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Using floristics, modern systematics and phylogenetics for disentangling biodiversity hotspots across scales: a Mediterranean case study

V. I. Simón-Porcar, M. Escudero, L. Navarro, A. de Castro, J. Lorite, R. Molina- Venegas & J. Arroyo

To cite this article: V. I. Simón-Porcar, M. Escudero, L. Navarro, A. de Castro, J. Lorite, R. Molina-Venegas & J. Arroyo (2018): Using floristics, modern systematics and phylogenetics for disentangling biodiversity hotspots across scales: a Mediterranean case study, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, DOI: 10.1080/11263504.2018.1445131 To link to this article: https://doi.org/10.1080/11263504.2018.1445131

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Using floristics, modern systematics and phylogenetics for disentangling biodiversity hotspots across scales: a Mediterranean case study

V. I. Simón-Porcara, M. Escuderoa, L. Navarrob, A. de Castroa, J. Loritec, R. Molina-Venegasa,d and J. Arroyoa

aDepartment Plant Biology and Ecology, School of Biology, University of Seville, Seville, Spain; bCenter for Technological Research and Innovation, University of Seville (CITIUSII), Seville, Spain; cFaculty of Science, Department of Botany, University of Granada, Granada, Spain; dInstitute of Plant Sciences, University of Bern, Bern, Switzerland

ABSTRACT ARTICLE HISTORY Biodiversity comprises various levels of biological organisation. Global patterns of biodiversity are well Received 8 February 2018 established based on species occurrence. However, functional and historical processes underlying Accepted 20 February 2018 biodiversity patterns have been only recently approached. The increasingly active field of phylogenetics KEYWORDS has allowed the rise of phylofloristic studies to help in elucidating the historical, evolutionary causes of Alpha diversity; Baetic– plant biodiversity in addition to most commonly approached ecological correlates. Here, we review recent Rifan range; barcoding; phylogeny-based studies disentangling the role of eco-geographic and historical factors on the biodiversity beta diversity; elevation; patterns of the Baetic–Rifan range and exemplify the use of barcoding to explore the evolutionary endemism; Sierra Nevada; assembly of woody flora in the Spanish Sierra Nevada mountain range super-hotspot. Our species-level substrate DNA barcoding approach resulted in a phylogeny potentially improving refinement of hypotheses in comparison with previous approaches based on phylogenies at genus level, and providing insights into the woody flora of Sierra Nevada subjected to systematic discussion. We show that the roles of elevation and substrate in biodiversity distribution vary according to the biodiversity component considered and also between endemic and non-endemic flora. We aim to illustrate how modern methods can further our understanding of the mechanisms underlying the assemblage of species and, consequently, the conservation of biodiversity.

Introduction Even long before the term was coined, biodiversity account had a spatially explicit nature. This has produced a vast litera- Biodiversity is a multidimensional concept, which encom- ture describing the patterns of distribution of the variety of passes the classical hierarchical view of biological organisation life on Earth and its environmental correlates, with a particular levels, from genes to ecosystems, but it also has a horizontal focus on biodiversity hotspots (Cowling et al. 1996; Myers et al. dimension, i.e. interactions between elements within those 2000; Gaston and Spicer 2009). Although this prospect has been levels, from gene interactions to species interactions and ulti- strongly dependent on how completely biotas are known, the mately to biome interdependence (Gaston 2000). This makes patterns of global spatial variation of diversity are long estab- biodiversity difficult to measure when trying to capture under- lished (i.e. diversity gradients: Gaston 2000), including correla- lying processes and mechanisms at different scales (Gaston tions between the abundances of different kinds of organisms, to 1996; Lawton 1999; but see also Hubbell 2001). Processes use some of them as proxies for total biodiversity (Caro 2010). As generating biodiversity are best accounted for at small bio- a consequence, we have a reasonable knowledge on the number logical scales (species and populations), where most interac- and distribution of biodiversity hotspots and coldspots (Gaston tions, which are a critical part of biodiversity build-up, occur et al. 1995). Patterns of biodiversity started to be determined in (Valiente-Banuet et al. 2015). These processes are difficult to a merely descriptive manner (the question usually being “how disentangle at the community level, where biotic interactions many species, or endemic species, are there?”) or through cor- and historical constraints intermingle. Nevertheless, all these relative inquiry (“what are the environmental correlates of bio- levels play a critical role in biodiversity and are a main target logical diversity?”), which was a starting point to explore causal for conservation. The mechanisms generating biodiversity drivers. However, functional and historical processes underlying can be inferred by studying communities in a landscape and biodiversity patterns have been only recently approached com- in larger spatial species assemblages, such as biotas where plementarily. Studies across communities, including ecological environmental and historical gradients are evident (Cavender- interactions (e.g. Trøjelsgaard et al. 2015), functional traits (Díaz Bares et al. 2009). et al. 2016) and historical setting of scenarios across large regions

CONTACT J. Arroyo [email protected] supplemental data for this article can be accessed https://doi.org/10.1080/11263504.2018.1445131. © 2018 Società Botanica Italiana

Published online 07 Mar 2018 2 V. I. SIMÓN-PORCAR ET AL.

