Do microclimatic conditions in two forest types on serpentine bedrock affect culturable microfungi in pine litter needles?

Abstract

Microfungi colonising coniferous needles in litter were intensively studied in previous decades, but forest stands on serpentine soils have been overlooked. Also, the effects of microclimatic conditions on fungal communities in coniferous litter are unknown. In our study, we aimed to characterise communities of culturable microfungi colonising pine litter needles collected from two types of Scots pine forest growing on serpentine bedrock, i.e. dense forest with relatively stable microclimatic conditions and open-canopy forest on exposed rock with highly variable conditions. The composition of their fungal communities was analysed in respect to microclimatic conditions at the collection sites.

Using a combination of phenotypic and molecular data sequences of ITS rDNA), 35 taxa were distinguished in 1078 fungal colonies recorded, out of which 25 were identified to the species level.
Fungal communities were most affected by needle type (litter vs. fermentation layer) followed by maximum temperature during the previous five months. Interestingly, a higher number and abundance of species were recorded at the warmer site, in the open-canopy forest. Dominant fungi recorded in this study (Desmazierella acicola, Phacidium lacerum and Scleroconidioma sphagnicola) were mostly identical to those recorded in previous studies and the occurrence of less abundant taxa previously not recorded in pine litter suggests that the uppermost litter layer represents an important reservoir of fungal diversity.

Full text

Do microclimatic conditions in two forest types
on serpentine bedrock affect culturable microfungi
in pine litter needles?
ONDŘEJ KOUKOL1*, ELENA MAGDALINOU1,HANA PÁNKOVÁ2,JAN BOROVIČKA3,
ZUZANA MÜNZBERGOVÁ1,2
1Department of Botany, Faculty of Science, Charles University, Benátská 2, CZ-128 00 Praha 2,
Czech Republic
2Institute of Botany of the Czech Academy of Sciences, Zámek 1, CZ-252 43 Průhonice,
Czech Republic
3Institute of Geology of the Czech Academy of Sciences, Rozvojová 269, CZ-165 00 Praha 6,
Czech Republic
*corresponding author: ondrej.koukol@natur.cuni.cz
Koukol O., Magdalinou E., Pánková H., Borovička J., Münzbergová Z. (2022):
Do microclimatic conditions in two forest types on serpentine bedrock affect
culturable microfungi in pine litter needles? – Czech Mycol. 74(2): 181–194.
Microfungi colonising coniferous needles in litter were intensively studied in previous decades,
but forest stands on serpentine soils have been overlooked. Also, the effects of microclimatic condi-
tions on fungal communities in coniferous litter are unknown. In our study, weaimed to characterise
communities of culturable microfungi colonising pine litter needles collected from two types of
Scots pine forest growing on serpentine bedrock, i.e. dense forest with relatively stable micro-
climatic conditions and open-canopy forest on exposed rock with highly variable conditions. The
composition of their fungal communities was analysed in respect to microclimatic conditions at the
collection sites.
Using a combination of phenotypic and molecular data (sequences of ITS rDNA), 35 taxa were
distinguished in 1078 fungal colonies recorded, out of which 25 were identified to the species level.
Fungal communities were most affected by needle type (litter vs. fermentation layer) followed by
maximum temperature during the previous five months. Interestingly, a higher number and abun-
dance of species were recorded at the warmer site, in the open-canopy forest. Dominant fungi re-
corded in this study (Desmazierella acicola,Phacidium lacerum and Scleroconidioma sphagni-
cola) were mostly identical to those recorded in previous studies and the occurrence of less abun-
dant taxa previously not recorded in pine litter suggests that the uppermost litter layer represents an
important reservoir of fungal diversity.
Key words: fungal diversity, ITS rDNA, temperature and humidity, Pinus sylvestris.
Article history: received 5 July 2022, revised 1 November 2022, accepted 4 November 2022, pub-
lished online 24 November 2022.
