Aquatic hyphomycetes occur worldwide on a wide range of plant substrates decomposing in freshwaters, and are known to play a key role in organic matter turnover. The presumed worldwide distribution of many aquatic hyphomycete species has been based on morphology-based taxonomy and identification, which may overlook cryptic species, and mask global-scale biogeographical patterns. This might be circumvented by using DNA sequence data. The internal transcribed spacer (ITS) region from rDNA was recently designated as the most suitable barcode for fungal identification. In this study, we generated ITS barcodes of 130 isolates belonging to 6 aquatic hyphomycete species (Anguillospora filiformis, Flagellospora penicillioides, Geniculospora grandis, Lunulospora curvula, Tetrachaetum elegans and Tricladium chaetocladium), and collected from streams of Southwest Europe (86 isolates) and East Australia (44 isolates). European and Australian populations of 4 species (A. filiformis, F. penicillioides, G. grandis and T. elegans) grouped into different clades, and molecular diversity indices supported significant differentiation. Continents did not share haplotypes, except for T. chaetocladium. Overall results show substantial population diversity for all tested species and suggests that the biogeography of aquatic hyphomycetes may be species-specific.
Citation: Duarte S, Seena S, Bärlocher F, Cássio F, Pascoal C (2012) Preliminary Insights into the Phylogeography of Six Aquatic Hyphomycete Species. PLoS ONE7(9): e45289. https://doi.org/10.1371/journal.pone.0045289
Editor: Jason E. Stajich, University of California Riverside, United States of America
Received: June 4, 2012; Accepted: August 14, 2012; Published: September 18, 2012
Copyright: © Duarte et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The European Regional Development Fund - Operational Competitiveness Programme (FEDER-POFC-COMPETE) and the Portuguese Foundation for Science and Technology supported this study (PEst-C/BIA/UI4050/2011 and PTDC/AAC-AMB/113746/2009) and SD (SFRH/BPD/47574/2008). The Natural Sciences and Engineering Research Council of Canada (NSERC) also supported this study and FB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Most large eukaryotic plants and animal species have relatively narrow geographic distributions. By contrast, it has long been assumed that prokaryotic microorganisms occur essentially everywhere and will grow and reproduce whenever conditions are suitable: “everything is everywhere, but the environment selects” . Fenchel and Finlay  and Finlay and Fenchel  extended the assumption of global distribution to eukaryotic microorganisms. They postulated a transition from ubiquity to biogeography at a body size between 1 and 10 mm. Many morphospecies (taxonomy based on morphological differences) of small aquatic animals and protists are indeed very widely distributed. However, molecular genetic evidence has shown that reliance on morphological data inflates the range of geographical distribution of many microorganisms, including prokaryotes, diatoms and protists . Taylor et al.  reached the same conclusion concerning fungi. For instance, strains of Neurospora discreta were resolved into several distinct phylogenetic species with different geographic distributions , . The genus Lentinula was thought to have 4 morphologically distinct species , but molecular analyses revealed that only 1 of the 4 was a single phylogenetic species. Each of the other 3 contained 2 phylogenetic species, all with more restricted geographic distributions . Among fungi, Aspergillus fumigatus seems to be an exception. Even though the single morphospecies was split into two phylospecies, both exhibited global distribution .
Aquatic hyphomycetes occur worldwide on a wide range of plant substrates, such as leaves and wood, decomposing in freshwaters, and are known to play a key role in organic matter turnover , . Phylogenetically, most species are affiliated with the Ascomycota . Historically, their classification has been based on the morphology and development of conidia (asexually produced spores). Their predominantly tetraradiate or sigmoid shapes suggest convergent evolution due to the common need for attachment to a suitable substrate in flowing water . Molecular data  support and extend the phylogenetic heterogeneity suggested by morphologies of the teleomorphs (the sexual reproductive stages) .
