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Keywords:

  • Acanthopagrus spp. complex;
  • admixture;
  • Bream;
  • dispersal;
  • hybrids;
  • introgression;
  • microsatellites;
  • mtDNA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

For free-spawning estuarine taxa, gene flow among estuaries may occur via hybridization with mobile congeners. This phenomenon has rarely been investigated, but is probably susceptible to anthropogenic disturbance. In eastern Australia, the estuarine Black Bream Acanthopagrus butcheri and marine Yellowfin Bream Acanthopagrus australis have overlapping distributions and the potential to hybridize. We used surveys of microsatellite and mtDNA variation in 565 adults from 25 estuaries spanning their distributional range to characterize the species and their putative hybrids. Hybrids were widespread (68% of estuaries) and hybrid frequencies varied greatly among estuaries (0–58%). Most (88%) were classed as advanced generation backcrosses with A. butcheri and displayed A. butcheri mtDNA haplotypes. We found most hybrids in the three estuaries within the zone of sympatry (57%). Our study highlights the underemphasized importance of estuaries as sites of hybridization and suggests that hybridization is driven both by opportunity for contact and human activity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Populations of species that complete their entire life cycle within estuaries (obligately estuarine) are potentially small and confined to isolated patches of suitable habitat. This creates conditions that promote rapid evolutionary divergence through the combined effects of drift, founder events and site-specific selection (Frankham, 1995, 2005). Given that such taxa may possess highly mobile adults or larvae, this situation may parallel that of populations restricted to mountain peaks or islands (Assefa et al., 2007; Lukoschek et al., 2007). However, for genetically compatible free-spawning taxa, the effects of isolation may be reduced by hybridization with wide-ranging congeners, resulting in gene flow both among estuaries and between species (Addison & Hart, 2005; Harper et al., 2007). These phenomena have rarely been investigated and both the likelihood of hybridization and its ecological and evolutionary consequences are potentially both estuary specific and susceptible to anthropogenic disturbance through processes such as harvesting and mechanical modification of estuarine hydrology (e.g. Scribner et al., 2000).

The consequences of hybridization can either enhance or reduce the potential for persistence and adaptive evolution of hybridizing species (Harrison, 1993; Levin et al., 1996; Rhymer & Simberloff, 1996; Arnold, 1997). In particular, low levels of hybridization may be beneficial for estuarine taxa as introgression may introduce genetic novelty and oppose the anticipated effects of genetic drift and/or inbreeding within otherwise isolated populations (Chaplin et al., 1998). However, when there is a high probability of interspecific matings, then demographic and genetic swamping may occur with potentially serious consequences if hybrids are either inviable or infertile and gametes are wasted, or if outbreeding depression results because hybrids are relatively less fit than their parents (Barton, 2001; Burke & Arnold, 2001). At the extreme, frequent backcrossing could contaminate the genome of one or other parental species causing loss of species identity as the genomes of the two species recombine (e.g. Woodruff & Gould, 1987). However, for estuarine-dependent species the net consequences of hybridization may reflect a complex function of the above factors and will be influenced by the site fidelity of the hybrid and the degree to which estuarine taxa and their hybrids compete for resources such as spawning sites and mating partners.

It seems likely that the consequences of hybridization between marine and estuarine taxa will be the greatest for locally rare, obligately estuarine taxa. In this circumstance, we would expect that the rare taxon is more likely to be involved in cross-species matings. Moreover, these effects may be exacerbated if hybrids are both inter-fertile and remain within estuaries leading to the production of later generation hybrids and backcrosses and the replacement of the estuarine taxa by a hybrid swarm (i.e. genetic swamping).

