Research Article Algae 2016, 31(2): 155-165 http://dx.doi.org/10.4490/algae.2016.31.4.14 Open Access tufA gene as molecular marker for freshwater Chlorophyceae Helena Henriques Vieira1,*, Inessa Lacativa Bagatini1, Carla Marques Guinart2 and Armando Augusto Henriques Vieira1 1Ecology and Natural Resources Post-graduation Program (PPGERN), Laboratory of Phycology, Botany Department, Federal University of São Carlos, São Carlos, SP 13565-905, Brazil 2Molecular Laboratory of Biodiversity and Conservation, Genetic and Evolution Department, Federal University of São Carlos, São Carlos, SP 13565-905, Brazil Green microalgae from the class Chlorophyceae represent a major biodiversity component of eukaryotic algae in con- tinental water. Identification and classification of this group through morphology is a hard task, since it may present cryptic species and phenotypic plasticity. Despite the increasing use of molecular methods for identification of micro- organisms, no single standard barcode marker is yet established for this important group of green microalgae. Some available studies present results with a limited number of chlorophycean genera or using markers that require many dif- ferent primers for different groups within the class. Thus, we aimed to find a single marker easily amplified and with wide coverage within Chlorophyceae using only one pair of primers. Here, we tested the universality of primers for different genes (tufA, ITS, rbcL, and UCP4) in 22 strains, comprising 18 different species from different orders of Chlorophyceae. The ITS primers sequenced only 3 strains and the UCP primer failed to amplify any strain. We tested two pairs of primers for rbcL and the best pair provided sequences for 10 strains whereas the second one provided sequences for only 7 strains. The pair of primers for the tufA gene presented good results for Chlorophyceae, successfully sequencing 21 strains and recovering the expected phylogeny relationships within the class. Thus, the tufA marker stands out as a good choice to be used as molecular marker for the class. Key Words: ITS; molecular marker; phylogeny; rbcL; tufA; UCP4 Abbreviations: BLAST, Basic Local alignment Search Tool; CBOL, Consortium for the Barcode of Life; CCMA, Freshwater Microalgae Culture Collection (in Portuguese acronymic); COXI, cytochrome oxidase I; GTR, general-time-reversible nucleotide substitution model; ISS, Index of Substitution Saturation; ISSc, Index of Substitution Saturations critic; ITS, internal transcribed spacer; MCMC, Monte Carlo Makov Chain; NCBI, National Center for Biotechnology Information; OCC, Oedogoniales Chaetopeltidales Chaetophorales; PCR, polymerase chain reaction; rbcL, large unit ribulose bispho- sphate carboxylase (gene); SC, Sphaeropleales Chlamydomonadales; tufA, elongation factor tu (gene); UCP, universal chlorophyte primers This is an Open Access article distributed under the Received December 20, 2015, Accepted April 14, 2016 terms of the Creative Commons Attribution Non-Com- Corresponding Author mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which * permits unrestricted non-commercial use, distribution, and reproduction E-mail: [email protected] in any medium, provided the original work is properly cited. Tel: +55-16-3351-8311, Fax: +55-16-3351-8308 Copyright © 2016 The Korean Society of Phycology 155 http://e-algae.org pISSN: 1226-2617 eISSN: 2093-0860 Algae 2016, 31(2): 155-165 INTRODUCTION The most constructive results achieved so far have focused in phylogenetic questions for genera within the The class Chlorophyceae comprises approximately class (Van Hannen et al. 2000, Hall et al. 2010, Fučíková et 3,496 described species, according to Algaebase and is al. 2011, McManus and Lewis 2011), therefore there is no one of the most relevant phytoplankton groups in con- known marker fulfilling the requirements of a universal tinental waters. The classification of this group is often barcode marker for Chlorophyceae. hampered by the predominance of microscopic cells, fre- Besides the universality, if the recovered marker has a quently lacking obvious structures used to discriminate good phylogenetic signal, it will allow a correct identifi- species or genera. Moreover, life habits, morphologic cation of a completely unknown organism, based on its convergence favored by the unicellular form, the occur- phylogeny among others organisms already described. rence of cryptic species and asexual reproduction, which Thus, although unknown or undescribed, organisms keeps mutations that can lead to a large morphologic can be classified in lower taxonomic levels if species dis- variability (Potter et al. 1997) are factors that make the crimination is not possible, helping in culturing indepen- classification task arduous (Krienitz et al. 2001, Fawley et dent community studies, such as studies using massive al. 2006, Krienitz and Bock 2012, Leliaert et al. 2012). sequencing platforms (Reyes et al. 2012, Salipante et al. The urgency of a faster and practical classification sys- 2013, Fumagalli et al. 2014). tem drives many investigations for an efficient molecular