START_REF] Catry | Post-fire tree mortality in mixed forests of central Portugal[END_REF].

Needle sampling

In each plot, we cut an unscorched branch from the top of the south-facing crown in the 9/10 trees selected per species for each sampling time studied: pre-burning, 24 h and 1 year postburning. Five twigs with unscorched healthy needles were cut immediately, covered with aluminium paper and stored in a portable refrigerator at 4 °C until being stored at -20 °C in the laboratory for terpene analysis. The time period between the field and the laboratory did not exceed 2 h. Additionally, about five twigs were transported to the laboratory, dried at 60 °C and stored in tins before δ 13 C and N concentration analysis.

Needle terpene concentration

In the studied pine species, needles reached up to 3 years old. Before terpene extraction, we collected the 1-year-old needles from each twig to control for the effect of age for each sampling time. Needles were cut in small parts (~5 mm) and placed in well-filled, tightly closed amber glass vials to avoid exposure to light and oxygen (Guenther 1949[START_REF] Farhat | Seasonal changes in the composition of the essential oil extract of East Mediterranean sage (Salvia libanotica) and its toxicity in mice[END_REF]. The extraction method consisted in dissolving 1 g of cut 1-year-old unscorched green needles in 5 ml of organic solvent (cyclohexane + dichloromethane, 1:9), containing a constant amount of undecane, a volatile internal standard to quantify terpene concentrations which was not naturally stored in the needles. Extraction occurred for 20 min, under constant shaking at room temperature, similar to extractions shown in [START_REF] Ormeño | Plant coexistence alters terpene emission and concentration of Mediterranean species[END_REF]. The extract was stored at -20 °C and then analysed within the following 3 weeks. Analyses were performed on a gas chromatograph (GS-Agilent 7890B, Agilent Technologies, Les Ulis, France) coupled to a mass selective detector (MSD 5977A, Agilent Technologies, Les Ulis, France). Compound separation was achieved on an HP-5MS (Agilent Technologies, Les Ulis, France) capillary column with helium as the carrier gas. After sample injection (1μl), the start temperature (40 °C for 5 min) was ramped up to 245 °C at a rate of 3 °C min -1 , and then until 300 °C a rate of 7 °C min -1 . Terpene identifications were based on the comparison of terpene retention times and mass spectra with those obtained from authentic reference samples (Sigma-Aldrich ® , Sigma-Aldrich, Saint-Quentin-Fallavier, France) when available, or from databases (NIST2008, Adams 2007) when samples were unavailable. Also, we calculated the Kovats retention index and compared it with bibliographical data. Terpenes were quantified based on the internal standard undecane (36.6 ng μl -1 of injected solution). Thus, based on calibrations of terpene standards of high purity (97-99%), also prepared using undecane as internal standard, chromatographic peak areas of an extracted terpene where converted into terpene masses based on the relative response factor of each calibrated terpene. Results were expressed on a needle dry mass (DM) basis. The identified terpenes were grouped in mono-, sesqui-and diterpenes. At each post-burning time, we calculated the relative change of terpene concentration as the difference between the pre-and post-burning concentration of each terpene group expressed as percentage.

Tree physiological condition: δ 13 C and N analysis δ 13 C and N analysis were carried out on 1-year-old unscorched needles in pre-burning and 1 year post-burning samples. For δ 13 C and N, needles were oven-dried at 60 °C for 48 h, ground and analysed at the Stable Isotope Facility of the University of California at Davis (USA) using an ANCA interfaced to a 20-20 Europa ® isotope ratio mass spectrometer (Sercon Ltd, Cheshire, UK).

Climatic data before and during sampling years

Monthly precipitation (P) and temperature (T) from March 2012 to August 2014 were downloaded from the three nearest meteorological stations to the sub-Mediterranean and the Mediterranean plots. Monthly potential evapotranspiration (PET) was estimated using the Thornthwaite (1948) method. For each sampling year (t), 2013 and 2014, accumulated values of P and PET of different periods were calculated for each meteorological station. Seven periods of accumulated climate data were compiled: annual, from June before the sampling year (t -1) to May of the sampling year (t); spring, summer, fall and winter before the sampling year (t -1); spring and summer of the sampling year (t). For each period, we calculated the difference between P and PET (P -PET) for each meteorological station and sampling year.

Linear mixed models (LMM), considering plot as a random factor, were used to:

(i) Analyse potential differences in pre-burning tree physiological condition and fire parameters among pine species. (ii) Test for differences in total terpene and terpene group concentrations (expressed in a needle mass basis and as the percentage of the terpene group from the total) between times since burning for each pine species. (iii) Model 24 h and 1 year impact of PB on the relative concentration change of mono-, sesqui-and diterpenes with respect to pre-burning concentration. The 24 h and 1 year post-burning models considered pine species as a fixed factor, needle δ 13 C and N concentration pre-burning, and the proportion of crown scorched and fire residence time above 60 °C as covariables. In addition, in the 1 year postburning model, δ 13 C and N concentration changes were also included (1 year post-burning minus pre-burning levels of δ 13 C and N concentration). Second interactions of pine species with each co-variable were included.

Terpene concentrations were log-transformed to accomplish normal distribution requirement. When the relative concentration change of terpenes was modelled, 100 was summed as a constant before taking the logarithm. Therefore, log-transformed data higher than 2 indicate higher terpene concentrations than pre-burning, while values lower than 2 mean lower terpene concentrations. Residuals presented no pattern and highly correlated explanatory variables were avoided. The variance explained for the fixed effects was obtained by comparing the final model with the null model (containing only the random structure). A Tukey post-hoc test was used for multiple comparisons when needed.

For each pine species, terpene profiles were evaluated using a principal component analysis to show potential qualitative and quantitative variation in needle terpene within and between plots and time since burning. Terpene concentrations were centred and the variance-covariance matrix used to understand how terpene profiles varied. Moreover, for each pine species, we used a multilevel sparse partial least squares discriminant analysis (sPLS-DA) to select the terpenes that best separated each time since burning in terms of their concentration. The sPLS-DA is a supervised technique that takes the class of the sample into account, in this case time since burning, and tries to reduce the dimension while maximizing the separation between classes. To conduct the analysis, we selected those compounds that were present in at least 75% of the sampled trees, resulting in a total of 48, 37 and 35 compounds in P. halepensis, P. nigra and P. sylvestris, respectively. We used the multilevel approach to account for the repeated measures on each tree to highlight the PB effects within trees separately from the biological variation between trees. The classification error rate was estimated with leave-one-out cross validation with respect to the number of selected terpenes on each dimension. Lastly, differences in P -PET between sampling years were tested by a Student's t-test for the Mediterranean and sub-Mediterranean plots. All analyses were conducted with the software R (v. 3.2.1, The R Foundation for Statistical Computing, Vienna, Austria) using the package nlme for linear mixed-effects modelling and the package mixOmics for the sPLS-DA analysis. The model variances explained by fixed effects (marginal R 2 ) and by both fixed and random effects (conditional R 2 ) are provided [START_REF] Nakagawa | A general and simple method for obtaining R2 from generalized linear mixed-effects models[END_REF].

Results

Tree, fire and climate characteristics

The proportion of crown scorched was significantly higher in P. halepensis than in the other species despite the fact that the three pine species presented similar height to live crown base (Table 3). By contrast, no differences in fire residence time above 60 °C were encountered among species (Table 3). Needle δ 13 C decreased significantly 1 year post-burning in the three species while N concentration was similar (Table 3).

This decrease in δ 13 C contrasted with the drier conditions found 1 year post-burning (P -PET = 200 mm and = 135 mm in Mediterranean and sub-Mediterranean plots, respectively) in comparison with pre-burning (P -PET = 481 mm and = 290 mm in Mediterranean and sub-Mediterranean plots, respectively; see Figure S1 available as Supplementary Data at Tree Physiology Online).

