Plant Syst Evol (2014) 300:535–547
DOI 10.1007/s00606-013-0902-y
ORIGINAL ARTICLE
Genetic diversity and genetic structure in wild populations
of Mexican oregano (Lippia graveolens H.B.K.) and its
relationship with the chemical composition of the essential oil
Daniela A. Martı́nez-Natarén • Vı́ctor Parra-Tabla •
Miriam M. Ferrer-Ortega • Luz Marı́a Calvo-Irabién
Received: 29 April 2013 / Accepted: 14 August 2013 / Published online: 28 August 2013
Ó Springer-Verlag Wien 2013
Abstract Mexican oregano is an aromatic plant tradi-
tionally harvested from wild populations by rural com-
munities; however, there is little information about
population genetics aspects of this species. Moreover,
considering that the variation in essential oil production of
aromatic plants has been attributed to several environ-
mental as well as genetic factors, in this study we estimated
the genetic diversity and genetic structure from 14 wild
populations of L. graveolens located in four different bio-
climatic regions in southeastern Mexico using AFLP
markers. The overall genetic diversity of L. graveolens
described as the percentage of polymorphic loci
(PPL = 60.9 %) and Nei’s gene diversity (Hj = 0.17) was
moderate, but not associated with the bioclimatic condi-
tions. Genetic variation was analyzed at chemotype and
population levels. Regarding chemotypes, thymol had the
highest genetic diversity (PPL = 82.8 % and Hj = 0.22).
PCoA revealed that chemotypes exhibit a certain level of
genetic differentiation. Maximum parsimony dendrogram
showed a grouping of individuals with a predominant
chemotype. Bayesian analyses revealed a low, but signifi-
cant differentiation among chemotypes (hII = 0.008).
Regarding populations, gene diversity showed significant
D. A. Martı́nez-Natarén  V. Parra-Tabla (&) 
M. M. Ferrer-Ortega
Departamento de Ecologı́a Tropical, Campus de Ciencias
Biológicas y Agropecuarias, Universidad Autónoma de Yucatán,
Km. 15.5, Carretera Mérida-Xmatkuil, 4-116, Mérida,
YUC 97315, Mexico
e-mail: ptabla@uady.mx
D. A. Martı́nez-Natarén  L. M. Calvo-Irabién
Centro de Investigación Cientı́fica de Yucatán, A. C., Calle 43
No.130. Col. Chuburná de Hidalgo, Mérida, YUC 97200,
Mexico
differences (F13,1204 = 22.8, P \ 0.001); populations
dominated by individuals from the thymol chemotype
showed the highest gene diversity (Hj = 0.31–0.25), while
populations with exclusively sesquiterpene chemotype
showed the lowest value (Hj = 0.058). Cluster and
Bayesian analyses (hII = 0.027) revealed a low level of
genetic differentiation among populations. Correlation
analysis showed a significant association between the dis-
tance matrices based on the genetic markers (AFLP) and
chemical compounds of essential oil (r = 0.06,
P \ 0.001). Our results suggest an important genetic
influence on the observed chemical profiles. Nevertheless,
other biotic and abiotic environmental pressures also play
an important role in determining the chemotype and
structure found in this aromatic species.
Keywords AFLP  Chemotype  Genetic
differentiation  Lippia graveolens  Yucatan
Introduction
Lippia graveolens H.B.K., known as Mexican oregano, is
an aromatic plant of high economic importance in this
country, because of its use as seasoning and in traditional
medicine (Hernández et al. 2003). L. graveolens is an
aromatic perennial shrub, with a height between 1.0 and
2.5 m (Huerta 1997). Leaf blades are 2–6 cm long, oblong
to elliptic in shape (Nash and Nee 1984), and covered by
peltate and capitate glandular trichomes on both leaf sur-
faces (Martı́nez-Natarén et al. 2011). This shrub has axil-
lary capitate spikes inflorescences, characterized by white
flowers, small (4 mm in size), sessile and zygomorphic,
hermaphrodite and self-compatible (Ocampo-Velázquez
et al. 2009). On average, 11.4 % of flowers produce fruits
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with one to two seeds (Ocampo-Velázquez et al. 2009).
L. graveolens is found mainly in xerophytic scrub
throughout the arid and semi-arid regions in Mexico, but it
is also found in tropical deciduous forest (Nash and Nee
1984; Soto et al. 2007).
Currently in some Mexican states, oregano leaves are
used as a source of oregano essential oil that is extracted
through steam distillation (Pino et al. 1990; Bulletin 2009).
The essential oil of L. graveolens has demonstrated sig-
nificant antibiotic (Arana-Sánchez et al. 2010; Rivero-Cruz
et al. 2011), antioxidant (Rocha-Guzmán et al. 2007) and
antiviral activities (Ribas et al. 2011) as well antiparasitic
effects (Martinez-Velazquez et al. 2011). These different
activities of oregano have been attributed mainly to the
presence of terpenes, carvacrol, and thymol in its essential
oil (Castillo-Herrera et al. 2007). Moreover, these com-
pounds are quality parameters for this essential oil and in
particular for L. graveolens in Mexico, where a 45–50 %
content of both compounds is considered as a good quality
oil (Bosabalidis 2002).
