Soil Nematodes in Alpine Meadows of the Tatra National Park (Slovak Republic)

HELMINTHOLOGIA, 54, 1: 48 – 67, 2017

Soil nematodes in alpine meadows of the Tatra National Park (Slovak Republic)

L. HÁNĚL

Biology Centre CAS, Institute of Soil Biology, Na Sádkách 7, CZ-370 05 České Budějovice, Czech Republic, E-mail: [email protected]

Article info Summary

Received September 13, 2016 The assemblages of soil nematodes were studied at fi ve alpine meadow sites, 1763 – 2200 m. a. Accepted December 19, 2016 s. l., in the Tatra National Park in the Slovak Republic. A total of 110 species were distinguished, 19 species were recorded in the Slovak Republic for the fi rst time. The interesting new records are the occurrence of Sphaeronema alni at the elevation of 2003 m a . s. l. and the populations of Coomansus menzeli at four sites. The total numbers of species at individual sites varied from 45 to 72. The most abundant nematode genera were Aglenchus, Plectus, Acrobeloides, Paratylenchus, Eudorylaimus, Helicotylechus, and Aphelenchoides. The total mean abundance ranged from 704 to 2054 x 103ind.m-2 and the total mean biomass from 442 to 1531 mg.m-2. The lowest values of the Maturity Indices (∑MI, MI) were found at the highest elevation. The signifi cantly highest values of the Plant Parasite Index were at the lowest elevation. The mean values of the of the Enrichment Index varied from 16.3 to 38.4, the mean values of the Structure Index from 64.1 to 85.4. The Structure metabolic footprints were signifi cantly greater at the lowest elevation than at the highest elevation. Cluster Analysis and Principal Component Analysis performed on species presence and absence, genera abundance and genera metabolic footprints showed nematode assemblages at sites of higher elevations different from those at sites of lower elevations. Keywords: soil nematodes; diversity, maturity; soil food web; alpine meadow

Introduction Nematode faunas in grasslands are diverse and abundant (Wa- silewska, 1979; Boag & Yeates, 1998; de Goede & Bongers, 1998) Grassland (including shrub steppes, savannahs and prairies) is and may in fact control total grassland primary production (Scott the potential natural vegetation on 33 – 45 million km2 covering et al., 1979; Verschoor, 2001). Nematological studies of Central about a quarter of the Earth’s land surface (Lauenroth, 1979; European grasslands are numerous but relatively few alpine and Bardgett & Cook 1998; Bonan, 2008). Besides climatically de- subalpine meadows above timberline were investigated for nema- termined grasslands there are semi-natural and agricultural todes. Nevertheless, the available data showed a great variety of grasslands on land formerly covered by forests and mires. The nematode faunas in these habitats. total area of semi-natural grasslands in the Central and Eastern Since about 1940’s it is known that the nematode abundance in European countries is estimated at around 7 million hectares, a alpine meadows of Central Europe usually varies in order of sev- part of them was ploughed and is currently being restored (Jonge- eral millions of individuals per square metre, rarely is greater than pierová, 2008). ten millions or less than one million of individuals (Franz, 1950).

48 The Table 13 in his book deals with 42 species at 17 sites and it is of nematode taxa tends to be lower at higher elevations. Tempera- interesting that 14 species belonged to the order Dorylaimida and ture can be limiting factor for nematodes in cold climates, however this order had the greatest species richness at 14 sites. The large Hoschitz & Kaufmann (2004a) pointed out that the apparent tem- predator Coomansus zschokkei occurred at fi ve sites. Vinciguerra perature sensitivity may be the result of indirect effects, e.g. by (1988) found 31 species in three alpine meadows in Italian Alps, resource availability. 16 species belonged to the order Dorylaimida and C. zschokkei A drop in the soil moisture does not usually limit alpine and subal- occurred in two meadows. Gerber (1991) reported on 33 species pine meadows and the surplus of water leads to the formation of at alpine habitats in Austria but only three were from the order peat meadows and peat bogs accompanied by complex changes Dorylaimida. in nematode faunas (Háněl, 2015a). At lower elevations in grass- The extensive survey of nematode communities in mountain lands on soils susceptible to desiccation the total number of nem- grasslands from Romania carried out by Popovici (1998) showed atode taxa can be reduced, nematode communities differ in xero- much more diverse nematode faunas. However, a part of nema- philic and mesophilic grasslands (Ciobanu & Popovici, 2015) and todes could be determined to the genus level only. The synthe- a general trend of increasing nematode richness with decreasing sis of the data published by Popovici & Ciobanu (2000) therefore altitude can be reversed. gives the numbers of taxa (genera + species) as a measure of The behaviour of various community indices across alpine-subal- nematode taxonomic richness. They found that the alpine grass- pine grasslands is little known. Popovici & Ciobanu (2000) found lands (above 2000 m a. s. l.) had 46 – 50 nematode taxa. Aglen- that the Shannon’s index of generic diversity (H’) of the 36 sites chus agricola, Filenchus spp. and Paratylenchus spp. were the ranged between 2.38 and 3.47, without clear differences between dominant nematodes and plant feeders composed 62 – 66 % of in- the different nematode communities, and in eight alpine-subalpine dividuals. In the subalpine grasslands at the 1700 – 1850 m a. s. l. sites between 2.65 and 3.07. The ratios of hyphal to bacterial feed- (according to Popovici, 1998) the genera Filenchus, Acrobeloides, ing nematodes showed a constant preponderance of the bacterial Gracilacus, Paratylenchus, Plectus and Rotylenchus were preva- feeding group. It is consistent with results of Hoschitz & Kaufmann lent. Plant feeding nematodes composed 28 – 52 % of individuals (2004b). The minimum values of the Maturity index (MI) on the and the number of taxa varied from 60 to 88. The percentage of southern faces of the alpine summits were statistically signifi cant omnivores in subalpine and alpine stands varied from 3 to 12 % but did not seem biologically relevant (Hoschitz & Kaufmann, while at lower elevations they composed 5 – 41 % of individuals. 2004a). On the hand, multivariate analyses were effective in dis- Authors concluded that the environmental variables, such soil pH, tinguishing different nematode communities. total nitrogen, humus content, exchangeable bases and soil type This study gives a detailed survey of nematode assemblages at could explain the variations in the composition of nematode com- fi ve high-mountain meadows in the Tatra National Park (the west- munities in grasslands, but no single factor could be selected as ern part of the Carpathians) in the Slovak Republic. The main aims being of overriding importance. of the study are as follows: Hoschitz & Kaufmann (2004a) studied soil nematodes (families (i) To evaluate the species composition of nematode faunas. and trophic groups) of Alpine summits in Austria and especially (ii) To study the abundance of nematode genera and trophic differences between plots facing the four compass directions. The groups and the community indices based on abundance. results showed that the nematode abundances on the south or (iii) To estimate the biomass and metabolic footprints of nematode east facing side were signifi cantly higher than on the northern assemblages. and western sides. The aspects of the sites also affected nem- Preliminary results and methods were presented by Háněl (2015b) atode families and trophic groups and nematode diversity (num- at the 13th Central European Workshop on Soil Zoology and they ber of families) and abundance responded to differences in soil will be published in the Proceedings of the workshop. Therefore microclimate. Authors therefore concluded that nematode as- some topics such as selection of indices and statistical methods semblages are potentially good bioindicators of climate change. and problems with determination of some nematode populations The most abundant family at two Carex fi rma swards (2214 and are not discussed in detail in this paper. 2255 m a. s. l.) were bacterivorous Cephalobidae. This contrasts with the alpine sites studied by Popovici & Ciobanu (2000) where Material and Methods plant feeding nematodes dominated. In another study Hoschitz & Kaufmann (2004b) found that bacterivorous Acrobeloides were The research was carried out in alpine parts of the High Tatra most abundant nematodes at Caricetum curvulae sedge mat Mountains (the Tatra National Park), in the Slovak Republic, above (2595 m a. s. l.) while in an alpine pasture with dominant Poaceae the timberline. The areas studied has cold alpine climate, parent (1961 m a. s. l.) the most abundant nematodes were plant feeding/ rocks are mostly granites. Soil types are rankers, podzols and plant-associated Rotylenchus and Tylenchidae. This indicates that cambic podzols, acid and rich in organic matter. vegetation is also an important factor determining taxonomic and Nematodes were studied at fi ve meadow sites in two mountain trophic composition of nematode assemblages while the richness valleys of two lake catchments:

