Variations in Wood Density, Annual Ring Width and Vessel Properties of Quercus Brantii Affected by Crown Dieback

Article Variations in Wood Density, Annual Ring Width and Vessel Properties of Quercus brantii Affected by Crown Dieback

Forough Soheili 1, Stephen Woodward 2, Isaac Almasi 3, Hazandy Abdul-Hamid 4,5 and Hamid Reza Naji 1,*

1 Department of Forest Sciences, Ilam University, Ilam 69315-516, Iran; [email protected] 2 School of Biological Sciences, University of Aberdeen, Aberdeen AB24 3UU, UK; [email protected] 3 Department of Statistics, Razi University, Kermanshah 67144-15111, Iran; [email protected] 4 Institute of Tropical Forestry and Forest Products (INTROP), Universiti Putra Malaysia, Serdang 43400, Malaysia; [email protected] 5 Faculty of Forestry and Environment, Universiti Putra Malaysia, Serdang 43400, Malaysia * Correspondence: [email protected]

Abstract: Tree decline due to climate change results in physiological weaknesses, attacks by harmful pests and pathogens and threats to forest ecosystem stability. In the work described here, the effects of drought on wood density, tree ring width and variations in vessel morphology are investigated in Persian oak (Quercus brantii) in the forest of the Zagros Mountains, Ilam Province, western Iran. Discs are cut from trunks of declined and healthy trees and woodblocks are cut radially from the sapwood near the bark, at a mid-point between the vascular cambium and the pith (middle) and from wood near the pith. Observations are made on transverse sections from the blocks using microscopy. In trees with decline symptoms, wood density is greater than in healthy trees. Furthermore, declining trees have the narrowest ring width, reduced vessel diameter and area and the highest numbers of vessels and tylose in pith towards the bark. It is concluded that changes in anatomical features are Citation: Soheili, F.; Woodward, S.; associated with the weakening of trees and are components of declining tree health. Almasi, I.; Abdul-Hamid, H.; Naji, H.R. Variations in Wood Density, Annual Ring Width and Vessel Keywords: climate change; tree decline; wood density; anatomy; Quercus brantii Properties of Quercus brantii Affected by Crown Dieback. Forests 2021, 12, 642. https://doi.org/10.3390/ f12050642 1. Introduction Forest dieback is a complex phenomenon characterized by decreasing tree growth, Academic Editor: Veronica De Micco defoliation and change in leaf size, shape and color, leading to the immediate or gradual death of trees [1]. Decline and mortality of oak species have been reported for more than Received: 12 April 2021 a century [2], sometimes occurring in vast areas of forest due to complex and interacting Accepted: 10 May 2021 reactions involving environmental stresses, pests and diseases [3]. The Zagros forest in Published: 19 May 2021 western Iran is the largest oak forest in the world, covering over 5 million ha, dominated by Persian oak (Quercus brantii L.). Forest decline has been reported in this oak forest and Publisher’s Note: MDPI stays neutral is ultimately resulting in extensive tree mortality [4]. In the past decade, the impacts of with regard to jurisdictional claims in climate change on the Zagros oak forest have led to serious forest disturbance, including published maps and institutional affil- dust storms, fires and pest and disease outbreaks, leading to a decline in the main forest iations. tree species of the region [5]. Knowledge of anatomy and function in secondary xylem is a key issue in understanding tree defenses against pathogens [6,7]. Tree ring width records, for example, can provide valuable information about the responses of trees to past environmental events [8,9]. Copyright: © 2021 by the authors. Regulation of vascular cambium activity over the years is crucial to the ability of Licensee MDPI, Basel, Switzerland. trees to adjust to fluctuating environmental conditions and, therefore, is highly responsive This article is an open access article to climate change. Annual ring structure, however, is stable compared to other morpho- distributed under the terms and logical characteristics of trees [10,11]. Although variations in tree-ring structure are well conditions of the Creative Commons understood, little is known of the intrinsic mechanisms responsible for variations [12]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ The water potential in the xylem is a key parameter that is widely used to monitor 4.0/). drought stress conditions throughout the plant [13]. The vessel features are influenced

Forests 2021, 12, 642. https://doi.org/10.3390/f12050642 https://www.mdpi.com/journal/forests Forests 2021, 12, x FOR PEER REVIEW 2 of 13

