Airborne myxomycete spores: detection using molecular techniques

Naturwissenschaften (2009) 96:147–151 DOI 10.1007/s00114-008-0454-0

SHORT COMMUNICATION

Airborne myxomycete spores: detection using molecular techniques Akiko Kamono & Hisaya Kojima & Jun Matsumoto & Kimitaka Kawamura & Manabu Fukui

Received: 20 February 2008 / Revised: 27 August 2008 / Accepted: 18 September 2008 / Published online: 3 October 2008 # Springer-Verlag 2008

Abstract Myxomycetes are organisms characterized by a life cycle that includes a fruiting body stage. Myxomycete fruiting bodies contain spores, and wind dispersal of the spores is considered important for this organism to colonize new areas. In this study, the presence of airborne myxomycetes and the temporal changes in the myxomycete composition of atmospheric particles (aerosols) were investigated with a polymerase chain reaction (PCR)-based method for Didymiaceae and Physaraceae. Twenty-one aerosol samples were collected on the roof of a three-story building located in Sapporo, Hokkaido Island, northern Japan. PCR analysis of DNA extracts from the aerosol samples indicated the presence of airborne myxomycetes in all the samples, except for the one collected during the snowfall season. Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products showed seasonally varying banding patterns. The detected DGGE bands were Electronic supplementary material The online version of this article (doi:10.1007/s00114-008-0454-0) contains supplementary material, which is available to authorized users. A. Kamono (*) : H. Kojima : M. Fukui (*) Basic Cryoscience Section, The Institute of Low Temperature Science, Hokkaido University, N19, W8, Kita-ku, Sapporo 060-0819, Japan e-mail: [email protected] e-mail: [email protected] J. Matsumoto Fukui Botanical Garden, Asahi 17-3-1, Echizen-cho, Fukui-ken 916-0146, Japan K. Kawamura Marine and Atmospheric Sciences Section, The Institute of Low Temperature Science, Hokkaido University, N19, W8, Kita-ku, Sapporo 060-0819, Japan

subjected to sequence analyses, and four out of nine obtained sequences were identical to those of fruiting body samples collected in Hokkaido Island. It appears that the difference in the fruiting period of each species was correlated with the seasonal changes in the myxomycete composition of the aerosols. Molecular evidence shows that newly formed spores are released and dispersed in the air, suggesting that winddriven dispersal of spores is an important process in the life history of myxomycetes. This study is the first to detect airborne myxomycetes with the use of molecular ecological analyses and to characterize their seasonal distribution. Keywords Myxomycetes . Polymerase chain reaction . Spore dispersal . True slime molds

Introduction Myxomycetes (also termed Myxogastria or true slime molds) are amoeboid organisms characterized by fruiting body production (Adl et al. 2005). Their distinctive life cycle has been mostly revealed through laboratory studies with culturable strains of some species such as Physarum polycephalum Schwein. (Aldrich and Daniel 1982). However, details of myxomycete life history in the field are poorly understood. Most field studies of myxomycetes are based on the fruiting body stage, which is the only stage of their life cycle where morphological identification of the species is possible. Many studies have been carried out on the ecology and distribution of myxomycetes on the basis of field surveys of fruiting bodies (e.g., Ing 1994; Stephenson et al. 2008). Wind dispersal of the spores, which are typically approximately 10 μm in diameter, is considered an important dispersal mechanism for these organisms (Alexopoulos 1963; Gray and Alexopoulos 1968; Ing 1994). On the

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basis of the structural features of the fruiting bodies, this mechanism is widely accepted. However, only few studies reported experimental evidence suggesting the presence of spores in the air. Spore morphology alone does not allow species identification of myxomycetes. Consequently, airborne myxomycetes have been detected by cultivation of atmospheric particles (aerosols), which led to the growth of plasmodia and the formation of fruiting bodies (Brown et al. 1964). However, universal cultivation techniques have not been established for myxomycetes, and therefore, this method applies only to a minority of species. In this study, a culture-independent technique was applied in order to detect airborne myxomycetes and to characterize their seasonal distribution. We used the polymerase chain reaction (PCR) with primers for Didymiaceae and Physaraceae (Kamono and Fukui 2006). The PCR fragments obtained from aerosol samples were analyzed by denaturing gradient gel electrophoresis (DGGE), which is suitable for monitoring the dynamics of microbial community structure (Muyzer et al. 1993). For species identification of airborne myxomycetes, nucleotide sequences retrieved from DGGE bands were compared against sequences in the databases, including those of myxomycete fruiting bodies collected in this study.