A total of 56, 59 and 49 terpenes were identified and quantified in P. halepensis, P. nigra and P. sylvestris, respectively (see Table S1 available as Supplementary Data at Tree Physiology Online). Preburning, P. nigra showed the highest terpene concentration (65.6 ± 7.1 mg g DM -1

) followed by P. halepensis and P. sylvestris (41.2 ± 5.8 mg g DM -1 and 21.4 ± 2.6 mg g DM -1 , respectively the diterpene thunbergol in P. halepensis, the sesquiterpene β-caryophyllene in P. nigra and the monoterpene α-pinene in P. sylvestris were the major compounds found, representing an average of 22%, 22% and 40% of the total terpene concentration, respectively (see Figure S2 available as Supplementary Data at Tree Physiology Online). Terpene concentration and composition strongly varied within plots in all species, with no clear differences in terpene composition among plots (see Figures S3a,S4a and S5a available as Supplementary Data at Tree Physiology Online). The variation in terpene concentrations was high within pre-and 24 h post-burning samples, while variation for 1 year post-burning concentrations was much lower (see Figures S3b,S4b and S5b available as Supplementary Data at Tree Physiology Online). In all species, the quantity of dominant compounds in pre-and 24 h post-burning samples were clearly different from those of 1 year post-burning samples (see Figures S3b,S4b and S5b available as Supplementary Data at Tree Physiology Online). For instance, the quantity of α-pinene was higher in pre-and 24 h post-burning times in all pine species in opposition with 1 year post-burning samples. Limonene was characteristic 24 h post-burning in the needles of P. halepensis and P. nigra, while the quantity of camphene and myrcene was higher in pre-and 24 h post-burning needles samples of P. sylvestris. Differences in total terpene concentration between pre-and 24 h post-burning were only detected in P. nigra, which decreased ∼39% (Figure 1a). When analysing terpene groups, the 24 h post-burning needle concentration of both mono-and sesquiterpenes were, in comparison with pre-burning, slightly higher in P. halepensis, lower in P. nigra and similar in P. sylvestris (Figure 1b andc). No differences were detected in the diterpene concentration between pre-and 24 h post-burning times (Figure 1d).

One year after burning, total terpene concentration was lower compared with the levels observed pre-and 24 h post-burning in the three species (Figure 1a). In P. halepensis this reduction was similar for each terpene group while, in the two sub-Mediterranean species, it was mostly due to a decrease in the proportion of monoterpenes (see Table S2 available as Supplementary Data at Tree Physiology Online). In contrast, an increase in the relative contribution of the sesquiterpene group to the total terpenes was found 1 year post-burning in both sub-Mediterranean species.

The relative changes of mono-and diterpene concentrations 24 h post-burning were directly related to the proportion of crown scorched (Table 4). However, crown scorch volume interacted with pine species to explain the relative changes in mono-and diterpene concentrations (Table 4). Thus, in both P. halepensis and P. sylvestris, the 24 h post-burning concentration of monoterpenes was higher than pre-burning and increased with crown scorched (Figure 2a.1 and a.3); only individual pines with low proportion of the crown scorched (<15--20%) showed similar or lower concentration than pre-burning. In for each pine species (P. halepensis, n = 20; P. sylvestris, n = 19; P. nigra, n = 19 and n = 18 in 1 year-post-burning). Differences in the concentration between TSB within each pine species were tested using LMM considering plot as a random factor. Within each pine species, different letters indicate differences between TSB using a Tukey post-hoc, where regular letters indicate significant differences at P < 0.05; italic letters represent a marginal significant difference (0.05 < P < 0.1). contrast, the relative change of monoterpene concentration in P. nigra was generally lower than pre-burning, at least in the range of crown scorch measured (0-50%) (Figure 2a.2). The relationship between the relative concentration change in diterpenes and crown scorch followed a similar trend as in monoterpenes for P. halepensis and P. sylvestris (Figure 2b.1 and b.2), while in P. nigra, the ratio of change in crown scorch was higher and shifted from lower to higher concentrations than pre-burning in the middle of the measured crown scorch range (Figure 2b.3).

The relative concentration change of monoterpene was also directly related to the needle N concentration and the height to live crown base (Table 4). In the case of sesquiterpenes, needle N concentration interacted with pine species (Table 4, Figure 2). Thus, the relative concentration change of sesquiterpenes 24 h post-burning was higher in P. halepensis and P. sylvestris, and augmented as needle N concentration increased (Figure 2c.1 and c.3), whereas it was always lower in P. nigra, decreasing inversely with increasing needle N concentration (Figure 2c.2). Finally, fire residence time above 60 °C directly affected the relative change of sesquiterpene concentration in all species (Table 4).

One year after PB, the relative change of mono-and sesquiterpene concentrations were always lower than pre-burning and inversely related with δ 13 C of pre-burning needles (Table 5, Figure 3a.1). The 1 year post-burning relative change concentration of diterpenes were also lower than pre-burning, but variations were associated with changes in δ 13 C or N concentration of needles (Figures 3a.2 and a.3).

Similar to 24 h post-burning, the proportion of crown scorched had a direct effect on the relative concentration change of all terpene groups, although marginally significant in mono-and sesquiterpene models (Table 5). This variable interacted with pine species in the case of diterpenes (Figure 3b) and showed that as crown scorch increased, the relative concentration change in P. nigra was more acute than in the other species (Table 5, Figure 3b.2).

Discriminant terpenes across time since burning for each pine species

The multilevel sPLS-DA in P. halepensis led to optimal selection of six and one terpenes on the first two dimensions with a classification error rate of 0.26 and 0.06, respectively, reflecting a clear separation between times since burning (Figure 4). Among compounds, terpinen-4-ol separated pre-burning (Cluster 2) from both post-burning times; whereas E-β-ocimene and α-thujene discriminated the 24 h post-burning sampling time from the others (Cluster 1). Four sesquiterpenes characterized the 1 year postburning needle samples (Cluster 3).

In P. nigra, we chose three dimensions and the corresponding terpenes selected for each were four, one and one (Figure 5). The classification error rates were 0.35, 0.33 and 0.18, respectively, for the first three dimensions. Two clusters were differentiated: pre-burning was discriminated, mainly, by three sesquiterpenes (Cluster 1) and bornyl acetate and β-springene represented, postburning samplings (Cluster 2) (Figure 5). Table 4. Summary of the models characterizing the impact of prescribed burning and tree vitality on the 24 h post-burning relative concentration change of mono-, sesqui-and diterpenes, calculated as the standardized difference between 24 h post-burning and pre-burning concentration expressed as percentage (logarithmically transformed). Only the significant interaction terms are shown. Bold characters indicate significant effects (P < 0.05). Finally, two dimensions were selected for P. sylvestris (Figure 6) with 11 terpenes on each component. The classification error rates were 0.66 and 0.33. As in P. nigra, two clusters were distinguished: sesquiterpenes characterized the pre-burning sampling time, whereas both post-burning times were characterized mainly by mono-and sesquiterpenes (Figure 6).

h post-burning relative concentration change

Monoterpenes

Discussion

Pinus nigra is a species considered to be resistant to mediumlow fire intensities, P. sylvestris a moderately fire-resistant species and P. halepensis a fire-sensitive species [START_REF] Agee | Fire and pine ecosystems[END_REF][START_REF] Fernandes | Fire resistance of European pines[END_REF]. While the concentration of the semi-Figure 2. Measured and predicted (line) relative concentration change (log-transformed) using 24 h post-burning models (see Table 4) of monoterpenes and diterpenes against crown scorched (a and b) and for sesquiterpenes against needle N (c). Before the log-transformation, 100 was summed. The dashed line indicates no changes between pre-and post-burning terpene concentrations: higher values indicate a higher terpene concentration than those of pre-burning, while the opposite is indicated by lower values. volatile diterpenes was not affected 24 h post-burning, the concentration of mono-and sesquiterpenes seems to decrease in P. nigra, was sustained in P. sylvestris and tended to increase in P. halepensis. Although massive needle terpene emissions have been reported at ambient temperatures often reached during PB [START_REF] Alessio | Direct and indirect impacts of fire on isoprenoid emissions from Mediterranean vegetation[END_REF][START_REF] Loreto | Abiotic stresses and induced BVOCs[END_REF][START_REF] Zhao | Terpenoid emissions from heated needles of Pinus sylvestris and their potential influences on forest fires[END_REF], various explanations may justify the different terpene contents observed 24 h post-burning between species. For instance, terpenes stored in needle resin ducts are likely to encounter different resistance to volatilization due to differences in the specific characteristics of the epistomatal chambers which are, respectively, unsealed, sealed and buried in needles of P. nigra, P. sylvestris and P. halepensis [START_REF] Hanover | Surface wax deposits on foliage of Picea pungens and other conifers[END_REF][START_REF] Boddi | Structure and ultrastructure of Pinus halepensis primary needles[END_REF][START_REF] Kim | Micromorphology of epicuticular waxes and epistomatal chambers of pine species by electron microscopy and white light scanning interferometry[END_REF]). These differences in needle morphology may contribute to explaining the reduction of terpenes observed 24 h post-burning in P. nigra. Another reason for variable terpene contents may be different respiration sensitivity between species. As the consumption of assimilates increases relative to the photosynthetic production at high temperatures [START_REF] Farrar | The effects of increased atmospheric carbon dioxide and temperature on carbon partitioning, source-sink relations and respiration[END_REF], this could bring about a decrease in the weight of carbohydrates and, thus, an apparent increase in needle terpene concentrations. If the respiration sensitivity to increasing temperature is higher in P. halepensis than in the other two species, this may explain the slight increase in terpene concentration in this species 24 h post-burning. Alternatively, the increase in monoterpene concentration in unscorched needles of P. halepensis 24 h post-burning may partly reflect systemic induced resistance, triggered by burning needles from lower parts of the canopy, although no data was found in literature to support this hypothesis. Finally, although we carefully selected only 1-year-old unscorched needles and from the same part of the crown, we cannot fully exclude that terpene variation between preand post-burning are reflecting differences in light availability between the sampled needles.

