Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region

This study investigated the phylogeographic structure of Cistus ladanifer, in order to locate its Quaternary refugia, reconstruct its recolonisation patterns and assess the role of geographical features (mountain ranges, rivers and the Strait of Gibraltar) as barriers to its seed flow and expansion through the Western Mediterranean. Thirty-eight populations were screened for length variation of polymorphic chloroplast simple sequence repeats (cpSSRs). Statistical analyses included estimation of haplotypic diversity, hierarchical analysis of molecular variation (amova) and fixation indices. Mantel tests, SAMOVA and BARRIER analyses were applied to evaluate the geographical partitioning of genetic diversity across the entire species range. Pollen data from bibliography were used to complement molecular inferences. Chlorotype diversity within populations was similar throughout the natural range of C. ladanifer (mean haplotypic diversity=0.32). High differentiation among populations was estimated (G(ST)=0.60). Our data suggest that the barriers of the Strait of Gibraltar and the Betic ranges may have favoured the divergence during glacial periods of four different lineages of populations inferred with SAMOVA. The main northward colonisation of in the Iberian Peninsula occurred from refugia in southwest Iberia. This process may have been influenced by human activities (forest clearance, livestock grazing and even commerce) in the Iberian Peninsula. In contrast, populations in the Betic area have conserved a specific haplotype.     

Tolerance and bioaccumulation of heavy metals in five populations of Cistus ladanifer L. subsp. ladanifer

The effects of heavy metals on the growth, mineral composition (P, K, Fe and Mn) and metal accumulation of five populations of Cistus ladanifer subsp. ladanifer from NE Portugal were investigated in hydroponic experiments. Plants were exposed to increasing concentrations (0–2000 M) of one of eight heavy metals: Cd, Co, Cr, Cu, Mn, Ni, Pb or Zn. Populations of C. ladanifer, whose origin was ultramafic soils (S and UB) or soils developed on basic rocks (B), showed a higher tolerance to the metals Cd, Co, Cr, and Mn, and a considerable degree of tolerance to Ni. In contrast, populations originating on acid-rock soils (M and SC) showed higher tolerance to the metals Cu and Zn. Populations showed different patterns of metal accumulation and distribution in the plant parts, suggesting different mechanisms of metal tolerance are used. The more Cd-, Co- and Mn-tolerant populations (S, UB, B and SC (Cd)) showed accumulation of these three metals in the shoots (shoot:root metal concentration ratios (S:R) > 1). Shoot concentrations of up to 309 g Cd g^–1, 2667 g Co g^–1 and 6214 g Mn g^–1 were found in these populations. The populations, UB and M, showed considerable tolerance to Ni and Zn, respectively. These populations accumulated up to 4164 g Ni g^–1 and 7695 g Zn g^–1 in their shoot tissues, and these metals were efficiently transported from the roots to aerial parts (S:R > 3 (Ni), S:R > 1 (Zn)). In contrast, the S and SC populations maintained higher growth rates in the presence of Ni and Zn, respectively, but showed exclusion mechanisms of metal tolerance: reduced Ni and Zn transport to shoots (S:R < 1). Cistus ladanifer was not able to efficiently transport Cr, Cu or Pb from its roots to its aerial parts (S:R ranged from 0–0.4). The more Cu-tolerant populations, M and SC, showed a greater restriction of Cu transport to the shoots than the ultramafic- or basic-rock populations. Significant changes in the plant mineral composition were found, however, concentrations were generally above mineral deficiency levels. Based on these preliminary results the possible usefulness of this plant for phytoremediation technologies is discussed. However, further investigations are necessary to evaluate its growth and metal accumulation under soil and field conditions.     

Marked differences in seed dormancy in two populations of the Mediterranean shrub,Cistus albidus L.

Background: Population structure and dynamics in natural ecosystems can be affected by seed viability and dormancy. However, how the endogenous contents of phytohormones and vitamin E in seeds relative to the environment affect viability and dormancy is not yet fully understood.

Aims: We studied seed viability and germination capacity in two populations of the Mediterranean shrub, Cistus albidus, exposed to contrasting environmental conditions.

Methods: We measured seed viability and germination capacity and endogenous contents of abscisic acid (ABA), gibberellins and vitamin E in seeds collected from two populations, in two environmentally contrasting environments in north-eastern Spain.

