Tropical Understory Piper Shrubs Maintain High Levels of Genotypic Diversity Despite
Frequent Asexual Recruitment
Eloisa Lasso1,3, James W. Dalling2, and Eldredge Bermingham1
1 Smithsonian Tropical Research Institute, Unit 0948 APO AA 34002?0948, Panama
2 University of Illinois at Urbana?Champaign, 265 Morrill Hall, Illinois 61801, U.S.A.
ABSTRACT
Many plant species have the capacity to regenerate asexually by resprouting from stem and leaf fragments. In the pan?tropical shrub
genus Piper, this tendency is thought to be higher in shade?tolerant than light?demanding species, and to represent a trade?off with
annual seed production. Here we use molecular markers to identify clones in ?ve Piper species varying in light requirements. We test pre-
dictions that (i) asexual recruitment success is highest in shade?tolerant species, and (ii) that consequently, shade?tolerant species are
characterized by lower genotypic diversity than light?demanding Piper. We found that two shade?tolerant Piper species recruited asexually
more frequently (36?42% of sampled shoots were of asexual origin) than, two light?demanding and one shade?tolerant species (0?26%).
Furthermore, as predicted, genotypic diversity was negatively correlated with the frequency of asexual recruitment in the population.
Nonetheless, genotypic diversity of Piper was high compared with other clonal plants. The proportion of unique genotypes found per
population ranged from 0.58 to 1.0 and the genotypic Simpson's diversity ranged from 0.93 to 1.0 for all ?ve species. Our results sug-
gest that even though asexual reproduction plays an important role in maintaining local populations of Piper in the understory, it does
not seem to reduce genotypic diversity to levels that will threaten these species ability to respond to environmental change.
Abstract in Spanish is available in the online version of this article.
Key words: AFLP; Barro Colorado Island; clonal reproduction; distinguishable genotypes; Panama; Piperacea; tropical wet forest; vegetative reproduction.
PIPER IS A PAN?TROPICAL GENUS CONTAINING OVER 1000 SPECIES
(Burger 1971) occupying a range of forest habitats, from treefall
gaps to deeply shaded forest understories. Piper is often a domi-
nant element in the shrub layer of tropical forests, and plays a
key role supporting frugivorous bat communities (Fleming 1985).
Piper is also a favorite model system for investigating how ecolog-
ically similar species coexist, and partition rain forest environ-
ments (Field & Vazquez?Yanes 1993). As a consequence, many
Piper species have been studied thoroughly in terms of their phys-
iology (Walters & Field 1987, Chazdon et al. 1988, Williams et al.
1989), phenology (Marquis 1988, Thies & Kalko 2004), seed
characteristics (V?zquez?Yanes & Smith 1982, Orozco?Segovia &
V?zquez?Yanes 1989, Daws et al. 2002), reproductive strategies
(Semple 1974, Fleming 1981, de Figueiredo & Sazima 2000) and
natural enemies (Greig 1993b, Marquis et al. 1997).
Experimental studies have revealed strong seed and estab-
lishment limitation of sexual recruitment, particularly among
shade?tolerant Piper species. Up to 76 percent of seeds are lost to
predators at the pre?dispersal stage (Greig 1993b), and remaining
seeds have exceptionally low emergence success. Piper have tiny
photoblastic seeds (<< 1 mg; V?zquez?Yanes 1974, V?zquez?
Yanes & Smith 1982, Orozco?Segovia & V?zquez?Yanes 1989,
Daws et al. 2002) that fail to emerge when sown in the under-
story, even when surface litter is removed. Only 0.2 percent of
8000 seeds sown in the understory emerged for ?ve Piper species
studied in Panama (Lasso et al. 2009). Likewise, Fleming (1981)
found only four surviving seedlings of 1600 seeds of Piper amalago,
and Greig (1993a) observed only ?ve surviving seedlings from
3000 seeds of ?ve Piper species, experimentally sown into both
gap and understory conditions. If seedling recruitment is strongly
limited, as published evidence suggests, then an alternative regen-
eration pathway through asexual reproduction could be critical
for Piper population persistence in the understory.
The most common methods of asexual reproduction
observed in Piper are layering and fragmentation. In layering, new
plants originate at nodes from branches and trunks that are tram-
pled, or pinned to the ground by litter fall. The original shoot
may eventually rot away leaving independent ramets. In fragmen-
tation, new plants resprout from portions of stem or leaf tissue
that remains alive on moist soil or leaf litter layers (Greig 1993b;
Lasso et al. 2009). In both cases, physiologically independent ra-
mets are produced that are dif?cult to identify as clones unless
molecular markers are used.
A consequence of asexual reproduction, which has both
bene?ts and costs, is that offspring are genetically identical to
each other and to their parent. Such genetic similarity may be
advantageous if a genotype is well adapted to a particular envi-
ronment, but disadvantageous if the environment changes. Thus,
an increase in the frequency of asexual recruitment could poten-
tially lead to a decrease in the genotypic diversity of the popula-
tion, which in the long term could limit a population's resilience
Received 7 July 2010; revision accepted 26 November 2010.
3Corresponding author; e?mail: lassoe@si.edu
? 2011 The Author(s) 35
Journal compilation ? 2011 by The Association for Tropical Biology and Conservation
BIOTROPICA 44(1): 35?43 2012 10.1111/j.1744-7429.2011.00763.x
to environmental change (Hamilton 1980, Burt 2000), and may
jeopardize its resistance to biotic enemies (Hamilton et al. 1990).
Even though, this assumption of reduced genotypic diversity has
prevailed for some time, a series of reviews indicate that popula-
tions in which asexual reproduction occurs can be as genotypi-
cally polymorphic as sexual ones (Ellstrand & Roose 1987,
Hamrick & Godt 1989, Wid?n et al. 1994, Silvertown 2008).