Mexican oregano is mainly obtained from wild popu-
lations. Previous studies have shown that the essential oil
of this species possesses chemical polymorphism among
different populations and even among individuals within
populations (Uribe-Hernández et al. 1992; Alaniz-Gut-
iérrez et al. 2000). This variation has been represented in
different chemotypes (carvacrol, thymol, carvacrol/thymol)
according to the main compounds present in their essential
oil (Castillo-Herrera et al. 2007; Jacinto et al. 2007).
Recent studies on wild populations from the Yucatan
Peninsula, a region where harvesting Mexican oregano
leaves represents an important activity, have revealed the
presence of two of the three chemotypes reported for this
species (carvacrol and thymol) and a chemotype designated
as sesquiterpene (with predominance of a and b-caryo-
phyllene) (Acosta-Arriola 2011). In Yucatan, L. graveolens
is not found under cultivation; however, its distribution
covers large areas within the state (spread out in small
populations) (Calvo-Irabién and Dzib 2006). In recent
studies, considerable spatial variation has been reported for
chemotypes (Acosta-Arriola 2011) and essential oil yield
of this species (Martı́nez-Natarén et al. 2012) in the
Yucatan Peninsula.
A major challenge in studies on essential oils has been
the characterization of the factors responsible for the
chemical polymorphism recorded within species. The
variation in the essential oil composition of aromatic plants
has been attributed to environmental (Ross and Sombrero
1991; Curado et al. 2006; Muñoz-Bertomeu et al. 2007) or
genetic factors (Vernet et al. 1986; Hannover 1992). In
recent studies, it was found that soil and climatic factors
played a relevant role in explaining the yield and chemical
variability of essential oil of L. graveolens in southeastern
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D. A. Martı́nez-Natarén et al.
Mexico (Acosta-Arriola 2011; Martı́nez-Natarén et al.
2012). However, studies regarding genetic diversity and
genetic structure are necessary to deepen our understanding
of the factors that determine essential oil quantity and
quality in this economically important species. There is
little information about population genetics aspects of
species of the genus Lippia (Suárez et al. 2008; Vega 2011)
and particularly of L. graveolens (Cazares et al. 2010;
Meléndez et al. 2010). Limited numbers of studies have
investigated the genetic variability within these species,
and even fewer studies have explored the correlation
between population genetic diversity and the essential oil
composition of aromatic species (but see Echeverrigaray
et al. 2001; Nan et al. 2003). On the other hand, Mexican
oregano is harvested from wild populations intensively in
different regions; however, the spatial distribution of
genetic diversity is unknown. This information would be
key to establishing harvest areas that allow the definition of
strategies to preserve their genetic diversity, along with the
interest in specific chemotypes.
DNA-based molecular markers, which are not affected
by environmental conditions, have become increasingly
important for surveying genetic diversity and genotype
identification of many plant species (Gounaris et al. 2002;
Katsiotis et al. 2009). In particular, the AFLP markers
based on amplification of polymorphic fragments obtained
by restriction enzymes of genomic DNA (Vos et al. 1995)
have proved useful to analyze inter- and intraspecific
genetic diversity in some oregano species (Ayanoğlu et al.
2006) and to discriminate between oregano subspecies
(Azizi et al. 2009).
Therefore, the objectives of this study were a) to assess
the genetic diversity and genetic structure of L. graveolens
at chemotype and population levels in Yucatan and b) to
explore the correlation between AFLP marker profiles
obtained in this study and the chemical polymorphism of
the essential oil of this species previously reported by
Acosta-Arriola (2011), in an effort to establish the relative
contribution of the genotype on the chemical profile.
Materials and methods
Study sites
We analyzed 95 adult individuals of L. graveolens col-
lected from 14 wild populations located in four different
bioclimatic regions of the Yucatan state (Olmsted et al.
1999; Orellana et al. 1999; Fig. 1). Between five and ten
individuals were collected for each population (Table 1).
For each individual, geographical coordinates were recor-
ded using a GPS (geographical positional system). For
genetic analysis, samples from each individual consisted of
Genetic diversity and genetic structure of Mexican oregano
10–15 young leaves that were preserved in zip-lock plastic
bags containing silica gel (Chase and Hills 1991) and
stored at -20 °C until DNA isolation. Essential oil com-
position from each individual had been previously char-
acterized (Acosta-Arriola 2011). Based on the definition of
chemotype by previous authors (Hendriks et al. 1990;
Keskitalo et al. 2001), three different chemotypes were
identified according to the compounds that exceeded 40 %
of the total peak chromatogram area: (1) thymol and (2)
carvacrol, characterized by high concentration of one of
these monoterpenes, and (3) sesquiterpene, with a pre-
dominance of a and b-caryophyllene (Table 2).
DNA isolation
The leaves were rinsed with 70 % ethanol, dried and then
ground to a fine powder under liquid nitrogen using a
mortar and pestle. Genomic DNA was isolated from
200 mg of the ground leaf tissue from each sample by a
modified CTAB method (Pirttilä et al. 2001) described for
aromatic plants. The quality and quantity of genomic DNA
were verified by 0.8 % (w/v) agarose gel electrophoresis in
1X TBE buffer (0.09 M Tris–borate, 2 mM EDTA, pH
8.3), run 45 min at 100 V, stained with ethidium bromide
Fig. 1 Distribution map of the 14 studied wild populations of Lippia graveolens located within different bioclimatic regions in Yucatan, Mexico
537
and visualized under UV light. DNA obtained was stored at
-20 °C in TE buffer (Tris–EDTA) until use.
