Shedding light on vampires: the phylogeny of vampyrellid amoebae revisited

Abstract

With the advent of molecular phylogenetic techniques the polyphyly of naked filose amoebae has been proven. They are interspersed in several supergroups of eukaryotes and most of them already found their place within the tree of life. Although the 'vampire amoebae' have attracted interest since the middle of the 19th century, the phylogenetic position and even the monophyly of this traditional group are still uncertain. In this study clonal co-cultures of eight algivorous vampyrellid amoebae and the respective food algae were established. Culture material was characterized morphologically and a molecular phylogeny was inferred using SSU rDNA sequence comparisons. We found that the limnetic, algivorous vampyrellid amoebae investigated in this study belong to a major clade within the Endomyxa Cavalier-Smith, 2002 (Cercozoa), grouping together with a few soil-dwelling taxa. They split into two robust clades, one containing species of the genus Vampyrella Cienkowski, 1865, the other containing the genus Leptophrys Hertwig & Lesser, 1874, together with terrestrial members. Supported by morphological data these clades are designated as the two families Vampyrellidae Zopf, 1885, and Leptophryidae fam. nov. Furthermore the order Vampyrellida West, 1901 was revised and now corresponds to the major vampyrellid clade within the Endomyxa, comprising the Vampyrellidae and Leptophryidae as well as several environmental sequences. In the light of the presented phylogenetic analyses morphological and ecological aspects, the feeding strategy and nutritional specialization within the vampyrellid amoebae are discussed.

Figure 1. Reconstructed phylogeny of the Vampyrellida in a cercozoan context based on SSU rDNA sequence comparisons.

Shown is the best maximum likelihood tree obtained by RAxML analyses of 81 sequences using 1640 aligned characters. The order Vampyrellida as well as the families Vampyrellidae and Leptophryidae are shaded in yellow/red colours, the remaining two deep-branching endomyxan clades in grey. The branch containing ‘Novel Clades’ 10–12 and some members of the ‘core Filosa’ are defined as outgroup. For previously published sequences taxonomic names and accession numbers are given. Sequences without taxonomic designations are environmental sequences and newly obtained sequences are combined with the strain designation only and are in bold (for accession numbers see Table 1). The support values of all methods applied are shown on the respective branches in the following order: ML/NJ/MP/BI. Support values less than 50% or 0.5 are not shown (−), whereas bold branches were maximally supported by all methods (100/100/100/1.00). Interrupted branches (//) show 50% of their original length. NC = ‘Novel Clade’ according to Bass & Cavalier-Smith (2004) and Bass et al. (2008) , .

Figure 2. Life history stages and morphological traits of Vampyrella lateritia.

2A. Advancing trophozoite. DIC. 2B. Large trophozoite likely a result from cell fusions. DIC. 2C. membranosomes moving along the pseudopodia. DIC. 2D. ‘pin-like’ pseudopodia, arrowheads: membranosomes. DIC. 2E. Numerous vacuoles in the cell periphery. 2F. Large, bulky plasmodium. DIC. 2G. Attached Vampyrella cell ingesting algal cell content. Brightfield. 2H. Ingestion pseudopodium (arrow) emptying an algal cell. DIC. 2I. Immobile Vampyrella cell with retracted pseudopodia and spiny surface turning into digestive cyst. DIC. 2J. Green motile cell after food uptake. Brightfield. 2K. Early digestive cyst stage with greenish contents. Brightfield. 2L. Mature digestive cyst with delicate outer cyst envelope (arrowhead). Brightfield. 2M. Digestive cyst with progeny after cell divisions in characteristic arrangement, arrowhead: outer cyst envelope. Brightfield. 2N. Digestive cysts at various stages in different colours. Dark field. 2O. Resting cyst with four envelopes (arrowheads). DIC. Scale bars: 2A, B, H, I, O = 10 µm; 2C–E = 5 µm; 2F = 50 µm; 2G, J–M = 20 µm.

Figure 3. Trophozoites and digestive cysts of Vampyrella pendula.

3A & B. Advancing trophozoites. Brightfield. 3C. Trophozoite and digestive cyst on a filament of Oedogonium. Brightfield. 3D. Trophozoite with clear pseudopodia lacking membranosomes. DIC. 3E & F. Trophozoite producing claviform pseudopodia (arrows). DIC. 3G. Early digestive cyst stage. DIC. 3H. Mature digestive cyst. DIC. 3I. Remaining digestive cyst envelopes with food remnant. DIC. Asterisk = very faint outer sheath, hollow arrows = outer cyst envelope, hollow arrowheads = delicate strands between outer and intermediate cyst envelope, arrowheads = central stand running from innermost cyst envelope through stalk. Scale bars: 10 µm.

Figure 4. Trophozoites of Leptophrys vorax and their variability.

4A & B. Small, compact trophozoites. DIC. 4C. Advancing cell with fan-like outline. DIC. 4D. Branched cell. DIC. 4E. Elongated cell with several attachment sites. DIC. 4F. Cell drawn out into thin cytoplasmic strand due to extending cell ends. DIC. 4G. Advancing trophozoite showing clear pseudopodia emerging from a hyaline fringe, a tail-like posterior projection and dendritic pseudopodial structures (arrowheads). DIC 4H. Vesicular nuclei in the cytoplasm of a dying individual (arrowheads). DIC. 4I. Isodiametric floating form with radiating pseudopodia. DIC. Scale bars: 4A–E, 4G, I = 20 µm; 4F = 100 µm; 4H = 10 µm.

Figure 5. Plasmodia and digestive cysts of Leptophrys vorax.

5A. Large extended Plasmodia. Phase contrast. 5B. Plasmodium showing several anastomoses (arrowheads) and a normal sized trophozoite for comparison of the sizes. Phase contrast. 5C. Small elliptical cyst. Brightfield. 5D. Roundish cyst. Brightfield. 5E. Irregular lobed cyst. Brightfield. 5F. Dumbbell-shaped cyst. Brightfield. 5G. Numerous digestive cysts in growing culture with Closterium sp. sometimes banana-shaped. Oblique illumination. 5H. Slender cyst containing Synedra sp. Brightfield. 5I. Moving trophozoite tightly packed with Closterium cells. Brightfield. 5J. Early digestive cyst stage with greenish food inclusions. Brightfield. 5K. Later digestive cyst stage with brownish food inclusions indicating the proceeding digestion. Brightfield. 5L. Digestive cysts after hatching of the trophozoites just containing brown food remnants. Brightfield. Scale bars: 5A, B = 100 µm; 5C–F, H–L = 20 µm; 5G = 200 µm.

Figure 6. The three distinct morphotypes of vampyrellid amoebae.

The shown morphotypes relate to the predominant morphology of locomotive trophozoites present in a growing vampyrellid population. Despite the existence of transitions from morphotypes II and III into a floating form resembling morphotype I (e.g. under food limitation) they serve well for describing the general appearance of the very variable vampyrellid taxa. Arrow = direction of movement, asterisk = side view.