Gonzalo GajardoLaboratory 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 togetherwith 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 past2. Morphology3. Ecology (habitat, food, temperature and salinity, preferred substrate 4. Biochemistry and physiology5. Reproductive cycle, chemical and behavioural isolation mechanisms6. 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

The size of PCR-amplified mtDNA segment (16SRNA) was c.535 bp.

Fragment patters generated and size (bp) by each enzyme are showed in Table 1.

Six different haplotypes (composite genotypes) were found in the populations analysed.

AluI A B BfaI A B DdeI A B DpnII A B HaeIII A B

340 --- 535 --- 383 --- 231 --- --- 468 ---260 --- 480 --- 359 --- 180 --- 234 ---195 --- 55 --- 152 --- --- 124 --- --- 234 ---155 --- 25 --- 110 --- 67 --- ---120 --- 70 ---

MspI A NotI A B RsaI A B TaqI A B

535 --- 535 --- 535 --- 261 ---299 --- 331 --- 251 ---236 --- 204 --- 170 ---

161 --- 54 --- --- 38 --- --- 21 --- ---

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 2Training/exchange 2

A 1500 bp mitochondrial rDNA, A 1500 bp mitochondrial rDNA, 12S + 16S12S + 16S

ResultsResults

100

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A. franciscana ?A. franciscanaA. franciscana ?A.franciscanaA.franciscanaA. franciscana A. franciscana A. franciscana A. franciscana A. franciscana A. franciscanaA. franciscanaA. franciscanaA.franciscanaA. franciscana A. franciscanaA. franciscana A. franciscana A. franciscana A. franciscanaA. franciscanaA. franciscanaA. franciscanaA. franciscana?A. franciscana?A. franciscana?A.franciscanaA. franciscanaA. persimilis?A. persimilis?A. persimilis

Bonaire Neth. AntillesPort Araya, Venezuela Curacao, Neth.AntillesSouth Arm GSL 2001GSL, USAGSL, USAGSL, USA GSL, USAGSL, USAVietnamVinh Chau, VietnamVinh Chau, VietnamVinh Chau, VietnamVinh Chau ,VietnamSFB, USAGSL, USASFB, USASFB,USASFB, USAIngebright North, CanadaIngebright North, CanadaVirrila, PeruMacau, BrazilLaguna Cejas, ChileChaxas, ChileSalar de Atacana, ChileGSL, USAGSL, USATorres del Paine, ChilePichilemu, ChileArgentina

285545021520152115081320128615091456145514571454130112581287147214701469140514064791300

15221519

1321

31 samples including 5 Chilean 31 samples including 5 Chilean ArtemiaArtemia samples and references samples of samples and references samples of A. franciscanaA. franciscana and and A. persimilisA. persimilis using using 8 restriction enzymes (Hae 8 restriction enzymes (Hae , Mse , Mse , Hpa , Hpa , Nde , Nde , Taq , Taq , Tsp 509 , Tsp 509 , Hinf , Hinf and Dde and Dde ).).

100

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unidentified Artemiaunidentified ArtemiaA. tibetianaA. parthenog.A. urmiana A. parthenogA. parthenog. A. sinicaA. sinicaA. sinicaA. salinaA. salinaA. salinaA. franciscana A. franciscana A. franciscanaA. franciscanaA. franciscana A. franciscanaA. franciscanaA. franciscana A. franciscanaA. monicaA. franciscana?A. franciscana?A. franciscana?A. franciscana?A. franciscana?A. persimilis?A. franciscana?A. persimilis

Tibet, Tibet Tibet,MadagascarUrmia Lake Aibi Lake Namibia Yuncheng Xiechi LakeYimeng TunisiaEgypt Larnace GSL, USAGSL, USA Virrila, PeruCuracao, Neth. Antil SFB, USAVinh Chau, Vietnam.Macau, Brazil Bonaire Neth. Antil.P. Araya,VenezuelaMono Lake, USALaguna Cejas, Chile.Llamara, Chile Chaxas, ChileSalar de Atacana,Chile.Los Vilos, ChileTorres del Paine, ChilePichilemu, ChileArgentina

1329 1461 1347 1314 1505 1236 1186 1206 1434 1188 1268 1290 1148 1508 1320 479

502 1258 1455 1300 28 554 1277

1321

31 samples including 7 Chilean 31 samples including 7 Chilean ArtemiaArtemia strains and references samples of strains and references samples of A. franciscanaA. franciscana, , A. monicaA. monica, , A. persimilisA. persimilis, , A. A. sinicasinica, , A. salinaA. salina, , A. urmianaA. urmiana, , A. tibetianaA. tibetiana and several parthenogenetic species using 5 restriction enzymes (Hpa and several parthenogenetic species using 5 restriction enzymes (Hpa , Hae , Hae , Nde , Nde , Tsp 509 , Tsp 509 and Taq and Taq ). ).

100

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unidentified Artemiaunidentified ArtemiaA. tibetianaA. parthenog.A. urmiana A. parthenog.A. parthenog A. sinicaA. sinicaA. sinicaA. franciscana?A. franciscanaA. franciscana A. franciscanaA. franciscana A. franciscana A. franciscanaA. franciscana ?A. franciscanaA. monicaA. franciscanaA. franciscanaA. franciscana?A. salinaA. salinaA. salinaA. persimilis?A. persimilis?A. persimilis

Tibet, Tibet Tibet,MadagascarUrmia Lake, Iran Namibia Aibi LakeYuncheng : Xiechi Lake, China.Yimeng, IM Curacao Fulk, Ne.Macau, BrazilSFB, USAVinh Chau, Vietn.GSL, USA.GSL, USA Virrila, PeruBonaire Neth. Antil.Port Araya, Vene.Mono Lake, USAChaxas, ChileSalar de Atacana,Chile.Laguna Cejas, ChileMegrine, TunisiaEgyptLARc Larnace Cy.Torres del Paine,ChilePichilemu, ChileArgentina

1329 1461 1347 1314 1505 1186 1236 1206 1434 1188 502 1300 1258 1455 1508 1320 479

285541277

126812901148

1321

29 samples including 5 Chilean 29 samples including 5 Chilean ArtemiaArtemia strains and references samples of strains and references samples of A. franciscanaA. franciscana, , A. monicaA. monica, , A. persimilisA. persimilis, , A. A. sinicasinica, , A. salinaA. salina, , A. monicaA. monica, , A. urmianaA. urmiana, , A. tibetianaA. tibetiana and several parthenogenetic strains using 6 restriction enzymes (Hae and several parthenogenetic strains using 6 restriction enzymes (Hae , Mse , Mse , Hpa , Hpa , Nde , Nde , Taq , Taq and Tsp 509). 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