Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue7.Aug2000:
Biochemical Systematics and Ecology 28 (2000) 619}632
Phenolic glycosides and condensed tannins in
Salix sericea, S. eriocephala and their F1 hybrids:
not all hybrids are created equal
Colin M. Orians!,*, Megan E. Gri$ths!, Bernadette M. Roche",1,
Robert S. Fritz"
!Department of Biology, Tufts University, Medford, MA 02155, USA
"Department of Biology, Vassar College, Poughkeepsie, NY 12601, USA
Received 26 May 1999; accepted 26 July 1999
Abstract
The performance of hybrids depends upon the inheritance and expression of resistance traits.
Secondary chemicals are one such resistance trait. In this study, we measured the concentrations of phenolic glycosides and condensed tannins in parental and F1 hybrid willows to
examine the sources of chemical variation among hybrids. S. sericea produces phenolic
glycosides, salicortin and 2@-cinnamoylsalicortin, and low concentrations of condensed tannin
in its leaves. In contrast, S. eriocephala produces no phenolic glycosides but high concentrations
of condensed tannins in its leaves. These traits are inherited quantitatively in hybrids. On
average, F1 hybrids are intermediate for condensed tannins, suggesting predominantly additive
inheritance or balanced ambidirectional dominance of this defensive chemical from the parental
species. In contrast, the concentration of phenolic glycosides is lower than the parental
midpoint, indicating directional dominance. However, there is extensive variation among F1
hybrids. The concentration of tannin and phenolic glycosides in F1 hybrid families is either (1)
lower than the midpoint, (2) higher than the midpoint, or (3) indistinguishable from the
midpoint of the two parental taxa. It appears that the production of the phenolic glycosides,
especially 2@-cinnamoylsalicortin, is controlled by one or more recessive alleles. We also
observed a two-fold or greater di!erence in concentration between some hybrid families. We
discuss how chemical variation may e!ect the relative susceptibility of hybrid willows to
herbivores. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Salicaceae; Willows; Hybridization; F1 hybrids; Phenolic glycosides; Condensed tannins
* Corresponding author. Tel.: #1-617-627-3543; fax: #1-617-627-3805.
E-mail address: [email protected] (C.M. Orians)
1 Current address: Department of Biology, Loyola College, Baltimore, MD 21210, USA.
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 0 1 - 5
620
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
1. Introduction
Hybridization is now regarded as a common feature of many plant species, and can
lead to speciation and introgression (Rieseberg, 1991, 1995; Rieseberg and Brunsfeld,
1992; Rieseberg and Ellstrand, 1993; Ellstrand et al., 1996; Arnold, 1997). In the last
10 yr, there have been numerous studies suggesting that hybridization, via changes in
resistance, also a!ects the ecology and evolution of plant-herbivore interactions (e.g.,
Whitham, 1989; Boecklen and Spellenberg, 1990; Whitham et al., 1991, 1994, 1999;
Aguilar and Boecklen, 1992; Floate and Whitham, 1993; Paige and Capman, 1993;
Fritz et al., 1994, 1996; Strauss, 1994; Messina et al., 1996; Orians et al., 1997; Fritz,
1999; Pilson, 1999). We know that the relative resistance of hybrids and parental taxa
to herbivores is highly variable (reviewed by Strauss, 1994; Fritz, 1999), but what
determines resistance is not well understood. Although multiple traits are involved,
secondary chemicals, such as phenolics, terpenoids, alkaloids and glucosinolates, are
often key components of the resistance of plants to herbivores (Rosenthal and Janzen,
1979; Roitberg and Isman, 1992; Bernays and Chapman, 1994; CardeH and Bell, 1995;
Chew and Renwick, 1995). Given the general importance of secondary chemistry to
plant}herbivore interactions, it is surprising that relatively few researchers have
studied the secondary chemistry of hybrids in relation to plant}herbivore interactions
(Huesing et al., 1989; Weber et al., 1994; Orians et al., 1997). We believe that
knowledge of how secondary chemicals are expressed in hybrids will help researchers
understand patterns of hybrid resistance.
The concentration of secondary chemicals in hybrids typically di!ers qualitatively
and quantitatively from their parents (Connor and Purdie, 1976; Harborne and
Turner, 1984; Meier et al., 1989; Altman et al., 1990; Levy and Milo, 1991; Rieseberg
and Ellstrand, 1993; Orians and Fritz, 1995). Qualitatively, novel chemicals may be
found or parental chemicals may be lost. Quantitatively, patterns of expression
depend upon the genetic control of production. Parental chemicals may be overexpressed (over-dominance), underexpressed (incomplete dominance), intermediate (no
dominance), similar to one of the parental species (dominance), or similar to
both parents (co-dominance) (Van Brederode et al. 1974; Falconer, 1989). Quantitative chemical di!erences can a!ect the resistance of a plant (Huesing et al., 1989;
Orians et al., 1997). What is lacking is an understanding of how secondary
chemistry varies among hybrids in ways that could a!ect patterns of resistance to
herbivores.
We have been studying the consequences of hybridization in willow for a number of
years. Willows and other Salicaceous plant species produce two main secondary
chemicals: phenolic glycosides and condensed tannins, both of which are known to
in#uence the susceptibility of plants to insect and mammalian herbivores (Zucker,
1983; Rowell-Rahier, 1984; Tahvanainen et al., 1985a,b; Basey et al., 1988; Lindroth
and Peterson, 1988; Lindroth et al., 1988; Clausen et al., 1989; Schultz, 1989; Denno
et al., 1990; Kolehmainen et al., 1994; Orians et al., 1997). Some willow species
produce only condensed tannins, others produce mostly phenolic glycosides, and
others produce both classes of compounds (Julkunen-Tiitto, 1986, 1989; Orians and
Fritz, 1995).
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
621
In our system, S. sericea produces high concentrations of phenolic glycosides and
low concentrations of condensed tannin, while S. eriocephala produces high concentrations of condensed tannin but no phenolic glycosides (Orians and Fritz, 1995). The
concentrations of both phenolic glycosides and condensed tannins are intermediate,
not signi"cantly di!erent than the midpoint, in naturally occurring hybrids of these
two species (Orians and Fritz, 1995). However, these hybrids were of unknown
pedigree. What is the chemistry of known F1 hybrids? Is there chemical variation
among F1 hybrids? How does the chemistry of experimental hybrids di!er from
natural hybrids? These are all questions that will help us understand how secondary
chemistry might determine patterns of hybrid survival. Here we report on the
concentrations of condensed tannin and phenolic glycosides in two year old seedlings
of Salix sericea, Salix eriocephala and their F1 hybrids.
2. Materials and methods
2.1. Plants
Both Salix sericea Marshall and Salix eriocephala Michx. are abundant in the
northeastern United States and eastern Canada (Argus, 1986). The species often
co-occur and hybridization between the two is common (Argus, 1986; Mosseler and
Papadopol, 1989; Fritz et al., 1994). Willows are dioecious and in this system the
initial hybridization event is unidirectional (S. eriocephala is the paternal parent and
S. sericea is the maternal parent), due to stigma-pollen incompatibility in female S.
eriocephala (Mosseler, 1990).
