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Plant Ecology and Evolution 151 (1): 61–76, 2018
https://doi.org/10.5091/plecevo.2018.1343
Genetic variation and dispersal patterns in three varieties
of Pinus caribaea (Pinaceae) in the Caribbean Basin
Virginia Rebolledo Camacho1,3, Lev Jardón Barbolla2, Ivón Ramírez Morillo3,
Alejandra Vázquez-Lobo4, Daniel Piñero5 & Patricia Delgado6,*
1Instituto de Investigaciones Forestales, Universidad Veracruzana, Parque Ecológico “El Haya”, Carretera antigua a Coatepec S/N, CP
91000 Xalapa, Veracruz, México
2Centro de Investigaciones Interdisciplinarias en Ciencias y Humanidades, Universidad Nacional Autónoma de México, Torre II de
Humanidades, 4º piso, Ciudad Universitaria, CP 04510 Ciudad de México, México
3Centro de Investigación Cientíca de Yucatán, A.C. Calle 43 #130 x 32 y 34, Colonia Chuburná de Hidalgo, CP 97205 Mérida, Yucatán,
México
4Centro de Investigación en Biodiversidad y Conservación, Universidad Autónoma del Estado de Morelos, Av. Universidad No. 1001,
Colonia Chamilpa, CP 62209 Cuernavaca, Morelos, México
5Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Tercer Circuito Exterior S/N,
Ciudad Universitaria, CP 04510 Ciudad de México, México
6Facultad de Agrobiología “Presidente Juárez”, Universidad Michoacana de San Nicolás de Hidalgo, Paseo de la Revolución esquina con
Berlín S/N, Colonia Viveros, CP 60170 Uruapan, Michoacán, México
*Author for correspondence: dvalerio@umich.mx
All rights reserved. © 2018 Botanic Garden Meise and Royal Botanical Society of Belgium
ISSN: 2032-3913 (print) – 2032-3921 (online)
REGULAR PAPER
Background – Pinus caribaea Morelet comprises three varieties of tropical pines distributed in the
Caribbean Basin: P. caribaea var. hondurensis, var. caribaea, and var. bahamensis. The insular and
continental distribution of these varieties, as well as the geological processes in the region, have been
important factors for analysing evolutionary processes implicated in the diversication of these lineages.
In this study, we evaluate the genetic and geographic structure within and between these three varieties in
order to infer the possible origin and dispersal routes of these taxa.
Methods – We used six polymorphic nuclear microsatellites (nSSR) in fteen representative populations of
the three pine varieties, sampled throughout their natural range in Central America, Cuba and the Bahamas
islands.
Results – The varieties contain similar levels of genetic variation (mean He = 0.571), with several populations
out of Hardy-Weinberg equilibrium, and signicant levels of inbreeding (0.097–0.184, P ≤ 0.05). A slight
but signicant genetic dierentiation was found between the varieties (RST = 0.088) and populations (RST=
0.082), and genetic dierentiation increased with geographic distance (r2 = 0.263). Distance and Bayesian
BAPS analyses generated seven groups; two represented by the two island varieties and the remainder by
the Central American populations of var. hondurensis. Migration rate estimates between pairs of groups
ranged from M = 0.47 to M = 20.16. Estimates were generally higher from the continent to islands, with
the highest migration rate estimated from a continental genetic group to the Cuba island group of var.
hondurensis (M = 20.16).
Conclusions – This study supports the hypothesis of a recent origin of these pine taxa through the
migration of an ancestor from Central America, where the historical demography is associated with events
of colonization, expansion and contraction of populations. The genetic variation and dierentiation suggest
that the three varieties are divergent lineages that currently share allelic variants, indicating that their
speciation has not yet completed.
Key words – Pinus caribaea varieties, genetic variation, microsatellites, lineage divergence, migration
routes, Caribbean Basin.
62
Pl. Ecol. Evol. 151 (1), 2018
INTRODUCTION
Mexico is considered a secondary centre of diversication of
the genus Pinus L., represented by 52 species, two subspe-
cies, 14 varieties and four forms (Perry 1991, Gernandt &
Pérez de la Rosa 2014). This high diversity originated dur-
ing the early Oligocene (≈37 Mya) due to multiple migra-
tions of species from the North American temperate zone to
Mexico, with subsequent local speciation events and adapta-
tions to dierent climatic and geological conditions (Millar
1993, Farjon 1996, Dvorak et al. 2009), including species
capable of establishing at the lowest altitude (sea level) (Far-
jon 2005). The species Pinus caribaea Morelet is naturally
distributed at altitudes from sea-level to 700 metres above
sea-level (m a.s.l) (rarely reaching 1000 m a.s.l) in tropical
forests susceptible to seasonal ooding (lowland and coastal
populations) and on a variety of soil types. Soils range from
sandy, acidic, nutrient-poor to deep sandy clays with good
drainage and to pure clays (Van 2002, Farjon & Styles 1997).
Pinus caribaea is subdivided into three varieties with a wide
distribution in the Caribbean Basin: Pinus caribaea var. hon-
durensis (Sénécl.) W.H.Barret & Golfari (Mexico and Cen-
tral America), Pinus caribaea var. caribaea (Western Cuba
and Juventud Island), and Pinus caribaea var. bahamensis
(Griseb.) W.H.Barret (Bahamas, Turks and Caicos Islands)
(Nikles 1966, Farjon & Styles 1997). Taxonomically, P. cari-
baea and its varieties belong to Pinus subsection Australes,
together with P. occidentalis Swartz, P. maestrensis Bisse,
P. cubensis Griseb, P. elliottii Engelm, P. echinata Mill., P.
palustris Mill., P. pungens Lamb., P. taeda L., P. oocarpa
Schiede ex Schltdl. and P. tecunumani F. Schwerdtf. ex Egui-
luz & J.P.Perry (Dvorak et al. 2000a, Gernandt et al. 2005,
Hernández-León et al. 2013). Most of the populations are
monospecic, but sometimes P. caribaea var. hondurensis
is in sympatry with P. oocarpa or P. tecunumani (Hondu-
ras, Belize, Guatemala, El Salvador and Nicaragua) (Perry
1991, Farjon 1996, Dvorak et al. 2000b). Pinus caribaea var.
hondurensis is the taxon with the most southern distribution
in America (Nicaragua) and, while this variety has a wider
distribution and its conservation status is considered as least
concern (LC, IUCN 2016), present logging activities threat-
en to fragment and reduce populations in some areas (Farjon
2013). Pinus caribaea var. caribaea and P. caribaea var. ba-
hamensis are threatened throughout their natural distribution;
P. caribaea var. caribaea is considered as endangered (EN)
because of logging and conversion to pasture, especially on
the mainland of Cuba, while P. caribaea var. bahamensis is
now considered as vulnerable (VU) and could be at risk due
to population decline in the Turks and Caicos Islands as re-
sult of attack by the pine tortoise scale, Toumeyella parvi-
cornis (Cockerell, 1897) (Farjon 2013, Sánchez et al. 2014,
IUCN 2016).
Morphologically, the three varieties are very similar, with
2–5 needles per fascicle, but they dier mainly by a seedling
stage phase, the number and position of resin canals on the
leaves and certain seed wing features (Farjon & Styles 1997).
Some authors have found molecular dierences among the
three varieties, which has an evolutionary implication. Using
isoenzyme markers, Zheng & Ennos (1999) analysed varia-
tion and genetic relationships in populations of the varieties
caribaea and bahamensis, while Matheson et al. (1989) ex-
amined populations of the varieties hondurensis and baha-
mensis. These studies reported signicant dierences in the
presence and frequency of alleles between the varieties, sup-
porting the notion that the varieties are dierent evolutionary
entities or units. When the three varieties of P. caribaea and
three additional species of pines distributed in the Caribbean
Basin (P. cubensis, P. maestrensis and P. occidentalis) were
analysed using a phylogeographic approach with plastid mi-
crosatellites, the highest genetic diversity was observed in
the populations of P. caribaea var. hondurensis distributed in
Central America (He=0.951) and dierent degrees of genetic
dierentiation were found among the varieties and species
(RST = 0.230 among varieties, RST = 0.110 among species)
(Jardón-Barbolla et al. 2011).
