Cross-species characterisation of polymorphic microsatellite loci in the giant otter (Pteronura brasiliensis)

Nineteen microsatellite loci developed for the Eurasian otter (Lutra lutra) and 15 loci developed for the North American river otter (Lontra canadensis) were tested for ease of amplification and degree of polymorphism on a set of 20 giant otter (Pteronura brasiliensis) faecal samples from the Bolivian Amazon basin. Nineteen loci amplified consistently well, with polymorphisms ranging from two to nine alleles and observed heterozygosity ranging from 0.15 to 0.85.     

Establishing the foundation for an applied molecular taxonomy of otters in Southeast Asia

Four species of otters (Mustelidae, Lutrinae) occur in Southeast Asia and are considered to be of conservation concern: Aonyx cinerea (Asian small-clawed otter), Lutra lutra (Eurasian otter), Lutra sumatrana (Hairy-nosed otter), and Lutrogale perspicillata (Smooth-coated otter). Among these, L. sumatrana is endemic to the region, yet little is known about its biology, and the precise distribution of all four species in Southeast Asia is not well known. Furthermore, the taxonomy and systematics of L. sumatrana and L. perspicillata have been the subject of controversy, which has implications for the legal protection and for conservation programs of these taxa. To resolve these controversies, we used a multigene data set comprised of segments from 13 nuclear and 5 mitochondrial loci (11,180 nucleotides) to evaluate the phylogenetic relationships of Asian Old World otters. Phylogenies were also estimated using two mitochondrial loci (1,832 nucleotides) obtained from two or more individuals of the four Southeast Asian species. The results from maximum parsimony, maximum likelihood and Bayesian inference showed that L. sumatrana and L. lutra are sister taxa, whereas L. perspicillata is sister to A. cinerea. Furthermore, the results from the two-mitochondrial gene analyses indicate that L. sumatrana is reciprocally monophyletic with respect to L. lutra, supporting the specific validity of the former taxon. Signs such as tracks and feces are often used in field surveys to provide information on the distribution and abundance of otters, but the accuracy of these methods may be compromised when several closely related species occur sympatrically. Therefore, the two-gene data set was used to develop a provisional set of diagnostic nucleotides that can be potentially used to identify the four species of Southeast Asian otters from noninvasively collected biological samples, such as feces. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Applied zooarchaeology and Oregon Coast sea otters (Enhydra lutris)

The sea otter (Enhydra lutris) was nearly driven to extinction on the Pacific Coast in the 19th century due to intensive commercial hunting and the maritime fur trade. Despite successful reintroduction efforts elsewhere in North America, the Oregon sea otter population remains locally extirpated and listed as endangered. Prior study addressed precontact sea otter teeth from Oregon and found they were not significantly different in absolute size from modern California sea otter (Enhydra lutris nereis) teeth, and smaller than modern Alaska sea otter (Enhydra lutris lutris) teeth. These geographic groupings were later confirmed by an ancient DNA study. The conclusion that distinct geographic populations exist based on tooth size was founded on small samples. Larger samples of teeth, as well as new data on humeri and femora, indicate dimensions vary significantly along a latitudinal cline from California to Alaska. Morphometric analyses of ancient animal remains can be used to examine spatial relationships of phenotypic features and inform conservation biology decisions.     

Molecular ecology of the Neotropical otter (Lontra longicaudis): non‐invasive sampling yields insights into local population dynamics

Non‐invasive genetic analysis has been frequently employed to estimate ecological and population parameters for many secretive and/or threatened species. However, Neotropical carnivores have so far been scarcely targeted by such studies. The Neotropical otter (Lontra longicaudis) is a poorly‐known species for which local levels of genetic diversity and demographic parameters are virtually absent. We employed non‐invasive sampling and amplification of microsatellite loci to investigate population size and density, spatial organization, and relatedness of a wild Neotropical otter population in an Atlantic forest area in southern Brazil. We directly identified 28 individuals and estimate a rather high population density at the study site. Spatial organization analysis indicated that male cumulative displacement was higher than that of females, with the latter sex showing evidence of philopatric behaviour. Also, the reconstruction of genealogical relationships suggests that spatial organization in this otter appears to be influenced by relatedness. By allowing the testing of specific hypothesis targeting these issues, our results provided important glimpses into the Neotropical otter's population biology. Moreover, the findings of the present study reaffirm the power of non‐invasive genetics to investigate the biology of this elusive species, and open up new avenues for ecological and demographic studies of other Neotropical carnivores. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 932–948.     

