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