Terpene dynamics within the species were modulated by fire severity. Thus, relative concentration changes of mono-and diterpenes increased with the proportion of crown scorched 24 h post-burning. This trend was evident 1 year post-burning, suggesting that the damaged pines were still investing in chemical defences. According to the GDBH (Herms andMattson 1992, Stamp 2003) and the reduction in radial growth detected in P. halepensis and P. sylvestris [START_REF] Valor | Assessing the impact of prescribed burning on the growth of European pines[END_REF], we hypothesized that the increase in monoterpenes by P. halepensis and, to a lesser extent, in P. sylvestris, may constrain primary metabolism. Although the rate of increase in diterpenes post-burning was greater in P. nigra than in the other two species, P. nigra required a greater proportion of scorched crown in order to achieve higher concentrations than those observed pre-burning. Therefore, trees with a greater proportion of scorched crown could be investing in secondary metabolism rather than primary metabolism, although this potential trade-off on carbon investment deserves further research.

Table 5. Summary of the models characterizing the impact of prescribed burning and tree vitality on the 1 year post-burning relative concentration change of mono-, sesqui-and diterpenes, calculated as the standardized difference between 1 year post-burning and pre-burning content expressed as percentage (logarithmic transformed). Only the significant interaction terms are shown. Bold characters indicate significant effects (P < 0.05). 2 HLCB, height to live crown base (m).

3 Change δ 13 C, change in δ 13 C (difference between 1 year post-burning and pre-burning δ 13 C). 4 Change N, change in foliar N content (difference between 1 year post-burning and pre-burning N content).

Needle N concentration was positively associated with the relative concentration change of monoterpenes in the three species and of sesquiterpenes in the case of P. halepensis and P. sylvestris. As resin canal ducts are limited by N [START_REF] Björkman | Different responses of two carbon-based defences in Scots pine needles to nitrogen fertilization[END_REF]), these positive relationships may be explained by an increase in the number and size of the ducts in needles with higher N content. In contrast, we did not detect any effect of pre-burning water status, as estimated by δ 13 C, for 24 h post-burning terpene concentration change in individual pines.

According to our study, tree-to-tree variation in terpene concentration is known to be naturally high, even over short spatial distances, or when plants grow in the same soil in the same geographic area [START_REF] Ormeño | Production and diversity of volatile terpenes from plants on Calcareous and Siliceous soils: effect of soil nutrients[END_REF][START_REF] Kännaste | Highly variable chemical signatures over short spatial distances among Scots pine (Pinus sylvestris) populations[END_REF]). Our study reveals, however, that this variation is reduced 1 year postburning within and between plots. One year post-burning, the terpene concentration was lower than pre-burning, while an increase could be expected given the drier meteorological conditions during the year after burning [START_REF] Loreto | Abiotic stresses and induced BVOCs[END_REF]. In contrast, lower needle δ 13 C values, compared with pre-burning, suggest a decrease in water competition 1 year post-burning, an increase in the photosynthetic rate or stomatal conductance [START_REF] Battipaglia | The effects of prescribed burning on Pinus halepensis Mill. as revealed by dendrochronological and isotopic analyses[END_REF], or an improvement in water conditions in the remaining needles of highly scorched trees [START_REF] Wallin | Effects of crown scorch on ponderosa pine resistance to bark beetles in Northern Arizona[END_REF]. A lower terpene concentration 1 year after burning differs from other studies [START_REF] Cannac | Phenolic compounds of Pinus laricio needles: a bioindicator of the effects of prescribed burning in function of season[END_REF][START_REF] Lavoir | Does prescribed burning affect leaf secondary metabolites in pine stands?[END_REF]) comparing burned versus unburned plots. These studies concluded that needle terpene concentration returns to normal values 1 year after fire. They suggested that short-term increases in nutrient availability had minor effects on terpene concentration. The discrepancies with our investigation may be explained by the higher burning intensity in our study, which impacted water availability as indicated by δ 13 C values. In agreement with the GDBH (Herms andMattson 1992, Stamp 2003), our results showed that the relative concentration change of diterpenes was lower in trees that had an improvement in their physiological condition 1 year post-burning, as suggested by needle δ 13 C change and changes in needle N concentration. Despite the fact that no relationships Figure 3. Measured and predicted (line) relative concentration change (log-transformed) using 1 year post-burning models (see Table 5) for monoterpenes against δ 13 C (a.1), for diterpenes against change in δ 13 C (a.2), change in needles N (a.3), and the interaction between species and crown scorch (b). Before the log-transformation, 100 was summed. The dashed line indicates no changes between pre-and post-burning terpene concentrations: higher values indicate a higher terpene concentration than those of pre-burning while the opposite is indicated by lower values.

were found between mono-or sesquiterpenes regarding the change in δ 13 C or N, the direct relationship between the relative terpene concentration change and the pre-burning δ 13 C suggested that the decrease in both terpene groups occurred in pines that were more stressed pre-burning.

The ecological functions of many mono-, sesqui-and diterpene compounds are still not well understood, although in recent years significant achievements have been made via genetic engineering (Cheng et al. 2007, Loreto and[START_REF] Loreto | Abiotic stresses and induced BVOCs[END_REF]. Likewise, research on terpenes and flammability is generally scarce, though there are some studies that have shown a correlation between both variables [START_REF] Owens | Seasonal patterns of plant flammability and monoterpenoid concentration in Juniperus ashei[END_REF][START_REF] Alessio | Direct and indirect impacts of fire on isoprenoid emissions from Mediterranean vegetation[END_REF][START_REF] Ormeño | The relationship between terpenes and flammability of leaf litter[END_REF]. The reduction in terpene concentration 24 h postburning in the fire-resister P. nigra could imply a reduction of needle flammability with respect to pre-burning, strengthened by a reduction in the highly flammable α-caryophyllene (also known as α-humulene) and the increase in bornyl acetate, which is inversely related to flammability [START_REF] Owens | Seasonal patterns of plant flammability and monoterpenoid concentration in Juniperus ashei[END_REF]. By contrast, increases of mono-and sesquiterpene concentrations in P. halepensis may involve greater flammability, which would favour fire reaching the canopy to effectively open the serotinous cones. Specifically, the sPLS-DA showed E-β-ocimene, which is correlated with flammability [START_REF] Page | Mountain pine beetle attack alters the chemistry and flammability of lodgepole pine foliage[END_REF], as representative of 24 h postburning samples. In P. sylvestris, the poor terpene discrimination in relation to time since burning limits the interpretation of any compound in terms of flammability.

