Genetic variation and evolutionary origins of parthenogenetic Artemia (Crustacea: Anostraca) with different ploidies

Using two nuclear (ITS1 and Na⁺/K⁺ ATPase) and three mitochondrial (COI, 16S and 12S) markers, we determined the genetic variation and evolutionary relationship of parthenogenetic and bisexual Artemia. Our analyses revealed that mitochondrial genes had higher genetic variation than nuclear genes and that the 16S showed more variety than the other mitochondrial genes in parthenogenetic populations. Triploid parthenogens showed lower genetic variation than diploid ones, whereas the tetra‐ and pentaploids had greater genetic distance than diploid parthenogens. No shared haplotype was found between individuals of parthenogenetic populations and Asian bisexual species with the exception of Na⁺/K⁺ ATPase (Artemia tibetiana). Only mitochondrial markers can demonstrate phylogenetic relationships, and showed that the parthenogenetic Artemia is a polyphyletic group in which the diploid lineages share a common ancestor with Artemia urmiana while tetraploids are closely related to Artemia sinica. The triploid and pentaploid linages are likely to be directly derived from diploid and tetraploid parthenogens, respectively. Subsequently, west Asia is origin for di‐/triploids, and tetra‐/pentaploids rose from East Asia.     

Sediment cores from Lake Urmia (Iran) suggest the inhabitation by parthenogenetic Artemia around 5,000?years ago

In Lake Urmia area, northwestern Iran, parthenogenetic Artemia and the bisexual Artemia urmiana Günther 1890 are found to occupy different ecological niches determined by salinity. Given the fluctuations of the lake over geological times, we thus hypothesized that species identification of Artemia cysts, buried in the sediments, can provide information on lake conditions in the past. Therefore, encysted embryos of Artemia were recovered from lake sediments by augering at a site near the present shoreline. Cysts and associated plant remains from two studied levels yielded radiocarbon ages in the range 5,000–6,700 YBP. For determination of the type of Artemia, the constant synonym mutation in exon-7 of the Na/K ATPase gene was verified, and the diameter of the recovered cysts was compared with that of modern cysts from the Lake Urmia region. The results show that the cysts represent a parthenogenetic type of Artemia, whose cyst diameter is somewhat different from that of present-day local parthenogenetic Artemia. The present study firstly confirms the stability of DNA in ancient Artemia cysts for molecular analysis. Moreover, it suggests variation in Lake Urmia’s conditions over time, and based on comparison with salinity preferences of contemporary Artemia populations, it more specifically suggests that Lake Urmia was a brackish lake dominated by a parthenogenetic Artemia population in the geological period sampled. It finally illustrates how, like in the study of freshwater propagule banks, paleogenetic analysis of Artemia DNA recovered from sediment cores can be used as a tool in the paleoecological study of generally highly fluctuating saline habitats.     

Salinity effects on maturation, reproductive and life span characteristics of four Egyptian Artemia populations (International Study on Artemia. LXVIII)

Three parthenogenetic Artemia populations, i.e. two coastal from Borg El-Arab and El-Max saltworks and one from the inland Qarun Lake, and a bisexual strain (Artemia salina) from the inland carbonate lake of Wadi El-Natrun, all from upper Egypt were assayed for their response in 5 salinities (i.e. 35, 80, 120, 150 and 200 g l^?1). The experimental procedure was carried out under laboratory conditions, where the effects of salinity on maturation and ten reproductive and life span characteristics were investigated. The parthenogenetic Egyptian populations are more euryhaline compared to the bisexual one. The two coastal parthenogenetic populations appeared to be very similar in maturation rate and reproductive output at all salinities tested. The inland asexual strain showed a different reproductive response to the elevation of salinity from the two coastal populations. Discriminant function analysis has proven to be a useful tool in determining the differential response of closely related Artemia populations. The bisexual population showed significantly lower reproductive output compared to the parthenogenetic ones and performed best at 35 g l^?1; this is the first record of an A. salina population inhabiting a carbonate lake. These findings may provide valuable information on Artemia biodiversity in an area where very little is known. %     