49 (i) the Furkotská dolina Valley of the Vyšné Wahlenbergovo Lake microscope Leica Leitz DMRB equipped with N PLAN 100x/1.25 (VWL) with soil-poor catchment. immersion oil objective and transmitted light interference contrast, V1: 49° 09’ 46.8’’ N, 20° 01’ 41.5’’ E; 2200 m a. s. l.; SW facing on maximum magniﬁ cation 1600x. As a basis for the determination of a 15° slope; Seslerietum distichae Krajina 1933 (dominant Ore- nematodes monographs written by Brzeski (1998) and Andrássy ochloa disticha (Wulf.) Link, Luzula alpinopilosa (Chaix) Breistr.) (2005, 2007, and 2009) were used plus publications dealing with

(Matějka, 2015); soil pH(H2O) 4.7, soil carbon 10.4 %. individual taxonomic groups mostly cited in the books mentioned V2: 49° 09’ 22.3’’ N, 20° 01’ 40.4’’ E; 2003 m a. s. l.; S facing on above and latest papers if necessary. Nematode numbers were a 5° slope; Juncetum triﬁ di Szafer et al. 1923 em. Krajina 1933 adjusted to give the total number per samples and then converted 2 (dominant Juncus triﬁ dus L.); soil pH(H2O) 4.5, soil carbon 17.0 %. to a per m basis. The mean abundance of genera at each site and V3: 49° 08’ 48.5’’ N, 20° 01’ 40.8’’ E; 1766 m a. s. l.; S facing sampling date were used for the calculation of community indices on a 30° slope; Agrostio pyrenaicae-Nardetum strictae (Sillinger (except for SR and GR) and multivariate analyses. 1933) Šomšák 1971 corr. Dúbravcová in Mucina et Maglocký 1985 The species richness was evaluated using the number of species

(dominant Nardus stricta L., Agrostis rupestris All.); soil pH(H2O) and the Species Richness index SR = (S–1)/loge(N), S = the num- 4.3, soil carbon 7.9 %. ber of species identifi ed, N = the number of individuals identifi ed. (ii) the Veľká studená dolina Valley of the Starolesnianske Lake The Genera Richness index GR was calculated accordingly with (SL) with soil-rich catchment S = the number of genera identifi ed. The Shannon index (H’gen) S1: 49° 10’ 56.5’’ N, 20° 09’ 50.8’’ E; 1994 m a. s. l.; SE facing on was calculated using natural logarithms and nematode genera a 18° slope; Agrostietum pyrenaicae Krajina 1933 corr. Paclová et abundance (Yeates & Bongers, 1999). The Maturity Index MI and al. in Mucina et Maglocký 1985 (dominant Agrostis rupestris All.; the Plant Parasite Index PPI were calculated according to Bongers with Senecio carniolicus Willd. and Salix retusa L.); soil pH(H2O) (1990) and cp-values according to Bongers & Bongers (1998) to- 4.5, soil carbon 3.6 %. gether with the Sum Maturity Index ∑MI (Yeates, 1994) that in- S2: 49° 10’ 41.7’’ N, 20° 10’ 33.3’’ E; 1763 m a. s. l.; S facing on a cludes all trophic groups (Wasilewska, 1994). 25° slope; Ranunculo platanifolii-Adenostyletum alliariae (Krajina To asses participation of nematodes (Table 1) in detritus and 1933) Důbravcová et Hadač ex Kočí 2001 (dominant Calamagros- grazing (direct consumption of primary production) food webs the tis villosa (Chaix) J. F. Gmel., Mulgedium alpinum (L.) Less.); soil ratios (B+F)/PP and (B+F+RFF)/PP were evaluated according to pH(H2O) 4.5, soil carbon 7.6 %. Wasilewska (1997). PP (plant parasites) are Wasilewska’s OPP Six soil samples were taken at random at each site (sampling area (obligatory plant parasites, which cause damage to host plants) of 5 x 5 m) using a cylindrical corer of cross-sectional area 10 cm2 and RFF (root-fungal feeders) are Wasilewska’s FPP (facultative inserted down to a depth of 10 cm (if possible). Sampling dates plant parasites, which cause no or little damage to host plants and were 20 – 21 September 2013 (a), 21 – 22 September 2014 (b) at least some can reproduce on fungi). Because of high population and 8 – 9 September 2015 (c). The soil in each individual sample densities of Aglenchus agricola that is an epidermal cell and root was weighed, carefully hand-mixed, stones taken out and sub- hair feeder but cause no evident damage to plants (Yeates et al., tracted from the sample, and a part of soil was dried to determine 1993; Brzeski, 1998) the ratio (B+F)/(PP+RFF) was also calculat- the water content. Soil moisture on individual sampling dates was ed. These ratios were constrained to have values between 1 (ma- determined gravimetrically, soil was dried for 48 h at 25 °C and jority of nematode individuals participate in the detritus food web) then for 4 h at 105 °C. The mean soil moistures at V1, V2, V3, S1 and 0 (majority of nematode individuals participate in the grazing and S2 were 48.5, 47.2, 32.2, 37.6 and 42.4 % of water in fresh food web) as follows: soil, respectively. Soil temperature was taken over the period of (i) Nematode Food-web Ratio 1: NFR1 = (B+F)/(PP+B+F). investigation by means of data-loggers placed at 5 cm of the soil (ii) Nematode Food-web Ratio 2: NFR2 = (B+F+RFF)/ profi le. The mean soil temperatures at V1, V2, V3, S1 and S2 were (PP+B+F+RFF). This ratio refl ects situations where fungivorous 1.4, 2.8, 5.1, 3.7 and 3.8 °C, respectively. Tylenchidae prevail in the RFF group, such as small Filenchus Nematodes were isolated from approximately 15 ml of mixed species (Okada et al., 2005). soil, which according to soil properties represented 3.75 to 10.02 (iii) Nematode Food-web Ratio 3: NFR3 = (B+F)/(PP+RFF+B+F). grams of the substrate per sample. Thus, a total of 90 soil samples This ratio refl ects situations when plant feeders, such as Aglen- were collected, which represented 1350 ml (628 g) of fresh soil chus, prevail in the RFF. examined for nematodes. The isolation of nematodes was carried The Nematode Channel Ratio NCR1 = B/(B+F) was calculated out using modifi ed Baermann funnels. Nematodes were killed and according to Yeates (2003) and NCR2 = B/(B+F+RFF) according preserved in a 3.5 % solution of formaldehyde and mounted and to Háněl (2010) to asses the relative activity of bacterial-based studied in glycerol on slides (Háněl, 1995). energy channel (pathway) and slower fungal based channel in Altogether 13,005 nematode specimens were extracted and deter- decomposition processes in soil. The values of those indices are mined to species/genus level. The studies of anatomy, morphology constrained to have values between 1 (totally bacterial-mediated and morphometry and determinations were performed using light decomposition) and 0 (totally fungal-mediated decomposition).

50 3 -2 Table 1. The mean abundance (x 10 ind.m ) of nematode trophic groups and genera (n = 18 individual samples) at the sites studied with the S.E., F(4,85) and p values of one-way ANOVA for trophic groups. The same letters (a, b and c) indicate homogeneous groups of means detected using Fisher LSD post-hoc test, alpha = 0.05.

Because Levene’s test detected heterogeneity of variances in predators and insect parasites Kruskal-Wallis test H(4,N=90) and p values are also calculated, followed by post-hoc multiple comparison of mean ranks of all pairs of groups and homogeneous groups (p > 0.05) are indicated by the same letters (x, y and z). Underlined ﬁ gures denote that this particular nematode genus makes up more than 4.5 % of the total nematode population at that site. For the key to abbreviated names see Fig. 3 and 5.

