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Moore, G.I. and Chaplin, J.A. (2013) Population genetic structures of three congeneric species of coastal pelagic fishes (Arripis: Arripidae) with extensive larval, post-settlement and adult movements. Environmental Biology of Fishes, 96 (9). pp. 1087-1099.
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1 Population genetic structures of three congeneric species of coastal pelagic fishes (Arripis:
2 Arripidae) with extensive larval, post-settlement and adult movements.
3
4 G.I. Moore1,2 and J.A. Chaplin1
5
6 1 Centre for Fish and Fisheries Research, School of Biological Sciences and Biotechnology, Murdoch
7 University, South St, Murdoch, Western Australia, Australia, 6150.
8 2 Fish Section, Department of Aquatic Zoology, Western Australian Museum, 49 Kew St., Welshpool,
9 Western Australia, Australia, 6106.
10
11 Author to whom correspondence should be addressed:
12 G.I. Moore
13 e-mail: [email protected]
14 phone: +61 08 9212 3744 15
1 16 Abstract
17
18 The population genetic structures of three congeneric coastal pelagic marine fishes (Arripis trutta, A.
19 truttaceus and A. georgianus) were investigated to determine whether these structures were consistent
20 with the apparently high gene flow life histories of these species. This investigation used fragment
21 length polymorphisms at two to four nDNA intron loci (amplified by EPIC-PCR) in samples of each
22 species collected from across their entire Australian distributions. The results revealed no evidence of
23 genetic subdivision in any species based on an analysis of variation either across samples (with
24 multilocus FST values ranging from effectively zero to 0.005), or between pairs of samples. These
25 findings, when considered in combination with other available evidence, are consistent with the view
26 that each of these species represents a rare example of a coastal fish species that is genetically
27 homogeneous over a vast area, including the entire geographic range (A. truttaceus and A. georgianus)
28 or Australian distribution (A. trutta).
29
30 Keywords: EPIC-PCR, Leeuwin Current, East Australian Current, migratory, high gene-flow,
31 Australian Salmon
2 32 Introduction
33 It was often traditionally assumed that coastal pelagic marine fishes exhibit genetic homogeneity, and
34 possibly even panmixia (random mating), over their geographic distributions (see DeLigny &
35 Pantelouris 1973; Waples 1987; Ward et al. 1994; Hauser & Ward 1998 for discussion). However,
36 most genetic studies of coastal fishes with a seemingly highly dispersive life history have detected the
37 presence of population subdivision (e.g. Gold et al. 2002; Knutsen et al. 2003; Rohfritsch & Borsa
38 2005; Ruzzante et al. 2006; Atarhouch et al. 2007; Chairi et al. 2007; Chlaida et al. 2008; Wang et al.
39 2008; Andre´ et al. 2011, but see Hoolihan et al. 2006; Cárdenas et al. 2009; Palm et al. 2009). Indeed,
40 in view of the results of recent genetic studies, it has been argued that panmixia is unexpected even in
41 widespread fish species that are distributed across different environments (Als et al. 2011).
42
43 Pelagic species with a life history that involves high dispersal potential at every life stage (larval,
44 juvenile and adult) could be expected to be the most likely candidates to exhibit extensive gene flow,
45 generating a considerable homogenising effect, if not panmixia (Waples 1987; Ward et al. 1994; Siegel
46 et al. 2003). Although this type of life history is uncommon among coastal fishes (Gillanders et al.
47 2003), members of the family Arripidae (Australian Salmon) exhibit impressive vagility throughout
48 their lives. In fact, few coastal marine fishes appear to share the dispersal potential of Arripis species
49 (Ward et al. 1994; Gillanders et al. 2003) and A. truttaceus, in particular, may well have the greatest
50 documented migration from juvenile to adult distribution of any marine fish (Gillanders et al. 2003).
51
52 The family Arripidae is endemic to temperate Australasian seas and contains a single genus Arripis,
53 and four currently recognised species. In Australian mainland waters, the family is represented by the
54 Eastern Australian Salmon Arripis trutta (Bloch & Schneider 1801), the Western Australian Salmon
55 Arripis truttaceus (Cuvier 1829) and the Australian Herring Arripis georgianus (Valenciennes 1831).
56 The latter two species are endemic to these waters, while the former also occurs in New Zealand waters
57 (Paulin 1993). A fourth species, the Northern Kahawai Arripis xylabion Paulin 1993 occurs around
58 islands north of New Zealand (and possibly also in New Zealand waters), but is not included in the
59 present study. All species are targeted in long-term, low financial value recreational and commercial
60 fisheries and there is a long research history for each in the context of fisheries management (e.g.
