Available via license: CC BY-NC 4.0
Content may be subject to copyright.
Research Article
Genetic Diversity and Structure
From Antillean Manatee
(Trichechus manatus manatus)
in the Southern Gulf of Me
´xico:
Comparison Between Connected
and Isolated Populations
Guadalupe G
omez-Carrasco
1
, Julia Mar
ıa Lesher-Gordillo
1
,
Le
on David Olivera-G
omez
1
, Robert K Bonde
2
,
Stefan Arriaga-Weiss
1
, Raymundo Herna
´ndez-Mart
ınez
1
,
Guillermo Casta~
n
on-Na
´jera
1
, Darwin Jime
´nez-Dom
ınguez
1
,
Armando Romo-L
opez
1
, and Alberto Delgado-Estrella
3
Abstract
Antillean manatees (Trichechus manatus manatus), a subspecies of the West Indian manatee, is listed as endangered species in
the Red List of Threatened Species of the International Union for Conservation of Nature. The aims of this research were to
survey on the possible regional genetic structure in the southern Gulf of Mexico and to compare genetic status of a
landlocked population in Laguna de las Ilusiones (IL) with individuals from localities with no barriers to displacement and
breed (open population [OP]). We analyzed 45 manatee skin samples collected from different locations in Tabasco (n¼38,
including 19 from IL), Veracruz (n¼3), Campeche (n¼2), and Chiapas (n¼2). The genomic DNA was isolated and PCR
amplifications were performed for each sample using 28 microsatellite loci, previously designed for West Indian manatees
and described as polymorphic for this species. Two clusters (k¼2) were identified by STRUCTURE. The analysis of both a
priori populations (IL and OP) indicate that the global values of F
ST
and R
ST
(F
ST
¼0.049, R
ST
¼0.077) were significant. The H
E
for IL was 0.38 0.03 and for OP was 0.49 0.01. The average number of alleles N
A
for IL was 2.21 0.09 and for OP was
2.32 0.09. The overall inbreeding coefficient was F
IS
¼0.013 for analyzed populations. Genetic diversity was low. The IL
population had slightly lower genetic diversity compared with OP, which could be explained by isolation of that small group,
so conservation plans for IL should be considered as priority.
Keywords
Microsatellites, gene flow, bottleneck, inbreeding, Laguna de las Ilusiones
Introduction
Determining population structure in large mammals is
important to facilitate the effective conservation man-
agement and help advance our understanding of the
mechanisms that drive the evolution of populations
(Hoelzer, Wallman, & Melnick, 1998). Microsatellites
had been used to differentiate between stocks of mana-
tees (Bonde, McGuire, & Hunter, 2012) and to achieve
alternative estimates of genetic diversity. Antillean
manatees (Trichechus manatus manatus), a subspecies
1
Centro de Investigaci
on para la Conservaci
on y Aprovechamiento de los
Recursos Tropicales (CICART) de la DACBiol; Universidad Jua
´rez
Aut
onoma de Tabasco, Villahermosa, Centro, Tabasco, Me
´xico
2
U.S. Geological Survey, Wetland and Aquatic Research Center, Gainesville,
FL, USA
3
Universidad Aut
onoma del Carmen, Ciudad del Carmen, Mexico
Received 11 May 2018; Revised 19 July 2018; Accepted 25 July 2018
Corresponding Author:
Julia Mar
ıa Lesher-Gordillo, Centro de Investigaci
on para la Conservaci
on y
Aprovechamiento de los Recursos Tropicales (CICART) de la DACBiol;
Universidad Jua
´rez Aut
onoma de Tabasco, Carretera Villahermosa-
Ca
´rdenas km 0.5, entronque Bosques de Saloya; Villahermosa, Tabasco
C
odigo Postal: 86039. Villahermosa, Centro, Tabasco, Me
´xico.
Email: lesher23@yahoo.com
Tropical Conservation Science
Volume 11: 1–10
!The Author(s) 2018
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/1940082918795560
journals.sagepub.com/home/trc
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-
NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and dis-
tribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.
sagepub.com/en-us/nam/open-access-at-sage).
of the West Indian manatee, is listed as endangered spe-
cies in the Red List of Threatened Species of the
International Union for Conservation of Nature (Self-
Sullivan & Mignucci-Giannoni, 2008). It is suspected
that habitat degradation and other anthropogenic
threats have driven native manatee populations to be
considered as endangered in Mexico (Secretar
ıa de
Medio Naturales Ambiente y Recursos [SEMARNAT],
2010); however, knowledge on manatee status and hab-
itat requirements in Mexico is still scarce. Previous
genetic studies have defined two populations in
Mexico, the Gulf of Mexico and the southeastern
Yucatan peninsula, and suggest that the Caribbean
coasts may benefit from higher gene flow from neighbor-
ing populations in Belize and Florida, where the Gulf of
Mexico is more isolated (Nourisson et al., 2011).
The southern Gulf of Mexico is a complex habitat for
manatees, which inhabits mostly freshwater systems on
extensive flood plains (Lefebvre, Marmontel, Reid,
Rathbun, & Domning, 2001). Populations of manatees
in this region are considered to be 1,000–1,500 individ-
uals (United Nations Environment Programme, 2010),
based on the best guessed estimation by specialists,
but recent effort suggest that this is an overestimate
(Puc-Carrasco, Morales-Vela, Olivera-Gomez, &
Gonza
´lez-Solis, 2017). The riverine environment is
not homogeneous, and manatees seems to be more
associated with lake systems connected to main rivers
by secondary waterways, which involve seasonal
movements in and out of these systems, following
flood pulses (Puc-Carrasco, Olivera-G
omez, Arriaga-
Hernandez, & Jimenez-Dominguez, 2016). This seasonal
movement promotes discrete groups of manatees
along the Usumacinta and Grijalva rivers, the largest
regional river basins. Information on the population
structure of manatees in Southern Gulf of Mexico by
Nourisson et al. (2011) is not conclusive and suggests
that more samples for this region are necessary to accu-
rately identify the genetic structure of the population as
a whole.
