Mycologia ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: https://www.tandfonline.com/loi/umyc20 Morphology and ecology of Didymium subreticulosporum Juan Mosquera, Carlos Lado & Esperanza Beltrán Tejera To cite this article: Juan Mosquera, Carlos Lado & Esperanza Beltrán Tejera (2000) Morphology and ecology of Didymium�subreticulosporum, Mycologia, 92:5, 978-983, DOI: 10.1080/00275514.2000.12061241 To link to this article: https://doi.org/10.1080/00275514.2000.12061241 Published online: 04 Jun 2019. Submit your article to this journal Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=umyc20 Mycologia, 92(5), 2000, pp. 978-983. © 2000 by The Mycological Society of America, Lawrence, KS 66044-8897 Morphology and ecology of Didymium subreticulosporum Juan Mosquera1 MATERIALS AND METHODS Department of Medicine, University of Manchester, CSB, Hope Hospital, Manchester M6 8HD, UK The study is based on material deposited in the herbaria TFC Mic., MA-Fungi and AH. All microscopic measurements and observations were made with material mounted directly in Hoyer's medium. Cross sections were made of sporophores soaked in nail-lacquer and dried. For all the SEM-photographs the critical-point dried material technique was employed. Values of pH were determined with pH-indicator strips (Merck). Cultures were prepared on oatmeal agar (see Lado et al 1999). Color notations in parentheses are from the ISCC-NBS Color-Name Charts Illustrated with Centroid Colors (Anonymous 1976). Carlos Lado Real Jardin Botanico, CSIC, Plaza de Murillo 2, 28014 Madrid, Spain Esperanza Beltran Tejera Departamento de Biolo{!;ia Thgetal (Botanica), Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain Specimens examined.-SPAIN. Leg. M. Oltra, 10-III-1994, det. G. Moreno, Oltra, Illana, AH 19047 (160 Oltra) [reported as: SPAIN. Valencia, Ctra. R6tova-Albaida, 30S)j4315, 40 m.s.m., on fallen cladodes of Dpuntia maxima Miller, 12VIII-1993, 160 Oltra (AH 19047) HOLOTYPE (Moreno et al1996: 57)]; Ibidem, 1-IX-1993, 168 Oltra (AH 19048). CANARY ISLANDS: Tenerife. 28RCS7455, 850 m, 15-XI-1995, TFC Mic. 7282 (MA-Fungi 40471); Ibidem, 9-XII-1995, TFC Mic. 7300, 7302, 7303; Ibidem, 10-VI-1996, TFC Mic. 7725, 7726; Ibidem, 4-VIII-1996, TFC Mic. 7825, 7831 (MA-Fungi 40472), 7832,7833,7836,7837,7838,7840,7847, 7849;Ibidem, 25-XI-1996, TFC Mic. 7915 (MA-Fungi 40473), 7918, 7927; Ibidem, 27-IV-1997, TFC Mic. 8126; Ibidem, 24-1-98, TFC Mic. 8205. 28RCS7355, 700 m, 15-11-1997, TFC Mic. 7988, 7989, 7990, 7993, 7994, 7998, 8127; Ibidem, 16-11-1997, TFC Mic. 8005 (MA-Fungi 40474), 8006 (MA-Fungi 40475), 8007, 8013, 8015. 28RCS2338, 275 m, 19-1-1998, TFC Mic. 8232, 8233. 28RCS6453, 525 m, 21-11-1998, TFC Mic. 8420, 8421, 8423, 8424. 28RCS3840, 475 m, 21-11-1998, TFC Mic. 8432, 8433, 8435. 28RCS4341, 100 m, 21-11-1998, TFC Mic. 8439, 8440. 28RCS1938, 375m, 14-III-1998, TFC Mic. 8592. MEXICO. BAJA CALIFORNIA: Tijuana, 13-XI-1994, AH 21014. Rosarito, 16-III-1997, AH 22610 (MA-Fungi 36743). Ensenada, 31-V-1997, AH 22450. Unless otherwise indicated, specimens were harvested from the inner tissue and/ or epidermis of decaying Dpuntia maxima cladodia. Abstract: A new description of Didymium subreticulosporum is provided that highlights its unique taxonomic characters-a discontinuous peridial wall, lack of didymioid capillitium, and the presence of an irregular membranous structure inside the sporotheca, filled with crystals. This species is considered succulenticolous due to its consistent fruiting on opuntia cladodia in early stages of decay and restriction to this microhabitat. Spore dispersal is apparently by cactophilic Drosophila flies. Key Words: Canary Islands, Drosophila, Mexico, Myxomycetes, Dpuntia, Spain, taxonomy INTRODUCTION Didymium subreticulosporum Oltra, G. Moreno & 11lana was described by Moreno et al (1996), based on three specimens from Spain collected on decaying Dpuntia maxima Miller [ = 0. ficus-indica] cladodia. A more detailed description with comments and illustrations was given by Oltra et al (1997), based on the same collections. Later, the same authors reported it from Baja California, Mexico, on the same type of substrate and made additional comments about its taxonomic characters (Lizarraga et al1998). We have examined the type material and more than 40 collections of this species from Tenerife, Canary Islands, on decaying cladodia of Dpuntia ficus-indica (L.) Mill. We studied the morphology of the sporocarp and reinterpret some of its features, as well as comment on the ecology and a possible method of spore dispersal. RESULTS Didymium subreticulosporum is a distinct morphospecies with unique and stable morphological characters that set it apart as an atypical member of the genus. In previous publications, its morphology was misinterpreted and species descriptions are not completely accurate. Using the description provided by Oltra et al ( 1997), one would assume that the capillitium is didymioid, with crystals inside the tubules (and the au- Accepted for publication March 8, 2000. 