Stomatal vs. genome size in angiosperms: the somatic tail - CSIC

12 Pages·2010·0.5 MB·English

Annalsof Botany105:573–584, 2010
doi:10.1093/aob/mcq011,availableonlineat www.aob.oxfordjournals.org
Stomatal vs. genome size in angiosperms: the somatic tail wagging
the genomic dog?
J.G.Hodgson1,†,*,M.Sharafi2,A.Jalili3,S.D´ıaz4,G.Montserrat-Mart´ı5,C.Palmer6,B.Cerabolini7,S.Pierce7,
B. Hamzehee3, Y. Asri3, Z. Jamzad3, P. Wilson8, J. A. Raven9, S. R. Band8, S. Basconcelo10, A. Bogard6,
G. Carter6, M. Charles6, P. Castro-D´ıez5, J. H. C. Cornelissen11, G. Funes4, G. Jones6, M. Khoshnevis3,
N. Pe´rez-Harguindeguy4, M. C. Pe´rez-Rontome´5, F. A. Shirvany3, F. Vendramini4, S. Yazdani3,
R. Abbas-Azimi3, S. Boustani3, M. Dehghan3, J. Guerrero-Campo4, A. Hynd6, E. Kowsary3,
F. Kazemi-Saeed3, B. Siavash3, P. Villar-Salvador5, R. Craigie6, A. Naqinezhad2, A. Romo-D´ıez12,
L. de Torres Espuny5 and E. Simmons6
1Peak Science and Environment, Station House, Leadmill, Hathersage, Hope Valley S32 1BA, UK, 2Department of Biology,
FacultyofSciences, UniversityofMazandaran,Babolsar,Iran,3ResearchInstituteofForestsandRangelands,POBox13185-
116,Tehran,Iran,4InstitutoMultidisciplinariodeBiolog´ıaVegetal(CONICET – UNC)andF.C.E.F.yN.,UniversidadNacional
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†ecBoalocgkigcraolucnidrcaunmdstAanimces.GTehneonmaetusreizoefi‘sthaisfuenccotlioogni,caanldcitrhceumprsotadnuccet,’oisf,cheolwlveovelur,mheo.tlAysdseubcahteidt.iHsecroen,twinegeinnvteosn- ación y Docum
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†UofMKtheaetnhdroe,dlusatsSiiontongmshPaiCptaAlb,aesntwodmeleeenaefgmcehenarogrmaecnett-esreiiczsoteilcovsgawliuceaerlseaomnbdetaatisanuxerodendforfmoomric1tp4ha4et2tPesrlpnaensctiidDeesNnftAriofimeCd-A.vSarlguuebenssteiqdnuaate,anIbrtalaysn,e,aSannpdaaisnmseaesnasdmsuterhene-t ientífica on A
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leaf specialization.Moreover,itis positively correlated with genome sizeacrossawide range ofmajor taxa.
†ConclusionsStomatalsizepredictsgenomesizewithinangiosperms.Correlationisnot,however,proofofcaus-
alityandhereourinterpretationishamperedbyunexpecteddeficienciesinthescientificliterature.Firstly,there
are discrepancies between ourown observations and established ideas about the ecological significance of sto-
matal size; very large stomata, theoretically facilitating photosynthesis in deep shade, were, in this study (and
in other studies), primarily associated with vernal geophytes of unshaded habitats. Secondly, the lower size
limit at which stomata can function efficiently, and the ecological circumstances under which these minute
stomata might occur, have not been satisfactorally resolved. Thus, ourhypothesis, that the optimization ofsto-
matalsize forfunctionalefficiency isa majorecologicaldeterminant ofgenome size,remains unproven.
Keywords: Stomatal size, genome size, seed size, life history, photosynthesis, allometry, ecology, evolution,
SLA,leafstructure, CAM,C .
4
INTRODUCTION of values remains uncertain (Knight et al., 2005). High DNA
amount is not associated with evolutionary advancement or
Within flowering plants, nuclear DNA content, or genome
organizational complexity; much takes the form of highly
size, varies almost 2000-fold (Bennett and Leitch, 2005;
repeated sequences of non-genic DNA (Davidson and
Greilhuber et al., 2006). The significance of this wide range
Britten, 1973). Processes have been identified both for redu-
†Presentaddress:DepartmentofArchaeology,UniversityofSheffield, cing genome size (Kirik et al., 2000; Orel et al., 2003;
SheffieldS14ET,UK. Bennettzen et al., 2005) and for its increase (Kidwell, 2002;
#The Author2010.Publishedby OxfordUniversity Presson behalfofthe AnnalsofBotanyCompany.Allrights reserved.
ForPermissions, please email: [email protected]
574 Hodgson et al. — Stomatal vs. genome size in angiosperms
Bennettzen et al., 2005). These mechanisms have doubtless mechanism controlling the exchange of gases, particularly
contributed to the major increases and decreases in genome the influx of carbon dioxide and the efflux of water vapour,
size reported within evolutionary lineages (Leitch et al., between the interior of the leaf and the atmosphere
1998; Soltis et al., 2003; Caetano-Anolle´s, 2005; Johnston (Woodward, 1998; Raven, 2002; Hetherington and
et al., 2005; Leitch et al., 2005) and the large differences in Woodward, 2003). The efficiency with which carbon
nuclear DNA amount recorded between closely related dioxide, a key raw material for photosynthesis, is taken up
species (Bennett and Leitch, 2005). andwaterlossrestrictedappearstobeinpartafunctionofsto-
TheexactroleofthisextraDNAisamatterofdebate.Some matal size (Allen and Pearcy, 2000; Aasamaa et al., 2001;
have argued that the additional DNA generally has little Hetherington and Woodward, 2003). Gaseous exchange is
impact on function and relates to the ‘selfish’ nature of most regulated through changes in the size of the stomatal pore.
