Phytotaxa 260 (1): 075–082 ISSN 1179-3155 (print edition) http://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2016 Magnolia Press Article ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.260.1.8 Phylogenetic Position and Morphological Observation of the Ctenocladus circinnatus Borzi, a rare green alga from Changtang Plateau, China BENWEN LIU1, 2, XUDONG LIU1, 2, ZHENGYU HU3, HUAN ZHU1 & GUOXIANG LIU1* 1Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, People’s Republic of China 2University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China 3State key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, People’s Republic of China * Corresponding author ([email protected]) Abstract Two microfilamentous green algal specimens from Tibet were identified as Ctenocladus circinnatus Borzi, due to their unique morphology and saline habitat. The phylogenetic evidence based on DNA sequence data from the nucleus (SSU) and chloroplast elongation factor TU (tufA) sequences clearly revealed that the genus Ctenocladus, which has been classified in the Chaetophorales (Chlorophyceae) or Trebouxiophyceae incertae sedis by most phycologists, should be included in the Ulvales (Ulvophyceae) instead, and has a close relationship with the genera Pseudendoclonium and Phaeophila. We specu- late that there may be undescribed or cryptic species especially in freshwater and other non-marine habitats. A phylogenetic re-evaluation based on large samples of microfilamentous ulvophycean algae especially freshwater specimens is needed. Keywords: Ctenocladus circinnatus, Changtang Plateau, Phaeophila, Pseudendoclonium, Ulvophyceae Introduction The relatively rare green algae Ctenocladus was first described in Italy (Borzi 1883). Since then, the distribution of Ctenocladus has been recorded in several sites (Ariño et al. 1996, Blinn 1971, Li et al. 1992). This branched filamentous alga is mainly found in inland water, with high salinity, pH, temperature, Na+/Mg2+ ratio and other ecological factors. The taxonomic position of Ctenocladus varies according to different phycologists. Ctenocladus circinnatus was separated into Lochmiopsis sibirica and Lochmiopsis printzii by Woronochin (Woronochin & Popova 1929). While Smith (1950) and Bourrelly (1966) combined Ctenocladus and Lochmiopsis, adopting the original name, Ctenocladus. Printz (1964) classified Ctenocladus as a section of the Gongrosira. According to the system of Bourrelly (Bourrelly 1990), Li & Bi (1998) classified the genus Ctenocladus based on specimens collected from Tibet in the family Chaetophoraceae. Both Tsarenko (2011) and Guiry & Guiry (2015) placed it in Chlorellales (Trebouxiophyceae), and Wehr et al. (2015) also classified in Trebouxiophyceae but with uncertain position. Ultrastructural evidence provided new taxonomic clues in this genus. Blinn & Morrison (1974) found pit-like intercellular connections in Ctenocladus which resembled those in Trentepohlia. Mattox & Stewart (1984) classified Ctenocladus in Ulvophyceae based on the ultrastructure of flagellar apparatus. Although this green algal classification based on ultrastructure of the basal body in flagellated cells and cytokinesis was proved by molecular phylogeny, few studies focused on the molecular phylogeny of Ctenocladus. Moreover, the relationship between Ctenocladus and other algal groups in Chlorophyta is unknown. Recent studies have shown that there may be cryptic lineages in microfilamentous algae with similar morphology in Ulvophyceae such as Hazenia and Pseudendoclonium (Škaloud et al. 2013), and that morphology may not be a good indicator of phylogenetic relatedness such as in Ulvella (O’Kelly et al. 2004, Nielsen et al. 2013). Moreover, molecular data for Ctenocladus are absent. Taking all these facts into account, the present study is aimed to determine the phylogenetic relationship of Ctenocladus with other Chlorophytes based on specimens from two saline lakes in Tibet. Accepted by Marina Aboal: 12 Apr. 2016; published: 9 May 2016 75 MATERIALS AND METHODS Sampling The Ctenocladus samples used in this study were collected in June 2014 from two endorheic lakes, Dong tso (31° 35’ 37” N, 91° 07’ 30” E) and Dum tso (32° 07’ 29” N, 84° 53’ 51” E ), in the west of Tibet, China. Each sample was preserved in 100% alcohol and frozen at –20℃ for DNA extraction and 4% formalin for morphological study. These two voucher specimens were deposited in the Freshwater Algal Herbarium (IHB), under the accession number TB2014012 and TB2014062 . The water parameters of the two lakes were measured following the standard protocols (SEPA 2002). DNA extraction, PCR amplification and Sequencing Genomic DNA was extracted using an Axygen DNeasy