Fire-damaged trees are more vulnerable to insects, especially bark beetles, and infections by root fungus, which contribute to trees susceptibility to beetle attack [START_REF] Sullivan | Association between severity of prescribed burns and subsequent activity of conifer-infesting beetles in stands of longleaf pine[END_REF][START_REF] Parker | Interactions among fire, insects, and pathogens in coniferous forests of the interior western United States and Canada[END_REF]. The accumulation of high amounts of monoterpenes 24 h post-burning in the lower fire resistant species (P. halepensis and P. sylvestris) when fire partially scorches the crowns, might accomplish several functions, such as effective transport of diterpenes to the affected tissues [START_REF] Phillips | Resin-based defenses in conifers[END_REF], better protection of the photosynthetic apparatus [START_REF] Vickers | Isoprene synthesis protects transgenic tobacco plants from oxidative stress[END_REF] or ensuring the needs for chemical defence against pathogens [START_REF] Phillips | Resin-based defenses in conifers[END_REF]. According with this last function, E-β-ocimene and α-thujene with antifungal activity [START_REF] Bajpai | Chemical composition and antifungal properties of the essential oil and crude extracts of Metasequoia glyptostroboides Miki ex Hu[END_REF][START_REF] Deba | Chemical composition and antioxidant, antibacterial and antifungal activities of the essential oils from Bidens pilosa Linn. var. radiata[END_REF] appear to correctly classify 24 h post-burning needle samples of P. halepensis. Although the discriminant analysis in P. sylvestris showed poor classification power, the presence of E-β-ocimene and ϒ-terpinene also suggests that trees possess a higher resistance to fungus compared with pre-burning [START_REF] Espinosa-García | Dosedependent effects in vitro of essential oils on the growth of two endophytic fungi in coastal redwood leaves[END_REF]). In the case of the fire-resistant P. nigra, the pre-burning concentration of monoterpenes may be sufficient to cope with biotic stresses related with medium intensity fires. Nonetheless, bornyl acetate seems to represent 24 h post-burning samples conferring resistance to defoliators immediately after fire [START_REF] Zou | Foliage constituents of Douglas fir (Pseudotsuga menziesii (Mirb.) Franco): their seasonal variation and potential role in Douglas fir resistance and silviculture management[END_REF]. The high accumulation of diterpenes 24 h post-burning in P. nigra as the proportion of the scorched crown increases in respect to the other species, and possibly indicates a better chemical protection against xylophagus insects [START_REF] Lafever | Diterpenoid resin acid biosynthesis in conifers: enzymatic cyclization of geranylgeranyl pyrophosphate to abietadiene, the precursor of abietic acid[END_REF]). In P. nigra and P. sylvestris, the fact that the percentage of sesquiterpenes was augmented significantly 1 year postburning with respect to pre-burning, together with the increase in the relative concentration change as crown scorch augmented, might indicate the importance of sesquiterpenes as indirect defences to a wide range of biotic stressors [START_REF] Phillips | Resin-based defenses in conifers[END_REF]Croteau 1999, Schnee et al. 2006) and, as reported in [START_REF] Lavoir | Does prescribed burning affect leaf secondary metabolites in pine stands?[END_REF], were representative in repeatedly burned plots. Similarly, our classification found the sesquiterpenes guaiol, α-muurolene and δ-elemene as being characteristic in 1 year post-burning P. halepensis needle samples. These compounds might have defensive roles in defoliated trees against insects [START_REF] Wallis | Systemic induction of phloem secondary metabolism and its relationship to resistance to a canker pathogen in Austrian pine[END_REF][START_REF] Liu | Guaiol-a naturally occurring insecticidal sesquiterpene[END_REF].

After fire, bark beetles pose a significant threat to trees, especially when a significant amount of the crown has been scorched [START_REF] Lombardero | Effects of fire and mechanical wounding on Pinus resinosa resin defenses, beetle attacks, and pathogens[END_REF]. Several volatile terpenes such as α-pinene, camphene and myrcene can be released during PB and facilitate the attack of bark beetles [START_REF] Coyne | Toxicity of substances in pine oleoresin to southern pine beetles[END_REF]. Twenty-four hour post-burning P. sylvestris tended to present higher amounts of these terpene compounds, suggesting higher susceptibility to bark beetle attack with respect to the other species. Finally, limonene, which is highly toxic for several types of beetle [START_REF] Raffa | Interactions among conifer terpenoids and bark beetles across multiple levels of scale: an attempt to understand links between population patterns and physiological processes[END_REF], was present in higher amounts in P. nigra and P. halepensis, suggesting a higher resistance to bark beetle attack for both species 24 h post-burning.

The concentration of mono-and sesquiterpenes 24 h post-burning was similar to the pre-burning ones in the more fire-sensitive species (P. halepensis and P. sylvestris) and lower in the fire-resistant P. nigra species. Terpene dynamics were modulated within the species by fire severity, as indicated by the direct relation between the proportion of scorched crown and the concentration of terpenes 24 h post-burning. As discussed, a combination of morphological and physiological mechanisms may be operating during and in the short-term after PB, but no clear conclusions may be stated. However, differences in terpene contents as a function of the pine species sensitivity to fire suggest that terpenic metabolites could have adaptive importance in fire-prone ecosystems, in terms of flammability and defence against biotic agents short-term after fire. In agreement with the GDBH (Herms andMattson 1992, Stamp 2003) trees may be allocating assimilates to growth rather than to defence, as suggested by the remarkable decrease in terpene concentration and the negative relation between terpene concentration and the change in needle δ 13 C. This decrease in terpene concentration, in turn, could imply a higher susceptibility to fire-related pathogens and insects.

Figure 1 .

 1 Figure 1. Concentration (mean ± SE) of total terpene (a), monoterpenes (b), sesquiterpenes (c) and diterpenes (d) across time since burning (TSB)for each pine species (P. halepensis, n = 20; P. sylvestris, n = 19; P. nigra, n = 19 and n = 18 in 1 year-post-burning). Differences in the concentration between TSB within each pine species were tested using LMM considering plot as a random factor. Within each pine species, different letters indicate differences between TSB using a Tukey post-hoc, where regular letters indicate significant differences at P < 0.05; italic letters represent a marginal significant difference (0.05 < P < 0.1).

Figure 4 .

 4 Figure 4. Hierarchical clustering for P. halepensis of the seven terpenes selected with multilevel sPLS-DA using terpene content. Samples are represented in columns and terpenes in rows. MHT, monoterpene hydrocarbon; SHT, sesquiterpene hydrocarbon; O, oxygenated compounds; der, derivative compounds.

Figure 5 .

 5 Figure 5. Hierarchical clustering for P. nigra of the six terpenes selected with multilevel sPLS-DA using terpene content. Samples are represented in columns and terpenes in rows. MT, monoterpene hydrocarbon; SHT, sesquiterpene hydrocarbon; DHT, diterpene hydrocarbon; der, derivative compounds; others, compounds other than terpenes.

Table 1 .

 1 Topographical and climate characteristics of the study localities.

	Study sites			Topography			Climate 1
	Localities	Lat. (°)	Long. (°)	Aspect	Slope	Elevation	Annual	Mean annual
					(%)	(m.a.s.l.)	rainfall (mm)	temperature (°C)
	Lloreda	1.5706	42.0569	N	30	715	731.6	11.7
	Miravé	1.4494	41.9515	NE	25	723	677.3	11.5
	El Perelló	0.6816	40.9068	NW	10	244	609.9	15.5

Table 2 .

 2 Characteristics of prescribed burnings and forest experimental units (mean

	± std).

1

Wind speed was measured outside the forest.

2 Range of maximum temperatures (Tmax) and residence time above 60 °C (RT60) in 10 trees in each of the Perelló experimental units and in 20 trees in Miravé and Lloreda.

3 Ph, Pinus halepensis; Pn/Ps, P. nigra and P. sylvestris; phytovolume calculated using the cover and height of the understory shrubs; diameter at breast height (DBH), density and basal area of trees with DBH ≥7.5 cm.

Table 3 .

 3 Studied pine trees and fire characteristics (mean ± std) before and after prescribed burnings grouped by species.

	Tree and fire characteristics	P. halepensis	P. nigra	P. sylvestris
	n (trees)		20	19	19 1
	DBH (cm)		20.0 ± 6.9a	13.6 ± 5.5b	12.7 ± 5.3b
	Total height (m)		9.1 ± 2.4a	8.3 ± 2.4a	8.6 ± 1.9a
	Height to live crown base (m)	5.2 ± 1.0a	4.8 ± 1.3a	6.6 ± 13.2b
	Crown scorched (%)		44.0 ± 32.1a	6.6 ± 13.2b	5.5 ± 9.5b
	Fire residence time >60 °C (min)	38.2 ± 54.1a	16.6 ± 6.9a	15.2 ± 6.4a
	Needle δ 13 C (‰)
	Pre-burning		-25.8 ± 0.5Aa	-26.6 ± 1.0 Ab	-26.5 ± 0.6Ab
	1 year post-burning		-27.6 ± 0.9Ba	-28.5 ± 1.4Ba	-28.0 ± 0.8Ba
	Needle N content (mg g DM	-1 )
	Pre-burning		14.8 ± 1.9Aa	10.1 ± 0.8Ab	12.3 ± 1.6Ac
	1 year post-burning		14.9 ± 3.2Aa	9.1 ± 2.7Ab	11.0 ± 3.1Aa

). Before PB, more than 45% of total terpene concentration was represented by diterpenes in P. halepensis while sesquiterpenes represented about 59% in P. nigra and monoterpenes represented 83% in P. sylvestris (see Table

S2

available as Supplementary Data at Tree Physiology Online). Considering all sampling times,

Climate variables, annual rainfall and annual mean temperature, were estimated using a georeferenced model(Ninyerola et al.