This dierentiation is probably associated with geologi-
cal and climatic processes of the Caribbean Basin that have
acted either as barriers or as corridors of dispersion. While
the geological history of this region has been widely studied,
nonetheless, it remains poorly understood. It has been pro-
posed that the Greater Antilles and the Bahamas have been
separated from North and Central America since the Mio-
cene (≈23 Mya) (Iturralde-Vinent 2006, Pindell et al. 2006).
However, paleogeographic studies show that changes in sea
level during recent glacial periods could have left extensive
areas of emerged land with temporary connections (coral
reef land bridges) between the islands and Central America
(Schuchert 1935, Hedges 1996a, 1996b). This theory, along
with the distribution of the related species of Pinus subsec-
tion Australes in Central America, suggests that the origin
and distribution of contemporary biota in this region is a re-
sult of species migration events from the continent to the is-
lands at a geological time of less than 20 Mya (Mirov 1967,
Nikles 1966, Hedges 1996b, Iturralde-Vinent 2004–2005,
Moonlight et al. 2015). Considering that Pinus arrived to
Mexico and Central America between 35 and 20 Mya (Millar
1993, Hedges 1996b), we can assume that Pinus caribaea
varieties, and subsection Australes in general, are of relative-
ly recent origin.
To date, two hypotheses have been proposed concern-
ing the origin of the species classied in Pinus subsection
Australes. The rst is based on a phylogenetic reconstruction
obtained from morphological data (Adams & Jackson 1997),
which proposed that the ancestor originated in the southeast-
ern region of North America, migrated from southern Florida
to the Caribbean islands and then to Central America. The
second hypothesis, initially suggested by Nikles (1966) and
Mirov (1967), was based on nuclear dominant RAPD (Ran-
dom Amplied Polymorphic DNA) markers (Dvorak et al.
2000a) and chloroplast DNA microsatellite data (Jardón-Bar-
bolla et al. 2011), and proposed a Central American origin
of the ancestor: the ancestral P. caribaea would have been
separated prior to the divergence of Australes, then migrat-
ed to Central America through the Western Gulf of Mexico.
This early form is suggested to have migrated to the Carib-
bean and eventually to the Florida peninsula, from where the
ancestor of P. caribaea var. caribaea would have dispersed
(probably infrequently over an extended period after the
Pleistocene) to Cuba and the Bahamas (Dvorak et al. 2000a).
It has also been inferred that populations of caribaea and ba-
63
Rebolledo Camacho et al., Genetic variation and dispersal of Pinus caribaea varieties
hamensis varieties have had recent colonization events to the
islands of Cuba and the Bahamas (97 900 years BP), whereas
P. caribaea var. hondurensis populations are older, associ-
ated with expansion processes during lower temperatures at
the beginning of a glacial period (326 300 years BP; Jardón-
Barbolla et al. 2011). Interpretations of these data should
consider that chloroplast markers are haploid and inherited
parentally through pollen, thus their coalescence time infer-
ences are shorter than those estimated with diploid nuclear
DNA (Rosenberg & Nordborg 2002, Avise 2009). It is there-
fore relevant to study the nuclear genetic variation in order
to achieve a more comprehensive historical reconstruction of
evolutionary processes. For example, in a phylogeographic
study of Pinus strobus L. (Zinck & Rajora 2016), inferences
with both nuclear and chloroplast markers were consistent,
revealing a single refuge, two re-colonization routes and
three genetically distinguishable lineages. In contrast to the
chloroplast markers, nuclear markers allowed the detection
of higher genetic diversity and more pronounced levels of
genetic structure. Similarly, joint use of mitochondrial DNA
sequences and nuclear loci allowed the detection of histori-
cal introgression events in Picea obovata Ledeb. and Picea
abies (L.) H.Karst. that were missed when using maternally
inherited markers alone (Tsuda et al. 2016).
In this sense, nuclear codominant microsatellites (nuclear
simple sequence repeats, nSSR) are suitable for providing
complementary information with which contrast the dier-
ent hypotheses about the origin of P. caribaea, considering
their high mutation rates from 10-5 to 10-3 mutations per site
per generation (Schlötterer 2000, Boys et al. 2005). Nuclear
SSRs are selectively neutral, conserved across species and
have been widely used to evaluate genetic variation and
structure, gene ow, inbreeding and eective population siz-
es (Karhu 2001, Boys et al. 2005, Furlan et al. 2007). More-
over, nSSRs allow the inference of demographic processes
such as dispersal, migration, expansion, or fragmentation
of populations (Adams & Jackson 1997, Rajora et al. 2000,
Williams et al. 2000, Mariette et al. 2001, Shepherd et al.
2002, Al-Rabab’ah 2003, Boys et al. 2005, Karhu et al. 2006,
Dvorak et al. 2009, Wang et al. 2009, Sánchez et al. 2014,
Zinck & Rajora 2016, Tsuda et al. 2016). Currently, only
three studies have been conducted on patterns of genetic var-
iation within the varieties hondurensis and bahamensis with
nSSRs. In three experimental P. caribaea var. hondurensis
populations established in Brazil with individuals from Pop-
tún, Guatemala, very low levels of variation and genetic dif-
ferentiation were detected (He= 0.249, FST = 0.021; Furlan
et al. 2007). Likewise, low levels of genetic variation and
dierentiation were found for two populations of the same
variety distributed in Mexico (He = 0.465, RST =0.033) and
this was attributed to a recent reduction of eective popula-
tion sizes (8100 to 35 000 years ago; Delgado et al. 2011).
On the other hand, var. bahamensis showed high levels of
genetic variation and structure among populations from the
Bahamas, and Turks and Caicos islands (TCI), which was at-
tributed to the eect of geographic isolation of the popula-
tion distributed in the latter region (Sánchez et al. 2014). The
joint study of the three varieties is necessary both for a bet-
ter understanding of the evolutionary history of this taxon, as
well as to outline management and conservation schemes of
this important complex of Caribbean pines.
In this study, nuclear microsatellites were used to test the
hypothesis that the three varieties of P. caribaea (var. cari-
baea, hondurensis and bahamensis) distributed in the Car-
ibbean Basin, represent independent evolutionary lineages
originating from one ancestor distributed in Central America.
Our aims were (i) to estimate levels of genetic variation in the
varieties and populations, inbreeding indices and eective
population sizes, and (ii) to determine the geographic struc-
ture of genetic variation across populations to infer possible
migration routes (gene ow). Finally, we analysed and dis-
cussed the geographic and demographic processes that may
underlie the distribution of genetic variation in this group of
pines.
MATERIALS AND METHODS
Study populations and sampling
Fifteen populations of the three varieties of Pinus caribaea
were sampled and geo-referenced throughout the Caribbean
Basin (g. 1). Leaf material was collected from two popula-
tions of P. caribaea var. caribaea located at the western end
of the island of Cuba (Viñales and Mil Cumbres), from two
populations of P. caribaea var. bahamensis in the Bahamas
(islands of Andros and New Providence), and from eleven
populations of P. caribaea var. hondurensis, one in Mexico
and one in Guatemala, four in Belize, three in Honduras, and
two in Nicaragua. Of these, seven were located inland (H1,
H2, H4, H6, H8, H9 and H10) and the other four were coast-
al lowland sources (table 1). Needles were collected from 13
to 30 trees in each of the population respecting a minimum
distance of 50 m between trees, in order to reduce the prob-
ability of parentage (Flores et al. 2005). A total of 316 in-
dividuals were used in the study. Plant tissue was stored in
plastic bags at -80°C for subsequent DNA extraction.
DNA extraction, amplication and genotyping
Genomic DNA was extracted with a CTAB miniprep meth-
od (Vázquez-Lobo 1996). The seven nSSRs assayed were
derived from Pinus taeda (Elsik et al. 2000); PtTX3025,
PtTX3013, PtTX3020, PtTX2146, PtTX2123, PtTX3029
and PtTX2037. The PCR amplication reaction conditions
described below were performed using a MasterCycler Gra-
dient thermocycler (Eppendorf Inc), according to Elsik et al.
