Low genetic variation and evidence of limited dispersal in the regionally important Belize manatee

Low genetic variation and evidence of limited dispersal inthe regionally important Belize manateeM. E. Hunter1,2, N. E. Auil-Gomez3,4, K. P. Tucker5, R. K. Bonde1,2, J. Powell4 & P. M. McGuire2

1 Sirenia Project, Southeast Ecological Science Center, U.S. Geological Survey, Gainesville, FL, USA

2 Department of Physiological Sciences, College of Veterinary Medicine, University of Florida,Gainesville, FL, USA

3 Wildlife Trust, Belize City, Belize

4 Sea to Shore Alliance, St Petersburg, FL, USA

5 College of marine Science, University of South Florida, St. Petersburg, FL, USA

Keywords

conservation genetics; low diversity;

microsatellite; mitochondria; marine

mammal; West Indian manatee.

Correspondence

Margaret E. Hunter, Sirenia Project,

Southeast Ecological Science Center, US

Geological Survey, 2201 NW 40th Terrace,

Gainesville, FL 32605, USA. Tel: +1 352

264 3484; Fax: +1 352 378 4956

Email: [email protected]

Received 26 October 2009; accepted 13 June

2010

doi:10.1111/j.1469-1795.2010.00383.x

Abstract

The Antillean subspecies of the West Indian manatee Trichechus manatus is foundthroughout Central and South America and the Caribbean. Because of severehunting pressure during the 17th through 19th centuries, only small populations ofthe once widespread aquatic mammal remain. Fortunately, protections in Belizereduced hunting in the 1930s and allowed the country’s manatee population tobecome the largest breeding population in the Wider Caribbean. However,increasing and emerging anthropogenic threats such as coastal development,pollution, watercraft collision and net entanglement represent challenges to thisecologically important population. To inform conservation and managementdecisions, a comprehensive molecular investigation of the genetic diversity,relatedness and population structure of the Belize manatee population wasconducted using mitochondrial and microsatellite DNA. Compared with othermammal populations, a low degree of genetic diversity was detected (HE=0.455;NA=3.4), corresponding to the small population size and long-term exploitation.Manatees from the Belize City Cayes and Southern Lagoon system were geneti-cally different, with microsatellite and mitochondrial FST values of 0.029 and0.078, respectively (P ! 0.05). This, along with the distinct habitats and threats,indicates that separate protection of these two groups would best preserve theregion’s diversity. The Belize population and Florida subspecies appear to beunrelated with microsatellite and mitochondrial FST values of 0.141 and 0.63,respectively (P ! 0.001), supporting the subspecies designations and suggestinglow vagility throughout the northern Caribbean habitat. Further monitoring andprotection may allow an increase in the Belize manatee genetic diversity andpopulation size. A large and expanding Belize population could potential assist inthe recovery of other threatened or functionally extinct Central AmericanAntillean manatee populations.

Introduction

The West Indian manatee Trichechus manatus is a threatenedaquatic mammal found throughout the south-eastern UnitedStates, Central and South America and the Caribbean. TheFlorida manatee Trichechus manatus latirostris and Antilleanmanatee Trichechus manatus manatus are the two recognizedsubspecies of the West Indian manatee. A low reproductiverate, environmental impacts and direct threats from the hu-man population have historically limited manatee populationgrowth. In 1982, the International Union for Conservation ofNature classified all West Indian manatee populations asvulnerable to extinction (IUCN, 2007).

During the 17th through 19th centuries, the Spaniards inBelize and throughout the region severely exploited the

Antillean subspecies for sustenance (Lefebvre et al., 2001).By 1936, the population decline was so severe in Belize thatManatee Protection Ordinances were introduced to preservethe population (McCarthy, 1986). Today, the Belize mana-tee is listed as endangered by the Belize Wildlife ProtectionAct of 1981, Part II, Section 3(a) (Auil, 1998, 2004). AManatee Recovery Plan was also proposed requestinginformation on habitat use and movement patterns to aidthe development of conservation policies for the protectionof the Belize manatee (Auil, 1998).