Results: Plants growing in the natural site produced seeds with lower germination percentage but similar viability, indicating higher seed dormancy. Enhanced seed dormancy was paralleled with higher contents of ABA and lower contents of gibberellins (GAs). Contents of tocopherols and tocotrienols were higher in seeds of the natural population. Tocopherol contents in seeds correlated positively with the ratio living:total aerial biomass.

Conclusions: Two Mediterranean populations of C. albidus growing in two sites with contrasting environmental conditions showed marked differences in seed dormancy and germination, which may be explained, at least in part, by differences in seed hormonal contents.     

Interpopulational variation in the flavonoid composition of Cistus ladanifer L. exudate

A study was made in different populations of Cistus ladanifer of the quantitative and qualitative variation of the exudate of the leaves and stems, including the exudate's aglycone flavonoid content. Two groups of populations were established according to different climate conditions, and the results showed the existence of interpopulational variability both quantitatively and qualitatively. The populations subject to the greatest thermal stress and moisture stress (Group I) synthesized more exudate and total flavonoids. The amounts of the different flavonoids synthesized also differed between the two groups of populations: Group I secreted more 3-O-methylkaempferol, 3,4′-di-O-methylkaempferol and 3,7-di-O-methylkaempferol and Group II more 4′-O-methylapigenin. Quantifying the proportion of each apigenin and kaempferol relative to their totals, it was observed that the synthesis of position 7 methylated flavonoids are enhanced in Group I, and position 4′ methylated flavonoids in Group II. The indication is that the climatic conditions to which a population of C. ladanifer is subject may lead to diversification in the flavonoid composition of the leaf and stem exudate, with the implication that flavonoids could have various ecological functions in this species.     

Effect of allelopathic compounds produced by Cistus ladanifer on germination of 20 Mediterranean taxa

In order to analyze the extent to which allelopathic action of Cistus ladanifer may influence the successional progression towards Mediterranean sub-climacic shrublands and the geographical distribution of other species, the inhibitory effect of Cistus ladanifer extracts on the germination of 20 Mediterranean species was analyzed. Five of the species tested were characteristic of maquis sub-climacic shrublands: Arbutus unedo, Adenocarpus argyrophyllus, Phillyrea angustifolia, Phillyrea latifolia, and Rhamnus alaternus. Tests were also carried out on 6 Cistaceae species in order to evaluate the auto-toxicity rate of allelopathic extracts: Halimium umbellatum subsp. viscosum, Halimium ocymoides, Cistus ladanifer, Cistus salvifolius, Cistus monspeliensis, and Cistus populifolius. Nine herbaceous species sharing the natural habitat with C. ladanifer were also examined. Results confirmed a clear inhibitory and delaying effect of aqueous C. ladanifer-leaf extracts on the germination of P. angustifolia, P. latifolia, R. alaternus, H. ocymoides, C. populifolius, Erysimum lagascae, Brassica barrelieri, Silene tridentata, and Moricandia moricandioides. Assays with soil collected below the canopy of the C. ladanifer community showed more pronounced inhibitory effect on sub-climacic shrub species than the aqueous extracts. In contrast, the opposite pattern was detected when analyzing the allelopathic effect of natural soil on Cistaceae and herbaceous species. Allelopathic compounds produced by C. ladanifer showed little auto-toxicity. The inhibitory effect of phytotoxic compounds accumulated in the jaral soil upon germination of A. unedo, B.␣barrelieri, and M. moricandioides was eliminated by heating soil at 150 °C for 10 min. Phenolic compounds (i.e. ferulic, p-hydroxybenzoic, vanillic, p-coumaric, and caffeic acids) and terpenes (i.e. α and β-pinene) with allelopathic documented effect were detected in the aqueous extracts and soil samples used in the analyses. We suggest that the allelopathic effect of C. ladanifer may influence the composition and structure of Mediterranean communities where the species is present, since it hinders the establishment of some sub-climacic species and may reduce the area occupied by numerous herbaceous species.     

