Host identities and other exciting updates

Hey everyone,

I’m getting closer to answering several of my main questions. Here are the ones I can begin to address with this blog post:

1) What are the hosts of Hypomyces lactifluorum, the lobster mushroom mold parasite?

2) Do genetic lineages of the parasite specialize on different host species?

3) Is the white lobster mushroom, named as the separate species Hypomyces macrosporus without DNA data, really its own species or just a color morph of H. lactifluorum?

I’ve had to use a hodgepodge of techniques to obtain enough parasite and host DNA to use for this project, including Sanger sequencing (the more “old-fashioned” sequencing technique) of the ITS gene, Illumina high-throughput sequencing to metabarcode the ITS gene, and whole-genome Illumina high-throughput sequencing. Different techniques worked for different samples, and I have very few clues as to why I found this variation. Just a few more mysteries to throw on the huge pile of fascinating questions in fungal genetics!

Question 1: Host range

This is the question that citizen science contributors seemed to be the most invested in—and of course people want to know what they’re eating under that beautiful orange exterior! 

In my last blog post, I provided some host ITS sequences for individual specimens and instructions for how to identify them via the NCBI BLAST webpage. This is a straightforward way to accomplish genetic identification, but not a very accurate one. There are several reasons for this: a) NCBI is a database of RNA, DNA, and protein sequences uploaded and labeled by its users, which means that if a sequence is mislabeled when it’s uploaded, it will likely stay mislabeled and cause inaccurate BLAST hits. b) BLAST is a sequence similarity-based approach, not an evolutionary approach, to comparing sequences. Only a phylogenetic, or tree-building approach, takes into the account the evolutionary process that produced the variation in the sequences.

So, in addition to BLAST identification, I built a phylogenetic tree of all the host ITS sequences I’ve been able to obtain so far. This tree will be continually updated as I’m able to wrest ITS from more specimens:

This phylogeny is based off of only one gene (ITS) because it’s the only gene I’ve been able to consistently find from the host genome in the lobster mushroom samples (and sometimes I can’t even find ITS). This means the branching order of the tree doesn’t have very good statistical support; however, the results do hold up to common sense. 

Every labeled tip of a branch represents an individual: either from my personally collected and donated dataset, or a reliably labeled mushroom in the family Russulaceae from the NCBI database. (I know they’re “reliably labeled” because I’ve only been using publicly available sequences from the guy who is on the cutting edge of Russulaceae evolutionary relationships.) Donated/personally collected samples have labels beginning with “LM” or “OL”. 

Clusters of individuals that are more closely related to each other (have a more recent common ancestor/node connecting their branches on the tree) than to any other individual are called a clade. This is an evolutionarily distinct group, and the main underlying principle when designating separate species.

In my lobster host mushroom ITS tree, we see that all my samples fall within one of two clades: one containing a reliably identified Russula delica sequence and one containing a reliably identified Russula brevipes sequence. This is a good way of identifying the hosts as belonging to whichever species is in its clade on the tree, although using more genes in the analysis would make the results more robust.

Both R. delica and R. brevipes are previously identified hosts of Hypomyces lactifluorum (Rochon et al. 2009), so no surprises there. Russula chloroides is another previously identified host, although none of my samples fall into its clade. My samples come from all over the United States, especially the coasts. So far there doesn’t seem to be a geographic pattern, but that’s something to check back on once I have more host sequences on my tree.

Current conclusion: No new hosts identified for Hypomyces lactifluorum so far, but I still have some samples left to sequence!

If you would like to know if your sample is on the tree yet and what its ID is, please let me know over Facebook or email.

Question 2: Specialization on hosts

Some parasites evolve in a manner that follows, or even reciprocally influences, the evolution of their hosts. When parasites and hosts, or any other pair of symbiotic organisms, have corresponding evolutionary trees, it’s called codivergence or cophylogeny. 

Here’s a comparison of the ITS phylogenies of hosts (left) and parasites (right) from the same specimen. I used software called PAco to mathematically compare the shapes of the two phylogenies. The PAco analysis found no congruence between the two trees, meaning I have no evidence that genetic groups of Hypomyces lactifluorum specialize on genetic groups of their hosts. This is an interesting result in itself because it implies that whatever strategy H. lactifluorum uses to invade and overtake its host, it’s likely not dependent on specific genotype combinations of the parasite and host and is more generalized instead.

Question 3: The Mysterious Case of the White Lobster

Is Hypomyces macrosporus really a different species from Hypomyces lactifluorum, or the same species with a superficial difference in color? 

Remember the idea of a clade from Question 1? If H. lactifluorum and H. macrosporus form distinct clades—two different clusters on the phylogeny, where within each cluster all individuals are more closely related to each other than to any individual in the other cluster—then that’s good evidence that they may be separate species. 

Unfortunately, I only received one white lobster specimen from my generous donations, and this specimen was from Canada. My fear was that even if the white specimen fell outside of the cluster of orange specimens on the tree, it could be due to genetic distance caused by geography, not a species boundary, and wouldn’t provide enough evidence one way or another. However, here are my results:

LM5 is my one white specimen. All of the individuals surrounding it are orange lobsters! This is the opposite of the “one orange clade, one white clade” scenario, because the white specimen falls smack dab in the middle of a bunch of orange specimens.

Things aren’t looking good for H. macrosporus, but I want to get DNA from more white lobsters before I start feeling too smug, and luckily I’ve been able to request some from museum collections. These specimens will be added to a larger tree once I’ve been able to obtain their Hypomyces ITS sequence.

That’s all for now, thanks again for your interest and support!

Kendra

Citations:

Rochon, C., Pare, D., Khasa, D. P., & Fortin, J. A. (2009). Ecology and management of the lobster mushroom in an eastern Canadian jack pine stand. Canadian journal of forest research, 39(11), 2080-2091.