37 Les/Wood, Vol. 72, No. 2, December 2023 ANATOMY OF XYLEM AND PHLOEM IN STEMS AND ROOTS OF POPULUS SIBIRICA AND ULMUS PUMILA FROM SEMI-ARID STEPPE IN MONGOLIA ANATOMIJA KSILEMA IN FLOEMA DEBEL IN KORENIN DREVESNIH VRST POPULUS SIBIRICA IN ULMUS PUMILA IZ POLSUHE STEPE V MONGOLIJI Anastazija Dimitrova 1* , Angela Balzano 2* , Katarina Čufar 2 , Gabriella S. Scippa 1 , Maks Merela 2 , Antonio Montagnoli 3 , Batkhuu Nyam-Osor 4 , Enkhchimeg Tsedensodnom Chimgee 4 , Donato Chiatante 3 UDK članka: 630*811.18:630*811.28 Received / Prispelo: 13.7.2023 Original scientific article / Izvirni znanstveni članek Accepted / Sprejeto: 29.8.2023 . Abstract / Izvleč ek Abstract: The present study focuses on the cambium and the anatomy of secondary tissues (xylem – wood and phloem) of Siberian poplar (Populus sibirica) and Siberian elm (Ulmus pumila) grown in a plantation in the semi- arid Mongolian steppe. Stem and root microcores from both species were collected and subsequently processed to obtain cross-sections for light microscopy by paraffin embedding, sectioning with a rotary microtome, and staining with safranin and astra blue. The results present the anatomy of the secondary xylem and phloem of stems and roots of both species, along with the characteristics of the youngest xylem and phloem annual rings. We discuss the critical aspects which need to be considered when using the microcoring methodology, along with the need for further studies on wood and phloem formation of less-commonly studied tree species and their characteristics when grown in semi-arid environments. Keywords: Siberian poplar (Populus sibirica), Siberian elm (Ulmus pumila), cambium, xylem, phloem, stem, root, microcore Izvleček: Raziskali smo kambij in anatomijo sekundarnih tkiv (ksilema–lesa in floema) sibirskega topola (Populus sibirica) in sibirskega bresta (Ulmus pumila) s plantaže v polsuhi stepi v Mongoliji. Tkivo za analize (mikro izvrtke) smo odvzeli iz debel in korenin dreves. Mikro izvrtke smo vklopili v parafin in z rotacijskim mikrotomom narezali preparate prečnih prerezov, ki smo jih obarvali z barviloma safranin in astra modro in pregledali pod svetlobnim mikroskopom. Rezultati predstavljajo anatomijo ksilema in floema debel in korenin obeh vrst. Posebno pozornost smo posvetili prikazu značilnosti najmlajših letnih prirastkov ksilema in floema. V razpravi poudarjamo kritične vidike metodologije odvzema, priprave mikro izvrtkov in anatomskih preparatov, ter potrebo po nadaljevanju raziskav nastajanja ter lastnosti lesa in floema manj znanih drevesnih vrst iz polsuhih okolij. Ključne besede: sibirski topol (Populus sibirica), sibirski brest (Ulmus pumila), kambij, ksilem, floem, deblo, korenina, mikro izvrtek 1 INTRODUCTION 1 UVOD Secondary tissues in trees, secondary xylem and secondary phloem, are produced by the vascu- lar cambium. Studies of cambial activity and xylem and phloem formation are therefore essential for a better understanding of cambial phenology, the subsequent formation processes and characteris- tics of the secondary tissues. Combined, they pro- vide information on the general health and growth potential of trees, the tree responses to changing Vol. 72, No. 2, 37-48 DOI: https://doi.org/10.26614/les-wood.2023.v72n02a02 1 University of Molise, Department of Biosciences and Territory, Contrada Fonte Lappone SnC, 86090 Pesche, Italy ² Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za lesarstvo, Jamnikarjeva 101, 1000 Ljubljana, Slovenia ³ University of Insubria, Department of Biotechnology and Life Science, Via Dunant 3, 21100 Varese, Italy 4 National University of Mongolia, Laboratory of Forest Genetics and Ecophysiology, School of Engineering and Applied Sciences, 14201 Ulaanbaatar, Mongolia * e-mail: anastazijadimitrova@hotmail.com, angela.balzano@bf.uni-lj.si * A. Dimitrova and A. Balzano contributed equally to this work 38 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomija ksilema in floema debel in korenin drevesnih vrst Populus sibirica in Ulmus pumila iz polsuhe stepe v Mongoliji environmental conditions, and tree plasticity, in terms of adapting their growth and the structure of xylem and phloem to the given environmental conditions and ensuring their optimal functioning (Rossi et al., 2006a; Gričar & Čufar, 2008; Prislan et al., 2013b; Balzano et al., 2018). In the present context, the term ‘cambium‘ is used to describe the cambial zone, which con- sists of several cell layers, including actively divid- ing cambial initials with phloem and xylem mother cells (Catesson et al., 1994; Larson, 1994; Lachaud et al., 1999; Gričar & Čufar, 2007). The study of cam- bial activity, and its productivity, usually involves collecting tissues from a living tree, fixation and preparation of tissues, cutting and staining of thin