Xylogenesis reveals the genesis and ecological signal of IADFs in Pinus pinea L. and Arbutus unedo L.

Xylogenesis reveals the genesis and ecological signal of IADFs in Pinus pinea L. and Arbutus... Abstract Background and Aims Mediterranean trees have patterns of cambial activity with one or more pauses per year, leading to intra-annual density fluctuations (IADFs) in tree rings. We analysed xylogenesis (January 2015–January 2016) in Pinus pinea L. and Arbutus unedo L., co-occurring at a site on Mt. Vesuvius (southern Italy), to identify the cambial productivity and timing of IADF formation. Methods Dendrochronological methods and quantitative wood anatomy were applied and enabled IADF identification and classification. Key Results We showed that cambium in P. pinea was productive throughout the calendar year. From January to March 2015, post-cambial (enlarging) earlywood-like tracheids were observed, which were similar to transition tracheids. The beginning of the tree ring was therefore not marked by a sharp boundary between latewood of the previous year and the new xylem produced. True earlywood tracheids were formed in April. L-IADFs were formed in autumn, with earlywood-like cells in latewood. In A. unedo, a double pause in cell production was observed, in summer and winter, leading to L-IADFs in autumn as well. Moreover, the formation of more than one IADF was observed in A. unedo. Conclusions Despite having completely different wood formation models and different life strategies, the production of earlywood, latewood and IADF cells was strongly controlled by climatic factors in the two species. Such cambial production patterns need to be taken into account in dendroecological studies to interpret climatic signals in wood from Mediterranean trees. Cambial activity, functional wood traits, intra-annual density fluctuations (IADFs), Pinus pinea L, Arbutus unedo L, tree rings, xylogenesis INTRODUCTION Environmental signals are incorporated in anatomical features of xylem rings, whose formation is driven by genetic and climatic factors affecting different phases of cambial cell division, cell differentiation and programmed cell death (Scarpella and Meijer, 2004; Fonti et al., 2010; Ružička et al., 2015). The general increase in drought forecast for the Mediterranean basin (Stocker et al., 2013) is expected to alter cambial phenology and xylogenesis, thus affecting the growth and productivity of forests (Sarris et al., 2007), vegetation dynamics (Martinez del Castillo et al., 2016) and, ultimately, influencing biogeochemical cycles (Xia et al., 2015). Mechanistic models of xylogenesis have been proposed to link the kinetics of tracheid formation in ‘typical’ tree rings formed by conifer species in temperate climates under optimal growth conditions (Cuny et al., 2014). Such models, based on a unimodal pattern of xylogenesis, are not directly transferrable to softwoods and hardwoods growing in Mediterranean climates. In fact, in Mediterranean woods, the plasticity of secondary growth in trees, which is responsible for their adaptation to specific environmental changes, is evident in the variable patterns of cambial activity in Mediterranean trees and shrubs. Such patterns are species and site specific (Camarero et al., 2010); they result in the formation of intra-annual density fluctuations (IADFs) in tree rings, mainly driven by seasonal variations in temperature and water availability (De Micco et al., 2007,2016a; Battipaglia et al., 2016). IADFs appear as regions in xylem increments in which abrupt, unexpected changes in density occur and have been recently re-classified according to their position and anatomical traits in the latewood or earlywood of tree rings (De Micco et al., 2016a). L-IADFs are frequent in Mediterranean trees and consist of the occurrence of a band of earlywood-like cells within latewood (Campelo et al., 2007, 2013; Battipaglia et al., 2010, 2016; de Luis et al., 2011b; Novak et al., 2013a, b; De Micco et al., 2014,2016a). L-IADFs, characterized by the presence of conducting cells with larger lumina than in true latewood, have been related to an extended period of wood production due to the return of favourable conditions for tree growth after the onset of latewood formation. L-IADFs are therefore primed by a bimodal pattern of wood formation, with two peaks of cambial activity: in spring and autumn (De Micco et al., 2016a; Zalloni et al., 2016). Analysis of IADF genesis can help to uncover the factors triggering their formation. The division of cambial cells and cell differentiation are influenced not only by genetic factors but also by the physiological status of plants. They depend on the availability of water and sugar, which, in turn, are affected by climatic conditions (Deslauriers and Morin, 2005; Cuny et al., 2015; Steppe et al., 2015; Vieira et al., 2015; Prislan et al., 2016). All the phases of cambial cell production and differentiation can be affected by climatic factors, and there is evidence that the onset, duration and ending of the different phenological phases of xylogenesis are variable among study sites and years (Martinez del Castillo et al., 2016). This phenomenon has clearly emerged according to several studies conducted on Pinus sylvestris and Fagus sylvatica, throughout their geographical distribution and in different biomes; it has been linked to variations in temperature, water availability, photoperiod and leaf phenology (Čufar et al., 2008; Gruber et al., 2012; Cuny et al., 2012, 2014; Prislan et al., 2013, 2016; Rossi et al., 2013; Swidrak et al., 2014; Martinez del Castillo et al., 2016; Ziaco et al., 2016). Interest in analysing xylogenesis in tree rings prone to form IADFs has recently increased in the context of understanding the capacity of species to adapt to expected climate changes in Mediterranean forest and shrub biomes (Camarero et al., 2010). Intra-annual formation of IADFs has been recorded in Pinus halepensis in Mediterranean sites in Spain and Italy (de Luis et al., 2011a; De Micco et al., 2016b; Novak et al., 2016a). In both cases, L-IADFs were formed in latewood after a period of reduced cambial activity due to summer drought, which was followed by the restoration of active cambial production induced by precipitation occurring either in autumn or in summer at Spanish and Italian sites, respectively (de Luis et al., 2011a; De Micco et al., 2016b). Restoring cambial activity allows an extension of the wood formation period, which may continue until the end of December (Novak et al., 2016b) if the conditions (i.e. temperature above a certain threshold) are favourable. Whatever the mechanism inducing their formation, IADFs influence the balance between efficiency and safety of water transport. While the formation of narrow conduits in latewood formed in summer allows slow, but still continuous, water transport under conditions of soil aridity, the formation of an extra band of earlywood-like cells in latewood guarantees increased the efficiency of water transport, according to the physical laws of conductivity, when water is widely available again after autumn rain (Sperry et al., 2006). Such a strategy is in line with anatomical peculiarities of Mediterranean species whose tree rings contain regions with conduits specialized for high conductivity, accounting for most of the water flow, but also regions/conduits characterized by high safety traits which are capable of maintaining water flow when larger conduits are embolized (De Micco et al., 2008). Genesis of IADFs in Mediterranean hardwoods is still poorly explored, and the relationships between IADF formation and environmental factors have been examined based on retrospective analyses of anatomical and isotopic traits in tree-ring series (Battipaglia et al., 2010, 2014; De Micco et al., 2012, 2014, 2016c). De Micco et al. (2016b) recently described for the first time the formation of IADFs in tree rings of the Mediterranean small tree Arbutus unedo at a site in southern Italy, through analysis of xylogenesis focused on summer months. As shown for P. halepensis growing at the same site, the formation of L-IADFs in A. unedo did not occur in autumn, but happened earlier, due to rain in summer (July–August) after a period of drought. To the best of our knowledge, cambial production throughout the year in Mediterranean hardwoods, which are also prone to form frequent IADFs, still remains unexplored. The aim of this study was to analyse cambial productivity, xylem formation and differentiation in two Mediterranean species, the softwood Pinus pinea L. (stone pine) and the hardwood Arbutus unedo L. (strawberry tree), co-occurring at the same site in southern Italy, characterized by a period of summer drought. We monitored xylogenesis in the two species throughout 1 year in order to identify the genesis of IADFs in their tree rings. We aimed to: (1) identify the onset of cambial activity, earlywood and latewood formation; (2) evaluate the phenology of cambium in the two species; and (3) analyse how many, when and what type of IADFs are formed in the two species. Cambium phenology and IADF genesis were compared between the two species and with climatic data to evaluate which factors play a role in IADF formation. The findings are also discussed in reference to hypotheses raised from previous studies based on retrospective analyses of tree-ring series showing IADFs throughout the Mediterranean region. MATERIALS AND METHODS Species, study site and climatic conditions The study was conducted on trees of stone pine (Pinus pinea L.) and strawberry tree (Arbutus unedo L.), co-occurring at a site in southern Italy. The study area, located on Mt. Vesuvius (40°78′N, 14°42′E, 200 m asl) (Fig. 1A) consists of a stone pine forest bordered by an area dominated by strawberry tree bushes. Stand density is 2500 P. pinea and 5000 A. unedo trees per hectare. The climate at the site is Mediterranean, with hot and dry summers followed by mild and wet winters. Sampling of microcores to analyse cambial productivity and xylem formation in the two species was carried out in 2015, which was characterized by an annual mean temperature of 17.8 °C. The hottest month was July (monthly average mean temperature of 28.5 °C), while the coldest month was February (monthly average mean temperature of 9.4 °C). The cumulative annual precipitation was 1054 mm, the wettest month was October, with a cumulative monthly precipitation of 286 mm, while the lowest value was recorded in July (cumulative precipitation of 9.4 mm). The arid period lasted from May to July (Fig. 1B). A review of long-term meteorological series from the same site, obtained from KNMI Climate Explorer (Trouet and Van Oldenborgh, 2013), indicated that meteorological conditions in 2015 did not deviate significantly from those of the long-term series (1995–2014). Fig. 1. View largeDownload slide (A) Geographic location of the study site in southern Italy. (B) Climatic diagram of monthly average mean temperatures (T) and total monthly precipitation (P) for 2015 elaborated from the gridded data set E-OBS (14°25′–14°50′E, 40°75′–41°00′N). Fig. 1. View largeDownload slide (A) Geographic location of the study site in southern Italy. (B) Climatic diagram of monthly average mean temperatures (T) and total monthly precipitation (P) for 2015 elaborated from the gridded data set E-OBS (14°25′–14°50′E, 40°75′–41°00′N). Tree-ring identification Dendrochronological techniques were applied to build tree-ring chronologies to allow the cross-dating of tree rings in order to identify the xylem increment formed in 2015. Core sampling was performed on 20 trees of P. pinea and 20 plants (not multistem) of A. unedo, by taking two cores at breast height from each tree (west and east directions) with an increment borer (diameter 5 mm). Cores were air-dried, mounted in a grooved wooden block, and their surface was polished with a graded series of sandpapers. Tree-ring series were visually cross-dated by comparison of signature rings and calendar dates assigned to the year of formation of each ring to facilitate the cross-dating of tree rings through dendrochronological techniques (Stokes and Smiley, 1968). IADFs were identified according to De Micco et al. (2012, 2014). Tree-ring width (TRW) measurement was done to the nearest 0.01 mm, using LINTAB measuring equipment and TSAP Win software (Frank Rinn, Heidelberg, Germany). The quality of cross-dating was checked using the R package dplR (Bunn, 2008), and two master chronologies were built, one for each species. The selection of six trees for xylogenesis and wood anatomical analyses was done taking into account the cores with the highest correlation with the master chronology of each species. Descriptive statistics of raw data of the six selected trees were calculated, as well as the expressed population signal (EPS), which indicates the level of coherence of the constructed chronology and how it portrays the hypothetical perfect population chronology (Wigley et al., 1984; Buras, 2017), the mean sensitivity (MS), which is a measure of the mean percentage change from each measured yearly ring value to the next, and the Gleichläufigkeit (GLK), which is a measure of the year to year agreement between the interval trends of two chronologies based upon the sign of agreement, were calculated for the chronologies. Microcore sampling and microscopy For the collection of microcores, six healthy trees/plants per species were selected according to the dendrochronological parameters. For P. pinea, the stem diameter at breast height was 53.3 ± 5.9 cm (mean ± s.d.), the height was about 16 m and age was about 95 years. For A. unedo, the stem diameter at breast height was 15.6 ± 4.9 cm, the height was 4–8 m and age was about 20 years. Microcores (1.8 mm diameter) were collected using a Trephor tool (Rossi et al., 2006) at weekly intervals from January 2015 until January 2016. In order to avoid wound effects, the points of microcore extraction were chosen following a spiral with a distance of 3–4 cm between consecutive samples. Each microcore contained inner phloem, cambium and xylem (at least three of the last-formed xylem rings). The extracted microcores were immediately fixed in 70 % ethanol and stored at 4 °C. The samples were dehydrated in a graded ethanol series (70, 90, 95 and 100 %), infiltrated with bio-clear (D-limonene) and embedded in paraffin blocks using a Leica TP1020-1 (Nussloch, Germany) tissue processor. Cross-sections (9 μm thick) were cut with a semi-automatic rotary microtome RM 2245 (Leica, Nussloch), using low profile microtome blades. The sections were placed on glass slides pre-treated with albumin and dried at 70 °C for 30 min. After removing the residual paraffin through bio-clear and ethanol, the sections were stained with a safranin (0.04 %) and astra blue (0.15 %) water solution (van der Werf et al., 2007) and mounted in Euparal (Bioquip Rancho Dominguez, CA, USA). The sections were observed under a Nikon Eclipse 800 light microscope, and microphotographs were taken by means of a DS-Fi1 digital camera with the NIS-Elements BR 3 image analysis system (Melville, NY, USA). The sections were analysed to identify and measure cambial and differentiating xylem cells, using visual criteria of proportional dimension and wall thickness (de Luis et al., 2007, 2011a; Čufar et al., 2008, 2011; De Micco et al., 2016b). Sections from samples collected every 2 weeks were measured, while for periods of high cambial production, sections collected every week were used. Although the transition from earlywood to latewood is generally abrupt in Pinus species, we applied the criteria to define earlywood and latewood based on the proportion of tracheid cell wall thickness and lumen diameter (Denne, 1989; Antony et al., 2012) to classify tracheids in P. pinea as earlywood or latewood as well as to define earlywood-like and latewood-like cells at the IADF level. More specifically, all tracheids whose common double cell wall is equal to or greater than the cell lumen (parameters always measured in the radial direction) are considered latewood or latewood-like. In A. unedo, the transition between earlywood and latewood was not always sharp, but latewood was characterized by the presence of narrower (half lumen diameter) and less frequent vessels than earlywood (De Micco et al., 2016c). On transverse sections, cambium cells (CCs) and cells in different differentiation phases were identified and counted, i.e. post-cambial or enlarging cells (PCs), cells undergoing secondary thickening and lignification of the cell walls (SWs) and mature cells (MTs) (Čufar et al., 2011; de Luis et al., 2011a; De Micco et al., 2016b) (Fig. 2). CCs were radially flattened, while PCs were characterized by larger radial dimensions. SWs had thicker cell walls, stained blue or light red, and could be discriminated from PCs by using polarized light (Rossi et al., 2006). MTs were cells with completely thickened and lignified cell walls, staining completely red and without any trace of protoplast in the lumen. Active production of xylem cells by the cambium was indicated by an increase in the number of cells and width of CCs and the presence of PC phases compared with the dormant state. Fig. 2. View largeDownload slide (A) Developing xylem in P. pinea and (B–D) A. unedo. Moving from the cambial zone towards the centre of the stem, the following cells are encountered: cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall and no protoplast in the lumen (MTs). Arrowheads in (D) mark the transition between SWs and MTs. Scale bars = 100 µm. Fig. 2. View largeDownload slide (A) Developing xylem in P. pinea and (B–D) A. unedo. Moving from the cambial zone towards the centre of the stem, the following cells are encountered: cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall and no protoplast in the lumen (MTs). Arrowheads in (D) mark the transition between SWs and MTs. Scale bars = 100 µm. In P. pinea, the number of cells was counted along three radial rows for all phenological phases (CCs, PCs, SWs and MTs). Since the boundary limiting the ending of wood formed in 2014 and the beginning of wood formed in 2015 was not always clear, counting MTs formed in 2015 was not straightforward and required the definition of a sort of point zero. This was established in sections of the first microcore sampling by counting the number of all MTs, starting from the beginning of the tree ring formed in 2014 (the transition between 2013 and 2014 tree rings was abrupt). This point zero number was subtracted from all MTs counted in successive microcores using the same principle (all MTs starting from the beginning of the 2014 tree ring). In A. unedo, in which vessels and fibres are not arranged in ordered radial rows, the width of all zones occupied by the various cell types in the different phenological phases was measured according to De Micco et al. (2016b). Values, i.e. measurements and counting along three radial rows of different xylem differentiation phases, were averaged. To avoid the influence of variability around the circumference, the number for P. pinea, and the width for A. unedo, of cells and tissues in the previous xylem ring were quantified to normalize the measurements (Rossi et al., 2003; Prislan et al., 2013). To detect the timing of IADF occurrence, we analysed the microsections from subsequent microcores by focusing on SWs and MTs, to highlight changes in cell lumen size and wall thickness marking the transition from earlywood to latewood, and vice versa, as reported in De Micco et al. (2016b). RESULTS Tree-ring series The statistics of the tree-ring chronologies are reported in Table 1 and refer to the six trees for each species selected for xylogenesis analyses. Arbutus unedo plants were much younger than P. pinea trees. The TRW chronologies of the two species showed different tree-ring patterns (Fig. 3), with no significant correlations between them. Table 1. Summary of the statistics of tree-ring chronologies for the analysed trees of P. pinea and A. unedo Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 TRW, tree-ring width; EPS, expressed population signal; GLK, Gleichläufigkeit, MS, mean sensitivity. EPS and r-bar are calculated for the period 2000–2015 for A. unedo and 1940–2015 for P. pinea. View Large Table 1. Summary of the statistics of tree-ring chronologies for the analysed trees of P. pinea and A. unedo Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 TRW, tree-ring width; EPS, expressed population signal; GLK, Gleichläufigkeit, MS, mean sensitivity. EPS and r-bar are calculated for the period 2000–2015 for A. unedo and 1940–2015 for P. pinea. View Large Fig. 3. View largeDownload slide Tree-ring width (TRW) chronologies of six P. pinea (black line) and six A. unedo (grey line). Bars indicate the s.d. Fig. 3. View largeDownload slide Tree-ring width (TRW) chronologies of six P. pinea (black line) and six A. unedo (grey line). Bars indicate the s.d. The mean ring widths (Table 1) of A. unedo were higher than those of P. pinea for the whole tree-ring chronologies (P < 0.05) and for 2015 (P < 0.001). Both chronologies have an EPS value >0.85, indicating that the chronologies were representative of radial growth variations of the whole population of trees. All the statistics listed in Table 1 showed a strong common tree-ring signal among individuals of the same species and high year to year radial growth variability associated with inter-annual changes in climatic conditions, especially for A. unedo, which represents the highest mean sensitivity. Cambial productivity and phenology of xylem formation The status of cambial productivity on the first sampling date [29 January 2015, day of year (DOY) 29] was different in the two analysed species, being active in P. pinea (Fig. 4A) and dormant in A. unedo (Fig. 5A). Curves showing the number of CCs (Fig. 6A, E), PCs (Fig. 6B, F) and SWs (Fig. 6C, G) during the sampling period followed a characteristic bimodal shape, with the second peak more evident in P. pinea than in A. unedo. The curve representing the number of MTs showed a typical sigmoid shape in A. unedo (Fig. 6H), while in P. pinea both SWs and new MTs were also present during the period January–March 2015 (Fig. 6D). Fig. 4. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea trees forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 43, (C) DOY 72, (D) DOY 100, (E) DOY 127, (F) DOY169, (G) DOY 189, (H) DOY 240, (I) DOY 268, (L) DOY 296, (M) DOY 317 and (N) DOY 345. Arrowheads point to the boundary between the 2014 and 2015 tree rings; the non-abrupt tree-ring boundary is evident in (F), (H) and (N). Images are taken at the same magnification. Scale bar = 100 µm. Fig. 4. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea trees forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 43, (C) DOY 72, (D) DOY 100, (E) DOY 127, (F) DOY169, (G) DOY 189, (H) DOY 240, (I) DOY 268, (L) DOY 296, (M) DOY 317 and (N) DOY 345. Arrowheads point to the boundary between the 2014 and 2015 tree rings; the non-abrupt tree-ring boundary is evident in (F), (H) and (N). Images are taken at the same magnification. Scale bar = 100 µm. Fig. 5. View largeDownload slide Light microscopy views of microsections of microcores of A. unedo plants forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 106, (C) DOY 168, (D) DOY 239 and (E) DOY 301. Images are taken at the same magnification. Scale bar = 50 µm. Fig. 5. View largeDownload slide Light microscopy views of microsections of microcores of A. unedo plants forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 106, (C) DOY 168, (D) DOY 239 and (E) DOY 301. Images are taken at the same magnification. Scale bar = 50 µm. Fig. 6. View largeDownload slide Number of cells in various phases of xylem formation in P. pinea trees (A–D) and width of different developmental xylem zones in A. unedo plants (E–H): cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall (MTs). Mean values and standard errors are shown. DOY, number of days from 1 January 2015 to 22 January 2016. Fig. 6. View largeDownload slide Number of cells in various phases of xylem formation in P. pinea trees (A–D) and width of different developmental xylem zones in A. unedo plants (E–H): cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall (MTs). Mean values and standard errors are shown. DOY, number of days from 1 January 2015 to 22 January 2016. In more detail, in the developing xylem of P. pinea, CCs consisted of 7.30 ± 0.66 cells (mean ± s.d.), while PCs consisted of 1.94 ± 0.82 cells in the period from the end of January until the end of March (DOY 29–86) (Figs 4A–E and 6A, B). At the end of March (DOY 86), the number of CCs and PCs started to increase (8.46 ± 1.06 and 3.94 ± 1.65, respectively), reaching the peak between DOY 100 and 112 (Figs 4F, G and 6A, B). The number of CCs and PCs then declined, reaching minimum values during the summer months (Figs 4H–Q and 6A, B). A second peak in the number of CCs (9.11 ± 1.00) occurred on 8 October (DOY 282) and of PCs (3.20 ± 1.57) on 22 October (DOY 296), after which they decreased again, reaching a minimum value on 4 January 2016 (6.22 ± 0.45 and 0.88 ± 0.40, respectively) (Fig. 6A, B). The curve of SWs showed a similar pattern to those of CCs and PCs, although shifted by a couple of weeks (Fig. 6C). The number of SWs started to increase in the middle of April, reaching the maximum value (12.22 ± 3.27) on 21 May (DOY 142). A second peak of SWs was detected in November (DOY 317). MTs were detected from the first sampling onwards: their number was low (6.49 ± 4.78) and started to increase in mid-May (DOY 142) (10.64 ± 1.87), reaching a maximum value (37.47 ± 13.71) in August (DOY 225), which was maintained until the end of the year (Fig. 6D). The cambium in A. unedo was still dormant on the first sampling date (CC width was 14.76 ± 2.78 µm) (Figs 5A and 6E). The first signs of activity were observed on 10 April (DOY 100), with the production of PCs (22.93 ± 6.33 µm) (Figs 5B and 6F). Cambial production reached a peak in May (DOY 142) (37.73 ± 8.55). A high presence of PCs was also found in April–June, with a peak on 7 May (DOY 127) (103.60 ± 61.95) (Figs 5C and 6F). At the beginning of July (DOY 189), we observed a decrease in CCs (22.24 ± 2.23), and such low values were observed until the beginning of October (DOY 282) (Figs 5D and 6E). In the second half of July and until the beginning of October, the width of the PC zone was lower than at the beginning of the year, suggesting that the cambium was not productive (Fig. 6F). An increased width of the PC zone (23.6 ± 18.30) was again observed around 22 October (DOY 296) (Figs 5E and 6F). Cambial productivity stopped again in December (DOY 345), when the ring consisted of MTs only (Fig. 6H). Genesis of IADFs Microscopy observation of the cross-sections of the microcores sampled at the beginning of 2016 showed that, in the period of the investigation (January 2015–January 2016) and in the two previous years, 2014 and 2013, tree rings showed IADFs in both P. pinea (Fig. 7A–C) and A. unedo (Fig. 7D–F). Such IADFs were of type L, being characterized by the presence of earlywood-like cells in latewood at the end of the tree ring (Fig. 7, arrows). Fig. 7. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea and A. unedo plants sampled in January 2016. IADFs (arrows) were observed in the years 2015 (A, D), 2014 (B, E) and 2013 (C, F). Scale bars = 100 µm. Fig. 7. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea and A. unedo plants sampled in January 2016. IADFs (arrows) were observed in the years 2015 (A, D), 2014 (B, E) and 2013 (C, F). Scale bars = 100 µm. In P. pinea, from the first sampling in 2015, cambium was productive and the boundary of the previous tree ring (corresponding to 2014) was not characterized by an abrupt transition from true latewood to earlywood tracheids: in the months January–March 2015 (DOY 29–71), ‘earlywood-like cells’ were formed, but with narrower lumina and thicker cell walls, corresponding to so-called transition tracheids (Fig. 4A–C). True earlywood MTs were evident at the end of March until the beginning of July (Fig. 4C–G); thereafter, the production of latewood cells started and led to mature latewood cells in the second half of July (DOY 211). Latewood tracheids were formed and differentiated until the beginning of October (DOY 281) (Fig. 4G–L). The genesis of L-IADFs occurred in mid-October with the production of earlywood-like tracheids lasting about 60 d; the first mature earlywood-like cells were evident on 22 October (DOY 295) and their production continued until the middle of December (Fig. 4L–N). The production of latewood then restarted, and mature true latewood cells were evident on 22 December (DOY 356). In A. unedo, no cambial cell production was found before April (DOY 100), the production of earlywood started later and lasted until the beginning of July, when mature latewood cells became evident (DOY 183). The production of latewood cells continued until the middle of October. All samples showed the formation of L-type IADFs, with the occurrence of mature earlywood-like cells on 22 October (DOY 295); such a production of earlywood cells lasted >90 d. Latewood maturation was evident again on 10 December (DOY 344), while cambium production stopped again at the beginning of January 2016. Moreover, in three out of six samples, the formation of more than one IADF was found in the year of investigation, with additional earlywood-like cells maturing in August (DOY 239) and new production of latewood cells in September (DOY 257) (Fig. 7D). DISCUSSION In Mediterranean climates, different patterns of cambial activity occur in different species and in various microclimatic conditions (Camarero et al., 2010; Martinez del Castillo et al., 2016). Martinez del Castillo et al. (2016) recently showed that different tree species, namely Pinus sylvestris and Fagus sylvatica, although growing at the same site, have different phenology of xylem formation, probably depending on their different life strategies and adaptive capacity under limiting environmental conditions. In our study, analysis of xylogenesis in two phylogenetically distant plant species, specifically a conifer tree and a hardwood shrub/tree, partially supported this idea. The two species showed a different timing of cambial production and different patterns of tree-ring chronologies (as underlined by low correlation between the two series), although bimodal xylogenesis leading to the formation of the same type of IADF in latewood (L-IADF) was found in both of them. The duration of cambial production was longer in P. pinea than in A. unedo under the same climatic conditions, confirming that the duration of xylogenesis is species specific. Indeed, P. pinea did not show a complete halt in cambial activity in winter, as assumed for Pinus spp. and other species, in which microcoring studies have been focused from spring to autumn (de Luis et al., 2007; Čufar et al., 2011; Martinez del Castillo et al., 2016). It has recently been shown that cambial production of PCs in P. halepensis can continue until the end of December and that its reactivation can be observed as early as February or March, indicating that the cambium in P. halepensis from a Mediterranean site was clearly not dormant (Novak et al., 2016a). In P. pinea growing on Mt. Vesuvius, we found that, although slow, xylogenesis took place from January until March, while in A. unedo cambial production restarted in April after the winter pause. However, in the two species, the first peak of cambial production (highest incidence of CCs and PCs) occurred in the same period, between the end of April and the beginning of May, when the air temperature increased. This agrees with several studies, also based on experiments of artificial heating of the stem, which indicate that the increase in spring temperature is the main factor triggering the onset of xylogenesis (Gričar et al., 2005, 2006; Begum et al., 2010; Prislan et al., 2011; Vieira et al., 2014). On the other hand, the onset of latewood in the two analysed species seemed to be synchronized with a strong decrease in precipitation, coinciding with the highest values of average mean temperature (July). In A. unedo, the appearance of mature latewood conduits occurred 1–2 weeks earlier than in P. pinea. A similar temporal shift in the onset of latewood was also found between A. unedo and P. halepensis co-occurring at the same site in a previous study in southern Italy, suggesting that A. unedo has a faster, more plastic control of cambial processes than Pinus spp. (De Micco et al., 2016b). The second peak of cambial production seemed to be strongly correlated with precipitation in October, which was very abundant in the year of investigation (millimetres of rain were more than double in October compared with September). The production of earlywood-like cells in latewood continued until the middle of December, when the temperature decreased again, reaching minimum values. The occurrence of L-IADFs in P. pinea and A. unedo is in line with findings in other Mediterranean species, such as Juniperus thurifera and A. unedo itself, developing in Mediterranean ecosystems in Spain, Portugal and Italy; in these studies, L-IADFs were ascribed to an extended period of growth due to autumn precipitation creating favourable conditions for wood growth (Camarero et al., 2010; de Luis et al., 2011b; Vieira et al., 2015; Zalloni et al., 2016). The mechanism triggering L-IADFs is linked with increased cell enlargement because of high turgor pressure in enlarging cells due to high water availability after abundant autumn rain (Sperry et al., 2006; De Micco et al., 2016a; Pacheco et al., 2016). The same mechanism has been reported for the formation of L-IADFs in P. halepensis and A. unedo, although in a different period of the year: a second burst of growth in summer after episodes of rain (De Micco et al., 2016b). In A. unedo, the high sensitivity of the rhythm of cambium production in response to changing environmental conditions may be responsible for extended xylogenesis and the frequent occurrence of more than a single IADF per calendar year, as found in this investigation and in agreement with previous observations (Cherubini et al., 2003; De Micco et al., 2016b). The high predisposition of A. unedo to form IADFs may also be due to the young plant age, which is correlated to larger tree rings which, in turn, are positively correlated with IADF frequency (Rigling et al., 2001, 2002; Cherubini et al., 2003; Campelo et al., 2015; Pacheco et al., 2016; Zalloni et al., 2016). An extension of xylogenesis in P. sylvestris has also been ascribed to mild autumn temperatures (Martinez del Castillo et al., 2016). Latewood is then produced again in winter, when low temperatures limit the processes of cell production and differentiation, and can lead to cessation of cell production or dormancy, as found in A. unedo. The presence of two unfavourable periods has been reported to be responsible for the spread of evergreen sclerophyllous shrubs in the Mediterranean environment (Mitrakos, 1980). Arbutus unedo, like other Mediterranean shrubs, has also adapted its phenology and reproduction to seasonal fluctuations (Aronne and Wilcock, 1997). The low sensitivity of P. pinea to winter temperature would explain the onset of latewood delayed by 1 week compared with A. unedo, and its lack of true cambium dormancy, leading to the formation of a few rows of transition wood made of earlywood-like tracheids before the production of true earlywood tracheids. Such a phenomenon explains why the boundary between successive tree rings is not always abrupt. This is probably due to the winter temperature (reaching minimum values of about 9°C), which is not low enough to induce cambium dormancy (Prislan et al., 2016). The cells that are considered to be the last ones in the tree ring are therefore not formed at the end of the calendar year. In P. pinea, the lack of cambium dormancy due to mild winter conditions is in agreement with findings reported for P. radiata growing in New Zealand and P. halepensis at a Mediterranean site in Spain (Barnett, 1987; Prislan et al., 2016). In a recent study by Prislan et al. (2016), a temperature of 10 °C in January did not induce cambial dormancy in P. halepensis. The production of earlywood-like tracheids has been also found in tree-ring chronologies of P. pinea and P. pinaster growing in Portugal: classified as L+-IADF. Such a wood type has been associated with above-average precipitation in early autumn (Campelo et al., 2007), leading to decreased deposition of cell walls (Antonova and Stasova, 1997). In our case, we prefer to refer to these earlywood-like cells as transition wood, according to a more recent re-classification of IADFs (De Micco et al., 2016a), but from a genesis viewpoint they may be assimilated to L+-IADF reported by Campelo et al. (2007), because they were formed at the beginning of the calendar year when rain was abundant after a dry period in December. Such transition wood makes the boundary between tree rings less abrupt, and care should be taken to refer to it as wood formed at the beginning of the calendar year (January–March in our case) and not in the last months of the previous year. This becomes a critical point in tree-ring dating and a warning to avoid errors in the case of interpretation of dendroclimatological analysis. Indeed, such findings suggest that a new perspective should be assumed when applying a dendrochronological approach to Mediterranean species, dismissing the idea of synchronizing visually identified tree rings with calendar years. Explorative xylogenesis analyses should be performed to synchronize sectors of tree rings to specific periods of the year, thus moving from a yearly resolution dendroclimatological analysis to a seasonal resolution analysis, also analysing in detail intra-annual wood anatomical functional traits. Indeed, such an approach has been recently applied to several species, allowing more accurate ecological information to be extracted from long tree-ring series (Carrer et al., 2017). Whatever the triggering factors, the occurrence of IADFs, or of transition wood, in tree rings is responsible for adjustment of the balance between efficiency and safety of water transport. Optimization of the efficiency/safety of water transport can be achieved through plastic control of conduit size during wood formation and through the occurrence of particular anatomical traits, especially in hardwoods (e.g. presence of tracheids, vessel redundancy, occurrence of vessels with different class diameters, etc.) (De Micco et al., 2008, 2009). The presence of IADFs can be interpreted as the maintenance of a sapwood characterized by different sectors: those showing anatomical traits promoting efficient water transport (earlywood and earlywood-like regions accounting for most water flow when water is available) and those showing traits of safety (latewood and latewood-like regions) which are critical to maintain water flow when larger conduits embolize. In the scenario of increasing drought and changes in the precipitation frequency, the occurrence of a plastic response at wood formation level, resulting in increased IADF frequency, would confer an adaptive advantage to species having bimodal cambial activity because the resulting wood anatomical peculiarities would allow the exploitation of water availability at best also maximizing water use efficiency (Pacheco et al., 2016). In conclusion, the occurrence of true earlywood in P. pinea was detected earlier than the onset of cambial activity in A. unedo, but in both species the appearances of mature latewood, of earlywood-like cells and of latewood again after the IADF were fairly well synchronized. This indicates that, despite having completely different wood formation models and different life strategies, the production of earlywood, latewood and earlywood-like cells is strongly controlled by climatic factors in the two species. Moreover, the lack of true winter dormancy, the slightly delayed onset of latewood in P. pinea compared with A. unedo, the occurrence of more than one IADF per year in A. unedo and the delayed onset of latewood after the IADF are all signs of a lower sensitivity of the softwood species and the higher plasticity of the hardwood species in response to variations in temperature and water availability. Indeed, the presence of IADFs in tree rings could be considered a consequence of plant plasticity in response to environmental fluctuations typical of Mediterranean climates and would indicate a more adaptive capability to cope with changing water availability in species forming frequent IADFs compared with species less prone to form IADFs. ACKNOWLEDGEMENTS This study profited from discussions within the COST Action STReESS (COST-FP1106), it was supported by COST (European Cooperation in Science and Technology) and by the Slovenian Research Agency (ARRS), programme P4-0015 and project Z4-7318. We thank Martin Cregeen for editing the English text. A.B., G.B. and V.D.M. conceived and designed the study. A.B. performed sampling. A.B. and M.M. performed sample preparation. V.D.M., A.B., P.P., K.C. and G.B. made a substantial contribution to the analysis of tree-ring series and anatomical signals in microsections and in data analysis. V.D.M., A.B., K.C. and G.A. contributed to interpretation of the overall data. V.D.M., K.C., G.A. and G.B. contributed to the analysis tools. A.B. and V.D.M. wrote the main part of the manuscript. K.C. and G.B. contributed to writing specific parts of the text. All authors made a critical revision of the manuscript, and read and approved the submitted version of the manuscript. LITERATURE CITED Antonova G , Stasova V . 1997 . Effects of environmental factors on wood formation in larch (Larix sibirica Ldb.) stems . Trees 11 : 462 – 468 . Antony F , Schimleck LR , Daniels RF . 2012 . A comparison of earlywood–latewood demarcation methods – a case study in loblolly pine . IAWA Journal 33 : 187 – 195 . Aronne G , Wilcock CC . 1997 . Reproductive phenology in Mediterranean macchia vegetation . Lagascalia 19 : 445 – 454 . Barnett JR . 1987 . The development of fibre-tracheid pit membranes in Pyrus communis L . IAWA Journal 8 : 134 – 142 . Google Scholar CrossRef Search ADS Battipaglia G , De Micco V , Brand WA et al. 2010 . Variations of vessel diameter and δ13C in false rings of Arbutus unedo L. reflect different environmental conditions . New Phytologist 188 : 1099 – 1112 . Google Scholar CrossRef Search ADS PubMed Battipaglia G , Campelo F , Vieira J et al. 2016 . Structure and function of intra-annual density fluctuations: mind the gaps . Frontiers in Plant Science 7 : 595 . doi: 10.3389/fpls.2016.01170 . Google Scholar CrossRef Search ADS PubMed Begum S , Nakaba S , Oribe Y , Kubo T , Funada R . 