plant Kit (Axygen Biotechnology, Hangzhou, China) according to the manufacturer’s specifications after approximately 15 mg of filaments were added to 1 mL of 0.5mm glass beads and 350 μl of phosphate buffer solution (PBS, pH 7.0). The algal cells were lysed by bead beating at 4800 rpm for 2 min in a mini-beadbeater (Model 3110BX, Biospec Products, Bartlesville, Oklahoma USA). Universal primers (Honda et al. 1999, Famà et al. 2002) were used to amplify the partial nuclear-encoded SSU rDNA and tufA sequences, respectively. The sequence amplification profile consisted of an initial 5 min denaturing at 94℃, 34 cycles of denaturing at 94℃ for 1 min each cycle, 50 s annealing at 56°C (SSU rDNA) and 53°C (tufA), 80s extension at 72℃ and a final extension of 5 min at 72℃. The excised PCR products were cloned into a pMD18-T vector and transferred into E. coli competent cells DH5α (Takara Bio Inc., Otsu, Shiga, Japan). Twenty clones of SSU rDNA sequence and tufA sequence were sent to WuHan Tsingke BioTech Co., Ltd. (WuHan, China) for sequencing, respectively. Universal sequencing primers were M13F and M13F (Vieira & Messing 1982). Phylogenetic analyses Sequences were selected from GenBank (http://www.ncbi.nlm.nih.gov/) for nuclear SSU rDNA and chloroplast tufA analyses. Together with 53 (SSU rDNA) and 47 (tufA) published sequences representing the Ulvophyceae, four new Ctenocladus SSU rDNA and tufA sequences were subjected to mafft7.2 (Katoh & Standley 2013) for initial alignments, and refined manually with Seaview v. 4.32 (Gouy et al. 2010). Base composition and transition/transversion ratio were calculated by MEGA5.0 (Tamura et al. 2011). ModelTest3.72 (Posada & Crandall 1998) was used to select the evolutionary best-fit model according to hierarchical likelihood ratio tests and Akaike information criterion. The best-fit model for SSU rDNA and tufA was GTR+I+G. Phylogenetic trees using maximum likelihood (ML) and Bayesian were constructed with RAxML8.0 (Stamatakis 2014) and with MrBayes3.1.2 (Huelsenbeck & Ronquist- 2001). Bootstrap analyses with 1000 replicates of the dataset for ML were performed to estimate statistical reliability. Bayesian analyses of both SSU rDNA and tufA sequences were performed with 2.0×106 generations of Markov chain Monte Carlo iterations and trees were sampled every 1×103 generations. It was assumed that the stationary distribution was reached when average standard deviation of split frequencies between two runs was lower than 0.01. The first 25% of the calculated trees was discarded as burn- in, and the remaining samples were used to construct a Bayesian consensus tree and to infer posterior probabilities. The bootstrap values and posterior probabilities were presented on the nodes. The resulting phylogenetic trees were edited using Figtree 1.4.2 (http://tree.bio.ed. ac.uk/software/figtree/). RESULTS Morphological Observation Ctenocladus circinnatus A. Borzi. Saggio di ricerche Sulla biologia delle alghe. Ctenocladus, gen. nov. Studi Algologici. 1883, 1: 27–50. Description: The thalli of C. circinnatus were composed of numerous radially arranged filaments with unilateral branching, without mucilage. The cells were cylindrical, 6–8 μm wide and 28–85 μm long, uninucleate, with a parietal plastid and one to three pyrenoids. Terminal vegetative cells usually produced thick-walled akinetes, which were spherical or approximately spherical with a diameter of 10–21 μm, giving rise to chain-like rows. Zoosporangia were 76 • Phytotaxa 260 (1) © 2016 Magnolia Press LIU ET AL. irregularly spherical, containing eight or more zoospores and were released at the apical end of the cell (Fig. 1). The C. circinnatus specimens sampled from Tibet, China were slightly different from the original description and illustrations (Printz 1964, Starmach 1972, Ariño 1996). Printz (1964) and Starmach (1972) reported cells 10–15 μm in diameter and Ariño (1996) reported cells 3–5 μm in diameter and 20–150 μm long. A previous study showed that C. circinnatus from Tibet was consistent with our observations (Li et al. 1992). FIGURE 1. Morphological observation of Ctenocladus specimens. A. Cytoplasm concentration on the top of the filaments; B. Random arrangement of filaments and numerous zoosporangia; C. Chains of thick-walled akinetes; D. E. Zoosporangia; F. Detail of the branching pattern. Scale bars: A–C = 40 μm, D = 10 μm, E = 20 μm, F = 40 μm. Arrows in B and D are presumptive zoosporangia and in C is presumptive zkinetes. A, akinetes; Z, zoosporangia. Distribution: Ctenocladus has been recorded world-wide (Russia, USA, Canada, Italy, Peru and China) (Blinn & Stein 1970, Li et al. 1992, Hu & Wei, 2006), but only restricted to inland habitats. In present