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INCO project Artemia biodiversityBeijing Workshop, September 23-26, 2002
ArtemiaArtemia characterisation based mainly on characterisation based mainly on
mtDNA RFLP analyses and mtDNA RFLP analyses and
progress in Mediterranean Basin Regional progress in Mediterranean Basin Regional
ActionAction
A. Triantafyllidis, A.D. Baxevanis, A. Tzika, E. A. Triantafyllidis, A.D. Baxevanis, A. Tzika, E.
Markatzinou & T.J. AbatzopoulosMarkatzinou & T.J. Abatzopoulos
AARISTOTLE RISTOTLE UUNIVERSITY OF NIVERSITY OF THTHESSALONIKIESSALONIKI
(AUTH)(AUTH)INCO Partner: 3INCO Partner: 3
Current research in AUTH – Objectives of INCO projectCurrent research in AUTH – Objectives of INCO project
Visiting scientistsVisiting scientists
Description of existing gene pools focusing on Description of existing gene pools focusing on
Artemia Artemia populations from:populations from:
1.1. North and South AmericaNorth and South America
2.2. South AfricaSouth Africa
3.3. Mediterranean BasinMediterranean Basin
Evolutionary history of parthenogenetic Evolutionary history of parthenogenetic ArtemiaArtemia
Molecular markers (identifying clones)Molecular markers (identifying clones)
Life-history traitsLife-history traits
MorphometryMorphometry
Differentiation of populations – Database Differentiation of populations – Database
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Visiting ScientistsVisiting Scientists
University de Los Lagos, Chile (INCO partner 14)University de Los Lagos, Chile (INCO partner 14)
Rhodes University, South Africa (INCO partner 9) Rhodes University, South Africa (INCO partner 9)
• Mr. Julio Crespo• 2,5 months
• Dr. Horst Kaiser• 1,5 months
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Molecular techniques used at AUTHMolecular techniques used at AUTH
DNA extraction: Phenol extraction, DNA extraction: Phenol extraction, ChelexChelex
PCR amplification of a mitochondrial PCR amplification of a mitochondrial 16S rRNA16S rRNA
regionregion
Analysis of data: REAP, PHYLIP, ARLEQUINAnalysis of data: REAP, PHYLIP, ARLEQUIN
Restriction Fragment Length Polymorphism (RFLP) Restriction Fragment Length Polymorphism (RFLP)
analysis with an initial screening of analysis with an initial screening of 1616 restriction restriction
enzymesenzymes
• All analyses were done at INDIVIDUAL level
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Chilean populations analysed in AUTH Chilean populations analysed in AUTH
SAMPLING SITESSAMPLING SITES NUMBER OF NUMBER OF INDIVIDUALS INDIVIDUALS
Laguna Laguna Cejas Cejas 2727
Laguna Laguna Chaxas Chaxas 2525
Los Los VilosVilos 2626
El El ConventoConvento 2020
PichilemuPichilemu 2525
TorresTorres Del Paine Del Paine 2222
IquiqueIquique 2424
LlamaraLlamara 2525
Artemia franciscanaArtemia franciscana ULAULA 1111
Artemia persimilisArtemia persimilis ULA ULA 1212
Artemia franciscanaArtemia franciscana AUTHAUTH 1212
Artemia persimilisArtemia persimilis AUTH AUTH 1111
TOTAL TOTAL 240240
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
UPGMA Dendrogram of ChileanUPGMA Dendrogram of Chilean ArtemiaArtemia populations 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)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Species/population specific markers (restriction Species/population specific markers (restriction
enzymes) were identified in order to distinguish: 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)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Analysis of parthenogenetic and bisexual Analysis of parthenogenetic and bisexual
populationspopulations
Populations scored by Dr. H. Kaiser Populations scored by Dr. H. Kaiser
Populations studied
Origin DescriptionSample size
CitrosMacedonia (Greece)
Parthenogenetic
9
Kalloni Lesbos (Greece)Parthenogenetic
9
M. EmbolonMacedonia (Greece)
Parthenogenetic
7
Madagascar S. AfricaParthenogenetic
10
Namibia S. AfricaParthenogenetic
8
Coega S. Africa Mixed 20
TOTAL 63Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Populations studied
Origin DescriptionSample size
PolychnitosLesbos (Greece)
Parthenogenetic
21
KesaniThrace (Greece)
Parthenogenetic
16
MesiThrace (Greece)
Parthenogenetic
17
Coega S. Africa Mixed 47
Artemia salinaTunis (Tunisia)
Bisexual 24
Artemia urmiana
Urmia L. (Iran)
Bisexual 20
TOTAL 145Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Additional populations characterized at AUTHAdditional populations characterized at AUTH
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Results of restriction analyses Results of restriction analyses
AluI BfaI
salina kesani mesi urmia madkal kesani mesi urmia salinapolich
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
UPGMA Phylogenetic tree of UPGMA Phylogenetic tree of ArtemiaArtemia haplotypes haplotypes
0.01
FRANC1 FRANC2 FRANC4 FRANC3 SALINA COEGA1 PARTH1 URMIA PARTH4 PARTH2 COEGA2 PARTH3 PERS2PERS3PERS1
98
75
70
95
0.01