‘junk’ DNA (Doolittle and Sapienza, 1980; Orgel and Crick, Because of their more rapid opening and closure, small
1980). Others have contended that the extra DNA has func- stomata afford greater water-use efficiency in dry habitats,
tional importance (Bennett, 1971; Cavalier-Smith, 1985, whereas in cool, moist and shaded habitats large stomata
2005) and many evolutionary and ecological correlates with may be advantageous.
genome size have been identified (e.g. Bennett, 1976; Jones Ageneralrequirementinautotrophicplantstocombinehigh
and Brown, 1976; Grime and Mowforth, 1982; Thompson, photosynthetic capacity and water-use efficiency represents a
1990; Vinogradov, 2003; Knight et al., 2005; Beaulieu strong ecological driver for optimizing stomatal size.
et al., 2007). However,stomatalsizeisnotsimplyanecologicallyimportant
D
Cells with a large genome exhibit disproportionately slow characteristic. The maximum size of the stomatal aperture is ow
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tively correlated (Jones and Brown, 1976; Thompson, 1990; size of the angiosperm genome are identified and, by means
Maranon and Grubb, 1993; Knight and Ackerly, 2002; of a literature review, the claims of stomatal size to be the
Knight et al., 2005; Beaulieu et al., 2007). The relationship ‘missing link’, the primary determinant of genome size in
is, however, inexact, perhaps in part because of the develop- the angiosperms, is assessed for a first time.
mental complexity and the nutritional and structural diversity
of seeds (Martin, 1947; Johansen, 1950; Hodgson and
Mackey, 1986; Raven, 1999). Species with small genomes MATERIALS AND METHODS
may produce large seeds by, for example, producing a small
Studyareas
embryo but an abundance of endosperm (e.g. Fraxinus excel-
sior). Moreover, the seeds of species withlarge genomes may Theinvestigationcentresontheflorasoffourclimaticallycon-
be minute (e.g. Orchidaceae, with obligate mycotrophy and trastedandgeographically disparateareas:theCo´rdobaregion
ripe seeds containing only a small undifferentiated embryo of central western Argentina (55 species measured), the
and no endosperm). Thus, in practice, genome size accounts Sheffield region of central England (745 species), the
for only a small amount (variously estimated at 3% and Arazbaran Protected Area in northern upland Iran (463
6%) of the recorded variation in seed mass (Beaulieu et al., species) and the Zaragoza region of north-east Spain (278
2007). species). The climatic characteristics of each region are
Another relationship with genome size involves stomata. briefly outlined in Table 1 and the areas described more
Stomata consist of small pores at the leaf surface, each fully in D´ıaz et al. (2004). The species studied represent an
bounded by two guard cells. They provide the primary ecologically balanced subset of their respective floras, except
Hodgson et al. — Stomatal vs. genome size in angiosperms 575
TABLE 1. Aclimaticcomparisonofthefourmainstudyareas(dataabstractedfromD´ıazetal.,2004)
Argentina Spain Iran England
Meanannualrainfall(mm)range, 85–912,confinedto 300–350,mainlyin 316–686,throughouttheyear 565–1800,throughouttheyear
distribution warmseason springandautumn withwintermaximum withwintermaximum
Meanannualtemperaturerange(8C) 8–20 6–24 5–14 9–11
No.ofmonthsinwhichevaporation 1–12 6 2–4 0–2
exceedsprecipitation(range)
No.offrost-freemonths(range) 0–8 3–5 6 3–6
for England, where a disproportionately large number of following leaf traits were assessed: maximum leaf size
species with large genomes have been included. Data for a (mm2); leaf dry-matter content (100(cid:2)dry mass of leaf/satu-
further 161 species collected from south-east Europe and the rated mass of leaf); leaf thickness (mm); specific leaf area
NearEastduringotherecologicalprojectswerealsoincorpor- [leaf area (mm2)/leaf mass (mg)]. The procedures used are
ated into this study. described in detail in Charles et al. (1997). They conform to
the general recommendations of Garnier et al. (2001) and
Cornelissen et al. (2003).
Attributes measured D
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morerecentpublications.Chromosomenumberswerecollated ance’ grouping. Vernals geophytes are, variously, woodland ocum
fawtiwohnrnfoaeecdsrmlecfuas,hBdmuarioaobnfiimrsgllayeenonyqeFasgucenoeee(ddm1sneos9ttarilhf9otlyay8vst,a)aoosln(ues1fadrefn9ct/tshod6esoers9eu,)BdgtgieeanieonnnncnfundlorusecemdteuRltiraesntat-iaaigvnsoiniegnnzdnFeaaetLond(v1daeeat9alrirhytot7icaeesv5h.et)ybs.(Wa1(ots2Mh9feo0r6prao09nsunu5t)ubyg,)m.lhaiGscbfPpfaaoeeletrrocilcnoaitfiadecneoldkyssrl hifcto‘anyrgonefetprrndodeobeslowMsyelroeqtmtofhaduhw’fareaganbtnfraioocycnncywreeodtchdtm(eoho,plfplf1hlitalet9aneehnxbn8tetpewi2stla)aalhttna.tthhisersWceag.ihsiotereuTncsmaghsieiunmeesmlgnnilrdiguoeldeareameilr–slvxalteyiyatlsrtiuehneifadotemaeuvntcumoceevniylosndeytccsir)lalcndelarueaarynrarolbsdsgrueflecofygdraoarhusspnrtrtapeiiobdrnimsitnegnrpveagaeereatdogcas(idceGpaktciaelatroyirriitzeoimmioevonddeaeer- entación Científica on April 27, 2011
was the Argentinian flora, with 90% of species lacking
allyviewedasan‘avoidance’groupbecausethelowwater-use
genome-size measurements. The Angiosperm Phylogeny
efficiency inevitably associated with their exceptionally large
Group (APG) has redefined the species composition of a
stomata precludes an extension of the period of growth into
number of major angiosperm taxa. The classification of
hotter, drier summer conditions (see Hetherington and
Angiosperm Phylogeny Group (APG III, 2009) has been fol-
Woodward, 2003).
lowed and families have been ordered in the sequence set
out by Haston et al. (2007, 2009).
