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Acta Oecologica 37 (2011) 239e248

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Acta Oecologica

journal homepage: www.elsevier .com/locate/actoec

Original article

An integrated approach for the conservation of threatened plants:The case of Arabis kennedyae (Brassicaceae)

M. Andreou a, P. Delipetrou a, C. Kadis b, G. Tsiamis c, K. Bourtzis c, K. Georghiou a,*

aDepartment of Botany, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15784 Athens, GreecebNature Conservation Unit, Frederick University, 1036 Nicosia, CypruscDepartment of Environmental and Natural Resources Management, University of Ioannina, 2 Seferi Street, 30100 Agrinio, Greece

a r t i c l e i n f o

Article history:Received 26 August 2010Accepted 10 February 2011Available online 10 March 2011

Keywords:Genetic diversityHabitats’ DirectiveMonitoringPlant conservationReproductive biologyThreatened speciesCyprus

* Corresponding author. Tel./fax: þ30 210 7274656E-mail addresses: andreoum@gmail.com (M. A

(P. Delipetrou), pre.kc@fit.ac.cy (C. Kadis), gtsiamikbourtz@uoi.gr (K. Bourtzis), kgeorghi@biol.uoa.gr (K

1146-609X/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.actao.2011.02.007

a b s t r a c t

The aim of this paper is to propose an integrated approach (including population and habitat monitoringand the study of reproductive biology and genetic diversity) for the comprehensive study of threatenedplants, for which conservation measures are imperative. We applied this model to the plant speciesArabis kennedyaewhich is classified as endangered according to the IUCN criteria. The current populationof the species consists of three small subpopulations (AR1, AR2, and AR3) at three locations. Populationsize was characterized by considerable annual fluctuations. The distribution pattern of the plant followedhabitat availability. Relative Reproductive Success remained stable but moderate. Germination ofdormant seeds was promoted by light and was optimal at 15 and 20 �C. Genetic analysis showed lowinterpopulation variability and detected two groups: haplotype I (AR1 and AR3) and haplotype II (AR2),which may represent two altitudinal ecotypes. The direct threats identified were related to recreationactivities, road construction and fire. The subpopulations of the plant are regulated by density anddepend on fecundity and on the soil seedbank while their persistence depends mainly on habitatavailability. Low genetic diversity combined with small population size and a possible reduction in fitnesssuggest increased susceptibility to loss of genetic variation. The overall results suggest that ex situconservation in a seed bank, and in situ conservation in the form of population restoration, are suitableconservation measures and the study of the different aspects of the species’ biology has provided thedata required for their implementation.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

During the second half of the 20th century species extinctionrates reached an almost unprecedented level in Earth’s history(Currie 2003; Frankham, 2003). The progressive loss and degrada-tion of European natural habitats has led to the adoption of theCouncil Directive 92/43/EEC (Habitats’ Directive) as the mainlegislative tool for biodiversity conservation through the conser-vation of natural habitats in a protected site network. The rare andendangered plant of Cyprus, Arabis kennedyae (Troodos Rockcress,Brassicaceae) (Christodoulou et al., 2007), is listed in Annex II of theHabitats’ Directive as a priority species and is also included inAppendix I of the strictly protected plants of Europe (BernConvention). It has a small population size and thus increased risk ofextinction due to environmental, demographic, or genetic factors

.ndreou), pindel@biol.uoa.grs1@gmail.com (G. Tsiamis),. Georghiou).

son SAS. All rights reserved.

(e.g., Frankham, 2003). Evidently, A. kennedyae is a priority speciesfor the application of conservationmeasures. These should be basedon the knowledge of certain critical aspects of the species’ biology.

Monitoring of plant populations is one of the core activities ofconservation biology that can be particularly useful for conservingspecies with small populations. Monitoring data are used to iden-tify species in decline or at risk of extinction, to track the spread ofinvasive species (Marsh and Trenham, 2008), and to assess whetherspecific management strategies work (Field et al., 2007; Marsh andTrenham, 2008). Such data can lead to the development of effectiveconservation plans for rare species (Pino and de Roa, 2007).In addition, monitoring of priority species is an obligation formember-states of the EU, in accordance with Article 11 of theHabitats Directive. Nevertheless, only a very small proportion ofpriority plant species (i.e., 16%) is being monitored due to the long-term and large-scale efforts required for sound species monitoring(Kull et al., 2008).

Investigation and understanding of traits associated withreproductive biology is an important aspect for developing soundguidelines for the conservation of threatened species (e.g., Evans

M. Andreou et al. / Acta Oecologica 37 (2011) 239e248240

et al., 2003). The knowledge of seed germination behaviour isindispensable in developing effective procedures and protocols forex situ conservation (e.g., Francisco-Ortega et al., 1994; Estrelleset al., 2004; Brusa et al., 2007; Flores et al., 2008). Furthermore,the study of seed germination provides a clue to the understandingof the survival strategy of the species, which can be defined as thetiming of seed dispersal along with dormancy/germination char-acteristics that result in germination during the optimumperiod forseedling establishment (Baskin and Baskin, 2001).

