Marine species with relatively low migratory capacity are threatened by habitat alterations derived from human activities. In November 2002 the tanker Prestige sank off the Spanish northwest coast releasing 70,000 tons of fuel and damaging biota in the area. Despite efforts to clean the damaged areas, fuel remnants have affected marine species over the last nine years. This study is focused on two flatfish, Lepidorhombus boscii (four-spotted megrim) and L. whiffiagonis (megrim), whose spawning areas are located at the edge of the continental platform. We have analyzed megrim samples from North Spanish and French waters obtained before and after the oil spill. Genotypes at the nuclear marker 5S rDNA indicate a significant increase in interspecific hybridization after the Prestige accident, likely due to forced spawning overlap. The mitochondrial D-Loop region was employed for determining the direction of hybrid crosses, which were most frequently L. boscii female x L. whiffiagonis male. Reduced ability of L. boscii females to select conspecific mates would explain such asymmetric hybridization. To our knowledge this is the first time that increased hybridization between fish species can be associated to an oil spill. These results illustrate the potential long-term effect of petrol wastes on wild fish species.
Citation: Crego-Prieto V, Martinez JL, Roca A, Garcia-Vazquez E (2012) Interspecific Hybridization Increased in Congeneric Flatfishes after the Prestige Oil Spill. PLoS ONE 7(4): e34485. https://doi.org/10.1371/journal.pone.0034485
Editor: Howard Browman, Institute of Marine Research, Norway
Received: November 28, 2011; Accepted: March 5, 2012; Published: April 12, 2012
Copyright: © 2012 Crego-Prieto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by the Spanish National project MICINN CGL2009-08279. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Many human activities endanger survival of marine species. For example, wild populations are threatened by high ship traffic , overfishing  and many others, such as oil spill accidents –, whose long term consequences have not yet been evaluated. Oil spills cause severe damage to marine wildlife due to polycyclic aromatic hydrocarbons (PAHs) released from fuel remnants causing oxidative stress, tissue alterations and cell death among other injuries by increasing oxygen-derived free radicals , ( and references therein).
The Prestige oil spill occurred off the Galician coast (Northwest of Spain; Fig. 1) on 13th of November 2002, and was classified as one of the worst ecological catastrophes of the century in Europe . The Prestige tanker carried about 77,000 tons of fuel type M-100 (one of the most toxic petroleum derivatives), of which 20,000 tons were dumped directly into the sea at the time of the accident affecting an area of 30,000 km2 . From then until the summer of 2003, 40,000 more tons were spilled  affecting marine communities of the Cantabric Sea and the Bay of Biscay along 2,600 coastal km , reaching the oyster farms in the Bay of Arcachon (France). Oil pollution caused a great damage to both marine biodiversity and the local economy . Eight years after the disaster there were still at least 202 beaches contaminated by fuel in Galicia, and many others in Asturias, Cantabria and the Basque Country (www.wwf.panda.org).
Many marine vertebrates were affected by the oil spill including birds, mammals and fish ,  and a review by . Due to the high density of the fuel, it accumulated on the sandy bottom of the continental shelf. Species that swim and live on the bottom where the fuel accumulated were consequently most affected by the oil spill , , . Previous studies carried out in the Mediterranean Sea demonstrated that the megrim L. boscii is a very sensitive species to PAHs exposure , and it was also especially affected by Prestige PAHs, which altered its DNA integrity and increased levels of stress and genotoxicity biomarkers .
L. boscii and its congeneric sympatric species L. whiffiagonis (Scophthalmidae, Pleuronectiformes) are distributed in the Atlantic Ocean from Iceland to Cape Bojador (26°N), and in the Mediterranean Sea. Both species spawn on the continental shelf from March to June. Little is known about the biology of megrims populations. Sanchez et al.  suggested low migratory capacity of both megrim species with aggregation and disaggregation movements. Surveys have shown that larvae do not move much from their spawning sites during the first year of life . Spatial genetic differences have been described within their Atlantic area of distribution, megrims inhabiting the Bay of Biscay, Cantabric Sea, Galician and Portuguese coasts belonging to the same population cluster in the two species . Studies on juveniles have been focused on development and growth patterns , , , but megrims' spawning areas have not been studied in depth and the reproductive barriers between the two species are unknown. The age at first maturity is 1.5 years for L. boscii  and two years could be reasonably considered their generation time; therefore approximately four generations (nine years) were affected since 2002 when the Prestige sank. The oil spill was quickly displaced during the 2002–2003 winter and in to a lesser extent until spring and summer 2003  by both surface and deepwater currents, and fuel was deposited on the seabed, most likely reducing suitable megrim spawning areas and thus increasing reproductive interactions between the two species. Furthermore, the seabed was not cleaned up and the oil deposited likely has continued to affect megrim spawning since the spill until the present day. Interspecific hybridization can follow habitat alterations . Our hypothesis is that the habitat alteration due to the Prestige accident has forced the two megrim species towards a closer interaction due to the reduction of “clean” spawning areas, especially L. boscii because of its higher sensitivity to fuel toxicity  and maybe altering their mating behaviour. As a consequence of these factors, together or separately, we would expect increased interspecific hybridization between the two megrim species.