AFLP-PCR amplification
AFLP analysis was performed according to Vos et al.
(1995) with minor modifications. Double digestion of
250 ng of genomic DNA was carried out in a 20 lL
reaction mixture containing 0.25 lL of EcoRI (10 U/lL)
and 0.20 lL of MseI (10 U/lL) restriction enzymes (New
England labs), 0.2 lL BSA (1 mg/mL), 2 lL Buffer 2
(New England labs) and Milli-Q water. Digestion was
performed during 2 h at 36 °C. Ligation was carried out in
a 6 lL reaction mixture containing 0.22 lL of EcoRI
(5 lM) and 0.22 lL of MseI (50 lM) adapters (Invitro-
gen), 0.15 lL T4-DNA ligase (5 U/lL), 0.6 lL Buffer T4-
DNA ligase (10X, New England Labs), 3 lL of DNA
digested and Milli-Q water. Ligation was performed during
1 h at 24 °C and then at 65 °C for 15 min to inactivate the
enzyme. Digestion-ligation products quality was verified
by 1.5 % (w/v) agarose gel electrophoresis in 1X TBE
buffer, run 45 min at 60 V, stained with ethidium bromide
and visualized under UV light. An aliquot of 3 lL (1:10
dilution of digested-ligated DNA with TE) was used to
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538 D. A. Martı́nez-Natarén et al.

Table 1 Environmental characteristics for each sampled wild populations of Lippia graveolens in Yucatan
Population Longitude Latitude Climate typeb Mean temperature Annual precipitation Vegetation
W Na °C mm type

1 89.573 21.253 Very warm semi-arid 25.5 538 CSV

2 89.115 21.338 25.7 684 CSV
3 89.807 21.163 25.7 614 LDF
4 88.189 21.561 The driest warm sub-humid 25.5 650 CSV
5 88.084 21.564 25.4 664 CSV
6 88.707 21.341 25.7 893 LDF
7 88.983 21.140 Warm sub-humid with summer 25.9 979 LDF
rains
8 89.652 20.634 26.1 1,007 LDF
9 89.599 20.601 26.1 1,014 LDF
10 90.148 20.616 26.7 1,021 LDF
11 89.857 20.539 26.3 1,069 LDF
12 90.004 20.576 26.6 1,099 LDF
13 88.909 20.500 Intermediate warm sub-humid 26.1 1,151 MSF
14 88.946 20.514 26.2 1,156 MSF
CSV coastal scrub vegetation, LDF low-height deciduous forest, MSF medium-height subdeciduous forest
a
Longitude and latitude are expressed as decimals
b
Köppen climate classification modified by Garcı́a (1988): very warm semi-arid (BS(h0 )), the driest warm sub-humid (Ax0 (w0)(x0 )(i0 )), warm
sub-humid with summer rains (Aw0(i0 )g), intermediate warm sub-humid (Ax0 w1(i0 )g)

amplify pre-selectively the fragments. Pre-selective
amplification reactions were carried out in a 15 lL volume
containing 0.9 lL of EcoRI ? A (20 lM) and 0.9 lL of
MseI ? C (20 lM) primers (Invitrogen), 5.52 lL GoTaqÒ
Plus Green 2X Master Mix (Promega) and Milli-Q water.
Reactions were performed on a TECHNE thermocycler
(TC-512) using the following conditions: 20 cycles of 30 s
at 94 °C, 30 s at 56 °C and 2 min at 72 °C; 10 min at
72 °C. From a preliminary screen of 16 primer combina-
tions EcoRI ? ANN/MseI ? CNN using four individuals
of L. graveolens, two combinations EcoRI ? ACA/
MseI ? CCC and EcoRI ? ACC/MseI ? CAC were
selected based on the presence of polymorphism, the ability
to discriminate between individuals and reproducibility of
amplifications to perform further selective amplifications.
Selective amplification reactions were carried out in a
20 lL volume containing 1 lL of EcoRI ? ANN (5 lM,
50 FAM labeled) and 1 lL of MseI ? CNN (1 lM) primers
(Invitrogen), 7.5 lL GoTaqÒ Plus Green 2X Master Mix
(Promega) and 3 lL pre-amplified PCR-product diluted in
Milli-Q water (1:2). Reactions were performed on a
TECHNE thermocycler (TC-512) using the following
conditions: 12 cycles of 30 s at 94 °C, 30 s at 65 °C and
1 min at 72 °C; 23 cycles of 30 s at 94 °C, 30 s at 56 °C
and 1 min at 72 °C; and 30 min at 60 °C. Amplification
products were purified with a spin column for PCR prod-
ucts purification kit following the instructions of the ID-
PURETM manufacturer. Then 2 lL of these products was
mixed with 2.8 lL of 98 % (v/v) formamide and denatured
at 95 °C for 5 min. Amplification products (4 lL) were
separated by electrophoresis on 6 % (w/v) polyacrylamide
gel with 5 M urea in 1X TBE buffer and run at 1900 V at
40 °C over 3 h, using a BaseStationTM DNA Sequencer
and Fragment Analyzer (MJ-GeneSys). A GeneScanTM 500
ROXTM size standard (Applied Biosystems) was used to
measure band size in base pairs, adding 0.2 lL to every
fifth sample.
