SEQUESTRATE BEN - IT STARTED WITH MACOWANITES

On October 11 and 12, 1997, the South Vancouver Island Mycological Society organized a mushroom foray to the Pacific Rim National Park near Tofino, British Columbia. With the help of mushroom experts (Bryce Kendrick, Paul Kroeger, Ian Gibbson, Oluna Ceska, Tony Trofimow, et al.) we compiled a list of about 140 mushroom species. One of the most interesting finds, at least for me, was Macowanites chlorinosmus.

Macowanites looks like Russula that has never made it: an unopened ball with contorted gills inside. Dr. Bryce Kendrick explained to us that Macowanites is a member of the so-called sequestrate fungi (also called secotioid or gastroid fungi), mushrooms that follow the example of truffles and remain buried underground, or grow close to the soil surface. They don't release their spores, but rely on animals for spreading their spores around.

I asked Dr. Bryce Kendrick to write me a short note on these mushrooms for BEN, and he sent me two articles that he published in McIlvainea. I read these articles and realized that a short note would not do justice to those interesting fungi. I have decided to post them in full and the next three issues of BEN (177, 178, and 179) will be fully devoted to this topic.

I apologize to all of you who believe that mushrooms really do not belong to the botanical realm. I will post these BEN issues in short intervals of two or three days. This will ensure some degree of continuity, and at the same time, it won't fill your mail boxes entirely. I hope you will enjoy this sequestrate diversion. - Adolf Ceska

EVOLUTION IN ACTION: FROM MUSHROOMS TO TRUFFLES? PART 1

[Kendrick, B. 1994.

Evolution in action: from mushrooms to truffles. I. McIlvainea 11 (2): 34-38.]

The fungi are very old. Their history extends over hundreds of millions of years. Yet their origins, and the major evolutionary pathways they have followed, are still cloaked in mystery. This is largely because the fossil record of the fungi is fragmentary and disconnected. Organisms that live on land, and particularly such ephemera as most fungal fructifications, are much less likely to be fossilized than are marine organisms with hard parts. The paucity of fossil evidence has not deterred the cognoscenti among mycologists from a little judicious speculation, inevitably based largely on what we know about fungi that are alive today. This speculation is probably wrong in many respects and is usually heavily laced with the prejudices of its authors, but it is not necessarily a bad thing: students of the fungi need a conceptual framework on which to cut their teeth, and at which to aim their more mature criticisms. But we are still on shaky ground when we try to look into the evolutionary history of most modern fungi.

It is, then, all the more exciting to encounter an area of mycology in which not only the results of evolution, but also the starting points, and the steps in the process, can still be seen in living organisms. And all this has happened, not in obscure microscopic fungi, but in conspicuous and fairly common mushrooms that can be held in the hand and compared. We now know that some mushrooms have given rise to radically changed but still viable descendants: relatives which, although often looking very different from their forebears, clearly betray their ancestry at the microscopic and molecular level. Let us see how this has happened in the well-known and easily recognized mushroom genus Lactarius (the "milky-caps": family Russulaceae, order Agaricales).

But before I describe the exciting changes we have seen, I must establish a base-line or starting point by describing how mushrooms usually develop, and what they do: how their form and function are interrelated. First, the thread-like mycelium, which permeates the soil and is often involved in an intimate and mutually beneficial mycorrhizal association with tree roots, must accumulate considerable reserves of food energy. Then conditions of temperature and moisture must be favourable. Finally, the mushroom begins its development underground, forming a clump of mycelium which differentiates into a "button," then rapidly expands upward and emerges from the earth as a characteristic structure with a central stalk or stipe, bearing an expanding circular cap or pileus. The top of the cap is covered by a skin or cutis. The thin, plate-like gills of Lactarius normally develop in a neat radial pattern (like the spokes of a wheel) on the under side of the expanding cap. The basidiospores will form on these gills. As the cap opens out like an umbrella, the gills assume a precise vertical orientation and are now ready to make and liberate spores.

The flat surfaces of the gills are covered by a fertile layer called a hymenium. This contains huge numbers of special cells called basidia, which produce and liberate astronomical numbers of spores. Each basidium bears four spores (sometimes more, occasionally fewer). These develop asymmetrically, in an offset manner, at the tips of four sterigmata, which are tiny projections from the mother cell. When ripe, the spores are delicately but deliberately launched into the air between the gills. They float slowly and gently downward until they emerge from the gills, and are then carried away like dust by air movement. In this way the fungus broadcasts its spores far and wide. The different genera and families of agarics often follow significantly different developmental pathways, some with gills exposed from the beginning, others with gills enclosed almost until maturity, but they all eventually arrive at the same endpoint, with vertical, exposed gills dropping spores into the air.

One of the ways in which we identify agarics is by placing a cap on a piece of white paper in a draftfree place and letting it drop millions of spores onto the paper overnight. The deposit will form a visible radiating pattern, which reflects the arrangement of the gills from which the spores came. This spore print may be white, cream, pink, brown or black, according to the mushroom genus which produces it. The spore print of Lactarius is white or cream coloured.

Everything I have said so far applies not just to Lactarius, but also to many other genera of mushrooms. So how does Lactarius differ from the rest? That's easy: it has a unique combination of three features which are not found together in any other genus of agarics.

[1] The cap and gills of Lactarius contain special cells filled with a milky juice or latex (white, yellow, orange or red) that oozes out in visible drops when the tissues are cut (and sometimes change colour after exposure to air).
[2] The flesh of Lactarius contains large numbers of swollen, thin-walled cells called sphaerocysts: these make the flesh extremely and characteristically brittle and granular.
[3] The spores of Lactarius are ornamented with conspicuous warts and spines, lines and ridges, which often join up to form a network. These ornamentations are chemically different from the rest of the spore wall, because they stain darkly (grey, blue, purple or black) in iodine, while the spore wall itself remains unstained, or stains only slightly. Ornamentation that gives this colour reaction is often described as iodine-positive, or amyloid.