2.2. Generation of seedlings (parental and F1 hybrid)
We performed intraspeci"c and interspeci"c crosses using known pure parents,
based on RAPD analysis, to generate pure parental and F1 hybrid progenies. From
a larger set of crosses (Fritz et al., 1998), we selected seven groups of progenies where
the parents of each F1 hybrid cross were used in the matched intraspeci"c cross (Table
1). Our goal was to avoid using a parental clone twice. However, one S. sericea parent
(s17) was involved in two crosses.
To ensure purity of each family, the female catkins were covered with mesh bags
prior to stigma emergence to prevent visitation by insect pollinators. Bags were
removed only while making crosses. The resulting seeds were mass planted in trays of
MetroMix 360TM in late May, and, covered with white, shear seed cloth to maintain
a high moisture level and to prevent accidental colonization of the trays by naturally
dispersing willow seeds. The cloth was removed after about two weeks, when natural
seed dispersal had ended. Four-week-old seedlings were transplanted into 3.7 l pots
"lled with our standard soil mixture (4 parts topsoil, 1 part peat moss, and 1 part
vermiculite). Due to mortality, the number of siblings per cross varied (Table 1). The
next spring the seedlings were transplanted into 7.4 l pots containing the same soil
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C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
Table 1
Crosses performed to generate related parental and F1 hybrid progeny
Group
S. sericea
$]#
F1 Hybrids
$]#
S. eriocephala
$]#
I
II
III
IV
V
VI
VII
s17]s22 (n"5)
s17]s16 (n"5)
s28]s27 (n"7)
s37]s55 (n"3)
s83]s14 (n"4)
s1]s95 (n"5)
s78]S60 (n"5)
s17]e18 (n"4)
s17]ne36 (n"5)
s28]ne35 (n"2)
s37]ne33 (n"9)
s83]ne37 (n"5)
s1]hhe46 (n"4)
s78]hhe12 (n"4)
e15]e18 (n"4)
e23]ne36 (n"4)
e23]ne35 (n"4)
e29]e115 (n"5)
e3]ne37 (n"5)
e64]hhe46 (n"4)
e60]hhe12 (n"4)
Note: F1 hybrids within each group have a female in common with the Salix sericea progeny and male in
common with the S. eriocephala progeny.
mixture mixed with 13 g of slow-release fertilizer (10 : 10 : 10 NPK). Plants were
randomized within blocks.
Leaf samples for chemical analyses were collected on 18 July. Twelve fully expanded
leaves were collected from each plant (the "rst two fully expanded leaves from each of
six shoots). Leaves were cut at the petiole, placed on ice, transported to the lab, and
vacuum-dried for at least 24 h. Vacuum-drying leaves has been shown to prevent the
loss of both condensed tannin and phenolic glycosides (Orians, 1995). Once dry, leaves
were ground to a "ne powder using a Wiley Mill equipped with a size 30 mesh and
then stored in a !203C freezer.
2.3. Chemical analyses
The phenolic glycosides were assayed using standard techniques (Orians, 1995).
Brie#y, leaf powder (30$3 mg) was extracted in cold MeOH (10 mg leaf powder/1 ml
MeOH) with sonication for 10 min. Cold water was constantly #ushed through the
sonicator to prevent the heating of samples. We centrifuged and "ltered (0.45 lm
"lter) each sample before placing extracts in crimp-top vials. Extracts were kept in the
freezer until analysis (48 h or less). We quanti"ed the concentration of glycosides with
an HPLC equipped with an autosampler and a UV detector set at 274 nm (HewlettPackard). A reverse-phase NOVA-PAK C (4 lm) column (Waters) and a gradient
18
system of distilled water and MeOH was used. 1,3-dimethoxybenzene was used as the
internal standard. Standard curves were determined for salicortin and 2@-cinnamoylsalicortin.
The condensed tannins also were extracted using standard techniques (Orians,
1995). Brie#y, approximately 300 mg leaf powder was washed with ether and extracted
with 70% acetone for 3 h in a 403C water bath. Acetone was removed under reduced
pressure and all extracts were diluted to 8 ml with distilled water. The butanol/HCl
method was used to quantify tannin concentrations (Hagerman and Butler, 1989). The
concentration of the condensed tannin (mg/g dry leaf) was determined using puri"ed
tannins collected from the two willow species and the hybrids.
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
623
2.4. Statistical analyses
A one way analysis of variance was used to determine the overall e!ects of taxon on
condensed tannins (using the means of each of the 7 families/taxon). The natural log of
condensed tannin concentration was used in the analysis because variance increased
with the mean. For the phenolic glycosides only S. sericea and hybrids were included
in the model, because S. eriocephala generally does not contain phenolic glycosides.
Contrasts were performed to determine if the hybrids were signi"cantly di!erent
from the midpoint of the two parental taxa (non-transformed data were used for the
contrasts), that is, if one averages the parental values do the hybrids di!er from that
average. This was done using the means comparison contrasts in SuperANOVA
(Abacus Concepts, 1989). These comparisons were performed with all taxa included in
the model.
Separate analyses were done for each group of families (Table 1). As above, an
analysis of variance was used to determine the e!ects of family (or taxon) on
chemistry, and contrasts, as described above, were performed to determine if the
concentrations of the chemicals in the hybrid family was signi"cantly di!erent from
the average of the two parental families.
Regression analysis was used to determine the relationship between parental and
hybrid chemistry, and between the production of phenolic glycosides and condensed
tannins.
3. Results
In general, the concentrations of condensed tannins is high in S. eriocephala, low in
S. sericea and intermediate in hybrids, while the concentration of the two phenolic
glycosides shows the opposite pattern (Table 2). The concentrations measured in these
plants are similar to that measured in "eld plants (unpublished data), and the patterns
are similar to those reported previously (Orians and Fritz, 1995). In general, S.
eriocephala does not produce phenolic glycosides in its leaves (see also Orians and
Fritz, 1995). However, two of the four siblings of one S. eriocephala cross (e60]hhe12)
produced phenolic glycosides (data not shown in Table 2), and the four s78]hhe12
siblings had levels of phenolic glycosides and tannins similar to S. sericea. Although
these results suggest there is something unusual about hhe12, other crosses involving
hhe12 (e64]hhe12 and s57]hhe12) were not unusual (unpublished data). Currently,
we are unable to explain the anomalous results obtained with the hhe12 crosses.
Because of these anomalous results, we excluded crosses involving hhe12 before
testing for taxon e!ects. Overall, there was a signi"cant e!ect of taxon on chemistry
and all three taxa were signi"cantly di!erent from one another (ANOVA, F*125.6,
p)0.0001) (Table 2). Contrasts revealed signi"cant deviation from the average (or
midpoint) of the two parental taxa for salicortin (p(0.01) and 2@-cinnamoylsalicortin
(p(0.001), but not for condensed tannins (p"0.99). For both phenolic glycosides,
the concentration was lower than the midpoint, indicating a small dominance deviation toward reduced expression.