2000).

Sample size is 18 for 1 year post-burning data because of death of one tree. Different small letters within a row indicate statistical significant differences (P < 0.05) among pine species using LME (where fixed factor = species, random factor = plot) followed by Tukey post-hoc test. Different capital letters within a column indicate statistical significant differences (P < 0.05) between pre-burning and 1 year post-burning for each pine species using LMM (where fixed factor = time since burning, random factor = plot) followed by Tukey post-hoc test.

Acknowledgments

We wish to thank GRAF (Bombers, Generalitat de Catalunya) who kindly executed the PB; the EPAF team, Dani Estruch, Ana I. Ríos and Alba Mora for their technical assistance in the field, and Carol Lecareux and Amelie Saunier for their help in the laboratory. Finally, we would like to thank Miquel De Cáceres for his invaluable comments.

Funding Ministerio de Economía, Industria y Competitividad (projects AGL2012-40098-CO3, AGL2015 70425R; EEBB I 15 09703 and BES 2013 065031 to T.V.; RYC2011 09489 to P.C.). CERCA Programme/Generalitat de Catalunya.

Conflict of interest

None declared.


nom. et stat. nov Verlaque Louiza-Nesrine

Aurelie Sellam

Charles-François Blanfuné

Thierry Boudouresque

C Thibaut

Marc Rebzani-Zahaf

Verlaque

J G Agardh

C Spinosa Sauvageau

stat. nov Al Nom Louiza

Nesrine Sellam

Aurélie Blanfuné

Charles F Boudouresque

Thierry Thibaut

Rebzani Chafika

Zahaf

Marc Verlaque

C Adriatica Sauvageau

G Agardh

Pc0535490

Roussel

C Turneri Montagne

C Montagnei

Pc0535491

P C Monnard

) Montagne

C Sp Nov

C Platyclada Sauvageau

Fig. 22 Montagne

Jean Bart

Thuret

J Feldmann

Ld00526

C Abies

Cystoseira montagnei

come

INTRODUCTION

In the Mediterranean Sea, the species of the genus Cystoseira C. Agardh, 1820, nom. cons., are the main forest-forming species of the photophilous rocky substrates from the littoral fringe down to the lower euphotic zone (down to 70-80 m depth in the clearest waters) [START_REF] Giaccone | Le Cistoseire e la vegetazione sommersa del Mediterraneo[END_REF][START_REF] Giaccone | -La vegetazione marina bentonica fotofila del Mediterraneo: II. Infralitorale e Circalitorale. Proposte di aggiornamento[END_REF][START_REF] Blanfuné | Decline and local extinction of Fucales in the French Riviera: the harbinger of future extinctions?[END_REF]Blanfuné et al., 2016a,b;[START_REF] Boudouresque | Where seaweed forests meet animal forests: the examples of macroalgae in coral reefs and the Mediterranean coralligenous ecosystem[END_REF]. Out of 289 taxa of Cystoseira listed worldwide (including homotypic and heterotypic synonyms and names of uncertain taxonomic status), 32 species and more than fifteen infra-specific taxa are currently accepted taxonomically in the Mediterranean Sea [START_REF] Guiry | their discussion is beyond the scope of the present study[END_REF]. However, in spite of their importance as habitat formers, the delimitation and distribution of a number of species are still not well known [START_REF] Roberts | Active speciation in the taxonomy of the genus Cystoseira C. Ag[END_REF][START_REF] Ribera | -Check-list of Mediterranean seaweeds. I. Fucophyceae (Warming, 1884)[END_REF][START_REF] Draisma | -DNA sequence data demonstrate the polyphyly of the genus Cystoseira and other Sargassaceae Genera (Phaeophyceae)[END_REF][START_REF] Cormaci | -Flora marina bentonica del Mediterraneo: Phaeophyceae[END_REF][START_REF] Berov | -Reinstatement of species rank for Cystoseira bosphorica Sauvageau (Sargassaceae, Phaeophyceae)[END_REF][START_REF] Bouafif | -Cystoseira taxa new for the marine flora of Tunisia[END_REF][START_REF] Bouafif | -New contribution to the knowledge of the genus Cystoseira C. Agardh in the Mediterranean Sea, with the reinstatement of species rank for C. schiffneri Hamel[END_REF]. The reason for this is that the species of Cystoseira offer few unambiguous diagnostic characters and that some of these characters are more or less overlapping. Genetic tools will probably help to disentangle their taxonomic value. Montagne (1838) described from Algeria (Cherchell, west of Algiers, Mediterranean Sea) a taxon he regarded as a new variety of the Atlantic species Cystoseira granulata C. Agardh, as C. granulata var. turneri Montagne. This taxon represents one of the least well known Cystoseira taxa; in addition, it became subsequently a source of confusion. Agardh (1842: 47-48), on the basis of distinct specimens from the north-western Mediterranean and the Adriatic Sea, raised Montagne's taxon to species level, under the name of C. montagnei J. Agardh, actually a new species. Cystoseira montagnei was widely recorded in the Mediterranean Sea until several authors expressed doubts regarding its taxonomic value, considering it as a mixture of distinct taxa (e.g. [START_REF] Sauvageau | A propos des Cystoseira de Banyuls et de Guéthary[END_REF][START_REF] Sauvageau | -A propos des Cystoseira[END_REF][START_REF] Papenfuss | Taxonomic and nomenclatural notes on three species of brown algae. In: Travaux de Biologie végétale dédiés au Professeur P. Dangeard[END_REF][START_REF] Roberts | Active speciation in the taxonomy of the genus Cystoseira C. Ag[END_REF]. Following [START_REF] Sauvageau | A propos des Cystoseira de Banyuls et de Guéthary[END_REF], Mediterranean authors replaced the name 'C. montagnei' by those of C. spinosa Sauvageau and C. adriatica Sauvageau. Cystoseira montagnei is now often treated as a taxon inquirendum in the updated Mediterranean checklists and floras [START_REF] Ribera | -Check-list of Mediterranean seaweeds. I. Fucophyceae (Warming, 1884)[END_REF][START_REF] Cormaci | -Flora marina bentonica del Mediterraneo: Phaeophyceae[END_REF]; but see Perret- [START_REF] Perret-Boudouresque | Inventaire des algues marines benthiques d'Algérie[END_REF].

In 2014 and 2015, we collected specimens corresponding to Montagne's taxon, C. granulata var. turneri, from the regions of Tipaza and Algiers (Algeria) [START_REF] Sellam | -Rediscovery of a forgotten seaweed forest in the Mediterranean Sea, the Cystoseira montagnei (Fucales) forest. Rapports et procès-verbaux de la commission internationale pour l'Exploration[END_REF]. The morphological study of these specimens showed that they belonged to a taxon quite distinct from C. spinosa. The aim of this study was (i) to reassess the status of C. granulata var. turneri, C. montagnei and C. spinosa, made obscure by taxonomic ambiguities and misuses (ii) to propose the lectotypification of C. granulata var. turneri and of C. montagnei, (iii) to propose a new name for Montagne's C. granulata var. turneri (C. michaelae nom. et stat. nov.) and (iv) to provide information concerning the ecology and distribution range of the latter species.

MATERIAL AND METHODS

Sampling and observations were undertaken using SCUBA diving, between the sea surface and 25 m depth, at different localities in the regions of Tipaza and Algiers (Algeria), from La Corne d'Or to Bounetah Island, between August 2014 and November 2015 (Fig. 1). The populations were studied all year round, at twoweek intervals.

Samples were transferred to the laboratory (in Algiers), then rinsed with seawater and cleaned of epiphytes. The samples were either preserved in 4% buffered formalin/seawater or pressed and prepared as herbarium specimens. A subsample of some specimens was preserved in silica gel for further DNA analyses. The material studied has been deposited in HCOM, the Herbarium of the Mediterranean Institute of Oceanography, Aix-Marseille University (Herbarium abbreviation follows [START_REF] Thiers | Index Herbariorum: A global directory of public herbaria and associated staff[END_REF].

