- Email: [email protected]
Molecular diversity of myxomycetes near Siegen (Germany)
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Short communication
Molecular diversity of myxomycetes near Siegen (Germany) Thomas Hoppe* Department of Natural Sciences and Engineering, Biology and Didactics, University of Siegen, Adolf-Reichwein-Str. 2, 57068 Siegen, Germany
article info
abstract
Article history:
Genetic variations of myxomycetes were seldom studied. Different probes were collected
Received 20 July 2012
in the general vicinity of the city of Siegen, Germany, with a special focus on Fuligo septica.
Received in revised form
Three different habitats around Siegen were screened for myxomycetes. Fuligo septica var.
7 November 2012
flava was examined for intraspecific variation using DNA sequence analysis. A 142 bp
Accepted 16 November 2012
region of the mitochondrial small subunit was analyzed. Although the sequence analysis
Available online 27 December 2012
did not strictly assign individuals to separate groups related to their geographical regions. Herewith I present the first molecular genetic tree revealing intraspecific variation that
Keywords: Geographical distribution
occurs within the morphospecies F. septica. ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Intraspecific variation mtDNA
Since the 15th century, the landscape around the Western German city of Siegen has been profoundly changed due to extraction of iron ore (Becker 1991), resulting in transformation of the original deciduous forest dominated by beech into a low timber forest, locally called Hauberg. The Hauberg forest has been coppiced every 15e20 years, resulting in a dominance of species resistant to coppice like oak (Quercus robur) and several species of birch (Betula). Since 1830, Hauberg timber forest has been replaced by plantations of spruce (Picea abies) and European beech (Fagus sylvatica). In order to sample characteristic forest types that form the current landscape, three different habitats were chosen (Obersetzen e OS, Faule Birke e FB and Oberholzklau e OK) and myxomycetes were collected in 2-day intervals from July to October 2011 (Table 1). In the study areas 1 and 2 oak is the prevailing tree, whereas in area 3 spruce also occurs as a dominant tree species (Fig. 1). Area 2 is a typical low forest without a distinctive shrub layer. This location is on a slope with a southern exposure. To allow comparisons between the locally collected specimens with those from some distance
away from Siegen, specimens of Fuligo collected in Madeira (Portugal) were included in the genetic analysis. The myxomycetes collected belong primarily to the order Physarales, and Fuligo septica var. flava (Pers.) Morgan turned out to be the most conspicuous species encountered (Fig. 2), since as its bright yellow plasmodium (i.e., an example of a phaneroplasmodia) is often more than 2 cm in diameter. Fuligo septica is known to grow near tanner yards on piles of rotting oak bark and for that reason its trivial name is “Lohblu¨te” (flowers of tan). The species concept in mycomycetes has been recently discussed by Fiore-Donno et al. (2009), Hoppe and Kutschera (2010) and Stephenson (2011). These organisms occur in many different habitats worldwide. These different habitats influence the morphology and complicate precise determination of some fruitings (Lister 1901; Alexopoulos 1969; Clark 2000; Stephenson 2011; Winsett and Stephenson 2011). The life cycle of these microorganisms involves two trophic stages (one haploid and the other diploid) and a reproductive stage in which meiosis takes place to restore the initial haploid trophic stage. The relatively large diploid plasmodium (the second of
* Tel.: þ49 271 740 3183. E-mail address: [email protected]. 1340-3540/$ e see front matter ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2012.11.001
310
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
Table 1 e Collections of Fuligo septica var. flava (specific geographical location and environmental conditions) used in the present study. Reference Number 47 47.2 47.3 47.4 412 452 458 459 462 466 467 469 471 482 494 501 502 520 525
Location F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F.
septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var.
flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava
Madeira (Portugal) Vo¨hl (Hessen, Germany) Madeira (Portugal) Vo¨hl (Hessen, Germany) Hohenebra (Thuringia, Germany) Obersetzen (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Oberholzklau (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Obersetzen (NRW, Germany) Obersetzen (NRW, Germany)
the two trophic stages) gives rise to spore-producing structures (fruiting bodies). After germination, spores develop into one to more unicellular myxamoebae (the first of the two tropic stages). The myxamoebae further divide via mitosis and are the major life form in the life cycle, albeit microscopic in size and easily overlooked. However, myxamoebae may fuse, producing non-identical mitotic cells due to crossover phenomena. A population of genetically very similar individuals can arise within short time. Further genetic divergencies may appear as some species of myxomycetes are characterized by both heterothallic lines and non-heterothallic lines (Wheals 1970; Clark and Haskins 2010). After sporegermination, two swarm cells of F. septica fuse to form a zygote (2n). This diploid cell starts to feed, which is a process
Coordinates Ma Vhl Ma Vhl AT OS FB FB FB FB FB FB FB FB OK FB FB OS OS
N51 130 /E8 540 N51 130 /E8 540 N51 320 86/E10 820 36 N50 930 70.1/E8 030 92.2 N50 830 90.7/E8 030 55.3 N50 830 91.8/E8 030 58.9 N50 030 63.7/E8 030 63.7 N50 080 39.2/E8 020 69.7 N50 830 94.3/E8 030 56.0 N50 830 92.9/E8 020 64.9 N50 830 92.1/E8 020 64.1 N50 830 98.9/E8 030 50.5 N50 550 30.8/E7 560 40.0 N50 500 35.3/E8 020 24.1 N50 500 35.5/E8 020 24.1 N50 560 70.4/E8 020 65.5 N50 560 70.3/E8 020 69.6
Collection date 2007 2003 2007 2003 27.07.2010 08.05.2011 10.07.2011 10.07.2011 10.07.2011 31.08.2011 31.08.2011 03.09.2011 03.09.2011 05.09.2011 17.09.2011 18.09.2011 18.09.2011 15.10.2011 15.10.2011
NCBI-number JX125020 JX125021 JX125022 JX125023 JX125024 JX125025 JX125026 JX125027 JX125028 JX125029 JX125030 JX125031 JX125032 JX125033 JX125034 JX125035 JX125036 JX125037 JX125038
that initiates mitosis (first karyogenesis and then some time later cytokinesis). As a result, a multinucleate, amorphous plasmodium develops. It feeds on bacteria and other microorganisms associated with decaying organic material. Stimulated by endogenous and exogenous signals, the mature diploid plasmodium produces fruiting bodies. Fuligo septica and the varieties that have been recognized are distributed worldwide. The small size of the spores (a mean size of about 10 mm) allows long-distance wind dispersal (Schnittler and Tesmer 2008; Stephenson et al. 2008). Given a few exceptions, the data presented herein show geographical incongruity. We analyzed local populations of a cosmopolitan morphospecies and could show heterogeneous sequences at the investigated locus.
Fig. 1 e Map of investigated locations around Siegen, Germany. A: Map of Germany. B: Map of the investigated areas: 1, Faule Birke; 2, Obersetzen; 3, Oberholzklau.
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
311
Fig. 2 e Fruiting body of Fuligo septica var. flava. A: Morphology of an aethalium. B: SEM picture of a single spore.
Field expeditions were carried out during summer and autumn of 2011. Our study areas were mainly three forests around Siegen, Germany (N 50 520 ; E 8 020 ; Fig. 1). More than 70 myxomycetes were collected, including 14 individuals of F. septica var. flava. Newly collected myxomycetes and material from different collections from other locations were included in our molecular analysis (Table 1). All samples were air-dried and stored in cardboard or PS-boxes at room temperature. To perform deoxyribonucleic-acid (DNA) extractions, spores were shredded by a homogenizer (Ribolyzer, Hybaid Ashford) first. We incubated the solution with Proteinase K at 50 C for 24 h. In the following process, the material was prepared according to the manufacturer’s protocol of the DNeasy plant mini kit (Qiagen, Germany). A variable region of the mitochondrial genome (mtDNA) was amplified. Primers were used for a region of the mtDNA according to Winsett and Stephenson (2011) and synthesized new ones (Table 2). The PCR protocol consisted of an initial step of 94 C for 4 min and 42 repetitions, denaturation (94 C) for 45 s, primer-linking (40 C) for 90 s and elongation (72 C) for 90 s, with a final elongation for 6 min. The amplification was examined by gel electrophoresis. All PCR amplifications were sequenced by SEQLAB (Germany) on an ABI 310 (Applied Biosystems, USA). They were aligned electronically using MEGA 5. New sequences have been deposited in the GenBank database (Table 1). Phylogenetic trees were constructed using maximum likelihood and Bayesian interference using the
Table 2 e A variable region of the mitochondrial genome (mtDNA). Primers were used for a region of the mtDNA according to Winsett and Stephenson (2011) and synthesized new ones. Primer
