Myxomycetes associated with the bark, cones and leaves of Australian cypress pines (Callitris spp.)

Keywords: amoebozoans, conifer myxomycete ecology, Cupressaceae, microbial ecology, moist chamber cultures, myxogastrids, plasmodial slime moulds, slime moulds.

One of the earliest branches of the eukaryote tree of life consists of an assemblage of amoeboid protists referred to as the supergroup Amoebozoa (Fiore-Donno et al. 2010). The most diverse members of the Amoebozoa are the eumycetozoans, commonly referred to as slime molds (Stephenson and Stempen 1994). Of these, the myxomycetes are the best known group, with approximately 1200 morphologically recognisable species (Lado 2005–2022). Myxomycetes are free-living predators of other eukaryote protists and bacteria and have been recorded from every terrestrial habitat investigated (Stephenson 2021). The two trophic stages (amoeboflagellates and plasmodia) in the life cycle are usually cryptic, but the fruiting bodies are often large enough to be observed directly in nature. Fruiting bodies release spores that are dispersed by air or (more rarely) by animal vectors (Stephenson and Stempen 1994). Under favorable conditions, these spores germinate and give rise to amoeboflagellates, from which the plasmodium is ultimately derived (Martin et al. 1983).

Myxomycetes are associated with a wide variety of substrates that occur in a wide range of microhabitats. The most important substrates are considered to be coarse woody debris, ground litter, aerial litter and the bark surface of living trees (Stephenson 1988). Specimens for study can be obtained as fruiting bodies that have developed in the field under natural conditions or were cultured in the laboratory. A substantial body of data on the worldwide biodiversity and distribution of myxomycetes has been assembled over the past 200 years. More recently, an appreciable body of data has become available on various aspects of the biology, ecology, phylogeny, and genetics of these organisms (Rojas and Stephenson 2021). However, there are still various aspects of their distribution and ecology that remain understudied. Considerable research has been conducted on the assemblages of myxomycetes associated with conifers and forest communities dominated by conifers around the world (e.g. Novozhilov et al. 2010; Takahashi and Hada 2010; Takahashi and Harakon 2012; Adamonytė et al. 2016; Stephenson et al. 2020a). Because most species of conifers occur in the northern hemisphere, studies of conifer–myxomycete associations have typically focused on locations outside of Australia, unless they involved plantations of imported pines. Very limited sampling conducted in western Queensland by the present authors in 2019 suggested that Callitris provides a favourable substrate for these organisms (Table 1). The genus Callitris (known as cypress pine) is the most diverse group of conifers native to Australia. In all, 13 of the 16 recognised species are endemic to Australia; the remaining three are endemic to New Caledonia (Piggin and Bruhl 2010; Crisp et al. 2019). Previous studies of myxomycetes in Australia have incidentally reported a diversity of species to occur on substrates (mostly samples of bark) derived from Callitris (Table 1), but the present study is the first to focus on these associations.

Table 1.  List of myxomycetes reported to occur in association with Callitris compiled from our study and a comprehensive review of the literature.

Samples of the non-living outer bark, fallen female cones, and litter (dead leaves/needles from the ground) were collected from four species of Callitris (C. columellaris F.Muell., C. glaucophylla J.Thomp. & L.A.S.Johnson, C. intratropica R.T.Baker & H.G.Smith, and C. rhomboidei R.Br. ex Rich. & A.Rich.; Fig. 1). Samples were sourced from Callitris populations at 13 localities in New South Wales, Queensland, and the Northern Territory (Table 2). Samples were from areas of high mean annual rainfall for Australia, ranging from the temperate to tropical climate zones to the transitional to arid climate zone. Each sample was placed in a paper bag, air dried, and sent to the Eumycetozoan Laboratory at the University of Arkansas in the United States. These samples were used to prepare three series of moist-chamber cultures (bark, cones, and litter) in the manner described by Stephenson and Stempen (1994). The series prepared with bark consisted of 40 cultures, the series prepared with litter consisted of 38 cultures, and the series prepared with cones consisted of 38 cultures. Each culture consisted of a 90 mm plastic disposable Petri dish, with the bottom lined with filter paper. Enough sample material was placed in each dish to cover most of the filter paper. Once prepared, enough distilled water was added to each culture to cover most of the sample material. After approximately 24 h, the pH of each culture was determined and recorded and then most of the water was poured off. All cultures were set aside out of direct sunlight and then examined with the aid of a dissecting microscope at weekly intervals over a period of 3 months. Any fruiting bodies of myxomycetes appearing in these cultures were collected or recorded. Those collected were placed in small pasteboard boxes for permanent storage in the herbarium of the University of Arkansas (UARK). Standard references for myxomycetes (e.g. Martin and Alexopoulos 1969) were used to identify the collections.