While studying Caribbean manatee populations, O’Shea& Salisbury (1991) concluded that ‘Belize remains one of thelast strongholds for the species in this part of the world.’From 1977 to 1991, the Belize manatee population sizeappeared stable, however, an overall negative trend in

Animal Conservation ]] (2010) 1–11 c" 2010 The Authors. Journal compilation c" 2010 The Zoological Society of London 1

Animal Conservation. Print ISSN 1367-9430

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numbers was detected from 1997 to 2002 (Auil, 2004).Although today Belize is considered the largest breedingAntillean population, it is still under pressure from anthro-pogenic threats and the census size of #1000 individuals iswell below that recommended for the long-term geneticsustainability of a population (Frankham, Ballou & Briscoe,2002; Quintana-Rizzo & Reynolds III, 2007).

Little is known of the abundance or distribution ofmanatees in the other Central American populations,although they are considered elusive and thought to be rare(Quintana-Rizzo & Reynolds III, 2007). However, supple-mentation from Belize or Mexico manatees in concert withcoordinated regional efforts could potentially assist in therecovery of the Guatemala populations and those furtherthe south. The large Mexico population is also protected

and manatees fromMexico have been documented travelingto Belize and even participating in a mating herd (Auil et al.,2007; Morales-Vela et al., 2007). Quintana-Rizzo & Rey-nolds III (2007) suggest that safeguarding the Belize andMexico populations as a resource of regional importanceshould be a high conservation priority.

Long-term exploitation and small population sizescan lower genetic diversity and decrease fitness-relatedtraits, such as fecundity and survival (Hoelzel et al., 1993;Frankham et al., 2002; Dixon et al., 2007). Genetic varia-tion often acts in concert with demographic stochasticity insmall populations. Loss of diversity can ultimately lead toan ‘extinction vortex,’ or a cyclic reduction in populationsize, resulting in loss of the population (Gilpin & Soule,1986).

Figure 1 Geographic map of Belize with the

three study sites inset, Belize City Cayes

(north), Northern & Southern Lagoons (cen-

ter), and Placencia Lagoon (south).

Animal Conservation ]] (2010) 1–11 c" 2010 The Authors. Journal compilation c" 2010 The Zoological Society of London2

Reduced Belize manatee dispersal and genetic variation M. E. Hunter et al.

A previous mitochondrial DNA (mtDNA) study identifiedthree haplotypes (A03, A04 and J01) in 43 animals in Belize(Vianna et al., 2006). The resultant haplotype parametersincluded 28 polymorphic sites and nucleotide substitutionswith h=0.558 and p=0.037. The distribution and geneticstructure of the mtDNA haplotypes was not addressed, butcould provide valuable information on the population struc-ture, especially coupled with biparentally inherited nucleardata. Presented here are mitochondrial and nuclear micro-satellite DNA studies addressing the genetic diversity,relatedness and population structure in Belize manatees.The Antillean Belize population is also compared withT. m. latirostris to provide information on the extent of WestIndian manatee migration and relatedness.

Materials and methods

Sample collection and DNA extraction

Manatee epidermis tissue and/or white blood cells werecollected from recovered carcasses or during wild manateehealth assessments in Belize from 1997 to 2007. Geneticsamples were collected from a total of 118 unique animalsin 213 health assessments (50% female and 50% male; Auilet al., 2007) and 12 carcass samples. Genomic DNA wasisolated using Qiagen’s DNeasy blood and tissue kits(Valencia, CA, USA). Within the Florida dataset, 96 geno-types were randomly chosen, proportionally representingthe four demographically defined management units(K.P. Tucker et al., unpubl. data).

Study locations

The Belize City Cayes (BCC), including the near shore andoutlying cayes, are utilized by the tourist industry and experi-ence heavy boat traffic and recreational activities from BelizeCity, the largest port in the country (Fig. 1). Manatee tours arelargely conducted around Swallow Caye within the BCC.

The Southern Lagoon system (SLS) consists of the South-ern and Northern Lagoons and has the highest recordednumber of manatees, with 55 individuals sighted in a singleaerial survey (O’Shea & Salisbury, 1991). While the popula-tion is not heavily affected by watercraft, it is potentiallyimpacted by salinity changes due to increased rainfall,influencing the abundance of aquatic vegetation, the pri-mary food source for manatees.

