Genome-based reclassification of Lentzea roselyniae (Okoro et al. 2010) Nouioui et al. 2018 and Lentzea terrae Li et al. 2018 as later heterotypic synonyms of Lentzea atacamensis (Okoro et al. 2010) Nouioui et al. 2018 and Lentzea soli Li et al. 2018, respectively

Technical Summary: Genome-Based Reclassification of Lentzea Species

Problem Statement
The taxonomy of the genus Lentzea (family Pseudonocardiaceae) has undergone significant revision due to the integration of genomic data, which often reveals that species described based on phenotypic characteristics are actually later heterotypic synonyms. This study addresses the taxonomic ambiguity surrounding two specific pairs of closely related Lentzea strains: Lentzea atacamensis DSM 45479^T versus Lentzea roselyniae JCM 17494^T, and Lentzea soli JCM 32384^T versus Lentzea terrae NEAU-LZS 42^T. While these strains were originally described as distinct species, preliminary observations suggested high genomic similarity, necessitating a comprehensive re-evaluation to determine if they represent the same species.

Methodology
The authors employed a multi-faceted approach combining phylogenomic, genomic, phenotypic, and chemotaxonomic analyses:

1. Dataset Curation: High-quality genome sequences for 29 Lentzea type species were retrieved from NCBI and filtered using CheckM2 to ensure ≥95% completeness and ≤5% contamination.
2. Phylogenetic Analysis:
   • 16S rRNA: Pairwise sequence identities were calculated, and phylogenetic trees were reconstructed using Neighbor-Joining (NJ), Maximum-Likelihood (ML), and Maximum-Parsimony (MP) algorithms.
   • Phylogenomics: Two independent frameworks were utilized: the Genome Taxonomy Database Toolkit (GTDB-Tk) using 120 conserved single-copy bacterial marker genes (bac120) and the Type (Strain) Genome Server (TYGS) using the Genome BLAST Distance Phylogeny (GBDP) method.
3. Genome Relatedness Indices (OGRIs): Pairwise comparisons were conducted to calculate Average Nucleotide Identity (ANI) via OrthoANI, digital DNA–DNA hybridization (dDDH) via GGDC, and Average Amino Acid Identity (AAI) via CompareM.
4. Comparative Genomics: Gene content was analyzed using OrthoVenn3 to identify orthologous clusters. Metabolic potential was assessed via KEGG pathway module analysis, and secondary metabolite biosynthetic gene clusters (BGCs) were predicted using antiSMASH.
5. Phenotypic and Chemotaxonomic Review: Original descriptions were reviewed to compare phenotypic traits (e.g., mycelial color, carbon source utilization, hydrolysis activities) and chemotaxonomic markers (menaquinones, polar lipids, fatty acids, whole-cell sugars).

Key Results

• Phylogenetic Congruence:
  • L. atacamensis and L. roselyniae shared 99.59% 16S rRNA gene similarity.
  • L. soli and L. terrae shared 99.07% 16S rRNA gene similarity.
  • Both pairs formed highly supported monophyletic lineages (bootstrap values ≥95% for 16S; 100% for phylogenomic trees) in both GTDB and GBDP frameworks.
• Genome Relatedness:
  • The L. atacamensis / L. roselyniae pair exhibited ANI, dDDH, and AAI values of 98.67%, 87.90%, and 98.42%, respectively.
  • The L. soli / L. terrae pair showed even higher similarity with ANI, dDDH, and AAI values of 99.76%, 98.7%, and 99.81%, respectively.
  • All values significantly exceeded the standard species delineation thresholds (ANI >95–96%, dDDH >70%, AAI >95%).
• Comparative Genomics:
  • The strain pairs shared extensive orthologous gene repertoires (7,488 and 8,630 shared clusters, respectively) with very few strain-specific clusters.
  • Metabolic profiles were highly conserved; L. soli and L. terrae possessed identical KEGG module profiles. L. atacamensis and L. roselyniae showed minor differences in specific biosynthetic pathways (e.g., amino acid vs. lipid metabolism) but shared a broadly conserved metabolic framework.
  • Secondary metabolite BGCs were largely conserved between pairs, with minor variations typical of strain-level diversity.
• Phenotypic Discrepancies:
  • Despite high genomic similarity, the original descriptions noted phenotypic differences (e.g., mycelial color, H₂S production, carbon source utilization). The authors attribute these to known phenotypic heterogeneity within Lentzea caused by cultivation conditions or accessory genomic variation, concluding they are insufficient to define separate species.

Key Contributions and Claims
Based on the convergence of genomic, phylogenomic, and chemotaxonomic evidence, the study proposes the following taxonomic reclassifications:

1. Lentzea roselyniae (Okoro et al. 2010) Nouioui et al. 2018 is reclassified as a later heterotypic synonym of Lentzea atacamensis (Okoro et al. 2010) Nouioui et al. 2018.
2. Lentzea terrae Li et al. 2018 is reclassified as a later heterotypic synonym of Lentzea soli Li et al. 2018.

The authors provide emended descriptions for both L. atacamensis and L. soli to incorporate the characteristics of the newly synonymized strains. These emendations expand the known ranges of phenotypic variability (e.g., mycelial color, carbon source utilization, temperature tolerance) and genomic features (genome size and G+C content) for these species.

Significance
The paper demonstrates that genome-based analyses are essential for resolving taxonomic ambiguities in Lentzea where phenotypic differentiation is limited or variable. By applying the principle of nomenclatural priority, the study clarifies the species boundaries within the genus, reducing redundancy in the taxonomic list and providing a more accurate framework for future ecological and physiological studies of these actinomycetes. The work underscores that observed phenotypic differences between type strains do not necessarily indicate species-level separation when supported by high genome relatedness indices.