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C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
Table 2
Mean concentration (MG/G dry leaf weight), standard errors (in parentheses), and coe$cients of variation
(cv) for condensed tannin and the phenolic glycosides in S. sericea, F1 hybrids and S. eriocephala leaves
Taxon
S. sericea
F1 hybrid
S. eriocephala
Chemical type
Condensed tannin
Salicortin
2@-cinnslctn
27.8 (1.8) a
cv"7.6
71.7 (11.9) b
cv"19.7
126.6 (4.2) c
cv"10.6
104.8 (4.1) a
cv"10.2
48.0 (10.7) b
cv"33.5
None
18.5 (1.4) a
cv"20.0
4.5 (2.4) b
cv"44.1
None
Note: 2@-cinnslctn "2@-cinnamoylsalicortin. Di!erent letters denote signi"cant di!erences among taxa
(p(0.001).
In addition, there was more among-family variation for hybrids than for the
parental taxa (see Coe$cients of Variation in Table 2). This was especially evident for
the phenolic glycosides. Some hybrid families produced very low concentrations of
salicortin and 2@-cinnamoylsalicortin (26.3 and 0.6 mg/g dry leaf weight, respectively
for Group IV) while other families produced relatively high concentrations (60.6 and
2.1 mg/g dry leaf weight, respectively for Group VI). Thus some families produce over
twice the concentration of other families. If we include the s78]hhe12 cross salicortin
and 2@-cinnamoylsalicortin concentrations were even higher 106.7 and 18.4 mg/g dry
leaf weight, respectively.
For each group of crosses, there was a highly signi"cant di!erence among families
(or taxa) (F*19.5; p(0.001) (Table 3). Based on our previous results, we expected
that F1 chemistry would be indistinguishable from the midpoint of the parental taxa.
However, contrasts revealed that some F1 hybrids were lower than the midpoint,
while others were higher than the midpoint (Fig. 1). Excluding the cross involving
hhe12 (Group VII), condensed tannin concentration was signi"cantly di!erent than
the parental midpoint for 2 of 6 hybrid families, one higher and one lower. Salicortin
concentration was signi"cantly lower than the midpoint for 3 of 6 families. In contrast,
2@-cinnamoylsalicortin concentrations were signi"cantly lower for all 6 hybrid families. For Group VII, condensed tannin was lower and the phenolic glycosides were
higher than the parental midpoint (Fig. 1). Generally, the production of phenolic
glycosides, especially 2@-cinnamoylsalicortin, showed directional dominance or recessive deviations.
There were signi"cant positive correlations between parental and hybrid chemistry.
The concentration of condensed tannins in the hybrids was positively correlated with
the concentration in the S. sericea parent (R2"0.79, p"0.02), but not with the
S. eriocephala parent (R2"0.08, p"0.59). There was a positive correlation for
2@-cinnamoylsalicortin (R2"0.64, p"0.05). However, there was no correlation for
salicortin (R2"0.04, p"0.33).
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
625
Table 3
Family mean (standard error) concentration, MG/G dry leaf weight, for condensed tannin and phenolic
glycosides in S. sericea (SS), F1 hybrids and S. eriocephala (SE)
Group
Family
Tannin
Salicortin
2@cinnslct
I
SS
F1
SE
s17]s22
s17]e18
e15]e18
22.8(3.3) a
66.8 (10.3) b
122.4(4.7) c
110.8 (7.2) a
43.9 (6.7) b
NONE
18.8 (2.4) a
2.2 (0.5) b
NONE
II
SS
F1
SE
s17]s16
s17]ne36
e23]ne36
20.8(0.6) a
48.6 (10.1) b
106.4(7.0) c
121.2 (4.4) a
39.3 (8.1) b
NONE
13.4 (1.0) a
1.8 (0.5) b
NONE
III
SS
F1
SE
s28]s27
s28]ne35
e23]ne35
24.7(2.2) a
87.4(7.1) b
113.0(3.0) c
98.7 (6.2) a
31.0 (9.5) b
NONE
17.9 (1.9) a
2.4 (1.0) b
NONE
IV
SS
F1
SE
s37]s55
s37]ne33
e29]e115
25.3(4.6) a
86.1(8.0) b
122.4(5.5) c
97.4 (6.3) a
26.3 (3.9) b
NONE
14.2 (1.0) a
1.1 (0.2) b
NONE
V
SS
F1
SE
s83]s14
s83]ne37
e3]ne37
24.7(3.5) a
70.5 (10.8) b
117.5(3.3) c
88.9 (8.9) a
28.6 (4.5) b
NONE
19.9 (0.9) a
1.7 (0.5) b
NONE
VI
SS
F1
SE
s1]s95
s1]hhe46
e64]hhe46
22.6(1.6) a
72.8(4.4) b
143.9(4.8) c
105.4 (7.6) a
60.6 (6.6) b
NONE
23.3 (2.5) a
3.9 (1.0) b
NONE
VII
SS
F1
SE
s78]S60
s78]hhe12
e60]hhe12
21.4(0.9) a
15.8(1.1) a
116.1 (13.5) b
111.2(7.9) a
106.7(4.2) a
24.9 (17.3) b
22.0 (2.1) a
18.5 (1.1) a
0.2 (0.2) b
Note: 2@cinnslct "2@-cinnamoylsalicortin. Di!erent letters denote signi"cant di!erences among families
within each group (p(0.001).
Among hybrids we found an inverse correlation between the concentration of
tannin and salicortin (Fig. 2) and between tannin and 2@-cinnamoylsalicortin
(>"78.5!3.3X, R2"0.41, p(0.001). These negative correlations suggest that
hybrids may not be able to produce high concentrations of both phenolic glycosides
and condensed tannins (see also Orians and Fritz, 1995). Interestingly, many F1
hybrids contained low concentrations of both condensed tannin and salicortin
(Fig. 2).
4. Discussion
Most secondary chemicals are under polygenic control and inherited in a quantitative fashion (Berenbaum and Zangerl, 1992), as we have found for both salicortin and
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C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
Fig. 1. The proportional deviation from the parental midpoint. (A) Values above the midpoint indicate that
condensed tannin concentrations deviate toward S. eriocephala. (B) and (C) Values above the midpoint
indicate salicortin and 2@-cinnamoylsalicortin concentrations deviate toward S. sericea. NS: not signi"cant
(*) p(0.05; (**) p(0.01.