climate change. Annual ring structure, however, is stable compared to other morphological characteristics of trees [10,11]. Although variations in tree-ring structure are well understood, little is known of the intrinsic mechanisms responsible for variations [12]. Forests 2021, 12, 642 2 of 13 The water potential in the xylem is a key parameter that is widely used to monitor drought stress conditions throughout the plant [13]. The vessel features are influenced by dryness and drought, as demonstrated previously in oaks [14,15]. Reducing vessel size— byand, dryness as a consequence, and drought, water as demonstratedconductivity— previouslymakes the vessels in oaks more [14,15 vulnerable]. Reducing to vesselcavita- size—and,tion [16]. Anderegg as a consequence, et al. [17] waterand Giagli conductivity—makes et al. [18] demonstrated the vessels decreased more vulnerable cambium ac- to cavitationtivity, shortened [16]. Anderegg periods etof al.cambial [17] and activity, Giagli formation et al. [18] demonstrated of narrower growth decreased rings cambium and in- activity,tra-annual shortened wood density periods fluctuations of cambial, resulting activity, formationfrom abiotic of stresses narrower on growth the vascular rings cam- and intra-annualbium. Abiotic wood stresses density such fluctuations,as dust storms, resulting as consequences from abiotic of stressesclimate onchange, the vascular are also cambium.known to affect Abiotic annual stresses ring such widths as dust and storms, vessel diameters as consequences and widths of climate in Q. change,brantii [19], are alsopossibly known due to to affect disruptions annual ringto hormonal widths and regulation vessel diameters during wood and widthsformation in Q. [20]. brantii [19], possiblyTo prepare due to disruptions evidence-based to hormonal management regulation plans during for the wood protection formation of the [20 forest]. in the ZagrosTo preparemountains, evidence-based it is crucial to management understand plansthe importance for the protection of biotic ofand the abiotic forest factors in the Zagrosand the mountains, relationship it between is crucial the to understandtwo in forest the decline. importance To our of knowledge, biotic and abioticthere has factors been andno specific the relationship study examining between this the issue. two in The forest aim decline. of the work To our reported knowledge, here, theretherefore, has been is to noimprove specific understanding study examining of the this impacts issue. Theof drought aim of thestress work on the reported wood here,structure therefore, of Q. bran- is to improvetii, focusing understanding on annual growth of the impacts increment of drought and vessel stress features. on the Improving wood structure our understand- of Q. brantii, focusinging of how on the annual environment growth increment influences and host vessel plants features. and the associated Improving pests our understanding and pathogens ofwill how help the to environment predict the influencesperformance host of plants the forest and the during associated drought pests, to and provide pathogens practical will helpsolutions to predict for forest the performance management. of the forest during drought, to provide practical solutions for forest management. 2. Material and Methods 2. Material and Methods 2.1.2.1. StudyStudy AreasAreas ThisThis studystudy waswas conductedconducted onon deaddead andand healthyhealthy PersianPersian oakoak treestrees ((QuercusQuercus brantiibrantii L.)L.) affectedaffected by by drought drought stress stress in three in three forest forest regions: regions: Dareh Dareh shahr shahr (A), Sheshdar (A), Sheshdar (B) and Chavar(B) and (C),Chavar all in (C) Ilam, all Province, in Ilam P Westernrovince, IranWestern (Figure Iran1). (Figure The trees 1). had The grown trees had as a grown result ofas naturala result regenerationof natural regeneration and were sampledand were from sampled areas from with areas uniform with conditionsuniform conditions of habitat, of altitude habitat, andaltitude topography, and topography, all in the all same in the biosocial same biosocial classes. The classes. dominant The dominant trees in the trees forest in the were forestQ. brantiiwere Q., Crataegus brantii, Crsp.,ataegusAcer monspessulanumsp., Acer monspessulanumand a shrub, andDaphne a shrub,sp. Daphne Important sp. Important information in- fromformation the study from areas the study is given areas in Tableis given1. in Table 1.

FigureFigure 1.1. LocationLocation ofof thethe forestforest sampledsampled inin thethe IlamIlam ProvinceProvince inin westernwestern Iran.Iran. Forests 2021, 12, 642 3 of 13

Table 1. Basic characteristics of the Quercus brantii forest sampled for this work.

Av. Precip. Av. Temp. Av. RH Forest Area Altitude (m) Longitude Latitude mm ◦C% Dareh shahr (A) 933 33◦3030” N 47◦19030” E 465.1 19.5 41 Sheshdar (B) 2230 33◦38055” N 46◦30037” E 582.2 16.9 40 Chavar (C) 1680 33◦43003” N 46◦14036” E Av. Precip.: Average precipitation; Av. Temp.: Average temperature; Av. RH: Average relative humidity. Climate data were based on the synoptic meteorological station of Ilam.

2.2. Sampling Method The sampled trees in each area were located close to each other. As the forest is protected, no permission to cut trees in the stands could be obtained. Wood samples, therefore, were collected from the main trunks of trees previously felled by the Department of Natural Resources of Ilam Province under the plan to remove trees showing severe decline from the forest. Eighteen naturally regenerated trees (six trees from each area) of 70–90 years old, with DBH (diameter at breast height) ranging from 30–40 cm were selected. From each sample tree, a disc of approximately ﬁve cm in thickness was taken from breast height for further measurements. In the laboratory, each disc was carefully halved transversely, to give two discs for examination of wood density and anatomical properties. For healthy trees, core samples were taken using an increment borer at breast height. Wounds created on the trees were treated with fungicides and pesticides.

2.3. Wood Density From the first part of the disc, blocks with dimensions of 1 × 1 × 1 cm3 were cut continuously (no interval) from the sapwood near the bark, towards the wood near the pith, using a rotary saw. For the samples from healthy trees, the woodblocks were prepared based on the size of the wood cores. Oven-dry wood densities of the samples were calculated based on ISO 3131-1975 (E) [21]. Wood samples were first oven-dried at 103 ± 2 ◦C to reach 0% moisture content and were weighed. Sample blocks were subsequently soaked in distilled water and block volumes were obtained with an accuracy of ±0.01 g. Prior to immersion, the samples were covered with a thin layer of paraffin wax. The wood density index (WD) was calculated as follows:

M g ρ = = (1) V cm3 where ρ, M and V are density, mass and volume, respectively.

2.4. Preparation for Macroscopic Measurement The second part of each disc was prepared using a series of sandpapers (80, 240 and 400) on an orbital sanding machine to attain a smooth, perfectly clear surface [22], before scanning at 2400 dpi resolution. Annual ring widths from 1987–2016 from each disc were measured radially from the pith to the most recent sapwood under a binocular microscope attached to a LINTAB 5 ring-width measuring system (Rinntech Company, Heidelberg, Germany).

