Ecological aspects of anatomical and morphological ...

Grass research Edited by: Ludwik Frey W. Szafer Institute of Botany, Polish Academy of Sciences Kraków, 2009

Ecological aspects of anatomical and morphological variation of Elymus hispidus, E. repens and E. ×mucronatus

MAGDALENA SZCZEPANIAK

Abstract: Species boundaries among Elymus hispidus (Opiz) Melderis, E. repens (L.) Gould and their interspecific hybrid, E. ×mucronatus (Opiz) Conert, are assessed using the results of multivariate analyses of anatomical and morphological characters of leaves and glumes. Interand intraspecific anatomical diversity is analysed as ecological adaptations to different habitats. Anatomical descriptions of leaves and glumes are provided. The results of principal coordinate analyses support significant anatomical differences among the species. Characterized by sunken stomata, much sclerenchymatous tissue, heavy cutinization and often dense pubescence, leaves of E. hispidus suggest a great xeromorphic adaptation. While E. repens is basically a mesophyte, it exhibits anatomical plasticity supporting its adaptive possibilities to different habitats. The coastal ecotype of E. repens was recognized as having some xeromorphic characters such as distinctly adaxially ribbed leaf blades with large bulliform cells, deeply sunken stomata and much sclerenchymatous tissue as well as glumes covered by a thick cuticle. E. ×mucronatus is clearly distinguished by a parental combination of anatomical characters of the leaf blade and glumes, which confirms its hybrid origin. Key words: anatomical plasticity, Elymus repens, Elymus hispidus, Elymus ×mucronatus, glume anatomy, leaf anatomy, glume morphology, leaf morphology, taxonomy

INTRODUCTION Adaptation to specific environmental conditions is very clearly displayed in the differences of a plant’s internal structure. Anatomical adaptation of leaves and wood essentially contributes to plant survival in extreme habitats of intense sun or shade, cold and heat, wetness or dryness, and soil mineral nutrient deficiencies (FAHN & CUTLER 1992; DICKISON 2000). Some characters of plant anatomy are associated with resistance to air pollution (stomatal distribution, trichome frequency, etc.) (DICKISON 2000). Phenotypic plasticity in the anatomical leaf structure can change to suit different temperature and water conditions

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(HLWATIKA & BHAT 2002; CHEN et al. 2006). Plastic responses of grass leaves to drought conditions have been studied in the anatomical structure as well as physiological and biochemical aspects (VAN ARENDONK & POORTER 1994; RYSER & LAMBERS 1995; VANHALA et al. 2004; CHEN et al. 2006). Elymus hispidus (Opiz) Melderis occurs in the area from Central Asia to Southern and Western Europe, reaching France and Spain (MELDERIS 1980). It is a xerothermic species that grows in grasslands on dry and sun-exposed slopes, gypsum rock outcrops, in abandoned quarries, very often on stony calcareous or chalk soils derived from gypsum or loess. It is rare in thermophilous scrubs and on mid-field and roadside scarps (SZCZEPANIAK 2001). Five subspecies of Elymus hispidus are distinguished in Flora Europaea (MELDERIS 1980). Subsp. hispidus and subsp. barbulatus (Schur) Melderis have the widest Euro-Asiatic distribution, while subsp. graecus Melderis is endemic to south-eastern Greece, subsp. pouzolzii (Godron) Melderis is endemic to southern and western France and subsp. varnensis (Velen.) Melderis is endemic to the eastern part of the Balkan Peninsula. Recently, var. hispidus and var. villosus (Hack.) Assadi are distinguished in the Polish flora based on the morphological and genetic variation within Elymus hispidus (SZCZEPANIAK 2003; SZCZEPANIAK & CIEŚLAK 2003). Specimens of E. hispidus var. villosus are generally more pubescent and more xerotermophilous (ASSADI 1998). In the present paper, the anatomical structure of E. hispidus is examined to determine xeric characters that would allow the species to survive in dry and sunny grasslands. Elymus repens (L.) Gould is a cospomopolitan species closely associated with human agricultural activities. It is a creeping, aggressive perennial grass that is a serious weed in the majority of northern temperature regions worldwide (HOLM et al. 1977). In Flora Europaea, MELDERIS (1980) distinguished five subspecies of Elymus repens of which only subsp. repens occurs throughout almost entire Europe and is unique in Poland. The distribution of the other subspecies which are found in north-western Europe [subsp. arenosus (Petif) Melderis], southern Europe [subsp. calcareus (Černjavski) Melderis] and in Ukraine and Russia [subsp. pseudocaesius (Pacz.) Melderis and subsp. elongatiformis (Drobov) Melderis] is limited. Elymus repens is characterized by wide phenotypic plasticity (TAYLOR & AARSSEN 1988), which allows it to occur in different habitats. Recently, a relatively continuous pattern of morphological variation of E. repens in the geographical scale of Poland has been documented and intraspecific varieties, var. repens, var. aristatus (Schreb. ex Baumg.) Melderis & D. C. Mc Clint. and var. subulatus (Roem. & Schult.) Szczepaniak have been recognized (SZCZEPANIAK 2009). The presence of E. repens in periodically flooded, saline, polluted or extremely dry sites may suggest an intraspecific anatomical and, consequently, physiological differentiation, which enables the plant to adapt to specific habitat conditions. Elymus hispidus and E. repens can easily crossbreed between each other and hybrids are relatively common in natural habitats (MAHELKA et al. 2005; SZCZEPANIAK et al. 2007). E. ×mucronatus (Opiz ex Bercht.) Conert are robust and competitive in relation to its parental species, particularly to the competitively weak E. hispidus (own field observations). Genetic, morphological and pollen fertility studies showed the hybrid origin of

