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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 diversification 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 fifteen 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 significant levels of inbreeding (0.097–0.184, P ≤ 0.05). A slight but significant genetic differentiation was found between the varieties (RST = 0.088) and populations (RST = 0.082), and genetic differentiation 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 differentiation suggest that the three varieties are divergent lineages that currently share allelic variants, indicating that their speciation has not yet completed. © 2018 Botanic Garden Meise and Royal Botanical Society of Belgium.
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Plant Ecology and Evolution 151 (1): 61–76, 2018
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 Cientíca de Yucatán, A.C. Calle 43 #130 x 32 y 34, Colonia Chuburná de Hidalgo, CP 97205 Mérida, Yucatán,
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:
All rights reserved. © 2018 Botanic Garden Meise and Royal Botanical Society of Belgium
ISSN: 2032-3913 (print) – 2032-3921 (online)
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 diversication 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.
MethodsWe 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
Results – The varieties contain similar levels of genetic variation (mean He = 0.571), with several populations
out of Hardy-Weinberg equilibrium, and signicant levels of inbreeding (0.097–0.184, P0.05). A slight
but signicant genetic dierentiation was found between the varieties (RST = 0.088) and populations (RST=
0.082), and genetic dierentiation 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 dierentiation suggest
that the three varieties are divergent lineages that currently share allelic variants, indicating that their
speciation has not yet completed.
Key wordsPinus caribaea varieties, genetic variation, microsatellites, lineage divergence, migration
routes, Caribbean Basin.
Pl. Ecol. Evol. 151 (1), 2018
Mexico is considered a secondary centre of diversication 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 dierent 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
monospecic, 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 dier 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 dierences 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 signicant dierences in the
presence and frequency of alleles between the varieties, sup-
porting the notion that the varieties are dierent 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 dierent degrees of genetic
dierentiation were found among the varieties and species
(RST = 0.230 among varieties, RST = 0.110 among species)
(Jardón-Barbolla et al. 2011).
This dierentiation 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 classied 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 Amplied 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-
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 dier-
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 eective 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
dierentiation 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 eective 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 eect 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 eective
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
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, amplication 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 amplication reaction conditions
described below were performed using a MasterCycler Gra-
dient thermocycler (Eppendorf Inc), according to Elsik et al.
(2000), with modications in the concentration of magne-
sium chloride (4 mM). Specic 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 specic 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] buer
at 0.5%), and run at 50–60 W for 1.5–3.5 hours, depending
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 conrm 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-amplied 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 Brookeld (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 eect (detecting a signicant
heterozygosity decit 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 eective 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
Rebolledo Camacho et al., Genetic variation and dispersal of Pinus caribaea varieties
State, District or
(m s.a.l)
Pinus caribaea var. hondurensis
H1-Caobas Mexico/
Quintana Roo
88º57′60.4″W 35 17 18 3 2.5 2.1 0.421
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
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
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
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
H6-Dolores Guatemala/
El Petén
89°25′34.9″W 438 20 25 4.2 3.2 2.9 0.537
H7-Mezapa Honduras/
87°36′38.2″W 306 25 24 4 3.0 2.9 0.521
H8-Trinidad Honduras/
Santa Bárbara
88°15′10.0″W 200 16 22 3.7 2.9 2.6 0.453
H9-Leimus Honduras/
Gracias a Dios
84°08′08.7″W 90 21 22 3.7 2.7 2.3 0.516
H10-Waspam Nicaragua/
North Atlantic
83°58′47.1″W 87 24 22 3.7 2.9 2.6 0.537
H11-Moss Nicaragua/
North Atlantic
83°54′14.4″W 128 21 23 3.8 3.0 2.5 0.516
Average 22 3.7 2.8 2.6 0.502 0.575 0.124**
Pinus caribaea var. caribaea
C1-Viñales Cuba/
Pinar del Río
83°42′29.5″W 239 19 20 3.3 2.7 2.4 0.522
C2-Mil Cumbres Cuba/
Pinar del Río
83°21′57.4″W 185 18 19 3.2 2.7 2.3 0.384
Average 19.5 3.2 2.7 2.3 0.476 0.564 0.184*
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
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
Average 19 3.2 2.7 2.3 0.512 0.576. 0.097
Table 1Geographical 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 eective
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%, condence intervals in brackets.
