Revision of Archaeosporomycetes with two old and two new fungal orders: Archaeosporales, Geosiphonales, Polonosporales, and Ambisporales

This study revises the fungal class Archaeosporomycetes by proposing a new taxonomic structure that divides the previously single order Archaeosporales into four distinct orders—Archaeosporales, Ambisporales, Geosiphonales, and Polonosporales—based on robust phylogenetic support and significant morphological and ecological differences.

The Gist

Imagine the fungal world as a massive, ancient library. For a long time, librarians thought that a specific section of this library, called the Archaeosporomycetes (let's call it the "Ancient Spore Wing"), only had one single, massive room called Archaeosporales. Inside this room, they shelved four different families of fungi together, assuming they were all close cousins living under the same roof.

But in this new study, a team of fungal detectives (led by Fritz Oehl and Gladstone Silva) decided to take a closer look. They used two powerful tools: DNA sequencing (reading the fungi's genetic "instruction manuals") and microscopes (looking at their physical "architecture").

What they found was shocking: The "Ancient Spore Wing" wasn't one big room at all. It was actually four completely different buildings, each with its own unique lifestyle and family history. They decided to renovate the library and split the single order into four distinct orders.

Here is the breakdown of the four new "buildings" using simple analogies:

1. The "Old Guard" (Archaeosporales)

• The Vibe: These are the classic, traditionalists.
• The Lifestyle: They are like gardeners who help plants grow by forming a partnership (symbiosis) with plant roots.
• The Look: Their spores (reproductive cells) are like delicate, thin-walled bubbles. They are small and often don't stain dark blue when tested with a special dye (Trypan blue), meaning they are a bit more "invisible" or faint compared to their cousins.
• The Twist: The researchers realized this group actually contains two very different families. One family (the Antiquisporaceae) is the "dark stain" version that forms little storage bubbles (vesicles) inside roots, while the other (the Archaeosporaceae) is the "faint stain" version that rarely does. They are like two different branches of the same old family tree that grew apart long ago.

2. The "Double-Act" Performers (Ambisporales)

• The Vibe: These are the versatile acrobats.
• The Lifestyle: They are also plant partners, but they are famous for being bimorphic. Think of them as actors who can play two completely different roles. They can produce two very different types of spores:
  1. The "Acaulosporoid" type: Large, thick-walled, and colorful (like a heavy-duty armored tank).
  2. The "Glomoid" type: Simpler, single-walled, and often smaller.
• The Difference: Compared to the "Old Guard," these guys are bigger, tougher, and their walls are much thicker. They are the heavy lifters of the group.

3. The "Alien" (Geosiphonales)

• The Vibe: The weirdo of the family who lives in a different universe.
• The Lifestyle: This is the most unique one. While all the other fungi in this group help plants, Geosiphon helps a cyanobacterium (a type of blue-green algae) called Nostoc.
• The Analogy: Imagine a fungus that doesn't shake hands with a plant, but instead swallows a tiny, glowing blue-green balloon (the algae) and keeps it inside its own body to do photosynthesis for it. It's a "roommate" situation rather than a "garden" situation.
• History: Scientists actually knew about this weirdo decades ago and gave it its own name, but it got lost in the shuffle. This paper officially moves it back to its own special order, recognizing that its life story is totally different from the plant-loving fungi.

4. The "Polish Specialist" (Polonosporales)

• The Vibe: The mysterious newcomer from Poland.
• The Lifestyle: This order is currently known by just one species (Polonospora polonica).
• The Look: This one is a construction marvel. It builds its spores directly on the neck of a tiny sac. Its walls are unique because it builds three permanent, thick walls from scratch.
• The Analogy: While the other fungi might build a temporary tent that falls apart after a while, this one builds a permanent, three-layered fortress. It's the only one in the group that doesn't seem to have the "two-spore" trick (bimorphism) of the others.

Why does this matter?

Think of it like realizing that whales, bats, and humans are all mammals, but you wouldn't put them in the same "order" just because they are warm-blooded. You'd separate them because whales swim, bats fly, and humans walk.

Similarly, these fungi all share a common ancestor, but they have evolved such different strategies for survival (some help plants, some eat algae, some build thick walls, some build thin ones) that they need their own distinct categories.

The Bottom Line:
The authors took a messy, one-size-fits-all classification and cleaned it up. They said, "Hey, these four groups are so different in their DNA, their looks, and how they live, that they deserve to be in four separate 'orders'." This helps scientists understand the true history and diversity of these ancient fungi, which are crucial for healthy soil and plant life on Earth.

Technical Summary

1. Problem Statement

The fungal class Archaeosporomycetes (within the phylum Glomeromycota/Mucoromyceta) was historically classified as a single order, Archaeosporales, containing four families: Archaeosporaceae, Ambisporaceae, Geosiphonaceae, and Polonosporaceae.

• Taxonomic Ambiguity: Despite significant morphological and ecological differences among these taxa (e.g., spore formation, staining properties, and symbiotic strategies), they remained grouped under one order.
• Phylogenetic Discrepancy: Previous molecular studies suggested distinct evolutionary lineages, but a formal taxonomic revision to reflect these deep divergences at the order level had not been completed.
• Specific Issues:
  • Geosiphon forms a unique endocytobiosis with cyanobacteria (Nostoc) rather than arbuscular mycorrhiza with plants, yet it was retained within Archaeosporales.
  • Recent discoveries (e.g., Antiquispora) and re-evaluations of existing genera (Intraspora, Palaeospora) indicated that the current classification does not align with phylogenetic clades.
  • The genus Polonospora possessed unique morphological traits (spore wall formation) not seen in other members of the order.