Recent conservation efforts have focused on genetic eventsin species with small populations, such as threatened species(Shibayama and Kadono, 2008; González-Pérez et al., 2009;Suárez-García et al., 2009), which are usually inbred and havelow levels of genetic diversity (Frankham et al., 2002). Individualfitnesswithin populations is affected by relative rates of inbreeding,diversity among individuals and degree of local adaptation (Aviseand Hamrick 1997; Neel, 2008; Provan et al., 2008). Intraspecificgenetic diversity is important for species persistence because itincreases individual fitness (Avise and Hamrick 1997; Neel, 2008).In addition to these immediate effects, genetic diversity providesthe raw material for evolutionary change and is essential foradaptation to new environmental conditions that invariably arisedue to natural and anthropogenic changes (Frankham et al., 2004).A species without an appropriate amount of genetic diversityis thought to be unable to cope with changes in environmentalconditions, evolving competitors, parasites (Frankham, 2003;Honnay and Jacquemyn, 2007; Zhao et al., 2008) or climatechange (Honnay and Jacquemyn, 2007).

Finally, investigations of the population’s genetic diversity maynot only illustrate the evolutionary process and mechanismbut also provide useful information for conservation of the species(Avise and Hamrick, 1997; Zhao et al., 2008), and contribute tothe evaluation of the risk of species extinction. Genetic factorsshould be considered seriously in any appropriate managementplan for a threatened-rare species (Avise and Hamrick, 1997;Frankham, 2003).

Several authors highlight the need for accumulating multidi-mensional knowledge regarding the biology and habitat charac-teristics of plant species, in order to develop sound measures fortheir conservation (e.g., Lozano et al., 2005). However, most plantconservation studies focus only on specific aspects of speciesbiology, such as genetic diversity (Barnaud and Houliston, 2010;Rahimmalek et al., 2009), reproductive success (Burne et al.,2003; Gaudeul and Till-Bottraud, 2004), monitoring of populationsize (Mondragon, 2009) and the extent and quality of the specieshabitat (Lozano et al., 2005). Consequently, management andprotection measures are most often suggested based on datarelating only to certain aspects of the species biology (Rahimmaleket al., 2009). Our work addresses this gap by compiling andassessing scientific information on geographical distribution,population dynamics, reproductive biology, and genetic diversity inorder to produce and implement sound conservation measures forthe European priority species A. kennedyae.

2. Materials and methods

2.1. Study species

A. kennedyae is an erect annual herb, 5e30 cm high. The fruit isa silique, 25e40� 1.5 mm, with seeds 1.3� 1mm, in a single row ateach loculus. Historical knowledge about the species is scarce:A. kennedyaewas described in 1962 (Meikle, 1977) from specimenscollected from the localities of Xerokolympos (1938) and KryosPotamos (1938, 1962). Since 1962, a few plants were found in Xer-okolympos (location AR4) in 1994 and at Kryos Potamos (location

AR1) in 2004, while two new locations were discovered: at Tripylos(location AR3) in 1998 and at Chionistra peak (location AR2) in2005 (Christodoulou et al., 2007). All locations lie about Troodosrange, in Natura 2000 sites.

2.2. Definitions and conservation status assessment

The terms population size, subpopulation, location, Area ofOccupancy (AOO) and Extent of Occurrence (EOO) are usedaccording to the definitions established by the International Unionfor the Conservation of Nature (IUCN) for the application of thecriteria for conservation status assessment (IUCN Standardsand Petitions Subcommittee, 2010). The Ramas Red List V.3 soft-ware package was used for the conservation status assessmentof A. kennedyae according to the IUCN criteria (Akcakaya andFerson, 2007).

2.3. Monitoring e reproductive biology

The wider area of all known locations of A. kennedyae wassurveyed for three consecutive years (2006e2008). Detailedmapping was done using a GPS device and occasionally a tapemeasure, once a year during the peak of the flowering season of thespecies. Polygons of the local extent of occurrence of eachsubpopulation (hereby termed local extent), i.e., the minimum areapolygon or polygons including all the plant colonies not separatedby unsuitable habitat at each location, were constructed. The ArcView 3.1� (ESRI) software package was used for data digitizationand area estimations. Population size was measured by countingevery individual that was flowering and/or fruiting. Field observa-tions on reproductive phenology were carried out weekly from thebeginning of the flowering season until the dispersal of the seeds.

Reproductive biology was studied in subpopulation AR2 (poly-gon AR2a) for 3 years. Fecundity and RRS were studied by taggingrandomly selected plants at the beginning of the flowering season.The number of flower stalks and the number of flowers and fruitsper stalk were recorded weekly over the duration of flowering andfruiting. The number of ovules was estimated as the sum of soundand aborted seeds per silique. Fecundity was estimated as thenumber of flowers and fruits per plant and of seeds per fruit and perplant (Reekie and Bazzaz, 2005). Relative Reproductive Success(RRS), i.e., the total percentage of all ovules maturing into seeds,was estimated bymultiplying the Fruits/Flowers ratio by the Seeds/Ovules ratio (Wiens, 1984). Seed mass was measured using ananalytical digital balance. Seed rain was estimated by dividingthe estimated total number of sound seeds by the local extent ofthe colonies. Seedling survival was studied by tagging randomlyselected seedlings during November or December and checkingtheir viability in the following spring, after the snow had melted.