2@-cinnamoylsalicortin (Orians et al., 1996). As a consequence most parental chemicals
are found in hybrids (Table 2; Rieseberg and Ellstrand, 1993). However, the concentration in the hybrids can be quite variable (Harborne and Turner, 1984). Our results
show that on average F1 hybrids contain intermediate concentrations of condensed
tannin (equal to the parental midpoint), suggesting predominantly additive inheritance or a lack of dominance of this defensive chemical from the parental species. In
contrast, the concentration of the phenolic glycosides, salicortin and 2@-cinnamoylsalicortin, is lower than the parental midpoint, indicating incomplete dominance over
production. This incomplete dominance suggests that one or more alleles that control
phenolic glycoside production, especially 2@-cinnamoylsalicortin, are recessive in the
F1 hybrid. Altman et al. (1990) also found recessive control of the production of the
terpenoid raimondal in cotton hybrids; compared to the parental taxon, the concentration in hybrids was 9}12%.
There have been a number of studies evaluating the genetics of secondary chemical
production by F1 hybrids of contrasting species. These studies have shown that the
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
627
Fig. 2. The relationship between condensed tannin and salicortin concentration in F1 hybrids of S. sericea
and S. eriocephala.
patterns of secondary chemical expression is variable. Of the chemicals present in
both parents and hybrids, the concentration of 38% were similar to one or both of the
two parental taxa (dominance or co-dominance), 29% were intermediate to the two
parental taxa (incomplete dominance or no dominance), 19% were greater than either
parent (over-dominance), and 14% were less than either parent (underexpression)
(Fahselt and Ownbey, 1968; Belzer and Ownbey, 1971; McMillan et al., 1975; Harborne and Turner, 1984; Spring and Schilling, 1990; Buschmann and Spring, 1995;
Orians and Fritz, 1995). Parental chemicals are not always expressed in the F1
hybrids. Current estimates suggest that parental chemicals are found only 68% of the
time, lost 27% of the time and novel 5% of the time in the hybrids (Rieseberg and
Ellstrand, 1993).
However, none of the above-cited studies looked at variation among F1 hybrids.
We found extensive variation among F1 hybrid families (Table 2). Some hybrid
families produced concentrations higher or lower than the midpoint (incomplete
dominance), concentrations similar to one of the parental taxa (dominance,
Group VII), or levels indistinguishable from the parental midpoint (no dominance).
Furthermore, there was extensive variation among hybrid families. For example, the
concentration of salicortin in one family was over twice that of a second family (Group
VI vs. IV, Table 3). Parental production explained some of the di!erences in production of condensed tannin and 2@-cinnamoylsalicortin by hybrids families; there was
a positive correlation between related parent and hybrid families. Surprisingly, the
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C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
concentration of condensed tannin in hybrids was not correlated with S. eriocephala,
but with S. sericea, the parental taxon that produces low concentrations. It is not clear
why this occurred. No correlation was observed for related parents and hybrids for
salicortin. Although genetic di!erences among parental individuals are the most likely
explanation for the variation in concentration, non-genetic maternal factors could be
important as well. Further work is required to di!erentiate between genetic and
maternal e!ects.
The negative correlation between condensed tannin and phenolic glycosides in
hybrids suggests that F1 hybrid o!spring are not able to produce high concentrations
of both condensed tannin and phenolic glycosides (Fig. 2). We reported a similar
trade-o! previously (Orians and Fritz, 1995). The basis of this trade-o! between
tannins and phenolic glycosides remains unknown. There are two possible explanations. First, there may be a resource allocation trade-o! such that only a certain
amount of carbon is allocated to these carbon-based defenses. Production of one
would limit the production of the other. However, this explanation does not explain
the low production of both chemicals in some hybrids. Second, there may be a genetic
tradeo!. If speci"c alleles control the production of both chemicals, one chemical
may be produced at the expense of the other ("negative pleiotropy). Again,
this explanation does not explain the reduced production of both chemical types
in some individuals. Perhaps the reduced production is due to heterozygosity
in some parents. This could lead to low production of one or both chemicals in some
hybrids.
Additional work is required to determine if this trade-o! is due to resource
allocation constraints or to genetic constraints. The analysis of later generation
hybrids could provide critical information. Unless there is strong genomic selection
preventing reorganization (Rieseberg et al., 1995, 1996), F2 recombinants could
contain all the alleles responsible for the production of both chemicals. These
individuals would be able to produce high concentrations of both chemicals, unless
there is a resource allocation constraint. We are currently designing experiments
with F2 recombinants to test these alternative hypotheses. Furthermore, if we do
identify F2 recombinants that produce high concentrations of both, we could examine
the costs of chemical production (defense). Reduced growth of these individuals
relative to those with lower concentrations would suggest a trade-o! between growth
and defense.
4.1. Implications
This study clearly documents extensive variation among F1 hybrids, and this
variation may a!ect the hybrid survival. Phenolic glycosides, for example, are important deterrents of herbivores, especially generalist herbivores (Rowell-Rahier, 1984;
Tahvanainen et al., 1985a; Orians et al., 1997). Slugs and Japanese beetles, for
example, are dominant generalist herbivores of seedlings and adults, respectively, and
both are inhibited by phenolic glycosides (Orians et al., 1997; Fritz et al., unpublished
data). Therefore, selection may favor hybrids that produce higher concentrations of
phenolic glycosides.
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
629
Unfortunately, few studies have evaluated the importance of secondary chemistry.
Previously, we showed the phenolic glycoside chemistry of naturally occurring
hybrids was not signi"cantly di!erent than the midpoint of the two parental taxa
(Orians and Fritz, 1995; Orians, unpublished data). In contrast, in this study, we show
that the concentration of phenolic glycosides in F1 hybrids was lower on average than
the midpoint. Also, the concentrations reported here are lower than in our previous
studies of naturally occurring hybrids (Orians and Fritz, 1995; Orians, unpublished
data). Taken together, these results suggests that recombinant hybrids that produce
higher concentrations of phenolic glycosides may be more likely to survive. If true, it
would be the "rst example of selection on hybrid chemistry. We are currently
experimentally testing the role of chemistry in the resistance of hybrids.
Are herbivores generally selective "lters on the "tness of hybrids? Unfortunately,
there is little empirical work on this question. Our work and that of others has
demonstrated extensive variation in the secondary chemistry of hybrid plants, and we
believe that such variation can a!ect hybrid "tness.
Overall, our understanding of the e!ects of hybridization on plant}herbivore
interactions is in its infancy. There is debate over the role of herbivores in plant
hybridization, and the e!ects of hybridization on community structure, host range
expansion, and evolution of herbivore virulence (Whitham, 1989; Floate and
Whitham, 1993; Fritz, 1999; Pilson, 1999; Whitham et al., 1999). Secondary chemistry
is unquestionably an important determinant of plant}herbivore interactions, and we
believe more careful attention to chemistry will help predict the ecological and
evolutionary consequences of hybridization.
Acknowledgements
We give thanks to Len and Ellie Sosnowski for permitting us to conduct experiments on their property, Rachel Samberg for help with the chemical analyses, and
Diana Pilson for her thoughtful discussion of the results. This research was supported
by the National Science Foundation (Grant No. DEB 92-07363), a Science Education
Grant from the Howard Hughes Medical Institute, the Faculty Research Awards
Committee (Tufts University). This work was completed while CMO was a Mellon
Grant Research Fellow at Tufts University.