Origin and Genetic Diversity of Diploid Parthenogenetic Artemia in Eurasia

There is wide interest in understanding how genetic diversity is generated and maintained in parthenogenetic lineages, as it will help clarify the debate of the evolution and maintenance of sexual reproduction. There are three mechanisms that can be responsible for the generation of genetic diversity of parthenogenetic lineages: contagious parthenogenesis, repeated hybridization and microorganism infections (e.g. Wolbachia). Brine shrimps of the genus Artemia (Crustacea, Branchiopoda, Anostraca) are a good model system to investigate evolutionary transitions between reproductive systems as they include sexual species and lineages of obligate parthenogenetic populations of different ploidy level, which often co-occur. Diploid parthenogenetic lineages produce occasional fully functional rare males, interspecific hybridization is known to occur, but the mechanisms of origin of asexual lineages are not completely understood. Here we sequenced and analysed fragments of one mitochondrial and two nuclear genes from an extensive set of populations of diploid parthenogenetic Artemia and sexual species from Central and East Asia to investigate the evolutionary origin of diploid parthenogenetic Artemia, and geographic origin of the parental taxa. Our results indicate that there are at least two, possibly three independent and recent maternal origins of parthenogenetic lineages, related to A. urmiana and Artemia sp. from Kazakhstan, but that the nuclear genes are very closely related in all the sexual species and parthenogegetic lineages except for A. sinica, who presumable took no part on the origin of diploid parthenogenetic strains. Our data cannot rule out either hybridization between any of the very closely related Asiatic sexual species or rare events of contagious parthenogenesis via rare males as the contributing mechanisms to the generation of genetic diversity in diploid parthenogenetic Artemia lineages.     

The morphological characteristics of Artemia shrimps from Siberian populations

Morphological analysis of characteristics of shrimps from parthenogenetic and bisexual Artemia populations from a vast territory from the Urals to the Sayan Mountains was made. A normal distribution of the majority of parameters and a different degree of their interrelation and dependence on some environmental factors (salinity, ion composition, and ratio) were recorded. The analysis included seasonal, interannual, and sexual variability of Artemia. The greatest influence of salinity and separately of main ions (except for carbonates, hydrocarbonates, and calcium) on the abdomen and furca structure is shown. No correlation was found between water salinity and body length. The cluster analysis of a complex of morphological parameters showed grouping of populations by salinity of lakes instead of their geographical position and sexual structure of populations.     

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.     

Variations in repetitive DNA and heterochromatin in the genus Artemia

The genus Artemia (Crustacea, Phyllopoda) is widely distributed all over the world as a result partly of natural colonization and partly of spread by birds and man. Artemia offers a very interesting model for speciation studies, since the genus comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, heteroploidy and polyploidy). The finding of the clustered repetitive AluI DNA family in the heterochromatin of A. franciscana can provide a useful tool for investigating the relationship between the members of the genus Artemia at the molecular level. Sixteen strains of Artemia, comprising sibling species and parthenogenetic populations, were analysed for the presence of AluI repetitive DNA by dot-blot hybridization. The observed variation in the content of repetitive DNA together with genetical, biological and geological data, support the hypothesis that Artemia living in the New World are derived from ancestral species that evolved in the Mediterranean area.     

A study of karyotypes and heterochromatic associations in Artemia,with special reference to two N. Greek populations

The chromosomes of three stains of Artemia were studied, a bisexual diploid (A. franciscana, strain GSL), and two parthenogenetic tetraploids from Northern Greece. Routine karyotypes were constructed for the diploid and tetraploid forms, as well as a Giemsa C-banding karyotype of the bisexual form; some data are also presented on the chromosomal C-banding patterns of the parthenogenetic populations. The possible significance of various chromosomal configurations that have been observed, and their relationships to heterochromatin, are discussed.This paper is dedicated to Professor C. Barigozzi for introducing us to Artemia research.Supported by a grant from the Greek Ministry of Agriculture, Request offprints from: Th. Abatzopoulos, Lab. of General Biology, School of Sciences, Aristotelian University, Thessaloniki, 54006, Greece.     