Nematodes and sites Names V1 V2 V3 S1 S2 Bacterivores B 652.6 249.7 239.3 284.1 334.1 S.E. 135.5 64.0 65.4 45.0 60.7 F=6.021, p<0.001 a b b b b Plectus Plectus 294.9 94.1 144.0 84.7 215.4 Acrobeloides Acrdes 279.1 54.4 69.6 127.0 56.6 Teratocephalus Terato 28.7 39.2 1.4 12.4 3.3 Heterocephalobus Heceph 11.5 23.9 6.8 28.3 6.6 Panagrolaimus Panagr 11.7 17.6 0 0 22.1 Rhabdolaimus Rhbdol 8.6 2.4 0 17.5 0 Rhabditis Rhabdi 0.4 2.2 9.6 2.2 11.1 Prismatolaimus Prisma 5.8 8.1 0.6 8.1 0.6 Metateratocephalus Metate 4.6 4.3 5.9 0 6.3 Bastiania Bastia 3.0 0 0 2.9 1.4 Eumonhystera Eumonh 0.9 1.2 0.6 0 1.9 Geomonhystera Geomon 00003.4 Bursilla Bursil 0 1.3 0.8 0 1.3 Monhystrella Monrel 0.5 0 0 1.1 1.7 Bunonema Bunone 1.2 0.5 0 0 1.4 Pristionchus Pristi 0.4 0 0 0 0.9 Alaimus Alaimu 1.2 0 0 0 0 Ceratoplectus Cerato 0 0.5 0 0 0 Fungivores F 102.7 241.5 93.1 139.7 81.3 S.E. 45.3 64.5 40.0 39.7 14.2 F=3.378, p=0.013 b a b ab b Aphelenchoides Aphdes 95.7 118.1 67.1 40.1 62.0 Tylencholaimus Tylmus 0 101.6 8.0 87.5 0.5 Ditylenchus1) Dityle 3.6 20.9 18.0 11.0 15.6 Diphtherophora Diphth 1.0 0 0 1.1 2.2 Deladenus Delade 2.4 0.9 0 0 0.9 Root-fungal feeders RFF 1089.7 437.9 48.3 258.0 66.5 S.E. 210.2 115.1 13.8 68.3 14.6 F=34.915, p<0.001 a b c b c Aglenchus Aglenc 1041.9 322.1 4.7 199.7 6.3 Filenchus Filenc 11.8 85.6 43.6 41.4 56.4 Lelenchus Lelenc 1.4 26.3 0 10.6 0 Cephalenchus Celenc 26.0 0.9 0 6.3 0 Tylenchus Tylenc 8.6 3.0 0 0 0 Coslenchus Colenc 00002.3 Malenchus Malenc 00001.5 Plant parasites PP 10.8 125.8 108.7 221.8 711.1 S.E. 3.8 60.2 50.6 116.9 173.6 F=21.393, p<0.001 c b b b a Paratylenchus (Paratylenchus) Paraty 0.7 75.4 94.7 76.2 182.5 Helicotylenchus Helico 1.0 1.4 9.7 14.1 359.3 Rotylenchus Rotyle 00096.6 54.2

51 Nagelus Nagelu 000099.4 Sphaeronema juv. Sphaer 0 47.0 0 0 0 Paratylenchus (Gracilacus) Gracil 0 2.0 0 30.5 0 Pratylenchus Pratyl 9.2 0 4.4 4.4 2.8 Tylenchida juv. Tyjuvs 0 0 0 0 12.7 Omnivores O 151.4 180.0 81.8 223.3 136.6 S.E. 26.1 22.5 11.1 27.5 18.3 F=6.456, p<0.001 b ab c a b Eudorylaimus Eudory 113.8 55.5 66.7 120.4 60.1 Epidorylaimus Epidor 31.6 15.4 1.8 56.7 12.1 Aporcelaimellus Aprcll 0 68.9 3.9 1.3 0.6 Allodorylaimus Allodo 0 1.8 0 0 56.1 Enchodelus Enchdu 2.8 8.8 3.1 33.2 2.8 Heterodorus Herodo 0 21.9 2.4 8.3 3.5 Metaporcelaimus Metapo 3.3 7.7 3.7 3.5 1.5 Predators P 46.1 31.8 103.6 8.5 127.0 S.E. 15.6 12.3 21.5 4.0 22.4 F=20.362, p<0.001 b bc a c a H=42.732, p<0.001 yz z xy z x Tripyla Tripyl 43.8 6.8 47.2 8.5 60.4 Coomansus Cooman 2.3 23.9 54.0 0 62.5 Paravulvus Paravu 0 1.1 2.4 0 0.5 Clarkus Clarku 00003.7 Insect parasites IP 0.4 1.8 29.6 0.5 13.3 S.E. 0.4 1.8 9.8 0.5 8.6 F=8.221, p<0.001 b b a b b H=27.290, p<0.001 y y x y xy Steinernema juv. Steinl 0.4 1.8 29.6 0.5 13.3 All nematodes 2053.9 1268.5 704.4 1136.0 1470.0 S.E. 347.0 200.9 146.0 166.3 214.8 F=7.606, p<0.001 a b c b ab 1) Ditylenchus dipsaci was very rare, therefore all Ditylenchus species were included in the group of fungivores.

The NCR2 takes fungal-feeders in Tylenchidae (RFF) into account. tween trophic groups metabolic footprints of nematodes in the soil To evaluate the soil food web condition the Enrichment Index (EI), food web were computed as suggested by Ferris (2010). Metabol- the Structure Index (SI) and the Channel Index (CI) were calcu- ic footprints were calculated at genus level and to take species and lated according to Ferris et al. (2001) and the Basal Index (BI) adult-juvenile variations into account the mean genus biomass according to Berkelmans et al. (2003). A high BI indicates poor for each site and sampling date was calculated separately. The ecosystem health, while a high SI indicates a well-regulated, metabolic footprints of individual genera were further used for the healthy ecosystem. calculation of the metabolic footprints of functional groups of nem- The biomass (wet mass, fresh weight in μg) of adult nematode atodes. For example, the Herbivore metabolic footprint is the sum species occurring in the area studied was calculated using the of the footprints of root-fungal feeder and plant parasite genera in method described by Andrássy (1956). The biomass of juveniles the Table 1. Enrichment footprint was calculated using guilds indi- was assumed to be one half of that of the adults, except for the cating enrichment (Ba1 and Fu2) as deﬁ ned by Ferris et al. (2001). dauer-stage juveniles of the families Rhabditidae, Neodiplogastri- However, the Table 4 in Ferris (2010) takes only Rhabditidae (cp 1) dae and Steinernematidae and unidentiﬁ ed Tylenchida juveniles but not Aphelenchidae (cp 2) for the calculation of the Enrichment for which biomass was calculated separately. Biomasses were footprint and labels Qudsianematidae with the cp-value 5 while estimated for each individual soil sample (n = 6 at a site and sam- in the Table 1 of his paper and in Bongers & Bongers (1998) this pling date) and then mean values for each site and sampling date family has the cp-value 4. Ferris et al. (2001) take Qudsianemati- were calculated. But instead of simple biomass proportions be- dae as Om5 guild. This can make interpretation of EI and SI along

52 Table 2. The mean values of nematode community indices and metabolic footprints (n = 3 sampling dates) at the sites studied with the F(4,10) and p values of one-way ANOVA. The same letters (a, b and c) indicate homogeneous groups of means detected using Fisher LSD post-hoc test, alpha = 0.05.

