ARTICLE IN PRESS European Journal of PROTISTOLOGY European Journal of Protistology 40 (2004) 317–328 www.elsevier.de/ejop Comparative morphology and molecular phylogeny of aplanochytrids (Labyrinthulomycota) Celeste A. Leandera,Ã, DavidPorter b, Brian S. Leanderc aDepartment of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 bDepartment of Plant Sciences, University of Georgia, Athens, GA 30605, USA cProgram in Evolutionary Biology, Departments of Botany and Zoology, Canadian Institute for Advanced Research, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 Received19 April 2004; accepted30 July 2004 Abstract Aplanochytrids comprise one of three major subgroups within the Labyrinthulomycota. We have surveyed the diversity of aplanochytrids and have discovered that most isolates are difficult to identify to species because of character plasticity andambiguity. Ten isolates were studiedusing molecular phylogenies basedon small subunit ribosomal gene sequences (SSU rDNA) andmorphological characters derivedfrom light microscopy, SEM andTEM (e.g., colony size, colony shape, colony pattern, agar penetration, cell shape, cell surface patterns, cell inclusion characteristics andectoplasmic net morphology). Of these isolates, we couldpositively identifytwo of them to species, namely Aplanochytrium yorkensis (Perkins, 1973) Leander and Porter, 2000 and A. minuta (Watson andRaper, 1957) Leander and Porter, 2000. We used standardized conditions for growing aplanochytrid isolates in order to minimize environmentally induced phenotypic plasticity in our comparative studies of morphology. By mapping the morphological characters listedabove onto a conservative phylogenetic topology derivedfrom SSU rDNA sequences, we were able to identify several synapomorphies (e.g., gross colony characteristics and cell surface patterns) that serve as valuable taxonomic characters for the identification of species and specific clades of aplanochytrids. r 2004 Elsevier GmbH. All rights reserved. Keywords: Aplanochytrium; Character evolution; Labyrinthulomycota; Marine fungi; Stramenopiles; Molecular phylogeny Introduction andthraustochytridspossess globose sporangia and multi-laminar scaly walls (Darley et al. 1973; Perkins The Labyrinthulomycota is comprisedof three 1973; Porter 1989). However, the vegetative cells of distinct groups of marine heterotrophic stramenopiles aplanochytrids are capable of crawling movement using that are readily distinguished on the basis of gross ectoplasmic filaments that do not completely enrobe the morphological characters andmolecular phylogenetic cells, a characteristic that distinguishes aplanochytrids data: labyrinthulids (slime nets), thraustochytrids and from labyrinthulids (which are enrobed by and glide aplanochytrids (Honda et al. 1999; Leander and Porter through the ectoplasmic network) andthraustochytrids 2000, 2001; Raghukumar 2002)(Fig. 1). Aplanochytrids (which are immobile, except for the biflagellate spore stage) (Leander and Porter 2000, 2001). ÃCorresponding author. Fax: +1 604 822 6089. Aplanochytrids are classified within a single genus, E-mail address: [email protected] (C.A. Leander). Aplanochytrium, which is defined by the crawling 0932-4739/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2004.07.003 ARTICLE IN PRESS 318 C.A. Leander et al. / European Journal of Protistology 40 (2004) 317–328 depending on growth conditions, such as nutrients, temperature andsalinity ( Booth andMiller 1968 ; WetheredandJennings 1985 ). Therefore, as an intro- duction to the overall diversity of the group, it is prudent to first summarize the diagnostic characteristics andculture conditions associatedwith the eight species described so far. The type species of Aplanochytrium, A. kerguelensis Bahnweg andSparrow, 1972 was originally isolated from sub-Antarctic waters andwas grown in culture on pine pollen. The isolate was classifiedas a new genus because of the formation of spores that crawledalong substrates, rather than highly motile biflagellatedspores as seen in other members of the Labyrinthulomycota. Bahnweg andSparrow (1972) usedthe term ‘aplanos- pores’ to describe the relative lack of motility present in A. kerguelensis, which formedthe basis of the new genus name. However, the term ‘aplanospores’ generally refers to spores that are non-motile, so the term is perhaps inappropriate to describe the crawling spores of aplanochytrids. Accordingly, we have chosen to use the term ‘crawling spores’ from this point forwardto refer to the defining property of aplanochytrids. More- over, the definition of Aplanochytrium has since been modified to allow the inclusion of taxa that make biflagellate spores in addition to crawling spores (Leander and Porter 2000). The crawling spores