Skate-ing on thin ice: Molecular ecology of longnose skates in the Southeast Pacific Ocean

Carolina Andrea Vargas Caro Bachelor of Science (Hon)

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Faculty of Medicine Abstract

Longnose skates may be one of the most vulnerable taxa of elasmobranch fishes, with documented local extinctions and population declines worldwide. Longnose skates are the main component of the commercial elasmobranch fisheries in the south-east Pacific Ocean, especially in Chilean waters where target and bycatch fisheries have led two species to the brink of collapse. The yellownose skate Zearaja chilensis and the roughskin skate Dipturus trachyderma are endemic of southern South America yet little is known about population ecology and stock structure. Additionally, the external morphology of longnose skates is remarkably similar, especially in early life stages, and a lack of accurate species identification has compromised official landings records which in turn has impacted fishery management. Overall abundance of longnose skates has declined substantially over the last decade due to intensive fishing pressure and the fishery is considered to be `fully exploited'.

Based on all available information in peer-reviewed and grey literature, a comparative synthesis of the biology and ecology was conducted on Z. chilensis and D. trachyderma. A positive increase in relation to scientific knowledge over the past decade was noticeable, although several basic aspects of their biology and ecology are still missing. There is an urgent need to fill knowledge gaps on, for example, movement patterns, feeding ecology and habitat use, population size and structure, and levels of connectivity. In this study, taxonomic clarity was provided, and relevant information with regard to the biology, ecology and fisheries that interact with longnose skates was collated.

Confusion of identity between longnose skates occurs to the present day. In order to address this issue, morphometric and genetic tools are provided to aid in species identification of early-life stage specimens. Thirty-seven morphometric measurements and three meristic characters were used to identify specimens. These results suggest that the number of midline, nuchal and inter-dorsal thorns could be used to discriminate between specimens of Z. chilensis and D. trachyderma. Previously, the presence of a single nuchal thorn was considered to be a feature that could be used to separate species, however, this morphological feature has proven to be variable in Z. chilensis. Additionally, partial sequences of the 16S, cox1, nadh2 and the control regions of the mitochondrial DNA were amplified and analysed for intraspecific and interspecific divergence. Amplified fragments of cox1 gene and the control region contained information to separate the target species; however, specimens of D. trachyderma and Z. chilensis were grouped indistinctly within a single clade using 16S and nadh2 fragments.

–ii– The evolutionary history of Zearaja was also explored, in a broad taxonomic context by using whole mitochondrial genomes (mitogenomes) to understand higher-level phylogenetic relationships among batoids. A data set of 56 mitogenomes of 47 batoids and four outgroups was analysed. Overall, these results showed consensus phylogenomic trees that support previous phyletic reconstructions based on morphological characters, and consequently recovers all extant Orders within the Batoidea in two higher-level reciprocally monophyletic clades: Myliobatiformes + Rhinopristiformes, and Torpediniformes + Rajiformes. Intraspecific divergence is extremely low among the three Zearaja species and may suggest a radiation from a potential ancestral form (Z. chilensis) towards a derived species (Z. maugeana). The evolution of the genus Zearaja could be linked to major geological events, such as the terminal Eocene event and the sinking of Zealandia; which may explain the current restricted geographical distribution to the Southern Hemisphere.

Longnose skates in Chilean waters are considered to be a single stock by the fishery management in Chile however, little is known about the level of demographic connectivity within the fishery. Here, population connectivity at five locations along the Chilean coast was explored by using the mitochondrial (control region) and nuclear (microsatellite loci) DNA. Analysis of Z. chilensis populations revealed significant genetic structure among off-shore locations (San Antonio, Valdivia), two locations in the Chiloe Interior Sea (Puerto Montt and Aysén), and Punta Arenas in southern Chile. An important research outcome was lack of connectivity among individuals from the Chiloe Interior Sea and the other two proposed management units. These results provide evidence for three management units for Z. chilensis, and recommendations of separate harvest strategies were made considering each of these units. However, there is no evidence to discriminate the extant population of D. trachyderma as separate management units.

This thesis has expanded our general knowledge and has provided management guidelines for sustainable fishery practices in Chile; however, appropriate management and enforcement actions from the Chilean Government are urgently needed to avoid population collapse and extirpation of stocks.

–iii– Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

–iv– Publications during candidature

Conference abstracts

Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2016). Population structure and genetic diversity of commercial skates in Chile: Tools towards sustainable fishery management. International Postgraduate Symposium in Biomedical Sciences, 31st October and 1st November 2016, The University of Queensland, Australia.

Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2016). Estructura poblacional y diversidad genética de las rayas comerciales en chile: Herramientas para un manejo pesquero sustentable. XXXVI Congreso de Ciencias del Mar: Sustentabilidad y Multidisciplina en Ciencias del Mar, 23-27th of May 2016, Universidad de Concepción, Chile.

Vargas-Caro, C., Bustamante, C. & Bennett, M. B. (2016). Fishing the twilight zone: Catch composition of the southeast Pacific Ocean demersal longline fishery. XXXVI Congreso de Ciencias del Mar: Sustentabilidad y Multidisciplina en Ciencias del Mar, 23-27th of May 2016, Universidad de Concepción, Chile.

Bustamante, C., Vargas-Caro, C., Ovenden, J. R. & Bennett, M. B. (2016). Más allá de los genes: Aplicación de la filogenómica en la taxonomía de peces elasmobranquios. XXXVI Congreso de Ciencias del Mar: Sustentabilidad y Multidisciplina en Ciencias del Mar, 23-27th of May 2016, Universidad de Concepción, Chile.

Vargas-Caro, C., Bustamante, C. & Bennett, M. B. (2015). Fishing the twilight zone: Catch composition of the southeast Pacific Ocean demersal longline fishery. International Postgraduate Symposium in Biomedical Sciences, 3-5th November 2015, The University of Queensland, Australia.

Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2015). Phylogeny of longnose skates (genus Zearaja) inferred from complete mitochondrial genomes. 1st Meeting of Chilean scientists in Brisbane: Strengthening ties between Chile and Australia, 31st of July 2015, The University of Queensland, Australia.

Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2015). Phylogeny of longnose skates (genus Zearaja) inferred from complete mitochondrial genomes. Conference of the New Zealand Marine Sciences Society & Oceania Chondrichthyan Society, 6-9th of July 2015, The University of Auckland, New Zealand.

–v– Vargas-Caro, C., Bennett, M. B. & Ovenden, J. R. (2014). Skate-ing on thin ice: How to resolve identification issues for future management. Conference of the Shark International symposium, 2- 6th of June 2014, the Southern Sun Elangeni-Maharani Hotel, Durban, South Africa.

Listed journals with editorial committee

Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2017). Towards sustainable fishery management: The genetic population structure of Zearaja chilensis and Dipturus trachyderma (Chondrichthyes, Rajiformes) in the south-east Pacific Ocean. PLoS One 12, e0172255. DOI: 10.1371/journal.pone.0172255.

Bustamante, C., Barría, C., Vargas-Caro, C., Ovenden, J. R. & Bennett, M. B. (2016). The phylogenetic position of the giant devil ray Mobula mobular (Bonnaterre, 1788) (Myliobatiformes, Myliobatidae) inferred from the mitochondrial genome. Mitochondrial DNA Part A 27, 3540– 3541. DOI: 10.3109/19401736.2015.1074208.

Vargas-Caro, C., Bustamante, C., Lamilla, J., Bennett, M. B. & Ovenden, J. R. (2016). The phylogenetic position of the roughskin skate Dipturus trachyderma (Krefft & Stehmann 1975) (Rajiformes, Rajidae) inferred from the mitochondrial genome. Mitochondrial DNA Part A 27, 2965–2966. DOI: 10.3109/19401736.2015.1060462.

Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2016). The complete validated mitochondrial genome of the yellownose skate Zearaja chilensis (Guichenot 1848) (Rajiformes, Rajidae). Mitochondrial DNA Part A 27, 1227–1228. DOI: 10.3109/19401736.2014.945530.

Vargas-Caro, C., Bustamante, C., Lamilla, J. & Bennett, M. B. (2015). A review of longnose skates Zearaja chilensis and Dipturus trachyderma. Universitas Scientiarum 20, 321–359. DOI: 10.11144/Javeriana.SC20-3.arol.

Bowden, D. L., Vargas-Caro, C., Ovenden, J. R., Bennett, M. B., & Bustamante, C. (2016). The phylogenetic position of the grey nurse shark Carcharias taurus Rafinesque, 1810 (Lamniformes, Odontaspididae) inferred from the mitochondrial genome. Mitochondrial DNA A 27, 4328–4330. DOI: 10.3109/19401736.2015.1089486.

Bustamante, C., Vargas-Caro, C. & Bennett, M. B. (2014). Biogeographic patterns in the cartilaginous fauna (Pisces: Elasmobranchii and Holocephali) in the southeast Pacific Ocean. PeerJ 2, e416. DOI: 10.7717/peerj.416.

–vi– Bustamante, C., Vargas-Caro, C. & Bennett, M. B. (2014). Not all fish are equal: Functional biodiversity of cartilaginous fishes (Elasmobranchii and Holocephali) in Chile. Journal of Fish Biology 85, 1617–1633. DOI: 10.1111/jfb.12517.

–vii– Publications included in this thesis

The following publications have been included into my thesis as per UQ policy (PPL 4.60.07 Alternative Thesis Format Options). Detailed contribution of all authors is described below as per the requirements in the UQ Authorship Policy (PPL 4.20.04 Authorship).

Chapter II Vargas-Caro, C., Bustamante, C., Lamilla, J. & Bennett, M. B. (2015). A review of longnose skates Zearaja chilensis and Dipturus trachyderma. Universitas Scientiarum 20, 321–359.

Contributor Statement of contribution Vargas-Caro, Carolina (Candidate) Designed experiments (30%) Wrote the paper (70%) Bustamante, Carlos Designed experiments (30%) Wrote and edited paper (15%) Lamilla, Julio Wrote and edited paper (1%) Bennett, Michael B. Designed experiments (40%) Wrote and edited paper (14%)

Chapter IV Vargas-Caro, C., Bustamante, C., Lamilla, J., Bennett, M. B. & Ovenden, J. R. (2016). The phylogenetic position of the roughskin skate Dipturus trachyderma (Krefft & Stehmann 1975) (Rajiformes, Rajidae) inferred from the mitochondrial genome. Mitochondrial DNA Part A 27, 2965–2966.

Contributor Statement of contribution Vargas-Caro, Carolina (Candidate) Designed experiments (70%) Wrote the paper (70%) Bustamante, Carlos Designed experiments (10%) Wrote and edited paper (19%) Lamilla, Julio Wrote and edited paper (1%) Bennett, Michael B. Designed experiments (10%) Wrote and edited paper (5%) Ovenden, Jennifer R. Designed experiments (10%) Wrote and edited paper (5%)

–viii– Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2016). The complete validated mitochondrial genome of the yellownose skate Zearaja chilensis (Guichenot 1848) (Rajiformes, Rajidae). Mitochondrial DNA Part A 27, 1227–1228.

Contributor Statement of contribution Vargas-Caro, Carolina (Candidate) Designed experiments (70%) Data collection and analysis (70%) Wrote the paper (70%) Bustamante, Carlos Designed experiments (10%) Data collection and analysis (20%) Wrote and edited paper (15%) Bennett, Michael B. Designed experiments (10%) Wrote and edited paper (10%) Ovenden, Jennifer R. Designed experiments (10%) Data collection and analysis (10%) Wrote and edited paper (5%)

Chapter V Vargas-Caro, C., Bustamante, C., Bennett, M. B. & Ovenden, J. R. (2017). Towards sustainable fishery management: The genetic population structure of Zearaja chilensis and Dipturus trachyderma (Chondrichthyes, Rajiformes) in the south-east Pacific Ocean. PLoS One 12, e0172255.

Contributor Statement of contribution Vargas-Caro, Carolina (Candidate) Designed experiments (80%) Data collection (80%) Data analysis (70%) Wrote the paper (70%) Bustamante, Carlos Designed experiments (10%) Data collection (20%) Data analysis (10%) Wrote and edited paper (10%) Bennett, Michael B. Wrote and edited paper (10%) Ovenden, Jennifer R. Designed experiments (10%) Data analysis (20%) Wrote and edited paper (10%)

–ix– Contributions by others to the thesis

Chapter I –The general introduction is my own work. My supervisor Michael B. Bennett reviewed and provided important editorial comments.

Chapter II – I am the primary author and Michael B. Bennett, Carlos Bustamante and Julio Lamilla are co-authors. All co-authors assisted with important editorial comments and final manuscript editing. Jennifer Ovenden and Ian Tibbetts assisted with editorial comments to earlier versions of the chapter. Claudio Barría, Dirk Hovestadt, Jürgen Pollerspöck and María Teresa Bravo provided important citations. Anonymous reviewers also provided critical revisions to the manuscript.

Chapter III – I am the primary author and Michael B. Bennett and Carlos Bustamante are co-authors. All co-authors assisted with important editorial comments and final manuscript editing. Astrid Isla, Cynthia Díaz, Yhon Concha-Perez, Julio Lamilla and students of ELASMOLAB from Universidad Austral de Chile and Programa de Conservación de Tiburones, provided support during fieldwork and samples collection. Sam Williams and Jennifer Ovenden, provided advice on laboratory procedures.

Chapter IV – I am the primary author and Michael B. Bennett, Carlos Bustamante, Julio Lamilla and Jennifer Ovenden are co-authors. All co-authors assisted with important editorial comments and final manuscript editing. Andrew Stewart, Claudio Barria, Malcolm Francis and, Astrid Isla, Julio Lamilla and Yhon Concha-Perez, provided samples.

Chapter V – I am the primary author and Michael B. Bennett, Carlos Bustamante and Jennifer Ovenden are co-authors. All co-authors assisted with important editorial comments and final manuscript editing. Astrid Isla, Cynthia Díaz, Yhon Concha-Perez, Julio Lamilla and students of Elasmobranchs Laboratory from Universidad Austral de Chile provided support during fieldwork and samples collection. Sam Williams, Chris Dudgeon and Jessica Morgan provided advice on laboratory procedures.

Chapter VI –The general discussion was my own work. My supervisor Michael B. Bennett and Carlos Bustamante reviewed and provided important editorial comments.

Statement of parts of the thesis submitted to qualify for the award of another degree

None.

–x– Acknowledgements

I remember the mad day I decided to pursue a PhD and today I reckon, I was more frightened than determined. And, here I am today with a beautiful manuscript about conservation of longnose skates. This project could not have been possible without the support/help/advise from many people that I will like to thank here. Most importantly, to my family support: Anita and Rodrigo, and my little brother back in Chile. Their love, care and enthusiasm got me through all this process. From the day I left the country, you guys kept telling me to keep chasing my dreams so, I obviously wouldn’t be here if it wasn’t for you.

To my beautiful daughter Matilda, you were born when I was trying to finish this thesis. You don’t even realise how important you were for us in this period of our lives. You simply gave me the strength to finish even when I thought this was not possible and I’m extremely grateful for that. To Carlos, my husband, my best friend, my person. I just have no words to thank you enough. You were there for me day and night through this journey…to support me, to give me your love, knowledge and OMG your patience, to tell me that everything was going to be ok when I most needed. Thank you for believing in me!

I would like to thank my supervisor team, Mike Bennett who from the very first day showed his enthusiasm for getting involved with non-emblematic or charismatic animals. You taught me so much on all our meetings and over sudoku’s lunch-time breaks; but most important, you were always there for me with a smile, a confort word or power-talk to kick-my-arse and put me back on track. Thanks for sharing all your knowledge and when I say “all” I’m talking even bird-watching knowledge. To Jenny Ovenden, I still have those notes you made for me about “Genetics 101” in our first meeting. Thank you for taking the time to teach me step by step every procedure at the lab. You made me laugh and cry during this PhD but I’m a better scientist now so thank you for that. To my former supervisor, Julio Lamilla. You might not be here today but this research was possible thanks to you too! You gave me the opportunity of working at Elasmolab as an undergrad and made me dream. You loved the elasmobranch world but your passion, were the skates. I know this little project was hard to complete but it was very rewarding. On the same, I should thank every member of the Elasmolab team, specially to Astrid, Cynthia and Yhon. Without your help during fieldwork, the inmensity of samples wouldn’t have been possible to collect. Also, I would like to thank to my friends, Caroll, Loreto and Monica, somehow you have always been with me through the years, I just love you girls!

–xi– Last, but not least, to all members of the Bennett lab. Guys, you were there pretty much every day, and whether was beer and/or coffee you help me so much with those conversations. Deb, Kate and Bonnie, you are beautiful people and I owe you so much for all your love, care and friendship.

Academic Supervision

Michael B. Bennett, Jennifer R. Ovenden (The University of Queensland).

External and Internal Examiners

Ian Tibbetts, Christine L. Dudgeon (The University of Queensland).

Financial Support

CONICYT–Becas Chile RHD scholarship; Top-Up Assistance Program (TUAP) of the Graduate School of The University of Queensland.

Assistance with specific methodologies and analyses

Carlos Bustamante (The University of Queensland).

Assistance with samples and sampling

Carlos Bustamante (The University of Queensland), Julio Lamilla, Astrid Isla, Cynthia Diaz, Yhon Concha-Perez, Tania Ponce (Universidad Austral de Chile).

Other

Fiona Gilloway and James Mather (SMBS Postgraduate Administration Officers), and Deborah McCamley and Don Weerheim (SBMS Finance); for their help and patience surfing through UQ’s paperwork. To all doctors and doctor-to-be on the Bennett’s Lab: Carlos Bustamante, Chris Dudgeon, Bonnie Holmes, Deb Bowden, Kate Burgess, Sam Williams, Amelia Armstrong, Asia Armstrong, Arnault Gauthier and Tom Kashiwagi.

–xii– Keywords

Longnose skates, yellownose skate, roughskin skate, South America, Chile, population management units, fisheries, mitochondrial DNA, mitogenomes, microsatellites.

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060207, Population Ecology, 20% ANZSRC code: 060411, Population, Ecological and Evolutionary Genetics, 60% ANZSRC code: 060205, Marine and Estuarine Ecology (incl. Marine Ichthyology), 20%

Fields of Research (FoR) Classification

FoR code: 0602, Ecology, 40% FoR code: 0604, Genetics, 50% FoR code: 0699, Other Biological Sciences, 10%

–xiii– Table of Contents

Abstract ii Declaration by author iv Publications during candidature v Publications included in this thesis viii Contributions by others to the thesis x Acknowledgements xi Table of Contents xiii List of Tables xvi List of Figures xviii

Chapter I Research context 1 1.1. General Introduction 2

Application of genetic in fishery management and conservation of skates 3

Aims and significance 5 1.2. General methodology 6

Chapter II Literature review 9 2. A review of longnose skates Zearaja chilensis and Dipturus trachyderma 10

Abstract 10

Introduction 11

Materials and methods 13

Results and discussion 14

Conclusion 50

Chapter III Species identification 51 3. Morphological and molecular identification of commercially important longnose skates (Chondrichthyes, Rajiformes) in the southeast Pacific Ocean 52

Abstract 52

Introduction 53

Materials and methods 55

Results 68

Discussion 78

–xiv– Chapter IV Molecular taxonomy 83 4.1. The complete validated mitochondrial genome of the yellownose skate Zearaja chilensis (Guichenot 1848) (Rajiformes, Rajidae) 84

Abstract 84

Introduction 85

Materials and methods 85

Results and discussion 85 4.2. The phylogenetic position of the roughskin skate Dipturus trachyderma (Krefft & Stehmann 1975) (Rajiformes, Rajidae) inferred from the mitochondrial genome 87

Abstract 87

Introduction 88

Materials and methods 88

Results and discussion 89 4.3. Phylogeny of longnose skates (genus Zearaja): species-level relationships among major lineages of skates (Chondrichthyes, Batoidea) inferred from whole mitochondrial genomes 91

Abstract 91

Introduction 92

Materials and methods 94

Results 102 Discussion 110

Chapter V Genetic population structure 114 5. Towards sustainable fishery management: The genetic population structure of Zearaja chilensis and Dipturus trachyderma (Chondrichthyes, Rajiformes) in the south-east

Pacific Ocean 115

Abstract 115 Introduction 116 Materials and methods 118 Results 126 Discussion 138

–xv– Chapter VI General Discussion 143

Chapter VII References 150

Chapter VIII Appendices 196

–xvi– List of Tables

Table 1. Variation of the number on dermal denticles for the yellownose skate Zearaja chilensis and roughskin skate Dipturus trachyderma (Modified after Guichenot, 1848; Philippi, 1892; Delfín, 1902; Norman, 1937; De Buen, 1959; Menni, 1973; Leible, 1987; Leible & Stehmann, 1987; Lloris & Rucabado, 1991; Bizikov et al., 2004). 30 Table 2. Summary of age and growth studies for Zearaja chilensis and Dipturus trachyderma.

Key: L∞ = length at infinite age, k = growth coefficient, t0 = approximate time at which the

animal was size 0, tmax = estimated longevity, t50 = age at which the 50% of the population was

mature, L50 = length at which the 50% of the population was mature. 40 Table 3. Parasites reported to be infecting the yellownose skate Zearaja chilensis. 48 Table 4. Parasites reported to be infecting the rough skate Dipturus trachyderma. 49 Table 5. Definitions of morphometric and meristic variables taken on commercial longnose skates in Chile. Methodology to record for each variable is explained in Last et al. (2008b). 58 Table 6. List of primers used for PCR amplification and sequencing of commercial longnose skates in Chile. 60 Table 7. List of haplotypes of Zearaja chilensis and Dipturus trachyderma used for comparison analysis. Accessions numbers for GenBank are presented for each specimen and gene region amplified. 60 Table 8. List of species and haplotypes used for phylogenetic analysis, including accessions numbers for GenBank. 65

Table 9. Number of specimens (n) and size structure (as total length, LT) of sampled commercial longnose skates, by sex and locality. 68 Table 10. Morphometric and meristic data of Zearaja chilensis (n = 406) and Dipturus trachyderma (n = 177). Range and mean values are expressed in % of total length (Disc–1) with the exception of meristic variables. Definition for each variable is presented in Table 5. Length of clasper (Disc–13) is only applicable for males. 70 Table 11. Overall contribution (%) of morphometric and meristic variables to the differentiation between groups after SIMPER test. Definition for each variable is presented on text. 74 Table 12. Taxonomic list of species included in the present study. Accession numbers are indicated for new (eFish) and previously published mitogenomes (GenBank). The absence of deposited BioVoucher is indicated (–). 96 Table 13. Maximum Likelihood fits of 24 different nucleotide substitution models tested for the longnose skates mitogenomic dataset. 100

–xvii– Table 14. Pairwise patristic distances (n-distance; below the diagonal) and the number of non- identical bases (differences, SNPs; above the diagonal) in the mitogenome of Zearaja species. 103 Table 15. Amount of primer stock per microsatellite locus added to primer mix to set up multiplexed PCRs for genotyping Z. chilensis and D. trachyderma. Multiplex numbers are indicated in brackets. 125 Table 16. Geographic distribution of mtDNA Control Region haplotypes found in Zearaja chilensis and Dipturus trachyderma. Samples per locality and haplotypes are indicated as the proportion of the total number of specimens with that haplotype. Sampling localities: SA, San Antonio; VA, Valdivia; PM, Puerto Montt; AY Aysén; and PA, Punta Arenas. 128 Table 17. Genetic diversity per sampling locality from mtDNA control region and microsatellite loci for Zearaja chilensis and Dipturus trachyderma. Number of individuals (N); number of

mtCR haplotypes (Nh); haplotype diversity ± standard deviation (h); nucleotide diversity ±

S.D. (π); maximum number of alleles observed per locus (NA); observed (HO) and expected

heterozygosity (HE); Wright’s inbreeding coefficient of individuals relative to the

subpopulation (FIS). Values for microsatellite data are presented as the average across loci. Sampling locations as in Table 15. 129

Table 18. Estimates of pair-wise genetic distance (FST) (below diagonal) among locations for mtDNA control region (mtCR) sequences and microsatellites loci for Zearaja chilensis and

Dipturus trachyderma. FST P-values are indicated above diagonal. Significant ФST and FST values are presented in bold. Significance accepted at P < 0.05. Sampling locations are described in Table 15. 131 Table 19. Characterisation of polymorphic microsatellite loci used for Zearaja chilensis (n = 9) and Dipturus trachyderma (n = 7) in this study. Locus name, primer sequences, repeat motif,

allele size, maximum number of alleles (NA) and expected (HE) and observed (HO) heterozygosities. (―) Indicates microsatellite loci not used on the species. 134 Table 20. Genetic diversity per locality and microsatellite locus for Zearaja chilensis and Dipturus trachyderma. Sampling localities are as in Table 15. 135

–xviii– List of Figures

Figure 1. Map of Chile indicating main landing localities of longnose skates fisheries. 8 Figure 2. Cumulative number of peer-reviewed papers, books, government reports, separated into seven categories for (A) Zearaja chilensis and (B) Dipturus trachyderma. 15 Figure 3. Number of published studies for Zearaja chilensis (1848–2014) (black bars) and Dipturus trachyderma (1975–2014) (grey bars) by research category. 16 Figure 4. Total annual numbers of published studies for (A) Zearaja chilensis and (B) Dipturus trachyderma. 17

Figure 5. Morphotypes for Zearaja chilensis. A. Original description, female, 750 mm LT, Chile

(Philippi, 1982). B. Mature male, 975 mm LT, Chile (Delfín, 1902). C. Adult male, Argentina

(Norman, 1937). D. Immature male, 374 mm LT, Argentina (Bigelow & Schroeder, 1958). E. Mature male, Argentina (Lloris & Rucabado, 1991). F. Female, Falkland Island, British

Overseas Territory (Bizikov et al., 2004). G. Female, 690 mm LT, Los Vilos, Chile (Melo et

al., 2007). H. Mature female, 770 mm LT, Valdivia, Chile. 20

Figure 6. Morphotypes of Dipturus trachyderma. A. Original description, female, 1135 mm LT,

off Argentina (Krefft & Stehman, 1975). B. Male, 2000 mm LT, Chile (Leible & Stehmann,

1987). C. Male, 2075 mm LT, Beagle Channel, Argentina (Lloris & Rucabado, 1991). D. Female, Falkland Islands, British Overseas Territory (Bizikov et al., 2004). E. Male, 2007 mm

LT, Valdivia, Chile. 24 Figure 7. Distribution of the yellownose skate Zearaja chilensis in South America. The numbers represent documented records following (1) Philippi (1892), (2) Delfín (1902), (3) Lohnnberg (1907), (4) Gotschlich (1913), (5) Norman (1937), (6) De Buen (1959), (7) Carvajal (1971), (8) Menni (1973), (9) Sadowsky (1973), (10) Ojeda (1983), (11) Raschi (1984), (12) Fernández & Villalba (1985), (13) Leible (1987), (14) Leible et al. (1990), (15) Lloris & Rucabado (1991), (16) Lucifora et al. (2000), (17) Gomes & Picado (2001), (18) Koen Alonso et al. (2001), (19) Céspedes et al. (2005), (20) Licandeo et al. (2006), (21) Cousseau et al. (2007), (22) Licandeo & Cerna (2007), (23) Aburto et al. (2008), (24) Díaz de Astarloa et al. (2008), (25) Quiroz et al. (2009), (26) Silveira (2009), (27) Arkhipkin et al. (2012), (28) Bustamante et al. (2012), (29) Deli Antoni et al. (2012), (30) Bustamante et al. (2014b). Filled circle indicates the holotype locality. 35 Figure 8. Distribution of the roughskin skate Dipturus trachyderma in South America. The numbers represent documented records following (1) Krefft & Stehmann (1975), (2) Menni & Gosztonyi (1977), (3) FRV “Walther Herwing” (1978), (4) FRV “Holmberg” (1983), (5) Menni et al. (1984), (6) Leible & Stehmann (1987), (7) Lloris & Rucabado (1991), (8)

–xix– Cousseau et al. (2000), (9) Gomes & Picado (2001), (10) Knoff et al. (2001b), (11) Cedrola et al. (2005), (12) Céspedes et al. (2005), (13) Díaz de Astarloa et al. (2008), (14) Lamilla et al. (2010), (15) Perier et al. (2011), (16) Arkhipkin et al. (2012), (17) Lamilla et al. (2012a), (18) Lamilla et al. (2012b), (19) Bustamante et al. (2014b). Filled circle indicates the holotype locality. 36 Figure 9. Spatial distribution of the Zearaja chilensis and Dipturus trachyderma fishery in Chile, indicating main fishing locations and the extension of the artisanal (dark green) and industrial fisheries (light green). 43 Figure 10. Total longnose skate landing in Chile between 1979 and 2012 by fishery and species. Total catch is represented by the solid line. Artisanal (dashed line) and industrial (dotted line) landing are indicated for the ‘skates’ landing category (black), Dipturus trachyderma (green) and Zearaja chilensis (blue). [Note: Z. chilensis and D. trachyderma were officially separated into different landing categories by the Fishing Authority in 2004]. 44 Figure 11. Total landings of Dipturus trachyderma between 2004 and 2012 by fishery type: artisanal= black, industrial= green. 45 Figure 12. Map showing landing locations of longnose skates in Chile surveyed in this study. 55 Figure 13. Standardized photographs used to confirm species identification of longnose skates landed in Chile. Dorsal and ventral views of Zearaja chilensis (A, B). Dorsal and ventral views of Dipturus trachyderma (C, D). Colorimetric scale bar = 25 cm. 57 Figure 14. Non-metric multidimensional scaling (nMDS) ordination plot of morphometric measurements and meristic counts recorded on (A) whole population of commercial longnose skates from Chile (n =583). Differences by sex are presented for (B) Zearaja chilensis and (C) Dipturus trachyderma. 73 Figure 15. Neighbour-joining distance phylogenetic tree inferred from K2P distance model on mitochondrial DNA haplotypes of (A) 16S and (B) the control region, for Zearaja chilensis and Dipturus trachyderma, and close-related species within the Family Rajidae. Branches with identical haplotypes have been collapsed and the number of sequences is indicated in between brackets. Estimation of the nodes stability is expressed as a percentage (1,000 bootstrap replicates) and is only indicated (*) on nodes with >80%. 76 Figure 16. Neighbour-joining distance phylogenetic tree inferred from K2P distance model on mitochondrial DNA haplotypes of (A) cox1 and (B) nadh2, for Zearaja chilensis and Dipturus trachyderma, and close-related species within the Family Rajidae. Branches with identical haplotypes have been collapsed and the number of sequences is indicated in between brackets. Estimation of the nodes stability is expressed as a percentage (1,000 bootstrap replicates) and is only indicated (*) on nodes with >80%. 77

–xx– Figure 17. Morphology of adult (A) male and (B) female of Zearaja chilensis. Secondary sexual characters are indicated for each sex. For males, (a) hypertrophy of the mandibular abductor muscle and (b) enlargement of the pelvic fin base. 80 Figure 18. Comparison of the average pairwise identity (%) of Zearaja chilensis (Grey line) and Zearaja chilensis (Jeong & Lee, 2014) (Dashed line) mtDNA genes. The number of SNPs per genes is indicated in brackets. 86 Figure 19. Phylogenetic relationships of Dipturus trachyderma (in bold) inferred from the complete mitogenome, using closely related species of the family Rajidae. Taxonomic subdivisions are indicated for each major clade. Bootstrap values obtained by 1,000 steps are indicated for each node. GenBank accession numbers are provided following species names. Scale bar represent the expected substitution per site. 90 Figure 20. Phylogeny of longnose skates of the genus Zearaja (Rajidae, Rajiformes), inferred by maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) method. Consensus trees are based on the General Time Reversible model (16,720 aligned positions; 56 taxa; tree size: 5.306; Γ: 0.956; I: 0.336). Species names in bold, indicates new mitogenomes. Nodes with capital letters indicate a major lineage discussed in text, and represents (A) Batoids, (B) Myliobatiformes + Rhinopristiformes clade, (C) Torpediniformes + Rajiformes clade, (D) Order Rajiformes, (E) Family Anacanthobatidae, (F) Arhynchobatidae + Rajidae clade, (G) Family Arhynchobatidae, (H) Family Rajidae, (I) Tribe Amblyrajini, (J) Tribe Rajini, (K) shortnose skates clade, and (L) longnose skates clade. Bootstrap values (ML, MP) and posterior probability (BI) are represented for each node and are colour-coded. Scale bar represents number of substitution per site. 106 Figure 21. Evolutionary history of longnose skates of the genus Zearaja inferred from mitogenomes. Timetree was generated using the RelTime method and divergence times for all branching points in the topology were calculated using the General Time Reversible model. A total of four nodes with 95% credible intervals of divergence times from fossil records were used for the time constraints (see text). The dotted lines indicate the major marine extinction events during the Phanerozoic (Rohde & Muller 2005). 108 Figure 22. Map showing landing locations of Zearaja chilensis and Dipturus trachyderma surveyed in this study. San Antonio (blue) and Valdivia (grey) represent off-shore fishing grounds. Puerto Montt (yellow) and Aysén (orange) represent fishing grounds within the Chiloé Interior Sea. Punta Arenas (red) represent an admixture of fishing grounds between oceanic and fjord ecosystems. 119

–xxi– Figure 23. Standardized photographs used to confirm species identification of longnose skates. Dorsal and ventral views of Zearaja chilensis (female A, B; male C, D). Dorsal and ventral views of Dipturus trachyderma (female E, F; male G, H). Colorimetric scale bar = 25 cm. 120 Figure 24. Median-joining network of mtCR haplotypes for (A) Zearaja chilensis and (B) Dipturus trachyderma. Haplotypes are represented by circles with size proportional to frequency in the total sample. All branches correspond to one mutation except where indicated otherwise. Sampling locations: San Antonio (SA, blue), Valdivia (VA, grey), Puerto Montt (PM, yellow), Aysén (AY, orange) and Punta Arenas (PA, red). 130 Figure 25. Correlation between genetic and geographic distance, for populations of (A) Zearaja chilensis and (B) Dipturus trachyderma. Linear adjustment (solid line) and 95% confidence interval (dashed line) are specified. Geographic distance are indicated by paired locations, (a) SA and VA; (b) SA and PM; (c) SA and AY; (d) SA and PA; (e) VA and PM; (f) VA and AY; (g) VA and PA; (h) PM and AY; (i) PM and PA; (j) AY and PA. Sampling locations abbreviations as in Fig 22. 136 Figure 26. Scatterplots of the Discriminant Analysis of Principal Components (DAPC) from microsatellite genotypes for populations of (A) Zearaja chilensis and (B) Dipturus trachyderma. Dots represent individuals, whereas coloured ellipses correspond to geographical populations. Sampling locations: San Antonio (SA, blue), Valdivia (VA, grey), Puerto Montt (PM, yellow), Aysén (AY, orange) and Punta Arenas (PA, red). 137 Figure 27. Scatterplots of the Discriminant Analysis of Principal Components (DAPC) from microsatellites genotypes comparing (A) Zearaja chilensis and (B) Dipturus trachyderma. Dots represent individuals, whereas coloured ellipses correspond to geographical populations. Sampling locations: San Antonio (SA, blue), Valdivia (VA, grey), Puerto Montt (PM, yellow), Aysén (AY, orange) and Punta Arenas (PA, red). 141

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–1– CHAPTER I: RESEARCH CONTEXT

1.1. General introduction

The Class Chondrichthyes includes most of the extant fishes with a cartilaginous skeleton, such as sharks, rays, skates, and chimeras (Eschmeyer & Fong, 2011). Worldwide approximately 1,207 living species have been described, with fewer than 50 species in the Subclass Holocephali (Didier, 2004; Eschmeyer & Fong, 2011) and an overwhelming majority in the Subclass Euselachii\Elasmobranchii (Nelson, 2016) that comprises sharks, skates, and rays (McEachran & Aschliman, 2004; Ebert & Compagno, 2007). Conservation threats to chondrichthyan fishes due to direct and indirect fishing have been documented worldwide (Pauly et al., 2002; Worm et al., 2006; Dulvy et al., 2014), and cases of population level declines of large-bodied and less resilient species are increasing in the literature (Stevens et al., 2000; Baum et al., 2003; Myers et al., 2007; Dulvy et al., 2008; Ferretti et al., 2008; Dulvy et al., 2014). Skates belonging to the Family Rajidae (hardnose skates) inhabit mainly marine environments from the sublittoral zone up to 3,000 m in depth. They comprise the most diverse group of batoids, with about 27 genera and 245 species distributed worldwide (McEachran & Miyake, 1990; Ebert & Compagno, 2007; Weigman, 2016). This taxon represents about 20% of all extant chondrichthyans (Compagno, 2005a, b), and is the only group of the cartilaginous fishes that exhibits high species diversity in high latitudes and deep waters (McEachran & Miyake, 1990; Ebert & Compagno, 2007). Five genera of the Family Rajidae are present in Chilean waters: Amblyraja Malm 1877, Dipturus Rafinesque 1810, Gurgesiella de Buen 1959, Rajella Stehmann 1970 and Zearaja Whitley 1939 (Leible & Stehmann, 1987; Pequeño, 1989; Pequeño & Lamilla, 1993; Lamilla & Bustamante, 2005; Bustamante et al., 2014b), but only two genera, Dipturus and Zearaja, are caught and landed in commercial fisheries (Lamilla et al., 2010). In general longnose skates, a subgroup of within the Family Rajidae that comprises the genera Dipturus, Zearaja, have unique challenges for fishery management (Stevens et al., 2000) and conservation (Robert & Hawkins, 1999; Dulvy & Reynolds, 2002) to the species’ life histories, particularly late age-at-maturity, and low fecundity due to considerable maternal investment in egg production (Du Buit, 1968; Holden, 1977; Dulvy et al., 2000; Frisk et al., 2001; Dulvy & Reynolds, 2002; Myers & Worm, 2005). Despite being a large taxon within the skates (Compagno, 1999b; Ebert & Compagno, 2007; Séret & Last, 2008), the biology and ecology of longnose skates is poorly known and requires morphological, taxonomic and molecular research; particularly as many longnose skate species are subject to intense fishery exploitation worldwide (Dulvy et al., 2000; Myers & Worm 2005; Dulvy et al., 2014). Intense local fisheries with unmanaged landings are often accompanied by a lack of the biological information required to maintain a healthy fishing stock (Ferretti et al., 2008;

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2010). Agnew et al. (2000) suggested that longnose skates are generally vulnerable to stock collapse even when subjected to low levels of fishing pressure. Large population declines and local extinctions of longnose skates have been documented in the last 10 years. The common skate Dipturus batis (L. 1758), barndoor skate Dipturus laevis (Mitchill 1818), white skate Rostroraja alba (Lacepède 1803), thornback ray Raja clavata L. 1758, smooth skate Dipturus innominatus (Garrick & Paul 1974) and sharpnose skate Dipturus oxyrinchus (L. 1758), are the best documented examples of local local declines in cartilaginous fishes as a result of fishing activities (Casey & Myer, 1998; Dulvy et al., 2008; Francis et al., 2001; Dulvy & Forrest, 2010). In the southeast Pacific Ocean, there are only a few cartilaginous fishes that are landed as target species, and the yellownose skate Zearaja chilensis (Guichenot 1848) is one of the most valuable chondrichthyans in Chilean fisheries (Lamilla et al., 2010; SERNAP, 2012). While there is a directed fishery for yellownose skate, the roughskin skate Dipturus trachyderma (Krefft & Stehmann 1975) is often caught in smaller numbers as bycatch. Declining population sizes, habitat restriction, and extinction probability resulted in a Red List of Threatened SpeciesTM classification of Vulnerable for both Zearaja chilensis and Dipturus trachyderma, with an extinction probability of 10% (Kyne et al., 2007; Lamilla & Massa, 2007). This risk assessment provided a basis for management of the species, and as consequence the Chilean Government issued restricted catch quotas to regulate target fisheries as a “conservation” measure. However, a review of Z. chilensis fishery biology provided the first evidence of a decrease in landing volumes and symptoms of population collapse (Bustamante et al., 2012). As Z. chilensis and D. trachyderma are caught in the same fisheries there is a clear requirement for re-evaluation of both species, especially as the Red List assessment was conducted when information available in relation to the species’ biology, ecology and fisheries-interactions was very limited.

