Nickel Hyperaccumulation in the Serpentine Flora of Albania

NICKEL HYPERACCUMULATION IN THE SERPENTINE FLORA OF ALBANIA

Aida Bani1, *, Alma Imeri2, Guillaume Echevarria3, Dolja Pavlova4, Roger D. Reeves5, Jean Louis Morel3 and Sulejman Sulçe1

1Agro-Environmental Department, Faculty of Agronomy and Environment, Agricultural University of Tirana, Koder-Kamez, Tirana, Albania 2Department of Plant Production, Faculty of Agronomy and Environment, Agricultural University of Tirana, Koder-Kamez, Tirana, Albania 3Laboratoire Sols et Environnement, Nancy-Université, INRA, 2 avenue de la Forêt de Haye, B.P. 172 F-54505, Vandoeuvre-lès-Nancy, France 4Department of Botany, Faculty of Biology, University of Sofia, blvd. Dragan Tzankov 8, 1164 Sofia, Bulgaria 5School of Botany, University of Melbourne, Parkville, VIC 3010, Australia

ABSTRACT They are characterized by low calcium-to-magnesium ratios with Ca at significantly lower concentrations rela- Serpentine soils are widespread in Albania, they host tive than in surrounding areas. Serpentine soils are often some widespread species and several nickel hyperaccu- deficient in essential plant nutrients such as nitrogen, potas- mulating species. The objectives of the study were the sium, and phosphorus [1-3] and their physical conditions identification and collection of new and rare species, the also are inhospitable for many plants. These characteris- discovery of new instances of Ni hyperaccumulation, in tics support a unique serpentine flora, a flora which pre- which plant species can accumulate Ni to concentrations sents taxonomical, phytogeographical and ecological phe- exceeding 0.1% of the plant dry weight. In Albania, there nomena of great interest. Brady et al. (2005) [4] empha- has already been special interest in Ni accumulation by sized that plant adaptation to different soil types is evidence Alyssum murale in Pojska (Pogradec). We now report in- of the strong natural selection imposed by ecological dis- stances of hyperaccumulation of Ni (reaching shoot con- continuities. The influence of ultramafic soils on the speci- centrations of 0.5-1.26% d.w.) in plants of some serpentine ation process and species richness has been the subject of soils i.e. in Kukës, Qafë Shtamë, Gramsh and Librazhd. much investigation [1, 3, 5, 6]. The high number of endem- Most plants from these soils showed slightly elevated Ni ics indicates the importance of serpentine habitats as cen- concentrations in comparison with those from other soil ters for floristic differentiation and speciation, and this types: about 11-100 mg kg-1, rather than 0.5-10 mg kg-1. endemism ranges from strictly local (a single outcrop) to In some species Ni content ranged between 145-275 mg regional and widespread occurrences [5, 7, 8]. Serpentine kg-1, while the same species from the non-serpentine areas also appear to be important refuges for many non- substrata contained very low Ni concentrations as ex- endemic native species, since they are limitedly invaded by pected. Full knowledge of Albanian Ni hyperaccumulator other plant communities. Serpentine soils account for 10% plants and their properties is of interest because of their of the land area of Albania, on which some plant taxa are possible use in phytomining. This study was undertaken serpentine endemic. The species on serpentine soils can be in the framework of the need for further exploration of the serpentine-tolerant (ST) or serpentine-facultative (SF) plants, natural resources and the associated conservation issues. which are able to survive on serpentine but grow better else-

where and serpentine-endemic or serpentine-obligate (SO) plants, which are not found on other substrata [9]. Early KEYWORDS: work on the serpentine flora of the region has been sum- Ni hyperaccumulation; flora; endemic plants; high altitude marized by Brooks (1987) [1], and other notable contribu-

tions are those of Babalonas et al. (1984) [10], Pavlova, Shuka and Hallaçi (2010) [11-13]. According to Stevano- 1 INTRODUCTION vić et al. (2003) [8] there are 335 Balkan endemic vascular plant taxa growing on serpentine substrates, of which Due to their physical and chemical composition, ser- 123 are obligate serpentinophytes. Confirmation of the pentine soils are known for their distinctive flora and hyperaccumulation by a number of Albanian serpentine vegetation, as compared to adjacent non-serpentine areas. species has been subject of some studies [14-17], but it is Serpentine soils frequently contain elevated levels of still insufficiently investigated. Serpentine outcrops can be heavy metals, such as iron, manganese, nickel, chromium, regarded as inland formations in mainland environments and cobalt, some of which are toxic to most plants. and biogeographic theory may help understand their species richness and species distribution [18-20]. Species of * Corresponding author particular interest include a number of endemics, suben-

1792 © by PSP Volume 22 – No 6. 2013 Fresenius Environmental Bulletin

demics, and Ni hyperaccumulators such as Bornmuellera ranean climate. The annual average temperature was 13 ºC baldaccii (Degen) Heywood subsp. markgrafii O.E. with maximum from 25º-30ºC (from June to August), and Schulz, Alyssum markgrafii O.E. Schulz ex Markgraf, A. minimum 0 and 2.5ºC. The average annual precipitation bertolonii Desv., and the most widespread A. murale was 1600 mm. In the first sampling station (site 1) we Waldst. & Kit. Geographic isolation by altitude in high collected the Ni hyperaccumulator Alyssum bertoloni Desv. mountains and edaphic differentiation by soil types may subsp. scutarinum (silicula 2.8-5.5 mm wide, seed-wing underlie plant endemism in such regions [21]. Moreover, 0.5 mm wide) and plant taxa that accompanied Ni-hyper- some serpentine endemics of the Balkans, including hyperaccumulator present on the site. In site 2 we opened a accumulator species (e.g. B. baldaccii), are only reported at profile and sampled soil and plant. Ni hyperaccumulator high elevation in literature [15]. Alyssum bertoloni Desv. was collected also in site 3 at an Metal hyperaccumulators have recently gained con- altitude of 1044 m. siderable interest because of their potential use in phy- The third region was Kukës near the tunnel of Kali- toremediation [22, 23], phytomining [24, 25] and food crop mash along the road for Surroj village. The region is part biofortification [26, 27]. They also constitute an excep- of the north Albanian mountain climatic region, with Medi- tional biological material and gene reservoir that can be terranean influence. The average temperature of January exploited to understand metal homoeostasis and adapta- throughout the entire site reaches low values. Average tion to extremely hostile environments. temperature oscillate from a minimum -2º to -3ºC (January) to maximum 19º-21ºC (from June to August), and annual This concentration of Ni hyperaccumulators in Alba- average between 9º and 12ºC. The average annual precipi- nia led us to investigate their distribution and behavior tation ranges from 1300 up to 1600 mm, with rainfall more closely, especially at some sites that are know for occurring mainly from the end of October to the end of hyperaccumulators [14-16, 28, 29]. The objectives of the May. Ni hyperaccumulator Alyssum markgrafii O.E. Schulz study were the identification and the collection of rare and all accompanying plants taxa were sampled on site to taxa in serpentine habitats and the scanning for potential study the Ni hiperacumulation. Ni hyperaccumulation in relation to the soil characteristics and to these environments. The study region Librazhd belongs to the hilly Medi- terranean climate sub zone (up to 1100 m) where mean January temperature is 5.6 °C, mean July temperature 22 °C,