Experimental studies of Piper species consistently indicate
greater resprouting capacity for shade?tolerant than light?
demanding species (Greig 1993a, Lasso et al. 2009). This differ-
ence may result from variation in resource allocation patterns or
from variation in the frequency of stem breakage resulting from
greater debris fall in the forest understory (Aide 1987, Clark &
Clark 1991). For Piper, ?eld experiments carried out on 22 species
in Costa Rica (Greig 1993a), and eight species in Panama (Lasso
et al. 2009), indicate that shade?tolerant species have a higher
capacity to resprout and regenerate by layering and fragmentation
relative to congeneric light?demanding species. These studies have
also shown that shade?tolerant species tend to have lower sexual
reproductive success, suggesting that asexual regeneration may be
critical for population persistence.
Less clear, is whether differences in resprouting capacity
translate to actual differences in asexual recruitment rate, and if
the magnitude of asexual recruitment is suf?cient to signi?cantly
impact genotypic diversity. In two previous genetic studies of
Piper, three species, P. amalago, Piper pseudofuligineum, and Piper jac-
quemontianum, showed extremely low values of genetic diversity
(Heywood & Fleming 1986), whereas one species Piper cernuum,
showed high values of genetic diversity (Mariot et al. 2002). But
the relationship of genetic diversity to either light environment or
regeneration capacity was not explicitly studied in these four Piper
species.
The objective of this study was to connect experimental
studies of resprouting capacity and sexual reproductive success in
Piper with measurements of the frequency of asexual recruitment
in the ?eld based on the identi?cation of clones using genetic
markers. Speci?cally, we test the prediction that light?demanding
species restricted to forest gaps and clearings recruit asexually less
frequently than do species restricted to the forest understory.
Additionally, we test the prediction that asexual recruitment in
understory species is suf?cient to signi?cantly reduce genotypic
diversity. By linking molecular assessments of asexual reproduc-
tive success with experimental assessments of asexual reproduc-
tive capacity, we provide an experimental approach aimed at
assessing the importance of asexual recruitment in tropical for-
ests.
METHODS
STUDY SITE AND STUDY SPECIES.?The study was conducted in
tropical semi?deciduous forest on Barro Colorado Island (BCI),
Panama (9100 N, 79510 W), which is described in detail else-
where (Leigh 1999). Annual rainfall on BCI averages 2600 mm,
with a pronounced dry season between January and April. The
genus Piper is represented by 22 species on BCI (Croat 1978),
including both light?demanding and shade?tolerant species. Here
we focus on ?ve species; three are commonly found in the
understory (Piper darienensis C.DC., Piper cordulatum C.DC. and
Piper aequale Vahl.) and two are commonly found in gaps and
clearings (Piper dilatatum L.C.Rich., and Piper marginatum Jacq.).
Based on physiological data, fecundity data and the results
of fragmentation experiments in the ?eld (Lasso et al. 2009),
these Piper species were classi?ed according to three life?history
strategies. Piper marginatum and P. dilatatum are pioneer species
that live exclusively in gaps, produce large numbers of seeds, and
show little asexual reproductive ability. Piper darienensis and P. cor-
dulatum are shade?tolerant species with low annual seed output
and a high capacity to resprout. Piper aequale is shade?tolerant, but
resembles light?demanding species because it produces large
numbers of seeds and has intermediate capacity to resprout.
Here, we use the term individual to refer to stems, or plants
with apparent no connections to other plants. Once the outcome
of molecular analysis is known, these can be classi?ed as genets if
they have unique genetic ?ngerprints or as ramets or clones if they
share the same genetic ?ngerprint with other stems or individuals.
SAMPLING METHOD.?Differences in the distribution of shade?
tolerant and light?demanding species required different sampling
strategies. For P. dilatatum and P. marginatum, individuals were
concentrated close to the forest edge, and the size of gaps and
clearings did not permit the establishment of large plots. In con-
trast, aggregations of P. darienensis, P. cordulatum and P. aequale,
were more sparsely distributed; thus, larger areas needed to be
sampled to include a suf?cient number of individuals to obtained
unbiased estimates of genotypic diversity.
Collections of the three shade?tolerant Piper species were
made in two 1?ha plots located 754 m apart (Plot 1 next to Sny-
der Molino trail marker 2 and Plot 2 next to Lake trail marker 4).
Collections of the two light?demanding species were made in
three 35 m 9 35 m plots located in the edge of forest surround-
ing buildings and laboratories. In each plot, we collected leaves
from all stems not obviously connected to other stems and
spatially separated by at least 5 cm from other stems. In total, we
collected samples from 892 stems (range 26?182 stems per
species per plot; Table 1). A grid was established in each plot
to obtain x and y coordinates for each stem to calculate the
geographic distance among them.
DNA ISOLATION AND AMPLIFIED FRAGMENT LENGTH POLYMORPHISM
(AFLP) PROCEDURE.?Leaves were collected and kept on ice until
they were processed later the same day. In the laboratory, leaves
were surface cleaned with pure ethanol (95%) and dried in silica
gel for 1 wk. Twenty mg of dry tissue were ground using Fast-
Prep FP120 (Qbiogene). DNA was extracted using a DNeasy 96
plant extraction kit (Qiagen) following the manufacturer's proto-
col. DNA concentrations were estimated by running DNA
samples on agarose gels (1.5%) with a Low DNA MassTM Lad-
der (Invitrogen) of known concentrations.
