Characterization of a New Species in the Genus Didymosphenia and of Cymbella Janischii (Bacillariophyta) from Connecticut, USA

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

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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]

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 concentrate 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 prepare the Didymosphenia sp. tion for these two diatoms. tufts for SEM analyses. The tufts were placed in the mixture and centrifuged at 600 × g to guard against stalk material damage. The supernatant was discarded and the tufts were MATERIALS AND METHODS resuspended in BBM combined with 4% glutaraldehyde The West Branch of the Farmington River (WBFR) is overnight. The samples were placed in 2% osmium tetroxide located in Northwestern Connecticut, USA. The WBFR is for 2 h. The samples were dehydrated through a graded impounded twice, first at the Colebrook River Reservoir and ethanol (C2H5OH) series of 30, 50, 70, 95 and 100% on then at the West Branch Reservoir. The Metropolitan District ice, with the sample remaining in a shallow glass Petri dish Commission (MDC) operates West Branch Reservoir and bath for 15 m per step. Critical point drying using a Tousimis has a contractual agreement to maintain a minimum dis- 931.GL apparatus was followed by sputter coating. The charge from the oligotrophic hypolimnion (C. Bellucci CT stubs were viewed with the field emission Leo/Zeiss DSM DEEP, pers. comm.; MDC, 2013). The WBFR has stable 982 and a field emission FEI Nova Nano 450 scanning flow regimes and substrate stability because discharge from electron microscope. LM and SEM image plates were cre- this reservoir is managed. It also has very cold and oligo- ated using Adobe® Creative Suite® 6 Photoshop. trophic water, making it conducive for the growth of D. Also, the 0.2 ml Eppendorf® tube residual from the geminata as reported by Kilroy (2004), Spaulding & original PCR product (see below) was saved for SEM ver- Elwell (2007), Bothwell & Kilroy (2011) and ification of the isolated cell for identification purposes. The Didymosphenia sp. (Khan-Bureau et al., 2014). single frustule was retrieved from several individual PCR A new species of Didymosphenia from Connecticut, USA 3

