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Plant Systematics and Evolution
ISSN 0378-2697
Volume 297
Combined 3-4
Plant Syst Evol (2011) 297:237-251
DOI 10.1007/s00606-011-0511-6
Genetic structure at patch level of the
terrestrial orchid Cyclopogon luteoalbus
(Orchidaceae) in a fragmented cloud forest
Lilian Juárez, Carlos Montaña & Miriam
M.Ferrer
1 23
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ORIGINAL ARTICLE
Genetic structure at patch level of the terrestrial orchid
Cyclopogon luteoalbus (Orchidaceae) in a fragmented cloud forest
Lilian Jua
´rez •Carlos Montan
˜a•Miriam M. Ferrer
Received: 7 March 2011 / Accepted: 13 July 2011 / Published online: 9 August 2011
ÓSpringer-Verlag 2011
Abstract Genetic differentiation in space can be detected
at various scales. First, habitat fragmentation can produce a
mosaic genetic structure. Second, life history aspects of a
species such as dispersion, mating system, and pollination
can generate a genetic structure at a finer level. The
interplay of these levels has rarely been studied together. In
order to assess the effects of forest fragmentation we
analyzed the genetic structure at two spatial scales of the
terrestrial orchid Cyclopogon luteoalbus, which lives in
patches inside forest fragments in a cloud forest of eastern
Mexico. We hypothesized high differentiation between
forest fragments and strong spatial genetic structure within
fragments under this scenario of strong fragmentation and
restricted dispersal patterns. Using 11 allozymic loci we
found high genetic diversity at fragment level with mod-
erate differentiation among fragments, and at patch level,
strong and variable spatial genetic structure among life
cycle stages with high inbreeding coefficients. We also
found bottlenecks indicating recent population size reduc-
tions. While both inbreeding and restricted seed dispersal
may explain the strong spatial genetic structure at patch
level, reduction in population size may explain the genetic
structure at fragment level. However, the levels of genetic
diversity indicate that some between-fragment gene flow
has occurred. Bottlenecks and high inbreeding at patch
level may result in local extinctions, but as long as an
important number of fragments remain, patch recoloniza-
tion through immigration is possible in C. luteoalbus.
Keywords Allozymes Autocorrelation
Heterozygosity Inbreeding Outcrossing rate
Spatial genetic structure
Introduction
Genetic structure is defined by differences in allele fre-
quency within and among populations (Loveless and
Hamrick 1984). It can be detected at landscape, population,
or patch level (Epperson 1993; Fenster et al. 2003; Manel
et al. 2003) and is influenced by ecological factors (Kalisz
et al. 2001) such as pollinator availability (Van Rossum
and Triest 2006), pollination mechanism (Hamrick and
Godt 1996), habitat availability (Sun and Wong 2001), and
breeding system and dispersal mechanisms (Hamrick et al.
1993; Kalisz et al. 2001; Wright 1943). At landscape and
population scales, isolation by distance (due to genetic drift
associated with limited gene flow) may also influence
genetic structure (Wright 1943). Another factor that is
becoming important for genetic structure is habitat frag-
mentation, as isolation contributes to genetic differentia-
tion between fragmented populations and diminishes
genetic variation (Aguilar et al. 2008).
At fine scale or patch level, spatial genetic structure
(SGS) is promoted when relatedness decreases with dis-
tance and genotypes are not randomly distributed within a
population (Epperson 1993). SGS can be the result of
limited gene flow, genetic drift, microenvironmental
selection, breeding system, dispersal mechanisms, and
inherently ecological proximal causes acting at that level
(Hamrick et al. 1993; Kalisz et al. 2001; Wright 1943).
L. Jua
´rez C. Montan
˜a(&)
Red de Biologı
´a Evolutiva, Instituto de Ecologı
´a A.C.,
Apartado Postal 63, 91000 Xalapa, Veracruz, Mexico
e-mail: carlos.montana@inecol.edu.mx
M. M. Ferrer
Departamento de Ecologı
´a Tropical, Universidad Auto
´noma
de Yucata
´n, Km. 15.5 Carretera Me
´rida Xmatkuil,
97000 Me
´rida, Yucata
´n, Mexico
123
Plant Syst Evol (2011) 297:237–251
DOI 10.1007/s00606-011-0511-6
Author's personal copy
SGS in terrestrial orchids has been attributed to: (1) the
presence of pollinaria, because this ensures that seeds
within a capsule are full sibs, (2) seedling establishment
aggregated around mother plants where the necessary
mycorrhiza are found (this mechanism was inferred by
Chung et al. 2004a for Cephalanthera longibracteata, and
by Jacquemyn et al. 2006 for Orchis purpurea), (3) polli-
nation among near neighbors or flowers of the same plant
promoting inbreeding (e.g., Caladenia tentaculata, Peakall
and Beattie 1996 and Cymbidium goeringii, Chung et al.
1998), (4) clonal propagation (e.g., Cremastra appendicu-
lata, Chung et al. 2004b), and (5) recent founding of the
population (e.g., Spiranthes spiralis, Machon et al. 2003
and Cymbidium goeringii, Chung et al. 1998). Also, SGS
may vary with life cycle stage or cohort age (A
´lvarez-
Buylla et al. 1996; Epperson 1993; Kalisz et al. 2001); for
instance, SGS in seedlings may be lower than in adults
when after-establishment thinning preferentially eliminates
full sibs or highly related seedlings (Kalisz et al. 2001;
Tonsor et al. 1993). In these cases, seeds produced by
adults are highly inbred because seeds produced in polli-
naria are full siblings and seedlings mostly establish near
established plants.
Distributed mostly in the mountains of eastern Mexico,
cloud forest is home to half of the orchids known in Mexico
(IUCN/SSC 1996), but this vegetation type has been sub-
jected to a long and strong deforestation process to make
grazing lands, coffee plantations, and urban settlements,
particularly since the 19th century until now. In cloud forest,
as in most tropical forests, fragmentation may disrupt
genetic processes of some species, diminishing the genetic
diversity of remnant populations as local drift and/or
inbreeding increases and gene flow decreases (Aguilar et al.
2008; Honnay and Jacquemyn 2007). Orchidaceae is one of
the largest angiosperm families (17,000–35,000 species),
with around one-third of the species displaying terrestrial
growth habit (Dressler 1993). Cyclopogon luteoalbus
(A. Rich. & Galleoti) Schltr. is a locally abundant terrestrial
orchid patchily distributed in remnant cloud forest fragments
of very variable sizes but larger than 2 ha that are immersed
in a matrix of coffee plantations, pasturelands, and urban
settlements in the mountains of eastern Mexico. As its
generational time is 25 years, as shown by a demographic
study from 2005 to 2009 (Jua
´rez and Montan
˜a, unpublished
data), it is expected that its current genetic structure has been
affected by the long and strong fragmentation process.