2.5. Sample Preparation for Sectioning The disc was cut radially into blocks of 1 cm3 at three positions between the bark and the pith: sapwood near the bark (near-bark), at a mid-point between the vascular cambium and pith (middle) and in the wood immediately adjacent to the pith (near-pith). Each sample block contained approximately 10 growth rings. Blocks were labelled with the area, dieback class and their position on the disc. To better conserve the woodblocks, samples were ﬁxed in FAA solution (50% ethanol: acetic acid: formaldehyde at 1:1:1, v:v:v). To Forests 2021, 12, 642 4 of 13

soften the blocks for better sectioning and to improve section quality, samples were boiled at 100 ◦C for 60 min.

2.6. Sectioning Cross-sections with thicknesses of 20–25 µm were cut using a rotary microtome (POOYAN MK 1110, Iran), transferred to 10% sodium hypochlorite solution for 5 min until totally bleached and then to distilled water until no bleach odor was detectable. Sections were stained in 0.1% (w/v) safranin O, washed twice in distilled water for approximately 5 min and dehydrated in an ethanol series of 60%, 85%, 95% and absolute for 15 min each. Dehydrated sections were ﬁxed on slides using Canada balsam [23], prior to observation under an Olympus cx22LED microscope. Photographs of sections were captured using a digital camera (True chrome metrics, Fuzhou, China) attached to a computer. Tangential and radial diameters of earlywood vessels (DEV, µm), earlywood vessels’ area (EVA, µm2), vessels’ density (number of vessels per mm2, NV) and the total number of tyloses (NT, mm−1) visible in 30 standard observation ﬁelds of 500 µm2 were measured from each decline class from each sampling area.

2.7. Statistical Analysis Data were subjected to one-way analysis of variance (ANOVA). The normality and homogeneity of the data were evaluated using Shapiro–Wilk’s and Levene’s tests, respectively. Independent sample t-tests were carried out to determine differences in wood properties among healthy and declined trees at 1% and 5% levels of probability. The data were analyzed in SPSS software, version 21.

3. Results 3.1. Physical-Anatomical Variables 3.1.1. Wood Density (WD) There were signiﬁcant overall differences in WD between healthy and declined trees from the sampled areas (p < 0.01; Table2). The oven-dry WD of declined trees was higher than the WD in healthy trees (Table3). A decreasing trend in WD from near-pith to near- bark was found in declined trees sampled in all three areas. Comparing healthy trees Forests 2021, 12, x FOR PEER REVIEW in areas B and C against declined trees, there was a noticeable reduction in WD in the 5 of 13 near-pith vs. middle, with no signiﬁcant difference in the middle vs. near-bark blocks (Figure2).

) 0.9 3 D1 0.8 H1 D2 0.7 H2 0.6 D3

Wood density (g/cm density Wood H3 0.5 P M B

Radial sampling position FigureFigure 2. 2. WoodWood density density of declineddeclined (D1–D3) (D1–D3 and) and healthy healthy trees trees (H1–H3) (H1–H3 of )Quercus of Quercus brantii brantiiin three in three areasareas of of the the Iranian Iranian forest forest:: DarehDareh shahr, shahr, Sheshdar Sheshdar and and Chavar. Chavar. Density Density was measuredwas measured in the in wood the wood samplessamples from from near near-pith-pith towardstowards near-bark. near-bark H:. H: Healthy Healthy trees; trees; D: Declined D: Declined trees; 1:trees Area; 1: A; A 2:rea Area A; 2: B; Area B; 3:3: AreaArea C; C P:; P: Near-pith; Near-pith M:; M: Middle; Middle B: Near-bark.; B: Near-bark).

3.1.2. Annual Ring Width (ARW) Comparing ARW over time (Table 2), there was a significant difference (p < 0.01) between the means of declined compared with healthy trees across all three areas. The ARWs of declined trees from all sampling areas decreased over time, compared with healthy trees (Table 3). The decreases of ARW in declined trees were very similar in areas A and C. In healthy trees in both areas A and C, there was a similar trend in ARW changes between 2000–2016. Furthermore, in area B, ARWs decreased in declined trees compared to healthy trees (Figure 3).

12 D1 10 H1 8 D2 H2 6 D3 4 H3 Tree ring width (mm) width ring Tree 2

Year

Figure 3. Time series of annual ring width of Quercus brantii for declined (D1–D3) and healthy trees (H1–H3).

Forests 2021, 12, 642 5 of 13

Forests 2021, 12, x FOR PEER REVIEW Table 2. One-way ANOVA summarizing the signiﬁcance of wood-anatomical variables (WD:5 woodof 13 density; ARW: annual ring width) between healthy and declined Quercus brantii trees affected by drought stress in three oak forest areas in Iran.

