INCO project Artemia biodiversity Beijing Workshop, September 23-26, 2002 Artemia characterisation based mainly on mtDNA RFLP analyses and progress in Mediterranean Basin Regional Action A. Triantafyllidis, A.D. Baxevanis, A. Tzika, E. Markatzinou & T.J. Abatzopoulos A RISTOTLE U NIVERSITY OF TH ESSALONIKI (AUTH) INCO Partner: 3

Current research in AUTH – Objectives of INCO project  Visiting scientists  Description of existing gene pools focusing on Artemia populations from: 1.North and South America 2.South Africa 3.Mediterranean Basin  Evolutionary history of parthenogenetic Artemia Molecular markers (identifying clones) Life-history traits  Morphometry Differentiation of populations – Database Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Visiting Scientists  University de Los Lagos, Chile (INCO partner 14)  Rhodes University, South Africa (INCO partner 9) Mr. Julio Crespo 2,5 months Dr. Horst Kaiser 1,5 months Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Molecular techniques used at AUTH  DNA extraction: Phenol extraction, Chelex  PCR amplification of a mitochondrial 16S rRNA region  Analysis of data: REAP, PHYLIP, ARLEQUIN  Restriction Fragment Length Polymorphism (RFLP) analysis with an initial screening of 16 restriction enzymes All analyses were done at INDIVIDUAL level Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Chilean populations analysed in AUTH SAMPLING SITES NUMBER OF INDIVIDUALS Laguna Cejas 27 Laguna Chaxas 25 Los Vilos 26 El Convento 20 Pichilemu25 Torres Del Paine 22 Iquique24 Llamara25 Artemia franciscana ULA 11 Artemia persimilis ULA 12 Artemia franciscana AUTH 12 Artemia persimilis AUTH 11 TOTAL240 Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

UPGMA Dendrogram of Chilean Artemia populations Artemia populations 0.01 EL CONVENTO LLAMARA CHAXAS CEJAS LOS VILOS A. franciscana ULA A. franciscana AUTH IQUIQUE A. persimilis AUTH A. persimilis ULA TORRES DEL PAINE PICHILEMU A. persimilis A. franciscana Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

 Species/population specific markers (restriction enzymes) were identified in order to distinguish: 1. A. fransciscana from A. persimilis 2. Within A. fransciscana Northern from southern populations 3. Within A. persimilis Pichilemu from Torres del Paine population Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Analysis of parthenogenetic and bisexual populations Populations scored by Dr. H. Kaiser Populations studied OriginDescriptionSample size CitrosMacedonia (Greece)Parthenogenetic9 KalloniLesbos (Greece)Parthenogenetic9 M. EmbolonMacedonia (Greece)Parthenogenetic7 MadagascarS. AfricaParthenogenetic10 NamibiaS. AfricaParthenogenetic8 CoegaS. AfricaMixed20 TOTAL63 Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Populations studied OriginDescriptionSample size PolychnitosLesbos (Greece)Parthenogenetic21 KesaniThrace (Greece)Parthenogenetic16 MesiThrace (Greece)Parthenogenetic17 CoegaS. AfricaMixed47 Artemia salinaTunis (Tunisia)Bisexual24 Artemia urmianaUrmia L. (Iran)Bisexual20 TOTAL145 Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China Additional populations characterized at AUTH

Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China Results of restriction analyses AluI BfaI salinakesanimesiurmiamadkalkesanimesiurmiasalinapolich

Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China UPGMA Phylogenetic tree of Artemia haplotypes 0.01 FRANC1 FRANC2 FRANC4 FRANC3 SALINA COEGA1 PARTH1 URMIA PARTH4 PARTH2 COEGA2 PARTH3 PERS2 PERS3 PERS

0.01 El Convento Llamara Chaxas Cejas Los Vilos A. franciscana ULA A. franciscana AUTH Iquique A. salina Coega1 M. Embolon Madagascar Namibia A. urmiana Kalloni Polychnitos Kessani Mesi Coega2 Citros A. persimilis ULA A. persimilis AUTH Torres del Paine Pichilemu franciscana salina parthen. clade A clade B persimilis Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China UPGMA Phylogenetic tree of Artemia populations

 Two major clades were identified within parthenogetic populations No evident geographic structure found. Both clades coexist in nearby or even same site e.g. Polychnitos  A. urmiana genetic fingerprint identical to one of the two clades Genetic distance among these clades is similar to the genetic distance among A. franciscana and A. salina INCO meeting, September, Beijing, China Aristotle University of Thessaloniki (AUTH)

 The Coega population comprises of two different strains: a bisexual and an asexual one. a bisexual and an asexual one. The parthenogenetic strain clusters with one of the two The parthenogenetic strain clusters with one of the two parthenogenetic clades parthenogenetic clades The bisexual strain is grouped with A. salina INCO meeting, September, Beijing, China Aristotle University of Thessaloniki (AUTH)

INCO meeting, September, Beijing, China PCR product obtained from the amplification of mitochondrial 16S rRNA amplification of mitochondrial 16S rRNA Artemia cystsArtemia adult PCR successful even with CYSTS

Discriminant function analysis based on morphometry of bisexual Artemia populations Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China Discriminant function analysis based on morphometry of parthenogenetic Artemia populations (clade A) MAD

Discriminant function analysis based on morphometry of parthenogenetic Artemia populations Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China Contributors  INCO 3: AUTH, Greece A.D. Baxevanis A. Tzika E. Markatzinou A.C. Triantafyllidis T.J. Abatzopoulos  INCO 14: ULA, Chile J. Crespo G. Gajardo  INCO 9: RU, South Africa H. Kaiser T. Hecht

Mediterranean Basin Regional Action Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China  INCO 3: AUTH, Greece, A.D. Baxevanis, A.C. Triantafyllidis, T.J. Abatzopoulos (Moderator)  INCO 4: CSIC, Spain, F. Amat  INCO 5: La Sapienza, Italy, G. Mura  INCO 8: INAT, Tunisia, M. Romdhane (Additional contribution from ARC)

 Description of the present status Mediterranean Basin Regional Action  Cyst hatchability Information on samples existing in cyst banks Sampling data, storage conditions, hatching characteristics  NEW sampling Report on 2 new Greek parthenogenetic populations  Characterization of old and new Artemia populations Use of multidisciplinary approaches (A. salina, Greek parthenogens, Egyptian populations) Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China  Tracking down invasive species

Mediterranean Basin Total number of samples: 384 Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China

Mediterranean Basin Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China Geographical distribution of the recorded samples

Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China Mediterranean Basin (Number of samples per country) Sites not shown Morocco: 1 Portugal: 31 Unknown:

Mediterranean Basin 1. Report of A. fransciscana in Mediterranean Basin (El Estanquillo saltern, Spain)  Invasive species? samples need identification 3. Coexistence of A. salina and parthenogenetic strains Aristotle University of Thessaloniki (AUTH) INCO meeting, September, Beijing, China