Fungi
Jou nal o
A icle
Lack o Linkages among F ui ing Dep h, Weigh , and Ma u i y
in I iga ed T u le Fungi Ma ks he Complexi y o
Rela ionships among Mo phogene ic S ages
Se gi Ga cia-Ba eda 1,2,* , Se gio Sánchez 1, Ped o Ma co 1, Gian Ma ia NiccolòBenucci 3
and Vicen e González 4


Ci a ion: Ga cia-Ba eda, S.; Sánchez,
S.; Ma co, P.; Benucci, G.M.N.;
González, V. Lack o Linkages among
F ui ing Dep h, Weigh , and Ma u i y
in I iga ed T u le Fungi Ma ks he
Complexi y o Rela ionships among
Mo phogene ic S ages. J. Fungi 2021,
7, 102. h ps://doi.o g/10.3390/
jo 7020102
Academic Edi o s: Ra aella
Ma ia Bales ini and
Pie e-Emmanuel Cou y
Recei ed: 18 Janua y 2021
Accep ed: 28 Janua y 2021
Published: 1 Feb ua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Unidad de Recu sos Fo es ales, Cen o de In es igación y Tecnología Ag oalimen a ia de A agón (CITA),
Ins i u o Ag oalimen a io de A agón—IA2 (CITA-Uni e sidad de Za agoza), A da. Mon añana 930,
50059 Za agoza, Spain; [email p o ec ed] (S.S.); [email p o ec ed] (P.M.)
2Cen o de In es igación y Expe imen ación en T u icul u a de la Dipu ación de Huesca (CIET),
Polígono Faba do s/n, 22430 G aus, Spain
3Depa men o Plan s, Soil and Mic obial Sciences, Michigan S a e Uni e si y, Eas Lansing, MI 48824, USA;
[email p o ec ed]
4Unidad de P o ección Vege al, Cen o de In es igación y Tecnología Ag oalimen a ia de A agón (CITA),
Ins i u o Ag oalimen a io de A agón—IA2 (CITA-Uni e sidad de Za agoza), A da. Mon añana 930,
50059 Za agoza, Spain; [email p o ec ed]
*Co espondence: sga [email p o ec ed]
Abs ac :
The highly p ized black u le (Tube melanospo um) has become a model species o
ec omyco hizal ungi biology. Howe e , se e al ques ions conce ning i s ep oduc i e phase emain
unanswe ed. To p o ide new hypo heses on he ui body o ma ion p ocess, we ha e explo ed
he causal links among de elopmen cha ac e s o black u le ui bodies ha a e p ima ily linked
o ei he he ma ing p ocess, ui body g owing s age, o ma u a ion. Pa h analysis was applied
o es causal models ou lining he ela ionships among ui body de elopmen cha ac e s such
as ui ing dep h, weigh , shape, and spo e ma u i y. These cha ac e s we e in es iga ed o e a
wo-season su ey and h ee soil ypologies (plus pea -based subs a e) unde i iga ed condi ions.
We ound a clea and gene alized ela ionship be ween ui body weigh and shape. Among clus e s
o ui bodies we ound a posi i e ela ionship be ween he weigh o he la ges ui body and
he weigh o he emaining ui bodies. Howe e , no gene alized ela ionships among cha ac e s
linked o di e en de elopmen s ages appea ed. Ou esul s we e no iceably consis en ac oss soil
ypologies, bo h o ui bodies g owing singly and in clus e s, indica ing ha ea ly-de eloping
ui body cha ac e s did no in luence cha ac e s linked o subsequen mo phogene ic s ages. The lack
o links among s ages opens new pe spec i es o p e-ha es quali y managemen wi h s age-speci ic
cul i a ion p ac ices.
