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

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.

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

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).

A glimpse to the Chilean populations

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

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

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?

Interesting to know is there is congruence at different levels of the biological organisation: from DNA to phenotypes. Polymorphism at this level could help: Studies at DNA level (exchange 1, Greece): another approach 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.

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. Methodology Third: Assessment of genetic divergence and phylogenetic relationships among populations)

Results 3The size of PCR-amplified mtDNA segment (16SRNA) was c.535 bp. 3Fragment patters generated and size (bp) by each enzyme are showed in Table 1. 3Six different haplotypes (composite genotypes) were found in the populations analysed.

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

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

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

Results A. franciscana ? A. franciscana A. franciscana ? A.franciscana A. franciscana? A.franciscana A. persimilis? A. persimilis Bonaire Neth. Antilles Port Araya, Venezuela Curacao, Neth.Antilles South Arm GSL 2001 GSL, USA Vietnam Vinh Chau, Vietnam SFB, USA GSL, USA SFB, USA Ingebright North, Canada Virrila, Peru Macau, Brazil Laguna Cejas, Chile Chaxas, Chile Salar de Atacana, Chile GSL, USA Torres del Paine, Chile Pichilemu, Chile Argentina 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  ).

unidentified Artemia A. tibetiana A. parthenog. A. urmiana A. parthenog A. parthenog. A. sinica A. salina A. franciscana A. monica A. franciscana? A. persimilis? A. franciscana? A. persimilis Tibet, Tibet Tibet, Madagascar Urmia Lake Aibi Lake Namibia Yuncheng Xiechi Lake Yimeng Tunisia Egypt Larnace GSL, USA Virrila, Peru Curacao, Neth. Antil SFB, USA Vinh Chau, Vietnam. Macau, Brazil Bonaire Neth. Antil. P. Araya,Venezuela Mono Lake, USA Laguna Cejas, Chile. Llamara, Chile Chaxas, Chile Salar de Atacana,Chile. Los Vilos, Chile Torres del Paine, Chile Pichilemu, Chile Argentina 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  ).

unidentified Artemia A. tibetiana A. parthenog. A. urmiana A. parthenog. A. parthenog A. sinica A. franciscana? A. franciscana A. franciscana ? A. franciscana A. monica A. franciscana A. franciscana? A. salina A. persimilis? A. persimilis Tibet, Tibet Tibet, Madagascar Urmia Lake, Iran Namibia Aibi Lake Yuncheng : Xiechi Lake, China. Yimeng, IM Curacao Fulk, Ne. Macau, Brazil SFB, USA Vinh Chau, Vietn. GSL, USA. GSL, USA Virrila, Peru Bonaire Neth. Antil. Port Araya, Vene. Mono Lake, USA Chaxas, Chile Salar de Atacana,Chile. Laguna Cejas, Chile Megrine, Tunisia Egypt LARc Larnace Cy. Torres del Paine,Chile Pichilemu, Chile Argentina 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).

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