168 ACTA BOT. CROAT. 81 (2), 2022 Acta Bot. Croat. 81 (2), 168–176, 2022 CODEN: ABCRA 25 DOI: 10.37427/botcro-2022-014 ISSN 0365-0588 eISSN 1847-8476 Morpho-anatomical diversity of five species of the genus Asparagus (Asparagaceae) from Algeria Kenza Boubetra1*, Nabila Amirouche2, Rachid Amirouche2 1 National Institute of Forest Research, INRF, PO Box 37, Cheraga 16014 Algiers, Algeria 2 University of Sciences and Technology Houari Boumediene, Faculty of Biological Sciences, Laboratory for Biology and Physiology of Organisms, USTHB, Bab Ezzouar, 16111, Algiers, Algeria Abstract – Five species of the genus Asparagus are recognized in the flora of Algeria: A. acutifolius L., A. albus L., A. horridus L., A. officinalis L., and the endemic A. altissimus Munby. The chorology of each of these species is fairly well known. In this study, morphological variation and the anatomical features of the cladodes have been evaluated in re- spect to each taxonomic unit and ecogeographical distribution and they suggest distinct adaptive strategies. Analyses have been performed on twenty-nine natural populations sampled along the east-west bioclimatic gradient of North- ern Algeria. Multivariate analysis based on the main diagnostic descriptors underlines the interspecific differentiation particularly with respect to the stigma type, bifid versus trifid, shape of flowers, color of berry, and the number of cladodes in a fascicle. For each species, the anatomy of the cladodes is unique, unlike that of stems and roots. Interspe- cific differentiation was observed in the form of cross-sections of the cladode, thickness of the cuticle, shape of epider- mal cells, number of vascular bundles and presence of raphides. Morphological and anatomical traits of the cladode constitute important interspecific criteria within the genus Asparagus. Keywords: adaptation, Algeria, anatomy, Asparagus, cladodes, morphology, taxonomy Introduction The genus Asparagus (Asparagaceae) includes more than 200 species distributed in the arid and subarid regions of Africa, Europe, Asia and Australia (Chen and Tamanian 2000). Most species of Asparagus have great economic value, particularly A. officinalis L., cultivated as a vegetable crop all over the world. Some wild Mediterranean species such as A. acutifolius L., A. horridus L., A. aphyllus L. and A. albus L., are traditionally collected and sold in local markets (Pieroni 2005, Boubetra et al. 2017a, Mantovani et al. 2019). The genus is noteworthy for its diversity of herbaceous, shrubby and climbing forms. Plants are provided with un- derground rhizomes from which the aerial shoots arise. All species are characterized by cladodes that correspond to green photosynthetic stems; the true leaves are reduced to small scales. In Mediterranean forests, the lianascent spe- cies grow preferentially in shady and wet ecological niches (Schnitzler and Arnold 2010). In contrast, shrubby species are encountered in open habitats especially in steppic areas, exhibiting tolerance to high temperatures and aridity. Among the Asparagaceae (sensu Angiosperm Phylogeny group III 2009), species may display different phylloclad- odes from flattened or cylindrical forms (Kubitzki and Rudall 1998, Fukuda et al. 2005). For instance, species of the genus Ruscus, develop a leaf-like organs, termed precisely phylloclades. They differ from cladodes in consisting of a stem with multiple internodes (Bell 2008). It is through the variation of these photosynthetic organs (cladodes leaves, stems, phyllocladodes) that species express adaptation and physiological responses to environmental changes. Molecu- lar phylogenetic studies on Asparagus, indicated that clad- odes evolve from a leaf-like (flattened) to a rod-like (cylin- drical) form, suggesting a rapid radiation particularly in arid regions (Fukuda et al. 2005, Kubota et al. 2012). The genes involved in the evolutionary pathway of the transfor- mation of the leaf-like form to axillary shoots were identi- fied by Nakayama et al. (2012). Their cooption into a gene network is well documented in Nakayama et al. (2013). Although numerous studies have been devoted to the biochemical characteristics of some Asparagus species, the morpho-anatomical variations in response to changes in * Corresponding author e-mail: kenzaboubetra@yahoo.fr GENUS ASPARAGUS IN ALGERIA ACTA BOT. CROAT. 