Fig. 1.  Examples of Australian cypress pines and the different portions that were sampled for associated myxomycetes. (a) Callitris glaucophylla woodland, showing a typical habitat representing one of the types of habitat where the three substrates were collected. (b) A representative stand of several C. intratropica in the tropics of Cape York, Queensland. This species seldom formed the near-monodominant stands of some of the other species. (c) The bark of C. glaucophylla that was a substrate in this study. Only the outer flakey layers of bark were collected (the cambium of trees was not damaged). (d) Cones of C. glaucophylla still present in this tree. We selected these cones as a substrate only once they had fallen to the ground. (e) Fallen litter (composed of needles/leaves), such as the ones shown in these images, were collected as our third substrate. All images in Fig. 1 © Todd F. Elliott.

Table 2.  Localities where samples of Callitris needles, cones and bark were collected in the present study.

Of the 116 moist-chamber cultures prepared in this study, 90 (78%) yielded evidence (plasmodia or fruiting bodies) of myxomycetes. Twenty-three species in 15 genera were recorded. The percentage of positive cultures was 87% (cones), 83% (bark), and 63% (litter). The differences among the three substrates were more appreciable when numbers of species were considered. Seventeen species were recorded from bark, 10 from litter, and six from cones. Only two species (Arcyria cinerea and S. fusca) were recorded from all three substrates. As is the case for other studies of myxomycetes, a record is based on the occurrence of at least one specimen of the species in question. However, it is possible for a record to be based on many hundreds of specimens. This is especially true for some of the smaller species.

Acryria cinerea (18 specimens) was the most abundant species, but Cribraria confusa (eight specimens), Diderma effusum (eight specimens), C. minutissima (five specimens), Perichaena vermicularis (five specimens), and Physarum decipiens (five specimens) were also relatively common. Mean values of pH recorded for the three sets of cultures displayed little variation, with a range of only 5.7–5.9. The values recorded for cultures prepared with bark were the most variable (3.9–6.8), with the highest value a conspicuous outlier.

Species of myxomycetes recorded in the present study are listed alphabetically by genus and then species. Nomenclature used follows Lado (2005–2022). The total number of specimens recorded for each species, their distribution with respect to the three substrates, the species of Callitris and the specific localities from which the species was recorded, and comments on their abundance and ecology are also provided. The latter is based on Stephenson (2021) and the Atlas of Living Australia (https://ror.org/018n2ja79). Numbers given in parentheses are those of the first author.

Specimens: 18 specimens in total, with three from bark, 11 (including 34 522 and 34 613) from litter, and four (including 34 463) from cones [pH 4.1–6.3]. A. cinerea was recorded from eight localities (1, 2, 3, 4, 6, 8, 9 and 10) and all four species of Callitris.

Fig. 2.  Examples of four species of myxomycetes that we found in association with Callitris in this study. (a) Arcyria cinerea. (b) Comatricha laxa. (c) Cribraria minutissima. (d) Licea operculata. Image b © Joe Takano. Images a, c and d © Yuri Novozhilov.

Comments: A. cinerea was considered to be cosmopolitan by Martin and Alexopoulos (1969) and is very common in Australia. Wellman (2019) previously reported the species from the bark of Callitris (Table 1).

Specimens: three specimens (including 34 500) on bark [pH 5.6–5.7]. C. metallica was recorded from two localities (3 and 11) on Callitris glaucophylla.

Comments: Martin and Alexopoulos (1969) reported C. metallica as widely distributed in Europe and temperate North America, with additional records from scattered localities throughout the rest the world. The species has been reported from a number of localities in Australia, but does not appear to be common. Wellman (2019) previously reported the species from the bark of Callitris.

