KRAFTIONEMA ALLANTOIDEUM, A NEW GENUS AND FAMILY OF ULOTRICHALES(CHLOROPHYTA) ADAPTED FOR SURVIVAL IN HIGH INTERTIDAL POOLS1

Richard Wetherbee2 and Heroen Verbruggen

School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia

The marine, sand-dwelling green alga Kraftionemaallantoideum gen. et sp. nov. is described fromclonal cultures established from samples collectedin coastal, high intertidal pools from south easternAustralia. The species forms microscopic, uniseriate,unbranched, 6–8 lm wide filaments surrounded bya gelatinous capsule of varying thickness. Filamentsare twisted, knotted, and variable in length from 4to 50 cells in field samples but straighter and muchlonger in culture, up to 1.5 mm in length. Celldivision occurs in several planes, resulting indaughter cells of varying shape, from square torectangular to triangular, giving rise to gnarledfilaments. Mature cells become allantoid, elongatewith rounded ends, before dividing one time toform bicells comprised of two domed cells.Adjacent bicells separate from one another andmature filaments appeared as a string of looselyarranged sausages. A massive, single, bandedchloroplast covered 3/4 of the wall circumference,and contained a single large pyrenoid encased in astarch envelope that measures 1.5–2.5 lm.Filaments were not adhesive nor did they producespecialized adhesive cells or structures.Reproduction was by fragmentation with all cellscapable of producing a new filament. No motile orreproductive cells were observed. Filaments inculture grew equally well in freshwater or marinemedia, as well as at high salinity, and cells quicklyrecovered from desiccation. Phylogenetic analysisbased on the nuclear-encoded small subunitribosomal RNA (18S) shows the early branchingnature of the Kraftionema lineage amongUlotrichales, warranting its recognition as a family(Kraftionemaceae).

Key index words: 18S nuclear ribosomal DNA; Chloro-phyta; Kraftionema; Kraftionemaceae; molecular phy-logeny; sand dwelling; Ulotrichales

The Ulvophyceae (Chlorophyta) is a diverse classof eight orders classically distinguished by their lifecycles and ultrastructure, and more recentlythrough molecular phylogenetics using a range of

markers. The Ulotrichales and Ulvales form a clearlineage within the Ulvophyceae based on nuclearand plastid multigene data sets (Cocquyt et al. 2010,Fucikova et al. 2014). The two orders were classicallydistinguished by features of their life histories butmolecular phylogenies have not supported theorders as monophyletic (reviewed by Leliaert et al.2012).The Ulotrichales-Ulvales are highly diverse in

their cytology, morphology and ecology. Their func-tional types range from single-celled organisms tofilaments and colonies to larger multicellular sea-weeds such as Ulva and Monostroma. They have mar-ine species as well as species living in freshwaterhabitats. Some species grow on rock or on sand,while some are subaerial and others burrow intomarine limestone.Molecular phylogenies suggest that the origin of

the macroscopic growth forms has been achievedindependently in the major orders of the Ulvo-phyceae from ancestral unicellular and filamentousforms (Cocquyt et al. 2010). The genera Rhizoclo-nium, Ulothrix and Oedogonium, along with the newgenus Kraftionema described here, are some com-mon examples of Ulvophyceae characterized byuniseriate, unbranched filaments that undergo vege-tative reproduction by cell division and fragmenta-tion into segments of one or more cells. Thesefeatures are also found in several lineages of theChlorophyta, including the Trebouxiophyceae (e.g.,Stichococcus, Geminella and Koliellopsis) and Chloro-phyceae (e.g., Schizomeris, Cylindrocapsa, Radiofilum,and the siphonous Sphaeroplea). The green algaebelonging to the Streptophyta also have thesegrowth forms, most noticeably in Klebsormidium(Klebsormidiophyceae), as well as Spirogyra, Zygnema,Mougeotia, and other related genera from the Zygne-matophyceae.We have a research program studying the diversity

and phylogeny of microalgae adapted to life in mar-ine tide pools, a relatively unexplored, oftendynamic habitat where organisms typically requireadhesive structures and/or mechanism to survive.Several groups of sand-dwelling algae have beenstudied in detail, including species of diatoms (e.g.,Hay et al. 1993), pelagophytes (Wetherbee et al.2015) and euglenids (e.g., Kingston 1999) thatmigrate within sand in response to light, tidal, anddiurnal rhythms. In addition, benthic diatoms can

1Received 16 March 2016. Accepted 28 May 2016.2Author for correspondence: e-mail richardw@unimelb.edu.au.Editorial Responsibility: L. Graham (Associate Editor)

J. Phycol. 52, 704–715 (2016)© 2016 Phycological Society of AmericaDOI: 10.1111/jpy.12447

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have a range of adhesive structures to secure theirplace in benthic surfaces, including adhesive stalks,tubes, and pads (for review, Molino and Wetherbee2008). Unicellular dinoflagellates have also beenstudied to some degree, and can attach with stalksor pads, or adhere and flatten out over the surfaceof sand grains (e.g., Tamura and Horiguchi 2005).Benthic cell rafting is another classic mechanism foradhesion to sand. Motile unicells attach adjacent toone another and become amoeboid and crowdtogether to form rafts that appear to increase theoverall strength of adhesion. Rafting has also beenobserved for the zoospores of the green seaweedUlva (Callow et al. 1997) as well as unicellular gen-era of the Prymnesiophyceae (Haptophyta; Grantet al. 2011) and Raphidophyceae (Heterokonta;Grant et al. 2013) among others. Many species thatoccupy sand are observed to divide only in the ben-thic stage, producing motile cells that are short-livedbut allow for colonization of new surfaces. Theshorter the exposure of motile cells in a life history,the seemingly greater chance for survival in adynamic habitat.