using Arlequin v. (Excoer & 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 eects of population demographic decline were
examined using the T2 statistic (Cornuet & Luikart 1996),
which reects the deviation from expectations at demograph-
ic equilibrium (Budde et al. 2017). The test was performed
using the innite 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). Signicance 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
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 signicance was ob-
tained with 1000 non-parametric permutations (Excoer &
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
denes 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).
Eective 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).
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 eective 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 dierent 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 signicant heterozygosity decit 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 diered signicantly 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
signicant 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 signicant. In general, most popu-
lations showed dierent 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-
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 signicantly 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 signicant dierentiation 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 dierentia-
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 signicant (P < 0.001). Similarly, the AMO-
VA between the seven groups obtained with the BAPS analy-
sis reected hierarchical population structure (RST = 0.077; P
< 0.002; 95% CI: 0.042–0.109) (table 3, electronic appendix
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
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 2Bottleneck 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.
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
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 3RST 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 signicance was obtained with 1000 non-parametric permutations (Excoer &
Lischer 2010).
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).
Eective size, migration rates and isolation by distance
The average estimate of historical eective 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
signicant relationship between genetic and geographic dis-
tances (r2 = 0.263; P = 0.005) (g. 4), whereas no association
was detected between dierentiation 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).
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 eective 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 aected 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
P. caribaea var. hondurensis
K1 (H3 and H5) 0.0 2.10 1.67 3.03 2.27 9.18 4.00 1.2924
K2 (H2, H8, H9, H10) 4.38 0.0 3.64 1.53 0.77 3.45 0.84 1.7731
K3 (H1) 1.50 1.40 0.0 0.45 1.02 3.53 0.47 0.7989
K4 (H7) 1.74 4.16 1.40 0.0 1.01 1.71 1.71 1.2172
K5 (H4, H6, H11) 8.32 4.57 7.96 10.90 0.0 20.16 5.56 2.1463
P. caribaea var. caribaea
K6 (C1 and C2) 1.26 1.30 7.54 2.02 3.43 0.0 2.37 0.6456
P. caribaea var. bahamensis
K7 (B1 and B2) 2.63 1.52 0.970 4.11 1.10 4.85 0.0 0.8328
Table 4 Migration rates (M) and historical eective 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%, Condence intervals are given in
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 dened 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
signicant (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 signicant 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 signicantly 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 coecient was higher and signicant (FIS = 0.097, P <
0.05; Delgado et al. 2011). These dierences could be due
to sample size dierences 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 signicant
(-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
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 signicant (r2 = 0.263; P = 0.005), where 26% of the observed dierences 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 eective population size
due to fragmentation and geographical isolation. Recent pro-
cesses of colonization could be also plausible, at least from
the island populations with signicant 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 eective 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, reect-
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 identied the varieties as a signi-
cant level of genetic dierentiation. 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
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 dierent 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 dierentiation 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 dier-
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 dierentiation 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 eects 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 dierentiation (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 specic 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 signicant 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 dierent regions. However, when the analysis was con-
ducted on populations of var. hondurensis alone, IBD was
not signicant, which once again suggests that the genetic
dierences 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 dierent 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
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 condence 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 aected 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.
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 signicant 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 are available in pdf at Plant Ecology
and Evolution, Supplementary Data Site (http://www.ingen- 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 dierent groups of data from
three Caribbean pine varieties.
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
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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Manuscript received 8 Mar. 2017; accepted in revised version 12
Dec. 2017.
Communicating Editor: Myriam Heuertz.