This genetic study of C. luteoalbus was conducted, at
both fragment and patch (within-fragment) levels, in the
cloud forest of central Veracruz. In this highly fragmented
landscape we hypothesized high genetic differentiation
between fragments as a consequence of limited gene flow,
and strong spatial genetic structure at patch level because
seedling establishment occurs nearby already established
plants. We also expected to find differences among the
SGS of the different life stages due to nonrandom thinning
of individuals in transitions from each life stage to the next
one.
Materials and methods
Species and study sites
Cyclopogon luteoalbus (Orchidoideae: Cranichideae) is a
Neotropical species distributed from Mexico to El Salva-
dor. It is composed of a rosette (fewer than eight leaves)
and a simple system of tuberoid roots. Clonal propagation
is rare (5 out of 500 revised individuals in a 5-year
demographic study had connecting roots; Jua
´rez and
Montan
˜a, unpublished data). Flowering occurs synchro-
nously in winter (February), while fruits are set in March.
Nectar-producing flowers last 10 days. Fruits contain
2,510 ±1,593 SD seeds (N=8 fruits), which are wind-
dispersed during the dry season. The species has a mixed
mating system: autonomous self-pollination is possible, as
corroborated experimentally (0.83 ±0.23 SD fruit set in
bagged inflorescences, N=14 and 0.85 ±0.13 SD fruit
set in nonbagged inflorescences, N=17), and outcrossing
is promoted by sequential opening of flower within inflo-
rescences. Two halictid bees (Caenaugochlora cupriventris
and Augochlora sp.) are potential pollinators. Five-year
(2005–2009) demographic studies conducted in two
populations (where the fate of a total of 950 individuals
was monitored) showed that the generational time of
C. luteoalbus is around 25 years (Jua
´rez and Montan
˜a,
unpublished data).
The species lives in unevenly distributed patches inside
forests fragments of eastern Mexico. In a surface area of
approximately 15 km 915 km (which included the SBN
and Martinica study sites), Dı
´az-Toribio (2009) reported
the presence of C. luteoalbus in 14 cloud forest fragments
of more than 2 ha surface area. The mean surface area of
and the mean distance between the 14 fragments were
13.3 ±10.7 SD ha and 4.8 ±2.7 SD km. The values of
some soil parameters were: litter depth 5.7 ±1.8 SD cm,
pH 4.6 ±0.8 SD, herbaceous cover 52.6 ±22.2 SD%,
total N 1.2 ±0.6 SD%, total C 18.7 ±12.7 SD%, C/N
14.2 ±2.5 SD, available P 3.9 ±3.4 SD ppm, and K
0.8 ±0.4 SD cmol/kg (N=14 in all cases). Most proba-
bly the patchy distribution inside fragments is due to the
very special soil conditions needed for the development of
the mycorrhizae required for seed germination (Rasmussen
1995). These soil conditions under forest canopies may be
locally absent for natural reasons or may have been elim-
inated by management in forested areas used for shadow
coffee plantations, where under a canopy of native and
238 L. Jua
´rez et al.
123
Author's personal copy
introduced trees, the understory is permanently eliminated
with consequent loss of the upper soil layer. The eventual
use of fertilizers (mainly phosphorus) and/or pesticides also
affects soil conditions. Even if coffee plantation is aban-
doned, reconstruction of these upper soil layers may take
several decades (or even centuries).
According to Rzedowski (1978), cloud forest in the
mountains of eastern Mexico has been occupied and
exploited by man during centuries. Forest clearing for
maize and bean cultivation was done during pre-Colum-
bian times, and for cane sugar cultivation since colonial
times (16th century). Challenger (1998) mentions that
deforestation for introduction of coffee plantations was
very pronounced between the last quarter of the 19th
century and 1960. Using satellite images of a watershed
which included two of our three study sites, Mun
˜oz-Villers
and Lo
´pez-Blanco (2008) documented a process of forest
conversion to grazing and agriculture lands that occurred in
central Veracruz between 1990 and 2003 (this process
began by the mid 20th century and is still going on). In this
1,325 km
2
watershed (1,316 of which has the potential to
sustain several types of forest, but mainly cloud forest)
these authors report that there remained only 561 km
2
of
forests in 1990 and that this figure had decreased to
487 km
2
in 2003. This variation was due to a small
increase in oak–pine (25 km
2
) and conifer (48 km
2
) forests
due to reforestation programmes and to a decrease from
427 to 279 km
2
of cloud forest cleared to be used as pas-
ture and agriculture lands. These anthropogenic changes in
natural habits had led to fragmentation (a complex process
that involves habitat loss, an increase in number of patches,
a decrease in patch size, and patch isolation; Fahrig 2003)
of the original continuous habitat for cloud forest orchids,
affecting the connection between them.
The study was conducted in three cloud forest fragments
in the central part of the State of Veracruz, Mexico [San-
tuario del Bosque de Niebla (SBN), Martinica, and Zon-
golica sites]. SBN is a 30-ha protected area (since 1975)
located 2.5 km southwest from the City of Xalapa
(19°3100500N, 96°5600300W, 1,350 m a.s.l., 1,517 mm rain-
fall, 18°C mean annual temperature). The dominant species
at the forest canopy are: Liquidambar styraciflua,Quercus
xalapensis, and Carpinus caroliniana, and at the under-
story are Palicourea padifolia and Piper auritum with
some remnants of farming such as coffee, orange, loquat,
and guava. Martinica is located 8 km northwest from SBN
at the outskirts of the City of Banderilla (19°3404900N,
96°5602800W, 1,550 m a.s.l., 1,470 mm rainfall, 16°C mean
annual temperature), comprising ca. 10 ha of privately
owned land that has not been farmed for the last 20 years.
The vegetation is similar to SBN.
Zongolica is a ca. 50 ha nonprotected area located ca.
200 km northwest from Martinica and SBN, in the Sierra
Zongolica, in western Veracruz (18°3900000N, 96°4904900W,
1,330 m a.s.l., 2,270 mm rainfall, 17°C mean annual
temperature). Cloud forest in this zone is mixed with
tropical semi-evergreen forest and is subjected to illegal
wood extraction, clearing for farming purposes, and
browsing by goats at the understory. Canopy dominant
species include Alchornea latifolia,Cupania dentata, and
Guettarda elliptica.
The size of the studied populations is variable (Marti-
nica ca. 1,500, SBN ca. 1,000, and Zongolica ca. 200
plants/ha).