2010 . Cambial sensitivity to rising temperatures by natural condition and artificial heating from late winter to early spring in the evergreen conifer Cryptomeria japonica . Trees 24 : 43 – 52 . Google Scholar CrossRef Search ADS Bunn AG . 2008 . A dendrochronology program library in R (dplR) . Dendrochronologia 26 : 115 – 124 . Google Scholar CrossRef Search ADS Buras A . 2017 . A comment on the expressed population signal . Dendrochronologia 44 : 130 – 132 . Google Scholar CrossRef Search ADS Camarero JJ , Olano JM , Parras A . 2010 . Plastic bimodal xylogenesis in conifers from continental Mediterranean climates . New Phytologist 185 : 471 – 480 . Google Scholar CrossRef Search ADS PubMed Campelo F , Vieira J , Battipaglia G et al. 2015 . Which matters most for the formation of intra-annual density fluctuations in Pinus pinaster: age or size ? Trees 29 : 237 – 245 . Google Scholar CrossRef Search ADS Campelo F , Vieira J , Nabais C . 2013 . Tree-ring growth and intra-annual density fluctuations of Pinus pinaster responses to climate: does size matter ? Trees 27 : 763 – 772 . Google Scholar CrossRef Search ADS Campelo F , Nabais C , Freitas H , Gutiérrez E . 2007 . Climatic significance of tree-ring width and intra-annual density fluctuations in Pinus pinea from a dry Mediterranean area in Portugal . Annals of Forest Science 64 : 229 – 238 . Google Scholar CrossRef Search ADS Carrer M , Castagneri D , Prendin AL , Petit G , von Arx G . 2017 . Retrospective analysis of wood anatomical traits reveals a recent extension in tree cambial activity in two high-elevation conifers . Frontiers in Plant Science 8 : 737 . doi: 10.3389/fpls.2017.00737 . Google Scholar CrossRef Search ADS PubMed Cherubini P , Gartner BL , Tognetti R , Bräker OU , Schoch W , Innes JL . 2003 . Identification, measurement and interpretation of tree rings in woody species from Mediterranean climates . Biological Review 78 : 119 – 148 . Google Scholar CrossRef Search ADS Cuny HE , Rathgeber CBK , Lebourgeois F , Fortin M , Fournier M . 2012 . Life strategies in intra-annual dynamics of wood formation: example of three conifer species in a temperate forest in north-east France . Tree Physiology 32 : 612 – 625 . Google Scholar CrossRef Search ADS PubMed Cuny HE , Rathgeber CBK , Frank D , Fonti P , Fournier M . 2014 . Kinetics of tracheid development explain conifer tree-ring structure . New Phytologist 203 : 1231 – 1241 . Google Scholar CrossRef Search ADS PubMed Cuny HE , Rathgeber CBK , Frank D et al. 2015 . Woody biomass production lags stem-girth increase by over one month in coniferous forests . Nature Plants 1 : 1 – 6 . Google Scholar CrossRef Search ADS Čufar K , Prislan P , Gričar J . 2008 . Cambial activity and wood formation in beech (Fagus sylvatica) during the 2006 growth season . Wood Research 53 : 1 – 10 . Čufar K , Cherubini M , Gričar J , Prislan P , Spina S , Romagnoli M . 2011 . Xylem and phloem formation in chestnut (Castanea sativa Mill.) during the 2008 growing season . Dendrochronologia 29 : 127 – 134 . Google Scholar CrossRef Search ADS De Micco V , Aronne G . 2009 . Seasonal dimorphism in wood anatomy of the Mediterranean Cistus incanus L. subsp. incanus . Trees 23 : 981 – 989 . Google Scholar CrossRef Search ADS De Micco V , Saurer M , Aronne G , Tognetti R , Cherubini P . 2007 . Variations of wood anatomy and δ13C within tree rings of coastal Pinus pinaster showing intra-annual density fluctuations . IAWA Journal 28 : 61 – 74 . Google Scholar CrossRef Search ADS De Micco V , Aronne G , Baas P . 2008 . Wood anatomy and hydraulic architecture of stems and twigs of some Mediterranean trees and shrubs along a mesic–xeric gradient . Trees 22 : 643 – 655 . Google Scholar CrossRef Search ADS De Micco V , Battipaglia G , Cherubini P , Aronne G . 2014 . Comparing methods to analyse anatomical features of tree rings with and without intra-annual density fluctuations (IADFs) . Dendrochronologia 32 : 1 – 6 . Google Scholar CrossRef Search ADS De Micco V , Campelo F , De Luis M et al. 2016a. Intra-annual density fluctuations in tree rings: how, when, where, and why ? IAWA Journal 37 : 232 – 259 . Google Scholar CrossRef Search ADS De Micco V , Balzano A , Čufar K et al. 2016b. Timing of false ring formation in Pinus halepensis and Arbutus unedo in Southern Italy: outlook from an analysis of xylogenesis and tree-ring chronologies . Frontiers in Plant Science 7 : 705 . doi: 10.3389/fpls.2016.00705 . Google Scholar CrossRef Search ADS PubMed De Micco V , Battipaglia G , Balzano A , Cherubini P , Aronne G . 2016c. Are wood fibres as sensitive to environmental conditions as vessels in tree rings with intra-annual density fluctuations (IADFs) in Mediterranean species ? Trees – Structure and Function 30 : 971 – 983 Google Scholar CrossRef Search ADS Denne MP . 1989 . Definition of latewood according to Mork (1928) . IAWA Journal 10 : 59 – 62 . Google Scholar CrossRef Search ADS Deslauriers A , Morin H . 2005 . Intra-annual tracheid production in balsam fir stems and the effect of meteorological variables . Trees 19 : 402 – 408 . Google Scholar CrossRef Search ADS Fonti P , von Arx G , Garcia-Gonzalez I et al. 2010 . Studying global change through investigation of the plastic responses of xylem anatomy in tree rings . New Phytologist 185 : 42 – 53 . Google Scholar CrossRef Search ADS PubMed Gričar J , Čufar K , Oven P , Schmitt U . 2005 . Differentiation of terminal latewood tracheids in Silver fir trees during autumn . Annals of Botany 95 : 959 – 965 . Google Scholar CrossRef Search ADS PubMed Gričar J , Zupančič M , Čufar K , Koch G , Schmitt U , Oven P . 2006 . Effect of local heating and controlling on cambial activity and cell differentiation in the stem of Norway spruce (Picea abies) . Annals of Botany 97 : 943 – 951 . Google Scholar CrossRef Search ADS PubMed Gruber A , Pirkebner D , Florian C , Oberhuber W . 2012 . No evidence for depletion of carbohydrate pools in Scots pine (Pinus sylvestris L.) under drought stress . Plant Biology 14 : 142 – 148 . Google Scholar PubMed de Luis M , Gričar J , Čufar K , Raventós J . 2007 . Seasonal dynamics of wood formation in Pinus halepensis from dry and semi-arid ecosystems in Spain . IAWA Journal 28 : 389 – 404 . Google Scholar CrossRef Search ADS de Luis M , Novak K , Raventós J , Gričar J , Prislan P , Čufar K . 2011a. Cambial activity, wood formation and sapling survival of Pinus halepensis exposed to different irrigation regimes . Forest Ecology Management 262 : 1630 – 1638 . Google Scholar CrossRef Search ADS de Luis M , Novak K , Raventós J , Gričar J , Prislan P , Čufar K . 2011b. Climate factors promoting intra-annual density fluctuations in Aleppo pine (Pinus halepensis) from semiarid sites . Dendrochronologia 29 : 163 – 169 . Google Scholar CrossRef Search ADS Martinez del Castillo EM , Longares LA , Gričar J et al. 2016 . Living on the edge: contrasted wood-formation dynamics in Fagus sylvatica and Pinus sylvestris under Mediterranean conditions . Frontiers in Plant Science 7 : 370 . doi: 10.3389/fpls.2016.00370 . Google Scholar CrossRef Search ADS PubMed Mitrakos K . 1980 . A theory for Mediterranean plant life . Acta Oecologica 1 : 245 – 252 . Novak K , de Luis M , Raventós J , Čufar K . 2013a. Climatic signals in tree-ring widths and wood structure of Pinus halepensis in contrasted environmental conditions . Trees 27 : 927 – 936 . Google Scholar CrossRef Search ADS Novak K , Saz Sánchez MA , Čufar K , Raventós J , de Luis M . 2013b. Age, climate and intra-annual density fluctuations in Pinus halepensis in Spain . IAWA Journal 34 : 459 – 474 . Google Scholar CrossRef Search ADS Novak K , De Luis M , Gričar J et al. 2016a. Missing and dark rings associated with drought in Pinus halepensis . IAWA Journal 37 : 260 – 274 Google Scholar CrossRef Search ADS Novak K , De Luis M , Saz MA et al. 2016b. Missing rings in Pinus halepensis – the missing link to relate the tree-ring record to extreme climatic events . Frontiers in Plant Science 7 : 727 . doi: 10.3389/fpls.2016.00727 . Google Scholar CrossRef Search ADS PubMed Pacheco A , Camarero JJ , Carrer M . 2016 . Linking wood anatomy and xylogenesis allows pinpointing of climate and drought influences on growth of coexisting conifers in continental Mediterranean climate . Tree Physiology 36 : 502 – 512 . Google Scholar CrossRef Search ADS PubMed Prislan P , Schmitt U , Koch G , Gričar J , Čufar K . 2011 . Seasonal ultrastructural changes in the cambial zone of beech (Fagus sylvatica) grown at two different altitudes . IAWA Journal 32 : 443 – 459 . Prislan P , Gričar J , de Luis M , Smith KT , Čufar K . 2013 . Phenological variation in xylem and phloem formation in Fagus sylvatica from two contrasting sites . Agricultural and Forest Meteorology 180 : 142 – 151 . Google Scholar CrossRef Search ADS Prislan P , Gričar J , de Luis M et al. 2016 . Annual cambial rhythm in Pinus halepensis and Pinus sylvestris as indicator for climate adaptation . Frontiers in Plant Science 7 : 1923 . doi: 10.3389/fpls.2016.01923 . Google Scholar CrossRef Search ADS PubMed Rigling A , Waldner PO , Forster T , Bräker O , Pouttu A . 2001 . Ecological interpretation of tree-ring width and intra annual density fluctuations in Pinus sylvestris on dry sites in the central Alps and Siberia . Canadian Journal of Forest Research 31 : 18 – 31 . Google Scholar CrossRef Search ADS Rigling A , Bräker OU , Schneiter G , Schweingruber FH . 2002 . Intra-annual tree-ring parameters indicating differences in drought stress of Pinus sylvestris forests within the Erico–Pinion in the Valais (Switzerland) . Plant Ecology 163 : 105 – 121 . Google Scholar CrossRef Search ADS Rossi S , Deslauriers A , Morin H . 2003 . Application of the Gompertz equation for the study of xylem cell development . Dendrochronologia 21 : 33 – 39 . Google Scholar CrossRef Search ADS Rossi S , Deslauriers A , Anfodillo T et al. 2006 . Conifers in cold environments synchronize maximum growth rate of tree-ring formation with day length . New Phytologist 170 : 301 – 310 . Google Scholar CrossRef Search ADS PubMed Rossi S , Anfodillo T , Čufar K et al. 2013 . A meta-analysis of cambium phenology and growth: linear and non-linear patterns in conifers of the northern hemisphere . Annals of Botany 112 : 1911 – 1920 . Google Scholar CrossRef Search ADS PubMed Ružička K , Ursache R , Hejátko J , Helariutta Y . 2015 . Xylem development – from the cradle to the grave . New Phytologist 207 : 519 – 535 . Google Scholar CrossRef Search ADS PubMed Sarris D , Christodoulakis D , Körner C . 2007 . Recent decline in precipitation and tree growth in the eastern Mediterranean . Global Change Biology 13 : 1187 – 1200 . Google Scholar CrossRef Search ADS Scarpella E , Meijer AH . 2004 . Pattern formation in the vascular system of monocot and dicot plant species . New Phytologist 164 : 209 – 242 . Google Scholar CrossRef Search ADS Sperry JS , Hacke UG , Pittermann J . 2006 . Size and function of conifer tracheids and angiosperm vessels . American Journal of Botany 93 : 1490 – 1500 . Google Scholar CrossRef Search ADS PubMed Steppe K , Sterck F , Deslauriers A . 2015 . Diel growth dynamics in tree stems: linking anatomy and ecophysiology . Trends in plant science 20 : 335 – 343 . Google Scholar CrossRef Search ADS PubMed Stocker TF , Qin D , Plattner GK et al. 2013 . IPCC, 2013: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change . Cambridge : Cambridge University Press . Stokes MA , Smiley TL . 1968 . Introduction to tree-ring dating . Chicago, IL : University of Chicago Press . Swidrak I , Gruber A , Oberhuber W . 2014 . Xylem and phloem phenology in co-occurring conifers exposed to drought . Trees 28 : 1161 – 1171 . Google Scholar CrossRef Search ADS PubMed Trouet V , Van Oldenborgh GJ . 2013 . KNMI Climate Explorer: a web-based research tool for high-resolution paleoclimatology . Tree-Ring Research 69 : 3 – 13 . Google Scholar CrossRef Search ADS Van der Werf GW , Sass-Klaassen UG , Mohren GM J . 2007 . The impact of the 2003 summer drought on the intra-annual growth pattern of beech (Fagus sylvatica L.) and oak (Quercus robur L.) on a dry site in the Netherlands . Dendrochronologia 25 : 103 – 112 . Google Scholar CrossRef Search ADS Vieira J , Rossi S , Campelo F , Freitas H , Nabais C . 2014 . Xylogenesis of Pinuspinaster under a Mediterranean climate . Annals of Forest Science 71 : 71 – 80 . Google Scholar CrossRef Search ADS Vieira J , Campelo F , Rossi S , Carvalho A , Freitas H , Nabais C . 2015 . Adjustment capacity of maritime pine cambial activity in drought-prone environments . PLoS One 10 : e0126223 . doi: 10.1371/journal.pone.0126223 . Google Scholar CrossRef Search ADS PubMed Wigley TM , Briffa KR , Jones PD . 1984 . On the average value of correlated time series, with applications in dendroclimatology and hydrometeorology . Journal of Climate and Applied Meteorology 23 : 201 – 213 . Google Scholar CrossRef Search ADS Xia J , Niu S , Ciais P et al. 2015 . Joint control of terrestrial gross primary productivity by plant phenology and physiology . Proceedings of the National Academy of Sciences, USA 112 : 2788 – 2793 . Google Scholar CrossRef Search ADS Zalloni E , de Luis M , Campelo F et al. 2016 . Climatic signals from intra-annual density fluctuation frequency in Mediterranean pines at a regional scale . Frontiers in Plant Science 7 : 579 . doi: 10.3389/fpls.2016.00579 . Google Scholar CrossRef Search ADS PubMed Ziaco E , Biondi F , Rossi S , Deslauriers A . 2016 . Environmental drivers of cambial phenology in Great Basin bristlecone pine . Tree Physiology 36 : 818 – 831 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Botany Oxford University Press

Xylogenesis reveals the genesis and ecological signal of IADFs in Pinus pinea L. and Arbutus unedo L.

Loading next page...
 
/lp/ou_press/xylogenesis-reveals-the-genesis-and-ecological-signal-of-iadfs-in-XVaHlXP83X
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
ISSN
0305-7364
eISSN
1095-8290
D.O.I.
