- Email: [email protected]
The risk of pollen-mediated gene flow into a vulnerable eucalypt species
Forest Ecology and Management 381 (2016) 297–304
Contents lists available at ScienceDirect
Forest Ecology and Management journal homepage: www.elsevier.com/locate/foreco
The risk of pollen-mediated gene flow into a vulnerable eucalypt species Bruce W. Randall a, David A. Walton a,⇑, David J. Lee a,b, Helen M. Wallace a a b
University of the Sunshine Coast, Maroochydore DC, Queensland 4558, Australia Department of Primary Industries and Fisheries, Gympie, Queensland 4570, Australia
a r t i c l e
i n f o
Article history: Received 11 April 2016 Received in revised form 22 September 2016 Accepted 25 September 2016
Keywords: Eucalyptus argophloia Hybridisation Pollination Gene flow Pollen tube growth Eucalypts
a b s t r a c t Hybridisation is recognized as a threat to the genetic integrity of rare, threatened and vulnerable species. Eucalypts are widely planted for plantation forestry and many species readily hybridise making ‘‘vulnerable”, locally-occurring species potentially at risk of genetic swamping. Eucalyptus argophloia (Queensland western gum or Chinchilla white gum) is a vulnerable species with limited distribution in South Eastern Queensland, Australia. The risks to this species from gene flow from surrounding forest or plantations are poorly understood. We investigated the breeding system and hybridisation potential of E. argophloia by controlled pollinations with selected sympatric and allopatric species. Treatments included self-pollination, outcross within species and hybridisation with E. crebra, E. microcarpa and E. moluccana (from within the same section, Adnataria) and E. pellita and E. resinifera (from a more distantly related section, Latoangulatae). The aim was to determine the degree of self-pollination and gene flow into E. argophloia from closely-related and more distantly-related sympatric and allopatric species. Eucalyptus argophloia is readily self-fertile. It also hybridises with a broad range of intra-sectional and inter-sectional eucalypt species. All crosses produced some pollen tubes, capsules and seed, and open pollination produced significantly more capsules and seeds than all controlled pollinations attempted. The number of seeds per capsule pollinated was not significantly different between pollen treatments. There is a potential risk of gene flow into E. argophloia populations from adjacent natural populations of the species tested as pollen parents, or from planted Eucalyptus species. This risk of foreign gene flow could be greater with open pollination than indicated by controlled pollination in this study because of the limiting effect of bud damage incurred during controlled pollinations. The ability to self-pollinate in the small population of E. argophloia raises the potential risk of genetic swamping from foreign pollen and elevated evolutionary pressure. These results will be useful for guiding management of this vulnerable species such as limiting plantings of cross-compatible species close to existing populations. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Hybridisation between species is an important evolutionary process that can drive plant adaptations and can also present a threat to the genetic integrity of plant populations (Schierenbeck and Ellstrand, 2009; Ellstrand et al., 2010; Larcombe et al., 2014). Hybridisation is a particular threat to rare, threatened and vulnerable species and in extreme cases there is a danger of extinction due to genetic swamping (Levin et al., 1996; Vila et al., 2000; Butcher et al., 2005; Barbour et al., 2010). Hybridisation is now an urgent threat to species integrity because environmental disturbance and human activities such as forest plantations are bringing
⇑ Corresponding author at: Genecology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland 4519, Australia. E-mail address: [email protected] (D.A. Walton). http://dx.doi.org/10.1016/j.foreco.2016.09.042 0378-1127/Ó 2016 Elsevier B.V. All rights reserved.
together cross-compatible species that previously were geographically separated (Rhymer and Simberloff, 1996; Ellstrand et al., 2010). The threat of hybridisation commonly occurs when pure species are pollinated by foreign pollen, i.e. from pollen-mediated gene flow (da Silva et al., 2015; Shepherd and Lee, 2016). Further, when hybrids survive to flowering, even with low fecundity, there is a potentially serious risk to native populations of introgression of genes and genetic swamping if the hybrid progeny are as fit as or more fit than the parent population (Dickinson et al., 2013; Shepherd and Lee, 2016). Introgression can lead to extinction of small restricted populations if reduced fitness of subpopulations allows swamping of the gene pool or introduction of new genes (Linacre and Ades, 2004). On the positive side, hybridisation may help some species adapt to rapidly changing conditions such as climate change (Broadhurst et al., 2008; Nevill et al., 2014).
298
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
Eucalypts are one of the most widely planted taxa for plantation forestry with over 20 million ha globally (Whyte et al., 2016). Eucalypts are also the dominant trees throughout most non-arid plant communities in the Australian continent (Brooker and Kleinig, 1999). There are nearly 900 eucalypt taxa which are endemic to Australia and its surrounding islands (CPBR, 2006) and 77 taxa are currently listed as endangered or vulnerable at the national level in Australia and require special attention (Environment Australia, 2009). Gene flow via hybridisation is a particular risk for rare eucalypt species in Australia (Barbour et al., 2010). Eucalypt diversity can be viewed as a consequence of substantial outbreeding and eucalypts often demonstrate weak reproductive barriers between closely related taxa (Griffin et al., 1988; Boland et al., 1984; Dickinson et al., 2012, 2013). Within the eucalypts, hybrids are establishing in the wild in some circumstances (Griffin et al., 1988; Barbour et al., 2003). Predicting the extent of gene flow necessitates understanding pollen dispersal and flowering overlap (Barbour et al., 2003, 2005, 2006). Most records of manipulated hybrids derive from the commercially important subgenus Symphyomyrtus (Griffin et al., 1988; Larcombe et al., 2015). Examples of successful manipulated hybrids used in forestry plantations are E. grandis E. urophylla, widely used for forestry plantations in Brazil and China, and Corymbia torelliana C. citriodora, increasingly planted in Australia and other subtropical regions around the world (Barbour et al., 2008; Lee et al., 2010; Dickinson et al., 2013; da Silva et al., 2015; Xie et al., 2015; Shepherd and Lee, 2016). The risk of natural hybridisation between species in different subgenera depends on the degree of spatial separation, taxonomic separation and flowering synchronicity between parents (Ellstrand, 1992; Barbour et al., 2002, 2006, 2008; Horsley et al., 2010). Generally, the risk of hybridisation with surrounding native populations decreases with increasing taxonomic distance between parents (Potts et al., 2003; Potts and Dungey, 2004; Larcombe et al., 2015). E. argophloia (Queensland Western White Gum or Chinchilla White Gum) is a vulnerable eucalypt endemic to Southeast Queensland, Australia with a restricted distribution (Brooker and Kleinig, 2004). Eucalyptus argophloia is of interest for both forestry and conservation, similar to Eucalyptus benthamii (Butcher et al., 2005; Ngugi et al., 2004a, 2004b; Merchant et al., 2007) Droughts are expected to increase in frequency and magnitude in many parts of the world (IPCC, 2014). Eucalypt plantations are already experiencing more frequent droughts and species that can tolerate long periods of drought are likely to be more critical as the impacts of climate change intensify (Whyte et al., 2016). E. argophloia has commercial potential as a forestry species due to fast growth rates, strong durable timber, drought tolerance (600–900 mm mean annual rainfall) and salinity tolerance (Ngugi et al., 2004a,b; Merchant et al., 2007). Hybrids of the species may also be useful as is the case for other eucalypts (Lee, 2007; Barbour et al., 2008; Dickinson et al., 2013; da Silva et al., 2015; Xie et al., 2015; Shepherd and Lee, 2016). The natural range of the species is an area only 30 km east to west and 20 km north to south, with three small disjunct sub-populations named ‘‘Burncluith”, ‘‘Burra Burri” and ‘‘Fairyland” (Lee et al., 2015). Land clearing has fragmented the sub-populations of E. argophloia and the total remaining wild population is only around 1000 individuals (Lee et al., 2015). Consequently, E. argophloia is listed as ‘‘vulnerable” (needing conservation, protection, and implementation of recovery plans to ensure that it remains a viable population) by the Environment Protection and Biodiversity Conservation Act 1999 (Australia) (Environment Australia, 2009) and the Nature Conservation Act 1992 (Queensland Government, 1992). Small, fragmented remnant populations such as E. argophloia may be at risk of being genetically swamped by other species because the rate of gene flow may increase as the size of the population decreases (Ellstrand et al.,
1989; Ellstrand and Elam, 1993; Linacre and Ades, 2004; Barbour et al., 2006). In addition in small, fragmented eucalypt populations higher levels of selfing and inbreeding are also a risk (Butcher et al., 2005). E. argophloia is a member of the subgenus Symphyomyrtus (Brooker, 2000), therefore it may be at risk of genetic swamping due to hybridisation with other eucalypts (Griffin et al., 1988; Strauss, 2001). Other common, endemic eucalypt populations sympatric with E. argophloia include E. crebra, E. moluccana and E. microcarpa. The risk to E. argophloia of gene flow and genetic swamping from surrounding Eucalyptus species is poorly understood. We examined the breeding system and hybridisation potential of E. argophloia. Our aims were to examine (1) the breeding system of E. argophloia and in particular the degree of self-compatibility, (2) hybridisation potential with closely related sympatric species, (3) hybridisation potential with allopatric and more distantly related inter-sectional species. In-vivo pollen tube growth, capsule set and seed set were examined to determine pollination success and ability to hybridise. We hypothesized that E. argophloia would not hybridise readily with more distantly related species such as E. resinifera and E. pellita although anecdotal evidence suggests that it may readily cross with closely related Eucalyptus species such as E. moluccana, E. microcarpa and E. crebra. The results of this study will increase understanding of the risks posed by gene flow into E. argophloia populations from adjacent ecosystems, information that will aid conservation and management of this species. In addition, knowledge of compatible species may be useful if hybrids of E. argophloia prove to be useful for forestry, as has been the case for other Eucalyptus and Corymbia species. 2. Materials and methods 2.1. Study Site Pollination studies were conducted in an E. argophloia seed orchard at the Dunmore State Forest Station on the Darling Downs, southeast Queensland (27.34°S; 151.04°E) in the flowering season April to October 2007 and 2009. This seed orchard contains trees from three locations in the natural range of the species. Two of these locations, ‘‘Fairyland” and ‘‘Burra Burri”, contain relatively undisturbed blocks of several hundred trees and the other location, approximately 11 km away (Burncluith), consists of isolated trees in cleared agricultural land. Because E. argophloia is a small population the entire population is considered to be one provenance, and the three locations to be sub-provenances. All experiments were conducted using an elevated platform to access flowers. 2.2. Breeding system and pollination methods We selected four ten-year-old E. argophloia maternal parent trees from the seed orchard, two from the Burncluith subprovenance and two from the Burra Burri sub-provenance. Pollen parents were: (1) self-pollen from the same tree; (2) E. argophloia pollen from an unrelated tree of the Burncluith sub-provenance; (3) a second E. argophloia pollen from an unrelated tree of the Burra Burri sub-provenance; (4) E. moluccana pollen (a closely-related sympatric species). We emasculated individual buds on the maternal trees using a curved scalpel blade just prior to anthesis, when the operculum changed from green to brown as described by Randall et al. (2012). We collected flower buds from pollen parents just prior to anthesis, removed anthers with forceps and desiccated them over silica gel for 24–48 h to promote dehiscence. The anther-pollen mix was stored in gelatine capsules at 5 °C in jars with a desiccant. We employed two pollination methods, the ‘three-stop’ method
299
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
(Sedgley and Smith, 1989) and the ‘one-stop’ method (Trindade et al., 2001). Three replicate bunches (groups of umbellasters) were emasculated for each treatment on each maternal tree to assess capsule set and seed production (4 pollen parents 2 pollination methods 3 replicate bunches per tree 3 trees, total 72 bunches). 20 unopened female flowers were emasculated on each bunch and all other flower buds were removed. After emasculation, we bagged each bunch with a polyester pollen exclusion bag. A moistened match head was used to apply pollen to flower buds receiving the three-stop method 3–7 days after emasculation, discarding each match after use. For the one-stop method we pollinated flower buds on the day of emasculation and bagged them immediately afterwards.