annual average 11.5 °C and total annual rainfall 1360 mm. 2 MATERIALS AND METHODS Up to 2% Ni has been recorded in the iron-type laterites of 2.1 Study area the Librazhd-Pogradec region. There are also Cr mines that are still mining for chromite in this area. Ni hyperaccumu- Four serpentine regions in Albania: Gramsh, Qafë lator Alyssum markgrafii O.E. Schulz and all accompany- Shtamë, Kukës and Librazhd were investigated. The study ing plants were sampled only in site 1 of this region. area belongs mainly to two climate sub zones: the pre- mountainous Mediterranean and mountainous Mediterra- 2.2 Herbarium specimens investigated for Ni hyperaccumula- nean climate in the highest peaks. tion Gramsh (Grabovë e Sipërme) is situated in the Eastern Plant samples for Brassicaceae species reported as Ni mountain climatic region of Albania, within the Mediterra- hyperaccumulators from each of the four selected regions nean mountain Climatic Zone at an elevation of 1594 m were taken from the specimens of the National Herbarium (40°47 824’N, 20°25 864"E). This mountain region dis- of Albania at the University of Tirana to investigate Ni plays cold winter and fresh summer temperature. Average and to compare them with our collected samples. The spe- temperature is 14.3ºC, with minimum 4.6 ºC in January cies were: A. bertolonii subsp. scutarinum, A. markgrafii, and maximum 26.4 ºC in July. The average annual pre- A. murale and B. baldaccii subsp. Markgrafii. cipitation attains 1350 mm. Three sites were studied for Ni hyperaccumulators. In site 1 together with Ni hyperac- 2.3 Soil and plant analyses cumulators Bornmuellera baldaccii subsp. markgrafii (Bras- The plant identification and nomenclature follows sicaceae) and Alyssum markgrafii O.E. Schulz all plant taxa Tutin et al. (1993) [30]. Voucher specimens of the plants that accompanied already known Ni-hyperaccumulators collected were deposited in the Herbarium of the Univer- were also collected. This site was a pasture with low pro- sity of Tirana, Albania. One to three specimens for each ductivity which was currently being grazed. The other Ni plant were collected from each site. Specimens were pooled hyperaccumulator, Alyssum murale Waldst. & Kit was to make a composite sample when there was more than sampled in site 2 (i.e. a rocky area) and in site 3 (i.e. one. All plant samples were washed, dried and ground to a Komjan). We selected three sampling points for each re- fine powder. Trace metal concentrations in leaves were gion to obtain representative results for that region. Qafë determined by atomic absorption spectrophotometry (AAS) Shtamë is situated at about 2 km in the northeast of the after microwave digestion of plant samples: A 0.25-g DM historical town of Kruja in the center of Albania. This plant aliquot was digested by adding 8 ml of 69% HNO3 2 protected area of 20 km belongs to two climatic Sub-zones: and 2 ml of H2O2. Solutions were filtered and adjusted to pre-mountainous climate and at the top is mountain Mediter- 25 ml with 0.1 M HNO3.

1793 © by PSP Volume 22 – No 6. 2013 Fresenius Environmental Bulletin

TABLE 1 - Localisation of Ni-hyperaccumulator plant taxa sampled in serpentine sites of Albania.

Sampled plants Sampling sites Site Altitude (m asl) Geographical coordinates Alyssum markgrafii O.E. Schulz Gramsh Site 1 1594 40°47 824’N, 20°25 864"E Bornmuellera baldaccii subsp. markgrafii O.E. Schulz Gramsh Site 1 1594 40°47 824’N, 20°25 864"E Alyssum murale Waldst. & Kit Gramsh Site 2 1512 40° 47 449' N 20°25 001’E Gramsh Site 3 1300 40° 46 530' N, 20°20 546’ E Alyssum bertolonii Desv. subsp. scutarinum E. I. Nyàràdy Qafë Shtamë Site 1 1400 41° 51' 99"N, 19°88' 240"E Qafë Shtamë Site2 1235 41° 52' 280"N, 19°89' 886"E Qafë Shtamë Site 3 1044 41° 52' 05"N, 19°89' 319"E Alyssum markgrafii O.E. Schulz Kukës Site 1 1300 42° 00 35"N , 20° 17.44" E Kukës Site 2 1454 42° 01 161' N, 20° 03.84' E Kukës Site 3 1460 42° 01 766" N, 20° 19’ 558"E. Librazhd Site 1 1150 41° 10' 59.944"N, 20° 19' 00"E Alyssum saxatile L. Kukës Site 1 1300 42° 00 35"N , 20° 17.44" E

