Population Genetics and Conservation of the Florida Manatee: Past, Present, and Future

POPULATION GENETICS AND CONSERVATION OF THE FLORIDA MANATEE: PAST, PRESENT, AND FUTURE

ROBERT KNUDSEN BONDE

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009

To the perpetuation of mermaid myths

ACKNOWLEDGMENTS

I would like to thank my committee, especially Dr. Peter McGuire whose ideas, guidance, and direction were a major contributor for all our present day understanding of sirenian genetics.

It was through his unselfish determination and fortitude that all of his enthusiastic graduate students, me included, found their strengths. Dr. Roger Reep was the guiding light that led me into graduate school so many years after establishing my career with the Sirenia Project. Roger’s

encouragement, perseverance, and compassion helped mold my personality and way of

approaching problems too. Dr. Lynn Lefebvre was my mentor and direct-line supervisor for

much of my professional career with U. S. Geological Survey (USGS) Sirenia Project. Through

her support, I was able to juggle the responsibilities of my job with those required of my

academic career. Dr. Tim King of the USGS Leetown Science Center whose expertise on

population genetics is rivaled by few in the world; Tim your guidance and advice are ingrained

and treasured by all immersed in wildlife genetics today. Finally, Dr. Ruth Francis-Floyd, who

joined my committee in the later stages and still provided upbeat enthusiasm, even when times

appeared discouraging. Thanks to all.

Of particular importance to me and my career is Dr. Maggie Kellogg Hunter (fellow

graduate student) who guided me through the final stages of my work and who will help lead

future genetic efforts at the USGS Sirenia Project. Other stellar graduate students from UF who

shared their knowledge are Dr. Angela Garcia-Rodriguez who designed the first studies and

thinking relative to trichechid biogeography and laid the foundation for development of Florida

manatee population genetics. Thanks to Dr. Kim Pause Tucker (University of South Florida)

who carried the torch further and helped establish our present day understanding of Florida

manatee genetics. None of these studies would have been possible without the knowledge and

tutoring provided by AnnMarie “Ginger” Clark of the University of Florida (UF)

Interdisciplinary Center for Biotechnology Research genetics lab. Ginger is a great friend and

resource that we have all become accustomed to use during times of uncertainty. Fellow student,

Coralie Nourisson, who is conducting similar studies on manatee genetics in Mexico, also is

thanked. Good luck with your research Coralie. I also thank Marta Rodriguez-Lopez from

Puerto Rico who developed a pioneering genetic study on Antillean manatees a number of years

ago. Dr. Juliana Vianna was also very helpful in sharing her study of manatee populations from

throughout their range using mtDNA and microsatellite analysis in Brazil. The critical thinking

about evolution by Dr. Daryl Domning of Howard University, who was responsible for much of

the formulation leading to the marriage (disparities) between paleontology and genetics, is

greatly appreciated.

International researchers that have aided the sirenian genetics programs with valuable

sample collection include Nicole Auil, Belize; Dr. Miriam Marmontel, Brazil; Dr. Antonio

Mignucci-Giannoni, Puerto Rico; Drs. Janet Lanyon and Helene Marsh, Australia; and Dr.

Benjamin Morales-Vela, Mexico. A sincere thanks to my colleague and dear friend, Dr. James

“Buddy” Powell with Sea to Shore Alliance. All the manatee rescue teams and rehabilitation facilities also are owed a great deal of gratitude for assistance with genetic sample collection, as well as Kit Curtin for her efforts in South Florida. Federal research permits and encouragement was extended through many of our colleagues in management, especially the U.S. Fish and

Wildlife Service. Particular thanks are extended to Monica Farris, Nicole Adimey, Dawn

Jennings, and Jim Valade (thanks Jim, who purchased my first PCR machine which is still in use today), as well as all the members of the Coordinated Florida Manatee Genetics Working Group.

This project would not have been possible without the foresight and stalwart efforts of many dedicated people, especially the staff of friends and colleagues at the USGS, Sirenia

Project. Cathy Beck, Jim Reid, Susan Butler, Amy Teague, Gaia Meigs-Friend, Howard

Kochman, Dr. Cathy “KB” Langtimm, Dr. Tom O’Shea, Dr. Galen Rathbun, Dr. Lynn Lefebvre, and Dr. Jeff Keay. Thanks to Dr. Kay Briggs (USGS), Dr. Daryl Domning (Howard University),

Dr. Bill Farmerie (UF), Sean McCann (UF), and Dr. John Reynolds III (Mote Marine Lab). A special thanks to all our colleagues at the Florida Fish and Wildlife Conservation Commission, especially Michelle Davis, Dr. Charles “Chip” Deutsch, Dr. Martine deWit, Andy Garrett, Tom

Pitchford, Dr. Mike Tringali, and Leslie Ward-Geiger.

A special thanks to my wife, best friend, and companion during our journey with the manatees, Cathy Beck, and to our children, Michael and Julie, that through their eyes we hope to be able to continue to carry on the pursuit of science and understanding.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF FIGURES ...... 11

LIST OF ABBREVIATIONS ...... 13

Organizations ...... 13 General Abbreviations ...... 13 Laboratory ...... 14 Genetics ...... 14 Genetic Analyses ...... 15

ABSTRACT ...... 17

CHAPTER

1 INTRODUCTION ...... 19

Project Summary ...... 19 The Manatee ...... 20 Sirenian Evolution ...... 20 Florida Manatee Biology ...... 21 Overview of Manatee Status, Research, and Management ...... 24 Why Do We Need to Study Manatee Genetics? ...... 29

2 TRICHECHID EVOLUTION ...... 35

Systematic Characters ...... 35 Manatee Evolution ...... 36 Population Expansion ...... 41 Molecular Status ...... 44 Extinction Processes ...... 45

3 CRYSTAL RIVER MANATEE FINE-SCALE Population ANALYSIS ...... 53

Introduction ...... 53 Materials and Methods ...... 58 Sample Collection and DNA Extraction ...... 58 DNA Isolation ...... 58 Microsatellite Analysis ...... 58 Statistical Analysis ...... 59 Neighbor-joining trees ...... 60 Analysis using STRUCTURE ...... 61 Multivariate analysis ...... 61

Results ...... 62 Microsatellite Marker Analysis ...... 62 Cluster Analysis Using Multi-locus Genotypes ...... 62 Neighbor-joining trees ...... 63 Analysis using STRUCTURE ...... 64 Multivariate analysis ...... 64 Individual identity ...... 64 Cow/Calf Relationships ...... 65 Grandmother Pedigrees ...... 66 Discussion ...... 66 Population Biology ...... 66 Individual Identity ...... 68 Cow/Calf Relationships ...... 70 Potential for Adoption ...... 70 Grandmother Pedigrees ...... 72 Conclusion ...... 72

4 MOLECULAR TOOLS FOR MANATEE CONSERVATION ...... 89

Sirenian Conservation ...... 89 Why Study Manatees? ...... 91 Genetic Markers ...... 92 Allozymes ...... 92 Mitochondrial DNA ...... 93 Microsatellites ...... 93 SNPs ...... 94 Gene Expression ...... 95 Genomes ...... 96 Implications for Manatee Conservation ...... 97 Genetic Samples ...... 97 Molecular Tools ...... 97

5 CONCLUSIONS AND FUTURE DIRECTIONS ...... 101

Recovery of an Endangered Species in Florida ...... 101 Manatee Analysis using 454 GS-20TM Pyrosequencing Technology ...... 103 Future Directions ...... 105 Sequence the Manatee Transcriptome using Massively Parallel Pyrosequencing ...... 105 Genetically Identify Individual Crystal River Manatees and Integrate Data with the Manatee Individual Photo-identification System for Capture-Recapture Studies .....108 Examine the Effective Population Size (Ne) of the Current and Past Florida Manatee Population ...... 109 Examining the Evolution and Relatedness of Trichechids ...... 110 Continue Collaboration on Wildlife Species with USGS Scientists and International Researchers to Further Genetic Capabilities ...... 113 Conclusions ...... 114

APPENDIX: TAIL NOTCH PROTOCOL ...... 123

Protocol for Collection of Manatee Tissue Samples for Genetic Research – March 2009 ..123

LIST OF REFERENCES ...... 128

BIOGRAPHICAL SKETCH ...... 144

LIST OF TABLES

Table page

3-1 Manatee sample collection information...... 74

3-2 Characteristics of the 11 polymorphic microsatellite loci utilized for the analysis of Crystal River manatee (T. m. latirostris) samples...... 80

5-1 List of di-, tri-, and tetra-nucleotides identified using FLX technology on manatee DNA with number of repeats for each sequence...... 118

LIST OF FIGURES

Figure page

1-1 Timeline of genetic research studies conducted on the Florida manatee...... 32

1-2 Pregnant Florida manatee at Crystal River, Florida. (Photo by R.K. Bonde, USGS, Sirenia Project)...... 33

1-3 Sirenian global distribution. Red is Steller’s sea cow, pink is dugong, light blue is Amazonian manatee, dark blue is West Indian manatee, and green is West African manatee...... 34

2-1 Evolution of sirenians with comparisons of cranial morphology; adapted from Domning 2005 (Reep & Bonde 2006)...... 48

2-2 Sirenian rostral deflection from least to greatest. For species designations, Ts = Trichechus senegalensis; Ti = T. inunguis; Tmm = T. manatus manatus; Tml = T. m. latirostris; and Dd = Dugong dugon...... 49

2-3 High sea level. During a warming of climate, water is released from sea ice resulting in an increase in sea level. The orange color is land mass at high sea level, outline is land mass, and water level is depicted in light blue...... 50

2-4 Moderate sea level. This is the present day condition. The orange color is land mass at middle sea level and water level is depicted in light blue...... 51

2-5 Low sea level. During very cold climatic conditions, ocean water is tied up in sea ice resulting in a lowering of sea level. The orange color is land mass at low sea level, outline is land mass today, and water level is depicted in light blue...... 52

3-1 Map of Caribbean region depicting manatee haplotypes, from Vianna et al. 2006...... 81

3-2 Notcher used to remove a small piece of skin from the tail margin of manatees...... 82

3-3 Probability of identity (P(ID)) is illustrated for the manatee population calculated for sibling association (in red), for Hardy Weinberg equilibrium (in blue), and for values observed in this study (in green) with step-wise addition for each allele in order of decreasing identity using GENALEX 6...... 82

3-4 Neighbor-joining tree describing the relationships among individuals from the Crystal River, Florida manatee population. Known family units are depicted by similar colors. Two distinct clusters are apparent and labeled A and B with an outlier group C. This neighbor-joining tree was rooted for clarity to help visualize clusters...... 83

3-5 Summary plot of q estimates generated by the sequential cluster analysis of the program STRUCTURE using a K = 3 value performed on the Crystal River, Florida population. Individuals assigned to neighbor-joining tree clusters are indicated by A, B, or C...... 84

3-6 Crystal River, Florida manatees depicted in a neighbor-joining tree using the program, PHYLIP and color-coded to assigned clusters generated from STRUCTURE using a K = 3 value with q estimate > 60...... 85

3-7 Summary plot of q estimates generated by the sequential cluster analysis of the program Structure using a K = 2 value performed on the Crystal River, Florida manatee population and compared to the rest of Florida. At top is the structure output generated by Pause 2007 for all four management areas of Florida...... 86

3-8 Scatter plot of genetic distance versus relatedness of Crystal River manatee samples. As genetic distance decreases, relatedness increases (P < 0.001)...... 87

3-9 Crystal River, Florida manatees compared with PAST multivariate analyses using non-metric multidimensional scaling for relatedness (on left) and genetic distance (on right). Each graph was color-coded with six known individual family groups observed over time with life history observations. Lines correspond to convex hulls, or polygons, containing all points of that color...... 88

5-1 Population size as compared to genetic diversity and future trends (adapted from Wahlund 1928). Population size and time are not scaled to specific events. How will the Florida manatee population respond with an anticipated loss of allelic diversity? ...... 119

5-2 Current winter Florida manatee subpopulation designations (shaded area). Blue stars are natural warm water sources, yellow stars are passive (alternate) warm water sources, and red stars are artifical sources of warm water. Four management units are represented by shaded areas; Northwest (pink), Southwest (blue), upper St. Johns River (green), and Atlantic coast (yellow)...... 120

5-3 Total number of di,- tri-, and tetra-nucleotides identified using FLX technology on manatee DNA, of 346 nucleotide repeats detected during one titration run...... 121

5-4 Number of putative microsatellites with di,- tri-, and tetra-nucleotides with at least 40 bp of flanking region at each end using FLX technology on manatee DNA...... 121

5-5 Potential winter Florida manatee subpopulation designations (shaded area) after the loss of artifical sources of warm water in winter. Blue stars are natural sources and yellow stars are passive (alternate) sources of warm water. Note the loss of artifical sources of warm water previously depicted by red stars and the anticipated shift in wintering population patterns...... 122

LIST OF ABBREVIATIONS

Organizations

ABI Applied Biosystems

FWC Florida Fish and Wildlife Conservation Commission

FWRI Fish and Wildlife Research Institute

SESC Southeast Ecological Science Center

UF University of Florida

UF ICBR University of Florida, Interdisciplinary Center for Biotechnology Research

USFWS United States Fish and Wildlife Service

USGS United States Geological Survey

General Abbreviations

Bp A single base pair

CR Crystal River, Florida

DPSs Distinct population segments

ESA Endangered Species Act

FLXTM Roche 454 standard technology

GS-20 Roche 454 Genome Sequencer

MUs Management units

Mbp Million base pairs

MIGS Manatee Individual Genetic-identification System

MIPS Manatee Individual Photo-identification System

MMPA Marine Mammal Protection Act mya Million years ago

Laboratory

BSA Bovine serum albumin

DMSO Dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid

EtOH Ethyl alcohol

MgCl Magnesium chloride 2

NaCl Sodium chloride

PCR Polymerase chain reaction

SDS Sodium dodecyl sulfate

Tris-HCl Tris(hydroxymethyl)aminomethane hydrochloride

Genetics cDNA Complementary DNA

CO1 Cytochrome c oxidase 1 d-loop Displacement loop dNTP Deoxynucleotide triphosphate

DNA Deoxyribonucleic acid mtDNA Mitochondrial DNA ncRNA Non-coding RNA

PCR Polymerase chain reaction

PSA Proportion of shared alleles

RNA Ribonucleic acid

SAA Serum amyloid-A

SNP Single nucleotide polymorphism repeat

SSRs Simple sequence repeats

STRs Short tandem repeats

VNTRs Variable number of tandem repeats

Genetic Analyses

FST Fixation index. The proportion of genetic variation distributed among subdivided populations. This is used as an index for estimating gene flow and population subdivision.

HE Expected heterozygosity. A measure of the expected proportion of heterozygotes in the population based on the principle of Hardy-Weinberg equilibrium.

Ho Observed heterozygosity. The actual proportion of heterozygotes in the population calculated from the dataset.

HWE Hardy-Weinberg equilibrium. A law stating an idealized population of diploid organisms that reproduces sexually in a random fashion with non- overlapping generations. The model population of infinite size experiences no mutation, migration, and selection processes. In population genetic data analysis, this principle states that allele frequencies will remain the same, but genotype frequencies will change over time. Values for Ho and HE are used to test if allele frequencies meet

HWE expectations.

K Number of genetic population clusters assigned by the program STRUCTURE.

MCMC Markov Chain Monte Carlo. An analog statistical technique used to estimate the probability of distribution for all possible combinations of parameter values in equilibrium distribution (Excoffier & Heckel 2006).

N Number of samples

NA Number of alleles

NE Effective number of alleles. The measure of allelic diversity that takes into account the homozygosity as well as the number of alleles (Yeh & Boyle 1997).

Ne Effective population size

NJ Neighbor-joining tree

P(ID) Probability of Identity. Probability of choosing two individuals from a population that has identical genotypes at the loci examined.

P(ID)HW Standard probability of identity assuming sample is in Hardy-Weinberg equilibrium (Waits et al. 2001).

P(ID)sib Probability of identity of siblings which accounts for related individuals in the dataset (Waits et al. 2001).

PR(X/K) Log probability of data q estimate Amount of admixture associated with assignment of an individual to a population cluster using the program STRUCTURE. Admixture of individuals within a population corresponds to mixed ancestry.

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

POPULATION GENETICS AND CONSERVATION OF THE FLORIDA MANATEE: PAST, PRESENT, AND FUTURE

Robert Knudsen Bonde

December 2009

Chair: Peter M. McGuire Major: Veterinary Medical Sciences

Manatees have the ability to establish new populations within their subtropical range, as evidenced by their evolutionary history and genetic traits. Although vicariance separated the taxa over time, the vagility of this unique group of aquatic mammals enabled populations to

disperse through deliberate migration or stochastic events. Although novel habitats are not

always suitable, the trichechines exhibit adaptive plasticity, as shown by behavioral modification

and distinct, subtle morphological characteristics among populations (Domning 2005). The

Florida manatee population was established recently, within the last 12-15,000 years. Genetic

profiling examining relatedness and genetic distance among family units of manatees in the

Crystal River subpopulation provides clues of behavioral traits that support familial distribution

and potential mate selection patterns. Thus, within peninsular Florida, where populations are

well established, distinct habitat types require manatees to maintain different survival strategies

that are passed on from generation to generation.

Although there has been an increase in the population of Florida manatees in recent

decades, genetic evidence of prior founder events affecting the population is still evident. A

reduction in manatee numbers due to anthropogenic loss occurred for centuries, and it will likely

take many generations to resolve the genetic consequences. Scarcity of warm water habitat in

the future will have severe effects on the recovery of the species. Knowledge of the genetic composition of the population will determine whether, and to what extent, breeding between geographic areas is occurring. Linking genetic tools with individual photo-identification will assist in understanding the population structure by complementing efforts to model various manatee life history traits.

There is low to average genetic variation in the Florida population. However, the genetic data suggest that even with adequate gene flow, there may be concerns regarding low allelic diversity within manatee population units due to inbreeding and bottleneck events. Employment of genetic tools will clarify stochastic demography of the Florida population. The best remedy for this low genetic diversity in the population would be to encourage growth and address conservation practices to allow breeding between population units throughout all regions of

Florida.

CHAPTER 1 INTRODUCTION

Project Summary

This project is divided into five primary chapters: Chapter 1, Project Introduction with an introduction to manatee biology and conservation; Chapter 2, Trichechid Evolution, which details the evolution of the manatees; Chapter 3, Crystal River Manatee Fine-scale Genetic

Analysis and how the Florida manatee persists in today’s environment; Chapter 4, Molecular

Tools for Manatee Conservation and where we as researchers are directing future genetic research efforts; and Chapter 5, Conclusions and Future Directions. More specifically, in

Chapter 1 there is a discussion on the status and the unique biological capabilities of the manatee, and sirenians in general. In Chapter 2, an evaluation of trichechid evolution is illustrated with possible scenarios to explain present day distribution patterns. Chapter 3 covers the present day trends in the Florida manatee population, as well as a review of genetic studies conducted to date, and presents new information on the fine-scale population structure of the manatee population in Crystal River, Florida. This information will interface with the existing

Manatee Individual Photo-identification System (MIPS) database and thus provide information to support assumptions that predict rates and trends in the population. Chapter 4 is an examination of current molecular technologies, capabilities, and tools suggested for better understanding manatee populations and conservation. Current information on manatee life history, coupled with 454 “Next Generation” sequencing technology, will direct future research efforts. Lastly, the final chapter assimilates this information and makes concluding recommendations to help guide management’s recovery efforts and improve the understanding of modern evolutionary synthesis.

The Manatee

Manatees are large aquatic mammals that can attain a maximum length of over 4 meters

and weigh up to 2,000 kilograms (Figure 1-2). Detailed accounts of sirenian biology can be

found in “Manatees and Dugongs” (Reynolds & Odell 1991), “Manatees” (Powell 2002),

“Mysterious Manatee” (Glaser & Reynolds 2003), and “The Florida Manatee: Biology and

Conservation” (Reep & Bonde 2006). A summary of life history characteristics is provided in

Table 1-1. Global distribution of all sirenians is portrayed in Figure 1-3.

Sirenian Evolution

The mammalian order Sirenia encompasses two unique families, the dugongids and the trichechids (Reep & Bonde 2006). The dugongids are marine adapted and composed of two species, the dugong (Dugong dugon) and the recently extirpated Steller’s sea cow (Hydrodamalis

gigas). The trichechids on the other hand are aquatically adapted and represented by the

Amazonian manatee (Trichechus inunguis), the West African manatee (Trichechus

senegalensis), and the West Indian manatee (Trichechus manatus). There are two recognized

subspecies of the West Indian manatee, the Antillean manatee (T. m. manatus) and the Florida

manatee (T. m. latirostris).

Sirenian distribution has been centered along the warm equatorial band, with the exception

of the Steller’s sea cow that inhabited the Bering Sea off the Commander Islands of present day

Russia. Due to hunting by Russian fur sealers for food in the mid 1700s, the Steller’s sea cow

was completely eradicated a short 27 years following their “discovery” (Stejneger 1887). The

Steller’s sea cow was larger than present day sirenians, up to 8 meters long and weighed

approximately 8-10 tons (Scheffer 1972). Their mass gave them a physiological advantage and

provided insulation necessary for life in arctic conditions. Alternatively, the extant sirenians of

semitropical habitats have had an evolutionary tract that met the challenges of removing excess

body heat, not conserving it. It is this mechanism alone that predominantly controls the

boundaries of their distribution.

Based on phylogenetic analysis the sirenians evolved through a complex series of taxa that originated with the founders of the mammalian superorder Afrotheria, approximately 100 million years ago (mya). Afrotheria also comprised the ancestral bridges to the elephants, hyraxes, aardvarks, tenrecs, golden moles, and elephant shrews. The clade Paenungulata branched from

Afrotheria approximately 20 million years after the appearance of that superorder and includes the ancestors of extant sirenians, elephants, and hyraxes. Protosirenians, such as Pezosiren, were present 70 mya and still retained evidence of hind limbs. This genus provided the evolutionary link tying the dugongids and the trichechids together, although these families split about 30 mya

(Domning 2005). More recently, within the last 5 million years, trichechids started evolving into the present day manatee species (Domning 2005). Much of the evolutionary adaptations evident today were influenced by glacial events that altered habitat types, resulting in specialization of different taxa and species. Today many of the differences among these extraordinary mammals can be traced through unique morphological characters, such as tooth development, bone processing, and more specifically, rostral deflection, all derived by adaptive behaviors and fixed through natural selection (Domning & Hayek 1986).

Florida Manatee Biology

Florida manatees are semitropical, herbivorous, K-selected, aquatic mammals (Reep &

Bonde 2006). Apart from r-selected animals, the K-selected manatees are generally large, have only one or two (~1.5%) offspring at birth, put a great deal of energy into multiple pregnancies, spend long periods nurturing their young, reach sexual maturity late, and have long life expectancies. Manatee anatomy is quite different compared to their most closely related terrestrial relatives and their physiology restricts them to a life in warm tropical waters. Suited

for a shallow-water existence, manatees generally spend much of their time in and around coastal

communities, often bringing them into direct contact with humans. Along with other myriad

threats associated with coexisting with humans, very often manatees are struck and killed by

water vessels (Lightsey et al. 2006; O’Shea et al. 1985). As more humans and boats are

introduced into the habitat, the compatibility between civilization and all living diversity,

manatees included, is increasingly challenged.

The Florida manatee is a unique aquatic mammal living in diverse habitats throughout its

distribution. Florida is at the northern limit of the species’ distribution and this subspecies has

been subjected to restrictions of population expansion primarily due to temperature barriers.

Between glacial episodic events, manatees in the southeastern United States have pulsed in and

out of geographic and temperature limited boundaries. During glacial events, land formations

were in closer proximity, and allowed trichechids the mobility to cross traditional vicariate

barriers into suitable habitats. Behaviorally, manatees in Florida quickly adapted to utilize

natural warm water springs to meet their thermal needs in winter. By the 1950s they also had

adapted to utilize artificial warm water sources, such as power plant effluents, to survive cold

winters, a major physiological challenge for this subspecies existing on the energetic fringe at

their northern range limit. Being obligate, generalist herbivores much of their adaptability to

occupy these areas has been aided by the presence of abundant aquatic plants and expansive

seagrass beds. They are not dependent on any one type of food source, but rather to a host of

species, both native and exotic. Their life history traits have mediated this ability to meet

challenges afforded by the environment throughout their evolutionary history. The factors that

make manatees so resilient lie in their biological and behavioral traits, which are discussed here in detail (Table 1-1).

Florida manatees have an uncanny ability to move over extended areas when necessary, although the motives may not be apparent. Their counterparts in the Caribbean probably have less migratory drive as these animals have their biological needs met within their restricted tropical range, but would consequently be limited in mate selection and reproductive strategies.

On the other extreme, Florida manatees must travel great distances to meet challenges related to temperature and food resources. Home ranges even vary among groups of individuals. Some may travel only a few kilometers during the year, whereas others will span greater distances.

