Fungal reproduction: a complex topic

Before diving into the specific mechanisms of fungal reproduction, it is essential to frame the topic within the broader context of the fungal kingdom. Fungi are not plants, nor animals; they represent a kingdom of their own, with unique evolutionary and biological strategies. Their reproduction is a complex ballet of cellular fusions, nuclear exchanges, and spore production, whose ultimate purpose is survival and dissemination. Understanding how fungi reproduce means appreciating the extraordinary diversity of this kingdom, from unicellular yeasts to the imposing basidiomycetes of our woods.

The basics of fungal reproduction: not just spores

When we think of fungal reproduction, our minds immediately go to spores. And although these are the disseminating elements par excellence, they represent only the final phase of a much more articulated process. Fungal reproduction can be asexual (or vegetative) or sexual. Asexual reproduction, such as mycelial fragmentation or the production of asexual spores (conidia), generates clones genetically identical to the parent. Sexual reproduction, on the other hand, involves the fusion of two parental nuclei and the subsequent genetic reshuffling, creating offspring with new DNA combinations. It is in this second scenario that the monokaryotic and dikaryotic phases play a leading role, especially in higher fungi (Ascomycota and Basidiomycota).

Monokaryon: the solitary life

What defines a monokaryotic mycelium

A monokaryotic mycelium is characterized by the presence of only one nucleus per cellular compartment (hyphae). These nuclei are haploid, meaning they contain a single set of chromosomes (denoted "n"). This mycelium is the direct product of the germination of a haploid spore and, as such, represents a unique genotype. Its growth is typically slower and less vigorous compared to a dikaryotic mycelium, as it possesses only half the genetic potential and must rely on a single set of instructions.

Its main function is to explore the substrate, grow, and, above all, meet another genetically compatible monokaryotic mycelium to initiate the traditional reproductive process. Without this encounter, the monokaryotic mycelium might simply exhaust the available resources and die, or in some cases, reproduce asexually.

Origin and development of the primary mycelium

It all starts with a spore. Whether it's a basidiospore or an ascospore, inside it holds a haploid nucleus. Under favorable environmental conditions (humidity, temperature, suitable substrate), the spore germinates. Through a process of mitosis, the nucleus duplicates and the cell begins to produce a germ tube, which elongates and branches, forming the first hypha. This network of septate hyphae, each containing identical nuclei, is the primary or monokaryotic mycelium.

Mating types 

Perhaps the most fascinating aspect of the monokaryon is the mating type system. To avoid self-fertilization and promote outcrossing, fungi have evolved sophisticated genetic mechanisms. Instead of having distinct sexes (male/female), they possess specific gene loci that determine sexual compatibility.

In Basidiomycetes, this system is often very complex. The tetrapolar system, common in many cap fungi, involves two independent loci: A and B. Each locus has multiple allelic variants. For two monokaryotic mycelia to be compatible and able to form a fertile dikaryon, they must possess different alleles at both the A and B loci.

Dikaryon: a unique nuclear symbiosis

The nature of the dikaryotic mycelium: two nuclei in one cell

The dikaryotic mycelium is a condition unique in the biological kingdom. It is not diploid (2n), where two chromosome sets fuse into a single nucleus, but dikaryotic (n + n), where two genetically distinct haploid nuclei coexist and divide in tandem within each hyphal cell. This partnership is maintained by specialized structures called clamp connections, which meticulously coordinate cell division to ensure that each new compartment receives a pair of nuclei, one from each parent.

The presence of the dikaryon, for the mycologist and the grower, is the clearest signal that the fungus is mature, genetically complete, and potentially ready to fruit. It is the mycelium we see colonizing a straw substrate, a log, or a cultivation block with incredible speed and efficiency.

Formation and structure of clamp connections

Clamp connections are the iconic morphological feature that visually distinguishes a dikaryotic mycelium of a Basidiomycete from a monokaryotic one. They are hook-shaped structures that resemble a miniature fishing hook. Their task is to ensure proper segregation of the two nuclei during mitosis.

