Present or Absent? About a Threatened Fern, Asplenium adulterinum Milde, in South-Eastern Carpathians (Romania)

Asplenium adulterinum Milde is one of the rarest and most threatened representatives of the Aspleniaceae family in the Carpathians. Botanical literature mentioned the ladder spleenwort in many localities in South-Eastern Carpathians in the past, but during the last decades the species has not been collected and deposited in public herbaria by any botanist. All existing herbarium material in Romanian herbaria (including Asplenium trichomanes and A. trichomanes-ramosum) was revised and all available information from botanical literature was critically compiled in order to clarify the distribution of Asplenium adulterinum in the South-Eastern Carpathians. After almost 80 years since the first mention of the species in the Carpathians, this paper reports the recent identification of Asplenium adulterinum in three new locations in South-Eastern Carpathians (Ţesna Valley, Vânturătoarea Waterfall and Şugăului Gorges).


Introduction
Asplenium L. is a relatively large genus of Aspleniaceae family with about 700 species. They are also one of the most widespread fern groups and occur in temperate and tropical regions of all continents, except Antarctica (Schneider et al., 2004). Thirtyone species of this genus are present in European flora (Crabbe et al., 1993), ten of these species occurring in Romanian flora (Asplenium scolopendrium L., A. septentrionale (L.) Hoffm., A. trichomanes L., A. trichomanes-ramosum L., A. adulterinum Milde, A. ruta-muraria L., A. lepidum C. Presl, A. cuneifolium Viv., A. adiantum-nigrum L., A. onopteris L.) .
Some fern species of Asplenium genus (Asplenium adulterinum, A. adiantum-nigrum and A. cuneifolium) are often called serpentine ferns, because their occurrence is almost strictly related to serpentine rocks (Hayek, 1916). Serpentinites is a rock composed of one or more serpentine group minerals. Serpentinization is a geological low-temperature metamorphic process involving heat and water, in which low-silica mafic and ultramafic rocks are oxidized and hydrolysed with water into serpentinite. Serpentinite is formed from olivine via several reactions, some of which are complementary (Sleep et al., 2004).
Serpentinites have a limited number of characteristic plants, which are confined exclusively to magnesium silicates or carbonates (serpentine, magnesite) and these species may be considered strictly serpentine species. In Europe, serpentinites occur in the Alps, Carpathians, Balkan Peninsula, Southeast Portugal and Great Britain (Roberts and Proctor, 1992). In Romania they grow in South-Eastern Carpathians (Southern Carpathians and Apuseni Mountains, less in the Eastern Carpathians) (Corvin Papiu, 1963). Serpentine ferns are extremely rare and have been declining at an alarming rate.
The present study focuses on Asplenium adulterinum Milde, one of the rarest and most threatened plant species in the Romanian flora. Asplenium adulterinum was included in the list of species as an extension to Annex II of the Habitat Directive (Directive 92/43/EEC) so that its protection requires the designation of a Special Area of conservation. It was also listed together with Asplenium cuneifolium in Annex IV of The Convention of European Wildlife and Natural Habitats (Bern Convention) concerning plant species demanding strict protection in all European Union countries.
The species occurs on ledges and crevices of ultrabasic rocks (serpentine, magnesite) or rarely on basic rocks. Asplenium adulterinum Milde subsp. presolanense Mokry, Rasbach & Reichst. occurs in North of Italy and South of Switzerland on limestone or mica-schist (Crabbe et al., 1993).
In the Romanian botanical literature Asplenium adulterinum is considered a rare species (Boşcaiu et al., 1994;Oltean et al., 1994;Oprea, 2005) or placed in the "Insufficiently Known" (K) sozological category . The species was included in the Red Book of Vascular Plants of Romania (Dihoru and Negrean, 2009). Inclusion in a sozological category of species in Romania was difficult, considering that the fern has not been seen by botanists in recent decades. Since 1931 no botanist has collected Asplenium adulterinum from the Romanian Carpathians and deposited in a public herbarium (in reality there is a single herbarium voucher in BUCA Herbarium (BUCA no. 004147), whereas the plant was collected by Grinţescu on 3 July 1931, near Băile Herculane, but the data have never been published.
According to the Red Book of Vascular Plants of Romania (Dihoru and Negrean, 2009) it seems that there is also a specimen deposited in the CL herbarium: "Custura Mătaniei, 12 VII 1964, N. Boşcaiu [CL]". The authors hereby checked this information in CL herbarium (on 15 and 16 January 2015) and did not find any specimens of Asplenium adulterinum.