Keywo ds:
Tube melanospo um; hypogeous ui bodies; ui body o ma ion; mo phological ai s;
pa h analysis; u le cul i a ion
1. In oduc ion
The Eu opean black u le (Tube melanospo um Vi ad., Pezizales) is an ec omyco -
hizal ungus ha in na u e mos ly g ows in associa ion wi h Angiospe m plan s (e.g.,
Fagaceae). In cul i a ed o cha ds, he mos common hos s a e Que cus species. T u le
cul i a ion has ad anced g ea ly in ecen yea s, al hough i is no comple ely domes ica ed
ye , and many biological and ecological aspec s o he se e al p ocesses in ol ed s ill need
cla i ica ion [
1
,
2
]. Black u le has also a ac ed a en ion as a model ec omyco hizal
ascomyce ous species o genomic s udies, esea ch on he ma ing p ocess and popula ion
gene ic s uc u e, on ui body (FB) nu i ion o on a oma [
2
–
5
]. Black u le ui ing is a
mul igene-media ed p ocess ha ollows speci ic and o ganized di e en ia ion pa e ns
J. Fungi 2021,7, 102. h ps://doi.o g/10.3390/jo 7020102 h ps://www.mdpi.com/jou nal/jo
J. Fungi 2021,7, 102 2 o 16
and equi es se e al mon hs o each comple ion [
3
,
6
,
7
]. The sequen ial mo phogene ic
s ages leading o he FB o ma ion can be classi ied in o ma ing p ocess, FB g owing s age,
and ma u a ion [
1
,
6
–
8
]. Howe e , e y li le is known abou he in insic o en i onmen al
signaling pa hways egula ing u le FB mo phogenesis [1,7,9,10].
The ma ing p ocess ( om he s imula ion o he o ma ion o he ma ing s uc u es o
he ma ing i sel ) ypically happens h oughou la e sp ing, appa en ly in se e al lushes [
1
]
(Table 1). The p ecise loca ion along he mycelial ne wo k whe e he ma ing e en be ween
mycelia o opposi e ma ing ypes happens will de e mine he soil dep h o he ull-g own
FB [
2
,
11
]. A e he ma ing e en , he FB s a s o de elop and i s s uc u e becomes
g adually complex as he weigh apidly inc eases [
7
,
8
]. G owing below g ound, he FB
swelling and i s inal shape will be in luenced by he soil mechanical cons ain s, wi h
a ele an ole o he cha ac e is ic py amidal wa s o he pe idium [
6
]. A he end o
he in ense g ow h s age, he FB has p ac ically achie ed i s inal size. I is hen, ha he
ma u a ion s age begins, wi h he spo es acqui ing hei cha ac e is ic pigmen a ion and
he FB de eloping i s unique a oma [
7
,
12
,
13
]. Ma u a ion begins in la e au umn, and he
subsequen senescence p ocesses se he momen in which dogs can localize he ipe FB.
The FBs a e ha es ed du ing se e al mon hs h oughou he win e , e idencing ha he
ola ile compounds ha a ac dogs a e no o med simul aneously in all FBs [
5
] (Table 1).
Each one o he spo s localized by a dog is exca a ed by he ha es e : in mos o he digs
only one FB appea s (single FBs), whe eas in o he s, a clus e o FBs g ow in e y close
p oximi y. Li le scien i ic a en ion is usually paid o u le FBs g owing in clus e s [
14
,
15
],
al hough g owing wi hin hese clus e s could ei he a ec FB o ma ion pa e ns o could
ade o wi h size due o localized esou ce deple ion o inhibi ion mechanisms.
Table 1.
Rela ions be ween mo phogene ic s ages and ui body de elopmen cha ac e s (based on
Za i i e al. [7]).
S age o
F ui body Mo phogenesis Associa ed De elopmen Cha ac e s Pe iod
Fo ma ion o ma ing
s uc u es (game es),
ma ing, and ea ly s age o
ui body di e en ia ion
(hyphal s age)
F ui ing dep h May–June
F ui body g ow h:
de elopmen and swelling
(pe idial, eined, ascal and
spo al s ages)
Weigh , shape July–ea ly No embe
Ma u a ion (pigmen ed
s age). Ripening
(a oma de elopmen )
Spo e ma u i y, ha es ing da e No embe –Ma ch
The ou come o he FB o ma ion p ocess elies upon how his sequence o s ages
(ma ing, g owing and ma u a ion) p oceeds. The de elopmen al pa e ns o FBs a e
a ec ed no only by en i onmen al bu also by endogenous ac o s a di e en s ages,
such as he exp ession o ce ain enzymes ela ed wi h melanin-syn hesis pa hways [
10
,
16
].