tubes, washed in deionized water, and centrifuged to ensure Terminator Cycle Sequencing Reaction Kit (Applied that the cell was not discarded and could later be found at the Biosystems, Foster City, California, USA). Products of cycle bottom of the tube for SEM verification. The supernatant sequencing were cleaned using ethanol precipitation and ana- was removed and replaced with 25 µl of deionized water and lysedonanABI3100DNASequencer™ (Applied transferred onto a 25 mm, 3 μm pore size polycarbonate Biosystems, Foster City, California, USA). Contigs of indivi- Millipore filter (Lang & Kaczmarska, 2011). The filter was dual reads were assembled in Geneious© (Geneious, 2013), to placed on a SEM stub with double-sided tape. For produce consensus sequences. These were compared with C. janischii we followed the methodology of Morales et al. published data in GenBank, using the BLASTn tool, to obtain (2001), and the stubs were coated for 1 min at 1.8 kV with information on the closest matches. The sequences were gold/palladium using a Polaron sputter coater. aligned against a sampling of the sequences presented in Kermarrec et al.(2011) and the sequence of Cocconeis staur- oneiformis (W. Sm.) Okuno AB430614 (Sato et al., 2008). DNA extraction, PCR and cloning of Cymbella Sequences were aligned manually using Geneious© janischii (Geneious, 2013). Confidence of branch support was assessed One water sample from July 2012 was used for molecular using MrBayes (Huelsenbeck & Ronquist, 2001; Ronquist & 6 characterization of C. janischii. This sample was centri- Huelsenbeck, 2003). Bayesian analyses were run for 10 ×3 fuged, rinsed with deionized water, and then split into three generations with one cold chain and 3 heated chains, and fi replicate 50 µl microtubes. DNA extraction was accom- sampling every 1000 generations. The rst 10% of the trees plished using the MoBio PowerLyser™ Soil Extraction kit. were discarded as burn-in. Alignments will be available from PCR amplification of the V4 region of the 18S rDNA gene www.treebase.org. was achieved using primers D512F and D978R fi (Zimmermann et al., 2011). The PCR temperature pro le RESULTS included an initial denaturation step at 9°C (2 min), then five cycles consisting of denaturation at 94°C (45 s), annealing at LM and SEM analysis 52°C (45 s) and elongation at 72°C (1 min), followed by 35 Cells of C. janischii were an average of 130 μm long, cycles in which the annealing temperature was lowered to – μ 50°C, and a final elongation at 72°C (10 min). Resulting consistent with the published range of 105 383 m fi – PCR products were visualized on a Syber Safe stained agar- (Bahls, 2007) (Supplementary gs 1 4). Several LM ose gel, then quantified with a Nano Drop spectrophot- images were taken of live single and recently divided ometer. Cloning of PCR products was performed using cells of Didymosphenia sp. prior to preparing the Invitrogen TOPO® TA Cloning® Kit. Plasmid Prep fol- samples for acid cleaning (Figs 1–6). Sexual repro- lowed using the QIAprep® Spin Miniprep Kit. duction was not observed over this eight month time period. The valve and girdle views of Didymosphenia sp. illustrate the cell size variation of this population Direct PCR of single cells of Didymosphenia (Figs 7–16). Comparisons were made of LM images To best match DNA sequences with a specificmorphotype, of smaller D. geminata cells, which ranged in size direct PCR was performed on cells that later were used for from 57–161 μm from plates in Spaulding (2010) and SEM imaging. Initially several cells from fresh samples, were Metzeltin & Lange Bertalot (2014), and isolated using a micropipette and placed in a 0.2 ml PCR tube. Didymosphenia sp. cells (Figs 17–25) as shown in From these tubes, one individual cell of Didymosphenia sp. Supplementary table 1. The smaller D. geminata cells Downloaded by [159.247.3.230] at 08:09 03 March 2016 – was placed in three replicate PCR tubes and washed 3 5 fi times (Lang & Kaczmarska, 2011). After the final wash and vary signi cantly in size whereas the Didymosphenia centrifugation, the supernatant was removed and replaced by sp. valves in all observed cells of this population – μ 1 μl of sterile water. The samples were then heated at 95°C on ranged consistently from 40 60 m over eight a thermocycler for 10 min prior to PCR to open the frustules months (Figs 7–16). for DNA extraction (Lang & Kaczmarska, 2011). PCR ampli- Didymosphenia sp. frustules were successfully fication of the V4 region of the 18S rDNA gene was achieved retreived after the single-cell PCR reactions were using the primers D512F and D978R of Zimmermann et al. completed, placed on a Millipore filter on a stub (2011). The PCR mix consisted of 10 μl GoTaq® Green and viewed with SEM. Although the recovered frus- Master Mix, 0.5 μl of each primer (Zimmermann et al., tules were fractured, they were adequate for identifi- fi 2011), and sterile deionized water for a nal volume of 20 cation (Figs 26–28). These frustules correspond to μ l in the PCR tubes, each containing a single Didymosphenia the cells used to generate sequences of sp. cell. The PCR temperature profile used for the amplifica- Didymosphenia sp. in the phylogenetic tree tion of C. janischii DNA was also employed here. (Fig. 29, see below). Also, SEM images of the Didymosphenia sp. cells attached to their stalks Sequencing of Didymosphenia sp. and C. janischii further demonstrate the small cell size and the com- – The cloned fragments and cleaned PCR fragments were pressed valve morphology (Figs 30 38). This is in directly sequenced using the amplification primers of contrast to cells of D. geminata found at other North Zimmermann et al.(2011). The sequencing cycle comprised American sites, including Massachusetts, which are 27 cycles of denaturing at 96°C for 30 s, annealing at 50°C for more robust and up to 137 µm (Kilroy, 2004; 15 s, and extension at 60°C for 4 min, using the Big Dye™ Spaulding, 2010). SEM features of the apical pore D. A. Khan-Bureau et al. 4 Downloaded by [159.247.3.230] at 08:09 03 March 2016

Figs 1–6. LM images of ﬁeld collected Didymosphenia hullii. Figs 1, 5, 6: Cells attached to their stalks illustrate cell division. Scale bars = 20 μm(Figs 1, 2, 5); 10 μm(Fig. 3); 50 μm(Fig. 4); 40 μm(Fig. 6).