During the summer of 2013, a sand-dwellinggreen alga comprised of thin, unbranched filamentswas isolated from sand samples taken from a highintertidal pool near Point Lonsdale Pier, Victoria,Australia. Another strain was isolated from sandobtained from an upper intertidal tide pond fromBittangabee Bay, NSW, Australia in April, 2014 andagain in March, 2015. Not subjected to excessivewave action, this alga does not feature any of theadhesive cells or structures observed in other sand-dwelling algae, but appears to flourish by producingfast growing, entangled filaments that readily repro-duce by fragmentation. Our goal in this study is todescribe the morphology, ecology and evolutionaryrelationships of these strains, which turn out to rep-resent a new species, genus, and family. Ourapproach consisted of a combination of cultureobservations, molecular phylogenetic analysis, lightand transmission electron microscopy.

MATERIALS AND METHODS

Sampling, isolation and culture. Sand samples were collectedin December 2013 from deep, high intertidal tide pools in asandstone platform at the base of a sandstone cliff ~100 mSSW of the Point Lonsdale Pier, Victoria (38°170 S, 144°360E). The pools would be replenished at virtually all high tidesand on our numerous visits to this site the salinity has alwaysbeen between 28& and 30&. On two separate occasions,sand samples were also collected from a large tidal pond onthe foreshore at Bittangabee Bay, 300 m north of the camp-ground, New South Wales (37°120 57 S, 150°000 53 E). Thispond would only be replenished at very high tides and dur-ing storm surges. The first samples were collected in March,2014 when the pond was reduced in size due to dry condi-tions and the salinity was 45. Sand samples were taken fromthe bottom of the pond as well as the dried out sand on thepond edge. Further samples were collected from the samesite in April, 2015 when significant rain and run off from the

surrounding area had collected in the pond, and salinity wasmeasured at between 15 to 20. Samples consisted of ~0.5–1.0 cm of sand taken from the bottom of the tide pools plusseawater that was placed into 60 mL plastic jars and the salin-ity determined. The sand only samples taken in April, 2015were returned to the lab where sterile filtered seawater (salin-ity of 30) was added. Clonal cultures of the filamentous greenalga that is the focus of this study were established by isolat-ing filaments from field samples by micro-pipetting intoeither K-enriched seawater medium (salinity of 30) (Kelleret al. 1987) or the freshwater media DM (Cohn and Pickett-Heaps 1988), and were maintained in 60 mL plastic contain-ers at 21°C under Sylvania 58 Luxline Plus and Gro-Luxflourescent lamps with a daily 14:10 h light:dark cycle; stockcultures were transferred into new K- or DM medium onceevery 2 weeks. Two strains (CS-1143, CS-1144) were isolatedinto culture, one from each sample site. The cultures usedfor microscopy were taken from the K-culture isolated fromPoint Lonsdale.

Salinity and desiccation experiments. Filaments in log phasegrowth in both K- (salinity of 30) and freshwater DM media(salinity of 2–3), were placed in 10 mL test tubes and concen-trated into a pellet with a hand centrifuge and the mediaremoved. Filaments grown in the K-marine media were resus-pended into freshwater DM media and cells grown in the DMmedia resuspended into K- media. Filaments were thenobserved immediately and subsequently over 72 h. Five millil-itres of culture medium with a low concentration of K. allan-toideum filaments was allowed to dry in Greiner 6-well plastictrays and placed in a constant temperature room at 21°C withthe lids off and allowed to dry out. Wells were rehydratedafter 30 and 60 d by the addition of either K- or DM mediaand filaments observed during rehydration and over the next72 h.

Light, fluorescence, and electron microscopy. Filaments of bothstrains were mounted onto microscope slides with coverslipssealed with a 1:1:1 ratio of Vaseline, lanoline, and paraffinwax. Live cells were observed and recorded using a ZeissAxioPlan 2 microscope (Carl Zeiss, Oberkochen, Germany)and photographs were taken using a Canon EOS 60D digitalsingle-lens reflex camera (Canon USA, Melville, NY, USA).Live cells in K-media were first stained with Hoechst stain(1.0 lg of stain per 1.0 mL; Lyndon et al. 1980) for one min-ute, washed in K-media and then stained with SYBRGreennucleic acid stain (Suzuki et al. 1997) for another minute,then mounted as above. Confocal images were taken on aLeica SP2, Laser Scanning Confocal Microscope. For trans-mission electron microscopy, 5 mL of a 2% solution of glu-taraldehyde in K- seawater media was mixed with 5 mL ofcells from a thick culture and left for 1 h, washed 39 with K-media, post fixed in 1% osmium tetroxide in K- media, fol-lowed by 39 washes in K- media. Cells were then broughtinto distilled water in steps: 75:25, 50:50, and 25:70 K-media:distilled water. Cells were washed three times in distilledwater and placed in a solution of 2% uranyl acetate in dis-tilled water for 1 h before gradual dehydration in EtOH(10%, 25%, 50%, 75%, 90%, and 39 at 100%). Cells werethen gradually embedded in Spurr’s medium (Spurr 1969).

