THE GENUS DRYOPTERIS ( PTERIDOPHYTA : DRYOPTERIDACEAE ) IN THE FLORA OF THE REPUBLIC OF MACEDONIA – 30 YEARS AFTER MICEVSKI ’ S FLORA –

This article presents the records about four new Buckler fern (Dryopteris) species for the territory of the Republic of Macedonia and new data about the distribution of some rare Buckler fern species. Dryopteris is one of the most complex fern genera in Europe. Hybridization and apomixis among some of the species contribute to difficulties and uncertainties for identification of individual species. That, coupled with lack of research on ferns in Macedonia for the last 30 years, resulted in a low level of knowledge within this vascular plants’ group. The field research of the flora of Macedonia and thorough check of two herbaria (MKMEL and MKNH) in Skopje revealed presence of four new Buckler fern species in Macedonia: Dryopteris affinis subsp. jessenii, D. oreades, D. mindshelkensis and D. expansa. Additionlly, several other localities for rare species: D. borreri, D. dilatata and D. carthusiana were reported. Further research is needed to fill the remaining gaps, particularly in relation to identification of hybrids and more detailed study of the distribution pattern of rare Buckler fern species.


INTRODUCTION
Buckler fern (Dryopteris Adans.)stands for one of the most complex genera among the European pteridophytes.According to Plant List [1], genus Dryopteris includes 1,458 accepted and unassessed names of species rank and 35 names of infraspecific rank worldwide (http://www.theplantlist.org/).At the same time, Euro+Med Plant Base [2] recognizes 30 Dryopteris species and 11 subspecies in Europe and Mediterranean region [2].Beside the relatively high number of species in the genus, the complexity comes from the high level of hybridization among many different species and subsequent apomictic reproduction characteristics for some of the species (Dryopteris affinis group in particular), Fraser-Jenkins [3,4].Hybridization and apomixis are the main reasons for the origin of some species, morphologically separated by fine discrete characters [4].Owing to above reasons, there was considerable confusion about Dryopteris taxonomy, thus resulting with complex synonymy and different treatment in existing standard floras, which makes identification of the particular species difficult and in some cases problematic.This particularly stands for the taxa of D. affinis group for which the concept of Fraser-Jenkins [4] is now accepted by the majority of researcher (see Euro+Med PlantBase) [2].
The aim of this work was to explore the Dryopteris collections in MKMEL! and MKNH! [19], to identify the species in the light of the new concept for Dryopteris taxonomy and to distinguish potential new taxa.The final goal was to assess the status of the genus Dryopteris in the flora of the Republic of Macedonia.

METHODS
The study was based on the fieldwork on the territory of the Republic of Macedonia during the last two decades and the collected and stored plant material in the Herbarium Melovskiorum (MKMEL!), as well as on the check of the Macedonian National Herbarium (MKNH!).The abbreviations of the herbaria follow Index Herbariorum [19].Besides the field surveys, relevant literature sources were used for supplementation of the distribution records.
Identification of the specimens was performed on the basis of the keys from [10; 15], as well as Fraser-Jenkins [4] for D. affinis group.The detailed morphological analyses and descriptions of individual species in [20][21][22][23][24][25][26][27] were valuable for proper identification.Beside the analysis of standard morphological characters, light microscopy was applied for identification of members of the D. dilatata group.The spores were analyzed for perispore ornamentation rather than for dimensions of the spores.At least 10 spores from at least three herbarium specimens (D. dilatata and D. carthusiana) or two specimens in case of D. expansa were analyzed.An effort was done to avoid spore contamination during preparation of glycerol slides (by taking spores only directly from the sporangia).Only few spores were photographed and presented in Figure 3 for comparison.
We followed the nomenclature of the taxa used in Euro+Med PlantBase [2].The distribution of the species was mapped on 10 × 10 km using the UTM grid system (UTM Zone 34T) and Military Grid Reference System (MGRS) projection [28], modified by Avukatov (unpublished) to 5 km × 5 km for the purpose of this paper.The quotation of the distribution locations and habitats for all records in this article follows the MAKMEL data base pattern.The regional division of Melovski et al. [29] was used to present the general geographic position of the localities.