1 Mean Square Source of Variation df WD (g/cm3) ARW (µm)

) 0.9 3 Decline 1 0.57 ** 115.408 ** D1 Areas 2 0.13 ** 201.184 ** 0.8 H1 Decline × Areas 2 0.005 * 47.531 ** D2 0.7Error 594 0.002 1.439 H2 WD: wood density; ARW: annual ring width; * Signiﬁcance at 0.05; ** Signiﬁcance at 0.01. 0.6 D3

Table (g/cm density Wood 3. Summary of Student’s t-test analyses of differences in wood characteristicsH3 (means ± SE) between0.5 healthy and declined Quercus brantii affected by drought stress. P M B Attributes Sampling Group Mean Difference t-test Radial sampling position Declined 0.92 ± 0.02 WD (g/cm3) 0.048 5.181 * Figure 2. Wood density of declinedHealthy (D1–D3) and healthy 0.87 ±trees0.06 (H1–H3) of Quercus brantii in three areas of the Iranian forest: Dareh shahr, Sheshdar and Chavar. Density was measured in the wood Declined 0.78 ± 0.24 ARW (µm) 1.60 5.241 * samples from near-pith towardsHealthy near-bark. H: Healthy 2.38 trees;± 0.54 D: Declined trees; 1: Area A; 2: Area B; 3: Area C; P: Near-pith; M: Middle; B: Near-bark). WD: wood density; ARW: annual ring width; * p < 0.01. 3.1.2. Annual Ring Width (ARW) 3.1.2. Annual Ring Width (ARW) Comparing ARW over time (Table 2), there was a significant difference (p < 0.01) be- Comparing ARW over time (Table2), there was a signiﬁcant difference ( p < 0.01) tweenbetween the themeans means of ofdeclined declined compared compared with with healthy healthy trees trees across across all all three three area areas.s. Th Thee ARWARWss of of declined declined trees trees from from all all sampling sampling area areass decreased decreased over over time, time, compared compared with with healthyhealthy trees trees (Table (Table 3).3). The The decreases decreases of of ARW ARW in in declined declined trees trees were were very very similar similar in in area areass AA and and C. C. In In healthy healthy trees trees in in both both area areass A A and and C, C, there there was was a asimilar similar trend trend in in ARW ARW changes changes betweenbetween 2000 2000–2016.–2016. Furthermore, Furthermore, in in area area B, B, ARW ARWss decreased decreased in in declined declined trees trees compared compared toto healthy healthy trees trees (Figure (Figure 3).3).

12 D1 10 H1 8 D2 H2 6 D3 4 H3 Tree ring width (mm) width ring Tree 2

Year

Figure 3. Time series of annual ring width of Quercus brantii for declined (D1–D3) and healthy trees Figure 3. Time series of annual ring width of Quercus brantii for declined (D1–D3) and healthy trees(H1–H3). (H1–H3).

Forests 2021, 12, 642 6 of 13

3.2. Wood Anatomical Features 3.2.1. Diameter of Earlywood Vessels (DEV) Variance analysis (Table4) showed the effect of decline on DEV in the three parts of the stem cross-section: near-pith, middle and near-bark. The sampling area also had a significant impact on DEV in the middle. The interaction effects of decline and area were also significant in the three sampled parts of the trees. The mean DEV of declined trees showed a descending trend from the near-pith to the bark in areas A and C and was constant and similar in healthy trees. In the declined trees from area B, the trend in radial direction was constant and similar, while in healthy trees from area B, there was a decreasing trend from near-pith to near-bark (285.6 down to 262.1 µm, respectively) (Figure4). In general, the mean DEVs in declined trees from all three sampled parts of the stem were lower than in healthy trees. There were significant differences in DEV between healthy and declined trees in the near-pith, middle and near-bark (Table5).

Table 4. One-way ANOVA of wood-anatomical variables between healthy trees and those with decline at the three study areas.

Mean Square The Source of Variation df DEV (µm) EVA (µm−2) NV (mm−2) NT (mm−2) Pith 38.977 ** 27.906 ** 1.533 ns 14.092** Decline Middle1 762.045 ** 127.558 ** 2.219 ns 8.477 ** Bark 508.746 ** 64.223 ** 11.486 ** 111.562 ** Pith 1.571 ns 1.71 ns 3.633 * 40.897 ** Area Middle2 9.400 ** 3.984 * 6.040 ** 96.150 ** Bark 1.125 ns 4.442 * 1.332 ns 52.205 ** Pith 13.842 ** 6.941 ** 0.722 ns 6.764 ** × Decline Area Middle2 32.288 ** 1.330 ns 0.367 ns 2.898 ns Bark 67.281 ** 4.221 * 1.964 ns 10.815 ** Pith 12,267.234 2.622 × 1011 2.010 0.918 Error Middle594 2808.444 2,165,248,861 1.730 1.387 Bark 3290.675 4,289,042,107 2.104 1.396 DEV: Diameter of earlywood vessels; EVA: Earlywood vessels’ area; NV: Number of vessels; NT: Number of tyloses; * Significantly different at p < 0.05; ** Significantly different at p < 0.01; ns No significant difference.

Table 5. Differences in wood characteristics (means ± SE) between healthy and declined trees based on Student’s t-test.

Anatomical Sampling Pith Middle Bark Group Features Declined 228.43 ± 15.54 ** 171.74 ± 6.72 ** 173.85 ± 7.9 ** DEV (µm) Healthy 284.88 ± 10.00 291.19 ± 6.28 282.93 ± 7.18 Declined 38,754 ± 3391.46 ** 21,669 ± 2852.86 ** 22,720 ± 2646.88 ** µ 2 EVA ( m ) Healthy 74,562.83 ± 13,280.79 64,579.56 ± 7088.08 65,573.28 ± 10,484.12 ns ns −2 Declined 1.62 ± 0.16 2.09 ± 0.16 2.24 ± 0.18 ** NV (mm ) Healthy 1.48 ± 0.14 1.93 ± 0.12 1.79 ± 0.18 ns ns −2 Declined 0.67 ± 0.12 1.32 ± 0.16 1.15 ± 0.16 ** NT (mm ) Healthy 1.02 ± 0.14 1.04 ± 0.12 0.28 ± 0.06 DEV: Diameter of earlywood vessels; EVA: Earlywood vessels’ area; NV: Number of vessels; NT: Number of tyloses; ** p < 0.01; ns No signiﬁcant difference. Forests 2021, 12, x FOR PEER REVIEW 6 of 13

significant in the three sampled parts of the trees. The mean DEV of declined trees showed a descending trend from the near-pith to the bark in areas A and C and was constant and similar in healthy trees. In the declined trees from area B, the trend in radial direction was constant and similar, while in healthy trees from area B, there was a decreasing trend from near-pith to near-bark (285.6 down to 262.1 μm, respectively) (Figure 4). In general, the mean DEVs in declined trees from all three sampled parts of the stem were lower than in Forests 2021, 12, 642 healthy trees. There were significant differences in DEV between healthy and declined7 of 13 trees in the near-pith, middle and near-bark (Table 5).