Coexistence of Sexual and Parthenogenetic Artemia Populations in Lake Urmia and Neighbouring Lagoons

We studied the Artemia populations existing in Lake Urmia (north‐western Iran), one of the largest habitats of Artemia in the world, in order to settle the long‐standing controversy over the sexual status of the endemic Artemia populations. Experiments were carried out in the laboratory and in the field. Cysts, collected from different sites of the lake and peripheral lagoons, were hatched and cultured to adults in the laboratory. Adult sexual and parthenogenetic animals were isolated and newly hatched nauplii from them were cultured to maturity in different salinities, ranging from 15–80 ppt. Survival levels and percentage of animals attaining adulthood were measured over a period of 30 days. In the field experiment, cysts taken from Lake Urmia were hatched and the resulting nauplii were inoculated into six earthen ponds (80–140 ppt) constructed in the vicinity of the lake. Population composition in each pond was determined over a period of two years. Results indicated that both sexual and parthenogenetic Artemia coexist in Lake Urmia. While the lake itself is dominated by sexual Artemia, the asexual populations were found to be restricted to particular areas in or near the lake. Artemia appearing seasonally in the lagoons adjacent to the lake were exclusively parthenogenetic. Parthenogens could grow, mature and reproduce at very low salinities (15–33 ppt), whereas higher salinities (above 50 ppt) were required for A. urmiana to attain sexual maturity. We consider salinity to be a major abiotic factor determining the distribution of these sexually different populations within and outside the lake. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

May parthenogenesis in Artemia be attributed to Wolbachia?

Rickettsia-like maternally inherited bacteria are commonly associated with the induction of severe modifications of the reproductive behaviour of their hosts, such as parthenogenesis. Among them, the endosymbiont Wolbachia is the best known because it is widespread in many arthropods, including insects, terrestrial isopods and the exclusively aquatic ostracods. The genus Artemia (Crustacea, Anostraca) that consists of both bisexual species and parthenogenetic strains was—to our knowledge—for the first time tested for infection with Wolbachia. We screened 38 Artemia populations (parthenogenetic and bisexual) using a PCR assay. Our results clearly show that Wolbachia is not involved in the induction of parthenogenesis in Artemia.     

Highly repetitive DNA sequence in parthenogeneticArtemia

Summary The study of the structural organization of the eukaryotic genome is one of the most important tools for disclosing the evolutionary relationships between species.Artemia (Crustacea, Phyllopoda) offers a very interesting model for speciation studies. The genus, distributed all over the world, comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, polyploidy, and heteroploidy).Digestion of genomic DNA of the parthenogeneticArtemia sp. from Tsing-Tao (China) with the restriction enzymes Eco RI and Alu I reveals that a highly repetitive sequence of 133 bp is present. The Eco RI fragment has been cloned and characterized by genomic organization. The distribution of the Eco RI family of repeats was also studied in several bisexual and parthenogeneticArtemia populations and compared with an Alu I repetitive fragment previously identified inArtemia franciscana.     

Characterisation of high-altitude Artemia populations from the Qinghai-Tibet Plateau,PR China

The brine shrimp Artemia was discovered in a number of saline lakes on the Qinghai-Tibet Plateau, widely diverging in chemical composition. Several lakes were athalassohaline, with relatively high amounts of trace elements. Common environmental factors are their high altitude (exceeding 4500 m) and the low average annual temperatures. A number of Artemia populations in this area were analysed to assess their preference for low temperatures and an athalassohaline medium. Furthermore, their characteristics were compared with Artemia tibetiana, the species recently described for one lake in this area. All samples contained a variable mixture of parthenogenetic and bisexual individuals. A cross-breeding test of the sample from Jingyu Lake showed cross-fertility both with A. tibetiana and A. sinica. All populations showed similarities to A. tibetiana: a large cyst diameter and naupliar length, high HUFA content and a high tolerance to low temperatures, as compared to the control A. franciscana samples. These can thus be considered as recurrent characteristics of the populations from the high-altitude low-temperature environment on the Qinghai-Tibet Plateau, although further research is needed to identify their exact species status.     