Natural and man-made modifications to the hydrol-
ogy of particular areas have created isolated groups of
manatees, as well as translocation efforts of individuals
into closed lakes with touristic and waterway manage-
ment intentions. The genetic health of these isolated
populations is a matter of concern due to the small
number of individuals and the time they have been iso-
lated. One of these isolated populations is located in
“Laguna de las Ilusiones” (IL), an area that was origi-
nally connected to the Carrizal River but access is no
longer possible due to construction of a dam 40 years
ago, preventing manatee movement in and out of the
lake. After IL was landlocked, wild manatees from
other areas were rescued and released into IL, as we
know from “anecdotic” talks with people inhabited the
lake borders, but they failed to provide exact dates and
there are not any published record from environmental
authorities. As far as we know, no new animals were
translocated into the lake in the last 20 years.
The extension of the 219 ha lake made this population
of 18 to 39 manatees isolated and without proper man-
agement (Pablo-Rodr
ıguez & Olivera-G
omez, 2012;
Pe
´rez-Garduza, 2013). Direct urban pressure on the IL
population (Arag
on-Mart
ınez, Olivera-G
omez, &
Jimenez-Dominguez, 2014) and concern about the
health and fitness of the manatees provided an opportu-
nity to conduct a survey of the genetic diversity of this
population and compare the findings with opportunistic
samples collected from wild manatees from other local-
ities throughout the southern Gulf of Mexico.
The aims of this research were to survey the fine-scale
regional genetic structure of manatees based on all the
available samples and compare the genetic status of the
wild OP in the southern Gulf of Mexico with the land-
locked population of IL. This information will serve as a
baseline for management strategies and to discuss poten-
tial threats.
Methods
Sampling
We analyzed 45 manatee samples of T. m. manatus col-
lected from different locations in Tabasco (n¼38),
Veracruz (n¼3), Campeche (n¼2), and Chiapas (n¼2;
Figure 1). Skin samples, collected between 2006 and 2015,
were used for DNA isolation. Small skin samples were
taken from the tip of the caudal tail during health assess-
ments of live manatees captured for radio tagging and
from recently dead individuals. Samples were separated
into two groups according to their origin. The first group
corresponds to individuals from “IL” (n¼19), a land-
locked lake located within the urbanized area of the city
of Villahermosa (Figure 1(b)) where a small closed pop-
ulation (Pe
´rez-Garduza, 2013) was isolated from the rest
of the regional manatee population several decades ago.
Animals in IL were most likely come from nearby areas in
Tabasco, but their real origin is unknown. IL was not
considered a priori as a genetically isolated population
but spatially isolated. The second group of samples was
taken from manatees in areas where there are no fixed
barriers to displacement, so considered an OP (n¼26).
We assumed that animals from this OP can move and
breed freely along the study region.
Genomic DNA Extraction and
Microsatellite Genotyping
The genomic DNA was isolated using QIAGEN’S
DNeasy Blood and Tissue DNA Isolation kits
2Tropical Conservation Science
(Valencia, CA, USA). Polymerase chain reaction (PCR)
amplifications were performed for each sample using 28
microsatellite loci, previously designed for West Indian
manatees and described as polymorphic: TmaH13
(Nourisson et al., 2011; Pause et al., 2007), TmaE14
(Nourisson et al., 2011; Pause et al., 2007), and TmaSC5
(Pause et al., 2007). The annealing temperature (Tm)was
modified for the following microsatellites loci: TmaE02,
TmaE26, TmaM79 (59C; Garc
ıa-Rodr
ıguez, Moraga-
Amador, Farmerie, McGuire, & King, 2000; Nourisson
et al., 2011); TmaE11 (57C; Garc
ıa-Rodr
ıguez et al.,
2000; Nourisson et al., 2011); TmaE1 (54C; Pause
et al., 2007); TmaK01 (54C; Nourisson et al., 2011;
Pause et al., 2007); TmaE4 and TmaE07 (57C; Pause
et al., 2007); TmaJ02 (60C; Nourisson et al., 2011;
Pause et al., 2007); TmaKb60 (60C; Pause et al., 2007);
Tma-FWC01, Tma-FWC03, Tma-FWC04, Tma-FWC05,
Tma-FWC07, Tma-FWC09, Tma-FWC10, Tma-FWC11,
Tma-FWC12, Tma-FWC13, Tma-FWC15, Tma-FWC16,
Tma-FWC17, Tma-FWC18 (58C; Tringali et al., 2008);
and Tma-FWC14 (57C; Tringali et al., 2008).
Amplifications were performed in a T100 Thermal
Cycler (Biorad) or a MyCycler thermocycler (Biorad)
using the conditions as listed in the original publication
for each primer, with the exception of the Tm, indicat-
ed earlier.
Amplifications were performed in a total volume of
22 mL, with 20 ng template DNA, 15 mLPlatinum
V
R
PCR SuperMix (22 U/mL recombinant Taq DNA poly-
merase with PlatinumV
RTaq Antibody, 22 mM Tris-HCl
[pH 8.4], 55 mM KCl, 1.65 mM MgCl2, 220 mMdGTP,
220 mM dATP, 220 mMdTTP,220mM dCTP, and stabil-
izers; Invitrogen), and 200 nM per primer. To visualize
the PCR products, 2% agarose gel electrophoresis,
stained with ethidium bromide, was used (Barril &
Nates, 2012; Porras Murillo, Bola~
nos Montero, & Barr,
2008). Gels were photo-documented using Molecular
ImagerV
RGel Doc
TM
(Bio-Rad). The positive bands
were analyzed using the automatic gel documentation
and image analyzer UVband software (UVITEC, UK;
Yazdian-Robati et al., 2015). In a random subsample,
we verified number of alleles by automatic electrophoresis
(Experion-Bio-Rad) and we found the same pattern than
using conventional agarose gel electrophoresis. We com-
pared our results with those of Nourisson et al. (2011),
resulting in similar number of alleles. Noninformative loci
were eliminated for posterior analysis.