... modificada para microsatélites (Excoffier y Lischer, 2010). Dado que el parámetro θ= 4N e µ (Kimura, 1968), y al asumir una tasa de mutación (µ) de 10 -3 por generación, el N e se estimó como θ/4X10 -3 (Boys et al., 2005;Delgado et al., 2011;2013;Rebolledo et al., 2018). ...
... Los valores altos de endogamia, y por lo tanto, el deficit de heterócigos observados en las población, se pueden deber a la presencia de alelos nulos (alelos no expresados) que dan lugar a valores erroneos que desvían a las poblaciones de la panmixia (Shinde et al., 2003). En el análisis para la detección de alelos nulos, (Rebolledo et al., 2018). ...
... Moreno y Piñero, 2009;Jardón et al., 2011).Solo se han consignado tres trabajos con el uso de SSRn en pinos, que muestran una asociación baja pero significativa: Pinus pinaster Ait en la cuenca del mediterráneo en Europa (r=0.360)(Mariette et al., 2001); P. resinosa que se ubica en el Noreste de EUA (r= 0.381)(Boys et al., 2005); y en el complejo de pinos caribeños para la cuenca del Caribe (r= 0.263)(Rebolledo et al., 2018). Estos resultados son similares al obtenido en el presente estudio, por lo que una gran parte (≈60 %) de la distribución de la variación genética podría estar vinculada a otros factores, como la fragmentación espacial de las poblaciones, demarcada tanto por la geomorfología natural de la cuenca, como por al cambio de uso de suelo, de forestal a huertos de aguacate y de urbanización(Bravo et al., 2009). ...
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La conservación de los hábitats terrestres asociados a cuencas hidrográficas tiene como componente intrínseco la conservación de los recursos genéticos de las especies que conforman la cubierta vegetal de estos sistemas. El objetivo del presente estudio fue evaluar los niveles de variación y estructura genética de las poblaciones de pino distribuidas en la cuenca del río Cupatitzio, en Michoacán, con el uso de cuatro microsatélites de núcleo. El estudio incluye ocho poblaciones de cuatro especies de pino distribuidas en las zonas altas, media y baja de la cuenca. Los resultados indican que las poblaciones SL3 de P. pseudostrobus y RB6 de P. douglasiana contienen los valores más altos de variación genética (HE =0.674 y HE= 0.615, respectivamente). Las poblaciones presentan importantes niveles de endogamia (FIS entre 0.057-0.544) y una diferenciación genética significativa (FST entre 0.094-0.152), la cual se asocia de manera moderada con la distribución geográfica de las poblaciones (r= 0.443) y se agrupa de acuerdo con las especies. La parte alta de la Reserva, en Quinceo y la Tzaráracua, presenta los niveles más bajos de variación genética y los mayores niveles de endogamia, por lo que se recomienda hacer actividades de restauración en estas localidades. Así mismo, se sugieren actividades de conservación in situ en San Lorenzo y la parte baja de la Reserva, ya que ambas poblaciones presentan los tamaños efectivos más grandes y son representativas del acervo genético de los bosques de pino en la cuenca del Cupatitzio.
... Uno de los marcadores genéticos más utilizados para reconstruir la historia evolutiva de las poblaciones y especies son los microsatélites de núcleo (SSRn), pues son marcadores codominantes con un alto grado de polimorfismo, reproductibilidad y especificidad (Oliveira et al., 2006;Delgado y Piñero, 2008). Los SSRn han sido ampliamente utilizados en especies de pinos para estimar los niveles de variación y estructura genética, el flujo genético y los niveles de endogamia, para hacer inferencias de la demografía histórica de las poblaciones, en la delimitación de taxa infraespecíficos y para analizar las tasas de entrecruzamiento, entre otros parámetros genéticos de las poblaciones (Marquardt y Epperson, 2004;Boys et al., 2005;Dvorak et al., 2009;Chhatre y Rajora, 2014;Naydenov et al., 2015;Zinck y Rajora, 2016;Rebolledo Camacho et al., 2018). ...
... (Excoffier y Lischer, 2010). La tasa de mutación que se asumió fue de 10 -3 por generación (Boys et al., 2005;Delgado Valerio et al., 2013;Rebolledo Camacho et al., 2018). ...