We performed AFLP analysis following the method of Vos
et al. (1995) with some modi?cations. The polymerase chain
36 Lasso, Dalling, and Bermingham
reaction conditions and genotyping procedures are described in
detail in Lasso (2008). We used four primer combinations per
species to obtain AFLP ?ngerprints. Three combinations were
used across all species; EcoRI?ACG/MseI?CTG, EcoRI?AGG/
MseI?CCA, EcoRI?ATG/MseI?CAG. The fourth primer pair used
was as follows: EcoRI?ACG/MseI?CTA for P. darienensis and
P. dilatatum, EcoRI?AGT/MseI?CTG for P. aequale, EcoRI?AGG/
MseI?CAG for P. cordulatum, and EcoRI?AGT/MseI?CAT for
P. marginatum. For P. dilatatum we used two additional primer
pairs, EcoRI?ACG/MseI?CTT and EcoRI?ACG/MseI?CCA, because
many of the loci were monomorphic. The four primer combina-
tions yielded 559?770 clearly identi?able bands per species. After
the ?ltering of loci, only 15?27 percent of fragments were
selected as follows: 120 loci (87 polymorphic loci) for P. darienen-
sis, 102 (68 polymorphic loci) for P. cordulatum, 124 (95 poly-
morphic loci) for P. aequale, 140 (49 polymorphic loci) for
P. dilatatum, 153 (110 polymorphic loci) for P. marginatum (Lasso
2008). Fingerprint data were obtained by running the ampli?ed
samples in an ABI Prism 3130 capillary sequencer instrument,
and presence or absence of fragments was scored using Gene-
scan and Genotyper software (version 3.7, Applied Biosystems).
CLONE IDENTIFICATION, CLONAL SIZE, AGGREGATION AND DISPERSAL
DISTANCES.?Each sample was either classi?ed as a member of a
clone (ramet) or as a unique genotype (genet) using the software
package Genotype (Meirmans & Van Tienderen 2004). Genotype
uses pairwise genetic distances to classify samples as ramets or as
unique genotypes based on a user?speci?ed threshold. In Genotype,
the threshold determines the maximum dissimilarity allowed
between two ?individuals? to still be considered ramets of the
same clone. We calculated this threshold after inspecting the fre-
quency distribution of pairwise genetic distances among three
replicate samples from 11?24 known clones; threshold values
ranged from 0 to 5 percent (Lasso 2008).
The frequency of sexual reproduction was assessed for each
plot by dividing the number of unique genotypes found in the
plot by the number of samples from that plot and from there we
calculated the frequency of asexual recruitment (1  frequency
of sex recruitment). To estimate the size of clones or dispersal
distances by asexual means, we calculated the pairwise geographic
distances among ramets from plot coordinates using GenAlEx
6.0 (Peakall & Smouse 2006). To assess the spatial distribution of
clones we calculated Morisita's index of dispersion (d)(Morisita
1959) after splitting sampling plots in 16 quadrats (see Fig. 1)
Id ?
q
Pq
i?1 xi?xi  1?
N?N  1?
;
where q is the number of quadrats, xi is the number of individu-
als in the i th quadrat and N is the number of plants in the whole
plot. Values of I > 1 indicate patchy distribution, I < 1 uniform
distribution and I = 0 random distribution.
CLONAL GENOTYPIC DIVERSITY INDICES.?Different metrics of
genotypic diversity have been calculated for clonal plants, the
most commonly used being ?proportion distinguishable? (PD) and
Simpson's diversity index (D) (Ellstrand & Roose 1987). We cal-
culated these same two indices using the software Genodive (Meir-
mans & Van Tienderen 2004). The PD was calculated as G
(number of genotypes)/N (number of samples), which is the pro-
portion of distinguishable genotypes or the proportion of individ-
uals in the plot that were recruited sexually. The Simpson
genotypic diversity index corrected for sample size (Pielou 1969)
was calculated as
D ?
n
n  1
 ?1 
X
s
i?1
p2i ?;
TABLE 1. Asexual recruitment, genetic diversity and spatial dispersion in Piper populations at Barro Colorado Island, Panama. The number of stems sampled (N), the number of
genets identi?ed with ampli?ed fragment length polymorphism (AFLP) markers (G), the proportion of distinguishable genotypes (G/N), Simpson's genotypic diversity (D),
the percentage of asexual recruitment (Asex), and the Morisita's index of dispersion for genets ( Igenets) and clones ( Iclones), are presented.
Species Plot N G G/N D Asex (%) Igenets Iclones
Shade?tolerant
Piper darienensis 1 182 105 0.58 0.93 42 4.69 7.22
2 167 98 0.59 0.98 41 3.43 4.50
Piper cordulatum 1 72 46 0.64 0.98 36 2.96 3.71
2 60 38 0.63 0.93 37 2.55 5.12
Piper aequale 1 166 156 0.94 0.99 6 4.44 3.33
2 59 55 0.93 0.99 7 5.90 2.29
Light?demanding
Piper dilatatum 3 26 26 1.0 1.0 0 5.81 ?
4 43 39 0.91 0.99 9 3.87 6.40
5 33 30 0.91 0.99 9 3.50 7.47
Piper marginatum 3 41 41 1.0 1.0 0 7.43 ?
4 43 32 0.74 0.97 26 2.75 6.15
Asexual Recruitment in Piper 37
where n = sample size and pi = frequency of genotype i. To test
whether the populations sampled were of suf?cient size to pro-
vide unbiased estimates of genotypic diversity, we plotted jack?
knife estimates of diversity against sample size for each species
and plot combination. In all cases, diversity estimates were
asymptotic. To determine whether genotypic diversity is negatively
associated with asexual recruitment, we carried out a Pearson
correlation analysis between the Simpson genotypic diversity
index calculated for each plot and the percent of individuals that
were recruited asexually in each plot.
CORRESPONDENCE BETWEEN SPECIES? RESPROUTING ABILITY AND
MOLECULAR DATA.?In our previous work, we created an index of
asexual reproductive ability (ARAI) for Piper species included in
FIGURE 1. Location of all genets (black circles) and clones (white circles) in 1 ha plots established in the understory and 35 m 9 35 m plots located in clear-
ings. (A) and (B) Piper darienensis Plot 1 and 2, respectively; (C) and (D) Piper cordulatum Plots 1 and 2, respectively; (E) and (F) Piper aequale Plots 1 and 2, respec-
tively; (G) and (H) Piper marginatum Plots 3 and 4, respectively; (I), (J) and (K) Piper dilatatum Plots 3, 4 and 5, respectively.