2. Methodology

The authors employed an integrative approach combining molecular phylogenetics and detailed morphological analysis.

• Phylogenetic Analysis:

  • Datasets: Two datasets were constructed:
    1. Dataset 1: Concatenated partial SSU, ITS, and partial LSU nrDNA sequences (78 sequences, 1763 sites).
    2. Dataset 2: Complete SSU nrDNA sequences (57 sequences, 1671 sites).
  • Outgroup: Paraglomus brasilianum was used to root the trees.
  • Algorithms: Maximum Likelihood (ML) analyses were performed using RAxML-NG. Support values were calculated using Felsenstein Bootstrap Proportion (FBP) and Transfer Bootstrap Expectation (TBE).
  • Models: Nucleotide substitution models (GTR+I+G and TIM3+I+G) were selected via ModelTest-NG.

• Morphological Analysis:

  • Specimens: The authors examined specimens of nearly all species in Ambisporaceae, Archaeosporaceae, and Polonosporaceae. This included historical slides (pre-1990, mounted in lactophenol) and modern preparations (PVLG or PVLG + Melzer's reagent).
  • Focus: Analysis focused on spore wall architecture (number of layers, permanence, formation mechanisms), staining reactions (Trypan blue, Melzer's reagent), germination structures, and mycorrhizal symbiosis types.
  • Comparison: Morphological data were cross-referenced with phylogenetic clades to validate taxonomic boundaries.

3. Key Contributions

The study proposes a major taxonomic restructuring of the class Archaeosporomycetes, elevating four distinct clades to the rank of Order.

1. Resurrection of Geosiphonales: The order Geosiphonales (originally described by Cavalier-Smith in 1998 but later subsumed) is formally reinstated within Archaeosporomycetes to accommodate the unique genus Geosiphon and its cyanobacterial symbiosis.
2. Establishment of New Orders:
   • Ambisporales ord. nov.: Contains the family Ambisporaceae (genera: Ambispora, Appendiculaspora, Ephemerapareta, Pelotaspora).
   • Polonosporales ord. nov.: Contains the monogeneric family Polonosporaceae (genus: Polonospora).
3. Revision of Archaeosporales: The original order is retained but redefined to include only two families:
   • Archaeosporaceae: Genera Archaeospora, Intraspora, Palaeospora, and Andinospora.
   • Antiquisporaceae fam. nov.: A new family created for the genus Antiquispora, which was previously grouped within Archaeosporaceae but is phylogenetically distinct.
4. Taxonomic Clarification: The study clarifies the status of Intraspora (confirming it as a distinct genus, not a synonym of Archaeospora) and validates the separation of Antiquispora based on ~10% genetic divergence (Maximum Identity) and distinct morphological traits.

4. Results

• Phylogenetic Support:

  • The four proposed orders form well-supported monophyletic clades.
  • Bootstrap support values were generally >95% across all analyses for the major clades.
  • Ambisporales showed slightly lower support in the SSU-only tree (75% FBP) but remained robust in the combined dataset.
  • Antiquispora formed a distinct clade sister to Archaeosporaceae with >95% support.

• Morphological Differentiation:

  • Geosiphonales: Unique for forming endocytobiosis with Nostoc; spores are glomoid-like but lack the complex wall structure of AMF.
  • Polonosporales: Distinctive for forming spores directly on the neck of sporiferous saccules. They possess three permanent, thick spore walls (Outer, Middle, Inner), with two forming de novo. This contrasts with other orders where the outer wall is often semi-permanent or evanescent.
  • Ambisporales: Characterized by spore bimorphism (acaulosporoid and glomoid morphs). Acaulosporoid spores have three thick walls and form on pedicels. They stain pale blue in Trypan blue.
  • Archaeosporales:
    • Archaeosporaceae: Hyphae and structures generally do not stain or stain faintly in Trypan blue. Spores are small (<100 µm), thin-walled, and often lack vesicles.
    • Antiquisporaceae: Forms intraradical hyphae, vesicles, and spores that stain darkly in Trypan blue. Spores are small and hyaline.

• Wall Architecture: The study provides a detailed breakdown of spore wall layers (OW, MW, IW) and their formation mechanisms (e.g., separation from hyphal wall vs. de novo formation), which serves as a key diagnostic feature for the new orders.

5. Significance

• Evolutionary Insight: The revision aligns the taxonomy of Archaeosporomycetes with its evolutionary history, separating lineages that diverged early in the Glomeromycota radiation.
• Ecological Context: It formally recognizes the unique ecological strategy of Geosiphon (cyanobacterial symbiosis) as distinct from the plant-arbuscular mycorrhizal symbiosis of the other orders, justifying its separation at the order level.
• Diagnostic Clarity: By defining clear morphological criteria (spore wall permanence, staining reactions, bimorphism), the study provides robust tools for identifying these fungi in soil and root samples, resolving previous confusion regarding genera like Intraspora and Antiquispora.
• Foundation for Future Research: The establishment of these four orders creates a stable framework for future phylogenomic studies, ecological surveys, and the description of new species within this ancestral clade of arbuscular mycorrhizal fungi.

In conclusion, this paper successfully transitions the classification of Archaeosporomycetes from a morphology-based, single-order system to a phylogenetically robust, multi-order system that accurately reflects the diverse evolutionary pathways and ecological strategies of these fungi.