SBN was partially used (away from the sites occupied
by patches of C. luteoalbus) for coffee and citrus planta-
tions from the 19th century until the 1970s. In contrast, at
Martinica and particularly Zongolica, deforestation was
triggered by conversion of cloud forest into pasturelands
that began in the second half of the 20th century.
Sample collection
At fragment level, leaf tissue from 30 reproductive adults
from each of the three fragments (SBN, Martinica, and
Zongolica) was collected.
At patch level, SGS was analyzed in the largest patch
within SBN (SBN-1 henceforth) containing 258 individuals
in a plot of 30 m 940 m (Fig. 1). At SBN-1 all individ-
uals were sampled and mapped using Cartesian coordi-
nates. Four life cycle stages were used to categorize
individuals as follows: 31 recruits or seedlings (nonrepro-
ductive with only one tuberoid root), 60 juveniles (nonre-
productive individuals with foliar area \25.0 cm
2
), 106
nonreproductive adults (nonreproductive individuals with
foliar area [25.1 cm
2
), and 61 reproductive adults (dis-
playing reproductive structures at least once during
2005–2007 with foliar area [25.1 cm
2
). No additional
C. luteoalbus plants were observed in the immediate
neighborhood of the patch. Maximum distance between
individuals was 8.3 m, and spatial analysis (not shown)
using the Kstatistic (Ripley 1976) after 19 Monte Carlo
simulations indicated that individuals were aggregated.
Enzymatic extraction and electrophoresis
Three hundred milligrams of foliar tissue from sampled
individuals (transported in ice from field and stored at
-70°C) were ground in a mortar and pestle, adding 250 ll
extraction buffer consisting of 0.05 mM Tris–HCl pH 7.5,
2% PVP-40, 5% sucrose, and 0.1% b-mercaptoethanol,
modified from Soltis et al. (1983). Homogenized tissue
embedded in wicks (2 920 mm) was placed in starch gels
(11.5% w/v) and run in an electrophoresis chamber with
constant current of 35 and 50 mA for the R (Chao-Luan
et al. 1999) and PK buffer systems (Soltis et al. 1983)at
Genetic structure at patch level of the terrestrial orchid Cyclopogon luteoalbus 239
123
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4°C during 9 and 7 h, respectively. Six legible loci were
found in the R system: anodic peroxidase (Apx-1,Apx-2,
and Apx-3), phospoglucoisomerase (Pgi), esterase (Est),
and acid phosphatase (Acph) and five in the PK system:
malate dehydrogenase (Mdh), malic enzyme (Me), diaph-
orase (Dia), and glutamate oxalacetate transaminase (Got-1
and Got–2). Loci and alleles were designated with
ascending ordered numbers and letters, respectively, from
the most anodally to the most cathodally migrating. No
genotype was identical, thus clonal propagation was ruled
out in the C. luteoalbus sampled individuals.
Genetic diversity measurements
For all fragments and SBN-1 we measured: (1) percentage
of polymorphic loci (P), (2) mean number of alleles per
locus (A), (3) observed mean heterozygosity (H
O
), and (4)
expected mean heterozygosity (H
E
) (Hartl and Clark 2007).
Hardy–Weinberg equilibrium for genotype frequency was
tested using the Monte Carlo method (Weir 1990). Com-
parisons of H
O
and H
E
among life cycle stages and popu-
lations were done with Kruskal–Wallis nonparametric
analysis of variance (ANOVA) (Zar 1999). Additionally,
inbreeding coefficients (F
IS
) were estimated for each of the
life cycle stages and populations, and significant differ-
ences from 0 for each estimator were determined by chi-
squared tests following Weir (1990).
Genetic structure at fragment level
Genetic variation among and within fragments was
assessed with the Weir and Cockerham (1984) modified
estimators of Wright (1965)Fstatistics. Departures from
0 for each locus were determined by chi-squared tests
following Weir (1990) for F
IS
and F
IT
and Workman
and Niswander (1970) for F
ST
. Confidence intervals
(95%) of Fstatistics were obtained from bootstrapping
for all loci, and from jackknifing for single-locus esti-
mates. All these analyses were done using TFPGA 1.3
(Miller 1997). To test for isolation by distance, the
coefficient of regression between pairwise F
ST
values
and pairwise geographical distances was calculated and
compared with those obtained from 1,000 permutations
of individual genotypes among fragments (Hardy and
Vekemans 2002).
Evidence of recent bottlenecks
After a bottleneck, the heterozygosity expected under
Hardy–Weinberg equilibrium (H
E
) should be greater than
that expected under mutation–drift equilibrium (H
eq
)in
more than 50% of loci (Cornuet and Luikart 1996). This
hypothesis was assessed at each fragment and for each life
cycle stage at SBN-1 using the BOTTLENECK program
1.2 (Cornuet and Luikart 1996).
Fig. 1 Spatial distribution of
Cyclopogon luteoalbus plants
within patch SBN-1 with inset
showing SBN fragment and the
four patches existing inside it
(full squares, the upper of them
being the studied SBN-1 patch).
Reproductive adults (white
triangles), nonreproductive
adults (black triangles),
juveniles (circles), and
seedlings (stars). Axes scale in
meters
240 L. Jua
´rez et al.
123
Author's personal copy
Spatial genetic structure at patch level
To analyze SGS by life cycle stage, the number of dis-
tance classes to be used for kinship analyses was
obtained using Sturge’s rule (Legendre and Legendre
1998). These distance intervals were chosen to maximize
the number of pairs of plants (at each life cycle stage)
contained in the distance interval. The chosen lags were
2 m for the first 11 classes and 25, 30, and 40 m for the
last three distance classes. Pairs of seedlings were found
only in 8 distance classes, while pairs of individuals of
the remaining life cycle stages were found in all 14
distance classes.
The mean kinship coefficients (F
ij
) proposed by Loiselle
et al. (1995) were estimated for pairs of individuals within
each of the 14 distance intervals using the program SPA-
GeDI 1.3 (Hardy and Vekemans 2002). The coefficients of
the regressions (b
F
) between the kinship coefficients and
the logarithm of the distances between paired individuals
were estimated within each life cycle stage and also for all
individuals in the pooled dataset. SGS is significant when
the slopes of these regressions are negative and different
from 0. The hypothesis that SGS is significant was tested
by comparing the values of the slopes with those obtained
from 1,000 permutations of individual genotypes. To allow
nonstatistical assessment of differences between different
studies, the S
p
statistics were estimated using the formula
S
p
=-b
F
/(F
1
-1), where F
1
is the average kinship
coefficient for the shortest distance class (Vekemans and
Hardy 2004).
