Acta Protozool. (2012) 51: 291–304 http://www.eko.uj.edu.pl/ap ACTA doi:10.4467/16890027AP.12.023.0783 PROTOZOOLOGICA Ultrastructure of Diplophrys parva, a New Small Freshwater Species, and a Revised Analysis of Labyrinthulea (Heterokonta) O. Roger ANDERSON1 and Thomas CAVALIER-SMITH2 1Biology and Paleo Environment, Lamont-Doherty Earth Observatory of Columbia University, Palisades, New York, U.S.A.; 2Department of Zoology, University of Oxford, South Parks Road, Oxford, UK Abstract. We describe Diplophrys parva n. sp., a freshwater heterotroph, using fi ne structural and sequence evidence. Cells are small (L = 6.5 ± 0.08 μm, W = 5.5 ± 0.06 μm; mean ± SE) enclosed by an envelope/theca of overlapping scales, slightly oval to elongated-oval with rounded ends, (1.0 × 0.5–0.7 μm), one to several intracellular refractive granules (~ 1.0–2.0 μm), smaller hyaline peripheral vacuoles, a nucleus with central nucleolus, tubulo-cristate mitochondria, and a prominent Golgi apparatus with multiple stacked saccules (≥ 10). It is smaller than pub- lished sizes of Diplophrys archeri (~ 10–20 μm), modestly less than Diplophrys marina (~ 5–9 μm), and differs in scale size and morphology from D. marina. No cysts were observed. We transfer D. marina to a new genus Amphifi la as it falls within a molecular phylogenetic clade extremely distant from that including D. parva. Based on morphological and molecular phylogenetic evidence, Labyrinthulea are revised to include six new families, including Diplophryidae for Diplophrys and Amphifi lidae containing Amphifi la. The other new families have dis- tinctive morphology: Oblongichytriidae and Aplanochytriidae are distinct clades on the rDNA tree, but Sorodiplophryidae and Althorniidae lack sequence data. Aplanochytriidae is in Labyrinthulida; the rest are in Thraustochytrida; Labyrinthomyxa is excluded. Key words: Diplophryidae, fi ne structure, molecular genetics, Labyrinthomyxa, labyrinthulean taxonomy. INTRODUCTION scription of any Diplophrys, so D. archeri is the type species. Subsequent studies (e.g. Hertwig and Lesser 1874, Penard 1902) reported that this non-fl agellate or- Barker (1868), in a brief report to the Dublin Micro- ganism, containing a prominent refractive granule and scopical Club (1867), originally described Diplophrys emergent polar fi lopodia, occurred as small mounds of archeri as an ‘exceedingly minute, nearly orbicular or cells on submerged aquatic plants. The cells appeared broadly elliptic’ freshwater rhizopod bearing at ‘two to be enclosed by a thin, hyaline test, and for some time opposite points … a tuft of fi liform pseudopodia’, and Diplophrys, though apparently non-phagotrophic, was containing within the body ‘an oil-like refractive glob- considered to be a rhizopod, now belonging either to ule of an orange or amber color’. That was the fi rst de- the cercozoan family Amphitremidae of fi lose testate amoebae with bipolar tufts of branching fi lopodia (e.g. Address for correspondence: O. R. Anderson, Biology and Paleo En- Calkins 1926) or the foraminiferan family Allogromi- vironment, Lamont-Doherty Earth Observatory, Palisades, New York 10964; Tel.: 845-365-8452; E-mail: [email protected] idae, containing phagotrophic unicells enclosed by an 292 O. R. Anderson and T. Cavalier-Smith organic test and having reticulose pseudopodia (e.g. water Diplophrys sp. deposited in the American Type Grassé 1953, p. 140). A second nominal species from Culture Collection (ATCC 50360) is genetically ex- the early literature, Diplophrys stercorea (Cienkowski tremely distant from D. marina and does not group with 1876, Olive 1903), was later split off as a separate genus it, but very weakly instead with an Aplanochytrium/ Sorodiplophrys because it makes multicellular fruiting Labyrinthuloides clade (Cavalier-Smith and Chao bodies like a slime mold (Dykstra and Olive 1975), but 2006); that cast doubt on whether strain ATCC 50360, its vegetative cells have the bipolar fi lopodial pheno- for which there is no published morphology, really be- type that led Cienkowski to put it in Diplophrys. Dyk- longs to the same genus as D. marina and suggested stra and Porter (1984), however, considered that both that Diplophrys-like organisms had much greater phy- Sorodiplophrys and Diplophrys might be distantly re- logenetic depth than previously assumed. lated to labyrinthulids and thraustochytrids as all have To clarify more fully this putative deep diversity of tests of thin organic scales and naked thread-like ab- Diplophrys-like species, we examined the light micro- sorptive projections and none of them are phagotrophs, scopic and fi ne structural morphology of strain ATCC in marked contrast to fi lose amoebae. 