All relevant data are within the manuscript and its Supporting Information files.

Introduction {#sec001}
============

Brazil is the richest country in terms of biodiversity in the world. Currently, the Brazilian flora harbors at least 46,500 species, of which 4,754 are algae, 33,158 are angiosperms, 1,567 bryophytes, 5,719 fungi, 30 gymnosperms and 1,345 are ferns and lycophytes \[[@pone.0224406.ref001]\]. As living organisms, plants communicate with the environment, and some of their strategies are related to defense \[[@pone.0224406.ref002]\] and others to reproduction \[[@pone.0224406.ref003]\].

EOs are an organic mixture of compounds occurring in specialized plant cells or glands, that play a fundamental role in plant-biotic/abiotic environment interactions \[[@pone.0224406.ref004]\]. They originate from intermediate products of the main metabolic pathways involved in the production of sugars, lipids and proteins. Characteristically, EOs have high genetic plasticity and wide chemical diversity, which allows the plants to adapt to the environmental demands, which are in continuously changing \[[@pone.0224406.ref005]\]. Among the functions exerted by EOs in plants, defense against bacteria, virus and fungi, as well as herbivorous insects or animals have been well-described \[[@pone.0224406.ref006]\].

Plants within the Myristicaceae have been known to produce EOs. Ethnopharmacological interest in this family emerged in the 1950's, when Richard Schultes disclosed the discovery that some species of the genus *Virola* were sources of snuffs *yakee*, *parika* or *epena*, a powerful hallucinogenic narcotic used by Indian people from the Amazon region \[[@pone.0224406.ref007]\]. Myristicaceae species are still used for many purposes by native Amazonians, for example, in the preparation of arrows and for the treatment of dermatological or stomach infections, rheumatism, tumors, among others \[[@pone.0224406.ref008], [@pone.0224406.ref009], [@pone.0224406.ref010]\]. Some species of the genus *Iryanthera* have been popularly used as hallucinogenic, such as the bark of *I*. *macrophylla* (Benth.) Warb., or used as antidiarrhoeic, as the bark of *I*. *tessmanii* Markgr.; moreover, the wood of several species has been used in construction \[[@pone.0224406.ref011]\]. A phytochemical study made with the fruits of *I*. *lancifolia* Ducke revealed the presence of dihydrochalcones and flavonolignans which showed higher antioxidant activity in relation to α-tocopherol and quercetin \[[@pone.0224406.ref012]\]. *I*. *polyneura* were chemically accessed \[[@pone.0224406.ref013]\], and diarylpropanoids were isolated from the wood of their trunks.

Due to the lack of chemical and biological information regarding *I*. *polyneura*, the aim of the present work was to assess the chemical composition of the EOs obtained from the leaves of *I*. *polyneura* throughout a two-year span. Further, researchers aimed at the comprehension of how climatic factors such as the relative humidity, precipitation, minimum, average and maximum daily temperatures, insolation, wind and evaporation, and the seasonal variation might affect the chemical composition of the oils.

Material and methods {#sec002}
====================

Collection of botanical material {#sec003}
--------------------------------

The collection of botanical material was carried out in a *terra-firme* forest, within the Amazon Basin. The collections were made under licenses \# 14895 (ICMBio/MMA/Brazil) and 12A/2008 (IBAMA/CGen/MMA), as required by the Brazilian Government. A voucher from each individual was collected as described in [Table 1](#pone.0224406.t001){ref-type="table"}. The present study was based on a previous one made with *Osteophloeum platyspermum* (Spruce ex A.DC.) Warb., Myristicaceae \[[@pone.0224406.ref014]\].

10.1371/journal.pone.0224406.t001

###### Indexation of botanical material collected from *Iryanthera polyneura*.

DBH = diameter at breast height.

![](pone.0224406.t001){#pone.0224406.t001g}

  ----------------------------------------------------------------------------------------------------------------------------------------
  Individual (Field Number)   Herbarium Voucher      Determined by   DBH (cm)   Universal Transverse Marcator (UTM Zone 20)   
  --------------------------- ---------------------- --------------- ---------- --------------------------------------------- ------------
  22                          Oliveira A.A. 4064;\   Paciencia, M.   14.1       780103.21                                     9686375.79
                              UNIP 5170                                                                                       

  80                          Oliveira 4144;\        Paciencia, M.   17.4       780073.14                                     9686558.12
                              UNIP 5279                                                                                       

  530                         Paciencia, M. 3609;\   Paciencia, M.   14.5       780141.24                                     9686608.38
                              UNIP 8566                                                                                       
  ----------------------------------------------------------------------------------------------------------------------------------------

Collections were made from trees having DBH≥10cm (diameter at breast height), closely located in the same hectare plot, containing enough leaf biomass to allow for a periodical collection spanning two years, from October 2009 to December 2011. The individuals were identified as 22EO, 80EO and 530EO. Individuals 22EO and 530EO provided 15 material collections, while 80EO provided 14. The collection dates were determined in a random way, depending on the expedition logistics.