SGS differences between life cycle stages were made
using autocorrelation coefficients (which are another
measure of SGS) between geographical distance and
genetic similarity using the heterogeneity test proposed by
Smouse et al. (2008, GENEALEX 6.4). Significance of the
between life cycle stages differences in autocorrelation
coefficients within each distance class were tested with the
t
2
statistic using sequential Bonferroni correction, while the
significance of between life cycle stages differences in
autocorrelation coefficients in the pooled dataset was tested
using the multiclass test statistic x(Peakall and Smouse
2006).
Results
Genetic diversity
At both fragment and patch level all loci were polymorphic
(P=100%), with mean number of alleles per locus of
2.12 ±0.13 and 2.15 ±0.10 (Table 1), respectively.
Genetic diversity was high with mean H
O
of 0.46 ±0.04
and 0.40 ±0.02 at fragment and patch level, respectively
(Table 1). The observed heterozygosity at fragment level
was higher than expected, but at patch level the reverse was
true.
Table 1 Genetic diversity statistics for patch SBN-1 and for three fragments in Veracruz (SBN, Martinica, and Zongolica) of Cyclopogon
luteoalbus
NPAH
O
H
E
F
IS
–?
Life cycle stage
Seedlings 28.36 100 2.00 0.365 0.443 0.176** 3 0
Juveniles 51.18 100 2.20 0.404 0.463 0.127*** 5 0
Nonreproductive
adults
92.18 100 2.20 0.425 0.473 0.101*** 3 2
Reproductive adults 53.18 100 2.20 0.410 0.453 0.095*** 3 2
Mean 56.22 100 2.15 0.401 0.458 0.125
SD 26.48 0.10 0.026 0.013 0.037
Fragments
SBN 27.70 100 2.27 0.415 0.442 0.061 ns 2 1
Martinica 26.30 100 2.00 0.509 0.461 -0.104* 1 2
Zongolica 25.30 100 2.10 0.479 0.433 -0.106* 1 2
Mean 26.43 100 2.12 0.468 0.445 -0.05
SD 1.20 0.13 0.048 0.014 0.09
If all loci are found in every plant, Nwill equal the number of plants, but it will decrease if some loci are missing in some individuals. Number of loci
(total 11 loci) displaying significant deviations from Hardy–Weinberg equilibrium: heterozygosity deficiency (-) and heterozygosity excess (?)
Naverage number of genotypes analyzed, Ppercentage of polymorphic loci, Anumber of alleles per locus, H
O
and H
E
mean observed and
expected heterozygosity, respectively, F
IS
inbreeding coefficient
Asterisks indicate the probability of v
2
under the null hypothesis that F
IS
is equal to zero (Weir 1990), * P\0.05, ** P\0.01, *** P\0.001
Genetic structure at patch level of the terrestrial orchid Cyclopogon luteoalbus 241
123
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Hardy–Weinberg equilibrium was found in only 2 out of
11 loci in the patch- and fragment-level analysis. At frag-
ment level, 4 and 5 loci had deficiency and excess of
heterozygotes, respectively, while at patch level these
figures were 14 and 4, respectively. There were no sig-
nificant differences in genetic diversity among fragments
[Kruskal–Wallis tests: H
(2,31)
=1.19, P=0.55 and H
(2,31)
=
0.21, P=0.89 for H
O
and H
E
, respectively]. Similarly, no
significant differences were found for genetic diversity
among different life cycle stages [Kruskal–Wallis tests:
H
(3,44)
=0.64, P=0.88 and H
(3,44)
=1.06, P=0.78 for
H
O
and H
E
, respectively].
At fragment level H
O
was 0.415, 0.509, and 0.479 (SBN,
Martinica, and Zongolica fragments, respectively). The
average of H
O
over life cycle stages at patch level was
0.401, decreasing from 0.41 in reproductive adults to 0.365
in seedlings.
At fragment level, F
IS
was negative and different from 0
(except for at SBN, Table 1). At patch level, all F
IS
values
for life cycle stages were positive and significantly differ-
ent from 0 (Table 1). The value of F
IS
for seedlings was
1.86 times higher than that of reproductive adults, while the
F
IS
values of juveniles and nonreproductive adults were
1.34 and 1.07 times higher than that of reproductive adults
(Table 1). Allele frequencies are given in Appendix 1.
Genetic structure at fragment level
CIs (95%) for the mean values of F
IT
and F
IS
included
zero, while mean values of F
ST
were positive and signifi-
cantly different from zero, indicating that ca. 12% of
genetic diversity was distributed among fragments
(Table 2). The F
ST
value between the SBN and Zongolica
fragments was the highest (F
ST
=0.208), followed by the
F
ST
value between the SBN and Martinica fragments,
which was two times higher than the F
ST
value between
Martinica and Zongolica (F
ST
=0.128 and 0.066, respec-
tively). The distance between the SBN and Martinica
fragments is ca. 8 km, while Martinica and Zongolica are
more than 100 km apart. No isolation by distance was
found: the slope (b=0.000013) of the regression between
paired F
ST
values and geographic distance did not differ
from that obtained from permutation test (P=0.67).
Evidence of recent bottlenecks
Both within fragments and within all life cycle stages at
SBN-1, most of the 11 loci analyzed had higher expected
heterozygosity under Hardy–Weinberg (HW) equilibrium
than under drift–mutation equilibrium (Table 3). Similar
results were obtained using the infinite allele model and the
stepwise mutation model.
Spatial genetic structure at patch level
Spatial genetic structure analyses indicated significant
positive autocorrelation for all life cycle stages (and for the
pooled data set) at the shortest distance class (Fig. 2a–e).
Different life cycle stages showed different levels of
relatedness at the shortest distance class, which includes
the following significant kinship coefficients (F
ij
):
0.142 ±0.023 SE for all life cycles pooled (Fig. 2a),
0.03 ±0.025 SE (marginally significant, P=0.052,
Fig. 2b) for seedlings, 0.192 ±0.041 SE (Fig. 2c) for
juveniles, 0.22 ±0.044 SE (Fig. 2d) for nonreproductive
adults, and 0.154 ±0.027 SE (Fig. 2e) for reproductive
adults. Significant slopes of the regression between the
logarithm of distances and the kinship coefficients for the
pooled data set (b
F
=-0.045, P\0.001), for seedlings
(b
F
=-0.023, P\0.05), and for juveniles, nonreproduc-
tive adults, and reproductive adults (b
F
=-0.067, -0.062,
and -0.059, P\0.001, respectively; Table 4) confirmed
that genetic relatedness decreases with distance. The S
p
value for the pooled data set was 0.053, increasing from
seedlings to juveniles to reproductive adults and then
decreasing slightly in nonreproductive adults (S
p
=0.024,
0.084, 0.08, and 0.069, respectively; Table 4), suggesting
that spatial genetic structure increases with age.
The heterogeneity test was not significant after Bon-
ferroni sequential correction for within distance–class
comparisons between the life cycle stages (Appendix 2).
