In many aquatic plant taxa, classification and identification based on morphology has always been difficult. Molecular markers revealed that the complexity in several of these aquatic taxa could be addressed to recurrent hybridization events and cryptic species diversity. The submerged macrophyte genus Ruppia is one of these aquatic genera with a complex taxonomy due to the absence of clear distinguishable traits and several hybridization events. Two species co-exist throughout Europe, R. maritima and R. spiralis (previously known as R. cirrhosa), but recent molecular studies also found several indications of hybridization, introgression and chloroplast capture between these species. However, the full extent and frequency of hybridization and introgression in this genus has not been studied so far, nor is it clear how these hybrid lineages can co-exist locally with their parental species. In this paper, we wanted to detect whether a single coastal wetland where both species co-exist can act as a Ruppia hybrid zone. As a case study, we chose the Camargue, a Mediterranean coastal wetland that harbours a wide diversity in aquatic habitats, especially in terms of salinity and hydro regime. We sampled several Ruppia populations within this wetland. To identify each sample and reconstruct the local genetic structure of the two parental species and their hybrids, we used both chloroplast and nuclear microsatellite markers. Afterwards, we tested whether different species had different habitat preferences. Our results confirmed that R. maritima and R. spiralis are two strongly divergent species with different reproductive ecologies and different habitat preferences. This prevents frequent hybridization and consequently we could not detect any trace of a recent hybridization event. However, we found several populations of later-generation hybrids, including a population of R. maritima x hybrid backcrosses. These hybrid populations occupy a different habitat and are genetically distinct from their parental species, although they tend to be morphological similar to parental R. maritima. Although hybridization and introgression in Ruppia is less frequent than we expected, the taxonomy of Ruppia is complicated due to ancient hybridizations and several back-crossings.  

• Ruppia plants were sampled in May 2014 from 20 locations within the French regional park of the Camargue (Figure 1A). In each location, we collected 30 different ramets at 1.5m intervals along a linear transect. 

• Plant DNA was extracted from dried leaf tissue using the E.Z.N.A (R) HP Plant DNA Mini Kit Protocols (Omega bio-tek, Norcross GA, USA).  

• We used two sets of microsatellites, each set was combined in a multiplex: one comprised species-specific microsatellites for R. maritima, the second multiplex comprised species-specific microsatellites for R. spiralis. Based on species assignment (R. maritima, R. spiralis  or hybrid), we used respectively the first, the second or both multiplexes. 

• We used a QIAGEN multiplex PCR Plus kit to generate DNA-fragments with a PCR reaction in a thermal cycler (MJ research PTC-200 and Bio-Rad My Cycler). These fragments were run on an ABI3730XL sequencer (Macrogen, Seoul, Korea).

• We manually scored the results with GeneMarker V2.20 (SoftGenetics LLC®)

• All samples with more than 50% missing values are removed from the dataset, except for the hybrid populations