Ecologicalattributes.HetheringtonandWoodward(2003)have
Leaf traits. Values relate to healthy, sexually mature plants suggestedthatselectionforoptimalstomatalsizerelatestosur-
growing in unshaded habitats and are usually an average of vivalinshadedandindroughtedhabitats,largestomatabeing
at least six replicate measurements. Prior to measurement, favoured in the former and small in the latter. With a view to
leaves were enclosed within a moistened paper towel and confirmingtheserelationships,habitattypewasincludedinthe
kept refrigerated overnight in a sealed polythene bag to analyses. This was assessed from published sources, particu-
ensure that they are fully imbibed. Subsequently, the area of larly Braun Blanquet and de Bolo´s (1953) and Grime et al.
the leaf lamina (using a leaf area machine or scanner), leaf (2007), and from unpublished vegetation surveys and field
fresh weight, leaf dry weight and intervenal leaf thickness observations. Some species from Iran were too ecologically
(to the nearest 0.01mm, using a dial thickness gauge) were wide-ranging for a confident assessment of habitat type and
measured. Because of their ecological importance (Givnish, there were too few data to include Argentina in the analyses.
1988; Reich et al., 1992; Bolha`r-Nordenkampf and Draxler, The following life-history classes were also separated:
1993; Garnier and Laurent, 1994; D´ıaz et al., 2004), the annual, monocarpic perennial (‘biennial’), herbaceous
576 Hodgson et al. — Stomatal vs. genome size in angiosperms
polycarpicperennial(excludingvernalgeophytes),vernalgeo- %) 40 A
phytes and woody species (trees, shrubs and subshrubs). s ( 30
e
ci
e 20
p
Analyses s
of 10
The statistical properties of guard-cell length, genome size, o.
N 0
leaf size, leaf thickness, leaf dry-matter content and amphist-
omy were checked. It was found necessary to log -transform
the first four variables prior to statistical analysis1a0nd present %) 40 B
leafdry-mattercontentasitssquare-root.Nosatisfactorytrans- s ( 30
e
formation for amphistomy was identified and species were, eci 20
p
therefore, grouped into the following five subequal classes: s
1, 0%; 2, ,25%; 3, 25–40%; 4, 40–45%; 5, 45–50% (% o. of 10
stomata on the surface with lower density). Except where N 0
otherwise stated, statistical tests were performed using SPSS
for WindowsTM (Version 14.0).
%) 40 C
Threesetsofanalyseswerecarriedout.Inthefirst,stomatal s ( 30
length values from different geographical regions, families, e
life-history groupings and habitats were compared using one- eci 20 Dow
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fGaemoiglriaesphaincdaliannfrda-tfaaxmoniloimaRlicEgrvSoaUurpiLaitnTigoSns).in stomatal size 5F(aEnAn9nIodG;)gt.l(AisaBn1tnra)g.dtteihScsSn:eptittacio1rniame.na4lmalaa7yb:ta:+alisoln1iggi.l04ne1n.n30g1i(fig3+sfitct,hoag0mnnud.tri1al¼esy1ttsar,7dilabn4ignfu5u¼fdt.eiaorrt2eAadn8nb-Nw4tcle;eOaisltt(lVh,CPliAgen)r,noIegFruaat03cphn,.h1i,0bn5cm5o5g:9fmsi1n¼t.)wh4+Te17itu3fh+kos.1.eutd,hy0r.e.m11P(.1psa4,a,oi2mnsn+t0e-s¼.ht0uso00d4uc.11y7f).fi35atx,;eHrnse(aetaDs¼rrs.ee). ocumentación Científica on A
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of guard-cell lengths, however, differed little between online). Species with the largest stomata were almost
regions and was similar to the 10–80mm range cited by exclusively monocotyledonous vernal geophytes [Fritillaria
Hetherington and Woodward (2003) for the world flora. meleagris (Liliaceae), 100.8mm; Lilium martagon
Values ranged from 15.3mm (Trichloris crinita, Poaceae) to (Liliaceae), 83.4mm; Gagea lutea (Liliaceae),76.3mm;
71.3mm (Baccharis articulata, Asteraceae) in Argentina, Orchis mascula (Orchidaceae), 75.4mm; Orchis anthropo-
from 12.5mm (Medicago orbicularis, Fabaceae) to 61.0mm phora (Orchidaceae), 70.9mm; Ophrys apifera
(Adonis annua, Ranunculaceae) in Spain, from 14.9mm (Orchidaceae), 68.3mm, Ophrys punctulata (Orchidaceae),
(Punica granatum, Lythraceae, formerly Punicaceae) to 67.4mm; Ophrys insectifera (Orchidaceae), 66.1mm; Scilla
67.4mm (Ophrys punctulata, Orchidaceae) in northern Iran mischtschenkoana (Asparagaceae), 65.3mm; Dactylorhiza
and from 12.1mm (Salix repens, Salicaceae) to 100.8mm praetermissa (Orchidaceae), 64.2mm]. The only exceptions
(Fritillaria meleagris, Liliaceae) in England. were Baccharis articulata (Asteraceae), a stem succulent,
71.3mm, and Caltha palustris (Ranunculaceae), an early-
flowering, wetland herb, 65.6mm. By contrast, and consistent
Variation in stomatal size in relation to life history and habitat
with the findings of Beaulieu et al. (2008), species with the
In each life-history class there was at least a 3-fold differ- smallest stomata were predominately woody species [Salix
ence between the largest and smallest average stomatal sizes repens (Salicaceae), 12.1mm; Pistacia terebinthus
(Fig. 2). Nevertheless, despite this high level of variation (Anacardiaceae), 12.8mm; Arthrocnemum macrostachyum
within groupings, stomatal size appeared to be very much a (Amaranthaceae, formerly Chenopodiaceae), 13.2mm; Salix
function of life history (Fig. 2). Moreover, general trends caprea (Salicaceae), 14.3mm; Punica granatum
Hodgson et al. — Stomatal vs. genome size in angiosperms 577
%) 5600 A cwoonotdralsatneddwliifteh-htriesetosrwyitthypsemsadllifsfteorminagtaianndstosmmaaltlalvesrinzaelg(ee.og-.
