Genetic variability and interpopulational differentiation of Artemia strains from South America

Seven Artemia samples from three South American countries (Chile, Brazil, Peru) were studied by starch electrophoresis with the aim of comparing levels of genetic variation and genetic similarity to representative populations of A. franciscana (San Francisco Bay, California, USA) and A. persimilis (Buenos Aires, Argentina), which are species endemic to the New World. Based on the analysis of 22 loci, parameters measuring genetic variability were, for some populations, found to be among the highest reported for Artemia so far. The percentage of polymorphic loci varied from 31.8% (Piura, Peru; Buenos Aires) to 50% (Los Vilos and Salar de Atacama, Chile), while the observed heterozygosity varied from 0.025 (Piura) to 0.165 (Los Vilos, Chile). A dendrogram based on Nei's genetic distance (D) produced four major groups. The Argentinian form, A. persimilis; the San Francisco Bay strain together with samples from Brazil (Macau and Rio Grande do Norte) and Chile (Pichilemu and Salar de Atacama); two coastal populations from Chile (Los Vilos and Iquique) and the sample from Peru (Piura). These four groups have inter-group D values that are, in some cases, far above those normally associated with conspecific populations.     

Species identification of Chilean Artemia populations based on mitochondrial DNA RFLP analysis

Aim Two species of the brine shrimp, namely Artemia franciscana Kellogg and A. persimilis Piccinelli and Prosdocimi, inhabit Chile. Most studies so far have shown that A. franciscana is the most widely distributed species in Chile, with A. persimilis present only in Chilean Patagonia. In general, there is good agreement between morphological and genetic comparisons of Chilean populations with respect to species discrimination. However, a number of results indicate an overlap with some populations tending to diverge from A. franciscana and/or resembling A. persimilis. Prior to the mid 90's the use of DNA markers in Artemia was rather limited, despite their successful application in numerous other species. In this study, we investigate whether the conclusions drawn from traditional comparative tools are congruent with the pattern of genetic divergence depicted by DNA analysis at the mitochondrial level. Location Eight sites in Chile and two reference samples of A. franciscana and A. persimilis from San Francisco Bay (USA) and Buenos Aires (Argentina), respectively. Methods Restriction fragment length polymorphism (RFLP) analysis of a 535 bp segment of the mitochondrial 16S rRNA gene with nine restriction enzymes in 240 individuals. Results No haplotype was shared between the two species. Five restriction enzymes produced species‐specific patterns, enabling the unambiguous assignment of populations to species. Very high (100%) bootstrap values supported the clustering of haplotypes in two groups corresponding to the two species. The two species were clearly differentiated with average sequence divergence of 12.3%. High genetic differentiation was also found among con‐specific populations of A. franciscana with an F_ST estimate of 91%. Main conclusions The mitochondrial DNA (mtDNA) results of this study show a broadly similar pattern to those of previous allozyme and nuclear DNA analyses, with the two New World species appearing as highly divergent. The presence of A. persimilis in southern Chile (Chilean Patagonia) was confirmed. Hence, a species previously regarded as geographically restricted mainly to Argentina, appears to have expanded its range. Populations of A. franciscana appear highly structured with a level of inter‐population genetic differentiation much higher for mtDNA than previously reported with allozymes. Clustering of these populations does not follow a clear geographic pattern. The identification of population‐specific genetic markers for A. persimilis and A. franciscana will help to tackle further aspects of the speciation patterns of these species.     

Genetic characterization and intra-generic relationships of Artemia monica Verrill and A. urmiana Günther

Allozyme variation encoded for by 22 enzyme loci analyzed with starch-gel electrophoresis from samples of gonochoristic Artemia monica and A. urmiana collected from Lake Urmia, Iran and from Mono Lake, CA, USA, respectively, are compared and contrasted with data from representative gonochoristic populations of A. franciscana, A. persimilis and A. salina, as well as diploid, triploid, tetraploid, and pentaploid parthenogenetic forms. Values for parameters measuring degree of genetic variability (number of alleles per locus, proportion of polymorphic loci, and expected heterozygosity) in A. monica and A. urmiana were among the highest in the genus (1.76, 0.46, 0.19, and 2.19, 0.55, and 0.14, respectively). For A. monica, possible factors promoting its high genetic variability are discussed. Mean values for Nei's D between A. monica and A. franciscana were very low (0.09) and consistent with reported cytological and morphological similarities, thus the physiological differentiation which distinguishes the former must involve differences at very few loci. D values from A. urmiana to rest of the gonochoristic species (0.95) are consistent with its taxonomic status and distinctive morphology, while its low genetic distance to the monophyletic Artemia parthenogenetic lineage (mean D = 0.48) suggests a recent common ancestry.     