81 (2), 2022 169 environmental conditions have been poorly documented. In addition, current researches are more focused on roots and rhizomes for the detection of bioactive molecules. In the flora of Algeria, five species have been recorded: A. acutifolius L., A. albus L., A. horridus L., (= A. stipularis Forsk.), A. officinalis L., and the endemic A. altissimus Munby. All these species are diploids with 2n = 2x = 20, and grow in contrasting ecological conditions in northern parts of Algeria, except the endemic A. altissimus which is hexa- ploid with 2n = 6x = 60 (Boubetra et al. 2017b). The most widespread species, A. acutifolius, is encountered from humid to arid bioclimates, while A. horridus and A. albus, are less common and have a predilection for open habitats, dry, sandy and stony soils. A. altissimus, is a narrow endem- ic to NW Algeria and Morocco. Asparagus officinalis is very rare and seems a remnant of ancient cultures. However, spears of wild asparagus (A. acutifolius, A. horridus, A. albus) are widely harvested for food by local people. Therefore, wild species constitute a very interesting potential as a genetic resource for breeding programs. The aims of this study were to evaluate morphological, floral and anatomical variations among the natural popula- tions of the five Asparagus species occurring in Algeria. An- atomical examinations have been performed on cross sec- tions of the cladodes in order to understand the ecological affinities and adaptive strategies of these species. Material and methods Examined specimens Twenty-four locations were selected in northern Algeria in humid, subhumid, semiarid and arid zones correspond- ing to the main vegetation types of forest, shrub formation, open habitat and steppic highlands. The taxonomic deter- mination of the specimens was made according to the Flora of North Africa (Maire 1958) and Algeria (Quézel and Santa 1962). Overall, twenty-nine populations representative of the five studied species, recognized in the flora of Algeria, were sampled, some of them occurring in sympatry (Fig. 1, On-line Suppl. Tab. 1). Specimens for morphological and anatomical analysis were sampled in reproductive stage with their whole vegeta- tive structures. Seeds collected in the field were sown in the experimental station of the National Institute of Forest Re- search (Baraki, Algiers) in order to start a living collection of the studied species. Vouchers were deposited in the Of- ficial Herbarium of ENSA (Algiers, Algeria). Fig.1. Locations of the sampled populations of the five species of the genus Asparagus in northern Algeria. Tab. 1. Morphological and floral characters used in the multi- variate analyses of five species of the genus Asparagus from Algeria. Abbreviations Characters Qualitative values and measurement units 1 CL Shape of cladodes Smooth Spiny Strongly spiny 0 1 2 2 LCL Lenght of cladodes mm 3 NCL Number of cladodes in a fascicle 4 CFL Color of flower Yellow White Purplish 0 1 2 5 TFL Type of flower Dioecious Hermaphrodite 0 1 6 FFL Shape of flower Campanulate Stelate Tubular 0 1 2 7 NFL Number of flowers 8 STI Type of stigma Bifid Trifid 0 1 9 LPE Pedicel length mm 10 PER Perianth length mm 11 CBA Color of berry Red Black Purple 0 1 2 12 NGR Number of seed per berry 13 DIG Diameter of seed mm BOUBETRA K., AMIROUCHE N., AMIROUCHE R. 170 ACTA BOT. CROAT. 81 (2), 2022 Analysis of the variation of the vegetative and floral characters The morphological evaluation was carried out on three or four individuals per population directly taken in their natural environment. A total of five quantitative and eight qualitative morphological and floral characters were select- ed following the diagnostic criteria for species delimitation, and self-observations (Tab. 1). The global raw data matrix consisting of 120 individuals belonging to the five studied species, and 13 variables, was first subjected to principal component analysis. This PCA was performed on the basis of the correlation matrix of the variables, generated after standardization of the raw matrix data. This analysis was used to estimate the relative contribution of each variable to the main principal components. To detect the phenetic grouping and relationships among the species studied, a UPGMA dendrogram was constructed using the Euclidean distances of the mean values per population. In order to assess the impact of the bioclimatic condi- tions on the morphological variability, another PCA was focused on the populations of A. acutifolius which have the largest biogeographical distribution. This analysis was based on the average values of each variable for each popu- lation and included the main parameters of the Mediterra- nean bioclimate and the altitude (On-line Suppl. Tab. 1). For all the analyses we used Statistica software (ver. 12.0). Anatomical analyses Anatomical examinations were performed on cross sec- tions of cladodes freshly collected and conserved in ethanol 70°. The cross sections were made using a microtome. The technique consists of making