2.5. Pollen tube harvest and microscopy Buds for pollen tube examination were collected seven to fourteen days after pollination, fixed in acetic ethanol (1 part glacial acetic acid: 3 parts ethanol) and processed as previously described (Randall et al., 2012). 2.6. Capsule set and seed production The capsules remained on the trees until they matured, and were collected at 210–220 days after emasculation, just prior to valves opening. The number of capsules set for each treatment, total number of seeds per bunch and total seed weight per bunch were recorded. 2.7. Viability of seed from crosses with sympatric species
2.3. Hybridisation with sympatric species We applied four pollen treatments on each of four E. argophloia maternal trees, one from Burncluith sub-provenence, one from Burra Burri sub-provenence, one from Fairyland sub-provenence and one of unknown identity from an isolation row. Pollen treatments were (1) E. moluccana, (2) E. crebra, (3) E. microcarpa (all from the same section as E. argophloia, Adnataria) (Brooker, 2000); and (4) cross within species using pollen from an unrelated tree from Burra Burri sub-provenence (Table 1). Three bunches, each with 20 flower buds for each treatment for each tree were pollinated using the three stop method as described above (3 bunches 4 treatments 4 trees, total 48 bunches). Three additional bunches with 10 flower buds per pollen parent treatment on each tree were emasculated and harvested to examine pollen tube growth. Each replicate was bagged and pollinated as described above.
Seeds obtained from maternal E. argophloia trees crossed with E. argophloia pollen and with pollens from E. moluccana, E. crebra, and E. microcarpa were sown in standard 40-cell ‘Hyco V93 trays’ used to grow eucalypts for plantation forestry in Australia, in a glasshouse, during January 2009. Each cell in the tray had a 70 ml plastic insert, to facilitate sorting and seedling assessment. The potting medium consisted of 50% pine bark fines (0–10 mm), 25% pine bark peat, 25% coarse perlite, and a mix of 12–14 month slow release OsmocoteÒ (N 17.9: P 0.8: K 7.3) fertiliser at a rate of 4 kg/m3, gypsum (1 kg/m3), MicromaxÒ (1 kg/m3) and a granular wetting agent Hydroflo2Ò (1 kg/m3). Seed were irrigated five times a day for three minutes at each irrigation time using an overhead spray and fertilised with 2 ml/l of both NitrosolÒ (N 10.5%, P 2.3%, K 6.8% and some trace elements) (Yates Pty Ltd) and SeasolÒ (primarily trace elements) (Seasol International Pty Ltd) liquid fertiliser every two weeks. At three months the number of seedlings for each cross type were counted and viabilities determined based on the number of seed planted.
2.4. Hybridisation with allopatric species
2.8. Statistical analysis
We applied five pollen treatments to bunches of flower buds on each of four maternal E. argophloia trees, one from the Burncluith sub-provenance, one from the Burra Burri sub-provenance and two from the Fairyland sub-provenance. Pollen treatments were: (1) open pollination (as a control), (2) cross within species (using a pollen polymix to prevent self-fertilisation), (3) sympatric species E. microcarpa (from the same section as E. argophloia, Adnataria), (4) allopatric species E. pellita (from a more distant section, Latoangulatae) and (5) allopatric species E. resinifera, also from Latoangulatae (Brooker, 2000) (Table 1). The three-stop pollination method was used in all treatments except for open pollination. Three bunches of 20 flower buds for each pollen treatment for each tree were pollinated to assess fruit set and seed production (3 bunches 5 treatments 4 trees, total 60 bunches) and three bunches of 10 flower buds per treatment for each tree were pollinated for pollen tube analysis as described above.
The percentage of capsules set, seeds per capsule pollinated and seed weight per capsule set were calculated for each bunch for each experiment. Data for 2.2 ‘‘Breeding system and pollination methods” were normally distributed and were analysed as a 2way ANOVA with pollination method (one stop or three stop method) and pollen source (self, Burncluith, Burra Burri and E. moluccana) as factors followed by a Tukey’s HSD test where differences were significant. One tree was excluded from the analysis due to failure to set capsules in any treatment. An additional analysis was carried out to determine whether selfing rates differed between female trees. The number of seeds per capsule pollinated was analysed as a 3 way ANOVA with pollen source, female and pollination methods as factors (main effects only). Data for percentage of capsule pollinated and seeds per capsule pollinated from experiment 2.3 ‘‘Hybridisation with sympatric species” were square root transformed where necessary and
Table 1 Classification of species used for hybridisation. Occurrence
Species
Subgenus
Section
Series
Subseries
Sympatric
E. E. E. E.
Symphyomyrtus Symphyomyrtus Symphyomyrtus Symphyomyrtus
Adnataria Adnataria Adnataria Adnataria
Submelliodorae Siderophloiae Buxeales Buxeales
Continentes Continentes
Symphyomyrtus Symphyomyrtus
Latoangulatae Latoangulatae
Annulares Annulares
Allopatric
Brooker (2000).
argophloia crebra microcarpa moluccana
E. pellita E. resinifera
300
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
analysed with a 1-way ANOVA to test for significant differences in capsules and seed production between pollen sources. Data for seed weight per capsule set for experiment 2.4 ‘‘Hybridisation with allopatric species” were also analysed with a 1 way ANOVA to test for significant differences between pollen sources for the parameter ‘‘seed weight per capsule set”. In experiment 2.3 ‘‘Hybridisation with sympatric species” data for pollen tubes and seed weight, and experiment 2.4 ‘‘Hybridisation with allopatric species” data for pollen tubes” capsule set and seeds per capsule pollinated were non-parametric and did not meet the assumptions of ANOVA. These were therefore analysed with a Kruskal-Wallis tests followed by Mann Whitney -U tests where differences were significant. A Bonferroni correction factor was applied to determine the appropriate level of significance (Sokal and Rohlf, 1995). All data were analysed using SPSS version 17 (SPSS Science, Chicago)
Table 2 Mean pollen tubes in upper and lower style, for each pollination treatment for crosses with sympatric eucalypt species and cross within species. There were no significant differences between treatments, but all crosses produced pollen tubes. Pollination treatment
Pollen tubes upper style
Pollen tubes lower style
E. E. E. E.
0.3 1.0 11.8 0.5
0.5 1.3 0.5 0.8
argophloia crebra microcarpa moluccana
There were no significant difference in selfing rates (seeds produced per capsule pollinated) between female trees (P < 0.05) and they ranged from 0.5 to 1.35 for 1 stop treatments and 3.74 to 4.73 for 3 stop pollination treatments.
3.2. Hybridisation with sympatric species 3. Results 3.1. Breeding system and pollination methods Our results show that three stop pollination produced significantly higher percentage capsule set and seeds per capsule pollinated (P < 0.001, Fig. 1). All crosses and self-pollination treatments produced some capsules and seed, and there were no significant differences between any of the pollen sources tested.
All crosses tested in this hybridisation study produced some pollen tubes in the upper and lower style, although differences were not significant (Table 2). All crosses also produced both capsules and seed. However there were no significant differences between pollen treatments for percentage capsule set, number of seeds per capsule pollinated or seed weight per capsule set (Table 3). Examples of putative hybrids from the Dunmore seed orchard are presented in Fig. 3.
3.3. Hybridisation with allopatric species All crosses produced pollen tubes in the upper and lower style, although there were no significant differences (P < 0.05) between pollen treatments (Table 4). All pollen parents produced some capsules and some seed (Fig. 2a, b). Intraspecific pollen produced a similar number of seeds to interspecific pollen, irrespective of how closely or distantly related the species were. Open-pollination produced significantly more capsules than all the control-cross pollinations, however there was no significant difference between the other pollen parents (Fig. 2a). There were significant differences in seeds per capsule pollinated between treatments (Fig. 2b, P = 0.007). Open pollination produced significantly more seeds per capsule pollinated (P = 0.017) than the other pollinations, however there was no significant difference between the other crosses. Open pollination produced five seeds per capsule (very high compared with one to four considered more normal for a eucalypt) and there were one to two per capsule for the cross-pollinations attempted.
3.4. Viability of seed from crosses with sympatric species Mean viability of seed ranged from 0.41 to 0.51, and there was no significant difference between a E. argophloia outcross and crosses with E. moluccana, E. microcarpa and E. crebra (Table 6).
Table 3 Mean percentage of capsules set and mean number of seeds per capsule pollinated and standard errors (SE), for each pollination treatment for crosses with sympatric eucalypt species and cross within species. There were no significant differences between treatments, but all crosses produced seed.
Fig. 1. (a) Capsules set (%) and (b) seeds per capsule pollinated from E. argophloia using one-stop and three-stop pollination treatments. Self = self-pollination using pollen from the same tree, Burra Burri = within species cross using pollen from an unrelated tree ‘‘Burra Burri”, Burncluith = within species cross using pollen from an unrelated tree ‘‘Burncluith”,moluccana = cross pollination using pollen from E. moluccana. Means with different letters are significantly different (P < 0.05).
Pollination treatment
Capsule set % (SE)
Seeds per capsule pollinated (SE)
E. E. E. E.
6.1 3.3 2.5 4.1
1.4 1.5 1.9 1.6
argophloia crebra microcarpa moluccana
(1.3) (0.8) (0.8) (1.3)
(0.2) (0.3) (0.3) (0.3)
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
301
4. Discussion
Fig. 2. (a) Capsules set (%) and (b) Seeds per capsule pollinated for E. argophloia for cross within-species, cross with closely related sympatric species (E. microcarpa), cross with more distantly related allopatric species (E. resinifera and E. pellita), and open pollination. Treatment means with different letters are significantly different (P < 0.05) (Mann-Whitney U tests with a Bonferroni correction factor). E. argo = E. argophloia;E. micro = E. microcarpa;E. resin = E. resinifera;Open = open pollination.
These results show that E. argophloia readily forms hybrids not only with species both within its own section Adnataria (E. moluccana, E. crebra and E. microcarpa,) but also with species in a more distant section, Latoangulatae (E. pellita and E. resinifera). In addition, E. argophloia readily self-pollinates. Previous studies have shown that many Eucalyptus species readily hybridise within the subgenus Symphomyrtus (Potts and Reid, 1985; Potts, 1986; Potts et al., 1987; Potts and Reid, 1988; Field et al., 2010), but not those from different subgenera (Ellis et al., 1991). In the current study controlled inter-specific hybridisation of E. argophloia with closely related species E. microcarpa produced no significant differences for pollen tubes, capsule set and seed set compared with self-pollination or crossing with the more distantly related species E. resinifera and E. pellita. This is contrary to the hypothesis of reduced seed set with increasing genetic distance (Griffin et al., 1988), as observed in several previous studies (Meddings et al., 2003; Barbour et al., 2005; Dickinson et al., 2012). Seed set decreased with increasing genetic distance for Corymbia hybrids (Dickinson et al., 2012) and seven Tasmanian eucalypts pollinated with E. nitens (Barbour et al., 2005). Similarly, E. camaldulensis (section Exsertaria) as the female parent crossed with E. globulus (section Maidenaria) exhibited substantial barriers to hybridisation compared with outcrossed E. camaldulensis (Meddings et al., 2003). When E. globulus pollen was applied to 99 eucalypt species there was a decline in compatibility with increasing genetic distance between species (Larcombe et al., 2015). The current results indicate that E. argophloia may readily accept pollen from a wide variety of eucalypt species within the subgenus Symphomyrtus. The implication of this is the risk of genetic pollution of the remaining vulnerable, natural stands of E. argophloia. These results demonstrate an important principle with wide application, that ability to hybridise should never be assumed from relationships, but can only be determined by experimentation. Natural hybridisation is now recognized as an important component of evolution of species (e.g. Ellstrand and Schierenbeck, 2000; Abbot et al., 2003), however, hybridisation also threatens a substantial number of plant and animal species with extinction
Fig. 3. Eucalyptus argophloia (Chinchilla White Gum) and putative hybrids. a, Eucalyptus argophloia trees of ‘‘Fairyland” sub-provenence; b, c, atypical trees in the Dunmore State Forest Station seed orchard grown from E. argophloia seed. Note characteristic white bark in ‘a’, and on background trees in ‘c’, compared with rough, dark bark in ‘b’, and tessellated box-type bark in foreground in ‘c’.