Process of clamp connection formation (step by step):
1. The two nuclei within a dikaryotic hypha begin to divide (mitosis).
2. A small lateral protrusion shaped like a hook (the initial clamp) forms from the cell.
3. One of the nuclei migrates into this hook.
4. The nuclei orient themselves: one set of daughter nuclei positions itself at the apex of the hypha, the other set in the central part of the cell and the nucleus migrated into the hook.
5. Transverse septa form that separate the new cellular compartments: one between the apex and the cell, one between the cell and the hook, and one inside the hook.
6. The tip of the hook (which contains a nucleus) fuses with the underlying cell, donating its nucleus and thus creating a new dikaryotic cell (with two nuclei of different origin).
7. The final result is two dikaryotic daughter cells, each containing a pair of nuclei from the two original parents.

This process, repeated millions of times, is what allows the dikaryotic mycelium to grow while maintaining its dual nuclear identity intact. The absence of clamp connections in a mycelium that should have them is an indicator of genetic instability or a mycelium that is still monokaryotic.

Hybrid vigor and efficiency of the dikaryon

The dikaryotic mycelium is not just a biological curiosity; it is an ecological war machine. It displays a phenomenon known as heterosis or hybrid vigor. The combination of two different genomes in a single organism confers a series of advantages:

• Superior growth speed: it colonizes the substrate much faster than a monokaryon, overcoming potential bacterial or fungal competitors.
• Greater enzymatic efficiency: it possesses a wider repertoire of lignocellulosic enzymes (laccase, manganese peroxidase, etc.) to degrade complex substrates.
• Stress resistance: it is generally more resistant to environmental fluctuations (temperature, humidity, pH) and parasite attacks.
• Fruiting capacity: only the dikaryotic mycelium, in the vast majority of species, is able to form fruiting bodies (the mushrooms we collect).

The transition from mono to di: plasmogamy

Recognition and fusion mechanisms

The beginning of everything is a chemical dialogue. The two compatible monokaryotic mycelia recognize each other through the exchange of pheromones and the interaction between surface receptors encoded by the mating type loci. In fungi with a tetrapolar system, the A and B loci control different parts of this complex process.

• Locus B (controlling somatic incompatibility): regulates the initial recognition and the fusion of hyphae (anastomosis) and nuclear migration. It must be different for plasmogamy to occur.
• Locus A (controlling nuclear incompatibility): regulates the formation of clamp connections and the subsequent coordinated nuclear division. It must be different for a stable and fertile dikaryon to form.

When the hyphae of two compatible monokaryons approach each other, the pheromones bound to their respective receptors trigger a morphogenetic response, often leading to directional growth towards each other. This is followed by anastomosis: the cell walls of the two hyphae fuse, creating a cytoplasmic bridge through which cellular contents, including nuclei, can mix.

Nuclear migration and stabilization of the dikaryon

After the initial fusion, a spectacular event occurs: nuclear migration. The nuclei from one of the two monokaryons (the donor) actively migrate through the hyphae of the recipient, using the cytoskeleton (microtubules) of the host cell. This process is energetically costly and rapidly transforms the entire network of the recipient mycelium from monokaryotic to dikaryotic.

Once migration is complete and the nuclei are paired, the mycelium stabilizes its new dikaryotic condition. It begins to produce clamp connections at each cell division, consolidating the partnership and officially initiating the phase of vigorous growth that will lead, under appropriate conditions, to fruiting.

For a technical deep dive into the molecular mechanisms behind these processes, resources like The Fungal Genetics Stock Center are valuable.

From karyogamy to spore formation

Fruiting: the stage for nuclear fusion

The fruiting body (the actual mushroom) is a complex dikaryotic entity. Its task is to elevate the spores above the substrate to facilitate their dispersal. Within the hymenium, the fertile layer located under the cap (in basidiomycetes) or inside asci (in ascomycetes), specialized cells called basidia or asci complete the cycle.

Inside these cells, the crucial event occurs: karyogamy. The two haploid nuclei that have coexisted peacefully throughout the dikaryotic phase finally fuse, forming a transient diploid nucleus (2n). This nucleus represents the only diploid phase in the entire life cycle of the fungus.

