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\DOI{10.5802/crchim.236}
\datereceived{2023-04-22}
\daterevised{2023-06-06}
\datererevised{2023-06-14}
\dateaccepted{2023-06-14}
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\dateposted{2023-09-19}
\begin{document}

\begin{noXML}

%\TopicEF{Chemical Ecology -- Chemical Mediation in the
%Environment}{\'Ecologie chimique -- M\'ediation chimique dans
%l'environnement}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Chemical Ecology -- Chemical Mediation in the Environment}
%\def\cdr@specialtitle@french{\'Ecologie chimique -- M\'ediation chimique dans l'environnement}
%\makeatother

\title{Mediterranean island endemic \protect\textit{Arum pictum} 
emits isomyocorene as a major component of floral scent}

\alttitle{\textit{Arum pictum}, end\'{e}mique des \^{i}les
m\'{e}diterran\'{e}ennes,  \'{e}met de l'isomyocor\`{e}ne comme
composant volatil floral majoritaire}

\author{\firstname{Marc} \lastname{Gibernau}\CDRorcid{0000-0003-3866-3099}\IsCorresp}
\address{CNRS-University of Corsica Pascal Paoli, UMR 6134 SPE, Projet
Phytochimie \&amp; \'{E}cologie chimique, Route des Sanguinaires - Vignola,
20000 Ajaccio, France}
\email[M. Gibernau]{gibernau\_m@univ-corse.fr}

\author{\firstname{Arianna} \lastname{Amadori}\CDRorcid{0000-0001-5493-1335}}
\address{Department of Chemical, Physical, Mathematical and Natural
Sciences, University of Sassari, via Piandanna 4 -- 07100
Sassari, Italy}
\email[A. Amadori]{ariannaamadori81@gmail.com}

\author{\firstname{Adrienne} \lastname{Godschalx}}
\addressSameAs{1}{CNRS-University of Corsica Pascal Paoli, UMR 6134 SPE, Projet
Phytochimie \&amp; \'{E}cologie chimique, Route des Sanguinaires - Vignola,
20000 Ajaccio, France}
\address{Lower Columbia College, 1600 Maple St, Longview, WA 98632,
USA}
\email[A. Godschalx]{adrienne.godschalx@gmail.com}

\author{\firstname{J\'{e}r\^{o}me} \lastname{Albre}}
\addressSameAs{1}{CNRS-University of Corsica Pascal Paoli, UMR 6134 SPE, Projet
Phytochimie \&amp; \'{E}cologie chimique, Route des Sanguinaires - Vignola,
20000 Ajaccio, France}
\email[J. Albre]{albrej@gmail.com}

\author{\firstname{F\'{e}lix} \lastname{Tomi}\CDRorcid{0000-0003-4233-4845}}
\addressSameAs{1}{CNRS-University of Corsica Pascal Paoli, UMR 6134 SPE, Projet
Phytochimie \&amp; \'{E}cologie chimique, Route des Sanguinaires - Vignola,
20000 Ajaccio, France}
\email[F. Tomi]{tomi\_f@univ-corse.fr}

\author{\firstname{Paolo} \lastname{Marcia}}
\address{Department of Veterinary, University of Sassari, via 
Piandanna 4 -- 07100 Sassari, Italy}
\email[P. Marcia]{pmarcia@uniss.it}

\author{\firstname{Emmanuele} \lastname{Farris}\CDRorcid{0000-0002-9843-5998}}
\addressSameAs{2}{Department of Chemical, Physical, Mathematical and Natural
Sciences, University of Sassari, via Piandanna 4 -- 07100
Sassari, Italy}
\address{NBFC, National Biodiversity Future Center, Palermo 90133, Italy}
\email[E. Farris]{emfa@uniss.it}

\shortrunauthors

\keywords{\kwd{Araceae}
\kwd{Floral thermogenesis}
\kwd{$p$-cresol}
\kwd{Skatole}
\kwd{Indole}
\kwd{2,6-dimethylocta-1,3,7-triene}}

\altkeywords{\kwd{Araceae}
\kwd{Thermogen\`{e}se florale}
\kwd{$p$-cr\'{e}sol}
\kwd{Scatole}
\kwd{Indole}
\kwd{2,6-dim\'{e}thylocta-1,3,7-tri\`{e}ne}}

\thanks{Financial support was supported by the Laboratory Sciences for
the Environment (UMR-CNRS 6134) through the CPER program between the
University of Corsica, the Cullettivit\`{a} di Corsica and the French
Ministry of Territorial Cohesion and Relations with Local Authorities.}

\begin{abstract}
The west Mediterranean \textit{Arum pictum} L.f., endemic from Corsica,
Sardinia and Balearic Islands, is the only autumn-flowering
\textit{Arum} species. This species is pollinated by deceptively
attracting coprophilous and saprophilous Diptera and staphylinid
Coleoptera. Four populations from Sardinia and Corsica were studied.
The inflorescence produces heat during anthesis from the appendix and
stamens; the appendix is able to increase its temperature twice within
the same day, a trait unique in \textit{Arum}. \textit{A. pictum}
floral scents likely mimic horse/donkey dung by releasing several
dung-related volatile organic compounds (VOCs: $p$-cresol,
skatole, or indole). Surprisingly, the main floral VOC was
2,6-dimethylocta-1,3,7-triene or isomyocorene.\vspace*{-2pt}
\end{abstract}

\begin{altabstract}
\textit{Arum pictum} L.f., esp\`{e}ce insulaire end\'{e}mique de la
Corse, de la Sardaigne et des \^{i}les Bal\'{e}ares, est la seule
esp\`{e}ce d'{\textit{Arum}} {\`{a} floraison automnale. Cette
esp\`{e}ce est pollinis\'{e}e en dupant des Dipt\`{e}res coprophiles et
saprophiles et des Col\'{e}opt\`{e}res staphylinid\'{e}s. Nous avons
\'{e}tudi\'{e} quatre populations de Sardaigne et de Corse.
L'inflorescence produit de la chaleur durant l'anth\`{e}se au niveau de
l'appendice et des \'{e}tamines. L'appendice est capable d'augmenter sa
temp\'{e}rature deux fois dans la m\^{e}me journ\'{e}e, un trait unique
pour les} {\textit{Arum}}{. Les parfums floraux d'}{\textit{A. pictum}}
{imitent probablement le crottin de cheval ou d'\^{a}ne en lib\'{e}rant
plusieurs compos\'{e}s organiques volatils li\'{e}s aux crottins (COV:}
$p$-cr\'{e}sol, scatole ou indole). \'{E}tonnamment, le
principal COV floral \'{e}tait le 2,6-dim\'{e}thylocta-1,3,7-tri\`{e}ne
ou isomyocor\`{e}ne.
\end{altabstract}