ﬁeld, stigmata, striae, shape, areolae and length of the stigmata and 9–11 striae in 10 µm. The areolae frustule can be used to distinguish this taxon from have deep inclined walls and are surrounded by other members of Didymosphenia (Figs 39–44, spine-like projections (spines) with dendritic slits Table 1). Didymosphenia sp. is 40–60 μm in length, below the spines, and are most similar to the areolae with cells of 38 µm and 68 µm occuring less fre- of D. geminata, D. clavaherculis (Ehrenb.) Metzeltin quently. The width is 26.5–30.5 μm with 1–4 & Lange-Bert., and D. laticollis Metzeltin & Lange- A new species of Didymosphenia from Connecticut, USA 5

Figs 7–15. LM images of Didymosphenia hullii cells in valve view showing size variation. Fig. 16: LM image of D. hullii cell in girdle view. Scale bars = 10 μm.

Bert. and as described by Metzeltin & Lange-Bertalot sequences of C. janischii from Idaho, USA, and C. (1995, 2014), although the cell shape and size of D. mexicana (Ehrenb.) Cleve (Fig. 29). These sequences clavaherculis and D. laticollis are distinct from that grouped next most closely with those of C. tumida of Didymosphenia sp. (Bréb. ex Kütz.) Van Heurck, C. proxima Reimer, and The D. geminata-like diatom that occurs in the D. geminata, but were distinct from other Cymbella West Branch of the Farmington River has similar representatives. The three new V4 sequences from Downloaded by [159.247.3.230] at 08:09 03 March 2016 autecology and physico-chemical preferences to D. Didymosphenia sp. differed by just one nucleotide. geminata (Khan-Bureau et al., 2014). Unlike the These were identified as close matches to three pub- diatom that was found in Massachusetts lished sequences of D. geminata from Italy (Supplementary fig. 5), Didymosphenia sp. is mor- (JN790293), New Zealand (JN680079), Colorado, phologically distinct from D. geminata and other USA (KJ011637), D. dentata Skvortsov & C.I. species in this genus, warranting unique species- Meyer (Skvortzow & Meyer, 1928) from Russia level status. (KJ011635) and D. siberica (Grunow) M. Schmidt from Russia (KJ011637), and were more distantly related to C. janischii from Idaho, USA (KJ011622) DNA barcode analysis and from this study, C. mexicana from Texas USA Three independent V4 rDNA sequences of (KJ011624), and C. tumida from Spain (JN790274). Didymosphenia sp., resulting from the three single Didymosphenia hullii Khan-Bureau, sp. nov. (Figs 1–16, cell isolations, plus four sequences from the cloned 25–28, 30–44) material of C. janischii were obtained. The V4 sequences were 334–410 bp in length, and were DESCRIPTION: Didymosphenia hullii Khan-Bureau sp. compiled into a final alignment with selected related nov. forms colonies of cells on long stalks. The cells published diatom sequences to produce an alignment are heteropolar, with a rounder, shorter and more of 1816 nucleotides in length. The four sequences of compressed headpole and footpole than the distinctly C. janischii from Connecticut were identical across capitate D. geminata. Valves of D. hullii (Figs 7–16) the barcode region and were resolved closest to are compact with transapically expanded headpoles D. A. Khan-Bureau et al. 6 Downloaded by [159.247.3.230] at 08:09 03 March 2016

Figs 17–25. LM images of Didymosphenia geminata cells in valve view showing size variation. Fig. 17: Courtesy of S. Spaulding, Figs 18–24: Courtesy of D. Metzeltin. Reprinted from Metzeltin & Lange-Bertalot (2014, Iconographia Diatomologica 25) with the permission of Koeltz Scientiﬁc Books. Fig. 25: D. hullii cell in valve view. Scale bars = 10 μm.

and the concavity at the constriction of the headpole length consistently in the range of 40–60 μm with is shallow and short. The footpole is slightly capitate, cells slightly smaller at 38 μm and larger to 68 μm though blunt. The footpole has an apical pore ﬁeld of occassionally observed, the width range of 26.5–30.5 very small spherical perforations that are present μm, and 1–4 stigmata present. The central area is where the stalk growth originates. The frustule com- inﬂated and elliptical with 9–11 striae in 10 μm that prises the epivalve and hypovalve through four girdle are radiate and have irregular short and long lengths. bands patterned with raised pustules. From the girdle Large pentagonal and square shaped depressions with view, the headpole is broad and tapers to the footpole pores (areolae) are present throughout the valve in similar to a wedge or a V shape. The valve has a complex deep wells that are surrounded by spines. Downloaded by [159.247.3.230] at 08:09 03 March 2016

Table 1. Comparison of traits of selected Didymosphenia taxa and their localities. Morphology of areolae determined using images and descriptions in Metzeltin & Lange-Bertalot (1995, 2014) and of species new A Mrozińska et al.(2006).