Sequence (50 -30 )
mtCore1 TAG TGT TAT TCG TGA TGA CT mtCore2 CTC GAA TTA AAC CAC AT C3F C5F C4R
Reference Winsett and Stephenson 2011 Winsett and Stephenson 2011 In this study
TTC ACT GAT TAT TTT GAG TGC WAR AGA CG ACC TCT TTA ATC TGC GTT CAC In this study GTT TCK CGC GTT ARC TTY AAA CC In this study
methodology described by Fiore-Donno et al. (2005, 2008). Sequences were aligned by using Muscle in MEGA 5. Different molecular trees were constructed with homologous results (Fig. 3). A total of 207 sites of the mtSSU were analyzed, but only 142 nucleotides were considered in the construction of the molecular tree. In addition, individuals from other countries were used, including two collections from Kassel (Germany) and two collections from Madeira (Portugal, Mu¨ller 2006a). The amplified region was 780 bp apart from the start of the gene (by reference to GenBank: X75591.1). Based on these sequences, three clades could be distinguished with heterogeneous affiliation. One singleton haplotype was detected for each area. The second clade was represented by only one collection from Obersetzen (Area 2, Table 2). The third clade includes 8 sequences from all areas. The nucleotide diversity was 0.168767 (Table 3). The mtDNA investigation showed the presence of three different groups in the study. Clade 1 is represented by seven isolates from all investigated areas in Germany and consists of several subgroups, whereas Clade 3 is represented by one group of six isolates from a single stand, the Faule Birke area (Fig. 2). In this clade, only one nucleotide sequence is separated from the main group (remaining sequences) by eleven nucleotides. Clade 2 is only represented by a single isolate. The molecular data identified a distribution pattern of F. septica var. flava in local geographically distinct regions. It showed that individuals from other distant localities are also molecular-genetically distinct from the Siegen-Fuligo-group (SFG). But genetic divergence is not related to geographic distance to SFG as the Madeira specimen showed no significant differences to the Kassel specimens. There is no consistency in relation to the investigated isolates. Clade 3 is genetically very distended to the two other clades but consists up to now only of one sequence type. Further studies could show more individuals of this group. The mtDNA region examined herein shows more resemblance between Siegen myxomycetes and those found near Kassel than those specimens found in Madeira island (Table 4). This feature may be explicated by dispersal mode of most myxomycetes, although empiric data is limited. Wind distribution (autochory) has been experimentally tested and likely the preferential method for spreading the spores of many
312
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
Fig. 3 e Molecular-genetic analysis by ML of mtDNA-sequences. The maximum likelihood method based on the Kimura 2parameter model was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 19 nucleotide sequences. There were a total of 142 positions in the final dataset (Vhl e Vo¨hl; FB e Faule Birke; OS e Obersetzen; AT e Ahnatal; OK e Oberholzklau; Ma e Madeira).
species (Schnittler and Tesmer 2008), even though zoochory may be an important mechanism on a local or regional scale (Russell 1979; Mu¨ller 2006b). Whether distribution by another organism is a specific form of the spore-distribution processes still has to be elucidated. The low nucleotide variation revealed already relatively significant changes have taken place, whereas numerically low differences demonstrate an accidental resemblance between some clades. Future investigations should include extended fragments of more individuals. Fuligo septica is a widespread myxomycete with relatively large fruiting bodies. The description of the microhabitat is not well-known. Likewise, a huge number of spores, which
can disperse easily, are a distinct feature of Fuligo and the species is likely to be one in which the spores are dispersed mainly by a wind. Winsett and Stephenson (2008, 2011) also found different genetic types of the same species in adjacent locations when studying Didymium. The data presented above are geographically incongruous with isolates of a cosmopolitan morphospecies at the mtDNA locus. The arboreous landscape around Siegen is strongly fragmented. Therefore, the distribution of many different types could be found there. Changes could assert themselves rapidly by partial isolation in a confined region. In addition, the reproduction strategy may be a possible reason for formation of different genetic types. It is possible that further investigations will identify other genetic types of F. septica and other myxomycetes species in isolated habitats.