302
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
Table 4 Mean pollen tubes in the upper and lower style for open pollination, cross within species (E. argophloia), cross with a closely related sympatric species (E.microcarpa) and crosses with more distantly related allopatric eucalypt species (E. pellita and E. resinifera). Pollination treatment
Pollen tubes upper style
Pollen tubes lower style
Open E. argophloia E. microcarpa E. pellita E. resinifera
0.6 4.2 2.2 5.3 0.7
0.6 2.1 1.5 1.2 0.2
Table 5 Viability of seed obtained from Eucalyptus argophloia maternal trees by crossing with E. argophloia, and crossing with pollen from closely related sympatric species. Cross type
Mean viability
Number of crosses tested
E. E. E. E.
0.43 0.49 0.51 0.41
4 3 3 3
argophloia moluccana crebra microcarpa
(Levin et al., 1996; Ellstrand et al., 2010). Hybridisation is perhaps the most rapidly acting genetic threat to endangered species (Wolf et al., 2001). Hybridisation may also confer new characters that may in some cases facilitate hybrids establishing as weeds (Wallace and Leonhardt, 2015). Human activities such as removing habitat and planting foreign species are increasing the opportunities for hybridisation (Ellstrand et al., 2010). Any activity that exposes native populations of E. argophloia to foreign pollen (such as plantations or nearby amenity plantings) would be a threat to the genetic integrity of the species, particularly in view of its ability to readily hybridise. In addition the ability of the species to selfpollinate is a concern for maintenance of genetic diversity. As an example, the genetic diversity of the rare mallee E. absita was significantly compromised by a high rate of selfing and potential hybridisation (Bradbury et al., 2015). Our results show that E. argophloia crosses readily with intrasectional and inter-sectional species within the Symphomyrtus subgenus. Gene flow from different species can be detrimental to small populations such as this species due to fitness reduction of hybrids from the breakdown of co-adaptation to local conditions (Waser and Price, 1989, 1991). Small populations can be expected to receive gene flow at a higher rate than large populations, and are more likely to receive gene flow from large populations than small ones, even if the latter is in closer proximity (Ellstrand et al., 1989). The potential for hybridisation often depends on how closely related a species is to adjacent native forest species (Potts and Reid, 1985; Griffin et al., 1988). Because E. argophloia hybridises readily with closely related and distantly related Eucalyptus species there are concerns about the risk of gene flow by pollen from naturally occurring closely related congeners such as E. crebra, E. moluccana and E. microcarpa, especially if these are more numer-
ous. The presence of putative hybrids in the seed orchard suggests that hybridisation with sympatric species is already occurring. The threat from the other hybridizing species, E. resinifera and E. pellita may not be such a concern as they are not sympatric with E. argophloia, and different climatic requirements suggest that they would not be suitable for plantations near natural populations of E. argophloia (Brawner et al., 2013). Flowering synchronicity is a major factor in the potential for hybridisation of species. E. argophloia has been reported to have a flowering range from January to August (Boland et al., 1984; Brooker and Kleinig, 2004; Randall et al., 2012, 2015). This means that due to flowering synchronicity it could hybridise with sympatric intra-sectional pollen from E. moluccana, E. microcarpa and E. crebra, and even with allopatric inter-sectional pollen from E. pellita and E. resinifera if the latter two became adjacent (Table 5). The inflow of foreign pollen presents a risk to small populations such as E. argophloia. There may be a risk of introgression with more numerous common species to produce hybrid cohorts in which the characters of the rare species are eventually swamped by one of the parent species (Ellstrand and Elam, 1993; Abbot et al., 2003; Vanden-Broeck et al., 2005). This is often called genetic swamping, but could be termed ‘‘pollen swamping”, i.e. repeated backcrosses with incoming pollen (Petit et al., 2004). Introgression involves many steps that involve several hybrid generations (F1, F2, BC1, BC2 etc. (Vanden-Broeck et al., 2005). The end result is that one species is replaced by the other, or alternatively both hybridizing parents are eventually replaced by swamping from their hybrid-derived descendants (Hedge et al., 2006). Hybridisation could lead to introgression if F1 hybrids backcross (Dickinson et al., 2012; Vanden-Broeck et al., 2012). In eucalypts, the integrity of the rare species Eucalyptus aggregata is at risk from preferential backcrossing with hybrids with the common E. rubida (Field et al., 2010). Similarly, the more common species E. amygdalina is seen to be invading the range of the rare species E. risdonii by both seed and pollen migration (Potts, 1986). The vulnerable species E. argophloia is potentially at risk because of its ability to readily hybridise with a wide range of species. There are other factors which increase the risk for the species. It is a small population, highly fragmented and has a high degree of susceptibility to Myrtle Rust (Puccinia psidii) (Lee et al., 2015). Threatened, rare and vulnerable species such as E. argophloia are often particularly at risk from hybridisation because of small population size and fragmentation. This would be true for many species in a global context. There are many forest species such as E. argophloia, that while not commercially important, are nonetheless important in an ecological context or as an iconic species (Yu et al., 2014; Chandler and McGraw, 2015; Taylor et al., 2016). Viable seed has been produced from crossing E. argophloia with closely related species. Crosses with E. moluccana, E. crebra and E. microcarpa had similar viability to outcrossing within species. There is strong anecdotal evidence of natural hybrids of E. argophloia, for example, 5% putative hybrids have been reported in an
Table 6 Flowering times of Eucalyptus argophilia and species used as pollen parents with E. argophloia. Species
Jan
Feb.
March
April
May
June
July
Aug.
argophloia crebra microcarpa moluccana pellita resinifera
#
#
/
/
x* x
x* x
x* x* x*
x x x*
x x x*
x*
x* x*
x* x* x*
Sept.
Oct.
Nov.
Dec.
x* x
*
*
x
x
x
x
*
*
Flowering time is identified by x (Brooker and Kleinig, 2004). * Boland et al. (1984). # Randall et al. (2012) and / Randall et al. (2015).
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
E. argophloia plantation (Lee, personal comment). A few putative hybrids also exist in the seed orchard that hosted the current study (Fig. 3). However, the ability of hybrids to persist and backcross with E. argophloia is yet to be tested. Hybrids may have low rates of establishment compared with wildlings. For example E. nitens, E. ovata and E. viminalis had open pollination wildling establishment rates of 94–100% compared with 0.3–5.5% for hybrids of E. nitens with E. ovata and E. viminalis (Barbour et al., 2003). While there are putative hybrids in the seed orchard in the current study, and viable seed has been produced, at present there is little evidence that E. argophloia hybrids are establishing in the wild. For this to occur, hybrid progeny must be at least as fit as or fitter than the parent population (Dickinson et al., 2013; Shepherd and Lee, 2016). The risk is in proportion to the proximity of donor populations. Pollination in hybrid eucalypts is mostly over relatively short distances of less than 200 m, but low levels of pollination success are also to be expected over longer distances (Barbour et al., 2003; da Silva et al., 2015). The small E. argophloia bud is very difficult to hand pollinate without damage that affects retention of capsules (Randall et al., 2012, 2015). The buds of E. argophloia are only 0.4 0.4 cm (Brooker and Kleinig, 2004), smaller than those of many eucalypts and are much smaller than those evaluated in previous pollination studies (e.g. E. grandis 0.8 0.7 cm, E. nitens 0.7 0.6 cm, E. macarthurii 0.5 0.6 cm (Brooker and Kleinig, 2004). Higher seed set per bunch indicates that open pollination is much more successful. Hybrid seed set from open pollinations could be higher than the current study indicates without the limiting effects of emasculation during controlled pollination. Hybridisation risks from foreign gene flow to E. argophloia could be substantial. This was suggested by the pollen tube data which showed that there were no differences between E. argophloia intra-species and inter-species pollination.
5. Conclusions This study has shown that E. argophloia freely hybridises with genetically close, intra-sectional relatives E. crebra, E. microcarpa and E. moluccana, as well as with more distant inter-sectional species E. pellita and E. resinifera. The species is potentially at risk of genetic swamping by foreign pollen because of its ability to hybridise with a wide range of species and because of its very small and fragmented population structure. E. argophloia also readily self-pollinates; self-compatibility exposes this species to elevated evolutionary pressure. As a consequence, the genetic integrity of this iconic species may be at risk. The extent of hybridisation of this species with sympatric species is unknown, however the results of the current study demonstrate the need for genetic studies to determine the extent of foreign pollen flow into E. argophloia. Acknowledgements The authors wish to thank the Australian Research Council linkage grant number LP0562678 for financial assistance, Department of Primary Industries and Fisheries (formerly AgriScience Queensland) for technical support, and HQ Plantations (formerly DPI-Forestry) for financial assistance and access to the trial sites. References Abbot, R.J., James, J.K., Milne, R.I., Gillies, A.C.M., 2003. Plant introductions, hybridisation and gene flow. Philosoph. Trans. Royal Soc. London 358, 1123– 1132. Barbour, R., Potts, B., Vaillancourt, R., Tibbits, W., Wiltshire, R., 2002. Gene flow between introduced and native Eucalyptus species. New For. 23, 177–191.