This aises he ques ion o whe he he momen and condi ions in which a mo phogene ic
s age occu s migh in luence he ollowing ones. Resea ch on he ela ionships among
FB de elopmen cha ac e s could help shed ligh on his aspec . As ou lined abo e, a
numbe o FB de elopmen cha ac e s ha include ui ing dep h, weigh , shape, and
spo e ma u i y can be p ima ily linked o pa icula mo phogene ic s ages (Table 1). Since
hese cha ac e s de ine o in luence he comme cial quali y s anda ds o u le FBs [
17
],
unde s anding he ela ionships among de elopmen cha ac e s and he p ocesses ha
shape hem may also open new pe spec i es o p e-ha es quali y managemen h ough
imp o ed a ming p ac ices.
J. Fungi 2021,7, 102 3 o 16
He e, we aimed o: (i) build a causal model o explain how de elopmen cha ac e s
o u le FB in luence one ano he , and (ii) es whe he hese ela ionships a e consis en
ac oss di e en soils and dig ypologies (single FBs and FB clus e s). We es ed se e al
al e na i e models o each dig ypology in h ee eplica e blocks along a soil ex u e
g adien ha is ep esen a i e o common u le o cha d soils, and compa ed hese mine al
soils wi h he FBs g owing wi hin a pea -based subs a e amendmen . The causal models
we e buil conside ing he linkages be ween he s udied cha ac e s and he sequen ial
mo phogene ic s ages o u le FBs: ui ing dep h linked o he ma ing p ocess, weigh
and shape linked o he g owing s age, and spo e ma u i y linked o ma u a ion [
1
,
7
,
8
]
(Table 1). We hypo hesized ha : (i) ui ing dep h would ha e a posi i e e ec on weigh
and ma u i y, because soil dep h bu e s ex eme alues in empe a u e and wa e con en ,
which a e pa icula ly a iable in Medi e anean clima es [
18
,
19
]; (ii) no ela ionship
be ween weigh and ma u i y would appea , because dogs usually localize ull-de eloped
ipe FBs o sizes om less han 10 g o mo e han 100 g; (iii) shape o small FBs would
we mo e ounded, because hey need o make and occupy less soil olume and a e less
likely o ace mechanical cons ain s du ing g ow h; (i ) in FB clus e s, he weigh o he
la ges FB would show a nega i e ela ionship wi h he weigh o he emaining FBs, due
o he local esou ce deple ion o inhibi ion mechanisms hypo hesized by Moo e e al. [
20
];
and ( ) di e ences among soils and wi h subs a e would a ec ela ionships among FB
de elopmen cha ac e s, since soil p ope ies and localized subs a e amendmen s a e able
o in luence hese cha ac e s [21].
2. Ma e ials and Me hods
2.1. Expe imen al Si e
The s udy was conduc ed in a 15-ha u le o cha d es ablished in 2001 wi h
Que cus ilex
subsp. ballo a and Que cus aginea seedlings (a anged in ows 2:1) inocula ed wi h T.
melanospo um [
21
]. The expe imen al si e is loca ed in Gúda -Ja alamb e coun y (Te uel
p o ince, eas e n Spain, 1150 m a. s. l.). The clima e is Con inen al Medi e anean, wi h a
mean annual ain all o 519 mm and a mean annual empe a u e o 11.1
◦
C, ypical o Span-
ish u le-p oducing egions [
22
]. The expe imen al si e is placed in he piedmon o Gúda
moun ain ange, wi h calca eous soils de eloped on C e acic clayey limes one in he uppe
pa (block 3) and on Te ia y sil s ones/sands ones in he lowe pa
(block 1) (Table S1).