Table 3 e Results from Tajima’s Neutrality Test. The analysis involved 19 nucleotide sequences. There were a total of 142 positions in the final dataset. m
S
ps
Q
p
D
19
91
0.640845
0.183355
0.168767
0.329662
Abbreviations: m ¼ number of sequences, S ¼ Number of segregating sites, ps ¼ S/m, Q ¼ ps/a1, p ¼ nucleotide diversity, and D is the Tajima test statistic.
Table 4 e Relative distances to the main area (FB) in km. Obersetzen_ Oberholzklau_ Vo¨hl_ Madeira_ OS OK Vhl Ma Faule Birke_FB
12
10
150
2885
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
Disclosure There are no conflicts of interests. All the experiments carried out in this study comply with the current laws of Japan.
Acknowledgments I thank SL Stephenson (University of Arkansas), A. Feest (University of Bristol), Ch. Kuschal (NIH), A. Kupfer (University of Potsdam) and other colleagues for their helpful remarks on an earlier version of the manuscript.
references
Alexopoulos CJ, 1969. The experimental approach to the taxonomy of the myxomycetes. Mycologia 61: 219e239. Becker A, 1991. Der Siegerla¨nder Hauberg. Vergangenheit, Gegenwart und Zukunft der Waldwirtschaftsform. Verlag die Waldschmiede, Kreuztal. Clark J, 2000. The species problem in the myxomycetes. Stapfia 73: 39e53. Clark J, Haskins EF, 2010. Reproductive systems in the myxomycetes: a review. Mycosphere 1: 337e353. Fiore-Donno AM, Berney C, Pawlowski J, Baldauf SL, 2005. Higherorder phylogeny of plasmodial slime molds (Myxogastria) based on EF1A and SSU rRNA sequences. Journal of Eukaryotic Microbiology 52: 201e210.
313
Fiore-Donno AM, Meyer M, Baldauf SL, Pawlowsi J, 2008. Evolution of dark-spored Myxomycetes (slime-molds): molecules versus morphology. Molecular Phylogenetic Evolution 46: 878e889. Fiore-Donno AM, Nikolaev SI, Nelson M, Pawlowski J, CavalierSmith T, Baldauf SL, 2009. Deep phylogeny and evolution of slime moulds (mycetozoa). Protist 161: 55e70. Hoppe T, Kutschera U, 2010. In the shadow of Darwin: Anton de Bary’s origin of myxomycetology and a molecular phylogeny of the plasmodial slime molds. Theory Bioscience 129: 15e23. Lister A, 1901. Notes on mycetozoa. Journal of Botany 39: 81e91. Mu¨ller H, 2006a. Assoziationen von Coleoptera mit Myxomycetes in Thu¨ringen. Entomologische Zeitschrift 116: 75e78. Mu¨ller H, 2006b. Myxomycetenfunde auf der Insel Madeira (Port.). Zeitschrift fu¨r Mykologie 72: 35e46. Russell LK, 1979. Beetles associated with slime molds (Mycetozoa) in Oregon and California (Coleoptera: Leiodidae, Sphindidae, Lathridiidae). Pan-Pacific Entomologist 55: 1e9. Schnittler M, Tesmer J, 2008. A habitat colonization model for spore-dispersed organisms e does it work eumycetozoans? Mycological Research 112: 697e707. Stephenson SL, Schnittler M, Novozhilov YK, 2008. Myxomycete diversity and distribution from the fossil record to the present. Biodiversity and Conservation 17: 285e301. Stephenson SL, 2011. From morphological to molecular: studies of myxomycetes since the publication of the Martin and Alexopoulos (1969) monograph. Fungal Diversity 50: 21e34. Wheals AE, 1970. A homothallic strain of the myxomycete Physarum polycephlum. Genetics 66: 623e633. Winsett KE, Stephenson SL, 2008. Using ITS sequences to assess intraspecific genetic relationships among geographically separated collections of the myxomycete Didymium squamulosum. Revista Mexicana de Micologia 27: 59e65. Winsett KE, Stephenson SL, 2011. Global distribution and molecular diversity of Didymium difforme. Mycospehere 2: 135e146.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Short communication
Molecular diversity of myxomycetes near Siegen (Germany) Thomas Hoppe* Department of Natural Sciences and Engineering, Biology and Didactics, University of Siegen, Adolf-Reichwein-Str. 2, 57068 Siegen, Germany
article info
abstract
Article history:
Genetic variations of myxomycetes were seldom studied. Different probes were collected
Received 20 July 2012
in the general vicinity of the city of Siegen, Germany, with a special focus on Fuligo septica.
