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 speculate 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 investigation, the two specimens were also collected from endorheic saline lakes, Dum tso and Dong tso. The main water parameters of the two lakes were compiled in Table1.

Phylogenetic analyses (Figs. 2, 3) The nuclear SSU rDNA sequences aligned in this study consisted of 1670 base pairs. A total of 500 sites in these nucleotides were variable, in which 395 sites were parsimoniously informative and 102 sites were singleton sites. The average contents of A, T, C, and G were 24.91%, 25.80%, 21.60%, and 27.69%, respectively, of which A + T (50.71%) was greater than G + C (49.29%). The transition/transversion ratio was 1.5. The nuclear tufA sequences aligned in this study consisted of 763 base pairs. A total of 437 sites in these nucleotides were variable, in which 371 sites were parsimoniously informative and 66 sites were singleton sites. The average contents of A, T, C, and G were 34.98%, 31.01%, 13.59%, and 20.42%, respectively, of which A + T (65.99%) was greater than G + C (34.01%). The transition/ transversion ratio was 0.85.

CTENOCLADUS CIRCINNATUS FROM CHINA Phytotaxa 260 (1) © 2016 Magnolia Press • 77 TABLE 1. Water chemical parameters of two sampling sites Charaters Dum tso Dong tso TP (mg/L) 0.0998 0.1396 SRP (mg/L) 0.1006 0.0258 TN (mg/L) 0.4418 1.0898 CODMn (mg/L) 6.1008 1.3978 NH3+-N (mg/L) 0.0884 0.0000 NO2 N (mg/L) 0.0030 0.0030 Cond. (ìS/cm) 17.8100 - TDS (mg/L) 10.1400 - Salinity (%) 10.5500 - pH 9.9000 - T (°C) 10.6 12.3

+ “-” = no data; TP =total phosphorus; SRP = soluble reactive phosphorus TN = total nitrogen; CODMn = chemical oxygen demand; NH3 -N - = ammonium; NO2 -N = nitrite; Cond. = conductivity; TDS = total dissolved solids.

FIGURE 2. ML and Bayesian phylogenetic tree constructed for the SSU rDNA sequences of the Ulvophyceae. The numbers on the nodes represent the posterior probabilities (PP)/bootstrap support values(BP) above 50/0.50. The sequences obtained in our study are shaded in gray.

78 • Phytotaxa 260 (1) © 2016 Magnolia Press LIU ET AL. FIGURE 3. ML and Bayesian phylogenetic tree constructed for the tufA sequences of the Ulvophyceae. The numbers on the nodes represent the posterior probabilities (PP)/bootstrap support values(BP) above 50/0.50. The sequences obtained in our study are shaded in gray.

Phylogenetic trees using Bayesian and ML showed similar topology to previous studies (Carlile et al. 2011, Škaloud et al. 2013), and resolved both orders as reciprocally monophyletic, with moderate-strong support. Phylogenetic analyses of these sequences resolved six currently recognized families in the Ulvales, plus six lineages within the Ulotrichales. The alignment of 53 SSU rDNA sequences and 47 tufA sequences were used to determine the unambiguous phylogenetic placement of the genus Ctenocladus within the order Ulvales (Ulvophyceae) with a high support value (BP/PP, 100/1.00). Phylogenetic trees of SSU rDNA showed that the genus Ctenocladus and genus Pseudendoclonium (paraphyletic) formed a discrete clade, which had a close relationship with the genus Phaeophila, and formed a lineage separated earlier from other lineages (Fig. 2). To further resolve the placement of Ctenocladus in the Ulvales, analyses were also conducted on the tufA sequence data, which were different from the SSU rDNA sequence data (Fig. 3). The genus Ctenocladus as a separate clade had a low support value (BP/PP, 54/0.64). The genus Ctenocladus and Phaeophila did not form a sister clade within the Ulvales.