The Bream species complex (Acanthopagrus spp.) of eastern Australia is potentially an excellent model system within which to investigate hybridization at the marine/estuarine interface. Acanthopagrus australis (Günther) (Yellowfin Bream) uses estuaries for feeding and as a nursery for juveniles, but is not restricted to this habitat as it moves between coastal and estuarine waters. The morphologically similar Acanthopagrus butcheri (Munro) (Black Bream) completes its entire life cycle within estuaries. Where these species are sympatric, they are thought to hybridize occasionally (with the presence of at least some hybrids confirmed by allozyme data) (Rowland, 1984). However, the population dynamics of both species may have been altered substantially in recent years with anthropogenic impacts potentially increasing the likelihood of hybridization. Both species are the basis of a valuable and heavily exploited commercial and recreational fishery in eastern Australia (Kailola, 1993; New South Wales Department of Primary Industries, unpublished data) and the hydrodynamics of many estuaries is frequently modified by artificial opening of estuary mouths (New South Wales Department of Natural Resources, unpublished data).

Many of the estuaries of eastern Australia that are reported to support populations of A. butcheri are small, isolated and have intermittent patterns of opening to the adjoining ocean (indeed some estuaries may be landlocked for several years, Roy et al., 2001; NSW DNR, unpublished data). These characteristics may potentially increase the effects of hybridization especially if hybrids remain in residence for extended periods. We currently do not know the frequency of occurrence of hybridization or the distribution of hybrids. Here, we use a combination of microsatellite and mitochondrial DNA markers and morphology to initially characterize pure species and various classes of hybrids, and then to utilize a broad-scale genetic survey to investigate the geographical extent of hybridization and to test whether hybrids are indeed more common in the area of sympatry.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Species background

Acanthopagrus butcheri has a disjunct distribution within estuaries from central NSW to WA, including Tasmania. Acanthopagrus australis is distributed along the entire east coast of Australia from northern Queensland to about central Victoria, overlapping part of its range with A. butcheri (Edgar, 2000) (Fig. 1). Acanthopagrus australis inhabits a range of areas encompassing rocky headlands, offshore reefs and ocean beaches, as well as estuaries.

image

Figure 1.  Map showing the location of study estuaries (asterisks indicate Acanthopagrus butcheri sampling sites) (see Table 1 for more detail).

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Both species have annual reproductive cycles with asynchronous gonad development and group spawning occurring over a period of 2–5 months. Spawning time in A. butcheri varies between estuaries, but generally peaks in September or October, and takes place in the extreme upper estuary, near the interface of fresh and salt water, well away from any influence of coastal currents. Fertilized eggs are pelagic, hatching ∼36–48 h (depending on water temperature) after fertilization (Haddy & Pankhurst, 1998). Larvae recruit to seagrass meadows and other submerged structures for growth and development. By partial contrast, the peak spawning of A. australis occurs in July and August in the vicinity of estuary entrances and the adjacent surf zones of ocean beaches. Some A. australis males may remain inside estuaries during the spawning season and, perhaps more significantly, large numbers may be trapped within estuaries for several years by closure of estuary mouths. However, little is known about how A. australis males behave in the presence of A. butcheri spawning females. As for A. butcheri, larvae recruit to shallow seagrass meadows and submerged structures in estuaries (Griffiths, 2001); however, larvae can spend a significant time (∼30 days) in the coastal ocean plankton under the influence of the East Australian Current and are therefore thought to disperse great distances before recruiting to seagrass meadows in the lower reaches of estuaries (Neira et al., 1998).

Molecular markers and population sampling

We genotyped on average 22.6 ± 4.0 (SE) adult fish (collected randomly, i.e. not knowing their specific species status) from each of 25 estuaries between Queensland and Western Australia (Table 1) using eight microsatellite loci from a selection that were developed for Acanthopagrus schlegelii (Jeong et al., 2003) and A. butcheri (Yap et al., 2000). The microsatellite loci (accession number) we used were: pAb2B7 (AF284352), pAb2A5 (AF284354), pAb2D11 (AF284355) (Yap et al., 2000), Acs1* (AB102864), Acs3* (AB095008), Acs6* (AB095010) (Jeong et al., 2003), Acs-16* (AB095012) and Acs-21* (AB095014) (D. Jeong & T. Umino, unpublished data). In addition, we amplified ∼400 bp of the 3′-domain of the mitochondrial (mtDNA) control region using primers LPW and HPX of Jean et al. (1995), and subsequently performed restriction digests (RFLP) on the fragment. Details of the reaction conditions used to amplify each locus, the RFLP analysis of mtDNA and genotyping are described in an online appendix.

