AUS TRALIAN MUSEUM Biochemi cal Genetics and Stock Assessment of Common Gemf ish and Ocean Perch Final Report , FRDC Project 91/35 John R. Paxton and Donald J. Colgan November 1993 Australian Museum 6-8 Col lege St . Sydney , N.S.W. 2000 tel . 02 339-8111 A Fisheries Research and Development Corporation Project BIOCHEMICAL GENETICS AND STOCK ASSESSMENT OF COMMON GEMF ISH AND OCEAN PERCH Final Report , FRDC Pro ject 91/35 John R. Paxton and Donald J. Colgan November 1993 TABLE OF CONTENTS Table of Contents ..................................2 Sununary ............................................4 Introduction .......................................5 Ob jectives .........................................8 Materials ..........................................8 Methods ............................................9 Results ...........................................14 Discussion ........................................23 Reconunendations ...................................27 Technical Sununary .................................29 Acknowledgements ..................................32 Literature Cited ..................................33 Tables ............................................ 41 Figures ...........................................61 Appendices ........................................6 9 Tables 1. Enzymes scored 2. Morphometrics of Rexea solandri 3. Allozyme frequencies of Rexea solandri by 10 areas 4. Al lozyme frequencies of Rexea solandri by 3 regions 5. Genetic distances of Rexea solandri between 3 regions 6. Fsr statistics of Rexea solandri between 3 regions 7. Migration rate estimates of Rexea solandri between 3 regions 8. Genetic dis tances of Rexea solandri between 10 areas 9. Electrophoretic phenotypes of 3 species of Rexea 10. Mitochondrial DNA restriction digestion haplotypes of Rexea solandri by three regions 11. Mitochondrial DNA restriction digestion fragment sizes of Rexea solandri 12 . Mitochondrial DNA estimated sequence divergences for Rexea solandri 13 . 12S rDNA sequence of Rexea solandri specimen NI409 from New Zealand . 14 . DNA sequence comparison of Eas tern and Western Australian gemf ish 15 . Allozyme frequencies of ocean perch He licolenus percoides Figures 1. Map of gemfish Rexea solandri analysed 2. Map of ocean perch Helicolenus percoides available 3. Morphometrics of Rexea solandri 4. Phenetic relationships of Rexea solandri 5. Southern Blot of Hinfl -digested Rexea solandri mitochondrial DNA 6. Part of DNA sequencing gel for Rexea solandri 7. Map of Rexea solandri stocks and known breeding areas 8. Phenetic relationships of He l icolenus percoides Appendices 1. Covering letter 2. Wanted poster 3. Table of Rexea specimens sampled 4. Table of He licolenus percoides material examined 4 SUMMARY Recognition of separate breeding stocks and/or simi lar species is important if commercial fisheries are to be effectively managed . Two targets of the deepwater trawl fishery in southeas tern Au stralia , the common genfish Rexea solandri and the ocean perch He l icolenus percoides have unanswered questions regarding the number of separate breeding stocks and similar species , respectively . Both species occur around the southern half of Australia and New Zealand . Thi s report describes two years of FRDC funded research on the biochemical genetics of these two fishes in an attempt to resolve these questions . Fresh or frozen specimens were obtained directly from commercial fishers , from the markets, from fisheries biologists in Australia and New Zealand and on research vesse ls . Mus cle , liver and gonad tissues were removed and kept frozen at -80°C until analysed . A series of counts (fin rays , vertebrae ) and measurements of body parts were made on gemf ish from five areas to see if different stocks were morphologically identifiable . A similar morphological study of ocean perch has been undertaken by N.S.W. Fisheries . The biochemical analyses were of three kinds . 1) Enzymes may have a number of distinct forms (called allozymes ) whi ch are genetically-determined . Individuals can differ in their allozymic constitution . The enzymes were solubilized by homogeniz ing the tissue and subjected to electrophoresis , a process which separates allozymes on the basis of their electric charge as shown by differential migration through an inert matrix under an electric field . After electrophoresis , enzyme-specific stains were used to reveal the position of allozymes , enabling the genetic make-up of individual fish to be inferred . 2) The mitochondria of animal cells contain a circular DNA molecule about 15, 000 base pairs in length . Restriction endonuclease enzymes were used to make sequence­ specific cuts in this DNA . Variation between indviduals or populations in the position of these cuts was assayed by Southern Blotting after agarose gel electrophoresis to separate the mtDNA fragments on the basis of their molecular weight . A cloned fragment of lake trout (Salvelin us namaycush ) mtDNA was used as a probe in these experiments. 