Application of genetic in fishery management and conservation of skates

Current research has provided strong evidence that many chondrichthyan fishes have a high potential risk for future extinction (García et al., 2008). Given this growing concern for skate populations worldwide, there is a strong ongoing need for science to help improve community understanding and the conservation management of these organisms (White & Last, 2012). Many wildlife conservation and management measures fail in their ultimate goal, as authorities misunderstand the concept of conservation to, for example, modify fishing quotas (Jordan, 1995). Conservation is the act of preserving, guarding or protecting biodiversity, environment, and natural resources, including protection and management (Dobson, 1996), however, defining a taxonomic entity is a crucial first step for conservation (Mace, 2014). Taxonomic distinction plays an important role in conservation, determining whether or not the effort is adequately directed: Erroneous

–3– CHAPTER I: RESEARCH CONTEXT classification, e.g. grouping multiple cryptic species into one taxonomic entity, may deny species adequate protection even when conservation management is applied (Mace, 2014). Recently, the important role of chondrichthyan taxonomy for conservation management has been highlighted with a taxonomic resolution of a number of threatened species (White & Last, 2012), but when taxonomic differentiation is unclear, it often can be resolved using molecular markers (Dudgeon et al., 2012). The recent development of molecular tools to assist with species identification has also highlighted cryptic species and species complexes, leading to undescribed taxa being identified and described (White & Last, 2012; Cerutti-Pereyra et al., 2012; Tillett et al., 2012). Genetics also can allow researchers to gain greater insight into population dynamics and can help to answer important questions that previously may not have been possible to address with traditional field techniques (Schwartz et al., 2007; Ovenden, 2013). Understanding genetic diversity, population connectivity, and trends of abundance are crucial to the development of conservation measures for vulnerable species (Reed & Frankham, 2003). Genetic studies on elasmobranch populations often allow for inferences to be made about historical and contemporary processes responsible for observed patterns of spatial genetic differentiation, and can identify operational taxonomic units (OTUs) of importance for fisheries management and conservation purposes (Beheregaray, 2008; Ovenden, 2013). The integrating concept commonly used for the management of threatened species, the OTU, is not widespread in marine populations, but can be a basic tool for defining a species (Allendorf & Luikart, 2007). The ‘species’ concept can have different meanings according to the field of work or research scope. The biological species concept (BSC) defines a species as groups of interbreeding (or potentially interbreeding) natural populations that are reproductively isolated from other such groups (Mayr, 2000); the morphological species concept (MSC) is based on individuals sharing a set of morphological characters and are therefore able to be distinguished on the basis of discontinuities in these phenotypic characteristics (Keogh, 1999); the genetic species concept (GSC) invokes genetically compatible interbreeding natural populations that are genetically isolated from other such groups (Baker & Bradley, 2006); and the evolutionary species concept (ESC) defines a species as a "lineage (an ancestral descendant sequence of populations) evolving separately from others and with its own unitary evolutionary role and tendencies" (Simpson, 1961). Aware of the gaps and criticisms about the use of these concepts alone, and for the purposes of this study, ‘species’ are defined as our OTUs, linking every concept of species. Operational taxonomic units (or species) require management as separate units. However, populations within species may be on the path to speciation. If they show significant adaptive differentiation to different habitats or significant genetic differentiation, then they may justify management as separate evolutionary lineages for conservation purposes (Frankham et al., 2004).

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This assumption that populations may be on independent evolutionary paths has shifted the efforts of conservation away from species as the essential taxonomic units, instead population genetic data can greatly contribute to determining whether populations can be considered independent and therefore as separate management units, such as evolutionary significant units (ESUs) (Moritz, 1994, 1995; Paetkau, 1999; Crandall et al., 2000; Fraser & Bernatchez, 2001; Hey et al., 2003). The ESU concept was developed to provide an objective approach to prioritizing units for protection below taxonomic levels (Ryder,1986), given that existing taxonomy may not amply reflect the underlying genetic diversity and that resources are limited (Avise, 1989). An evolutionary significant unit (ESU) can be defined broadly as a subspecies, population or group of populations that merit separate management or priority for conservation because of high distinctiveness (both genetic and ecological) (Allendorf & Luikart, 2007). Moritz (1994) proposed that genetic markers be used to define management units within species. If mtDNA genotypes show no overlap between populations, and nuclear loci show the significant divergence of allele frequencies, then they should be defined as separate ESUs and managed separately. More accurately, there has been ongoing discussion about how it should be defined (e.g. Moritz, 1994; Nielsen & Powers, 1995; Legge et al., 1996; Pennock & Dimmick, 1997; Bowen, 1998; Duvernell & Turner, 1998; Waples, 1998; Bowen, 1999; Dimmick et al., 1999; Karl & Bowen, 1999; Moritz, 1999; Paetkau, 1999; Crandall et al., 2000; Goldstein et al., 2000). Some authors have pointed out that an ESU can be divided into several management units (MUs) (Taberlet et al., 1998; Waits et al., 2001; Palsbøll et al., 2007). The MUs usually are defined as populations, stocks or group of stocks that are demographically independent. The identification of these units would be useful for short-term management – such as delineating hunting or fishing areas, setting local harvest quotas, and monitoring habitat and population status (Allendorf & Luikart, 2007). MUs, unlike ESUs, generally do not show long-term independent evolution or strong adaptive differentiation. The conservation of multiple populations, not just one or two, is critical for ensuring the long-term persistence of species (Hughes et al. 1997; Hobbs & Mooney, 1998).

Aims and significance

Management and conservation of skates arise as a necessary duty for fishing countries in order to maintain biodiversity and ecosystem structures (Agnew et al., 2000; Dulvy et al., 2008). This research thesis provides the basic information required to clarify taxonomic and systematic issues within the Family Rajidae. Solving the genus re-assignment between Dipturus and Zearaja may lead to fishing quotas in Chilean waters being re-assessed, leading to improved resource allocation for sustainable management of these endemic species of conservation concern. While some biological

–5– CHAPTER I: RESEARCH CONTEXT aspects of Zearaja chilensis have been reasonably well studied, information available for Dipturus trachyderma is very limited. However, sustainable management of a species requires knowledge of basic aspects of life history as well as the ecological significance of the impact of fishing, so the results of this research will unify the information available to produce a baseline for future research. Research in this thesis aimed to address knowledge gaps on the biology, taxonomy, and genetics of skates in the South East Pacific Ocean; to define OTUs and describe the population structure of Z. chilensis and D. trachyderma to permit recommendations on sustainable management for South American skates in the Pacific to be made. The following chapters aimed to (a) perform an exhaustive literature review about Zearaja chilensis and Dipturus trachyderma, (b) identify the operational taxonomic units (OTUs) through morphological and molecular methods, (c) describe the evolutionary history and phyletic relation of longnose skates and, (d) identify the population genetic structure of the OTUs.

1.2. General methodology

The present thesis is separated into chapters, each of them addresses a particular aspect of the biology, taxonomy, and genetics of commercial longnose skates in the southeast Pacific Ocean. Taken together, the chapters provide a comprehensive basis for conservation-concerned fisheries management in the region. Samples were collected during research surveys in different localities along the Chilean Coast, as part of a joint project between The University of Queensland and the Universidad Austral de Chile. The project -Population units of the yellownose and roughskin skates in Chile, was funded by the Fisheries Research Fund (a Chilean entity analogous to Australia’s Fisheries Research and Development Corporation). I provide insight on the use of genetics to identify population structure, and how that information can be used in new ways to investigate the extent of the movement and interbreeding between populations. Novel methods to investigate connectivity, and estimate movement and interbreeding across different types of marine species could fine-tune fisheries models and promote further collaboration. However, mutual understanding between geneticists and scientists who work in the field of fisheries is needed yet, currently lacking nowadays. Given the increased level of interest in the use of genetics in wild fisheries management (Dichmont et al., 2012), and the technological and theoretical improvements in the field of population genetics that have occurred over the past five- years (Allendorf et al., 2010; Helyar et al., 2011; Dudgeon et al., 2012; Hoban et al., 2012). Specimens of Z. chilensis and D. trachyderma used in the current study were sourced from within Chilean territorial waters. Collection locations (Fig. 1) were selected based on the geographical

–6– CHAPTER I: RESEARCH CONTEXT distribution, the fisheries, and the main landing sites (based on historical landings records sensu SERNAP, 2013) of the two species: San Antonio (33°35.5'S 71°37'W) and Valdivia (39°52.5'S 73°24'W) represent off-shore fishing grounds in the north; Puerto Montt (41°28.4'S 72°56.4'W) and Aysén (45°25'S 72°49'W) represent the Chiloé Interior Sea and; Punta Arenas (53°10.2'S 70°54.5'W) represents an admixture of oceanic and fjord fishing grounds in the south of Chile. Sampling was associated with the commercial fishery, and specimens of both Z. chilensis and D. trachyderma were retained at random. Morphometric and demographic data (length, mass, sex, and maturity) were determined for each specimen. Tissue samples were collected from specimens collected in each of the five localities for subsequent genomic sequencing and microsatellite genotyping for population structure analysis. Following the general aims of the thesis, each specific objective is presented separately and consecutively as part of a logical progression of research in sequence, i.e., literature review, genetic and demographic analysis, and determination of origins. Each specific objective has its own methodology and hypothesis, as appropriate. The linear, step-wise structure of the thesis allows for discrete stand-alone outputs that are subsequently integrated into a conceptual population model for the two-studied species.

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Figure 1. Map of Chile indicating main landing localities of longnose skates fisheries.

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2

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2. A review of longnose skates Zearaja chilensis and Dipturus trachyderma

Originally published in Universitas Scientiarum (ISSN: 0122-7483)

Copyright © Carolina Vargas-Caro, 2015

Published online: 27 Aug 2015

License Number: Open Access

Abstract

Longnose skates may have a high intrinsic vulnerability among fishes due to their large body size, slow growth rates and relatively low fecundity, and their exploitation as fisheries target-species places their populations under considerable pressure. These skates are found circumglobally in subtropical and temperate coastal waters. Although longnose skates have been recorded for over 150 years in South America, the ability to assess the status of these species is still compromised by critical knowledge gaps. Based on a review of 185 publications, a comparative synthesis of the biology and ecology was conducted on two commercially important elasmobranchs in South American waters, the yellownose skate Zearaja chilensis and the roughskin skate Dipturus trachyderma; in order to examine and compare their taxonomy, distribution, fisheries, feeding habitats, reproduction, growth and longevity. There has been a marked increase in the number of published studies for both species since 2000, and especially after 2005, although some research topics remain poorly understood. Considering the external morphological similarities of longnose skates, especially when juvenile, and the potential niche overlap in both, depth and latitude it is recommended that reproductive seasonality, connectivity and population structure be assessed to ensure their long-term sustainability.

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INTRODUCTION

Global threats to sharks, skates and rays have been increasing due to direct and indirect fishing effects (Pauly et al., 2002; Worm et al., 2006; Dulvy et al., 2014). Important declines in exploited population and species have been documented, particularly in larger and less resilient species (Stevens et al., 2000; Baum et al., 2003; Myers et al., 2007; Dulvy et al., 2008; Ferretti et al., 2008; Dulvy et al., 2014).

Skates (Family Rajidae) are the most diverse group of batoids, with 27 genera and about 245 species distributed worldwide (McEachran & Miyake, 1990; Ebert & Compagno, 2007) representing about 25% of all extant chondrichthyan species (Compagno, 2005 a, b). Rajids mostly inhabits marine environments from the sublittoral zone to depths of about 3,000 m and exhibits high species diversity in high latitudes and deep waters (McEachran & Miyake, 1990; Ebert & Compagno, 2007).

In general, longnose skates (Tribe Rajini) present unique challenges for fishery management (Stevens et al., 2000) and conservation (Robert & Hawkins, 1999; Dulvy & Reynolds, 2002) due to large adult body size, low fecundity, late age at sexual maturity, relatively high longevity and large offspring size (Du Buit, 1968; Holden, 1977; Frisk et al., 2001; Dulvy & Reynolds, 2002; Myers & Worm, 2005; Dulvy et al., 2014). The biology and ecology of longnose skates is poorly known and requires further morphological, taxonomic and molecular research in spite of being one of the largest taxa within batoids (Compagno, 1999a; Ebert & Compagno, 2007) and have been subject to intense fishery exploitation (Baum et al., 2003; Myers & Worm, 2005; Myers et al., 2007; Dulvy et al., 2008, 2014; Ferretti et al., 2008). Intense local fisheries with unmanaged landings are often accompanied by a lack of biological information required to maintain a healthy fishing stock (Ferretti et al., 2008, 2010), and it has been suggested that longnose skates are generally vulnerable to stock collapse even when subjected to low levels of fishing pressure (Agnew et al., 2000). Large population declines and local extinctions of longnose skates have been documented over recent years. The common skate Dipturus batis (L. 1758), barndoor skate D. laevis (Mitchill 1818), white skate Rostroraja alba (Lacepède 1803), thornback ray Raja clavata L. 1758, smooth skate D. innominatus (Garrick & Paul 1974) and longnosed skate D. oxyrinchus (L. 1758), are among the best documented examples of local declines in cartilaginous fishes as a result of fishing activities (Casey & Myer, 1998; Francis et al., 2001; Dulvy et al., 2008; Dulvy & Forrest, 2010).

Relatively few fisheries specifically target elasmobranch fishes in Chilean waters and among those, the yellownose skate Zearaja chilensis (Guichenot 1848) and the roughskin skate Dipturus trachyderma (Krefft & Stehmann 1975) are the most valuable species (Bustamante et al., 2012; SERNAP, 2012). Although there are basic identification keys and field guides for longnose skates of

–11– CHAPTER II: LITERATURE REVIEW the southeast Pacific, poor species identification and the lack of specific landing records are big issues for fishery management and quota allocation, impairing conservation management. In particular, these two commercially important species, Z. chilensis and D. trachyderma, have an additional vulnerability as both are endemic to southern South America, occurring in temperate-cold waters (De Buen, 1959; Leible & Stehmann, 1987; Agnew et al., 2000; Menni & Stehmann, 2000; Gomes & Picado, 2001; Bustamante et al., 2014b).

From the taxonomic perspective, the genus Zearaja was described in 1939 from a monotypic species, Z. nasuta (Muller & Henle 1841), which was relocated from the genus Raja. Later, Stehmann (1990) synonymised this taxon within Dipturus, but Last & Yearsley (2002) resurrected Zearaja as subgenus by the relocation of two species (Z. chilensis and Z. nasuta) and suggested that zearajids are likely to be generically distinct from rajids due to skeletal differences, e.g. in clasper, neurocranium, pelvic and pectoral girdle morphologies. Last & Gledhill (2007) suggested the upgrade of Zearaja to a genus based on key features of the external and internal anatomy, i.e., disc width/length ratio (giving it the familiar sub-rhomboid shape), presence of a strong rostral cartilage, orbital and midline thorn patterns and all the skeletal characteristic previously mentioned. Currently, the genus Zearaja includes three valid species: Z. nasuta from New Zealand, Z. chilensis from Chile and Argentina, and Z. maugeana Last & Gledhill 2007, recently described from Tasmanian waters in Australia. However, these changes to the genus Zearaja are one part of the taxonomic and systematic issues of genera within the Family Rajidae (Eschmeyer & Fong, 2011).

Longnose skates of the genus Dipturus have a widespread distribution including waters of the Caribbean (Compagno, 1999a; Gomes & Picado, 2001), northwestern Atlantic Ocean (McEachran & Carvalho, 2002; Gedamke et al., 2005; Schwartz, 2012), off South America (Gomes & Picado, 2001; Soto & Mincarone, 2001; Díaz de Astarloa et al., 2008; Bustamante et al., 2014b), Australasia (Last et al., 2008a; Last & Alava, 2013), western Europe and Africa (Ellis et al., 2005a, b; Ebert & Compagno, 2007; Cannas et al., 2010). The genus Dipturus Rafinesque 1810 was described using Raja batis (Linnaeus 1758) as the type species and initially was considered a subgenus within genus Raja (Stehmann, 1973; Ishihara & Ishiyama, 1986; Ishihara, 1987; Pequeño, 1989; Séret, 1989; Pequeño & Lamilla, 1993; Jacob & McEachran, 1994; López et al., 1996; Bizikov et al., 2004), but was resurrected to generic level by McEachran & Dunn (1998). The genus includes 45 valid and several undescribed species (Séret, 1989; McEachran & Dunn, 1998; Ebert & Compagno, 2007; Last, 2008; Last et al., 2008a; Séret & Last, 2008; Last & Alava, 2013; Ebert et al., 2013). Eight species of the genus Dipturus are present in South American waters: D. ecuadoriensis (Beebe & Tee-Van 1941) in the Pacific Ocean, D. argentinensis Díaz de Astarloa, Mabragaña, Hanner & Figueroa 2008, D. bullisi (Bigelow & Schroeder 1962), D. diehli Soto & Mincarone 2001, D. leptocauda (Krefft &

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Stehmann 1975), D. mennii Gomes & Paragó 2001 and D. teevani (Bigelow & Schroeder 1951) in the Atlantic Ocean, and D. trachyderma in both Pacific and Atlantic Oceans. All South American longnose skates have been reported on the continental shelf and slope between 100 – 450 m and are heavily fished over the entire range (Leible & Stehmann, 1987; Lloris & Rucabado, 1991; Gomes & Picado, 2001; Soto & Mincarone, 2001; Díaz de Astarloa et al., 2008).

Overall, peer-reviewed literature regarding the basic biology and taxonomy of longnose skates is scarce. For example, no additional information (molecular or morphological) has been produced to appraise the genus re-assignment of Last & Gledhill (2007). Recent changes in the taxonomic status of longnose skates has resulted in confusion that is of considerable importance in the context of South American fisheries management, where two sympatric and morphologically similar species, originally in the genus Dipturus, are now in separate genera, Dipturus and Zearaja, (Menni & López, 1984; Agnew et al., 2000; Bustamante et al., 2012).

MATERIALS AND METHODS

A reference matrix was constructed using specialised search engines, i.e., Google Scholar, ScienceDirect, SharkReferences and worldwide academic libraries by a combination of fuzzy matching keywords for any scientific and common names regarding the species Zearaja chilensis and Dipturus trachyderma (in Spanish and English), e.g., ‘Raja’, ‘Zearaja’, ‘Dipturus’, ‘Trachyderma’, ‘Trachydermus’, ‘Chilensis’, ‘Flavirostris’, ‘Roughskin skate’, ‘Yellownose skate’, ‘Raya volantín’, ‘Raya espinosa’, ‘Raya de vientre áspero’. Query matches were filtered by content and only those focused somehow on the target species were included in the matrix. An additional filter was applied to exclude references that lacked observational data or analysis. Remaining matches were counted only once and separated into seven categories based on their content. The seven categories (and sections) are: Anatomy and morphology (body size, colouration, dermal denticles, dentition, skeletal components, egg cases); Checklists and biological inventories (distribution ranges); Habitats and feeding habits; Life-history patterns (reproduction, age, growth and longevity); Fisheries (southeast Pacific, southwest Atlantic); Genetics; and Parasites.

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RESULTS AND DISCUSSION

A total number of 185 articles were found regarding the biological entities Zearaja chilensis (Fig. 2A) and Dipturus trachyderma (Fig. 2B). An additional 32 articles were excluded from the matrix as the species received only nominal mention. For Z. chilensis, 77% of the articles correspond to scientific publications and 23% to ‘grey literature’ (e.g. non peer-reviewed and ‘popular’ media) that comprised mostly fishery reports, covering almost 170 years since the original description. A similar division between published scientific and grey literature was found for D. trachyderma, 75% and 24% respectively.

The most studied category for both species was ‘Checklist and biological inventories’ with at least one article per year, with 54 publications for Z. chilensis and 33 for D. trachyderma (Fig. 3). While some publications contained specific information about taxonomic identity and sampling data, most papers reported historical and observation records without mention of the diagnostic features used for species recognition, and therefore were considered as doubtful reports.

‘Genetics’, was the least represented categories for both species (Fig. 3), which may reflect that relevant and accessible genetic approaches for species identification, stock structure and population analysis do not have a long research history (Dudgeon et al., 2012), and hence research has yet to address these topics. ‘Habitat and feeding habits’ was also poorly represented in the literature (Fig. 3), which may indicate a lack of resources or imperative to conduct research in these areas even though both species are of high economic value to South American economies. Undoubtedly, the study of many aspects of the biology of longnose skates is challenging due mainly to difficulties in access to these deep-water species, a dependency on fishing activity for sample collection across their extensive ranges and associated issues with the handling of large, economically valuable animals. There was a marked increase in the number of published studies for both species from 2000 and especially after 2005, when both species became an important asset to Chilean economy as fishing interest increased along with the creation of new landing species-specific categories for Z. chilensis and D. trachyderma (Fig. 4). However, opportunities for future research in many of the categories remain, especially on population size and structure, short and long-term movements or migrations, feeding ecology and habitat use, and reproduction. To avoid near-future population collapse due to incomplete or inappropriate management measures, there is an urgent need to fill these information gaps.

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Figure 2. Cumulative number of peer-reviewed papers, books, government reports, separated into seven categories for (A) Zearaja chilensis and (B) Dipturus trachyderma.

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Figure 3. Number of published studies for Zearaja chilensis (1848‒2014) (black bars) and Dipturus trachyderma (1975‒2014) (grey bars) by research category.

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Figure 4. Total annual numbers of published studies for (A) Zearaja chilensis and (B) Dipturus trachyderma.

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Systematics

Order RAJIFORMES Berg 1940 Family RAJIDAE Blainville 1816 Genus Zearaja Whitley 1939 Zearaja chilensis (Guichenot 1848) (Fig. 5)

Raia chilensis Guichenot, 1848.

Raja chilensis Philippi, 1892; Delfín, 1900; De Buen, 1959; De Buen, 1960; Carvajal, 1971; Carvajal & Goldstein, 1971; Bahamonde & Pequeño, 1975; Carvajal & Dailey, 1975; Pequeño, 1975; Pequeño, 1977; Ojeda, 1983; Fernández & Villalba, 1985; Pequeño & Lamilla, 1985; Villalba & Fernández, 1985; Sielfeld & Vargas, 1999; Gili et al., 1999; Buschmann et al., 2005.

Raja flavirostris Philippi, 1892; Delfín, 1900; Garman, 1913; Gotschlich, 1913; Quijada, 1913; Fowler, 1916; Fowler, 1926; Norman, 1937; Fowler, 1941; Oliver, 1943; Fowler, 1945; Hart, 1947; Fowler, 1951; Bahamonde, 1953; Bigelow & Schroeder, 1953; Mann, 1954; De Buen, 1957; Angelescu et al., 1958; Bigelow & Schroeder, 1958; Ringuelet & Aramburu, 1960; López, 1963a, b; Hulley, 1966; Olivier et al., 1968; Hulley, 1970; Menni, 1971; Ringuelet & Aramburu, 1971; Hulley, 1972; Castello & Tapia-Vera, 1973; Sadowsky, 1973; Gosztonyi, 1979; Menni & López, 1979; Gosztonyi, 1981; Menni, 1981; Menni et al., 1981, Menni & Gosztonyi, 1982; Menni & López, 1984; Menni et al., 1984; Raschi, 1986; Threlfal & Carvajal, 1986; Prenski & Sánchez, 1988; Sánchez & Prenski, 1996; García de la Rosa, 1998; Agnew et al., 2000; Jo et al., 2004; Wakeford et al., 2005; Buschmann et al., 2005.

Raja oxyptera Philippi, 1892; Delfín, 1900; Lönnberg, 1907; Garman, 1913; Gotschlich, 1913; Quijada, 1913.

Raja latastei Delfín, 1902.

Raja brevicaudata Marini, 1933.

Raja (Dipturus) flavirostris Stehmann, 1970; Menni, 1972; Menni, 1973; Stehmann, 1978; Leible, 1984; Leible, 1987; Leible & Stehmann, 1987; Leible, 1988; Fuentealba & Leible, 1990; Fuentealba et al., 1990; Leible et al., 1990; López et al., 1996.

Raja (Dipturus) chilensis Pequeño, 1989; Lloris & Rucabado, 1991; Pequeño & Lamilla, 1993; Sáez & Lamilla, 1997; Bizikov et al., 2004.

Dipturus chilensis Bahamonde et al., 1994; Bahamonde et al., 1996; Mould, 1997; McEachran & Dunn, 1998; Meneses & Paesch, 1999; Paesch, 1999; Paesch & Meneses, 1999; Cousseau &

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Perrotta, 2000, 2004; Cousseau et al., 2000; Lucifora et al., 2000; Koen Alonso et al., 2001; Gomes & Picado, 2001; Soto & Mincarone, 2001; Lamilla et al., 2002; Last & Yearsley, 2002; Nion et al., 2002; Sánchez & Mabragaña, 2002; Calderón et al., 2003; Lamilla & Sáez, 2003; Meneses & Paesch, 2003; Buren, 2004; García de la Rosa et al., 2004; Massa et al., 2004; Smith et al., 2004; Acuña et al., 2005; Céspedes et al., 2005; Compagno, 2005a, b; Lamilla & Bustamante, 2005; Quiroz & Wiff, 2005; Oddone et al., 2005; Araya & Cubillos, 2006; Lamilla et al., 2006; Licandeo et al., 2006; Concha et al., 2007; Cousseau et al., 2007; Ebert & Bizarro, 2007; Ebert & Compagno, 2007; Licandeo & Cerna, 2007; Licandeo et al., 2007; Melo et al., 2007; Aburto et al., 2008; Andrade & Pequeño, 2008; Arkhipkin et al., 2008; Díaz de Astarloa et al., 2008; Domingo et al., 2008; Lamilla et al., 2008; Paesch & Oddone, 2008; Quiroz et al., 2008; Reyes & Torres-Flores, 2008; Mabragaña et al., 2009; Quiroz et al., 2009; Ruarte et al., 2009; Silveira, 2009; Quiroz et al., 2010; Zavatteri, 2010; Aversa et al., 2011; Mabragaña et al., 2011a; Quiroz et al., 2011; Cortes & Cueto 2012; San Martin & Trucco, 2012; Colonello & Cortes, 2014; Silveira et al., 2014.

Dipturus flavirostris Menni & Stehmann, 2000; Cedrola et al., 2005; Bovcon & Cochia, 2007.

Dipturus (Zearaja) chilensis Estalles et al., 2011; Kyne & Sympfendorfer, 2007; Lamilla et al., 2009.

Zearaja chilensis Kyne et al., 2007; Last & Gledhill, 2007; Menni & Lucifora, 2007; Kyne & Simpfendorfer, 2010; Lamilla et al., 2010; Vargas-Caro, 2010; Lamilla & Flores, 2011; Lamilla et al., 2011; Mabragaña et al., 2011b; Perier et al., 2011; Queirolo et al., 2011; Arkhipkin et al., 2012; Bustamante et al., 2012; Concha et al., 2012; Deli Antoni et al., 2012; Lamilla et al., 2012a, b; Naylor et al., 2012; Bovcon et al., 2013; Lamilla et al., 2013; Bustamante et al., 2014a, b; Espindola et al., 2014, Jeong & Lee, 2014; Sáez et al., 2014; Vargas-Caro et al., 2016a.

Zearaja flavirostris Menni et al., 2010; Naylor et al., 2012.

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Figure 5. Morphotypes for Zearaja chilensis. A. Original description, female, 750 mm LT, Chile

(Philippi, 1982). B. Mature male, 975 mm LT, Chile (Delfín, 1902). C. Adult male, Argentina

(Norman, 1937). D. Immature male, 374 mm LT, Argentina (Bigelow & Schroeder, 1958). E. Mature male, Argentina (Lloris & Rucabado, 1991). F. Female, Falkland Island, British Overseas Territory

(Bizikov et al., 2004). G. Female, 690 mm LT, Los Vilos, Chile (Melo et al., 2007). H. Mature female,

770 mm LT, Valdivia, Chile.

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The yellownose skate (Zearaja chilensis) was first described in 1848 and since then the species’ identity has caused confusion within the scientific community. Guichenot (1848) described the species from a male of 836 mm total length (LT) collected in Quinteros Bay, Chile. Although this report may be considered brief, the author provided information about external features and highlighted the fact that it was very abundant on local markets. Later, Philippi (1892) described Raja flavirostris (Fig. 5A) based on two females of 750 mm average LT and Raja oxyptera, from a mature male of 1080 mm LT from the same locality (Quinteros Bay), and provided a comprehensive description including morphometric measurements and detailed external morphology. On this occasion, the author considered R. flavirostris and R. oxyptera as separate species mainly for having different thorn patterns on the caudal region, but did not include a comparison with the previously described R. chilensis. Delfín (1902) provided a description of a new species, Raja latastei (Fig. 5B), based on male (795 mm LT) and a female (567 mm LT) specimen of an ostensible R. chilensis from Coquimbo, Chile. Garman (1913) synonymised R. latastei as R. flavirostris and provide a comparison between R. flavirostris and R. oxyptera suggesting that apparent changes in external morphology due to sexual maturity and ontogenetic changes may be used to separate two morphotypes of the same species, with the relocation of R. flavirostris and R. oxyptera to the same nominal species. Norman (1937) subsequently published a comprehensive review of skates (rajids) of the Argentinian Patagonia, supporting Garman hypothesis and suggested that this ‘nominal species’ is R. flavirostris and relocating R. chilensis as a major synonymy. Interestingly, De Buen (1959) provided a description and comparisons of Chilean longnose skates using specimens (males and females) collected from the R. chilensis type locality, and concluded that R. flavirostris, R. oxyptera and R. latastei should be synonyms of R. chilensis supplementing Guichenot’s original description with different morphotypes. De Buen’s suggestions were supported by Leible (1984, 1987) and Leible et al. (1990), who described R. chilensis as a polymorphic species and distinguished at least three clear morphotypes in central and southern Chile, agreeing with observations by Pequeño & Lamilla (1985), Pequeño (1989), and Lloris & Rucabado (1991) (Fig. 5E). Following the Principle of Priority of the International Code of Zoological Nomenclature (ICZN), it is appropiate to use and apply the name R. chilensis, rather than R. flavirostris, to the nominal species recognised as the ‘yellownose skate’ in Chilean waters. The use of change of ranks and combination (Article 23c, ICZN, 1964) and plenary power (Article 79b, ICZN, 1964) to consider R. chilensis as a senior synonym of R. flavirostris (Menni & Stehmann, 2000) was incorrect, as a modification of the Code that would have seen a valid change from chilensis to flavirostris, based on 50-years without references to a species name, occurred after the reference to R. chilensis by De Buen (1959).

The phylogenetic analysis provided by McEachran & Dunn (1998) proposed the elevation of the subgenus Dipturus (formerly under Raja) to genus level by using internal anatomy and external

–21– CHAPTER II: LITERATURE REVIEW morphology of the Family Rajidae, as suggested by Stehmann (1970). Recently, Last & Yearsley (2002), resurrected Zearaja as a subgenus (from Dipturus), and Last & Gledhill (2007) raised Zearaja to genus level based on key features of the external morphology and internal anatomy of the clasper. As consequence, the name Zearaja chilensis (Guichenot 1848) is valid to date, and is suggested the relocation of Raja latastei, R. flavirostris and R. oxyptera as junior synonyms.

Order RAJIFORMES Berg 1940 Family RAJIDAE Blainville 1816 Genus Dipturus Rafinesque 1810 Dipturus trachyderma Krefft & Stehmann 1975 (Fig. 6)

Raja (Dipturus) trachyderma Krefft & Stehmann, 1975; Krefft, 1978; Leible, 1984; Leible & Stehmann, 1987; Leible, 1988; Pequeño, 1989; Lloris & Rucabado, 1991; Pequeño & Lamilla, 1993; López et al., 1996; Bizikov et al., 2004.

Raja trachyderma Menni & Gosztonyi, 1977; Menni & Gosztonyi, 1982; Sielfeld & Vargas, 1992; Sielfeld & Vargas, 1999; Gili et al., 1999; Buschmann et al., 2005; Thiel et al., 2009.

Dipturus trachydermus López et al., 1989; Compagno, 1999b; Knoff et al., 2001b; Knoff et al., 2002; López et al., 2002; Oyarzún et al., 2003; Knoff et al., 2004; Cedrola et al., 2005; Compagno, 2005a; Kyne & Simpfendorfer, 2007; Lamilla & Massa, 2007; Menni & Lucifora, 2007; Reyes & Torres-Flores, 2008; Muniz-Pereira et al., 2009; Quiroga et al., 2009; Kyne & Simpfendorfer, 2010; Menni et al., 2010.

Dipturus trachyderma Stehmann, 1978; Menni et al., 1984; Mould, 1997; McEachran & Dunn, 1998; Menni & Stehmann, 2000; Cousseau et al., 2000; Gomes & Picado, 2001; Soto & Mincarone, 2001; Knoff et al., 2001a; Nion et al., 2002; Lamilla & Sáez, 2003; Smith et al., 2004; Céspedes et al., 2005; Compagno, 2005b; Lamilla, 2005; Lamilla & Bustamante, 2005; Lamilla et al., 2006; Licandeo et al., 2006; Bovcon & Cochia, 2007; Ebert & Compagno, 2007; Cousseau et al., 2007; Licandeo et al., 2007; Melo et al., 2007; Arkhipkin et al., 2008; Díaz de Astarloa et al., 2008; Domingo et al., 2008; Jeong & Nakabo, 2008; Lamilla et al., 2008; Quiroz et al., 2008; Santos et al., 2008; Góngora et al., 2009; Lamilla et al., 2009; Mabragaña et al., 2009; Lamilla et al., 2010; Quiroz et al., 2010; Vargas-Caro 2010; Estalles et al., 2011; Lamilla & Flores, 2011; Lamilla et al., 2011; Mabragaña et al., 2011a, b; Moreira et al., 2011; Perier et al., 2011; Queirolo et al., 2011; Sáez & Lamilla, 2012; Arkhipkin et al., 2012; Concha et al., 2012; Lamilla et al., 2012a, b; Bovcon et al., 2013; Lamilla et al., 2013; Bustamante et al., 2014a, b.

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The roughskin skate (Dipturus trachyderma) was described by Krefft & Stehmann (1975)

(Fig. 6A), using an immature male of 1135 mm LT as the holotype. This specimen was collected during the cruises of R/V “Walter Herving” to South America in 1971, particularly off southern Argentina (49° S, 60° 52' W) at the outer shelf edge (195 – 200 m depth). Authors provided general information about the external morphology, neurocranium and pelvic girdle of the species. Considering that the specimen was an immature, a detailed description of secondary sexual characters, i.e. clasper morphology, and of dorsal thorns in general, is absent. Additional specimens from Argentinian waters were collected, but unfortunately due to the large size of these animals only general external measurements (e.g., LT) were recorded, and in some cases body parts were preserved and kept for reference and further investigations (Menni & Gosztonyi, 1977, 1982). The species description was supplemented through specimens collected in Chile (Leible & Stehmann, 1987) (Fig. 6B), Even with some incomplete specimens, the authors were able to provide a detailed description of the neurocranium and pelvic girdle, including information on meristics, morphometrics, and variations of shape and squamation. Also, Leible & Stehmann (1987) provided the first description of the scapulocoracoid cartilage and external and internal morphology of claspers. Unfortunately, since then no other study has addressed morphological characters. General additional information about descriptive demography can be found in checklists, reference books and field guides (Lloris & Rucabado, 1991; Cousseau et al., 2000; Bizikov et al., 2004; Lamilla & Bustamante, 2005; Cousseau et al., 2007).

The roughskin skate had been subject of misspelling errors similarly to Z. chilensis. Several authors have maintained and amplified the misspelling of Dipturus trachyderma as D. trachydermus originally made by López et al (1989). Also, the year of first publication have been addressed wrongly as 1974 rather than the original year of first description. These issues are currently leading to confusion in the literature (Compagno, 1999b; Compagno, 2005a, b Kyne & Simpfendorfer, 2007, 2010; Naylor et al. 2012a, b; Last et al., 2016). Nomenclature spelling emendation in an attempt to make the genus and the species epithet to agree in gender, is ruled in the Code of Zoological. However, Krefft & Stehmann (1975), introduced trachyderma as a noun in apposition, rather than an adjective, therefore, indeclinable to trachydermus. Following the Principle of Priority and Principle of the First Reviser (after Stehmann, 1978) of the ICZN, it is appropriate to use and apply the name D. trachyderma, rather than D. trachydermus, to the nominal species recognised as the ‘roughskin skate’ in South American waters.

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Figure 6. Morphotypes of Dipturus trachyderma. A. Original description, female, 1135 mm LT, off

Argentina (Krefft & Stehman, 1975). B. Male, 2000 mm LT, Chile (Leible & Stehmann, 1987). C.

