Taxonomic Reconsideration of some spp. Germplasm Maintained in World Genebank Collections

I. Doležalová, A. Lebeda and E. Křístková I. Tiefenbachová Research Institute of Crop Production Palacký University Praha-Ruzyně Faculty of Science Division of Genetics and Breeding Department of Botany Department of Gene Bank Šlechtitelů 11 Workplace Olomouc, Šlechtitelů 11 783 71 Olomouc-Holice 783 71 Olomouc-Holice Czech Republic Czech Republic

Keywords: genetic resources, isozyme polymorphism, morphology, reference herbarium specimens, taxonomic revision, wild

Abstract In total 49 accessions of 24 wild Lactuca spp., a hybrid between L. serriola × L. sativa and the related species Mycelis muralis were evaluated with the aim to validate their taxonomic status. Based on morphological and other characters (e.g. chromo- some number, relative DNA content, isozymes polymorphism) 17 germplasm acces- sions were reclassified. The majority of these 17 accessions was classified to the species L. serriola and as primitive forms of L. sativa. The taxonomic status of the other questionable Lactuca accessions is discussed in relation to their morphology. It was found after taxonomic revision, that the genetic variability of the Lactuca maintained in the genebank collection, represents 18 wild Lactuca species and cultivated L. sativa. Due to absence of passport data and reference herbarium material, the generic name Lactuca sp. was used for two accessions. The species L. alpina (L.) Gray, L. dentata L. (Thunb.) Robins., L. denticulata (Houtt.) Maxim., L. homblei De Wild., L. livida Boiss. & Reut., L. quercina L. and L. squarrosa (Thunb.) Miq. (syn L. indica L.) reported previously in the Czech National Germplasm Collection are not present. It was not possible to identify the accession named as L. homblei unambiguously because of a lack of available reference herbarium speci- mens. Preliminary conclusions suggest, it may be L. schweinfurthii Oliv. & Hiern or L. logenspicata De Wild. Lactuca angustana was treated as L. sativa L. var. angustana Irish. The accession classified previously to the genus Lactuca is actually Sonchus oleraceus and was excluded from Lactuca collection.

INTRODUCTION The problems connected with the preservation of genetic variability and germplasm conservation have become a focus for many scientists throughout the world. The breeding of crop cultivars is dependent on the acquisition and utilization of that variability (Hancock, 1998). The maintenance of plant germplasm requires a basic knowledge of the taxonomic status and other important morphological and biological features, which are essential for their practical utilization (Lebeda and Astley, 1999). From a total of 97 wild Lactuca spp. reported in the literature (Lebeda et al., 2004), only 27 Lactuca species and related genera are represented in world genebank collections according to the “International Lactuca database” (Boukema et al., 2002; ILDB, 2001; Stavelíková et al., 2002). There have been 24 Lactuca species and related genera included in the Czech National Collection of Lactuca genetic resources (Křístková and Lebeda, 1999; Lebeda and Astley, 1999). However, the actual number of Lactuca spp. in the collection is lower due to their incorrect or incomplete taxonomic determination (Lebeda et al., 1999; Doležalová et al., 2002; Lebeda et al., 2001b; 2004). A broad morphological variability, the spontaneous hybridization of some species in natural localities and the absence of an uniform classification for the genus Lactuca make the precise taxonomical determination of individual lettuce germplasm accessions difficult

Proc. XXVI IHC – IVth Int. Symp. of Cultivated Ed. C.G. Davidson and P. Trehane Acta Hort. 634, ISHS 2004 193 Publication supported by Can. Int. Dev. Agency (CIDA) (Cronquist, 1985; Rulkens, 1987; Zohary, 1990; Koopman et al., 1998; Koopman, 1999; Lebeda and Astley, 1999; Lebeda et al., 2001b). Recently within an international cooperation in the field of detailed characterization of wild Lactuca spp. genetic resources (GENE-MINE project, Fifth Framework Programme of European Union) the list of basic morphological descriptors for wild Lactuca spp. germplasm was developed (Doležalová et al., 2003) for the precise characterization of genebank material. The main aim of this study was to validate the taxonomic status of the accessions in the Czech National collection of Lactuca spp. germplasm to avoid the misinterpretation of results in the use of this material for scientific and breeding purposes.

MATERIALS AND METHODS

Plant Material Forty nine accessions of 24 Lactuca species, a hybrid between L. serriola × L. sativa and Mycelis muralis were studied. These accessions are maintained by the Gene Bank in Olomouc (Research Institute of Crop Production /RICP/ Praha-Ruzyně, Czech Republic), however most have been obtained from various germplasm collections around the world (Doležalová et al., 2002).

