A taxonomic summary of Aphelidiaceae

Aphelids are parasitoids of various algae and diatoms, and in a recent classification are contained in family Aphelidiaceae, phylum Aphelidiomycota, kingdom Fungi. Family Aphelidiaceae (the only family in the phylum) is composed of four genera: Aphelidium, Paraphelidium, Amoeboaphelidium, and Pseudaphelidium. All species are known morphologically, and most have been illustrated. Few have been examined ultrastructurally, and even fewer have been sequenced for molecular comparisons. Recent studies in molecular phylogenetics have revealed an abundance of related environmental sequences that indicate unrealized biodiversity within the group. Herein, we briefly summarize the history of aphelids and acknowledge the controversy of placement of the group with related organisms. With light microscopic images and transmission electron micrographs, we illustrate typical life cycle stages for aphelids, provide updated descriptions and taxonomy for all described species, and provide a key to the species.


INTRODUCTION
Aphelids (Aphelidiaceae, Aphelidiomycota) are a group of obligate endoparasitoids of various common algae and diatoms. We employ the term "parasitoid" for these organisms, as eventually the infected host cell is consumed and killed, although in multicellular hosts, uninfected cells adjacent to those with infection remain viable. The type, Aphelidium deformans, was described more than 130 years ago (Zopf 1885). Among the four described genera, Aphelidium, Amoeboaphelidium, and Paraphelidium occur in freshwater habitats, while Pseudaphelidium is found in marine environments (Karpov et al. 2017a;Scherffel 1925;Schweikert and Schnepf 1996;Zopf 1885). Currently, Aphelidium is composed of seven species, Amoeboaphelidium of five, Paraphelidium of two, and Pseudaphelidium is monotypic. Thallus morphology has been illustrated for all taxa except Am. achnanthis, for which only a written description exists. A minority of taxa have been examined for their zoospore and thallus ultrastructure. Although even fewer have been sequenced for molecular comparisons, recent advances in molecular phylogenetics have revealed an abundance of related environmental sequences that indicate hitherto unrealized biodiversity within the group (e.g. Karpov et al. 2014aKarpov et al. , 2017a. In a recent high-level classification (Tedersoo et al. 2018), aphelids are placed as an early-diverging lineage in kingdom Fungi, and we adhere to this classification here. Although aphelids are considered opisthokonts because of their posteriorly uniflagellate zoospores, the classification of aphelids as Fungi (Tedersoo et al. 2018) is not without controversy. Gromov (2000) and Karpov et al. (2013) thoroughly discussed historical interpretations of the phylogenetic affinities of aphelids, the organisms originally having been considered extremely divergent "fungal animals"-organisms demonstrating a fungal-like life-cycle, but having an amoeboid trophic stage. Later, aphelids were for a time considered protists (class Rhizopoda, order Proteomyxida) (e.g. Hall 1953). With molecular phylogeny analyses, Karpov et al. (2013) showed that Aphelidea was sister to both Microsporidia and Cryptomycota, and all three phyla form a separate monophyletic lineage sister to traditional fungi, which include Dikarya (Ascomycota and Basidiomycota), paraphyletic Zygomycota, and Chytridiomycota. Karpov et al. (2013) erected a superphylum Opisthosporidia "… named by word combination of Opisthokont and sporae, making reference to the specialized penetration apparatus of the spore (in Microsporidia) and cyst (in the two other phyla) characteristic for all three phyla Microsporidia, Cryptomycota, and Aphelida" (Karpov et al. 2013). Opisthosporidia is sister to the traditional fungi. Most recently, Torruella et al. (2018), analyzing various protein datasets in multi-gene phylogenomic analyses, place aphelids as the closest relatives of Fungi to the exclusion of Cryptomycota and Microsporidia, suggesting that Fungi evolved from an aphelid-like ancestor that lost phagotrophy and became osmotrophic. Nonetheless, a clear and convincing taxonomic repository for the aphelids remains to be determined. Alternatively, Adl et al. (2019), in a classification of eukaryotes that adopted a hierarchal system without formal rank designations, retained Aphelidea in the Opisthosporidia (Fungi), but noted "… the placement of Aphelidea in Opisthosporidia is unstable and may change".
