Annals of Botany 118: 1293–1306, 2016

doi:10.1093/aob/mcw183, available online at www.aob.oxfordjournals.org

Genetic diversity and structure of wild populations of Carica papaya in

Northern Mesoamerica inferred by nuclear microsatellites and
chloroplast markers
Mariana Chavez-Pesqueira and Juan N
un~ez-Farfan*
Laboratorio de Genética Ecol
ogica y Evoluci
on, Departamento de Ecologıa Evolutiva, Instituto de Ecologıa, Universidad
Nacional Autonoma de México, Apartado Postal 70-275, México 04510 Distrito Federal, México
*For correspondence. E-mail [email protected]

Received: 26 January 2016 Returned for revision: 31 May 2016 Accepted: 4 July 2016 Published electronically: 6 October 2016

 Background and aims Few studies have evaluated the genetic structure and evolutionary history of wild varieties
of important crop species. The wild papaya (Carica papaya) is a key element of early successional tropical and
sub-tropical forests in Mexico, and constitutes the genetic reservoir for evolutionary potential of the species. In this
study we aimed to determine how diverse and structured is the genetic variability of wild populations of C. papaya
in Northern Mesoamerica. Moreover, we assessed if genetic structure and evolutionary history coincide with
hypothetized (1) pre-Pleistocene events (Isthmus of Tehuantepec sinking), (2) Pleistocene refugia or (3) recent
patterns.
 Methods We used six nuclear and two chloroplast (cp) DNA markers to assess the genetic diversity and phylo-
geographical structure of 19 wild populations of C. papaya in its natural distribution in Northern Mesoamerica.
 Key Results We found high genetic diversity (Ho ¼ 0681 for nuclear markers, and h ¼ 0701 for cpDNA
markers) and gene flow between populations of C. papaya (migration r up to 420 km). A lack of phylogeographical
structure was found with the cpDNA markers (NST < GST), whereas a recent population structure was inferred
with the nuclear markers. Evidence indicates that pre-Pleistocene events or refugia did not play an important role in
the genetic structuring of wild papaya.
 Conclusions Because of its life history characteristics and lack of an ancient phylogeographical structure found
with the cpDNA markers, we suggest that C. papaya was dispersed throughout the lowland rain forests of Mexico
(along the coastal plains and foothills of Sierras). This scenario supports the hypothesis that tropical forests in
Northern Mesoamerica did not experience important climate fluctuations during the Pleistocene, and that the life
history of C. papaya could have promoted long-distance dispersal and rapid colonization of lowland rainforests.
Moreover, the results obtained with the nuclear markers suggest recent human disturbances. The fragmentation of
tropical habitats in Northern Mesoamerica appears to be the main driver of genetic structuring, and the major threat
to the dispersion and survival of the species in the wild.

Key words: Carica papaya, Northern Mesoamerica, nuclear microsatellites, chloroplast DNA, genetic diversity,
genetic structure, phylogeography, gene flow, barriers, habitat fragmentation, wild papaya.

INTRODUCTION (Davis et al., 2005) and some authors believe that these have re-
mained relatively stable since the climatic fluctuations of the
Mexico is one of the highest ranked countries in terms of biodi- Pliocene–Pleistocene (Colinvaux et al., 1996; Fine and Ree,
versity, comprising approx. 12 % of the world’s biota (Toledo 2006), preventing genetic structuring of tropical species.
and Ord o~
nez, 1993). Its biological diversity is due partially to However, longer periods of climatic stability in the tropics may
its geographical position between the Nearctic and the reflect range disjunctions around geological barriers that are
Neotropics, and to its highly complex topography that has given much older than the Pleistocene vegetation changes that shaped
rise to a diverse combination of environmental conditions that genetic patterns at higher latitudes (Dick, 2010). Other authors
host numerous ecosystems (Coates and Obando, 1996). South- suggest that the areas of concentrated species diversity and spe-
eastern Mexico covers a large part of the Mesoamerican terri- cies endemism in mountainous regions of tropical America rep-
tory (hereafter Northern Mesoamerica), one of Earth’s most im- resent zones that acted as tropical refugia for rain forest taxa
portant biodiversity ‘hotspots’ (Myers et al., 2000). during the cool, dry Pleistocene period; these Pleistocene refu-
Genetic structure and genetic divergence within and among gia may have received high rainfall and remained continually
populations of tropical trees in Mesoamerica result from a num- warm while savannas and tropical dry forests expanded
ber of contemporary and historical factors acting at different (Haffer, 1969; Graham, 1973; Toledo, 1982; Prance, 1982;
temporal and spatial scales. For tropical forest species a variety Pennington et al., 2000), leaving a high genetic structure signa-
of scenarios can be expected to explain the structuring of their ture in populations from different refugia. In addition, because
genetic diversity. Tropical forests date back to Cretaceous times most tropical tree species occur at low population densities,

C The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company.
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1294 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica

they may be more susceptible to genetic drift, showing higher plant species (Vavilov, 1926), so natural populations of impor-
inbreeding than the relatively common tree species studied in tant crops species and their wild relative species inhabit this re-
temperate and boreal forests (Fedorov, 1966). Moreover, low- gion. In spite of their importance, few studies have addressed
land tropical plants are thought to be physiologically sensitive the phylogeographical history and genetic structure and diver-
to mild stress of drought and cold, especially to low tempera- sity of wild populations of important tropical crop species
tures associated with increasing elevations (Janzen, 1967). This (Fukunaga et al., 2005; Londo et al., 2006; Serrano-Serrano
could promote a latitudinal gradient with a stronger phylogeo- et al., 2010).
graphical structure in tropical lowland species than in temperate Wild papaya (Carica papaya) is a tropical nomadic pioneer
plants (Dick and Heuertz, 2008), and several studies have docu- tree that occurs naturally in lowland tropical and sub-tropical
mented phylogeographical structure of Neotropical trees sam- forests of Mesoamerica. The cultivated varieties of C. papaya
pled around geographical barriers (Aide and Rivera, 1998; represent the third most cultivated tropical crop worldwide
Cavers et al., 2003; Dick et al., 2003; Novick et al., 2003), and (FAO, 2012). Natural populations of C. papaya occur in
higher FST values in tropical trees than in trees from temperate Northern Mesoamerica; however, the wide geographical distri-
and/or boreal forests (Dick and Heuertz, 2008). Finally, current bution of wild populations and the lack of collections in many
environmental features may drive patterns of gene flow and ge- areas of the region have prevented a precise assessment of their
netic structure of tropical plants (Poelchau and Hamrick, 2012); diversity and genetic structure for conservation purposes. Few
in particular, habitat fragmentation in recent times may also studies have evaluated the genetic structure and evolutionary
have played a role in structuring genetic diversity (Twyford history of wild varieties of important crops, with relevance to
et al., 2013). conservation and management. Moreover, Mesoamerica (par-
In Northern Mesoamerica two historical events are known to ticularly Mexico) represents one of the regions where Vavilov
have reduced dispersal and generated a spatial structuring of ge- (1926) suggested that many cultivated species originated, diver-
netic diversity in different species: the Isthmus of Tehuantepec sified and were domesticated, such as maize, tomato, beans,
(pre-Pleistocene event) and the Pleistocene glacial refugia cotton, cacao and vanilla. Thus, the aims of the present study
(Bryson et al., 2011; Gutiérrez-Rodrıguez et al., 2011; were (1) to assess the actual state of natural undomesticated
Rodrıguez-G omez et al., 2013). The Isthmus of Tehuantepec populations of C. papaya regarding its distribution and genetic
forms a narrow piece of lowland that separates the south- diversity in Northern Mesoamerica, using nuclear and chloro-
central Mexican highlands (Sierra de Juarez and Sierra Madre plast markers; (2) to evaluate the genetic structure and evolu-
del Sur) from the highlands of Chiapas (Ornelas et al., 2010). It tionary history in its Mesoamerican distribution and whether it
has been suggested that the isthmus was a seaway for much of is consistent with three hypothetical scenarios: pre-Pleistocene
the Pliocene (Morrone, 2006), and therefore a major barrier to events (sea-level oscillations in the Isthmus of Tehuantepec),
gene flow during this period (Twyford et al., 2013). Moreover, patterns of ancient Pleistocene refugia or more recent events;
if the Pleistocene refugia theory is correct, some regions in (3) to define the possible barriers to gene flow in recent and an-
south-eastern Mexico might have acted as Pleistocene refuges cestral times; and (4) to estimate recent migration rates. Finally,
in the past, as suggested by Toledo (1982). Thus, for tropical in light of our results we discuss some conservation remarks for
species in Northern Mesoamerica, we could expect the follow- this important species.
ing scenarios: high levels of population differentiation among
populations of tropical species inhabiting different regions pro-
posed as Pleistocene refugia; a genetic subdivision west and MATERIALS AND METHODS
east of the Isthmus of Tehuantepec; and high genetic structure
Study system
promoted by recent anthropogenic disturbances that have modi-
fied the Northern Mesoamerican landscape. The current rate of Carica papaya L. is a tropical, nomadic, short-lived tree (Fig.
deforestation and habitat fragmentation in Northern 1) that belongs to the family Caricaceae and is the only member
Mesoamerica is high enough to interrupt potential gene flow of the genus after removal from the Vasconcella group, until re-
and endanger the existence of tree species (Novick et al., cently considered as a section within the genus Carica (Badillo,
2003). 2000). Carica papaya is part of a small clade confined to
There is a relative dearth of detailed phylogeographical infor- Mexico and Guatemala that also includes three perennial herbs
mation on Neotropical lowland plant species (but see: Cavers (Jarilla chocola, J. heterophylla and J. nana) and a treelet with
et al., 2003; Novick et al., 2003; Poelchau and Hamrick, 2012; spongy thin stems (Horovitzia cnidoscoloides) (Carvalho and
Twyford et al., 2013), even though lowland tropical vegetation Renner, 2012).
covers most of the Mesoamerican landscape. Although the vast There are many cultivated varieties of papaya that differ in
majority of the world’s tree diversity lies in the tropics, most of traits such as fruit size, colour and flavour, and tree size.
the evolutionary history and genetic knowledge comes from Nowadays, only two varieties are cultivated in Mexico:
temperate and boreal forests, with a marked emphasis on pines, ‘Maradol’, introduced from Cuba, and ‘Mexicana/Amarilla’,
poplars and oaks (Dick, 2010). In Northern Mesoamerica, phy- domesticated in Mexico (SAGARPA, 2009). The two main dif-
logeographical studies have focused mainly on montane tem- ferences between wild and domesticated papayas relate to
perate species or tropical species inhabiting cloud forest flower morphology and fruit size. Wild (undomesticated) popu-
(Jaramillo Correa et al., 2008; Cavender-Bares et al., 2011; lations of papaya are characterized by a strictly dioecious
Gutiérrez-Rodrıguez et al., 2011; Ornelas et al., 2013; Ruız- breeding system (Chavez-Pesqueira et al., 2014) (rather than
Sanchez and Ornelas, 2014). Moreover, Mesoamerica has been being trioecious like most varieties of cultivated papaya, where
recognized as one of the centres of origin of many domesticated hermaphroditic individuals are preferred); and female trees
 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica 1295

A B

C D E

FIG. 1. (A) Natural population of Carica papaya at El Cielo, Tamaulipas, Mexico. (B) Female individual of C. papaya with fruits. (C) Male inflorescence of a wild
C. papaya plant. (D) Female flower of a wild C. papaya plant. (E) Mature fruits of a wild C. papaya plant.

produce small (no more than 15 cm in diameter), seedy fruits The geographical distribution of the C. papaya clade and the
with a thin mesocarp and almost without pulp (Carvalho and occurrence of wild papayas in Mesoamerica are consistent with
Renner, 2012) (Fig. 1). Sexual expression in C. papaya is ge- a domestication of papaya in that region (Carvalho and Renner,
netically determined by a pair of homomorphic sex chromo- 2012). Carica papaya diverged from its sister clade approx. 25
somes (Yu et al., 2007). Carica papaya is pollinated by Mya. The biogeographical history of Caricaceae involves long-
Lepidoptera, mainly by sphingid hawk moths (Vega-Frutis & distance dispersal from Africa to Central America 35 Mya, and
Guevara, 2009). Wild C. papaya blooms and fruits year round; expansion across the Panamanian land bridge between 27 and
the fruits are consumed and probably dispersed mainly by birds 19 Mya (Carvalho and Renner, 2012).
and small mammals that climb the stem to consume the fruits Wild papayas inhabit many parts of south-eastern Mexico,
(our pers. observ.). usually associated with lowland humid and sub-humid tropical
1296 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica

1 Populations:
N 1. Cielo
17
19 2. Huasteca
2 16 3. Tamazunchale
4. Poza Rica
3 5. Tuxtlas
4 Gulf of Mexico 18
Yucatán 6. Acayucan
Peninsula 7. Matías Romero
8. Santiago Astata
15 9. Ventanilla
5 14 10. Marquelia
13 11. Villa Guadalupe
12. Palenque
6 Isthmus 11 13. Mamantel
of 12 14. Caobas
7 Tehuantepec 15. Oxtankah
16. Dzibilchaltún
10 17. Río Lagartos
Pacific Ocean 8 18. Chichén Itzá
9 19. Cancún
0 500 km

FIG. 2. Distribution of the 19 sample locations of wild Carica papaya in Northern Mesoamerica. Yellow areas represent the proposed Pleistocene refugia for Mexico
(Toledo, 1982).