61 Fairbridge 1950; Malcolm 1960; Nicholls 1971; Lenanton & Hall 1976; Stanley 1988a; Cappo et al.
62 2000; Fairclough et al. 2000a; Stewart et al. 2011).
3 63
64 The three Australian species of Arripis have complex life histories involving impressive migrations
65 (see Fig. 1). For example, adult A. truttaceus are distributed around the south-west corner of Australia.
66 Breeding adults migrate to, and concentrate in, the western-most parts of this area to spawn (Malcolm
67 1960; Stanley 1988b). The planktonic larvae are then transported hundreds or thousands of kilometres
68 to the south-east by the Leeuwin Current and wind-generated water flow (Petrusevics & Bye 1995;
69 Caputi et al. 1996). Post-settlement and first-year A. truttaceus occur along much of the southern coast
70 of Australia but are most abundant in south-eastern Australian waters (Malcolm 1960). Juvenile and
71 sub-adult fish migrate progressively westward, and enter the adult distribution in their fourth year of
72 life (Malcolm 1960; Hoedt & Dimmlich 1994). The migratory life history of A. trutta reflects that of
73 A. truttaceus, except that the individuals follow a north-south route along the east coast of Australia
74 (Stanley & Malcolm 1977; Stanley 1988c; Stewart et al. 2011; Fig. 1). Likewise, the migratory life
75 history of A. georgianus is very similar to that of A. truttaceus (Fig. 1), except that A. georgianus may
76 have more local larval retention (Lenanton 1982; Fairclough et al. 2000b), and individuals enter the
77 adult distribution at two years of age (Fairclough et al. 2000a).
78
79 On the basis of possessing a high dispersal potential at every life stage, it is tempting to hypothesize
80 that the species of Arripis provide some rare examples of coastal pelagic species that are genetically
81 homogeneous over their entire range (see Waples 1987; Ward et al. 1994; Siegel et al. 2003). The only
82 published test of this hypothesis was an allozyme-based study of the population structure of A.
83 georgianus that was conducted by Ayvazian et al. (2004), as part of a multi-disciplinary stock
84 assessment of this species. Ayvazian et al. (2004) found no evidence of population genetic subdivision
85 in this species, but based on a test with very limited power due to a very low level of allozyme
86 polymorphism. There is clearly a need to find more polymorphic markers to improve the power of the
87 analyses and to study the population genetics of the other Arripis species.
88
89 The present study used nDNA markers in the form of fragment length polymorphism at several intron
90 loci to investigate the population genetic structures of Arripis species. These polymorphisms are
91 assayed using ‘universal’ primers in exon-primed, intron-crossing PCR (EPIC-PCR, Palumbi and
92 Baker 1994) and have been used to investigate population and evolutionary questions in other fishes
93 and also invertebrate species (e.g. Bierne et al. 2000; Berrebi et al. 2005; Atarhouch et al. 2007;
4 94 Fauvelot et al. 2007; Rolland et al. 2007; Li et al. 2010; Boissin et al. 2011). This type of nDNA
95 marker was used because it provides many of the advantageous qualities of microsatellite markers (e.g.
96 highly polymorphic, single locus, co-dominant markers; e.g. Li et al. 2002), but avoids the time and
97 cost required to developed customised primers, as would have been the case for a microsatellite-based
98 study. This latter point is particularly relevant in the context of this study because the species of
99 Arripis are of limited financial value and have a relatively low profile from a conservation perspective,
100 and hence attracting funding for molecular work is difficult. In addition, there is currently no published
101 information on microsatellite loci in the Arripidae and, given the uncertainty of the systematic position
102 of this family (e.g. Johnson and Fritzche 1989; Yagishita et al. 2009), there are no obviously related
103 groups to target for cross-species transfer of microsatellite primers (see Selkoe & Toonen 2006).
104 Furthermore, transferability of microsatellite loci between fish families is generally poor (e.g. Barbará
105 et al. 2007).