The soil profile was described and samples were taken all the sites were characterized by elevated levels of met- from the two horizons identified in the field (0–20 cm, 20– als such as Ni, Cr, Co that are typical of ultramafic envi- 35 cm). From each site one to three soil samples were taken ronments (Table 2). The total Ni concentrations varied in from the upper horizon at a depth of 0-20 cm where pos- the range 1772- 3077 mg kg-1. All concentrations were sible, near the roots of the sampled plants. Again, a com- rather higher than those considered toxic to normal plants posite sample was made when there was more than one. by Allen et al. (1974) [34] and Kabata-Pendias (1984) Sampling areas are indicated by the four serpentine regions [35], although toxicity depends on Ni bioavailability [31]. shown in Table 1. Soil samples were air-dried and sieved The total concentrations of Cr at all sites were high lying in to 2 mm. Samples were mineralized with a microwave the range 295-1035 mg kg-1 exceeding the upper limit giv- digester. Conditions for mineralization were 6 ml HCl, 2 ml en for normal soils by Allen et al. (1974) and Brooks -1 HNO3, and 3 ml H2O2, per 0.5-g soil. Total major (Ca, (1987) [1, 34], except Gramsh rock that is 178 mg kg . Mg, and Fe) and trace elements (Co, Cr, Ni, Zn) were measured in mineralization solution by atomic absorption spec- The Zn concentrations fell within the ranges for nor- trophotometry (AAS) in the Public Health Laboratory of mal soils lying in the range 20-152 mg kg1 [34, 35]. The Tirana, Albania. Nickel availability in different soil sam- Co concentrations, ranging from 62 -260 mg kg-1, were ples was characterised by the DTPA-TEA method [31, 32]. also higher than the values indicated for normal soils by DTPA-extractable Ni was determined using the method of Allen et al. (1974), Kabata-Pendias & Pendias (1984) and Lindsay and Norvell (1978) [33]. Concentration of Ni in Brooks (1987) [1, 34, 35]. All soils had Fe concentrations soil extracts was also determined by atomic absorption spec- higher than 8.5 % as expected for soils of ultramafic ori- trophotometry (AAS). Soil Ca:Mg ratios were calculated gin. The pH of serpentine soils can vary quite widely, and soil pH (in water) was also measured. mainly in the range 6.1-8.8 [1, 5], although values as low as 4.6 have occasionally been reported (Proctor 1992) [36]. All the soils values in this study were neutral to alkaline 3 RESULTS AND DISCUSSION despite the high elevation and high rainfall. The range of pH values among the soils was quite narrow. The most 3.1 Soil characteristics acidic soil had a value of 6.7 (Gramsh), which was also the For the first time, we have analyzed some of the high- soil at the highest elevation (about 1600 m above sea level). altitude serpentine soils of Albania. The serpentine soils at The soil of Kukës was the most alkaline soil. As expected

TABLE 2 - pH, total major elements and total trace elements in some Albanian serpentine soils.

Total trace elements Sampling site pH Ca Mg Fe Co Cr Ni Zn % mg kg-1 Gramsh, site 1, profile 0- 20cm 6,71 0,23 5,4 10,3 85 295 1772 36 Gramsh, site 1, profile 20 – 40cm 6,85 0,18 4,7 10,3 195 377 2237 31 Gramsh, site 2(0-20cm) 6,89 0,40 3,7 10 156 178 1978 20,1 Gramsh, site 3(0-20cm) 6,87 0,52 5,8 9,8 156 488 2947 30,1 Qafë Shtamë, site 1(0-20cm), 7,04 0,3 12,3 9,81 260 617 2680 18,8 Qafë Shtamë site 2, profile 0- 20cm 6,96 0,15 3,6 9,6 101 670 2658 27 Qafë Shtamë site 2, profile 20 – 40cm 7,08 0,11 3,4 9,05 62 1035 3077 53 Qafë Shtamë, Site 3(0-20cm) 7,36 0,3 4,5 8,5 78 262 1658 25 Kukës, Site 1, profile 0- 20 cm 7,23 0,45 16,8 12,2 150 503 2455 109 Kukës, Site 1, profile 20 – 40cm 7,45 0,40 16,2 11,7 164 487 2715 80 Kukës, site 2 (0-20cm) 7,1 0,40 15,7 11,3 158,4 338,5 1787 152 Kukës, site 3(0-20cm) 7,42 0,38 12,7 12 140 578 1926 161 Librazhd W(0-20cm) 6,9 0,661 5,09 9,82 250 773 2989 175

1794 © by PSP Volume 22 – No 6. 2013 Fresenius Environmental Bulletin

FIGURE 1 - Soil Ni availability assessed by DTPA-TEA extraction in some serpentine soils from Albania.

FIGURE 2 - Concentration of Ni, Ca and Mg in plants of the different taxa of Ni hyperaccumulators from Albania.

1795 © by PSP Volume 22 – No 6. 2013 Fresenius Environmental Bulletin

FIGURE 3 - Spectra of the main floristic elements on study ultramafic sites.

for serpentine soils, all were characterized by extremely 3.3 Uptake of Ni and other elements by hyperaccumulator low Ca:Mg quotients of 0.024-0.12. Soil Ca ranged from plants 0.11-0.66%, whilst total Mg concentrations were 3.4- Analysis of metals performed on plant bulk shoots 16.8%. (i.e., stems, leaves and flowers/ fruits) and on leaf material showed different plant responses to the presence of Ni, Ca, 3.2 Variability of Ni availability along the high-altitude serpen- Mg in soils collected at the different sites (Figure 2). The tine soils of Albania highest Ni values in plants were recorded for A. markgrafii, The total concentration of trace elements in soils pro- which is a serpentine-endemic and Ni hyperaccumulator vides limited information on their bioavailability, as this species, but Ni concentration in this species was highly de- does not show how strongly the element is bound to the pendent on the site of collection. The highest Ni concentra- surface of the soil constituents [37]. DTPA-extractable Ni tion in bulk shoots of A. markgrafii was observed in study was chosen as an estimate of soil Ni chemical availability. sites from Kukës (ranging from 8,830 to 12,691 mg kg-1), Although it only reflects the potential pool of available Ni followed by Gramsh (site 1) (6,074 mg kg-1) and the lower (R. Chaney, pers. comm.), it allows relative comparison values were recorded in Librazhd. Ni content in A. mark- of soils. We used this measurement as the indicator of the grafii shoots from herbarium specimens (Table 3) was much relative variability of Ni availability of the different ser- lower and reached 8,089 mg kg-1 in Kukës as a maximum. pentine soils (Fig. 3). The amounts of DTPA-extracted Ni A. murale, which is the species with higher Ni content ever were high in high-altitude Albanian soils. recorded in Albania and the most common Ni hyperac- DTPA-extracted Ni in soils varied from 280 mg kg-1 cumulator in the Balkans [15, 16], was found only at one (Site 1 Gramsh) to 21 mg kg-1 (Site 3, Qafë Shtamë). site in Gramsh. Its Ni concentration in shoots ranged from -1 They were higher for the soils of site 1 and site 3 in 5,866 to 9,300 mg kg depending on pedogenetic proc- Gramsh (respectively 280 and 260 mg kg-1), site 2 in esses, soil functioning and elevation. It was very similar to Kukës (151 mg kg-1), and sites 1 and 2 in Qafë Shtamë the concentration of Herbarium specimens from this site. (140 and 136 mg kg-1 respectively). The amounts of Qafë Shtamë is the serpentine region where we have DTPA-extracted Ni were lower in soils of site 3 in Qafë collected Alyssum bertolonii Desv. subsp. scutarinum an Shtamë (24 mg kg-1) and of site 2 in Gramsh (28 mg kg-1). endemic and Ni hyperaccumulator species which appears The differences in availability of Ni in our soils and its on some serpentine localities in North Western part of subsequent transfer to plants depend mainly on its minera- Balkan peninsusla [41] and Greece [42]. However, Hartvig logical origin and therefore on the local pedogenetic prop- (2002) [43] considers this taxon as doubtfully recorded for erties [38, 39] which result from climatic conditions and Greece and represented taxa included in synonymy of A. weathering history of the soil as well as on soil characteris- bertolonii subsp. scutarinum more closely related to A. tics such as pH [40]. Ni availability in the soils is gener- murale than to A. bertolonii s.str. from Italy. The popula- ally higher when pH values are lower [31]. tions of A. bertolonii from Apennines (Tuscany region)