(2000), with modications in the concentration of magne-
sium chloride (4 mM). Specic touchdown PCR conditions
were as follows: one cycle at 94°C for 5 min; two cycles of
94°C for 1 min, 60°C for 1 min and 72°C for 35 s; 20 cy-
cles of 94°C for 45 s, 45 s at specic annealing temperature
(TM) of the primer pair, decreasing by 0.5°C each cycle, and
72°C for 1 min; 20 cycles at 94°C for 1 min, nal TM for 1
min and 72°C for 1 min; nal extension at 72°C for 5 min.
The annealing temperatures for each primer pair were; 63°C
for PtTX3013, PtTX3029, PtTX2037, 59°C for PtTX3025,
57°C for PtTX2146, PtTX2123, and 65°C for PtTX3020.
The fragments were separated by electrophoresis on 5% pol-
yacrylamide gels (7M Urea; Tris-Borate-EDTA [TBE] buer
at 0.5%), and run at 50–60 W for 1.5–3.5 hours, depending
64
Pl. Ecol. Evol. 151 (1), 2018
Figure 1 – Geographical location of fteen populations for Pinus caribaea varieties distributed in the Caribbean Basin. The graphics
represent the proportion of individuals for each population in accord to the best clustering (K seven groups) obtained with BAPS analysis
(logML = -3612; P = 1.000). Population codes according to table 1. Blue colour on the map represents the geographic distribution range of
the varieties (adapted from Francis 1992). Date and projection of the map: WGS 1984, Latitude/Longitude. Map created with QGIS version
2.14 (QGIS Development Team 2017).
on fragment size. A positive control (genotype) was used for
each nSSR to conrm and standardize the allele sizes. Frag-
ments were revealed with the silver nitrate staining method
(Echt et al. 1996), and fragment size was determined visually
using a 10 bp DNA ladder (Invitrogen).
The presence of null alleles (non-amplied alleles) has
been reported in two of seven microsatellites used here
(PtTX2037 and PtTX3020; Williams et al. 2000, Shepherd
et al. 2002); therefore, the frequency of null alleles for all
of the loci was estimated using Micro-Checker v. 2.2.3 and
the genotypes adjusted according to the correction algorithm
of Brookeld (van Oosterhout et al. 2004). A low proportion
of null alleles was determined for the loci PtTX2146 (0.171)
and PtTX2037 (0.204) and a high proportion for PtTX3029
(0.743). According to studies in which null alleles were pre-
sent, it is possible to correct their eect (detecting a signicant
heterozygosity decit relative to Hardy-Weinberg equilib-
rium, which could be misconstrued as evidence of inbreed-
ing) by eliminating some individuals with high proportions
of missing data, and repeating the analysis (Williams et al.
2000, Dakin & Avise 2004). Therefore, 17 individuals were
excluded for all loci; null alleles for the locus PtTX2146 were
corrected in ve populations (H5, H6, H8, H10 and H11), and
for the locus PtTX2037 in four populations (H1, H10, C2 and
B2). However, it was not possible to correct this problem in
locus PtTX3029 in eight populations (H2, H4, H5, H6, H7,
H8, C1, C2), which maintained high proportions of null al-
leles (0.214 to 0.373), and this locus was therefore eliminated
from the study altogether. As a result of this correction, all of
the analyses of variation and genetic structure were carried
out with six loci and a total of 299 individuals.
Data analyses
Genetic variation was estimated using the following pa-
rameters: number of alleles per locus (A), average number
of alleles per locus (n), number of eective alleles per locus
(Ae), observed (Ho) and expected (He) heterozygosity. The
inbreeding index (FIS) was estimated according to Wright
(1965), and deviations from Hardy-Weinberg equilibrium
were assessed with an unbiased estimation using the Mark-
ov chain Monte Carlo (MCMC) method with 100 000 steps
(Guo & Thompson 1992). These analyses were performed
65
Rebolledo Camacho et al., Genetic variation and dispersal of Pinus caribaea varieties
Population
Country/
State, District or
Department
Latitude
Longitude
Altitude
N n A ArAeHoHeFIS
(m s.a.l)
Pinus caribaea var. hondurensis
H1-Caobas Mexico/
Quintana Roo
18º14′77.5″N
88º57′60.4″W 35 17 18 3 2.5 2.1 0.421
(0.264)
0.488
(0.219)
0.057
[0.001–0.221]
H2-Carmelitas Belize/Belize 17°48′12.2″N
88°32′55.0″W 13 19 19 3.2 2.7 2.4 0.441
(0.252)
0.568
(0.132)
0.233**
[0.006–0.282]
H3-Rock Belize/Belize 17°24′43.1″N
88°26′02.4″W 16 18 21 3.5 2.8 2.6 0.523
(0.182)
0.593
(0.147)
0.124
[0.013–0.253]
H4-Mountain Pine Belize/Cayo 16°59′35.0″N
88°57′50.1″W 501 30 24 4 2.8 2.6 0.566
(0.106)
0.581
(0.138)
0.028
[0.000–0.125]
H5-Deep River Belize/Toledo 16°29′16.0″N
88°41′08.3″W 31 22 23 3.8 2.9 2.7 0.491
(0.238)
0.578
(0.246)
0.154*
[0.051–0.289]
H6-Dolores Guatemala/
El Petén
16°29′07.7″N
89°25′34.9″W 438 20 25 4.2 3.2 2.9 0.537
(0.111)
0.634
(0.140)
0.157*
[0.004–0.253]
H7-Mezapa Honduras/
Atlántida
15°34′18.6″N
87°36′38.2″W 306 25 24 4 3.0 2.9 0.521
(0.254)
0.600
(0.238)
0.135*
[0.020–0.245]
H8-Trinidad Honduras/
Santa Bárbara
15°05′54.0″N
88°15′10.0″W 200 16 22 3.7 2.9 2.6 0.453
(0.188)
0.589
(0.172)
0.239*
[0.078–0.352]
H9-Leimus Honduras/
Gracias a Dios
14°45′57.0″N
84°08′08.7″W 90 21 22 3.7 2.7 2.3 0.516
(0.157)
0.553
(0.141)
0.068
[0.000–0.146]
H10-Waspam Nicaragua/
North Atlantic
14°43′09.8″N
83°58′47.1″W 87 24 22 3.7 2.9 2.6 0.537
(0.124)
0.621
(0.073)
0.140*
[0.007–0.205]
H11-Moss Nicaragua/
North Atlantic
14º27′13.8″N
83°54′14.4″W 128 21 23 3.8 3.0 2.5 0.516
(0.153)
0.616
(0.070)
0.177*
[0.005–0.246]
Average 22 3.7 2.8 2.6 0.502 0.575 0.124**
[0.034–0.225]
Pinus caribaea var. caribaea
C1-Viñales Cuba/
Pinar del Río
22°32′48.4″N
83°42′29.5″W 239 19 20 3.3 2.7 2.4 0.522
(0.194)
0.573
(0.123)
0.092
[0.000–0.126]
C2-Mil Cumbres Cuba/
Pinar del Río
22°47′45.9″N
83°21′57.4″W 185 18 19 3.2 2.7 2.3 0.384
(0.161)
0.555
(0.173)
0.319**
[0.246–0.518]
Average 19.5 3.2 2.7 2.3 0.476 0.564 0.184*
[0.025–0.349]
Pinus caribaea var. bahamensis
B1-Andros Bahamas 25°00′31.2″N
77°30′06.9″W 9 16 19 3.2 2.6 2.3 0.591
(0.145)
0.551
(0.112)
-0.063
[0.000–0.112]
B2-New Providence Bahamas 24°55′13.1″N
78°00′49.8″W 4 13 19 3.2 2.7 2.3 0.433
(0.195)
0.593
(0.104)
0.280**
[0.017–0.368]
Average 19 3.2 2.7 2.3 0.512 0.576. 0.097
[-0.079–0.201]
Table 1 – Geographical location and genetic parameters estimated in fteen populations of the three Pinus caribaea varieties
distributed in the Caribbean Basin.
