Botanical Journal of the Linnean Society (1994), 116: 13" 31. With 3 figures
Reproductive isolation mechanisms among
four closely-related species of Conospennum
(Proteaceae)
DAVID A. MORRISON, MARGARET McDONALD, PETER BANKOFF,
PAUL QUIRICO
Department if Applied Biology, Universiry
NS. W 2007, Australia
if Technology,
Sydney, P. 0. Box 123, Broadway,
AND
DAVID MACKAY
Botany Building (A12), Universiry
if Sydney, NS. W
2006, Australia
Received January 1994, acceptedfor publication June 1994
The effectiveness of geographical isolation, ecological isolation, temporal isolation, mechanical
isolation, ethological isolation, cross-incompatibility, hybrid inviability, hybrid sterility and hybrid
breakdown as practical barriers to gene flow in the field between Conospennum taxijolium, C. ericijolium,
C. ellipticum and C. longijolium has been quantified. The barriers to gene flow between C. ericifolium
and C. ellipticum are completely effective, owing to their allopatric distributions" The barriers to
gene flow between C. taxifolium and these two species are only partially effective, as their ecological
separation breaks down in intermediate habitats, and partially-fertile F 1 plants can grow in the
areas of overlap. The barriers to gene flow between C. longifolium and the other three species are
almost completely effective, as cross-incompatibility is very high and the F 1 plants are femalesterile.
ADDITIONAL KEY WORDS:-allopatry -- gene flow barriers - hybrids - hybrid zones incompatibility - speciation - sympatry.
CONTENTS
14
Introduction
Material and methods
The species
Geographical isolation
Ecological isolation
Temporal isolation "
Mechanical isolation
Ethological isolation
Cross-incompatibility
Hybrid inviability .
Hybrid sterility
Hybrid breakdown .
Results
15
15
15
16
16
17
17
17
18
19
20
20
David Mackay's present address: National Herbarium of New South Wales, Royal Botanic Gardens, Mrs
Macquaries Road, Sydney, N.S"W. 2000, Australia.
13
0024-4074/94/009013+ 19$08.00/0
© 1994 The Linne an Society of London
D. A. MORRISON ET AL.
14
Geographical isolation
Ecological isolation
Temporal isolation .
Mechanical isolation
Ethological isolation
Cross-incompatibility
Hybrid inviability .
Hybrid sterility
Hybrid breakdown .
Discussion .
Acknowledgements .
References .
20
20
22
22
22
23
24
25
26
26
30
30
INTRODUCTION
The basis of speciation is usually taken to be the establishment of intrinsic
barriers to gene flow between closely-related populations by the development
of reproductive isolation (Littlejohn, 1981; Mayr, 1982). It is usually impossible
to study speciation as it actually happens (Mayr, 1982; Arthur, 1984), but
comparisons of gene flow between closely-related species are useful in
complementing direct studies. Comparative studies are open to the criticism
that it is impossible to tell whether the observed differences between species
evolved before, during or after speciation (Arthur, 1984), but these studies do
allow an examination of the taxonomic distribution and effectiveness of the
various known reproductive isolation mechanisms. The study reported here is
therefore an attempt to quantifY the extent to which the potential reproductive
isolation mechanisms are effective in impeding gene flow between a group of
closely-related species in their natural habitats.
Conospermum (Proteaceae) is a genus of about 40 endemic Australian species
of perennial sclerophyllous shrubs Uohnson & Briggs, 1975), six of which occur
in New South Wales (Mackay, 1991). Two of these New South Wales
species are morphologically quite distinct from the others, but morphological
intermediates are known to occur rarely in the Sydney region between the
other four species:- C. taxifolium Smith, C. ericifolium Smith, C. ellipticum Smith
and C. longifolium Smith Uohnson & McGillivray, 1975; Beadle, Evans & Carolin,
1982; Mackay & Morrison, 1989; Mackay, 1991 ). These intermediates are
presumed to be hybrids, which form because of incomplete reproductive isolation
between the four gene pools or when these isolation mechanisms break down
(Mackay & Morrison, 1989). Mackay & Morrison (1989) hypothesized that
geographical isolation may be primarily responsible for preventing gene flow
between C. ericifolium and C. ellipticum, while ecological segregation isolates C.
taxifolium from these two species. They suggested that C. longifolium is isolated
from the other three species by a post-fertilization mechanism. Our experiments
were designed to test these hypotheses.
Our study therefore sought to quantify the extent to which each of the
following potential reproductive isolation mechanisms is effective in impeding
gene flow between the four Conospermum species (adapted from Levin, 1978;
Grant, 1981);
(1) Pre-pollination mechanisms (prevent heterospecific pollen from reaching
stigma)
(a) geographical isolation (large-scale spatial separation)
(b) ecological isolation (habitat specialization)
REPRODUCTIVE ISOLATION IN CONOSPERMUM
15
(c) temporal isolation (differences in flowering times)
(d) mechanical isolation (differences in floral structure)
(e) ethological isolation (specialization or spatial separation of pollen vectors)
(2) Post-pollination mechanisms (genetic incompatibility among heterospecifics)
(i) Pre-zygotic mechanisms (prevent fusion of gametes)
(a) cross-incompatibility (fertilization not successful)
(ii) Post-zygotic mechanisms (prevent hybrid offspring from reproducing)
(a) hybrid inviability (F 1 seeds do not germinate or grow)
(b) hybrid sterility (F 1 plants do not produce viable seeds)
(c) hybrid breakdown (F 2 plants do not produce viable seeds).