M. Szczepaniak: Anatomical and morphological variation of Elymus species

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E. ×mucronatus (SZCZEPANIAK et al. 2007) and nvar. mucronatus Szczepaniak with usually glabrous or sparsely hairy glumes and nvar. tesquicolus (Czerniak.) Szczepaniak with densely pubescent glumes have been distinguished (SZCZEPANIAK 2003). It was expected that additional characters which would confirm the hybrid origin of the species could found in the anatomical structure of E. ×mucronatus. Anatomical characters of the grasses, especially pertinent to ultrastructural features of leaf, have made essential contributions to the classification of the family Poaceae and are used to distinguish species, intraspecific taxa or hybrids (HANSEN 1959; METCALFE 1960; CAROLIN et al. 1973; ELLIS 1986). RUNEMARK and HENEEN (1968) examined cross-sectional blade anatomy as part of investigations on the generic border between Elymus L. and Agropyron Gaertn. Using the presence of “crown cells”, primary rib type and hypodermis, they distinguished three anatomical types of leaves in the Agropyron-Elymus complex, among which species of Elytrigia Desv. and Elymus L. were characterized by the same leaf type. JARVIE and BARKWORTH (1992a) showed the correlation between leaf and glume anatomical characters and genomic constitution (based on combinations of the E, J, and St haploid genome) within some perennial Triticeae. According to their results, leaves and glumes of heterogenomic species, Elymus repens and Elymus hispidus, are similar and differ only in leaf blade convolution. Additionally, micro-morphological features of the leaf were used as taxonomical characters in the Agropyron-Elymus complex (WEBB & ALMEIDA 1990; WANG & HENWOOD 1999). The above studies deal mainly with intergeneric differences while examinations of the level of intraspecific anatomical variation within Elymus repens and E. hispidus, especially ecological adaptations in the internal structure, are lacking. The aims of the present paper are to analyze (i) the ecological significance of anatomical and morphological variation of leaves and glumes within Elymus hispidus and E. repens, (ii) the usefulness of anatomical characters to distinguish E. repens and E. hispidus and (iii) to support the hybrid origin of E. ×mucronatus. The results are part of biosystematics studies conducted on E. repens and E. hispidus, including macro-morphological, anatomical, cytological, and chemotaxonomical studies as well as AFLP analyses (SZCZEPANIAK 2002a, b, 2003, 2009; SZCZEPANIAK et al. 2002, 2007; SZCZEPANIAK & CIEŚLAK 2003).

MATERIAL AND

METHODS

Analyses of the anatomical structure and morphological variation of leaves and glumes were performed for one plant representing each population i. e. ten chosen populations of Elymus hispidus (var. hispidus and var. villosus), including E. ×mucronatus (nvar. mucronatus and nvar. tesquicolus), and ten populations of E. repens (var. repens, var. aristatus and var. subulatus) (Appendix). Individual plants were sampled from different ecological habitats in Poland. To standardize growth conditions, the plants grew in homogeneous experimental cultures in the Botanical Garden, Jagiellonian University, for two years. Mature leaves and glumes where then collected for anatomical analysis. Voucher specimens were deposited in the Herbarium of the W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków (KRAM). Cross sections were made in the middle part of blades from the second node of the culm and in the mid-part of upper glumes of a spikelet half way along the spike. Fresh leaves were fixed in a 70% solution of ethanol. Sections were hand-cut in black lilac’s stem with a razor blade and twice stained, first in alum

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Table 1. Anatomical and morphological variation of leaves and glumes of Elymus hispidus, E. repens and E. ×mucronatus. All characters were used in PCoA, excluding two characters marked with asterisks * E. hispidus

No. leaf characters 1 blade thickness across rib in mid-blade [μm] 2 *

3 *

4

5 6 7 8 9 10

210–320 (258 ± 34) depth of furrows between vascular bundles in mid- 120–195 blade [μm] (156 ± 27) presence of nodular crystals in epidermis; present absent – 0, present – 1 number of vascular bundles 29–45 (37 ± 5) presence of primary ribs; mildly promildly pronounced – 1, pronounced – 2 nounced rib shape; tall with sloping not tall with rounded sides – 1, tall with rounded sides, flat at the sides – 2, tall with sloping sides, flat at the top – 3 top pubescence of adaxial epidermis; glabrous / hairy glabrous – 0, hairy – 1 pubescence of abaxial epidermis; glabrous / hairy glabrous – 0, scabrous – 1, hairy – 2 number of sclerenchyma layers at the xylem pole 3–6 (5 ± 1) number of sclerenchyma layers at the phloem pole 3–4 (3) presence of cuticle; present absent – 0, present – 1 stoma location; sunken superficial – 1, sunken – 2

E. ×mucronatus min–max (mean ± SD) 200–255 (223 ± 28) 120–150 (132 ± 16) absent / present

E. repens

170–285 (214 ± 41) 100–165 (134 ± 23) absent / present

31–38 (35 ± 4) 28–50 (38 ± 8) mildly pronounced mildly pronounced or pronounced tall with rounded shallow with sides rounded sides glabrous / hairy

glabrous / hairy

glabrous

glabrous / scabrous

3–4 (3) 3 present

2–4 (3 ± 1) 2–5 (3 ± 1) absent / present

sunken

11 number of bulliform cells 12 bulliform cells; superficial – 1, sunken – 2

3–5 (4) sunken

13 blade; flat – 1, convolute at margins – 2, convolute – 3

convolute at mar- convolute at margins or convolute gins or convolute

superficial (occasionally slightly sunken) 3–5 (4) superficial (occasionally slightly sunken) flat or convolute at margins