sections, observation under a microscope, image analysis and quantification (counting and measur- ing) of cambial cells in radial rows (e.g., Prislan et al., 2009; Balzano et al., 2022; Prislan et al., 2022). Studies can also include an analysis of cambial cell morphology, cell content and the developmental stages of their derivatives. Studying actively divid- ing or dormant cambium cells can be also based on observation of their ultrastructure, focusing on the presence, appearance, and frequency of cell orga- nelles. Such analyses require a demanding fixation with solutions like glutaraldehyde and osmium te- troxide and observation with a transmission elec- tron microscope (Prislan et al., 2011; 2013a; 2016). The secondary xylem in hardwoods (dycotile- dons) consists of vessels, various types of fibres, ax- ial parenchyma, and rays. These major components vary widely among species (Wheeler et al., 1989). During wood formation, the new cells formed by the cambium go through several stages of differen- tiation. In the first phase, they have a thin, non-lig- nified primary cell wall and undergo expansion to reach the final form and size. This phase is followed by deposition and lignification of the cell walls, and finally, programmed cell death (Prislan et al., 2009). The secondary phloem in hardwoods consists of sieve tubes with companion cells, axial parenchy- ma, phloem rays and phloem fibres (Čufar, 2006; Prislan et al., 2012; Crivellaro & Schweingruber, 2015; Gričar et al., 2016). Some taxa, e.g., Fagus, do not form phloem fibres but contain sclereids which are formed during secondary processes in older bark (Čufar, 2006; Prislan et al., 2012). After the cambium produces the phloem, we can follow the cell differentiation, maturation and secondary changes, and observe the non-collapsed and con- ducting phloem which is able to transport prod- ucts of photosynthesis. The older (outer) phloem is identified as collapsed and non-conductive, and over time it undergoes considerable changes (Pris- lan et al., 2019). In temperate species we can usual- ly define at least one annual growth ring in phloem, with early and late phloem and the phloem ring boundary (Prislan et al., 2012). When studies focus on the characteristics and dynamics of the cambial activity, xylem and phloem formation in response to climatic and environmen- tal changes, it is important to prepare an adequate sampling design, along with an appropriate tech- nique for tissue collection and sample preparation, as described by Prislan et al. (2014a; 2014b; 2022) and Balzano et al. (2022). It is also important to have a thorough knowledge of the anatomy of the secondary tissues of the species in question. Recently, the need for such knowledge arose in activities focused on species selection for envi- ronmental restoration in the semi-arid steppe are- as suffering from increasing aridity. In particular, as part of the Green Belt project in Mongolia, planta- tions of the Siberian poplar (Populus sibirica Hort. Ex. Taush) and Siberian elm (Ulmus pumila L.), have been used to study the effects of irrigation and fer- tilization on several plant characteristics, i.e., tree growth – stem height and root collar diameter (Byambadorj et al., 2021a), morphophysiological traits – leaf area and leaf biomass (Byambadorj et al., 2021b), above and belowground biomass par- titioning (Byambadorj et al., 2022), root biomass distribution (Nyam-Osor et al., 2021), and plasticity of root architecture in Ulmus pumila (Montagnoli et al., 2022). The aim of this study is thus to present the basic anatomy of the vascular cambium, second- ary xylem, and secondary phloem from microcores taken from stems and roots of Populus sibirica and Ulmus pumila, grown under natural conditions (without irrigation or fertilization) in the semi-arid Mongolian steppe. As such, the study presents the first attempt to underline the anatomical and struc- tural characteristics of xylem and phloem of the two species grown in semi-arid conditions. Further- more, we underline critical aspects related to tissue collection and sample preparation, as part of future 39 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomy of xylem and phloem in stems and roots of Populus sibirica and Ulmus pumila from semi-arid steppe in Mongolia recommendations for more in depth studies on cambial activity and the development of secondary tissues in challenging environmental conditions. 