Phylogeography and population genetics of the endangered Amazonian manatee, Trichechus inunguis Natterer, 1883 (Mammalia, Sirenia)

We used mitochondrial DNA control region sequences to examine phylogeography and population differentiation of the endangered Amazonian manatee Trichechus inunguis. We observe lack of molecular differentiation among localities and we find weak association between geographical and genetic distances. However, nested clade analysis supports restricted gene flow and/or dispersal with some long-distance dispersal. Although this species has a history of extensive hunting, genetic diversity and effective population sizes are relatively high when compared to the West Indian manatee Trichechus manatus. Patterns of mtDNA haplotype diversity in T. inunguis suggest a genetic disequilibrium most likely explained by demographic expansion resulting from secession of hunting and enforcement of conservation and protective measures. Phylogenetic analysis of T. manatus and T. inunguis haplotypes suggests that T. inunguis is nested within T. manatus, effectively making T. manatus a paraphyletic entity. Paraphyly of T. manatus and recent divergence times of T. inunguis and the three main T. manatus lineages suggest a possible need for a taxonomic re-evaluation of the western Atlantic Trichechus.     

INTERSPECIFIC AND INTRASPECIFIC MORPHOLOGICAL VARIATION IN MANATEES (SIRENIA: TRICHECHUS)

The three living species of Trichechus are clearly defined and well exemplify the degree of variability and taxonomic value of morphological characters in a well-understood mammalian genus. Statistical analysis of the largest sample of manatee skulls yet studied has allowed us to identify small suites of characters that effectively distinguish these species. The two subspecies of T. manatus proposed by Hatt (1934) can likewise be distinguished, and their use as taxonomic categories seems justified. This suggests that the cool winters of the northern Gulf Coast, on the one hand, and the deep water and strong currents of the Straits of Florida, on the other, are effective barriers to gene flow between Florida and Antillean manatees. Alleged taxonomic distinctions within T. senegalensis , however, have no demonstrated basis. No significant sexual dimorphism was detected in skulls of any of the species. Many interspecific differences can be correlated with feeding ecology, but others remain unexplained. Features of tooth crown morphology are among the most constant characters examined, but some osteological characters are equally good. T. manatas and T. senegalensis (which are phenetically the most similar) also seem to share a more recent common ancestor than either does with T. inunguis. However, the three species probably separated from each other at nearly the same time. T. inunguis has since become the most derived species, while T. senegalensis has changed the least.     

Phylogeography of the West Indian manatee (Trichechus manatus): how many populations and how many taxa?

To resolve the population genetic structure and phylogeography of the West Indian manatee ( Trichechus manatus ), mitochondrial (mt) DNA control region sequences were compared among eight locations across the western Atlantic region. Fifteen haplotypes were identified among 86 individuals from Florida, Puerto Rico, the Dominican Republic, Mexico, Colombia, Venezuela, Guyana and Brazil. Despite the manatee's ability to move thousands of kilometres along continental margins, strong population separations between most locations were demonstrated with significant haplotype frequency shifts. These findings are consistent with tagging studies which indicate that stretches of open water and unsuitable coastal habitats constitute substantial barriers to gene flow and colonization. Low levels of genetic diversity within Florida and Brazilian samples might be explained by recent colonization into high latitudes or bottleneck effects. Three distinctive mtDNA lineages were observed in an intraspecific phylogeny of T. manatus , corresponding approximately to: (i) Florida and the West Indies; (ii) the Gulf of Mexico to the Caribbean rivers of South America; and (iii) the northeast Atlantic coast of South America. These lineages, which are not concordant with previous subspecies designations, are separated by sequence divergence estimates of d = 0.04–0.07, approximately the same level of divergence observed between T. manatus and the Amazonian manatee ( T. inunguis , n = 16). Three individuals from Guyana, identified as T. manatus , had mtDNA haplotypes which are affiliated with the endemic Amazon form T. inunguis . The three primary T. manatus lineages and the T. inunguis lineage may represent relatively deep phylogeographic partitions which have been bridged recently due to changes in habitat availability (after the Wisconsin glacial period, 10 000 BP ), natural colonization, and human-mediated transplantation.     