However, the global test (x) was significant for all
Table 2 Wright’s Fstatistics for three Cyclopogon luteoalbus pop-
ulations in Veracruz cloud forest
Loci F
IT
F
ST
F
IS
Got-1 0.363*** 0.167* 0.235***
Got-2 -0.432*** -0.006 ns -0.423***
Apx-1 0.331* 0.374*** -0.067 ns
Apx-2 0.123* 0.068 ns 0.059 ns
Apx-3 0.102*** -0.001 ns 0.103*
Me 0.388*** 0.092* 0.326***
Pgi 0.250*** 0.266*** -0.022 ns
Acph -0.272*** 0.054 ns -0.344***
Dia 0.419*** 0.249*** 0.226***
Mdh -0.214*** 0.029 ns -0.251***
Est -0.493*** 0.006 ns -0.502***
Mean 0.068 ns 0.128* -0.069 ns
SD 0.10 0.04 0.09
Upper limit 95% CI 0.240 0.029 0.087
Lower limit 95% CI -0.121 0.053 -0.223
ns not significant, CIs confidence intervals
Probability of the v
2
goodness-of-fit test with respect to Hardy–
Weinberg equilibrium: * P\0.05; ** P\0.01; *** P\0.001
242 L. Jua
´rez et al.
123
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comparisons between life cycle stages except seedlings
versus reproductive adults, suggesting differences in
strength for SGS (Appendix 2).
Discussion
Genetic diversity
The high genetic diversity found in C. luteoalbus is par-
tially explained by the fact that only polymorphic loci were
considered here. Comparing the average values of H
O
(0.46
and 0.40 in the fragments and at patch SBN-1) with those
obtained in other studies in which only polymorphic
loci were analyzed and \5 populations were studied,
C. luteoalbus genetic diversity was higher than that observed
for a narrowly distributed and obligated outcrossing orchid
(H
O
=0.33 ±0.02, N=2, Pleurothallis fabiobarossii,
Borba et al. 2001), similar to a widespread and preferen-
tially outcrossing orchid (H
O
=0.46 ±0.15, N=3,
Govenia superba, Garcı
´a-Cruz et al. 2009) but lower than
another widespread and preferentially outcrossing orchid
(H
O
=0.52 ±0.18, N=2, Govenia mutica, Garcı
´a-Cruz
et al. 2009), lower than that for orchids with mechanical
barriers to self-pollination (H
O
=0.61, N=1, Bulbo-
phyllum bidentata, and H
O
=0.49, N=1, B. rupicolum,
Azevedo et al. 2007), but higher than that for orchids
where pollination by deceit prevents inbreeding (H
O
=
0.21 ±0.06, N=5, Sophronitis sincorana, Borba et al.
2007). However, some caution about these comparisons
should be applied, because only polymorphic loci were
analyzed and only between 36% and 55% of those found in
the cited species were shared with those used in C. lute-
oalbus. High genetic diversity seems to be related with
cross-pollination, and it has also been related to high out-
crossing rates and floral synchrony as a result of increased
gene flow via pollen (Domı
´nguez et al. 2005; Ferrer et al.
2004), and this could be true in C. luteoalbus, a species
with synchronous blooming and whose negative F
IS
values
indicate high outcrossing rates.
Between-fragment gene flow through pollen exchange
is expected in species with a mechanism of pollination
by deceit which promotes long-distance pollen flow. In
food-rewarding species such as C. luteoalbus, pollinator
foraging pattern is spatially limited as pollinators spend
more time foraging within the same patch (Cozzolino
and Widmer 2005). Although this foraging pattern pro-
motes local mating and positive inbreeding coefficients
at patch level (as was found in C. luteoalbus), the high
genetic diversity and negative F
IS
values (indicating high
outcrossing rates) found at fragment level suggest that
some interpatch and interfragment pollen exchange is
Table 3 Expected (Exp.) and observed (Obs.) values for the number
of loci showing higher expected heterozygosity under HW than under
drift–mutation equilibrium for the infinite allele model (IAM) and the
stepwise mutation model (SMM), and Pvalues for the Wilcoxon test
comparing the observed and expected values
IAM SMM
Number of loci with heterozygosity
excess
Pvalue Number of loci with heterozygosity
excess
Pvalue
Exp. Obs. Wilcoxon test Exp. Obs. Wilcoxon test
Life cycle stage
Seedlings 4.67 11
0.0002
5.24 11 0.0002
Juveniles 4.60 11
0.0002
5.37 10 0.0005
Nonreproductive adults 4.39 11
0.0002
5.14 11 0.0002
Reproductive adults 4.64 11
0.0002
5.25 10 0.0007
Fragments
SBN 4.99 11
0.0002
5.66 9 0.004
Martinica 4.27 10
0.0005
4.77 10 0.0005
Zongolica 4.38 9
0.0009
4.94 8 0.0068
Results from bottleneck analyses in SBN-1 patch, and in three fragments of Cyclopogon luteoalbus from cloud forest of Veracruz. Only the
Wilcoxon test is reported, as all other tests used by BOTTLENECK program (sign, standardized departure, and Wilcoxon tests) gave similar
results
Genetic structure at patch level of the terrestrial orchid Cyclopogon luteoalbus 243
123
Author's personal copy
occurring as in other terrestrial orchids (Caladenia ten-
taculata, Peakall and Beattie 1996). Also, even if seed
dispersal is leptokurtic, some between-fragment gene
flow through wind-dispersed seeds (that may travel long
distances) has been found (Jersakova and Malinova
2007). This is also possible in C. luteoalbus,asa
between-fragment distance of 4.8 ±2.7 SD km has been
reported by Dı
´az-Toribio (2009) for 14 fragments in a
15 km 915 km area which included the SBN and
Martinica fragments.
Fig. 2 Correlograms showing
kinship coefficients F
ij
and their
95% confidence intervals
(vertical bars), calculated by
1,000 permutations within each
distance class. Kinship
coefficients for all life cycle
stages pooled (a), for seedlings
(b), for juveniles (c), for
nonreproductive adults (d), and
for reproductive adults (e). Data
from Cyclopogon luteoalbus
plants found at patch SBN-1
244 L. Jua
´rez et al.
