PERMANENT GENETIC RESOURCES: Eighteen new polymorphic microsatellite markers for the endangered Florida manatee, Trichechus manatus latirostris

Here we describe 18 polymorphic microsatellite loci for Trichechus manatus latirostris (Florida manatee), isolated using a polymerase chain reaction‐based technique. The number of alleles at each locus ranged from two to four (mean = 2.5) in specimens from southwest (n = 58) and northeast (n = 58) Florida. Expected and observed heterozygosities ranged from 0.11 to 0.67 (mean = 0.35) and from 0.02 to 0.78 (mean = 0.34), respectively. Departures from Hardy–Weinberg equilibrium occurred at two loci. There was no evidence of genotypic disequilibrium for any pair of loci. For individual identification, mean random‐mating and θ‐corrected match probabilities were 9.36 × 10^?7 and 1.95 × 10^?6, respectively.     

Signature information and individual recognition in the isolation calls of Amazonian manatees,Trichechus inunguis (Mammalia: Sirenia)

Acoustic signals are assumed to form the basis of manatee communication. Empirical evidence of individual vocal recognition has been reported. If manatees can recognize one another by acoustical means, it should be possible to identify individual vocal patterns. We recorded vocalizations of 14 individually housed Amazonian manatees and then digitized selected vocalizations, allowing seven variables to be measured and subjected to multivariate statistical treatment. Discriminant function analysis indicated that individuals can be separated on the basis of variables related to the fundamental frequency and signal duration. We observed significant differences in the vocal patterns between sexes and age classes. Females tended to have greater fundamental frequency and shorter note duration than males. Calves had shorter note durations and greater values for the fundamental frequency range than subadults and adults. An inverse relationship between total body length and fundamental frequency range suggests that the fundamental frequency becomes more defined as the animal ages. The similar individual patterns in the vocalizations of a mother and calf pair are discussed. Individual recognition by Amazonian manatees according to their vocal patterns is suggested through a preliminary playback experiment.     

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.     

Physical and morphological aspects of the eye of the manatee trichechus inunguis natterer 1883: (Sirenia: Mammalia)

The eyes and aspects of vision of the Amazonian manatee Trichechus inunguis NATTERER 1883: (Sirenia: mammalia) have been examined in five animals, in relation to previous findings for sirenians. This paper presents a review of previous findings confirming those upon gross ocular anatomy, retinal histology and ocular refraction. The confirmation of a primarily rod retina, high receptor: ganglion cell ratio and low refractive error under water were of note. A visual pigment based upon vitamin A₁ and a difference spectrum of λ max at 505 nm was in agreement with Dartnalls nomogram. The findings suggest a retina more suited to low light levels and at best, moderate visual acuity, though motion perception appears present and a low degree of binocular vision possible.     

Phylogeography and population genetics of Acanthophyllum squarrosum complex (Caryophyllaceae) in the Irano-Turanian region

Acanthophyllum squarrosum and two closely related species, A. heratense and A. laxiusculum (Caryophyllaceae), form a complex that covers parts of subalpine steppes of the Irano-Turanian (IT) region. In this study, we explored the genetic structure and phylogeography of this complex based on partial sequences of two chloroplasts (psbA–trnH and rpl32–trnL (UAG)) and two nuclear (EST24 and nrITS) DNA regions. We analysed 80 individuals from eight populations and detected 12 chloroplast haplotypes, 16 and eight nuclear alleles in EST24 and nrITS sequences, respectively. Phylogenetic trees and haplotype networks did not show distinct genetic groups in the complex and this could be explained by incomplete lineage sorting or introgression between species. Divergence time analysis revealed a Quaternary origin for A. squarrosum complex at approximately 1.8 million years ago (Mya) and the neutrality test results indicated that this complex experienced a recent population expansion. AMOVA analysis of the chloroplast regions showed a significant genetic differentiation among populations and low genetic differentiation within populations, but opposite results were found with nuclear markers, implying introgression between A. squarrosum complex populations.     