2 MATERIALS AND METHODS 2 MATERIAL IN METODE 2.1 MATERIALS 2.1 MATERIAL The Populus sibirica Hort. ex Tausch and Ulmus pumila L. trees used in this study originate from a plantation–experimental site (area 2 ha) – located in Mongolia (latitude 47°52’15.43” N, longitude 105°10’46.4” E, elevation 1130 m a.s.l.) within the forest nursery of the South Korea-Mongolia Joint Green Belt Plantation Project in Lun soum, Tuv aim- ag, Mongolia, 135 km west of Ulaanbaatar, Mon- golia (Byambadorj et al., 2021a). In particular, the nursery is located on the right bank of the Tuul Riv- er, in a dry-steppe area which is densely populated and greatly degraded by intense livestock grazing. The site had a mean annual temperature of 0.7°C and total annual sum of precipitation 196 mm (period 2000-2019) as recorded by the Lun soum weather station (Byambadorj et al., 2021a). The mean monthly temperatures were below zero from November to March, with a mean air temperature of the warmest month (July) of 16°C, while that of the coldest month (January) of -22°C. Detailed me- teorological data measured during the experiments showed that July-September 2019 was character- ized by below average precipitation and above av- erage temperatures (Byambadorj et al., 2021a). As part of the Green Belt project, an experi- mental design aiming to evaluate the performance of Populus sibirica and Ulmus pumila under differ- ent combination of fertilization and irrigation treat- ments was established (for detailed overview of the experimental design, see Byambadorja et al., 2021a; 2021b). Briefly, as preparatory work, after growing them in a greenhouse, two-year-old Pop- ulus sibirica saplings (grown from 20 cm cuttings) and Ulmus pumila seedlings (grown from seeds) were acclimated in an open nursery and trans- planted in open plantations in May 2011. After one month of open-field acclimatization with sufficient watering, the pre-selected combination of fertili- zation and irrigation treatment was applied. In the autumn of 2019, the trees had reached 10 years of age with a height of 180 cm and 190 cm, and root collar diameter of 3.7 cm and 5.6 cm, for Ulmus and Populus, respectively (Byambadorj et al., 2021a). 2.1 SAMPLING AND SAMPLE PREPARATION 2.1 VZORČENJE IN PRIPRAVA VZORCEV In the present study, we investigated the sam- ples coming from eight trees (four from the poplar and four from the elm) which were grown under natural conditions, i.e., without any fertilization and irrigation treatment. For each tree, four micro- cores were sampled on 20 October 2019; two were taken from the stem (5 cm above ground) and two from the root (5 cm below ground) using a Trephor tool (Rossi et al., 2006b). The microcores were initially stored in FAA (mixture of formalin, acetic acid and ethanol) and then dehydrated in gradient ethanol series (70, 90, 95 and 100%), infiltrated with bio-clear (D-li- monene) and embedded in paraffin blocks using a Leica TP1020-1 tissue processor (Nussloch, Germa- ny). Using a semi-automatic rotary microtome (RM 2245, Leica, Nussloch), cross-sections (9 µm thick) were obtained, and subsequently stained with sa- franin (0.04%) and astra blue (0.15%) water solution (Prislan et al., 2013a; Balzano et al., 2022; Prislan et al., 2022). The samples were mounted in Euparal (Bioquip Rancho Domingez, CA, USA) and observed under a light microscope – transmitted light mode Zeiss Axio Imager A.2 light microscope (Carl Zeiss Microscopy, White Plains, NY , USA), while images were acquired with a Zeiss Axiocam 712 color (Carl Zeiss Microscopy GmbH, Jena, Germany). On the transverse sections of each specimen, we identified the non-collapsed phloem and the phloem growth rings including early and late phlo- em and measured their respective widths. We also recorded the number of cambium cells, the width of the cambium and made observations on the stage of its productivity. On the xylem side, we ex- amined and measured the tissues with the newly formed cells in the enlargement stage, with cells in the secondary cell wall deposition and lignification phase, and the tissue where the cells were mature. We also measured the width of the most recently formed xylem growth ring and the rings formed in one or more previous years. 40 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomija ksilema in floema debel in korenin drevesnih vrst Populus sibirica in Ulmus pumila iz polsuhe stepe v Mongoliji 3 RESULTS 3 REZULTATI 3.1 XYLEM AND PHLOEM STRUCTURE OF Populus sibirica 3.1 ZGRADBA KSILEMA IN FLOEMA SIBIRSKEGA TOPOLA Populus sibirica The wood of the stem of Populus sibirica has the typical anatomy of poplar with the following features observed on cross-section: wood diffuse porous, growth ring boundaries distinct, tangential diameter of vessel lumina 50 – 100 µm, fibres gen- erally thin-walled, and rays exclusively uniseriate (Wheeler, 2011; InsideWood, 2023) (Fig. 1a, 2a). The growth ring boundaries were characterized by radially flattened fibres arranged in radial rows. Al- though axial parenchyma is usually absent, rare, or appears in marginal or seemingly marginal bands in poplars (InsideWood, 2023), we noted abundant paratracheal scanty axial parenchyma (Fig. 2). How- ever, its presence would need to be further con- firmed in radial or tangential sections. The wood of the roots had slightly larger vessels and less distinct growth ring boundaries (Fig. 1 b, 2b) than the wood of the stem, likely due to the occurrence of density fluctuations. In the stem, the width of the xylem ring averaged 986 µm in 2019 and 740 µm in 2018; the corresponding values in the root were 268 and 212 µm. Tension wood was present in the xylem of both stems and roots, consisting of fibres with cell walls containing a blue stained gelatinous layer. The cambium of stems and roots had an aver- age of four and three cambium cells per radial file, respectively, and the cambium was not productive. The phloem of stems of Populus sibirica con- sists of sieve tubes with companion cells, axial pa- Figure 1. Secondary tissues of Populus sibirica, overview: (a) stem, (b) root. CC- cambial cells, XR1… XR5–xylem rings, arrows pointing to growth ring boundaries, Ph-phloem, EP-early phloem (sieve tubes), PF-phloem fibres, NPh – non-collapsed (conductive) phloem, CPh – collapsed phloem. Slika 1. Populus sibirica, pregled sekundarnih tkiv: (a) deblo, (b) korenina. CC – celice kambija, XR1… XR5 – ksilemske branike, puščice kažejo letnice – meje med branikami, Ph – floem, EP – rani floem (sitaste cevi), PF – floemska vlakna, NPh – nekolabi- rani (prevodni) floem, CPh – kolabirani floem. Figure 2. Cambium and secondary tissues of Popu- lus sibirica, detailed view: (a) stem and (b) root. CC – cambial cells, Xy – xylem, AP – axial parenchyma, V – vessel, XF – xylem fibre, Ph – phloem, EP – early phloem, LP – late phloem, PF – phloem fibres, S – sieve tubes. Slika 2. Populus sibirica, kambij in sekundarna tkiva, podroben pogled: (a) deblo in (b) korenina. CC – ce- lice kambija, Xy – ksilem, AP – aksialni parenhim, V – traheja, XF – ksilemsko vlakno, Ph – floem, EP – rani floem, LP – kasni floem, PF – floemska vlakna, S – sitaste cevi. 41 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomy of xylem and phloem in stems and roots of Populus sibirica and Ulmus pumila from semi-arid steppe in Mongolia renchyma, phloem fibres between the early and late phloem, and phloem rays. We could distinguish between non-collapsed (conducting) phloem and collapsed (non-conducting) phloem (Fig. 1 and 2). In the non-collapsed phloem, we could distinguish between early phloem with larger sieve tubes and late phloem in which the sieve tubes had smaller diameters. This is consistent with the phloem struc- ture of Populus tremula described by Gričar (2019) where early and late phloem were divided by groups of fibres arranged in tangential bands. Moreover, it was possible to detect the phloem growth ring boundary that delineates the youngest increment in Populus tremula (Gričar, 2019), whereas in our case the growth ring boundary could not be detect- ed in the phloem of Populus sibirica. We could not detect late phloem in the phloem of the roots of Populus sibirica, while the non-collapsed phloem was narrower than that in the stem (Fig. 2). 3.2 XYLEM AND PHLOEM STRUCTURE OF Ulmus pumila 3.2 ZGRADBA KSILEMA IN FLOEMA SIBIRSKEGA BRESTA Ulmus pumila The wood of the stem of Ulmus pumila has the typical anatomy of elms with the following features observed on cross-sections: wood ring porous, growth ring boundaries distinct, tangential diame- ter of early wood vessel lumina 100 – 200 µm, late wood vessels in tangential bands, vessel clusters common, tyloses common, vascular/vasicentric tracheids present, fibres thin- to thick-walled, axial parenchyma vasicentric, confluent and in margin- al or in seemingly marginal bands with 3 – 4 cells per parenchyma strand, larger rays commonly 4 –10-seriate, rays of two distinct sizes (InsideWood, 2023) (Fig. 3, 4). Most of the fibres in the wood of the stem and root stained blue, possibly indicating the abun- dant presence of tension wood (Fig. 3, 4) which is formed to reinforce or change the position of the stem, branch or the root. The wood of the roots had less distinct growth ring boundaries and more earlywood vessels than the wood of the stem (Fig. 3 a, 4 a, b). In the stems of trees of Ulmus, the width of the last xylem ring was around 1.5 mm. In the roots intra-annual density fluctuations (IADFs) cannot be excluded (Fig. 4b). The cambium of stems and roots had an aver- age of 6.7 and 6 cells per radial file, and the cambi- um was not productive. The phloem of Ulmus pumila consists of early and late phloem sieve tubes with companion cells, axial parenchyma, and phloem rays (Fig. 3). Fibre sclereids (Fig. 3b) and mucilage (slime) cells (Fig. 3a) are observed in the older phloem, as described by Holdheide (1951) in Ulmus scabra. In Ulmus pumila Figure 3. Secondary tissues of Ulmus pumila stem: (a) overview, (b) detail