Crop to wild gene flow and genetic diversity in a vulnerable Macadamia (Proteaceae) species in New South Wales, Australia

Biological Conservation 191 (2015) 504–511

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Crop to wild gene flow and genetic diversity in a vulnerable Macadamia (Proteaceae) species in New South Wales, Australia Katie O'Connor a, Michael Powell a, Catherine Nock b, Alison Shapcott a,⁎ a b

Genecology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD 4558, Australia Southern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, Australia

a r t i c l e

i n f o

Article history: Received 27 April 2015 Received in revised form 26 July 2015 Accepted 2 August 2015 Available online 15 August 2015 Keywords: Wild relative Pollination Hybrid Rainforest Genetic diversity Microsatellite

a b s t r a c t Habitat fragmentation is a leading threat to biodiversity, with extinction rates increasing as anthropogenic alteration of the environment increases. Crop to wild hybridisation is a threat to biodiversity resulting from native vegetation being replaced with agriculture and crops. Remnant populations of Australia's vulnerable subtropical rainforest tree Macadamia tetraphylla are potentially threatened by hybridisation with M. integrifolia orchard crops. Leaf samples were taken from ten crop-wild population sites across the distribution of M. tetraphylla in New South Wales, Australia. Microsatellite markers were used to investigate the presence of marker alleles from cultivars in wild populations, and genetic diversity within and among wild M. tetraphylla populations. Despite the small size of the wild M. tetraphylla populations, relatively high genetic diversity and low inbreeding were observed. This study found that M. integrifolia orchard trees pollinated wild M. tetraphylla trees, and that orchard seeds dispersed into wild populations, providing the first evidence of crop-wild gene flow in macadamia. Pollen flow between relatively close wild populations maintains genetic diversity, reduces inbreeding, and also enables gene flow from nearby M. integrifolia orchards. The potential for crop to wild gene flow and hybridisation risks the integrity and persistence of wild populations. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Plant species exist in heterogeneous and fragmented landscapes naturally, but populations may become reduced in size and isolated from each other by anthropogenic activities (Young et al., 1996; Frankham, 2003; Oostermeijer et al., 2003). Gene flow among populations is often affected by habitat fragmentation, which is predicted to reduce genetic diversity, and lead to genetic drift and inbreeding in plant populations (Hamrick and Godt, 1996; Leimu et al., 2010). Habitat fragmentation can alter forest ecology, and affect survival and mortality rates of populations and species directly by land clearing, and indirectly through edge effects and environment degradation (Young et al., 1996; Laurance et al., 1998; Frankham, 2003). Loss of habitat is also likely to affect gene flow and population density, as well as growth, reproduction, fecundity and recruitment, as competition for resources changes (Alvarez-Buylla et al., 1996; Oostermeijer et al., 2003). Due to spatio-temporal increases in both habitat fragmentation and agriculture, many crops are now cultivated in areas that contain wild relatives within the surrounding modified matrix (Ellstrand et al., 1999; Young and Boyle, 2000; Cornille et al., 2013). Natural barriers to pollination may be disrupted, creating new pathways for pollinators to move between previously separated patches, such as between crops ⁎ Corresponding author. E-mail address: [email protected] (A. Shapcott).

http://dx.doi.org/10.1016/j.biocon.2015.08.001 0006-3207/© 2015 Elsevier Ltd. All rights reserved.

and wild relatives (Hengstum et al., 2012). Crop to wild gene flow and consequent hybridisation is of particular concern where fragmented areas contain crop species within the natural range of native congeners (Barbour et al., 2008a; Delplancke et al., 2012; Cornille et al., 2013). This issue is receiving increased attention, as it occurs in twelve of the world's thirteen most important food crops including wheat, maize and rice (Ellstrand et al., 1999; Delplancke et al., 2012). Crop to wild gene flow can lead to significant genetic effects through hybridisation and introgression (Ellstrand et al., 1999; Barbour et al., 2008b). Crop cultivars often have low levels of genetic diversity due to domestication-enforced bottlenecks (Delplancke et al., 2012). Gene flow from crop cultivars to wild populations of the same species or genus can, therefore, facilitate an influx of genetically depauperate offspring resulting in genetic swamping (Ellstrand et al., 1999; Booy et al., 2000). Other potential hybridisation issues include reduced fitness due to outbreeding depression, or hybrid vigour in which first generation crop-wild hybrids have advantages over wild seedlings (Charlesworth and Willis, 2009; Frankham et al., 2011). Hybridisation may lead to loss of genetic distinctiveness, and the wild relatives of some domesticated species are now at risk of extinction, particularly endangered species, or those with small, isolated populations and low genetic diversity (Small, 1984; Levin et al., 1996; Ellstrand et al., 1999; Dickinson et al., 2012). The extent of crop to wild gene flow is expected to vary among species, populations, pollinators, and distance between the crop and wild population (Arriola and Ellstrand, 1996; Ellstrand