Functional morphology of the taste buds of Florida manatee,Trichechus manatus latirostris

The Florida manatee, Trichechus manatus latirostris, is a fully aquatic, threatened marine mammal for which increased understanding of their physiology, reproduction, and nutrition supports management decisions. Manatees may use taste to distinguish saltwater gradients, toxin detection, food assessment, and social interactions. This study sought to locate and characterize manatee taste buds comparing location, structure, number, and size to other species. Entire heads from manatees (6 males, 4 females) of various ages were obtained. The muzzle, tissue surrounding the nares, oral cavity, and epiglottis were examined grossly for pits and papillae. Tissues were examined using light and transmission electron microscopy. Within the predominant taste bud location, the tongue root, taste bud number was estimated using samples from four animals. The average number of taste buds within the tongue root was 11,534 (range 2,711–23,237) with sparse taste buds located on the soft palate and epiglottis. The location along the lateral surface of the tongue root and bordered by grooves, through which tastants could be easily transported, has functional significance. Large numbers of taste buds within the tongue root suggest that taste is an important component of manatee sensory systems and behavioral research would clarify this.     

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.     

Anatomy of the cecum of the West Indian manatee,Trichechus manatus (Mammalia,Sirenia)

Summary The anatomy of the cecum of the West Indian manatee (Trichechus manatus) is described at the macroscopic and microscopic levels. The cecum possesses a voluminous corpus ceci with two lateral outpocketings; two diverticulae (cecal horns) extend from the corpus at the vicinity of the ileocecal junction. An ampulla ceci is found distal to the corpus ceci, the two compartments communicating via a wide-opened orifice. The most striking microscopic finding is the mucosal covering of a keratinized stratified squamous epithelium overlying the crypts of Lieberkühn.An attempt is made to interpret the morphological observations in terms of the functional digestive process in the manatee, giving alternative suggestions concerning the significance of the cecum and colon in the nutritional status of the manatee.Abbreviations A Ampulla ceci - CC Corpus ceci - CH Cecal horns - I Ileum - C Colon The present investigation was supported by a grant from the Deutsche Forschungsgemeinschaft (La 325/7-1)The author is greatly indebted to the members of the Fish and Wildlife Service, Sirenia Project, Gainesville Station, Gainesville Florida: special thanks are extended to Dr. Thomas O'Shea for organizing this undertaking, to Cathy A. Beck and Robert K. Bonde for their generous hospitality, for allowing the author to participate in the salvaging project, which they carried out with great efficiency, and for help in collecting the material. Without this aid, this study could not have been completed     

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.     

Phylogeography and population history of the endangered golden sun moth (Synemon plana) revealed by allozymes and mitochondrial DNA analysis

A combination of allozyme and mitochondrial DNA markers were used to determine the contribution of recent and ancient causes of patterns of genetic variation within and among 46 populations of the endangered golden sun moth, Synemon plana. Allozyme analysis grouped the 46 populations into 5 major genetic clusters that corresponded closely with geographic location following a classic isolation-by-distance model. Phylogenetic analysis of 14 mtDNA haplotypes revealed two reciprocally monophyletic groups. One of these groups (containing 4 geographically distant populations) was clearly identified by allozyme analysis and represents a distinct evolutionary unit. The remaining 4 allozyme groups were not distinguishable by mtDNA analysis. The evidence suggests that the populations within these groups derived from a small founding population that underwent rapid demographic expansion in ancient times. This was followed by more recent population bottlenecks resulting from habitat fragmentation associated with the widespread introduction of agriculture into the region. The generally low levels of allozyme and nucleotide diversity within these populations support this hypothesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogeography and population genetics of the maize stalk borer Busseola fusca (Lepidoptera, Noctuidae) in sub-Saharan Africa

The population genetics and phylogeography of African phytophagous insects have received little attention. Some, such as the maize stalk borer Busseola fusca, display significant geographic differences in ecological preferences that may be congruent with patterns of molecular variation. To test this, we collected 307 individuals of this species from maize and cultivated sorghum at 52 localities in West, Central and East Africa during the growing season. For all collected individuals, we sequenced a fragment of the mitochondrial cytochrome b. We tested hypotheses concerning the history and demographic structure of this species. Phylogenetic analyses and nested clade phylogeographic analyses (NCPA) separated the populations into three mitochondrial clades, one from West Africa, and two--Kenya I and Kenya II--from East and Central Africa. The similar nucleotide divergence between clades and nucleotide diversity within clades suggest that they became isolated at about the same time in three different refuges in sub-Saharan Africa and have similar demographic histories. The results of mismatch distribution analyses were consistent with the demographic expansion of these clades. Analysis of molecular variance (amova) indicated a high level of geographic differentiation at different hierarchical levels. NCPA suggested that the observed distribution of haplotypes at several hierarchical levels within the three major clades is best accounted for by restricted gene flow with isolation by distance. The domestication of sorghum and the introduction of maize in Africa had no visible effect on the geographic patterns observed in the B. fusca mitochondrial genome.     