\maketitle

\vspace*{8pt}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

The \textit{Arum} genus is composed of 29 described
species~\cite{1,2,3} but estimated to be comprised of 43
species~\cite{4}. Regardless of the genus diversity, one stable
subdivision within the genus, based on morpho/ecological characters and
molecular data, consistently groups all \textit{Arum} species
separately from \textit{Arum pictum}. \textit{A. pictum} is the only
species of the subgenus 
{\textit{Gymnomesium} (L.f.) Schott,}
which \mbox{appears} to be sister to all other \textit{Arum} species~\cite{1,2,5,6}.
Thus, \textit{A. pictum} appears to be a unique taxon within the genus
\textit{Arum}, endemic to West Mediterranean islands: Corsica,
Sardinia, Tuscan Archipelago (Montecristo) and Balearic
Islands~\cite{1}. This species presents some distinguishing characters
such as (1) autumn flowering (versus spring for all other species) and
(2)~leaves produced after or simultaneously with inflorescences (versus
before for all other species,~\cite{2}). For all of the above-cited
reasons, it has been proposed to elevate the subgenus
\textit{Gymnomesium} to the rank of a monospecific genus~\cite{4}.
Since a proper taxonomical synonymy has not yet been published, the
name \textit{Arum pictum} will be used in this\break manuscript.

The inflorescence of \textit{A. pictum} consists of a spadix (e.g.
reproductive spike) and a spathe (e.g. modified bract). The anthesis
lasts two days and is protogynous, preventing spontaneous
self-pollination~\cite{7}. The spathe, which completely encloses the
spadix during floral development, opens in its upper half during
anthesis to reveal the sterile appendix of the apical part of the
spadix. This appendix produces heat and releases scent for pollinator
attraction. The basal half of the spathe forms a pollination chamber
around the basal part of the spadix, which bears unisexual flowers,
stamens and ovaries. Attracted insects are trapped on the first day of
anthesis during the female stage when they slip and fall into the
floral chamber, and eventually pollinate the female flowers. On the
next morning, during the male stage, they are dusted with pollen before
being able to leave the pollination chamber. After pollination in
autumn, red berry-like fruits develop throughout the winter as an
infructescence that mature by the following spring~\cite{7}. 
\looseness=-1

The deceptive pollination strategy of \textit{A. pictum} has been
studied more than a decade ago in one Corsican population~\cite{7}. In
summary, the anthesis cycle of \textit{A. pictum} occurred during the
day, attracting mainly sphaerocerid flies (Brachycera) and staphylinid
beetles early in the morning and late in the afternoon of the first
day. The pollen was released from the anthers early in the morning of
the second day before insects depart. \textit{A. pictum}'s
thermogenic cycle matched the anthesis cycle and the pollination events
with an original and unique, bimodal temperature pattern of the
appendix (early morning ${\sim}$8:00 am and late afternoon
${\sim}$07:30 pm), contrary to the unimodal pattern found in
all other studied \textit{Arum} species~\cite{7,8}. As mentioned
earlier, the main pollinators of \textit{Arum pictum} were sphaerocerid
flies (Brachycera) and staphylinid beetles and not psychodid,
chironomid or ceratopogonid flies (all nematoceran) known to be the
main pollinators of \textit{Arum} species in West
Europe~\cite{8,9,10,11,12,13}. Most of the \textit{Coproica}
(Sphaeroceridae) flies captured by \textit{A. pictum} inflorescences
were females (more than 70\%) suggesting that the floral scent of
\textit{A. pictum} may have sex-specific properties and/or may mimic 
the fly oviposition sites~\cite{7}. \textit{A. pictum} is characterised
by a dung odour close to horse/donkey dung, described as
pungent~\cite{1}. The floral scent was described as been dominated by 5
volatile compounds namely: benzyl alcohol, indole, dihydroocimene,
ocimene and skatole~\cite{7}. The comparative study of the volatiles
compounds (VOCs) emitted by four different types of dung showed that
the scent of the horse dung was dominated by $p$-cresol,
${\upbeta}$-citronellene, and 4-methyl-3-heptanone~\cite{14}. Other
notables VOCs were dihydrolimonene, \textit{cis}-pinane, indole,
skatole, and various alkanes~\cite{14}.

The aim of this paper is to document some regional aspects of the
pollination ecology of \textit{Arum pictum} across the island of
Sardinia (Italy) in comparison to Corsica. We measured the floral
thermogenic patterns, collected the floral volatiles during the most
scented phase of the anthesis according to human nose, and the insects
trapped within pollination chambers of inflorescences from several
populations.\looseness=-1

\section{Materials and methods}\label{sec2}
\subsection{Study species and populations}\label{sec21}

\textit{Arum pictum} L. fil. is a Tyrrhenian islands endemic, found on
Sardinia, Corsica, Mallorca, Menorca
and Montecristo in the Tuscan Archipelago~\cite{15,16}, including some minor
islets surrounding the large islands. In Sardinia, it is distributed in
all sectors of the islands~\cite{16}, usually below 1000~masl, but
sometimes above~\cite{17, 18}. The plant thrives on every kind of soil,
but prefers shady or semi-shady habitats under the canopy of evergreen
trees and shrubs~\cite{19} in the thermo- and meso-Mediterranean
bioclimatic belts~\cite{20}. This species is not threatened in Italy
and its conservation status is considered as LC~\cite{21}.