Identification of metalliferous ecotypes of Cistus ladanifer L. using RAPD markers

The genetic diversity of Cistus ladanifer ssp. ladanifer (Cistaceae) growing on ultramafic and non-ultramafic (basic and schists) soils in the NE of Portugal was studied in order to identify molecular markers that could distinguish the metal-tolerant ecotypes of this species. Random Amplified Polymorphic DNA (RAPD) markers were used in order to estimate genetic variation and differences between populations. The RAPD dataset was analysed by means of a cluster analysis and an analysis of molecular variance (AMOVA). Our results indicate a significant partitioning of molecular variance between ultramafic and non-ultramafic populations of Cistus ladanifer, although the highest percentage of this variance was found at the intra-population level. Mantel's test showed no relationship between inter-population genetic and geographic distances. A series of RAPD bands that could be related to heavy metal tolerance were observed. The identification of such markers will enable the use of Cistus ladanifer in phytoremediation procedures.     

Anatomical features, monomer lignin composition and accumulation of phenolics in 1-year-old branches of the Mediterranean Cistus ladanifer L.

Mediterranean shrub species are described as having phenology, habitus , reproductive biology and anatomical alterations in certain tissues, allowing their survival during the dry season and protecting them from herbivory. Anatomical and chemical analyses were conducted in 1-year-old branches of Cistus ladanifer L. in order to investigate the role played by shoot structure in the adaptive strategies of this species in the Mediterranean environment. Results showed that both xylem and pith underwent lignification. Pith parenchyma cells had thickened walls, higher lignin content than xylem and different monomer composition. Xylem presented features aiding safe water transport. A large accumulation of phenolic substances was found in xylem, pith and cortical parenchyma. Observations reported in this paper suggest the occurrence of adaptive strategies in 1-year-old branches of C. ladanifer whose structural features: (1) allow mechanical reinforcement of tissues to withstand drought without suffering permanent damage; (2) favour safety rather than efficiency in water transport; (3) defend the plants from animal predation and pathogens by accumulating phenolics in various tissues, and (4) protect inner tissues against UV-B radiation through deposition of phenolic compounds in cortical layers.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155 , 361–371.     

Loss of flower bud vigour in the Mediterranean shrub,Cistus albidus L. at advanced developmental stages

To better understand aging in perennials, age‐related changes in the physiology of leaves and flower buds of the Mediterranean shrub, Cistus albidus L. were evaluated. Two groups of different ages (5 and 10 years old), both at advanced developmental stages but of similar size, were compared. Total plant biomass, biomass produced per apical meristem and levels of cytokinins, abscisic acid and jasmonic acid in leaves and flower buds, as well as flower production, were measured. No differences in plant size, vegetative growth rates and levels of phytohormones in leaves were observed between 5‐ and 10‐year‐old plants. However, they showed significant differences in flower bud development; the older plants having reduced vigour, with 29.6% of flowers reaching anthesis compared to 52.5% in the younger plants. Furthermore, endogenous concentrations of zeatin and abscisic acid in flower buds at stage I (start of flower organ formation) were 61% and 41%, respectively, smaller in 10‐ than in 5‐year‐old plants. At stage II (with all flower organs formed), zeatin and abscisic acid concentrations decreased by ca. 90% and 80%, respectively, but differences between age groups were still evident (60% and 29% for zeatin and abscisic acid, respectively). Jasmonic acid levels in flower buds decreased by 80% from stage I to II, but did not differ between age groups. Despite reductions in flower bud vigour, total number of flowers per individual was not significantly different between age groups, so that an age‐related loss in reproductive vigour at the organ level did not lead to a decrease in flower production at the whole plant level.     

Hydrogen peroxide is involved in the acclimation of the Mediterranean shrub, Cistus albidus L., to summer drought

