Pl. Syst. Evol. 265: 165–177 (2007) Plant Systematics DOI 10.1007/s00606-007-0519-0 and Evolution Printed in The Netherlands Morphological systematics of Serapias L. (Orchidaceae) in Southwest Europe C. Venhuis, P. Venhuis, J. G. B. Oostermeijer, and P. H. van Tienderen Institute for Biodiversity and Ecosystem Dynamics, Universiteit van Amsterdam, Amsterdam, The Netherlands Received January 27, 2006; accepted January 18, 2007 Published online: April 30, 2007 Ó Springer-Verlag 2007 Abstract. We measured morphological characters while Sundermann (1980) only mentioned and relative DNA contents to assess variation and three. The distribution is predominantly Med- phylogenetic relationships among Serapias species in iterranean. Its range extends from the Azores three populations of each of the 10 putative taxa that and the Canaries in the west to the Caucasus in occur in Southwest Europe. DNA contents indicated the east, and as far north as Brittany (France) diploidy for most species, except for tetraploid S. (Go¨lz and Reinhard 1980; Perez Chiscano lingua and hexaploid S. olbia. Multivariate (discrimi- et al. 1991; Delforge 1995, 2002). The genus nant) analyses yielded two main groups: a small- flowered S. parviflora group and a large-flowered is considered monophyletic, based on genetic S. vomeracea group. Within the S. parviflora group, S. (Pridgeon et al. 1997; Bateman et al. 1997, elsae should be considered a large-flowered variation 2003) and morphological differences (Delforge of S. strictiflora. The geographically disjunct S. gre- 1995, 2002). Serapias is represented in south- garia and S. strictiflora are probably different taxa. In west Europe (Iberian Peninsula and southern the S. vomeracea group, analyses suggest that S. France) by ten species (Delforge 2002) (Ta- neglecta and S. cordigera are closely related. Serapias ble 1). Taxa within the genus are morpholog- cordigera from the southwestern coast of the Iberian ically still poorly defined. Systematic studies Peninsula is probably a subspecies, S. perez-chiscanoi based on morphology have utilized both was separated from all other species and S. occiden- quantitative and qualitative characters (e.g. talis was morphologically intermediate between S. Baumann and Ku¨nkele 1989, Martine and cordigera and S. vomeracea,suggestingahybrid Gerbaud 1998). However, a detailed study of origin, with the latter two taxa as parents. the morphological variation, using multivari- Key words: Mediterranean, morphology, orchids, ate analysis of many traits, has not previously phylogeny, self-pollination, systematics, taxonomy. been undertaken. Serapias is also poorly studied at the molecular genetic level. Aceto et al. (1999) concluded that, although variation for floral The number of recognised species in the orchid characters and molecular markers corre- genus Serapias L. varies strongly among sponded closely, the phylogenetic patterns authors. Delforge (2002) recognised 26 species, within the genus could not be resolved with 166 C. Venhuis et al.: Morphological variation in Serapias the molecular markers used (ITS). Pellegrino Morphology. A total of 23 vegetative and et al. (2001) isolated and characterised micro- floral characters were recorded for each specimen. satellite loci from S. vomeracea that could Fifteen of these characters were measured quanti- prove applicable to all Serapias species. tatively (Table 2) and eight qualitatively (epichile The aims of the present study are: (1) to -curvation, -hair distribution, -shape, -position determine the taxonomic status of the Serapias and lamellae position, hood postion, petal shape and the bract/hood length ratio), using pre-made species, as described by Delforge (2002, 2004), categorical verification lists to avoid subjective and from the Iberian Peninsula and southern gradual changes over time in scoring procedures. France on the basis of a large number of Multivariate statistical analyses. To determine morphological characters, (2) to determine whether the measured morphological characters variation in quantitative and qualitative char- could significantly discriminate among the taxa acters within and among the putative taxa, (3) presented by Delforge (2002) we used a Discrimi- to determine the relative DNA content and nant Analysis. This approach maximises the vari- thereby infer information on their ploidy level, ance among relative to within predefined groups and (4) to explore the phylogenetic relation- and tests which of the measured characters contrib- ships among the species based on morpholog- utes significantly to the discriminating functions. ical characters. DNA content. We sampled leaves of five plants per population, which were analysed with a Partec Flow Cytometer, following the standard prepara- Materials and methods tion and staining