DOI: https://doi.org/10.33585/cmy.74205
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Koukol O., Magdalinou E., Pánková H., Borovička J., Münzbergová Z. (2022):
Mají mikroklimatické podmínky ve dvou typech lesa na hadcovém podloží vliv
na kultivovatelné mikroskopické houby v jehlicích borovic v opadu? – Czech Mycol.
74(2): 181–194.
Mikroskopické houby kolonizující jehlice v opadu byly intenzivně studovány v předchozí dekádě,
avšak stanoviště na hadcových půdách byla opomíjena. Také vliv mikroklimatických podmínek na hou-
bová společenstva v jehličnatém opadu není znám. V naší studii jsme se zaměřili na popis společenstev
kultivovatelných mikroskopických hub kolonizujících jehlice borovic v opadu ve dvou typech lesa tvo-
řeného borovicí lesní, rostoucích na hadcovém podloží – hustý les s relativně stabilními mikroklimatic-
kými podmínkami a otevřený les na exponované skále s dynamicky se měnícími podmínkami. Složení
společenstev bylo analyzováno ve vztahu k mikroklimatickým podmínkám na daném stanovišti.
Za použití kombinace fenotypových a molekulárních dat (sekvence oblasti ITS rDNA) bylo rozliše-
no 35 taxonů mezi 1078 získanými izoláty, z nichž 25 bylo určeno do druhu. Společenstva hub byla nej-
více ovlivněna typem jehlic (z hrabanky vs. z fermentační vrstvy), následně maximální teplotou během
předchozích pěti měsíců. Zajímavé je, že větší množství druhů a větší abundance jednotlivých druhů
byla zaznamenána na teplejší lokalitě, v otevřeném typu lesa. Dominantní druhy nalezené v této studii
(Desmazierella acicola,Phacidium lacerum aScleroconidioma sphagnicola) byly většinou shodné
jako v předchozích studiích; výskyt několika méně hojných druhů, které dosud nebyly zaznamenány
v jehličnatém opadu, naznačuje, že hrabanka představuje důležitý zdroj diverzity hub.
INTRODUCTION
Coniferous needles in litter harbour rich fungal communities. A high diversity
of microfungi in pine needles has been documented since the first systematic sur-
veys aimed at culturable fungi (Mitchell et al. 1978, Tokumasu et al. 1994). Al-
though recent studies using a metagenomic approach have mostly ignored fungal
diversity in the uppermost soil horizon (Buée et al. 2009, Buscardo et al. 2014),
Santalahti et al. (2016) observed the highest fungal richness and also the highest
number of saprotrophic species in the uppermost soil layer as compared to
deeper soil horizons. Fungal communities in the litter are influenced by numer-
ous factors including chemical composition of the needles and local climatic con-
ditions. Seasonal patterns were already observed in the past, with some fungi
changing frequency and others disappearing for a particular season (Tokumasu
et al. 1994, Hirose et Osono 2006), but recent surveys indicate a stable commu-
nity in the uppermost soil horizon across different seasons (Santalahti et al.
2016). These large-scale surveys may however not reveal subtle changes on a fine
scale, as different fungal communities may exist at sites with contrasting micro-
climatic conditions due to e.g. different openness or exposure of the stands.
In our study, we primarily aimed to characterise fungal communities colonising
pine litter needles in Scots pine (Pinus sylvestris) forest on two types of sites with
contrasting microclimatic conditions (temperature and humidity), i.e. sites cov-
ered by dense pine forest with relatively stable microclimatic conditions versus
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sites with sparse pine growth on exposed rock and highly variable conditions.
The composition of the fungal communities was analysed with respect to both
microclimatic conditions and vegetation cover in the moss layer (i.e. mosses and
lichens). All sampling sites were located in forests on serpentine bedrock. Ser-
pentine soils are stressful environments for plants due to multiple limitations
posed by their physical characteristics and chemical composition. The low soil
water-holding capacity and low Ca/Mg ratio combined with relatively high con-
centrations of toxic metals such as Ni may pose a considerable challenge to local
plant species and result in a high proportion of endemic species with adaptive
morphologies and a distinctive structure of serpentine plant communities (Brady
et al. 2005, Pánková et Münzbergová 2011). Our secondary aim was to reveal if lit-
ter needles on serpentinite harbour a distinct fungal community.