303
Barbour, R.C., Potts, B.M., Vaillancourt, R.E., 2003. Gene flow between introduced and native Eucalyptus species: exotic hybrids are establishing in the wild. Aust. J. Bot. 51, 429–439. Barbour, R., Potts, B., Vaillancourt, R., 2005. Gene flow between introduced and native Eucalypt species: crossability of native Tasmanian species with exotic E. nitens. Aust. J. Bot. 53, 465–477. Barbour, R., Potts, B., Vaillancourt, R., Tibbits, W., 2006. Gene flow between introduced and native Eucalyptus species: flowering asynchrony as a barrier to F1 hybridisation between exotic E. nitens and Tasmanian Symphyomyrtus species. For. Ecol. Manage. 226, 9–21. Barbour, R., Otahal, Y., Vaillancourt, R., Potts, B., 2008. Assessing the risk of pollenmediated gene flow from exotic Eucalyptus globulus plantations into native eucalypt populations of Australia. Biol. Conserv. 141, 896–907. Barbour, R.C., Wise, S.L., McKinnon, G.E., Vaillancourt, R.E., Williamson, G.J., Potts, B. M., 2010. The potential for gene flow from exotic eucalypt plantations into Australia’s rare native eucalypts. For. Ecol. Manage. 260, 2079–2087. Boland, D.J., Brooker, M.I.H., Chippendale, G.M., Hall, N., Hyland, B.P.M., Johnson, R. D., Kleinig, D.A., McDonald, M.W., Turner, J.D., 1984. Forest Trees of Australia. CSIRO, Melbourne. Bradbury, D., Grayling, P.M., MacDonald, B., Hankinson, M., Byrne, M., 2015. Clonality, interspecific hybridisation and inbreeding in a rare mallee eucalypt, Eucalyptus absita (Myrtaceae), and implications for conservation. Conserv. Genet. http://dx.doi.org/10.1007/s10592-015-0771-8. Brawner, J.T., Lee, D.J., Meder, A.R., Almeida, A., Dieters, M.J., 2013. Classifying genotype by environment interactions for targeted germplasm deployment with a focus on Eucalyptus. Euphytica 191, 403–414. Broadhurst, L.M., Lowe, A., Coates, D.J., Cunningham, S.A., McDonald, M., Vesk, P.A., Yates, P., 2008. Seed supply for broadscale restoration: maximizing evolutionary potential. Evol. Appl. 1, 587–597. Brooker, M., 2000. A new classification system of the Genus Eucalyptus L’Her. (Myrtaceae). Aust. System. Bot. 13, 79–148. Brooker, M., Kleinig, D., 1999. Field Guide to Eucalypts—Volume 1 Southeastern Australia. Bloomings Books, Melbourne, pp. 70–186. Brooker, M., Kleinig, D., 2004. Field Guide to Eucalypts – Northern Australia. Inkata Press, Sydney. Butcher, P.A., Skinner, A.K., Gardiner, C.A., 2005. Increased inbreeding and interspecies gene flow in remnant populations of the rare Eucalyptus benthamii. Conserv. Genet. 6, 213–226. Chandler, J.L., McGraw, J.B., 2015. Variable effects of timber harvest on the survival, growth, and reproduction of American ginseng (Panax quinquefolius L.). For. Ecol. Manage. 344, 1–9. CPBR, 2006. EUCLID: Eucalypts of Australia. CSIRO Publishing, Collingwood, Victoria. da Silva, P.H.M., Sebbenn, A.M., Grattapaglia, D., 2015. Pollen-mediated gene flow across fragmented clonal stands of hybrid eucalypts in an exotic environment. For. Ecol. Manage. 356, 293–298. Dickinson, G.R., Lee, D.J., Wallace, H.M., 2012. The influence of pre- and postzygotic barriers on hybridisation between Corymbia sections. Ann. Bot. 109, 1215–1226. Dickinson, G., Lee, D., Wallace, H., 2013. Reciprocal and advanced generation hybrids between Corymbia citriodora and C. torelliana: forestry breeding and the risk of gene flow. Ann. Forest. Sci. 70, 1–10. Ellis, M.F., Sedgley, M., Gardner, J.A., 1991. Interspecific pollen-pistil interaction in Eucalyptus L’Hér. (Myrtaceae): the effect of taxonomic distance. Ann. Bot. 68, 185–194. Ellstrand, N., 1992. Gene flow by pollen: implications for plant conservation genetics. Oikos 63, 77–86. Ellstrand, N., Elam, D., 1993. Population genetics consequences of small population size: implications for plant conservation. Annu. Rev. Ecol. Syst. 24, 217–242. Ellstrand, N.C., Schierenbeck, K.A., 2000. Hybridisation as a stimulus for the evolution of invasiveness in plants? Proc. Nat. Acad. Sci. USA 97, 7043–7050. Ellstrand, N.C., Devlin, B., Marshall, D.L., 1989. Gene flow by pollen into small populations: data from experimental and natural stands of wild radish. Proc. Nat. Acad. Sci. USA 86, 9044–9047. Ellstrand, N.C., Biggs, D., Kaus, A., Lubinsky, P., McDade, L.A., Preston, K., Prince, L.M., Regan, H.M., Roriver, V., Ryde, K.A., Schierenbeck, K.S., 2010. Got hybridisation? A multidisciplinary approach for informing science policy. Bioscience 60, 384–388. Environment Australia, 2009. Environment Protection and Biodiversity Conservation Act, 1999. Flora that are Endangered (accessed 26th November 2014). Field, D.L., Ayre, D.J., Whelan, R.J., Young, A.G., 2010. Patterns of hybridisation and asymmetrical gene flow in hybrid zones of the rare Eucalyptus aggregata and common E. rubida. Heredity 2010, 1–13. Griffin, A., Burgess, I., Wolf, L., 1988. Patterns of natural and manipulated hybridisation in the genus Eucalyptus L’Herit.: a review. Aust. J. Bot. 36, 41–66. Hedge, S.G., Nason, J.D., Clegg, J.M., Ellstrand, N.C., 2006. The evolution of California’s wild radish has resulted in the extinction of its progenitors. Evolution 60, 1187–1197. Horsley, T., Johnson, S., Myburg, A., 2010. Comparison of different controlpollination techniques for small-flowered eucalypts. New For. 39, 75–88. IPCC, 2014. Climate change 2014: synthesis report. In: Pachauri, R.K., Meyer, L.A. (Eds.), Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge.
304
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
Larcombe, M.J., Barbour, R.C., Vaillancourt, R.E., Potts, B.M., 2014. Assessing the risk of exotic gene flow from Eucalyptus globulus plantations to native E. ovata forests. For. Ecol. Manage. 312, 193–202. Larcombe, M.J., Holland, B., Steane, D.A., Jones, R.C., Nicolle, D., Vaillancourt, R.E., Potts, B.M., 2015. Patterns of reproductive isolation in Eucalyptus. Mol. Biol. Evol. 32, 1833–1846. Lee, D.J., 2007. Achievements in forest tree genetic improvement in Australia and New Zealand 2: Development of Corymbia species and hybrids for plantations in eastern Australia. Aust. For. 70, 11–16. Lee, D.J., Huth, J.R., Osborne, D.O., Hogg, B.W., 2010. Selecting hardwood taxa for wood and fibre production in Queensland’s subtropics. Aust. Forest. 73, 106– 114. Lee, D.J., Brawner, J.T., Pegg, G.S., 2015. Screening Eucalyptus cloeziana and E. argophloia populations for resistance to Puccinia psidii. Plant Dis. 99, 71–79. Levin, D.A., Francisco-Ortega, J., Jansen, R.K., 1996. Hybridisation and the extinction of rare plant species. Con. Biol. 10, 10–16. Linacre, N., Ades, P., 2004. Estimating isolation distances for genetically modified trees in plantation forestry. Ecol. Model. 179, 247–257. Meddings, R.A., McComb, J.A., Calver, M.C., Thomas, S.R., Mazanec, R.A., 2003. Eucalyptus camaldulensis globulus hybrids. Aust. J. Bot. 51, 319–331. Merchant, A., Callister, A., Arndt, S., Tausz, M., Adams, M., 2007. Contrasting physiological responses of six Eucalyptus species to water deficit. Ann. Bot. 100, 1507–1515. Nevill, P.G., Després, T., Bayly, M.J., Bossinger, G., Ades, P.K., 2014. Shared geographic patterns and widespread chloroplast haplotype sharing in Eucalyptus species with different ecological tolerances. Tree Genet. Genomes 10, 1079–1092. Ngugi, M., Hunt, M., Doley, D., Ryan, P., Dart, P., 2004a. Selection of species and provenances for low-rainfall areas: physiological responses of Eucalyptus argophloia to seasonal conditions in subtropical Queensland. Forest Ecol. and Manage. 193, 141–156. Ngugi, M., Hunt, M., Doley, D., Ryan, P., 2004b. Physiological responses to water stress in Eucalyptus cloeziana and E. argophloia seedlings. Trees 18, 381–389. Petit, R.J., Bodenes, C., Ducousso, G.R., Kremer, A., 2004. Hybridisation as a mechanism of invasion in oaks. New Phytol. 161, 131–164. Potts, B.M., 1986. Population dynamics and regeneration of a hybrid zone between Eucalyptus risdonii Hook. f. and E. amygdalina Labill. Aust. J. Bot. 34, 305–329. Potts, B., Dungey, H., 2004. Interspecific hybridisation of Eucalyptus: key issues for breeders and geneticists. New For. 27, 115–138. Potts, B.M., Reid, J.B., 1985. Analysis of a hybrid swarm between Eucalyptus risdonii Hook.f. and E. amygdalina Labill. Aust. J. Bot. 33, 543–562. Potts, B.M., Reid, J.B., 1988. Hybridisation as a dispersal mechanism. Evolution 42, 1245–1255. Potts, B.M., Potts, W.C., Cauvin, B., 1987. Inbreeding and interspecific hybridisation in Eucalyptus gunnii. Silvae Genet. 36, 194–199. Potts, B.M., Barbour, R.C., Hingston, A.B., Vaillancourt, R.E., 2003. Genetic pollution of native eucalypt gene pools – identifying the risks. Aust. J. Bot. 51, 1–25. Queensland Government, 1992. Nature Conservation Act, 1992 (accessed August, 2015). Randall, B.W., Walton, D.A., Lee, D.J., Wallace, H.M., 2012. Fertilisation and pollen tube development in Eucalyptus argophloia. Silvae Genet. 61, 121–126.
Randall, B.W., Walton, D.A., Lee, D.J., Wallace, H.M., 2015. Comparison of three pollination methods for Eucalyptus argophloia, a small-flowered eucalypt. Ann. For. Sci. 72, 127–133. Rhymer, J.M., Simberloff, D., 1996. Extinction by hybridisation and introgression. Ann. Rev. Ecol. Syst. 27, 83–109. Schierenbeck, K.A., Ellstrand, N.C., 2009. Hybridisation and the evolution of invasiveness in plants and other organisms. Biol. Invasions 11, 1093–1105. Sedgley, M., Smith, R.M., 1989. Pistil receptivity and pollen tube growth in relation to the breeding systems of Eucalyptus woodwardii Maiden (Symphyomyrtus). Ann. Bot. 64, 21–31. Shepherd, M., Lee, D.J., 2016. Gene flow from Corymbia hybrids in northern New South Wales. For. Ecol. Manage. 362, 205–217. Sokal, R., Rohlf, F., 1995. Biometry: The Principles and Practice of Statistics in Biological Research. W. H. Freeman and Company, New York. Strauss, S., 2001. Benefits and risks of biotic exchange between Eucalyptus plantations and native Australian forests. Austral Ecol. 26, 447–457. Taylor, J.E., Ellis, M.V., Rayner, L., Ross, K.A., 2016. Variability in allometric relationships for temperate woodland Eucalyptus trees. For. Ecol. Manage. 360, 122–132. Trindade, H., Boavid, L.C., Borralho, N., Feijo, J.A., 2001. Successful fertilization and seed set from pollination on immature non-dehisced flowers of Eucalyptus globulus. Ann. Bot. 87, 469–475. Vanden-Broeck, A., Villar, M., Van Bockstaele, E., Van Slycken, J., 2005. Natural hybridisation between cultivated poplars and their wild relatives: evidence and consequences for native populations. Ann. For. Sci. 62, 601–613. Vanden-Broeck, A., Cox, K., Michiels, B., Verschule, P., Villar, M., 2012. With a little help from my friends: hybrid fertility of exotic Populus x canadensis enhanced by related native Populus nigra. Biol. Invasions 14, 1683–1696. Vila, M., Weber, E., D’Antonio, C.M., 2000. Conservation implications of invasion by plant hybridisation. Biol. Invasions 2, 207–217. Wallace, H.M., Leonhardt, S.D., 2015. Do hybrid trees inherit invasive characteristics? Fruits of Corymbia torelliana X C. citriodora hybrids and potential for seed dispersal by bees. PLOS ONE. http://dx.doi.org/10.1371/ journal.pone.0138868. Waser, N.M., Price, M.V., 1989. Optimal outcrossing in Ipomopsis aggregata: seed set and offspring fitness. Evolution 43, 1097–1109. Waser, N.M., Price, M.V., 1991. Outcrossing distance effects in Delphinium nelsonii: pollen loads, pollen tubes and seed set. Ecology 72, 171–179. Whyte, G., Howard, G.E.St., Hardy, T.I., Burgess, T.I., 2016. The tree decline recovery seewaw; a conceptual model of the decline and recovery of drought stressed plantation trees. For. Ecol. Manage. 370, 102–113. Wolf, D.E., Takebayashi, N., Rieseberg, L.H., 2001. Predicting the risk of extinction through hybridisation. Conserv. Biol. 15, 1039–1053. Yu, J., Wang, C., Wan, J., Han, S., Wang, Q., Nie, S., 2014. A model-based method to evaluate the ability of nature reserves to protect endangered tree species in the context of climate change. For. Ecol. Manage. 327, 48–54. Xie, Q., Li, Z., Yang, L., Lv, J., Jobe, T.O., Wang, Q., 2015. A newly identified passive hyperaccumulator Eucalyptus grandis x E. urophylla under manganese stress. PLoS ONE 1, 2015. http://dx.doi.org/10.1371/journal.pone.0136606.