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Antecedentes y Objetivos: El decremento y fragmentación de las poblaciones resulta en la pérdida de variación e incremento de la diferenciación genética. Pinus remota es una especie arbórea-arbustiva de distribución restringida con poblaciones dispersas y fragmentadas. Los objetivos del presente estudio fueron: evaluar los niveles de variación genética, la endogamia y el tamaño efectivo de las poblaciones, probar la hipótesis de la existencia de cuellos de botella asociados a la disminución de la variación genética de las poblaciones y analizar la estructura genética y su asociación con la distribución geográfica de las poblaciones. Métodos: El estudio se realizó en siete poblaciones representativas de su distribución natural en México, con muestras de acículas de 112 árboles analizadas con cinco microsatélites de núcleo. Los datos se evaluaron con parámetros de la genética de poblaciones y métodos de aproximación Bayesiana. Resultados clave: La heterocigosidad promedio esperada (He=0.557) fue más alta que la observada (Ho=0.375). Las poblaciones no se encuentran en equilibro Hardy-Weinberg, con una endogamia significativa (FIS=0.259) y tamaños efectivos reducidos (Ne entre 375-425); están estructuradas en tres grupos genéticos (FST=0.158), con valores altos del índice Mc (0.186-0.283), sugiriendo eventos de declinamiento demográfico ancestral (entre 11,910 y 23,820 años atrás), asociados con los cambios climáticos del Pleistoceno. Conclusiones: El estudio demuestra que P. remota contiene una variación genética considerable, pero estructurada espacialmente y asociada a cuellos de botella ancestrales. Además, se confirma que P. catarinae es su sinónimo. La especie ha sido capaz de subsistir y adaptarse a condiciones ambientales locales. Con este conocimiento se plantean estrategias para la conservación de las poblaciones remanentes de la especie.
... The three varieties slightly differ in their morphology, suggesting that they diverged only recently or the existence of abundant gene flow among them (Jardón- Barbolla et al. 2011). Furthermore, the genetic differentiation among varieties and populations is slight (Sanchez et al. 2014;Rebolledo Camacho et al. 2018). Caribbean pine is a fastgrowing wind-and insect-pollinated species that can tolerate drought and maritime exposure. ...
... This temperature threshold for reproduction has also been observed in Mediterranean conifers and might be related with the flowering temperature cumulative thresholds (Mutke et al. 2003). As expected (Rebolledo Camacho et al. 2018), the differentiation among populations was slight, although it was significant for other fitness-related traits not considering in the final mixed-effects model of reproduction, suggesting that populations are locally adapted (Appendix Table 6). Contrarily, straightness did not show a provenance effect and was exclusively influenced by the trial temperature, in agreement with other analyses performed on the species (Moura and Dvorak 2001), but unexpected for a breeding trait (Cameron et al. 2012). ...
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• Key message The combination of structural equation modelling and linear mixed-effects models opens a new perspective to investigate trait adaptation syndromes through phenotypic integration prediction at large geographical scales, a necessary step to understand the future of organisms under climate change. In the case of Pinus caribaea Morelet, reproduction limits the species suitability, decreasing towards southernmost latitudes where dry conditions increase.ContextCaribbean pine is an ecologically and economically important species planted in all the tropical regions of the world, where it shows optimal growth and survival but low reproduction rates.AimsThis study investigates Caribbean pine fitness-related traits, accounting for phenotypic plasticity and local adaptation, to detect co-variation among traits and predict their relationship across the tropics.MethodsI re-analysed earlier data of survival, growth, reproduction, stem quality and development stage from 25 provenances of Caribbean pine planted in 16 trials in the tropical regions in a two-step modelling approach including (i) structural equation modelling (SEM) based on the current knowledge of the species and theoretical expectations coming from other species; (ii) mixed-effects model accounting for trait-relationships as defined by SEM and allowing for trait prediction.ResultsGrowth, survival and reproduction showed a slight but significant provenance effect indicating population differentiation and a positive co-variation between growth and reproduction, suggesting that trees reached optimal growth before they reproduced. Models predicted low reproduction rates of Caribbean pine across the tropics, decreasing towards southern latitudes where dry conditions increased.Conclusion This study opens new perspectives to investigate trait adaptation syndromes through phenotypic integration prediction at large geographical scales.