Cytogenetic study of Artemia from Urmiah, Maharloo and Incheborun Lakes

Three populations of Artemia from Urmiah (West-Azarbaijan Province), Maharloo (Fars Province) and Incheborun (Golestan Province) Lakes in Iran were cytogenetically analyzed. In order to study the reproduction mode in Artemia populations, cyst samples reared for three successive generations (F₁--F₃). The results obtained from rearing until maturation indicated that, there are two subpopulations of Artemia in Urmiah lake (bisexual and parthenogenetic). These two subpopulations have the same diploid chromosome number (2 n =42). The results obtained from rearing and cytogenetic analysis of Artemia from Maharloo and Incheborun Lakes showed that these two populations are parthenogenetic and their chromosome number is also 2 n=42. So far, no cytogenetic differences in ploidy level have been detected among the populations investigated in this study. Very small chromosomes with metacentric, submetacentric and telocentric morphology were observed in mitotic prometaphase cells, implying that chromosomes of Artemia may be monocentric.     

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.     

Geographical distribution and evolutionary divergence times of Asian populations of the brine shrimp Artemia (Crustacea,Anostraca)

The brine shrimp Artemia represents a widespread genus of microcrustaceans adapted to hypersaline environments. The species of this genus have been the subject of numerous phylogenetic studies, but many open questions remain, especially for Eurasian Artemia lineages. Artemia sinica Cai, 1989 and Artemia tibetiana have a restricted geographical distribution, whereas the Eurasian haplotype complex (EHC) and especially Artemia urmiana Günther, 1899 show wider ranges. We examined the geographic distribution, evolutionary age, and historical demography of the Asian Artemia lineages (A. urmiana, A. sinica, A. tibetiana, and the Eurasian haplotype complex) using samples from 39 geographical localities and based on the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Asian Artemia taxa clusters into four distinctive clades with high nodal support, consisting of 69 unique haplotypes. A star‐like haplotype pattern was visible in EHC lineages (comprising pathenogenetic populations), which were genetically close to two sexual species, A. urmiana and A. tibetiana. The Bayesian approach of molecular clock estimation indicated that A. sinica had already diverged in the late Miocene (19.99 Mya), whereas A. urmiana, A. tibetiana, and EHC shared a common ancestor in the late Pliocene (5.41 Mya). Neutrality tests indicated a recent population expansion in A. urmiana and EHC lineages. The diversification within A. urmiana and EHC lineages occurred in the Pleistocene (1.72 Mya) and Holocene (0.84 Mya), respectively. Overall, these results suggest a much longer evolutionary history of A. sinica and the possible evolutionary origin of EHC lineages from Asian sexual ancestors. Our findings point to the importance of species structure and divergence time variations of Asian Artemia, highlighting interspecific diversification and range expansion of local species in Asia. © 2015 The Linnean Society of London     

Potential for parthenogenesis of virgin females in a bisexual population of the geographically parthenogenetic mayfly Ephoron shigae (Insecta: Ephemeroptera,Polymitarcyidae)

The burrowing polymitarcyid mayfly Ephoron shigae is a geographically parthenogenetic species. Interestingly, the distributions of the bisexual and unisexual populations overlap broadly in their respective geographic ranges. In this mayfly, obligatory diploid thelytoky appears within unisexual populations. In the present study, we examined the potential for parthenogenesis or the parthenogenetic ability of females in a bisexual population aiming to understand the emergence of unisexual populations. The results obtained revealed that females in the examined bisexual populations showed a potential for diploid thelytoky as also seen in the unisexual populations, although, in females from bisexual populations, the development success rates of their unfertilized eggs were considerably lower than those of virgin females from unisexual populations. In the three bisexual reproducing species (Ephemera japonica, Ephemera strigata, and Ephemera orientalis) in the closely‐related family Ephemeridae, diploid thelytoky (i.e. tychoparthenogenesis; < 3%) was also observed. However, in this case, the parthenogenetic development success rates of unfertilized eggs were significantly lower than those of virgin females in the bisexual (Hino‐yosui Irrigation Canal) population of E. shigae. Accordingly, we suggest that parthenogenetic ability (i.e. tychoparthenogenesis or facultative parthenogenesis) in bisexual populations of E. shigae may facilitate the evolutionary transition to unisexual populations with fully obligatory parthenogenesis. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 326–334.     

