The genus Nigritella ( Orchidaceae ) in the Iberian Peninsula by Llorenç Sáez

As a result of the revision of Nigritella L.C.M. Richard in the Iberian Peninsula, here we recompile information of its variability, taxonomy, nomenclature and chorology. Two taxa are recognized: Nigritella austriaca subsp. iberica (Teppner & E. Klein) L. Sáez, comb. nov. and N. gabasiana Teppner & Klein, and the presence of N. corneliana is excluded. Detailed phytodermologic analysis showed that size of guard cells is useful for species identification.

Most of the Nigritella species have a similar habit and exhibit marked similarities in macromorphological traits, which makes it difficult to identify them in the field (Bournérias & al., 1998;Brütsch, 2000).Here we clarify some taxonomic and chorologic aspects of the genus Nigritella, since there is still information lacking on the identity and the distribution of several species in the Iberian Peninsula.

Material and methods
This revision was based mainly on herbarium material (102 specimens), including types, from the following herbaria (abbreviations according to Holmgren &al., 1990 andHolmgren &Holmgren 1993): ARAN, BC, BCB, BCF, BCN, JACA, and MA.Features of gross morphology were studied under a binocular stereoscopic microscope.Those concerning floral pieces were examined on flowers from the basal 1/3 of the inflorescence.Measurements of flowers were made from fresh plants (18 specimens) or on rehydrated herbarium material (flowers were submerged 5 minutes in boiling water).We have not been able to found significant differences between measures made from fresh plants and those made on rehydrated specimens.
For scanning electron microscopy (SEM) observations, the seeds samples were glued to aluminium stubs, coated with 50 nm gold film and examined in a Hitachi-2300S scanning electron microscope at 15 kV.
For epidermic studies, dried leaves were rehydrated in water (leaves were submerged in water for 24-48 hours, depending on their conservation condition) and decolored for 3-8 hours in a commercial solution of sodium hypochlorite until totally transparent (Stace, 1965).After washed with distilled water, the adaxial and abaxial epidermic surfaces were separated and stained in an alcoholic solution of Bismarck brown at 2% for 12 hours.They were then dehydrated through an alcohol series, placed in xylol, mounted in Canada Balsam and studied under a Zeiss-Universal microscope equipped with a camera lucida.Twenty measures of stomatal guard cells were done for each accession.Epidermic data on N. gabasiana and N. austriaca subsp.iberica were obtained from 53 and 19 individuals respectively (Table 1).
For the geographical distribution, data is given from herbarium sheets which document the occurrence of each species for each of the provinces of the study area, according to the criteria used in Flora iberica (Castroviejo & al., 1986).Nevertheless, additionally reliable chorological data can be consulted in Teppner & Klein (1993).

Taxonomic characters
The main morphological characters studied are listed below.Their potential taxonomical value is discussed.

Macromorphologic features
Inflorescences.All taxa have flowers in a dense terminal spike.In N. gabasiana, the inflorescence is subconical in flower and ovate to subcylindrical in fruit.In N. austriaca subsp.iberica the spikes are subconical or hemispherical in flower, and become subovate in fruit.Nevertheless, the size and shape vary among individuals and populations, and in my opinion this character cannot be used for species identification.
Flower colour.The basic flower colour of these two species is dark red-brown to reddish-violet.Nevertheless, some plants differ, e.g., a yellow-flowered specimen of N. gabasiana from the province of Huesca, Central Pyrenees (JACA 142698).
Labellum.The shape and size of the labellum has been used very often as a discriminatory feature in identifying several species of the genus (Teppner & Klein, 1985a, 1985b, 1990;1993;Brütsch, 2000;Delforge, 2001).Thus, Nigritella gabasiana is characterised by its labellum, which is only slightly open, narrow at its basal third, and has recurved margins that almost touch each other (Fig. 4).Regarding the size of the labellum, N. austriaca subsp.iberica and N. gabasiana show few differences and noticeably overlap.Nevertheless, the labellum of N. austriaca subsp.iberica can reach 10.2 mm in length, whereas in N. gabasiana it is less than 9.1 mm long.
Bracts.The occurrence of denticles (and their size and shape) in the margin of the lowermost bracts of the inflorescence has been used as a diagnostic character (Teppner & Klein, 1990;Bournérias & al., 1998;Brütsch, 2000;Delforge, 2001).On the basis of my observations, I conclude that bract denticulation is a character that should be used with caution.Smooth basal bracts (sometimes irregularly denticulate) are common in N. austriaca subsp.iberica.However, some specimens (MA 295050) bear irregularly denticulate lowermost bracts (denticles up to 0.03 mm long).Bracts of N. gabasiana are usually denticulate, with subconical denticles up to 0.05 mm long.The presence of this denticulation, at least in the middle of the bract, is a characteristic feature of this species according to several authors (Bournérias & al., 1998;Delforge, 2001).A detailed study of the bracts from herbarium material and 27 live plants revealed that some specimens of N. gabasiana bear slightly denticulate (JACA 10097272) or even smooth bracts (MA 622648, JACA 787987, JACA 141097).Furthermore, I found that this character is variable even within a single population.In the localities of Serra d'Ensija and Coll de la Creueta (Eastern Pre-Pyrenees) plants with denticulate basal bracts, and other specimens with smooth or slightly denticulate ones are found in the same locality.The plants of Coll de la Creueta were cytologically determined as diploids and had a chromosome number of 2n = 40.