123
Author's personal copy
Bottleneck analyses showed evidence of recent reduc-
tions in effective population size of C. luteoalbus at both
fragment and patch level. A decrease in genetic diversity is
expected after a reduction of effective population size
(Aguilar et al. 2008; Ghazoul 2005; Honnay and Jacque-
myn 2007) due to random fixation and loss of alleles and
consequent reduction of heterozygosity, but this would be
evident in C. luteoalbus if monomorphic loci were also
included in this analysis. Genetic diversity is also affected
if the efficiency of pollinator services decreases as a con-
sequence of fragmentation (Aguilar et al. 2008; Ghazoul
2005; Honnay and Jacquemyn 2007). In C. luteoalbus the
effect of reduction of population size and genetic drift in
the remnant fragments are counterbalanced by gene flow
among them. Also, the high genetic diversity in C. lute-
oalbus could be partially due to selection favoring het-
erozygotes, as discussed in the next subsection.
It is interesting to note that the inbreeding coefficient F
IS
is negative at the Martinica and Zongolica fragments (but
not significant at the SBN fragment), while it is positive
and significant at the SBN-1 patch (Table 1). This suggests
that within-fragment heterozygotes are favored by natural
selection (Lewontin 1974; Mitton and Grant 1984) while at
patch level local mating inside family groups favors a
Wahlund effect.
Genetic structure at fragment level
Genetic differences between fragments were found to be
moderate (F
ST
=0.12 ±0.04 SD). However, no evi-
dence of isolation by distance was found. First, the
F
ST
=value for C. luteoalbus falls within the range of
F
ST
values for data reported for allozyme studies made in
70 orchid species (G
ST
or F
ST
) of 0.012–0.75 (Forrest
et al. 2004). This considerable variation has been attrib-
uted to a high variability in life histories characteristics
such as reproductive strategies and generation times, and
to the degree of isolation (Forrest et al. 2004). Bottle-
necks and isolation have been found to increase genetic
structure among populations, resulting in among-popula-
tion differentiation in allelic frequencies (Honnay and
Jacquemyn 2007).
Fig. 2 continued
Genetic structure at patch level of the terrestrial orchid Cyclopogon luteoalbus 245
123
Author's personal copy
SBN fragment, which is immersed in an urban matrix
and surrounded by coffee plantations, is the more differ-
entiated fragment, as shown by the larger F
ST
value
between this fragment and Martinica and Zongolica
(F
ST
=0.128 and F
ST
=0.208, respectively). Moreover,
larger portions of SBN (away from the sites where
C. luteoalbus is present) were used for coffee and citrus
plantations from the 19th century until the 1970s. In con-
trast, at Martinica and particularly Zongolica, deforestation
began more recently (the second half of the 20th century)
by the conversion of cloud forest into pasturelands.
This moderate but significant genetic differentiation
suggests that, even if C. luteoalbus is a locally abundant
species, colonization of new fragments depends on (1)
specific habitat requirements (see ‘‘Species and study
sites’’), and (2) even if abandoned, coffee plantation
physicochemical soil properties may remain unsuitable for
orchid colonization as the continuous removal of under-
story during coffee cultivation eliminates soil surface lay-
ers. The lack of suitable sites for recruitment of
C. luteoalbus individuals in grazing lands, urban environ-
ments, and present or past coffee plantations confined this
species to settle on relatively undisturbed cloud forest
fragments, which are becoming less frequent.
Despite the fragmentation process, there is still some
between-fragment gene flow that hinders the development
of stronger differentiation between them. This is suggested
by the small F
ST
values and by the lack of evidence of
isolation by distance (i.e., there was no progressive
increase of genetic differentiation with geographic dis-
tance). In this sense, it is important to note that the frag-
mentation process has developed unevenly in space and
time and so its probable effect on F
ST
values is by now
spatially variable. Taking into account that the generational
time of C. luteoalbus is 25 years, it can be expected that, in
places where fragmentation started in the 18th or early 19th
century for coffee cultivation, its current effect should be
stronger than in places that were cleared during the last
decades of the 20th century to be used as pasturelands.
Spatial genetic structure at patch level
As we expected, strong spatial genetic structure was found at
patch SBN-1. The S
p
statistic for all life cycle stages pooled
(S
p
=0.05 ±0.02 SD) was similar to that found for mixed
mating species (S
p
=0.03 ±0.03 SD, N=7) and herba-
ceous plants (S
p
=0.04 ±0.06 SD, N=24) but higher
than those expected for species with gravity seed dispersal
and animal-dispersed pollen (S
p
=0.02 ±0.01 SD,
N=17 and S
p
=0.01 ±0.01 SD, N=6, respectively), as
reported by Vekemans and Hardy (2004).
The short distance at which SGS was detected (\6m)
falls within the range recorded for five other terrestrial
orchids (range of minimal distance at which SGS equals
zero in the correlograms: 5–16 m; Peakall and Beattie
1996; Chung et al. 1998,2005; Chung et al. 2004a,b).
This distance reflects the spatial structure of the family
group derived from restricted seed and pollen dispersal
(Sokal and Wartenberg 1983).
Inbreeding in SBN-1 (F
IS
=0.12) could be a conse-
quence of the foraging behavior of the pollinators that
promotes local mating. In C. congestus, the halictid bee
Pseudoaugochloropsis graminea visits 1–4 flowers per
inflorescence and 1–2 inflorescences per visit on a patch
(Singer and Sazima 1999). As a similar foraging pattern
was observed in bee visitors to C. luteoalbus (Caenaug-
ochlora cupriventis and Augochlora sp.; Jua
´rez and
Montan
˜a, personal observation), capsules set within an
inflorescence could be derived from both geitonogamous
pollination and within-patch pollination. On the other hand,
experimental and observational studies show that seed
dispersal is highly leptokurtic in orchids, as most of the
seeds are dispersed by gravity near to the mother plant
(Chung et al. 2005; Jersakova and Malinova 2007). Both
the pollination mechanism and the leptokurtic seed dis-
persal may lead to outcrossed, biparental-inbred, and self-
fertilized seeds (and also to a Wahlund effect derived from
within-patch breeding). All these factors may serve to
explain the strong spatial genetic structure.
Four scenarios involving proximal causes such as pollen
and seed dispersal have been envisaged in the literature for
SGS development: (1) strong SGS and inbreeding, when
dispersal of both pollen and seeds is limited (Caujape
´-
Castells and Pedrola-Monfort 1997; Chung et al. 2004a),
(2) weak SGS without inbreeding, when dispersal of both
pollen and seeds is widespread (Epperson and Allard
1984), (3) SGS without inbreeding, when pollen dispersal
is random and seed dispersal limited (Hamrick and Nason
1996), and (4) SGS absent, when pollen dispersal is limited
but seed dispersal is not (Chung et al. 2003a). The case of
Table 4 Statistics of spatial genetic structure in Cyclopogon
luteoalbus within SBN-1
Life cycle stage F
ij
at 2 m b
F
(slope) S
p
Seedlings 0.039 -0.023* 0.024
Juveniles 0.192 -0.067* 0.084
Nonreproductive adults 0.220 -0.062* 0.080
Reproductive adults 0.154 -0.059* 0.069
All samples 0.142 -0.045* 0.053
The kinship coefficient (F
ij
) between individuals in the shortest dis-
tance class, and the slope (b
F
) of the regression between the kinship
coefficients and the logarithm of the distances between paired
individuals
*P\0.05
246 L. Jua
´rez et al.