K. O'Connor et al. / Biological Conservation 191 (2015) 504–511

et al., 1999). Evidently, the potential occurrence of crop to wild gene flow needs to be investigated in a diverse range of species. Macadamia (Proteaceae) is an iconic subtropical rainforest genus endemic to south-eastern Queensland and north-eastern New South Wales and is the only native Australian tree commercialised for its fruit (Hardner et al., 2009). Two of the four species in the genus, Macadamia integrifolia Maiden and Betche and M. tetraphylla L.A.S. Johnson and their hybrids are cultivated in Australia and overseas and breeding programmes have developed a set of cultivars used in commercial orchards (Hardner et al., 2009). Both species are classified as vulnerable in the wild by the Environment Protection and Biodiversity Act (1999). M. integrifolia is the most widespread species, ranging from Maryborough in south-east Queensland (SE Qld), to the north-east New South Wales (NE NSW) border (Fig. 1). Macadamia tetraphylla naturally occurs from just north of the Qld-NSW border and south to near Lismore in NE NSW (Fig. 1). These two species co-occur naturally in parts of SE Qld but only M. tetraphylla occurs naturally in NSW (Hardner et al., 2009, Fig. 1). This genus depends mainly on native Trigona (Tetragonula) spp. bees and introduced Apis mellifera honeybees for pollination, and is partially self-incompatible (Wallace et al., 1996; Blanche et al., 2006). Seeds are dispersed by gravity and water, and by the black rat Rattus rattus and native rodents (Neal, 2007; Hardner et al., 2009). In SE Qld and NE NSW, 92% of the lowland subtropical rainforest has been cleared since European settlement, and most remnant patches are less than 10 ha (Department of Sustainability, 2013). Of the Macadamia species in the region, M. tetraphylla has been impacted the most by habitat fragmentation, and many wild populations are small and surrounded by agricultural land (Costello et al., 2009; Parkes et al., 2012). In NE NSW, many orchards containing M. integrifolia cultivars are established close to wild M. tetraphylla populations (Hardner et al., 2009), primarily in the northern Tweed area and southern Lismore area. Macadamia species have overlapping flowering periods, and are reported to hybridise in the wild and in orchards (Trueman and Turnbull, 1994; Peace et al., 2008; Hardner et al., 2009), thus there is the potential for orchard grown cultivars of M. integrifolia to hybridise

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with wild M. tetraphylla. Macadamia juveniles cannot accurately be classified to species level by their leaf morphology (Hardner et al., 2009). Molecular tools are thus useful for the identification of potential hybrids. The occurrence and extent of crop to wild gene flow and hybridisation in Macadamia are unknown, but there are many potential scenarios across the distribution of the three wild species where they are grown in close proximity to cultivars grown in orchards given the known pollen dispersal distances. This preliminary study aims to determine if crop to wild gene flow and hence hybridisation has occurred from M. integrifolia in orchards to wild M. tetraphylla populations in the geographically closest wild populations to cultivated orchards. We used microsatellite markers to test for the presence of cultivar alleles in wild populations, and to determine genetic diversity and composition in wild M. tetraphylla populations and M. integrifolia orchards. 2. Methods 2.1. Sampling design and study area The study was undertaken outside the natural range of M. integrifolia between Tweed Heads and Lismore, NSW, to facilitate the detection of alleles derived from orchard grown cultivars of this species in wild populations of the related M. tetraphylla and thus avoid issues of natural gene flow complicating the results (Hardner et al., 2009, Fig. 1). This area, particularly the northern and southern regions, contains the most intensive areas of commercial macadamia cultivation in Australia, improving the chance of detecting crop to wild pollen flow. The orchards contained predominantly M. integrifolia cultivars, with some M. integrifolia × M. tetraphylla hybrids. The trees in these orchards are approximately eight to 30+ years old while wild trees may live to greater than 100 years. The sampling design focussed on wild populations that are the closest in proximity to commercial orchards as those are most likely to have experienced hybridisation due to gene flow from commercial orchards.

Fig. 1. Locations of Macadamia tetraphylla sites MT1 to MT10 and nearby towns in north-east New South Wales, Australia. Approximate range of Macadamia integrifolia and M. tetraphylla, and the overlapping hybrid zone in south-eastern Queensland are also indicated.

