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.     

Morphometric and Preliminary Genetic Characteristics of Artemia Populations from Iran

Six Artemia populations, one bisexual and five parthenogenetic, from different parts of Iran were compared using morphometric and genetic characteristics. The discriminant analysis based on 19 morphometric variables showed that there are significant differences between the studied populations based on their morphological characteristics, where 85.9% of original grouped cases were correctly classified. The bisexual Artemia urmiana however exhibited a 100% separation from the parthenogenetic populations. However, a 1500 bp mitochondrial rDNA fragment showed similar RFLP patterns for all Iranian populations confirming earlier reports of a close genetic relationship between A. urmiana and parthenogenetic Artemia. (© 2009 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.     

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.     

Genetic Polymorphism and Evolution in Parthenogenetic Animals. II. Diploid and Polyploid SOLENOBIA TRIQUETRELLA (Lepidoptera: Psychidae)

Genic polymorphism at sixteen enzyme loci of four different chromosomal races of Solenobia triquetrella (bisexual, two diploid parthenogenetic races and tetraploid parthenogenetic) has been studied by starch gel electrophoresis. Isolated small diploid bisexual populations have rather uniform allele frequencies at all loci which we have studied. Diploid and tetraploid parthenogenetic individuals of this species are in general as heterozygous as bisexual ones. All parthenogenetic local populations are different from each other in the Alps. These parthenogenetic genotypes cannot be derived from a common ancestor through single mutations but rather bear evidence for a polyphyletic origin of parthenogenesis in Solenobia triquetrella. In the marginal distribution areas of the species in northern Europe single genotypes are spread over far larger areas than in the mountain regions of central Europe. This may be due to the old origin of parthenogenesis and polyploidy in northern Europe. No new parthenogenetic and polyploid strains have lately arisen in the regions outside of the Alps.     

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.     

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.     

Discovery of a diploid population of theHemidactylus garnotii-vietnamensis complex (Reptilia: Gekkonidae)

A diploid member of the parthenogenetic gekkonid species complexHemidactylus garnotii-vietnamensis was discovered for the first time from Thailand. This gecko, seemingly unisexual and parthenogenetic, possesses 2n=2x=38 chromosomes, showing distinct heteromorphisms. The absence of bisexual congeneric species with a combination of karyomorphs to produce this karyotype indicates the occurrence of chromosomal rearrangements after the initial estabilishment of a diploid clonal lineage of hybrid origin. Results of karyotypic comparisons of the present sample and the three known triploid species belonging to theH. garnotii-vietnamensis complex suggest that a triploid karyomorph similar to that ofH. vietnamensis has first emerged through an insemination of the diploid parthenogen's egg by the sperm from a bisexual species having 44 chromosomes (all telocentric), and that the karyomorph subsequently experienced some minor chromosomal aberrations to produce the karyomorphs ofH. vietnamensis andH. garnotii. The origin of theH. stejnegeri karyotype still remains an open question for future studies.     

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.     

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.     

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.     

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. %     

The use of a multidisciplinary approach for the characterization of a diploid parthenogenetic Artemia population from Torre Colimena (Apulia, Italy)

A parthenogenetic Artemia population from Torre Colimena, southernItaly, originally reported in 1998, is characterized by a multidisciplinaryapproach including cyst and naupliar biometry, morphometry ofadults, scanning electron microscopy (SEM) of brood pouch, cytogeneticsand 16S rRNA PCR-RFLP analysis. We confirmed parthenogeneticstatus, inferred ploidy level and determined phenotypic andmolecular relationships of this population through comparisonswith other asexual Artemia strains as well as bisexual speciesfound (A. salina) or introduced (A. franciscana) in the Mediterraneanbasin. Cyst and naupliar sizes for Torre Colimena are amongthe smallest recorded for asexual Artemia while the oppositeis true for chorion thickness. Discriminant analysis of adultbody measurements shows increased differentiation (89.5% forthe first four out of the twelve functions produced) from tetraploidparthenogenetic strains and bisexual species (A. salina andA. franciscana). Scanning electron micrographs of brood pouchreveal the characteristic morphology of asexual strains, whilechromosome observations of instar-I nauplii unequivocally establishdiploidy. Restriction patterns give evidence that the TorreColimena population shares an identical set of mitochondrialDNA (mtDNA) polymorphisms with a diploid Spanish parthenogeneticstrain and it is well differentiated from other tetraploid aswell as bisexual auto- and allochthonous strains (A. salinaand A. franciscana) from the Mediterranean. The present studymay serve as a reference methodological framework for multidisciplinarycharacterizations and biodiversity assessments in the genusArtemia.     