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Phenolic glycosides and condensed tannins in
Salix sericea, S. eriocephala and their F1 hybrids:
not all hybrids are created equal
Colin M. Orians!,*, Megan E. Gri$ths!, Bernadette M. Roche",1,
Robert S. Fritz"
!Department of Biology, Tufts University, Medford, MA 02155, USA
"Department of Biology, Vassar College, Poughkeepsie, NY 12601, USA
Received 26 May 1999; accepted 26 July 1999
Abstract
The performance of hybrids depends upon the inheritance and expression of resistance traits.
Secondary chemicals are one such resistance trait. In this study, we measured the concentrations of phenolic glycosides and condensed tannins in parental and F1 hybrid willows to
examine the sources of chemical variation among hybrids. S. sericea produces phenolic
glycosides, salicortin and 2@-cinnamoylsalicortin, and low concentrations of condensed tannin
in its leaves. In contrast, S. eriocephala produces no phenolic glycosides but high concentrations
of condensed tannins in its leaves. These traits are inherited quantitatively in hybrids. On
average, F1 hybrids are intermediate for condensed tannins, suggesting predominantly additive
inheritance or balanced ambidirectional dominance of this defensive chemical from the parental
species. In contrast, the concentration of phenolic glycosides is lower than the parental
midpoint, indicating directional dominance. However, there is extensive variation among F1
hybrids. The concentration of tannin and phenolic glycosides in F1 hybrid families is either (1)
lower than the midpoint, (2) higher than the midpoint, or (3) indistinguishable from the
midpoint of the two parental taxa. It appears that the production of the phenolic glycosides,
especially 2@-cinnamoylsalicortin, is controlled by one or more recessive alleles. We also
observed a two-fold or greater di!erence in concentration between some hybrid families. We
discuss how chemical variation may e!ect the relative susceptibility of hybrid willows to
herbivores. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Salicaceae; Willows; Hybridization; F1 hybrids; Phenolic glycosides; Condensed tannins
* Corresponding author. Tel.: #1-617-627-3543; fax: #1-617-627-3805.
E-mail address: [email protected] (C.M. Orians)
1 Current address: Department of Biology, Loyola College, Baltimore, MD 21210, USA.
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 0 1 - 5
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C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
1. Introduction
Hybridization is now regarded as a common feature of many plant species, and can
lead to speciation and introgression (Rieseberg, 1991, 1995; Rieseberg and Brunsfeld,
1992; Rieseberg and Ellstrand, 1993; Ellstrand et al., 1996; Arnold, 1997). In the last
10 yr, there have been numerous studies suggesting that hybridization, via changes in
resistance, also a!ects the ecology and evolution of plant-herbivore interactions (e.g.,
Whitham, 1989; Boecklen and Spellenberg, 1990; Whitham et al., 1991, 1994, 1999;
Aguilar and Boecklen, 1992; Floate and Whitham, 1993; Paige and Capman, 1993;
Fritz et al., 1994, 1996; Strauss, 1994; Messina et al., 1996; Orians et al., 1997; Fritz,
1999; Pilson, 1999). We know that the relative resistance of hybrids and parental taxa
to herbivores is highly variable (reviewed by Strauss, 1994; Fritz, 1999), but what
determines resistance is not well understood. Although multiple traits are involved,
secondary chemicals, such as phenolics, terpenoids, alkaloids and glucosinolates, are
often key components of the resistance of plants to herbivores (Rosenthal and Janzen,
1979; Roitberg and Isman, 1992; Bernays and Chapman, 1994; CardeH and Bell, 1995;
Chew and Renwick, 1995). Given the general importance of secondary chemistry to
plant}herbivore interactions, it is surprising that relatively few researchers have
studied the secondary chemistry of hybrids in relation to plant}herbivore interactions
(Huesing et al., 1989; Weber et al., 1994; Orians et al., 1997). We believe that
knowledge of how secondary chemicals are expressed in hybrids will help researchers
understand patterns of hybrid resistance.
The concentration of secondary chemicals in hybrids typically di!ers qualitatively
and quantitatively from their parents (Connor and Purdie, 1976; Harborne and
Turner, 1984; Meier et al., 1989; Altman et al., 1990; Levy and Milo, 1991; Rieseberg
and Ellstrand, 1993; Orians and Fritz, 1995). Qualitatively, novel chemicals may be
found or parental chemicals may be lost. Quantitatively, patterns of expression
depend upon the genetic control of production. Parental chemicals may be overexpressed (over-dominance), underexpressed (incomplete dominance), intermediate (no
dominance), similar to one of the parental species (dominance), or similar to
both parents (co-dominance) (Van Brederode et al. 1974; Falconer, 1989). Quantitative chemical di!erences can a!ect the resistance of a plant (Huesing et al., 1989;
Orians et al., 1997). What is lacking is an understanding of how secondary
chemistry varies among hybrids in ways that could a!ect patterns of resistance to
herbivores.
We have been studying the consequences of hybridization in willow for a number of
years. Willows and other Salicaceous plant species produce two main secondary
chemicals: phenolic glycosides and condensed tannins, both of which are known to
in#uence the susceptibility of plants to insect and mammalian herbivores (Zucker,
1983; Rowell-Rahier, 1984; Tahvanainen et al., 1985a,b; Basey et al., 1988; Lindroth
and Peterson, 1988; Lindroth et al., 1988; Clausen et al., 1989; Schultz, 1989; Denno
et al., 1990; Kolehmainen et al., 1994; Orians et al., 1997). Some willow species
produce only condensed tannins, others produce mostly phenolic glycosides, and
others produce both classes of compounds (Julkunen-Tiitto, 1986, 1989; Orians and
Fritz, 1995).
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
621
In our system, S. sericea produces high concentrations of phenolic glycosides and
low concentrations of condensed tannin, while S. eriocephala produces high concentrations of condensed tannin but no phenolic glycosides (Orians and Fritz, 1995). The
concentrations of both phenolic glycosides and condensed tannins are intermediate,
not signi"cantly di!erent than the midpoint, in naturally occurring hybrids of these
two species (Orians and Fritz, 1995). However, these hybrids were of unknown
pedigree. What is the chemistry of known F1 hybrids? Is there chemical variation
among F1 hybrids? How does the chemistry of experimental hybrids di!er from
natural hybrids? These are all questions that will help us understand how secondary
chemistry might determine patterns of hybrid survival. Here we report on the
concentrations of condensed tannin and phenolic glycosides in two year old seedlings
of Salix sericea, Salix eriocephala and their F1 hybrids.
2. Materials and methods
2.1. Plants
Both Salix sericea Marshall and Salix eriocephala Michx. are abundant in the
northeastern United States and eastern Canada (Argus, 1986). The species often
co-occur and hybridization between the two is common (Argus, 1986; Mosseler and
Papadopol, 1989; Fritz et al., 1994). Willows are dioecious and in this system the
initial hybridization event is unidirectional (S. eriocephala is the paternal parent and
S. sericea is the maternal parent), due to stigma-pollen incompatibility in female S.
eriocephala (Mosseler, 1990).