Male, 2075 mm LT, Beagle Channel, Argentina (Lloris & Rucabado, 1991). D. Female, Falkland

Islands, British Overseas Territory (Bizikov et al., 2004). E. Male, 2007 mm LT, Valdivia, Chile.

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Diagnosis

Zearaja chilensis is characterized by the combination of the following characters: Disc broader than long, anterior margins undulated. Long and strong rostral cartilage with a white-yellow rostrum. Dorsal surface of disc dark brown, ocelli are present on each pectoral fin. Dorsal side of the disc smooth, with denticles irregularly scattered over body, thorns on orbital, rostrum, nuchal, tail, and alar and malar (males) areas. Ventral surface with whitish and brownish areas, reddish brown colour at posterior disc margins; most of the disc, pelvic fins and tail pale to medium brown. Alar thorn patch well-developed with 1 – 3 rows on each pectoral fin. Tail with 1 – 3 rows (one median and two laterals) in males, up to 5 rows in females with a maximum of 47 thorns; 1 – 5 interdorsal thorns. Tooth rows in upper jaw 30 – 46.

Dipturus trachyderma is characterized by the combination of the following characters: Disc broadly rhombic; anterior margin of disc concave and posterior margin convex with rounded inner corner to level of pelvic fins. Snout hard, long and pointed. Dorsal surface of disc dark brown without a pattern or distinct ocelli; only mucus pores coloured black. Ventral surface as dark as dorsal side in large areas, reddish brown colour at posterior disc margins; gill slits and cloaca creamy white. Both dorsal and ventral sides completely covered by dermal denticles, by the exception of a smooth belly. Ocular and spiracular thorns present, nuchal thorn absent, no other thorns on the disc. Alar thorn patch very well developed with 4 – 5 rows on each pectoral fin. Tail with 1 – 3 rows (one median and two laterals) in males, up to 5 rows in females with a maximum of 43 thorns; 1 – 4 interdorsal thorns. Tooth rows in upper jaw 36 – 44.

Description

Most studies on the taxonomy, growth, maturity, maximum size, and species demography rely on measurement of all or part of the body (Francis, 2006). Hubbs & Ishiyama (1968) found that skates commonly have interspecific differences in the relative size of the head, trunk, tail and fins as well as between sexes. As a consequence, researches have used a variety of measurement, making the accurate comparison among studies almost impossible and in some cases leading to invalid data comparison and incorrect conclusions (De Buen, 1959; Francis, 2006; Last et al., 2008b).

As skate body morphology is likely to vary inter and intra specifically (between sexes and with body size), a standardised measurement protocol is necessary for specimen comparisons. Hubbs & Ishiyama (1968) defined a large number of body measurements for skate identification; including disc, tail, fins, head, trunk and cranial areas. They recommended that disc width (DW) be used as the independent variable when calculating relative proportions of body measurements, as tail length,

–25– CHAPTER II: LITERATURE REVIEW which is part of LT, exhibited negative allometry. The use of total length was considered problematic in embryonic and small juvenile skates because of a non-linear scaling with body mass (Templeman,

1987). However, LT has been used as the standard reference measurement for body proportions of the

Rajidae, as greater variation in proportions was noted if DW was used (Hulley, 1970; Stehmann, 1970). Disc width measurements of Bathyraja richardsoni (Garrick 1961) in fresh condition have been shown to be highly variable, lack repeatability and are sensitive to inter-observer error

(Templeman, 1973). Regarding preserved material, DW depends on the shape in which the specimen was preserved, which highlights the importance of making measurements on unfixed specimens (Ishiyama, 1950). A comprehensive review of the methods used for measuring chondrichthyans recommended the use of LT as the reference for batoids (Francis, 2006), and a standard methodology for measuring skates proposed the use of 46 measurements and the use of LT as reference for body size (Last et al., 2008b).

The literature that pre-dates this proposal has, however, variably used LT or DW, in seven studies on Z. chilensis between 13 and 23 variables were measured (Philippi, 1982; Delfín, 1902; De Buen, 1959; Menni, 1973; Pequeño & Lamilla, 1985; Leible, 1987; Lloris & Rucabado, 1991) and 15 ‒ 20 variables were used in three studies of D. trachyderma (Krefft & Stehmann, 1975; Leible & Stehmann, 1987; Lloris & Rucabado, 1991). Many of these authors used a variety of measurements as ‘body descriptors’ and not only LT was used as the standard reference, making comparison between studies difficult or virtually impossible.

The minimum and maximum sizes reported for Z. chilensis are 156 mm and 1580 mm LT respectively, with females growing to a larger sizes than males (Norman, 1937; Bahamonde, 1953; De Buen, 1959; Leible, 1987; Fuentealba & Leible, 1990; Leible et al., 1990; García de la Rosa, 1998; Cousseau et al., 2000, 2007; Gomes & Picado, 2001; Cedrola et al., 2005; Licandeo et al., 2006; Licandeo & Cerna, 2007; Paesch & Oddone, 2008; Estalles et al., 2011; Lamilla et al., 2012a, b; Bustamante et al., 2012). In comparison, D. trachyderma reaches 2540 mm LT for females and

2320 mm LT for males (Leible & Stehmann, 1987; Cousseau et al., 2000, 2007; Gomes & Picado, 2001; Cedrola et al., 2005; Licandeo et al., 2007; Lamilla et al., 2012a, b). A single reference that suggested Z. chilensis could attain a maximum size of 2480 mm LT for females and 2220 mm LT for males in Argentinian waters (Koen Alonso et al., 2001) is likely to be a misidentification of D. trachyderma (Leible, 1987; Cedrola et al., 2005; Iglésias et al., 2010; Estalles et al., 2011).

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Colouration

The dorsal body surface of batoids may be plain or patterned and is generally brown, grey, black, or yellowish, although red, blue, and green colours also occur in some species, whereas the ventral skin is usually lighter and of a uniform colour (Kemp, 1999). Colour pattern of skates may reflect different habitat occupancy within both horizontal and vertical ranges, with the possibility that interspecific colour variation may be influenced by environmental factors additional to phyletic relationships. Overall, multiple factors have to be considered before an assumption of intraspecific and ontogenetic colour variations can be made, such as whether (or not) colour of the integument is uniform or spotty, variation of dorsal and ventral surfaces when fresh and preserved, presence of colour markings or patterns on either surface of the body and, presence of pectoral ocelli (Ishiyama, 1958b). Body coloration can be an important feature for species identification, as colour patterns in different body regions on both the dorsal and ventral surfaces are often diagnostic (Hubbs & Ishiyama, 1968).

Colouration of Z. chilensis is red-brownish when fresh, usually with irregular dark spots on the dorsal surface which appear to fade after a few hours post-capture. Both sides of the rostral cartilage are yellowish and translucent, and in some individuals a large circular dark ocellus is present at the base of each pectoral fin. The ventral surface is dark brown with a creamy white abdomen, and pelvic fins have a white base and are reddish around their edges. Numerous blackish pores are also present grouped around the mouth and gill opening on the ventral surface. In mature males, the terminal section of the claspers has a reddish-brown colour (Norman, 1937; De Buen, 1959; Leible, 1987; Lloris & Rucabado, 1991; Bizikov et al., 2004; Cousseau et al., 2007). In D. trachyderma the dorsal surface is uniformly dark brown and supports numerous pores that appear blackish. The ventral surface has a base colour reddish to dark brown especially at posterior disc margins, abdomen, posterior pelvic lobes and underside of tail. However, in fresh specimens large areas may appear blackish, due to a layer of mucus over the skin. Jaws, gill slits and cloaca are creamy white in appearance. Ventral mucus-filled pores appear black, although they are indistinct due to dark base colour of the skin. In mature males, the terminal section of the claspers has a light reddish-brown colour (Krefft & Stehmann, 1975; Leible & Stehmann, 1987; Lloris & Rucabado, 1991; Cousseau et al., 2000, 2007). Some colour variations are suggested to relate to ontogenetic development, sex of individuals and environmental conditions (Ishiyama, 1958b; Leible, 1988).

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Dermal denticles

Dermal denticles (placoid scales) are a characteristic of the skin of chondrichthyan fishes and cover the entire body in sharks, but are usually distributed discontinuously in batoids and chimaeras (Reif, 1985; Kemp, 1999). Each dermal denticle comprises a basal plate embedded in the dermis, a neck, and an exposed spiny crown (Kemp, 1999). Overall, dermal denticles can differ dramatically among species and even body regions of an individual and their morphology is usually associated with functional roles such as protection from predators, reduction of mechanical abrasion and hydrodynamic drag (Reif, 1985; Bushnell & Moore, 1991; Raschi & Tabit, 1992; Lang et al., 2012). Skates have an impressive array of dermal denticles on the dorsal and ventral disc, around the head, trunk, pectoral and pelvic fins, and the tail. Small denticles < 3 mm in length are referred as prickles and may cover either the dorsal or ventral disc surfaces, or both (Ishiyama, 1958b; Stehmann & Bürkel, 1984; Leible, 1988; Deynat, 2000a; Gravendeel et al., 2002). Medium-sized denticles or thorns < 3 mm long have a circular or star-like basal plate. Thorns are generally located on the dorsal surface and can be separated into ‘series’ according to the body region, i.e., orbital, rostral, nuchal, scapular and caudal series (Hubbs & Ishiyama, 1968; Stehmann & Bürkel, 1984; Gravendeel et al., 2002). The distribution, number and basal plate edges morphologies of thorns are usually diagnostic features for species identification (Hubb & Ishiyama, 1968; Leible, 1988; Gravendeel et al., 2002; Last et al., 2008b). The number of thorns reported to occur in different body areas of longnose skates from Chilean waters is summarised in Table 1, although values may vary according to sex and maturity of individuals. For example, mature males develop their alar series, while mature females have more thorns in the caudal zone than males (Leible, 1988). The presence of a nuchal thorn in Z. chilensis was originally considered to be a diagnostic character that separated it from D. trachyderma (Leible, 1987), but Z. chilensis was later recognised as a polymorphic species and the nuchal thorn was not always present (Leible et al., 1990). However, the fact that the presence of a nuchal thorn is not a diagnostic character has been overlooked in several field guides (Cousseau et al., 2000; Lamilla & Sáez, 2003; Lamilla & Bustamante, 2005; Cousseau et al., 2007), which generates an unfortunate uncertainty for rapid species identification. Adult male series of large and curved denticles, or hooks, in the alar and malar regions can vary among species in the number of transverse and lengthwise rows. Alar hooks are erectile and are grouped near the pectoral fin margins, while the non-erectile malar hooks are aligned near the margin of the disc lateral to the eyes (Bigelow & Schroeder, 1953; Hubb & Ishiyama, 1968). While caudal thorns and other dermal denticles have been used for species identification of some extant and fossilised rajids (Stehmann & Bürkel, 1984; Gravendeel et al., 2002), few studies have been used dermal denticles morphology for taxonomic and phylogenetic purposes (Deynat & Séret, 1996; McEachran & Konstantinou, 1996; Deynat, 1998, 2000a, b). Caudal thorns have also been sectioned and used to age some skates and rays (Gallagher & Nolan, 1999;

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Henderson et al., 2004; Gallagher et al., 2006; Davis et al., 2007; Matta & Gunderson, 2007; Moura et al., 2007).

The body surfaces of Z. chilensis are relatively smooth (excluding the rostral area), with sparse prickles on the dorsal surface and without obvious denticles on the ventral surface. In contrast, both dorsal and ventral surfaces in D. trachyderma are covered with prickles when mature, except for a smooth patch around the abdominal area (in general Dipturus species are covered with dermal denticles with the dorsal surface rougher than the ventral surface) (Krefft & Stehmann, 1975; Menni & Gosztonyi, 1977; Leible & Stehmann, 1987; Leible, 1988).

Dentition

Dental morphology is widely used in taxonomic, biological and fossil studies of cartilaginous fishes (e.g. Herman et al., 1995, 1996; Kemp, 1999; Adnet & Cappetta, 2001; Lamilla & Sáez, 2003; Sáez & Lamilla, 2012), and while most studies provide descriptions of tooth morphology (Du Buit, 1978; Pequeño & Lamilla, 1985; Leible, 1987, 1988; Zorzi & Anderson, 1988; Herman et al., 1995, 1996), relatively few address variation in tooth number and arrangement, mono- or dignathic heterodonty, malformation, dental sexual dimorphism and ontogenetic change or their relation to dietary intake (Bigelow & Schroeder, 1953; Leible, 1988; Motta, 2004; Gutteridge & Bennett, 2014; Belleggia et al., 2016).

Description of the dentition of Z. chilensis and D. trachyderma includes the number of tooth rows: 30 ‒ 46 rows in Z. chilensis (Philippi, 1892; Delfín, 1902; Norman, 1937; Leible, 1987, 1988) and 36‒44 in D. trachyderma (Krefft & Stehmann, 1975; Leible & Stehmann, 1987; Lloris & Rucabado, 1991). The dental formulae of Z. chilensis, has been reported for both sexes in Chile: 20– 20/19–20 for females and 19–19/20–18 for males (Sáez & Lamilla, 1997). Information about how teeth vary with ontogeny is not available, although this will impact the usefulness of row count for taxonomic purposes. Sexually dimorphic dentition has been reported in Z. chilensis, with sharp and conical (spike-shaped) teeth in mature male skates, while females have blunt ‘oval shaped’ teeth (Leible, 1987; Sáez & Lamilla, 1997; Belleggia et al., 2016). Such sexual dimorphism dentition was hypothesised to correlate with different diets between the sexes in batoids (Feduccia & Slaughter, 1974), but has since shown to facilitate the male grasp of the female pectoral fins to aid in the act of copulation (McEachran, 1977; Gutteridge & Bennett, 2014). Although no further studies have been published regarding dental morphology or dental formulae for longnose skates in South America, both features are widely, if uncritically, used as diagnostic characters in cartilaginous fishes.

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Table 1. Variation of the number on dermal denticles for the yellownose skate Zearaja chilensis and roughskin skate Dipturus trachyderma (Modified after Guichenot, 1848; Philippi, 1892; Delfín, 1902; Norman, 1937; De Buen, 1959; Menni, 1973; Leible, 1987; Leible & Stehmann, 1987; Lloris & Rucabado, 1991; Bizikov et al., 2004). † There is no available information in the literature that mention this series.

Range Body series Z. chilensis D. trachyderma Orbital 1 ‒ 9 1 ‒ 7 Spiracular 1 ‒ 2 n/a† Nuchal 0 ‒ 1 0 Alar 9 ‒ 33 17 ‒ 48 Alar rows 1‒3 4 ‒ 5 Caudal 10 ‒ 47 11 ‒ 43 Interdorsal 1 ‒ 5 0 ‒ 4 Caudal rows 3 ‒ 5 3 ‒ 5

Skeletal components

Studies of the anatomy and systematics of batoids have revealed significant variation among chondrichthyans (Compagno, 1977; Miyake, 1988; Miyake & McEachran, 1991). The neurocranium, pectoral and pelvic girdles and the male reproductive organs may provide useful diagnostic skeletal features for species identification (Ishiyama & Hubbs, 1968; Hulley, 1970, 1972; Stehmann, 1970; McEachran, 1982, 1983). According to Ishiyama (1958b), differences in clasper skeletal components “provide excellent generic and specific features and insights about phylogenetic relationship within groups and fully discriminative analyses even between closely related species”. Clasper morphology was the main feature used to resurrect Zearaja to genus level (Last & Yearsley, 2002), and used to relocate Z. chilensis and Z. nasuta to the genus (Last & Gledhill, 2007). External differences in clasper morphology occurs between longnose skate; for example, the shield is short and robust in Zearaja, but long and slender in Dipturus; and the distal lobe is spatulated in Zearaja, but rounded in Dipturus. Internally, claspers may differ in their structural components by the presence or absence of cartilages (Last & Gledhill, 2007). Menni (1971) described the internal components of the clasper of Z. chilensis which was later supplemented with an external anatomical comparison (Leible, 1987). However, the presence of the accessory terminal I cartilage (Atr1) was erroneously reported by Menni (1971) and

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Leible (1987), as there is no equivalent structure in Zearaja species (Last & Gledhill, 2007). Leible (1987) also described the neurocranium, pelvic girdles and scapulocoracoid of Z. chilensis, and suggested that intra-specific variations occur among Chilean longnose skates populations, highlighting the need of further investigation. For D. trachyderma, the original (Krefft & Stehmann, 1975) and subsequent description (Leible & Stehmann, 1987) included details of external and internal structures, such as neurocranium, pelvic girdles, scapulocoracoid and clasper; and where the presence of Atr1 and the sentinel (both absent in Zearaja) were reported. Moreira et al., (2011) found differences in the neurocranium, scapulocoracoid and clasper between D. menni and D. trachyderma, which was consistent with there being two separate species, even though some specimens of D. menni were originally identified as D. trachyderma (Gomes & Picado, 2001). The taxonomy revision of the South American batoids by Leible in 1988 highlight that most species exhibit marked morphological variation, mainly related to sex and maturity. However, as clasper morphology and that of other skeletal components has not been addressed in the past 25 years, and given the relative importance of these structures in taxonomic and phylogenetic studies, a revision of the internal skeletal components of longnose skates is deemed necessary.

Egg cases

All skates utilise extended oviparity in which fertilised eggs are encapsulated in a structurally complex egg case that protects the embryo during an often-long development, which occurs primarily outside of the mother (Hamlett & Koob, 1999; Hamlett, 2005). Longnose skates are single oviparous, bearing one egg capsule per oviduct at a time (Mabragaña et al., 2011b; Concha et al., 2012), with a marked synthetic synchrony of the nidamental gland activity, consistent with other rajoid species (McEachran & Aschliman, 2004; Ebert & Compagno, 2007). The morphology of egg cases is considered to be genus- and species-specific, and has been used as a taxonomic tool for species identification and systematics (e.g. Ishiyama, 1950, 1958a, b; Ebert, 2005; Ebert & Davis, 2007; Ishihara et al., 2012). It is, however, imperative that morphological descriptions of egg cases are performed where the species identity is known unambiguously; such as egg cases collected from a mother’s uterus or those that have been laid in an aquarium that contains a single oviparous species (Ebert & Davis, 2007; Bustamante et al., 2013). Variations in egg case morphology among oviparous species may relate to the structure of the habitat in which they are deposited, for example, the presence of long tendrils may suit deposition in a structurally complex habitat (Ebert et al., 2006) and provide information about the reproductive strategy (Dulvy & Reynolds, 1997; Didier, 2004; Kyne & Simpfendorfer, 2010).

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Egg cases of longnose skates are generally rectangular in shape with a horny process in each corner (Mabragaña et al., 2011b). The egg cases of Z. chilensis and D. trachyderma have been described from specimens from the southwest Atlantic (Paesch & Oddone, 2008; Mabragaña et al., 2011b) and southeast Pacific (Concha et al., 2012). For Z. chilensis the egg case is dark brown in colour with a smooth surface, finely striated and without ridges, and has a capsule length (CL) of 94 – 158 mm and capsule width (CW) of 64 – 76 mm. For D. trachyderma the deposited egg case is a golden-brown colour, and is possibly the largest of any cartilaginous fish at 197 – 230 mm CL and 100 – 163 mm CW (Mabragaña et al., 2011b; Concha et al., 2012). The egg case has well developed aprons and a strong lateral keel, similar to the shape of egg cases from other Dipturus species e.g. giant skate D. gigas (Ishiyama 1958), longnosed skate D. oxyrinchus (L. 1758) (Ishiyama, 1958b), and wedgenose skate D. whitleyi (Iredale 1938) (Treloar et al., 2006). In comparison to the egg case of Z. chilensis, that of D. trachyderma has smaller horns, located closer to the terminal zone of the apron (Concha et al., 2012).

Checklists

Although Z. chilensis was described in 1848 (as R. chilensis), there are no further records of its presence in Chile until after 1900. Several authors documented the presence of Z. chilensis (but identified as R. flavirostris) in the South Pacific and South Atlantic between 1900 and 1960 (Delfín, 1900, 1902; Lönnberg, 1907; Garman, 1913; Gotschlich, 1913; Quijada, 1913; Fowler, 1926; Norman, 1937; Oliver, 1943; De Buen, 1960). In Chilean waters, checklists and field guides reported the presence of R. chilensis from 1959 onwards (De Buen, 1960; Bahamonde & Pequeño, 1975; Pequeño, 1977, 1989). Dipturus trachyderma was added subsequently to the Chilean ichthyofauna (Pequeño, 1989), and both species of longnose skates were documented to occur in Chilean waters by Pequeño & Lamilla (1993), Sielfeld & Vargas (1999), Lamilla & Sáez (2003), Lamilla & Bustamante (2005) and Bustamante et al. (2014a, b). In the southwest Atlantic Z. chilensis is reported in systematic lists and field guides for marine fishes of Argentina and Uruguay (Ringuelet & Aramburu, 1960; López, 1963b; Menni et al., 1984; López et al., 1989, 1996; Lloris & Rucabado, 1991; Nion et al., 2002; Meneses & Paesch, 2003; Cousseau & Perrota, 2004; Bovcon & Cochia, 2007; Ruarte et al., 2009; Figueroa, 2011). Both Z. chilensis and D. trachyderma were identified as part of the chondrichthyan fauna of Argentina and Uruguay (Menni & Lucifora, 2007; Cousseau et al., 2000, 2007; Perier et al., 2011), and from Falkland Island waters (Bizikov et al., 2004). In Brazilian waters, the presence of D. trachyderma has been made indirectly, through taxonomic lists (Gomes & Picado, 2001) and parasite studies (Knoff et al., 2001a, b, 2002, 2004). The misspelling of D. trachyderma (as D. trachydermus by López et al., 1989) and the incorrect citation of the year

–32– CHAPTER II: LITERATURE REVIEW of its original description may lead to confusion in the literature (Compagno, 1999b; Compagno, 2005a, b; Kyne & Simpfendorfer, 2007, 2010).

Distribution

Of the two genera of longnose skates, Zearaja is restricted to the southern Hemisphere (Beentjes et al., 2002; Cousseau et al., 2007; Last & Gledhill, 2007; Bustamante et al., 2014b), while Dipturus has a worldwide distribution (Gomes & Picado, 2001; Soto & Mincarone, 2001; McEachran & Carvalho, 2002; Gedamke et al., 2005; Ellis et al., 2005a, b; Díaz de Astarloa et al., 2008; Last et al., 2008a; Cannas et al., 2010; Schwartz, 2012; Last & Alava, 2013; Bustamante et al., 2014b). The greatest species diversity of longnose skates occurs on the continental slope, but they are also present in demersal areas of the continental shelf worldwide (Ebert & Compagno, 2007).

In the southeast Pacific, Z. chilensis occurs in cold-temperate waters off Chile between 32° S and 56° S (Fig. 7), although there are dubious records around 29° S (Delfín, 1902), and the type locality Quinteros Bay (32° S), central Chile (Philippi, 1892). The historical absence of records between 1848 and 1892 may be explained by the relative undeveloped fishing activities in the region where the skates may occur that later provided most of the observations. The known range of Z. chilensis in Chile currently includes the central and austral zones (Fig. 7). The presence of D. trachyderma in Chile was documented from specimens collected in central and south Chile by Leible & Stehmann (1987), although its current range also includes waters off Patagonia and Austral Chile (Fig. 8). Overall, Z. chilensis and D. trachyderma seems to have the same distribution in Chilean waters (32° S – 56° S), but this range may be biased considering that most documented records comes from the demersal longline fishery which operates over this latitudinal range. Fishery independent surveys northwards 32° S and southwards of 56° are required to confirm latitudinal boundaries (Bustamante et al., 2014a). No longnose skates have been reported from around Chile’s oceanic island in the southeast Pacific although the areas have been sampled (Andrade & Pequeño, 2008).

In the southwest Atlantic, the presence of Z. chilensis is documented along the Argentinian coast, from 34° S to 54° S, and including the Falkland Islands (Fig. 7). Early records suggested that D. trachyderma in the southern Atlantic was restricted to Argentinian waters (Krefft & Stehmann, 1975; Menni & Gosztonyi, 1977; Menni et al., 1984), but recent records show that it co-occurs with Z. chilensis around the Falkland Islands (Fig. 8), and D. trachyderma has also been documented off south Brazil (Gomes & Picado, 2001; Knoff et al., 2001b), although the occurrence of the species north of 40° S needs to be further explored.

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Dipturus trachyderma has been characterised as an endemic species of the Magellanic Province (see Bustamante et al., 2014b), which is a relatively stable biogeographic region with cold water masses distributed from latitude 42° to 56° S in Chile and Argentina (Camus, 2001), but with an influence across the continental slope up to 36° S (Balech & Ehrlich, 2008). The thermal barrier produced by the warm-tropical water masses of the Argentinian Province (Menni et al., 2010), which extends from Rio de Janeiro (23° S) to Valdes Peninsula (42° S), may restrain ‘Magellanic Province species’ to the continental shelf of Argentina. The scarce records of D. trachyderma from latitudes 32° S to 41° S in the southwest Atlantic raises questions about its proposed presence in Brazilian waters (Gomes & Picado, 2001), 1,500 km north of confirmed Argentinian records. Paesch (2000), reported 44 relatively small specimens (between 39 and 113 cm) from the Argentina and Uruguay Common Fishing Zone (35.5°S and 39.5°S) at depths of 50–240 m. However, size range and capture depth may raise questionings regarding the species identity. As such, records of D. trachyderma northwards Rio de La Plata are considered of doubtful validity and specimens from Brazil may represent a misidentification of species of similar appearance, such as D. mennii.

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Figure 7. Distribution of the yellownose skate Zearaja chilensis in South America. The numbers represent documented records following (1) Philippi (1892), (2) Delfín (1902), (3) Lohnnberg (1907), (4) Gotschlich (1913), (5) Norman (1937), (6) De Buen (1959), (7) Carvajal (1971), (8) Menni (1973), (9) Sadowsky (1973), (10) Ojeda (1983), (11) Raschi (1984), (12) Fernández & Villalba (1985), (13) Leible (1987), (14) Leible et al. (1990), (15) Lloris & Rucabado (1991), (16) Lucifora et al. (2000), (17) Gomes & Picado (2001), (18) Koen Alonso et al. (2001), (19) Céspedes et al. (2005), (20) Licandeo et al. (2006), (21) Cousseau et al. (2007), (22) Licandeo & Cerna (2007), (23) Aburto et al. (2008), (24) Díaz de Astarloa et al. (2008), (25) Quiroz et al. (2009), (26) Silveira (2009), (27) Arkhipkin et al. (2012), (28) Bustamante et al. (2012), (29) Deli Antoni et al. (2012), (30) Bustamante et al. (2014b). Filled circle indicates the holotype locality.

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Figure 8. Distribution of the roughskin skate Dipturus trachyderma in South America. The numbers represent documented records following (1) Krefft & Stehmann (1975), (2) Menni & Gosztonyi (1977), (3) FRV “Walther Herwing” (1978), (4) FRV “Holmberg” (1983), (5) Menni et al. (1984), (6) Leible & Stehmann (1987), (7) Lloris & Rucabado (1991), (8) Cousseau et al. (2000), (9) Gomes & Picado (2001), (10) Knoff et al. (2001b), (11) Cedrola et al. (2005), (12) Céspedes et al. (2005), (13) Díaz de Astarloa et al. (2008), (14) Lamilla et al. (2010), (15) Perier et al. (2011), (16) Arkhipkin et al. (2012), (17) Lamilla et al. (2012a), (18) Lamilla et al. (2012 b), (19) Bustamante et al. (2014b). Filled circle indicates the holotype locality.

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Habitat

The longnose skates, Z. chilensis and D. trachyderma, are distributed in cold-temperate waters of South America, inhabiting the continental shelf and slope of central Chile to southern Brazil at depths of 14 – 477 m (Norman, 1937; Leible & Stehmann, 1987; Agnew et al., 2000; Menni & Stehmann, 2000; Gomes & Picado, 2001; Bustamante et al., 2014b). Sea temperature and salinity preference for these species in the southwest Atlantic are reported to be 3.5 –14 °C and 32.2 –34.3 parts per thousand (ppt) respectively for Z. chilensis (Menni, 1973; Menni & López, 1984; García de la Rosa, 1998); 4.0 –8.2 °C and 33.1‒33.6 ppm for D. trachyderma (Krefft & Stehmann, 1975; Menni & Gosztonyi, 1977; Menni et al., 2010; Arkhipkin et al., 2012).

Zearaja chilensis appears to be relatively common at depths of between 14 ‒ 100 m depth (Menni & López, 1984; Leible et al., 1990; García de la Rosa, 1998; Concha et al., 2012), is abundant between 150 to 350 m (Menni & Gosztonyi, 1977; Menni & López, 1984; Leible, 1987; Leible et al., 1990; Licandeo et al., 2006; Quiroz et al., 2009; Arkhipkin et al., 2012), but can be found at depths of up to 450 m (Ojeda, 1983; Menni & López, 1984; Fuentealba & Leible, 1990; García de la Rosa, 1998). In contrast, D. trachyderma appears less abundant in shallow waters (a single specimen reported from 20 – 22 m in the Beagle Channel (Lloris & Rucabado, 1991), and catches reported from 87 – 97 m in San Jorge Gulf (Cedrola et al., 2005), seems most common at 180 m – 350 m (Krefft & Stehmann, 1975; Menni & Gosztonyi, 1977; Bizikov et al., 2004; Lamilla et al., 2010; Menni et al., 2010; Arkhipkin et al., 2012), but there are records from 400 ‒ 500 m (Leible & Stehmann, 1987; Gomes & Picado, 2001).

Feeding habits

Diet, food consumption and feeding habits of longnose skates have been studied for a number of species, mainly from by-catch fisheries of Patagonia (Koen Alonso et al., 2001, Belleggia et al., 2016), Tasmania (Treloar et al., 2007), Barents Sea (Dolgov et al., 2005), South Africa (Ebert et al., 1991), the north-west Atlantic Ocean (Templeman, 1982), and south-west Atlantic Ocean (Lucifora et al., 2000). In their analysis of skate diets, Ebert & Bizzarro (2007) showed that five species of Dipturus had estimated trophic levels of between 3.52 and 4.06, whereas Z. chilensis had a trophic level of 4.22, with the prey category ‘teleost fishes’ of the greatest dietary importance (81.8%) followed by ‘squid’ (11.6%). Although Z. chilensis had the highest trophic level of all of the 60 skate species examined, the estimated level probably reflected a sampling bias for larger specimens as it is well-known that the diet and trophic level of many elasmobranchs often changes with body size. For

Z. chilensis it has been shown that crustaceans form the main prey category for skates of < 50 cm LT

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(Bahamonde, 1953; De Buen, 1959; Sánchez & Prenski, 1996; Koen Alonso et al., 2001), which would result in a trophic level of about 3.5. However, Belleggia et al. (2016), reassessed the trophic level of Z. chilensis from 776 specimens caught in the south-western Atlantic Ocean and reported ontogenetic and dietary shifts that contrast previous findings. Trophic level varies with body lengh, from 4.29 to 4.59 (mean 4.53). Whereas, as Z. chilensis increases in size the importance of ‘teleost fishes’ (particularly, Argentine hake Merluccius hubbsi and longtail southern cod Patagonotothen ramsayi) in the diet increases dramatically (Sánchez & Prenski, 1996; García de la Rosa, 1998; Lucifora et al., 2000; Paesch, 2000; Koen Alonso et al., 2001; Sánchez & Mabragaña, 2002; Belleggia et al., 2016), confirming their ecological role as a top predator.

While there is information on the diets of various species in the genus Dipturus that suggests that they feed primarily on decapod crustaceans and, secondarily, on teleost fishes (Ebert & Bizarro, 2007; Treloar et al., 2007; Kyne et al., 2008; Simon et al., 2009; Yigin & Ismen, 2010; Forman &

Dunn, 2012), information for D. trachyderma comes from a single individual of 242 cm LT in which four hake M. hubbsi, one sandskate (Psammobatis sp.) and the remains of Antarctic King Crab (Lithodes sp.) were found (Cousseau et al., 2007). Paesch (2000) examined 44 specimens off Rio de La Plata, and found that teleost fishes (M. hubbsi and Patagonotothen sp) are the main dietary component and secondarily, on crustaceans (Amphiphoda and Euphausiacea).

Reproduction

All members of the family Rajidae are oviparous (Wourms, 1977; Carrier et al., 2004), and mature females of both Z. chilensis and D. trachyderma have two functional ovaries and paired uteri. For Z. chilensis the ovaries initiate development of vitellogenic oocytes, indicative of maturity, when females reach 940 – 1039 mm LT, whereas males mature at 760 – 900 mm LT (Fuentealba & Leible, 1990; Oddone et al., 2005; Licandeo et al., 2006; Paesch & Oddone, 2008; Colonello, 2009; Quiroz et al., 2009; Bustamante et al., 2012; Colonello & Cortes, 2014; Wehitt et al., 2015). For D. trachyderma, the oviducal glands and uteri enlarge when female mature at about 2000 mm LT. In males, maturity occurs at around 1860 mm LT based on clasper size and calcification (Licandeo & Cerna, 2007).

Oviparity as it occurs on skates is a specialized strategy since it is based on the advantages of producing small numbers of large eggs and competent newborns (Wourms, 1977). Information related to ovarian fecundity is rare, but oocyte counts for Z. chilensis of 70, 22 – 62, 24 – 84 have been made by Fuentealba & Leible (1990), Licandeo et al. (2006) and Licandeo & Cerna (2007) respectively. For D. trachyderma, ovarian fecundity is 28 – 68 follicles (Licandeo et al., 2007). Only

–38– CHAPTER II: LITERATURE REVIEW one egg capsule per oviduct has been documented for both species, and it is assumed that there is a single embryo per egg case as there are only rare exceptions to this (big skate Raja binoculata Girard 1855 and mottled skate R. pulchra Liu 1932 which have up to four embryos per egg case (Ebert & Winton, 2010). Gestation period for Z. chilensis and D. trachyderma has not been reported, but incubation periods for oviparous batoids may last from a few months to over a year (Carrier et al., 2004). There are no reports on the mating behaviours of Z. chilensis or D. trachyderma.

Age, growth and longevity

Estimation of age-at-maturity, longevity, and growth rate, coupled with knowledge of reproductive output, are mainstays for stock assessment and demographic models that allow for the estimation of intrinsic productivity, resilience, vulnerability, and how populations may change over time (Ricker, 1975; Cailliet et al., 1986; Cailliet & Goldman, 2004). A variety of mineralised structures may be used for age and growth estimation in chondrichthyans, i.e., vertebrae (Francis et al., 2001; Gedamke et al., 2005; Ainsley et al., 2014; Natanson et al., 2014), dermal denticles (Moura et al., 2004; Campana et al., 2006; Serra-Pereira et al., 2008), and caudal thorns (Henderson et al., 2004; Francis & Ó Maolagáin, 2005; Arkhipkin et al., 2008; Francis & Gallagher, 2009). A popular ageing technique in elasmobranch is the sagittal-sectioning of vertebral centra from the thoracic region (Cailliet et al., 2006). Generally, growth band pairs are visible, to a greater or lesser degree, within each centrum from anterior vertebrae of sharks (Goldman, 2004) and posterior vertebrae of batoids (Francis et al., 2001; White et al., 2001; Licandeo et al., 2006; Pierce & Bennett, 2010).

Age and growth of Z. chilensis may be influenced by the geographic location, based on differences in life-history traits differences found for this species in southern Chile (Licandeo & Cerna, 2007) and Argentina (Zavatteri. 2010; Colonello & Cortes, 2014). Estimated maximum age for females is 21 – 27 years, and 17 – 23 years for males (see Table 2), which is considerably older than the 9 years reported for the congeneric species Z. nasuta (Francis et al., 2001). However, estimates of age for Z. chilensis conducted prior to 2000 may have been confounded by inclusion of D. trachyderma specimens among on the samples examined (Fuentealba & Leible, 1990; Fuentealba et al., 1990; Bahamonde et al., 1994, 1996; Gili et al., 1999). The estimated maximum age of D. trachyderma in Chilean waters, determined through the use of the same vertebral analytical approach, was 26 years for females and 25 years for males (Licandeo et al., 2007), which is consistent with an estimated maximum age of 27 years for the sexes combined (Céspedes et al., 2005) (Table 2). Dipturus trachyderma grows to a large size for a skate and has a preference for cold-water habitats, the combination of which might be expected to be reflected in considerable longevity. The estimated maximum age of D. trachyderma in Chilean waters, determined through the use of the same vertebral

–39– CHAPTER II: LITERATURE REVIEW analytical approach as for Z. chilensis is 26 years for females and 25 years for males (Licandeo et al., 2007), which is consistent with an estimated maximum age of 27 years for the sexes combined (Céspedes et al., 2005) (Table 2). Other large skate species, such as Bathyraja griseocauda (Arkhipkin et al., 2008), D. batis (Du Buit, 1972), D. innominatus (Francis et al., 2001), Raja binoculata and R. rhina (McFarlane & King, 2006) also live to over 20 years of age. The concern is that larger species that have slow growth rates and are long lived are particularly vulnerable to population collapse, as has been suggested for Z. chilensis (Agnew et al., 2000; Bustamante et al., 2012).

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Table 2. Summary of age and growth studies for Zearaja chilensis and Dipturus trachyderma. Key: L∞ = length at infinite age, k = growth coefficient, t0 = approximate time at which the animal was size 0, tmax = estimated longevity, t50 = age at which the 50% of the population was mature, L50 = length at which the 50% of the population was mature.