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The study sites were selected in cooperation with orchards associated with the Australian Macadamia Society and private landholders. Compiled databases of M. tetraphylla location records and macadamia orchards were used in combination with local knowledge to locate potential wild populations with nearby commercial orchards. The estimated age of each orchard was determined based on farmer knowledge. Sites were selected that had wild populations containing both mature adults estimated to be older than the age of nearby orchards, and seedlings. This was to ensure that at least one mature wild tree was present prior to the orchard establishment, and that hybrid offspring could have potentially arisen since the maturation of the orchard. Ten M. tetraphylla population and M. integrifolia orchards were thus sampled across the geographic distribution of M. tetraphylla and selected to cover a range in orchard abundances (Fig. 1). For this study, all wild populations were within 10–1600 m of the nearest orchard, within the potential cross pollination distance of 2800 m (Neal, 2007). In some cases, there was more than one orchard nearby. 2.2. Field methods In wild populations where possible, all M. tetraphylla trees were sampled, up to about 50 per site. At site MT8, only those closest to the nearby orchard were sampled. Site MT10 had a larger than expected population for the area, so all 66 individuals were sampled. Leaf material was collected from each plant and dried with silica gel prior to DNA extraction. In addition, up to four replicates of each of 15 reported orchard cultivars were sampled, giving a total of 37 cultivar samples. The known cultivars present in each orchard were documented so that later hybrid analysis could cross check that the M. integrifolia alleles found in the wild M. tetraphylla populations could have arisen from nearly orchards. In wild populations the relative locations of each plant were mapped. Depending on the nature of the terrain, either a belt transect or GPS readings for each plant was used and converted to XY coordinates (m). The height of each M. tetraphylla plant was recorded using a Leica Disto D5 laser distance metre. The distance from each wild population to the nearest Macadamia orchard was determined using the Google Earth 7 (2013) ruler tool, using the furthest wild tree. 2.3. Laboratory methods Approximately 30 mg of leaf sample was frozen in liquid nitrogen before disruption using a Retsch MM200 Tissue Lyser. Total genomic DNA was extracted using DNeasy® Plant Mini Kits following the manufacturer's protocol (QIAGEN Valencia, CA, USA). DNA extracts were checked for quality on a 1.5% agarose gel and viewed under UV light using a Syngene gel documentation system and GeneSnap software (Syngene). Microsatellite markers were used for this study due to their highly mutative, co-dominant nature, which is useful in parentage analysis and the existence of previously developed variable markers (Cupertino et al., 2009). These were considered to be superior for the purposes of this study compared to chloroplast markers. Fifteen available primers were selected for loci with known private alleles for M. integrifolia and M. tetraphylla (Schmidt et al., 2006; Nock et al., 2014) to maximise the chance of detecting orchard alleles from M. integrifolia cultivars in wild seedlings of M. tetraphylla. These 15 primers were tested to confirm their amplification in both species and optimised. Fourteen primer pairs amplified consistently: MinμS1, MinμS2, MinμS4, MinμS5, MinμS7, MinμS16 (Schmidt et al., 2006), and Mac001, Mac002, Mac003, Mac004, Mac005, Mac006, Mac008 and Mac011 (Nock et al., 2014). Forward primers were end-labelled with one of four fluorescent dyes (FAM, Geneworks; VIC, NED, PET, Applied Biosystems). PCR reactions for MinμS primers followed methods of Shapcott and Powell (2011): approximately 25 ng of genomic DNA, 1 × PCR reaction buffer (Fisher Biotech), 0.2 mM dNTPs, 1.5 mM MgCl2, 0.5 units Taq F1