Indices and sites V1 V2 V3 S1 S2 Nematode diversity indices Species F=4.186, p=0.030 38.33 ab 48.00 a 31.67 b 40.00 ab 47.67 a SR F=6.431, p=0.008 5.10 b 7.07 a 5.13 b 6.04 ab 6.88 a Genera F=4.044, p=0.033 24.67 bc 29.33 ab 22.33 c 24.67 bc 30.67 a GR hv) F=6.649, p=0.007 3.24 b 4.27 a 3.57 b 3.66 b 4.38 a H'gen F=17.258, p<0.001 1.70 b 2.58 a 2.46 a 2.50 a 2.47 a Nematode maturity indices ∑MI F=7.298, p=0.005 2.22 b 2.62 a 2.61 a 2.68 a 2.67 a MI F=4.540, p=0.024 2.46 b 3.08 a 2.75 ab 3.02 a 2.70 ab PPI hv) F=14.037, p<0.001 2.01 b 2.09 b 2.09 b 2.16 b 2.67 a PPI/MI F=8.127, p=0.003 0.82 b 0.68 c 0.77 bc 0.72 bc 0.99 a Nematode food-web and channel ratios NFR1 hv) F=22.346, p<0.001 0.99 a 0.80 b 0.76 b 0.71 b 0.35 c NFR2 hv) F=31.668, p<0.001 0.99 a 0.89 ab 0.79 b 0.79 b 0.39 c NFR3 F=3.983, p=0.035 0.43 b 0.45 b 0.67 a 0.50 ab 0.33 b NCR1 hv) F=6.579, p=0.007 0.87 a 0.50 c 0.72 ab 0.68 b 0.79 ab NCR2 F=5.127, p=0.017 0.38 bc 0.26 c 0.61 ab 0.45 abc 0.67 a Nematode food-web indices EI F=4.454, p=0.025 16.28 c 38.36 a 30.53 ab 17.21 bc 36.76 a SI hv) F=3.028, p=0.071 64.12 84.77 76.05 85.42 78.60 CI F=3.406, p=0.053 68.78 75.44 67.27 87.64 40.52 BI F=4.043, p=0.033 33.02 a 13.83 b 21.36 ab 14.18 b 18.89 ab Nematode metabolic footprints in mgC.m-2 Bacterivore F=1.942, p=0.180 59.40 26.01 32.86 22.81 76.45 Fungivore1) F=1.927, p=0.182 5.86 19.62 4.44 13.67 5.08 Root-fungal feeder RFF2) F=34.459, p<0.001 56.15 a 17.50 b 0.24 c 10.73 b 0.46 c Plant parasite PP hv) F=8.167, p=0.003 0.50 c 3.33 bc 2.66 bc 28.62 b 63.42 a Plant feeder (RFF2)+PP) F=7.525, p=0.005 56.65 a 20.83 a 2.90 b 39.36 a 63.89 a Omnivore F=7.874, p=0.004 34.99 bc 99.09 a 21.63 c 67.11 ab 64.34 ab Predator F=10.959, p=0.001 21.00 b 57.48 ab 136.26 a 3.16 c 156.84 a Insect parasite hv) F=5.539, p=0.013 0.07 b 0.32 b 5.25 a 0.09 b 2.36 ab Composite F=2.130, p=0.151 177.97 223.35 203.34 146.20 368.97 Herbivore1) F=6.986, p=0.006 56.93 a 23.42 a 3.56 b 40.03 a 64.97 a Basal F=1.945, p=0.179 61.29 25.64 34.76 23.84 74.80 Enrichment F=1.608, p=0.247 7.51 8.99 3.63 2.70 8.69 Structure F=4.847, p=0.020 57.60 c 169.87 ab 159.00 ab 81.86 bc 221.80 a Functional F=2.879, p=0.080 218.25 966.74 270.79 112.10 954.53 1) with Filenchus included 2) exclusive of Filenchus hv) Levene‘s test detected heterogeneous variances but n = 3 was too low number of samples for Kruskal-Wallis test and the test was able to detect only the most contrasting differences.

53 with Enrichment and Structure footprints uncertain. Therefore (and lenchida (33 species) and Dorylaimida (23 species). The greatest having original biomass data of the nematode populations in the species richness had the genera Plectus (11 species) and Aphel- area studied), I did not use NINJA automated calculation system enchoides (11 species). Alaimus arcuatus occurred at V1 and was (Sieriebriennikov et al., 2014) but have done calculations manually one and only representative of the order Alaimida. Some species with cp-values according to Bongers & Bongers (1998). could not be satisfactorily identiﬁ ed with any available description STATISTICA (StatSoft, 2001) was used to perform the Cluster and may represent locally variable populations of the known spe- Analysis and the Analysis of Variance (ANOVA) with Fisher LSD cies or species new to science. The dendrogram based on a Clus- post-hoc test at alpha = 0.05. The nematode data were ln(x+1) ter Analysis of species presence and absence showed two clus- transformed. The assumption of normality was evaluated with Kol- ters. The larger cluster in the upper part of the chart consisted of mogorov-Smirnov & Lilliefors test for normality, and Shapiro-Wilk’s the samples from higher elevations and the smaller cluster in the W test. If the Levene’s test detected heterogeneity of variances a lower part consisted of the samples from lower elevations (Fig. 1). Kruskal-Wallis ANOVA was also calculated followed by post-hoc multiple comparison of mean ranks of all pairs of groups. Nematode abundance and indices According to Detrended Correspondence Analysis (DCA) by seg- The total mean abundance of nematodes ranged from 704 to ments (performed on the abundance and the metabolic footprints 2054 x 103ind.m-2 (Table 1). The genera Plectus and Acrobeloi- of nematode genera as primary data) the ﬁ rst ordination axes had des were most abundant bacterivores at all sites and the greatest lengths of gradient < 3 S.D. This indicated a linear (monotonous) abundance of bacterivores was at V1. The bacterivorous genera response model along the data gradient as in preliminary analyses Teratocephalus, Heterocephalobus, Rhabditis, and Prismatolai- (Háněl 2015b). Therefore, nematode data were analysed using mus occurred at all sites but their abundance was relatively low. Principal Component Analysis (PCA) (ter Braak & Šmilauer, 2002). Fungivores reached the greatest abundance at V2 with the genera Aphelenchoides and Tylencholaimus, Ditylenchus occurred at all Results sites but was less abundant. Among root-fungal feeders the genus Aglenchus reached the greatest abundance and strongly dominat- Nematode species composition ed at sites of higher elevations. The abundance of the genus Fi- A total of 110 nematode species, belonging to 50 genera, were dis- lenchus was generally lower than that of Aglenchus except for the tinguished. At individual sites their numbers varied from 45 to 72 sites V3 and S2. The greatest abundance of plant parasites was (Appendix 1). Most nematodes species belonged to the orders Ty- at S2 and the genera Helicotylenchus, Paratylenchus and Nagelus

Species of nematodes Ward's method Euclidean distances

V1a V1b V1c S1a S1c S1b V2a V2c V2b V3a V3b V3c S2a S2b S2c

3 4567891011 Linkage distance Fig. 1. Dendrogram based on Cluster Analysis of the presence and absence of nematode species.

54 Table 3. The mean biomass (fresh weight) of nematode trophic groups (n = 18) and the mean individual biomass in nematode trophic groups (n = 5 – 18) ± S.E. at the sites studied with the F and p values of one-way ANOVA, except for individual biomass of insect parasites. The same letters indicate homogeneous groups of means detected using Fisher LSD post-hoc test, alpha = 0.05. Because Levene’s test mostly detected heterogeneity of variances Kruskal-Wallis test H and p values are also calculated, followed by post-hoc multiple comparison of mean ranks of all pairs of groups and homogeneous groups (p > 0.05) are indicated by the same letters. Nematodes and sites V1 V2 V3 S1 S2 Mean biomass of trophic groups in mg.m-2 Bacterivores 147.1 ± 76.9 62.1 ± 25.8 82.8 ± 19.9 48.2 ± 12.6 233.3 ± 51.0

F(4,85)=8.703, p<0.001 b c bc c a

H(4,N=90)=26.259, p<0.001 xy y xy y x Fungivores 11.2 ± 5.4 39.3 ± 16.9 7.0 ± 3.2 31.3 ± 11.6 7.5 ± 1.7

F(4,85)=3.704, p=0.008 bc a c ab c

H(4,N=90)=11.833, p=0.019 xy x y xy xy Root-fungal feeders 104.5 ± 20.5 36.1 ± 10.5 1.3 ± 0.4 20.5 ± 7.2 2.3 ± 0.5

F(4,85)=36.836, p<0.001 a b c b c

H(4,N=90)=68.309, p<0.001 xxyxy Plant parasites 0.9 ± 0.3 5.3 ± 2.0 4.5 ± 2.1 83.2 ± 75.8 155.2 ± 50.7

F(4,85)=17.121, p<0.001 c bc bc b a

H(4,N=90)=41.007, p<0.001 z yz yz y x Omnivores 117.1 ± 22.5 455.3 ± 101.9 79.6 ±16.8 247.8 ± 46.6 255.7 ± 44.9

F(4,85)=12.326, p<0.001 c a c b ab

H(4,N=90)=31.338, p<0.001 yz x z xy xy Predators 83.6 ± 29.1 337.0 ±140.1 762.8 ± 192.1 11.2 ± 5.3 871.8 ± 229.7

F(4,85)=24.607, p<0.001 b b a c a

H(4,N=90)=47.609, p<0.001 yyxyx Insect parasites 0.2 ± 0.2 0.7 ± 0.7 11.8 ± 3.9 0.2 ± 0.2 5.3 ± 3.4

F(4,85)=7.236, p<0.001 b b a b b

H(4,N=90)=27.290, p<0.001 yyxyxy All nematodes 464.6 ± 131.8 935.9 ± 164.5 949.7 ± 202.9 442.4 ± 87.6 1531.1 ± 258.6

F(4,85)=10.482, p<0.001 c b b c a

H(4,N=90)=32.210, p<0.001 z xy xy yz x Mean individual biomass in trophic groups in μg.ind-1 Bacterivores 0.155 ± 0.027 0.203 ± 0.028 0.374 ± 0.050 0.154 ± 0.036 0.735 ± 0.068

