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
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