thin paraffin sections (about 10 μm). Several successive steps are required: fixation, in- clusion in paraffin, microtome sections, staining, assembly and observation. The technique of double staining with suc- cessively methyl green (7’) and Congo red (10’) was used. The methyl green stains the lignified walls and the Congo red stains the cellulosic walls. Cross sections were photo- graphed with a Zeiss Axiostar-Plus microscope equipped with a Canon digital camera. Results Interspecific variation for morphological and floral traits In aim to assess the morphological characters involved in the variability and the interspecific relationships, a PCA Fig. 2. Principal components analysis of 29 natural populations of five Asparagus species from Algeria based on 13 morphological and floral traits. A – loading of the 13 variables on the principal components (see Table 1 for abbreviations), B – overall scatter plot of 120 individuals representative of all species. Tab. 2. Loading of the morphological and floral characters on the first three PCA axes of five species of genus Asparagus from Algeria (see Table 1 for abbreviations). PCA loadings > 0.60 are indicated in bold. Characters PC1 PC2 PC3 LCL 0.65 -0.47 0.05 NCL 0.02 0.78 0.36 CL -0.40 -0.85 0.14 TFL 0.74 0.54 0.26 CFL 0.79 -0.49 0.28 FFL 0.78 -0.20 -0.44 NFL 0.79 0.37 0.32 STI 0.97 0.08 -0.03 LPE 0.56 0.62 -0.16 PER 0.22 0.05 0.84 DIG 0.22 -0.45 0.29 CBA 0.81 -0.49 0.04 NGR 0.18 0.08 -0.54 Eigenvalue 5.08 3.16 1.72 Total variance (%) 39.13 24.35 13.29 Cumulative variance (%) 39.13 63.49 76.79 GENUS ASPARAGUS IN ALGERIA ACTA BOT. CROAT. 81 (2), 2022 171 followed by UPGMA analysis, were applied to all popula- tions In the overall PCA, we first examined the relative con- tribution of each variable on the first two principal compo- nents, then the distribution of individuals (Fig. 2). The load- ing of the variables, the eigenvalues and the cumulative variance to the principal components are given in Tab. 2. Together, the two first components describe 63.58% of the overall variances with 39.13% and 25.35% for PC1 and PC2 respectively. As shown in Fig. 2A, eight variables displayed a high ab- solute contribution to PC1 in respect to their correlation: pedicel length (LPE), type of flower (TFL), number of flow- ers (NFL), type of stigma (STI), shape of flower (FFL), color of berries (CBA), color of flowers (CFL) and length of clad- ode (LCL). The highest contribution being that of the type of stigma bifid versus trifid. Compared to the second axis, only the number of cladodes (NCL) and the seed diameter (DIG) are discriminative. The length of the perianth (PER) and the number of seeds per berry (NGR) show no signifi- cance in either PC1 or PC2. The scatterplot of individuals on the two first PCs dis- play three main well-separated groups (Fig. 2B). Compared to PC1, a first group (I) is isolated in the negative part and corresponds to A. acutifolius. By contrast, towards the pos- itive values of axis 1, the next two groups (II, III) concern individuals of other species. These last two groups are dis- tinguished from each other in respect to PC2. One corre- sponds to the individuals of A. horridus, the other to those of A. albus. The individuals belonging to A. altissimus and A. officinalis occupy a neighboring position to A. albus. This principal component analysis makes it possible to discriminate the relevant diagnostic criteria of the five stud- ied species. For instance, the color of berries and type of flowers discriminate all the species. The berries are red in A. albus, A. officinalis and A. altissimus, black in A. acutifolius and purple in A. horridus. The flowers are campanulate in A. acutifolius, stellate in A. albus, A. horridus and A. altissimus, and tubular in A. officinalis. The last two species, A. altissimus and A. officinalis, show affinities in relation to their smooth cladodes. On the other hand, A. horridus and A. acutifolius are outstanding, by one strongly spiny cladode and by bifid stigmas, respectively. A hierarchical analysis using the matrix of the means values by population of the 13 morphological and floral traits, brings out the same grouping of populations as in the previous PCA (Fig. 3). At the distance d < 100, two main clades are clearly distinct from each other. The first clade brings together the populations belonging to A. albus and A. horridus which constitute small distinctive groups. Al- though developing in open and semi-arid habitats, the pop- ulations of these two species show unexpected inter-popu- lation variability. The other two species A. officinalis and A. altissimus, show close relationships with A. albus due to the smooth cladodes. Within A. acutifolius, two clusters are quite well sepa- rated (at the distance d < 55). The first one is mainly com- posed of coastal populations from Tipaza, El Aouana and Fig. 3. Dendrogram analysis showing relationships among 29 Algerian populations of the genus Asparagus. Analysis was performed on the matrix of mean values of 13 morphological and floral traits. The dotted lines correspond to the distances allowing the main clusters to be distinguished. BOUBETRA K., AMIROUCHE N., AMIROUCHE R. 172 ACTA BOT. CROAT. 