of image a. CC – cambial cells, Xy – xylem, arrows pointing to growth ring boundaries, Ph – phloem, LV – latewood vessel, F – xylem fibre, EP – early phloem (sieve cell), R – xy- lem ray, NPh – non-collapsed (conductive) phloem, CPh – collapsed phloem, LP – late phloem, FS – fi- bre sclereid, PR– phloem ray, MC – mucilage (slime) cell. Slika 3. Sekundarna tkiva debla Ulmus pumila: (a) pregled, (b) podroben pogled slike a. CC – celice kambija, Xy – ksilem, puščice kažejo na letnice, Ph – floem, LV – traheje kasnega lesa, F – ksilemsko vlakno, R – ksilemski trak, NPh – nekolabirani (pre- vodni) floem, CPh – kolabirani floem, LP – kasni flo- em, EP – rani floem (sitasta cev), PR – floemski trak, FS – vlaknasta sklereida, MC – sluzna celica. 42 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomija ksilema in floema debel in korenin drevesnih vrst Populus sibirica in Ulmus pumila iz polsuhe stepe v Mongoliji we could distinguish between non-collapsed (con- ducting) phloem and collapsed (non-conducting) phloem (Fig. 3, 4). In the non-collapsed phloem, we could recognize early phloem with larger sieve tubes and late phloem where the sieve tubes had smaller diameters. However, we were unable to identify clear growth ring boundaries and there- fore could not accurately determine the width of the last formed phloem annual increment. The first thick walled fibre sclereids were observed at a dis- tance of about 300 μm from the cambium (Fig. 3b) in phloem tissue that may have been formed in the previous year. 4 DISCUSSION 4 RAZPRAVA The microcoring technique has proven effec- tive for the study of wood, cambium, and phloem, including their anatomy and stage of development at the end of the vegetation period in both species and in the particular environment. The method is time consuming and requires a sophisticated pro- cedure from the removal from the tree to sample preparation and production of sections of appro- priate quality for microscopy and image analysis. As a result, numerous technical challenges can be en- countered during all steps of the analysis. Samples are typically collected from forest trees, which are often located in remote areas. Therefore, tissues must be efficiently fixed and appropriately stored before section preparation can begin. Cutting is usually complicated by the fact that plant tissue is generally heterogeneous and fragile (Balzano et al., 2022). These limitations can be exacerbated when we sample species for which there are few or no studies of wood and bark anatomy, and when we work in difficult, remote environments, as in our case in semi-arid areas in the Mongolian steppe, far from the laboratory. The amount of material available for analyses may also limit our ability to make high quality microscopic preparations, as we usually do not have replicates to replace damaged microcores. In addition, we rely mainly on cross sections, as we do not have enough material to make radial or tangential sections as well. All these factors may affect the results of the study. The present study was conducted on two tree species, Populus sibirica and Ulmus pumila, whose wood and bark anatomy are less well known, espe- cially when the trees grow in a harsh environment under semi-arid conditions, in addition to occasion- al very cold winter and very hot summer conditions. The wood and phloem anatomy could be compared with the anatomy of other species of Populus and Ulmus like InsideWood (2023) for wood, and Hold- heide (1951) and Gričar (2019) for phloem. The study involved sampling in remote areas with a relatively large team assigned to perform the various steps of the experimental work. Although two technical replicates of microcores from the stem and root of each tree were taken, the mate- rial was limited and the samples often did not con- tain enough wood due to the thicker bark. This also Figure 4. Secondary tissues of Ulmus pumila root: (a) normal structure of xylem ring (XR1) below cambium (CC) with clear tree ring boundary, (b) overview (cross-section of the microcore) where some growth ring boundaries are not clear (?), (c) detail from b. EV – early wood vessel, LV late wood vessel, R – ray, F – fibre, AP – axial parenchyma; for other abbreviations see Figs. 1 to 3. Slika 4. Sekundarna tkiva korenine Ulmus pumila: (a) normalna struktura ksilemske branike (XR1) pod kambijem (CC) z razločno letnico (TRB), (b) pregled tkiv (prečni prerez mikro izvrtka), kjer nekatere meje med branikami niso jasne (?), (c) podrobnosti iz slike b (okvir). EV – traheja ranega lesa, LV traheja kasnega lesa, R – trak, F – vlakno, AP – aksialni pa- renhim; za ostale okrajšave glej slike 1–3. 