Autophagic activity in the midgut gland of the overwintering harvestmen Gyas annulatus (Phalangiidae, Opiliones)

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Arthropod Structure & Development 43 (2014) 493e500

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Arthropod Structure & Development

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

Autophagic activity in the midgut gland of the overwinteringharvestmen Gyas annulatus (Phalangiidae, Opiliones)

Sa�ska Lipov�sek a, b, c, *, Franc Jan�zekovi�c b, Tone Novak b

a Medical Faculty, University of Maribor, Taborska ulica 8, SIe2000 Maribor, Sloveniab Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koro�ska cesta 160, SIe2000 Maribor, Sloveniac Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova ulica 17, 2000 Maribor, Slovenia

a r t i c l e i n f o

Article history:Received 21 May 2013Received in revised form23 April 2014Accepted 3 June 2014Available online 11 June 2014

Keywords:ArachnidsAutophagosomesDegradation processesDormancyMidgut gland

* Corresponding author. Department of Biology, FaMathematics, University of Maribor, Koro�ska cesta 160Tel.: þ386 2 22 93 709; fax: þ386 2 2518 180.

E-mail addresses: [email protected],(S. Lipov�sek).

http://dx.doi.org/10.1016/j.asd.2014.06.0011467-8039/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Juvenile harvestmen Gyas annulatus overwinter in dormancy in hypogean habitats for 4e5 months. Theultrastructure of the autophagic structures in their midgut epithelium cells was studied by lightmicroscopy, transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) duringthis non-feeding period. Before overwintering (November), autophagic structures were scarce. In themiddle (January) and at the end of overwintering (March), phagophores, autophagosomes and autoly-sosomes were present in the cytoplasm of both the secretory and the digestive midgut epithelium cells,gradually increasing their abundance during overwintering. In addition, vacuolization of the cytoplasmintensified. Both processes are induced by starvation. Autophagic structures and cytoplasm vacuolizationenable the reuse of the cell's own components required for the maintenance of vital processes duringdormancy. While TEM is a much more convenient method for recognition of the autophagic structuretypes and their ultrastructure, IFM enables exact counting of these structures.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Juveniles of the Alpine harvestmen Gyas annulatus hatch in latesummer or in early autumn. After the first autumn frost, usually inNovember, they enter hypogean habitats, such as water caves andwater current-adjacent pebble and gravel accumulations, andoverwinter there in quiescence (Martens, 1978; Novak et al., 1984,2004). In March or April they return to the epigean habitats. Gyasannulatusmature and mate in late spring and die in late summer orearly autumn (Martens, 1978; Novak et al., 2004).

Autophagy is a complex intracellular process that results in thedegradation of cytoplasmic components within lysosomes(Lockshin and Zakeri, 2004; Mizushima and Klionsky, 2007). Auto-phagic processes have been studied in most Eucaryota, from yeaststo vertebrates (Klionsky and Emr, 2000; Levraud et al., 2004;Mizushima et al., 2004; Picazarri et al., 2008; Yang and Klionsky,2010; Suzuki et al., 2011; Rost-Roszkowska et al., 2012). In general,autophagy has been described as a cellular response to nutrientstarvation in invertebrates and vertebrates that becomes activatedin response to low nutrient availability (Levine and Klionsky, 2004;

culty of Natural Sciences and, SIe2000 Maribor, Slovenia.

[email protected]

(Mizushima et al., 2004; Mizushima, 2007; Kristensen et al., 2008;Yang and Klionsky, 2010; Malagoli et al., 2010; Yin et al., 2010;Lionaki et al., 2013). The physiological roles of autophagy arevarious. It plays a part in homeostasis by regulating the turnover ofmany proteins and organelles (Yoshimori, 2004), and the removal ofaggregated proteins and damaged organelles, while protectingagainst cellular stress caused byextracellular or intracellular factors,like hypoxia, reactive oxygen species or heat (Lee and Baehrecke,2001; Levine and Klionsky, 2004; Kourtis and Tavernarakis, 2009).Autophagy plays a role in developmental remodeling (Yang andKlionsky, 2010; Malagoli et al., 2010), e. g., in metamorphosing in-sects (Lockshin and Zakeri, 2004; Tettamanti et al., 2008), as well asin regeneration processes, e. g., in Planaria (Tettamanti et al., 2008).In the vinegar fly Drosophila, it has been observed in the larval fatbody in response to starvation (Scott et al., 2004), in the ovariesduring oogenesis and in the muscles (Hou et al., 2008; Barth et al.,2011). In the midgut epitelium of Isohypsibius granulifer, Eutardi-grada, it appears during oogenesis (Rost-Roszkowska et al., 2011a)and in response to a microsporidian infection (Rost-Roszkowskaet al., 2011b). In adult Acheta domesticus, it was observed duringdegeneration processes in the midgut epithelium (Rost-Roszkowska, 2008, 2010).