1796 © by PSP Volume 22 – No 6. 2013 Fresenius Environmental Bulletin

are considered as a typical subspecies of A. bertolonii [41]. collection. It was higher for A. markgrafii Kukës, site 2 Also, according to Hartvig (2002) [43] the Italian popula- (7.1) and B. baldaccii, Gramsh site 1 (6.8). This ratio rep- tions of A. bertolonii differs from Balkan populations in resented the transfer factor since soil was sampled around several significant characters,, very small, multi-rayed, the roots of plant species [48, 49]. This ratio can be inter- sublepidote stellate hairs on the leaves, distinctly and un- preted as an indication of optimal hyperaccumulation con- evenly inflated silicula valves, and narrowly winged seeds. ditions due to high availability of Ni in the soil of origin Differences between two taxa are obvious comparing size and high accumulation potential of the plant. If available of the siliculas which are always higher in subspecies soil Ni is low, the transfer factor still reaches values higher scutarinum [41]. On the basis of molecular and morpho- than 1 due to the hyperaccumulating physiology of plants, logical evidence from native populations, Chechi et al. like the case of A murale in Domosdova field and A. (2010) [17] indicate that A. bertolonii subsp. scutarinum, markgrafii in Gjegjan, Fushe-Arraz in Albania [15]. A. chlorocarpum, A. jankenii, A. markgrafii and A. chal- Calcium concentrations in shoots of hyperaccumulator cidicum, previously regarded as endemic hyperaccumula- species were very high despite the low soil Ca levels. This tors, can hardly be differentiated t from A. murale. Also, property of Alyssum species on serpentine has been marked he considers that most populations of this species growing previously [14-16]. In the present work we found higher in Albania and Greece have a clear preference for serpen- values for A. murale (2.2%) and A. markgrafii (2.1%) and tine soils and ability for Ni hyperaccumulation and con- lower for B. baldaccii (0.69%). This observation has been cluding that slight morphological differentiation may sup- reiterated by others [14, 50]. Ca hyperaccumulation in port, at most, their recognition at the varietal rank. Brassicaceae endemic from serpentine is a typical trait of High Ni values were found for A. bertolonii subsp. this taxonomic group of hyperaccumulators [15]. -1 scutarinum (9,938 mg kg ) from Qafë Shtamë. Hyperaccumulator plants showed a different uptake Genus Bornmuellera Hausskn. is one of the smallest pattern in response to the high concentration of Mg and in European flora [30, 44] presented with three species (B. the low concentration of Ca in soils than the other non- baldaccii, B dieckii and B tymphaea) distributed in the accumulator species [25], especially the species from the Balkan peninsula. Variable species B. baldaccii has two genus Alyssum (Table 3). The mean Mg concentrations for subspecies (subsp. baldaccii and subsp. rechingeri Greuter) the Alyssum species were in the range 0.12-0.5%, while according to Akeroyd, 1993 [44] distributed in Albania and those for Bornmuellera were higher (0.62%). Distributions Greece, respectively. All three species are known from Ni- of Ni hyperacumulators depend on the biology of the spe- rich ultramafic (serpentine) soils and are probably endemic cies and edaphic factors. So A. markgrafii was found in to such areas [45]. Bornmuellera baldaccii which was found the colder sites and the Ni hyperaccumulation was higher in some of the study sites demonstrates remarkable Ni con- in Kukës where pH is higher than other sites. This study centration in bulk shoots of 12,171 mg kg-1. This is the confirms previous data [15] and shows that Ni hyperac- second Ni-richest species in this study. The Ni concentra- cumulator and endemic A. markgrafii is in best condition tions for B. baldaccii, were similar to that of the specimens for hyperaccumulation in the mountains of the north of collected in Gramsh from the Herbarium, but lower that the Albania, Kukës. A. bertolonii was found only in one re- specimen collected at an altitude of 1700m near Kamja in gion, in the center of Albania. B. baldaccii, an serpentine southeastern Albania (27300 mg kg -1) [46]. We confirm data endemic and Ni hyperaccumulator plants was also found of Reeves et al. (1983) [47] who reported high Ni concentra- only in one site where the pH is lower in comparisons tions in B. baldaccii (Degen) Heywood from Albania and with other sites and only at elevations higher than 1500 m from Greece. The same authors report extremely high Ni above sea level. –1 concentrations in leaf dry matter (31 200 mg kg ) in other The serpentine flora of Albania is characterized by Balkan endemic species B. tymphaea (Hausskn.) Hausskn. plant diversity specific for each study site. The most com- from northern Greece as well. mon species in the study serpentine area are the perennial The ratio of Ni concentration in plant shoots to its grasses (hemicryptophytes). The number of species in the concentration in soil varied depending on species and site sites varies from 15 to 20. The dominant species in sites are

TABLE 3 - Concentration of Ni, Ca, and Mg in plants of the different species of Ni hyperaccumulators from Albanian serpentine sites collected from dry material kept in National Herbarium. Results are given as mean values of three replicates ± standard deviations.

