Fast and cost-effective single nucleotide polymorphism (SNP) detection in the absence of a reference genome using semideep next-generation Random Amplicon Sequencing (RAMseq).

Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing (RAMseq), a novel approach for fast and cost-effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction (PCR) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNPs produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.

Phylogeography of the smooth-coated otter (Lutrogale perspicillata): distinct evolutionary lineages and hybridization with the Asian small-clawed otter (Aonyx cinereus).

We investigated the phylogeography of the smooth-coated otter (Lutrogale perspicillata) to determine its spatial genetic structure for aiding an adaptive conservation management of the species. Fifty-eight modern and 11 archival (dated 1882-1970) otters sampled from Iraq to Malaysian Borneo were genotyped (mtDNA Cytochrome-b, 10 microsatellite DNA loci). Moreover, 16 Aonyx cinereus (Asian small-clawed otter) and seven Lutra lutra (Eurasian otter) were sequenced to increase information available for phylogenetic reconstructions. As reported in previous studies, we found that L. perspicillata, A. cinereus and A. capensis (African clawless otter) grouped in a clade sister to the genus Lutra, with L. perspicillata and A. cinereus being reciprocally monophyletic. Within L. perspicillata, we uncovered three Evolutionarily Significant Units and proved that L. p. maxwelli is not only endemic to Iraq but also the most recent subspecies. We suggest a revision of the distribution range limits of easternmost L. perspicillata subspecies. We show that smooth-coated otters in Singapore are L. perspicillata x A. cinereus hybrids with A. cinereus mtDNA, the first reported case of hybridization in the wild among otters. This result also provides evidence supporting the inclusion of L. perspicillata and A. cinereus in the genus Amblonyx, thus avoiding the paraphyly of the genus Aonyx.

wisepair: a computer program for individual matching in genetic tracking studies.

Individual-based data sets tracking organisms over space and time are fundamental to answering broad questions in ecology and evolution. A 'permanent' genetic tag circumvents a need to invasively mark or tag animals, especially if there are little phenotypic differences among individuals. However, genetic tracking of individuals does not come without its limits; correctly matching genotypes and error rates associated with laboratory work can make it difficult to parse out matched individuals. In addition, defining a sampling design that effectively matches individuals in the wild can be a challenge for researchers. Here, we combine the two objectives of defining sampling design and reducing genotyping error through an efficient Python-based computer-modelling program, wisepair. We describe the methods used to develop the computer program and assess its effectiveness through three empirical data sets, with and without reference genotypes. Our results show that wisepair outperformed similar genotype matching programs using previously published from reference genotype data of diurnal poison frogs (Allobates femoralis) and without-reference (faecal) genotype sample data sets of harbour seals (Phoca vitulina) and Eurasian otters (Lutra lutra). In addition, due to limited sampling effort in the harbour seal data, we present optimal sampling designs for future projects. wisepair allows for minimal sacrifice in the available methods as it incorporates sample rerun error data, allelic pairwise comparisons and probabilistic simulations to determine matching thresholds. Our program is the lone tool available to researchers to define parameters a priori for genetic tracking studies.

Evaluating the Phylogenetic Status of the Extinct Japanese Otter on the Basis of Mitochondrial Genome Analysis.