Species Sex L∞ k t0 tmax t50 L50 Reference Zearaja chilensis

♀ 1456 mm LT 0.084 -1.534 24 – 1068 mm LT Céspedes et al. (2005)

♂ 1248 mm LT 0.972 -1.655 21 – 822 mm LT Céspedes et al. (2005)

♀ 1283 mm LT 0.112 -0.514 27 14 1060 mm LT Licandeo et al. (2006)

♂ 1078 mm LT 0.134 -0.862 23 11 860 mm LT Licandeo et al. (2006)

♀ 1364 mm LT 0.104 -0.669 21 13.49 1031 mm LT Licandeo & Cerna (2007)

♂ 1179 mm LT 0.116 -1.056 17 10.74 879 mm LT Licandeo & Cerna (2007)

♀ 1496 mm LT 0.087 -1.266 22 12.75 1035 mm LT Licandeo & Cerna (2007)

♂ 1220 mm LT 0.11 -1.263 19 10.31 871 mm LT Licandeo & Cerna (2007) Dipturus trachyderma

♀/♂ 2848 mm LT 0.067 -0.443 27 – 1940 mm LT Céspedes et al. (2005)

♂ – – – – – 1540 mm LT Céspedes et al. (2005)

♀ 2650 mm LT 0.079 -1.438 26 17 2150 mm LT Licandeo et al. (2006)

♂ 2465 mm LT 0.087 -1.157 25 15 1951 mm LT Licandeo et al. (2006)

–41– CHAPTER II: LITERATURE REVIEW Fisheries in the southeast Pacific

In Chile, the directed fishery for skates started in 1979 and initially targeted Z. chilensis. However, until 2004 at least six skate species including Z. chilensis and D. trachyderma were landed under the generic category of ‘skate’ in official records (Gili et al., 1999; Roa & Ernest, 2000). Recently, the take of skates as bycatch in demersal fisheries for southern hake Merluccius australis (Hutton 1872) and pink ling Genypterus blacodes (Forster 1801) has become an attractive economic activity for many artisanal fishermen as a result of the commercial value and market demand for large skates. Overall, the skate fishery is geographically extensive, covering around 20 degrees of latitude from San Antonio to Punta Arenas (Fig. 9), but with most of the fishing effort between Valdivia and Aisén. The Chilean Fishing Authority identifies two main components within the fishery: (1) Industrial vessels (over 18 m length) that have restricted fishing zones outside of five nautical miles off the coast. These vessels operate southward 41°28.6´ S and can only land skates as bycatch; (2) Artisanal vessels (12 – 17 m length), that are managed through fishing quotas.

Landings and fishing effort have increased steadily since 1993, when the fishery was opened to the Asian market. The artisanal fleet expanded due to international investment and, together with the industrial fishery, reported 3000 tonnes (t) in landings in 1994 (Fig. 10). This artisanal fleet with approximately 1900 vessels accounts for 70% of national landings, whereas only 5 vessels in the industrial trawling fleet that targets hake are authorised to land skates as bycatch, which represents 20 – 25% of total catch. In 1997, fishing management measures were established that included annual catch quotas between the Concepción and the parallel 41°28.6´ S (Fig. 9). During the Asian financial crisis in 1998, skate landings increased considerably peaking at 4000 t and 5193 t in 2000 and 2003 respectively, where approximately 85% was caught solely by the artisanal fleet (Fig. 10). Skate landings subsequently decreased by about 40% (to about 1500 t per annum) over the following decade (SERNAP, 2012).

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Figure 9. Spatial distribution of the Zearaja chilensis and Dipturus trachyderma fishery in Chile, indicating main fishing locations and the extension of the artisanal (dark green) and industrial fisheries (light green).

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Figure 10. Total longnose skate landing in Chile between 1979 and 2012 by fishery and species. Total catch is represented by the solid line. Artisanal (dashed line) and industrial (dotted line) landing are indicated for the ‘skates’ landing category (black), Dipturus trachyderma (green) and Zearaja chilensis (blue). [Note: Z. chilensis and D. trachyderma were officially separated into different landing categories by the Fishing Authority in 2004].

The yellownose skate Z. chilensis is caught in Chile as a target species, but also as a secondary target species in teleost and shark fisheries, i.e. South Pacific hake Merluccius gayi (Guichenot 1848), pink ling G. blacodes, sand flounder Paralichthys microps (Günther 1881), corvina drum Cilus gilberti (Abbott 1899), black ling Genypterus maculatus (Tschudi 1846) and spiny dogfish Squalus acanthias L. 1758, (Lamilla et al., 2008, 2010; Bustamante et al., 2012). However, the roughskin skate D. trachyderma is only observed as a ‘secondary target’ species (>25% of total catch by mass per fishing trip) in yellownose skate and pink ling fisheries, and apparently peaked at about 60 t in 2006 (Fig. 11). However, landing records of D. trachyderma are underestimated as adult skates (>10 kg) are processed on-board and only their fins are landed (Lamilla, 2005), while Z. chilensis is landed and sold complete (ungutted). Although only target skates (Z. chilensis and D. trachyderma) are identified and recorded in landing records, at least another four skates, Bathyraja albomaculata (Norman 1937), B. brachyurops (Fowler 1910), B. griseocauda and Rajella sadowskii (Krefft & Stehmann 1974) are landed as bycatch and reported in a generic category without taxonomic identity which represents 5% of the national landings (Lamilla et al., 2010).

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Figure 11. Total landings of Dipturus trachyderma between 2004 and 2012 by fishery type: artisanal= black, industrial= green.

After 2006, a temporal fishing closure was imposed on the artisanal fleet which operates during the austral summer months (December – February) to protect ‘possible reproductive events’ (Lamilla, 2005). Subsequently, in 2009 – 2011, a total fishing closure was imposed on the entire fishery in response to landing size and overall biomass decline, but despite these ‘closures’, the government continues to allocate national catch quotas of up to 700 t annually as a result of political pressures and lobbying.

The current skate fishery in Chile is considered to be ‘fully exploited’; however, information on stock structure of longnose skates is lacking, which confounds assessment, management and resource allocation. The high percentage of juveniles present in catches, the decreasing trend of the catch size, as well as the constant fishing effort and landings, are symptoms of a fishing collapse as suggested for Z. chilensis (Bustamante et al., 2012). Sustainable harvest requires management strategies based on comprehensive knowledge of the biology and ecology of biological stocks, especially for longnose skates that have intrinsic life history patterns that make them highly susceptible to commercial over-exploitation (Dulvy et al., 2000, 2014; Dulvy & Reynolds, 2002; Frisk, 2010).

–45– CHAPTER II: LITERATURE REVIEW Fisheries in the southwest Atlantic

A different situation is observed for the zone that includes waters off Argentina, Uruguay and the Falkland Islands where longnose skates are caught as bycatch in several trawl and longline fisheries (Colonello et al., 2002; Paesch & Oddone, 2008). An analysis of landing records of marine fishes in Argentina from 1898 to 2010 conducted by Sánchez et al. (2012) has little information about Z. chilensis which only appears after 2008, and nothing about D. trachyderma which is not reported in any landing category. Up today (2017), landing categories in Argentina encompass 90% of skate species landings under a generic label (“rayas nep”), which averaged 17,000 tonnes landed between 2014 and 2017 (MINAGRI, 2017). Between 2009 and 2010, 1.331 and 1.459 t of Z. chilensis were landed in Argentina (MINAGRI, 2017); however, annual landings after 2010 averaged 514 t.

Additional information is available, from incidental capture records for Z. chilensis and D. trachyderma in the Patagonian red shrimp Pleoticus muelleri (Spence Bate 1888) and the Argentine hake M. hubbsi (Marini 1933) fisheries (Angelescu et al., 1958; Buratti, 2004; Cedrola et al., 2005; Estalles et al., 2011). Falkland Islands fisheries have reported Z. chilensis as an abundant species since 1993, where it is caught in the multi-species skate fishery, with approximately 16 other skate species (Agnew et al., 2000; Falkland Islands Government, 2014). Cedrola et al. (2005), reported that the skate bycatch (1.6 – 1.9% of the total catch) in the P. muelleri fishery in San Jorge Gulf comprise seven species, included Z. chilensis as the most frequently caught, with a total biomass of 2,135 kg (55.6% of the total bycatch). Dipturus trachyderma was only present in 9.3% of hauls, with a total biomass of 829 kg (Pettovello, 1999; Cedrola et al., 2005). Overall, the frequency of occurrence of Z. chilensis is 49% in the shrimp (P. muelleri) fishery and 74% in the hake (M. hubbsi) fishery, while the presence of D. trachyderma in the hake fishery is 25% (Bovcon et al., 2013).

The yellownose skate, is the skate with the highest value in Argentina's fisheries with all its catches are exported in total to “Asian markets” (Sánchez et al., 2011). In the early 21st century there was a directed fishery towards skates that operated off north Argentina, whose target and most valuable catch was Z. chilensis. This fishery caught a great number of juveniles and adult females of this species. Currently, Z. chilensis is still the most valuable skate and continues to be exported to Asia (CNP, 2009; Sánchez et al., 2012).

Estalles et al. (2011), reported that Z. chilensis is one of the most abundant species (21.5% relative abundance) in the demersal trawl fishery of M. hubbsi. Dipturus trachyderma was also present as 1.5% of the total bycatch, although the record is of dubious veracity and may relate to a different species given the reported small size-at-maturity (400 mm LT) when compared to specimens in the Pacific (2150 mm LT, Licandeo et al., 2007). However, all authors categorised D. trachyderma as a rare species of minor commercial importance (Góngora et al., 2009; Bovcon et al., 2013).

–46– CHAPTER II: LITERATURE REVIEW Genetics Molecular analysis has become a standard tool in the study of elasmobranch populations, and is used predominantly to estimate phylogenetic relationships and to define population structure (Heist, 2004; Portnoy, 2010; Dudgeon et al., 2012). Genetic methods have been increasingly used to analyse stock structure and connectivity, to identify specimens to species level (considering the presence of sister and cryptic species) and to identify body parts, such as shark fins from markets (Lavery & Shaklee, 1991; Shivji et al., 2002, 2005; Heist, 2004; Clarke et al., 2006; Griffiths et al., 2010), aiding fisheries management and conservation (Heist, 2005; Ovenden et al., 2009, 2010). The first application of molecular/genetic methods in relation to Z. chilensis and D. trachyderma was to identify species through protein electrophoresis and DNA molecular markers, based on 100 individuals from Chilean waters (Céspedes et al., 2005). Using protein electrophoresis, the authors were able to match species to field identification in most cases (88%), but failed to amplify nuclear and mitochondrial regions through PCR, arguing a lack of species-specific primers and that the species are very closely related. Later, Díaz de Astarloa et al. (2008) attempted to sequence the cox1 region of the mitochondrial DNA (mtDNA) from five specimens of Z. chilensis and two of D. trachyderma from Argentinian waters. The authors were able to distinguish Z. chilensis from other Dipturus species, but cox1 gene amplification for D. trachyderma failed ‘as a result of mutational differences within the primer sites’, indicating that D. trachyderma possessed a significantly divergent cox1 haplotype of its own. Using primers for nadh2, eight specimens of Z. chilensis were sequenced from a single locality in Chile (Naylor et al., 2012a), and they were able to differentiate them from the congeneric species Z. nasuta, and also from the biological entity identified as ‘Z. flavirostris’ from the Falkland Islands (despite of being a secondary homonym of Z. chilensis) (Last & Gledhill, 2007). Possible mismatch between Argentinian specimens may be related to the species’ geographical distribution polymorphism (Leible et al., 1990).

Recently, Jeong & Lee (2014), provided what was ostensibly a description of the complete mitochondrial genome of Z. chilensis. The structure of the mitogenome is similar to those of other skates, with 16,909 base-pairs (bp) in length and comprising 13 protein-coding regions, 22 tRNA genes, 2 rRNA genes and 2 non-coding areas (origin of replication and control region). However, Vargas-Caro et al., (2016a) compared the mitochondrial genome Z. chilensis by Jeong & Lee (2014) with one from Chilean waters and found 97.4% similarity (instead of close to 100% as might be expected). As the tissue sample used by Jeong & Lee (2014) was taken from a raw fillet in a Korean restaurant (without examination of the specimen), the 2.6% of difference may indicate the presence of two separate stocks of Z. chilensis in South America, or two different species. The uncertainties highlight the need for caution when using genetic resources without a taxonomic reference or voucher specimen (Vargas-Caro et al., 2016a).

–47– CHAPTER II: LITERATURE REVIEW Parasites

Parasites have been widely used as biological ‘tags’ to provide information for fisheries management on the movements and population discreteness of their fish hosts (Lester, 1990; Moser, 1991; Williams et al., 1992; MacKenzie, 2002). Basically, a fish can become infected by a parasite only when it is within the endemic area of that parasite. The more parasites with different endemic areas that can be used as biological ‘tags’, the better the information that will be obtained about the past movements of fish populations and, hence, about the stock structure (MacKenzie & Abaunza, 2013). Parasite load studies have particular value in deep waters, where standard tagging and recapture methods can be difficult (Mosquera et al., 2003). The use of parasites as an auxiliary tool is well documented for teleost fishes; however, there is little information for chondrichthyans (Moore, 2001). Approximately 20 parasites have been described to generic or species level from specimens of Z. chilensis (n = 12) and D. trachyderma (n = 8) and these have been summarized in Table 3 and 4 respectively. Sites of infection are mostly the stomach and the spiral valve, but mouth and gills are also documented (Villalba & Fernández, 1985).

Table 3. Parasites reported to be infecting the yellownose skate Zearaja chilensis.

Species Reference Platyhelminthes: Cestoda Order Tetraphyllidea; Family Onchobothridae Acanthobothrium annapinkiensis Carvajal & Goldstein (1971) Family Phyllobothriidae Echeneibothrium magalosoma Carvajal & Dailey (1975), Leible et al. (1990) Echeneibothrium multiloculatum Carvajal & Dailey (1975), Carvajal et al. (1985), Echeneibothrium williamsi Leible et al. (1990) Phyllobothrium sp. Leible et al. (1990) Order Trypanorhyncha; Family Grillotiidae Grillotia dollfusi Carvajal & Goldstein (1971), Leible et al. (1990) Platyhelminthes: Monogenea Order Monocotylidea; Family Monocotylidae Dendromonocotyle rajidicola Irigoitia et al. (2016) Merizocotyle euzeti Irigoitia et al. (2014)

–48– CHAPTER II: LITERATURE REVIEW Table 3. continued.

Species Reference Platyhelminthes: Trematoda Order Plagiorchiida; Family Azygiidae Otodistomum cestoides Threlfall & Carvajal (1986), Aburto et al. (2008) Phylum Nematoda: Rhabditea Order Ascaridida; Family Acanthocheilidae Pseudanisakis argentinensis Irigoitia et al. (2017) Pseudanisakis tricupola Fernández & Villalba (1985) Order Ascaridida; Family Anisakidae Anisakis sp. (type I) Fernández & Villalba (1985) Pseudoterranova sp. Order Spirurida; Family Physalopteridae Proleptus Carvajali Fernández & Villalba (1985) Arthropoda: Maxillopoda Order Cyclopoida; Family Chondracanthidae Acanthochondrites sp. Villalba & Fernández (1985)

Table 4. Parasites reported to be infecting the rough skate Dipturus trachyderma.

Parasite species Reference

Platyhelminthes: Cestoda Order Trypanorhyncha; Family Tentaculariidae Myxonybelinia beveridgei Knoff et al. (2002, 2004) Order Trypanorhyncha; Family Lacistorhynchidae Paragrillotia sp. Leible et al. (1990) Order Tetraphyllidea; Family Phyllobothriidae Phyllobothrium c.f. lactuca Leible et al. (1990)

Platyhelminthes: Trematoda Order Plagiorchiida; Family Azygiidae Otodistomum veliporum Knoff et al. (2001b), Kohn et al. (2007)

–49– CHAPTER II: LITERATURE REVIEW Table 4. continued.

Parasite species Reference

Nematoda: Rhabditea

Order Ascaridida; Family Anisakinae Anisakis sp. Knoff et al. (2001a) Contracaecum sp. Raphidascaris sp. Acanthocephala: Palaecanthocephala Order Echinorhynchida; Family Rhadinorhymchidae Gorgorhynchus sp. Knoff et al. (2001b), Santos et al. (2008)

CONCLUSIONS

For the first time, available information concerning Zearaja chilensis and Dipturus trachyderma has been reviewed in a single assessment. Conservation is the act of guarding and protecting biodiversity, and involves management, but defining a taxonomic entity is the first step for conservation. Here we provide taxonomic clarity, and collate all relevant information with regard to the biology, ecology and fisheries that impact these species.

The yellownose skate Z. chilensis and the roughskin skate D. trachyderma require particular attention to mitigate the chance of population collapse in South American waters. Population decline and symptoms of fishing collapse have already been documented for Z. chilensis and current research has provided strong evidence that longnose skates have a high potential risk of near-future extinction. Given the growing concern for longnose skate populations, there is a strong ongoing need for science to help improve community understanding and the conservation management of the species to ensure their long-term sustainability. In this review, we have highlighted the knowledge gaps that need to be addressed in order to better understand the biology and ecology of the species and the key threatening processes.

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3. Morphological and molecular identification of commercially important longnose skates (Chondrichthyes, Rajiformes) in the southeast Pacific Ocean

Manuscript to be submitted for consideration at Journal of Fish Biology (ISSN: 1095-8649)

Copyright © Carolina Vargas-Caro, 2017

License Number: n/a

Abstract

During October 2014 and January 2015, 583 longnose skates were obtained from the commercial fishery at four locations along the Chilean coast. Specimens were identified in the field as Zearaja chilensis and Dipturus trachyderma, but species identity was confirmed posteriorly using morphological characters and DNA analysis. Thirty-seven morphometric measurements and 3 meristic characters of the body of each skate were used to identify specimens and analyse potential variations an across species and locations. At the population level, morphological variations were manly associated with the reproductive condition of each specimen. No effect of the sampling location was detected yet, sexual dimorphism of Z. chilensis is evident. Overall, no significant differences were observed in morphometric variation of D. trachyderma due to the absence of mature individuals in both sexes. The number of midline, nuchal and inter-dorsal thorns could be used to discriminate specimens of Z. chilensis from D. trachyderma. Additionally, partial sequences of the 16S, cox1, nadh2 and the control regions of the mitochondrial DNA were amplified and analysed for K2P intraspecific and interspecific divergences, through Neighbour-Joining tree-reconstruction method. Amplified fragments of cox1 gene and the control region contained information to separate the target species; however, specimens of D. trachyderma and Z. chilensis were grouped indistinctly within a single clade using 16S and nadh2 fragments. Overall, the molecular analysis agreed with the morphological identification of specimens. Morphometric and mitochondrial DNA may therefore be a useful supporting tool for identifying longnose skates and may contribute to future fishery management strategies, especially to enforce species-specific quota allocation.

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INTRODUCTION

Many aspects of fisheries management are based on the accurate identification of the species involved and those affected directly and indirectly by this operation (Bonfil, 1994; Thorpe et al., 1995; Crandall et al., 2000). Landed specimens must be identified in order to maintain detailed fishery records, and to support administration and management processes (Pawson & Jennings, 1996; Musick et al., 2000; Musick & Bonfil, 2005). The biological definition of stocks, species’ distribution ranges, the discovery of cryptic species, identification of juveniles stages, identification of bycatch and construction of trophic models; assume that species can be, and are, accurately identified (Musick et al., 2000; Cadrin et al., 2014). Cryptic species and species-complexes are particularly difficult issues for fisheries management, however, genetic data may provide for reliable identification and can supplement species diagnosis (e.g., Smith et al., 2008, 2011).

The accurate taxonomic identification of a species plays an important role in conservation, determining the appropriate assessment of sampling effort; misidentification of several species into a single taxonomic entity might deny an appropriate conservation management to a particular species (Mace, 2014). One of the major issues associated with identifying marine fishes is that many taxonomic descriptions are based on visible morphology and therefore relies on phenotypic characters (Hanner et al., 2011). The correct identification of a species provides critical basal information to support any biological research (Last et al., 2008a, 2016); however, among elasmobranch fishes, skates have been the subject of numerous taxonomic revisions leading to a high level of uncertainty in the identification of species (Stevens et al., 2000; Dulvy & Reynolds, 2002; Ebert & Compagno, 2007; Last et al. 2016). For commercially targeted species, identification issues are usually solved by grouping several species at higher taxonomic levels, e.g. family level; or generic categories e.g., “skate” or “rays”, that lacks taxonomic value for a species complex, (Bonfil, 1994; Ebert & Compagno, 2007; Lamilla et al., 2010; Dulvy et al., 2014).

In batoids, morphometric variation in body shape may indicate differences in growth rates and the level of sexual development (Ishiyama, 1950; Du Buit, 1968; Carrier et al., 2004), which are part of the ontogeny of all living beings (Cadrin et al. 2014). Morphometric research is commonly used to investigate species and stock identification (Cadrin & Friedland, 2005), and is presented as a practical analysis of natural morphological variation and allows a mathematical approach to detect changes in the shape from a mosaic of forms, such as their transformation (variation) and their interaction (co-variation) with the environment (Francis, 2006; Klingenberg, 2009). The role of morphometrics, as applies to elasmobranch fishes, has been fundamental in the resolution of the identities of threatened species, especially in relation to their management and conservation (White

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& Last, 2012); however, morphologically-based taxonomic differentiation is not always clear, and under such circumstances molecular markers might provide a solution to the problem (Dudgeon et al., 2012). The recent development of molecular tools to assist in species identification has focused mainly on species complexes and cryptic species (White & Last, 2012; Cerutti-Pereyra et al., 2012; Tillett et al., 2012; Griffiths et al., 2013). The use of genetic markers has also allowed a better understanding of species dynamics and provided answers to questionings that have not been possible to resolve using traditional morphological methods (Schwartz et al., 2007; Ovenden, 2013).

There are few species of cartilaginous fishes considered target species in coastal and oceanic fisheries in the southeast Pacific Ocean (Bustamante et al., 2014b), although the longnose skates Zearaja chilensis and Dipturus trachyderma are of considerable commercial importance in Chilean waters (Lamilla et al., 2010; Vargas-Caro et al., 2015; SERNAP, 2015). Several authors have carried out taxonomic revisions and produced identification guides which include aspects of the morphology and biology of both species (see Vargas-Caro et al., 2015); however, the identities of Z. chilensis and D. trachyderma are still confused in landing records due to similarities in body shape and colour (Vargas-Caro et al., 2015). Juveniles of both species are caught indiscriminately and in high proportions by fisheries (Bustamante et al., 2012), and the recorded identity of the species landed usually relies on the expertise of the fishers. Immature longnose skates may lack (or may have lost) the morphological characters required for accurate species identification, such as the presence of a single nuchal thorn, or the number of thorns on the tail (Vargas-Caro et al., 2015). The general lack of rigor in ensuring that landing records are correct can be a major issue for fishery management and quota allocation, and may undermine any conservation measures enacted for these species.

There is a clear requirement to better understand the morphological variability of longnose skates, and the present research aims to identify morphological and molecular features to aid species identification in the early-life stages of Z. chilensis and D. trachyderma caught in Chilean fisheries. The use morphometric analysis is explored as a tool to assess the potential variations in body proportions of Z. chilensis and D. trachyderma. Additionally, genetic markers based on mitochondrial DNA analysis are explored as a potential tool for identification of body parts and processed, unidentified specimens. The inclusion of new morphological and genetic characters for species diagnosis may improve the reliability of fishery landings records, especially those from commercial fisheries. With this additional and correct information comes the means to improve fishery management, which could have a positive impact on these two species of conservation concern.

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MATERIALS AND METHODS

Sample collection

Skates were sampled at four landing sites along the coast of Chile during October 2014 and January 2015 (Fig. 12). Specimens were caught by artisanal vessels operating less than five nautical miles offshore, using bottom longline fishing gear. Skates caught during commercial activities were collected under a scientific research permit (R. Ex. 2878-14, 3442-14 and 1482-15) issued by the Fisheries Undersecretariat. Skates were identified to species level using external morphological characters as described by Lamilla & Bustamante (2005). Digital photographs were taken of each specimen following a standardised routine. For each specimen, the species’ preliminary identification, total length (LT) and sex was recorded in the field. Direct measurements were made to the nearest mm, and sex was based on the presence or absence of claspers.

Figure 12. Map showing landing locations of longnose skates in Chile surveyed in this study.

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Morphometric and meristic analyses

The identity of each specimen recorded in the field was confirmed post hoc by using the population range. Standardised photographs of the stretched dorsal and ventral side of each specimen, while placed on a flat surface, were taken perpendicular to the disc-midpoint using a digital camera with fixed lens, tripod and a two-axis clinometer. Each photo included a linear 25 cm scale as a calibration reference (Fig. 13). The number of thorns in the median dorsal row (TM), and the presence and number of nuchal thorns (TN) and thorns between dorsal fins (TD) were also registered in the field. A total of 36 (37 for males) morphometric measurements (after Last et al., 2008b) were obtained from the photos of each skate (Table 5), using the PointPicker plugin for ImageJ/Fiji 1.5f (NIH, Bethesda, MD, Schneider et al., 2012). All morphometric measures were expressed as a percentage of total length (LT).

To verify species identification, a non-metric multidimensional scaling analysis (nMDS) was performed as an ordination method to visualise differences between species. Potentially significant differences of body shape by sex and species were explored by using the complete dataset of morphometric measurements and meristic data. Differences within and between species by sex were explored using a similarity matrix on two-dimensional nMDS ordination plots. An analysis of similarity (ANOSIM), was used to identify the level of significance among potential groups. Also, a similarity percentages analysis (SIMPER) was used to identify the contribution of each variable (in this case, a morphometric or meristic variable) to the overall similarity within and between groups. All multivariate analyses were conducted in Primer v6 (Clarke & Gorley, 2006) with significance accepted at P < 0.05.

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Figure 13. Standardized photographs used to confirm species identification of longnose skates landed in Chile. Dorsal and ventral views of Zearaja chilensis (A, B). Dorsal and ventral views of Dipturus trachyderma (C, D). Colorimetric scale bar = 25 cm.

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Table 5. Definitions of morphometric and meristic variables taken on commercial longnose skates in Chile. Methodology to record for each variable is explained in Last et al. (2008b).

Variable Definition Disc–1 Total length, in mm (LT). Disc–2 Disc width. Disc–3 Disc length. Disc–4 Snout to maximum width. Disc–5 Snout length. Disc–6 Snout to spiracle. Disc–7 Dorsal head length. Disc–8 Cloaca to caudal fin length. Disc–9 Ventral snout length. Disc–10 Pre-nasal length. Disc–11 Ventral head length. Disc–12 Cloaca to pelvic-clasper insertion. Disc–13 Post-cloacal length of clasper (clasper outer length). Head–1 Orbit diameter. Head–2 Orbit and spiracle length. Head–3 Spiracle length. Head–4 Distance between orbits. Head–5 Distance between spiracles. Head–6 Snout to cloaca length. Head–7 Mouth width. Head–8 Distance between nostrils. Head–9 Nasal curtain length. Head–10 Total width of nasal curtain. Head–11 Width of first gill opening. Head–12 Width of fifth gill opening. Head–13 Distance between first gill openings. Head–14 Distance between fifth gill openings. Tail–1 Length of anterior pelvic lobe. Tail–2 Length of posterior pelvic lobe. Tail–3 Pelvic base width. Tail–4 Width of tail at axil of pelvic fins. Tail–5 Width of tail at mid-length. Tail–6 Width of tail at first dorsal fin origin. Tail–7 First dorsal base. Tail–9 First dorsal fin origin to caudal-fin tip. Tail–10 Second dorsal fin origin to caudal-fin tip. Tail–11 Caudal-fin length.

TN Number of thorns in the nuchal area. TM Number of thorns in the disc midline. TD Number of thorns between dorsal fins.

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Mitochondrial DNA amplification and analyses

Four mitochondrial DNA regions, traditionally used for species identification and phylogenetic analysis in marine fishes, were selected to confirm the species identity in the laboratory. A fin-clip was obtained from specimens of each species selected at random from each locality. Samples were preserved in absolute ethanol at -20°C until DNA was extracted. Total genomic DNA was extracted from ~25 mg of tissue following a modified salting-out method (Miller et al., 1988). DNA sequences for the 16S, nadh2, cox1 and control region were obtained for each individual via PCR amplification using primers listed in Table 6. For the regions 16S and cox1, universal primers designed for skates were used (Palumbi, 1996; Ward et al., 2005). Primers for the nadh2 gene and mtCR were designed ‘de novo’ with Primer3 v2.3.4 plugin of Geneious v9 (Biomatters, Auckland, NZ), using, as reference, the whole mitochondrial genome of Zearaja chilensis (Vargas-Caro et al., 2016a). PCR amplifications were performed in 10 µl reactions containing 1 µl of genomic DNA (20

– 50 ng), 5.9 µl of Milli-Q H2O, 1 µl of 10x buffer MgCl2 (15 mM), 1 µl of dNTPs (2 mM), 0.5 µl of each primer set, and 0.1 µl Taq DNA polymerase (5 U/µl). Thermal cycling consisted of an initial denaturation at 94–95 °C for 30–60 s, followed by 35 cycles at 94 °C for 30–60 s, 51–62 °C for 1 min, and 72 °C for 1 min. A final extension step was added at 72 °C for 10 s. Amplified PCR products were treated with 1 U ExoSAP-IT (USB® Products Affymetrix, Inc) at 37° C for 45 min, followed by an inactivation step at 80 °C for 15 min. The cleaned PCR product was sequenced in both directions using BigDye Kit v3.1 on an ABI Prism 3130xl Genetic Analyser (Applied Biosystems). Sequences were manually reviewed for uncalled and miscalled bases, and all variable positions were confirmed by comparing sequence reads produced by the forward and reverse primers on each individual. Primer sequences were removed, and once the sequences had been checked for discrepancies, a consensus sequence was produced for each specimen.

Intraspecific and interspecific genetic distances (Kimura, 1980) were calculated by pairwise comparison of the sequences of all the individuals using the Kimura 2-parameter (K2P) method with the Mega 7 (Kumar et al., 2016). The K2P distance matrix was used to reconstruct a Neighbour- Joining (NJ) tree for each amplified region in Geneious with bootstrap values for 1,000 replicates. Novel haplotype sequences were deposited in GenBank (Table 7). Final alignments for each mitochondrial region included related species within the family Rajidae as outgroups, providing a robust phylogenetic context for their analysis (Table 8).

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Table 6. List of primers used for PCR amplification and sequencing of commercial longnose skates in Chile.

Primer Sequence Reference 16S AR 5'-CGC CTG TTT AAC AAA AAC AT-3' Palumbi (1996) 16S BR 5'-CCG GTC TGA ACT CAG ATC ACG T-3' Palumbi (1996) cox1 FishF1 5'- TCT ACT AAT CAC AAA GAT ATC GGC AC -3' Ward et al. (2005) cox1 FishR2 5'- ACT TCA GGG TGA CCG AAG AAT CAG AA -3' Ward et al. (2005) nadh2 F4 5'-TCA AGC TTT TGG GCC CAT ACC-3' Present study nadh 2 R4 5'-AGA GGA TGT GAG ATA GAG TCT TGC-3' Present study CRzch F 5'-TGA ACT CCC ATC CTT GGC TC-3' Present study CRzch R 5'-GTA TTG GTC GGT TCT CGC CA-3' Present study

Table 7. List of haplotypes of Zearaja chilensis and Dipturus trachyderma used for comparison analysis. Accessions numbers for GenBank are presented for each specimen and gene region amplified.

Locality Specimen Id 16S cox1 nadh2 Control Region

Zearaja chilensis San Antonio Zch_001_S MF360252 MF360412 MF360572 KX708934 Zch_002_S MF360253 MF360413 MF360573 KX708935 Zch_003_S MF360254 MF360414 MF360574 KX708936 Zch_004_S MF360255 MF360415 MF360575 KX708937 Zch_005_S MF360256 MF360416 MF360576 KX708938 Zch_006_S MF360257 MF360417 MF360577 KX708939 Zch_007_S MF360258 MF360418 MF360578 KX708940 Zch_008_S MF360259 MF360419 MF360579 KX708941 Zch_009_S MF360260 MF360420 MF360580 KX708942 Zch_010_S MF360261 MF360421 MF360581 KX708943 Zch_011_S MF360262 MF360422 MF360582 KX708944 Zch_012_S MF360263 MF360423 MF360583 KX708945 Zch_013_S MF360264 MF360424 MF360584 KX708946 Zch_014_S MF360265 MF360425 MF360585 KX708947 Zch_015_S MF360266 MF360426 MF360586 KX708948 Zch_050_S MF360267 MF360427 MF360587 KX708949 Zch_051_S MF360268 MF360428 MF360588 KX708950 Zch_052_S MF360269 MF360429 MF360589 KX708951 Zch_053_S MF360270 MF360430 MF360590 KX708952 Zch_054_S MF360271 MF360431 MF360591 KX708953

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Table 7. Continued.

Locality Specimen Id 16S cox1 nadh2 Control Region Valdivia Zch_070_V MF360272 MF360432 MF360592 KX709004 Zch_071_V MF360273 MF360433 MF360593 KX709005 Zch_072_V MF360274 MF360434 MF360594 KX709006 Zch_073_V MF360275 MF360435 MF360595 KX709007 Zch_074_V MF360276 MF360436 MF360596 KX709008 Zch_075_V MF360277 MF360437 MF360597 KX709009 Zch_077_V MF360278 MF360438 MF360598 KX709010 Zch_078_V MF360279 MF360439 MF360599 KX709011 Zch_079_V MF360280 MF360440 MF360600 KX709012 Zch_080_V MF360281 MF360441 MF360601 KX709013 Zch_081_V MF360282 MF360442 MF360602 KX709014 Zch_082_V MF360283 MF360443 MF360603 KX709015 Zch_1001_V MF360284 MF360444 MF360604 KX709016 Zch_1002_V MF360285 MF360445 MF360605 KX709017 Zch_1003_V MF360286 MF360446 MF360606 KX709018 Zch_1005_V MF360287 MF360447 MF360607 KX709019 Zch_1006_V MF360288 MF360448 MF360608 KX709020 Zch_1007_V MF360289 MF360449 MF360609 KX709021 Zch_1008_V MF360290 MF360450 MF360610 KX709022 Zch_1009_V MF360291 MF360451 MF360611 KX709023 Puerto Zch_1201_P MF360292 MF360452 MF360612 KX709065 Montt Zch_1203_P MF360293 MF360453 MF360613 KX709066 Zch_1204_P MF360294 MF360454 MF360614 KX709067 Zch_1205_P MF360295 MF360455 MF360615 KX709068 Zch_1206_P MF360296 MF360456 MF360616 KX709069 Zch_1207_P MF360297 MF360457 MF360617 KX709070 Zch_1208_P MF360298 MF360458 MF360618 KX709071 Zch_1210_P MF360299 MF360459 MF360619 KX709072 Zch_1211_P MF360300 MF360460 MF360620 KX709073 Zch_1212_P MF360301 MF360461 MF360621 KX709074 Zch_1215_P MF360302 MF360462 MF360622 KX709075 Zch_1216_P MF360303 MF360463 MF360623 KX709076 Zch_1219_P MF360304 MF360464 MF360624 KX709077 Zch_1221_P MF360305 MF360465 MF360625 KX709078 Zch_1222_P MF360306 MF360466 MF360626 KX709079 Zch_1223_P MF360307 MF360467 MF360627 KX709080 Zch_1230_P MF360308 MF360468 MF360628 KX709081 Zch_1234_P MF360309 MF360469 MF360629 KX709082 Zch_1236_P MF360310 MF360470 MF360630 KX709083 Zch_1239_P MF360311 MF360471 MF360631 KX709084

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Table 7. Continued.

Locality Specimen Id 16S cox1 nadh2 Control Region Punta Zch_1601_Z MF360312 MF360472 MF360632 KX709160 Arenas Zch_1602_Z MF360313 MF360473 MF360633 KX709161 Zch_1603_Z MF360314 MF360474 MF360634 KX709162 Zch_1604_Z MF360315 MF360475 MF360635 KX709163 Zch_1605_Z MF360316 MF360476 MF360636 KX709164 Zch_1606_Z MF360317 MF360477 MF360637 KX709165 Zch_1607_Z MF360318 MF360478 MF360638 KX709166 Zch_1608_Z MF360319 MF360479 MF360639 KX709167 Zch_1609_Z MF360320 MF360480 MF360640 KX709168 Zch_1610_Z MF360321 MF360481 MF360641 KX709169 Zch_1611_Z MF360322 MF360482 MF360642 KX709170 Zch_1612_Z MF360323 MF360483 MF360643 KX709171 Zch_1613_Z MF360324 MF360484 MF360644 KX709172 Zch_1614_Z MF360325 MF360485 MF360645 KX709173 Zch_1616_Z MF360326 MF360486 MF360646 KX709174 Zch_1619_Z MF360327 MF360487 MF360647 KX709175 Zch_1621_Z MF360328 MF360488 MF360648 KX709176 Zch_1625_Z MF360329 MF360489 MF360649 KX709177 Zch_1626_Z MF360330 MF360490 MF360650 KX709178 Zch_1628_Z MF360331 MF360491 MF360651 KX709179

Dipturus trachyderma San Antonio Dtr_005_S MF360332 MF360492 MF360652 KX709205 Dtr_006_S MF360333 MF360493 MF360653 KX709206 Dtr_007_S MF360334 MF360494 MF360654 KX709207 Dtr_056_S MF360335 MF360495 MF360655 KX709208 Dtr_1401_S MF360336 MF360496 MF360656 KX709209 Dtr_1402_S MF360337 MF360497 MF360657 KX709210 Dtr_1403_S MF360338 MF360498 MF360658 KX709211 Dtr_1404_S MF360339 MF360499 MF360659 KX709212 Dtr_1405_S MF360340 MF360500 MF360660 KX709213 Dtr_1406_S MF360341 MF360501 MF360661 KX709214 Dtr_1407_S MF360342 MF360502 MF360662 KX709215 Dtr_1408_S MF360343 MF360503 MF360663 KX709216 Dtr_1409_S MF360344 MF360504 MF360664 KX709217 Dtr_1410_S MF360345 MF360505 MF360665 KX709218 Dtr_1411_S MF360346 MF360506 MF360666 KX709219 Dtr_1412_S MF360347 MF360507 MF360667 KX709220 Dtr_1413_S MF360348 MF360508 MF360668 KX709221 Dtr_1414_S MF360349 MF360509 MF360669 KX709222 Dtr_1415_S MF360350 MF360510 MF360670 KX709223 Dtr_1428_S MF360351 MF360511 MF360671 KX709224

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Table 7. Continued.