Data Analysis
Genetic structure. The software STRUCTURE v2.3.1
(Pritchard, Stephens, & Donnelly, 2000) was used to
Figure 1. Study area in Southern Gulf of Mexico. The points represent the sampling areas. (a) sampling region in Tabasco, Chiapas, and
Campeche, (b) lake of “Laguna de las Ilusiones,” IL, which is a lake currently landlocked with a small manatee population, around 260 ha in
extension. (c) The Gulf of Mexico region and location of samples in Veracruz, a small box shows extension of Figure 1(a).
G
omez-Carrasco et al. 3
identify the genetic subdivision of manatee populations
in Tabasco. The Admixture model was applied; the
number of populations (K) was set from 1 to 5 with a
burn-in period of 100,000 reiterations followed by
1,000,000 Markow chain Monte Carlo (MCMC) itera-
tions. The most probable number of populations, K,
was determined using STRUCTURE HARVESTER
(Earl & vonHoldt, 2011). The global values of genetic
differentiation F
ST
, as well pair wise F
ST
and R
ST
values,
were calculated with GENALEX 6.502 (Peakall &
Smouse, 2006) using an analysis of molecular variance.
To identify possible bottleneck events, the null hypoth-
esis was tested for excess of heterozygosity using
BOTTLENECK Software 1.2.02 (Cornuet & Luikart,
1996; Piry, Luikart, & Cornuet, 1999), applying the
Wilcoxon test for one tail under two models: stepwise
mutation model (SMM) and the two-phase mutation
model (TPM).
Genetic diversity. For genetic diversity, allelic richness
(N
A
), effective population size (N
E
), observed heterozy-
gosity (H
O
), expected heterozygosity (H
E
), inbreeding
coefficient (F
IS
), the deviation from Hardy–Weinberg
equilibrium, and linkage disequilibrium were estimated
using GENALEX 6.502 (Peakall & Smouse, 2006) and
GENEPOP 4.0.10 (Raymond & Rousset, 1995). The
presence of null alleles and the index of the polymorphic
content were estimated with CERVUS v. 3.0.7
(Kalinowski, Taper, & Marshall, 2007).
Results
All 45 samples from manatee skin tissue were used to
isolate genomic DNA. Each sample was successfully
amplified for 28 microsatellite loci and, from these, 67
alleles were obtained. Genetic diversity estimates
obtained with software GENALEX 6.502 and
GENEPOP 4.0.10 yield similar results. Therefore, we
only present the results obtained from GENALEX
6.502 (Table 1), because of the extended use of this pro-
gram in genetic diversity studies.
Genetic Structure
Two clusters (k¼2) were identified by the program
STRUCTURE 2.3.4 as the appropriate number of clus-
ters for assignment (Figure 2(a)). Figure 2(b) shows the
samples reassigned by Q from each cluster. Analysis of a
priori populations (IL and OP) indicate that the global
values of F
ST
and R
ST
(F
ST
¼0.049, R
ST
¼0.077) were
significant, as well as those which were analyzed by
STRUCTURE clusters, F
ST
¼0.071, R
ST
¼0.111, and
both indicated moderate differentiation among groups
(Frankham, Ballou, & Briscoe, 2004; Freeland, 2005).
A significant heterozygote excess was found in IL
(Wilcoxon signed rank test, P
TPM
¼0.006 and
P
SMM
¼0.031) and OP (P
TPM
and P
SMM
<0.001) on
the basis of mutational models. In IL, a strong normal
L-shaped distribution was fitted.
Genetic Diversity
Values of average number of different alleles (N
A
), aver-
age number of effective alleles (N
E
), observed heterozy-
gosity (H
O
), and expected heterozygosity (H
E
) estimated
by GENALEX 6.502 for each group and for the entire
region, are presented in Table 1. All microsatellite loci
were not significant for Hardy–Weinberg equilibrium.
No linkage disequilibrium was detected. The overall
inbreeding coefficient F
IS
¼0.013 was low but signifi-
cant. Error proportion was estimated by the verification
of 12% of samples using automatic electrophoresis and
no error was found. Null alleles were not observed in any
of the analyzed groups. Average polymorphism informa-
tion content (PIC) was 0.372, with locus TmaFWCL15
(Tringali et al., 2008) which had the higher PIC (0.560)
and locus TmaJ02 (Nourisson et al., 2011; Pause et al.,
2007) which had the lowest PIC (0.192).
Discussion
Manatees were historically harvested in the southern
Gulf of Mexico since pre-Hispanic times, but important
hunting began in the colony and continued into 1920s.
Table 1. Average Allelic Frequency and Standard Deviation by Population and Total.
nN
A
N
E
H
O
H
E
A priori groups
IL 19 2.21 0.09 1.71 0.08 0.50 0.06 0.38 0.03
OP 26 2.32 0.09 1.99 0.05 0.55 0.04 0.49 0.01
Global 45 2.27 0.06 1.85 0.05 0.52 0.04 0.43 0.02
STRUCTURE assigned groups
Group red 19 2.29 0.09 2.00 0.04 0.61 0.03 0.50 0.01
Group green 26 2.29 0.10 1.74 0.07 0.46 0.06 0.39 0.03
Global 45 2.29 0.07 1.87 0.05 0.54 0.04 0.44 0.02
n¼number of samples tested, N
A
¼average number of alleles, N
E
¼average number of effective alleles, H
O
¼observed
heterozygosity and H
E
¼expected heterozygosity.