We wished to quantify what appear to be only partially successful reproductive
barriers between these four species, because each species appears to retain its
morphological identity in the presence of hybrids (Mackay & Morrison, 1989).
We also wanted to explain the distribution and abundance of these hybrids as
a consequence of the types of reproductive isolation mechanisms that are
operating between the four parent species.
MATERIAL AND METHODS
The species
Conospermum taxijolium is widespread along the eastern Australian coast, from
Elliot River in Queensland to Wilsons Promontory in Victoria, as well as
occurring in Tasmania. It is morphologically fairly variable in leaf characteristics,
and Johnson & McGillivray (1975) recognized a Tasmanian form and a Blue
Mountains form as well as the common form. The Tasmanian and Blue
Mountains forms were excluded from our study because their taxonomic status
is uncertain, and moreover they are allopatric to the common form. C. ericijolium
and C. ellipticum are closely related to C. taxijolium (Mackay & Morrison, 1989),
and are confined to the Sydney region of New South Wales.
C. longifolium is more distantly related to the other three species Uohnson &
McGillivray, 197 5; Mackay & Morrison, 1989), and occurs along the New
South Wales coast between Newcastle and Jervis Bay. It is morphologically
more variable than the other three species, especially in leaf width, and Johnson
& McGillivray (1975) recognized three subspecies, which were not distinguished
in our study.
Plants that are morphologically intermediate between those of C. taxijolium
and C. ericijolium and between those of C. taxijolium and C. ellipticum usually
occur as rare discrete populations of morphologically homogeneous individuals
(Mackay & Morrison, 1989). No populations of intermediates between C.
ericijolium and C. ellipticum have been reported (Mackay & Morrison, 1989).
Plants that are morphologically intermediate between those of C. longijolium and
those of the other three species only occur as rare individuals within a
population of one of the putative parents (Mackay & Morrison, 1989). The
artificial cross-pollinations and subsequent cultivation of the offspring described
below confirm that all of these morphological intermediates are of hybrid origin.
Geographical isolation
Data from dried specimens housed in the National Herbarium of New South
Wales (NSW) and the John Ray Herbarium (SYD), plus extensive field surveys
16
D. A. MORRISON ET AL.
during the spnng of 1988, were used to compile a detailed distribution map
of C. taxifolium, C. ericifolium and C. ellipticum. A detailed distribution map of C.
longifolium (76 records) was presented by Johnson & McGillivray (1975).
Ecological isolation
The degree of ecological separation between the species was quantified in
two ways:
(1) the physical and chemical characteristics of the soils in which the plants
occur; and
(2) the floristic composition of the communities in which the plants occur.
Climatic, topographical and geological differences were not quantified because
none of these factors appeared to be important in ecological isolation within
the restricted geographical area in which any of the four species are sympatric.
Ten areas were chosen for these analyses: two replicate areas containing a
population of each of the four Conospermum species (C. taxifolium: La Perouse,
O'Hares Creek; C. ericifolium: Patonga, Terry Hills; C. ellipticum: Kurnell, O'Hares
Creek; C. longifolium: Patonga, O'Hares Creek), plus one area with a population
of C. taxifolium-ericifolium intermediates (Bouddi National Park) and one with a
population of C. taxifolium-ellipticum intermediates (O'Hares Creek).
For the soil analyses, two random soil samples, each from the top 20 em
of the soil profile (excluding the A 0 horizon), were collected from each site
with an auger. Two replicate analyses were performed on each soil sample for
each of the following characteristics, using the methods of Allen (1989): percent
sand (particles 0.05-2.0 mm diam.), per cent silt (0.002-0.05 mm diam.), and
per cent clay (> 0.002 mm diam.) by the gravimetric method; per cent air-dry
moisture; pH in a water solution; per cent soil organic matter by the wet oxidation
method; total phosphorus by the ascorbic acid method; and exchangeable calcium,
magnesium, potassium and sodium by the ammonium acetate method. For data
analysis, the mean value of each characteristic at each site was calculated.
For the floristic composition, all vascular plant species were recorded as
present or absent in each of 20 contiguous 2 m square quadrats along a
transect through each site. A total of 166 species were identified at the ten
study sites, varying from 23 to 51 species per site. For analysis, the mean
frequency of each species at each site was calculated.
The data were analysed by redundancy analysis (ter Braak, 1988). This is a
constrained ordination technique based on principal components analysis that
assesses the degree to which two data sets show co-variation (ter Braak &
Prentice, 1989). The two data sets analysed were the floristic data and the soil
data.
Temporal isolation
Only overlap in flowering season
separation of reproductive maturity
can remain open and receptive for
Flowering time was monitored in
was investigated for these species. Diurnal
was not investigated because each flower
several days (personal observation).
1985 in two replicate populations of each
REPRODUCTIVE ISOLATION IN CONOSPERMUM
17
of the four Conospennum species (C. taxifolium: Kurnell, O'Hares Creek; C.
ericifolium: Patonga, Terry Hills; C. ellipticum: Kurnell, O'Hares Creek; C.
longifolium: Patonga, O'Hares Creek), plus one population of C. taxifolium-ericifolium
intermediates (Bouddi National Park). A sample of 30 plants was taken in each
of these populations every one to three weeks between July and October, and
the number of these plants with open flowers was recorded.