The Japanese otter lived throughout four main Japanese islands, but it has not been observed in the wild since 1979 and was declared extinct in 2012. Although recent taxonomic and molecular phylogenetic studies suggest that it should be treated as an independent species, International Union for Conservation of Nature Red List considers it as subspecies of Lutra lutra. Therefore, the taxonomic status of this species needs to be resolved. Here we determined the complete mitochondrial genome of two Japanese otters caught in Kanagawa and Kochi prefectures and five Eurasian otters (L. lutra). We reconstructed a molecular phylogenetic tree to estimate the phylogenetic position of the Japanese otter in Lutrinae using the Japanese otters and the other 11 Lutrinae species on the basis of ND5 (692 bp) and cytochrome b (1,140 bp) sequences. We observed that the two Japanese otters had close relationships with Eurasian otters, forming a monophyletic group (100% bootstrap probability). To elucidate detailed phylogenetic relationships among the Japanese and Eurasian otters, we reconstructed a maximum likelihood tree according to mitochondrial genome sequences (14,740 bp). The Japanese otter (JO1) collected in Kanagawa was deeply nested in the Eurasian otter clade, whereas the Japanese otter (JO2) collected in Kochi formed a distinct independent lineage in the Lutra clade. The estimated molecular divergences time for the ancestral lineages of the Japanese otters was 0.10 Ma (95%: 0.06-0.16 Ma) and 1.27 Ma (95%: 0.98-1.59 Ma) for JO1 and JO2 lineages, respectively. Thus, JO1 was identified as a member of L. lutra; JO2 represented the old Japanese otter lineage, which may be a distinct new species or subspecies of Lutra. We suggest that the ancestral population of the JO2 lineage migrated to Japan via the land bridge that existed between western Japanese islands and Asian continent at 1.27 Ma.

Non-Invasive Genetic Mark-Recapture as a Means to Study Population Sizes and Marking Behaviour of the Elusive Eurasian Otter (Lutra lutra).

Quantifying population status is a key objective in many ecological studies, but is often difficult to achieve for cryptic or elusive species. Here, non-invasive genetic capture-mark-recapture (CMR) methods have become a very important tool to estimate population parameters, such as population size and sex ratio. The Eurasian otter (Lutra lutra) is such an elusive species of management concern and is increasingly studied using faecal-based genetic sampling. For unbiased sex ratios or population size estimates, the marking behaviour of otters has to be taken into account. Using 2132 otter faeces of a wild otter population in Upper Lusatia (Saxony, Germany) collected over six years (2006-2012), we studied the marking behaviour and applied closed population CMR models accounting for genetic misidentification to estimate population sizes and sex ratios. We detected a sex difference in the marking behaviour of otters with jelly samples being more often defecated by males and placed actively exposed on frequently used marking sites. Since jelly samples are of higher DNA quality, it is important to not only concentrate on this kind of samples or marking sites and to invest in sufficiently high numbers of repetitions of non-jelly samples to ensure an unbiased sex ratio. Furthermore, otters seemed to increase marking intensity due to the handling of their spraints, hence accounting for this behavioural response could be important. We provided the first precise population size estimate with confidence intervals for Upper Lusatia (for 2012: N = 20 ± 2.1, 95% CI = 16-25) and showed that spraint densities are not a reliable index for abundances. We further demonstrated that when minks live in sympatry with otters and have comparably high densities, a non-negligible number of supposed otter samples are actually of mink origin. This could severely bias results of otter monitoring if samples are not genetically identified.

Efficacy of two canine distemper vaccines in wild Nearctic river otters (Lontra canadensis).

Canine distemper virus (CDV), a contagious morbillivirus, infects families in the order Carnivora, including Nearctic river otters (Lontra canadensis). As a preventative measure, vaccinations against CDV are frequently given to mustelids in captive environments. The Pennsylvania River Otter Reintroduction Project (PRORP) used wild-caught river otters to evaluate the efficacy and need for vaccinations against CDV as part of any reintroduction project. The objectives of this study were to: 1) evaluate the prevalence of exposure to CDV in wild river otters, 2) determine the immunologic response of river otters (i.e., seroconversion) after vaccination with a single (primary) vaccine dose compared to a second (booster) dose of Galaxy-D, a modified live-virus canine distemper (CD) vaccine (MLV CDV), and 3) determine the immunologic response after being vaccinated with a primary vaccination compared to a booster dose of Fervac-D, an MLV CDV. River otters were injected subcutaneously in the nape of the neck with their designated vaccine. Timeframes for collection of blood samples and/or injection of booster vaccines varied depending on the parameters of PRORP. Ten of the 22 river otters had positive prevaccination titer levels to CD. Both vaccines, Galaxy-D and Fervac-D, produced sufficient seroconversion or rise of titer levels (86% and 57%, respectively) to recommend the use of vaccines in wild river otters. Future studies are recommended to evaluate currently produced CD vaccines. Future research should also focus on the number of days required between administration of primary and booster vaccines to achieve sufficient immune response. If only a primary dose is required, then hard-release reintroduction projects for river otters could be recommended. If primary and booster vaccines are required then soft-release reintroduction projects should be recommended. Soft-release projects should include captive management periods that allow for appropriate vaccination intervals and boosters needed to maximize the probability of protection against CDV.