In previous research, most phycologists have mainly focused on morphological features, such as the size of thalli, akinetes and zoosporangia, the shape of cells and the obvious chain-like akinetes (Printz 1964, Starmach 1972). According to our observations, the C. circinnatus specimens sampled from Tibet were similar to 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, slightly different to our specimens. Importantly, those studies showed C. circinnatus with different habitats, such as saline lakes or archaeological sites. This means this species may have a wide adaptability or those may be different species with very similar morphology. A previous study showed that observations on C. circinnatus also from Tibet, were consistent with our findings (Li et al. 1992). All the available evidence suggested that Ctenocladus consists of a very polymorphic species (Tsarenko 2011, Wehr et al. 2015). In our opinion, the environmental characteristics of saline lakes, same of original description,, are important for growth and development of the species and such slight differences in morphology may be attributed to its high demands for salinity and pH values, and its unique habitat at an altitude of about 4750 m. Examination of the ultrastructural characteristics supported the placement of this genus in Ulvophyceae (Stewart et al. 1973, Blinn & Morrison 1974). Reproductive cells (zoosporangia) of Ctenocladus contained eight or more zoospores (Figs. 1D, 1E), released through a papilla at the apical end of the cell. This morphological evidence may confirm the findings from ultrastructural analysis that Ctenocladus should be classified in Ulvophyceae. Molecular data including SSU rDNA and the chloroplast encoded tufA, and ultrastructural features with strong support showed that the genus Ctenocladus is a member of Ulvales (Ulvophyceae). Furthermore, Ctenocladus and some Pseudendoclonium species (HF570952, KM020043) formed a discrete clade which had a close relationship with the genus Phaeophila, and formed a lineage separated earlier from other lineages (Fig. 2). However, analyses conducted on the tufA sequence data were different from the SSU rDNA sequence data (Fig. 3). The genus Ctenocladus and Phaeophila did not form a sister clade in Ulvales. To date, molecular phylogenetic reconstructions have revealed six main lineages within Ulvales, but the branching order Ulvales among the six lineages is currently unresolved (O’Kelly et al. 2004). The limited DNA sequences of genus Phaeophila may account for the differences between our SSU rDNA phylogeny and tufA phylogeny. We speculate that there are still undescribed or cryptic species, especially in freshwater and other non-marine habitats, thus it is premature to make judgments regarding higher level classification, although Ctenocladus has a close relationship with Pseudendoclonium and Phaeophila. Our study clearly showed that the genus Ctenocladus should be classified in Ulvales, has a close relationship with the genera Pseudendoclonium and Phaeophila, and formed a lineage separated earlier from other lineages, which were consistent with ultrastructural analysis. A further investigation based on large samples of microfilamentous Ulvophycean algae, especially freshwater specimens, is necessary for a detailed molecular phylogenetic re-evaluation.

ACKNOWLEDGMENTS

We would like to gratefully thank professor Dekui He and Dr. Xiong Xiong for their technical help. Special thanks also go to two anonymous reviewers. This research was funded by the Special foundment of Science and technology basic work of China (Grant No. 2014FY210700) and the National Natural Science Foundation of China (Grant No. 31270252).

REFERENCES

Ariño, X., Hernández-Mariné, M. & Saiz-Jiménez, C. (1996) Ctenocladus circinnatus (Chlorophyta) in stuccos from archaeological sites of southern Spain. Phycologia 35: 183–189. http://dx.doi.org/10.2216/i0031-8884-35-3-183.1 Borzi, A. (1883) Saggio di ricerche Sulla biologia delle alghe. Ctenocladus, gen. nov. Studi Algologici 1: 27–50. http://dx.doi.org/10.2216/i0031-8884-35-3-183.1 Blinn, D.W. (1971) Autecology of a filamentous alga, Ctenocladus circinnatus (Chlorophyceae), in saline environments. Canadian Journal of Botany 49: 735–743.