Preliminary genetic data on some Caribbean Artemia franciscana strains based on RAPD's

A total of fourteen Artemia samples from Colombia, Venezuela, Curaçao (Netherlands Antilles), Puerto Rico, and reference samples from U.S.A. (San Francisco Bay, SFB) belonging to the superspecies Artemia franciscana, and Argentina (A. persimilis), were analysed with the RAPD technique in order to demonstrate genetic dissimilarities. Pearson's correlation coefficients between the DNA banding patterns were calculated. They served as input values for the construction of UPGMA dendrograms. The results indicate that, within the collection of Colombian, Venezuelan and the two Netherlands Antilles Artemiacyst samples examined, two different groups seem to exist. Geographically, the mountainous area of Sierra Nevada de Santa Marta separates these two groups (lower Caribbean to the South and middle Caribbean to the North). Although the Caribbean, North and South American populations belong to A. franciscana, genetic discontinuities are to be expected due to habitat differences and geographic isolation. The Sierra Nevada (with an altitude of about 5800 m) emerges as the barrier very likely to explain the observed RAPD differences. Little genetic variability was present in the Colombian samples from Manaure that were collected almost every ten years, nor in the samples from Galerazamba collected almost two decades apart, although these samples were more likely subjected to different prevailing environmental conditions. The SFB population did not show a very close relation with all Caribbean populations analyzed, including the Puerto Rican. All A. franciscana populations analyzed were divergent from A. persimilis(Argentina).     

Observations on future sea level changes in the Venice lagoon

The identification of the environmental conditions inducing different ecophysiological responses in the different strains and populations of the brine shrimp Artemia should improve the understanding of their biogeographic distribution. Nauplii from two Argentinean brine shrimp populations, Artemia persimilis from Salinas Grandes de Hidalgo (province of La Pampa) and Artemia franciscana from Laguna Mar Chiquita (province of Cordoba), were grown up until adulthood at different salinities (30, 60, 90, 120 gL⁻¹) and temperatures (12, 21, 28°C). The aim was to assess the effects of these different conditions on prereproductive life span and reproductive traits. Results evidenced that at 21 and 28°C, at any salinity, A. franciscana from Laguna Mar Chiquita attained higher survival and fecundity, after a shorter prereproductive period, than A. persimilis from Salinas Grandes de Hidalgo. These data support that A. franciscana, considered a superspecies, exhibits higher phenotypic plasticity than A. persimilis, and that A. persimilis is better adapted to lower temperatures than A. franciscana. These differences in temperature and salinity tolerance could explain the present distribution of these two species in the South Cone in South America. Handling Editor: J. Melack     

Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from contrasting hypersaline habitats express a conserved stress protein complement

The brine shrimp Artemia is a well known animal extremophile adapted to survive in very harsh hypersaline environments. We compared the small stress proteins artemin and p26, and the chaperone hsc70 in encysted embryos (cysts) of the New World species, A. franciscana and A. persimilis. Cysts of the former, from San Francisco Bay, USA (SFB), were used essentially as a reference for these proteins, while both species were from locations in Chile where they occur in habitats at latitudinal extremes, the Atacama desert and Patagonia. These two species are phylogenetically distant, A. persimilis being closer to the Old World species, whilst A. franciscana is considered younger and undergoing evolutionary expansion. Using western blotting we found all three stress proteins in cysts from these five populations in substantial although variable amounts. The protein profiles revealed by Coomassie staining after electrophoresis (SDS-PAGE) were similar qualitatively, in spite of marked differences in the habitats from which these populations originated, and the long time since they diverged. We interpret these findings as further evidence for the adaptive importance of these three conserved proteins in coping with the variable, but severe stresses these encysted embryos endure.     