106
107 The objective of this study was to use length frequency polymorphisms at intron loci to investigate the
108 population genetic structures of Arripis trutta, A. truttaceus and A. georgianus, across their entire
109 ranges in Australian mainland waters. This was done to determine whether these structures are
110 consistent with the hypothesized high gene-flow life histories of these species.
111
112 Materials and Methods
113
114 Sample Collection
115
116 For each species, the collection locations were distributed across their Australian range (Fig. 1). For A.
117 trutta, 29 – 30 individuals were sampled from each of three locations in 2007. For A truttaceus, 13 –
118 45 individuals were sampled from each of five locations in 2007 or 2008, while for A. georgianus 9 –
119 44 individuals were collected from each of seven locations in 2006 or 2008 (see Table 2). For each
120 species, a combination of juveniles and adults was sampled.
121
122 Genetic Assays
123 Total genomic DNA was isolated from approximately 5 mg of muscle tissue from Arripis individuals
124 using a MasterPureTM extraction kit according to the manufacturer’s instructions.
5 125
126 Fragment length polymorphism at two to four intron loci, depending on the species, was assayed. The
127 loci were: Aldolase B, intron 4 (AldoB4); Calmodulin, intron 4 (CaM4); S7 ribosomal protein gene,
128 intron 2 (S72), and Creatin kinase (CK7). The loci were amplified by EPIC-PCR using published
129 primers (from Chow & Hazama 1998; Chow & Takeyama 1998; Hassan et al. 2002). These four loci
130 were chosen from a pool of 28 intron loci because, in preliminary trials, they were the only loci that
131 amplified and exhibited polymorphism in at least one species of Arripis (Moore 2012).
132
133 PCR reactions were carried out in a GeneAmp 9700 Thermal Cycler (Applied Biosystems) with: (i) an
134 initial denaturation phase of 5 min at 94˚C, (ii) 38 amplification cycles, with each cycle consisting of
135 30 s of denaturation at 94°C, 30 s of annealing at optimised temperatures (Table 1), 30 s of extension at
136 72°C, and (iii) a final 20 min extension at 72°C (to standardise poly-A additions). The only exceptions
137 were: (i) the locus CK7 in A. georgianus, which was amplified using a touch-down PCR of 57
138 amplification cycles, with annealing starting at 58°C for 60 s and decreasing by 0.3°C and 1 s each
139 cycle; and (ii) the locus CaM4 in A. georgianus, which was amplified using a touch-down PCR of 50
140 amplification cycles, with annealing starting at 65°C for 60 s and decreasing by 0.4°C each cycle
141 (Table 1). The PCR reaction mixture contained 0.1 mM of each dNTP (Promega), 0.25U Taq DNA
142 polymerase (Roche), 1.5 µl Taq Buffer (100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3;
143 Roche), 0.04-0.06 µM of each primer (Table 1; forward primers were fluorescently labelled), and
144 approximately 10 ng of DNA template, adjusted to a volume of 15 µl with PCR grade H2O.
145
146 PCR products were assayed either singularly or multiplexed for one large and one small locus, using
147 optimised volumes for each locus (Table 1). Fragment length was determined with an ABI 3730
148 Automated Sequencer (Applied Biosystems) using LIZ1200 (Applied Biosystems) as the size standard.
149 Allele peaks were manually scored to one decimal place using the software GENEMARKER v. 1.71
150 (SoftGenetics Inc.). The data points for the allele scores of all individuals of all species at a locus
151 grouped into a series of discrete clusters, with variability around each allele and gaps between each
152 allele cluster, as is typical for automated sizing of DNA fragments (see Idury & Cardon 1997; Anon.
153 2004). Each allele was named by the modal integer of the associated cluster of data points. It is
154 possible that some similar but different sized alleles have inadvertently been placed into the same allele
6 155 cluster. Both positive (individuals for which the genotype was already known) and negative (no added
156 DNA template) controls were included in assays.
157
158 Data Analyses
159 The program MICRO-CHECKER (v. 2.2.3; van Oosterhout et al. 2004) was used to test for the
160 presence of null alleles at each locus in each sample of each species. For each species, the number of
161 alleles per locus (NA), observed heterozygosity (HO) and expected heterozygosity (HE, Nei 1987) were
162 calculated for each sample and over all samples. Exact tests were used to assess departures from
163 Hardy-Weinberg Equilibrium (HWE) conditions at each locus in each sample. Fisher’s exact
164 probability tests were used to test for departures from linkage disequilibrium for each pairwise
165 combination of loci, in each sample of each species. An exact chi-square test with Fisher’s combined
166 probability method was used as a global test of linkage disequilibrium for all samples within a species.