Contents lists available at ScienceDirect
Forest Ecology and Management journal homepage: www.elsevier.com/locate/foreco
The risk of pollen-mediated gene flow into a vulnerable eucalypt species Bruce W. Randall a, David A. Walton a,⇑, David J. Lee a,b, Helen M. Wallace a a b
University of the Sunshine Coast, Maroochydore DC, Queensland 4558, Australia Department of Primary Industries and Fisheries, Gympie, Queensland 4570, Australia
a r t i c l e
i n f o
Article history: Received 11 April 2016 Received in revised form 22 September 2016 Accepted 25 September 2016
Keywords: Eucalyptus argophloia Hybridisation Pollination Gene flow Pollen tube growth Eucalypts
a b s t r a c t Hybridisation is recognized as a threat to the genetic integrity of rare, threatened and vulnerable species. Eucalypts are widely planted for plantation forestry and many species readily hybridise making ‘‘vulnerable”, locally-occurring species potentially at risk of genetic swamping. Eucalyptus argophloia (Queensland western gum or Chinchilla white gum) is a vulnerable species with limited distribution in South Eastern Queensland, Australia. The risks to this species from gene flow from surrounding forest or plantations are poorly understood. We investigated the breeding system and hybridisation potential of E. argophloia by controlled pollinations with selected sympatric and allopatric species. Treatments included self-pollination, outcross within species and hybridisation with E. crebra, E. microcarpa and E. moluccana (from within the same section, Adnataria) and E. pellita and E. resinifera (from a more distantly related section, Latoangulatae). The aim was to determine the degree of self-pollination and gene flow into E. argophloia from closely-related and more distantly-related sympatric and allopatric species. Eucalyptus argophloia is readily self-fertile. It also hybridises with a broad range of intra-sectional and inter-sectional eucalypt species. All crosses produced some pollen tubes, capsules and seed, and open pollination produced significantly more capsules and seeds than all controlled pollinations attempted. The number of seeds per capsule pollinated was not significantly different between pollen treatments. There is a potential risk of gene flow into E. argophloia populations from adjacent natural populations of the species tested as pollen parents, or from planted Eucalyptus species. This risk of foreign gene flow could be greater with open pollination than indicated by controlled pollination in this study because of the limiting effect of bud damage incurred during controlled pollinations. The ability to self-pollinate in the small population of E. argophloia raises the potential risk of genetic swamping from foreign pollen and elevated evolutionary pressure. These results will be useful for guiding management of this vulnerable species such as limiting plantings of cross-compatible species close to existing populations. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Hybridisation between species is an important evolutionary process that can drive plant adaptations and can also present a threat to the genetic integrity of plant populations (Schierenbeck and Ellstrand, 2009; Ellstrand et al., 2010; Larcombe et al., 2014). Hybridisation is a particular threat to rare, threatened and vulnerable species and in extreme cases there is a danger of extinction due to genetic swamping (Levin et al., 1996; Vila et al., 2000; Butcher et al., 2005; Barbour et al., 2010). Hybridisation is now an urgent threat to species integrity because environmental disturbance and human activities such as forest plantations are bringing
⇑ Corresponding author at: Genecology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland 4519, Australia. E-mail address: [email protected] (D.A. Walton). http://dx.doi.org/10.1016/j.foreco.2016.09.042 0378-1127/Ó 2016 Elsevier B.V. All rights reserved.
together cross-compatible species that previously were geographically separated (Rhymer and Simberloff, 1996; Ellstrand et al., 2010). The threat of hybridisation commonly occurs when pure species are pollinated by foreign pollen, i.e. from pollen-mediated gene flow (da Silva et al., 2015; Shepherd and Lee, 2016). Further, when hybrids survive to flowering, even with low fecundity, there is a potentially serious risk to native populations of introgression of genes and genetic swamping if the hybrid progeny are as fit as or more fit than the parent population (Dickinson et al., 2013; Shepherd and Lee, 2016). Introgression can lead to extinction of small restricted populations if reduced fitness of subpopulations allows swamping of the gene pool or introduction of new genes (Linacre and Ades, 2004). On the positive side, hybridisation may help some species adapt to rapidly changing conditions such as climate change (Broadhurst et al., 2008; Nevill et al., 2014).
298
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
Eucalypts are one of the most widely planted taxa for plantation forestry with over 20 million ha globally (Whyte et al., 2016). Eucalypts are also the dominant trees throughout most non-arid plant communities in the Australian continent (Brooker and Kleinig, 1999). There are nearly 900 eucalypt taxa which are endemic to Australia and its surrounding islands (CPBR, 2006) and 77 taxa are currently listed as endangered or vulnerable at the national level in Australia and require special attention (Environment Australia, 2009). Gene flow via hybridisation is a particular risk for rare eucalypt species in Australia (Barbour et al., 2010). Eucalypt diversity can be viewed as a consequence of substantial outbreeding and eucalypts often demonstrate weak reproductive barriers between closely related taxa (Griffin et al., 1988; Boland et al., 1984; Dickinson et al., 2012, 2013). Within the eucalypts, hybrids are establishing in the wild in some circumstances (Griffin et al., 1988; Barbour et al., 2003). Predicting the extent of gene flow necessitates understanding pollen dispersal and flowering overlap (Barbour et al., 2003, 2005, 2006). Most records of manipulated hybrids derive from the commercially important subgenus Symphyomyrtus (Griffin et al., 1988; Larcombe et al., 2015). Examples of successful manipulated hybrids used in forestry plantations are E. grandis E. urophylla, widely used for forestry plantations in Brazil and China, and Corymbia torelliana C. citriodora, increasingly planted in Australia and other subtropical regions around the world (Barbour et al., 2008; Lee et al., 2010; Dickinson et al., 2013; da Silva et al., 2015; Xie et al., 2015; Shepherd and Lee, 2016). The risk of natural hybridisation between species in different subgenera depends on the degree of spatial separation, taxonomic separation and flowering synchronicity between parents (Ellstrand, 1992; Barbour et al., 2002, 2006, 2008; Horsley et al., 2010). Generally, the risk of hybridisation with surrounding native populations decreases with increasing taxonomic distance between parents (Potts et al., 2003; Potts and Dungey, 2004; Larcombe et al., 2015). E. argophloia (Queensland Western White Gum or Chinchilla White Gum) is a vulnerable eucalypt endemic to Southeast Queensland, Australia with a restricted distribution (Brooker and Kleinig, 2004). Eucalyptus argophloia is of interest for both forestry and conservation, similar to Eucalyptus benthamii (Butcher et al., 2005; Ngugi et al., 2004a, 2004b; Merchant et al., 2007) Droughts are expected to increase in frequency and magnitude in many parts of the world (IPCC, 2014). Eucalypt plantations are already experiencing more frequent droughts and species that can tolerate long periods of drought are likely to be more critical as the impacts of climate change intensify (Whyte et al., 2016). E. argophloia has commercial potential as a forestry species due to fast growth rates, strong durable timber, drought tolerance (600–900 mm mean annual rainfall) and salinity tolerance (Ngugi et al., 2004a,b; Merchant et al., 2007). Hybrids of the species may also be useful as is the case for other eucalypts (Lee, 2007; Barbour et al., 2008; Dickinson et al., 2013; da Silva et al., 2015; Xie et al., 2015; Shepherd and Lee, 2016). The natural range of the species is an area only 30 km east to west and 20 km north to south, with three small disjunct sub-populations named ‘‘Burncluith”, ‘‘Burra Burri” and ‘‘Fairyland” (Lee et al., 2015). Land clearing has fragmented the sub-populations of E. argophloia and the total remaining wild population is only around 1000 individuals (Lee et al., 2015). Consequently, E. argophloia is listed as ‘‘vulnerable” (needing conservation, protection, and implementation of recovery plans to ensure that it remains a viable population) by the Environment Protection and Biodiversity Conservation Act 1999 (Australia) (Environment Australia, 2009) and the Nature Conservation Act 1992 (Queensland Government, 1992). Small, fragmented remnant populations such as E. argophloia may be at risk of being genetically swamped by other species because the rate of gene flow may increase as the size of the population decreases (Ellstrand et al.,
1989; Ellstrand and Elam, 1993; Linacre and Ades, 2004; Barbour et al., 2006). In addition in small, fragmented eucalypt populations higher levels of selfing and inbreeding are also a risk (Butcher et al., 2005). E. argophloia is a member of the subgenus Symphyomyrtus (Brooker, 2000), therefore it may be at risk of genetic swamping due to hybridisation with other eucalypts (Griffin et al., 1988; Strauss, 2001). Other common, endemic eucalypt populations sympatric with E. argophloia include E. crebra, E. moluccana and E. microcarpa. The risk to E. argophloia of gene flow and genetic swamping from surrounding Eucalyptus species is poorly understood. We examined the breeding system and hybridisation potential of E. argophloia. Our aims were to examine (1) the breeding system of E. argophloia and in particular the degree of self-compatibility, (2) hybridisation potential with closely related sympatric species, (3) hybridisation potential with allopatric and more distantly related inter-sectional species. In-vivo pollen tube growth, capsule set and seed set were examined to determine pollination success and ability to hybridise. We hypothesized that E. argophloia would not hybridise readily with more distantly related species such as E. resinifera and E. pellita although anecdotal evidence suggests that it may readily cross with closely related Eucalyptus species such as E. moluccana, E. microcarpa and E. crebra. The results of this study will increase understanding of the risks posed by gene flow into E. argophloia populations from adjacent ecosystems, information that will aid conservation and management of this species. In addition, knowledge of compatible species may be useful if hybrids of E. argophloia prove to be useful for forestry, as has been the case for other Eucalyptus and Corymbia species. 2. Materials and methods 2.1. Study Site Pollination studies were conducted in an E. argophloia seed orchard at the Dunmore State Forest Station on the Darling Downs, southeast Queensland (27.34°S; 151.04°E) in the flowering season April to October 2007 and 2009. This seed orchard contains trees from three locations in the natural range of the species. Two of these locations, ‘‘Fairyland” and ‘‘Burra Burri”, contain relatively undisturbed blocks of several hundred trees and the other location, approximately 11 km away (Burncluith), consists of isolated trees in cleared agricultural land. Because E. argophloia is a small population the entire population is considered to be one provenance, and the three locations to be sub-provenances. All experiments were conducted using an elevated platform to access flowers. 2.2. Breeding system and pollination methods We selected four ten-year-old E. argophloia maternal parent trees from the seed orchard, two from the Burncluith subprovenance and two from the Burra Burri sub-provenance. Pollen parents were: (1) self-pollen from the same tree; (2) E. argophloia pollen from an unrelated tree of the Burncluith sub-provenance; (3) a second E. argophloia pollen from an unrelated tree of the Burra Burri sub-provenance; (4) E. moluccana pollen (a closely-related sympatric species). We emasculated individual buds on the maternal trees using a curved scalpel blade just prior to anthesis, when the operculum changed from green to brown as described by Randall et al. (2012). We collected flower buds from pollen parents just prior to anthesis, removed anthers with forceps and desiccated them over silica gel for 24–48 h to promote dehiscence. The anther-pollen mix was stored in gelatine capsules at 5 °C in jars with a desiccant. We employed two pollination methods, the ‘three-stop’ method
299
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
(Sedgley and Smith, 1989) and the ‘one-stop’ method (Trindade et al., 2001). Three replicate bunches (groups of umbellasters) were emasculated for each treatment on each maternal tree to assess capsule set and seed production (4 pollen parents 2 pollination methods 3 replicate bunches per tree 3 trees, total 72 bunches). 20 unopened female flowers were emasculated on each bunch and all other flower buds were removed. After emasculation, we bagged each bunch with a polyester pollen exclusion bag. A moistened match head was used to apply pollen to flower buds receiving the three-stop method 3–7 days after emasculation, discarding each match after use. For the one-stop method we pollinated flower buds on the day of emasculation and bagged them immediately afterwards.
