INCO project Artemia biodiversity

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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 ARISTOTLE UNIVERSITY OF
THESSALONIKI (AUTH) INCO Partner 3
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Current research in AUTH Objectives of INCO
project

• Description of existing gene pools focusing on
  Artemia populations from
• North and South America
• South Africa
• Mediterranean Basin

• Evolutionary history of parthenogenetic Artemia
• Molecular markers (identifying clones)
• Life-history traits

• Morphometry
• Differentiation of populations Database

• Visiting scientists

Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Visiting Scientists

• University de Los Lagos, Chile (INCO partner 14)

• Mr. Julio Crespo
• 2,5 months

• Rhodes University, South Africa (INCO partner 9)

• Dr. Horst Kaiser
• 1,5 months

Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Molecular techniques used at AUTH

• DNA extraction Phenol extraction, Chelex

• All analyses were done at INDIVIDUAL level

• PCR amplification of a mitochondrial 16S rRNA
  region

• Restriction Fragment Length Polymorphism (RFLP)
  analysis with an initial screening of 16
  restriction enzymes

• Analysis of data REAP, PHYLIP, ARLEQUIN

Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Chilean populations analysed in AUTH
SAMPLING SITES NUMBER OF INDIVIDUALS
Laguna Cejas 27
Laguna Chaxas 25
Los Vilos 26
El Convento 20
Pichilemu 25
Torres Del Paine 22
Iquique 24
Llamara 25
Artemia franciscana ULA 11
Artemia persimilis ULA 12
Artemia franciscana AUTH 12
Artemia persimilis AUTH 11
TOTAL 240
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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UPGMA Dendrogram of Chilean Artemia populations
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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• 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, 23-26 September, Beijing, China
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Populations scored by Dr. H. Kaiser
Analysis of parthenogenetic and bisexual
populations
Populations studied Origin Description Sample size
Citros Macedonia (Greece) Parthenogenetic 9
Kalloni Lesbos (Greece) Parthenogenetic 9
M. Embolon Macedonia (Greece) Parthenogenetic 7
Madagascar S. Africa Parthenogenetic 10
Namibia S. Africa Parthenogenetic 8
Coega S. Africa Mixed 20
TOTAL 63
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Additional populations characterized at AUTH
Populations studied Origin Description Sample size
Polychnitos Lesbos (Greece) Parthenogenetic 21
Kesani Thrace (Greece) Parthenogenetic 16
Mesi Thrace (Greece) Parthenogenetic 17
Coega S. Africa Mixed 47
Artemia salina Tunis (Tunisia) Bisexual 24
Artemia urmiana Urmia L. (Iran) Bisexual 20
TOTAL 145
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Results of restriction analyses
BfaI
AluI
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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UPGMA Phylogenetic tree of Artemia haplotypes
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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UPGMA Phylogenetic tree of Artemia populations
franciscana
salina
clade A
parthen.
clade B
persimilis
0.01
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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• Two major clades were identified within
  parthenogetic populations

• Genetic distance among these clades is similar
  to the
• genetic distance among A. franciscana and A.
  salina

• 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

INCO meeting, 23-26 September, Beijing, China
Aristotle University of Thessaloniki (AUTH)
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• The Coega population comprises of two different
  strains
• a bisexual and an asexual one.

• The bisexual strain is grouped with A. salina

• The parthenogenetic strain clusters with one of
  the two
• parthenogenetic clades

INCO meeting, 23-26 September, Beijing, China
Aristotle University of Thessaloniki (AUTH)
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PCR product obtained from the amplification of
mitochondrial 16S rRNA
PCR successful even with CYSTS
Artemia cysts
Artemia adult
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Discriminant function analysis based on
morphometry of bisexual Artemia populations
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Discriminant function analysis based on
morphometry of parthenogenetic Artemia
populations (clade A)
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Discriminant function analysis based on
morphometry of parthenogenetic Artemia
populations
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Contributors

• INCO 3 AUTH, Greece

• A.D. Baxevanis
• A. Tzika
• E. Markatzinou
• A.C. Triantafyllidis
• T.J. Abatzopoulos

• INCO 14 ULA, Chile

• INCO 9 RU, South Africa

• J. Crespo
• G. Gajardo

• H. Kaiser
• T. Hecht

Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Mediterranean Basin Regional Action

• 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)

Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Mediterranean Basin Regional Action

• Description of the present status

• Information on samples existing in cyst banks

• Cyst hatchability

• 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)

• Tracking down invasive species

Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Mediterranean Basin
Total number of samples 384
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Mediterranean Basin
Geographical distribution of the recorded samples
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Mediterranean Basin (Number of samples per
country)
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China
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Mediterranean Basin
1. Report of A. fransciscana in Mediterranean
Basin (El Estanquillo saltern, Spain) ? Invasive
species? 2. 224 samples need identification 3.
Coexistence of A. salina and parthenogenetic
strains
Aristotle University of Thessaloniki (AUTH)
INCO meeting, 23-26 September, Beijing, China