4Tropical Conservation Science
Quantitative records of this hunting are not available in
any documents, although the dimension of this hunting
could be inferred because the first laws against commer-
cial use of manatees in Mexico did not exist until the
second decade of 20th century (Diario Oficial de la
Federaci
on, 1922). Current hunting is not significant
but still occurs in some inaccessible regions in Mexico
(L. D. Olivera-G
omez, 2008 personal communication).
Rovirosa (1885) mentions that manatees were abundant
in rivers and lakes of Tabasco. River runoff in this
regional coastal plain represents about 30% for all of
Mexico, thereby feeding a large and complex wetland
area and hydrological system. Different from other
Mexican states along the Gulf of Mexico (Veracruz,
Tamaulipas, and Campeche), Tabasco remained without
extensive agriculture or cattle farming until the middle of
the 20th century (Sa
´nchez-Mungu
ıa, 2005) which
brought relative protection from hunting of manatees
for food. However, Tabasco has focused in agriculture
growth since the 1940s leading to current deforestation
of 98% (Sa
´nchez-Mungu
ıa, 2005) of the original vegeta-
tion. Tabasco was also important for oil and gas extrac-
tion since the 1940s, both along the shore and inland at
areas associated with wetland productivity, which
caused heavy changes in land use and in the hydrological
drainage network.
Cold fronts north of Veracruz and Tamaulipas in
Mexico and in Texas in the USA could act as a barrier
of dispersion to and from northern Gulf of Mexico
resulting in movements of manatees favoring eastward
travel into the Yucatan Peninsula and along the
Caribbean coast. Nourisson et al. (2011) estimated lim-
ited genetic mixing with individuals from the Mexican
Caribbean population, resulting in the southern Gulf of
Mexico as a possible source of individuals to prevent
extensive inbreeding. Currently, the Antillean manatees
from Tabasco, in the southern Gulf of Mexico coastal
plain, is associated mainly with the Grijalva and
Usumacinta river basins (Colmenero & Hoz-Zavala,
1986; Jime
´nez-Dom
ınguez & Olivera-G
omez, 2014).
This region is thought to host the largest manatee pop-
ulation in Mexico (Marsh, O’Shea, & Reynolds III,
2011). Unfortunately, genetic diversity in this population
is lower when compared to that of the Caribbean coasts
of Mexico (Nourisson et al., 2011).
Genetic diversity increases the chance of populations
to adapt to environmental changes (Geffen, Luikart, &
Waples, 2007). In this study, the genetic diversity, esti-
mated on the heterozygosity and the number of alleles,
was low (Table 1). Table 2 presents a comparison of this
study with the results of previous works on West Indian
manatees. The average expected heterozygosity was
lower, although not significant, than that reported for
manatees in Belize (Hunter et al., 2010), for all of
Mexico (Nourisson, 2011), Florida (Pause Tucker
et al., 2012), and Puerto Rico (Hunter et al., 2012).
The expected heterozygosity in this study was similar
to the number estimated by Nourisson et al. (2011) for
the Gulf of Mexico, but lesser than what they estimated
for the Mexican Caribbean, however, despite the fact
that we used common loci, the total number of loci
detected was different. In general, manatee heterozygos-
ity had been lower than that from placental mammals
(Garner, Rachlow, & Hicks, 2005) and from threatened
mammals (DiBattista, 2008). The number of average
alleles (N
A
) observed was similar (Table 2), compared
with studies on Florida and Antillean manatees from
Belize and the Mexican Caribbean. In the Caribbean
coasts, it increases the chance to account for more alleles
shared with other Caribbean or Central America
Figure 2. Genetic groups estimated from a Bayesian assessment of genetic partition applying STRUCTURE. (a) Genetic identity for each
of the two populations: Cluster 1 (red) and Cluster 2 (green). (b) Graphical representation of K¼2 which assigned samples to each group;
the values were assigned by Q.
G
omez-Carrasco et al. 5
populations and even Florida. In the Gulf of Mexico,
however, the average number of alleles was similar
to that of the previous study (Nourisson et al., 2011).
By specific loci, we have the same number of alleles
at nine loci, and we have even more in some of the
others. Furthermore, low diversity is also reported
using mtDNA from manatees in Mexico (Casta~
neda &
Morales-Vela, 2005; Garc
ıa-Rodr
ıguez et al., 1998;
Vianna et al., 2006).
Low genetic diversity in the Gulf of Mexico could be
attributed to past bottleneck events even following his-
toric hunting, habitat loss (Lefebvre et al., 2001), and
low gene flow among populations (Nourisson et al.,
2011). This could also be observed in the global values
of F
IS
which imply a low but significant level of inbreed-
ing. The difference in H
E
from OP and IL was impor-
tant, approximately 0.1, considering that these are
animals from the same geographic area and the time of
isolation, which could be attributed to a founder effect,
or it also could be derived by the process of isolation in
the small population of IL. These isolated populations
present a greater risk of loss of genetic diversity, as well
as potential for extinction (Frankham, Bradshaw, &
Brook, 2014). Therefore, the management of manatees
in IL is critical and hence inbreeding is likely to be very
different from the rest of the region.
Examining the samples from IL and the OP, we iden-
tified a bottleneck effect in both groups, suggesting that
this effect in IL is derived from the founder individuals
that were introduced from the OP population that was
already under bottleneck effect as well. Nourisson et al.
(2011) also detected recent bottleneck events in manatees
from the Gulf of Mexico, and Hunter et al. (2012) dis-
covered a possible bottleneck for manatees examined
from Puerto Rico. Bottlenecks have been reported in
other mammal populations, such as Ursus arctos
(Xenikoudakis et al., 2015) and Panthera onca (Roques
et al., 2016), attributed to recent anthropogenic influen-
ces upon natural distribution and isolation events.
