2012 COURSE DATES: AUGUST 4 – 17, 2012 - Sirenian International

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Reduced Belize manatee dispersal and genetic variation M. E. Hunter et al. A previous mitochondrial DNA (mtDNA) study identified three haplotypes (A03, A04 and J01) in 43 animals in Belize (Vianna et al., 2006). The resultant haplotype parameters included 28 polymorphic sites and nucleotide substitutions with h=0.558 and p=0.037. The distribution and genetic structure of the mtDNA haplotypes was not addressed, but could provide valuable information on the population structure, especially coupled with biparentally inherited nuclear data. Presented here are mitochondrial and nuclear microsatellite DNA studies addressing the genetic diversity, relatedness and population structure in Belize manatees. The Antillean Belize population is also compared with T. m. latirostris to provide information on the extent of West Indian manatee migration and relatedness. Materials and methods Sample collection and DNA extraction Manatee epidermis tissue and/or white blood cells were collected from recovered carcasses or during wild manatee health assessments in Belize from 1997 to 2007. Genetic samples were collected from a total of 118 unique animals in 213 health assessments (50% female and 50% male; Auil et al., 2007) and 12 carcass samples. Genomic DNA was isolated using Qiagen’s DNeasy blood and tissue kits (Valencia, CA, USA). Within the Florida dataset, 96 genotypes were randomly chosen, proportionally representing the four demographically defined management units (K.P. Tucker et al., unpubl. data). Study locations The Belize City Cayes (BCC), including the near shore and outlying cayes, are utilized by the tourist industry and experience heavy boat traffic and recreational activities from Belize City, the largest port in the country (Fig. 1). Manatee tours are largely conducted around Swallow Caye within the BCC. The Southern Lagoon system (SLS) consists of the Southern and Northern Lagoons and has the highest recorded number of manatees, with 55 individuals sighted in a single aerial survey (O’Shea & Salisbury, 1991). While the population is not heavily affected by watercraft, it is potentially impacted by salinity changes due to increased rainfall, influencing the abundance of aquatic vegetation, the primary food source for manatees. Placencia Lagoon (PL) is located 75 miles south of the SLS (Fig. 1). Placencia Village is a popular tourist destination, with a large amount of coastal development and agricultural runoff severely altering the environment and aquatic vegetation. Nitrification from shrimp farm effluent causes extensive algae blooms that can compromise the quality and growth of seagrass (Thornton, Shanahan & Shanahan, 2003). Nitrification also provides a food source for a marine snail, thought to be an intermediate host of a respiratory fluke Pulmonicola cochleotrema (Blair, 2005), which is known to parasitize manatee lungs (Beck & Forrester, 1988). Manatees in PL present at necropsy with 594 large loads of P. cochleotrema and copious mucoid discharge that could hinder respiration (Auil et al., 2007). The carcass samples utilized here were recovered from Corazol (n=1), Stan Creek (n=1), the Belize River mouth in Belize City (an area of high watercraft activity), or south of the river, possibly caught in the ocean current (n=10). The northern most manatee reserve in Belize is a section of Chetumal Bay, which continues into Mexico (Fig. 1). Samples collected in Chetumal Bay are not included in this study. Laboratory procedures Mitochondrial DNA analysis Primers CR-4 and CR-5 from García-Rodríguez et al. (1998) amplified a 410 bp portion of the mtDNA control region displacement loop for 113 individuals; 101 live captures and 12 carcasses. All PCR amplifications were carried out on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA). The PCR reaction conditions followed Kellogg (2008). Representatives from each haplotype and any ambiguous sequences were sequenced in both directions to ensure the accuracy of nucleotide designations. Microsatellite DNA analysis A panel of 16 polymorphic microsatellite primers (García- Rodríguez et al., 2000; Pause et al., 2007) was PCR amplified using 118 individuals. The study included 88 individuals from the SLS system (12 Northern Lagoon and 76 Southern Lagoon), 21 from BCC, two from PL and seven amplifiable carcass samples. PCR conditions followed Kellogg (2008; Table 1). MgCl2 concentrations were 3 mM, except for TmaH13, TmaKb60 and TmaSC5, which required 2 mM. Fragment analysis was performed on an Applied Biosystems (Foster City, CA, USA) ABI 3730 Genetic Analyzer. All individuals amplified at 14 or more loci. The Florida data were kindly provided for use in this paper (K.P. Tucker et al., unpubl. data). Statistical analysis Mitochondrial DNA analysis The degree of differentiation, F ST and F ST, betweentheBCC and SLS and between Belize and Florida was calculated using ARLEQUIN 3.1 (Excoffier, Laval & Schneider, 2005). Comparisons with PL were not conducted due to the small sample size. Estimates of sequence divergence used the Kimura twoparameter genetic distance model (Kimura, 1980; Jin & Nei, 1990). Lastly, Tajima’s D of selective neutrality, genetic diversity (h), nucleotide diversity (p), number of polymorphic sites (S) and number of nucleotide substitutions (NS) were calculated (Nei, 1987; Tajima, 1993). Microsatellite DNA analysis The level of polymorphism was estimated by the observed (HO) and expected heterozygosity (HE) and the average Animal Conservation 13 (2010) 592–602 c 2010 The Authors. Animal Conservation c 2010 The Zoological Society of London
