Inferring evolutionary relationships among Crenotia species (Bacillariophyta): Evidence from natural populations and monoclonal strains from Slovakia

This study establishes the first molecular framework for the genus *Crenotia* by combining morphological and multilocus phylogenetic analyses of Slovakian populations to confirm the monophyly of three distinct species and clarify their evolutionary relationships within monoraphid diatoms.

The Gist

Imagine a bustling, microscopic city built by tiny, single-celled architects called diatoms. These architects build intricate glass houses (their shells, or "frustules") with amazing patterns. For a long time, scientists tried to organize these architects into families based solely on the shape of their glass houses.

However, just like in human history, families sometimes split, merge, or change their appearance so drastically that it's hard to tell who is related to whom. This is the story of a specific group of diatoms called Crenotia.

Here is the story of this paper, broken down into simple concepts:

1. The Mystery of the "One-Door" House

Most diatoms have a special feature called a raphe, which is like a zipper or a door that allows them to move. Usually, they have two doors (one on each side of their shell). But some diatoms, like the Crenotia, lost one of their doors during evolution. They became "monoraphid" (one-door) diatoms.

For years, scientists thought all these "one-door" diatoms were distant cousins. But recent DNA detective work showed that losing a door happened many times independently. It's like how birds and bats both have wings, but they aren't closely related; they just both figured out how to fly. The question was: Where exactly does Crenotia fit on the family tree?

2. The Detective Work: DNA vs. The Eye

The researchers (Alica, Pavla, and Jana) went to Slovakia to find these diatoms. They didn't just look at them under a microscope; they went to their "homes":

• Hot Springs: Some lived in warm, mineral-rich water bubbling up from the earth (like natural hot tubs).
• Gravel Pits: Others lived in a man-made lake with cooler, cleaner water.

They collected samples and grew them in the lab (like taking a single seed and growing a whole garden to study it). Then, they did two things:

1. The Microscope Check: They looked at the tiny glass houses to see their shape, size, and patterns.
2. The DNA Check: They read the genetic code (the "instruction manual") of the diatoms to see who their real relatives were.

3. The Big Discovery: Three Distinct Families

The study confirmed that there are three distinct species of Crenotia, and they are all closely related to each other, forming their own unique family branch.

Think of it like a family with three siblings who look similar but have different personalities:

• Sibling 1 (C. thermalis): The "Big Brother." He is the largest of the three and lives in the hot, mineral-rich springs. He has a smooth, rounded look.
• Sibling 2 (C. rumrichorum): The "Artistic Cousin." He is smaller and has a unique feature: a little "rimmed depression" on his shell (like a tiny bowl carved into the glass). He lives in the cooler gravel pit lake.
• Sibling 3 (Crenotia sp.): The "Mystery Sibling." He looks very similar to an old, unnamed species from a textbook. He lives in a very hot, mineral-heavy spring. The scientists are still figuring out his exact name because they need to check the original "blueprints" from 100 years ago.

4. The Family Tree Surprise

When the scientists built the family tree using DNA, they found something fascinating.

• The Old Theory: They thought Crenotia might be related to a very common group of diatoms called Achnanthidium (the "neighborhood" diatoms).
• The New Reality: The DNA proved that Crenotia is actually a cousin to a different group called Planothidium and some other "two-door" diatoms.

It's like discovering that your family isn't related to the neighbors you've known for years, but rather to a distant, wealthy clan you never met before. The "one-door" trait evolved separately, making them look similar to other one-door diatoms, but their DNA tells a different story.

5. Why Does This Matter?

This paper is important because it clears up the confusion.

• It's not just about looks: You can't always tell species apart just by looking at their shells; you need their DNA.
• They are good indicators: Because these diatoms are picky about where they live (some love hot springs, others love cool lakes), finding them tells us a lot about the health and chemistry of the water.
• Evolution is tricky: It shows that nature often finds the same solution (losing a door) in different ways, creating "look-alikes" that aren't actually related.

The Takeaway

Imagine a group of tiny glass artists who all decided to remove one of their doors. For a long time, we thought they were all part of the same guild. This study is like finding their birth certificates and realizing: "Ah! These three specific artists are actually a tight-knit family, they are related to a different group of artists, and they each have their own unique style and favorite neighborhood."

The researchers have now drawn the correct map of this family, ensuring that future scientists know exactly who belongs where in the microscopic world.

Technical Summary

1. Problem Statement

• Taxonomic Ambiguity: The classification of diatoms, particularly monoraphid genera (those with a raphe on only one valve), has historically relied on morphology. The genus Crenotia (established by Wojtal in 2013 from Achnanthidium thermale) is defined by specific morphological traits (zig-zag areolae arrangement, bent frustules) but lacks molecular validation.
• Phylogenetic Uncertainty: Monoraphid diatoms have evolved independently multiple times from biraphid ancestors, leading to polyphyletic groups and blurred taxonomic boundaries. The phylogenetic placement of Crenotia relative to other monoraphid genera (e.g., Achnanthidium, Planothidium) and biraphid orders (e.g., Cymbellales) remained unresolved.
• Species Delimitation: It was unclear whether observed morphological variations in Crenotia represented distinct species or merely intraspecific allometry (life-cycle changes) or environmental plasticity.

