Taxonomic differences in the needle epidermis characteristics of Pinus sylvestris L. and Pinus uncinata Ramond ex DC. from two Iberian populations were sought; such information could help identify these species when pollen analysis and the inspection of wood anatomy fails. The features of the cuticle are commonly well preserved in the fossil record. Although the epidermal patterns of the examined taxa were similar, qualitative differences were seen in the subsidiary and guard cells. P. sylvestris showed small subsidiary cells homogenously arranged around the opening of the epistomatal chamber, while P. uncinata showed small, lateral subsidiary cells and non-differentiated subsidiary cells in the polar position. The aperture of the epistomatic chamber of P. uncinata was also larger in diameter (15.1 ± 1.8 µm P. sylvestris ; 21.1 ± 2.8 µm P. uncinata ). Principal components analysis and discriminant analysis was performed on the features of the guard cells characterising the size and shape of the cuticular thickenings — all the variables analysed can be measured in disperse stomata in microscope preparations for pollen analysis. Significant differences were found in the upper woody lamellae width and the coefficient associated with the shape of the medial lamellae borders (discriminant analysis weighting 0.739 and 0.826 respectively). Other significant parameters included the coefficient associated with the relative size of the medial lamellae border width of the guard cells with respect to the distance between the external limits of the medial lamellae borders, and the length of the upper woody lamella. Different light regimens appeared not to significantly affect the variability of the studied features. Keywords plant taxonomy plant variation Iberian Peninsula epidermal characteristics needles Pinus 1 Introduction The cuticle preserves epidermal features of the leaf that are potentially important from a taxonomic point of view ( Florin, 1939; Stace, 1965; Theobald et al., 1979; Kerp, 1990 ). The analysis of the shape and arrangement of the epidermal cells and other foliar structures such as trichomes and papillae has provided new evidence for phylogenetic and taxonomic discussion ( Alvin et al., 1980; Barrón and Buades, 2002 ). Cuticle analysis has been used to revise the phylogeny of the genus Pinus L. and to classify it into sections, subsections and subgenera ( Yoshie and Sakai, 1985; Kim et al., 1999; Whang et al., 2001, 2004 ). Owing to the cuticle's strong resistance to degradation, its structures are frequently well conserved in samples from palaeobotanical sites ( Florin, 1939; Stace, 1965; Boulter, 1971; Theobald et al., 1979; Kerp, 1990 ). This, has led to substantial interest in its study. The analysis of cuticles is of particular value in the case of Pinus , as pollen commonly does not allow identification at the species level in this genus ( Huntley and Birks, 1983 ). In addition, wood anatomy often cannot be used to distinguish between Pinus sylvestris L. and Pinus uncinata Ramond ex DC. ( Schweingruber, 1990 ). The importance of the genus Pinus in the forest dynamics of Iberia's vegetation history is relatively well known, but our knowledge of the actual species involved remains poor. Taxonomic precision with respect to fossil Pinus material, however, is of considerable palaeophytogeographic interest ( Bennett and Parducci, 2006 ). The identification of species in the fossil record via the analysis of needle fragment cuticles and isolated stomata may help reveal forest history and the dynamics of species distribution in more detail. Peat sediments used in palynological investigations commonly harbour the remains of cuticles belonging to trees of the genus Pinus ( Del Río Merino, 2000; Rubiales et al., 2007 ). Analysis of these remains allows the identification of subsidiary cells of the stomatal complex, and reveals the structure and dimensions of the aperture of the epistomatic chamber (pore). Moreover, isolated stomata are frequently preserved in palynological preparations ( Aubert et al., 2004; Franco Múgica et al., 2001 ). Fossil remains of both types have been found at palaeobotanical sites in the mountains of southwestern Europe ( Del Río Merino, 2000; Aubert et al., 2004 ). In this area, P. sylvestris and/or P. uncinata were abundant throughout the Quaternary. Being able to identify these species from their stoma and/or cuticle characteristics would improve the interpretation of palynological and palaeobotanical datasets. Any study on the morphological characteristics of widespread species, such as P. sylvestris ( Gaussen et al., 1964; Farjon, 1984 ), will necessarily run into difficulties when it tries to be complete and rigorous. However, if the information provided by local or regional studies is integrated with that of studies from other areas of Europe that used the same methodology (e.g., those of Stružková, 2002; Sweeney, 2003 ), conclusions can be reached that are applicable to extensive territories. The selection of P. sylvestris and P. uncinata for the present study is the consequence of their being the only two Pinus species compatible with the environmental conditions prevailing in the high mountain areas of the Iberian Peninsula after the Wurmian glacial maximum ( Costa et al., 1997 ). Palaeoremains that can be analysed are found in these areas ( Rubiales et al., 2007; Turner and Hannon, 1988 ). The aim of the present investigation was to examine the diagnostic value of the epidermal features of the needles of contemporary P. sylvestris and P. uncinata , such as the stomatal complex, the subsidiary cells, the epistomatic chamber and the cuticular thickening of the guard cells, and to assess the possibility of using these to distinguish the species represented in fossil materials. 2 Materials and methods 2.1 Material and pilot study The studied material came from two natural...

Palaeoecological evidence indicates that highland pines were dominant in extensive areas of the mountains of Central and Northern Iberia during the first half of the Holocene. However, following several millennia of anthropogenic pressure, their natural ranges are now severely reduced. Although pines have been frequently viewed as first-stage successional species responding positively to human disturbance, some recent palaeobotanical work has proposed fire disturbance and human deforestation as the main drivers of this vegetation turnover. To assess the strength of the evidence for this hypothesis and to identify other possible explanations for this scenario, we review the available information on past vegetation change in the mountains of northern inland Iberia. We have chosen data from several sites that offer good chronological control, including palynological records with microscopic charcoal data and sites with plant macro- and megafossil occurrence. We conclude that although the available long-term data are still fragmentary and that new methods are needed for a better understanding of the ecological history of Iberia, fire events and human activities (probably modulated by climate) have triggered the pine demise at different locations and different temporal scales. In addition, all palaeoxylological, palynological and charcoal results obtained so far are fully compatible with a rapid human-induced ecological change that could have caused a range contraction of highland pines in western Iberia.

This study reports the value of leaf cuticle characteristics in the identification and classification of Iberian Mediterranean species of the genus Pinus (P. nigra subsp. salzmannii, P. pinaster, P. pinea and P. halepensis), with the aim of using these characters to identify isolated cuticles and stomata in palynology slides. Preparations were made of the cuticles of pine needles belonging to one natural Iberian population of each of the above species. A number of epidermal morphological characteristics were then recorded with the aim of distinguishing these species from one another. The structure of the stomatal complex (the shape and arrangement of the subsidiary cells) was different in each species. The aperture of the epistomatal chamber was significantly smaller in P. pinea than in the other species examined, and the variables recorded for the thickening of the guard cells provided relationships that clearly distinguished all four taxa. The width and length of the stomata and the upper woody lamellae, the central distance between the external limits of the medial lamellae borders and the length of the stem were the most useful variables in this respect. The present results contribute to the ongoing discussion regarding the taxonomic classification of the members of Pinus, and provide valuable clues for the identification of Iberian Mediterranean pine species from small pine needle fragments or isolated stomata. After validation of the present results for multiple populations, these results could also be used to help identify fossil leaf macroremains and the scattered/ isolated stomata commonly observed in palaeopalynological samples