38 Lasso, Dalling, and Bermingham
this study (Lasso et al. 2009). ARAI was calculated by averaging
the percentage of cuttings and pinned?down branches that sur-
vived in growing house and ?eld experiments. To determine how
this index of species? ability to reproduce asexually corresponds
with the estimated level of asexual recruitment from ?eldwork,
we computed a Pearson correlation analysis and a two sample
paired t?test between species ARAI scores and the percentage of
individuals recruited asexually estimated from the molecular data.
RESULTS
Asexual recruitment was more frequent in shade?tolerant species
than in light?demanding species (X2 = 7.08, df = 2; P = 0.03).
The frequency of asexual recruitment was remarkably similar
across the two sample plots for each species. For the two
shade?tolerant species, 41?42 percent of individuals of P. darienensis,
recruited asexually in the plots sampled, while in P. cordulatum
36?37 percent of individuals recruited asexually (Table 1). Piper
aequale, had much lower values of asexual recruitment than shade?
tolerant species, ranging from 6 to 7 percent asexual recruitment
across the plots. These values are similar to the two light?
demanding species, for which three of four populations had
asexual recruitment of 0?9 percent. One population of the light?
demanding P. marginatum recruited 26 percent of individuals
asexually (Table 1).
Individuals belonging to the same clone tended to be tightly
aggregated in localized patches (Fig. 1). Both clones and genets
exhibited clumped distributions as Morisita's index of dispersion
was always > 1 (Table 1). For all species, most clones were
< 10 m apart (Fig. 2A), although a few clones of the shade?
FIGURE 2. Frequency distribution of (A) the pairwise geographic distances among ramets that belong to the same clone and (B) the number of ramets per
clone.
Asexual Recruitment in Piper 39
tolerant species were distributed over remarkably large distances;
up to 45 m in P. darienensis and 78 m in P. cordulatum (Fig. 2A).
These two cases could probably represent long?distance dispersal
events or just the remnants of a clone that once occupied a larger
area since the probability that these genotypes could arise through
random recombination during sexual reproduction is very low
(probability of identity = 3.5 9 105 and 1 9 104 for P. darienensis
and P. cordulatum, respectively). One possible mechanism for long?
distance dispersal is through surface runoff during heavy rains,
especially in slopes. Populations of P. aequale had fewer clones, but
they tended to be more widespread (Fig. 2A). For all species, clones
were typically small with 2?6 ramets (Fig. 2B), though two shade?
tolerant species had several clones represented by 7?15 ramets (Fig.
2B) and P. darienensis had one clone containing 45 ramets.
Genotypic diversity in Piper decreased as asexual recruitment
increased (Pearson correlation = 0.83; P = 0.0014). Values of
genotypic diversity were in the range of D = 0.93?1.0 (Table 1).
Estimates of asexual recruitment frequency obtained from
molecular data were positively associated with estimates of species'
abilities to reproduce asexually based on transplant experiments
(one?tailed Pearson correlation = 0.85; P = 0.03). Nonetheless,
transplant experiments signi?cantly overestimated asexual recruit-
ment success (t = 3.34; df = 5; P = 0.029; Fig. 3).
DISCUSSION
HABITAT DIFFERENCES IN ASEXUAL RECRUITMENT.?Our data indi-
cate that asexual recruitment occurs in all Piper species regardless
of shade tolerance. We did, however, ?nd a marked difference in
the frequency of asexual recruitment between shade?tolerant Piper
species (42% clonal) and Piper species from gaps and clearings
(< 26% clonal). These differences are in agreement with ?ndings
from previous fragment survival and seedling recruitment experi-
ments (Greig 1993a, Lasso et al. 2009). Thus, our data support
the notion that asexual recruitment is an important alternative
regeneration pathway for species growing in the light?limited
understory, where sexual reproductive success is strongly
impacted by pre?dispersal seed predation (Greig 1993b), and
where both seed production and seedling establishment success
are low (Fleming 1981, Greig 1993a, Lasso et al. 2009). Whether
asexual reproduction is important for other understory genera
remains an open question. Several characteristics of the shade?
tolerant syndrome, and of the understory environment may, how-
ever, select for traits that promote asexual recruitment in other
understory species as well.
In the understory, high rates of debris fall increase the
chances that individuals of shade?tolerant species resprout from
fallen stems and stem fragments (Aide 1987, Gartner 1989, Clark
& Clark 1991, Guariguata 1998, Paciorek et al. 2000). Continually
moist conditions in the litter layer may also be critical to frag-
ment survival in the period before rooting systems develop. Fur-
thermore, because resprouting success is correlated to the
capacity to store resources in vegetative organs (Sagers 1993,
Verdaguer & Ojeda 2002, Bond & Midgley 2003), shade?tolerant
species may be better able to resprout than light?demanding spe-
cies because they store greater quantities of nonstructural carbo-
hydrate reserves in stems and roots (Kobe 1997, Poorter &
Kitajima 2007, Myers & Kitajima 2007). Thus differential respro-
uting ability among species groups may be an indirect conse-
quence of traits that confer shade tolerance. Nonetheless, the two
species with the lowest proportion of sexually recruited individu-
als, P. darienensis and P. cordulatum, were also the species with the
lowest annual seed production, lowest seed viability and lowest
seedling emergence rates in the ?eld (Lasso et al. 2009). Piper
aequale, although a shade?tolerant species, resembles more light?
demanding species with high annual seed production and a low
resprouting capacity (Lasso et al. 2009). Our molecular data con-
?rm that this species groups with the two light?demanding spe-
cies, P. dilatatum, and P. marginatum.
Our results fall within in the lowest range of reported fre-
quencies of asexual recruitment (i.e., 1  G/N 9 100) for tropi-
cal species obtained using molecular markers. Murawski &
Hamrick (1990) reported frequencies of 29?89 percent asexual
recruitment in populations of Aechmea magdalenae on Panama.