Samples of the collected material for botanical research were deposited at the UNIP Herbarium ([Table 1](#pone.0224406.t001){ref-type="table"}), São Paulo, Brazil. The material was cleaned so as to remove contaminants such as insects or other animals, other organs of the same plant, other plants, sand and earth. The cleaned material was kept in a cold room until it was used to obtain the volatile oils.

Preparation of the EOs {#sec004}
----------------------

Fresh leaves of *I*. *polyneura* were periodically collected from the same individuals in order to obtain EOs. EOs were obtained by hydrodistillation in a Clevenger apparatus \[[@pone.0224406.ref015]\] for four hours. The oils were collected from the Clevenger apparatus with the aid of pentane, and were evaporated using a rotary evaporator apparatuses (Buchi, Switzerland). The remaining water was then removed with the addition of anhydrous sodium sulfate. The yields (mg/mg) of all EO samples (15 samples obtained from individuals 22EO, and, 14 samples obtained from individual 80EO and 530EO) were evaluated and then stored at -10°C, until use. To perform gas chromatography coupled to mass spectrometer analyses (GC-MS), 20μL of each oil were diluted in 980μL of acetone.

Analysis of volatile oils by gas chromatography coupled to mass spectrometry (GC-MS) {#sec005}
------------------------------------------------------------------------------------

GC-MS analyses were performed on a Shimadzu 14B/QP5050A apparatus, with quadrupole type analyzer of the same brand. The column used was BPX5 (non-polar 5% Phenyl Polisylphenlene Siloxane), 30m, with internal diameter of 0.25mm. The running conditions of the gas chromatography were: initial oven temperature 60°C (6 min), final temperature 320°C (8 min), temperature increase of 10°C/min and a total running time of 40 min, column pressure was 150.0kPa with a column flow of 2.5mL/min, linear velocity of 58, split ratio of 9 and total flow of 30.0mL/min. Each of the 44 acetone-diluted samples were injected at a volume of 1μL. The substances were identified by comparing the obtained mass spectra with libraries, such as Willey229, NIST107, SHIM1607 and NIST21, and with a reference books and retention indices \[[@pone.0224406.ref016]\].

The retention indices (RI) were obtained from the injection of a mixture of n-alcanes (C~9~H~20~--C~25~H~52~, Sigma Aldrich, 99%) using Van den Dool and Kratz equation \[[@pone.0224406.ref017]\].

Experimental design {#sec006}
-------------------

With three exceptions, yields (mg/mg) of all EO samples were obtained. Fifteen samples were obtained from individuals 22EO and 530EO and 14 samples obtained from individual 80EO.

The chemical and statistical variation were calculated from 41 samples of EOs (15 from 22EO, 12 from 80EO and 14 from 530EO) and were based on terpene percentages. Two EOs from individual 80EO and one EO from individual 530EO were discarded because the number of terpenes detected after GC-MS analysis was very low. Therefore, the samples were considered outliers, since they created trends in the analyses in a very significant way. For the multivariate analyses, 41 EOs were considered as the cases and the 59 terpenes as the variables. Climatic variables used for running the analyses were relative humidity, precipitation, daily temperatures (minimum, average and maximum), insolation, wind speed and evaporation, where data obtained from the day of collection was considered ([www.inpe.gov.br](http://www.inpe.gov.br)). Seasons in the Amazon rain forest are defined according to the rain intensity, so the period of the year when the rain is more intense (accumulated precipitation as high as 3,500mm/year) is defined as the rainy season (RS), popularly known as the "winter", and spans from January to July. The period of the year known as "summer", or dry season (DS), is a period in which the precipitation drops down to 900mm/year and spans from July to December \[[@pone.0224406.ref018], [@pone.0224406.ref019]\].

Statistical analyses {#sec007}
--------------------

One-way ANOVAs and t-tests were performed in order to compare the occurrence of the terpene levels among individuals and between seasons. Differences were considered significant if α\<0.05 \[[@pone.0224406.ref020]\]. NMDS analysis was performed in the association with ANOSIM test of hypotheses, which is similar to the non-parametric analysis of variance based on similarities among samples \[[@pone.0224406.ref021]\]. In the present case we used ANOSIM to test two different null hypotheses (H~0~). The first was that there were no differences in the percentual-abundance and the composition of terpenes of the three *Iryanthera* individual plants (22EO, 80EO, and 530EO). The second was that there was no difference between EO samples obtained during dry and rainy seasons. Correspondence analysis (CA) and canonical correspondence analysis (CCA) were also conducted using the software MVSP, Multivariate Statistical Package version 3.22 (Kovach Computing Services) and Primer 6 version 6.1.6 (Primer-E Ltd.).