In all he blocks, he 0–30 cm soil ho izon in which almos all u les g ow is a homoge-
neous plow laye c ea ed a e epea ed illage ope a ions (du ing u le cul i a ion and
p e iously du ing many decades o ce eal cul i a ion).
T u les a e ha es ed by he owne once a week h oughou he ui ing season
(No embe o Ma ch). Each yea , when he ui ing season is o e , he soil shallowly illed,
and a pea -based subs a e is applied in en spo s a ound each ee [
21
]. The o cha d is
i iga ed wi h a sp inkling sys em om Ap il o Oc obe du ing he d y pe iods wi h
sca ce ain all.
Pea -based amendmen is aimed a inc easing ui ing dep h and shape o FBs [
21
].
In he expe imen al si e, he pea -based amendmen is being applied by he g owe ollow-
ing he mos common p ocedu e used in Spain. The localized applica ion o pea -based
subs a e a ound he hos ees in ol es digging onconical holes abou 25 cm deep, illing
hem wi h abou 1.5 L o a Eu opean Sphagnum pea -based subs a e (Tu ba u
®
om
P oja , Qua de Poble , Spain): A black pea —whi e pea —coi —pe li e mix 11–5–3–1,
wi h pH aised o 7.5) and e-co e ing he subs a e wi h soil [
21
]. G inded ipe u le FBs
a e mixed wi h he subs a e be o e being inco po a ed in o he soil. Annually, pa o he
FBs g ow in he bulk soil whils ano he pa appea wi hin he subs a e spo s.
J. Fungi 2021,7, 102 4 o 16
2.2. Expe imen al Design and Da a Collec ion
In he expe imen al si e, h ee eplica e blocks o 0.25 ha wi h di e en soil ex u es
we e selec ed in a soil g adien along a 300-m-leng h ansec line, wi h block 1 ha ing
sandy loam ex u e, block 2 ha ing loam ex u e and block 3 ha ing loam/clay loam ex u e
(Table S1). Al hough in he wild black u le is ound in almos e e y ype o ex u e,
his ex u e g adien is ep esen a i e o he ange o common soil ex u es in black u le
cul i a ion [23]. The h ee blocks we e managed wi h he same ag onomic p ac ices.
Du ing he 2016–2017 and he 2017–2018 ui ing seasons each block was su eyed
se en imes om No embe o Ma ch. A o al o 604 single FBs and 308 FB clus e s
we e measu ed a e being sys ema ically localized and ha es ed by he g owe wi h
he aid o ained dogs (Tables S2 and S3). F ui ing dep h was eco ded as he dep h in
he soil o he bo om pa o he deepes FB in he dig, a 10 cm in e als. The shape
was e alua ed as a combina ion o sphe ici y ( a io be ween measu ed diame e s), and
isually-es ima ed lobula i y (pe cen su ace occupied by lobules) and a e age heigh o
lobules (in ela ion o FB size). This esul ed in a shape index wi h nine ca ego ies, wi h
highe alues indica ing highe -p iced ound, egula shapes (Table S4). A spo e ma u i y
index was calcula ed as he p opo ion ( om 0 o 1) o asci con aining ma u e (i.e., da k
b own) spo es, bu his index is only a ailable o single FBs. Fo each FB, a hymenial
sample eaching 5–10 mm unde he pe idium was aken wi h a scalpel, and a minimum o
50 andomly selec ed asci we e coun ed in each sample unde ligh mic oscope, ollowing
Zeppa e al. [
24
]. F esh weigh was measu ed o he nea es 0.1 g a e gen ly emo ing
soil and subs a e wi h a b ush. Weigh was measu ed in e e y FB in season 2017–2018,
whe eas o 2016–2017 only he weigh o single FBs is a ailable. The esea ch da ase is
a ailable as Supplemen a y File S2.
