Patterns of Reproductive Isolation in Eucalyptus—
A Phylogenetic Perspective
Matthew J. Larcombe,*,1 Barbara Holland,2 Dorothy A. Steane,1,3 Rebecca C. Jones,1 Dean Nicolle,4
Rene E. Vaillancourt,1 and Brad M. Potts1
1
School of Biological Sciences, University of Tasmania, Hobart, TAS, Australia
School of Physical Sciences, University of Tasmania, Hobart, TAS, Australia
3
Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, Australia
4
Currency Creek Arboretum, Currency Creek, SA, Australia
*Corresponding author: E-mail: [email protected].
Associate editor: Juliette de Meaux
2
Abstract
We assess phylogenetic patterns of hybridization in the speciose, ecologically and economically important genus
Eucalyptus, in order to better understand the evolution of reproductive isolation. Eucalyptus globulus pollen was applied
to 99 eucalypt species, mainly from the large commercially important subgenus, Symphyomyrtus. In the 64 species that
produce seeds, hybrid compatibility was assessed at two stages, hybrid-production (at approximately 1 month) and
hybrid-survival (at 9 months), and compared with phylogenies based on 8,350 genome-wide DArT (diversity arrays
technology) markers. Model fitting was used to assess the relationship between compatibility and genetic distance, and
whether or not the strength of incompatibility “snowballs” with divergence. There was a decline in compatibility with
increasing genetic distance between species. Hybridization was common within two closely related clades (one including
E. globulus), but rare between E. globulus and species in two phylogenetically distant clades. Of three alternative models
tested (linear, slowdown, and snowball), we found consistent support for a snowball model, indicating that the strength
of incompatibility accelerates relative to genetic distance. Although we can only speculate about the genetic basis of this
pattern, it is consistent with a Dobzhansky–Muller-model prediction that incompatibilities should snowball with divergence due to negative epistasis. Different rates of compatibility decline in the hybrid-production and hybrid-survival
measures suggest that early-acting postmating barriers developed first and are stronger than later-acting barriers. We
estimated that complete reproductive isolation can take up to 21–31 My in Eucalyptus. Practical implications for hybrid
eucalypt breeding and genetic risk assessment in Australia are discussed.
Key words: reproductive isolation, speciation, Dobzhansky–Muller incompatibilities, hybridization, Eucalyptus globulus,
DArT markers.
Reproductive barriers that prevent hybridization between
previously cross-compatible taxa are fundamental drivers of
speciation (Coyne and Orr 2004; Abbott et al. 2013).
Comparative studies that assess patterns of reproductive isolation among groups of related taxa have been important in
identifying when and how these barriers evolve (Coyne and
Orr 1989; Orr and Turelli 2001; Moyle et al. 2004; Widmer
et al. 2009). It has been shown that premating barriers, such as
mate choice or flowering time, usually develop first, are often
responsible for most of the observed isolation and, because
they experience steep selection gradients associated with
sexual-selection, they tend to evolve more quickly than
postmating barriers (Coyne and Orr 2004; Lowry et al. 2008;
Widmer et al. 2009). However, premating barriers also tend to
be “leaky,” and it is usually a combination of pre- and
postmating barriers that result in complete reproductive isolation (Coyne and Orr 2004; Widmer et al. 2009).
Postmating barriers include both prezygotic and postzygotic mechanisms that are thought to arise principally through
drift (Hogenboom and Mather 1975; Orr and Turelli 2001;
Coyne and Orr 2004). As populations diverge, minor allelic
changes develop that are neutral or adaptive in the population of origin, but cause so-called Dobzhansky–Muller incompatibilities (DMI’s) when brought together in interpopulation
hybrids, leading to inviability or sterility (Bateson 1909;
Dobzhansky 1937; Muller 1942; Orr and Turelli 2001). DMIs
are widely accepted as a major cause of postmating reproductive isolation (Hogenboom and Mather 1975; Orr and
Turelli 2001; Coyne and Orr 2004; Turelli and Moyle 2007).
For example, the fact that hybrid compatibility often decreases as genetic distance increases (Coyne and Orr 1989;
Lee 2000; Presgraves 2002; Malone and Fontenot 2008), supports the basic drift model underlying the theory (Coyne and
Orr 1997). However, comparative studies have been generally
inconclusive regarding one prediction of the DMI model, that
is, that the number of DMIs should accelerate relative to
genetic distance because of epistasis—producing the so
called “snowball effect” (Orr and Turelli 2001; Johnson 2006;
Turelli and Moyle 2007; Gourbière and Mallet 2010; Giraud
and Gourbière 2012). This is despite evidence from detailed
ß The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
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Mol. Biol. Evol. 32(7):1833–1846 doi:10.1093/molbev/msv063 Advance Access publication March 16, 2015
1833
Article
Introduction
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Larcombe et al. . doi:10.1093/molbev/msv063
genetic mapping studies that incompatibilities can snowball
(Matute et al. 2010; Moyle and Nakazato 2010).
Another possible issue in comparative studies of reproductive isolation is that postmating barriers are often assessed at
only one, usually early, ontogenetic stage (but see Scopece
et al. 2007, 2008; Stelkens et al. 2010). This potentially ignores
hybrid incompatibilities that are expressed later in development—affecting overall estimates of both the strength and
rate of evolution of postzygotic barriers (Stelkens et al. 2010).
Related to this, studies investigating the phylogenetic basis of
reproductive isolation in plants have, to date, focused on
herbs (Widmer et al. 2009; Levin 2012). But, as noted recently
by Levin (2012), future studies need to include trees, because
long generation times extend the period when hybrid incompatibilities can be expressed prior to reproductive maturity
(often 5–10 years), which potentially represents a life-history
barrier that annual flowering herbs do not experience.
Eucalyptus is a large and diverse genus of around 700 species, the majority of which are trees native to Australia
(Grattapaglia et al. 2012). They are often foundation species
and dominate most Australian forests and woodlands,
making Eucalyptus one of the most ecologically important
genera in Australia (Pryor and Johnson 1981). There is great
morphological diversity in the genus, including the world’s
tallest angiosperm E. regnans (99.6 m), and the sometimes
prostrate mountain-top shrub E. vernicosa (Grattapaglia
et al. 2012). It is a taxonomically complex group that is divided
into ten subgenera, with the most speciose being subgenus
Symphyomyrtus (c. 484 species) and subgenus Eucalyptus
(formerly Monocalyptus, approximately 108 species)
(Grattapaglia et al. 2012). There is no evidence for variation
in chromosome number in the genus (2n = 22 in all 135 species assessed to date), making it an ideal lineage for investigating patterns of diploid speciation (Grattapaglia et al. 2012).
There is a well-recognized complete barrier to hybridization between the major Eucalyptus subgenera (Pryor and
Johnson 1981; Griffin et al. 1988). However, widely reported
hybridization within subgenera has led to eucalypts being
renowned for having weak reproductive barriers between
species (Field et al. 2009; Grattapaglia et al. 2012). Despite
this reputation, a major review of hybridization in the
genus by Griffin et al. (1988) found that barriers clearly
exist. They showed that, even within subgenera, only 15%
of potential hybrid combinations (expected based on natural
range overlap) have actually been found, and that hybridization within taxonomic sections was more common than between sections (Griffin et al. 1988). There is clearly variation in
the strength of barriers to hybridization in Eucalyptus, making
it a good candidate for investigating the phylogenetic basis of
postmating reproductive isolation.
