Geographical variation in cone volatile compositionamong populations of the African cycadEncephalartos villosus
TERENCE N. SUINYUY1,3,4*, JOHN S. DONALDSON1,2,4 and STEVEN D. JOHNSON3
1Kirstenbosch Research Centre, South African National Biodiversity Institute, P/Bag X7, Claremont7735, Cape Town, South Africa2Research Associate, Fairchild Tropical Botanic Garden, 10901 Old Cutler Road, Miami, FL 33156,USA3School of Life Sciences, University of KwaZulu Natal, P/Bag X01, Scottsville 3201,Pietermaritzburg, South Africa4Department of Botany, University of Cape Town, P/Bag Rondebosch 7701, Cape Town, South Africa
Received 20 December 2011; revised 6 February 2012; accepted for publication 6 February 2012bij_1905 514..527
Variation in traits across species distribution ranges is often indicative of diversifying evolution that can lead tospeciation. Of particular interest is whether traits vary clinally or abruptly because the latter pattern can beindicative of incipient speciation. Understanding of intraspecific variation in chemical traits is still in its infancybecause studies of population variation have tended to focus on morphology or neutral genetic markers. To addressthese issues, the composition of cone volatile odours was examined in ten populations of the South African cycadEncephalartos villosus across its range in the Eastern Cape and KwaZulu Natal using headspace sampling andanalysis by gas chromatography-mass spectrometry. Because volatiles play a key role in attracting pollinators tocones of Encephalartos cycads and may thus reflect local adaptation to pollinators, pollinator assemblages were alsoinvestigated in the ten populations of E. villosus. Volatile compounds from populations in the north of thedistribution range were dominated by unsaturated hydrocarbons, whereas, in the southern populations, nitrogen-containing compound and terpenoids were the major compounds. A shift between southern and northern popula-tions appeared to occur at the Umtamvuna River, where populations had odour profiles with components of boththe northern and southern populations. However, one population in the north (Vernon Crookes Nature Reserve)had a quantitatively similar odour profile to the populations in the extreme south of the range. These results revealstrong interpopulation variation in the cone scent of E. villosus, including variation in the relative emission ofdominant compounds that may play key functional role in this pollination system. However, pollinator assemblagesdid not differ across the different populations, which suggest that these patterns were produced by co-evolution ordrift, rather than by pollinator shifts. © 2012 The Linnean Society of London, Biological Journal of the LinneanSociety, 2012, 106, 514–527.
ADDITIONAL KEYWORDS: Eastern Cape – gas chromatography-mass spectrometry – insect pollinators –KwaZulu-Natal – nitrogen-containing compounds – odour profile – unsaturated hydrocarbons.
INTRODUCTION
Floral odour can play a key role in plant–pollinatorinteractions through its influence on the compositionand behaviour of pollinators that use this trait as acue (Dobson, 2006; Raguso, 2008). Odours are usuallyblends of compounds belonging to several chemical
classes, typically fatty acid derivatives, benzenoids,terpenoids, and sometimes nitrogen-containing com-pounds, and may vary in the number, composition,and relative amounts of the different constituents,and in their temporal and spatial emission patterns(Raguso, 2004; Knudsen et al., 2006). The particularconstituents and pattern of odour emission comprisethe signals that influence the composition and behav-iour of pollinators (Pellmyr, 1986a; Raguso, 2008). In*Corresponding author. E-mail: [email protected]
Biological Journal of the Linnean Society, 2012, 106, 514–527. With 3 figures
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© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527514
some cases, highly specialized plant–pollinator inter-actions are mediated by floral odours (Pellmyr, 1992;Jürgens, 2009) and these can provide a species spe-cific signal (‘private channel’) that elicits the requiredbehavioural response from the pollinating organism(Schiestl & Peakall, 2005; Raguso, 2008; Chen et al.,2009). The implication is that floral odour should be aspecies-wide attribute with the expectation thatchanges in floral odours may be associated with shiftsin pollinators that may lead to speciation in plants(Johnson, 1996; Johnson & Steiner, 1997). It has beenargued that such evolutionary changes in floralodours played a role in the diversification of bothangiosperms and pollinating insects (Pellmyr &Thien, 1986).
Although the majority of studies of floral odourhave focused on flowering plants, work on cycadsshows that cone odour is also an important factorinfluencing behaviour of pollinators in this ancientplant lineage (Terry et al., 2007a, b). Limited avail-able information shows that differences in odour pro-files between cycad species can be associated withdifferences in pollinators, as illustrated by Mac-rozamia species being pollinated by either thrips(Cycadothrips) or weevils (Tranes) (Terry et al., 2004a,b). The expectation may therefore be that the vola-tiles which influence pollinator behaviour will also beinvariant species-wide attributes in cycads. To date,there have been no studies of volatiles across the fulldistribution range of any cycad species to test thishypothesis.
Preliminary investigations of the African cycadEncephalartos villosus showed that cone odoursappeared to be inconsistent with the model of a stableodour profile throughout the species distribution.Plants originating from populations in the EasternCape (EC) had cone odour profiles characterizedby eucalyptol and 2-isopropyl-3-methoxypyrazine,whereas those from KwaZulu Natal (KZN) werecharacterized by (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene (T. N. Suinyuy, unpubl. data). Thesedifferences raise questions about possible shifts inpollination systems across the range of E. villosus andthe relative influence of different compounds on thebehaviour of pollinating insects. The present studytherefore aimed to determine the extent and patternof variation in cone odour chemistry across the fulldistribution range of E. villosus and to analyzewhether cone volatile variation between populationsis associated with different insect visitors. Twoalternative hypotheses were tested to explain theobserved variation in cone volatile composition amongpopulations of E. villosus.