DNA sequencing and analysis. DNA was isolated from freshcultures either using DNeasy Plant Mini Kit (Qiagen) or by fol-lowing an adapted CTAB, chloroform:isoamyl alcohol protocol(Kooistra et al. 2002). The SSU rRNA (18S) region was ampli-fied by PCR using primers described in Grant et al. (2011).PCR fragments were purified using the Isolate II PCR and GelKit Bioline (Alexandria, NSW, Australia). Sanger sequencingwas outsourced to the Australian Genome Research Facilityand reads were obtained with the PCR primers and four addi-tional primers: EukC (50-GCGGTAATTCCAGCTCCAATAGC-30), EukD (50-GTRAGTTTYCCCGTGTTGAGTC-30), BgR (50-

KRAFTIONEMA ALLANTOIDEUM GEN. ET SP. NOV 705

CAAGAACGAAAGTTAGGGG-30) and ENDF (50-GCTATTG-GAGCYGGAATTAC-30). Individual reads were assembled inGeneious R6 (Kearse et al. 2012) and the consensus sequencessubmitted to GenBank (KU862658-KU862659).

New 18S sequences were aligned with those from previoustaxonomic work on the order Ulotrichales (Friedl andO’Kelly 2002, O’Kelly et al. 2004a,b, �Skaloud et al. 2013, Sch-midt and Darcy 2015), and sequences of four Ulvales specieswere used as outgroups. Alignment was done with MUSCLEv3.8.31 using default settings (Edgar 2004) and the resultvisually verified. Phylogenetic trees were inferred from the1,823 positions long alignment using maximum likelihood(ML) and Bayesian inference (BI). Analyses were done with aGTR+Γ model. For ML, we performed 20 likelihood searchesstarting from different starting trees and 1,000 replicates ofthe rapid bootstrapping algorithm in RAxML v7.3.0 (Sta-matakis 2006). For BI, we used the birth-death tree prior andthe log-normal relaxed clock model implemented in BEASTv1.8.2 (Drummond et al. 2012). The chain was run for 10million generations, saving every 1,000th generation.Convergence of the chain was visually assessed in Tracer anda burn-in of one million generations was used to calculate amaximum clade credibility consensus tree with median nodeheights in TreeAnnotator.

We opted not to present other gene phylogenies becausethere are very few Ulotrichales sequences available for com-parison. Nonetheless, we have generated rbcL and tufAsequences for the species using high-throughput sequencingof a total DNA extract using previously described methods(Verbruggen and Costa 2015). These sequences are availableon GenBank (KX268523-KX268524) for future multigenestudies.

RESULTS

The phylogenetic analyses of 18S sequence datashow that the green alga we isolated forms a lineagedistinct from other sequenced Ulotrichales (Fig. 1,our new lineage flagged with a star). The families ofUlotrichales that were recognized in recent phyloge-netic studies are indicated with different shading,along with other family level lineages (�Skaloud et al.2013, Schmidt and Darcy 2015). This comparisonwith other family level lineages shows that our algais distinct at the family level. The combination ofthis distinctive molecular phylogenetic nature andits unique morphology among Ulotrichales leads usto recognize our alga as a new species, genus andfamily, Kraftionema allantoideum in the Kraftionema-ceae. The new family is situated in the “core Ulotri-chales” (O’Kelly et al. 2004a) indicated with ahexagon in the phylogeny (Fig. 1). While it is foundas a sister clade to Gomontiaceae in the Bayesianand ML trees, this position is not statistically sup-ported in either of the analyses.

Kraftionema allantoideum Wetherbee & Verbruggengen. et sp. nov.

Description—Filaments thin, uniseriate, unbranched,twisted, and knotted, variable in length from a fewcells in field samples up to 1.5 mm in culture,6–8 lm in width surrounded by a mucilagenous cap-sule between 1 and 3 lm thick (Figs. 2 and 3). Cellshighly variable in shape when actively dividing,from square to rectangular to triangular

2–6 lm 9 6–8 lm wide. Following division, all cellsenlarge and become allantoid (sausage-shaped) withrounded edges, 10–18 lm 9 6–8 lm wide. Allantoidcells typically divide once and produce bicells con-sisting of two dome-shaped cells. During most ofthe light/dark cycle, filaments comprised largely ofloosely associated allantoid cells and bicells that hadthe appearance of a string of sausages. A single,dark green, broad band chloroplast covered 3/4 ofthe wall circumference and contained a single pyre-noid encased in a starch envelope with an overalldiameter of 1.5–2.5 lm (Fig. 4). Filaments have noadhesive cells or structures. Reproduction was byfragmentation only with all cells capable of produc-ing new filaments. No motile zoospores or gameteswere observed. The species is characterized by a sub-stantial divergence of the 18S ribosomal RNA genefrom that of other known lineages of Ulotrichales,and its 18S sequence can be consulted on GenBank(accession KU862658).Holotype—MELU A 120812A, a mounted specimen

derived from strain CS-1143.Type locality—Approximately 100 m SSW of the

Point Lonsdale Pier, Victoria, Australia (38°170 S,144°360 E) in a small (2 m 9 1 m), deep (1.5 m)tide pool lined by sand in the upper intertidal; col-lected by Heroen Verbruggen in December 2013.GenBank accession numbers—Strain CS-1143:

KU862658; CS-1144: KU862659.Etymology—The genus is named in honour of Dr.

