Settling taxonomic and nomenclatural problems in brine shrimps, Artemia (Crustacea: Branchiopoda: Anostraca), by integrating mitogenomics, marker discordances and nomenclature rules

High morphological plasticity in populations of brine shrimp subjected to different environmental conditions, mainly salinity, hindered for centuries the identification of the taxonomic entities encompassed within Artemia. In addition, the mismatch between molecular and morphological evolution rates complicates the characterization of evolutionary lineages, generating taxonomic problems. Here, we propose a phylogenetic hypothesis for Artemia based on two new complete mitogenomes, and determine levels of congruence in the definition of evolutionary units using nuclear and mtDNA data. We used a fossil of Artemia to calibrate the molecular clock and discuss divergence times within the genus. The hypothesis proposed herein suggests a more recent time frame for lineage splitting than previously considered. Phylogeographic analyses were performed using GenBank available mitochondrial and nuclear markers. Evidence of gen e flow, identified through discordances between nuclear and mtDNA markers, was used to reconsider the specific status of some taxa. As a result, we consider Artemia to be represented by five evolutionary units: Southern Cone, Mediterranean—South African, New World, Western Asian, and Eastern Asian Lineages. After an exhaustive bibliographical revision, unavailable names for nomenclatural purposes were discarded. The remaining available names have been assigned to their respective evolutionary lineage. The proper names for the evolutionary units in which brine shrimps are structured remain as follows: Artemia persimilis Piccinelli & Prosdocimi, 1968 for the Southern Cone Lineage, Artemia salina (Linnaeus, 1758) for the Mediterranean-SouthAfrican Lineage, Artemia urmiana Günther, 1899 for the Western Asian Lineage, and Artemia sinica Cai, 1989 for the Eastern Asian Lineage. The name Artemia monica Verrill, 1869 has nomenclatural priority over A. franciscana Kellogg, 1906 for naming the New World Lineage. New synonymies are proposed for A. salina (= C. dybowskii Grochowski, 1896 n. syn., and A. tunisiana Bowen & Sterling, 1978 n. syn.), A. monica (= A. franciscana Kellogg, 1906 n. syn., and A. salina var. pacifica Sars, 1904 n. syn.); A. urmiana (= B. milhausenii Fischer de Waldheim, 1834 n. syn., A. koeppeniana Fischer, 1851 n. syn., A. proxima King, 1855 n. syn., A. s. var. biloba Entz, 1886 n. syn., A. s. var. furcata Entz, 1886 n. syn., A. asiatica Walter, 1887 n. syn., A. parthenogenetica Bowen & Sterling, 1978 n. syn., A. ebinurica Qian & Wang, 1992 n. syn., A. murae Naganawa, 2017 n. syn., and A. frameshifta Naganawa & Mura, 2017 n. syn.). Internal deep nuclear structuring within the A. monica and A. salina clades, might suggest the existence of additional evolutionary units within these taxa.

Investigation of the Possible Role of RAD9 in Post-Diapaused Embryonic Development of the Brine Shrimp Artemia sinica

Background: The cell cycle checkpoint protein RAD9 is a vital cell cycle regulator in eukaryotic cells. RAD9 is involved in diverse cellular functions by oligomer or monomer. However, the specific mechanism of its activity remains unknown in crustaceans, especially in embryonic diapause resumption of the brine shrimp Artemia sinica. Methods and Results: In the present article, a 1238 bp full-length cDNA of As–RAD9 gene, encoding 376 amino acids, was obtained from A. sinica. The expression pattern of As–RAD9 was analyzed by qPCR and Western blot. The mRNA expression level climbs to the top at the 10 h stage of embryo development, while the protein expression pattern is generally consistent with qPCR results. Moreover, the As–RADd9 related signaling proteins, As–RAD1, As–HUS1, As–RAD17, and As–CHK1, were also detected. Immunofluorescence assay showed that the location of As–RAD9 did not show tissue or organ specificity, and the intracellular expression was concentrated in the cytoplasm more than in the nucleus. We also explored the amount of As–RAD9 under the stresses of cold and high salinity, and the results indicate that As–RAD9 is a stress-related factor, though the mechanisms may be different in response to different stresses. Knocking down of the As–RAD9 gene led to embryonic development delay in A. sinica. Conclusions: All these results reveal that As–RAD9 is necessary for post-diapaused embryonic development in A. sinica.