Mechanical isolation
Mackay & Morrison (1989) presented a detailed morphological analysis of
the size and structure of the flowers of these four species and their intermediates,
analysing intra- and inter-population variation. This work is not repeated here.
Ethological isolation
The identity and constancy of floral visitors to plants were recorded during
the 1985 and 1987 flowering seasons on an ad hoc basis. No detailed investigation
of pollinator behaviour was undertaken to examine, for example, assortative
pollination, because of the unspecialized nature of the pollinators observed.
Cross-incompatibility
This was examined by reciprocal (diallel) pollination experiments m the field
in 1987, between plants from populations of each of the four species (C.
taxifolium: La Perouse; C. ericifolium: Patonga; C. ellipticum: O'Hares Creek; C.
longifolium: Patonga).
There were seven experimental treatments per population, with three replicate
plants per treatment:
( l) unmanipulated control, to test for natural pollination
(2) untriggered control, to test for effect of bags
(3) triggered control, to test for self-fertilization
(4) intra-population cross-pollination
(5-7) inter-population cross-pollination with each of the other species.
For each plant in treatments 2-7 a flowering shoot was bagged with fine cotton
mesh (0.5 mm diam. holes) two weeks before treatment. Upon treatment, each
flower head was culled to an average of ten mature but unpollinated flowers
(Table 1), which were then treated. After treatment, the flower heads were rebagged. The number of fruits (achenes) formed was counted three weeks later.
Each flower contains only one ovule.
Conospennum flowers are bilaterally symmetrical, and have an active pollination
mechanism (Carolin, 1961 ). Only two of the four anthers are fertile, and the
style is bent into a swan's-neck shape with the lower bend towards the fertile
pair of anthers and the upper bend towards the infertile pair. The flower is
in a state of tension when it opens, and when pressure is applied to the base
of the style it flicks away from the fertile anthers, striking the pollinator. The
two fertile anthers then dehisce explosively, each releasing its pollen in one
dusty mass. Thus, the stigma picks up pollen from the pollinator when it is
18
TABLE
D. A. MORRISON ET AL.
1. Results of the cross-pollinations within and between the populations of the four Conospermum species
Ovule source
Pollen source
Average no.
flowers treated
per plant
C. taxifolium
unmanipulated
untriggered
triggered
C. taxifolium
C. ericifolium
C. ellipticum
C. longijolium
unmanipulated
untriggered
triggered
C. taxifolium
C. ericijolium
C. ellipticum
C. longijolium
unmanipulated
untriggered
triggered
C. taxifolium
C. ericijolium
C. ellipticum
C. longijolium
unmanipulated
untriggered
triggered
C. taxijolium
C. ericijolium
C. ellipticum
C. longijolium
10.7
12.3
9.7
10.3
9.7
10.0
10.0
10.3
8.0
12.7
9.3
11.7
9.0
8.3
8.0
8.0
7.7
9.7
10.0
9.7
10.3
8.7
11.3
9.7
9.7
10.0
9.0
10.0
C. ericifolium
C. ellipticum
C. longifolium
Average percent
flowers forming
fruits (s)
Multiple
comparisons
test*
50.0 (4.5)
0.0 (0.0)
0.0 (0.0)
54.5 (12.7)
54.8 (9.0)
50.3 (5.1)
10.0 (0.0)
56.1 (5.4)
0.0 (0.0)
0.0 (0.0)
54.2 (12.3)
55.6 (7.7)
48.1 (6.4)
7.0 (6.1)
48.5 (9.4)
0.0 (0.0)
0.0 (0.0)
51.9 (3.2)
46.7 (5.8)
51.9 (I 0.5)
6.4 (5.5)
53.7 (11.6)
0.0 (0.0)
0.0 (0.0)
10.4 (10.0)
16.7 (5.8)
10.4 (10.0)
46.6 (3.0)
a
b
b
a
a
a
b
a
b
b
a
a
a
b
a
b
b
a
a
a
b
a
b
b
b
b
b
a
* Values with the same letters are not significantly different at P < 0.05.
triggered, and then the new pollen is immediately deposited on the pollinator.
Untriggered flowers are thus assumed to be unpollinated.
For treatments 3~ 7, the flowers were triggered using watch-maker's forceps.
For the cross-pollinations, the pollen mass was removed on the pre-moistened
forceps, and the treatment pollen mass was then deposited on the stigma. For
the inter-population crosses, mature but untriggered flowers were collected from
random individuals in the source population, and transported to the receiving
population either on the same day or on the following day.
The fruit-set data were analysed by a 2-factor orthogonal analysis of variance
(factor I: four populations; factor 2: seven treatments) followed by StudentNewman-Keuls tests (Wilkinson, 1987). Homogeneity of the variances was tested
using Cochran's test.
Hybrid inviabiliry
All of the fruits produced by the reciprocal cross-pollination experiment
(which tests for cross-incompatibility) were collected when they matured, by
leaving the bags on the shoots after the number of fruits had been counted.
The fruits were planted in unwashed river sand in foam containers 6 em diam.
REPRODUCTIVE ISOLATION IN CONOSPERMUM
19
in a glasshouse, with all of the fruits from any one plant in the same container.
The germination, and the growth of the seedlings, was followed for six months
in the glasshouse, and the seedlings were then planted out and followed until
they flowered.