Many species of aquatic hyphomycetes appear to be cosmopolitan; others are more common in certain climatic zones regardless of longitude. Wood-Eggenschwiler and Bärlocher  concluded that species occurrence is rarely restricted by geological barriers or by distance. On a worldwide scale, temperature, together with its influence on vegetation has been suggested as the main factor determining their distribution. The observation of identical morphospecies on geologically young islands far from mainlands, such as the Hawaiian Islands , suggests efficient long-distance transport of viable inocula. However, these conclusions are based on morphospecies, which ignores cryptic species whose characterization requires molecular data. In the few phylogenetic studies on this group of fungi no conclusions concerning biogeography were drawn due to the lack of isolates from diverse and distant geographic areas , , , , . By chance, in a study conducted by Seena et al.  the genotypes of Articulospora tetracladia were found to be geographically widespread with the exception of Malaysian isolates. In the largest study to date, Anderson and Shearer  observed high genotypic diversity among 391 isolates of Tetracladium marchalianum from rivers in Illinois and Wisconsin. Stable genetic differentiation was only observed between the most distant rivers (∼ 450 km). Analysis of partial β-tubulin sequence data suggested that all isolates belonged to a single species, and did not support the existence of cryptic species within T. marchalianum.
To provide further insight into the biogeography of aquatic hyphomycetes, we analyzed the internal transcribed spacer (ITS) sequences of 6 species isolated from streams in Southwest Europe and East Australia. We hypothesized that strains of these 6 morphospecies would exhibit very distinct biogeographic patterns due to the distance between the two continents and the lack of any landbridge. Although little is known about the evolutionary origins of aquatic hyphomycetes, freshwater Ascomycota are believed to have originated about 390 million years ago , . Continental drift separated Australia from what was left of Pangea ca. 130 million years ago; in the absence of frequent exchange between the two continents, this should provide ample time for genetic (and morphological) differentiation. We selected the ITS region, which shows considerable variation among and within aquatic hyphomycete species , ,  and was recently declared the most suitable barcode for fungal species (http://www.ecbol.org). The selected taxa (Anguillospora filiformis, Flagellospora penicillioides, Geniculospora grandis, Lunulospora curvula, Tetrachaetum elegans and Tricladium chaetocladium) are commonly found on decomposing plant-litter in freshwaters , , .
Materials and Methods
The dataset consists of 130 ITS sequences belonging to 6 aquatic hyphomycete species, A. filiformis, F. penicillioides, G. grandis, L. curvula, T. elegans and T. chaetocladium, sampled from streams of Europe (86 isolates) and Australia (44 isolates) between 2009 and 2011 and separated by ca. 18000 km. The European isolates originated in continental Portugal (58), the Azores archipelago (7), Spain (19) and Italy (2). All isolates got in this study were deposited in the culture collection of the Centre of Molecular and Environmental Biology (CBMA), Department of Biology of the University of Minho prior to sequencing. Eighteen ITS sequences of the Portuguese isolates were obtained in a previous study  and had already been deposited in the NCBI. Table S1 shows an overview of all sequences used in our analyses.
Sampling, Isolation and Culture Conditions
Isolates are maintained in the culture collection of the Centre of Molecular and Environmental Biology (CBMA), Department of Biology of the University of Minho. The methodology of fungal isolation is described in Pascoal et al. . All cultures were grown at room temperature on 1% malt agar extract during ca. 15 days before DNA extraction.
DNA was extracted with the MoBio Ultraclean Soil DNA Isolation kit according to the manufacturer’s instructions and stored at −20°C. For PCR reactions, 14 µL of Accuzyme mix (2x) (Bioline), ITS1 and ITS4 primers (1.6 µM) , MgCl2 (3 mM) and 2 µL of DNA (1–10 ng µL−1) were used in a final volume of 25 µL. PCR reactions were carried out in a Doppio thermocycler (VWR) as follows: 1) initial denaturation for 2 minutes at 94°C; 2) 40 cycles of denaturation for 45 seconds at 94°C; annealing for 45 seconds at 55°C and extension for 1 minute and 30 seconds at 72°C and 3) final elongation for 10 minutes at 72°C. The PCR products were run on a 2% agarose gel at 80V for 45 minutes to check the presence of the desired band. The PCR products were cleaned with a PureLink™ PCR purification Kit according to the manufacturer’s instructions (Invitrogen) and DNA concentration was checked with a nanodrop instrument (Spectrophotometer ND-1000, VWR). The amplicons were sequenced at StabVida (Oeiras, Portugal) using ITS1 and ITS4 primers .
