leaf morphology and anatomy in two contrasting...

Acta Botanica Hungarica 50(1–2), pp. 97–113, 2008DOI: 10.1556/ABot.50.2008.1–2.7

LEAF MORPHOLOGY AND ANATOMY IN TWOCONTRASTING ENVIRONMENTS FOR C3 AND C4GRASSES OF DIFFERENT INVASION POTENTIAL

L. J. HAN1, A. MOJZES2,3 and T. KALAPOS2

1School of Life Sciences, Changchun Normal University, 3 North Line, Jichang HighwayChangchun 130032, Jilin, China

2Department of Plant Taxonomy and Ecology, Eötvös Loránd UniversityH-1117 Budapest, Pázmány P. s. 1/C, Hungary; E-mail: [email protected]

3Institute of Ecology and Botany, Hungarian Academy of SciencesH-2163 Vácrátót, Alkotmány u. 2–4, Hungary

(Received 28 March, 2007; Accepted 30 July, 2007)

Leaf morphology, coarse structure and anatomy were compared for two invasive C4, twonon-invasive C4, and two expanding native C3 grass species grown in their original,high-light semiarid temperate habitat, and in a growth room under variable moderate lightand favourable supply of water and nutrients. It was hypothesised that (H1) among C4grasses leaf structural response will be greater for invasive than for non-invasive species,and (H2) for plants of high spreading capacity C4 species will be less responsive than C3 spe-cies. Leaf mass per area was lower in the growth room than in the field by 43.4–54% and5.7–21.2% for grasses of high spreading capacity and for non-invasive C4 species, respec-tively. Little or no response was observed in the proportion of epidermis and mesophyll,but the proportional area of veins plus sclerenchyma was greater in the field than in thegrowth room for the invasive C4 Sorghum halepense, and the spreading C3 Bromus inermisand Calamagrostis epigeios, while it did not differ for the two non-invasive C4 grasses and theinvasive C4 Cynodon dactylon. Leaf intervenial distance was invariant for C4 grasses (exceptfor the non-invasive Chrysopogon gryllus) and the C3 C. epigeios, but changed by 25.1% for theC3 B. inermis. These results suggest that among C4 grasses invasive species exceed non-inva-sive ones in the plasticity of leaf coarse structure, but not that of leaf morphology and anat-omy. However, leaf structure was not less plastic in invasive C4 than in expanding C3grasses except for intervenial distance.

Key words: Bothriochloa ischaemum, Bromus inermis, Calamagrostis epigeios, Chrysopogon gryl-lus, Cynodon dactylon, leaf intervenial distance, leaf mass per area, leaf structure, plasticity,Sorghum halepense

0236–6495/$ 20.00 © 2008 Akadémiai Kiadó, Budapest

INTRODUCTION

Grasses (Poaceae) usually inhabit high-light environments and theirabundance declines in biotopes with moderate to low light intensity (e.g. inforest understorey, Chapman (1996), Bredenkamp et al. (2002)). In addition totheir characteristic shoot morphology with erect leaves, morphology and ana-tomical structure of individual leaves must play an important role in this habi-tat preference. There is considerable variation among grasses in the capacity topersist in shaded environments. Grasses with the C4 photosynthetic pathwayare considered to have lower tolerance of low irradiance than grasses with theC3 photosynthetic metabolism, possibly due to the extra energy requirementof the C4 dicarboxylic pathway, to insufficient time for evolutionary adapta-tion to shaded environments (Pearcy and Ehleringer 1984) or to a limited plas-ticity or acclimation potential to low light intensity at leaf structural and bio-chemical levels (Sage and McKown 2006). A possible additional source of vari-ation among grasses in the tolerance of low light is the invasive potential of thespecies. We hypothesise that invasive grasses (particularly those with the C4photosynthetic pathway) capable of rapid spreading across a wide spectrumof environments should better tolerate heterogeneous light environments – in-cluding low-light habitats – than related species with low capacity for colonis-ation.