Eucalypts are the world’s most widely grown hardwood
trees, with 20 million ha of plantations in cultivation globally
(Myburg et al. 2014). The global eucalypt plantation estate is
dominated by nine species and their hybrids, all of which
come from subgenus Symphyomyrtus (Harwood 2011).
Eucalyptus globulus (subgenus Symphyomyrtus) is the most
widely grown species in temperate zones (Stackpole et al.
2010), and an important component of hybrid breeding
1834
programs in other areas (Bison et al. 2007; Hardner et al.
2011). The ecological and economic importance of
Eucalyptus has seen the development of significant genomic
resources (Grattapaglia et al. 2012), including diversity arrays
technology (DArT), which is a marker-based system providing
thousands of genome-wide polymorphic loci (Sansaloni et al.
2010; Steane et al. 2011; Petroli et al. 2012). It has recently
been pointed out that the small number of genes used to
estimate divergence in studies of reproductive isolation may
not be representative of divergence across the genome as a
whole, possibly causing problems in interpretation (Levin
2012; St€adler et al. 2012). The use of DArT markers largely
overcomes this problem.
This study uses over 8,300 DArT markers to build a distance-based phylogeny in order to investigate the phylogenetic basis of cross-compatibility between E. globulus and 64
eucalypt species belonging to 13 sections, mainly from subgenus Symphyomyrtus. We have chosen E. globulus because of
its global economic importance, and because it is the most
widely grown eucalypt in Australia, where it is often locally
exotic, and may cause genetic contamination of indigenous
eucalypt populations through hybridization and introgression
(Potts et al. 2003; Barbour et al. 2008). We assess two stages of
postmating compatibility: The number of hybrids produced
(hybrid-production) and F1 survival at 9 months (hybridsurvival). The following specific questions are addressed: 1)
Which species and groups of species are compatible with E.
globulus, and do phylogenetic barriers to hybridization exist?
2) does incompatibility increase with genetic distance? 3) is it
possible to detect a snowball effect in the strength of incompatibility? and 4) are isolating barriers to hybrid-production
and hybrid-survival similar in strength, indicating that they
evolve at a similar rates?
Results
Across all 99 species, 7,057 flowers were crossed with E.
globulus pollen, and compared with 2,917 control flowers
(see Materials and Methods). Two main crossing approaches
were used; supplementary and cut-style pollination. The cutstyle pollination technique was more efficient at producing E.
globulus hybrids, with a 25% success rate (number of hybrids/
number of plants produced), compared with the supplementary pollination at 14%. The “hybrid-production” data only
consider crosses done using the supplementary pollination
technique which aims to mimic natural pollination. The
“hybrid-survival” data include hybrids produced under any
of the crossing approaches (see Materials and Methods for
details). In most treatments (except treatment 5, see below),
no postpollination isolation was used so in compatible combinations mixed seedlots were produced including both pure
maternal species and hybrid seedlings. The pure species seedlings could arise from outcrossing with neighboring conspecific trees (species are planted in four tree blocks in the
arboretum) or from self-pollination. Hybrid seedlings could
arise from the applied E. globulus pollen or natural pollination
with other species in the arboretum.
Of the 99 species crossed, 64 produced viable seed (treatment or control), and from the successful crossing treatments
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Table 1. Summary of Crossing between Eucalyptus globulus (pollen parent in all crossing) and 99 Eucalypt Species from 13 Taxonomic Sections,
Mainly from Subgenus Symphyomyrtus (species information is provided in supplementary material S1, Supplementary Material online).
Cladea
n of Sections
Representedb
n Species
Crossed
n Flowers
Crossedc
n Species Producing
Viable Seed
n Species
Producing Hybrids
Ie
II
III
IV
Vg
Total
1
4
4
1
3
13
21
11
48
15
4
99
1,391
1,058
3,330
1,107
171
7,057
13
11
31
8
1
64
12
10
3
1
0
26
n Hybrids
Sup
495 (72)f
21
4
0
0
520 (97)
CS
49
34
5
8
0
96
% Hybrid
Survivald
Total
544 (121)
55
9
8
0
616
80
62
0
25
—
—
NOTE.—The number of species, count of species producing hybrids with E. globulus, and the number of E. globulus hybrid seedlings obtained are shown in this table. Sup,
supplementary pollination; CS, cut-style pollination; see Materials and Methods for details.
a
Clades are shown in figure 1.
b
Taxonomy according to Brooker (2000), but also recognizing the split in Bisectae identified by Steane et al. (2002).
c
Not including controls (2,917).
d
Survival was assessed at least 9 months after germination.
e
Eucalyptus goniocalyx (an outlier, see text) and E. globulus, both belong to Clade I.
f
n hybrids excluding E. goniocalyx in parenthesis.
g
Species outside subgenus Symphyomyrtus.
4,571 progeny were grown and assessed for hybrids. In all, 616
hybrids with E. globulus were identified (table 1); however, 423
were from one cross combination, E. globulus E. goniocalyx.
This result came from a single tree, which was a significant
outlier, producing 99% E. globulus hybrids. Eucalyptus goniocalyx and E. globulus are closely related, both belonging to
section Maidenaria series Globulares. There was a noted lack
of flowering in the conspecific trees neighboring this outlier
and it is likely that high self-incompatibility combined with an
absence of conspecific pollen competition may have contributed to the high level of hybridization in this tree. Across the
study there were also 42 natural hybrids found, from interspecific pollination from surrounding species. These were
identified where the morphology deviated from the maternal
type but was inconsistent with that of E. globulus hybrids.
Eucalyptus globulus hybrids were found among progeny of
26 (41%) of the 64 species that produced seed; these were
all verified using microsatellite-based parentage analysis (see
supplementary materials S1 and S2, Supplementary Material
online, for details). Hybrid survival was assessed for 215 of the
hybrids representing all hybrids from the 26 species, except
E. globulus E. goniocalyx, where 22 hybrids were randomly
selected for the survival study. Thirty-five percent of the hybrids died by the time they were evaluated, and complete
hybrid mortality occurred in five species (fig. 1). In comparison, mortality of the pure species seedlings was low (6%).
The phylogenetic network showed four main clades within
subgenus Symphyomyrtus (fig. 1). These clades had previously
been identified in a similar analysis (Steane et al. 2011). Clade I
was made up of species from section Maidenaria, which includes E. globulus. Clade II was closely associated with Clade I
and included species from sections Exsertaria, Incognitae,
Latoangulatae, and Racemus. There were relatively long
branches between Clades I/II and Clades III/IV. Clade III included species from sections Adnataria, Bisectae I (Steane
et al. 2002) and Dumaria, whereas Clade IV was made up
of species from Bisectae II (Steane et al. 2002).
