Site 2: slides BUL–2–1, BUL–2–6, BUL-2-7 (zygo.), BUL–2–10; Site 3: slides BUL–3–1; site 4: slides BUL–4–10; Site 7: slides BUL–7–1, BUL–7–2, BUL–7–3; site 8: slides BUL–8–3; site 12: slide BUL–12–5.

Genistellospora homothallica is a cosmopolitan species and its Simuliidae hosts are widespread and common in varied environments (Lichtwardt et al. 2001), especially in fast flowing waters. This species has been previously documented from many countries in the Northern Hemisphere, including USA (Lichtwardt 1972), Canada (Moss and Lichtwardt 1976), United Kingdom (Lichtwardt 1986), Spain (Santamaria and Girbal 1998), France (Valle 2004), Italy (Valle et al. 2013) and Portugal (Valle 2013a). The species has also been recorded from Southern tropical regions, including Costa Rica (Lichtwardt 1997), Puerto Rico (White et al. 2000), Argentina (López-Lastra et al. 2005), Dominican Republic (Valle and Cafaro 2010), Chile (Lichtwardt and Arenas 1996) and Colombia (Barón and Valle 2018). Trichospores of G. homothallica are typically ovate-elongated, slightly asymmetrical, measuring 34–40 × 10.5–12 μm in our collections. Young zygospores were observed in one Bulgarian specimen (BUL–2–7) with the characteristic zygosporophore of the species, bearing a straight or reflexed thumb-like terminal cell measuring 43–58 μm length. Often, G. homothallica thalli were covered with thalli of the epithallic Simuliomyces microsporus Lichtw., as seen in Fig. 2 (arrows).

Figures 1, 2. 

Genistellospora homothallica and Simuliomyces microsporus from Simuliidae larvae. 1 Fertile branches of G. homothallica with terminal trichospores 2 fertile branches and trichospores of G. homothallica with an attached thallus of S. microsporus showing a trichospore (arrow) and conjugation tubes (arrowhead). Scale bars: 25 μm in all figures.

Bulgaria, Sofia, Pasarel Village, Iskar River, 42.535885N, 23.508824E; 712 m a.s.l.; 19 Aug 2016; LG Valle and D Stoianova Leg; In the hindgut of Baetis melanonyx Pictet (Baetidae, Ephemeroptera); microscope slide BCB–BUL–2–2.

Same locality and date as the holotype; microscope slide BUL–2–3, BCB–BUL–2–4. Bulgaria, Sofia capital city, Darvenishka River, Park Vartopo, 42.645710N, 23.364568E; 585 m a.s.l.; 23 Aug 2016; LG Valle and D Stoianova Leg; In the hindgut of Baetis melanonyx (Baetidae, Ephemeroptera); microscope slide BCB-BUL–12–3.

Balkanensis, from the Balkan Peninsula.

Thalli measuring up to 600 μm long. Basal cell broadly inflated (18–30 μm diam.) and often branched (Fig. 3), bearing a small discoid secreted holdfast at the base or laterally to the basal cell axis (Fig. 4). Dichotomous branching above the basal cell; distal branches bearing spores (Figs 2, 8). Trichospores cylindrical, with a terminal refractive cap (not always visible), measuring 44–56 × 4.5–5.5 μm, with 3 appendages, one central long filiform appendage, coiled around two shorter (about 15–20 μm) and broader lateral appendages (Fig. 10). These appendages can be seen within the generative cell while still attached (Figs 7, 8 arrowhead). Fertile branches bearing 3–4 generative cells, measuring 20–35 × 4–6 μm. Zygospores biconical, 48–60 × 7.5–9.5 μm, with a collar 5–10 (–16) × 4 μm, attached eccentrically and laterally (Type II) to a zygosporophore 20–30 μm long, arising from the conjugation tube in series of scalariform conjugations (Fig. 9). In the hindgut of Baetidae (Ephemeroptera) nymphs.