Phytodermologic features
Main phytodermologic features of Iberian Nigritella leaves are as follows: Shape of cells.The epidermic cells are, in most cases, polygonal, with straight anticlinal walls on adaxial leaf surface (more or less straight on abaxial face).The thickness of these anticlinal walls varies from 8 to 13 m on the adaxial surface (4 to 10 m on abaxial surface) in Nigritella gabasiana.In N. austriaca subsp.iberica these values range from 7 to 14 m (4-12 m on abaxial surface).
Stomata.The Nigritella species bear hypoamphistomatic leafs.All the samples present anomocytic stomata (Fig. 1).On the adaxial surface, stomata are restricted to the apical area of the leaf (5-15 mm from the top) and near the margins on the upper part of the leaf.
Size of guard cells.For each species, the mean values of stomatal guard cell length from the abaxial surface are presented in Table 1.The guard cells of Nigritella austriaca subsp.iberica are longer than those of N. gabasiana (Fig. 2, Tab. 1).The use of guard cell length to infer the ploidy level of specimens has been successfully applied in many groups of plants (Evans, 1955;Barrington & al., 1986;Borrino & Powell, 1988;Viane, 1990).However, to our knowledge, this character has never been used in Nigritella.The predictive value of the phytodermological data to check the ploidy level is useful in the species of Nigritella found in the Iberian Peninsula, and probably in the whole genus.
In some Pyrenean specimens (Candanchú, JACA 270272; Sallent de Gallego, ARAN 59604; Añisclo, JACA 610874; Torla, JACA 135578) the results obtained from the stomatal guard cells and cell frequency are difficult to evaluate.These samples bear stoma-ta with intermediate lengths between those of the two species examined in this study.In addition, cell density also shows intermediate values.Thus, we cannot rule out that some of these specimens may be hybrids, presumely triploids, since the putative parents occur in the same location or at least in neighbouring areas.Partial sexuality in apomictic plants of the N. nigra group has been reported by Teppner (1996).
Cell frequency.Cell frequency (cells/mm 2 ) is higher in the diploid N. gabasiana (Table 1).This can be explained by the polyploidy nature of N. austriaca subsp.iberica: polyploids generally present larger cells as the level of ploidy increases.Cell frequency can be used to differentiate between diploid and tetraploid populations, at least in the Pyrenees.

Seeds
The seeds of the two species are very similar in shape and size (Fig. 3,Tab. 2).Nigritella gabasiana seeds measure 0.25-0.37× 0.15-0.25 mm, with testa cells corresponding to 1/3 or 1/2 of the chalaza side, nearly isodiametrical or up to twice as long as width, with anticlinal walls that are not sinuous.Seeds of N. austriaca subsp.iberica are slightly smaller (0.23-0.36 × 0.15-0.22mm), with testa cells corresponding to 1/3 or 1/2 of the chalaza side, usually twice as long as width, and with sinuous anticlinal walls.
The splitting of the N. nigra group into discrete geo-graphical entities (usually recognized at the subspecific level) is supported by constant, karyological, morphological and molecular features (Teppner & Klein, 1993;Hedrén & al., 2000).Nigritella nigra subsp.iberica is closely related to N. nigra subsp.austriaca, which occurs in the middle part of the Eastern Alps (Teppner & Klein, 1990).Some authors merged these two tetraploids within a single taxon, N. austriaca (Delforge & al., 1991;Bournérias, 1998;Brütsch, 2000), or recognized them within N. nigra at subspecific level (Breiner & Breiner, 1993;Teppner & Klein, 1993) or within N. austriaca at varietal rank (Delforge, 2001) Morphological discontinuities (mainly of quan-  titative nature) and the allopatric distribution of N. nigra s. str., N. nigra subsp.iberica and N. nigra subsp.austriaca, and molecular data on the origin of the tetraploid plants through hybridization between a triploid taxon closely related to N. nigra subsp.nigra and several diploid species (cf.Hedrén & al., 2000: 263) supports the recognition of a triploid (N.nigra) and a tetraploid taxon (N.austriaca) at specific rank.I conclude that this division better reflects the phylogenetic relationships of this polyploid complex.The two tetraploid taxa probably originated separately within their present distribution.The western populations of N. austriaca were probably the result of hybridization between a triploid species and a western diploid species, whereas in eastern populations, the diploid was genetically different (Hedrén & al., 2000).Accordingly, the western and eastern populations constitute morphologically recognisable groups, despite their putative hybrid origin.In this context, recognition of subspecific taxa within a single species (N.austriaca) is a better reflection of the morphological variation according to biogeographic patterns.Therefore, we propose the following change in nomenclature: Illustrations: Fig. 4.
Habitat: Montane to alpine meadows; clearings in open Pinus uncinata and Betula woodland, sometimes in rocky limestone sites; 1250 to 2550 m.

Species which presence is doubtful
Nigritella corneliana (Beauverd) P. Gölz & H.R. Reinhard in Jahresber.Naturwiss.Ver.Wuppertal 39: 39. 1986 (N. nigra subsp.corneliana Beauverd in Bull.Soc.Bot.Genève ser.2, 17: 336. 1926) has been reported in the eastern Pyrenees (Sanz & Nuet, 1997).This is a diploid species endemic of the southwestern Alps, characterised by its red-cinnabar to carminepink flowers, generally fading towards the base of the inflorescence, where they can be almost whitish (Bournérias & al., 1998).Because we did not find the species after several visits to the Pyrenean locality where it was cited and photographed, and no appropriate herbarium material from that site is known, we consider the occurrence of this species in the Iberian Peninsula doubtful.

Fig. 7 .
Fig. 7. Distribution map of Nigritella austriaca subsp.iberica in the Iberian Peninsula on the basis of herbarium specimens.