Functional rare males in diploid parthenogenetic Artemia

Functional males that are produced occasionally in some asexual taxa – called ‘rare males’ – raise considerable evolutionary interest, as they might be involved in the origin of new parthenogenetic lineages. Diploid parthenogenetic Artemia produce rare males, which may retain the ability to mate with females of related sexual lineages. Here, we (i) describe the frequency of male progeny in populations of diploid parthenogenetic Artemia, (ii) characterize rare males morphologically, (iii) assess their reproductive role, using cross‐mating experiments with sexual females of related species from Central Asia and characterize the F1 hybrid offspring viability and (iv) confirm genetically both the identity and functionality of rare males using DNA barcoding and microsatellite loci. Our result suggests that these males may have an evolutionary role through genetic exchange with related sexual species and that diploid parthenogenetic Artemia is a good model system to investigate the evolutionary transitions between sexual species and parthenogenetic strains.     

Reproduction characteristics,survival rate and sex-ratio of four brine shrimp Artemia salina (Linnaeus, 1758) populations from Tunisia cultured under laboratory conditions

Artemia has a remarkable genetic variability that can be expressed in various phenotypic characteristics, such as morphometry, growth rate, reproductive isolation or molecular composition. This study presents reproduction characteristics, survival rate and sex-ratio of four Artemia salina populations from Tunisia cultured under standard conditions. Results show that both low (17.8% and 30.6% in Megrine saltwork and Sabkhet Sijoumi, respectively) and high (83.9% for Sahline saltwork) values were recorded for the percentage of total oviparous offspring. The offspring per brood varied from 70.3?±?41.9 (Megrine saltwork) to 73.6?±?51.4 (Sabkhet El Adhibet). Broods per female varied between 3.1?±?0.9 (Sahline saltwork) and 4.5?±?1.2 (Megrine saltwork), and time between broods ranged from 5.7?±?1.1 to 6.5?±?2.2 days for Megrine and Sahline, respectively. Statistical analysis (one-way analysis of variance, Tukey's HSD test, p?<?0.05) revealed no significant differences between reproductive traits of the four studied populations except for mean of oviparous and oviviparous offspring per female (F?=?9.158, p?<?0.05), and brood per female (F?=?4.779, p?<?0.05). The survival rate of the four studied A. salina populations fluctuated between 31.4% and 64.5% for Megrine and Sahline saltwork, respectively. However, the sex-ratio showed that for Megrine saltwork and Sabkhet El Adhibet, males predominated with 1?:?0.90 and 1?:?0.97 (males?:?females), respectively. The comparison between our results and those reported of other Artemia populations showed that the offspring per brood of Tunisian Artemia are comparable to A. persimilis but different to A. salina from Abu Kammash (Libya) and A. urmiana (Urmia Lake, Iran), and that days between brood, are similar to A. salina from Abu Kammash, A. sinica and polyploid A. parthenogenetica, but different from A. persimilis and A. franciscana.     

Genetic differentiation of Artemia urmiana from various ecological populations of Urmia Lake assessed by PCR amplified RFLP analysis

This study concentrated on the mitochondrial DNA diversity in adult Artemia urmiana populations. A total of 210 individual specimens were collected from the surface and bottom layers from three different geographical areas, comprising six sampling sites in Urmia Lake (Iran). The mitochondrial rDNA gene region was amplified using the PCR technique followed by RFLP analysis based on using eleven restriction endonucleases. Analysis at the intrapopulation level indicated that the two median and south area bottom layers have somewhat higher proportion of polymorphic sites compare to others. Although floating and sinking cysts did not consistently show genetic differences, there was significant genetic variation among all samples (F_ST = 0.019, P = 0.03) and 98% of the variation was within samples. Our results clearly distinguished nucleotide divergence and genetic structuring patterns, suggest that may be genetic differentiation existed in Artemia populations from Urmia Lake. Pairwise comparisons of the samples showed that the south area surface layer location was the most genetically divergent of the six sampling sites. Genetic characterizations of the various Artemia populations in two defined depths (surface and bottom) revealed differentiation that may be important in understanding the ecology of this commercially important species.