F(4,85)=30.843, p<0.001 c c b c a

H(4,N=90)=45.581, p<0.001 z yz xy z x Fungivores 0.110 ± 0.025 0.121 ± 0.020 0.076 ± 0.013 0.169 ± 0.022 0.082 ± 0.010

F(4,81)=4.219, p=0.004 b ab b a b

H(4,N=86)=12.439, p=0.014 xy xy y x xy Root-fungal feeders 0.093 ± 0.002 0.078 ± 0.005 0.029 ± 0.008 0.071 ± 0.004 0.037 ± 0.006

F(4,84)=27.148, p<0.001 a ab c b c

H(4,N=89)=50.128, p<0.001 x x z xy yz Plant parasites 0.090 ± 0.019 0.048 ± 0.010 0.047 ± 0.009 0.094 ± 0.036 0.168 ± 0.027

F(4,70)=5.153, p=0.001 b b b b a

H(4,N=75)=21.397, p<0.001 yyyyx Omnivores 0.889 ± 0.113 2.320 ± 0.293 0.955 ± 0.153 1.047 ± 0.115 1.839 ± 0.185

F(4,85)=13.218, p<0.001 b a b b a

H(4,N=90)=32.845, p<0.001 z x z yz xy Predators 1.488 ± 0.161 8.669 ± 1.514 7.226 ± 0.772 1.274 ± 0.123 5.677 ± 0.744

F(4,60)=16.727, p<0.001 b a a b a

H(4,N=65)=32.232, p<0.001 yxxyx Insect parasites 0.400 0.400 0.400 0.400 0.400 All nematodes 0.222 ± 0.031 0.774 ± 0.094 1.785 ± 0.344 0.402 ± 0.058 1.376 ± 0.282

F(4,85)=15.556, p<0.001 c b a c a

H(4,N=90)=48.156, p<0.001 z xy x yz x

55 occurred on all sampling dates at S2. The representatives of the dominance of A. agricola in nematode assemblages. The SI val- genera Paratylenchus and Helicotylenchus were found at all sites. ues were high everywhere and no signifi cant differences between The genus Eudorylaimus was the most abundant omnivore except sites were found. The high values of the CI at V1, V2, V3 and S1 for V2 with the most abundant genus Aporcelaimellus. The genera were caused by a low abundance (or absence) of Ba1 guild. Epidorylaimus, Enchodelus and Metaporcelaimus occurred at all sites. A low abundance of predators was at S1 because of the Nematode biomass and metabolic footprints absence of the species Coomansus menzeli but at V3 the genus The total mean biomass of nematode assemblages ranged from Coomansus belonged to the dominant genera. The abundant po- 442 to 1531 mg.m-2. The greatest biomass was found at S2, the pulations the genus Tripyla were found at all sites. lowest values were at V1 and S1 (Table 3). The signifi cantly great- The Cluster Analysis of the ln(x+1) transformed genera abun- est biomass of bacterivores was at S2. The biomass of fungivores dance in Table 1 gave a similar dendrogram (Fig. 2) as the Cluster varied across sites. The signifi cantly greatest biomass of root-fun- Analysis of the species (Fig. 1). The Principal Component Analysis gal feeders was at V1 with the greatest abundance of Aglenchus showed gradient with the nematode faunas at the sites of higher agricola (Table 1). The signifi cantly greatest biomass of plant par- elevations on the left-hand side of the chart and those at the sites asites was at S2. The greatest biomass of omnivores occurred at of lower elevations on the right-hand side of the chart (Fig. 3). V2. The signifi cantly greatest biomass of predators was at S2 and The diversity indices of nematode assemblages reached greatest V3, the sites of the lowest elevations. values at S2 and V2 (Table 2). Maturity indices (∑MI, MI) had the The signifi cantly greatest biomass of an individual in the whole lowest values at V1. The greatest values of PPI and PPI/MI ra- nematode assemblage was at V3 and S2. As concerns the tropic tio were at S2. Nematode food-web ratios were lower than 0.5 at groups the signifi cantly greatest individual biomass had predators S2 because the plant parasites were the most abundant trophic (at V2, V3 and S2) followed by omnivores (at V2 and S2). Bac- group. NFR3 was also low at V1, V2 and S1 with abundant Aglen- terivores had the signifi cantly greatest individual biomass at S2 chus agricola. NCR1 was mostly greater than 0.5. The decrease in because of abundant populations of Plectus parietinus. Plant par- the values of NCR2 refl ected the increase in the abundance and asites had the signifi cantly greatest individual biomass at S2, too, Abundance of nematode genera Ward's method Euclidean distances

V1a V1b V1c V2a V2c V2b S1a S1b S1c V3a V3b V3c S2a S2b S2c

0 5101520 Linkage distance Fig. 2. Dendrogram based on Cluster Analysis of the ln(x+1) transformed abundance of nematode genera.

56 V2b

1.0 Tylmus Filenc Abundance Aprcll V2c Lelenc Sphaer S1c Paraty V2a Gracil Enchdu Rotyle S1a Cooman Aphdes

V3b V3c S2c Steinl Helico Axis 2 Nagelu Aglenc Epidor V3a S1b S2a S2b

Eudory

Acrdes Plectu Tripyl

V1c V1a

V1b -1.0 -1.0Axis 1 1.0

Fig. 3. Biplot of nematode genera and samples based on Principal Component Analysis using log(x+1) transformed abundance of genera. Eigenvalues for ordination axes 1, 2, 3 and 4 were 0.400, 0.175, 0.115 and 0.101, respectively. Cumulative percentage variance of genus data for ordination axes 1, 2, 3 and 4 were 40.0, 57.5, 68.9 and 79.1, respectively. All genera were included in this analysis but only the genera with a dominance ≥ 4.5 % at least on one sampling date at a site are shown in the biplot. Abbreviated names of these genera are in Table 1. because of Rotylenchus robustus, Helicotylenchus varicaudatus, The dendrogram based on Cluster Analysis of the ln(x+1) trans- H. pseudorobustus and Nagelus leptus. formed metabolic footprints of nematode genera showed samples The Composite nematode metabolic footprints did not differ be- from S2 in a separate cluster and similar nematode assemblages tween sites (Table 2). There were no signifi cant differences in at V1 and S1 (Fig. 4). The PCA of the genera metabolic footprints Bacterivore and Fungivore metabolic footprints, too. The Basal showed nematode assemblages at higher elevations (V1, V2 and and Enrichment metabolic footprints did not differ between sites S1) on the left-hand side of the gradient and those at lower eleva- and this contrasts with signifi cant differences found for the BI and tions (V3 and S2) on the right-hand side (Fig. 5). EI. On the other hand, the Structure metabolic footprints differed between sites while the SI values did not. The signifi cantly lowest Discussion Herbivore metabolic footprint was at V3. At the other sites the Her- bivore metabolic footprints did not differ signifi cantly but it must be Nematode species composition taken into account that Pl2 (RFF) and Pl3 (PP) components contrib- Among the 110 species distinguished, 19 nematode species that uted differently to the total Plant feeder footprint. Infective juveniles are not listed in the book by Lišková & Čerevková (2011) were of insect parasites (Steinernema) were also formally included to found. These formally represent new nematode species records obtain the Composite metabolic footprint although they develop to (N) in Slovakia. But there is a question whether they are actual- adults in insect bodies, not in soil. ly new to the fauna of the Slovak Republic because they might