230–380 (291 ± 52) 135–290 (186 ± 55) 5–6 (5) present

240–320 (268 ± 45) 130–150 (138 ± 10) 5–6 (5) absent / present

155–305 (207 ± 44) 80–170 (127 ± 25) 5–9 (6) absent / present

2–8 (4)

3

absent

glume characters 1 thickness across keel [μm] 2 depth of furrows between vascular bundles near keel [μm] 3 number of vascular bundles 4 presence of nodular crystals in epidermis; absent – 0, present – 1 5 number of continuous parenchyma layers adjacent to adaxial epidermis 6 chlorenchyma; not reaching adaxial epidermis – 1, reaching adaxial epidermis – 2, partially reaching adaxial epidermis 7 presence of cuticle; absent – 0, present – 1 8 pubescence of abaxial epidermis; glabrous – 0, scabrous – 1, hairy – 2 9 pubescence of adaxial epidermis; glabrous – 0, scabrous – 1 10 presence of sclerified epidermal cells; absent – 0, present – 1

4 sunken

adjacent to partially reaching abaxial epiadaxial epidermis dermis, crescentshaped present present glabrous or with dense long hairs scabrous

glabrous or with dense long hairs scabrous

present

present

compactly between abaxial and adaxial epidermis absent / occasional glabrous / scabrous scabrous or with short hairs absent


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carmine for about 10–20 minutes and second, after exact rinse, in green iodic for about 1 minute. Stained scraps were placed on basic glasses in a glicero-gelatine drop and covered by covering glasses. Thirteen leaf characters and ten glume characters were studied to evaluate inter- and intraspecific anatomical and morphological diversity (Table 1). Plant sections were examined and characters were measured using a light microscope, Nikon Eclipse E600. Relationships among species, varieties and hybrids were estimated by principal coordinate analysis (PCoA) based on Euclidean distances matrices. PCoA was performed separately on leaf and glume data sets with MVSP 3.13q (KOVACH 2005).

RESULTS Comparative leaf morphology and anatomy Elymus hispidus PCoA results clearly demonstrate differences in ultrastructural characters of leaf anatomy and morphology between Elymus hispidus and E. repens (Fig. 1). The type of hairness, the presence of epicuticular wax, the volume of sclerenchymatous tissue, the stoma location and the presence of silica crystals in epidermis significantly differentiate the species (Table 1). 3.6

Rr4

2.9

Rr2 Ra1

2.2

Axis 2 (16.24%)

1.5

Hv3 Rr3 Hh2

0.7

Ra3

Hh4 Hv2

0.0

Hh1

-0.7

Hv1

-1.5

Rr5 Rr1 Ra2

Rs ×Mm Hh3

×Mt2

Ra4

×Mt1

-2.2 -2.9 -4.9

-3.9

-2.9

-2.0

-1.0

0.0

1.0

2.0

2.9

3.9

4.9

Axis 1 (45.83%)

Fig. 1. Principal coordinate analysis (PCoA) of Elymus hispidus; var. hispidus ( ), var. villosus (∇); E. repens; var. repens (■), var. aristatus (▲), var. subulatus (●) and E. ×mucronatus; nvar. mucronatus ( ), nvar. tesquicolus ( ) specimens based on 13 anatomical characters of leaves (see Table 1). For specimens abbreviations see Appendix. Characters correlated (r ≥ 0.6) with first principal coordinate (Axis 1): 4, 9, 10, 12, 13; with second principal coordinate (Axis 2): 1, 2, 3, 5, 7, 8; for characters abbreviations see Table 1. The first three principal coordinate accounted for 74.35% (Axis 1 – 45.83%; Axis 2 – 16.24% and Axis 3 – 12.28%) of the total variance of the character set

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Fig. 2. Transverse sections of leaf blades of Elymus hispidus (A, Hv1), typical E. repens (B, Ra3), E. ×mucronatus (C, ×Mm) and coastal ecotype of E. repens (D, Rs). Bars = 200 μm. For specimens abbreviations see Appendix

Leaf blades of Elymus hispidus are more or less convolute, relatively thick and stiff, and with pronounced ribs arranged adaxially. In transverse section, they are with 29–45 (on average 37) vascular bundles, which are usually associated with the ribs. The ribs are irregular in size, tall, from rounded to flat at the top and with sloping sides. The midrib is slightly distinct or sometimes not distinguished. Adaxial furrows are deep and present between all the vascular bundles. The abaxial side is slightly wavy with the ribs not pronounced. The edge of the leaf blade contains a thick sclerenchyma girder, oval or more triangular in transverse section (Fig. 2A). Some adaptations to xeric habitats are present in the anatomical ultrastructure of Elymus hispidus leaves, e.g. the adaxial epidermis is covered with a cuticle and often with wax forming a bluish covering. Epidermal cells are rectangular and 2–3 times wider than long with a narrow lumen; outer walls are thick or very thick, anticlinal walls are straight and thick, and inner walls are moderately thickened. Adaxial and abaxial epidermal cells of E. hispidus have numerous nodular silica crystals. Stomata occur in both epidermises and are adaxially sunken in the mesophyll near bulliform cells (Figs 3A, B). Large bulliform cells are fan-shaped, 3–5 grouped in the deep furrows between vascular bundles and sunken in the mesophyll. The chlorenchyma tissue is tightly arranged and present in 3–4 layers between bundle sheaths. In Elymus hispidus, two bundle sheaths are present: an interrupted outer parenchymatous bundle sheath (PBS) as one layer of big cells with chloroplasts on flanks at the xylem pole, often reaching the phloem pole, and an inner sclerenchymatous mestome sheath (MS) as one layer of cells without chloroplasts, completely encircling the bundles (Figs 3A, B). In some cases, the PBS encloses small vascular bundles as a continuous layer at the xylem pole (Fig. 3A). Sclerenchymatous tissues adjacent to the vascular bundles are developed better in E. hispidus than in E. repens and