For germination experiments, seeds collected from subpopula-tions AR2 and AR3 were placed in Petri dishes lined with filterpaper and moistened with distilled water. The dishes were placedin temperature controlled plant growth cabinets. Brief, 10 min, Redlight irradiations for the activation of phytochrome, were admin-istered 72 h after the onset of imbibition. All manipulations ofimbibed seeds were carried out under a dim green safelight. Finalgermination and T50 (the time required to reach half of the finalgermination level) values are the means of 5 replicates of 20 seeds.

For habitat characterization and monitoring, the locations AR2and AR3 were stratified according to habitat type (Juniperus foeti-dissima canopy and stony openings in AR2 and Cedrus brevifolia andPinus brutia forest margins and openings in AR3). Permanentquadrats 5� 5m2were established so thatA. kennedyaewas presentin about half of them. All species in each quadrat were recordedusing the modified Braun-Blanquet 9-grade cover-abundance scale

M. Andreou et al. / Acta Oecologica 37 (2011) 239e248 241

(Wikumand Shanholtzer,1978) during the period of population sizemeasurements. A total of 61 samples were recorded, 15 quadrats(C1eC15) for 3 years at AR2 and 8 quadrats (T1eT8) for 2 yearsat AR3.

The direct threats in the environment where A. kennedyaeoccurs and the stresses they cause to the plant were recorded andclassified according to the IUCN categories (IUCN and CMP, 2006).

2.4. Genetic analysis

For the genetic analysis, 7e8 leaves, from 10 plants of eachsubpopulation were collected in May 2007 and placed in liquidnitrogen for transfer to the laboratory, where they were storedat �80 �C until analysed. Total genomic DNA was extracted andpurified from the leaves using the Qiagen DNAeasy mini-kit(Qiagen, USA) according to the manufacturer’s instructions.One fragment from the cpDNA (matK) and one from the nrDNA(ITS) were amplified by PCR using the following pairs ofprimers: matKF 50-aatttacgatcwattcattcaayatttc-30 and matKR50-tcgaagtatatactttattcgatac-30, ITS1 50-tccgtaggtgaacctgcgg-30

and ITS4 50-tcctccgcttattgatatgc-30 (Baldwin, 1992; Winkworthet al., 2002).

PCR amplifications were performed in a thermal minicycler�(MJ Research). A reactionmixture (20 ml) containing 10� PCR buffer(Minotech, Greece), 1.5 mM MgCl2 (Minotech, Greece), 0.25 mMdeoxynucleoside triphosphate (Invitrogen, USA), the appropriateprimers, 0.3 mM each (Invitrogen, USA), and 1 U Taq polymerase(Minotech, Greece) was prepared. PCR reactions were performedusing an initial step at 94 �C for 10 min, followed by 35 cycles of1 min denaturation at 94 �C, 1 min primer annealing at 50 �C formatK and 54 �C for ITS amplification and 90 s DNA chain extensionat 72 �C. The PCR was completed by a final extension at 72 �Cfor 10 min.

PCR products were purified by PEG precipitation. Sequencingreactions were performed by Macrogen (South Korea).

2.5. Data analysis

Comparisons of data on reproductive biology were performedby One Way ANOVA. Differences among pairs of means werechecked by the Tukey’s Method (Bartz, 2001) using the Statistica(StatSoft, 2008) software.

Plant community analysis was performed by modified TWIN-SPAN (Two-way Indicator Species Analysis) using the Juice softwarepackage (Tichý, 2002). Floristic composition changes and correla-tions to environmental parameters were investigated using theCANOCO software package (ter Braak and Smilauer, 1998) byCanonical Correspondence Analysis (CCA). The environmentalvariables tested were: altitude, aspect, slope, tree cover (a measureof canopy cover), geological substrate (dunite, harzburgite, diabase)and year of sampling (2006, 2007, 2008). The CCA model and thesignificance of the fitted environmental variables were evaluatedby the Monte Carlo permutation test.

For the genetic analysis, the gene sequences were assembledusing the DNAstar software (DNASTAR Inc.,WI). Sequence similarity

Table 1Subpopulations and locations of Arabis kennedyae.

