Genetic characterisation of Chilean Artemia strains based on RFLPs

Patricia Beristain1, Gonzalo Gajardo1, Peter Bossier2 & Patrick Sorgeloos3

1Laboratory of Genetics & Aquaculture, Universidad de Los Lagos,P.O. Box 933, Osorno, Chile2Departement Zeevisserij, Ministerie van Middenstand en Landbouw, Centrum voor Landbouwkundig Onderzoek-Gent Departement ZeevisserijAnkerstraat 1, B-8400m, Oostende, Belgium3Laboratory of Aquaculture & Artemia Reference Center, Ghent University Rozier 44, B-9000 Ghent, Belgium

1

Abstract

Seven Artemia samples from coastal and inland Chilean lakes and reference samples of the

bisexual New World species, A. franciscana (USA) and A. persimilis (Argentina) were

preliminarily characterised by using the PCR-RFLP fingerprinting technique. The study

considered a conserved 1500bp-mitochondrial rDNA fragment, and eight restriction

enzymes (Hae , Mse , Hpa , Nde , Taq , Tsp 509 , Hinf and Dde ). Results

confirm the presence in Chile of A. franciscana and A. persimilis, as allozyme and

chromosome traits had previously suggested. The first and more inclusive group of

populations, which cluster together with A. franciscana, includes all inland and one coastal

population. Two samples from coastal areas cluster with A. persimilis. Despite the fact that

the mitochondrial rDNA fragment is thought to be rather conservative, inter-individual

variation is high enough to reveal, with few individuals, the inter-population pattern

depicted with more classic techniques requiring larger samples. These markers, which had

been mainly used for systematic purposes so far, will help in evaluating the effect of

particular environments on Artemia gene pools, and monitoring the geographic expansion

of A. persimilis, a species originally restricted to Argentina, and its genetic consequences.

Keywords: Chile; Artemia; Polymerase Chain Reaction (PCR), Restriction Fragment

Length Polymorphism (RFLP), Restriction enzymes.

2

Introduction

The populations of the brine shrimp Artemia found in the American continent are bisexual,

being A. franciscana the dominant and most studied species. Its sibling species in the

continent, A. persimilis, is closely related to the original group of species that evolved in the

Mediterranean area (Abreu-Grobois, 1987; Gajardo et al. 2002). After a probable

colonisation event, A. persimilis settled in Argentina and remained restricted to this part of

the continent up to very recently (Vanhaecke et al., 1986; Triantaphyllidis et al., 1998;

Cohen et al. 1999). At certain point in time A. franciscana originated from A. persimilis

and evolved in complete isolation, hence is considered a derivate state and an expanding

species from an evolutionary viewpoint. It exhibits high genetic diversity at protein coding-

loci (Gajardo & Beardmore, 1989; Zapata et al. 1990) and non-coding genomic regions. For

example, the species shows great inter-population variation in the satellite I region, a repeat

of 113 bp, which is absent in Eurasian populations, and less relevant in A. persimilis

(Gajardo et al. 2001). Artemia franciscana is widely distributed in North, central and South

America, whereas A. persimilis, which used to be considered as a narrowly distributed

species, restricted to Argentina, has been found recently in southern Chile in a very unusual

environment (Gajardo et al. 1998, 1999, 2001).

This work is aimed at understanding at the DNA level, i.e. a 1500 bp mitochondrial rDNA

fragment, the evolutionary pattern of A. franciscana in Chile and adjacent countries. New

information produced at this level is expected to complement the scenario already depicted

by more classic techniques, such as allozymes (Gajardo & Beardmore, 1993; Gajardo et al.

1995, 1999) and cytogenetics (Colihueque & Gajardo, 1996; Gajardo et al. 2001). There are

indications of the occurrence of genomic changes in A. franciscana, such as a gradual

increase in repetitive heterochromatin deduced by the clinal variation in chromocentre

number north and south of the equator. Chromocentres are heavily stained

hetercochromatic areas with highly repetitive DNA, and the intraspecific variation observed

could indicate chromosome reorganisation. Although the function of satellite DNA

associated to chromocentres is unclear, observation in other animals invertebrates, for

example Drosophila, suggest that change in heterochromatin conformation can affect

(silence) wild type genes, and hence directly affect the phenotype.