polymerase (Fisher Biotech), 0.1 μM of each primer. Primer MinμS7 used 2 mM MgCl2. PCRs were performed with: 94 °C 1 min; 40 cycles 94 °C 30 s, annealing at 52 °C 30 s, 72 °C 40 s; and final elongation 72 °C for 2 min. Reactions for Mac primers used methods of Nock et al. (2014): 1 × PCR reaction buffer (Fisher Biotech), 0.1 mM dNTPs, 2.5 mM MgCl2, 0.5 units Taq F1 polymerase (Fisher Biotech), and 0.2 μM of each primer. PCR mix for Mac005, Mac006 and Mac011 contained 2 mM MgCl2. Thermocylcing conditions used were: 94 °C for 2 min; 30 cycles, 92 °C 10 s, annealing 60 °C 10 s, 70 °C 1 min; and final elongation 70 °C for 5 min and 30 °C for 3 min. Optimisation of cycling conditions for individual loci was as follows: 35 cycles for Mac005, Mac006 and Mac011, annealing temperature of 55 °C for Mac006 (Nock et al., 2014). The PCR products were visualised under UV light as above to test for successful amplification. Products were then processed on an AB 3500 Genetic Analyser (Applied Biosystems) using LIZ 600 as a size standard. Products were diluted for each locus after test analyses, and pooled according to allelic lengths and fluorescent dye to prevent overlapping signatures. Pool one Mac001 FAM, Mac002 FAM, Mac004 NED, Mac005 VIC, Mac009 PET; pool two Mac003 FAM, MinμS4 FAM, MinμS7 NED, MinμS16 VIC, Mac006 PET; pool three MinμS2 FAM, Mac007 NED, MinμS1 VIC, MinμS5 PET. Microsatellites for each primer locus were scored as alleles using GeneMarker 1.95 (SoftGenetics) software and manually checked and corrected to ensure consistency with expected repeat patterns. Loci that were not scored consistently were excluded from further analysis, leaving a total of 11 loci: MinμS2, MinμS5, MinμS7, Mac001, Mac002, Mac003, Mac004, Mac005, Mac006, Mac008 and Mac011. Multilocus genotypes were thus assigned to each individual. Private alleles unique to orchards were used as markers to determine possible hybrid offspring in wild populations. We discarded both alleles per locus for bad signatures/null alleles, so any individual identified as a hybrid had one crop parent and one wild parent. Thus our identification of hybrid individuals is an unambiguous one. 2.4. Hybrid detection and parentage analysis Parentage analysis was performed using CERVUS 3.0.3 (Kalinowski et al., 2007) to test cultivars as parents of young (juvenile or seedling) plants, thus identifying hybrids among the young plants from M. tetraphylla populations. Individuals less than 2 m in height were classified as ‘offspring’. All trees over 2 m in height as well as nearby cultivated trees, were potential ‘parents’. Individuals were identified as having one or both parents as cultivars, and the natural log of the likelihood ratio (logarithm of odds; LOD) scores, confidence (80% relaxed, 95% strict) and number of matching loci out of 11 typed loci were recorded. A Bayesian model-based clustering method was performed using the programme STRUCTURE 2.3.4 (Pritchard et al., 2000; Falush et al., 2003) based on multilocus (11 loci) genotype data to assign individuals to K clusters or populations. In order to illustrate admixture between M. tetraphylla and M. integrifolia in graphical format, analysis was conducted using K = 2. STRUCTURE analysis was run with a burn-in period of 500,000 steps, 500,000 Markov Chain Monte Carlo (MCMC) repetitions, admixture model and correlated allele frequencies. Individual M. tetraphylla trees were grouped according to their height (b1 m tall, 1.1–3 m, 3.1–5 m, 5.1–7 m, 7.1–9 m, N 9 m) consistent with Macadamia jansenii height classifications (Shapcott and Powell, 2011), and genetic diversity measures and allelic frequencies were calculated accordingly. Allelic frequencies without rarefaction corrections were used to calculate standard genetic diversity and inbreeding measures using GenAlEx 6.5 (Peakall and Smouse, 2012) including number of alleles per locus (A), number of effective alleles per locus (Ae), observed heterozygosity (Ho), expected heterozygosity (He), and percentage of polymorphic loci (P). Wright's mean allelic fixation indices (F) were calculated to estimate inbreeding (Frankham et al., 2002).

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Principal coordinates analysis (PCoA) was performed with 9999 permutations using Nei's (1978) genetic distance matrix to investigate genetic distance between cultivars and wild M. tetraphylla trees grouped by height. 2.5. Genetic analysis of wild populations Further genetic analyses excluded individuals from populations MT2 and MT6 due to their small populations, and all individuals identified as having orchard parents. Null allelic frequency (FN) estimations for each locus were obtained using CERVUS 3.0.3 (Kalinowski et al., 2007). Mean diversity measures were calculated as above using GenAlEx 6.5 (Peakall and Smouse, 2012). Spearman's rank correlations were undertaken in SPSS v21 (IBM 2012) to test for relationships between population size, genetic diversity and inbreeding measures (n, A, Ae, Ho, He, P and F). Mann–Whitney U tests were performed in SPSS v21 (IBM 2012) to compare genetic diversity and inbreeding measures between small (n = 7–16) and large (n = 46–59) populations, excluding hybrids. Analysis of molecular variance (AMOVA) was performed using 9999 permutations by incorporating the northern region (sites MT4, MT5 and MT8) and southern region (sites MT1, MT3, MT7, MT9 and MT10; Fig. 1) as a grouping variable, to produce the percentage of variation within and between populations as well as among regions. Phi-statistics and the distribution of genetic variation across the whole species (FIT), within populations (FIS), and between populations (FST), as well as number of migrants per generation (Nm) were calculated using GenAlEx 6.5 (Peakall and Smouse, 2012). A Mantel's test was performed to determine the correlation between geographic distance and genetic distance for each population using Nei's (1978) genetic distance matrix with 9999 permutations in GenAlEx 6.5 (Peakall and Smouse, 2012). STRUCTURE 2.3.4 (Pritchard et al., 2000; Falush et al., 2003) was used to determine the structure and admixture of wild populations across multiple loci. In order to detect population structure within M. tetraphylla, ten independent simulations were performed for each K value of K = 2–10 for all non-hybrid individuals. The simulations used a burn-in period of 100,000 steps, and 100,000 MCMC repetitions, along with an admixture model and correlated allele frequencies. These data were uploaded to Structure Harvester to summarise STRUCTURE outputs and identify the true number of clusters using the Evanno method (Evanno et al., 2005; Earl and von Holdt, 2012; http:// taylor0.biology.ucla.edu/structureHarvester/). Structure Harvester determined the optimal K value for M. tetraphylla populations as 3 clusters, with a delta K value of 261.20. Final analyses were run using the determined K value of K = 3 with a burn-in period of 500,000 steps, 500,000 MCMC repetitions, admixture model and correlated allele frequencies. 3. Results 3.1. Population demographics M. tetraphylla populations ranged from one individual at site MT2 (3.7 m tall), and three at MT6 to 66 trees at MT10. Across all sites, 44% of M. tetraphylla plants were seedlings (≤1 m), while almost 10% were over 9 m tall (Table 1). The area occupied by populations varied from 0.03 ha (MT3 and MT6) to 1.28 ha in MT9, and the mean density across all sites was 144 plants/ha (range 10 to 567 plants/ha; Table 1). 3.2. Parentage analysis A total of 30 plants (10.6% of all wild plants sampled) were identified as M. tetraphylla × M. integrifolia hybrids or M. integrifolia with orchard parents, across all wild sites, with the exception of MT2. Of these plants, 23 were seedlings ≤ 1 m tall and five were juveniles 1–2 m tall. Over all sites, 20 young plants (18%) up to 2 m tall were hybrids. The largest number of hybrids was seven in MT10, while only one hybrid was found in MT3 (Table 1). Seedlings and juveniles heterozygous for