MATERIAL AND METHODS
L o c a l i t i e s a n d c o l l e c t i o n. Litter needles were sampled in two forest types in Hadce
u Želivky National Nature Monument (Central Bohemia, Czech Republic) on 13 March 2018. The first
one is formed by dense Boreo-continental pine forest (Dicrano-Pinion sylvestris alliance) with a high
occurrence of self-seeded trees such as Frangula alnus,Picea abies and Betula pendula growing on
the top plateau and gentle slopes. The soil is generally shallow, but deeper than in the other habitat,
with a thicker humus layer. The vegetation cover is dense with a high abundance of grasses. The other
type includes open Peri-Alpidic serpentine pine forest (Erico carneae-Pinion alliance) located on ex-
posed rock platforms with shallow soil and sparse vegetation (Asplenion cuneifolii and Festucion
valesiacae alliances) but rich lichen cover (mostly Cladonia spp.). Vegetation cover in the moss layer
(E0) was evaluated for all localities and summary data for the coverage of mosses and lichens is pre-
sented in Tab. 1. Microclimatic conditions at the localities were determined with a TMS datalogger
(Wild et al. 2019) continuously for the period of five months (17 October 2017 to 18 March 2018) prior to
sample collection. Humidity and temperature on the surface were recorded every 15 min.
Tab . 1. Geographic location and characteristics of microclimatic conditions and moss and lichen
covers at three localities in the Boreo-continental forest and in the Peri-Alpidic forest type.
Mean humidity is expressed in relative values; T-min., T-mean and T-max. reflect minimum, mean and
maximum surface temperature (in °C) in the previous five months. Lichen and moss coverages are
expressedin%.
Locality Coordinates Humidity T-min. T-mean T-max. Lichen
coverage
Moss
coverage
Boreo-continental
Loc. 1 49°41'18.8" N, 15°05'57.1" E 1590.5 –14.4 1.8 19.6 0.0 88.0
Loc. 2 49°41'19.2" N, 15°06'11.9" E 1649.4 –13.9 3.3 23.8 0.0 18.0
Loc. 3 49°41'01.9" N, 15°07'53.3" E 1801.0 –13.3 2.3 19.0 0.0 68.0
Peri-Alpidic
Loc. 1 49°41'13.5" N, 15°06'09.3" E 1321.9 –11.8 2.7 17.1 0.0 38.0
Loc. 2 49°41'25.7" N, 15°06'17.8" E 1708.1 –14.1 3.2 25.2 3.0 8.0
Loc. 3 49°41'00.8" N, 15°07'59.2" E 1922.6 –11.3 3.1 29.2 3.0 2.0
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For each forest type, three localities were selected, each with three sampling sites, where pine
needles were collected from the organic soil horizon. Two soil horizons (layers) were distinguished:
the uppermost layer consisting of brown to yellow-brown needles still retaining their integrity (litter
needles in strict sense, Oi) and the layer beneath composed of black, partially fragmented and soft-
ened needles (fermentation layer, Oe). Needles were sampled into sterile plastic bags and processed
in the laboratory within 48 h. In the laboratory, 25 randomly selected needle pairs were cut with scis-
sors, and 2 cm long fragments from the middle part of each needle were surface-sterilised in 30% hy-
drogen peroxide for 90 s on a horizontal shaker (Koukol 2011). Fragments were placed in a Petri dish
(10 fragments per dish) with malt agar prepared from brewers wort with a final sucrose content ad-
justed to 2% w/v and with 18 g·l–1 agar (2MA) (Fassatiová 1986). Petri dishes were cultivated at room
temperature (23–25 °C). Fifty fragments were cultivated for each sampling site and horizon, render-
ing altogether 1800 fragments cultivated.