Bush & Mulcahy (1999) reported frequencies of 58?81 percent
asexual recruitment in populations of Poikilacanthus macranthus, an
understory montane forest shrub from Costa Rica. Miwa et al.
(2001) reported 79 percent asexual recruitment in Melaleuca cajup-
uti, a pioneer tree from Taiwan. Because those studies were con-
ducted on species from diverse taxa and habitats, and were not
accompanied by measures of the species' reproductive ecology,
generalizations about the importance of asexual reproduction for
regeneration in tropical forests and how it relates to other life?
history traits has not been possible. Our data, the ?rst systematic
assessment of frequency of asexual recruitment in a series of clo-
sely related species indicates that, at least for Piper, asexual
recruitment is more frequent in the understory than in gaps and
clearings, and in species with lower sexual success from seeds.
FIGURE 3. Correspondence between estimates of species ability to repro-
duce asexually from experiments (percent of fragments that resprout and sur-
vive) and measurements of the percentage of individuals in the population
that recruited asexually obtained using ampli?ed fragment length polymor-
phism markers.
40 Lasso, Dalling, and Bermingham
RECONCILING EXPERIMENTAL AND MOLECULAR ESTIMATES OF THE
FREQUENCY OF ASEXUAL RECRUITMENT.?In the absence of molecu-
lar data, ?eld experiments that have measured fragment survival
(Gartner 1989, Kinsman 1990, Greig 1993a) or estimated of the
frequency of asexual recruitment based on the presence of callus
tissue growth (Sagers 1993) may also help clarify the importance
of asexual recruitment. With this in mind, we compared our pre-
vious estimates of asexual reproduction obtained from experi-
mental approaches (i.e., tracking cutting survival) to those we
obtained using molecular markers. We found that our previous
assessments of species' ability to reproduce asexually based on
?eld experiments (Lasso et al. 2009) signi?cantly over?estimate
the frequency of asexual recruitment compared with data using
molecular markers. This may be because high survivorship of
fragments will translate to high asexual recruitment only if the
frequency of fragment formation is high. For example, about 80
percent of P. darienensis and P. cordulatum stem fragments and pin-
ned?down branches survived in previous experiments (Lasso et al.
2009), but the molecular data indicate that < 50 percent of the
individuals (disconnected shoots) in both populations recruited
asexually. Therefore, conclusions from previous studies, in which
levels of asexual recruitment were determined only from frag-
ment survival, should be revisited.
Estimates of asexual recruitment success based on callus tis-
sue growth may also be unreliable. For example, Bush & Mulcahy
(1999) who documented that 99 percent of ?eld?sampled plants
of the shrub P. macranthus had callus tissue; found that 58?81
percent individuals had actually recruited asexually when they
used molecular markers to identify clones. Sagers (1993) sug-
gested that 92 percent of newly established Psychotria horizontalis
plants originated asexually based on the presence of callus tissue
growth, but no molecular studies have con?rmed this frequency
of asexuality. Given that experimental approaches overestimate
actual asexual recruitment success, with the molecular data avail-
able for tropical shrubs we can only conclude that asexual
recruitment is probably taking place in many understory species,
possibly more often in shade?tolerant species than in light?
demanding species. Our data and previous data on tropical plants
indicate that none of the species are completely clonal and seed-
ling recruitment is probably taking place in all species.
CONSEQUENCES OF ASEXUAL RECRUITMENT ON GENOTYPIC
DIVERSITY.?Previous values of genotypic diversity reported for
Piper are in agreement with our ?ndings and prediction that
shade?tolerant species should be less genetically variable than
light?demanding species. The diversity indices found in the light?
demanding species P. cernuum (Mariot et al. 2002) are very high
compared with the values found in other shade?tolerant Piper spe-
cies studied in Costa Rica: P. amalago, P. pseudofuligineum, and P. jac-
quemontianum (Heywood & Fleming 1986). In these, three
sympatric populations from Costa Rica only three polymorphic
loci were detected in more than 20 loci analyzed, indicating that
this population may have been founded recently by a small num-
ber of individuals or may have suffered a recent bottleneck in the
number of individuals (Heywood & Fleming 1986) and that asex-
ual recruitment is probably not to blame for their reduced
diversity. Even though we found that genotypic diversity
decreases in populations with higher asexual recruitment, our
results are consistent with previous ?ndings for other clonal
plants that show that asexual species are genetically diverse
(Ellstrand & Roose 1987, Hamrick & Godt 1989, Wid?n et al.
1994, Silvertown 2008).
It has been suggested that the genotypic diversity of tropical
clonal species should be lower than in temperate clonal species,
because the more favorable year?round growing conditions could
lead to more extensive clonal spread (Bush & Mulcahy 1999).
Current data, including ours, are in disagreement with this predic-
tion. Values of Simpson?s genotypic diversity reported for tropical
species so far are in the range of 0.72?1.0 (Murawski & Hamrick
1990, Bush & Mulcahy 1999, Miwa et al. 2001), whereas the
range observed in temperate species is 0.13?1.0 (Ellstrand &
Roose 1987, Wid?n et al. 1994, Honnay & Jacqyemyn 2008).
Indicating that either clonal spread is not more frequent in the
tropics than in temperate zones or/and that other factors main-
tain high levels of genotypic diversity in spite of asexual recruit-
ment.
