European Journal of Phycology ISSN: 0967-0262 (Print) 1469-4433 (Online) Journal homepage: http://www.tandfonline.com/loi/tejp20 Characterization of a new species in the genus Didymosphenia and of Cymbella janischii (Bacillariophyta) from Connecticut, USA Diba A. Khan-Bureau, Eduardo A. Morales, Luc Ector, Michael S. Beauchene & Louise A. Lewis To cite this article: Diba A. Khan-Bureau, Eduardo A. Morales, Luc Ector, Michael S. Beauchene & Louise A. Lewis (2016): Characterization of a new species in the genus Didymosphenia and of Cymbella janischii (Bacillariophyta) from Connecticut, USA, European Journal of Phycology, DOI: 10.1080/09670262.2015.1126361 To link to this article: http://dx.doi.org/10.1080/09670262.2015.1126361 View supplementary material Published online: 03 Mar 2016. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tejp20 Download by: [159.247.3.230] Date: 03 March 2016, At: 08:09 Eur. J. Phycol. (2016), 1–14 Characterization of a new species in the genus Didymosphenia and of Cymbella janischii (Bacillariophyta) from Connecticut, USA DIBA A. KHAN-BUREAU1,2, EDUARDO A. MORALES3, LUC ECTOR4, MICHAEL S. BEAUCHENE5 AND LOUISE A. LEWIS6 1Department of Natural Resources and the Environment, University of Connecticut, 75 North Eagleville Road, Storrs, Connecticut, 06269, USA 2Three Rivers Community College, 574 New London Turnpike, Norwich, Connecticut, 06360, USA 3Herbario Criptogámico, Universidad Católica Boliviana “San Pablo”, Calle M. Márquez esq. Plaza Jorge Trigo s/n, P.O. Box 5381, Cochabamba, Bolivia 4Luxembourg Institute of Science and Technology (LIST), Environmental Research & Innovation (ERIN) Department, 41 rue du Brill, L-4422 Belvaux, Grand-duchy of Luxembourg 5Inland Fisheries Division, Department of Energy and Environmental Protection, 79 Elm Street, Hartford, Connecticut, 06106-5127, USA 6Department of Ecology and Evolutionary Biology, University of Connecticut, 75 North Eagleville Road, Storrs, Connecticut, 06269, USA (Received 20 July 2015; revised 15 October 2015; accepted 27 October 2015) Two non-native stalk-forming diatoms that were recently observed in the West Branch of the Farmington River, a tributary of the Connecticut River in Connecticut (USA), are characterized morphologically and barcode marker sequences were obtained for each of them. Cymbella janischii, the dominant stalk-forming species during the summer of 2012, previously had not been found in the northeastern USA. Samples of C. janischii were examined microscopically and used to obtain four sequences of the barcode marker, the V4 region of the 18S rDNA gene. Phylogenetic analysis indicated that the four independent sequences of C. janischii were distinct from, but most closely related to, published sequences of C. janischii from Idaho and C. mexicana from Texas, USA. A second non-native stalk-producing diatom, resembling Didymosphenia geminata, was found in November 2012 – June 2013 and first reported as Didymosphenia sp. Over this period, the observed cells had a compressed morphology and were consistently small compared with D. geminata. Sequences of the V4 region, obtained from three independent direct polymerase chain reactions (PCR) of single cells isolated from the Connecticut samples, indicated a close relationship to three published sequences of D. geminata from Italy, New Zealand and the USA, and to D. siberica and D. dentata from Russia. Frustules of the cells used in the PCR reactions were recovered and examined using scanning electron microscopy, providing a direct link Downloaded by [159.247.3.230] at 08:09 03 March 2016 between the observed morphology and sequence data. The morphology of the novel Connecticut Didymosphenia taxon was compared with that of other Didymosphenia taxa, being most similar to D. pumila, D. laticollis, D. grunowii and smaller cells of D. geminata. Didymosphenia sp. had a triundulate morphology with a consistent length of 40–60 µm. Given the unique morphological features of this diatom, it is proposed as a new species, Didymosphenia hullii Khan-Bureau, sp. nov. Key words: 18S V4 rDNA, benthic diatoms, cymbelloid diatoms, Didymosphenia geminata, gomphonemoid diatoms, invasive species, nuisance species, USA INTRODUCTION growth is often mistaken for D. geminata at the macroscopic level with tufts that are similar in The freshwater stalk-forming diatom Didymosphenia appearance (Pite et al., 2009; Whitton et al., 2009). geminata (Lyngb.) M. Schmidt is a well-known inva- Both species are commonly referred to as rock snot. sive and nuisance species with an ability to produce Under certain environmental conditions these species copious extracellular polymeric substances that form grow prolifically, forming thick