Consensus sequences of ITS region were obtained with CodonCode Aligner 2.0.6 (Codon Co., USA). Sequences were aligned using ClustalW , divergence was analyzed using Kimura 2-parameter (K2P) distance  and dendograms were generated with Neighbour-joining (NJ) method , using MEGA4 software . Branch support was assessed with bootstrap analysis (1000 replicates) . The ITS sequence of Articulospora tetracladia UMB-014.00 (GQ411288) from GenBank was used to root the trees. Sequence data obtained during this study were deposited in GenBank (Table S1).
Standard indices of molecular diversity, namely theta S (θS) and theta pi (θπ), and pairwise Fst values were obtained using Arlequin 220.127.116.11 . Theta (θ) represents the distribution of variation within or among populations when samples are considered to represent characteristics of the larger group from which they are sampled. Theta S exhibits the infinite site equilibrium relationship between polymorphic sites, sample size and θ, for non-recombining DNA sample, while θπ estimates the infinite site equilibrium relationship between the mean number of pairwise differences and θ , .
The phylogeography of 130 isolates belonging to 6 aquatic hyphomycete species was inferred from ITS sequences. Total sequence length varied from 513 to 577 bp (A. filiformis, 513–514 bp; T. elegans, 513–520 bp; T. chaetocladium, 577 bp; Table 1). Isolates of T. elegans showed the highest variation in sequence length, while those of G. grandis (519 bp) and T. chaetocladium did not vary. Anguillospora filiformis showed the highest nucleotide percentage of A+T, while L. curvula and T. chaetocladium showed the highest percentage of G+C (Table 1).
Except for L. curvula and T. chaetocladium, NJ trees supported considerable population diversity within fungal species (Fig. 1). Isolates of A. filiformis, G. grandis and T. elegans from Portugal and Australia clustered into distinct clades (Fig. 1A, 1B and 1D), while isolates of L. curvula and T. chaetocladium did not exhibit geographic cohesiveness (Fig. 1C and 1F). Flagellospora penicillioides yielded 4 geographic clades (Fig. 1E): clade I consisted of isolates from Australia, clade II and III included isolates from Spain, Italy and the Azores archipelago (Portuguese Islands) and clade IV isolates from continental Portugal.
Neighbour joining trees based on ITS sequences using Kimura 2-parameter distances for A. filiformis (A), G. grandis (B), L. curvula (C), T. elegans (D), F. penicillioides (E) and T. chaetocladium (F); bootstrap values above 50% calculated from 1000 full heuristic replicates are shown at the nodes. Scale bar indicates one base change per 100 nucleotides. The sequence of Articulospora tetracladia UMB-014.00 (GQ411288) from GenBank was used to root the trees.
The average evolutionary divergence was lowest for T. chaetocladium (0.1±0.1%) and greatest for T. elegans (2.0±0.4%). Geniculospora grandis and T. elegans did not exhibit any sequence divergence within countries, while maximum divergence was found among Australian L. curvula isolates (0.5±0.2%) (Table 1). Divergence between countries was lowest for European (Portugal and Spain) isolates of G. grandis (0%) and highest for isolates of T. elegans from Portugal and Australia (4.2±0.9%) (Table 1).
Molecular diversity indices θs and θΠ were higher for isolates from European than from Australian streams, except for L. curvula (Fig. 2). Australian isolates of L. curvula showed the highest molecular diversity indices within all species. On the other hand, G. grandis isolates did not exhibit any genetic variability within isolates of Portugal or any other country (Fig. 2). The number of haplotypes varied between 2 (G. grandis) and 8 (L. curvula) (Fig. 3). Australia did not share haplotypes with European countries except for T. chaetocladium (Fig. 3). On the other hand, European countries shared haplotypes of F. penicilloides, G. grandis and L. curvula (Fig. 3).