Placencia Lagoon (PL) is located 75miles south of theSLS (Fig. 1). Placencia Village is a popular tourist destina-tion, with a large amount of coastal development andagricultural runoff severely altering the environment andaquatic vegetation. Nitrification from shrimp farm effluentcauses extensive algae blooms that can compromise thequality and growth of seagrass (Thornton, Shanahan &Shanahan, 2003). Nitrification also provides a food sourcefor a marine snail, thought to be an intermediate host of arespiratory fluke Pulmonicola cochleotrema (Blair, 2005),which is known to parasitize manatee lungs (Beck &Forrester, 1988). Manatees in PL present at necropsy with

large loads of P. cochleotrema and copious mucoid dis-charge that could hinder respiration (Auil et al., 2007).

The carcass samples utilized here were recovered fromCorazol (n=1), Stan Creek (n=1), the Belize River mouthin Belize City (an area of high watercraft activity), or south ofthe river, possibly caught in the ocean current (n=10). Thenorthern most manatee reserve in Belize is a section ofChetumal Bay, which continues intoMexico (Fig. 1). Samplescollected in Chetumal bay are not included in this study.

Laboratory procedures

Mitochondrial DNA analysis

Primers CR-4 and CR-5 from Garcıa-Rodrıguez et al.(1998) amplified a 410 bp portion of the mtDNA controlregion displacement loop for 113 individuals; 101 livecaptures and 12 carcasses. All PCR amplifications werecarried out on a PTC-200 thermal cycler (MJ Research,Waltham, MA, USA). The PCR reaction conditions fol-lowed Kellogg (2008). Representatives from each haplotypeand any ambiguous sequences were sequenced in bothdirections to ensure the accuracy of nucleotide designations.

Microsatellite DNA analysis

A panel of 16 polymorphic microsatellite primers (Garcıa-Rodrıguez et al., 2000; Pause et al., 2007) was PCR ampli-fied using 118 individuals. The study included 88 individualsfrom the SLS system (12 Northern Lagoon and 76 SouthernLagoon), 21 from BCC, two from PL and seven amplifiablecarcass samples. PCR conditions followed Kellogg (2008;Table 1). MgCl2 concentrations were 3mM, except forTmaH13, TmaKb60 and TmaSC5, which required 2mM.Fragment analysis was performed on an Applied Biosystems(Foster City, CA, USA) ABI 3730 Genetic Analyzer. Allindividuals amplified at 14 or more loci. The Florida datawere kindly provided for use in this paper (K.P. Tuckeret al., unpubl. data).

Statistical analysis


The degree of differentiation, FST and FST, between the BCCand SLS and between Belize and Florida was calculated usingARLEQUIN 3.1 (Excoffier, Laval & Schneider, 2005). Compar-isons with PLwere not conducted due to the small sample size.Estimates of sequence divergence used the Kimura two-parameter genetic distance model (Kimura, 1980; Jin & Nei,1990). Lastly, Tajima’s D of selective neutrality, geneticdiversity (h), nucleotide diversity (p), number of polymorphicsites (S) and number of nucleotide substitutions (NS) werecalculated (Nei, 1987; Tajima, 1993).


The level of polymorphism was estimated by the observed(HO) and expected heterozygosity (HE) and the average


Reduced Belize manatee dispersal and genetic variationM. E. Hunter et al.

number of alleles per locus (NA) using GenAlEx 6.2 (Peakall& Smouse, 2006). Departures from linkage disequilibriumand Hardy–Weinberg equilibrium (HWE) were tested (de-memorization 10 000, batches 100, iterations per batch5000) in GENEPOP 4.0 (Raymond & Rousset, 1995) andBonferroni’s corrections were applied for multiple compar-isons. To assess overall genetic differentiation at the popula-tion level, GenAlEx 6.2 calculated FST using the infinitealleles model and RST using the stepwise mutation modelthrough an AMOVA. Comparisons included SLS, BCC andrecovered carcasses, and Belize and Florida.