Numerous plant traits have been identified so far to be associated withthe invasiveness of plants, including life history traits (Burke and Grime 1996,Kolar and Lodge 2001, Radford and Cousens 2000, Rejmánek and Richardson1996, White et al. 2001), present geographical distribution, reflecting the toler-ance of the range of climates (Burke and Grime 1996, Dukes and Mooney1999), as well as attributes associated with carbon gain and resource-use effi-ciency (McDowell 2002, Pattison et al. 1998, Williams et al. 1995, Yamashita etal. 2000, 2002). The role of leaf morphology and anatomy in a species’ invasionpotential, however, is not sufficiently investigated yet, particularly for thegroup of grasses (Molnár et al. 2000, Yamashita et al. 2000, 2002). In addition tochanges in leaf biochemistry, ultrastructure and plant biomass allocation, leafmorphological and anatomical modifications are frequent components ofplant adjustment to contrasting habitat light conditions (Boardman 1977,Fekete and Szujkó-Lacza 1973, Givnish 1988, Lambers et al. 1998, Mendes et al.2001, Mojzes et al. 2005, Oguchi et al. 2003, Sims and Pearcy 1992).

In this study, we aimed at comparing leaf morphological and anatomicalstructure of six grass species grown in two contrasting environments: underfull sun in their natural semiarid temperate habitats and in a growth room un-der variable moderate light and good supply of water and nutrients. Condi-tions with a lower radiation load in the growth room can be considered as

98 HAN, L. J., MOJZES, A. and KALAPOS, T.

Acta Bot. Hung. 50, 2008

analogous to (but do not mimicking) those in shade compared to full sunpatches of forest steppe vegetation mosaic. In addition to lower light intensity– as its main component – it also includes lower frequency or shorter periodsof high temperature and temporal water shortage, and is associated withlower mechanical stress caused by wind. Thus, rather than focusing on the ef-fect of light intensity exclusively, we explored phenotypical responses tochanges in several associated environmental factors. Two hypotheses weretested:

H1: invasive C4 grasses have a higher capacity to adjust leaf morphologyand anatomy to contrasting environments than non-invasive C4 grasses do.This is presumed because spreading species probably encounter heteroge-neous environments more often than their stationary relatives.

H2: Among grasses of high colonisation capacity (either invasive aliens orexpanding natives) C4 species possess a lesser degree of plasticity in leaf mor-phology and anatomy than C3 species do. In a recent paper Sage and McKown(2006) argues on such difference among photosynthetic pathways.

MATERIALS AND METHODS

Species studied

Six perennial grass species were included in this study: four C4 and twoC3 species. Among the C4 grasses bermudagrass (Cynodon dactylon (L.) Pers.)and Johnsongrass (Sorghum halepense (L.) Pers.) are invasive (Grace et al. 2001,Holm et al. 1977), while gold beard grass (Chrysopogon gryllus (Torn.) Trin.) andyellow bluestem (Bothriochloa ischaemum (L.) Keng) are non-invasive natives.The latter species may reach local dominance as the canopy of sympatricgrasses opens up on disturbance (Zólyomi and Fekete 1994). Each C4 speciesstudied belongs to the NADP-ME biochemical subtype except C. dactylon,which is a NAD-ME C4 plant. Both C3 species studied here are native and pos-sess high capacity for spatial expansion. Smooth brome (Bromus inermisLeyss.) is a mid-successional species native in Europe, frequent in semiariddisturbed areas, and is an invasive alien throughout North America (e.g.Grace et al. 2001, Grilz and Romo 1994, Willson and Stubbendieck 2000). Thenoxious weed chee reedgrass (Calamagrostis epigeios (L.) Roth) is able to colo-nise rapidly a wide range of disturbed habitats, particularly forest clearings,abandoned fields and wastelands (Rebele and Lehmann 2001).