M. E. Hunter et al. 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 (T m), BSA requirement of 0.4 mg mL 1 , number of alleles (N A), the observed and expected heterozygosity (H O and H E), polymorphic information content (PIC), and effective number of alleles (N E). number of alleles per locus (NA) using GenAlEx 6.2 (Peakall & Smouse, 2006). Departures from linkage disequilibrium and Hardy–Weinberg equilibrium (HWE) were tested (dememorization 10 000, batches 100, iterations per batch 5000) in GENEPOP 4.0 (Raymond & Rousset, 1995) and Bonferroni’s corrections were applied for multiple comparisons. To assess overall genetic differentiation at the population level, GenAlEx 6.2 calculated F ST using the infinite alleles model and RST using the stepwise mutation model through an AMOVA. Comparisons included SLS, BCC and recovered carcasses, and Belize and Florida. GENECAP (Wilberg & Dreher, 2004) calculated the unbiased probability of identity (PID), which is the probability that two individuals drawn at random from a population will have the same genotype at the assessed loci (Paetkau & Strobeck, 1994) and P(ID)sib, a related more conservative statistic for calculating PID among siblings (Evett & Weir, 1998). The program additionally searched for duplicate genotypes. GenAlEx 6.2 was used to estimate the shadow effect and P(ID)observed to determine whether unbiased P(ID) or P(ID)sib is the more accurate probability (Mills et al., 2000; Waits, Luikart & Taberlet, 2001) following Hunter et al. (2010). The relationship between genetic and geographic distance was evaluated to test for isolation by distance within Belize and for Belize and Florida based on 10 000 randomizations in GenAlEx 6.2. BOTTLENECK 1.2.02 evaluated heterozygote excess under the two-phased model using the Wilcoxon signranked test (Piry, Luikart & Cornuet, 1999). The M-ratio test used M_P_Val.ex to measure the proportion of unoccupied allelic states, which is lowered during a population reduction (Garza & Williamson, 2001). Input values included a mutation rate of 5 10 4 and y=0.274; and the recommended values of Dg=3.5 and ps=0.9. Datasets using seven or more loci and the appropriate mutation model can be assumed to have experienced a reduction in Reduced Belize manatee dispersal and genetic variation population size with an Mo0.68. The coefficient of genetic relatedness, r xy (Queller & Goodnight, 1989), was used to test the genetic variability within Belize using 95% confidence intervals in GenAlEx 6.2. Intra-populational relatedness should be significantly higher in populations that have undergone bottlenecks or founding events. To test the distribution of rxy between all pairs of individuals, KINGROUP 2 simulated expected unrelated and full-sib pairwise relatedness (10 000 pairs) from observed allele frequencies and the rxy values were plotted to compare the distribution (Konovalov, Manning & Manning, 2004). A deviation from random expectation could result from nonrandom mating among related individuals and/or overrepresentation from a few families, suggesting an excess of inbred individuals in the population. The program STRUCTURE 2.3.2 was used to identify the genetic subdivision within Belize and the genetic relationship and ancestral source populations of Belize and Florida manatees (Pritchard, Stephens & Donnelly, 2000). The most probable number of populations, K, was determined by evaluating the likelihood of the posterior probability [L(K); Pritchard, Wen & Falush, 2007] and by calculating DK, an ad hoc quantity related to the change in posterior probabilities between runs of different K values (Evanno, Regnaut & Goudet, 2005) in STRUCTURE HARVESTER (Earl, 2009). Simulations were conducted using the admixture model of K=1–10 with 10 000 repetitions of MCMC, following the burn-in period of 10 000 iterations with and without a priori ‘population’ information using the admixture model. The LOCPRIOR setting was used to detect cryptic structure by providing priors for the Bayesian assignment process based on the sample location. The LOCPRIOR model allows structure to be detected with lower levels of divergence and is not biased towards detecting structure when it is not present (Hubisz et al., 2009). Individual assignment success was Animal Conservation 13 (2010) 592–602 c 2010 The Authors. Animal Conservation c 2010 The Zoological Society of London 595
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ECOLOGY, BEHAVIOR & CONSERVATION OF
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discomfort without proper protectio
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ABOUT BELIZE Research Site: The pro
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FIELD TRAINING AND ASSIGNMENTS Duri
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ENVIRONMENTAL CONDITIONS SBCRC is s
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HEALTH INFORMATION Routine Immuniza
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• Consult the CDC for immunizatio
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Oil or oil-based repellant (e.g. ol
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Ecology, Behavior, and Conservation
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MINIMUM / MAXIMUM CLASS SIZE: 6-24
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COURSE FEE & ADDITIONAL INFORMATION
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Ecology, Behavior & Conservation of
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2012 Field Course Policy & Liabilit
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2012 Field Course Policy & Liabilit
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FIELD JOURNALS You are required to
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OUR INVESTIGATION Figure 2. G. crut
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OUR INVESTIGATION Introduction The
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OUR INVESTIGATION Introduction On a
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Method We selected a patch reef loc
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OUR INVESTIGATION Introduction We f
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OUR RESEARCH FINDINGS Introduction