Results and discussion {#sec008}
======================

Yields of the volatile oils obtained from the leaves of three individuals of *Iryanthera polyneura* spanned from 0.03% to 0.43%, with the lowest yield observed for 22EO1 and the highest for the 530EO9 ([Table 2](#pone.0224406.t002){ref-type="table"}, [Fig 1](#pone.0224406.g001){ref-type="fig"}). The yields of the oils from individuals 80EO and 530EO were significantly higher than the oils from the individual 22EO (*F*~(2,39)~ = 5.238; *P* = 0.0097).

10.1371/journal.pone.0224406.t002

###### Yields (%) obtained for the essential oils obtained from the leaves of three different *Iryanthera polyneura* individual plants.

\#22, \#80 and \#530 identifies different individuals, while EO1 toEO15 identifies dates of collection. Essential oil yield means for each individual was compared using one-way ANOVA, followed by Tukey post-test, considered significant if α\<0.05.

![](pone.0224406.t002){#pone.0224406.t002g}

  Essential oil ID                     Yield (%)                                         Essential oil ID   Yield (%)                                          Essential oil ID   Yield (%)                                         Date of collection
  ------------------------------------ ------------------------------------------------- ------------------ -------------------------------------------------- ------------------ ------------------------------------------------- --------------------
  22EO1                                0.033                                             80EO1              0.0889                                             530EO1             0.1041                                            10/01/2009
  22EO2                                0.0916                                            80EO2              0.1332                                             530EO2             0.1487                                            11/01/2009
  22EO3                                0.2821                                            80EO3              0.1124                                             530EO3             0.3483                                            02/03/2010
  22EO4                                0.0769                                            80EO4              0.0799                                             530EO4             0.1325                                            03/12/2010
  22EO5                                0.1745                                            80EO5              NT                                                 530EO5             0.3635                                            05/15/2010
  22EO6                                0.0907                                            80EO6              0.1104                                             530EO6             0.1708                                            05/28/2010
  22EO7                                0.1232                                            80EO7              NT                                                 530EO7             0.2657                                            08/29/2010
  22EO8                                0.1313                                            80EO8              0.0767                                             530EO8             0.1201                                            11/05/2010
  22EO9                                0.1195                                            80EO9              0.3378                                             530EO9             0.4362                                            12/14/2010
  22EO10                               0.0617                                            80EO10             0.0828                                             530EO10            0.191                                             02/11/2011
  22EO11                               0.0631                                            80EO11             0.115                                              530EO11            0.1419                                            04/15/2011
  22EO12                               0.0561                                            80EO12             0.0774                                             530EO12            0.1058                                            07/08/2011
  22EO13                               0.0723                                            80EO13             0.0767                                             530EO13            0.1346                                            08/20/2011
  22EO14                               0.0817                                            80EO14             0.0814                                             530EO14            0.107                                             10/21/2011
  22EO15                               0.0633                                                                                                                  530EO15            0.1348                                            12/16/2011
  mean±SD                              0.10±0,02[^A^](#t002fn001){ref-type="table-fn"}   mean±SD            0.11±0,02 [^A^](#t002fn001){ref-type="table-fn"}   mean±SD            0.19±0,03[^B^](#t002fn002){ref-type="table-fn"}   
  **Essential oil yield comparison**                                                                                                                                                                                                
  Barlett's statistic corrected        4.479                                             *P* = 0.1065                                                                                                                               
  One-way ANOVA/Tukey                  *F*~(2,39)~ = 5.238                               ***P* = 0.0097**                                                                                                                           
  Multiple comparisons                 22EO vs. 80EO                                     *P* = 0.9154                                                                                                                               
  22EO vs. 530EO                       ***P* = 0.0120**                                                                                                                                                                             
  80EO vs. 530EO                       ***P* = 0.0487**                                                                                                                                                                             

^A^ = no differences among means;

^B^ = differences among means. NT = not tested.

![Yields from the essential oils obtained from the *Iryanthera polyneura* leaves and the percentage of the major compounds spathulenol, α-cadinol and τ-muurolol present.](pone.0224406.g001){#pone.0224406.g001}

GC-MS analyses of the 44 EOs showed that terpenes were the only type of compound that was identified for this species in the present analysis ([Table 3](#pone.0224406.t003){ref-type="table"}, [Fig 2](#pone.0224406.g002){ref-type="fig"}), unlike the possible occurrence of phenylpropanoids in some species of Myristicaceae, such as *Myristica fragrans* \[[@pone.0224406.ref022]\]. Sesquiterpene hydrocarbons prevailed in the oils of the three *I*. *polyneura* individual plants, followed by the presence of oxygenated sesquiterpenes. Forty four terpenes, or 74.5%, were identified, and 15 compounds were not determined (25.4%). Spathulenol (6.42 ± 1.02%), α-cadinol (5.82 ± 0.40%) and τ-muurolol (5.24 ± 0.03%) were the major compounds found in the EOs (*N* = 44). Suffredini et al. (2016) has already reported the highest percentages of spathulenol and α-cadinol in EOs from another species of Myristicaceae, such as *Osteophloeum platyspermum* Warb.