LEAF STRUCTURAL RESPONSE OF INVASIVE GRASSES 99


Field sampling and growth conditions

Our aim was to compare the morphology and anatomy of leaves devel-oped under two contrasting conditions: in the field with semiarid climate andhigh-light conditions, and in a growth room of low-light environment with ad-equate water and nutrient supply. Whole plants in soil monoliths were col-lected in the field and transferred to laboratory for growing plants undersemicontrolled conditions. Except for B. inermis, species were sampled in for-est steppe vegetation and adjacent arable land (cornfield) near the villageIsaszeg, 25 km East of Budapest in the summers of 2002–2004. Annual meantemperature is 9 °C, yearly precipitation is about 600 mm. Chernozem soil cov-ers the loess bedrock typically (Fekete et al. 2000). Bromus inermis was trans-planted from forest steppe vegetation on calcareous sandy soil near the villageFülöpháza, on the Great Hungarian Plain. Annual mean temperature is 10.4°C, yearly precipitation is 505 mm (Kovács-Láng et al. 2000). Plants with theiroriginal soil were placed in 4-litre pots, shoots were cut back to 1 cm above soilsurface and newly emerged shoots were grown in the growth room of theEötvös Loránd University. In the growth room, plants received natural sun-light supplemented with a 1,000 W halogen lamp over a photoperiod of 12 h insummer and 9 h in winter. Pots were rotated every 3 weeks so that we canminimise spatial heterogeneity of light environment in the growth room. Themean maximum photosynthetic photon flux density (PPFD) at the height ofshoots on clear days was 810 µmol m–2 s–1 in summer and 180 µmol m–2 s–1 inwinter (i.e. 40% and 9% of full sunlight, respectively). Air temperature and rel-ative humidity were automatically recorded hourly by using an HOBO ProRH/Temp sensor (Onset Computers Inc., Bourne, MA, USA), which was puton the table where pots were placed. Daily mean air temperature was 24.0 ± 4.4°C in summer and 18.5 ± 2.3 °C in winter. Relative air humidity ranged fromabout 20% to 80% during the day. Plants were watered adequately and sup-plied with 0.5 ml nutrient solution per pot (containing 13% N, 4.5% P2O5, 6.5%K2O and micronutrients) in three-week intervals. The second fully developedleaf from the top of 10 shoots per species was sampled in October 2004. Fiveleaves per species were used for morphological and another five for anatomi-cal measurements (n = 5). For comparison with field-grown plants, leaveswere sampled in the same manner from plants in their original habitats (or forS. halepense from a similar degraded biotope in Budapest) in June 2005. Aftersampling, leaves were transported to the laboratory immediately in closedcontainers with the cut leaf base immersed in water. Morphological measure-ments were completed on the same or following day.



Data collection

Leaf morphology and coarse structure – Leaf length was measured by using aruler read to the nearest mm, while leaf width was determined in the middle ofthe leaf blade under a binocular microscope equipped with a measuring lensto the accuracy of 0.1 mm. One-sided leaf surface area was determined by aLeaf Area Meter (LI-COR 3000A, LI-COR Inc., Lincoln, Nebraska). Leaf thick-ness (T*, mm) was measured in the middle portion of the leaf lamina, halfwaybetween the leaf edge and the central vein by using a thickness meter (Dial In-dicator and Magnetic Base, a division of Siechert and Wood, Inc. Pasadena,USA, accuracy 0.01 mm). Leaf samples were dried to constant weight at 90 °Cand than dry mass was measured. Leaf mass per area (dry mass per unit leafarea, LMA, g m–2) and leaf bulk tissue density (dry mass per unit leaf volume,D = LMA/T, g cm–3) were calculated from these data.

Leaf anatomy – The middle portion of the leaf blade was severed and fixedin a 1:1:1 mixture of 96% alcohol, glycerine and distilled water until process-ing. Leaf blade cross sections were obtained by hand cutting without embed-ding by using elderpith and razor blades. Leaf cross-sections were perma-nently mounted in the same solute used for sample storage, observed withoutstaining and photographed under a light microscope (Nikon Eclipse E400,Nikon Inc., Yokohama, Japan) using a digital camera (Nikon CoolPix 4500).Quantitative leaf anatomical measurements were made on A4 sized printoutsof digital photographs. Distance data were measured by using a ruler, the var-ious tissue components were cut out, their mass was measured on an analyti-cal balance and their area was calculated by using the mass per area ratio ofthat sort of paper determined beforehand. For the sake of accuracy, scalingwas also photographed and printed out in the same way as leaf micrographs.Distance between vein centres (intervenial distance) were measured, and theproportional area of three component tissues (epidermis, mesophyll and thesum of vascular tissue and sclerenchyma) were calculated on a leaf blade crosssection halfway between the central vein and the leaf edge by using a magnifi-cation of 180 and 435 for C3 species and C4 species, respectively. The outer andinner bundle sheaths were included in the mesophyll and the vascular tissues,respectively. The thickness of mesophyll (for C3 species only) and epidermisthickness were determined at two points of an intervenial region. The thicknessof the outer, parenchymatous bundle sheath was measured on 3–4 randomlyselected bundle sheath cells per primary vein. These measurements were per-formed at a magnification of 850. Five replicates per environment were usedfor each species. For thickness parameters and for intervenial distance, aver-age values measured on the same cross section was used as replicates.