The hypotheses were: (1) that changes in volatilecomposition reflect a change in pollinators and mayindicate that E. villosus, as currently circumscribed,
comprises at least two cryptic species and (2) thatvariation is related to geographical separationbetween populations, such that plants in populationsthat are furthest apart would differ the most inemitted volatile compounds.
MATERIAL AND METHODSPLANT MATERIAL AND LOCALITY
Encephalartos villosus is distributed in a relativelynarrow band along the east coast of South Africa(Fig. 1) with a linear distance of approximately900 km between the southern-most and northern-most populations. There is some morphological varia-tion in E. villosus: plants from the EC tend to haveshorter heavily spined leaflets and cones with toothededges, whereas those from KZN tend to have longeralmost entire leaflets and cones with lightly toothededges (Goode, 1989). Across its distribution, E. villo-sus occurs in patches of Scarp Forest includingEastern Scarp Forest, Pondoland Scarp Forest, andTranskei Coastal Scarp Forest. The forest patches areembedded within three different vegetation types: theKZN Coastal Belt, Pondoland-Ugu Sandstone CoastalSourveld, and Transkei Coastal Belt (Mucina et al.,2006). Volatile odour samples were collected from tenlocalities spread across the range of the species,including populations near to the southern and north-ern limits of its distribution (Fig. 1). In total, sampleswere obtained from 59 male plants and 14 femaleplants. The sampling intensity for each localitydepended on the availability of cones because E. vil-losus does not cone regularly and cones may be scarceor absent in particular populations (Donaldson, 1997).Cones that were sampled were either elongated andshedding pollen in the case of males or receptive topollen with clearly open sporophylls in the case offemales.
SAMPLING OF VOLATILE COMPOUNDS
Headspace sampling was used to collect volatiles frommale and female cones during pollen release andreceptivity respectively. Polyacetate bags (Nalo Brat-folie Kalle GmbH) were placed over the entire cone justprior to sampling to concentrate the volatile com-pounds. Air from inside the bags was suctioned for30 min into an adsorbent trap using a portable battery-operated pump (Spectrex Personal Air Sampler PAS500) calibrated at 200 mL min-1. Air samples weresimultaneously collected from empty polyacetate bagsplaced way from the plant as controls to identifybackground contamination. The trap samples werestored at -20 °C in a sealed vial until analysis. Thetraps contained 2 mg of a 50 : 50 mixture of Tenax TA(Alltech Associates) and activated charcoal (Carbotrap,
VARIATION IN CONE VOLATILE COMPOSITION 515
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527
Supelco) in a glass tube closed on both ends with glasswool. The activated charcoal is highly retentive andsmall quantities can be used as a result of its highadsorbing capacity (Millar & Sims, 1998; Tholl & Röse,2006). The adsorbent Tenax TA used in the presentstudy is commonly used to trap volatile compounds andhas a high thermal stability up to 350 °C, which allowsfor thermal desorption in the gas chomatographyanalysis.
CHEMICAL ANALYSIS AND COMPOUND
IDENTIFICATION
Volatile samples were analyzed by gaschomatography-mass spectrometry using a coupledVarian 3800 gas chomatograph (Varian Palo Alto,
California, USA) and a Varian 1200 mass spectrom-eter. The gas chomatograph was equipped with aCarbowax column (DB-wax) of 30 ¥ 0.32 mm internaldiameter ¥ 0.25 mm film thickness (Alltech). Heliumwas used as the carrier gas at a flow rate of1 mL min-1. After sampling, traps were placed in aVarian 1079 injector by means of a ‘Chromatoprobe’fitting and thermally desorbed. After a 3 min hold at40 °C, the gas chomatograph oven was ramped upto 240 °C at 10 °C min-1 and held there for 12 min.Compound identification was carried out using theNIST05 mass spectral library and comparisons withretention times of chemical standards, where avail-able, as well as comparisons between calculatedKovats retention indices and those published in theliterature. An homologous series of alkanes (C8-C20)
Figure 1. Geographical variation in cone odour composition in 10 populations (green patches with labels) across therange of Encephalartos villosus (dotted area) in South Africa. Pie charts depict the percentage of total emission for eachpopulation. A, Umtiza Nature Reserve (UNR). B, Ocean View Guest Farm (OVGF). C, Dwesa Nature Reserve (DNR).D, Mpande area, Port St Johns (MPD). E, Mount Sullivan area, Port St Johns (MTS). F, Umtamvuna Nature Reserve(UMNR). G, Oribi Gorge Reserve (OGNR). H, Vernon Crookes Nature Reserve (VCNR). I, Kranzkloof Nature Reserve(KKNR). J, Nkandla Forest Reserve (NFR).
516 T. N. SUINYUY ET AL.
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527
was used to determine Kovats retention indices.All reference compounds used for retention time com-parisons were obtained from Sigma Aldrich Inc.GmbH, except (3E)-1,3-octadiene, which was obtainedfrom ChemSampco. Compounds present at higher orsimilar percentages in controls were considered ascontaminants and excluded from the analysis.
INSECT VISITORS TO MALE AND FEMALE CONES
To determine insect pollinator assemblages on differ-ent populations of E. villosus, male and female coneswere sampled during pollen shed and pollen receptiv-ity. Cones were surveyed at the same time that thevolatiles were sampled. Insects from male cones weresurveyed by placing a beating sheet under the cone,which was then tapped to dislodge all the insects ontothe sheet. Only insects crawling on the surface offemale cones were sampled because these could becollected and stored in alcohol without damaging thefemale cone. The insects were counted and recordedbefore storing in alcohol and later identified based onkeys provided in Endrödy-Younga (1991), Donaldson(1991), and Oberprieler (1996). Voucher specimens ofinsects have been deposited in the entomological col-lection of the school of Biological and ConservationSciences of the University of KwaZulu Natal, Pieter-maritzburg campus, South Africa.