The weigh o FBs g owing in clus e s du ing season 2016–2017 was es ima ed h ough
a pa ial leas squa es eg ession model i ed wi h he comple e da ase om season
2017–2018
(n= 1047). This model was i ed wi h se en componen s, mainly based on FB
maximum and minimum diame e . I accoun ed o 97% o he a iabili y in he FB weigh
o season 2017–2018 (Table S5, Figu e S1). I was alida ed wi h he a ailable 2016–2017
measu emen s (single FBs, n= 275). The eg ession be ween log- ans o med p edic ed
and ac ual alues o season 2016–2017 was highly signi ican (p< 0.001) and p esen ed a R
2
alue o 0.96.
2.3. S a is ical Analysis
The causal ela ionships among FB de elopmen cha ac e s we e e alua ed using he
d-sep me hod o pa h analysis [
25
], wi h he aid o he R package ggm [
26
]. Pa h analysis
has been applied o s udy causal pa e ns be ween mo phological, physiological, and
ecological a ibu es in plan biology and ag onomy [
25
,
27
]. The d-sep me hod judges i
a pa icula model is consis en wi h he expe imen al da a. Fo each model, i in ol es:
(i) speci ying a causal hypo hesis in he o m o a di ec ed acyclic g aph, (ii) iden i ying
he se o independence claims (basis se ) implied in he model, (iii) calcula ing he null
p obabili y associa ed wi h each claim, (i ) combining hese p obabili ies using Fishe ’s
C s a is ic, and ( ) compa ing his C wi h he ixed signi icance le el [
25
]. I a pa h
model exhibi ed a p- alue o Fishe ’s C highe han 0.05, i was conside ed consis en
wi h he da a [
25
]. When mo e han one pa h model was consis en wi h he da a, hey
we e compa ed wi h he Akaike’s In o ma ion C i e ion co ec ed o small sample size,
AICc[28].
We sepa a ely analyzed single FBs and FB clus e s, o assess whe he he s eng h
and pa e n o he ela ionships among de elopmen cha ac e s was consis en be ween
bo h dig ypologies. Since pea shows dis inc i e and unique ea u es in compa ison wi h
mine al soils [
29
]— ha p o oking di e ences in u le ui ing dep h, FB weigh , shape
and occu ence o clus e s [
21
]—FBs g owing wi hin he pea -based subs a e ac oss he
h ee blocks we e g ouped and analyzed sepa a ely om mine al soils. The bulk soil o each
eplica e block (BS1, BS2 and BS3) was analyzed sepa a ely o assess whe he he na u e o
J. Fungi 2021,7, 102 5 o 16
he ela ionships among cha ac e s was gene al ac oss soils, since he weigh and shape o
FBs can be in luenced by soil p ope ies [
21
]. Since ou s udy is no aimed a cha ac e izing
yea - o-yea a iabili y, FBs om bo h sampled ui ing seasons we e combined.
Fo single FBs, we buil h ee al e na i e pa h models o es he ela ionships among
he day o he season in which he FB was ha es ed (ha es ing da e, HD), ui ing dep h,
weigh , shape and spo e ma u i y. The h ee al e na i e models assumed a ela ionship
be ween weigh and shape, as well as an e ec o HD on weigh and ma u i y, which a e
widely accep ed by g owe s and esea che s. Model A assumed ha cha ac e s linked o
a pa icula mo phogene ic s age a e no in luenced by hose linked o p e ious s ages
(Table 1). Model B assumed ha weigh is in luenced by ui ing dep h, and ha spo e
ma u i y is in luenced by ui ing dep h and weigh . Model C assumed ha weigh and
shape a e in luenced by ui ing dep h, and ha ma u i y is in luenced by ui ing dep h
and weigh (Figu e S2).
The h ee models we e compa ed ollowing he d-sep me hod ou lined abo e. Once
selec ed a bes - i model, each o i s cons i uen pa hs was modelled wi h gene alized
addi i e models, in o de o allow o non-linea ela ionships and di e en ypes o e o
dis ibu ion [
30
]. A Poisson e o dis ibu ion was used o ui ing dep h and shape,
assessing he model i h ough o e dispe sion. A Gaussian (no mal) dis ibu ion was
used o weigh and ma u i y. In hese models, he assump ions o no mal dis ibu ion and
cons an a iance we e assessed, wi h weigh being log- ans o med o mo e closely mee
he assump ions. The analyses we e conduc ed wi h he R package mgc [31,32].