In both invertebrates and vertebrates, macroautophagy, micro-autophagyand chaperone-mediated autophagy have been described(Kourtis and Tavernarakis, 2009). Macroautophagy�referred to as

S. Lipov�sek et al. / Arthropod Structure & Development 43 (2014) 493e500494

autophagy by numerous authors (e. g., Mizushima et al., 2002; Xieand Klionsky, 2007)�is the best studied form of autophagy, bywhich a portion of the cytosol is surrounded by a double-membranephagophore, forming a double-membrane organelle�the autopha-gosome. When an autophagosome fuses with a lysosome, theytogether form the autophagolysosome, or autolysosome, which is adouble- or single-membrane structure, containing electron-denseamorphous material (Xie and Klionsky, 2007).

Quantitative electron microscopy is one of the most sensitivemethods for detecting the accumulation of autophagic vacuoles(Eskelinen, 2008). The LC3 proteins are reported to specificallyassociate with autophagic structures (Tanida et al., 2008). At thebeginning of the autophagy, the LC3-I protein, localized in thecytoplasm, is cleaved, lipidated and inserted as LC3-II into themembranes of the phagophores. An increase in the amount of LC3-II correlates with an increased number of autophagosomes (Tanidaet al., 2008; Klionsky et al., 2012).

Our previous research on G. annulatus focused on the generalstructure of the epithelial cells in the midgut gland�which is theprincipal metabolic organ�and the importance of their glycogenand lipid stores (Lipov�sek et al., 2004), as well as on seasonal- andage-dependent changes in the structure and chemical compositionof the spherites (Lipov�sek et al., 2002). As in other harvestmen, e. g.,Phalangium opilio (Alberti and Storch, 1983; Becker and Peters,1985; Ludwig and Alberti, 1990), the midgut gland of G. annulatusis composed of diverticula, most of them in the opisthosoma. Themidgut gland epithelium consists of secretory and digestive cellswith their apical plasma membrane differentiated into numerousmicrovilli. A secretory cell is characterized by an abundant roughendoplasmic reticulum (RER) and secretory granules. The digestivecell contains an apical tubulovesicular system, lipid droplets andelectron-dense protein granules (Lipov�sek et al., 2004).

During short periods of starvation, the amino acids seem to relyprimarily on the ubiquitin-proteasome system (Vabulas and Hartl,2005), while several hours after a meal, the amino acids arereleased by autophagy (Mizushima, 2007). In the arthropod lifecycle, autophagic processes could thus have an important influenceon their survival during dormancy. For invertebrates overwinteringin hypogean habitats, harvestmen included, the correlation be-tween the course of this non-feeding period and the autophagicprocesses has not been investigated. The abundance of autopha-gosomes is a good measure for estimating the relative amounts ofamino acids released. In this study we asked how accurately theabundance of autolysosomes in the midgut gland of G. annulatusresponds to the ongoing starvation during winter dormancy undernatural conditions in hypogean habitats. We hypothesized that thisabundance would increase along with the prolonged duration ofdormancy. For this purpose, autophagy in the epithelium of themidgut gland was studied by transmission electron microscopy(TEM) and immunofluorescence microscopy (IFM) for LC3 locali-zation in different time frames during overwintering.

2. Material and methods

2.1. Material

Juvenile G. annulatus were collected in the Babja luknja cavenear Gori�cane, Slovenia, at the beginning (November), the middle(January) and the end of the winter dormancy (March).