Materials and Methods
The investigations were based on recent field studies and analysis of herbarium material stored at CL, BUCA, BUCF, BVS, SIB, I, IAGB, IASI, B, W, WU, P, CRAI (acronyms according to Thiers, 2015), as well as literature data. All existing herbarium material (including Asplenium trichomanes and A. trichomanes-ramosum) was revised and all available information from botanical literature was critically compiled in order to clarify the distribution of Asplenium adulterinum in the South-Eastern Carpathians.
Phytosociological characteristics of possible sites with Asplenium adulterinum were studied according to the Braun-Blanquet approach (Braun-Blanquet, 1932).
The collected material has been lodged in the personal herbarium of A. Bartók.

Results and Discussion
Distribution of Asplenium adulterinum Milde in South-Eastern Carpathians Rarău Mountains, Eastern Carpathians Pietrele Doamnei (Lady's Stones) and their surroundings represent maybe the most intensively investigated region by the botanists, considering the whole range of Rarău Mountains.
In this mountain range the presence of Asplenium adulterinum was reported by some authors (Mititelu et al., 1989;Oprea andSârbu, 2012, 2013;Bădărău, 2013), but without exact localisation and never confirmed by herbarium material. The main botanists (Morariu, 1965;Raclaru, 1973Raclaru, , 1976 who investigated in detail the mountain and subalpine flora of Rarău Mountains did not mention Asplenium adulterinum; they both refer only to Asplenium trichomanes-ramosum and Asplenium trichomanes.
During the current research surveys, the species was not found near Pietrele Doamnei or in other parts of this mountain group (Bartók A, pers. obs. 2007(Bartók A, pers. obs. , 2010(Bartók A, pers. obs. , 2011(Bartók A, pers. obs. , 2014. It can be said that certainly it was confused here with Asplenium trichomanes-ramosum or Asplenium trichomanes, since these two species are frequently encountered in the region.

Călimani Mountains, Eastern Carpathians
Neither Csűrös (1951) nor Höhn (1998a) mentioned Asplenium adulterinum in the area of Călimani Mountains. However, Mititelu et al. (1986) and Chifu et al. (2008) reported ladder spleenwort in the flora of this mountain range, but without exact localisation or citation. Although Höhn (1998a) did not mention Asplenium adulterinum in the flora of Călimani Mountains, the same author highlights in another publication (Höhn, 1998b) (according to personal observations) that species with affinity for basic soils (e.g. Asplenium adulterinum Milde, Carex sempervirens L., Phyteuma orbiculare L., Saxifraga adscendens L.) do not occur on the south part of Călimani Mountains, but on the north face.
Yet, the most recent checklist of Călimani National Park (ICB Iaşi, 1994) does not mention Asplenium adulterinum in Călimani Mountains. Since it is known that the list of Mititelu et al. (1986) is partly based on older literature without citations, Asplenium adulterinum could not be observed in the field (Bartók A, pers. obs. 2007(Bartók A, pers. obs. , 2010(Bartók A, pers. obs. , 2011 and therefore it can be concluded that is was confused with Asplenium trichomanesramosum or Asplenium trichomanes, since these two species are frequently encountered in this region. There are no herbarium specimens of Asplenium adulterinum from the floristically well explored Călimani Mountains, in all herbaria checked.