What determines the genotypic diversity of clonal popula-
tions? Simulation models suggest that sexual recruitment rates of
only 1 percent per year can be suf?cient to maintain genotypic
diversity (Watkinson & Powell 1993). Beyond the balance
between sexual and asexual reproduction, some additional factors
may in?uence genotypic diversity in a local population, including
the number of founding individuals, the age of the population,
the life span of ramets, habitat heterogeneity, size of the clones
and frequency of somatic mutations. For example, populations
with more founding individuals should have more unique geno-
types and therefore higher genotypic diversity. If individual genets
are long?lived, then the genotypic diversity of the initial popula-
tion may persist through clonal growth (Yeh et al. 1995), even in
the absence of novel genotype recruitment. Another mechanism
that could maintain genotypic diversity is density?dependent mor-
tality driven by natural enemies (pathogens and herbivores). This
is a well?known mechanism acting on seeds, seedlings and
saplings that thins clumps of plants (Janzen 1970, Connell
1971, Augspurger 1983, S?nchez?Hidalgo et al. 1999). Density?
dependent processes may operate even more strongly on clonal
aggregations sharing exactly the same genotype. Conversely, large
clones may maintain genotypic diversity if genets with indepen-
dent ramets spread the risk of mortality among individuals, thus
reducing the probability of that genotype disappearing from the
population (Charlesworth 1980, Cook 1983). Habitat heterogene-
ity may also select for different genotypes, and therefore contrib-
ute to the maintenance of higher genotypic diversity. Finally, the
accumulation of somatic mutations could potentially add geno-
typic diversity to the population (e.g., Mes et al. 2002, van der
Hulst et al. 2003, Houliston & Chapman 2004, Paun et al. 2006).
Although no estimates of the frequency of somatic mutation
exists for tropical systems, data from elsewhere suggest that it is
frequent and could potentially lead to evolutionary changes (Gill
et al. 1995, Lian et al. 2003, Michel et al. 2004, Nagamitsu et al.
Asexual Recruitment in Piper 41
2004, O?Connell & Ritland 2004, Vaughan et al. 2007). How
populations of Piper and other clonal species maintain their geno-
typic diversity remains to be investigated, however, the high level
of genotypic diversity found suggests that even populations with
frequent asexual recruitment are probably well equipped to
respond to environmental change.
Asexual reproduction seems to play an important role in
maintaining local populations of Piper in the understory. Neverthe-
less, sporadic windows of opportunity for sexual reproduction,
resulting from occasional favorable conditions of light and mois-
ture conditions probably permit regeneration from seedlings,
which seem to be suf?cient to maintain high levels of genotypic
diversity. For gap species, for which forest regrowth limits the
period when conditions are favorable for recruitment, sexual
reproduction probably provides the only opportunity for long?
distance transport of genotypes to new suitable habitat. Regardless
of the reproductive mode used by Piper species from different
habitats, all seem capable of sustaining high levels of genotypic
diversity that enhances their resilience in a changing world.
ACKNOWLEDGMENTS
The authors thank J. Wright and C. Augspurger for their advice,
suggestions and comments on an earlier draft of this manuscript.
Thanks to O. Sanjur, M. Gonzales, G. Grajales and C. Vergara
for all their support and help in the lab, and to D. Kikuchi for
help with ?eldwork. Financial support for this work came from
an STRI short?term and a pre?doctoral fellowship, a dissertation
Improvement Grant?NSF (grant DEB 05?08471), the Francis M.
and Harlie M. Clark Research Support Grant from University of
Illinois, and a SENACYT?IFARHU doctoral fellowship.
LITERATURE CITED
AIDE, T. M. 1987. Limbfalls: A major cause of sapling mortality for tropical
forest plants. Biotropica 19: 284?285.
AUGSPURGER, C. K. 1983. Offspring recruitment around tropical trees:
Changes in cohort distance with time. Oikos 40: 189?196.
BOND, W. J., AND J. J. MIDGLEY. 2003. The evolutionary ecology of sprouting
in woody plants. Int. J. Plant Sci. 164(Suppl.): S103?S114.
BURGER, W. C. 1971. Piperaceae. Flora Costaricensis. Fieldiana Botany 35: 5?
218.
BURT, A. 2000. Perspective: Sex, recombination, and the ef?cacy of selec-
tion?was Weismann right? Evolution 54: 337?351.
BUSH, S. P., AND D. L. MULCAHY. 1999. The effects of regeneration by frag-
mentation upon clonal diversity in the tropical forest shrub Poikilacan-
thus macranthus: Random ampli?ed polymorphic DNA (RAPD) results.
Mol. Ecol. 8: 865?870.
CHARLESWORTH, B. 1980. Evolution in Age?Structured Populations. Cambridge
University Press, Cambridge, U.K.
CHAZDON, R. L., K. WILLIAMS, AND C. B. FIELD. 1988. Interactions between
crown structure and light environment in ?ve rain forest piper species.
Am. J. Bot. 75: 1459?1471.
CLARK, D. B., AND D. A. CLARK. 1991. The impact of physical damage on
canopy tree regeneration in tropical rain forest. J. Ecol. 79: 446?457.
CONNELL, J. H. 1971. On the role of natural enemies in preventing competi-
tive exclusion in some marine animals and in rain forest trees. In
P. J. Den Boer and G. Gradwell (Eds.). Dynamics of populations.
PUDOC, Wageningen, The Netherlands.
COOK, R. E. 1983. Clonal plant populations. Am. Sci. 71: 244?253.
CROAT, T. 1978. Flora of Barro Colorado Island. Stanford University Press,
Stanford, California, 943pp.
DAWS, M. I., D. F. R. P. BURSLEM, L. M. CRABTREE, P. KIRKMAN, C. E. MUL-
LINS, AND J. W. DALLING. 2002. Differences in seed germination
responses may promote coexistence of four sympatric Piper species.
Funct. Ecol. 16: 258?267.
DE FIGUEIREDO, R. A., AND M. SAZIMA. 2000. Pollination biology of Piperaceae
species in southeastern Brazil. Ann. Bot. 85: 455?460.
ELLSTRAND, N. C., AND M. L. ROOSE. 1987. Patterns of genotypic diversity in
clonal plant species. Am. J. Bot. 74: 123?131.