This study evaluated the possible role of hydrogen peroxide(H₂O₂) in the acclimation of a Mediterranean shrub, Cistus albidusL., to summer drought growing under Mediterranean field conditions.For this purpose, changes in H₂O₂ concentrations and localizationthroughout a year were analysed. H₂O₂ changes in response toenvironmental conditions in parallel with changes in abscisicacid (ABA) and oxidative stress markers, together with ligninaccumulation, xylem and sclerenchyma differentiation, and leafarea were also investigated. During the summer drought, leafH₂O₂ concentrations increased 11-fold, reaching values of 10µmol g^–1 dry weight (DW). This increase occurredmainly in mesophyll cell walls, xylem vessels, and sclerenchymacells in the differentiation stage. An increase in ABA levelspreceded that of H₂O₂, but both peaked at the same time in conditionsof prolonged stress. C. albidus plants tolerated high concentrationsof H₂O₂ because of its localization in the apoplast of mesophyllcells, xylem vessels, and in differentiating sclerenchyma cells.The increase in ABA, and consequently of H₂O₂, in plants subjectedto drought stress might induce a 3.5-fold increase in ascorbicacid (AA), which maintained and even decreased its oxidativestatus, thus protecting plants from oxidative damage. Afterrecovery from drought following late-summer and autumn rainfall,a decrease in ABA, H₂O₂, and AA to their basal levels (60 pmolg^–1 DW, 1 µmol g^–1 DW, and 20 µmol g^–1DW) was observed. Key words: Abscisic acid, ascorbate, ascorbate oxidative status, Cistus albidus, hydrogen peroxide, leaf plasticity, lignin, Mediterranean shrubs, oxidative markers, summer drought Received 29 July 2008; Revised 15 September 2008 Accepted 8 October 2008     

Pollinator‐mediated phenotypic selection does not always modulate flower size and number in the large‐flowered Mediterranean shrub Cistus ladanifer (Cistaceae)

Flower size and number usually evolve under pollinator‐mediated selection. However, hot, dry environments can also modulate display, counteracting pollinator attraction. Increased pollen deposition on larger flower displays may not involve higher female fitness. Consequently, stressful conditions may constrain flower size, favouring smaller‐sized flowers. The large‐flowered, self‐incompatible Mediterranean shrub Cistus ladanifer was used to test that: (1) this species suffers pollen limitation; (2) pollinators are spatially–temporally variable and differentially visit plants with more/larger flowers; (3) increased visits enhance reproduction under pollen limitation; (4) stressful conditions reduce female fitness of larger displays; and (5) phenotypic selection on floral display is not just pollinator‐mediated. We evaluated pollen limitation, related floral display to pollinator visits and fruit and seed production and estimated phenotypic selection. Flower size was 7.2–10.5 cm and varied spatially–temporally. Visitation rates (total visits/50 min) ranged from 0.26 to 0.43 and increased with flower size. Fruit set averaged 80% and seed number averaged 855, but only fruit set varied between populations and years. Selection towards larger flowers was detected under conditions of pollen limitation. Otherwise, we detected stabilizing selection on flower size and negative selection on flower number. Our results suggest that selection on floral display is not only pollinator‐dependent through female fitness in C. ladanifer. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 540–555.     

Climatic and fire determinants of early life-history stages in the Mediterranean shrub Cistus albidus

在伊比利亚半岛东北部沿水分梯度的典型地中海灌丛中对白毛岩蔷薇（Cistus albidus）的繁殖性状进行研究。火对该物种的萌发非常有利。此外,地中海的物种特别依赖于水分有效性。因此,我们建立了一个假说,即除火灾干扰外,该地中海植物的幼苗更新将因干旱事件得到改善,主要通过干旱事件引发冠层大量死亡。本研究收集了白毛岩蔷薇几个种群的个体,并对果实和种子的大小、重量和数量进行测定。同时也对五种发芽前处理进行了发芽测试：热激、吸胀、两个吸胀/干燥循环,以及热激和吸胀与吸胀/干燥循环的组合。此外,在野外对干旱事件后的幼苗数量进行了调查,并与冠层死亡进行相关分析。我们的研究表明,沿水分利用梯度,白毛岩蔷薇繁殖性状（例如发芽率和果实产量）具有变异性。这种变异性导致干旱条件下果实产量减少而发芽率增加。白毛岩蔷薇种子会因受热增加发芽率,表明它们具有在火灾后能够成功更新的能力。然而,白毛岩蔷薇种群的增长并不完全取决于火,因为在极端干旱后死亡的白毛岩蔷薇冠层下的幼苗更新率更高。最后,成年个体密度增加了白毛岩蔷薇的死亡以及幼苗的更新。这些结果表明,该物种在不同的繁殖结果（即种子产量与存活率）间表现出权衡,而这种权衡又取决于气候。这项研究也为种内竞争、气候（尤其是干旱事件和火干扰）如何影响常见且具有代表性的地中海易火灌木的生活史关键早期阶段的成功提供了证据。     