STATISTICAL ANALYSIS
PRIMER 6 (Clarke & Gorley, 2006) was used to assessthe variation in odour between different populations.Relative proportions of different compounds in eachsample were used for analyses. Non-metric multidi-mensional scaling (NMDS), based on Bray–Curtissimilarities of square root transformed data, was usedto detect similarities among samples. The stress valueis given to indicate how well the distance matrix isreproduced. The significance of differences in chemi-cal composition in samples from plants in differentpopulations was assessed using one-way analysis ofsimilarities (ANOSIM) with 10 000 permutations(factor: population) and the resulting test statisticR was taken as a relative measure of separationbetween defined groups, based on mean ranksbetween and within groups (0 means no separation,whereas 1 indicates complete separation) (Clarke &Gorley, 2006).
Simple Mantel tests were performed using ZT soft-ware to determine whether cone odour composition iscorrelated with the actual geographical distancesbetween populations (Bonnet & van der Peer, 2002).The odour similarity matrices were calculated usingthe Bray–Curtis similarity coefficient (Clarke &Warwick, 2001). The geographical distance matrix
was calculated from actual geographical distancesbetween the different populations sensu Hughes et al.(2006). Mantel tests with 10 000 permutations wereperformed for the complete data set.
RESULTSCOMPOUND CLASS PATTERNS IN CONE ODOURS
The chemical composition of cone odours emitted bymale and female E. villosus plants is given in Table 1.In total, 88 compounds were detected in all the odoursamples and 87 were identified (Table 1). The identi-fied compounds included 19 fatty acid derivatives (fourunsaturated hydrocarbons, six aldehydes, two ketones,five alcohols, and two esters), nine benzenoids, 54terpenoids (48 monoterpenes and six sesquiterpenes),and four nitrogen-containing compounds.
The populations of E. villosus can be distinguishedclearly from the composition of volatile emissions atthe level of compound class. The most dominant com-pound classes were monoterpenes and unsaturatedhydrocarbons which contributed 43.7% and 31% of thetotal emissions, respectively (Fig. 1, Table 1). Monot-erpenes were present in almost all E. villosus popula-tions but dominated the volatile profiles of populationsin the southern part of the distribution range in theEC, including Umtiza Nature Reserve (UNR), OceanView Guest Farm (OVGF), Dwesa Nature Reserve(DNR), Mpande area (MPD), and Mount Sullivanarea (MTS). By contrast, unsaturated hydrocarbonsoccurred exclusively in populations in the northernpart of the distribution range in KZN, notably atUmtamvuna Nature Reserve (UMNR), Oribi GorgeNature Reserve (OGNR), Vernon Crookes NatureReserve (VCNR), Kranzkloof Nature Reserve (KKNR),and Nkandla Forest Reserve (NFR) and were domi-nant components of volatiles in four populations.
Other compound classes comprised < 10% of totalemissions, although there were notable differences inthese compound classes between populations. Twopopulations in the north (NNR, KNR) had almostno minor compounds (G and I in Fig. 1), whereasnitrogen-containing compounds made up between 5%and 18% of emissions in three populations in the south(C, D, E in Fig. 1) that were otherwise dominatedby monoterpenes. The remaining five populations(A, B, F, H, I in Fig. 1) all had a greater diversity ofcompound classes typically with higher proportions ofaldehydes, benzenoids, and alcohols (Fig. 1), with ben-zenoids comprising almost 50% of emissions from UNR(A in Fig. 1) and aldehydes making up approximnately35% of emissions in the VCNR population (H in Fig. 1).A greater diversity of compound classes was observedin populations dominated by monoterpenes than thosedominated by unsaturated hydrocarbons.
VARIATION IN CONE VOLATILE COMPOSITION 517
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527
Tab
le1.
Ave
rage
rela
tive
amou
nts
(%)
ofco
mpo
un
dsin
the
con
eod
ours
ofE
nce
phal
arto
svi
llos
us
from
ten
popu
lati
ons
acro
ssth
esp
ecie
sra
nge
inth
eE
aste
rnC
ape
and
Kw
aZu
luN
atal
Com
pou
nd
CA
SK
RI
UN
RM
OV
GF
MO
VG
FF
DN
RM
MP
DM
MT
SM
MT
SF
UM
NR
MO
GN
RM
VC
NR
MK
KN
RM
KK
NR
FN
FR
M
(N=
5)(N
=6)
(N=
4)(N
=8)
(N=
5)(N
=8)
(N=
5)(N
=4)
(N=
6)(N
=5)
(N=
8)(N
=5)
(N=
4)
AL
IPH
AT
ICS
Alk
enes
(3E
)-1,
3-O
ctad
ien
ea10
02-3
3-1
1062
––
––
––
–7.
04(4
)9.
76(6
)0.
02(5
)64
.89
(8)
48.0
7(5
)47
.46
(4)
(3E
,5Z
)-1,
3,5-
Oct
atri
eneb
4008
7-61
-411
48–
––
––
––
30.4
6(4
)89
.53
(6)
0.02
(5)
33.2
0(8
)12
.60
(5)
51.3
9(4
)(E
,E,E
)-2,
4,6-
Oct
atri
eneb
1519
2-80
-011
98–
––
––
––
–0.
36(6
)16
.95
(5)
tr(4
)tr
(4)
0.22
(4)
1,2-
Dim
eth
yl-1
,4-c
yclo
hex
adie
neb
1735
1-28
-912
15–
––
––
––
–0.
08(4
)5.
70(5
)tr
(3)
tr(4
)0.