Locality Specimen Id 16S cox1 nadh2 Control Region Valdivia Dtr_017_V MF360352 MF360512 MF360672 KX709257 Dtr_018_V MF360353 MF360513 MF360673 KX709258 Dtr_019_V MF360354 MF360514 MF360674 KX709259 Dtr_020_V MF360355 MF360515 MF360675 KX709260 Dtr_021_V MF360356 MF360516 MF360676 KX709261 Dtr_022_V MF360357 MF360517 MF360677 KX709262 Dtr_023_V MF360358 MF360518 MF360678 KX709263 Dtr_024_V MF360359 MF360519 MF360679 KX709264 Dtr_025_V MF360360 MF360520 MF360680 KX709265 Dtr_026_V MF360361 MF360521 MF360681 KX709266 Dtr_1004_V MF360362 MF360522 MF360682 KX709267 Dtr_1011_V MF360363 MF360523 MF360683 KX709268 Dtr_1020_V MF360364 MF360524 MF360684 KX709269 Dtr_1026_V MF360365 MF360525 MF360685 KX709270 Dtr_1030_V MF360366 MF360526 MF360686 KX709271 Dtr_1066_V MF360367 MF360527 MF360687 KX709272 Dtr_1069_V MF360368 MF360528 MF360688 KX709273 Dtr_1082_V MF360369 MF360529 MF360689 KX709274 Dtr_1087_V MF360370 MF360530 MF360690 KX709275 Dtr_1097_V MF360371 MF360531 MF360691 KX709276 Puerto Dtr_1209_P MF360372 MF360532 MF360692 KX709310 Montt Dtr_1213_P MF360373 MF360533 MF360693 KX709311 Dtr_1214_P MF360374 MF360534 MF360694 KX709312 Dtr_1269_P MF360375 MF360535 MF360695 KX709313 Dtr_1270_P MF360376 MF360536 MF360696 KX709314 Dtr_1274_P MF360377 MF360537 MF360697 KX709315 Dtr_1275_P MF360378 MF360538 MF360698 KX709316 Dtr_1277_P MF360379 MF360539 MF360699 KX709317 Dtr_1282_P MF360380 MF360540 MF360700 KX709318 Dtr_1284_P MF360381 MF360541 MF360701 KX709319 Dtr_1292_P MF360382 MF360542 MF360702 KX709320 Dtr_1295_P MF360383 MF360543 MF360703 KX709321 Dtr_1296_P MF360384 MF360544 MF360704 KX709322 Dtr_1297_P MF360385 MF360545 MF360705 KX709323 Dtr_041_P MF360386 MF360546 MF360706 KX709324 Dtr_042_P MF360387 MF360547 MF360707 KX709325 Dtr_043_P MF360388 MF360548 MF360708 KX709326 Dtr_044_P MF360389 MF360549 MF360709 KX709327 Dtr_045_P MF360390 MF360550 MF360710 KX709328 Dtr_046_P MF360391 MF360551 MF360711 KX709329

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Table 7. Continued.

Locality Specimen Id 16S cox1 nadh2 Control Region Punta Dtr_1617_Z MF360392 MF360552 MF360712 KX709335 Arenas Dtr_1627_Z MF360393 MF360553 MF360713 KX709336 Dtr_1635_Z MF360394 MF360554 MF360714 KX709337 Dtr_1692_Z MF360395 MF360555 MF360715 KX709338 Dtr_1708_Z MF360396 MF360556 MF360716 KX709339 Dtr_1714_Z MF360397 MF360557 MF360717 KX709340 Dtr_1799_Z MF360398 MF360558 MF360718 KX709341 Dtr_1800_Z MF360399 MF360559 MF360719 MF360732 Dtr_1801_Z MF360400 MF360560 MF360720 MF360733 Dtr_1802_Z MF360401 MF360561 MF360721 MF360734 Dtr_1803_Z MF360402 MF360562 MF360722 MF360735 Dtr_1804_Z MF360403 MF360563 MF360723 MF360736 Dtr_1805_Z MF360404 MF360564 MF360724 MF360737 Dtr_1806_Z MF360405 MF360565 MF360725 MF360738 Dtr_1807_Z MF360406 MF360566 MF360726 MF360739 Dtr_1808_Z MF360407 MF360567 MF360727 MF360740 Dtr_1809_Z MF360408 MF360568 MF360728 MF360741 Dtr_1810_Z MF360409 MF360569 MF360729 MF360742 Dtr_1811_Z MF360410 MF360570 MF360730 MF360743 Dtr_1812_Z MF360411 MF360571 MF360731 MF360744

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Table 8. List of species and haplotypes used for phylogenetic analysis, including accessions numbers for GenBank database.

Species 16S cox1 nadh2 Control Region

Family Arhynchobatidae Atlantoraja castelnaui KM507724 KM507724 KM507724 KM507724 Bathyraja albomaculata MF278964 MF278964 MF278964 MF278964 Bathyraja maccaini MF278977 MF278977 MF278977 MF278977 Bathyraja richardsoni MF278979 MF278979 MF278979 MF278979 Bathyraja shuntovi MF278980 MF278980 MF278980 MF278980 Beringraja pulchra KF623030 KF623030 KF623030 KF623030

Family Rajidae

Dipturus argentinensis EU074406, EU074408, EU074410

Dipturus australis DQ108186, DQ108188 JQ518873

Dipturus batis EF081271, EF081272, JN184275 EF081273, EF081274, EF081275, EF081276, EF081277, EF081278, EF081280

Dipturus campbelli GU804912, JF493383

Dipturus canutus EU398775, EU398776

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Table 8. Continued.

Species 16S cox1 nadh2 Control Region

Dipturus cerva DQ108189

Dipturus confuses EU398771, EU398773 JQ518867

Dipturus flossada KF604219, KF604220

Dipturus gudgeri EU398763, EU398767 JN184276 Dipturus innominatus MF278961, MF278981 MF278961, MF278981 MF278961, MF278981, MF278961, MF278981 JQ518871

Dipturus intermedia KF604221, KF604222 Dipturus kwangtungensis KF318309 EU339344, EU339346, KF927820, KF927821, KF319309 KF318309 KF319309

Dipturus laevis JF895055, JF895056

Dipturus lemprieri EU848453

Dipturus leptocauda JQ518866

Dipturus linteus KF604225, KF604229 JN184277

Dipturus nidarosiensis EF081266, EF081267, EF081268 Dipturus oxyrinchus EF081269, EF081270, HM043217, MF278968 JQ518865, MF278968 MF278968 MF278968

Dipturus pullopunctatus HM140434, HM140436 JF493385, JF493387 JQ518875

Dipturus springeri GU805029, HQ945978 JQ518876

Dipturus tengu EF081265 JQ518863, KF927828

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Table 8. Continued.

Species 16S cox1 nadh2 Control Region Dipturus trachyderma KR152643 KR152643 KR152643, JQ400131 KR152643 Raja Asterias MF278971, FJ825436 MF278971 MF278971, JQ518887 MF278971

Raja binoculata JQ518885

Raja brachyura FJ825437 Raja clavate MF278973, FJ825435 MF278973 MF278973, JN184284 MF278973

Raja eglanteria JN184285, JQ518889 Raja miraletus MF278974, FJ825439 MF278974 MF278974, JQ518891 MF278974

Raja montagui JQ518886 Raja polystigma MF278972 MF278972 MF278972 MF278972

Raja rhina JN184286, JQ519188

Raja velezi JQ518888

Raja whitleyi DQ108178, DQ108181 Zearaja chilensis MF278962, KF648508, MF278962, KF648508, MF278962, KF648508, MF278962, KF648508, KJ913073 KJ913073, EU074402, KJ913073, JQ518904 KJ913073 EU074404 Zearaja maugeana MF278966, MF278967 MF278966, MF278967 MF278966, MF278967 MF278966, MF278967 Zearaja nasuta MF278960 MF278960 MF278960, JQ518903 MF278960

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RESULTS

A total of 583 specimens were sampled at the four locations along the Chilean coast (Table

9). In the field, 406 specimens were identified as Zearaja chilensis, 161 males of 726 – 1250 mm LT and 245 females of 652 – 1498 mm LT. The remaining 177 specimens were identified as Dipturus trachyderma, 74 males of 901 – 2224 mm LT and 103 females of 974 – 2053 mm LT.

Table 9. Number of specimens (n) and size structure (as total length, TL in mm) of sampled commercial longnose skates, by sex and locality.

Zearaja chilensis Dipturus trachyderma n Min TL Max TL n Min TL Max TL Sex Male 161 738 1228 74 890 2075 Female 245 250 1830 103 982 2425 Locality San Antonio 79 764 1134 89 976 2200 Valdivia 117 250 1141 46 890 2143 Puerto Montt 99 821 1198 22 1105 1678 Punta Arenas 111 872 1830 20 1203 2425 Total 406 250 1830 177 890 2425

Morphometric and meristic analyses

The complete morphometric and meristic dataset of 583 skates included 40 variables (Table 10). Orbital diameter, pre-nasal length, and the ventral head length had overlapping ranges; while the spiracle length, the distance between first and fifth gill openings, the tail width at the axil of pelvic fins displayed the most divergent values between species. Disc width and length was proportionally smaller in D. trachyderma than in Z. chilensis; and this difference in disc length/disc width ratio gives the characteristic sub-rhomboidal shape of D. trachyderma.

The post-cloacal length of clasper was excluded from any comparison to avoid bias related to sex. However, significant differences in morphology (P < 0.05) between males and females of Z. chilensis were found (ANOSIMZ. chilensis: R = 0.554). The multivariate plot of the

–68– CHAPTER III: SPECIES IDENTIFICATION morphometric/meristic matrix evidenced a clear separation between species (Fig. 14A). Sexual dimorphism in body shape was noticeable for Z. chilensis, but was not evident for D. trachyderma (Fig. 14B – C), possibly due to the low proportion of mature specimens in the sample. No significant differences in morphology between the sexes were detected within the D. trachyderma population

(ANOSIMD. trachyderma: R = 0.016; P = 0.128). The morphometric and meristic characters contributed largely to the separation of species as analysed by SIMPER (Table 11). A cumulative difference of over 55% was reached when five variables were included. Overall, the number of thorns present in the median dorsal row and disc width to total length ratio were the main variables responsible for the separation of Z. chilensis and D. trachyderma. The distance from the cloaca to pelvic fin insertion of Z. chilensis males also is presented as a discriminant factor. Additionally, the snout to cloaca distance contributed to the separation of male Z. chilensis from female Z. chilensis. No significant differences were observed in morphometry in individuals between localities Z. chilensis (ANOSIMZ. chilensis:

RGlobal = 0.167; P = 0.762) and D. trachyderma (ANOSIMD. trachyderma: RGlobal = 0.125; P = 0.2).

Of the sampled specimens, 16 females were incorrectly identified in the field due to the absence of reported discriminant morphological features and small body size. The identities of six Z. chilensis and ten D. trachyderma were corrected after the morphometric analysis. The misidentification of these specimens was mainly due the absence of the nuchal thorn, which is traditionally used to separate Z. chilensis from D. trachyderma, and all individuals that lacked this character are routinely identified as D. trachyderma.

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Table 10. Morphometric and meristic data of Zearaja chilensis (n =406) and Dipturus trachyderma (n = 177). Range and mean values are expressed in % of total length (Disc–1) with the exception of meristic variables. Definition for each variable is presented in Table 5. Length of clasper (Disc–13) is only applicable for males.

Z. chilensis Female Z. chilensis Male D. trachyderma Female D. trachyderma Male Variable Mean Range Mean Range Mean Range Mean Range

Disc–1 1041.6 (622.8 – 1498.7) 934.4 (726.9 – 1250.0) 1504.9 (974.4 – 2052.3) 1378.9 (901.5 – 2224.3) Disc–2 76.5 (68.9 – 84.2) 76.4 (71.5 – 83.5) 72.4 (68.5 – 76.4) 72.3 (68.7 – 77.5) Disc–3 61.5 (56.7 – 67.0) 60.7 (57.5 – 64.6) 59.3 (56.1 – 61.7) 59.6 (56.9 – 62.7) Disc–4 38.1 (32.4 – 46.8) 37.0 (32.4 – 45.4) 34.3 (29.0 – 38.3) 34.5 (31.0 – 40.6) Disc–5 19.8 (17.8 – 22.7) 18.1 (15.2 – 21.5) 20.6 (18.3 – 32.3) 20.5 (16.1 – 23.1) Disc–6 24.5 (22.4 – 28.1) 23.0 (19.0 – 26.4) 24.4 (20.0 – 26.1) 24.6 (20.7 – 26.9) Disc–7 26.8 (22.0 – 33.9) 25.4 (21.3 – 37.6) 27.1 (23.7 – 34.1) 27.6 (24.4 – 33.9) Disc–8 38.5 (32.5 – 53.9) 40.6 (34.1 – 47.2) 43.0 (37.6 – 45.6) 43.1 (36.5 – 46.9) Disc–9 18.3 (15.7 – 22.0) 16.3 (13.4 – 19.8) 20.0 (15.9 – 22.6) 20.0 (16.0 – 22.4) Disc–10 16.8 (14.7 – 19.8) 15.1 (12.5 – 18.1) 18.1 (15.0 – 20.4) 18.1 (15.0 – 20.6) Disc–11 35.7 (32.3 – 41.0) 35.0 (19.5 – 39.5) 33.9 (31.0 – 36.4) 34.1 (31.7 – 38.0) Disc–12 7.1 (2.5 – 5.3) 10.0 (4.4 – 13.1) 4.5 (2.4 – 6.2) 4.8 (3.3 – 6.8) Disc–13 * * 27.5 (8.6 – 34.7) * * 8.3 (2.1 – 21.7) Head–1 2.1 (1.4 – 3.9) 2.1 (1.4 – 3.7) 2.6 (1.7 – 3.4) 2.7 (1.5 – 3.8) Head–2 4.7 (3.5 – 5.7) 4.9 (4.3 – 6.1) 4.2 (3.7 – 5.1) 4.3 (3.3 – 5.4) Head–3 2.2 (1.5 – 3.3) 2.2 (1.2 – 2.8) 2.2 (1.5 – 2.7) 2.1 (1.5 – 2.7) Head–4 7.7 (5.6 – 10.2) 7.3 (6.1 – 9.5) 6.4 (5.3 – 8.2) 6.2 (5.0 – 8.4)

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Table 10. Continued.

Z. chilensis Female Z. chilensis Male D. trachyderma Female D. trachyderma Male Variable Mean Range Mean Range Mean Range Mean Range

Head–5 8.0 (6.2 – 9.0) 7.8 (7.0 – 8.7) 6.6 (6.0 – 8.7) 6.6 (6.1 – 8.4) Head–6 61.6 (53.7 – 67.6) 59.4 (56.6 – 64.3) 56.9 (45.0 – 62.5) 57.0 (54.6 – 63.1) Head–7 9.1 (6.6 – 10.9) 9.2 (6.3 – 11.9) 7.6 (5.3 – 10.3) 7.6 (5.9 – 10.4) Head–8 10.9 (9.2 – 12.0) 10.3 (9.3 – 11.4) 9.3 (8.5 – 10.8) 9.2 (8.6 – 11.2) Head–9 4.0 (2.7 – 5.7) 3.9 (2.3 – 6.0) 4.1 (2.6 – 5.3) 4.2 (2.3 – 5.2) Head–10 10.9 (8.2 – 13.5) 11.0 (3.9 – 13.1) 9.6 (8.6 – 11.0) 9.6 (9.0 – 11.6) Head–11 1.7 (0.9 – 2.5) 1.6 (1.0 – 2.5) 1.6 (1.1 – 2.9) 1.6 (1.2 – 2.0) Head–12 1.7 (1.1 – 2.3) 1.6 (0.8 – 2.2) 1.7 (1.2 – 2.3) 1.6 (1.1 – 2.1) Head–13 18.7 (15.9 – 21.9) 18.3 (15.7 – 22.7) 17.1 (15.3 – 20.0) 16.8 (14.8 – 20.8) Head–14 12.1 (8.5 – 13.8) 10.6 (8.8 – 13.1) 10.8 (9.6 – 12.7) 10.6 (9.5 – 13.4) Tail–1 9.2 (6.4 – 15.7) 9.6 (7.5 – 12.0) 10.6 (7.7 – 13.5) 11.1 (8.4 – 15.3) Tail–2 14.9 (12.6 – 17.5) 17.4 (12.3 – 21.3) 13.8 (12.6 – 16.1) 13.9 (12.7 – 15.7) Tail–3 16.4 (10.0 – 20.7) 14.9 (11.2 – 17.4) 11.8 (9.1 – 15.9) 11.4 (8.8 – 15.7) Tail–4 4.8 (3.5 – 6.4) 4.7 (3.5 – 5.7) 4.1 (3.3 – 5.5) 4.1 (3.2 – 5.0) Tail–5 3.1 (1.8 – 4.2) 3.0 (2.1 – 3.9) 2.7 (2.2 – 3.9) 2.7 (2.2 – 3.5) Tail–6 3.0 (1.9 – 3.9) 2.9 (2.2 – 4.0) 2.6 (1.8 – 3.4) 2.6 (2.0 – 3.8) Tail–7 3.1 (2.0 – 5.3) 3.3 (2.1 – 5.1) 3.3 (2.0 – 4.8) 3.5 (1.7 – 16.2) Tail–8 14.7 (11.3 – 18.3) 15.4 (11.4 – 19.0) 17.1 (14.8 – 19.9) 16.9 (13.3 – 19.7) Tail–9 8.6 (6.1 – 11.5) 9.0 (6.0 – 11.3) 10.0 (7.9 – 11.5) 9.8 (4.5 – 11.5)

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Table 10. Continued.

Z. chilensis Female Z. chilensis Male D. trachyderma Female D. trachyderma Male Variable Mean Range Mean Range Mean Range Mean Range

Tail–10 5.5 (2.9 – 8.5) 5.8 (3.6 – 8.2) 6.8 (4.4 – 8.7) 6.6 (3.2 – 8.2)

TN 1 (0 – 1) 1 (0 – 3) 0 (0) 0 (0)

TM 35 (20 – 39) 29 (16 – 37) 30 (25 – 45) 29 (27 – 45)

TD 1 (0 – 3) 1 (0 – 3) 2 (1 – 3) 2 (1 – 3)

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Figure 14. Non-metric multidimensional scaling (nMDS) ordination plot of morphometric measurements and meristic counts recorded on (A) whole population of commercial longnose skates from Chile (n =583). Differences by sex are presented for (B) Zearaja chilensis and (C) Dipturus trachyderma.

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Table 11. Overall contribution (%) of morphometric and meristic variables to the differentiation between groups after SIMPER test. Definition for each variable is presented on Table 5.

D. trachyderma Z. chilensis (male) Contrib. (%) Cum. (%) TM 30.5 29 14.98 14.98 Disc–12 4.74 10.14 11.84 26.82 Disc–2 72.26 76.39 10.1 36.92 Disc–9 20.17 16.33 9.22 46.14 Tail–3 11.47 14.91 9.02 55.16 Disc–4 34.42 37.02 8.36 63.52

D. trachyderma Z. chilensis (female) Contrib. (%) Cum. (%) TM 30.5 35 28.74 28.74 Disc–2 72.26 76.74 7.03 35.77 Disc–8 43.33 38.17 6.97 42.74 Tail–3 11.47 16.6 6.88 49.62 Disc–4 34.42 38.19 6.19 55.81

Z. chilensis (female) Z. chilensis (male) Contrib. (%) Cum. (%) TM 35 29 44.43 44.43 Disc–12 5.09 10.14 8.78 53.21 Disc–8 38.17 40.64 5.04 58.25 Disc–4 38.2 37.02 4.78 63.03 Head–6 61.87 59.43 4.76 67.79

Mitochondrial DNA and data analyses

Amplification of the four target regions of the mitochondrial DNA was successful in all cases with observable bands between 500 and 1,000 base pairs (bp). For the 16S region, a fragment of 580 bp was amplified for 160 specimens and included three parsimony informative sites (haplotypes). All sequences of Z. chilensis were identical and represented a single haplotype that was, however, shared with D. trachyderma (in 12.5% of specimens). Two additional haplotypes were detected in D. trachyderma (5% and 82.5%). The K2P sequence divergence for intraspecific comparisons was 0.000 – 0.002 in D. trachyderma (average = 0.001).

For the cox1 gene, a 652 bp fragment was amplified and contained seven haplotypes, and between 99.1 – 100% identical sites. Low nucleotide diversity was evident with overall K2P values of 0.001 (0.000 to 0.003) and 0.002 (0.000 to 0.002) for Z. chilensis and D. trachyderma, respectively. Four haplotypes were detected for D. trachyderma (12.5%, 15%, 20% and 52.5%) and three for Z. chilensis (8.75%, 15% and 76.25%).

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For the nadh2 gene, a 1,044 bp fragment was amplified and contained eight polymorphic sites, all were found in D. trachyderma specimens. Five haplotypes were detected in all specimens with K2P values of 0.00 – 0.006 (average = 0.002) in D. trachyderma and 0.00 for Z. chilensis. Similarly, to the 16S region, one single haplotype was detected in Z. chilensis, however, four evenly-distributed haplotypes were observed in D. trachyderma (17.5%, 18.75%, 30% and 33.75%).

For the control region, the first 557 bp were amplified, which contained 25 polymorphic sites and eight haplotypes. Four haplotypes were detected for each species and K2P values ranged from 0.00 to 0.009 in Z. chilensis (average = 0.002) and 0.00 to 0.004 in D. trachyderma (average = 0.003)

Final alignments included sequences from 42 additional close related species, however, the number of sequences included varied according to data availability (Fig. 15 – 16). In two of the four loci examined, phylogenetic trees did not provide a consistent and expected separation between the target species. In all cases, amplified fragments were contained in a reciprocally monophyletic clade with high bootstrap support (>85%), which represents longnose skates (genera Dipturus and Zearaja). However, specimens of D. trachyderma and Z. chilensis were arranged and clustered indistinctly within a single clade for the 16S and nadh2 fragments (Fig. 16). These two regions did not provide information on species identity for longnose skates. Yet, a different situation was observed within the cox1 and control region (Fig. 15). Fragments analysed had over 98% of identical sites, but species were successfully arranged in sister clades with high bootstrap support (>90%), and clearly separated specimens of Z. chilensis and D. trachyderma. However, phylogenetic analysis of cox1 situate specimens of Z. chilensis from Argentina in a paraphyletic clade with respect of Chilean specimens.

Figure 15 (next page). Neighbour-joining distance phylogenetic tree inferred from K2P distance model on mitochondrial DNA haplotypes of (A) 16S and (B) the control region, for Zearaja chilensis and Dipturus trachyderma, and close-related species within the Family Rajidae. Branches with identical haplotypes have been collapsed and the number of sequences is indicated in between brackets. Estimation of the nodes stability is expressed as a percentage (1,000 bootstrap replicates) and is only indicated (*) on nodes with >80%.

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Figure 16 (previous page). Neighbour-joining distance phylogenetic tree inferred from K2P distance model on mitochondrial DNA haplotypes of (A) cox1 and (B) nadh2, for Zearaja chilensis and Dipturus trachyderma, and close-related species within the Family Rajidae. Branches with identical haplotypes have been collapsed and the number of sequences is indicated in between brackets

DISCUSSION

Worldwide, skate fisheries are often accompanied by a lack of biological information necessary for adequate management (Ferretti et al., 2008, 2010; Griffiths et al., 2013; Dulvy et al., 2014). In particular, longnose skate fisheries have unique challenges for conservation due to the convergence of morphological characters that hinder their identification (Robert & Hawkins, 1999; Stevens et al., 2000; Dulvy & Reynolds, 2002; Griffiths et al., 2013). Deep-water elasmobranchs, and particularly the skates, are among the marine organisms most vulnerable to extinction as a result of overfishing (Fowler et al., 2005; Dulvy et al., 2014). The relatively low fecundity, late age of sexual maturity and high longevity of longnose skates increase their intrinsic risk and vulnerability to population collapse and local extinctions, even when subjected to low levels of fishing pressure (Frisk et al., 2001; Dulvy & Reynolds, 2002; Myers & Worm, 2005; Frisk, 2010; Dulvy et al., 2014).

The present research has emphasised the importance of individualise catch and landing records (e.g., derived from fisheries) of longnose skates, and highlights the phenotypic plasticity and natural variability of morphological features, often used as diagnostic characters for species identification. In Chilean waters, fishing records have been collected historically using categories that lack species-level resolution – an issue that has been discussed extensively (Lamilla et al., 2010; Bustamante et al., 2012, 2014b). Information extracted from official fishing records usually requires data mining and interpretation to separate out particular species records (if possible). For example, fishing records in relation to D. trachyderma were unavailable until 2004, as both longnose skates landed in Chile (Z. chilensis and D. trachyderma) were reported under a single category (Bustamante et al., 2012; SERNAP, 2015). This lack of discrimination between the species is generally ascribed to an inability to separate the species based on ‘casual observation’, due to their similar appearance (Lamilla et al., 2010), particularly when specimens are of a similar size (D. trachyderma grows to a larger body size than does Z. chilensis, making large individuals easy to ascribe to species). However, the relatively low abundance of D. trachyderma and the plasticity of some diagnostic features, such as the nuchal thorn in Z. chilensis, promotes lumping of species into a category that denies species-

–78– CHAPTER III: SPECIES IDENTIFICATION specific landing records. Although longnose skates may be morphologically similar to the untrained eye, the species have different life cycles and ecological niches, which are currently ignored by managers and legislation. In Chile, there is an urgent need for changes in fishing management to ensure sustainability and conservation of longnose skates (SUBPESCA, 2007), such as a revision of the allocation of fishing quotas, and enforcement of species-specific landing records.

Morphological cues of longnose skates

Morphometric analysis has been significantly enhanced by image processing techniques and this powerful tool may supplement traditional approaches to stock identification. Here, differences in body proportion and shape of two commercial skates were successfully used to separate and confirm species identity. Each morphological measurement and meristic count contribute to characterise the phenotypical change and degree of plasticity of each feature, which depict the natural variation of a species. By investigating these ranges, the variability due to local, geographically-linked adaptations or ontogenetic changes related to growth and maturation may be acknowledged and eventually improve the predictive power for species identification.

Sexual dimorphism in Zearaja chilensis is clearly seen, with clear morphological changes occurring in males due to sexual maturation. In both skates and rays, a change dental morphology occurs as a secondary sexual characteristic that is related to the males’ need to be able to firmly grip the females’ pectoral fin in their mouth for mating (Kajiura & Tricas, 1996; Sáez & Lamilla, 1997; Gutteridge & Bennett, 2014; Belleggia et al., 2016; Delpiani et al., 2017). The change in jaw and dental morphology is presumably related, functionally to hypertrophy of the mandibular abductor muscle. Also, sexual maturation coincides with the development of alar thorns, and the combination of these ontogenetic developments produces the characteristic concavity of the mid-anterior margin of the disc at the level of the mouth (Fig. 17). The characteristic elongation of the claspers as a consequence of sexual maturation, produces a deformation at the base of the pelvic fins of mature males which differentiates them from female Z. chilensis (Fig. 17).

The position and number of thorns on the disc surface is presented here as a discriminant character for species and sex. In Z. chilensis the number of thorns in the midline region does not exceed 39 in females and 37 in males, whereas in D. trachyderma the number of midline thorns does not exceed 44 in either sex. In D. trachyderma there is always one, and sometimes up to three thorns present between the dorsal fins, whereas inter-dorsal thorns may not be present in Z. chilensis, and while up to three nuchal thorns can be found in male Z. chilensis, one nuchal thorn is usually present in both sexes, although it may be easily overlooked or may not be present. None of the examined

–79– CHAPTER III: SPECIES IDENTIFICATION specimens of D. trachyderma had thorns in the nuchal area. The presence of nuchal thorns has been reported as a diagnostic feature for genus Zearaja (Lamilla & Sáez, 2003; Last & Gledhill, 2007); however, the variability observed in the current study of Z. chilensis may devalue its use as a discriminant character for identification between the two longnose skates of commercial importance in Chile.

Figure 17. Morphology of adult (A) male and (B) female of Zearaja chilensis. Secondary sexual characters are indicated for each sex. For males, (a) hypertrophy of the mandibular abductor muscle and (b) enlargement of the pelvic fin base.

At the population level, no noticeable differences between morphologies based on geographical location, considering the geographical scale of the study. In general, morphological variations in marine fishes may be associated with changes in the reproductive condition (Cadrin & Friedland., 2005). No significant differences in body size were observed within D. trachyderma population, and the lack of apparent sexual dimorphism (claspers aside) may have been a function of the relatively low proportion of mature individuals (> 10%) in the overall sample. In the case of Valdivia and Punta Arenas, differences in morphology between both stocks may be driven by the smaller body size of the specimens from Valdivia. Overall, the body size of specimens from Punta Arenas was the largest across sampling localities, which is consistent with the latitudinal increase of body size, as described for elasmobranch fishes (Taylor et al., 2016), but may be related to the lower

–80– CHAPTER III: SPECIES IDENTIFICATION level of fisheries exploitation of the resident stock in localities southward of Puerto Montt (Bustamante et al., 2012).

Considering empirical differences in body shape, the proportion of disc length with respect of total length have been suggested as a proxy to separate specimens of Z. chilensis from D. trachyderma (Lamilla & Sáez, 2003). However, the present results suggest the opposite. For this particular set of measurements (disc length/total length), there is considerable intra-specific variability that may be related to the reproductive condition of some specimens, and not to a species-specific morphological trait.

Molecular tools for longnose skates identification

The analysis of the mitochondrial DNA has been shown to be potentially useful as an alternative identification method for longnose skates, especially when morphological clues are not available, such as when dealing with the identification of processed body parts (Holmes et al., 2009; Coulson et al., 2011; Cawthorn et al., 2012; Griffiths et al., 2013). Here, through the amplification of fragments of mitochondrial DNA, it has been possible to observe diagnostic molecular characters that could be used for species determination in longnose skate fisheries. The effectiveness of identification of species from tissue samples in the laboratory was successfully made by exploring traditional and non-traditional regions of the DNA used for species identification, and novel primers. However, the characterisation of four loci from the mitochondrial DNA produced unexpected results. Authors from Chile (Céspedes et al., 2005) and Argentina (Díaz de Astarloa et al., 2008) unsuccessfully attempted, to establish patterns in mitochondrial DNA that could be used to identify commercial longnose skate species in South America. In these cases, the amplified fragments from cox1 gene did not provided enough information to establish phylogenetic relationships in the species studied; mainly due to the use of technologies now considered obsolete, their inability to develop species-specific primers, and the inherent evolutionary close-proximity of longnose skates (Díaz de Astarloa et al., 2008; Naylor et al. 2012a, b; Vargas-Caro et al., 2015, 2016, 2017). Sequences of all mitochondrial loci amplified in the present study showed over 98% identity and a low number of polymorphic sites for both, Z. chilensis and D. trachyderma. The close proximity and low genetic divergence of longnose skates may confound species separation within the Family Rajidae, as inter- specific comparisons based on specific genes or gene regions could suggest a single taxon, depending on what is compared.

Over the last decade, methods for identifying species based on DNA sequences through molecular taxonomy, such as 'DNA barcodes' have been gradually integrated with traditional methods

–81– CHAPTER III: SPECIES IDENTIFICATION based on morphology (Holmes et al., 2009; Moura et al., 2008; Toffoli et al., 2008; Ward et al., 2005, 2007, 2008, 2009; Wong et al., 2009; Zemlak et al., 2009). Numerous studies have demonstrated the efficacy of using cox1 and nadh2 as diagnostic sequences for universal species identification in animal lineages, and the discordance between cox1 and nadh2 analysis was unexpected. The nadh2 gene has been used widely for species identification and its use, rather than the use of cox1 has been justified based on an argument that it provides for better resolution among closely related elasmobranch species (Naylor et al., 2012a). However, the tree topology inferred from the phylogenetic analysis of the nadh2 gene proposed by Naylor et al. (2012a, b) has been impossible to replicate as this gene failed to separate Z. chilensis from D. trachyderma.

The development of species-specific markers for longnose skates provides an advance in knowledge that may contribute to future research and fishery management, and provides a molecular baseline for the comparison of population structures and genetic flow in Chilean longnose skates, and an ability to assess stock ranges and potential migrations to the Atlantic Ocean.

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4.1. The complete validated mitochondrial genome of the yellownose skate Zearaja chilensis (Guichenot 1848) (Rajiformes, Rajidae)

Originally submitted in Mitochondrial DNA Part A (ISSN: 2470-1394)

Copyright © Carolina Vargas-Caro, 2014

Published online: 04 Aug 2014

License Number: 4127980132019

Abstract

The yellownose skate Zearaja chilensis is endemic to South America. The species is the target of a valuable commercial fishery in Chile, but is highly susceptible to over-exploitation. The complete mitochondrial genome was described from 694,593 sequences obtained using Ion Torrent Next Generation Sequencing. The total length of the mitogenome was 16,909 bp, comprising 2 rRNAs, 13 protein-coding genes, 22 tRNAs and 2 non-coding regions. Comparison between the proposed mitogenome and one previously described from “raw fish fillets from a skate speciality restaurant in Seoul, Korea” resulted in 97.4% similarity, rather than approaching 100% similarity as might be expected. The 2.6% dissimilarity may indicate the presence of two separate stocks or two different species of, ostensibly, Z. chilensis in South America and highlights the need for caution when using genetic resources without a taxonomic reference or a voucher specimen.

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INTRODUCTION

The yellownose skate Zearaja chilensis is endemic to South America occurring from Chile (southeast Pacific) to Uruguay and the Falkland Islands (southwest Atlantic) (Menni & Stehmann, 2000). The species is a commercially valuable batoid caught as target and bycatch, especially in Chile. An assessment of the species’ demographic status in relation to the fishery has provided an indication of a near-future fishery collapse (Bustamante et al., 2012).

MATERIAL AND METHODS

A tissue sample was obtained from a single specimen from San Antonio, Chile (33° 35.168' S, 71° 37.809' W), and was identified using field guides to ensure taxonomic identity (Lamilla & Sáez, 2003; Lamilla & Bustamante, 2005). Biological and morphometric data taken during sampling are presented in the Appendix. Total genomic DNA was extracted using QIAgen DNeasy Blood and Tissue kit (Qiagen Inc., Valencia, CA) and the mitogenome was assembled from an Ion Torrent sequencing run on an ION PGM (Life Technologies, Carlsbad, CA) using a 318v2 chip. Exactly 694,593 sequences were produced with a mean read length of 268.8 bp (min: 8 bp; max: 616 bp). Sequences were mapped against the most similar skate available, Dipturus kwangtungensis (GenBank

Accession number: KF318309) using the software GENEIOUS v7.1.4 (Biomatters Ltd., Auckland). The 17,487 matching reads, with 100% coverage (min: 3 seq; max: 45 seq), were assembled de novo producing a 16,909 bp mitogenome similar in size to other skates (range 16,724 – 16,972; n = 7). Indels were validated on the depth and quality of sequences, and annotations were confirmed using the Mitochondrial Genome Annotation Server (Bernt et al., 2013).

RESULTS AND DISCUSSION

Gene order and structure of the Z. chilensis mitogenome (Genbank Accession number: KJ913073) was consistent to other fishes comprising 13 protein-coding regions: 22 tRNA genes, 2 rRNA genes and 2 non-coding areas (origin of replication and control region). The mitogenome had an A+T (69.6%) bias as seen in many other marine fishes, and an overall nucleotide composition of A, 37.7%, T, 31.9%, C, 18.8%, and G, 11.6%.

The proposed mitogenome was validated by Sanger sequencing as suggested by Bustamante & Ovenden (2016), using 622 bp length fragments of 16S region amplified from five additional

–85– CHAPTER IV: MOLECULAR TAXONOMY specimens from the same collection (forward primer: 16sAR, 5’- CGC CTG TTT AAC AAA AAC AT -3’; reverse primer: 16sBR, 5’- CCG GTC TGA ACT CAG ATC ACG T -3’). Fragments (KJ913068–KJ913072) were 100% identical to the expected region in the mitogenome without indels (insertion-deletions) or single nucleotide polymorphisms (SNP). The total number of SNPs was estimated compared to the recently described Z. chilensis mitogenome (KF648508, Jeong & Lee, 2014). The pairwise identity was 97.9% (447 SNP, Fig. 18), rather than being close to 100% as expected. An overall value of 99.7% of similarity has been reported for cryptic and sister species, and intraspecific variation (Botero-Castro et al., 2016). The low pairwise identity observed between the two samples and the fact that the origin of the Korean sample is unknown (“collected from a raw fish fillet from a specialty restaurant”, Jeong & Lee, 2014), may indicate the presence of two distinct populations or even the presence of two different, but closely-related species.

Figure 18. Comparison of the average pairwise identity (%) of Zearaja chilensis (Grey line) and Zearaja chilensis (Jeong & Lee, 2014) (Dashed line) mtDNA genes. The number of SNPs per genes is indicated in brackets.

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4.2. The phylogenetic position of the roughskin skate Dipturus trachyderma (Krefft & Stehmann 1975) (Rajiformes, Rajidae) inferred from the mitochondrial genome

Originally submitted in Mitochondrial DNA Part A (ISSN: 2470-1394)

Copyright © Carolina Vargas-Caro, 2015

Published online: 30 Jun 2015

License Number: 4127980491957

Abstract

The complete mitochondrial genome of the roughskin skate Dipturus trachyderma is described from 1,455,724 sequences obtained using Illumina NGS technology. Total length of the mitogenome was 16,909 base pairs, comprising 2 rRNAs, 13 protein-coding genes, 22 tRNAs and 2 non-coding regions. Phylogenetic analysis based on mtDNA revealed low genetic divergence among longnose skates, in particular those dwelling the continental shelf and slope off the coasts of Chile and Argentina.

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INTRODUCTION

The roughskin skate Dipturus trachyderma is a commercially valuable elasmobranch endemic to the continental shelf and slope off southern Chile and Argentina at depths of 180–350 m (Bustamante et al., 2012). It is one of the largest marine skates, reaching up to 240 cm total length. There is considerable concern for its long-term survival and it has been listed as Vulnerable on the IUCN Red List of Threatened SpeciesTM (Lamilla & Massa, 2007).

MATERIAL AND METHODS

The mitogenome of D. trachyderma (GenBank Accession number: KR152643) is described here from a single specimen collected in Aysén, Chile (45°26'S, 72°56' W) during commercial landings. Biological and morphometric data taken during sampling are presented in the Appendix. A genomic library for sequencing was developed with the TruSeq Nano DNA sample prep kit (Illumina, San Diego) and sequenced using the Miseq 600v3 (Illumina, San Diego) cartridge that supported the acquisition of 2 × 300 bp pair-end reads. Resulting reads (1,455,724 sequences) were merged using FLASH software (Magoč & Salzberg, 2011) with a minimum overlap of 25 bp and a 0.2 mismatch ratio. The mean length of the merged sequences was 544.8 bp (min: 300 bp, max: 575 bp) and were mapped against the closely related species, Dipturus kwangtungensis (GenBank Accession number:

KF318309) using GENEIOUS v8.1.2 (Biomatters Ltd., Auckland); generating 1,647 matching reads with 100% coverage (min: 21, max: 79) and a 16,907 bp mitogenome. Genes were annotated using the web-based tool MITOANNOTATOR (Iwasaki et al., 2013). The complete Genomic DNA sequence library was lodge at the Genomic Database Repository, eFish (BioVoucher: 2015-DTR-004).