IUCN subpopulation

IUCN location Area ofoccupancy (km2)

Altitude (m) Aspect (�) S

AR1 Kryos Potamos 4 1236 e

AR2 Chionistra 8 1821e1886 190AR3 Tripylos 4 1324e1407 44e275 3AR4 Xerokolympos 4 1306 e

searches were performed using the online sequence analysisresources “BLAST” and the sequences with the highest homologywere obtained for phylogenetic analyses. Alignment of sequenceswas carried out using the CLUSTALX 1.83 program (Larkin et al.,2007). Genetic diversity was examined by calculating total haplo-type diversity (Hd) and nucleotide diversity (Pi) using DNAsp v.5(Librado and Rozas, 2009). The same programwas used to estimateFst values and their significance for each locus was determined bybootstrapping over samples using 10 000 permutations and boot-straps for all sample-pairwise comparisons. Furthermore, phylo-genetic trees based on distancematrixmethodwere constructed foreach locus using PAUP* 4b10 (Swofford, 2002). The phylogenetictrees were created based on the evolutionary distances using themethod according to Kimura (1980) and Jukes and Cantor (1969).The phylogenetic analysis was performed using neighbor-joining(NJ) and maximum parsimony (MP) and was based on bootstrapanalysis of 10 000 trees (Fitch, 1971; Saitou and Nei, 1987). Cleomehassleriana sequences were used as outgroups since this taxon isclosely related to Arabis but not classified within this genus. In thephylogenetic analysis, all nucleotide sites and substitution classeswere weighted equally.

3. Results

3.1. Geographical distribution

The current and past distribution area of A. kennedyae is shownin Table 1 and Fig. 1. The entire population of the species consists ofthree subpopulations (AR1, AR2 and AR3), corresponding to thelocations Kryos Potamos, Chionistra and Tripylos, respectively. Thefourth site at Xerokolympos (AR4) was searched in 2005e2008 butno plants were found. The fact that A. kennedyae has not beenreported from this location since 1998, suggests that this subpop-ulation may be extinct.

During the three years of monitoring (2006e2008), the EOO ofthe whole population of A. kennedyae was 32.7 km2 and the AOObased on a 2 � 2 km grid- was 16 km2 (Fig. 1). Assuming that therecently discovered subpopulation (AR2) did exist in 1998, duringthe decade 1998e2008 the EOO of the species remained stable, butthe AOO was reduced by 4 km2, due to the apparent loss ofsubpopulation AR4.

3.2. Population size

Population size, expressed as the total number of mature indi-viduals in all subpopulations, as well as the size of each subpopu-lation, exhibited considerable annual fluctuation (Table 2). Totalpopulation size and the number of plants perm2, a rough estimationof plant density, were highest in 2007 in the two largest subpopu-lations (AR2 andAR3), which are located in areaswith neighbouringhabitat patches suitable for A. kennedyae. Subpopulation AR1,whichis located in an area with lack of neighbouring suitable habitatpatches, had minimal local extent and its size decreased. Subpop-ulation AR2 had the largest number of plants and local extent andconsisted of plant colonies scattered in two large, adjacent patches

lope (�) Habitat

0 Pinus-Quercus forest margin under Q. alnifolia adjacent to nature trail.30 Thin J. foetidissima forest canopy and openings flanking nature trail.

0e70 Cedrus and Pinus forest openings and margins adjacent to road.0 Pinus-Quercus forest opening under Q. alnifolia.

Fig. 1. Geographical distribution and habitat availability map of A. kennedyae inCyprus. Red dots: presence of A. kennedyae in 2006e2008; Black dot: presence ofA. kennedyae up to 1998.

M. Andreou et al. / Acta Oecologica 37 (2011) 239e248242

in an extended opening with scattered trees: AR2a with smallerlocal extent but higher number of plants and AR2b with a far largerlocal extent but lower number of plants. Although the number ofplants recorded each year varied up to 5-fold, the total local extentdid not change. On the other hand, as shown by the cover-abun-dance of A. kennedyae in the habitat monitoring plots, in 2007 and2008 the plant appeared in more plots than in 2006 and plantdensity increased, especially in 2007, to up to 25e50 plants per m2

at the juniper canopy plots. Subpopulation AR3 consisted of sixplant colonies which occupied distinct patches of 4e90 m2. Thecolonies were located at forest openings or adjacent to a road, atdistances of 70e300 m among them so that the whole subpopula-tion was scattered in an area of 35 000 m2. Both the number ofindividuals and local extentfluctuated greatly in each patch and twoof the smallest colonies went extinct in 2007.

Table 2Number of mature individuals, actual subpopulation cover and plants per m2 per subpo

Sub-population 2006 2007

No ofindividuals

Localextent (m2)

Plants/m2 No ofindividuals

AR1 61 1 61.1 48AR2 AR2a 1125 1757 0.6 5200

AR2ba e e e 150AR3 AR33 14 4 3.5 0

AR34 20 4 5.0 0AR35 7 20 0.4 30AR36 171 15 11.4 3AR37 e e e 879AR38 90 12 7.5 761

TOTAL 1488 7071

a The colonies in the part AR2b of subpopulation AR2, adjacent to AR2a and in the pa

3.3. Reproductive biology

The reproductive characteristics of A. kennedyae at location AR2are shown in Table 3. Fecundity was highest in 2006, i.e., the year ofthe minimum number of adult individuals and minimum pop-ulation density. In contrast, the preliminary study of seedlingsurvival showed that the transition from juvenile to adult plantshad the same pattern as population size and density, being lower in2006 and higher in 2007 and 2008. Also, seed rain was higher in2007 and 2008, due to the larger number of plants. On the otherhand, seed mass and RRS did not differ significantly among the 3years and approximately half of the flowers transformed to fruitseach year.