2.2. Generation of seedlings (parental and F1 hybrid)
We performed intraspeci"c and interspeci"c crosses using known pure parents,
based on RAPD analysis, to generate pure parental and F1 hybrid progenies. From
a larger set of crosses (Fritz et al., 1998), we selected seven groups of progenies where
the parents of each F1 hybrid cross were used in the matched intraspeci"c cross (Table
1). Our goal was to avoid using a parental clone twice. However, one S. sericea parent
(s17) was involved in two crosses.
To ensure purity of each family, the female catkins were covered with mesh bags
prior to stigma emergence to prevent visitation by insect pollinators. Bags were
removed only while making crosses. The resulting seeds were mass planted in trays of
MetroMix 360TM in late May, and, covered with white, shear seed cloth to maintain
a high moisture level and to prevent accidental colonization of the trays by naturally
dispersing willow seeds. The cloth was removed after about two weeks, when natural
seed dispersal had ended. Four-week-old seedlings were transplanted into 3.7 l pots
"lled with our standard soil mixture (4 parts topsoil, 1 part peat moss, and 1 part
vermiculite). Due to mortality, the number of siblings per cross varied (Table 1). The
next spring the seedlings were transplanted into 7.4 l pots containing the same soil
622
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
Table 1
Crosses performed to generate related parental and F1 hybrid progeny
Group
S. sericea
$]#
F1 Hybrids
$]#
S. eriocephala
$]#
I
II
III
IV
V
VI
VII
s17]s22 (n"5)
s17]s16 (n"5)
s28]s27 (n"7)
s37]s55 (n"3)
s83]s14 (n"4)
s1]s95 (n"5)
s78]S60 (n"5)
s17]e18 (n"4)
s17]ne36 (n"5)
s28]ne35 (n"2)
s37]ne33 (n"9)
s83]ne37 (n"5)
s1]hhe46 (n"4)
s78]hhe12 (n"4)
e15]e18 (n"4)
e23]ne36 (n"4)
e23]ne35 (n"4)
e29]e115 (n"5)
e3]ne37 (n"5)
e64]hhe46 (n"4)
e60]hhe12 (n"4)
Note: F1 hybrids within each group have a female in common with the Salix sericea progeny and male in
common with the S. eriocephala progeny.
mixture mixed with 13 g of slow-release fertilizer (10 : 10 : 10 NPK). Plants were
randomized within blocks.
Leaf samples for chemical analyses were collected on 18 July. Twelve fully expanded
leaves were collected from each plant (the "rst two fully expanded leaves from each of
six shoots). Leaves were cut at the petiole, placed on ice, transported to the lab, and
vacuum-dried for at least 24 h. Vacuum-drying leaves has been shown to prevent the
loss of both condensed tannin and phenolic glycosides (Orians, 1995). Once dry, leaves
were ground to a "ne powder using a Wiley Mill equipped with a size 30 mesh and
then stored in a !203C freezer.
2.3. Chemical analyses
The phenolic glycosides were assayed using standard techniques (Orians, 1995).
Brie#y, leaf powder (30$3 mg) was extracted in cold MeOH (10 mg leaf powder/1 ml
MeOH) with sonication for 10 min. Cold water was constantly #ushed through the
sonicator to prevent the heating of samples. We centrifuged and "ltered (0.45 lm
"lter) each sample before placing extracts in crimp-top vials. Extracts were kept in the
freezer until analysis (48 h or less). We quanti"ed the concentration of glycosides with
an HPLC equipped with an autosampler and a UV detector set at 274 nm (HewlettPackard). A reverse-phase NOVA-PAK C (4 lm) column (Waters) and a gradient
18
system of distilled water and MeOH was used. 1,3-dimethoxybenzene was used as the
internal standard. Standard curves were determined for salicortin and 2@-cinnamoylsalicortin.
The condensed tannins also were extracted using standard techniques (Orians,
1995). Brie#y, approximately 300 mg leaf powder was washed with ether and extracted
with 70% acetone for 3 h in a 403C water bath. Acetone was removed under reduced
pressure and all extracts were diluted to 8 ml with distilled water. The butanol/HCl
method was used to quantify tannin concentrations (Hagerman and Butler, 1989). The
concentration of the condensed tannin (mg/g dry leaf) was determined using puri"ed
tannins collected from the two willow species and the hybrids.
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
623
2.4. Statistical analyses
A one way analysis of variance was used to determine the overall e!ects of taxon on
condensed tannins (using the means of each of the 7 families/taxon). The natural log of
condensed tannin concentration was used in the analysis because variance increased
with the mean. For the phenolic glycosides only S. sericea and hybrids were included
in the model, because S. eriocephala generally does not contain phenolic glycosides.
Contrasts were performed to determine if the hybrids were signi"cantly di!erent
from the midpoint of the two parental taxa (non-transformed data were used for the
contrasts), that is, if one averages the parental values do the hybrids di!er from that
average. This was done using the means comparison contrasts in SuperANOVA
(Abacus Concepts, 1989). These comparisons were performed with all taxa included in
the model.
Separate analyses were done for each group of families (Table 1). As above, an
analysis of variance was used to determine the e!ects of family (or taxon) on
chemistry, and contrasts, as described above, were performed to determine if the
concentrations of the chemicals in the hybrid family was signi"cantly di!erent from
the average of the two parental families.
Regression analysis was used to determine the relationship between parental and
hybrid chemistry, and between the production of phenolic glycosides and condensed
tannins.
3. Results
In general, the concentrations of condensed tannins is high in S. eriocephala, low in
S. sericea and intermediate in hybrids, while the concentration of the two phenolic
glycosides shows the opposite pattern (Table 2). The concentrations measured in these
plants are similar to that measured in "eld plants (unpublished data), and the patterns
are similar to those reported previously (Orians and Fritz, 1995). In general, S.
eriocephala does not produce phenolic glycosides in its leaves (see also Orians and
Fritz, 1995). However, two of the four siblings of one S. eriocephala cross (e60]hhe12)
produced phenolic glycosides (data not shown in Table 2), and the four s78]hhe12
siblings had levels of phenolic glycosides and tannins similar to S. sericea. Although
these results suggest there is something unusual about hhe12, other crosses involving
hhe12 (e64]hhe12 and s57]hhe12) were not unusual (unpublished data). Currently,
we are unable to explain the anomalous results obtained with the hhe12 crosses.
Because of these anomalous results, we excluded crosses involving hhe12 before
testing for taxon e!ects. Overall, there was a signi"cant e!ect of taxon on chemistry
and all three taxa were signi"cantly di!erent from one another (ANOVA, F*125.6,
p)0.0001) (Table 2). Contrasts revealed signi"cant deviation from the average (or
midpoint) of the two parental taxa for salicortin (p(0.01) and 2@-cinnamoylsalicortin
(p(0.001), but not for condensed tannins (p"0.99). For both phenolic glycosides,
the concentration was lower than the midpoint, indicating a small dominance deviation toward reduced expression.