Materials and methods Sampling Aerosol sampling was conducted as described previously (Kawamura et al. 2004) on the roof of the three-story building of the Institute of Low Temperature Science (43°03′ 56″ N, 141°21′27″ E), which is located on the campus of Hokkaido University, at ~15 m above ground level. Twentyone samples were collected from April 2001 to March 2002 and in April 2005, and the collection period for each sample was 2 or 3 days (see S1, Electronic supplementary material). After the sampling, each filter on which the aerosols had been collected was stored at −20°C in a clean glass bottle with a Teflon-lined screw cap until the analyses. Myxomycete fruiting body samples were collected on the campus of Hokkaido University and in Nopporo Forest Park and Uryu Experimental Forest (Hokkaido, Japan) in 2005 and 2006. The fruiting body samples were taxonomically identified mainly with reference to Yamamoto (1998, 2006) and Martin and Alexopoulos (1969; see S2, Electronic supplementary material). DNA preparation DNA extraction and purification from the fruiting body samples were performed as described previously (Kamono

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and Fukui 2006). With regard to the aerosol samples, DNA was extracted from a circular disk (diameter, 1.5 cm) taken from each of 21 stored filters. DNA preparation from the filter disks was carried out with the abovementioned procedure except for the first homogenization step. The filter disk samples were subjected to bead beating performed with 0.25 g (each) of 1.00-, 0.10-, and 0.05-mm baked glass beads using a Mikro-Dismembrator U (Braun Biotech, Germany). The pellets finally obtained by these procedures were resuspended in 20 μl (fruiting body samples) or 15 μl (filter disk samples) of Tris-EDTA buffer. Polymerase chain reaction PCR was conducted with the primers designed for the small subunit ribosomal RNA gene (SSU rDNA) of Didymiaceae and Physaraceae (Kamono and Fukui 2006). The primer pair phf1b/phr2b was used for the fruiting body samples, and the resultant PCR products were subjected to sequence analysis. The primer pair phf1b-GC/phr2b was used for the aerosol samples, and the resultant products were subjected to DGGE analysis. DGGE and nucleotide sequence analyses PCR products amplified from the aerosol samples were subjected to DGGE analysis. The procedures and conditions for DGGE, excision of bands, and reamplification were essentially as described previously (Kamono and Fukui 2006; Muyzer et al. 1996), except for the use of a 20–50% denaturant gradient. In the DGGE analysis of the samples collected from spring to summer (Fig. 1), approximately 300 ng of the PCR product was loaded onto each lane.

Fig. 1 DGGE analysis of PCR-amplified SSU rDNA fragments of Didymiaceae and Physaraceae in aerosols collected in the period from April through September, 2001. Lanes were marked with uppercase letters corresponding to the name of the aerosol samples (see S1, Electronic supplementary material). Markers are present on both sides of the gel. All marked bands were subjected to sequence analysis. Bands marked with the same symbols had identical sequences except for the bands indicated with a solid circle: open circle, as1; open square, as2; solid triangle, as3; open triangle, as4


PCR products from the fruiting body samples and reamplified products recovered from the DGGE bands were subjected to sequence analysis as described previously (Kamono and Fukui 2006). The obtained sequences were compared to sequences in a database constructed independently with the sequences of the fruiting body samples with the use of the stand-alone BLAST program. They were also compared to online databases with the BLAST program (Altschul et al. 1990) located at the NCBI website (www. ncbi.nlm.nih.gov). Nucleotide sequence accession numbers All nucleotide sequences were submitted to the DDBJ/EMBL/ GenBank database and were assigned accession numbers AB435320 to AB435363.

Results PCR products were obtained from all the aerosol samples except for sample S, which had been collected in January

Fig. 2 DGGE analysis of PCRamplified SSU rDNA fragments of Didymiaceae and Physaraceae in the aerosols collected in the period from May 2001 through March 2002. a DGGE gel image. Lanes were marked with uppercase letters corresponding to the names of the aerosol samples (see S1, Electronic supplementary material). Markers are present on both sides of the gel. All marked bands were subjected to sequence analysis. Bands marked with the same symbols had identical sequences except for bands indicated with a solid circle: open circle, as1; solid triangle, as3. b– e Fruiting body samples from which nucleotide sequences identical to those of the DGGE bands were derived: Didymium dubium (specimen no. AK-06007) (b); D. squamulosum (AK-06119) (c); D. saundersii (AK-06120) (d); and D. squamulosum (AK-06085) (e). Scale bars 1 mm

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(see S1, Electronic supplementary material). As for this sample, no product was detected by agarose gel electrophoresis despite additional amplification tests that were conducted by changing the amount of template DNA. Temporal changes in the DGGE banding patterns were almost negligible during April and May 2001 in comparison with the patterns in the following season (Fig. 1). Seasonal changes in the myxomycete composition of the aerosol samples were more apparent in the DGGE profiles of the additional samples, i.e., samples R and T (Fig. 2). Although a nucleotide sequence could not be obtained for all the detected bands, nine sequences were recovered from the aerosol samples. Sequence comparison suggested that all the obtained sequences were derived from myxomycetes (Table 1). Four sequences, i.e., as1, as5, as6, and as7, were identical to those of the fruiting bodies collected during the field research. Band as1 corresponded to Didymium dubium Rostaf. and the remaining bands to Didymium squamulosum (Alb. & Schwein.) Fr. (as5 and as6) and Diderma saundersii (Massee) Lado (as7). One more spring sample, i.e., sample U, was collected in April 2005 and analyzed by DGGE (data not shown). Two

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Table 1 Partial SSU rDNA sequences of the DGGE bands Name

as1 as2 as3 as4 as5 as6 as7 as8 as9 a b

Accession no.