Further evidence and characterization of Artemia franciscana (Kellogg, 1906) populations in Argentina

Aim This paper reports the presence of Artemia franciscana ( Kellogg, 1906 ) in Mar Chiquita (CHI) salt lake and in Las Tunas (TUN) lagoon, as well as at a shallow lake in Salinas Grandes (Province of Córdoba) in Argentina. To date, this species has been considered absent from Argentina. This study also provides further data on the characterization of the A. franciscana populations from this area. Location Province of Córdoba (Argentina). Methods The cyst samples collected at the three hypersaline environments were measured to assess its mean diameter with a dissecting microscope. The length of the nauplii hatched from the cysts was also determined with the microscope. The adults raised from these nauplii under strictly controlled conditions were analysed for 12 morphological parameters and compared through multivariate discriminant analysis with other American populations. Fatty acids from the total lipids were analysed by gas chromatography. Reproductive compatibility was evaluated from single‐pair intraspecific and interspecific crosses of adult specimens. Results The results endorse the morphometric assimilation of the Artemia populations of this area to other American populations of the A. franciscana‘super‐species’, together with its morphometric differentiation from the species A. persimilis ( Piccinelli & Prosdocimi, 1968 ). The more conspicuous population from CHI shows cross‐fertility with A. franciscana original from San Francisco Bay (California, USA). Both populations from CHI and TUN show cross‐fertility between them and reproductive isolation with A. persimilis from Hidalgo (Province of La Pampa), which to date is considered to be endemic or exclusive of hypersaline ecosystems in Argentina. The reproductive characteristics displayed by the population from TUN lagoon suggest the existence of an occasional hybridization between A. franciscana and A. persimilis ( Papeschi et al., 2000 ). Artemia cysts from CHI and TUN show a fatty acid profile rich in eicosapentaenoic acid differing markedly from the fatty acid profiles found in A. persimilis cysts. Main conclusions: Artemia franciscana is present in Argentina at 36° S and north of this latitude. Artemia persimilis is confined to the south of latitude 37°10′ S. There are different pieces of evidence that point to a certain level of hybridization of the two species taking place in the land belt between these parallels.     

A biometric and ecologic comparison between Artemia from Mexico and Chile

Background

A preliminary biometric and ecologic database for the brine shrimp Artemia from Mexico and Chile is presented. The area abounds in small and seasonal ponds and large inland lakes, the latter mainly located in Mexico, although relatively large and isolated lakes are found in complex hydrological settings in pre-high plateau areas of Chile. This paper summarizes research efforts aimed at the localization, characterization, and evaluation of the aquaculture potential of Artemia populations in Mexico and Chile, which exhibit great habitat diversity (ponds, salterns, coastal lagoons, sea arms, coastal and inland lakes), contrasting weather conditions and different levels of isolation and human intervention.

Results

This study covered locations between 29° north latitude (Baja California, Mexico) to 50° south latitude (Puerto Natales, Chile). Biological characteristics considered are species name, reproductive mode, cyst diameter, chorion thickness, and nauplius length, whereas ecological data include pond size, pH, salinity, temperature, and water ionic composition. Artemia franciscana is the only species found in Mexico, it exists together with A. persimilis in Chile, though separated geographically. Ecological differences in habitat exist between both regions but also within countries, a pattern particularly clear with regard to water composition. Surprisingly, a Mexican (Cuatro Ciénegas, A. franciscana) and a Chilean location (Torres del Paine, A. persimilis) share habitat characteristics, at least for the period when data were collected. The discriminat analysis for cyst diameter and nauplius length shows that Artemia from only one location match in cyst diameter with those from San Francisco Bay (SFB) (Point Lobos), and one (Marquez) is far apart from SFB and all the others. The Chilean locations (Pampilla, Cejar, Cahuil, Llamara, Yape) share cyst diameter, but tend to differ from SFB. The remaining Mexican locations (Juchitan, Ohuira, Yavaros) are well separated from all the others. With regard to nauplii length, populations tend to distribute in a relatively random manner, being Marquez the location differing the most in cyst diameter from SFB.