3

The use of the fingerprinting technique is expected to help in further investigating the

intraspecific genomic change revealed by chromocentres in A. franciscana. It s also

expected in tracking down their effect in relation to environmental conditions, and evaluate

genetic differences in relation to its sibling species A. persimilis.

Material and Methods:

Artemia populations

Adults of Chilean Artemia populations were collected in the field and kept under laboratory

conditions in aquarium containing 5l of artificial seawater. Samples of the following

populations were fixed in ethanol (95 %): Llamara, Chaxas, Laguna Cejas, Salar de

Atacama, Los Vilos, Pichilemu and Torres del Paine (Table 1). Reference samples of A.

persimilis, A. franciscana, A. monica, A. sinica, A. tibetiana, A. urmiana, A. salina and

some parthenogenetic types, were kindly provided as cysts by the Laboratory of

Aquaculture & Artemia Reference Center, Ghent, Belgium.

DNA extraction

a) Adults samples

Different protocols were utilised in order to extract DNA such as: Wizard Genomic DNA

Purification kit (Promega, USA) combined with phenol and chloroform-isoamyl alcohol

(1:24); CTAB buffer (hexadecyltrimethylammonioum bromide); Chelex compounds, and

SDS buffer (sodium dodecyl sulfate) with a saline EDTA solution (SE) (750mM NaCl; 250

mM Na2EDTA). Each individual was crushed on ice, with sterile material, with 500µl SE

and 5µl of Tris (pH 8, 50mM). After centrifugation, 20µl of proteinase -K (10mg/ml) and

50µl SDS (10%) were added to the sample. After 30 min. of incubation at 65°C, the sample

was extracted with 250 µl of phenol and 250 µl chloroform-isoamyl alcohol (24:1) as well

as with 250μl chloroform-isoamyl alcohol (24:1). After centifugation (10.000 rpm, 5 min)

650µl of 100% ethanol (EtOH) was added to the supernatant and DNA precipitated at –

20°C during 2 hours. EtOH was removed and isopropanol (80%; -20°C) was used to rinse

each sample. The pellet obtained was dried at room temperature and the DNA obtained was

resuspended by using 50µl of sterilised and deionised water.

4

b) CystFor each strain 10 mg of crushed cysts were treated with 600µl CTAB buffer

(hexadecyltrimethylammonioum bromide: Tris-HCl 100mM, pH 8.0, NaCl 1.4M, EDTA

20mM, CTAB 2%) and incubated in a thermal bath at 60°C for 30 min. After centrifugation

(14.000rpm, 15 min), the supernatant was treated with phenol and chloroform-isoamyl

alcohol (1:24). The samples were treated again adding 300l chloroform-isoamyl alcohol

(24 :1) individually, then shaken during 2 minutes and centrifuged at 14.000 during 15

minutes. The supernatant was treated with 2.0 2.5 fold of the sample volume of EtOH

100% then kept at –20°C overnight. After centrifuge, at 14.000 rpm during 15 minutes, the

EtOH was poured off and the pellet were rinsed with 300l of 70% EtOH. The EtOH was

poured away and the pellets were dried at room temperature and then dissolved in 25 µl in

distilled water overnight (4°C).

Polymerse chain reaction (PCR)

PCR was performed in a Hybaid PCR express machine. The amplification reactions were

carried out in a final volume of 50l containing a mixture of 50-100 ng of gDNA, 5mM

Tris-HCl (pH 8.3), 2.5 mM Mg Cl2, 0.2 mM dNTP’s, 1.75 I.U DNA polymerase mixture

(Expand high Fidelity PCR System, Roche Molecular Biochemicals) and 25M of

12S-SP (5’- CTAGGATTAGATACCCTA- 3’) and 16S-SP (5’-

CCGGTCTGAACTCAGATC- 3’) primers. Another pair of primers, 12S-R and 16S-F

were utilised in order to get effective and successful DNA amplification reactions. In both

cases, PCR was initiated with a simulated hot start at 94°C during 2 min, continued with 34

cycles 1 min and 15 sec at 94°C, 45 sec at 52°C, 2 min at 72°C and ended with an

extension at 72°C during 4 min.