Received in revised form
Three different habitats around Siegen were screened for myxomycetes. Fuligo septica var.
7 November 2012
flava was examined for intraspecific variation using DNA sequence analysis. A 142 bp
Accepted 16 November 2012
region of the mitochondrial small subunit was analyzed. Although the sequence analysis
Available online 27 December 2012
did not strictly assign individuals to separate groups related to their geographical regions. Herewith I present the first molecular genetic tree revealing intraspecific variation that
Keywords: Geographical distribution
occurs within the morphospecies F. septica. ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Intraspecific variation mtDNA
Since the 15th century, the landscape around the Western German city of Siegen has been profoundly changed due to extraction of iron ore (Becker 1991), resulting in transformation of the original deciduous forest dominated by beech into a low timber forest, locally called Hauberg. The Hauberg forest has been coppiced every 15e20 years, resulting in a dominance of species resistant to coppice like oak (Quercus robur) and several species of birch (Betula). Since 1830, Hauberg timber forest has been replaced by plantations of spruce (Picea abies) and European beech (Fagus sylvatica). In order to sample characteristic forest types that form the current landscape, three different habitats were chosen (Obersetzen e OS, Faule Birke e FB and Oberholzklau e OK) and myxomycetes were collected in 2-day intervals from July to October 2011 (Table 1). In the study areas 1 and 2 oak is the prevailing tree, whereas in area 3 spruce also occurs as a dominant tree species (Fig. 1). Area 2 is a typical low forest without a distinctive shrub layer. This location is on a slope with a southern exposure. To allow comparisons between the locally collected specimens with those from some distance
away from Siegen, specimens of Fuligo collected in Madeira (Portugal) were included in the genetic analysis. The myxomycetes collected belong primarily to the order Physarales, and Fuligo septica var. flava (Pers.) Morgan turned out to be the most conspicuous species encountered (Fig. 2), since as its bright yellow plasmodium (i.e., an example of a phaneroplasmodia) is often more than 2 cm in diameter. Fuligo septica is known to grow near tanner yards on piles of rotting oak bark and for that reason its trivial name is “Lohblu¨te” (flowers of tan). The species concept in mycomycetes has been recently discussed by Fiore-Donno et al. (2009), Hoppe and Kutschera (2010) and Stephenson (2011). These organisms occur in many different habitats worldwide. These different habitats influence the morphology and complicate precise determination of some fruitings (Lister 1901; Alexopoulos 1969; Clark 2000; Stephenson 2011; Winsett and Stephenson 2011). The life cycle of these microorganisms involves two trophic stages (one haploid and the other diploid) and a reproductive stage in which meiosis takes place to restore the initial haploid trophic stage. The relatively large diploid plasmodium (the second of
* Tel.: þ49 271 740 3183. E-mail address: [email protected]. 1340-3540/$ e see front matter ª 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2012.11.001
310
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
Table 1 e Collections of Fuligo septica var. flava (specific geographical location and environmental conditions) used in the present study. Reference Number 47 47.2 47.3 47.4 412 452 458 459 462 466 467 469 471 482 494 501 502 520 525
Location F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F.
septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var. septica var.
flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava flava
Madeira (Portugal) Vo¨hl (Hessen, Germany) Madeira (Portugal) Vo¨hl (Hessen, Germany) Hohenebra (Thuringia, Germany) Obersetzen (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Oberholzklau (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Faule Birke/Eisern (NRW, Germany) Obersetzen (NRW, Germany) Obersetzen (NRW, Germany)
the two trophic stages) gives rise to spore-producing structures (fruiting bodies). After germination, spores develop into one to more unicellular myxamoebae (the first of the two tropic stages). The myxamoebae further divide via mitosis and are the major life form in the life cycle, albeit microscopic in size and easily overlooked. However, myxamoebae may fuse, producing non-identical mitotic cells due to crossover phenomena. A population of genetically very similar individuals can arise within short time. Further genetic divergencies may appear as some species of myxomycetes are characterized by both heterothallic lines and non-heterothallic lines (Wheals 1970; Clark and Haskins 2010). After sporegermination, two swarm cells of F. septica fuse to form a zygote (2n). This diploid cell starts to feed, which is a process
Coordinates Ma Vhl Ma Vhl AT OS FB FB FB FB FB FB FB FB OK FB FB OS OS
N51 130 /E8 540 N51 130 /E8 540 N51 320 86/E10 820 36 N50 930 70.1/E8 030 92.2 N50 830 90.7/E8 030 55.3 N50 830 91.8/E8 030 58.9 N50 030 63.7/E8 030 63.7 N50 080 39.2/E8 020 69.7 N50 830 94.3/E8 030 56.0 N50 830 92.9/E8 020 64.9 N50 830 92.1/E8 020 64.1 N50 830 98.9/E8 030 50.5 N50 550 30.8/E7 560 40.0 N50 500 35.3/E8 020 24.1 N50 500 35.5/E8 020 24.1 N50 560 70.4/E8 020 65.5 N50 560 70.3/E8 020 69.6
Collection date 2007 2003 2007 2003 27.07.2010 08.05.2011 10.07.2011 10.07.2011 10.07.2011 31.08.2011 31.08.2011 03.09.2011 03.09.2011 05.09.2011 17.09.2011 18.09.2011 18.09.2011 15.10.2011 15.10.2011
NCBI-number JX125020 JX125021 JX125022 JX125023 JX125024 JX125025 JX125026 JX125027 JX125028 JX125029 JX125030 JX125031 JX125032 JX125033 JX125034 JX125035 JX125036 JX125037 JX125038
that initiates mitosis (first karyogenesis and then some time later cytokinesis). As a result, a multinucleate, amorphous plasmodium develops. It feeds on bacteria and other microorganisms associated with decaying organic material. Stimulated by endogenous and exogenous signals, the mature diploid plasmodium produces fruiting bodies. Fuligo septica and the varieties that have been recognized are distributed worldwide. The small size of the spores (a mean size of about 10 mm) allows long-distance wind dispersal (Schnittler and Tesmer 2008; Stephenson et al. 2008). Given a few exceptions, the data presented herein show geographical incongruity. We analyzed local populations of a cosmopolitan morphospecies and could show heterogeneous sequences at the investigated locus.
Fig. 1 e Map of investigated locations around Siegen, Germany. A: Map of Germany. B: Map of the investigated areas: 1, Faule Birke; 2, Obersetzen; 3, Oberholzklau.
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
311
Fig. 2 e Fruiting body of Fuligo septica var. flava. A: Morphology of an aethalium. B: SEM picture of a single spore.
Field expeditions were carried out during summer and autumn of 2011. Our study areas were mainly three forests around Siegen, Germany (N 50 520 ; E 8 020 ; Fig. 1). More than 70 myxomycetes were collected, including 14 individuals of F. septica var. flava. Newly collected myxomycetes and material from different collections from other locations were included in our molecular analysis (Table 1). All samples were air-dried and stored in cardboard or PS-boxes at room temperature. To perform deoxyribonucleic-acid (DNA) extractions, spores were shredded by a homogenizer (Ribolyzer, Hybaid Ashford) first. We incubated the solution with Proteinase K at 50 C for 24 h. In the following process, the material was prepared according to the manufacturer’s protocol of the DNeasy plant mini kit (Qiagen, Germany). A variable region of the mitochondrial genome (mtDNA) was amplified. Primers were used for a region of the mtDNA according to Winsett and Stephenson (2011) and synthesized new ones (Table 2). The PCR protocol consisted of an initial step of 94 C for 4 min and 42 repetitions, denaturation (94 C) for 45 s, primer-linking (40 C) for 90 s and elongation (72 C) for 90 s, with a final elongation for 6 min. The amplification was examined by gel electrophoresis. All PCR amplifications were sequenced by SEQLAB (Germany) on an ABI 310 (Applied Biosystems, USA). They were aligned electronically using MEGA 5. New sequences have been deposited in the GenBank database (Table 1). Phylogenetic trees were constructed using maximum likelihood and Bayesian interference using the