A new subspecies identification and population study of the Asian small-clawed otter (Aonyx cinereus) in Malay Peninsula and southern Thailand based on fecal DNA method.

Three species of otter can be found throughout Malay Peninsula: Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata. In this study, we focused on the A. cinereus population that ranges from the southern and the east coast to the northern regions of Malay Peninsula up to southern Thailand to review the relationships between the populations based on the mitochondrial D-loop region. Forty-eight samples from six populations were recognized as Johor, Perak, Terengganu, Kelantan, Ranong, and Thale Noi. Among the 48 samples, 33 were identified as A. cinereus, seven as L. sumatrana, and eight as L. perspicillata. Phylogenetically, two subclades formed for A. cinereus. The first subclade grouped all Malay Peninsula samples except for samples from Kelantan, and the second subclade grouped Kelantan samples with Thai sample. Genetic distance analysis supported the close relationships between Thai and Kelantan samples compared to the samples from Terengganu and the other Malaysian states. A minimum-spanning network showed that Kelantan and Thailand formed a haplogroup distinct from the other populations. Our results show that Thai subspecies A. cinereus may have migrated to Kelantan from Thai mainland. We also suggest the classification of a new subspecies from Malay Peninsula, the small-clawed otter named A. cinereus kecilensis.

Impact of population expansion on genetic diversity and structure of river otters (Lontra canadensis) in Central North America.

Populations of North American river otters (Lontra canadensis) declined throughout large portions of the continent during the early 1900s due to habitat degradation and unregulated trapping. River otters had been extirpated in North Dakota (ND), but the Red River Valley has since been recolonized, with potential source populations including the neighboring states of Minnesota or South Dakota, or the Canadian province of Manitoba (MB). We genotyped 9 microsatellite loci in 121 samples to determine the source population of river otters in the Red River Valley of ND, as well as to assess population structure and diversity of river otters in central North America. Overall, genetic diversity was high, with an average observed heterozygosity of 0.58. Genetic differentiation was low (F ST < 0.05) between river otters in ND and those of Minnesota, suggesting that eastern ND was recolonized by river otters from Minnesota. River otters from MB were genetically distinct from all other sampled populations. Low genetic differentiation (F ST = 0.044) between South Dakota and Louisiana (LA) suggested that reintroductions using LA stock were successful. The genetic distinctiveness of river otters from different geographic regions should be considered when deciding on source populations for future translocations.

Development of novel real-time TaqMan(®) PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring.

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real-time polymerase chain reaction assays using fluorescently labelled TaqMan(®) MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost-saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.

Feeding ecology of the Neotropical otter Lontra longicaudis in the Lower Arroio Grande River, southern Brazil.