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Fig. 3. Transverse sections of leaf blades of Elymus hispidus; var. hispidus (A, Hh4), var. villosus (B, Hv3) and E. ×mucronatus; nvar. tesquicolus (C, ×Mt2), nvar. mucronatus (D, ×Mm). Ph, phloem; Xy, xylem; Sc, sclerenchyma; PBS, parenchymatous bundle sheath; MS, mestome sheath; Bc, bulliform cells, Tr, trichome; Ha, hair; St, stoma. Bars = 100 μm. For specimens abbreviations see Appendix

form thick-walled girders. Large bundles are adaxially accompanied by T-shape girders, up to 6-layers, and abaxially by wide I-shape girders, up to 4-layers of sclerenchyma. Sometimes small vascular bundles with well-marked girders are observed only either abaxial or adaxial (Fig. 3A).

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The extensive variation of the adaxial and abaxial surfaces of leaves in Elymus hispidus from hairless to sparsely hairy to densely pubescent was affirmed. The costal unicellular hairs are diverse on prickles, trichomes, shorter and longer hairs. Angular prickle-hairs are observed at leaf margin. There are no significant differences in anatomical and morphological leaf characters between Elymus hispidus var. hispidus and var. villosus (Fig. 1). Densely pubescent leaf blades occur relatively more often on both surfaces in var. villosus; however, more or less hairy leaves were also observed in the typical variety. Elymus repens The leaf blade of Elymus repens is flat or slightly convolute at margin. The lamina exhibits adaxial ribs only in transverse section and primary ribs are more or less pronounced (Fig. 2B). Adaxial ribs are roughly uniform in size, round-topped and shallower than in E. hispidus. Leaves usually have 28–50 (on average 38) vascular bundles and each leaf rib is formed by

Fig. 4. Transverse sections of leaf blades of Elymus repens; var. repens (A, Rr1), var. aristatus (B, Ra3) and coastal ecotype of E. repens (C, Rs). Ph, phloem; Xy, xylem; Sc, sclerenchyma; PBS, parenchymatous bundle sheath; MS, mestome sheath; Bc, bulliform cells; St, stoma. Bars = 100 μm. For specimens abbreviations see Appendix


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one vascular bundle. The abaxial side is almost completely flat. The sharp leaf blade margin is formed by several sclerenchymatous layers, triangular in transverse section (Fig. 2B). Epidermises usually lack epicuticular waxes, but a thin cuticle layer was present in plants from dry habitats (Rs, Rr4), which produced a blue-green leaf colouration. The shape of epidermal cells in Elymus repens is similar to that in E. hispidus and they are rectangular and twice as wide as long, with thick outer walls and thin anticlinal walls. Superficial stomata are present in the adaxial and abaxial epidermises in great numbers (Fig. 4). Fan-shaped, bulliform cells are arranged in groups of 3–5 and are regularly situated between vascular bundles, superficial and considerably larger than adjacent adaxial epidermal cells (Figs 4A, B). The homogeneous chlorenchyma consists of similar cells and forms a compact layer without lacunae between vascular bundles. The parenchymatous bundle sheath (PBS) occurs as one interrupted layer of cells on flanks at the xylem pole in primary vascular bundles in Elymus repens. The PBS forms a continuous layer adjacent to the xylem pole in some small vascular bundles. The mestome sheath (MS) is always present as a radial file of sclerified cells around the vascular bundle. Sclerenchymatous tissues adjacent to the adaxial and abaxial parts of primary bundles form I-shape girders, up to 4-layers and up to 5-layers, respectively. Small bundles are often devoid of sclerenchyma. No significant differences in leaf anatomy were found among Elymus repens varieties (Fig. 1). Specimens from individual populations differ by the presence or lack of unicellular hairs on the adaxial and abaxial leaf sides, but this character is not unique to one variety. Trichomes show a range of forms within E. repens, but they usually occur as elongated and irregularily scattered hairs on both leaf surfaces or short prickles on the rib top. Although Elymus repens is basically a mesophyte, some specimens (Rs, Rr4) collected on a Baltic Sea beach, are characterized by a few anatomical xeromorphic characters, such as more rolled-up leaves with prominent ribs, a relatively thicker cuticle and more sunken stomata arranged near large buliform cells (Figs 2D, 4C). Multivariate analyses supported the anatomical distinction of these specimens from the typical E. repens and showed that Rs and Rr4 were joined with the E. hispidus subgroup in cluster analysis (graph not shown). Additionally, Rs and Rr4 were also placed within the E. hispidus range of variation along axis 1, correlated with xeromorphic characters, in PCoA (Fig. 1). The coastal ecotype was recognized within E. repens using these results. Elymus ×mucronatus The anatomical structure of Elymus ×mucronatus leaves exhibits a greater similarity to one parental species, E. hispidus, than to E. repens (Figs 1, 2C, Table 1). Leaf thickness is intermediate between the parents; hybrid leaf blades are somewhat thicker than in E. repens, but not as thick as in E. hispidus, with well-marked adaxial ribs and 31–38 (on average 35) vascular bundles. Leaf blade margin is thickened and formed by several sclerenchymatous layers, oval or triangular on cross section and similar to E. hispidus. The adaxial epidermis of Elymus ×mucronatus is often covered with a thick cuticle. Epidermal cells are longitudinal and contain nodular siliceous crystals. Bulliform cells are large and sunken in the mesophyll. Interrupted parenchymatous bundle sheaths (PBS) and very sclerified mestome sheaths (MS) enclose primary vascular bundles. The sclerenchyma