Molecular diversity indices estimate the level of diversity existing within each country for each aquatic hyphomycete species. NA, not available.
In the present study, we assessed the intraspecific diversity of 6 aquatic hyphomycete species by comparing strains from Southwest Europe and East Australia. Internal transcribed spacer regions had been used successfully to identify Portuguese isolates of A. filiformis, F. penicillioides, G. grandis, L. curvula and T. chaetocladium . However, since the previous study was limited to fungal strains from a geographically restricted area (Northwest and Central Portugal), it did not allow any conclusions concerning cohesiveness between isolates from widely dispersed regions. Aquatic hyphomycete species might form genetic continua between a sequence of geographically distant connected locations. However, in sparsely populated or geographically isolated areas, genetically discrete taxa may be formed and assessed through the analysis of gene regions with high mutation rates (e.g. ITS). In the current study, we compared the ITS region in aquatic fungal isolates from Southwest Europe (continental Portugal, Portuguese Azores archipelago, Spain and Italy) and East Australia. The 6 selected species are common in all these geographically distant locations , . Neighbour-Joining trees based on ITS showed substantial intrapopulation diversity for A. filiformis, F. penicillioides, G. grandis and T.elegans, and intraspecific clades were supported by bootstrap values generally ≥70%. By using computer simulations and a laboratory-generated phylogeny, Hillis and Bull  found that bootstrap values ≥70% usually correspond to a probability ≥95% that the corresponding clades are real. Four of the species (A. filiformis, F. penicillioides, G. grandis and T. elegans) displayed higher evolutionary divergence between isolates from different continents (Australia and Europe) than within continents. However, we should point out that some sampling sites belonging to the same continent were very close (e.g. sampling sites in Portugal and Spain for G. grandis), and thus these differences are expected to occur.
Although European and Australian isolates from A. filiformis, F. penicillioides, G. grandis and T. elegans grouped into different clades, there was no linear relationship between geographic distance and evolutionary divergence (p = 0.26, r2 = 0.065). For example, isolates of F. penicillioides from Portugal and Spain (ca. 450 km apart) diverged by 1.7%, while from Spain and Australia (ca. 18000 km apart) diverged only by 0.3%.
The maximum divergence of 4.2% was found between isolates of T. elegans from Australia and Portugal (ca. 18000 km apart), which exceeds the values reported for fungal intraspecific variability (0–3%) inferred from ITS sequences . This may suggest the presence of cryptic species within the T. elegans species and deserves further investigation. Considerable intraspecific diversity was previously reported for T. elegans by random amplified polymorphic DNA (RAPD, ) and by amplified fragment length polymorphism (AFLP, ). However, no significant correlation was found between geographical and genetic distances, probably because streams were geographically close (0.5–18.5 km) .
Molecular diversity indices, θs and θΠ, of isolates within a country were higher for Portugal than for Australia, except for L. curvula. On the other hand, G. grandis did not show any difference within isolates of the same country, but it should be noted that the number of isolates was low (2 to 5 per country contrasting with 3 to 10 per country for L. curvula or 2 to 13 per country for F. penicillioides, respectively). In addition, isolates of G. grandis were sampled at unique sites within each country, except for Portugal. The same pattern was found for Australian isolates of A. filiformis and T. elegans that were also sampled at unique sites. On the other hand, intraspecific diversity was greatest for Azores isolates of F. penicillioides that were sampled at two stream sites in S. Miguel Island.