Data analysis
CartographerTM software (MJ-GeneSys) was used to score
the fluorescent AFLP patterns generated for each individual
plant of L. graveolens analyzed. The fluorescent AFLP
patterns were visualized as electropherograms, in which
each peak of fluorescence corresponded to an amplified
DNA fragment. Presence (1) and absence (0) matrixes were
constructed scoring each individual of L. graveolens for 74
fragments for EcoRI ? ACA/MseI ? CCC and for 120
fragments for EcoRI ? ACC/MseI ? CAC combinations.
Allelic frequencies were estimated according to Bayesian
method with a non-uniform prior distribution of allele fre-
quencies, assuming Hardy–Weinberg equilibrium (HWE
hereafter) genotypic frequencies (Zhivotovsky 1999). A
correlation between the allelic frequencies and their frag-
ment weights was estimated for each primer combination
using AFLP-SURV v. 1.0 (Vekemans et al. 2002). Because a

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Genetic diversity and genetic structure of Mexican oregano 539

Table 2 Genetic diversity statistics estimated at species, chemotype Genetic diversity

and population level of Lippia graveolens from 87 AFLP loci
Level PPL Hj ± S.E. (FIS = 0) Chemotypea Genetic diversity for species, chemotype and population
level was measured by calculating the percentage of poly-
Species 60.9 0.169 ± 0.01 morphic loci (PPL) at the 5 % level and the expected het-
Chemotype erozygosity or Nei’s gene diversity (Hj) and its standard
Thymol (T) 82.8 0.218 ± 0.01a error with the program AFLP-SURV v. 1.0 (Vekemans et al.
Carvacrol (C) 72.4 0.154 ± 0.01b 2002), using the approach of Lynch and Milligan (1994) and
Sesquiterpene (S) 51.7 0.143 ± 0.01b the Bayesian method with non-uniform prior distribution to
Population PPL Hj ± S.E. (FIS = 0) Chemotypea calculate allelic frequencies (Zhivotovsky 1999) assuming
either HWE, or some deviation (FIS = 0.1 and 0.25) from
C T S
HWE genotypic frequencies. The results for different FIS
4 66.7 0.306 ± 0.02a 5 values were very similar; therefore, only results assuming
5 88.5 0.259 ± 0.01a 9 1 HWE are presented. Univariate analysis of variance and
8 71.3 0.249 ± 0.02a 7 3 Tukey post hoc test were used to evaluate differences in Hj
2 49.4 0.176 ± 0.01b 5 among chemotypes and populations (Zar 1984).
13 50.6 0.172 ± 0.01bc 5
7 44.8 0.160 ± 0.01bc 5 Differentiation and genetic structure
10 42.5 0.156 ± 0.02bc 5
14 58.6 0.155 ± 0.01bc 5 Genetic differentiation at the chemotype and population
11 58.6 0.154 ± 0.01bc 3 7 levels was assessed with Bayesian methods to estimate hII
12 66.7 0.146 ± 0.01bc 2 6 2 (analogous to Wright FST) from dominant markers without
1 47.1 0.143 ± 0.01bc 5 assuming HWE frequencies (Holsinger et al. 2002). Bayes
6 47.1 0.123 ± 0.01bc 5 posteriors were obtained for a full model (f-inbreeding
9 49.4 0.106 ± 0.01c 2 8 coefficient and h -population differentiation were esti-
3 23.0 0.058 ± 0.01d 5 mated), a model in which f = 0, a model in which hII = 0
and an f-free model (estimates hII without estimating
PPL percentage of polymorphic loci at the 5 % level, Hj expected f) using Hickory v. 1.1 (Holsinger and Lewis 2001–2007).
heterozygosity or Nei’s gene diversity
To prevent bias in gene differentiation at the population
Different letters indicate significant differences among chemotypes
and populations (Tukey, P \ 0.05)
level (i.e., changes in allele frequencies) due to the dif-
a ference in population size, the analysis was also performed
Number of individuals from each chemotype
by standardizing the population sizes (i.e., with five ran-
significant negative correlation between the allelic fre- domly selected individuals). The deviance information
quency and the fragment weight was found in both matrixes, criterion (DIC hereafter) was used to estimate the fit of the
fragments displaying lower weight than 100 bp in which different models and those with lower DIC values and
homoplasy is likely to occur (Vekemans et al. 2002) were differences no larger than 6 units between them were
removed from further analyses. A merged matrix with chosen (Spiegelhalter et al. 2002). For both levels analyzed
EcoRI ? ACA/MseI ? CCC and EcoRI ? ACC/MseI ? (chemotype and population), the full model and the
CAC fragment scoring was generated and analyzed with f = 0 model were equally likely, since their DIC values
FAMD v. 1.25 (Schlüter and Harris 2006) to identify the were lower and the difference between these values was\6
presence of private alleles at chemotype (i.e., markers units (at chemotype level, the DIC values were 1,179.37 for
present in one chemotype, but absent in others) and popu- the full model and 1,180.64 for the f = 0 model; at pop-
lation (i.e., markers present in one population, but absent in ulation level, the DIC values for full and f = 0 models
others) levels. No private allele was found for both chemo- were 3,182.28 and 3,185.97, respectively); therefore, only
type and population levels. Monomorphic loci in which results for the full model are presented. Identical significant
fragment presence or absence were higher than 95 % were hII values were also recorded for both population levels
removed from the matrix. After removal of monomorphic analyzed.