Endonuclease digestion of DNA

To purify PCR products the Wizard DNA purification System (Promega) was utilised and

the following eight restriction enzymes were employed in a final volume of 25 µl.,

according to the manufacture suggested protocol, to display DNA polymorphism: Hae ,

Tru 9 , Hpa , Nde , Taq , Tsp509 , Hif and Dde . Purified products were checked

using agarose gel.

5

Data analysis

Digested products were electrophoretically analysed through an agarose gel (2.5%) in 1x

TAE buffer (Tris acetate EDTA) during 7 hours at 80V, visualised with ethidium bromide

staining and photographed on a UV table, using a Polaroid camera and Polaroid negative

black and white film 667. A 100-bp ladder (Promega) was loaded as a reference. The

images, scanned by a flatbed scanner (HP Scanjet cx) were processed with the

Gelcompar software (Applied Math, Kortrijk, Belgium). The UPGMA (unweighted

pair-group method with arithmetic averages) dendogram of composite matrix of Dice

distance were constructed based on the RFLP polymorphism using the same software.

Results

Mitochondrial DNA from all Chilean Artemia strains was digested using 5 restriction

enzymes, whereas five samples (SAT, CEJ, CHA, PIC and TPA) were treated with 8

restriction enzymes (RE). Nevertheless, the pattern emerging from the RFLP analysis is

quite consistently, irrespective of the number of restriction enzymes considered. Figure 1

shows the dendrogram based on eight RE for five Chilean Artemia strains (CHA, CEJ,

SAT, PIC and TPA) and references samples of A. franciscana and A. persimilis. The

American bisexual populations cluster in two groups. The first one corresponds to A.

franciscana, which is subdivided in several sub-groups, with strains from USA and

Vietnam (a recent introduction of A. franciscana) displaying high similarity. Inland Chilean

populations are very close to A. franciscana, the highest similarities being between CHA

and CEJ (83%) while similarity tend to decrease between either two and SAT (72%). Two

coastal populations from the central (Pichilemu) and the southern-most region (Torres del

Paine) (separated by about 1500 Kilometers), group together with A. persimilis. While both

strains exhibit close to 85 % of similarity, in spite of their geographical distance, they share

over 55% of similarity with A. persimilis.

Although Caribbean samples from Venezuela and Netherlands Antilles clustered with A.

franciscana, they are divergent groups. Likewise, although samples from Brazil and Peru

are within A. franciscana, they are genetically divergent.

6

Dendogram of Fig. 2, based on 5 RE, shows the relationship between the Chilean samples

and all Artemia species, including parthenogentic types. This picture confirms A.

franciscana as the most common species in the area. The highest similarities are among

inland populations from northern Chile such as CHA and SAT (higher than 90%) and

between them and CEJ (82%). One coastal population (LVI) found further south also

cluster with A. franciscana though with a lower similarity (64%). On the other hand, two

other coastal strains found at latitude X South (PICH) and y south (TPA) share high

similarity to A. persimilis. The comparison of South American strains, including the

Chilean ones, show that A. franciscana and A. persimilis are the closest relatives, whereas

all other diverge significantly.

