Four-marker plastid analysis places two unusual South American spleenworts within Asplenium

A molecular phylogenetic study found that the South American spleenworts Asplenium brasiliense and A. douglasii form a strongly supported sister pair within the A. schaffneria clade of Asplenium . Maximum-likelihood and Bayesian analyses of four chloroplast markers recovered the pair with 100% bootstrap support and a Bayesian posterior probability of 1.00, placing both species within the broadly circumscribed genus rather than supporting their segregation as a separate genus. Both species were originally described in Asplenium , in 1817 and 1829, but their simple fronds, anastomosing venation and paired indusia that open toward one another prompted transfers among genera including Hemidictyum , Antigramma , Phyllitis and Scolopendrium .

Subsequent work on stelar anatomy and spore ornamentation supported recognition of Antigramma as an independent genus, while a broader treatment of Asplenium eventually returned the species to their original generic placement. For the molecular analysis, researchers extracted DNA from silica-gel-dried leaf material of A. brasiliense and A. douglasii held in the Missouri Botanical Garden herbarium. They amplified four chloroplast markers: the atpB and rbcL genes, the rps4 and rps4–trnS regions, and the trnL and trnL–trnF regions.

Seven sequences for the two target species were newly generated. Initial BLAST comparisons associated the new sequences with the A. varians subclade of the A. schaffneria clade. The researchers then assembled available sequences from members of that clade and representatives of the remaining major Asplenium lineages.

Following alignment with MAFFT and manual adjustment in BioEdit, the concatenated matrix contained 3,599 base pairs and 115 samples representing 85 species. Maximum-likelihood inference used RAxML-HPC2 on the CIPRES Science Gateway, with 1,000 bootstrap replicates used to assess nodal support. Bayesian inference used MrBayes in two independent runs, each with four Markov chains and one million generations; samples were collected every 1,000 generations.

The first 25% were discarded as burn-in, and the reported average standard deviation of split frequencies was below 0.01, indicating convergence under the study’s criterion. Both approaches placed the target species within the A. schaffneria clade and recovered nine well-supported subclades. Asplenium brasiliense and A. douglasii formed a monophyletic group, with the two species sister to one another.

The authors designated this lineage the Antigramma subclade and recovered it as sister to the A. varians subclade. The molecular result places the pair firmly within Asplenium sensu lato despite their unusual combination of simple fronds, anastomosing venation and paired indusia, features that differ from those common in many other members of the genus. The study also compared seven morphological characters: degree of frond division, overall frond shape, the ratio of petiole length to lamina length, venation type, sorus morphology, sorus length relative to the vein, and the presence or absence of auriculate lobes or short basal pinnae.

Scanning electron microscopy was used to examine spore ornamentation in two individuals per species. A. brasiliense from Argentina, Brazil and Paraguay generally has lanceolate fronds, short petioles, linear sori densely arranged near the midrib, and cristate spores. A. douglasii has narrowly cordiform fronds, petioles equal to or longer than the lamina, narrow sori between the midrib and margin, and lophate spores.

Peruvian specimens tentatively assigned to A. brasiliense differed from the described pattern by having elliptic or, rarely, rhombiform laminae and sori confined mainly to the upper half of the lamina, generally midway between the costa and margin. The authors stated that this material may represent a new species but retained it under A. brasiliense because molecular evidence was lacking. That uncertainty applies specifically to the Peruvian material; it does not establish that all populations assigned to A. brasiliense have an unresolved taxonomic identity.

Both focal species are lowland ferns that grow terrestrially or among rocks. Their mapped distributions include the Atlantic Forest and neighboring regions extending into western Amazonia. The authors proposed that reduced leaf dissection might improve light capture under deep shade and that anastomosing venation might provide hydraulic redundancy against embolism, but these functional explanations were not directly tested by the phylogenetic and morphological analyses.

Simple fronds with anastomosing venation and paired indusia opening toward one another also occur in other Asplenium lineages, including the A. hemionitis subclade, the A. nidus complex and species formerly placed in Phyllitis and Scolopendrium . The authors interpreted this recurrence as evidence that these traits evolved independently multiple times within the genus.

Subject of Research: Plastid phylogenetic placement and morphology of two South American Asplenium species

Article Title: Phylogenetic placement of two taxonomically controversial South American spleenworts with unusual morphology: Asplenium brasiliense Sw. and Asplenium douglasii Hook. & Grev. ( Aspleniaceae )

Article References: Zhang, J.-S., Du, M.-H., Zhang, L.-B., & Xu, K.-W. (2026). Phylogenetic placement of two taxonomically controversial South American spleenworts with unusual morphology: Asplenium brasiliense Sw. and Asplenium douglasii Hook. & Grev. (Aspleniaceae). PhytoKeys, 278, 15-26. https://doi.org/10.3897/phytokeys.278.198424

Image Credits: AI Generated

DOI: 10.3897/phytokeys.278.198424

Keywords: Asplenium, plastid phylogeny, fern molecular systematics, A. brasiliense, A. douglasii, Antigramma, chloroplast markers, fern morphology, spore micromorphology, Phylogenetic, placement, taxonomically