fragments and other data to avoid fragment size homoplasy, The genetic structure of L. graveolens at the chemotype
a total of 87 fragments were included as polymorphic loci and population level was explored by means of a principal
(51 and 36 loci for EcoRI ? ACA/MseI ? CCC and coordinate analysis (PCoA) using Jaccard’s similarity
EcoRI ? ACC/MseI ? CAC combinations, respectively). coefficient and the first two axes were plotted graphically
The new matrix was used to perform all statistical analysis using PAST v. 2.08 (Hammer et al. 2001). Also, a con-
described in the following sections. sensus dendrogram using similarities of haplotypes and

123
540
maximum parsimony criteria was constructed with Phylip
v. 3.6 (Felsenstein 1993).
Isolation by distance was tested by assessing the sig-
nificance of the correlation between Nei’s genetic distance
and geographical distance among populations performing
10,000 permutations using the program TFPGA v. 1.3 to
conduct Mantel test (Miller 1997). Pairwise Nei’s genetic
distances (Nei 1978) were calculated for all populations
after Lynch and Milligan (Lynch and Milligan 1994) and
10,000 random permutations were performed using AFLP-
SURV v. 1.0 (Vekemans et al. 2002).
Additionally, fine-scale genetic structure within popu-
lations was obtained by examining the slope of the
regression between the geographical distance between
individuals, within each population, and the average kin-
ship coefficient between individuals estimator (Fij) for
dominant markers (Hardy 2003). To test if the slope is
significantly different from zero, 1,000 random permuta-
tions of genotypes between individuals were performed
using SpaGeDi v. 1.3 (Hardy and Vekemans 2002).
Correlation between genetic and chemical distance
matrices
Euclidean distances based on the content (%) of major
components in the essential oil (carvacrol, thymol,
p-cymene, c-terpinene, a and b-caryophyllene, data pre-
viously reported by Acosta-Arriola (2011) and genetic
distances based on Jaccard’s similarity coefficient were
calculated for each pair of individuals using PAST v. 2.08
(Hammer et al. 2001). To investigate the correlation
between molecular (AFLP) and chemical data, the genetic
distance and the Euclidean distance matrices were analyzed
using a Pearson correlation coefficient (Zar 1984).
Results
Genetic diversity of Lippia graveolens
Overall, the percentage of polymorphic loci obtained for
L. graveolens in Yucatan was 60.9 % and Nei’s gene
diversity was 0.169 ± 0.011 (Table 2).
After analyzing the genetic variation at different levels
(i.e., chemotype and population), diverse estimators showed
high intraspecific variability for this species (Table 2).
Genetic diversity among chemotypes
The results showed different values of polymorphism
between chemotypes; the thymol chemotype had the
highest percentage of polymorphic loci (PPL = 82.8),
followed by carvacrol (PPL = 72.4), while the
123
D. A. Martı́nez-Natarén et al.
sesquiterpene presented the lowest PPL (51.7 %, Table 2).
Also, significant differences in Nei’s gene diversity were
observed (F2,258 = 12.6, P \ 0.001), being significantly
higher in the thymol chemotype than in the carvacrol and
sesquiterpene chemotypes (Tukey test P \ 0.001, in both
cases). Both the carvacrol and sesquiterpene chemotypes
did not vary significantly in terms of gene diversity (Tukey
test P [ 0.05, Table 2).
Genetic differentiation among chemotypes
The results of the Bayesian analyses indicated that
L. graveolens showed significant differentiation among
chemotypes. The hII index for the full model at the
chemotype level had a value of 0.008 (±0.004 S.E.), which
was significantly different from zero, indicating a low but
significant differentiation (0.8 % of the total genetic vari-
ation is due to differentiation among chemotypes).
Similarly, PCoA revealed that individuals of L. graveo-
lens exhibit a certain level of genetic differentiation at the
chemotype level (Fig. 2). The first two axes explained
24 % of the total variation in the data. Although the second
axis explained a lower percentage of variability (8.8 %), it
allowed separating some individuals according to their
chemotype. A large number of individuals from the ses-
quiterpene and carvacrol chemotypes (i.e., 78 and 64 %
respectively) were grouped into the positive values of Axis
2, while 75 % of individuals from the thymol chemotype
were grouped into negative values (Fig. 2).