Alt, altitude; N, sample size; n, total number of alleles; A, average number of alleles; Ar, allelic richness; Ae, average number of eective
alleles per locus; Ho and He, average of heterozygosis observed and expected; FIS, inbreeding index, * P= 0.05, **P= 0.01. SD, standard
deviations are given in parentheses and CI-95%, condence intervals in brackets.
using Arlequin v. 3.5.1.2 (Excoer & Lischer 2010). Fur-
ther, ADZ v.1 (Szpiech et al. 2008) was used to estimate al-
lelic richness, Ar estimation, using a rarefaction approach to
standardize estimates to the smallest population sample size
of the data set (El Mousadik & Petit 1996).
Genetic eects of population demographic decline were
examined using the T2 statistic (Cornuet & Luikart 1996),
which reects the deviation from expectations at demograph-
ic equilibrium (Budde et al. 2017). The test was performed
using the innite allele model (IAM), the stepwise mutation
model (SMM) and the two-phase model (TPM; 70% of mu-
tations under the SMM model and 30% under IAM) with
Bottleneck v.1.2.02 (Piry et al. 1999). Signicance in the
three mutation models was tested using Wilcoxon´s signed
rank test, with 10 000 replicates.
Genetic structure (RST), was estimated with a hierarchical
analysis of molecular variance (AMOVA) assuming a step-
wise mutation model (SMM; Slatkin 1995). The analysis
was divided into four components: among the three varieties
(FCT), between populations within varieties (FSC), between
66
Pl. Ecol. Evol. 151 (1), 2018
individuals within populations of each variety (FIS) and
within individuals (FIT). This analysis was also conducted
between all populations and between the K groups obtained
with BAPS analysis (see next paragraph), assessing three
components; between populations or K Groups (FCT), be-
tween individuals within populations or K groups (FIS) and
within individuals (FIT). All statistical signicance was ob-
tained with 1000 non-parametric permutations (Excoer &
Lischer 2010).
The association between geographic structure and ge-
netic variation of populations and varieties was estimated
with the Bayesian inference algorithm implemented in the
program BAPS v. 5.4 (Corander et al. 2008). The algorithm
denes groups of populations using information pertaining
to their spatial distribution in order to detect the most likely
genetic structure. The estimates were obtained with a spatial
clustering method, assuming 1 to 15 groups (K), with 10 rep-
licates per K, using 10 000 iterations for estimates, preceded
by 1000 iterations discarded as burn-in. The partition with
the highest marginal probability (LogML; natural logarithm
of likelihood) was selected as the one that best describes
the genetic structure of the data. In order to analyse the ge-
netic distance between populations, a distance tree using the
neighbor-joining method was constructed with the POP-
TREE2 software (Takezaki et al. 2010), based on standard-
ized genetic distances (Da) (Nei et al. 1983). Support of this
distance tree was evaluated by bootstrap analysis, using 1000
replicates (Takezaki & Nei 1996).
Eective population size (Ne) and historical migration
rates (M) were estimated for the groups of populations ob-
tained with BAPS. Estimations were carried out with Mi-
grate version 3.4.2 (Beerli 2008, software accessed in Janu-
ary 2013), under a Bayesian approach (Beerli & Palczewski
2010). Uniform priors were used for all parameters with three
independent runs to verify the convergence. Markov chains
were obtained with 500 000 iterations, after a burn-in pe-
riod of 10 000 steps, and a thinning interval of 0.0 to 100
(Beerli 2008). A mutation rate (µ) of 10-3 per generation was
assumed; this rate has been used with nSSRs in other pine
species (Boys et al. 2005, Delgado et al. 2011). Theta (θ) and
M parameters were generated with the FST calculation (FST =
1/1 + 4Nem; Beerli 1998, 2008), and since θ = 4Neµ, Ne was
estimated as θ/4 × 10-3 (Boys et al. 2005). In addition, an
analysis of isolation by distance (IBD) was performed, re-
gressing the genetic distance between pairs of populations on
their geographic distance and testing the relationships using a
Mantel test, with 10 000 permutations (Mantel 1967, Sokal &
Rohlf 1995). Standardized genetic distances (Da) were used
for the genetic data (Nei et al. 1983), and absolute distances
(in kilometres) through Mercator transformation were used
for geographic distances (ESRI 1992–2000). This analysis
was run with the IBD program (Bohonak 2002).
RESULTS
Genetic variation
The six loci analysed were polymorphic; four had high lev-
els of genetic diversity (expected heterozygosity): PtTX2146
(He = 0.732), PtTX2123 (He = 0.679), PtTX3020 (He =
0.659) and PtTX3025 (He = 0.564). All loci presented ge-
netic diversity values of He > 0.5. A total of n = 35 alleles
were obtained with an average of 3.4 alleles per locus. For
var. hondurensis, the average number of alleles per popula-
tion was n = 22, ranging from 18 (H1) to 25 alleles (H6);
the number of eective alleles was Ae = 2.6 and allelic rich-
ness was Ar = 2.8 (table 1). The diversity estimates for the
other two varieties were lower than those obtained for var.
hondurensis: n = 19 in var. bahamensis and n = 20 in var.
caribaea, with values of Ae = 2.37 and Ar = 2.7 for both va-
rieties (table 1). Two unique alleles were observed, one in
population H4 of var. hondurensis (locus PtTX2146, 159pb)
and another in population B2 of var. bahamensis (PtTX3025,
263pb). The average estimates of expected (He) and observed
(Ho) heterozygosity were very similar for var. hondurensis
(He = 0.575, Ho = 0.502) and var. bahamensis He = 0.576,
Ho = 0.512), whereas the average estimates for var. caribaea
were lower (He = 0.564, Ho = 0.476), although not statistical-
ly dierent from those of the other two varieties (P > 0.05).
Three populations of var. hondurensis presented the high-
est genetic diversity values: population H6 from Guatemala
(He = 0.634) and populations H10 and H11 from Nicaragua
(He = 0.621 and He = 0.616, respectively). The lowest values
were found in two populations of var. hondurensis, H1 (He =
0.488) and H9 (He = 0.553) sampled in Mexico and Hondu-
ras, respectively, and in population C2 of var. caribaea (He =
0.555) from Cuba (table 1). All the He values were higher
than those of Ho (except in population B1 from the Baha-
mas), with a signicant heterozygosity decit of two or more
loci per population in 9 of the 11 populations of var. hondu-
rensis, the two populations (C1 and C2) of var. caribaea and
population B2 of var. bahamensis. Most of the populations
therefore contained fewer heterozygotes than expected under
mutation-drift equilibrium.
The average inbreeding index for the varieties was posi-
tive and diered signicantly from random mating expecta-
tions (FIS = 0.131, P = 0.05, 95% CI: 0.043–0.203). Pinus
caribaea var. caribaea displayed the highest value, FIS =
0.184 (P = 0.001, 95% CI: 0.025–0.349), followed by var.
hondurensis, FIS = 0.124 (P = 0.001, 95% CI: 0.034–0.225)
and var. bahamensis, FIS = 0.097 (P = 0.09, 95% CI: -0.130–
0.223) (table 1). Most populations of var. hondurensis had
signicant levels of inbreeding, with the exception of two
populations distributed in Belize (H3 and H4), and one in
Honduras (H9). Population C2 of Mil Cumbres, var. cari-
baea, had the highest inbreeding index of all populations,
FIS = 0.319 (P = 0.001), together with B2 of var. bahamen-
sis FIS = 0.280 (P = 0.001). The other population of var. ba-
hamensis (B1) presented a negative inbreeding index (FIS =
-0.063), but this was not signicant. In general, most popu-
lations showed dierent degrees of inbreeding, indicating
population isolation throughout their evolution.
