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.