Development of polymorphic microsatellite loci for North American river otters (Lontra canadensis) and amplification in related Mustelids

In order to complement ecological research on native and reintroduced populations of the North American river otter (Lontra canadensis), we developed 10 polymorphic microsatellite loci that would permit us to evaluate population genetic structure and conduct fecal DNA analyses. The number of alleles per locus ranged from 3 to 14 and observed heterozygosity ranged from 0.23 to 0.68. Preliminary screening revealed that primers were polymorphic in a subset of other Mustelids.     

Sex identification of Eurasian otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) non-invasive DNA samples using ZFX/ZFY sequences

We developed a new PCR/RFLP system targeted to amplify and cut a segment of the ZFX/ZFY gene in non-invasive otter (Lutra lutra) samples. This assay produced one sex-specific fragment in females (XX genotypes) and two fragments in males (XY genotypes), and is intrinsically more reliable than alternative systems (e.g., SRY genes) that are based on the amplification of a single Y-linked fragment. The new primer pair correctly identified the sex of 23 DNA extracted from sexed otter tissues. This procedure was used to sex unknown DNA extracted from otter scats. A multi-tube approach led to identify the sex of 72/91 (79%) samples, using a minimum of two PCR replicates. This procedure is currently used in non-invasive genetic monitoring of Italian otter populations.     

Relative spraint density and genetic structure of otter (Lutra lutra) along the Drava River in Hungary

In this study, we used genetic-based approaches to estimate population size and structure of Eurasian otter along the Drava River in Hungary, and compared these results to traditional survey-based methods. The relative spraint density of otter was estimated based on the number of fresh (D_f) and total number (D_t) of spraints collected on standard routes over a 2-year period. Nine microsatellite loci were screened, generating 17 individual otter genotypes composed of 45 different alleles. The expected heterozygosity ranged from 0.53 to 0.89 and observed heterozygosity from 0.25 to 0.92. The mean density (D_g) estimated over six different sites was 0.17 individuals per km of shoreline. A close correlation was found between the number of genotypes and spraint counts along a standard route (fresh spraints: r_P=0.85, P<0.01; total spraints r_P=0.76, P<0.05). All genotypes found within the 50 km-long study area were closely related (D_m ranged between 0.08 and 0.21).     

River otter population size estimation using noninvasive latrine surveys

Across much of North America, river otter (Lontra canadensis) populations were extirpated or greatly reduced by the early 20th century. More recently, reintroductions have resulted in restored populations and the recommencement of managed trapping. Perhaps the best example of these river otter reintroductions occurred in Missouri, regarded as one of the most successful carnivore recovery programs in history. However, abundance estimates for river otter populations are difficult to obtain and often contentious when used to underpin management activities. We assessed the value of latrine site monitoring as a mechanism for quantifying river otter abundance. Analyses of fecal DNA to identify individual animals may result in an improved population estimate and have been used for a variety of mammal species. We optimized laboratory protocols, redesigned existing microsatellite primers, and calculated genotyping error rates to enhance genotyping success for a large quantity of river otter scat samples. We also developed a method for molecular sexing. We then extracted DNA from 1,421 scat samples and anal sac secretions (anal jelly) collected during latrine site counts along 22–34-km stretches representing 8–77% of 8 rivers in southern Missouri in 2009. Error rates were low for the redesigned microsatellites. We obtained genotypes at 7–10 microsatellite loci for 24% of samples, observing highest success for anal jelly samples (71%) and lowest for fresh samples (collected within 1 day of defecation). We identified 63 otters (41 M, 22 F) in the 8 rivers, ranging from 2 to 14 otters per river. Analyses using program CAPWIRE resulted in population estimates similar to the minimum genotyping estimate. Density estimates averaged 0.24 otters/km. We used linear regression to develop and contrast models predicting population size based on latrine site and scat count indices, which are easily collected in the field. Population size was best predicted by a combination of scats per latrine and latrines per kilometer. Our results provide methodological approaches to guide wildlife managers seeking to initiate similar river otter fecal genotyping studies, as well as to estimate and monitor river otter population sizes. © 2011 The Wildlife Society.     