Table 1.   Collections of adult Acanthopagrus spp. (Acanthopagrus australis, Acanthopagrus butcheri or their hybrids) from Australian estuaries*.
EstuaryGeographical locationSpeciesMsatNumber mtDNAMorph
  1. *The number of samples genotyped using microsatellites (Msat), mitochondrial DNA (mtDNA) and examined for morphology (Morph) is indicated.

  2. †Pooled samples of between 5 and 18 Bream collected from 3 and 10 estuaries respectively.

Gold Coast (Go)QueenslandA. australis413320
Forster (Fo)New South Wales 7459 
Botany Bay (Bo)  3028 
Port Hacking (Po)  3937 
Meroo (Me) A. butcheri2511 
Coila (Co)  502120
Cuttege (Cu)  248 
Scamander (Sc)Tasmania 2020 
Swan (Sw)  4948 
Derwent (De)  2121 
Northwest (No)  4944 
Victoria (VIC)†Victoria 7947 
South Australia (SA)†South Australia 1512 
Western Australia (WA)Western Australia 4932 
Total  56542140

Genetic analyses

Factorial correspondence analysis (FCA) (performed in genetix 4.03, Belkhir et al., 2002) based on the eight-locus nuclear genotypes of all 565 samples was used to visualize the genetic relationship among individuals to see if there was an obvious separation between species. This type of analysis is particularly suited to describing the genetic structure of hybrid zones because the genetic differentiation is decomposed in a nested fashion across the axes: the between-species differentiation is apparent on the first axis of the FCA, whereas differentiation between populations of the same species emerges on the secondary axes (see Daguin et al., 2001; Bierne et al., 2003 for application of FCA to hybrid zones).

As FCA suggested that hybrids were present within our samples, we tested all Bream for evidence of mixed ancestry using an assignment test. The assignment approach used population allele frequencies under the assumptions of both linkage and Hardy–Weinberg equilibrium while simultaneously assigning individuals to populations based on their eight-locus microsatellite genotype. Assignment testing was conducted using the admixture model implemented in structure (version 2.0) (Pritchard et al., 2000; Falush et al., 2003) to calculate qi (the mean posterior proportion of ancestry ± 95% CIs). We used a two-population model, after an initial burn-in period of 100 000 iterations, and collected data for 1 000 000 iterations. Default values were used for all other parameters. We present qi ± 95% CIs as the inferred proportion of A. butcheri ancestry. An arbitrarily chosen qi-value threshold of 0–0.05 and 0.95–1.0 was used to identify pure A. australis and A. butcheri respectively. Any individual with 0.95 > qi > 0.05 was classified as a hybrid. We performed additional analyses varying both the q threshold (q = 0.1–0.2) and the number of loci (four most diagnostic). Three independent runs confirmed the consistency of the inference.

Mitochondrial markers: inferring the pattern of mating and direction of hybridization

Mitochondrial DNA is maternally inherited and so by determining the mtDNA haplotypes of hybrid fish we can determine whether hybridization and backcrossing involve particular species × gender combinations, i.e. if backcrossing or hybridization always involve pure species females or both males and females (e.g. Rosenfield et al., 2000). The direction of hybridization can potentially be inferred from the presence and frequency of hybrid individuals with genotypes more similar to one or other parental species. In this instance, we used mtDNA (in conjunction with information from the microsatellites) to infer both the pattern of mating and the likely direction of hybridization. This approach could potentially be confounded if A. butcheri undergoes frequent sex change as is seen in some fish species. There is little evidence that sex change occurs in this species, although Rowland & Snape (1994) reported finding specimens (8%) with ovotestis; however, the ovarian component was nonfunctional.