mats that cover sec- the stalks (Blanco & Ector, 2009; Aboal et al., 2012). tions of the river substrate, negatively impacting Cymbella janischii (A.W.F. Schmidt) De Toni, other aquatic organisms (Kilroy, 2004; Spaulding & another stalk-forming diatom with abundant stalk Elwell, 2007; Kumar et al., 2009; Morales et al., Correspondence to: Diba A. Khan-Bureau. E-mail: diba. 2012;Zgłobicka, 2013; Kuhajek & Wood, 2014). [email protected] ISSN 0967-0262 (print)/ISSN 1469-4433 (online)/16/000001-14 © 2016 British Phycological Society http://dx.doi.org/10.1080/09670262.2015.1126361 D. A. Khan-Bureau et al. 2 Unlike C. janischii, D. geminata prefers oligotrophic, Benthic samples were collected from the West Branch of cold and low soluble reactive phosphorus environ- the Farmington River in July 2012 – June 2013 (Khan-Bureau ments, which may in part cause unusual overgrowth et al., 2014). Samples of the mucilaginous tufts were taken conditions (Krammer & Lange-Bertalot, 1986; from rock substrate, submerged vegetation, and overhanging Kilroy & Bothwell, 2011; Bothwell et al., 2012, tree branches, and placed in Whirlpac® bags. The latter were 2014). In addition, D. geminata establishment is placed on ice, and transported to the lab for processing. influenced by a structurally suitable substrate, the development of a pad which adheres to the substrate, Light microscopy (LM) and the orientation of the cell upon attachment (Kilroy & Bothwell, 2014; Kuhajek & Wood, 2014; Prior to acid-washing the samples, live samples were placed on a microscope slide with a coverslip overlain and then Kuhajek et al., 2014). viewed at 200 and 400× magnifications using a BX 60 Many states throughout the USA are monitoring Olympus microscope. Images were digitally captured using their waterways for D. geminata because of its an Olympus DP 25 camera and cellSens software then expanding geographic range (Kuhajek & Wood, viewed to identify the taxa according to Krammer & 2014). In the USA, D. geminata was transported Lange-Bertalot (1986), Round et al.(1990), and three online from the western states into several southeastern databases, the ANSP Algae Image Database (http://diatom. states, and more recently to northeastern states ansp.org/algae_image/), Diatoms of the United States (Bothwell & Spaulding, 2008; Blanco & Ector, (http://westerndiatoms.colorado.edu/) and the Great Lakes 2009; Spaulding, 2010). In May 2013, Image Database (http://www.umich.edu/~phytolab/ Massachusetts first recorded and confirmed an occur- GreatLakesDiatomHomePage/top.html). For permanent rence of D. geminata with growth lasting two months slide preparation the river samples were centrifuged to con- centrate the diatom cells to the bottom of the microtube. The (A. Madden, MA. Div. Fisheries and Wildlife, pers. supernatant was poured off and distilled water was added. comm.). Samples were then simmered on a hot plate in a 1:1 ratio of The Connecticut Department of Energy and water and 68% nitric acid to oxidize organic matter, then Environmental Protection (CT DEEP, 2011) started taken off the hotplate and allowed to cool for several min- monitoring the West Branch of the Farmington River utes. Deionized water was used to rinse the samples of the after purported D. geminata tufts were observed in acid, 4–5 times to neutralize samples, and then centrifuged 2011. In July 2012 reports of mucilaginous tufts to concentrate the diatom frustules (following the protocol occurring downstream of the original location in of R. Lowe, pers. comm.). After air-drying the diatom sam- 2011 were later confirmed to be substantial growths ples overnight on coverslips, frustules were mounted on of C. janischii. In addition, an unusual morphological glass microscope slides in the mounting medium population of Didymosphenia sp. was found in NAPHRAX®, heated on a hot plate and then cooled to November 2012 (Khan-Bureau et al., 2014)at produce permanent vouchers. The diatom frustules were examined at 600 and 1000× magnifications with a BX 60 another location along the river. The present study Olympus microscope. 125 valves were measured. Images characterizes the morphology of these two diatoms were captured using an Olympus DP 25 colour camera (2560 from the West Branch of the Farmington River in × 1920 pixels). Connecticut. We show that Didymosphenia sp. is Downloaded by [159.247.3.230] at 08:09 03 March 2016 morphologically distinct from other Didymosphenia species and propose it as a new taxon. In addition, we Scanning electron microscopy (SEM) present new sequence data of the V4 rDNA region in A mixture of glutaraldehyde and Bold Basal Medium order to link the morphological and genetic informa- (BBM) was used to fix and