Cladocera: Daphniidae
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Zoological Studies 51(2): 222-231 (2012) Systematic Study of the Simocephalus Sensu Stricto Species Group (Cladocera: Daphniidae) from Taiwan by Morphometric and Molecular Analyses Shuh-Sen Young1,*, Mei-Hui Ni2, and Min-Yun Liu3 1Department of Applied Science, National Hsinchu University of Education, Hsinchu 300, Taiwan 2Hsinchu Municipal Hsinchu Elementary School, Hsinchu 300, Taiwan 3National Applied Research Laboratories, Taiwan Ocean Research Institute, Taipei 106, Taiwan (Accepted September 9, 2011) Shuh-Sen Young, Mei-Hui Ni, and Ming-Yun Liu (2012) Systematic study of the Simocephalus sensu stricto species group (Cladocera: Daphniidae) from Taiwan by morphometric and molecular analyses. Zoological Studies 51(2): 222-231. There is some controversy regarding the traditional taxonomy of the Simocephalus sensu stricto species group. We conducted molecular and morphometric analyses to differentiate the 3 species from this group found in Taiwan: S. vetulus (O.F. Müller, 1776), S. vetuloides Sars, 1898, and S. mixtus Sars, 1903. The landmark method was employed, followed by a transfer into 24 characteristic values for a principal component analysis (PCA), the results of which indicated morphometric overlap among these species. The dorsal angle, brood size, and body length were smallest in S. vetulus, medium in S. vetuloides, and largest in S. mixtus. In the Simocephalus sensu stricto group from Taiwan, the dorsal angle and body length were significantly correlated with brood size in a quadratic manner. In the molecular analysis, 98 specimens of Simocephalus were used, and the 641-bp mitochondrial DNA cytochrome oxidase subunit 1 sequence was employed as a marker to analyze the genetics of S. vetulus, S. vetuloides, S. mixtus, S. serrulatus (Koch, 1841), and S. heilongjiangensis Shi and Shi, 1994. Simocephalus vetulus, S. vetuloides, and S. mixtus shared several haplotypes, and the interspecific genetic distance was merely 0.00671-0.00785, which is within the range of intraspecific differences. We concluded that S. vetulus, S. vetuloides, and S. mixtus in Taiwan belong to the same species and should be treated as S. cf. vetulus. The number of species of Simocephalus in Taiwan is thus reduced to 3: S. cf. vetulus, S. serrulatus, and S. heilongjiangensis. http://zoolstud.sinica.edu.tw/Journals/51.2/222.pdf Key words: Systematics, Biodiversity, Simocephalus, Freshwater zooplankton. The general morphologies of Simocephalus 1998, Orlova-Bienkowskaja 2001, Tuo 2002). vetulus (O.F. Müller, 1776), S. vetuloides Sars Other authors treated S. vetuloides as a local form 1898, and S. mixtus Sars 1903 are very similar. (Johnson 1953) or as a synonym of S. vetulus Sars (1916) first discriminated S. vetulus and (Fryer 1957, Harding 1961, Sharma 1978, Negrea S. vetuloides based on the dorsoposterior valve 1983, Michael and Sharma 1988). Sars (1903) angle. After that, many authors defined S. described S. mixtus as having a more-protruding (to vetuloides by a more-protruding dorsal valve the rear) dorsal valve margin and larger denticles margin and more-numerous and larger denticles on the posterior dorsal valve margin compared to on the posterior dorsal valve margin compared S. vetulus and S. vetuloides. Flössner (1972) and to S. vetulus (Uéno 1966, Chiang and Du 1979, Negrea (1983) treated S. mixtus as a synonym of Yoon and Kim 1987 2000, Shi and Shi 1996, Kim S. vetulus. After that, Orlova-Bienkowskaja (1998) *To whom correspondence and reprint requests should be addressed. E-mail:[email protected] 222 Young et al. – Study of Simocephalus from Taiwan 223 made a more-detailed revision and treated S. large, with the exception of the shape of the dorsal mixtus as a valid species. valve. However, the shape of the dorsal valve Orlova-Bienkowskaja (2001) proposed a of cladocerans may be affected by the brooding different method of discriminating S. vetulus, S. status, with growing embryos pushing the valve vetuloides, and S. mixtus. She drew an inner circle more prominently outwards, than in individuals along the shell posterior, the diameter of which without eggs. and the prominence of the dorsal valve being key The species level is recognized as the features for identification. The shell posterior of basic unit of biodiversity (Mayer and Ashlock S. vetulus ends without an extending shell spine, 1991). Nowadays, alpha taxonomy is still based the inner circle is larger than in S. vetuloides and mainly on morphology. Morphometry is one of S. mixtus, while S. mixtus has more-protruding several possible methods to determine species dorsal valves than S. vetuloides. The diameter and analyze morphological differences between of the inner circle of S. mixtus is larger than the closely related species and populations (Chen et prominence portion, and S. vetuloides differs from al. 2010). With the advent of molecular technology S. mixtus in that the diameter of the inner circle of S. for DNA sequencing, morphologically cryptic vetuloides is smaller than the prominence portion. species have