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with very thick-walled cells forms adaxial T-shape griders as 3–4 layers adjacent to the vascular bundles (Figs 3C, D). Different types of unicellular trichomes such as prickles and short or elongated hairs are present adaxially (Figs 3C, D). Hairy leaves are characteristic of Elymus ×mucronatus nvar. tesquicolus, but the type of hairiness is not a diagnostic character of the variety and single, sparse long hairs also occur in E. ×mucronatus nvar. mucronatus. Comparative glume morphology and anatomy Elymus hispidus Morphologically, glumes of Elymus hispidus are slightly unequal, wide, stiff, asymmetric, oblong or lanceolate-oblong, obliquely or horizontally truncate, obtuse on the apex and with a wide membranous margin. PCoA analysis showed significant differences in glume anatomy between Elymus hispidus and E. repens along axis 1 (Fig. 5). E. hispidus glumes are thick (c. 290 μm across the keel region), with a mildly pronounced keel. Nodular crystals are always visible in sclerified epidermal cells. Continuous 2–3 layers (up to 8 layers in Hv2 specimen) of parenchyma cells with mostly thick walls and large lumina are present adjacent to the 3.0

Hh1

2.4

Hh3

1.8

×Mm

Hh2

Axis 2 (14.44%)

1.2

Rr5

0.6

Rs

0.0 -0.6

Rr4 Rr3

Ra1

Ra4

Rr2

Hv1 Hh4

Hv2

Ra3

Hv3

-1.2

Ra2 ×Mt1 ×Mt2

Rr1

-1.8 -2.4 -3.0 -5.7

-4.6

-3.4

-2.3

-1.1

0.0

1.1

2.3

3.4

4.6

Axis 1 (51.49%)

Fig. 5. Principal coordinate analysis (PCoA) of Elymus hispidus; var. hispidus ( ), var. villosus (∇); E. repens; var. repens (■), var. aristatus (▲), var. subulatus (●) and E. ×mucronatus; nvar. mucronatus ( ), nvar. tesquicolus ( ) specimens based on 10 anatomical characters of glumes (see Table 1). For specimens abbreviations see Appendix. Characters correlated (r ≥ 0.6) with first principal coordinate (Axis 1): 1, 2, 5, 6, 7, 9, 10; with second principal coordinate (Axis 2): 4, 8; for characters abbreviations see Table 1. The first three principal coordinate accounted for 75.86% (Axis 1 – 51.49%; Axis 2 – 14.44% and Axis 3 – 9.93%) of the total variance of the character set


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adaxial epidermis. The value of chlorenchyma is relatively small. Chlorenchyma cells form crescent-shaped assemblages arranged between the abaxial epidermis and parenchyma and between 5–6 (on average 5) vascular bundles (Figs 6A, B). The results of PCoA also displayed differences of the abaxial epidermis hairiness of glumes between Elymus hispidus varieties which were separated along axis 2 (except Hh4) (Fig. 5). E. hispidus var. hispidus usually has a glabrous abaxial epidermis of glumes or single, long hairs at margins in the upper part. Epidermal cells of var. villosus glumes are variable in several types of unicellular trichomes and types of hairs. Long, soft, dense and ascending hairs are present abaxially on the entire surface of the glume in var. villosus. Elymus repens The glumes of Elymus repens are similar, nearly equal, lanceolate to lanceolate-oblong, stiff, acute, glabrous, unawned or with rough awn up to 3.5 mm, and with a wide membraneous margin, usually up to 3/4 of the length of the spikelet. PCoA analysis showed some anatomical variation of the glume structure within Elymus repens (Fig. 5). In transverse section glumes are bent, relatively thin (c. 207 μm across the keel region), with a pronounced keel and 5–9 (on average 6) rough veins, formed by single vascular bundles. Vascular bundles are abaxially and adaxially strengthened by 3–4-seriate sclerenchyma girders. The parenchyma is not present or is present as assemblages of several cells with walls thinner than in E. hispidus and with large lumina, and it is adjacent to the adaxial epidermis in the keel (Figs 6E–G). The chlorenchyma is mainly 4-6 layered, with small intercellular spaces and a compactly filled space among vascular bundles and between the epidermises. The Rs individual is clearly different from Elymus repens in having more xeromorphic anatomical characters of glumes on the PCoA scatter diagram (Fig. 5). It is characterized by glumes with a thick cuticle, more sclerified veins and a greater ratio value of the parenchyma to chlorenchyma tissue (Fig. 6G). In the cluster analysis (diagram not shown) performed on all the glume characters, the Rs specimen was joined with the E. hispidus subclaster, which suggests its anatomical similarity to the xeromorphic E. hispidus. Glumes of the Rr4 specimen are more similar to the typical E. repens. PCoA results showed that Elymus repens specimens differ in the presence of nodular crystals in the glume epidermis and in the type of hairiness or its lack, but these characters do not distinguish intraspecific varieties (var. repens, var. aristatus and var. subulatus) (Fig. 5). Short, sharp, unicellular trichomes usually occur on the keel while somewhat smaller and sparser trichomes are observed on lateral nerves. Trichomes and short hairs on the adaxial epidermis are present in all the specimens. Elymus ×mucronatus The morphological and anatomical structure of Elymus ×mucronatus glumes generally showed a greater similarity to E. hispidus than to E. repens (Figs 5, 6C, D, Table 1). Glumes of the hybrid are nearly equal, stiff, asymmetric, oblong or lanceolate-oblong, obliquely or horizontaly truncate, obtuse on the apex and covered with epicuticular waxes, and similar to E. hispidus in these characters. Characters such as acute glumes or a short mucro apex