Figures 3–10. 

Glotzia balkanensis sp. nov. from Baetidae nymphs. 3 thallus overview, note the inflated and branched basal cell structures 4, 5 detail of various swollen basal cells 6 loose zygospore with a collar 7, 8 trichospores on fertile branches, see the appendages inside the generative cell (arrowheads) 9 Zygospore arising from a conjugation tube 10 loose trichospore with a central long filiform appendage and two smaller lateral appendages. Scale bars: 50 µm (3), 25 µm (4–10).

The genus Glotzia has nine species (including that described here), all of them sharing the characteristic cylindrical trichospores with a slightly globose cap and the peculiar 3-appendage arrangement observed also in G. balkanensis. This new species mostly resembles the type species G. centroptili described by Gauthier (1936) in French pools and streams of Dauphiné province (south-eastern France) from Centroptilum luteolum nymphs (Baetidae). This French species was recently rediscovered in Catalonia (Spain) also within Centroptilum sp. nymphs (Busquets et al. 2018). This second observation in Spain was important for providing new material to complete the original description, which was scant and had no photographs, only a drawing of a single specimen (Gauthier 1936). The specimens from Bulgaria can be differentiated from G. centroptili by spore characteristics. Trichospores of the Bulgarian species are longer than those observed in France or Spain (40 × 4 μm according to Gauthier (1936); 35–43 × 4–6 μm according to Busquets et al. (2018), up to 56 μm in the specimens reported here). All the fertile branches observed had a maximum of four generative cells in G. balkanensis, while up to seven have been reported in G. centroptili. Zygospores of G. balkanensis are quite similar to those of G. centroptili in length, but they have significantly larger diameter in the French species, 15 μm diameter (according to Gauthier 1936), while only 7.5–9.5 μm (8.4 μm average) in G. balkanensis. Unfortunately, the specimens of Glotzia centroptili collected from Spain, had no zygospores to compare with the new species, only trichospores were observed and, thus, we do not have a broad perspective of the zygosporic variation in this species, because apparently, the description of the type species was based on just a few specimens (Busquets et al. 2018). The presence of a collar on released zygospores was not described by Gauthier in G. centroptili. The species described here has a quite variable collar length, but in most zygospores, it is rather short (5–10 μm). Regarding thallial characteristics, both species are quite similar, but there are major differences in their fertile branches, generative cells and in the basal cell. The basal cell is much more swollen in the Bulgarian species, resembling (but not identical to) that of the Italian species Glotzia distorta LG Valle, Santam. & W Rossi which has different spore features (Valle et al. 2014). Most species of Glotzia are associated with Baetidae (Ephemeroptera), except for one species recorded in a New Zealand Plecoptera nymph, Glotzia plecopterorum Lichtw. (Williams and Lichtwardt 1990) and another species living within Dixidae (Diptera) larvae Glotzia incilis Strongman & MM White, (Strongman and White 2008). Actually, Glotzia is one of the Harpellid genera with a wider host range. Glotzia centroptili was recorded from Centroptilum (Baetidae) in France and Spain and G. distorta from the related Procleon pennulatum (= Centroptilum pennulatum) (Valle et al. 2014). However, Glotzia balkanensis has been recorded from a different host, Baetis melanonyx, but in the same family Baetidae. In fact, this is the first record of a Harpellid fungus within this host species.

Site 2: slide BUL–2–5; Site 8: slides BUL–8–1, BUL–8–2.