Species Sampling Site Ni Ca Mg mg kg-1 Alyssum murale Gramsh 1300 m 10354±527 21250±334 4937±150 Librazhd 1150 m 4973±280 21157±875 3762±240 Alyssum bertoloni subsp. scutarinum Qafë Shtamë 1200 – 1400 m 6984±1250 30555±5400 3398±849 Qafë Shtamë 600 m 8567±740 20000±2498 4500±1297 Alyssum markgrafii Kukës 1200 m 8089±580 30740±6589 6587±1279 Gramsh 1600 m 3731±1177 20132±245 2244±1456 Bornmuellera baldaccii subsp. markgrafii Gramsh 1600 m 12115±28 6741±165 6089±564

1797 © by PSP Volume 22 – No 6. 2013 Fresenius Environmental Bulletin

representatives of Asteraceae, Poaceae, Lamiaceae, etc. the Balkans. The ophiolithic areas of Albania like the ophio- The flora is mainly composed of Euro-Mediterranean lithic areas of all the Balkans [8] are undoubtedly important (17.4%), Eurasian and Paleotemp (11.11 %), Endemic centers of endemism. (17.46%), Balcanic (7.93%) and Eur, S-Eur (12.69) taxa (Figure 3). The mountains of Albania represent the impor- 3.4 Ni uptake by the different plant species tant centre of ophiolithic endemism [8] where the major- Approximately 63 specimens were analyzed for, Ni ity of regional endemics occur (8 in northern and 4 in concentration. Data for these elements are shown in Ta- central Albania). We collected eight endemics in northern ble 4, which includes examples of typical behavior and Albania in Kukës, two in Qafë Shtamë and two in different response for Ni in the soil. Most plants of serpen- Gramsh, in central Albania. tine area of Albania showed slightly elevated Ni concentra- In spite of the location of Albania the percentage of tions in comparison with those on other soil types about 11- typical Mediterranean species in the study area is quite 100 mg kg-1, rather than 0.5-10 mg kg-1 [52]. In some low (6.34 %). Edaphic isolation caused by the physical and species in our sites, Ni content was elevated whilst sam- chemical nature of serpentine bedrock, as well as ‘‘moun- ples of the non-serpentine substrata, they contain the tain island’’ isolation of ancestral populations, have both expected very low Ni concentrations [52]. The highest Ni contributed to the creation of the great number regional concentration was found in Festuca glauca Vill. (275 mg (Aster albanicus Degen subsp. albanicus, Polygala doer- kg-1), Campanula glomerata L. (229 mg kg-1), Pinguicula fleri Hayek, Festucopsis serpentini L.) and trans-regional hirtiflora Ten. (198 mg kg-1), Dianthus petraeus Waldst & (Minuartia baldaccii (Halácsy) Mattf. subsp. doerfleri Kit. (157 mg kg-1), Centaurium erythraea Rafin. (154 mg Hayek, Alyssum markgrafii O.E. Schulz, Bornmmuellera kg-1), Thymus longicaulis C. Presl (146 mg kg-1), and baldaccii (Degen) Heywood subsp. markgrafii endemics in Lotus corniculatus L. (145 mg kg-1). We confirm Brooks the mountains [8]. Within both Alyssum and Bornmuellera, [1] and Konstantinou and Babalonas (1996) [53] that the close morphological relationships among the Ni ac- plants from families like Caryophyllaceae, Polygonaceae, cumulators makes it likely that these are endemic species Poaceae, Fabaceae (Genisteae) and Ericaceae have poten- that have evolved in the locality in which they are pres- tial to grow in the hostile edaphic environment of serpen- ently found, and have not succeeded in becoming more tine soils and accumulate Ni in their tissues. In Kukës, it widely distributed [17]. The presence of 8 endemics in was found a new Ni hyperaccumulator, i.e. Alyssum saxa- Kukës demonstrated the effect of edaphic and geographi- tile L., where the Ni content was 4,125 mg kg-1. Thymus cal mechanisms of isolation and confirmed previous data longicaulis, present in 3 study regions is a local indicator [13]. The species Alyssum markgrafii O.E.Schulz, Aster species, with a high affinity but not restricted to serpen- albanicus subsp. albanicus Degen, Minuartia baldaccii tine substrates. High-altitude serpentine areas are impor- (Halaćsy) Mattfeld, and Polygala doerfleri Hayek, in- tant for the flora and vegetation, where the obligate ser- cluded in IUCN [51] Red Book of Valter and Gillet pentinophytes plants are conditioned by the different cli- (1998), were found in some serpentine sites and make matic regions and substrate composition. These areas sup- those sites with great importance for the biodiversity in port a distinctive flora with numerous endemic species

TABLE 4 - Floristical composition and concentrations of Ni in harvested shoots of the species collected in high-altitude serpentine area of Albania(abcd). List of plant taxa sampled in serpentine area of high elevation in Albania a) Gramsh

Site of Species Family Floristical Elements Ni concentration Collection mg kg-1 Site 1 Lotus corniculatus L. Fabaceae Paleotemp 145 Festuca glauca Vill. Poaceae Eur 275 Thymus longicaulis C. Presl Lamiaceae EurMed 146 Galium verum L. Rubiaceae EurAs 123 Trifolium repens L. Fabaceae Paleotemp 45.5 Dianthus petraeus Waldst. &Kit. Caryophyllaceae Balcan 157 Trifolium nigrescens Viv. Fabaceae EurMed 61.2 Campanula cervicaria L. Campanulaceae Eur 85.5 Campanula rotundifolia L. Campanulaceae Eur 44.4 Verbascum alpinum Turra Scrophulariaceae Med 30.7 Pinguicula hirtiflora Ten. Lentibulariaceae Balcan 198 Scarbiosa graminifolia L. Caprifoliaceae S-Eur 30.4 Euphorbia myrsinites L. Euphorbiaceae S-Eur 40.4 Alyssum markgrafii O.E.Schulz Brassicaceae Neoendemic 6072 Bornmuellera baldaccii (Degen) Heywood Brassicaceae Endem 12171 subsp. markgrafii Site 2 Alyssum murale Waldst. & Kit Brassicaceae Eur-subMed 5866 Site 3 Alyssum murale Waldst. & Kit Brassicaceae Eur-subMed 9300