Discussion

During the last years several efforts have been performed to characterise South American

Artemia populations by means of different techniques, i.e. morphometric, cytogenetic,

allozyme and cross-breeding analysis. These studies confirmed the presence of the bisexual

species A. franciscana and A. persimilis in the continent, and have shown great genetic

heterogeneity among A. franciscana populations, which is considered an expanding species

from an evolutionary viewpoint (see Gajardo et al. 2002 for a review). The use of different

DNA-based tools developed in recent years offers opportunities for fine-tunning previous

findings at other level of the biological organisation. This preliminary work in which we

standardised RFLP markers for the Chilean populations is aimed at evaluating our previous

findings based on less sensitive tools. Hence we compared the Chilean samples with A.

franciscana and A. persimilis, which are the two species found in the continent, to test if the

former is the dominant species in the continent. Similarly, we sought confirmation of the

presence of A. persimilis in Chile as previously reported, and to evaluate the genetic

relationship of the Chilean and some other south American samples with regard to other

Artemia species. Until very recently the idea that A. franciscana predominated in the

Americas with the exception of Argentina, whilst A. persimilis was restricted to Argentina

was common in the Artemia literature (Abreu-Grobois, 1987). Nevertheless, the finding of

A. persimilis in Chile (Gajardo et al. 1999, 2001) and A. franciscana in Argentina shows

that the Andes barrier could be not so effective as a natural barrier between the two

7

countries. The use of RFLP markers and others are likely to provide more reliable

information in this regard. Hence in this work we studied in a 1500 bp mitochondrial rDNA

fragment which is quite conservative according to XXX. Results at this level roughly

confirm our previous findings. In northern Chile, between 21°-23°S latitude, amid the

Atacama desert, A. franciscana is the sole species present, though divergence with samples

from other latitudes is evident. Based on allozymes Gajardo et al (1995) reported high

degree of allozyme divergence among certain Chilean populations and between them and

the San Francisco Bay sample from which most of the South American localities could

have been derived. Genetic distance values were reported to be at the upper limit for

conspecific populations observed for Artemia by Abreu-Grobois (1987). In this work,

populations from the north which are mainly found in inland salares, appear quite

genetically similar, and this is likely to be related to their geographic proximity and

similarity of water characteristics (Gajardo et al., 1992, 1998; Zuñiga et al., 1999). Further

south Artemia habitats are coastal, and often ephemeral ponds as it is the case of Los Vilos.

In spite of the habitat differences and geographical distance to northern samples of this

location, it appears quite similar or divergent to populations in the north. Instead, a sample

from Central Chile (Pichilemu) (34º25’S; 72º10’W) and one in the extreme south (Torres

del Paine) (50º29’S; 73º45’W) cluster with A. persimilis. This finding had been previously

reported for Pichilemu based on cytogenetic data (Colihueque & Gajardo, 1996) and for

Torres del Paine in an allozyme study (Gajardo et al., 1999).

Samples from Caribbean countries form subclusters in the bigger A. franciscana cluster as

also was reported previously (Camargo et al., 2002), as well as morphometric similarities

among them and differences between these and A. persimilis (Hontoria and Amat, 1992).

Peruvian and Brazilian strains are A. franciscana (introduced by artisanal saltworker

several years ago) seem to have evolve quite fast respect to the parental strain

The RFLP fingerprinting technique applied on a 1500 bp mitochondrial rDNA fragment

resulted in a powerful tool to group Artemia strains. Despite the rDNA from the

mitochondrion is a rather conservative marker, it showed highly variable to the point that

dendrogrames based on one individual reconstruct the genetic relationships obtained with

more traditional approaches usually based on larger samples.

8

GelCompar II results enable to compare large numbers of complex patterns in a short

period of time. However, the program is not completely automatic but require the use to

make critical decisions that affect the way in which the analysis is done and the final

results. It may be useful to include DNA from at least two identical strains on each gel to

verify the ability of the software to recognise identical patterns at the chosen settings. It

should be stressed that computer programs may be used as an aid in the analysis of complex

banding patterns; they do not provide an undisputedly correct analysis (Gerner-Smith et al.,

1998).

9

References:

Abreu-Grobois, F.A., 1987. A review of the genetics of Artemia. In Sorgeloos,P., D.A. Bengstone, W. Decleir & Jaspers (eds), Artemia Research and its applications. Vol 1. Universa Press, Wetteren, Belgium 1: 61-69.