Standard population genetic parameters

We used popgene (Yeh et al., 1999) and GENALEX (Peakall & Smouse, 2006) to calculate standard population genetic parameters for the total sample of each estuary and the separate groupings identified by the admixture analysis.

Classifying hybrids into distinct classes

Although our initial analyses demonstrate the presence of pure species and hybrids within our samples, the potentially complex breeding biology of these fish raises the possibility that estuaries contain not just simple F1 hybrids but also second or later generation hybrids or backcrosses between hybrids and either parental species. We therefore used the program newhybrids to estimate the probability that a specific individual belongs to each of the six different genotypic classes of parentals and hybrids that are possible for first and second generation matings between two species (Anderson & Thompson, 2002). Importantly, these analyses confirmed that we were able to distinguish hybrids from parentals (i.e. the results were generally in agreement with those obtained using structure), although, as for most hybrid zones (see Boecklen & Howard, 1997 for discussion), it was clear that our data had relatively low power to accurately classify individual fish into distinct classes of hybrid (i.e. F1s, F2s and BC1) (data not presented).

Morphological analysis

Rowland (1984) detected relatively few hybrid Bream in earlier allozyme surveys, but argued that hybrids could be distinguished from pure parentals using meristic and morphological measurements. We used principal coordinates analysis (PCA) (using the program pco, Anderson, 2003) to determine if a sample of 40 adult Bream again formed discrete clusters of putatively pure species and hybrid fish and then compared morphological assignments with those based on our more sensitive mtDNA and microsatellite genetic data. The analysis was based on the range-standardized Euclidean distance matrix of four meristic and 26 body ratio measurements (online appendix).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Microsatellite diversity

There was variation in every estuary for seven of the eight microsatellite loci. Overall, numbers of alleles per locus ranged between seven (pAb2D1) and 30 (Acs-16*), with six of the eight loci displaying greater than 15 alleles per locus, the average (±SE) was 18.4 (±2.5). Across all loci, we detected a total of 147 alleles (Table S1, online appendix). Often null alleles are encountered in cross-species amplification of microsatellite loci. Controlled breeding experiments revealed simple Mendelian inheritance and no evidence of null alleles (four pairs; 30 larvae per pair) (D. G. Roberts, unpublished data).

Separation of pure species and the identification of hybrids using microsatellite genotypes

Factorial correspondence analysis revealed the presence of two genotypic clusters that correspond generally to the sets of fish collected as A. australis and A. butcheri. Although there were relatively few fish within the entire sample that were truly genetically intermediate between the species (i.e. F1 hybrid), there was a large set of fish with genotypes that were outside, but most closely aligned with the cluster of pure species A. butcheri (black unbroken circle), whereas very few fish outside the two species groups were similar to the A. australis cluster (grey unbroken circle) (Fig. 2). Importantly, the fish that clustered inside the unbroken black circle (pure A. butcheri) were the Tasmanian, western Victorian and South and Western Australian fish that were expected to be beyond the range of hybridization (i.e. outside the described range of A. australis). The set of fish outside the two species groups, i.e. putative hybrids, were, as expected, largely fish caught in the area of known sympatry in southern NSW and eastern Victoria.

image

Figure 2.  Factorial correspondence analysis based on the eight locus genotype of all 565 Acanthopagrus spp. Fish were essentially sampled ‘blindly’ with little indication of their likely species status (the two species have extremely similar morphology). However, we expected pure species genotypes would be found within fish caught in marine-dominated northern areas (i.e. putative A. australis, closed black circles inside the grey unbroken circle, Queensland and New South Wales) and fish from the far south and west beyond the described range of A. australis (i.e. putative A. butcheri, closed black squares, Tasmania; closed triangles, South Australia; open hexagons, Western Australia, all inside the black unbroken circle). In addition, we sampled fish within estuaries in the area of known sympatry (open triangles, New South Wales; open diamonds, Victoria). These fish (putative backcrossed hybrids) scattered outside the two species groups but with genotypes more closely aligned with the cluster of pure species A. butcheri.

