Fermentation part I

IMG_20161002_170626Well, here we have some fermenting sugar cane juice from a cachaça maker in Brazil.


And here is the total genomic DNA (and RNA smear at the bottom) from the above juice.


So, I loaded it into one of these portable sequencing contraptions from Oxford Nanopore.


And generated not very much sequence data, probably because the flow cell and library kit was very old. But still, some interesting preliminary results using WIMP…



ITS sequences of some Chilean boletes

There are currently 6 boletes (Boletaceae; Boletus loyo, B. loyita, B. auracarianus, B. bresinskyanus, B. putidus, and Tylopilus temucensis) endemic to the southern temperate forests of Chile, and probably also Argentina, comprising the Patagonia region (herein called ‘Patagonian boletes’) . They are likely mycorrhizal, forming associations with southern beech (Nothofagus spp.).  Very little documentation exists of the ectomycorrhizal (ECM) fungi associated with southern beech in South America, although diversity appears to be low relative to other ECM forests. Some speculation on the biogeographic affinities and origins of these fungi exist, but not much rigorous work has been done and it is not clear if they are relicts of an ancient Gondwanan distribution when South America was connected to Australia/New Zealand and Africa (unlikely) or if they are recent arrivals, either through ECM corridors (such as Alnus or Juglans?) connecting northern oak forests with South America (presently ending in Colombia) or through long-distance spore dispersal. In any case, no DNA sequences exist of Patagonian boletes. I’ve never been to Patagonia, so I’ve not had the privilege to study and collect these beautiful organisms myself (here would be the perfect place for a nice photo…), but, luckily, preserved specimens collected by other intrepid mycologists are available for study.

DNA was from 12 specimens of Chilean boletes borrowed from Zurich (herbarium code ZT) using the enzymatic digestion method in Dentinger et al. (2009). The ITS region was PCR-amplified with standard recipes using primer combinations ITS1F, ITS2 and ITS3, ITS4 (White et al. 1990, Gardes & Bruns 1993 — for more info, see this overview provided by the Vilgalys lab of traditional rDNA primers  http://sites.biology.duke.edu/fungi/mycolab/primers.htm). The amplicons were cleaned and Sanger sequenced, then edited with Sequencher. Only two specimens (ZTMyc3876: Boletus putidus Horak; ZTMyc3883: Boletus bresinskyanus Garrido) had clean sequences. I then BLASTed each sequence against the INSDC non-redundant nt database (http://blast.ncbi.nlm.nih.gov/) and downloaded the top 100 hits for each, as well as some outgroup taxa (Chalciporus, Rubinoboletus).  I also downloaded the top hits in UNITE (Kõljalg et al. 2005) that have not been submitted to the INSDC database. In total the dataset consisted of 261 ITS sequences, including the two from the Chilean specimens. I first aligned the sequences using the Q-INS-i algorithm in MAFFT (Katoh et al. 2002; Katoh & Toh 2008; Katoh & Standley 2013). I then ran a maximum likelihood analysis under a GTRGAMMA model in RAxML v8.2.4 (Stamatakis 2006, Ott et al. 2007) with automatic bootstopping based on the MRE criterion (i.e. $raxmlHPC-PTHREADS-SSE3 -T 16 -f a -m GTRGAMMA -n Chile_xerocomoids -s ./Chile_xerocomelloids_QINSI.fas -N autoMRE -x 12345 -p 12345). Bootstopping terminated after 400 reps. Here’s the tree:


The two Chilean seqs are in RED. Bootstrap supports on the branches are colored from blue (low) to pink (100%). Both are in unsupported placements, but not each others closest relatives. The take home here is that the ITS sequences of both Boletus putidus and Boletus bresinskyanus are not particularly closely related to any of the other Boletaceae sequences that are most similar. While this evidence does not contradict the hypothesis that they are derived from North American ancestors that migrated south with oaks following the formation of the Panamanian isthmus, you might expect there to be less divergence in the ITS for taxa that shared a common ancestor relatively recently (3 million years ago). Of course, it may simply be a consequence of data deficiency resulting in Type II errors rather than true evidence: the reference sequence databases are depauperate and many species remain undocumented. Maybe their close relatives in North America/Central America/Colombia just haven’t been sequenced yet.

Vicariance following the disarticulation of Gondwana would probably not be the first explanation, either. First, that was a really long time ago (85 million years), and with only 6 existing species in all of Patagonia (unless there are many that haven’t been seen), that would be a long time without speciation or with frequent extinction. Also, where are the characteristic austral relicts, i.e. Austroboletus? I’m not saying it’s impossible, but it’s not the most parsimonious explanation.

Could they have arrived from long-distance spore dispersal events? At this point, it’s still anyone’s guess and this exercise doesn’t seem to have shed any light on this mystery ,but maybe it brings us one step closer to understanding how they got there.

Incomplete sampling of both genes and taxa is the 800 pound gorilla that continues to plague fungal ecologists and taxonomists, so here at least are some new ITS sequences to help lighten the load on all of us to fill in the gap between the known and the unknown.

Special thanks to Laura Martínez-Suz who did all the lab work for this.

GenBank Accession numbers:

ZTMyc3877 Boletus putidus      KX002262

ZTMyc3883 Boletus bresinskyanus KX002263

I’ve also put all the data (sequence chromatograms) and datasets on figshare, which is citable (doi:10.6084/m9.figshare.3126199)


Dentinger, B. T. M., Margaritescu S., Moncalvo J.-M. (2009). Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematic of mushrooms. Molec. Ecol. Resources 10: 628 – 633.

Gardes, M. & Bruns T. D. (1993). ITS primers with enhanced specificity for basidiomycetes — application to the identification of mycorrhizae and rusts. Molec. Ecol. 2: 113 – 118.

Katoh, K., Misawa, K., Kuma, K. & Miyata, T. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30: 3059 – 3066.

Katoh, K. & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molec. Biol. Evol. 30: 772 – 780.

Katoh, K. & Toh, H. (2008). Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework. BMC Bioinformatics 9: 212.

Ott, M., Zola, J., Aluru, S. & Stamatakis, A. (2007). Large-scale maximum likelihood-based phylogenetic analysis on the IBM BlueGene/L. Proceedings of ACM/IEEE Supercomputing conference. Article No. 4.

Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688.

White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White (eds), PCR Protocols: A Guide to Methods and Applications, pp. 315 – 322. Academic Press Inc., New York.