title: Co-occurring Mediterranean orchids: insights from morphometrics, genetic diversity and introgression in two species: Ophrys fusca and Ophrys dyris
Creators: name: Alberto Abreu, Joana Isabel
Publisher: name: University of Reading
Year: 2022

description: Ophrys is amongst the best known orchid genera and is an established system for the study of 
pollinator mediated floral evolution. Two species, Ophrys fusca s.l. and Ophrys dyris (= O. omegaifera 
subsp. dyris), belonging to Ophrys section Pseudophrys are the focus of this study. An integrative study 
of morphological and genetic diversity of O. fusca and O. dyris was made, based on data collected 
from six populations in Central Portugal in five years. The populations were characterized a priori as 
“mixed” or pure”. The data included morpho-anatomical traits (272 plants) and phenological records 
and pollination success assessments (887 flowers from 260 plants). Additionally, sampling for DNA 
(165 plants) and leaf and pollinarium collection for cytological study (67 plants) was carried out. 
Multivariate analysis of the whole morphological dataset recovered two groups. When including only 
the plants common both to morphological and genetic datasets and a reduced number of diagnostic 
characters, three groups were found. Traits that better discriminate morphological groups are 
qualitative ones, namely the pilosity of the different lobes of the labellum. Univariate analysis of 
morpho-anatomical traits showed that vigour characters and depth of labellum indentation were the 
most variable, including between populations. Labellum indentation also showed greatest differences 
between the three types of populations (O. fusca/mixed/O. dyris). The study of flowering behaviour
revealed incomplete floral isolation between the two taxa. Discrepancy between morphotypes in the 
average time for the flowering period in different years might threaten the synchrony with the 
supposed specialist pollinator, required for an effective pollination by sexual deception. This type of 
disruption being confirmed, it could represent an advantage for the fusca plants regarding the 
availability of pollinators. Percentages of pollination success obtained range from 2% to 20%. No 
significant differences in pollination success were found between plants confirmed as parental species 
or hybrids based on genetic data. Flow cytometry methods revealed constancy of nuclear DNA content 
(1C = 11.19 pg) in all the specimens analysed, including species and putative hybrids. Constancy of 
cytotypes was also confirmed, based on chromosome counts from the roots of two specimens, one of 
each species (2n = 4x = 72, 74). 72 for fusca, 74 for dyris. Following microsatellite data analysis, all the 
populations considered included individuals with mixed genotypes, representing a total of 44.8% of 
the plants. Genetic hybrids were found in all but one population, refuting the initial assumption of 
“pure” (one taxon) versus “mixed” populations. When comparing genetic and morphological datasets, 
despite a positive correlation, strong discrepancies have been found between the composition of the 
morphological groups and the genetic clusters. Implications are considered, in terms of predicting the 
persistence of putative hybrids and the conservation of evolutionary processes.