Length: Striae Stigmata Areolae (in 10 µm), Taxon Length (µm) Width (µm) Width (in 10 µm) number morphology Pole shape Locality References

D. clavaherculis (Ehrenberg) 130–135 33–38 3.9–3.5 8–93 10–12 Narrower foot-pole, capitate head-pole Northern Metzeltin & Lange-

Metzeltin & Lange-Bertalot Deep inclined walls surrounded though less than D. geminata, distinct Ireland, Bertalot (1995, Didymosphenia by spine-like projections, constriction of apices and central part Mourne 2014) dendritic slits below spines Mountains D. curvata (Skvortsov & K.I. 53–120 28–35 1.9–3.4 8–10 1 rarely 2 11–14 Head-pole broadly rounded, foot-pole Russia – Siberia, Metzeltin & Lange- Meyer) Metzeltin & Lange- Deep inclined walls and narrower though rounded Lake Baikal Bertalot (1995, Bertalot dendritic slits with no spine- 2014) like projections D. dentata (Dorogostaïsky) 76–178 35–54 2.1–3.2 7–90 10–11 Capitate though smaller bulbous poles, Russia – Siberia, Skvortzow & Meyer Skvortzow & K.I. Meyer Deep inclined walls and no with inflated central part, distinct Lake Baikal (1928) USA Connecticut, from dendritic slits with large constriction of apices and central part semicircular apertures, vimines lacking D. geminata (Lyngbye) M. 48–132 25–45 1.9–2.9 8–10 1–79–12 Distinct capitate head-pole, foot-pole Europe, Asia, Metzeltin & Lange- Schmidt 65–161 36–41 1.8–3.9 7–92–5 Deep inclined walls surrounded rounded to slightly subcapitate, and North Bertalot (1995, by spine-like projections, distinct constriction of apices and America 2014) Spaulding dendritic slits below spines central part (2010) D. grunowii Lange-Bertalot & 55–85 27–35 2–2.4 10–13 1 15–17 Head-pole blunt and rounded, Russia – Siberia, Metzeltin & Lange- Metzeltin Deep inclined walls and constriction between central inflation reservoir in Bertalot (2014) elongated dendritic slits with and head-pole poorly defined, foot- Irkutsk no spine-like projections pole not bulbous D. hullii Khan- Bureau sp. nov. 40–60 26.5–30.5 1.5–1.9 9–11 1–4 8-10 Head-pole round, blunt, constriction U.S.A., CT, This study Deep inclined walls between central inflation and head West surrounded by spine-like pole poorly defined, foot-pole bulb Branch, projections, dendritic slits shaped Farmington below spines River D. laticollis Metzeltin & Lange- 57–123 32–42 1.7–2.9 7–92–58–12 Head-pole round, blunt, constriction North European Metzeltin & Lange- Bertalot Deep inclined walls surrounded between central inflation and head pole Russia, Lake Bertalot (2014) by spine-like projections, poorly developed, foot-pole narrower Onega dendritic slits below spines D. pumila Metzeltin & Lange- 40–56 25–30 1.6–1.9 8–10 1–214 Head-end weakly protracted from the Russia – Siberia, Metzeltin & Lange- Bertalot Deep inclined walls, no spine- central part, foot-poles broadly reservoir in Bertalot (1995, like projections, dendroid rounded Irkutsk 2014) slits nearly flush to mantle D. siberica (Grunow) M. 70–85 34–40 2–2.1 10–12 1 11–12 Poles very bluntly rounded, foot-pole Russia – Siberia, Dawson (1973), Schmidt 75–173 33–50 2.2–3.4 10–13 1 Deep inclined walls, no spine- narrower, constriction between head- Jenissey Stoermer et al. like projections, dendritic pole and central inflation poorly River (1986), Metzeltin & slits defined Lange- Bertalot (2014) D. tatrensis Mrozińska, 60–99 28–41 2–2.4 13–14.5 1 10–13 Head-pole bluntly rounded, constriction Poland, Mrozińska et al.(2006) Czerwik- Marcinkowska & Depressed inclined walls and between head-pole and central Slovakia