Phylogeography of the endangered Cathaya argyrophylla (Pinaceae) inferred from sequence variation of mitochondrial and nuclear DNA

Cathaya argyrophylla is an endangered conifer restricted to subtropical mountains of China. To study phylogeographical pattern and demographic history of C. argyrophylla, species-wide genetic variation was investigated using sequences of maternally inherited mtDNA and biparentally inherited nuclear DNA. Of 15 populations sampled from all four distinct regions, only three mitotypes were detected at two loci, without single region having a mixed composition (G(ST) = 1). Average nucleotide diversity (theta(ws) = 0.0024; pi(s) = 0.0029) across eight nuclear loci is significantly lower than those found for other conifers (theta(ws) = 0.003 approximately 0.015; pi(s) = 0.002 approximately 0.012) based on estimates of multiple loci. Because of its highest diversity among the eight nuclear loci and evolving neutrally, one locus (2009) was further used for phylogeographical studies and eight haplotypes resulting from 12 polymorphic sites were obtained from 98 individuals. All the four distinct regions had at least four haplotypes, with the Dalou region (DL) having the highest diversity and the Bamian region (BM) the lowest, paralleling the result of the eight nuclear loci. An AMOVA revealed significant proportion of diversity attributable to differences among regions (13.4%) and among populations within regions (8.9%). F(ST) analysis also indicated significantly high differentiation among populations (F(ST) = 0.22) and between regions (F(ST) = 0.12-0.38). Non-overlapping distribution of mitotypes and high genetic differentiation among the distinct geographical groups suggest the existence of at least four separate glacial refugia. Based on network and mismatch distribution analyses, we do not find evidence of long distance dispersal and population expansion in C. argyrophylla. Ex situ conservation and artificial crossing are recommended for the management of this endangered species.     

Phylogeography and population genetics of the cryptic bonnethead shark Sphyrna aff. tiburo in Brazil and the Caribbean inferred from mtDNA markers

Resolving the identity, phylogeny and distribution of cryptic species within species complexes is an essential precursor to management. The bonnethead shark, Sphyrna tiburo, is a small coastal shark distributed in the Western Atlantic from North Carolina (U.S.A.) to southern Brazil. Genetic analyses based on mitochondrial markers revealed that bonnethead sharks comprise a species complex with at least two lineages in the Northwestern Atlantic and the Caribbean (S. tiburo and Sphyrna aff. tiburo, respectively). The phylogeographic and phylogenetic analysis of two mitochondrial markers [control region (mtCR) and cytochrome oxidase I (COI)] showed that bonnethead sharks from southeastern Brazil correspond to S. aff. tiburo, extending the distribution of this cryptic species >5000 km. Bonnethead shark populations are only managed in the U.S.A. and in the 2000s were considered to be regionally extinct or collapsed in southeast Brazil. The results indicate that there is significant genetic differentiation between S. aff. tiburo from Brazil and other populations from the Caribbean (Φ_ST = 0.9053, P < 0.000), which means that collapsed populations in the former are unlikely to be replenished from Caribbean immigration. The species identity of bonnethead sharks in the Southwest Atlantic and their relationship to North Atlantic and Caribbean populations still remains unresolved. Taxonomic revision and further sampling are required to reevaluate the status of the bonnethead shark complex through its distribution range.     

Functional properties of hemoglobin and whole blood in an aquatic mammal,the Amazonian manatee (Trichechus inunguis)

• 1. Hematocrit (43%) and O₂ binding capacity (18.8 ml O₂/100 ml blood) ofTrichechus inunguis blood are low compared to the values for other diving mammals but are similar to those for land mammals.
• 2. Stripped manatee Hb is similar to human Hb A in its sensitivity to pH, 2,3-diphosphoglycerate and CO₂, but less sensitive to temperature and more prone to dissociate into dimers.
• 3. The unique Hill plots exhibit no cooperativity below 30% O₂-saturation indicating a highly stabilized T or low-affinity state(s); such asymmetric Hill plots together with biphasic O₂-binding kinetics could mean chain heterogeneity.
• 4. The pH dependence of oxygen binding by the apparent T state, hemoglobin as seen in the Hill plots, is enhanced by 2,3-diphosphoglycerate but eliminated by CO₂.

