Recurrent hybridisation events between Primula vulgaris, P. veris and P. elatior (Primulaceae, Ericales) challenge the species boundaries: using molecular markers to re‐evaluate morphological identifications

Abstract

Three Primula species, Primula vulgaris, P. veris and P. elatior, have been objects of fascination for gardeners and botanists over several centuries. The species are able to hybridise, and where they co‐occur, hybrids are commonly found. In Denmark, Møns Klint on the island of Møn and Købelev Skov on Lolland are examples of localities where all three species occur and where the hybrids P. × digenea, the hybrid between P. vulgaris and P. elatior, and P. × polyantha, the hybrid between P. veris and P. vulgaris, can also be found. To investigate relationships between the species and their hybrids, we sampled 168 specimens from 10 geographical locations and subjected them to genetic analysis. The samples were also identified based on morphological traits: primarily inflorescense structure, the size, shape, colour and markings of corolla and leaf basis, leaf blade texture and hairiness. After identifying species‐specific SNPs in the Internal Transcribed Spacer sequence, these were used to resolve species and hybrid boundaries and status through a cleaved amplified polymorphic sequence assay. Polymorphisms in the chloroplast trnL sequence were used as a high‐throughput marker and used to determine the maternal parent of hybrids. Ten simple sequence repeat markers were applied to obtain further insight into the genetic makeup of the accessions using structure and Introgress, providing information of genetic variability within and between populations. Our results indicated that backcrossing of P. × digenea hybrids with parental species has occurred, and that many of the P. × digenea sampled were later‐generation hybrids rather than F1s. Analyses of P. × polyantha specimens show mostly the expected pattern for primary hybrids but indications of P. veris ancestry of a P. vulgaris plant was discovered. Our results further indicate that some of the specimens initially identified as P. elatior include P. vulgaris among their progenitors and thus challenge currently accepted species boundaries.

Asymmetries of reproductive isolation are reflected in directionalities of hybridization: integrative evidence on the complexity of species boundaries

“…2b) than in a Hungarian contact site (Kálmán et al, 2003), implying stronger isolation in the latter at this postmating stage. Finally, a molecular analysis of a Danish contact site detected more introgression (Tendal et al, 2018) than in the Swiss site (Fig. 4), implying weaker postmating isolation in the former.…”

“…The total number of nuclear markers used to genotype the contact site was 20: seven microsatellite and 13 RAD-based markers (Table S2). Additionally, we used the maternally inherited plastid trnL (Tendal et al, 2018) to identify the maternal species of the hybrids and exon 3 of the CYP734A50 gene (CYP T ) to identify the maternal morph of the hybrids; CYP T encoding a cytochrome P450 that controls style length and is located in the hemizygous heterostyly supergene present only in S-morphs (Huu et al, 2016;Li et al, 2016). We also checked whether some of the generated trnL and CYP T sequences could be assigned to Primula elatior (whose sequences are available from GenBank and Huu et al (2016), respectively), because this species is known to hybridize readily with P. vulgaris but rarely with P. veris (Jacquemyn et al, 2009), and it is not reciprocally monophyletic to them (Schmidt-Lebuhn et al, 2012).…”

“…Additionally, patterns of floral variation in British and Hungarian contact sites prompted researchers to suggest that most phenotypically intermediate plants are F 1 hybrids, whereas backcrosses are rare (Clifford, 1958; Mowat, 1961; Woodell, 1965; Kálmán et al ., 2004). However, the only available genetic study, using one nuclear (ITS), one plastid ( trnL ), and 10 microsatellite markers, supported introgression in Danish contact sites (Tendal et al ., 2018). Lastly, no genetic studies have examined whether floral morphs impose directionalities on hybridization, although morph‐dependent asymmetries of reproductive barriers were shown in a different primrose species pair (Keller et al ., 2016).…”

“…Botanists and horticulturists alike have long known that hybridization frequently occurs between primrose species, both in nature and under cultivation (Richards, 2003), and phylogenetic studies show that hybridization plays a pivotal role in the evolutionary history of Primula (Guggisberg et al, 2008(Guggisberg et al, , 2009Casazza et al, 2012Casazza et al, , 2013Schmidt-Lebuhn et al, 2012;Cianchi et al, 2015;Boucher et al, 2016). Reports of natural hybridization in Primula are usually based on morphological identification of hybrids (Kerner, 1875;Lotsy, 1925;Woodell, 1965;Kálmán et al, 2004), but detailed population genetic analyses are scarce (but see Ma et al, 2014;Xie et al, 2017;Tendal et al, 2018).…”

Genetic Variability in Balkan Paleoendemic Resurrection Plants Ramonda serbica and R. nathaliae Across Their Range and in the Zone of Sympatry

“…However, it is already clear that hybridization between the two species is occurring and that, at the very least, backcrossing (in which tetraploid hybrids give rise to pentaploids) is possible. Unfortunately, backcrosses with one or both parental species often result in the appearance of individuals whose morphology is difficult to distinguish from the parental species, and identification of hybrids based on morphology becomes very difficult or impossible ( Lihová et al, 2007 ; Čertner et al, 2015 ; Tendal et al, 2018 ; Beirinckx et al, 2020 ), as seems to be the case with both sites of sympatry for Ramonda ( Lazarević et al, 2014 ).…”