Camargo, W., Bossier, P., Sorgeloos, P., Sun, Y., 2002. Preliminary genetic data on some Caribean Artemia franciscana strains based on RAPD’s. Hydrobiologia 468, 245-249.

Cohen, R., Amat, F., Hontoria, F., Navarro, F., 1999. Preliminary characterization of some Argentinean Artemia populations from La Pampa and Buenos Aires province. International Journal of Salt Lake Research 8, 329-340.

Colihueque, N., Gajardo, G., 1996. Chromosomal analysis in Artemia populations from South America. Cytobios 88, 141-148.

Gajardo, G., Beardmore, J., 1989. Ability to switch reproductive mode in Artemia is related to maternal heterozygosity. Mar. Ecol. Prog. Ser. 55, 191-195.

Gajardo, G., Wilson, R., Zuñiga O., 1992. Report on the occurrence of Artemia in a saline deposit of the Chilean Andes (Branchiopoda, Anostraca). Crustacea 63 (2), 169-174.

Gajardo, G., da Conceicao, M., Weber, L., Beardmore, J., 1995. Genetic variability and interpopulational differentiation of Artemia strains from South America. Hydrobiologia 301, 21-29.

Gajardo, G., Colihueque, N., Parraguez, M., Sorgeloos, P., 1998. International Study on Artemia LVIII. Morphologic differentiation and reproductive isolation of Artemia populations from South America. International Journal of Salt Lake Research 7, 133-151.

Gajardo, G., Mercado, C., Beardmore, J., Sorgeloos, P., 1999. International study on Artemia. Allozyme data suggest that a new Artemia population in southern Chile (50°29’S; 73°45’W) is A. persimilis. Hydrobiologia 405, 117-123.

Gajardo, G., Parraguez, M., Beardmore, J., Sorgeloos, P., 2001. Reproduction in the brine shrimp Artemia: evolutionary relevance of laboratory cross-breeding tests. J. Zool., Lon. 253, 25-32.

Gajardo, G., Beardmore, J. and Sorgeloos, P., 2001. International study on Artemia. LXII. Genomic relationship between Artemia frannciscana and A. persimilis, inferred from chromocentre numbers. Heridity 87, 172-175.

Gerner-Smidt, P., M. Graves, L.M., Hunter, S. Swaminathan, B., 1998. Computerized Analysis of Restriction Fragment Length Polymorphism Patterns: Comparative Evaluation of Two Commercial Software Packages. Journal of Clinical Microbiology, 36(5), 1318-

10

1323.

Hontoria, F., Amat, F., 1992. Morphological characterization of adult Artemia (Crustacea, Branchiopoda) from different geographical origins, American populations. Journal of Plankton Research 14 (10), 1461-1471.

Triantaphyllidis, G., Abatzopoulos, T., Sorgeloos, P., 1998. Review of the biogeography of the genus Artemia (Crustacea, Anostraca). Journal of Biogeography 25, 213-226.

Vanhaecke, P., Tackaert, W., Sorgeloos, P., 1987. The biogeography of Artemia: an updated review. Artemia Research and its Applications. Vol. I. Morphology, genetics, strain characterization, Toxicology. P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers (Eds). Universa Press, Wetteren, Belgium. 380 pp.

Zapata, C., Gajardo, G., Beardmore, J., 1990. Multilocus heterozygosity and sexual selection in the brine shrimp Artemia franciscana. Mar. Ecol. Prog. Ser. 62, 211-217.

Zuñiga, O., Wilson, R., Amat, F., Hontoria, F., 1999. Distribution and characterization of Chilean populations of the brine shrimp Artemia (Crustacea, Branchiopoda, Anostraca). International Journal of Salt Lake Research 8, 33-40.

11

Table 1: Chilean Artemia populations, abbreviations and geographical coordinates, used during this study.

Figure 1: The UPGMA dendogram obtained from RFLP analysis of mitochondrial rDNA on 29 samples including 5 Chilean Artemia strains and references samples of A. franciscana and A. persimilis, using 8 restriction enzymes (Hae , Mse , Hpa , Nde , Taq , Tsp 509 , Hinf and Dde ).