80 • Phytotaxa 260 (1) © 2016 Magnolia Press LIU ET AL. http://dx.doi.org/10.1139/b71-112 Blinn, D.W. & Morrison, E. (1974) Intercellular cytoplasmatic connections in Ctenocladus circinnatus Borzi (Chlorophyceae) with possible ecological significance. Phycologia 13: 95–97. http://dx.doi.org/10.2216/i0031-8884-13-2-95.1 Blinn, D.W. & Stein, J.R. (1970) Distribution and taxonomic reappraisal of Ctenocladus (Chlorophyceae: Chaetophorales). Journal of Phycology 6: 101–105. http://dx.doi.org/10.1111/j.0022-3646.1970.00101.x Bourrelly, P. (1966) Les Algues d’ Eau Douce I. Les Algues Vertes. Boubée, Paris, 511 pp. Bourrelly, P. (1990) Les Algues d’ Eau Douce I. Les Algues Vertes. Boubée, Paris, 511 pp. Carlile, A.L., O’Kelly, C.J. & Sherwood, A.R. (2011) The green algal genus Cloniophora represents a novel lineage in the Ulvales: a proposal for Cloniophoeaceae fam. nova. Journal of Phycology 7: 1379–1387. http://dx.doi.org/10.1111/j.1529-8817.2011.01065.x Famà, P., Wysor, B., Kooistra, W.H.C.F. & Zuccarello, G.C. (2002) Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. Journal of Phycology 38: 1040–1050. http://dx.doi.org/10.1046/j.1529-8817.2002.t01-1-01237.x Gouy, M., Guindon, S. & Gascuel, O. (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Molecular Biology and Evolution 27: 221–224. http://dx.doi.org/10.1093/molbev/msp259 Guiry, M.D. & Guiry, G.M. (2015) AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. Available from: http://www.algaebase.org (accessed 19 December 2015) Honda, D., Yokochi, T., Nakahara, T., Raghukumar, S., Nakagiri, A., Schaumann, K. & Higashihara, T. (1999) Molecular phylogeny of Labyrinthulids and Thraustochytrids based on sequencing of 18S ribosomal RNA gene. Journal of Eukaryotic Microbiology 46: 637–647. http://dx.doi.org/10.1111/j.1550-7408.1999.tb05141.x Hu, H.J. & Wei, Y.X. (2006) The freshwater algae of China: Systematics, Taxonomy and Ecology. Science Press, Beijing, 1023 pp. Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755. http://dx.doi.org/10.1093/bioinformatics/17.8.754 Katoh, K. & Standley, D.M. (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30: 772–780. http://dx.doi.org/10.1093/molbev/mst010 Li, Y.Y., Wei,Y.X., Shi, Z.X. & Hu, H.J. (1992) The Series of scientific expedition to Qinghai-Xizang plateau-the algae of the Xixang plateau. Academic Press, Beijing, 509 pp. Li, S.H. & Bi, L.J. (1998) Flora Algarum Sinicarum Aquae Dulcis-Tomus V Charophyta. Academic Press, Beijing, 136 pp. Mattox, K.R. & Steward, K.D. (1984) Classification of the Green Algae: A concept based on Comparative Cytology. In: Irvine, D.E. & John, D.M. (Eds.) Systematics of the Green Algae. Academic Press, London, pp. 29–72. Nielsen, R., Petersen, G., Seberg, O., Daugbjerg, N., O’Kelly, C.J. & Wysor, B. (2013) Revision of the genus Ulvella (Ulvellaceae, Ulvophyceae) based on morphology and tufA gene sequences of species in culture, with Acrochaete and Pringsheimiella placed in synonymy. Phycologia 52: 37–56. http://dx.doi.org/10.2216/11-067.1 O’Kelly, C.J., Wysor, B. & Bellows, W.K. (2004) Gene sequence diversity and the phylogenetic position of algae assigned to the genera Phaeophila and Ochlochaete (Ulvophyceae, Chlorophyta). Journal of Phycology 40: 789–799. http://dx.doi.org/10.1111/j.1529-8817.2004.03204.x Posada, D. & Crandall, K.A. (1998) Modeltest: Testing the model of DNA substitution. Bioinformatics 14: 817–818. http://dx.doi.org/10.1093/bioinformatics/14.9.81 Printz, H. (1964) Die Chaetophoralen der Binnengewässer. Hydrobiologia 24: 1–376. Škaloud, P., Nedbalová, L., Elster, J. & Komárek, J. (2013) A curious occurrence of Hazenia broadyi spec. nova in Antarctica and the review of the genus Hazenia (Ulotrichales, Chlorophyceae). Polar Biology 36: 1281–1291. http://dx.doi.org/10.1007/s00300-013-1347-z Smith, G.M. (1950) The Fresh-Water Algae of the United States. McGraw-Hill, New York, 719 pp. Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312–1313. http://dx.doi.org/10.1093/bioinformatics/btu033 Starmach, K. (1972) Clorophyta 3, Zielenice Nitkowate. In: Starmach, K. & Sieminska, J. (Eds.) Flora slodkovodna Polski. Warsawa- Krakow, pp. 450–452.

CTENOCLADUS CIRCINNATUS FROM CHINA Phytotaxa 260 (1) © 2016 Magnolia Press • 81 Stewart, K.D., Mattox, K.R. & Floyd, G.L. (1973) Mitosis, cytokinesis, the distribution of plasmodesmata and other cytological characteristics in the Ulotrichales, Ulvales and Chaetophorales. Phylogenetic and taxonomic considerations. Journal of Phycology 9: 128–14l. http://dx.doi.org/10.1111/j.1529-8817.1973.tb04068.x SEPA. (2002) Water and wastewater monitoring and analysis method (4th ed.). Environmental Science Press, Beijing, 836 pp. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739. http://dx.doi.org/10.1093/molbev/msr121 Tsarenko, P.M. (2011) Chlorophyta. In: Tsarenko, P.M., Wasser, S.P. & Nevo, E. (Eds.) Algae of Ukraine: diversity, nomenclature, taxonomy, ecology and geography. A.R.G. Gantner, Ruggell, Liechtenstein, 713 pp. Vieira, J. & Messing, J. (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268. http://dx.doi.org/10.1016/0378-1119(82)90015-4 Woronochin, N.N. & Popova, T.G. (1929) Lochmiopsis a new genus of alga from the family Leptosireae (in Russian). Botanical Oksch 3: 1–9. Wehr, J.D., Sheath, R.G. & Kociolek, P.J. (2015) Freshwater algae of North America: Ecology and Classification. Elsevier, London, 1050 pp.