* Abbreviations: LMA = leaf mass per area; T = leaf thickness; D = leaf bulk tissue density

Statistical analyses

To test our hypotheses, we analysed intraspecific differences for eachvariable between means under the two growth conditions. Multiple compari-sons among species in the same environment were performed as well, but dueto the low number of species, these results were discussed only for robust dif-ferences among photosynthesis types or C4 subtypes regardless of the growthenvironment. Two-way ANOVA with growth conditions and species asgrouping variables was used with subsequent least significant difference(LSD) test to analyse significant differences among means. For variables wherethe homoscedasticity assumption of ANOVA was fulfilled within the samespecies or within the same light conditions only, unpaired t-test (with separatevariance estimates if necessary) was applied for comparisons of the means ofgrowth conditions, and one-way ANOVA with LSD test was used to test sig-nificant differences among the means of species, respectively. When data didnot meet the assumptions of ANOVA, nonparametric Kruskal-Wallis test withsubsequent post hoc test was used instead for multiple comparisons. Each sta-tistics was performed by using the Statistica version 7.0 software (StatSoft Inc.2004), and differences were considered significant at p < 0.05 level.

RESULTS

Leaf coarse structure

Leaf mass per area (LMA) was significantly greater in the field than in thegrowth room for each species except for C. gryllus (Fig. 1A). Variation in thisparameter was greater for S. halepense and C. dactylon than for B. ischaemum,while both C3 grasses showed about twofold differences (Table 1). For C.epigeios, S. halepense and C. dactylon, these differences in LMA resulted fromhigher leaf thickness (T) of plants grown in full sun compared with those de-veloped under moderate light conditions (Fig. 1B), while leaf bulk tissue den-sity (D) did not differ significantly between the two light environments forthese species (Fig. 1C). For B. inermis, both components of LMA (i.e. T and D)were significantly lower for leaves developed in the growth room than forthose grown in the field. Greater leaf bulk tissue density was the main deter-minant of higher LMA of field-grown leaves for B. ischaemum only, but the dif-ference was only marginally significant. Chrysopogon gryllus showed remark-able invariance in each leaf coarse structural parameter (Fig. 1, Table 1). Undermoderate irradiance, leaf thickness of this grass was the greatest among thespecies studied, and under field conditions it was similar to that of B. inermis.





0

20

40

60

80

Leaf

mas

spe

rar

ea(L

MA,g

m–2

)

Field Growth roomBC

ab

*

C

ab

*

B bA

*

A

a

*

A

a

*

ab

0

0.07

0.14

0.21

0.28

Leaf

thic

knes

s(T

,mm

)

C

b

*

D

b

*

C c

A aB

a

*

B

a

*

0

0.2

0.4

0.6

0.8

Leafb

ulk

tissu

ede

nsity

(D,g

cm-3)

ABa

*

A abAB b

C

c

m

B b B b

B. inermis C.epigeios C.gryllus B.ischaemumS.halepense C.dactylon

C3 expanding C4 non-invasive C4 invasive

A

B

C

Fig. 1. Leaf coarse structure for six grasses of different photosynthetic pathway type andcapacity for area expansion developed in the field in full sun semiarid habitat or undervariable moderate light and good supply of water and nutrients in a growth room. A) Leafmass per area (LMA, g m–2); B) leaf thickness (T, mm); C) leaf bulk tissue density(D = LMA/T, g cm–3). Asterisks indicate significant differences (p < 0.05) between growthconditions, “m” denotes marginal significance (p = 0.0538). Within a given growthcondition, the same letters indicate statistically insignificant differences among means ofspecies (lower case letters for growth room, upper case characters for field conditions). For

each variable mean ± 1 SE of 5 replicates are shown



Tabl

e1

Var

iati

onin

leaf

coar

sest

ruct

ure,

anat

omy

and

mor

phol

ogy

fors

ixgr

ass

spec

ies

ofdi

ffer

entp

hoto

synt

heti

cpa

thw

ayty

pean

dca

pac-

ity

fora

rea

expa

nsio

nm

easu

red

unde

rcon

tras

ting

grow

thco

ndit

ions

(in

the

fiel

din

full

sun

sem

iari

dha

bita

tori

na

grow

thro

omun

-de

rvar

iabl

em

oder

ate

light

and

good

supp

lyof

wat

eran

dnu

trie

nts)

expr

esse

das

perc

enta

geof

the

low

erm

ean

valu

e[(

high

erm

ean

valu

e–

low

erm

ean

valu

e)/l

ower

mea

nva

lue]