2.5. Pollen tube harvest and microscopy Buds for pollen tube examination were collected seven to fourteen days after pollination, fixed in acetic ethanol (1 part glacial acetic acid: 3 parts ethanol) and processed as previously described (Randall et al., 2012). 2.6. Capsule set and seed production The capsules remained on the trees until they matured, and were collected at 210–220 days after emasculation, just prior to valves opening. The number of capsules set for each treatment, total number of seeds per bunch and total seed weight per bunch were recorded. 2.7. Viability of seed from crosses with sympatric species
2.3. Hybridisation with sympatric species We applied four pollen treatments on each of four E. argophloia maternal trees, one from Burncluith sub-provenence, one from Burra Burri sub-provenence, one from Fairyland sub-provenence and one of unknown identity from an isolation row. Pollen treatments were (1) E. moluccana, (2) E. crebra, (3) E. microcarpa (all from the same section as E. argophloia, Adnataria) (Brooker, 2000); and (4) cross within species using pollen from an unrelated tree from Burra Burri sub-provenence (Table 1). Three bunches, each with 20 flower buds for each treatment for each tree were pollinated using the three stop method as described above (3 bunches 4 treatments 4 trees, total 48 bunches). Three additional bunches with 10 flower buds per pollen parent treatment on each tree were emasculated and harvested to examine pollen tube growth. Each replicate was bagged and pollinated as described above.
Seeds obtained from maternal E. argophloia trees crossed with E. argophloia pollen and with pollens from E. moluccana, E. crebra, and E. microcarpa were sown in standard 40-cell ‘Hyco V93 trays’ used to grow eucalypts for plantation forestry in Australia, in a glasshouse, during January 2009. Each cell in the tray had a 70 ml plastic insert, to facilitate sorting and seedling assessment. The potting medium consisted of 50% pine bark fines (0–10 mm), 25% pine bark peat, 25% coarse perlite, and a mix of 12–14 month slow release OsmocoteÒ (N 17.9: P 0.8: K 7.3) fertiliser at a rate of 4 kg/m3, gypsum (1 kg/m3), MicromaxÒ (1 kg/m3) and a granular wetting agent Hydroflo2Ò (1 kg/m3). Seed were irrigated five times a day for three minutes at each irrigation time using an overhead spray and fertilised with 2 ml/l of both NitrosolÒ (N 10.5%, P 2.3%, K 6.8% and some trace elements) (Yates Pty Ltd) and SeasolÒ (primarily trace elements) (Seasol International Pty Ltd) liquid fertiliser every two weeks. At three months the number of seedlings for each cross type were counted and viabilities determined based on the number of seed planted.
2.4. Hybridisation with allopatric species
2.8. Statistical analysis
We applied five pollen treatments to bunches of flower buds on each of four maternal E. argophloia trees, one from the Burncluith sub-provenance, one from the Burra Burri sub-provenance and two from the Fairyland sub-provenance. Pollen treatments were: (1) open pollination (as a control), (2) cross within species (using a pollen polymix to prevent self-fertilisation), (3) sympatric species E. microcarpa (from the same section as E. argophloia, Adnataria), (4) allopatric species E. pellita (from a more distant section, Latoangulatae) and (5) allopatric species E. resinifera, also from Latoangulatae (Brooker, 2000) (Table 1). The three-stop pollination method was used in all treatments except for open pollination. Three bunches of 20 flower buds for each pollen treatment for each tree were pollinated to assess fruit set and seed production (3 bunches 5 treatments 4 trees, total 60 bunches) and three bunches of 10 flower buds per treatment for each tree were pollinated for pollen tube analysis as described above.
The percentage of capsules set, seeds per capsule pollinated and seed weight per capsule set were calculated for each bunch for each experiment. Data for 2.2 ‘‘Breeding system and pollination methods” were normally distributed and were analysed as a 2way ANOVA with pollination method (one stop or three stop method) and pollen source (self, Burncluith, Burra Burri and E. moluccana) as factors followed by a Tukey’s HSD test where differences were significant. One tree was excluded from the analysis due to failure to set capsules in any treatment. An additional analysis was carried out to determine whether selfing rates differed between female trees. The number of seeds per capsule pollinated was analysed as a 3 way ANOVA with pollen source, female and pollination methods as factors (main effects only). Data for percentage of capsule pollinated and seeds per capsule pollinated from experiment 2.3 ‘‘Hybridisation with sympatric species” were square root transformed where necessary and
Table 1 Classification of species used for hybridisation. Occurrence
Species
Subgenus
Section
Series
Subseries
Sympatric
E. E. E. E.
Symphyomyrtus Symphyomyrtus Symphyomyrtus Symphyomyrtus
Adnataria Adnataria Adnataria Adnataria
Submelliodorae Siderophloiae Buxeales Buxeales
Continentes Continentes
Symphyomyrtus Symphyomyrtus
Latoangulatae Latoangulatae
Annulares Annulares
Allopatric
Brooker (2000).
argophloia crebra microcarpa moluccana
E. pellita E. resinifera
300
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
analysed with a 1-way ANOVA to test for significant differences in capsules and seed production between pollen sources. Data for seed weight per capsule set for experiment 2.4 ‘‘Hybridisation with allopatric species” were also analysed with a 1 way ANOVA to test for significant differences between pollen sources for the parameter ‘‘seed weight per capsule set”. In experiment 2.3 ‘‘Hybridisation with sympatric species” data for pollen tubes and seed weight, and experiment 2.4 ‘‘Hybridisation with allopatric species” data for pollen tubes” capsule set and seeds per capsule pollinated were non-parametric and did not meet the assumptions of ANOVA. These were therefore analysed with a Kruskal-Wallis tests followed by Mann Whitney -U tests where differences were significant. A Bonferroni correction factor was applied to determine the appropriate level of significance (Sokal and Rohlf, 1995). All data were analysed using SPSS version 17 (SPSS Science, Chicago)
Table 2 Mean pollen tubes in upper and lower style, for each pollination treatment for crosses with sympatric eucalypt species and cross within species. There were no significant differences between treatments, but all crosses produced pollen tubes. Pollination treatment
Pollen tubes upper style
Pollen tubes lower style
E. E. E. E.
0.3 1.0 11.8 0.5
0.5 1.3 0.5 0.8
argophloia crebra microcarpa moluccana
There were no significant difference in selfing rates (seeds produced per capsule pollinated) between female trees (P < 0.05) and they ranged from 0.5 to 1.35 for 1 stop treatments and 3.74 to 4.73 for 3 stop pollination treatments.
3.2. Hybridisation with sympatric species 3. Results 3.1. Breeding system and pollination methods Our results show that three stop pollination produced significantly higher percentage capsule set and seeds per capsule pollinated (P < 0.001, Fig. 1). All crosses and self-pollination treatments produced some capsules and seed, and there were no significant differences between any of the pollen sources tested.
All crosses tested in this hybridisation study produced some pollen tubes in the upper and lower style, although differences were not significant (Table 2). All crosses also produced both capsules and seed. However there were no significant differences between pollen treatments for percentage capsule set, number of seeds per capsule pollinated or seed weight per capsule set (Table 3). Examples of putative hybrids from the Dunmore seed orchard are presented in Fig. 3.
3.3. Hybridisation with allopatric species All crosses produced pollen tubes in the upper and lower style, although there were no significant differences (P < 0.05) between pollen treatments (Table 4). All pollen parents produced some capsules and some seed (Fig. 2a, b). Intraspecific pollen produced a similar number of seeds to interspecific pollen, irrespective of how closely or distantly related the species were. Open-pollination produced significantly more capsules than all the control-cross pollinations, however there was no significant difference between the other pollen parents (Fig. 2a). There were significant differences in seeds per capsule pollinated between treatments (Fig. 2b, P = 0.007). Open pollination produced significantly more seeds per capsule pollinated (P = 0.017) than the other pollinations, however there was no significant difference between the other crosses. Open pollination produced five seeds per capsule (very high compared with one to four considered more normal for a eucalypt) and there were one to two per capsule for the cross-pollinations attempted.
3.4. Viability of seed from crosses with sympatric species Mean viability of seed ranged from 0.41 to 0.51, and there was no significant difference between a E. argophloia outcross and crosses with E. moluccana, E. microcarpa and E. crebra (Table 6).
Table 3 Mean percentage of capsules set and mean number of seeds per capsule pollinated and standard errors (SE), for each pollination treatment for crosses with sympatric eucalypt species and cross within species. There were no significant differences between treatments, but all crosses produced seed.
Fig. 1. (a) Capsules set (%) and (b) seeds per capsule pollinated from E. argophloia using one-stop and three-stop pollination treatments. Self = self-pollination using pollen from the same tree, Burra Burri = within species cross using pollen from an unrelated tree ‘‘Burra Burri”, Burncluith = within species cross using pollen from an unrelated tree ‘‘Burncluith”,moluccana = cross pollination using pollen from E. moluccana. Means with different letters are significantly different (P < 0.05).
Pollination treatment
Capsule set % (SE)
Seeds per capsule pollinated (SE)
E. E. E. E.
6.1 3.3 2.5 4.1
1.4 1.5 1.9 1.6
argophloia crebra microcarpa moluccana
(1.3) (0.8) (0.8) (1.3)
(0.2) (0.3) (0.3) (0.3)
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
301
4. Discussion
Fig. 2. (a) Capsules set (%) and (b) Seeds per capsule pollinated for E. argophloia for cross within-species, cross with closely related sympatric species (E. microcarpa), cross with more distantly related allopatric species (E. resinifera and E. pellita), and open pollination. Treatment means with different letters are significantly different (P < 0.05) (Mann-Whitney U tests with a Bonferroni correction factor). E. argo = E. argophloia;E. micro = E. microcarpa;E. resin = E. resinifera;Open = open pollination.