     

Phylogeography and conservation genetics of Eld's deer (Cervus eldi)

Eld's deer (Cervus eldi) is a highly endangered cervid, distributed historically throughout much of South Asia and Indochina. We analysed variation in the mitochondrial DNA (mtDNA) control region for representatives of all three Eld's deer subspecies to gain a better understanding of the genetic population structure and evolutionary history of this species. A phylogeny of mtDNA haplotypes indicates that the critically endangered and ecologically divergent C. eldi eldi is related more closely to C. e. thamin than to C. e. siamensis, a result that is consistent with biogeographic considerations. The results also suggest a strong degree of phylogeographic structure both between subspecies and among populations within subspecies, suggesting that dispersal of individuals between populations has been very limited historically. Haplotype diversity was relatively high for two of the three subspecies (thamin and siamensis), indicating that recent population declines have not yet substantially eroded genetic diversity. In contrast, we found no haplotype variation within C. eldi eldi or the Hainan Island population of C. eldi siamensis, two populations which are known to have suffered severe population bottlenecks. We also compared levels of haplotype and nucleotide diversity in an unmanaged captive population, a managed captive population and a relatively healthy wild population. Diversity indices were higher in the latter two, suggesting the efficacy of well-designed breeding programmes for maintaining genetic diversity in captivity. Based on significant genetic differentiation among Eld's deer subspecies, we recommend the continued management of this species in three distinct evolutionarily significant units (ESUs). Where possible, it may be advisable to translocate individuals between isolated populations within a subspecies to maintain levels of genetic variation in remaining Eld's deer populations.     

Description of the Oocysts of Two New Species of Eimeria (Apicomplexa: Eimerüdae) from the Florida Manatee, Trichechus manatus (Sirenia: Trichechidae)

Two new species of Eimeria (Apicomplexa: Eimerüdae) are described from the feces of the Florida manatee, Trichechus manatus latirostrts (Sirenia: Trichechidae). Oocysts of Eimeria manatus n. sp. are spherical to subsphencal, 11.8 × 10.7 (10.5–13.5 × 9.0–13.5) μ m , with a smooth, thin, bilayered wall; shape index (length/width) 1.1 (1.0–1.3). Micropyle and oocyst residuum absent; polar granule(s) usually present. Sporocysts are ovoid, 8.6 × 5.1 (8.0–9.5 × 5.0–5.5) μm, with thin, membrane–like walls and a knoblike Stieda body; shape index 1.7 (1.4–1.8). Sporozoites elongate, each with a large posterior refractile body. The sporocyst residuum consists of a small cluster or row of few to many small granules. Oocysts of Eimeria nodulosa n. sp. are spherical to subspherical, 15.6 × 14.7 (14.5–17.5 × 13.0–16.0) μm, with a distinctly bilayered wall; shape index 1.1 (1.0–1.2). Unsporulated and freshly sporulated oocysts often possess large, knob–like structures on the external surface of the oocyst wall that support a thin membrane or filament. Micropyle, oocyst residuum and polar granule absent. Sporocysts are ovoid, 10.6 × 5.9 (9.5–12.0 × 5.0–6.5) μm, with a smooth, thin wall and knob–like Stieda body; shape index 1.8 (1.5–2.1). Sporozoites granular and elongate, each with a large posterior refractile body. The sporocyst residuum consists of a loose aggregate or scattered mass of moderately sized granules.     

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.     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.     

Phylogeography and conservation genetics of the Columbia spotted frog (Rana luteiventris; Amphibia, Ranidae)

The Columbia spotted frog (Rana luteiventris) has a widespread distribution in western Canada and the western US, although the southern reach of its range is highly fragmented into several isolated populations. Threats from various factors have raised concerns regarding the long-term survival of many small, isolated populations. Here, we report a study designed to determine the phylogeographic and conservation genetic parameters of R. luteiventris in the western US. Mitochondrial DNA (mtDNA) sequences were examined for phylogeographic structuring using phylogenetic reconstruction methods, coupled with networking and nested clade analyses. These methods permitted a distinction to be made between historic and demographic forces acting to generate geographical patterning of genetic variation. Phylogenetic analysis revealed four geographically correlated monophyletic clades. Three of these clades correspond to well-defined, nonoverlapping geographical locations in the fragmented portion of the range. The other is comprised of all samples collected from the contiguous range and includes one isolate from northern Wyoming. Networking and nested clade analyses confirmed these results and revealed that historical processes, such as range expansion and vicariance, rather than recurrent gene flow are likely responsible for observed patterns of genetic variation. A measure of genetic variation (theta = 4N(e)mu) revealed that R. luteiventris populations in Utah have a relatively low amount of genetic variation compared with populations in the continuous portion of the range.