43 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomy of xylem and phloem in stems and roots of Populus sibirica and Ulmus pumila from semi-arid steppe in Mongolia caused challenges during the fixation of the sam- ples. In view of the above, we recommend for fu- ture studies a rapid evaluation of the material and a prioritization of high-quality cross-sections that can provide sufficient information. This is espe- cially important when working with less frequent- ly studied species. Indeed, most previous studies have focused on the more widespread tree species (e.g., Fagus sylvatica L., Pinus sylvestris L., Quercus robur L., etc.) from less remote sites in temperate climates (e.g., Van der Werf et al., 2007; Michelot et al., 2012; Giagli et al., 2016; Martinez del Castillo et al., 2016). However, our study has shown that the ana- lysed tree tissues contain a lot of information if we know how to extract and interpret it. Impending climatic challenges require expan- sion of studies to areas of increasing aridity (e.g., Liu et al., 2022), as such changes will inevitably af- fect cambium productivity and wood and phloem formation. This raises the need for better under- standing of how a particular species behaves in a semiarid climate and how this climate affects tissue development and structure. Another aspect of in- terest is the selection of plant tissues. Microcoring is usually performed on stems, often focusing on the anatomy and formation of the wood. However, the phloem is often not included in such studies, as most focus on the aboveground portion of the tree, such as the stem at breast height, which is more accessible. Root sampling presents a list of techni- cal and logistical challenges (Freschet et al., 2021). However, as the above- and belowground tree parts, i.e., stem and root, play distinct roles in plant establishment and performance (Byambadorj et al., 2022), understanding their combined response to growing conditions could provide more in-depth information about plant plasticity and adaptive po- tential. Given the projected climatic challenges, af- forestation and reforestation measures are consid- ered extremely important. However, these costly and complicated actions require appropriate and context-specific species selection. Therefore, in order to understand the anatomy of wood, which represents a large proportion of biomass, and bark, which is a smaller proportion of the biomass but performs critical functions for photosynthate transport (non-collapsed phloem) and protective function (collapsed phloem and outer bark), it is of particular value to know their anatomical charac- teristics as well as the potential changes in tissue due to management actions and/or changing cli- matic conditions. The present study highlights the similarities and differences between the wood anatomy of the stem and roots of Populus sibirica and Ulmus pumila. Although certain technical challenges have hindered a more thorough analysis, i.e., small num- ber of replicates, damage to some samples due to extended storage in FAA, thick bark, and the in- creased presence of tension wood, the descriptive approach and characterization of the wood, cam- bium, and phloem that we have used provides the basis for expanding the analyses. To the best of our knowledge, this is the first study to attempt to char- acterize the secondary tissues of Populus sibirica and Ulmus pumila growing in semi-arid areas. 5 CONCLUSIONS 5 ZAKLJUČKI The present study summarizes the anatomy of secondary xylem and phloem in the stems and roots of two tree species less known in this context, Populus sibirica and Ulmus pumila. Observations were made on samples collected from the trees in the plantation in the Mongolian semi-arid steppe on October 20, 2019, when temperatures dropped below zero and the growing season had ended. The samples contained cambium that was no longer producing new cells and a narrow cambial zone. Most of the cells in the secondary xylem and phlo- em produced during the current growing season were already mature. In the wood of the stems of Populus we found some tension wood where the cell walls of the fibres contained a non-lignified G-layer. In both species the phloem increment con- tained early and late phloem, but no clear growth ring boundaries. Because of the timing of tissue sampling, it was possible to see tissues formed in the last growth period, while the process of xylem and phloem formation was not evident. Despite the difficulty of sampling in the remote areas and some technical problems in preparing samples for microscopy, we have shown that mi- croscopic examination of the basic anatomy of the 44 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomija ksilema in floema debel in korenin drevesnih vrst Populus sibirica in Ulmus pumila iz polsuhe stepe v Mongoliji tissues of interest was possible in the stems and roots of the two species. As far as we know, the results presented here are the first attempt to characterize xylem and phlo- em structure in a specific time of wood and phloem formation of Populus sibirica and Ulmus pumila at the end of the vegetation period in the semi-arid region of Mongolia. This study also provides infor- mation for planning future studies focusing on the seasonal dynamics of secondary xylem and phloem formation of the two species, which would provide valuable information on their potential for adapta- tion to the challenging climatic conditions. 