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Fig. 6. Transverse sections of glumes of Elymus hispidus; var. hispidus (A, Hh2), var. villosus (B, Hv3), E. ×mucronatus; nvar. mucronatus (C, ×Mm), nvar. tesquicolus (D, ×Mt2), E. repens; var. repens (E, Rr5), var. aristatus (F, Ra4) and coastal ecotype of E. repens (G, Rs). Pa, parenchyma; Ch, chlorenchyma; Sc, sclerenchyma; ab, abaxial epidermis; ad, adaxial epidermis. Bars = 100 μm. For specimens abbreviations see Appendix 1. Notice the chlorenchyma partially reaching to adaxial epidermis in E. ×mucronatus glumes

of glumes that are sometimes present in hybrids are similar those in E. repens. Glumes of the hybrid are thicker than in E. repens (c. 270 μm in keel), have pronounced, asymmetric keels and 5–6 (on average 5) vascular bundles.


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Siliceous, nodular crystals are often present in epidermal cells of the hybrid. Parenchyma cells do not create layers as compact and continuous as in Elymus hispidus and are usually divided by the chlorenchyma tissue. The chlorenchyma value is generally greater than in E. hispidus. Crescent-shaped assemblages of the chlorenchyma are arranged adjacently to the abaxial epidermis as in E. hispidus but chlorenchyma cells fill the entire space between the epidermises in some parts of glumes as in E. repens (Figs 6C, D). The type of the parenchyma arrangement exhibits an intermediate character between the parental species and can be used as a diagnostic character to distinguish the hybrids. Abaxial and adaxial epidermal cells are elongated and form unicellular trichomes and long hairs. The abaxially densely pubescent glumes are characteristic of Elymus ×mucronatus nvar. tesquicolus, however, specimens of E. ×mucronatus nvar. mucronatus with single, long hairs at the top and margins of glumes were sometimes observed.

DISCUSSION The ecological significance of anatomical variation within grass species has long been recognized (e.g. GARNIER & LAURENT 1994; WAHL et al. 2001; CHEN et al. 2006). The present paper shows that Elymus hispidus and E. repens exhibit anatomical adaptive characters to different habitats. Anatomical structure revealing species adaptation to habitats Evidence from leaf anatomy shows that Elymus hispidus has a few xeromorphic characters and is adapted to dry, xerothermic habitats. Leaf blades of E. hispidus are more or less convolute, often densely pubescent, with distinctly pronounced adaxial ribs and large bulliform cells with adjoining cells of the sunken stomatal complex. Well-developed bulliform cells make leaf involution and folding easy and in effect limit excessive evaporation (ESAU 1967). Elymus hispidus differs from E. repens in the volume of chlorenchyma cells in leaves and especially in glumes, which is also an adaptive xeromorphic character. In dry habitat conditions in which E. hispidus occurs, plants develop a photosynthetic apparatus with xeromorphic characters based on a decreased volume of chlorenchyma cells, but with increased numbers of chloroplasts per leaf blade. In this way, the photosynthetic activity in wheat is more effective with a concurrent increase of transpiration per unit of leaf area (POZDEEV 1998). Glumes of Elymus hispidus exhibit tightly packed chlorenchyma tissues of which they have less in comparison with typical, mesophytic specimens of E. repens. This contributes to the photosynthetic function of glumes under conditions of inadequate water and high temperature. Other morphological and anatomical characters of E. hispidus glumes also indicate the typical character of xeromorphism. Glumes are stiff due to a thick, continuous parenchymatous layer and are covered with epicuticular wax. Dense pubescence of glumes (usually in var. villosus) and a thick cuticle in E. hispidus may be a mechanism to minimize cuticular transpiration, thereby aiding water conservation in drought habitats.