Gerald Thompson Kraft for his outstanding contri-butions to our knowledge of seaweeds from Aus-tralia and around the world, including the greenalgae of which Kraftionema is a new genus and fam-ily. Gerry was a legendary teacher of marine botanyat the University of Melbourne for three decades,and inspired generations of undergraduate, post-graduate and postdoctoral students with his unparal-leled scholarship, passion, and expertise in marinephycology. The specific epithet indicates the allan-toid shape of the mature cells, plus the appearanceof the filaments as a loose string of sausages.Habitat—marine, sand-dwelling.Culture lodgement—ANACC code: Point Lonsdale

strain CS-1143; New South Wales strain CS-1144;CSIRO, Hobart, Tasmania, AustraliaKraftionemaceae Wetherbee & Verbruggen fam.

nov.Type species—K. allantoideum Wetherbee & Ver-

bruggenDescription—Family forms a distinct lineage in

molecular phylogenies of 18S sequences, with thesequence of the type strain of the type species serv-ing as a reference (GenBank accession KU862658).Only known representative of the family formsmicroscopic, uniseriate, unbranched, knotted fila-ments variable in length surrounded by a mucilage-nous capsule. Cells are allantoid and have a single,dark green, broad band chloroplast contained a sin-gle, large pyrenoid encased in a starch envelope.

706 RICHARD WETHERBEE AND HEROEN VERBRUGGEN

Light microscopic and TEM observations. Uniseriate,unbranched, twisted, and knotted filaments werefound growing among sand grains in high intertidaltide pools. Filaments were not sticky nor were theyattached to the substratum by any differentiated celland/or adhesive structure. In field samples, theknotted filaments were relatively short, between 4and 50 cells and individual cells were rarely in focus(Fig. 2, a–c). The longer, entangled filaments fromcultures were best observed when tightly pressedbetween the slide and coverslip where sequences ofcells could be observed in clear focus (Fig. 2, d ande). In field samples, filaments of strain CS-1143from Point Lonsdale were consistently longer, up to50 cells in length, while strain CS-1144 from Bit-tangabee Bay had shorter filaments to a maximumof 8–15 cells. No other differences were observed inthe sequencing data, light, or electron microscopy,so the strains were regarded as the same species.

Cell shape and size varied considerably, depend-ing on the stage of the cell cycle and the age of theculture. In log phase cultures, most cells dividedduring the first 2–6 h of the light period, the result-ing cells being square, slightly rectangular or trian-gular in shape measuring 2–6 lm 9 6–8 lm wide(Figs. 2, d, f, g; 3, a and b). The planes of cell divi-sion often varied considerably, accounting for thetwisted, knotted filaments (Figs. 2g; 3, a and b).The large banded chloroplast occupied much of thecell volume and contained a large pyrenoid sur-rounded by a starch envelope that was obvious inthe light microscope (Figs. 2, e–g; 3, a–c).

Following the division cycle, cell growth resultedin elongate cells with rounded ends that eventuallypulled away from adjacent cells in the filament(Figs. 2, e and f; 3c). These allantoid cells (sausage-shaped) were 10–18 lm 9 6–8 lm wide and typi-cally divided once, forming bicells consisting of twodome-shaped cells that shared a cross wall (Figs. 2,e and f; 3c). For most of the cell cycle, allantoidcells and bicells were aligned in a haphazard wayalong each filament, looking like a string of sau-sages (Figs. 2, e and f; 3, c, d and g). Filamentsoften fragmented at this stage and formed frag-ments of one or more cells/bicells retaining a por-tion of the parental capsule (Fig. 3b). During thenext division cycle, the allantoid cells and bicellsfrom each fragment divided and developed into thenext generation of filaments, with the rounded endsof the parental dome cells defining the ends ofeach new filament (Figs. 2, f and g; 3, a and b).The 1–3 lm thick capsule could be observed at anystage of development (Figs. 2g; 3, a and b), but wasmost obvious during fragmentation (Fig. 3b).

Stains-All (MP Biomedicals, Cole and Narine1984) showed two distinct extracellular layers, thecell wall adjacent the plasma membrane and thecapsule that encased filaments (Fig. 3, d–f). The cellwall was best observed by staining live cells withHoechst stain (Lyndon et al. 1980). The dye did

not enter the cells or stain the capsule material, butaccumulated at the cell surface (blue in Fig. 3, g–i),presumably within the fibrous cell wall observed intransmission EM images (Fig. 4, a–c). The samecells were then stained with SYBRGreen nucleic acidstain (Suzuki et al. 1997) that penetrated the livecells and stained the nuclei (yellow green). Redauto fluorescence revealed the chloroplast, whichoccupied most of the cell cytoplasm, plus the pyre-noid, which was surrounded by large starch grainsthat do not autoflouresce. Allantoid bicells are seenas a loose chain, surrounded by a cell wall that doesnot abut adjacent bicells in the filament (Fig. 3g).However, as the allantoid cells divide, the cell wallsof recently divided cells are appressed against oneanother (Fig. 3, h and i), and the progeny of a sin-gle allantoid bicell can be discerned by the roundeddome cells that defined the parental bicell. An eightcell fragment resulting from two divisions of a bicellwas observed (Fig. 3i), with six square cells sand-wiched between the two parental dome cells.Transmission electron micrographs further