Seed germination experiments performed either immediatelyafter collection or three months later produced the same results.The majority of A. kennedyae seeds were dormant. Final germina-tion in the dark was lower than 10% at all temperatures tested(5e25 �C). Dormancy was broken by a single irradiation with Redlight. The study of temperature dependence after Red light irradi-ation showed maximum final germination and germination rate at15 �C (98%, T50 ¼ 2.5 d) and 20 �C (80%, T50 ¼ 2.5 d). Germinabilityfell steeply at 10 �C, and less steeply at 25 �C. The high germinabilityobserved at the optimal conditions indicated that the majority ofthe seeds produced were sound.

The phenology of A. kennedyae presented similar patterns in the3 years of monitoring (Fig. 2). Germination and the subsequentseedling emergence and establishment coincided with the mainrainy season on the Troodos range and the favourable temperatureregime. The first phase of seed germination occurred in autumn,when maximum daily temperatures were below 25 �C and meandaily temperatures kept above 10 �C. A fairly high percentage ofthese seedlings at location AR2 survived (Table 3) the harsh winterat the hardy rosette phase, usually in protected microsites, underthe canopy or in the vicinity of juniper trees or between small rocks.A second phase of seed germination was observed in early spring,especially at lower altitudes, when maximum daily temperatureswere above 10 �C. Flowering and fruiting started in late March orApril andwere completed in a period of 2.5months. The life cycle ofthe plant was completed with the seed dispersal in summer.Initiation of flowering and fruiting in 2006 were delayed comparedto 2007 and 2008, but the completion of these stages was notdelayed. Spring germination and seedling emergence were alsodelayed in 2006.

3.4. Habitat characterization and monitoring

A. kennedyae inhabits openings and margins of forests that aretypical of the Troodos range at altitudes above 1200 m (Table 1).The floristic composition of the habitat patches studied was

pulation per year.

2008

Localextent (m2)

Plants/m2 No ofindividuals

Localextent (m2)

Plants/m2

1 48.0 21 1 21.01757 3.0 4355 1757 2.5

21 551 0.01 170 21 551 0.010 0 00 0 06 5.0 84 6 14.01 6.0 30 4 7.5

40 22.0 150 24 6.390 8.5 360 90 4.0

5170

rt AR37 of subpopulation AR3 were discovered in 2007.

Table 3Reproductive characteristics of A. kennedyae subpopulation AR2, polygon AR2, during three consecutive years (2006e2008).

2006 2007 2008

n n n

Flowers (Fl) per plant (�SE) 27 � 3.5a, * 35 18 � 2.3b 45 14 � 1.4b 30Siliques (Fr) per plant (�SE) 14 � 2.1a 35 9 � 1.3b 45 8 � 1.2b 30Ovules (O) per flower (�SE) 33 � 0.5a 100 30 � 0.5b 100 28 � 0.7b 95Seeds (S) per silique (�SE) 33 � 0.5a 100 29 � 0.6b 100 27 � 0.7b 95Seeds (FrxS) per plant 465 247 210Seed rain (seeds/m2) 298 730 521Seed mass (mg) (�SE) 0.2 � 0.00 100 0.2 � 0.00 100 0.2 � 0.00 100RRS (%) (FrxS)/(FlxO) � 100 51 46 54Seedling survival (%) 31 15 81 15 70 15

*Means followed by the same letter in each row do not differ significantly (p < 0.05).

M. Andreou et al. / Acta Oecologica 37 (2011) 239e248 243

characterised by a low number of species (42 in total). TheTWINSPAN analysis (data not shown) resulted in 4 vegetationgroups, corresponding to the initial habitat stratification: open-ings of P. brutia (T7) and C. brevifolia (T1eT6, T8) forest, canopy ofJ. foetidissima open stands (C1eC10) and herb communities onopen rocky substrate among the stands (C11eC15). The full CCAmodel explained 1.710 of the total inertia of the species data(3.162) and selected seven significant variables (p ¼ 0.005), indecreasing order of significance: altitude, diabase, dunite, treecover, slope, aspect, rock. In the CCA plot (Fig. 3), the quadrats ofeach location were grouped together along the 1st axis in parallelto the vectors of geological substrate, altitude and slope: T1eT8(AR3) at increasing values for diabase and slope, and C1eC15(AR2) at increasing values for altitude and dunite. Since year ofsampling was not a significant variable, the points representingthe annual samplings at each quadrat overlapped. QuadratsC1eC10 were grouped at increased values of tree cover anddecreased values of rock cover and C11eC15 at increased valuesof rock cover. Quadrat T7 with P. brutia was separated from allothers. Quadrats T1eT8 were separated along the 2nd axisdepending on rock cover, tree cover and aspect. A. kennedyae wasnot correlated strongly with any of the variables and was locatedclose to the axes intersection, among the juniper canopy andcedar forest quadrats, where it was more abundant, and awayfrom the rocks herb communities where it was less abundant.This was because its niche breadth includes most of the range ofthe variables examined and it occurs in all of the habitat groupsidentified except from P. brutia forest. In fact, in 2007 or 2008 itappeared even in some quadrats where it was not present in2006.