Specimen: one specimen (34 505) on cones [pH 5.4]. C. debaryanum was recorded from one locality (4) on Callitris intratropica.

Comments: Martin and Alexopoulos (1969) reported C. debaryanum as abundant in the tropics, but also known from scattered non-tropical localities. The species has been recorded from scattered localities in Australia, but is not especially common.

Specimens: four specimens, with three (including 34 501) on bark and one (34 540) on litter [4.5–5.7]. C. laxa was recorded from three localities (1, 3 and 12) on C. glaucophylla, C. intratropica, and C. rhomboidea.

Comments: Martin and Alexopoulos (1969) listed C. laxa from only Europe, Asia, temperate North America, and Tahiti. However, Ing (1999) considered the species as probably cosmopolitan. It is known from scattered localities in Australia, but is not particularly common.

Specimens: three specimens (including 34 503), all on bark [pH 3.9–6.3]. C. nigra was recorded from two localities (8 and 10) on C. glaucophylla and C. intratropica.

Comments: Martin and Alexopoulos (1969) considered C. nigra to be cosmopolitan. It is known from scattered localities throughout Australia, but small forms of this species commonly appear in moist-chamber cultures.

Specimens: eight (including 34 508), all on bark [pH 4.1–4.7]. C. confusa was recorded from three localities (2, 4 and 5) on Callitris columellaris and C. intratropica.

Comments: C. confusa was not recognised as distinct from C. minutissima at the time when the monograph by Martin and Alexopoulos (1969) was published. On the basis of Australian records, the species is relatively common.

Specimens: five specimens (including 34 507), all on bark [pH 4.1–5.0]. C. minutissima was recorded from three localities (5, 8 and 10) on C. glaucophylla and C. intratropica.

Comments: Martin and Alexopoulos (1969) listed this species as widely distributed in the United States and as infrequently recorded elsewhere. Like for the morphologically similar C. confusa, there are enough records of C. minutissima from Australia to consider it as relatively common.

Specimens: eight specimens (including 34 557 and 34 539), all on bark [pH 5.6–6.7]. D. effusum was recorded from three localities (1, 3 and 11) on C. glaucophylla and C. rhomboidea.

Comments: this species was considered as cosmopolitan by Martin and Alexopoulos (1969). It is widespread in Australia and often not uncommon on dead leaves and other similar types of plant debris.

Specimens: two specimens, with one (34 553) on cones [pH 3.9] and one (34 618) on litter [pH 5.7]. D. nigripes was recorded from two localities (5 and 6) on C. intratropica.

Comments: Martin and Alexopoulos (1969) considered this species to be cosmopolitan. However, there are relatively few records of D. nigripes from Australia, but it appears to be more common than the morphologically similar D. iridis (Ditmar) Fr., with which it is sometimes confused (Stephenson 2021).

Specimens: four specimens, with two (including 34 520) on cones and two (including 34 554) on bark [pH 4.4–5.9]. E. minutum was recorded from four localities (3, 4, 10 and 13) on C. glaucophylla and C. intratropica.

Comments: E. minutum was considered as cosmopolitan by Martin and Alexopoulos (1969), and it has been recorded in virtually every survey for myxomycetes ever undertaken. This situation applies to Australia (Stephenson 2021)

Specimen: one observed but not collected, on bark [pH 3.9]. L. kleistobolus was recorded from one locality (8) on C. intratropica.

Comments: Martin and Alexopoulos (1969) listed on a few localities for this species, but Ing (1999) considered it as probably cosmopolitan. Like for the vast majority of species in the genus Licea, the fruiting bodies are small and easily overlooked. However, L. kleistobolus is distinct enough so that it is unlikely to be confused with any other species, and there are enough records from Australia to consider it as relatively common. Wellman (2019) previously reported the species from the bark of Callitris.

Specimens: four, with two (including 34 510) on bark and two (including 34 499) on cones [pH 4.9–5.7]. L. operculata was recorded from three localities (2, 3 and 4) on C. columellaris, C. glaucophylla, and C. intratropica.

Comments: Martin and Alexopoulos (1969) listed L. operculata from scattered localities in the northern hemisphere, with most of these from North America. Since then, the species has been reported from the southern hemisphere, although it does not appear to be common. There are relatively few records from Australia.