RESULTS AND DISCUSSION
Among the known Buckler fern taxa in the Republic of Macedonia, D. filix-mas is the most frequent and abundant and D. villarii and D. pallida are frequently found on limestone mountains in western Macedonia, while D. borreri, D. carthusiana and D.dilatata are very rare species [5].
The present work adds four new Buckler fern species to the flora of the Republic of Macedonia and new distribution data for rare species.
Dryopteris affinis (Lowe) Fraser-Jenk.subsp.jessenii (Fraser-Jenk.)Fraser-Jenk., Fern Gaz.18 (1): 5 (2007) D. affinis is a diploid apomict which is nominal species of the complex Dryopteris affinis aggregate together with other seven species [4,17].Up to now, five subspecies, including the nominal one, were described [4], out of which three can be found across the Balkan Peninsula: subsp.affinis in Slovenia, Croatia, Greece, Bulgaria (doubtful) and Romania; subsp.punctata in Slovenia and Croatia and subsp.jessenii in Serbia and Romania [2, but on the basis of 13; 4].The results of this article show that D. affinis subsp.jessenii is distributed in the Republic of Macedonia as well, where it was recorded only on Plačkovica Mt. (Figure 1).It was also recently reported for Poland [30].
The finding of D. affinis subsp.jessenii far from its main range is curious.However the specimens found on Plačkovica Mt. have typical morphological characteristics of D. affinis subsp.jessenii (Fig. 2) as described by Fraser-Jenkins [4].Besides, it is not a rare case that Carpathian species spread to the south in the north-eastern Macedonian mountains (e.g.Viola dacica Borb., [32]).The new finding in the southern (Carpathian) part of Poland [30] shows that the subspecies is spreading its range to the north as well.This triploid apomict is the most widespread species in the D. affinis group [4].However, it is a rare species in Macedoniathe only known locality up to now was Osogovo Mts.[5] (Fig. 1).It can be relatively easily distinguished from D. affinis by its indusia which look more like those of D. filixmas, however in concordance with other morphological characters (Fig. 2).

Existing chorological record
Osogovo-Sasa.Beside the rocks close to the river.1050 m.n.v. 15 The species remains rare (Figure 1) which is probably realistic since Macedonia is at the southern border of its range in Europe (excluding Turkey) [2].It is also comparatively rare in Bulgaria and was only recently reported [24] if one considers that it is a common species in Europe.
Dryopteris oreades Fomin, Monit.Jard.Bot.Tiflis 18: 20 (1910) Mountain male fern (D. oreades) has interesting, rather disjunct distribution range.It can be found in west and west-central Europe, extending to N.C.Italy [10], in the Caucasus region and Turkey as well as in Romania [2].It is possible that it has been overlooked in the past researches in the fern floras, although it is easily recognizable by its sori (high indusium which lifts slightly and remains embracing sporangia even after spores' ripening, see Figure 2).Future research can reveal new findings.Taxonomic remarks: the 'scattered minute glands' on the underside of the pinnae of Macedonian plants cannot be seen, except for the youngest fronds; some of the plants have more or less acute teeth at the tips of the pinnules instead of blunt.In the Republic of Macedonia, this species is found on silicate rocky ground on the western mountains (Figure 1).

First chorological records
Šar Planina: Mengulova Kulaabove v. Gorno Jelovce (above Ramen Kamen), silicate rocks, 2000 m a.s.l., 14 [2].Micevski [5] did not list this species in his Flora.However, two years after Micevski's flora has been published, Fraser-Jenkins (during his revision of Dryopteris material from MKNH!) identified three specimens as D. submontana wrongly identified as D. villarii by Micevski.In the Republic of Macedonia, D. mindshelkensis is found on calcareous rocky ground on the western mountains (Figure 1).