The present study was based on the analysis of spraints (n = 294) collected between December 2005 and November 2007. The importance of each type of prey was determined by absolute and relative frequency. Niche breadth was also estimated. The results were reported as mean (± SD) of relative frequencies. Fish were the main prey found in spraints (57.8 ± 7.0%). Fish from Callichthyidae family were the main prey, followed by Cichlidae, the species Hoplias malabaricus and the family Characidae. The frequencies of other fish and taxa were lower than 13.9% (absolute frequency) and 5.6% (relative frequency). Despite significant seasonal variations, (P = 0.001), niche breadth values were low in all seasons (0.39 ± 0.11). Therefore, Lontra longicaudis presented a narrow niche breadth as a result of consistent fish predation. Based on these data, fish with rapid movement presenting territorial behavior and benthic fish with slow movements are the most consumed. Seasonal fluctuations are caused by the increase of secondary prey in the diet during some seasons.

Gene transcription in sea otters (Enhydra lutris); development of a diagnostic tool for sea otter and ecosystem health.

Gene transcription analysis for diagnosing or monitoring wildlife health requires the ability to distinguish pathophysiological change from natural variation. Herein, we describe methodology for the development of quantitative real-time polymerase chain reaction (qPCR) assays to measure differential transcript levels of multiple immune function genes in the sea otter (Enhydra lutris); sea otter-specific qPCR primer sequences for the genes of interest are defined. We establish a 'reference' range of transcripts for each gene in a group of clinically healthy captive and free-ranging sea otters. The 10 genes of interest represent multiple physiological systems that play a role in immuno-modulation, inflammation, cell protection, tumour suppression, cellular stress response, xenobiotic metabolizing enzymes, antioxidant enzymes and cell-cell adhesion. The cycle threshold (C(T)) measures for most genes were normally distributed; the complement cytolysis inhibitor was the exception. The relative enumeration of multiple gene transcripts in simple peripheral blood samples expands the diagnostic capability currently available to assess the health of sea otters in situ and provides a better understanding of the state of their environment.

Characterization of basal seminal traits and reproductive endocrine profiles in North American river otters and Asian small-clawed otters.

In this study, fecal samples were collected from 24 North American river (NARO) and 17 Asian small-clawed otters (ASCO) for 6-36 months and semen collected seasonally from NARO males (n=4/season) via electroejaculation. Our main objectives were to: (1) characterize endocrine parameters by longitudinal monitoring of fecal hormone metabolites and (2) investigate semen collection and basal seminal traits in NARO. NARO demonstrated a distinct seasonality in the spring, with females having a monoestrual estrogen elevation lasting 15.33+/-1.98 (mean+/-SEM) days and males peaking in testosterone production for 25.50+/-7.51 days. Pregnancy was characterized by 7-9 months of basal fecal progesterone, presumably corresponding to embryonic diapause, followed by a rapid increase over the final 68-73 days to term. Pseudopregnancy exhibited a similar late winter progesterone peak of 68-72 days, which could not be differentiated from pregnancy. Geographic latitude possibly influenced the timing of increased testosterone in males and increased progesterone in pregnant/pseudopregnant females. In ASCO, monitoring of fecal estrogens did not allow consistent detection of peak values associated with behavioral estrus. Both pregnancy and pseudopregnancy were characterized by a moderate rise in fecal progesterone for 14-16 days postovulation followed by a marked increase. Total gestation length was 67-77 days compared with 62-84 days for pseudopregnancy. In NARO, optimal sperm recovery and quality occurred only in the spring, corresponding with seasonal increases in testicular volume and fecal testosterone. These findings represent the first comprehensive information on normative endocrine and seminal traits in freshwater otter species.

Conservation genetics of the giant otter (Pteronura brasiliensis (Zimmerman, 1780)) (Carnivora, Mustelidae)