Manatees in Florida may travel up to several hundred kilometers during the summer months, and in some cases warm season sightings have even been in excess of over 1,000 kilometers from a known wintering site (Reep & Bonde 2006). Much of this migratory behavior is probably hardwired as instinctual behavior or is reminiscent behavior taught to individuals by their mothers. Much like elephants, manatees have a very specialized ability to replicate distribution patterns taught to them during their period of dependency (Deutsch et al. 2003).

Cow/calf bonding is very strong and deliberate. Some calves will remain with their mothers for just one year, but most will stay two, three and rarely four years depending on the complexity of meeting challenges related to the habitat within their specific home range. For example, mothers in fresh water systems like the St. Johns River may only need one year to bond with and teach their young, as there is ample forage material and adequate warm water available in the springs of that riverine system to meet their needs year round. Manatees nurtured in conditions that are brackish to saline generally have a longer period of dependence to allow more time for training related to finding food, fresh water, and warm water refugia during winter.

Mothers also teach their calves the migratory routes and sites that best meet their needs, regardless of the sex of the calf. Female natal philopatry is also common in elephants (Okello et

al. 2008a). However, female manatee calves are more likely to follow the mother’s pattern than

their male counterparts who tend to deviate from their taught site fidelity soon after weaning.

This deviation amongst the young males is influenced by loose association with other male

counterparts, each possibly with different site fidelity and movement patterns imparted by their

mothers. The ranges of young males may therefore be influenced by hormonal activity and the

motivation to breed. Males have adapted to find receptive females for breeding, whereas weaned

females are driven by nutritional requirements to meet the challenges of pregnancy.

General social organization in manatees is loose, except for the tight bond between the mother and her dependent calf. Single calves are common, while twinning is rare. Although many manatees are often sighted together, these groups are generally associated with an environmental catalyst that makes the site suitable for meeting basic biological needs, such as feeding, drinking fresh water, cavorting, breeding, resting, or thermoregulating. Through these interactions among cohorts, post-weaned manatees start to learn other patterns and strategies that have been passed on by their mothers. This information continues to build on their life experiences and adaptability so integral for survival in these ever-changing environments.

People have also directly affected the habitats, and manatees have, thus far, responded accordingly.

Overview of Manatee Status, Research, and Management

The trichechids have been resilient throughout their history, but the ability of Florida manatees to survive perturbations due to habitat changes and recent anthropogenic threats has made them an appropriate model for championing the success of the U.S. Endangered Species

Act (ESA). Population expansion events have been common in this family’s 5 million years of evolution, despite the loss of some local populations and some trichechid species. This unique animal has been valued in Florida since early protections for the Florida manatee were

implemented at the turn of the 19th century. Following the implementation of the ESA in the

United States in 1967, manatees have been afforded protections that have undoubtedly allowed

growth in the population. Protections analogous to the ESA were recommended by the United

Nation’s International Union for the Conservation of Nature (IUCN) for manatee populations throughout their range. Hunting during the last couple of centuries significantly reduced the size of the Florida manatee population. However protection measures, coupled with the advent of artificial warm water sites used as cold weather refugia, the accidental introduction of exotic vegetation used as a food source by manatees, and abundant natural vegetation, enabled the population to rebound (Hartman 1974; O’Shea 1988).

The historic genetic diversity of the population is unknown, but anthropogenic deaths caused a severe decline in manatee numbers during the last century and affected their distribution. Protected by state and local laws in Florida as far back as 1893, manatees were recognized early on as a keystone species, bringing not only their plight to the forefront of the community consciousness, but extending that protection on to many other types of wildlife and habitats. This appreciation of natural, wild areas and diverse speciation was expressed via additional legislation through the decades that followed (O’Shea 1988; O’Shea et al. 2001).

Public awareness of the environment during the times of Archie Carr’s treatise “The Sea Turtle:

So Excellent a Fishe” was growing, along with public interest in wildlife (Carr 1986). Most significantly, the advent of power plants allowed the manatee population to better survive winters and increase in the southeastern United States from the mid-1900’s to present (Laist &

Reynolds 2005).

Long-term field observations, aided by photo-identification and radio-tracking technology, have provided invaluable data on the life history of the Florida manatee. The first pioneering

study resulting in baseline information on the biology of the manatee was begun in Florida

during the late 1960s with the groundbreaking work of Daniel “Woody” Hartman. In his

dissertation, Hartman outlined much of the biological character of the manatees wintering in

Crystal River, Florida (Hartman 1971), and later published a book that remains a highly cited

reference on manatee life history (Hartman 1979). Since that time, scientists with the USGS

Sirenia Project have spent over 30 years studying Florida manatees and have taken the

opportunity to examine in fine-scale the genetic attributes of the Crystal River population. Since

donning wet suit, mask, snorkel, and flippers and entering the underwater environment, we have

learned quite a bit about the Florida manatee. Understanding the population genetics of the

manatee will help us be better stewards and better grasp the fundamentals of recovery of this

endangered species.

Sustainable protection of manatees requires a better understanding of the genetic background of the species. Early genetic studies of the manatee included allozyme analysis

(McClenaghan & O’Shea 1988) and partial sequencing of the cytochrome b gene (Bradley et al.

1993). Results from the McClenaghan and O’Shea study indicated that the 59 manatee samples

examined from the Florida population exhibited evidence of population heterozygosity with

ample gene flow among five selected geographic areas. The first DNA-based study to follow by

Bradley and colleagues provided mitochondrial DNA (mtDNA) cytochrome b sequence data for

225 base pairs to identify 75 amino acid codons. These were then compared to published genes

from proboscideans. This study only examined three manatee samples, all sharing the same

haplotype, but suggested intra- and inter-species evolutionary evidence linking sirenians to

proboscideans. It was not until later when studies on the mtDNA d-loop and microsatellites were

initiated that scientists started to understand the genetic picture of the present day Florida manatee population.

Detailed analysis of mtDNA d-loop sequences led to the conclusion that the Florida manatee may have little haplotype diversity (i.e., all individuals have the same haplotype), but permitted the definition of population structure throughout the range of Trichechus manatus.

Three distinct clusters were identified: (I) Florida to Puerto Rico and the Dominican Republic,

(II) along the Gulf and Caribbean coasts of Mexico, and Central America, and (III) South

America and the Atlantic coast of Brazil to the area around the mouth of the Amazon River

(Garcia-Rodriguez et al. 1998; Vianna et al. 2006). More precise delineation of manatee population structure within Florida required the development of microsatellite fingerprinting for this species, which soon followed (Garcia-Rodriguez et al. 2001; Nourisson 2005; Pause et al.

2007; Tringali et al. 2008b; Hunter et al. 2009b). To date several polymorphic loci have been used to identify individuals and examine population structure. Each of those studies observed little genetic diversity in Florida. Recently, however, distinct population structure has been detected among the populations in Florida, Puerto Rico and Belize (Kellogg 2008). This distinctness should be expected, as there is no known reproductively effective movement occurring among any of these populations. More detailed studies are being employed now to look at finer scale population structure within the endangered manatee population in Florida

(Figure 1-1). This research will allow studies of kinship groups within geographic populations and facilitate genetic analyses of inbreeding, mating success, and paternity/maternity, as well as post-mortem identification of carcasses. These genetic data will identify and determine the extent of family structures and allow modeling of demography based on field observations.

They also may lead to better-informed management decisions regarding conservation of

manatees and assist researchers with model development to predict potential for long-term

sustainability, longevity, and population growth of this endangered species.

In 1974, Hartman predicted the manatee population in Florida based on aerial survey counts to be between 750-800 individuals, with a possible calculated maximum of 1,000 and a conceivable minimum of 600. Soon after, Irvine and Campbell in 1978 conducted additional aerial surveys and counted 738 manatees in Florida, and Brownell et al. (1981) concurred with a range of 800 to 1,000 animals. By 1985 the estimated population number was 1,200, which appears to have increased ever since. Trends in positive population growth have been observed at intensively monitored sites, such as in Blue Spring and Crystal River, Florida, with the use of

aerial surveys and ground censuses. The last synoptic statewide aerial survey conducted in

January 2009 counted 3,807 manatees (FWC 2009a; USFWS 2009a). This number is not an

estimate of the population as it is dependent on observer conditions, but merely reflects the actual number of individuals recorded on one day of survey effort. Care should be taken when trying to predict estimates for population size or minimum population size as these are very difficult to justify statistically using current methodologies and are often misunderstood and quoted out of context by the lay person (O’Shea 1988). Use of current genetic data coupled with other evaluation tools in understanding manatee population demographics will give a clearer picture of manatee recovery. Efforts to calculate effective population (Ne) size in manatees using

genetic samples holds great promise as long as the appropriate number of samples can be

obtained and the potential for sampling bias is avoided.

Recent protection measures, such as the ESA, the U.S. Marine Mammal Protection Act

(MMPA) of 1972, and the Florida Manatee Sanctuary Act of 1978, provided much needed

regulatory authority for manatee protection to state and federal agencies (USFWS 1979). These

protections not only afforded the opportunity for the Florida manatee population to reach levels

of presumed recovery, but also recently influenced State managers to consider down-listing the

species to threatened status. In 2007, the U.S. Fish and Wildlife Service published the ESA

mandated 5-Year Status Review (USFWS 2007) which also suggested down-listing the manatee

to threatened. In 2007, a petition to down-list the manatee from endangered to threatened status

on the State’s endangered species list was denied despite recent scientific findings. The outcry

from public sources prompted the Florida Fish and Wildlife Conservation Commission (FWC) delegates to disregard scientific data and management opinion, likening the decision to the one made by the judge in the classic movie “Miracle on 34th Street” (wherein the judge claimed that

there really is a Santa Claus) and rejected the petition to down-list (Pittman 2007). Soon after

the decision was rejected by the Commission, the State of Florida embraced their recently

completed Manatee Management Plan (FWC 2009b) focusing and redirecting their research

efforts to obtain an accurate census to determine the size of the population. Manatee population

status in Florida is still not completely understood, and their fate lies not only in the commitment

to anticipate and eliminate future threats, but also in the ability to look ahead and secure a place

for them in the decades to come.

Why Do We Need to Study Manatee Genetics?

Humankind has the responsibility to be better stewards of our environment and resources.

Manatees are an excellent example of how we might effectively manage and conserve a

population through strategic habitat conservation efforts. The species’ resilience to perturbations

and their short-term adaptability make them excellent candidates for recovery given that

appropriate and adequate measures are in place to protect them and their habitat. Knowledge of

their genetic make-up, coupled with understanding the limitations of the environment, will assist

managers in developing conservation strategies to ensure continued success and survival of this unique endangered species.

Table 1-1. List of life history characteristics for the Florida manatee (Modified from Reep & Bonde 2006). Population and Habitat Feeding

Population size ~4000; listed as Endangered under ESA Preferred water depth of 1-4 meters Marine and freshwater distribution, primarily coastal Snout deflection consistent with generalist feeding strategy Availability of fresh water and warm water sources required Consume 4-10% of body weight/day from a wide variety of plants Generalist obligate aquatic herbivore Feed for ~6 hours/day Abundant food resources Prehensile use of perioral bristles during feeding No natural predators Resting manatees breathe ~every 10 minutes Adapted for tropics-subtropics; Florida at northern extreme of winter range Long gut transit time, ~7 days Low metabolic rate; susceptibility to cold stress Hindgut fermentation like horses Seasonal migrations for thermoregulation Continual molar tooth replacement Seasonal site fidelity Low genetic diversity Anatomy Little genetic intermingling between East and West coast populations Evolved ~5 mya from Caribbean stock Large body size (adults usually 400-550 kg; 2.7-3.0 m long) Anthropogenic causes account for ~30% of annual mortality Record body size (up to 2000 kg; 4.0 m long) Females tend to be larger and heavier

31 Life History Pachyostosis/osteosclerosis bone; may aid hydrostasis Two hemidiaphragms, oriented longitudinally Sexual maturity at 3-5 years of age Lungs elongated Mating herds and scramble polygyny Internal testes Long gestation time ~12-14 months Paddle shaped tail Single offspring, twinning rare (~1.5%) Fingernails (3-4) on pectoral flippers Newborns weigh 18-45 kg; are ~1.2 m long Skin gray-brown with numerous pits 1-4 year calf dependency period Skin is shed often Calves suckle from two axillary teats Suckling underwater for 3-5 min every few hours Behavior and Brain Long period of early learning Maternal-calf bond main social unit Slow movements, 0 - 7 m/sec No territoriality or aggression Low encephalization quotient Vocalizations rare; chirps and squeaks Reduced visual, olfactory, and taste systems 2.5-3.0 year calving interval Expanded sensory hair system Tactile exploration using oral disk Kissing, nuzzling behavior Hearing best at 10-18 KHz

Figure 1-1. Timeline of genetic research studies conducted on the Florida manatee.

Figure 1-2. Pregnant Florida manatee at Crystal River, Florida. (Photo by R.K. Bonde, USGS, Sirenia Project).

Figure 1-3. Sirenian global distribution. Red is Steller’s sea cow, pink is dugong, light blue is Amazonian manatee, dark blue is West Indian manatee, and green is West African manatee.

CHAPTER 2 TRICHECHID EVOLUTION

Systematic Characters

Phylogenetically, sirenians (manatees and dugongs) are classified with the Paenungulata in

Afrotheria (Mammalia) (Stanhope et al. 1996; Springer et al. 1999; Parr 2000; Hedges 2001).

This group of taxa places sirenians in a genetic relationship with the Proboscidea (elephants) and

Hyracoidea (hyraxes) (Pardini et al. 2007). Studies to detect these evolutionary associations

explore gene similarities by employing chromosome painting techniques with sophisticated

cross-species fluorescent in situ hybridization (FISH) probe technology (Kellogg et al. 2007;

Pardini et al. 2007). Much of the previous amino acid and gene identification in sirenians has

been conducted on dugongid samples (De Jong et al. 1981; Rainey et al. 2984; Shosgani 1986;

Wyss et al. 1987; Ozawa et al. 1997; Springer et al. 1999; Novacek 2001; Gheerbrant et al.

2003; Yang et al. 2003; Redi et al. 2007; Springer & Murphy 2007; Tabuce et al. 2008), but

recently researchers have hybridized chromosome segments in trichechid tissues with other

species (Kellogg et al. 2007; Pardini et al. 2007). Additional scrutiny of the segments is useful

to identify relationships of taxa within the Paenungulata and clarify the processes encountered

during evolution.

It should be noted that early studies on morphology associate the sirenians within the

Ungulata group in contrast to recent genetic evidence for classification. This discrepancy

remains unresolved today, but as more genetic pieces to the puzzle are introduced, bridges between these camps are being crossed. Osteologic and dentitional evidence also associates sirenians with the extinct embrithopods, which were rhinoceros-like herbivorous mammals with

plantigrade feet, and the desmostylians, which were hippopotamus-like amphibious creatures

(Simpson 1945; Domning 2001a).

Manatee Evolution

According to the fossil record, the delineation of present-day sirenians can be traced back to the most primitive taxon Prorastomus (Figure 2-1; Savage et al. 1994; Domning 2001a).

Later, the Pezosiren was recognized as a primitive four-legged ancestral member of the Sirenia that appeared in the Eocene, about 40 mya. Pezosiren exhibited some of the characters observed in all sirenian species that followed (Domning 2001b). Certainly the present forms, both marine/aquatic (trichechids) and completely marine (dugongids), have lost their hind limbs, but other characters in common with Pezosiren, such as their dentition, mandibular structure, and rostral deflection, are retained today in extant species. The Pezosiren type specimen was recovered from a dig site in Jamaica and many of the other 35 known extinct species of sirenians that followed were also recovered from fossil sites in warm, tropical environments (Domning

2005). An intermediary taxon that evolved between Pezosiren and extant sirenians was

Protosiren (Domning 2005). This genus occurred in the Middle Eocene.

Two families of sirenians, the Dugongidae and the Trichechidae, comprise today’s four surviving species (Hatt 1934; Domning & Hayek 1986; Domning 1994a): the dugong (Dugong dugon), the Amazonian manatee (Trichechus inunguis), the West African manatee (T. senegalensis), and the West Indian manatee (T. manatus). The latter species is divided into two subspecies, with the vernacular names Antillean manatee (T. m. manatus) and Florida manatee

(T. m. latirostris). It is not certain when the transitional or intermediary steps between

Protosiren and the two branches composing the dugongids and trichechids actually took place, but it was probably sometime during the mid-Eocene to Oligocene periods (~30-40 mya)

(Domning 2005). At that time, ancestral lineages for the present day dugongs and manatees separated into two distinct clades.

The more recent evolution of the trichechids can be directly traced back to Potamosiren

during the Middle Miocene (~12-14 mya), a genus in which horizontal molar tooth replacement

was not evident (Domning 2005). The horizontal tooth replacement seen in all extant trichechines followed later (Domning 1983; 1994b; 1997). Trichechid evolution was further refined with the appearance of Ribodon from South America which followed in the Early

Pliocene (~5 mya). Both Potamosiren and Ribodon exhibited apomorphic characters defining the trichechids (Domning 2005). The Amazonian manatee (T. inunguis) appears in some ways to be the most primitive present day species but still has the largest number of uniquely derived autapomorphic (a derived trait that is unique to a related group) characters. These include

(compared to other trichechids): smaller body size, smooth skin surface, fewer ribs (n = 14-16 pairs), no nails on flippers (hence the specific epithet), and more chromosomes (diploid n = 56)

(Domning & Hayek 1986). Characters include mid-range rostral deflection of the premaxillary bones (25°-41°). On the other hand, the West African manatee (T. senegalensis) has the fewest derived characters and the least rostral deflection of the premaxillary bones (15°-40°), though very little is known about this species (Domning & Hayek 1986). Analysis of their morphological features places them as more primitive than, but more closely related to, the West

Indian manatee (T. manatus) than to the Amazonian manatee (T. inunguis). This divergence between West African and West Indian manatees must not have resulted from the opening of the

Atlantic Ocean that occurred during the Mesozoic period, but instead can be attributed to dispersal that occurred more recently. It is likely that at least one transatlantic dispersal event must have taken place sometime later, perhaps as recently as five mya (Domning 2005). The T. manatus autapomorphic characters include a larger body mass, rougher skin surface, greatest

rostral deflection of the premaxillary bones (29°-52°), and fewer chromosomes (diploid n = 48)

(Domning & Hayek 1986; Gray et al. 2002).

Distinction between the subspecies of West Indian manatees includes craniometrical characters and slight but significant differences between the rostral deflection (Figure 2-2) of the premaxillary bones (T. m. m. 29°-48°and T. m. l. 30°-52°) as described by Hatt (1934). The type specimen of T. m. latirostris was described by Harlan (1824). Certain cranial morphological characters are diagnostic of the Antillean subspecies. In T. m. manatus the angle of rostral deflection of the premaxillary is comparatively less than measured in T. m. latirostris (Domning

& Hayek 1986). With respect to classification, a subspecies has been defined by Mayr (1970) as

“an aggregate of phenotypically similar populations of a species, inhabiting a geographic subdivision of the range of a species, and differing taxonomically from other populations of the species. Additionally, the species should differ taxonomically by exhibiting distinct morphological characters and the subspecies should not be a unit of evolution, except when it corresponds to a genetic isolate.” Domning and Hayek (1986) also caution that there are overlapping ranges of variation in morphological characters within the populations. The information on morphology, along with other considerations such as isolation between geographic populations and the low potential for reproductively effective movement, has substantiated the subspecies designation.

Recent studies have associated the actual shifting of populations with rapid climatic change (Carstens & Knowles 2007). Through the course of time, changes in sea level have occurred ranging from high (Figure 2-3) making movement across seas more difficult, to the moderate (Figure 2-4) status of today’s sea level, to low (Figure 2-5), more conducive for species to bridge vicariance barriers. Timing of these geological epochs has resulted in changes in the

potential for manatees to make population expansion moves. These climatic and stochastic

events during the last 5 million years have resulted in changes that allowed trichechine

populations to pulse in and out of critical habitats (Domning 1994b). During a period 2-3 mya

there was a severe ecological upheaval that influenced much of the flora and fauna in the

Caribbean and Florida region (Bianucci et al. 2008; Domning 1994b). During this period

dugongs, which were well established in the region, were extirpated due to loss of old forms of abundant vegetation. Within a couple of million years, new forms of vegetation became established, favoring newly evolved manatees to occupy the range (Domning 2005). A similar phenomenon may have occurred with T. senegalensis even more recently. As sirenians did not exist until much later, it would have been impossible for the West African manatee to have taken advantage of the continental drift initiated in the Mesozoic. Instead, much more recently, individuals ancestral to T. senegalensis probably dispersed from the New World via ocean currents to establish a population in distant continental Africa. A plausible possibility was proposed by Boekschoten and Best (1988): approximately 18,000 years ago a high-velocity current is thought to have developed in the Atlantic Ocean between South America and Africa.

Salinity gradients could have allowed a lens of fresh water to persist at the surface to provide drinking water for long-distance traveling manatees. Generally, a physiologic need for fresh water (Ortiz et al. 1999) has been one of the primary constraints on dispersal of manatees.

Perhaps wayward manatees used that current and were cast into the nearshore habitats to colonize West Africa during the Pleistocene. The trip would obviously have been one-way - west to east - and not intentional, but afforded the opportunity for settlement into a new niche. It was the predecessors to present-day West African manatees that were the best candidates for this dispersal event (Domning 2005). Having recently arrived into the Caribbean from South

America, these manatees were well-suited for feeding on floating vegetation. It is therefore plausible that these individuals made the first translocation across the Atlantic Ocean, sustained during the crossing by rafting vegetation on a lens of fresh water (Doming 2005).

Conversely, the remaining manatees in the Caribbean were free to develop the greater rostral deflection observed today which was better adapted for surviving in more marine habitats when necessary, versus their relatives in the West African manatees (Figure 2-2). Domning

(2005) reports another sister taxon, T. m. bakerorum, within the T. manatus group. T. m. bakerorum appears to have established itself in the southeastern United States within the last

125,000 years. Specimens of this subspecies have been discovered in North Carolina and

Florida. It is likely that this subspecies was extirpated during glacial events when temperatures became so cold that these tropical descendents were not able to survive. A cold period exists in the geologic record approximately 110,000 years ago. The presence of this subspecies may exemplify how groups of manatees from the Caribbean dispersed into peninsular Florida during interglacial warm periods. Such a dispersal event may have resulted from the propensity that manatees have for colonization through natural population expansion events. At present, we do not know much about the population structure of manatees in neighboring Cuba; studies on these manatees could shed some light on this hypothesis of dispersal from neighboring communities.

As ecological (temperature, salinity, vegetation type) and physical (water, land) barriers would pose a challenge to dispersal, these events were more likely to have occurred during large-scale climatic or stochastic events (e.g., ice ages, climatic upheavals, hurricanes). Sensitivity to climate-induced habitat change has affected several species of marine mammals (Laidre et al.

2008).

Manatees also have been detected in fossil records well up the Mississippi River and Ohio

River basins (Williams & Domning 2004). These manatees were probably opportunistic wanderers during periods when their dispersal was only limited by weather and not human hunting pressures. When the weather was good, manatees would travel great distances to exploit

available habitats in new frontiers. Recently, a free-ranging manatee was observed, but died, in

the Mississippi River near Memphis, Tennessee (USGS Sirenia Project files). Radio-tagged

manatees from Florida have traveled successfully within a season along the entire Atlantic

seaboard all the way up to Rhode Island and back (Deutsch et al. 2003), and sighting events

outside of Florida during summer are now quite common, with recent sightings from as far north

as Cape Cod Bay, Massachusetts (USGS Sirenia Project files).

Population Expansion

It is possible that some individuals, by nature of their morphology and preferred feeding

strategy, may be adapted to forage on floating vegetation as well as submerged vegetation, as

was suspected for manatees that initially populated the peninsular Florida area. This could allow

for differences in subpopulation dispersal patterns by some groups of manatees. Manatees have

been observed “rafting” along with mats of floating vegetation in marine waters off Puerto Rico

(USGS Sirenia Project files). If the vegetation flotsam is large and becomes displaced it could

effectively get carried great distances by drift currents. Once the vegetation is depleted,

manatees may be left victims of the current. Such cases have been reported for manatees in

Florida that have been in the Gulf Stream and essentially carried out to sea (USGS Sirenia

Project files). In one case, a male manatee (Mo), was released with a radio tag in Crystal River,

FL but was later rescued after he drifted 300 miles south to the Dry Tortugas. Similarly, a

female manatee (Xoshi), released in Miami with a radio tag, also required rescue some 30 miles

off Merritt Island, Florida, before she was carried further out, irretrievably, into deeper Atlantic

Ocean waters.