×10

0,(%

)fo

rea

chva

riab

le.S

tati

stic

ally

sign

ific

ant

diff

eren

ces

(p<

0.05

)be

twee

ngr

owth

cond

itio

nsar

ein

bold

type

.“m

”de

note

sm

argi

nals

igni

fica

nce

(p=

0.05

38)

Phot

osyn

thet

icty

pean

dsp

read

ing

capa

city

Leaf

trai

tV

aria

ble

C3

expa

ndin

gC

4no

n-in

vasi

veC

4in

vasi

ve

B.in

erm

isC

.epi

geio

sC

.gry

llus

B.is

chae

mum

S.ha

lepe

nse

C.d

acty

lon

Coa

rse

stru

ctur

e

Leaf

mas

spe

rar

ea11

7.2

99.6

6.1

26.9

86.3

76.7

Leaf

thic

knes

s68

.811

0.0

4.3

5.6

72.4

57.6

Leaf

bulk

tiss

uede

nsit

y28

.50.

10.

124

.0m

7.0

14.1

Ana

tom

y

Thic

knes

sof

mes

ophy

ll8.

038

.4

epid

erm

is0.

08.

734

.752

.028

.72.

5

bund

lesh

eath

24.3

11.9

15.1

28.0

3.3

31.0

Inte

rven

iald

ista

nce

25.1

6.9

18.0

0.1

0.1

9.9

Are

apr

opor

tion

of

mes

ophy

ll8.

65.

45.

30.

80.

79.

6

vein

spl

ussc

hler

ench

yma

76.4

33.4

7.1

10.5

103.

31.

5

epid

erm

is6.

56.

26.

63.

321

.115

.4

Mor

phol

ogy

Leaf

area

148.

56.

338

.210

0.7

84.1

29.6

Leaf

leng

th82

.212

.646

.152

.377

.341

.8

Leaf

wid

th15

.82.

040

.329

.846

.01.

5

In contrast, leaves of the other three C4 grasses were significantly thinner thanthose of the two C3 species under both light regimes.

Leaf anatomy

The mesophyll was significantly thinner in the growth room than in fullsun for C. epigeios, but remained similar for B. inermis (Table 2, this variablewas obtained for C3 species only). Epidermis thickness was significantly lowerin the growth room than in the field for C. gryllus, B. ischaemum and S.halepense, but did not vary significantly between light conditions for C. epigeios,B. inermis and C. dactylon. Field-grown leaves of B. inermis, B. ischaemum and C.dactylon possessed significantly thinner parenchymatous bundle sheath thanthose developed under moderate light conditions; an opposite pattern wasfound for C. gryllus, whereas C. epigeios and S. halepense did not show signifi-cant differences in this trait between the two environments. Among species, C.dactylon had the thickest parenchymatous bundle sheath under both irradian-ce levels. Average distance between veins was remarkably invariant for the C4grasses in the two contrasting environments. This trait differed significantlyonly for C. gryllus and B. inermis, but in an opposite way: it was greater undergrowth room conditions for B. inermis, but was lower for C. gryllus. When thesix species are compared concerning their photosynthesis types, mean inter-venial distance was significantly shorter for the C4 grasses than for the two C3grasses, and among the C4 types, this was greater for the NAD-ME C. dactylonthan for the other three NADP-ME type C4 species, regardless of the growthenvironment. Area proportion of mesophyll decreased slightly, but signifi-cantly under lower light conditions for C. gryllus, while increased for C.dactylon, and did not change significantly for the other species. Proportionalarea of veins plus sclerenchyma was significantly greater in field-grownleaves of B. inermis, C. epigeios and S. halepense than in those developed in thegrowth room, whereas did not vary significantly between light conditions forthe other three species. For S. halepense, this was at the expense of the area ofepidermis, which was proportionally lower in leaves exposed to full sun thanin leaves grown under moderate light level, while in the two C3 grasses bothmesophyll and epidermis areas tended to be lower under field conditions thanin the growth room.