forests (Paz and Vazquez-Yanes, 1998). In many cases, wild markers exhibit different modes of inheritance that help to elu-
varieties are also found in home gardens of ethnic groups from cidate the evolutionary history of species. DNA microsatellites
south-eastern Mexico. In some regions such as Yucatan (Teran show high variability and allow genome-wide information that
and Rasmussen, 1995), San Luis Potosı and Veracruz in reflects both contemporary pollen and seed dispersal. In con-
Mexico, people use wild papaya fruits to make sweet glaze (our trast, chloroplast DNA (cpDNA) is maternally inherited in
pers. observ.). In tropical rain forests wild papaya behaves like many plants (including C. papaya) and is dispersed only by
a typical fast-growing, short-lived, pioneer tree; they establish seeds, thus providing information about ancient seed dispersal
rapidly and grow only in recent and relatively large canopy and reflecting a different evolutionary history from nuclear
light-gaps in mature forest and in early secondary forest DNA because of its small effective population size. Because of
(Chavez-Pesqueira et al., 2014). Wild papaya is relatively abun- this, we used both nuclear (DNA microsatellites) and chloro-
dant in recent large, man-made, clearings that are not ploughed plast (cpDNA) markers to assess the genetic structure and phy-
(called acahuales). Large natural forest gaps 1–5 years old can logeographical history of natural populations of C. papaya.
sometimes contain several wild papaya trees (Paz and Six microsatellite primers from Ocampo et al. (2006), previ-
Vazquez-Yanes, 1998). In Mexico wild papayas occur within ously used for wild C. papaya (Chavez-Pesqueira et al., 2014),
pollen/seed dispersal range from cultivars (our pers. observ.) were amplified using a Multiplex approach. Each multiplex
(10 lL) contained 20 ng of DNA template (2 ll), 02 lM
fluorescently labelled forward primers, 02 lM reverse
Sampling primers, RNase/DNase-free water and Reaction Mix (1).
Amplifications reactions were carried on a Veriti 96-well ther-
A total of 355 individuals from 19 populations were sampled,
mal cycler. The PCRs were performed through touchdown re-
covering the natural distribution of C. papaya in Mexico
actions, starting with an initial heat activation at 95  C for
(Fig. 2). Between 11 and 35 individuals were sampled in each
10 min followed by 31 cycles with a denaturation at 94  C for
population (Table 1). To find these populations, extensive
1 min, an annealing for 1 min and 1 min of extension at 72  C.
searches were done throughout tropical and subtropical regions
Annealing cycling temperature began at 57  C dropping one de-
of south-eastern Mexico. Populations were chosen and col-
gree every cycle until reaching 51  C; this temperature was
lected only if they were strictly wild (dioecious individuals,
held for six cycles, followed by two stages of 12 cycles, at 55
small fruits), distant of human populations and had a significant
and 54  C, respectively. PCR products were run on ABI Prism
number of individuals to sample.
310 and ABI 3730xl automated capillary sequencers, and allele
sizes were scored manually using LIZ-500 size standard in
GeneMarker v2.4.0 (SoftGenetics, LLC, State College, PA,
DNA extraction, amplification and sequencing
USA).
DNA was extracted using the CTAB method (Doyle and Additionally, nine chloroplast markers (ndhJ-trnF, trnQ-
Doyle, 1987) with some modifications. Nuclear and chloroplast 50 rps16, ndhF-rp132, psbJ-petA, trnH-psbA, trnL-F, rbcL800f-
 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica 1297

TABLE 1. Population number, sample sites, geographical location subdivisions for multilocus genotypic data using a Bayesian
and sample size (n) of wild populations of Carica papaya in its procedure, considering spatial proximity when assigning indi-
natural distribution in Northern Mesoamerica viduals to clusters. Ten independent runs were conducted for
each analysis using 1 000 000 Markov chain Montel Carlo
Population Sample site Geographical location n
no.
(MCMC) iterations with a thinning value of 100. Uncorrelated
and null allele model options were activated. The number of ge-
1 Cielo, Tamaulipas 23 010 016000 N, 99 070 315000 W 35 netic clusters was set to unknown, but the maximum possible
2 Huasteca, San Luis Potosı 21 500 352000 N, 99 090 065000 W 20
3 Tamazunchale, San Luis Potosı 21 140 36800 0 N, 98 440 36900 0 W 30 number of clusters was limited to 19 to offer a large enough
4 Poza Rica, Veracruz 20 280 49610 0 N, 97 390 22210 0 W 16 search space for the MCMC algorithm. Geographical coordina-
5 Tuxtlas, Veracruz 18 340 56940 0 N, 95 040 44000 0 W 30
6 Acayucan, Veracruz 17 300 512500 N, 95 050 127500 W 25
tes were decimal degrees of the sampling locations with an un-
7 Matıas Romero, Oaxaca 16 540 24420 0 N, 95 000 35640 0 W 30 certainty of 400 km (based on the long migration rates found
8 Santiago Astata, Oaxaca 15 590 01100 0 N, 95 390 02170 0 W 11 with BAYESASS among populations; see Results). MCMC
9 Ventanilla, Oaxaca 15 400 43150 0 N, 96 340 10810 0 W 30
10 Marquelia, Guerrero 16 340 51260 0 N, 98 470 17210 0 W 23 post-processing was done with a burn-in of 1000 iterations, and
11 Villa Guadalupe, Tabasco 17 220 10780 0 N, 93 370 13350 0 W 30 the average posterior probability was used to select the best-
12 Palenque, Chiapas 17 290 39980 0 N, 92 010 09130 0 W 26 suited run.
13 Mamantel, Campeche 18 320 49580 0 N, 91 050 09000 0 W 30
14 Caobas, Campeche 18 300 476500 N, 89 280 152100 W 32 We further evaluated the genetic differentiation of popula-
15 Oxtankah, Quintana Roo 18 340 58400 0 N, 88 140 40060 0 W 25 tions by assuming a step-wise mutation model with Slatkin’s
16 Dzibilchaltun, Yucatan 21 050 283400 N, 89 350 510000 W 28
17 Rıo Lagartos, Yucatan 21 310 02660 0 N, 88 080 25620 0 W 27
RST (Slatkin, 1995). Partitioning of genetic variability within
18 Chichén Itza, Yucatan 20 400 561700 N, 88 340 065300 W 26 and among population groups was tested by analysis of molecu-
19 Cancun, Quintana Roo 20 540 336100 N, 87 250 516200 W 18 lar variance (AMOVA; Excoffier et al., 1992) among the ge-
netic clusters derived from Geneland, and between the
populations east and west from the Isthmus of Tehuantepec, us-
600r, matKF1-R1 and rpl20-rps12), as well as the internal tran- ing GenAlex 6.5 (Peakall and Smouse, 2012). In addition, the
scribed spacer (ITS1–2), were tested for amplification and se- isolation-by-distance model (Wright, 1943) was tested using a
quence variation in C. papaya. trnH-psbA and rpl20-rps12 Mantel test (Mantel, 1967), to determine whether matrices of
were chosen because of their high amplification quality and pairwise population genetic distances [RST/(1  RST)] were cor-
variability among populations of C. papaya. The remaining related to the logarithm of the Euclidean geographical dis-
tested markers were poorly amplified or were invariant across tances. We calculated Mantel’s correlation coefficients (r)
populations. PCRs were performed in a volume of 15 lL, con- using the ade4 package (Dray and Dufour, 2007) in R (R
taining 1 lL DNA, 2 lL buffer, 125 lL MgCl2 (125 mM), 2 lL Development Core Team, 2011). The statistical significance of
dNTP and 1 lL of each primer. The amplification condition for the estimators was determined with 9999 permutations.
trnH-psbA was 94  C for 5 min, followed by 35 cycles at 94  C The geographical location of genetic discontinuities among
for 1 min, 56  C for 1 min and 72  C for 2 min, and a final exten- populations was assessed with the Monmonier’s maximum dif-
sion at 72  C for 8 min. For rpl20-rps12 the same conditions ference algorithm implemented in BARRIER version 2.2
were used, but the annealing temperature was set to 53  C. PCR (Manni et al., 2004). This program first creates a map of the
products were visualized via 1 % agarose gel electrophoresis. sampling locations from geographical coordinates. From a ma-
All forward and reverse strands were sequenced and then edited trix of pairwise genetic distances (FST) between populations,
in Sequencher 5.0 (Gene Codes Corp., Ann Arbor, MI, USA); barriers are then represented on the map by identifying the
nucleotide substitutions, indels (i.e. insertions or deletions) and edges of polygons where the maximum distances occur.
inversions were visually checked. Sequences were aligned online Population pairwise FST comparisons were calculated using
with MAFFT version 7 (Katoh and Standley, 2012). The se- ARLEQUIN version 3.5.1.2 (Excoffier et al., 2005). To obtain
quences reported in this study are available from GenBank (ac- statistical confidence values for the barriers, 100 replicates of
cession numbers: KX451371–KX451661 for psbA-trnH and both distance matrices were calculated by resampling individ-
KX451662–KX451837 for rpl20-rps12). uals within populations.
The program BAYESASS edition 3 (Wilson and Rannala,
2003) was used to estimate short-term migration rates.
BAYESASS uses a genetic assignment to estimate short-term
Data analyses for nuclear microsatellites
dispersal rates, providing an estimate of migration rates over
Parameters of genetic diversity for each population [percent- the past two generations. We performed five runs (each with
age of polymorphic loci (%P), the number of alleles (A), ob- different starting seed value) of 10 million generations, with a
served heterozygosity (Ho), expected heterozygosity (He) and burn-in of 1 million generations, and sampled the chain every
inbreeding coefficient (f)] were calculated using GenAlex 6.5 2000 generations.
(Peakall and Smouse, 2012). We used Microchecker 2.2.3 (van
Oosterhout et al., 2004) to detect null alleles.
The genetic structure of populations was inferred using
Data analyses of cpDNA
Geneland (version 4.0; Guillot et al., 2008) in R (R
Development Core Team, 2011). We preferred Geneland over Haplotype and nucleotide diversities, as well as neutrality
Structure because Geneland includes a null allele model and, in test statistics of Tajima’s D (Tajima, 1989) and Fu’s F (Fu,
particular, because it takes into account the spatial distribution 1997) were calculated for each population using ARLEQUIN
of populations. Geneland determines the number of population version 3.5.1.2 (Excoffier et al., 2005). Tajima’s D takes into
1298 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica

account the genetic diversity and the number of variable sites Euclidean geographical distances. The statistical significance of
in a sequence, to test for demographic range expansion. the estimators was determined with 9999 permutations.
Significant D values can be due to bottlenecks, selective effects, Because nuclear microsatellites reflect a recent history of
population expansion or heterogeneity of mutation rates populations, we also used BARRIER version 2.2 (Manni et al.,
(Tajima, 1996). Fu’s F (Fu, 1997) uses information of the distri- 2004) for the psba-trnH region to account for more ancient bar-
bution of haplotypes to test for demographic expansion and it is riers to gene flow.
more sensitive to population growth than Tajima’s D. Large Genetic differentiation among populations was estimated by
negative F values generally are indicative of sudden and signifi- computing a distance matrix based on the number of mutational
cant population growth. steps between haplotypes (NST) and by using haplotype fre-
We were not able to concatenate the two studied regions quencies (GST). The occurrence of phylogeographical structure
of cpDNA because of inconsistency in the amplification of was inferred by testing for significant differences between GST
many individuals. The genealogical relationship between and NST using PERMUT 2.0 (Pons and Petit, 1996) with 1000
haplotypes was estimated independently for trnH-psbA and permutations. In contrast to GST, NST considers sequence differ-
rpl20-rps12 sequences using statistical parsimony in TCS ences between the haplotypes. Thus, NST > GST indicates that
1.2.1 (Clement et al., 2000), using an algorithm for clado- closely related haplotypes are observed more often in a given
grams estimated by maximum parsimony, with indels coded geographical area than would be expected by chance (Pons and
as a fifth state. Petit 1996).
Spatial structuring of variation in psbA-trnH was examined
using spatial analysis of molecular variance (SAMOVA;
Dupanloup et al., 2002), to infer population structure without RESULTS
any prior knowledge. SAMOVA attempts to identify groups of
Nuclear microsatellite diversity and population structure
locations that are geographically homogeneous and genetically
differentiated from each other, maximizing the proportion of to- All loci were polymorphic and moderate to high values of neu-
tal genetic variance due to differences between groups of popu- tral genetic diversity were estimated in all natural populations
lations (FCT). We considered values of K (group number) of C. papaya (Ho values from 0409 to 0783 and He values
between 2 and 19 using 100 initial conditions for each run. from 0634 to 0806) (Table 2). Populations 3, 4, 6, 7, 11, 13,
AMOVAs (Excoffier et al., 1992) for psbA-trnH were per- 14 and 15 (Tamazunchale, Poza Rica, Acayucan, Matıas
formed to determine how genetic variability is distributed Romero, Villa Guadalupe, Mamantel, Caobas and Oxtankah)
within and among localities, and between two pre-defined ge- showed He values higher than 07. In contrast, populations 5, 8
netic groups: east and west of the Isthmus of the Tehuantepec. and 19 (Tuxtlas, Santiago Astata and Cancun) showed the low-
A total of 1000 permutations were performed for each est values. Furthermore, f values were, in general, close to 0 but
AMOVA. A pattern of isolation by distance (Wright, 1943) with positive and negative values, implying populations with
was evaluated for the chloroplast marker psba-trnH, which can excess (–) and deficiency (þ) of heterozygotes (Table 2) at mi-
indicate restricted seed dispersal, with a Mantel test in R (pack- crosatellite loci. In particular, the Oxtankah population (popula-
age ade4; Dray and Dufour, 2007). We compared FST pairwise tion 15) showed the lowest value (f ¼ 0100) and Tuxtlas
genetic distance among populations and the logarithm of the population (population 5) the highest (0100). We detected null

TABLE 2. Genetic variability (6s.d.) at DNA microsatellite loci in 19 populations of Carica papaya along its natural distribution
in Mexico. n ¼ simple size; %P ¼ polymorphic loci percentage; A ¼ allele number; Ho ¼ observed heterozygosity; He ¼ expected
heterozygosity; f ¼ inbreeding coefficient