Table 2 e A variable region of the mitochondrial genome (mtDNA). Primers were used for a region of the mtDNA according to Winsett and Stephenson (2011) and synthesized new ones. Primer
Sequence (50 -30 )
mtCore1 TAG TGT TAT TCG TGA TGA CT mtCore2 CTC GAA TTA AAC CAC AT C3F C5F C4R
Reference Winsett and Stephenson 2011 Winsett and Stephenson 2011 In this study
TTC ACT GAT TAT TTT GAG TGC WAR AGA CG ACC TCT TTA ATC TGC GTT CAC In this study GTT TCK CGC GTT ARC TTY AAA CC In this study
methodology described by Fiore-Donno et al. (2005, 2008). Sequences were aligned by using Muscle in MEGA 5. Different molecular trees were constructed with homologous results (Fig. 3). A total of 207 sites of the mtSSU were analyzed, but only 142 nucleotides were considered in the construction of the molecular tree. In addition, individuals from other countries were used, including two collections from Kassel (Germany) and two collections from Madeira (Portugal, Mu¨ller 2006a). The amplified region was 780 bp apart from the start of the gene (by reference to GenBank: X75591.1). Based on these sequences, three clades could be distinguished with heterogeneous affiliation. One singleton haplotype was detected for each area. The second clade was represented by only one collection from Obersetzen (Area 2, Table 2). The third clade includes 8 sequences from all areas. The nucleotide diversity was 0.168767 (Table 3). The mtDNA investigation showed the presence of three different groups in the study. Clade 1 is represented by seven isolates from all investigated areas in Germany and consists of several subgroups, whereas Clade 3 is represented by one group of six isolates from a single stand, the Faule Birke area (Fig. 2). In this clade, only one nucleotide sequence is separated from the main group (remaining sequences) by eleven nucleotides. Clade 2 is only represented by a single isolate. The molecular data identified a distribution pattern of F. septica var. flava in local geographically distinct regions. It showed that individuals from other distant localities are also molecular-genetically distinct from the Siegen-Fuligo-group (SFG). But genetic divergence is not related to geographic distance to SFG as the Madeira specimen showed no significant differences to the Kassel specimens. There is no consistency in relation to the investigated isolates. Clade 3 is genetically very distended to the two other clades but consists up to now only of one sequence type. Further studies could show more individuals of this group. The mtDNA region examined herein shows more resemblance between Siegen myxomycetes and those found near Kassel than those specimens found in Madeira island (Table 4). This feature may be explicated by dispersal mode of most myxomycetes, although empiric data is limited. Wind distribution (autochory) has been experimentally tested and likely the preferential method for spreading the spores of many
312
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
Fig. 3 e Molecular-genetic analysis by ML of mtDNA-sequences. The maximum likelihood method based on the Kimura 2parameter model was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 19 nucleotide sequences. There were a total of 142 positions in the final dataset (Vhl e Vo¨hl; FB e Faule Birke; OS e Obersetzen; AT e Ahnatal; OK e Oberholzklau; Ma e Madeira).
species (Schnittler and Tesmer 2008), even though zoochory may be an important mechanism on a local or regional scale (Russell 1979; Mu¨ller 2006b). Whether distribution by another organism is a specific form of the spore-distribution processes still has to be elucidated. The low nucleotide variation revealed already relatively significant changes have taken place, whereas numerically low differences demonstrate an accidental resemblance between some clades. Future investigations should include extended fragments of more individuals. Fuligo septica is a widespread myxomycete with relatively large fruiting bodies. The description of the microhabitat is not well-known. Likewise, a huge number of spores, which
can disperse easily, are a distinct feature of Fuligo and the species is likely to be one in which the spores are dispersed mainly by a wind. Winsett and Stephenson (2008, 2011) also found different genetic types of the same species in adjacent locations when studying Didymium. The data presented above are geographically incongruous with isolates of a cosmopolitan morphospecies at the mtDNA locus. The arboreous landscape around Siegen is strongly fragmented. Therefore, the distribution of many different types could be found there. Changes could assert themselves rapidly by partial isolation in a confined region. In addition, the reproduction strategy may be a possible reason for formation of different genetic types. It is possible that further investigations will identify other genetic types of F. septica and other myxomycetes species in isolated habitats.