There was a phylogenetic pattern to hybridization success
(fig. 1). Contingency Chi-square tests showed that E. globulus
was significantly more likely to hybridize with taxa in Clades I
(its own clade) and II (hybrids in 22 of 32 species), than with
those in Clades III and IV (hybrids in 4 of 63 species; 2 1 = 23.9,
P < 0.0001; fig. 1). However, there was no difference between
Clades I and II in the number of species forming hybrids with
E. globulus (2 1 = 0.17, P 4 0.99).
Because of the E. goniocalyx E. globulus outlier (see
above) the model fitting (Compatibility ~ genetic distance)
was run with and without E. goniocalyx. The inclusion of
E. goniocalyx did change the magnitude of effects for
Maidenaria, but it did not affect the direction of effects, the
significance levels, or interpretation of the results. We have
presented the results including E. goniocalyx, and the alternative analysis is available in supplementary material S2,
Supplementary Material online. When assessing the rate of
hybrid-production (number of hybrids/number of plants produced) for both the section-level (fig. 2a) and species-level
data sets (fig. 2d), there was a strong negative effect of genetic
distance. The relationship is consistently negative, but shows
a strongly leptokurtic decline (particularly for the section-level
data set; fig. 2a/d). Hybrid-survival, measured for both the
section-level (fig. 2b) and the species-level data sets (fig. 2e),
declined with increasing genetic distance. However, survival of
the pure seedlings was not related to their genetic distance to
E. globulus (Z21 = 0.484, P = 0.629), arguing that the reductions
in hybrid-survival are not associated with maladaptation of
the maternal species to nursery conditions, but due to intrinsic incompatibilities. In the species level-hybrid survival data,
the results were based on hybrids from single mother trees in
all but two species (E. gunnii and E. viminalis), and these
showed no significant maternal variation (see supplementary
material S1, Supplementary Material online, for details). The
relationship between genetic distance and hybrid-survival was
more linear than it was for hybrid-production (fig. 2). In the
section-level data set, the shape of the hybrid-production
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Clade II:
Exsertaria
Incognitae
Latoangulatae
Racemus
Crossability = 0.045 (0.036)
Clade I:
Maidenaria
(E. globulus)
Clade IV:
Bisectae II
Crossability = 0.546 (0.464)
Cr
os
sa
bi
lit
y
p
ow
n
Hybrids (%)
90
80
70
Clade V:
Subgenus Eucalyptus
60
50
40
ba
rri
er
0.0
06
(0
)
Clade III:
Adnataria
Bisectae I
Domesticae
Dumaria
Kn
100
=
:c
ro
ss
ab
ilit
y=
0(
0)
30
20
10
†
††
† †
* *
*
* *
*
*
goniocalyx
arcana
crenulata
morrisbyi
mannifera
viminalis
cephalocarpa
cornuta
hallii
pulverulenta
botryoides
nubila
michaeliana
camaldulensis
gunnii
dwyeri
kabiana
scias
nortonii
ovata
scoparia
cosmophylla
parrama ensis
rudis
tere
rnis
angulosa
conglobata
exigua
oraria
torquata
albopurpurea
argophloia
baueriana
beyeriana
bosistoana
crebra
lansdowneana
largiflorens
leucoxylon
paniculata
polyanthemos
viridis
diversicolor
astringens
conferruminata
erythronema
gardneri
laeliae
megacornuta
occidentalis
tenera
thamnoides
varia
vesiculosa
caesia
calycogona
decipiens
gillii
minniritchi
pyriformis
socialis
insularis
0
FIG. 1. Top: The phylogenetic network based on 78 species and 8,350 genome-wide markers shows four main clades in subgenus Symphyomyrtus (I–IV,
above the solid line) annotated with the most recent sectional taxonomy (Brooker 2000; Steane et al. 2002). The estimated crossability between
Eucalyptus globulus and various parts of the phylogenetic network is shown (arrows and dashed lines), the first number gives the probability of
producing hybrids and the number in parentheses is the combined probability of hybrid-production and -survival. Bottom: The histogram shows the
percentage of hybrids found in the 62 species pollinated with E. globulus pollen (two species not shown belong to sections outside the phylogeny and
produced no hybrids). The species are colored according to their phylogenetic affinities to the color-coded clades above (species are ranked by
hybridization rate, and then by the clades genetic distance to E. globulus). *No hybrids with supplementary pollination but hybrids produced with cutstyle pollination (see Materials and Methods). †Complete hybrid mortality.
compared with hybrid-survival curves indicates that earlyacting postmating barriers develop more quickly (i.e., at
lower genetic distances) and are stronger (i.e., result in
lower mean probability of hybrids) than later-acting barriers
to survival. For the species-level data set a similar relationship
is evident, with stronger reproductive isolation occurring for
hybrid-production, although the E. goniocalyx outlier is
1836
increasing estimates of compatibility at low genetic distances
(see above and supplementary SI5 fig. S1, Supplementary
Material online, for a comparison with this outlier removed).
This more rapid development is also evident in the
higher accumulation rates (K) obtained in the mode of evolution modeling for the hybrid-production barriers (table 2).
Combining the hybrid-production and hybrid-survival
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Species-level dataset
(a) Hybrid-production
0.00
0.10
0.20
0.4
0.6
0.8
(b) Hybrid-survival
D = 75.4 % ***
0.2
0.4
(c) Combined
0.6
0.8
0.00
0.10
0.20
D = 99.2 % ***
0.2
0.4
0.6
0.8
Genetic distance (ADD1)
(d) Hybrid-production
D = 69.3 % *
0.4
0.0 0.2 0.4 0.6 0.8 1.0
0.0 0.2 0.4 0.6 0.8 1.0
0.30
Probability
0.2
0.0 0.2 0.4 0.6 0.8 1.0
0.30
D = 96.8 % ***
0.0 0.2 0.4 0.6 0.8 1.0
Section-level dataset
0.5
0.6
0.7
0.8
0.9
(e) Hybrid-survival
D = 42.8 % ***
0.4
0.5
(f) Combined
0.6
0.7
0.8
0.9
D = 76.1% ***
0.4
0.5
0.6
0.7
0.8
0.9
Genetic distance (ADD2)
FIG. 2. The relationship (mean [solid line] and its 95% confidence intervals [dashed lines]) between genetic distance (ADD) and hybridization rate
between Eucalyptus globulus and the section-level and species-level data sets. D, deviance explained by genetic distance. For both datasets hybrid
compatibility is shown at two stages, hybrid production measured at the early seedling stage (a and d), and hybrid survival at nine months (b and e).
Plots c and f are combined estimates of both hybrid production and survival. ***P < 0.001, *P < 0.05. The two genetic distances are derived from
estimates multiplicatively (see Materials and Methods)
improved the fit for both data sets (fig. 2c/f). There
was no detectable effect of style length (t1 = 0.003,
P = 0.99), or style length + ovary depth (t1 = 0.166, P = 0.87)
on compatibility, and these were not included in the final
model.
In order to test for evidence of snowballing incompatibilities, we followed the approach of Gourbière and Mallet
(2010), which tests three alternative models of evolution,
the snowball, linear and slowdown models. We fitted equivalent models to Gourbière and Mallet (2010), but used a
maximum-likelihood framework (see Materials and
Methods for details), rather than the previous least squares
approach. The modeling relies on the genetic distance
measure having a linear relationship with divergence time,
which appears to be the case here (fig. 3). Of the three
models assessed, we found overwhelming support for the
snowball model which provided the best fit in all situations
(the lowest Akaike’s information criterion [AIC]; table 2).