A dataset consisting of all available mitogenomes from closely related taxa was constructed in order to assess validity of the mitogenome and level of inter-specific divergence, as recommended by DeSalle & Kolokotronis (2015). This also serves to identify potential sequencing errors of the mitogenome (Botero-Castro et al., 2016). The mitogenomic dataset was aligned using GENEIOUS and resulting alignment was trimmed with TRIMAL using the automated1 option in the PHYLEMON web suite (Sánchez et al., 2011), in order to remove ambiguously aligned positions. Final alignment included 12 taxa and 16,912 sites. A maximum likelihood (ML) tree was inferred under the

GTR+GAMMA model implemented in PHYML (Guindon & Gascuel, 2003). Node robustness was evaluated with 1,000 bootstrap replicates. The resulting topology and branch length was used to validate the phylogenetic position and divergence of the draft genome with respect to other skates within the family Rajidae.

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RESULTS AND DISCUSSION

The mitogenome of D. trachyderma had an A+T (58.4%) bias as seen in many other marine fishes, and relative proportions (%) of bases in DNA of A, 29.7%, T, 28.7%, G, 14.7%, and C, 27.0%. Gene order and structure of the D. trachyderma mitogenome was consistent with other elasmobranchs, consisting of 13 protein-coding regions (PCGs), 22 tRNA genes, 12S and 16S ribosomal RNAs and 2 non-coding areas. Most of the genes were encoded on the H-strand, except for ND6 and eight tRNAs genes that were encoded on the L-strand. Sequence length of tRNA genes varied from 68 to 75 bp, while PCGs varied in length, from 168 bp (ATP8) to 1,836 bp (nadh5). All PCGs used ATG as the starting codon, except for cox1, which used GTG. Only nadh6 gene used TAG as the stop codon, all the other genes used TAA, or had incomplete codons (TA or T). Gene overlaps were identified at six locations; the longest was 10 bp between ATP8 and ATP6.

The phylogenetic tree inferred from the complete mitochondrial genome (Fig. 19), supports the inclusion of longnose skates among the clade Rajini and accordingly, validates the mitogenome of D. trachyderma. The proposed phylogeny clearly separate hardnose skates (subfamily Rajinae) and softnose skates (subfamily Arhynchobatinae) and locate D. trachyderma among longnose skates of the genera Dipturus and Zearaja, in agreement with traditional taxonomy. A low degree of identity, ranging from 93.4% to 97.3% is observed among the clade Rajini, which includes at least six genera (McEachran & Dunn, 1998). However, the taxonomic identity of Zearaja chilensis (KF648508) is questionable in the absence of a voucher specimen and the lack of validated specimen identification (see Vargas-Caro et al., 2016b). Considering the tree topology and pairwise identity (97.4%), this particular clade may represent a sister-cryptic species complex (>3% genetic distance) or may suggest the potential hybridization between D. trachyderma and Z. chilensis.

The mitogenome of D. trachyderma may represent a useful resource to understand the evolutionary traits and speciation of close-related species that may face extinction due unsustainable exploitation. Also, the use of complete mitogenomes to infer phylogenetic relationships is presented as powerful instrument to resolve confusions and untangle taxonomic-problematic taxa.

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Figure 19. Phylogenetic relationships of Dipturus trachyderma (in bold) inferred from the complete mitogenome, using closely related species of the family Rajidae. Taxonomic subdivisions are indicated for each major clade. Bootstrap values obtained by 1,000 steps are indicated for each node. GenBank accession numbers are provided following species names. Scale bar represent the expected substitution per site.

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4.3. Phylogeny of longnose skates (genus Zearaja): species-level relationships among major lineages of skates (Chondrichthyes, Batoidea) inferred from whole mitochondrial genomes

Manuscript to be submitted for consideration at Zoologica Scripta (ISSN: 1463-6409)

Copyright © Carolina Vargas-Caro, 2017

License Number: n/a

Abstract

Longnose skates are one of the most vulnerable taxa of elasmobranch fishes, with documented local extinctions and population declines worldwide. Between longnose skates, species of the genus Zearaja are of particular conservation concern due to targeted fisheries, bycatch and habitat degradation. Here, the evolutionary history of the genus Zearaja and higher-level phylogenetic relationships among elasmobranch fishes were investigated by using complete mitochondrial genomes (mitogenomes). A dataset with 16,720 bp as informative characters from 56 mitogenomes of 47 batoids and four outgroups, was aligned and interrogated using probabilistic approaches. Including 22 new mitogenomes and published records, this represents the largest dataset of elasmobranch phylogeny analysed to date. Intraspecific divergence is extremely low among the three Zearaja species (n-distance = 0.001) and may suggest a radiation from a potential ancestral form (Z. chilensis) towards a derived species (Z. maugeana). The evolution of the genus Zearaja could be linked to major geological events, such as the terminal Eocene event and the sinking of Zealandia; which may explain the current restricted geographical distribution to the Southern Hemisphere. Overall, the resulting consensus tree supports previous phyletic reconstructions and recovers all extant Orders within the Batoidea in two higher-level reciprocally monophyletic clades: Myliobatiformes + Rhinopristiformes and Torpediniformes + Rajiformes. A classification of major lineages for the Order Rajiformes is revised to incorporate molecular insights to the current cladistic classification.

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INTRODUCTION

Understanding the phylogenetic relationships between organisms is essential for biological studies as a baseline for taxonomy and empirical data collection (Delsuc et al., 2005). Phylogenetic studies are based on the assumption that shared characteristics between species are signposts to common ancestry. Recent research has explored the phyletic relationships among Chondrichthyes (e.g., Naylor et al., 2012a, b), yet little attention has been paid to the interrelationships of skates and rays (batoids) due to the unresolved and incomplete taxonomy (Last et al., 2016). However, there is a growing interest in the evolution of batoids, which comprise more than half of the chondrichthyan diversity (630 of 1,188 species sensu Weigman, 2016); with a well-supported phylogeny needed for proper interpretation of comparative studies of life history (Dulvy et al., 2014). To date, there have been few attempts to use molecular data to resolve batoids phylogenies (e.g., Douady et al., 2003; Aschliman et al., 2012b; Naylor et al., 2012a, b). However, there is no consensus on the interrelationships of major lineages using morphological characters due in part, to the high degree of specialization and a low degree of synapomorphies which challenges finding informative characters between extant orders (McEachran et al., 1996; Shirai, 1996; McEachran & Aschliman, 2004; Aschliman et al., 2012a).

Studies based on mitochondrial DNA have proved to be more informative than those based on nuclear DNA in reconstructing phylogenetic relationships among closely related species, due to the rapid evolution in the former and the challenge of finding orthologous nuclear gene regions that contain evolutionarily informative polymorphisms (Delsuc et al., 2005; Inoue et al., 2003, 2010). Many caveats regarding the use of mitochondrial sequences in phylogenetic analyses could be discussed, such as the lack of lineage sorting and potential hybridisation among target species (Dudgeon et al., 2012). However, by increasing the number of characters analysed in a single dataset, mitogenomes may have the potential to alleviate and resolving these intrinsic problematic issues in taxonomy (Mueller et al., 2004; Yamanoue et al., 2008; Tanaka et al., 2013; Poortvliet et al., 2015).

Molecular studies were expected to be decisive in resolving persistent controversies in the elasmobranch phylogeny (Compagno, 1973, 1999; Dudgeon et al., 2012; Nelson, 2016), and phylogenetic analyses of mitogenomes have successfully resolved many controversial issues in systematic ichthyology (Miya & Nishida, 2000; Inoue et al., 2003), and have provided new insights into the evolutionary history at higher taxonomic levels in several vertebrate models, including elasmobranch fishes (Eisen & Fraser, 2003; Miya et al., 2003; Delsuc et al., 2005; Inoue et al., 2010).

However, phylogenetic reconstructions have failed to obtain a harmonizing hypothesis to examine evolution and relationships between Rajiformes (Naylor et al., 2012a, b). In general,

–92– CHAPTER IV: MOLECULAR TAXONOMY sequence-based phylogenetic reconstructions rely on mathematical models to obtain multiple alignments of orthologous genes (Snel et al., 1999; Tekaia et al., 1999), and the evolutionary history may be inferred from the comparison of homologous characters (Eisen & Fraser, 2003; Delsuc et al., 2005). Usually, phylogenetic reconstructions are inferred from a “supertree” (Gribaldo & Philippe, 2002; Holder & Lewis, 2003; Driskell et al., 2004), which combines the optimal trees obtained from the analysis of individual markers (i.e., incomplete sequences from a single gene) o concatenated sequences of mitochondrial and nuclear loci (Philippe & Laurent, 1998; Murphy et al., 2001; Delsuc et al., 2005). As more genes are analysed, topological conflicts between phylogenies based on individual genes were revealed. Moreover, information from a single gene is often insufficient to obtain a consistent hypothesis with the natural history (de Carvalho, 1996; Compagno, 1999b; Dunn et al., 2003; McEachran & Aschliman, 2004; Ebert et al., 2013) and firm statistical support (Miyamoto & Fitch, 1995; López et al., 2006; Inoue et al., 2010; Aschliman et al., 2012a; Smith et al., 2015). As a consequence, batoids phylogeny have remained overlooked and poorly resolved simply because of the limited availability of data (Aschliman et al., 2012a; Last et al., 2016). Today, access to genomic information could potentially alleviate previous problems due to sampling effects by expanding the number of characters that can be used in phylogenetic analysis, from a few thousand bp to tens of thousands base pairs (bp), from genes to genomes; with an integrative methodology from the phylogenomics - the reconstitution of evolutionary history using genomes (Eisen & Fraser, 2003; Delsuc et al., 2005; Botero-Castro et al., 2016).

The Superorder Batoidea is generally regarded as a monophyletic clade and a sister group to sharks (Superorder Selachimorpha) based on morphological and molecular characters (McEachran & Aschliman, 2004; Aschliman et al., 2012a). Typically, four major taxonomic Orders are recognised (Nelson et al. 2016), including Rajiformes (skates), Myliobatiformes (panray, thornbacks, stingrays, eagle rays and mobulids), Torpediniformes (electric rays) and sawfishes (Pristiformes). Of these four orders, only the Rajiformes comprising 3 families, 30 genera, and 285 species; uses an oviparous reproductive strategy (Ishiyama, 1958) and is regarded as a reciprocally phylogenetic clade strongly supported by their morphology and life history (Ishihara et al., 2012). The longnose skates (Order Rajiformes, Family Rajidae, Tribe Rajini, genus Dipturus, 44 species) are among the main chondrichthyan species subject to targeted fishing (Dulvy et al., 2014), with documented local extinctions and population declines due to incidental capture in fisheries targeting other species (Dulvy et al., 2000). Longnose skates have a worldwide distribution, inhabiting mostly marine environments from the sublittoral zone to depths of about 3,000 m (McEachran & Miyake, 1990; Ebert & Compagno, 2007). Until recently, the genus Zearaja was considered a junior synonym of Dipturus (Stehmann 1990); however, Last & Gledhill (2007) proposed Zearaja be promoted to genus- level based on key features of their external and internal anatomy, i.e., disc width to length ratio

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(giving it the familiar sub-rhomboid shape), presence of a strong rostral cartilage, orbital and midline thorn patterns, and skeletal differences, e.g. in clasper, neurocranium, pelvic and pectoral girdle morphologies. Currently, the genus Zearaja (Whitley 1939) is represented by three species, all with conservation concerns due to targeted fisheries, bycatch and habitat degradation. The rough skate Zearaja nasuta (Müller & Henle 1841) inhabits New Zealand waters and the Chatham Rise; the yellownose skate Z. chilensis (Guichenot 1848) is distributed in south South American waters; and the Maugean skate Z. maugeana Last & Gledhill 2007, is found in semi-enclosed harbours in southwestern Tasmania. Given this geographical distribution in the Southern Hemisphere, it is hypothesised that Zearaja became endemic to these separate regions during the late Mesozoic as a result of continental drift (Last & Gledhill, 2007).

Under the expectation that Zearaja is a reciprocally monophyletic group within Dipturus, the present study explores the evolutionary history of Zearaja in a broader taxonomic context by using whole mitochondrial genomes to understand higher-level phylogenetic relationships among batoids. Furthermore, this study includes a greater number of skate species and DNA sequence data than all previous studies. Complete mitochondrial genomes and a set of calibration fossils have been used to perform divergence time estimations to provide further understanding into the current biogeographic evolution of Zearaja skates.

MATERIALS AND METHODS

Mitogenome reconstruction

The complete mitochondrial genome of all three Zearaja species was analysed from specimens collected in Chile (Z. chilensis), Australia (Z. maugeana) and New Zealand (Z. nasuta). Tissue was preserved in absolute ethanol and genomic DNA was extracted using the salting-out procedure of Miller et al. (1988) and then stored at -20°C. Briefly, a small piece of tissue (ca. 100 mg) was digested overnight at 56°C in a 1.5 mL microcentrifuge tube containing 600 μL of lysis buffer (0.05 M Tris-HCl, pH 8; 0.1 M EDTA; 5 M NaCl and 5 M SDS) and 20 μL of proteinase K (10 mg mL-1). The DNA was fragmented by acoustic shearing using an ultrasonicator (Covaris S2, Woburn, MA), and genomic libraries for sequencing were developed for each specimen with the TruSeq PCR-Free DNA sample preparation kit (Illumina, San Diego, CA). Samples were sequenced using an Illumina HiSeq 2000 platform (San Diego, CA) that supported the acquisition of 2 × 125 base pair (bp) pair-end reads, following the protocols supplied by the manufacturer. Resulting sequences were merged using FLASH (Magoč & Salzberg 2011) with a minimum overlap of 25 bp and a 0.2 mismatch ratio. Using GENEIOUS 8.1.2 (Biomatters, Auckland, NZ), contigs were mapped against

–94– CHAPTER IV: MOLECULAR TAXONOMY two closely related species, the little skate Leucoraja erinacea (GenBank Accession Number JQ034406, Wang et al. 2012) and the mottled skate Beringraja pulchra (KF623030, Hwang et al. 2016), and a single consensus sequence was obtained from references. The assembled mitogenomes were checked for erroneous/ambiguous indels (insertion-deletions) and single nucleotide polymorphisms (SNPs) that could have been introduced by homopolymer sequencing errors and ambiguities on the reference genomes. Potential ambiguities and SNPs were validated based on depth and sequence quality. Genes were annotated using the web-based tool MITOANNOTATOR (Iwasaki et al. 2013). The complete genomic DNA sequence libraries were lodged at the Genomic Database Repository (Table 12).

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Table 12. Taxonomic list of species included in the present study. Accession numbers are indicated for new (eFish) and previously published mitogenomes (GenBank). The absence of deposited BioVoucher is indicated (–).

Mitogenome Species GenBank Accession Number eFish BioVoucher Reference length (bp) Bony fishes (Osteichthyes) Istiompax indica 16,532 KJ510416–17 2011-IIN-1002, 1003 Williams et al. (2016) Rexea solandri 16,350 KJ408216 2014-RSO-1004 Bustamante & Ovenden (2016) Sharks Carcharias taurus 16,715 KT337317 2015-CTA-001 Bowden et al. (2016) Chlamydoselachus anguineus 17,313 KU159431 2015-CAN-1061 Bustamante et al. (2016b) Skates and rays Amblyraja georgiana 16,734 MF278975 2016-AGE-1217 Present study Amblyraja hyperborea 16,732 MF278976 2016-AHY-1219 Present study Amblyraja radiata 16,725 AF106038 – Rasmussen & Arnason (1999) Anoxypristis cuspidate 17,243 KP233202 – Chen et al. (2016b) Atlantoraja castelnaui 16,750 KM507724 – Duckett & Naylor (2016) Bathyraja albomaculata 16,742 MF278964 2016-BAL-1087 Present study Bathyraja griseocauda 16,744 MF278965 2016-BGR-1088 Present study Bathyraja maccaini 16,744 MF278977 2016-BMA-1221 Present study Bathyraja meridionalis 16,742 MF278978 2016-BME-1223 Present study Bathyraja multispinis 16,742 MF278963 2016-BMU-1086 Present study Bathyraja richardsoni 16,742 MF278979 2016-BRI-1224 Present study

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Table 12. Continued.

Mitogenome Species GenBank Accession Number eFish BioVoucher Reference length (bp) Bathyraja shuntovi 16,744 MF278980 2016-BSH-1227 Present study

Beringraja pulchra 16,907 KF623030, KR676446–48 – Hwang et al. (2016) Dipturus innominatus 16,913 MF278961, MF278981 2016-DIN-1067, 1242 Present study Dipturus oxyrinchus 16,912 MF278968 2016-DOX-1170 Present study Dipturus trachyderma 16,907 KR152643 2015-DTR-004 Vargas-Caro et al. (2016b) Hongeo koreana 16,905 KC914433 – Jeong et al. (2014) Leucoraja circularis 16,719 MF278970 2016-LCI-1183 Present study Leucoraja erinacea 16,724 JQ034406 – Wang et al. (2012) Leucoraja naevus 16,719 MF278969 2016-LNA-1181 Present study Mobula japanica 18,880 JX392983 – Poortvliet & Hoarau (2013) Mobula mobular 18,913 KT203434 2015-MMO-1001 Bustamante et al. (2016a) Narcine bancroftii 16,971 KT119411 – Gaitán-Espitia et al. (2016) Narcine brasiliensis 16,997 KT119410 – Gaitán-Espitia et al. (2016) Narcine entemedor 17,081 KM386678 – Castillo-Páez et al. (2016) Notoraja tobitukai 16,799 KX150853 – Si et al. (2016b) Okamejei hollandi 16,974 KP756687 – Li et al. (2016) Okamejei kenojei 16,972 AY525783 – Kim et al. (2005) Pavoraja nitida 16,760 KJ741403 – Yang & Naylor (2016) Pristis clavata 16,804 KF381507 – Feutry et al. (2015) Pristis pectinate 16,802 KP400584 – Chen et al. (2016c)

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Table 12. Continued.

Mitogenome Species GenBank Accession Number eFish BioVoucher Reference length (bp) Raja asterias 16,920 MF278971 2016-RAS-1193 Present study Raja clavata 16,920 MF278973 2016-RCL-1197 Present study Raja miraletus 16,920 MF278974 2016-RCL-1201 Present study Raja polystigma 16,918 MF278972 2016-RPO-1196 Present study Rhina ancylostomus 17,217 KU721837 – Si et al. (2016a) Rhinobatos hynnicephalus 16,776 KF534708 – Chen et al. (2015) Rhinobatos schlegelii 16,780 KJ140136 – Chen et al. (2016a) Rhynchobatus australiae 16,804 KU746824 – Si et al. (2016d) Sinobatis borneensis 16,701 KX014715 – Si et al. (2016c) Zearaja chilensis 16,909 KJ913073, 2014-ZCH-1004, Vargas et al. (2016a), MF278962 2016-ZCH-1084 Present study Zearaja maugeana 16,908 MF278966, MF278967 2016-ZMA-1106, 1134 Present study Zearaja nasuta 16,909 MF278960 2016-ZNA-1065 Present study

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Phylogenomic analysis

Evolutionary relationships of longnose skates were explored using complete mitogenomes of related taxa in the Superorder Batoidea, including sharks and bony fishes as outgroups. All available mitogenomes in public databases of the Order Rajiformes were sought in order to provide a robust phylogenetic context and examine species-level relationships with emphasis on the Family Rajidae. Considering the relatively low data availability on rajids, the mitogenome of 15 additional skates were assembled following the same methodology used to produce Zearaja mitogenomes and were included in post hoc analyses, which includes 6 of the 9 extant genera within Tribe Rajini (Aschliman et al. 2012a).

A dataset was constructed from records deposited at the National Centre for Biotechnology Information server, GenBank (Benson et al. 2013) and new mitogenomes, comprising a total of 56 specimens of 43 skates and rays within Order Batoidea and, two sharks and two bony fishes used as outgroup (Table 12). Mitogenomes were linearized and aligned using default parameters in

GENEIOUS. The alignment utility, TRIMAL using the automated1 option PHYLEMON 2.0 (Capella- Gutiérrez et al. 2009; Sánchez et al. 2011), was implemented to identify potential gaps and ambiguities in the resulting alignment as consequence of the hypervariable regions of the DNA (e.g., control region).

Phylogenomic analyses were conducted using three different probabilistic methods, and the general time-reversible (GTR) substitution matrix (Lanave et al., 1984; Nei & Kumar, 2000) was implemented using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) approaches. The GTR+Γ+I model had the lowest scores of the Bayesian Information Criterion (BIC) and Akaike Information Criterion corrected (AICc) among 24 different nucleotide substitution models and accordingly was considered to be the best to describe the substitution pattern in the dataset. BIC and AICc were calculated using MEGA7 and presented in Table 13. Maximum likelihood and Bayesian inference analyses were conducted in PHYML (Guindon & Gascuel, 2003) and

MRBAYES 3.2.6 (Huelsenbeck & Ronquist, 2001) plugins in GENEIOUS. Bayesian inference consisted of four Markov chains of 10,000,000 generations with a sampling frequency of 1 tree every 200 generations. A consensus tree was then calculated after omitting the first 25% trees as burn-in. Likelihood analysis considered a Neighbour-Joining tree and use the Nearest Neighbour Interchange approach to search for a consensus tree topology. Parsimony analyses were conducted in MEGA7 (Kumar et al., 2015) using the Subtree-Pruning-Regrafting search method to produce a strict consensus tree. Node robustness in all trees was evaluated with 1,000 bootstrap replicates. The evolutionary divergence of lineages was estimated by the pairwise patristic distance (n-distance), commonly used as an objective measure of molecular evolution (Cohen & Pisera, 2016),

–99– CHAPTER IV: MOLECULAR TAXONOMY incorporating the number of apomorphic characters and the consensus tree arrangement, following the recommendations of Stuessy & König (2008).

Table 13. Maximum Likelihood fits of 24 different nucleotide substitution models tested for the longnose skates mitogenomic dataset.

Model Parameters BIC AICc GTR+G+I 115 406679.9 405334.4 GTR+G 114 407423.2 406089.5 TN93+G+I 112 407654.4 406344 HKY+G+I 111 408162.1 406863.4 TN93+G 111 408589.6 407291 HKY+G 110 408944.7 407657.7 T92+G+I 109 414379.7 413104.4 T92+G 108 415087.1 413823.5 GTR+I 114 417044.3 415710.5 TN93+I 111 418858.4 417559.7 K2+G+I 108 418911.5 417648 K2+G 107 419315.6 418063.7 HKY+I 110 419698.2 418411.2 T92+I 108 424998.7 423735.1 K2+I 107 429175.1 427923.2 JC+G+I 107 441330.8 440078.9 JC+G 106 441914 440673.8 JC+I 106 449950.3 448710.2 GTR 113 452610.8 451288.7 TN93 110 454914.6 453627.6 HKY 109 456472.4 455197.1 T92 107 460308.7 459056.9 K2 106 463698.1 462457.9 JC 105 483079 481850.5

GTR: General Time Reversible; HKY: Hasegawa-Kishino-Yano; TN93: Tamura-Nei; T92: Tamura 3-parameter; K2: Kimura 2-parameter; JC: Jukes-Cantor.

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Divergence time estimation

A relaxed molecular clock method was inferred from the timetree analysis using RELTIME method (Tamura et al., 2012) implemented MEGA7 (Kumar et al., 2015). Divergence time for each node was computed using the consensus ML/MP/BI tree and previously estimated divergence times. Fossil records were used to constrain minimum and maximum divergence times for their associated nodes, in order to provide realistic divergence time estimates (Benton & Donoghue 2007; Donoghue & Benton 2007). Four calibration points, expressed as time before present (million years ago, Mya), were sought from literature (Benton et al., 2009; Heinicke et al., 2009; Inoue et al., 2010; Aschliman et al., 2012b; Tanaka et al., 2013), to obtain an optimal hierarchical coverage across the ML/MP/BI tree, using divergence time between Osteichthyes and Chondrichthyes (443 ∼ 464 Mya in the 95% confidence interval), Selachii and Batoidea (251 ∼ 318 Mya), Plesiobatidae and Rajidae (178 ∼ 247 Mya), and Amblyraja and Okamejei (43 ∼ 97 Mya).

Taxon sampling

For each species, specimens are listed alphabetically within genera. Voucher specimens are housed in the Museum of New Zealand Te Papa Tongarewa (MONZ), and authors’ private collection (eFish): Amblyraja georgiana, Cuvier Island, New Zealand (MONZ-P.045774). Amblyraja hyperborea, Stewart Island, New Zealand (MONZ-P.054654). Bathyraja albomaculata, San Antonio, Chile (eFish-1102). Bathyraja griseocauda, Valdivia, Chile (eFish-1088). Bathyraja maccaini, Chatham Islands, New Zealand (MONZ-P.045949). Bathyraja meridionalis, White Island, New Zealand (MONZ-P.043957). Bathyraja multispinis, Valdivia, Chile (eFish-1086). Bathyraja richardsoni, Chatham Islands, New Zealand (MONZ-P.054311). Bathyraja shuntovi, Canterbury Bight, New Zealand (MONZ-P.042419). Dipturus innominatus, Kaikoura Peninsula, New Zealand (MONZ-P.045587); the Chatham Islands, New Zealand (eFish-1067). Dipturus oxyrinchus, the Catalan Sea, Spain (eFish-1170). Leucoraja circularis, the Catalan Sea, Spain (eFish-1183). Leucoraja naevus, the Catalan Sea, Spain (eFish-1181). Raja asterias, the Catalan Sea, Spain (eFish- 1193). Raja clavata, Catalan Sea, Spain (eFish-1197). Raja miraletus, the Catalan Sea, Spain (eFish- 1201). Raja polystigma, the Catalan Sea, Spain (eFish-1196). Zearaja chilensis, Valdivia, Chile (eFish-1084). Zearaja maugeana, Tasmania, Australia (eFish-1106; eFish-1134). Zearaja nasuta, Chatham Islands, New Zealand (eFish-1065).

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RESULTS

Mitogenome of longnose skates

Genomic libraries yielded approximately 6.5 million DNA sequences per sample. For each specimen, over 10,000 sequences matched the reference mitogenomes, with 100% coverage and an average of 250 matches in depth (min. 123, max. 347). The complete mitochondrial genome sequence was 16,908 bp for Z. maugeana and 16,909 bp for Z. chilensis and for Z. nasuta, and have been deposited in GenBank (Table 12). The mitogenomes of the three Zearaja species have an A+T bias (58.8%) as seen in other marine fishes, and a nucleotide composition of A, 29.9–30%, C, 26.9%, G, 14.3–14.4%, and T, 28.8%. Gene order and structure were consistent with other elasmobranchs, comprising 13 protein-coding regions (PCR), 22 tRNA genes, 12S and 16S ribosomal RNAs and 2 non-coding regions (control region and origin of L-strand replication). For the mitogenome of Z. chilensis, 99.89% (n-distance = 0.001) of the sites are identical to those of a previously reported mitogenome from San Antonio, Chile (Vargas-Caro et al., 2016a). For Z. maugeana there was 99.94% identity between two mitogenomes. For Z. chilensis, non- synonymous mutations in the third-codon position were observed on the genes nadh4 (pos: 10,941) and nadh5 (pos: 13,173). Neutral (synonymous) mutations were observed in several genes, ranging between 1 SNP (12S, nadh4L, nadh5, and cytB), 2 SNPs (nadh2), 4 SNPs (nadh4) and 9 SNPs in the control region. For Z. maugeana, non-synonymous mutations were observed on the genes nadh1 (pos: 3,002) and coxIII (pos: 9,334); while eight neutral SNPs were observed on 12S (3 SNPs), nadh1 (3 SNPs), nadh2 (1 SNPs) and nadh5 (1 SNPs). As only one mitogenome was available for Z. nasuta no intraspecific comparison is presented. While the overall intraspecific divergence is extremely low (n-distance = 0.001), the interspecies divergence is quite conservative, ranging between 0.010 and 0.012 (Table 14) with a clear radiation from a potential ancestral form (Z. chilensis) towards a derived species (Z. maugeana).

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Table 14. Pairwise patristic distances (n-distance; below the diagonal) and the number of non- identical bases (differences, SNPs; above the diagonal) in the mitogenome of Zearaja species.

n-distance / differences Species Genbank KJ913073 MF278962 MF278966 MF278967 MF278960 Z. chilensis KJ913073 17 162 156 136 Z. chilensis MF278962 0.001 159 153 131 Z. maugeana MF278966 0.012 0.012 10 46 Z. maugeana MF278967 0.012 0.011 0.001 40 Z. nasuta MF278960 0.010 0.010 0.003 0.003

Phylogenetic analysis

The analysis of the complete mitogenome of 56 specimens of 47 species, included 16,720 informative sites and produced fully bifurcated trees inferred from the ML/MP/BI approaches. All three methods produced identical topology and a single, strict consensus tree is presented in Fig. 20. In general, posterior probabilities and bootstrap values were very high (90–100%) for all nodes. The overall tree shape evidences several lineages with short internal branches subtending species-rich crowns. The consensus tree topology supports the monophyly of the Batoidea as a natural group, in agreement with traditional taxonomy (Ebert & Compagno 2007; Ebert et al. 2013; Last et al. 2016). The proposed phylogeny clearly separated all extant Orders within the Batoidea, in two higher-level reciprocally monophyletic clades: Myliobatiformes + Rhinopristiformes and Torpediniformes + Rajiformes supporting previous phylogenetic reconstructions inferred from nuclear and mitochondrial DNA markers (Aschliman et al., 2012a; Chiquillo et al., 2014; Last et al., 2016). However, the genera included within the order Torpediniformes appear not to be monophyletic to all rays and skates, as previously suggested by Gaitán-Espitia et al., (2016). Node “A” (Fig. 20), shows the studied batoids as a monophyletic clade and support the reciprocal monophyly of batoids and Selachimorpha (sharks). This node suggests a divergence of two major lineages at approximately 226 Mya (Fig. 21), unfolding the four extant orders within batoids: stingrays and mantas rays (Myliobatiformes), sawfishes and wedgefishes (Rhinopristiformes), electric rays (Torpediniformes), and skates (Rajiformes). Node “B” shows the divergence Myliobatiformes and Rhinopristiformes about 199.31 Mya, emerging as two reciprocally monophyletic sister clades with relatively high differentiation (n-distance = 0.786–0.847). The orders Torpediniformes and Rajiformes are represented by node “C”, with an average divergence time of 218.66 Mya. However, the average

–103– CHAPTER IV: MOLECULAR TAXONOMY genetic distance between electric rays and skates is lower (n-distance = 0.338–0.585) than the observed between stingrays and sawfishes (and their allies).

Node “D” contains all studied Rajiformes and supports the current morphology-based phylogeny (McEachran & Dunn, 1998; McEachran & Miyake, 1990; McEachran et al., 1996; McEachran & Aschliman, 2004; Nelson, 2016; Weigmann, 2016). All extant families are represented with the exception of family Gurgesiellidae, due to the lack of comparative material. This family (Gurgesiellidae), includes the genera Cruriraja and Fenestraja (ex-Tribe Crurirajiini), and Gurgesiella (ex- Tribe Gurgesiellini); however, their systematic relationships may undergo further revisions due to ongoing taxonomic uncertainties within this family (Weigmann, 2016). The family Anacanthobatidae (node “E”), represented by the Borneo leg skate Sianobatis borneensis, appears to be monophyletic and in a basal position with an estimated divergence time of 143.88 Mya. Node “F” comprises the families Arhynchobatidae (softnose skates) and Rajidae (hardnose skates) as reciprocally monophyletic clades, sister to leg skates (Anacanthobatidae) with an average divergence time of 115.65 Mya. Among softnose skates (Arhynchobatidae, node “G”), the tribes Riorajini, Bathyrajini and Arhynchobatini (McEachran & Aschliman, 2004; Last et al., 2016) are fully recovered and supported by the proposed phylogeny. Divergence time between tribes is approximately 74.16 Mya, with an average n-distance of 0.3907. The tribe Riorajini is presented as a basal clade to the family and includes the genus Atlantoraja, with an extension to Rioraja (not sampled). Two additional tribes are retrieved as reciprocally monophyletic, which contains the former taxa grouped under Arhynchobatini (McEachran & Aschliman, 2004; Aschliman et al., 2012a). The tribe Bathyrajini is represented by the genus Bathyraja plus Rhinoraja, Psammobatis, and Symperygia (subgroup Ia of McEachran & Miyake, 1990); which is clearly separated from the tribe Arhynchobatini, formerly known as “pavorajines” (subgroup Ib of McEachran & Miyake, 1990). The Arhynchobatini comprises the genera Pavoraja and Notoraja, plus Arhinchobatus, Brochiraja, Irolita and Pseudoraja (McEachran, 1984, Last & McEachran, 2006). Divergence time between these two tribes is 56.97 Mya yet, n-distance ranges are inconclusive due to a high number of non-sampled genera. However, genetic distance within the examined Bathyraja species is low (average n-distance = 0.034), and may reflect the Pliocene radiation of Bathyrajini in the Southern Hemisphere (Valsecchi et al., 2004).

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Figure 20 (next page). Phylogeny of longnose skates of the genus Zearaja (Rajidae, Rajiformes), inferred by maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) method. Consensus trees are based on the General Time Reversible model (16,720 aligned positions; 56 taxa; tree size: 5.306; Γ: 0.956; I: 0.336). Species names in bold, indicates new mitogenomes. Nodes with capital letters indicate a major lineage discussed in text, and represents (A) Batoids, (B) Myliobatiformes + Rhinopristiformes clade, (C) Torpediniformes + Rajiformes clade, (D) Order Rajiformes, (E) Family Anacanthobatidae, (F) Arhynchobatidae + Rajidae clade, (G) Family Arhynchobatidae, (H) Family Rajidae, (I) Tribe Amblyrajini, (J) Tribe Rajini, (K) shortnose skates clade, and (L) longnose skates clade. Bootstrap values (ML, MP) and posterior probability (BI) are represented for each node and are colour-coded. Scale bar represents number of substitution per site.

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Figure 21 (next page). Evolutionary history of longnose skates of the genus Zearaja inferred from mitogenomes. Timetree was generated using the RelTime method and divergence times for all branching points in the topology were calculated using the General Time Reversible model. A total of four nodes with 95% credible intervals of divergence times from fossil records were used for the time constraints (see text). The dotted lines indicate the major marine extinction events during the Phanerozoic (Rohde & Muller 2005).

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Hardnose skates (Rajidae) are contained in node “H” and are represented by two tribes, Amblyrajini and Rajini with an estimated divergence of 77.3 Mya and average n-distance of 0.348. The phylogenomic analysis within the Amblyrajini (Node “I”), supports the separation of the genus Amblyraja and Leucoraja, as reciprocally monophyletic clades with an estimated divergence time of 40.74 Mya. Pairwise patristic distance is lower among Amblyraja (n-distance = 0.028) than Leucoraja (n-distance = 0.131). Cryptic speciation and highly-conserved morphology have been suggested for Amblyraja species (McEachran & Aschliman, 2004; Aschliman et al., 2012a; Weigmann, 2016), which may suggest a potential explanation for the low genetic divergence values among the examined species (ranging between 0.005 and 0.041). However, misidentification due to morphological convergences cannot be out ruled. Examined specimens of Amblyraja georgiana and A. hyperborea come from New Zealand waters; however, the presence of A. hyperborea in the Southern Hemisphere is doubtful (Weigmann, 2016), and may be a misidentification of A. georgiana. The high identity of the whole mitogenome (99.6 %) provides support for the misidentification hypothesis, but further examination of the genus is recommended to resolve the taxonomic uncertainties. Within the tribe Rajini (Node “J”), two clades are recovered representing short- and longnose skates. The shortnose skates (Node “K”) are in a basal position and are exemplified by north-east Atlantic species of Raja and may include Dentiraja and Spiniraja (not sampled) as suggested by Chiquillo et al. (2014) and Last et al. (2016). Divergence times between clades is approximately 55 Mya, with an average pairwise patristic distance of 0.215. The intra-genera genetic distance of Raja is low (n-distance = 0.066), which may reflect the geographical constraints of the species examined.

Longnose skates (Node “L”) are subdivided into two reciprocally monophyletic clades, with an estimated divergence time of 44.9 Mya. Skates from the northwest Pacific of the genus Hongeo, Okamejei, and Beringraja are grouped in a single clade with low average inter-genus patristic distance (n-distance = 0.138 – 0.249). A sister clade diverged 36.48 Mya and comprises the genus Dipturus and Zearaja. Genetic distance between these two genera is low (average n-distance = 0.054), yet the genetic distance within Zearaja is lower (n-distance = 0.008) when compared to Dipturus (n-distance = 0.033). The low divergence of Zearaja species may be a reflection of a recent speciation event, occurring approximately 4 Mya and to the restricted distribution across the Southern Hemisphere. The intra-genus distance is similar in all cases studied. Interestingly, the intraspecies divergence is low in all genera, ranging from 0.001 in Z. chilensis, Z. maugeana, D. innominatus, to 0.06 in B. pulchra. This variation represents 99.87–99.94% of identical bases, equating to 13 – 21 SNPs across the whole mitogenome.

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DISCUSSION

A phylogenomic reconstruction (the inference of phyletic relationships using mitogenomes) of the Superorder Batoidea was used here to investigate the evolutionary position and phylogeny of longnose skates of the genus Zearaja. The inclusion of the complete sequence of the mitochondrial genome, not only protein-codding genes but ribosomal subunits, tRNAs and non-coding regions, have shown to increase our capacity (and statistical support) to understand overlooked empirical morphological convergences as discussed by Compagno (1999), and to untangle previous conflicting signals present previous phylogenetic reconstructions (Dunn et al., 2003; Aschliman et al., 2012a; Naylor et al., 2012a, b; Chiquillo et al., 2014; Gaitán-Espitia et al., 2016).

The molecular data present a consistent signal of batoid interrelationships and speciation of Zearaja skates. In many aspects, the molecular taxonomy (Fig. 20–21) supports hypotheses of batoid evolution proposed in the context of morphology-based phylogenies. Here, all major clades of batoids were recovered as monophyletic, in agreement with morphological phylogenies. However, a number of novel clade arrangements are recovered in addition to those anticipated by morphological cladistics.

The proposed phylogeny of Batoidea is characterised by a topology in which lineages have short internal branches subtending species-rich crowns. This pattern may indicate a group’s rapid radiation after periods of slow diversification, with proposed explanations including the advent of a key innovation, environmental change, accelerated molecular evolution, or others (Crisp & Cook, 2009).