of A. kerguelensis contain a large eccentric vacuole, which is a distinguishing character of the species, and a granular cytoplasm. The vacuole is also a conspicuous part of the developing sporangium. Ten to fifty crawling spores are Fig. 1. Illustrations of the three major subgroups within the releasedthrough rupture in the sporangial wall, or the Labyrinthulomycota. (a) Labyrinthulids, as represented by spores germinate within the oldsporangial wall to make Labyrinthula sp. with spindle-shaped vegetative cells enrobed clusters of sporangia. The ectoplasmic net is formed by the ectoplasmic net. (b) Thraustochytrids, as represented by from several places on the spore body and extends in all a non-proliferous Thraustochytrium sp. with unilateral non- directions. motile ectoplasmic net. (c) Aplanochytrids, as represented by A. yorkensis (Perkins, 1973) Leander and Porter, 2000 an Aplanochytrium sp. gliding via an ectoplasmic net. was isolatedfrom oyster mantle, water samples, sedi- ment anddetritusandwas maintainedin axenic culture on a glucose/gelatin hydrolysate medium (Perkins 1973). movements andectoplasmic net characteristics de- Unlike A. kerguelensis, A. yorkensis makes biflagellate scribedabove ( Leander and Porter 2000). Aplanochy- spores in addition to crawling spores. Perkins also trids are most often associated with dead and decaying described various membrane-bound inclusions within plant material, but some species are known to be the cytoplasm, which might be the same structures as pathogens of molluscs (Bower 1987). Although Apla- those causing the granular appearance in A. kerguelen- nochytrium remainedmonotypic for 30 years ( Bahnweg sis. A. yorkensis forms clumpy, cream-colouredcolonies, andSparrow 1972 ), the transfer of five species from the andmature sporangia are rarely motile. The ectoplasmic genus Labyrinthuloides andone species from Labyr- net has two major radiating filaments that subsequently inthula to Aplanochytrium resultedin a total of seven branch into finer threads. species (Leander and Porter 2000). One new species, A. A. minuta (Watson andRaper, 1957) Leanderand stocchinoi Andreoli and Moro, 2003, has since been Porter, 2000 was originally isolatedfrom a green alga, added (Moro et al. 2003), bringing the total number of Ulva sp. collectedfrom the undersurface of a boat recognizedaplanochytridspecies to eight. Nonetheless, (Watson andRaper 1957 ). We have subsequently identification of aplanochytrid species is difficult due to isolated A. minuta from many other substrates, includ- the plastic nature of fundamental morphological fea- ing chlorophytes, rhodophytes, Zostera marina and tures. Important identification characters can change sediments. The cells are more oblong than those of ARTICLE IN PRESS C.A. Leander et al. / European Journal of Protistology 40 (2004) 317–328 319 A. yorkensis and A. kerguelensis and divide into tetrads. typical. Unlike most aplanochytrids, crawling spores are Biflagellate spores were not described in the original not formed. Sporangia are readily made with the description, but have been reported since (Perkins 1974). addition of seawater and three to ten biflagellate spores A. minuta tends to spread in distinct rays as a monolayer are releasedthrough a tear in the wall. over agar surfaces. Vegetative cells remain motile for the A. thaisii (Cox andMackin, 1974; Leanderand entire lifecycle, except during daughter cell formation, Porter, 2000) was described as a labyrinthulid. A. thaisii andhave very fine ectoplasmic net elements ( Watson was isolatedfrom the marine gastropod Thais haemas- andRaper 1957 ). Movement involves alternate reversal toma floridana, andwas grown on beef serum agar ( Cox of direction. andMackin 1974 ). Like A. saliens and A. schizochy- A. saliens (Quick, 1974) Leander and Porter, 2000 was trops, this species is characterizedby sporangia-rich described from the marine grass Halophila englemannii;it colonies alternating with vegetative cell-rich colonies. was originally grown on a modifiedbloodserum agar Both types of colonies occur in mono-layers. The (Quick 1974a). A. saliens is relatively rare (foundin two vegetative colonies seem to be determinate, because of 12 host plants), andhas not been reportedsince the they never return to a sporangial stage. Vegetative cells original description. The sporangia are spherical, but may divide by binary or quaternary divisions, producing be compartmentalizedmaking the sporangium appear to tetrads that are enveloped by a mucilaginous sheath. have a rough texture. Four to twenty crawling spores are Sporangia are immobile with small reflective drops. releasedvia fissures in the wall of the sporangium andare Biflagellate spores are readily produced in the host characterizedby