revealed the chloroplast and pyrenoid surroundedby a starch envelope (Fig. 4, a–d). Chloroplast thy-lakoids intersected the pyrenoid matrix from severallocations through the starch envelope (Fig. 4d).Starch grains were also common within the chloro-plast matrix (Fig. 4, a–d). The fibrous cell walls areobserved while the capsules are electron opaque.When the filament orientation is correct, the nucleiand starch coated pyrenoids in daughter cells hadopposite polarity. This was most obvious in recentlydivided cells (Fig. 3h) and was also observed inelectron microscope images (Fig. 4c).Salinity and desiccation trials. Filaments of

K. allantoideum grew well in both K-marine media ata salinity of 30 or in freshwater media DM (salinityof 2–3). In order to assess resistance to changes insalinity, filaments grown in K-marine media (salinityof 30) were concentrated by centrifugation andresuspended immediately into DM freshwater media(salinity of 2–3), or the reverse with filaments grownin DM concentrated and resuspended into K-media.No obvious impact on filament/cell survival orgrowth was observed, as cells continued to divide insubsequent division cycles. Filaments grown in bothDM and K-media were allowed to dry out in openPetri dishes and left uncovered in a constant tem-perature room with a 10:14 light:dark cycle at 21°Cfor 30 and 60 d. Filaments were surrounded byenlarged capsules and never appeared to shrivel orlose color. On rehydration the filaments regainedtheir normal morphology and within 24 h dividingcells were obvious as the filaments resumed growth.

DISCUSSION

The phylogeny clearly shows that Kraftionema is anearly branching lineage within the Ulotrichales. Italso shows very limited statistical support for many

KRAFTIONEMA ALLANTOIDEUM GEN. ET SP. NOV 707

of the deeper relationships among Ulotrichales lin-eages. This includes the sister relationship betweenKraftionema and the Gomontiaceae shown in thefigure, which has very low statistical support andshould not be interpreted as indicative of these twofamilies forming a natural lineage.

The lack of support in our phylogenetic treepoints to the 18S gene not providing muchinformation about early relationships among Ulotri-chales lineages. For future studies of the phyloge-netic history of the order, a different source ofdata will thus be needed. In the related classTrebouxiophyceae, chloroplast phylogenomics havedrastically improved resolution at the level of rela-tionships among genera and families, and in manycases also among the orders (Lemieux et al. 2014,

Turmel et al. 2015), and we anticipate that thiswould also improve the resolution of the relation-ships among the higher level taxa of the Ulvales-Ulotrichales lineage.Our choice to recognize Kraftionemaceae as a

family is primarily based on the early branching nat-ure of the lineage in the phylogeny. The level ofmolecular divergence between Kraftionema and theGomontiaceae is similar to that between Ulotricha-ceae and Monostromataceae (Fig. 1). A secondargument to recognize the family is that not doingso would result in the genus not being assigned toany family, because the low support for earlybranches does not allow its assignment to any of theother families. We hope that future studies basedon larger data sets will resolve Ulotrichales radiation

0.0040

Gloeotilopsis paucicellulare Z47997

Gomontia polyrhiza AY278216

Kraftionema allantoideum CS-1143

Monostroma grevillei AF015279

Halochlorococcum moorei AY198122

Protoderma sarcinoidea GQ121000

Ulothrix zonata AY278217

Collinsiella tuberculata AY198125

Hazenia basiliensis Z47996

Urospora penicilliformis AB049417

Pseudendoclonium akinetum DQ011230

Chamaetrichon capsulatum GQ121003

Hazenia broadyi HF570951

Acrosiphonia arcta AY303600

Gloeotilopsis paucicellulare GQ121001

Ulothrix zonata Z47999

Gloeotilopsis planctonica Z28970

Gayralia sp. JF680952

Gloeotilopsis sarcinoidea Z47998

Uncultured clone E108.06D HQ188958

Eugomontia sacculata AY198123

Bolbocoleon piliferum AY303596

Pseudendocloniopsis botryoides AJ416103

Chamaetrichon capsulatum GQ121002

Desmochloris halophila FM882216

Kraftionema allantoideum CS-1144

Chamaetrichon capsulatum GQ120998

Trichosarcina mucosa AM109906

Pseudoneochloris marina U41102

Capsosiphon groenlandicus DQ821514

Monostroma nitidum AF499665

Planophila laetevirens AJ416102

Hazenia mirabilis AF387156

1.00/91

1.00/95

0.97/51

1.00/100

0.99/100

1.00/100

1.0080

1.00/100

1.00/100

1.00/92

1.00/100

1.00100

1.00/80

-/71

Ulvales

Acrosiphoniaceae

Gayraliaceae

Planophila lineage

Kraftionemaceae

Gomontiaceae

Nepal clade

Ulotrichaceae

Trichosarcina lineage

Monostromataceae

Hazenia lineage

FIG. 1. Phylogenetic tree ofUlotrichalesinferredfromadatasetof 18S ribosomal RNA sequences.The new species Kraftionemaallantoideum—indicated with a star—is shown to be an early branchinglineage of the “core Ulotrichales”(indicated with a hexagon). Thetree was inferred by BI and showsBayesian posterior probabilitiesand ML bootstrap values.