In the early 1990’s a dependent female manatee was photographed in the Homosassa

River, Florida. In February 2000, a manatee was sighted in the Bahamas, well afield of natural manatee habitat. When a USGS researcher traveled to the Bahamas to radio-tag the manatee it was positively identified as the manatee previously documented in the Homosassa River (near

Crystal River) population (USGS files). At long-term study sites in Florida, there is a decrease in the estimated annual adult survival rates following extreme storm events (Langtimm & Beck

2003). It is possible that this manatee was displaced from the Homosassa River area during a storm event, or was driven away by increased human disturbance or behavioral conflicts, and was entrained by the Gulf Current, and subsequently found a niche in the Bahamas (USGS

Sirenia Project files). Continued monitoring in the area of her Bahamian sighting subsequently documented her with a calf.

In 2006, scientists received a report of a lone calf in the surf off the Cayman Islands.

Habitat in and around the Cayman Islands is not suitable for meeting the needs of manatees. The

116 cm male newborn orphan calf was rescued and sent to Florida for rehabilitation. Genetic analysis of the manatee’s tissues determined that this animal’s parents may have originated from

Antillean manatee stock. Could this manatee be the result of a translocation event of the pregnant mother? Drift patterns throughout the Caribbean could explain how a wayward manatee might end up on shores of several Caribbean islands, or on the coastal beaches of

Mexico.

Other events have shed an interesting light on the subject of manatee dispersal. Recently another manatee (CR131) from Crystal River, Florida was photographed and positively

identified in Cuba by her distinct scar patterns (Alvarez-Aleman et al. 2007). This manatee was a well-known adult female that had a more than 25 year sighting history in Florida with the

Manatee Individual Photo-identification System (MIPS). She had also been documented during several successful calving events in her monitoring history. Her last sighting in Florida was in

July 2006, with a calf. In January 2007 she was sighted with a calf and positively identified by photographs taken in the Camilo Cienfuegos power plant intake canal, just east of Havana, Cuba.

Although the number of extra-limital reports is increasing annually, out-of-state sighting events have led to greater public awareness. It is possible that these events are the result of natural population expansion. Presently, there are many out-of-state sightings along the Gulf and

Atlantic seaboards ranging from Texas to Massachusetts (Fertl et al. 2005; USGS Sirenia Project files). In addition to the manatee in 2006 that was observed well up the Mississippi River near

Memphis, sightings have been documented in Massachusetts during the early fall of 2008 and

2009, and increasingly in the Carolinas, Chesapeake Bay, and Texas. Fossil and recent records have placed manatees in these areas during fluctuating geological epochs and prior to human occupation (Barber 1982; Williams & Domning 2004; Domning 2005). Today, it is presumed that most of these animals continue to use these extra-limital habitats but return to overwinter in

Florida, as did the well-documented manatee Chessie (TBC-42) on several occasions, but some fall victim to the temperature limitations encountered by all Florida manatees found outside their expected winter range.

Other interesting dispersal events have been taking place in the last couple of years as well and may be influenced by behavioral traits. In March 2009 a large well-known female manatee

(RB050) with an extensive sighting history from Riviera Beach, Florida, was sighted off Rum

Key in the eastern Bahamas. Researchers hypothesize that she was caught in the Gulf Stream off

the coast of eastern Florida and drifted north. As she reached the northern end of the Bahamas,

she was carried around the north of the island chain then southward into the eastern reaches of

the islands some 400 miles southeast of Florida. By August 2009 she was re-located further

southwest off Long Key where she was rescued, but died a short time later from dehydration and

starvation imposed by lack of appropriate habitat and the stress of the journey. Manatee distribution could be limited by the need for fresh water. Without an available freshwater source, manatees in marine habitats are not likely able to adequately digest seagrasses consumed.

A trend in the dispersal pattern of this manatee toward the west due to drift currents is also supported by island lizard distribution patterns in the Bahamas (Calbeek & Smith 2003).

Molecular Status

At present, Florida manatees exhibit low genetic diversity, most likely due to a founder effect. This is not that surprising for such a recent population as we have in Florida and given their isolation from other manatee populations by distance and vicariance. If the Florida population has only been isolated for a short 12-15,000 years (moving here during the last low sea level conditions (Figure 2-5), then this current resident population has not had adequate time to fix a large number of neutral alleles. The Florida subspecies does, however, have unique alleles that, when compared with the Puerto Rico and Belize populations, distinguish it from these Antillean manatees (Hunter et al. 2009a; 2009c). This information, coupled with the morphological evidence, continues to support sub-speciation between the two populations of T. manatus.

It is interesting that manatee populations throughout their range can become so small that they become difficult to locate and economically unfeasible to hunt. Many manatee populations have been hunted for long periods of time and numbers have been reduced (Lefebvre et al.

2001). However, those remaining in reduced populations continue to have a unique ability to

find each other and successfully breed. It may be through an ability to detect breeding partners that manatees in Florida are more apt or pressured to disperse. Alternatively, as populations increase, detection among individuals is even more likely to lead to excessive breeding activity by many individuals, which may tend to drive manatees out of their established normal home ranges. Recent observations involving aggressive pursuit by males during mating has resulted in cases where the target adult female has died (deWit 2009 pers. comm.). Male elephants will adopt different behavioral and migratory patterns in an attempt to avoid opportunities that lead to breeding with related individuals (Archie et al. 2007). This elephant fission-fusion society enables random breeding by dominant males, resulting in gene flow among strongly aligned matrilineal groups (Archie et al. 2008). Future studies examining fitness need to be implemented before we fully understand whether this phenomenon is affecting manatee dispersal.

Extinction Processes

Scientists agree that there has been great opportunity for inbreeding and a founder effect in manatees from Florida, especially if we agree with Domning and others on the likelihood of recent dispersal events. The genetic uniqueness of the Florida manatee, coupled with specialized morphologic characters, has formed the framework for speciation. The possibility arises that perhaps the manatees, through human inroads and perturbations, have altered their ability to pass beneficial genes among the population successfully, while at the same time eliminating deleterious genes. Natural events have ensured the survival of manatees in the niche they now occupy, but human interaction has also tampered with the normal adaptive processes. Natural selection processes have been disrupted by recent historical events that have dictated manatee population decline as well as growth over relatively short time periods.

Past fluctuations in population size have been common throughout the manatee’s range, primarily due to gradual climate change. Nevertheless, anthropogenic effects have impacted population size during the last 10,000 years. The shift toward rapid population decreases due to hunting has forced issues related to population stability. The result, outside of Florida, has been the survival of more stealthy manatees better equipped to coexist among humans undetected.

We have already observed how hunting of a population can lead to rapid extinction as observed with the Steller’s sea cow (Stejneger 1887). Recently, however, in Florida, this has changed with advent of protective measures and development of artificial warm water sources, and manatees are gaining an unparalleled trust in humans. Are there deleterious implications associated with that trust?

Seldom do species perform well with low allelic diversity. We have noted that there is low genetic diversity in the Florida manatee. Nonetheless, some species like elephant seals (Hoelzel et al. 1993; Slade et al. 1998) and flightless cormorants (Duffie et al. 2009) have maintained healthy attributes despite low diversity. However, other populations like those of the cheetah,

Florida panther, whale species, and lions (O’Brien et al. 1985; O’Brien 2003) have shown severe loss of fitness and even genetic anomalies due to reduced population size, low genetic diversity, and inbreeding.

Finding appropriate habitat for populations is important for securing their establishment and survival (Mayr 1954; Palumbi 1994; Rocha & Bowen 2008). Recent findings in surgeonfishes from the Hawaiian Islands show that certain individuals separated by currents show specialized adaptations that make them dependent upon local habitats while at the same time unable to survive in an adjacent habitat occupied by very similar individuals of the same species (Eble et al. 2009).

With regard to Florida manatees, we also see another conundrum. Manatees have become dependent on artificial sources of warm water during cold weather. This has influenced their winter distribution and dispersal, and to a certain degree limited their selection of mates. In the very near future these aging power plants will become obsolete, will close, and will no longer be available as a warm water habitat for manatees (Laist & Reynolds 2005). When that occurs, the existing population of manatees will once again undergo modification of their routines. Home ranges will invariably change as manatees look for other over-wintering options. Population numbers will be reduced, winter range will constrict, and possibly several smaller populations might be isolated, causing genetic harm to the manatee’s fitness through reduced gene flow. We can only hope that their previous adaptive resilience, honed over millions of years, will help them during this trying period.

Figure 2-1. Evolution of sirenians with comparisons of cranial morphology; adapted from Domning 2005 (Reep & Bonde 2006).

Figure 2-2. Sirenian rostral deflection from least to greatest. For species designations, Ts = Trichechus senegalensis; Ti = T. inunguis; Tmm = T. manatus manatus; Tml = T. m. latirostris; and Dd = Dugong dugon.

Figure 2-3. High sea level. During a warming of climate, water is released from sea ice resulting in an increase in sea level. The orange color is land mass at high sea level, outline is land mass, and water level is depicted in light blue.

Figure 2-4. Moderate sea level. This is the present day condition. The orange color is land mass at middle sea level and water level is depicted in light blue.

Figure 2-5. Low sea level. During very cold climatic conditions, ocean water is tied up in sea ice resulting in a lowering of sea level. The orange color is land mass at low sea level, outline is land mass today, and water level is depicted in light blue.

CHAPTER 3 CRYSTAL RIVER MANATEE FINE-SCALE POPULATION ANALYSIS

Introduction

In 1978, a long-term research project was initiated by Department of the Interior federal scientists to look at the life history parameters of the winter resident population of manatees

(Trichechus manatus latirostris) in Crystal River (CR), Florida. It built upon pioneering studies by Daniel Hartman and James Powell (Hartman 1974) begun in CR in 1968, when the winter CR population estimate was a mere 63 manatees, and was the first research to lay the foundation for the present day Manatee Individual Photo-identification System (MIPS) managed by the USGS,

Sirenia Project. The MIPS relies on a compilation of photographs and ancillary information on manatee biology and reproduction collected on the date of each sighting, obtained from staff swimming amongst manatees in their natural habitat. Individual manatees are identified from their photographic documentation, allowing maternal pedigrees through association to be formulated from field notes. In 1989, the first manatee biopsy tissue samples were collected by

USGS, Sirenia Project staff from free-ranging manatees (mostly calves) and archived for future genetic analysis. To date over 1,000 biopsy samples have been collected from manatees in the

CR population, forming the source of material enabling the research of Peter McGuire, Angela

Garcia-Rodriguez, Kimberly Pause Tucker, Margaret Kellogg Hunter, myself, and others.

This project was initiated to complement photo-identification efforts by utilizing genetic capabilities to improve data collection techniques and reduce uncertainty in the estimation of vital rates. Presently manatee vital rates obtained from photographs have been employed in population models to estimate population growth rate (Runge et al. 2004) and project population viability (Runge et al. 2007b). Possible sources of error with photo-identification, which can be reduced with additional genetic data include, but are not limited to, (1) difference in survivability

between sexes, (2) ability to re-identify individually scarred manatees accurately, (3) accuracy of

visual observations to associate cow/calf pairs, and (4) reliance on visual observation to assign sex (Langtimm et al. 1998; Kendall et al. 2004; Runge et al. 2004). Furthermore, genetic information can provide additional information currently not possible with photo-id data for incorporation into more complex population models, such as identification at various life stages, sex, age, age at first reproduction, mate selection and success, identification of unscarred individuals, and links from individuals to various mortality factors. Though future research efforts will address most of the points mentioned above, this study will establish criteria for examining a couple of those capabilities, including identification of specific individuals, age, age at first reproduction, accuracy of assigned relatedness between cow/calf associations, and maternal lineages.

This study seeks to identify genetically distinct individuals of the CR manatee population, address the genetic or familial structure, match genotypes of samples taken from animals as calves and adults, confirm pedigrees assigned by MIPS using microsatellite loci, and strengthen

tools for determining survival estimates and mortality information. A panel of 11 markers was

selected and analyzed against a subset of individuals with maternal associations, i.e., cows and

their dependent calves. The fine-scale genetic information on relatedness will enhance the

understanding of the biology and reproductive capabilities of the individuals within this primary manatee population center in Florida.

Previous studies using cytochrome-b (Bradley et al. 1993) and d-loop mitochondrial DNA

(Garcia-Rodriguez et al. 1998; Vianna et al. 2006), determined that the Florida manatee population had only one haplotype and provided no information on relatedness or gene flow.

However, allozyme studies of 59 samples and 24 loci conducted in 1988 (McClenaghan &

O’Shea) suggested normal levels of heterozygosity and ample population mixing (implying gene flow) for all Florida manatees, prompting further research. The wave of genetic studies that followed focused on developing microsatellite primers to identify highly variable sites in the nuclear DNA. To date, there has been marked success in developing primers (Garcia-Rodriguez

2000; Garcia-Rodriguez et al. 2001; Pause et al. 2007; Pause 2007; Kellogg 2008; Tringali et al.

2008a; Hunter et al. 2009b) useful for analysis of polymorphic loci in the manatee genome.

A molecular clock of separation (based on one mutation every 1.25 million years) was proposed by Garcia-Rodriguez et al. (1998) wherein mitochondrial DNA (mtDNA) was examined among several populations of T. manatus throughout the species’ range. This information formulated the theory that manatees in Florida were more closely related to present day populations located in adjoining islands of the Caribbean, while more distant to counterparts in Central and South America. It is likely that these Caribbean manatees have found their way into Florida many times over the last 1.25 million years (Domning 2005). The last migration into peninsular Florida probably occurred about 15-20 thousand years ago during the last interglacial period and fits the molecular clock mutation rate (Garcia-Rodriguez et al. 1998; Laist

& Reynolds 2005).

The single d-loop haplotype identified in the Florida population (A01) is shared with the

Mexico, Dominican Republic and Puerto Rico Antillean manatee populations in the Caribbean

(Garcia-Rodriguez et al. 1998). The low mtDNA diversity in the Florida manatee population is thought to be the result of a genetic bottleneck or founder event (Garcia-Rodriguez et al. 1998).

After the work of Garcia-Rodriguez and colleagues in 1998, additional mtDNA studies were incorporated into a study involving many more samples (Rodriguez-Lopez 2004; Cantanhede

2005; Vianna et al. 2006), but still confirming the single haplotype, A01, in Florida. Vianna and

colleagues also supported the Garcia-Rodriguez proposal of three distinct clusters of West Indian

manatees: (I) the Caribbean and Florida, (II) coastal Central America, and (III) coastal South

America (Figure 3-1). The identification of the mtDNA haplotype A01 in manatees occupying

the Antilles resulted in some confusion with respect to the subspecies designation between T.

manatus manatus and T. manatus latirostris that had been based on morphological characters

(Domning & Hayek 1986). Since the Florida population is recent relative to other T. manatus

populations, and has not had time to undergo mutation events for differentiation, the use of more informative tools such as microsatellites became necessary to advance research capabilities.

Regarding the manatee genetic population structure within Florida, preliminary results using microsatellite primers (Pause 2007) identified a high degree of gene flow, in agreement with the allozyme study conducted in 1988. The lack of genetic separation of manatees within the Florida population provides evidence that there is enough movement for ample gene flow among individuals throughout management units or between discrete geographical regions. In an effort to examine the effectiveness of the 18 polymorphic primers on 400 manatees in the study by Tucker et al. (2009), Hunter et al. (2009c) examined approximately 100 manatees from

Puerto Rico and compared those data with the Florida data. The results to date indicate clear differentiation between these two populations, signifying geographic isolation between the

Florida and the Puerto Rico populations of manatees. This certainly has been due to vicariance

and limitations to dispersal and would support the subspecies designations based on unique

morphology (Domning & Hayek 1986).

The panmictic population structure also indicates a recent colonization to Florida, resulting

in genetic founder effect influences. This period of 12-15,000 years ago corresponds to the last

ice age when the earth’s temperature was dramatically reduced and continental shelves were

exposed as the water level receded. This lowered water level brought coastlines closer together,

making deep ocean barriers less significant and population dispersal more likely. Natural springs

(now many miles off shore) and shallow natural structures and marshes like the present day

“River of Grass” in the Everglades, might have helped supply solar-warmed water to assist

manatees during cold periods, thereby providing suitable habitat to allow establishment of a new population. These conditions additionally provided adequate access to and availability of food resources for manatees.

This study incorporated tissue samples from 145 individual manatees. All of these adult individuals were known from scar patterns or were calves of well-known, related mothers from the overwintering population in CR. The manatee population in CR is unique in that it has been extensively studied for over four decades and is undoubtedly the most thoroughly researched population of sirenians in the world. The data set represents a significant portion of the suspected total overwintering population using CR. Best estimates based on aerial surveys for this area are 480 manatees, with increases in the counts each year since 1978 (CRNWR files).

Additionally, there is a large amount of ancillary reproduction and cow-calf association data taken at the time of sample collection. This ancillary data can be compared to the genetic results

and used to help strengthen the understanding of the manatee population and provide insight into

the biological capabilities of the CR population. These genetic data will tighten the assumptions

of reproductive potential, benefit tracking of individuals through the Manatee Individual

Genetic-identification System (MIGS) and the MIPS, and result in better estimates of age-

specific survival. Future research and new innovative modeling efforts are focusing on

determining population structure based on sex, season, and range within the CR area and the

entire Florida population using more informative markers (Bonde & Hunter in progress).

Materials and Methods

Sample Collection and DNA Extraction

Dermis tissue used in this study was collected from the tail margin of wild, free-ranging

manatees by swimmers in the water using a cattle ear notcher (Figure 3-2) (Appendix). All

samples were stored in a high salt buffer solution (SED buffer, 0.24 M EDTA, pH 7.5, 2.99 M

NaCl, 20% DMSO) at room temperature (Amos & Hoelzel 1991; Proebstel et al. 1993).

Geographic location and date of sample collection, sex, and other basic information were

obtained from biologists' field notes and are listed in Table 3-1. All samples used in this study

were collected by the USGS, Sirenia Project and obtained from their tissue archive maintained

under USFWS Wildlife Research Permit MA791721, issued to the USGS, Sirenia Project. From

over 700 samples, 145 were selected for genetic analysis, greater than 25% of the current

minimum CR population estimate of 480.

DNA Isolation

Florida manatee genomic DNA was isolated from these tissues using QIAGEN’s DNeasy

Blood and Tissue kits (Valencia, California) for the 145 individuals, consisting of 47 well-known

mothers and their 98 calves (54 males and 44 females). DNA was isolated from the skin samples

using the QIAGEN DNeasy Tissue Kit protocol, eluting the DNA in 30 μl AE (10 mM Tris-HCl,

0.5 mM EDTA, pH 9.0) in the final step (QIAGEN, Valencia, CA). All isolations were quantified using a spectrophotometer (Nanodrop™, Thermo Scientific, Wilmington, DE). Each

DNA isolation was diluted to a final concentration of 10 ng/μl for use in polymerase chain

reaction (PCR) amplification.

Microsatellite Analysis

The eleven most informative manatee microsatellite loci (Garcia-Rodriguez et al. 2001;

Pause et al. 2007) were selected for use on the CR, Florida samples (Hunter et al. 2009b). All

microsatellites are di-nucleotides except for TmaH13, which is a tetra-nucleotide. Isolated DNA

was PCR amplified using: 14 ng DNA, 0.8 mM dNTPs, 1x Sigma PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.001% gelatin), 0.04 units Sigma Jump Start Taq polymerase, 0.24 µM of each primer and bovine serum albumin (BSA) where needed (Table 3-2). MgCl2 concentrations

were 3 mM, except for TmaH13, TmaKb60, and TmaSC5, which required 2 mM.

Amplifications were carried out on a PTC-200 (MJ Research, Waltham, MA) or a Touchgene

Gradient thermocycler (Techne, USA) using the following conditions: initial denaturing at 95°C

for 5 min, 35 cycles at 94°C for 30 s, annealing temp for 1 min, 72°C for 1 min, final extension

10 min at 72°C. Most samples were successfully amplified at 11 loci, however a few tissue

samples were problematic and only partially amplified using the marker set. Fragment analysis

was performed on an Applied Biosystems ABI 3130 XL Genetic Analyzer. GENEMARKER,

version 1.75 (Soft Genetics, State College, PA), was used to analyze the microsatellite fragment

data. A Microsoft Access database (MIGS) was used to store allelic information for each

individual.

Statistical Analysis

The level of polymorphism was estimated by the observed (Ho) and expected (HE) heterozygosity, polymorphic information content (PIC), and the number of alleles per locus

(Table 3-2) calculated using GENALEX 6 (Peakall & Smouse 2006). Departures from the

expected genotypic frequencies in Hardy-Weinberg equilibrium (HWE) were tested using the

Markov chain method (dememorization 10,000, batches of 100, iterations per batch 5,000) in

GENEPOP 3.4 (Raymond & Rousset 1995). Additionally, linkage disequilibrium was tested for

non-random associations between alleles of different loci. The Markov chain method was

implemented and the P-values were adjusted using Bonferroni sequential correction for multiple

comparisons (Rice 1989). Confirmation of twins related to their cows included sighting

corroboration within the MIPS database, analysis of genotypes, as well as relatedness tests using

CERVUS (Marshall et al. 1998), ML-RELATE (Kalinowski et al. 2007), and an EXCEL spreadsheet developed by M. Tringali (2009 pers. comm.).

CERVUS and ML-RELATE was used to confirm cow/calf relationships reported in this study.

CERVUS and ML-RELATE are computer programs that use genetic markers for assignment of

parents to their offspring. Both use maximum likelihood, a well-established statistical method

employed to assign parentage. Milligan (2003) recently showed that maximum likelihood

estimates of relatedness usually have a lower root mean squared error than other estimators.

CERVUS introduces two key enhancements to this process: (1) likelihood ratios are calculated

allowing for the possibility that the genotypes of parents and offspring may be mistyped, and (2)

simulation determines the level of confidence in the parentages it assigns. ML-RELATE makes

compensation for null alleles, common in microsatellite data that can easily lead to errors in

estimating relatedness or relationship if their potential presence is not accommodated during

calculations (Dakin & Avise 2004; Wagner et al. 2006).

Neighbor-joining trees

Neighbor-joining (NJ) trees based on individual genetic distances were used to visualize

relationships among suspected related individuals. MICROSAT version 1.5d (Minch et al. 1997)

estimated genetic distances based on pairwise Calvalli-Sforza and Edwards chord distance, DC.

DC is based on allele frequencies and provides accurate microsatellite tree topology (Takezaki &

Nei 1996). Trees were constructed by comparing individual genotypes without a priori

partitioning. The distance matrix was analyzed with the Neighbor algorithm in the Phylogeny

Inference Package (PHYLIP) version 3.6 (Felsenstein 2004) to produce the NJ tree. Genetic differentiation statistic (FST) was calculated from clusters identified in the NJ tree. Suspected

related individuals were analyzed from known individual sighting records obtained from a MIPS

dataset for the CR population.

Analysis using STRUCTURE

The program STRUCTURE version 2.3.1 was used to illustrate relationships between

individuals within the CR population of manatees. STRUCTURE employs principles of Hardy-

Weinberg equilibrium and linkage equilibrium to create subgroupings of unique genotypes that meet the equilibrium expectations. Multilocus genotypes were used to identify the most probable number of genetic clusters in the CR dataset. The probability of the data, Pr(X|K) was

estimated and a value of K (number of clusters, 1-7 averaged over three runs) was chosen for the

best fit approach. The probability was estimated using a burn-in of 10,000 iterations and

100,000 repetitions of a Markov chain Monte Carlo (MCMC) analysis. Posterior probabilities,

Pr(K|X), can be calculated following Bayes’ rule. The best fit value of K with the highest

posterior probability based on ad hoc approximation is the most likely number of clusters

(Pritchard & Wen 2004), however this can deviate from biological interpretation for selecting the

appropriate K (Evanno et al. 2005; Pritchard et al. 2009). Prichard and colleagues suggest that

researchers focus on selection of values of K that capture most of the structure in the data set,

while still remaining biologically sensible. A more detailed explanation of K value can be found

in STRUCTURE help documentation (Pritchard et al. 2009). Individuals were assigned by

proportion of membership (q estimates) to the assigned STRUCTURE cluster when they had > 60%

of that character.

Multivariate analysis

GENALEX was used to compare coefficients between relatedness in a Mantel test as

determined by Lynch and Ritland (1999) analyses and the proportion of shared alleles (PSA)

derived from MICROSAT. The program PAST (Paleontological Statistics) version 1.95 will be

used for multivariate analyses to detect differences based on relatedness and allelic distances

(Hammer et al. 2001). Principal coordinates, non-metric multi-dimensional scaling, correspondence, and detrended correspondence will be examined in PAST to determine

significance.

Results

Of the 145 individual samples collected, 139 were suitable for comparative analysis. Two

of the samples did not provide adequate DNA for allelic detection. The remaining four were

discarded since slight errors were detected that could be explained by errors in field data

documentation, accidental re-sampling of the same individual, or improper maternal association

made through MIPS assignments. These errors were checked against the MIPS data and

subsequent sighting information gathered after time of initial sample collection was corrected.