167 The above exact tests were conducted in GENEPOP v. 4.0 (Raymond & Rousset 1995) with 10,000
168 dememorisation steps, 1,000 batches and 10,000 iterations. Where appropriate, a sequential Bonferroni
169 correction (Rice 1989) was applied to control for group-wide Type I error.
170
171 For each species, single-locus FST estimates were made using FSTAT v. 2.9.3.2 (Goudet 1995),
172 assuming samples in HWE and using 10,000 permutations to test statistical significance. The value of
173 FST over all loci was calculated using the software GENETIX v. 4.05.2 (Belkhir et al. 2004) and again
174 using 10,000 permutations to test the statistical significance. Pairwise FST tests between samples
175 within each species were conducted in FSTAT, with statistical significance determined as above.
176 Finally, the factorial correspondence analysis (FCA) of GENETIX was used to search for structure in
177 the multilocus genotype data, based on individuals rather than samples, following Guinand (1996).
178
179 A retrospective power analysis was conducted with 1000 runs using the method described by Theisen
180 et al. (2008) and the software POWSIM v. 4.0 (Ryman & Palm 2006). Briefly, this involved simulated
181 populations that were allowed to drift to various levels of genetic divergence (quantified by FST, as a
182 product of the number of generations, t, and population size, Ne). Power was determined by finding
183 the least number of generations of drift that could be detected among the sampled populations of each
184 species with a power ≥ 95%, while holding the population size constant at 1000. This analysis only
185 included the individuals of a species that were typed for all polymorphic loci for that species (A. trutta
7 186 N = 82, A. truttaceus N = 119, A. georgianus N = 188), because this software cannot accept different
187 sample sizes for different loci within a sample.
188
189 Results
190
191 The AldoB4 and S72 loci were polymorphic in all three species, while CK7 was polymorphic only in
192 Arripis trutta and A. georgianus and CaM4 was polymorphic only in A. georgianus (Table 2). The
193 alleles ranged in size from 169-191 bp at locus AldoB4, from 469-504 bp at CaM4, 844-984 bp at CK7
194 and 816-931 bp at S72 (Table 2; details in Moore 2012). The overall level of polymorphism was
195 greatest in the samples of A. georgianus (four polymorphic loci, with a total of 32 different alleles),
196 followed by those of A. trutta (three polymorphic loci, with a total of 19 different alleles), while those
197 of A. truttaceus contained only two polymorphic loci, with 7 different alleles (Table 2). Mean expected
198 heterozygosity per sample per locus, when the locus was polymorphic in the sample, was typically low
199 to moderate (e.g. between 0.083 and 0.655; Table 2). The results from MICRO-CHECKER revealed
200 no evidence for the presence of null alleles or large allele dropout at any locus in any sample in any
201 species. In fact, only a single departure from HWE was detected (locus CK7, HO sample of A.
202 georgianus) and even this was not statistically significant following Bonferroni correction (Table 2).
203 No cases of significant linkage disequilibrium were detected, after Bonferroni correction, for any pair
204 of loci in any sample of any species, suggesting independent loci (data not presented).
205
206 Within each species, single locus estimates of (global) FST were consistently low (Table 3).
207 Accordingly, the (global) multi-locus values of FST for each species were also low (e.g. ≤ 0.005) and
208 not significantly different from that expected by chance (Table 3). Similarly, the values of FST
209 between each pair of samples within each of the three study species were invariably low (e.g.
210 effectively zero for A. trutta, FST < 0.018 for A. truttaceus, and FST < 0.030 for A. georgianus) and
211 none were significantly different from that expected by chance (Table 4). For each species, no spatial
212 patterns were discernable in the FCA, as there was little to no clustering of individuals from the same
213 collection site in any species and the distribution of the multi-locus genotype data was not strongly
214 explained by either axis (Fig. 2).