GENECAP (Wilberg & Dreher, 2004) calculated the unbiasedprobability of identity (PID), which is the probability that twoindividuals drawn at random from a population will have thesame genotype at the assessed loci (Paetkau & Strobeck, 1994)and P(ID)sib, a related more conservative statistic for calculat-ing PID among siblings (Evett & Weir, 1998). The programadditionally searched for duplicate genotypes. GenAlEx 6.2was used to estimate the shadow effect and P(ID)observed todetermine whether unbiased P(ID) or P(ID)sib is the moreaccurate probability (Mills et al., 2000; Waits, Luikart &Taberlet, 2001) following Hunter et al. (2010).

The relationship between genetic and geographic distancewas evaluated to test for isolation by distance within Belizeand for Belize and Florida based on 10 000 randomizationsin GenAlEx 6.2. BOTTLENECK 1.2.02 evaluated heterozygoteexcess under the two-phased model using theWilcoxon sign-ranked test (Piry, Luikart & Cornuet, 1999). The M-ratiotest used M_P_Val.ex to measure the proportion of unoccu-pied allelic states, which is lowered during a populationreduction (Garza & Williamson, 2001). Input values in-cluded a mutation rate of 5$ 10%4 and y=0.274; and therecommended values of Dg=3.5 and ps=0.9. Datasetsusing seven or more loci and the appropriate mutationmodel can be assumed to have experienced a reduction in

population size with an Mo0.68. The coefficient of geneticrelatedness, rxy (Queller & Goodnight, 1989), was used totest the genetic variability within Belize using 95% confi-dence intervals in GenAlEx 6.2. Intra-populational related-ness should be significantly higher in populations that haveundergone bottlenecks or founding events.

To test the distribution of rxy between all pairs ofindividuals, KINGROUP 2 simulated expected unrelated andfull-sib pairwise relatedness (10 000 pairs) from observedallele frequencies and the rxy values were plotted to comparethe distribution (Konovalov, Manning & Manning, 2004).A deviation from random expectation could result fromnonrandom mating among related individuals and/or over-representation from a few families, suggesting an excess ofinbred individuals in the population.

The program STRUCTURE 2.3.2 was used to identify thegenetic subdivision within Belize and the genetic relation-ship and ancestral source populations of Belize and Floridamanatees (Pritchard, Stephens & Donnelly, 2000). The mostprobable number of populations, K, was determined byevaluating the likelihood of the posterior probability [L(K);Pritchard, Wen & Falush, 2007] and by calculating DK, anad hoc quantity related to the change in posterior probabil-ities between runs of different K values (Evanno, Regnaut &Goudet, 2005) in STRUCTURE HARVESTER (Earl, 2009). Simu-lations were conducted using the admixture model ofK=1–10 with 10 000 repetitions of MCMC, following theburn-in period of 10 000 iterations with and without a priori‘population’ information using the admixture model. TheLOCPRIOR setting was used to detect cryptic structure byproviding priors for the Bayesian assignment process basedon the sample location. The LOCPRIOR model allows struc-ture to be detected with lower levels of divergence and is notbiased towards detecting structure when it is not present(Hubisz et al., 2009). Individual assignment success was

Table 1 Characteristics and PCR conditions for the 16 microsatellite loci implemented in the Belize manatee samples

Locus name Tm ( 1C) BSA NA HO HE PIC NE

TmaA02 56 % 2 0.310 0.349 0.534 1.537

TmaE1 60 + 4 0.615 0.619 1.121 2.622

TmaE02 55 % 2 0.265 0.300 0.477 1.429

TmaE7 62 + 4 0.299 0.323 0.627 1.477

TmaE08 58 % 3 0.701 0.657 1.084 2.917

TmaE11 56 % 5 0.761 0.757 1.488 4.123

TmaE14 62 + 4 0.564 0.573 1.035 2.340

TmaE26 60 % 4 0.381 0.341 0.622 1.518

TmaF14 58 % 2 0.390 0.403 0.593 1.675

TmaH13 58 + 2 0.359 0.355 0.540 1.550

TmaJ02 60 % 2 0.504 0.482 0.675 1.932

TmaK01 54 % 2 0.530 0.390 0.578 1.638

TmaKb60 56 % 5 0.416 0.483 0.782 1.935

TmaM79 56 + 3 0.627 0.593 0.974 2.454

TmaSC5 58 % 3 0.410 0.366 0.663 1.577

TmaSC13 58 % 2 0.162 0.230 0.391 1.298

Mean % % 3.1 0.456 0.451 0.762 2.001

The optimized annealing temperature (Tm), BSA requirement of 0.4mgmL%1, number of alleles (NA), the observed and expected heterozygosity

(HO and HE), polymorphic information content (PIC), and effective number of alleles (NE).



recorded as the likelihood of assignment (Q). Individualswere assigned to a cluster with QZ60%.