Bistriţei Mountains, Eastern Carpathians
Asoltani (2007) listed Asplenium adulterinum in this mountain range from Şaru Bucovinei and Şeştină, but it was not confirmed by herbarium voucher. Seghedin in his thesis and in floristically synthesis of Suceava County (Seghedin, 1985(Seghedin, , 1987 did not mention Asplenium adulterinum, but Asplenium trichomanes-ramosum and Asplenium trichomanes instead, in this mountain group. Also Mardari (2008) in a floristical synthesis of Bistriţei Mountains flora could not confirm the presence of ladder spleenwort in this area.
The flora of this mountain range is not well studied, and the presence of Asplenium adulterinum remains doubtful (there was no herbarium voucher in all checked herbaria).
However, Nechita and Bliderişanu (2006) listed ladder spleenwort in Bicazului Mts. but without exact locality or citation. The species was reticently mentioned to appear in Stânişoarei Mountains (Oprea andSârbu, 2008, 2009). It might be supposed that the mentioned coronim (Bicazului Mts.) refers to the Northeastern part of Hăşmaş Mountains, which is situated near Stânişoarei Mountains. Bădărău (2013) mentioned Asplenium adulterinum in "Cheile Bicazului-Hăşmaş" (ROSCI 0027) Natura 2000 network, but without exact locality or citation. Also, ladder spleenwort is listed in the Management Plan of Cheile Bicazului-Hăşmaş National Park, but without exact locations or citation. There are no herbarium vouchers of Asplenium adulterinum in all checked herbaria.
Moreover, ladder spleenwort was recently reported on Munticelu-Cheile Şugăului Nature Reserve near Şugăului Gorges by Associate Professor Dr. Ioana Popescu (Fig. 1, photo A). Herbarium material was not collected, but in the pictures taken it is clear that the colour of rachis is green in distal part. Asplenium adulterinum is growing here in a non-typical habitat on limestone rocks (the studied area does not include serpentine rocks) (Corvin Papiu, 1963).
Ladder spleenwort is listed in the Management Plan of Ceahlău National Park, but without exact data. There are no herbarium specimens of Asplenium adulterinum from the floristically well explored Ceahlău Mountains in all checked herbaria.

Mehedinţi Mountains, Southern Carpathians
This mountain range was not well explored floristically; only Domogled Peak with its surroundings and Ţesna Valley were studied more rigorously from the botanical point of view. Borbás (1874) and Simkovics (1878) followed by Degen (1901) studied the flora of Domogled Massif, without any report about the occurrence of Asplenium adulterinum in this mountain group, but Asplenium trichomanes-ramosum and Asplenium trichomanes instead. Nor the more recent research of Resmeriţă (1970Resmeriţă ( , 1971Resmeriţă ( , 1972 did not demonstrate the occurrence of ladder spleenwort in the floristically explored Ţesna Valley. Neither Georgescu (1934) nor Buia (1959) or Bujorean and Popescu (1966) mentioned Asplenium adulterinum for the Mehedinţi Mountains.
In the document compiled by Faculty of Biology and Geography -Babeş-Bolyai University Cluj-Napoca -" Research about flora and vegetation of the parcels with Banat black pine from the Domogled-Valea Cernei National Park" (2006) the occurrence of Asplenium adulterinum was mentioned in this mountain range, but without any specific locality or citation. Moreover, the authors of this publication recognized the absence of the species on field. Bădărău (2013) mentioned Asplenium adulterinum in "Domogled-Valea Cernei" (ROSCI 0069) Natura 2000 network, but without exact locality or citation. A previously unknown citation about a population of Asplenium adulterinum was noted in Mehedinţi Mountains (Southern Carpathians) on 1 June 2013, reffering to the middle part of the ŢesnaValley, in semi shady rocky place, exposed to the north, at approximately 600 m a.s.l. Asplenium adulterinum was growing there in a non-typical habitat: on limestone rocks, in crevices of rocks, not far from the tourist path. Although the authors of the current study researched in detail a great part of the area, a single individual was found. Accordingly, it may be supposed there was a very small population in that place. Because a single specimen was found, herbarium material was not collected. This discovery was documented by photos ( Fig. 1. B, C).
Additionally, it is worth mentioning that only one voucher specimen of Asplenium adulterinum from Romanian Carpathians was found in all herbaria consulted (BUCA no. 004147). The plant was collected by Grinţescu on 3 July 1931 near Băile Herculane (in humid rock crevices), the only specimen previously known from the Romanian Carpathian, but the data have never been published.
Mehedinţi Mountains are one of the few mountains in Romanian Carpathians wherein the presence of this rare fern was confirmed.

Cernei Mountains, Southern Carpathians
The area of Arjana Peak and Vânturătoarea Waterfall from Cernei Mountains, shelter a very interesting flora, with rare species e.g. Minuartia graminifolia (Ard.) Jáv. subsp. hungarica Jáv., considered endemic for Southern Carpathians (Conti, 2003). There are no previous collected herbarium specimens of Asplenium adulterinum or floristic data about this species in the Cernei Mountains.
During the current study field research, a population of Asplenium adulterinum was identified on 27 April 2014, near Vânturătoarea Waterfall, in adumbral rocky place, exposed to the north, at 550 m a.s.l. Asplenium adulterinum was growing there in a non-typical habitat: on limestone rocks, in crevices of rocks. The area is protected; it is included in Domogled-Valea Cernei National Park. The population was poor, counting only three specimens of plants. The population was probably more numerous, but it was not possible to examine the whole area in detail. The collected material was lodged in the personal herbarium of A. Bartók.
There are no herbarium specimens from the Ţarcu Mountains in all checked herbaria. Moreover, the authors could not find Asplenium adulterinum in the area of Custura Mătaniei nor in other parts of this mountain range (Bartók A, pers. obs. 2013(Bartók A, pers. obs. , 2014, but Asplenium trichomanes-ramosum instead.
Unfortunately, the occurrence of Asplenium adulterinum in Ţarcu Mountains remains doubtful.