b) Qafë Shtamë Site of Species Family Floristical Elements Ni concentration Collection mg kg-1 Site 1 Thymus longicaulis C Presl Lamiaceae EuroMed 36.4 Daphne oleoides Schreber Thymelaeaceae Med 7.82 Euphorbia spinosa L. Euphorbiaceae Med 19.5 Achillea millefolium L. Asteraceae EurSib 8.12 Stachys officinalis (L.) Trev. Lamiaceae EurCauc 60.5 Minuartia recurva (All.)Schinz & Thell. Caryophyllaceae EurCauc 3.05 Polygala nicaeensis Risso ex Koch Polygalaceae EurMed 16.6 Carlina acanthifolia All. Asteraceae Palaeoendemic 8.06 Pinus nigra Arnold Pinaceae NE-Euri-Medit 0.95 Rosa cinnamomea L. Rosaceae EurAs 62.6 Juniperus oxycedrus L. subsp. oxycedrus Cupressaceae EurMed 46.3 Galium verum L. Rubiaceae EurAs 30.2 Geranium dissectum L. Geraniaceae EurAs 63.9 Poa pratensis L. Poaceae Circumbor 118 Alyssum bertoloni Desv. Brassicaceae endemic-Eur 9938 Site 2 Alyssum bertoloni Desv. Brassicaceae endemic-Eur 6145 Site 3 Alyssum bertoloni Desv. Brassicaceae endemic-Eur 6526 c) Kukës Site of Species Family Floristical Elements Ni concentration Collection mg kg-1 Site 1 Linum flavum L. Linaceae S-Eur 15.7 Potentilla erecta L. Rosaceae EurAs 39.1 Dioscorea balcanica Kosanin. Dioscoreaceae Balcan-endemic 30.8 Linum bienne Mill. Linaceae Med 39.4 Polygala doerfleri Hayek Polygalaceae Palaeoendemics 20.8 Epimedium alpinum L. Berberidaceae S-Eur 11.5 Campanula glomerata L. Campanulaceae Eur-OrTuran 46 Vaccinum myrtillus L. Ericaceae Circumbor 104 Prunella vulgaris L. Lamiaceae Kosm 101 Artemisia absinthium L. Asteraceae EurMed 9.16 Festucopsis serpentini L. Poaceae Palaeoendemics 31.6 Minuartia baldaccii (Halácsy) Mattf. subsp. Caryophyllaceae Balcan-Endemic 63.5 doerfleri Hayek Aster albanicus Degen subsp. albanicus Asteraceae Endemic(Neo) 31.9 Salvia ringens Sibth. & Sm. Lamiaceae Balcanic 103 Plantago argentea Chaix Plantaginaceae subMed 11 Asyneuma comosiforme Hayek & Janch. Campanulaceae Endemic(Neo) 40.3 Alysum saxatile L. Brassicaceae Balcanic 4125 Primula elatior (L.) Mill. Primulaceae Eur 40.8 Centaurea vllachorum Turra Asteraceae BalcEndemic(Neo) 80.1 Alyssum markgrafii O.E. Schulz ex Markgraf Brassicaceae Neoendemic 8833 Site 2 Alyssum markgrafii O.E. Schulz ex Markgraf Brassicaceae Neoendemic 12691 Site 3 Alyssum markgrafii O.E. Schulz ex Markgraf Brassicaceae Neoendemic 9547 d) Librazhd Site of collection Species Family Floristical elements Ni concentration mg kg-1 Site 1 Dorycnium pentaphyllum Scop. Fabaceae S-EurSib 41.2 Centaurium erythraea Rafin. Gentianaceae Paleotemp 154 Linum hologynum Rchb. Linaceae EurAs 13.9 Filago germanica (L.) Hudson Asteraceae Paleotemp 147 Lembotropis nigricans (L.) Griseb. Fabaceae S-EurS-Sib 55.1 Campanula glomerata L. Campanulaceae EurAs 229 Sanguisorba minor Scop. Rosaceae Paleotemp 26.1 Anthemis cotula L. Asteraceae EuroMed 80.2 Trifolium campestre Schreb. Fabaceae Paleotemp 97.3 Aegilops triuncialis L. Poaceae EuroMed 9.81 Asperula aristata L. fil Rubiaceae EuroMed 17.2 Lotus angustissimus L. Fabaceae EuroMed 109 Stachys scardica (Griseb.) Hayek Lamiaceae Paleotemp 52.1 Trifolium angustifolium L. Fabaceae EuroMed 44.6 Alyssum markgrafii O.E.Schulz Brassicaceae Neoendemic 5234

such as Bornmuellera baldaccii (Degen) Heywood subsp bertolonii Desv. subsp. scutarinum E.I. Nyàràdy (Qafë markgrafii O.E. Schulz (Gramsh), Alyssum markgrafii O.E. Shtamë), Festucopsis serpentini L., Aster albanicus subsp. Schulz ex Markgraf (Gramsh, Librazhd, Kukës), Alyssum albanicus Degen, Minuartia baldaccii (Halácsy) Mattf.,