41(4
)A
ldeh
ydes
Hex
anal
a66
-25-
111
25–
––
––
–tr
(2)
––
––
3.34
(5)
–H
epta
nal
b11
1-71
-712
0910
.12
(4)
30.5
0(5
)14
.58
(2)
––
––
––
34.5
8(5
)–
7.50
(4)
–(Z
)-2-
Hep
ten
ala
5726
6-86
-113
40–
––
––
––
––
–tr
(1)
––
(E)-
2-O
cten
alb
2548
-87-
014
46–
––
––
––
–0.
99(5
)–
––
(2E
,4E
)-H
epta
-2,4
-die
nal
b43
13-5
-315
13–
––
––
––
tr(4
)–
0.01
(2)
0.27
(1)
–2,
4-O
ctad
ien
alb
3036
1-28
-516
10–
––
––
––
tr(5
)–
tr(2
)0.
04(1
)–
Ket
ones
3-O
ctan
oneb
106-
68-3
1274
–1.
97(2
)–
––
––
––
3.13
(5)
0.02
(6)
–2,
2,6-
Trim
eth
yl-6
-vin
yldi
hyd
ro-
2H-p
yran
-3(4
H)-
onec
3393
3-72
-114
89–
––
––
––
–0.
05(3
)–
––
Alc
ohol
(Z)-
3-H
exen
-1-o
lb92
8-96
-113
90–
––
––
––
0.41
(4)
––
––
–3-
Oct
anol
a58
9-98
-013
86–
3.67
(3)
––
––
––
0.02
(5)
2.66
(5)
0.35
(6)
––
1-O
cten
-3-o
la33
91-8
6-4
1456
––
7.09
(4)
––
––
0.65
(3)
tr(5
)2.
70(5
)0.
35(6
)16
.04
(3)
0.02
(4)
Lav
andu
lolb
498-
16-8
1690
––
–tr
(1)
tr(4
)–
––
––
––
–1,
7-O
ctad
ien
-3-o
lc30
385-
19-4
2086
––
––
––
––
–0.
59(5
)–
––
Est
er2-
Ph
enet
hyl
hex
anoa
tec
6290
-37-
512
70–
––
––
––
––
––
1.88
(4)
–M
eth
yl2,
4-h
exad
ien
oate
c15
15-8
0-6
1358
––
––
––
––
–1.
08(5
)–
––
BE
NZ
EN
OID
An
isol
ea10
0-66
-313
57–
––
––
––
––
–0.
02(2
)1.
40(1
)–
Ben
zald
ehyd
ea10
0-52
-715
5315
.78
(5)
14.6
2(6
)16
.09
(4)
0.02
(8)
0.54
(5)
––
1.03
(4)
0.01
(6)
2.62
(5)
0.01
(8)
2.89
(5)
0.03
(4)
1-Is
opro
pyl-
2-m
eth
oxy-
4-m
eth
ylbe
nze
neb
1076
-56-
816
16–
––
0.01
(4)
––
––
––
––
–
Met
hyl
ben
zoat
ea93
-58-
316
461.
02(5
)0.
65(3
)0.
70(4
)–
tr(2
)–
––
––
––
–M
eth
ylsa
licy
late
a11
9-36
-818
08–
––
––
––
–tr
(1)
––
a-M
eth
yl-b
enzy
lal
coh
olb
98-8
5-1
1832
––
––
––
––
tr(5
)0.
30(5
)–
––
Ben
zyl
alco
hol
a10
0-51
-618
961.
12(4
)4.
51(6
)8.
40(4
)–
0.01
(1)
––
–tr
(2)
–tr
(4)
––
Ph
enol
a10
8-95
-220
322.
04(5
)1.
54(5
)1.
46(4
)–
0.11
(3)
0.03
(8)
0.43
(3)
–tr
(3)
–tr
(3)
0.16
(2)
0.01
(4)
p-A
nis
alde
hyd
ea12
3-11
-520
6129
.27
(5)
15.1
7(6
)2.
27(4
)0.
23(7
)0.
79(5
)0.
62(8
)tr
(2)
5.58
(4)
0.03
(6)
7.20
(5)
0.02
(8)
tr(2
)0.
09(4
)
TE
RP
EN
OID
SM
onot
erp
enes
a-P
inen
ea77
85-7
0-8
1095
tr(4
)–
–7.
35(8
)23
.48
(4)
14.0
7(8
)21
.11
(5)
––
0.01
(2)
0.03
(1)
tr(1
)–
b-T
hu
jen
ea28
634-
89-1
1102
––
––
–tr
(2)
––
––
–0.
22(1
)–
Cam
phen
ea79
–92-
511
12–
––
7.92
(8)
10.3
9(4
)8.
99(8
)6.
43(4
)–
––
––
–U
nkn
own
1154
––
–0.
29(7
)0.
09(3
)–
–0.
09(3
)–
––
––
b-P
inen
ea12
7-91
-311
9434
.05
(4)
0.01
(4)
10.0
6(2
)16
.55
(8)
2.09
(3)
5.60
(5)
7.01
(4)
2.98
(4)
0.15
(5)
–tr
(1)
tr(1
)–
b-M
yrce
nea
123-
35-3
1199
tr(4
)–
––
–0.
22(4
)tr
(4)
tr(3
)–
–0.
14(2
)1.
23(4
)–
Un
know
n12
13–
––
10.3
3(4
)2.
43(5
)–
–6.
50(4
)–
–0.
04(3
)–
–a-
Terp
inen
ea99
-86-
512
20–
––
24.9
6(8
)11
.16
(5)
12.2
5(7
)–
21.4
7(4
)–
–0.
09(1
)–
–L
imon
enea
138-
86-3
1224
––
7.25
(2)
–0.