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Epidermis hairs assist in reflecting strong sunshine to a lower temperature. As glumes pubescence is correlated with ecological preferences, it was used to dinstinguish varieties within E. hispidus; var. villosus occurs in xeric, drier habitats more often than var. hispidus (ASSADI 1998; SZCZEPANIAK & CIEŚLAK 2003). The majority of leaves and glumes anatomical structures observed in Elymus repens are those typically associated with mesophytes. Mesophytes generally avoid extremes of moisture and drought. E. repens usually has slightly thinner leaves compared to the xeric E. hispidus, with a greater number of stomata on the abaxial epidermis. Typical specimens of E. repens have only basic features for water conservation, such as a relatively thin waxy cuticle and superficial stomata guard cells. Glumes of E. repens mostly have the chlorenchyma tissue and either completely lack or have small assemblages of the parenchymatous tissue, considerably fewer than in E. hispidus. The adaptive nature of plasticity plays a remarkable role in the ecological distribution and evolutionary diversification of plants (SULTAN 2000). Phenotypic plasticity, i.e. the capacity of a given genotype to express different phenotypes in different environments, can change specimen development, morphology, anatomy and physiology depending on environmental factors. Previous studies suggest that morphological diversity higher than genetic diversity within Elymus repens can be mainly caused by plasticity and the outbreeding mating system (TAYLOR & AARSSEN 1988; KOSINA 1995). TAYLOR and AARSSEN (1988) proved that older populations of E. repens tended to show a greater degree of plasticity than younger populations, which may be a consequence of the past selection of more plastic genotypes in a variable or unpredictable environment. An alternative interpretation of greater variance of performance in older populations may suggest that natural selection favoured genotypes which are specialized to particular environmental conditions (TAYLOR & AARSSEN 1988). Additionally, the extensive morphological variation of E. repens in relation to the relatively low level of genetic variation also proves phenotypic plasticity of the species, where different morphological patterns are realized on the same genome basis (SZCZEPANIAK et al. 2002; SZCZEPANIAK & CIEŚLAK 2003). Various anatomical adaptive characters shown in Elymus repens support a wide ecological tolerance of the species and the possibility of its occurrence in a range of habitats from mesic to xeric. The taxon occurs in a continuous series of overlapping habitats in nature (SZCZEPANIAK 2002b). E. repens can creates physiological ecotypes, resistant to soil pollution, and exerts variability along the ecological gradient (BREJ 2001). The anatomical ultrastructure of E. repens leaves and glumes shows some intraspecific variation, most of which can also probably be caused by phenotypic plasticity. Morphological and anatomical characters of Elymus repens specimens collected on a beach, that is a saline and dry site, indicated a habitat-modifying effect. The coastal ecotype of E. repens, described in the present paper, is characterized by a few xeromorphic characters such as distinctly adaxially ribbed leaf blades with large bulliform cells (which decreases the area of transpiration), more sunken stomata and generally more sclerenchymatous tissue than in typical specimens of the species. Convolute, stiff and thicker leaves were observed in E. repens in dry natural habitats. Moreover, anatomical characters of glumes of the coastal ecotype, such as more sclerenchyma and a covering of a thick cuticle, also showed adaptation to dry, more stressful conditions. The presence of a thicker cuticle in


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some individuals of E. repens may have been significant with regard to water conservation. It has been shown elsewhere that grass leaves produced under low nutrient availability tend to contain a greater amount of sclerenchymatous tissue than those produced under a high nutrient supply (van ARENDONK & POORTER 1994), which increased their robustness and reduced damage and loss. Another study showed that native Agrostis species of sub-antarctic islands, with leaf thickness greater and the proportion of support tissue higher than introduced Agrostis species, were more able to survive in exposed sites (PAMMENTER et al. 1986). Anatomical and chemical investigations of Phragmites communis showed that foliar vascular bundle cell walls differed greatly among the tissues of different ecotypes of this species. The adaptation of Ph. communis, a typically hydrophytic species, to saline or drought-prone dunes triggers changes in higher percentages of bundle sheath areas and lower percentages of xylem and phloem areas (CHEN et al. 2006). The intraspecific variation of E. repens in the mesophyll to sclerenchyma ratio observed in the present study is important in determining the potential success of the species in stressful environments. The coastal ecotype of E. repens was subjected to long-term environmental stresses such as drought and salinity, which has caused anatomical differentiation from typical specimens. Characters of anatomical structure useful in taxonomy Significant taxonomical differences in leaf and glume anatomical and morphological characters among Elymus hispidus, E. repens and E. ×mucronatus were supported by multivariate analysis. The anatomical ultrastructural pattern of leaves was frequently used in taxonomical treatments within the Poaceae family (e.g. BROWN 1958; METCALFE 1960; CAROLIN et al. 1973; ELLIS 1986). The Elymus species examined in this paper are included in the Pooideae sub-family which is characterized by presence of two vascular bundle sheaths. An inner mestome sheath contains cells with thickened walls without chloroplasts and an outer parenchymatous bundle sheath is formed by cells with thinner walls and containing plastids (CAROLIN et al. 1973). The high level of correlation between anatomical variation and genomic constitution in the genera of the Triticeae tribe was shown by JARVIE and BARKWORTH (1992a). In their study, one pattern of the anatomical structure was ascertained for both blade and glume of Elymus repens and E. hispidus although the species were included in two different genera, Elytrigia and Trichopyrum, respectively. The present results provide clear differences between E. repens and E. hispidus, especially in the volume and distribution of the support tissue and the chlorenchyma tissue in leaves and glumes. Other glume and blade anatomical characters of E. repens are similar to those of E. hispidus, which indicates some relationship between genomes of this species (ASSADI & RUNEMARK 1995). The association between morphological variation and genome constitution within Triticeae was significantly smaller than between anatomical variation and genome constitution (JARVIE & BARKWORTH 1992b). Leaves and glumes of Elymus hispidus and E. repens display considerable variation in trichome types and their distribution among specimens from the same population. E. hispidus var. villosus is distinguished by densely pubescent glumes and leaves, but more or less