The pollination ecology of \textit{Arum pictum} was studied in October
2018 in three populations from \mbox{northern} Sardinia (Italy). The first two 
populations were located in north-western Sardinia, namely: near the
lake Baratz (Sassari, GPS coordinates: 40.676729, 8.225554) and
Gutierrez (Alghero, GPS coordinates: 40.625107, 8.273025) at almost sea
level (24~masl), in the thermo-Mediterranean dry bioclimatic belt,
the first on aeolian sand, the second on Mesozoic limestone. Both
populations grew in \textit{Pinus pinea} and \textit{P.~halepensis}
plantations, but the first is characterized by \textit{Juniperus
phoenicea} vegetation, whereas the second by \textit{Olea
europaea} vegetation. The third population was located in north-eastern
Sardinia at Diga del Liscia (Sant'Antonio di Gallura-Luras, GPS
coordinates: 41.001154, 9.144749, named Diga afterwards) at an
elevation of 242 masl, corresponding to a meso-Mediterranean dry
bioclimatic belt. This population grew on acidic soil (on schists and
granites) in an open wood of \textit{Quercus suber}. In autumn 2018,
these three \textit{Arum} populations were large with more than 100
individual plants. For a broader geographical comparison, one
south-west Corsican population was also studied in October 2018,
located in Coti-Chiavari (GPS coordinates: 41.793473, 8.769976, named
Coti afterwards) on the south bank of the Ajaccio Gulf in a former
penitentiary area. This population, large of ${\sim}$60 individual
plants, grows in an open wood of \textit{Quercus ilex}.

\subsection{Thermogenesis and anthesis}\label{sec22}

Thermogenesis in \textit{A. pictum} was studied by measuring the
temperatures of 8 inflorescences belonging to two different Sardinian
populations (Baratz in the west and Diga in the east). During the
entire anthesis, temperatures were recorded every 10~min from two
different zones of the spadix (the appendix and the male flowers) with
simultaneous measurements of ambient air, using type-K probes
connected to Digi-Sense$^{\mbox{\textregistered}}$ 
DualLogR$^{\mbox{\textregistered}}$
thermocouple thermometers (Testo model 175T3$^{\mbox{\textregistered}}$). The
female flowers (e.g. gynoecia) are known to be non-thermogenic~\cite{7}
and were thus not recorded. The two probes recording the spadix
temperatures were inserted about 5 mm deep into either the appendix or
the stamen zone. Ambient air temperature was recorded near the studied
inflorescence in the shade (e.g. under an \textit{Arum} leaf). Twice
per day {(i.e.}
in the morning and in the \mbox{afternoon)} the monitored
inflorescences were observed for insect presence and floral
observations.

{\vspace*{-6pt}}

\subsection{Floral scent analyses}\label{sec23}

{\vspace*{-2pt}}

Floral volatiles were collected during the morning between 09:00--10:30
am in the four studied populations from Coti ($N=9$ inflorescences)
between October 4{th} and 7{th}, Gutierrez ($N=3$) on October 13{th},
Baratz ($N=2$) on October 14{th}, and Diga ($N=4$) on October 19{th}.
Floral scent samples were collected \textit{in situ} from scented
inflorescences that were enclosed within Nalophan (NA colourless,
calibre 90, ETS Charles-Fr\`{e}res, Saint-Etienne, France) bags 
{opened in their upper part to create a headspace.}
The emitted volatile
organic compounds (VOCs) were adsorbed by Solid Phase Micro Extraction
(SPME) Fibers (StableFlexTM SPME Fiber, 65~${\upmu}$m PDMS-DVB
Coating, Supelco, Darmstadt, Germany) for 20~min, then stored at 
5~\textdegree C until analysis by thermal desorption. To identify possible
ambient contaminants, control samples were simultaneously collected
from an empty bag {for}
each scent sample, following the same protocols
described above.

The trapped volatiles were analysed by combined gas chromatography-mass
spectrometry (GC-MS). The SPME fibers were manually injected and
desorbed into the split/splitless injector of a Clarus 580 Perkin-Elmer
directly coupled to a Clarus SQ8S Perkin Elmer TurboMass detector
(quadrupole). The gas chromatographer was equipped with two
fused-silica capillary columns (50 m $\times$ 0.22 mm, film thickness 
0.25~$\upmu$m), a non-polar BP-1 (polydimethylsiloxane) and a polar
BP-20 (polyethylene glycol). The oven temperature was programmed from
60 to 220~\textdegree C at 2~\textdegree C${\cdot}$min$^{-1}$ and then held
isothermal at 220~\textdegree C for 20~min; injector temperature at
270~\textdegree C; carrier gas was helium (0.8~mL${\cdot}$min$^{-1}$); split
ratio of 1:15; ionisation energy at 70 eV. The electron ionisation (EI)
mass spectra were acquired over the mass range 40--350~Da. The relative
proportions of the constituents were expressed as percentages obtained
by peak-area normalisation, without using correcting factors. Kovats
retention indices (RI) were determined relative to the retention times
of a series of \textit{n}-alkanes with linear interpolation (Target
Compounds software from Perkin Elmer). Compounds were tentatively
identified by comparing their mass spectra and retention times to those
of authentic standards and from the reference libraries Adams, NIST20,
Perkin FF2013, Wiley FFNSC Library 2{nd} Edition and Wiley
Registry{\texttrademark} 8{th} Edition.

\subsection{Isomyocorene NMR identification}\label{sec24}

In order to confirm {mass} identification of the main unidentified
floral VOCs, floral scent samples were collected \textit{in situ} from
four inflorescences during the peak of thermogenic activity and floral
scent emission on the female phase of anthesis. The inflorescences were
enclosed within Nalophan (NA colourless, calibre 90, ETS
Charles-Fr\`{e}res, Saint-Etienne, France) bags 
{opened in their upper part to create a headspace.}
Sampling was conducted by dynamic
headspace, scented air was drawn for 30~min through adsorbent traps
connected with silicone tubing to a membrane pump (ASF Thomas Inc,
Memmingen, Germany) that worked at a constant flow of 
$250~\mathrm{mL}{\cdot}\mathrm{min}^{-1}$. The
adsorbent traps (10 cm long, 0.5 i.d.), made out of silanised glass
cartridges, were filled with a 1:1 mixture of Tenax{\texttrademark} TA
(50 mg, 80/100, Macherey-Nagel 706318, D\"{u}ren, Germany) and
Carbopack{\texttrademark} X (20/40, Supelco 1-0435), fixed in each tube
with silanised glass wool plugs.