Microsatellite Marker Analysis

The 11 nuclear microsatellite markers (Table 3-2) had similar levels of polymorphism (HE

= 0.5451 (ranged from 0.384-0.656); HO = 0.5313 (0.401-0.654); NA = 2.9 (2-5)), when

compared to Florida manatee population analyses that used 11 loci (HE = 0.410 (0.186-0.645),

NA = 4.1 (2-7)), detailed in Tucker et al. (2009). No evidence of heterozygote deficiency due to

null alleles was detected. Hardy-Weinberg (HW) disequilibrium was observed at only one locus

TmK01 (P < 0.05) using 55 pairwise comparisons in GENEPOP and a sequential Bonferroni

correction. There was linkage observed after a Bonferroni correction between TmE1 and

TmE14.

Cluster Analysis Using Multi-locus Genotypes

The GeneCap genetic P(ID) result for P(ID)sib was 1.618E-03 and translated to a probability

match in a population of 618 individuals, whereas the P(ID)HW was 1.472E-06 and resulted in a

population of 679,346 individuals that could be individually identified. Since the winter

population in CR is approximately 480 individuals, with an upper estimate of 600 (based on

MIPS identities and potential for errors in aerial survey techniques), both of these calculations

afforded acceptable ranges for significant individual identification. Furthermore, the P(ID)observed was calculated using GENALEX, which indicated that it was in closer approximation to the

P(ID)HW calculations than to the P(ID)sib calculations (Figure 3-3). Therefore, the upper end of the estimate would fall closer to 1 in 679,346. With this estimate, the ability to correctly differentiate individuals from this population falls well within acceptable limits.

Neighbor-joining trees

The individual NJ tree identified two unique clusters, labeled A and B, and a more distant group labeled C (Figure 3-4). This tree was rooted to visualize the genetic distance between potential groups in the population. In the tree, suspected lineages were found within and between each group. The genetic distance separating the two main groups was modest. When compared to the K = 3 cluster (Figure 3-5) in STRUCTURE, most of the individuals in the A

cluster of the NJ tree were also found in the red cluster in STRUCTURE. This was also the case for

the individuals in the B cluster of the NJ tree and the blue cluster in STRUCTURE. The green

cluster appeared to be the result of mixing of the two NJ clusters (A & B) and also contained

many of the individuals that had the largest genetic distances from the two main clusters. This

information is also presented in an un-rooted NJ tree labeled with STRUCTURE assigned clusters

greater than 60% of the q estimate (Figure 3-6).

Genetic differentiation statistics investigated the separation of the clusters (A & B)

identified in the NJ tree. Genetic differentiation was observed in the CR data set after estimating

FST, which was significant at 0.035. This finding suggests low differentiation but supports two

significant groups within the population examined.

Analysis using STRUCTURE

The STRUCTURE Bayesian assignment test suggests K = 1 (ln(PD)AVE = -2604.3) was the

appropriate cluster since the lowest number of K should be used with the highest posterior

probability (Pritchard & Wen 2004). However three of the K-values (1-3) had similar

ln(PD)AVE values. The ln(PD)AVE values for K = 2 (-2573.6) and K = 3 (-2557.3) were also

investigated as these clusters appeared to capture biologically feasible structure (Prichard et al.

2009). The clusters indicated by the NJ tree and the K = 3 cluster in STRUCTURE corresponded to one another as discussed above. From the 139 individuals examined, 48 (35 > 60% q estimate)

were assigned to cluster A, 47 (28 > 60%) were assigned to cluster B, and 44 (32 > 60%) were

assigned to cluster C. A data set consisting of 119 manatees from CR was compared to a set of

84 Florida manatees from outside the CR region. The program STRUCTURE identified K=2

(ln(PD)AVE = -4126.0), separating CR from the rest of Florida (Figure 3-7).

Multivariate analysis

The 139 CR individuals exhibited a relatively strong correlation between genetic distance and relatedness. As genetic distance decreased, relatedness increased (Figure 3-8). This was significant to an R2 value of 0.5791, with a probability of <0.001. However, multivariate

analyses using relatedness and PSA showed similar trends when compared. Color labels were

added to the data set for six known family units for visual comparison and displayed using the

non-metric multidimensional scaling analysis (Figure 3-9).

Individual identity

Of the resultant 139 individual samples, 45 were MIPS identified mothers and 94 were

calves, of which 53 were male and 41 were female. Each of three calf genotypes matched three

samples taken later from adults (CR458, CR485, and CR506; two of which were collected in

1995, one in 2001). Additionally, a set of suspected twins of CR032 (CR032c4a & CR032c4b)

appeared to be duplicate samples of the same individual, later confirmed with sighting data in the

MIPS archive. In the 94 cases of suspect cow/calf pairs, four pairs (CR130c2b, CR104c4,

CR271c2a, CR506c1) were identified by genetics to be unrelated. It is interesting to note that two of those cases involved one of the calves from a suspected set of twins.

Cow/Calf Relationships

Successful assignment of genetically fraternal twins was evident in one cow/twin set.

CR205 was observed with two calves, a male (CR205c1a) and a female (CR205c1b). Both calves were about the same estimated length and weight and were sampled on the same day in

1996. Subsequently, they were re-sighted and recorded in the MIPS database with their biological mother on numerous occasions, validating the finding.

In the 94 cases of suspect cow/calf pairs, two cases (CR130c2a & CR130c2b and

CR271c2a & CR271c2b) were suspected to be sets of twins but were not genetically related. In these cases, it is presumed that the lactating mother was behaviorally willing to provide for a stray calf. One of the cow/calf pairs (CR506 & CR506c1) was visually identified and convincingly associated by their observed behavior at the time of sample collection. However, the genetic samples did not relate the two, the mother was not observed with a calf in subsequent sightings, and no identifying photographs of the cow with the suspect calf exist.

In some cases, individuals from one family unit (e.g., CR271, and her five calves) matched precisely to clusters identified by both NJ tree and STRUCTURE assignment. This cluster association pattern suggests familial related breeding among individuals. Generally, most related offspring in this analysis were associated within the same cluster. This is, however, not always the case. One example to illustrate this exception involves members of the largest related group

(CR071-CR104, and their seven calves) who apparently bred with males outside their NJ tree and STRUCTURE clusters.

Grandmother Pedigrees

The data set supplied by MIPS also identified four maternal lineages that extend from

grandmothers to calves and subsequently on to grandcalves, which were not excluded by the

genetic data. One individual, CR071 with eight related offspring and grandcalves, gave birth to

three calves, one of which in 1978 was a female (CR104) who successfully reached sexual

maturity and gave birth to five calves during the sampling period. A sixth calf (CR104c4) was assigned to CR104 by MIPS but was genetically proven not to be hers, through mismatching alleles and relatedness analyses in CERVUS and ML-RELATE. Evidence of apparent calf adoption has been observed in manatees in Belize as well (Hunter & Bonde unpublished data). Other genetically feasible grandparent lineages included: CR046 and two of her four calves, one of which was a female CR506 who birthed two calves; CR070 who is the known mother of CR130 who in turn gave birth to three calves; and CR321 who had four calves of which one, CR458, successfully gave birth to a calf.

Discussion

Population Biology

Minimal genetic difference was detected within CR, indicating no barriers to gene flow within the subpopulation and that breeding is occurring throughout the region. Biologically this is quite possible and is not surprising. NJ trees indicated two primary clusters (Figure 3-4) that reflected the division shown by STRUCTURE in the K = 3 scenario (Figure 3-5). The individuals

outside of the two main clusters with the largest genetic distances from the others in the NJ trees

may have been due to missing data or parental breeding success with partners outside of their

assigned cluster. These observations could be explained by pair breeding between the two

groups, resulting in an offspring with one parent from each group. However, after review of the

data it appears that the preponderance of breeding takes place between related individuals within

their assigned group. This is contradictory to results of previous evidence that has suggested

very slight population clustering (Pause 2007; Kellogg 2008), but could be explained by the

uniqueness of the CR population versus the remainder of Florida. Other populations share this

type of low genetic variability as seen in flightless cormorants (Duffy et al. 2009), Mediterranean

monk seals (Pastor et al. 2004), and Guadeloupe fur seals (Weber et al. 2004).

The STRUCTURE program identified one primary cluster. This finding also is not

surprising, as strong site-fidelity is suspected within this region of Florida and has been well documented by the MIPS for over 30 years. When CR was compared to areas throughout

Florida, a K = 2 value was suggested, supporting differentiation between the two populations

(Figure 3-7). However, it also implies that individuals from both groups were reproductively mixing. Adult female manatees pass on their migratory and site fidelity habits through a strong association with their calves during the long period of dependency. After weaning, young manatees may adopt similar site fidelity patterns and use sites in common with their moms

(Deutsch et al. 2003). Through these familial traits, the likelihood of breeding within one’s

assigned group is high. The STRUCTURE and NJ tree analyses sometimes placed strong

association between family groups and clusters. This familial cluster association pattern

suggests that breeding among individuals is probably influenced by both manatee behavior and

site fidelity patterns. Maternal migratory patterns, strong winter site fidelity, and routine

repeated habitat usage most likely places related manatees in proximity during potential breeding

periods thereby increasing the probability of mating between these more closely related

individuals.

STRUCTURE also suggested two other K-values, 2 and 3, which displayed similar ln(PD)

results as K = 1. When examined between individuals, the K = 3 model was concordant within

the two separate groups (A&B) assigned during NJ tree analysis. Support of genetic distance with the allelic relatedness strengthens the association between relatedness and possible family units or clans. This was additionally supported by correlation analyses where individuals were linked by association of relatedness over values obtained from genetic distance alone in 58% of the time (Figure 3-8). In an attempt to explain the other 42%, suspected known related individuals through field observation were compared using multivariate analyses and there was no evidence that individuals were more closely associated either by relatedness or by genetic distance (Figure 3-9). This can be explained by the low genetic diversity in the population and by breeding with genetically unique males outside of the family unit.

Use of these genetic data will help in the development of more robust survival rate estimation models. Strengthening of biological assumptions, such as sex determination, age class assignment, age at first reproduction, calving rate and interval, and genetic identification of specific individuals not dependent on visual scar pattern acquisition will improve capture-mark- recapture analyses of Florida manatee populations. Similar mark-recapture models (Besbeas et al. 2004; McKelvey & Schwartz 2004a; 2004b) have been employed for populations of grizzly bears (Boulanger et al. 2004; Kendall et al. 2008; Kendall et al. 2009) and whales (Baker et al.

2007).

Individual Identity

Informative markers were selected for these analyses based on the Hunter et al. 2009 study. Examination of these samples with the sensitive marker panel provided support for the individuals identified in the MIPS data set. In a Florida manatee population, the selected 11 loci used the fewest number of markers necessary for positive identification and accurate genotyping of individuals, thereby decreasing cost and improving time effectiveness while achieving satisfactory PID values (Hunter et al. 2009b). There was sufficient power in the panel to

determine match probabilities confidently, while taking into account the bias of known relatedness and current population size.

One manatee (CR485) was uniquely scarred by propeller lacerations at a young age and entered into the MIPS database while still a dependent calf. She also was genetically sampled as a calf in 2001, then re-sampled as an adult in 2008. The comparison of the genetic samples collected in 2001 and 2008, along with the identifying photographs, verified the correct MIPS assignment of this individual, illustrating the utility of merging MIPS visual techniques with genotyping. Under conservative guidelines, the probability of a genetic mismatch is low when allowing for the small size of the population and the reduced diversity of the loci used in this study. Another individual (CR458) was genetically matched to a sample collected when she was a scarred calf (CR321c1) of MIPS manatee CR321 in 1995. Matching of this genotype and

MIPS sighting data yielded a great deal of information on the animal’s long-term reproductive contributions. CR458 was documented with a calf in 2000. Today’s age of CR458 is estimated at 14 years and therefore the age of first known reproduction for this individual is estimated at 5 years, supporting the estimated age. In another case, adult CR506 was linked to an indistinct calf of MIPS manatee CR046 also sampled in 1995 (CR046c1). This confirmed the age of that animal as well, but also allowed researchers to make inference about her relatedness to her mother and siblings in the data set. Notes on her visual sightings indicated she also had her first calf at age 5. All these examples helped validate MIPS assumptions based on samples collected from unscarred calves and later matched to distinct scarred adults, thus providing additional data for survivability estimates.

The genetic matching of individuals also identified mistakes that were made during sample collection or handling. Inadvertently, one sample appeared to be mislabeled. The match

revealed that one of the samples labeled for manatee CR049 was actually a sample from CR046.

Genetic mismatch of suspected mother and calves verified the error. Validation of this mistaken genotype will be conducted in the lab to determine if the sample was mislabeled at time of collection, or if an error occurred during handling on the bench. Similar mistakes in recording of field data from CR using molecular tools were recently published by Lanyon and colleagues

(2009).

Cow/Calf Relationships

This study illustrates the importance of genetic data to validate field data collected for the

MIPS. Especially when twins are suspected, genetics can confirm field observation assumptions obtained by short-term sighting associations between cow/calf pairs. At present, much of the discrepancy between cow/calf pairing with the genetic data can be confirmed by analysis of the long-term archived files residing in the MIPS database. Better field collection protocols and continued effort to document and monitor individuals may help to avoid this discrepancy.

Manatees have unique behavioral traits that allow the skilled biologist to record cow-calf bonding with fairly good accuracy (Hartman 1979; Reynolds 1977). However, observed cases in the field (Hartman 1974; Bonde 2009 pers. obs.; Curtin 2009 pers. comm..; Hartley 2009 pers. comm.) have documented that some adult female manatees have been known to accept orphaned or stray calves, which needs to be factored in as a possible reason for mistaken associations in the field. Although adoption is probably a rare occurrence, a healthy lactating mother, even with her natural calf, could supply necessary milk and nourishment to the adopted calf, thus reinforcing pair bonding with the mother.

Potential for Adoption

From these data there is genetic evidence using ML-RELATE (Kalinowski et al. 2007), NJ trees, and mismatched alleles to suggest that adoption is possible between receptive, lactating

females and stray orphaned calves. Even adult manatees in confined quarters may try to suckle from receptive females, as has been observed by this author on many occasions. In captivity, subadult and adult manatees, and even biological mothers of lactating adult offspring, may routinely nurse from each other for extended periods of time. This nurturing seems ingrained in manatee behavior, but acceptance is not always the norm. Body condition and nutrition can affect the ability of a female to produce ample milk to provide for her biological calf, let alone another individual.

Field observations have been made of lactating cows with non-biological, but related strays, orphan calves, or recently-weaned juveniles associating closely with and nursing from the cow. For example, BS196 (Georgia), nursed both her dependent calf, BS213 (Peaches) and another known male calf, BS200 (John) for several months. John had been previously weaned by his mother, BS092 (June) and was nutritionally independent at the time. Small calves attempting to nurse with multiple lactating females also have been observed. On one such occasion in CR, a small stray calf was seen nursing within 30 minutes from two adult female manatees, each with her own biological calf. In one instance both the stray and biological calf were simultaneously feeding from the target female. The stray calf also was rejected numerous times by other females in and around the warm-water resting site in CR as it tried to initiate bouts of nursing.

Another interesting case involved a radio-tagged manatee, BC288 (Ruth) whose small two- month-old calf was observed suckling from a different female while still in the company of Ruth.

Within a week, the manatee Ruth died from a massive, chronic infection of her uterus presumed to result from a complication during parturition. It was probably due to this considerable internal infection that the moribund manatee stopped producing milk and her calf started looking

elsewhere for nourishment. After Ruth’s carcass was recovered, field crews monitored reports of

stray live or dead calves from the area. None was reported, and it was presumed that Ruth’s

dependent calf was successful in finding an accepting female to meet her nursing demands.

It should be noted that larger calves or juveniles are less behaviorally and nutritionally dependent on their mothers. Lactating females also have been observed rejecting attempts at nursing by either related or unrelated individuals. There has not been any documented aggression displayed in these manatees towards nursing attempts, only annoyance resulting in retreat being the most common negative response observed. Notably, in the examples mentioned above, field observers would likely assume relatedness between a cow and calf when nursing was observed, although that may not necessarily be the case. Genetics will be an effective tool to validate these processes and assumptions.

Grandmother Pedigrees

Individuals belonging to the four documented maternal lineages identified in this study were not excluded through genotype comparisons, validating the MIPS assumptions. Through these lineages, we are able to acquire better estimates of age and obtain accurate age at stage events, like first reproduction in young females. Coupled with size, behavior, and reproductive history, this information could yield potentially important insight about fitness and nutritional state. Complete examination of the genotype data with plans to process 700 additional samples from CR and another 2,000 samples from carcasses collected statewide will allow researchers to relate these known individuals in mark-recapture models.

Conclusion

Since the Florida population was recently founded and experienced a genetic bottleneck, the use of highly variable tools such as microsatellites became necessary for a better understanding population biology in the Florida manatee. However, the lack of variation and

genetic relatedness of manatees using these microsatellite tools limits the ability to use pedigree

programs with any certainty. With more primers and integration of more variable markers such

as single nucleotide polymorphisms (SNPs), we hope to initiate pedigree studies in the near

future. Continuation of genetic screening of archived manatee samples from the CR population

coupled with predictive modeling efforts and MIPS data will illuminate issues related to basic

manatee biology. Future research efforts should focus on utilizing SNPs, microarrays, and

functional gene sequencing in the arsenal for fine-scale population relatedness studies. These molecular tools, coupled with more robust modeling efforts, will permit better interpretation of

recovery efforts for this unique endangered species.

Table 3-1. Manatee sample collection information. Gen Gen Process Field Mom Calf Calf Number Sample Date IDNUM Sequence IDNUM Size(cm) Sex CR01 06-318-6 14-Nov-06 CR026 CR026 Mom 300 F CR107 05-362-1 28-Dec-05 CR026 CR026c1 05-362-1 230 M CR02 06-326-15 22-Nov-06 CR027 CR027 Mom 300 F CR60 97-336-3 02-Dec-97 CR027 CR027c1 CR027c 230 F CR70 99-320-3 16-Nov-99 CR027 CR027c2 99-320-3 175 F CR103 02-330-3 26-Nov-02 CR027 CR027c3 CR027e 220 M CR117 07-332-3 28-Nov-07 CR032 CR032 Mom 350 F CR118 92-043-3 12-Feb-92 CR032 CR032c1 92-043-3 220 F CR119 93-335-2 1-Dec-93 CR032 CR032c2 93-335-2 230 F CR120 97-344-2 10-Dec-97 CR032 CR032c3 97-344-2 180 F CR126 99-362-2 28-Dec-99 CR032 CR032c4a 99-362-2 170 M CR127 99-363-3 29-Dec-99 CR032 CR032c4b 99-363-3 170 M 74 CR03 07-015-2 15-Jan-07 CR045 CR045 Mom 340 F CR04 06-326-14 22-Nov-06 CR046 CR046 Mom 300 F CR50 95-032-4 01-Feb-95 CR046 CR046c1 CR046d 180 F CR61 97-344-1 10-Dec-97 CR046 CR046c2 CR046e 230 F CR05 06-281-3 8-Oct-06 CR049 CR049 Mom 300 F CR74 00-333-2 28-Nov-00 CR049 CR049c1 CR049f 210 M CR132 06-340-1 6-Dec-06 CR054 CR054 Mom 340 F CR133 92-043-2 12-Feb-92 CR054 CR054c1 92-043-2 230 F CR134 96-341-5 6-Dec-96 CR054 CR054c2 96-341-5 220 M CR135 02-350-4 16-Dec-02 CR054 CR054c3 02-350-4 190 F CR06 06-320-5 16-Nov-06 CR060 CR060 Mom 320 F CR68 99-055-6 24-Feb-99 CR060 CR060c1 CR060d 160 M CR82 01-303-2 30-Oct-01 CR060 CR060c2 CR060h 210 M Gen Process Number, laboratory identification number, Gen Field Sample, number assigned to the sample in the field, Date, date collected, Mom ID, suspected MIPS number of the mom, Calf Sequence, sequence number of calves for suspected mom, Calf IDNUM, MIPS ID number for the calf, Size, total estimate length, and Sex, sex of the animal. Shaded areas represent samples not utilized in the analysis.

Table 3-1. Continued. Gen Gen Process Field Mom Calf Calf Number Sample Date IDNUM Sequence IDNUM Size(cm) Sex CR07 06-320-6 16-Nov-06 CR060 CR060c3 06-320-6 160 F CR08 06-320-3 16-Nov-06 CR061 CR061 Mom 290 F CR80 01-011-2 11-Jan-01 CR061 CR061c1 CR061e 220 F CR09 07-029-7 29-Jan-07 CR070 CR070 Mom 320 F CR10 07-108-3 18-Apr-07 CR071 CR071 Mom LA F CR53 96-068-1 8-Mar-96 CR071 CR071c1 96-068-1 230 F CR88 03-008-1 08-Jan-03 CR071 CR071c2 CR071i 180 M CR114 07-318-5 14-Nov-07 CR093 CR093 Mom 320 F CR102 02-323-3 19-Nov-02 CR093 CR093c1 02-323-3 230 M CR11 07-015-1 15-Jan-07 CR104 CR104 Mom 320 F CR46 93-020-2 20-Jan-93 CR104 CR104c1 CR104d 240 M CR57 97-017-1 17-Jan-97 CR104 CR104c2 CR104f 230 M 75 CR66 98-114-1 24-Apr-98 CR104 CR104c3 CR104g 160 M CR71 00-035-1 04-Feb-00 CR104 CR104c4 CR104h 220 M CR79 00-325-3 20-Nov-00 CR104 CR104c5 CR104i 180 F CR85 02-017-1 17-Jan-02 CR104 CR104c6 CR104k 180 M CR12 07-029-10 29-Jan-07 CR123 CR123 Mom 320 F CR45 92-043-1 12-Feb-92 CR123 CR123c1 92-043-1 240 M CR54 96-330-1 25-Nov-96 CR123 CR123c2 CR123d 235 F CR69 99-055-4 24-Feb-99 CR123 CR123c3 CR123e 200 F CR72 00-325-2 20-Nov-00 CR123 CR123c4 CR123f 200 M CR13 06-320-2 16-Nov-06 CR125 CR125 Mom 280 F CR49 95-031-4 31-Jan-95 CR125 CR125c1 CR125e 210 M CR58 97-308-1 04-Nov-97 CR125 CR125c2 97-308-1 230 F CR76 00-335-4 01-Dec-00 CR125 CR125c3 CR125h 200 M CR14 06-281-4 8-Oct-06 CR130 CR130 Mom 300 F CR78 98-012-1 12-Jan-98 CR130 CR130c1 98-012-1 220 M CR89 03-008-2 08-Jan-03 CR130 CR130c2a CR130g 230 M

Table 3-1. Continued. Gen Gen Process Field Mom Calf Calf Number Sample Date IDNUM Sequence IDNUM Size(cm) Sex CR90 03-017-3 17-Jan-03 CR130 CR130c2b CR130h 210 F CR15 06-308-5 4-Nov-06 CR133 CR133 Mom 300 F CR63 97-356-1 22-Dec-97 CR133 CR133c1 CR133c 200 F CR95 00-336-6 01-Dec-00 CR133 CR133c2 CR133d 160 M CR16 06-308-4 4-Nov-06 CR157 CR157 Mom 340 F CR100 01-311-2 07-Nov-01 CR157 CR157c1 CR157b 160 F CR17 06-308-8 4-Nov-06 CR164 CR164 Mom 290 F CR64 97-363-3 29-Dec-97 CR164 CR164c1 CR164b 170 F CR84 01-317-1 13-Nov-01 CR164 CR164c2 CR164c 190 M CR116 07-332-2 28-Nov-07 CR171 CR171 Mom 320 F CR124 99-055-2 24-Feb-99 CR171 CR171c1 99-055-2 180 M CR115 07-332-1 28-Nov-07 CR171 CR171c2 07-332-1 170 F 76 CR18 07-038-5 7-Feb-07 CR205 CR205 Mom 290 F CR55 96-341-3 06-Dec-96 CR205 CR205c1a CR205c 140 M CR56 96-341-4 06-Dec-96 CR205 CR205c1b CR205d 140 F CR92 05-022-6 22-Jan-05 CR205 CR205c2 05-022-6 220 M CR98 01-005-1 05-Jan-01 CR205 CR205c3 CR205g 200 F CR113 07-318-3 14-Nov-07 CR235 CR235 Mom 290 F CR122 98-050-1 19Feb98 CR235 CR235c1 98-050-1 190 M CR105 03-317-1 13-Nov-03 CR235 CR235c2 03-317-1 210 F CR19 06-308-2 4-Nov-06 CR251 CR251 Mom 300 F CR65 98-040-1 9-Feb-98 CR251 CR251c1 98-040-1 190 M CR37 06-326-4 4-Nov-06 CR251 CR251c2 06-326-4 220 F CR20 07-031-10 31-Jan-07 CR263 CR263 Mom 280 F CR83 01-310-2 06-Nov-01 CR263 CR263c1 CR263c 210 F CR142 08-017-2 17-Jan-08 CR266 CR266 Mom 300 F CR143 95-032-1 1-Feb-95 CR266 CR266c1 95-032-1 200 F CR144 98-014-2 14-Jan-98 CR266 CR266c2 98-014-2 160 M