The snowball model received consistently strong support,
with wi (the probability of being the best model) ranging
from 0.75 to 1.00 (table 2). The only other model to receive
minor support was the slowdown model fitted to the hybridsurvival species level data (wi = 0.25; table 2). However, the
negative value of the a parameter in this model indicates a
slowdown model with a snowball shape (table 2; Gourbière
and Mallet 2010). There was no support for the linear model
being the best fit for any of the data sets (table 2). When
fitting the same data sets using the method of Gourbière and
Mallet (2010) we could not find statistical support for any
model over another, in any of the data sets (see supplementary material S2, Supplementary Material online). As in the
previous analysis, these results hold with the outlier
E. goniocalyx removed.
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Table 2. Comparison of Mode of Evolution Models (linear, snowball, and slowdown; see text) Based on Patterns of Hybrid Compatibility between
Eucalyptus globulus and Taxa in the Section- and Species-Level Data Sets (see Materials and Methods), at Different Stages of Reproductive Isolation.
Comparison/Data Set
Hybrid-production/section-level
Hybrid-survival/section-level
Combined/section-level
Hybrid-production/species-level
Hybrid-survival/species-level
Combined/species-level
ln L
1,007.6
876.0
974.0
106.5
97.7
102.4
905.4
753.0
868.7
1,008.7
777.3
785.3
103.9
94.3
94.4
996.6
744.5
749.1
Model
Linear
Snowball
Slowdown
Linear
Snowball
Slowdown
Linear
Snowball
Slowdown
Linear
Snowball
Slowdown
Linear
Snowball
Slowdown
Linear
Snowball
Slowdown
AIC
2,017.2
1,754.0
1,952.0
214.9
197.4
208.9
1,812.7
1,508.1
1,741.4
2,019.5
1,556.7
1,574.7
209.8
190.7
192.9
1,995.1
1,491.0
1,502.3
i
263.2
0.0
198.0
17.5
0.0
11.4
304.6
0.0
233.4
462.8
0.0
18.0
19.2
0.0
2.2
504.1
0.0
11.3
wi
0.000
1.000
0.000
0.000
0.997
0.003
0.000
1.000
0.000
0.000
1.000
0.000
0.000
0.754
0.246
0.000
0.996
0.004
K
832.7
3,789.8
704.6
218.9
933.8
161.6
909.5
4,456.1
780.7
243.6
405.1
123.3
64.5
112.6
34.3
243.1
407.8
123.7
a
NA
NA
1.097
NA
NA
1.556
NA
NA
1.097
NA
NA
1.109
NA
NA
1.100
NA
NA
1.111
Genec distance (ADD)
NOTE.—The best model is underlined. i = AICi AICmin, where AICmin is the AIC value for the best model; wi, Akaike weight, the probability of that model being the best fit; K,
accumulation rate; a, model optimization parameters (see Materials and Methods).
1
R² = 0.96
0.8
0.6
0.4
0.2
0
0
10
20
30
40
Divergence me (mya)
FIG. 3. The relationship between genetic distance among the five clades
in the phylogenetic network shown in figure 1, and the divergence time
between those clades estimated from the most recent dated phylogeny
(Crisp et al. 2011).
Discussion
Patterns of Reproductive Isolation
To our knowledge, this is the first large comparative study to
investigate the relationship between genetic distance and reproductive isolation in trees. We have found strong evidence
of a reduction in cross-compatibility with increasing genetic
distance in Eucalyptus, which is consistent with similar studies
across a range of taxa (Coyne and Orr 1989; Lee 2000;
Presgraves 2002; Malone and Fontenot 2008). We also
found consistent support for a “snowball effect” in the
strength of incompatibilities as genetic distance increased.
This finding is consistent with detailed genetic mapping studies assessing the number of genetic incompatibilities between
taxa (Matute et al. 2010; Moyle and Nakazato 2010), but is
inconsistent with previous comparative studies that look for a
1838
snowball effect in the strength of incompatibilities between
groups of taxa (Johnson 2006; Turelli and Moyle 2007;
Gourbière and Mallet 2010; Giraud and Gourbière 2012).
In investigating the snowball model, we employed a similar
approach to Gourbière and Mallet (2010) but addressed some
of the statistical shortcomings that they identified in their
modeling (see supplementary material S2, Supplementary
Material online). The maximum-likelihood approach we
used did change the outcome, providing support for the
snowball model, whereas with the least squares method of
Gourbière and Mallet (2010) we could not significantly differentiate between the models (see supplementary material
S2, Supplementary Material online). One possible problem
with this type of modeling is that crosses in this study were
not phylogenetically independent (i.e., all our crosses include
E. globulus). It is possible that a critical incompatibility could
exist between Clade I (including our focal taxa E. globulus) and
the rest of the phylogeny, and this would not be detected
with the current modeling approach. Although we cannot
rule this out, we believe that it is unlikely for two reasons. First,
there is evidence in the literature of similar reductions in
compatibility between species from Clades II and Clades III/
IV (Griffin et al. 1988; Ellis et al. 1991) indicating that the sharp
loss in compatibility between clades is not restricted to Clade
I. Second, evidence from a genetic mapping study showed
that postzygotic isolation between E. globulus (Clade I) and E.
grandis (Clade II) is a consequence of multiple genic incompatibilities rather than one or a small number of large changes
(Myburg et al. 2004), arguing against the presence of a major
branch/Clade I specific incompatibility.
The snowball theory suggests that, as the number of
incompatibilities between taxa increases, so does the
Reproductive Isolation in Eucalyptus . doi:10.1093/molbev/msv063
opportunity for negative epistatic interactions. This negative
epistasis subsequently causes the rate of DMI accumulation
to accelerate relative to genetic distance (Orr and Turelli
2001). Presgraves (2010), pointed out that the apparent absence of the snowball effect in comparative studies (Johnson
2006; Turelli and Moyle 2007; Gourbière and Mallet 2010;
Giraud and Gourbière 2012), was likely due to that fact that
the theory refers explicitly to the number of gene incompatibilities, not their magnitude. Hence, because the strength of
any single incompatibility can vary considerably, the overall
degree of incompatibility between pairs of taxa is a poor
proxy for the number of incompatibilities between those
taxa (Presgraves 2010).
As in previous comparative studies of hybrid compatibility
(Coyne and Orr 1989, 1997; Johnson 2006; Turelli and Moyle
2007; Gourbière and Mallet 2010; Giraud and Gourbière
2012), we cannot identify the number of incompatibilities
that underlie the patterns of reproductive isolation found
here in Eucalyptus. However, we can say that the strength
of incompatibility appears to snowball as divergence increases. This relationship may, or may not, be associated
with snowballing DMIs. Nevertheless, there is some genomic
evidence in Eucalyptus that is indicative of DMI-type processes playing a role in speciation. For example, despite significant genome size variation between E. grandis (Clade II)
and E. globulus (Clade I), the two species show high synteny
and colinearity, they share the same karyotype number
(2n = 22), and show no evidence of large inversions or deletions (Myburg et al. 2004; Hudson et al. 2012). In fact, the
variation in genome size is associated with thousands of small
dynamic changes (loses and gains) across the genomes of
both species (Myburg et al. 2014). Furthermore, genomewide scans show that there are thousands of allelic changes
differentiating the genomes of these species (Hudson et al.