215
8 216 Power analyses suggested that genetic divergence would be detected with ≥ 95% confidence, at least at
217 FST ≥ 0.034 for A. trutta (Ne = 1000, t = 70), FST ≥ 0.039 for A. truttaceus (Ne = 1000, t = 80) and FST
218 ≥ 0.009 for A. georgianus (Ne = 1000, t = 20).
219
220 Discussion
221
222 This research did not reveal any evidence of genetic structuring in any of Arripis trutta, A. truttaceus or
223 A. georgianus in Australian waters. For one or more species, this could potentially be due to: (i) errors
224 in the genetic data; (ii) inadequate sampling; and/or (iii) the effects of selection and/or (iv) inadequate
225 genetic data or (v) no genetic structure. Each of these alternatives is evaluated below. As is explained,
226 when the genetic data are considered along with other evidence, alternative (v) seems particularly
227 likely.
228
229 Errors in the genetic data. There was no evidence of methodological errors, such as PCR artefacts,
230 impacting on the Arripis genetic data sets and regardless most such errors typically produce
231 heterogeneity rather than homogeneity in genetic data (Pompanon et al. 2005). The conservative
232 approach to allele scoring (see Methods) could conceivably have masked some variation through a
233 failure to discriminate alleles of similar but different size. However, the adopted approach was
234 preferable to other methods of allele scoring of fragment length variation at intron loci where, for
235 example, arbitrary size categories have been imposed on the data (e.g. Gomulski et al. 1998; Berrebi et
236 al. 2005; Hubert et al. 2006; Atarhouch et al. 2007).
237
238 Inadequate sampling. The results were based on samples obtained from across virtually the entire
239 Australian distribution of all three study species. Thus, it seems highly unlikely that any genetic
240 differentiation in any of the species in Australian waters was missed due to, for example, sampling all
241 individuals from a single, localised breeding unit within a subdivided species (see Page and Holmes
242 1998).
243
244 Extensive sampling for temporal or cohort effects (see Johnson and Black 1984; Hedgecock 1994) was
245 not undertaken, partly because preliminary analyses revealed no evidence of significant spatial genetic
9 246 heterogeneity in any of the three study species. In addition, limited testing found no evidence that
247 these factors had any effect on genetic variation in A. georgianus (Moore 2012).
248
249 Selection. Although uniform selection can maintain genetic homogeneity across a species’ range (see
250 Ehrlich and Raven 1969; Lamy et al. 2011), it is unlikely to account for the absence of genetic structure
251 at multiple, independent intron loci (see Avise 2000; Hare 2001; but see Amos and Harwood 1998), as
252 was observed in the Arripis species.
253
254 Inadequate genetic data.
255 Resolving the finer details of population genetic structure in high gene-flow situations, such as in the
256 arripids, is difficult (see Shaklee & Currens 2003). Evidence in support of the hypothesis of genetic
257 homogeneity is relatively strong for A. georgianus, where, for example, the results of the power
258 analysis indicate that the current data set could detect modest levels of genetic subdivision (FST ≥
259 0.009). Furthermore, a prior allozyme study, based on four loci, similarly failed to detect any evidence
260 of genetic subdivision in this species (based on 646 individuals sampled from 11 sites between Victoria
261 and Western Australia; Ayvazian et al. 2004), although the levels of polymorphism at the allozyme loci
262 were admittedly very low. The number of polymorphic intron loci assayed for A. trutta and A.
263 truttaceus was too small to provide compelling evidence of a lack of structure for either species.
264 However, in this context, it is worth noting that an unpublished study of variation at the esterase locus
265 revealed no evidence of subdivision in the latter species (based 580 individuals from nine sites between
266 Victoria and Western Australia; MacDonald 1980). There are no prior population genetic studies for A.
267 trutta in Australian waters. Furthermore, all Arripis species, particularly A. truttaceus, exhibit
268 unusually low levels of polymorphism genome wide (Ayvazian et al. 2004; MacDonald 1980; Moore
269 2012), making it unusually difficult to find sufficient polymorphic markers to test a hypothesis of
270 genetic homogeneity with a high degree of power (see Moore 2012).