Furthermore, the dendrogram based on similarities of
haplotypes grouped most individuals of L. graveolens into
four major clusters (Fig. 3), showing a predominance of a
given chemotype in each cluster. Cluster-I, forming a large
group (50 individuals), contained mostly individuals from
the sesquiterpene chemotype (i.e., 66 % of all sesquiter-
pene individuals); individuals from the thymol and carva-
crol chemotypes were also present in that cluster, but to a
lesser extent (26 and 20 %, respectively). Cluster-II,
comprising 14 individuals, included a similar number of
individuals from the sesquiterpene and carvacrol chemo-
types (six and five, respectively). Cluster-III consisted of
five individuals from the carvacrol and sesquiterpene
chemotypes, and none of the individuals from the thymol
chemotype were found in that cluster. Cluster-IV com-
prising 18 individuals contained mainly individuals from
the thymol chemotype (78 %). However, four individuals
(3.1, 3.3, 8.10 and 7.7) were not grouped at all, so these
plants were the most dissimilar.
Similar to cluster analysis, correlation analysis also
showed that there was a low, but significant association
between the two distance matrices based on the genetic
markers (AFLP) and the chemical compounds of essential
oils (r = 0.06, P \ 0.001).
Genetic diversity and genetic structure of Mexican oregano
Fig. 2 Principal coordinates
analysis plot using Jaccard’s
similarity coefficient from 87
AFLP loci, showing the
relationships among 95
individuals of Lippia graveolens
from different chemotypes and
different geographical location.
The same number indicates
individuals from the same
population (1–14). Numbers in
brackets represent the
percentage of the variation
explained by each axis
Genetic diversity among populations
Polymorphism rates among populations were variable,
showing a range between 88.5 and 23.0 % for population 5
(with a predominance of the thymol chemotype) and popu-
lation 3 (exclusively sesquiterpene chemotype), respectively
(Table 2). Also, gene diversity among populations showed
significant differences (F13,1204 = 22.8, P \ 0.001); popu-
lations 4, 5 and 8 (predominantly thymol chemotype)
showed the highest gene diversity, while population 3
(exclusively sesquiterpene chemotype) showed the lowest
value (Table 2).
Population differentiation and spatial genetic structure
The Bayesian analyses indicated a low, but significant
differentiation among populations of L. graveolens. The
significant value of hII for the full model at the population
level showed that 2.7 % of global genetic variation was due
to differentiation among populations.
Also, the PCoA indicated that individuals of L. graveo-
lens presented some level of genetic differentiation with
respect to their population of origin (Fig. 2). Similarly to
what was observed at the chemotype level, the second axis
allowed grouping some individuals according to their
location. All individuals from population 10 and most
individuals (between 60 and 80 %) from populations 1, 2,
3, 7, 8, 9, 13 and 14 were grouped into the positive values
of the second axis. In contrast, all individuals from
541
population 4 and a high proportion of individuals from
populations 5, 6 and 12 were grouped into the negative
values of the second axis.
Likewise in the dendrogram based on similarities of
haplotypes, some individuals of L. graveolens were grouped
according to their location (Fig. 3). All individuals from
population 4 and most individuals from population 5 were
grouped very closely in the cluster-IV, while the majority of
individuals from populations 6 were grouped together in
cluster-II. Similar to PCoA results, most individuals
(between 60 and 100 %) from populations 1, 2, 8, 9, 10, 13
and 14 were grouped into the same cluster-I (Fig. 3) and
particularly some individuals from populations 8, 10 and 13
were grouped very closely. Nevertheless, the considerable
overlap of individuals from different populations showed a
pattern of low differentiation among populations, and even
lower according to regions defined by the bioclimatic region.
The results of the Mantel test revealed that the genetic
distance between these populations was not significantly
correlated with their geographical distance (r = 0.092,
P = 0.175). On the other hand, fine-scale genetic structure
analyses showed significant positive correlation between
individuals distributed within 0–10 m from each other,
indicating that individuals within these distances were
more likely to be genetically related. Also, a significant
negative correlation was observed between individuals
distributed at a distance [25 m from each other, indicating
that individuals separated further than these distances were
likely to be genetically unrelated (Fig. 4).
123
542
Fig. 3 Maximum parsimony dendrogram using similarities of hap-
lotypes from 87 AFLP loci, representing genetic relationships among
individuals of Lippia graveolens from different chemotypes and
different geographical location. Individuals from thymol chemotype
are shown in green color, from carvacrol chemotype in red color and
from sesquiterpene chemotype in blue color
123
D. A. Martı́nez-Natarén et al.
Discussion
Genetic diversity among populations of Lippia
graveolens
Allelic and gene diversity of L. graveolens in Yucatan were
moderate (PLP = 60.9, H = 0.169) compared with those
reported for this species in Chihuahua (PLP = 76.9,
H = 0.219; Meléndez et al. 2010) where AFLP markers
were also used. The discrepancy in genetic diversity among
the Yucatan and the Chihuahua populations could be
attributed to the differences in the number and the area size
for sample collections (i.e., regions and populations) and to
the higher variability of genetic diversity detected among
populations for this species in Yucatan (PPL = 23.0–88.5
and Hj = 0.058–0.306). In contrast, variability among the
populations in Chihuahua was lower (PPL = 41.3–62.0
and H = 0.160–0.238; Meléndez et al. 2010). The wide
range of values observed in genetic diversity among pop-
ulations in Yucatan also contrasts with those reported
among populations of the close relative species Lippia
origanoides Kunth from Colombia (PPL = 75.4–93.0 and
H = 0.268–0.319), using AFLP markers (Vega 2011). The
observed differences in the levels of genetic diversity
among L. origanoides populations have been attributed to
different ecological factors and, particularly, lower gene
diversity was associated with restricted gene flow due to
elevation gradients (Vega 2011). Contrary to these results,
in this study as well as those reported by Meléndez et al.