51(5
)tr
(3)
tr(2
)–
––
–0.
59(1
)–
Eu
caly
ptol
a47
0-82
-612
312.
55(5
)11
.87
(5)
11.5
0(3
)13
.88
(8)
17.9
4(5
)31
.63
(8)
30.9
4(5
)1.
80(4
)–
1.27
(5)
0.76
(2)
0.02
(1)
0.04
(2)
tran
s-b-
Oci
men
ea50
2-99
-812
670.
83(2
)–
––
–tr
(1)
–1.
06(4
)–
–0.
02(1
)–
0.08
(2)
g-Te
rpin
enea
99-8
5-4
1269
––
–2.
66(8
)3.
17(5
)2.
37(6
)–
1.50
(4)
––
––
–ci
s-b-
Oci
men
ea33
38-5
5-4
1275
––
––
tr(1
)–
–0.
05(4
)–
0.02
(1)
tr(1
)–
p-C
ymen
ea99
-87-
612
941.
56(3
)4.
30(2
)12
.65
(3)
5.14
(8)
4.87
(5)
––
8.14
(4)
––
––
–a-
Terp
inol
enea
586-
62-9
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518 T. N. SUINYUY ET AL.
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527
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VARIATION IN CONE VOLATILE COMPOSITION 519
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527
GENERAL OVERVIEW OF PATTERN OF
VOLATILE EMISSIONS
The number of compounds emitted by cones variedmarkedly between populations, ranging from 16 inUNR to 45 in KKNR (Table 1) and also betweensexes. In female plants, as few as ten compoundswere emitted in the MTS population and up to 24compounds were emitted in KKNR. In male andfemale plants, the most commonly occurring com-pounds in sampled populations were p-anisaldehyde(all ten populations); benzaldehyde, eucalyptol andlinalool in nine populations; b-pinene and a-terpinene in eight populations; and phenol anda-pinene in seven and six populations, respectively.Fatty acid derivatives in the EC populations werecomposed of only six compounds compared to 19compounds in the KZN populations (Table 1). It is
noteworthy that all the nitrogen-containing com-pounds were pyrazine derivatives (Table 1).
CONE ODOUR VARIATION BETWEEN POPULATIONS
A Bray–Curtis NMDS analysis of cone odour com-pounds of E. villosus (Fig. 2) showed a significantseparation between the different populations (NMDSstress value 0.12; one-way ANOSIM, factor popula-tion: global R = 0.835, P < 0.01). Out of 45 pairwisecomparisons between the different populations,significant separation was found between 17 of them(Table 2). The clustering of populations based onchemical profiles (Fig. 2) tended to follow the overallgeographical pattern of separation of EC andKZN populations. The only exceptions were indivi-duals from VCNR in KZN in which volatiles were
Figure 2. Non-metric multidimensional scaling (NMDS) based on Bray–Curtis similarities of male cone odour com-position comprising 89 compounds from 73 samples of Encephalartos villosus from ten populations across the speciesrange in the Eastern Cape and KwaZulu Natal. [two-dimensional stress value: 0.12; one-way analysis of similarities,global R (population) = 0.835, P < 0.01]. FR, Forest Reserve; GF, Guest Farm; NR, Nature Reserve; PSJ, Port StJohns.
520 T. N. SUINYUY ET AL.
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characterized by heptanal, linalool, p-anisaldehyde,and benzaldehyde, and had closer affinities to popu-lations from the south of the range (OVGF and UNR;Fig. 2). Individuals from the UMNR population, situ-ated at the boundary between EC (south) and KZN(north), had volatile compounds characterized by(3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene thatwere typically dominant in KZN populations (OGNR,KKNR, and NFR), as well as a-terpinene and2-isopropyl-3-methoxypyrazine that occurred in ECpopulations. In the EC, populations in the extremesouth (UNR, OVGF) clustered together as a resultof the presence of eucalyptol, benzaldehyde, heptanal,b-pinene, linalool, and p-anisaldehyde. By contrast,other EC populations (DNR, MPD, and MTS)clustered together as a result of the prevalence of2-isopropyl-3-methoxypyrazine, a-pinene, camphene,and a-terpinene.
Mantel tests were performed to compare cone odourmatrices calculated using the Bray–Curtis similaritycoefficient (Clark and Warwick, 2001) with the matrixof geographical distance (km) between populations.The results showed a significant correlation (r = 0.39;P = 0.001) between changes in cone volatiles and geo-graphical distance across the full range of E. villosus.When the data were analyzed in subsets, changesin volatile composition were strongly correlatedwith distance between populations in EC (r = 0.78;P = 0.008) and might be indicative of restricted geneflow between the populations. This is observed in thechange in composition across populations from UNRto MTS (Fig. 1). There was no correlation betweenvolatile composition and geographical separation inKZN populations (r = 0.05; P = 0.51). This is indicatedby the lack of variation in the dominant compoundsacross the populations, especially at OGNR, KKNR,and NFR (Fig. 1).