Demographic reduction of population size considering
the intermediate TPM mutation model showed eight out of
15 populations with signals of a bottleneck (deviation from
mutation-drift equilibrium). The extreme models IAM and
SMM, showed 13 populations and one population respec-
tively (table 2). Based only on the TPM mutation model,
populations H10 (Waspam) from Nicaragua, H7 (Mezapa)
from Honduras, H2 (Carmelitas) and H3 (Rock) from Be-
67
Rebolledo Camacho et al., Genetic variation and dispersal of Pinus caribaea varieties
lize and H6 (Dolores) from Guatemala of var. hondurensis,
showed heterozygosity excess indicating recent bottlenecks
(P < 0.05). In island populations, the two Cuban populations
were signicantly bottlenecked, along with one population of
Bahamas islands (B2-New Providence). These results clearly
suggested that most populations of the three varieties had re-
duced population sizes in the recent past.
Genetic relationships between populations and varieties
The best clustering solution of populations obtained with
BAPS comprised K=7 groups (logML = -3612; P = 1.000)
(g. 1, electronic appendix 1). The rst ve groups com-
prised populations of var. hondurensis, the variety was thus
spatially and genetically sub-structured: the K1 group con-
sisted of two populations (H3 and H5 from Belize), the K2
group had four populations (H2 from Belize, H8, H9 from
Honduras and H10 from Nicaragua), the K3 and K4 groups
were represented by only one population each (H1 from
Mexico; H7 from Nicaragua) and the K5 group included
three populations (H4 from Belize, H6 from Guatemala, and
H11 from Nicaragua). The K6 group included the two popu-
lations of var. caribaea (C1 and C2 from Cuba), and the K7
group the two populations of var. bahamensis (B1 and B2
from the Bahamas). In this way, we observed a tendency of
the populations to cluster together in accordance with vari-
eties and geographical distribution. The AMOVA analysis
indicated a signicant dierentiation between varieties as
estimated with RST (RST or FCT = 0.088, P < 0.001; 95% CI:
0.024–0.103), which was of similar magnitude to dierentia-
tion between populations (RST = 0.082, P < 0.001; 95% CI:
0.064–0.132). The highest variance was found within indi-
viduals (FIT = 0.178; 95% CI: 0.110–0.307), followed by var-
iance between individuals within populations of each variety
(FIS = 0.058; 95% CI: 0.045–0.123) and between populations
within varieties (FSC = 0.043; 95% CI: 0.025–0.129). All of
the values were signicant (P < 0.001). Similarly, the AMO-
VA between the seven groups obtained with the BAPS analy-
sis reected hierarchical population structure (RST = 0.077; P
< 0.002; 95% CI: 0.042–0.109) (table 3, electronic appendix
2A–C).
The neighbour-joining tree based on Nei’s standardized
genetic distance (Da) showed two large groups supported by
100% of the bootstraps (g. 2). The rst comprised seven
populations of var. hondurensis, indicating that the popula-
tions distributed in Belize (H3 and H5) were the most basal,
while the most derived populations were H9 from Honduras
and H10 from Nicaragua (g. 2). The second group included
the remaining populations of this variety and those of the
varieties caribaea and bahamensis. The most basal distant
Population
T2, statistic
IAM P-value TPM P-value SMM P-value
H1-Caobas 1.278 0.039 0.685 0.218 0.015 0.421
H2-Carmelitas 2.158 0.015 1.653 0.015 1.046 0.023
H3-Rock 2.027 0.007 1.410 0.015 0.778 0.053
H4-Mountain Pine 2.729 0.023 1.328 0.078 0.202 0.421
H5-DeepRiver 1.83 0.218 1.143 0.218 0.225 0.218
H6-Dolores 2.016 0.007 1.237 0.039 0.361 0.343
H7-Mezapa 2.218 0.015 1.607 0.039 0.915 0.078
H8-Trinidad 1.802 0.023 1.198 0.054 0.406 0.343
H9-Leimus 1.52 0.023 0.766 0.078 -0.341 0.656
H10-Waspam 2.345 0.007 1.715 0.007 0.896 0.078
H11-Moss 1.598 0.023 0.782 0.078 -0.315 0.578
C2-Mil Cumbres 1.967 0.007 1.415 0.023 0.776 0.218
C1-Viñales 2.018 0.015 1.422 0.039 0.705 0.078
B1-Andros 1.098 0.078 0.392 0.421 0.322 0.500
B2-New Providence 2.495 0.007 1.893 0.015 1.314 0.053
Table 2 – Bottleneck tests estimated for the three Pinus caribaea varieties distributed in the Caribbean Basin.
The T2, bottleneck statistic (Cornuet & Luikart 1996) and P-values of the Wilcoxon signed rank test (one tail for heterozygosity excess)
under the IAM, TPM and SMM mutation models, are shown for each population.
68
Pl. Ecol. Evol. 151 (1), 2018
Figure 2 – Distance tree using neighbour-joining method, based on standardized genetic distances Da (Nei et al. 1983), between fteen
populations representing all three varieties of Pinus caribaea. Bootstrap support values of 1000 replicates are indicated at the base of the
branches (in italics). Coloured clusters represent the K seven population groups obtained with Bayesian analysis (BAPS). Dashed line
indicates the origin of the two large lineages (clusters) obtained.
Source of variation d.f. Sum of
squares
Variance
components
Percentage of
variation RST Fixation index P-value
Among populations 14 8882.512 14.4117 8.2907 0.0829 < 0.001
Among groups of varieties 2 3963.351 16.1625 8.8417 0.0884 < 0.001
Among K7 groups 6 6477.252 13.5866 7.7547 0.0775 < 0.002
Table 3 – RST Fixation indices obtained with AMOVA analysis on three levels of grouping of Pinus caribaea varieties.
In bold type the highest value of FST obtained. Statistical signicance was obtained with 1000 non-parametric permutations (Excoer &
Lischer 2010).
69
Rebolledo Camacho et al., Genetic variation and dispersal of Pinus caribaea varieties
populations were those of the var. hondurensis, distributed
in Honduras (H7), Guatemala (H6) and Nicaragua (H11).
The island populations of var. caribaea and var. bahamensis
represented derived populations within two independent sub-
groups (g. 2).
Eective size, migration rates and isolation by distance
The average estimate of historical eective population size
(Ne) for the seven BAPS groups of P. caribaea was 362 indi-
viduals. The highest Ne was for group K5 of var. honduren-
sis (Ne = 537), comprising two populations from Belize (H4,
H6) and one from Nicaragua (H11), followed by group K2 of
the same variety (Ne = 443; H2, H8, H9 and H10) (table 4).
Group K6 formed by two populations of var. caribaea (C1
and C2 of Cuba), had the lowest value (Ne = 161 individu-
als), followed by var. hondurensis (Ne = 201) and var. ba-
hamensis (Ne = 208) from K3 (H1, Mexico) and K6 (B1 and
B2, Bahamas) groups, respectively. Migration rate estimates
between pairs of BAPS groups were comprised between M =
0.47 and M = 20.16. Inferred migration was predominantly
from the continent to the islands, departing from groups K5
and K1 (M = 20.16 and 9.18) of var. hondurensis, and within
the continent between the populations of group K5 towards
groups K1 (M = 8.32), K2 (M = 4.57), K3 (M = 7.96), and
K4 (10.90) of the same variety (see table 4 and g. 3). Low
M values were observed between the island varieties despite
their relative geographic proximity (M = 4.85). The highest
M value of var. caribaea was found towards population H1
of the K3 group (Mexico) of var. hondurensis (M = 7.54).
Thus, we observed that the highest migration rate estimates
were associated with dispersion from the distribution range
of var. hondurensis. This indicates that P. caribaea possibly
originated on the continent.
The IBD analysis among all of the populations showed a
signicant relationship between genetic and geographic dis-
tances (r2 = 0.263; P = 0.005) (g. 4), whereas no association
was detected between dierentiation among the seven BAPS
groups and geographic distance (r2 = 0.215; P = 0.139), or
among the populations of var. hondurensis and geographic
distance (r2 = -0.065, P = 0.611).