2.2. Methods

The ultrastructure of the epithelial cells in the midgut gland andthe presence of autophagic structures were studied by light mi-croscopy, TEM and IFM in the secretory and the digestive midgut

gland epithelium cells. Between these two cell types, there were nosignificant differences (ManneWhitney U test, p >> 0.05) in theabundance of autophagic structures; the description, therefore,relates to both cell types. Ten specimens were analyzed at thebeginning, the middle and the end of overwintering.

2.2.1. Light microscopy and TEMThe midgut gland was extracted from the harvestman opistho-

soma under a stereomicroscope, dissected into small fragments andfixed in 2.45% glutaraldehyde (GA) and 2.45% paraformaldehyde(PFA) in a 0.1 M sodium cacodylate buffer (pH 7.4) at room tem-perature for 3 h and then at 4 �C for 12 h. For this purpose, weprepared 8% PFA (8 g PFA þ 100 mL aqua bidist.) and 8% GA (3.2 mL25% GA þ 6.8 mL aqua bidist.). For 10 mL of the fixative, 3.1 mL 8%PFA and 3.1mL 8% GAwere put into 3.3mL 0.3M sodium cacodylatebuffer, resulting in end concentrations of 2.45% PFA and 2.45% GA.The tissue was washed in a 0.1 M sodium cacodylate buffer (pH 7.4)at room temperature for 4 h and postfixed with 2% OsO4 at roomtemperature for 2 h. Afterwards, the sampleswerewashed in a 0.1Msodium cacodylate buffer (pH 7.4) at room temperature for 3 h anddehydrated with a graded series of ethanol (50%, 70%, 90%, 96%,100%, each for 30 min at room temperature). The samples wereembedded inTAAB epoxy resin (Agar Scientific Ltd., Essex, England).For light microscopy, semi-thin sections (5 mm) were obtained andstained with 0.5% toluidine blue in aqueous solution to localize theepithelial cells within the midgut gland. For electron microscopy,ultra-thin sections (70�75 nm) of the tissue were transferred tocopper grids, stained with uranyl acetate and lead citrate andanalyzedwith a Zeiss EM902 transmission electronmicroscope. Foreach time frame�i. e., the beginning of overwintering in November,the middle in January and the end of overwintering in March�theultrastructure of the epithelial cells of the midgut gland wasanalyzed, with special attention to the morphology of the auto-phagic structures.

2.2.2. IFM for LC3 localizationIn order to visualize autophagic structures in the epithelial cells

of the midgut gland, we used the marker LC3B primary antibody,widely used in studies of autophagy (Klionsky et al., 2012). Thelocalization of LC3 by immunofluorescence was vizualized as greenfluorescent dots.

The midgut gland was dissected in PBS and fixed in 3.7% form-aldehyde (in PBS as diluent) for 20 min at room temperature. Thetissue was rinsed with PBS-TX (PBS containing 0.2% Triton X 100)three times for 5 min each buffer, and permeabilized in 0.2% TritonX 100 in PBS for 20 min at room temperature. To block possiblenon-specific binding, the tissue was incubated in BlockAid™Blocking Solution (Molecular Probes) for 1 h at room temperature.The samples were rinsed again with PBS-TX three times for 5 mineach and treated with primary antibodies LC3B (rabbit polyclonalantibody, Molecular Probes) diluted in PBS-TX (1:250) for 14 h at4 �C. The pieces of the midgut gland were washed with PBS-TXthree times for 5 min each, and incubated with secondary goatanti-rabbit IgG antibodies (Molecular Probes; Alexa Fluor 488 goatanti-rabbit IgG (H þ L)) diluted in PBS-TX (1:100) for 2 h at roomtemperature. After incubation with secondary antibodies, themidgut glands were washed again with PBS-TX three times for5 min each and examined. Fluorescence images of the sampleswere obtained using an inverted confocal laserscanning fluores-cencemicroscope Leica TCS SP5 II. Samples were excitedwith argonlaser line at 488 nm. All images were acquired with a 40� oil im-mersion objective (NA¼ 1.30) at 800�magnification. To control forpotential false-positive signals from the immunofluorescence an-alyses, the pieces of the midgut gland were incubated with sec-ondary antibodies without the primary antibody.