European countries did not share any haplotypes with Australia, except for T. chaetocladium. It has been suggested that meiospores may be responsible for long-distance dispersal of aquatic hyphomycetes  and their production by Hydrocina chaetocladia (teleomorphic phase of T. chaetocladium) and Nectria penicillioides (teleomorph of F. penicillioides) have been documented . Haplotypes of F. penicillioides from continental Portugal were also distinct from the ones in the Azores Islands, but the latter shared haplotypes with Spain and Italy. Flagellospora penicillioides has been reported as endophyte of plant roots , as all other species of this study with the exception of T. elegans . Plant trading events in the 18th and 19th centuries and other historical or current commercial interactions , may have facilitated the genetic exchange between European countries and Azores. In some fungi, asexual reproductive structures (mitospores) have been reported to disperse within (e.g. continental Europe to Great Britain, ) and between continents (e.g. from South Africa to Australia, ; for other examples, see ). The relatively delicate conidia of aquatic hyphomycetes seem ill suited for longer distance dispersal. Nevertheless, in the current study, some widely distributed morphologically defined aquatic hyphomycete species were divided into genetically distinct populations with more narrow geographical distributions. A similar pattern was recently found for Articulospora tetracladia, whose genotypes appear to be widespread with the exception of Malaysian haplotypes . Additional samples are needed to decide whether or not these differences are sufficiently consistent to define phylogenetic species. There is a growing concern that accelerating species loss may jeopardize ecosystem function and services. To evaluate this threat, we need to know intra- and interspecific variability of ecological functions and which species concept (morphospecies, phylospecies) is more relevant for capturing functional variability. An earlier review concluded that morphospecies of aquatic hyphomycetes have broadly overlapping functions . On the other hand, conspecific strains, isolated from geographically close streams, can differ significantly in their ability to tolerate heavy metals and other pollutants (e.g., , ). It seems unlikely, though untested, that in these cases genetic differences have resulted in the formation of new phylospecies. Several examples from other fungal groups have been discussed by Taylor et al. . The non-congruence of morphospecies and phylogenetic species is more pronounced in microorganisms than in macroorganisms. Taylor et al.  offer two non-exclusive reasons. Smaller organisms are morphologically less complex, providing less information to differentiate among forms. In addition, the rate of morphological change is slower for organisms with less elaborate development and fewer cells. The expected course of events in geographically widespread microbial species is therefore genetic differentiation, followed by reproductive and eventually morphological differentiation.
Our study suggests that there is no consistent inter- and intra-continental phylogeographic structure in aquatic fungi. The preliminary conclusion is that the biogeography or the extent of geographic distribution of aquatic hyphomycetes may be species-specific. Unfortunately, our sample size was small, since we relied on DNA from pure cultures that had been established from single conidia. This conventional approach is time-consuming and its success is greatly affected by chance. Analyses based on DNA extracted from individual spores potentially lower this hurdle , as will emerging next-generation sequencing techniques. Both nevertheless depend on a rich reference library of sequences from described fungal strains, which does not exist for aquatic hyphomycetes. Pyrosequencing potentially recovers all sequences in an environmental sample . Access to these extensive datasets will vastly expand our capability of addressing phylogeography at a multi-species level, providing a better foundation to investigate biogeographic patterns in aquatic hyphomycetes.
Conceived and designed the experiments: SD SS FB FC CP. Performed the experiments: SD. Analyzed the data: SD SS. Contributed reagents/materials/analysis tools: SD FC CP. Wrote the paper: SD SS FB FC CP.
- 1. Baas-Becking LGM (1934) Geobilogie of inleiding tot de milieukunde. W.P. van Stockum and Zoon, The Hague, The Netherlands.
- 2. Fenchel T, Finlay BJ (2004) The ubiquity of small species: patterns of local and global diversity. BioScience 54: 777–784.
- 3. Finlay BJ, Fenchel T (2004) Cosmopolitan metapopulations of free-living microbial eukaryotes. Protist 155: 237–244.
- 4. LaChance MA (2004) Here and there or everywhere? BioScience 54: 884.
- 5. Taylor JW, Turner E, Townsend JP, Dettman JR, Jacobson D (2006) Eukaryotic microbes, species recognition and the geographic limits of species: examples from the kingdom Fungi. Phil Trans R Soc B 361: 1947–1963.
- 6. Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, et al. (2000) Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31: 21–32.