350 a a a 300 a a a a a D1 250 b H1 b b b b b 200 b D2 b H2 150 D3 100 H3

50 Diameter of earlywood vessel (µm) vessel earlywood of Diameter 0 P M B Radial sampling position

Figure 4. Mean diameter of earlywood vessels in declined and healthy trees of Quercus brantii. Figure 4. Mean diameter of earlywood vessels in declined and healthy trees of Quercus brantii. H: HH:ealthy Healthy trees trees;; D: Declined D: Declined trees; trees;1: Area 1: A Area; 2: A A;rea 2: B;Area 3: Area B; 3:C; AreaP: Near C;-pith P: Near-pith;; M: Middle M:; B: Middle; Near- B: bark.Near-bark. The bar The on bareach on column each column represents represents the SE ( thestandard SE (standard error). Columns error). Columns with no letters with no on letters them on indicatethem indicate insignificance. insigniﬁcance.

Table 4. One-way ANOVA of3.2.2. wood Earlywood-anatomical Vessels’variables Area between (EVA) healthy trees and those with decline at the three study areas. Decline and sampling area had significant effects on EVA in the middle and near-bark, with no significant differences in the near-pith. In addition, interactionMean Square effects between The Source ofdecline Variation× area were significantdf in near-barkDEV (μm) samples,EVA (μm but−2) not significantNV (mm−2) in theNT (mm middle−2) or ns near-pith (TablePith 4). Significant differences38.977 ** were found,27.906 ** however,1.533 in the middle14.092** samples Decline Middle 1 762.045 ** 127.558 ** 2.219 ns 8.477 ** (Table4). TheBark mean EVA of declined508.746 trees ** showed64.223 a decreasing ** 11.486 trend ** from111.562 near-pith ** to near-bark atPith all three sampling areas.1.571 Healthy ns trees1.71 in areasns A and3.633 B showed* a40.897 decreasing ** Area trend in EVAMiddle from near-pith to2 near-bark,9.400 ** while an3.984 increasing * trend6.040 was** observed96.150 in** trees from areaBark C (53,816 to 82,628 µm2, respectively)1.125 ns (Figure4.442 * 5). In general,1.332 ns EVA52.205 in declined ** trees from thePith three sampling positions13.842 in stems** was6.941 lower ** than in0.722 healthy ns trees.6.764 Significant ** Decline × Area differencesMiddle in EVA were found2 between32.288 healthy ** and1.330 declined ns trees0.367 in ns the near pith,2.898 ns middle ns and near-barkBark xylem (Table5). 67.281 ** 4.221 * 1.964 10.815 ** Pith 12,267.234 2.622 × 1011 2.010 0.918 Error 3.2.3. NumberMiddleof Vessels (NV)594 2808.444 2,165,248,861 1.730 1.387 Bark 3290.675 4,289,042,107 2.104 1.396 DEV: Diameter of earlywood vesselsDecline; EVA: had Earlywood a significant vessels’effect area; NV: on NVNumber only of invessels near-bark; NT: N xylemumber of tissues tyloses; (Table * 4). Significantly different at p < Sampling0.05; ** Signifi area,cantly however, different hadat p < a 0.01; significant ns No significant effect on difference. NV inall three sampling positions. There was no significant interaction between decline × area in the near bark, middle and near-pith tissues. The highest NV was in the near-bark in declined trees from all three areas. In the healthy trees, an increasing trend in NV occurred from near-pith to near- bark in samples from area A (1.3 to 1.62 mm−1, respectively). In contrast, healthy trees from area B had an increasing trend in NV from near-pith to near-bark (1.39 to 2 mm−2, respectively). However, healthy trees in area C showed a fixed trend from near-pith to near-bark (Figure6). In general, NV in declined trees from the three sampled parts of the xylem tissues (near-pith, middle and near-bark) were higher than in healthy trees. Significant differences in NV were observed between healthy and declined trees only in near-bark xylem (Table5). Forests 2021, 12, x FOR PEER REVIEW 7 of 13

Table 5. Differences in wood characteristics (means ± SE) between healthy and declined trees based on Student’s t-test.

Anatomical Sampling Pith Middle Bark Group Features Declined 228.43 ± 15.54 ** 171.74 ± 6.72 ** 173.85 ± 7.9 ** DEV (μm) Healthy 284.88 ± 10.00 291.19 ± 6.28 282.93 ± 7.18

Declined 38,754 ± 3391.46 ** 21,669 ± 2852.86 ** 22,720 ± 2646.88 ** EVA (μm2) Healthy 74,562.83 ± 13,280.79 64,579.56 ± 7088.08 65,573.28 ± 10,484.12 Declined 1.62 ± 0.16 ns 2.09 ± 0.16 ns 2.24 ± 0.18 ** NV (mm−2) Healthy 1.48 ± 0.14 1.93 ± 0.12 1.79 ± 0.18 Declined 0.67 ± 0.12 ns 1.32 ± 0.16 ns 1.15 ± 0.16 ** NT (mm−2) Healthy 1.02 ± 0.14 1.04 ± 0.12 0.28 ± 0.06 DEV: Diameter of earlywood vessels; EVA: Earlywood vessels’ area; NV: Number of vessels; NT: Number of tyloses; ** p < 0.01; ns No significant difference.