Hybrid sterility
A number of experiments were conducted on the intermediates observed in
the field. It was assumed that they were hybrids originating from the Conospermum
species occurring in their vicinity. These hybrids appeared to be morphologically
perfectly normal, with an abundance of normally-formed flowers.
Firstly, reciprocal (diallel) pollination experiments were carried out in the
field in 1987 involving plants from populations of the C. taxifolium-ericifolium
intermediates (Bouddi National Park) and the C. taxifolium-ellipticum intermediates
(O'Hares Creek).
There were 13 experimental treatments per population, with three replicate
plants per treatment:
( 1) unmanipulated control, to test for natural pollination
(2) untriggered control, to test for effect of bags
(3) triggered control, to test for self-fertilization
(4) intra-population cross-pollination
(5) inter-population cross-pollination with the other intermediate
(6-9) inter-population cross-pollination with each of the species
(10-13) inter-population reciprocal cross-pollination with each of the species.
The techniques and other populations used were as described for the previous
cross-pollination experiment. The fruit-set data were analysed by a 2-factor
orthogonal analysis of variance (factor 1: two populations; factor 2: 13 treatments)
followed by Student-Newman-Keuls tests (Wilkinson, 1987). Homogeneity of the
variances was tested using Cochran's test.
Secondly, reciprocal pollination experiments were carried out in the field in
1987 between individual plants of the C. longifolium-taxifolium intermediates
(O'Hares Creek), C. longijolium-ericifolium intermediates (Mount White) and C.
longifolium-ellipticum intermediates (O'Hares Creek). These plants are rare and do
not occur in large populations, thus limiting the number of manipulative
treatments available.
There were six experimental treatments per intermediate, with only one plant
per treatment:
(1) unmanipulated control
(2) intra-population cross-pollination
(3 & 4) inter-population cross-pollination with each parent
(5 & 6) inter-population reciprocal cross-pollination with each parent. The
techniques and other populations used were as described for the previous crosspollination experiments.
Thirdly, pollen fertility was examined for each species and for each of the
intermediates. Pollen was collected from three flowers from each of three plants
in each population. The pollen was stained with lactophenol cotton blue (a
cytoplasmic stain), and 30 grains from each flower were examined. The
20
D. A. MORRISON ET AL.
populations used were as described above. The data were analysed by a 2factor nested analysis of variance (factor 1: populations; factor 2: plants) followed
by Student-Newman-Keuls tests (Wilkinson, 1987). Homogeneity of the variances
was tested using Cochran's test, with the data needing to be arcsin transformed.
Hybrid breakdown
All of the fruits produced by the reciprocal cross-pollination experiments
testing for hybrid sterility were collected and cultivated, as described above for
the hybrid inviability test.
RESULTS
Geographical isolation
Conospermum taxifolium is widespread throughout the Sydney region (Fig. 1), as
well as occurring north and south of there. It usually occurs within 20 km of
the coast, except for isolated groups of populations in the Hill Top to Thirlmere
area, near Agnes Banks, and in the Putty area. It occurs sympatrically with C.
longifolium (see Map 1 in Johnson & McGillivray, 197 5), and both species are
sympatric with C. ericifolium and C. ellipticum. Conospermum ericifolium is confined
to the area between Terrigal and Port Jackson (Fig. 1), occurring inland as far
as Mangrove Mountain and Dural. C. ellipticum is confined to the area between
Port Jackson and Bulli (Fig. 1), occurring inland as far as Appin. These two
species are thus completely allopatric.
Only three extensive populations of intermediates between C. taxifolium and
C. ericifolium were located during the survey, and only two extensive populations
of intermediates between C. taxifolium and C. ellipticum. These intermediates
always occurred as spatially discrete populations of several dozen plants, usually
several hundred metres from either of their putative parents. Intermediates
between C. longijolium and the other three species are geographically widespread,
but they only ever occurred as a group of 1-3 plants within a population of
one of their putative parents.
Ecological isolation
The first two axes from the redundancy analysis account for 45% of the
variance of the species-environment biplot. This analysis clearly shows the
floristic separation of the habitats of C. taxifolium and C. longifolium (dry heaths
and woodlands) from those of C. ericifolium and C. ellipticum (wet heaths) (Fig.
2). This floristic separation is associated with several soil characteristics, as C.
ericifolium and C. ellipticum both occur in wetter sites, with more organic matter
and a lower pH, compared with the other two species (Fig. 2).
Furthermore, there is considerable inter-site floristic variation within both C.
taxifolium and C. ellipticum. This is related to the mechanical composition of
their soils (Fig. 2), as both species occur on coastal sand dunes as well as rockbased soils.
The two sites containing intermediates occupy ecologically intermediate
positions in the analysis (Fig. 2), although the C. taxifolium-ericifolium intermediates
REPROD UCTIVE ISOLATI ON IN CONOSPERMUM
A
A
A
21
e
Somersby
A
Hornsby •
Parramatta•
Liverpool•
"
"
"ov
"
0
eAppin
0
"
N
"
Oo
""
0
0
20
km
0
Figure I. Distribution of Conospermum taxifolium (0 ), C. ericifolium (6)
and C. ellipticum ('\7) in the Sydney
region, based on herbarium records and field surveys. See Map I in
Johnson & McGillivray (1975) for the
distribution of C. longi.folium.
22
D. A. MORRISON ETAL
1.0.--------.------,..-------.------,..------,
...