81 (2), 2022 Zemmouri. The second cluster concerns populations from the inland collecting sites at higher altitudes such as Senal- ba (southern slope of the Saharan Atlas) Mezloug, Ain Sma- ra (east of the Tellian Atlas), Redjredj and Keddara (central part of the Tellian Atlas). In order to estimate the impact of climatic factors on morphological variability, we performed another PCA fo- cused on the 16 populations of A. acutifolius. Indeed, unlike A. albus and A. horridus, populations of A. acutifolius are encountered in very diverse habitats from wetlands to semi- arid and arid areas. This PCA takes into account the five main parameters of the Mediterranean bioclimate (Fig. 4). In contrast to the two species A. albus and A. horridus, populations of A. acutifolius are encountered in very diverse habitats of undergrowth and open areas from wetlands to semi-arid and arid zones. In this aim, a second PCA was focused on the 16 populations of A. acutifolius taking into account the five main parameters of the Mediterranean bio- climate (Fig. 4). The PCA has been carried out on the aver- age of the values of each variable for each population as well as the annual precipitation (P), the altitude (Alt.), the aver- age of the maximum in summer (M °C) and the minimal temperatures in winter (m °C) (On-line Suppl. Tab. 1). The first two axes PC1 and PC2 describe 55.26% of the total in- formation with 34.45% and 21.81%, respectively (Fig. 4A). The length of the cladodes, LCL, and, to a lesser extent NGR, NCL and NFL, are located on the positive part of PC1 and highly correlated with the precipitation P. In contrast, the altitude (Alt) is located on the negative part of this axis. PC2 shows, on one hand, a positive correlation between the diameter of the seeds (DIG) and the maximum temperature in summer M °C and, on the other hand, a negative corre- lation with the minimum temperature in winter (m °C). Two groups of populations are opposed with respect to PC1 (Fig. 4B). The populations from continental, semi-arid to arid bioclimates and higher altitudes (Senalba, Redjredj, Ain Smara and from Mezloug in the East to Mansourah in the West area) constitute a first group. The second one con- cerns populations from northeast coastal stations as Zem- mouri and El Aouana located in subhumid and humid bio- climate. All other populations are from subhumid bioclimate (Keddara, Bainem, Bouchaoui, Tipaza, Souida- nia) and are distributed in an intermediate situation be- tween the two previous groups within the biogeographical sector of Algiers. Anatomical analysis of the cladodes The cross sections of the cladodes of the five studied species show a different structure. Interspecific variations have been observed relatively to the shape of section, the thickness of the cuticle, the shape of epidermal cells, the number of layers of palisade cells, the number of vascular bundles and the presence of raphides (Fig. 5, On-line Suppl. Tab. 2). Circular cross sections are found in three species, A. acutifolius, A. officinalis and A. altissimus (Fig. 5A, I, K). The first one is characterized by uniseriate epidermis with iso- diametric cells strongly cutinized (Fig. 5B) with the pres- ence of stomata along this epidermis (Fig. 5C). Just below the epidermis, the palisade parenchyma consists of two lay- ers of elongated cells rich in chloroplasts. Spongy parenchy- ma varies highly in shape, and the cells may be isodiametric or elongated. At this level, some cells, called idioblasts, con- tain raphides (calcium oxalate crystal) in the form of rods (Fig. 5D). In the pith, the cells are strongly sclerified, and only two vascular bundles have been observed. In A. officinalis, the cuticle is thin and the epidermal cells are rounded and not of the same size with some larger cells (Fig. 5J). Stomata were observed all around the cross section. The palisade parenchyma is composed of two lay- Fig. 4. Principal components analysis focused on 16 populations of A. acutifolius showing the relationships between the morphological and floral traits and the main bioclimatic parameters. A – loading of the variables on the circle of correlations, B – scatter of the mean values of each population. GENUS ASPARAGUS IN ALGERIA ACTA BOT. CROAT. 