Contrastingly, Gonza
´lez-Sua
´rez, Aurioles-Gamboa,
and Gerber (2010) found no evidence of a bottleneck
in Zalophus californianus despite high hunting pressure
from the past. And most notable is the case of the ele-
phant seal where they recovered from a practically extinct
population from Isla Guadalupe, Baja California,
Mexico, to the current population of 225,000 animals,
with little evidence of loss of fitness (Abad
ıa-Cardoso,
Freimer, Deiner, & Garza, 2017). However, they sug-
gested that social structure, fitness, and population
dynamics could have affected their findings. Although
still under research, manatees in this region appear to
be associated with specific river-lake systems adjacent to
large rivers (Puc-Carrasco et al., 2017), where females
have discovered localities that offered food and shelter
to their calves and they continue to use those sites in a
fashion of temporal movements in and out of the lake
systems following flood pulses. This behavior is acting
against large-scale dispersal of individuals and promoting
regional bottlenecks that promote limited genetic flow.
The Grijalva and Usumacinta river basins are con-
nected to each other nearly 12 km from the coast line
forcing more interchange between individuals than they
would have in other river basins along the coastal plain.
In a healthy population, manatees need to travel along
the high energy coast, with limited submerged vegetation
because of plumes and siltation discharge of large
rivers. We identified two genetic clusters in our samples
(Figure 2) which indicate more structure in the region
when compared with Nourisson et al. (2011).
The few samples we had from Veracruz and
Campeche (n¼5) are from the red cluster, with ancestry
levels in STRUCTURE higher than 0.9. This genetic
cluster was also found in some samples from the most
coastal sites of Tabasco, but high ancestry of this cluster
is shown in individuals from Catazaja
´Lake in northern
Chiapas and one individual from the upper Usumacinta
River. In Las Ilusiones Lake, we found a mixed ancestry
in the individuals, which suggest that the previous trans-
location of individuals occurred with individuals from
several source populations.
There are just a few reports of sightings of manatees
along the coast in the southern Gulf of Mexico and came
from sparse opportunistic observations by local fisher-
men, park guards, or researchers working in other
Table 2. Comparison of the Total Allele Number, N
A
, in Different Studies of the Genetic Diversity of
West Indian Manatees Using Microsatellites.
Loci
microsatellites
N
A
average allele
number (range) Region Author
28 2.27 0.06 (2–4) Gulf of Mexico This article
16 3.1 (2–5) Belize Hunter et al. (2010)
13 2.62 0.24 Gulf of Mexico Nourisson et al. (2011)
3.00 0.32 Chetumal Bay
3.62 0.48 Florida
18 4.77 0.51 Florida Pause Tucker et al. (2012)
15 3.9 (2–6) Puerto Rico Hunter et al. (2012)
6Tropical Conservation Science
projects. A female manatee equipped with a satellite
transmitter and tagged in 2015 at the confluence of
Grijalva and Usumacinta river basins traveled about
50 km along the coast in a single night to other nearby
freshwater systems (L. D. Olivera-G
omez, personal
communication). In general, there are no geographical
barriers to dispersal of animals within the region of
southern Gulf of Mexico, but behavior could influence
individuals to be more concentrated in particular areas
within river basins (Puc-Carrasco et al., 2017). Hunter
et al. (2010) identified two genetically distinct groups of
manatees in Belize, where no visible barriers are present.
In killer whales, from northeastern Pacific, genetic stud-
ies clearly differentiated between two ecotypes, transi-
ents and residents, which have been separated by diet
and predatory behavior, even when they are currently
sympatric (Hoelzel, Dahlheim, & Stern, 1998; Morin
et al., 2010; Moura et al., 2015). In Florida, despite
having evidences to winter separate between manage-
ment units along the east and west coasts, genetic differ-
entiation was weak (Pause Tucker et al., 2012).
Implications for Conservation
The origin of individuals within the IL is not clear; if
they were occupying the lake when it was landlocked or
if they were introduced from other sites, the original
number of the population is unknown. The best popu-
lation estimate in this lake today produced a range of 18
to 39 individuals (Pe
´rez-Garduza, 2013). In IL, we iden-
tified slightly lower genetic diversity compared with OP,
which could be explained by isolation. The amount of
time of isolation in IL population is not known but esti-
mated to be only three to four decades (Pablo-Rodr
ıguez
& Olivera-G
omez, 2012). In the Florida subspecies
(T. manatus latirostris), the generation time is estimated
in a range of 16 to 23 years (Haubold, Deutsch, &
Fonnesbeck, 2006; Marmontel, O’Shea, Kochman, &
Humphrey, 1996), where it would not likely be enough
time to produce high levels of random genetic differen-
tiation. However, as we include samples from animals
from all ages, we have mixed generations in the study.
Without alleles from new individuals, and if habitat
quality decreased in this lake, genetic problems will be
more evident in the future. Therefore, based on our find-
ings, IL-specific management actions should be
addressed to assure survival and fitness of this small iso-
lated manatee population. The first step could be the
decision of keeping this isolated population as it current-
ly exists or translocate all the individuals to other areas.
However, the younger individuals, with the red cluster
genetic profile or with a higher number of alleles, could
be introduced into the lake system. For OP, a travel
corridor would help insure gene flow within river
basins and among other basins to protect the species
and promote increased genetic diversity. Access from
the sea coast to large river basins could be restored
with considerations to promote health genetic popula-
tion management and involve local human communities
in conservation actions.
Acknowledgments
The authors thank Dr Margaret Hunter of the USGS Wetland
and Aquatic Research Center in Gainesville, FL, for providing
us with manatee-specific primers used in this study. The
authors also thank Dr Michelle Davis for her advice and
help in the laboratory, as well as Dr Coralie Nourisson, for
her guidance for applying for financial support from the SMM.