(2010), there was no clear trend showing that L. graveolens
populations from a particular environment had higher or
lower genetic diversity.
The remarkable polymorphism and gene diversity vari-
ation detected at the population level showed that AFLP
markers could be used as a powerful tool for estimating
infraspecific genetic diversity for L. graveolens, in partic-
ular, and also for other aromatic plants.
On the other hand, we suggest that the level of genetic
diversity found in this species could indicate a considerable
influence of outcrossing in the reproduction of L. graveo-
lens, a perennial species that is both self- and cross-polli-
nated (Ocampo-Velázquez et al. 2009). Flowers of
L. graveolens remain open for about 7 days before senes-
cence (Ocampo-Velázquez et al. 2009) making these
attractive to pollinators and favoring a high rate of cross-
pollination between plants. It has been reported that out-
crossing species usually have relatively high levels of
genetic diversity (Hamrick and Godt 1996; Nybom 2004).
However, this prediction needs to be confirmed by further
studies.
Genetic diversity and genetic structure of Mexican oregano
Fig. 4 Spatial genetic structure
of Lippia graveolens in
Yucatan. Black dots represent
significant (P \ 0.05) Fij values
Genetic diversity among chemotypes
Our results showed significant differences in genetic
diversity among chemotypes of L. graveolens. Individuals
from the thymol chemotype had the highest genetic
diversity, compared with individuals from the carvacrol
and sesquiterpene chemotypes, which exhibited similar
levels of gene diversity. To our knowledge, this is the first
study that has explored the potential relationship between
genetic diversity and the expression of different chemo-
types. Further studies on this economically important aro-
matic species are needed to assess whether the different
levels of genetic variation are associated with different
pathways of gene expression or biosynthesis of the dif-
ferent components of the essential oils, as reported in
Thymus vulgaris where biosynthesis of carvacrol and thy-
mol are controlled by an epistatic series of several bio-
synthetic loci (Vernet et al. 1986).
Differentiation and genetic structure
among chemotypes
Cluster (PCoA and dendrogram) and Bayesian analyses
revealed a certain level of genetic differentiation regarding
chemotypes among individuals of L. graveolens. Similar
results have been obtained in other aromatic species such
as Salvia fruticosa Mill. (Skoula et al. 1999) and Cunila
galioides Benth. (Fracaro et al. 2005), where individuals
grouped in the same molecular cluster had similar terpene
543
composition, suggesting genetic differences among indi-
viduals from different chemotypes.
The positive and significant correlation between the
distance matrix based on AFLP markers and the distance
matrix based on the chemical compounds of essential oils
(terpenes) confirms that there is some congruence of rela-
tionships between individuals of L. graveolens, revealed by
the chemical and molecular markers. Significant correla-
tion between genetic and terpenoid distance matrices has
also been reported in some aromatic plants using different
molecular markers, e.g., Thymus vulgaris L. (Echeverrig-
aray et al. 2001), Tanacetum vulgare L. (Keskitalo et al.
2001), Ocimum gratissimum L. (Vieira et al. 2001),
Primula ovalifolia L. (Nan et al. 2003) and Origanum
vulgare L. (Azizi 2010). However, there are several
examples of aromatic species in which no association has
been found between genetic similarity and chemical com-
position data, such as Ocimum basilicum L. (Labra et al.
2004; De Masi et al. 2006), Thymus caespititius (Trindade
et al. 2008), Lippia alba Mill. (Manica-Cattani et al. 2009)
and Origanum onites L. (Tonk et al. 2010). A significant
aspect of this study was that the AFLP analysis discrimi-
nated several individuals of L. graveolens regarding their
chemotype by using only two primer combinations (87
polymorphic loci), particularly taking into account that
molecular markers that show differences in the whole plant
genome are not necessarily related to a specific secondary
compound or morphological trait (Nan et al. 2003; Labra
et al. 2004; De Masi et al. 2006).
123
544
Although there are reports that have provided evidence
that terpene chemotypes are strongly controlled by genetic
factors (Vernet et al. 1986; Hannover 1992; Thompson
et al. 2003), Hannover (1992) also reported the effect of
environmental variation in terpene expression under
extreme habitat conditions. In the case of L. graveolens, the
influence of environmental factors on the chemical com-
position of the essential oil has also been reported. Indi-
viduals from the carvacrol and thymol chemotypes are
dominant in dry climates, whereas in humid climates
individuals from the sesquiterpene chemotype are more
abundant (Acosta-Arriola 2011). Similar results have been
reported for Thymus vulgaris (Thompson et al. 1998).