FIG. 2. Light micrographs in DIC of Kraftionema allantoideum gen. et sp. nov during stages of its cell cycle. (a) Several knotted, entangled fil-aments during the division cycle as seen in field samples. (b, c) Single, knotted filaments of between 30 and 50 cells as seen in field samples.(d) Entangled filaments from cultures during the division cycle, flattened between the slide and coverslip to get the filaments more in focus,cells appear mainly square to rectangular. (e) Following the division cycle, maturing filaments are twisted and entangled and comprised ofallantoid cells (A) and bicells (Bi) held in a loose association within the capsule. Pyrenoids and starch envelopes are observed when cells arein focus (arrows). (f) A maturing filament shows a string of allantoid cells (A) and bicells (Bi) at the bottom of the image. During the divisionstage, allantoid cells, and bicells divide further (arrowheads at the top of the image) to produce short fragments within the parental filament.Pyrenoids are encased in a starch envelope (arrows). (g) Short filament resulting from fragmentation is surrounded by the capsule (arrows)and dividing in several planes (arrowheads) resulting in cells of various shape. Scale bars = 40 lm (a), 25 lm (b–d), 15 lm (e), 10 lm (f, g).

708 RICHARD WETHERBEE AND HEROEN VERBRUGGEN

and discover how Kraftionema relates to other fami-lies. Such studies would also facilitate subdividingthe order into monophyletic lineages at the

suborder and family level. We have not presentedother gene phylogenies because few Ulotrichalessequences are available for comparison, but

KRAFTIONEMA ALLANTOIDEUM GEN. ET SP. NOV 709

710 RICHARD WETHERBEE AND HEROEN VERBRUGGEN

deposited rbcL and tufA sequences of Kraftionema forfuture work.

Besides the molecular data, a combination ofmorphological features delineates K. allantoideumfrom other green algae with uniseriate, unbranchedfilaments that undergo fragmentation as a form ofasexual reproduction (examples in Table 1). Fea-tures include twisted and knotted filaments, a dis-tinct capsule, cells with a massive, single-bandedchloroplast containing one large pyrenoid encasedin a starch envelope and allantoid cells and bicellsloosely arranged in mature filaments. Unusual for asand-dwelling protist, filaments lack any obviousmechanism for adhesion, neither a sticky surfacenor a specialized basal cell/adhesive pad for attach-ment. In addition to the algae listed in Table 1,there are a range of macroscopic algae (seaweeds)that have uniseriate, unbranched filaments repro-ducing by fragmentation, but we have not included

them in Table 1 as their size clearly sets them apartfrom the microscopic species we need to compareKraftionema to. These macroscopic algae lack thecombination of fine details found in K. allantoideumand generally possess complex morphologies andlife histories.Based on morphology alone, the new genus has

characteristics that might place it in a number ofclasses in two different divisions of green algae. Ofall those genera investigated, including those listedin Table 1, some species of the freshwater genusGeminella Turpin (Tsarenko 2011) are known to pro-duce somewhat bent, twisted filaments encased inmucilage with a single pyrenoid, thereby showing amorphological resemblance to Kraftionema. How-ever, a combination of morphological features,including the absence of a pyrenoid starch envel-ope, make Kraftionema distinctive. Only a few speciesof Geminella have been sequenced with all now

FIG. 3. Light micrographs of filaments of Kraftionema allantoideum gen. et sp. nov. in DIC (a–c), stained with Stains-All (d–f)) and fluorescentlystained (g–i). (a) Dividing fragment with different division planes (arrowheads) resulting in cells of different shape. Pyrenoids plus starch envelopes(p) and the capsule (arrow) are observed. (b) Recently fragmented, dividing filaments, revealing division planes (arrowheads) resulting in cells ofdifferent size and shape. The capsule (arrows) is obvious. (c) Several hours after the division cycle, mature allantoid bicells are loosely associatedwithin the capsule. (d–f) DIC images following staining with StainsAll showing the cell wall (cw) and capsule (arrows). (g–i) Fluorescent imagesshowing the cell wall (blue) from Hoechst stain, the nuclei (yellow/green) from SYBRGreen stain and chloroplast auto-fluorescence (red). (g) Fila-ment of bicells showing their loose arrangement in the filament. (h) Dividing filament with blue walls and showing the opposing polarity of pyre-noids (p) and nuclei (n) in adjacent daughter cells. (i) An allantoid bicell has undergone two division cycles to produce an eight-celled fragment ofsix square cells and the two terminal dome cells (stars) that defined the original cell. Scale bars = 5 lm (a, e, f, h, i), 10 lm (b, c, g), 15 lm (d, g).

FIG. 4. Transmission electronmicroscope images (a–d) of cellsfrom Kraftionema allantoideum gen.et sp. nov. showing the nucleus(n), pyrenoid (p) and surroundingstarch envelope. The chloroplast(c) dominates the cell cytoplasm,and contains many small starchgrains. (a) Section through amature allantoid cell with closelyappressed cell wall (cw). (b, c)Recently divided cells showingtheir cell walls (cw) and theopposite polarity of the pyrenoids(p). (d) Chloroplast thylakoids (t)intersected the pyrenoid matrixthrough the starch envelope. Scalebars = 1 lm (a, d), 1.5 lm (b, c),5 lm (a, e, f, h), 10 lm (b, c, g),15 lm (d).