Samples were collected under scientific collection permits
SGPA/DGVS/d0/06; SGPA/DGVS–01103/07; SGPA/DGVS/
00263/08; SGPA/DGVS/01754/09; SGPA/DGVS/04675/10;
SGPA/DGVS/02901/11; SGPA/DGVS/03562/12; SGPA/
DGVS/05846/13; SGPA/DGVS/11519/13; SGPA/DGVS/
00808/14. Any use of trade, product, or firm names is for
descriptive purposes only and does not imply endorsement by
the U.S. Government.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of
this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This study was funded by a grant from the Mexican Program for
Faculty Improvement (Programa de Mejoramiento del
Profesorado PROMEP). Project UJAT-CA-219. Financial sup-
port for training in Florida was provided by the Society for
Marine Mammalogy (SMM) to first author. The first author
wants to thank the Consejo Nacional para la Ciencia y la
Tecnolog
ıa (CONACYT; scholarship number: 399891) and to
PISA (UJAT) for the financial support during her Master’s
degree studies.
ORCID iD
Julia Mar
ıa Lesher-Gordillo http://orcid.org/0000-0001-
6973-2304
References
Abad
ıa-Cardoso, A., Freimer, N. B., Deiner, K., & Garza,
J. C. (2017). Molecular population genetics of the
Northern Elephant Seal Mirounga angustirostris. Journal
of Heredity,108(6), 618–627.
Arag
on-Mart
ınez, A., Olivera-G
omez, L. D., & Jimenez-
Dominguez, D. (2014). Seasonal prevalence of antibodies
to Leptospira interrogans in Antillean manatees from a
landlocked lake in Tabasco, Me
´xico. Journal of Wildlife
Diseases,50(3), 505–511.
G
omez-Carrasco et al. 7
Barril, P., & Nates, S. (2012). Introduction to agarose and
polyacrylamide gel electrophoresis matrices with respect to
their detection sensitivities. S. Magdeldin (Ed.), Gel electro-
phoresis principles and basic (pp. 3–14). Croatia: InTech.
Bonde, R. K., McGuire, P. M., & Hunter, M. E. (2012). A
review of the key genetic tools to assist imperiled species
conservation: Analyzing West Indian manatee populations.
Journal of Marine Animals and Their Ecology,5(1), 8–19.
Casta~
neda, A., & Morales-Vela, B. (2005). Informe te
´cnico sobre
los ana
´lisis de DNA de las muestras de sangre de los manat
ıes
de Quintana Roo, Tabasco y Campeche [Technical report on
the DNA analysis of the blood samples of the manatees of
Quintana Roo, Tabasco and Campeche] (ECOSUR,
Chetumal). Retrieved from http://www.conabio.gob.mx/insti
tucion/proyectos/resultados/InfH164.pdf.pdf
Colmenero, L. C., & Hoz-Zavala, M. E. (1986). Distribuci
on
de los manat
ıes, situaci
on y su conservaci
on en Me
´xico
[Distribution of manatees, situation and their conservation
in Mexico]. Serie Zoolog
ıa,56, 955–1020.
Cornuet, J. M., & Luikart, G. (1996). Description and power
analysis of two tests for inferring recent population bottle-
necks from allele frequency data. Genetics,144, 2001–2014.
Diario Oficial de la Federaci
on. (1922). Diario Oficial de la
Federaci
on 19 de enero de 1922, Secretar
ıa de Agricultura y
Fomento: PREVENCIONES reglamentarias estableciendo
veda absoluta para la pesca del “manat
ı”, en aguas federales
[Official Gazette of the Federation January 19, 1922,
Ministry of Agriculture and Development:
REGULATORY PREVENTIONS establishing an absolute
ban for the fishing of the “manatee” in federal waters] (p.
280). Ciudad de Me
´xico, Me
´xico.
DiBattista, J. D. (2008). Patterns of genetic variation in
anthropogenically impacted populations. Conservation
Genetics,9, 141–156.
Earl, D. A., & vonHoldt, B. M. (2011). STRUCTURE
HARVESTER: A website and program for visualizing
STRUCTURE output and implementing the Evanno
method. Conservation Genetic Resources,4, 359–361.
Frankham, R., Ballou, J., & Briscoe, D. (2004). Introduction to
conservation genetics. Edn. Cambridge, England:
Cambridge University Press.
Frankham, R., Bradshaw, C. J. A., & Brook, B. W. (2014).
Genetics in conservation management: Revised recommen-
dations for the 50/500 rules, Red List criteria and popula-
tion viability analyses. Biological Conservation,17, 56–63.
Freeland, J. R. (2005). Molecular ecology (402 pp.). West
Sussex, England: John Wiley & Sons Ltd.
Garc
ıa-Rodr
ıguez, A. I., Bowen, B. W., Domning, D.,
Mignucci-Giannoni, A. A., Marmontel, M., Montoya-
Ospina, R. A.,... McGuire, P. M. (1998). Phylogeography
of the West Indian manatee (Trichechus manatus): How
many populations and how many taxa? Molecular
Ecology,7(9), 1137–1149.
Garc
ıa-Rodr
ıguez, A. I., Moraga-Amador, D., Farmerie, W.,
McGuire, P., & King, T. L. (2000). Isolation and
characterization of microsatellite DNA markers in the
Florida manatee (Trichechus manatus latirostris) and their
application in selected Sirenian species. Molecular Ecology,
9, 2161–2163.
Garner, A., Rachlow, J. L., & Hicks, J. F. (2005). Patterns of
genetic diversity and its loss in mammalian populations.
Conservation Biology,19, 1215–1221.
Geffen, E., Luikart, G., & Waples, R. (2007). Impacts of
modern molecular genetic techniques on conservation biology.