... caribaea para el occidente de la isla de Cuba e Isla de la Juventud. En la actualidad dicha clasificación es acertada para referirse a una u otra variedad, dadas las diferencias en crecimiento (Francis 1992), comportamiento en plantaciones fuera de su rango natural como parte de pruebas de especies o procedencias (Graves 1981, Gibson 1982, Wang & al. 1999) y desde el punto de vista molecular (Zheng & Ennos 1999, Geada-López & al. 2004, Jardón-Barbolla & al. 2011, Rebolledo-Camacho & al. 2018. ...
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Variation of anatomical characters is crucial in the recognition of ecological adaptability, especially in Pinus. Pinus caribaea var. caribaea is an endemic taxon of Western Cuba that grows in pure populations or sympatry with Pinus tropicalis and occupies a great variety of ecotopes that are also distinguished by the characteristics of the edatope. The objective of this research is to determine the anatomical variation of the needles as an adaptive differential response to the environmental conditions determined by lithology, altitude and slope. From 20 to 30 individuals from all the ecotopes where the taxon grows naturally were sampled. Cross sections were made of 10 needles from each tree and 12 anatomical variables, related to water regulation, transport and storage of metabolites, were assessed. The results of the statistical analysis revealed significant differences between ecotopes. The principal component analysis showed a relationship between anatomical variables that follow a functional pattern of water regulation and assimilation. The cluster and discriminant analysis made possible to distinguish the formation of groups by the relationship of the anatomical variables, mainly due to the effect of lithology, and those that contributed the most to differentiate them were those of water regulation, primary metabolism together with cuticle thickness. The results are a contribution to the local conservation of the taxon since the structure of the anatomical variation is a consequence of the genetic evolution of the populations and are very important in ecological and for silvicultural management. Citation: Geada-López, G., Sotolongo-Sospedra, R., Pérez-del Valle, L. & Ramírez-Hernández, R. 2021. Diferenciación anatómica foliar en poblaciones naturales de Pinus caribaea var. caribaea (Pinaceae) en Pinar del Río y Artemisa, Cuba. Revista Jard. Bot. Nac. Univ. Habana 42: 175-188. Received: 23 March 2021. Accepted: 13 May 2020. Online: 21 July 2021. Editor: José Angel García-Beltrán.
... Dicho autor reconoció las discretas diferencias entre las poblaciones (localidades) de P. tropicalis, que consideró una especie altamente adaptada en relación con P. caribaea. En esta última son más notables las diferencias entre las localidades (Figuras 2-3, Tabla III), lo que demuestra su capacidad de ocupar varios ecótopos y una posible gran plasticidad fenotípica, buena capacidad competitiva e invasividad (Badik & al. 2018, Rebolledo-Camacho & al. 2018. Estudios previos han referido una mayor variación a nivel de procedencias en P. caribaea (García-Quintana & al. 2007) que en P. tropicalis. ...
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Las variaciones en la anatomía foliar pueden ser respuestas adaptativas o de aclimatación interpoblacional al estrés edafoclimático, sobre todo de especies que se desarrollan en ambientes extremos. Pinus caribaea var. caribaea y P. tropicalis se distribuyen en el occidente de Cuba, principalmente en la provincia de Pinar del Río donde forman poblaciones puras o en simpatría. Es objetivo de este trabajo comparar las características anatómicas distintivas de las acículas de ambos taxones en diferentes localidades donde se asocian simpátricamente. Para ello se realizaron cortes transversales de las acículas y se evaluaron 14 variables anatómicas. Los análisis estadísticos empleados permitieron diferenciar claramente los dos taxones y ambos presentan variaciones propias para adaptarse a un mismo ambiente. El análisis de componentes principales mostró que dentro de cada taxón las poblaciones se segregan en relación al edátopo donde se desarrollan. Para P. caribaea var. caribaea las variables anatómicas que más contribuyeron a la variación y ordenación fueron el número de estomas, grosor y número de capas de células de la hipodermis; en P. tropicalis el grosor de la cutícula y el parénquima clorofílico, y para ambos taxones el tipo de canal fue inequívocamente endonales y marginal.