81 (2), 2022 173 ers of elongated cells. In the pith, two vascular bundles are observed and surrounded by a single layer of spongy paren- chyma. In the endemic A. altissimus, the epidermis is made up of a single layer of square cells also covered by thick cuticle with numerous stomata. The palisade parenchyma is com- posed of two layers of elongated cells. The cells of spongy parenchyma are irregular and some of them contain raph- ides (Fig. 5L). Four vascular bundles are observed. In the two other species, i.e. A. albus and A. horridus, the cross section is triangular. (Fig. 5E, G). The epidermal cells in A. albus, are rounded to irregular in shape with vari- able size. They are covered by a thin cuticle (Fig. 5E). Stomata were also observed. The palisade parenchyma contains elongated cells arranged in three layers whereas the spong y parenchyma consists of relatively large, thin-walled cells. In the pith, the cells are sclerified with four vascular bundles (Fig. 5F). In A. horridus, the epider- mal cells are rounded with a thick cuticle (Fig. 5H). In the palisade parenchyma, the cells were rearranged in several layers with small intercellular spaces. The pith occupies the greatest part and contains numerous vascular bundles arranged in a ring. Here, in each bundle, the xylem is directed toward the center of the cladode. Discussion Taxonomic relationships Algeria occupies a central biogeographic position in North Africa, characterized by an impressive east-west bio- climatic gradient from humid to arid lands. In this Medi- terranean region, strong topographic and soil heterogene- ity explains the highly contrasting habitats and the floristic richness. In this ecogeographical context, wild species of the genus Asparagus occur in various ecological condi- tions, under forest cover as well as in open and dry habitats of the steppe vegetation of the highlands, showing high tol- erance to drought and high temperatures (Boubetra et al. 2017a, b). Asparagus acutifolius is widespread and quite common in moist and shady biotopes of woodlands and shrublands of humid and semiarid bioclimates. Asparagus albus and A. horridus were less common in this research; both are linked to dry and stony soils mostly in arid and semiarid habitats of NW Algeria. Sometimes, they occur in sympat- ry in steppic highlands making high xerophytic shrubs. Asparagus altissimus is endemic to northwestern Algeria consisting of small populations of scattered individuals along hedges on saline and dry rather sandy soils. Asparagus Fig. 5. Cross sections of cladodes of the five Algerian Asparagus species. A. acutifolius: A – circular section, B – epidermis and cuticle, C – stomata, D – raphides. A. albus: E – triangular section, F – vascular bundles. A. horridus: G – triangular section, H – epidermis and cuticle. A. officinalis: I – circular section, J – epidermis. A. altissimus: K – circular section, L – raphides. Cu: cuticle, Ep: epidermis, Pp: palissadic parenchyma, St: stomata, Gc: guard cell, Sc: substomatal chamber, R: raphides, Pi: pith, Vb: vascular bundles, Xy: xylem, Ph: phloem. BOUBETRA K., AMIROUCHE N., AMIROUCHE R. 174 ACTA BOT. CROAT. 81 (2), 2022 officinalis is very rare and appears to have naturalized as isolated individuals on the edges of cultivated fields. Despite the wide distribution of the species of the genus Asparagus in the Mediterranean region and their ecological and economic interest, few studies have been performed to elucidate the morphological variability, particularly for wild A. acutifolius, A. albus and A. horridus. Current researches are focused mainly on the cultivated species A. officinalis for its economic importance. Studies based on molecular markers, aim to assess genetic diversity among germplasm including the related wild species from Europe and Asia (Mousavizadeh et al. 2021). Other studies combine both morphological and molecular data (Irshad et al. 2019, Chen et al. 2020). Morphological variability, growth and produc- tion of spears may be correlated to environmental factors (Altunel 2021). Concerning wild species, a multivariate analysis per- formed on Indian populations of A. racemosus Willd. (Chithra and Siril 2017), showed that significant characters were relative to plant height, the diameter and color of stem, length and number of cladodes in fascicle. In the Iranian species, A. officinalis, A. persicus Baker, A. verticillatus L., and A. breslerianus Schult. & Schult., the length of cladodes and seed number per fruit are the most discriminating characters (Mousavizadeh et al. 2015). In these species, the number of seeds per berry varies from 1 to 6, unlike the Al- gerian