Considering the different results found in the literature
and those described in this study, the question regarding the
relative contribution of the genetic background versus the
environment control on the essential oils composition of
aromatic plants is still not fully understood. To fully
understand the influence of both environmental and genetic
factors on volatiles composition, other specific studies
(e.g., quantitative trait loci: QTL) as well as common
garden experiments to test if the oil composition of plants
from different environment (propagated by stem cutting
and cultivated in a field experiment) was not significantly
different from that of the original plants should be
explored.
Population differentiation and genetic structure
The level of population differentiation found in this study
(hII = 0.027) was similar to those reported for popula-
tions of a related species, such as L. origanoides
(hII = 0.032; Suárez et al. 2008), but lower than those
reported in other studies among populations of the same
L. origanoides species from Colombia (hII = 0.156) by
Vega (2011) and among O. vulgare populations
(Ast = 0.24) by Van Looy et al. (2009). This result may
suggest that in Yucatan, L. graveolens has a high gene
flow among populations. The plain topography in Yuca-
tan, together with pollinators presenting a high capacity of
flight to forage (e.g., Apis mellifera; Dyer and Seeley
1991; Blight et al. 1997), could partially be an explana-
tion for the low genetic differentiation among populations
in this species (Levin 1981). In contrast, it has been
reported that L. origanoides populations from Colombia
are subject to important barriers to gene flow (e.g.,
mountains) that influence the genetic differentiation in
these populations (Vega 2011). In O. vulgare, Van Looy
et al. (2009) suggested that the relatively high level of
genetic differentiation among these populations can be
explained by founder effects, rather than by genetic drift
in isolated populations, due to continuous extinction and
colonization of habitats (for a very small number of
123
D. A. Martı́nez-Natarén et al.
colonists), to which this typical river corridor species of
calcareous floodplain soils is subjected.
To explain the low, but significant differentiation among
L. graveolens populations, we consider that isolation by
distance among populations could be a factor promoting
the differentiation among these populations as expected
under Wright’s (Wright 1946) isolation-by-distance model
of population structure. However, the lack of significance
of the correlation between Nei’s genetic distance and
geographical distance among populations suggests that
genetic variation in L. graveolens is not spatially structured
(i.e., nearby populations can be genetically very different
or very similar and vice versa). Nevertheless, at a finer
scale, analyses using the average kinship coefficient (Fij)
revealed significant genetic structure of L. graveolens
suggesting that the distribution of genetic diversity among
individuals of this species is not random. Based on the
spatial autocorrelation analysis, individual plants located
within the spatial range of \10 m are likely related, while
individuals separated for more than 25 m are likely unre-
lated. These results may suggest that L. graveolens is a
species with limited seed dispersal and, therefore, family
groups are expected close in space. Studies suggesting that
seeds show a localized dispersal rate resulting in patches of
genetically related individuals plants have been reported
(Wright 1946; Seidler and Plotkin 2006).
Overall, our results showed a high genetic variation in
Mexican oregano (L. graveolens); however, the level of
genetic differentiation among the chemotypes and the
populations was low. In general, individuals from the
carvacrol and sequiterpene chemotypes seem to be genet-
ically more similar to each other than with individuals from
the thymol chemotype. The significant correlation between
the distance matrices based on genetic and chemical
markers suggested that there may be a genetic basis for the
chemical profiles observed. Nevertheless, biotic and abiotic
environmental pressures also play an important role in
determining genetic structure and diversity in this aromatic
species.
Implications for conservation
In this study we found that L. graveolens in Yucatan
exhibit a moderate level of genetic diversity and can be
associated with some factors that can define their
exploitation.
Although L. graveolens is distributed in large areas
within the state, in numerous Mayan communities of the
Yucatan Peninsula, harvesting Mexican oregano leaves
from wild populations is a traditional practice and repre-
sents an important source of income (Osorno-Sánchez et al.
2009; Calvo-Irabién 2009). Due to the essential oil quality
produced, in particular wild populations from the carvacrol
Genetic diversity and genetic structure of Mexican oregano
and thymol chemotypes would become vulnerable due to
overharvesting. Because the harvest of L. graveolens
involves removing the leaves and stems, an intensive har-
vest can have important consequences for the rates of
survival, growth and reproduction of the individual plants
involved (Ticktin 2004). This should be taken into account
in the oregano management plan, since it has been sug-
gested that a reduction in population size can lead to the
loss of alleles and reduction of genetic variation (Ellstrand
and Elam 1993). Therefore, it would be important to
monitor changes in genetic diversity of these populations.
Additionally, habitat fragmentation by human distur-
bance and deforestation that are taking place in regions
where L. graveolens is naturally distributed could lead to
significant concerns regarding the conservation of this
species. Therefore, the collection of different genotypes of
this species could help preserve the levels of genetic
diversity in a propagation program and some appropriate
conservation and breeding strategies should be imple-
mented if a massive harvest and production of essential oils
is considered in the future.
Acknowledgments This study was financially supported by CO-
NACyT through a doctoral scholarship and a grant for an academic
stay at the University of Winnipeg to D.M.N., and a grant given to
L.C.I. (CB-2008-01, 106389). We thank Sara V. Good, University of
Winnipeg, for laboratory facilities and Dulce Linares-Beltrán and
Felix Martı́nez-Nuñez for technical assistance in laboratory work.
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