(Anacker and Harrison 2012; Wulff et al.2013 ) have been particu- deeply determined by the Strait of Gibraltar, which divides the larly informative. hotspot into two landmasses belonging to two main tectonic Of particular interest to unravel the evolutionary grounds plates, Eurasian and African. The geomorphological and historical of hotspots is the widespread use of phylogenies for particular setting is well known and thus, for example, we know that the lineages and for species assemblages in territories. If a biodiver- Strait was open and closed intermittently across the geological sity account should include evolutionary relationships, then it periods, being open for the last time at the end of the Messinian is necessary that sound phylogenetic relationships are recov- period in the Miocene c. 4.5 Ma (Duggen et al. 2003). Thereafter, ered and used to determine the importance of taxa and, more the most relevant geomorphological changes concerned sea level critically, processes in specific territories. There are a number of oscillations due to Ice Ages (Lambeck and Chappell 2001), which potential problematic situations that phylogenetic information made the Strait somewhat narrower (Collina-Girard 2001). may help to elucidate. They include proper taxon delimitation, avoiding over-splitting of sister taxa, which add comparatively Phylofloristics of the Baetic–Rifan hotspot little diversity, leading to biodiversity inflation particularly in ter- ritories rich in neo-endemic lineages. Indeed, the identification The increasingly active field of phylogenetics is making of appropriate independent monophyletic evolutionary units vast amounts of phylogenies and DNA sequences available. helps explain the evolution of phenotypes, such as independent Moreover, novel analytical tools enable their use for elaborating reproductive lineages and pollination niches in N. papyraceus-N. mega-phylogenies for species assemblages at different scales, tazetta (Pérez-Barrales et al. 2014) in the Mediterranean Basin. from local communities to wide-ranging biodiversity hotspots. Similarly, phylogeographical and genetic analyses may uncover This approach can provide strong insights into the historical pro- hidden patterns blind to former purely morphological analy- cesses that made up the emergence of a given hotspot. Swenson ses, as happened to some Mediterranean Tertiary relicts (e.g. and Umaña (2014) coined the term “phylofloristics”, which helps Frangula alnus: Hampe et al. 2003; Laurus nobilis: Rodríguez- in elucidating the historical evolutionary causes of biodiversity Sánchez et al. 2009). in addition to the most commonly studied ecological correlates. In addition to its intrinsic systematic value, studies on specific In this sense, coordinated efforts to use sound phylogenetic lineages have been a necessary starting point to account for evo- information on hotspots will help in optimal resource manage- lutionary processes in a territory. Although these phylogenies ment for conservation of biodiversity singularities in hotspots may well exemplify processes if groups are representative of the and sub-hotspots, and ultimately to predict responses of line- hotspots, their usual lack of a particular geographic frame and the ages to future global changes. limited availability for numerous groups in a given region restricts The Baetic–Rifan hotspot in the Western Mediterranean has their applicability for biodiversity account. Similar to the ecologi- several characteristics that make it suitable for such an approach. cal theory on diversity measurements, including alpha, beta and As mentioned above, there is a sound previous floristic work and, gamma diversities (Magurran 1988), phylogenetic studies at com- in addition, many of the hotspot species have been represented munity level should also be considered, since the evolutionary in recent phylogenetic studies of either Iberian or more gener- history of lineages within a hotspot is critical to establish its bio- ally Holarctic plant groups. As a matter of fact, it was possible to diversity level (May 1990; Forest et al. 2007). In practical terms, the recover a genus-level and time-calibrated mega-phylogeny of evaluation of phylogenetic diversity is increasingly been claimed the vascular flora of Andalusia and northern Morocco, where the within the criteria for nature conservation (Sechrest et al. 2002; Baetic–Rifan hotspot is located. This phylogeny included 898 gen- Winter et al. 2013; Pollock et al. 2015). era (99% of all existing genera) based on available DNA sequences Amongst the world hotspots, the Mediterranean Basin stands extracted from GenBank (see Molina-Venegas and Roquet 2014 out because of its extension and pronounced spatial heteroge- for further details on the phylogenetic procedure). The number neity (Thompson 2005), highly diverse flora (about 10% of the of sequences available in these repositories was far too low to world flora in 1.6% of land surface (Médail and Quézel 1997) and resolve a tree at the species-level. Thus, species where inserted endemism level (about 50% of the species; Médail and Quézel either using a Yule-Harding branching process with constant birth 1997). Some in-depth analyses of floristics, species range (ende- rates (e.g. Molina-Venegas and Roquet 2014) or as soft polytomies mism) and historical account have allowed the identification of in corresponding genera (e.g. Molina-Venegas, Aparicio, Slingsby, a number of sub-hotspots (Médail and Quézel 1997, 1999) and et al. 2015). This has been critical for having a complete phyloge- refuge spots in the Mediterranean area (Médail and Diadema netic hypothesis for the entire flora of this hotspot. Given the 2009). Here, we will focus on the woody flora of one of the hottest available information on the distribution of species in ecoregions, subspots: the Baetic-Rifan mountain range (Médail and Quezel elevation ranges, and substrates, specific hypotheses could be 1999), whose comparatively well-known floristics and biogeog- developed concerning the structuring of biodiversity across spa- raphy permits exploring processes and mechanisms underlying tial scales and ecological gradients, from local assemblages to the biodiversity and how it is currently structured. The flora of the entire flora, including historical information (Molina-Venegas et al. Baetic-Rifan mountain range is well known due to the work of 2013, 2016, 2017; Molina-Venegas, Aparicio, Slingsby, et al. 2015; coordinated modern botanists using similar criteria, who made Molina-Venegas, Aparicio, Lavergne, et al. 2015). available three standard floras of non-overlapping territories A first analysis of phylogenetic alpha- and beta-diversity (PAD (Valdés et al. 1987, 2002; Blanca et al. 2009), which embrace and PBD, respectively) across ecoregions of the entire hotspot most of the floristic diversity of the hostspot. The Baetic–Rifan revealed that highest PAD, and hence high phylogenetic over-dis- hotspot has a remarkable geomorphological, substrate and cli- persion, tends to occur at the wettest ecoregions at the western- mate heterogeneity, as well as a complex biogeographical history most side of the hotspot (close to Atlantic Ocean), whereas high PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 3 phylogenetic clustering, i.e. lower PAD, is found on the eastern, a lower dispersal efficiency. Local diversification, and thus ende- drier regions. Temperature apparently did not show any signifi- mism, was higher in those families with lower migration rate cant relationship (Figure 1(a); Molina-Venegas, Aparicio, Lavergne, across the Strait (Lavergne et al. 2013). It is unknown if these et al. 2015). This probably reflects some niche conservatism of mechanisms are also responsible for the differentiation between older lineages typical of more humid, tropical climates prior to other regions across the two landmasses. The larger scale of the the onset of Mediterranean climates, which have a relict presence whole Baetic–Rifan hotspot might probably involve many more close to the shores of the Strait of Gibraltar at the western end possible concomitant causes than migration interruption and of the region. Similarly, phylogenetic clustering linked to drier differentiation, and probably would require a lineage-by-lineage climates is probably associated to in situ diversification of some analysis. lineages under more arid, Mediterranean conditions typical of the eastern end of the region. In fact a higher level of palaeoen- The role of elevation and substrates demism (Relative Phylogenetic Endemism, RPE, as determined by branch lengths of narrow range species) in western ecoregions Elevation and substrate are regarded as isolation mechanisms and of neo-endemism in eastern regions is completely consist- with a putative role on determining the floristic and phylogenetic ent with this pattern (Molina-Venegas et al. 2017). These results diversity in Mediterranean areas and particularly in the Baetic– should be regarded with some caution, as these correlates are Rifan hotspot (Favarger 1972; Goldblatt 1997; Lobo et al. 2001; with current climate, and the relationship with pre- and eu-Medi- Thompson 2005; Mota et al. 2008; Anacker et al. 2011). While terranean climate since its onset is unknown. Unfortunately, a his- the insufficiency of North African sources of detailed informa- torical reconstruction of past climates from independent source tion in floristics, distribution and phylogenetics has precluded models for critical Last Glacial Maximum or Pliocene periods does gaining more knowledge of these associations, recent work on not have sufficient resolution at the hotspot scale for historical the Baetic range has provided insight into the structuring of correlative purposes. Interestingly, low RPE (that is, high neo-en- plant diversity in relation to elevation gradients and lithology. demism) is associated with elevation, pointing to a recent differ- To explicitly test the role of elevation on plant diversity, Molina- entiation linked to the recent uplift of Baetic–Rifan mountains Venegas, Aparicio, Slingsby, et al. (2015) compared patterns of (about 8 Myr; Braga et al. 2003). taxonomic, phylogenetic and functional diversity of the eudicot flora between three elevation belts (700–1300, 1300–1800 and 1800–2700 m) in eight Andalusian mountain ranges. The authors A split hotspot? compiled a list of 1982 species and subspecies and their pheno- The division of the Baetic–Rifan hotspot into two landmasses typic information in order to classify taxa according to several separated by the Strait of Gibraltar offers the opportunity to functional traits (e.g. life-form, seed dispersal, pollination mode, test its effect on the differentiation of lineages. First approaches leaf type; Herrera 1992). Results showed that phylogenetic and based on taxonomic and floristic (species lists) information functional beta diversity between elevation belts within sier- (Valdés 1991; Arroyo 1997; Rodríguez-Sánchez et al. 2008; ras were higher than between sierras, whereas taxonomic beta Molina-Venegas et al. 2013) suggested unity of the flora from diversity was similar or even lower within sierras than between both sides of the Strait. Subsequent availability of phylogenetic sierras (Figure 1(c); Molina-Venegas, Aparicio, Slingsby, et al. information led to an explicit evolutionary analysis of the unity 2015). However, this study had little opportunity to explore the of this flora through the computation of the PBD, which rep- role of substrates because most of the sierras have a dominant resents the turnover of lineages across territories. The results limestone substrate, and mainly because elevation belts may showed that turnover of terminal branches mostly occur across include locally differentiated substrates, which the floristic data- these landmasses, whereas turnover of deep lineages does set could not discriminate. Moreover, particular substrates are so within landmasses (Figure 1(b); Molina-Venegas, Aparicio, often associated to outcrops and thus to elevation. Lavergne, et al. 2015). This was interpreted as an effect of rel- In order to overcome this limitation, Molina-Venegas et al. atively recent lineage diversification between landmasses after (2016) designed a study based on a zoom-approach at a more the opening of the Strait of Gibraltar (and posterior parallel local scale: the woody plant communities of the Sierra Nevada diversification within landmasses), whereas deeper lineages range, considered as the hottest “jewel of the crown” of the would have diversified in older periods probably due to climatic Baetic–Rifan hotspot (Castro-Parga et al. 1996; Blanca et al. 1998). events equally affecting both landmasses, as mentioned above A full factorial design was set to account simultaneously for ele- for wet climates. vation and substrate effects on structuring phylogenetic and In order to unravel mechanisms for diversification of lineages functional diversity in these communities. Two transects in each across the Strait, Lavergne et al. (2013) explored the effect of combination of substrate (limestone, dolomites and mica-schist, several plant traits related with dispersal and establishment. This which are dominant in the range) and elevation (1300, 1650 and study could ascertain that lineages with propagules dispersed 1950 m a.s.l.) were set. It was unpractical to go up to the top by wind and externally on animals (epizoochory), but not those of the mountain (3482 m a.s.l. of Mulhacén peak, the highest of ingested (endozoochory), tend to occur at both sides of the Strait, the whole hotspot) as this elevation is reached only by the most as a probable result of higher dispersal. It similarly occurred with abundant substrate (mica-schist). Abundance of each woody spe- plants having a short life-cycle, for which establishment should cies as well as functional traits related with the different response be less limited. The apparent lack of effect of endozoochory was to edaphic conditions and response to low temperature, as pro- attributed to its association with a woody life form and longer voked by elevation, were measured. These transects harboured 77 life span, and thus to lower establishment capacity, rather than woody species, and the local community phylogeny was obtained 4 V. I. SIMÓN-PORCAR ET AL.