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Table 2
Mean concentration (MG/G dry leaf weight), standard errors (in parentheses), and coe$cients of variation
(cv) for condensed tannin and the phenolic glycosides in S. sericea, F1 hybrids and S. eriocephala leaves
Taxon
S. sericea
F1 hybrid
S. eriocephala
Chemical type
Condensed tannin
Salicortin
2@-cinnslctn
27.8 (1.8) a
cv"7.6
71.7 (11.9) b
cv"19.7
126.6 (4.2) c
cv"10.6
104.8 (4.1) a
cv"10.2
48.0 (10.7) b
cv"33.5
None
18.5 (1.4) a
cv"20.0
4.5 (2.4) b
cv"44.1
None
Note: 2@-cinnslctn "2@-cinnamoylsalicortin. Di!erent letters denote signi"cant di!erences among taxa
(p(0.001).
In addition, there was more among-family variation for hybrids than for the
parental taxa (see Coe$cients of Variation in Table 2). This was especially evident for
the phenolic glycosides. Some hybrid families produced very low concentrations of
salicortin and 2@-cinnamoylsalicortin (26.3 and 0.6 mg/g dry leaf weight, respectively
for Group IV) while other families produced relatively high concentrations (60.6 and
2.1 mg/g dry leaf weight, respectively for Group VI). Thus some families produce over
twice the concentration of other families. If we include the s78]hhe12 cross salicortin
and 2@-cinnamoylsalicortin concentrations were even higher 106.7 and 18.4 mg/g dry
leaf weight, respectively.
For each group of crosses, there was a highly signi"cant di!erence among families
(or taxa) (F*19.5; p(0.001) (Table 3). Based on our previous results, we expected
that F1 chemistry would be indistinguishable from the midpoint of the parental taxa.
However, contrasts revealed that some F1 hybrids were lower than the midpoint,
while others were higher than the midpoint (Fig. 1). Excluding the cross involving
hhe12 (Group VII), condensed tannin concentration was signi"cantly di!erent than
the parental midpoint for 2 of 6 hybrid families, one higher and one lower. Salicortin
concentration was signi"cantly lower than the midpoint for 3 of 6 families. In contrast,
2@-cinnamoylsalicortin concentrations were signi"cantly lower for all 6 hybrid families. For Group VII, condensed tannin was lower and the phenolic glycosides were
higher than the parental midpoint (Fig. 1). Generally, the production of phenolic
glycosides, especially 2@-cinnamoylsalicortin, showed directional dominance or recessive deviations.
There were signi"cant positive correlations between parental and hybrid chemistry.
The concentration of condensed tannins in the hybrids was positively correlated with
the concentration in the S. sericea parent (R2"0.79, p"0.02), but not with the
S. eriocephala parent (R2"0.08, p"0.59). There was a positive correlation for
2@-cinnamoylsalicortin (R2"0.64, p"0.05). However, there was no correlation for
salicortin (R2"0.04, p"0.33).
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
625
Table 3
Family mean (standard error) concentration, MG/G dry leaf weight, for condensed tannin and phenolic
glycosides in S. sericea (SS), F1 hybrids and S. eriocephala (SE)
Group
Family
Tannin
Salicortin
2@cinnslct
I
SS
F1
SE
s17]s22
s17]e18
e15]e18
22.8(3.3) a
66.8 (10.3) b
122.4(4.7) c
110.8 (7.2) a
43.9 (6.7) b
NONE
18.8 (2.4) a
2.2 (0.5) b
NONE
II
SS
F1
SE
s17]s16
s17]ne36
e23]ne36
20.8(0.6) a
48.6 (10.1) b
106.4(7.0) c
121.2 (4.4) a
39.3 (8.1) b
NONE
13.4 (1.0) a
1.8 (0.5) b
NONE
III
SS
F1
SE
s28]s27
s28]ne35
e23]ne35
24.7(2.2) a
87.4(7.1) b
113.0(3.0) c
98.7 (6.2) a
31.0 (9.5) b
NONE
17.9 (1.9) a
2.4 (1.0) b
NONE
IV
SS
F1
SE
s37]s55
s37]ne33
e29]e115
25.3(4.6) a
86.1(8.0) b
122.4(5.5) c
97.4 (6.3) a
26.3 (3.9) b
NONE
14.2 (1.0) a
1.1 (0.2) b
NONE
V
SS
F1
SE
s83]s14
s83]ne37
e3]ne37
24.7(3.5) a
70.5 (10.8) b
117.5(3.3) c
88.9 (8.9) a
28.6 (4.5) b
NONE
19.9 (0.9) a
1.7 (0.5) b
NONE
VI
SS
F1
SE
s1]s95
s1]hhe46
e64]hhe46
22.6(1.6) a
72.8(4.4) b
143.9(4.8) c
105.4 (7.6) a
60.6 (6.6) b
NONE
23.3 (2.5) a
3.9 (1.0) b
NONE
VII
SS
F1
SE
s78]S60
s78]hhe12
e60]hhe12
21.4(0.9) a
15.8(1.1) a
116.1 (13.5) b
111.2(7.9) a
106.7(4.2) a
24.9 (17.3) b
22.0 (2.1) a
18.5 (1.1) a
0.2 (0.2) b
Note: 2@cinnslct "2@-cinnamoylsalicortin. Di!erent letters denote signi"cant di!erences among families
within each group (p(0.001).
Among hybrids we found an inverse correlation between the concentration of
tannin and salicortin (Fig. 2) and between tannin and 2@-cinnamoylsalicortin
(>"78.5!3.3X, R2"0.41, p(0.001). These negative correlations suggest that
hybrids may not be able to produce high concentrations of both phenolic glycosides
and condensed tannins (see also Orians and Fritz, 1995). Interestingly, many F1
hybrids contained low concentrations of both condensed tannin and salicortin
(Fig. 2).
4. Discussion
Most secondary chemicals are under polygenic control and inherited in a quantitative fashion (Berenbaum and Zangerl, 1992), as we have found for both salicortin and
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C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
Fig. 1. The proportional deviation from the parental midpoint. (A) Values above the midpoint indicate that
condensed tannin concentrations deviate toward S. eriocephala. (B) and (C) Values above the midpoint
indicate salicortin and 2@-cinnamoylsalicortin concentrations deviate toward S. sericea. NS: not signi"cant
(*) p(0.05; (**) p(0.01.
2@-cinnamoylsalicortin (Orians et al., 1996). As a consequence most parental chemicals
are found in hybrids (Table 2; Rieseberg and Ellstrand, 1993). However, the concentration in the hybrids can be quite variable (Harborne and Turner, 1984). Our results
show that on average F1 hybrids contain intermediate concentrations of condensed
tannin (equal to the parental midpoint), suggesting predominantly additive inheritance or a lack of dominance of this defensive chemical from the parental species. In
contrast, the concentration of the phenolic glycosides, salicortin and 2@-cinnamoylsalicortin, is lower than the parental midpoint, indicating incomplete dominance over
production. This incomplete dominance suggests that one or more alleles that control
phenolic glycoside production, especially 2@-cinnamoylsalicortin, are recessive in the
F1 hybrid. Altman et al. (1990) also found recessive control of the production of the
terpenoid raimondal in cotton hybrids; compared to the parental taxon, the concentration in hybrids was 9}12%.