Leaf morphology

Except for C. gryllus and C. epigeios, leaf area was significantly greater inthe growth room than in natural habitats (Fig. 2A). This was because each ofthe four species possessed significantly longer leaves in moderate than under





Tabl

e2

Qua

ntita

tive

light

mic

rosc

opic

leaf

anat

omic

alch

arac

teri

stic

s(m

ean

±1

SE,n

=5)

for

six

gras

ses

ofdi

ffer

entp

hoto

synt

hetic

path

way

type

and

capa

city

fora

rea

expa

nsio

nde

velo

ped

inth

efie

ldin

full

sun

sem

iari

dha

bita

toru

nder

vari

able

mod

erat

elig

htan

dgo

odsu

p-pl

yof

wat

eran

dnu

trie

nts

ina

grow

thro

om.F

orea

chsp

ecie

s,st

atis

tical

lysi

gnifi

cant

diff

eren

ces

(p<

0.05

)bet

wee

ngr

owth

cond

ition

sar

ein

bold

type

.For

each

vari

able

,sig

nific

anti

nter

spec

ific

diff

eren

ces

with

ina

give

ngr

owth

cond

ition

are

indi

cate

das

inFi

gure

1

Phot

osyn

thet

icty

pean

dsp

read

ing

capa

city

Var

iabl

eG

row

thco

ndit

ions

C3

expa

ndin

gC

4no

n-in

vasi

veC

4in

vasi

veB.

iner

mis

C.e

pige

ios

C.g

ryllu

sB.

isch

aem

umS.

hale

pens

eC

.dac

tylo

n

Thic

knes

sof

mes

ophy

ll(µ

m)

Gro

wth

room

117.

8±2.

8a

108.

6±9.

2a

Fiel

d10

9.1±

4.5

A15

0.2±

9.8

B

epid

erm

is(µ

m)

Gro

wth

room

17.8

±0.5

c13

.5±0

.4b

13.1

±0.5

b15

.0±0

.7b

17.8

±0.8

c10

.1±0

.4a

Fiel

d17

.8±1

.9C

14.7

±0.6

B17

.7±0

.9C

22.8

±0.8

D22

.9±1

.3D

10.4

±0.5

A

bund

lesh

eath

(µm

)G

row

thro

om17

.3±1

.0d

12.5

±0.6

bc10

.2±0

.6a

12.1

±0.3

b14

.0±0

.7c

28.5

±1.5

eFi

eld

13.9

±0.8

C14

.0±0

.6C

11.7

±0.6

B9.

5±0.

3A

14.5

±0.8

C21

.7±1

.2D

Inte

rven

iald

ista

nce

(µm

)G

row

thro

om27

5.8±

10.7

d28

3.1±

19.2

d67

.8±2

.7a

65.4

±1.7

a10

8.1±

5.2

b13

0.3±

6.9

cFi

eld

220.

5±10

.5E

265.

0±8.

6F

80.0

±3.7

B65

.4±4

.1A

108.

0±7.

0C

143.

2±7.

4D

Are

apr

opor

-ti

onof

mes

ophy

ll(%

)G

row

thro

om59

.2±2

.6b

56.0

±1.3

ab54

.4±0

.8ab

50.7

±0.8

a58

.7±0

.8b

56.3

±1.7

abFi

eld

54.5

±1.1

B53

.1±0

.9A

B57

.3±0

.6C

51.1

±1.4

A58

.3±0

.6C

51.4

±0.9

Ave

ins

plus

schl

eren

chym

a(%

)

Gro

wth

room

8.7±

0.5

a13

.8±0

.5b

17.8

±1.5

c8.

6±1.

0a

6.3±

1.1

a13

.0±1

.1b

Fiel

d15

.4±0

.8BC

18.5

±1.5

D16

.6±1

.4C

D9.

5±0.

9A

12.8

±0.7

B13

.1±0

.8B

epid

erm

is(%

)G

row

thro

om32

.1±2

.7ab

30.2

±1.1

ab27

.8±1

.3a

40.7

±1.6

c35

.0±1

.6b

30.7

±2.0

abFi

eld

30.1

±1.6

B28

.4±1

.0A

B26

.1±1

.3A

39.4

±1.0

D28

.9±0

.9A

B35

.4±0

.5C



0

18

36

54

72

Leaf

are

a(c

m2)

Field Growth room

B

bc

*

Cabc

Babc

Aab

C

c

*A

**

a

0

18

36

54

72

Le

af

length

(cm

)

C

c

*

E

d

*

DE

d

B

b

*

D

d

*

A

a

*

0

5

10

15

20

Le

af

wid

th(m

m)