Results


The Belize samples contained three haplotypes (A03, A04 andJ01). The BCC samples were comprised of the A04 (n=4) andJ01 (n=15) haplotypes. The SLS individuals contained theA04(n=52), J01 (n=22) and A03 (n=6) haplotypes. A04 (n=1)and J01 (n=1) were identified in the PL samples. Recoveredcarcasses contained the A04 (n=6) and J01 (n=6) haplotypes.MtDNA sequence divergence estimates were h=0.534& 0.025and p=0.031& 0.015.Within Belize, Tajima’sDwas large andpositive (D=4.118) consistent with a population bottleneck,however it was not significant (Po1.000); therefore thenull hypothesis of selective neutrality cannot be rejected.Genetic differentiation estimates between BCC and SLSwere FST=0.078 (Po0.045& 0.015) and FST=%0.036(Po0.712& 0.052). Twenty-eight variable sites and nucleotidesubstitutions (6.8%) were identified in the three haplotypes.Within Florida, one haplotype was observed (A01, p=0.000;unpubl. data; Garcıa-Rodrıguez et al., 1998). Belize andFloridamtDNA sequence divergence estimates were FST=0.626(Po0.00001& 0.0) andFST=0.302 (Po0.00001& 0.0).


The 16 nuclear microsatellite markers had lower levels ofvariation [HE=0.453 (0.230–0.757); HO=0.456 (0.162–0.761), NA=3.1 (2–5); Table 1] than the Florida populationover 14 loci (K.P. Tucker et al., unpubl. data). In Belize,TmaKb60 and TmaSC13 had evidence of null alleles due to aheterozyogote deficiency. Deviation from HWE was foundfor TmaKb60 and TmaK01, even after a sequential Bonfer-roni adjustment (Po0.001). After 120 comparisons, linkagedisequilibrium was observed between TmaE1 and TmaE14

(Po0.05). FIS was 0.012 overall, suggesting slight inbreed-ing in the population. The error rate was determined byre-genotyping 11% of the individuals; no errors or duplicategenotypes were detected.

Genetic differentiation between the BCC, SLS and recov-ered carcass samples was significant (Po0.05), although low(Table 2). The majority of private alleles were detected inSLS (n=5) as compared with BCC (n=1). The geneticdistances showed a positive relationship with geographicaldistances between the BCC and SLS [r=0.20, (Po0.001)]and Belize and Florida [(r=0.48, (Po0.001)]. Pairwise FST

and RST values for Belize and Florida were 0.141 and 0.082(Po0.001), respectively, supporting the subspecies distinc-tion. Private alleles were detected for Florida (n=17) andBelize (n=1).

The loci produced an unbiased P(ID) estimate of4.55E%08 and a P(ID)sib estimate of 3.60E%04, in whichunrelated individuals could be identified in a populationsize of 4.7E+03 and 53, respectively, with a shadoweffect of ! 1.00. Calculations in GenAlEx determined thatP(ID)observed was nearly identical to that of unbiased P(ID),demonstrating that relatedness in this population is unlikelyto affect match probability, because the population iso4.7E+03.

The BCC and SLS Mantel test was positive and significant(Po0.001). All mutation model heterozygosity excess testswere significant (P ! 0.05), with low heterozygosity deficiencyto excess ratios, SMM (4:12), IAM (2:14) and TPM (3:13).However, the mode shift test was L-shaped with no distortionof allele frequencies. The M-ratio was 0.54, indicativeof a recent reduction in population size. The Belize intra-populational pairwise relatedness was rxy=0.184 (95%CI=0.174–0.185), and fell above of the 95% CI (%0.022–0.015) for permutations assuming random mating.