When possible, outgrowing fungi were directly identified based on their morphology. If sterile
colonies were formed, they were grouped into morphotypes according to phenotypic characteristics
(e.g. growth rate, colour, colony profile and margin). A representative strain from each morphotype
was transferred onto a new plate with 2MA. Sterile cultures were kept for 6 months at lowered tem-
perature (18 °C) combined with near-ultraviolet light (UVA) and on nutrient-poor medium potato car-
rot agar (Fassatiová 1986) to promote sporulation.
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Fig. 1. Temperature fluctuations during the five-month period prior to the collection of litter needles.
The red line indicates records from the Boreo-Continental sites, the blue line those from the Peri-
Alpidic sites (means of three measurements).

F u n g a l id e n t i f i c a t i o n. In order to identify sterile as well as sporulating strains of problem-
atic genera (e.g. Trichoderma), genomic DNA was isolated from 7–14 d old cultures using a Zymo
Research Fungal/Bacterial kit (Zymo Research, Orange, USA). Nuclear rDNA containing internal
transcribed spacers (ITS1 and ITS2), 5.8S and D1/D2 domains of the 28S region were amplified with
primer sets ITS1/NL4 or ITS1/ITS4 (White et al. 1990, O’Donnell 1993). PCR products were purified
using a GenElute PCR Clean-Up Kit (Sigma-Aldrich, St. Louis, USA). For identification of some
morphotypes in which the ITS region was not sufficient, a fragment of the genes encoding the elonga-
tion factor 1a(EF1-a) was amplified using primer sets 983F/2218R (O’Donnell et Cigelnik 1997). The
same primers were used for sequencing at the Sequencing Laboratory of the OMICS Core Facility,
BIOCEV (Vestec, Czech Republic). Sequences were compared for homology in GenBank using the
BLAST algorithm (Altschul et al. 1997). Only 99–100% matches with reliable sources (ex-type se-
quences, taxonomic studies) were taken as identification evidence. Fungal species names were
checked with Index Fungorum (http://www.indexfungorum.org/names/Names.asp).
C h e m i c a l a n a l y s e s. For chemical characterisation of the substrates, both needles from lit-
ter (Oi) and fermentation (Oe) soil horizons were collected from each sampling site. Specimens were
pooled together for each locality, air-dried, milled to fine powder followed by extraction in 1M HCl
according to Borovička et al. (2019). Concentrations of extractable macronutrients (Ca, Mg, K, P)
and metals (Ni, Cr) were determined in the laboratory of the Institute of Geology of the Czech Acad-
emy of Sciences using Element 2 high resolution inductively coupled plasma mass spectrometry
(ICP-MS).Control soil samples collected from four pine forests on sandstone, calcareous sandstone,
or schist in Central Bohemia, Czech Republic were processed for comparison.
S t a t i s t i c a l a n a l y s e s. Abundances of each morphotype were calculated as the number of
needle fragments with (at least one) colony of a particular morphotype. Morphotypes recorded in
a single colony were excluded from the analyses of species composition (see below). Abundances
were joined for morphotypes later shown to represent identical species based on morphological or
molecular data.
First, factors determining numbers of fungal species and their abundances in the samples were
explored using generalised linear mixed effect models with sampling site and locality as random fac-
tors. Both variables were modelled with a Poisson distribution and log-link function, as they are both
non-negative small integer numbers. The predictors were needle type (litter or fermentation layer),
temperature and humidity in the soil (minimum, maximum and mean values for the previous five
months) and vegetation cover on the sampling sites. Chemical composition of the soil was not used
in the tests, as most variation in soil chemistry was explained by needle type and so soil chemistry
would not add any new information to the tests. In addition, the sample size in this study was too low
to test the effects of many predictors. Because we had too many predictors for a limited number of
samples even when excluding litter chemistry, the effect of needle type, which was expected to play
a dominant role, was tested first. Subsequently, forward step-wise selection of the other predictors
was performed with needle type as a covariate in case it was significant. The mixed effect models
were run in the package lme4 for R (Bates et al. 2015).