Procedures to genotype problematic non-invasive otter (Lutra lutra) samples

Non-invasive genetics is a powerful tool in wildlife research and monitoring, especially when dealing with elusive and rare species such as the Eurasian otter (Lutra lutra). Nevertheless, otter DNA obtained from scats and anal secretions appears to be exposed to very quick degradation processes, and the success rate in DNA amplification is lower than in other carnivores. We collected 191 samples from April to September 2011 along the river Sangro basin (Italy) which was recently re-colonized by the Eurasian otter. Using two sets of microsatellite loci (six Lut and seven OT loci), we investigated the influence of sample type and age, collection time, storage time, temperature and humidity on genotyping success and amplification success. We also tested the efficacy of different DNA extraction kits and storage buffer mediums. Finally, we compared amplification success rate, allelic dropout and false allele rates for each locus. We obtained a mean amplification success rate of 79.0 % and a genotyping success rate of 35.1 %. Fresh pure jellies yielded the highest amplification success and genotyping rate. Six microsatellite loci should be theoretically sufficient to distinguish the individual unrelated otters (PID?=?0.001), while 13 loci were needed to distinguish sibling otters (PID_sibs?=?0.002) in our population. We identified 11 otters, and molecular sexing ascertained the presence of five males, four females and two uncertain individuals. Generalized linear models highlighted a significant influence of sample type and age, temperature and humidity both on genotyping and amplification success.     

Characterization of eight microsatellite loci in Sea Otter, Enhydra lutris, and cross-species amplification in other Mustelidae

Herein we describe the development of eight microsatellite markers for the northern sea otter, Enhydra lutris kenyoni. A total of 45 primer pairs were developed and screened from enriched AAAT, CATC, TACA, and TAGA libraries derived from genomic DNA of E. lutris kenyoni. Of these, eight amplified successfully. The average observed heterozygosity, expected heterozygosity, and number of alleles per locus was 0.506, 0.537, and 3.4, respectively. These eight loci were tested across three additional genra; Vulpes lagopus, Martes americana, and Mustela nivalis. Based on the success of our results these loci will be useful for future studies across all sub-species of E. lutris.     

Influence of occupation history and habitat on Washington sea otter diet

Habitat characteristics are primary determinants of nearshore marine communities. However, biological drivers like predation can also be important for community composition. Sea otters (Enhydra lutris ssp.) are a salient example of a keystone species exerting top‐down control on ecosystem community structure. The translocation and subsequent population growth and range expansion of the northern sea otter (Enhydra lutris kenyoni) in Washington State over the last five decades has created a spatio‐temporal gradient in sea otter occupation time and density, and acts as a natural experiment to quantify how sea otter population status and habitat type influence sea otter diet. We collected focal observations of sea otters foraging at sites across the gradient in varying habitat types between 2010 and 2017. We quantified sea otter diet composition and diversity, and long‐term rates of energy gain across the gradient. We found that sea otter diet diversity was positively correlated with cumulative sea otter density, while rate of energy gain was negatively correlated with cumulative density. Additionally, we found that habitat type explained 1.77 times more variance in sea otter diet composition than sea otter cumulative density. Long‐term diet studies can provide a broader picture of sea otter population status in Washington State.     

Ten new polymorphic microsatellite loci for North American river otters (Lontra canadensis) and their utility in related mustelids

We describe 10 novel North American river otter (Lontra canadensis) polymorphic microsatellite loci. Individuals from two river drainages in New York were sampled, and the number of alleles per locus ranged from two to 12 (Drainage 1) and two to 10 (Drainage 2). Observed heterozygosities ranged from 0.21 to 0.83 (Drainage 1) and 0.20 to 0.80 (Drainage 2). Preliminary screening revealed that loci amplified in five other mustelids [Martes pennanti (n = 6), Martes fiona (n = 8), Mustela frenata (n = 8), Mustela erminea (n = 8) and Mustela vison (n = 5)].     

Determining Observer Reliability in Counts of River Otter Tracks

ABSTRACT In many research projects, reliability of collected data is dependent on reliability of field observers. However, it is uncommon for observer reliability to be either measured or reported in wildlife research. We tested whether observer skill affected outcomes of a northern river otter (Lontra canadensis) track survey conducted by the Texas Parks and Wildlife Department. Observers recorded presence of tracks at bridge sites (n = 250) throughout a 27-county region in east Texas, USA. Logistic regression indicated that observers were significantly associated with frequency of reported otter tracks. Because observers were not assigned to bridges at random, we tested and found associations between the bridges surveyed by each observer (SURVEY ROUTE) and habitat variables (WATERSHED, VEGETATION-TYPE, WATER-TYPE, BRIDGE-AREA) that may have influenced otter presence and probability of detection. A standardized tracker evaluation procedure indicated that experienced observers (n = 7) misidentified 37% of otter tracks. Additionally, 26% of tracks from species determined to be “otter-like” were misidentified as otter tracks. We recommend that observer skill in identification of animal tracks and other indirect signs be measured to detect and reduce observer errors in wildlife monitoring.     