Even beginners can easily identify Lactarius by the milky juice it exudes when the brittle flesh is broken: no other ordinary mushroom has anything like it.

But in addition to specimens of Lactarius as I have just described it, we occasionally find extraordinary specimens. Specimens which have a few important differences from the milky caps we are used to seeing. They are similar (and theoretically could therefore be included in the genus) because they have all three characters listed above: brittle flesh full of sphaerocysts; latex exuded when the tissues are ruptured; and spores with ornamentation that is iodine-positive. Yet they are different because their cap develops in such a way as to enclose their gills, and the gills are no longer vertical plates, but have become crumpled or convoluted to form a spongy, chambered mass. Since the cap remains closed, the spores obviously cannot escape. This sounds as if it would be a serious problem for the fungus: after all, have not mushrooms evolved to be spore-making and spore-launching machines? And if the spores are not released into the atmosphere, how will they be dispersed? Yet if we remember that the lungs of land vertebrates evolved from the swim-bladders of their fish ancestors, and the wings of birds from the forearms of their earthbound reptilian ancestors, we will appreciate that evolution, guided by environmental forces, often drives organisms in unforeseen directions. Something of this kind appears to be happening to the Lactarius, and we must assume that some other way of dispersing the spores has been evolved.

If we now cut away the edge of the unopened cap which is obscuring the gills, and try to make a spore print, we will not succeed. No spores will be deposited. This is not because the mushroom is either unripe or overmature. If we examine some of the basidia under a microscope, we will see that they have produced mature basidiospores. But the basidia have subtly changed. The four spores tend to develop symmetrically (not offset) on the sterigmata, and they tend to remain attached to the sterigmata: they are never forcibly discharged, as they were in normal Lactarius fruit bodies.

These differences are important enough for taxonomists to conclude that the fungus can no longer be called a Lactarius, and it has been placed in a different genus, named Arcangeliella. This genus has sometimes been excluded from the family Russulaceae and even from the order Agaricales, and has instead been put in a separate order, the Hymenogastrales. But there is no doubt that it has evolved from Lactarius in relatively recent times, that it is still closely related to that genus, and that it should be retained in the Agaricales, and even in the Russulaceae.

Arcangeliella still looks very like a mushroom, even if its behaviour is a little strange. But we have found other specimens which have evolved even further away from Lactarius. These specimens develop, and remain, just below the surface of the ground, looking rather like truffles. They are rounded or irregular in shape. The skin that covered the Lactarius now completely surrounds the truffle-like specimens. They have no stalk. There are no gills: the hymenium lines labyrinthine chambers. And the basidiospores, now sitting straight on the sterigmata of the basidia, are not actively shot away.

Note that the outer skin and often the walls of the labyrinthine spore-bearing tissues contain sphaerocysts; latex oozes from the cut surfaces of fresh specimens; and the spores have spiny or ridged ornamentation that stains dark in iodine. Once again, the three diagnostic characters of Lactarius. A vestige of a stalk may even occur in the form of a pad of sterile tissue inside the base of the fruit body; the walls of the labyrinthine chambers could be derived from crumpled gills; and the presence of sterigmata on the basidia is a reminder that these structures were originally evolved as part of a mechanism to launch spores into the air.

Yet it would be stretching the concept of Lactarius beyond the breaking point to include these specimens in it: surely no-one would call them agarics. It is also clear that they are considerably more "reduced" even than those placed in Arcangeliella. So mycologists put them in another new genus, called Zelleromyces.

Although Zelleromyces differs from both Arcangeliella and Lactarius in important ways, the fact that it has latex, sphaerocysts and iodine-positive (amyloid) spore ornamentation is a compelling argument for keeping it in the family Russulaceae of the order Agaricales. After all, this disposition seems to best reflect its true relationships. Arcangeliella and Zelleromyces are what we now call sequestrate (see the note below) derivatives of the original agaric. The word sequestrate implies that they sequester or retain their spores, rather than broadcasting them into the air. This retentive habit, diagnosed by spores sitting symmetrically on the sterigmata of non-shooting basidia, is clearly characteristic of both genera.

Before drawing the first part of this discussion to a close, I must address one final issue. If these sequestrate genera share all the essential diagnostic features of Lactarius, how are we to distinguish the Lactarius we all know from its sequestrate derivatives? It is apparent that the three diagnostic characters I described earlier must be supplemented by three more, as follows:

[4] the cap of a true Lactarius expands at maturity and the gills are exposed.
[5] its gills are vertically oriented.
[6] its basidiospores are asymmetrically mounted on the sterigmata and are forcibly discharged at maturity.

If the Lactarius -> Arcangeliella -> Zelleromyces sequence was the only case in which this strange evolutionary sequence had been observed, we might be able to dismiss it as a quirk of evolution, a freak. But we have evidence that similar pathways have evolved in other mushroom genera. These will be explored in the second part of this article, in the next two issues of BEN.

The term "sequestrate" has recently been introduced (Kendrick 1992) to describe all these closed or hypogeous offshoots of regular fungi. It means that the spores are sequestered or hidden away, kept from contact with the outside world, at least until the fruit body decays or is eaten. The term sequestrate appears to be a more useful and more widely applicable term than such frequently-used words as 'gastroid' (which inappropriately implies close relationship with gasteromycetes) and 'secotioid,' an arcane word suggesting similarity with the genus Secotium (which is a sequestrate derivative of Agaricus). Most amateur and many professional mycologists have never seen Secotium, so the term derived from that name conveys little or no meaning.

[Continuation in BEN # 178]

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