1 Email: jmosquer@fsl.ho.man.ac.uk 978 Published online 04 Jun 2019 MOSQUERA ET AL: DIDYMIUM SUBRETICULOSPORUM thors compared it to the capillitium of D. tubi-crystallinum Nann.-Bremek. & R.L. Critchf.). Nevertheless, in their SEM micrographs of the capillitium (01tra et al 1997 Figs. 9, 10) no didymioid threads can be observed, only a line of crystals. Lizarraga et al (1998) commented that the capillitium is "formed not by hyaline filaments, as is common in the genus Didymium, but by crystals of calcium carbonate, united to form 'lines' which project radially from the central part of the sporocarp." In our opinion, D. subreticulosporum lacks a capillitium formed by didymioid tubules or lines of crystals. We think what Oltra et al (1997) and Lizarraga et al (1998) thus interpreted, are actually pieces of an atypical structure present inside the sporotheca. We prepared cross sections of sporophores soaked in nail-lacquer, which show a very irregularly shaped structure (FIG. 1), formed by a continuous wall filled internally with crystals. When a sporophore dehisces, the delicate structure ruptures (see FIG. 8), giving a white calcareous, spiky appearance when it is observed by reflected light (FIG. 2). When a sporophore is mounted, this membranous structure breaks up into numerous platelets of similar material and color as the stalk apex: these are covered by crystalline lime on one side. These features were interpreted as the calcareous capillitium in the former descriptions. Only occasionally can any crystal alignment without connection with the platelets be observed. If the sporocarp is mounted in lactophenol, the calcareous crystals disappear and the platelets can be observed as isolated elements, or adjoined at the stalk apex as the remains of the crystalline membranous structure. We have never seen capillitial tubules with or without crystals inside, and we do not think this structure is typical didymioid capillitium. Regarding the peridium, in all the material examined by us, including the type collection, the sporothecae does not have a continuous peridial wall. We have observed only isolated membranous brownish platelets and a uniform, porous crust cover of crystals, which are not just "sprinkled" (FIGs. 3, 4). We think these platelets were interpreted as the areolae of a membranaceum peridium by Oltra et al (1997), but they are isolated, not forming darker parts of a continuous peridial wall as in D. melanospermum (Pers.) T. Macbr. Since the sporotheca lacks a continuous peridial wall, the crust of crystals works like a wall, keeping the sporophore intact with the spores inside until it dehisces. We have not observed any cartilaginous material with a slightly granular, deepbrown basal plate, as mentioned by Oltra et al (1997) and Lizarraga et al (1998). None of these authors mentions that the spores are 979 paler and possess a less marked and often a broken reticulation on one side, something we have observed in all material (FIGS. 5-7). The spore wall is probably thinner there, as spores tend to collapse (FIG. 5). When observed on agar, spores germinated through that area by means of a wedge-shaped split. Efforts to observe further stages of the life cycle on agar were unsuccessful. These observations merit a revised description, as follows. Didymium subreticulosporum Oltra, G. Moreno & 11lana in G. Moreno, Castillo, Illana & Lizarraga, in Lado & Hernandez (eds.), II Congreso Internacional de Sistematica y Ecologia de Myxomycetes, ICSEM2 [Abstr. 2nd Intern. Congr. Syst. Ecol. FIGS. 1-8 Myxomycetes, ICSEM2]: 57 (1996). I con. 01 tra et al (1997: Figs. 1-1 7) ; Lizarraga et al (1998: Figs. 1-6). References. Moreno et al ( 1996); Oltra et al ( 1997); Lizarraga et al (1998). Sporophore sporocarpic, grouped to crowded, stalked. Sporocarps erect, 1-2(-3) mm in total height, often fused and subreniform, occasionally gyrose-confluent. Sporotheca subglobose, 0.5-1 mm diam, to subhemisphaerical, 0.3-0.5 X 0.3-0.8 mm, covered by calcareous crystals forming a porous crust, white (263. White) to light bluish grey (190. l. b Gray). Hypo thallus membranous, irregular, sometimes common to a group of sporophores, deep brown (56. deep Br). Stalk 0.2-0.9 mm in height, erect, longitudinally striate, often compressed laterally, sometimes expanded at the apex, up to 0.4 mm diam, not calcareous, shining, black (267. Black), turning dark brown (59. d. Br) towards the apex, brownish orange (54. br 