0
0
D
D
-1.0 L _ . . - - - - J . . . . __ _ _ _J....__ _ _ _J....__ _ _ _J....__ _ _
-1.0
~
1.5
Axis 1
Figure 2. Projection of the Conospennum sites onto axes representing the first two mmponents of the spccic~­
environmf'nt biplot fi-om the redundancy analysis of the floristic and soil data. C. taxifoLium (0), C rricjfoLium
(6:, C. ellipticum (\71, C. longi]Olium (0), C. ta:rffofium-ericifolium intermediates (.&..,: and C. taxifolium-t!liptirum
intermediates (.... ). The floristic similarities arc indicated by the spatial relationship of the symbols, whilf" thrinflucncc of the soil characteristics is indicated by the direction and length of the arrows.
were on a coastal sand dune whereas the C. taxifolium-ellipticum intermediates
were not. This indicates that the intermediates occur m areas where the
ecological separation between their parent species breaks down.
Temporal isolation
There is considerable inter-population phenological variation in each of these
species, but the peak flowering times all overlap (Fig. 3). There is thus no
temporal isolation between these speoes.
Mechanical isolation
Mackay & Morrison ( 1989) showed that these four species and their
intermediates cannot be distinguished by morphological floral attributes. There
is thus no mechanical isolation between these species.
Ethological isolation
A number of species of flies (Diptera: ?Syrphidae; see also Carolin, 1961 ),
beetles (Coleoptera: Apionidae, Cleridae, Curculionidae) and ants (Hymenoptera:
REPRODUCTIVE ISOLATION IN CONOSPERMUM
23
C. ericifolium
C. ellipticum
C. taxifolium
C. longifolium
C. tax-eric hybrid
0
10
20
30
40
50
60
70
80
Time (days)
Figure 3. flowering phenology of the Conospermum species, expressed as the proportion of plants flowering
out of a sample of 30 plants from each population.
Formicidae) were observed to visit the flowers regurlarly. Of these insects, the
only ones likely to be effective pollinators are the flies and clerids, and neither
of these was exclusive to any one Conospermum species. They visited only three
or four flowers per inflorescence, before moving to another inflorescence on
the same or a nearby plant. They did not appear to visit flowers of other
species. There is thus unlikely to be any ethological isolation between these
Conospermum species.
Cross-incompatibility
The analysis of variance of the reciprocal pollination experiment indicates a
significant interaction term (F = 15.227, P < 0.001 ). The Student-Newman-Keuls
tests indicate that this is because the inter-population crosses involving C.
longifolium had a significantly lower fertilization success than did any of the
other crosses (Table 1).
Both the triggered and the untriggered controls did not produce any fruits
(Table 1), indicating that the flowers do not fertilize themselves in any of these
species, and that the bags themselves did not influence the fruit set. The
unmanipulated controls produced about 50% fruit set in all species (Table 1),
as did all of the intra-population crosses, indicating that our pollination
manipulations were not detrimental to fruit set. All of the reciprocal
interpopulation crosses among C. taxifolium, C. ericifolium and C. ellipticum also
produced about 50% fruit set (Table 1), indicating that there is no more cross-
24
TABLE
D. A. MORRISON ET AL.
2. Results of the cross-pollinations within and between the populations of the intermediates with
Conospermum taxifO!ium
Ovule source+
Pollen source+
Average no.
flowers treated
per plant
C. tax-eric
unmanipulated
untriggered
triggered
C. tax-eric
C. tax-ellip
C. taxijolium
C. ericifOlium
C. ellipticum
C. longijolium
unmanipulated
untriggered
triggered
C. tax-eric
C. tax-ellip
C. taxifOlium
C. ericifOlium
C. ellipticum
C. longijolium
c. tax-eric
c. tax-ellip
c. tax-eric
c. tax-ellip
c. tax-eric
C. tax-ellip
c. tax-eric
C. tax-ellip
11.7
16.0
12.3
9.3
9.0
10.0
10.3
10.0
10.3
8.3
10.3
14.0
9.7
10.3
8.7
10.0
9.3
10.0
10.7
9.7
8.7
11.0
9.0
10.0
10.0
9.7
C. tax-ellip
C. taxifOlium
C. ericifOlium
C. ellipticum
C. longifolium
Average percent
flowers forming
fruits (s)
31.6
0.0
0.0
3!.7
4.2
6.7
8.5
6.7
9.1
29.2
0.0
0.0
6.7
29.4
10.4
13.4
10.8
13.3
18.1
23.6
22.2
23.4
17.9
23.3
10.0
10.0
Multiple
comparisons
test*
(5.9)
(0.0)
(0.0)
(7.6)
(7.2)
(5.8)
(7.8)
(5.8)
(9.1)
(8.6)
(0.0)
(0.0)
(11.5)
(4.2)
(10.0)
(5.8)
(1.4)
(11.5)
(6.9)
(10.5)
(7.9)
(6.7)
(10.5)
(11.5)
(10.0)
(10.0)
a
b
b
a
b
b
b
b
b
a
b
b
b
a
b
b
b
b
ab
ab
ab
ab
ab
ab
b
b
*
Values with the same letters are not significantly different at P < 0.05.
+ C. tax-eric, C. taxifOlium-ericifOlium intermediates; C. tax-ellip, C. taxifOlium-ellipticum intermediates.
incompatibility between these three species than there is within any one of
them. However, all of the reciprocal interpopulation crosses involving C.
longifolium only produced about 10% fruit set (Table 1). There is therefore a
considerably greater degree of cross-incompatibility between this species and the
other three.
