Integrative Molecular, Cytological, and Anatomical Analyses Reveal Two Distinct Clusters in Fittonia Cultivars

Fittonia Coem. is a widely cultivated ornamental foliage genus, but the taxonomy and relationships of cultivated forms remain poorly resolved because traditional classifications rely heavily on variable foliar traits. In this study, we investigated the genetic relationships and phenotypic variation among 14 commercially available Fittonia cultivars using amplified fragment length polymorphism (AFLP), methylation-sensitive amplified fragment length polymorphism (MSAP), DNA flow cytometry, chromosome counting, and comparative morphological and anatomical analyses. Morphological character states were further mapped onto AFLP- and MSAP-derived phylograms to assess their evolutionary patterns and taxonomic relevance. AFLP and MSAP analyses consistently recovered two well-supported clusters within the cultivated material examined: Clade I, comprising 'Titanic' and 'Angel Snow', and Clade II, comprising the remaining 12 cultivars. These clades were independently supported by stable differences in trichome morphology, cystolith distribution, epidermal cell form, leaf venation architecture, and stem anatomy. In contrast, several traditionally used diagnostic characters, including leaf vein color and petiole length, were found to be homoplastic or continuously variable. Chromosome counts and flow cytometry indicated that all cultivars were diploid (2n = 36) and exhibited limited variation in genome size. Together, these results provide integrative evidence for two distinct clusters within cultivated Fittonia. However, because sampling was limited to commercially available cultivars, the relationship of these clusters to currently recognized species remains unresolved. Our findings highlight the limitations of traditional trait-based classifications and underscore the need for broader sampling, including wild populations, to reassess species boundaries in the genus.