Morphological Assessment The set of accessions included 12 European Lactuca species, one Lactuca species from Middle East (L. aculeata), six from Asia (L. altaica, L. indica, L. squarrosa, L. taraxacifolia, L. dentata and L. denticulata), three from South Africa (L. capensis, L. dregeana, L. homblei) and two from North America (L. biennis, L. canadensis), as well as one representative of the related taxon Mycelis muralis (Doležalová et al., 2002). Each accession was represented by 16 plants which were cultivated in plastic pots (19 x 19 cm) in a greenhouse from April to September (1998-2001) under controlled conditions (temperatures by day 18-30°C, by night 12-16°C). The biennial (L. biennis, L. canadensis, L. virosa) and perennial species (L. viminea, L. perennis, L. tatarica) were maintained overwinter at the stage of basal rosette at temperatures of 5-7°C in a glass- house. The evaluation of all accessions was carried out throughout the growing period according to the basic morphological descriptors (Doležalová et al., 2003). Voucher specimens of all accessions were deposited in the herbarium of the Department of Botany of Palacký University (OL) at Olomouc (Czech Republic) and supplemented with photo- graphs of all developmental stages of the plants. Data based on morphological observations of Lactuca spp. accessions during the vegetative period (Doležalová et al., unpubl. results; Lebeda et al., 1999; 2001a) together with morphological data available in the world literature (Jeffrey, 1966; 1975; Feráková, 1976; 1977; Dethier, 1982; Iwatsuki et al., 1995; Stace, 1997) and a comparative study in the Herbarium Vadense (WAG), Wageningen University at Wageningen (the Netherlands) were used as primary sources for precise taxonomic determination (Table 1). Data on chromosome numbers and relative DNA contents (Doležalová et al., 2002), and isozyme analysis served as complementary evidence to support the morpho- logical observations and subsequently the taxonomic status of individual Lactuca spp. accessions.

Isozyme Analysis Three young basal rosette leaves were taken (4-5 weeks after transplanting) from three plants per accession and a mixed sample used for analysis. Approximately 300 mg aliquots of tissues frozen by liquid nitrogen were crushed and about 900 µl of 0.1 M Tris- HCl sample buffer (pH = 7.2) containing 0.1% 2-mercaptoethanol, 2 mg/l PMSF (Phenyl- methylsulfonyl fluorid) (Aung and Evans, 1987) was added. The extracts were centri- fuged (10 000 rpm, 10 min, 4°C) and the supernatant stored at a temperature of -80°C until use. Before electrophoresis, the protein extract was solidified with 40% surcose solution (enriched with bromophenol blue) and after mixing, samples were loaded

194 into slots in the stacking gel (4.2% stacking gel incorporating Tris-HCl pH 6.8 (0.5 M)). Analyses were performed on 10% vertical discontinuous native polyacrylamide gels (7.5 x 135 x 100 mm) (Luhový and Reňáková, 1989) incorporating Tris-HCl pH 8.8 (1.5 M). Samples were separated by electrophoresis (30 mA, 390 V, 2 h, 4°C) in Laemmli (1970) discontinuous buffer system using a Standard Vertical Gel Electrophoresis Unit (Sigma) and EC3000P power supply (E-C Apparatus Corporation). The voltage was maintained at 120 V until the samples had entered the stacking gel, and then the voltage was raised to 390 V. Tris-glycine buffer pH 8.3 was used as the electrode buffer. PAGE gels were specifically stained for esterases (Est). Before staining gels were soaked for 10-15 min in the staining buffer, incubated in the staining solution (Manchenko, 1994) for 1-2 h at room temperature and, then fixed in 8% acetic acid.