No. Population n %P A Ho He f

1 Cielo 21 100 6167 (0946) 0667 (0082) 0655 (0076) 0017 (0039)
2 Huasteca 16 100 6167 (0703) 0698 (0030) 0710 (0450) 0006 (0044)
3 Tamazunchale 15 100 5500 (0617) 0744 (0036) 0720 (0047) 0041 (0028)
4 Poza Rica 17 100 7667 (0760) 0735 (0062) 0806 (0009) 0089 (0073)
5 Tuxtlas 20 100 6167 (0477) 0600 (0058) 0671 (0018) 0100 (0090)
6 Acayucan 20 100 7167 (0601) 0742 (0024) 0726 (0026) 0025 (0037)
7 Matıas Romero 19 100 7667 (0989) 0711 (0033) 0740 (0030) 0034 (0050)
8 Santiago Astata 11 100 4167 (0307) 0409 (0080) 0654 (0029) 0387 (0110)
9 Ventanilla 18 100 6167 (0401) 0630 (0031) 0688 (0028) 0083 (0030)
10 Marquelia 19 100 4500 (0428) 0693 (0066) 0656 (0036) 0064 (0093)
11 Villa Guadalupe 18 100 7167 (0792) 0750 (0087) 0762 (0036) 0035 (0083)
12 Palenque 18 100 6667 (0333) 0667 (0059) 0705 (0046) 0059 (0042)
13 Mamantel 20 100 8000 (1095) 0783 (0069) 0788 (0021) 0013 (0070)
14 Caobas 20 100 7833 (0792) 0758 (0033) 0748 (0030) 0019 (0050)
15 Oxtankah 20 100 6667 (0715) 0733 (0063) 0669 (0046) 0100 (0070)
16 Dzibilchaltun 20 100 5500 (0563) 0692 (0054) 0655 (0018) 0054 (0074)
17 Rıo Lagartos 20 100 5667 (0615) 0658 (0082) 0652 (0052) 0004 (0100)
18 Chichén Itza 23 100 6617 (0477) 0692 (0046) 0690 (0039) 0005 (0041)
19 Cancun 20 100 5167 (0601) 0583 (0076) 0634 (0077) 0075 (0055)
Mean 355 100 6325 (0175) 0681 (0015) 0701 (0010) 0029 (0017)
 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica 1299

FIG. 3. The six genetic clusters (in blue) inferred from GENELAND using DNA microsatellite loci of natural populations of Carica papaya in Northern
Mesoamerica. Solid black lines represent the location of the most probable barriers obtained with BARRIER for DNA microsatellite loci, and dotted black lines for
the psbA-trnH chloroplast region.

alleles in all loci but their estimated frequency was relatively the most prominent barriers for C. papaya dispersion.
low (<10 %) (Microchecker 2.2.3; van Oosterhout et al., 2004). Interestingly, some barriers were identified in the Yucatan
Six clusters were found by posterior cluster membership Peninsula, between the Cancun population and its surroundings
(GENELAND; K ¼ 6): (1) Cielo, Huasteca, Tamazunchale and and north from the Mamantel and Caobas populations, although
Poza Rica (populations 1, 2, 3 and 4); (2) Ventanilla and no visible physical barriers are present in this area.
Marquelia (populations 9 and 10); (3) Tuxtlas, Acayucan, Villa Recent migration rates among populations, as estimated with
Guadalupe and Palenque (populations 5, 6, 11 and 12); (4) BAYESASS, showed moderate recent migration rates among
Matıas Romero and Santiago Astata (populations 7 and 8); (5) populations that were separated by as much as approx. 420 km
Mamantel and Caobas (populations 13 and 14); and (6) [between Poza Rica (population 4) and Acayucan (population
Oxtankah, Dzibichaltun, Rıo Lagartos, Chichén Itza and 6)] (Table 3). Three populations [Rıo Lagartos (population 17),
Canc un (populations 15, 16, 17, 18 and 19) (Fig. 3). The in- Acayucan (population 6) and Tuxtlas (population 5)] were the
ferred clusters contained closely distributed populations, with principal source with higher migration rates than other popula-
the exception of Oxtankah (population 15), which was grouped tions. Migration rates between Rıo Lagartos (population 17)
with the northern populations of the Yucatan Peninsula. and Chichén Itza (population 18) (migration rate ¼ 01955),
Genetic differentiation among all populations was moderate Rıo Largartos and Oxtankah (population 15) (migration rate ¼
(FST ¼ 0148 and RST ¼ 0149) whereas differentiation among 01834), and Rıo Lagartos and Caobas (population 14) (migra-
Geneland clusters was lower (FST ¼ 0112 and RST ¼ 0082). tion rate ¼ 01725) were the highest. Some populations such as
Hierarchical partitioning of molecular variance (AMOVA) for Cancun, Cielo, Marquelia, Rıo Lagartos and Tuxtlas did not re-
the Geneland groups revealed that the highest proportion of vari- ceive significant migration from any other population. In addi-
ance was located within populations (8445 %; P < 00001) and tion, 12 populations did not represent important sources of gene
lower proportions among populations within groups (1064 %; flow (Caobas, Chichén Itza, Dzibilchaltun, Huasteca,
P < 00001) or among groups (491 %; P ¼ 00293). For the east/ Mamantel, Marquelia, Matıas Romero, Oxtankah, Poza Rica,
west partition, we found the same pattern. Pairwise genetic dif- Santiago Astata, Tamazunchale and Villa Guadalupe).
ferentiation was significantly correlated to geographical distance,
according to the Mantel test for all sites (r ¼ 0171, P ¼ 00189).
Chloroplast genetic diversity and structure
BARRIER suggested that the largest genetic breaks were in
many cases concordant with mountainous areas (Fig. 3), with We obtained 291 cpDNA sequences for the psbA-trnH region
the Sierra Madre del Sur and the Sierra de Juarez Oaxaca being and 176 for the rpl20-rps12 region. No individuals of the
1300 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica

Huasteca population amplified for rpl20-rps12. The psbA-trnH reflecting very low within-site diversity and the lack of varia-
region was 423 bp long with 34 variable sites, 20 substitutions tion between sequences from the same population. Only the
and 14 indels, while the rpl20-rps12 region was 721 bp long Tamazunchale population showed a moderate haplotype and
with 15 variable sites. Indels more than 2 bp long were treated nucleotide diversity (h ¼ 06762, p ¼ 00022). Stochasticity in
as a fifth character state. For psbA-trnH, an inversion of 5 bp polymorphisms, i.e. fewer mutations producing homoplasy,
was also treated as a fifth character state. Haplotype diversity could explain the differences in variation between the two
(h) for psbA-trnH was high for most localities, ranging from cpDNA markers. Discordant patterns may arise from hybridiza-
0307 to 0934, reflecting the presence of different haplotypes tion/introgression between different species (or even between
within each site (Table 4). Nucleotide diversity (p) was moder- highly differentiated populations in time and space), by homo-
ate (00009–00211) for most populations, indicating some vari- plasy or even heteroplasmy (reviewed by Wheeler et al., 2014).
ation between sequences within the same population (Table 4). Yet for papaya, little is known about these processes.
Within-population average haplotype diversity (hs) was 0701 Moreover, the psbA-trnH region has been recognized to show
(0089) and nucleotide diversity (ps) was 0014 (0005). For high levels of variation compared to other cpDNA loci and
rpl20-rps12, h and p were 0 for ten out of the 18 populations, therefore has been widely used for barcoding (Kress et al.,
2005).
TABLE 3. Recent migration rates (6s.d.) and distance (km) be- The statistical parsimony network of natural populations of
tween recipient and source populations of Carica papaya in its C. papaya in Northern Mesoamerica recovered 50 haplotypes
natural distribution in Northern Mesoamerica; migration rates for the psbA-trnH region and ten for the rpl20-rps12 region
higher than 005 are reported (Figs 3 and 4). For psbA-trnH, ten out of 50 haplotypes were
shared between populations, and 40 were private. The most
Recipient population Source population Migration rate (s.d.) Distance (km) widespread haplotypes were H1, H2 and H3, which were found
Acayucan Tuxtlas 01439 (00311) 11248 in 11, ten and ten populations, respectively, and were shared be-
Caobas Rıo Lagartos 01725 (00286) 35910 tween distant populations. Huasteca (population 2) and Tuxtlas
Chichén Itza Rıo Lagartos 01955 (00247) 9864 (population 5) were the populations with more private haplo-
Dzibilchaltun Canc un 01657 (00282) 22813 types, seven (H24, H25, H26, H34, H35, H36, H37) and six
Huasteca Cielo 01640 (00283) 13234
Mamantel Palenque 00574 (00644) 15815 (H17, H18, H42, H43, H44, H45), respectively (Fig. 4). For
Matıas Romero Acayucan 01079 (00538) 7411 rpl20-rps12, less variation was found, and most individuals
Oxtankah Rıo Lagartos 01834 (00272) 32360 (86 %) bore haplotype HA, while six out of nine haplotypes
Palenque Tuxtlas 01340 (00391) 34162 were private. We found that Tamazunchale (population 3) was
Poza Rica Acayucan 00568 (00329) 42350
PozaRica Cielo 00674 (00301) 32211
the population with the highest number of haplotypes (five), and
Santiago Astata Ventanilla 00556 (00254) 10716 was also the one with more private haplotypes, together with
Tamazunchale Cielo 01601 (00307) 20557 Chichén Itza (population 18) (HC and HJ for Tamazunchale and
Ventanilla Acayucan 01299 (00329) 25169 HG and HH for Chichen Itza) (Fig. 5). Due to low variation at
Villa Guadalupe Tuxtlas 01696 (00287) 20420 rpl20-rps12, this marker was not analysed further.