El ConventoLlamaraChaxasCejasLos VilosA. franciscana ULAA. franciscana AUTHIquiqueA. salinaCoega1M. EmbolonMadagascarNamibiaA. urmianaKalloniPolychnitosKessaniMesiCoega2CitrosA. persimilis ULAA. persimilis AUTHTorres del PainePichilemu
franciscana
salina
parthen.clade A
clade B
persimilis
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
UPGMA Phylogenetic tree of UPGMA Phylogenetic tree of ArtemiaArtemia populations populations
Two major clades were identified within parthenogetic Two major clades were identified within parthenogetic
populationspopulations
• No evident geographic structure found. Both clades coexist in nearby or even same site e.g. Polychnitos
A. urmianaA. urmiana genetic fingerprint identical to one of the genetic fingerprint identical to one of the
two cladestwo clades
• Genetic distance among these clades is similar to the genetic distance among A. franciscana and A. salina
INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, ChinaAristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH)
The Coega population comprises of two different The Coega population comprises of two different
strains: 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 cladesparthenogenetic clades
• The bisexual strain is grouped with The bisexual strain is grouped with A. salinaA. salina
INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, ChinaAristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH)
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
PCR product obtained from thePCR product obtained from the amplification of mitochondrial 16S rRNAamplification of mitochondrial 16S rRNA
Artemia cysts Artemia adult
PCR successful even with CYSTS
Discriminant function analysis based on morphometry of Discriminant function analysis based on morphometry of bisexual bisexual ArtemiaArtemia populations populations
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Discriminant function analysis based on morphometry of Discriminant function analysis based on morphometry of parthenogenetic parthenogenetic ArtemiaArtemia populations populations (clade A)(clade A)
MAD
MAD
Discriminant function analysis based on morphometry of Discriminant function analysis based on morphometry of parthenogenetic parthenogenetic ArtemiaArtemia populations populations
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
ContributorsContributors
INCO 3: AUTH, INCO 3: AUTH,
GreeceGreece• A.D. Baxevanis• A. Tzika• E. Markatzinou• A.C. Triantafyllidis• T.J. Abatzopoulos
INCO 14: ULA, ChileINCO 14: ULA, Chile• J. Crespo• G. Gajardo
INCO 9: RU, South AfricaINCO 9: RU, South Africa• H. Kaiser• T. Hecht
Mediterranean Basin Regional ActionMediterranean Basin Regional Action
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
INCO 3: AUTH, Greece, A.D. Baxevanis, A.C. INCO 3: AUTH, Greece, A.D. Baxevanis, A.C.
Triantafyllidis, Triantafyllidis, T.J. Abatzopoulos T.J. Abatzopoulos
(Moderator)(Moderator) INCO 4: CSIC, Spain, F. AmatINCO 4: CSIC, Spain, F. Amat INCO 5: La Sapienza, Italy, G. MuraINCO 5: La Sapienza, Italy, G. Mura INCO 8: INAT, Tunisia, M. RomdhaneINCO 8: INAT, Tunisia, M. Romdhane
(Additional contribution from ARC)(Additional contribution from ARC)
Description of the present statusDescription of the present status
Mediterranean Basin Regional ActionMediterranean Basin Regional Action
Cyst hatchabilityCyst hatchability
• Information on samples existing in cyst banksInformation on samples existing in cyst banks
• Sampling data, storage conditions, hatching characteristicsSampling data, storage conditions, hatching characteristics
NEW samplingNEW sampling• Report on 2 new Greek parthenogenetic populations
Characterization of old and new Characterization of old and new ArtemiaArtemia populations populations• Use of multidisciplinary approaches (A. salina, Greek parthenogens, Egyptian populations)
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Tracking down invasive species Tracking down invasive species
Mediterranean Basin Mediterranean Basin
Total number of samples: 384
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Mediterranean Basin Mediterranean Basin
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Geographical distribution of the recorded samplesGeographical distribution of the recorded samples
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China
Mediterranean Basin (Number of samples per country) Mediterranean Basin (Number of samples per country)
Sites not shown Morocco: 1 Portugal: 31 Unknown: 5
1
137
271615
42
18
1
25
6
48
10
Mediterranean Basin Mediterranean Basin
1. Report of A. fransciscana in Mediterranean Basin(El Estanquillo saltern, Spain) Invasive species?
2. 224 samples need identification3. Coexistence of A. salina and parthenogenetic
strains
Aristotle University of Thessaloniki (AUTH)Aristotle University of Thessaloniki (AUTH) INCO meeting, 23-26 September, Beijing, ChinaINCO meeting, 23-26 September, Beijing, China