The empirical distribution of pairwise relatedness valueswas low (mean=0.002, range=0.852–0.815), but not sig-nificantly different from the simulated distribution underrandom mating (mean=0.144; Mann–Whitney U-test,

Table 2Genetic differentiation and haplotype proportions of Belize manatees in the Southern Lagoon System (SLS), Belize City Cayes (BCC), and

recovered carcasses (CAR)

Geographic region SLS BCC CAR

(a) Genetic differentiation

SLS – 0.031 0.045

BCC 0.041 – 0.025

CAR 0.215 0.033 –

(b) Haplotype proportions

FST values (above diagonal) and RST values (below diagonal) generated from a survey of 16 microsatellite loci. Statistically significant values are in

italics (P ! 0.01).

Pie charts indicating the haplotype proportions of the A03 (black), A04 (dark gray), and J01 (light gray) in the SLS (n=80), BCC (n=19) and CAR

(n=12) samples.



P40.05) or the full-sib distribution (Mann–Whitney U-test,P40.05; Fig. 2). However, the full-sib pairwise relatednessindicated a full-sib detection threshold of 0.21 with 19.9% ofthe pairs potentially representing full-sibs under this criter-ion, suggesting a strong representation of a few families inthe population.

The Bayesian and resulting Evanno et al. (2005) analysesstrongly supported two genetic clusters (P#1; Fig. 3a and c).Belize (QAVE=98.5%) and Florida (QAVE=98.6%) had noadmixture and strong assignment of individuals to theirrespective populations. When Belize was analyzed alonewithout a priori ‘population’ information, one populationwas supported (P#1). Alternatively, under the LOCPRIOR

model, K=2 was supported (P#1; Fig. 3b) and a singlegenetic cluster or more than two were rejected (P#0, K=1,3–10). DK=2 also had weak support as the largest modal

value (Fig. 3d). The two clusters corresponded closely withthe sampled geographic regions, the BCC (95% correctassignment, QAVE=91.6%) and SLS (86% correct assign-ment, QAVE=81.0%).

Discussion

Genetic diversity

Trichechus manatus manatus is believed to have inhabitedthe majority of the coastal habitat of Central and SouthAmerica historically, although no specific census size esti-mates are available (Husar, 1978; IUCN, 2007). A fewthousand manatees were estimated in the 18th century inBelize (Auil, 1998). Today, the subspecies is severely re-duced, rare, or absent in many areas where manatees werepreviously identified as abundant (Husar, 1978). Currentpopulation estimates place the Antillean manatee subspeciesat o2500 mature individuals (IUCN, 2007) and Belize atapproximately 1000 individuals after recovering from asevere decline (Quintana-Rizzo & Reynolds III, 2007). Thelow levels of haplotype diversity, microsatellite heterozyg-osity and allelic variation found in the Belize manateepopulation are characteristic of small, isolated populationsenduring bottlenecks or long-term persecution (Jamieson,Wallis & Briskie, 2006).

Belize manatees have lower levels of diversity than otherendangered or bottlenecked populations (Gebremedhinet al., 2009), including a koala population on French Island,Australia, which was colonized by three animals (HE=0.48,NA=3.8, 15 loci; Cristescu et al., 2009), the Wanglang giantpanda (HE=0.68,NA=6.2, 13 loci; He et al., 2008), and theEast African cheetah (HE=0.47, NA=3.55, 82 loci;

Figure 3 Plot of mean and standard deviation of the posterior probabilities, L(K), among STRUCTURE 2.3.3 runs for each value of K (1–10) for (a)

Florida and Belize and (b) Belize alone. Plot of DK versus K for (c) Florida and Belize and (d) Belize alone.

Figure 2 Distributions of pairwise relatedness values rxy (Queller &

Goodnight, 1989) for the Belize manatee population (gray bars).

Simulated unrelated (solid line) and full-sib (dashed line) curves are

based on 10 000 iterations.



Driscoll et al., 2002). Examples of species with lowerdiversity than the Belize manatee include the Mediterraneanmonk seal (HE=0.41, NA=2.3, 15 loci; Paster et al., 2004)and the critically endangered Amur leopard (HE=0.37,NA=2.6, 25 loci; Uphyrkina et al., 2002).