Polygala doerfleri Hayek, Dioscorea balcanica Kosanin, [3] Roberts, B. and Proctor, J. (eds). (1992) The Ecology of Areas with Serpentinized Rocks: a World View. Kluwer Acad. Publish- Asyneuma comosiforme Hayek & Janch., Centaurea vlla- ers, Dordrecht. chorum Turra (Kukës). Results from these investigations [4] Brady, K.U., Kruckeberg A.R., dhe Bradshaw, H.D. (2005) Evo- illustrate the need to protect serpentine landscapes that con- lutionary ecology of plant adaptation to serpentine soils. Annual tain multiple local populations and the full range of micro- Review of Ecology, Evolution and Systematics 36, 243–266. habitats that are inhabited by serpentine endemic plants. [5] Kruckeberg, A.R. (1984) California Serpentines: Flora, Vegeta- tion, Geology, Soils and Management Problems. California Univ. Press, California 4 CONCLUSIONS [6] Harrison, S., Rajakaruna, N. (2011) Serpentine: Evolution and Ecology in a Model System. Berkely (CA), USA: University of Soil samples collected at the present study showed California Press. characteristics of limited weathering compared to soils at [7] Rajakaruna, N., Baker, A.J.M. (2004) Serpentine: A model habi- lower altitude and contained high levels of metals typical tat for botanical research in Sri Lanka. Ceylon Journal of Science from ultramafic environments: e.g. Ni, Cr and Co were 32,1-19. present at high concentration. The plant species collected [8] Stevanović, V., Tan, K. and Iatrou, G. (2003) Distribution of the at the sites exhibited different concentrations in metals. endemic Balkan fl ora on serpentine I. – obligate serpentine endemics. Plant Syst. Evol., 242, 149-170. Also, at a given site, different species had different concentrations of metals and hyperaccumulator plants were [9] Reeves, R.D., Baker, A.J.M., Borhidi, A. and Berazain, R. (1999). Nickel Hyperaccumulation in the Serpentine Flora of Cu- found along with plants with low metal content. Distribu- ba. Annals of Botany 83, 29-38, tions of Ni hyperacumulators depend on the biology of the [10] Babalonas, D., Karataglis, S. and Kabassakalis., V. (1984) The species and edaphic factors. The hyperaccumulator species ecology of plant populations growing on serpentine soils. III. preserved in the National Herbarium from the University of Some plant species from North Greece in relation to the serpen- Tirana were all found at the same serpentine area. A new tine problem. Phyton (Austria) 24, 225-238. Ni-hyperaccumulator was reported in Albania, i.e. Alys- [11] Pavlova, D. (2004) The serpentine flora in the Central Rhodopes sum saxatile. Mountains, South Bulgaria. – In: Melovski, L. (ed.), Proc. Sec- ond Congr. Ecol. R Macedonia. Pp. 450-457. Macedonian Ecol. Ultramafic areas in Albania, such as those visited for Soc., Ohrid. the present study, have been floristically studied in less [12] Pavlova, D. (2001) Mountain vegetation on serpentine areas in detail than many others, at least until recently, and deserve the Bulgarian Eastern and Central Rhodopes Mts. – ecology and much more attention. These sites are remarkable because it conservation. In: Radoglou K (ed.), Proceedings of International Conference: Forest Research: a challenge for an integrated Euro- is possible to see a lot of Ni hyperaccumulators in Albania, pean approach. Thessaloniki, 1, 227-232. representatives of Brassicaceae, growing together in a [13] Shuka, L., Hallaçi, B. (2010) Is Determined Flora and Vegetation very small area. Following our analyses we conclude that of Mirusha (Kosovo) and Kolshi (Albania) area from the Serpen- the serpentine sites at high-altitude are characterized by tine Substrate? BALWOIS 2010 - Ohrid, Republic of Macedonia endemism and host relict species, making it with particu- - 25, 29 May 2010, 1-6. lar importance for the flora and vegetation of the country [14] Shallari, S., Schwartz, C., Hasko, A. and Morel, J.L. (1998) and the wider Balkan area. The results of our study clear- Heavy metal in soils and plants of serpentine and industrial sites ly indicate the areas occupied by a rich and diverse, obli- of Albania. Sci. Tot. Environ., 209, 133–142. gate serpentine endemic flora. According to the criteria [15] Bani, A., Pavlova, D., Echevarria, G., Mullaj, A., Reeves, R.D., set out in IUCN – WWFPCP and IUCNTPU (1987), the Morel, J.L. and Sulçe, S. (2010) Nickel Hyperaccumulation by Species of Alyssum and Thlaspi (Brassicaceae) from Ultramafic Balkan mountains including Albanian mountains are quali- Soils of the Balkans. Botanica Serbica, 34(1), 3-14 fied as a prime European candidate representing hotspots of [16] Bani, A., Echevarria, G., Mullaj, A., Reeves, R.D, Morel, J.L, biodiversity global in magnitude. We suggest that conser- and Sulçe, S. (2009) Ni hyperaccumulation by Brassicaceae in vation measures should be actively taken to protect some of serpentine soils of Albania and NW Greece. Northeastern Natu- these floristically rich serpentine sites. Also in Albanian ralist 16 (Special Issue 5), 385–404 flora there is a large number of Ni hyperaccumulators that [17] Cecchi, L., Gabbrielli, R., Arnetoli, A., Gonnelli, C., Hasko, A., displayed sufficient Ni-efficiency for use in phytomining. Selvi, F. (2010) Evolutionary lineages of nickel hyperaccumula- Further exploration of some of the lesser known serpentine tion and systematics in European Alysseae (Brassicaceae): evidence from nrDNA sequence data. Annals of Botany, 1-17 areas should be conducted before there is any further habi- tat modification for agriculture, forestry, conventional [18] Kruckeberg, A.R. (1991) An essay: geoedaphics and island bio- geography for vascular plants. Aliso, 13, 225-238. mining, urban development, or other activities. [19] Chiarucci, A. and De Dominicis, V. (2001) The diversity and richness of the serpentine flora of Tuscany. Bocconea, 13, 557- 560. REFERENCES [20] Selvi, F. (2007) Diversity, geographic variation and conservation of the serpentine flora of Tuskany (Italy). Biodivers. & Conserva- [1] Brooks, R. (1987) Serpentine and its Vegetation (A Multidisci- tion, 16, 1423-1439. plinary Approach). Dioscorides Press, Portland, Oregon [21] Médail, F. and Quézel, P. (1997) Hot-spots analysis for conserva- [2] Proctor, J. and Woodell, S. (1975) The ecology of serpentine tion of plant diversity in the Mediterranean Basin. Annals of Mis- soils. Advances Ecol. Res., 9, 255-366. souri Botanical Garden, 84, 112–127.