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Four Pinus kesiya natural populations in the Central Highland region of Vietnam, separated from one another by distances of 75 to 380km, were examined using tetranucleotide microsatellite markers to evaluate their genetic diversity and population structure. The surveyed populations displayed relatively high level of genetic variation ( H E = 0.671). Only between the Kon Tum and Dak Nong populations was the pairwise value F ST significant. These two populations, separated by 300 km, also showed the greatest separation in the UPGMA cluster analysis using Nei’s pairwise genetic distance. The UPGMA analysis clustered the four populations into two geographic groups (1) the Kon Tum population, which is located in the North of the Central Highlands and (2) the remaining three populations (Gia Lai, Dak Nong and Dak Lak). Within group 2, Gia Lai and Dak Lak located in the center area of the Central Highland, clustered into the same subgroup with the southern Dak Nong population a single subgroup. This topology was essentially in agreement with the geographic distribution of the studied populations. The implications for conservation and development programs for this species are also reported and discussed.
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Plants offer excellent models to investigate how gene flow shapes the organization of genetic diversity. Their three genomes can have different modes of transmission and will hence experience varying levels of gene flow. We have compiled studies of genetic structure based on chloroplast DNA (cpDNA), mitochondrial DNA (mtDNA) and nuclear markers in seed plants. Based on a data set of 183 species belonging to 103 genera and 52 families, we show that the precision of estimates of genetic differentiation (GST) used to infer gene flow is mostly constrained by the sampling of populations. Mode of inheritance appears to have a major effect on GST. Maternally inherited genomes experience considerably more subdivision (median value of 0.67) than paternally or biparentally inherited genomes (~0.10). GST at cpDNA and mtDNA markers covary narrowly when both genomes are maternally inherited, whereas GST at paternally and biparentally inherited markers also covary positively but more loosely and GST at maternally inherited markers are largely independent of values based on nuclear markers. A model-based gross estimate suggests that, at the rangewide scale, historical levels of pollen flow are generally at least an order of magnitude larger than levels of seed flow (median of the pollen-to-seed migration ratio: 17) and that pollen and seed gene flow vary independently across species. Finally, we show that measures of subdivision that take into account the degree of similarity between haplotypes (NST or RST) make better use of the information inherent in haplotype data than standard measures based on allele frequencies only
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Background and Aims The recurrence of wildfires is predicted to increase due to global climate change, resulting in severe impacts on biodiversity and ecosystem functioning. Recurrent fires can drive plant adaptation and reduce genetic diversity; however, the underlying population genetic processes have not been studied in detail. In this study, the neutral and adaptive evolutionary effects of contrasting fire regimes were examined in the keystone tree species Pinus halepensis Mill. (Aleppo pine), a fire-adapted conifer. The genetic diversity, demographic history and spatial genetic structure were assessed at local (within-population) and regional scales for populations exposed to different crown fire frequencies. Methods Eight natural P. halepensis stands were sampled in the east of the Iberian Peninsula, five of them in a region exposed to frequent crown fires (HiFi) and three of them in an adjacent region with a low frequency of crown fires (LoFi). Samples were genotyped at nine neutral simple sequence repeats (SSRs) and at 251 single nucleotide polymorphisms (SNPs) from coding regions, some of them potentially important for fire adaptation. Key Results Fire regime had no effects on genetic diversity or demographic history. Three high-differentiation outlier SNPs were identified between HiFi and LoFi stands, suggesting fire-related selection at the regional scale. At the local scale, fine-scale spatial genetic structure (SGS) was overall weak as expected for a wind-pollinated and wind-dispersed tree species. HiFi stands displayed a stronger SGS than LoFi stands at SNPs, which probably reflected the simultaneous post-fire recruitment of co-dispersed related seeds. SNPs with exceptionally strong SGS, a proxy for microenvironmental selection, were only reliably identified under the HiFi regime. Conclusions An increasing fire frequency as predicted due to global change can promote increased SGS with stronger family structures and alter natural selection in P. halepensis and in plants with similar life history traits.