Individual identification and sex determination of Eurasian otters (Lutra lutra) in Daegu city based on genetic analysis of otter spraint

The Eurasian otter, Lutra lutra, is a well known endangered species in South Korea that experienced rapid population decreases through poaching and industrialization until the 1980’s. To evaluate the genetic diversity and the existing number of otters recently found in Daegu City, 81 fecal samples collected from the Gumho River and Shincheon stream were subjected to DNA extraction for sex determination and genetic analysis using nuclear genetic markers. Individual identification and relatedness between individuals were detemined by genotypic data using twelve microsatellite loci, and sex identification was also determined based on sequence variation of the zinc finger protein gene on sex chromosomes. Our results showed that at least seven otter individuals were identified and the kinship relationships of seven individual pairs were determined. It was concluded that otters distributed widely in Daegu City have moderate levels of genetic diversity, and close monitoring of the small-sized otter population is necessary to promote successful settlement of the otters in the area.     

Patterns of otter Lutra lutra road mortality in a landscape abundant in lakes

In order to reduce otter (Lutra lutra) road mortality and to predict accident sites, we assessed the localities of traffic accidents involving otters in the Uckermark County, Brandenburg, Northeast Germany. Eighty-eight otter accident sites from the years 1990 to 2003—as recorded by the Naturschutzstation Zippelsförde des Landesumweltamtes Brandenburg (Brandenburg State Office for Environment)—were categorised according to traffic and road patterns, otter habitat suitability, as well as presence and type of watercourse crossings. From these numerical data, we compiled a list of characteristics to identify other potentially dangerous sites where otter road traffic accidents are likely to occur using a geographic information system. With a principal component analysis, we categorised three groups of accident sites. Firstly, most otter road accidents were located close to either one or two lakes. Secondly, otters were frequently killed at places where no body of water occurred within a distance of 200 m to the road. Thirdly, and in contrast to other locations, in the Uckermark County, fewest accidents happened where a road crossed a watercourse. Mitigation measures were suggested and discussed for the recorded and potential accident sites.

     

Natural,human and spatial constraints to expanding populations of otters in the Iberian Peninsula

Aim To determine the relationships between otter (Lutra lutra) distribution dynamics and environmental and spatial constraints over a 20‐year period. Location Andalusia, southern Iberian Peninsula. Methods We synthesized otter distribution data from three otter surveys (1985, 1995 and 2005) using subcatchment areas defined by hydrological barriers. Subcatchments were characterized by two ‘natural’ (climatic and orographic variables) and two ‘human’ (land use and population density) gradients. In addition, we calculated two contagion variables (the distance to previously occupied subcatchments and the percentage of occupied subcatchments within a 50 km buffer) for consecutively surveyed subcatchments. Results Between 1985 and 2005 the percentage of subcatchments with otters present increased from 42% to 72%. Otters tended to be rare or absent from human‐dominated areas. Anthropogenic gradients were better predictors of otter distribution than natural ones. Human and natural gradients showed strong covariation, but for any value of the natural gradients otters tended to be present in subcatchments with lower human impacts. Colonization of new subcatchments was found to be strongly related to contagion variables and expansion rates were slower than those estimated in other studies. Newly colonized areas tended to be located in areas with intermediate human influence, while repeated absences occurred mainly in areas where human impact was most severe. Main conclusions Our results suggest that recent otter expansion across Andalusia is a reflection of large‐scale improvement in environmental conditions. Otter populations that survived the period of strong and generalized declines appear to be acting as sources from which neighbouring areas are colonized, probably aided by improved water quality and increases in food availability. However, the further expansion of otters into their full original range is likely to be constrained by human‐impacted landscapes.     

Genetic pattern of the recent recovery of European otters in southern France

We investigated how landscape affects the population genetic structure and the dispersal of the elusive European otter Lutra lutra in a contemporary colonization context, over several generations and at the level of hydrographic basins. Our study area included 10 basins located in the Cévennes National Park (CNP), at the southern front of the natural otter recovery in France. Each basin comprised 50 to 300 km of permanent rivers that were surveyed for otter presence from 1991 to 2005. Faecal samples collected in 2004 and 2005 in this area were genotyped at 9 microsatellite loci, resulting in the identification of 70 genetically distinct individuals. Bayesian clustering methods were used to infer genetic structure of the populations and to compare recent gene flow to the observed colonization. At the regional level, we identified 2 distinct genetic clusters (NE and SW; FST=0.102) partially separated by ridges, suggesting that the CNP was recolonized by 2 genetically distinct otter populations. At the basin level, the genetic distance between groups of individuals in different basins was positively correlated to the mean slope separating these basins. The probable origins and directions of individual movements (i.e. migration between clusters and basin colonization inside clusters) were inferred from assignment tests. This approach shows that steep and dry lands can stop, impede or divert the dispersal of a mobile carnivore such as the otter.     