Table 3-1. Continued. Gen Gen Process Field Mom Calf Calf Number Sample Date IDNUM Sequence IDNUM Size(cm) Sex CR145 03-022-1 22-Jan-03 CR266 CR266c3 03-022-1 180 F CR21 06-326-2 22-Nov-06 CR271 CR271 Mom 300 F CR44 92-041-2 10-Feb-92 CR271 CR271c1 92-041-2 140 M CR73 00-326-2 21-Nov-00 CR271 CR271c2a CR271b 180 F CR75 00-335-3 01-Dec-00 CR271 CR271c2b 00-335-3 200 M CR87 02-364-1 30-Dec-02 CR271 CR271c3 CR271c 170 M CR38 06-326-7 22-Nov-06 CR271 CR271c4 06-326-7 180 M CR22 06-309-5 5-Nov-06 CR272 CR272 Mom 300 F CR121 98-040-2 09-Feb-98 CR272 CR272c1 CR272c 240 M CR106 04-357-1 22-Dec-04 CR272 CR272c2 CR272b 190 M CR23 06-308-1 4-Nov-06 CR277 CR277 Mom 340 F CR52 96-023-4 23-Jan-96 CR277 CR277c1 96-023-4 180 M 77 CR91 03-315-1 11-Nov-03 CR277 CR277c2 03-315-1 190 M CR24 06-326-10 22-Nov-06 CR278 CR278 Mom 290 F CR47 93-035-1 4-Feb-93 CR278 CR278c1 93-035-1 180 F CR39 06-326-11 22-Nov-06 CR278 CR278c2 06-326-11 200 M CR25 06-318-2 14-Nov-06 CR321 CR321 Mom 300 F CR48 95-027-1 27-Jan-95 CR321 CR321c1 95-027-1 230 F CR59 97-312-9 08-Nov-97 CR321 CR321c2 CR321b 200 F CR81 01-302-4 29-Oct-01 CR321 CR321c3 CR321c 200 M CR93 05-022-2 22-Jan-05 CR321 CR321c4 05-022-1 190 M CR109 07-309-1 05-Nov-07 CR321 CR321c5 07-309-1 210 M CR36 06-281-2 08-Oct-06 CR333 CR333 Mom 290 F CR40 06-281-1 08-Oct-06 CR333 CR333c1 06-261-1 190 M CR26 06-326-18 22-Nov-06 CR339 CR339 Mom 280 F CR86 02-330-1 26-Nov-02 CR339 CR339c1 CR339a 220 M CR27 07-029-5 29-Jan-07 CR341 CR341 Mom 300 F CR51 95-052-1 21-Feb-95 CR341 CR341c1 CR341a 195 F

Table 3-1. Continued. Gen Gen Process Field Mom Calf Calf Number Sample Date IDNUM Sequence IDNUM Size(cm) Sex CR67 98-352-1 18-Dec-98 CR341 CR341c2 CR341b 200 M CR28 06-309-3 5-Nov-06 CR354 CR354 Mom 300 F CR99 01-008-2 08-Jan-01 CR354 CR354c1 CR354a 200 M CR101 02-016-1 16-Jan-02 CR354 CR354c2 CR354b 220 M CR41 06-309-4 5-Nov-06 CR354 CR354c3 06-309-4 180 F CR29 06-308-7 4-Nov-06 CR358 CR358 Mom 290 F CR62 97-344-5 10-Dec-97 CR358 CR358c1 CR358a 160 F CR77 01-010-1 10-Jan-01 CR358 CR358c2 CR358c 190 F CR42 06-308-6 4-Nov-06 CR358 CR358c3 06-308-6 160 F CR137 07-064-2 5-Mar-07 CR360 CR360 Mom 280 F CR139 99-004-1 4-Jan-99 CR360 CR360c1 99-004-1 200 M CR138 07-064-3 5-Mar-07 CR360 CR360c2 07-064-3 180 M 78 CR128 07-065-1 6-Mar-07 CR363 CR363 Mom 320 F CR129 97-016-1 16-Jan-97 CR363 CR363c1 97-016-1 180 F CR130 00-039-1 8-Feb-00 CR363 CR363c2 00-039-1 180 F CR131 01-310-4 6-Nov-01 CR363 CR363c3 01-310-4 210 M CR30 06-308-3 4-Nov-06 CR385 CR385 Mom 300 F CR123 99-007-2 07-Jan-99 CR385 CR385c1 CR385b 200 M CR136 06-298-1 25-Oct-06 CR385 CR385c2 06-299-1 170 F CR31 06-309-7 5-Nov-06 CR401 CR401 Mom 300 F CR125 99-320-1 16-Nov-99 CR401 CR401c1 CR401b 190 F CR112 07-315-3 11-Nov-07 CR413 CR413 Mom 320 F CR104 02-336-6 2-Dec-02 CR413 CR413c1 02-336-6 220 F CR110 07-313-1 9-Nov-07 CR443 CR443 Mom 340 F CR111 07-313-4 9-Nov-07 CR443 CR443c1 07-313-4 160 M CR32 07-031-14 31-Jan-07 CR458 CR458 Mom 280 F CR94 05-022-1 22-Jan-05 CR458 CR458c1 05-022-1 180 F CR33 06-326-3 22-Nov-06 CR474 CR474 Mom 300 F

Table 3-1. Continued. Gen Gen Process Field Mom Calf Calf Number Sample Date IDNUM Sequence IDNUM Size(cm) Sex CR97 00-342-4 07-Dec-00 CR474 CR474c1 CR474b 180 M CR140 01-016-1 16-Jan-01 CR485 CR485 Mom 230 F CR141 08-017-1 17-Jan-08 CR485 CR485 Mom 280 F CR34 06-318-3 14-Nov-06 CR506 CR506 Mom 280 F CR96 00-336-7 01-Dec-00 CR506 CR506c1 CR506a 200 M CR108 06-067-2 08-Mar-06 CR506 CR506c2 06-067-2 220 F CR35 06-326-12 22-Nov-06 CR509 CR509 Mom 270 F CR43 06-326-13 22-Nov-06 CR509 CR509c1 06-326-13 170 M Gen Process Number, laboratory identification number, Gen Field Sample, number assigned to the sample in the field, Date, date collected, Mom ID, suspected MIPS number of the mom, Calf Sequence, sequence number of calves for suspected mom, Calf IDNUM, MIPS ID number for the calf, Size, total estimate length, and Sex, sex of the animal. Shaded areas represent samples not utilized in the analysis.

Table 3-2. Characteristics of the 11 polymorphic microsatellite loci utilized for the analysis of Crystal River manatee (T. m. latirostris) samples. Locus N Tm(°C) BSA NA NE PIC Ho HE TmaE1 136 55 + 4.000 2.907 1.181 0.551 0.495 TmaE02 135 62 2.000 1.969 0.685 0.401 0.384 TmaE7 136 56 + 3.000 2.338 0.954 0.552 0.496 TmaE08 137 60 3.000 1.978 0.706 0.401 0.391 TmaE11 136 58 5.000 2.824 1.154 0.474 0.481 TmaE14 134 56 + 4.000 2.582 1.020 0.618 0.613 TmaH13 137 60 3.000 1.624 0.672 0.562 0.572 TmaJ02 137 62 2.000 1.642 0.580 0.632 0.646 TmaKb60 137 62 2.000 1.985 0.689 0.654 0.656 TmaK01 138 58 3.000 1.926 0.840 0.630 0.619 TmaSC5 136 58 3.000 2.622 1.018 0.518 0.492 80 Locus name (Locus), number of samples (N), Annealing temperature (Tm(°C)), BSA requirement (0.4 mg/mL), number of alleles (NA), effective number of alleles (NE), polymorphic information content (PIC), and observed and expected heterozygosity (Ho and HE) for the Crystal River T. m. latirostris population.

Figure 3-1. Map of Caribbean region depicting manatee haplotypes, from Vianna et al. 2006.

Figure 3-2. Notcher used to remove a small piece of skin from the tail margin of manatees.

Figure 3-3. Probability of identity (P(ID)) is illustrated for the manatee population calculated for sibling association (in red), for Hardy Weinberg equilibrium (in blue), and for values observed in this study (in green) with step-wise addition for each allele in order of decreasing identity using GENALEX 6.

Figure 3-4. Neighbor-joining tree describing the relationships among individuals from the Crystal River, Florida manatee population. Known family units are depicted by similar colors. Two distinct clusters are apparent and labeled A and B with an outlier group C. This neighbor-joining tree was rooted for clarity to help visualize clusters.

Figure 3-5. Summary plot of q estimates generated by the sequential cluster analysis of the program STRUCTURE using a K = 3 value performed on the Crystal River, Florida population. Individuals assigned to neighbor-joining tree clusters are indicated by A, B, or C.

CR363 CR271c2b CR358c2 CR363c1 CR266c1 CR506c1 CR093c1 CR341c1 CR125c1 CR157c1 CR046c2 CR104c4 CR506c2 CR130 CR235c2 CR171c1 CR443 CR093 CR443c1 CR251 CR272 CR278c2 CR358c3 CR130c2a CR251c1 CR235c1 CR061 CR133c2 CR049c1 CR341 CR358 CR235 CR506 CR278 CR266c2 CR130c1 CR046c1 CR071c2 CR263 CR060c1 CR266 CR333 CR123c4 CR272c1 CR026c1 CR341c2 CR278c1 CR272c2 CR060c3 CR060c2 CR271c2a CR049 CR358c1 CR413c1 CR070 CR032c4b CR157 CR032c4a CR046 CR123c2 CR360c2 CR277 CR164c1 CR061c1 CR205c2 CR277c1 CR509c1 CR060 CR385c2 CR125c2 CR104c2 CR026 CR263c1 CR363c3 CR385c1 CR385 CR485 CR360c1 CR125 CR360 CR205c1b CR205c3

CR205c1a CR354 CR401c1 CR164c2 CR054c2 CR413 CR401 CR271c1 CR164 CR277c2

85 CR104c5 CR104c3 CR104 CR509 CR321 CR027c2 CR205 CR071c1 CR104c6 CR071 CR027c3 CR054c3 CR054 CR104c1 CR027c1 CR123c3 CR130c2b CR125c3 CR027 CR333c1 CR458c1 CR133 CR321c3 CR032c3 CR054c1 CR354c2 CR123c1 CR251c2 CR321c5 CR321c1 CR458 CR354c3 CR354c1 CR321c2 CR271c4 CR171c2 CR321c4 CR123 CR171 CR032 CR045 CR363c2 CR339 CR474c1 CR474 CR032c1 CR271

CR133c1 CR032c2

CR271c3 CR339c1

Figure 3-6. Crystal River, Florida manatees depicted in a neighbor-joining tree using the program, PHYLIP and color-coded to assigned clusters generated from STRUCTURE using a K = 3 value with q estimate > 60.

Figure 3-7. Summary plot of q estimates generated by the sequential cluster analysis of the program Structure using a K = 2 value performed on the Crystal River, Florida manatee population and compared to the rest of Florida. At top is the structure output generated by Pause 2007 for all four management areas of Florida.

Figure 3-8. Scatter plot of genetic distance versus relatedness of Crystal River manatee samples. As genetic distance decreases, relatedness increases (P < 0.001).

Figure 3-9. Crystal River, Florida manatees compared with PAST multivariate analyses using non-metric multidimensional scaling for relatedness (on left) and genetic distance (on right). Each graph was color-coded with six known individual family groups observed over time with life history observations. Lines correspond to convex hulls, or polygons, containing all points of that color.

CHAPTER 4 MOLECULAR TOOLS FOR MANATEE CONSERVATION

Sirenian Conservation

Management and conservation of West Indian manatee populations would benefit from multiple methods of genetic population analyses. Manatee behavior is unique and not well understood, and genetic data could provide insights into stochastic demography. A variety of tools is available for investigating population genetics. Mitochondrial DNA (mtDNA) sequencing, multi-locus analyses utilizing microsatellites and single nucleotide polymorphisms

(SNPs), gene expression analysis, genomic sequencing, and identification of protein variants are the major techniques employed. Utilizing multiple genetic tools has strengthened the position for implementation of conservation measures in other threatened and endangered species (Fallon

2007).

In the Florida manatee polymorphic microsatellite loci are difficult to identify in manatee samples. Currently 36 microsatellite loci have been developed for manatee genotyping, however only a limited number of alleles have been identified (Garcia-Rodriguez et al. 2001; Pause et al.

2007; Tringali et al. 2008b). This finding of low allelic diversity in the Florida manatee and

other manatee populations decreases the accuracy of parental assignments, and the identification

of more diverse microsatellite sites may allow for pedigree fingerprinting studies that are more

precise. An additional 17 primers, designed originally for dugong populations hold potential for

manatee population applications as well (Hunter et al. 2009b).

Examination of neutral and functional markers will enhance genetic research by providing information on whole genomic assemblies, individual population distinctiveness, identification of specific genes and their regulation, and the organism’s response to environmental change.

“Next Generation” sequencing technology can identify genes for molecular systematics and

functional genomic analysis, as well as marker development. Darwin’s “On the Origin of

Species” (1859) identified basic rules of evolution that proceed randomly during natural

selection. Conservation genetics can instruct us on how to preserve species that are successful at

meeting adaptive challenges to environmental change (Frankham et al. 2002). Acting upon

those random events are contemporary anthropogenic processes. The effects of human-caused

environmental changes as well as society’s efforts to manage a population may conflict with the

natural evolutionary processes of a wildlife population (Munoz-Vinas 2004). Society often must

make decisions regarding wildlife populations based upon the best available science. More

rigorous and detailed studies will assist managers in making policy decisions that could have

long-term consequences. The protection of wildlife may require delineation of management

units (MUs), distinct population segments (DPSs), subspecies and species designations that are

compatible with laws to preserve biologically intact communities (Haig et al. 2006; USFWS

2007). The Manatee Core Biological Model (Runge et al. 2007a) is a useful tool for assessing

manatee population status based upon current understanding of annual variability in survival and

reproductive rates, demographic stochasticity (including changes in effective population size),

effects of changes in warm-water capacity, and catastrophes. A reduction in effective population

size will result in loss of genetic diversity (Frankham et al. 2002; Allendorf & Luikart 2007).

This loss of diversity will likely result in loss of the ability to adapt to environmental changes

(Frankham 1995; Franklin & Frankham 1998). Therefore, greater efforts should be maintained

to improve effective population size in the manatee (Runge et al. 2007b). As the human

population continues to grow, its impact on the environment and resources becomes even

greater, creating a need for rigorous scientific information on affected wildlife species (Reep &

Bonde 2006).

Why Study Manatees?

Migration has played an important role in manatees’ ability to adapt and respond to survival challenges, and accounts for much of their geographic distribution. Migration also is necessary for gene flow and can increase the genetic diversity. Manatees have persisted in environments where other fragile wildlife populations like the Florida panther, Florida red wolf, pallid beach mouse, and Caribbean monk seal have not thrived or have been completely extirpated (IUCN 2009; USFWS 2009b). Florida manatees have adapted recently to survival

among human populations, whereas their Antillean manatee counterparts are much more

secretive due to direct threats related to historical and continued hunting (O’Shea 1988; Deutsch

et al. 2007; Quintana-Rizzo & Reynolds 2007).

Typically, manatee populations have undergone significant fluctuations that have resulted

in low allelic diversity when compared to other wildlife populations (O’Brien et al. 1985;

O’Brien 2003). The observed low genetic diversity might have placed limits on their ability to

cope with some environmental and anthropogenic changes, but those limitations appear not to

have affected fecundity and survival of the Florida manatee. The Florida manatee’s resilience to

environmental pressures, coupled with the separation of populations by distance and vicariance,

has lead to interesting biological responses on the organismal level. For example, the patterns of

winter site fidelity of manatees in response to cold temperatures have lead to large numbers

utilizing warm water sites. Manatees in large aggregations spend time in close proximity to one

another, increasing the potential for disease transmission, through direct contact and through

coprophagy. Understanding the relationship among manatee genetics and manatee behavioral

responses and adaptive capabilities will help managers direct the recovery of the species.

The consensus among researchers is that the Florida manatee population has grown in

numbers in recent decades. The initial reaction of managers is that the Florida manatee

population is doing well. However, evidence of limited genetic diversity suggests that the manatee population may not be as healthy as predicted based on just the number of individuals

(Hunter 2009 pers. comm.). Lack of allelic diversity could have deleterious effects. Still, some wildlife populations, such as the Northern elephant seal, have been able to thrive despite severe historical bottlenecks and low allelic diversity (Hoelzel et al. 1993; Slade et al. 1998). Whether this will be the case for manatees has yet to be determined.

Genetic data have been instrumental in the determination of listing criteria for several endangered species (Fallon 2007). A recent study on the genetic tools used to assess threatened and endangered populations of animals aptly illustrated that when multiple sources of genetic data were used in concert, there was a higher probability for the organisms to receive protection

(Fallon 2007). The data from genetics, coupled with other demographic sources of information, are necessary for determining which population units or stocks will benefit optimally with applied management (Dizon et al. 1992).

Genetic Markers

A host of genetic markers and tools are available to researchers to aid in determining population status. The selection of the appropriate marker depends on several variables and on the animal under investigation. Considerations for selection criteria include: (1) Is the selected marker appropriate to answer the questions at hand? (2) Are there limitations related to sample size and method of preservation? and, (3) What are the budget restrictions if any? Many analyses are expensive, but with recent technological advances, the cost has been reduced.

Several techniques are discussed in more detail below.

Allozymes

Historically, allozyme analysis was performed on the Florida manatee to examine geographic distribution patterns in five areas of Florida corresponding to carcass recovery

locations (McClenaghan & O’Shea 1988). High rates of gene flow accounted for the

homozygosity observed across all five geographic regions analyzed in the study.

Mitochondrial DNA

Matrilineal haplotypes from sequenced mitochondrial DNA can yield information on

phylogenetics and evolutionary processes. The mitochondrial displacement loop (d-loop) has

been used consistently on manatee populations to date (Garcia-Rodriguez et al. 1998; Vianna et

al. 2006). A 410 bp segment has been most widely used. Cytochrome-b analysis also has been

implemented, and illustrated little variation in the Florida population, but the sample size was

small (Bradley et al. 1993). Sequencing other regions of the mitochondrial genome, such as the

cytochrome c oxidase 1 (CO1) gene, has been proposed. The CO1 gene has displayed typical

variation in many animal systems and is used in DNA barcode identification of species (Stoeckle

2003; Herbert et al. 2004). New sequencing technology will make mapping of the entire Florida manatee mitogenome possible in the near future, which will provide additional characters for

determining genetic variation among populations. Complete mapping of genomic mtDNA in

killer whales (Orcinus orca), for example, has provided information on variations among

populations that has lead to the discrimination of three ecotypes within the species (Morin et al.

2009). The Antillean manatee mitogenome was sequenced recently and will have value in

comparative research (Arnason et al. 2008).

Microsatellites

Microsatellites, also known as simple sequence repeats (SSRs), short tandem repeats

(STRs), or variable number of tandem repeats (VNTRs), are sequences of di, tri, tetra, or larger

tandem nucleotides and are an excellent tool for determining relatedness among individuals and

populations. These sites can vary in length at each locus among individuals. Microsatellites are

neutral markers passed down from both parents that provide contemporary information on

relatedness and population structure. They also can be used to identify individuals in

fingerprinting studies. This trait has been particularly beneficial in mark-recapture studies,

where genetic information from individuals is matched to life-history observations and additional

samples that are acquired through time. This technique has been applied in the “cradle to grave”

tracking concept, modeling survivability and cause of death, wherein the analysis can identify

individuals’ genetic diversity in relation to health status classification and mortality events

(Tringali 2009 pers. comm.). Microsatellites are helpful in comparing allelic diversity among

populations, determining effective population size, and calculating the size of an original founder

population (Frankham et al. 2002; Allendorf & Luikart 2007).

Microsatellite studies have been conducted extensively on the Florida manatee population

to determine structure (Garcia-Rodriguez 2000; Pause 2007). Microsatellites also have been

used to compare Florida manatees with other manatee populations (Cantanhede et al. 2005;

Vianna et al. 2006; Kellogg 2008). Microsatellite studies of other threatened West Indian manatee populations are needed.

SNPs

Single nucleotide polymorphisms (SNPs) are nucleotide variations at a single base which

also can aid in population genetic studies. SNPs are bi-parentally inherited and can be expressed

in both intron (noncoding) and exon (coding) regions. The presence and uniqueness of

polymorphic loci can yield information on taxonomy and individual identification, and have

proven useful in identifying populations with low genetic diversity (Vignal et al. 2002).

Furthermore, Restriction-site Associated DNA (RAD) studies can identify gene sequences and

large numbers of individual SNP sites (Baird et al. 2008). Identification of SNPs from genes

could provide functional information in addition to detecting subpopulations for demographic

analysis, enabling managers to implement meaningful conservation measures.

Gene Expression

Examination of gene expression and regulation in manatees will provide insight to health,

reproductive fitness, and early disease detection. SAGE (Serial Analysis of Gene Expression) is

an approach for analyzing the observed variation in gene expression among individuals and

populations (Velculescu et al. 1995). Understanding manatee immunology, susceptibility to disease, wound healing, bone resorption, dental or other adaptations, and response to cold stress

or red tide will inform researchers and managers. Also, examination of major histocompatibility

complex (MHC) genes can increase our understanding of population differences, genetic

diversity, disease defense, mate selection, and reproductive fitness (Bernatchez & Landry 2003;

Piertney & Oliver 2006). The variations in gene expression among individuals and populations

also can provide information on demographics and adaptive responses. Behavioral responses to

density-dependent pressures may affect breeding success, reproduction, dispersal, and disease

susceptibility. These tools can help us gauge the cumulative impacts of climate change (Thomas

et al. 2004). This advanced form of health assessment will allow scientists to develop predictive

modeling programs and study target animal response, which will enable a more informed

approach for management.

The development of manatee specific assays can provide information on the levels of gene

expression of manatees encountering various environmental stressors, such as red tide (Bossart et

al. 1998), cold stress (Bossart et al. 2003), and exposure to pollutants (Stockwell et al. 2002).

Such transcriptome or gene expression analyses can be used to develop on-site tests to evaluate

the health status of injured or impaired manatees. Research to detect gene expression can utilize

special microarray chip sets with unique sequences. These chips are coated with several

thousand DNA probes and when linked with a sample will generate a signal if hybridization

occurs. Gene expression research becomes even more pressing as the manatee population in

Florida responds to lower carrying capacity due to a reduction in available warm water sites for

artificial winter refugia with the imminent closing of power plants (Laist & Reynolds 2005;

Runge et al. 2007b). It is uncertain when artificial sources of warm water currently available for

Florida manatee use will disappear, but all agree that this will happen within the next couple of

decades.

Use of field tests for wild manatees could benefit diagnostic studies during manatee health

assessments. For example, a manatee caught in 2004 in Port of the Islands, Florida suffered

from a massive internal infection undetected at the time of capture. Tools have been designed clinically to detect inflammation in manatees using levels of serum amyloid-A (SAA), but assays are expensive and must be performed later in the lab (Harr et al. 2006). The manatee captured in

2004 was released with a radio tag but, unfortunately, died a month later. Had real-time

information on the acute level of SAA been available during the processing of the manatee, that

manatee could have been taken to a rehabilitation facility for monitoring and treatment (Rember et al. 2009).

Genomes

The Genome 10K Project provides whole-genome sequences from multiple vertebrate species for comparative purposes (Haussler et al. 2009). To date, 32 species of mammals have been genotyped and entered into a comparative database (Haussler et al. 2009). Proposed by the

Genome 10K Project, manatees have not been included in the species sequenced to date, but having total genome coverage would be extremely beneficial for the species and collaborative research.

Implications for Manatee Conservation

Genetic Samples

Current genetic analyses of many manatee populations are incomplete because of inadequate sample size. Manatee sample collection has evolved from opportunistic carcass collection to live animal biopsy. Biopsy techniques have improved from cattle ear notchers applied to tail margins (Appendix 1), to dermal skin scrapers (Carney et al. 2007), to biopsy needles (Davis et al. 2009). The collection of fecal samples also has led to innovative genetic applications in very remote areas of sirenian distribution where sample collection is extremely difficult (Tikel et al. 1994; Muschett et al. 2009). However, use of fecal material has limitations because the possibility of contamination and collection of multiple samples from the same individual may bias results. Additional samples obtained from remote areas throughout the range of manatees and analyzed with current genetic methods for assessing population structure also will be useful. Once a cache of samples is available, a host of options for genetic analyses exists.

