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Gonzalo Gajardo Laboratory of Genetics & Aquaculture Osorno. Chile

INCO Concerted Action on Artemia Biodiversity. Artemia diversity and evolution in South America: new results and ideas in the framework of the Artemia Biodiversity project. Gonzalo Gajardo Laboratory of Genetics & Aquaculture Osorno. Chile. Thematic partnership.

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Gonzalo Gajardo Laboratory of Genetics & Aquaculture Osorno. Chile

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  1. INCO Concerted Action on Artemia Biodiversity Artemia diversity and evolution in South America: new results and ideas in the framework of the Artemia Biodiversity project Gonzalo Gajardo Laboratory of Genetics & Aquaculture Osorno. Chile

  2. Thematic partnership • Artemia adaptation and speciation: considering genetic and environmental factors. • Following aspects are considered relevant: i) standardisation of methodologies (supply cyst material and published info. For the ARC cyst Bank and project DataBase); ii) develop regional or specific collaborations. Our laboratory gave priority to the standardisation of protocols to produce genetic markers for use in speciation and adaptation. These markers together with other already considered in our studies are expected to broaden the evolutionary scenario of Artemia in South America.

  3. Speciation: a multi-disciplinary and multi-dimensional problem • 1. Geographic distribution, present and past • 2. Morphology • 3. Ecology (habitat, food, temperature and salinity, preferred substrate • 4. Biochemistry and physiology • 5. Reproductive cycle, chemical and behavioural isolation mechanisms • 6. Genetics (allozymes, nuclear (n) and mitDNA), cytogenetics (2n, morphology, karyotype), immunology, hybridization (natural o artificial) White 1978

  4. What is our current knowledge on Artemia from Chile? (1) ecology (physicochemical & biological parameters) (2) biometry (multivariate discriminant analysis) (3) reproduction (reproductive performance & isolation) (4) cytogenetics (chromosomes & chromocentres) (5) electrophoretic studies (allozyme analysis).

  5. A glimpse to the Chilean populations

  6. Chile Iquique Yape y Llamara Antofagasta Salar de Atacama y Chaxa La Serena Los Vilos Pichilemu Rancagua Punta Arenas Torres del Paine

  7. Emphasis on intraspecific genetic change • Speciation: how intra-specific differences turn out into inter-specific differences

  8. A. franciscana • The best studied species so far • expanding from an evolutionary viewpoint • High genetic diversity at coding and non-coding genomic regions • Great inter-population variation in the satellite I region: amplification of heterochromatin or DNA content? • Allopatric mode of speciation relevant?

  9. Studies at DNA level (exchange 1, Greece): another approach Interesting to know is there is congruence at different levels of the biological organisation: from DNA to phenotypes. Polymorphism at this level could help: • To extend the knowledge we previously had of Artemia populations from the New World. • To clarify if Chilean populations belong to the species inhabiting the continent, i.e.A. franciscana or A. persimilis. • To evaluate the genetic divergence, and phylogenetic relationships among them as well as in relation to the other species in the genus. • Increase tool sensitivity to tackle other problems: • influence of environment in promoting genetic differentiation, etc.

  10. Methodology First step: Training on techniques based in DNA extraction, amplification by PCR, and electrophoresis in agarose gels. Second: Standardisation on mtDNA RFLP technique (9 restriction enzyme ) using 5 Chilean populations and 2 reference samples. Third: Assessment of genetic divergence and phylogenetic relationships among populations)

  11. Results • The size of PCR-amplified mtDNA segment (16SRNA) was c.535 bp. • Six different haplotypes (composite genotypes) were found in the populations analysed. • Fragment patters generated and size (bp) by each enzyme are showed in Table 1.

  12. TABLE 1. Fragment patterns observed among Artemia populations in part of the 16SRNA region. Haplotypes in capitals letters and size in bp. ________________________________________________________________

  13. Laguna Cejas Los Vilos Laguna Chaxas El Convento A. f. - ULL A. f. - AUTH Pichilemu A. p. - ULL 0.01 A. p. - AUTH Figure 1. Phylogenetic tree based on the presence or absence of restriction sites.