These results show that E. argophloia readily forms hybrids not only with species both within its own section Adnataria (E. moluccana, E. crebra and E. microcarpa,) but also with species in a more distant section, Latoangulatae (E. pellita and E. resinifera). In addition, E. argophloia readily self-pollinates. Previous studies have shown that many Eucalyptus species readily hybridise within the subgenus Symphomyrtus (Potts and Reid, 1985; Potts, 1986; Potts et al., 1987; Potts and Reid, 1988; Field et al., 2010), but not those from different subgenera (Ellis et al., 1991). In the current study controlled inter-specific hybridisation of E. argophloia with closely related species E. microcarpa produced no significant differences for pollen tubes, capsule set and seed set compared with self-pollination or crossing with the more distantly related species E. resinifera and E. pellita. This is contrary to the hypothesis of reduced seed set with increasing genetic distance (Griffin et al., 1988), as observed in several previous studies (Meddings et al., 2003; Barbour et al., 2005; Dickinson et al., 2012). Seed set decreased with increasing genetic distance for Corymbia hybrids (Dickinson et al., 2012) and seven Tasmanian eucalypts pollinated with E. nitens (Barbour et al., 2005). Similarly, E. camaldulensis (section Exsertaria) as the female parent crossed with E. globulus (section Maidenaria) exhibited substantial barriers to hybridisation compared with outcrossed E. camaldulensis (Meddings et al., 2003). When E. globulus pollen was applied to 99 eucalypt species there was a decline in compatibility with increasing genetic distance between species (Larcombe et al., 2015). The current results indicate that E. argophloia may readily accept pollen from a wide variety of eucalypt species within the subgenus Symphomyrtus. The implication of this is the risk of genetic pollution of the remaining vulnerable, natural stands of E. argophloia. These results demonstrate an important principle with wide application, that ability to hybridise should never be assumed from relationships, but can only be determined by experimentation. Natural hybridisation is now recognized as an important component of evolution of species (e.g. Ellstrand and Schierenbeck, 2000; Abbot et al., 2003), however, hybridisation also threatens a substantial number of plant and animal species with extinction
Fig. 3. Eucalyptus argophloia (Chinchilla White Gum) and putative hybrids. a, Eucalyptus argophloia trees of ‘‘Fairyland” sub-provenence; b, c, atypical trees in the Dunmore State Forest Station seed orchard grown from E. argophloia seed. Note characteristic white bark in ‘a’, and on background trees in ‘c’, compared with rough, dark bark in ‘b’, and tessellated box-type bark in foreground in ‘c’.
302
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
Table 4 Mean pollen tubes in the upper and lower style for open pollination, cross within species (E. argophloia), cross with a closely related sympatric species (E.microcarpa) and crosses with more distantly related allopatric eucalypt species (E. pellita and E. resinifera). Pollination treatment
Pollen tubes upper style
Pollen tubes lower style
Open E. argophloia E. microcarpa E. pellita E. resinifera
0.6 4.2 2.2 5.3 0.7
0.6 2.1 1.5 1.2 0.2
Table 5 Viability of seed obtained from Eucalyptus argophloia maternal trees by crossing with E. argophloia, and crossing with pollen from closely related sympatric species. Cross type
Mean viability
Number of crosses tested
E. E. E. E.
0.43 0.49 0.51 0.41
4 3 3 3
argophloia moluccana crebra microcarpa
(Levin et al., 1996; Ellstrand et al., 2010). Hybridisation is perhaps the most rapidly acting genetic threat to endangered species (Wolf et al., 2001). Hybridisation may also confer new characters that may in some cases facilitate hybrids establishing as weeds (Wallace and Leonhardt, 2015). Human activities such as removing habitat and planting foreign species are increasing the opportunities for hybridisation (Ellstrand et al., 2010). Any activity that exposes native populations of E. argophloia to foreign pollen (such as plantations or nearby amenity plantings) would be a threat to the genetic integrity of the species, particularly in view of its ability to readily hybridise. In addition the ability of the species to selfpollinate is a concern for maintenance of genetic diversity. As an example, the genetic diversity of the rare mallee E. absita was significantly compromised by a high rate of selfing and potential hybridisation (Bradbury et al., 2015). Our results show that E. argophloia crosses readily with intrasectional and inter-sectional species within the Symphomyrtus subgenus. Gene flow from different species can be detrimental to small populations such as this species due to fitness reduction of hybrids from the breakdown of co-adaptation to local conditions (Waser and Price, 1989, 1991). Small populations can be expected to receive gene flow at a higher rate than large populations, and are more likely to receive gene flow from large populations than small ones, even if the latter is in closer proximity (Ellstrand et al., 1989). The potential for hybridisation often depends on how closely related a species is to adjacent native forest species (Potts and Reid, 1985; Griffin et al., 1988). Because E. argophloia hybridises readily with closely related and distantly related Eucalyptus species there are concerns about the risk of gene flow by pollen from naturally occurring closely related congeners such as E. crebra, E. moluccana and E. microcarpa, especially if these are more numer-
ous. The presence of putative hybrids in the seed orchard suggests that hybridisation with sympatric species is already occurring. The threat from the other hybridizing species, E. resinifera and E. pellita may not be such a concern as they are not sympatric with E. argophloia, and different climatic requirements suggest that they would not be suitable for plantations near natural populations of E. argophloia (Brawner et al., 2013). Flowering synchronicity is a major factor in the potential for hybridisation of species. E. argophloia has been reported to have a flowering range from January to August (Boland et al., 1984; Brooker and Kleinig, 2004; Randall et al., 2012, 2015). This means that due to flowering synchronicity it could hybridise with sympatric intra-sectional pollen from E. moluccana, E. microcarpa and E. crebra, and even with allopatric inter-sectional pollen from E. pellita and E. resinifera if the latter two became adjacent (Table 5). The inflow of foreign pollen presents a risk to small populations such as E. argophloia. There may be a risk of introgression with more numerous common species to produce hybrid cohorts in which the characters of the rare species are eventually swamped by one of the parent species (Ellstrand and Elam, 1993; Abbot et al., 2003; Vanden-Broeck et al., 2005). This is often called genetic swamping, but could be termed ‘‘pollen swamping”, i.e. repeated backcrosses with incoming pollen (Petit et al., 2004). Introgression involves many steps that involve several hybrid generations (F1, F2, BC1, BC2 etc. (Vanden-Broeck et al., 2005). The end result is that one species is replaced by the other, or alternatively both hybridizing parents are eventually replaced by swamping from their hybrid-derived descendants (Hedge et al., 2006). Hybridisation could lead to introgression if F1 hybrids backcross (Dickinson et al., 2012; Vanden-Broeck et al., 2012). In eucalypts, the integrity of the rare species Eucalyptus aggregata is at risk from preferential backcrossing with hybrids with the common E. rubida (Field et al., 2010). Similarly, the more common species E. amygdalina is seen to be invading the range of the rare species E. risdonii by both seed and pollen migration (Potts, 1986). The vulnerable species E. argophloia is potentially at risk because of its ability to readily hybridise with a wide range of species. There are other factors which increase the risk for the species. It is a small population, highly fragmented and has a high degree of susceptibility to Myrtle Rust (Puccinia psidii) (Lee et al., 2015). Threatened, rare and vulnerable species such as E. argophloia are often particularly at risk from hybridisation because of small population size and fragmentation. This would be true for many species in a global context. There are many forest species such as E. argophloia, that while not commercially important, are nonetheless important in an ecological context or as an iconic species (Yu et al., 2014; Chandler and McGraw, 2015; Taylor et al., 2016). Viable seed has been produced from crossing E. argophloia with closely related species. Crosses with E. moluccana, E. crebra and E. microcarpa had similar viability to outcrossing within species. There is strong anecdotal evidence of natural hybrids of E. argophloia, for example, 5% putative hybrids have been reported in an
Table 6 Flowering times of Eucalyptus argophilia and species used as pollen parents with E. argophloia. Species
Jan
Feb.
March
April
May
June
July
Aug.
argophloia crebra microcarpa moluccana pellita resinifera
#
#
/
/
x* x
x* x
x* x* x*
x x x*
x x x*
x*
x* x*
x* x* x*
Sept.
Oct.
Nov.
Dec.
x* x
*
*
x
x
x
x
*
*
Flowering time is identified by x (Brooker and Kleinig, 2004). * Boland et al. (1984). # Randall et al. (2012) and / Randall et al. (2015).
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
E. argophloia plantation (Lee, personal comment). A few putative hybrids also exist in the seed orchard that hosted the current study (Fig. 3). However, the ability of hybrids to persist and backcross with E. argophloia is yet to be tested. Hybrids may have low rates of establishment compared with wildlings. For example E. nitens, E. ovata and E. viminalis had open pollination wildling establishment rates of 94–100% compared with 0.3–5.5% for hybrids of E. nitens with E. ovata and E. viminalis (Barbour et al., 2003). While there are putative hybrids in the seed orchard in the current study, and viable seed has been produced, at present there is little evidence that E. argophloia hybrids are establishing in the wild. For this to occur, hybrid progeny must be at least as fit as or fitter than the parent population (Dickinson et al., 2013; Shepherd and Lee, 2016). The risk is in proportion to the proximity of donor populations. Pollination in hybrid eucalypts is mostly over relatively short distances of less than 200 m, but low levels of pollination success are also to be expected over longer distances (Barbour et al., 2003; da Silva et al., 2015). The small E. argophloia bud is very difficult to hand pollinate without damage that affects retention of capsules (Randall et al., 2012, 2015). The buds of E. argophloia are only 0.4 0.4 cm (Brooker and Kleinig, 2004), smaller than those of many eucalypts and are much smaller than those evaluated in previous pollination studies (e.g. E. grandis 0.8 0.7 cm, E. nitens 0.7 0.6 cm, E. macarthurii 0.5 0.6 cm (Brooker and Kleinig, 2004). Higher seed set per bunch indicates that open pollination is much more successful. Hybrid seed set from open pollinations could be higher than the current study indicates without the limiting effects of emasculation during controlled pollination. Hybridisation risks from foreign gene flow to E. argophloia could be substantial. This was suggested by the pollen tube data which showed that there were no differences between E. argophloia intra-species and inter-species pollination.
5. Conclusions This study has shown that E. argophloia freely hybridises with genetically close, intra-sectional relatives E. crebra, E. microcarpa and E. moluccana, as well as with more distant inter-sectional species E. pellita and E. resinifera. The species is potentially at risk of genetic swamping by foreign pollen because of its ability to hybridise with a wide range of species and because of its very small and fragmented population structure. E. argophloia also readily self-pollinates; self-compatibility exposes this species to elevated evolutionary pressure. As a consequence, the genetic integrity of this iconic species may be at risk. The extent of hybridisation of this species with sympatric species is unknown, however the results of the current study demonstrate the need for genetic studies to determine the extent of foreign pollen flow into E. argophloia. Acknowledgements The authors wish to thank the Australian Research Council linkage grant number LP0562678 for financial assistance, Department of Primary Industries and Fisheries (formerly AgriScience Queensland) for technical support, and HQ Plantations (formerly DPI-Forestry) for financial assistance and access to the trial sites. References Abbot, R.J., James, J.K., Milne, R.I., Gillies, A.C.M., 2003. Plant introductions, hybridisation and gene flow. Philosoph. Trans. Royal Soc. London 358, 1123– 1132. Barbour, R., Potts, B., Vaillancourt, R., Tibbits, W., Wiltshire, R., 2002. Gene flow between introduced and native Eucalyptus species. New For. 23, 177–191.
