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Aberystwyth University DNA Barcoding and Microsatellites Help View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Aberystwyth Research Portal Aberystwyth University DNA barcoding and microsatellites help species delimitation and hybrid identification in endangered galaxiid fishes Vanhaecke, Delphine; Leaniz, Carlos Garcia De; Gajardo, Gonzalo; Young, Kyle; Sanzana, Jose; Orellana, Gabriel; Fowler, Daniel; Howes, Paul; Monzon-Arguello, Catalina ; Olmo, Sofia Consuegra Del Published in: PLoS One DOI: 10.1371/journal.pone.0032939 Publication date: 2012 Citation for published version (APA): Vanhaecke, D., Leaniz, C. G. D., Gajardo, G., Young, K., Sanzana, J., Orellana, G., ... Olmo, S. C. D. (2012). DNA barcoding and microsatellites help species delimitation and hybrid identification in endangered galaxiid fishes. PLoS One, 7(3), [e32939]. https://doi.org/10.1371/journal.pone.0032939 General rights Copyright and moral rights for the publications made accessible in the Aberystwyth Research Portal (the Institutional Repository) are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the Aberystwyth Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the Aberystwyth Research Portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. tel: +44 1970 62 2400 email: [email protected] Download date: 03. Oct. 2019 DNA Barcoding and Microsatellites Help Species Delimitation and Hybrid Identification in Endangered Galaxiid Fishes Delphine Vanhaecke1, Carlos Garcia de Leaniz2, Gonzalo Gajardo3, Kyle Young3, Jose Sanzana3, Gabriel Orellana3, Daniel Fowler4, Paul Howes2, Catalina Monzon-Arguello2,1, Sofia Consuegra1* 1 Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom, 2 Department of BioSciences, College of Science, Swansea University, Swansea, United Kingdom, 3 Laboratorio de Gene´tica, Acuicultura y Biodiversidad, Universidad de Los Lagos, Osorno, Chile, 4 Falkland Islands Fisheries Department, Stanley, Falkland Islands Abstract The conservation of data deficient species is often hampered by inaccurate species delimitation. The galaxiid fishes Aplochiton zebra and Aplochiton taeniatus are endemic to Patagonia (and for A. zebra the Falkland Islands), where they are threatened by invasive salmonids. Conservation of Aplochiton is complicated because species identification is hampered by the presence of resident as well as migratory ecotypes that may confound morphological discrimination. We used DNA barcoding (COI, cytochrome b) and a new developed set of microsatellite markers to investigate the relationships between A. zebra and A. taeniatus and to assess their distributions and relative abundances in Chilean Patagonia and the Falkland Islands. Results from both DNA markers were 100% congruent and revealed that phenotypic misidentification was widespread, size-dependent, and highly asymmetric. While all the genetically classified A. zebra were correctly identified as such, 74% of A. taeniatus were incorrectly identified as A. zebra, the former species being more widespread than previously thought. Our results reveal, for the first time, the presence in sympatry of both species, not only in Chilean Patagonia, but also in the Falkland Islands, where A. taeniatus had not been previously described. We also found evidence of asymmetric hybridisation between female A. taeniatus and male A. zebra in areas where invasive salmonids have become widespread. Given the potential consequences that species misidentification and hybridisation can have for the conservation of these endangered species, we advocate the use of molecular markers in order to reduce epistemic uncertainty. Citation: Vanhaecke D, Garcia de Leaniz C, Gajardo G, Young K, Sanzana J, et al. (2012) DNA Barcoding and Microsatellites Help Species Delimitation and Hybrid Identification in Endangered Galaxiid Fishes. PLoS ONE 7(3): e32939. doi:10.1371/journal.pone.0032939 Editor: Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, Canada Received September 19, 2011; Accepted February 2, 2012; Published March 6, 2012 Copyright: ß 2012 Vanhaecke 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: Funding for this study was provided by a DEFRA Darwin Initiative (Grant No. 162/15/020) and a Darwin post-project award ‘Protecting galaxiids from salmonid invasions in Chile and the Falklands’ (Grant No. EIDPO-041; www.biodiversity.cl); with additional support from the University of Los Lagos (Chile) and Falkland Islands Development Corporation. 