Fig. 1. A. Semithin frontal planeesection of a juvenile G. annulatus (November). In the opisthosoma (O), the midgut (M) and the diverticula of the midgut gland (MG) are seen. P,prosoma. Bar: 350 mm. B. Semithin frontal planeesection of the midgut gland in G. annulatus (January). SC, secretory cells; DC, digestive cells; / microvilli of the apical plasmamembrane. L, lipids; P, protein granula. Bar: 20 mm.

Table 12

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2.2.3. Abundance of autophagosomesCounting of autophagosomes in the midgut epithelial cells was

performed on unitary image areas (Eskelinen, 2008) in November,January and March using two methods: 1. the TEM images of theultrathin sections of epithelial cells at 3000� magnification, and 2.the IFM images of the thin sections at 800�magnification taken byan inverted Leica confocal laserscanning fluorescence microscopeafter applying an immunofluorescence method to localize LC3 onthe autophagosomes. At first counting, the image area measured14 mm � 14 mm, and at second counting, 21 mm � 21 mm.

Descriptive statistics of abundance of autophagosomes (N/100 mm per time frame)in the midgut gland epithelial cells of G. annulatus, observed by TEM. Number ofsamples: 100 in each time frame.

Time frame Beforeoverwintering(November)

Middle ofoverwintering(January)

End ofoverwintering(March)

Mean ± StDmin�max

0.2 ± 0.330.0e1.5

1.5 ± 0.960.0e4.1

3.2 ± 1.161.0e6.1

2.2.4. Statistical analysisThe figures for the abundance of autophagosomes were

tested for normality using the KolmogoroveSmirnov test. Thedistribution in November was not normal; we therefore used theManneWhitney U test (M�W) to test differences between theabundance of autophagosomes in the midgut gland cells during

different time frames. The data analysis was carried out withSPSS 21.0 statistical software.

3. Results

The frontal plane section of the branched system of the midgutdiverticula in G. annulatus is shown in Fig. 1A, and the epithelium inFig. 1B. The abundance of autophagosomes within the epithelial

S. Lipov�sek et al. / Arthropod Structure & Development 43 (2014) 493e500496

cells gradually increased from a low to a high rate during over-wintering (Table 1).

3.1. Autophagy in the midgut epithelial cells

3.1.1. Before overwintering (November)Before overwintering, the ultrastructure of the secretory and

the digestive cells (Fig. 2A, B) was in accordance with the

Fig. 2. 2: The midgut epithelial cells. A, B: Before overwintering (November). A. The apicainto numerous microvilli (MV). Beneath the microvilli, in the apical cytoplasm of the cell,endoplasmic reticulum. S, spherite. Bar: 2 mm. B. The secretory cell (SC) contains protein gcontaining many mitochondria in the apical cytoplasm, and lipid droplets (L) and glycogenwintering (January). An autolysosome (AL) in the digestive cell (DC). SC, secretory cell; Pgranules. Bar: 500 nm. D. The end of overwintering (March). An autolysosome (AL) in thsecretory cell; L, lipid droplet; LU, lumen; P, protein granules. Bar: 2 mm.

description in the previous study (Lipov�sek et al., 2004). Auto-phagic activity was rarely observed (Table 1). In the cells only afew structures immuno-positive to LC3 were present (Fig. 5A;Table 2).

3.1.2. Middle of overwintering (January)Phagophores, autophagosomes and autolysosomes were pre-

sent in the digestive cells (Figs. 2C and 3BeD) and the secretory

l plasma membrane of the digestive cell (DS) and the secretory cell (SC) is differentiatedmany mitochondria (M) and some protein granules (P) can be observed. RER, roughranules (P) and numerous mitochondria. The digestive cells (DC) are electron-lucent,granules (arrow). LU, lumen of the midgut gland. Bar: 2 mm. C. The middle of over-, protein granules; RER, rough endoplasmic reticulum; the arrow indicates glycogene digestive cell (DC). The cytoplasm of the DC shows a vacuolizated appearance. SC,

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cells (Fig. 3A). Individual vacuoles were present, as well (Fig. 2C).The cytoplasm of the digestive cells was rich with phagophoresengulfing parts of the cytoplasm (Fig. 3C). Locally, only fragments ofa double membrane were seen (Fig. 3D, arrows), engulfing theelectron-lucent material. Vesicles with either electron-lucent(Fig. 3D, arrowhead) or electron-dense material were enclosed bythe membrane of a phagophore (Fig. 3D, asterisks). In the digestivecell, electron-dense vesicles appeared inside the autophagosome