507

Table 1 Summary of the characteristics of each wild M. tetraphylla site, excluding MT2 (N = 1). The population codes, population size (N), number of seedlings, area of patch, Macadamia density, number of hybrid plants detected (one cultivar parent) and number of plants derived from seed dispersed from orchards (two cultivar parents) are given. Population

N

No. of seedlings (≤1 m)

Area (ha)

Tree density (plants/ha)

No. of hybrid plants

No. of M. integrifolia seeds dispersed

MT1 MT3 MT4 MT5 MT6 MT7 MT8 MT9 MT10 Mean or total

16 17 52 49 3 14 53 13 66 31

12 7 19 26 2 7 12 3 38 14

0.075 0.030 0.525 0.329 0.030 1.080 0.851 1.280 0.780 0.553

213 567 99 149 100 13 62 10 85 144

3 1 3 1 2 0 2 1 7 20

0 0 0 2 0 7 0 1 0 10

cultivar alleles were assumed to have received these from nearby orchards. Those homozygous for cultivar alleles were assumed to be seed dispersed by gravity, water or rodents. Ten plants were detected as having two cultivar parents at MT5, MT7 and MT9 (Table 1). MT7 had the greatest seed dispersal distance at more than 1000 m. Consequently, seeds are assumed to have been dispersed in three ways, and over three different distances: dispersed by gravity was about 25 m, dispersal by water was more than 1000 m, and dispersal by rodents was about 80 m. Overall, 82.1% of seedlings and juveniles b 2 m tall in M. tetraphylla populations were assigned a parent pair from their wild population and/or nearby orchard. Results from CERVUS 3.0.3 were concordant with those from STRUCTURE 2.3.4 (Pritchard et al., 2000; Falush et al., 2003; Fig. 2a). Full-length dark grey lines represent M. integrifolia, while half-length dark grey lines represent hybrids in M. tetraphylla populations (Fig. 2a). A number of lines were only a quarter-length, which may be attributed to these seedlings having a M. tetraphylla × M. integrifolia cultivar parent. 3.3. Hybrid detection A single diagnostic locus with species-specific alleles is invaluable for identifying F1 hybrids created from gene flow between two distinct species (Ellstrand et al., 1999). Both Mac004 and MinμS2 markers had null allelic frequency estimates above 0.600. However, these two loci had species-specific alleles useful in detecting hybrids, and the data acquired through the use of these loci were included in analyses. Comparison of allelic frequencies between height classes revealed alleles present in M. tetraphylla seedlings and juveniles that were not present in any mature plants (Table 2). Twenty-two alleles across 9 loci were found only in cultivar plants and not in adult (3 m +) wild M tetraphylla plants (see examples Table 2), indicating that their source was orchard trees, not wild trees. Consequently, these alleles provided markers for detecting hybrids. When grouped by height, the majority of M. tetraphylla individuals were distinct from the cultivars in PCoA analysis (Fig. 3). There was clustering of some wild plants in height classes 1 and 2 (b3.1 m) with cultivars. These individuals had alleles found in cultivars and not in wild M. tetraphylla parents (Table 2, Fig. 3). The cultivars in the centre of the PCoA of height classes were A-type cultivars of M. integrifolia × M. tetraphylla. This would explain the intermediate location of these cultivars between the M. tetraphylla individuals and M. integrifolia orchard cultivars. 3.4. Genetic analysis A total of 99 alleles were scored across 11 loci from 251 M. tetraphylla samples. The mean number of alleles per locus was 9, ranging from 1 allele in MinμS2 to 27 in MinμS7. With small populations