303
Barbour, R.C., Potts, B.M., Vaillancourt, R.E., 2003. Gene flow between introduced and native Eucalyptus species: exotic hybrids are establishing in the wild. Aust. J. Bot. 51, 429–439. Barbour, R., Potts, B., Vaillancourt, R., 2005. Gene flow between introduced and native Eucalypt species: crossability of native Tasmanian species with exotic E. nitens. Aust. J. Bot. 53, 465–477. Barbour, R., Potts, B., Vaillancourt, R., Tibbits, W., 2006. Gene flow between introduced and native Eucalyptus species: flowering asynchrony as a barrier to F1 hybridisation between exotic E. nitens and Tasmanian Symphyomyrtus species. For. Ecol. Manage. 226, 9–21. Barbour, R., Otahal, Y., Vaillancourt, R., Potts, B., 2008. Assessing the risk of pollenmediated gene flow from exotic Eucalyptus globulus plantations into native eucalypt populations of Australia. Biol. Conserv. 141, 896–907. Barbour, R.C., Wise, S.L., McKinnon, G.E., Vaillancourt, R.E., Williamson, G.J., Potts, B. M., 2010. The potential for gene flow from exotic eucalypt plantations into Australia’s rare native eucalypts. For. Ecol. Manage. 260, 2079–2087. Boland, D.J., Brooker, M.I.H., Chippendale, G.M., Hall, N., Hyland, B.P.M., Johnson, R. D., Kleinig, D.A., McDonald, M.W., Turner, J.D., 1984. Forest Trees of Australia. CSIRO, Melbourne. Bradbury, D., Grayling, P.M., MacDonald, B., Hankinson, M., Byrne, M., 2015. Clonality, interspecific hybridisation and inbreeding in a rare mallee eucalypt, Eucalyptus absita (Myrtaceae), and implications for conservation. Conserv. Genet. http://dx.doi.org/10.1007/s10592-015-0771-8. Brawner, J.T., Lee, D.J., Meder, A.R., Almeida, A., Dieters, M.J., 2013. Classifying genotype by environment interactions for targeted germplasm deployment with a focus on Eucalyptus. Euphytica 191, 403–414. Broadhurst, L.M., Lowe, A., Coates, D.J., Cunningham, S.A., McDonald, M., Vesk, P.A., Yates, P., 2008. Seed supply for broadscale restoration: maximizing evolutionary potential. Evol. Appl. 1, 587–597. Brooker, M., 2000. A new classification system of the Genus Eucalyptus L’Her. (Myrtaceae). Aust. System. Bot. 13, 79–148. Brooker, M., Kleinig, D., 1999. Field Guide to Eucalypts—Volume 1 Southeastern Australia. Bloomings Books, Melbourne, pp. 70–186. Brooker, M., Kleinig, D., 2004. Field Guide to Eucalypts – Northern Australia. Inkata Press, Sydney. Butcher, P.A., Skinner, A.K., Gardiner, C.A., 2005. Increased inbreeding and interspecies gene flow in remnant populations of the rare Eucalyptus benthamii. Conserv. Genet. 6, 213–226. Chandler, J.L., McGraw, J.B., 2015. Variable effects of timber harvest on the survival, growth, and reproduction of American ginseng (Panax quinquefolius L.). For. Ecol. Manage. 344, 1–9. CPBR, 2006. EUCLID: Eucalypts of Australia. CSIRO Publishing, Collingwood, Victoria. da Silva, P.H.M., Sebbenn, A.M., Grattapaglia, D., 2015. Pollen-mediated gene flow across fragmented clonal stands of hybrid eucalypts in an exotic environment. For. Ecol. Manage. 356, 293–298. Dickinson, G.R., Lee, D.J., Wallace, H.M., 2012. The influence of pre- and postzygotic barriers on hybridisation between Corymbia sections. Ann. Bot. 109, 1215–1226. Dickinson, G., Lee, D., Wallace, H., 2013. Reciprocal and advanced generation hybrids between Corymbia citriodora and C. torelliana: forestry breeding and the risk of gene flow. Ann. Forest. Sci. 70, 1–10. Ellis, M.F., Sedgley, M., Gardner, J.A., 1991. Interspecific pollen-pistil interaction in Eucalyptus L’Hér. (Myrtaceae): the effect of taxonomic distance. Ann. Bot. 68, 185–194. Ellstrand, N., 1992. Gene flow by pollen: implications for plant conservation genetics. Oikos 63, 77–86. Ellstrand, N., Elam, D., 1993. Population genetics consequences of small population size: implications for plant conservation. Annu. Rev. Ecol. Syst. 24, 217–242. Ellstrand, N.C., Schierenbeck, K.A., 2000. Hybridisation as a stimulus for the evolution of invasiveness in plants? Proc. Nat. Acad. Sci. USA 97, 7043–7050. Ellstrand, N.C., Devlin, B., Marshall, D.L., 1989. Gene flow by pollen into small populations: data from experimental and natural stands of wild radish. Proc. Nat. Acad. Sci. USA 86, 9044–9047. Ellstrand, N.C., Biggs, D., Kaus, A., Lubinsky, P., McDade, L.A., Preston, K., Prince, L.M., Regan, H.M., Roriver, V., Ryde, K.A., Schierenbeck, K.S., 2010. Got hybridisation? A multidisciplinary approach for informing science policy. Bioscience 60, 384–388. Environment Australia, 2009. Environment Protection and Biodiversity Conservation Act, 1999. Flora that are Endangered
304
B.W. Randall et al. / Forest Ecology and Management 381 (2016) 297–304
Larcombe, M.J., Barbour, R.C., Vaillancourt, R.E., Potts, B.M., 2014. Assessing the risk of exotic gene flow from Eucalyptus globulus plantations to native E. ovata forests. For. Ecol. Manage. 312, 193–202. Larcombe, M.J., Holland, B., Steane, D.A., Jones, R.C., Nicolle, D., Vaillancourt, R.E., Potts, B.M., 2015. Patterns of reproductive isolation in Eucalyptus. Mol. Biol. Evol. 32, 1833–1846. Lee, D.J., 2007. Achievements in forest tree genetic improvement in Australia and New Zealand 2: Development of Corymbia species and hybrids for plantations in eastern Australia. Aust. For. 70, 11–16. Lee, D.J., Huth, J.R., Osborne, D.O., Hogg, B.W., 2010. Selecting hardwood taxa for wood and fibre production in Queensland’s subtropics. Aust. Forest. 73, 106– 114. Lee, D.J., Brawner, J.T., Pegg, G.S., 2015. Screening Eucalyptus cloeziana and E. argophloia populations for resistance to Puccinia psidii. Plant Dis. 99, 71–79. Levin, D.A., Francisco-Ortega, J., Jansen, R.K., 1996. Hybridisation and the extinction of rare plant species. Con. Biol. 10, 10–16. Linacre, N., Ades, P., 2004. Estimating isolation distances for genetically modified trees in plantation forestry. Ecol. Model. 179, 247–257. Meddings, R.A., McComb, J.A., Calver, M.C., Thomas, S.R., Mazanec, R.A., 2003. Eucalyptus camaldulensis globulus hybrids. Aust. J. Bot. 51, 319–331. Merchant, A., Callister, A., Arndt, S., Tausz, M., Adams, M., 2007. Contrasting physiological responses of six Eucalyptus species to water deficit. Ann. Bot. 100, 1507–1515. Nevill, P.G., Després, T., Bayly, M.J., Bossinger, G., Ades, P.K., 2014. Shared geographic patterns and widespread chloroplast haplotype sharing in Eucalyptus species with different ecological tolerances. Tree Genet. Genomes 10, 1079–1092. Ngugi, M., Hunt, M., Doley, D., Ryan, P., Dart, P., 2004a. Selection of species and provenances for low-rainfall areas: physiological responses of Eucalyptus argophloia to seasonal conditions in subtropical Queensland. Forest Ecol. and Manage. 193, 141–156. Ngugi, M., Hunt, M., Doley, D., Ryan, P., 2004b. Physiological responses to water stress in Eucalyptus cloeziana and E. argophloia seedlings. Trees 18, 381–389. Petit, R.J., Bodenes, C., Ducousso, G.R., Kremer, A., 2004. Hybridisation as a mechanism of invasion in oaks. New Phytol. 161, 131–164. Potts, B.M., 1986. Population dynamics and regeneration of a hybrid zone between Eucalyptus risdonii Hook. f. and E. amygdalina Labill. Aust. J. Bot. 34, 305–329. Potts, B., Dungey, H., 2004. Interspecific hybridisation of Eucalyptus: key issues for breeders and geneticists. New For. 27, 115–138. Potts, B.M., Reid, J.B., 1985. Analysis of a hybrid swarm between Eucalyptus risdonii Hook.f. and E. amygdalina Labill. Aust. J. Bot. 33, 543–562. Potts, B.M., Reid, J.B., 1988. Hybridisation as a dispersal mechanism. Evolution 42, 1245–1255. Potts, B.M., Potts, W.C., Cauvin, B., 1987. Inbreeding and interspecific hybridisation in Eucalyptus gunnii. Silvae Genet. 36, 194–199. Potts, B.M., Barbour, R.C., Hingston, A.B., Vaillancourt, R.E., 2003. Genetic pollution of native eucalypt gene pools – identifying the risks. Aust. J. Bot. 51, 1–25. Queensland Government, 1992. Nature Conservation Act, 1992
Randall, B.W., Walton, D.A., Lee, D.J., Wallace, H.M., 2015. Comparison of three pollination methods for Eucalyptus argophloia, a small-flowered eucalypt. Ann. For. Sci. 72, 127–133. Rhymer, J.M., Simberloff, D., 1996. Extinction by hybridisation and introgression. Ann. Rev. Ecol. Syst. 27, 83–109. Schierenbeck, K.A., Ellstrand, N.C., 2009. Hybridisation and the evolution of invasiveness in plants and other organisms. Biol. Invasions 11, 1093–1105. Sedgley, M., Smith, R.M., 1989. Pistil receptivity and pollen tube growth in relation to the breeding systems of Eucalyptus woodwardii Maiden (Symphyomyrtus). Ann. Bot. 64, 21–31. Shepherd, M., Lee, D.J., 2016. Gene flow from Corymbia hybrids in northern New South Wales. For. Ecol. Manage. 362, 205–217. Sokal, R., Rohlf, F., 1995. Biometry: The Principles and Practice of Statistics in Biological Research. W. H. Freeman and Company, New York. Strauss, S., 2001. Benefits and risks of biotic exchange between Eucalyptus plantations and native Australian forests. Austral Ecol. 26, 447–457. Taylor, J.E., Ellis, M.V., Rayner, L., Ross, K.A., 2016. Variability in allometric relationships for temperate woodland Eucalyptus trees. For. Ecol. Manage. 360, 122–132. Trindade, H., Boavid, L.C., Borralho, N., Feijo, J.A., 2001. Successful fertilization and seed set from pollination on immature non-dehisced flowers of Eucalyptus globulus. Ann. Bot. 87, 469–475. Vanden-Broeck, A., Villar, M., Van Bockstaele, E., Van Slycken, J., 2005. Natural hybridisation between cultivated poplars and their wild relatives: evidence and consequences for native populations. Ann. For. Sci. 62, 601–613. Vanden-Broeck, A., Cox, K., Michiels, B., Verschule, P., Villar, M., 2012. With a little help from my friends: hybrid fertility of exotic Populus x canadensis enhanced by related native Populus nigra. Biol. Invasions 14, 1683–1696. Vila, M., Weber, E., D’Antonio, C.M., 2000. Conservation implications of invasion by plant hybridisation. Biol. Invasions 2, 207–217. Wallace, H.M., Leonhardt, S.D., 2015. Do hybrid trees inherit invasive characteristics? Fruits of Corymbia torelliana X C. citriodora hybrids and potential for seed dispersal by bees. PLOS ONE. http://dx.doi.org/10.1371/ journal.pone.0138868. Waser, N.M., Price, M.V., 1989. Optimal outcrossing in Ipomopsis aggregata: seed set and offspring fitness. Evolution 43, 1097–1109. Waser, N.M., Price, M.V., 1991. Outcrossing distance effects in Delphinium nelsonii: pollen loads, pollen tubes and seed set. Ecology 72, 171–179. Whyte, G., Howard, G.E.St., Hardy, T.I., Burgess, T.I., 2016. The tree decline recovery seewaw; a conceptual model of the decline and recovery of drought stressed plantation trees. For. Ecol. Manage. 370, 102–113. Wolf, D.E., Takebayashi, N., Rieseberg, L.H., 2001. Predicting the risk of extinction through hybridisation. Conserv. Biol. 15, 1039–1053. Yu, J., Wang, C., Wan, J., Han, S., Wang, Q., Nie, S., 2014. A model-based method to evaluate the ability of nature reserves to protect endangered tree species in the context of climate change. For. Ecol. Manage. 327, 48–54. Xie, Q., Li, Z., Yang, L., Lv, J., Jobe, T.O., Wang, Q., 2015. A newly identified passive hyperaccumulator Eucalyptus grandis x E. urophylla under manganese stress. PLoS ONE 1, 2015. http://dx.doi.org/10.1371/journal.pone.0136606.