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Boreal species were repeatedly exposed to ice ages and went through cycles of contraction and expansion while sister species alternated periods of contact and isolation. The resulting genetic structure is consequently complex and demographic inferences are intrinsically challenging. The range of Norway spruce (Picea abies) and Siberian spruce (Picea obovata) covers most of northern Eurasia; yet their geographical limits and histories remain poorly understood. To delineate the hybrid zone between the two species and reconstruct their joint demographic history, we analyzed variation at nuclear SSR and mitochondrial DNA in 102 and 88 populations, respectively. The dynamics of the hybrid zone was analyzed with Approximate Bayesian Computation (ABC) followed by posterior predictive STRUCTURE plot reconstruction and the presence of barriers across the range tested with Estimated effective migration surfaces (EEMS). To estimate the divergence time between the two species nuclear sequences from two well-separated populations of each species were analyzed with ABC. Two main barriers divide the range of the two species: one corresponds to the hybrid zone between them, and the other separates the southern and northern domains of Norway spruce. The hybrid zone is centered on the Urals, but the genetic impact of Siberian spruce extends further west. The joint distribution of mitochondrial and nuclear variation indicates an introgression of mitochondrial DNA from Norway spruce into Siberian spruce. Overall, our data reveal a demographic history where the two species interacted frequently and where migrants originating from the Urals and the West Siberian Plain recolonized Northern Russia and Scandinavia using scattered refugial populations of Norway spruce as stepping-stones towards the west. This article is protected by copyright. All rights reserved.
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Background Knowledge of the historical distribution and postglacial phylogeography and evolution of a species is important to better understand its current distribution and population structure and potential fate in the future, especially under climate change conditions, and conservation of its genetic resources. We have addressed this issue in a wide-ranging and heavily exploited keystone forest tree species of eastern North America, eastern white pine (Pinus strobus). We examined the range-wide population genetic structure, tested various hypothetical population history and evolutionary scenarios and inferred the location of glacial refugium and post-glacial recolonization routes. Our hypothesis was that eastern white pine survived in a single glacial refugium and expanded through multiple post-glacial recolonization routes. Results We studied the range-wide genetic diversity and population structure of 33 eastern white pine populations using 12 nuclear and 3 chloroplast microsatellite DNA markers. We used Approximate Bayesian Computation approach to test various evolutionary scenarios. We observed high levels of genetic diversity, and significant genetic differentiation (FST = 0.104) and population structure among eastern white pine populations across its range. A south to north trend of declining genetic diversity existed, consistent with repeated founder effects during post-glaciation migration northwards. We observed broad consensus from nuclear and chloroplast genetic markers supporting the presence of two main post-glacial recolonization routes that originated from a single southern refugium in the mid-Atlantic plain. One route gave rise to populations at the western margin of the species’ range in Minnesota and western Ontario. The second route gave rise to central-eastern populations, which branched into two subgroups: central and eastern. We observed minimal sharing of chloroplast haplotypes between recolonization routes but there was evidence of admixture between the western and west-central populations. Conclusions Our study reveals a single southern refugium, two recolonization routes and three genetically distinguishable lineages in eastern white pine that we suggest to be treated as separate Evolutionarily Significant Units. Like many wide-ranging North American species, eastern white pine retains the genetic signatures of post-glacial recolonization and evolution, and its contemporary population genetic structure reflects not just the modern distribution and effects of heavy exploitation but also routes northward from its glacial refugium. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0624-1) contains supplementary material, which is available to authorized users.