Molecular Tools

Care should be exercised when selecting the appropriate set of markers. Studies have demonstrated that a more robust assessment is obtained when more markers are employed (Latta

2006). The discreteness (amount) and significance (type) of genetic analysis must be appropriate for the questions to be addressed (Fallon 2007). Microsatellites have been useful for delimiting population differences in many species (Quellar et al. 1993; Jarne & Lagoda 1996; Goldstein &

Pollock 1997). Species that have been separated for long periods of time generally require smaller data sets for analysis, whereas recent separation of species (such as the Florida manatee) require more samples and genetic characters (Fallon 2007). Generally, multiple genetic markers versus a single type of marker are favored. Neutral markers, versus genes that code for adaptive

variation under selection in the environment, change more quickly, making them good indicators

of population uniqueness (Merila & Crnokrak 2001; McKay & Latta 2002; Hoekstra et al. 2004).

Care should be taken to make sure that the genetic data are used in conjunction with ecologic,

geographic, morphologic, or life history data sets (Haig et al. 2006).

Technologies and tools for molecular analyses continue to evolve and many currently are

applied to understand the Florida manatee population. Allozymes, mtDNA, and microsatellites

demonstrated little to no genetic differences among the four existing MUs (McClenaghan &

O’Shea 1988; Bradley et al. 1993; Garcia-Rodriguez et al. 1998; Vianna et al. 2006; Pause

2007), however recent analyses using new statistical techniques are suggesting significant

differences among the four Florida MUs and the populations inhabiting the east and west coasts

(Tucker et al. 2009; Tringali 2009 pers. comm.). This also was supported when the Crystal

River manatee population was compared to the rest of Florida and cluster analysis identified two distinct groupings. When samples were compared between the Florida and Puerto Rico manatee populations, very distinct structure was observed (Hunter et al. 2009c), as expected, since the two groups represent sub-species. However, the Endangered Species Act only recognizes the

West Indian manatee throughout its range as one all-inclusive group for legal protection

(USFWS 2007). Thus, one benefit of genetic scrutiny may be the development of separate management strategies for each subspecies.

Genetic information currently is used when selecting release sites for captive manatees, to ensure that manatees from one area of Florida are not introduced into another. Presently, manatees typically are not moved intentionally from one coast to another, although through misguided efforts in the past this has occurred. Generally, captive manatees are released to

populations that, to the best of our knowledge, have similar genetic characteristics, with the

expectation of preserving beneficial local adaptations.

Recent genetic findings have suggested that there is low allelic diversity in the manatee

population in Florida (Pause 2007), likely the result of a founder event in the Florida manatees’

history. Additionally, Florida manatees have a low effective population (Ne) size (Hunter 2009

pers. comm.). This raises concerns for the well-being of the manatee population as a whole in

Florida, as many policies regarding their protection are based primarily on predicted population size. Additional studies are needed to examine population fitness and the impacts of inbreeding in the Florida manatee. Studies have shown that once a population drops below inbreeding

thresholds, detrimental effects can occur during stochastic change (Frankham et al. 1995).

Examination of genetic markers would be very useful in determining the resiliency of manatee

populations to perturbations leading to dramatic population fluctuations. Genetic markers also

can be utilized to determine evolutionary lineages with predictions of founder population size

and subsequent coalescent time calculation dating back to the event (Beaumont 2003).

Currently, no standards or guidelines are in place to assist researchers and managers with

the appropriate selection of genetic tools to study the status of wild populations (Fallon 2007).

As each wildlife population is different and warrants different considerations, standardization

among species is problematic. The information gained through additional genetic analyses,

however, would greatly enhance the possibilities for preservation of the populations of West

Indian manatees throughout their range. These tools would be useful for studies of other

trichechids and dugongids as well. The future direction of genetic research will utilize

sequencing modalities, implementation of large volumes of data, and will design tools to gauge

the health and fitness of individuals within populations of sirenians. New information will be provided to managers for implementation in manatee protection strategies.

100

CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS

Recovery of an Endangered Species in Florida

Research has paved the way for a better understanding of the Florida manatee. Research

legacies have formed the cornerstones for much of the past, continuing, and proposed research

directions. Several long-term data sets are proving to be very valuable, not only for interpreting the species’ biology, but also for designing tools to predict future scenarios through trend analysis. Genetics is one of those tools. Coupled with traditional field applications, molecular science is providing another discipline to enhance the knowledge of manatee biology.

Manatees have low genetic diversity, a long generation time, a low rate of reproductive replacement, and in Florida, as elsewhere, there are significant anthropogenic threats. The challenges confronting the manatee make recovery of the species even more tenuous.

Population viability trends are easily influenced by slight changes in life history parameters. The lack of diversity in the Florida manatee population suggests that manatees have gone through a bottleneck in recent evolutionary time (Soule 1985; Amos and Harwood 1998; Waldick et al.

2002), and likely reflects the recent colonization of Florida from stocks established in the

Caribbean Islands, as suggested by Domning (2001a).

One consequence of population reduction is loss of genetic diversity (Figure 5-1). Several

generations of severe inbreeding in a small subpopulation, or repeated crashes of the numbers of

animals in a subgroup, will result in a depletion of most of the genetic variation originally

present in the initially larger population (Wahlund 1928; Soule 1985; Okello et al. 2008b). It is

generally recognized that genetic variability is necessary for both adaptation to changing

environments and long-term survival of the species (Ralls et al. 1979; Frankel & Soule 1981;

Frankham et al. 2002; Allendorf & Luikart 2007). Strategies to preserve or increase genetic

101

diversity require knowledge of the distribution of variation within subpopulations, levels of gene

flow between these segments of the population, and mitigation of threats to the population.

West Indian manatee mitochondrial DNA (mtDNA) haplotypes indicate patterns of

evolution and phylogeography on a grand scale. Through mutations, molecular clocks give

inference as to the length of time populations have been separated from each other. However, mtDNA cannot answer the finer-scale questions regarding the diversity and movements of the

Florida manatee population (Garcia-Rodriguez et al. 1998; Garcia-Rodriguez 2000).

Furthermore, no population structure among geographic locations was detected in Florida using

mtDNA, as only one haplotype has been identified (Garcia-Rodriguez et al. 1998; Vianna et al.

2006; Hunter et al. 2009c). Additionally, mtDNA is maternally inherited, representing only the

female lineage, and therefore does not yield all the evidence to piece together sex-biased

dispersal influences.

Microsatellite nuclear DNA (nDNA) analyses reveal higher individual resolution in genetic

diversity and population structure as compared to mtDNA analyses. Microsatellite markers are

biparental and provide a contemporary vs. historic perspective of the population. The allelic variability at microsatellite loci is generally high enough to distinguish genetic relationships among closely-related populations (Allendorf & Luikart 2007). Microsatellite analyses can also ascertain paternity and reproductive success, determine pedigrees, mate selection, levels of inbreeding, effective population size (Ne), local gene flow, and aid in forensic analyses (Budowle

et al. 1991; Edwards et al. 1992; Bruford & Wayne 1993; Queller et al. 1993; Morin et al. 1994;

Allen et al. 1995; McConnell et al. 1995; Blouin 2003).

Building on previous work initiated by the USGS Sirenia Project, a panel of 18 primers

(Garcia-Rodriguez et al. 2001; Pause et al. 2007) has been utilized to conduct fine-scale genetic

102

structure and relatedness studies on the manatee populations in Florida (Tucker et al. 2009),

Belize (Hunter et al. 2009a), Mexico (Nourisson et al. 2009), and Puerto Rico (Hunter et al.

2009c). The fine-scale manatee population studies found low levels of genetic diversity and little sub-structuring among the four management units in Florida (Figure 5-2). The Puerto Rico study (Hunter et al. 2009c) compared the Puerto Rico population to the Florida population, since the populations are listed and managed together under the U.S. Endangered Species Act (ESA) by the U.S. Fish and Wildlife Service (USFWS 1982). The 2007 ESA Status Review suggested that the West Indian manatee be downlisted from endangered to threatened, primarily due to successful recovery efforts in Florida which have resulted in a recent increase in population size.

However, a highly significant differentiation was identified between Florida and Puerto Rico, suggesting little, if any, breeding and/or migration between the populations. This study by

Hunter and colleages indicated that the Puerto Rico population would benefit from a separate management plan that also considered the unique threats, habitats, and significantly smaller number of individuals.

In conjunction with the primers mentioned above, future studies will utilize an additional

18 primers (Tringali et al. 2008b) developed for Florida manatees (Trichechus manatus latirostris) and 17 primers (Broderick et al. 2007) developed for dugongs (Dugong dugon) which were determined to be polymorphic in the Florida manatee (Hunter et al. 2009b). These panels of microsatellites will be used to perform in-depth population and conservation genetic studies detailed below.

Manatee Analysis using 454 GS-20TM Pyrosequencing Technology

To illustrate the utility of new molecular technology, a sample of manatee blood was collected from a 34 year-old captive-born female manatee, Lorelei, from Homosassa Springs

Wildlife State Park, Florida. Lorelei was born in 1975, conceived by two long-term captive

103

manatees, Romeo and Juliet, at Miami Seaquarium. Her blood sample was centrifuged and the

buffy coat containing white blood cells was isolated and placed into blood lysis buffer solution

(100 mM Tris-HCl, 100 mM EDTA, pH 8.0, 10 mM NaCl, 1% SDS) at room temperature for

storage (White and Densmore 1992). Genomic DNA was isolated and submitted to the

University of Florida, Interdisciplinary Center for Biotechnology Research (UF, ICBR), to be

prepped for pyrosequencing with the Roche 454 Genome Sequencer (GS-20) using FLXTM

standard technology (Roche 2009). Titration sequence data produced segments of approximately

250 bases in length, which were generated and stored in massive text files. These data were run

through a based algorithm bioinformatics program, PERL SCRIPT (Temnykh et al. 2001; modified

by W. Farmerie, University of Florida, ICBR). The PERL SCRIPT program identifies segments of

nucleotide repeats in the sequence data based on parameters set by the operator. Further data

manipulation analysis allows investigators to sort the data and identify unique sequences with

adjoining flanking regions.

Using this stochastic approach in the GS-FLX titration analysis, over 700,000 bases of the

manatee genome were identified. PERL SCRIPT examined 2,200 reads of approximately 250 bases in length recording 347 di-, tri-, or tetra-nucleotide repeat sequences (Figure 5-3).

Screening only of available nucleotide data identified 120 di- (5-14 repeats), 71 tri- (4-7 repeats),

and 12 tetra- (4-6 repeats) nucleotide sequences that contained at least 40 bp of flanking region at

both the 5’ and 3’ ends (Figure 5-4; Table 5-1). In follow-up studies, flanking primers can then

be designed for the putative loci and used with representative samples from a manatee population

to detect the level of polymorphism. In one 10-hour run, this capability amassed more potential

polymorphic microsatellite loci than researchers have produced during the last 10 years using

enriched libraries, conventional cloning, and capillary electrophoresis-based sequencing tools.

104

Future Directions

The aims presented here are directly related to longstanding efforts by federal, state, and

private agencies to ensure the continued health and survival of the endangered Florida manatee.

These future research endeavors are supported by the collaborative efforts outlined in the stated

goals of the Cooperative Florida Manatee Genetics Working Group. This group guides genetic

research efforts to address concerns and priorities outlined in the Florida Manatee Recovery Plan

(USFWS 2001) by avoiding duplication of resources and efforts and provides a forum to

facilitate exchange of ideas.

By establishing the degree and pattern of genetic variation in the Florida population,

informed judgments can be made about the ability of manatees to withstand periodic catastrophic

losses, which have occurred or may be expected to occur due to assorted stresses, including prolonged cold weather, red tide events, or viral diseases. Manatee use of Florida habitat by the current population in winter and their dependence on warm-water refugia (Figure 5-2) are expected to change in the near future as artificial sources are reduced and natural springs and passive thermal refugia are utilized (Figure 5-5). Until now, management decisions, including establishment of habitat sanctuaries (USFWS 1979), have been based largely on increasing the population, but future recovery plans could be guided by data on the effective population size, the fitness of individuals and populations, the genetic diversity present in each geographic area, and the level of gene flow between areas. The studies detailed here will be conducted by the

USGS, Southeast Ecological Science Center (SESC) Conservation Genetics Laboratory.

Sequence the Manatee Transcriptome using Massively Parallel Pyrosequencing

The microsatellite studies conducted to date have revealed reduced genetic differentiation and poor resolution of structure in the West Indian manatee populations. Therefore, management and conservation of manatee populations would greatly benefit from the

105

comprehensive information provided by massively parallel pyrosequencing technologies. Most

wildlife species, including manatees, do not have genomic or expressed gene sequences

available. Massively parallel Roche 454 pyrosequencing is a highly useful technique to identify

expressed genes in non-model species like the manatee. Gene sequences and markers gleaned

from 454 pyrosequencing can be used in a variety of high-resolution studies, including the

taxonomic classification of species and subspecies and determining the genetic structure of

populations or geographically distributed groups. Comprehensive gene expression profiles can

be used to address the overall health and fitness of individuals within a population and the impact

of environmental stressors.

West Indian manatees lack genetic diversity, both at mitochondrial and nuclear loci,

making fine-scale population studies difficult. In fact, all T. manatus populations investigated to

date have lower microsatellite diversity than the average demographically challenged

mammalian population (HE = 0.5 to 0.6 and Aave = 6.9) affected by historical or long-term

harvesting, fragmentation, or pollution (DiBattista 2007). In comparison, large, undisturbed and

healthy mammalian populations have higher average diversity values (HE = 0.6 to 0.7 and Aave =

8.8) (DiBattista 2007; Garner et al. 2005). Therefore, more variable and informative tools are needed to address population structure, diversity, and fitness. As discussed, parallel pyrosequencing will be used to develop a transcriptome (or identify actively expressed nuclear genes) for the West Indian manatee to allow for the identification of large-scale functional genomic tools sensitive to adaptive population differences. This technique also can be used to study the genetics of the dugong and other manatee species (Tikel 1997; Cantanhede et al. 2005;

McDonald 2005; Vianna et al. 2006). Pyrosequencing, or sequencing-by-synthesis, using a

Roche 454 pyrosequencer, is a new technique detecting the release of pyrophosphate during the

106

nucleotide incorporation rather than traditional Sanger chain-terminator-based sequencing. This

method increases DNA sequence depth and transcriptome coverage, while decreasing costs,

labor, and time.

The development of a manatee transcriptome-based analysis on mass gene expression

would facilitate functional genomic studies (relating genetic sequence and biological function)

and gene expression profiling. Comparative gene expression profiles can identify adaptive

differences between recently diverged populations, candidate genes associated with the current

states of fitness, or differences in transcriptome expression in response to physical stressors such

as cold temperatures, brevetoxin poisoning, debilitation, or infection due to traumatic injury.

From this information, on-site health tests could quantify the degree of physical or

immunological stress the animal is experiencing in order to triage sick animals effectively.

Transcriptome-based information would enable molecular phylogenetic studies and single

nucleotide polymorphism (SNP) marker development. SNPs are often based on functional

genes, sensitive to fitness conditions or adaptive differences that can identify phylogenetic or

evolutionary divergence of groups. SNP markers are especially useful as they are less expensive

and easier to standardize than microsatellites, enabling more markers to be used and resulting in

greater resolution of diversity.

Improved population differentiation will permit evaluation of the relative importance and

validity of regions used for demographic benchmark criteria in the Florida Manatee Recovery

Plan (USFWS 2001). Additionally, functional markers with improved resolution will enable biologists to determine the factors which affect gene flow in the population and the possible

impacts of inbreeding. Health-related issues with regional significance could be identified.

107

Understanding these factors will allow us to ascertain how differential mortality could affect

population structure and genetic diversity.

Genetically Identify Individual Crystal River Manatees and Integrate Data with the Manatee Individual Photo-identification System for Capture-Recapture Studies

The integration of genotyping with the Manatee Individual Photo-identification System

(MIPS) data can improve the tools to examine manatee population parameters. The genetic

signature from microsatellite markers and SNPs can complement the MIPS (Beck & Reid 1995)

by improving the ability to estimate adult survival and reproductive rates and to assess population growth rates and trends in models. Specifically, multiple identification photographs

and genetic samples collected from individual manatees during different life stages and at death will benefit the program. Genetic identification of manatees without physical identifying features, such as scars, particularly for young calves, will allow capture-recapture analysis through subsequent sample collection opportunities. This also will enable determination of the identity of unscarred manatees, decomposed carcasses, and to a lesser extent manatees with scar patterns that have changed since initial documentation to the extent that a photographic match is difficult or impossible. Genetics can also be used to estimate an error rate for MIPS-based identifications. As determined by our current acceptable estimates of probability of identity

(P(ID)sib = 1.618E-03, P(ID)HW = 1.472E-06), researchers will have the ability to identify,

confidently and accurately, individual manatees in the Florida population using 18 microsatellite

markers (Hunter et al. 2009b). Sex of the animal if unknown also can be determined with

specially designed probes (McHale et al. 2008; Tringali et al. 2008b; Lanyon et al. 2009).

Working closely with researchers at the USGS, Sirenia Project, a conceptual capture-

recapture model developed by William Kendall (USGS) will jointly analyze the MIPS and

genetic data. This novel effort builds on the manatee stage-based matrix model (Runge et al.

108

2004) and provides the ability to track individuals from their first identification as a calf, through

the life stages, and terminating ultimately as a recovered carcass. The model is capable of

estimating survival rates at each life stage. It will also be capable of determining individual

breeding rates for females. Analysis will also allow for the partitioning of cause of death of

individuals into relative sources of mortality. Additionally, by integrating genetics, pedigree

studies can address factors such as the frequency of twins, whether twins are fraternal or

identical, the frequency of adoption, and the potential paternal component. Selected samples,

such as those from rehabilitated animals scheduled for release or animals rescued from areas

outside of Florida, will be subjected to microsatellite studies to identify their location of origin.

Since the manatee genetics sample collecting effort in Crystal River, Florida was begun by

the U. S. Geological Survey (USGS) Sirenia Project in 1989, more than 1,000 genetic samples from free-ranging manatees (mostly calves, but more recently adults) have been collected and maintained in archives (Appendix). To date, 300 samples have been genetically identified, and two calf samples have been successfully linked by genetics to known MIPS individuals. Genetic data collected from carcasses provided by the Fish and Wildlife Research Institute (FWRI) in

Florida are also available for future matching.

Examine the Effective Population Size (Ne) of the Current and Past Florida Manatee Population

Genetic diversity is critical to maintaining evolutionary potential and individual fitness.

The loss of genetic diversity can result in reduced adaptability and decreased population

persistence. Health or viability of the population or long-term persistence of the species is

quickly decreased in populations with low densities, limited genetic diversity, or high

anthropogenic impact. Indeed, many T. manatus populations have been determined to have low

mitochondrial DNA variation, and Florida has no variation (Vianna et al. 2006). Similarly, a

109

survey of 18 nuclear microsatellite loci by Hunter and colleagues (2009a; 2009c) has identified

limited diversity within Florida, Belize, and Puerto Rico (expected heterozygosity, HE = 0.41,

0.46, 0.45 and average number of alleles, Aave = 4.1, 3.4, and 3.9, respectively).

Another indicator of genetic diversity is the effective population size (Ne). Ne was proposed by Sewall Wright who helped to define theoretical population genetics and the current paradigm of evolutionary biology. Ne is defined as the number of breeding individuals in an

idealized population that would show the same amount of dispersion of allele frequencies under

random genetic drift or the same amount of inbreeding as the population under consideration

(Wright 1931). Ne can be calculated by the variance in allele frequencies or by fluctuations in

the inbreeding coefficient; the two are closely related. Many population parameters reduce Ne,

including variations in population size, unequal sex ratios, and overlapping generations.

Although the Florida manatee census size has had a recent positive trend, the low haplotype diversity and nuclear diversity values, A and HE, indicate that the population was

founded by only a few individuals, or has recently undergone a bottleneck (a severe reduction in

population size). It is therefore important to calculate the Ne to determine the overall genetic

health and long-term sustainability of the population. The Florida manatee Ne will be compared

to the Belize and Puerto Rico manatee populations to provide a reference point that may be a

more appropriate (i.e., biologically meaningful) number for managers to strive to maintain or

increase through management actions.

Examining the Evolution and Relatedness of Trichechids

The range of the West Indian manatee extends throughout tropical habitats where there is

unobstructed access to fresh water sources. Generally this is from Brazil through the Caribbean

and Central America to Florida. Throughout this range, manatees are negatively impacted by

habitat destruction and anthropogenically induced mortality. Effective management of imperiled

110

species requires detailed genetic and taxonomic information to identify species, subspecies, and

distinct populations. A study looking at mitochondrial DNA dispersal and relatedness in the

West Indian manatee identified three evolutionarily related clades (Vianna et al. 2006); however,

information addressing contemporary movement patterns is needed in order to promote healthy

ecological and evolutionary processes.

Conservation practices that protect and maximize the existing genetic diversity are needed

for manatee populations to recover. The Florida, Belize, and Puerto Rico populations have

reduced genetic diversity (Aave = 4.1, 3.4, and 3.9, respectively) as compared to the average hunted or fragmented mammalian population (Hunter et al. 2009a; 2009c). Presumably, other

manatee populations, which are smaller and have unsustainable conservation practices, possess

even lower diversity. The data generated in this study can help to identify imperiled manatee

populations in need of enhanced conservation efforts.

A proposed genetic study will genetically investigate West Indian manatee samples from

collaborators in at least 16 countries throughout the species’ range and compare their relatedness

and genetic diversity to existing data sets established from the Florida, Belize, Mexico, and

Puerto Rico populations. Effort is being employed to gather new samples and utilize the existing

200 archived samples held at the USGS Conservation Genetics Laboratory. Samples currently in

the USGS archive to include in the study are from the Bahamas, Brazil, Cayman Islands,

Colombia, Costa Rica, Dominican Republic, French Guiana, Guatemala, Guyana, Honduras,

Panama, and Venezuela. DNA extracted from fecal samples can also be used for genetic

analysis (Tikel et al. 1994; Muschett 2008; Muschett 2009). Microsatellite nDNA and mtDNA

will identify unique populations and genotype analyses will explore the genetic diversity and

identify regions in need of protection. Information also will be provided on the relationships of

111

populations, phylogeographical distributions, and taxonomic status for the identification of appropriate stocks or management units.

By examining the connectivity of threatened West Indian manatees we will better understand the connectivity of populations throughout the species’ range. This information will greatly assist in determining the proper management units for improved manatee conservation and recovery efforts. For example, the Belize manatee population is the largest in the Wider

Caribbean and may serve as a source for repopulation of other Caribbean countries. However, increased anthropogenic threats, such as pollution and development, could cause the population to decline, limiting the number of individuals able to migrate to other countries. Investigations in southern Belize, near the Guatemalan border, have detected diseased manatees and a severe loss of sea grass beds (a primary food source), possibly attributed to the intensifying shrimp farming industry along the coastal habitat utilized by manatees. With recent evidence of manatee poaching in Guatemala, information on local movements could launch additional protections for populations in both southern Belize and Guatemala (Quintana-Rizzo & Reynolds

2007).

Previous genetic studies have assisted in identifying management units for the conservation of manatee populations. With the Florida (T. m. latirostris) and Puerto Rico (T. m. manatus) manatees currently listed together under the U.S. Endangered Species Act, actions are being implemented to identify unique characters between these two populations that will lead to different management recovery plans (USFWS 2009a). A survey of microsatellite DNA identified highly significant differentiation between the Florida and Puerto Rico populations (FST

= 0.16, P < 0.001), indicating that each population should be considered a unique unit of management (Hunter et al. 2009c). This information has lead to consideration by the USFWS to

112

treat the Florida and Puerto Rico manatee populations as two separate distinct population segments when evaluating strategic management actions (USFWS 2009a). Similar analyses will be utilized to identify the correct unit of management and identification of stocks for other T. manatus populations.

Continue Collaboration on Wildlife Species with USGS Scientists and International Researchers to Further Genetic Capabilities

Our genetics capabilities provide the opportunity for collaborations among researchers within the USGS and with institutions outside of the Southeast Ecological Science Center

(SESC). In addition to the manatee component, the SESC Conservation Genetics Laboratory is working on other related projects in conjunction with fellow scientists. Two invasive species, the black carp and Burmese python, currently are being characterized genetically to determine relatedness and define the population structures of these species. Important questions, such as source and sink population dynamics and potential release sites will be addressed. These findings have significant management implications.

Currently, two graduate students are working with USGS staff on the population dynamics of manatees; one on the West African manatee (Trichechus senegalensis) and another on West

Indian (T. m. manatus) and Amazonian (T. inunguis) manatees. Additional collaborations with international sirenian researchers will provide much needed data to advance our understanding of sirenian biology. These collaborators include Drs. Janet Lanyon from the University of

Queensland, Australia; Helene Marsh, James Cook University, Australia; Benjamin Morales,

ECOSUR, Mexico; and Miriam Marmontel, Instituto de Desenvolvimento Sustentável

Mamirauá, Brazil.

113

Conclusions

An increase in habitat loss and high mortality are factors which threaten the future of the

Florida manatee. A low intrinsic reproductive rate, low natural population density, and high use of urbanized habitats make this species particularly vulnerable to human perturbations. One consequence of population reduction is loss of genetic diversity, and the reduced diversity leads to more reduction in population size. These factors combined synergistically will hasten extinction processes. Several generations of severe inbreeding in a small population or repeated crashes of the population leaving a small number of individuals can deplete most of the genetic variation from an initially larger population.