DISCUSSION
Genetic variation and demographic history
We showed that populations of the three varieties of Pinus
caribaea studied here had intermediate levels of genetic di-
versity when compared to other pine species studied with nS-
SRs (Williams et al. 2000, Shepherd et al. 2002, Dvorak et al.
2009, Sánchez et al. 2014, Zinck & Rajora 2016, Budde et al.
2017). The mean number of alleles (A = 3.4), allelic richness
(Ar = 2.7–3.2) and eective number of alleles (Ae = 2.1–2.6)
were similar to earlier ndings in P. caribaea var. bahamen-
sis (Ar = 3.2, Sánchez et al. 2014) and lower than values ob-
tained for other pine species, such as P. resinosa Aiton (A =
9.0, Boys et al. 2005), P. pinaster Aiton (Ar = 8.3–10.2, Ae =
3.1–4.2, Mariette et al. 2001; A = 9.8, Ar = 9.2, De-Lucas et
al. 2009), P. taeda (A = 4.9, Williams et al. 2000; A = 5.4,
Al Rabah’ah & Williams 2004) and P. oocarpa (Ar =11.86,
Dvorak et al. 2009). Diversity levels in P. caribaea were
similar to those found in P. halepensis (Ar = 2.6–3.8, Budde
et al. 2017). These comparisons should be taken with caution
due to variation in sample sizes, marker polymorphism and
completeness of geographic range sampled. Also, He is more
robust than Ar because He is less aected by marker poly-
morphism. In this sense, the mean He across all populations
(He = 0.571) was within the reported range for pine species
(0.341 to 0.800, Karhu 2001, De-Lucas et al. 2009, Zinck
& Rajora 2016, Budde et al. 2017), and was very similar to
that obtained in P. taeda (He = 0.520, Al Rabah’ah & Wil-
liams 2004) and P. patula (He = 0.586, Dvorak et al. 2009).
Populations of the var. hondurensis distributed in Guatemala
(H6) and Nicaragua (H10 and H11) had the highest values
M (migration rate) K1K2K3K4K5K6K7θ
(4Neµ)
Ne
Historical
P. caribaea var. hondurensis
K1 (H3 and H5) 0.0 2.10 1.67 3.03 2.27 9.18 4.00 1.2924
[0.5333–3.0666]
323.17
[132.22–766.67]
K2 (H2, H8, H9, H10) 4.38 0.0 3.64 1.53 0.77 3.45 0.84 1.7731
[1.0000–3.5333]
443.27
[250.00–883.33]
K3 (H1) 1.50 1.40 0.0 0.45 1.02 3.53 0.47 0.7989
[0.0000–2.4666]
200.72
[0.00–616.66]
K4 (H7) 1.74 4.16 1.40 0.0 1.01 1.71 1.71 1.2172
[0.4000–2.9333]
304.31
[100.00–733.33]
K5 (H4, H6, H11) 8.32 4.57 7.96 10.90 0.0 20.16 5.56 2.1463
[1.1333–4.3333]
537.00
[288.33–1083.32]
P. caribaea var. caribaea
K6 (C1 and C2) 1.26 1.30 7.54 2.02 3.43 0.0 2.37 0.6456
[0.0000–2.2666]
161.41
[0.00–566.66]
P. caribaea var. bahamensis
K7 (B1 and B2) 2.63 1.52 0.970 4.11 1.10 4.85 0.0 0.8328
[0.0000–2.5333]
208.19
[0.00–633.33]
Table 4 – Migration rates (M) and historical eective size (Ne) estimates in K = 7 groups of Pinus caribaea varieties obtained with
BAPS analysis.
In bold type the highest values of M obtained between pairs of K groups. Readings of the values of M from left (row) to right (column).
Markov chains were obtained with 10 000 burn-in steps and 500 000 iterations (Beerli 2008). CI-95%, Condence intervals are given in
brackets.
70
Pl. Ecol. Evol. 151 (1), 2018
Figure 3 – Diagrammatic map with the high dispersal routes of the fteen populations for Pinus caribaea varieties, obtained with Migrate
analysis among the K seven groups dened by Bayesian analysis (BAPS). The gure shows only the migration rates (M) greater than four
units, indicated inside of rectangles. Coloured lines represent the dispersal routes; continuous lines are the migration routes from continent-
continent and continent-island groups. Dashed lines represent the dispersal routes among islands and islands-continent.
of He, whereas H3 of Mexico, and B1 of Bahamas Islands
displayed the lowest values. However, for some of the pop-
ulations, Ho was lower than expected under mutation–drift
equilibrium, suggesting non-random mating.
In fact, the inbreeding levels of nine populations were
signicant (table 1). Inbreeding has been more drastic for
the population Mil Cumbres (C2) of var. caribaea, which
showed the highest value (FIS = 0.319). This population
is restricted to a small area in Cajálbana in western Cuba
(70 km2), growing only on ferrous serpentine soils (Marrero
et al. 1998). Similarly, the Dolores (H6) population of var.
hondurensis, located in Guatemala, presented high inbreed-
ing (FIS = 0.157). This small population is restricted to sa-
vanna forest and represents the most westerly distribution
of this variety. Also, the population New Providence (B2)
of var. bahamensis, the smallest forest area of all the Baha-
mas Islands, presented a high FIS value (0.280). From this
population, a study with ve nSSRs also showed a signi-
cant FIS (0.090) and a low He (0.487) (Sánchez et al. 2014).
This population has lost around 64% of its initial extension
due to deforestation and urbanization during the last century
(Sánchez 2012), which could have led to a loss of genetic di-
versity with a consequent increase in inbreeding. In contrast,
other populations with small size did not show signicant in-
breeding. For example, Caobas (H1) a small stand in Mexico
surrounded by tropical semi-perennial forest and located in
the northernmost distribution of var. hondurensis, displayed
the lowest value of He (0.488) and did not signicantly devi-
ate from Hardy-Weinberg equilibrium (FIS =0.057). A previ-
ous work using six nSSRs (four of them were used in this
study) showed a similar value of He (0.471), but the inbreed-
ing coecient was higher and signicant (FIS = 0.097, P <
0.05; Delgado et al. 2011). These dierences could be due
to sample size dierences or marker choice; in this work the
estimations were obtained based on 17 individuals whereas
the work of Delgado et al. (2011) used 60 individuals. It has
been suggested that this population might be a remnant one
(Dvorak et al. 2005, Delgado et al. 2011). Another particular
example is the northeast population of Andros Island (B1)
of the var. bahamensis, where the FIS was not signicant
(-0.063). This result is similar to that obtained by Sánchez et
al. (2014) for the same population (FIS = 0.019) and another
seven populations studied in the Bahamas Islands (0.063 to
-0.063). Also, population B1 did not show evidence of a re-
cent population size decline (see table 2). Therefore, these
results suggest that these populations from the Bahamas are
in demographic equilibrium, where long distance gene ow
through pollen dispersal, soil seed bank and wind-dispersed
71
Rebolledo Camacho et al., Genetic variation and dispersal of Pinus caribaea varieties
Figure 4 – Pattern of isolation by distance (IBD) among Pinus caribaea population pairs distributed in the Caribbean Basin. The correlation
value was low but signicant (r2 = 0.263; P = 0.005), where 26% of the observed dierences on the genetic distance can be attributed to
geographical distance between populations. P-value was obtained with 10 000 permutation using Mantel test. Black symbols indicate the
association among population pairs of varieties hondurensis/caribaea (●), hondurensis/bahamensis (▲), and caribaea/bahamensis (■).
Open symbols indicate the association within var. hondurensis (◊), var. caribaea (○) and var. bahamensis (∆) populations.
seeds of scattered mature individuals could have contributed
to the maintenance of genetic variation (Sánchez et al. 2014).