Chloroplast microsatellites reveal colonization and metapopulation dynamics in the Canary Island pine

Chloroplast microsatellites are becoming increasingly popular markers for population genetic studies in plants, but there has been little focus on their potential for demographic inference. In this work the utility of chloroplast microsatellites for the study of population expansions was explored. First, we investigated the power of mismatch distribution analysis and the F(S) test with coalescent simulations of different demographic scenarios. We then applied these methods to empirical data obtained for the Canary Island pine (Pinus canariensis). The results of the simulations showed that chloroplast microsatellites are sensitive to sudden population growth. The power of the F(S) test and accuracy of demographic parameter estimates, such as the time of expansion, were reduced proportionally to the level of homoplasy within the data. The analysis of Canary Island pine chloroplast microsatellite data indicated population expansions for almost all sample localities. Demographic expansions at the island level can be explained by the colonization of the archipelago by the pine, while population expansions of different ages in different localities within an island could be the result of local extinctions and recolonization dynamics. Comparable mitochondrial DNA sequence data from a parasite of P. canariensis, the weevil Brachyderes rugatus, supports this scenario, suggesting a key role for volcanism in the evolution of pine forest communities in the Canary Islands.     

Long-distance colonization of the Western Mediterranean by Cistus ladanifer (Cistaceae) despite the absence of special dispersal mechanisms

Aim There are few biogeographical and evolutionary studies that address plant colonization and lineage origins in the Mediterranean. Cistus serves as an excellent model with which to study diaspore dispersal and distribution patterns of plants exhibiting no special long‐distance dispersal mechanisms. Here we analyse the pattern of genetic variation and divergence times to infer whether the African–European disjunction of C. ladanifer L. is the result of long‐distance dispersal or of vicariance events. Location Principally the Western Mediterranean region, with a focus on the Strait of Gibraltar. Methods We used DNA sequence phylogenetic approaches, based on plastid (rbcL/trnK‐matK) and nuclear (ITS) sequence data sets, and the penalized likelihood method, to date the diversification of the 21 species of Cistus. Phylogenetic relationships and phylogeographical patterns in 47 populations of C. ladanifer were also analysed using two plastid DNA regions (trnS‐trnG, trnK‐matK). These sequence data were analysed using maximum parsimony, Bayesian inference and statistical parsimony. Results Dating estimates indicated divergence dates of the C. ladanifer lineage in the Pleistocene. Eight nucleotide‐substitution haplotypes distributed on the European (four haplotypes) and African (five haplotypes) sides of the Strait of Gibraltar were revealed from C. ladanifer sequences. Both the haplotype network and the phylogenetic analyses depicted two main Cistus lineages distributed in both Europe and North Africa. An Iberian haplotype forms part of the North African lineage, and another haplotype distributed on both continents is related to the European lineage. Haplotype relationships with respect to outgroup sequences supported the hypothesis that the centre of genetic diversity is in northern Africa. Main conclusions Based on lineage divergence‐time estimates and disassociation between geographical and lineage haplotype distributions, we inferred at least two intercontinental colonization events of C. ladanifer post‐dating the opening of the Strait of Gibraltar (c. 5 Ma). This result supports a hypothesis of long‐distance dispersal rather than a hypothesis of vicariance. We argue that, despite limited dispersal abilities, preference for disturbed habitats was integral to historical colonization after the advent of the Mediterranean climate (c. 3.2 Ma), when Cistus species diverged and became established as a dominant element in the Mediterranean scrub.     

Enhanced UV-B radiation increases the reproductive effort in the Mediterranean shrub Cistus creticus under field conditions

Seedlings of the Mediterranean shrub Cistus creticus L. were grown in the field under ambient or ambient plus supplemental UV-B radiation (simulating a 15% ozone depletion over Patras, 38.3°W, 29.1°E) for 20 months. During this period, measurements of photosynthetic capacity, photochemical efficiency of PS II, chlorophylls and carotenoids were performed once per season. Supplemental UV-B radiation had no significant effect on these parameters nor on the total, above ground biomass accumulation, plant height and leaf specific mass measured at plant harvest. It was observed, however, that UV-B supplementation increased the number of seeds per fruit as well as mean individual seed mass. As a result, seed number and total seed mass per plant were considerably increased. Germination rates of produced seeds were not affected. We may conclude that C. creticus is a UV-B resistant plant whose competitive ability may be improved by enhanced UV-B radiation through an increase in its reproductive effort and a higher contribution to the seed bank.     