10.1371/journal.pone.0224406.t003

###### Chemical composition (in percentage, %) of essential oils obtained from individual *I*. *polyneura* leaves from 22EO, 80EO and 530EO individuals and a summary of the class of compounds identified.

![](pone.0224406.t003){#pone.0224406.t003g}

                                      22EO     80EO                                    530EO                                                          
  ----------------------------------- -------- --------------------------------------- -------- ---------------- -------------- -------------- ------ ------
  p-Cymene                            1027     [\*](#t003fn002){ref-type="table-fn"}   0.01     0.01             0.02           0.01           0.01   0.01
  Linalool                            1109     [\*](#t003fn002){ref-type="table-fn"}   0.06     0.03             0.03           0.03                  
  Terpinen-4-ol                       1182     [\*](#t003fn002){ref-type="table-fn"}   0.03     0.03             0.02           0.02                  
  p-Cimen-8-ol                        1190     [\*](#t003fn002){ref-type="table-fn"}   0.02     0.01                                                  
  α-Terpineol                         1195     [\*](#t003fn002){ref-type="table-fn"}   0.06     0.02             0.03           0.02           0.01   0.01
  δ-Elemene                           1337     1335                                    1.08     0.17             0.97           0.10           1.44   0.22
  α-Cubebene                          1349     1345                                    0.10     0.03             0.21           0.15           0.17   0.04
  Cycloisosativene                    1364     [\*](#t003fn002){ref-type="table-fn"}   0.35     0.10             0.45           0.09           0.52   0.09
  Cyclosativene                       1366     1369                                    0.98     0.26             0.46           0.10           1.20   0.25
  α-Yllangene                         1370     1373                                    0.12     0.04             0.06           0.03           0.24   0.06
  α-Copaene                           1375     1374                                    0.43     0.09             0.51           0.05           0.68   0.12
  β-Bourbonene                        1383     1387                                    0.63     0.14             0.33           0.06           0.37   0.07
  β-Elemene                           1391     1389                                    2.56     0.28             3.84           0.25           3.35   0.18
  β-Caryophyllene                     1417     1417                                    0.64     0.09             0.63           0.10           0.81   0.11
  Sesq1                               1428     [\*](#t003fn002){ref-type="table-fn"}   0.72     0.13             1.26           0.20           0.98   0.19
  γ-Elemene                           1433     1434                                    0.31     0.05             0.50           0.08           0.59   0.08
  Aromadendrene                       1437     1439                                    0.65     0.08             1.36           0.11           1.03   0.10
  α-Humulene                          1452     1452                                    0.13     0.04             0.16           0.05           0.17   0.03
  α-Gurjunene                         1408     1409                                    0.01     0.01             0.06           0.03           0.08   0.03
  α-Patchoulene                       1454     1454                                    0.01     0.01                                           0.08   0.07
  Allo-Aromadendrene                  1459     1458                                    0.12     0.03             0.30           0.05           0.26   0.04
  γ-Muurolene                         1475     1478                                    2.85     0.28             2.90           0.15           3.90   0.33
  α-Amorphene                         1479     1483                                    1.25     0.15             1.28           0.12           1.85   0.23
  Sesq2                               1484     [\*](#t003fn002){ref-type="table-fn"}   0.65     0.09             0.98           0.05           0.93   0.07
  α-Elemene                           1493     [\*](#t003fn002){ref-type="table-fn"}   1.36     0.12             1.70           0.11           1.94   0.15
  α-Muurolene                         1499     1500                                    2.80     0.30             3.12           0.28           4.03   0.41
  β-Bisabolene                        1508     1505                                                                                            0.01   0.01
  α -Cadinene                         1513     1513                                    1.53     0.32             1.73           0.07           2.10   0.23
  Sesq3                               1514     [\*](#t003fn002){ref-type="table-fn"}   0.69     0.27             0.09           0.09           0.33   0.21
  Calamenene                          1522     1521                                    1.92     0.15             1.25           0.14           2.41   0.27
  Valencene                           1533     1532                                    0.61     0.07             0.85           0.06           1.00   0.07
  α-Cadinene                          1536     1537                                    0.10     0.06                                                  
  α-Calacorene                        1541     1544                                    0.60     0.14             0.31           0.08           0.30   0.10
  Elemol                              1549     1548                                    2.08     0.24             1.13           0.16           1.11   0.12
  Germacrene B                        1555     1559                                    0.72     0.13             1.07           0.28           0.97   0.15
  Longifolene                         1558     1555                                    0.35     0.03             0.80           0.08           0.41   0.07
  Sesq4                               1565     [\*](#t003fn002){ref-type="table-fn"}   3.00     0.22             4.15           0.34           4.17   0.32
  Spathulenol                         1577     1577                                    6.51     0.47             8.13           0.42           4.61   0.37
  Sesq5                               1582     [\*](#t003fn002){ref-type="table-fn"}   4.79     0.21             7.14           0.40           5.09   0.43
  γ-Selinene                          1590     [\*](#t003fn002){ref-type="table-fn"}   1.03     0.13             2.00           0.17           1.04   0.14
  Guaiol                              1597     1600                                    0.79     0.13             0.13           0.04           0.18   0.03
  Globulol                            1601     1590                                    1.30     0.15             2.62           0.21           0.74   0.24
  Eremophyllene                       1601     [\*](#t003fn002){ref-type="table-fn"}   0.05     0.05                                           1.05   0.21
  Sesq6                               1611                                                                       0.78           0.11                  
  Sesq7                               1624     [\*](#t003fn002){ref-type="table-fn"}   1.87     0.26             1.89           0.15           1.91   0.22
  Sesq8                               1628     [\*](#t003fn002){ref-type="table-fn"}   5.19     0.47             3.58           0.30           4.33   0.25
  Sesq9                               1631     [\*](#t003fn002){ref-type="table-fn"}   1.82     0.26             1.29           0.06           1.37   0.12
  τ-Muurololl                         1641     [\*](#t003fn002){ref-type="table-fn"}   5.28     0.29             5.19           0.26           5.26   0.37
  Sesq10                              1646     [\*](#t003fn002){ref-type="table-fn"}   2.46     0.25             2.45           0.11           2.87   0.19
  β-Eudesmol                          1649     1649                                    1.28     0.22             0.89           0.04           0.76   0.05
  α-Cadinol                           1654     1654                                    5.35     0.65             6.61           0.41           5.49   0.45
  γ-Eudesmol                          1664     1630                                    0.07     0.04                                           0.16   0.06
  Sesq11                              1667     [\*](#t003fn002){ref-type="table-fn"}   0.19     0.13             0.74           0.17           1.16   0.18
  Cadalene                            1673     1675                                    1.13     0.12             0.98           0.09           1.41   0.09
  Sesq12                              1693     [\*](#t003fn002){ref-type="table-fn"}   0.15     0.06             0.81           0.10           0.86   0.11
  Sesq13                              1706     [\*](#t003fn002){ref-type="table-fn"}   0.23     0.08             0.46           0.02           0.31   0.03
  Sesq14                              1716     [\*](#t003fn002){ref-type="table-fn"}   0.67     0.16             0.88           0.15           0.70   0.16
  cis-Jasmone                         1725     [\*](#t003fn002){ref-type="table-fn"}   0.01     0.01                                                  
  Sesq15                              1748     [\*](#t003fn002){ref-type="table-fn"}   0.09     0.05             0.30           0.03           0.24   0.03
  Monoterpene hydrocarbon (%, MH)     0.69                                             0.85                      0.47                                 
  Oxigenated monoterpene (%, OM)      3.70                                             1.42                      0.23                                 
  Sesquiterpene hydrocarbon (%, SM)   78.24                                            80.74                     81.82                                
  Oxigenated sesquiterpene (%, OS)    17.36                                            17.00                     17.48                                
  *MH OM SH OS amounts*                                                                                                                               
                                      MH (A)   0.67                                    0.1102   F (3,8) = 2580   **\<0.0001**                         
                                      OM (B)   1.783                                   1.018                     A-B            0.7197                
                                      SH (C)   80.27                                   1.06                      A-C            **\<0.0001**          
                                      OS (D)   17.28                                   0.1442                    A-D            **\<0.0001**          
                                                                                                                 B-C            **\<0.0001**          
                                                                                                                 B-D            **\<0.0001**          
                                                                                                                 C-D            **\<0.0001**          