KRAFTIONEMA ALLANTOIDEUM GEN. ET SP. NOV 711

TABLE1.

Morphologicalch

aracteristicsforsomeuniseriate,unbranch

ed,filamen

tousmicroscopic

gree

nalgaefrom

theUlvophyceaean

dTrebouxiophyceae

(Chlorophyta)

andKlebsorm

idiophyceae(Strep

tophyta).

Taxon

Referen

ceFilam

ent

diameter

Filam

ents

knotted

Basal

attach

men

tCell

size

(lm)

Nuclei

per

cell

Cap

sule/

sheath

Plastid

no.

andtype

Pyren

oids

Pyren

oid

starch

envelope

Motile

asex

.stage

Hab

itat

Ulvophyceae(C

hlorophyta)

Kraftionem

aallantoideum

Thispap

er6–

8lm

Yes

None

6–89

2–18

1Ye

s1ban

ded

,massive

1Ye

sNo

Marine

sand

Binuclearia

tatran

aDay

etal.

1995

8–12

lm

No

Basal

cell

holdfast

8–12

920

–35

1Ye

s1girdle

shap

ed1

Yes

Zoospores

Freshwater

Coccothrix

chlorolobata

Broad

yan

dLokh

orst

2000

3.5–5

lm

No

None

3.5–5

92–

71

No

1lobed

,parietal

1Ye

sNo

Terrestrial

Fottea

pyrenoides

Broad

y19

766lm

No

None

69

9–18

1Mucilage

nous

colonies

1parietal

plate-like

1No

No

Terrestrial

Gloeotilopsis

plan

ctonica

Deason

1969

3–7lm

No

None

3–7lm

98–

241

No

1plate

like

1or2

No

Zoospores

Freshwater

planktonic;

terrestrial

Heterothrichopsis

viridis

Iyen

garan

dKan

tham

ma

1941

6–8lm

No

None

6–89

14–1

61

No

1–8 plate-like

or

disco

id

1ormore

per

plastid

Yes

No

Freshwater

and

terrestrial

Hormidiopsis

crenulata

Burova

etal.20

1114

–16lm

No

None

8–10

98–

101

Yes

1plate-

like

None

No

No

Freshwater

and

terrestrial

Hormidiospora

verrucosa

Lokh

orst

AlgaeBase

4–6lm

No

None

4–69

15–3

01

No

1–2 parietal,

laminate

None

No

Zoospores

Freshwater

Okellya

curvata

Leliaert

etal.20

097–

10lm

No

Basal

disco

idholdfast

4–10 940

–80

1,2,4–8

No

1parietal

None

No

Zoospores

Marine

Pearsoniella

variabilis

Sarm

aan

dKeshri

1995

20–4

0lm

No

Lobed

holdfast

20–4

09

20–9

01

No

1ring-

shap

edMan

yNo

Zoospores

Freshwater

Rhizoclonium

tortuosiumi

Koster

1955

50–6

0lm

No

Basal

cells

orholfast

40–6

09

80–1

20Multiple

Yes

1parietal,

reticu

late

2- man

yNo

Zoospores

Freshwater

andmarine

Ulothrix

zonata

John20

0225

–40lm

No

Simple

or

rhizoidal

basal

cells

25–4

09

10–2

01

No

1girdle-

like

,parietal

1- man

yOccasionally

Zoospores

Freshwater

andmarine

Trebouxiophyceae

Catenaviridis

Chodat

1900

6–10

lm

No

None

6–10

914

–20

1Ye

s1parietal,

ban

d-shap

edNone

No

No

Freshwater

Gem

inella

interrupta

Tsarenko

2011

10–2

0lm

Some

species

Sessileor

free

floating

4–89

4–10

1Ye

s1parietal,

girdle

shap

ed

1No

No

Freshwater

and

terrestrial

Gloeotila

vesiculosa

Tsarenko

2011

10–2

0lm

No

None

6–10

915

–30

1Ye

s1ban

dshap

ed1

No

No

Freshwater

Koliellopsis

inundata

Lokh

orst

etal.20

043–

4lm

No

None

3–49

12–3

21

No

1parietal

None

No

Zoospores

Terrestrial

and

freshwater

(continued

)

712 RICHARD WETHERBEE AND HEROEN VERBRUGGEN

belonging to the Trebouxiophyceae. The single spe-cies Geminella terricola J.B.Petersen was subsequentlyfound to be Interfilum terricola (J.B. Petersen)Mikhailyuk, Sluiman, Massalski, Mudimu, Dem-chenko, Friedl & Kondratyuk, and transferred tothe Klebsormidiophyceae (Mikhailyuk et al. 2008).Some species of Klebsormidium P.C.Silva, Mattox &