Seasonal Dimorphism in the Mediterranean Cistus incanus L. subsp. incanus

Mediterranean perennial species are described as being sclerophyllous,or summer deciduous, or seasonally dimorphic. Field observationin the coastal maquis of Castelvolturno Nature Reserve, southernItaly, showed thatCistus incanus L. subsp. incanus is a seasonallydimorphic species as it develops brachyblasts with small leavesin summer, and dolichoblasts with large leaves in winter. Fieldbiometric data confirmed that winter shoots were 14-times longerthan those developed in summer and had many more leaves. Thearea of single winter leaves was five-times that of summer leaves.Anatomical leaf structure also changed with the season: winterleaves were flat while summer leaves had a crimped lamina whichwas partially rolled to form crypts in the lower surface. Leaveswere covered by considerably more trichomes in summer than inwinter. Stomata were uniformly distributed along the lower epidermisof winter leaves but were only present in the crypts of summerleaves. In summer leaves, a palisade layer was often found onboth sides of the lamina, the mesophyll cells were generallysmaller and the intercellular spaces were reduced. Winter leaveshad a dorsiventral structure and larger intercellular spaces.Seasonal dimorphism is generally reported to be an adaptationto summer drought. However, the morphology and anatomy of C.incanus L. subsp.incanus showed that the subspecies has notonly developed a strategy to survive summer drought, but hasevolved two different habits, one more xerophytic than the other,to optimize adaptation to the seasonal climatic changes occurringin Mediterranean environments. Copyright 2001 Annals of BotanyCompany Cistus, Cistus incanus L. subsp. incanus, climatic changes, leaf anatomy, leaf dimorphism, Mediterranean shrubs, phenology, seasonal dimorphism     

Chloroplast and nuclear evidence for multiple origins of polyploids and diploids of Hedera (Araliaceae) in the Mediterranean basin

Chloroplast (trnT-L) and nuclear rDNA (ITS) sequence analyses of the Araliaceae provide strong molecular evidence for the monophyly of the genus Hedera. Phylogenetic reconstructions suggest multiple origins and an active polyploidization process not only in the formation of tetraploids (2n = 96), hexaploids (2n = 144), and octoploids (2n = 192), but also of diploids (2n = 48). A high basic chromosome number of x = 24, extensive polyphyly in widespread diploids, and terminal placement of Hedera in phylogenies of the Araliaceae reveal that extant diploid taxa may be, in fact, assemblages of ancestral polyploids from plants of n = 12. Four major lineages containing four types of chloroplast (chlorotypes I, II, III, and IV), which are defined by different trnT-L nucleotide substitutions and two large insertions (50- and 30-bp), provide evidence for evolutionary processes and historical biogeography in Hedera. We propose a scenario where an initial colonization in the Mediterranean basin by Asian ancestors (carrying the ancestral Araliaceae chlorotype I) is followed by differentiation into the four chlorotypes of the Mediterranean region, and then recolonization of Asia and northern Europe only by chlorotype III. The Macaronesian taxa (Hedera azorica, Hedera maderensis ssp. maderensis, and Hedera canariensis) appear to have originated from a single-colonization event to each archipelago with no further contact either with continental or insular species.     

Earlier summer drought affects leaf functioning of the Mediterranean species Cistus monspeliensis L.

The Mediterranean vegetation is characterized by a high diversity of growth forms, habits and phenology that enable it to endure under harsh environmental conditions. It is however unclear whether these adaptations may allow plant survival under more extreme conditions, as predicted by climatic models under the perspective of climate change. A manipulative experiment aiming at anticipating summer aridity has been run to analyse the effects of the experimental drought on spring-leaf functioning and characteristics of the leaf-dimorphic Mediterranean shrub Cistus monspeliensis L.Assimilation rates were reduced under anticipated summer aridity due to a decrease of stomatal conductance, but only before morphological adaptations to drought (an increase of leaf mass per area) occurred. These adaptations were anticipated under experimental dry conditions, and causes photosynthetic performances to recover compared to previous dates. When natural summer aridity occurred, the leaf mass per area also changed in the control. However, this causes no recovery of the photosynthetic performances, because of the decrease of stomatal conductance due to low soil water content and leaf water potential values. Moreover, under experimental drought, leaf shedding was anticipated to reduce water losses, causing an overall reduction of leaf lifespan.     