Meiosis and dispersal: the return to haploid

The diploid phase is very brief. The newly formed nucleus immediately undergoes meiosis, the process of reductional cell division that restores the haploid state and, at the same time, reshuffles the genetic heritage of the two parents.

• In Basidiomycetes: the diploid nucleus in the basidium undergoes meiosis, producing 4 haploid nuclei. These migrate into equally many sterigmata and form the external basidiospores.
• In Ascomycetes: the diploid nucleus in the ascus undergoes meiosis, often followed by a mitosis, producing 8 haploid nuclei that will be incorporated into the ascospores inside the ascus.

These mature spores are then violently expelled (actively) or passively released into the air. Carried by wind, rain, or animals, they will land in a new location. If conditions are favorable, they will germinate, giving rise to new monokaryotic mycelia, closing and at the same time reopening the endless circle of fungal reproduction.

Practical implications for the mycoculturist

Strain selection and preservation

An experienced grower doesn't just work with "spores of Pleurotus ostreatus". They work with a specific strain, which is a genetically unique isolate with desirable characteristics (yield, colonization speed, resistance, color, flavor). To preserve a strain long-term without genetic variations, techniques for preserving the dikaryotic mycelium are used (on agar slants, in sterile water, in cryopreservation) because propagation via spores (sexual reproduction) would reintroduce genetic variability, producing mushrooms potentially different from the parent.

Cloning fruiting bodies by taking sterile internal tissue (tissue culture) is a method to bypass sexual reproduction and preserve exactly the dikaryotic genotype of the mushroom we appreciated.

Isolation and breeding

To create new strains, breeders actively exploit this cycle. They cross two different dikaryotic strains by collecting the spores of their offspring. From these spores, hundreds of different monokaryotic mycelia germinate, each with a unique combination of the grandparents' genes. The breeder must then test the compatibility and performance of these monokaryons, strategically crossing them to generate new dikaryons with, hopefully, the best characteristics of both parents.

Diagnosis and contamination control

Knowing how to visually recognize the difference between a monokaryotic mycelium (rare in cultivation) and a dikaryotic one (what should normally be used) helps diagnose problems. A fungal colony that grows very slowly and without the typical dikaryotic structures (rhizomorphs, clamps visible under the microscope) might be a contaminant or an unwanted primary mycelium.

Research, curiosities, and the future of mycology

Recent research and notable discoveries

Modern genetics is unveiling the most intimate secrets of the mating type loci. Research on the famous model fungus Schizophyllum commune has revealed that the A and B loci are actually giant gene complexes with dozens of different alleles in nature, explaining the enormous genetic diversity.

Studies on plant pathogenic fungi (e.g., Magnaporthe oryzae, the fungus that causes rice blast) focus on their reproduction to predict the emergence of new virulent strains and develop control strategies.

In medicine, understanding how pathogenic fungi like Candida albicans reproduce (which has both a clonal and a sexual cycle) is crucial for understanding its ability to develop drug resistance.

Curiosities from the fungal world

• Killer fungi: some species of fungi in the genus Agaricus have up to 20,000 different alleles for their mating type loci, making statistical incompatibility almost impossible. Practically every encounter is a success!
• Fungi that change sex: the mating type in some fungi is not fixed. They can undergo mutations or chromosomal rearrangements that change their type, a strategy to increase the chances of finding a compatible partner in isolated environments.
• Dikaryon without sex: in some fungi, the formation of the dikaryon can occur without actual mating, through fusions between hyphae of the same strain (vegetative anastomosis), although this does not lead to genetic novelty.

The future: genetic editing and beyond

The advent of technologies like CRISPR-Cas9 is opening unimaginable doors. Researchers can now "turn off" specific genes in the mating type loci to study their function, or even design strains with specific mating characteristics for highly advanced breeding programs. The possibility of creating synthetic dikaryons between different species to confer new abilities, such as the degradation of particularly recalcitrant pollutants, is being explored.