Specimens were compared with the almost exhaustive collection of Mediterranean Cystoseira species deposited in the HCOM and with the syntype of C. granulata C. Agardh var. turneri Montagne (deposited in the herbaria of the Muséum National d'Histoire Naturelle, Paris, PC), the syntype of C. montagnei J. Agardh (deposited in the J.G. Agardh herbarium, Botanical Museum, Lund University, LD) and the lectotypes of C. spinosa Sauvageau and C. adriatica Sauvageau (herbaria of the Muséum National d'Histoire Naturelle, Paris, PC). They were also compared with other specimens housed in the herbaria of the Université Montpellier 2 (MPU) and of PC (Table 1). Identification criteria, in the genus Cystoseira, are based on the mode of attachment to the substratum, the number and the form of axes, the aspect of apices and tophules (when present), the phyllotaxy and the morphology of branches, the occurrence and the arrangement of cryptostomata and aerocysts, and the location and the morphology of reproductive structures (see [START_REF] Sauvageau | A propos des Cystoseira de Banyuls et de Guéthary[END_REF][START_REF] Sauvageau | -A propos des Cystoseira[END_REF][START_REF] Hamel | Phéophycées de France[END_REF][START_REF] Ercegović | -Fauna i Flora Jadrana. Jadranske cistozire. Njihova morfologija, ekologija i razvitak / Fauna et Flora Adriatica. Sur les Cystoseira adriatiques[END_REF]Gómez Garreta et al., 2001;[START_REF] Mannino | Guida all' identificazione delle Cistoseire. Area Marina Protetta "Capo Gallo -Isola delle Femmine[END_REF][START_REF] Cormaci | -Flora marina bentonica del Mediterraneo: Phaeophyceae[END_REF][START_REF] Taşkin | The Mediterranean Cystoseira (with photographs)[END_REF]. The initial nomenclature (before the changes resulting from the present investigation) followed that adopted by [START_REF] Guiry | their discussion is beyond the scope of the present study[END_REF].

Literature dealing with C. granulata var. turneri, C. montagnei, C. spinosa and C. adriatica was exhaustively searched and analyzed.

RESULTS

Hereafter, we describe the morphology, phenology and habitat of the specimens we collected in Algeria. Morphological description. Plants stiff, up to 30 cm high, not caespitose, yellowbrown in colour when alive and darker when dried, attached to the substrate by a robust discoid holdfast (Figs 2-4); main axis up to 20 cm high, branched, with apices not protruding, surrounded by spinose tophules (Figs 5, 7); spinose tophules ovoid, 5-13 mm × 5-7 mm, becoming smooth-tuberculate when older (Figs 5-7); primary branches, up to 18-19 cm long, of two different types: either slightly complanate, up to 2.5 mm wide, with an inconspicuous rib and irregularly alternately branched in one plane, or cylindrical and branched in all directions, with spaced short simple to bifid spine-like appendages . The habit varies with depth: shallow specimens have cylindrical branches while the deeper ones have complanate branches. Cryptostomata scattered along branches; specimens monoecious; receptacles both intercalary basal, compact to more or less loosely arranged, attached just above the tophule (Figs 11-13), and terminal, cylindrical and more or less diffuse on branchlets ; conceptacles male, female or hermaphroditic, differentiated in the branch and at the base of spine-like appendages . No obvious relationship was found between the location of the conceptacles, their type (male, female, hermaphroditic) and the receptacles. Phenology. The annual growth cycle is similar to that of other lower infralittoral species of Cystoseira. The plant grows from the early spring to the summer. While terminal receptacles were observed in late summer and autumn, basal receptacles were present all year round. Plants shed their primary branches in late autumn and they are almost devoid of primary branches in winter (Fig. 4). Habitat. The species thrives in the lower infralittoral zone (sensu [START_REF] Pérès | Major benthic assemblages[END_REF], between 10 m and 25 m depth (limit of our investigations), and on sub-horizontal to gently sloping photophilous rocky substrates (0 to 45°). It is always heavily covered with epiphytes such as other macroalgae, bryozoans, hydroids and sponges. The specimens we collected from the region of Algiers correspond well with Montagne's description and to his herbarium specimens (syntype) housed in the Muséum National d'Histoire Naturelle (Paris: PC) (Table 1). The taxon is very easily distinguishable from all other Cystoseira species through a panel of characters: (i) a single axis with spinose (when young) to smooth-tuberculate (when old) tophules, (ii) primary branches either slightly compressed, with an inconspicuous rib, and irregularly alternately branched in one plane, or cylindrical and branched in all directions, with spaced short spine-like appendages (iii) receptacles either intercalary basal, just above the tophule, or terminal, cylindrical and diffuse on branchlets.

The fate of Cystoseira granulata var. turneri and the confusing story of C. montagnei and C. spinosa [START_REF] Meneghini | Alghe italiane e dalmatiche[END_REF] reported C. granulata var. turneri from Naples, Toulon, the northern Adriatic Sea and Dalmatia. J.G. [START_REF] Agardh | Algae maris Mediterranei et Adriatici, observationes in diagnosin specierum et dispositionem generum[END_REF], receiving several specimens of Cystoseira with spinose tophules from France (Cette, now Sète, and Marseille) and the northern Adriatic Sea (Trieste, Italy), concluded that they all belonged to Montagne's taxon. Considering C. granulata var. turneri to be quite distinct from C. granulata C. Agardh [currently C. usneoides (Linnaeus) M.Roberts [START_REF] Roberts | -Taxonomic and nomenclatural notes on the genus Cystoseira C[END_REF][START_REF] Spencer | -Typification of Linnaean names relevant to algal nomenclature[END_REF]] and from all the other species known at that time ('Species distinctissima, a Montagne primum bene descripta, sed cum C. granulata male confusa': 'very distinct species…, well described for the first time by Montagne, but with C. granulata badly confused'), J.G. Agardh raised the var. turneri to species rank under the name C. montagnei J. Agardh. However, it is worth noting that, in his description, J.G. Agardh mentioned the spinose tophules and the compressed branches but omitted the major diagnostic character of Montagne's taxon, i.e. the basal intercalary receptacles. Montagne (1846) followed J.G. Agardh and re-described his alga from Algeria under the name C. montagnei J. Agardh [START_REF] Sauvageau | A propos des Cystoseira de Banyuls et de Guéthary[END_REF] published his impressive revision of the genus Cystoseira, with descriptions of new species with spinose tophules (C. adriatica Sauvageau and C. spinosa Sauvageau), the first taxonomic questions about the real identity of C. montagnei J. Agardh were raised. Considering all the previous records of C. montagnei with, when possible, re-examination of samples, [START_REF] Sauvageau | A propos des Cystoseira de Banyuls et de Guéthary[END_REF][START_REF] Sauvageau | -A propos des Cystoseira[END_REF] concluded that C. montagnei J. Agardh differed from C. montagnei sensu Montagne and was a mixture of species. Except for specimens from Algeria that possessed all the characteristics of C. montagnei Montagne non J. Agardh (sic) [here: C. michaelae], all the other records from France, Corsica, Sardinia, Italy and Adriatic Sea were doubtful because devoid of basal intercalary receptacles: C. montagnei sensu Va liante from Naples would probably be C. spinosa and C. montagnei sensu Hauck from the Adriatic sea would probably be C. adriatica (subsequently synonymised with C. spinosa; see [START_REF] Cormaci | Observations taxonomiques et biogéographiques sur quelques espèces du genre Cystoseira C.Agardh[END_REF]. In the Adriatic Sea, [START_REF] Ercegović | -Fauna i Flora Jadrana. Jadranske cistozire. Njihova morfologija, ekologija i razvitak / Fauna et Flora Adriatica. Sur les Cystoseira adriatiques[END_REF] followed the conclusions of [START_REF] Sauvageau | A propos des Cystoseira de Banyuls et de Guéthary[END_REF][START_REF] Sauvageau | -A propos des Cystoseira[END_REF] and treated (i) C. montagnei J. Agardh sensu Hauck as a synonym of C. adriatica, (ii) 'C. montagnei J. Agardh (ex parte)' (sic) as a synonym of C. platyramosa Ercegović [currently C. spinosa var. compressa (Ercegović) Cormaci et al.], and (iii) 'C. montagnei J. Agardh (pro parte)' (sic) and C. montagnei J. Agardh sensu Va liante as synonyms of C. spinosa. At Naples, [START_REF] Funk | Beiträge zur Kenntnis der Meeresalgen von Neapel: Zugleich mikrophotographischer Atlas[END_REF] did the opposite and recorded C. montagnei J. Agardh, with C. spinosa Sauvageau as synonym. According to [START_REF] Papenfuss | Taxonomic and nomenclatural notes on three species of brown algae. In: Travaux de Biologie végétale dédiés au Professeur P. Dangeard[END_REF], 'until J. G. Agardh's material has been examined and C. montagnei lectotypified, it will not be possible to settle the status of the species'. Our re-examination of the syntype of C. montagnei J. Agardh dating before 1842 and deposited in the J.G. Agardh Herbarium (LD) showed that no specimen originates from Algeria and confirmed the conclusions of Sauvageau and Ercegović: all the specimens of J.G. Agardh do not differ from C. spinosa Sauvageau (synonyms: C. adriatica Sauvageau and C. platyramosa Ercegović) [here: C. montagnei]. In Spain, C. montagnei was recorded from the Balearic Islands before being excluded from the flora [START_REF] Gallardo | -A preliminary checklist of Iberian benthic marine algae[END_REF]Ribera Siguan & Gómez Garreta, 1985;Gómez Garreta et al., 2001). The species was never recorded from Morocco (see [START_REF] Benhissoune | -A checklist of the seaweeds of the Mediterranean and Atlantic coasts of Morocco. II. Phaeophyceae[END_REF], Libya, in spite of extensive research on the genus Cystoseira [START_REF] Nizamuddin | Cystoseira gerloffi, a new species from the coast of Libya[END_REF][START_REF] Nizamuddin | -A new species of Cystoseira C. Ag. (Phaeophyta) from the Eastern part of Libya[END_REF][START_REF] Nizamuddin | A caespitose-tophulose Cystoseira species from Tripoli, Libya[END_REF], and from Egypt [START_REF] Aleem | Marine algae of Alexandria, Egypt[END_REF]. Subsequently, C. montagnei was definitely considered as taxon inquirendum [START_REF] Ribera | -Check-list of Mediterranean seaweeds. I. Fucophyceae (Warming, 1884)[END_REF][START_REF] Furnari | Catalogue of the benthic marine macroalgae of the Italian coast of the Adriatic Sea[END_REF][START_REF] Furnari | -Biodiversità marina delle coste italiane: catalogo del macrofitobenthos[END_REF][START_REF] Giaccone | -Biodiversità vegetale marina dell'arcipelago 'Isole Eolie[END_REF][START_REF] Cormaci | -Flora marina bentonica del Mediterraneo: Phaeophyceae[END_REF][START_REF] Taşkin | The Mediterranean Cystoseira (with photographs)[END_REF][START_REF] Tsiamis | -Seaweeds of the Greek coasts. I. Phaeophyceae[END_REF], including in Algeria [START_REF] Ould- | -Checklist of the benthic marine macroalgae from Algeria. I. Phaeophyceae[END_REF]. See Table 2 for further records of C. montagnei, C. spinosa and C. adriatica. the Algerian coast. Currently, C. michaelae seems to be an endemic species restricted to Algeria and northern Tunisia (Cyrine Bouafif, pers. com.).