Sesq = unidentified sesquiterpenes;

\* = not found

![Structure of the compounds identified in the essential oils from the leaves of *Iryanthera polyneura*.\
MH = monoterpene hydrocarbon; OM = oxygenated monoterpene; SH = sesquiterpene hydrocarbon; OS = oxygenated sesquiterpene.](pone.0224406.g002){#pone.0224406.g002}

[Table 3](#pone.0224406.t003){ref-type="table"} shows the qualitative and quantitative terpene variation in individuals 22EO, 80EO and 530EO. Cis-jasmone and α-cadinene were solely found in 22EO, while sesq6 exclusively occurred in 80EO and β-bisabolene in 530EO. This difference may suggest that each individual has genetic variability or that each one may be in a particular physiological phase influenced by intrinsic or extrinsic factors. Spathulenol, elemol, α-muurolene, τ-muurolene, sesq2, sesq3, and τ-muurolol were commonly found in all individuals. Hydrogenated and oxygenated monoterpenes (MH and OM) and sesquiterpenes (SH and OS) were identified in the oils obtained from the three individuals. The terpene content in each of the four groups was summed and used in the statistical evaluation. According to the ANOVA, the SH-group was significantly more prevalent in the oils, in relation to the other three groups (*F*~(3,8)~ = 2.580; *P* \< 0.001), as was the group OS.