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Fig. 2. Bar plot showing STRUCTURE 2.3.4 (Pritchard et al., 2000; Falush et al., 2003) Bayesian assignment of individuals to M. tetraphylla populations and M. integrifolia cultivars. Each line represents one individual; length of coloured segments for each line is proportional to the assignment of that individual to each ancestry. Individuals grouped by populations labelled beneath. a, K = 2, two species; all individuals from M. tetraphylla populations and M. integrifolia cultivars. b, K = 3, M. tetraphylla populations, excluding hybrids.

(MT2, MT6) and putative hybrids excluded, the wild M. tetraphylla populations contained considerable genetic diversity. Levels varied among populations but were on average relatively high (A = 4.602; Ae = 2.675; He = 0.512; P = 87.5) across all sites despite their small size (Tables 1 and 3). Site MT8 possessed the highest number of alleles and effective alleles per locus (A = 6.000 and Ae = 3.322). Diversity was significantly higher in large compared to small populations (p = 0.036) and allelic diversity was significantly correlated with increasing population size (rs = 0.762, p = 0.028). Overall, wild populations had low levels of inbreeding despite their small sizes (mean F = 0.160; Tables 1 and 3). MT5 was the most inbred, (F = 0.331), whereas MT1 had an excess of heterozygotes (F = − 0.116). Low levels of inbreeding are probably due to selfincompatibility, coupled with pollen flow from nearby M. tetraphylla populations. Table 2 Frequency of cultivar-specific alleles across various loci. Alleles found only in cultivars and M. tetraphylla seedling and juvenile height are shown in boldfaced. Height classes were 1 = ≤1 m tall; 2 = 1.1–3 m; 3 = 3.1–5 m; 4 = 5.1–7 m; 5 = 7.1–9 m; and 6 = ≥9 m. Locus

Allele

Class 1

Class 2

Classes 3–6

Cultivars

Mac002

281 288 294 259 222 224 232 327 337 342 320 326 391 180 186 188 146 150 99 103 105 140

0.055 0.100 0.010 0.012 0.024 0.012 0.000 0.000 0.052 0.065 0.000 0.004 0.012 0.036 0.032 0.012 0.008 0.052 0.000 0.012 0.004 0.008

0.028 0.038 0.000 0.008 0.008 0.000 0.008 0.008 0.023 0.008 0.008 0.000 0.008 0.008 0.016 0.031 0.000 0.031 0.008 0.008 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.230 0.649 0.122 0.351 0.162 0.054 0.027 0.027 0.500 0.270 0.027 0.081 0.068 0.068 0.176 0.176 0.054 0.608 0.068 0.297 0.027 0.014

Mac003 Mac004

Mac005

Mac006 Mac008 Mac011

MinμS2 MinμS7

While most genetic variation was found within populations, 21% of genetic diversity was attributed to differences among M. tetraphylla populations (PhiPR = 0.211, FST = 0.175, Table 4). Average estimates of gene flow among populations (Nm 1.2, Table 4) suggest sufficient historic gene flow to counter the effects of drift among populations. Genetic distance was weakly but significantly correlated with geographic distance among wild individuals (RXY = 0.184, p = 0.000). The STRUCTURE analysis assigned wild individuals to three clusters. Some of these contained geographically proximate populations but some admixture among clusters was apparent (Fig. 2b). 4. Discussion 4.1. Crop to wild gene flow in macadamia This is the first study to document hybridisation in Macadamia due to crop to wild gene flow. Genetic and parentage analyses in this study suggest that M. integrifolia cultivar alleles have entered wild M. tetraphylla populations through both pollen and seed dispersal within a 30 year time frame since orchard establishment. In Eucalyptus, cropwild hybrids were an order of magnitude higher in open-pollination experiments than in natural hybrids, and increased with proximity to plantations (Barbour et al., 2003, 2005). This level of hybridisation, however, is low compared with that found in M. tetraphylla populations. Seven percent of the trees from M. tetraphylla populations were hybrids, produced by pollen dispersal from orchards. In comparison, 37% of wild apple Malus sylvestris trees were hybrids due to crop to wild gene flow from the cultivated apple M. domestica in Europe, in the absence or weakness of barriers to gene flow (Cornille et al., 2013). It is thought that this process may threaten the integrity of wild species (Cornille et al., 2013). Thus the levels of crop to wild gene flow found in this study are higher than expected probably due to the close proximity of orchards and the effectiveness of pollinators within these distances. Further studies are needed to investigate if this is occurring in other wild Macadamia species in other areas where wild populations and commercial orchards are in close proximity to assess the potential implications for loss of genetic distinctiveness among the three species that overlap in distribution. The flow of crop genes varies with different pollinators and the distance between the crop and the wild populations (Arriola and Ellstrand,