0) to brownish black (65. br Black) and strong yellowish brown (74. s. y Br) towards the apex by LM. Peridium membranous, not continuous but formed by isolated platelets, light orange yellow (70. l. OY) by LM, covered with a continuous crust of crystals. A continuous membranous structure with a very irregular shape, filled with calcareous crystals, is present inside the sporotheca. Dehiscence irregular, showing spores and a broken calcareous, spiky membranous structure. Spores brownish black (65. br Black) in mass, light greyish brown (60. l. gy. Br) to dark greyish brown (62. d. gy. Br) by LM, lighter on one side, subglobose, reticulate, with the reticulum less marked and often broken on the paler side, 8.4-11.6 IJ.m diam. Plasmodium a phaneroplasmodium, whitish brown. DISCUSSION Taxonomy.-The membranous structure filled with calcareous crystals and present inside the sporotheca 980 MYCOLOGIA FIGS. 1-7. Didymium subreticulosporum. 1. Cross section of sporophore soaked in nail-lacquer. The irregular shape of the structure internal to the sporotheca, filled with crystals, can be observed (arrows). 2. Dehisced sporophores showing a broken irregular calcareous structure. 3, 4. Scanning electron micrographs (SEM) of an inner view of the outer crust of crystals. The peridial platelets can be observed, with well defined, not torn, edges (arrow). 5-7. SEM of spores. 5. Spore partially collapsed at the lighter side, with a partial reticulum. 6, 7. View of the lighter side. with a very irregular shape, could be interpreted morphologically as a columella. Nevertheless, functionally this structure is capillitium. A study of its morphogenesis is needed to determine if it is also capillitium in a developmental sense. This morphological character of crystalline membranous elements is unique within the genus. On the other hand, lack of typical capillitium has been already observed in other Didymium species such as Didymium atrichum Henney & Alexop. (Henney et al 1980) and D. eremophilum M. Blackw. & Gilb. (Blackwell and Gilbertson 1980) and is quite a variable character in some other genera (Alexopoulos 1976). The occurrence of a noncontinuous peridial wall in this species has been repeatedly observed in all material we have studied. When an undehisced sporophore is treated with a dilute aqueous solution of hydrochloric acid, the lime crystals disappear and, at MOSQUERA ET AL: DIDYMIUM SUBRETICULOSPORUM FIG. 8. Didymium subreticulosporum. Dehisced sporophore showing an irregular structure. the same time, the spore mass disperses because of the gas bubbles formed and the lack of a continuous peridial wall to retain it. According to Gilert ( 1995), the peridium, in the sense of Blackwell (1974), corresponds to the electron dense peridial layer newly formed during the development of the sporophore, under a slime coat that covers the plasmodium during early stages of differentiation. That slime coat is apparently the electron transparent layer that covers the mature peridium. Charvat et al (1973) showed that the peridial wall forms directly outside the plasma membrane in Perichaena vermicularis (Schwein.) Rostaf. The peridial platelets of D. subreticulosporum would represent that electron dense but discontinuous peridial layer of material deposited outside the plasma membrane and under the slime layer. This layer contains the substance that will give rise to the outer crystal crust, during the maturation of the sporotheca. It is continuous and seems to be even in thickness over the entire surface. The peridial darker areas of species such as D. melanospermum (Pers.) T. Macbr., D. minus (Lister) Morgan, and D. nigripes (Link) Fr. may appear similar to the peridial platelets that we have described for D. subreticulosporum. In all three species, however, the peridial wall is continuous. The differential formation of the peridium, in the way that we have described it, occurs in other species of myxomycetes belonging to different orders, such as Echinostelium arboreum H.W. Keller & T.E. Brooks (Haskins and McGuinness 1989), Licea kleistobolus G.W. Martin (Gilert 1997), Cribraria spp. (Lado et al1999), Anyria spp. (Gilert 1997) and Trichia crateriformis G.W. Martin (Frederick et al 1983). We do not know of any species with a similar feature in the order Physarales, apart from D. subreticulosporum. 981 Ecology.