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Assignment tests allowed us to estimate the proportion of A. butcheri ancestry (q) for each fish. This meant that we could determine both the specific status of each individual and the overall genetic composition of each estuary (i.e. hybrids vs. pure species). We classified 178 and 304 fish as pure A. australis and A. butcheri, respectively, while there were 83 hybrids (15% of the total). Hybrids were found in 68% of estuaries (range of % hybrids per estuary 0–58). However, ∼70% of hybrids (= 58) were in NSW, with the majority of hybrids in Meroo, Coila and Cuttege Lakes (57%). Varying q and the number of loci made no substantive difference to our conclusions. Analyses revealed that for four and eight loci and application of q of 0.1 between 52% and 64% of estuaries supported hybrids and that the range of the percentage of hybrid fish per estuary was 0–42 or 0–56%. Even when applying an extremely relaxed q of 0.2, we still detected hybrids in between 44% and 56% of estuaries, whereas the percentage of hybrids per estuary was between 0–21% and 0–32% (Table S2, online appendix).

Inferring the direction of hybridization

Strikingly few of the 83 hybrids were seemingly simple F1s that displayed both A. australis and A. butcheri alleles at each locus. Instead, most were either later generation hybrids or backcrosses. Most strikingly, however, the genotypes of most of these fish (70 of 83) were more similar to A. butcheri than A. australis (qi > 0.5) implying that backcrossing most often involves the obligately estuarine A. butcheri, although we did identify some 13 hybrids whose genotypes were more similar to A. australis (0.05 < qi < 0.50) (Fig. 3).

image

Figure 3.  Ancestry (qi) of all 565 Acanthopagrus spp. measured as the average (±95% CIs) proportion of A. butcheri genome (based on eight microsatellite loci) (a). Estimates of ancestry between 0 and 0.05, and 0.95 and 1.0 (the lower and upper broken black line), we classed as pure A. australis and A. butcheri, respectively, whereas all others were hybrids. The geographical distribution of hybrids (b); the majority (58/83) were in New South Wales estuaries (closed black squares, Meroo, Coila and Cuttege; open triangles, Forster, Port Hacking and Botany Bay). Hybrids were also present in Queensland (closed circles), Tasmania (open diamonds, Swan and north-west), Victoria (closed grey triangles) and South Australia (open hexagons).

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Inferring the pattern of mating

Examination of the mtDNA haplotypes of the 83 hybrid fish revealed that 95% of fish displayed the mtDNA haplotype of the species with which they shared the greater nuclear DNA similarity, i.e. fish that received 0.95 > qi > 0.5 possessed mtDNA of A. butcheri. The three exceptions were two fish that on the basis of their microsatellite genotypes were A. australis-like hybrids (qi < 0.5) that displayed an A. butcheri mtDNA haplotype and one A. butcheri-like hybrid (qi > 0.5) that displayed an A. australis mtDNA haplotype (all from NSW – Cuttege Lake). For the majority of hybrids, this implies that backcrossing usually involves the estuarine-resident A. butcheri females and either hybrid or A. australis males. Restriction digests of mitochondrial DNA (mtDNA) revealed five composite haplotypes, of which two were specific to fish previously assigned to A. butcheri and three were specific to fish assigned to A. australis. For each species, however, more than 90% of fish displayed only one of these haplotypes (Table S1, online appendix).

