Aetokthonos hydrillicola Wilde et Johansen, 2014

Wilde, Susan B., Johansen, Jeffrey R., Wilde, H. Dayton, Jiang, Peng, Bartelme, Bradley A. & Haynie, Rebecca S., 2014, Aetokthonos hydrillicola gen. et sp. nov.: Epiphytic cyanobacteria on invasive aquatic plants implicated in Avian Vacuolar Myelinopathy, Phytotaxa 181 (5), pp. 243-260 : 247-255

Aetokthonos hydrillicola Wilde et Johansen , sp. nov. ( Figs. 2–4 )

Main filaments creeping, forming attached irregular disks on the underside of leaves of aquatic plants, more or less radiating from the center of the colony, sometimes biseriate to multiseriate, typically branching mostly to one side, subtorulose, 9.1–14.0 μm wide. Sheath absent, thin, or firm and slightly widened, colorless, homogenous, sometimes extending beyond tips of branches following hormogonia release. Trichomes mostly monoseriate, biseriate to multiseriate in older parts of colony, tapering abruptly towards ends of main filaments, tapering more gradually in terminal parts of branches, 6.0–8.0 μm wide. Cells in main filaments often with their own envelopes, compressed spherical to almost spherical, after division semi-quadratic, with minutely granular dull blue-green cell contents, 5.0–10.0 μm long, 10.0–14.0 μm wide. Cells in branches becoming longer than wide and cylindrical, not strongly constricted at the crosswalls, ungranulated, 8.0–10.0 μm long, 9.5–11.0 μm wide. Cytoplasm with irregular thylakoids showing a characteristic pattern with phycobilisomes, with polyphosphate bodies and cyanophycin. End cells separated from subterminal cells, forming a cap like structure at the end of branches (possibly functioning as a hormocyte), typically with reduced thylakoid development, becoming colorless in LM, sometimes remaining attached to branch after separation and consequently appearing to rise mid-trichome. Heterocytes mostly intercalary with two polar nodules, rarely lateral or terminal with a single polar nodule, light tan-colored to almost clear, 7.0–9.0 μm long, 9.0–10.5 μm wide. Akinetes enlarged, with thickened walls, granular, dark yellowish olive in color, in masses within the main filaments, shorter than wide, 10.5–13.0 μm wide. Hormogonia short, few celled, released from tips of branches.

Type:— UNITED STATES. South Carolina, Lake Thurmond (holotype: US Algal collection #217986, Algal Herbarium, Smithsonian National Museum of Natural History , Washington, DC.) . Reference strain: CCALA 1050 , Culture Collection of Autotrophic Organisms , Třeboň Czech Republic .

Habitat:— Growing on the underside of aquatic plants in subtropical regions.

Etymology:— hydrillicola = ‘living on Hydrilla ’.

Observations:— Aetokthonos hydrillicola is morphologically and ecologically closest to Fischerella reptans Geitler (1933: 629) var. reptans and F. reptans var. stigonematoides ( Geitler 1933: 629) . Both varieties of F. reptans are tropical and occur growing on aquatic plants. They differ in cell size, in particular in having branches that are considerably thinner (4–6 μm wide).Akinetes were not recorded for this species. The apical caps were also not reported. Further study (i.e. molecular sequencing) of these very poorly known taxa must be completed before we know if they belong to Fischerella or Aetokthonos . Nineteen sites from North Carolina to Arkansas have been confirmed as AVM positive sites and all sites contain invasive aquatic plants (primarily hydrilla) and dense colonies of A. hydrillicola (>50% leaf coverage). These colonies appear as dark spots on the underside of the leaf and can be been with the naked eye while still in the field collecting plant material. While Aetokthonos colonies are large enough to be seen on the leaf in sunlight, these colonies are quite cryptic under normal light microscopic illumination. Epifluorescent lighting and a rhodamine filter allowed optimal discernment of cyanopigments within the cyanobacterial cells on the surface of the hydrilla leaf. The species assemblage on the hydrilla leaves containing Aetokthonos includes both diatoms and additional cyanobacterial species, and obtaining pure cultures was challenging. Our final successful cultures were isolated from Lake Thurmond, GA/SC during October 2012 using 10% BG11 media under low light conditions (<20 µmol s-1 m-2). Colony growth overall was slow in culture, and was markedly higher in the lower nutrient agar (10% BG11). Aetokthonos hydrillicola could be identified genetically by a CAPS assay, in which a 247 bp restriction fragment was diagnostic for the species ( Fig. 5 ). In field sample analyses, a fragment of 314 bp was often observed that resulted from the presence of other cyanobacteria or possibly from incomplete Rsa I digestion.