INSECT POLLINATORS ON MALE AND FEMALE
CONES OF E. VILLOSUS
Pollen dehiscent cones of E. villosus in all the popu-lations were visited by an undescribed species ofPorthetes (Fig. 3C, Table 3). Oberprieler (1996) gavethe latter species a manuscript name of P. pearsoniibut the description has not been formally published.Two other beetle species (Coleoptera), namely anundescribed Erotylidae sp. nov. and Metacucujusgoodei Endrödy-Younga (Boganiidae) (Fig. 3A, B),were collected from all the populations except UMNRand NFR (Table 3). Their absence from UMNR andNFR may be a result of the small number of cones(four male cones) in each of these populations. Por-thetes sp. occurred in between the sporophylls, alongthe cone axis, and on the cone surface, whereas Ero-tylidae sp. nov. and M. goodei were moving inbetween the sporophylls and along the cone axis.Female cones of E. villosus in the three populationssampled were visited by Erotylidae sp. nov. andPorthetes sp. (the same taxa as on male cones)(Table 3) and Antliarhinus zamiae (Thunberg)(Fig. 3D, E, Table 3). Female A. zamiae were observedpiercing the cone sporophylls with their rostrums andcrawling over the cone surface. Similarly, maleA. zamiae were seen crawling on the cone surface,with some forcing their way in between the tightmegasporophylls. In some cases, male and femaleA. zamiae were observed mating on the cone surface(Fig. 3F). A few Erotylidae sp. nov. and Porthetes sp.were actively crawling on the female cone surface,whereas some were forcing their way in betweenthe tightly packed megasporophylls. Overall, themean ± SE number of Porthetes sp. individuals perpopulation (833.8 ± 115.) was significantly greaterthan that for Erotylidae sp. nov. (294.0 ± 58.9),
Table 2. Test statistics (R) of pairwise comparisons (one-way analysis of similarities) between populations of Enceph-alartos villosus
UNRa OVGF DNR MPD MTS UMNR OGNR VCNR KKNR NFR
UNROVGF 0.41DNR 1.00 0.98b**MPD 0.97 0.92* 0.41MTS 1.00** 0.97** 0.42 0.13UMNR 1.00 0.96 1.00 0.88 0.95*OGNR 1.00 1.00** 1.00* 1.00 1.00* 1.00VCNR 0.90 0.76* 1.00 1.00 1.00** 1.00 1.00KKNR 1.00** 0.98** 1.00** 1.00** 1.00** 0.70 0.44 1.00**NFR 1.00 1.00 1.00 1.00 1.00* 1.00 1.00 1.00 -0.08
aNames of populations are the same as given in Table 1. bBold values indicate populations that are significantly different.**P < 0.01; *P < 0.05.
VARIATION IN CONE VOLATILE COMPOSITION 521
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527
M. goodei (171.2 ± 32.3), and A. zamiae (43.8 ± 9.7)(analysis of variance: F3,16 = 26.8, P < 0.01). This trendwas evident in all of the study populations (Table 3).
DISCUSSION
The present study provides a detailed investigationof geographical variation in volatile composition forE. villosus and is the first detailed geographicalanalysis of volatile composition in any cycad species.The results obtained show that there is considerablevariation in the chemical composition of cone volatileemissions between populations of E. villosus includ-ing a shift from dominance of monoterpenes in thesouthern part of the range (e.g. DNR, MPD, and MTSaround Port St Johns) to dominance of unsaturatedhydrocarbons in the northern part of the range (e.g.
KKNR, NFR). The UMNR population, situated in thecentre of the range, appears to be the transition point(Fig. 1). The results further show that despite thevariation in volatile composition, the same insectspecies, namely A. zamiae, Erotylidae sp. nov.,M. goodei, and Porthetes sp., are common across allthe sampled E. villosus populations.
Studies of geographical variation in plant traitshave shown several outcomes, including lack of struc-tured variation across the range (Svensson et al.,2005), clinal variation (Knudsen, 2002), and discreteor saltational variation reflecting adaptation to differ-ent pollinators (Schlumpberger & Raguso, 2008). TheMantel test for E. villosus data provides statisticalevidence for volatile profiles being associated withgeographical separation, whereas the cluster analysisindicates that this variation is more consistent with
Figure 3. Insects that visit male and/or female cones of Encephalartos villosus. A, an undescribed Erotylidae sp. nov.(male and female cones). B, Metacucujus goodei (male cones). C, Porthetes sp (male and female cones). D, femaleAntliarhinus zamiae (female cones). E, male Antliarhinus zamiae (female cones). F, male and female Antliarhinus zamiaemating. Scale bars = 1000 mm.
522 T. N. SUINYUY ET AL.
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 514–527
discrete or saltational changes than with clinalchanges across the range. Further studies arerequired to explore genetic structure across the dif-ferent populations of E. villosus that may be linked todifferences in cone volatiles.
CHEMICAL COMPOSITION OF CONE VOLATILES
Many of the volatile compounds and compoundclasses identified in this study are known to occurin other cycads (Pellmyr et al., 1991; Terry et al.,2004a, b; Azuma & Kono, 2006, Proches & Johnson,2009; Suinyuy, Donaldson & Johnson, 2010) and otherplants (Knudsen et al., 2006). Out of 87 identifiedcompounds, only five compounds occurred in highrelative amounts (� 30%) in at least one population(Table 1). The majority of the compounds wereemitted in small relative amounts ranging from traceamounts to just above 20%. Schlumpberger & Raguso(2008) suggest that compounds that occur in smallrelative amounts should not be ignored because theycan serve critical functions in plant–pollinator rela-tions. Terpenoids, particularly the monoterpenes, arethe most numerous compounds in the volatile blend ofE. villosus and some of them together with somebenzenoids occur in almost all the populations(Table 1). Their occurrence in almost all populationssuggests that they could be critical compounds thatserve different functions and require further investi-gation. Sampling pollen dehiscent and receptive conesat different times of the day will establish whetherthese compounds are emitted in different concentra-tions that can affect insect behaviour in a similar
manner to b-myrcene in some Macrozamia cycads(Terry et al., 2004a, 2007a, b). There were relativelyfew unsaturated hydrocarbons but they included (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene, the mostabundant compounds emitted by plants from KZNpopulations. These compounds have been recorded inthe volatile profile of few plants and have been iden-tified as possible insect attractants (Skubatz et al.,1996; T. N. Suinyuy, unpubl. data). Four pyrazinecompounds occurred in varying amounts, mostly inplants from EC populations, with 2-isopropyl-3-methoxypyrazine as a dominant compound. Generallypyrazines have distinct sensory properties and havebeen associated with warning signals, alerting signalsto predators, aggregation pheromones of insects, ovi-position stimulants (Rothschild, Moore & Brown,1984; Abassi et al., 1998), and insect attractants(Ervik, Tollsten & Knudsen, 1999). The differentpyrazines could therefore fulfill different functions incycads.