es ( 40 phytes with large stomata). When comparisons were focused
eci 30 more narrowly, a negative relationship between stomatal size
p
o. of s 1200 aronodteadridanitnyucaolsuladndbedeceopn-sriosotetnetdlytrdeeestecatnedd.taFlolrshbroutbhs,shsaplelcoiwes-
N from more droughted environments had smaller stomata than
0
comparable ones from mesic habitats (Table 2Bi). It was not
%) 60 B possible, however, to find any evidence supporting another
s ( 50 suggestion of Hetherington and Woodward (2003), namely
e 40 thatthepossessionoflargestomataisanimportantcomponent
ci
e 30 of specialization for shade-tolerance. Summer-green species
p
of s 20 from the woodland floor (i.e. shade-tolerant species) have
o. 10 smaller stomata than woodland vernal geophytes, ‘shade-
N
0 avoiders’, which exploit only the open phase of thewoodland
before canopy closure (Table 2Bii).
%) 60 C
50
s (
e 40 Stomatal size and leaf structure D
ci ow
No. of spepecies (%) 123345600000000 D 7a(t1i(hvFFr39aeeirTgt%l%ieyht.pi,elrsa3olepanAwfsterhedthicrnhaeaie3teeenr8sesdmtaPt%owuseCtB,dalieAotyl)ha.ffvog(aTtasrSrhxmrhiaeieseatla,shinmlxelsci1dapers0xeeets0inoicppnm.itm8eeeifiunctsahmimsetee,amdws,d1fif)otra,2wwhort.ah1mitPtthihhmmcCeehtmaAhttovsah)rmsicaaexehclxau,oaelwleeduslasefnirfs1tgvthetw–eacfsls3tloeutiat,rrvoereegmrssaeltepyoas26pptmo18asreneoa%%cpeixttnas--,,. at Caob.oxfordjournals.orgnloaded from
No. of s 12000 8sop3cec%cuipeysanwddiiftfh7er1Ce%nAt.MpToashinetdiotnh‘csroeoeonlsstpeheaecsioathnlir’setevegPrrnCoauAlpgsa,exoeCpsh4y(Ftsepisge.cail3esCso, entro de Inform
%) 5600 E andPCDA).aOxnisly1,Ce3xpsplaeicniiensgwaeprperowxi.d2e8ly%scoaftttehreedv.ariance,wasa ación y D
es ( 40 ‘xeromorphic-mesomorphic axis’ of leaf structure and has ocum
No. of speci 1230000 1·0– 1·1– 1·2– 1·3– 1·4– 1·5– 1·6– 1·7– 1·8– 1·9– stii(otnAosmpsoteloocAmwyercneagoarteaclnoleetgaisnineicd)zaa,,el wieLnq[aeeurrH.eigrvee.aatshlepenerAdcicinseivpegasittrdo,oioncstmahpateaaenirdndmlr(yWayZ-yosfghorooadqdpmwueheadybrlardlaaraxicc(dih2esoa0eh-0rbe)a3llb)aa.itantianAnctdogst entación Científica on A
1·1 1·2 1·3 Lo1g·140 sto1·m5ata1l ·l6engt1h· 7(µm1)·8 1·9 2·0 Llsemiathvalrelas,esathoimgtheartand.irfyoAlimtaattht(eeArnhcaiocgnahtreedrniat,ecxeltaorewe)m],sepewwciietfirhce lssepmaefacliale,rseatfhraoincmdk pril 27, 2011
FIG. 2. Stomatallengthdistributionwithindifferentlife-historyclasses:(A) mesic and from shaded, habitats, [e.g. Anthriscus cerefolium
woody polycarpic perennialsa [log (stomatal guard-cell length, mm)+s.d.
1.41+0.10, n¼205]; (B) monoc1a0rpic perennialsab (1.45+0.09, n¼89); (Apiaceae) and Valeriana officinalis (Caprifoliaceae)], with
(C)annualsab(1.45+0.11,n¼451);(D)herbaceouspolycarpic perennialsb large, thin leaves, a low dry matter content, high specific
(1.47+0.11, n¼658); (E) vernal geophytes (1.68+0.16, n¼46). leaf area and larger stomata. Fast-growing species of pro-
ANOVAF4,1444¼58.6,P,0.001. ductivehabitats(seeGrimeandHunt,1975)occupiedaninter-
mediate position along PCA axis 1 [Chenopodium album
(Lythraceae), 14.9mm; Ficus carica (Moraceae), 15.3mm; (Amaranthaceae), value 51% of maximum; Urtica dioica
Myrica gale (Myricaceae), 15.4mm; Robinia pseudoacacia (Urticaceae), 56%; Holcus lanatus (Poaceae), 65%].