Figure 1. Results of previous studies on the taxonomic, phylogenetic and functional diversity of flora in the Baetic-Rifan hotspot in western Mediterranean. (a) Alpha phylogenetic diversity of angiosperms in ecoregions of Andalusia and North Africa with a climatic gradient in temperature and precipitation. PD: Phylogenetic Diversity; MPD: Mean Phylogenetic Distance. (b) PBD: Phylogenetic beta diversity (turnover component) between the angiosperm flora of ecoregions ofA ndalusia and North Africa. Red lines connect regions which showed significant turnover in terminal lineages (i.e. lower than expected values of the turnover component of PBD for the given species composition) and blue lines connect regions which showed significant turnover in deep lineages. (c)T axonomic (CBD) and phylogenetic (SESpbd) beta diversity between the eudicot flora of elevation belts (belt 1, 700–1300 m; belt 2, 1300–1800 m; belt 3, 1800–2700 m) of various Baetic sierras of Andalusia. Horizontal dotted lines represent threshold values for SESpbd scores (−1.96 and +1.96, respectively). (d) Phylogenetic and functional alpha and beta diversity patterns in shrub communities along elevation and soil gradients in Sierra Nevada. SLA specific leaf area; C:N leaf carbon:nitrogen ratio. Figures modified with permission from Molina-Venegas, Aparicio, Slingsby, et al. (2015), Molina-Venegas, Aparicio, Lavergne, et al. 2015, Molina-Venegas et al. 2016). PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 5 from those formerly resolved for the whole flora of the hotspot. at the species and subspecies level to explore the taxonomic PAD was positively correlated with elevation whereas functional and phylogenetic patterns of diversity, including the endemic alpha diversity (i.e. plant height and phenology-related traits) element in relation to elevation and substrate, based on a com- showed the opposite pattern. Thus, communities at the highest plete updated list (Lorite 2016). Our DNA barcoding approach elevations tended to show a narrow set of functional traits, which resulted in a totally resolved molecular phylogeny, including ter- are present in distantly related taxa. Meanwhile, a wider array of minal branches, for the 74% of species and 85% of genera present, functional trait values was present in phylogenetically clustered respectively, thereby potentially allowing refinement of hypothe- communities at low elevations. Therefore, functional and phy- ses and increased statistical robustness in comparison with previ- logenetic diversity patterns were opposite across the elevational ously used approaches based on genus-level phylogenies (Kress gradient. Functional beta diversity was also associated with soil et al. 2009; Swenson 2009). Furthermore, the refinement in the composition (Figure 1(d); Molina-Venegas et al. 2016). By contrast, measurement of phylogenetic diversity might help to more accu- phylogenetic beta diversity was mostly indifferent to changes rately assess the conservation value of species assemblages (Faith in elevation and soil composition, this result being incongruent 1992; Crozier 1997; Crozier et al. 2005; Davies et al. 2008). We aim with the one obtained at the sierra scale. This might be explained to illustrate how modern methods can help to further our under- by the different metrics of beta diversity employed and by differ- standing of the mechanisms underlying species assemblage and, ences in the species arrays between the two scales, as the studied consequently, the conservation of biodiversity. communities of Sierra Nevada presented a dominance of small chamaephytes, many of them recently evolved endemics, and a Material and Methods virtual absence of pre-Mediterranean species. Study area and taxon sampling. The Sierra Nevada mountain range is a European and Mediterranean biodiversity super-hot- New uses for new phylogenies: a pilot barcoding spot (a core within a hotspot) located in the SE Iberian Peninsula study of the woody flora of Sierra Nevada hotspot (Figure 2). With a surface of 2100 km2, it has a complex orogra- To date, studies of biodiversity patterns considering the phy- phy and soil composition. The average annual rainfall is highly logenetic relatedness of taxa depend on (i) how many phy- irregular, with values ranging between 350 and 1200 mm per logenies of representative groups are available, (ii) how many year, depending mostly on altitude. The average temperatures sampled species there are, and (iii) how informative are available are below 0 °C during winter with a snow cover that can persist sequences. Phylogenetic studies of systematic interest for par- up to 8 months in the highest areas. Geologically, the study area ticular taxonomical groups are still scarce, albeit steadily increas- is composed of siliceous rocks, mainly mica-schists, surrounded ing, whereas sequence information for the whole flora is still by limestone and, to a lesser extent, dolomites (Jabaloy et al. insufficient, as it depends on information coming from different 2008). Sierra Nevada is an isolated high-mountain range, more regions and taxa. This is why previously the phylofloristic study than 700 km from other comparable mountain ranges, such of the whole Baetic-Rifan hot-spot could only be performed at as the Pyrenees (N Iberian Peninsula) in the north or the High the genus level. Using phylogenetic information at this level and Middle Atlas (N Morocco) in the south. This range marks might fail to detect existing patterns when exploring local or the southernmost limit of the influence of the Quaternary gla- regional diversity patterns at species level (Molina-Venegas and ciations in Europe, when it was covered with glaciers only in Roquet 2014), and may contain inaccuracies when estimating areas above 2500 m a.s.l., while large areas of it remained free of the phylogenetic signal. As such, incompletely resolved trees permanent ice. This turned Sierra Nevada into a refuge for many can inflate phylogenetic signal estimation (Davies et al. 2011), plant species during glacial ages (Blanca et al. 1998) as well as for and non-accurate branch lengths can lead to false patterns in isolated populations that have evolved under particular condi- phylogenetic signal tests (Molina-Venegas and Rodríguez 2017). tions (e.g. soil type or isolated summit areas). As a result of this In addition, local species sequences may depart from those uniqueness, Sierra Nevada harbours 2353 taxa of vascular plants available from other regions. This might be critical for relatively (about one-third of the entire flora of the Iberian Peninsula), and recent processes of diversification, such as those derived from comprises 80 taxa endemic to the mountain range (Lorite 2016). recent uplift of the Baetic Mountains (8 Myr; Braga et al. 2003). The study area includes both National and Natural Parks, which Modern barcoding approaches aim to solve these shortcomings cover about 1726 km2. by extensive species sequencing, and they have the added value An exhaustive list of the native woody flora of the Sierra of providing information when species identification is prob- Nevada Natural Park, including species and subspecies, was lematic due to taxonomic uncertainties or lack of morphological compiled from available catalogues that have been updated characters of taxonomic significance during sampling. To date, progressively (N = 440; Lorite 2016). Species were considered few attempts at barcoding whole floras have been performed, as woody if at least some of the aboveground parts are woody, including some relatively simple temperate floras (Burgess et al. which includes phanerophytes and chamaephytes and excludes 2011; de Vere et al. 2012; Elliott and Davies 2014; Braukmann hemicryptophytes. One individual per listed taxon (N = 326) was et al. 2017; see also Bruni et al. 2012) but also some tropical areas geo-referenced in the field and collected as herbarium speci- (Kress et al. 2009; Huang et al. 2015). men, and a leaf sample was dried with silica gel for subsequent Here, we report a comprehensive approach to the diversity DNA extraction. In some cases, specimens were collected in the patterns of the entire woody flora of the Sierra Nevada National “Hoya de Pedraza” Botanical Garden, which is located in the cen- and Natural Parks (Spain). In contrast with previous work, we tral massif of Sierra Nevada and harbours a good representation approached the barcoding of the whole woody flora in the area of its flora. Woody plants in this Botanical Garden all originated 6 V. I. SIMÓN-PORCAR ET AL.