Map of Northwest Iberia (Cantabric Sea) showing the location where the Prestige sunk (triangle off Galician coast) and its last trajectory (black line with dots) from the location of the accident and mayday alarm (cross). Arrows represent marine currents in the area and season of the accident (taken from http://oceancurrents.rsmas.miami.edu/). Their sizes are representative of their strength. The area affected by the oil spill is marked in light grey. The sampling area is marked as a dotted rectangle.
The aim of this study was to test whether interspecific hybridization between the two megrim species increased after the Prestige oil spill in the Cantabric Sea, which contained the coastal regions most affected by the catastrophe , . The nuclear 5S rDNA locus, frequently used as species-specific marker in fish , and RFLPs (Restriction Fragment Length Polymorphisms) at the mitochondrial D-loop sequence for determining the maternal species of hybrids were employed as molecular markers for identifying interspecific hybrids.
Amplification of the 5S rDNA locus yielded different fragments for each species. Lepidorhombus boscii exhibits one main fragment 233 bp long and a secondary fragment of 330 bp (much weaker in the gels). For L. whiffiagonis we obtained two fragments of 217 (main) and 472 (secondary, also weaker) bp long, as described in  for Atlantic megrim. Before the Prestige accident, only one individual (0.75%) from the sampled area exhibited a pattern of amplification fragments which corresponded to an interspecific hybrid, containing the two main fragments of each species: 217 and 233 bp long (Fig. 2). The weaker species-specific secondary fragments also appeared but are less clear in agarose gels and were not considered in this study. The hybrid specimen had been classified de visu as L. whiffiagonis. After the accident the situation changed drastically (Table 1). A total of 38 individuals (25.67%) exhibited hybrid genotypes. Three of them exhibited a typical L. whiffiagonis phenotype and 35 were L. boscii-like. A Chi-Square analysis confirmed that the proportion of interspecific hybrids increased significantly after the Prestige accident (χ2 = 36.54, 1 degree of freedom, P<<0.001). The species composition of the samples was also different, with more L. whiffiagonis in the after-Prestige sample and more L. boscii in the before-Prestige one.
Agarose gel (2.5%) stained with ethidium bromide containing PCR amplification products of the 5S rDNA of Lepidorhombus boscii (B), the interspecific hybrid (H) and L. whiffiagonis (W). M: 100 bp ladder as DNA fragment size marker.
The direction of hybrid crosses was assessed from D-loop RFLP. Amplification of this region with D-loopDF and D-loopDR primers yielded one fragment 535 bp long for both Lepidorhombus species. After digestion with Dra I, individuals with L. boscii mitochondrial D-loop (without restriction targets for this enzyme) yielded the same uncut fragment of 535 bp, as expected. Individuals with L. whiffiagonis mitochondrial DNA, with one Dra I restriction target, provided two fragments 230 and 305 bp long (Fig. 3) as also expected from the sequences. The 35 hybrids classified de visu as L. boscii exhibited a L. boscii D-loop pattern, and the three hybrids morphologically identified as L. whiffiagonis possessed a L. whiffiagonis D-loop (Table 1). As mitochondrial DNA is maternally inherited, we can conclude that most of the hybrid crosses that occurred after the Prestige accident (92.1% of the hybrids found in this survey) corresponded to L. boscii females mating with L. whiffiagonis males. All the hybrids were classified de visu as belonging to the maternal species, indicating that the external phenotype could be heavily influenced by the mother in interspecific crosses.
As the first long-term investigation of the area, the results found in this study reveal an increase in hybridization between the two megrim species in an area especially affected by the Prestige oil spill , . The proportion of hybrids changed from less than 1% to more than 25% in only nine years and, according to the D-loop region, most hybrid crosses involved L. boscii females and L. whiffiagonis males. Increased hybridization between sympatric species following environmental disturbances has been observed in a wide range of plant and animal taxa . For example, the proportion of interspecific hybrids between stickleback species from Enos Lake (Victoria, British Columbia, Canada) increased from 1%  to 12%  or 24%  following an anthropogenic-derived ecological change (introduction of an exotic predator) . A species breakdown has been suggested for sticklebacks , , ; however this not seems to be the case for megrims, in which only part of one area/population of the whole distribution was affected, similar to a unique event of hybridization increase. The appearance of many hybrids in the studied area may not be directly attributed to habitat loss caused by the petrol waste because interspecific matings on affected sea bottoms have not been physically observed. However, it seems to be at least one of the causative factors because loss or alterations of habitats are frequently implicated as contributing factors in hybridization in fishes . In other species such as cichlids, loss of water transparency has caused the rupture of pre-mating barriers based on body coloration, increasing the proportion of hybrids up to 88% .