Fig. 3. The midgut epithelial cells in the middle of overwintering (January). A. In the secrgranula. Bar: 2 mm. B. The autophagosome (AP) composed of electron-dense granules, enindicating the processes of degeneration are present in the cytoplasm. L, lipid droplet. Bar:indicate two locations where a longer fragment of the membrane surrounds the cytoplasmobserved. Bar: 1 mm. D. In the very close vicinity to the autolysosome (AL), numerous granulea double-membrane engulfing the electron-lucent material. The vesicles with electron-luccases, the granules are composed of electron-dense material enclosed by the membrane, s

(Fig. 3B). Autophagic activity was intensified with respect to thecells before overwintering (Fig. 5B, Tables 1 and 2).

3.1.3. End of overwintering (March)At the end of overwintering, more or less intensive vacuolization

of the cytoplasm appeared (Figs. 2D and 4A). The autophagicprocesses intensified, as established in the case of numerousautophagic structures (for autophagosomes, see Table 1) and

etory cell, electron-dense autolysosomes (arrows) are present. L, lipid droplet; P, proteinveloped by a double membrane in the digestive cell (DC). Other structures (arrows)1 mm. C. Double-membrane phagophore (PH) formation can be observed. The arrowsand the degenerated organelles. Beside the phagophores, an autolysosome (AL) can bes are present in the cytoplasm of the digestive cell (DC). The arrows show fragments ofent contents are enclosed by the membrane of the phagophore (arrowhead). In othero the whole granule has an electron-dense appearance (asterisks). Bar: 500 nm.

Fig. 4. The midgut epithelial cells at the end of overwintering (March). A. Apical regions of the digestive cell (DC) and the secretory cell (SC). In SC some autolysosomes (AL)containing intermediately electron-dense and electron-dense material can be seen. MV, microvilli. Bar: 2 mm. B. Double-membrane autophagosome (AP) contains cytoplasmicmaterial and electron dense residual components of the cell compartments. DC, digestive cell. Bar: 500 nm. C., D. The residual bodies (RB) in the digestive cell are composed ofmaterial of varied electron density. Locally, electron-dense material (>) can be seen. L lipid droplet. P, protein granule. S, spherite. Bar: 1 mm.

S. Lipov�sek et al. / Arthropod Structure & Development 43 (2014) 493e500498

vacuoles containing degraded material (Figs. 2D and 4). Manyautolysosomes (Fig. 4A) and autophagosomes (Fig. 4B) werepresent. The electron-lucent and electron-dense material accu-mulated in large, irregularly-shaped residual bodies (Fig. 4C, D).Autophagic activity was twice as intense as in January (Tables 1 and2, Fig. 5C).

3.2. Abundance of autophagosomes

According to both the TEM and the IFM, before overwintering,abundance of autophagosomes was very low (means: TEM 0.2/

100 mm2, and IFM 0.3/100 mm2). The autophagosomes were moreabundant in the middle and most abundant at the end of over-wintering. During dormancy, the numberof autophagosomes variedfrom between 0 and 6/100 mm2 (Table 1, TEM) to between 0 and 10/100 mm2 (Table 2, IFM). The differences between the abundance ofautophagosomes differed significantly among the three time frames(Table 1, for TEM,M�W p< 0.001; Table 2, for IFM,M�W p< 0.001).In November, autophagy triggered by unknown reasons wasdetected in only a few cells. In January and March, the mean valuesfor the number of autophagosomes in the IFM sampling amountedto twice those in the TEM samples (Tables 1 and 2).

Fig. 5. Detection of autophagosomes labeled with antiserum against LC3. Representative images show autophagosomes (arrows) in the epithelial cells of the midgut gland ofG. annulatus in November (A), January (B) and March (C). Bar: 5 mm.