2015). Such circumstances would be conducive to the development of DMI-like genic differences (Seehausen et al. 2014).
Costa e Silva et al. (2012) also showed that negative epistasis
causes hybrid incompatibility between the closely related E.
nitens (Clade I) and E. globulus (Clade I). Interestingly that
study also found that reduced hybrid survival was associated
with higher-order epistatic interactions, as would be expected
if more than two loci were interacting to reduce survival
(Costa e Silva et al. 2012), which is in line with the mechanism
that is predicted to drive the snowball effect. Therefore, although we cannot comment explicitly on the genetic mechanisms driving the patterns found, when our results are
viewed in the context of the existing literature, it appears
that the evolution of reproductive isolation in Eucalyptus is
generally consistent with the snowball theory.
Despite the overall trend for reduced compatibility with
increasing genetic distance, the shape of the response curves
at the different developmental stages indicates that early
acting postmating barriers (including prezygotic barriers)
evolve more quickly and are stronger than later acting postzygotic barriers (fig. 2; table 2). This finding is consistent with
the general picture in animal systems (Coyne and Orr 1989,
1997; Presgraves 2002; Stelkens et al. 2010), and with nonphylogenetic studies assessing barriers between pairs of plant taxa
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(Ramsey et al. 2003; Martin and Willis 2007; Lowry et al. 2008).
However, our findings are inconsistent with the few plantbased studies that address the relationship between early and
late acting barriers in a comparative phylogenetic framework
as we have done (Widmer et al. 2009). Some of these studies
found no clear predominance of prezygotic over postzygotic
barriers (Scopece et al. 2013), whereas others reported that
postzygotic barriers were stronger (Jewell et al. 2012; Meiners
and Winkelmann 2012) and evolve more quickly than prezygotic barriers (Jewell et al. 2012). Although our hybridproduction measure confounds postmating prezygotic
barriers with early acting postzygotic barriers, there is a
body of evidence suggesting that postmating prezygotic barriers are important mechanisms of reproductive isolation in
eucalypts (Sedgley et al. 1989; Sedgley and Smith 1989; Gore
et al. 1990; Ellis et al. 1991; Dickinson et al. 2012). For example,
Dickinson et al. (2012) recently found that prezygotic barriers
operating on pollen in the stigma and style were significantly
stronger than postzygotic barriers to seed development in the
eucalypt groups studied. Therefore, although not conclusive,
our results provide some of the first comparative support for
postmating, prezygotic barriers being stronger and evolving
more quickly than postzygotic barriers in a speciose plant
group, as seems to be the case in animals (Coyne and Orr
2004).
Long-lived perennial plants, including trees, often have an
extended juvenile stage where barriers—to survival for
example—might act to prevent hybrids contributing to the
next generation. Because previous comparative studies of
reproductive isolation in plants have focused on annual flowering herbs (Widmer et al. 2009; Levin 2012), this extendedpre-reproductive barrier is relatively poorly understood (Levin
2012). Although informative, our assessment of hybrid-survival
is still likely to underestimate the true strength of this prereproductive barrier. Several studies have found that selection
against interspecific eucalypt hybrids also occurs later in development (2–10 years after field planting; Lopez et al. 2000;
Barbour, Potts, and Vaillancourt 2006; Costa e Silva et al.
2012). Even within section Maidenaria (Clade I) this reduced
juvenile fitness in hybrids is thought to limit gene flow and help
maintain allopatric/sympatric species boundaries in nature
(Lopez et al. 2000; Costa e Silva et al. 2012; Larcombe et al.
2014). Despite the difficulties in gathering long-term survival
data for hybrids between multiple species, a proper understanding of the pre-reproductive hybrid-survival barrier in
long-lived perennial plants will require data on survival from
germination to reproductive maturity in species with extended
juvenile stages.
Hybridization and the Timing of Speciation in
Eucalyptus
In addition to the previously recognized complete barrier to
hybridization between the major subgenera in Eucalyptus
(Griffin et al. 1988), this study has identified that significant
postmating barriers extended to phylogenetic clades within
subgenera. These barriers become stronger as genetic divergence increases and are likely to result in a parallel increase in
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Larcombe et al. . doi:10.1093/molbev/msv063
reproductive isolation. The results here clarify the genus-wide
taxonomic patterns in hybridization identified by Griffin et al.
(1988), and intersectional patterns of pollen–pistil interactions found by Ellis et al. (1991). For example Ellis et al.
(1991) found a pollen–pistil barrier between sections
Bisectae I (Clade III) and Exsertaria (Clade II) that was not
present between Bisectae I and Adnataria (both Clade III).
Griffin et al. (1988) also found higher levels of natural hybridization between co-occurring species from sections
Maidenaria (Clade I), than between co-occurring species
from sections Maidenaria and Exsertaria (Clade II). The phylogenetic approach taken here supports, quantifies, and clarifies these barriers identified on the basis of taxonomic
relationships.
The absence of strong reproductive isolation in closely related species is also consistent with evidence of historical introgression and possible reticulate evolution in eucalypts
(McKinnon et al. 2004; Nevill et al. 2014). For example,
there is evidence of geographically structured chloroplast
sharing between species within sections in both of the
main Eucalyptus subgenera (Symphyomyrtus and
Eucalyptus), which is indicative of locally common historical
introgressive hybridization between closely related species
(McKinnon et al. 2004; Nevill et al. 2014). There is also
some evidence of less-common reticulate evolution between
sections within Clade II, and between Clade I and Clade II
(Poke et al. 2006). However, we are unaware of any evidence
of introgression between more distantly related species (i.e.,
between Clades I/II and Clades III/IV). Therefore, our results
seem consistent with both contemporary and historical patterns of hybridization in Eucalyptus. It is worth noting that the
effect of possible reticulate evolution on the estimates of divergence used in our analysis is not completely clear.
However, the strong linear relationship between our estimates of divergence (Additive Dollo Distance [ADD]), and
independent estimates of divergence time (fig. 3; Crisp et al.
2011), suggests any effect across the phylogeny is relatively
minor.
The time needed for pairs of taxa to achieve complete
reproductive isolation is a poorly understood, but important,
aspect of plant speciation (Rieseberg and Willis 2007; Levin
2012). Our ability to quantify crossability at various positions
on the phylogenetic network allows us to estimate the timing
of reproductive isolation from dated phylogenies. The most
recent molecular clock approach in eucalyptus (Crisp et al.
2011) suggests that species divergence within sections of
Symphyomyrtus occurred approximately 3–10 Ma, implying
that it can take at least 3 My for crossability to decline by 54%
(fig. 1). Divergence between Clades I/II and Clades III/IV is
dated to 21–31 Ma (Crisp et al. 2011), implying that complete
reproductive isolation (100%; fig. 1) can take at least 21 My in
Eucalyptus. These comparisons are obviously approximate,
and do not take into account premating barriers that
might operate to prevent mating (Moyle et al. 2014).