This species is characterised by the unusual formation of vegetative propagules from the bulbous basal cells (Fig. 12), a feature only shared with the related genus Gauthieromyces (Lichtwardt 1983). Graminella bulbosa was described from France (Léger and Gauthier 1937; Manier 1962). The species is also known from Spain (Valle 2007), Portugal (Valle 2013a) and Italy (Valle et al. 2013). Graminella bulbosa has been reported associated with various species of Baetis and related genera, very frequently within the hindgut of B. rhodani (Pictet). This species of mayfly is common and widespread in Europe and it also hosted Bulgarian specimens of G. bulbosa in the surveyed rivers, together with B. alpinus (Pictet). In fact, the genus Baetis bears different Harpellid species, including the more common Legeriomyces ramosus, occasionally sharing the same gut lumen with Graminella bulbosa. Bulgarian specimens of G. bulbosa show the typical small and numerous trichospores (Fig. 13), measuring 8 –11 × 2 µm in our collections. These measurements are midway between G. bulbosa and G. microsporus (see discussion for further information). Unfortunately, only immature zygospores were observed (Fig. 11 arrowhead).

Site 2: slides BUL–2–1, BUL–2–6, BUL–2–7, BUL–2–10; site 4: slides BUL–4–10; Site 7: slides BUL–7–1, BUL–7–2, BUL–7–3; Site 8: slides BUL–8–3; site 12 slide: BUL–12–5

A cosmopolitan or sub-cosmopolitan species, widespread in the Northern Hemisphere, it is common in European localities where their hosts were available. It has been found attached to the peritrophic matrix of Simuliidae larvae in different Bulgarian localities. Our specimens have the typical characteristics of the species, distinguishable on the basis of trichospore morphometry (Fig. 14) and holdfast structure (Fig. 15) (Léger and Duboscq 1929a). The trichospores in our specimens measured 50–60 × 6–7 μm and were variable in shape, from nearly straight to allantoid or slightly coiled.

Site 2: slide BUL–2–9; Site 11: slides BUL–11–1 (zygo.), BUL–11–2, BUL–11–3.

This species was found on the hindgut lining of Baetidae hosts (Baetis rhodani Pictet, Baetis melanonyx (Pictet) and B. alpinus (Pictet). The species seems to have a cosmopolitan distribution, at least in the Northern Hemisphere (Lichtwardt et al. 2001). In Europe, the species is common, with records from France, where it was originally described under the name of Genistella ramosa (Léger and Gauthier 1932) (Pouzar 1972), from United Kingdom (Moss 1979), Switzerland (Lichtwardt 1986), Spain (Valle and Santamaria 2002), Norway (White and Lichtwardt 2004), Sweden (Lichtwardt 1984), Portugal (Valle 2013a) and Italy (Valle et al. 2013). Legeriomyces ramosus has been reported also from India (Misra and Tiwari 2008), China (Strongman et al. 2010), from several localities in USA (Lichtwardt 1986) and Canada (Strongman 2010). Bulgarian specimens have, as is usual in this species, a broad range of trichospore variability, measuring 30–40 ×7–8.5 µm, with two appendages differing in length. Zygospores measure 50–61 × 9–12 µm in our collections (Fig. 16).

Figures 11–16. 

Various species of Harpellales. 11–13 Graminella microspora from Baetidae nymphs: 11 thallus overview, with inflated basal cell structures from which propagules arise or are extruded (note: one empty basal cell which has extruded its cellular content, arrow); young zygospores can also be observed (arrowhead) 12 a cluster of basal cells 13 fertile branch with a long series of minute trichospores; 14, 15 Harpella melusinae from Simuliidae larvae: 14 generative cells and allantoids or curved trichospores 15 basal cell and small conic holdfast 16 Legeriomyces ramosus from Baetidae nymphs, zygospores and zygosporophores. Scale bars: 25 μm in all figures.

site 2: slides BUL–2–1, BUL–2–6, BUL–2–10; site 3: slides BUL–3–1; Site 7: slide BUL–7–3(zygo.); Site 12: slide BUL–12–5.