The giant otter (Pteronura brasiliensis) is an aquatic mammal of the Mustelidae family, endemic to South America. Its original distribution corresponds to the region from the Guyanas to Central-North Argentina, but it is extinct or on the verge of extinction in most of its historical range. Currently, the species is considered endangered by the World Conservation Union (IUCN). Based on its geographic distribution in the South American continent and on some morphological characters, two subspecies were suggested: P. brasiliensis brasiliensis, occurring in the Amazon and Orinoco River Basins, and P. brasiliensis paranensis, in the Paraná and Paraguai River Basins. However, there is no consensus on assuming this subspecies division and no detailed studies have been carried out to elucidate this question. This study aims to evaluate the genetic diversity and population structure of Pteronura brasiliensis along its range in Brazil to check the possibility of the existence of two distinct subspecies using also a reciprocal monophyly criterion. We analyzed the control region, and the Cytochrome b and Cytochrome c Oxidase subunit I genes of the mitochondrial DNA in several giant otter populations from the Amazon and Paraguai River Basins. Analyses have indicated some degree of geographic correlation and a high level of inter-population divergence, although the subspecies division is not highly supported. As we observed strong population structure, we cannot rule out the existence of further divisions shaping the species distribution. The results suggest that a more complex population structure occurs in P. brasiliensis, and the conservation practice should concentrate on preserving all remaining local populations.

A ariranha (Pteronura brasiliensis) é um mamífero aquático da família Mustelidae, endêmico da América do Sul. Sua distribuição original se estendia desde as Guianas até o centro-norte da Argentina, mas está extinta ou à beira da extinção na maior parte de sua distribuição histórica. Atualmente a espécie é considerada como ameaçada de extinção pela World Conservation Union (IUCN). Em função de sua distribuição no continente sul-americano e de algumas características morfológicas, duas subespécies foram sugeridas: P. brasiliensis brasiliensis, com ocorrência nas bacias do Amazonas e Orinoco, e P. brasiliensis paranensis, ocorrendo nas bacias dos Rios Paraná e Paraguai. Inexiste, contudo, um consenso sobre a validade da divisão em subespécies e nenhum estudo detalhado foi realizado para elucidar esta questão. Este trabalho tem o objetivo de avaliar a diversidade genética e a estrutura populacional de P. brasiliensis ao longo de sua distribuição no Brasil para verificar a existência de duas subespécies baseando-se também em um critério de monofilia recíproca. A região controle e os genes do Citocromo b e da Subunidade I da Citocromo c Oxidase do DNA mitocondrial foram analisados em diversas populações de ariranha que ocorrem nas bacias dos rios Amazonas e Paraguai. As análises indicaram um grau moderado de correlação geográfica e um alto nível de divergência inter-populacional, embora a divisão em subespécies não seja bem sustentada. Como uma forte estruturação populacional foi observada, não é possível descartar a existência de outras subdivisões nesta espécie. Os resultados indicam a presença de uma estrutura populacional mais complexa em P. brasiliensis, o que implica que medidas de conservação deveriam concentrar seus esforços preservando todas as populações locais remanescentes.

Conservation genetics of the giant otter (Pteronura brasiliensis (Zimmerman, 1780)) (Carnivora, Mustelidae).

The giant otter (Pteronura brasiliensis) is an aquatic mammal of the Mustelidae family, endemic to South America. Its original distribution corresponds to the region from the Guyanas to Central-North Argentina, but it is extinct or on the verge of extinction in most of its historical range. Currently, the species is considered endangered by the World Conservation Union (IUCN). Based on its geographic distribution in the South American continent and on some morphological characters, two subspecies were suggested: P. brasiliensis brasiliensis, occurring in the Amazon and Orinoco River Basins, and P. brasiliensis paranensis, in the Paraná and Paraguai River Basins. However, there is no consensus on assuming this subspecies division and no detailed studies have been carried out to elucidate this question. This study aims to evaluate the genetic diversity and population structure of Pteronura brasiliensis along its range in Brazil to check the possibility of the existence of two distinct subspecies using also a reciprocal monophyly criterion. We analyzed the control region, and the Cytochrome b and Cytochrome c Oxidase subunit I genes of the mitochondrial DNA in several giant otter populations from the Amazon and Paraguai River Basins. Analyses have indicated some degree of geographic correlation and a high level of inter-population divergence, although the subspecies division is not highly supported. As we observed strong population structure, we cannot rule out the existence of further divisions shaping the species distribution. The results suggest that a more complex population structure occurs in P. brasiliensis, and the conservation practice should concentrate on preserving all remaining local populations.