According to the CBOL criteria of barcode applicabil- marker attending the premises of barcode concept from ity, the first step is to find primers that can recover those Consortium for the Barcode of Life (CBOL). This concept candidate molecular markers from the largest possible comprises the idea that molecular identifications should number of taxa. Thus, we aimed to test the universal- be conducted using a single pair of primers applicable in ity of primers from published studies, already tested in the most diverse groups of organisms, recovering a short other groups, for molecular markers in different orders of marker (~700 bp) with enough variation for specific dis- freshwater Chlorophyceae. Furthermore, we have built a crimination (CBOL Plant Working Group et al. 2009). phylogenetic tree with successfully sequenced marker, in There are many markers proposed for different groups, order to investigate the possibilities of its application in such as the widely used cytochrome oxidase I (COX I), the class. an official marker for some groups of animals, like fishes (Ward et al. 2005), red (Sherwood et al. 2008, Le Gall and Saunders 2010), and brown algae (McDevit and Saunders MATERIALS AND METHODS 2010), as well as diatoms (Evans et al. 2007). In green algae, COX I is too variable requiring specific Strain cultures primers to be recovered in different taxa (Fučíková et al. 2011). The amplification of this gene has failed for some All organisms are maintained in pure cultures in the chlorophycean taxa (Hall et al. 2010). Furthermore, it may Microalgae Collection at the Phycology Laboratory of the present introns (Turmel et al. 2002), hindering the design Federal University of São Carlos–Freshwater Microalgae of new primers (Saunders and Kucera 2010). Culture Collection (CCMA) (Portuguese acronym). Most Other markers are frequently used for phylogeny and strains were cultured in axenic conditions. The strains identification studies of some algal groups, such asrbc L used in this study were classified and identified according (rubisco large subunit), ITS (internal transcribe spacer), to Algaebase sensu Komárek and Fott (Komárek and Fott tufA (plastid elongation factor). Although widely used in 1983) (Table 1). Chaetophora sp. (CCMA-UFSCar 548) phylogeny of green algae, 18S rDNA (Baldauf et al. 1990, and Oedogonium sp. (CCMA-UFSCar 570) strains could Buchheim and Chapman 1991, An et al. 1999, Krienitz et not be classified further than genera. The only order from al. 2001, 2002, Shoup and Lewis 2003, Hall et al. 2010, Bu- the Chlorophyceae that could not be tested was the Chae- chheim et al. 2011) is a conserved gene (Luo et al. 2010, topeltidales, due to the lack of isolates from this order in Fučíková et al. 2011) requiring other genes to solve closely the culture collection. phylogenetic relations in green algae. Moreover, many Microalgae strains were cultivated in 100 mL Erlen- primers are necessary to recover it from different taxa meyer flasks, with Wright’s Chryptophyte medium (Guil- (Garcia et al. in press), for example, used 12 primers to lard and Lorenzen 1972), pH 7.0, 25 ± 1°C, light intensity recover 18S rRNA gene from strains of one family within of 300 µmol photons m-2 s-1 and a 12 : 12 light : dark cycle. Chlorophyceae. Cultures in exponential growth phase, determined by op- http://dx.doi.org/10.4490/algae.2016.31.4.14 156 Vieira et al. Molecular Marker for Chlorophyceae Table 1. Microalgae strains from CCMA–UFSCar collection and the amplification and sequencing results for each marker Strain tufA GrbcL rbcLFP ITS Chlorophyceae Ankistrodesmus densus Korshikov 1953 CCMA-UFSCar 003 KT003399 n/a n/a n/a Ankistrodesmus densus Korshikov 1953 CCMA-UFSCar 128 KT003380 n/a KT003371 n/a Ankistrodesmus densus Korshikov 1953 CCMA-UFSCar 239 KT003398 n/a n/a n/a Selenastrum bibraianum Reinsch 1866 CCMA-UFSCar 047 KT003389 KT003379a KT003379a n/a Selenastrum gracile Reinsch 1866 CCMA-UFSCar 005 KT003390 n/a n/a n/a Selenastrum gracile Reinsch 1866 CCMA-UFSCar 350 KT003391 n/a n/a n/a Kirchneriella aperta Teiling 1912 CCMA-UFSCar 123 KT003385 KT003372 n/a KT003363 Monoraphidium komarkovae Nygaard 1979 CCMA-UFSCar 353 KT003386 KT003377a KT003377a n/a Raphidocelis subcaptata (Korshikov) Nygaard, Komárek, J. Kristiansen & CCMA-UFSCar 048 KT003400 n/a n/a n/a O. M. Skulberg 1987 Scenedesmus ecornis (Ehrenberg) Chodat 1926 CCMA-UFSCar 439 KT003393 KT003373a KT003373a n/a 157 Scenedesmus sp. E. Hegewald 1978 CCMA-UFSCar 088 KT003392 n/a n/a n/a Coealastrum sphaericum Nägeli 1849 CCMA-UFSCar 060 KT003382 n/a n/a n/a Desmodesmus communis (Hegewald) E. Hegewald 2000 CCMA-UFSCar 030 KT003384 KT003374a KT003374a KT003361 Desmodesmus spinosus (Chodat) E. Hegewald 2000 CCMA-UFSCar 046 KT003394 KT003375a KT003375a n/a Desmodesmus spinosus (Chodat) E. Hegewald 2000 CCMA-UFSCar 062 KT003395 KT003376a KT003376a n/a Chlamydomonas chlorastera Ettl 1968 CCMA-UFSCar 009 KT003381 n/a n/a n/a Pandorina morum (O. F. Müller) Bory de Saint-Vincent 1824 CCMA-UFSCar 095 KT003388 n/a n/a n/a Pediastrum duplex Meyen 1829 CCMA-UFSCar 055 KT003387 n/a KT003378 KT003362 Volvox sp.