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pubescent glumes and leaves are sometimes present in var. hispidus. Most of the variation of E. repens is due to phenotypic plasticity and therefore the continuous character of leaf pubescence has no taxonomical significance (SZCZEPANIAK 2009). Interspecific hybrids between Elymus hispidus and E. repens [=E. ×mucronatus] often occur in natural communities (PROKUDIN & DRULEVA 1971, 1972; MAHELKA et al. 2005; SZCZEPANIAK et al. 2007). Specimens of E. ×mucronatus are partially fertile and more vigorous than its parental species which usually disappear (SZCZEPANIAK et al. 2007). Analyses of anatomical structure of the E. ×mucronatus revealed intermediate glume character, which has taxonomical implications. The presence of discontinuous layers of the parenchyma which is broken by the chlorenchyma in glumes is a diagnostic character of hybrid. Generally, hybrid specimens were situated in the E. hispidus range of variation in multivariate analyses of the anatomical characters examined here (Figs 1, 5). E. ×mucronatus is anatomically more similar to E. hispidus than to E. repens, which is also supported by morphological data (SZCZEPANIAK 2003). This anatomical and morphological pattern of variation shows one-way introgression and cross-breeding of F1 hybrids with E. hispidus, which is indicated by AFLP analyses (SZCZEPANIAK et al. 2007). The analysis of anatomical and morphological variation of leaves and glumes confirms the distinction between Elymus hispidus and E. repens and reveals their ecological differentiation. E. hispidus is a typical xeric grass, whereas E. repens exhibits high anatomical plasticity and its leaf and glume structures can change to adapt to a particular environment. The examination of anatomical characters supports the claim that E. hispidus and E. repens are parental species of E. ×mucronatus. Acknowledgements. This research was supported by the State Committee for Scientific Research, grant no. 6 P04C 076 19 (Poland).

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VANHALA T. K., VAN RIJN C. P. E., BUNTJER J., STAM P., NEVO E., POORTER H. & VAN EEUWIJK F. A. 2004. Environmental, phenotypic and genetic variation of wild barley (Hordeum spontaneum) from Israel. Euphytica 137: 297–309. WAHL S., RYSER P. & EDWARDS P. J. 2001. Phenotypic plasticity of grass root anatomy in response to light intensity and nutrient supply. Ann. Bot. 88: 1071–1078. WANG S. & HENWOOD M. J. 1999. The taxonomic utility of micromorphological characters in Australian and New Zealand Elymus species (Poaceae). Telopea 8(3): 351–362. WEBB M. E. & ALMEIDA M. T. 1990. Micromorphology of the leaf epidermis in taxa of the AgropyronElymus complex (Poaceae). Bot. J. Linn. Soc. 103: 153–158. Author’s address: MAGDALENA SZCZEPANIAK, W. Szafer Institute of Botany, Polish Academy of Sciences, Department of Vascular Plants Systematics, Lubicz Str. 46, PL-31-512 Kraków, Poland; e-mail: [email protected]


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Appendix. Collection data for specimens of Elymus repens, E. hispidus and E. ×mucronatus used in anatomical studies Taxon / specimen abbreviation

Geographic area1: locality

E. repens var. aristatus Ra1 ML: Błonie near Łęczyca, overgrown peatbog Ra2 PLD: Krąg near Starogard Gdański, thickets on the Wierzyca river Ra3 PLD: Gorzędziej near Tczew, on the Vistula river, on sand Ra4 LU: Józefów near Zamość, on sand E. repens var. repens Rr1 KU: Suchedniów near Skarżysko Kamienna, edge of deciduous forest, on ferruginous soil Rr2 WL: Stawiszyn, in forest on sand Rr3 MLD: on the Szeląg Wielki lake near Ostróda, on sand Rr4 KC: Gąski, on dunes, coastal ecotype Rr5 LU: Udrycze near Zamość, field with oat, on loamy soil E. repens var. subulatus Rs GC: the Hel Peninsula, on the beach near road between Jastarnia and Chałupy, coastal ecotype

Latitude (N) / longitude (E)

52°05′ / 19°08′ 54°01′ / 18°27′ 54°02′ / 18°50′ 50°29′ / 23°04′ 51°03′ / 20°50′ 51°55′ / 18°07′ 53°42′ / 20°06′ 54°14′ / 15°55′ 50°18′ / 23°16′ 54°36′ / 18°48′

E. hispidus var. hispidus Hh1 NB: Racławice, gypsum reserve Hh2 NB: Skorocice, steppe reserve Hh3 VU: Gródek on the Bug river, riverside of the Bug and edge Hh4 LU: Kazimierz Dolny on the Vistula river, Trzy Krzyże Mt., in scrubs

50°20′ / 20°14′ 50°25′ / 20°40′ 50°48′ / 23°58′ 51°19′ / 21°58′

E. hispidus var. villosus Hv1 NB: Szczepanowice near Słomniki, loess and calcareous hills Hv2 NB: Skorocice, steppe reserve Hv3 NB: Smoniowice near Miechów, loess slope along road

50°19′ / 20°03′ 50°25′ / 20°40′ 50°17′ / 14°20′

E. ×mucronatus nvar. mucronatus ×Mm PW: Rożdżałów, in thickets

51°04′ / 23°31′

E. ×mucronatus nvar. tesquicolus ×Mt1 NB: Śladków Mały near Pińczów, calcareous rocks at the road to Chmielnik ×Mt2 NB: Chotel Czerwony near Busko Zdrój, steppe reserve “Przęślin”

50°35′ / 20°45′ 50°22′ / 20°43′

1 Geographic area (according to KONDRACKI (1981): GC – the Gdańsk Coastline, KC – the Koszalin Coastline, KU – the Kielce Upland, LU – the Lublin Upland, ML – the Central Mazovian Lowland, MLD – the Mazurian Lake District, NB – the Niecka Nidziańska Basin, PLD – the Eastern Pomeranian Lake District, PW – the Polesie Wołyńskie region, VU – the Western Volhynia Upland, WL – the Southern Wielkopolska Lowland.