This species was obtained from the hindgut lining of Simuliidae larvae. Most of the observed specimens of S. microsporus were attached to thalli of both Genistellospora homothallica (Fig. 2) and Paramoebidium chattoni. All the individuals had trichospores and one of the specimens showed also the typical type-I zygospores’ young (Fig. 2). All characteristics and measurements of Bulgarian S. microsporus match that of previous descriptions of the species in Europe (Moss 1970; Valle 2004, White and Lichtwardt 2004). This species, discovered in the USA (Lichtwardt 1972), has a sub-cosmopolitan distribution (see Lichtwardt et al. 2001), with a patchy distribution in the Northern Hemisphere and also Australia (Lichtwardt and Williams 1992).

site 12: slides BUL–12-1; BUL–12-4.

This species was previously known from Costa Rica (Lichtwardt1997), Spain (Valle and Santamaria 2004), Dominican Republic (Valle and Cafaro, 2010) and Mexico (Valle et al. 2011) in the tract of Simuliidae and Chironomidae. Bulgarian specimens were associated with Chironomidae midges (Chironomus sp.). They had cylindrical-elongated trichospores measuring 16–19 × 3–4 µm, with a short collar about 2.5 µm, slightly flared outwards. The thallus is branched at the base, with verticillate apical branching (Fig. 17). Each fertile branch has 4–6 generative cells. Specimens from Bulgaria resemble most of those described from Spain.

Site 7: slide BUL–7–4; Site 10: slide BUL–10–3.

This species was described from France (Tuzet and Manier 1950) in the hindgut of Baetidae nymphs. Spartiella barbata is distinguished by its lobulate basal cell, obpyriform trichospores, measuring 21–26.5 × 6.5–7.5 µm in our specimens (Fig. 18) and the presence of one appendage tightly coiled just after release and then eventually uncoiling into a long delicate filiform structure. Spartiella barbata seems to be more common in Europe, where it has been recorded from France (Tuzet and Manier 1950, Manier 1962b), United Kingdom (Lichtwardt 1986) and Spain (Valle 2007), with only one report of the species from North America, in Canada (Strongman 2010).

Site 11: slide BUL–11–4.

Stachylina nana was found in the mid-gut of Chironomidae (Chironomus sp.) in the same host as Smittium dipterorum. Specimens of S. nana in Bulgaria had a small thallus, 60–70 × 7–8 µm, with 1–4(-5) generative cells. Trichospores measure 20–24 × 7–7.5 µm, without a collar. All measurements agree with the original description of the species (Lichtwardt 1984). This species was recorded before from Europe, including France (Lichtwardt 1984) and Spain (Santamaria and Girbal 1997); Asia, in Thailand (Hapsari et al. 2009), China (Strongman and Wang 2015) and also America, including Canada (Strongman 2007, 2010; Strongman and White 2008) and USA (Beach and White 2012). Probably, S. nana has a cosmopolitan distribution, although we only have patchy data from few surveyed countries.

Site 4: slides BUL–4–3, BUL–4–11.

Stipella vigilans was originally described from the French Alps in the hindgut of Simuliidae, together with the protozoan Paramoebidium sp. (Léger and Gauthier 1932). This species was also reported from Spain (Valle 2007), England (Moss 1970), Armenia (Nelder et al. 2005), Thailand (Hapsari et al. 2009). The species is easily distinguished by the particular basal cell, simple or forked, verrucose and narrowing in the most basal section, attached to the hindgut by means of a mucilaginous adhesive substance (holdfast) (Fig. 20). The trichospores of S. vigilans are also very characteristic, almost cylindrical, measuring 35–50 × 3–4.5 µm in our collections, although probably somewhat young, they fit the original description, according to Léger and Gauthier (1932). We did not see released trichospores, since none of them was mature enough in our collections. Trichospores in this species bear three petaloid appendages, which are visible inside the generative cells before detachment.

Figures 17–20. 

Various species of Harpellales. 17 Smittium dipterorum from Chironomidae larvae; thallus overview with fertile branches and trichospores 18 Spartiella barbata from Baetidae nymphs, fertile branches with trichospores 19 Stachylina nana from Chironomidae larvae, thallus overview with trichospores 20 Stipella vigilans from Simuliidae larvae, thallus with mucilaginous material at the basal cell (arrow). Scale bars: 25 μm in all figures.