TABLE 4. Population size (n), number of haplotypes, number of polymorphic sites, haplotype diversity (h) 6 s.d., nucleotide diversity
(p) 6 s.d., Tajima’s D and Fu’s F for the plastid trnH-psbA marker in 19 wild Carica papaya populations in Northern Mesoamerica;
Tajima’s D and Fu’s F (P-values are not shown because in all cases P > 01)

Population n Haplotype no. Polymorphic sites h p D F

1. Cielo 18 3 13 03072601316 00062600038 11157 40306

2. Huasteca 14 9 13 09341600448 00152600086 22854 06617
3. Tamazunchale 15 4 14 07333600669 00159600089 21334 59607
4. Poza Rica 13 4 13 07692600724 00164600092 23307 53750
5. Tuxtlas 11 6 7 08364600887 00052600035 10291 12888
6. Acayucan 17 8 21 08382600675 00211600115 06628 21509
7. Matıas Romero 12 4 16 07121601053 00128600075 02247 39722
8. Santiago Astata 10 3 2 05111601643 00013600013 11117 05938
9. Ventanilla 15 9 18 09048600544 00192600106 12114 03064
10. Marquelia 20 5 17 07526600615 00091600053 02945 28275
11. Villa Guadalupe 14 6 7 07912600894 00061600039 09683 02864
12. Palenque 19 8 18 08304600657 00113600064 05600 04672
13. Mamantel 19 4 5 05556601030 00047600031 03456 17388
14. Caobas 15 5 6 08095600589 00050600033 00290 03408
15. Oxtankah 18 5 6 07190600910 00068600042 15282 15639
16.Dzibilchaltun 16 3 2 04250601326 00012600012 03301 02898
17. Rıo Lagartos 16 6 6 08417600534 00071600043 20978 04179
18. Chichén Itza 10 4 5 06444601518 00031600023 08222 03120
19. Cancun 19 2 1 04094601002 00009600010 07937 10079
Mean 516 10 07013600896 00149600049 0465461206 14067621249
Total 291 50 30
 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica 1301

H22 H23 H30

A
H27 H39
H26 H33
H12 H21
H40 H20
H36 H35 H13 H29 H11
H4 H32 H14
H45
H18
H34 H10 H49
H7 H46 H44 H42
H25 H48
H37 H6
H3 H2 H1 H41
H16 H43
H47 H24 H17
H9 H19 H8

H5 H28 H15
H31 H50 H38

0 500 Km

FIG. 4. (A) Statistical parsimony haplotype network for the psbA-trnH chloroplast region of Carica papaya. The size of circles is proportional to the frequency of
each haplotype, and small black circles represent non-sampled haplotypes. (B) Geographical distribution of 19 natural populations of C. papaya cpDNA haplotypes
in south-eastern Mexico. Pie charts represent the haplotypes found in each sampling locality. The area of the pie chart represents the size of the population and the
size of sections is proportional to the haplotypic frequency.

The analysis of spatial genetic structure for psbA-trnH using with the nuclear microsatellite data, using BARRIER for
SAMOVA showed that the highest FCT value was for K ¼ 2; psbA-trnH (Fig. 3). As for the nuclear markers, the most likely
however, one group was formed by only one population (popu- barriers were found in the Yucatan Peninsula separating
lation 1). The K with the highest FCT increment was 9, but Chichén Itza (population 18) and Caobas (population 14)
again, some groups included only one sampling site. When the from all populations nearby. BARRIER also detected two bar-
AMOVA was performed between the populations east and west riers in southern Oaxaca, isolating the Santiago Astata popula-
of the Isthmus of Tehuantepec, we found that most variation tion (population 8).
was contained within stands (512 %; P < 00001), followed by The results from PERMUT showed that the level of NST
among populations within groups (349 %; P < 00001) and (0224, s.e. 00410) was not significantly higher (P > 005) than
finally among groups (139 %; P ¼ 00117). A pattern of isola- GST (0273, s.e. 00571), indicating a lack of phylogeographical
tion by distance was also detected (Mantel test; r ¼ 01663, structure for C. papaya.
P ¼ 00255). Finally, demographic analyses for psbA-trnH showed no sig-
We assessed the potential of genetic breaks that could sug- nificant expansion for any of the populations of C. papaya
gest more ancient barriers among populations, in comparison (Table 4).
1302 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica

A B

N
HE
HJ

HF
HD
Gulf of
Mexico Yucatán
Peninsula
HI HG
HA

HH

HC

HB

Pacific
Ocean 500 Km
0

FIG. 5. (A) Statistical parsimony haplotype network for the rpl20-rps12 chloroplast region of Carica papaya. Haplotype designations in the network correspond to
those in Table 4. The size of circles is proportional to the frequency of each haplotype, and small black circles represent non-sampled haplotypes. (B) Geographical
distribution of 19 natural populations of C. papaya cpDNA haplotypes in south-eastern Mexico. Pie charts represent the haplotypes found in each sampling locality.
The area of the pie chart represents the size of the population and the size of sections is proportional to the haplotypic frequency.

DISCUSSION that could suggest an ancient or recent barrier in the zone.