The aphelid life-cycle is similar among the included taxa. When viewed with light or transmission electron microscopy, the motile zoospore may be amoeboid (Figs. 1 and 2a,b), with one or more pseudopodia that may be either broad (e.g. "lamellipodium", Pa. tribonematis, see Karpov et al. 2017a, Fig. 2c, d) or thin (e.g. "filopodia", Aph. desmodesmi, see Letcher et al. 2017; "stiletto pseudopodium", Aph. chlorococcorum f. majus, see Gromov 1976, Figs. 1 and 2). The zoospore may also be round or oval and without pseudopodia ( Fig. 2c) (e.g. Ps. drebesii, see Schweikert and Schnepf 1996). The motile zoospore approaches the host and often contours its surface to that of the host (Fig.  2d), encysts on the host, attaches with an appressorium (Fig. 2e, Fig. 3a), and penetrates the host with a penetration tube (Fig. 1b, Fig. 3a). A posterior vacuole within the cyst (Fig. 2e) pushes the cyst contents into the host through the penetration tube. The endobiotic parasitoid becomes a phagotrophic amoeba. As the parasitoid grows it becomes a plasmodium that engulfs host cytoplasm (Fig. 2f, Fig. 3a), finally containing one or two residual bodies ( Fig. 3b-d, f). At maturity the plasmodium is multinucleate, with a central vacuole and a residual excretion body. The plasmodium divides into numerous uni-nucleate cells (Fig. 3b, c), which are subsequently released from the empty host cell to further infect other host cells. An unreleased zoospore occasionally may remain in an evacuated sporangium (Fig. 3d), and the empty zoospore cyst may or may not persist (Fig. 3d, e). A resting spore may or may not be formed.
Variation in character states that may be taxonomically informative are: spore size and shape, flagellum length, and nature of pseudopodia; morphology of the zoospore cyst; size of residual body in the plasmodium; presence or absence of a resting spore; and shape and wall construction of the resting spore. Character states and hosts of taxa are summarized (Table 1).
Diversity within the group is indicated by 18S rRNA gene sequence molecular affinity of members of three genera, with numerous environmental sequences from diverse habitats (e.g., Karpov et al. 2017b). Molecular data for Pseudaphelidium are not available.  Synonym: Aphelidea B.V. Gromov, Zool. Zhurn. 79: 521 (2000).
Description: "Amoeboid endobiotic parasitoids of algae. Dispersal zoospores or amoebae attach to a new host cell and encyst, (either sessile on the substrate or producing a stalk; "apophyse"; Gromov 2000). Amoeboid body penetrates into the host's cell through a cyst stalk. The intracellular amoeba engulfs the contents of the host's cell, forming food vacuoles which transport food into the central digestive vacuole. An excretory body is formed in the digestive vacuole. The amoeboid trophont grows into a plasmodium, which totally replaces the cytoplasm of a host cell; the multinuclear plasmodium develops into an unwalled sporangium and divides into uninucleate amoeboid cells or flagellated zoospores. No specialized cell wall is formed by the parasitoid around the sporangium. Some species form intracellular resting spores" (Karpov et al. 2014a  Synonym: Aphelididae B.V. Gromov, Zool. Zhurn. 79: 521 (2000).
Note: The family comprises the genera: Aphelidium, Paraphelidium, Amoeboaphelidium, and Pseudaphelidium. Diagnosis: "Parasitoid of various algae, forming round or oval zoospores with one posterior flagellum with an acroneme and one or several lipid grains. Vegetative development as described for the class. Resting spores round or oval, with a thick smooth cell wall. The excretory body is ejected from the spore into the space between the walls of the spore and the destroyed cell" (Gromov 2000). Diagnosis: Scherffel (1925) observed neither formation of a zoospore cyst nor penetration of the parasite into the host cell. He did observe the parasite plasmodium within the host, with multiple digestive vacuoles. The parasite often caused hypertrophy of the infected cell. In the sporangium zoospores were initially spherical,~2.7 μm diam, with a single flagel-lum~9 μm long, and the zoospore may have possessed a posterior cavity and 2-3 contractile vacuoles. Prior to discharge zoospores became ovoid, 3-4 μm in length; zoospores were passively discharged, quiescent after exit, and then suddenly became motile, like many chytrids. Resting spores were not observed.

Aphelidium chaetophorae
Note: Gromov (1976) wrote "The parasite develops in the same way as the other species described by Scherffel (1925)". However, Gromov (2000) noted  that the morphology of this species "does not correspond to the presented diagnosis of the genus", without providing specifics. In our opinion, Aph. chaetophorae is not a doubtful species because Aph. deformans (the type species) is similar to Aph. chaetophorae, and in neither were encysted zoospores and empty cysts observed.