The effect of the same predictors (i.e. needle type, soil temperature and humidity, and vegetation
cover) on the composition of the fungal communities was tested using Redundancy analysis (RDA)
in the vegan package for R (Oksanen et al. 2019). Again, the effect of needle type was tested first. Sub-
sequently, forward step-wise selection of the other predictors with needle type as a covariate was
used in case it was significant. Sampling site was used to define the structure of the data. Significance
of the effects was assessed using permutation tests (999 permutations) permuting only within the
sampling site (for litter type) or only between the sampling sites (for the other predictors).
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Tab . 2. Fungal taxa isolated from pine needles in litter, their putative identification and abundance
(number of colonised needle fragments) at the three studied localities for each forest type.
Taxon Boreo-continental Peri-Alpidic
Loc. 1 Loc. 2 Loc. 3 Loc. 1 Loc. 2 Loc. 3
Desmazierella acicola Lib. 36 41 16 50 55 29
Phacidium lacerum Fr. 363935352228
Scleroconidioma sphagnicola Tsuneda, Currah et Thormann 4 21 4 25 66 8
Trichoderma polysporum (Link) Rifai 14 3 15 18 3 25
Gymnopus androsaceus (L.) J.L. Mata et R.H. Petersen 5 16 15 9 12 7
Cladosporium cladosporioides (Fresen.) G.A. de Vries 14 9 13 3 10 8
Trichoderma koningii Oudem. 7151087
Alternaria alternata (Fr.)Keissl. 5414267
Truncatella conorum-piceae (Tubeuf)Steyaert 916631
Herpotrichia pinetorum (Fuckel)G.Winter 0060414
Angustimassarina sp. 039121
Phoma herbarum Westend. 351511
Helotiales sp.020270
Corticiales sp. 105210
Epicoccum nigrum Link 312003
Mycena sp. 000801
Plectania melastoma (Sowerby)Fuckel 300412
Pseudocamaropycnis pini Crous 011007
Fusarium sp.1 200041
Sistotrema efibulatum (J.Erikss.)Hjortstam 001060
Hormonema dematioides Lagerb.etMelin 110310
Ceratobasidium sp.1 030020
Cladosporium herbarum (Pers.)Link 101021
Lophodermium pinastri (Schrad.)Chevall. 100111
Erythricium hypnophilum (P.Karst.)J.Erikss.etHjortstam300000
Knufia tsunedae Madrid,GuarroetCrous 000003
Pleosporales sp. 003000
Anthostomella formosa Kirchst. 001000
Beauveria pseudobassiana S.A.RehneretR.A.Humber 000001
Ceratobasidium sp.2 000010
Dothideomycetes sp. 001000
Fusarium sp.2 010000
Mycena epipterygia (Scop.)Gray 010000
Roridomyces roridus (Fr.)Rexer 001000
Trametes versicolor (L.)Lloyd 000020
Total number of species 18 18 21 16 23 21
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RESULTS
The isolations from pine litter needles yielded a total of 1078 colonies growing
from 56% of needles (790 needle fragments remaining sterile). Colonies were as-
signed to 98 morphotypes, which were later reduced to 35 taxa after comparing
phenotypic and molecular data. Twenty-five taxa were identified to species, while
the remaining ten morphotypes did not match in their rDNA sequences with
those present in GenBank and were assigned a higher taxonomic rank (Tab. 2).
The communities of culturable fungi were dominated by ascomycetes (25 spe-
cies), while basidiomycetes were represented by 10 species. The three most
abundant species were Desmazierella acicola (Pezizales, recorded as the Verti-
cicladium stage, overall abundance 227), Phacidium lacerum (Helotiales,re
-
corded as the Ceuthospora stage, overall abundance 195) and Scleroconidioma
sphagnicola (Dothideomycetes inc. sed., overall abundance 128).