After collection, the four traps were eluted with deuterated chloroform
(150~$\upmu$L CDCl$_{3}$) and pooled together to attain a
sufficient quantity for the NMR analysis. Nuclear Magnetic Resonance
spectra were recorded on a Bruker AVANCE 400 Fourier Transform
spectrometer (Wissembourg, France) operating at 400.132 MHz for
$^{1}$H and 100.623 MHz for $^{13}\mathrm{C}$,
equipped with a 5 mm probe, in CDCl$_{3}$, with all shifts
referenced to internal tetramethylsilane (TMS). The
$^{1}$H-NMR spectrum was recorded with the following
parameters: pulse width (PW) 4.3~$\upmu$s; relaxation delay 1~s,
acquisition time: 2.6 s for 32 K data table with a spectral width (SW)
of 6000 Hz; accumulation scans: 32. The $^{13}\mathrm{C}$-NMR
spectrum was recorded with the following parameters: 
$\mathrm{PW}= 4~\upmu$s (flip angle 45\textdegree); acquisition time:
2.7~s for 128 K data table with a spectral width of 25,000~Hz 
(250~ppm); accumulated scans: 512. For the 2D spectra, sequences from Bruker
Topspin\texttrademark~(Bruker, Wissembourg, France) library (DEPT,
COSY, HMBC) and gradient-enhanced sequences were used.

\subsection{Visiting entomofauna}\label{sec25}

Arthropods attracted to the inflorescences and temporarily trapped in
the pollination chamber were collected in ethanol 60\% with an
aspirator in three populations Coti ($N=26$ inflorescences), Baratz
($N=30$) and Diga ($N=8$). Arthropods per inflorescence were counted
and identified at the order rank (and family level for Staphylinidae
and Braconidae). Based on known pollinators in the genus \textit{Arum},
we grouped non-insect arthropods plus scarce insects (Formicidae,
Blattoidea, Lepidoptera, Hemiptera) in the category
``Others''~\cite{8}.

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig01}
{\vspace*{2pt}}
\caption{\label{fig1}Temperature difference curves from one
inflorescence from Baratz representative of the typical thermogenic
pattern in \textit{Arum pictum}: between the appendix and ambient air
(black line) and between the male flowers and ambient air (brown line).
The grey boxes indicate the night period. The temperature peaks are
numbered by chronological order for both the stamens ``sta'' and the
appendix ``app''.}
{\vspace*{2pt}}
\end{figure*}

\begin{table*}
\caption{\label{tab1}Thermogenic data of Sardinian \textit{Arum pictum}
recorded from 10 inflorescences{\vspace*{2pt}}}
\begin{tabular}{cccc}
\thead
\parbox{2cm}{\centering \vspace*{.2pc}Temperature peaks} &amp; 
\parbox{2cm}{\centering Anthesis time (days)} &amp; 
\parbox{3cm}{\centering Mean duration (h) (min--max)} &amp;
\parbox{3cm}{\centering Temp. diff. max (\textdegree C) (min--max)}\vspace*{2pt}\\
\endthead
Stamens 1 &amp; D-2/D-1 &amp; $&gt;$17h25$^{\mathrm{a}}$ &amp; 6.9 (6.7--7) \\
Stamens 2 &amp; D-1 &amp; 6h10 (4h30--8h00) &amp; 3.5 (2.5--5.1) \\
Stamens 3 &amp; D-1/D &amp; 15h30 (14h20--17h30) &amp; 11.4 (7.1--12.6) \\
Appendix 1 &amp; D &amp; 10h56 (8h50--12h30) &amp; 14.6 (9.3--20.2) \\
Appendix 2 &amp; D &amp; 8h32 (6h20--10h10) &amp; 6.7 (4.7--12.7) \\
Stamens 4 &amp; D/D+1 &amp; 17h56 (17h00--19h40) &amp; 7.5 (3.7--9)
\botline
\end{tabular}
\tabnote{For each temperature peak: corresponding day(s) during the
anthesis, mean duration of the thermogenic event (range), and mean maximum
temperature difference (range). D: pollination day.}
\tabnote{$^{\mathrm{a}}$ Temperatures were recorded after the temperature
increase started.}
{\vspace*{2pt}}
\end{table*}

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Structure of the 2,6-dimethyl-1,3E,7-octatriene
also known as isomyocorene. Right. Corresponding mass spectra.}
\end{figure*}