57 Metabolic footprints of nematode genera Ward's method Euclidean distances

V1a V1c V1b S1b S1a S1c V2a V2c V2b V3a V3b V3c S2a S2b S2c

02 4681012 14 1618 20 Linkage distance Fig. 4. Dendrogram based on Cluster Analysis of the ln(x+1) transformed metabolic footprints of nematode genera. be overlooked or misidentifi ed with very similar species in older at V2, 2003 m a. s. l., host plant was probably Salix herbacea L. papers. (the genus Salix is among host plants; see Brzeski, 1998), which Plectus magadani (N) was originally described as a subspecies occurred at this site (Matějka, 1915). Alaimus arcuatus (N) and of Plectus acuminatus and is similar to Plectus communis. Plec- Coomansus menzeli (N) occur in Europe (Andrássy, 2009). It is tus rotundilabiatus (N) and Plectus velox (N) are very similar to interesting that C. menzeli was abundant at V2, V3 and S2 while it Plectus parietinus. All these species are common in Europe (Zell, was absent from S1 and only one female of Coomasus zschokkei 1993; Andrássy, 2005) and there is hardly a reason for their ab- was found at S2. Juveniles of the two species are diffi cult to tell sence from the Slovak Republic. But the determination requires apart and some records of large specimens of C. zschokkei may in permanent slides and high-resolution immersion oil objective. fact be C. menzeli (Loof & Winiszewska-Ślipińska, 1993). Lišková & Čerevková (2011) report on Tylenchus davainei in many Epidorylaimus leptosoma (N) can be easily determined according localities but Brzeski (1998) writes that this species is rare, most to the key in Andrássy (2009) and his earlier keys cited in his book. often found in moss and perhaps some records of T. davainei ac- On the other hand, in the Andrássy’s (2009) key the species Eu- tually should be corrected to other species. Tylenchus elegans (N) dorylaimus pseudobokori (N) is in the group of species with “Tail is common in Europe (Andrássy, 2007; Geraert, 2008). Filenchus longer, 1.2 – 2 anal body diameters, its ventral contour arcuate hamatus (N) is a rare species and probably actually new to the (paragraph 19) and Lip region continuous with body, lips hardly Slovak Republic. Aphelenchoides and Ditylenchus are genera separate, rounded (paragraph 25)”. Zell (1986) writes “Lippen ab- with a lot of small and medium-sized species diffi cult to tell apart gesetzt, getrennt.” and the Figures 12 – 14 show lip region at least (Brzeski, 1991, 1998; Andrássy, 2007). Lišková & Čerevková partly offset. The female specimens of E. pseudobokori with more (2011) report on 25 species of Aphelenchoides and 15 species of offset lips can be misidentifi ed with Eudorylaimus altherri Tjepke- Ditylenchus and this study adds two and two species that also live ma, Ferris & Ferris, 1971. However, c = 15.0 – 27.8 in E. pseu- in the Europe to the two genera, respectively. dobokori (see Zell, 1986) and c = 27 – 38 in E. altherri as given An interesting fi nding is the occurrence of Sphaeronema alni (N) in Andrássy (2009). The male of E. pseudobokori described by

58 Aprcll V2b

1.0 Metabolic footprints V2c

Tylmus Metapo V2a Cooman V3c

V3b

Enchdu V3a

Axis 2 Aglenc S2a S1a Allodo Eudory Helico Rotyle S2c S1c Tripyl Plectu S2b V1a Acrdes V1c V1b Epidor S1b -1.0 -1.0Axis 1 1.0

Fig. 5. Biplot of nematode genera and samples based on Principal Component Analysis using log(x+1) transformed metabolic footprints of genera. Eigenvalues for ordination axes 1, 2, 3 and 4 were 0.375, 0.199, 0.128 and 0.099, respectively. Cumulative percentage variance of genus data for ordination axes 1, 2, 3 and 4 were 37.5, 57.3, 70.1 and 80.1, respectively. All genera were included in this analysis but only the genera with a proportion ≥ 4.5 % of the Composite footprint at least on one sampling date at a site are shown in the biplot. Abbreviated names of these genera are in Table 1.

Zell (1986) has ﬁ ve supplements as the male found in the present cf. baldaccii, Diphtherophora cf. kirjanovae and Metaporcelaimus study at V1 in September 2015. The male of E. altherri is unknown cf. monohystera but on close examination the available specimens (Andrássy, 2009). Eudorylaimus stefanskii (N) is a rare European proved to be somewhat different from original descriptions. There nematode and was transferred to the genus Eudorylaimus by An- were also nematodes that could be determined to the genus level drássy (1991). only (Appendix 1) but morphologically represented separate spe- Aporcelaimellus alius (N) and A. medius (N) were described in cies. 2002 and some records of A. obtusicaudatus listed in Lišková This is consistent with the results of the studies mentioned in the & Čerevková (2011) may cover the two species. Enchodelus lu- Introduction – a lot of nematode populations could be determined cinensis (N) was described from Moldavia and was also found in to genus or to the family level only. No surprise, only about 30,000 Western Romanian Carpathians (Ciobanu et al., 2010) and can nematode species were described. With an estimated number of actually be a new species to the Slovak Republic. species extending to about 106 most nematode species remain Some populations (Q) shared diagnostic characters of two spe- undescribed, the proportion of cryptic species may be high and cies and this variability does not allow for unequivocal placement. some authors assume that failing adequately to delimit a species Those were Teratocephalus lirellus / T. stratumus, Epidorylaimus is preferable to falsely delimiting entities (Hugot et al., 2001; Palo- humilior / E. humilis, Eudorylaimus maritus / E. carteri and Encho- mares-Rius et al., 2014). Alpine habitats do not form a continuous delus macrodorus / E. carpaticus and they are discussed in anoth- landscape. They are rather islands in a non-alpine ocean and as er study mentioned in the introduction (Háněl, 2015b). Andrássy’s such they can be inhabited by a high number of endemic species (2007, 2009) keys also lead to the determination of Paratylenchus to be described in future.

59 The nematode taxonomic richness was greater at lower elevations of Paratylenchus with cp-value 2 and various species of Paratylen- than at higher elevations, a trend described in the Introduction, ex- chus can be also dominant in meadows at lower elevations along cept for V3. The numbers of species and genera at V3 were lower with the cp-3 plant parasites. than in mountainous Carpathian meadows in Romania (Popovici & Bacterivorous nematodes are usually the second most abundant Ciobanu, 2000) and Austrian Alps (Hoschitz & Kaufmann, 2004b) and dominant trophic groups following plant feeders (PP+RFF) in but evidently greater than in Italian Alps studied by Vinciguerra alpine and subalpine meadows in Central Europe and the majority (1988). Also the total SR and GR indices (Appendix 1) were lowest of bacterivores belong to Plectus and Acrobeloides (Háněl, 1994; among the meadows studied. Therefore, the low species-generic Popovici & Ciobanu, 2000; Hoschitz & Kaufmann, 2004b). The richness at V3 seems to have been an exception to a rule of great- two genera also dominated in the sites studied and together with er nematode richness at elevation below 2000 m a. s. l. than above Aglenchus decreased the values of H’gen and ∑MI at V1. The 2000 m a. s. l. in subalpine-alpine meadows. low nematode taxonomic richness and abundance at V3 contrasts The lowest taxonomic richness at V3 coincided with the lowest with the high taxonomic richness and abundance at S2. Both sites total abundance (Table 1). But the abundance of predators was are at similar elevations (1763 – 1766 m a. s. l.) but differ in soil high and they represented 14.7 % of all nematodes at V3. Such a microclimate and vegetation. Nevertheless, their overall nematode high percentage of predators was not observed at any meadow in species and generic combinations were similar (Figs 1, 2 and 3). Romania (Popovici & Ciobanu, 2000). This may indicate that the The Shannon diversity index (H’gen) was signifi cantly lowest at nematode populations at V3 were “overgrazed”, species richness V1. At other sites the values of the H’gen were close to those cal- and consequently abundance were reduced by too strong biolog- culated by Popovici & Ciobanu (2000) but a direct comparison is ical top-down control. problematic because they combined species and genera. The values of MI and PPI were similar to those in Hoschitz & Kaufmann Nematode abundance and indices (2004ab) and PPI/MI ratios calculated from the mean values in Multivariate analyses clearly showed that nematode assemblages their papers did not exceed the value of 0.9, except for an alpine in meadows at higher elevation (1994 – 2200 m a. s. l.) differed pasture (PPI/MI = 0.93). In the present study the PPI/MI ratio had from those at lower elevations (1763 – 1766 m a. s. l.). The most signifi cantly greatest value (0.99) at S2 and indicated a slight nu- striking signifi cant differences were in the abundance of A. agrico- trient disturbance (enrichment) to soil food web (Bongers et al., la. A. agricola was the dominant nematode in alpine grasslands 1997). (2050 – 2270 m a. s. l.) from Romania (Popovici & Ciobanu, 2000) Nematode food-web ratios suggested that the majority of nema- accompanied by Filenchus and Paratylenchus and plant feeding tode individuals at S2 participated in grazing food web. This can nematodes generally dominated in mountainous grasslands. At be also true for V1 and V2 because the values of NFR3 were low meadow sites of elevations lower than 2000 m a. s. l. the domi- (due to abundant A. agricola). Participation of nematodes in de- nance of A. agricola decreases (Popovici, 1998). This is also in the tritus and grazing food webs at V3 and S1 appears to have been present study and in other meadows at lower elevations in Slova- more balanced. The assessment of the role of omnivores in soil kia (Šály & Žuffa, 1980; Šály, 1983, 1985; Lišková & Čerevková, food web still remains problematic (McSorley, 2012) as they link 2005; Čerevková, 2006; Háněl & Čerevková, 2006) where plant several levels of food chains (Wasilewska, 1997) but Enchodelus parasites with cp-values 3 become dominant. with long odontostylet may feed on plant roots in a similar way A bit different situation is in the Bohemian Massif in the Czech like Pungentus (Trudgill, 1976). NCR1 values were largely in the Republic where alpine zone is almost missing. In the Krkonoše favour of bacterial feeding group and indicated predominance of Mountains A. agricola represented 26 % (P. microdorus 33 %) of bacteria in detritus food web. This is consistent with results of all nematodes at a mountain meadow (1280 m a. s. l.) and 47 % Popovici & Ciobanu (2000). Low values of NCR2 at V1, V2 and at a grassy clear-cut (1200 m a. s. l.) left after spruce die-back S1 were caused by high population densities of plant-feeding A. and salvage logging (Háněl, 1994, 1998). In the Bohemian Forest agricola. High values of CI (except for S2 but the difference was