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Resumen Las coníferas (Pinophyta) son árboles o arbustos con hojas simples y estructuras fértiles arregladas en conos polínicos simples y conos ovulados compuestos, excepto en Taxaceae. Las coníferas son los componentes dominantes de diversos tipos de vegetación. En México crecen desde el nivel del mar hasta por encima de los 4 000 m; la mayor diversidad se encuentra en los bosques montañosos de la Sierra Madre Occidental y Sierra Madre Oriental. Están representadas por 4 familias: Pinaceae (4 géneros y 61 especies), Cupressaceae (4 géneros y 29 especies), Podocarpaceae (1 género y 3 especies) y Taxaceae (1 especie). De las 94 especies de coníferas mexicanas, 43 son endémicas del país, de éstas 18 tienen un rango de distribución limitado a 3 o menos estados.
The Bahaman archipelago contains large expanses of pine forests, where the endemic Caribbean pine Pinus caribaea var. bahamensis is the dominant species. This pine forest ecosystem is rich in species and also a valuable resource for the local economy. Small areas of old-growth forest still remain in the Turks and Caicos islands (TCI) and in some of the islands in the Bahamas; despite on-going severe infestation by pine tortoise scale insect Toumeyella parvicornis and high pine mortality in the former and intensive past commercial logging activities in the latter. For the first time integrated research on the genetics, morphology, ecology and biogeography of this variety was carried out throughout its whole distribution range. Past and present forest areas were mapped using historical physical maps and modern satellite imagery, showing forest loss due to urbanisation, pests and storm surges and expansions resulting mainly from dry-season human induced fires. Population genetic analysis using plastid and nuclear microsatellites revealed high ancient gene flow and recent genetic distance between populations of the Bahamas and the TCI; in addition to genetic structure within regions. Morphological differences were also observed and discussed. The variety showed high individual genetic and morphological variance and high plasticity. Despite the observation of good forest regeneration in normal circumstances, stochastic events did cause severe reductions in forest area and effective population size. A predominantly random and outcrossing breeding system was also inferred from the data, despite detection of some inbreeding in the smaller populations. Suggestions for the future conservation and management of the species included fire management and the creation or extension of in-situ conservation areas and ex-situ collections. Available online:
Phylogenetic analysis of plastid DNA restriction site and rearrangement mutations suggests a number of major revisions to taxonomy and phylogenetic concepts in the hard pines. Total genomic DNA from 18 species that sampled all nine subsections was digested with 19 restriction enzymes, blotted, and probed with 70% of the Douglas-fir (Pseudotsuga menziesii) chloroplast genome, or, with clones encompassing the entire chloroplast genome of Pinus contorta. A total of 204 site mutations and five rearrangement mutations were generated, of which 126 were phylogenetically informative. Wagner parsimony analyses revealed 11 clades that were strongly supported by bootstrap and decay index analyses. All North American species except P. resinosa formed a distinct monophyletic group that was strongly separated from the Eurasian species. Within the Eurasian clade subsect. Sylvestres was polyphyletic; its Mediterranean species were closely allied with members of sect. Pinea. Sect. Pinea appeared polyphyletic as well; both species of its subsect. Leiophyllae showed a close affinity to Mesoamerican pines of subsect. Oocarpae in sect. Pinus. Within the North American pines subsects. Ponderosae and Oocarpae were polyphyletic. Despite its shallow fossil record, subsect. Contortae emerged as a sister group to all of the North American pines apart from P. resinosa, which was allied with Eurasian species of subsect. Sylvestres. The remaining North American subsections formed two groups: a poorly resolved clade with subsects. Ponderosae and Sabinianae, and sequentially nested clades represented by: P. radiata; P. taeda; representatives of subsects. Oocarpae and Ponderosae from Mesoamerica, and subsect. Leiophyllae. We present estimates of divergence times for each of these major clades based on molecular clocks calibrated using two hard pine fossil observations.