\section{Results and discussion}\label{sec3}

\subsection{Thermogenesis and anthesis}\label{sec31}

The timing and intensity of the temperature peaks varied among
inflorescences due to asynchronous flowering, inflorescence size, and
physiology differences but the daily thermogenic pattern (e.g.
sequence) was the same in individuals and populations. The
appendix and the male flowers appeared to be responsible for heat
production in inflorescences of \textit{Arum pictum} but the two
regions of the spadix were not synchronised with each other resulting
in a complex thermogenic pattern (Figure~\ref{fig1}). The first temperature
rise, caused by the male flowers, was recorded the night before the
opening of the spathe from 6:30 pm until noon the next day 
(Figure~\ref{fig1}---sta1). Between 09:00--10:00 am, the temperature of the flowers reached
a maximum of 23.2~\textdegree C, on average, 7~\textdegree C warmer
than ambient air (Table~\ref{tab1}). Later, on the same day, a second
thermogenic phase occurred. The stamen temperature increased again
between 3:30 and 9:30 pm (Figure~\ref{fig1}---sta2) coinciding with the spathe
opening (upper half). The temperature of the male flowers reached a
maximum of 19--24~\textdegree C between 6:00--8:00 pm, which was, on
average, 3.5~\textdegree C warmer than ambient air (Table~\ref{tab1}). In the
same evening, the stamen temperature increased again and with greater
intensity between 9:30 pm and 3:30 pm on the following day 
(Figure~\ref{fig1}---sta3). The temperature of the flowers reached a maximum of
22--30~\textdegree C between 01:30--04:30 am, which was, on average,
11.4~\textdegree C warmer than ambient air (Table~\ref{tab1}). The stigmas
looked wet with turgescent papillae (e.g. receptive) in early morning.
The appendix started heating up at about the same time as the male flowers
reached maximum temperatures (sta3), of 28--33~\textdegree C between
06:10--09:20 am (Figure~\ref{fig1}---app1), corresponding to a maximum
temperature difference of 9--20~\textdegree C above ambient air 
(Table~\ref{tab1}).
Insects were \mbox{attracted} to the scented inflorescences and eventually
slipped or walked down inside the pollination chamber. Note that the
stamen's temperature increased again during the appendix thermogenic
peak to a tissue temperature of 30--31~\textdegree C at 08:20--08:35 am
representing a maximum temperature difference of 8.5--9.5~\textdegree C
above ambient air (Figure~\ref{fig1}). Afterwards, both stamen and appendix
temperatures declined within two hours to reach ambient temperature at
around noon. A third thermogenic phase began at the end of this first
day of flowering: the appendix started heating up at about 1:40--4:00
pm (Figure~\ref{fig1}---app2), while the male flowers started heating up at
around 3:00--6:00~pm (Figure~\ref{fig1}---sta4). Insects could also be attracted in
the afternoon during this \mbox{second} scented phase but in somewhat lower
numbers. Appendix temperature quickly reached a maximum temperature
around 21--30~\textdegree C between 5:00--7:30 pm
(4.7--12.7~\textdegree C above ambient air). After 6:00--7:00 pm, the
appendix temperature decreased gradually until reaching ambient level
between 9:20 pm and midnight (Figure~\ref{fig1}). The male flowers'
temperature was still increasing and reached a maximum temperature of
21--26~\textdegree C (3.75--9~\textdegree C above ambient air, Table~\ref{tab1})
around 00:30--03:00 am on the morning of the second day (Figure~\ref{fig1}---sta4).
Later in the morning, the pollen was released from the
mature stamens and the trapped insects were able to leave the
pollination chamber. Afterwards, the temperature declined, returning
abruptly to ambient level between 10:30 am and noon.

The floral thermogenic pattern of \textit{Arum pictum} individuals in
Sardinia appeared to be consistent with the thermogenic pattern
obtained from Corsican plants (see Figures~1B and 1C in~\cite{7}). A
complex thermogenic pattern resulting from non-synchronised heat
productions by two different regions of the spadix (e.g. the appendix
and the male flowers) appears to be the rule in the genus \textit{Arum}
\textit{sensu lato}~\cite{8} as it has been previously documented in
\textit{Arum maculatum}~\cite{22}, \textit{A. italicum}~\cite{23} and
\textit{A. concinnatum}~\cite{24}. Floral heat production in
\textit{Arum} results from an alternative mitochondrial respiration,
the cyanide-insensitive pathway, catalysed by the alternative oxidase
(AOX) resulting in heat dissipation instead of ATP
production~\cite{24,25}. The thermogenic pattern should be considered
as a floral trait such as colour, shape or scent as the
floral temperature peaks are synchronised with the main anthesis events
in relation to the pollination process. The timing of insect attraction
(e.g. scent emission and appendix temperature peak) occurred during the
early morning and at the end of the afternoon. {\unskip\break}Morning temperature
floral peak has been reported in some \textit{Arum} species from East
Mediterranean such as \textit{A. dioscoridis}, \textit{A. creticum} and
\textit{A. palaestinum}~\cite{24}; whereas in other species such as
\textit{A. italicum}, \textit{A. maculatum}, \textit{A. rupicola} and
\textit{A. jacquemontii} the temperature appendix peak has been
recorded during the first evening or night of anthesis (review
in~\cite{8}). The main originality of the thermogenic pattern in
\textit{Arum pictum}, in contrast with all other documented
\textit{Arum}  species, is that the appendix is able to increase its
temperature twice within the same day in a manner similar to fertile
male flowers~\cite{7,8,23,24}. This double temperature increase may be
related to maximise day insect attraction in autumn, a potentially very
temperamental season.

\subsection{Isomyocorene identification}\label{sec32}

The major VOC was identified as 2,6-dimethyl-1,3,7-octatriene
(Figure~\ref{fig2},  supplementary materiel) by comparison of the
proton and carbon chemical shift values with those reported in
the literature~\cite{26}.


$^{1}$H NMR (CDCl$_{3}$, 400 MHz): 6.14 (d, H3); 5.77 (ddd, H7); 5.62
(dt, H4); 4.94--5.00 (m, 2H, H8); 4.87 (bs, 2H, H1); 2.22 (m, H6); 2.11
(m, 2H, H5); 1.83 (s, Me, H9); 1.00 (d, Me, H10). 

$^{13}\mathrm{C}$ NMR (CDCl$_{3}$, 100 MHz): 144.09 (C7); 142.16 (C2);
134.14 (C3); 128.90 (C4); 114.40 (C1); 112.60 (C8); 39.90 (C5); 37.78
(C6); 19.50 (C9); 18.72 (C10).

EI-MS 70 eV, \textit{m/z} (rel.int): 136 (1), 121 (12), 107 (12), 79
(57), 77 (10), 81 (100), 55 (27), 53 (22), 41 (22), 39 (16), 40 (11).

Kovats retention Indices: non-polar (BP-1): 960; polar (BP-20): 1119.