3.2.2. Earlywood Vessels’ Area (EVA) Decline and sampling area had significant effects on EVA in the middle and near- bark, with no significant differences in the near-pith. In addition, interaction effects between decline × area were significant in near-bark samples, but not significant in the middle or near-pith (Table 4). Significant differences were found, however, in the middle samples (Table 4). The mean EVA of declined trees showed a decreasing trend from near-pith to near-bark at all three sampling areas. Healthy trees in areas A and B showed a decreasing trend in EVA from near-pith to near-bark, while an increasing trend was observed in trees from area C (53,816 to 82,628 μm2, respectively) (Figure 5). In general, EVA in de- Forests 2021, 12, 642 8 of 13 clined trees from the three sampling positions in stems was lower than in healthy trees. Significant differences in EVA were found between healthy and declined trees in the near pith, middle and near-bark xylem (Table 5).

120,000.00 a ) 2 - 100,000.00 a μm a D1 80,000.00 a a a a a H1 60,000.00 b a D2 b b b 40,000.00 b b H2 Forests 2021, 12, x FOR PEER REVIEW b 8 of 13 b 20,000.00 b D3 Earlywood Vessels area( Earlywood Vessels H3 the healthy trees,0.00 an increasing trend in NV occurred from near-pith to near-bark in samples from area A (1.3 to 1.62P mm−1, respectively).M In contrast, healthyB trees from area B had an increasing trend in NV from nearRadial-pith sampling to near- barkposition (1.39 to 2 mm−2, respectively). How- ever, healthy trees in area C showed a fixed trend from near-pith to near-bark (Figure 6). In general, NV in declined trees from the three sampled parts of the xylem tissues (near- pith,FigureFigure middle 5. 5.Comparisons Comparisons and near ofof-bark) meanmean were areaarea of ofhigher earlywoodearlywood than vessels vesselsin healthy inin declineddeclined trees. andandSignificant healthyhealthy treestreesdifferences ofof QuercusQuercus in NVbrantii.brantii were.H: H: Healthyobserved Healthy trees; trees between; D: D: Declined Declined healthy trees; trees and; 1: 1: declined Area Area A; A; 2: 2: trees Area Area only B; B; 3: 3: Area inArea near C; C; P:- P:bark Near-pith; Near xylem-pith; M: (TableM: Middle; Mid- 5). B:dle Near-bark.; B: Near-ba Therk. bar The on bar each on columneach column represents represents the SE the (standard SE (standard error). error).

3.2.3. Number3 of Vessels (NV) a Decline had a significant effect on NV only in near-bark axylem tissues (Table 4). Sam- 2.5 )

pling2 area, however, had a significant effect on NV in all three sampling positions. There

- b was no significant interaction between decline × area in the near bark, middleD1 and near- pith tissues.2 Thea highest NV was in the near-bark in declined trees from all three areas. In b H1 1.5 b D2 H2

1 D3

Number of vessels (µm vessels of Number H3 0.5

0 P M B Radial sampling position

Figure 6. Mean number of vessels in declined and healthy trees of Quercus brantii. H: Healthy trees; D: Figure 6. Mean number of vessels in declined and healthy trees of Quercus brantii. H: Healthy treesDeclined; D: D trees;eclined 1: Area trees A;; 1: 2: A Arearea A B;; 2: 3: A Arearea B C;; 3: P: A Near-pith;rea C; P: N M:ear Middle;-pith; M: B: Near-bark.Middle; B: N Theear bar-bark on. eachThe barcolumn on each represents column the represents SE (standard the SE error). (standard Columns error). with Columns no letters with on no them letters indicate on them insignificance. indicate insignificance. 3.2.4. Number of Tyloses (NT) 3.2.4.The Number effect of of Tyloses decline on(NT) the NT was significant in vessels in the near bark, middle and near-pith xylem of stem sections (Table4). NT varied significantly between the three forest The effect of decline on the NT was significant in vessels in the near bark, middle and areas in all sampled xylem. Interaction effects, however, was no significantly different only near-pith xylem of stem sections (Table 4). NT varied significantly between the three forest in the middle. In declined trees, NT showed an increasing trend from near-pith to near-bark areas in all sampled xylem. Interaction effects, however, was no significantly different in trees from all three areas. Healthy trees in areas A and B had the lowest NT in near-bark, only in the middle. In declined trees, NT showed an increasing trend from near-pith to increasing towards the pith. Furthermore, NT in healthy trees showed a decreasing trend near-bark in trees from all three areas. Healthy trees in areas A and B had the lowest NT from near-pith to near-bark in trees from area C (1.8 to 0.65 mm−2) (Figures7 and8). Thus, inhigher near- NTsbark, were increasing found intowards declined the trees pith. from Furthermore, the near-pith, NT in middle healthy and trees near-bark showed xylem, a de- −2 creasingcompared trend with from healthy near trees.-pith to Finally, near-bark there in weretrees significantfrom area C differences (1.8 to 0.65 betweenmm ) (Figure NT ins 7 and 8). Thus, higher NTs were found in declined trees from the near-pith, middle and near-bark xylem, compared with healthy trees. Finally, there were significant differences between NT in healthy and declined trees in near-bark, with no significant differences between the middle and near-pith vessels (Table 5).