In contrast to the previous example, the results of bot-
tleneck tests obtained with two of the three mutation models
(IAM and TPM) supports the hypothesis that most popula-
tions of the P. caribaea varieties showed signals of recent
population bottlenecks, where allele number is reduced
faster than heterozygosity (Cornuet & Luikart 1996). Sev-
eral populations with isolated distribution and/or small Ne,
presented the highest deviation of genetic diversity from ex-
pectations under demographic equilibrium (T2), such as H10
from Nicaragua, in the southernmost part of the distribution
of var. hondurensis; H6, a fragmented population from Gua-
temala, the two populations from Cuba, and B2, the New
Providence population from the Bahamas (table 2). Our re-
sults indicate that most P. caribaea populations have experi-
enced a historic bottleneck in their eective population size
due to fragmentation and geographical isolation. Recent pro-
cesses of colonization could be also plausible, at least from
the island populations with signicant signals of bottlenecks.
Events of colonization have also been demonstrated with the
use of cpSSR, from the same island pine varieties (Jardón-
Barbolla et al. 2011).
The historical eective population size according to the
groups obtained with the BAPS analysis was higher for P.
caribaea var. hondurensis in which a total of ve genetic
clusters were observed (each with Ne of between 201 and
537 individuals) than for var. bahamensis and var. caribaea
which each harbored a single genetic cluster (Ne = 208 and
Ne = 161, respectively). Also, the Ne estimates within clusters
(gene pools) of var. hondurensis were heterogeneous, reect-
ing the degree of population fragmentation or isolation. In
pines, higher Ne estimates are associated with high values of
genetic diversity and larger census population sizes (Ledig
1998, Rajora et al. 2000, Delgado et al. 2002, Ma. et al. 2006,
Naydenov et al. 2014). For example, in P. densata Masters,
an ancestral hybrid species with a large distribution in the
Tibetan Plateau, the estimated Ne with seven loci was 73 200
(Ma et al. 2006). In contrast, in P. pinaster, a species with
fragmented populations distributed in the Mediterranean re-
gion, a small Ne of 86.8 was obtained using eight nSSR (range
of 42.5–359.1), suggesting signals of a demographic decline
due to a recent bottleneck (Naydenov et al. 2014). In the same
sense, for P. resinosa, with a larger and fragmented distribu-
tion in the northern USA and southern Canada, a small Ne of
142 was estimated (range of 62–222), using four nSSR and
the same Ne estimator as this study (Beerli 2008), probably
caused by an extreme genetic bottleneck (Boys et al. 2005).
These values of Ne are more similar to those obtained for the
island varieties of P. caribaea with a restricted distribution
and some marginal land populations of var. hondurensis,
most of them showing signals of recent bottlenecks.
Genetic relationships between populations and varieties
The analyses performed to assess the genetic structure of the
populations of P. caribaea identied the varieties as a signi-
cant level of genetic dierentiation. The average value of RST
among the varieties was 0.088, indicating that 8.8% of the
genetic variation is distributed among varieties. This value
72
Pl. Ecol. Evol. 151 (1), 2018
was relatively low, but lay within the values obtained for
other pine species with nSSR (P. pinaster, RST = 0.111, Ma-
riette et al. 2001; P. resinosa, RST = 0.280, Boys et al. 2005;
P. radiata D.Don, RST = 0.145, Karhu et al. 2006; P. taeda,
RST = 0.041, Al-Rabab’ah 2003; P. oocarpa, RST = 0.130, P.
tecunumani, RST = 0.075 and P. patula Schltdl. & Cham.,
RST = 0.083, Dvorak et al. 2009) and was in fact higher than
those obtained for some studied populations of P. caribaea
var. hondurensis (RST = 0.021, Furlan et al. 2007; RST = 0.033,
Delgado et al. 2011). While it may not be convenient to com-
pare dierent markers, studies based on isoenzyme variation
indicated weak genetic structure among the populations of
the varieties caribaea and hondurensis (FST = 0.020, Zheng
& Ennos 1999, and FST = 0.023, Dvorak et al. 2005, respec-
tively), and moderate genetic structure in var. bahamensis
(FST = 0.078, Zheng & Ennos 1999). In the present study,
the highest genetic dierentiation was found among popula-
tions of var. hondurensis (FST = 0.085), followed by var. ba-
hamensis (FST = 0.076), with an FST similar to the one found
by Zheng & Ennos (1999), whereas weaker genetic dier-
entiation was obtained among populations of var. caribaea
(FST = 0.059) (electronic appendix 2D–F). More recently, a
phylogeographic study of the subsection Australes obtained
higher levels of genetic dierentiation for the three varieties
using plastid microsatellites (cpSSRs) (RST = 0.230; Jardón-
Barbolla et al. 2011). Since cpSSRs are haploid markers,
they are of course more susceptible to the eects of genetic
drift, and the absence of recombination in cpDNA does not
obscure the geographic structure associated to gene genealo-
gies as may be the case for nSSRs. It is therefore expected
to obtain higher values of RST, generating a more marked ge-
netic dierentiation (Rosenberg & Nordborg 2002, Petit et
al. 2005, Avise 2009).
The results obtained with the Bayesian analysis of popu-
lation structure (BAPS) and the tree topology were dependent
on variety, distinguishing the populations of the two insular
varieties (group K6 of the var. caribaea and K7 of the var.
bahamensis) from the populations of var. hondurensis. This
may suggest that the varieties represent three independent
evolutionary lineages. The populations of var. bahamensis
(B1 on New Providence and B2 on Andros Island) are sepa-
rated by a few kilometres (53.16 km), and the two populations
of var. caribaea are located in the northern Cuba, at a rela-
tively short distance apart (44.27 km). Geographical distance
therefore does not seem to have played a major role in the
genetic structure within each variety and each of these two
varieties conforms to a specic genetic cluster. In contrast,
populations of var. hondurensis show genetic structure within
their geographical range; populations H9 and H10, located in
the southern extreme of the distribution in Central America
(Honduras and Nicaragua), are the most derived, while the
populations H6 of Guatemala, H11 of Nicaragua and H7
of Honduras, are closer to the other varieties. These results
are comparable to those obtained by Jardón-Barbolla et al.
(2011), using cpSSRs, in which a marked phylogeographic
structure was obtained, since haplotypes were not shared
among the three varieties and a more signicant relationship
between the haplotypes of the varieties bahamensis and hon-
durensis was found. The latter variety also had substructure
between its populations that comprised two groups; group I
was distributed in the north (various populations of var. hon-
durensis and the two populations of the var. bahamensis), and
group II in southern Central America. In this study, the sub-
structure obtained for var. hondurensis was greater, given that
ve genetic groups were present. This is most likely due to
the larger population size (2N) of nSSR relative to cpSSR
(N), providing information from both progenitors (pollen and
ovules). Isolation of groups could therefore be related to poor
movement of seeds and/or pollen between some of the popu-
lations of these taxa.
Isolation by distance and dispersal routes
The IBD analysis showed a moderate correlation between
genetic and geographic distances among all populations of
the three varieties, indicating that nearby populations are
less genetically isolated from each other than populations
from dierent regions. However, when the analysis was con-
ducted on populations of var. hondurensis alone, IBD was
not signicant, which once again suggests that the genetic
dierences between the populations of this variety are due to
ecological factors. In this sense, a regional metapopulation
dynamic has been stated by Jardón-Barbolla et al. (2011),
where some populations are of recent formation while others
tend towards extinction (Slatkin 1977). Populations that do
not adjust to the IBD model are located in dierent areas of
the species distribution. For example, the population Moss
(H11) in southern Nicaragua has high values of RST with its
neighboring population Leimus (H9; RST = 0.136) of Hon-
duras, and is very similar to other, more geographically dis-
tant, populations; in the genetic distance tree, it clusters with
Mountain Pine (H4) and Dolores (H6) from the north, and
with Mezapa (H7) distributed in central Honduras (g. 2).