271
272 No genetic structure.
273 The results of this study are consistent with the hypothesis that the amount of gene flow across the
274 entire and vast Australian distribution of each Arripis species is sufficient to prevent genetic
275 structuring. This was hypothesised in view of the high migratory life-history of these species (see
276 Introduction). While additional genetic data are required to confirm this interpretation (see above), the
10 277 genetic results are consistent with the results of studies based on other types of evidence, specifically
278 otolith microchemistry and tagging, as follows.
279
280 Otolith microchemistry analyses have provided evidence of extensive movements and mixing of
281 individuals in both A. trutta and A. georgianus (Ayvazian et al. 2004; Hughes et al. 2011). Using
282 analysis of δ18O and δ13C from 559 fish across 10 sites between Adelaide (South Australia) and
283 Dongara (Western Australia), Ayvazian et al. (2004) found that, in general, the sampled individuals of
284 A. georgianus had not spent their entire lives in the waters in which they were captured. This is in
285 contrast to a range of other species with similar geographic distributions (e.g. Edmonds and Fletcher
286 1997; Edmonds et al. 1999). Similar conclusions were reached for A. trutta by Hughes et al. (2011)
287 based on 115 individuals sampled across eight sites between northern New South Wales and Tasmania.
288 Elemental signatures (Li, Na, Mg, Mn, Sr, Ba) were strongly influenced by age, reflecting the extensive
289 movements and geographical age stratification of maturing fish. The data from both studies supported
290 the idea that A. trutta comprised a single, well-mixed population. Both studies also reported limited
291 evidence of partial residency in some populations, but not in sexually mature fish. No otolith
292 microchemistry studies have been conducted on A. truttaceus.
293
294 Tagging studies have also demonstrated a progressive age-based movement of maturing fish from
295 nursery areas in the south-east to respective adult distributions along the east coast (A. trutta) and the
296 south-west coast (A. truttaceus and A. georgianus) of Australia. These studies, which were based on
297 hundreds of tagged fish, also showed that adults of each species make multi-directional movements
298 within their adult distributions with no evidence for site-fidelity or a return to nursery areas (e.g.
299 Stanley 1986; 1988a; 1988b; 1988c; Ayvazian et al. 2004). The conclusions from all of these studies
300 were that each of the species represents a single homogenous population.
301
302 The results of this study add to a small list of coastal pelagic species that could be genetically
303 homogeneous and possibly even panmictic over a vast distance (e.g. see Hoolihan et al. 2006; Cárdenas
304 et al. 2009; Palm et al. 2009). It is also noteworthy that the results were effectively the same for the
305 eastern and western sibling-species of Australian Salmon (A. trutta and A. truttaceus, respectively),
306 indicating that environmental conditions on both the east and west coasts of Australia are similarly
307 conducive to high gene flow in these species. Three factors appear to be critical in the high gene flow
11 308 life history of Arripis. Firstly, almost all of the spawning individuals of each species converge on a
309 relatively spatially-restricted area over a short time-frame, which will facilitate random mating (see
310 also White et al. 2009). Secondly, pelagic spawning is closely coincident (and possibly linked) with
311 oceanographic conditions, especially major currents. Specifically, spawning in both A. truttaceus and
312 A. georgianus occurs in south-western Australia, peaking in April and May when the south-east-
313 flowing Leeuwin Current is at its strongest (Caputi et al. 1996), while spawning in A. trutta occurs in
314 eastern Australia, peaking in January when the south-flowing East Australian Current is strongest
315 (Stewart et al. 2011). Thirdly, there is a progressive migration of early life history stages back to the
316 adult distribution, which presumably provides an extensive homogenising effect in all species of
317 Arripis (see also Ward et al. 2003).
318
319 Conclusions.
320 Firmly proving or disproving a hypothesis of genetic homogeneity in a marine species is difficult, as is
321 exemplified by a series of studies of the European Eel (Anguilla anguilla), which have variously
322 proposed and rejected a model of homogeneity (see DeLigny & Pantelouris 1973; Daemen et al. 2001;
323 Wirth & Bernatchez 2001; Maes & Volckaert 2002; Lintas et al. 1998; Dannewitz et al. 2005; Palm et
324 al. 2009; Als et al. 2011). This study provides the strongest evidence to date that, as predicted, each of
325 Arripis trutta, A. truttaceus and A. georgianus is genetically homogenous in Australian waters.
326 Information from additional genetic markers is, however, required to improve the power of the analysis
327 of the population genetic structures of these species.