[22] Chaney, R.L, Angle, J.S, McIntosh, M.S. (2005) Using hyperac- [42] Greuter, W., Burdet, H., Long, G. (1986) Med-Checklist, 3, Con- cumulator plants to phytoextract soil Ni and Cd. Zeitschrift servaroir et jardin boraniques, ville de Geneve, 395p. Naturforschung, 60, 190-198. [43] Hartvig, P. (2002) Alyssum, - In: Strid, A., Tan, K. (eds.) Flora [23] Pilon-Smits, E. (2005) Phytoremediation. Annual Review of Hellenica, 2, 199-224. Plant Biology, 56,15-39. [44] Akeroyd, J.R .(1993) Bornmuellera Hausskn. In: Tutin, T.G. et [24] Li, Y.M., Chaney, R.L, Brewer, E., Roseberg, R., Angle, J.S., al. (eds.). Flora Europaea, 2nd ed., Vol. 1, p. 52. Cambridge: Baker, A.J.M, Reeves, R.D. and Nelkin, J. (2003) Development Cambridge Univ. Press. of a technology for commercial phytoextraction of nickel: eco- [45] Reeves, R.D., Adigüzel, N. and Baker, A. (2009) Nickel Hyper- nomic and technical considerations. Plant and Soil, 249, 107-115. accumulation in Bornmuellera kiyakii Aytaç & Aksoy and Asso- [25] Bani, A., Echevarria, G., Sulçe, S., Mullaj, A., and Morel, J.L. ciated Plants of the Brassicaceae from Kızıldağ (Derebucak, (2007). In-situ phytoextraction of nickel by native population of Konya-Turkey), Turk. J Bot. 33, 33-40. A. murale on ultramafi c site in Albania. Plant and Soil 293, 79- [46] Dudley, T. R. (1964) Studies in Alyssum: near Easter representa- 89. tives and their allies. I.J. Arnold Arbor,45,57-100 [26] Broadley, M.R, White, P.J, Hammond JP, Zelko I, Lux A. 2007. [47] Reeves R.D., Brooks, R.R. and Dudley, T.R. (1983) Uptake of Zinc in plants. New Phytologist 173,677-702. nickel by species of Alyssum, Bommuellera and other genera of Old World tribus Alysseae. Taxon 32, 184-192. [27] Palmgren, M.G., Clemens, S., Williams, L.E., (2008) Zinc biofortification of cereals: problems and solutions. Trends in Plant Sci- [48] Kumar, N., Dushenkov, P.B.A., Motto, H. and Raskin. I. (1995) ence, 13,464-73. Phytoextraction: The use of plants to remove heavy metals from soils. Environmental Science and Technology 29(5), 1232–1238. [28] Markgraf, F. (1926) Bemerkungswerteneue Pflanzenarten aus Albanien. Ber. Deutsch Bot. Ges 44, 420-432. [49] Revees, R.D. (2002) Hyperaccumulation of trace elements by plants. Pp. 29, In J.L. Morel, G. Echevarria, and N. Goncharova [29] Markgraf, F. (1931) Pflanzen aus Albanien 1928. Akad. Wiss. (Eds.). Phytoremediation of Metal- Contaminated Soils, NATO Wien math. Naturwiss. Kl.Denkschr, 102, 317-360. Science Series (IV): Earth and Environmental sciences Volume 68.

[30] Tutin, T.G, Burges, N.A., Chater, A.O., Edmondson, J.R., Hey- [50] Lombini, A., Dinelli, E., Ferrari, C. and Simoni, A. (1998) Plant- wood, V.H., Moor, D.M., Valentine, D.H., Walters, S.M. and soil relationships in the serpentinite screes of Mt Prinzera (North- Webb, D.A. (eds.) 1993. Flora Europaea 1, 2nd edn, Cambridge ern Apennines, Italy). Journal of Geochemical Exploration, 64, University Press, Cambridge. 19– 33.

[31] Echevarria, G., Massoura, S.T., Sterckeman, T., Becquer, T., [51] Walter. S and Gillet, J.H. (1998) IUCN Red List of Globally Schwartz, C. and Morel, J.L., (2006) Assessment and control of Threatened Specie,. IUCN. the bioavailability of nickel in soils. Environmental Toxicology [52] Reeves, R.D. (1992) The hyperaccumulation of nickel by serpen- and Chemistry, 25, 643-651. tine plants. PP. 253–277, In A.J.M. Baker, J. Proctor, and R.D. [32] Topi, T., Bani, A., Malltezi, J., Sulce, S. (2012) Heavy metals in Reeves (Eds.). The Vegetation of Ultramafic (Serpentine) Soils. soil, sediments, mussels, and water from Butrinti lagoon (Alba- Intercept Ltd., Andover, UK. nia). Fresenius Environmental Bulletin, Vol 21. No 10a, 3042- [53] Konstantinou, M. and Babalonas, D. (1996) Metal uptake by 3051 Caryophyllaceae species from metalliferous soils in Northern Greece. Plant Systematics and Evolution 203,1-10. [33] Lindsay, W.L., and W.A. Norvell. (1978) Development of DTPA soil test for zinc, iron, manganese, and copper. Soil Science Soci- ety of America Journal 42, 421–428

[34] Allen, S.E., Grimshaw, H.M., Parkinson, J.A. and Quarmby, C. (1974) Chemical Analysis of Ecological Materials. Blackwell Scientifi c Publications, Oxford, UK.

[35] Kabata-Pendias, A. and Pendias, H. (1984) Trace elements in soils and plants. Boca Raton. FL, CRC Press, 315 pp. [36] Proctor, J. (1992) Chemical and ecological studies on the vegetation of ultramafic sites in Britain. In: Roberts, B.S. & Proctor, J. Received: December 10, 2012 (eds.) The ecology of areas with serpentinized rocks: a world Accepted: February 25, 2013 view, pp.135–167, Kluwer Academic Publishers, Dordrecht. [37] Manceau, A., Boisset, M.C., Sarret, G., Hazemann, J.L., Mench, M., Cambier, P. and Prost, R. (1996) Direct determination of CORRESPONDING AUTHOR lead speciation in contaminated soils by EXAFS spectroscopy. Environmental Science and Technology, 30, 540–1552 Aida Bani [38] Kabata-Pendias, A. (1993) Behavioural properties of traces met- Agro-Environmental Department als in soils, Applied Geochemistry, 2, 3–9. Agricultural University of Tirana [39] Massoura, S.T., Echevarria, G., Becquer, T., Ghanbaja, J., Le- University Campus No.1 clerc-Cessac, E. and Morel, J.L., (2006) Nickel bearing phases and availability in natural and anthropogenic soils. Geoderma, Koder Kamez, Tirana 136, 28–37. ALBANIA

[40] Baker, A.J.M. and Walker, P.L. (1989) Physiological responses of plants to heavy metals and the quantification of tolerance and Phone: + 355692467488 toxicity. Chem. Speciat. Bioavailab,, 1 pp. 7–17. Fax: + 35542321857 [41] Ball, P. and Dudley, T. (1993) Alyssum, In: Tutin, T., Burges, N., E-mail: [email protected] Chater, A., Edmond son, J.Heywood, V., Moore, D. Valentine, D., Walters, S.,Webb, D. (eds.) Flora Europaea 1, 359-369. FEB/ Vol 22/ No 6/ 2013 – pages 1792 - 1801

1801