123
Author's personal copy
C. luteoalbus seems to points to the first scenario where
SGS is promoted by restricted pollen and seed dispersal.
SGS varies across life cycle stages because demographic
processes affect each life cycle stage differently (Jacque-
myn et al. 2007; Kalisz et al. 2001). If reproductive adults
have strong SGS, within-patch mating will result in marked
SGS in the other stages (Kalisz et al. 2001). According to
Kalisz et al. (2001) the lower SGS of seedlings as com-
pared with other life cycle stages can be due to historical
contingencies, local selection, or random processes. The
current reproductive cohort could have been the product of
a single population founding event (that triggered a genetic
bottleneck; Nei et al. 1975; Cornuet and Luikart 1996),
creating a pattern of genetic structure in adults that is
decaying in offspring cohorts. Microlocal environmental
conditions (such as fine-scale genetic interactions of
mycorrhizal associations; Taylor and Bruns 1999) may
favor survival of spatial aggregates of relatives. Kalisz
et al. (2001) mention that also balancing selection favoring
increasing levels of heterozygosity with age could partially
explain differences between offspring and adults.
Also the differences in SGS among life cycle stages
suggest a differential reproductive contribution over time
which is due to the fact that a different set of adults
reproduce each year. In other words, each year seedlings
descend from the same set of adults while juveniles and
adults (recruited from different cohorts of seedlings) des-
cend from a different set of adults, and are established
close to their mother plant. Five-year demographic data of
C. luteoalbus show that only 0.4% of adults set fruits all
years while 50% set fruits only 1 out of the 5 years (7, 18,
and 25% set fruit in 4, 3, and 2 out of 5 years, respectively;
Jua
´rez and Montan
˜a, unpublished data).
Because inbreeding and the consanguinity level affect the
kinship coefficient (F
ij
) (coancestry coefficient sensu Crow
and Kimura 1970), the expected values of 0.250 for full sibs
and 0.125 for half-sibs under random mating (Loiselle et al.
1995) must be corrected (Weir et al. 2006). Seedlings and
juveniles at SBN-1 are not originated by random mating
(F
IS
=0.176 and 0.127, respectively; Table 1), and their
expected F
ij
values are 0.213 and 0.222 assuming mating
between full sibs, 0.106 and 0.111 assuming mating between
half-sibs, 0.053 and 0.055 assuming mating between first
cousins, and 0.027 and 0.028 assuming mating between
second cousins (estimated from Crow and Kimura 1970,
eqn 3.3.1, p. 69). The observed values of the mean kinship
coefficient for seedlings and juveniles at 0–2 m distance
class were F
ij
=0.039 and 0.192, respectively, suggesting
that recruited seedlings are most likely progeny from first or
second cousins while juveniles are most likely progeny from
full siblings. The kinship coefficient of seedlings is lower
than that of juveniles, which suggests differential survival
favoring closely related individuals.
Differences in the length of residence in each life cycle
stage can contribute to between life cycle stages SGS
changes: seedlings came from only one cohort and remain
at this stage only 1 year, while juveniles came from at least
five cohorts (minimum age of first reproduction is 5 years)
and may remain more than 5 years at this stage. The
accumulation of juveniles during this time interval arising
from within-patch mating may reinforce the juvenile SGS,
and a similar reasoning applies for adult stages.
The heterogeneity test shows that SGS of seedlings and
reproductive adults do not differ (Appendix 2), most
probably due to the genetic similarity between reproductive
adults and their progeny and to the fact that seedlings of
different mother plants are more of less evenly distributed
in space because their seed shadows largely overlap inside
the patch. Afterwards, the mechanisms described above
may be responsible for creating the difference between
SGS of seedlings and the other life cycle stages (Tonsor
et al. 1993; Kalisz et al. 2001).
After fragmentation, plants from a formerly continuous
population remain isolated in patches and inbreeding
becomes the predominant mechanism of mating. As plants
of these patches senesce and die, genetic variability is
eroded and inbreeding depression may appear. Seedlings
produced in these populations have a high inbreeding
coefficient which decreases as life cycle develops, probably
due to the expression of deleterious allele combinations at
different life cycle stages or due to the negative effects of
fragmentation, as Aguilar et al. (2008) suggests.
Nevertheless, the genetic viability of populations with a
history of bottlenecks and isolation may not be severely
affected by inbreeding because deleterious alleles in the
homozygous condition are purged by natural selection
(Wallace 2003).
In conclusion, this study shows the existence of spatial
genetic structure inside patches, and this coincides with a
scenario where gene flow via pollen and seed dispersal is
restricted. Also, there are high levels of genetic diversity
and moderate between-fragment genetic differentiation that
points to a scenario of moderate gene flow via between-
fragment pollen and seed dispersal. At patch level, local
mating may result in high levels of autogamy, geitonogamy
or biparental inbreeding. By contrast, all the few between-
fragment matings are between unrelated plants. The neg-
ative effects of fragmentation revealed by recent bottle-
necks and the high inbreeding coefficients (that decrease as
the life cycle develops) may result in local extinctions at
patch or even fragment level (when there are few and/or
small patches in the fragment).
However, as long as pollinator services remain in an
important number of fragments bearing suitable habitats,
long-distance pollen and seed dispersal (even if not
very frequent) will counterbalance negative effects of
Genetic structure at patch level of the terrestrial orchid Cyclopogon luteoalbus 247
123
Author's personal copy
fragmentation by promoting gene flow and fragment recol-
onization. Our information was collected in one element of
the current landscape mosaic (forest fragments), but man-
agement of other elements of the mosaic (shadow coffee
plantations) may put in motion another process of coloni-
zation and local extinctions. In these plantations the canopy
is never removed but the understory is regularly cleared to
improve coffee production. However, the vagaries of coffee
prices trigger temporary abandonment of these clearing
practices in periods of low prices, when coffee grains are not
collected. During these periods (whose timing and duration
are unpredictable, but can last for a couple of decades)
C. luteoalbus (and many other herbaceous species) can
colonize the understory and contribute to regional gene flow
until the clearing practices are reestablished.
As the current structure of the fragmented landscape is
sufficient to maintain adequate genetic diversity, stopping
the fragmentation process seems necessary to improve the
viability of C. luteoalbus populations in the cloud forests
of central Veracruz.