10.1093/aob/mcy008
Publisher site
See Article on Publisher Site

Abstract

Abstract Background and Aims Mediterranean trees have patterns of cambial activity with one or more pauses per year, leading to intra-annual density fluctuations (IADFs) in tree rings. We analysed xylogenesis (January 2015–January 2016) in Pinus pinea L. and Arbutus unedo L., co-occurring at a site on Mt. Vesuvius (southern Italy), to identify the cambial productivity and timing of IADF formation. Methods Dendrochronological methods and quantitative wood anatomy were applied and enabled IADF identification and classification. Key Results We showed that cambium in P. pinea was productive throughout the calendar year. From January to March 2015, post-cambial (enlarging) earlywood-like tracheids were observed, which were similar to transition tracheids. The beginning of the tree ring was therefore not marked by a sharp boundary between latewood of the previous year and the new xylem produced. True earlywood tracheids were formed in April. L-IADFs were formed in autumn, with earlywood-like cells in latewood. In A. unedo, a double pause in cell production was observed, in summer and winter, leading to L-IADFs in autumn as well. Moreover, the formation of more than one IADF was observed in A. unedo. Conclusions Despite having completely different wood formation models and different life strategies, the production of earlywood, latewood and IADF cells was strongly controlled by climatic factors in the two species. Such cambial production patterns need to be taken into account in dendroecological studies to interpret climatic signals in wood from Mediterranean trees. Cambial activity, functional wood traits, intra-annual density fluctuations (IADFs), Pinus pinea L, Arbutus unedo L, tree rings, xylogenesis INTRODUCTION Environmental signals are incorporated in anatomical features of xylem rings, whose formation is driven by genetic and climatic factors affecting different phases of cambial cell division, cell differentiation and programmed cell death (Scarpella and Meijer, 2004; Fonti et al., 2010; Ružička et al., 2015). The general increase in drought forecast for the Mediterranean basin (Stocker et al., 2013) is expected to alter cambial phenology and xylogenesis, thus affecting the growth and productivity of forests (Sarris et al., 2007), vegetation dynamics (Martinez del Castillo et al., 2016) and, ultimately, influencing biogeochemical cycles (Xia et al., 2015). Mechanistic models of xylogenesis have been proposed to link the kinetics of tracheid formation in ‘typical’ tree rings formed by conifer species in temperate climates under optimal growth conditions (Cuny et al., 2014). Such models, based on a unimodal pattern of xylogenesis, are not directly transferrable to softwoods and hardwoods growing in Mediterranean climates. In fact, in Mediterranean woods, the plasticity of secondary growth in trees, which is responsible for their adaptation to specific environmental changes, is evident in the variable patterns of cambial activity in Mediterranean trees and shrubs. Such patterns are species and site specific (Camarero et al., 2010); they result in the formation of intra-annual density fluctuations (IADFs) in tree rings, mainly driven by seasonal variations in temperature and water availability (De Micco et al., 2007,2016a; Battipaglia et al., 2016). IADFs appear as regions in xylem increments in which abrupt, unexpected changes in density occur and have been recently re-classified according to their position and anatomical traits in the latewood or earlywood of tree rings (De Micco et al., 2016a). L-IADFs are frequent in Mediterranean trees and consist of the occurrence of a band of earlywood-like cells within latewood (Campelo et al., 2007, 2013; Battipaglia et al., 2010, 2016; de Luis et al., 2011b; Novak et al., 2013a, b; De Micco et al., 2014,2016a). L-IADFs, characterized by the presence of conducting cells with larger lumina than in true latewood, have been related to an extended period of wood production due to the return of favourable conditions for tree growth after the onset of latewood formation. L-IADFs are therefore primed by a bimodal pattern of wood formation, with two peaks of cambial activity: in spring and autumn (De Micco et al., 2016a; Zalloni et al., 2016). Analysis of IADF genesis can help to uncover the factors triggering their formation. The division of cambial cells and cell differentiation are influenced not only by genetic factors but also by the physiological status of plants. They depend on the availability of water and sugar, which, in turn, are affected by climatic conditions (Deslauriers and Morin, 2005; Cuny et al., 2015; Steppe et al., 2015; Vieira et al., 2015; Prislan et al., 2016). All the phases of cambial cell production and differentiation can be affected by climatic factors, and there is evidence that the onset, duration and ending of the different phenological phases of xylogenesis are variable among study sites and years (Martinez del Castillo et al., 2016). This phenomenon has clearly emerged according to several studies conducted on Pinus sylvestris and Fagus sylvatica, throughout their geographical distribution and in different biomes; it has been linked to variations in temperature, water availability, photoperiod and leaf phenology (Čufar et al., 2008; Gruber et al., 2012; Cuny et al., 2012, 2014; Prislan et al., 2013, 2016; Rossi et al., 2013; Swidrak et al., 2014; Martinez del Castillo et al., 2016; Ziaco et al., 2016). Interest in analysing xylogenesis in tree rings prone to form IADFs has recently increased in the context of understanding the capacity of species to adapt to expected climate changes in Mediterranean forest and shrub biomes (Camarero et al., 2010). Intra-annual formation of IADFs has been recorded in Pinus halepensis in Mediterranean sites in Spain and Italy (de Luis et al., 2011a; De Micco et al., 2016b; Novak et al., 2016a). In both cases, L-IADFs were formed in latewood after a period of reduced cambial activity due to summer drought, which was followed by the restoration of active cambial production induced by precipitation occurring either in autumn or in summer at Spanish and Italian sites, respectively (de Luis et al., 2011a; De Micco et al., 2016b). Restoring cambial activity allows an extension of the wood formation period, which may continue until the end of December (Novak et al., 2016b) if the conditions (i.e. temperature above a certain threshold) are favourable. Whatever the mechanism inducing their formation, IADFs influence the balance between efficiency and safety of water transport. While the formation of narrow conduits in latewood formed in summer allows slow, but still continuous, water transport under conditions of soil aridity, the formation of an extra band of earlywood-like cells in latewood guarantees increased the efficiency of water transport, according to the physical laws of conductivity, when water is widely available again after autumn rain (Sperry et al., 2006). Such a strategy is in line with anatomical peculiarities of Mediterranean species whose tree rings contain regions with conduits specialized for high conductivity, accounting for most of the water flow, but also regions/conduits characterized by high safety traits which are capable of maintaining water flow when larger conduits are embolized (De Micco et al., 2008). Genesis of IADFs in Mediterranean hardwoods is still poorly explored, and the relationships between IADF formation and environmental factors have been examined based on retrospective analyses of anatomical and isotopic traits in tree-ring series (Battipaglia et al., 2010, 2014; De Micco et al., 2012, 2014, 2016c). De Micco et al. (2016b) recently described for the first time the formation of IADFs in tree rings of the Mediterranean small tree Arbutus unedo at a site in southern Italy, through analysis of xylogenesis focused on summer months. As shown for P. halepensis growing at the same site, the formation of L-IADFs in A. unedo did not occur in autumn, but happened earlier, due to rain in summer (July–August) after a period of drought. To the best of our knowledge, cambial production throughout the year in Mediterranean hardwoods, which are also prone to form frequent IADFs, still remains unexplored. The aim of this study was to analyse cambial productivity, xylem formation and differentiation in two Mediterranean species, the softwood Pinus pinea L. (stone pine) and the hardwood Arbutus unedo L. (strawberry tree), co-occurring at the same site in southern Italy, characterized by a period of summer drought. We monitored xylogenesis in the two species throughout 1 year in order to identify the genesis of IADFs in their tree rings. We aimed to: (1) identify the onset of cambial activity, earlywood and latewood formation; (2) evaluate the phenology of cambium in the two species; and (3) analyse how many, when and what type of IADFs are formed in the two species. Cambium phenology and IADF genesis were compared between the two species and with climatic data to evaluate which factors play a role in IADF formation. The findings are also discussed in reference to hypotheses raised from previous studies based on retrospective analyses of tree-ring series showing IADFs throughout the Mediterranean region. MATERIALS AND METHODS Species, study site and climatic conditions The study was conducted on trees of stone pine (Pinus pinea L.) and strawberry tree (Arbutus unedo L.), co-occurring at a site in southern Italy. The study area, located on Mt. Vesuvius (40°78′N, 14°42′E, 200 m asl) (Fig. 1A) consists of a stone pine forest bordered by an area dominated by strawberry tree bushes. Stand density is 2500 P. pinea and 5000 A. unedo trees per hectare. The climate at the site is Mediterranean, with hot and dry summers followed by mild and wet winters. Sampling of microcores to analyse cambial productivity and xylem formation in the two species was carried out in 2015, which was characterized by an annual mean temperature of 17.8 °C. The hottest month was July (monthly average mean temperature of 28.5 °C), while the coldest month was February (monthly average mean temperature of 9.4 °C). The cumulative annual precipitation was 1054 mm, the wettest month was October, with a cumulative monthly precipitation of 286 mm, while the lowest value was recorded in July (cumulative precipitation of 9.4 mm). The arid period lasted from May to July (Fig. 1B). A review of long-term meteorological series from the same site, obtained from KNMI Climate Explorer (Trouet and Van Oldenborgh, 2013), indicated that meteorological conditions in 2015 did not deviate significantly from those of the long-term series (1995–2014). Fig. 1. View largeDownload slide (A) Geographic location of the study site in southern Italy. (B) Climatic diagram of monthly average mean temperatures (T) and total monthly precipitation (P) for 2015 elaborated from the gridded data set E-OBS (14°25′–14°50′E, 40°75′–41°00′N). Fig. 1. View largeDownload slide (A) Geographic location of the study site in southern Italy. (B) Climatic diagram of monthly average mean temperatures (T) and total monthly precipitation (P) for 2015 elaborated from the gridded data set E-OBS (14°25′–14°50′E, 40°75′–41°00′N). Tree-ring identification Dendrochronological techniques were applied to build tree-ring chronologies to allow the cross-dating of tree rings in order to identify the xylem increment formed in 2015. Core sampling was performed on 20 trees of P. pinea and 20 plants (not multistem) of A. unedo, by taking two cores at breast height from each tree (west and east directions) with an increment borer (diameter 5 mm). Cores were air-dried, mounted in a grooved wooden block, and their surface was polished with a graded series of sandpapers. Tree-ring series were visually cross-dated by comparison of signature rings and calendar dates assigned to the year of formation of each ring to facilitate the cross-dating of tree rings through dendrochronological techniques (Stokes and Smiley, 1968). IADFs were identified according to De Micco et al. (2012, 2014). Tree-ring width (TRW) measurement was done to the nearest 0.01 mm, using LINTAB measuring equipment and TSAP Win software (Frank Rinn, Heidelberg, Germany). The quality of cross-dating was checked using the R package dplR (Bunn, 2008), and two master chronologies were built, one for each species. The selection of six trees for xylogenesis and wood anatomical analyses was done taking into account the cores with the highest correlation with the master chronology of each species. Descriptive statistics of raw data of the six selected trees were calculated, as well as the expressed population signal (EPS), which indicates the level of coherence of the constructed chronology and how it portrays the hypothetical perfect population chronology (Wigley et al., 1984; Buras, 2017), the mean sensitivity (MS), which is a measure of the mean percentage change from each measured yearly ring value to the next, and the Gleichläufigkeit (GLK), which is a measure of the year to year agreement between the interval trends of two chronologies based upon the sign of agreement, were calculated for the chronologies. Microcore sampling and microscopy For the collection of microcores, six healthy trees/plants per species were selected according to the dendrochronological parameters. For P. pinea, the stem diameter at breast height was 53.3 ± 5.9 cm (mean ± s.d.), the height was about 16 m and age was about 95 years. For A. unedo, the stem diameter at breast height was 15.6 ± 4.9 cm, the height was 4–8 m and age was about 20 years. Microcores (1.8 mm diameter) were collected using a Trephor tool (Rossi et al., 2006) at weekly intervals from January 2015 until January 2016. In order to avoid wound effects, the points of microcore extraction were chosen following a spiral with a distance of 3–4 cm between consecutive samples. Each microcore contained inner phloem, cambium and xylem (at least three of the last-formed xylem rings). The extracted microcores were immediately fixed in 70 % ethanol and stored at 4 °C. The samples were dehydrated in a graded ethanol series (70, 90, 95 and 100 %), infiltrated with bio-clear (D-limonene) and embedded in paraffin blocks using a Leica TP1020-1 (Nussloch, Germany) tissue processor. Cross-sections (9 μm thick) were cut with a semi-automatic rotary microtome RM 2245 (Leica, Nussloch), using low profile microtome blades. The sections were placed on glass slides pre-treated with albumin and dried at 70 °C for 30 min. After removing the residual paraffin through bio-clear and ethanol, the sections were stained with a safranin (0.04 %) and astra blue (0.15 %) water solution (van der Werf et al., 2007) and mounted in Euparal (Bioquip Rancho Dominguez, CA, USA). The sections were observed under a Nikon Eclipse 800 light microscope, and microphotographs were taken by means of a DS-Fi1 digital camera with the NIS-Elements BR 3 image analysis system (Melville, NY, USA). The sections were analysed to identify and measure cambial and differentiating xylem cells, using visual criteria of proportional dimension and wall thickness (de Luis et al., 2007, 2011a; Čufar et al., 2008, 2011; De Micco et al., 2016b). Sections from samples collected every 2 weeks were measured, while for periods of high cambial production, sections collected every week were used. Although the transition from earlywood to latewood is generally abrupt in Pinus species, we applied the criteria to define earlywood and latewood based on the proportion of tracheid cell wall thickness and lumen diameter (Denne, 1989; Antony et al., 2012) to classify tracheids in P. pinea as earlywood or latewood as well as to define earlywood-like and latewood-like cells at the IADF level. More specifically, all tracheids whose common double cell wall is equal to or greater than the cell lumen (parameters always measured in the radial direction) are considered latewood or latewood-like. In A. unedo, the transition between earlywood and latewood was not always sharp, but latewood was characterized by the presence of narrower (half lumen diameter) and less frequent vessels than earlywood (De Micco et al., 2016c). On transverse sections, cambium cells (CCs) and cells in different differentiation phases were identified and counted, i.e. post-cambial or enlarging cells (PCs), cells undergoing secondary thickening and lignification of the cell walls (SWs) and mature cells (MTs) (Čufar et al., 2011; de Luis et al., 2011a; De Micco et al., 2016b) (Fig. 2). CCs were radially flattened, while PCs were characterized by larger radial dimensions. SWs had thicker cell walls, stained blue or light red, and could be discriminated from PCs by using polarized light (Rossi et al., 2006). MTs were cells with completely thickened and lignified cell walls, staining completely red and without any trace of protoplast in the lumen. Active production of xylem cells by the cambium was indicated by an increase in the number of cells and width of CCs and the presence of PC phases compared with the dormant state. Fig. 2. View largeDownload slide (A) Developing xylem in P. pinea and (B–D) A. unedo. Moving from the cambial zone towards the centre of the stem, the following cells are encountered: cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall and no protoplast in the lumen (MTs). Arrowheads in (D) mark the transition between SWs and MTs. Scale bars = 100 µm. Fig. 2. View largeDownload slide (A) Developing xylem in P. pinea and (B–D) A. unedo. Moving from the cambial zone towards the centre of the stem, the following cells are encountered: cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall and no protoplast in the lumen (MTs). Arrowheads in (D) mark the transition between SWs and MTs. Scale bars = 100 µm. In P. pinea, the number of cells was counted along three radial rows for all phenological phases (CCs, PCs, SWs and MTs). Since the boundary limiting the ending of wood formed in 2014 and the beginning of wood formed in 2015 was not always clear, counting MTs formed in 2015 was not straightforward and required the definition of a sort of point zero. This was established in sections of the first microcore sampling by counting the number of all MTs, starting from the beginning of the tree ring formed in 2014 (the transition between 2013 and 2014 tree rings was abrupt). This point zero number was subtracted from all MTs counted in successive microcores using the same principle (all MTs starting from the beginning of the 2014 tree ring). In A. unedo, in which vessels and fibres are not arranged in ordered radial rows, the width of all zones occupied by the various cell types in the different phenological phases was measured according to De Micco et al. (2016b). Values, i.e. measurements and counting along three radial rows of different xylem differentiation phases, were averaged. To avoid the influence of variability around the circumference, the number for P. pinea, and the width for A. unedo, of cells and tissues in the previous xylem ring were quantified to normalize the measurements (Rossi et al., 2003; Prislan et al., 2013). To detect the timing of IADF occurrence, we analysed the microsections from subsequent microcores by focusing on SWs and MTs, to highlight changes in cell lumen size and wall thickness marking the transition from earlywood to latewood, and vice versa, as reported in De Micco et al. (2016b). RESULTS Tree-ring series The statistics of the tree-ring chronologies are reported in Table 1 and refer to the six trees for each species selected for xylogenesis analyses. Arbutus unedo plants were much younger than P. pinea trees. The TRW chronologies of the two species showed different tree-ring patterns (Fig. 3), with no significant correlations between them. Table 1. Summary of the statistics of tree-ring chronologies for the analysed trees of P. pinea and A. unedo Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 TRW, tree-ring width; EPS, expressed population signal; GLK, Gleichläufigkeit, MS, mean sensitivity. EPS and r-bar are calculated for the period 2000–2015 for A. unedo and 1940–2015 for P. pinea. View Large Table 1. Summary of the statistics of tree-ring chronologies for the analysed trees of P. pinea and A. unedo Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 Pinus pinea Arbutus unedo TRW mean (1/100 mm) 222 ± 106 372 ± 141 TRW mean 2015 (1/100 mm) 175 ± 57 317 ± 115 EPS 0.950 0.957 GLK 96 % 84 % r-bar 0.717 0.312 MS 0.13 0.28 TRW, tree-ring width; EPS, expressed population signal; GLK, Gleichläufigkeit, MS, mean sensitivity. EPS and r-bar are calculated for the period 2000–2015 for A. unedo and 1940–2015 for P. pinea. View Large Fig. 3. View largeDownload slide Tree-ring width (TRW) chronologies of six P. pinea (black line) and six A. unedo (grey line). Bars indicate the s.d. Fig. 3. View largeDownload slide Tree-ring width (TRW) chronologies of six P. pinea (black line) and six A. unedo (grey line). Bars indicate the s.d. The mean ring widths (Table 1) of A. unedo were higher than those of P. pinea for the whole tree-ring chronologies (P < 0.05) and for 2015 (P < 0.001). Both chronologies have an EPS value >0.85, indicating that the chronologies were representative of radial growth variations of the whole population of trees. All the statistics listed in Table 1 showed a strong common tree-ring signal among individuals of the same species and high year to year radial growth variability associated with inter-annual changes in climatic conditions, especially for A. unedo, which represents the highest mean sensitivity. Cambial productivity and phenology of xylem formation The status of cambial productivity on the first sampling date [29 January 2015, day of year (DOY) 29] was different in the two analysed species, being active in P. pinea (Fig. 4A) and dormant in A. unedo (Fig. 5A). Curves showing the number of CCs (Fig. 6A, E), PCs (Fig. 6B, F) and SWs (Fig. 6C, G) during the sampling period followed a characteristic bimodal shape, with the second peak more evident in P. pinea than in A. unedo. The curve representing the number of MTs showed a typical sigmoid shape in A. unedo (Fig. 6H), while in P. pinea both SWs and new MTs were also present during the period January–March 2015 (Fig. 6D). Fig. 4. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea trees forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 43, (C) DOY 72, (D) DOY 100, (E) DOY 127, (F) DOY169, (G) DOY 189, (H) DOY 240, (I) DOY 268, (L) DOY 296, (M) DOY 317 and (N) DOY 345. Arrowheads point to the boundary between the 2014 and 2015 tree rings; the non-abrupt tree-ring boundary is evident in (F), (H) and (N). Images are taken at the same magnification. Scale bar = 100 µm. Fig. 4. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea trees forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 43, (C) DOY 72, (D) DOY 100, (E) DOY 127, (F) DOY169, (G) DOY 189, (H) DOY 240, (I) DOY 268, (L) DOY 296, (M) DOY 317 and (N) DOY 345. Arrowheads point to the boundary between the 2014 and 2015 tree rings; the non-abrupt tree-ring boundary is evident in (F), (H) and (N). Images are taken at the same magnification. Scale bar = 100 µm. Fig. 5. View largeDownload slide Light microscopy views of microsections of microcores of A. unedo plants forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 106, (C) DOY 168, (D) DOY 239 and (E) DOY 301. Images are taken at the same magnification. Scale bar = 50 µm. Fig. 5. View largeDownload slide Light microscopy views of microsections of microcores of A. unedo plants forming IADFs in the 2015 tree ring. (A) DOY 29, (B) DOY 106, (C) DOY 168, (D) DOY 239 and (E) DOY 301. Images are taken at the same magnification. Scale bar = 50 µm. Fig. 6. View largeDownload slide Number of cells in various phases of xylem formation in P. pinea trees (A–D) and width of different developmental xylem zones in A. unedo plants (E–H): cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall (MTs). Mean values and standard errors are shown. DOY, number of days from 1 January 2015 to 22 January 2016. Fig. 6. View largeDownload slide Number of cells in various phases of xylem formation in P. pinea trees (A–D) and width of different developmental xylem zones in A. unedo plants (E–H): cambial cells (CCs), enlarging post-cambial cells (PCs), cells developing secondary walls (SWs) and mature cells with lignified secondary wall (MTs). Mean values and standard errors are shown. DOY, number of days from 1 January 2015 to 22 January 2016. In more detail, in the developing xylem of P. pinea, CCs consisted of 7.30 ± 0.66 cells (mean ± s.d.), while PCs consisted of 1.94 ± 0.82 cells in the period from the end of January until the end of March (DOY 29–86) (Figs 4A–E and 6A, B). At the end of March (DOY 86), the number of CCs and PCs started to increase (8.46 ± 1.06 and 3.94 ± 1.65, respectively), reaching the peak between DOY 100 and 112 (Figs 4F, G and 6A, B). The number of CCs and PCs then declined, reaching minimum values during the summer months (Figs 4H–Q and 6A, B). A second peak in the number of CCs (9.11 ± 1.00) occurred on 8 October (DOY 282) and of PCs (3.20 ± 1.57) on 22 October (DOY 296), after which they decreased again, reaching a minimum value on 4 January 2016 (6.22 ± 0.45 and 0.88 ± 0.40, respectively) (Fig. 6A, B). The curve of SWs showed a similar pattern to those of CCs and PCs, although shifted by a couple of weeks (Fig. 6C). The number of SWs started to increase in the middle of April, reaching the maximum value (12.22 ± 3.27) on 21 May (DOY 142). A second peak of SWs was detected in November (DOY 317). MTs were detected from the first sampling onwards: their number was low (6.49 ± 4.78) and started to increase in mid-May (DOY 142) (10.64 ± 1.87), reaching a maximum value (37.47 ± 13.71) in August (DOY 225), which was maintained until the end of the year (Fig. 6D). The cambium in A. unedo was still dormant on the first sampling date (CC width was 14.76 ± 2.78 µm) (Figs 5A and 6E). The first signs of activity were observed on 10 April (DOY 100), with the production of PCs (22.93 ± 6.33 µm) (Figs 5B and 6F). Cambial production reached a peak in May (DOY 142) (37.73 ± 8.55). A high presence of PCs was also found in April–June, with a peak on 7 May (DOY 127) (103.60 ± 61.95) (Figs 5C and 6F). At the beginning of July (DOY 189), we observed a decrease in CCs (22.24 ± 2.23), and such low values were observed until the beginning of October (DOY 282) (Figs 5D and 6E). In the second half of July and until the beginning of October, the width of the PC zone was lower than at the beginning of the year, suggesting that the cambium was not productive (Fig. 6F). An increased width of the PC zone (23.6 ± 18.30) was again observed around 22 October (DOY 296) (Figs 5E and 6F). Cambial productivity stopped again in December (DOY 345), when the ring consisted of MTs only (Fig. 6H). Genesis of IADFs Microscopy observation of the cross-sections of the microcores sampled at the beginning of 2016 showed that, in the period of the investigation (January 2015–January 2016) and in the two previous years, 2014 and 2013, tree rings showed IADFs in both P. pinea (Fig. 7A–C) and A. unedo (Fig. 7D–F). Such IADFs were of type L, being characterized by the presence of earlywood-like cells in latewood at the end of the tree ring (Fig. 7, arrows). Fig. 7. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea and A. unedo plants sampled in January 2016. IADFs (arrows) were observed in the years 2015 (A, D), 2014 (B, E) and 2013 (C, F). Scale bars = 100 µm. Fig. 7. View largeDownload slide Light microscopy views of microsections of microcores of P. pinea and A. unedo plants sampled in January 2016. IADFs (arrows) were observed in the years 2015 (A, D), 2014 (B, E) and 2013 (C, F). Scale bars = 100 µm. In P. pinea, from the first sampling in 2015, cambium was productive and the boundary of the previous tree ring (corresponding to 2014) was not characterized by an abrupt transition from true latewood to earlywood tracheids: in the months January–March 2015 (DOY 29–71), ‘earlywood-like cells’ were formed, but with narrower lumina and thicker cell walls, corresponding to so-called transition tracheids (Fig. 4A–C). True earlywood MTs were evident at the end of March until the beginning of July (Fig. 4C–G); thereafter, the production of latewood cells started and led to mature latewood cells in the second half of July (DOY 211). Latewood tracheids were formed and differentiated until the beginning of October (DOY 281) (Fig. 4G–L). The genesis of L-IADFs occurred in mid-October with the production of earlywood-like tracheids lasting about 60 d; the first mature earlywood-like cells were evident on 22 October (DOY 295) and their production continued until the middle of December (Fig. 4L–N). The production of latewood then restarted, and mature true latewood cells were evident on 22 December (DOY 356). In A. unedo, no cambial cell production was found before April (DOY 100), the production of earlywood started later and lasted until the beginning of July, when mature latewood cells became evident (DOY 183). The production of latewood cells continued until the middle of October. All samples showed the formation of L-type IADFs, with the occurrence of mature earlywood-like cells on 22 October (DOY 295); such a production of earlywood cells lasted >90 d. Latewood maturation was evident again on 10 December (DOY 344), while cambium production stopped again at the beginning of January 2016. Moreover, in three out of six samples, the formation of more than one IADF was found in the year of investigation, with additional earlywood-like cells maturing in August (DOY 239) and new production of latewood cells in September (DOY 257) (Fig. 7D). DISCUSSION In Mediterranean climates, different patterns of cambial activity occur in different species and in various microclimatic conditions (Camarero et al., 2010; Martinez del Castillo et al., 2016). Martinez del Castillo et al. (2016) recently showed that different tree species, namely Pinus sylvestris and Fagus sylvatica, although growing at the same site, have different phenology of xylem formation, probably depending on their different life strategies and adaptive capacity under limiting environmental conditions. In our study, analysis of xylogenesis in two phylogenetically distant plant species, specifically a conifer tree and a hardwood shrub/tree, partially supported this idea. The two species showed a different timing of cambial production and different patterns of tree-ring chronologies (as underlined by low correlation between the two series), although bimodal xylogenesis leading to the formation of the same type of IADF in latewood (L-IADF) was found in both of them. The duration of cambial production was longer in P. pinea than in A. unedo under the same climatic conditions, confirming that the duration of xylogenesis is species specific. Indeed, P. pinea did not show a complete halt in cambial activity in winter, as assumed for Pinus spp. and other species, in which microcoring studies have been focused from spring to autumn (de Luis et al., 2007; Čufar et al., 2011; Martinez del Castillo et al., 2016). It has recently been shown that cambial production of PCs in P. halepensis can continue until the end of December and that its reactivation can be observed as early as February or March, indicating that the cambium in P. halepensis from a Mediterranean site was clearly not dormant (Novak et al., 2016a). In P. pinea growing on Mt. Vesuvius, we found that, although slow, xylogenesis took place from January until March, while in A. unedo cambial production restarted in April after the winter pause. However, in the two species, the first peak of cambial production (highest incidence of CCs and PCs) occurred in the same period, between the end of April and the beginning of May, when the air temperature increased. This agrees with several studies, also based on experiments of artificial heating of the stem, which indicate that the increase in spring temperature is the main factor triggering the onset of xylogenesis (Gričar et al., 2005, 2006; Begum et al., 2010; Prislan et al., 2011; Vieira et al., 2014). On the other hand, the onset of latewood in the two analysed species seemed to be synchronized with a strong decrease in precipitation, coinciding with the highest values of average mean temperature (July). In A. unedo, the appearance of mature latewood conduits occurred 1–2 weeks earlier than in P. pinea. A similar temporal shift in the onset of latewood was also found between A. unedo and P. halepensis co-occurring at the same site in a previous study in southern Italy, suggesting that A. unedo has a faster, more plastic control of cambial processes than Pinus spp. (De Micco et al., 2016b). The second peak of cambial production seemed to be strongly correlated with precipitation in October, which was very abundant in the year of investigation (millimetres of rain were more than double in October compared with September). The production of earlywood-like cells in latewood continued until the middle of December, when the temperature decreased again, reaching minimum values. The occurrence of L-IADFs in P. pinea and A. unedo is in line with findings in other Mediterranean species, such as Juniperus thurifera and A. unedo itself, developing in Mediterranean ecosystems in Spain, Portugal and Italy; in these studies, L-IADFs were ascribed to an extended period of growth due to autumn precipitation creating favourable conditions for wood growth (Camarero et al., 2010; de Luis et al., 2011b; Vieira et al., 2015; Zalloni et al., 2016). The mechanism triggering L-IADFs is linked with increased cell enlargement because of high turgor pressure in enlarging cells due to high water availability after abundant autumn rain (Sperry et al., 2006; De Micco et al., 2016a; Pacheco et al., 2016). The same mechanism has been reported for the formation of L-IADFs in P. halepensis and A. unedo, although in a different period of the year: a second burst of growth in summer after episodes of rain (De Micco et al., 2016b). In A. unedo, the high sensitivity of the rhythm of cambium production in response to changing environmental conditions may be responsible for extended xylogenesis and the frequent occurrence of more than a single IADF per calendar year, as found in this investigation and in agreement with previous observations (Cherubini et al., 2003; De Micco et al., 2016b). The high predisposition of A. unedo to form IADFs may also be due to the young plant age, which is correlated to larger tree rings which, in turn, are positively correlated with IADF frequency (Rigling et al., 2001, 2002; Cherubini et al., 2003; Campelo et al., 2015; Pacheco et al., 2016; Zalloni et al., 2016). An extension of xylogenesis in P. sylvestris has also been ascribed to mild autumn temperatures (Martinez del Castillo et al., 2016). Latewood is then produced again in winter, when low temperatures limit the processes of cell production and differentiation, and can lead to cessation of cell production or dormancy, as found in A. unedo. The presence of two unfavourable periods has been reported to be responsible for the spread of evergreen sclerophyllous shrubs in the Mediterranean environment (Mitrakos, 1980). Arbutus unedo, like other Mediterranean shrubs, has also adapted its phenology and reproduction to seasonal fluctuations (Aronne and Wilcock, 1997). The low sensitivity of P. pinea to winter temperature would explain the onset of latewood delayed by 1 week compared with A. unedo, and its lack of true cambium dormancy, leading to the formation of a few rows of transition wood made of earlywood-like tracheids before the production of true earlywood tracheids. Such a phenomenon explains why the boundary between successive tree rings is not always abrupt. This is probably due to the winter temperature (reaching minimum values of about 9°C), which is not low enough to induce cambium dormancy (Prislan et al., 2016). The cells that are considered to be the last ones in the tree ring are therefore not formed at the end of the calendar year. In P. pinea, the lack of cambium dormancy due to mild winter conditions is in agreement with findings reported for P. radiata growing in New Zealand and P. halepensis at a Mediterranean site in Spain (Barnett, 1987; Prislan et al., 2016). In a recent study by Prislan et al. (2016), a temperature of 10 °C in January did not induce cambial dormancy in P. halepensis. The production of earlywood-like tracheids has been also found in tree-ring chronologies of P. pinea and P. pinaster growing in Portugal: classified as L+-IADF. Such a wood type has been associated with above-average precipitation in early autumn (Campelo et al., 2007), leading to decreased deposition of cell walls (Antonova and Stasova, 1997). In our case, we prefer to refer to these earlywood-like cells as transition wood, according to a more recent re-classification of IADFs (De Micco et al., 2016a), but from a genesis viewpoint they may be assimilated to L+-IADF reported by Campelo et al. (2007), because they were formed at the beginning of the calendar year when rain was abundant after a dry period in December. Such transition wood makes the boundary between tree rings less abrupt, and care should be taken to refer to it as wood formed at the beginning of the calendar year (January–March in our case) and not in the last months of the previous year. This becomes a critical point in tree-ring dating and a warning to avoid errors in the case of interpretation of dendroclimatological analysis. Indeed, such findings suggest that a new perspective should be assumed when applying a dendrochronological approach to Mediterranean species, dismissing the idea of synchronizing visually identified tree rings with calendar years. Explorative xylogenesis analyses should be performed to synchronize sectors of tree rings to specific periods of the year, thus moving from a yearly resolution dendroclimatological analysis to a seasonal resolution analysis, also analysing in detail intra-annual wood anatomical functional traits. Indeed, such an approach has been recently applied to several species, allowing more accurate ecological information to be extracted from long tree-ring series (Carrer et al., 2017). Whatever the triggering factors, the occurrence of IADFs, or of transition wood, in tree rings is responsible for adjustment of the balance between efficiency and safety of water transport. Optimization of the efficiency/safety of water transport can be achieved through plastic control of conduit size during wood formation and through the occurrence of particular anatomical traits, especially in hardwoods (e.g. presence of tracheids, vessel redundancy, occurrence of vessels with different class diameters, etc.) (De Micco et al., 2008, 2009). The presence of IADFs can be interpreted as the maintenance of a sapwood characterized by different sectors: those showing anatomical traits promoting efficient water transport (earlywood and earlywood-like regions accounting for most water flow when water is available) and those showing traits of safety (latewood and latewood-like regions) which are critical to maintain water flow when larger conduits embolize. In the scenario of increasing drought and changes in the precipitation frequency, the occurrence of a plastic response at wood formation level, resulting in increased IADF frequency, would confer an adaptive advantage to species having bimodal cambial activity because the resulting wood anatomical peculiarities would allow the exploitation of water availability at best also maximizing water use efficiency (Pacheco et al., 2016). In conclusion, the occurrence of true earlywood in P. pinea was detected earlier than the onset of cambial activity in A. unedo, but in both species the appearances of mature latewood, of earlywood-like cells and of latewood again after the IADF were fairly well synchronized. This indicates that, despite having completely different wood formation models and different life strategies, the production of earlywood, latewood and earlywood-like cells is strongly controlled by climatic factors in the two species. Moreover, the lack of true winter dormancy, the slightly delayed onset of latewood in P. pinea compared with A. unedo, the occurrence of more than one IADF per year in A. unedo and the delayed onset of latewood after the IADF are all signs of a lower sensitivity of the softwood species and the higher plasticity of the hardwood species in response to variations in temperature and water availability. Indeed, the presence of IADFs in tree rings could be considered a consequence of plant plasticity in response to environmental fluctuations typical of Mediterranean climates and would indicate a more adaptive capability to cope with changing water availability in species forming frequent IADFs compared with species less prone to form IADFs. ACKNOWLEDGEMENTS This study profited from discussions within the COST Action STReESS (COST-FP1106), it was supported by COST (European Cooperation in Science and Technology) and by the Slovenian Research Agency (ARRS), programme P4-0015 and project Z4-7318. We thank Martin Cregeen for editing the English text. A.B., G.B. and V.D.M. conceived and designed the study. A.B. performed sampling. A.B. and M.M. performed sample preparation. V.D.M., A.B., P.P., K.C. and G.B. made a substantial contribution to the analysis of tree-ring series and anatomical signals in microsections and in data analysis. V.D.M., A.B., K.C. and G.A. contributed to interpretation of the overall data. V.D.M., K.C., G.A. and G.B. contributed to the analysis tools. A.B. and V.D.M. wrote the main part of the manuscript. K.C. and G.B. contributed to writing specific parts of the text. All authors made a critical revision of the manuscript, and read and approved the submitted version of the manuscript. LITERATURE CITED Antonova G , Stasova V . 1997 . Effects of environmental factors on wood formation in larch (Larix sibirica Ldb.) stems . Trees 11 : 462 – 468 . Antony F , Schimleck LR , Daniels RF . 2012 . A comparison of earlywood–latewood demarcation methods – a case study in loblolly pine . IAWA Journal 33 : 187 – 195 . Aronne G , Wilcock CC . 1997 . Reproductive phenology in Mediterranean macchia vegetation . Lagascalia 19 : 445 – 454 . Barnett JR . 