(Fabaceae), 15.4mm]. Only four herbaceous species occurred PCAaxis2,the‘succulenceaxis’[highestscores:Grahamia
in ‘the bottom twelve’: the perennial C grass, Trichloris bracteata(Portulacaceae)andSedumrupestre(Crassulaceae)],
4
crinita (15.3mm), one herbaceous perennial (Trifolium fragi- accounted for a further approx. 26% of the variance. Species
ferum, 15.2mm) and two annual legumes (Medicago orbicu- with high values had thick, amphistomatous leaves with a
laris, 12.5mm; Medicago radiata, 14.5mm). More typically, low dry matter content and moderately large stomata. As is
representatives of these other life-history categories (annual, characteristic of succulents (Vendramini et al.,2002), specific
monocarpic perennial, herbaceous polycarpic perennial) were leaf area was relatively high. The stem succulents, Baccharis
of intermediate stomatal size (Fig. 2). articulata and Cereus validus (Cactaceae), not included in
Stomatal size also varied according to habitat, but with the analysis, also have large stomata.
lesser statistical significance (Table 2A). In part, this relates PCA axis 3, which explained a further approx. 19% of the
to the frequent co-existence within the same habitat of variance, was a ‘size axis’ relating to the dimensions of both
578 Hodgson et al. — Stomatal vs. genome size in angiosperms
TABLE 2. A regional comparison of stomatal length for major habitats: (A) compares all habitats and (B) ecological species
groupingssubjecttodifferinglevelsofdroughtandshade
Habitat n Meanlog guard-celllength+s.d.(mm)
10
(A)Allhabitats
England
Skeletal 79 1.43+0.11a
Wasteland 152 1.45+0.12ab
Arable 109 1.47+0.10ab
Maritime 23 1.47+0.10ab
Woodland 148 1.48+0.14ab
Wetland 167 1.49+0.12b
Pasture 98 1.49+0.14b
F ¼3.5P,0.01
6,769
(R2¼0.23;F ¼7.0,P,0.001;
31,744
lifehistoryF ¼28.7,P,0.001)
4,744
Iran
Secondarywoodland(altitudinalzone2) 100 1.43+0.10a
Drypasture(altitudinalzone4) 69 1.45+0.10ab
Primarywoodland(altitudinalzone1) 125 1.47+0.11b
Pasture(altitudinalzone3) 55 1.51+0.09 Dow
Fl(iRf3e,234¼h5is¼0to.1r5y9.9;FFP41,3,82,3020¼0.0¼50.146.,2P,P,,0.00.0010)1; nloaded from
SpD‘PWAWaSiarrnaeosaylttobiulpndalreaneleas’dntau(ndordenwagnyadsptsewulaamsntdesolainlsd) 631843738451 111111......444443312577++++++000000......111111302103aaaaabbbbb at Caob.oxfordjournals.org
(B)Droughtandshade Fl[iRf5e,227¼h2is¼0to.1r3y4.8;FFP42,2,15,2650¼6.0¼31.26..0P.P,,0.00.10]1; entro de Inform
(Ai)nnDuraolusg:hatridvs.‘mesic’habitats ación y D
EnASgklraaenbledltealhabitats 9496 t11..¼44603++.5,00P..11,02 0.001 ocumentación C
ETSpanAADlaglilrrrnawagynbeodplnoeatdisnytuasrep(aerciide)s(canopyheight.3m):aridvs.temperateclimates 172056 t111...¼3434683+++.8,000P...111,101b0.001 ientífica on April 27, 2011
Spain 26 1.36+0.12b
Iran 30 1.42+0.08ab
England(temperate) 47 1.45+0.11a
F ¼6.3,P,0.001
3,109
(ii)Shade
England:woodlandground-floorvegetation
Summer-greenperennialherbs 64 1.46+0.11
‘Shade-avoiding’vernalgeophytes 16 1.69+0.16
t¼6.9,P,0.001
SpeciesgroupingswiththesamelettersarenotstatisticallysignificantlydifferentatP,0.05inTukey(post-hoc)tests.
In(A)statisticalanalysesinparenthesisrelatetotwo-wayANOVAswhereadditionallylife-historyattributesareincluded;allstatisticallysignificant
treatmenteffectsofhabitatorlifehistoryareappended.
theleafanditscomponentparts.AttheupperendofPCAaxis Taxonomic variation in stomatal size
3 were species with large, relatively thick, amphistomatous
Stomatal size further relates to both cytological status and
leaves and large stomata [e.g. Petasites hybridus
phylogeny. Intrageneric polyploids tended to have larger
(Asteraceae)] and at the lower end, with small, thin, hyposto-
stomata than their close diploid relatives (Fig. 4A).
matous leaves, were Aphanes arvensis (Rosaceae),
Nevertheless, even when this increase in size through poly-
Callitriche stagnalis (Plantaginaceae), and other similarly
ploidy is factored out by including only familial or ‘clade’
small and/or short-lived species.
Hodgson et al. — Stomatal vs. genome size in angiosperms 579
4 4
A B
2 + + + 2
+ + +
y
m
PCA axis 2–02 Leaf thicknessAmphistomyStomatal sizeSLADMC PCA axis 3–20 Leaf areaStomatal sizeLeaf thicknessSLAAmphisto
+ + + +
SLA + –4 SLA +
–4 Leaf area + Leaf area +
Large stomata Stomatal size + Stomatal size +
Small stomata + Leaf thickness + Leaf thickness D
+ DMC + DMC ow
24–4 C –2 0PCA axis 12 +4+ + 24–4D –2 0PCA axis 12 + 4+ + at Caob.oxfordjournals.orgnloaded from
PCA axis 2–20 Leaf thicknessAmphistomyStomatal sizeSLA+DMC+ PCA axis 3–20 Leaf areaStomatal sizeLeaf thicknessSLA+Amphistomy+ entro de Información y Documentación C
–4 CCCVe34ArMnal geophyte + LSeatoLf methaaSicft LakaAlnr seeiaszse +++ –4 + LSetaoLfm ethaaSiftc LaakAlr nseeiazses +++ ientífica on April 27, 2011
+ DMC + DMC
–4 –2 0 2 4 –4 –2 0 2 4
PCA axis 1 PCA axis 1
FIG.3. PCAordinationof1186angiospermspeciesfromArgentina,England,IranandSpain,onthebasisofsixleaftraits.Labelsdisplaytraitswiththehighest
eigenvectorscoresonPCAaxes1,2and3,withthelabelwiththehighestscorepresentednearesttheaxis.In(A)and(B),thedistributionofspecieswithlarge
stomata(.40mm)andthosewithsmallstomata(,20mm)isshown,asindicatedin(A),andin(C)and(D)thedistributionofC,C,CAMandvernalgeophyte
3 4
speciesisshownasindicatedin(C).