Results obtained from the statistical analyses of the chemical composition percentage and seasonal variation were assessed. A *t*-test was performed in order to evaluate variation between terpene levels in relation to seasonality, and a F test to compare variances was applied to all *t*-tests (*P* \> 0.05). For that analysis, the total terpene percentages were summed for each of the 41 oils, which produced two groups, those EOs from the DS and those from the RS. The analyses were made for each set of oils, 22EO, 80EO and 530EO. The total terpene percentage of oils occurring in 22EO was significantly lower in DS samples (*t*~0.05(2),12~ = 3.603; *P* = 0.0036), but higher for the oils from 80EO (*t*~0.05(2),9~ = 2.305; *P* = 0.0467), while no differences were observed in the terpenes from 530EO (*t*~0.05(2),12~ = 1.295; *P* = 0.2197). When considering the total amount of terpenes of all individuals, which were pooled together to evaluate a variation for the species, no differences were observed between the RS and DS (*t*~0.05(2),13~ = 0.6985; *P* = 0.4972).

Statistical analyses were also made considering only the terpenes that were common to all of the 41 EOs, τ-muurolene, α-muurolene, elemol, spathulenol, τ-muurolol, sesq 2 and sesq 3. The analysis revealed that the common terpenes occurred in higher amounts in the DS (*t*~0.05(2),37~ = 2.078; *P* = 0.0447).

Spathulenol, τ-muurolol and α-cadinol were the major compounds identified. As seen in [Table 4](#pone.0224406.t004){ref-type="table"}, the occurrence of spathulenol was measured in each individual, according to the seasonality. It was observed that spathulenol was higher in the RS for individual 22EO (*t*~0.05(2),11~ = 2.345; *P* = 0.0388), but in individuals 80EO and 530EO, levels were not affected by the time of year of collection. The occurrence of spathulenol among the three individuals and the differences between the RS and DS were evaluated. In the analyses, the content of spathulenol was significantly higher in both the RS (*F*~(2,19)~ = 10.82; *P* = 0.0007) and the DS (*F*~(2,16)~ = 5.911; *P* = 0.012), in the oils derived from individual 80EO. The occurrence of τ-muurolol was also evaluated for each individual according to the seasonality, and it was observed that the occurrence of this terpene was significantly higher during DS, for 22EO (*t*~0.05(2),11~ = 2.311; *P* = 0.0412), 80EO (*t*~0.05(2),10~ = 3.012; *P* = 0.0131) and 530EO (*t*~0.05(2),11~ = 2.349; *P* = 0.0385). The total amount of τ-muurolol calculated for the three individuals together was seasonally compared, and a significantly higher level of production of the terpene during the DS vs the RS was confirmed (*t*~0.05(2),46~ = 2.22; *P* = 0.0314). Finally, the seasonal variation of α-cadinol differed from what was observed for spathulenol and for τ-muurolol. The percentage of α-cadinol was higher in the DS for individuals 22EO (*t*~0.05(2),11~ = 2.436; *P* = 0.0331) and 80EO (*t*~0.05(2),10~ = 2.912; *P* = 0.0155), while the compound was predominant in RS in individual 530EO (*t*~0.05(2),11~ = 2.257; *P* = 0.0453). The total amount of α-cadinol produced by all three individuals together, when seasonally compared, resulted in a significant higher occurrence of α-cadinol in DS (*t*~0.05(2),39~ = 2.674; *P* = 0.0109).

10.1371/journal.pone.0224406.t004

###### Statistical analyses based on the chemical composition (in percentage, %) of the essential oils obtained from the leaves of individuals 22EO, 80EO and 530EO of *Iryanthera polyneura* in either the dry season (DS) or the rainy season (RS).

![](pone.0224406.t004){#pone.0224406.t004g}

  ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Analysis                                                                                                                                factors          mean           SE                 F / t         comparison       P/adjusted P   Analysis    factors            mean         SE               F / t        comparison   P/adjusted P         Analysis   factors          mean         SE           F / t   comparison   P/adjusted P
  --------------------------------------------------------------------------------------------------------------------------------------- ---------------- -------------- ------------------ ------------- ---------------- -------------- ----------- ------------------ ------------ ---------------- ------------ ------------ -------------------- ---------- ---------------- ------------ ------------ ------- ------------ --------------
  *Comparison of the percentage means of the major compounds occurring in the rainy and the dry seasons of each individual plant*                                                                                                                                                                                                                                                                                                 