been increasingly revealed, and the In the past, many authors proposed S. vetulus use of DNA markers as a new tool to overcome to be a cosmopolitan species first des-cribed from morphological impediments was suggested (Tautz the Old World, as it was found in many areas, et al. 2003). The ideal DNA-based identification with the exception of New Zealand and Australia system (DNA barcodes) would employ a single (Werestschagin 1923, Uéno 1927, Rylov 1930, gene, and be suitable for any organism in the Hemsen 1952, Harding 1961, Manuilova 1964, taxonomic hierarchy. Folmer et al. (1994) de- Uéno 1966, Chiang and Du 1979, Rajapaksa and signed a universal primer for the mitochondrial Fernando 1982, Boonsom 1984, Yoon and Kim cytochrome oxidase subunit I (COI) gene, which 1987, Kim 1998, Mizuno and Takahashi 1991, Du subsequently became a popular marker to study 1993, Hann 1995, Shi and Shi 1996, Michael and invertebrates. Hebert et al. (2003), Tautz et al. Sharm 1998, Tuo 2002). Orlova-Bienkowskaja (2003), Blaxter (2004), Lefébure et al. (2006), (2001) indicated that the distribution of S. vetulus and Costa et al. (2007) suggested that the COI was limited to northern Africa and Europe, gene appears to be an appropriate molecular while S. vetuloides had a limited distribution in marker (as a DNA barcode) on several taxonomic eastern Siberia. Outside of Africa, Europe, and scales, but particularly at the species level. We eastern Siberia, Simocephalus sensu stricto attempted to clarify the taxonomic status of S. comprises S. punctatus Orlova-Bienkowskaja, vetulus, S. vetuloides, and S. mixtus in Taiwan 1998, S. elizabethae (King, 1853), and S. by morphometric comparisons and used the mixtus. Simocephalus mixtus is a cosmopolitan mitochondrial (mt)DNA COI gene marker as a new species distributed in Asia, Eastern Europe, character. North Africa, and North America. Simocephalus This paper is our 1st step dealing with (Coronocephalus) serrulatus (Koch, 1841) is vetulus-like populations of Simocephalus in Taiwan, also regarded as a cosmopolitan species, as which are currently regarded as conspecific to it is distributed in Asia, Europe, Africa, North the Palaearctic cosmopolitan species. We thus America, South America, and Australia (Orlova- attempted to improve the taxonomy of the genus Bienkowskaja 2001). Simocephalus by solving a small piece of the Based on the description by Orlova- puzzle from the overall picture. Bienkowskaja (2001) and other morphological comparisons, Tuo (2002) described 3 species of Simocephalus from Taiwan, S. serrulatus, MATERIALS AND METHODS S. vetulus, and S. vetuloides. Since then, this extensive collection has increased to include S. Samples were taken from many temporary heilongjiangensis Shi and Shi, 1994 and S. mixtus freshwater bodies throughout Taiwan using a Sars from southern Taiwan (Ni 2005). At some plankton net. Each sample was fixed in 70% collection sites, S. vetulus and S. vetuloides were ethanol (EtOH), later preserved in 95% EtOH and found simultaneously as were S. vetuloides and stored at a low temperature (< -20°C). Within 72 h, S. mixtus (Ni 2005). Morphological similarities each raw sample was sorted and identified under among S. vetulus, S. vetuloides, and S. mixtus are a stereomicroscope. In total, 187 individuals (170 224 Zoological Studies 51(2): 222-231 (2012) with eggs) were collected in 2003 and 2004 and Hudec, 1991 (Daphniidae) and Diaphanosoma used for the morphometric analysis: 45 individuals dubium Manuilova, 1964 (Sididae) from Taiwan of S. vetulus from 8 sites, 72 individuals of S. were analyzed in order to obtain outgroup vetuloides from 11 sites, and 70 individuals of sequences. S. mixtus from 10 sites. From this set of 187 individuals, 72 individuals, including 22 individuals Morphometric analysis of S. vetulus from 8 sites, 28 individuals of S. vetuloides from 11 sites, and 22 individuals of S. Lateral-view images of S. vetulus, S. mixtus from 10 sites, were selected for the DNA vetuloides, and S. mixtus were taken using a analysis. Additionally, 7 individuals of S. serrulatus digital camera under a stereomicroscope for the (Fig. 1) from 3 sites and 19 individuals of S. morphometric study. Morphometric characters heilongjiangensis (Fig. 1) from 5 sites were also were extracted from the photographic images, and included in the DNA analysis. Daphnia similoides 8 morphometric data points were used to construct (A) (B) (C) (D) (E) Fig. 1. General morphology of female Simocephalus with summer eggs found in Taiwan (all drawings are original). (A) S. vetulus; (B) S. vetuloides; (C) S. mixtus; (D) S. serrulatus; (E) S. heilongjiangensis. The valve shape is the major difference among S. vetulus, S. vetuloides, and S. mixtus; S. serrulatus has teeth on the top of its head, and S. heilongjiangensis has a different posterior end of the valve. Scale bars = 0.1 mm. Young et al. – Study of Simocephalus from Taiwan 225 24 length measurements, each of which was animals. Each animal was taken from 95% EtOH divided by body length to obtain size-free ratios and placed into pure water for 1 h for cleaning.