(the Šumava Mountains) A. agricola represented 79 % of all nem- not signiﬁ cant) are rather result of a low abundance of Ba1 guild, atodes at a natural grassy stand (ca 1340 m a. s. l.) and occurred as deﬁ ned by Ferris et al. (2001), than an indication of a high ac- in small populations in Norway spruce forests. After clear-cut of tivity of fungal decomposition channel. The high SI and the low EI spruce trees damaged by bark beetles and succession of grass at values delimited Quadrant C with structured food web, but unlike sites of 1180 – 1240 m a. s. l. A. agricola became the eudominant the Table 4 in Ferris et al. (2001), having decomposition channel species (Háněl, 1999, 2004). Below 1000 m a. s. l. the populations rather bacterial than fungal. of A. agricola in meadows mostly decrease and plant parasites with cp-values 3 often dominate (Háněl, 1997, 2010). Thus in the Nematode biomass and metabolic footprints Carpathians and the Bohemian Massif A. agricola dominates in The total biomass of nematodes (in mg.m-2) and the biomass of alpine meadows and subalpine/mountainous meadows, respec- a nematode individual (in μg) at the sites studied are generally tively. This species can be accompanied by abundant populations within values established in grasslands as reported, for example,

60 by Wasilewska (1979), Sohlenius (1980) and Háněl (2010). Nev- respiration is suspended. This, and variations in soil temperature, ertheless, nematodes at higher elevations (V1 and S1) had lower should be taken into consideration when estimating nematode res- total and individual biomass than those at lower elevations (V3 and piration in the fi eld (e.g. Persson et al., 1980; Verschoor, 2001). S2) where Coomansus menzeli was abundant and increased the Yeates (1979) reports on greater dry weight (42 – 58.5 % of the biomass values of predators. This species has not been reported fresh weight) of nematode bodies dried at temperatures lower than from Slovakia (Lišková & Čerevková, 2011) but occurs in some 75ºC. Ferris (2010) uses the b regressions coeffi cient of 0.75 in mountain grasslands in the Romanian Carpathians (Popovici, the nematode metabolic body size (Wb in μg.ind-1). In general, the 1998). As a consequence of a high abundance and the adult indi- mean value of b = 0.75 is acceptable for poikilotherms (Wright, vidual biomass (24.008 μg) of C. menzeli the metabolic footprints 1998) and is not signifi cantly different from b = 0.72 ± 0.092 calcu- of predators at V3 and S2 reached the greatest values across all lated by Klekowski et al. (1972) for the whole group of nematodes. sites and trophic groups. This suggests strong top-down control of But the b coeffi cient in individual species and trophic groups can nematode assemblages by predacious nematodes similar to that vary (e.g. Schiemer & Duncan, 1974; Klekowski & Wasilewska, in some mountain peat bogs (Háněl, 2015a) with abundant Jense- 1982). The mean metabolic (carbon) footprints of RFF at V1 and nonchus sphagni (Brzeski, 1960) indicating undisturbed natural those of PP at S2 were practically equal in size. A question arises condition of the site. whether it is actually true to reality because dominant nematode The lowest values of the Herbivore footprint occurred at V3 and species, plant species and soil temperatures at the sites differed. were about 10.5 times lower that Bacterivore + Fungivore foot- Klekowski et al. (1972), page 401, discuss irregularities in the prints. This is in agreement with nematode food-web ratios indicat- intensity of oxygen consumption among plant feeders. Power ing that nematodes at this site mainly participated in the detritus regression based on the data in their paper gives equation for 0.11 food web. At V1, S1 and S2 the metabolic footprints of nematodes weight-respiratory rate RR = 0.58W (r = 0.187, n = 16) nlO2. involved in detritus food web and grazing food web seem to have ind-1.hr-1. It is a bit strange result and may indicate that the pa- been in a balanced proportion although nematode food-web ratios rameters of oxygen consumption are different in different guilds based on abundance suggest greater participation of nematodes of plant feeders. For omnivores RR = 0.90W1.01 (r = 0.982, n = 7) in the grazing food web at S2. But in the detritus food web predom- and b = 1 suggests weight-independent metabolism (Wright, inance of bacteria-mediated decomposition is indicated by both 1998). For predators the equation is RR = 3.03W0.47 (r = 0.623, criteria. It is also evident that the Structure footprint contributed n = 7), which may indicate a surface area-dependent metabolism most to the Composite footprint, except for V1 with the lowest val- and a high metabolic (respiration) intensity (Schiemer & Duncan, ues of H’gen, ∑MI and SI. 1974; Wright, 1998). The knowledge of guild-specifi c respiration An advantage of Ferris’ (2010) approach to the subject is that he could improve the estimate of metabolic footprints. The production takes metabolic activity of nematodes into account but there are component is adjusted using cp-values so why not to be particular also some problematic points. On the page 100 he writes that “The about nematode guilds in the respiration component? However, functional metabolic footprint is maximized when the rhomboid it would need a lot of work and funds to measure respiration of a shape becomes a square ... so that the system is in metabolic wide spectrum of nematode species. balance.”. But in the present study the enrichment component of Results of multivariate analyses on the metabolic footprints of the metabolic footprint is very low at all sites, which results in very genera were similar to those based on the abundance of nema- narrow rhomboids. Multivariate analyses (Figs 1, 2, 3, 4, and 5) tode genera. But soil temperatures on individual sampling dates showed little variations in nematode faunas at individual sites (with across all sites varied from 1.3 to 10.6 °C and Q10 temperature an exception for S1), which is rather an indication of a balanced coeffi cient in nematodes differs for temperatures below 5 °C and state in climax systems than unbalanced. Soil temperature at the above 5 °C being assumed to be 5 and 3, respectively (Persson sites studied is probably too low to stimulate fast-growing bacteria et al., 1980). Ferris (2010) may be right that the sets of predomi- reach population densities suffi cient for abundant populations of nant species under one set of ambient conditions will have similar the r-strategist Ba1 nematodes. temperature-specifi c coeffi cient values to each other but different The equation for the calculation of the metabolic footprints of nem- from those of species predominating under alternate conditions. atodes is a very neat technique to evaluate nematode function in This may explain similarity of the results of multivariate analyses, soil but inevitably includes simplifi cations that can be a source of position of the nematode assemblage at S2 (with abundant cp-3 errors when very different nematode faunas are analyzed. Freck- plant parasites) as an outgroup in the dendrogram of Cluster Ana- man (1982) reports that more detailed mathematical formula to lysis based on metabolic footprints (Fig. 4) and variable position determine individual nematode weight gave values that differed of the nematode assemblage at S1 in PCA biplots (Figs 3 and 5). by 5 – 31 % from Andrassy’s (1956) short formula and she con- Metabolic footprints are recently being used in nematode ecology siders this difference as serious problem for determining biomass, studies (e.g., Renčo et al., 2015; Zhang et al., 2015), however, as respiration and production of nematodes. Another problem she every new method of measures of ecosystem services they should lists is the occurrence of periods of cryptobiosis when nematode be interpreted with caution.