Hybrid inviability
Germination was extremely low .in all cases. Only five seedlings were produced
from the 230 fruits: two C. taxifolium (from 33 fruits), one C. ericifolium (from
36 fruits), one C. taxifolium-ericifolium intermediate (from 31 fruits), and one C.
longifolium-ellipticum intermediate (from seven fruits). The seedlings produced by
the inter-population crosses are morphologically identical to the intermediates
observed in the field, confirming that the field plants are hybrids. All of these
seedlings grew normally and survived to reproductive maturity, indicating that
hybrid inviability is unlikely to be an effective isolation mechanism between
any of these species.
REPRODUCTIVE ISOLATION IN CONOSPERMUM
TABLE
25
3. Results of the cross-pollinations within and between the individuals of the
intermediates with Conospermum longifolium
Ovule source+
Pollen source+
C. long-tax
unmanipulated
C. long-tax
C. longifolium
C. taxifolium
unmanipulated
C. long-eric
C. longi.folium
C. ericifolium
unmanipulated
C. long-ellip
C. longifolium
C. ellipticum
C. long-tax
C. long-eric
C. long-ellip
C. long-tax
C. long-eric
C. long-ellip
C. long-eric
C. long-ellip
C. longi.folium
C. taxifolium
C. ericifolium
C. ellipticum
Number of
flowers treated
per plant
Percent flowers
forming fruits
II
12
10
II
15
9
10
10
10
0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
8
7
0.0
0.0
20.0
18.2
12.5
10.0
20.0
14.3
10
II
8
10
10
7
+ C. long-tax, C. longifolium-taxifolium intermediates; C. long-eric, C. longifolium-ericifolium
intermediates; C. long-ellip, C. longi.folium-ellipticum intermediates.
Hybrid sterility
The analysis of variance of the reciprocal pollination experiment for the C.
taxifolium-ericifolium and C. taxifolium-ellipticum intermediates indicates a significant
interaction term (F = 2.885, P = 0.004). The Student-Newman-Keuls tests
indicate that this is because the reciprocal inter-population crosses were not
equally successful reproductively (Table 2).
Both the triggered and the untriggered controls did not produce any fruits
(Table 2), indicating that the flowers do not fertilize themselves in either of
these intermediates, and that the bags themselves did not influence the fruit
set. The unmanipulated controls produced about 30% fruit set in both
populations (Table 2), indicating some degree of hybrid sterility since this is
lower than the fruit set for any of the parent species. Both of the intrapopulation crosses also produced about 30% fruit set (Table 2), indicating that
our pollination manipulations were successful.
All of the inter-population crosses involving C. taxifolium, C. ericijolium or C.
ellipticum as the female parent produced about 20% fruit set (Table 2), while
the reciprocal crosses with the intermediates as the female parent only produced
about 10% fruit set (Table 2). This indicates that there is some form of
incomplete barrier to back-crossing with the parents, and that the intermediates
are more female-sterile than they are male-sterile in this regard. All of the
reciprocal inter-population crosses involving C. longifolium only produced about
10% fruit set (Table 2).
The reciprocal pollination experiment for the C. longi.folium-taxifolium, C.
longifolium-ericifolium and C. longifolium-ellipticum intermediates indicates a
D. A. MORRISON ET AL.
26
TABLE
4.
Results of the pollen fertility analysis of the
Conospermum plants
Pollen source+
C. taxifolium
C. ericifolium
C. ellipticum
C. longijolium
C. tax-eric
C. tax-ellip
C. long-tax
C. long-eric
C. long-ellip
Average percent
pollen fertility (s)
98.5 (1.6)
98.3 (1.6)
98.8 (1.3)
95.1 (3.7)
98.0 (3.2)
98.0 (0.9)
28.7 (11.4)
31.0 (16.4)
16.3 (5.5)
Multiple
comparisons
test*
a
a
a
a
a
a
b
b
b
* Values
with the same letters are not significantly different
at P< 0.05.
+ C. tax-eric, C. taxijolium-ericifolium intermediates; C. tax-ellip,
C. taxifolium-ellipticum intermediates; C. long-tax, C. longifoliumtaxifolium intermediates; C. long-eric, C. longijolium-ericifolium
intermediates; C. long-ellip, C. longijolium-ellipticum intermediates.
considerable degree of hybrid sterility. The unmanipulated parent controls
produced about 50% fruit set (Table 1), while all of the inter-population crosses
involving one of the four species as the female parent produced only about
15% fruit set (Table 3), and all of the inter- and intra-population crosses
involving the intermediates as the female parent produced no fruits at all (Table
3). These intermediates are thus completely female-sterile but only partially
male-sterile.
The results of the pollen staining confirm the lack of male sterility in the
C. taxifolium-ericifolium and C. taxifolium-ellipticum intermediates (Table 4), as well
as confirming the partial male sterility of the C. longifolium-taxifolium, C. longifoliumericifolium and C. longijolium-ellipticum intermediates (Table 4). The pollen grains
from the C. longifolium intermediates are also about 30% smaller than are the
grains from the other plants.
Hybrid breakdown
No germination occurred in this experiment, out of the I 07 fruits from the
first pollination experiment and the nine fruits from the second experiment.
This result is equivocal, as it may indicate that hybrid breakdown exists between
these species, or it may simply be a product of the small number fruits obtained
(see Discussion).