Figure 2. Location of Sierra Nevada protected area in the western Mediterranean area. Note: Dark and light grey indicate the limits of the National Park and the Natural Park of Sierra Nevada, respectively, which have different levels of protection. from natural populations; coordinates of the original sites are also approach (Sanderson 2003), as implemented in treePL (Smith and available. The subset of taxa sampled was representative of the O’Meara 2012). Four calibration points were used (crown node of whole woody flora of Sierra Nevada with respect to their taxo- seed plants, min = 315 Myr, max = 350 Myr; crown node of angio- nomic classification in families and their occurrence in elevation sperms, min = 140 Myr, max = 165 Myr; crown node of eudicots, belts and substrates (p > 0.4, χ2 test). min = 120 Myr, max = 129 Myr; and crown node of monocots, min = 91 Myr, max = 101 Myr; Bell et al. 2010). The rate smooth- Barcoding ing parameter g was set to 1000 on the basis of cross-validation We sequenced three plastid DNA regions (rbcL, matK and psbA- and the chi-square test in treePL (five smoothing values between trnH spacer) and one nuclear DNA region (ITS) for each sampled 0.1 and 1000 were compared). These phylogenetic analyses were taxon. Details on DNA extraction, primers and PCR conditions performed at the computer cluster located in the Andalusian are available in Supplemental online material 1. Scientific Information Technology Center (CICA, Seville, Spain).