The use of a tannin crude extract from Cistus ladanifer L. to protect soya-bean protein from degradation in the rumen

Cistus ladanifer L. (CL) is a perennial shrub abundant in dry woods and dry land of Mediterranean zone, with high level of tannins. Tannins bind to protein, preventing its degradation in the digestive compartments. This tannin/protein complex may be advantageous when partially protecting good-quality feed protein from excessive rumen protein degradation. The objective of this trial was to use a CL phenol crude extract to prevent excessive rumen degradation of soya-bean meal protein. The phenolic compounds were extracted using an acetone/water solution (70:30, v/v). Soya-bean meal was then treated with this crude CL extract, containing 640 g of total phenols (TP) per kg of dry matter (DM), in order to obtain mixtures with 0, 12.5, 25, 50, 100 and 150 g of TP per kg DM. Three rumen-cannulated rams were used to assess in sacco rumen degradability of DM and nitrogen (N). The three-step in vitro procedure was used to determine intestinal digestibility. Increasing extract concentrations quadratically decreased the N-soluble fraction a (R² = 0.96, P = 0.0001) and increased the non-soluble degradable fraction b (R² = 0.92, P = 0.005). The rate of degradation c linearly decreased with CL extract doses (R² = 0.44, P = 0.0065). For the effective rumen degradability of N, a linear reduction (R² = 0.94, P < 0.0001) was observed. The in vitro intestinal digestibility of protein (ivID) quadratically decreased (R² = 0.99, P < 0.0001) with TP inclusion and the rumen undegradable protein (RUP) showed a quadratic increase (R² = 0.94, P = 0.0417). Total intestinal protein availability, computed from the RUP and ivID, linearly decreased with TP inclusion level (R² = 0.45, P = 0.0033).     

Comparison of the regeneration after burning,cutting and ploughing in a Cistus ladanifer shrubland

The recovery of a Cistus shrubland after experimental treatments (cutting, burning and ploughing) was studied to determine qualitative and quantitative differences in the secondary succession. The study was carried out in three experimental plots of 100 m² each, in which five 1 m² sampling units were fixed and surveyed for five years, estimating cover percentage of each plant species. The data were analysed by means of affinity indexes (Sorensen and Steinhaus Indexes). The results indicated that the cover of the woody species increased progressively, but the degree of recovery depended on the treatment, as well as on the time afterwards. The number of these species remained almost constant throughout the whole period. The herbaceous species diversity was higher in the two first years in all treatments. When treatments were compared, the greatest diversity occurred after cutting. The regeneration of the community occurred by autosuccession in which the diversity decreased when the dominance of Cistus increased, unlike the general trend of the classical theories. The recovery was very quick after all three treatments, which can be interpreted as a great resilience of this ecosystem.     

Chloroplast sequences reveal a diversity gradient in the Mediterranean Ruppia cirrhosa species complex

Two subcosmopolitan species Ruppia maritima and Ruppia cirrhosa are recognized throughout Europe, whereas Ruppia drepanensis is endemic to SW Europe. We aimed at characterizing the geographic structure of the chloroplast DNA haplotype diversity of 56 Ruppia populations across the European part of the Mediterranean. On the basis of five cpDNA markers (total length of 2300 bp) 16 haplotypes were obtained for 1546 individuals. Three populations of R. maritima showed a single haplotype and differed in five insertions/deletions and 16 substitutions from a highly variable R. cirrhosa species complex, which included R. drepanensis. The haplotype diversity of this species complex was much higher in the western Mediterranean basin than in the eastern basin. Analysis of molecular variance (AMOVA) showed significant differentiation of R. cirrhosa between the two basins and also within the western Mediterranean thereby suggesting the latter as an important centre of Ruppia diversity. An isolation-by-distance (IBD) pattern was stronger between the West-East basin populations than within basins. A PCO analysis of the western basin populations indicated a diversity gradient with those of Sardinia as polymorph intermediates. The low diversity within the eastern basin suggests that the observed gradient could be hypothesized as a historical dispersal of only a limited number of haplotypes from west to east.