Evolutionary research of lowland tropical tree species is at an Probably, because of the pioneer/nomadic behaviour of wild C.
early stage of development (Dick and Heuertz, 2008). For papaya, and its short life cycle, populations could have estab-
C. papaya, we found contrasting results between neutral (nu- lished and expanded rapidly after the Isthmus of Tehuantepec
clear microsatellites) and chloroplast (cpDNA) markers. arose, erasing any sign of past genetic break. Another study has
Microsatellite data are known to resolve patterns of recent gene shown similar results; Twyford et al. (2013) found low FCT val-
flow and pollen dispersal, due to their high mutational rate, ues between populations east and west from the isthmus for
whereas chloroplast markers tend to reflect ancient patterns be- Begonia heracleifolia, a neotropical lowland plant. In contrast,
cause lack of recombination, low effective population size and Gutiérrez-Rodrıguez et al. (2011) found a phylogeographical
their conservative mutation rate (Provan et al., 2001). Our re- break at the Isthmus of Tehuantepec, with private haplotypes at
sults suggest a lack of phylogeographical structure for C. pa- either side of the isthmus for Palicourea padifolia, a cloud for-
paya inferred by cpDNA, but a recent structuring derived from est herb. For a hummingbird species, Amazilia cyanocephala,
the microsatellite data. Rodrıguez-Gomez et al. (2013) found two genetic groups sepa-
rated by the Isthmus of Tehuantepec in the late Pleistocene,
with the split occurring in the presence of gene flow. It is inter-
esting to note that neither lowland species (C. papaya and B.
Isthmus of Tehuantepec and Pleistocene refugia hypothesis
heracleifolia) showed genetic signals by the Isthmus of
We evaluated whether two hypothetical biogeographical Tehunatepec, and in contrast the shrub P. padifolia and the
events documented for other tropical species have influenced hummingbird, both from cloud forests, showed a genetic break.
the genetic structure of C. papaya in Northern Mesoamerica: This suggests that this event, in contrast to species that inhabit
the Isthmus of Tehuantepec and putative Pleistocene glacial re- cloud forest at higher altitudes, did not represent a strong bar-
fugia. As a result of the several sea-level oscillations and conti- rier for gene flow of lowland tropical species and that these spe-
nental uplifts that took place in the Isthmus of Tehuantepec cies were able to recolonize once the isthmus emerged.
during the Pliocene (Lambeck and Chappell, 2001), isolation On the other hand, we could not test properly the Pleistocene
and reductions to gene flow among populations of lowland refugia hypothesis given that we did not find natural popula-
tropical species located at each side of the isthmus could be ex- tions of C. papaya in most of the proposed refugia in Mexico
pected. However, we found little evidence of a genetic break (La Lacandona, Soconusco, Los Tuxtlas, Sierra de Juarez and
corresponding to the isthmus as indicated by the low FCT value Cordoba) (Fig. 2); we found wild papaya populations only at
in the AMOVA for both markers. Furthermore, the genetic rela- the Los Tuxtlas region (population 5) and at the La Lacandona
tionships depicted by the haplotype network obscure the role of rain forest [population 12 (Palenque) lies in the region of La
the Isthmus of Tehuantepec as a biogeographical barrier as it Lacandona rain forest]. However, we did find signs of genetic
supports that populations between west and east share haplo- structuring in the Tuxtlas population, which bore many private
types. Finally, with both nuclear and chloroplast genetic data, haplotypes for the chloroplast marker, suggesting a long-term
no genetic break was detected in the Isthmus of Tehuantepec accumulation of variation and therefore a possible refugia role
 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica 1303

of this region. Despite this, few studies have found evidence are important resources for frugivores, mainly small mammals
supporting the refugial hypothesis, particularly for lowland tree and birds (Chavez-Pesqueira et al., 2014), which can cross frag-
species. For temperate and tropical cloud forests, evidence has mented habitats efficiently (Diffendorfer et al., 1995; Levey
suggested that the great endemism of Mesoamerican highlands et al., 2005). We could expect that, in the past, tropical and
is the result of persistence throughout glacial cycles of the relict sub-tropical forests in Northern Mesoamerica were continuous,
montane taxa that survived through the interglacials rather than allowing efficient dispersal between populations of wild pa-
the persistence of relict lowland tropical species that migrated paya, whereas now, fragmentation of the natural habitat of wild
to the highlands (Colinvaux et al., 2000). However, Gutiérrez- papaya may decrease the efficiency of dispersers with low mo-
Rodrıguez et al. (2011) did not find evidence of genetic struc- bility. Moreover, although we found many private haplotypes
ture by Pleisotcene refugia in the cloud forest species P. padifo- for natural populations of C. papaya, which could suggest a
lia. For tropical lowland species like C. papaya, responses to high phylogeographical structure, the most common haplotypes
historical climatic fluctuations may depend on species-specific were widely represented among populations and covered most
adaptations, therefore making the identification of refugia for of the distribution of this species in Northern Mesoamerica.
complex tropical species assemblages difficult (Poelchau and This occurrence of widespread plastid haplotypes in the range
Hamrick, 2012). Generalizations about refugia may apply only of C. papaya is consistent with the effective seed dispersal in
for species with similar ecological preferences, rather than at the past that we propose.
broad taxonomic scales (Twyford et al., 2013). Weak population genetic structure spanning tropical
America has been also reported for pioneer, drought-tolerant
trees such as Ceiba pentandra (Dick et al., 2007), Cordia allio-
dora (Rymer et al., 2013), Jacaranda copaia (Scotti-Saintagne
Genetic diversity and structure
et al., 2013), Trema micrantha (Dick et al., 2013) and Begonia
Overall, we found high values of genetic diversity. With the heracleifolia (Twyford et al., 2013), suggesting an apparent as-
microsatellite data, we found values of observed heterozygosity sociation between drought tolerance and low levels of phylo-
(Ho) above 07 for many populations across the entire distribu- geographical structure (Honorio Coronado et al., 2014). This
tion of wild papaya in Mexico. Only three populations showed suggests that populations survived in situ through historical cli-
values of Ho below 06: Santiago Astata (population 8), Tuxtlas matic fluctuations, having enough time to differentiate by drift
(population 5) and Cancun (population 19). Santiago Astata has and selection for local adaptation, and limited dispersal of the
the smaller population size and the highest inbreeding coeffi- accumulated new mutations across the range of the species.
cient (f ¼ 0387). Wild C. papaya combines small population Carica papaya is a light-demanding tree that tolerates sub-
sizes (M.C.-P., pers. observ.) and strict dioecy, which can com- tropical forest conditions, and can tolerate drought to some ex-
promise their amount and maintenance of genetic diversity in tent. This could explain, in part, the lack of phylogeographical
the long term. The Los Tuxtlas population lies in a very frag- structure we found for wild papaya.
mented rain forest area that could affect genetic contact among
sub-populations, thus decreasing genetic diversity, as previ-
ously shown (Chavez-Pesqueira et al., 2014). A similar sce-
Barriers to gene flow and migration rates
nario could be the case for the Cancun population, which lies
near to a human-populated and tourist area. Important mountain chains were recognized as the most
Regarding the chloroplast genetic data, we found high likely barriers to gene flow for the nuclear microsatellites. In
variability for psbA-trnH. The high level of haplotype diversity fact, the Sierra Madre del Sur, Sierra Madre Oriental and Sierra
(h ¼ 07013) was surprising, as single haplotypes may be ex- de Oaxaca greatly exceed the current elevational limits of low-
pected to be fixed by genetic drift in the typical small popula- land rain forest trees (1000 m a.s.l. for C. papaya). For Cancun
tions of C. papaya. However, the papaya populations may have the inferred barrier may be due to the species low genetic diver-
acted as a meta-population, maintaining haplotype diversity via sity and anthropogenic fragmentation rather to a physiographi-
extinction–colonization dynamics (Ray, 2001) if dispersal was cal feature preventing gene flow. For the chloroplast marker, no
effective in the past. The most frequent haplotypes (H1, H2 and geographical features such as mountain chains were recognized
H3) were widely represented across the C. papaya range although as barriers. The barriers inferred were mainly in the Yucatan
40 haplotypes (80 %) were private. In particular, Huasteca and Peninsula, suggesting isolation of the Chichén Itza and Caobas
Tuxtlas were the populations with more private haplotypes, sug- populations; although there are no actual climatic or geographi-
gesting particular cases of ancestral polymorphisms. cal events associated with this isolation, palaeobotanical studies
Genetic structure for the nuclear microsatellites was associ- have documented dramatic changes in the vegetation cover of
ated with six groups. All groups were geographically close, the peninsula along its biogeographical history. For instance,
showing moderate values of genetic differentiation among extensive savannas existed in what is now covered with tropical
them. For the chloroplast marker, we found an almost lack of rain forest (Vazquez-Domınguez and Arita, 2010). Moreover,
phylogeographical structure, suggesting ancient seed dispersal barriers could arise not only if gene flow is interrupted but also
across the distribution of C. papaya in Northern Mesoamerica. if populations are undergoing an adaptive isolation process in
This could be explained by a more effective dispersal of fruits. which migrants are selected against.
Although wild papayas are mainly pollinated by nocturnal In contrast to the barrier analysis, migration rates obtained
sphingids, which are known to be efficient, and long-distance from microsatellite data showed that, in recent times, the
pollinators (Dafni, 1992), moving pollen as far as 20 km Yucatan Peninsula may not represent a barrier and that the Rıo
(Amorim et al., 2014), fruits may have longer dispersal. Fruits Lagartos population, the most northern population in the
1304 Ch un~ez-Farf
avez-Pesqueira and N an — Genetic diversity of C. papaya in Northern Mesoamerica