TLR4 and TLR8 variability in Amazonian and West Indian manatee species from Brazil

Amazonian (Trichechus inunguis) and West Indian (Trichechus manatus) manatees are aquatic mammals vulnerable to extinction found in the Amazon basin and the coastal western Atlantic. Toll-like receptors (TLR) play a key role in recognizing pathogen-associated molecular patterns using leucine-rich repeats (LRRs). We described the diversity of TLR4 and TLR8 genes in these two species of manatee. Amazonian manatee showed seven SNPs in TLR4 and the eight in TLR8, while West Indian manatee shared four and six of those SNPs, respectively. In our analysis, TLR4 showed one non-conservative amino acid replacement substitution in LRR7 and LRR8, on the other hand, TLR8 was less variable and showed only conserved amino acid substitutions. Selection analysis showed that only one TLR4 site was subjected to positive selection and none in TLR8. TLR4 in manatees did not show any evidence of convergent evolution compared to species of the cetacean lineage. Differences in TLR4 and TLR8 polymorphism may be related to distinct selection by pathogens, population reduction of West Indian manatees, or an expected consequence of population expansion in Amazonian manatees. Future studies combining pathogen association and TLR polymorphism may clarify possible roles of these genes and be used for conservation purposes of manatee species.     

Phylogeography and Sex-Biased Dispersal across Riverine Manatee Populations (Trichechus inunguis and Trichechus manatus) in South America

Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian (Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop (Control Region, Mitochondrial DNA) sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments.     

MORBILLIVIRUS INFECTION IN MANATEES

The first evidence of exposure of free-ranging Florida manatees ( Trichechus manatees latirostris ) to a morbillivirus is reported. Blood samples were collected from 148 Florida manatees between 1977 and 1994. The sample included manatees that were under rehabilitation (n = 97), free-ranging (n = 40) and fresh necropsy specimens (n = 11). Serum from six animals (4%) neutralized porpoise and dolphin morbilliviruses to a higher titer than any other member of the Morbillivirus genus. Antibodies were not detected in sera from 12 free-ranging Antillean manatees ( T. manatus manatus ) from Guyana sampled in 1992 or from 12 hand-reared Amazonian manatees ( T. inunguis ) sampled in 1984. Immunoprecipitation studies using radio-labelled canine distemper virus protein and serum from Florida manatees showed precipitation of the nucleo-capsid (N) protein. The combination of low antibody titers and absence of clinical disease suggest that the Florida manatee is a dead-end host. Sporadic infection may occur following contact with another species in which infection is enzootic. Morbillivirus could, either by fatal infection or more insidious effects on the immune system or reproduction, pose a problem to this already threatened species.     

A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus)

Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure.     

Cranial and chromosomal geographic variation in manatees (Mammalia: Sirenia: Trichechidae) with the description of the Antillean manatee karyotype in Brazil

We employed landmark‐based 3D geometric morphometrics to assess cranial size and shape diversification in Trichechus manatus and T. inunguis to compare it with patterns among all manatee taxa (T. manatus latirostris, T. m. manatus, T. inunguis and T. senegalensis), and to analyse geographic variation within American manatee populations, using a sample of 189 skulls. Chromosome G‐ and C‐banding techniques were performed in T. m. manatus from Brazil. All taxa were statistically discriminated by skull shape. Trichechus m. manatus and T. m. latirostris have larger skulls than T. inunguis. A morphological discontinuity was noted within T. m. manatus, with the Brazilian population south of the Amazon discriminated from the T. m. manatus Caribbean and T. m. latirostris USA populations. Specimens from Suriname and Guyana had a skull shape more similar to the one from the Caribbean population. The Brazil Antillean manatee population exhibited morphological differences similar in magnitude to those found between the Amazonian and African species. Additionally, structural chromosome differences were detected between that population (chromosome pair 4 is metacentric and 10 is submetacentric) and manatees from Puerto Rico and Florida. Based on such morphological discontinuity and chromosomal divergence, we hypothesize that the Amazon River mouth may be acting as a reproductive barrier for the T. m. manatus population in Brazil, thus indicating that its taxonomic status and conservation strategies need an urgent reassessment.     