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. * E-mail: [email protected] Introduction 9], but its taxonomic status remains unclear [11]. Little is known about the ecology and biogeography of Aplochiton [9,12], which Given the current ratio of species discovery to extinction rates, it tend to display a patchy, restricted distribution [9], and whose is likely that many species will go extinct before they are properly reproductive ecology has only recently been described [13]. described [1]. Accurate classification is thus an essential first step Aplochiton are believed to have an amphidromous life cycle with a towards effective conservation of local biodiversity [2–3]. This is marine larval phase followed by juvenile growth and spawning in particularly critical in the Southern Hemisphere, where a high freshwater [12,14], although landlocked populations are also proportion of endemic species are poorly known, and are now known [12]. Apparent declines in the abundance of Aplochiton have threatened by non-native introductions [4]. Biological invasions been attributed to a number of stressors [15–16], most notably are a leading cause of animal extinctions [5] and one of the main predation and/or competition from invasive salmonids, which are threats to aquatic biodiversity [6], particularly for fishes [7], which widespread and dominate freshwater fish communities throughout tend to display high rates of endemism. much of Aplochiton’s range [17–20]. A. zebra has been classified as The diversity of fish species in Patagonia is very low, with only ‘in danger of extinction’ in parts of its range, while the five families represented (Galaxiidae, Trichomycteridae, Diplo- conservation status of A. taeniatus remains unclear due to data mystidae, Atherinopsidae and Percichthyidae) [8]. The genus deficiency [17]. Aplochiton is one of the three genera representing the family Fishes from the family Galaxiidae are morphologically very Galaxiidae in South America [9] and has two recognised species A. diverse, particularly in relation to buccal arrangements and shape zebra and A. taeniatus (Jenyns 1842) restricted to Patagonia [9], and of caudal fin [21–23]. Although both A. zebra and A. taeniatus have in the case of A. zebra, also present in the Falkland Islands [10]. elongate, fusiform bodies with a slender, deeply forked tail [12], The existence of a third species, A. marinus, has been suggested [8– they exhibit morphological differences that appear to be related to PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32939 Misidentification and Hybridisation of Galaxiids their different trophic positions. Thus, A. taeniatus seems to attain a measure of body shape [33]. Size comparisons between locations larger size than A. zebra and is considered an specialist that preys and species were carried out in SYSTAT v.11. mostly on fish, displaying particular adaptations for piscivory such as a large mouth, enlarged teeth and elongated stomach. In mtDNA analysis contrast, A. zebra is considered a generalist that feeds mainly on DNA was extracted using the WizardH SV 96 DNA Purification aquatic invertebrates [12], and resembles more other galaxiids that Kit following manufacturer’s instructions. For mtDNA analysis, are also generalized invertebrate predators [17,24–25]. Differences regions of the COI (cytochrome c oxidase subunit I) and cyt b in body size and trophic ecology probably reflect differences in (cytochrome b) genes were amplified. A region of 515 bp of the 59 niche breadth, which could ultimately result in different region of the mitochondrial COI gene was amplified for 367 fish vulnerability to salmonid invasions through predation and using primers FishF1 and FishR1 [30]. In addition, the primers resource competition. L14724 [34] and H15149 [35] were used to amplify a region of Discrimination between A. zebra and A. taeniatus is currently 354 bp of the cyt b gene for 105 fish. PCR was conducted using an based on variation in meristic (number of vertebrae, gillrakers and initial denaturation step at 95uC for 5 minutes, followed by fin rays) and morphological traits (body depth, and relative size of 35 cycles at 94uC for 30 seconds, 55uC for
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