Structure of the mitochondrial control region of the Eurasian otter (Lutra lutra; Carnivora, Mustelidae): patterns of genetic heterogeneity and implications for conservation of the species in Italy.

In this study we determined the complete sequence of the mitochondrial DNA (mtDNA) control region of the Eurasian otter (Lutra lutra). We then compared these new sequences with orthologues of nine carnivores belonging to six families (Mustelidae, Mephitidae, Canidae, Hyaenidae, Ursidae, and Felidae). The comparative analyses identified all the conserved regions previously found in mammals. The Eurasian otter and seven other species have a single location with tandem repeats in the right domain, while the spotted hyena (Hyaenidae) and the tiger (Felidae) have repeated sequences in both the right and left domains. To assess the degree of genetic heterogeneity of the Eurasian otter in Italy we sequenced two fragments of the gene and analyzed length polymorphisms of repeated sequences and heteroplasmy in 32 specimens. The study includes 23 museum specimens collected in northern, central, and southern Italy; most of these specimens are from extinct populations, while the southern Italian samples belong to the sole extant Italian population of the Eurasian otter. The study also includes all the captive-reared animals living in the colony "Centro Lontra, Caramanico Terme" (Pescara, central Italy). The colony is maintained for reintroduction of the species. We found a low level of genetic polymorphism; a single haplotype is dominant, but our data indicate the presence in central and southern Italy of two slightly divergent haplotypes. One haplotype belongs to an extinct population, the other is present in the single extant Italian population. Analyses of length polymorphisms and heteroplasmy indicate that the autochthonous Italian samples are characterized by a distinct array of repeated sequences from captive-reared animals.

Conservation genetics of carnivores in Italy.

Pleistocene climatic changes shaped the patterns of biodiversity in Europe and around the Mediterranean. Describing the phylogeographic structure of animal populations and inferring past population dynamics is essential to develop a framework for conservation biology in Europe. Direct persecution, habitat loss, population fragmentation and hybridization with domesticated conspecifics, are the main threats to the survival of large mammalian species. In this paper I will summarize the available information on phylogeography and population genetics of brown bear, wolf, wildcat and otters in Italy and in Europe.

Phylogenetic position of the Japanese river otter Lutra nippon inferred from the nucleotide sequence of 224 bp of the mitochondrial cytochrome b gene.

A 224 bp fragment of the mitochondrial cytochrome b gene has been amplified from a 30-year-old mummy-like specimen of the Japanese river otter Lutra nippon by polymerase chain reaction (PCR). The amplified products were subcloned in the Smal site of pUC 18 and sequenced. The sequence was different from those of the congeneric Eurasian otters Lutra lutra (Latvia) and Lutra lutra (China) in 7-9 nucleotides, all of which were located at the third position of a codon and identified as transitional differences A<-->G or C<-->T. The phylogenetic analysis using the 224 bp sequences of Lutra nippon, Lutra lutra (Lativa), Lutra lutra (China), Aonyx cinerea (Asian small-clawed otter), Mustela sibirica and Mustela itatsi (weasels) supports the recent morphological study that the Japanese river otter is not a subspecies of Lutra lutra, but a distinct species, Lutra nippon. We found that Lutra nippon and Lutra lutra contain the cytochrome b-like sequences, that appear to be a pseudo-form of cytochrome b gene. The sequences are characterized by the presence of deletion and termination codons by the presence of several types of sequences with minor variations, and by the faster evolutionary rate compared with that of the mitochondrial cytochrome b gene. The genes would present in the nuclear DNA rather than in the mitochondrial DNA, as in the case of the nonfunctional cytochrome b-like sequences previously reported in a rodent.