\onecolumngrid
\pagebreak
\begin{sidewaystable}
\caption{\label{tab2}Floral scent composition as blend relative
proportion (\%): mean, standard deviation, minimum and maximum from
four populations (Corsica: Coti; Sardinia: Baratz, Gutierrez, Diga ---
see materials and methods for full details) of \textit{Arum pictum}}
\fontsize{8.7}{10.6}\selectfont
\tabcolsep=2.2pt
\begin{tabular}{cclcccccccc}
\thead
\xmorerows{1}{RI polar} &amp; \xmorerows{1}{RI non-polar} &amp;  \xmorerows{1}{Name} &amp; 
\multicolumn{2}{c}{Coti ($N=9$)} &amp; \multicolumn{2}{c}{Baratz
($N=2$)} &amp;  \multicolumn{2}{c}{Gutierrez ($N=3$)} &amp;
\multicolumn{2}{c}{Diga ($N=4$)} \\
 \cline{4-5}\cline{6-7} \cline{8-9} \cline{10-11}
&amp; &amp; &amp; mean $\pm$  stdev &amp; min--max &amp; mean
$\pm$  stdev &amp; min--max &amp; mean $\pm$  stdev &amp; min--max &amp; mean $\pm$ 
stdev &amp; min--max \\
\endthead
1030 &amp; \0937 &amp; ${\upbeta}$-citronellene &amp; $\mathbf{6.3}  \pm
\mathbf{3.3}$  &amp; 3.4--12 &amp; $\mathbf{4.4} \pm   \mathbf{4.5}$ &amp; 1.2--7.6
&amp; $\mathbf{2.4}\pm \mathbf{2.4}$ &amp; 0.5--5.2 &amp; $\mathbf{9} \pm   
\mathbf{2.7}$ &amp; 5.4--11.1 \\
1119 &amp; \0960 &amp; Isomyocorene &amp; $\mathbf{71.5} \pm   \mathbf{11.2}$ &amp; 
57.5--87.4 &amp; $\mathbf{62} \pm    \mathbf{26.5}$ &amp; 43.2--80.7 &amp; 
$\mathbf{71.3}  \pm \mathbf{12.2}$ &amp; 60.1--84.3 &amp; 
$\mathbf{82} \pm    \mathbf{2.1}$ &amp; 79.2--83.9 \\
1156 &amp; \0979 &amp; Myrcene &amp; 0.2 $\pm$  0.2 &amp; 0--0.4 &amp; 0.1 $\pm$  0.2 &amp; 
0--0.2 &amp; 0 &amp; 0 &amp; 0.1 $\pm$  0.1 &amp; 0--0.2 \\
1168 &amp; \0984 &amp; Unknown1$^{\mathrm{b}}$ &amp; $\mathbf{1.8} \pm    \mathbf{0.3}$ &amp; 
1.3--2.3 &amp; $\mathbf{1.2} \pm    \mathbf{1.7}$ &amp; 0--2.4 &amp; 0.2 $\pm$ 0.3
&amp; 0--0.6 &amp; $\mathbf{1.4} \pm    \mathbf{0.7}$ &amp; 0.6--2.1 \\
1181 &amp; \0997 &amp; Unknown2$^{\mathrm{b}}$ &amp; 0.9 $\pm$  0.4 &amp; 0.4--1.6 &amp; 0.5 $\pm$ 
0.7 &amp; 0--1 &amp; 0 &amp; 0 &amp; 0.2 $\pm$  0.3 &amp; 0--0.5 \\
1205 &amp; \0736 &amp; 3-methyl-1-butanol$^{\mathrm{a}}$ &amp; 0 &amp; 0 &amp; 0.1 $\pm$  0.1 &amp; 0--0.1 &amp; 
$\mathbf{1.2} \pm    \mathbf{1.7}$ &amp; 0.1--3.1 &amp; 0 &amp; 0 \\
1267 &amp; 1006 &amp; 3,6-dimethyl-1,7-octadiene$^{\mathrm{a}}$ 
&amp; 0.1 $\pm$  0.1 &amp; 0--0.2 &amp; 0 &amp; 0 &amp; 0.1 $\pm$  0.1 &amp; 0--0.2 &amp; 0 &amp; 0 \\
1445 &amp; 1091 &amp; Unknown3$^{\mathrm{b}}$ &amp; $0.2 \pm  0.2$ &amp; 0--0.4 &amp; tr &amp; 0--0.1 &amp; 
$\mathbf{2.4} \pm    \mathbf{4.2}$ &amp; 0--7.2 &amp; 0 &amp; 0 \\
1456 &amp; 1380 &amp; Unknown4$^{\mathrm{b}}$ &amp; $\mathbf{1.8} \pm    \mathbf{2.9}$ &amp; 
0--7.6 &amp; 0 &amp; 0 &amp; 0 &amp; 0 &amp; 0.4 $\pm$  0.7 &amp; 0--1.4 \\
1517 &amp; 1398 &amp; 2,6,10-trimethyl-1,5,9-undecatriene$^{\mathrm{a}}$ &amp; 
0.5 $\pm$  0.5 &amp; 0--1.1 &amp; 0.1 $\pm$  0.1 &amp; 0--0.2 &amp; 0 &amp; 0 &amp; 0 &amp; 0 \\
1604 &amp; 1086 &amp; Hotrienol$^{\mathrm{a}}$ &amp; 0.3 $\pm$  0.3 &amp; 0--0.9 &amp; tr 
&amp; 0--0.1 &amp; 0 &amp; 0 &amp; 0 &amp; 0 \\
1632 &amp; 1420 &amp; Unknown5 &amp; 0.3 $\pm$  0.3 &amp; 0--0.7 &amp; tr &amp; 0--0.1 &amp; 0 &amp; 0 &amp; 0 &amp; 0 \\
1637 &amp; 1451 &amp; Alloaromadendrene &amp; 0.5 $\pm$  0.6 &amp; 0--1.5 &amp; 0.1 $\pm$ 0.1 &amp; 0--0.1 &amp; 0 &amp; 0 &amp; 0 &amp; 0 \\
1655 &amp; 1180 &amp; 3,6-dimethyl-1-octanol$^{\mathrm{a}}$ 
&amp; 0.1 $\pm$ 0.2 &amp; 0--0.4 &amp; tr &amp; 0--0.1 &amp; 0.1 $\pm$ 0.1 &amp; 0--0.2 &amp; 0 &amp; 0 \\
1666 &amp; \0863 &amp; 3-methylbutanoic acid$^{\mathrm{a}}$ 
&amp; 0.2 $\pm$ 0.2 &amp; 0--0.4 &amp; $\mathbf{3.7} \pm \mathbf{5.1}$ &amp; 
0.1--7.3 &amp; $\mathbf{3.3} \pm  \mathbf{1.6}$ &amp; 2.3--5.2 &amp; 0.2 $\pm$ 0.2 &amp; 0--0.5 \\
1701 &amp; 1472 &amp; Germacrene D &amp; 1 $\pm$ 1.4 &amp; 0--3.6 &amp; 0 &amp; 0 &amp; 0 &amp; 0 &amp; 0 &amp; 0 \\
2079 &amp; 1052 &amp; $p$-cresol &amp; $\mathbf{7.6} \pm    \mathbf{4.1}$ &amp; 
3.3--14.1 &amp; $\mathbf{22.9} \pm    \mathbf{17.6}$ &amp; 6.2--39.6 &amp; 
$\mathbf{17} \pm    \mathbf{12.3}$ &amp; 5.4--29.9 &amp; $\mathbf{5.6} \pm   \mathbf{3.2}$ &amp;
3.2--10.3 \\
2439 &amp; 1252 &amp; Indole &amp; $\mathbf{3} \pm    \mathbf{2.9}$ &amp; 0.6--10.1 &amp; 
$\mathbf{1.4} \pm  \mathbf{1.8}$ &amp; 0.1--2.7 &amp; $1 \pm    1$ &amp; 
0.1--1.9 &amp; 0.2 $\pm$    0.3 &amp; 0--0.5 \\
2485 &amp; 1344 &amp; Skatole &amp; $\mathbf{3} \pm   \mathbf{2.4}$ &amp; 0--6.8 &amp; 
$\mathbf{3.4} \pm \mathbf{3.7}$ &amp; 0.8--6 &amp; $\mathbf{1.2} \pm  \mathbf{1.5}$ &amp;
0.1--3 &amp; 0.7 $\pm$ 0.2 &amp; 0.6--1.1
\botline
\end{tabular}
\xxtabnote{Volatile compounds are classified by increasing order of
their Kovats retention index (RI) on the polar column (BP-20), RI on
the non-polar column (BP-1) are also given. Those with an average
relative proportion above 1\% are indicated in bold, and tr those with
an average relative proportion below 0.1\%.}
\tabnote{$^{\mathrm{a}}$Tentative compound identification.}
\tabnote{$^{\mathrm{b}}$Mass spectra given in the Supplementary Material.}
\end{sidewaystable}
\twocolumngrid