Interspecific megrim hybrids may have accumulated in the studied region after the environmental degradation during the four generations elapsed since the accident. In addition to reduced and deteriorated habitat, and although interspecific mating barriers have not been studied for megrims, stress conditions in the area could have affected the mating behaviour of megrims, which is the second part of our hypothesis. In some amphibians stress affects the quality of male vocalization which is a determinant of female choice , . Altered behaviour of females, particularly the rejection of allospecific males, may explain many cases of unidirectional hybridization . For example, rodents of both sexes prefer conspecific over congeneric individuals in normal non-stress conditions, but mate choice may change if the hormonal balance is altered, as happens in stressful conditions . Other studies show that males of high body condition are often preferred by females due to the relationship between body size and male quality or fitness , . This could explain the higher proportion of hybrids with a L. whiffiagonis father and L. boscii mother as L. whiffiagonis are larger than L. boscii. Although we don't understand the particular underlying mechanism, it is possible that L. boscii females were more receptive to accepting males of the other species due to hormonal changes (probably pheromones) in response to the ecological stress caused by the spillage and that those pheromones could act as a pre-mating barrier in non-stressful conditions.
An alternative explanation is that hybrid crosses have not increased but instead the fitness of hybrids has been enhanced. Changes in the fitness of hybrids before and after environmental perturbations have been reported in the scientific literature for both plants and animals , . In fish, as in other species, hybrids may constitute a mechanism by which species deal with marginal habitats and environmental deterioration  and may even enable colonization of new habitats , . Whether hybrid crosses increased directly by forced spawning overlap of the two species when changes in hormones relaxed mating choice, and/or hybrid fitness has increased due to environmental deterioration, the final result was an increase in interspecific hybridization in the area affected by the petroleum.
The difference between pre- and post-Prestige samples in the proportion of pure individuals of each species could be explained by differential sensitivity of the two species. Martínez-Gómez et al.  showed that L. boscii was strongly sensitive to the Prestige toxic wastes, and the decrease of this species in post-Prestige megrim samples is consistent with such high sensitivity, indicating a possible decline in its population size. In addition, the hybridization that occurred after the oil spill was asymmetrical, with a higher proportion of hybrids resulting from L. boscii females. The rare species frequently provides the female in hybrid crosses , , consistent with what we observed in our study with L. boscii.
In conclusion, a high increase in interspecific megrim hybrids in the northern Spanish area affected by the Prestige oil spill may suggest that the accident could have increased the interspecific hybridization rate between megrims, likely due to a combination of altered mating behaviour and reduction of suitable spawning habitat. These hypotheses should be verified with future work and, if proven correct, the consequences of such interspecific introgression should be examined in further surveys. Although the trace of alien introduced genomes will likely remain for generations, measures for helping the most affected species L. boscii including a reduction in fishing mortality by increasing the allowable megrim size at catch, should be considered for future conservation of these valuable flatfish.
Materials and Methods
One year before the Prestige accident (August 2001) muscle fragments from adults of the two Lepidorhombus species were collected during research cruises and identified de visu by technical staff of the Spanish research institutions AZTI (a technological centre specialised in marine and food research) and IEO (Spanish Institute of Oceanography) from the Cantabric Sea and Bay of Biscay (corresponding with the ICES area VIIIc). Species classification was made according to head differences between species and the four characteristic spots typical of L. boscii's fins (see Fig. 4). L. whiffiagonis individuals present a sharp snout, which is also approximately two-times bigger than their eye diameter, and the dorsal fin origins closer to the tip of the snout than to the anterior edge of the eye. Otherwise, L. boscii individuals' dorsal fin originates closer to the anterior edge of eye and presents a smaller snout length than L. whiffiagonis. In total 39 L. whiffiagonis and 95 L. boscii were sampled.
In July 2011, nine years after the oil spill, new adult samples (heads or whole individuals) of the two megrim species were collected randomly from fishing vessels operating across Cantabric Sea and Bay of Biscay waters (similar locations as in 2001). In total we sampled 101 L. whiffiagonis and 47 L. boscii. They were visually classified as explained above. Only two phenotypic morphs were found across all individuals (corresponding with L. boscii and L. whiffiagonis “typical” individuals) and no morphological differences were found among individuals belonging to the same species.
A piece of gill or muscle tissue (approx. 3 g) was taken from each sample and stored in 100% ethanol for genetic analyses.