Almăjului Mountains, Western Romanian Carpathians, Banat Mountains
The flora of Almăjului Mountains was relatively well studied, but the presence of Asplenium adulterinum was not yet demonstrated by herbarium material.
Volume I of Romanian flora (Grinţescu, 1952) mentioned the occurrence of ladder spleenwort in Danube Gorges on the serpentine massif between Poiana Mraconia and Baia Nouă, but without any specific location or citation. It is curious that Grinţescu did not specify Cernei-valley coronim in Flora of Romania, although he collected herbarium material in 1931. There is no publication (before 1952) which listed the presence of this rare fern in that mountain range. Matacă (2005) mentioned the occurrence of Asplenium adulterinum in Porţile de Fier Nature Park, but she only cites the Romanian flora (Grinţescu, 1952). Other publications (Borbás, 1874;Simkovics, 1878;Hayek, 1916;Borza, 1931;Resmeriţă et al., 1968;Csűrös et al., 1968;Pop et al., 1969;Dihoru et al., 1972) did not list the presence of ladder spleenwort in this mountain group. Bădărău (2013) mentioned Asplenium adulterinum in "Porţile de Fier" (ROSCI 0206) Natura 2000 network, but without exact locality or citation.
The area of "Cazanele Mari" is dominated by serpentine rocks (the ideal substrate for Asplenium adulterinum) and in the authors' opinion this is the explanation for undocumented citation of this rare fern in the mountain group.
Ladder spleenwort could not be found in Aninei Mountains (Bartók A, pers. obs. 2011(Bartók A, pers. obs. , 2013. There are no herbarium specimens of Asplenium adulterinum from that mountain range in all checked herbaria.

Codru-Moma Mountains, Western Romanian Carpathians, Apuseni Mountains
In the area of Codru-Moma Mountains, Asplenium adulterinum was mentioned in Izbuc Brook (Briheni village) by Burescu and Păşcuţ (2010) in a phytosociological paper. The authors only mentioned the presence of ladder spleenwort within Asplenium trichomano-rutae murariae Kuhn 1937 association, but without other comments of this important biogeographical and floristical finding. In this plant association, the ladder spleenwort would be present together with Poa nemoralis L. and Doronicum columnae Ten.
The flora of this mountain group was studied by Paucă (1936Paucă ( , 1941aPaucă ( , 1941b, but the occurrence of this rare fern was not mentioned. There are no herbarium specimens of Asplenium adulterinum from that mountain group in all checked herbaria.
In contrast, Asplenium trichomanes coming from this mountain range was well documented in the Romanian botanical literature.
Based on the current study field research, analysis of stored herbarium material and literature data, it was developed the chorological map of distribution of Asplenium adulterinum in Romanian Carpathians (Fig. 2).

Recommended IUCN threat category
Only three population of Asplenium adulterinum are certainly known in the Romanian Carpathians, in restricted area of Hăşmaş, in Mehedinţi and Cernei Mountains. The places where the species occurs are apparently protected (Domogled-Valea Cernei and Cheile Bicazului-Hăşmaş National Park), but in reality the area is exposed to human activities (e.g. tourism, grazing).
On the basis of new chorological data and estimation of the number of individuals and population condition, Asplenium adulterinum can be define as IUCN CR C2a(i) (IUCN, 2012) in Romania.

Conclusions
All available information from Romanian botanical literature was critically compiled in order to clarify the distribution of Asplenium adulterinum in the South-Eastern Carpathians.
Based on field studies, three new localities of Asplenium adulterinum (one important threatened species in Europe) were discovered. The occurrence of Asplenium adulterinum in Romanian Carpathians was recorded and the threatened status according to criteria and categories of IUCN was determined.