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Reproductive barriers that evolve to prevent hybridisation between previously cross- compatible taxa are fundamental drivers of speciation (Coyne and Orr 2004). Comparative studies that assess patterns of reproductive isolation among groups of related taxa have been important in identifying when these barriers develop, and determining the relative contribution of different barriers to reproductive isolation (Coyne and Orr 1989; Moyle et al. 2004; Orr and Turelli 2001; Widmer et al. 2009). It has been shown that pre-mating barriers, such as mate choice or flowering time, usually develop first, are often responsible for most of the observed isolation between taxa, and because they experience steep selection gradients as a result of their direct impact on reproductive success, they tend to evolve more quickly than post-mating barriers (Coyne and Orr 2004; Lowry et al. 2008; Widmer et al. 2009). However, pre-mating barriers also tend to be “leaky”, and it is often a combination of pre- and post-mating barriers that result in actual isolation (Coyne and Orr 2004; Widmer et al. 2009).

Post-mating barriers include both pre- and postzygotic mechanisms that are thought to arise principally via drift in a clock-like fashion (Coyne and Orr 2004; Hogenboom and Mather 1975; Orr and Turelli 2001). Theory suggests that as populations diverge minor allelic changes develop that are neutral or adaptive in the population of origin, but cause epistatic incompatibilities when brought together in inter-population hybrids – leading to inviability or sterility (a process widely known as the Dobzhansky-Muller model; Bateson 1909; Dobzhansky 1937; Muller 1942; Orr and Turelli 2001). In animal systems there is abundant evidence for Dobzhansky-Muller incompatibilities. For example, reproductive isolation consistently increases with genetic distance (Coyne and Orr 1989; Lee 2000; Malone and Fontenot 2008; Presgraves 2002), and male hybrid sterility has been found to involve hundreds of genes (Lu et al. 2010), both of which are indicative of accumulating incompatibilities. However, the situation in plants is less clear-cut

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(Lowry et al. 2008; Widmer et al. 2009). Some studies show a correlation between genetic distance and postzygotic isolation consistent with Dobzhansky-Muller type incompatibles (Jewell et al. 2012; Meiners and Winkelmann 2012; Scopece et al.

2007), while others studies have found no clear evidence for epistatic mechanisms (Widmer et al. 2009). For example, isolation can developed quickly as a result of selection acting on post-mating prezygotic barriers (Giraud and Gourbière 2012; Scopece et al. 2007), or genomic rearrangements and very simple gene incompatibilities can cause instantaneous and strong reproductive isolation (Nosrati

et al. 2011; Ramsey et al. 2003; Sweigart et al. 2006). Additionally, allopolyploidy can lead to rapid unpredictable patterns of speciation in plants (Abbott et al. 2013; Levin 2013). It has recently been suggested that simple Dobzhansky-Muller models are inadequate for explaining all the patterns of post-mating isolation emerging, particularly in plants, and more research is needed identify how the model should be broadened (Abbott et al. 2013; Giraud and Gourbière 2012).

Another key point of difference between animal and plant systems is the speed at which pre- and post-mating isolation develops, with pre-mating barriers evolving more quickly than post-mating barriers in animals (Coyne and Orr 1989; Coyne and Orr 1997; Mendelson 2003), but not plants (Moyle et al. 2004; Scopece et al. 2007; Scopece et al. 2008). It has been suggested that there may be more barriers affecting isolation between plant taxa (Widmer et al. 2009), but it may also be related to the fact that post-mating barriers are typically assessed only at one, often early, ontogenetic stage (but see: Scopece et al. 2007; Scopece et al. 2008; Stelkens et al.

2010). This may significantly underestimate hybrid incompatibilities that are expressed later in development – affecting estimates of both their strength and rate of evolution (Stelkens et al. 2010). Related to this, studies investigating the phylogenetic basis of reproductive isolation in plants have to date focused on herbs. But, as noted recently by Levin (2012), future studies need to include trees, because long generation times extend the period when hybrid incompatibilities can be expressed prior to reproduction (often 5-10 years), which potentially represents a life-history barrier that annual flowering herbs do not experience.

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Recently methods have been developed to use model fitting in order to determine support for different modes of evolution, including the Dobzhansky-Muller model (Giraud and Gourbière 2012; Gourbière and Mallet 2010). Gourbière and Mallet (2010) proposed three relationships between the log of compatibility and time since divergence that provide support for different modes of evolution. The first provides support for Dobzhansky-Muller “snowball model” where the number of incompatibilities accelerate relative to the number of mutations because of epitasis (Gourbière and Mallet 2010; Orr and Turelli 2001). The second produces a “linear model” supporting a mode of evolution where epitasis does not influenced the rate of increase in incompatibilities through time (Gourbière and Mallet 2010). The third produces a “slowdown model” which is indicative of natural selection, specifically reinforcement, acting to cause reproductive isolation, and produces the opposite pattern to the “snowball model” (Gourbière and Mallet 2010). This method has now been deployed across a range of taxa, and there is often support for the “slowdown” and “linear” models but only rarely is there evidence for the “snowball” effect (Giraud and Gourbière 2012; Gourbière and Mallet 2010).