DISCUSSION
At least some of the potential reproductive isolation mechanisms are effective
between each of the possible pairs of these four Conospermum species. However,
temporal, mechanical and ethological isolation appear to play no part in
preventing gene flow between any of these species.
No hybrids between C. ericifolium and C. ellipticum have been found in the
field, and so their reproductive isolation is effectively complete. Geographical
REPRODUCTIVE ISOLATION IN CONOSPERMUM
27
isolation appears to be the main barrier to gene flow, as C. ericifolium only
occurs north of Port Jackson and C. ellipticum only occurs south of Port Jackson.
In fact, very few populations now exist between the southern tip of Broken
Bay and the northern tip of Botany Bay, due to the urban expansion of
metropolitan Sydney. Therefore, these taxa are currently more isolated than is
indicated in Figure 1. There is no ecological isolation, cross-incompatibility or
hybrid inviability between these species. Hybrid sterility and breakdown were
not tested.
Hybrids between C. taxifolium and both C. ericifolium and C. ellipticum are
known (but rare) in the field, occurring as relatively large but spatially discrete
populations, and so C. taxifolium is not completely isolated from either of the
other two species. Ecological isolation appears to be one of the main barriers
to gene flow, with C. ericifolium and C. ellipticum being restricted to wet heaths
while C. taxifolium only occurs in dry heaths and woodlands. This separation
breaks down in ecologically intermediate habitats, and this is where the hybrids
occur. There appears to be no geographical isolation, cross-incompatibility or
hybrid inviability between C. taxifolium and the other two species. The hybrids
are about 60% fertile relative to their parent species, providing a partiallyeffective barrier, and back-crossing to the parents is only about 20-40% effective
depending on whether the hybrids are the female or male parent. The test of
hybrid breakdown was equivocal, but there is no evidence that offspring of the
back-crosses occur in the field.
Hybrids between C. longi.folium and all three of C. taxifolium, C. ericifolium and
C. ellipticum are known (but very rare) in the field, occurring as scattered
individuals within a population of one of their parents, and so C. longi.folium is
not completely isolated from any of the other three species. Cross-incompatibility
appears to be one of the main barriers to gene flow, as inter-species crosspollinations are only about 20% successful relative to intra-species crosses. C.
longifolium is also ecologically isolated from C. ericifolium and C. ellipticum, but as
noted above this is not a particularly effective barrier to gene flow in these
species. There appears to be no geographical isolation or hybrid inviability
between C. longi.folium and the other three species. The hybrids are femalesterile, and back-crossing to the parents is only about 30% effective, providing
a considerable barrier to gene flow between the species. It is unclear whether
the offspring of these back-crosses are viable, but there is no evidence that
they occur in the field.
The barriers to gene flow between C. ericifolium and C. ellipticum are thus
completely effective in the field, relying on their allopatric distributions backed
up by reduced hybrid fertility. The barriers to gene flow between C. taxifolium
and these two species are only partially effective, as their ecological separation
breaks down in intermediate habitats and the healthy partially-fertile F 1 plants
can grow there. The barriers to gene flow between C. longifolium and the other
three species are almost completely effective, as cross-incompatibility is very
high and the F 1 plants are vegetatively healthy but partially male-sterile and
completely female-sterile.
Most plant species are not isolated by single reproductive barriers but by
combinations of different mechanisms working in temporal concert, and it is
the combined strength of the numerous barriers that renders hybridization and
introgression unlikely (Levin, 1978). Among the four species of Conospermum
28
D. A. MORRISON ET AL.
studied here, only the geographical barrier is completely effective alone. Spatial
isolation accrues if the distance between populations is greater than the dispersal
range of the populations (Levin, 1978). Consequently, large-scale geographical
isolation can be an extremely effective barrier to gene flow, because pollen and
seed dispersal are usually very restricted in space (Levin & Kerster, 19 74;
Handel, 1983; Slatkin, 1985).
Ecological isolation on its own is a relatively ineffective barrier to gene
exchange among the Conospermum species. However, our data concerning the
ecological isolation of C. taxifolium and C. longifolium from C. ericifolium and C.
ellipticum are based on descriptive rather than manipulative tests. The nature
and effectiveness of this ecological isolation could perhaps be further examined
by reciprocal transplants in the field.
There are three commonly-reported ecological characteristics of hybrids (Levin,
1978): a narrow ecological distribution, occupation of intermediate habitats, and
presence in disturbed areas. The first two of these characteristics appear to be
true for the C. taxifolium-ericifolium and C. taxifolium-ellipticum hybrids, as they are
restricted to a particular habitat type and populations of C. taxifolium and C.
ellipticum are known to occur within 250 m of each other with no sign of
intermediates if the appropriate habitat type is not available. Hybrids are often
restricted to the central portion of morphological and ecological clines between
the parent species (i.e. hybrid zones, Hewitt, 1988), but the intermediate habitats
of the C. taxifolium hybrids are spatially separate. Although these hybrids are
known from disturbed areas this does not seem to be a requirement. The C.
longifolium hybrids do not occur in intermediate habitats, but simply occur within
one of the parent populations.
Cross-incompatibility and hybrid sterility often occur together as isolation
mechanisms (Ornduff, 1969), as they do in Conospermum. They are usually
related to the degree of morphological divergence between the taxa (Levin,
1978), and in Conospermum the more distantly-related C. longifolium (Mackay &
Morrison, 1989) has a higher degree of incompatibility and sterility when
crossed with the more closely-related C. taxifolium, C. ericifolium and C. ellipticum.