Fo each pa h in he bes - i model, we p esen he p- alue, he shape o he es i-
ma ed ela ionship and he pe cen de iance explained by each a iable, calcula ed as he
educ ion in de iance a e d opping ha e m while main aining he same smoo hing
pa ame e s h oughou . The la e is aimed a compa ing he ela i e con ibu ion o each
a iable, because we a oided s anda diza ion o keep he ela ionships be ween cha ac e s
wi h i s o iginal shape.
Fo FB clus e s, we buil i e al e na i e pa h models o es he ela ionships among
HD, ui ing dep h, weigh and shape o he la ges FB in he clus e , and combined weigh
o all he o he FBs in he clus e (Figu e S3). Ma u i y was no included due o da a
una ailabili y. All he models assumed a weigh -shape ela ionship o he la ges FB, as
well as an e ec o HD on weigh . Models A–C assumed ha cha ac e s o he la ges FB a e
no in luenced by he weigh o he emaining FBs, whe eas models D and E assumed ha
he weigh and shape o he la ges FB a e in luenced by he weigh o he emaining FBs.
Models A and D assumed ha nei he weigh no shape is in luenced by ui ing dep h
(which is linked o a p e ious s age); models B and E assumed ha weigh is in luenced
by ui ing dep h; and model C assumed ha bo h weigh and shape a e in luenced by
ui ing dep h. When analyzing he bes - i pa h models, a Gamma e o dis ibu ion was
used o he weigh o he emaining FBs.
3. Resul s
3.1. Single F ui bodies
The h ee pa h models p oposed (Figu e S2) we e consis en wi h he collec ed da a
o he pea -based subs a e amendmen (he ea e called subs a e) and he bulk soil o
he h ee eplica e blocks (p> 0.05, Table 2). Howe e , in he subs a e, BS2 and BS3, model
A eached a much lowe AIC
c
alue and a much highe weigh , indica ing ha , acco ding
o Shipley [
28
] c i e ion, model A allowed a much be e i o he da a (Table 2). In BS1,
models B and C p esen ed simila AIC
c
alues, much lowe han ha o model A (Table 2).
Howe e , only model B is shown as he bes - i ing model because he equa ion pa ame e s
a e e y simila , and in bo h cases he pa hs linking ui ing dep h wi h o he a iables a e
no s a is ically signi ican (Figu e 1).

J. Fungi 2021,7, 102 6 o 16
Table 2.
Model i o he h ee compe ing pa h models (Figu e S2) o ui bodies g owing singly in
he pea -based subs a e and in he bulk soil o each block. Bold le e s indica e he models selec ed
acco ding o he model weigh (C: Fische ’s C s a is ic, d : deg ees o eedom, P: null p obabili y, K:
numbe o pa ame e s needed o i he model, AICc: Akaike alue, W: model weigh ).
Model C (d , P) K AICc W
Subs a e
A 12.5 (14, 0.56) 11.6 36.9 0.80
B 9.7 (8, 0.28) 14.5 40.7 0.12
C 8.2 (6, 0.23) 15.5 41.4 0.08
Bulk soil o block 1
A 22.3 (14, 0.07) 10.5 45.6 0.05
B 9.9 (8, 0.27) 13.5 40.8 0.50
C 7.5 (6, 0.28) 14.5 41.1 0.45
Bulk soil o block 2
A 7.9 (14, 0.90) 10.6 31.0 0.83
B 5.2 (8, 0.74) 13.6 35.6 0.08
C 3.3 (6, 0.77) 14.3 35.7 0.08
Bulk soil o block 3
A 10.9 (14, 0.69) 10.5 34.6 0.89
B 9.0 (8, 0.35) 13.5 40.3 0.05
C 6.7 (6, 0.35) 14.2 40.1 0.06
Figu e 1.