species where the number of seeds is 1-2, exception- ally 3 in an individual of A. officinalis. Despite the morphological similarities among some spe- cies of the genus Asparagus, Chen et al. (2020) have shown that individual variations could be linked to environmental factors. The analysis of the genetic variation of Italian pop- ulations of A. acutifolius, evaluated by the ISSR markers (Sica et al. 2005), indicated an inter-population diversity ac- cording to their geographical origin. A study on the system- atics and the chorology of A. acutifolius, A. albus and A. Fig. 6. Illustrations of some cross sections of stems and roots from Algerian specimens of Asparagus. Stem of A. horridus: A – global view of the section, B – arrangement of cribro-vascular bundles. C – trichomes in the stem of A. acutifolius. D – stomata in the stem of A. altissimus. E – vascular bundle in stem of A. horridus. Root of A. acutifolius: F – general view of the section, G – root hairs, H – raphides within cells of the cortex, I – general view of vascular cylinder. Cu: cuticle, Ep: epidermis, St: stomata, Co: collenchyma, Pi: pith, Vb: vascular bundles, Xy: xylem, Ph: phloem, Cr: cortex (parenchyma cells), Rh: root hairs, R: raphides, Pi: pith, Ed: endodermis, Pr: pericycle. GENUS ASPARAGUS IN ALGERIA ACTA BOT. CROAT. 81 (2), 2022 175 horridus in Sardinia, allowed Urbani et al. (2007) to confirm the presence of these species and to exclude A. aphyllus L., from the flora of this island on the basis of anatomical cri- teria of cladodes. In Iran, the wild asparagus polyploids (8x, 10x) are adapted to saline and dry lands (Mousavizadeh et al. 2022). These results agree with the repartition of the en- demic hexaploid (6x) A. altissimus which occurs preferen- tially on saline soils. Anatomical diversity of the cladodes Compared to the stems and roots (Fig. 6), the morphol- ogy and anatomy of the cladode in the genus Asparagus, show a striking diversity and distinctive interspecific fea- tures. Undoubtedly the structure of the cladodes has a tax- onomic significance. It would also be linked to the ecologi- cal conditions with regard to the chorology and geographic distribution of each species (Boubetra et al. 2017a, b). The cross section is circular in all the species, except A. albus and A. horridus, which show a triangular shape. Cir- cular sections have also been observed in Asparagus species from Bulgaria such as A. tenuifolius Lam., and A. acutifolius (Raycheva and Stojanov 2013). The triangular shape is rare, having been reported only in A. adscendens Roxb., (Kawale et al. 2014). Various other shapes are specific to this genus such as the oval in A. brachyphyllus Turcz., and A. schoberioides Kunth (Ito et al. 2006), irregular elliptic in A. lycicus P.H. Davis and A. persicus (Güvenç and Koyuncu 2002). In A. officinalis, A. maritimus (L.) Mill., and A. verticillatus, the shape varies from irregular elliptic to stellar with unclear angles (Raycheva and Stojanov 2013). Samples of A. officinalis from Algeria are remarkable for their circular shape. The interspecific variability of cross-section of the clad- odes was also observed within other genera such as Ruscus where the shape is rectangular in R. aculeatus L., and ellipti- cal in R. colchicus Yeo (Güvenç et al. 2011). Furthermore, the epidermal cells also exhibit a variability. For the Algerian specimens of A. acutifolius and A. officinalis, the cells are re- spectively isodiametric and rounded, whereas they are rect- angular in Bulgarian populations (Raycheva and Stojanov 2013), barrel-shaped in A. brachyphyllus (Bercu 2008) and longitudinal in A. racemosus (Durai Prabakaran et al. 2015). In addition, these cells are covered with a cuticle vary- ing in thickness depending on the species. Anatomical stud- ies performed on A. asparagoides (L.) Druce show that the cuticle thickness correlates with the shape of the section of the cladode (Coles et al. 2006). According to Tamanian (1982), this variability expresses adaptation to ecological conditions. Under the epidermis of each species two or three layers of palisade cells with different lengths are situated. These results are consistent with those obtained on A. adscendens (Kawale et al. 2014) and A. racemosus (Durai Prabakaran et al. 2015). The most numerous and the longest palisade cells were observed in A. verticillatus, with four rows (Raycheva and Stojanov 2013). In A. tenuifolius, A. maritimus and A. officinalis, the pa- renchyma is represented by two rows of slightly prolonged cells (Raycheva and Stojanov 2013). These results correlate with our studied species except for A. albus and A. horridus whose show three and several layers respectively. Compared