A study of seed micromorphology in the genus Ophrys (Orchidaceae) ; Un estudio de la micromorfología de las semillas en el género Ophrys (Orchidaceae)

Galán Cela, P., Seligrat, I., Ortúñez, E., Gamarra, R., Vivar, A. & Scrugli, A. 2014. A study of seed micromorphology in the genus Ophrys (Orchidaceae). Anales Jard. Bot. Madrid 71(2): e008 Seed micromorphology of 19 taxa of the genus Ophrys have been studied using SEM and light microscope. Quantitative data (length and width of seed and embryo, number of testa cells along the longitudinal axis, volume of seed and embryo, and percentage of free air space), as well as qualitative characters (seed shape, features of the anticlinal and periclinal walls, ornamentation and colour) were analysed. All the seeds are fusiform, with an asymmetrical basal pole, the periclinal walls of the medial cells have parallel and transverse to slanting ridges, and raised anticlinal walls. Statistical analyses show two large clusters according to the volumes of seed and embryo. Our results support the monophyly of the genus and their recent diversification, however, seed features are not congruent with the recognition of sections and groups within Ophrys.


INTRODUCTION
The genus Ophrys L. (Orchidaceae, Orchidinae) is distributed from the Canary islands to the Near Orient, with great diversification in the Mediterranean basin (Pridgeon & al., 2001;Delforge, 2006;Pedersen & Faurholdt, 2007). It is well characterized by their labellum, which resembles the body or some organs of different insects, and it is linked with the mechanism of pollination (pseudocopulation).
Their monophyly is strongly supported by morphological and molecular data (Soliva & al., 2001;Bateman & al., 2003;Bernardos & al., 2005), with Serapias L. and Anacamptis Rich. as the closest relatives genera. No intergeneric hybrids with other representatives of the subtribe Orchidinae have been found, however, interspecific hybridization is widespread in the field, and perhaps, several taxa may be of hybrid origin (Pedersen & Faurholdt, 2007).
The taxonomy of this genus remains difficult, with a profusion of taxa along the distribution range, mainly in Greece and Turkey. Several authors have contributed to the taxonomy of the genus (Godfery, 1928;Nelson, 1962;Del Prete, 1984;Baumann & Künkele, 1986;Devillers & Devillers-Terschuren, 1994;Delforge, 2006;Pedersen & Faurholdt, 2007). The lack of consensus between orchidologists has resulted in discrepancies as to the number of taxa, for instance, Delforge (2006) recognised 252 species throughout their distribution range, whilst Pedersen & Faurholdt (2007) mentioned 19 species in Europe, many of them including infraspecific taxa (subspecies and varieties), and several hybrids.
Preceding studies on orchid seeds have demonstrated the phylogenetic value of certain quantitative and qualitative characters (Clifford & Smith, 1969;Barthlott & Ziegler, 1981;Tohda, 1983;Molvray & Kores, 1995). In addition, recent publications (Gamarra & al., 2007;Gamarra & al., 2008;Gamarra & al., 2012) emphasize the strong support between the seed micromorphology and the molecular analyses published in the subtribe Orchidinae (Bateman & al., 2003). Barthlott (1976) compared the seeds of the genera Ophrys and Serapias, demonstrating the taxonomic value of the seed shape and the tipology of the trabeculae in the testa cells to distinguish both genera. Also, he mentioned the difficulty of distinguishing the seeds within Ophrys. Mrkvicka (1994) Aybeke (2007) has studied 8 taxa from southeastern mediterranean countries, concluding that all taxa showed reticulated surfaces in the testa cells.
The main aim of our survey is to study and analyse the seed micromorphology of selected species of Ophrys, based on qualitative and quantitative data. A statistical and a SEM survey of seeds were conducted to investigate patterns of infrageneric variability in the genus. Finally, to demonstrate a correlation between our results and Ophrys lineages recognised in previous publications on the basis of morphological and molecular data.

MATERIAL AND METHODS
Seeds of 19 species were obtained from mature capsules collected in the field. Fresh seeds were dried for at least one month and stored in small paper envelopes. Specimens for seed morphological analyses were deposited in the herbaria MAUAM and CAG (acronyms according to Thiers, 2012). Voucher specimens, with their circumscription to sections and groups according to Delforge (2006), are listed in Table 1.
For scanning electron microscopy observations, the sam ples were mounted on SEM stubs and coated with gold in a sputter-coater (SEM Coating System, Bio-Rad SC 502). The seeds were examined with a Philips XL30, with a filament voltage of 20 kV. Qualitative data such as seed shape and testa cell features (sculpturing, morphology of cells and the anticlinal and periclinal walls) were analysed. In addition, an average of 30 seeds from each specimen were analysed using a light microscope, previously mounted with PVA (polyvinilic alcohol). Quantitative variables like seed size (length and width), number of cells along the longitudinal axis, embryo morphology and size (length and width), and seed colour (in subjective terms) were recorded. Furthermore, the length/width ratio (L/W), the percentage of free air space and the seed and embryo volumes were estimated. The terminology and methods adopted were those of Arditti & al. (1979), Barthlott & Ziegler (1981), Molvray & Kores (1995), Chase & Pippen (1988), Arditti & Ghani (2000). Statistical ana lyses were performed by SPSS 17 for Windows, after logarithmic transformation of the volume data. Ward's method with Euclidean distances was conducted for cluster analysis.