S. Lipov�sek et al. / Arthropod Structure & Development 43 (2014) 493e500 499

4. Discussion

In Gyas annulatus, overwintering is relatively long-lasting, up tofour months of the 16-month life cycle (Lipov�sek et al., 2004).During this non-feeding period, the midgut gland epithelial cellsundergo characteristic starvation processes. The gradual augmen-tation of the number of autophagosomes within the cells clearlyindicates that these structures gradually increase their role in themaintenance of vital processes during dormancy. This implies acellular response to nutrient starvation, as reported for many in-vertebrates and vertebrates (Mizushima et al., 2004; Yang andKlionsky, 2010; Malagoli et al., 2010; Yin et al., 2010; Lionakiet al., 2013). All these processes contribute to the homeostasis(Lee and Baehrecke, 2001; Levine and Klionsky, 2004; Kourtis and

Table 2Descriptive statistics of abundance of autophagosomes (N/100 mm2 per time frame)in the midgut gland epithelial cells of G. annulatus, as observed by IFM. Number ofsamples: 200 in each time frame.

Time frame Beforeoverwintering(November)

Middle ofoverwintering(January)

End ofoverwintering(March)

Mean ± StDmin�max

0.3 ± 0.300.0e1.4

3.1 ± 0.611.1e5.4

6.4 ± 1.343.4e10.0

Tavernarakis, 2009). During overwintering, G. annulatus spendsabout 5.20 J g�1 dry mass$day�1 lipids and 0.026 mg g�1 drymass$day�1 glycogen (Novak et al., 2004), which implies thatintracellular resources are required. Once macromolecules havebeen degraded within autolysosomes, monomers, such as aminoacids, are released to the cytosol at disposal for reuse (Franzettiet al., 2012). Autophagy can recycle molecules from the degener-ating structures and supply nutrients during the non-feedingperiod, as was established for the silkworm (Franzetti et al.,2012). In overwintering G. annulatus, amino acids released fromthe autophagic structures are probably reused for the synthesis ofenzymes and other proteins required in, e. g., cell respiration, cellcycle control, and the recycling of various membranes (cf. Finn andDice, 2006).

In overwintering G. annulatus, the number of autophagicstructures showed a gradual but marked increase indicating thatthe autophagy intensifies with the duration of starvation. This iscongruent with the intensification of autophagic processes asstarvation progresses, because of their role in the turnover of cellconstituents (Yoshimori, 2004). Amino acids are probably releasedfrom the reserve proteins by autophagy, since this process beginsseveral hours after a meal (Mizushima, 2007). A similar decrease inprotein amounts has been detected, e. g., in diapausing cavecrickets Troglophilus cavicola (Lipov�sek et al., 2011). Towards theend of overwintering, in addition to compounds released in

S. Lipov�sek et al. / Arthropod Structure & Development 43 (2014) 493e500500

autophagy, intensified vacuolization in the cytoplasm of themidgutgland epithelial cells demonstrates the increasing decomposition ofthe cytoplasm, which probably supplies the required compounds(Lipov�sek et al., 2012).

Autophagosomes are appropriate structures for evaluating theprocess of autophagy because they can undoubtedly be identifiedusing the specific immuno-reaction against the LC3 protein(Klionsky et al., 2012). The results of TEM and IFM were congruent,while IFM turned out to be much more efficacious in detectingautophagosomes. This is because IFM also allows fragments ofautophagosomes to be definitely visualized and counted. WhileTEM is much more convenient for recognizing the types and ul-trastructural details of the autophagic structures, IFM enables exactcounting.

5. Conclusion

In juvenile G. annulatus, before their winter dormancy, thesecretory and digestive cells of the midgut gland exhibit a normalultrastructure. Starvation triggers autophagy, as has been demon-strated by TEM and IFM. Autophagy is an important mechanism inthe maintenance of these harvestmen during overwintering.Autophagy, along with vacuolization, provides intracellularnutrient resources during this starvation period.

Acknowledgments

We would like to thank Elisabeth Bock, Rudi Schmied (MedicalUniversity Graz) and Rudi Mlakar (Faculty of Medicine, Universityof Maribor) for their excellent technical assistance. Michelle Gad-paille made valuable linguistic correction to themanuscript. We areindebted to two anonymous reviewers for their insightful com-ments and suggestions. This study was partly supported by theSlovenian Research Agency within the Biodiversity Research Pro-gramme (Grant No. P1-0078).

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