# Shape and Size Variability of the Gynostemium in Epipactis helleborine (L.) Crantz (Orchidaceae)

## TL;DR

This study examines the shape and size variability of the gynostemium in Epipactis helleborine to improve species identification.

## Contribution

The study introduces a detailed geometric morphometric analysis of gynostemium variability for taxonomic identification of E. helleborine.

## Key findings

- Gynostemium shape and size variation is primarily driven by population differences.
- Ventral view of the gynostemium is most informative for distinguishing morphological differences.
- Statistical analyses confirm significant correlations between gynostemium width and height.

## Abstract

Morphological variability of the gynostemium structure in Epipactis helleborine (L.) Crantz complicates species identification. A total of 122 flowers of E. helleborine were collected, prepared, and analysed for gynostemium morphological variation. Geometric morphometric methods (GMMs) and statistical tests were used to assess variation in the shape, size, and stigma inclination angle among populations, individual plants (ramets), and years of research. The results indicate that most variations in gynostemium shape and size show a correlation with population, with relatively little variation within populations or between research years. Both morphometric parameters and the overall structural pattern of gynostemium morphology, including dorsal, frontal, ventral, and right lateral views, should be considered for taxonomic identification of E. helleborine.

Epipactis helleborine (L.) Crantz is considered a challenging and phenotypically difficult species to identify due to its wide range of morphological variability. This variability is mainly observed in the perianth parts but also extends to the gynostemium structure, which has so far been considered one of the most useful diagnostic characteristics. As a result, a simple graphic illustrating the structural pattern of gynostemium morphology has appeared in 10 different forms in available European taxonomic keys, which significantly complicates the identification of this species. A total of 122 flowers of E. helleborine were collected from four natural populations in the Lower Silesia region (Poland) between 2017 and 2019 and analysed for gynostemium morphological variation. Geometric morphometric analyses, including Procrustes ANOVA, PCA, and CVA, were used to examine gynostemium shape, with statistical tests assessing variation in size and stigma inclination angle among populations, individual plants (ramets), and years of research. Statistical analysis revealed significant positive correlations between gynostemium width and height, with significant variation in size and angle of stigma inclination, primarily driven by population, while ramet and year of research had a lesser impact. Geometric morphometric analyses indicated significant population-level variation in gynostemium shape, with principal component analysis identifying the ventral view as the most informative for discriminating these differences. The first two principal components explained the major shape variation, and canonical variate analysis confirmed that this view is most important for species identification.