Forests 2021, 12, 642 9 of 13

ForestsForests 2021 2021, 12, 12, x, xFOR FOR PEER PEER REVIEW REVIEW 9 9of of 13 13 healthy and declined trees in near-bark, with no signiﬁcant differences between the middle and near-pith vessels (Table5).

2.52.5 ) ) 2 2 - - aa D1D1 22 aa aa H1H1 1.51.5 D2D2 aa H2H2 bb bb 11 D3D3 bb aa H3H3 Number of tyloses (mm tyloses of Number Number of tyloses (mm tyloses of Number 0.50.5 bb bb 00 PP MM BB RadialRadial sampling sampling position position

Figure 7. Mean number of tyloses in xylem vessels of Quercus brantii. H: Healthy trees; D: Declined FigureFiguretrees; 1:7. 7. AreaMean Mean A; n number 2:umber Area of B;of tyloses 3:tyloses Area in C;in xylem P:xylem Near-pith; vessels vessels of M: of Quercus Middle;Quercus brantii B:brantii Near-bark.. H:. H: H Healthyealthy The bartrees trees on; D:; each D: D Declined columneclined treestrees; 1:; 1: A Arearea A A; 2:; 2: A Arearea B B; 3:; 3: A Arearea C C; P:; P: N Nearear-pith-pith; M:; M: M Middleiddle; B:; B: N Nearear-bark-bark. The. The bar bar on on each each col- col- represents the SE (standard error). Columns with no letters on them indicate insigniﬁcance. umnumn represents represents the the SE SE ( s(tandardstandard e rror).error). Columns Columns with with no no le letterstters on on them them indicate indicate insignificance. insignificance.

FigureFigure 8. 8. Tyloses TylosesTyloses (T) (T) (T) occluding occluding occluding vessel vessel vessel lumen lumen lumen in in transverse intransverse transverse section section section of of a adeclined of declined a declined tree tree from treefrom near from near- near-- barkbark tissues. tissues.

4.4. Discussion Discussion InIn this this this work, work, work, variations variations variations in in annual in annual annual growth growth growth ring ring ring and and andvessel vessel vessel sizes sizes sizesand and other andother otherwood wood woodana- ana- tomicaltomicalanatomical f eaturesfeatures features were were investigated were investigated investigated in in Quercus Quercus in Quercus brantii brantii affected brantii affectedaffected by by drought drought by droughtstress. stress. In In stress.general, general, In increasesincreasesgeneral, inincreases in NV NV and and in NT NT NV led led and to to increased NTincreased led to WD increasedWD in in declined declined WD intrees, trees, declined agreeing agreeing trees, with with agreeing previously previously with publishedpreviouslypublished work work published on on oak oak workshowing showing on oakthat that showingwood wood density density that wood varies varies density as as vessel vessel varies features features as vessel change change features [ 24[24].]. SantiagoSantiagochange [et24 et al.]. al. Santiago[ 25[25] ]indicated indicated et al. [that 25that] indicatedan an increase increase that in in anthe the increase total total surface surface in the area area total of of surface the the vessels vessels area leads ofleads the tovesselsto lower lower leadswood wood todensit densit lowery.y. woodThe The occurrence occurrence density. The of of vascular occurrencevascular anomalies anomalies of vascular as as reactions reactions anomalies to to mechanical asmechanical reactions ororto physiological mechanicalphysiological or disturbance disturbance physiological emphasizes emphasizes disturbance the the emphasizescrucial crucial importance importance the crucial of of xylem importancexylem vessels vessels of in in xylem tree tree physiology,vesselsphysiology, in tree as as it physiology, it is is thought thoughtas to to it play isplay thought a arole role in to in a play atrading trading a role triangle triangle in a trading comprising comprising triangle three comprisingthree competing competing three functions:functions:competing water water functions: transport, transport, water mechanical mechanical transport, mechanicalsupport support and and support resistance resistance and to resistance to embolisms embolisms to embolisms [ 26[26].]. [26]. DensityDensity is is isan an an important important important attribute attribute attribute in in inwood wood wood quality quality quality and and and is is associat isassociat associatededed with with with other other other deter- deter- de- minativeminativeterminative properties. properties. properties. Wood Wood Wood density density density mainly mainly mainly varies varies variesthrough through through differences differences differences in in anatomical anatomical in anatomical prop- prop- erties,properties,erties, such such as such as size size as and sizeand distribution and distribution distribution of of different different of different cells cells cells [ 27[27 [],27], and ],and and is is isstrongly strongly strongly related related related to to cell cell sizesize and and vessel vessel proportions proportions [ 28[28,29,29].]. In In the the work work on on Q. Q. b rantiibrantii presented presented here, here, the the DEV DEV and and EVAEVA of of declined declined trees trees from from the the three three sampled sampled forest forest a reasareas showed showed a adescending descending trend trend fromfrom near near-pith-pith to to near near-bark.-bark. Due Due to to the the decrease decrease in in the the DEV DEV and and EVA EVA and and increase increase in in the the