Cystoseira forests are highly impacted due to the cumulative effects of increasing human pressure (e.g. destruction of habitats, pollution, non-indigenous species, overfishing, coastal aquaculture and global warming). Losses have been reported throughout the Mediterranean Sea caused by habitat destruction, eutrophication and overgrazing by herbivores (fish, sea urchins), leading to a shift to lesser structural complexity, such as turf-forming seaweed assemblages or barren grounds where sea urchins are the drivers of habitat homogenization [START_REF] Pinedo | -Long-term decline of the populations of Fucales (Cystoseira, Sargassum) in the Albères coast (northwestern Mediterranean)[END_REF][START_REF] Blanfuné | Decline and local extinction of Fucales in the French Riviera: the harbinger of future extinctions?[END_REF]Blanfuné et al., 2016a,b). Protective measures should be taken so that the C. michaelae forests do not suffer the same decline as many Cystoseira forests of the Mediterranean Sea.

  Specimens studied: H8287-8288 -Cap Caxine (36' 49' 4" N & 2°57' 19" E), September 2014, 17 m depth, rocky substrates; H8289-8290 -Aïn Benian, close to the harbor (36°48' 45" N & 2°53' 29" E), August 2014, 19 m depth, rocky substrates; H8291-8294 and H8300-8301 -Tipaza, La Corne d'Or (36°35' 45" N & 2°26' 44" E), September 2014, 16 m depth, rocky substrates; H8295 -Aïn Benian, close to the harbour, September 2014, 18 m depth, rocky substrates; H8296-8297 -Aïn Benian, close to the harbour, April 2015, 14 m depth, rocky substrates; H8298 -Cap Caxine, April 2015, 16 m depth, rocky substrates; H8299 -Islets of Tipaza (36°35' 52" N & 2°27' 40" E), March 2015, 11 m depth, rocky substrates; H8302 -Islets of Tipaza, August 2015, 10 m depth, rocky substrates; H8303 -Bounetah Island (36°47' 46" N & 3°21' 19" E), August 2015, 14 m depth, rocky substrates; H8304 -Cap Caxine, August 2015, 13 m depth, rocky substrates; H8305 -Islets of Tipaza, October 2015, 12 m depth, rocky substrates; H8306 -Cap Caxine, November 2015, 13 m depth, rocky substrates (Fig. 1).

  The species forms sparse algal forests (< 5 individuals.m -2 ), in association with other large macroalgae such as Cystoseira zosteroides (Turner) C. Agardh, Dictyopteris lucida M.A.Ribera Siguán et al., Dictyota cyanoloma Tronholm et al., Dictyota spp., Flabellia petiolata (Turra) Nizamuddin, Phyllariopsis sp., Sargassum sp., Zonaria tournefortii (J.V. Lamouroux) Montagne, and a rich sessile fauna dominated by Eunicella singularis (Esper, 1791) and large species of sponges, bryozoans and hydroids.DISCUSSION AND CONCLUSIONSA good fit between Cystoseira granulata C. Agardh var. turneri Montagne and the specimens collected in the Algiers regionMontagne (1838: 340-342) described from Algeria, 'prope Juliam Caesaream' (now Cherchell, ~80 km west of Algiers) a taxon he regarded as a variety of the Atlantic species Cystoseira granulata C. Agardh, as C. granulata var. turneri Montagne. Montagne (1846: 13-14, plate 4) re-described and nicely illustrated

Fig. 1 .

 1 Fig. 1. Locations with collection dates of Cystoseira michaelae Ve rlaque et al., nom. et stat. nov. (C. granulata C. Agardh var. turneri Montagne) in Algeria (Tipaza and Algiers regions) -Historical data: Herbarium specimens and references (light circles) and newly collected specimens (this work) (dark circles). *: Debray (1897): specimens not found.

Figs 2- 4 .

 4 Figs 2-4. Cystoseira michaelae Ve rlaque et al., nom. et stat. nov. from Algeria (newly collected specimens, this work). 2-3. Habit of specimens H8300 and H8291, respectively, from Tipaza, La Corne d'Or, September 2014. 4. Habit of an old individual, specimen H8299 from Islets of Tipaza, March 2015. Bars = 5 cm.

Figs 5 -

 5 Figs 5-10. Cystoseira michaelae Ve rlaque et al., nom. et stat. nov. from Algeria (newly collected specimens, this work). 5-6. Apical views of axes showing spinose (black arrows) and smooth-tuberculate (white arrows) tophules (specimen H8300); bars = 5 mm. 7. Spinose and smooth-tuberculate tophules, specimen H8288 from Cap Caxine, September 2014; bar = 1 cm. 8. Complanate branch with inconspicuous midrib, specimen H8300; bar = 1 cm. 9. Complanate to cylindrical branch with short spine-like appendages, specimen H8291; bar = 1 cm. 10. Detail of a complanate branch with inconspicuous midrib, specimen H8300; bar = 1 cm.

Figs

  Figs 11-20. Cystoseira michaelae Ve rlaque et al., nom. et stat. nov. from Algeria (newly collected specimens, this work). 11-12. Compact tuberculate-spinose basal intercalary receptacles (arrows) close to the tophule (arrow heads), specimen H8300: bars = 5 mm. 13. Diffuse spinose basal intercalary receptacles (arrows) close to the tophule (arrow head), specimen H8290 from Aïn Benian, August 2014; bar = 5 mm. 14. Transverse section of a female basal receptacle, specimen H8291; bar = 200 µm. 15. Transverse section of a male basal receptacle, specimen H8300; bar = 200 µm. 16. Transverse section of a female basal conceptacle, specimen H8291; bar = 100 µm. 17. Transverse section of a male basal conceptacle, specimen H8300; bar = 100 µm. 18-19. Diffuse spinose terminal receptacles, specimen H8291; bars = 5 mm. 20. Transverse section of a hermaphroditic terminal conceptacle, specimen H8291; bar = 100 µm.