  *spathulenol*                                                                                                                           *α-cadinol*      *τ-muurolol*                                                                                                                                                                                                                                                           

  t-tests                                                                                                                                 22EO (A)                                           t = 2.345\    **0.0388**       t-tests        22EO (A)                                    t = 2.436\       **0.0331**   t-tests      22EO (A)                                         t = 2.311\   **0.0412**                        
                                                                                                                                                                                             df = 11                                                                                   df = 11                                                                                     df = 11                                        

                                                                                                                                          RS               7.307          0.6993                                                                       RS                 5.466        0.4597                                                                     RS               4.793        0.3727                            

                                                                                                                                          DS               5.428          0.2749                                                                       DS                 7.012        0.4258                                                                     DS               5.993        0.3538                            

                                                                                                                                          80EO (B)                                           t = 0.8917\   0.3935                          80EO (B)                                    t = 2.912\       **0.0155**                80EO (B)                                         t = 3.012\   **0.0131**                        
                                                                                                                                                                                             df = 10                                                                                   df = 10                                                                                     df = 10                                        

                                                                                                                                          RS               8.51           0.8227                                                                       RS                 5.695        0.5491                                                                     RS               4.593        0.3467                            

                                                                                                                                          DS               7.748          0.2299                                                                       DS                 7.533        0.3114                                                                     DS               5.79         0.194                             

                                                                                                                                          530EO (C)                                          t = 1.314\    0.2135                          530EO (C)                                   t = 2.257\       **0.0453**                530EO (C)                                        t = 2.349\   **0.0385**                        
                                                                                                                                                                                             df = 12                                                                                   df = 11                                                                                     df = 11                                        

                                                                                                                                          RS               4.199          0.4534                                                                       RS                 4.83         0.6215                                                                     RS               4.439        0.46                              

                                                                                                                                          DS               5.162          0.5943                                                                       DS                 2.948        0.309                                                                      DS               5.833        0.3482                            

  *Comparison of the percentage means of major compounds occurring in the rainy season among the three individual plants*                                                                                                                                                                                                                                                                                                         

  *spathulenol*                                                                                                                           *α-cadinol*      *τ-muurolol*                                                                                                                                                                                                                                                           

  ANOVA 1-factor                                                                                                                                                          F (2,19) = 10.82   **0.0007**    ANOVA 1-factor                              F (2,17) = 2.068   0.1571       ANOVA 1-factor                             F (2,19) = 0.1554    0.8572                                                                     

                                                                                                                                          22EO (A)         6.948          0.7043                           A-B              0.2564                     22EO (A)           5.466        0.4597                        A-B          0.9444                          22EO (A)         4.959        0.3629               A-B          0.8446

                                                                                                                                          80EO (B)         8.51           0.8227                           A-C              **0.0154**                 80EO (B)           5.695        0.5491                        A-C          0.2702                          80EO (B)         4.593        0.3467               A-C          0.9728

                                                                                                                                          530EO (C)        4.199          0.4534                           B-C              **0.0007**                 530EO (C)          4.37         0.4825                        B-C          0.179                           530EO (C)        4.823        0.5533               B-C          0.9354

  *Comparison of the percentage means of major compounds occurring in the dry season among the three individual plants*                                                                                                                                                                                                                                                                                                           

  *spathulenol*                                                                                                                           *α-cadinol*      *τ-muurolol*                                                                                                                                                                                                                                                           

  ANOVA 1-factor                                                                                                                                                          F (2,16) = 5.911   **0.012**     ANOVA 1-factor                              F (2,12) = 7.055   **0.0094**   ANOVA 1-factor                             F (2,16) = 0.06998   0.9327                                                                     

                                                                                                                                          22EO (A)         6.004          0.621                            A-B              0.0767                     22EO (A)           7.334        0.3408                        A-B          0.6579                          22EO (A)         5.653        0.4532               A-B          0.9609

                                                                                                                                          80EO (B)         7.748          0.2299                           A-C              0.506                      80EO (B)           7.76         0.2616                        A-C          **0.0458**                      80EO (B)         5.79         0.194                A-C          0.9333

                                                                                                                                          530EO (C)        5.162          0.5943                           B-C              **0.0104**                 530EO (C)          6.03         0.4011                        B-C          **0.0094**                      530EO (C)        5.833        0.3482               B-C          0.9963

  *Comparison of the percentage means of the major compounds occurring in the rainy and the dry seasons of the three individual plants*                                                                                                                                                                                                                                                                                           

  *spathulenol*                                                                                                                           *α-cadinol*      *τ-muurolol*                                                                                                                                                                                                                                                           

  t-tests                                                                                                                                                                                    t = 0.1277\   0.8991           t-tests                                                    t = 2.674\       **0.0109**   t-tests                                                       t = 2.22\    **0.0314**                        
                                                                                                                                                                                             df = 39                                                                                   df = 39                                                                                     df = 46                                        

                                                                                                                                          Individuals RS   6.374          0.5252                                                                       Individuals RS     5.049        0.3869                                                                     Individuals RS   4.838        0.2323                            