6 SUMMARY 6 POVZETEK Proučili smo kambij (v tem prispevku z besedo kambij označujemo kambijevo cono s kambijevimi inicialkami ter ksilemskimi in floemskimi materin- skimi celicami) in anatomijo ksilema – lesa in floe- ma sibirskega topola (Populus sibirica) in sibirske- ga bresta (Ulmus pumila) v okviru širše raziskave, namenjene preučevanju plastičnosti in sposobnos- ti prilagajanja obeh vrst na podnebne in okoljske spremembe. Vzorčenje oz. odvzem mikro izvrtkov je bilo opravljeno na plantaži v Mongoliji, v stepi s polsuhim podnebjem. Tkivo za analize (mikro izvrt- ke) smo odvzeli iz debel in korenin dreves obeh vrst 19.10. 2019, pri čemer smo v tej študiji raziskali samo vzorce iz kontrolne skupine (brez gnojenja in namakanja). Preparati prečnih prerezov tkiv za svetlobno mikroskopijo so bili po vklapljanju mi- kro izvrtkov v parafin narezani z rotacijskim mikro- tomom ter obarvani z barviloma safranin in astra modro. Les debla Populus sibirica ima anatomijo lesa, kot je značilna za rod Populus z naslednjimi znaki, vidnimi na prečnem prerezu: les je difuzno po- rozen, prirastne plasti so razločne, tangencialni premer trahej je 50–100 µm, vlakna so večinoma tankostenska, trakovi so izključno enoredni (Whe- eler et al., 2011; InsideWood, 2023) (sliki 1a in 2a). Proučeni topol je imel v nasprotju z opisi v literaturi tudi dokaj obilen aksialni parenhim (slika 2). Les ko- renin je imel nekoliko večje traheje in manj izrazite branike (sliki 1b in 2b) kot les debla. Les debla je imel okoli 1 mm široke branike, v lesu korenin pa so bile te široke okoli 0,25 mm. V ksilemu korenin so bile letnice manj izrazite, zato ne izključujemo pri- sotnosti »lažnih branik« oz. gostotnih variacij znot- raj letnih prirastnih plasti. V kambiju debla smo v povprečju našteli 4 in v kambiju korenin 3 celice na radialni niz. V nobenem primeru nismo zasledili produkcije novih celic. Floem debla Populus sibirica je sestavljen iz sitastih cevi s celicami spremljevalkami, aksialnega parenhima, floemskih vlaken in floemskih trakov. Razlikovali smo lahko med nekolabiranim (prevo- dnim) floemom in kolabiranim (neprevodnim) flo- emom (sliki 1 in 2). V nekolabiranem floemu smo lahko prepoznali rani floem s sitastimi cevmi večjih premerov in kasni floem, v katerem so imele sitaste cevi manjši premer. Meje med prirastnimi plastmi (floemske letnice) nismo mogli določiti. Anatomija floema Populus sibirica je večinoma podobna kot pri trepetliki (Populus tremula), ki jo je podrobno opisala Gričar (2019), kjer so rani in kasni floem delile skupine vlaken, razporejene v tangencialnih pasovih. Poleg tega je bilo pri Populus tremula mo- goče zaznati mejo floemske branike (Gričar, 2019), medtem ko v našem primeru meje prirastnih plasti v najmlajšem floemu Populus sibirica ni bilo mogo- če zaznati. V floemu korenin Populus sibirica nismo mogli zaznati kasnega floema, nekolabirani floem pa je bil ožji od tistega v floemu debla (slika 2). Les debla sibirskega bresta (Ulmus pumila) ima značilno anatomijo lesa brestov z naslednjimi zna- ki, ki jih lahko vidimo na prečnem prerezu: les je venčasto porozen, letnice so izrazite, tangencialni premer trahej ranega lesa je 100–200 µm, traheje kasnega lesa so grupirane v tangencialnih skupinah, traheje so pogosto otiljene, prisotne so vaskularne/ vazicentrične traheide, vlakna so tankostena do de- belostena, aksialni parenhim je paratrahealen, va- zicentričen, zlivajoč in v marginalnih ali navidezno marginalnih pasovih s 3–4 celicami na parenhimski pramen, trakovi so dveh različnih velikosti, večji tra- kovi so običajno 4 do 10-redni (InsideWood, 2023) (sliki 3 in 4). Večina vlaken v lesu debla in korenine se je pri sibirskem brestu obarvala modro, kar verjetno kaže na obilno prisotnost tenzijskega lesa (sliki 3 in 4), ki omogoča usmerjanje položaja debla, veje ali kore- nine. Les korenin Ulmus pumila je imel manj izra- zite letnice in več trahej ranega lesa kot les debla (slike 3a, 4a, 4b). V deblih preiskanih dreves je bila povprečna širina zadnje branike približno 1,5 mm. 45 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomy of xylem and phloem in stems and roots of Populus sibirica and Ulmus pumila from semi-arid steppe in Mongolia V koreninah je bil prirastek nekoliko manjši, znotraj branik pa so se pojavljale gostotne variacije (IADF) (slika 