Gradziński dendroid apertures with no inﬂation poorly deﬁned 7 spine-like projections D. A. Khan-Bureau et al. 8

Figs 26–28. SEM images of single valves of Didymosphenia hullii retrieved after PCR reactions. Fig. 26: The valve is fractured but still identiﬁable. Fig. 27: High magniﬁcation image of a cell with two stigmata. Scale bars = 20 μm(Figs 26, 28); 5 μm(Fig. 27).

At the distal end, the raphe quickly curves or forms a M. Schmidt but differs in lacking a smooth external hook shape, but does not go through the apical pore valve surface as is characteristic of these species. ﬁeld. This taxon has both asymmetrical apical and Didymosphenia hullii also differs in stria density, num- transpical axis. ber of stigmata, and areola structure (Metzeltin & Lange-Bertalot, 1995, 2014;Mrozińska et al., 2006) HOLOTYPE: Fig. 14 (LM) and Fig. 28 (SEM) are made (Table 1). The rough external valve face and areolae of from the holotype from the population on slide D. hullii are most similar to that of D. geminata and D. CONN00178537, partially illustrated here in clavaherculis, the only two Didymosphenia species Figs 1–16 (University of Connecticut Herbarium), currently found in the USA, and D. laticollis known and Figs 26–28. Samples were collected by D. from Russia. Valves of D. hullii fall into the lower end Khan-Bureau, 29 November 2012. of the size range of D. geminata but valves of D. hullii ISOTYPES: Population on slide HCUCB D-00791 observed from cells collected across eight months (Herbario Criptogámico, Universidad Católica, San (Figs 7–16) were compact with transapically expanded

Downloaded by [159.247.3.230] at 08:09 03 March 2016 Pablo, Cochabamba, Bolivia). head poles, and the concavity at the constriction of the head pole is shallow and shorter in D. hullii compared TYPE LOCALITY: The West Branch of the Farmington with D. geminata and other Didymosphenia taxa. River, a tributary of the Connecticut River in Unlike D. tatrensis, D. siberica, D. pumila and D. Barkhamsted, Connecticut, USA (41.960° N, grunowii the areolae of D. hullii have deep inclined 73.017° W). Samples were taken from the epilithon. walls with dendritic slits within the valves which are HABITAT: Abundant growth occurred on a wide range surrounded by spines below the interior walls of large cobbles and boulders covering the river sub- (Mrozińska et al., 2006; Metzeltin & Lange-Bertalot, strate, bank to bank. 2014). These species are the smaller of the Didymosphenia taxa. They are compressed and their ETYMOLOGY: The species is named in honour of the late David Hull MD, Director of Transplant at basal poles are much less elongated than D. geminata, Hartford Hospital in Connecticut. He enjoyed nature and they differ in valve length and width, stria density, and aspired to understand the many facets of science. and number of stigmata (Table 1). Didymosphenia siberica was reported by Dawson (1973)and Didymosphenia hullii is similar in size to D. pumila Stoermer et al.(1986) as having only one isolated Metzeltin & Lange-Bert., D. grunowii Lange-Bert. & stigma internally and referred to this as a raised con- Metzeltin, D. laticollis Metzeltin & Lange-Bert. volution. Subsequently, Metzeltin & Lange-Bertalot (2014) (the Sweden population), D. tatrensis (1995)describedD. siberica with 1–3stigmataand Mrozińska, Czerwik-Marcinkowska & Gradziński, D. pumila having 1–2 stigmata, whereas D. hullii has and smaller populations of D. siberica (Grunow) 1–4 stigmata internally. A new species of Didymosphenia from Connecticut, USA 9 Downloaded by [159.247.3.230] at 08:09 03 March 2016