When compared to the control sites, both Oi and Oe soil horizons from the
serpentine bedrock were considerably enriched with Mg and Ni, and their Ca/Mg
ratios were remarkably lower than those at the control sites (Tab. 3). On average,
concentrations of Mg, Ni and Cr in the Oe horizon were approx. 2×, 5× and 5×
higher, respectively, than those in the Oi horizon.
Tab . 3. Chemical characteristics of the uppermost litter (Oi) and fermentation (Oe) soil horizons col-
lected at serpentinite and control sites. The values represent extractable element fractions (mg·kg-1)
expressed as mean ± standard deviation out of six serpentinite and four control samples.
Element Serpentinite Control
Oi Oe Oi Oe
Ca 7847 ± 605 8105 ± 868 7461 ± 781 5089 ± 584
Mg 2086 ± 563 3703 ± 2004 748 ± 61.0 512 ± 162
Ca/Mg 3.96 ± 0.80 3.18 ± 2.01 10.0 ± 1.25 10.8 ± 2.63
K 685 ± 74.1 867 ± 44.1 1697 ± 668 839 ± 95.7
P 277 ± 38.6 416 ± 65.3 605 ± 106 515 ± 48.8
Ni 13.6 ± 4.84 67.8 ± 41.2 0.99 ± 0.18 9.20 ± 3.37
Cr 1.25 ± 0.79 6.55 ± 4.93 1.09 ± 0.18 17.4 ± 9.40
Both the number and abundances of species were significantly affected by
soil horizon (F = 30.155, p < 0.001 and F = 44.39, p < 0.001, respectively), with
higher values in the uppermost litter (Oi) than in the fermentation layer (Oe).
After accounting for this effect, we detected a significant effect of maximum
surface temperature in the previous five months (Fig. 1), with a slightly higher
number of species at sites in the Peri-Alpidic forest type (Tab. 2).
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Similarly to species number and abundance, also species composition signifi-
cantly differed between the two layers, explaining 10.5% of variation in the data.
In a subsequent step-wise selection using layer as a covariate, maximum surface
temperature in the previous five months was detected as the only significant pre-
dictor explaining an additional 4.1% of variation (Fig. 2).
DISCUSSION
In our study we primarily focused on how communities of culturable micro-
fungi in pine needles in the litter depend on local microclimatic conditions. There-
fore, two contrasting Scots pine forest types (open and dense pine forest) differ-
ing in vegetation cover, but situated close to each other were selected and de-
tailed characteristics of the conditions on the surface were determined. To our
knowledge, this was the first study in which fungal communities in litter were
compared based on detailed microclimatic measurements rather than mean an-
nual rainfall (Prihatini et al. 2015) or seasonal differences (Santalahti et al. 2016).
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Fig. 2. Affinity of fungal species to warm conditions at the locality expressed as their scores along
the first RDA axis. Only 13 species with the highest scores (threshold 0.04) are shown.

Factors affecting fungal communities
Out of all the factors tested, we only identified soil horizon (litter and fermen-
tation layer) to be the most important determinant of fungal diversity and abun-
dance observed. Although this difference could be partly attributed to the sterili-
sation technique used (higher penetration of hydrogen peroxide into the more
fragile needles in the Oe layer), a similar pattern was also observed in pyro-
sequence data by Santalahti et al. (2016) and Solly et al. (2017), who recorded
a distinct community in the Oi layer differing from deeper soil horizons. Also, the
higher abundance of fast-growing Trichoderma species observed on the Oi nee-
dles (data not shown) might have prevented other fungi from growing.
