View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Plant Syst Evol (2008) 273:179–189 DOI 10.1007/s00606-008-0010-6 ORIGINAL ARTICLE Do they really hybridize? A field study in artificially established mixed populations of Euphrasia minima and E. salisburgensis (Orobanchaceae) in the Swiss Alps Burgi Liebst Received: 20 June 2006 / Accepted: 21 December 2007 / Published online: 15 May 2008 Ó Springer-Verlag 2008 Abstract Hybridization and introgression in the Euro- subsections taxonomy is difficult due to large intraspecific pean species of Euphrasia depend on the relationships variability, to small interspecific differences and to the between the species, on flower size and habitat. Hybrid- occurrence of hybrids (Yeo 1968). The close relatedness of ization between Euphrasia minima and Euphrasia some of the species within the same subsection is reflected salisburgensis was investigated in their natural habitat by successful interspecific cross-pollination (Liebst and using artificial sympatric populations of both species in the Schneller 2005; Yeo 1966, 1976). Artificial crosses of taxa Swiss Alps. The insect behavior in the populations sug- of different subsections may result either in low seed set or gests, that cross-pollination is likely to occur. A number of in a seed set similar to that resulting from intraspecific putative hybrids were detected by morphological charac- crossing or selfing. The F1 hybrids of such crosses are either teristics, and their hybrid origin was verified using RAPD sterile or bear only a few seeds (Liebst 2006; Yeo 1966). In analysis. The predominance of RAPD bands in one of the a few cases, hybridization between diploid and tetraploid species and the occurrence of these bands in some plants of Euphrasia species has been observed (Liebst and Schneller the second species point to earlier introgression events. The 2005; Pugsley 1930; Yeo 1956). number of hybrids found in the artificial populations Artificial pollination may illustrate the interfertility of together with results of earlier studies indicate that insect species, but cannot be used to estimate the probability or visits and cross-pollination in small-flowered Euphrasia the frequency of interspecific cross-pollination in nature. species in lower alpine regions may be more common than Preconditions for hybridization between insect-pollinated has been suggested in the past. species include the occurrence of at least two species in the immediate neighborhood and of overlapping flowering Keywords Euphrasia minima Á periods. Mixed populations of Euphrasia species have Euphrasia salisburgensis Á Field experiment Á frequently been found in Europe (von Wettstein 1893; Insect behavior Á Hybridization Á Introgression Á Yeo 1966). The probability of interspecific cross-polli- RAPD Á Discriminant analyses nation in these populations depends on the availability of pollinators and on the breeding system of the taxa, which is strongly associated with the size of the corolla (von Introduction Wettstein 1896; Yeo 1966, 1978a; French et al. 2005). Cross-pollination is common in large-flowered species, The Euphrasia species of the Northern Hemisphere (section whereas the small-flowered species are predominantly Euphrasia) are hemiparasitic herbs of either subsection selfing. In alpine populations of the small-flowered Ciliatae or subsection Angustifoliae. Within both E. minima and E. willkommii few or no flower visitors have been detected (Kreisch 1996; Gomez 2002). How- ever, a few hybrids of small flowered alpine Euphrasia & B. Liebst ( ) taxa have been found in the last two centuries suggesting Institut fu¨r systematische Botanik, Universita¨tZu¨rich, Zollikerstr. 107, 8008 Zu¨rich, Switzerland that cross-pollination at least occasionally occurs (see e-mail: [email protected] references in von Wettstein 1896; Vitek 1986). 123 180 Plant Syst Evol (2008) 273:179–189 Both the small and the large flowers of Euphrasia are Euphrasia, there are strong differences in their quality as adapted to the same type of pollinators (Yeo 1968) and are host plants (Yeo 1964; Matthies 1998). mainly visited and pollinated by flies, hover flies (Diptera, Both E. minima and E. salisburgensis are widespread in Syrphidae) and bees (Hymenoptera, Apidae s.l.). Accord- the Alps. In Switzerland, the altitudinal distribution of E. ing to Schultz (see Knuth 1909) the nectary is well salisburgensis ranges from colline to alpine regions, while developed in the larger-flowered species and less well E. minima is usually restricted to subalpine and alpine developed or absent in the smaller-flowered ones. Pollen regions (Hess et al. 1972). E. salisburgensis grows mainly seems to be at least as much an attraction as the nectar, on basic soils, E. minima prefers acidic substrates. The particularly for Syrphidae (Yeo 1968). The mechanisms of species can be morphologically separated by the two main pollination in Euphrasia and the flower biology have been characters used for the separation of the subsections: in described in detail by von Wettstein (1896) and Yeo subsection Ciliatae ciliate capsules and leaves with con- (1968). tiguous teeth; in subsection Angustifoliae glabrous capsules The tetraploid, small-flowered species E. minima (sub- (or capsules with few small cilia) and leaves with at least section Ciliatae) and E. salisburgensis (subsection some teeth distant (Yeo 1978). The color of the corolla is Angustifoliae) are among the most common Euphrasia yellow or white in E. minima and white or lilac in E. species in the Swiss Alps. Despite their different ecological salisburgensis. Like most Euphrasia flowers they also have preferences, sympatric and parapatric populations occur. violet longitudinal veins forming guide marks that con- Both species are successfully selfing (Liebst 2006). It is verge to the throat (Yeo 1966) and yellow spots on the unlikely that their flowers may attract many insects, how- lower lip and throat. ever, Yeo (1966) argued that even between small-flowered Euphrasia species crosses may be common. Because E. Establishment of the artificial populations minima and E. salisburgensis belong to different subsec- tions, it is expected that hybrids are highly sterile (Yeo Two to three ripe fruits from about 400 individuals of E. 1968). Nevertheless, an artificial F2 generation was raised minima and E. salisburgensis were collected at three in a garden experiment from seed resulting from artificial locations in the Swiss Alps in large sympatric or parapatric selfing and crossing of F1 hybrids of E. minima and E. populations in autumn 2001: (1) Canton Tessin, Piora (PI); salisburgensis, and from artificial back-crossing of the F1 Alpe Tom (2,049 m, 46°32051.6900N8°41019.6500E) to hybrids with the parental species (Liebst 2006). Cadagno di fuori, Cadagno di dentro and Alpe di Piora So far, inter- and intraspecific crossing and selfing in (2,013 m, 46°32050.6200N8°42056.3300E); (2) Canton Uri, Euphrasia have been investigated exclusively by artificial Andermatt (AM); Na¨tschen (rail stop Matterhorn–Gotthard pollination (Liebst 2006; Yeo 1976). In the present study, Bahn, 1,890 m, 46°38038.6600N8°36037.0500E) to Gu¨tsch for the first time pollination by insects and hybridization in (Oberstafel, about 2,399 m, 46°39026.2000N8°37014.7700E); the natural habitat of the species were investigated. Artifi- (3) Canton Tessin, Valle Bedretto (VB); Alpe Cruina cially established, mixed populations of E. minima and E. (2,050 m, 46°28021.1700N8°25034.9300E). From here salisburgensis were used to answer the following questions: onward these populations are named ‘‘origin populations’’. (1) Do the flowering periods of E. minima and E. salis- To facilitate the discrimination of the species in the arti- burgensis overlap sufficiently to allow interspecific ficial populations, yellow flowered E. minima individuals pollination? (2) Are the flowers of E. minima and E. salis- were chosen as seed donors. Fruit collecting resulted in burgensis visited by insects and does the insect’s behavior about 12,000 seeds per species and population, except for potentially allow pollen transfer between the species? (3) E. salisburgensis in VB, where only 7,000 seeds were Can hybrids establish in a natural habitat? collected (for details see Liebst 2006). The experimental area was a 15 9 7 m plot in a pasture in the Pian Murinascia in Val Piora (Canton TI, about Materials and methods 1,980 m, 46°32040.9800N8°43046.8000E). In this area single E. minima and E. salisburgensis plants and also some Species E. alpina (diploid, atypical forms) and E. hirtella plants (diploid) naturally occurred. Within the experimental area Euphrasia minima Jacq. ex DC., subsection Ciliatae, and for each origin population four plots of 75 9 75 cm E. salisburgensis, subsection Angustifoliae (Wettst.) Joerg. (without Euphrasia plants) were prepared for seeding by are annual, hemiparasitic herbs. E. minima is a facultative cutting away grass and herbs and then removing the plant hemiparasite but grows much more vigorously when it is litter. For a more even distribution of the seeds, each plot attached to a suitable host plant (Heinricher 1924; Matthies was divided in nine sub plots (25 9 25 cm). The seeds 1998). Although many species are suitable hosts for from each origin population were mixed. About 300 of 123 Plant Syst Evol (2008) 273:179–189 181 each E. minima and E. salisburgensis seeds were sown into yellow to white, it was considered as plant of hybrid origin each subplot of the populations PI and AM, and about 300 and is referred to as such. E. minima and 190 E. salisburgensis seeds were sown in The flower color of each plant was recorded and then all each subplot of the population VB. After sowing, the seeds plants were pressed and dried. A calyx and the largest of were covered with a fine layer of quartz sand. the bracts with the maximum number of teeth were mounted on a sheet of paper using transparent adhesive Record of flowering plants tape, and photographed with a digital camera.