The Belize population was also found to have reducedvariation compared with other marine mammal populations.Trichechus manatus latirostris currently has greater diversityand a quickly recovering large population, possibly owing toa shorter and/or less intense bottleneck period. Hunting ofother marine mammal populations, such as the large ceta-ceans, occurred for a similar duration and intensity as for theAntillean manatee, and also resulted in the commercialextinction of many populations in the 1800s or 1900s.

Populations with intense hunting and smaller historicalsizes, such as the North Atlantic right whale (NARW), appearto have lost much of their diversity. The NARW was huntedfor an extended period (11th–20th century) and was reducedfrom #12 000 individuals to the low hundreds, where itremains today (Waldick et al., 2002). The NARW geneticdiversity reflects the extended and severe bottleneck thepopulation has endured (NA=4.1, HE=0.46, 11 poly-morphic loci). Alternatively, the related South Atlantic rightwhale population experienced less intense whaling that al-lowed the population to remain in the thousands and hashigher genetic diversity (Waldick et al., 2002). Genetic studieshave found large diversity, pre-whaling population estimatesand recovered population sizes for fin (Berube et al., 1998),minke (Pastene, Goto&Kanda, 2009) and humpbacks whales(Garrigue et al., 2004), and suggest that the cessation ofhunting in the early 20th century, allowed these populationsto remain above 1000 individuals (Roman & Palumbi, 2003).

Though it is reported as once being abundant in theCaribbean, T. m. manatus historical population sizes werenot likely as large as some whale populations (Husar, 1977,1978). Further, a protracted period of severe hunting re-duced the regional populations too1000 individuals and in

some cases to effective extinction (O’Shea & Salisbury, 1991;UNEP/CEP, 1995). This population contraction could havereduced the genetic diversity in Belize, which is below eventhat of the critically endangered NARW. However, lowerdispersal and immigration capabilities in manatees maylimit the exchange and development of inherent diversity inthe taxa.

The detected heterozygosity excess, mean pairwise values,and recent reduction in the population size suggests that abottleneck/founder event, inbreeding, genetic drift and/or areproductive skew has increased the relatedness amongBelize manatees. Because diversity is considered necessaryfor adaptation to diseases and environmental changes,erosion of the currently low variation could negatively affectthe population in the future (Reusch & Wood, 2007).Populations characterized by low diversity and past inbreed-ing are at risk for further losses and those not intenselymonitored can become inbred with little warning (Frank-ham, 1995; Bijlsma, Bundgaard & Boerema, 2000). Alter-natively, inbreeding accumulating gradually in populationscan provide the opportunity for natural selection to removedeleterious alleles. To date, there are no indications ofdecreased fitness due to inbreeding depression in the Belizepopulation. However, further reduction of population sizecould increase inbreeding above the ‘inbreeding threshold,’resulting in loss of fitness and risk of extinction (Frankham,1995). Further monitoring could help to detect inbreedingdepression or declines in population size (Schwartz, Luikart& Waples, 2007).

The immigration of genetically distinct individuals cansubstantially reduce the deleterious effects of inbreeding(Frankham et al., 2002). However, the enhancement ofdiversity through this mechanism is limited since manymanatee populations are small, isolated and movement isrestricted by their dependence on fresh water. Manateesprefer near-shore habitat, have limited coastal ranges andinfrequently move long distances, highlighting the need for

Figure 4 Plot of posterior probability of as-

signment for individuals (vertical lines) based

on Bayesian analysis of variation at 16 micro-

satellite loci. Individuals are grouped by popu-

lation, (a) Belize and Florida and (b) the Belize

City Cayes and Southern Lagoon System. Red

indicates genetic cluster 1 and green indicates

genetic cluster 2 in each plot.



detection and management of genetically isolated popula-tions (Lefebvre et al., 2001; Vianna et al., 2006; Quintana-Rizzo & Reynolds III, 2007).

Genetic population structure

Genetic differentiation, private alleles and a strong discre-pancy in haplotype proportions indicate restricted admix-ture between the BCC and SLS regions (Table 2). Asignificant Mantel test suggests a potential barrier to geneflow and isolation by distance. The BCC and SLS haddisparate proportions of haplotypes, J01 (79%) and A04(65%), respectively, and greater mtDNA FST values thanmicrosatellite FST values, indicating female philopatry andmale-biased gene flow. These data also suggest a largernumber of individuals moving from SLS to BCC. Limitinghuman activity in the Bar River and Belize River corridorsmight encourage movement and breeding between the SLSand BCC, leading to greater genetic diversity.