Morphological characterization of adult Artemia (Crustacea, Branchiopoda) from different geographical origin. Mediterranean populations

Twelve morphological parameters have been measured in Artemiaindividuals belonging to 27 different populations located aroundthe Western Mediterranean basin. An analysis, through multivariatediscriminant procedures, allows us to establish relationshipsamong different populations. The three different types of populationstudied (bisexual diploid, parthenogenetic diploid and parthenogenetictetraploid) are thoroughly characterized by their morphologicalcharacteristics. This simple method is shown to be useful ingrouping different populations and to have predictive valuein assigning new populations to the groups previously analyzed.     

The origin and evolution of parthenogenesis in Heteronotia binoei (Gekkonidae)

Within Australia, the gekkonid lizard Heteronotia binoei exists as diploid bisexual and triploid all-female populations. Three bisexual cytotypes and three triploid clones can be described on the basis of the morphology of gross karyotypes. This paper reports the results of a C-banding analysis that revealed both intrapopulation polymorphism and interpopulation polytypic variation within the most widespread bisexual cytotype (A6). A C-band variant that defined a ZW sex chromosome system in populations from the MacDonnell Ranges in central Australia was also identified. Silver staining confirmed that in all populations the nucleolus organising region always occurred distally on chromosome 6. Examination of C-banding and silver-staining patterns of triploid populations provided strong support for a hybrid origin of the parthenogens that involved the central and western A6 populations. It is proposed that the hybridisation of these populations with the other chromosomally distinct bisexual cytotypes (SM6) resulted in the triploid clones. At present, seven clones have been karyotypically defined, and all the chromosomal variants that were present in the triploids can be accounted for by multiple hybridisation events between the bisexual populations. The analysis also provided evidence that rare sterile tetraploid females are the result of insemination of the triploid parthenogens by male H. binoei. This paper is dedicated to the memory of M.J.D. White (1910–1983)     

Cytogenetics of ticks (Acari: Ixodoidea)

Haemaphysalis longicornis consists of diploid bisexual races (20+ XX; 20+X), triploid obligatory parthenogenetic races (30–35 chromosomes) and an aneuploid race capable of bisexual and parthenogenetic reproduction (22–28 chromosomes). Karyotypes were analyzed for each race. Hybridization failed between diploid and triploid races, but succeeded between bisexual diploid males and parthenogenetic aneuploid females. F₁ and F₂ progeny were produced and their chromosomes studied. Crossing of F₁ progeny to a bisexual race was successful. Parthenogenetic ability was almost completely lost in F₁ and F₂ females. Several possible modes of evolution from diploid bisexual individuals to triploid parthenogenetic ones are discussed as is species characterization in taxa with races reproducing bisexually, parthenogenetically and by a combination of both methods.Supported in part by National Science Foundation Research Grant GB-21008, National Institute of Allergy and Infectious Diseases (NIH) Grant 09556 and the United States Army Medical Research and Development Command, Office of The Surgeon General, Department of the Army, Washington, D. C. 20315, U.S.A.     

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.     

Life histories of two clonal populations of Stratiotes aloides L.

Cysts of parthenogenetic Artemia strains collected from the Citros and M. Embolon saltworks in Northern Greece are evaluated for their potential use in aquaculture. The following characterizations were performed: cyst and naupliar biometrics, cyst hatching characteristics, fatty acid profile of the nauplii, caloric content of nauplii stored at 25 °C and in a refrigerator (4–8 °C). The above evaluation reveals that the two Artemia strains studied exhibit good qualities for use in aquaculture, especially in culturing fresh-water species. The biometrical analysis of cysts, nauplii and adults shows a high degree of similarity with other parthenogenetic strains from various geographical sources, but especially with tetraploid Artemia from Spain. The Greek Artemia strains cannot be considered as sources for aquacultural uses unless proper management of the saltworks is assured.     

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.     

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.