Melosirae is considered similar to (has an affinity with) Aph. melosirae, whose host is Melosira varians, because, of the six species known for the genus, strain P-1 appears to be morphologically most similar to Aph. melosirae. Aphelidium aff. Melosirae is therefore an undescribed strain, and to decide whether strain P-1 belongs to Aph. melosirae or not, the morphology and molecular phylogeny of Aph. melosirae parasitizing Melosira varians need to be studied (Karpov et al. 2014b (Gromov 2000). Thallus morphology has been studied (Gromov 1972;Scherffel 1925;Karpov et al. 2016). Thallus ultrastructure has not been studied. GenBank accession: KY129663 (partial SSU rDNA; Karpov et al. 2016).
Diagnosis: Zoospores swim with a posteriorly oriented flagellum or move like amoebae with an immobile flagellum. Amoeboid zoospore can produce a short, broad anterior lamellipodium with subfilopodia from the lamellipodium and separate filopodia. Mature resting spore (sporocyst) is ellipsoid and covered with one or two walls. The two-walled morphology of the resting spore is present only in the type species (Karpov et al. 2017a), the resting spore of the second described species P. letcheri having only a single wall (Karpov et al. 2017b). Diagnosis: "Crawling flagellated zoospores have a body up to 4 μm long and able to produce a lamellipodium with subfilopodia up to 1.8 μm in length; swimming zoospores with spherical body 2-2.5 μm in diameter, and a flagellum 8-10 μm including an acroneme of 4 μm. Large residual body associated with one or two lipid globules totally occupies a central vacuole of plasmodium. Sporocyst spherical 6-8 μm in diameter with smooth wall" (Karpov et al. 2017b). Parasitoid of Tribonema gayanum. Thallus morphology has been studied (Karpov et al. 2017b), but not the ultrastructure. Gen-Bank accession: KY412789 (partial SSU rDNA).

Paraphelidium letcheri
Note: Paraphelidium letcheri is distinguishable from the type species, P. tribonematis, by a much larger residual body associated with big colorless lipid globules in the plasmodium, and by the single-walled resting spore.
Diagnosis: "Parasitoids of various species of algae. Amoeboid zoospores with or without posterior pseudocilium, forming flat hyaline pseudopodium with subfilopodia, or filopodia of different length. Vegetative development as described for the class. Resting spores rounded to oval, with a thick cell wall" (Karpov et al. 2014a).
Diagnosis: Thallus morphology is descriptive only, as Scherffel (1925) did not illustrate this taxon. "Parasitoid of the diatom alga Achnanthes, amoebae about 2 μm long" (Gromov 2000). Thallus ultrastructure has not been studied, and molecular sequence data are not available.
The amoebae form numerous pseudopodia, thin trichipodia (hair-like), and thick lobopodia. The amoeba cyst is attached to the host by a short stalk (e.g. Letcher et al. 2015: Fig. 3d-, e). Gromov and Mamkaeva (1969c) stated the "diameter of amoeba with retracted pseudopodia~2-4 μm; parasite grows well on surface of solid media; contrast with Amoeboaphelidium radiatum, which grows only in semi-solid or liquid media; contrast with Aphelidium chlorococcorum that grows only in algae in liquid medium".
Diagnosis: "Parasitoid of the chlorococcus algae Kirchneriella and Ankistrodesmus. Amoebae 1-3 μm in diameter with limited motility, have very thin and long filopodia (10-12 μm). Development of the surface of solid culture media not observed" (Karpov et al. 2014a). Thallus morphology has been studied (Gromov and Mamkaeva 1969a), but ultrastructure details and molecular sequence data are not available.
Note: Gromov and Mamkaeva (1969a) stated that the motility of amoeboid spores was limited. Gromov and Mamkaeva (1969c) reported that the organism grew only in semi-solid or liquid media.
Diagnosis: "Parasitoids of diatoms. Zoospores colourless, Letcher and Powell IMA Fungus (2019) 10:4 lacking conspicuous refractive granules, with a single opisthokont flagellum. A zoospore attaches to a host cell, encysts, penetrates into the cell interior, and develops into a phagocytotic plasmodium which ingests portions of host cytoplasm and includes them in a single big digestion vacuole. At the end of the trophic phase the plasmodium cleaves to form uni-nucleate amoeboid cells which encyst and give rise to new zoospores" (Schweikert and Schnepf 1996).