Figure 2: The UPGMA dendogram obtained from RFLP analysis of mitochondrial rDNA on 31 samples including 7 Chilean Artemia strains and references samples of A. franciscana, A. monica, A. persimilis, A. sinica, A. salina, A. urmiana, A. tibetiana and several parthenogenetic species using 5 restriction enzymes (Hpa , Hae , Nde , Tsp 509 and Taq ).

Figure 3: The UPGMA dendogram obtained from RFLP analysis of mitochondrial rDNA on 29 samples including 5 Chilean Artemia strains and references samples of A. franciscana, A. monica, A. persimilis, A. sinica, A. salina, A. monica, A. urmiana, A. tibetiana and several parthenogenetic strains using 6 restriction enzymes (Hae , Mse , Hpa , Nde , Taq and Tsp 509).

Figure 4: The UPGMA dendogram obtained from RFLP analysis of mitochondrial rDNA on 29 samples including 5 Chilean Artemia strians and references samples of A. franciscana, A. monica, A. persimilis, A. sinica, A. salina, A. monica, A. urmiana, A. tibetiana and several parthenogenetic strains, using 8 restriction enzymes (Hae , Mse , Hpa , Nde , Taq , Tsp 509 , Hinf and Dde ).

12

Locality Abbreviation Geographical co-ordinates Type of habitat

Llamara LLA 21º18’S; 69º37’W Inland

Chaxas CHA 22º47’S; 67º58’W Inland

Laguna Cejas CEJ 23º02’S; 68º13’W Inland

Salar de Atacama SAT 23º10’S; 68º10’W Inland

Los Vilos LVI 31º58’S; 71º25’W Coastal

Pichilemu PIC 34º25’S; 72º10’W Coastal

Torres del Paine TPA 50º29’S; 73º45’W Coastal

13

14RFLP HaeIII+RFLP mseI+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I+RFLP HinfI+RFLP DdeI8 RE Chile+francisc

100

959085807570656055504540353025

A. franciscana ?

A. franciscana

A. franciscana ?

A.franciscana

A.franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A.franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana?

A. franciscana?

A. franciscana?

A.franciscana

A. franciscana

A. persimilis?

A. franciscana?

A. persimilis

Bonaire Neth. Antilles

Port Araya, Venezuela

Curacao Fulk, Neth.Antilles

South Arm GSL 2001

North Arm GSL 2000

GSL '99 ( N.A.) >90%

GSL, USA

GSL, USA

GSL '00 ( N.A.) DOUG 11038

Vietnam

Vinh Chau, Vietnam

Vinh Chau, Vietnam

Vinh Chau, Vietnam

Vinh Chau , Vietnam(ISA study)

SFB, USA

GSL, USA

SFB location 5, USA

SFB location 3, USA

SFB location 2, USA

Ingebright North, Canada

Ingebright North, Canada

Virrila, Peru

Macau, Brazil (ISA study)

Laguna Cejas, Chile

Chaxas, Chile

Salar de Atacana, Chile

South Arm GSL 1995

GSL, USA

Torres del Paine, Chile

Pichilemu, Chile

Argentina

28

554

502

1520

1521

1508

1320

1286

1509

1456

1455

1457

1454

1301

1258

1287

1472

1470

1469

1405

1406

479

1300

1522

1519

1321

15

RFLP HaeIII+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I

100

95908580757065605550454035302520

unidentified Artemia

unidentified Artemia

A. tibetiana

A. parthenog. (3n)

A. urmiana

A. parthenog

A. parthenog. (2n)

A. sinica

A. sinica

A. sinica

A. salina

A. salina

A. salina

A. franciscana

A. franciscana

A. franciscana

A. franciscana ?

A. franciscana

A. franciscana

A. franciscana

A. franciscana ?

A. franciscana

A. monica

A. franciscana?

A. franciscana?

A. franciscana?

A. franciscana?

A. franciscana?

A. persimilis?

A. franciscana?

A. persimilis

Tibet,

Tibet - Bozi Co

Tibet, China

Madagascar

Urmia Lake 2001

Aibi Lake

Namibia 2n

Yuncheng :

Xiechi Lake, PR .