This latter population shares the highest number of alleles
with populations distributed in the northern and southern re-
gions; in the tree topology it is located in the early diverg-
ing part of the second group and thus could represent one of
the most ancestral populations. The population Caobas (H1),
distributed in the northern region, presents high values of dif-
ferentiation with the populations Rock (H3; RST = 0.157) and
Deep River (H5; RST = 0.091) distributed at a close distance
(Belize) and is clustered with two of the southern populations
(H10; Waspam and H9; Leimus). Furthermore, these popula-
tions have low values of He (H1, 0.421; H5, 0.491), and high
values of inbreeding (H5, 0.154; H11, 0.177). These results
give partial support to the hypothesis of a metapopulation dy-
namic (Jardón-Barbolla et al. 2011), since some populations
located on the periphery or coastal lowland of the distribu-
tion area of this variety contain the lowest values of genetic
variability and the smallest Ne (e.g. H1, Caobas in Mexico).
Other populations located in the center of the distribution
area of the variety (most of which exceed 300 individuals)
contain alleles that are representative of the gene pool of the
species and have a large Ne (e.g. H4, Mountain Pine, Belize
or H7, Mezapa, Honduras). Therefore, the possible evolu-
tionary scenario of var. hondurensis could be associated with
expansion and contraction events of its populations. In par-
ticular, populations included in the K5 group (H11 and H4/
H6) could support this hypothesis: these populations are geo-
graphically distant, though genetically similar, which could
be explained by a metapopulation dynamic, in which some
73
Rebolledo Camacho et al., Genetic variation and dispersal of Pinus caribaea varieties
populations retain ancestral allelic variants (probably in pro-
cess of expansion) while others do not (due to population
contraction or extinction). This scenario is likely consider-
ing that pine savannas are frequently subjected to forest res
(Dvorak et al. 2005, Jardón-Barbolla et al. 2011).
Currently, as explained in the introduction, there are two
general hypotheses regarding the dispersal routes of pine spe-
cies in the Caribbean Basin. The rst postulates that the initial
migration involved an ancestor from Southern Florida to the
Caribbean Islands (Adams & Jackson 1997), while the second
hypothesis proposes that this dispersion could have occurred
from Central America to the islands of Cuba and the Antilles
(Mirov 1967, Dvorak et al. 2000a, 2005, Jardón-Barbolla et
al. 2011). The results obtained in this study concur with the
second hypothesis; the tree topology shows a closer associa-
tion between the populations of var. hondurensis distributed
in the central and southern regions of Central America and
the two island varieties. The populations of var. caribaea and
var. bahamensis, are nested within continent populations,
suggesting that the dispersion initiated from Central America
(Honduras, Nicaragua and Guatemala) towards the islands.
The Migrate analysis corroborates these results and indicates
that the main dispersal routes depart from the K5 and K1
groups from Central America towards all the populations of
var. hondurensis and to those of the two island varieties (K6
and K7) with M values of between 4.57 and 20.16. The latter
value is the migration rate obtained from var. hondurensis
(K5) toward var. caribaea (K6) (table 4 and g. 3). Three
additional migration routes were suggested: (1) from the
populations of var. bahamensis (K7 of the Bahamas) to var.
caribaea (K6; M= 4.85, in Cuba), (2) from var. bahamensis
(K7) to var. hondurensis (K4; M = 4.11, Mezapa, Honduras)
and (3) from var. caribaea (K6) to var. hondurensis (K3; M =
7.54, Caobas, Mexico). These results support the hypothesis
of demographic processes of expansion and contraction of
population of var. hondurensis, and recent colonization to the
two islands (Jardón-Barbolla et al. 2011), as well as sporadic
events of migration from the islands to Central America.
Coalescence times of the Caribbean pine genetic clusters
Fossil-based divergence times calculated by Krupkin et al.
(1996), as well as a recent phylogenetic reconstruction us-
ing chloroplast sequences calibrated with the fossil record
(Hernández-León et al. 2013), suggested that the Australes
group separated from its ancestors (Oocarpae) approximate-
ly 10 to 12 Mya; whereas Willyard et al. (2007), with evi-
dence from nuclear and chloroplast loci and calibration with
the fossil record, suggested a wider time interval (5 to 18
Mya). These divergence times may not be entirely correct;
however, they do serve as a point of reference indicating that
the ancestral clade of P. caribaea separated before or during
this time. Taking into consideration the average value of the
parameter θ in each gene pool obtained in the Migrate analy-
sis, where θ = 4Neµ for diploid DNA (Hartl & Clark 1997),
and where, mutation rate was assumed as 10-3 (Boys et al.
2005), the expected coalescence time within each gene pool
can be obtained from Ne data of the table 4 as 2Ne. Estimates
vary between 323 generations for P. caribaea var. caribaea
(K6) to 1074 for one of the gene pools of P. caribaea var.
hondurensis (K5). Considering time to rst reproduction
between 10 (Okoro 1984) and 15 years (Jardón-Barbolla
et al. 2011), one could consider a generation time of c. 30
years. The mean coalescence time within clusters would thus
be on the order of between 9700 and 32 000 years, bearing
in mind wide condence intervals associated to the highly
stochastic coalescent process. Divergence between varieties
should a priori precede within-cluster coalescence, which is
congruent with a speciation time of P. caribaea dated to the
later Pliocene or early Pleistocene based on chloroplast and
nuclear sequences data (Hernández-León et al. 2013). Since
our coalescence time estimates for genetic clusters were re-
cent, we inferred that demographic processes detected within
clusters probably aected the last tens or hundreds of gen-
erations. Evidence from pollen records in Guatemala (Pe-
tén) have shown that the forests in the region included pines,
oaks and elms, along with certain rainforest elements that
were dated to between 8000 and 7000 years before present
(Leyden 1984, Dvorak et al. 2005). Therefore, the distribu-
tion and abundance of the var. hondurensis in this region cer-
tainly expanded and contracted along with climatic changes
over the last 10 000 years (Dvorak et al. 2005), which is par-
tially consistent with the chronological times obtained in this
study and the bottleneck detected for some populations of
this complex of P. caribaea.
CONCLUSIONS
Our results support the hypothesis of the recent origin of this
taxon from an ancestor of Central America (Honduras); the
inferred migrations were predominantly from the continent
to islands with sporadic migration events from the islands
to continent. Thus, we deduce that the greatest source of ge-
netic diversity is Central America, the area of distribution of
var. hondurensis. Moreover, similar values of genetic diver-
sity and shared genetic variation between the three varieties
indicate that their speciation is not yet concluded. Most pop-
ulations of var. hondurensis, and one of the two populations
of each island variety, showed signicant levels of inbreed-
ing, with the highest levels found in those populations with
marginal and coastal lowland distribution and small Ne. The
historical demography of this species could be associated
with long distance colonization events, followed by expan-
sion and contraction of their populations.
SUPPLEMENTARY DATA
Supplementary data are available in pdf at Plant Ecology
and Evolution, Supplementary Data Site (http://www.ingen-
taconnect.com/content/botbel/plecevo/supp-data) and con-
sist of the following: (1) summary of Bayesian BAPS results
of three Caribbean pine varieties with the K-groups generat-
ed, and (2) AMOVA results for dierent groups of data from
three Caribbean pine varieties.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the comments and
suggestions of David Gernandt, Keith MacMillan, the editor
Myriam Heuertz and two anonymous reviewers that greatly
74
Pl. Ecol. Evol. 151 (1), 2018
improved the quality of the manuscript. We also thank the
Leon University Herbarium of Nicaragua, Pinar del Río
University of Cuba, Belize Wildlife Service, Honduras Uni-
versity Herbarium (TEFH) and Santo Domingo Botanical
Garden, F. Chi, P. Simá, R. Balam, N. Soltero, D. Escudero
and Gretel Geada, for their help and cooperation in the col-
lection of samples. We are grateful to G. Castillo, J. Coello,
and A. Quiróz for their assistance with molecular work, and
to Silvia Hernández-Aguilar and Germán Carnevali, for pro-
viding information of CICY herbarium database and regis-
tration of some samples. This work was nanced by the Doc-
toral scholarship CONACYT-203335 to V. Rebolledo, and
the projects CONACYT-44373 to P. Delgado and CONA-
CYT-46925 to D. Piñero.
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Communicating Editor: Myriam Heuertz.