Total genomic DNA was extracted following a Chelex based protocol  and stored at 4°C. We amplified the 5S rDNA locus employing the primers A (5′-TACGCCCGATCTCGTCCGATC-3′) and B (5′-CAGGCTGGTATGGCCGTAAGC-3′) designed by  in 20 µl of total volume containing 4 µl of 5× Promega Green Buffer, 2 µl of 25 mM MgCl2, 2 µl of a 2.5 mM dNTPs mixture, 1 µl of each primer at 20 µM, 0.1 µl of GoTaq polymerase at 5 U/µl (Promega), 2 µl of sample DNA and 7.9 µl of bidistilled water. PCR amplification cycles were: 5 min of initial denaturing at 95°C, followed by 35 cycles of denaturing at 95°C for 20 s, annealing at 65°C for 20 s and extension at 72°C for 30 s, plus a final extension at 72°C for 20 min. Amplification products were run in 2.5% w/v agarose gels at 100 V, and stained with 2 µl ethidium bromide (10 mg/ml) to visualize them. Fragment sizes were estimated by comparison with a standard 100 bp DNA marker (Promega).
The maternal species of hybrids was identified by a species-specific RFLP within the mitochondrial D-loop sequence. To develop the method, D-loop sequences of the two megrim species were obtained from GenBank (accession numbers FJ590680-FJ590700 and FJ590730-FJ590750) and aligned with the ClustalW application  included in BioEdit. Invariant (monomorphic) regions were visually identified within each megrim species with the BioEdit Sequence Alignment Editor software  after sequences alignment. Restriction enzyme targets within the invariant regions of each species were detected with the NEBcutter ver. 2.0 software. The enzyme Dra I recognizes the sequence 5′-TTTAAA-3′ and makes a blunt cut 5′-TTT/AAA-3′ . Such sequence is present in L. whiffiagonis and absent in L. boscii D-loop sequences.
D-loop amplification was carried out employing the primers D-loopDF (5′-GTCGCCACCATTAACTTATGC-3′) and D-loopDR (5′-CCCAAACTCCCAAAGCTAAG-3′) described by . The amplification mixture, of a total volume of 20 µl, contained 4 µl of 5× Promega Green Buffer, 1.2 µl of 25 mM MgCl2, 2 µl of a 2.5 mM dNTPs mixture, 1 µl of each primer at 20 µM, 0.12 µl of GoTaq polymerase at 5 U/µl (Promega), 2 µl of sample DNA and 8.68 µl of bidistilled water. Eight µl of PCR amplification were loaded in a 2% agarose gel stained with 2 µl of 10 mg ml−1 ethidium bromide to verify that only one band was amplified. Then 10 µl of the PCR product were mixed with 2.5 µl of 10× Buffer M (Roche), 0.1 µl of Dra I enzyme (Roche) at 10 ud/µl and 12.4 µl of bidistilled water making a total volume of 25 µl, and incubated at 37°C for one hour. After incubation, the products were loaded in a 2.5% agarose gel, run at 80 v for 40 min and stained with 2 µl of 10 mg ml−1 ethidium bromide. Fragment sizes were estimated with a standard 100 bp DNA marker (Promega).
We are indebted to I. G. Pola for helping in laboratory tasks. Thanks to C. R. Sparrevohn for his helpful comments on our results. Thanks to F. Juanes and two anonymous reviewers for kindly revising the article. We wish also to thank Paula Alvarez from the AZTI (Institute of Technology, Basque Country) and Francisco Sanchez from the I.E.O. (Spanish Institute of Oceanography), as well as the staff from Asturian markets and fishing vessels (Cofradia Virgen de las Mareas, Aviles, Spain) for kindly providing megrim samples.
Conceived and designed the experiments: VCP EGV AR. Performed the experiments: VCP. Analyzed the data: VCP. Contributed reagents/materials/analysis tools: JLM AR. Wrote the paper: VCP EGV.
- 1. Perry RI, Cury P, Brander K, Jennings S, Möllmann C, et al. (2010) Sensitivity of marine systems to climate and fishing: Concepts, issues and management responses. J. Marine Syst. 79: 427–435.RI PerryP. CuryK. BranderS. JenningsC. Möllmann2010Sensitivity of marine systems to climate and fishing: Concepts, issues and management responses. J. Marine Syst.79427435
- 2. Jackson JBC, Kirby MX, Berger WH, Bjordal KA, Botsford LW, et al. (2001) Historical Overfishing and the Recent Collapse of Coastal Ecosystems. Science 293(5530): 629–637.JBC JacksonMX KirbyWH BergerKA BjordalLW Botsford2001Historical Overfishing and the Recent Collapse of Coastal Ecosystems.Science2935530629637
- 3. Gilbert TD (2000) TD Gilbert2000Oil Spills in the Australian Marine Environment: Environmental Consequences and Response Technologies. Pub. AMSA, 1–12. Available at www.amsa.gov.au/marine_environment_protection/national_plan/Reports-Fact_Sheets-Brochures/. Last access 12/07/2011. Oil Spills in the Australian Marine Environment: Environmental Consequences and Response Technologies. Pub. AMSA, 1–12. Available at www.amsa.gov.au/marine_environment_protection/national_plan/Reports-Fact_Sheets-Brochures/. Last access 12/07/2011.