FIELD, C. B., AND C. VAZQUEZ?YANES. 1993. Species of the genus Piper provide
a model to study how plants can grow in different kinds of rainforest
habitats. Interciencia 18: 230?236.
FLEMING, T. H. 1981. Fecundity, fruiting pattern, and seed dispersal in Piper
amalago (Piperaceae), a bat?dispersed tropical shrub. Oecologia 51: 42?
46.
FLEMING, T. H. 1985. Coexistence of ?ve sympatric Piper (Piperaceae) species
in a tropical dry forest. Ecology 66: 688?700.
GARTNER, B. L. 1989. Breakage and regrowth of piper species in rain forest
understory. Biotropica 21: 303?307.
GILL, D. E., L. CHAO, S. L. PERKINS, AND J. B. WOLF. 1995. Genetic mosaicism
in plants and clonal animals. Annu. Rev. Ecol. Syst. 26: 423?444.
GREIG, N. 1993a. Regeneration mode in Neotropical Piper: habitat and species
comparisons. Ecology 94: 2125?2135.
GREIG, N. 1993b. Predispersal seed predation on ?ve Piper species in tropical
rainforest. Oecologia 93: 412?420.
GUARIGUATA, M. R. 1998. Response of forest tree saplings to experimental
mechanical damage in lowland Panama. For. Ecol. Manage. 102: 103?
111.
HAMILTON, W. D. 1980. Sex versus non?sex versus parasite. Oikos 35: 282?
290.
HAMILTON, W. D., R. AXELROD, AND R. TANESE. 1990. Sexual reproduction as
an adaptation to resist parasites (a review). Proc. Natl. Acad. Sci. 87:
3566?3573.
HAMRICK, J. L., AND M. J. GODT. 1989. Allozyme diversity in plant species.
InA. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir (Eds.).
Plant population genetics, breeding, and genetic resources, pp. 43?63.
Sinauer, Sunderland, Massachusetts.
HEYWOOD, J. S., AND T. H. FLEMING. 1986. Patterns of allozyme variation in
three Costa Rican species of Piper. Biotropica 18: 208?213.
HONNAY, O., AND H. JACQUEMYN. 2008. A meta?analysis of the relation
between mating system, growth form and genotypic diversity in clonal
plant species. Evol. Ecol. 22: 299?312.
HOULISTON, G. J., AND H. M. CHAPMAN. 2004. Reproductive strategy and pop-
ulation variability in the facultative apomict Hieracium pilosella (Astera-
ceae). Am. J. Bot. 91: 37?44.
JANZEN, D. H. 1970. Herbivores and the number of tree species in tropical
forest. Am. Nat. 104: 501?529.
KINSMAN, S. 1990. Regeneration by fragmentation in tropical montane forest
shrubs. Am. J. Bot. 77: 1626?1633.
KOBE, R. K. 1997. Carbohydrate allocation to storage as a basis of interspe-
ci?c variation in sapling survivorship and growth. Oikos 80: 226?233.
LASSO, E. 2008. The importance of setting the right genetic distance threshold
for identi?cation of clones using ampli?ed fragment length polymor-
phism: A case study with ?ve species in the tropical plant genus Piper.
Mol. Ecol. Resour. 8: 74?82.
LASSO, E., B. ENGELBRECHT, AND J. DALLING. 2009. When sex is not enough:
Ecological correlates of resprouting capacity in congeneric tropical for-
est shrubs. Oecologia 161: 43?56.
LEIGH, E. G. J. (Ed.). 1999. Tropical forest ecology: A view from Barro Colo-
rado Island. Oxford University Press, Oxford, U.K.
LIAN, C., R. OISHI, N. MIYASHITA, AND T. HOGETSU. 2003. High somatic insta-
bility of a microsatellite locus in a clonal tree, Robinia pseudoacacia.
Theor. Appl. Genet. 108: 836?841.
42 Lasso, Dalling, and Bermingham
MARIOT, A., L. C. DI STASI, AND M. S. DOS REIS. 2002. Genetic diversity in
natural populations of Piper cernuum. J. Hered. 93: 365?369.
MARQUIS, R. J. 1988. Phenological variation in the neotropical understory
shrub Piper arieianum: Causes and consequences. Ecology 69: 1552?
1565.
MARQUIS, R. J., E. A. NEWELL, AND A. C. VILLEGAS. 1997. Non?structural car-
bohydrates accumulation and use in an understorey rain?forest shrub
and relevance for the impact on leaf herbivory. Funct. Ecol. 11: 636?
643.
MEIRMANS, P. G., AND P. H. VAN TIENDEREN. 2004. Genotype and genodive:
Two programs for the analysis of genetic diversity of asexual organ-
isms. Mol. Ecol. Notes 4: 792?794.
MES, T. H. M., P. KUPERUS, J. KIRSCHNER, J. STEPANEK, H. STORCHOVA,
P. OOSTERVELD, AND J. C. M. DEN NIJS. 2002. Detection of genetically
divergent clone mates in apomictic dandelions. Mol. Ecol. 11: 253?
265.
MICHEL, A., R. S. ARIAS, B. E. SCHEFFLER, S. O. DUKE, M. NETHERLAND, AND
F. E. DAYAN. 2004. Somatic mutation?mediated evolution of herbicide
resistance in the nonindigenous invasive plant hydrilla (Hydrilla verticillata).
Mol. Ecol. 13: 3229?3237.
MIWA, M., R. TANAKA, T. YAMANOSHITA, M. NORISADA, K. KOJIMA, AND
T. HOGETSU. 2001. Analysis of clonal structure of Melaleuca cajuputi
(Myrtaceae) at a barren sandy site in Thailand using microsatellite
polymorphism. Trees Struct. Funct. 15: 242?248.
MORISITA, M. 1959. Measuring of interspeci?c association and similarity
between communities. Memoirs of the Faculty of Science Kyushu Uni-
versity Series E 3, pp. 65?80.