Toxicity:— Laboratory feeding trials have been conducted to confirm the presence of the AVM neurotoxin within the hydrilla/ A. hydrillicola complex. Farm raised mallards and chickens have been used to investigate the potential toxicity of field-collected hydrilla containing the epiphytic cyanobacteria A. hydrillicola . Trial duration varied from 12–28 days, and total quantity of the hydrilla material varied, but in all cases, only birds receiving the hydrilla material with the A. hydrillicola epiphytes developed the characteristic AVM lesions ( Table 2). The hydrilla from control sites (Lake Seminole, Harris Lake, and Lake Marion) did have numerous epiphytic diatoms, chlorophytes and cyanobacterial species, but the novel A. hydrillicola species was not present. All of the trials were conducted using hydrilla collected during late fall (October–December) when densities of A. hydrillicola are maximal on the hydrilla leaves at AVM sites. Since 2001, we have had the capability to screen for A. hydrillicola , and have been able to confirm the presence of dense colonies of this species growing on invasive plants during the AVM mortality events in Arkansas, North Carolina, South Carolina and Georgia ( Fig. 1 , Table 1).

Phylogeny:— Aetokthonos is phylogenetically isolated from all other true-branching clades ( Fig. 6 , arrowed clades). It clustered with an uncultured bacterium (MIZ23) sequence, which is so similar in sequence (98.2%) that we assume that this sequence belongs to an undescribed and uncharacterized Aetokthonos species ( Table 3). Of the true branching clades ( Stigonemataceae , Hapalosiphonaceae , Chlorogloeopsidaceae , Symphyonemataceae ), Aetokthonos is closest to strains in the Stigonemataceae (e.g. Stigonema ocellatum Thuret ex Bornet & Flahault (1887: 69) , Petalonema Berkeley ex Correns (1889: 321) in Table 3). However, in none of our numerous phylogenetic analyses did the genus cluster with the Stigonemataceae with any support. It was generally distant, as shown in the Bayesian Analysis we present. It appears it is distant from all true-branching forms, and is consequently likely in its own family with Cylindrospermum -like strains from the PMC collection (only one shown in Fig. 6 ). These strains do not belong to Cylindrospermum sensu stricto ( Fig. 6 ), which has been studied in some detail ( Johansen et al. 2014). Morphologically, Aetokthonos is completely different than Cylindrospermum Kützing ex Bornet et Flahault (1888: 249) , and we do not doubt that it is both a separate species and genus from these problematic strains. The position of most of the clades in the Nostocales were not resolved with respect to each other, and Aetokthonos was in an unresolved position between the Nostocaceae / Aphanizomenonaceae and the Rivulariaceae . From our phylogenetic analyses, we lack sufficient evidence to make a family-level assignment. It is morphologically most similar to the Hapalosiphonaceae , but very dissimilar in 16S rRNA gene sequence to any members of that family (e.g. Fischerella , Mastigocladus Cohn ex Kirchner (1898: 81) in Table 3).

Comparative Analysis of 16S-23S ITS Region:— The two clones of the ITS sequenced were identical in sequence, and did not contain any tRNA genes. The members of Nostocales can have up to four ribosomal operons, with 2–3 markedly different ITS regions in these operons sometimes being found ( Iteman et al. 2000, Boyer et al. 2001, Flechtner et al. 2002). Typically, the operons more easily recovered are those with two tRNA genes, tRNAIle and tRNAAla. However, there is some evidence that in some genera the operons containing the tRNA genes may be missing. For example, in all three Mojavia Řeháková et Johansen in Řeháková et al. (2007: 488) strains sequenced thus far only the operon lacking tRNA genes was recovered. A more systematic effort (such as obtaining whole-genome sequence) will need to be made in both Aetokthonos and Mojavia to know if there are indeed no ribosomal operons containing tRNA genes in these genera. For purposes of comparison, a representative set of heterocytous taxa were chosen to compare lengths of conserved domains, with the operons lacking tRNA genes generally being chosen for comparison ( Table 4). This analysis showed that the length of some conserved domains (leader, D1–D1’ helix, Box- B helix, Post Box-B spacer, Box A antiterminator) were similar in length to the same structures in a number of taxa. The D4, V3 helix, and D5 had lengths similar to some taxa in solitary comparisons, but no comparison taxa had the same combination of sequence lengths as Aetokthonos ( Table 4). The V2 spacer was markedly different in length than all other V2 spacers in operons lacking the tRNA genes ( Table 4). The secondary structure for the conserved domains of the 16S-23S ITS region in Aetokthonos (D1-D1’, BoxB, and V3 helices) were markedly different from those observed in the morphologically similar genus Fischerella ( Fig. 7 ). A comparison with published secondary structures for Nostoc Vaucher ex Bornet & Flahault (1886: 181) , Mojavia , Cylindrospermum , Aulosira Kirchner ex Bornet & Flahault (1886: 256) , Rexia Casamatta et al. (2006: 23) and Spirirestis Flechtner & Johansen in Flechtner et al. (2002: 6) ( Casamatta et al. 2006, Řeháková et al. 2007, Lukešová et al. 2009, Johansen et al. 2014), as well as other unpublished structures sequenced in our research group but not yet published demonstrated that the three helices in A. hydrillicola are unique among all ITS secondary structures recovered thus far in the heterocytous cyanobacteria. Only one strain, Cylindrospermum marchicum ( Lemmermann 1905: 148) Lemmermann (1907: 196) CCALA 1001 , had a structure identical to that found in Aetokthonos (the Box-B helix, see Johansen et al. 2014, fig. 3c), but the sequence was different (70% similar), particularly in the terminal portion of the helix. The terminus of the D1–D1’ helix was unusual in that only a single C-G base pair formed between the terminal loop and subterminal bilateral bulge ( Fig. 7A ). Typically, the energetics of rRNA folding does not allow the formation of such a singleton pair, but the program we used (Mfold) gave only this structure for Aetokthonos .