1987 . The development of fibre-tracheid pit membranes in Pyrus communis L . IAWA Journal 8 : 134 – 142 . Google Scholar CrossRef Search ADS Battipaglia G , De Micco V , Brand WA et al. 2010 . Variations of vessel diameter and δ13C in false rings of Arbutus unedo L. reflect different environmental conditions . New Phytologist 188 : 1099 – 1112 . Google Scholar CrossRef Search ADS PubMed Battipaglia G , Campelo F , Vieira J et al. 2016 . Structure and function of intra-annual density fluctuations: mind the gaps . Frontiers in Plant Science 7 : 595 . doi: 10.3389/fpls.2016.01170 . Google Scholar CrossRef Search ADS PubMed Begum S , Nakaba S , Oribe Y , Kubo T , Funada R . 2010 . Cambial sensitivity to rising temperatures by natural condition and artificial heating from late winter to early spring in the evergreen conifer Cryptomeria japonica . Trees 24 : 43 – 52 . Google Scholar CrossRef Search ADS Bunn AG . 2008 . A dendrochronology program library in R (dplR) . Dendrochronologia 26 : 115 – 124 . Google Scholar CrossRef Search ADS Buras A . 2017 . A comment on the expressed population signal . Dendrochronologia 44 : 130 – 132 . Google Scholar CrossRef Search ADS Camarero JJ , Olano JM , Parras A . 2010 . Plastic bimodal xylogenesis in conifers from continental Mediterranean climates . New Phytologist 185 : 471 – 480 . Google Scholar CrossRef Search ADS PubMed Campelo F , Vieira J , Battipaglia G et al. 2015 . Which matters most for the formation of intra-annual density fluctuations in Pinus pinaster: age or size ? Trees 29 : 237 – 245 . Google Scholar CrossRef Search ADS Campelo F , Vieira J , Nabais C . 2013 . Tree-ring growth and intra-annual density fluctuations of Pinus pinaster responses to climate: does size matter ? Trees 27 : 763 – 772 . Google Scholar CrossRef Search ADS Campelo F , Nabais C , Freitas H , Gutiérrez E . 2007 . Climatic significance of tree-ring width and intra-annual density fluctuations in Pinus pinea from a dry Mediterranean area in Portugal . Annals of Forest Science 64 : 229 – 238 . Google Scholar CrossRef Search ADS Carrer M , Castagneri D , Prendin AL , Petit G , von Arx G . 2017 . Retrospective analysis of wood anatomical traits reveals a recent extension in tree cambial activity in two high-elevation conifers . Frontiers in Plant Science 8 : 737 . doi: 10.3389/fpls.2017.00737 . Google Scholar CrossRef Search ADS PubMed Cherubini P , Gartner BL , Tognetti R , Bräker OU , Schoch W , Innes JL . 2003 . Identification, measurement and interpretation of tree rings in woody species from Mediterranean climates . Biological Review 78 : 119 – 148 . Google Scholar CrossRef Search ADS Cuny HE , Rathgeber CBK , Lebourgeois F , Fortin M , Fournier M . 2012 . Life strategies in intra-annual dynamics of wood formation: example of three conifer species in a temperate forest in north-east France . Tree Physiology 32 : 612 – 625 . Google Scholar CrossRef Search ADS PubMed Cuny HE , Rathgeber CBK , Frank D , Fonti P , Fournier M . 2014 . Kinetics of tracheid development explain conifer tree-ring structure . New Phytologist 203 : 1231 – 1241 . Google Scholar CrossRef Search ADS PubMed Cuny HE , Rathgeber CBK , Frank D et al. 2015 . Woody biomass production lags stem-girth increase by over one month in coniferous forests . Nature Plants 1 : 1 – 6 . Google Scholar CrossRef Search ADS Čufar K , Prislan P , Gričar J . 2008 . Cambial activity and wood formation in beech (Fagus sylvatica) during the 2006 growth season . Wood Research 53 : 1 – 10 . Čufar K , Cherubini M , Gričar J , Prislan P , Spina S , Romagnoli M . 2011 . Xylem and phloem formation in chestnut (Castanea sativa Mill.) during the 2008 growing season . Dendrochronologia 29 : 127 – 134 . Google Scholar CrossRef Search ADS De Micco V , Aronne G . 2009 . Seasonal dimorphism in wood anatomy of the Mediterranean Cistus incanus L. subsp. incanus . Trees 23 : 981 – 989 . Google Scholar CrossRef Search ADS De Micco V , Saurer M , Aronne G , Tognetti R , Cherubini P . 2007 . Variations of wood anatomy and δ13C within tree rings of coastal Pinus pinaster showing intra-annual density fluctuations . IAWA Journal 28 : 61 – 74 . Google Scholar CrossRef Search ADS De Micco V , Aronne G , Baas P . 2008 . Wood anatomy and hydraulic architecture of stems and twigs of some Mediterranean trees and shrubs along a mesic–xeric gradient . Trees 22 : 643 – 655 . Google Scholar CrossRef Search ADS De Micco V , Battipaglia G , Cherubini P , Aronne G . 2014 . Comparing methods to analyse anatomical features of tree rings with and without intra-annual density fluctuations (IADFs) . Dendrochronologia 32 : 1 – 6 . Google Scholar CrossRef Search ADS De Micco V , Campelo F , De Luis M et al. 2016a. Intra-annual density fluctuations in tree rings: how, when, where, and why ? IAWA Journal 37 : 232 – 259 . Google Scholar CrossRef Search ADS De Micco V , Balzano A , Čufar K et al. 2016b. Timing of false ring formation in Pinus halepensis and Arbutus unedo in Southern Italy: outlook from an analysis of xylogenesis and tree-ring chronologies . Frontiers in Plant Science 7 : 705 . doi: 10.3389/fpls.2016.00705 . Google Scholar CrossRef Search ADS PubMed De Micco V , Battipaglia G , Balzano A , Cherubini P , Aronne G . 2016c. Are wood fibres as sensitive to environmental conditions as vessels in tree rings with intra-annual density fluctuations (IADFs) in Mediterranean species ? Trees – Structure and Function 30 : 971 – 983 Google Scholar CrossRef Search ADS Denne MP . 1989 . Definition of latewood according to Mork (1928) . IAWA Journal 10 : 59 – 62 . Google Scholar CrossRef Search ADS Deslauriers A , Morin H . 2005 . Intra-annual tracheid production in balsam fir stems and the effect of meteorological variables . Trees 19 : 402 – 408 . Google Scholar CrossRef Search ADS Fonti P , von Arx G , Garcia-Gonzalez I et al. 2010 . Studying global change through investigation of the plastic responses of xylem anatomy in tree rings . New Phytologist 185 : 42 – 53 . Google Scholar CrossRef Search ADS PubMed Gričar J , Čufar K , Oven P , Schmitt U . 2005 . Differentiation of terminal latewood tracheids in Silver fir trees during autumn . Annals of Botany 95 : 959 – 965 . Google Scholar CrossRef Search ADS PubMed Gričar J , Zupančič M , Čufar K , Koch G , Schmitt U , Oven P . 2006 . Effect of local heating and controlling on cambial activity and cell differentiation in the stem of Norway spruce (Picea abies) . Annals of Botany 97 : 943 – 951 . Google Scholar CrossRef Search ADS PubMed Gruber A , Pirkebner D , Florian C , Oberhuber W . 2012 . No evidence for depletion of carbohydrate pools in Scots pine (Pinus sylvestris L.) under drought stress . Plant Biology 14 : 142 – 148 . Google Scholar PubMed de Luis M , Gričar J , Čufar K , Raventós J . 2007 . Seasonal dynamics of wood formation in Pinus halepensis from dry and semi-arid ecosystems in Spain . IAWA Journal 28 : 389 – 404 . Google Scholar CrossRef Search ADS de Luis M , Novak K , Raventós J , Gričar J , Prislan P , Čufar K . 2011a. Cambial activity, wood formation and sapling survival of Pinus halepensis exposed to different irrigation regimes . Forest Ecology Management 262 : 1630 – 1638 . Google Scholar CrossRef Search ADS de Luis M , Novak K , Raventós J , Gričar J , Prislan P , Čufar K . 2011b. Climate factors promoting intra-annual density fluctuations in Aleppo pine (Pinus halepensis) from semiarid sites . Dendrochronologia 29 : 163 – 169 . Google Scholar CrossRef Search ADS Martinez del Castillo EM , Longares LA , Gričar J et al. 2016 . Living on the edge: contrasted wood-formation dynamics in Fagus sylvatica and Pinus sylvestris under Mediterranean conditions . Frontiers in Plant Science 7 : 370 . doi: 10.3389/fpls.2016.00370 . Google Scholar CrossRef Search ADS PubMed Mitrakos K . 1980 . A theory for Mediterranean plant life . Acta Oecologica 1 : 245 – 252 . Novak K , de Luis M , Raventós J , Čufar K . 2013a. Climatic signals in tree-ring widths and wood structure of Pinus halepensis in contrasted environmental conditions . Trees 27 : 927 – 936 . Google Scholar CrossRef Search ADS Novak K , Saz Sánchez MA , Čufar K , Raventós J , de Luis M . 2013b. Age, climate and intra-annual density fluctuations in Pinus halepensis in Spain . IAWA Journal 34 : 459 – 474 . Google Scholar CrossRef Search ADS Novak K , De Luis M , Gričar J et al. 2016a. Missing and dark rings associated with drought in Pinus halepensis . IAWA Journal 37 : 260 – 274 Google Scholar CrossRef Search ADS Novak K , De Luis M , Saz MA et al. 2016b. Missing rings in Pinus halepensis – the missing link to relate the tree-ring record to extreme climatic events . Frontiers in Plant Science 7 : 727 . doi: 10.3389/fpls.2016.00727 . Google Scholar CrossRef Search ADS PubMed Pacheco A , Camarero JJ , Carrer M . 2016 . Linking wood anatomy and xylogenesis allows pinpointing of climate and drought influences on growth of coexisting conifers in continental Mediterranean climate . Tree Physiology 36 : 502 – 512 . Google Scholar CrossRef Search ADS PubMed Prislan P , Schmitt U , Koch G , Gričar J , Čufar K . 2011 . Seasonal ultrastructural changes in the cambial zone of beech (Fagus sylvatica) grown at two different altitudes . IAWA Journal 32 : 443 – 459 . Prislan P , Gričar J , de Luis M , Smith KT , Čufar K . 2013 . Phenological variation in xylem and phloem formation in Fagus sylvatica from two contrasting sites . Agricultural and Forest Meteorology 180 : 142 – 151 . Google Scholar CrossRef Search ADS Prislan P , Gričar J , de Luis M et al. 2016 . Annual cambial rhythm in Pinus halepensis and Pinus sylvestris as indicator for climate adaptation . Frontiers in Plant Science 7 : 1923 . doi: 10.3389/fpls.2016.01923 . Google Scholar CrossRef Search ADS PubMed Rigling A , Waldner PO , Forster T , Bräker O , Pouttu A . 2001 . Ecological interpretation of tree-ring width and intra annual density fluctuations in Pinus sylvestris on dry sites in the central Alps and Siberia . Canadian Journal of Forest Research 31 : 18 – 31 . Google Scholar CrossRef Search ADS Rigling A , Bräker OU , Schneiter G , Schweingruber FH . 2002 . Intra-annual tree-ring parameters indicating differences in drought stress of Pinus sylvestris forests within the Erico–Pinion in the Valais (Switzerland) . Plant Ecology 163 : 105 – 121 . Google Scholar CrossRef Search ADS Rossi S , Deslauriers A , Morin H . 2003 . Application of the Gompertz equation for the study of xylem cell development . Dendrochronologia 21 : 33 – 39 . Google Scholar CrossRef Search ADS Rossi S , Deslauriers A , Anfodillo T et al. 2006 . Conifers in cold environments synchronize maximum growth rate of tree-ring formation with day length . New Phytologist 170 : 301 – 310 . Google Scholar CrossRef Search ADS PubMed Rossi S , Anfodillo T , Čufar K et al. 2013 . A meta-analysis of cambium phenology and growth: linear and non-linear patterns in conifers of the northern hemisphere . Annals of Botany 112 : 1911 – 1920 . Google Scholar CrossRef Search ADS PubMed Ružička K , Ursache R , Hejátko J , Helariutta Y . 2015 . Xylem development – from the cradle to the grave . New Phytologist 207 : 519 – 535 . Google Scholar CrossRef Search ADS PubMed Sarris D , Christodoulakis D , Körner C . 2007 . Recent decline in precipitation and tree growth in the eastern Mediterranean . Global Change Biology 13 : 1187 – 1200 . Google Scholar CrossRef Search ADS Scarpella E , Meijer AH . 2004 . Pattern formation in the vascular system of monocot and dicot plant species . New Phytologist 164 : 209 – 242 . Google Scholar CrossRef Search ADS Sperry JS , Hacke UG , Pittermann J . 2006 . Size and function of conifer tracheids and angiosperm vessels . American Journal of Botany 93 : 1490 – 1500 . Google Scholar CrossRef Search ADS PubMed Steppe K , Sterck F , Deslauriers A . 2015 . Diel growth dynamics in tree stems: linking anatomy and ecophysiology . Trends in plant science 20 : 335 – 343 . Google Scholar CrossRef Search ADS PubMed Stocker TF , Qin D , Plattner GK et al. 2013 . IPCC, 2013: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change . Cambridge : Cambridge University Press . Stokes MA , Smiley TL . 1968 . Introduction to tree-ring dating . Chicago, IL : University of Chicago Press . Swidrak I , Gruber A , Oberhuber W . 2014 . Xylem and phloem phenology in co-occurring conifers exposed to drought . Trees 28 : 1161 – 1171 . Google Scholar CrossRef Search ADS PubMed Trouet V , Van Oldenborgh GJ . 2013 . KNMI Climate Explorer: a web-based research tool for high-resolution paleoclimatology . Tree-Ring Research 69 : 3 – 13 . Google Scholar CrossRef Search ADS Van der Werf GW , Sass-Klaassen UG , Mohren GM J . 2007 . The impact of the 2003 summer drought on the intra-annual growth pattern of beech (Fagus sylvatica L.) and oak (Quercus robur L.) on a dry site in the Netherlands . Dendrochronologia 25 : 103 – 112 . Google Scholar CrossRef Search ADS Vieira J , Rossi S , Campelo F , Freitas H , Nabais C . 2014 . Xylogenesis of Pinuspinaster under a Mediterranean climate . Annals of Forest Science 71 : 71 – 80 . Google Scholar CrossRef Search ADS Vieira J , Campelo F , Rossi S , Carvalho A , Freitas H , Nabais C . 2015 . Adjustment capacity of maritime pine cambial activity in drought-prone environments . PLoS One 10 : e0126223 . doi: 10.1371/journal.pone.0126223 . Google Scholar CrossRef Search ADS PubMed Wigley TM , Briffa KR , Jones PD . 1984 . On the average value of correlated time series, with applications in dendroclimatology and hydrometeorology . Journal of Climate and Applied Meteorology 23 : 201 – 213 . Google Scholar CrossRef Search ADS Xia J , Niu S , Ciais P et al. 2015 . Joint control of terrestrial gross primary productivity by plant phenology and physiology . Proceedings of the National Academy of Sciences, USA 112 : 2788 – 2793 . Google Scholar CrossRef Search ADS Zalloni E , de Luis M , Campelo F et al. 2016 . Climatic signals from intra-annual density fluctuation frequency in Mediterranean pines at a regional scale . Frontiers in Plant Science 7 : 579 . doi: 10.3389/fpls.2016.00579 . Google Scholar CrossRef Search ADS PubMed Ziaco E , Biondi F , Rossi S , Deslauriers A . 2016 . Environmental drivers of cambial phenology in Great Basin bristlecone pine . Tree Physiology 36 : 818 – 831 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Annals of BotanyOxford University Press

Published: Feb 3, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off