B. inermis C.epigeios C. gryllus B.ischaemum S.halepense C.dactylon

DE c

D b

C

a

*

A a

*

E

d

*

B a

C3expanding C4non-invasive C4invasive

A

B

C

Fig. 2. Leaf morphology for six grasses of different photosynthetic pathway type and capac-ity for area expansion developed in the field in full sun semiarid habitat or under variablemoderate light and good supply of water and nutrients in a growth room. A) Leaf area(cm2); B) leaf length (cm); C) leaf width (mm). For each variable mean ± 1 SE of 5 replicates

are shown. Significant differences are indicated as in Figure 1

high-light conditions (Fig. 2B), but for S. halepense and B. ischaemum, signifi-cantly greater leaf width also contributed to the variation in total leaf size (Fig.2C). Among species, B. inermis exhibited the greatest differences in leaf areaand length between the two light regimes (Table 1). Chrysopogon gryllus alsoproduced significantly longer, but narrower leaves in the growth room, result-ing in leaf area similar to that developed in the field. Neither the length nor thewidth of leaves showed significant differences between light conditions for C.epigeios. Under moderate light level, S. halepense tended to have larger leavesthan the other species; however, differences were significant in comparisonwith B. ischaemum and C. dactylon only.

DISCUSSION

Leaf responses to marked change in growth environment were heteroge-neous in morphology, coarse structure and anatomy, and within groups spe-cies often responded differently, that for certain variables makes difficult todraw unambiguous conclusions to the group as a whole. However, this alsoimplies an existing diversity in the mechanism of leaf structural responses toaltered growth environment even within a plant functional group (i.e. peren-nial grasses). In line with our hypothesis (H1), among C4 grasses the invasive S.halepense and C. dactylon exhibited a higher degree of plasticity in leaf coarsestructure between contrasting environments than the non-invasive C. gryllusand B. ischaemum. Both C4 invaders altered substantially leaf coarse structure(LMA and thickness) in response to moderate light environment, whereasboth traits remained invariable for the non-invasive C. gryllus, and LMAchanged much less for B. ischaemum. This is in line with prior evidence on theimportant role of leaf mass per area and its plasticity in the performance andsuccess of certain invasive exotic or expanding native species (Gloser andGloser 1996, Juraimi et al. 2004, McDowell 2002, Smith and Knapp 2001, Wil-liams et al. 1995). Leaf bulk tissue density appears to be the least responsive tosuch environmental change irrespective of the species’ invasion capacity. Forthe invasive S. halepense, thicker leaves in the field than in the growth roommust have resulted from a greater proportion of veins plus sclerenchyma, thatindicates larger investment into vascular and support tissues under temporalwater shortage and mechanical stress caused by wind in the natural habitat.Knapp and Gilliam (1985) also reported a greater area of the bundle sheath-primary vascular bundle complex associated with greater leaf thickness andleaf mass per area for Andropogon gerardii in the high-light (burned) environ-ment compared to the light-limited, unburned site in a tallgrass prairie. How-



ever, no such investment was observed for the other invasive C4 grass, C. dac-tylon and for the two non-invasive C4 grasses in our study.

Leaf morphology responded markedly to the change in growth environ-ment, although the response was not consistently different between invasiveand non-invasive C4 species. The greatest change in leaf length and width oc-curred for the invasive S. halepense closely followed by the two non-invasivespecies, while the invasive C. dactylon produced the smallest difference. Grassleaves usually grow larger, longer but narrower under moderate light inten-sity than in unshaded conditions (Langer 1979). Among C4 grasses in ourstudy, both invasive species and the non-invasive B. ischaemum developedlarger leaves in the growth room than in the field, that were longer, but notnarrower (wider instead for S. halepense and B. ischaemum). Only C. gryllus pro-duced narrower leaves in the growth room than in the original grassland, butsimultaneous increase in leaf length compensated for it, thus leaf area did notchange for this species.