Specimens: four, with two (including 34 559) on litter and two (including 34 525) on bark [pH 3.9–6.2]. M. decapillata was recorded from three localities (3, 8 and 11) on C. glaucophylla and C. intratropica.

Comments: Martin and Alexopoulos (1969) reported this species from a few localities in the United States and there was one record from Costa Rica. It is now known from scattered localities throughout the world, but is never very common. M. decapillata is known from just a few records in Australia.

Specimen: one specimen (34 573) on bark [pH 5.7]. P. rigida was recorded from one locality (3) on C. glaucophylla.

Comments: the concept of Paradiacheoplus ridida was not firmly established at the time when the Martin and Alexopolous monograph (1969) was published. This appears to be an uncommon species worldwide (Ing 1999), and there are very few records from Australia. It should be noted that the material of P. ridida obtained in the present study consisted of a single sporocarp and it is only provisionally referred to this species. However, it was clearly different from any other species recorded from Callitris.

Specimen: one specimen (34 558) on litter [pH 6.2]. P. corticalis was recorded from one locality (11) on Callitris glauophylla.

Comments: P. corticalis was considered to be cosmopolitan by Martin and Alexopoulos (1969). The typical form is relatively common in Australia (Stephenson 2021), but the small form with little or no capillitium present is apparently known from only a single record (McHugh et al. 2009). The latter is recognised as a distinct species (Perichaena liceoides Rosstaf.) in some treatments of the myxomycetes.

Specimens: five specimens, with three (including 34 651) on litter and two (including 34 504) on bark [pH 5.5–6.0]. P. vermicularis was recorded from two localities (1 and 3) on Callatris columellaris and C. glaucophylla.

Comments: P. vermicularis was considered as cosmopolitan by Martin and Alexopoulos (1969). This species is relatively common in Australia and appears to be somewhat more abundant than the somewhat similar P. corticalis.

Specimen: one specimen (recorded but not collected) on litter [pH 6.2]. P. cinereum was recorded from one locality (6) on C. intratropica.

Comments: Martin and Alexopoulos (1969) considered P. cinereum as cosmopolitan. It is common and sometimes abundant throughout Australia.

Specimens: five specimens, with all (including 34 657) on bark [pH 6.3–6.7]. P. decipiens was recorded from three localities (3, 10 and 11) on C. glaucophylla.

Comments: Martin and Alexopoulos (1969) listed P. decipiens from widely scattered localities throughout the world, but also commented that it is sometimes confused with P. auriscalpium Cooke and P. serpula Morgan, two morphologically similar species. P. decipiens is not common in Australia (2021), but was reported previously from Callitris bark by Wellman (2019).

Specimens: three specimens (including 34 523) on bark [pH 6.7]. P. leucophaeum was recorded from one locality (11) on C. glaucophylla.

Comments: Martin and Alexopoulos (1969) listed P. leucophaeum as not common, but Ing (1999) considered it as probably common. In Australia, this species is widely distributed but not particularly common. Wellman (2019) previously reported the species from the bark of Callitris.

Specimen: one specimen (34 617) on litter [pH 5.8]. P. pusillum was recorded from one locality (4) on C. intratropica.

Comments: P. pusillum was considered to be cosmopolitan by Martin and Alexopoulos (1969). The species is relatively common in Australia (Stephenson 2021).

Specimen: one specimen (34 615) on bark [pH 4.4]. P. viride was recorded from one locality (13) on C. intratropica.

Comments: this distinctive species was considered to be cosmopolitan by Martin and Alexopoulos (1969). P. viride is one of the more common species of Physarum found throughout Australia. Interestingly, the specimen from bark recorded in the present study consisted of only a single sporocarp.

Specimens: four specimens, with one (34 570) on bark, one (34 647) on litter, and two (including 3464) on cones [pH 4.9–5.1]. S. fusca was recorded from three localities (1, 2 and 9) on C. columellaris, C. intratropica, and C. rhomboidea.

Comments: S. fusca was considered to be cosmopolitan by Martin and Alexopoulos (1969). It is one of the most common members of the genus Stemonitis found in Australia. Because of their small size and the fact that they appeared in moist-chamber cultures, the specimens recorded in the present study would be referred to S. fusca var. nigrescens (Rex) Torrend. The latter is sometimes recognised as a distinct species (Stemonitis nigrescens Rex).