  14. Training/exchange 2 A 1500 bp mitochondrial rDNA, 12S + 16S

  15. Results 100 50 25 75 A. franciscana ? Bonaire Neth. Antilles 28 A. franciscana Port Araya, Venezuela 554 A. franciscana ? Curacao, Neth.Antilles 502 A.franciscana South Arm GSL 2001 1520 A.franciscana GSL, USA 1521 A. franciscana GSL, USA 1508 A. franciscana GSL, USA 1320 A. franciscana GSL, USA 1286 A. franciscana GSL, USA 1509 A. franciscana Vietnam 1456 A. franciscana Vinh Chau, Vietnam 1455 A. franciscana Vinh Chau, Vietnam 1457 A. franciscana Vinh Chau, Vietnam 1454 A.franciscana Vinh Chau ,Vietnam 1301 A. franciscana SFB, USA 1258 A. franciscana GSL, USA 1287 A. franciscana SFB, USA 1472 A. franciscana SFB,USA 1470 A. franciscana SFB, USA 1469 A. franciscana Ingebright North, Canada 1405 A. franciscana Ingebright North, Canada 1406 A. franciscana Virrila, Peru 479 A. franciscana Macau, Brazil 1300 A. franciscana? Laguna Cejas, Chile A. franciscana? Chaxas, Chile A. franciscana? Salar de Atacana, Chile A.franciscana GSL, USA 1522 A. franciscana GSL, USA 1519 A. persimilis? Torres del Paine, Chile A. persimilis? Pichilemu, Chile A. persimilis Argentina 1321 31 samples including 5 Chilean Artemia samples and references samples of A. franciscana and A. persimilis using 8 restriction enzymes (Hae , Mse , Hpa , Nde , Taq , Tsp 509 , Hinf  and Dde ).

  16. 100 25 50 75 unidentified Artemia Tibet, 1329 unidentified Artemia Tibet 1461 A. tibetiana Tibet, 1347 A. parthenog. Madagascar 1314 A. urmiana Urmia Lake 1505 1434 A. parthenog Aibi Lake 1236 1188 1268 A. parthenog. Namibia 1186 1290 A. sinica Yuncheng 1206 1148 A. sinica Xiechi Lake 1508 A. sinica Yimeng 1320 479 A. salina Tunisia 502 A. salina Egypt 1258 A. salina Larnace 1455 A. franciscana GSL, USA 1300 A. franciscana GSL, USA 28 554 A. franciscana Virrila, Peru 1277 A. franciscana Curacao, Neth. Antil A. franciscana SFB, USA A. franciscana Vinh Chau, Vietnam. A. franciscana Macau, Brazil A. franciscana Bonaire Neth. Antil. A. franciscana P. Araya,Venezuela A. monica Mono Lake, USA 1321 A. franciscana? Laguna Cejas, Chile. A. franciscana? Llamara, Chile A. franciscana? Chaxas, Chile A. franciscana? Salar de Atacana,Chile. A. franciscana? Los Vilos, Chile Torres del Paine, Chile A. persimilis? A. franciscana? Pichilemu, Chile Argentina A. persimilis 31 samples including 7 Chilean Artemia strains and references samples of A. franciscana, A. monica, A. persimilis, A. sinica, A. salina, A. urmiana, A. tibetiana and several parthenogenetic species using 5 restriction enzymes (Hpa , Hae , Nde , Tsp 509  and Taq ).

  17. 100 25 50 75 unidentified Artemia Tibet, 1329 unidentified Artemia Tibet 1461 A. tibetiana Tibet, 1347 A. parthenog. Madagascar 1314 A. urmiana Urmia Lake, Iran 1505 A. parthenog. Namibia 1186 A. parthenog Aibi Lake 1236 A. sinica Yuncheng : 1206 A. sinica Xiechi Lake, China. 1434 A. sinica Yimeng, IM 1188 A. franciscana? Curacao Fulk, Ne. 502 A. franciscana Macau, Brazil 1300 A. franciscana SFB, USA 1258 A. franciscana Vinh Chau, Vietn. 1455 A. franciscana GSL, USA. 1508 A. franciscana GSL, USA 1320 A. franciscana Virrila, Peru 479 A. franciscana ? Bonaire Neth. Antil. 28 A. franciscana Port Araya, Vene. 554 A. monica Mono Lake, USA 1277 A. franciscana Chaxas, Chile A. franciscana Salar de Atacana,Chile. A. franciscana? Laguna Cejas, Chile A. salina Megrine, Tunisia 1268 A. salina Egypt 1290 A. salina LARc Larnace Cy. 1148 A. persimilis? Torres del Paine,Chile A. persimilis? Pichilemu, Chile A. persimilis Argentina 1321 29 samples including 5 Chilean Artemia strains and references samples of A. franciscana, A. monica, A. persimilis, A. sinica, A. salina, A. monica, A. urmiana, A. tibetiana and several parthenogenetic strains using 6 restriction enzymes (Hae , Mse , Hpa , Nde , Taq  and Tsp 509).

  18. Artemia work in Chile and adjacent countries= a significant database at different levels • Morphology • Karyology • Allozyme • Distribution, ecology • Cross-fertility tests • Nuclear & • extra-nuclear DNA

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