                                                                                                                                          Individuals DS   6.289          0.3799                                                                       Individuals DS     6.602        0.4359                                                                     Individuals DS   5.695        0.3173                            
  ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

NMDS (*stress* = 0.12), in association with an ANOSIM, showed that the abundance and composition of terpenes in the samples varied significantly among the three *Iryanthera* individuals (R~Global~ = 0.284, *p* = 0.001) and also between DS and RS (R~DS\ x\ RS~ = 0.185; *p* = 0.038). Variation was determined according to calculations, because the values of the *R statistic* computed by ANOSIM vary from 0 to 1, with values closer to 0 representing indistinguishable groups of data. There were significant differences between abundance and the composition of terpenes between the two tested cases (all analyses were significant). However, the distinction between groups was not very evident. Considering only the three *Iryanthera* individuals tested, the groups that could be best distinguished (with respect to composition and abundance of terpenes) corresponded to individuals 80EO and 530EO (R~80EO\ vs.\ 530EO~ = 0.373, *p* = 0.001), followed by individuals 80EO and 22EO (R~80EO\ vs.\ 220EO~ = 0.342, *p* = 0.001). On the other hand, the group of samples of individuals 22EO and 530EO could scarcely by distinguished from each other (R~22EO\ vs.\ 530EO~ = 0.183, p = 0.008). These results are shown in [Fig 3](#pone.0224406.g003){ref-type="fig"}.

![Ordination (NMDS) analysis of the 41 essential oils derived from three *Iryanthera polyneura* individual plants, using a Bray-Curtis Index (upward triangles = 22EO; downward-facing triangles = 80EO; squares = 530EO; shaded figures = rain season; open figures = dry season).\
The matrix used for the analysis was based on the percentages of the 59 terpenes found in the essential oils.](pone.0224406.g003){#pone.0224406.g003}

CA and CCA were performed to characterize oil composition according to the percentage of the major compounds spathulenol, τ-muurolol and α-cadinol. The utilization of such statistical tools makes it possible to observe spatial and temporal variations in the chemical composition of the oils considered \[[@pone.0224406.ref014], [@pone.0224406.ref023]\]. Further analyses allowed researchers to correlate geographical and environmental factors \[[@pone.0224406.ref024]--[@pone.0224406.ref026]\].

[Fig 4](#pone.0224406.g004){ref-type="fig"} shows a CA analysis made with the EOs from the three individuals (22EO, 80EO and 530EO) that has been based on the major compounds present on the oils. A cumulative percentage of 100% is shown on the second axis. The analysis revealed that the major compounds from the EOs varied based on whether they were obtained from leaves collected in the DS or the RS. The CCA analysis, shown in [Fig 5](#pone.0224406.g005){ref-type="fig"}, resulted in a cumulative constraint percentage of 100% on the second axis, and also showed a gradient, which was formed on the first axis with the samples of EOs obtained from the leaves collected in the DS and RS. Also, the CCA ([Fig 5](#pone.0224406.g005){ref-type="fig"}) showed that τ-muurolol and α-cadinol, which were more present in higher concentrations in the DS, according to the ANOVA first performed, are particularly influenced by the evaporation, rather than other climatic factors. Although the total amount of spathulenol does not change between the DS and RS ([Table 4](#pone.0224406.t004){ref-type="table"}), the slight tendency of individual 22EO to have higher amounts of spathulenol during RS seemed to have been influenced by the precipitation and relative humidity (which are usually frequent during the RS), temperature and wind velocity.

![Correspondence analysis made with 41 essential oils (cases) obtained from the leaves of three *Iryanthera polyneura* individual plants (22EO, 80EO and 530EO).\
The major compounds spathulenol, τ-muurolol and α-cadinol are the variables. The numerical sequence (1 to 15) corresponds to different collection dates for each individual, fand increases according to chronology. Ellipses highlight the discriminated groups.](pone.0224406.g004){#pone.0224406.g004}

![Canonical correspondence analysis made using 41 essential oils (cases) and three major compounds spathulenol, τ-muurolol and α-cadinol (variables).\
Oils were derived from the leaves of three *Iryanthera polyneura* individual plants (22EO, 80EO and 530EO). The numerical sequence (1 to 15) corresponds to different collection dates for each individual, following a chronological order.](pone.0224406.g005){#pone.0224406.g005}

Conclusions {#sec009}
-----------

The findings presented have shown that there is a variation in the occurrence of the major compounds comprising EO from *I*. *polyneura* individual plants. The compounds spathulenol, τ-muurolol and α-cadinol varied among individuals and that the percentage variation was related to season and climatic changes.

Supporting information {#sec010}
======================

###### Crude data from essential oil percentage.

(XLSX)

###### 

Click here for additional data file.

###### Biological data resulted from *in vitro* assays.

(XLSX)

###### 

Click here for additional data file.

###### Climate data from site of plant collection.

(XLSX)

###### 

Click here for additional data file.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