Based on the attributes of the habitat of the plant, i.e., altitude,geological substrate and vegetation type, the available habitat forA. kennedyae is restricted to a total area of 56e70 km2 (dependingon the actual area of pure ormixed P. brutia forest compared) on theTroodos range (Fig. 1). The actual area of available habitat is evensmaller since forested slopes have been included, but the plantoccurs only in forest openings, the smaller of which could not bemapped.

Fig. 2. Phenology of A. kennedyae. Black: stage observed in this period in 2006, 2007,and 2008; Gray: stage observed in this period in 2007 and 2008; Diagonal grid: stageobserved in this period in 2006.

3.5. Threats

The direct threats identified were: a) the development ofrecreation areas (threat code 1.3) and activities therein (threat code4.1) at locations AR1 and AR2, which are close to nature trails andpicnic sites; b) the construction of roads (threat code 4.1), fortransportation at location AR4 and for forest management atlocation AR3; c) low to moderate frequency of human induced firesat all locations, despite the fire protection measures, due to theproximity of the inflammable pine forest and the high frequency ofvisitors. These threats pose the stresses of habitat loss (stress code1.3) and disturbance due to trampling (stress code 2.2), especially atthe tiny colony at AR1. Road widening has apparently resulted inthe extinction of subpopulation AR4.

3.6. Genetic analysis

Sequence analysis of both the 699 bp long amplified matKfragment (28 plants) and of the 611 bp long amplified ITS region(24 plants) showed evidence of genetic variation for A. kennedyae.The matK gene and ITS sequences reported in this study have beendeposited in GenBank under accession numbers GU458356 andGU458407, respectively. The analysis revealed two homozygoticalleles (haplotype I and II) for matK locus with three variables SNPs.Hd was 0.476 � 0.057 and Pi was 0.0020 � 0.000. Two haplotypeswere also revealed for ITS locus with two variable SNPs. Hd was0.507 � 0.045 and Pi was 0.0016 � 0.000. For both loci, twogenetically defined groups were detected: subpopulations AR1 andAR3 represent the first group and subpopulation AR2 representsthe second group. Pairwise Fst values were 0.0 between subpopu-lations AR1 and AR3 and 1.0 between subpopulation AR2 and eachof the two others, indicating that subpopulation AR2 is significantlydifferent (p < 0.05) from the other two subpopulations. Phyloge-netic analysis of the matK and of the ITS sequence data producedsimilar trees (the NJ and MP approaches had identical results andthe NJ trees are shown in Fig. 4). In each case, two well-supportedgroups were produced representing the two genetically definedgroups (group 1: AR1 and AR3; group 2: AR2) of A. kennedyae.These groups were supported by high bootstrap values (matK: 86%for group1 and 95% for group2; ITS: 72% for group1 and 88% forgroup2). A. kennedyae was clustered with Arabis alpina, which isapparently its closest relative.

3.7. Conservation status assessment

The data entered in the Ramas Red List software for AOO, EOOand number of subpopulations and locations were those shown inTable 2 and Fig. 1. Fluctuations of up to five-fold were observed intotal population size and greater than ten-fold in the size of distinct

Fig. 3. CCA plot with biplot scaling focused on inter-species distances, axes I (eigenvalue 0.645, p ¼ 0.005) and II (eigenvalue 0.288, p ¼ 0.005). The two axes explain 29.5% of thevariance of species data and 54.5% of the variance of specieseenvironment relation. Black dots: species; open circles: samples with A. kennedyae; open diamonds: samples withoutA. kennedyae. The legends C1 to C15 (quadrats at Chionistra) and T1 to T8 (quadrats at Tripylos) are located adjacent to the samples at each quadrat.

M. Andreou et al. / Acta Oecologica 37 (2011) 239e248244

plant colonies, but the monitoring period was not long enough toascertain the range of fluctuations. Therefore, total population sizewas estimated as theminimum count during themonitoring periodand extreme fluctuations (i.e., at least ten-fold) were consideredpossible (30e50%) with a high degree of uncertainty. Subpopula-tion AR1 is a fragment of the total population according to the IUCNterminology (IUCN Standards and Petitions Subcommittee, 2010),since it is small (smaller than the minimum of 50 individuals usedby IUCN) and relatively isolated. Subpopulations AR2 and AR3 arelarger but have a low genetic diversity among them. Therefore,these may not be viable, even though in the nearby areas there aresuitable habitats for the species. So fragmentation was consideredpossible (30%) with a high degree of uncertainty. Based on theabove data, A. kennedyae was classified as endangered (EN)according to the criteria B1a,c(iv) and B2 a,c(iv) with a range ofplausible categories from endangered to vulnerable (VU). The taxonalso fulfills criterion D2 for the characterisation as VU.