There have been a number of studies evaluating the genetics of secondary chemical
production by F1 hybrids of contrasting species. These studies have shown that the
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
627
Fig. 2. The relationship between condensed tannin and salicortin concentration in F1 hybrids of S. sericea
and S. eriocephala.
patterns of secondary chemical expression is variable. Of the chemicals present in
both parents and hybrids, the concentration of 38% were similar to one or both of the
two parental taxa (dominance or co-dominance), 29% were intermediate to the two
parental taxa (incomplete dominance or no dominance), 19% were greater than either
parent (over-dominance), and 14% were less than either parent (underexpression)
(Fahselt and Ownbey, 1968; Belzer and Ownbey, 1971; McMillan et al., 1975; Harborne and Turner, 1984; Spring and Schilling, 1990; Buschmann and Spring, 1995;
Orians and Fritz, 1995). Parental chemicals are not always expressed in the F1
hybrids. Current estimates suggest that parental chemicals are found only 68% of the
time, lost 27% of the time and novel 5% of the time in the hybrids (Rieseberg and
Ellstrand, 1993).
However, none of the above-cited studies looked at variation among F1 hybrids.
We found extensive variation among F1 hybrid families (Table 2). Some hybrid
families produced concentrations higher or lower than the midpoint (incomplete
dominance), concentrations similar to one of the parental taxa (dominance,
Group VII), or levels indistinguishable from the parental midpoint (no dominance).
Furthermore, there was extensive variation among hybrid families. For example, the
concentration of salicortin in one family was over twice that of a second family (Group
VI vs. IV, Table 3). Parental production explained some of the di!erences in production of condensed tannin and 2@-cinnamoylsalicortin by hybrids families; there was
a positive correlation between related parent and hybrid families. Surprisingly, the
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C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
concentration of condensed tannin in hybrids was not correlated with S. eriocephala,
but with S. sericea, the parental taxon that produces low concentrations. It is not clear
why this occurred. No correlation was observed for related parents and hybrids for
salicortin. Although genetic di!erences among parental individuals are the most likely
explanation for the variation in concentration, non-genetic maternal factors could be
important as well. Further work is required to di!erentiate between genetic and
maternal e!ects.
The negative correlation between condensed tannin and phenolic glycosides in
hybrids suggests that F1 hybrid o!spring are not able to produce high concentrations
of both condensed tannin and phenolic glycosides (Fig. 2). We reported a similar
trade-o! previously (Orians and Fritz, 1995). The basis of this trade-o! between
tannins and phenolic glycosides remains unknown. There are two possible explanations. First, there may be a resource allocation trade-o! such that only a certain
amount of carbon is allocated to these carbon-based defenses. Production of one
would limit the production of the other. However, this explanation does not explain
the low production of both chemicals in some hybrids. Second, there may be a genetic
tradeo!. If speci"c alleles control the production of both chemicals, one chemical
may be produced at the expense of the other ("negative pleiotropy). Again,
this explanation does not explain the reduced production of both chemical types
in some individuals. Perhaps the reduced production is due to heterozygosity
in some parents. This could lead to low production of one or both chemicals in some
hybrids.
Additional work is required to determine if this trade-o! is due to resource
allocation constraints or to genetic constraints. The analysis of later generation
hybrids could provide critical information. Unless there is strong genomic selection
preventing reorganization (Rieseberg et al., 1995, 1996), F2 recombinants could
contain all the alleles responsible for the production of both chemicals. These
individuals would be able to produce high concentrations of both chemicals, unless
there is a resource allocation constraint. We are currently designing experiments
with F2 recombinants to test these alternative hypotheses. Furthermore, if we do
identify F2 recombinants that produce high concentrations of both, we could examine
the costs of chemical production (defense). Reduced growth of these individuals
relative to those with lower concentrations would suggest a trade-o! between growth
and defense.
4.1. Implications
This study clearly documents extensive variation among F1 hybrids, and this
variation may a!ect the hybrid survival. Phenolic glycosides, for example, are important deterrents of herbivores, especially generalist herbivores (Rowell-Rahier, 1984;
Tahvanainen et al., 1985a; Orians et al., 1997). Slugs and Japanese beetles, for
example, are dominant generalist herbivores of seedlings and adults, respectively, and
both are inhibited by phenolic glycosides (Orians et al., 1997; Fritz et al., unpublished
data). Therefore, selection may favor hybrids that produce higher concentrations of
phenolic glycosides.
C.M. Orians et al. / Biochemical Systematics and Ecology 28 (2000) 619}632
629
Unfortunately, few studies have evaluated the importance of secondary chemistry.
Previously, we showed the phenolic glycoside chemistry of naturally occurring
hybrids was not signi"cantly di!erent than the midpoint of the two parental taxa
(Orians and Fritz, 1995; Orians, unpublished data). In contrast, in this study, we show
that the concentration of phenolic glycosides in F1 hybrids was lower on average than
the midpoint. Also, the concentrations reported here are lower than in our previous
studies of naturally occurring hybrids (Orians and Fritz, 1995; Orians, unpublished
data). Taken together, these results suggests that recombinant hybrids that produce
higher concentrations of phenolic glycosides may be more likely to survive. If true, it
would be the "rst example of selection on hybrid chemistry. We are currently
experimentally testing the role of chemistry in the resistance of hybrids.
Are herbivores generally selective "lters on the "tness of hybrids? Unfortunately,
there is little empirical work on this question. Our work and that of others has
demonstrated extensive variation in the secondary chemistry of hybrid plants, and we
believe that such variation can a!ect hybrid "tness.
Overall, our understanding of the e!ects of hybridization on plant}herbivore
interactions is in its infancy. There is debate over the role of herbivores in plant
hybridization, and the e!ects of hybridization on community structure, host range
expansion, and evolution of herbivore virulence (Whitham, 1989; Floate and
Whitham, 1993; Fritz, 1999; Pilson, 1999; Whitham et al., 1999). Secondary chemistry
is unquestionably an important determinant of plant}herbivore interactions, and we
believe more careful attention to chemistry will help predict the ecological and
evolutionary consequences of hybridization.
Acknowledgements
We give thanks to Len and Ellie Sosnowski for permitting us to conduct experiments on their property, Rachel Samberg for help with the chemical analyses, and
Diana Pilson for her thoughtful discussion of the results. This research was supported
by the National Science Foundation (Grant No. DEB 92-07363), a Science Education
Grant from the Howard Hughes Medical Institute, the Faculty Research Awards
Committee (Tufts University). This work was completed while CMO was a Mellon
Grant Research Fellow at Tufts University.
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