Mitochondrial DNA haplotypes have been analyzedthroughout the manatee range and the A03 haplotype hasbeen found exclusively in the SLS (presented here; Viannaet al., 2006), suggesting that A03 females remain isolated,although further analyses of samples from the region areneeded. Reduced movement has also been observed in mana-tees tracked using VHF, GPS or UHF transmitters. Themajority of manatees tagged had strong site fidelity, manyremaining within a 15mile radius of the tagging location (Auilet al., 2007). The distinct habitats and lack of movement andadmixture indicate that protection and preservation of thepopulation as at least two genetic populations would bestprotect the discrete diversity in each region.

The increasing incidence of boat strike near Belize Citycould quickly erode the BCC population size and diversity(O’Shea & Salisbury, 1991; Auil, 1998, 2007). The recoveredcarcasses from the Belize River mouth or to the south werenot statistically different from the BCC samples. The identi-fied cause of death in the carcasses was boat strike orundetermined due to decomposition, corresponding to thehigh boat traffic near the mouth of the Belize River.

Phylogeographic relationships

Recently, known Florida manatees have been documentedtraveling great distances from Florida, including, Cuba, theBahamas and Rhode Island (Reid, 2000; Deutsch et al.,2003; Alvarez-Aleman, Powell & Beck, 2007). Floridamanatees could potentially travel to Mexico along the GulfofMexico coast or by way of the Yucatan Peninsula. In fact,Mexico and Florida manatees share the A01 mitochondrialhaplotype, possibly relating the two populations. However,although gene flow between Florida and Belize throughMexico is possible, the nuclear and mitochondrial DNAdata do not support admixture, indicating possible barriersto gene flow (Domning & Hayek, 1986).

Radio-tracking technologies have documented manateestraveling between Mexico and Belize (Auil et al., 2007;Morales-Vela et al., 2007). Although these movements have

been tracked and the populations share two haplotypes, eachcountry also contains a private haplotype (A01 and A03,respectively; Vianna et al., 2006), suggesting a paucity offemale gene flow and incomplete genetic mixing. The limitedrelatedness of the threeWest Indian populations illustrates thehistorically low vagility and genetic exchange of the species.

Future directions

Quantifying the degree of migration and diversity in theWider Caribbean using genetic tools is recommended toclarify the current and future role Belize may play inmaintaining the Antillean subspecies in the region. Addi-tionally, a more thorough examination of genetic connect-edness among West Indian manatee populationsthroughout the range will assist in determining breedingpopulations and appropriate units of management for con-servation. Further nuclear studies are anticipated to providemore detail on the genetic relationships. Identification ofisolated or fragmented populations is important as lack ofgene flow has significant deleterious effects on inbreeding,fitness and population sustainability (Frankham, 1995;Frankham et al., 2002). The growth and expansion of theBelize manatee population could also assist in the repopula-tion of small or functionally extinct Antillean manateepopulations throughout the Wider Caribbean region(O’Shea & Salisbury, 1991; Bonde & Potter, 1995).

Acknowledgments

This work was conducted under the USFWS Wildlife Re-search Permit MA791721, issued to the USGS, Sirenia Pro-ject. Funding for this project was provided by the USGS, andthe University of Florida College of Veterinary MedicineAquatic Animal Health Program. We would like to thankthe Belize Manatee Project that provided access to geneticsamples, logistical support and permission to collaborativelywork under their Belize Forestry Department scientific collec-tion and research permits CD/72/02 and CD/60/3/03 withsubsequent amendments issued to N. Auil-Gomez and J.A.Powell. Additionally, Ginger Clark with the UF ICBR andSean McCann provided critical technical assistance andMartha Sudholt and Susana Caballero provided helpfuleditorial recommendations. Any use of trade, product, or firmnames is for descriptive purposes only and does not implyendorsement by the US Government.

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Low genetic variation and evidence of limited dispersal in the regionally important Belize manatee

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