Site 4: slides BUL–4–1, BUL–4–5, BUL–4–7; site 7: slide BUL–7–6.

We found this species associated with Leuctra hippopus (Kempny 1899) in two Bulgarian rivers and streams. The specimens examined had the typical characteristics of the species, including the straight trichospores measuring 47–56 × 5–7 µm in our collections, with generative cells 21–26 µm long and a supporting cell 6–8 µm length (Fig. 21). All the characters of trichospores and accompanying cells fit the description of the species (Santamaria and Girbal 1998). Zygospores were not seen on this occasion. This species was described from Catalonia, Spain (Santamaria and Girbal 1998, Valle and Santamaria 2005) and has been reported also from Norway (White and Lichtwardt 2004), France (Valle 2013b) and Italy (Valle et al. 2014).

Site 10: slide BUL–10–4 (zygo.)

Collections were made from the hindgut of Protonemura montana Kimmins nymphs, with a low infestation rate (2%). Orphella coronata has been reported from diverse localities in Europe, (e.g. France (Léger and Gauthier 1931, 1932), Norway (White and Lichtwardt 2004), Spain (Valle and Santamaria 2005), Portugal (Valle 2013a) and Italy (Valle et al. 2013)). Orphella coronata seems to be the rarest species of the genus in the Bulgarian streams surveyed, although many potential hosts were dissected from the Struma River. Fortunately, the only Protonemura infested specimen was carrying various mature thalli of O. coronata, so that we could observe trichospores and the typical heterothallically-formed helicoidal zygospores typical for the species, these being very important to discern and identify possible cryptic species (Valle and Santamaria 2005, Valle et al. 2014, White et al. 2018). Bulgarian specimens show the typical thallus with a bifurcate basal cell and allantoid trichospores (Fig. 22), measuring 36–41 × 5.5–6.5 µm in our collections, slightly smaller than previously reported (35–48 × 6–7.5 µm according to Valle & Santamaria 2004). Terminal cell measures 22–25 × 3–3.5 µm. Zygospores (Fig. 23) in our collections measure 26–32 × 6–7 µm, also somewhat smaller than those reported in the description of the zygospores (30–35 × 5–7 µm according to Valle & Santamaria 2004), but likely attributable to intraspecific variation; in fact, just a couple of thalli were found producing sexual spores in our collections and about 5 producing trichospores.

site 4: slide BUL-4-1; Site 7: slide BUL-7-6; site 10: slides BUL-10-2, BUL-10-5.

Species were obtained from the hindgut lining of Leuctridae nymphs (mainly Leuctra hippopus). We found several thalli, most of them producing trichospores and one also bearing helicoidal zygospores, formed homothallically, measuring 25–27 × 5.5–6.5 µm, growing on a fusiform zygosporophore measuring 20–23 × 7–8.5 µm, with a 3 µm long supporting cell and a sigmoid or reflexed intermediate cell about 20–24 µm long in the specimens seen (Fig. 24). All the characteristics of spores and accompanying cells fit those of the specimens reported from other localities in Europe, including Spain (Santamaria and Girbal 1998; Valle and Santamaria 2005), Norway (White and Lichtwardt 2004); Italy (Valle et al. 2013). Thallus has the characteristic basal cell (Fig. 25) and lateral subsidiary branches.

Figures 21–25. 

Various species of Orphellales. 21 Orphella catalaunica from Leuctridae nymphs, trichospores and accompanying cells 22, 23 Orphella coronata from Nemouridae nymphs 22 allantoid trichospores and accompanying cells 23 zygospores produced homothallically 24, 25 Orphella helicospora from Leuctridae nymphs: 24 homothallical zygospores and accompanying cells 25 basal cell and holdfast. Scale bars: 25 μm in all figures.

