Specimens of G. horridus and G. curticornis emitted the defensive secretion in the form of a jet posteriorly (Gnaspini and Hara 2007). Eventually the emitted secretion spread over the carapace, where it evaporated.
According to the mass spectrometry data and retention index calculations, we identified five compounds in total in the defensive secretion of G. curticornis and G. horridus. We identified compounds 1, 2, 4 and 5 in the chromatogram of the defensive secretion of G. curticornis (Table 1, Fig. 1). On the other hand, we identified compounds 2, 3, 4 and 5 in that of G. horridus (Table 1, Fig. 2).
The compound with retention time of 8.78 min (compound 1) in the analysis of the defensive secretion of G. curticornis showed the following fragments of m/z 112 M+ (15); 97(12); 84(35); 83(12);69(12);58(28); 56(23); 55(100); 41(29) in the mass spectrum. Comparing these data with those in the literature (Wouters et al. 2013) and the calculation of the retention index, resulted in the identification of this compound as 4-methyl-1-hexen-3-one. In the 1H NMR spectra, it was observed the presence of olefinic hydrogen signals of a terminal doublet by the signals of three doublets at δ 6.27 (J = 17.5, 1.4 Hz), δ 5.77 (J = 10.5, 1.4 Hz) and δ 6.44 (J = 17.5, 10.5 Hz). The doublet at δ 1.10 (3H) of J = 7.5 Hz and the multiplet at δ 2.89 (1H) indicate the presence of a methyl vicinal to a methine group. The triplet at δ 0.79 (3H) along with the signal at δ 1.59 (m, 2H) indicate the presence of a methyl coupling with the methyl hydrogens. These data set allowed to identify the compound 1 as 4-methyl-1-hexen-3-one (1) (Table 2, Fig. 1).
The compound with the retention peak at 12.47 min (compound 2) in the defensive secretion of G. horridus (Fig. 1) and G. curticornis (Fig. 2) showed the molecular ion with m/z 126 and fragments of m/z 111, 84 and 55 (Table 1). The fragment of m/z 55 corresponds to an α-carbonyl cleavage, while the fragment of m/z 84 results from McLafferty rearrangement (Silverstein et al. 2005). According to the 1H NMR analysis, the presence of a terminal double bond is evidenced by the presence of three double doublets at δ 6.25 (J = 18.0 and 1.2 Hz), δ 5.85 (J = 10.5 and 1.4 Hz) and δ 6.47 (J = 18 and 10.4 Hz) close to a ketone. The methyl vicinal to the methine carbon can be evidenced by the signals of a multiplet at δ 2.83 (1H) and the doublet at δ 0.98 (J = 7.5 Hz, 3H). The triplet at δ 0.79 (J = 7.8 Hz, 3H) indicates a terminal methyl coupling the methyl hydrogens. The multiplet at δ 1.59 (2H) refers to the methylene at position 5 of the molecule, which is unprotected as a function of the carbonyl, while the signal at δ 1.34 (m, 2H) refers to the methylene hydrogens of carbon 6. All these data set confirmed this compound as 4-methyl-1-heptan-3-one 2 (Table 2).
The compound with the retention time of 14.11 min (compound 3) in the defensive secretion of G. horridus showed molecular ion of m/z 144, which corresponds to the molecular formula of C9H2O. Based on the molecular ion, it is possible to visualize the loss of 15 units, corresponding to one methyl in m/z 129. The fragment of m/z 45 indicates the presence of an alcohol, whereas the fragments of m/z 57 and 87, altogether with the remaining data, and the calculated retention index value, allowed us to identify this compound as 7-methyl-2-octanol 3. So far, 7-methyl-2-octanol 3 has not been described for other opilionid defensive secretion.
The isomers 4 and 5 in the defensive secretion of both species showed the molecular ions of m/z 224 and its characteristic fragmentation pattern allowed the identification led us to compounds 1-(6-(1-methyl-propyl)3,4-dihydro-2H-pyran-2-yl)2-methylbutanone (6) and 1-(6-isopropyl-3,4-dihydro-2H-pyran-2-yl) isobutanone (7) (Wouters et al. 2013) (Table 1).
The defensive secretions of both species of Gonyleptes studied here are a mixture of compounds, as usual for Opiliones (Hara et al. 2005; Jones et al. 2009; Raspotnig et al. 2005, 2015; Rocha et al. 2011). The compounds in the defensive secretion of both Gonyleptes spp. are similar (Table 1). We mapped those compounds in the modified phylogeny of Pinto-da-Rocha et al. (2014) (Fig. 3). The list of characters and the character matrix are in Tables 3 and 4, respectively.
All the vinyl ketones identified in the defensive secretion of Gonyleptes spp. are restricted to the K92 clade, as expected (Kury 1992; Caetano and Machado 2013). This corroborates the current view that vinyl ketones are synapomorphic for K92, derived from alkyl phenols and benzoquinones in the remaining gonyleptids (Caetano and Machado 2013).