2. Methodology

The study employed an integrative approach combining detailed morphological analysis with multilocus phylogenetics.

• Sampling:
  • Locations: Five sites in Slovakia were sampled (2021–2022): four mineral/thermal springs (Močiar, Rojkov, Gánovce, Sivá Brada) and one gravel pit lake (Zlaté Piesky).
  • Habitats: Ranged from acidic, highly mineralized thermal springs (14.7–21.9°C) to alkaline, low-mineralization lake environments.
• Culture and Isolation:
  • Monoclonal strains were isolated via single-cell picking into WC medium.
  • Cultures were maintained for long-term study and DNA extraction.
• Morphological Analysis:
  • Light Microscopy (LM): Examined natural populations and cultures to assess frustule shape, size, and colony formation.
  • Scanning Electron Microscopy (SEM): Used to visualize ultrastructural details, including raphe structure, striae arrangement (uniseriate vs. biseriate), central area morphology, and the "rimmed depression" on rapheless valves.
• Molecular Phylogenetics:
  • Markers: Four molecular markers were sequenced: nuclear 18S rDNA, 28S rDNA (D1-D2 region), and plastidial rbcL and psbA.
  • Dataset: A concatenated alignment of 216 taxa (including 20 new Crenotia strains and GenBank sequences) totaling 5,748 base pairs.
  • Analysis: Maximum Likelihood (ML) phylogeny was constructed using IQ-TREE with ModelFinder for partition selection. Node support was assessed via Ultrafast Bootstrap (UFB) (1,000 replicates).
  • Topological Testing: An Approximately Unbiased (AU) test was performed to statistically evaluate alternative phylogenetic placements of Crenotia.

3. Key Results

A. Morphological Characterization

The study confirmed three distinct species within the genus Crenotia based on a combination of size, valve shape, and ultrastructure:

1. Crenotia thermalis: The largest species (9.8–34.7 µm). Valve ends are broadly rounded/subcapitate. It lacks a distinct rimmed depression on the rapheless valve. Found in thermal springs.
2. Crenotia sp. (Unnamed): Small valves (8.3–14.9 µm) with capitate to subrostrate ends. Morphologically similar to Achnanthes grimmei var. hyalina but distinct in habitat and ultrastructure. Found in high-ion springs.
3. Crenotia rumrichorum: Small valves (8.3–18.5 µm) with subrostrate ends. Key diagnostic feature: a distinct rimmed depression on the rapheless valve (visible as a shadow in LM). Found in the gravel pit lake.

• General Features: All species exhibit a characteristic "saddle-shaped" bending of the frustule (visible as a double image in LM) and biseriate striae arranged in a zig-zag pattern.

B. Phylogenetic Relationships

• Monophyly: The genus Crenotia forms a well-supported monophyletic clade (UFB = 100).
• Placement: Crenotia is placed within a subgroup of monoraphid genera closely related to Cymbellales and other biraphid diatoms.
• Sister Group: The AU test and ML analysis suggest Crenotia is most closely related to the genus Planothidium (UFB = 98), rather than the Achnanthidium minutissimum complex (which was statistically rejected).
• Intraspecific Variation: Low genetic variability was observed within species. In C. thermalis, a single base-pair difference in the rbcL marker was found, but this did not correlate with morphological differences or distinct ecological niches, confirming it as intraspecific variation.

C. Ecological Differentiation

• Habitat Specificity: Despite phylogenetic closeness, the species occupy distinct ecological niches.
  • C. thermalis and Crenotia sp. thrive in mineralized, slightly acidic thermal springs.
  • C. rumrichorum inhabits a man-made gravel pit lake with lower mineralization and higher pH.
• Convergence: The study highlights that phylogenetic proximity does not strictly dictate habitat similarity; closely related species can adapt to vastly different hydrochemical conditions.

4. Key Contributions

1. First Molecular Framework: This study provides the first multilocus phylogenetic framework for the genus Crenotia, confirming its monophyly and resolving its evolutionary position within the diatom tree of life.
2. Species Validation: It validates the existence of three distinct species using an integrative taxonomy approach (morphology + genetics), distinguishing them from historical synonyms and Achnanthidium complexes.
3. Refinement of Morphology: The study offers an emended description of the genus, detailing ultrastructural features (e.g., the rimmed depression in C. rumrichorum) that serve as reliable diagnostic characters.
4. Phylogenetic Placement: It challenges the assumption that Crenotia is closely related to Achnanthidium, instead linking it to Planothidium and the Cymbellales lineage.

5. Significance

• Taxonomic Clarity: The research resolves long-standing confusion regarding the classification of Crenotia and related taxa, providing a stable foundation for future biodiversity assessments.
• Evolutionary Insight: It contributes to the understanding of the repeated evolutionary loss of the raphe in diatoms, demonstrating that monoraphid lineages can be closely related to specific biraphid groups (like Cymbellales) rather than forming a single monophyletic group.
• Bioindication: Given the specific habitat preferences of these species (thermal springs vs. gravel lakes), Crenotia species are identified as valuable bioindicators for specific hydrochemical environments, particularly mineral springs.
• Methodological Benchmark: The study demonstrates the necessity of combining monoclonal cultures with natural population sampling to accurately distinguish between life-cycle allometry and true speciation in diatoms.