However, postmating barriers are of particular interest because they include intrinsic postzygotic barriers, which are
considered largely irreversible, and thus particularly effective
at promoting speciation (Matute et al. 2010). Our approach is
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also analogous to the one used by Levin (2012) to produce
the first wide-ranging assessment of the timing of hybrid
sterility in plants. That study found a pronounced reduction
in hybrid fertility after 4 My (Levin 2012), which is in line with
the approximately 50% reduction evident here within Clade I.
It also showed, as we have done, that partial cross-compatibility between tree species can be maintained for a very long
time, up to 50 My in Plantanus (Levin 2012).
Practical Implications
The phylogenetic patterns in crossing success identified here
in Eucalyptus have two major practical applications. First,
there are around 538,000 ha of E. globulus plantations growing
across southern Australia, mainly outside the species’ native
range (Gavran and Parsons 2011). Concerns have been raised
that these plantations may pose a genetic risk to indigenous
eucalypt species through pollen mediated gene flow and introgression (Potts et al. 2003; Barbour et al. 2008). Based on
the well-known complete barrier to hybridization between
subgenera, it was previously thought that any of the 484
Symphyomyrtus species that occurred within the pollen dispersal range of E. globulus plantations were potentially at risk
of introgression (Potts et al. 2003; Barbour et al. 2008).
Although advantageous alleles can overcome even strong
barriers to introgression (Goodman et al. 1999; Barton
2001), in Eucalyptus there is mounting evidence that the
combined effect of pre- and postzygotic barriers to hybrid
production, fitness and survival, limits gene flow between
even closely related species from the same section (Lopez
et al. 2000; Barbour et al. 2005a, 2005b; Barbour, Potts, and
Vaillancourt 2006; Barbour, Potts, Vaillancourt, and Tibbits
2006; Costa e Silva et al. 2012; Larcombe et al. 2014). This
may at least partially explain the common natural cooccurrence of eucalypt species from the same taxonomic
section (Lopez et al. 2000; Larcombe et al. 2014). In light of
these previous studies, our results suggest that species in
Clades III and IV of subgenus Symphyomyrtus are likely to
be reproductively isolated from E. globulus, probably reducing
the number of species potentially at-risk of hybridization to
138 (a 71% reduction), and these will only be at risk if they
occur within the pollen dispersal zone of a plantation
(Barbour et al. 2008). Furthermore species in Clade II
appear to be at a far lower risk of introgression than species
within Clade I, although the potential for introgression of
advantageous alleles (Barton 2001), as well as evidence of
historical-introgression between Clades I and II (Poke et al.
2006), means some risk remains. The crossability estimates
presented will therefore enable more informed assessment of
genetic risk during plantation planning and establishment in
Australia.
Second, the estimates of crossability will be of interest to
tree breeders. These are the first broad estimates of crossability between sections in the economically important subgenus
Symphyomyrtus (Grattapaglia et al. 2012), and they match
well the results from long-term hybrid breeding programs. For
example, hybrids between species in sections Latoangulatae
and Exsertaria (both Clade II, fig. 1) are utilized more widely,
Reproductive Isolation in Eucalyptus . doi:10.1093/molbev/msv063
and show fewer abnormalities than hybrids between species
in Clades I and II (Potts and Dungey 2004). In addition, some
crosses within Clade I (e.g., E. globulus gunnii) show very few
abnormalities (Potts and Dungey 2004), whereas others, that
are now known to be more divergent (E. globulus x nitens;
McKinnon et al. 2008), do show morphological abnormalities
(Potts and Dungey 2004) and outbreeding depression due to
negative epistasis (Costa e Silva et al. 2012). Therefore, the
estimates of cross-success and its relationship with genetic
distance may help guide species selection for hybridization
programs.
Conclusion
We found that hybrid compatibility in Eucalyptus declines as
genetic distance increases, and that the strength of incompatibility seems to snowball with divergence. This second
finding is consistent with detailed genetic mapping studies
that show that the number of incompatibilities can snowball
with increasing divergence due to compounding negative
epistasis, as predicted by the Dobzhansky–Muller model.
Conversely, our findings are inconsistent with previous comparative studies investigating the strength of hybrid incompatibilities, which have failed to detect a snowball effect. We
cannot comment on the specific genetic control of the pattern found here in Eucalyptus, but the results are broadly
consistent with a snowballing epistatic Dobzhansky–Muller
model. We also found that early acting barriers, including a
prezygotic component, develop first and are stronger than
later-acting postzygotic barriers. The phylogenetic approach
allowed us to estimate that complete reproductive isolation
can take 21–31 My to evolve in Eucalyptus. The study has also
provided strong evidence that significant barriers to hybridization operate within subgenera in Eucalyptus, including
some that result in effectively complete reproductive isolation. This has direct implications for assessing the risk of
exotic gene flow from E. globulus plantations in Australia,
and should be informative for hybrid eucalypt breeding in
forestry.
Materials and Methods
Crossing
Pollen was collected from 14 unrelated E. globulus trees to
provide a broad genetic base for the crossing program (supplementary material S1, Supplementary Material online).
Pollen was extracted, and viability tested using the techniques
of Potts and Marsden-Smedley (1989), except that the boric
acid was increased to 150 ppm in the agar medium. Only
pollen with over 5% viability was used. In total, 99 species
were crossed, with E. globulus always used as the pollen
parent, thus the results specifically pertain to crosses in this
one direction. Most of the crossing was undertaken at a specialist eucalypt research arboretum in South Australia
(Currency Creek Arboretum; www.dn.com.au, last accessed
March 21, 2015). Additionally, for 13 species, native forest
and/or ornamental trees in Tasmania, Western Australia,
and South Australia were used (supplementary material S1,
Supplementary Material online).
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Crossing was undertaken between May 2010 and May
2011, with one to five trees per species being crossed.
Eucalypts are bisexual preferential outcrossers (the average
outcrossing rate is 0.70; Byrne 2008) that display varying
levels of self-incompatibility (Pound et al. 2002). Two main
crossing approaches were used; supplementary pollination
and cut-style pollination (see treatment details below). The
supplementary pollination technique involves simply dabbing
pollen onto the receptive stigma of open flowers (“supplementary” pollination; Barbour et al. 2005a). The cut-style approach was aimed at circumventing unreceptive stigmas and
stigma incompatibilities (Boland and Sedgley 1986; de Sousa
and Pinto-Junior 1994; Oddie and McComb 1998). This involved removing the stigma with a razor blade and applying
pollen directly to the surface of the cut style (cut-style pollination; Cauvin 1988; Patterson, Gore, et al. 2004). On each
tree, seven treatments were applied, each randomly allocated
to a different branch (mean flowers/branch = 9.6, number of
treatment branches = 1,039). Each individual E. globulus
pollen was kept separate, with each treatment using single,
randomly selected E. globulus pollens, to enable subsequent
molecular validation of hybrid combinations using parentage
analysis. Treatments 1–3 were supplementary pollinations.