Forests 2021, 12, 642 10 of 13

size and vessel proportions [28,29]. In the work on Q. brantii presented here, the DEV and EVA of declined trees from the three sampled forest areas showed a descending trend from near-pith to near-bark. Due to the decrease in the DEV and EVA and increase in the NV in the wood in the near-bark of trees with decline, it appeared that vessel number and density were inversely correlated with average vessel area [30]. It is also known that an increased total surface area of vessels leads to lower wood density [25]. Annual rings are an important source of environmental information [31], enabling variations in climate to be tracked over thousands of years. Narrow rings indicate unde- sirable environmental conditions (drought or cold periods), whereas wide rings suggest good growth in reasonable conditions (warmer or wetter). In this work, the ARW of trees suffering from decline was significantly smaller than in healthy trees. Although the main driver of this decline in ARW was probably the drought suffered by the trees over a protracted time, it is also possible that changes in the life strategies of endophytic fungi present naturally in the plant tissues, becoming pathogenic under conditions stressful to the host, may further weaken the host plant [32]. The average DEV and EVA in declined trees were smaller than the same features in healthy Q. brantii. Decline due to drought could lead to higher NVs in affected trees, as the plants attempt to improve sap flow to the photosynthetic tissues in the crown. A simultaneous reduction of DEV and EVA will lead to a reduction in sap flow in the trees, especially those in decline. Thus, adverse changes in certain wood structural parameters such as vessel diameters are important for hydraulic conductivity. The changes may indicate that the tree genotypes most badly affected are poorly adapted to changing environmental conditions and will, therefore, begin to decline. Furthermore, in response to environmental changes, trees can regulate water conductance further by changing the size and density of the vessels [33,34]. Radial growth of declining Q. robur in Poland showed that the average tree ring width and diameter of earlywood vessels in trees that had died was lower than in healthy oaks [35]. Water conductance to leaves in ring-porous trees, such as oaks, is highly dependent on large, functional earlywood vessels. Usually, there is an inverse relationship between the size and the number of vessels, therefore, trees will optimize or increase hydraulic conductivity according to environmental conditions [36]. In trees affected by increasing environmental stresses, the diameter of earlywood vessels and, as a consequence, the hydraulic conductivity, decreases [37]. However, trees may form high numbers of vessels in an attempt to compensate for the problem [38,39]. Likewise, small vessel diameter is highly dependent on climate conditions, including drought [40,41]. Oaks (ring-porous species) usually have large and wide vessels, particularly in the earlywood, and embolism is relatively frequent [42]. An invading pathogen compromising the vascular system leads to the blocking of vessels by the expansion of tyloses from xylem companion cells, sometimes with the secretion of gums and gels to isolate the damaged tissues. The formation of tyloses is an important cause of vascular blockage, preventing sap flow. When the process occurs excessively, it can be a major cause of dieback in the plant. In this study, NT in declined trees from all three forest areas increased in near-bark vessels, compared to xylem in the near-pith. NT was directly related to NV and inversely related to DEV and EVA. Furthermore, there were significant differences in NT and NV in the near-bark of declined trees, but no significant differences in these features in the xylem in the near-pith of trees in decline. It is probable, therefore, that trees that eventually died from decline had the greatest weakness in hydraulic conductivity due to reductions in DEV and EVA and increases in NT and NV. It is well known that anatomical variations caused by abiotic stress decrease with increasing distance from the affected area (here, near-bark towards near-pith) [43]. The presence of tyloses in the earlywood vessels of the annual wood increment influences the reduction of hydraulic conductance in declining oaks [35]. Earlywood vessels contribute to large amounts of sap flow in the main stem and along branches but are vulnerable to embolization [44]. Decreased lumen diameters in severely stressed or infected Forests 2021, 12, 642 11 of 13

oak may assist in controlling water transport and reducing vulnerability to cavitation and embolism [45]. Large-scale and local climate change will have many different effects on individual trees, forest structure and growth performance, including the development and prevalence of fungal diseases [46]. Likely, the increasing occurrence of charcoal disease, caused by Biscogniauxia mediterranea, in oaks in the Zagros Mountains is related to recent drought phenomena, emphasizing the need to examine in-depth the relationship between abiotic climatic and biotic factors [7]. Many biotic and abiotic factors are certainly involved in the decline of oak trees, leading Henrique et al. [47] to suggest the complex interacting factors determining the roles of different factors in the survival of this tree.

5. Conclusions Recent studies have not provided comprehensive evidence quantifying the impact of drought stress on Persian oak’s anatomical features and wood density. The results presented here, however, have described the effects of abiotic stress on wood density, growth ring width, the diameter of vessels, vessels’ area and vessel and tyloses numbers. Notable variation was observed in ARW, DEV, EVA, NT and NV in trees from different sampling areas, in xylem from the near-pith to the near-bark of Q. brantii in decline. The WD of declined trees from the near-pith to the near-bark showed a decreasing trend. Water conductivity decreased in response to vessel size and NV increased in declined trees from near-pith to near-bark tissues, contributing to a loss of vitality, and ultimately, the death of trees. The results of this study provide a theoretical basis for future related research on hydraulic parameters in declined trees.

Author Contributions: F.S. and H.R.N. carried out the experiment and wrote the manuscript with support from S.W.; I.A. performed the computations and verified the analytical methods; H.A.-H. helped to supervise the project. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: Data Citation: Soheili, F., Woodward, S., Almasi, I., Abdul-Hamid, H., Naji, H.R., 2021; Total data Oak Decline Naji; https://figshare.com/s/4fc5300bf260bbad18bd, accessed on 12 April 2021; doi:10.6084/m9.figshare.14593422. Acknowledgments: We would like to thank A. Hawasi and S. Soltani for their kind help during this work. Conflicts of Interest: The authors declare no conflict of interest.

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