Fig. 29. Bayesian phylogenetic tree based the V4 region of the 18S rDNA of selected published sequences of Cymbellales, plus newly obtained sequences from three isolated single cells of Didymosphenia sp. and four sequences of Cymbella janischii (GenBank accession numbers are included in the taxon labels). The current taxonomic families are indicated. Bayesian posterior probabilities (BPP × 100) values indicate node support. Scale bar = expected number of substitutions/site. D. A. Khan-Bureau et al. 10

Figs 30–38. SEM images of Didymosphenia hullii on stalks from the West Branch of the Farmington River showing bifurcated stalks with cells attached. Scale bars = 20 µm (Figs 30, 31, 34); 50 µm (Figs 32, 33, 37); 40 µm (Fig. 35); 5 µm (Fig. 36); 200 µm (Fig. 38).

Downloaded by [159.247.3.230] at 08:09 03 March 2016 DISCUSSION sequences of C. janischii and C. mexicana,and more distantly related to C. tumida (Cymbellaceae) In this study we characterized two recently reported and D. geminata (Gomphonemataceae). The C. nuisance stalk-forming diatoms in Connecticut, thus janischii sequences also indicate that this species is contributing information on the morphology, varia- distantly related to most sequences from the family tion and phylogenetic relationships of cymbelloid Cymbellaceae, such as Cymbella affinis Kütz. and C. diatoms. The morphology of the Cymbella species cymbiformis (Ehrenb.) Grunow. These relationships from Connecticut is consistent with that of C. are consistent with a recent multi-locus investiga- janischii.IntheUSA,C. janischii is described as tion (Nakov et al., 2014) and provide additional endemic to the Pacific Northwest. Outside of the evidence that the taxonomy of the families PacificNorthwest,C. janischii had only been Gomphonemataceae and Cymbellaceae requires re- observed in four other states, Arizona, Colorado, evaluation (Kociolek & Stoermer, 1988;Nakov& New York and Oklahoma (Bahls, 2007), until Theriot, 2009; Kermarrec et al., 2011; Graeff & recently reported in Connecticut in July 2012 Kociolek, 2013;Nakovet al., 2014). (Khan-Bureau et al., 2014). Suzawa et al.(2011) We also described the new taxon, Didymosphenia confirmed blooms of the non-native C. janischii in hullii. Twenty-two species (and several varieties and Japan and reported that it was introduced from North subspecies of D. geminata) are currently known America. Phylogenetic analysis of four independent within the genus Didymosphenia (Metzeltin & V4 sequences of C. janischii from Connecticut indi- Lange-Bertalot, 2014). The type material of the cate that this species is related to published best-known species, D. geminata, was not readily A new species of Didymosphenia from Connecticut, USA 11 Downloaded by [159.247.3.230] at 08:09 03 March 2016

Figs 39–44. SEM images of Didymosphenia hullii. Fig. 39: Internal view of the valve displaying three stigmata. Fig. 40: View of apical pore ﬁeld, footpole. Figs 41, 42: Central valve view with striae, and 1 and 4 stigmata, respectively. Fig. 43: Internal view of central valve showing two stigmata and uniseriate striae. Fig. 44: External views of frustule girdle and valve morphology. Scale bars = 20 μm(Fig. 39); 5 μm(Figs 40, 43); 10 μm(Figs 41, 42); 30 μm(Fig. 44). D. A. Khan-Bureau et al. 12