Fig. 21 .

 21 Fig. 21. Illustration of Cystoseira michaelae Ve rlaque et al., nom. et stat. nov., as C. montagnei J. Agardh (sensu Montagne), in Montagne (1846, plate 4, figs 2a-h). a: Habit. b: Lower part of a branch with intercalary receptacles. c: Terminal receptacle. d: Detail of a terminal receptacle. e: Transverse section of a terminal receptacle showing the conceptacles. f: Oogonia. The original numbering of figures within the plate has been changed, but the original numbers have not been erased and can be seen in very small print.

  [here: C. michaelae]. At the same time, he excluded all the records of[START_REF] Meneghini | Alghe italiane e dalmatiche[END_REF]. J.G.[START_REF] Agardh | Species genera et ordines algarum, seu descriptiones succinctae specierum, generum et ordinum, quibus algarum regnum constituitur[END_REF] considered Montagne's illustrations of C. montagnei [here: C. michaelae] as excellent ('Montagne (1846) p.43. tab. IV.2 (eximie !)'), and completed the distribution of the species ('Hab. In mari mediterraneo ad littora Occitaniae et galloprovinciae (ipse! = J.G. Agardh), ad Algeriam (Montagne!); in Adriatico ad Trieste (Biasoletto! et C. Agardh!) et Ve netiam (Martens!); e Gadibus = Cadix (Cabrera!)', but always without any mention of basal intercalary receptacles. This shows that (i) J.G. Agardh probably never saw any genuine specimen of Montagne's taxon, (ii) the J.G. Agardh concept of C. montagnei is much broader than that of Montagne and (iii) C. montagnei cannot be treated as a replacement name for C. granulata var. turneri (according to Art. 6.11 of ICN; McNeill et al., 2012), but as a new species based upon the Sète, Marseille and Trieste specimens (syntype housed in the Lund herbarium, LD). Kützing (1849) transferred C. montagnei to the genus Phyllacantha Kützing (currently a junior synonym of Cystoseira). Later on, he published the illustrations of P. montagnei and P. montagnei var. cirrosa Kützing on the basis of Algerian specimens sent by Montagne (Kützing, 1860) [here: C. michaelae]. Hauck (1885) recorded C. montagnei (with Phyllacantha gracilis Kützing, P. pinnata Kützing and P. affinis Kützing as synonyms), from the Adriatic Sea, with no mention of basal intercalary receptacles; the illustrations of these species of Phyllacantha in Kützing (1860) agree more with the Sauvageau (1912) C. spinosa [here: C. montagnei] than with Montagne's taxon [here: C. michaelae]. In his 'Catalogue des algues du Maroc, de l'Algérie & de la Tunisie', Debray (1897) recorded C. montagnei [here: C. michaelae] only from Algeria, close to Algiers [Cherchell, Matifou and Saint Eugène (now Bologhine)]. When

Cystoseiramichaelae

  Fig. 22. Lectotype of Cystoseira granulata C. Agardh var. turneri Montagne (here: C. michaelae Ve rlaque et al., nom. et stat. nov.), Algiers [Algeria], PC (Herbarium Montagne), barcode PC0043663, by courtesy of the MNHN-Paris ©; Collection C. and P. Monnard; labelled 'Cystoseira granulata L. Turner var. turneri Montagne -Alger n°397 -Com. Class. Monnard'. The lectotype is the top left specimen. Isolated branches (top right and bottom) possibly belong to the same individual.

Figs

  Figs 23-26. Lectotype of Cystoseira montagnei J. Agardh, Cette (now Sète), France, May 1837, Botanical Museum, Lund University (Herbarium J. Agardh), barcode LD528, by courtesy of Lund University ©. 23. Habit; bar = 10 cm. 24. Detail of the label. 25. Detail of spinose tophules; bar = 5 mm. 26. Detail of the upper part of branchlets with receptacles; bar = 5 mm.

Figs

  Figs 27-29. Lectotype of Cystoseira spinosa Sauvageau, Banyuls-sur-Mer, Pyrénées-Orientales, France, 6 May 1907, PC (Herbarium Général, collection C. Sauvageau), barcode PC0525446, by courtesy of the MNHN-Paris ©. 27. Habit; bar = 2 cm. 28. Detail of spinose tophules; bar = 5 mm. 29. Detail of the upper part of branchlets with receptacles; bar = 5 mm.

Table 1 .

 1 Major herbarium specimens of Cystoseira examined. The syntypes of C. michaelae Ve rlaque et al. nom. et stat. nov. and of C. montagnei Ve rlaque et al. comb. nov.

	C. michaelae Ve rlaque	et al. nom. et stat. nov.
	C. granulata var. turneri	Montagne

Table 1. Major herbarium specimens of Cystoseira examined. The syntypes of C. michaelae Ve rlaque et al. nom. et stat. nov. and of C. montagnei J. Agardh are indicated with an asterisk. (coll.: collection); m.d.: missing data. Correct names according to the authors of the present study (continued)

Table 2 .

 2 Records of Cystoseira species with spinose tophules, referred to as C. spinosa, C. adriatica and C. montagnei, in the Mediterranean Sea (in addition to the records mentioned within the text), and probable correspondence with the taxonomic treatment in the present study (Cystoseira montagnei J.Agardh and C. michaelae Ve rlaque et al. nom. et stat. nov.)

			Name(s) and	Correct name(s)
	Reference	Location	authority used by	according to the	Comments
			the author(s)	present treatment
	Ardissone &	Liguria (Italy)	C. montagnei	C. montagnei ?	No description or
	Strafforello		J. Ag.		illustration
	(1877)
	Piccone	La Galite	C. montagnei	C. montagnei or	No description or
	(1879)	(Tunisia)	J. Agardh	C. michaelae ?	illustration
	Va liante	Gulf of Naples	C. montagnei	C. montagnei	Description and illustrations
	(1883)	(Italy)	J. Ag.		corresponding well to
					C. spinosa Sauvageau [here
					C. montagnei]
	Piccone	Sardinia (Italy)	C. montagnei	C. montagnei ?	No description or
	(1884)		J. Ag.		illustration
	Rodríguez	Balearic Islands	C. montagnei	C. montagnei ?	No description or
	y Femenías	(Spain)	J. Ag.		illustration
	(1889)
	Petersen	La Galite	C. montagnei	C. michaelae	As the 2 taxa are cited,
	(1918)	(Tunisia)	Montagne and	and	the possibility that they
			C. spinosa	C. montagnei,	could actually refer
			Sauvageau	respectively ?	to C. michaelae and
					C. montagnei must be
					considered

Acknowledgements. The authors wish to thank the Herbarium LD and Dr Patrik Froden, Assistant Curator at the Botanical Museum of Lund University, for sending photographs of J.G. Agardh specimens of Cystoseira montagnei (syntype); Dr V. Bourgade of the Université Montpellier 2 and Prof. Bruno de Reviers and Dr B. Dennetière of the Muséum National d'Histoire Naturelle, Paris, for permission to consult collections and notebooks of C. Montagne, and for permission to reproduce the photographs of the lectotypes of C. michaelae (C. granulata var. turneri), C. adriatica and C. spinosa; Michèle Perret-Boudouresque for documentation assistance; Oussalah Adel for diving assistance; and Michael Paul for revising the English text. Many thanks are due to the anonymous reviewers for their comments and constructive criticism of the manuscript.

Algérie / Cystoseira michaelae / Cystoseira montagnei / Cystoseira spinosa /

(7 specimens that predate the protologue) (Table 1), because it is the most similar to the fertile specimen illustrated by Montagne (1846: Plate 4, Fig. 2a-h) (reproduced here as Fig. 21). Type locality: On the sheet of the lectotype: Algiers (Algeria). However, in the protologue, Montagne (1838) mentions 'propè Juliam Caesaream' (near Cherchell, ~80 km West of Algiers). We therefore consider that the type locality is Cherchell. Illustrations: Montagne (1846, Algiers, Plate 4, Fig. 2a-h

Cystoseira montagnei J. Agardh