3) The development of the polymerase chain reaction ("PCR" ) has allowed the production of large amounts of any desired DNA sequence . We applied this technique to a mitochondrial gene of Rexea solandri to provide material for determining the sequence of bases in this segment of DNA . The results of all three of these sets of tests were statistically analysed to indicate how much interbreeding there is between fish from different areas . Gemf ish A total of 288 specimens of common gemf ish were analysed for protein electrophoresis (277), restriction fragment length 5 variation in mtDNA (13 6), DNA sequencing (14) and/or morphometrics (62). The area totals are : N.S.W. and eastern Victoria (79), Tasmania (26), western Victoria , South Australia and the Great Australian Bight (94) the west coast of Western Australia (47) and New Zealand (42). No external differences were found in the counts or body measurements from fish from different regions , although a number of measurements were found to vary with age (head length , snout length , eye diameter ). However, the biochemical results indicate that two dis tinct gemfish stocks occur in Au stralia with almost no interbreeding . All three biochemical analyses gave simi lar results , although the restriction fragment analysis of mtDNA in wh ich all 11 tested endonucleases revealed fixed (or nearly fixed) differences wa s more sensitive than protein electrophoresis where only one of 36 enzyme loci showed a nearly fixed difference . The boundary between the stocks is at the we stern edge of Bass Stra it, with the possibil ity of some limited mixing (and very limited interbreeding) on the west coast of Tasmania. More specimens are needed from this critical area . There is no indication of sub-populations in either the eas tern stock (Byron Bay, N.S.W. to southern Tasmania) or the southern/western stock (Westernport , Victoria to Shark Bay , Western Australia . In contrast, differences between populations from eas tern Australia and New Zealand were observed only in small frequency changes in allo zymes , restriction site presence or DNA sequences . Our conclusion is that there is some level of mixture of the two populations . In theory , low migration levels (<1%) should result in complete population homogenisation . Our results show that this has not occurred , implying that migration between the areas is not at a high enough level to require fisheries managers to treat them as a single stock . Ocean Perch N.S.W. Fisheries biologists have identified a shallow and a deep form of ocean perch . A total of 92 specimens (39 shallow , 53 deep) were obtained . From N.S.W. , there were 34 shallow and 15 deep ; from Tasmania , five shallow and 16 deep ; from Western Australia , 13 deep ; and from New Zealand , nine deep (Fig . 2). Protein electrophoresis wa s conducted on 72 specimens (31 shallow, 41 deep) and mtDNA restriction analysis on 11 specimens (5 shallow, 6 deep) . Our results show that the forms do not interbreed along the east coast of Australia , and that intermixing between populations within forms is limited . DNA analyses of many more specimens from throughout the range wi ll be required to obtain accurate estimates of the extent of this limitation . 6 INTRODUCTION Recognition of separate breeding stocks and differentiation of similar species are important if commercial fisheries are to be effectively managed . Two targets of the deepwater trawl fishery in southeast Australia , the common gemfish Rexea solandri ( Cuvier, 1832) and the ocean perch He licolenus percoides ( Richardson , 1842) , have unanswered questions regarding the number of breeding stocks and the number of similar species , respectively, involved in the commercial fishery . The present study was undertaken , with the financial support of FRDC , to determine if biochemical genetics could be utilised to separate breeding stocks/ species in the two fisheries . To simplify presentation , each section of this report is in two parts , A. Gemf ish and B. Ocean Perch. A. Gemf ish The common gemf ish is one of six species of the Indo-Pacif ic genus Rexea , four of wh ich are found in Australian waters (Parin , 1989; Parin and Paxton , 1990) , and one of 16 Australian species of the family Gempylidae ( Nakamura and Parin , 1993) . Other gempylids of commercial importance in Australia include the barracouta Thyrs ites atun , the escolar Ruvettus pretiosus and , more rarely, the oilfish Lepi docybium f lavobrunneum. The common gemfish has been recorded only from the