Phylogenetic tree: sequence alignments, phylogenetic Analyses of taxonomic and phylogenetic alpha and beta analyses and times of divergence diversity DNA sequences (rbcL, matK, psbA-trnH and ITS) were aligned First, we calculated the Phylogenetic Diversity (i.e. PD sensu Faith individually across all species and then concatenated in super- 1992) for the whole pool of species in our phylogeny and for the matrices for phylogenetic analyses. The rbcL, matK and ITS DNA subset of Baetic-North African endemic species present. The regions were aligned using Muscle (Edgar 2004). The hyper-var- phylogenetic signal of the endemic character was estimated iable DNA region psbA-trnH was aligned using PRANK aligner with the D-statistic, which equals 1 if the character has evolved (Löytynoja and Goldman 2005) as implemented in SATe (Liu following uncorrelated trajectories (i.e. it is randomly distributed et al. 2012). This is a “divide and conquer” approach: SATe per- in the phylogeny) and 0 if the trait fits a Brownian motion model forms iteratively many cycles of generating sub-alignments and of evolution (i.e. it is clumped; Fritz and Purvis 2010). merging to consensus alignment based on the likelihood score In order to test the influence of elevation and substrate on of an inferred phylogenetic tree which determines the optimal the taxonomic and phylogenetic diversity of the woody flora alignment state. A guide tree (inferred here using rbcL gene) was in Sierra Nevada, we codified the presence or absence of each provided to improve this alignment. species in each of five elevation belts (belt 0, <700 m; belt 1, The four DNA sequences were analysed individually (rbcL, 700–1500 m; belt 2, 1500–1900 m; belt 3, 1900–2800 m; belt 4, matK, psbA-trnH and ITS) and concatenated using RAxML ver. 8 > 2800 m) and three major types of substrate (CA, calcareous; (Satamatakis 2014), under GTRCAT model (partitioned for concat- NC, non-calcareous (mostly mica-schist); SS, special substrates enated matrices). We used rapid bootstrapping as implemented - mostly dolomites- which are thought to be highly influential in RAxML (Stamatakis et al. 2008). 1000 rapid bootstrap searches on floristic composition and vegetation structure, Mota et al. were performed. The rapid bootstrapping algorithm is faster than 2008). This characterisation was based on information available the standard bootstrapping algorithm (one order of magnitude). in the reference regional flora (Blanca et al. 2009) and expert Bootstrap trees are used as starting points for the ML searches knowledge according to the general patterns of occurrence of which reduces the computation time (Stamatakis et al. 2008). The each species. Together with the time-calibrated phylogenetic resulting maximum likelihood tree using the four concatenated tree, it was used to estimate the taxonomic (compositional) and DNA regions was time-calibrated using a penalised likelihood phylogenetic alpha diversities (CAD and PAD) of the species PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 7 assemblages of each elevation belt and substrate type, and the Results beta diversities (CBD and PBD) between elevation belts and Phylogenetic analyses of woody flora of Sierra Nevada between substrate types. To contextualise our results, we cal- We obtained a rbcL matrix of 309 samples and 604 pb, a matK culated the surface area of each elevation belt and substrate type matrix of 291 samples and 1160 bp, a psbA-trnH matrix of 285 in the studied area. To do this, we analysed cartographic data samples and 32019 bp and an ITS matrix of 271 samples and on elevation and substrates acquired from the Environmental 1294 bp. We obtained combined matrices: a rbcL-matK matrix of Information Network of Andalusia (REDIAM 2017) with Quantum 314 samples with only rbcL (5), only matK (23) or both (286) DNA GIS (Quantum GIS Development Team 2014). regions, a plastid matrix of 314 samples (sequences included in CAD was calculated as the number of taxa present in the plant rbcL-matK matrix plus additional 274 psbA-trnH sequences) and assemblages of each belt and substrate. PAD was computed using a nuclear-plastid matrix of 314 samples (sequences included in PD (Phylogenetic Diverstiy; Faith 1992) and the standardised ver- plastid matrix plus additional 271 ITS sequences). sions of PD, MPD (Mean Phylogenetic Distance between each Trees from individual DNA regions were in general congruent sampled taxa and every other tip in the phylogeny) and MNTD amongst them and with current taxonomic treatments (most of (Mean Nearest Taxon Distance: mean distance between each sam- the families and genera were monophyletic). Taxonomic congru- pled taxon and its own most closely related taxon in the phylog- ence was very high for rbcL, matK and ITS trees and lower for eny) indices (Webb et al. 2002). SES.PD, SES.MPD and SES.MNTD psbA-trnH tree (results not shown). The combined DNA regions indicate whether the observed values of PD, MPD and MNTD are displayed higher branch supports for families and genera, espe- higher or lower than expected given a null distribution of values cially when we combined the three plastid DNA regions and the of the indices, and were estimated as: nuclear ITS region (Figure 3). In this tree, main clades (gymno- SES = Xobs − xmean null∕Xsd null sperms, angiosperms, monocots, eudicots, rosids, asterids, etc.), families and most of the genera were monophyletic (Figure 3). where X and X are the mean and standard deviation mean null sd null Whereas the branch supports for clades of containing genera and of a null distribution of X values, respectively. The null distribu- families are in general very high, the branch support for deeper tions of X values were generated by randomising the tip labels clades is low (Figure 3). The tree showed a matching topology with of the phylogeny 999 times. Negative values indicate phyloge- the APG IV (Angiosperm Phylogeny Group 2016) family-level phy- netic clustering and positive values indicate phylogenetic over- logeny, except the position of Aristolochia, which was separated dispersion, with a significance threshold α( = 0.05, two-sided) from other Magnoliidae and located between monocots and eud- at −1.96 and +1.96, respectively. icots. This position is however congruent with recent evidence on CBD was calculated computing the Sørensen index (Sørensen the monophyly of Magnoliidae and eudictots (Zeng et al. 2014). 1948) between each belt and substrate, and PBD was analysed Our phylogenetic tree provided insights into the woody flora using the PhyloSor index (Bryant et al. 2008), which computes of Sierra Nevada subjected to systematic discussion. The sep- the fraction of shared phylogenetic branch length between two aration of a number of species and subspecies questioned at samples. Both indices were used as distance metrics (1 – Sørensen times appeared well supported. This was the case of (i) Juniperus index; 1 – PhyloSor index), to explore the two additive compo- communis subsp. alpina and J. communis subsp. hemisphaerica nents of PBD: nestedness and turnover (Leprieur et al. 2012). (Figure 4(a), (b)); (ii) Genista umbellata subsp. umbellata and While the nestedness component is related to differences in PD G. umbellata subsp. equisetiformis, despite the very slight or grad- between species assemblages, the turnover component is the ual morphological differentiation within both pairs of sister taxa fraction of PBD explained without an increasing in PD between (Blanca et al. 2009); and (iii) Helianthemum apeninun subsp. suf- assemblages (either positive or negative). We calculated the fruticosum and H. apeninun subsp. estevei (Figure 4(c), (d)), oppos- standardised versions of the total PBD and their nestedness and ing the hypothesis that both of them are hybridising soil ecotypes turnover components as explained above. Values above 1.96 and (Castroviejo et al. 2005). The three subspecies within Rhamnus lyci- below −1.96 indicate higher and lower values than expected for oides were also well supported, with and intermediate position of the given taxonomic beta diversity, respectively (see Leprieur R. lycioides subsp. velutina going against the separation of this et al. 2012). taxon as a different species (Rivas-Martínez and Pizarro 2015). The spatial distribution of different floristic elements such Finally, Sempervivum tectorum populations in Sierra Nevada, that as endemic and non-endemic taxa might be influenced differ- were overlooked (e.g. Roselló 1997) and considered as lowland ently by eco-geographical factors (Jiménez-Alfaro et al. 2014). In forms of S. minutum, appeared also well differentiated, supporting order to specifically assess the role of substrate and elevation in their classification as a different species (Blanca et al. 2009). This the endemism patterns, all the analyses described above were classification is relevant for conservation purposes, as S. minutum is repeated for the subset of Baetic-North African endemic spe- an endemic species and S. tectorum has only two small populations cies. Finally, it is known that contrasting lineage-specific phy- in Sierra Nevada which are unique in southern Iberian Peninsula, logenetic patterns may conceal the phylogenetic structure in being evaluated as Endangered at regional level (Cabezudo larger clades (Elliott et al. 2016). Thus, we repeated the analyses et al. 2005). for the largest monophyletic clusters of species: angiosperms On the other hand, the two subspecies within Thymus serpyl- and eudicots. All the analyses were performed in R 2.15.1 (R loides (subsp. serpylloides and subsp. gadorensis) were not sister Development Core Team 2012), using the packages PICANTE taxa in our tree, while the subspecies within Arenaria armerina (Kembel et al. 2010), VEGAN (Oksanen et al. 2012) and BETAPART and A. tetraquetra, appeared intermingled (Figure 4(e), (f)). Finally, (Baselga et al. 2013). 8 V. I. SIMÓN-PORCAR ET AL.