RESULTS AND DISCUSSION A detailed morphological study of macroscopic features (e.g. stem, leaves, inflorescence, ligules and achenes) of individual Lactuca spp. accessions highlighted incorrect determinations for many of them. The results of the validation of taxonomic status of individual Lactuca spp. accessions are summarized in Table 1. The accession 09-H58-0105 classified previously to the genus Lactuca is actually Sonchus oleraceus and was excluded from Lactuca collection. The accession identified originally as L. alpina (L.) Gray (syn. alpina (L.) Wallr. was reclassified as a L. serriola L. with possible introgression from L. sativa based on the characters: fresh green colour, high glossiness and richness of rosette leaves. However, the general habit of plants, the morphology of cauline leaves and inflorescence characters of this accession were identical with L. serriola f. serriola. The possible influence of L. sativa is supported by reports of spontaneous interspecific hybrids L. sativa × L. serriola and vice versa in the wild (Boukema et al., 1990; Křístková, 1998; pers. com.; Lebeda, 1998). The accessions LJ 74172, LJ 74179 and Sal 056 were originally identified as L. altaica Fisch. & Mey. On the basis of their morphology accessions LJ 74172 and LJ 74179 were reclassified as primitive forms of L. sativa (Table 1). Plants of these accessions were marked with rosettes of densely accumulated, fresh green leaves, they did not form heads as reported Ryder and Whitaker (1976). In addition, trichomes were recorded on the midrib of cauline leaves, thus supporting the classification to the primitive forms of L. sativa. The esterases zymograms show a banding pattern similar to L. sativa (Fig. 1). Accession Sal 056 previously described as L. altaica was reclassified as L. serriola f. integrifolia. This accession is characterized by entire undivided rosette and cauline leaves, large and plump seeds, and probably belongs to the group of oil seed . Boukema et al. (1990) reported that oil seed lettuces were related either to L. serriola or L. sativa, and recorded the existence of transitional types between both species. The presence of prickles on the leaf veins and stem, early bolting and the bitter taste of the leaves support the determination that this accession (Sal 056) is L. serriola. These features agree fairly well with the description of L. serriola by Harlan (1986). In spite of the morphological observations supporting the determination as L. serriola, the esterase zymogram indicate a determination rather closer to L. sativa than L. serriola (Fig. 1). Lactuca angustana (Sal 059) was treated as L. sativa var. angustana Irish (Table 1) according to Vries and Raamsdonk (1994). The basic morphological characters of the accessions named as L. dentata L. (Thunb.) Robins. (syn. Ixeris dentata (Thunb.) Nakai), L. denticulata (Houtt.) Maxim. (syn. Youngia denticulata (Houtt.) Kitam.) and L. squarrosa (Thunb.) Mig. did not correspond with their descriptions in local floras (Iwatsuki et al., 1995). Based on their morphology, isozyme variability (Fig. 1), chromosome number, and genome size analyses (Doležalová et al., 2002) these accessions were reclassified as L. serriola, oil seed lettuce type. These accessions were characterized by broad spathulate leaves that did not form rosettes, by early bolting and relatively large L. serriola-type seeds. The chromosome number of accession PI 234204 (originally received as a two different species, L. dentata

195 and L. denticulata (Table 1)) differs from the chromosome numbers for these species reported in the literature (Fujishima, 1980). This accession is morphologically almost identical with PI 236396 (incorrectly described as L. squarrosa) and they both may belong to a single species. There was found very similar relative DNA content in all these three accessions (Doležalová et al., 2002). Moreover, they show similar patterns in their esterases (Fig. 1) and acid phosphatases isozymes which confirm their close relationship (Lebeda et al., 2001a; Doležalová et al., unpubl. results). The accessions Sal 069 and Sal 070, originally determined as L. livida Boiss. & Reuter (Table 1) are annual. The plants did not form basal rosettes, were early bolting, and had narrow lanceolate leaves and brown achenes. Clearly these 2 accessions were not the biennial species L. livida, which is characterized by oblong or spathulate leaves and black winged achenes, and is considered to be closely related to L. virosa (Feráková, 1977). Even Velasco Negueruela (1981) treated L. livida as a subspecies of L. virosa. Isozyme analysis of esterases of Sal 069 and Sal 070 showed similar zymogram banding pattern (Lebeda et al., 2001a; Doležalová et al., unpubl. results), and similar relative DNA content with L. dregeana DC. (CGN 04790) (Doležalová et al., 2002). According to their morphology (Jeffrey, 1966), isozyme variability and analysis of relative DNA content (Doležalová et al., 2002) these accessions (Sal 069, Sal 070) were reclassified as L. dregeana. The morphology of accessions LJ 75009 and LJ 75150, previously determined as a L. livida, did not correspond with the botanical description of this species in local floras (Feráková, 1977; Rollán, 1985). The generic name Lactuca sp. (Table 1) has been applied to these accessions in the absence of passport data and available reference herbarium material. The accessions Sal 073 and CGN 05808, named as L. quercina L. (Table 1) differed from the viewpoint of botanical description of this species in many characters. According to their habit and leaf morphology they belong to L. sativa which confirm presence of brown seeds instead of those black typical for L. quercina (Hegi, 1987). The morphological assessment of CGN 05808 showed that this accession is probably a primitive form of L. sativa based on the by fresh green leaves which did not form head. The accession Sal 073 belongs to the group of oak leaf lettuces (Vries and Raamsdonk, 1994) with anthocyanin coloration in the leaves and prickles on the midrib. Based on the leaf morphology, the L. serriola f. integrifolia (09-H58-0113) with divided rosette and cauline leaves was reclassified as a L. serriola f. serriola (Table 1). The accession maintained as L. homblei De Wild. (CGN 11322) (Table 1) is more probably L. schweinfurtii Oliv. & Hiern or L. longespicata De Wild. as described by Jeffrey (1966) and Dethier (1982). According to the morphological assessment and a comparison with herbarium specimens in Herbarium Vadense (WAG), its classification as L. homblei cannot be confirmed. Since these Central African species are not available in gene banks collections, the future development of a larger number of reference herbarium specimens is needed. The same is true for L. taraxacifolia Chalk., a newly described species from the mountains of Central Asia (Chalkuziev, 1974), which has not been yet reported in local floras (Lebeda et al., 2004).