GEOGRAPHICAL VARIATION OF E. VILLOSUS
CONE VOLATILES
Although E. villosus exhibited geographical variationin cone volatile emissions, the present study showedthat eight of the 88 compounds occurred in almost allthe populations (Table 1) and may be of critical impor-tance in influencing insect behaviour because thesame insect assemblages occurred in all populations(Table 3). It is noteworthy that the pattern ofgeographical variation in E. villosus cone odour is
Table 3. Mean ± SE number of insects of different species collected from male and female cones of Encephalartos villosusin different populations across the distribution range in the Eastern Cape and KwaZulu Natal
Populationa Cone sexNumberof cones
Insect species and number of individuals
Antliarhinuszamiae
Erotylidaesp. nov.
Metacucujusgoodei Porthetes sp.
UNR Male 5 – 1.4 ± 0.5 1.8 ± 0.7 7.0 ± 0.9OVGF Male 5 – 2.6 ± 1.5 2.0 ± 0.9 15.8 ± 3.6
Female 5 15.5 ± 3.0 2.8 ± 1.2 – 1.4 ± 0.6DNR Male 4 – 86.0 ± 23.0 60.8 ± 11.3 168.0 ± 36.3MPD Male 4 – 101.0 ± 16.8 47.8 ± 9.6 191.8 ± 9.1MTS Male 5 – 11.0 ± 3.4 7.4 ± 2.3 37.6 ± 9.7
Female 3 18.0 ± 5.3 1.0 ± 0.6 – 3.0 ± 1.0UMNR Male 4 – – – 22.5 ± 4.3OGNR Male 5 – 43.4 ± 4.3 31.2 ± 3.1 93.4 ± 3.5VCNR Male 4 – 64.5 ± 4.8 31.7 ± 3.4 186.3 ± 15.5KKNR Male 5 – 34.4 ± 7.2 16.6 ± 4.3 213.2 ± 21.1
Female 4 17.8 ± 2.0 2.5 ± 0.6 – 11.0 ± 1.8NFR Male 4 – – – 65.5 ± 6.6
aNames of populations are the same as given in Table 1.
VARIATION IN CONE VOLATILE COMPOSITION 523
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explained best by changes in two compounds: 2-isopropyl-3-methoxypyrazine and (3E)-1,3-octadiene.The nitrogen-containing compound 2-isopropyl-3-methoxypyrazine occurs in all southern populationsfrom UNR to UMNR and increases in relativeamounts from UNR to MTS. By contrast (3E)-1,3-octadiene occurs in all northern populations startingfrom UMNR and tends to increase along the northerngradient to NFR (Fig. 1).
Samples from UMNR in the centre of the distribu-tion range were characterized by compounds fromboth the southern and northern populations. TheUMNR is situated in the eastern part of the Umtam-vuna river gorge and occurs within the Maputaland-Pondoland centre of endemism. Pondoland isdominated by ancient outcrops of nutrient-poorquartzite sandstone that appear to have acted asedaphic barriers to plant migration (Carbutt &Edwards, 2001) and support a resident flora that isapparently trapped by these barriers. It is not clearexactly why the UMNR population of E. villosus con-tains odour compounds that otherwise occur sepa-rately in populations to the north and south, althoughthe sharp transition in cone volatile composition inE. villosus across this region (Fig. 1) supports the ideaof a biogeographical barrier in the Umtamvuna areaas suggested by Carbutt & Edwards (2001).
The VCNR population, sandwiched between OGNRand KKNR, has a suite of volatile compounds similarto that of plants from the KKNR but present indifferent relative quantities (Fig. 1, Table 1). Thedominant compounds are monoterpenes, aldehydes,and benzenoids, and these are closest to those emittedby plants from the EC region (Fig. 1, Table 1). Thissuggests that these compounds occurred more widelyacross the range but that only plants from the VCNRpopulation have retained and expressed the genesfor biosynthesis of all the volatile compounds oncepresent in the different populations. Long distancedispersal of seeds from the southern part of the rangeis highly unlikely to account for the volatile compo-nents of the VCNR population. There is no knownlong distance dispersal mechanism and cycad dis-persal is typically within a short distance of theparent plant (Snow & Walter, 2007). Long rangepollen dispersal (> 5 km) also appears to be unlikelybecause Donaldson (1997) discovered that Porthetessp., Erotylidae sp. nov., M. goodei, and A. zamiae losta substantial amount of pollen within a few hoursafter they left the pollen shedding cones of E. villosus,and also that plants situated > 5 km from sourcepopulations never had insect pollinators present (J. S.Donaldson, unpubl. data).
Intraspecific variation in plant morphology iswell documented, although an increasing numberof studies are revealing similar variation in che-
mical traits (Dobson et al., 1997; Azuma, Toyota &Asakawa, 2001; Dötterl, Wolfe & Jürgens, 2005;Chess, Raguso & LeBuhn, 2008; Jhumur, Dötterl &Jürgens, 2008; Schlumpberger & Raguso, 2008).Hypotheses for intraspecific trait variation includephenotypic plasticity, neutral processes such as drift,adaptive processes such as co-evolution or pollinatorshifts, and local hybridization. The evidence for eachof these hypotheses is weighed up in relation tothe geographical variation in the cone odour ofE. villosus.