After the effect of soil layer had been filtered, fungal communities were af-
fected by the maximum temperature in the previous five months. Thus resistance
to high temperatures and potentially also to desiccation rate may be expected in
fungi recorded at sites in the Peri-Alpidic forest. This is particularly true for
Scleroconidioma sphagnicola, a species with a strongly melanised mycelium
able to withstand adverse abiotic conditions (Koukol et Kovářová 2007), which
had the highest score along the first axis in the RDA analysis (Fig. 2). Similarly,
Solly et al. (2017) observed that soil warming at the alpine treeline led to an in-
crease in abundance of several litter-associated ascomycetes. Effects of season
had already been documented, e.g. Zamora et al. (2008) observed a high diver-
gence of fungal communities isolated in spring vs. autumn from pine needles in
plantations in Northern Spain.
Fungi in serpentine soils
Our study pioneered the characterisation of the diversity of culturable sapro-
trophic microfungi from pine litter on serpentine soils. Previous studies were
only aimed at plant-symbiotic fungi and soil fungi, but indicated that mycobiota
may be less affected by nutrient limitation or excess of metal cations as com-
pared to the strong impact which serpentinite has on plant communities. Muller
et Hilger (2015) recorded a similar richness of fungal symbionts in the roots of
the perennial herb Onosma echioides (Boraginaceae) at serpentine and non-ser-
pentine sites, but their structure differed. The fungal community at non-serpen-
tine sites was dominated by arbuscular mycorrhizal members of Glomero-
mycota. Their occurrence on serpentine site was lower and basidiomycetous
species (potentially opportunistic symbionts) prevailed. Waseem et al. (2017)
found a high diversity of ectomycorrhizal fungi associated with different en-
demic species of Trist an iopsis (Myrtaceae) growing on both ultramafic and sed-
imentary sites. Rosenstock et al. (2016) observed a higher biomass of ectomyco-
rrhizal fungi in mesh bags placed on serpentinite compared to that on granite and
amphibolite. Differences in ectomycorrhizal fungal communities associated with
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these three parent materials were attributed to different pH levels. Another pos-
sible indirect effect is that vegetation cover seems to affect microbial communi-
ties in serpentine soils rather than soil properties. Bordez et al. (2016) found an
unexpectedly high richness of fungal species in ultramafic soils with different
successional stages of aboveground vegetation, which contrasts to the present
study, where vegetation cover did not affect the species composition of the fungal
communities. Cecchi et al. (2019) observed a lower fungal richness in soil micro-
fungi isolated from serpentinite-rich soil, but the most abundant microfungi were
not affected by geochemical factors of the soil. The addition of serpentinite may
even promote fungal diversity. For example, Małek et al. (2021) observed that out
of several treatments, soil in a serpentinite-fertilised temperate spruce forest har-
boured the second highest number of operational taxonomic units.
In our present study, we did not include collections of litter needles for fungal
isolation from non-serpentine soils as a negative control. However, we may com-
pare the results with those obtained by Koukol (2011), who collected needles of
Scots pine from a forest on sandstone rock in the same season (March) and sur-
veyed fungal communities using the same approach. Koukol (2011) isolated fungi
from 320 needles collected from the Oi layer only and obtained 35 species in 340
colonies. In the present study, 32 species were identified in 678 colonies growing
from 900 needles from the Oi layer. When fungal abundances in the different for-
est types are compared separately, needles from the Peri-Alpidic sites yielded
0.81 colony per needle, whilst those from the Boreo-continental sites only 0.69
colony compared to 1.06 colony per needle collected on sandstone rock (Koukol
2011). The diversity and abundance of fungi at the serpentine sites were thus
slightly lower, but the most abundant species were almost identical in both
studies. The only exception is the anamorphic ascomycete Sympodiella acicola
(Venturiales), which was absent in the present study, although it was the second
most frequently isolated species on sandy soils. Whether local absence rather
than the sensitivity of S. acicola to the specific conditions of the serpentine soil
plays a role here is not clear. In concordance to previous studies, fungi should be
able to balance the negative effect of serpentinite due to their phenotypic plastic-
ity (Waseem et al. 2017), and particularstrains, rather than species, may show dif-
ferent sensitivity and tolerance to the specific chemical composition of serpen-
tine soils similarly to the tolerance of heavy metals at polluted sites (Colpaert et
al. 2011). Although the concentrations of Ni, Cr, and the Ca/Mg ratio were even
more unfavourable in the Oe layer (Tab. 3), we assume that the statistically signif-
icant lower fungal diversity and abundances in this layer were rather related to
the effect of the sterilisation procedure, as already mentioned.