Treatment 4 was cut-style pollination, applied to open flowers at the same developmental stage as those pollinated in
treatments 1–3. Treatment 5 aimed to exclude pollen from
nontarget species and involved cut-style pollination with the
following additions: Pollination was carried out on flowers
prior to anthesis by removing the operculum (usually just
prior to it being shed naturally), cutting the style, applying
the pollen, and isolating the pollinated flowers in waxed
paper bags for at least 3 months (unless the bag failed).
Treatments 4 and 5 used one of the three pollens used in
treatments 1–3. Treatments 6 and 7 were open-pollinated
controls, which were flowers in the same state as the treatment branches, treated in the same way (e.g., nonopen buds
removed), except that E. globulus pollen was not applied.
These controls were used to judge whether any failure to
produce seed on the treatment branches was influenced by
maternal/environmental effects, and also to provide pure
species specimens for comparison with hybrid progeny
(pure specimens would be self or outcross from surrounding
con-specifics). To quantify differences in flower size and to
test for any size effect on crossing success, three flowers from
each species were dissected and two measurements were
made: 1) From the stigma to the top of the ovaries and 2)
from the stigma to the base of the ovaries.
Capsules were collected at maturity, when the valves were
well-developed (Barbour et al. 2005a), 10–26 months after
pollination. Capsules were dried and the seed extracted.
Eucalypt seeds are often difficult to identify because they
are small, variable in shape and color, and aborted ovaries
produce chaff that can be indistinguishable from true seeds
(Boland et al. 1980). Therefore, given the number of species
crossed and in line with previous studies (Barbour et al. 2005a;
Larcombe et al. 2014), the number of germinates/seedlot was
used to determine the number of viable seed produced. Due
to the large number of seedlots (519, down from the 1,039
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Larcombe et al. . doi:10.1093/molbev/msv063
due to failure to produce seed or capsules), sowing was done
in batches over 12 months between January 2012 and January
2013. Seedlots were randomized within and between batches,
except that each batch was designed to have as near as possible equal representation of each taxonomic section. Seedlots
(all extracted seed and chafe) from each tree were sown on
the same day into trays (450 295 250 mm) containing
moist potting mix, and the soil surface was covered with
vermiculite after sowing. Germination was undertaken in a
controlled environment facility under a light regime of 16 h
light to 8 h dark at 22 C and 70% humidity. Three weeks after
germination the boxes were moved into a conventional glasshouse and, after scoring (see below), moved outside.
Hybrids were identified initially on the basis of morphological deviation from the pure parental type (see supplementary material S2, Supplementary Material online)
approximately 2–6 weeks after germination (when hybrid
differences were first expressed) and monitored thereafter
to ensure consistency of developmental deviations from the
pure morphology. Seedling morphology in eucalypts is often
diagnostic (Pryor and Johnson 1981), and has been used
widely in the identification of hybrid eucalypts (see supplementary material S2, Supplementary Material online; and
Griffin et al. 1988; Barbour et al. 2005a, 2007; Larcombe
et al. 2014). Additionally, subsets of each putative hybrid
combination were validated with parentage analysis using
ten microsatellite loci (see supplementary materials S1 and
S2, Supplementary Material online). A conservative approach
was taken to the visual identification of hybrids so that, in the
case of any uncertainty, the seedlings were checked with parentage analysis. There was one cross type where the pure
parental species morphology made identification of hybrids
somewhat ambiguous (E. globulus E. mannifera). In that
case, all 28 ambiguous progeny were assessed with parentage
analysis. Given the conservative approach to hybrid detection
we assume a false negative rate close to zero. If overcrowding
was affecting the health of plants in a box, pure (nonhybrid)
samples were removed to reduce competition as soon as they
could be distinguished from hybrids. All surviving hybrids and
representatives of the pure species were transferred to individual pots after scoring. All pots were randomized
and moved outside. Survival of the hybrid and pure seedlings
was assessed in September 2013, which was at least 9 months
postgermination. Therefore, compatibility was assessed at
two different life history stages: First, the early seedling stage
which assessed barriers from pollination to viable seedling
production (hereafter termed hybrid-production); and second, hybrid survival at 9 months which assessed barriers to
F1 seedling survival (hereafter termed hybrid-survival).
The hybrid-production data only consider crosses done
using the supplementary pollination technique which aims
to mimic natural pollination. This makes biological sense because the removal of the style (under the cut-style approach)
potentially removes barriers that would operate in nature.
However, the hybrid-survival data include hybrids produced
under any of the crossing approaches (supplementary, cutstyle). The reason for this is that regardless of how these
hybrids were produced, we cannot rule out hybrids between
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these combinations arising in nature; thus, the question of
their survival is still relevant. It is possible that both crossing
techniques allow for subsequent (or even prior) nontarget
pollen deposition, because the flowers are open prior to crossing and no isolation is used postpollination (except in treatment 5, see above). The cut-style technique probably does
reduce the likelihood of nontarget pollination because the act
of removing the stigma is likely to reduce the length of time
the flower is receptive.
Phylogenetic and Statistical Methods
To investigate phylogenetic patterns in hybridization, as well
as the association between hybridization rate and genetic
distance, we used two data sets derived from genome-wide
markers (DArTs; Sansaloni et al. 2010), in which the divergence estimates for species coincident to both data sets are
strongly positively correlated (R2 = 0.85; supplementary material S2, Supplementary Material online). The DArT approach
provides data for thousands of dominant molecular markers
that assay genome wide patterns in genetic diversity (Petroli
et al. 2012). The first DArT data set was used to calculate the
average genetic distance between E. globulus and each of the
13 taxonomic sections covered in the crossing program, and is
referred to as the “section-level data set” from here-after. This
section-level data set included 8,350 markers and has been
published previously by Steane et al. (2011). Of the 94 species
used by Steane et al. (2011), 78 belong to the taxonomic
sections used in our experiment (supplementary material
S1, Supplementary Material online). Part of the distance
matrix produced by Steane et al. (2011) that corresponded
to the sections used in the crossing experiments here was
extracted and used to produce a phylogenetic network (see
below and fig. 1). It should be made clear that the species in
this phylogenetic analysis do not correspond exactly to the
species used in this study, with 22 of the 78 species being
coincident (supplementary material S1, Supplementary
Material online). However, sectional level taxonomy in this
group is relatively well-resolved (Byrne 2008), our phylogenetic reconstruction is congruent with other analyses using a
range of techniques (Steane et al. 2002; Crisp and Cook 2007;
Crisp et al. 2011; Steane et al. 2011) and the species used for
crossing were representative of each section. Therefore, we
think it is very unlikely that we have misassigned species and
their compatibility-estimates when calculating the sectionlevel estimates. This is supported by the fact that our genetic
distance explains 96% of the variation in hybrid compatibility
at the section-level (fig. 2). A second DArT data set was used
to calculate genetic distance between E. globulus and the 21
species that were crossed (and produced viable seed) from
the five taxonomic sections that are most closely related to E.
globulus (Grattapaglia et al. 2012) including Maidenaria (the
section to which E. globulus belongs), Exsertaria, Incognitae,
Latoangulatae, and Racemus (Brooker 2000). This data set is
here-after referred to as the “species-level data set.” The
species-level data set is a subset of a large data set that consists of 558 samples including 191 taxa genotyped with 5,050
DArT markers, which is to be published in full elsewhere
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Reproductive Isolation in Eucalyptus . doi:10.1093/molbev/msv063
(Jones RC, unpublished data). Pairwise genetic distances
(ADD, a measure developed specifically for DArT data that
is expected to increase linearly with time; Woodhams et al.