HYDROSTASIS IN THE SIRENIA: QUANTITATIVE DATA and FUNCTIONAL INTERPRETATIONS

It has long been assumed that the heavy bones and horizontal lungs of sirenians are adaptations for maintaining neutral buoyancy and horizontal trim. These assumptions are examined in the light of detailed measurements of skeletal weight distribution in the Florida manatee ( Trichechus manatus latirostris ), measurements of the positions of body and skeletal centers of gravity in the Amazonian manatee (T. inunguis ), and available data on lung morphology in sirenians. The detailed arrangement of bones, muscle masses, and viscera in a sirenian ( T. inunguis ) is illustrated in cross sections for the first time. The hypotheses that skeletal weight is appropriately distributed to serve as hydrostatic ballast, and that the lungs are properly designed to help maintain horizontal trim, are confirmed. We suggest that selection for maintenance of trim and maximization of turning moments of the flippers may help account, respectively, for hindlimb loss and shortening of the neck in both sirenians and cetaceans. We recommend that the term "pachyostosis" be used to refer only to thickening of bones regardless of their density; that "osteosclerosis" be used for the replacement of cancellous with compact bone; and that "pachyosteosclerosis" be used for the joint occurrence of these conditions. Pachyosteosclerosis in sirenians, which may be brought about during ontogeny by mechanisms involving thyroid and parathyroid hormones, is fully normal and adaptive in this order and in no sense "pathological."     

Erythropoiesis and granulopoiesis in the West Indian manatee, Trichechus manatus (Mammalia: Sirenia)

The West Indian manatee, Trichechus manatus, has amedullary, pachyostotic long bones. Since marrow cavities of the long bones typically are the locations of hemopoiesis in adult mammals, the manatee has evolved an alternative primary site of hemopoiesis. Histological examinations of spleens, livers, kidneys, and vertebral bodies indicate that the last-named location is the main site of erythropoiesis and granulopoiesis in T. manatus. This conclusion is based on the presence in the vertebrae of bone-marrow-containing diagnostic cell types including erythroblasts, megakaryocytes, and myelocytes, which are the precursor cells of erythrocytes, platelets, and granular leukocytes, respectively. No developing lymphocytes were found, so that the location of lymphopoiesis remains unknown.     

LONG-TERM TRENDS IN ABUNDANCE AND DISTRIBUTION OF MANATEES (TRICHECHUS MANATUS) IN THE NORTHERN BANANA RIVER, BREVARD COUNTY, FLORIDA

Four aerial survey projects were conducted between 1977 and 1986 to determine the abundance, density and distribution of West Indian manatees (Trichechus manatus), in the northern Banana River, Kennedy Space Center, Florida. Manatee density and distribution within selected portions of the 78.5 km² study area were determined. Peak numbers of manatees occurred in spring of each year. The maximum counts increased from 56 in 1978 to 297 in 1986. Manatee abundance was lowest in the winter of each year. Mean density per flight increased from 0.52 manatees/km2 in 1977–78 to 2.73/km² in 1984–86. This increase may reflect increases in the east coast population or shifts in the population distribution. Distributional changes were observed in the study area through time, with a lower percentage of manatees occurring in industrial areas and a correspondingly higher percentage of manatees in nonindustrial areas by 1985.     

Gross and microscopic anatomy of the kidney of the West Indian manatee, Trichechus manatus (Mammalia: Sirenia)

Examination of the gross and microscopic anatomy of the kidney of the West Indian manatee, Trichechus manatus, revealed that: (1) the medulla is about 6 times thicker than the cortex; (2) juxtamedullary glomeruli have a mean diameter 1.3 times greater than that of cortical glomeruli; (3) juxtamedullary glomeruli have 1.7 times as much volume as cortical glomeruli; (4) there are about twice as many cortical glomeruli as juxtamedullary glomeruli per square millimeter of cortical tissue, and (5) the vasa recta are closely juxtaposed to the thin loops of Henle in the outer medulla. Many of these results suggest an enhanced urine-concentrating ability in this species.     

Echeneid–sirenian associations, with information on sharksucker diet

Association of the sharksucker Echeneis naucrates and whitefin sharksucker E. neucratoides with the West Indian manatee Trichechus manatus and the dugong Dugong dugon is presented and discussed. Alimentary tract-content analysis and field observations suggest that coprophagy (feeding on host faecal material) may be the primary food source for echeneids associated with sirenian hosts.     