available until recently (D. Metzeltin, pers. comm.) taxa and facilitate identification of new or cryptic spe- and morphological data are still limited even though cies of diatoms. However, finding a suitable species it has been almost 200 years since Lyngbye first level marker has proven challenging for some algae described Didymosphenia geminata as Echinella and specifically the diatoms (Evans et al., 2007;Hall geminata (Lyngbye, 1819; Whitton et al., 2009; et al., 2010;Hamsheret al., 2011; Zimmermann et al., Blanco & Ector, 2013). Most early reports were 2011; Luddington et al., 2012;Kermarrecet al., 2014). based on drawings and light micrographs (Blanco & Nakov et al.(2014) recently examined phylogenetic Ector, 2013). Only one species of Didymosphenia, D. signal in alternative diatom barcode genes, including geminata, occurs commonly within the geographic the plastid rbcL gene (which we were unsuccessful in boundaries of the continental USA, specifically in the obtaining from our samples), for the Cymbellales. western states, although D. clavaherculis was docu- Although the selected markers provided resolution mented in Alaska (Spaulding, 2010). New reports of across the order it is not possible to assess if they D. geminata in the USA have come from midwestern would be useful within the genus Didymosphenia and eastern states (Kilroy, 2004; Spaulding & Elwell, because just one representative from each of three 2007; Blanco & Ector, 2009; Kumar et al., 2009; species of Didymosphenia was used in that study. Kilroy & Unwin, 2011; Bothwell et al., 2012), as Future work is needed to identify suitable phyloge- well as Canada (Kirkwood et al., 2007; Gillis & netic markers for the species of Didymosphenia. Chalifour, 2009; Lavery et al., 2014) and South Molecular data are not currently available for most America (Kilroy & Unwin, 2011; Segura, 2011; species, and most of the sequences available are not Morales et al., 2012; Rivera et al., 2013; Sastre associated with a specific morphology and do not repre- et al., 2013; Reid & Torres, 2014). The diatom sent the taxonomic type. Also needed are studies that reported from Connecticut is morphologically dis- include samples of single species from distinct geo- tinct from other species of Didymosphenia, including graphic locations in order to understand the levels of D. geminata reported from neighbouring sequence variation within and among species. Together, Massachusetts (Supplementary fig. 5), and is thus these studies would provide a better understanding of recognized as a distinct species. the geographic ranges and help connect physiological Despite the economic and ecological importance of preferences and tolerances to particular diatom species. Didymosphenia there were only two accessions of this genus in GenBank, both reporting sequences of the 18S ACKNOWLEDGEMENTS gene of D. geminata, until Nakov et al.(2014) provided additional sequences from D. geminata, D. dentata and We thank the Connecticut Institute of Water Resources D. siberica. None of the existing D. geminata sequences (CTIWR) for funding this research. are from the type locality, thus we cannot state which D. We thank K. Fučíková,S.Olm,C.Lo,M.Letsch,H. geminata sequence represents the species. We targeted McManus and acknowledge the late F. Trainor for helpful the V4 region of the 18S gene to make the new data discussion. This work made use of the computer cluster from D. hullii comparable to the published sequences of maintained by the Bioinformatics Facility (Biotechnology/ D. geminata and related taxa. The results of the present Bioservices Center) at the University of Connecticut. We are grateful for D. Metzeltin’s expert opinion and H.

Downloaded by [159.247.3.230] at 08:09 03 March 2016 study indicate that D. geminata and D. hullii are more closely related to C. janischii than to Gomphonema and Lange-Bertalot for the use of their D. geminata images. other genera in Gomphonemataceae, further illustrating Thanks to S. Spaulding for access to her D. geminata image. We thank Three Rivers Community College’s the non-monophyly of Cymbellaceae and Dean of Information Technology S. Goetchius, K. Gomphonemataceae as noted by other authors Barfield, S. Cohen, D. Jewett, and retired President G. S. (Kociolek & Stoermer, 1988; Nakov & Theriot, 2009; Jones for providing continual support for this project, J. Kermarrec et al., 2011; Graeff & Kociolek, 2013; Sgro, John Carroll University, for providing his input and Nakov et al., 2014). continual support, for the expert assistance of M. Cantino As was seen among sequences of the Cymbella and S. Daniels of the EM lab at UCONN, NSF 1126100 janischii clade (but not for other species of Cymbella), supporting the purchase of the Nova Nano SEM, V. Kask and from the data from the V4 region we were unable to scientific illustrator in Biological Central Services at separate D. hullii, D. geminata, D. siberica or D. den- UCONN, J. Morrison, East Hartford, USGS, L. tata sequences due to very low sequence variation, Matthews VT DEC, M. Becker and C. Bellucci, CT indicating that these taxa are very closely related and DEEP for their assistance. We thank the reviewers for perhaps evolutionarily young. The lack of variation their helpful comments. across this short barcode region, coupled with a lack of published sequences from the other Didymosphenia species means that we are currently unable to resolve DISCLOSURE STATEMENT the relationship among species of Didymosphenia.A No potential conflict of interest was reported by the more variable marker may better differentiate among author(s). A new species of Didymosphenia from Connecticut, USA 13