Yucatan Peninsula, is an important source of variation for the papaya, warn against the ecological and evolutionary effects
region. In general, high migration rates were found between of domesticated genes and transgenes contaminating wild
populations separated by as far as 420 km, suggesting that wild populations.
C. papaya has long dispersal ability. However, we did not find
significant migration rates between populations separated by
mountainous chains, thus validating them as important gene CONCLUSIONS
flow barriers for this species. Furthermore, non-sampled popu-
lations of wild papaya, or even cultivars, could act as gene flow Our study has revealed contrasting results depending on the ge-
corridors, contributing to the high migration rates we found. netic marker used. Given the life history characteristics of wild
Yet, we do not know to what extent the movement of seeds or C. papaya, and the lack of an ancient phylogeographical struc-
disturbance by ancient human cultures in the region have ture, we suggest that tropical forests in Northern Mesoamerica
played a role in the genetic structure of C. papaya. did not suffer important climate fluctuations, as other authors
have suggested in relation to the refugial hypothesis (Colinvaux
et al., 1996, 2000; Fine and Ree, 2006). Our results also suggest
that the life history of C. papaya promoted its long dispersal
Conservation remarks and rapid colonization of lowland rain forests, thus maintaining
Wild C. papaya is a key element of early-successional tropi- genetic diversity throughout its range. However, recent human
cal and sub-tropical forests in Mexico, and represents the ge- disturbance, mainly the fragmentation of tropical habitats in
netic reservoir for the evolutionary potential of the species Northern Mesoamerica, appear to represent a threat to its dis-
(Chavez-Pesqueira et al., 2014). Although we found overall persion and therefore to its genetic diversity and structure.
high levels of genetic variability, we also found that natural Further research in lowland tropical species of Mesoamerica
populations of this species are becoming structured, probably is necessary to understand the present distribution of genetic
because of human disturbance to its natural habitat. This result variability of species inhabiting this ecosystem, and their phylo-
is extremely important for generating adequate conservation geographical history. Moreover, assessing the actual situation
strategies for this important crop species. Moreover, the centre of wild varieties and relatives of crop species is fundamental to
of origin for papaya has not been appropriately defined. It has ensure the maintenance of their genetic reservoirs and evolu-
been generally suggested that papaya originated somewhere in tionary potential.
Mesoamerica (Vavilov, 1926; Storey, 1976). We found high
genetic diversity in the Isthums of Tehuantepec zone for the
plasmid marker, as well as highest number of haplotypes, ACKNOWLEDGEMENTS
suggesting a hotspot of diversity in that region. We propose, We thank R. Tapia-Lopez for assistance in obtaining genetic
based on these data, a tentative origin of the species in that re- data, and the members of the Laboratorio de Genética
gion. In addition, papaya is most closely related to four species Ecologica y Evolucion for logistical support and field assis-
from southern Mexico and Guatemala (Carvalho and Renner, tance. We thank Pilar Suarez-Montes, Diego Carmona,
2012), suggesting that C. papaya could have originated in Marisol De la Mora and Jorge Juarez for their help during the
southern Mexico. Moreover, this plant has been proposed as be- field trips. We are very grateful to Juan Pablo Jaramillo-
ing domesticated in this area by the Mayans (Carvalho and Correa for his valuable and insightful help through the whole
Renner, 2012). Finally, when comparing our estimates of ge- process of this study. Finally, we thank Daniel Pi~nero,
netic diversity with the few other studies of natural populations Mauricio Quesada, Antonio Gonzalez, Alejandro Casas and
of papaya, both in Costa Rica (Coppens d’Eeckenbrugge et al., Alejandra Vazquez-Lobo for suggested improvements to the
2007; Brown et al., 2012), we found higher values in Mexico. manuscript. This work was supported by a grant of the
However, these Costa Rican natural populations are considered Programa de Apoyo a Proyectos de Investigacion e
possible feral plants, i.e. descended from domesticated plants, Innovacion Tecnologica [PAPIIT IN 215111-3 awarded to
which may explain their lower diversity derived by domestica- J.N.F.] and a grant of Comision nacional para el conocimiento
tion genetic bottlenecks (Doebley et al., 2006). y uso de la biodiversidad [CONABIO DGAP003/WQ003/15
Although mating between wild and cultivated papayas has awarded to M.C.P]. This paper constitutes a partial fulfillment
not been assessed in Mexico, possible ‘hybrids’ have been seen of the Graduate Program in Biological Sciences of the
in tropical zones in the country (our pers. observ.) and evidence National Autonomous University of Mexico (UNAM) for
exists from Costa Rica for gene flow between feral and culti- M.C.P., who acknowledges a scholarship and financial support
vated plants (Brown et al., 2012). Crosses between wild and do- provided by the National Council of Science and Technology
mesticated papaya threaten the maintenance of the natural (CONACYT).
genetic pool of the species. Indeed, this genetic diversity forms
the capital for current and future improvements to crop plants
(Chavez-Pesqueira et al., 2014). Thus, our results point to po- LITERATURE CITED
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