- 4. Richardson N (2010) Deepwater Horizon and the Patchwork of Oil Spill Liability Law. Resources for the future, 1–6. 01: N. Richardson2010Deepwater Horizon and the Patchwork of Oil Spill Liability Law. Resources for the future, 1–6.01Available: http://www.rff.org/rff/documents/RFF-BCK-Richardson-OilLiability.pdf. Accessed 2011 July. Available: http://www.rff.org/rff/documents/RFF-BCK-Richardson-OilLiability.pdf. Accessed 2011 July.
- 5. Xia Y, Boufadel MC (2010) Lessons from the Exxon Valdez Oil Spill disaster in Alaska. Disaster Advances 3(4): 270–273.Y. XiaMC Boufadel2010Lessons from the Exxon Valdez Oil Spill disaster in Alaska.Disaster Advances34270273
- 6. Di Giulio RT, Habig C, Evan P, Gallagher P (1993) Effects of black Rock Harbor sediments on indices of biotransformation, oxidative stress and DNA integrity in channel catfish. Aquat. Toxicol. 26: 1–22.RT Di GiulioC. HabigP. EvanP. Gallagher1993Effects of black Rock Harbor sediments on indices of biotransformation, oxidative stress and DNA integrity in channel catfish. Aquat. Toxicol.26122
- 7. Myers MS, Johnson LL, Olson OP, Stehr CM, Horness BH, et al. (1998) Toxicopathic hepatic lesions as biomarkers of chemical contaminant exposure and effects in marine bottomfish species from the Northeast and Pacific coasts, USA. Mar. Pol. Bull. 37(1–2): 92–113.MS MyersLL JohnsonOP OlsonCM StehrBH Horness1998Toxicopathic hepatic lesions as biomarkers of chemical contaminant exposure and effects in marine bottomfish species from the Northeast and Pacific coasts, USA. Mar. Pol. Bull.371–292113
- 8. Albaigés J, Morales-Nin B, Vilas F (2006) The Prestige oil spill: A scientific response. Mar. Pol. Bull. 53: 205–207.J. AlbaigésB. Morales-NinF. Vilas2006The Prestige oil spill: A scientific response. Mar. Pol. Bull.53205207
- 9. Sánchez F, Velasco F, Cartes J, Olaso I, Preciado I, et al. (2006) Monitoring the Prestige oil spill impacts on some key species of the Northern Iberian shelf. Mar. Pol. Bull. 53: 332–349.F. SánchezF. VelascoJ. CartesI. OlasoI. Preciado2006Monitoring the Prestige oil spill impacts on some key species of the Northern Iberian shelf. Mar. Pol. Bull.53332349
- 10. Martínez-Gómez C, Campillo JA, Benedicto J, Fernández B, Valdés J, et al. (2006) Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill. Mar. Pol. Bull. 53: 305–314.C. Martínez-GómezJA CampilloJ. BenedictoB. FernándezJ. Valdés2006Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill. Mar. Pol. Bull.53305314
- 11. Surís-Regueiro JC, Garza-Gil MD, Varela-Lafuente MM (2007) The Prestige oil spill and its economic impact on the Galician fishing sector. Disasters 31(2): 201–15.JC Surís-RegueiroMD Garza-GilMM Varela-Lafuente2007The Prestige oil spill and its economic impact on the Galician fishing sector.Disasters31220115
- 12. Martínez-Abraín A, Velando A, Oro D, Genovart M, Gerique C, et al. (2006) Sex-specific mortality of European shags after the Prestige oil spill: demographic implications for the recovery of colonies. Mar. Ecol. Prog. Ser. 318: 271–276.A. Martínez-AbraínA. VelandoD. OroM. GenovartC. Gerique2006Sex-specific mortality of European shags after the Prestige oil spill: demographic implications for the recovery of colonies. Mar. Ecol. Prog. Ser.318271276
- 13. Zuberogoitia I, Martinez JA, Iraeta A, Azkona A, Zabala J, et al. (2006) Short-term effects of the prestige oil spill on the peregrine falcon (Falco peregrinus). Mar. Pol. Bull. 52: 1176–1181.I. ZuberogoitiaJA MartinezA. IraetaA. AzkonaJ. Zabala2006Short-term effects of the prestige oil spill on the peregrine falcon (Falco peregrinus). Mar. Pol. Bull.5211761181
- 14. Rogowska J, Namieśnik J (2010) Environmental Implications of Oil Spills from Shipping Accidents. Rev. Environ. Contam. Toxicol. 206: 95–114.J. RogowskaJ. Namieśnik2010Environmental Implications of Oil Spills from Shipping Accidents. Rev. Environ. Contam. Toxicol.20695114
- 15. “Ministerio de Ciencia y Tecnología y el IEO” (2003) “Informe 24: El vertido del Prestige. Situación un año después del accidente”. 29 p. (In Spanish). 07: IEO” “Ministerio de Ciencia y Tecnología y el2003“Informe 24: El vertido del Prestige. Situación un año después del accidente”. 29 p. (In Spanish).07Available: http://www.ieo.es/prestige/pdfs/Informe_IEO_24.pdf. Accessed 2011 July. Available: http://www.ieo.es/prestige/pdfs/Informe_IEO_24.pdf. Accessed 2011 July.