Morphological analysis

Although the overall appearance of A. australis and A. butcheri was similar, a PCA plot based on our detailed analysis of 26 morphological variables clearly distinguished two clusters of points corresponding to the species groupings identified using microsatellite data. There was considerable variation amongst individuals of A. australis reflected in a wide scatter of points along axis two. This is not entirely unexpected considering the diversity of environments A. australis inhabit; indeed, our collections encompassed various areas of the lower reaches of estuaries, at the extremes these included the highly marine influenced and exposed estuary bar and training wall, as well as protected seagrass meadows. As might be predicted from the genetic similarity of A. butcheri and the majority of the hybrids, there was little difference in morphology between hybrids and A. butcheri. Based on the PCA plot it would be difficult, perhaps impossible, to separate A. butcheri from their hybrids based solely on morphology (Fig. 4). Unfortunately, there were no A. australis-like hybrids in our collections preventing comparison.

image

Figure 4.  Principal coordinates analysis based on 26 meristic and body ratio measurements used to compare the morphology (closed symbols) of Acanthopagrus spp. (= 40). The ancestry was estimated and each fish was classified as either A. australis (circles), A. butcheri (triangles) or their hybrid (squares, A. butcheri backcrosses or later generation hybrids). The genetic relationship, based on FCA analysis of the eight-locus genotype of all individuals, is also displayed (open symbols).

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Genetic diversity: Acanthopagrus australis, A. butcheri and A. butcheri backcrossed hybrids

We calculated standard measures of genetic diversity for the separate groupings identified with our assignment test. Although the analysis of the microsatellite data set revealed clear allele frequency differences between species, 79 of 147 alleles across the eight loci were common to both species. Acanthopagrus australis was the most diverse with an average (±SE) expected heterozygosity of 0.815 (0.037) compared with 0.610 (0.084) for A. butcheri, and with nearly twice as many alleles per locus. Of 68 private alleles, i.e. alleles unique to a particular taxon, 57 were detected within A. australis. In general, A. butcheri backcrosses had both fewer alleles per locus and less heterozygosity than parental A. australis, but greater numbers of alleles and more heterozygosity than A. butcheri (Table S3, online appendix).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Our study demonstrates the importance of estuaries as sites of hybridization for a pair of migratory coastal and obligately estuarine species. Estuaries provide similar zones of contact for a range of species worldwide (Beheregaray & Sunnucks, 2001; Pampoulie et al., 2004; Watts & Johnson, 2004), leading us to predict that this phenomena is more widespread and is likely to be exacerbated by increased effects of anthropogenic disturbance (Waples et al., 2008). Indeed, this situation may bear remarkable similarity to hybridization and introgression that has recently been described for highly mobile invasive plant species, or introduced freshwater and anadromous fish species, that are impacting on native species through hybridization, with consequent demographic and genetic swamping (Ayres et al., 1999, 2004; Scribner et al., 2000; Allendorf et al., 2004, 2005).

Characterization of the hybrid zone

The combination of microsatellite and mitochondrial DNA markers allowed us to distinguish hybrids from each of their parental species. We were able to demonstrate that the great majority of hybrids are both more closely related to A. butcheri and display A. butcheri mtDNA haplotypes. Although much remains to be determined about the factors that facilitate hybridization in this system, these findings clearly imply that most hybridization and backcrossing involve the obligately estuarine A. butcheri females. This pattern matches what is known of the reproductive synchronicity of these species as ‘ripe’A. australis males are thought to have the potential to overlap the spawning period of A. butcheri, whereas A. australis females are typically ‘spent’ when A. butcheri spawning occurs. The presence of a small number of A. australis-like hybrids with A. australis mitochondrial haplotypes, however, also demonstrates that hybridization can proceed through A. australis females. The limitation of our data set, as is typical of most attempts to formally distinguish different classes of hybrids, is that we cannot with confidence distinguish the majority of F1, backcross and later generation hybrids (see Boecklen & Howard, 1997 for discussion).