Bes - i pa h models showing he causal links among de elopmen cha ac e s in FBs g owing singly in pea -based
subs a e (
a
), and he bulk soil o block 1 (
b
), block 2 (
c
) and block 3 (
d
). Solid lines indica e signi ican links be ween he
a iables, dashed lines indica e non-signi ican links included in he model, black lines indica e posi i e ela ionships and
g ey lines nega i e ela ionships. The hickness o an a ow is p opo ional o he pe cen age o de iance explained by a
pa icula a iable.
The bes - i pa h models o each soil ypology (Figu e 1) sha ed he ollowing ea u es:
(i) ui ing dep h did no show a signi ican ela ionship wi h any o he cha ac e , (ii) FB
weigh showed a s ong nega i e ela ionship wi h he shape index (i.e., bigge FBs ha ing
mo e i egula , less ounded shapes; Table S4), and (iii) he HD showed a s ong posi i e
ela ionship wi h spo e ma u i y (Tables 3and 4, Figu es S4–S7). The HD showed a
signi ican and nega i e ela ionship wi h FB weigh in he subs a e, BS2 and BS3, bu
no signi ican ela ionship in BS1. F ui body weigh showed a signi ican and nega i e
J. Fungi 2021,7, 102 7 o 16
ela ionship wi h ma u i y in BS1, which was no ound in any o he soil (Tables 3and 4;
Figu es S4–S7). The same associa ions be ween de elopmen cha ac e s we e obse ed in
he bi a ia e analyses (Figu e 2).
Table 3.
Null p obabili y (P) and pe cen de iance explained (D
2
) o each pa h in he bes - i model
o ui bodies g owing singly in subs a e and he bulk soil o Soil blocks 2 (BS2) and 3 (BS3).
Response P edic o Subs a e BS2 BS3
PD2PD2PD2
Weigh 1Ha es ing da e <0.001 5.8 0.03 3.6 0.04 4.1
Shape Weigh <0.001 16.4 <0.001 21.9 <0.001 28.7
Ma u i y Ha es ing da e <0.001 40.8 <0.001 35.5 <0.001 34.2
1Va iable log- ans o med.
Table 4.
Null p obabili y (P) and pe cen de iance explained by each a iable (D
2
) o each pa h in
he bes - i model o ui bodies g owing singly in he bulk soil o block 1.
Response P edic o P D2
Weigh 1Ha es ing da e 0.13 -
F ui ing dep h 0.20 -
Shape Weigh <0.001 23.6
Ma u i y Ha es ing da e <0.001 22.7
Weigh 0.01 4.2
F ui ing dep h 0.47 -
1Va iable log- ans o med.
The bes - i pa h models did no explain mo e han 6% o he a iabili y in FB weigh in
any soil ypology, while hey explained 16–29% o he a iabili y in he shape index and 23–
41% o he a iabili y in he spo e ma u i y (Tables 3and 4). In hese bes - i pa h models,
he a iabili y in FB weigh was exclusi ely explained by HD, whe eas he a iabili y
in shape was explained by he weigh and he a iabili y in spo e ma u i y was mainly
explained by HD, wi h weigh also con ibu ing o explain he a iabili y o ma u i y in
BS1 (Tables 3and 4). The ela ionship be ween weigh and shape was nega i e, wi h clea
di e ences be ween FBs smalle han 25 g and FBs la ge han
50 g (Figu es S4–S7).
The
ela ionship be ween weigh and ma u i y in BS1 was nega i e bu pla eauing abo e 10 g,
co esponding o a mean FB diame e o 2.5–3 cm (Figu e S5).
3.2. F ui body Clus e s
Among he i e al e na i e pa h models p oposed (Figu e S3), model D was he one
ha eached lowe AICc alue and highe weigh o all he analyzed soil ypologies,
indica ing ha , acco ding o Shipley [
28
] c i e ion, model D allowed he bes i o he da a
(Table 5).