to species of the genus Ruscus, the palisade and spongy pa- renchyma are replaced by layers of cells containing chloro- plasts (Güvenç et al. 2011). The vascular system is delimited by the assimilation pa- renchyma by one cell row. In our study, raphides are re- markable in this parenchyma, and are present only in clad- odes of A. acutifolius and A. altissimus. Their presence in some taxa may have a taxonomic significance as mentioned also by Prychid and Rudall (1999), also shown in the genus Ruscus (Güvenç et al. 2011). The cladodes of the Algerian samples of A. acutifolius are distinguished by the constant presence of two vascular bundles while in Turkish and Bul- garian specimens, this number were four and five respec- tively (Güvenç and Koyuncu 2002). Two vascular bundles have also been reported for A. brachyphyllus and A. officinalis (Begum et al. 2017). In A. horridus from Algeria, the vascu- lar bundles are can number up to 20 and occupy all the pith, as in the case of A. aphyllus (Güvenç and Koyuncu 2002). As suggested by Raycheva and Stojanov (2013), the anatom- ical characters of the cladodes particularly the number of vascular bundles can be correlated with the ecological en- vironments. Conclusion This study constitutes the first report on morpho-anat- omy for the genus Asparagus in Algeria. The results high- light the taxonomic importance of morphological and floral characters as well as the anatomical features of the cladodes, mainly the shape, the number of vascular bundles and the raphides. They also provide perspective for a better under- standing of the diversity of species and populations in relation to environmental conditions, particularly for A. acutifolius, which is widespread in highly contrasting bioclimatic con- ditions. Acknowledgements This work was carried out in the framework of the Proj- ect “Asparagales in Algeria”, between the University of Sci- ences and Techniques Houari Boumediene (USTHB, Algiers) and the National Institute of Forest Research (INRF, Algiers). The authors wish to thank the two anonymous reviewers for their suggestions and comments that have improved our manuscript. We are also grateful to S. Benhouhou, the manager of the Official Herbarium of ENSA (Algiers). References Altunel, T.A., 2021: Morphological and habitat characteristics of Asparagus (Asparagus officinalis L.) and socio-economic structure of producers. Turkish Journal of Agriculture - Food Science and Technology 9, 1092–1099. Angiosperm Phylogeny Group III, 2009: An update of the An- giosperm phylogeny group classification for the orders and BOUBETRA K., AMIROUCHE N., AMIROUCHE R. 176 ACTA BOT. CROAT. 81 (2), 2022 families of flowering plants: APG III. Botanical Journal of the Linnean Society 161, 105–121. Begum, A., Sindhu, K., Giri, K., Umera, F., Gauthami, G., Kumar, J.V., Naveen, N., Rao, K.N.V., Ali, S.S., Sri, K., Dutt, R., 2017: Pharmacognostical and Physio-Chemical Evaluation of Indian Asparagus officinalis L. International Journal of Pharmacognosy and Phytochemical Research 9, 327– 336. Bell, A.D., 2008: Plant form: An illustrated guide to flowering plant morphology. Timber Press, Portland. Bercu, R., 2008: Some aspects of Asparagus brachyphyllus Tucz. (Asparagaceae) anatomy. Research Journal of Agricultural Science 50(3), 7–12. Boubetra, K., Amirouche, N., Amirouche, R., 2017a: Le genre Asparagus L. en Algérie : Systématique, chorologie et impor- tance en écologie forestière. Biocénose 8, 77–81. Boubetra, K., Amirouche, N., Amirouche, R., 2017b: Compara- tive morphological and cytogenetic study of five Asparagus (Asparagaceae) species from Algeria including the endemic A. altissimus Munby. Turkish Journal of Botany 41, 588–599. Chen, H., Guo, A., Wang, J., Gao J., Zhang S., Zheng J., Huang, X., Xi, J., Yi, K., 2020: Evaluation of genetic diversity within asparagus germplasm based on morphological traits and ISSR markers. Physiology and Molecular Biology of Plants 26, 305–315. Chen, X.Q., Tamanian, K.G., 2000: Asparagus L. In: Wu, Z.Y., Raven, P.H. (eds.), Flora of China Vol. 24. (Flagellariaceae through Marantaceae), 209–216. Science Press, Beijing, Mis- souri Botanical Garden Press, St. Louis. Chithra, M.G., Siril, E.A., 2017: Morphological variability of aerial vegetative characters among 20 Shatavari (Asparagus racemosus Willd.) collections from Kerala, India. Journal of Root Crops 43(2), 21–32. Coles, R.B., Willing, K.L., Conran, J.C., Gannaway, O., 2006: The identification and distribution of western cape form bridal creeper (A. asparagoides) in the south east of south Australia and western Victoria. Plant Protection Quarterly 21, 104– 108. Durai Prabakaran, K., Vadivu, R., Jayshree, N., 2015: Pharma- cognostical standardization of leaves of Asparagus racemosus Willd. International Journal of Multidisciplinary Research and Development 2, 332–335. Fukuda, K., Ashizawa, H., Suzuki, R., Ochiaï, T., Nakamura, T., Kanno, A., Kameya, T., Yokoyama, J., 2005: Molecular phy- logeny of the genus Asparagus (Asparagaceae) inferred from Plastid petB intron and petD-rpoa intergenic spacer sequenc- es. Plant Species Biology 20, 121–132. Güvenç, A., Koyuncu, M., 2002: Studies on the anatomical struc- ture of cladodes of Asparagus L. species (Liliaceae) in Turkey. Israel Journal of Plant Sciences 50, 51–65. Güvenç, A., Coşkun M., Arihan, O., 2011: Anatomical structure of cladodes of Ruscus L. taxa (Liliaceae) in Turkey. FABAD Journal of Pharmaceutical Sciences 36, 119–128. Irshad, M., Idrees, M., Tariq, A., Pathak, M.L., Hanif, M., Naeem, R., 2019: Genetic diversity among Asparagus species using morphological characteristics and RAPD markers in Paki- stan. Journal of Biodiversity Conservation and Bioresource Management 5, 13–24. Ito, T., Ochiai, T., Ashizawa, H., Shimodate, T., Sonoda, T., Fukuda, T., Yokoyama, J., Kameya, T., Kanno, A., 2006: Production and analysis of reciprocal hybrids between Asparagus officinalis L. and A. schoberioides Kunth. Genetic Resources and Crop Evolution 54, 1063–1071. Kawale, M., Ankoliya, S., Saravanan, R., Dhanani, T., Manivel, P., 2014: Pharmacognostical and physicochemical analysis of Asparagus adscendens Buch. Ham. ex Roxb. (Shweta musali). Journal of Pharmacognosy and Phytochemistry 3, 131–139. Kubitzki, K., Rudall, P.J., 1998: Asparagaceae. In: Kubitzki, K. (ed.), The families and genera of vascular plants, 125–129. Springer, Berlin, Heidelberg, New York. Kubota, S., Konno, I., Kanno, A., 2012: Molecular phylogeny of the genus Asparagus (Asparagaceae) explains interspecific cross ability between the garden Asparagus (A. officinalis) and other Asparagus species. Theoretical and Applied Ge- netics 124, 345–354. Maire, R., 1958: Flore de l’Afrique du nord. Vol. 5, 215-232. Paul Lechevalier, Paris. Mantovani, D., Rosati, A., Perrone, D., 2019: Photosynthetic characterization and response to drought and temperature in wild Asparagus (Asparagus acutifolius L.). Horticultural Science 54, 1039–1043. Mousavizadeh, S.J., Hassandokht, M.R., Kashi, A., 2015: Multi- variate analysis of edible Asparagus species in Iran by mor- phological characters. Euphytica 206, 445–457. Mousavizadeh, S.J., Gil, J., Castro, P., Hassandokht, M.R., Moreno, R., 2021: Genetic diversity and phylogenetic analysis in Asian and European Asparagus subgenus species. Genetic Resources and Crop Evolution 68, 3115–3124. Mousavizadeh, S.J., Gil, J., Moreno, R., Mashayekhi, K., 2022: Asparagus ploidy distribution related to climates adaptation in Iran. Environment. Development and Sustainability, 24, 5582-5593. Nakayama, H., Yamaguchi, T., Tsukaya, H., 2012: Cladodes, leaf- like organs in Asparagus, show the significance of co-option of pre-existing genetic regulatory circuit for morphological diversity of plants. Plant Signaling & Behavior 7, 961–964. Nakayama, H., Yamaguchi, T., Tsukaya, H., 2013: Modification and co-option of leaf developmental programs for the acqui- sition of flat structures in monocots: unifacial leaves in Juncus and cladodes in Asparagus. Frontiers in Plant Sciences, 4, 248. Pieroni, A., 2005: Food for two seasons: culinary uses of non- cultivated local vegetables and mushrooms in a south Italian village. International Journal of Food Sciences and Nutrition 56, 245–272. Prychid, C.J., Rudall, P.J., 1999: Calcium oxalate crystals in monocotyledons: a review of their structure and systematics. Annals of Botany 84, 725–739. Quézel, P., Santa, S., 1962: Nouvelle Flore de l’Algérie et des Ré- gions Désertiques Méridionales. Vol. 1, 208–209. Éditions du Centre National de la Recherche Scientifique, Paris. Raycheva, T., Stojanov, K., 2013: Comparative anatomical study of five species of genus Asparagus in Bulgaria. Trakia Journal of Sciences 2, 104–109. Schnitzler, A., Arnold, C., 2010: Contribution of vines to forest biodiversity in the Mediterranean basin. Ecologia Mediter- ranea 36, 5–24. Sica, M., Gamba, G., Montieri, S., Gaudio, L., Aceto, S., 2005: ISSR markers show differentiation among Italian popula- tions of Asparagus acutifolius L. BMC Genetics 6, 17. Tamanian, K.G., 1982: Analysis of the taxonomic value of ana- tomic and morphologic characteristics of the cladodia of Caucasian species of the genus Asparagus L. USSR. 1. Bio- logical Journal of Armenia 35, 885– 892 (in Russian). Urbani, M., Becca, G., Ledda, M.G., 2007: Notes on systematics and chorology of Asparagus L. (Asparagaceae) in Sardinia (Italy). Bocconea 21, 267–271.