Leaf anatomy was less responsive than morphology or coarse structurefor the grasses studied, and no clear difference appeared between invasiveand non-invasive C4 species. Most leaf anatomical traits varied for the non-in-vasive C. gryllus, but the magnitude of response (5.3–34.7%) remained belowthat of the fewer anatomical variables changing significantly for the two inva-sive C4 grasses (9.6–103.3%). This suggests some anatomical adjustment abilityfor the non-invasive C. gryllus as well. Considering that anatomical changes ofthe other non-invasive grass, B. ischaemum was comparable with that of the in-vasive C. dactylon and S. halepense, invasives do not appear to exceed non-invasives in leaf anatomical response to the environment, at least for the C4grasses studied here. Thinner parenchymatous bundle sheath cell layer inleaves of C. gryllus in the growth room than in the field may partly be responsi-ble for shorter distances between veins in the growth room. In contrast, veindensity remained unchanged for the other C4 grasses in this study under bothlight conditions. For three of the four C4 species studied, thicker epidermis inthe field than in the growth room may provide greater protection againsthigher transpirational water loss, wind damage and wind-carried dust parti-cles in natural environment (Mauseth 1988). Plasticity of leaf traits for the na-tive B. ischaemum appeared to be intermediate between that of the non-inva-sive C. gryllus and the two invasive C4 species. This may be associated with thespecies’ ability to reach local dominance mainly in disturbed grassland vege-tation (Zólyomi and Fekete 1994). Among the two C3 species of high spreadingcapacity in this study, B. inermis showed particularly high responsiveness thatappears to be consistent with the species’ forest edge phytocoenological affin-ity (Szujkó-Lacza and Rajczy 1986, Zólyomi and Fekete 1994). Phenotypicplasticity in photosynthesis and leaf coarse structure has been documented to



be greater for species adapted to spatially or temporally heterogeneous or un-predictable environments (e.g. for early or middle successional species) thanfor those from more homogeneous and stable environments (e.g. for late suc-cessionals, Abrams and Mostoller 1995, Strauss-Debenedetti and Bazzaz 1991,Yamashita et al. 2000). Furthermore, early or mid-successional status of a spe-cies often couples with high potential for invasiveness (Grace et al. 2001,Yamashita et al. 2000, 2002). Thus, the flexibility of leaf morphology and struc-ture may also help B. inermis to successfully invade temperate grasslands inNorth America (Grace et al. 2001, Grilz and Romo 1994, Willson and Stubben-dieck 2000).

Our results do not support the hypothesis of greater ability for structuraladjustment to the growth environment for C3 than for C4 grasses (H2), a com-parison we could make for species of high capacity to spread. Overall, therewere similar changes in leaf morphology, coarse structure and anatomy in re-sponse to the growth environment, although certain variables behaved differ-ently in the two groups. Leaf intervenial distance was the most prominentvariable of this sort, which showed less variation among C4 than C3 species,most probably due to the strong need of close proximity of mesophyll andbundle sheath cells in C4 photosynthesis that may restrict the ability of shadedC4 plants to increase vein spacing (Sage and McKown 2006). Albeit leaf coarsestructure changed at a similar or greater extent for the two C3 species than forC4 invasives, in leaf morphology and anatomy the responsiveness of the C3 B.inermis and the C4 S. halepense was similarly high, while that of the C3 C. epigeiosand the C4 C. dactylon was comparably moderate. Thus, based on these dataneither C3 nor C4 grasses can be qualified as more plastic in leaf anatomy andmorphology. Similar to these results, no consistent differences were found inother comparisons of C3 and C4 species in the ability to reduce leaf thickness inresponse to shading (Sage and McKown 2006). Shorter intervenial distanceswe observed here for C4 grasses than for C3 grasses, and for C4 NADP-ME spe-cies than for the C4 NAD-ME C. dactylon are consistent with previous results(Dengler et al. 1994, Kawamitsu et al. 1985, Ogle 2003).

In conclusion, among C4 grasses studied here invasive species appear topossess greater leaf coarse structural, but not consistently higher morphologi-cal and anatomical plasticity between contrasting light environments if com-pared with non-invasive ones. A higher plasticity may contribute to the suc-cessful establishment and persistence of invasive C4 grasses in heterogeneouslight environments. Ecophysiological plasticity also proved to play an impor-tant role in the invasion success of some tropical and subtropical species(Pattison et al. 1998, Williams et al. 1995, Yamashita et al. 2000). However, ourresults do not support the hypothesis that among grasses of high capacity forcolonisation C4 species are inferior to C3 species in the capacity of adjusting



leaf morphology and structure to the environment. Nevertheless, further stud-ies involving a greater number of species are necessary to reach more generalconclusions.

*

Acknowledgements – Support from the Hungarian Scientific Research Fund (OTKAT038028, W15301) is acknowledged. This work is a result of the first author’s visit to Hun-gary supported by the intergovernmental scientific exchange program between China andHungary (administered by the Hungarian Scholarship Board).

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