61 Appendix 1. Check-list of nematode species at the sites studied.

Nematode species and sites V1 V2 V3 S1 S2 Monhysterida 1 Eumonhystera longicaudatula (Gerlach & Riemann, 1973) + + + - - 2 Eumonhystera vulgaris (de Man, 1880) - - - - + 3 Geomonhystera villosa (Bütschli, 1873) - - - - + 4 Monhystrella sp. + - - + + Araeolaimida 5 Bastiania uncinata Andrássy, 1991 + - - + + 6 Rhabdolaimus terrestris de Man, 1880 + +-+- 7 Plectus acuminatus Bastian, 1865 s.l. +++++ 8 Plectus amorphotelus Ebsary, 1985 - + + - + 9 Plectus communis Bütschli, 1873 +++++ 10 Plectus geophilus de Man, 1880 + + - - + 11 Plectus inquirendus Andrássy, 1958 - + - - - 12 Plectus longicaudatus Bütschli, 1873 +++++ N 13 Plectus magadani Kuzmin, 1979 + - + - - 14 Plectus parietinus Bastian, 1865 + + + - + 15 Plectus parvus Bastian, 1865 - - + + - N 16 Plectus rotundilabiatus Zell, 1993 - - - - + N 17 Plectus velox Bastian, 1865 - + + - - 18 Ceratoplectus armatus (Bütschli, 1873) - + - - - 19 Metateratocephalus crassidens (de Man, 1880) + + + - + Rhabditida 20 Teratocephalus dadayi Andrássy, 1968 + + - + + 21 Teratocephalus lirellus Anderson, 1969 - + - - - Q 22 Teratocephalus lirellus Anderson, 1969 / stratumus Eroshenko, 1973 + + - + + 23 Teratocephalus paratenuis Eroshenko, 1973 +++++ 24 Teratocephalus terrestris (Bütschli, 1873) + +-+- 25 Teratocephalus sp. + - - - - 26 Heterocephalobus elongatus (de Man, 1880) +++++ 27 Acrobeloides nanus (de Man, 1880) - ++++ 28 Acrobeloides tricornis (Thorne, 1925) ++++- 29 Panagrolaimus rigidus (Schneider, 1866) + + - - + 30 Rhabditis terricola Dujardin, 1845 +++++ 31 Protorhabditis ﬁ liformis (Bütschli, 1873) - - + - - 32 Bursilla monhystera (Bütschli, 1873) - + + - + 33 Bunonema reticulatum Richters, 1905 + + - - + 34 Steinernema sp. dauer larvae +++++ 35 Pristionchus lheritieri (Maupas, 1919) + - - - + Aphelenchida N 36 Aphelenchoides breviuteralis Eroshenko, 1968 + + - - + 37 Aphelenchoides conimucronatus Bessarabova, 1966 +++++ 38 Aphelenchoides curiolis Gritsenko, 1971 - - + + + N 39 Aphelenchoides editocaputis Shavrov, 1967 - - - - + 40 Aphelenchoides graminis Baranovskaya & Haque, 1968 - - - - + 41 Aphelenchoides lagenoferrus Baranovskaya, 1963 - + - - + 42 Aphelenchoides macronucleatus Baranovskaya, 1963 +++++ 43 Aphelenchoides parasubtenuis Shavrov, 1967 + +-+-

62 44 Aphelenchoides saprophilus Franklin, 1957 + + - + - 45 Aphelenchoides sp.1 - - - - + 46 Aphelenchoides sp.2 - - - - + Tylenchida 47 Aglenchus agricola (de Man, 1884) +++++ 48 Coslenchus costatus (de Man, 1921) - - - - + 49 Filenchus aquilonius (Wu, 1969) - + - - + 50 Filenchus discrepans (Andrássy, 1954) +++++ 51 Filenchus facultativus (Szczygieł, 1970) ++++- N 52 Filenchus hamatus (Thorne & Malek, 1968) - - - - + 53 Filenchus longicaudatulus Zell, 1988 - - - + - 54 Filenchus misellus (Andrássy, 1958) s.l. +++++ 55 Filenchus sp.1 + + - + - 56 Filenchus sp.2 - - - - + 57 Tylenchus davainei Bastian, 1865 - + - - - N 58 Tylenchus elegans de Man, 1876 + - - - - 59 Malenchus bryophilus (Steiner, 1914) - - - - + 60 Cephalenchus leptus Siddiqi, 1963 + + - + - 61 Lelenchus leptosoma (de Man, 1880) + + - + - 62 Ditylenchus dipsaci (Kühn, 1857) + + - + - 63 Ditylenchus ﬁ lenchulus Brzeski, 1991 +++++ N 64 Ditylenchus tenuidens Gritzenko, 1971 - - - - + 65 Ditylenchus terricola Brzeski, 1991 - + + - - N 66 Ditylenchus valveus Thorne & Malek, 1968 + - - - + 67 Ditylenchus sp. - - + + - 68 Nagelus leptus (Allen, 1955) - - - - + 69 Pratylenchus crenatus Loof, 1960 + - + + + 70 Helicotylenchus pseudorobustus (Steiner, 1914) - - + + + 71 Helicotylenchus varicaudatus Yuen, 1964 + + - + + 72 Rotylenchus robustus (de Man, 1876) apud Brzeski (1998) - - - + + Q 73 Paratylenchus (Paratylenchus) sp. cf. baldaccii Raski, 1975 - - - - + 74 Paratylenchus (Paratylenchus) microdorus Andrássy, 1959 +++++ 75 Paratylenchus (Paratylenchus) projectus Jenkins, 1956 - - - - + 76 Paratylenchus (Gracilacus) straeleni (De Coninck, 1931) - + - + - N 77 Sphaeronema alni Turkina & Chizhov, 1986 juv. - + - - - 78 Deladenus durus (Cobb, 1922) + + - - + 79 Tylenchida juv. - - - - + Enoplida 80 Prismatolaimus dolichurus de Man, 1880 +++++ 81 Prismatolaimus intermedius (Bütschli, 1873) s.l. + + - + - 82 Tripyla afﬁ nis de Man, 1880 +++++ Alaimida N 83 Alaimus arcuatus Thorne, 1939 + - - - - Diphtherophorida Q 84 Diphtherophora cf. kirjanovae Ivanova, 1958 + - - + + Mononchida 85 Clarkus papillatus (Bastian, 1965) - - - - + N 86 Coomansus menzeli Loof & Winiszewska-Ślipińska, 1993 + + + - + 87 Coomansus zschokkei (Menzel, 1913) - - - - + Dorylaimida

63 88 Paravulvus hartingii (de Man, 1880) juv. - + + - - 89 Allodorylaimus sp. - + - - + 90 Epidorylaimus agilis (de Man, 1880) +++++ 91 Epidorylaimus consobrinus (de Man, 1918) + - - + - Epidorylaimus humilior (Andrássy, 1959) / humilis (Thorne & Swanger, Q 92 +++++ 1936) N 93 Epidorylaimus leptosoma (Altherr, 1963) + + - + + 94 Eudorylaimus brevis (Altherr, 1952) + - + + - 95 Eudorylaimus discolaimioideus (Andrássy, 1958) +++++ Q 96 Eudorylaimus maritus Andrássy, 1959 / carteri (Bastian, 1965) - - - - + N 97 Eudorylaimus pseudobokori Zell, 1986 + + - - + N 98 Eudorylaimus stefanskii (Brzeski, 1960) - + + + - 99 Eudorylaimus sp.1 + - - - + 100 Eudorylaimus sp.2 -+-+- N 101 Aporcelaimellus alius Andrássy, 2002 - ++++ N 102 Aporcelaimellus amylovorus (Thorne & Swanger, 1936) - + - - - N 103 Aporcelaimellus medius Andrássy, 2002 - - - + - 104 Aporcelaimellus obtusicaudatus (Bastian, 1865) - + - - - Q 105 Metaporcelaimus cf. monohystera (Brzeski, 1964) +++++ N 106 Enchodelus lucinensis Popovici, 1978 - + + + - Enchodelus macrodorus (de Man, 1880) / carpaticus Ciobanu, Q 107 ---++ Popovici, Guerrero & Peña-Santiago, 2010 108 Enchodelus sp. (hopedorus (Thorne, 1929) group) + - - - - 109 Heterodorus sp. juv. - ++++ 110 Tylencholaimus mirabilis (Bütschli, 1873) - ++++

Total number of species 59 68 45 57 72 Total SR 6.87 8.62 6.10 7.40 8.98 Total number of genera 34 37 27 30 42 Total GR 3.91 4.63 3.60 3.83 5.19 N – species found in this study that are not listed in the book by Lišková & Čerevková (2011) and are probably recorded in the Slovak Republic for the ﬁ rst time. Q – species that were determined according to keys mentioned in the Material and Methods section but were partially different from the original descriptions or showed variability overlapping two species. Tylenchida juv. – damaged juveniles that could not be determined to a genus. Acknowledgements References

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