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Fungal identification
We identified our isolates primarily based on morphology. Only sterile isolates
were identified based on molecular data, mostly the sequence of the ITS region.
Even for the sequenced strains, this marker may not be sufficient to reveal ge-
netic divergence. In a study by Jourand et al. (2010), nickel-tolerant and nickel-
sensitive phenotypes of the ectomycorrhizal species Pisolithus albus isolated
from ultramafic and volcano-sediment soils, respectively, clustered in a single lin-
eage based on ITS. However, amplified fragment length polymorphism indicated
that they represent two distinct populations. Therefore, we may not exclude that
some of the species isolated in our study represent cryptic species or ecotypes
adapted to the specific chemical composition.
Our approach of identifying sterile strains based on molecular data has never-
theless produced interesting results including unusual fungal species isolated
from pine litter and species with unclear species delimitation. As an example, in
the Boreo-continental forest, we recorded Pseudocamaropycnis pini,which
was described only recently from pine needles in Hong Kong by Crous et
Groenewald (2016). At five out of six sites, we isolated slow-growing grey sterile
colonies, the ITS sequence obtained from a representative one of which showed
100% identity with sequences originating from types of five species of the recently
described genus Angustimassarina (Thambugala et al. 2015). They differed
from three other species by only a single insertion. The sequence of EF1-awas
100% identical with A. acerina, but differed in just a single mutation from two
other species described by Hyde et al. (2017). Giving that all Angustimassarina
species were described as lignicolous saprotrophs from twigs of several broad-
leaved trees, our record from pine needles in litter expands the niches known for
this genus. It also points to the difficult identification and unclear taxonomic
boundaries between species described based on single isolates, and calls for
a critical revision of this genus. Neither the identity of a strain originating from
both forest types and identified in our study as Truncatella conorum-piceae was
straightforward. This saprotrophic species described from spruce cones is fre-
quently found as a pine twig endophyte and may be also pathogenic to pine and
spruce needles and cones (Blumenstein et al. 2021). The morphology of its
conidia and identity of the ITS sequence to sequence MT790329 originated from
a study by Blumenstein et al. (2021) supported the identification. Rather surpris-
ingly, it was also almost 100% identical (496/498 bp) with sequence NR_154504
originating from the type of Truncatella spartii, a species described from
abranchofSpartium junceum (Senanayake et al. 2015). This species was later
combined into the new genus Heterotruncatella by Liu et al. (2019), who men-
tioned numerous isolates obtained from pines. Considering that T. conorum-
piceae was not represented by any ITS sequence in GenBank before 2018, these
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two species seem to be conspecific. The older epithet was overlooked by exces-
sive emphasis placed on molecular data in description of T. spartii (Koukol et
Delgado 2021).
CONCLUSIONS
To conclude, we determined soil horizon to be the main factor affecting the
species composition of microfungi in pine litter. However, this factor combines
the effect of the sterilisation method and potentially also the chemical composi-
tion and is thus difficult to interpret. Interestingly, seemingly less favourable con-
ditions (high temperature) at the locality may actually support a higher diversity
and abundance of fungi.
ACKNOWLEDGEMENTS
The project was supported by the Ministry of Education, Youth and Sports of the Czech Republic
and by the Nature Conservation Agency of the Czech Republic, Project PPK-435a/25/18. Institutional
support to the institutes of the Czech Academy of Sciences was provided by Long-term Development
Projects RVO67985831, RVO61389005 and RVO67985939.
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