Figure 3. Maximum likelihood phylogenetic tree of the woody flora of Sierra Nevada (Spain) resulted from the analysis of four concatenated DNA regions (rbcL, matK, psbA- trnH and ITS). >50% bootstrap supports are presented above the respective nodes of the tree. Baetic-North African endemic species are indicated in red. according to our results, two taxa might be changed in their status Chamaespartium undulatum (Figure 4(h)) within the polyphyletic regarding the treatment in Flora de Andalucía Oriental (Blanca et genus Genista, according to Pardo et al. (2004). Though the conclu- al. 2009): (i) Nevadensia purpurea (Figure 4(g)) could be included in sions should be confirmed by further analyses on specific clades, the genus Hormathophylla as in Flora Iberica (Küpfer 1993), and (ii) we show here that barcoding techniques have utility for solving PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 9

Figure 3. (Continued) taxonomic problems. We also suggest that they will be valuable Diversity of the woody flora of Sierra Nevada and its when identification of specimens is strongly needed for manage- endemic component ment purposes, especially when many of the woody species in our Total PD for the woody flora of Sierra Nevada was estimated list are considered as endangered (Cabezudo et al. 2005). as 9676.616 Myr and PD for the subset of endemic species was 10 V. I. SIMÓN-PORCAR ET AL.

Figure 3. (Continued)

3880.087 Myr. The endemic character approximated a random Diversity of woody flora in elevation belts distribution across the phylogeny although this was not statis- The surface area of Sierra Nevada mountain was essentially dis- tically significant, i.e. we did not find any phylogenetic signal tributed between 700 and 2800 m a.s.l. Elevation belts 1, 2 and 3

(D = 0.867; PD=1 = 0.032; PD=0 = 0.00). accounted for 28, 33 and 32% of the study area, respectively, PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 11

Figure 3. (Continued) while belts 0 and 4 occupied only 4 and 3% of the area, respec- CAD per unit area was much higher in elevation belt 0 (Ntax/ tively (Supplemental online material 2). The CAD of woody flora Km2 = 2.62) than in the other belts (Ntax/Km2 range 0.26–0.53). was the highest in elevation belt 1 (700–1500 m; Ntax = 260) and These results were repeated for all the monophyletic groups the lowest in elevation belt 4 (>2800 m; Ntax = 27). However, analysed (Figure 5(a); Supplemental online material 2). As 12 V. I. SIMÓN-PORCAR ET AL.

Figure 4 Some woody species of Sierra Nevada subjected to systematic discussion. (a) Juniperus communis subsp. alpina and (b) J. communis subsp. hemisphaerica, and (c) Helianthemum apeninun subsp. suffruticosum and (d) H. apeninun subsp. estevei appeared well separated in our tree. In contrast, (e) Arenaria armerina and (f) Arenaria tetraqueta appeared intermingled. (g) Nevadensia purpurea was included in the genus Hormathophylla, and (h) Chamaespartium undulatum within Genista. expected, PD values were highly correlated with taxonomic suggests that in belt 2 we can find clusters of phylogenetically diversity (R2 > 0.97; p < 0.01); SES.PD values were positive and close species, but these clusters appear randomly distributed nearly significant in belts 0 and 1, indicating greater PD than in the phylogeny (i.e. random and clumped MPD and MNTD expected given the observed CAD, and negative and signifi- values, respectively); this pattern occurs also in upper belts cant in belts 2 and 3, indicating lower PD than expected given although non-significantly. For the subset of endemic taxa, CAD the observed CAD. SES.MNTD values were positive in belts 0–1 was the highest in elevation belt 3 (Ntax = 60) and the lowest in (indicating phylogenetic overdispersion) and negative in belts elevation belt 1 (N = 15). The number of endemic taxa per unit 2–4 (indicating phylogenetic clustering) in each species group, area was the highest in elevation belt 4 (Ntax/Km2 = 0.34), fol- but they were only significant for belt 2. SES.MPD values were in lowed by elevation belt 0 (Ntax/Km2 = 0.23), and the lowest in general close to zero and did not show apparent patterns, indi- elevation belt 1 (Ntax/Km2 = 0.08). The proportion of endemic cating random phylogenetic structure of species assemblages in species increased with elevation (9, 16, 26, 43 and 67% of the each elevation belt. The interpretation of these indices together total number of species in elevation belts 0–4, respectively). PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 13

Figure 5. Taxonomic and phylogenetic alpha and beta diversities (CAD, PAD, CBD and PBD) of the whole woody flora (74% of species and subspecies sampled), and the subsets of angiosperm, eudicot and Baetic-North African endemic taxa, in elevation belts (belt 0,< 700 m; belt 1, 700–1500 m; belt 2, 1500–1900 m; belt 3, 1900–2800 m; belt 4, > 2800 m) and substrate types (CA: calcareous; NC: non-calcareous; SS: special substrates) of Sierra Nevada mountain (Spain). (a) Alpha diversity of woody taxa in each elevation belt and substrate type. Bars indicate CAD values as number of taxa. Asterisks indicate groups where PAD values were significantly negative, indicating phylogenetic clustering of species (SES.PD and SES.MNTD < −1.96 for elevation belts and SES.MPD < −1.96 for substrate types). (b) Beta diversity of woody taxa between pairs of elevation belts and substrate types. Bars indicate taxonomic beta diversity values as the Sørensen dissimilarity index, including their turnover and nestedness components. Asterisks indicate groups where PBD values were significantly positive, indicating greater PBD than expected for the given taxonomic beta diversity. Grey asterisks indicate nearly significant values. Asterisks are located either above the bars for de undecomposed beta phylogenetic diversity or inside the correspondent bar fragment for each component of beta diversity. Bars in each belt or substrate comparison correspond to the different groups analysed, from left to right: A“ ll taxa”, “Angiosperms”, “Eudicots” and “Endemics”.

