127 | Integrated taxonomy methodology applied to phycological herbarium samples from southern Madagascar

Valerio Avola¹, Marina Morabito¹, Line Le Gall², Antonio Manghisi¹ | ¹Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; ²Institut de Systématique, Évolution, Biodiversité ISYEB, Muséum National d’Histoire Naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Paris, France.

Our research group was entrusted with the study of Halymeniacean herbarium samples collected during the Atimo Vatae expedition [1], held in the spring 2010 in southern Madagascar, housed in the cryptogamic collection (PC) in the Herbarium of Muséum National d’Histoire Naturelle de Paris (MNHN), France, as silica-dried and herbarium samples. They were subjected to a preliminary DNA barcoding screening, which showed two putative new species of Austroepiphloea among ten herbarium specimens. Therefore, the samples of the putative new species were chosen for further in-depth analyses [2], which consist of an integrated genomic and morpho-anatomical approach. For the genetic study, effective protocols were formulated to address the poor efficiency of extraction and amplification conditions available in the literature caused by low DNA quantity or integrity. The extraction protocols for red algae combined liquid nitrogen sample disruption with home-made lysis solutions, using low risk chemicals, with commercially available silica filters for nucleic acid purification, followed by PCR amplification and sequencing of nuclear, mitochondrial, and plastid genes (LSU, COI-5P, rbcL), applying modulation of reagent concentration and specific additives in PCR mixtures, serial dilution of DNA templates, selection of taxon-specific PCR primers, design of step-up and step-down cycling programs. In order to verify the supposed species, a distance analysis was performed for COI-5P, using the Neighbor Joining algorithm with a Kimura-2 substitution model. Later, phylogenetic analyses were performed by a combined multigene approach: generated sequences were included in an alignment together with other sequences of Halymeniacean taxa from GenBank and analysed with RAxML (bootstrap 2000) to obtain a Maximum Likelihood tree and infer phylogenetic relations. The morpho-anatomical study of the herbarium specimens aimed to describe vegetative and reproductive features essential for the description of new taxa. It consists of the following steps: observation of herbarium sheets using a stereomicroscope (Leica MZ 12); hand-sections by obsidian chips of rehydrated fragments, coloration with aniline blue and Congo red, observed using an optical microscope (Leitz Laborlux 12); micro-photographs taken using a camera (Nikon D5100) mounted on the microscopes, coupled with the software digiCamControl V.2.1.6.0, focus stacking images of multiple shots elaborated with Adobe Photoshop 2025; measurement of the images made by comparison with micrometric slide. Our integrated taxonomy methodology confirmed the presence of two new species of Austroepiphloea, temporary named Austroepiphloea sp.1, Austroepiphloea sp.2 and revealed a new species of Halymenia, temporary named Halymenia sp. aff. plana, previously misidentified as Austroepiphloea [3].