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area on the north part of the islan
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Introduction Our Investigation Befo
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Introduction Sea stars have long be
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INVESTIGATION Introduction Retracti
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Introduction Shells serve as a limi
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Introduction Lunar reproductive rhy
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TRIP SUMMARY SHEET (page 1) Day of
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RECORD OF EFFORT DATA SHEET Event (
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MANATEE SIGHTINGS AND SCANS Page 2
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Date: Page 2 of ____ Sight/Scan Rec
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Sighting Log Continued from Page 1
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2010 VT: Scholar, BANNER, Safe Zone
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presentation to Horn Point Environm
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• Developed and managed education
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LaCommare, Katherine S. November 20
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• Comparative Vertebrate Anatomy,
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B. Ouranos, C. Bursey, and H. Kalb.
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2006. Holy Redeemer Day Camp. Conta
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18 NEWS AND VIEWS By 1995, in respo
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4842 J. M. BLUMENTHAL ET AL. are co
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4850 J. M. BLUMENTHAL ET AL. mixed
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The Elusive Manatee -- An ethologic
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during the summer months while othe
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polyandrous - often mating with sev
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in the summer. Chessie, a male Flor
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ecords indicate that manatees have
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Florida manatee. National Biologica
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parturition: the action or process
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Manatees on the Belize Barrier Reef
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(a) (b) Manatees on the Belize Barr
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direction, and (e) when ‘travelli
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Season signi cantly aVected mean se
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male travelled at least as far as B
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55. Ontario Fisheries Research Labo
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NEWS OF THE WEEK MEETINGBRIEFS>> 10
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Functional Ecology 2008, 22, 284-28
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286 S. R. Noren Fig. 3. Swimming ki
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288 S. R. Noren Kelly, H.R. (1959)
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630 MARINE MAMMAL SCIENCE, VOL. 23,
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36 LaCommare et al. to the Drowned
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40 LaCommare et al. points was 0.31
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172 NOTES Caribbean Journal of Scie
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174 FIG. 1. Drowned Cayes study are
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176 severe event occurred in 1998 (
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Coral Reefs (1997) 16, Suppl.: S23
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Table 1. Caribbean Diadema lore Sou
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Bradbury RH, Seymour RM, Antonelli
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Social Interactions in Captive Fema
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Captive Female Manatee Social Behav
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ut not random. Behaviors were not o
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Community Structure, Population Con
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422 THE AMERICAN NATURALIST the pla
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424 THE AMERICAN NATURALIST sarily
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194 C.M. Duarte well as more recent
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202 C.M. Duarte detecting losses of
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204 C.M. Duarte Coles, R. & Fortes,
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206 C.M. Duarte Ramos-Esplá, A., M
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$1.2 million (Moore et al. 2009a).
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and positively related to knowledge
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student’s ability to learn facts
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Conservation and Behaviour Richard
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animals may adopt microhabitat choi
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decision rules they use in response
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Gagnon JW, Theimer TC, Dodd NL, Boe
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Anim Cogn (2009) 12:43-53 DOI 10.10
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a. b. c. Power Power Power 100 80 6
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2012 COURSE DATES: AUGUST 4 – 17, 2012 - Sirenian International

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