Specimens: two specimens (including 34 555) on litter [pH 5.2–5.3]. T. erecta was recorded from one locality (6) on C. intratropica.

Comments: most of the records of T. erecta listed by Martin and Alexopoulos (1969) were from North America, and this species appears to be relatively uncommon throughout the rest of the world. There are relatively few records from Australia (Stephenson 2021).

Numerous previous studies have investigated the occurrence of myxomycetes in different types of habitats (e.g. tropical forests, temperate forests, grasslands, and deserts) or on different substrates (e.g. the bark of living trees, coarse woody debris, ground litter, and dung; Härkönen 1977; Blackwell and Gilbertson 1984; Stephenson 1988, 1989; Novozhilov et al. 2000; Schnittler and Stephenson 2000; Wrigley de Basanta 2000; Lado et al. 2003; Snell et al. 2003; Takahashi 2004; Estrada-Torres et al. 2009; Ndiritu et al. 2009; Adamonyte et al. 2011; Dagamac et al. 2012; Wellman 2019; Stephenson et al. 2020b, 2022; White et al. 2020). Many species of myxomycetes appear to be widely distributed worldwide and are found in virtually all habitats and microhabitats investigated. However, studies have indicated that it is often possible to define the ecological niche of a particular species, a concept first described for myxomycetes by Stephenson (1988). For example, on a large scale, there are some species that appear to be largely or completely restricted to tropical or temperate regions of the world. On a small scale, there are some species that appear to be specialists that primarily associate with coarse woody debris, whereas others almost exclusively associate with the bark of living trees. The ecological niche has been demonstrated to be relatively broad for some species and narrow for others. Among the species recorded in the present study, A. cinerea has a broad niche, because it can be found in virtually any habitat or microhabitat. In contrast, species such as C. confusa appear to have a narrow niche; virtually all available records suggest that it is largely or completely confined to tree bark (Keller et al. 1988). Just what factors determine the niche breadth of different species of myxomycetes is still incompletely known, but substrate pH is considered to be an important factor determining the natural distribution of myxomycetes. However, the mean values of pH recorded for cones (5.7), litter (5.8), and bark (5.9) in the present study were not significantly different.

Any consideration of the ecological or geographical distribution of myxomycetes must take into account the fact that a particular morphospecies (i.e. a species recognised solely on the basis of clearly evident morphological features) can sometimes consist of a complex of biotypes or biospecies (i.e. distinct taxonomic entities defined on the basis of genetic sequences). These biospecies have been demonstrated to display evidence of possible ecological differentiation with respect to substrate and habitat in a number of studies (e.g. Feng and Schnittler 2015; Dagamac et al. 2017). Such is the case for A. cinerea, the most abundant species recorded in the present study. Molecular approaches have only recently begun to be used to study myxomycete taxonomy and ecology; it is therefore plausible that many morphologically valid species that we report could be species complexes.

We are not aware of any previous studies that have examined the assemblages of myxomycetes associated with different portions of the same type of plant, and our data suggest that species of myxomycetes may ‘distinguish’ among the Callitris-derived substrates. Further studies with more replicates are needed to conduct a statistical analysis of these differences. The fact that the highest number of species was recorded from bark is not surprising, because it has long been known that bark is an especially favourable substrate for myxomycetes. The lower totals recorded for cones and ground litter suggest that these two substrates are less favourable; however, why this is the case is not possible to determine in the context of this study. As noted earlier, the fact that only two species (A. cinerea and S. fusca) were recorded from all three substrates was an unexpected result and provides evidence that there is still a lot to be learned about niche relationships in myxomycetes.

Any additional data are available upon reasonable request from the authors.

The authors declare no conflicts of interest.

TFE received funding for fieldwork for this project from two University of New England Robine Enid Wilson Grants and two Holsworth Wildlife Research Endowment Grants from the Ecological Society of Australia. The School of Environmental and Rural Science at the University of New England provided TFE with an International Postgraduate Research Fellowship and a 4WD to travel from Armidale, NSW, to Cape York, Queensland.