328
329 Acknowledgements
330
331 We are grateful to E. Sezmiş, N. Philips, F. Brigg and M. Hale for valuable advice on many aspects of
332 this work. We thank S. Hoeksema, B. Chuwen, D. Wheatcroft, K. Armstrong, J. Stewart, J. Hughes
333 and P. & M. Selim (Selim Fisheries) for assistance in sourcing specimens. This work complies with
334 the current laws of Australia and was conducted under Murdoch University Animal Ethics Approval
335 W1199/2006. Funding was provided by Murdoch University and the Centre for Fish and Fisheries
336 Research, Murdoch University.
337
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20 558 Fig. 1 Sampling sites and generalised map of the distribution and movements of Arripis trutta
559 (dashed arrows), A. truttaceus (black arrows), and A. georgianus (grey arrows) in Australian waters.
560 ‘I’ indicates presumed larval dispersal, including some local retention; ‘II’ represents direction of
561 movement of juvenile fish; ‘III’ represents movements of sub-adult fish; and ‘IV’ represents adult
562 distribution and movements. Sample sites are indicated with a species prefix: A. trutta (E), A.
563 truttaceus (W) and, A. georgianus (H), and location suffix: Fremantle (F), Canal Rocks (C), Augusta
564 (A), Yeagarup (Y), Peaceful Bay (P), Irwin Inlet (I), Oyster Harbour (O), Albany (L), Wellstead
565 Estuary (W), in Western Australia; The Coorong, South Australia (S); Flinders Island, Tasmania (T);
566 Lakes Entrance, Victoria (V); Port Stephens, New South Wales (N) 567
21
568 Fig. 2 Factorial correspondence analysis (FCA) illustrating the lack of a strong relationship
569 between the multi-locus genotypes and collection sites of individuals of Arripis trutta (top), A.
570 truttaceus (middle) and A. georgianus (bottom). The total variation explained by each axis is given as
571 a percentage. Sample codes are as per Fig. 1. For scaling purposes, outliers have been shifted and their
572 correct position indicated by an arrow and coordinates. Note, many points overlap and are obscured 573
22
574 Table 1 PCR and plate loading conditions for assays for polymorphic intron loci in the three
575 species of Arripis. In each case, the optimised annealing temperature and primer concentration used in
576 PCR reactions is given, along with the volume of PCR product loaded onto plates for fragment
577 analysis. A dash (-) indicates that the locus was monomorphic for a species, and not included in the
578 present study. An asterisk (*) indicates touchdown PCR
579
Arripis trutta Arripis truttaceus Arripis georgianus Anneal. Primer Load Anneal. Primer Load Anneal. Primer Load Locus temp. conc. vol. temp. conc. vol. temp. conc. vol. (°C) (µM) (µl) (°C) (µM) (µl) (°C) (µM) (µl)
AldoB4 56 0.04 2.0 56 0.04 2.0 57 0.04 0.5
Cam4 - - 65-45* 0.04 1.0
CK7 53 0.06 3.5 - 58-41* 0.04 2.0
S72 62 0.06 2.0 63 0.06 2.5 64 0.06 2.5 580
23 581 Table 2 Characteristics of four intron loci for samples of N individuals of three species of Arripis.