-The ecology of D. subreticulosporum has been poorly understood. This species has been found in the old and new world and always on decaying Dpuntia cladodia, which suggests its specificity to this substrate and the fact that it is a strictly succulenticolous species (Lado et al 1999, Mosquera et al 1999). In Tenerife (Canary Islands), despite the abundant presence of this substrate in the low areas and the numerous plots examined, we have collected it only on the north slope of the island, perhaps due to the excessive sunlight and the higher temperatures on the southern slopes. Mter several years studying the succulenticolous myxomycete biota of Tenerife, we have noticed D. subreticulosporum is normally the earliest myxomycete species that fruits during the decomposition process of 0. ficus-indica. It appears when the decaying cladodium, dark in color because of the rotting process, begins to lose its own moisture content but still retains a relatively high humidity. At this time, the cladodium is a dark, dense, moist, rotten mass, and when the epidermis is opened, immature stages of D. subreticulosporum sporophores are found fruiting with the dark stalks already formed supporting white, immature sporothecae. Other species such as Hemitrichia minor G. Lister or Physarum compressum Alb. & Schwein. can only occasionally be found at this early stage of decay but are very frequent and abundant in later stages. In these later stages of cladodium decomposition, sporocarps of D. subreticulosporum can be found, but normally as old weathered specimens, often invaded by filamentous fungi. A possible hypothesis for why this happens is that this species has become better adapted to the early decomposition conditions, producing more vegetative forms (myxamoebae and plasmodia) than other species. These conditions are a high and lasting moisture content from the succulence of the plant tissue, a high temperature, a basic pH (ranging from 8.5 to 9) and high levels of nutrients as organic substances (Fogleman and Abril1990), yeasts, fungi and bacteria (Starmer and Phaff 1983, Starmer and Aberdeen 1990). Their success is reflected in the predominance of sporophores of D. subreticulosporum at this stage. Competition for food with dipteran larvae, which feed on bacteria and yeasts in this microhabitat (Barker et al 1984), as do the vegetative forms of D. subreticulosporum, must be taken into consideration. Adaptation to early decomposition conditions would permit D. subreticulosporum to outcompete at this stage but also would prevent effective competition with other myxomycete species in the later stages of decomposition, characterised by lower moisture content conditions, less nutrient abundance, a different bacterial, yeast and fungal biota, and an even higher 982 MYCOLOGIA pH (albeit lower in the final stages). An additional observation is that D. subreticulosporum has never developed in the numerous moist chamber cultures we have established with opuntia in different stages of decomposition, unlike other species such as H. minor, Didymium vaccinum (Durieu & Mont.) Buchet, Badhamia gracilis (T. Macbr.) T. Macbr. or Physarum compressum. An intriguing question remains related to the process of colonization of new cladodia by D. subreticulosporum. The cladodia of opuntia spp. possess a thick and continuous epidermis, except where the cladodium joins others or in areas of injury. As such, it does not seem probable that colonization is by wind dispersed spores or soil myxomycete plasmodium-forming units (PFU). The probability that PFU will colonise at the exact moment of an injury or in time to allow the early fructification of this species is remote. Moreover, according to Feest and Madelin (1985), PFU seem to be less abundant in desert soils than in others with different vegetation types. Larvae of Drosophila buzzati Patterson & Wheeler were frequently observed in Tenerife on the same substrate as D. subreticulosporum. This species of Drosophila feeds and breeds on decaying opuntia cladodia (Monel us 1976), and it seems likely that these insects serve as vectors for D. subreticulosporum spores. This method of spore dispersal by invertebrates has been already suggested for a few species that feed on myxomycete spores, such as mites and nematodes for corticolous myxomycetes (Keller and Smith 1978, Ing 1994), beetles for aethalial myxomycetes (Blackwell et al 1982), or flies (Stephenson and Stempen 1994) for certain species with aethalia or pseudoaethalia. The important role played by dipterans, as vectors of bacteria and especially yeasts in rotting cacti, has been reported by Fogleman and Foster (1989) and Ganter (1988). Larvae that become adults and leave the cladodium, or simply adults that fly from one cladodium to another, could carry on their bodies, or in their digestive tracts, spores or PFU of myxomycetes, which would be transfered to other early decaying cladodia where the flies feed, mate or lay eggs. The fact that insects serve as vectors in this way has been shown for bacteria and yeasts by these same authors. 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