The primary structure of the hemoglobin of the Brazilian manatee (Trichechus inunguis, Sirenia)

The hemoglobin of the Brazilian Manatee (Trichechus inunguis, Sirenia) consists of one component. We present the primary structures of the alpha- and beta-chains which have been separated by chromatography on carboxymethyl-cellulose CM-52. The sequences have been determined by automatic Edman degradation with the film technique, using the native chains, tryptic peptides and the C-terminal prolyl-peptide obtained by acid hydrolysis of the Asp-Pro bond of the alpha-chains. Compared to the corresponding human chains we found 27 substitutions in the alpha- as well as in the beta-chains. Three heme contacts and four alpha 1/beta 1 contacts between the subunits are affected by exchanges. The hemoglobin of Trichechus inunguis is compared with those of Elephas maximus, Loxodonta africana, and Procavia habessinica and the monophyletic origin of the superorder Paenungulata is discussed.     

The former southern distribution of New World manatees (Trichechus spp.)

Records of New World manatees in 16th and 17th century literature extend the former distribution of Trichechus manatus to about 20° S. Early 19th century records confirm this range, but the species has since been exterminated from these southerly localities and is now rarely found to the south of Cayenne (French Guiana). Manatees probably did not occur at Rio de Janeiro or Santos, the Sao Vicente "monster" most likely being a pinnepede. The former distribution of T. manatus seems to have been bounded by the 24^° C mean annual isotherm.     

Biology, conservation and status of the Amazonian Manatee Trichechus inunguis

The Amazonian Manatee Trichechus inunguis (Natterer, 1883) is the only exclusively freshwater sirenian and the smallest of the living species. Its inability to reduce peripheral heat loss limits its distribution to tropical waters. The species is endemic to the Amazonian region, being distributed throughout the Amazon basin, occurring mainly in calm waters and lakes. The species has been commercially exploited since 1542, with the meat and hide being the main products extracted. This commercial exploitation, combined with their very low reproductive rate, has seriously reduced the manatee population. Therefore the Amazonian Manatee is now considered an endangered species. Although it is protected by most of the countries where it occurs, there is no law enforcement, and the species is still captured throughout the Amazon. Data on general biology, physiology and management of the Amazonian Manatee, as well as its status and recommendations for its conservation, are presented.     

AGE DETERMINATION IN MANATEES USING GROWTH-LAYER-GROUP COUNTS IN BONE

Growth layers were observed in histological preparations of bones of known-age, known minimum-age, and tetracycline-marked free-ranging and captive Florida manatees ( Trichechus manatus latirostris ), substantiating earlier preliminary findings of other studies. Detailed analysis of 17 new case histories showed that growth-layer group (GLG) counts in the periotic bone were consistent with known age, or time since tetracycline administration, but were less reliable in other bones. GLG counts were also made in periotic bones of 1,196 Florida manatees of unknown age found dead from 1974 through 1991. These counts were conducted in order to assess variability and to determine relationships among estimated age, size, sex, and degree of bone resorption. Resorption can interfere with accuracy of GLG counts. This effect does not occur until ages greater than about 15 yr and body lengths greater than 300 cm are attained. GLGs were also observed in periotic bones of Antillean manatees ( Trichechus manatus manatus ) but were not validated against known-age specimens. Use of GLG counts in the periotic bone is suitable for application to studies of population dynamics and other age-related aspects of manatee biology.     

Polymorphic microsatellite DNA markers for the Florida manatee (Trichechus manatus latirostris)

Florida manatees (Trichechus manatus latirostris) are marine mammals that inhabit the coastal waters and rivers of the southeastern USA, primarily Florida. Previous studies have shown that Florida manatees have low mitochondrial DNA variability, suggesting that nuclear DNA loci are necessary for discriminatory analyses. Here we report 10 polymorphic microsatellite loci with an average of 4.2 alleles per locus, and average heterozygosity of 50.1%. These loci have been developed for use in population studies, parentage assignment, and individual identification.     

Omphalitis and peritonitis in a young West Indian manatee (Trichechus manatus)

Mortality data for the West Indian manatee (Trichechus manatus) indicates that from 1979 to 1984 16% of the recorded deaths involved young juveniles. Necropsy of a young manatee from the west coast of Florida revealed an active infection of the umbilical area (omphalitis) extending down the umbilical artery and veins. A generalized peritonitis was present. Bacterial cultures revealed Streptococcus faecium, Plesiomonas shigelloides, Pseudomonas putrefaciens and Escherichia coli.