4b). Kambij debel in korenin je imel v povprečju 6–7 celic v radialnem nizu in ni bil več produktiven. Floem Ulmus pumila sestavljajo sitaste cevi s celicami spremljevalkami, aksialni parenhim in flo- emski trakovi (slika 3). Običajnih floemskih vlaken nismo zaznali, v starejšem floemu pa smo opazili sklereide (slika 3b) in sluzne celice (slika 3a). To se ujema z opažanji, ki jih je objavil (Holdheide, 1951), ki je poročal, da floem Ulmus scabra nima vlaken, ima pa številne vlaknaste sklereide s podobnim videzom, vendar različno ontogenijo kot floemska vlakna (Čufar, 2006; Prislan et al., 2019). Holdheide (1951) poroča tudi o številnih sluznih celicah v sta- rejšem floemskem tkivu Ulmus scabra. Pri Ulmus pumila smo lahko razlikovali med ne- kolabiranim (prevodnim) floemom in kolabiranim (neprevodnim) floemom (sliki 3 in 4). V nekolabi- ranem floemu smo lahko razlikovali med ranim flo- emom s sitastimi cevmi večjih premerov in kasnim floemom, v katerem so imele sitaste cevi manjši tangencialni premer. V floemskih prirastnih plasteh nismo mogli določiti letnic (mej med dvema floem- skima branikama) zato tudi nismo mogli natančno izmeriti širine zadnje nastale floemske branike. Prve debelostene vlaknaste sklereide smo opazili na razdalji približno 300 μm od kambija, najverje- tneje v floemu, nastalem v predhodnem letu 2018 (slika 3b), kar je v skladu z ugotovitvami, ki jih je objavil Holdheide (1951). Predstavljamo tudi nekatere ključne tehnične in logistične vidike, ki jih je treba upoštevati pri iz- vedbi terensko in laboratorijsko zahtevne raziska- ve. Čeprav smo odvzeli po več vzorcev iz debla in korenin posameznega drevesa, je bilo v nekaterih primerih tkivo poškodovano zaradi narave materi- ala, postopka vzorčenja in dolgotrajnega hranjenja mikroizvrtkov v fiksirnem sredstvu FAA (mešanice formalina, ocetne kisline in alkohola). Kljub tehnič- nim težavam so izdelani preparati omogočili preu- čevanje osnovne anatomije in razvojno fazo lesa in floema, nastalega v tekočem letu. Pri obeh vrstah so branike lesa vsebovale ve- činoma zrele celice. Floemski prirastek je pri obeh vrstah vseboval rani in kasni floem, nismo pa mog- li razmejiti prirastnih plasti v floemu. Zaradi časa odvzema je bilo mogoče videti le tkiva, ki so nastala v zadnji rastni sezoni, zato bo v prihodnje potrebno dobro časovno načrtovano vzorčenje, osredotoče- no na študij dinamike kambijeve aktivnosti ter pro- cesa nastajanja ksilema in floema. V predstavljeni študiji smo se osredotočili na kvalitativne podatke, vendar so proučeni prepara- ti uporabni tudi za merjenje oz. analizo slike tkiv, s čimer bi lahko pridobili tudi kvantitativne podatke, in s tem povečali vrednost pridobljenih informacij o lesu in skorji proučenih vrst. ACKNOWLEDGEMENTS ZAHVALA The study and the time spent at the University of Ljubljana were supported by the PhD fellowship for AD from the University of Molise. AM and DC acknowledge the Department of Biotechnology and Life Science at the University of Insubria for the support in the joint research project. The authors also thank the staff of the Korea-Mongolia Joint Green Belt Plantation Project and the members of the Laboratory of Forest Genetics and Ecophysiol- ogy from the National University of Mongolia for their assistance in the field and laboratory works. The work of AB, MM and KČ and the laboratory equipment were funded by the Programme P4- 0015 of the Slovenian Research and Innovation Agency ARIS. We thank Luka Krže for his great sup- port in the laboratory. We thank Darja Vranjek and Paul Steed for editing the Slovene and English lan- guage, and two reviewers whose comments helped us to improve the original version of the manu- script. Author contributions: AM, BN-O, GSS, and DC conceived and designed the project and the study plan. BN-O, ETC, DC, and AM were responsible for tissue collection; AD and AB, with the assistance of MM, performed the laboratory analyses (tissue preparation, microscopy, and measurements) and organized the results; KČ, AB, and AD drafted and wrote the manuscript. All co-authors contributed to the writing; they also read and approved the fi- nal version of the manuscript. AD and AB contribut- ed equally to this work. 46 Les/Wood, Vol. 72, No. 2, December 2023 Dimitrova, A., Balzano, A., Čufar, K., Scippa, G. S., Merela, M., Montagnoli, A., Osor-Nyam, B., Chimgee, E. T., & Chiatante D.: Anatomija ksilema in floema debel in korenin drevesnih vrst Populus sibirica in Ulmus pumila iz polsuhe stepe v Mongoliji REFERENCES VIRI Balzano, A., Čufar, K., Battipaglia, G., Merela, M., Prislan, P ., Aronne, G., & De Micco, V. (2018). Xylogenesis reveals the genesis and ecological signal of IADFs in Pinus pinea L. and Arbutus unedo L. Annals of Botany, 121(6), 1231–1242. 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