It is generally recognized that genetic variability is necessary for both adaptation to changing environments and long-term survival of a species. Strategies to preserve genetic diversity require knowledge of the distribution of variation within populations and among species. As the existing manatee population in Florida changes, allelic diversity is more difficult to predict (Figure 5-1). What lies in the future for the Florida manatee? Manatee numbers were reduced due to anthropogenic loss for centuries, and it will likely take many generations to resolve the genetic consequences. Scarcity of warm water habitat in the future may have severe consequences for the recovery of the species. As artificial sources of warm water are eliminated in the next few generations, local manatee distribution will be affected (Laist & Reynolds 2006).

The individual manatee will likely respond by adapting to smaller winter site fidelity clusters

(Figure 5-5). These tighter aggregations will be separated by greater distances, making breeding between individuals from each area less likely than it is today. Ensuring open travel corridors among these areas and providing ample habitat for manatees will help facilitate adequate gene flow.

114

The genetic tools described here will provide information on the frequency and success of

different lineages or family units in each geographic area, as well as the level of gene flow

among those areas. The studies described here will also establish a basis for examining disease

susceptibility among manatees. Knowledge of health and fitness of the manatee and associated

biological systems will help predict outcomes from exposure to anthropogenic sources, habitat

alterations, and climate change.

Definition of the Florida manatee population structure and reproductive parameters will have conservation implications. Management policies in-place and adopted for the future will benefit from recognition of family relationships within geographic or wintering groups as expressed by management units. For example, should depleted manatee populations be supplemented by translocations from other areas? Should a rehabilitated manatee be released in any appropriate location without regard to genetic consequences? Misdirected translocations could compromise the integrity of genetic differences which have accumulated over evolutionary time, however they can also benefit local populations. The definition of family units based on these genetic tools will help to avoid future inbreeding events, should animals be placed in captivity for long periods of time before they are returned into the wild.

The data gathered on manatee genetics over the last two decades are already being made available to managers for use in assessing future manatee recovery efforts. This molecular information is also helping reconstruct phylogenetic assumptions based on previous morphological characters and acquired traits. Similarities in elephant behavior and biology are also helping the field scientist better understand manatees and their abilities to respond to various challenges. Debates regarding the consequences of behavior (Bengtson 1981) and “scramble

promiscuity” (Anderson 2002) may yield information on extra-limital movements and changes in site

fidelity and home range boundaries. Recent evidence of over mating has occurred in Florida

115

manatees resulting in death of the target female. This possible density dependent event may have

influences on manatee social structure and migration. Misdirected captive releases and translocations of manatees may compromise the integrity of genetic differences established

between some areas. Certain depleted populations may require direct intervention through

supplementation and reintroduction programs to aid the fitness of the local gene pools, especially

in the Caribbean and Central and South America. Misdirected translocation without regard for maintenance of genetic diversity will have consequences and be detrimental to the local population. These activities should be closely monitored.

Is there a population separation between the east and west coasts of Florida and are they distinct evolutionary units? Data indicate that the amount of gene flow between the coasts is keeping populations from diverging into distinct evolutionary units. However, differences suggested by FST, and allelic frequencies show evidence of subtle population differentiation

between the east and west coasts of Florida (Garcia-Rodriguez 2000; Pause 2007; Tucker et al.

2009), but more information is needed. A reduction in effective population size will over time

affect the long-term maintenance of genetic diversity resulting in lower reproduction output and

survival rates (Frankham et al. 2002). Even with the resilience and plasticity of manatees, without management based intervention a low Ne will eventually lead to inbreeding and

extinction. Intervention will require human management responses to help ensure survival. This

may include removal of individuals to protect them from threats and potential disease contact,

thereby improving reproduction and survival. Some of these factors are currently being

employed, but with better understanding of complicated interactions between biological systems

and the environment, greater genetic diversity can be achieved with the reduction of threats and

translocation of individuals.

116

Manatee populations have evolved over geologic time and have persisted. Recent conditions and the anthropogenic pressures have changed some natural processes, thereby

threatening the manatee. Stringent conservation measures require our continued commitment and diligent attention. Especially, with their dependence on artificial warm water sources,

Florida manatees will need to alter their already compromised adaptive behaviors in order to

persist into the next century.

117

Table 5-1. List of di-, tri-, and tetra-nucleotides identified using FLX technology on manatee DNA with number of repeats for each sequence. Number of sequences Sequence Number of repeats 23 AC 5-14 13 AG 5-8 05 AT 5 14 CA 5-10 01 CG 6 09 CT 5-7 13 GA 5-7 08 GT 5-8 08 TA 5-8 06 TC 5-7 20 TG 5-9 13 AAC 4-6 03 AAT 4-5 02 AGA 4 01 AGC 4 01 ATC 4 02 ATT 4 04 CAA 4-5 03 CAC 4-5 01 CCG 4 02 CCT 4 01 CGC 4 01 CTA 4 04 CTC 4 01 CTT 4 01 GAA 4 02 GCC 4 04 GGA 4-7 03 GGT 4 01 GTA 4 01 GTG 5 05 GTT 4-6 01 TAA 4 01 TAT 4 01 TCC 4 01 TGC 6 04 TGT 4-6 01 TTA 6 02 TTC 4-5 04 TTG 4 05 AAAC 4-5 01 ATCC 6 01 CAAA 4 01 CATC 4 01 CTTC 5 01 TTAT 4 02 TTTG 4

118

Figure 5-1. Population size as compared to genetic diversity and future trends (adapted from Wahlund 1928). Population size and time are not scaled to specific events. How will the Florida manatee population respond with an anticipated loss of allelic diversity?

119

Figure 5-2. Current winter Florida manatee subpopulation designations (shaded area). Blue stars are natural warm water sources, yellow stars are passive (alternate) warm water sources, and red stars are artifical sources of warm water. Four management units are represented by shaded areas; Northwest (pink), Southwest (blue), upper St. Johns River (green), and Atlantic coast (yellow).

120

Number of sequences

250

200

150

100 Number of sequences containing repeats (n=346) 50

0 di‐ tri‐ tetra‐

Figure 5-3. Total number of di,- tri-, and tetra-nucleotides identified using FLX technology on manatee DNA, of 346 nucleotide repeats detected during one titration run.

Number of sequences 140

120

100

60 Number of sequences containing repeats with flanking regions 40 (n=203)

0 di‐ tri‐ tetra‐

Figure 5-4. Number of putative microsatellites with di,- tri-, and tetra-nucleotides with at least 40 bp of flanking region at each end using FLX technology on manatee DNA.

121

Figure 5-5. Potential winter Florida manatee subpopulation designations (shaded area) after the loss of artifical sources of warm water in winter. Blue stars are natural sources and yellow stars are passive (alternate) sources of warm water. Note the loss of artifical sources of warm water previously depicted by red stars and the anticipated shift in wintering population patterns.

122

APPENDIX TAIL NOTCH PROTOCOL

Protocol for Collection of Manatee Tissue Samples for Genetic Research – March 2009

Protocol used by research field staff to collect and preserve tissue samples while swimming with free-ranging manatees at Crystal River, Florida. Priorities are provided to rank the selection of target individuals to fit sampling design.

123

Protocol for Collection of Manatee Tissue Samples for Genetic Research – March 2009

Objectives 1) Provide support for the Manatee Individual Photo-identification System (MIPS) 2) Establish and maintain genotyping fingerprints for individual manatees 3) Document calves at early life stage for better age determination estimates 4) Provide basis for “cradle to grave” tracking 5) Evaluate age at first reproduction 6) Facilitate cow/calf dependency period through visible monitoring 7) Determine relatedness between cows and suspected calves 8) Assess male reproductive contribution through pedigree lines 9) Verify sex determination with molecular techniques

Animal Selection Priority for the collection of tissue samples from the tail margin of manatees is as follows: 1) Calves (helpful if sex of calf and MIPS ID of mother are determined) – see figure for site placement of cookie on calves 2) Adult manatees with scar features (MIPS) that have an old cookie – site is reserved 3) Adult manatees with scar features (MIPS) that do not have an old cookie – site is reserved 4) Adult female manatees with calf, with old cookie, and without scar features – site should correspond to location in figure 5) Adult female manatees with calf, without cookie and without scar features – site should correspond to location in figure

General rule is that it is always best to have samples from both calves and scarred cows when possible. Be discrete when collecting samples in areas populated with other divers. Though our efforts are research permitted, it is best to avoid situations where we would need to explain our actions under field scenarios.

124

Methodology All manatees should be completely photo-documented before collection of the sample. A cattle ear notcher, 20mm U-shaped, device should be used. Samples should be collected from the margin of the tail at a predetermined location (see figure – Tail Notch Locations above). If possible, a photograph of the tail should be taken after the sample has been collected. Once collected, the sample (cookie) should be removed from the notcher and taken to a dry place for processing (see figure 1). Remove a 4-5mm wedge of the sample from the inside edge of the cookie with a sterile blade, and place in a pencil labeled cryovial; be certain this piece includes white connective tissue (see figures 2&3). The sample should be kept on ice until it can be frozen. The remainder of the sample should be cut into two pieces and both halves placed into a labeled 20ml plastic vial containing prepared tissue buffer solution. That sample should be kept at a cool temperature until stored for long-term at room temperature. During any handling of samples it is best to wear gloves and take every precaution to ensure that you are not cross contaminating any tissues (see figure 4). Clean up any processing areas with diluted bleach solution after processing.

1) Clean preparation area 2) Slicing manatee tissue

3) Sliced manatee tissue 4) Placing sample in vial

125

Labeling Labels for any vials containing genetic material must consist of the sample ID number (2 digit year, 3 digit Julian date, and the sequential number). An example would be 09-055-1 for the first sample collected on 24 February 2009. Additional information on the size of the manatee, sex, site location abbreviation, and collector can also be on the label. More detailed notes must be written on the diver’s underwater slate, where information on the date and site location, cookie ID number, size of animal, sex, associated photographic frame numbers taken, and behaviors are recorded. To avoid confusion generated at different collection sites on the same day, when assigning sequential numbers make sure you coordinate with any other researchers before samples are finally labeled. Duplication of field ID numbers will complicate tracking identity of the sample.

Sampling Field Kits A field kit for sample collection should include the following supplies: a notcher, razor blades, sharps container, exam gloves, 1.0ml cryovials, 20ml tissue vials, pencil, permanent marker, cooler with ice, and diluted cleaner containing bleach. A copy of our federal research permit (MA791721) should be in the possession of each researcher. Any collected fecal samples should be placed in 100% EtOH (200 proof ethanol) in a labeled 50ml centrifuge tube for storage. Additional labeled 15ml centrifuge tubes are useful for freezing fecal samples as well. Kits should be well maintained and stored in environmentally stable conditions.

126

Successful Application Below are examples and scenarios for selection of application sites. The first picture is of a recently collected sample from a calf. The second is from a calf that has had a sample collected previously (note how the open area stretches out as the tail margin grows). The third example is of a MIPS distinguishable adult where the sample was previously collected from the reserved area. And the final image is of a distinct MIPS adult that had been sampled as a calf and has also more recently been sampled in the reserve area. Other notches in the tail evident in pictures 2 and 3 are unrelated to genetic sampling.

1) New sample from a calf 2) Manatee sampled previously as a calf

3) MIPS adult sampled in the reserved area 4) MIPS adult previously sampled as a calf (upper left margin) and sampled again from the reserve area

127

LIST OF REFERENCES

Allen PJ, Amos W, Pomeroy PP, Twiss SD (1995) Microsatellite variation in grey seals (Halichoerus grypus) shows evidence of genetic differentiation between two British breeding colonies. Molecular Ecology, 4, 653-662.

Alvarez-Aleman A, Powell JA, Beck CA (2007) First report of a Florida manatee documented on the North Coast of Cuba. SireNews (Newsletter of the IUCN/SSC Sirenia Specialist Group), 47, 9-10.

Allendorf FW, Luikart G (2007) Conservation and Genetics of Populations. Blackwell Publishing, Malden, Massachusetts. 642 pp.

Amos W, Harwood J (1998) Factors affecting levels of genetic diversity in natural populations. Philosophical Transactions of the Royal Society of London B, 353, 179-186.

Amos B, Hoelzel AR (1991) Long-term preservation of whale skin for DNA analysis. Report of the International Whaling Commission Special Issue, 13, 99-103.

Anderson PK (2002) Habitat, niche, and evolution of sirenian mating systems. Journal of Mammalian Evolution, 9, 55-98.

Arnason U, Adegoke JA, Gullberg A et al. (2008) Mitogenomic relationships of placental mammals and molecular estimates of their divergences. Gene, 421, 37-51.

Archie EA, Hollister-Smith JA, Poole JH (2007) Behavioural inbreeding avoidance in wild African elephants. Molecular Ecology, 16, 4138-4148.

Archie EA, Maldonado JE, Hollister-Smith JA (2008) Fine-scale population genetic structure in a fission-fusion society. Molecular Ecology, 17, 2666-2679.

Baird NA, Etter PD, Atwood TS et al. (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers. Public Library of Science One, 3, e3376.

Baker CS, Cooke JG, Lavery S et al. (2007) Estimating the number of whales entering trade using DNA profiling and capture-recapture analysis of market products. Molecular Ecology, 16, 2617-2626.

Barber EA (1982) Mound pipes. The American Naturalist, 16, 265-281.

Beaumont MA (2003) Estimation of population growth or decline in genetically monitored populations. Genetics, 164, 1139-1160.

Beck CA, Reid JP (1995) An automated photo-identification catalog for studies of the life history of the Florida manatee. In: Population biology of the Florida manatee. (eds. O’Shea TJ, Ackerman BB, Percival HF), pp. 120-134. National Biological Service Information and Technology Report 1. Washington, D. C. 128

Bengtson JL (1981) Ecology of manatees (Trichechus manatus) in the St. Johns River, Florida. PhD Dissertation, University of Minnesota, Duluth, Minnesota. 126 pp.

Bernatchez L, Landry C (2003) MHC studies in nonmodel vertebrates: what have we learned about natural selection in 15 years. Journal of Evolutionary Biology, 16, 363-377.

Besbeas P, Freenan SN, Morgan BJT, Catchpole EA (2004) Interating mark-recapture-recovery and census data to estimate animal abundance and demographic parameters. Biometrics, 58, 540-547.

Bianucci G, Carone G, Domning DP et al. (2008). Peri-Messinian dwarfing in Mediterranean Metaxytherium (Mammalia: Sirenia): evidence of habitat degradation related to the Messinian Salinity Crisis. Garyounis Scientific Bulletin, 5, 145-157.

Blouin MS (2003) DNA-based methods for pedigree reconstruction and kinship analysis in natural populations. Trends in Ecology & Evolution, 18, 503-511

Boekschoten GJ, Best MB (1988) Fossil and recent shallow water corals from the Atlantic islands off western Africa. Zoologishe Mededelingen (Leiden), 62, 99-112.

Bossart GD, Baden DG, Ewing RY, Roberts B, Wright SD (1998) Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: gross, histologic and immunohistochemical features. Toxicologic Pathology, 26, 276–282.

Bossart GD, Meisner RA, Rommel SA, Ghim S, Jenson AB (2002) Pathological features of the Florida manatee cold stress syndrome. Aquatic Mammals, 29, 9-17.

Boulanger J, McLellan BN, Woods JG, Proctor MF, Strobeck C (2004) Sampling design and bias in DNA-based capture-mark-recapture population and density estimates of grizzly bears. Journal of Wildlife Management, 68, 457-469.

Bradley JL, Wright SD, McGuire PM (1993) The Florida manatee - cytochrome-b DNA sequence. Marine Mammal Science, 9, 197-202.

Broderick D, Ovenden J, Slade R, Lanyon JM (2007) Characterization of 26 new microsatellite loci in the dugong (Dugong dugon). Molecular Ecology Notes, 7, 1275-1277.

Brownell RL, Ralls K, Reeves RR (1981) Report of the West Indian manatee workshop. In: The West Indian manatee in Florida. Proceedings of a workshop held in Orlando, FL, 27-29 March 1978 (eds. Brownell RL, Ralls K). Florida Department of Natural Resources, Tallahassee, Florida. l57 pp.

Bruford MW, Wayne RK (1993) Microsatellites and their application to population genetic studies. Current Opinion in Genetics & Development, 3, 939-943.

129

Budowle B, Chakraborty R, Giusti AM, Eisenberg AJ, Allen RC (1991) Analysis of the VNTR Locus D1S80 by the PCR followed by high-resolution PAGE. American Journal of Human Genetics, 48, 137-144.

Calbeek R, Smith TB (2003) Ocean currents mediate evolution in island lizards. Nature, 426, 552-555.

Cantanhede AM, da Silva VMF, Farias IP et al. (2005) Phylogeography and population genetics of the endangered Amazonian manatee, Trichechus inunguis Natterer, 1883 (Mammalia, Sirenia). Molecular Ecology, 14, 401-413.

Carney SL, Bolen EE, Barton SL et al. (2007) A minimally invasive method of field sampling for genetic analyses of the Florida manatee (Trichechus manatus latirostris). Marine Mammal Science, 23, 967-975.

Carr A (1986) The Sea Turtle: So Excellent a Fishe. University of Texas Press, Austin, Texas. 280 pp.

Carstens BC, Knowles LL (2007) Shifting distribution and speciation: species divergence during rapid climate change. Molecular Ecology, 16, 619-627.

Dakin EE, Avise JC (2004) Microsatellite null alleles in parentage analysis. Heredity, 93, 504- 509.

Darwin C (1859) On the Origin of Species. John Murray, London, England. 484 pp.

Davis MC, Seyoun S, Carney SL et al. (2009) This won't hurt a bit: new molecular tools for population assessment of the Florida manatee. Abstract. Eighteenth Biennial Conference on the Biology of Marine Mammals, 12-16 October 2009, Quebec, Canada.

Deutsch CJ, Reid JP, Bonde RK et al. (2003) Seasonal movements, migratory behavior, and site fidelity of West Indian manatees along the Atlantic Coast of the United States. The Wildlife Society, Wildlife Monographs, No. 151, 1-77.

Deutsch CJ, Self-Sullivan C, Mignucci-Giannoni A (2007) Trichechus manatus. In: IUCN 2007.2007IUCN Red List of Threatened Species. Accessed 13 September 2009. Available from www.iucnredlist.org.

De Jong WW, Zweers A, Goodman M (1981) Relationship of aardvaark to elephants, hyraxes, and sea cows from α-crystallin sequences. Nature, 292, 538-540.

DiBattista JD ( 2007) Patterns of genetic variation in anthropogenically impacted populations. Conservation Genetics, 9, 141-156.

Domning DP (1983) Marching teeth of the manatee. Natural History, 92, 8-11.

130

Domning DP (1994a) A phylogenetic analysis of the Sirenia. Pages 177-189 in Contributions in Marine Mammal Paleontology Honoring Frank C. Whitmore, Jr. (eds. Berta A, Demere TA). Proceedings of the San Diego Society of Natural History, 29, 177-189.

Domning DP (1994b) West Indian tuskers. Natural History, 103, 72-73.

Domning DP (1997) Sirenia. In: Vertebrate Paleontology in the Neotropics: The Miocene Fauna of La Venta, Colombia (eds. Kay RF, Madden RH, Cifelli RL, Flynn JJ), pp. 383–391. Smithsonian Institution Press, Washington, District of Columbia.

Domning DP (2001a) Sirenians, seagrasses, and Cenozoic ecological change in the Caribbean. Palaeogeography, Palaeoclimatology, Palaeoecology, 166, 27-50.

Domning DP (2001b) The earliest known fully quadrupedal sirenian. Nature, 413, 625-627.

Domning DP (2005) Fossil sirenia of the West Atlantic and Caribbean region. VII. Pleistocene Trichechus manatus Linnaeus, 1758. Journal of Vertebrate Paleontology, 25, 685-701.

Domning DP, Hayek L-A (1986) Interspecific and intraspecific morphological variation in manatees (Sirenia: Trichechus). Marine Mammal Science, 2, 87-144.

Duffy CV, Glenn TC, Vargus FH, Parker PG (2009) Genetic structure within and between island populations of the flightless cormorant (Phalacrocorax harrisi). Molecular Ecology, 18, 2103-2111.

Eble JA, Toonan RJ, Bowen BW (2009) Endemism and dispersal: comparative phylogeography of three surgeonfishes across the Hawaiian Archipelago. Marine Biology, published online: 06 January 2009, 1-10.

Edwards A, Hammond H, Jin L, Caskey CT, Chakraborty R (1992) Genetic variation at five trimeric and tetrameric repeat loci in four human population groups. Genomics, 12, 244- 253.

Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology, 14, 2611–2620.

Excoffier L, Heckel G (2006) Computer programs for population genetics data analysis: A survival guide. Nature Reviews Genetics, 7, 745-758.

FWC (2009a) FWC records high counts during statewide manatee survey. Accessed 13 September 2009. Available from http://myfwc.com/NEWSROOM/09/statewide/News_09_X_ManateeSynoptic.htm

FWC (2009b) FWC Manatee Management Plan. Accessed 13 September 2009. Available from http://myfwc.com/docs/WildlifeHabitats/Manatee_MgmtPlan.pdf

131

Fallon SM (2007) Genetic data and the listing of species under the U.S. Endangered Species Act. Conservation Biology, 21, 1186-1195.

Felsenstein J (2004) PHYLIP (Phylogeny Inference Package) version 3.6, p. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, Washington.

Fertl D, Schiro AJ, Regan GT et al. (2005) Manatee occurrence in the northern Gulf of Mexico, west of Florida. Gulf and Caribbean Science, 17, 69-94.

Frankel OH, Soule ME (1981) Conservation and Evolution. Cambridge University Press, Cambridge, UK. 366 pp.

Franklin IR, Frankham R (1998) How large must populations be to retain evolutionary potential? Animal Conservation, 1, 69-73.

Frankham R (1995) Inbreeding and extinction - a threshold effect. Conservation Biology, 9, 792- 799.

Frankham R, Ballou JD, Briscoe DA (2002) Introduction to Conservation Genetics. Cambridge University Press, Cambridge, UK.

Garcia-Rodriguez AI (2000) Genetic studies of the West Indian manatee (Trichechus manatus). PhD dissertation, University of Florida, Florida. 115 pp.

Garcia-Rodriguez AI, Bowen BW, Domning D et al. (1998) Phylogeography of the West Indian manatee (Trichechus manatus): how many populations and how many taxa? Molecular Ecology, 7, 1137–114.

Garcia-Rodriguez AI, Moraga-Amador D, Farmerie W, McGuire P, King TL (2001) Isolation and characterization of microsatellite DNA markers in the Florida manatee (Trichechus manatus latirostris) and their application in Sirenian species. Molecular Ecology, 9, 2161-2163.

Garner A, Rachlow JL, Hicks JF (2005) Patterns of genetic diversity and its loss in mammalian populations. Conservation Biology, 19, 1215-1221.

Gheerbrant E, Domning DP, Tassy P (2005) Paenungulata (Sirenia, Proboscidea, Hyracoidea, and relatives). In: The Rise of Placental Mammals. (eds. Rose KD, Archibald JD), pp. 84- 105. The Johns Hopkins University Press, Baltimore, Maryland.

Glaser K, Reynolds JE III (2003) Mysterious Manatees. University Press of Florida, Gainesville, Florida. 188 pp.

Goldstein DB, Pollock DD (1997) Launching microsatellites: a review of mutation processes and methods of phylogenetic inference. Journal of Heredity, 88, 335–342.

132

Gray BA, Zori RT, McGuire PM, Bonde RK (2002) A first generation cytogenetic ideogram for the Florida manatee (Trichechus manatus latirostris) based on multiple chromosome banding techniques. Hereditas, 137, 215-223.

Haig SM, Beever EA, Chambers SM et al. (2006) Taxonomic considerations in listing subspecies under the U.S. Endangered Species Act. Conservation Biology, 20, 1584– 1594.

Hammer Ø, Harper DAT, Ryan PD (2001) PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica, 4, 9 pp.

Harlan R (1824) On a species of lamantin resembling the Manatus Senegalensis (Cuvier) inhabiting the coast of East Florida. Journal, Academy of Natural Sciences of Philadelphia, 3, 390-394.

Hartman DS (1971) Ecology and behavior of the American manatee, Trichechus manatus Linnaeus, at Crystal River, Florida. PhD Dissertation, Cornell University, Ithaca, New York. 285 pp.

Hartman DS (1974) Distribution, status, and conservation of the manatee in the United States. U. S. Fish and Wildlife Service, National Fish and Wildlife Laboratory Contract Report No. 14160008748. National Technical Information Service, #PB81 140725, Springfield, Virginia. 246 pp.

Hartman DS (1979) Ecology and Behavior of the Manatee (Trichechus manatus) in Florida. The American Society of Mammalogists, Special Publication Number 5. 153 pp.