582 NA is the number of alleles for each sample and for the species (in parentheses). The range of allele
583 sizes for each locus is given for each species (in bp). HE and HO are expected and observed
584 heterozygosity, respectively. HWE(p) is the probability of a Type 1 error in exact tests for departures
585 from Hardy-Weinberg equilibrium expectations. Collection sites are ordered from the most northerly
586 to the most south-easterly (see Fig. 1). A dash (-) indicates that the locus was monomorphic for a
587 sample. An asterisk (*) indicates not significant after Bonferroni correction. ‘n/a’ indicates that a p-
588 value could not be calculated because only 2 alleles were present in the sample, one of which was
589 represented by a single copy
24 Arripis trutta Arripis truttaceus Arripis georgianus EN EV ET WA WY WL WW WS HF HC HP HI HO HW HS AldoB4 (170-191 bp) (178-182 bp) (170-182 bp) N 29 30 30 30 15 39 9 37 44 39 36 23 28 9 41
N 5 3 3 (6) 2 2 2 2 2 (2) 5 2 4 4 3 3 4 (5) A HE 0.419 0.347 0.326 0.480 0.391 0.474 0.345 0.382 0.316 0.120 0.305 0.361 0.194 0.291 0.159
HO 0.414 0.433 0.333 0.667 0.400 0.513 0.222 0.405 0.295 0.128 0.250 0.348 0.214 0.333 0.171 HWE(p) 0.33 0.45 1.00 0.06 1.00 0.74 0.34 1.00 0.50 1.00 0.43 0.71 1.00 1.00 1.00
CaM4 (477 bp) (477 bp) (469-504 bp) N monomorphic monomorphic 42 39 36 23 28 8 40
NA 5 5 3 3 3 2 6 (8)
HE 0.177 0.146 0.107 0.083 0.223 0.116 0.228
HO 0.190 0.154 0.111 0.087 0.179 0.125 0.200 HWE(p) 1.00 1.00 1.00 1.00 0.35 n/a 0.48
CK7 (943-984 bp) (952 bp) (844-976 bp) N 28 28 29 monomorphic 42 38 35 23 26 8 39
NA 3 4 3 (4) 6 7 4 3 2 1 6 (11)
HE 0.283 0.197 0.273 0.278 0.174 0.209 0.198 0.142 - 0.279
HO 0.321 0.214 0.321 0.214 0.184 0.229 0.217 0.154 - 0.231 HWE(p) 1.00 1.00 1.00 0.20 1.00 1.00 1.00 1.00 - 0.13
S72 (816-931 bp) (892-931 bp) (871-919 bp) N 29 25 28 27 15 38 13 45 32 36 34 23 26 8 36
NA 6 7 4 (9) 4 3 4 4 5 (5) 6 5 4 3 3 3 5 (8)
HE 0.655 0.594 0.535 0.444 0.558 0.465 0.426 0.579 0.466 0.450 0.286 0.294 0.272 0.531 0.336
HO 0.552 0.480 0.679 0.333 0.467 0.474 0.462 0.667 0.406 0.528 0.324 0.261 0.154 0.750 0.333 HWE(p) 0.31 0.31 0.51 0.12 0.56 1.00 0.43 0.24 0.44 1.00 1.00 0.52 0.01* 0.70 0.63
25 590 Table 3 Single locus and multilocus tests of population differentiation in each Arripis species.
591 FST values are given along with the probability of obtaining, by chance, a value as large as or larger
592 than the observed FST from 10,000 permutations. A dash (-) indicates that the locus was monomorphic
593 for a species
594
Arripis trutta Arripis truttaceus Arripis georgianus FST p-value FST p-value FST p-value (i) Single-locus AldoB4 -0.010 0.34 0.011 0.23 0.007 0.14 CaM4 - - - - -0.004 0.98 CK7 -0.006 0.52 - - -0.009 0.26 S72 -0.002 0.63 0.001 0.42 0.005 0.48
(ii) Overall -0.005 0.73 0.005 0.22 0.005 0.10 595
26 596 Table 4 Pairwise FST (below diagonal) and associated p-values (above diagonal) for samples of
597 Arripis trutta, A. truttaceus and A. georgianus. p-values are the probability of obtaining, by chance, a
598 value as large as or larger than the observed FST from 10,000 permutations. Sample codes are as per
599 Fig. 1
600
Arripis trutta EN EV ET EN 0.27 0.32 EV -0.004 0.97 ET -0.000 -0.013
Arripis truttaceus WA WY WL WW WS WA 0.40 0.83 0.49 0.25 WY 0.009 0.11 0.22 0.79 WL -0.011 0.012 0.76 0.08 WW 0.008 -0.011 0.000 0.32 WS 0.018 -0.019 0.018 -0.006
Arripis georgianus HF HC HP HI HO HW HS HF 0.35 0.90 0.34 0.37 0.87 0.72 HC 0.003 0.39 0.14 0.38 0.61 0.90 HP 0.005 0.012 0.92 0.68 0.24 0.73 HI 0.010 0.019 -0.013 0.30 0.07 0.28 HO 0.010 0.005 -0.005 -0.000 0.19 0.54 HW -0.016 0.000 0.029 0.030 0.026 0.42 HS 0.004 -0.002 0.002 0.009 -0.007 0.018 601
27