McEachran & Dunn (1998), divided the Rajiformes into three major lineages based on 31 taxa and 55 morphological characters, and suggest one fully resolved clade (tribe Gurgesiellini), two partially resolved clades (tribes Rajini and Amblyrajini) and several incertae sedis taxa. However, before McEachran & Dunn (1998), all skates were subsumed into an enormous, polyphyletic taxon. The current mitogenome-based analysis strongly supports rajids as monophyletic, in agreement with anatomical studies (McEachran & Miyake, 1990; McEachran et al., 1996; McEachran & Dunn, 1998; McEachran & Aschliman, 2004; Aschliman, et al. 2012a, b). The major lineages of batoids appear to have arisen relatively rapidly, with short internodes between branching events. Batoids are here estimated to have originated between about 200 and 230 Mya, followed by the derivation of their major lineages by about 160 Mya. Most groups of extant batoids occur in similar habitats or are thought to have originated in shallow environments before being competitively displaced to the deep sea (Siverson & Cappetta, 2001; McEachran & Aschliman, 2004; Aschliman et al. 2012a).

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The phylogenomic reconstruction inferred from mitogenomes suggests a fundamental split of batoids into two separate clades and four respectively monophyletic lineages: Rajiformes + Torpediniformes and Myliobatiformes + Rhinopristiformes. Compagno (1973), discussed the Pristiformes to be sister to all other extant batoids, with Torpediniformes sister to Rajiformes + Myliobatiformes. Recently, the Rhinopristiformes was described as a novel clade grouping all extant families previously under Pristiformes and Rhiniformes (Naylor et al., 2012a, b; Last et al., 2016). Our results recover the new proposed Order as a monophyletic clade, sister to Myliobatiformes and supports previous hypotheses regarding morphological synapomorphies among Pristiformes and Rhinopristiformes (as Rhiniformes and Myliobatiformes) (Kitamura et al., 1996; Dunn et al., 2003; Maisey et al., 2004; Naylor et al., 2005; Last et al., 2016).

Torpediniformes share no known synapomorphies with any other taxon within batoids and have contrasting theories regarding their monophyletic or paraphyletic position within batoids. Torpediniformes are usually presented as a separated, sister clade to other batoids (McEachran & Ashilman, 2004; Gaitán-Espitia et al., 2016) or as a clade sister to Rajiformes and Myliobatiformes (Compagno, 1973; Dunn et al., 2003). However, our results support the monophyly of the Order, as a sister clade to the Rajiformes. McEachran & Aschliman (2004), suggested that the depressed, disk- shaped morphology of batoids was achieved by two lineages through separate ancestral taxa, which is in general agreement with the currently proposed phylogeny.

Classification of major lineages for the Order Rajiformes

The cladistic analysis using mitogenomes confirm the position of the Rajiformes as the sister clade to Torpediniformes (Kitamura et al., 1996; Maisey et al., 2004; Naylor et al., 2005; Last et al., 2016). For the rajids, Hulley (1970, 1972) recognized six families: Rajidae, Anacanthobatidae, Arhynchobatidae, Pseudorajidae, Gurgesiellidae, and Crurirajidae; while Compagno (1999a), recognized three families, placing Gurgesiellidae, Pseudorajidae, and Crurirajidae under Rajidae. Aschliman et al. (2012a), proposed a provisional classification for batoids, which suggest the inclusion of all rajids under one single family (Rajidae) disregarding all other taxa. Recently, Weigmann (2016), presented a subdivision of four families which are supported with the proposed higher-level molecular phylogenies discussed here: Anacanthobatis, Gurgesiellidae, Arhynchobatidae and Rajidae. Family Anacanthobatis comprises 13 species of legskates in three genera: Anacanthobatis, Sinobatis, and Indobatis. The phyletic position of the family Gurgesiellidae could not be confirmed due to the absence of comparative material, but according to Weigmann (2016), it comprises 19 species, namely all eight species of Cruriraja, eight valid species of Fenestraja and three species of Gurgesiella. Family Arhynchobatidae includes 12 genera of softnose

–111– CHAPTER IV: MOLECULAR TAXONOMY skated distributed in two tribes, Riorajini (genera Atlantoraja and Rioraja), and Arhynchobatini. The later includes two subgroups (sensu McEachran & Miyake, 1990), clade Ia – including genera Bathyraja, Rhinoraja, Psammobatis and Sympterygia; and clade Ib – represented by genera Arhynchobatis, Brochiraja, Irolita, Pavoraja, Pseudoraja, and Notoraja.

The family Rajidae (hardnose skates) has 13 genera in three tribes: The tribe Amblyrajini, includes the genera Amblyraja and Leucoraja plus Breviraja, Dactylobatus and Rajella (not included in the current analysis). The Tribe Rajini contains Raja, Hongeo, Okamejei, Beringraja, Dipturus, and Zearaja plus Dentiraja and Spiniraja (not sampled). A third tribe, the Rostrorajini, includes the genera Malacoraja, Neoraja, Orbiraja and Rostroraja (Last et al., 2016) could not be confirmed due to the absence of comparative material.

Evolution of longnose skates

In contrast to Dipturus species, which have worldwide distribution, the restricted geographical distribution of the genus Zearaja to the Southern Hemisphere may be consistent with a recent speciation process as evidenced from the inferred divergence times. The glaciation of Antarctica as consequence of the drastic cooling of ocean basins during the terminal Eocene event (Veevers, 2000), may be responsible of the radiation between Dipturus and Zearaja, approximately 36 Mya. The formation of the Southern Ocean from the warming influences from the rest of the world (Stevens, 1989), could result in connectivity bottlenecks on an ancestral Dipturus form of Gondwanic origin driving the speciation of Zearaja. Overall changes in deep ocean circulation during Eocene (Storey et al., 1988), may explain the distribution range of extant Zearaja species. During this epoch (56 to 33.9 Mya), oceans shifted from a salinity-driven state to one dominated by temperature; and the terminal Eocene event may have reduced the overall temperature of the Pacific and Southern Oceans with the formation of the Humboldt and Circum-Antarctic Currents, respectively (Prothero, 1984). Interestingly, the speciation process of Z. maugeana appears to be a relatively recent event (approx. 4 Mya), in spite of the apparent lack of connectivity between Tasmania and New Zealand. The ancestral form Z. maugeana/nasuta could have been distributed around Zealandia and Australian waters, and population isolation may have led to the speciation event with the connectivity disrupted due to the sinking of Zealandia about 25 Mya (Campbell & Hutching, 2007). However, the evolution of Z. maugeana may require additional revisions upon the discovery and calibration of new fossil records. Divergence times inferred from the molecular clock and natural history, yet contrasting, may present additional hypotheses of Zearaja evolution. The age of separation of the southern continent and the recent speciation of Z. nasuta and Z. maugeana, suggests that habitat preferences (and therefore, a species’ habitat specialisation) may play an important role in skate speciation processes.

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The Maugean skate Z. maugeana, is the only known skate restricted to shallow, brackish estuarine waters (Pogonoski et al. 2002), and this habitat specialization may have forced a relative rapid radiation from Z. nasuta due to connectivity bottlenecks.

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5. Towards sustainable fishery management: The genetic population structure of Zearaja chilensis and Dipturus trachyderma (Chondrichthyes, Rajiformes) in the south-east Pacific Ocean

Originally published in PLoS ONE (eISSN: 1932-6203)

Copyright © Carolina Vargas-Caro, 2017

Published online: 16 Feb 2017

License Number: Open Access

Abstract

The longnose skates (Zearaja chilensis and Dipturus trachyderma) are the main component of the elasmobranch fisheries in the south-east Pacific Ocean. Both species are considered to be a single stock by the fishery management in Chile however, little is known about the level of demographic connectivity within the fishery. In this study, we used a genetic variation (560 bp of the control region of the mitochondrial genome and ten microsatellite loci) to explore population connectivity at five locations along the Chilean coast. Analysis of Z. chilensis populations revealed significant genetic structure among off-shore locations (San Antonio, Valdivia), two locations in the Chiloé Interior Sea (Puerto Montt and Aysén) and Punta Arenas in southern Chile. For example, mtDNA haplotype diversity was similar across off-shore locations and Punta Arenas (h = 0.46 – 0.50), it was significantly different to those in the Chiloé Interior Sea (h = 0.08). These results raise concerns about the long-term survival of the species within the interior sea, as population resilience will rely almost exclusively on self-recruitment. In contrast, little evidence of genetic structure was found for D. trachyderma. Our results provide evidence for three management units for Z. chilensis, and we recommend that separate management arrangements are required for each of these units. However, there are no evidence to discriminate the extant population of Dipturus trachyderma as separate management units. The lack of genetic population subdivision for D. trachyderma appears to correspond with their higher dispersal ability and more offshore habitat preference.

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INTRODUCTION

The skates (Family Rajidae) form one of the largest groups within the batoids, with 27 genera and about 245 species (McEachran & Miyake, 1990; Ebert & Compagno, 2007). Among these, longnose skates (Tribe Rajini) have a worldwide distribution, inhabiting mostly marine environments from the sublittoral zone to depths of about 3,000 m (McEachran & Miyake, 1990; Ebert & Compagno, 2007). Life history traits such as slow growth, late sexual maturity, and low fecundity make them highly susceptible to overexploitation (Stevens et al., 2000; Frisk et al., 2001; Dulvy & Reynolds, 2002; Myers & Worm, 2005; Dulvy et al., 2014). Appropriate management strategies require an understanding of the population structure of target species, especially as fishing pressure varies among species and fisheries (Dulvy et al., 2014). Vulnerability to collapse due to direct and indirect fishing effects has been well documented for several species, including the common skate Dipturus batis (L. 1758), smooth skate D. innominatus (Garrick & Paul 1974), barndoor skate D. laevis (Mitchill 1818), longnosed skate D. oxyrinchus (L. 1758), white skate Rostroraja alba (Lacepède 1803), and the thornback ray Raja clavata L. 1758; where local declines have occurred as a result of continuous, unregulated fisheries (Dulvy et al., 2000; Francis et al., 2001; Pauly et al., 2002; Myers & Worm, 2005; Worm et al., 2006; McPhie & Campana, 2009; Dulvy et al., 2014).

Most studies on skates rely on fishery catch data to define a species’ distribution, abundance, dispersal potential and to detect demographic fluctuations (Moritz, 1994; Lamilla et al., 2010). However, such data are generally insufficient to allow different stocks to be distinguished, as is required for sound fisheries management practices (Vargas-Caro et al., 2015). Molecular analysis has become an important tool in the study of exploited fish populations (Dudgeon et al., 2012), and is frequently applied to examine stock structure and connectivity, and to identify species from body parts, such as fins and carcasses (Lavery & Shaklee, 1991; Shivji et al., 2002; Heist 2004, 2005; Clarke et al., 2006, Griffiths et al., 2010; Portnoy, 2010), each of which can aid fisheries management and conservation efforts (Ovenden et al., 2009, 2010; Portnoy, 2010).

The yellownose skate Zearaja chilensis (Guichenot 1848) and the roughskin skate Dipturus trachyderma (Krefft & Stehmann 1975) are an important component of commercial elasmobranch fisheries in South American waters (Agnew et al., 2000; Estalles et al., 2011; Bustamante et al., 2012; Sánchez et al., 2012; SERNAP, 2012). Both of these longnose skates have a southern distribution from central Chile (32°S) to central Argentina (40°S), and includes waters off the Falkland Islands, although the Atlantic range of Z. chilensis extends northwards to the La Plata River (34°S) (Menni et al., 2010; Bustamante et al., 2014b). The external morphology of these two skates is remarkably similar, especially in early life stages. As a result, frequent confusion of the species has occurred in

–116– CHAPTER V: GENETIC POPULATION STRUCTURE landing data and official statistical records, which has impacted the effectiveness of fishery monitoring (Licandeo et al., 2006, 2007; Bustamante et al., 2012).

In Chile, a directed fishery for both species started in 1979, which initially targeted Z. chilensis (Bahamonde et al., 1996). However, until 2004 at least six species, including Z. chilensis and D. trachyderma, were landed under the generic category of ‘skate’ according to official records (Gili et al., 1999; Roa & Ernest, 2000; SERNAP, 2004). The fishing effort increased after 1993 when the fishery was opened to the Asian market. The artisanal fleet expanded due to international investment and, together with the industrial fishery, reported 3,000 tonnes (t) in landings in 1994. Once the Asian financial crisis ended in 1998, skate landings increased considerably, peaking 4,000 t and 5,193 t in 2000 and 2003 respectively (SERNAP, 2004; Vargas-Caro et al., 2015). After 2006, a temporary fishing closure was imposed on the artisanal fleet, which operates during the austral summer months (December – February) to protect ‘possible reproductive events’ (Licandeo et al., 2006, 2007). Subsequently, between 2009 and 2011, a total fishing closure was imposed on the entire fishery in response to a declining trend in the overall catch per unit effort (CPUE), including the decline in the size of individuals landed. In spite of these ‘closures’, the government continues to allocate national catch quotas of up to 700 t annually (Bustamante et al., 2012; Vargas-Caro et al., 2015).

The longnose skate fishery in Chile is geographically extensive (approximately 20° of latitude, or 2,400 km from north to south), and operates year-round along the outer continental shelf at 150 – 450 m depth using bottom-set longlines (Bustamante et al., 2012; Vargas-Caro et al., 2015). However, most of the fishing effort is located between central-south Chile, between 33.5°S and 45.5°S (Licandeo et al., 2006, 2007; Bustamante et al., 2012). The fishery captures mostly small, immature fishes which, as a gauntlet fishery, could potentially compromise the integrity of the stock (Licandeo et al., 2006; Bustamante et al., 2012). The overall abundance of skates has declined substantially over the last decade due to intensive fishing pressure, and as consequence, the fishery is considered to be ‘fully exploited’ (Lamilla et al., 2010; Bustamante et al., 2012; Vargas-Caro et al., 2015).

Considering the external morphological similarities between longnose skates, the current and past fishing pressures, and the overlap in both depth and latitudinal distribution; the aim of the present study was to assess genetic diversity and population structures of Z. chilensis and D. trachyderma in Chilean waters using mitochondrial and nuclear genetic markers. Population structure of each species was expected to be similar due to low fecundity and low dispersal potential. The research was carried out to provide relevant information to the single stock harvesting strategy for both species, currently used by the Chilean Government.

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MATERIALS AND METHODS

Sample collection, species identification, and DNA extraction

Zearaja chilensis and Dipturus trachyderma were collected from five locations in the south- east Pacific Ocean, off the coast of Chile (Fig. 22). The five locations represent landing sites for three separate fishing areas; San Antonio (SA, 33°35.5'S 71°37'W) and Valdivia (VA, 39°52.5'S 73°24'W) represent off-shore fishing grounds in the north; Puerto Montt (PM, 41°28.4'S 72°56.4'W) and Aysén (AY, 45°25'S 72°49'W) represent the Chiloé Interior Sea and; Punta Arenas (PA, 53°10.2'S 70°54.5'W) represents an admixture of oceanic and fjord fishing grounds in the south of Chile. Of the 408 specimens caught, 271 were Z. chilensis (101 males, 170 females) and 137 were D. trachyderma (67 males, 70 females). These specimens were used to explore the possibility of spatial genetic structure within their respective populations. This study did not involve endangered or protected species, and samples were obtained from deceased animals caught by a commercial fishery. Capture of skates was permitted under Fisheries Undersecretariat Research Permits (R. Ex.) 2878-14, 3442- 14 and 1482-15, issued by the Ministry of Economy, Development, and Tourism. Landed specimens were photographed using a standardised protocol (see Fig. 23) and initially identified using field guides (Lamilla & Bustamante, 2005; Lamilla & Sáez, 2003). In all cases, species identification was subsequently checked using the standardised photographs. Some specimens lacked the morphological features (e.g., spinulation pattern and head shape) necessary for unambiguous identification, such as nuchal thorns or, as in juvenile males (total length, LT <100 cm) (Vargas-Caro et al., 2015), secondary sexual characteristics. However, DNA analysis was used to confirm species identity of all specimens.

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Figure 22. Map showing landing locations of Zearaja chilensis and Dipturus trachyderma surveyed in this study. San Antonio (blue) and Valdivia (grey) represent off-shore fishing grounds. Puerto Montt (yellow) and Aysén (orange) represent fishing grounds within the Chiloé Interior Sea. Punta Arenas (red) represent an admixture of fishing grounds between oceanic and fjord ecosystems.

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Figure 23. Standardized photographs used to confirm species identification of longnose skates. Dorsal and ventral views of Zearaja chilensis (female A, B; male C, D). Dorsal and ventral views of Dipturus trachyderma (female E, F; male G, H). Colorimetric scale bar = 25 cm.

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The size (LT) and sex of each specimen were recorded following Last et al. (2008b). A Chi- square goodness-of-fit test (χ2) (Sokal & Rohlf, 1987) was conducted to examine whether the male to female ratio varied significantly from 1:1. Previously published size-at-maturity values were used to determine the number of mature individuals for both Z. chilensis (Bustamante et al., 2012) and D. trachyderma (Licandeo et al., 2007).

A muscle tissue sample was taken from each individual skate and stored in 100% ethanol at - 20°C until required for DNA-extraction. Total genomic DNA, extracted (from ~25 mg of tissue) following a modified salting-out method (Miller et al., 1988) was used for both mitochondrial DNA (mtDNA) and microsatellite genotyping.

Mitochondrial DNA amplification and data analysis

Mitochondrial DNA was one of two types of genetic marker used to assess genetic population structure in the two species. Novel primers were designed for the mitochondrial control region (mtCR) from the complete mitochondrial genome of Z. chilensis (Vargas-Caro et al., 2016a), using

Primer3 in GENEIOUS Software v.8.1 (Biomatters Ltd., Auckland). The forward primer sequence, Zch_CR_F (5'– TGA ACT CCC ATC CTT GGC TC –3'), was placed at tRNAPro between 15,610 bp and 15,629 bp, and the reverse primer, Zch_CR_R (5'–GTA TTG GTC GGT TCT CGC CA –3'), was positioned in the central control region between 16,186 base pairs (bp) and 16,205 bp. Primers were blasted against the complete mitochondrial genome and all available sequences on NCBI Genbank (Benson et al., 2013), to ensure that the sequences were unique and on target.

Initial screening of the selected primers was performed on 16 Z. chilensis individuals, and they were subsequently tested against the closely related species D. trachyderma. Approximately 600 bp of the mtCR was successfully amplified for both skate species. PCR amplifications were performed for all sampled individuals in 10 µl reactions containing 1 µl of genomic DNA (20 – 50 ng), 5.9 µl of Milli-Q H2O, 1 µl of 10x buffer MgCl2 (15 mM), 1 µl of dNTPs (2 mM), 0.5 µl of each primer (Zch_CR_F and Zch_CR_R), and 0.1 µl Taq DNA polymerase (5 U/µl). Thermal cycling consisted of an initial denaturation at 95 °C for 1 min, followed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. A final extension step was added at 72 °C for 10 s. Amplified PCR products were treated with 1 U ExoSAP-IT (USB® Products Affymetrix, Inc) at 37 °C for 45 min, followed by an inactivation step at 80 °C for 15 min. The cleaned PCR product was sequenced in both directions using BigDye Kit v3.1 on an ABI Prism 3130xl Genetic Analyser (Applied Biosystems).

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Sequences were analysed using GENEIOUS v.8.1 (Biomatters Ltd, Auckland). Each sequence was manually reviewed for uncalled and miscalled bases, and all variable positions were confirmed by comparing sequence reads produced by the forward and reverse sequences on each individual. Primer sequences were then removed, and once the sequences had been checked for discrepancies, a consensus sequence was produced for each individual. Aligned consensus sequences were used to determine the position of single nucleotide polymorphisms (SNPs) and insertion-deletion (indel) events across individuals. All sequences were deposited in GenBank under the accession numbers KX708934 to KX709341.

To characterise the mtDNA genetic diversity within and between populations, the number of haplotypes (Nh), polymorphic sites and, haplotype (h) and nucleotide (π) diversity indices were calculated using DNASP v.5.10.1 (Librado & Rozas, 2009) for each species. Genetic differentiation among populations and pair-wise ФST were assessed using ARLEQUIN v3.5.1.2 (Excoffier & Lischer, 2010). The genetic distance between haplotypes was estimated using the Tamura–Nei (Tamura & Nei, 1993) nucleotide substitution model with gamma set to 0.25. A median-joining network of haplotypes was constructed in NETWORK v.4.6.1.3 (Bandelt et al., 1999) to visualise haplotype clustering and diversity.

Microsatellite loci amplification and data analysis

To develop species-specific microsatellite genetic markers for each species, raw genomic DNA data from two voucher specimens (Vargas-Caro et al., 2016a, b) were retrieved from the Genomic Database Repository, eFish (Zearaja chilensis BioVoucher: 2014-ZCH-1004; Dipturus trachyderma BioVoucher: 2015-DTR-004). Sequences between 150 and 400 bp were explored for microsatellite motifs using the software QDD v.3.1 (Meglécz et al., 2014). Sequences having dinucleotide and imperfect repeat motifs were excluded. The remaining sequences were considered if the number of repeat units was greater than ten. Selected sequences were blasted against the NCBI Genbank dataset to identify and exclude loci located in potential coding regions. Finally, selected forward and reverse primers were custom-blasted against the original libraries using GENEIOUS v.8.1, to exclude primers with homology to regions outside the target flanking sequence and ensure no loci duplication. For each species, a set of 48 microsatellite loci was selected, and the 5’ end of the forward primers were tailed with a ‘CAG-tail’ to allow fluorescent labelling of PCR product while the reverse primers were tailed with ‘GTTT-tail’ to ensure complete adenylation (Peters et al., 2009).

To optimise the experimental design of population surveys with these loci, a power analysis

(POWSIM, Ryman & Palm, 2006) was used to simulate the probability of detecting population genetic

–122– CHAPTER V: GENETIC POPULATION STRUCTURE differentiation across a range of expected FST values in two theoretical populations. Allele frequencies for 16 loci were collected in order to perform a pilot testing in the power analysis. Loci were excluded if they had (1) a major allele frequency greater than 90%, and (2) potential lower scoring accuracy due to the presence of large numbers of alleles. As recommended by Ryman & Palm (2006), estimates of statistical power for a defined level of divergence was controlled by changing the generations of drift (t) instead of varying the effective population size (Ne), which can cause the loss of low- frequency alleles. Sample sizes per population were 50, 100 and 150 and the final number of loci considered was 10. Values of FST were set at 0.01, 0.005 and 0.0025, which was equivalent to migration rate of 2.5%, 5% and 10%. The following parameters were used: effective population size

(Ne) = 1,000; number of simulations = 1,000; and generations of drift (t) = 20 (FST = 0.01), 10 (FST =

0.005) and 5 (FST = 0.0025). The degree of significant differentiation (quantified as FST values) for each replicate run was tested using chi-square and Fisher’s exact probability to test the null hypothesis of genetic homogeneity.

Initial screening of the selected microsatellite primers was performed on 16 individuals of each species. Samples were amplified using a PCR reaction of 11 µl containing 1 µl of genomic DNA (10 – 15 ng), 0.05 µl forward primer, 0.25 µl reverse primer, 0.25 µl of ‘CAG-FAM’, 5.5 µl of 2 x MyTaq mix (Bioline Australia, containing about 0.15 µl of Taq) and 3.95 µl of Milli-Q H2O. Thermocycler conditions were 95 °C for 3 min, followed by 37 cycles of 94 °C for 15 s, 57 °C for 15 s and 72 °C for 10 s. A final extension was performed at 72 °C for 30 min and held at 15 °C. PCR products were diluted 200–fold and sequenced by capillary electrophoresis on an ABI Prism 3130xl Genetic Analyser following the manufacturer’s recommendation. Alleles were sized against an internal size standard (GeneScan-500 LIZ) before being scored using GENEIOUS v.8.1. Further characterisation of these loci was undertaken by genotyping 32 additional individuals of Z. chilensis; the best loci were then tested against D. trachyderma in order to have the final selection.

A total of ten microsatellite loci were found to be polymorphic and selected for genotyping the species in this study. Of these, nine were used for Z. chilensis and seven for D. trachyderma. Six out of the ten loci were common to both species in order to test for potential hybridisation, as the species overlap in latitudinal and bathymetric distribution, are morphologically similar and closely- related longnose skates of the Family Rajidae. One primer stock was set up for each locus, consisting of forward and reverse primer pairs and the corresponding fluorescently labelled CAG primer (namely Fam, Vic, Ned or Pet) and used in multiplex PCRs. Locus and colour combinations per multiplex PCR were chosen to minimise allele overlap in size. Primer stocks consisted of 6 µl of 100uM forward primer, 30 µl of 100uM reverse primer, 15 µl of 200uM of CAG-fluoro labelled primer and 99 µl Milli-Q H2O. For use in PCR amplifications, primer stocks were combined into a

–123– CHAPTER V: GENETIC POPULATION STRUCTURE primer mix in proportions that were determined experimentally to achieve optimal amplification across all loci. Microsatellite PCR amplifications were performed in 14 µl reaction containing 2 µl of genomic DNA (10 – 15 ng), 4 µl of primer mix and 8 µl of 2 x MyTaq mix (Bioline, Australia containing about 0.15ul of Taq) that was added once the reaction was at 95 °C in the PCR machine. Thermal conditions were the following: 95 °C for 10 min, followed by 37 cycles of 94 °C for 15 s, 57 °C for 15 s and 72 °C for 10 s. The reaction was then exposed to 72 °C for 30 min and held at 12 °C. Four (Z. chilensis) and three (D. trachyderma) multiplexed PCRs were performed to genotype each individual at these loci (Table 15). The PCR products from multiple amplification reactions (one of each fluorophore) were diluted and combined in a single lane (lane-plex). Fragment separation was performed on an ABI 3130XL, and alleles were sized against an internal size standard (GeneScan-500 LIZ).

Genotypes were scored and binned using the microsatellite plug-in in GENEIOUS software v.8.1. MICRO‐CHECKER v.2.2.3 (Van Oosterhout et al., 2004) was used to check for scoring errors revealed by heterozygote deficit across loci (caused by the presence of null alleles and/or large allele dropout) and heterozygote excess (caused by incorrectly scoring stutter bands). If deviations were detected, scoring was revised until deviations were eliminated or minimised. GENEPOP v.4 on-the- web (Raymond & Rousset, 1995; Rousset, 2008) was used to assess for departures from Hardy– Weinberg equilibrium (HWE) and linkage disequilibrium (LD) among different loci; significance was accepted at P < 0.05. The assessment used Markov-chain parameters of 1,000 dememorisation steps, 100 batches and 1,000 iterations per batch, for multiple comparisons using Bonferroni adjustment (α= 0.05). The number of alleles (NA) and their frequencies, expected (HE) and observed heterozygosity (HO) was estimated using GENALEX v.6.501 (Peakall & Smouse, 2006, 2012).

Population pair-wise FSTs and p-values of the FST between populations were estimated using

1000 permutations in ARLEQUIN v.3.5.2.2 (Excoffier & Lischer, 2010). Partial Mantel tests were implemented using the Isolation by Distance Web Service v.3.23 (Jensen et al., 2005), with significance determined via permutation tests.

The Bayesian clustering algorithm implemented in STRUCTURE v.2.3.4 (Pritchard et al., 2000; Falush et al., 2003, 2007; Hubisz et al., 2009) was used to examine possible population subdivision. To determine the optimal number of cohesive groups (k) was estimated with 20 independent runs of K = 1 – 10. The length of the burn-in period was set to 100,000 iterations, followed by 100,000 Markov Chain Monte Carlo (MCMC) repetitions under the admixture model with correlated allele frequencies. The optimal number of populations was estimated using the ∆K method (Evanno et al.,

2005) implemented in STRUCTURE HARVESTER v.0.6.94 web-based tool (Earl & VonHoldt, 2012).

Cluster Markov Packager Across K (CLUMPAK, Kopelman et al., 2015) was used to summarise results

–124– CHAPTER V: GENETIC POPULATION STRUCTURE from STRUCTURE and provide a graphical representation referring to individuals and populations through bar plots. To maximize discrimination between populations, discriminant analysis of principal components (DAPC, Jombart et al., 2010) was applied as implemented in R-package

ADEGENET v.1.4-2 (Jombart, 2008; Jombart & Ahmed, 2011). The cross-validation function xvalDapc was used to set the number of principal components to 50 (Jombart et al., 2010). DAPC uses a sequential K-means approach to determine the numbers of clusters in the data, assigning individuals to clusters independent of their collection site.

Table 15. Amount of primer stock per microsatellite locus added to primer mix to set up multiplexed PCRs for genotyping Z. chilensis and D. trachyderma. Multiplex numbers are indicated in brackets.

Locus Number Primer stock per single Multiplexed PCR ID Locus name (Lab Code) reaction (μL)

Zearaja chilensis

CAG-VIC (M_1) Zch_MS08 Zch_067 0.5

CAG-VIC (M_1) Zch_MS13 Zch_070 0.5

CAG-VIC (M_1) Zch_MS19 Zch_019 0.8

CAG-NED (M_2) Zch_MS15 Zch_059 0.2

CAG-NED (M_2) Zch_MS31 Zch_085 0.5

CAG-PET (M_3) Zch_MS06 Zch_007 0.5

CAG-PET (M_3) Zch_MS16 Zch_072 1.2

CAG-FAM (M_4) Zch_MS10 Zch_057 0.9

CAG-FAM (M_4) Zch_MS29 Zch_083 1

Dipturus trachyderma

CAG-VIC (M_1) Zch_MS08 Zch_067 0.6

CAG-VIC (M_1) Zch_MS19 Zch_019 0.9

CAG-NED (M_2) Zch_MS15 Zch_059 0.2

CAG-NED (M_2) Zch_MS31 Zch_085 0.5

CAG-NED (M_2) Dtr_MS08 Dtr_009 0.8

CAG-PET (M_3) Zch_MS06 Zch_007 0.5

CAG-PET (M_3) Zch_MS16 Zch_072 1.2

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RESULTS

Of the 408 specimens collected, there were 11 whose identity was uncertain based on external morphology. Although initially considered to be D. trachyderma they were all identified as Z. chilensis based on their mtCR (557 bp) sequences.

The sex ratio (males: females) for Z. chilensis was significantly biased towards females (1:2.17; χ2 = 6.357, d.f. = 1, P > 0.05) in all locations except VA, where the sex ratio (1:0.906) was not significantly different to 1:1 (χ2 = 0.262, d.f. = 1, P = 0.968). Overall, body size ranged between

61 and 117 cm LT for males and between 71 and 141.8 cm LT for females. Mature females were common at PM (57%) and PA (84%) but represented only 14% of specimens from the other locations. In the case of males, approximately half of the individuals were mature (53%), with PM and PA having the highest proportions (83% and 100% respectively).

No significant differences were observed in the overall sex ratio of D. trachyderma (1:1.04; χ2 = 0.029, d.f. = 1, P = 0.965), although there was a significant bias towards females in SA (1:2.058; χ2 = 5.557, d.f. = 1, P > 0.05). However, low sample sizes for PM (14), AY (11) and PA (7) preclude a definitive interpretation of the data. Males of D. trachyderma ranged from 89 to 207.5 cm LT and females 92.9 to 242.5 cm LT. Except for a low percentage of mature specimens in SA and VA (4.4% and 3.6% respectively), all males and females were immature.

Mitochondrial data

Zearaja chilensis

A total of 271 mtCR consensus sequences of 557 bp in length were successfully obtained for Zearaja chilensis. Analysis of these sequences revealed 10 polymorphic sites and one 1bp indel, which together defined 11 haplotypes (Table 16). About 90% of individuals were represented by the haplotypes Zch_CR1 (45.4%) and Zch_CR2 (44.6%). With the exception of one individual found in

PM, haplotype Zch_CR1 was only observed in samples from off-shore fishing grounds in the north

(SA and VA) and PA in the south; and occurred in 64 – 72% of individuals. While haplotype Zch_CR2 was found in Z. chilensis landed at all five locations, it occurred in 96 – 100% of individuals from the Chiloé Interior Sea that were landed at PM and AY, in 36% of individuals from PA and only 5 – 13% of individuals to the north (VA and SA) (Table 16). Haplotype Zch_CR3 occurred at both northern sites but was only found in 3 – 4% of individuals. The remaining eight haplotypes were only found at individual sites, and in five cases was represented by a single individual.

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To test whether mtDNA genetic diversity was homogeneous across populations, off-shore (SA and VA) and PA populations were compared to those in the Chiloé Interior Sea (PM and AY) (Table 17). Haplotype diversity was similar across off-shore locations and PA (h = 0.46 – 0.50), but was significantly different to those in the interior sea: PM (h = 0.08) and AY (h = 0.00). While nucleotide diversity was low for all locations, it was extremely low in the Chiloé Interior Sea compared to off-shore fishing grounds and PA.

The median-joining network for Z. chilensis subdivided the mtCR haplotypes into two main groups: one composed of haplotypes mostly from PM and AY and the other of haplotypes mostly from SA, VA, and PA (Fig. 24A). Each group was the centre of a star-like cluster with more haplotypes diverging from them by two (or more) mutations. Comparisons of ФST between locations suggest a significant genetic differentiation between the Chiloé Interior Sea (PM and AY), and off- shore locations (SA and VA) and PA (P < 0.05; Table 18).

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Table 16. Geographic distribution of mtDNA Control Region haplotypes found in Zearaja chilensis and Dipturus trachyderma. Samples per locality and haplotypes are indicated as the proportion of the total number of specimens with that haplotype. Sampling localities: SA, San Antonio; VA, Valdivia; PM, Puerto Montt; AY Aysén; and PA, Punta Arenas.

Position Sampling location 1 1 1 2 2 4 4 5 5 5 9 0 2 6 0 0 1 9 1 2 2 Z. chilensis 9 0 1 6 1 2 9 5 2 8 9 SA VA PM AY PA Total Zch_CR1 A G – G A T T C C C T 0.700 0.721 0.021 – 0.644 123 Zch_CR2 · · – · · · · · T · · 0.057 0.131 0.957 1.00 0.356 121 Zch_CR3 G · – · · · · · · · · 0.043 0.033 – – – 5 Zch_CR4 · A T · · · · · · T C 0.100 – – – – 7 Zch_CR5 · · – · G · · · T · · – 0.082 – – – 5 Zch_CR6 · · – A G · · · T · · 0.071 – – – – 5 Zch_CR7 · · – · · C · · · · · 0.014 – – – – 1 Zch_CR8 · · – · · · C · T · · – 0.016 – – – 1 Zch_CR9 · · – A · · · · · · · 0.014 – – – – 1 Zch_CR10 · · – · · · · T T · · – – 0.021 – – 1 Zch_CR11 · · – · · · C · · · · – 0.016 – – – 1 N= 70 61 47 48 45 271

1 1 1 2 2 2 3 4 5 4 7 7 0 4 7 0 0 4 D. trachyderma 9 6 8 1 5 1 5 2 9 SA VA PM AY PA Total Dtr_CR1 T T T G A T G T T 0.981 0.811 0.857 1.00 1.00 124 Dtr_CR2 C C C A T A A · G – 0.132 – – – 7 Dtr_CR3 C · · · · · · C · 0.019 0.038 0.071 – – 4 Dtr_CR4 C · · · · · · · · – 0.019 0.071 – – 2 N= 52 53 14 11 7 137

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Table 17. Genetic diversity per sampling locality from mtDNA control region and microsatellite loci for Zearaja chilensis and Dipturus trachyderma.

Number of individuals (N); number of mtCR haplotypes (Nh); haplotype diversity ± standard deviation (h); nucleotide diversity ± S.D. (π); maximum number of alleles observed per locus (NA); observed (HO) and expected heterozygosity (HE); Wright’s inbreeding coefficient of individuals relative to the subpopulation (FIS). Values for microsatellite data are presented as the average across loci. Sampling locations as in Table 15.

mtDNA haplotypes Microsatellites Z. chilensis N Nh h π N NA HO HE FIS SA 70 7 0.496 ± 0.069 0.0024 ± 0.0017 30 10 0.603 0.613 0.026 VA 61 6 0.462 ± 0.073 0.0012 ± 0.0010 32 9 0.576 0.593 0.021 PM 47 3 0.084 ± 0.055 0.0001 ± 0.0003 32 10 0.578 0.576 0.002 AY 48 1 0.000 0.000 32 7 0.534 0.546 0.004 PA 45 2 0.469 ± 0.044 0.0008 ± 0.0008 28 8 0.602 0.599 -0.023

D. trachyderma SA 52 2 0.038 ± 0.037 0.0001 ± 0.0003 32 5 0.421 0.432 0.049 VA 53 4 0.329 ± 0.077 0.0036 ± 0.0023 31 6 0.360 0.376 0.022 PM 14 3 0.275 ± 0.148 0.0007 ± 0.0008 14 3 0.471 0.440 -0.033 AY 11 1 0.000 0.000 11 3 0.414 0.390 0.015 PA 7 1 0.000 0.000 7 3 0.347 0.322 -0.096

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Figure 24. Median-joining network of mtCR haplotypes for (A) Zearaja chilensis and (B) Dipturus trachyderma. Haplotypes are represented by circles with size proportional to frequency in the total sample. All branches correspond to one mutation except where indicated otherwise. Sampling locations: San Antonio (SA, blue), Valdivia (VA, grey), Puerto Montt (PM, yellow), Aysén (AY, orange) and Punta Arenas (PA, red).

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Table 18. Estimates of pair-wise genetic distance (FST) (below diagonal) among locations for mtDNA control region (mtCR) sequences and microsatellites loci for Zearaja chilensis and Dipturus trachyderma. FST P-values are indicated above diagonal. Significant ФST and FST values are presented in bold. Significance accepted at P < 0.05. Sampling locations are described in Table 15.