Alpha phylogenetic diversity measures SES.PD, SES.MNTD, and pairs of belts 0–2, 0–4, 1–2 and 1–3. In the angiosperms, this result SES.MPD for endemic taxa were never significantly different was repeated by adding the pair 1–4 (Figure 5(b); Supplemental from random expectations (Figure 5(a); Supplemental online online material 2). The turnover component was positive and sig- material 2). nificant or nearly significant for pairs 0–2 and 0–4 in all groups, The patterns of taxonomic beta diversity were highly parallel indicating a major replacement of lineages in plant assemblages for all the monophyletic groups analysed and for the subset of between these belts (Figure 5(b); Supplemental online material endemic species. The Sørensen dissimilarity index increased with 2). On the other hand, the nestedness component was positive the difference between the elevation belts in comparison (i.e. it and significant (or nearly significant) in other belt pairs: 0–3, 1–2 was lower between belts 0–1 than between belts 0–3) and was and 1–3 when analysing either the whole phylogeny, the angio- the highest when belt 4 was involved in the comparison (Figure sperms or the eudicots (Figure 5(b); Supplemental online material 5(b); Supplemental online material 2). This pattern was highly par- 2). Taken together, these results suggest that the altitude deter- allel to that found for the turnover component of taxonomic beta mining the most striking differences in PBD in the woody flora of diversity, suggesting that the replacement of species is important Sierra Nevada may be established at 1500 m a.s.l., the boundary alongside the elevation gradient. Only the taxonomic beta diver- of elevation belts 1 and 2. For the subset of endemic taxa, PBD sity between belts 0 and 1 and 3 and 4 were better explained by showed random values and patterns. There was only a nearly the nestedness component than by turnover, indicating that the significant positive value in the comparison of elevation belts 3 species assemblages of belts 0 and 4 are a subset of those in belts and 4, which was totally explained by the nestedness component 1 and 3, respectively (Figure 5(b); Supplemental online material 2). (Figure 5(b); Supplemental online material 2). PBD and their turnover and nestedness components were higher than expected between several pairs of elevation belts Diversity of woody flora in substrate types for the whole woody flora and for the monophyletic groups, but Most of the surface studied area of Sierra Nevada (79%) was never between belts 0 and 1 nor between belts 2, 3 and 4. For the classified as non-calcareous (mica-schists). Calcareous and total of the phylogeny and for the eudicot group, PBD was signif- special substrates accounted for 18 and 3% of the study area, icantly or nearly significantly higher than expected between the respectively (Supplemental online material 2). The woody 14 V. I. SIMÓN-PORCAR ET AL.

