: de novo conserved regulatory motifs. Added file 13: Primer sequences for 5′ and 3′ RACE in D. galeata, C. dubia and M. macrocopa. Further file 14: Accession numbers of your sequences for phylogenetic evaluation. Further file 15: Primer sequences for quantitative PCR. More file 16: DapmaDsx1- TF-map. Added file 17: DappuDsx1- TF-map. More file 18: DapmaDsx1- TF-map. More file 19: DappuDsx1- TF-map. Additional file 20: DapmaDsx2 TF-map. Further file 21: DappuDsx2 TF-map.The 5 1.0 Kbp upstream area was extracted from all D. pulex gene models and utilized to create a background frequency model for eight bp length motifs in Daphnia promoter regions. WeederH [59] was utilised to search for conserved regulatory motifs of length eight present in all six dsx promoters in D.Nitroxoline magna and D. pulex. The WeederH algorithm measures conservation determined by the sequence conservation also because the motif occurrence’s positions relative for the TSS. WeederH produces a two score assessing how conserved the motif is compared to the rest on the homologous sequences. We applied a two score threshold of three, discarding motifs using a 2 score less than 3peting interests The authors declare that they have no competing interests.EGF Protein, Human Authors’ contributions KT, YK, HM, HW, NT and TI developed the experiments; KT, YK, MS, NS, SM, HM, SO, YO, CH and TM performed the experiments; KT, HM, JKC, TI, SP and CJ analyzed the data; KT, HM, CJ, JKC and TI wrote the paper. All authors have study and approved the final manuscript. Acknowledgments The D. magna and D. pulex sequence data had been supplied by The Center for Genomics and Bioinformatics at Indiana University, which is supported in part by the METACyt Initiative of Indiana University, funded in aspect via a significant grant in the Lilly Endowment, Inc.PMID:24103058 , and distributed by way of wFleaBase in collaboration with Dr. Donald Gilbert plus the Daphnia Genomics Consortium https://wiki.cgb.indiana.edu/display/DGC/Home. This work was partly supported by the Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists. to KT and CH, the Sasakawa Scientific Investigation Grant in the Japan Science Society to KT, the Saito Hoon Kai Scientific Research Grant from the Saito Gratitude Foundation to KT, grants in the Ministry of Education, Culture, Sports, Science and Technology (TI), the Ministry with the Environment of Japan (TI), a grant of Long-Range Analysis Initiative by Japan Chemical Business Association (TI) as well as a grant from National Institute for Standard Biology (TI). Author details 1 Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of All-natural Sciences, and Department of Basic Biology, Faculty of Life Science, Graduate University for Advanced Research (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. 2 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. 3Toyota Nishi High College, 14-65 Kosaka, Toyota, Aichi 471-0035, Japan. 4National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan. 5The Center for Genomics and Bioinformatics, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA. 6Present address: College of Biosciences, University of Birmingham, Birmingham B15 2TT, UK. Received: 29 September 2012 Accepted: 26 March 2013 Published: 10 April 2013 References 1. Marin I, Baker BS: The evolutionary dynamics of sex det.