Table 3 e Results from Tajima’s Neutrality Test. The analysis involved 19 nucleotide sequences. There were a total of 142 positions in the final dataset. m
S
ps
Q
p
D
19
91
0.640845
0.183355
0.168767
0.329662
Abbreviations: m ¼ number of sequences, S ¼ Number of segregating sites, ps ¼ S/m, Q ¼ ps/a1, p ¼ nucleotide diversity, and D is the Tajima test statistic.
Table 4 e Relative distances to the main area (FB) in km. Obersetzen_ Oberholzklau_ Vo¨hl_ Madeira_ OS OK Vhl Ma Faule Birke_FB
12
10
150
2885
m y c o s c i e n c e 5 4 ( 2 0 1 3 ) 3 0 9 e3 1 3
Disclosure There are no conflicts of interests. All the experiments carried out in this study comply with the current laws of Japan.
Acknowledgments I thank SL Stephenson (University of Arkansas), A. Feest (University of Bristol), Ch. Kuschal (NIH), A. Kupfer (University of Potsdam) and other colleagues for their helpful remarks on an earlier version of the manuscript.
references
Alexopoulos CJ, 1969. The experimental approach to the taxonomy of the myxomycetes. Mycologia 61: 219e239. Becker A, 1991. Der Siegerla¨nder Hauberg. Vergangenheit, Gegenwart und Zukunft der Waldwirtschaftsform. Verlag die Waldschmiede, Kreuztal. Clark J, 2000. The species problem in the myxomycetes. Stapfia 73: 39e53. Clark J, Haskins EF, 2010. Reproductive systems in the myxomycetes: a review. Mycosphere 1: 337e353. Fiore-Donno AM, Berney C, Pawlowski J, Baldauf SL, 2005. Higherorder phylogeny of plasmodial slime molds (Myxogastria) based on EF1A and SSU rRNA sequences. Journal of Eukaryotic Microbiology 52: 201e210.
313
Fiore-Donno AM, Meyer M, Baldauf SL, Pawlowsi J, 2008. Evolution of dark-spored Myxomycetes (slime-molds): molecules versus morphology. Molecular Phylogenetic Evolution 46: 878e889. Fiore-Donno AM, Nikolaev SI, Nelson M, Pawlowski J, CavalierSmith T, Baldauf SL, 2009. Deep phylogeny and evolution of slime moulds (mycetozoa). Protist 161: 55e70. Hoppe T, Kutschera U, 2010. In the shadow of Darwin: Anton de Bary’s origin of myxomycetology and a molecular phylogeny of the plasmodial slime molds. Theory Bioscience 129: 15e23. Lister A, 1901. Notes on mycetozoa. Journal of Botany 39: 81e91. Mu¨ller H, 2006a. Assoziationen von Coleoptera mit Myxomycetes in Thu¨ringen. Entomologische Zeitschrift 116: 75e78. Mu¨ller H, 2006b. Myxomycetenfunde auf der Insel Madeira (Port.). Zeitschrift fu¨r Mykologie 72: 35e46. Russell LK, 1979. Beetles associated with slime molds (Mycetozoa) in Oregon and California (Coleoptera: Leiodidae, Sphindidae, Lathridiidae). Pan-Pacific Entomologist 55: 1e9. Schnittler M, Tesmer J, 2008. A habitat colonization model for spore-dispersed organisms e does it work eumycetozoans? Mycological Research 112: 697e707. Stephenson SL, Schnittler M, Novozhilov YK, 2008. Myxomycete diversity and distribution from the fossil record to the present. Biodiversity and Conservation 17: 285e301. Stephenson SL, 2011. From morphological to molecular: studies of myxomycetes since the publication of the Martin and Alexopoulos (1969) monograph. Fungal Diversity 50: 21e34. Wheals AE, 1970. A homothallic strain of the myxomycete Physarum polycephlum. Genetics 66: 623e633. Winsett KE, Stephenson SL, 2008. Using ITS sequences to assess intraspecific genetic relationships among geographically separated collections of the myxomycete Didymium squamulosum. Revista Mexicana de Micologia 27: 59e65. Winsett KE, Stephenson SL, 2011. Global distribution and molecular diversity of Didymium difforme. Mycospehere 2: 135e146.