Eucalyptus is a large and diverse genus of around 700 species of trees and shrubs that are native mainly to Australia (Euclid 2006). They are often foundation species and dominate most Australian forests and woodlands, making Eucalyptus one ofthe most ecologically important genera in Australia (Pryor and Johnson 1981). There is great morphological diversity in the genus including the world’s tallest angiosperm

E. regnans (99.6m), and the prostrate mountain-top shrub E. vernicosa (Grattapaglia

et al. 2012). It is a taxonomically complex group that is divided into 10 subgenera, with the most speciose being Symphyomyrtus (c. 470 species) and Eucalyptus

(formally Monocalyptus, c. 108 species) (Grattapaglia et al. 2012). There is no evidence for polyploidy in the genus (2n = 22 in all 135 species assessed to date), making it an ideal lineage for investigating patterns of diploid speciation (Grattapaglia et al. 2012).

There is a well recognised complete barrier to hybridisation between the major

Eucalyptus subgenera (Griffin et al. 1988; Pryor and Johnson 1981). However, widely reported hybridisation within subgenera has led to Eucalyptus being

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renowned for having weak reproductive barriers between species (Field et al. 2009; Grattapaglia et al. 2012). Despite this reputation, a major review of hybridisation in the genus found that barriers clearly exist (Griffin et al. 1988). Griffin et al. (1988) showed that even within subgenera only 15 % of hybrid combinations (expected based on range overlap) have actually been found, and that hybridisation within taxonomic sections was more common than between sections. There is clearly variation in the strength of barriers to hybridisation in Eucalyptus,making it a good candidate for investigating the phylogenetic basis of post-mating isolation.

Eucalypts are also the world’s most widely grown hardwood trees, with 20 million ha of plantations under cultivation in over 100 countries across six continents (GIT 2013). Their adaptability and fast growth means eucalypts are likely to be a prominent source of biomass for future sustainable energy production (Shepherd et al. 2011) and eucalypt plantations already reduce anthropogenic pressure on native forests and biodiversity (Bauhus et al. 2010). Despite great taxonomic diversity, the global eucalypt plantation estate is dominated by nine species and their hybrids, all of which come from the subgenus Symphyomyrtus (Harwood 2011). The superior pulpwood properties of Eucalyptus globulus have seen it become the most widely grown species in temperate zones (Stackpole et al. 2010). As a result it has also become a significant component of hybrid breeding programs to improve wood properties in the tropics and subtropics (Bison et al. 2007; Hardner et al. 2011). A better understanding of phylogenetic barriers to hybridisation will benefit hybrid breeding programs in this economically important genus.

The ecological and economic significance of Eucalyptus has seen the development of significant genomic recourses (Grattapaglia et al. 2012). Recent developments include diversity arrays technology (DArT), which is a marker based system providing thousands of genome-wide polymorphic loci (Petroli et al. 2012; Sansaloni et al. 2010; Steane et al. 2011). Levin (2012) noted that the small number of genes used to estimate divergence in studies of plant reproductive isolation may not be representative of divergence across the genome as a whole, possibly causing problems in interpretation. The application of DArT markers would largely overcome this problem.

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This study uses over 8000 DArT markers to build a distance based phylogeny to investigate the phylogenetic basis of cross-compatibility between E. globulus and 100 eucalypt species belonging to 13 sections, mainly from subgenus

Symphyomyrtus. We have chosen E. globulus because of its global economic importance, and because it is the most widely grown eucalypt in Australia where it is often locally exotic, and could affect the genetic integrity of indigenous eucalypt populations through hybridisation and introgression (Barbour et al. 2008b; Potts et al. 2003). We assess two stages of post-mating compatibility: the number of viable hybrids seeds produced, herein termed pre-dispersal compatibility; and F1 survival at

nine months, herein termed post-dispersal compatibility (see methods for detailed definitions). The following specific questions are addressed: 1) does incompatibility increase with genetic distance, and if it does, is the increase consistent with a Dobzhansky-Muller model of isolation? 2) are patterns of pre- and post-dispersal isolation similar in strength indicating that they evolve at a similar rate?

2.3 Materials and Methods