4. Discussion

The distribution area of the plant, expressed in IUCN terms asEOO andAOO, did not change significantly in themonitoring period.The distribution pattern of the plant seems to follow habitat avail-ability: subpopulation AR2 is scattered in one continuous largeopening, while subpopulation AR3 consists of distinct colonies indiscontinuous suitable habitat patches. Apparently, the same pat-chy distributionwould be expected at locations AR1 and AR4where

the suitable habitat is restricted to few and discontinuous patches,too. The habitat aspects common at all locations, were therocky ophiolithic substrate and the open sites in the vicinity ofC. brevifolia and J. foeditissima trees. In arid areas, woody vegetationoffers favourable conditions to annuals (Holzapfel et al., 2006)whiletrees offer protection from snowcover (Fenner and Thompson,2005). On the other hand, the species never grows under closedcanopy. This implies the threat of population loss due to habitatsuccession, especially in transitory habitat patches (Johnson, 2000),as at location AR3. Closing of vegetation may be a primary cause ofpopulation reduction (Eisto et al., 2000). Consequently, habitatavailabilitymaywell be an important constraint for species survival.

Population size showed significant fluctuations which were notcaused by changes in the habitat or in the threats during themonitoring period. Fluctuations in population size are a commoncharacteristic in annual species (Kiviniemi and Lofgren, 2009) butimply that the effective population is smaller than the censuspopulation (Orians, 1997). A. kennedyae might be adapted to largefluctuations but for small populations fluctuations signify theincreased possibility for a drastic decline and lack of recovery,especially in the event of spatial synchrony (Hanski, 1999). Pop-ulation Viability Analysis would quantify the extinction risk butneeds more years of monitoring (Morris et al., 1999). In subpopu-lation AR2, the low adult population in 2006 may have been theresult of increased seedling mortality, which in turn might havebeen caused by adverse temperature conditions (in winter 2006there was a longer continuous period and a higher total number of

Fig. 4. Neighbor-joining phylograms showing the phylogenetic relation between A. kennedyae haplotypes. The numbers at each node represent bootstrap proportions. The treesrepresent the analysis of the matK (a) and ITS (b) sequences.

M. Andreou et al. / Acta Oecologica 37 (2011) 239e248 245

days with mean daily temperatures below 0 �C than in subsequentyears). Flowering was also delayed by a month in 2006, which mayalso have been due to low temperatures.

The fecundity of A. kennedyae at location AR2 declined in theyears with higher population size and higher plant densities. Thisimplies negatively density dependent population regulation,a phenomenon observed in annuals with high densities that hasbeen attributed to intraspecific competition (e.g., Watkinson, 1990)or to increased risk of herbivory or pathogen infections (Begonet al., 1996). Although the densities observed in A. kennedyaewere an order lower than the usual known thresholds of densitydependent reduction of fecundity (e.g., Kluth and Bruelheide, 2005;Schmalholz and Kiviniemi, 2007), there is evidence that in rockyenvironments, water shortage may trigger reduced fecundity atlower densities (Nilsson, 1995). In addition, on the low resource e

sparse vegetation cover environment of A. kennedyae, intraspecificinteractions may manifest as competition for space, e.g., for safe-sites for germination such as hollows among stones or protrudingjuniper roots. Density dependent population regulation at the stageof recruitment has been observed in other winter annuals (Kluthand Bruelheide, 2005; Nilsson, 1995). Density regulation of thepopulation might also explain the reductions in the number ofindividuals at subpopulation AR3 (Table 2).

The germination strategy of A. kennedyae is dependent ontemperature, which restricts germination in autumn and/or spring,and on dormancy which is broken by exposure to light. Thepromotion of germination by Red light was reversible by brief Far-Red light (data not shown), indicating the involvement of thephytochrome low energy reaction in the control of germination(e.g., Bewley and Black, 1982). The ecophysiological role of thisreaction is related to germination at or close to the soil surface,often exhibited by small seeded plants, and to the avoidance ofgermination under a canopy (e.g., Fenner and Thompson, 2005).This suggests the creation of a transient or persistent soil seed bankif seeds are buried or under closed canopy and is consistent withthe habitat of the plant, such as isolated J. foetidissima trees wherethere is sufficient red light to promote germination. The succes-sional closing of the canopy will inhibit germination (Rees andLong, 1992) and cause decline of the population. Exit from thesoil seed bank may be triggered by a disturbance that opens thecanopy or an increase in safe-sites for germination (Enright andLamont, 1989). This type of episodic recruitment may be impor-tant for the local persistence of this plant. Thus dormancy inA. kennedyae is a mechanism for spreading the germination in timeas well as for selecting suitable sites for germination. Temporalvariation in seed germination within the same year also reduces

M. Andreou et al. / Acta Oecologica 37 (2011) 239e248246

sibling competition (Venable, 1989) and provides a second oppor-tunity for successful recruitment which can be important forannual species relying on a yearly production of adults andoffspring. Moreover, a soil seed bank is a means of perennating forannuals or species with small population size and short life span(Begon et al., 1996; Quilichini and Debussche, 2000) and ofpersistence despite environmental stochasticity, a fact that reducesthe risk of extinction (Kalisz and McPeek, 1993).

In conclusion, the abundance of A. kennedyae is determined bydensity independent mortality (e.g., temperature and rain condi-tions) and by density dependent regulation. The distributionpattern and changes in patch occupancy in subpopulation AR3suggest a metapopulation structure in which the persistence ofplants at patch level is regulated by density and depends onfecundity and the seasonal or persistent soil seedbank. At distri-bution area level, the persistence of the plant depends on theavailability of neighbouring habitat patches and on a persistentseedbank.