- 16. Pietrapiana D, Modena M, Guidetti P, Falugi C, Vacchi M (2002) Evaluating the genotoxic damage and hepatic tissue alterations in demersal fish species: a case study in the Ligurian Sea (NW-Mediterranean). Mar. Pol. Bull. 44: 238–243.D. PietrapianaM. ModenaP. GuidettiC. FalugiM. Vacchi2002Evaluating the genotoxic damage and hepatic tissue alterations in demersal fish species: a case study in the Ligurian Sea (NW-Mediterranean). Mar. Pol. Bull.44238243
- 17. Sánchez F, Pérez N, Landa J (1998) Distribution and abundance of megrim (Lepidorhombus boscii and Lepidorhombus whiffiagonis) on the northern Spanish Shelf. ICES J. Mar. Sci. 55: 494–514.F. SánchezN. PérezJ. Landa1998Distribution and abundance of megrim (Lepidorhombus boscii and Lepidorhombus whiffiagonis) on the northern Spanish Shelf. ICES J. Mar. Sci.55494514
- 18. ICES (2008) Report of the Working Group on the Assessment of Southern Shelf Stocks of Hake, Monk and Megrim (WGHMM). 07: ICES2008Report of the Working Group on the Assessment of Southern Shelf Stocks of Hake, Monk and Megrim (WGHMM).07Available: http://www.ices.dk/reports/ACOM/2008/WGHMM/WGHMM08.pdf. Accessed 2011 July. Available: http://www.ices.dk/reports/ACOM/2008/WGHMM/WGHMM08.pdf. Accessed 2011 July.
- 19. Danancher D, Garcia-Vazquez E (2009) Population differentiation in megrim (Lepidorhombus whiffiagonis) and four spotted megrim (Lepidorhombus boscii) across Atlantic and Mediterranean waters and implications for wild stock management. Mar. Biol. 156: 1869–1880.D. DanancherE. Garcia-Vazquez2009Population differentiation in megrim (Lepidorhombus whiffiagonis) and four spotted megrim (Lepidorhombus boscii) across Atlantic and Mediterranean waters and implications for wild stock management. Mar. Biol.15618691880
- 20. Landa J, Piñeiro C (2000) Megrim (Lepidorhombus whiffiagonis) growth in the North-eastern Atlantic based on back-calculation of otolith rings. ICES J. Marine Science. 57: 1077–1090.J. LandaC. Piñeiro2000Megrim (Lepidorhombus whiffiagonis) growth in the North-eastern Atlantic based on back-calculation of otolith rings. ICES J. Marine Science.5710771090
- 21. Landa J, Perez N, Piñeiro C (2002) Growth patterns of the four spotted megrim (Lepidorhombus boscii) in the northeast Atlantic. Fish. Res. 55: 141–152.J. LandaN. PerezC. Piñeiro2002Growth patterns of the four spotted megrim (Lepidorhombus boscii) in the northeast Atlantic. Fish. Res.55141152
- 22. Perry AL, Low PJ, Ellis JR, Reynolds JD (2005) Climate Change and Distribution Shifts in Marine Fishes. Science 308: 1912–1915.AL PerryPJ LowJR EllisJD Reynolds2005Climate Change and Distribution Shifts in Marine Fishes.Science30819121915
- 23. Seehausen O, van Alphen JM, Witte F (1997) Cichlid Fish Diversity Threatened by Eutrophication That Curbs Sexual Selection. Science 277: 1808–1811.O. SeehausenJM van AlphenF. Witte1997Cichlid Fish Diversity Threatened by Eutrophication That Curbs Sexual Selection.Science27718081811
- 24. Pendas AM, Moran P, Martinez JL, Garcia-Vazquez E (1995) Applications of 5S rRNA in Atlantic salmon, brown trout, and in Atlantic salmon x brown trout hybrid identification. Mol. Ecol. 4: 275–276.AM PendasP. MoranJL MartinezE. Garcia-Vazquez1995Applications of 5S rRNA in Atlantic salmon, brown trout, and in Atlantic salmon x brown trout hybrid identification. Mol. Ecol.4275276
- 25. Garcia-Vazquez E, Izquierdo JI, Perez J (2006) Genetic variation at ribosomal genes supports the existence of two different European subspecies in the megrim Lepidorhombus whiffiagonis. J. Sea Res. 56: 59–64.E. Garcia-VazquezJI IzquierdoJ. Perez2006Genetic variation at ribosomal genes supports the existence of two different European subspecies in the megrim Lepidorhombus whiffiagonis. J. Sea Res.565964