To the best of our knowledge, 2,6-dimethylocta-1,3,7-triene, also known
as isomyocorene or alpha-menthrene, was obtained by
palladium-catalysed telomerisation of isoprene~\cite{26}{. It has been
recently} reported as a minor VOC emitted by scentless flowers of
\textit{Tillsandia bandensis}~\cite{27}. However, we have a doubt
concerning the assignations of isomyocorene and cosmene in~\cite{27}.
The authors reported isomyocorene with a Kovats retention index value
of 1017 and cosmene with a Kovats retention index value of 968
(DB-5MS). Indeed, the Kovats retention index value of 1017 appears more
adequate for cosmene isomers which are reported to elute after ocimenes
(3,7-dimethyl-octatrienes)~\cite{28}. On the other hand, the Kovats
retention index assigned to cosmene (RI: 968 DB-5MS) appears to be more
appropriate for isomyocorene according to our data (RI: 960; BP-1).
Then either these two compounds have been reversed or they have been
misidentified~\cite{27}.\looseness=1

\subsection{Floral scent composition}\label{sec33}

In all samples, isomyocorene (see above) was the main floral VOC
representing between 43\% and 87\% of the floral scent composition.
Its corresponding alcohol 2,6-dimethyl-1,3,7-octatrien-6-ol
(synonym hotrienol) was tentatively identified in small amounts in
a few samples. 

\begin{table*}
\caption{\label{tab3}Diversity of inflorescence-visiting arthropods
for three populations of \textit{Arum pictum}\vspace*{3pt}}
\tabcolsep10pt
\begin{tabular}{cccc}
\thead
Population &amp; Coti &amp; Baratz &amp; Diga \\
Region &amp; Corsica &amp; West Sardinia &amp; East Sardinia \\
Insect Orders$\backslash$Sample size &amp; $N=26$ &amp; $N=30$ &amp; $N=8$ \\
\endthead 
Brachycera (Diptera) &amp; 126 &amp; 93 &amp; 13 \\
Nematocera (Diptera) &amp; \010 &amp; \03 &amp; 36 \\
Coleoptera &amp; \012 &amp; \01 &amp; \00 \\
Staphylinidae (Coleoptera) &amp; \017 &amp; 19 &amp; \02 \\
Braconidae (Hymenoptera) &amp; \0\02 &amp; \00 &amp; \02 \\
Others$^{\mathrm{a}}$ &amp; \051 &amp; 21 &amp; 10
\botline
\end{tabular}
\tabnote{Total arthropod abundances per order or family.}
\tabnote{$^{\mathrm{a}}$Others represent the sum of unlikely \textit{Arum}
pollinators: Collembola ($N=8$), Acari ($N=34$), Salticids ($N=29$),
Blattodea ($N=1$), Hemiptera ($N=1$), Formicidae ($N=4$), Isopoda
($N=1$), Lepidoptera ($N=1$), Psocoptera ($N=1$), Thysanoptera
($N=2$).}
\vspace*{8pt}
\end{table*}