The phylogenetic study, by using combined plastid and nuclearDNA data, consolidates the position of A. kennedyae as a separatespecies in the A. alpina complex. Interestingly, the genetic markersused were able to differentiate the three subpopulations studied. Inthe near future, an AFLP and/or microsatellite-based approachwhich would provide a much higher resolution of the populationgenetic structure should be adopted. According to the geneticmarkers used, the lower altitude subpopulations AR1 and AR3 aregenetically identical and significantly different from the high alti-tude subpopulation AR2. Since contemporary propagule transferbetween AR1 and AR3 is physically improbable, it could be postu-lated that the two genomes studied represent two altitudinalecotypes. Most likely, this nucleotide diversity represents adapta-tion of the subpopulation AR2 to environmental conditions athigher altitudes. The observed low level of overall genetic variationin A. kennedyae could be a result of its evolutionary process but lackof actual samples from the pre-bottleneck subpopulations does notallow assessing the genetic variation from valid reference points.Genetic diversity surveys in endangered populations typicallydetermine the variation currently maintained in the populationrather than the magnitude or rate of loss of genetic diversity overtime (Gaudeul et al., 2000). While reduced genetic variation canlead to lower individual fitness and lower population adaptability(Newman and Pilson, 1997), low genetic diversity is a frequentfeature in localised endemics (Karron,1997) and such a species mayhave “adapted” its members to the condition of rarity (Gaston andKunin, 1997).

In subpopulation AR2, annual fecundity varied but the RRS,which indicates fertility rate and population fitness, remainedstable. However, the RRS value was moderate compared to theusual values for annuals (Wiens, 1984). RRS values of 80e90% wererecorded for another two annual Arabis species (Hoffmann et al.,2010) and also for A. kennedyae in subpopulation AR4 (Kadis,1995). This implies that fitness in the largest subpopulation mightbe reduced. The smallest subpopulation (AR1)might be particularlysusceptible to loss of genetic variation and thus to reduced fitness(Ellstrand and Elam, 1993; Fischer and Matthies, 1998). Populationslower than 50 individuals typically present lower genetic diversityand reduced fitness (Armstrong and De Lange, 2005; Karron, 1997)and it is argued that populations will have to be maintained atsizes over 2000 individuals to maintain their population fitness(Reed, 2005).

Ex situ conservation is an appropriate measure sinceA. kennedyae produces orthodox seeds which can be preserved ina seed bank (Roberts, 1992). Seeds to be stored should be collectedfrom both subpopulations AR2 and AR3 so that the genetic diversityof the species (two genomes) will be covered effectively.

In situ conservation requires management to eliminate anydirect threats and undertaking any interventions necessary toensure the survival of the species, based on population data. Thecurrent status of the plant is only partly related to the directthreats that were identified. Road construction and accidentaltrampling at locations AR2 and AR3 can be handled by informingthe relevant authorities and by signposting for the public, whilefencing can protect the small colony at location AR1. On the otherhand, the population data indicates an increased extinction riskdue to the small population size combined with large fluctuationsand low genetic diversity. Also, habitat availability was identifiedas a critical aspect for the persistence of the species. This indi-cates that in situ conservation should also focus on populationsize enhancement and on the maintenance of forest openings.Population restoration, i.e., the establishment of new plant colo-nies at suitable microsites, is a sound conservation measure atlocations AR1 and AR4. In both locations, the species has beenrepeatedly found for more than 70 years but the subpopulationsare currently too small or extinct. The genetic analysis indicatesthat seeds from subpopulation AR3 should be used as a source ofplants for location AR1 (same haplotype) and also for AR4 (similaraltitude). The studies of germination and reproductive phenologyhave provided the information needed for the production of newplants as well as the timing for seed collection, seed sowing andseedling establishment in the field. Habitat monitoring identifiedthe specifications of the plant habitat. At locations AR2 and AR3where population size is larger, the enhancement of bothsubpopulations with additional colonies could still be an appro-priate measure due to the fluctuations observed. It is possible thatlarge fluctuations are normal for this species but populationenhancement would be justified if further monitoring and pop-ulation viability analysis indicated increased extinction risk. Forthe establishment of new colonies at AR2 and AR3, seeds from therespective haplotypes should be used, since they probablyrepresent genetically distinct ecotypes within a single species(Linhart and Grant, 1996).

In conclusion, the investigation of each aspect of the biology ofA. kennedyae yielded the information needed in order to identifythe critical aspects that affect the survival of the species and toguide the proposal and implementation of sound conservationmeasures. We propose our methodology as a model for thecomprehensive study of threatened plants for which conservationmeasures are imperative.

Acknowledgements

Our researchwas funded by the Research Promotion Foundationof Cyprus (project PENEK 2006-0506/32) and by the EU LIFE-NATURE project LIFE04NAT/CY/000013. We greatly acknowledgethe co-operation of the Forestry Department of Cyprus in the fieldwork.

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