diploids, families showed significant differences in stomatal considerably higher than, and statistically different from,
size (Fig. 4B). Within the present dataset, some, particularly those from the less geographically and phylogenetically
Brassicaceae, Fabaceae and Rosaceae, had small stomata restricted subset examined in Beaulieu et al. (2008) [27.42
whereasothers(e.g.OrchidaceaeandRanunculaceae)hadcon- 62.7 (39.5) mm; n¼9; t¼4.6, P,001]. In contrast, for
sistently large stomata. A familial summary of stomatal size species common to both studies there was broad correspon-
for the 1442 species measured is presented as Table S2 in dence in measured values for stomatal length between the
Supplementary data, available online. These familial averages two studies (r¼0.61, n¼24, P,0.01; paired t¼0.7, n.s.).
must, however, be treated with caution. For example in Notwithstanding the results for Orchidaceae, and consistent
Orchidaceae, the values, originating only from England and withtheresultsinFig.4B,stomatalsizedoesappearconserva-
Iran [range (mean) 44.1275.4 (57.9) mm; n¼20], are tively expressed within major taxa (Fig. S1 in Supplementary
580 Hodgson et al. — Stomatal vs. genome size in angiosperms
A 2·5 A
1·8 1:1
Poaceae
d 2·0
oi Other monocots g)
m), polypl 1·6 Eudicots ar DNA (p 11··05
µ e
ength ( 1·4 2C nucl 0·5
al l 10 0·0 Eudicots
mat Log Basal dicots
o –0·5 Poaceae
st01·2 Other monocots
g1 –1·0
Lo 1·0 1·2 1·4 1·6 1·8 2·0
Log stomatal length (µm)
10
1·0 2·5 B
1·0 1·2 1·4 1·6 1·8
Log10 stomatal length (µm), diploid g) 2·0 Dow
Stomatal length (µm) 876543000000 B Log 2C nuclear DNA (p10––100011······0505051·0 1·2 1·4 1·6 1·8RFaabnaucnec2au·e0laceae at Centro de Informaob.oxfordjournals.orgnloaded from
20 1·5 C Log10 stomatal length (µm) ación y D
EFaIGch. d41a.t0u(mA)poDOinirpctlioPsaiobdaasnRitaanenntrdFacagdtbeonRehcor asivcBedcraispmlFPboaoaeilldlme–CribpalysrotAlocymdmpealaobtPcaidldaetphLaacaidnmr(rbAmedsleoattnCeobdacpoptcAosdp,leyiepxlcoliudds-. ear DNA (pg) 01··50 ocumentación Científica on A
i0‘snip.n0get0ceP1ireon;saar.cti(e¼oBane)0a;.Sl7P’t4ooa,amvcPeaert,aaagel;e0la.es0nnt0odg1mteh.auHtfdaoeilrcresodi,tzispae,nlaovdsiadilinunsedptiheicsecaituereessdem)dd.aiPffifnoaeirirnrsmegdboteatrteb¼wlwee5seid.n1aen,flydanmd¼fiiiglsi7uter0rsie,b.suPT,tahe,nde 2C nucl10 0·0 Fabeae pril 27, 2011
g
boxplotsincludethemedian(centralline),thefirstandthirdquarters(box) o –0·5 Trifolieae
L
and outliers. The 14 families illustrated (Amaranthaceae, Apiaceae, Other
Asteraceae, Brassicaceae, Caprifoliaceae, Caryophyllaceae, Fabaceae,
Lamiaceae, Orchidaceae, Plantaginaceae, Poaceae, Polygonaceae, –1·0
RanunculaceaeandRosaceae)areidentifiedbytheirfirstthreelettersandphy- 1·0 1·2 1·4 1·6
logenetically ordered as recommended by Haston et al. (2007). ANOVA
Log stomatal length (µm)
F ¼29.43, P,0.001. Here ‘diploidy’ relates to the familial base 10
13,305
chromosome number as given in Raven (1975) except for Orchidaceae,
where,usingBatemanetal.(2003),ploidywasassessedinrelationtoclade FIG. 5. Examplesoftherelationshipbetweenstomatalandgenomesize.(A)
basenumber.Familieswiththesamelettersarenotstatisticallysignificantly All species: r2¼0.36, P,0.001, n¼446. Eudicots (r2¼0.26, P,0.001,
differentatP,0.05inTukey(post-hoc)tests.Themeanvalueforstomatal n¼326); basal dicots (n¼3); monocots, excluding Poaceae (r2¼0.39,
sizeforalldiploidspeciesmeasuredisidentifiedbyabrokenline. P,0.001,n¼30);andPoaceae(r2¼0.53,P,0.001,n¼87),asindicated.
(B) Contrasted families: Ranunculaceae (r2¼0.64, P,0.001, n¼23), and
Fabaceae (r2¼0.64, P,0.001, n¼46), as indicated. Other families:
Data, available online). Similar values were recorded for Asteraceae (r2¼0.08, P,0.05, n¼51; minus Chrysanthemum segetum,
r2¼0.08); Caryophyllaceae (r2¼0.23, P,0.1, n¼15); Polygonaceae
familial subsets differing in life-history. Superimposed on (r2¼0.32, P,0.05, n¼13). (C) Contrasted tribes (Fabaceae): Fabeae
this a further ecological effect was noted for woody species (r2¼0.29, P,0.01, n¼26); Trifolieae (r2¼0.67, P,0.001, n¼13);
(Fig. S1A). These tended to have smaller stomata than and other (n¼7), as indicated. Tribes in other families: Asteraceae,
related herbaceous species (excluding vernal geophytes). For Anthemideae (r2¼0.13, n.s., n¼17; minus Chrysanthemum segetum, r2¼
0.30,P,0.05,n¼16);Lactuceae(r2¼0.50,P,0.001,n¼18);Poaceae,
themuchsmallervernalgeophytesdataset,nostatisticallysig-
Agrostideae (r2¼0.49, P,0.01, n¼13); Aveneae (r2¼0.72, P,0.001,
nificantdifferencewasobserved(Fig.S1B).Vernalgeophytes n¼11);Poeae(r2¼0.05,n.s.,n¼19).