Conclusion

This database will contribute to the knowledge of radiation centers and serves as a baseline for further biogeographic studies, population characterization, management, and monitoring of Artemia biodiversity. Likewise, the impact of colonization and translocations for aquaculture purposes can be better assessed with a baseline for reference. Mexico and Chile exemplify the need to increase and further integrate regional information to tackle fundamental problems underlying practical utilization of Artemia.     

Allozyme Variation and Phylogenetic Relationships in Picea jezoensis (Pinaceae) Populations of the Russian Far East

Genetic variation and differentiation of 12 populations of Picea jezoensis from the Russian Far East were studied using 20 allozyme loci. The mean number of alleles per locus was 2.63, the percent of polymorphic loci was 88.1%, the observed heterozygosity was 0.181, and the mean value of expected heterozygosity amounted to 0.189. The values of expected heterozygosity of the northern and central mainland populations were higher than in the southern part of the natural range. A significant bias of Hardy–Weinberg heterozygosity to equilibrium heterozygosity (H_eq) suggests that most of the mainland populations have recently experienced a severe expansion in population size while populations from Kamchatka Peninsula have undergone a reduction in population size. Unbiased Nei’s genetic distance values were low within and between the mainland and Sakhalin Island populations (D_N=0.008). The largest values (D_N=0.063) were found between the mainland/Sakhalin and Kamchatka Peninsula populations. Based on genetic distance, P. jezoensis and P. kamtschatkensis could be considered as distinct taxa, but P. ajanensis, P. microsperma, and P. komarovii do not warrant taxonomic recognition.     

Allozyme variation and divergence in the phyllotine rodent Calomys hummelincki (Husson, 1960)

The aim of the present study was to assess the degree of genetic variation and divergence among six populations of Calomys hummelincki, a phyllotine rodent distributed in northern South America. With this information we will try to evaluate the two hypotheses of possible colonization and differentiation of this group of rodents postulated by Baskin and Reig. We studied 34 loci by electrophoretic analysis: 21 were monomorphic for all populations and 13 were polymorphic in at least one population, being P _1% = 21.6% the mean value for all populations. The mean value of heterozygosity per locus was H = 0.075. Low values of genetic distance were observed among populations of the Llanos region (0.001 < D < 0.006). There was a larger genetic distance (D = 0.024) between the population from Isiro, in the northwestern semiarid region, and those from the Llanos region. The insular population of Aruba displayed the lowest value of genetic distance with the population from Isiro (D = 0.014). The specimens from Sipao, on the right side of the Orinoco river, displayed the highest values of genetic distances in comparison with other populations of C. hummelincki (0.070 < D < 0.095). The relatively high differentiation was due to the fixation of new alleles, not found in other populations of C. hummelincki, at loci Idh-1 and Est-2. F-statistics and Nm values indicated reduced gene flow among the populations sampled. Despite the limited data, the results seem to support Reig's hypothesis about south to north colonization of genus Calomys in South America. This revised version was published online in July 2006 with corrections to the Cover Date.     

International study on Artemia XLIX. Salinity effects on survival,maturity, growth,biometrics, reproductive and lifespan characteristics of a bisexual and a parthenogenetic population of Artemia

Two Artemia populations, a bisexual from San Francisco Bay (California, USA) and a parthenogenetic from Tanggu area (Tianjin province, People's Republic of China) are assayed for their tolerance and fitness in various salinity levels. This study was carried out under laboratory conditions where salinity effects upon special characteristics of the two Artemia populations, such as survival, growth rate, maturation, morphology, fecundity and life duration, were recorded. This evaluation revealed that the two populations examined exhibits significant differences in their response against elevated salinity levels. Furthermore, specific biometric parameters can be a useful tool for the discrimination and/or determination of their distribution in a mixed population, which, in fact, is the case in Tanggu salt works. Artemia franciscana seems to be a more effective colonizer at higher salinities. The data presented in this study may generate useful suggestions for proper management of the solar saltworks at Tanggu although further experimentation is needed.International interdisciplinary study on Artemia populations coordinated by the Laboratory of Aquaculture & Artemia Reference Center, University of Ghent, Belgium.     