W.H.Blackwell and Stichococcus N€ageli show a resem-blance to Kraftionema, particularly in old cultureswhere their filaments are more constricted at thecross walls, yet are quite distinct. Pyrenoid morphol-ogy is a major difference, as the pyrenoid of Kleb-sormidium is coated by a large number of smallstarch grains, so the large, distinct starch envelopseen in Kraftionema is not formed. Stichococcus has asmall, naked pyrenoid in all but one species, wherea number of starch grains surround the pyrenoid(Neustupa et al. 2007). Interestingly, Klebsormidiumand Stichococcus were once considered to be closelyrelated in classifications based on their similar vege-tative morphology and by ultrastructural characteris-tics such as the presence of open mitosis andcytokinesis by a cleavage furrow (e.g., Mattox & Ste-wart 1984, Ettl & G€artner 1995). Originally, Kleb-sormidium along with Stichococcus were placed in thecharophycean evolutionary lineage based on ultra-structural features, including the structure of theflagellar apparatus of motile zoospores, althoughmotile cells were not observed in Stichococcus.Despite all the similarities, molecular phylogeniesconstructed mainly on 18S rDNA gene sequenceshave shown these two genera to be only distantlyrelated, Klebsormidium was confirmed within thecharophycean lineage (Division Streptophyta) andStichococcus in the Trebouxiophyceae of the chloro-phycean lineage (Division Chlorophyta).In terms of pyrenoid morphology, Ulothrix (Ulvo-

phyceae) most closely resembles Kraftionema. How-ever, Ulothrix filaments consist of relatively large,cylindrical cells with no constriction of the crosswalls, and filaments are never twisted and knottedlike observed in the thin filaments of Kraftionema. Amucilaginous sheath is absent from Ulothrix and fila-ments are anchored to the substratum by an adhe-sive basal cell, additional features that distinguish itfrom Kraftionema.A combination of features distinguish the fila-

ments of K. allantoideum, but nothing is as com-pelling as the clear delineation determined by themolecular phylogeny. Morphologically, the uniseri-ate, unbranched filaments result from cells divid-ing in several different planes, resulting in cellsthat are variously shaped and therefore assembledinto markedly twisted and knotted filaments. Fol-lowing a cycle of divisions, the cells enlarge andbecome allantoid, and often divide into bicellsthat remain as such until the next division cycle.Allantoid cells and bicells do no share adjacentwalls, and have the unusual appearance of a stringof sausages. The large pyrenoid with thylakoidsT

ABLE1.

(continued

)

Taxon

Referen

ceFilam

ent

diameter

Filam

ents

knotted

Basal

attach

men

tCell

size

(lm)

Nuclei

per

cell

Cap

sule/

sheath

Plastid

no.

andtype

Pyren

oids

Pyren

oid

starch

envelope

Motile

asex

.stage

Hab

itat

Stichococcus

jenerensis

Neu

stupa

etal.20

074–

6lm

No

None

3–59

4–12

1No

1parietal

1(n

one

inmost

species)

Yes

No

Terrestrial

for

thisspecies

Klebsorm

idiophyceae(Strep

tophyta)

Klebsormidium

flaccidium

Silvaet

al.

1972

4–6lm

No

Unattach

edorbasal

cellhold

fast

4–69

8–10

1No

1parietal

1Ye

sZoospores

Terrestrial

Alllisted

speciesundergo

fragmen

tationas

aform

ofasex

ual

reproductionan

dwereselected

based

onpossessinganumber

ofmorphologicalfeaturesobserved

inKraftio-

nem

a.In

appearance

only,thege

nusGem

inella,as

wellas

Klebsormidium

andStichococcus,most

closely

resemble

Kraftionem

a,butthey

arein

differentclasses.

KRAFTIONEMA ALLANTOIDEUM GEN. ET SP. NOV 713

intersecting the matrix and surrounded by anenvelope of large starch grains is a distinctive fea-ture of cells, and these features may be considereda deeply conserved character in the evolutionarylineages of the Trebouxiophyceae and Chloro-phyceae.

Wave action would be limited in the high inter-tidal pools where K. allantoidium was collected,except during extreme tides or storm surges wheresome disturbance would occur. Adhesion is animport mechanism for survival in dynamic environ-ments such as sand subjected to constant waveaction. However, K. allantoideum filaments have nospecialized cells or mechanism for adhesion to asurface, nor do they have any known motile stage intheir life history in order to populate new sand. Fila-ment capsules were never observed to be sticky, notsurprising as that feature would have left themclogged with sand and detritus in their habitat.Likewise, filaments were never observed adhered tosand, and therefore there was no evidence to sug-gest that the capsule of growing filaments becamemomentarily adhesive on contact before then cur-ing. Direct adhesion to a substratum appears unnec-essary in a sand dwelling organism that is notsubjected to routine wave action, but rather main-tains itself by producing fast growing, knotted andentangled filaments that penetrate throughout thesubstratum. Filaments routinely fragment, particu-larly if subjected to any disturbance, increasing theirchances of establishing and maintaining themselvesin sand.

The ability of filaments to grow successfully in arange of salinities, as well as periods of desiccation,are consistent with a sand organism living in highintertidal tide pools or ponds just above the intertidalwhere only extreme tides or storm surges wouldreplenish the pools. Such ponds may occasionally dryout, or the salinity might increase with evaporation ordecrease drastically with the addition of rainwater.The protective capsule of K. allantoideum appears toallow the filaments to successfully survive periods ofosmotic stress and/or drying out. All of these featurestogether would appear to enhance the survival of thespecies in its natural habitat.

We thank Joana Costa for generating the 18S sequences andFabio Rindi for nomenclatural advice. Dr. Simon Crawfordhelped with the electron microscopy. Funding during thepreparation of this study was provided by the Australian Bio-logical Resources Study (RFL213-08) and the AustralianResearch Council (FT110100585, DP150100705).

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