CONCLUSIONS The data summarized in this paper showed that the species diversity of the genus Lactuca maintained in the Czech National Germplasm Collection is lower than was thought previously. After this taxonomic study we conclude that the collection of wild Lactuca genetic resources includes 18 species, hybrid L. serriola × L. sativa and one related species, Mycelis muralis. The majority of the reclassified accessions are L. serriola and primitive forms of L. sativa. For the remaining two accessions we used the generic botanical name (Lactuca sp.) to avoid confusion. Several species (L. alpina, L. dentata, L. denticulata, L. homblei, L. livida, L. quercina and L. squarrosa) which have been reported in the Czech National Germplasm Collection and in other international collections (Lebeda and Astley, 1999; Lebeda and Boukema, 2001) are no longer available due to the clarification of the taxonomic status of individual accessions. In

196 addition the situation in many gene bank collections is further complicated due the large extent of duplicated material (Hintum and Boukema, 1999). Therefore, a good knowledge of classical taxonomy combined with the comparative study of original herbarium specimens must be considered as a most important step for the efficient management and utilization of Lactuca genetic resources (Lebeda et al., 1999), and the correct interpretation of experimental data (Lebeda et al., 2002). The taxonomic validity of Lactuca genetic resources must become a part of the global activities in the field of genetic resources and biodiversity conservation (Lebeda et al., 2004).

ACKNOWLEDGEMENTS The authors express their sincere thanks to Mrs. D.Vondráková for her excellent technical assistance and Dr. K. Doležal for help with isozyme analyses. The authors are obliged to Dr. W.J.M. Koopman and staff members of the Herbarium Vadense, Plant Taxonomy, Wageningen University at Wageningen (the Netherlands) for providing the herbarium facilities and their kind support. Valuable comments of Assoc. Prof. V. Feráková to the taxonomic reidentification of some Lactuca spp. are greatly acknow- ledged. This research was supported by Internal grants of Palacký University (1999, 2000), partly by grant “Stress and pathological biology, biochemistry and bioenergetics of plants” (MSM 153100010) and “National programme on plant genetic resources con- servation and utilization”.

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199 Tables

Table 1. Taxonomic redetermination of some wild Lactuca spp. accessions.

International or Czech Original description Redetermination genebank number L. altaica Fisch. & Mey. LJ 74172 L. sativa L. (primitive form) L. altaica Fisch. & Mey. LJ 74179 L. sativa L. (primitive form) L. altaica Fisch. & Mey. Sal 056 L. serriola L. (oil seed lettuce) L. angustana All. Sal 059 L. sativa var. angustana Irish L. homblei De Wild. CGN 11322 probably L. schweinfurthii or L. logenspicata L. livida Boiss. & Reut. LJ 75009 Lactuca sp. L. livida Boiss. & Reut. LJ 75150 Lactuca sp. L. livida Boiss. & Reut. Sal 069 L. dregeana DC. L. livida Boiss. & Reut. Sal 070 L. dregeana DC. L. quercina L. Sal 073 L. sativa L. (probably oak leaf lettuce) L. quercina L. CGN 05808 L. sativa L. (primitive form) L. serriola f. integrifolia 09-H58-0113 L. serriola f. serriola L. squarrosa (Thunb.) Miq. (syn. L. indica L.) PI 236396 L. serriola L. (oil seed lettuce) L. squarrosa (Thunb.) Miq. (syn. L. indica L.) 09-H58-0105 Sonchus oleraceus L. (L.) Wallr. (syn. L. alpina (L.) Gray) 09-H58-0123 L. serriola L. Ixeris dentata (Thunb.) Nakai (syn. L. dentata L. (Thunb.) Robins.) PI 234204 L. serriola L. (oil seed lettuce) Youngia denticulata (Houtt.) Kitam. (syn. L. denticulata (Houtt.) Maxim.) PI 234204 L. serriola L. (oil seed lettuce)

200 Figurese

LJ 74172 Sal 056 L. sativa L. sativa L. serriola (oil seed lettuce)

PI 234204 PI 234204 L. serriola

Fig. 1. Comparison of L. sativa and L. serriola esterases zymograms.

201