- 26. Gilman RT, Behm JE (2011) Hybridization, species collapse, and species reemergence after disturbance to premating mechanisms of reproductive isolation. Evolution 65(9): 2592–2605.RT GilmanJE Behm2011Hybridization, species collapse, and species reemergence after disturbance to premating mechanisms of reproductive isolation.Evolution65925922605
- 27. McPhail JD (1992) Ecology and evolution of sympatric sticklebacks (Gasterosteus): morphological and genetic evidence for a species pair in Paxton Lake, British Columbia. Can. J. Zool. 70: 361–369.JD McPhail1992Ecology and evolution of sympatric sticklebacks (Gasterosteus): morphological and genetic evidence for a species pair in Paxton Lake, British Columbia. Can. J. Zool.70361369
- 28. Kraak SBM, Mundwiler B, Hart PJB (2001) Increased number of hybrids between benthic and limnetic threespined sticklebacks in Enos Lake, Canada: the collapse of a species pair? J. Fish Biol. 58: 1458–1464.SBM KraakB. MundwilerPJB Hart2001Increased number of hybrids between benthic and limnetic threespined sticklebacks in Enos Lake, Canada: the collapse of a species pair? J. Fish Biol.5814581464
- 29. Gow JL, Peichel CL, Taylor EB (2006) Contrasting hybridization rates between sympatric three-spined sticklebacks highlight the fragility of reproductive barriers between evolutionarily young species. Mol. Ecol. 15: 739–752.JL GowCL PeichelEB Taylor2006Contrasting hybridization rates between sympatric three-spined sticklebacks highlight the fragility of reproductive barriers between evolutionarily young species. Mol. Ecol.15739752
- 30. Taylor EB, Boughman JW, Groenenboom M, Sniatynski M, Schluter D, et al. (2006) Speciation in reverse: morphological and genetic evidence of the collapse of a three-spined stickleback (Gasterosteus aculeatus) species pair. Mol. Ecol. 15: 343–355.EB TaylorJW BoughmanM. GroenenboomM. SniatynskiD. Schluter2006Speciation in reverse: morphological and genetic evidence of the collapse of a three-spined stickleback (Gasterosteus aculeatus) species pair. Mol. Ecol.15343355
- 31. Behm JE, Ives AR, Boughman JW (2010) Breakdown in Postmating Isolation and the Collapse of a Species Pair through Hybridization. Am. Na. 175(1): 11–26.JE BehmAR IvesJW Boughman2010Breakdown in Postmating Isolation and the Collapse of a Species Pair through Hybridization. Am. Na.17511126
- 32. Scribner KT, Page KS, Bartron ML (2000) Hybridization in freshwater fishes: a review of case studies and cytonuclear methods of biological inference. Rev. Fish Biol. Fish. 10(3): 293–323.KT ScribnerKS PageML Bartron2000Hybridization in freshwater fishes: a review of case studies and cytonuclear methods of biological inference. Rev. Fish Biol. Fish.103293323
- 33. Leary CJ, Garcia AM, Knapp R (2006a) Elevated corticosterone levels elicit non-calling mating tactics in male toads independently of changes in circulating androgens. (4): Horm. Behav. 49. pp. 425–432.CJ LearyAM GarciaR. Knapp2006aElevated corticosterone levels elicit non-calling mating tactics in male toads independently of changes in circulating androgens.4Horm. Behav. 49425432
- 34. Leary CJ, Garcia AM, Knapp R (2006b) Stress hormone is implicated in satellite-caller associations and sexual selection in the Great Plains toad. (4): Am. Nat. 168. pp. 431–440.CJ LearyAM GarciaR. Knapp2006bStress hormone is implicated in satellite-caller associations and sexual selection in the Great Plains toad.4Am. Nat. 168431440
- 35. Wirtz P (1999) Mother species–father species: unidirectional hybridization in animals with female choice. Anim. Behav. 58: 1–12.P. Wirtz1999Mother species–father species: unidirectional hybridization in animals with female choice. Anim. Behav.58112
- 36. Adkins-Regan E (1998) Hormonal mechanism of mate choice. Amer. Zool. 38: 166–178.E. Adkins-Regan1998Hormonal mechanism of mate choice. Amer. Zool.38166178
- 37. Andersson M (1994) Sexual Selection. Princeton University Press. ISBN. 9780691000572 p.M. Andersson1994Sexual Selection.Princeton University Press. ISBN9780691000572