Genetic diversity and population structure in the endangered giant otter, Pteronura brasiliensis

We assessed levels of genetic diversity and investigated patterns of population structure in three remnant populations of the endangered giant otter, Pteronura brasiliensis, using microsatellite loci. All populations displayed moderate to low levels of heterozygosity and allelic richness (H _O 0.56–0.57, A _R 4.00–5.15) and effective population sizes were low (N _E 10.8–54) although only the Iténez population exhibited the signature of a genetic bottleneck. Population structure analyses revealed a pattern in which the populations of the Upper Amazon, Orinoco and Essequibo drainages comprised partially differentiated segments of a northern South American metapopulation, whereas the population of the Iténez appeared isolated. The observed patterns are congruent with previous mitochondrial DNA analysis which suggested the Iténez and northern South American groups constitute two evolutionary significant units. The results presented here should be considered in planning future policies aiming to manage the recovery of the giant otter across its range.     

Stream community richness predicts apex predator occupancy dynamics in riparian systems

Streams and adjacent riparian habitats represent linked terrestrial and aquatic ecosystems that exchange materials and energy. Recognized relationships among apex predators and ecosystem biodiversity led us to hypothesize that these predators in riparian‐stream systems were more likely to be found in sites with high stream quality, defined as increased ecosystem function and integrity. In our freshwater study system, river otter Lontra canadensis and mink Neovison vison play critical roles as apex predators. We used multi‐season occupancy modelling across three sampling years (2012–2014) to compare aspects of the stream communities that explain occupancy dynamics of river otter and mink, including their interactions with other semi‐aquatic mammals. We surveyed for semi‐aquatic mammals at 77 sites in 12 major watersheds in southern Illinois, USA (44 526 km²). Naïve occupancy differed among years but generally increased for river otter, and remained high (≥93.5%) for mink. Increasing substrate availability increased detectability of river otter, whereas mink detection varied by survey period. Occupancy of river otter during the initial survey period was higher in sites closer to reintroduction points. Probability of colonization of river otter was positively associated with macroinvertebrate index of biotic integrity, fish species richness, and beaver presence. Sites with high species richness of fish families preferred by river otter also had increased river otter persistence. Mink occupied sites with increased fish richness, muskrat presence and mussel community index. Taken together, our results show occupancy of both mink and river otter were predicted by aspects of prey diversity and presence, indicating the importance of community composition in occupancy dynamics of riparian predators. Ultimately, these relationships suggest that habitat heterogeneity and system stability are important to apex predator site use. However, the relative role of bottom–up and top–down forcing in stream systems remains to be resolved.     

What is the taxonomic status of East Asian otter species based on molecular evidence?: focus on the position of the Japanese otter holotype specimen from museum

The Japanese otter (Lutra nippon), once inhabited in most islands of Japan, is now considered as an extinct species. Although the Japanese otter is regarded as a distinct species from the Eurasian otter (L. lutra), its phylogeny and taxonomic status are based on limited information on morphological and genetic data, and thus further clarification is required. Here, we assessed the phylogenetic relationship among the genus Lutra and taxonomic status of L. nippon by using the complete sequences of cytochrome b gene of its holotype. The present phylogenic trees supported that the genus Lutra specimens largely formed monophyletic group, with L. sumatrana as a basal to other Lutra species. Within Lutra species, L. nippon was distantly related with L. lutra. The European otter population of L. l. lutra were clustered together with its subspecies, L. l. chinensis rather than the same subspecies, Korean otter population. The discrepancy between the genetic data and traditional taxonomy justifies the necessity of reexamination of the current subspecific classification system of Eurasian otters. Level of genetic divergence between the holotype of L. nippon and L. lutra was two to three-fold lower than those among the other sister species of the Lutrinae. Based on the level of divergence between the L. nippon and L. lutra, and insufficient evidence of morphological difference between them, it is suggested that designation of Japanese otter as a separate species from L. lutra will be reconsidered.