Confirmation through Genetic Analysis of the Existence of Many Local Phyloclades of the Genus Simocephalus (Crustacea, Cladocera) in China Xiaona Huang 1,2 *, Xinlu Shi 2 , Alexey A. Kotov 3 , Fukang Gu 1 1 School of Life Science, East China Normal University, Shanghai, China, 2 Hangzhou Key Laboratory for Animal Adaptation and Evolution, Hangzhou Normal University, Hangzhou, China, 3 A.N. Severtsov Institute of Ecology and Evolution, Leninsky Prospect 33, Moscow, Russia Abstract Previously, a series of Simocephalus taxa (Cladocera: Daphniidae) from China were described. Most were proposed to be junior synonyms in the last revision of the genus. Using original material from China and data from GenBank, we investigate the biodiversity and phylogeny of Simocephalus using sequences of the cytochrome c oxidase subunit I (COI) and the nuclear 18S genes. In both cases, neighbor-joining, maximum likelihood and Bayesian inference analyses led to highly congruent tree topologies. The grouping of the deeper clades agrees with the inter-generic classification of Orlova- Bienkowskaja (2001). Only the populations of S. serrulatus from Eurasia and North America seem to be closely related, and there are no other shared species between the two continents. Our study unambiguously confirms the existence of many lineages from the subgenera of Simocephalus (Echinocaudus) and Simocephalus s.str. in China, but their morphology needs to be reexamined by taking a wider range of characters (e.g., of female thoracic limbs and adult males) into consideration. Citation: Huang X, Shi X, Kotov AA, Gu F (2014) Confirmation through Genetic Analysis of the Existence of Many Local Phyloclades of the Genus Simocephalus (Crustacea, Cladocera) in China. PLoS ONE 9(11): e112808. doi:10.1371/journal.pone.0112808 Editor: Donald James Colgan, Australian Museum, Australia Received June 15, 2014; Accepted October 20, 2014; Published November 13, 2014 Copyright: ß 2014 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: The reagents, materials and analysis tools of this study was partly supported by the National Natural Science Foundation of China (Grant Nos. 31071880 and 31272262), Zhejiang Provincial Natural Science Foundation and Zhejiang Key Scientific & Technological Innovation Team Project (Grant Nos. 2010R50039-20), and Hangzhou Key Laboratory for Animal Adaptation and Evolution (Grant No. 20100333T05) and part of analysis tools of this study was partly supported by Russian foundation for Basic research (grants 12-04-00207-a and 14-04-01149-a). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: nxxiaona@163.com Introduction Cladocera (Crustacea: Branchiopoda) is an important group of micro-crustaceans predominantly inhabiting continental water bodies of different, if not all, types [1]. Among the most famous peculiarities of these animals are their sexually produced diapausing eggs, which are resistant to desiccation and other unfavourable conditions and are important propagules for passive dispersal by different modes, i.e. by birds [1], [2]. Their strong ability to survive passive dispersal was one reason why cladoceran species’ distributions were for a long time accepted as cosmopol- itan, but since the 1970’s this concept has changed radically to the so-called non-cosmopolitanism, or ‘‘continental endemism’’ [3], [4], [5], [6]. The correctness of this idea is now confirmed for some genera and species groups [6], [7], [8], [9], although the real diversity and distribution of taxa in other groups needs to be accurately studied. Some cladocerans, such as species of the genus Simocephalus Scho ¨dler, 1858 (family Daphniidae Straus, 1820), are used as environmental indicators and ‘‘standard’’ test objects in toxico- logical studies [10], [11]. Representatives of this genus are very common in vegetation, the open littoral zones of ponds and lakes, the semi-static affluents of rivers and pools and puddles of various types. Based on morphological characters, Orlova-Bienkowskaja [9] recognized 20 valid species in this genus belonging to five subgenera: Simocephalus s. str., Simocephalus (Coroncephalus), Simocephalus (Acutirostratus), Simocephalus (Echinocaudus), and Simocephalus (Aquipiculus). Many of the taxa were regarded by Orlova-Bienkowskaja [9] as junior synonyms of species described earlier. Several species of Simocephalus were identified and then re- described by Chinese authors [12], [13], [14], [15], [16], [17], [18]. Only Simocephalus heilongjiangensis Shi & Shi, 1994, which is widely distributed in the tropics, was regarded as a valid species by Orlova-Bienkowskaja [9]. Among the taxa synonymized by Orlova-Bienkowskaja [9], there were several, earlier-described species from China, such as S. himalayensis Chiang & Chen, 1974 and S. beianensis Shi & Shi, 1994. In addition, S. himalayensis microdus Chen, Shi & Shi, 1992 was not discussed by Orlova- Bienkowskaja [9], and its taxonomic status remains unclear. Therefore, there is a conflict that needs to be resolved in the understanding of the taxonomy of the genus between Western investigators, who mainly follow Orlova-Bienkowskaja [9], and Chinese researchers. Near the end of the 20 th century, a powerful new tool for testing taxonomic hypotheses, molecular phylogenetics, became available. In cladocerans, it was mainly applied to species of different Daphnia groups [5], [19]. However, molecular phylogenetic studies were subsequently conducted for some other genera and families of the cladocerans [6], [20], [21], [22]. PLOS ONE | www.plosone.org 1 November 2014 | Volume 9 | Issue 11 | e112808