MURAWSKI, D. A., AND J. L. HAMRICK. 1990. Local genetic and clonal structure
in the tropical terrestrial bromeliad, Aechmea magdalenae. Am. J. Bot.
77: 1201?1208.
MYERS, J. A., AND K. KITAJIMA. 2007. Carbohydrate storage enhances seedling
shade and stress tolerance in a neotropical forest. J. Ecol. 95: 383?
395.
NAGAMITSU, T., M. OGAWA, K. ISHIDA, AND H. TANOUCHI. 2004. Clonal diver-
sity, genetic structure, and mode of recruitment in a Prunus ssiori popu-
lation established after volcanic eruptions. Plant Ecol. 174: 1?10.
O?CONNELL, L. M., AND K. RITLAND. 2004. Somatic mutations at microsatellite
loci in western red cedar (Thuja plicata: Cupressaceae). J. Hered. 95:
172?176.
OROZCO?SEGOVIA, A., AND C. VAZQUEZ?YANES. 1989. Light effect on seed ger-
mination in Piper L.Acta Oecol./Oecol. Plantar. 10: 123?146.
PACIOREK, C. J., R. CONDIT, S. P. HUBBELL, AND R. B. FOSTER. 2000. The
demographics of resprouting in tree and shrub species of a moist
tropical forest. J. Ecol. 88: 765?777.
PAUN, O., J. GREILHUBER, E. M. TEMSCH, AND E. HORANDL. 2006. Patterns,
sources and ecological implications of clonal diversity in apomictic
Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae).
Mol. Ecol. 15: 897?910.
PEAKALL, R., AND P. E. SMOUSE. 2006. GENALEX 6: Genetic analysis in
excel. Population genetic software for teaching and research. Mol.
Ecol. Notes 6: 288?295.
PIELOU, E. C. 1969. An introduction to mathematical ecology. Wiley?Inter-
science, New York, New York.
POORTER, L., AND K. KITAJIMA. 2007. Carbohydrate storage and light require-
ments of tropical moist and dry forest tree species. Ecology 88: 1000?
1011.
SAGERS, C. L. 1993. Reproduction in neotropical shrubs: The occurrence and
some mechanisms of asexuality. Ecology 74: 615?618.
S?NCHEZ?HIDALGO, M. E., M. MART?NEZ?RAMOS, AND F. J. ESPINOSA?GARC?A.
1999. Chemical differentiation between leaves of seedlings and spatially
close adult trees from the tropical rain?forest species Nectandra ambigens
(Lauraceae): An alternative test of the Janzen?Connell model. Funct.
Ecol. 13(5): 725?732.
SEMPLE, K. 1974. Pollination in Piperaceae. Ann. MO. Bot. Gard. 61: 868?
871.
SILVERTOWN, J. 2008. The evolutionary maintenance of sexual reproduction:
Evidence from the ecological distribution of asexual reproduction in
clonal plants. Int. J. Plant Sci. 169: 157?168.
THIES, W., AND E. V. K. KALKO. 2004. Phenology of neotropical pepper plants
(Piperaceae) and their association with their main dispersers, two
short?tailed fruit bats, Carollia perspicillata and C. castanea (Phyllostomi-
dae). Oikos 104: 362?376.
VAN DER HULST, R. G., T. H. M. MES, M. FALQUE, P. STAM, J. C. M. DEN NIJS,
AND K. BACHMANN. 2003. Genetic structure of a population sample of
apomictic dandelions. Heredity 90: 326?335.
VAUGHAN, S. P., J. E. COTTRELL, D. J. MOODLEY, T. CONNOLLY, AND K. RUSSELL.
2007. Clonal structure and recruitment in British wild cherry (Prunus
avium L.). For. Ecol. Manage. 242: 419?430.
V?ZQUEZ?YANES, C. 1974. Estudios sobre la eco?siolog?a de la germinaci?n
en una zona c?lido h?meda en M?xico. PhD dissertation, UNAM,
M?xico, p. 140.
V?ZQUEZ?YANES, C., AND H. SMITH. 1982. Phytochrome control of seed ger-
mination in the tropical rain forest trees Cecropia obtusifolia and Piper
auritum and its ecological signi?cance. New Phytol. 92: 477?485.
VERDAGUER, D., AND F. OJEDA. 2002. Root starch storage and allocation pat-
terns in seeder and resprouter seedlings of two Cape Erica (Ericaceae)
species. Am. J. Bot. 89: 1189?1196.
VOS, P., R. HOGERS, M. BLEEKER, M. REIJANS, T. VANDELEE, M. HORNES,
A. FRIJTERS, J. POT, J. PELEMAN, M. KUIPER, AND M. ZABEAU. 1995.
AFLP?a new technique for DNA??ngerprinting. Nucleic Acids Res.
23: 4407?4414.
WALTERS, M. B., AND C. B. FIELDS. 1987. Photosynthetic light acclimation in
two rainforest Piper species with different ecological amplitudes. Oeco-
logia 72: 449?456.
WATKINSON, A. R., AND J. C. POWELL. 1993. Seedling recruitment and the
maintenance of clonal diversity in plant populations?a computer sim-
ulation of Ranunculus Repens. J. Ecol. 81: 707?717.
WID?N, B., N. CRONENBERG, AND M. WID?N. 1994. Genotypic diversity, molec-
ular markers and spatial distribution of genets in clonal plants, a litera-
ture survey. Folia Geobot. Phytotaxon. 29: 245?263.
WILLIAMS, K., C. B. FIELD, AND H. A. MOONEY. 1989. Relationships among
leaf construction cost, leaf longevity, and light environment in rain?for-
est plants of the genus Piper. Am. Nat. 133: 198?211.
YEH, F. C., D. K. X. CHONG, AND R.?C. YANG. 1995. RAPD variation within
and among natural populations of trembling aspen (Populus tremuloides
Michx.) from Alberta. J. Hered. 86: 454?460.
Asexual Recruitment in Piper 43