## 2 Results and Discussion
### Analysis of \"in Vivo\" VOCs
\begin{table}
\begin{tabular}{l c c c c c c c c c} \hline \hline  & \multicolumn{3}{c}{**Control**} & \multicolumn{3}{c}{_O. sphegodes_ subsp. _sphegodes_} & \multicolumn{3}{c}{_O. bertoloni_} **subsp. _bencencensis_**} & \multicolumn{3}{c}{_N. tridentuna_} \\ \hline
**Compounds** & **RT \({}^{*}\)** & **Mean (\(n=3\))\({}^{*}\)** & **SD \({}^{*}\)** & **Mean (\(n=3\))\({}^{*}\)** & **SD \({}^{*}\)** & **Mean (\(n=3\))\({}^{*}\)** & **SD \({}^{*}\)** & **Mean (\(n=3\))\({}^{*}\)** & **SD \({}^{*}\)** \\ \hline pivalic acid & 25.21 & ND & - & ND & - & 0.3 & 0.09 & ND & - \\ hexanoic acid & 30.34 & ND & - & 0.32 & 0.21 & 0.42 & 0.00 & 0.15 & 0.00 \\ heptanoic acid & 31.99 & 0.21 & 1.17 & ND & - & 0.45 & 0.29 & 0.35 & 0.11 \\ octanoic acid & 33.08 & ND & - & 0.03 & 0.00 & 0.19 & 1.01 & 0.67 & 0.03 \\ nonanoic acid & 34.14 & 0.14 & 0.49 & 0.39 & 0.00 & 1.33 & 0.74 & 0.25 & 0.12 \\ decanoic acid & 35.41 & ND & - & ND & - & 0.84 & 0.00 & 0.91 & 0.00 \\ benzoic acid & 37.74 & 0.12 & 0.41 & 0.49 & 0.49 & 0.42 & 0.13 & 0.40 & 0.02 \\ \hline
**Total** & & **1.77** & & **13.25** & & **4.66** & & **4.05** & \\ \hline _Miscellaneous_ & & & & & & & & & \\ acetonitrile & 5.42 & 8.04 & 0.32 & 0.42 & 0.36 & 11.95 & 0.12 & 17.26 & 0.98 \\ dimethyl sulfone & 31.18 & 0.45 & 0.00 & 1.24 & 0.18 & 0.30 & 0.21 & 0.06 & 0.00 \\ \hline
**Total** & & **8.49** & & **1.65** & & **12.25** & & **17.32** & \\ \hline \hline \end{tabular}  
* Retention time, \({}^{*}\) Normalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of control samples (\(n=3\));
* Standard deviation (\(\pm\)), \({}^{4}\) Normalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of _O. sphegodes_ subsp. _sphegodes_, _O. bertoloni_ subsp. _bencensis_ and _N. tridentata_ living plants (\(n=3\)); ND: not detected; The most representative compounds for all orchid species were labeled with a number.  
\end{table} 
Table 1: _Cont._  
**Figure 1.** Volatile fingerprint of _O. sphegodes_ subsp. _sphegodes_ (**A**), _O. bertolonii_ subsp. _benacensis_ (**B**) and _N. tridentata_ (**C**) \"_in vivo_\" plant (the most representative compound peaks were labeled with numbers). _Ophrys_ flowers usually produce complex bouquets of volatiles, but presumably not all components are important for the attraction of male pollinators [6]. The literature reports that only some hydrocarbons, in particular very long-chain alkanes and alkenes and terpenes act as chemical mimicry of the sex pheromone of the virgin female pollinators. In agreement with our results, other authors confirmed terpenes and hydrocarbons together with alcohols and aldehydes as major constituents of orchid volatile profiles [10,11,19,20] and among these chemical classes a high emission of nonanal and caryophyllene by orchid flowers was previously reported [19,21,22]. In particular, the chemical composition of orchids pollinated by bees (e.g., _Ophrys_) revealed an abundance of terpenes (mono- and sesquiterpenes) [23]. Not all hydrocarbons, aldehydes and alcohols make an important contribution to the aroma of the plants. Some of them are abundant in cuticular waves and are said to have the primary function of protecting the plant from dehydration. Other compounds seem to play an important role in plant-herbivore interaction and acting as a solvent for the male-attracting volatiles [24]. The aromatic profile of _N. tridentata_ was characterized by a high concentration of aldehydes, hydrocarbons, esters and terpenes and in particular, as reported in Table 1 and Figure 1, 2-methylbutanoic acid methyl ester (12.36%), nonanal (7.92%), decanal (7.42%), hexane (7.14%) and \(\beta\)-pinene (4.59%). The genus _Neotinea_ includes food-deceptive species and, in contrast with the _Ophrys_ genus, is thought to attract and deceive mostly naive pollinators by generally mimicking nectariferous plants [5]. _N.