Turkish Journal of Botany Turk J Bot (2016) 40: 1-16 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1409-7 Biogeographic divergences in the Iberian flora. A morpho-anatomic, ISSR-based, and environmental study of Iberian Buxus sempervirens L. 1 2 1,3 1,3 4 5 Márcia CARVALHO , João ROCHA , Valdemar CARNIDE , Sandra MARTINS , Maurici MUS , Francisco AMICH , 2 6 6 6 6, Rubim ALMEIDA , Cláudia MACHADO , Berta GONÇALVES , Eunice BACELAR , António L. CRESPÍ * 1 Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology (IBB/CGB-UTAD), University of Trás-os- Montes e Alto Douro, Vila Real, Portugal 2 Department of Botany, Faculty of Sciences, University of Porto, Porto, Portugal 3 Department of Genetics and Biotechnology, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal 4 Centre d’Estudis de Postgrau, University of Balearic Islands, Palma de Mallorca, Spain 5 Evolution, Taxonomy and Conservation Group (ECOMED), Department of Botany, University of Salamanca, Salamanca, Spain 6 Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, Portugal Received: 07.09.2014 Accepted/Published Online: 15.04.2015 Final Version: 01.01.2016 Abstract: Iberian Buxus spp. are represented by B. sempervirens, restricted to the Cantabrian, eastern Iberian Peninsula, and northeastern mountain systems, emerging scarcely in Portugal, and by B. balearica that occurs in the Betic deep river valleys and in the Balearic islands. The genetic affinity existent between fifteen populations ofB. sempervirens was evaluated by seven ISSRs primers. Moreover, leaf morpho- anatomical measurements indicative of leaf performance (such as leaf area, leaf mass per unit area, width, length, shape factor, tissues thickness, stomatal density, and quantification of epi- and intracuticular waxes) and environmental characterization were conducted to get insight into the functional ecology of the genus Buxus. Six populations of B. balearica and 15 of B. sempervirens, collected in the Iberian Peninsula and the Balearic islands, were included to establish the interspecific ranges. A functional ecological description by morpho-anatomical analysis of leaves and a genetic and environmental approach exposed differences between Pyrenaean–Cantabrian– Portuguese and Iberian B. sempervirens populations. Genetic relationships among Buxus populations were investigated using inter- simple sequence repeat (ISSR) markers. Seven ISSR primers generated a total of 159 unambiguous and repeatable bands, of which 156 (98.1%) were polymorphic. These divergences for Iberian B. sempervirens are explained by the isolation in their distributions since the Oligocene. Previsions for future climate change scenarios confirm those biogeographic divergences for the Iberian B. sempervirens. Key words: B. sempervirens, genetic flows, molecular marker, leaf anatomy, climatic characterization, Oligocene–Holocene evolution 1. Introduction the glacial/interglacial genetic flow between the Iberian Buxus L. is represented in the western Mediterranean Peninsula and the Western Mediterranean areas of several basin by evergreen shrubs, usually no more than 5 m tall plant species have stated the important concentration of (Benedí, 1997; Lázaro and Traveset, 2006; Lázaro et al., floristic diversity in the Iberian Peninsula, as a result of 2006; Roselló et al., 2007; Di Domenico et al., 2011). Both climatic change’s impact on the chorology of unrelated species are monoecious; the inflorescences contain one floristic groups, and revealed biogeographic similarities female flower surrounded by male flowers (usually four), of several taxa (Almeida da Silva et al., 2014). In this or in some cases individuals with just male flowers. They context, a better understanding of the natural migration are self-compatible and ambophilous (pollinization by flows from Eastern Europe and the Middle East insects or wind), and their fruits are dehiscent capsules. penetrating the Iberian Peninsula, and their implications Small black seeds are barochory, which explains the on diversification in situ highlighted by the genus Buxus concentration of individuals and the access to abiotic are of remarkable interest. The occurrence of this genus means for wider disseminations (Köhler, 2007). This is a in the western Mediterranean basin involves two species, genus of exogenous origin that reached Western Europe B. sempervirens L. and B. balearica Lam. (Benedí, 1997). by glacial dynamics (Di Domenico et al., 2011). Studies of Some authors refer to overlapping for both species in * Correspondence: [email protected] 1 CARVALHO et al. / Turk J Bot northwestern Africa (Tunisia, Algeria, and Morocco) Buxus found in the Iberian Peninsula and Balearic