K. O'Connor et al. / Biological Conservation 191 (2015) 504–511

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Fig. 3. Principal coordinates analysis showing genetic relationships among cultivars (Cult) and M. tetraphylla individuals grouped according to their height. Height classes were 1 = ≤1 m tall; 2 = 1.1–3 m; 3 = 3.1–5 m; 4 = 5.1–7 m; 5 = 7.1–9 m; and 6 = ≥9 m.

1996; Ellstrand et al., 1999). Understanding pollination and dispersal patterns and distances for different plant species is necessary in order to implement strategies to minimise pollen and seed dispersal into nearby wild populations (Jones et al., 2007). Wild Macadamia species have been shown to be linked at distances of 2 km by pollen dispersal, thus it is expected that cultivated orchards within this distance may hybridise with wild populations. Capurro et al. (2013) argued the importance of increasing the distance between cultivated potato Solanum tuberosum fields and wild potato populations to prevent potentially detrimental gene flow via pollen between the two. Forest trees generally have high dispersal capabilities, which increase the risk and effects of crop to wild gene flow in these species (Strauss, 2011). Now that it has been established in the closest proximity cases, further research is required to investigate the effect of distance between Macadamia orchards and wild populations on crop-wild gene flow. Seedling recruitment in M. integrifolia was higher in fragmented landscapes than continuous habitat, suggesting that habitat fragmentation did not have a detrimental affect on regeneration in that species (Neal et al., 2010). The absence of crop-wild hybrids over 2 m tall Table 3 Summary of mean genetic diversity measures for M. tetraphylla populations excluding hybrids, averaged over 11 loci. N = population size; distance to nearest orchard in metres; A = number of alleles; Ae = number of effective alleles; Ho = observed heterozygosity; He = expected heterozygosity; P = percentage polymorphic loci; F = Wright's allelic fixation index. Standard errors are given in parentheses. Site

N

MT1

16

560

MT3

17

195

MT4

52

50

MT5

49

125

MT7

14 1020

MT8

53

MT9

13

Dist. to orchard A (m)

115 155

MT10 66 1600 Mean

3.455 (0.493) 4.182 (0.672) 5.545 (1.090) 5.909 (1.124) 1.909 (0.251) 6.000 (1.044) 4.545 (0.743) 5.273 (1.019) 4.602 (0.323)

Ae

Ho

He

P

2.358 (0.294) 2.700 (0.315) 2.587 (0.440) 3.069 (0.485) 1.429 (0.140) 3.322 (0.461) 2.927 (0.412) 3.007 (0.387) 2.675 (0.142)

0.542 (0.085) 0.417 (0.103) 0.348 (0.098) 0.368 (0.100) 0.225 (0.068) 0.439 (0.081) 0.504 (0.081) 0.526 (0.093) 0.421 (0.032)

0.490 (0.073) 0.559 (0.068) 0.480 (0.092) 0.554 (0.085) 0.237 (0.066) 0.612 (0.073) 0.568 (0.072) 0.593 (0.068) 0.512 (0.028)

91

F

−0.116 (0.061) 91 0.301 (0.142) 91 0.242 (0.114) 91 0.331 (0.117) 64 −0.029 (0.128) 91 0.299 (0.089) 91 0.083 (0.096) 91 0.114 (0.117) 87.5 0.160 (0.040)

documented in this study may suggest that young hybrids have low survivorship, or that the young age of nearby orchards (up to 30 years) has not given enough time for mature hybrids to establish given the growth rate of wild Macadamia trees. However, rainforest seedlings may remain dormant for long periods and only grow when environmental conditions are favourable (Connell and Green, 2000). Liu et al. (2013) stated that investigating the fertility and behaviour of crop-wild hybrids is the next step for this type of study. Currently, many macadamia orchards grow more than one cultivar to promote cross-pollination and higher yield (O'Hare et al., 2004; Trueman, 2013). The practice of growing more than two cultivar varieties should be encouraged to reduce potential impacts of genetic swamping by single genotypes in adjacent wild populations, as this study showed that multiple cultivar alleles were present in wild populations and that this increased genetic diversity in the wild populations rather than decreasing it due to allelic swamping. Estimates of long-distance seed dispersal are difficult to achieve (Cain et al., 2000) and genetics may offer an approach to do so as shown by the results of this study. Macadamia nuts are dispersed by gravity, water and rodents (Hardner et al., 2009; Whitehouse et al., 2012). We found that seeds were dispersed into wild populations by all three of these vectors. The establishment of M. integrifolia seedlings downhill and downstream from macadamia orchards was detected at a number of sites, up to 1000 m away. Seed dispersed Eucalyptus nitens seedlings were found within a much smaller range of 30 m (Barbour et al., 2003), while Banksia hookeriana (Proteaceae) was Table 4 Summary of F-statistics, analysis of molecular variance (AMOVA) partitioning of genetic variation and number of migrants for M. tetraphylla, excluding hybrids. FIS = genetic diversity among individuals within populations; FIT = diversity among individuals the total species population; FST = diversity among populations. PhiPT = diversity among populations, percentage of total variation found among and within populations. * Significance p b 0.005. Statistic