SUPPLEMENTARY INFORMATION Dawson, P.A. (1973). Further observations on the genus Didymosphenia M. Schmidt–D. siberica (Grun.) M. Schm. The following supplementary material is accessible via the British Phycological Journal, 8: 197–201. Supplementary Content tab on the article’sonlinepageat Evans, K.M., Wortley, A.H. & Mann, D.G. (2007). An assessment http://dx.doi.org/10.1080/09670262.2015.1126361 of potential diatom “barcode” genes (cox1, rbcL, 18S and ITS Supplementary table 1: Taxon list and source page rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta). Protist, 158: 349–364. numbers of images used for morphological comparisons Geneious (2013). Version 7.0.5, created by Biomatters. Available of D. geminata and D. hullii. from http://www.geneious.com/. Supplementary figs 1–4: LM images offield-collected Gillis, C.-A. & Chalifour, M. (2009). Changes in the macrobenthic C. janischii cells showing size distribution. Fig. 3: Central community structure following the introduction of the invasive valve illustrates the distinct radiated striae with striae more algae Didymosphenia geminata in the Matapedia River (Québec, Canada). Hydrobiologia, 647:63–70. closely together and longer pointed areolae in the central Graeff, C.L. & Kociolek, J.P. (2013). New or rare species of area, differentiating between other larger Cymbella taxa cymbelloid diatoms (Bacillariophyceae) from Colorado (USA). and C. janischii.Figs1–3: Scale bars = 10 μm. Fig. 4: LM Nova Hedwigia, 97:87–116. image of cell on its stalk prior to acid cleaning. Scale bar = Hall, J.D., Fučíková, K., Lo, C., Lewis, L.A. & Karol, K.G. (2010). μ An assessment of proposed DNA barcodes in freshwater green 20 m. – fi algae. Cryptogamie, Algologie, 31: 529 555. Supplementary g. 5: LM image of D. geminata Hamsher, S.E., Evans, K.M., Mann, D.G., Poulíčková, A. & from Massachusetts. Scale bar = 10 μm. Saunders, G.W. (2011). Barcoding diatoms: exploring alterna- tives to COI-5P. Protist, 162: 405–422. Huelsenbeck, J.P. & Ronquist, F. (2001). MrBayes: Bayesian infer- – AUTHOR CONTRIBUTIONS ence of phylogeny. Bioinformatics, 17: 754 755. Kermarrec, L., Ector, L., Bouchez, A., Rimet, F. & Hoffmann, L. D.A. Khan-Bureau: original concept, LM and SEM and mol- (2011). A preliminary phylogenetic analysis of the Cymbellales ecular analyses, drafting and editing the manuscript; E.A. based on 18S rDNA gene sequencing. Diatom Research, 26: Morales: reviewed LM and SEM images and edited the manu- 305–315. script; L. Ector reviewed the LM and SEM images and edited the Kermarrec, L., Franc, A., Rimet, F., Chaumeil, P., Frigerio, J.M., manuscript; M.S. Beauchene, provided water quality data and Humbert, J.F. & Bouchez, A. (2014). A next-generation sequen- historical information and edited the manuscript; L.A. Lewis cing approach to river biomonitoring using benthic diatoms. reviewed molecular, SEM, and LM analyses, and edited the Freshwater Science, 33: 349–363. manuscript. Khan-Bureau, D.A., Beauchene, M.S., Ector, L., Morales, E.A. & Lewis, L.A. (2014). Observations of two nuisance stalk-forming REFERENCES diatoms (Bacillariophyta) from a river in Connecticut, Northeastern U.S.A.: Didymosphenia sp. and Cymbella janischii Aboal, M., Marco, S., Chaves, E., Mulero, I. & García-Ayala, A. (A. Schmidt) De Toni. BioInvasions Records, 3: 139–149. (2012). Ultrastructure and function of stalks of the diatom Kilroy, C. (2004). A new alien diatom, Didymosphenia geminata Didymosphenia geminata. Hydrobiologia, 695:17–24. (Lyngbye) Schmidt: its biology, distribution, effects and potential Bahls, L.L. (2007). Cymbella janischii – giant endemic diatom of risks for New Zealand fresh waters. 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