Qualitative data
In all the analyzed species, the seed shape is fusiform (Fig. 1A), slightly wider in the middle or towards the apical pole where the ellipsoidal embryo is located. Generally, the seeds show a more or less truncated apical pole (Fig. 1B) and an asymmetric basal pole (Fig. 1C). Cells of both poles are polygonal and shorter ( Fig. 1B-C), whereas medial cells are elongated (Fig. 1D).
Medial testa cells are longitudinally oriented. They have predominantly parallel ridges on their periclinal walls, ranging in orientation from transversal to slanting ( Fig. 2A-C). Adhesion zones between cells show a distinct lamella (Fig. 2B). Anticlinal walls are longitudinal and straight, strongly raised, and the outer periclinal surface is generally concave (Fig. 2C, D). Seed colour ranges from brown-yellowish to dark brown. The embryo is brown in colour. Table 2 shows the length and width of seed and embryo, the number of cells along the longitudinal axis, and the length/width ratio. Average values of the seed and embryo volumes, the percentage of free air space and the colour of testa are shown in Table 3.

Quantitative data
The maximum L/W ratio was observed in O. chestermanii, and the minimum in O. lutea, which agrees with the seed length measurements. The number of testa cells along the longitudinal axis varies from 4 to 8. In general, the longest seeds have the highest number of testa cells. The highest seed and embryo volume were measured in O. dyris and O. scolopax, respectively. The lowest average values for these variables were found in O. sicula. Fifteen of the species analysed had more than 70 % free air space, however, in O. funerea and O. lutea, the values did not exceed 50%.
The quantitative data do not verify the required hypothesis for analysis of variance, because significant differences in the standard deviations of the variables have been found. Even, the coefficients of skewness and kurtosis indicate the absence of normality in some variables. Analyzing the variables Sv (Seed volume) and Ev (Embryo volume), the statistical analysis revealed that the species fall into two groups. Higher volumes were found in Ophrys dyris, O. dianica, O. chestermanii, O. eleonorae, O. scolopax and O. morisii, and lower volumes in the rest of taxa (Fig. 3).
Using Ward's method with euclidean distance, two large clusters were produced by the dendrogram (Fig. 4)

DISCUSSION
Our micromorphological study reveals a strong concordance in the qualitative features of the seeds in the genus Ophrys. The fusiform shape has been observed in many genera of the subtribe Orchidinae (Barthlott & Ziegler, 1981;Mrkvicka, 1994;Gamarra & al., 2007;Gamarra & al., 2012). We have found a set of characters which allow us to recognise the seeds as Ophrys-type. These are characterized by the asymmetry of the basal pole, the strong concavity of the testa cells with raised anticlinal walls and a distinct lamella. If we compare with the variation observed in other genera, this suite of qualitative data support the monophyly of the genus. Furthermore, all the species show the same sculpture pattern of the periclinal walls, with parallel ridges, only varying Table 1. List of selected species with voucher specimens and codes. Sections and groups according to Delforge (2006).   in the orientation, from transverse to slanting. This feature is not congruent with the observation of the reticulate surface described by Aybeke (2007). Barthlott (1976) states that testa cells of Ophrys can be clearly distinguished of those of Serapias, which show slanting, dense and thin ridges in the periclinal walls. The clear separation with related genera is not mirrored in within-genus variation. In Ophrys, unlike other genera such as Dactylorhiza Neck. ex Nevski ( Averyanov, 1990), Anacamptis and Orchis (Gamarra & al., 2012), the qualitative data observed in seeds do not support the distinction of infrageneric levels. According to our statistical results two large clusters, based on the size of seed and embryo volumes, have been found. These clusters appear subdivided into six minor groups. Taxa of the sections Pseudophrys and Ophrys appear in both clusters, although a great number of the taxa analysed in the section Ophrys showed lower volumes. Furthermore, species ascribed to the same group by Delforge (2006) (Soliva & al., 2001;Bateman & al., 2003;Bernardos & al., 2005).

Species
According to previous molecular analyses (Bateman & al., 2003;Inda & al., 2012), genera of the subtribe Orchidinae such as Serapias or Neotinea Rchb. fil., with higher bootstrap values, show a common micromorphological pattern of testa Table 3. Average values of seed and embryo volumes, percentage of free air space, and colour of the testa of the taxa analysed. cells (Barthlott, 1976;Gamarra & al., 2007). On the opposite, in genera such as Anacamptis, Orchis and Dactylorhiza, with lower bootstrap values, the testa cells show qualitative differences which allow recognition of infrageneric groups (Averyanov, 1990;Gamarra & al., 2012). In the genus Ophrys, our results are congruent with the high bootstrap value obtained by Bateman & al. (2003) and Inda & al. (2010).

Species
In contrast with the species richness and the high diversity of floral characters, the homogeneous micromorphological pattern of testa cells found in Ophrys is also supportive of the recent radiation and rapid colonization proposed for the evolution of this genus along the Mediterranean basin (Soliva & al., 2001;Amich & al., 2007).