Mixture of parthenogenetic and zygogenetic brine shrimp Artemia (Branchiopoda: Anostraca) in commercial cyst lots from Great Salt Lake, UT, USA

The brine shrimp Artemia is one of the most studied animals in the world. A large part of the knowledge of this crustacean is based on cysts harvested from two main sources; the Great Salt Lake, UT (GSL), and the San Francisco Bay salterns, CA (SFB), USA. Artemia populations from these habitats are recognized to belong to a single zygogenetic species, Artemia franciscana Kellogg, 1906. However, the GSL Artemia has been in doubt for more than a century about the existence of parthenogenetic reproduction. By using morphological, reproductive, and molecular analyses, we report that commercial GSL cyst lots contained two different brine shrimp species; a parthenogenetic (60%) and a zygogenetic (A. franciscana) (40%). From this finding, at least three hypotheses can be drawn. The parthenogenetic Artemia is native of GSL, or it was introduced to GSL, or foreign parthenogenetic cysts were mixed with A. franciscana cysts and canned for commercial distribution. Researchers using brine shrimp cysts from GSL should therefore pay careful attention to the correct identity of the species under study. The potential of an easy and unnoticed introduction of parthenogenetic Artemia into America is discussed.     

Heterochromatin in the genus Artemia

A bisexual species of the genus Artemia (Crustacea, Phyllopoda), Artemia franciscana Barigozzi of San Francisco Bay and a parthenogenetic population of Artemia sp. of Tsing-Tao (China), both with 42 chromosomes, were compared with respect to the microscopic structure of the interphase larval nucleus, the microscopical structure of the prophase chromosomes and the DNA structure. — Artemia franciscana exhibits several chromocenters in the resting nucleus, heterochromatic blocks located at the end of the prophase chromosomes, and a large amount of repetitive DNA (Alu I 110-bp fragments). The other Artemia sp. lacks chromocenters, heterochromatic blocks in the chromosomes, and the Alu I DNA. The two populations thus differ by a remarkable amount of repetitive DNA.The authors dedicate this paper to Professor Hans Bauer, on the occasion of his 80th birthday     

Aspects of the population dynamics of Neosarmatium meinerti at Mgazana, a warm temperate mangrove swamp in the East Cape, South Africa, investigated using an indirect method

Encysted embryos (cysts) of the brine shrimp, Artemia provide an excellent model system for the study of biochemical adaptation to environmental extremes. Here, we describe an experiment in which cysts of A. franciscana from the San Francisco Bay (SFB), California, U.S.A., were inoculated into experimental ponds in the Mekong Delta region of Vietnam where water temperatures are much higher than the SFB. Cysts produced in each of three successive growing seasons (1996–1998) were collected and examined in the laboratory for resistance to high temperature and relative contents of three stress proteins (Hsp-70, artemin and p26). Thermal adaptation took place rapidly, during the first growing season. The increase in thermal tolerance was reflected in an overall increase in stress protein content, compared to SFB cysts used for the initial inoculation. Also examined were cysts of A. tibetiana collected from a lake on the high plateau of Tibet, PR China, almost 4.5 km above sea level. These cysts were very sensitive to high temperatures, and contained much lower levels of all stress proteins examined, compared to A. franciscana cysts from SFB and Vietnam. Cysts of A. sinica, collected from a hypersaline lake in Inner Mongolia, PR China, were examined in the same fashion and found to be similar to SFB cysts in terms of thermal resistance and stress protein content. The harsh environments in which Artemia are found, and the great diversity of its habitats, world-wide, provide excellent opportunities to relate the ecological setting of an organism to the underlying physiological and biochemical processes enabling its survival.     

Isolation and characterization of polymorphic microsatellite markers in the migratory freshwater fish Prochilodus costatus

We isolated six polymorphic microsatellite loci in the migratory freshwater fish Prochilodus costatus, which is an endemic species and important fisheries resource from São Francisco river basin, Brazil. We have evaluated the applicability of these loci to study genetic variation in wild population of this fish. Thus, based on the genotypes of 32–48 individuals, we detected two to 21 alleles per locus, observed and expected heterozygosities ranging from 0.19 to 0.89 and from 0.17 to 0.92, respectively. These polymorphic markers should provide efficient tools to study population genetic structure of this fish.     