Site 4: slide BUL-4-2

This species is characterised by having a thallus bent approximately at a right angle about one-quarter up the thallus from the holdfast; no other described species shares this feature. This species was originally described from the stonefly family Taeniopterygidae. Our specimens were observed in Nemouridae, a different family in the same Order of insects sharing all characteristics with P. angulatum, although most individuals were immature, measuring 350–380 × 29–33 µm, with the typical thallus with the right angle bend and discoid acellular holdfast, known previously only from France (Valle 2014a).

Site 2: slide BUL–2–6.

This species of Paramoebidium is common within Simuliidae hosts and is identifiable by having the widest diameter of thallus at the basal to middle sections, slightly tapering towards the distal end. It has a non-cellular holdfast located at the proximal end smaller in diameter (12–27 µm) than the thallus, cylindrical or slightly campanulate (Valle 2014b). Bulgarian species perfectly match with the original description (Léger and Duboscq 1948) and with that provided in the validation of the species by Valle (2014b). The species was recently reported from Colombia (Barón and Valle 2018). In our collections, Simuliomyces microsporus was often found attached to the thalli of this Paramoebidium (Fig. 27).

Site 2: slides BUL–2–1; BUL–2–6, BUL–2–7, BUL–2–10; Site 12: slide BUL–12–5.

This species was found attached to the posterior hindgut or anal gills of larval Simuliidae hosts, where we also found P. chattoni. In Bulgaria, the species was quite common in the black fly hosts dissected. All the individuals had the typical characteristics of the species (Dang and Lichtwardt 1979), with a holdfast placed on the incurved section of the thallus. This species is known from USA (Dang and Lichtwardt 1979), Sweden (Lichtwardt 1984), Armenia (Nelder et al. 2005), Canada (Strongman and White 2008), Spain (Valle and Santamaria 2009, Valle 2014b), Italy (Valle et al. 2014). P. curvum is probably very common and cosmopolitan as their hosts, but their thalli can be easily overlooked when attached to or near to the anal gills.

Site 1: slides BUL–1–1, BUL–1–2.

Paramoebidium hamatum was described originally from USA in Chironomidae, Ameletidae and Baetidae (Ephemeroptera). In Bulgaria, it is associated with Baetidae nymphs (Baetis sp. B. rhodani and B. melanonyx). The species was recorded before in Europe (Spain, Busquets et al. 2018). Bulgarian specimens measured 180–300 × 12–25 µm, with the broader diameter at the proximal end, near the holdfast, thinner at the distal end (Fig. 29); cystospores observed, measuring about 10 × 4–4.5 µm. The species was identified by the curved portion at the basal one-eighth to one-third of the thallial length and by its holdfast characteristics (Bench and White 2012).

Site 4: slide BUL–4–6.

Species observed attached to the hindgut lining of Protonemura montana, measuring 280–340 × 40–60 µm in our collections. This species was described from France, associated with Nemoura variegata nymphs, in the stonefly family Nemouridae. The species has three different thallial morphologies (Léger and Duboscq 1929b, Duboscq et al. 1948). Subsequent to the revision of the species by Duboscq et al. (1948), this endobiont was not reported again, until now, probably because of the thallus variability and, thus, relatively difficult identification. We only saw one of the thallial morphologies described for the species, this being the stouter and shorter thallus type. The other two morphological types show longer and incurved thalli, one type being wider than the other (Duboscq et al. 1948). Neither was observed in our collections.

Figures 26–30. 

Various species of Amoebidiales. 26 Paramoebidium angulatum from Nemouridae nymphs, thalli overview with typical angular shape at the upper section 27 Paramoebidium chattoni from Simuliidae larva, overview, with thin filaments of Simuliomyces microsporus 28 Paramoebidium curvum from Simuliidae larva, overview 29 Paramoebidium hamatum from Baetidae nymphs, overview of various thalli in different growth phases 30 Paramoebidium inflexum from Nemouridae nymphs, thallus overview. Scale bars: 50 μm (26, 29, 30), 100 μm (27), 25 μm (28).