helix spacer spacer gene gene spacer helix spacer Leader ’ - D 1 D 1 D 2 with 3 with D tRNAIle spacer 2 V tRNAAla Pre-Box-B Box-B Post-Box-B Box-A D 4 V 3 D 5

Aetokthonos hydrillicola 8 63 32 93 30 17 11 21 58 23

Fischerella ATCC 43239 8 71 33 17 74 88 73 28 30 17 11 28 67 24

Scytonema HTT-U-KK4 8 65 31 157 30 17 11 20 52 31 Mojavia pulchra JT 2-VF2 8 64 31 55 26 18 11 27 103 23 Nostoc commune EV 1-KK1 8 63 31 130 33 17 11 27 57 23

Nostoc desertorum CM 1-VF14 8 67 32 44 31 17 11 26 39 27 Nostoc indistinguendum CM 1-VF10 8 67 32 41 31 17 11 26 39 28 Nodularia PCC 73104 8 65 31 50 31 17 11 23 60 57 Cylindrospermum HA 4236-MV2 8 62 30 50 27 17 11 20 39 23 Hassallia CNP 1-B1-c09 8 63 31 242 33 17 11 27 39 27 Cyanocohniella HDL 9DIL2 7 65 30 55 30 17 11 27 35 22 Entophysalidaceae UFS-A4UI-NPMV4 7 67 33 158 27 17 11 27 34 31 Microcoleus steenstrupii FI-LIZ 3 8 65 31 156 30 17 11 21 59 23 Trichocoleus desertorum WJT 46-NPBG1 8 63 31 301 35 19 11 21 48 17 Trichocoleus desertorum WJT 16-NPBG1 8 62 31 390 35 19 11 21 48 17

cannot be supported based on clear phylogenetic evidence.

Figures

FIGURE 1. Locations (in order of confirmation) across the Southeastern United States where Aetokthonos hydrillicola, invasive plants (Hydrilla, Egeria, Myriophyllum), and bird deaths from AVM have been confirmed.

FIGURE 2. Light microscope views of Aetokthonos hydrillicola. A. Fluorescence microscopy of A. hydrillicola on leaf of Hydrilla, scale = 100 μm. B. Fluorescence microscopy of thallus morphology in culture, scale = 100 μm. C–D. Fluorescence microscopy of branching filaments, arrows show hormogonia production in sheath, Scale = 10 μm. E. Thallus showing apical caps (black arrow) and thick-walled enlarged cells possibly functioning as akinetes (white arrow), scale = 10 μm. F–G.Thallus showing typical branching pattern and dehiscent apical cells (hormocytes?) (arrows), scale=10 μm.

FIGURE 3. Electron Microscope views of Aetokthonos hydrillicola. A–C. Scanning electron microscope views showing dehiscent apical caps (white arrows). D. Cells embedded in firm, thick, mucilage. Dark inclusions are likely cyanophycin bodies. E. Dehiscent apical cell showing initiation of dehiscence. F. Four thylakoids with characteristic electron dense and electron light areas, with dark areas associated with thylakoids representing phycobilisomes. G. Cell showing characteristic thylakoids, arrowed thylakoids shown at greater magnitude in Fig. 3F. H. High magnification views of cyanophycin and polyphosphate bodies.

FIGURE 4. Line drawing showing branching pattern (artist: Bradley Bartelme). Scale = 10 μm.

FIGURE 5. CAPS assay for A. hydrillicola. RsaI digestion of a PCR-amplified fragment of the16S rRNA gene produces a 247 bp sequence that is diagnostic for the species. At top, the sizes of RsaI digestion products from A. hydrillicola and related cyanobacteria are shown. Lane 1: RsaI digestion products of PCR-amplified DNA from an axenic culture of A. hydrillicola. Lane 2: PCR product from DNA of a field sample. Lane 3: RsaI digestion products of PCR-amplified DNA from a field sample. Arrowheads indicate the size (in base pairs) of the products of PCR and RsaI digestion. M = 100 bp DNA markers. Closed circles = PCR primer binding sites. Open circles = RsaI restriction sites.

FIGURE 6. Bayesian Analysis based on alignment of 318 OTU’s. Posterior probabilities and bootstrap values from a parsimony analysis of the same alignment are shown above or in close proximity to the nodes that are supported at least at the 50% level.Asterisks (*) indicate 1.00 or 100% support, hyphens (-) indicate unsupported node. Nodes with no support from either analysis have no annotation. The six arrows indicate the clades in the tree consisting of true-branching taxa, and show that the former concept of Stigonematales or Section V

FIGURE 7. Secondary structure of conserved domains in the 16S-23S ITS of true-branching Aetokthonos and Fischerella. A–C. D1–D1’ helix, D–F. Box-B helix, G–I. V3 helix.