Aquatic Botany 105 (2013) 34–40 Contents lists available at SciVerse ScienceDirect Aquatic Botany jo u r n al hom ep age: www.elsevier.com/locate/aquabot Molecular identification of hybrids from a former hot spot of Potamogeton hybrid diversity Zdenek Kaplan ∗ , Judith Fehrer Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pr˚ uhonice, Czech Republic a r t i c l e i n f o Article history: Received 14 December 2011 Received in revised form 8 November 2012 Accepted 20 November 2012 Available online 20 December 2012 Keywords: Hybridization Taxonomy Potamogeton Molecular identification Diversity Aquatic vegetation a b s t r a c t More than a century ago, an extremely high diversity of Potamogeton hybrids was recorded in several rivers and streams in Jutland, Denmark. Accuracy of their identification was doubted by some later authors who were sceptical about the existence and co-occurrence of so many hybrids. Selected original local- ities were investigated for the presence of morphologically matching plants after more than 100 years. In spite of profound changes in landscape and considerable deterioration of aquatic habitats during the 20th century resulting in a significant decline of submerged vegetation, three of ten previously recorded hybrids were recently found that had persisted at their original localities. Two of them, whose exist- ence had not been proved previously, were subjected to molecular analyses. RFLP, direct sequencing and cloning of the ITS region confirmed their previous morphological identification: P. × undulatus as P. crispus × P. praelongus and P. × cognatus as P. perfoliatus × P. praelongus. Chloroplast DNA sequencing identified P. praelongus as the maternal parent in both crosses. The existence of most of the other hybrid combinations recorded from Jutland was proved by means of molecular analyses conducted on plants from other regions. Their morphologies perfectly correspond to old herbarium vouchers from Jutland and support their original identifications. These observations indicate that Jutland rivers and streams hosted a high species and hybrid diversity still in the late 19th century, but most of this richness has meanwhile disappeared. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Hybridization is one of the key processes contributing to the taxonomic complexity in Potamogetonaceae (Kaplan et al., 2009; Kaplan, 2010a; Kaplan and Fehrer, 2011; Kaplan and Uotila, 2011). Although the occurrence of hybrids in Potamogeton was first sug- gested more than a century ago (see the reviews in Wiegleb et al., 2008; Kaplan et al., 2009), molecular studies have only recently provided firm evidence for the existence of many of them (e.g., King et al., 2001; Fant et al., 2003, 2005; Kaplan and Fehrer, 2004, 2006, 2009; Ito et al., 2007; Wang et al., 2007; Du et al., 2009; Kaplan et al., 2009; Zalewska-Gałosz et al., 2009; Zalewska-Gałosz and Ronikier, 2010). DNA sequencing and RFLPs have contributed to the discovery and/or exact identification of several entirely new hybrid combinations (Kaplan et al., 2009, 2011; Du et al., 2010; Zalewska-Gałosz et al., 2010; Kaplan and Fehrer, 2011; Zalewska- Gałosz and Ronikier, 2011) and even confirmed the existence of a triple hybrid in Potamogeton (Kaplan and Fehrer, 2007). Hybridiza- tion thus markedly increases the taxonomy-relevant diversity in Potamogeton (Kaplan, 2010a). ∗ Corresponding author. Tel.: +420 271 015 253; fax: +420 267 750 031. E-mail address: kaplan@ibot.cas.cz (Z. Kaplan). The geographic distribution of Potamogeton hybrids is highly uneven. Wiegleb (1988) identified Great Britain, Denmark, Sweden, and Japan as centres of hybridization in Potamogeton. High hybrid diversity was recently identified also in the north-eastern U.S.A. (Kaplan et al., 2009). Although at least 86 hybrid combinations have been convincingly documented in the Potamogetonaceae, almost all were recorded in a relatively few regions of the Northern Hemisphere (for a review, see Kaplan, 2010a). Only recently, a Pota- mogeton hybrid was identified also in the Southern Hemisphere, in Australia (Kaplan et al., 2011). In Europe, there is a clear gradient of hybrid occurrence towards higher latitude: both hybrid diversity and the number of locali- ties with hybrids increase towards northern Europe (Kaplan, 2007, 2010a; Kaplan et al., 2009). This distribution pattern is typically associated with suitable habitats occurring in a previously glaciated landscape (Kaplan, 2007). The northern regions host the highest diversity with about 25–30 hybrids (Kaplan, 2010a). Some areas of Denmark used to be rich in Potamogeton hybrids in the past. Johannes Schönberg Baagøe (1838–1905), a pharmacist in Næstved, was an extremely active field botanist and particu- larly enthusiastic about Potamogeton. In the period from 1893 to 1905 he collected several thousands of specimens, professionally preserved them for his herbarium and identified many of them as hybrids. He widely distributed his duplicates to various herbaria 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2012.11.002