The mapping of the characters indicates that all the identified vinyl ketones are homoplastic. However, the combined presence of 4-methyl-1-heptan-3-one; 1-(6-(1-methyl-propyl)3,4-dihydro-2H-pyran-2-yl)2-methyl-butanone (4) and 1-(6-isopropyl-3,4-dihydro-2H-pyran-2-yl) isobutanone (5) are exclusive of Gonyeleptes spp., hence allowing to diagnose it. Likewise, Sodreaninae can be diagnosed by the combined presence of 5-methyl-1-hexen-3-one and 1-(6-isopropyl-3,4-dihydro-2H-pyran-2-yl) isobutanone (5). Those results hint that some compounds might be useful for diagnosing monophyletic groups in Gonyleptidae. However, our taxa sampling is reduced, and many other groups close to either Gonyleptes or Soderaninae cannot be diagnosable compound wise. Part of those issues might be because of the unsettled phylogenetic relationship among K92 taxa. There are many competing phylogenetic relationships of K92 (Pinto-da-Rocha et al. 2014; Kury et al. 2022) and they can differ quite considerably from one another. Those issues hamper attempts to make inferences of the evolution of the defensive secretion in Gonyleptidae. Soon, when those phylogenetic relationships become more stable, we might have a clearer picture of the usefulness of compounds to diagnose and infer taxonomic groupings in Gonyleptidae.
Because behavioral studies on the effectiveness of defensive chemicals and natural history are scarce in Opiliones, it is unfortunately not possible to comment on the use of such chemicals against predators or in other contexts. Finally, we report 7-methyl-2-octanol (3) that is present in G. horridus defensive secretion for the first time in Opiliones. It is autapomorphic for this species and the first alcohol in a gonyleptid secretion to our knowledge.
Table 3
List of modified chemical characters from Caetano and Machado (2013) of the clade K92 (Arachnida, Opiliones) used in the present analysis
Number
|
Chemical character present in the odoriferous secretion
|
State
|
01
|
2-ethyl-1,4-benzoquinone
|
[0] absent; [1] present
|
02
|
2-ethyl-3-methyl-1,4-benzoquinone
|
[0] absent; [1] present
|
03
|
2-ethyl-5-methyl-1,4-benzoquinone
|
[0] absent; [1] present
|
04
|
2-ethyl-3,5-dimethyl-1,4-benzoquinone
|
[0] absent; [1] present
|
05
|
5-ethyl-2-methyl-1,4-benzoquinone
|
[0] absent; [1] present
|
06
|
2-methyl-1,4-benzoquinone
|
[0] absent; [1] present
|
07
|
2,3-dimethyl-1,4-benzoquinone
|
[0] absent; [1] present
|
08
|
2,5-dimethyl-1,4-benzoquinone
|
[0] absent; [1] present
|
09
|
2,5-dimethyl-3-ethyl-1,4-benzoquinone
|
[0] absent; [1] present
|
10
|
2,3,5-trimethyl-1,4-benzoquinone
|
[0] absent; [1] present
|
11
|
hept-5-en-3-ona
|
[0] absent; [1] present
|
12
|
1-hexen-3-one
|
[0] absent; [1] present
|
13
|
1-hepten-3-one
|
[0] absent; [1] present
|
14
|
3-methylhexan-2-ona
|
[0] absent; [1] present
|
15
|
4-methylhexan-3-one
|
[0] absent; [1] present
|
16
|
4-methyl-1-hexen-3-one
|
[0] absent; [1] present
|
17
|
4-methyl-1-heptan-3-ona
|
[0] absent; [1] present
|
18
|
5-methyl-1-hexen-3-one
|
[0] absent; [1] present
|
19
|
7-metiloct-6-en-4-ona
|
[0] absent; [1] present
|
20
|
4,5- dimethylheptan-3-one
|
[0] absent; [1] present
|
21
|
1-(6-butyl-3,4-di-hydro-2H-pyran-2-yl) pentanone
|
[0] absent; [1] present
|
22
|
1-(6-(1-methyl-propyl)3,4-dihydro-2H-pyran-2-yl)2-methyl-butanone
|
[0] absent; [1] present
|
23
|
1-(6-(propyl)3,4-dihydro-2H-pyran-2-yl)butanone
|
[0] absent; [1] present
|
24
|
1-(6-propyl-3,4-dihydro-2H-pyran-2-yl)2-methyl-butanone
|
[0] absent; [1] present
|
25
|
1-(6-propyl)3,4-dihydro-2H-pyran-2-yl) isobutanone
|
[0] absent; [1] present
|
26
|
1-(6-isopropyl-3,4-dihydro-2H-pyran-2-yl) isobutanone
|
[0] absent; [1] present
|
27
|
1-(6-butyl-3,4-di-hydro-2H-pyran-2-yl) pentanone
|
[0] absent; [1] present
|
28
|
2-methyl-5-ethylphenol
|
[0] absent; [1] present
|
29
|
2,3-dimethylphenol
|
[0] absent; [1] present
|
30
|
2,5-dimethylphenol
|
[0] absent; [1] present
|
31
|
2,3,4-trimethylphenol
|
[0] absent; [1] present
|
32
|
2,3,6-trimethylphenol
|
[0] absent; [1] present
|
33
|
7-methyl-2-octanol
|
[0] absent; [1] present
|