islands (Leporatti and Ghedira, 2009) and Turkey (Davis, 1988), to evaluate their taxonomic differentiation and phylogeny. but no more common occurrences have been reported. B. The morpho-genetic differences will be decisive to discuss sempervirens is observed along the north, northwestern, the recent evolution of both species and their probable and eastern Iberian Peninsula, while B. balearica is biogeographic dynamic during the Late Quaternary restricted to Sardinia, Mallorca, the southeastern Iberian period (Di Domenico et al., 2012), i.e. since the Peninsula, northern Morocco, and southern Turkey. fragmentation and separation of their occurrence areas. This biogeographical characteristic suggests a tertiary Several examples with other genera and species are also divergence between these two species, with current applied to explain recent morpho-genetic differentiations geographic overlap only in the Iberian Peninsula and (Mazur et al., 2010; Fuertes Aguilar et al., 2011). On the Turkey (Pignatti, 1978). other hand, biogeographic dynamics data can help in It is accepted that coadaptative evolutionary changes in policy decisions for conservation of the taxa, in particular the physiology or morphology of any genus have occurred in the incorporation of B. sempervirens into the red list of on a local or regional scale basis, and that consequently threatened species. populations within a single species may diverge in their characteristics (Ferea et al., 1999). Nevertheless, as 2. Materials and methods responses to changes in environmental conditions occur 2.1. Study area and data collection at different time scales, several degrees of acclimation may The distribution ofBuxus in the Iberian Peninsula and result. Leaf gas exchanges rapidly respond to changes in Balearic islands (Figure 1) was determined based on light intensity, temperature, vapor pressure deficit, and herbaria information. The occurrence of B. sempervirens leaf water potential, but it can also acclimate to distinct is concentrated along the northeastern mountains and the environmental conditions more slowly, as a result of long- Cantabrian mountains systems, the Iberian mountains term changes in its mass per area (leaf mass per area (LMA)) systems, and there is a dispersed population on the western and leaf chemistry (Brooks et al., 1996). According to Letts Iberian Peninsula (Portugal and Occident of the Central et al. (2011), B. sempervirens exhibits high compensatory mountains system). B. balearica just occurs in the west of physiological and morphological acclimation to light Mallorca (Balearic islands), and occasionally in the eastern intensity. Therefore, it is also possible anatomically that mountains and on the Dragonera islet (palynological leaf modifications resulted from acclimation to water evidence also indicates the presence of this taxon on the stressed habitats for this species, such as increased island of Minorca (Yll et al., 1997)), and the southern mesophyll thickness and density as adaptive responses to Betic mountain system (provinces of Málaga and Almeria, stressful conditions (Poorter et al., 2009). Thus the search Iberian Peninsula), despite the Algerian and Moroccan for leaf anatomical traits indicative of leaf performance populations (Lázaro et al., 2006; Navarro-Cerrillo et al., seems crucial to get insight into the functional ecology of 2011). Twenty-one populations of the two Buxus species the genus Buxus. were selected for the present study. These included DNA-based markers are suitable tools for a reliable 15 populations of B. sempervirens from four different evaluation, characterization, and taxonomical analysis of regions (four populations from deep valleys of northern plants. They provide a good classification of plant species Portuguese rivers, six populations from the eastern Iberian because the genetic material may be directly compared Peninsula, three populations from the northeast, and two independently of environmental influences (Kumar et al., populations from the Cantabrian mountain system) and 2009). six populations of B. balerica (three populations from The inter-simple sequence repeats (ISSRs) are deep river valleys of the Betic mountain system and three considered useful markers in different areas, such as populations from the Balearic mountain system) (Table phylogenetics, genetic identity and diversity, gene tagging, 1; Figure 1). For every population five individuals were genome mapping, and evolutionary biology (Zietkiewicz collected. These individuals should be distant from each et al., 1994; Kumar et al., 2009; Sutkowska et al., 2014). other to guarantee a lack of recent kinship and, at the ISSRs are known to