The American brine shrimp as an exotic invasive species in the western Mediterranean

The hypersaline environments and salterns present in the western Mediterranean region (including Italy, southern France, the Iberian Peninsula and Morocco) contain autochthonous forms of the brine shrimp Artemia, with parthenogenetic diploid and tetraploid strains coexisting with the bisexual species A. salina. Introduced populations of the American brine shrimp A. franciscana have also been recorded in these Mediterranean environments since the 1980s. Based on brine shrimp cyst samples collected in these countries from 1980 until 2002, we were able to establish the present distribution of autochthonous brine shrimps and of A. franciscana, which is shown to be an expanding invasive species. The results obtained show that A. franciscana is now the dominant Artemia species in Portuguese salterns, along the French Mediterranean coast and in Cadiz bay (Spain). Co-occurrence of autochthonous (parthenogenetic) and American brine shrimp populations was observed in Morocco (Mar Chica) and France (Aigues Mortes), whereas A. franciscana was not found in Italian cyst samples. The results suggest these exotic A. franciscana populations originate as intentional or non-intentional inoculations through aquacultural (hatchery effluents) or pet market activities, and suggest that the native species can be rapidly replaced by the exotic species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variation in low elevation Douglas-fir of British Columbia and its relevance to gene conservation

Patterns of variation were studied at 20 isozyme loci in 49 coastal, low-elevation Douglas-fir populations in SW British Columbia and NW Washington State. Several components of variation were estimated for each population including the number of alleles per locus N _a, number of alleles per polymorphic locus N _a(95), inbreeding F, heterozygosity H, and population divergence D. F was near zero indicating nearly complete outcrossing within populations. H was quite high (16%) and in aecord with previous studies of Douglas-fir. D values were low (equivalent to Wrights F _ST of 0.08) indicating levels of gene flow sufficient to largely homogenize populations. The parameters of diversity N _a, N _a(95), H, and D showed little intercorrelation across populations. A homogenous pattern of genetic relationship among populations was shown by the clustering of populations based on their inferred relationship, and by the principal components of the matrix of inferred genetic relationship. Because of the complex nature of gene diversity and the continuous nature of population differentiation in Douglas-fir, it is difficult with isozyme markers to identify specific populations of value for genetic conservation in this species.This paper is dedicated to J.C. Heaman on the occasion of his retirement.     

Species-specific RFLP pattern in the Heat Shock Protein26 gene (Hsp26): a single-locus tool for species identification and experimental testing of habitat-induced isolation in the New World Artemia species

The brine shrimp Artemia (Crustacea, Branchiopoda), a paradigmatic inhabitant of hypersaline lakes, has molecular features to survive under stressful conditions, such as the p26 heat shock protein. We report the RFLP fingerprinting pattern (four restriction enzymes) of a 217 bp fragment of exon2 of the Hsp26 gene in six Artemia franciscana and four Artemia persimilis populations, the most genetically divergent Artemia species co-occurring in latitudinal extremes of Chile. The species-specific RFLP pattern observed is a simple and cost-effective single-locus tool for species delimitation and experimental testing the habitat-induced isolation barrier between them.     

Habitat diversity and adaptation to environmental stress in encysted embryos of the crustacean<Emphasis Type="Italic">Artemia</Emphasis>

Encysted embryos (cysts) of the brine shrimp,Artemia, provide excellent opportunities for the study of biochemical and biophysical adaptation to extremes of environmental stress in animals. Among other virtues, this organism is found in a wide variety of hypersaline habitats, ranging from deserts, to tropics, to mountains. One adaptation implicated in the ecological success ofArtemia is p26, a small heat shock protein that previous evidence indicates plays the role of a molecular chaperone in these embryos. We add to that evidence here. We summarize recently published work on thermal tolerance and stress protein levels in embryos from the San Francisco Bay (SFB) of California inoculated into experimental ponds in southern Vietnam where water temperatures are much higher. New results on the relative contents of three stress proteins (hsp70, artemin and p26) will be presented along with data on cysts of A.tibetiana collected from the high plateau of Tibet about 4.5 km above sea level. Unpublished results on the stress protein artemin are discussed briefly in the context of this paper, and its potential role as an RNA chaperone. Interestingly, we show that the substantial tolerance ofA. franciscana embryos to ultraviolet (UV) light does not seem to result from intracellular biochemistry but, rather, from their surrounding thick shell, a biophysical adaptation of considerable importance since these embryos receive heavy doses of UV in nature.