flora of Sierra Nevada showed the highest CAD in calcareous phylogenetic metrics for floristic analyses, which have been substrates (Ntax = 242) and the lowest in special substrates sometimes replaced by simpler approaches, such as the taxo- (Ntax = 166), and this result was equal for each of the mono- nomical arrangement of species as a proxy of their evolution- phyletic groups analysed (Figure 5(a); Supplemental online ary distinctness (e.g. Loidi et al. 2015). Phylogenetic diversity material 2). Nevertheless, CAD per unit area was much higher measures have been employed more frequently when analys- in special substrates (Ntax/Km2 = 3.63) than in calcareous (Ntax/ ing Mediterranean-type floras at the community level (Morlon Km2 = 0.77) and non-calcareous substrates (Ntax/Km2 = 0.14). et al. 2011; Anacker and Harrison 2012). To our knowledge, the Similar numbers of endemic species were found in each sub- unique previous example of the use of barcoding for under- strate type (Ntax = 38 for calcareous and non-calcareous sub- standing biodiversity distribution in an entire Mediterranean strates; Ntax = 30 for special substrates) rendering a proportion hotspot was provided by Forest et al. (2007), who barcoded the of endemism of 16, 20 and 18% in each substrate type, respec- entire angiosperm flora of the Cape of South Africa at the genus tively, and showing the same pattern than the complete woody level and claimed the decoupling of taxonomic and phyloge- flora for CAD values per unit area (SS Ntax/Km2 = 0.66; CA Ntax/ netic biodiversity measures and its importance for conserva- Km2 = 0.12; NC Ntax/Km2 = 0.03). PD values were significantly tion planning. These authors estimated an average PD per QDS correlated with alpha taxonomic diversity for the whole woody (quarter-degree square, a surface equivalent to one-third of our flora but not for the endemic element (R2 = 0.99; p < 0.05 for total study area) of 70.8 Myr, a value in accordance with the high the whole woody flora; R2 = 0.88; p = 0.3 for endemics). SES.PD, long-term lineage persistence of South African clades (Sauquet SES.MNTD, and SES.MPD values were random for almost every et al. 2009; Valente and Vargas 2013; Skeels and Cardillo 2017). substrate type and species group including the endemic subset, Unfortunately, our estimates are difficult to compare with lacking apparent trends or patterns. Only SES.MPD values were those of this study due to the different methods employed for significantly negative for eudicots in calcareous substrates, indi- the phylogenetic reconstruction (i.e. calibration points). Also, it cating clustering of species assemblages across the phylogeny should be underlined that PD values increase with number and of this group on this substrate (Figure 5(a); Supplemental online age of the species considered, with a likely substantial effect of material 2). gymnosperms in our phylogeny. The taxonomic beta diversity for each monophyletic group We also analysed the influence of elevation and substrate analysed was slightly higher when calcareous substrates were on the woody flora of Sierra Nevada and different lineages, an involved in the comparison (Figure 5(b); Supplemental online approach that allowed us to describe some general patterns of material 2). For each monophyletic group, the turnover compo- plant biodiversity in this hotspot. Our results indicate that taxo- nent was the highest between calcareous and non-calcareous nomic diversity of the woody flora in Sierra Nevada is the highest substrates and the nestedness component was highest between between 700 m and 1500 m a.s.l. Above 1500 m, the taxonomic calcareous and special substrates (Figure 5(b); Supplemental and, more strongly, the phylogenetic diversity of the woody flora online material 2). For the subset of endemic taxa, taxonomic decreases gradually; they are partly composed of small clusters beta diversity was similar between each substrate comparison of species spread across the phylogeny, which probably resulted and was mostly based on the turnover component. Values of PBD from radiations in taxa adapting to specific microhabitats at high were random for either the turnover or the nestedness compo- elevations, from ancestors with convergent adaptation to colo- nent. Only the turnover component of PBD between calcareous nise high-mountain habitats. Establishing what drove these radi- and special substrates was positive and nearly significant for the ations would require in-depth studies of niche differentiation, eudicot group. which is beyond our current aims. Interestingly, the endemic component showed an opposite pattern, increasing CAD with increasing elevation up to belt 3 and its relative abundance up to Discussion belt 4. The association between endemism richness and elevation On the effects of substrate and elevation in differentiation of and the special need for protection of the highest-lying areas of woody species in Sierra Nevada Sierra Nevada have long been claimed (Blanca et al. 1998; Mota We studied the biodiversity of the woody flora in the Sierra et al. 2002; Giménez et al. 2004; see also Steinbauer et al. 2016). Nevada mountain range. Our extensive sampling and barcod- Endemism richness may be promoted by orographic isolation ing approach enabled the reconstruction of a comprehensive without any confounding effect of substrate: dominant sub- phylogeny for this flora, providing some insight into taxa sub- strates in high elevation areas of Sierra Nevada are mica-schists, jected to systematic discussion, and facilitating estimates of the which, on the other hand, are the less biodiverse substrates (see existing taxonomic and phylogenetic diversity. Estimates and below). comparisons of taxonomic diversity between areas have a long Taxonomic beta diversity increased with the difference history, and the recognised importance of evolutionary his- between the elevation belts in comparison and was the highest tory in biodiversity analyses has promoted phylogenetic diver- when the less diverse belt 4 was involved. This was largely due sity studies in recent years (Sechrest et al. 2002; Winter et al. to the replacement of species between elevation belts except 2013; Pollock et al. 2015). Importantly, when analysing the role for pairs 0–1 and 3–4, where a high nestedness component indi- of eco-geographical factors in biodiversity, differences exist cated that the species assemblages of the two less diverse belts when using taxonomic or phylogenetic measures (e.g. Morlon (0 and 4) were subsets of those in the two more diverse belts et al. 2011). The difficulty in applying barcoding techniques (1 and 3). This means that the upper and lower elevation thresh- to complete floras and the unavailability of DNA sequences olds for lowland and alpine woody species in the study area are in public repositories has precluded the widespread use of located above 700 m and below 2800 m, respectively. The small PLANT BIOSYSTEMS – AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 15 area occupied by belts 0 and 4 may also influence this result, traits driven by the joint effects of environmental filtering and though it is noteworthy that the number of species occurring in facilitative interactions. Likewise, Ojeda et al. (1995) pointed out belt 0 is similar to those in belts 1 to 3. The PBD was higher than that acid soil islands within the basic soil area of southern Spain expected in several comparisons between pairs 0–1 and pairs might have promoted plant speciation. In contrast, the predom- 2–4, suggesting that a significant phylogenetic replacement in inant randomness of alpha and beta phylogenetic patterns in the woody flora of Sierra Nevada takes place at ca. 1500 m, at relation to substrate types suggests a minor role of lithology in the boundary of elevation belts 1 and 2. The significant patterns the phylogenetic structure of the woody flora in Sierra Nevada, of PAD and PBD in relation to elevation were lost in the analyses a result that agrees with previous studies conducted in the same of the endemic species group, whose assembly in relation to this region (Molina-Venegas, Aparicio, Slingsby, et al. 2015, Molina- eco-geographical factor was, instead, well explained by the tax- Venegas et al. 2016). onomic diversity patterns. Our results on the biodiversity of elevation belts showed par- Concluding remarks tial similarities to those obtained by Molina-Venegas, Aparicio, Slingsby, et al. (2015) for the entire eudicot flora in a conjunct Increasing floristic, biosystematic and phylogenetic knowledge of the Baetic Sierras (see also above). While PBD did not show of the flora of the Baetic–Rifan hotspot during the last two dec- differences between belts 3 and 4 in Sierra Nevada, in their study, ades has allowed to elaborate a multi-scale research programme these authors found significantly positive values for virtually on the factors associated with building this biodiversity hot- all elevation belt comparisons, a difference that might be due spot, from populations and their ecological current scenarios to either the inclusion of non-woody taxa or to the separation to complete floras and their historical settings. However, much of calcareous and siliceous areas of Sierra Nevada as different more research is still needed. It is particularly critical to acquire mountain ranges in their study. On the other hand, our results a homogeneous knowledge for different parts of the region. In were opposed to the results of Molina-Venegas et al. (2016), who this respect, the North African flora still needs some efforts in found alpha phylogenetic overdispersion at highest elevations all these aspects, starting with the floristic database. Whilst the and a null effect of elevation on beta phylogenetic diversity. The checklist is reasonably complete, the distribution maps should lesser elevation range and the remarkably smaller species data- be thoroughly improved and, more importantly, assembled in set considered in that study, which analysed spatial structure at a standard spatial network in order to readily detect patterns the community level, might be largely responsible of the different free from pre-established ecoregions of limited value for pure results obtained. Furthermore, additional studies in Sierra Nevada biogeographic analyses. Finally, sound phylogenetic studies of have shown that other factors influence the taxonomic and phy- critical groups centred in the region and representing different logenetic diversity at the community level, such as the presence histories, ecology and traits are needed to disentangle a com- of facilitating species (Pistón et al. 2016). mon history and the factors driving differentiation and diversity. Taxonomic diversity showed smaller differences amongst the Although barcoding has some drawbacks and correct taxo- three substrate types considered. In spite of the high dominance nomic identification of properly preserved specimens will be still of non-calcareous substrates in Sierra Nevada, this lithology har- necessary, these techniques will be increasingly useful for highly boured an intermediate number of woody taxa in comparison diverse and problematic floras, where many narrow endemics of with calcareous and special substrates. We hypothesise that the difficult identification are common. Obviously, this will require much lower geomorphological heterogeneity in mica-schists greater efforts in making appropriate markers available for the than in calcareous soils (more prone to erosion) of Sierra Nevada biological scale of interest. are responsible for the lower diversity relative to the area occu- pied in the former. More remarkably, special substrates, occu- Author contribution statement pying a very small fraction of the study area, harboured about half of the regional pool of taxa, a result likely influenced by the JA and VISP conceived ideas; JL and VISP carried out field work; abundance of edaphically indifferent species and by the pro- LN, AC and VISP conduced lab work; ME, VISP and JL analysed motion of processes of species differentiation (see below). The data; RMV provided technical support for biodiversity analyses; highest nestedness component of beta taxonomic diversity VISP and JA wrote the paper; all authors contributed to the final between calcareous and special substrates indicates that there text. is a higher relative proportion of species shared between these two substrate types, while the highest turnover of species occurs Acknowledgements between calcareous and non-calcareous substrates. Interestingly, the taxonomic beta diversity was much higher in the substrate We thank the “Hoya de Pedraza” Botanical Garden staff for their collaboration comparison when considering the subset of endemic species. in sampling the woody flora of Sierra Nevada. We thank Andalusian Scientific Information Technology Center (CICA, Seville, Spain) and the Biology and This result indicates that these species are more substrate-spe- Herbarium Services of the Center for Technological Research and Innovation cific and the lithology had likely a more important role in their of the University of Seville (CITIUS) for providing computational and research differentiation. Less abundant and stressful substrate types might resources. Juan Carlos Rubio (IGME, Granada) provided indispensable geo- have an especially important role, as indicated by the highest logical information for the field work in Sierra Nevada. The Sierra Nevada relative abundance of endemism in special substrates (see also National Park staff provided all logistic support and permits for making these studies possible. This contribution largely resulted from the invitation Mota et al. 2008). In this sense, Molina-Venegas et al. (2018) have of Lorenzo Peruzzi (University of Pisa) to contribute to the 111th Congress of recently shown that dolomitic soils in the area might promote the Italian Botanical Society and 3rd International Plant Science Conference plant adaptation and endemism through changes in functional (IPSC), held at Rome in September 2016. 16 V. I. SIMÓN-PORCAR ET AL.

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