The bes - i pa h models o each soil ypology (Figu e 3) sha ed he ollowing ea u es:
(i) ui ing dep h did no show a signi ican ela ionship wi h any o he cha ac e , (ii) he
weigh o he la ges FB in a clus e showed a s ong posi i e ela ionship wi h he com-
bined weigh o all he o he FBs in he clus e , and (iii) he HD did no show a signi ican
ela ionship wi h he weigh o he la ges FB in he
dig (Table 6, Figu es S8–S11
). In he
subs a e and BS2, he weigh o he la ges FB in he clus e showed a s ong nega i e
ela ionship wi h i s shape index (Table 6, Figu es S8 and S10). In he subs a e he HD
showed a signi ican and nega i e ela ionship wi h he weigh o he emaining FBs in
he clus e (Table 6; Figu e S8). Finally, in BS2 he shape index o he la ges FB o he
clus e showed a signi ican and posi i e ela ionship wi h he weigh o he emaining
FBs (Table 6; Figu e S10)
. The same associa ions be ween de elopmen cha ac e s a e sug-
ges ed by he bi a ia e analyses, al hough in some cases concealed by he ac ha o he
a iables as he HD a e also in ol ed in he ela ionship (Figu e 4).
J. Fungi 2021,7, 102 8 o 16
Figu e 2.
Sca e plo da a o he de elopmen cha ac e s in he single ui bodies o he s udied blocks. Pea son’s
co ela ion coe icien is epo ed o each bi a ia e ela ionship. BS1–BS3: bulk soil o blocks 1–3.
J. Fungi 2021,7, 102 9 o 16
Table 5.
Model i o he i e compe ing pa h models (Figu e S3) o ui body clus e s in he subs a e
and in he bulk soil o each block. Bold le e s indica e he models selec ed acco ding o he model
weigh (C: Fische ’s C s a is ic, d : deg ees o eedom, P: null p obabili y, K: numbe o pa ame e s
needed o i he model, AICc: Akaike alue, W: model weigh ).
Model C (d , P) K AICc W
Subs a e
A 74.9 (14, <0.001) - - -
B 60.0 (10, <0.001) - - -
C 61.7 (8, <0.001) - - -
D 6.6 (10, 0.76) 13.3 35.2 0.85
E 2.6 (6, 0.86) 16.4 38.6 0.15
Bulk soil o block 1
A 22.3 (14, 0.07) 8.4 43.6 0.11
B 18.0 (10, 0.06) 10.5 46.1 0.03
C 17.1 (8, 0.03) - - -
D 10.4 (10, 0.41) 10.8 39.7 0.81
E 7.4 (6, 0.29) 13.2 45.8 0.04
Bulk soil o block 2
A 29.4 (14, 0.009) - - -
B 26.7 (10, 0.003) - - -
C 20.7 (8, 0.008) - - -
D 7.6 (10, 0.67) 11.2 39.9 0.99
E 6.9 (6, 0.33) 13.7 50.0 0.01
Bulk soil o block 3
A 19.3 (14, 0.15) 8.2 87.7 <0.01
B 12.7 (10, 0.24) 10.2 59.4 0.39
C 12.2 (8, 0.14) 11.2 70.1 <0.01
D 9.5 (10, 0.49) 10.5 58.6 0.60
E 0.8 (6, 0.99) 12.6 78.9 <0.01
Figu e 3.
Bes - i pa h models showing he causal links among de elopmen cha ac e s in FBs g owing in clus e s in
pea -based subs a e (
a
), and he bulk soil o block 1 (
b
), block 2 (
c
) and block 3 (
d
). Solid lines indica e signi ican links
be ween he a iables, dashed lines indica e non-signi ican links included in he model, black lines indica e posi i e
ela ionships and g ey lines nega i e ela ionships. The hickness o an a ow is p opo ional o he pe cen age o de iance
explained by a pa icula a iable. FB: ui body.
J. Fungi 2021,7, 102 16 o 16
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