- 7. Dettman JR, Jacobson DJ, Taylor JW (2006) Multilocus sequence data reveal extensive phylogenetic species diversity within the Neurospora discreta complex. Mycologia 98: 436–446.
- 8. Pegler DN (1983) The genus Lentinula (Tricholomataceae tribe Collybieae). Sydowia 36: 227–239.
- 9. Hibbett DS (2001) Shiitake mushrooms and molecular clocks: historical biogeography of Lentinula. J Biogeogr 28: 231–241.
- 10. Rydholm C, Szakacs G, Lutzoni F (2006) Low genetic variation and no detectable population structure in Aspergillus fumigatus compared to closely related Neosartorya species. Eukaryot Cell 5: 650–657.
- 11. Bärlocher F (2005) Freshwater fungal communities. In: Dighton J, White JF, Oudemans P, editors. The fungal community: its organization and role in the ecosystem. Boca Raton: CRC. 39–59.
- 12. Gessner MO, Gulis V, Kuehn KA, Chauvet E, Suberkropp K (2007) Fungal decomposers of plant litter in aquatic ecosystems. In: Kubicek CP, Druzhinina IS, editors. The Mycota: environmental and microbial relationships. Springer, Berlin, 301–321.
- 13. Shearer CA, Descals E, Kohlmeyer B, Kohlmeyer J, Marvanová L, et al. (2007) Fungal biodiversity in aquatic habitats. Biodivers Conserv 16: 49–67.
- 14. Krauss G-J, Solé M, Krauss G, Schlosser D, Wesenberg D, et al. (2011) Fungi in freshwaters: ecology, physiology and biochemical potential. FEMS Microbiol Rev 35: 620–651.
- 15. Belliveau MJR, Bärlocher F (2005) Molecular evidence confirms multiple origins of aquatic hyphomycetes. Mycol Res 109: 1407–1417.
- 16. Webster J (1992) Anamorph-teleomorph relationships. In: Bärlocher F, editor. The Ecology of Aquatic Hyphomycetes. Springer-Verlag, Berlin.
- 17. Wood-Eggenschwiler S, Bärlocher F (1985) Geographical distribution of Ingoldian fungi. Verh. Internat. Verein. Limnol. 22: 2780–2785.
- 18. Ranzoni FV (1979) Aquatic hyphomycetes from hawaii. Mycologia 71: 786–795.
- 19. Nikolcheva LG, Bärlocher F (2002) Phylogeny of Tetracladium based on 18S rDNA Czech Mycol. 53: 285–295.
- 20. Baschien C, Marvanová L, Szewzyk U (2006) Phylogeny of selected aquatic hyphomycetes based on morphological and molecular data. Nova Hedwigia 83: 311–352.
- 21. Letourneau A, Seena S, Marvanová L, Bärlocher F (2010) Potential use of barcoding to identify aquatic hyphomycetes. Fungal Divers 40: 51–64.
- 22. Seena S, Pascoal C, Marvanová L, Cássio F (2010) DNA barcoding of fungi: a case study using ITS sequences for identifying aquatic hyphomycete species. Fungal Divers 44: 77–87.
- 23. Seena S, Duarte S, Pascoal C, Cássio F (2012) Intraspecific variation of the aquatic fungus Articulospora tetracladia: an ubiquitous perspective. PloS ONE 7: e35884.
- 24. Anderson JL, Shearer CA (2011) Population genetics of the aquatic fungus Tetracladium marchalianum over space and time. PLoS ONE 6: e15908.
- 25. Vijaykrishna D, Jeewon R, Hyde KD (2006) Molecular taxonomy, origins and evolution of freshwater Ascomycetes. Fungal Divers 23: 351–390.
- 26. Bärlocher F (2010) Molecular approaches promise a deeper and broader understanding of the evolutionary ecology of aquatic hyphomycetes. J N Am Benthol Soc 29: 1027–1041.
- 27. Bärlocher F, Stewart M, Ryder DS (2011) Analysing aquatic fungal communities in Australia: impacts of sample incubation and geographic distance of streams. Czech Mycol 63: 113–132.