Hatt RT (1934) A manatee collected by the American Museum Congo Expedition with observations on the recent manatees. Bulletin of the American Museum of Natural History, 66, 533-566.

Haussler D, Obrien SJ, Ryder OA (2009) Genome 10K: a proposal to obtain whole-genome sequence for 10,000 vertebrate species. Journal of Heredity, 100, 659-674.

Hedges SB (2001) Afrotheria: plate tectonics meets genomics. Proceedings of the National Academy of Sciences, 98, 1-2.

Herbert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of birds through DNA barcodes. Public Library of Science Biology, 2, e312.

Hoekstra HE, Drumm KE, Nachman MW (2004) Ecological genetics of adaptive color polymorphism in pocket mice: geographic variation in selected and neutral genes. Evolution, 58, 1329–1341.

133

Hoelzel AR, Halley J, O'Brien SJ et al. (1993) Elephant seal genetic variation and the use of simulation-models to investigate historical population bottlenecks. Journal of Heredity, 84, 443-449.

Hunter MK, Bonde RK, McCann S et al. (2009a) Conservation genetics of the Antillean manatee population in Belize: the last Caribbean stronghold. Biological Conservation, submitted.

Hunter MK, Broderick D, Ovenden J et al. (2009b) Characterization of highly informative cross- species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers. Molecular Ecology Resources, in press.

Hunter MK, King TL, Tucker KP et al. (2009c) Comprehensive genetic investigation recognizes evolutionary divergence in the Florida (Trichechus manatus latirostris) and Puerto Rico (T. m. manatus) manatee populations and subtle substructure in Puerto Rico. Molecular Ecology, submitted.

Irvine AB, Campbell HW (1978) Aerial census of the West Indian manatee, Trichechus manatus, in the southeastern United States. Journal of Mammalogy, 59, 613-617.

IUCN (2009) 2009.1 IUCN Red List of Threatened Species. Accessed 13 September 2009. Available from http://www.iucnredlist.org/

Jarne P, Lagoda PJL (1996) Microsatellites, from molecules to populations and back. Trends in Ecology & Evolution, 11, 424–429.

Kalinowski ST, Wagner AP, Taper ML (2007) ML-RELATE: a computer program for maximum likelihood estimation of relatedness and relationship. Molecular Ecology Notes, 6, 576- 579.

Kellogg ME (2008) Sirenian conservation genetics and Florida manatee (Trichechus manatus latirostris) cytogenetics. PhD Dissertation, University of Florida, Gainesville, Florida. 159 pp.

Kellogg ME, Burkett S, Dennis TR et al. (2007) Chromosome painting in the manatee supports Afrotheria and Paenungulata. BMC Evolutionary Biology, 7, 1-7.

Kendall WL, Langtimm CA, Beck CA, Runge MC (2004) Capture-recapture analysis for estimating manatee reproductive rates. Marine Mammal Science, 20, 424-437.

Kendall KC, Stetz JB, Roon DA et al. (2008) Grizzly bear density in Glacier National Park, Montana. Journal of Wildlife Management, 72, 1693-1705.

Kendall KC, Stetz JB, Boulanger JB, Macloed AC, Paetkau D, White GC (2009) Demography and genetic structure of a recovering grizzly bear population. Journal of Wildlife Management, 73, 3-17.

134

Laidre KL, Stirling I, Lowry LF et al. (2008) Quantifying the sensitivity of Arctic marine mammals to climate-induced habitat change. Ecological Applications, 18, S97-S125.

Laist DW, Reynolds JE (2005) Influence of power plants and other warm-water refuges on Florida manatees. Marine Mammal Science, 21, 739-764.

Langtimm CA, O’Shea TJ, Pradel R, Beck CA (1998) Estimates for annual survival probabilities for adult Florida manatees (Trichechus manatus latirostris). Ecology, 79, 981-997.

Langtimm CA, Beck CA (2003) Lower survival probabilities for adult Florida manatees in years with intense coastal storms. Ecological Applications, 13, 257-268.

Lanyon JM, Sneath H, Ovenden JR, Broderick D, Bonde RK (2009) Sexing sirenians: validation of visual and molecular sex determination in both wild dugongs (Dugong dugon) and Florida manatees (Trichechus manatus latirostris). Aquatic Mammals, 35, 187-192.

Latta RG (2006) Integrating patterns across multiple genetic markers to infer spatial processes. Landscape Ecology, 21, 809–820.

Lefebvre LW, Marmontel M, Reid JP, Rathbun GB, Domning DP (2001) Status and biogeography of the West Indian manatee. In: Biogeography of the West Indies: Patterns and Perspectives, Second Edition (eds. Woods CA, Sergile FE), pp. 425-474. CRC Press, Boca Raton, Florida.

Lightsey JD, Rommel SA, Costidis AM, Pitchford TD (2006) Methods used during gross necropsy to determine watercraft-related mortality in the Florida manatee (Trichechus manatus latirostris). Journal of Zoo and Wildlife Medicine, 37, 262-275.

Lynch M, Ritland K (1999) Estimation of pairwise relatedness with molecular markers. Genetics 152, 1753–1766.

Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statistical confidence for likelihood- based paternity inference in natural populations. Molecular Ecology, 7, 639-655.

Mayr E (1954) Geographic speciation in tropical echinoids. Evolution, 8, 1–18.

Mayr E (1970) Populations, species and evolution. Belknap 9, Cambridge, Massachusetts.

McClenaghan LR, O'Shea TJ (1988) Genetic variability in the Florida manatee (Trichechus manatus). Journal of Mammalogy, 69, 481-488.

McConnell SK, O’Reilly P, Hamilton L, Wright JM, Bentzen P (1995) Polymorphic microsatellite loci from Atlantic salmon (Salmo salar): genetic differentiation of North American and European populations. Canadian Journal of Fishery & Aquatic Science, 52,1863–1872

135

McDonald B (2005) Population genetics of dugongs around Australia: Implications of gene flow and migration. PhD Thesis, James Cook University, Townsville, Australia. 184 pp.

McHale M, Broderick D, Ovenden JR, Lanyon JM (2008) A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). Molecular Ecology Notes, 8, 669-670.

McKay JK, Latta RG (2002) Adaptive population divergence: markers, QTL and traits. Trends in Ecology & Evolution, 17, 285–291.

McKelvey KS, Schwartz MK (2004a) Genetics errors associated with population estimation using non-invasive molecular tagging: problems and new solutions. Journal of Wildlife Management, 68, 439-448.

McKelvey KS, Schwartz MK (2004b) Providing reliable and accurate genetic capture-mark- recapture estimates in a cost-effective way. Journal of Wildlife Management, 68, 453- 456.

Merila J, Crnokrak P (2001) Comparison of genetic differentiation at marker loci and quantitative traits. Journal of Evolutionary Biology, 14, 892–903.

Milligan BG (2003) Maximum-likelihood estimation of relatedness. Genetics, 163, 1153-1167.

Minch E, Ruiz-Linares A, Goldstein DB, Feldman M, Cavalli-Sforza LL (1997) MICROSAT, version 1.5d: A computer program for calculating various statistics on microsatellite allele data. Stanford University, Stanford, California.

Morin PA, Allen E, Pitman R et al. (2009) Population mitogenomics of killer whales: 2.5 million bp of DNA sequence yields global patterns of killer whale ecotype evolution. Abstract. Eighteenth Biennial Conference on the Biology of Marine Mammals, 12-16 October 2009, Quebec, Canada.

Morin PA, Moore JJ, Chakraborty R et al. (1994) Kin selection, social structure, gene flow and the evolution of chimpanzees. Science, 265, 1193-1201.

Munoz-Vinaz S (2004) Contemporary Theory of Conservation. Butterworth-Heinemann, second edition, Oxford, United Kingdom. 256 pp.

Muschett G (2008) Distribution and genetic studies of the manatee (Trichechus manatus) in the Panama Canal basin, Panama. MS thesis, Pontificia Universidad Catolica de Chili, Santiago, Chili. 83 pp.

Muschett G, Bonacic C, Vianna JA (2009) A noninvasive sampling method for genetic analysis of theWest Indian manatee (Trichechus manatus). Marine Mammal Science, 25, 955-963.

136

Nourisson C (2005) Development and utilization of microsatellite DNA markers on the Ten Thousand Islands population of the Florida manatee (Trichechus manatus latirostris). MS Thesis, Centre d’Océanologie de Marseille, Marseille, France. 32 pp.

Nourisson C, Morales-Vela B, Padilla-Saldivar J et al. (2009) Genetic diversity and population structure of manatee (Trichechus manatus manatus) in Mexico and implications for their conservation. Manuscript, in prep.

Novacek MJ (2001) Mammalian phylogeny: genes and supertrees. Current Biology, 11, R573- R575.

O’Brien SJ (2003) Tears of the Cheetah. Saint Martin’s Press, New York, New York. 287 pp.

O’Brien SJ, Roelke M, Marker L et al. (1985) Genetic basis for species vulnerability in the Cheetah. Science, 227, 1428-1434.

O'Shea TJ (1988) The past, present, and future of manatees in the southeastern United States: realities, misunderstandings, and enigmas. In: Proceedings of the Third Southeastern Nongame and Endangered Wildlife Symposium. Georgia Department of Natural Resources, Game and Fish Division, Social Circle, Georgia. 253 pp.

O'Shea TJ, Beck CA, Bonde RK, Kochman HI, Odell DK (1985) An analysis of manatee mortality patterns in Florida. Journal of Wildlife Management, 49, 1-11.

O'Shea TJ, Lefebvre LW, Beck CA (2001) Florida manatees: perspectives on populations, pain, and protection. In: Handbook of Marine Mammal Medicine, 2nd ed (eds Dierauf LA, Gulland FMD), pp. 31-43. CRC Press, Boca Raton, Florida.

Okello JBA, Masembe C, Rasmussen HB et al. (2008a) Population genetic structure of Savannah elephants in Kenya: conservation and management implications. Journal of Heredity, 99, 443-452.

Okello JBA, Wittemyer G, Rasmussen HB et al. (2008b) Effective population size dynamics reveal impacts of historic climatic events and recent anthropogenic pressure in African elephants. Molecular Ecology, 17, 3788-3799.

Ortiz RM, Worthy GAJ, Byers FM (1999) Estimation of water turnover rates of captive West Indian manatees (Trichechus manatus) held in fresh and salt water. The Journal of Experimental Biology, 202, 33-38.

Ozawa T, Hayashi S, Mikhelson VM (1997) Phylogenetic position of mammoth and Steller's sea cow within Tethytheria demonstrated by mitochondrial DNA sequences. Journal of Molecular Evolution, 44, 406-413.

Palumbi SR (1994) Genetic divergence, reproductive isolation, and marine speciation. Annual Review of Ecology and Systematics, 25, 547–572.

137

Pardini AT, O’Brien PCM, Fu B et al. (2007) Chromosome painting among Proboscidea, Hyracoidea and Sirenia: first molecular cytogenetic support for Paenungulata (Afrotheria, Mammalia). Proceedings of the Royal Society B-Biological Sciences, 274, 1333-1340.

Parr LA (2000) The position of Sirenia within the subungulates and comparisons of intra- and inter-specific mitochondrial DNA variation in extant species of manatees. PhD Dissertation, Portland State University, Portland, Oregon. 156 pp.

Pastor T, Garza JC, Allen P, Amos W, Aguilar A (2004) Low genetic variability in the highly endangered Mediterranean monk seal. Journal of Heredity, 95, 291-300.

Pause KC (2007) Conservation genetics of the Florida manatee, Trichechus manatus latirostris. PhD Dissertation, University of Florida, Gainesville, Florida. 177 pp.

Pause KC, Nourisson C, Clark A et al. (2007) Polymorphic microsatellite DNA markers for the Florida manatee (Trichechus manatus latirostris). Molecular Ecology Notes, 7, 1073- 1076.

Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in EXCEL. Population genetic software for teaching and research. Molecular Ecology Notes, 6, 288-295.

Piertney SB, Oliver MK (2006) The evolutionary ecology of the major histocompatibility complex. Heredity, 96, 7-21.

Pittman C (2007) Manatees get another reprieve. St. Petersburg Times news article accessed on 13 September 2009. Available at http://www.sptimes.com/2007/12/06/State/Manatees_get_another_.shtml

Powell JA (2002) Manatees. WorldLife Library, Voyageur Press, Stillwater, Minnesota. 72 pp.

Pritchard JK, Wen W (2004) Documentation for STRUCTURE software: Version 2. Department of Human Genetics, University of Chicago, Chicago, Illinois.

Pritchard JK, Wen X, Falush D (2009) Documentation for STRUCTURE software: Version 2.3. Available at http://pritch.bsd.uchicago.edu/software

Proebstel DS, Evans RP, Shiozawa DK, Williams RN (1993) Preservation of nonfrozen tissue samples from a salmonine fish Brachymystax lenok (Pallas) for DNA analysis. Journal of Ichthyology, 9, 9-17.

Quellar DC, Strassmann JE, Hughes CR (1993) Microsatellites and kinship. Trends in Ecology & Evolution, 8, 285–288.

138

Quintana-Rizzo E, Reynolds III J (2007) Regional management plan for the West Indian manatee (Trichechus manatus). Caribbean Environment Programme, United Nations Environment Programme, CEP Technical Report.

Rainey WE, Lowenstein JM, Sarich VM, Magor D (1984) Sirenian molecular systematics including the extinct Steller's sea cow (Hydrodamalis gigas). Naturwissenschaften, 71, 586-588.

Ralls K, Brugger K, Ballou J (1979) Inbreeding and juvenile mortality in samll populations of ungulates. Science, 206, 1101-1103.

Raymond M, Rousset F (1995) GENEPOP (version 1.2): Population genetics software for exact tests and ecumenicism. Journal of Heredity, 86, 248-249.

Rector A, Bossart GD, Ghim S-J et al. (2004) Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1. Journal of Virology, 78, 12698-12702.

Redi CA, Garagna S, Zuccotti M, Capanna E (2007) Genome size: a novel genomic signature in support of Afrotheria. Journal of Molecular Evolution, 64, 484-487.

Reep RL, Bonde RK (2006) The Florida Manatee: Biology and Conservation. University Press of Florida, Gainesville, Florida. xvi + 189 pp.

Rember R, Harr KE, Ginn PE et al. (2009) Boat strike and polycystic kidneys in a free-ranging Florida manatee (Trichechus manatus latirostris). Journal of American Veterinary Medical Association, submitted.

Reynolds JE III (1977) Aspects of the social behavior and ecology of a semi-isolated colony of the Florida manatee, Trichechus manatus. MS Thesis, University of Miami, Coral Gables, Florida. 206 pp.

Reynolds JE III, Odell DK (1991) Manatees and Dugongs. Facts on File Press, New York, New York. 192 pp.

Rice WR (1989) Analyzing tables of statistical tests. Evolution, 43, 223-225.

Rocha LA, Bowen BW (2008) Speciation in coral-reef fishes. Journal of Fish Biology, 72, 1101- 1121.

Roche (2009) 454 Life Sciences Technology. Accessed 13 September 2009. Available from http://www.454.com/

139

Rodriguez-Lopez MA (2004) Phylogeography of manatees (Trichechus manatus) in Puerto Rico: A management tool for species conservation. MS Thesis, Universidad Metropolitana, San Juan, Puerto Rico. 52 pp.

Runge MC, Langtimm CA, Kendall WL (2004) A stage-based model of manatee population dynamics. Marine Mammal Science, 20, 361-385.

Runge MC, Sanders-Reed CA, Fonnesbeck CJ (2007a) A core stochastic projection model for Florida manatees (Trichechus manatus latirostris). U.S. Geological Survey Open-File Report 2007-1082. 41 pp.

Runge MC, Sanders-Reed CA, Langtimm CA, Fonnesbeck CJ (2007b) A quantitative threats analysis for the Florida manatee (Trichechus manatus latirostris). U.S. Geological Survey Open-File Report 2007-1086. 34 pp.

Savage RJG, Domning DP, Thewissen JGM (1994) Fossil Sirenia of the West Atlantic and Caribbean region. V. The most primitive known sirenian, Prorastomus sirenoides Owen, 1855. Journal of Vertebrate Paleontology, 14, 427-449.

Scheffer V (1972) The weight of the Steller sea cow. Journal of Mammalogy, 53, 912–914.

Shoshani J (1986) Mammalian phylogeny: comparison of morphological and molecular results. Molecular Biology and Evolution, 3, 222-242.

Simpson GG (1945) The principles of classification and a classification of mammals. Bulletin of the American Museum of Natural History, 85, 1-350.

Slade RW, Moritz C, Hoelzel AR, Burton HR (1998) Molecular population genetics of the southern elephant seal Mirounga leonina. Genetics, 149, 1945-1957.

Slatkin M (1995) A measure of population subdivision based on microsatellite allele frequencies. Genetics, 139, 457-462.

Soule ME (1985) What is conservation biology? Bioscience, 35, 727-734.

Springer MS, Amrine HM, Burk A, Stanhope MJ (1999) Additional support for Afrotheria and Paenungulata, the performance of mitochondrial versus nuclear genes, and the impact of data partitions with heterogeneous base composition. Systematic Biology, 48, 65-75.

Springer MS, Murphy WJ (2007) Mammalian evolution and biomedicine: new views from phylogeny. Biological Reviews, 82, 375-392.

Stanhope MJ, Smith MR, Waddel VG et al. (1996) Mammalian evolution and the interphotoreceptor retinoid binding (IRBP) gene: Convincing evidence for several superordinal clades. Journal of Molecular Evolution, 43, 83-92.

140

Stejneger L (1887) How the great northern sea cow (Rytina) became exterminated. American Naturalist, 21, 1047-1056.

Stockwell BR (2003) Chemical genetic screening approaches to neurobiology. Neuron, 36, 559- 562.

Stoeckle M (2003) Taxonomy, DNA and the bar code of life. BioScience, 53, 2-3.

Tabuce R, Asher RJ, Lehmann T (2008) Afrotherian mammals: a review of current data. Mammalia, 72, 2-14.

Takezaki N, Nei M (1996) Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA. Genetics, 144, 389-399.

Temnykh S, DeClerck G, Lukashova A et al. (2001) Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Research, 11, 1441-1452.

Thomas CD, Cameron A, Green RE et al. (2004) Extinction risk from climate change. Nature, 427, 145-148.

Tikel D, Blair D, Marsh HD (1994) Marine mammal faeces as a source of DNA. Molecular Ecology, 5, 456-457.

Tikel D 1997 Using a genetic approach to optimize dugong (Dugong dugon) conservation and management. PhD Dissertation, James Cook University of North Queensland, Townsville, Australia. 273 pp.

Tringali MD, Davis MC, Rodriguez-Lopez MA et al. (2008a) Simultaneous use of the X- and Y- chromosome genes SMCX, SMCY, and DBY for sex determination in the Florida manatee (Trichechus manatus latirostris). Marine Mammal Science, 24, 218-224.

Tringali MD, Seyoum S, Carney SL et al. (2008b) Eighteen new polymorphic microsatellite markers for the endangered Florida manatee, Trichechus manatus latirostris. Molecular Ecology Resources, 8, 328-331.

Tucker KP, Hunter MK, Bonde RK et al. (2009) The endangered Florida manatee (Trichechus manatus latirostris) has little genetic differentiation. Conservation Genetics, submitted.

USFWS (1979) Establishment of Manatee Protection Areas. Federal Register 44:60962.

USFWS (1982) Endangered and Threatened Wildlife and Plants (50CFR 17.11 and 17.12). U.S. Government Printing Office, Washington, DC.

USFWS (2001) Florida manatee (Trichechus manatus latirostris) recovery plan, third revision. USFWS, Atlanta, Georgia. 144 pp + appendices.

141

USFWS (2007) West Indian manatee (Trichechus manatus) 5-Year Review: Summary and Evaluation. USFWS, Southeastern Region, Atlanta, Georgia. 79 pp.

USFWS (2009a) Manatee Stock Assessment Report. Accessed 13 September 2009. Available from http://www.fws.gov/northflorida/Manatee/SARS/20090518_rpt_Draft_Florida_Manatee_ SAR.pdf

USFWS (2009b) Endangered Species Program. Accessed 13 September 2009. Available from http://www.fws.gov/endangered/

Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene expression. Science, 270, 484-487.

Vianna JA, Bonde RK, Caballero S et al. (2006) Phylogeography, phylogeny and hybridization in trichechid sirenians: implications for manatee conservation. Molecular Ecology, 15, 433–447.

Vignal A, Milan D, SanCristobal M, Eggan A (2002) A review of SNP and other types of molecular markers and their use in animal genetics. Genetics Selection Evolution, 34, 275-305.

Wagner AP, Creel S, Kalinowski ST (2006) Estimating relatedness and relationships using microsatellite loci with null alleles. Journal of Heredity, 97, 336-345.

Waits LP, Luikart G, Taberlet P (2001) Estimating the probability of identity among genotypes in natural populations: Cautions and guidelines. Molecular Ecology, 10, 249-256.

Waldick RC, Kraus SS, Brown M, White BN (2002) Evaluating the effects of historic bottleneck events: An assessment of microsatellite variability in the endangered, North Atlantic right whale. Molecular Ecology, 11, 2241-2250.

Wahlund S (1928). Zusammensetzung von Population und Korrelationserscheinung vom Standpunkt der Vererbungslehre aus betrachtet. Hereditas, 11, 65–106.

Weber DS, Stewart BS, Lehman N (2004) Genetic consequences of a severe population bottleneck in the Guadalupe fur seal (Arctocephalus townsendi). Journal of Heredity, 95, 144-153.

Wellehan JFX, Johnson AJ, Childress AL, Harr KE, Isaza R (2007) Six novel gammaherpesviruses of Afrotheria provide insight into the early divergence of the Gammaherpesvirinae. Veterinary Microbiology, 127, 249-257.

142

White JR, Harkness DR, Isaacks RE, Duffield DA (1976) Some studies on blood of Florida manatee, Trichechus manatus latirostris. Comparative Biochemistry and Physiology A- Physiology, 55, 413-417.

White PS, Densmore LD (1992) Mitochondrial DNA isolation. In: Molecular Genetic Analysis of Populations: A Practical Approach. (ed. Hoezel AR), pp. 29-58. Oxford University Press, New York, New York.

Williams ME, Domning DP (2004) Pleistocene or post-Pleistocene manatees in the Mississippi and Ohio River valleys. Marine Mammal Science, 20, 167-171.

Woodruff RA, Bonde RK, Bonilla JA, Romero CH (2005) Molecular identification of a papilloma virus from cutaneous lesions of captive and free-ranging Florida manatees. Journal of Wildlife Diseases, 41, 437-441.

Wright S (1931) Evolution in Mendelian populations. Genetics, 16, 0097-0159.

Wyss AR, Novacek MJ, McKenna MC (1987) Amino acid sequence versus morphological data and the interordinal relationships of mammals. Molecular Biology and Evolution, 4, 99- 116.

Yang F, Alkalaeva EZ, Perelman PL (2003) Reciprocal chromosome painting among human, aardvark, and elephant (superorder Afrotheria) reveals the likely eutherian ancestral karyotype. PNAS, 100, 1062-1066.

Yeh FC, Boyle T (1997) Population genetic analysis of co-dominant and dominant markers and quantitative traits. Belgian Journal of Botany, 129, 157.

143

BIOGRAPHICAL SKETCH

Robert Bonde was born in Hawthorne, California in 1953. Bob earned his BA in History

in 1977 and a Certificate in Environmental Studies in 1978 at California State University Long

Beach. Bob grew up in southern California before moving to Gainesville, Florida with his wife,

Cathy in 1978 where he was employed by the U.S. Fish and Wildlife Service as part of the

Sirenia Project. He continues to work for the Sirenia Project, now under the U.S. Geological

Survey, where he has been studying manatee biology for more than 31 years.

Bob continues to participate in studies of the life history of the manatee population in

Crystal River, Florida. He consults with the NOAA-Fisheries Working Group for Unusual

Marine Mammal Mortality Events on issues related to necropsy assessment of the stranded marine mammals and participates in the USFWS Manatee Rescue, Rehabilitation, and Release

Program. He stays involved in field radio telemetry and tracking studies, manatee genetic studies and biomedical health assessments, and international research projects and study design.

Bob chairs the Florida Manatee Genetics Research Working Group. In addition to over 70 technical and semi-technical scientific publications, Bob coauthored a book in 2006 with Dr.

Roger Reep, entitled, The Florida Manatee: Biology and Conservation.

Bob entered graduate school in 2004 under the mentorship of Dr. Peter McGuire with the

College of Medicine. Enrolled in the College of Veterinary Medicine (CVM), Department of

Physiological Sciences, he has been an active member of the CVM Aquatic Animal Program and

serves through a courtesy faculty appointment. Bob is always happy to share his knowledge of the manatee and to get into the field to discover many new and interesting things about manatees and their intertwined connection with our planet. When chicken wings are not available, he enjoys breakfast, lunch, and dinner at Taco Bell.

144