Phenotypic plasticity is highly unlikely to accountfor the geographical odour patterns observed in E.villosus because plants from populations in EC thathave been growing under different environmentalconditions in the Kirstenbosch Botanic Garden inCape Town for close to 100 years emitted the samevolatile compounds as those from the natural popu-lations (T. N. Suinyuy, unpubl. data).
Drift also appears unlikely to account for variationin the major compounds as these compounds havebeen shown to play functional roles in pollinatorattraction (T. N. Suinyuy, unpubl. data). However,neutral and adaptive processes could apply to differ-ent compounds. For example, geographical variationin the volatile profile may occur only in compoundsthat are not used by pollinators to find and locate hostplants (Dötterl et al., 2005; Füssel, Dötterl & Jürgens,2007). Pollinator shifts have been invoked for caseswhere there are different pollinators in differentgeographical areas and these can result in quantita-tive shifts involving changes in assemblages forplant species which are visited by a number of differ-ent pollinators (Pellmyr, 1986b; Schlumpberger &Raguso, 2008), or involve complete transitions(Johnson & Steiner, 1997). However, variation infloral traits can also occur without pollinator shifts(Ellis & Johnson, 2009). This was evidently the casefor E. villosus, which showed no change in beetlespecies composition across the distribution range. Thesame insect visitors were recorded from all E. villosuspopulations (i.e. A. zamiae, Erotylidae sp. nov.,M. goodei, and Porthetes sp.) despite the difference involatile compounds. A recent molecular study of phy-logenetic relationships within Porthetes concludedthat the specimens from different E. villosus popula-tions across the range of distribution, as well as fromthe related cycads Encephalartos aplanatus andEncephalartos umbeluziensis (Treutlein, Vorster &Wink, 2005), comprised a single species (Downie,Donaldson & Oberprieler, 2008). This suggests thatchanges in cone volatiles are not associated withdifferent pollinator assemblages. However, it is stillpossible that there has been localized co-evolutionbetween these insects and E. villosus, which is notreflected in molecular markers or morphology of the
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beetles. Performing scent bioassays at different siteswill aid in understanding whether beetles, which areostensibly the same species, exhibit regional differ-ences in volatile preferences that could accountfor the geographical variation in the cone odours ofE. villosus.
It is noteworthy that the barrier between northernand southern populations of E. villosus at theUmtamvuna river is also the contact zone for twoother cycad species that are closely related to oneanother (Treutlein et al., 2005): Encephalartos natal-ensis north of the Umtamvuna and Encephalartosaltensteinii to the south. The odour compound (3E)-1,3-octadiene, which dominated the odour profile ofE. villosus north of the Umtamvuna, is also the domi-nant compound in cone volatiles of E. natalensis(Suinyuy et al., 2010). In addition, at least one of thepollinators of E. villosus also occurs on E. natalensis(Donaldson, 1997; T. N. Suinyuy, unpubl. data).Hybridization between E. villosus and other Enceph-alartos species such as E. natalensis could account forsome of the geographical variation in the scent ofE. villosus. If there are no other barriers and back-crosses occur, gene flow across species boundaries ispossible. Stökl et al. (2008) showed that overlap offlowering times in Ophrys iricolor and Ophrys luper-calis, which emit the same compounds and attract thesame insects results in extensive hybridization andintrogression. Although natural hybrids betweenE. villosus and E. natalensis have not been recorded,hybrids have been found to occur between E. villosusand Encephalartos senticosus (previously consideredpart of Encephalartos lebomboensis) (Dyer, 1965;Vorster, 1986), which also emits (3E)-1,3-octadiene(T. N. Suinyuy, unpubl. data). Hybrids have also beenfound in the south of the range between E. villosusand E. altensteinii in the EC where they occur insympatry (Dyer, 1965; Vorster, 1986). Although E. vil-losus and E. altensteinii have different major com-pounds, they emit similar minor volatile compoundsthat could be involved in pollinator attraction. Thegeographical variation in volatile compounds in E. vil-losus could therefore be affected by introgressionof odours traits through hybridization with otherEncephalartos species. Alternatively, the dominanceof (3E)-1,3-octadiene among cycads in KZN may rep-resent convergent evolution resulting from adaptationto a similar local suite of insects. Further investiga-tion of volatile compounds and pollinators in otherEncephalartos species is required to determine howthey vary in relation to E. villosus across a widegeographical area.
In conclusion, the widespread cycad E. villosus con-sists of a number of geographically structured chemo-types. The discontinuities between these chemotypesare not sufficiently pronounced to justify recognition
of distinct taxa, nor does it appear from preliminaryinvestigations that these chemotypes are associatedwith different pollinators. However, the patternssuggest ongoing evolutionary diversification in E. vil-losus, which makes this species suitable for furthermicroevolutionary studies on the role that insect pol-linators played in the evolution of Encephalartos.
ACKNOWLEDGEMENTS
We thank Jacques De Wet Bösenberg for his dedi-cated assistance in the field. We greatly appreciatethe assistance of Andreas Jürgens in the volatileanalysis and Sandy-Lynn Steenhuisen for technicalsupport in the laboratory. Special thanks go toFerozah Conrad for reading through the manuscriptand John Measey for helping with the data analysis.We also thank the two anonymous reviewers for theirhelpful comments on the manuscript. We are gratefulto Ian Smith and Johanne Van Ryneveld who grantedus access to their farm and to The Eastern CapeParks Board and Ezemvelo KwaZulu Natal Wildlifefor issuing permits to sample from plant populationsin nature reserves. The present study was financiallysupported by The Fairchild Tropical Botanic Garden,South African National Biodiversity Institute, theAndrew W. Mellon Foundation, University of CapeTown and the University of KwaZulu Natal.
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