AB435355 AB435356 AB435357 AB435358 AB435359 AB435360 AB435361 AB435362 AB435363

Closest relative Taxonomic name

Accession no.a

Didymium dubium Lepidoderma carestianum Leocarpus fragilis var. bisporus Lepidoderma carestianum Didymium squamulosum Didymium squamulosum Diderma saundersii Didymium dubium Lepidoderma carestianum

AB435325, AB435326, AB435327 AM231296b, AB435340b AB259485 AM231296b, AB435340b AB259432, AB259434, AB259436, AB435339 AB435338 AB435324 AM231295, AB435325, AB435326, AB435327 AM231296b, AB435340b

Similarity (%) 100 93 99 99 100 100 100 95 95

Accession numbers for the sequences obtained from the fruiting body samples of this study are in bold The sequence of the analyzed region is identical to that of Lepidoderma carestianum var. chailletii (AB259442 and AB259443)

bands were detected, and the sequence analysis showed that they corresponded to as1 and as2, which were also recognized in the spring samples of 2001 and 2002.

Discussion In this study, the seasonal changes in the composition of airborne myxomycetes were revealed using the PCR–DGGE profiles (Figs. 1 and 2). Moreover, similar DGGE profiles were observed for samples collected in the snow melting seasons of 2001, 2002 and 2005, highlighting the seasonal aspect of these changes. Among the four DGGE bands whose sequences were identical to those of known myxomycetes, band as1, corresponding to Didymium dubium, was observed only in spring. The fruiting bodies of this species have been reported to usually form on plant debris near melting snow in Japanese Islands (Yamamoto 1998). In fact, their occurrence in the snow melting period was confirmed by our field research. The remaining bands corresponding to D. squamulosum (as5 and as6) and D. saundersii (as7) were detected in sample P and/or Q (Fig. 2). These two species are known to form fruiting bodies from spring to autumn in the Japanese Islands (Yamamoto 1998). We found the fruiting bodies of D. squamulosum in July and August and D. saundersii in August. Hence, it appears that the difference in the fruiting period of each species was correlated with the seasonal changes in the myxomycete composition of the aerosols. This suggests that the newly formed spores are released and dispersed in the air. We could not identify the source of five DGGE bands (as2, as3, as4, as8, and as9). The main reason for this is that the myxomycete sequence databases are still incomplete. As shown in Fig. 2, band as3 was detected in two different seasons (spring and autumn). An explanation for this result is that the myxomycete species may form fruiting bodies during these two seasonally different periods. This result

can also be explained in terms of the time lag between the formation of spores and their detection in the air. For instance, spores may be suspended in the air for a long period or dispersed sometime after spore formation. The presence of airborne myxomycetes was confirmed with the use of a PCR-based approach. Exceptionally, no PCR product was obtained from Sample S, and therefore, airborne myxomycete spores would be small in number. As a preliminary quantitative experiment, a PCR detection test was performed using a Physarum bivalve Pers. spore suspension whose concentration was estimated (data not shown). A circular filter disk was spotted with each tenfold serial dilution of the suspension and then subjected to DNA extraction and PCR with the same procedure described for the analysis of aerosol samples. Approximately 100 spores could be detected as PCR-positive. Sample S was collected in January when snow accumulation was relatively high in Sapporo. Therefore, continuous snow cover on the ground may diminish or stop the supply of airborne myxomycetes, and this may explain why no amplification was observed in this sample. Another possible explanation is the filtering of airborne myxomycetes by snowfall. Our results provide evidence that newly formed myxomycete spores are dispersed by the wind. Considering that the aerosol samples were collected at ~15 m above the ground, it can be concluded that myxomycete spores are able to reach a certain height. This appears to be consistent with the findings that some species whose fruiting bodies normally occur on substrates such as decayed logs and dead leaves were found in tree canopies (Schnittler et al. 2006; Snell and Keller 2003). Although the germinating capacity of the spores could not be discussed on the basis of the results of this study, it appears that air dispersion of spores is an important strategy for myxomycetes to reach a suitable microhabitat. This study succeeded in detecting airborne myxomycetes and characterizing their seasonal distribution by molecular


ecological analyses of aerosols. This is an important step in understanding airborne myxomycete spore dispersal and their distribution patterns. Further application of such molecular ecological techniques will be quite useful for unveiling the ecological behavior of myxomycetes and will enhance our understanding of their specific role in the ecosystem. Acknowledgments We thank H. Hagiwara and Y. Kasahara for discussions or comments; Y. Higashioka, M. Fujii, M. Tsutsumi, and T. Nishi for assistance with field collections of fruiting body samples; M. Kobayashi, N. Tsubonuma, and K. Okuzawa for the help of aerosol sampling. We thank the Nopporo Forest Park, Hokkaido, Japan for permission to collect materials. This work was supported by a grant of Ministry of Education, Culture, Sports, Science and Technology to M.F. (16370014). The experiments comply with the current laws of the countries in which they were performed.

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