Dryopteris dilatata complex
The complex consists of three species (D. dilatata, D. expansa and D. carthusiana) which are known for difficult and uncertain identification [20, 3, 25, etc.] due to their high variability and interconnected origin.It is recommended that one should use a set of characters for identification rather than one or two usually used in standard keys.Many morphological characters were studied and involved (including dimensions and volume of the glands [33]), but still the perispore remains among the most important ones.In some keys [e.g.10] D. dilatata perispore is described as being covered with dense and obtuse tubercles, and D. expansa perispore with sparse acute tubercles, which is not sufficient for identification of the two taxa, especially if the spores are examined by the light microscopy ("acute" and "obtuse" is not easily visible).Both D. dilatata and D. expansa have acute tubercles (or echinulae as R. L. L. Viane [33] names them) at least on light microscope images and the main difference is the density [33]; (see also Figure 3).D. expansa has well-spaced echinulae unlike D. dilatata with dense echinulae.Plants from Macedonia confirm this finding (Figure 3).Yet, traditionally accepted morphological characters used to distinguish the species in the group (particularly the length of the basioscopic pinnule) are sufficient to identify the majority of the specimens growing in Macedonia.However, in the frame of the considerable variation in fronds' morphology in this buckler fern group there are some individuals with characters that approach border values.In these cases the spores' morphology is important for proper identification.

Bot. 631 (1848)
This species is distributed in the whole Europe excluding Montenegro and Albania where its presence is doubtful [2].In Macedonia there was much confusion in regard to this species in the past.D. dilatata is listed in the Flora of Macedonia [5] for several localities: Pelister, Dudica, Jakupica -Kadina Reka river, Osogovo.However, the revision of Buckler ferns in MKNH! by Fraser-Jenkins in 1987 (after the Flora has been published) showed that it was not present in Macedonia (all specimens from MKNH! were identified by Fraser-Jenkins as D. expansa).On the other hand, the research presented in this article showed that he misidentified the specimens from Dudica (Kožuf Mt.) and Mt.Jakupica (Kadina Reka river).These specimens certainly are D. dilatata, which was confirmed by microscopic analysis of the spores (Figure 3) and thorough analysis of the morphological characteristics, particularly the ratio between basioscopic and acroscopic pinnulae of the lowest pinna and the degree of the frond division.Spores are frequently pointed as quite certain character for separation of the two close species -D.dilatata and D. expansa, in some standard keys, including Flora Europaea [10].Present distribution of D. dilatata in Macedonia is shown on Fig. 1.
Further research is needed to check the accuracy of the data given by old botanists from the beginning of 19-th century and mentioned in Micevski [5].

Existing chorological records
Dudicaon humid ground, on andesite.1500 m.n.v., 24 1; see also notes above, under 'Dryopteris dilatata' subchapter).As a matter of fact, most of the specimens in MKNH! actually belong to D. expansa, which was proved by microscopic analysis of the spores (Figure 3), thorough morphological analysis and revision of Dryopteris in MKNH! by Fraser-Jenkins in 1987.

First chorological records
Šar Planina: Čaušica-Peskovi-Bistra, in the crevices of silicate rocks, 2600 m a.s.l., 24 [2].Up to this research it was considered as a very rare species in Macedonia, found only on Osogovo Mts.(Osogovski Planini) [5] (Figure 1).This research proved that it has broader distribution though it is still quite rare (Figure 1).It is confined to the localities where the last remnants of alder forests and woodlands are still preserved.Further research is needed to check the data given by old botanists from the beginning of the 19-th century and questioned by Micevski [5].

New chorological records
Bistra-Šar Planina: Children's resort "Bunec" below the resort, swampy site in alder woodland, 1240 m a.s.l., 14 It can be concluded that the flora of the Republic of Macedonia consists of 10 Dryopteris species: Dryopteris affinis with one subspecies -D.affinis subsp.jessenii, D. borreri, D. filix-mas, D. oreades, D. villarii, D. pallida, D. mindshelkensis, D. dilatata, D. carthusiana and D. expansa.However, the knowledge of the Buckler ferns in Macedonia is still incomplete.Presence of some other species cannot be rejected.Future research is needed to prove presence of several hybrid taxa for which there are indications that they can be found in the Republic of Macedonia.

Figure 1 .
Figure 1.Distribution map of the first records, new records and literature data of the presented Dryopteris taxa in the Republic of Macedonia