Location Species SA VA PM AY PA Z. chilensis mtCR SA – 0.009 0.000 0.000 0.000 VA 0.036 – 0.000 0.000 0.126 PM 0.500 0.583 – 0.756 0.000 AY 0.525 0.623 0.008 – 0.000 PA 0.079 0.023 0.584 0.644 – Microsatellite loci SA – 0.666 0.061 0.105 0.030 VA -0.002 – 0.406 0.016 0.003 PM 0.008 0.000 – 0.723 0.076 AY 0.007 0.012 -0.003 – 0.103 PA 0.011 0.019 0.007 0.006 –

D. trachyderma mtCR SA – 0.000 0.126 0.990 0.990 VA 0.110 – 0.369 0.198 0.432 PM 0.074 0.027 – 0.513 0.765 AY -0.045 0.037 0.029 – 0.990 PA -0.075 0.006 -0.015 0.000 – Microsatellite loci SA – 0.131 0.358 0.996 0.166 VA 0.011 – 0.028 0.608 0.436 PM 0.002 0.030 – 0.248 0.072 AY -0.035 -0.007 0.010 – 0.607 PA 0.020 0.0001 0.041 -0.017 –

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Dipturus trachyderma

Sequence data (556 bp of the mtCR) was obtained for 137 D. trachyderma. Among individuals, a total of nine polymorphic sites were detected, representing four haplotypes. All differences were due to substitutions and no indels were found (Table 16). Haplotype frequencies clearly showed that Dtr_CR1 was the dominant haplotype, found in 91% of individuals. This haplotype occurred at all collection sites and at high frequency; 81 – 100% of specimens within any given region. VA was the only location where all four haplotypes were observed, and haplotype

Dtr_CR2 was only detected at this location. Haplotypes Dtr_CR3 and Dtr_CR4 occurred at frequencies of 7% or less and were not present at AY or PA. Differences in frequencies may be related to the restricted sampling size of PM, AY, and PA.

The mean haplotype and nucleotide diversities for specimens from the three most northern sites followed a similar pattern: VA > PM >> SA, ranked from highest to lowest diversity (Table 17). AY and PA were excluded from this analysis as only a single haplotype was present in these locations.

The median-joining network of mtCR haplotypes for D. trachyderma shows a general lack of population structure (Fig. 24B). Four distinct haplotypes were detected with Dtr_CR1 being the most prevalent haplotype present in all locations. Only one mutation was observed between all haplotypes, with exception of Dtr_CR2. This haplotype was only found in 13% of individuals, exclusively found in VA and separated by seven mutations from all remaining haplotypes. Comparisons of pair-wise

ФST values revealed a low and non-significant pair-wise divergence among locations (Table 18).

Microsatellite data

Zearaja chilensis

A total of 154 individuals were genotyped with 9 microsatellite loci in the nuclear DNA

(nDNA). Tests for scoring error indicated the possibility of null alleles for only one locus (Zch_MS10) at a single location (AY) due to an excess of homozygotes. Overall, no loci showed significant departure from HWE or evidence for LD. However, three loci deviated from HWE (P < 0.05);

Zch_MS16 in VA, Zch_MS13 in PM and Zch_MS19 in PA. The exclusion of these loci at these sites did not change the pattern of differentiation between populations and therefore these loci were included in the study.

Across all sampled populations, the allelic diversity ranged from two (Zch_MS13) to 10 alleles

(Zch_MS31) (Table 19). Overall, expected (HE) and observed heterozygosity (HO) values ranged

–132– CHAPTER V: GENETIC POPULATION STRUCTURE between 0.300 and 0.810. The highest HO was registered for locus Zch_MS19 whereas locus Zch_MS13 showed the lowest HO and HE values (Table 19). A number of alleles, HO and HE varied among populations at different loci. Observed number of alleles within locations ranged between two in SA at locus Zch_MS13 and, 10 in SA and PM at locus Zch_MS31 (Table 20). The highest mean of HO and

HE was observed in SA (0.603 and 0.613 respectively) and the lowest in AY (HO 0.534; HE 0.546).

The power analysis for detecting genetic differentiation using ten microsatellite loci indicated that a pair-wise FST of 0.01 could be detected 89% of the time at a sample size of 50 or less; for smaller FSTs of 0.005 and 0.0025, the percentage fell to 52% and 21% respectively. However, the probability of detecting population subdivision could be increased at a higher sampling size. For example, with a sample size of 100 individual and FST-values as low as 0.0025 could be detected 77% of the time. Global FST is low and non-significant (FST 0.00907, P-value = 0.106). Pairwise FST tests indicated low, non-significant genetic differentiation among Z. chilensis sampling locations (-0.003 and 0.019) (Table 18). However, significant genetic differentiation was found between the off-shore locations (SA and VA) and PA, as well as between VA and AY. The correlation between genetic [FST -1 2 (1 - FST) ] and geographic distance was also significant (P < 0.05, r =0.049), supporting the hypothesis of isolation by distance of gene flow (Fig. 25A). Population assignment analyses using

STRUCTURE were inconclusive as no signal of genetic differentiation between collection locations was observed (results not shown). One main cluster was identified using DAPC (Fig. 26A) and, a subdivision within this cluster was observed. Individuals from off-shore locations (SA and VA) were most closely co-located; the Chiloé Interior Sea (PM and AY) centroids were also close, and the PA centroid sat off to one side. These relationships reflect the outcomes of the mtDNA analyses, but similarities should be interpreted cautiously as the microsatellite data ellipses have considerable overlap.

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Table 19. Characterisation of polymorphic microsatellite loci used for Zearaja chilensis (n = 9) and Dipturus trachyderma (n = 7) in this study. Locus

name, primer sequences, repeat motif, allele size, maximum number of alleles (NA) and expected (HE) and observed (HO) heterozygosities. (―) Indicates microsatellite loci not used on the species.

GenBank Z. chilensis D. trachyderma Repeat Motif Size (bp) Locus accession number NA HE HO NA HE HO 8 Zch_MS06 KX708924 (ATC) 137 8 0.452 0.474 2 0.253 0.289 10 Zch_MS08 KX708925 (AC) 143 5 0.540 0.545 4 0.387 0.445 7 Zch_MS10 KX708926 (AGC) 333 4 0.650 0.614 — — — 7 Zch_MS13 KX708927 (AC) 166 5 0.302 0.300 — — — 7 Zch_MS15 KX708928 (AATG) 141 5 0.609 0.599 2 0.475 0.463 10 Zch_MS16 KX708929 (AC) 301 7 0.622 0.578 4 0.209 0.142 8 Zch_MS19 KX708930 (AAT) 213 7 0.797 0.810 4 0.419 0.475 9 Zch_MS29 KX708931 (AC) 394 7 0.496 0.518 — — — 7 Zch_MS31 KX708932 (AC) 182 10 0.800 0.768 6 0.568 0.580 13 Dtr_MS08 KX708933 (ACTC) 329 — — — 6 0.468 0.434

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Table 20. Genetic diversity per locality and microsatellite locus for Zearaja chilensis and Dipturus trachyderma. Sampling localities are as in Table 15.

Z. chilensis D. trachyderma Locus SA VA PM AY PA SA VA PM AY PA Zch_MS06 NA 7 6 5 5 5 2 2 2 2 2 HE 0.550 0.515 0.451 0.353 0.464 0.285 0.144 0.293 0.298 0.245 HO 0.467 0.594 0.469 0.375 0.393 0.281 0.156 0.357 0.364 0.286 Zch_MS08 NA 5 5 5 5 4 2 4 2 2 2 HE 0.528 0.437 0.490 0.593 0.650 0.469 0.389 0.459 0.636 0.133 HO 0.500 0.438 0.484 0.625 0.679 0.438 0.438 0.571 0.483 0.143 Zch_MS10 NA 4 4 4 4 4 — — — — — HE 0.668 0.680 0.656 0.627 0.641 — — — — — HO 0.577 0.688 0.636 0.438 0.714 — — — — — Zch_MS13 NA 2 3 4 4 3 — — — — — HE 0.262 0.225 0.314 0.301 0.406 — — — — — HO 0.241 0.219 0.233 0.344 0.464 — — — — — Zch_MS15 NA 4 5 4 5 4 2 2 2 2 2 HE 0.587 0.602 0.639 0.626 0.571 0.458 0.451 0.497 0.469 0.500 HO 0.633 0.563 0.677 0.548 0.589 0.516 0.313 0.308 0.650 0.429 Zch_MS16 NA 4 5 6 4 5 2 4 2 2 2 HE 0.665 0.669 0.645 0.517 0.536 0.242 0.272 0.293 0.236 0.139 HO 0.600 0.563 0.710 0.548 0.551 0.156 0.250 0.214 0.091 0.147 Zch_MS19 NA 6 7 6 6 6 3 4 3 3 2 HE 0.787 0.797 0.797 0.811 0.667 0.467 0.484 0.589 0.310 0.245 HO 0.852 0.750 0.875 0.906 0.796 0.438 0.469 0.798 0.182 0.286 Zch_MS29 NA 6 6 3 5 5 — — — — — HE 0.647 0.625 0.355 0.387 0.541 — — — — — HO 0.690 0.625 0.306 0.359 0.536 — — — — — Zch_MS31 NA 10 9 10 7 8 4 6 3 3 3 HE 0.818 0.788 0.842 0.726 0.786 0.572 0.584 0.571 0.539 0.612 HO 0.867 0.750 0.806 0.633 0.828 0.567 0.567 0.533 0.625 0.571 Dtr_MS08 NA — — — — — 5 5 3 3 3 HE — — — — — 0.533 0.471 0.417 0.398 0.520 HO — — — — — 0.552 0.379 0.273 0.250 0.714

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Figure 25. Correlation between genetic and geographic distance, for populations of (A) Zearaja chilensis and (B) Dipturus trachyderma. Linear adjustment (solid line) and 95% confidence interval (dashed line) are specified. Geographic distance are indicated by paired locations, (a) SA and VA; (b) SA and PM; (c) SA and AY; (d) SA and PA; (e) VA and PM; (f) VA and AY; (g) VA and PA; (h) PM and AY; (i) PM and PA; (j) AY and PA. Sampling locations abbreviations as in Fig 22.

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Figure 26. Scatterplots of the Discriminant Analysis of Principal Components (DAPC) from microsatellite genotypes for populations of (A) Zearaja chilensis and (B) Dipturus trachyderma. Dots represent individuals, whereas coloured ellipses correspond to geographical populations. Sampling locations: San Antonio (SA, blue), Valdivia (VA, grey), Puerto Montt (PM, yellow), Aysén (AY, orange) and Punta Arenas (PA, red).

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Dipturus trachyderma

Microsatellites alleles were analysed for 10 microsatellite loci for 95 individuals. Initial scoring identified three loci, Zch_MS13, Zch_MS10 and Zch_MS29 as monomorphic in this species and was thus excluded from further analysis. Evidence of scoring error was detected due to an excess of homozygotes at two loci (Zch_MS16 in SA and Zch_MS15 in VA). Deviation from HWE (P < 0.05) was observed at these loci and at locus Zch_MS19 in PM. However, no evidence of linkage disequilibrium was detected at any loci.

In general, allelic diversity in D. trachyderma was lower than in Z. chilensis for the same loci ranging from two (Zch_MS6 and Zch_MS15) to six alleles (Zch_MS31 and Dtr_MS8) across all sampled locations (Table 19). Overall, HE and HO values ranged between 0.142 and 0.580. However, the highest and lowest HO and HE values were registered for locus Zch_MS31 and Zch_MS16 respectively

(Table 19). Among sampling locations, the observed number of alleles, HO and HE varied at different loci (Table 20). SA and VA were the most variable in terms of allele number, however most locations had an average of two alleles.

The POWSIM analysis indicated that a pair-wise FST of 0.01 could be detected in 85% whereas a lower FST of 0.0025 could only be detected 19% of the time. Overall, global FST is low and non-significant (FST 0.00951, P-value = 0.143) and pairwise FST test indicated no significant genetic differentiation among sampling locations (-0.035 to 0.041). However, significant differences were found between VA and PM (Table 18). Comparisons of pairwise FST values revealed no significant isolation by distance (IBD) (P = 0.651, r2 =0.040) (Fig. 25B). DAPC analysis grouped all locations into a single cluster in agreement with previous results supporting the lack of population structure of D. trachyderma in Chilean waters (Fig. 26B).

DISCUSSION

This study provides a genetic analysis of populations of Zearaja chilensis and Dipturus trachyderma, two economically important elasmobranch fishes in South America. Mitochondrial and nuclear markers were used to provide insight into connectivity, genetic diversity and population structure along the Chilean coast from San Antonio in the north to Punta Arenas in the south.

The microsatellite loci (nDNA) analysis was not informative enough as expected for a nuclear marker to assess the stock structure of longnose skates. However, it is interesting that mitochondrial genome (mtDNA) provides resolution on population structure, even though mtDNA variation in

–138– CHAPTER V: GENETIC POPULATION STRUCTURE elasmobranchs is notoriously low (Portnoy et al., 2010) and relatively low numbers of SNPs were found in the mtCR of these two longnose skates.

Our data revealed that populations of Z. chilensis might comprise three potential management units, whereas a single-unit harvest management strategy is currently employed in this fishery. For Z. chilensis, there were significant differences among all locations with the exception of Puerto Montt and Aysén. The latter two are located in the Chiloé Interior Sea and hence, might be expected to be a single, relatively isolated population. The pattern of mtDNA haplotype diversity among localities emphasises the likely susceptibility of longnose skates to local overexploitation. At Aysén, only mtCR haplotype Zch_CR2 was found in 100% of the individuals and at Puerto Montt it was found in 96%, emphasising the potential isolation of the Chiloé Interior Sea due to oceanographic features, such as cold subsurface currents, high freshwater contribution from precipitation and river discharges, and high depth profile (50 to 400 m) (Lara et al., 2010). Given the maternal inheritance of mtDNA these data indicate that there is little or no movement of males and females of Z. chilensis into the Chiloé Interior Sea and, as such, population resilience will rely almost exclusively on reproduction and self-recruiting by within- Chiloé Interior Sea adults.

Fuentealba & Leible (1990), reported that longnose skates in the Arauco Gulf (between San Antonio and Valdivia) represented a major fishing resource for the region between 1980 and 1990. However, this fishery is currently non-existent, with an apparent local depletion due to a lack of management leading to the loss of large, breeding females (Fuentealba & Leible, 1990). This outcome provides a cautionary lesson whereby local population recovery is highly dependent on the species’ intrinsic resilience rather than migration from adjacent populations or stocks. In the case of longnose skates, this resilience is considered to be low leading to slow recovery from over-exploitation. The longnose skates population inhabiting Chiloé Interior Sea may be on the brink of collapse if current exploitation levels are maintained.

On the other hand, there were fewer mtDNA breaks in gene flow for D. trachyderma. The main disruption is detected between San Antonio and Valdivia in the north of the sampling area. Interestingly, 13% of specimens from Valdivia possessed a highly divergent haplotype. However, no significant population structure was found with mtDNA or microsatellite markers. Unfortunately, the low sample sizes for the Chiloé Interior Sea and Punta Arenas impeded resolution of population structure, and further research is required to assess the dispersal and connectivity of D. trachyderma along the Chilean coast.

The finding of genetic breaks between populations with mtDNA, but not with microsatellites for Z. chilensis may suggest a lack of power in the microsatellite analysis. The power analysis conducted for this species shows that FST down to 0.01 could be detected with the sample sizes used

–139– CHAPTER V: GENETIC POPULATION STRUCTURE here (n = 50 or less). Indeed, three population pairs were separated by significant FST of this magnitude, but FST lower than 0.01 measured in this study were not significant.

Aspects of the biology of chondrichthyan fishes may have the potential to influence the genetic findings such as the possibility of female regional philopatry to inshore egg-laying grounds. The mtDNA subdivision among locations may have been the result of such movements, with our samples comprising mostly juveniles that result directly from this reproductive behaviour. Natal philopatry is well-recognised among various marine vertebrates including elasmobranch fishes, but while it has been shown definitively for sharks (Hueter et al., 2005; Dudgeon et al., 2012; Feldheim et al., 2014; Chapman et al., 2015; Hoff, 2016), the situation in skates is less clear. Tagging studies have indicated that skates may have the potential to travel hundreds of kilometres (Templeman, 1984; Hunter et al., 2005), however, 90% of individuals had a relatively small home range (within a 100 km2 area) (Walker et al., 1997; King et al., 2010; Farrugia et al., 2016). These observations are consistent with the regional population structure observed for Z. chilensis, although we cannot confirm whether these migrations reflect small-scale movements due to preference and selection of particular habitats (e.g. nursery sites) or to natal philopatry.

Longnose skates are oviparous, producing egg capsules that contain a single embryo which spends weeks in the environment before hatching (Aschliman et al., 2012a; Ishihara et al., 2012). Also, females are able to store sperm, and mating may occur months before the return to proposed egg-laying or nursery grounds (Carrier et al., 2004). The egg capsules of Z. chilensis and D. trachyderma are among the largest in marine fishes (Mabragaña et al., 2011b; Concha et al., 2012) and, probably as a result of this, fecundity is low (Bustamante et al., 2012; Vargas-Caro et al., 2015). The morphology of the egg capsules of both species show adaptations that suggest they are laid on soft substrates, such as coastal sediments or lodged in rocky inshore reefs (Mabragaña et al., 2011b; Concha et al., 2012). Given these biological traits and our results, it is highly probable that mature females do not inhabit in the fishing grounds surveyed along the Chilean coast all year round but move between inshore nursery and offshore “adult” habitats. Mature female longnose skates appear to be transient visitants of coastal areas to lay their egg capsules and it is during these inshore migrations they are particularly vulnerable to fishing activity.

The genotypes from microsatellite loci were not particularly informative regarding the spatial extent of stocks. We believe this is due to mating during the dispersal stage (non-egg-laying), where both sexes move into deeper water away from the coast as well as alongshore to seek suitable feeding habitats. Adult D. trachyderma inhabit deeper waters than Z. chilensis (Bustamante et al., 2014a) and have been rarely sampled in the area, i.e., the egg capsule of D. trachyderma was described from the only mature female observed in Valdivia over a 10-year period of sampling (CB, Pers. Obs.). The

–140– CHAPTER V: GENETIC POPULATION STRUCTURE smaller skate species (Z. chilensis) may have lower dispersal, with corresponding smaller home ranges and without extensive migrations; in contrast, the much larger D. trachyderma might be expected to have a greater dispersal potential. This proposed size of the home ranges is correlated with the pronounced genetic isolation by distance signature for Z. chilensis inferred from the microsatellite data. This relationship occurs when random mating does not occur within the entire population. Instead, there is an increased likelihood of mating among individuals in a local region as a consequence of the constrained dispersal potential of the species.

The set of microsatellite loci used was carefully designed to detect any hybrids between the species, and discard this plausible hypothesis (hybridisation) as individuals of cryptic morphotypes have been reported for Z. chilensis in the past (Leible et al., 1990). The use of the same set of microsatellite loci in both species has confirmed the distinctiveness of the species identity (Fig. 27) and provides an alternative to mtDNA sequencing for accurate species identification of, for example, unidentified ‘skate wings’. The separate species status has also been confirmed by phylogenomic reconstruction using whole mitochondrial genomes for both species (Vargas-Caro et al., 2016a, b).

Figure 27. Scatterplots of the Discriminant Analysis of Principal Components (DAPC) from microsatellites genotypes comparing (A) Zearaja chilensis and (B) Dipturus trachyderma. Dots represent individuals, whereas coloured ellipses correspond to geographical populations. Sampling locations: San Antonio (SA, blue), Valdivia (VA, grey), Puerto Montt (PM, yellow), Aysén (AY, orange) and Punta Arenas (PA, red).

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The synergy between genetic results and our understanding of biological features of longnose skates significantly increases the validity of our findings. For example, the addition of new genetic data has confirmed previous inferences about the reproductive and dispersal biology of longnose skates. The population dynamics uncovered represent an initial step towards an integrated stock and potential population connectivity assessment between South American countries. Our results elucidate new information for the sustainable management of the Chilean skate resources; however, appropriate management and enforcement actions from the Chilean government are needed to safeguard the species.

Our results provide evidence for three management units (San Antonio-Valdivia, Chiloé Interior Sea, and Punta Arenas), and the spatial stock status for Z. chilensis should be considered constrained and separate management arrangements are required for each of the management units detected. Currently, both longnose skates are caught by one single fishery widespread along the Chilean coast but only management measurements are enforced for Zearaja chilensis. There are no grounds to discriminate the extant population of Dipturus trachyderma as separate management units. However, as the fishery cannot positively separate any of the target species (Lamilla et al., 2010), we suggested that the same management units proposed for Z. chilensis to be considered for D. trachyderma following the precautionary principle. The actual fishery management strategy and the low genetic diversity for D. trachyderma, raise concern over the conservation status of this species. The lack of genetic evidence for population subdivision appears to correspond with their higher dispersal ability and more offshore habitat preference.

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–143– CHAPTER VI: DISCUSSION

6. General discussion

In the last two years, the Chilean Fishing Authority has prioritised research of longnose skates following international concerns on sustainability and conservation of these species (Dulvy et al., 2014). As a consequence, the Fisheries Research Fund on behalf of Ministry of Economy has provided funds to conduct the first integrated assessment to determinate population units of the yellownose skate Zearaja chilensis and roughskin skate Dipturus trachyderma. The present research thesis was an expected outcome from that integrated assessment as a novel attempt to fill extant knowledge gaps. The overall aim of this research was to examine aspects of the taxonomy, systematics, ecology and evolution of longnose skates in the southeast Pacific Ocean, as well as to provide management guidelines for a sustainable fishery. Each of the chapters of this thesis addressed particular issues about taxonomy, population genetic structure, genetic diversity and evolutionary history of Z. chilensis and D. trachyderma. Prior to this study, information on all these issues was scarce, incomplete or did not exist, which highlights the findings of this thesis.

Overall, the fishery of longnose skates in Chile remains an important economic resource since its start in 1979. In spite of the historic importance, and the fact that the Chilean people do not consume or trade (internally) any skate-derived product, the fishing effort towards skates has remained continuous and intense; especially after the longnose skates trade was opened to Asian markets in which consumption of skate is an ancestral tradition. From 1980, skate landings increased considerably in Chile, with the highest peak (of 5,193 tonnes) occurring in 2003. The increase in this unregulated but profitable business raised concerns within the Fishing Authority, and a seasonal fishing closure (applied during the Austral summer months) was imposed in 2006 to protect ‘possible reproductive events’ of longnose skates. This closure affected only target fisheries, and was directed towards the small-scale (artisanal) fishery which operates exclusively during months of December to March; however, the not insignificant bycatch by industrial vessels that interact with longnose skates was not considered in this closure. Subsequently, between 2009 and 2011, a total fishing closure was imposed for these skates in Chilean waters in response to a declining trend in the overall catch per unit effort, which included a decline in the size of specimens landed. After 2011, the fishery has been closed and opened again, according to the political weather in Chile. In spite of the efforts made to regulate access, the longnose skates fishery is considered to be ‘fully exploited’ and bycatch quotas are still issued. Information on the stock structure is absent in annual official fisheries reviews, which makes any attempt to manage and protect longnose skates almost impossible.

The current status of knowledge of longnose skates in Chilean waters was discussed through a comparative review (Chapter II). A positive increase in relation to scientific knowledge over the

–144– CHAPTER VI: DISCUSSION past decade is noticeable, although several basic aspects of the biology and ecology of longnose skates are still missing. There is an urgent need to fill knowledge gaps on, for example, movement patterns, feeding ecology and habitat use, population size and structure, and levels of connectivity. In some cases, it was observed that important data have been collected and partially analysed, yet never published. Since it is extremely important to publish and distribute their work following standardised protocols this is a ‘wake-up call’ for researchers. Often, the lack of communication within the scientific community forces researchers to use a variety of ‘novel’ measurements which only impedes comparison between published research and, in some cases, leads to invalid data comparisons and incorrect conclusions. It was also noticed that most of the studies are based on fishery-dependent surveys, which are a fundamental input to stock assessments. However, the fishery-independent surveys used here allowed a more extensive area to be explored, that included traditional commercial trawl zones and non-traditional fishing zones, which provided robust information about the health status of the populations. For example, it is relevant to note that large individuals of D. trachyderma were caught in the same geographical area as the traditional artisanal fishery operates, but at greater depths, i.e., further down the continental slope (Bustamante et al. 2014b). This observation of a deeper depth preference might be the reason why adult D. trachyderma specimens have rarely been reported in the area, i.e., the egg capsule of D. trachyderma was described from the only mature female observed in Valdivia over a 10-year period of sampling (CB, Pers. Obs.). Sampled individuals are only one fraction of a whole population, and hence the status of the un-sampled proportion of a population remains a mystery.

Taxonomic status of longnose skates has been always challenging, especially when landing records are used as a primary source of information, however, scientific efforts over the last decade have aimed to assess, clarify and provide new insights into conservation and fishing management of longnose skates. Confusion of identity between commercial longnose skates occurs to the present day. In order to address this issue, the present research provided morphometric and genetic tools to aid species identification of the early-life stages caught in Chilean fisheries (Chapter III). This thesis provides evidence about the importance of, and difficulty in, the identification of individuals within catch records (e.g., derived from fisheries) of longnose skates. Additionally, insights on the phenotypic plasticity and natural variability of morphological features, often used as diagnostic characters for species identification, are presented. Prior to this study, the presence of a nuchal thorn was considered to be a feature that could be used to separate Z. chilensis from D. trachyderma, and all individuals that lacked this character were identified as D. trachyderma. However, the presence of a nuchal thorn has proven to be variable in Z. chilensis, with one to three thorns observed in landed specimens, and in some instances nuchal thorns were even absent. The identity of specimens without nuchal thorns, but identified as Z. chilensis in the field, was confirmed through molecular methods

–145– CHAPTER VI: DISCUSSION applied a posteriori. The occurrence of specimens of Z. chilensis that lack nuchal thorns had not been reported previously, and identification of this morphological variant is an important outcome from the research. Also, in the case of Z. chilensis, an absence of sexual dimorphism has been reported, based on ratios of body mass to total length, and disc width or disc length to total length, which formed part of an investigation into the reproductive biology of the species (Bustamante et al., 2012). In contrast, sexual differences in body shape were clearly evident in the current study, and highlights that the potential value of using a different set of tools (in this particular case morphometric and meristic data) to address a particular question. The result of the combined methods approach was to successfully separate species and sexes in Z. chilensis, although not for D. trachyderma.

Mitochondrial DNA (mtDNA) is a useful and particularly popular marker in molecular ecology, population genetics, evolutionary biology, phylogeographic and phylogenetic studies of animals. Despite the general assumptions and its wide use, mtDNA sequence can in some cases be an uninformative marker for phylogenetics and species delineation if only portions of the genome are used (Galtier et al., 2009). Improvements in sequencing technologies have made genomic data increasingly available to researchers, and complete mitochondrial genomes have proven to be a particularly useful tool when attempting to alleviate and resolve taxonomic issues. Here, the evolutionary history of Zearaja was explored, in a broad taxonomic context by using whole mitochondrial genomes, to understand higher-level phylogenetic relationships among batoids (Chapter IV). Overall, results showed consensus phylogenomic trees that support previous phyletic reconstructions based on morphological characters, and consequently recovers all extant Orders within the Batoidea in two higher-level reciprocally monophyletic clades: Myliobatiformes + Rhinopristiformes, and Torpediniformes + Rajiformes.

Until recently, molecular data for South American species of longnose skates was scarce. Authors from Chile (Céspedes et al., 2005) and Argentina (Díaz de Astarloa et al., 2008) attempted, to establish patterns in mitochondrial DNA that could confirm the identification of commercially- caught longnose skates. However, where they failed, we found success. The development of species- specific markers for longnose skates in this study is an advance in knowledge that may contribute to future research and fishery management. Also, the current study has provided a molecular baseline of potential future use to compare population structure and genetic flow in Chile, and to assess stock distribution ranges and potential migrations to the Atlantic Ocean.

However, the proposed cladistic analysis is not written in stone and is expected to change in the future, with the addition of new tools and species to the longnose skate pool. Systematic and taxonomy are by definition, changing and challenging sciences that evolves with each discovery leaping forward our understanding of the natural world; and the same is expected to happen in the

–146– CHAPTER VI: DISCUSSION marine realm. Changes in taxonomy of longnose skates have been proposed recently, with the relocation of Dipturus argentinensis to Zearaja on molecular grounds (Last et al., 2016). The absence of comparative material and the lack of molecular data on public repositories, however, raises questions regarding the validity of this change; yet a review on the morphological and molecular traits of longnose skates from the SW Atlantic, is expected to resolve their tangled taxonomy.

The population genetic structure and diversity was also assessed (Chapter V). There was evidence of low genetic diversity for Z. chilensis and even lower for D. trachyderma, which raises concern over the conservation status of these highly exploited species. In Chile, a ‘single-unit’ harvest management strategy is currently employed in which both species are, in combination, treated as a single stock. However, the results presented here revealed that population of Z. chilensis seemingly comprises three potential management units, which should be managed separately in order to safeguard the species. A major finding was the lack of connectivity among individuals from the Chiloe Interior Sea (CIS) and the other two management units, which is of concern for the continuation of the CIS stock given the current levels of exploitation. On the other hand, no significant population structure was found for D. trachyderma with mtDNA analysis or microsatellite markers, and hence the species should be considered a single population. Therein lies a dilemma, as the relative inability to differentiate between the species in the field means that Z. chilensis in proposed management units could inadvertently be over-harvested as bycatch for D. trachyderma. However, further research is required to assess whether the low sample sizes in some locations could have impeded resolution of population structure, and this particular issue could be addressed by monitoring the population using fishery-independent data.

Current status and future directions

The current study has demonstrated that despite the increasing scientific interest in longnose skates over the past decade, there is still much to be achieved. This thesis has identified critical knowledge gaps, and has collected new genetic information allowing for a better understanding of the general ecology of Z. chilensis and D. trachyderma in Chile. However, this research is not just relevant in the Chilean context, as the methodologies used here are replicable and could be of value for researchers that face the similar questions about other species, or the same species in other localities. This study has also provided insights on why the current management strategies used by the Chilean Government might not be sufficient safeguard the species.

In order to protect the longnose skates and avoid ‘extinction due overexploitation’, a total fishing closure was imposed on the entire fishery during 2012. However, the government continues

–147– CHAPTER VI: DISCUSSION to allocate National Catch Quotas in a ‘research’ context. The Asian market still demands skates, and artisanal fishermen see this quota as a legal loophole and an opportunity for profit. According to the Chilean legislation, it is mandatory for fishermen who would like to use the special scientific research quota to be partnered with a university or research institute. However, only a few publications and grey literature (i.e., technical reports) have been produced over the last five-years from those sanctioned scientific research quota projects. The absence of meaningful and verifiable data, or peer- reviewed publications from this ‘research’ completely undermines the primary purpose of this quota – to help protect the long-term viability of the species’ populations. If this landing of longnose skates continues under the apparent guise of research, then it may provide the mechanism, with ‘scientific support’, that leads to fishery collapse.

The results of this thesis have indicated that population collapse of the longnose skates fishery is entirely possible because current management is neither species-specific nor recognizes the different management units of Z. chilensis. Hence, in the light of these new findings, a new biological assessment of Z. chilensis and D. trachyderma is strongly recommended. After consideration of the available literature, the recommendation is that the fishery should remain closed for all fisheries having longnose skates as target species, and extended to fisheries that may interact with longnose skates (i.e., those where longnose skates are among the unreported catches, or specimens are released or returned dead to the sea, or where bycatch and fining occurs), until new management arrangements are discussed, enacted and enforced. Currently, both species are caught in a single, widespread fishery along the Chilean coast, but management measurements are only enforced for Z. chilensis. However, Z. chilensis and D. trachyderma should really be managed as two separate species, and as separate stocks in the case of the former.

The entire population (fishery-dependant and fishery-independent fraction) of both species needs to be surveyed and analysed before granting access to the fishery and establishment of new fishing quotas. Modern, non-lethal methods can be used to survey endangered and conservation concerned species, such as small tissue biopsy collection and tag-release-recapture. Such approaches could be implemented to assess individuals’ movements, growth rates, and survival, as well as to estimate genetic population size and other molecular studies requiring DNA. On the other hand, researchers need to work together with the government and the community, in order to raise awareness about longnose skate conservation. In this matter, and following this year biological closure (April to November 2017) fishing authorities (SUBPESCA) have launched an innovative and colourful campaign to “protect longnose skates”, inviting the community to report illegal catch, landing, processing and storage of products derived from these species.

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Fishery management of longnose skates in South America is still incomplete and obsolete. Imposition of a closure to the artisanal fishery may have a positive impact on skate population recovery. However, bycatch of longnose skates is allowed in artisanal fisheries and have an annual quota, about 10–15% of previous year’s landings (D. Ex. 1838-2017). This quota does not include large industrial vessels that may process their catch at sea – a practise that hinders collection of accurate landing records - and with the current fishery management, allows them to operate on the fringes of the Law. On the other hand, landing of longnose skates in Argentinian waters is legal and is regulated by volumes contrary to several management recommendations (Massa et al., 2004; Crespi-Abril et al., 2013). However, since 2013, skate bycatch was set at 30% of the total catch of a vessel, which seems to aid catch and landings limits of longnose skates in Argentina (Resolución 4, CFP Argentina). Bycatch of longnose skates in Argentina (Cedrola et al., 2005; Tamini et al., 2006, Estalles et al., 2011; Schejter et al., 2012), Uruguay (Silveira et al., 2014) and southern Brazilian waters (Alvarez Perez & Wahrlich, 2005) is reported in coastal and depth water multi-specific fisheries, with similar signs of population collapse for Z. chilensis, as those found in Chile. These signs, such as the decreasing trend on overall landings and the shift towards a smaller maturity size in both sexes of Z. chilensis (have been attributed to the large fishing pressure in the area and the lack of applied research and management (Paesch & Oddone, 2008; Estalles et al., 2011; Crespi-Abril et al., 2013).

Longnose skates, especially Z. chilensis and D. trachyderma, need help to avoid population collapse. A common framework is required to protect and manage populations in Argentina and Chile, but we are a long way from that situation; especially as both countries lack clear enforcement of the conservation priorities in their own National Plans of Action (NPOA). Since 2009, Chile has a NPOA to ensure conservation of sharks and their relatives (SUBPESCA, 2009), however, conservation goals have remained unchanged in spite of the research efforts made to advance this matter.

Overall, the current scenario provides some hope for progress: New tools and novel research methods have been proposed that allow longnose skates’ population ‘health’ to be examined from a new perspective; As researchers, we have established research priorities, and a baseline of biological and molecular information is now available; A significant investment was made by the Chilean Government to have access to this information; The present doctoral research was sponsored by Chile and the main outcomes have been delivered directly to the Fishing Authority. It is hoped that a change in the management strategy for longnose skates in Chilean waters and a recovery strategy will be installed – for a sustainable fishery.

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7. List of References

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Zorzi, G. D. & Anderson, M. E. (1988). Records of the deep-sea skates, Raja (Amblyraja) badia Garman, 1899 and Bathyraja abyssicola (Gilbert, 1896) in the eastern north Pacific, with a new key to California skates. California Fish and Game 74, 87–105.

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EXAMINED MATERIAL (Chapter 4.1)

Genetype: Yellownose skate Zearaja chilensis

Examined material BioVoucher 2014-ZCH-1009 Locality San Antonio, Chile (33° 35.168' S, 71° 37.809' W) Date of collection 27/11/2012 Sex Female Maturity Immature Total length 113.5 cm Diagnosis Disc broader than long, anterior margins undulated. Long and strong rostral cartilage with white-yellow rostrum. Dorsal surface of disc dark brown, ocelli present on each pectoral fin. Dorsal side of disc smooth, denticles irregularly scattered over body, thorns on orbital, rostrum, nuchal and tail. Ventral surface with whitish and brownish areas, reddish brown colour at posterior disc margins; most of disc, pelvic fins and tail pale to medium brown.

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Morphometric and meristic data

Morphometric landmark Size (mm) Morphometric landmark Size (mm) Total length 1135 Mouth width 102 Disc width 785 Distance between nostrils 110 Disc length 617 Nasal curtain length 36 Snout to maximum width 397 Total width of nasal curtain 106 Snout length 189 Width of first gill opening 20 Snout to spiracle 236 Width of fifth gill opening 18 Dorsal head length 248 Distance between first gill openings 202 Cloaca to caudal fin length 380 Distance between fifth gill openings 131 Ventral snout length 178 Length of anterior pelvic lobe 95 Pre-nasal length 163 Length of posterior pelvic lobe 147 Ventral head length 383 Pelvic base width 167 Cloaca to pelvic-clasper insertion 55 Width of tail at axil of pelvic fins 53 Orbit diameter 22 Width of tail at mid-length 34 Orbit and spiracle length 47 Width of tail at first dorsal fin origin 37 Spiracle length 24 First dorsal base 33 Distance between orbits 82 First dorsal fin origin to caudal-fin tip 168 Distance between spiracles 84 Second dorsal fin origin to caudal-fin tip 100 Snout to cloaca length 621 Caudal-fin length 71

Number of thorns in the nuchal area 1 Number of thorns in the disc midline 35 Number of thorns between dorsal fins 3

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EXAMINED MATERIAL (Chapter 4.2)

Genetype: Roughskin skate Dipturus trachyderma

Examined material BioVoucher 2015-DTR-004 Locality Aysén, Chile (45°26'S, 72°56' W) Date of collection 22/02/2014 Sex Female Maturity Immature Total length 192 cm Diagnosis Disc broadly rhombic; anterior margin of disc concave and posterior margin convex with rounded inner corner to level of pelvic fins. Snout hard, long and pointed. Dorsal surface of disc dark brown. Ventral surface as dark as dorsal side in large areas, reddish brown colour at posterior disc margins; gill slits and cloaca creamy white. Dorsal and ventral sides covered by dermal denticles, with exception of a smooth belly.

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Morphometric and meristic data

Morphometric landmark Size (mm) Morphometric landmark Size (mm) Total length 1920 Mouth width 86 Disc width 723 Distance between nostrils 95 Disc length 593 Nasal curtain length 38 Snout to maximum width 374 Total width of nasal curtain 102 Snout length 215 Width of first gill opening 14 Snout to spiracle 251 Width of fifth gill opening 13 Dorsal head length 319 Distance between first gill openings 174 Cloaca to caudal fin length 445 Distance between fifth gill openings 104 Ventral snout length 206 Length of anterior pelvic lobe 106 Pre-nasal length 193 Length of posterior pelvic lobe 132 Ventral head length 348 Pelvic base width 115 Cloaca to pelvic-clasper insertion 53 Width of tail at axil of pelvic fins 36 Orbit diameter 17 Width of tail at mid-length 27 Orbit and spiracle length 37 Width of tail at first dorsal fin origin 29 Spiracle length 19 First dorsal base 29 Distance between orbits 57 First dorsal fin origin to caudal-fin tip 179 Distance between spiracles 62 Second dorsal fin origin to caudal-fin tip 102 Snout to cloaca length 553 Caudal-fin length 73

Number of thorns in the nuchal area 0 Number of thorns in the disc midline 20 Number of thorns between dorsal fins 2

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