Retrieved from https://digitalcommons.unl.edu/cgi/viewcon
tent.cgi?referer¼https://scholar.google.com.mx/&httpsredir
¼1&article¼1460&context¼usdeptcommercepub
Gonza
´lez-Sua
´rez, M., Aurioles-Gamboa, D., & Gerber, L. R.
(2010). Past exploitation of California sea lions did not lead
to a genetic bottleneck in the Gulf of California. Ciencias
Marinas,36(2), 199–211.
Haubold, E. M., Deutsch, C., & Fonnesbeck, C. (2006). Final
biological status review of the Florida manatee (Trichechus
manatus latirostris). Florida Fish and Wildlife Conservation
Commission. St. Petersburg, Florida,63, 2–63.
Hoelzel, A. R., Dahlheim, M., & Stern, S. J. (1998). Low genet-
ic variation among killer whales (Orcinus orca) in the east-
ern North Pacific and genetic differentiation between
foraging specialists. Journal of Heredity,89(2), 121–128.
Hoelzer, G. A., Wallman, J., & Melnick, D. J. (1998). The
effects of social structure, geographical structure, and pop-
ulation size on the evolution of mitochondrial DNA: II.
Molecular clocks and the lineage sorting period. Journal
of Molecular Evolution,47(1), 21–31.
Hunter, M. E., Auil-G
omez, N. E., Tucker, K. P., Bonde,
R. K., Powell, J., & McGuire, P. M. (2010). Low genetic
variation and evidence of limited dispersal in the regionally
important Belize manatee. Animal Conservation,
13, 592–602.
Hunter, M. E., Mignucci-Giannoni, A. A., Tucker, K. P.,
King, T. L., Bonde, R. K., Gray, B. A., & McGuire,
P. M. (2012). Puerto Rico and Florida manatees represent
genetically distinct groups. Conservation Genetic,
13(6), 1623–1635.
Jime
´nez-Dom
ınguez, D., & Olivera-G
omez, L. D. (2014).
Caracter
ısticas del ha
´bitat del Manat
ı antillano
(Trichechus manatus manatus) en sistemas fluviolagunares
del sur del Golfo de Me
´xico [Habitat characteristics of the
Antillean manatee (Trichechus manatus manatus) in fluvio-
lagunares systems of the southern Gulf of Mexico].
THERYA,5(2), 601–614.
Kalinowski, S. T., Taper, M. L., & Marshall, T. C. (2007).
Revising how the computer program CERVUS accommo-
dates genotyping error increases success in paternity assign-
ment. Molecular Ecology,16, 1099–1106.
Lefebvre, L. W., Marmontel, M., Reid, J. P., Rathbun, G. B.,
& Domning, D. P. (2001). Distribution, status, and biogeog-
raphy of the West Indian manatee. In C. A. Woods & F. E.
Sergile (Eds.), Biogeography of the West Indies: Patterns and
8Tropical Conservation Science
perspectives (2nd ed., pp. 425–474). Boca Raton, FL:
CRC Press.
Marmontel, M., O’Shea, T. J., Kochman, H. I., & Humphrey,
S. R. (1996). Age determination in manatees using growth-
layer-group counts in bone. Marine Mammal Science,
12, 54–88.
Marsh, H., O’Shea, T. J., & Reynolds, J. E. III. (2011). Ecology
and conservation of the Sirenia: Dugongs and Manatees (521
pp.). Cambridge, MA: Cambridge University Press.
Morin, P. A., Archer, F. I., Foote, A. D., Vilstrup, J., Allen,
E. E., Wade, P., & Harkins, T. (2010). Complete mitochon-
drial genome phylogeographic analysis of killer whales
(Orcinus orca) indicates multiple species. Genome
Research,20(7), 908–916.
Moura, A. E., Kenny, J. G., Chaudhuri, R. R., Hughes, M. A.,
Reisinger, R. R., De Bruyn, P. J. N., & Hoelzel, A. R.
(2015). Phylogenomics of the killer whale indicates ecotype
divergence in sympatry. Heredity,114(1), 48–55.
Nourisson, C., Morales-Vela, B., Padilla-Saldivar, J., Pause-
Tucker, K., Clark, A. M., Olivera-G
omez, L. D., ...
McGuire, P. (2011). Evidence of two genetic clusters of
manatees with low genetic diversity in Mexico and implica-
tions for their conservation. Gene
´tica,139, 833–842.
Nourisson, C. (2011). Estructura Gene
´tica de los manat
ıes en
Me
´xico (Tesis de Doctorado, 124 pp.). El Colegio de
Frontera Sur, Chetumal, Quintana Roo, Me
´xico.
Retrieved from https://ecosur.repositorioinstitucional.mx/
jspui/bitstream/1017/660/1/0000507881_documento.pdf
Pablo-Rodr
ıguez, N., & Olivera-G
omez, L. D. (2012).
Situaci
on de una poblaci
on aislada de manat
ıes Trichehus
manatus (Mammalia: Sirenia: Trichechidae) y conocimiento
de la gente, en una Laguna Urbana, en Tabasco, Me
´xico
[Situation of an isolated population of manatees Trichehus
manatus (Mammalia: Sirenia: Trichechidae) and knowledge
of the people, in a Laguna Urbana, in Tabasco, Mexico].
Universidad y Ciencia,28(1), 15–26.
Pause, K. C., Nourisson, C., Clark, A., Kelloggs, M. E.,
Bonde, R. K., & McGuire, P. M. (2007). Polymorphic
microsatellite DNA markers for the Florida manatee
(Trichechus manatus latirostris). Molecular Ecology Notes,
7, 1073–1076.
Pause Tucker, K., Hunter, M. E., Bonde, R. K., Austin, J. D.,
Clark, A. M., Beck, C. A., . . . Oli, M. K. (2012). Low
genetic diversity and minimal population substructure in
the endangered Florida manatee: Implications for conser-
vation. Journal of Mammalogy,93(6), 1504–1511.