In all cases the expression of the incompatibility and sterility is bilateral.
Cross-incompatibility may be the result of sporophytic incompatibility (failure
of pollen grains to germinate) or gametophytic incompatibility (abnormal pollen
tube growth, failure of the pollen tube to penetrate the micropyle, or premature
flower abscission) (Levin, 1978). These could be further investigated in the C.
longifolium hybrids by histochemical study of the pollen/stigma interactions, pollen
tube growth, and looking for chromosomal abnormalities.
Hybrid sterility may be due to abnormalities of the diploid or the haploid
tissues (Levin, 1978). In the case of the C. longifolium hybrids it appears to be
due to the haploid tissues, and to be more complete in the female
structures. The causes of these abnormalities could be investigated by cytogenetic
studies.
Our tests of hybrid inviability and hybrid breakdown were not particularly
effective, due to the low ( < 3%) germination. This is unlikely to have been
caused by insufficient time for germination to occur, as Kullmann (1981) notes
that a range of Conospermum species require a maximum of 25-110 days for
germination, and we waited 180 days. Conospermum in general has low germination
(Fox, Dixon & Monk, 1987), and many of the species produce copious achenes
REPRODUCTIVE ISOLATION IN CONOSPERMUM
29
that do not contain a viable seed (George, 1984). We did not section our
experimental fruits to see whether they contained seeds, as we needed them
for the experimental treatments, but our results suggest that about 50 fruits
per treatment per population would be needed to do the inviability and
breakdown tests effectively. This would be a mammoth undertaking to produce
the required number of cross-pollinations.
These four species maintain their morphological (and presumably genetic)
identity in spite of the presence of hybrids (Mackay & Morrison, 1989). C.
ericifolium and C. ellipticum are geographically allopatric, and C. taxifolium is locally
allopatric to these two species; and furthermore the hybrid fertility studies
suggest that there is likely to be little gene flow back to the parents. Moreover,
C. longifolium does not produce a pure F 2 generation. This suggests that actual
gene flow is likely to be more restricted than potential gene flow, and this
could be investigated by a study of pollen flow among these four species. This
could be done by studies of allozyme distribution among the hybrids, labelled
pollen, and pollinator movement (Richards, 1986).
The hybrids appear to be morphologically homogeneous in all cases (Mackay
& Morrison, 1989), with the C. longifolium hybrids needing to be formed anew
each generation while the C. taxifolium hybrids do not. The genetic makeup of
the hybrid populations could be investigated by allozyme analysis to determine
if they are predominantly F 1 plants, back-crosses, or F2 plants. There are
unlikely to be any F 2 plants for the hybrids with C. longifilium, but there may
be back-crosses. Pollen flow is unequal in the complex involving C. taxifolium,
as the F 1 X F 1 crosses are more productive than are the back-crosses to the
parents. This may explain why the hybrid populations are morphologically
homogeneous.
Reproductive isolation is usually considered to be the by-product of divergent
evolution (Levin, 1978), but isolation itself is amenable to both re-inforcement
and degradation, and therefore any one instance of partial isolation may be
the result of divergent or convergent evolution (Grant, 1981). However, if the
formation of pre-zygotic isolation mechanisms precedes or is concurrent with
the formation of post-zygotic mechanisms (Bush, 1975), then the most likely
scenario for these four species is that the relative strength of the isolation
mechanisms reflects the more recent divergence of C. ericifolium and C. ellipticum
from a common ancestor with C. taxifolium compared to the earlier divergence
from a common ancestor with C. longifilium Uohnson & McGillivray, 1975).
Thus, the divergence of C. ericifolium and C. ellipticum would be the result of
their invasion of a new habitat, and their consequent ecological isolation has
not yet been re-inforced by the formation of effective post-pollination barriers
to gene flow.
Our results thus confirm the hypotheses regarding reproductive isolation
mechanisms in Conospermum generated by Mackay & Morrison (1989). The first
three species are isolated primarily by pre-pollination barriers, while C. longifolium
is isolated by post-pollination barriers. These post-pollination barriers are usually
much more effective than are the pre-pollination ones. Therefore, C. ericifolium
and C. ellipticum represent geographical species (i.e. their allopatric distributions
are responsible for the genetic discontinuity), while C. taxifolium is an ecological
species (i.e. the genetic discontinuities are caused by differing ecological
amplitudes), and C. longifolium is a genetic species (i.e. there is a genetically-
D. A. MORRISON ET AL.
30
controlled reproductive barrier) (see Grant, 1960). Taxonomically, Mayr (1986)
suggests that it is the failure of reproductive isolation mechanisms to prevent
gradual merging of the populations that indicates conspecificity under the
biological species concept. Consequently, all four Conospermum taxa can still be
interpreted as representing good biological species, as each of the gene pools
appears to maintain its identity in spite of the incomplete nature of the isolation
mechanisms.
ACKNOWLEDGEMENTS
Thanks to Seana McAniff and Sharon Nash for help with the field work;
to the National Parks & Wildlife Service of N.S.W. and also the Metropolitan
Water Sewerage and Drainage Board for allowing us to conduct experiments
on land under their care; to David Keith for access to unpublished data;
to Jon Lovett Doust, Lucinda McDade and Robert Wyatt for helpful
discussions; and to Caroline Gross for commenting on an earlier draft of the
manuscript.
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