Value

FIS FIT FST Nm PhiPT Among populations Within populations

0.186 0.321 0.175 1.216 0.214* 21% 79%

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found to disperse seeds over 2500 m (He et al., 2004). Rodents may be responsible for the establishment of orchard seedlings in M. tetraphylla habitat approximately 80 m away at site MT5, since these seedlings were some distance uphill from the nearby creek. These results are similar to those for vertebrate-dispersed seeds of various Queensland rainforest species, which were found in forests as far as 80 m from the source (Willson and Crome, 1989). Seedlings from orchards may extend the historical range of M. integrifolia, which may have consequences for the genus' distribution, which may be significant for climate change adaptation (Hardner et al., 2009; Powell et al., 2010).

crop and wild populations. In this case the hybridisation of the wild populations by a different species of Macadamia could lead in the long term to loss of species identity for the M. tetraphylla populations if this process continues but this is expected to take a long time to have an effect given the tree's generation time. The results clearly indicate potential for long-term impacts of macadamia orchards in close proximity to wild populations, and future developments should bear this in mind. However, more research is needed to build on these data. Future studies should be conducted to find and investigate additional Macadamia cropwild sites at greater distances and different regional densities, including wild populations of other Macadamia species such as Macadamia ternifolia with adjacent macadamia plantations.

4.2. Genetic diversity, population differentiation and inbreeding Small populations of isolated endemic species often have low levels of genetic diversity (Young and Clarke, 2000; Leimu et al., 2006). However, relatively high diversity was found among our M. tetraphylla populations. Allelic diversity of the M. tetraphylla populations was similar to a previous study of the same species (Spain and Lowe, 2011). Comparatively, genetic diversity was higher in the congener M. integrifolia (Neal, 2007) and the rare Grevillea repens (Proteaceae; Holmes et al., 2009). This study identified a strong positive relationship between population size and genetic diversity in M. tetraphylla. In comparison, there was no relationship between population size and genetic diversity in fragmented populations of Grevillea macleayana (Proteaceae; England et al., 2002). This suggests that despite the small populations, M. tetraphylla is genetically diverse. This diversity, however, may be at risk with increasing fragmentation if populations become more geographically isolated. The nearest known neighbours of most M. tetraphylla populations were within pollination distance estimated for M. integrifolia (2800 m; Neal, 2007), and there may be closer unknown isolated trees. Thus, populations of M. tetraphylla are potentially connected by paternal gene flow. Genetic differentiation among the M. tetraphylla populations (FST = 0.175) concurred with a reported mean score for outcrossing in animal-pollinated plants (Hamrick and Godt, 1989). In comparison, Neal (2007) observed lower genetic differentiation (FST) among M. integrifolia populations while both G. macleayana and G. repens had higher differentiation due to limited inter-population gene flow (England et al., 2002; Holmes et al., 2009). However, estimated gene flow rates in M. tetraphylla were much lower than those reported in the congener M. integrifolia, and this may be attributed to the shorter distances between populations of this species. Since M. integrifolia private alleles were used to detect crop to wild gene flow in this study, gene flow among wild populations of M. tetraphylla would not have confounded our results. Inbreeding accumulates over time in small, isolated populations in the absence of gene flow migration among populations (Frankham et al., 2002). Inbreeding was limited in M. tetraphylla, despite the small population size, and may be due to pollenmediated gene flow between close populations (Ghazoul, 2005). Maintaining a network of connected populations through gene flow is therefore important for long-term survival of wild M. tetraphylla populations, as it sustains outcrossing and limits inbreeding. The presence of large populations of cultivars derived from a different Macadamia species within the M. tetraphylla metapopulation geographic region may promote both pollinator visitation and movement and also lead to increased crop to wild hybridisation between the two species. 5. Conclusions This study is the first to demonstrate crop to wild hybridisation in Macadamia when orchards are close to wild populations. We provided evidence of hybridisation, and both pollen and seed dispersal between

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