50360, isolated in 1992 from the intestinal tract of More recently, Dykstra and Porter (1984) isolated a goldfi sh (Carassius auratus) by S. A. Schaffer, and a new species (Diplophrys marina) from marine vas- describe it as a new species, D. parva. We also carried cular plants and described its transmission electron mi- out a more comprehensive phylogenetic analysis in- croscopic fi ne structure and light microscopic morphol- cluding 13 environmental 18S rDNA sequences related ogy. They noted that its external envelope was not an to D. marina or D. parva, and a comprehensive selec- organic membranous test characteristic of allogromids, tion of other Labyrinthulea, using improved methods but was composed of thin organic, overlapping scales, for 327 heterokonts, which reveals at least 15 geneti- reinforcing their idea that Diplophrys and Sorodiploph- cally diverse Diplophrys-related species spread across rys are related to Labyrinthulea similar to Labyrinthu- two anciently diverged clades. Given their deep genetic divergence and ultrastructural differences, we transfer loides (Labyrinthuloides was later synonymized with D. marina to a new genus Amphifi la and establish sep- Aplanochytrium: Leander and Porter 2000). Patterson arate new families: Diplophryidae for D. archeri and et al. (2000), however, considered Diplophrys to have D. parva, and Amphifi lidae for Amphifi la marina. In the only one species, D. archeri, and as an amoeba of un- light of our 18S rDNA tree, we also conduct a broader certain affi nities. Leander and Porter (2001) presented taxonomic revision of Labyrinthulea at the family level the fi rst molecular phylogenetic evidence, though with to harmonize their classifi cation better with deep ge- negligible bootstrap support, placing the non-fl agellate netic divergences revealed on this and other recent trees Diplophrys marina amongst the labyrinthulids and (Tsui et al. 2009, Lara et al. 2011) and with marked thraustochytrids, previously shown to be a very deep- morphological differences across the tree that are in- branching part of the chromistan Heterokonta (= stra- suffi ciently refl ected in current family demarcations. menopiles) (Cavalier-Smith et al. 1994). Labyrinthulids Altogether, we establish six new families within Laby- and thraustochytrids mostly have fl agellate zoospores rinthulea, plus a seventh for Labyrinthomyxa (Laby- and together constitute the heterokont class Labyrinthu- rinthomyxidae) which because of limited data (Dubosq lea and subphylum Sagenista (Olive 1975, Cavalier- 1921) cannot be included in Labyrinthulea: we place it Smith 1986), of the most deeply branching heterokont incertae sedis within the chromist subkingdom Harosa. phylum Bigyra, which otherwise consists predominant- ly of diverse phagotrophic fl agellates (Cavalier-Smith 1997, Cavalier-Smith and Chao 2006). Labyrinthulea MATERIALS AND METHODS has two orders, Labyrinthulida (labyrinthulids) and Thraustochytrida (thraustochytrids), each with a single family (Cavalier-Smith and Chao 2006, Porter 1990). Light microscopy More comprehensive trees placed D. marina with very Light microscopic observations were made on live cells us- strong support fi rmly within Labyrinthulea (Cavalier- ing the following equipment: (1) a Zeiss Axioskop compound mi- croscope equipped with an Optronics DEI-470 CCD camera, and -Smith and Chao 2006), but its position as sister to Laby- (2) a Zeiss Axioplan compound microscope equipped with a Zeiss rinthula was only weakly supported. However, a sepa- AxioCam digital camera. Images were captured electronically. rate 18S rDNA tree suggested that an undescribed fresh- Measurements were made from digital photographs. Ultrastructure of Diplophrys 293 Electron microscopy 307, 327, 333, 342 and 354 sequences of Harosa (i.e. the chrom- ist subkingdom comprising Heterokonta, Alveolata and Rhizaria: Samples were prepared for ultrathin sectioning and direct obser- Cavalier-Smith 2010a), which removed most Ochrophyta and vary- vation of the surface scales using negative staining. Cultures of ATCC ing numbers of others by excluding more closely related sequences. 50360 isolate (designated as Diplophrys sp.), maintained in ATCC To see which groupings were stable irrespective of method, we also medium 802: Sonneborn’s Paramecium medium, were fi xed for ran neighbor joining (NJ) distance trees for these and other taxon transmission electron microscopy as previously published (Ander- samples using the F84 gamma model of Phylip v. 3.68 (Felsenstein son et al. 1997). The medium also contained Aerobacter aerogenes http://evolution.genetics.washington.edu/phylip). and mixed bacteria as prey. A suspension of cells placed in a 15 ml graduated conical centrifuge tube was mixed with an equal volume of TEM-grade glutaraldehyde (4% (w/v) in 0.2 M cacodylate buffer, pH 7.2), to yield a fi nal fi xative of 2%