- 38. Arnold ML, Bulger MR, Burke JM, Hempel AL, Williams JH (1999) Natural hybridization: how low can you go and still be important? (2): Ecology 80. pp. 371–381.ML ArnoldMR BulgerJM BurkeAL HempelJH Williams1999Natural hybridization: how low can you go and still be important?2Ecology 80371381
- 39. Hochwender CG, Fritz RS, Orians CM (2000) Using hybrid systems to explore the evolution of tolerance to damage. (4–6): Evol. Ecol. 14. pp. 509–521.CG HochwenderRS FritzCM Orians2000Using hybrid systems to explore the evolution of tolerance to damage.4–6Evol. Ecol. 14509521
- 40. Edwards CJ, Suchard MA, Lemey P, Welch JJ, Barnes I, et al. (2011) Ancient hybridization and an Irish origin for the modern Polar bear matriline. Curr. Biol. 21(15): 1251–1258.CJ EdwardsMA SuchardP. LemeyJJ WelchI. Barnes2011Ancient hybridization and an Irish origin for the modern Polar bear matriline. Curr. Biol.211512511258
- 41. Nolte AW, Freyhof J, Stemshorn KC, Tautz D (2005) An invasive lineage of sculpins, Cottus sp. (1579): (Pisces, Teleostei) in the Rhine with new habitat adaptations has originated from hybridization between old phylogeographic groups. Proc. R. Soc. B 272. pp. 2379–2387.AW NolteJ. FreyhofKC StemshornD. Tautz2005An invasive lineage of sculpins, Cottus sp.1579(Pisces, Teleostei) in the Rhine with new habitat adaptations has originated from hybridization between old phylogeographic groups. Proc. R. Soc. B 27223792387
- 42. Rieseberg LH, Kim S-C, Randell RA, Whitney KD, Gross BL, et al. (2007) Hybridization and the colonization of novel habitats by annual sunflowers. (2): Genetica 129. pp. 149–165.LH RiesebergS-C KimRA RandellKD WhitneyBL Gross2007Hybridization and the colonization of novel habitats by annual sunflowers.2Genetica 129149165
- 43. Avise JC, Saunders NC (1984) Hybridization and introgression among species of sunfish (Lepomis): Analysis by mitochondrial DNA and allozyme markers. Genetics 108: 237–255.JC AviseNC Saunders1984Hybridization and introgression among species of sunfish (Lepomis): Analysis by mitochondrial DNA and allozyme markers.Genetics108237255
- 44. Estoup A, Largiadèr CR, Perrot E, Chourrout D (1996) Rapid one-tube DNA extraction for reliable PCR detection of fish polymorphic marker and transgenes. (4): Mol. Mar. Biol. Biotech. 5. pp. 295–298.A. EstoupCR LargiadèrE. PerrotD. Chourrout1996Rapid one-tube DNA extraction for reliable PCR detection of fish polymorphic marker and transgenes.4Mol. Mar. Biol. Biotech. 5295298
- 45. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. (22): Nucleic Acid Res. 22. pp. 4673–80.JD ThompsonDG HigginsTJ Gibson1994CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.22Nucleic Acid Res. 22467380
- 46. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41: 95–98.TA Hall1999BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser.419598
- 47. Purvis IJ, Moseley BEB (1983) Isolation and characterisation of DraI, a type II restriction endonuclase recognising a sequence containing only A:T basepairs, and inhibition of its activity by uv irradiation of substrate DNA. Nucleic Acid Res. 11: 5467–5474.IJ PurvisBEB Moseley1983Isolation and characterisation of DraI, a type II restriction endonuclase recognising a sequence containing only A:T basepairs, and inhibition of its activity by uv irradiation of substrate DNA. Nucleic Acid Res.1154675474
- 48. Campo D, Garcia-Vazquez E (2010) D. CampoE. Garcia-Vazquez2010Evolutionary history of the four-spotted megrim (Lepidorhombus boscii) and speciation time within the genus based on mitochondrial genes analysis. J. Sea Res. 64: 360–368. http://wwf.panda.org Last access: 01/07/2011. Evolutionary history of the four-spotted megrim (Lepidorhombus boscii) and speciation time within the genus based on mitochondrial genes analysis. J. Sea Res. 64: 360–368. http://wwf.panda.org Last access: 01/07/2011.