2013) between E. globulus and the species of interest were
extracted from the full data set (and averaged where multiple
samples of the same species were available; see supplementary material S1 [Database], Supplementary Material online).
For consistency with the DArT data sets, the crossing results
of three maternal subspecies were merged to the species level
(supplementary material S1 [Database], Supplementary
Material online). Many of the samples used to create these
phylogenetic data sets were collected from the arboretum
where most of the crossing was done. This resulted in
either the crossed tree or a sibling (from the same seed lot)
being used in the generation of the genetic distance for 26 of
the 99 species (supplementary material S1 [Database],
Supplementary Material online).
A phylogenetic network using ADD was produced from
the section-level data set (of 78 species) in Splitstree4 using
the neighbor-net method (Bryant and Moulton 2004; Huson
and Bryant 2006). Clades were identified based on the topology of the network and comparisons with a similar analysis
(Grattapaglia et al. 2012). The positions of taxonomic sections
within the clades were identified (fig. 1). The 64 species that
produced viable seed were assigned to positions on the phylogenetic network (i.e., their taxonomic section; supplementary material S1 [Database], Supplementary Material online)
to reveal patterns of hybridization (fig. 1). Species that failed
to produce any viable seed (treatment and control) were
excluded from the analysis because it is impossible to distinguish between maternal effects on fecundity and incompatibility in that situation. Contingency Chi-square tests were
used to compare the number of hybridizing species in the
different sections (pooling both supplementary and cut-style
pollination techniques). Predispersal crossability with E. globulus was calculated at various points on the phylogenetic
network by averaging the cross success (number of hybrids/
number of progeny arising from the treatments) for all species
in the clade(s) of interest. A second crossability estimate that
accounted for hybrid survival was produced by multiplying
the first estimate by the proportion of hybrids surviving in the
clade(s). Both these estimates were adjusted by the known
intraspecific cross-compatibility in E. globulus when using the
same supplementary crossing approach (95% success rate for
applied pollen; Patterson, Vaillancourt, et al. 2004). To test
whether ADD accumulates linearly with time across the phylogeny, we compared the genetic divergence between the five
clades in figure 1, with their divergence time (Ma) according
to the most recent dated phylogeny (Crisp et al. 2011).
Generalized Linear Models were used to assess the
relationship between genetic distance and hybridization.
The relationship between genetic distance (ADD) and
hybrid-production (number hybrids/number plants produced) was tested, as was the relationship between ADD
and hybrid survival, and the relationship between ADD and
pure seedlings survival (in independent models). Additionally
an estimate of the combined effect of hybridization rate and
survival of the hybrids was compared with ADD. The
combined rate was calculated by multiplying the number of
hybrids produced by the probability of survival and adjusting
that number by the known intraspecific cross-compatibility
in E. globulus (95%; Patterson, Vaillancourt, et al. 2004).
Logistic regression fits with logit link functions were used to
model all three isolation levels (i.e., hybrid-production, hybridsurvival, and combined) for both the section-level and species-level data sets. The two flower-size measurements
(stigma to the top of the ovaries, and stigma to the base of
the ovaries) and their interaction were included as covariates
in the logistic models for the species-level data sets. Model
simplification was undertaken by removing nonsignificant
terms starting with the higher order interactions. A quasibinomial distribution was used to account for overdispersion
where necessary (Zuur et al. 2009). The “t” distribution was
used for significance testing when fitting quasibinomial
models, whereas the “z” distribution was used when fitting
binomial models. All logistic model fitting was undertaken in
R version 2.14.1(R core development team) using the package
MASS (Venables and Ripley 2002).
The mode of evolution modeling used the general approach outlined by Gourbière and Mallet (2010), which
uses linear regression to test whether or not patterns in the
strength of incompatibility best fit a snowball, linear or slowdown curve, assuming that one of these models is correct (see
Gourbière and Mallet 2010 for a theoretical explanation of
these curves). We fitted models equivalent to those of
Gourbière and Mallet (2010) using a likelihood-based approach to overcome some statistical issues outlined in that
article (see supplementary material S2, Supplementary
Material online, for more details including a comparison of
the analysis using the least squares approach of Gourbière and
Mallet 2010).
In the linear model, incompatibilities (I) accumulate linearly with time (t) at rate k
IðtÞ ¼ kt:
The incompatibilities act multiplicatively. The model assumes that each incompatibility has a small deleterious effect
on fitness (s) so the compatibility, C (i.e., probability of successfully forming offspring) of two species that are separated
by time t is
CðtÞ ¼ ð1 sÞIðtÞ :
The likelihood is the probability of getting the observed
data expressed as a function of the parameters in the model
(in this case the only parameter is k).
Lðdata j kÞ ¼
Y
Cðti Þxi ð1 Cðti ÞÞni xi ;
i
where ni is the number of attempted crosses with species i, xi
is the number of successful crosses with species i, and ti is the
genetic distance (ADD) between E. globulus and species i. The
value of k that gives the largest likelihood is then chosen. s was
fixed to be 0.01 (testing different values showed that choice of
number here did not affect the likelihood score—i.e., different
1843
Larcombe et al. . doi:10.1093/molbev/msv063
values of s result in different values of k but the curve C(t) is
the same).
The only difference between the linear and the snowball
models is that the epistatic nature of the accumulating incompatibilities, and these are modeled using the square of
time IðtÞ ¼ kt2 ; apart from this, the expression for C(t) and
the likelihood remain unchanged.
Following the notation in Gourbière and Mallet (2010), the
slowdown model expressed in terms of compatibility is a twoparameter model
e3 lnð1 þ atÞ
;
CðtÞ ¼ Exp a
where the e3 parameter encapsulates both k, the rate at which
incompatibilities accumulate, and s, the deleterious effect of
incompatibilities; the a parameter determines the rate of
“slowdown.”
The hybrid-production, hybrid-survival, and combined
crossability estimates were again used as measures of compatibility, with both the section-level and species-level data
sets tested for all three models. All model fitting was undertaken in R version 2.14.1 (R Development Core Team 2011)
using the optim function. The Brent method (Brent 1973) was
selected for both the linear and snowball models (as it is
recommended for one-dimensional optimization) and the
Nelder and Mead method (Nelder and Mead 1965) was
used for the slowdown model. The three alternative models
were compared using the AIC under the framework suggested in Burnham and Anderson (1998) with an Akaike
weight (loosely interpreted as model probabilities) calculated
for each model for each of the six data sets. For comparison,
all data sets were also analyzed using the method of
Gourbière and Mallet (2010) (see supplementary material
S2, Supplementary Material online).
Supplementary Material
Supplementary materials S1 and S2 are available at Molecular
Biology and Evolution online (http://www.mbe.oxfordjournals.
org/).
Acknowledgments
For assistance in the field, the authors thank Guy and Simone
Russel, James Worth, and Paul Tilyard. They also thank Forest
and Wood Products Australia, as well as the Australian
Research Council (Discovery Grants DP0986491 and
DP0770506) for funding this research.
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