Hodgson et al. — Stomatal vs. genome size in angiosperms 581
were primarily restricted to families in which all or, at least contributetovariabilitywithinthedataset.Nevertheless,avail-
most, species had large stomata. able data indicate that cytoplasm occupies a sizeable pro-
portion of guard cell volume [e.g. 31–41% for Arabidopsis
thaliana (Brassicaceae); Tanaka et al., 2007; see also,
Stomatal and genome size
Fricker and White, 1990; Willmer and Fricker, 1995; Merkel
Stomatal length and genome size are positively correlated et al., 2007] and provides no evidence that the fraction of
(Fig. 5). This relationship appears to be a general feature guardcellvolumeatagivenproportionofthemaximumaper-
within the eudicots, Poaceae and the remaining monocots. ture is significantly greater in genotypically large as opposed
OnlyintribePoeae(Poaceae),withahighincidenceofintras- to genotypically small guard cells.
pecific polyploidy, was the relationship not detected.
Genome size and chromosome number. Two problems restrict
the usefulness of published data. The first is accuracy, both
DISCUSSION
inmeasurementandtaxonomy.Thesecondrelatestocytology.
Problems and conclusions A significant minority of species have cytotypes differing in
ploidy and genome size (for examples, see Bennett and
Beforeanyinterpretationoftheresults,thebasicdifficultiesin
Leitch, 2005). Where necessary, taxa have been identified to
acquiring and analysing data for a broad study of this type
their subspecies and known relationships between ploidy and
should be considered. These include the following.
geographical distribution assessed. However, some groups
D
Intraspecific variation in stomatal size. Plants grow in hetero- [e.g. tribe Poeae (Poaceae), with a high incidence of intraspe- ow
gsAl1Pwaeine9etsianydal8flar,e8csot;hrsyteeutaa,mrsBstvuiu2epseoclt0etntli,uih0crvnapraa0`ieethrrl;uvo-erNsinAanettnmaro,orebdortaenydlntnygmpenfttnuisehociknmsuapcaopnpmtnlfhidaadoepsectnrhtfrtiiDeieccnsdiaeegthrnayeuuMdss(ipmousLoinlDicaetnadscncrli,oalhttyiy,hxbomeluwfl2aeptnu0riaoc,dc0orrzt1intuw1t,a)lha9y.nam1t9ttiTe9un3ecrh8pg;toii4amcsltv;AiogpapplGhiollraleatenainsvcbieptninnioisltcaitiiiesintonnothytndy-f,.. clsmcfSaooiaafirarmlmlltcyyctperhplispibeoinsrgdeltogyon.bPpmibTlfiolieohaacemiitsaadsa.enalyAmtti(accaPlant.onyohdrdaWor,eceuxteeloggpaaehtlaesinauo)fliooesnnimpsnr,bseefeeeFcoarttircwvgeheta.xehxlteo5aueanfCtenm,stts.h,pdtoaeleaemnsrf,epeoatuiuthtapcerellyofllaotaioonudclrdryoka]gsgboiaeescfrntsaeuatoldlpmsyeiatefearfdttmoisi,csriiatztussine---, at Caob.oxfordjournals.orgnloaded from
Taapnhpderoinixnt.rtar4as0ppoe%pciufiolacftitcohonemmpraaenragisneoninosfbthesettowsmeaeamntaplcleossuinz(edtraietwasanisnodttiycspahitocewadlnlya) enecaoortllyothgedicisvaaellmrygeibnapglhaydnlicocegodetsnseuwtbiceserebtaoliafnncsclpueed.ceFideo.srMwexaoasrmecophvloeesr,e,not,nhlteyhreetrheraeriees entro de Inform
bPraoratidculcaorlryresepnocnoduernagceing(Fwige.reSv2aluines SfourppTlermifoelniutamryredpaetnas)., alilmmiotesdt nsoubdsaettaoffotrhtehewosprledcifleos-rraicchantreoapsiiclsy.lTehade tuosemoisflesuadchinga ación y D
the onlyspecies common to all studyareas. Its mean stomatal average values of stomatal size for major taxa [see above, the ocum
1le6n.g4thanidn1S7p.a3inm,mA,rrgeesnpteinctai,veElnyg.land and Iran was 15.3, 16.2, cBoemauplaieriusoent aolf. (o2u0r08re)]s.uRltsepfroersenOtracthiviednaceessaeisafnudrthtehrosreedfurcoemd entación C
scdwCthalrhatauaisrccasthicuofitrnlceaearlastwviloeewhnaseafotswhcfheellearraeacrfaksicsitnnottrregmour.lscalteWtauadnriedtow.hrIaenlrdrlefeiogsorsafsmerutdchantteekitdooeinnmwsotporanouunrccldtdtaeunrrptttaharlioebnbicoaiehcmbxahltrpyeeaomnchrtttieaacrvtnasoelt, iP2,itnsh0lea20unc5n%tso)kpmDnieosocpfNwiaaaeArnnsmisgCooffiooenn-rvasus,ptamurelberuereesmendptstwseacDlie(nteGaianvctrtaehehbligseyeato,vosreepm9ymr0eaaeest%ttaenalKn,tl.t.e7a,Nw7ns2etd%0uv(0deB,7gryet5)ehn4nafenorn%leoemdtmstseag,aneAnidnstdioriz5gnmeL3ec.enel%uitsTtidcnihzeohaeesf,, ientífica on April 27, 2011
been useful contributors to the ‘xeromorphic-mesomorphic
Iran, Spain and England.