- 28. Bärlocher F, Marvanová L (2010) Aquatic hyphomycetes (Deuteromycotina) of the Atlantic Maritime Ecozone. In: Assessment of Species Diversity in the Atlantic Maritime Ecozone. Ottawa: NRC Research Press. Canadian Science Publishing, ISBN 978-0-660-19835-4, 36 pp.
- 29. Pascoal C, Marvanová L, Cássio F (2005) Aquatic hyphomycete diversity in streams of Northwest Portugal. Fungal Divers 19: 109–128.
- 30. White T, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press Inc, New York.
- 31. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680.
- 32. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide-sequences. J Mol Evol 16: 111–120.
- 33. Saitou N, Nei M (1987) The neighbor-joining method - a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.
- 34. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599.
- 35. Felsenstein J (1985) Confidence-limits on phylogenies - an approach using the bootstrap. Evolution 39: 783–791.
- 36. Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10: 564–567.
- 37. Tajima F (1983) Evolutionary relationship of DNA-sequences in finite populations. Genetics 105: 437–460.
- 38. Tajima F (1989) Statistical-method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585–595.
- 39. Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42: 182–192.
- 40. Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson K-H (2008) Intraspecific ITS variability in the Kingdom Fungi as expressed in the International Sequence Databases and its implications for molecular species identification. Evol Bioinform 4: 193–201.
- 41. Charcosset J-Y, Gardes M (1999) Infraspecific genetic diversity and substrate preference in the aquatic hyphomycete Tetrachaetum elegans. Mycol Res 100: 736–742.
- 42. Laitung B, Chauvet E, Feau N, Feve K, Chikhi L, et al. (2004) Genetic diversity in Tetrachaetum elegans, a mitosporic aquatic fungus. Mol Ecol 13: 1679–1692.
- 43. Bärlocher F (2009) Reproduction and dispersal in aquatic hyphomycetes. Mycoscience 50: 3–8.
- 44. Webster J, Scheuer C, Om-Kalthoum Khattab S (1991) Hydrocina chaetocladia gen. et sp. nov., the teleomorph of Tricladium chaetocladium. Nova Hedwigia 52: 65–72.
- 45. Iqbal SH, Bareen F, Yousaf N (1995) Freshwater hyphomycete communities in a canal. 1. Endophytic hyphomycetes of submerged roots of trees sheltering a canal bank. Can J Bot 73: 538–543.
- 46. Bärlocher F (2006) Fungal endophytes in submerged roots. In: Schulz B, Boule C, Sieber TN, editors. Microbial root endophytes. Berlin & Heidelberg: Springer-Verlag.
- 47. Martin R (2011) Azores: the finest walks on the coast and in the mountains. Bergverlag Roother GmbH, Munich (2nd ed).
- 48. Brown JKM, Jessop AC, Rezanoor HN (1991) Genetic uniformity in barley and its powdery mildew pathogen. Proc R Soc B 246: 83–90.
- 49. Watson I, De Sousa C (1983) Long distance transport of spores of Puccinia graminis tritici in the southern hemisphere. Proc Linn Soc NSW 106: 311–321.
- 50. Bärlocher F (1992) Community organization. In: Bärlocher F, editor. The ecology of aquatic hyphomycetes. Springer-Verlag, Berlin.
- 51. Braha B, Tintenmann H, Krauss G, Ehrman J, Bärlocher F (2007) Stress response in two strains of the aquatic hyphomycete Heliscus lugdunensis after exposure to cadmium and copper ions. BioMetals 20: 93–105.
- 52. Fernandes I, Pascoal C, Cássio F (2011) Intraspecific traits change biodiversity effects on ecosystem functioning under metal stress. Oecologia 166: 1019–1028.
- 53. Bärlocher F, Charette N, Letourneau A, Nikolcheva LG, Sridhar KR (2010) Sequencing DNA extracted from single conidia of aquatic hyphomycetes. Fungal Ecol 3: 115–121.
- 54. Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, et al. (2006) Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7: 57.