Distribution and consequences of hybridization for A. butcheri

As might be expected the hybrids were not evenly distributed over the geographical range of the species, but rather were concentrated within the area of greatest sympatry. Interestingly, within this ‘hot spot’ of hybridization in southern NSW, three estuaries that were most similar in terms of their physical, hydrodynamic and habitat characteristics (NSW DNR, 2008; http://naturalresources.nsw.gov.au/estuaries/inventory/index_ns.shtml) accounted for 57% of the overall number of hybrids detected. Future work will be needed to determine whether the high incidence of hybrids in this zone of greatest species overlap reflects a greater opportunity for hybridization within this area or alternatively whether characteristics of these sites favour hybridization and hybrid survival. As in other hybrid zones, selection favouring certain genotypes or local adaptation to some environmental or ecological factor may play a role in the differential survival of genotypic classes (e.g. Harrison, 1986; Lexer et al., 2003; Gross et al., 2004). In this instance, the apparent A. butcheri backcrosses and/or later generation hybrids appear to be favoured as other classes, such as the putative A. australis backcrosses (i.e. fish that both had their qi based on microsatellites < 0.5 and possessed mtDNA characteristic of A. australis), were detected relatively rarely (= 13) and only at northern sites in strongly marine influenced lower reaches of permanently open estuaries.

In the absence of hybridization, the genetic structure of A. butcheri in NSW might be expected to resemble that of Western Australian populations in that they too would be highly genetically differentiated (FST = 0.17) with populations in different estuaries effectively isolated, self-seeding and potentially highly locally adapted (Chaplin et al., 1998, although see Farrington et al., 2000;Burridge et al., 2004;Burridge & Versace, 2007). However, in the presence of its migratory marine congener, NSW populations have experienced an overwhelming amount of interspecific gene flow and consequent genetic homogenization precluding any formal estimate of gene flow among A. butcheri populations. Indeed, the level of introgression detected in the zone of greatest species overlap is extensive and suggests that genetic and demographic swamping by hybrids is producing localized extinction of populations of the obligately estuarine A. butcheri. It seems less likely that introgression would have a major impact on the genomic integrity of A. australis– as they form a single vast panmictic population (D. G. Roberts, unpublished data) and are not confined to estuaries. Moreover, the majority of hybrids appear to have been formed by mating between A. australis males and A. butcheri females and subsequent backcrossing with A. butcheri. It seems critical that we now uncover the mechanism facilitating hybridization, at a minimum we need to understand if the relatively high incidence in more southern and western populations reflects widespread dispersal of A. australis or hybrids themselves. Moreover, more extensive sampling and temporal replication are needed to determine the persistence or general occurrence of hybridization within the sampled estuaries, and whether the zone of hybridization is stable or expanding.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank the New South Wales Department of Primary Industries for scientific collection permits. James Haddy and Chris Burridge provided Tasmanian and Queensland, and Victorian, South Australian and Western Australian Bream respectively. This work was supported by an Australian Research Council Linkage grant and NSW Recreational Fishing Trust grant to D.J.A., R.J.W. and C.A.G., and the Institute for Conservation Biology, University of Wollongong. Einar Nielsen, Chris Burridge, Dustan Marshall, Cecile Perrin, David Field, Laurance Clarke and Craig Sherman provided comments on an earlier version of the manuscript. We thank two anonymous reviewers for comments. This is contribution number 282 of the Ecology and Genetics Group at the University of Wollongong.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Table S1 Microsatellite allele and mtDNA haplotype frequencies for Acanthopagrus spp. in estuaries of Australia.*

Table S2 The effect of using different numbers of loci to estimate ancestry (qi = the average proportion of Acanthopagrus butcheri genome) and q-threshold values (q = 0.05–0.20) to distinguish pure species and hybrids, on classification (the overall proportion of hybrids) of 565 Acanthopagrus spp. from 25 estuaries. The proportion of estuaries with hybrids, the range of the proportion of hybrids per estuary and the geographic areas with the greatest proportions of hybrids are included.*

Table S3 Number of alleles (A), allelic richness (AR), number of private alleles (alleles unique to a particular group) (PA), observed heterozygosity (Ho) and Nei’s 1973 expected heterozygosity (He) for Acanthopagrus australis (= 178), Acanthopagrus butcheri (= 304) and their hybrids (= 73).

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