axis’ (PCA axis 1), and the amount of non-photosynthetic
water-storage tissue would have enhanced the ‘succulence
Insufficient sampling of vernal geophytes. Vernal geophytes are
axis’ (PCA axis 2).
poorlyrepresentedwithinthefourflorasstudiedandconstitute
Vacuolization of the guard cell. Genome size and nuclear and onlyaminorcomponentofthepresentdatabase.Moreover,in
cell volume are positively correlated for non-vacuolated cells only nine families were there data for both vernal and non-
(Bennett, 1972; Edwards and Endrizzi, 1975; Cavalier- vernal species. This is unfortunate. If we are to understand
Smith, 2005; Sugiyama, 2005; Jovtchev et al., 2006). the ecological significance of large stomata, vernal geophytes
However, the additional presence of a vacuole is essential for areakeyecologicalgrouping.Nevertheless,atpresentthedata
stomatal functioning: the opening and closure of the stomatal are lacking to separate the phylogenetic and ecological deter-
pore involves potentially large changes in vacuolar and cellu- minants of their stomatal size satisfactorily.
lar volume (Willmer and Fricker, 1995). A deterministic Nevertheless, despite these problems, the following con-
impact of vacuolar size on stomatal dimensions (i.e. the clusions can be drawn.
absence of a close allometric relationship between guard cell
size and the size of its cytoplasmic component) would (a) As with genome size (see Leitch et al., 1998), there are
seriously undermine our rationale for examining the relation- clear lineage-specific differences in stomatal size. These
ship between stomatal and genome size. Doubtless, there are interfamilial differences appear to dwarf those found
differences in vacuolization both between species and within polyploid series (Fig. 4; Table S1 in
between broader taxonomic groupings, and these will Supplementary Data, available online).
582 Hodgson et al. — Stomatal vs. genome size in angiosperms
(b) Variationwasdetectedinstomatalsizealongthreeimpor- adjustments resulting in predominantly nocturnal stomatal
tant ecological axes of leaf specialization: xeromorphic openinghavereducedtheimportanceofstomatalsizeasareg-
(stomata small) vs. mesomorphic (large), non-succulent ulator of water-use efficiency and even in arid habitats CAM
(small) vs. succulent (large) and ‘small’ (small) vs. species may have relatively large stomata.
‘largelamina’(large). Stomatalsizeisanimportantcom- C speciesgenerallyhavesmallerstomatareflectingdaytime
4
ponent of leaf specialization and patterns with respect to opening in arid habitats (Fig. 3), but the importance of size,
C , CAM and life history (woody species,herbaceous both here and in C species, may be similarly complicated
4 3
species,vernal geophytes). by their mode of function: e.g. the ‘dumb-bell’ shape of
(c) Becauseofitsrelativelyconsistentandstrongcorrelations stomata in Poaceae is a reflection of a mechanism by which
with 2C DNA amount and its ecological importance, the subsidiary cells also change turgor, effectively relaxing as
possibility that stomatal size is a key determinant of guardcellsinflate,offeringlessresistancetoguardcellmove-
genome size in angiosperms deserves consideration. ment and allowing stomatal responses an order of magnitude
faster than species with ‘kidney’ type stomata (Franks and
Farquhar, 2007).
Nonetheless, there remains a fundamental requirement
Stomatalsize, akeydeterminantofgenomesizeinangiosperms?
within the angiosperms for efficient stomatal conduction, and
Beaulieu et al. (2008) argue that stomatal size is a conse- size undoubtedly plays a large part in this (‘throughout
quence of genome size. However, since they do not identify biology size matters’; Cavalier-Smith, 2005). Genome size
D
whatactuallydeterminesgenomesize,wefindtheirarguments could potentially be an ultimate consequence of stomatal ow
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whichphotosynthesisisonlyone.Stomatal sizeisnotaneco- guard cell walls dominates diffusion through the aperture
logicallyandphysiologically‘standalone’character.Itwillbe (such ‘Knudsen diffusion’ becomes important for apertures
subject to trade-offs with other ecologically important plant ,0.5–1mm; Leuning, 1983). Similarly, maximum genome
attributes and may often have, at best, a subordinate impact size should be constrained by the speed of opening and
upon genome size. closure of very large stomata.
Take, for example, vernal geophytes, a grouping in which, Do such limits operate in practice? Unfortunately, it is not
we suspect, endopolyploidy is uncommon (see Barrow and possible to be sure. For example, there are discrepancies
Meister, 2003). Here, a large genome facilitates a form of between our own observations and established ideas on the
‘coolseasongrowth’inwhichcelldivisionandcellexpansion physiological and ecological significance of large stomata. It
are uncoupled (see Grime and Mowforth, 1982). These vernal hadbeensuggestedthatverylargestomatafacilitatephotosyn-
geophytes have large, ‘conductively inefficient’ stomata but thesis in deep shade (Hetherington and Woodward, 2003). In
the cell size issues relevant to vernal growth appear to rep- this study, very large stomata were primarily associated with
resent the main ecological determinant of genome size. vernal geophytes, which exploit either unshaded habitats or
Depending on their level of endopolyploidy, succulent the ‘light phase’ of deciduous woodland before closure of
CAM species may have similar ‘design conflicts’. Genome the canopy rather than with shade-tolerant, summer-green
sizepotentiallyrepresentsasimpletrade-offbetweenselection woodland herbs (Table 2Bii). A revised assessment of where
for large cells with large vacuoles (to store water and the and why large stomata occur is urgently required and, criti-
organic acids accumulated nocturnally) and that for small cally, it needs to take into account the findings of Grime and
stomata (to restrict transpiration). However, metabolic Mowforth (1982) – see above.