The floral scent of the Corsican population of Coti was mainly composed
of seven VOCs representing 87--99\% of the total blend (Table~\ref{tab2}),
namely: isomyocorene (average 72\%), $p$-cresol (8\%),
${\upbeta}$-citronellene (6\%), indole and skatole (3\% each),
unknown1 and unknown4 (2\% each). The floral scent of the west
Sardinian population of Baratz was also mainly composed of seven VOCs
representing 98--100\% of the total blend (Table~\ref{tab2}), namely:
isomyocorene (average 62\%), $p$-cresol (23\%),
${\upbeta}$-citronellene (4\%), 3-methylbutanoic acid (4\%), skatole
(3\%), indole (1\%), and unknown1 (1\%). The floral scent of the
west Sardinian population of Gutierrez was also mainly composed of
seven VOCs representing 98--100\% of the total blend (Table~\ref{tab2}), 
namely: isomyocorene (average 71\%), $p$-cresol (17\%),
3-methylbutanoic acid (3\%), ${\upbeta}$-citronellene and unknown3 
(2\% each), skatole and 3-methyl-1-butanol (1.2\% each). Finally, the
floral scent of the east Sardinian population of Diga was composed of
only four main VOCs representing 96--99\% of the total blend
(Table~\ref{tab2}), namely: isomyocorene (average 82\%),
${\upbeta}$-citronellene (9\%), $p$-cresol (6\%), and\break unknown1 (1\%).

Contrary to the previous published floral scent of \textit{A. pictum},
we found noticeable differences. Among the five major VOCs
found previously in Corsican inflorescences~\cite{7}, two have also been
found in the present study, namely indole and skatole. The three
others were not identified in any of our samples from Corsica or
Sardinia~\cite{7}, (Table~\ref{tab2}). The isomyocorene was probably mistakenly
identified as ocimene, because the two compounds have similar framework
and differ by a double bond, the first is 
2,6-dimethyl-1,3,7-octatriene whereas the second is 
3,7-dimethyl-1,3,6-octatriene. Also, $p$-cresol was probably
mistakenly identified as benzyl alcohol, and ${\upbeta}$-citronellene,
synonym of dihydromyrcene, as dihydroocimene. No Kovats retention
indexes were published and several tentative compound identifications
were performed by manual mass spectra interpretations~\cite{7}.\looseness=1

\textit{Arum pictum} is characterised by a dung odour close to
horse/donkey dung~\cite{1}. Several foul-smelling VOCs were identified
from the floral odour of \unskip\break \textit{A. pictum}, most related to dung (as
$p$-cresol, skatole, or indole) or fermentation/cheese
(3-methyl-1-butanol, 3-methylbutanoic acid) in combination with floral
VOCs (${\upbeta}$-citronellene, 3,6-dimethyl-1-octanol, germacrene D).
Interestingly several major VOCs found in the floral scent of
\textit{A. pictum} were identified as important VOCs emitted by horse
dung such as $p$-cresol, ${\upbeta}$-citronellene, indole or
skatole~\cite{14}. On the other hand, the major VOC,
4-methyl-3-heptanone, from horse dung was not identified in the floral
blend of \textit{A. pictum}~\cite{14}. Such diverse VOC combination is
probably linked to the deceptive pollination strategy of \textit{Arum}
in order to attract insects associated to dung and/or fermenting
matters~\cite{8,9,11,12,13}.

\subsection{Visiting entomofauna}\label{sec34}

The visiting entomofauna was dominated in all populations by Diptera,
representing 69--78\% when considering all the arthropods but 80--89\%
when considering only insects (Table~\ref{tab3}). 
Secondary insect visitors were
Coleoptera, particularly Staphylinidae (rove beetles, 4--16\%). The
average number of \mbox{insects} trapped per inflorescence varied among
populations, with fewer in Baratz (3.9) than in Coti (6.4) and Diga
(6.6). Interestingly, the floral visitors in the populations of Coti
and Baratz were both highly dominated by Brachycera flies (respectively
75 and 80\%) with Staphylinidae (respectively 10 and 16\%) being
secondary visitors (Table~\ref{tab3}). On the other hand, while the floral
visitors in the population of Diga were mainly Diptera, Nematocera were
attracted in greater proportions (68\%) and Brachycera were less
frequent~(25\%).


In a previous study, the main pollinators of \textit{Arum pictum} were
sphaerocerid flies (Brachycera) and staphylinid beetles~\cite{7}.
Although we did not identify the Diptera to family level, many of the
Dipteran Brachycera were sphaerocerids, confirming the same kind of
visiting insect community in \textit{A. pictum} inflorescences from
north-west Sardinia (Baratz) and south-west Corsica (Coti and Sagone
in~\cite{7}). On the other hand, the population of north-east Sardinia
(Diga) presented a different visiting insect community dominated by
Nematocera (Table~\ref{tab3}). Psychodid and Chironomid flies
(Nematocera) are
known to be the main pollinators of other European deceptive
\textit{Arum} species~\cite{8,9,10,11,12,13}. Interestingly, the floral
scent in the population of Diga was the one with the lowest proportion
of dung associated VOCs (${\sim}$6.5\% in average) compared to the three
other studied populations (14--28\% in average; Table~\ref{tab2}).

\section{Conclusions}\label{sec4}

The autumn flowering \textit{Arum pictum} has developed a deceptive
pollination strategy combining a dark brown-purple spathe, floral heat
production and scent emission mimicking probably horse/donkey dung, and
attracting a coprophilous/saprophilous insect community composed mainly
of diverse Diptera and staphylinid Coleoptera. Several foul-smelling
VOCs related to dung ($p$-cresol, skatole, or indole) were
reported in notable quantities. But surprisingly the main floral VOC
was 2,6-dimethylocta-1,3,7-triene, also known as isomyocorene. Its
odour is not known but probably not related to dung scent as other
documented dimethyl-octatrienes (ocimenes) or acyclic hydrocarbon
monoterpenes. Further studies are necessary to better understand the
relationship between the floral scent composition in \textit{Arum
pictum} and the diversity of the attracted insects as it has been
done recently for\break \textit{A. maculatum}~\cite{11,12,13}.

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgements}
AA thanks her parents for supporting her and AG
during this work, particularly by hosting them in Calangianus and
accompanying them to the east-Sardinian studied site.

\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-236-suppl.pdf}}

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