Yimeng, IM

Megrine, Tunisia

Egypt

LARc Larnace Cy.

GSL '99 ( N.A.) >.

GSL, USA

Virrila, Peru

Curacao Fulk, Ne.

SFB, USA

Vinh Chau, Vietn.

Macau, Brazil (IS.

Bonaire Neth. Antil.

Port Araya, Vene.

Mono Lake, USA

Laguna Cejas, Chi.

Llamara, Chile

Chaxas, Chile

Salar de Atacana,.

Los Vilos, Chile

Torres del Paine,.

Pichilemu, Chile

Argentina

1329

1461

1347

1314

1505

1236

1186

1206

1434

1188

1268

1290

1148

1508

1320

479

502

1258

1455

1300

28

554

1277

1321

16RFLP HaeIII+RFLP mseI+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I

100

959085807570656055504540353025

unidentified Artemia

unidentified Artemia

A. tibetiana

A. parthenog. (3n)

A. urmiana

A. parthenog. (2n)

A. parthenog

A. sinica

A. sinica

A. sinica

A. franciscana ?

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana ?

A. franciscana

A. monica

A. franciscana?

A. franciscana?

A. franciscana?

A. salina

A. salina

A. salina

A. persimilis?

A. franciscana?

A. persimilis

Tibet,

Tibet - Bozi Co

Tibet, China

Madagascar

Urmia Lake 2001

Namibia 2n

Aibi Lake

Yuncheng :

Xiechi Lake, PR .

Yimeng, IM

Curacao Fulk, Ne.

Macau, Brazil (IS.

SFB, USA

Vinh Chau, Vietn.

GSL '99 ( N.A.) >.

GSL, USA

Virrila, Peru

Bonaire Neth. Antil.

Port Araya, Vene.

Mono Lake, USA

Chaxas, Chile

Salar de Atacana,.

Laguna Cejas, Chi.

Megrine, Tunisia

Egypt

LARc Larnace Cy.

Torres del Paine,.

Pichilemu, Chile

Argentina

1329

1461

1347

1314

1505

1186

1236

1206

1434

1188

502

1300

1258

1455

1508

1320

479

28

554

1277

1268

1290

1148

1321

17

RFLP HaeIII+RFLP mseI+RFLP HpaII+RFLP NdeII+RFLP TaqI+RFLP Tsp509I+RFLP HinfI+RFLP DdeI

100

959085807570656055504540353025

A. parthenog. (3n)

A. urmiana

A. parthenog. (2n)

A. parthenog

unidentified Artem.

unidentified Artem.

A. tibetiana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. franciscana

A. monica

A. franciscana ?

A. franciscana

A. franciscana ?

A. franciscana?

A. franciscana?

A. franciscana?

A. salina

A. salina

A. salina

A. sinica

A. sinica

A. sinica

A. persimilis?

A. franciscana?

A. persimilis

Madagascar

Urmia Lake 2001

Namibia 2n

Aibi Lake

Tibet,

Tibet - Bozi Co

Tibet, China

SFB, USA

Vinh Chau, Vietnam

GSL '99 ( N.A.) >90%

GSL, USA

Virrila, Peru

Macau, Brazil (ISA st.

Mono Lake, USA

Bonaire Neth. Antilles

Port Araya, Venezuela

Curacao Fulk, Neth.A.

Chaxas, Chile

Salar de Atacana, Chi.

Laguna Cejas, Chile

Megrine, Tunisia

Egypt

LARc Larnace Cyprus

Yuncheng :

Xiechi Lake, PR China

Yimeng, IM

Torres del Paine, Chile

Pichilemu, Chile

Argentina

1314

1505

1186

1236

1329

1461

1347

1258

1455

1508

1320

479

1300

1277

28

554

502

1268

1290

1148

1206

1434

1188

1321