procedures. Trout fluorescent erythrocytes were added to the analysed solution as Approach. We sampled three populations, each a reference. The outcomes of the analyses were counting 25 specimens, of all ten presently recog- plotted in a frequency histogram. The histogram nised taxa by Delforge (2002, 2004) as occurring in displays relative fluorescence intensity versus the Spain, Portugal and southern France. In addition, number of cells. The results have been compared we sampled plants of the recently described S. with ploidy levels based on previous chromosome occidentalis C. Venhuis & P. Venhuis (Venhuis et counts on S. perez-chiscanoi (diploid, 2n = 36) by al. 2006). So, in total 75 specimens per taxon have Perez Chiscano (1988) and on S. lingua (tetraploid, been sampled. 2n = 72) by D’Emerico et al. (1998). Phylogeny. To explore the putative phylo- genetic relationships among the study species, we used the computer program Phylip 3.61. Within Table 1. Groups and species recognised in the Phylip, we used the procedure CONTML, which genus Serapias in southwest Europe by Delforge was especially designed for continuous characters (2002) and employs the Maximum Likelihood method, based on the Brownian motion model. The analysis Groups Species was based on averaged floral dimensions (length and width of bracts, hypochile and epichile, length S. parviflora group S. parviflora Parlatore of sepals and number of flowers) and on four S. vomeracea group S. vomeracea (Burman) qualitative flower characters (epichile position, Briquet S. cordigera L. shape of the lamellae, petal shape, and bract to Anacamptis palustris S. neglecta De Notaris hood length ratio). We used S. perez-chiscanoi Acedo (Jacq.) R.M. Bateman, Pridgeon and M.W. Chase, which is closest related to the genus Serapias (Aceto S. lingua group S. lingua L. et al. 1999, Bateman et al. 2003), as the outgroup in S. olbia Verguin our analyses. However, the long branch length S. gregaria Godfery subtending the outgroup makes the rooting of the S. strictiflora Welwitsch tree not very reliable. It was sufficient to use one ex da Veiga Serapias S. elsae Delforge outgroup only, because was already known to be monophyletic. C. Venhuis et al.: Morphological variation in Serapias 167 Table 2. Pooled within)group correlations between the quantitative morphological characters and stan- dardized canonical discriminant functions for the main analysis on all species, and the two separate analyses on each of the two main groups yielded by the first analysis. The largest absolute correlations between each variable and any discriminant function are indicated in bold. Analysis on all species Subanalysis Subanalysis S. parviflora group S. vomeracea group DA1 DA2 DA3 DA1 DA2 DA1 DA2 DA3 Eigenvalue 7.01 2.71 1.32 2.90 1.68 4.45 2.94 1.33 % Variance Explained 54.5 21.1 10.2 46.9 27.2 48.4 32.0 14.4 Epichile width 0.847 0.047 0.208 0.601 )0.091 )0.260 0.538 0.355 Hypochile width 0.607 )0.140 0.080 0.407 )0.109 )0.392 0.350 0.171 Epichile length 0.535 )0.237 0.335 0.486 0.272 )0.305 0.092 0.122 Hypochile length 0.296 )0.576 0.364 0.429 0.544 )0.609 )0.068 0.362 Bract width 0.189 )0.023 0.672 )0.075 0.420 )0.026 )0.276 0.261 Plant height 0.003 0.171 0.646 )0.179 0.464 0.215 )0.140 0.516 Bract length 0.139 0.040 0.558 )0.164 0.327 0.020 )0.156 0.201 Sepal length 0.355 )0.105 0.499 0.227 0.303 )0.121 )0.062 0.315 Mean length rosette leaves 0.049 0.156 0.471 )0.184 0.300 0.132 )0.010 0.520 Number of rosette leaves 0.076 0.248 0.368 )0.065 0.147 0.241 0.047 0.353 Number of flowers 0.151 0.272 0.215 )0.186 )0.028 0.188 0.047 0.036 Number of stem leaves 0.094 0.289 0.198 )0.100 0.021 0.304 0.054 0.024 Mean width rosette leaves 0.109 0.047 0.262 )0.191 0.030 )0.031 )0.141 0.056 Ovary length 0.016 0.041 0.312 )0.166 0.288 0.010 )0.044 0.179 Petal length 0.345 )0.133 0.446 0.228 0.267 )0.151 )0.078 0.248 Results divergent lamellae, while S. lingua had an entire or emarginated swelling. The stigmatic surface Morphology: qualitative characters. Most of S. olbia, S. gregaria, S. strictiflora and qualitative morphological character states were S. elsae contained a deeply grooved swelling, constant within populations and species. The which was slightly variable in shape and bract length to hood length ratio, however, serration (Fig. 1). varied strongly within and between popula- Morphology: discriminant analyses. Discri- tions and species. It was always less than 1 (i.e. minant analyses on morphometric data bract shorter than the hood) for S. lingua, and showed separation between two main groups: varied within all other sampled populations. On average, most species were characterised by bracts that were shorter than the hood. Excep- tions were two populations of S. parviflora from the Iberian Peninsula, all populations of S.