Acknowledgments This study is part of L.J.’s PhD dissertation
funded by a CONACyT PhD scholarship and a CONACyT research
grant to C.M. Daniel Pin
˜ero, Luis Eguiarte, Oscar Rı
´os, and Octavio
Rojas commented on early versions of the manuscript. Jorge Garcı
´a,
Francisco Reyes, Da
´nae Cabrera, and Juan Pablo Esparza helped with
laboratory and field work, Phil Brewster with pictures, and Ricardo
Ayala determined bee specimens. Julia Herna
´ndez helped in genetic
analysis performed at the Laboratorio de Gene
´tica de Poblaciones,
INECOL (Instituto de Ecologı
´a, A.C.).
Appendix 1
See Table 5.
Table 5 Allele frequency of 11 loci estimated for four life cycle stages (seedlings, juveniles, nonreproductive adults, and reproductive adults) of
Cyclopogon luteoalbus individuals found at patch SBN-1 and at three fragments in central Veracruz (SBN, Martinica, and Zongolica)
Locus Allele Life cycle stage Population
Seedlings Juveniles Nonreproductive adults Reproductive adults SBN Martinica Zongolica
Got-1 a 0.8462 0.8404 (-) 0.7118 0.8868 0.8519 (-) 0.5714 (-) 0.4231
b 0.15 0.1596 0.2882 0.1132 0.1481 0.4286 0.5769
Got-2 a 0.4750 0.5694 0.6250 0.6759 0.6304 – 0.6786 (?)
b 0.5250 0.4306b 0.3750 0.3241 0.3696 – 0.3214
Dia a 0.6667 0.4792 0.6118 (?) 0.6977 0.2778 0.7143 0.8833
b 0.3333 0.5208 0.3882 0.3023 0.4074 0.2857 0.1167
c 0.0000 0.0000 0.0000 0.0000 0.3148 0.0000 0.0000
Est a 0.7333 0.7203 0.6538 0.6639 (?) 0.5667 0.4833 (?)–
b 0.2667 0.2797 0.3462 0.3361 0.4333 0.5167 –
Mdh a 0.5323 0.5000 0.5051 0.4569 (?) 0.4655 (?) 0.6667 0.5862
b 0.4677 0.5000 0.4949 0.5431 0.5345 0.3333 0.4138
Apx-1 a 0.5769 (-) 0.5114 (-) 0.4247 (-) 0.4464 (-) 0.1667 0.7115 0.7353
b 0.4231 0.4886 0.5753 0.5536 0.8333 0.2885 0.2647
Apx-2 a 0.6935 (-) 0.5268 (-) 0.6857 (-) 0.5492 0.8000 0.6481 0.5200
b 0.3065 0.4732 0.3134 0.4508 0.2000 0.3519 0.4800
Apx-3 a 0.7097 0.5882 0.6404 0.5104 (-) 0.7321 0.6042 0.7045
b 0.2903 0.4118 0.3596 0.4896 0.2679 0.3958 0.2955
Acph a 0.6207 0.6724 (-) 0.5931 0.6356 0.6833 0.4333 (?) 0.5167 (?)
b 0.3793 0.3276 0.4069 0.3644 0.3167 0.5667 0.4833
Me a 0.7143 (-) 0.7143 (-) 0.6180 (-) 0.5700 (-) 0.5536 (-) 0.3333 0.2500 (-)
b 0.2857 0.2245 0.3539 0.3900 0.3929 0.6667 0.7500
c 0.0000 0.0612 0.0281 0.0400 0.0536 0.0000 0.0000
Pgi a 0.5500 0.4153 0.5316 (?) 0.6548 0.6538 0.7143 0.1500
b 0.4500 0.5508 0.4421 0.3333 0.3462 0.2813 0.7167
c 0.0000 0.0339 0.0263 0.0119 0.0000 0.0000 0.1333
Signs in parenthesis indicate significant excess (?) or deficiency (-) of heterozygotes
248 L. Jua
´rez et al.
123
Author's personal copy
Appendix 2
See Table 6.
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Table 6 Single-class (t
2
) and multiclass (x) test and associated Pvalues for the heterogeneity test of SGS between different life cycle stages of
Cyclopogon luteoalbus at patch SBN-1 in Veracruz, Mexico
Distance
class
123456 7 8 9 1011121314xP
Interval (m) 0–2 2–4 4–6 6–8 8–10 10–12 12–14 14–16 16–18 18–20 20–22 22–25 25–30 30–40
Life cycle stages pairs
Seedlings versus juveniles
t
2
11.14 1.71 2.35 9.64 2.23 2.60 1.72 1.31 2.48 1.52 3.75 1.67 11.14 1.71 70.51 0.01
P0.01 0.19 0.12 0.01 0.15 0.11 0.23 0.29 0.12 0.08 0.01 0.05 0.01 0.19
Seedlings versus reproductive adults
t
2
8.55 4.36 0.05 1.22 1.65 0.26 0.05 5.50 2.64 0.82 0.35 0.81 8.55 4.36 37.76 0.06
P0.01 0.02 0.85 0.24 0.22 0.67 0.78 0.04 0.12 0.77 0.98 0.87 0.01 0.02
Seedlings versus nonreproductive adults
t
2
4.08 0.17 2.91 1.16 4.29 0.15 1.81 6.55 1.69 0.84 2.07 0.27 4.08 0.17 41.76 0.02
P0.05 0.68 0.10 0.32 0.04 0.71 0.22 0.03 0.17 0.55 0.14 0.94 0.05 0.68
Juveniles versus reproductive adults
t
2
0.61 1.08 10.52 5.97 0.12 3.07 3.62 11.02 0.17 1.31 8.49 1.94 0.61 1.08 62.38 0.01
P0.40 0.35 0.01 0.01 0.78 0.09 0.06 0.01 0.68 0.31 0.01 0.15 0.40 0.35
Juveniles versus nonreproductive adults
t
2
4.05 2.02 25.82 20.47 20.29 6.25 12.33 14.35 0.41 0.90 0.60 3.08 4.05 2.02 73.57 0.01
P0.04 0.18 0.01 0.01 0.01 0.02 0.02 0.01 0.45 0.34 0.47 0.09 0.04 0.18
Reproductive adults versus nonreproductive adults
t
2
3.15 9.77 8.88 7.64 22.80 1.82 6.88 1.53 0.65 0.00 1.96 1.37 3.15 9.77 65.50 0.01
P0.07 0.01 0.02 0.01 0.01 0.20 0.02 0.22 0.37 0.90 0.16 0.27 0.07 0.01
xvalues in bold are significant at P\0.05. Pvalues of t
2
must be compared with 0.0035 =0.05/14, which is the Bonferroni-corrected
significance level (equivalent with 0.05 for only one comparison) for comparison within each of the 14 distance classes
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