Peakall, R., & Smouse, P. E. (2006). GENALEX 6: Genetic
analysis in excel. Population genetic software for teaching
and research. Molecular Ecology Notes,6, 288–295.
Pe
´rez-Garduza, F. (2013). Evaluaci
on de la eficiencia de cinco
te
´cnicas para determinar la abundancia de manat
ıes
Trichechus manatus en ambientes poco visibles [Evaluation
of the efficiency of five techniques to determine the abun-
dance of manatees Trichechus manatus in inconspicuous
environments] (MSc Thesis). Universidad Jua
´rez Aut
onoma
de Tabasco, Me
´xico, 45 pp. Available with the author.
Piry, S., Luikart, G., & Cornuet, J. M. (1999).
BOTTLENECK: A program for detecting recent effective
population size reductions from allele data frequencies.
Journal of Heredity,90, 502–503.
Pritchard, J. K., Stephens, M., & Donnelly, P. (2000).
Inference of population structure using multilocus genotype
data. Genetics,155(2), 945–959.
Porras Murillo, L. P., Bola~
nos Montero, J. R., & Barr, B. R.
(2008). Variaci
on gene
´tica y flujo de genes entre poblaciones
de Crocodylus acutus (Crocodylia: Crocodylidae) en tres
r
ıos del Pac
ıfico Central Costa Rica. Revista De Biolog
ıa
Tropical,56(3), 1471–1480.
Puc-Carrasco, G., Olivera-G
omez, L. D., Arriaga-Hernandez,
S., & Jimenez-Dominguez, D. (2016). Relative abundance
of Antillean manatees in the Pantanos de Centla Biosphere
Reserve in the coastal plain of Tabasco, Mexico. Ciencias
Marinas,42(4), 261–270.
Puc-Carrasco, G., Morales-Vela, B., Olivera-G
omez, L. D., &
Gonza
´lez-Solis, D. (2017). First field-based estimate of
Antillean manatee abundance in the San Pedro River
system suggests large errors in current estimates for
Mexico. Ciencias Marinas,43(4), 285–299.
Raymond, M., & Rousset, F. (1995). GENEPOP (version 1.2):
Population genetics software for exact tests and ecumeni-
cism. Journal of Heredity,86, 248–249.
Roques, S., Sollman, R., Ja
´como, A., Torres, N., Silveira, L.,
Cha
´vez, C., . . . Palomares, F. (2016). Effects of habitat
deterioration on the population genetics and conservation
of the jaguar. Conservation Genetics,17, 125–139.
Rovirosa, J. N. (1885). Apuntes para la zoolog
ıa de Tabasco.
Vertebrados observados en el territorio de Macuspana
[Notes for the zoology of Tabasco. Vertebrates observed
in the territory of Macuspana]. La Naturaleza,7, 345–389.
Sa
´nchez-Mungu
ıa, A. (2005). Uso del suelo agropecuario y
deforestaci
on en Tabasco 1950-2000 (123 pp.).
Villahermosa, Mexico: Divisi
on Acade
´mica de Ciencias
Biol
ogicas. Universidad Jua
´rez Aut
onoma de Tabasco.
Self-Sullivan, C., & Mignucci-Giannoni, A. (2008). Trichechus
manatus ssp. manatus.The IUCN Red List of Threatened
Species 2008:e.T22105A359161. Retrieved from http://dx.
doi.org/10.2305/IUCN.UK.2008.RLTS.T22105A93
59161.en.
Secretar
ıa de Medio Naturales Ambiente y Recursos. (2010).
Norma Oficial Mexicana 059, Protecci
on ambiental Especies
nativas de Me
´xico de flora y fauna silvestres-Categor
ıas de
riesgo y especificaciones para su inclusi
on, exclusi
on o
cambio-Lista de especies en riesgo [Official Mexican
Standard 059, Environmental protection Mexican native
species of wild flora and fauna-Risk categories and specifi-
cations for inclusion, exclusion or change-List of species at
risk]. Retrieved from http://www.profepa.gob.mx/innova
portal/file/435/1/NOM_059_SEMARNAT_2010.pdf
G
omez-Carrasco et al. 9
Tringali, M. D., Seyoum, S., Carney, S. L., Davis, M. C.,
Rodr
ıguez-L
opez, M. A., Reynolds, J. E., & Haubold, E.
(2008). Eighteen new polymorphic microsatellite markers
for the endangered Florida manatee, Trichechus manatus
latirostris.Molecular Ecology Resources,8(2), 328–331.
United Nations Environment Programme. (2010). Regional
Management Plan for the West Indian Manatee
(Trichechus manatus) (United Nations Environment
Program, CEP Technical Report 48). Kingston, Jamaica.
Retrieved from https://www.fws.gov/caribbean/PDF/
ManateeManagementPlan.pdf
Vianna, J. A., Bonde, R. K., Caballero, S., Giraldo, J. P.,
Lima, R. P., Clark, A., . . . Santos, F. R. (2006).
Phylogeography, phylogeny and hybridization in trichechid
sirenians: Implications for manatee conservation. Molecular
Ecology,15(2), 433–447.
Xenikoudakis, G., Ersmark, E., Tison, J. L., Waits, L.,
Kindberg, J., Swenson, J. E., & Dalen, L. (2015).
Consequences of a demographic bottleneck on genetic
structure and variation in the Scandinavian brown bear.
Molecular Ecology,24, 3441–3454.
Yazdian-Robati, R., Pourtaji, A., Rashedinia, M.,
Hosseinzadeh, H., Ghorbani, M., Razavi, B. M., &
Abnous, K. (2015). Screening and identification of SUMP-
proteins in sub-acute treatment with diazinon. Iranian
Journal of Basic Medical Sciences,18(12), 1240–1244.
10 Tropical Conservation Science
