Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize,Central America using cross‐species microsatellites and faecal DNA

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co‐occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross‐species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of H_E = 0.60 ± 0.18 (SD) for jaguars, H_E = 0.65 ± 0.21 (SD) for pumas and H_E = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field‐collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.     

Novel Y‐chromosome short tandem repeats in Sus scrofa and their variation in European wild boar and domestic pig populations

Y‐chromosome markers are important tools for studying male‐specific gene flow within and between populations, hybridization patterns and kinship. However, their use in non‐human mammals is often hampered by the lack of Y‐specific polymorphic markers. We identified new male‐specific short tandem repeats (STRs) in Sus scrofa using the available genome sequence. We selected four polymorphic loci (5–10 alleles per locus), falling in one duplicated and two single‐copy regions. A total of 32 haplotypes were found by screening 211 individuals from eight wild boar populations across Europe and five domestic pig populations. European wild boar were characterized by significantly higher levels of haplotype diversity compared to European domestic pigs (H_D = 0.904 ± 0.011 and H_D = 0.491 ± 0.077 respectively). Relationships among STR haplotypes were investigated by combining them with single nucleotide polymorphisms at two linked genes (AMELY and UTY) in a network analysis. A differentiation between wild and domestic populations was observed (F_ST = 0.229), with commercial breeds sharing no Y haplotype with the sampled wild boar. Similarly, a certain degree of geographic differentiation was observed across Europe, with a number of local private haplotypes and high diversity in northern populations. The described Y‐chromosome markers can be useful to track male inheritance and gene flow in wild and domestic populations, promising to provide insights into evolutionary and population genetics in Sus scrofa.     

Characterization of 27 novel microsatellite loci in Sinibotia superciliaris (Günther 1892) and cross‐species transferability in Sinibotia reevesae (Chang 1944)

The Chinese golden loach, Sinibotia superciliaris and its congener Sinibotia reevesae, are endemic to China and extraordinary similar in morphological traits. However, few genetic studies have been conducted on them, especially those based on nuclear markers. Here, we developed 27 novel polymorphic microsatellite markers from S. superciliaris and further tested the cross‐species transferability of those loci in S. reevesae. The cross‐species amplification test showed that 19 loci demonstrate polymorphism in S. reevesae. The mean number of alleles (N_A) were both 3 in S. superciliaris and S. reevesae, the mean expected heterozygosities (H_E) were 0.57 and 0.54, the Shannon‐Wiener Diversity Indices (SW) were 0.84 and 0.82, and the evenness (E) were 0.09 and 0.08, respectively. The polymorphic information content (PIC) showed a high level of polymorphism for the two species (mean 0.54 and 0.50, respectively). In addition, the cross‐species transferability (100%) of those markers was clearly high, which confirmed that the microsatellite markers developed here could be used effectively for other related species.     

ACTIVITY RHYTHMS OF WILD AND LABORATORY GOLDEN HAMSTERS (MESOCRICETUS AURATUS) UNDER ENTRAINED AND FREE-RUNNING CONDITIONS

The golden hamster (Mesocricetus auratus) is one of the most frequently used laboratory animals, particularly in chronobiological studies. One reason is its very robust and predictable rhythms, although the question arises whether this is an inbreeding effect or rather is typical for the species. We compared the daily (circadian) activity rhythms of wild and laboratory golden hamsters. The laboratory hamsters were derived from our own outbred stock (Zoh:GOHA). The wild hamsters included animals captured in Syria and their descendants (F1). Experiments were performed under entrained (light: dark [LD] 14h:10h) and under free-running (constant darkness, DD) conditions. Locomotor activity was recorded using passive infrared detectors. Under entrained conditions, the animals had access to a running wheel for a certain time to induce additional activity. After 3 weeks in constant darkness, a light pulse (15 min, 100 lux) was applied at circadian time 14 (CT14). Both laboratory and wild hamsters showed well-pronounced and very similar activity rhythms. Under entrained conditions, all hamsters manifested about 80% of their total 24h activity during the dark portion of the LD cycle. The robustness of the daily rhythms was also similar. However, interindividual variability was higher in wild hamsters for both measures. All animals used the running wheels almost exclusively during the dark portion of the LD cycle, although the wild hamsters were three times more active. The period length, measured in constant darkness, was significantly shorter in wild (23.93h ± 0.10h) than in laboratory hamsters (24.06 ± 0.07h). The light-induced phase changes were not different (about 1.5h). In summary, these results indicate that the laboratory hamster is not much different from the wild type. (Chronobiology International, 18(6), 921–932, 2001)     

New approaches to species delimitation and population structure of anthozoans: Two case studies of octocorals using ultraconserved elements and exons

As coral populations decline worldwide in the face of ongoing environmental change, documenting their distribution, diversity and conservation status is now more imperative than ever. Accurate delimitation and identification of species is a critical first step. This task, however, is not trivial as morphological variation and slowly evolving molecular markers confound species identification. New approaches to species delimitation in corals are needed to overcome these challenges. Here, we test whether target enrichment of ultraconserved elements (UCEs) and exons can be used for delimiting species boundaries and population structure within species of corals by focusing on two octocoral genera, Alcyonium and Sinularia, as exemplary case studies. We designed an updated bait set (29,181 baits) to target‐capture 3,023 UCE and exon loci, recovering a mean of 1,910 ± 168 SD per sample with a mean length of 1,055 ± 208 bp. Similar numbers of loci were recovered from Sinularia (1,946 ± 227 SD) and Alcyonium (1,863 ± 177 SD). Species‐level phylogenies were highly supported for both genera. Clustering methods based on filtered single nucleotide polymorphisms delimited species and populations that are congruent with previous allozyme, DNA barcoding, reproductive and ecological data for Alcyonium, and offered further evidence of hybridization among species. For Sinularia, results were congruent with those obtained from a previous study using restriction site associated DNA sequencing. Both case studies demonstrate the utility of target‐enrichment of UCEs and exons to address a wide range of evolutionary and taxonomic questions across deep to shallow timescales in corals.     

Microsatellite loci in the house fly,Musca domestica L. (Diptera: Muscidae)

Genomic libraries from house flies enriched for (CA)₁₅ and (CAG)₁₀ repeats were constructed by using biotinylated probes. Twenty‐five loci were isolated and evaluated for polymorphisms in wild flies representing two geographically diverse populations. Fourteen of 19 dinucleotide loci, and one of six trinucleotide loci were polymorphic. One hundred and twenty‐seven alleles were detected, 39 of which were private. Average number of alleles per polymorphic locus was 8.4 ± 2.5 and average heterozygosity was 72 ± 4%. F_ST by the private allele method was 0.73. Three of 15 loci showed significant heterozygote deficiencies, attributed to null alleles. Five of 15 loci were amplified in the face fly, Musca autumnalis.     

Retention of solute and particle markers in the digestive tract of captive Somali wild asses (<Emphasis Type="Italic">Equus africanus somaliensis</Emphasis>)

In contrast to the domestic horse, whose digestive physiology has been thoroughly investigated, knowledge on the digestive physiology of wild equids is scarce. Comparisons between the domestic horse and the domestic donkey suggest that wild asses might achieve higher digestibilities. This could derive from longer retention times or a greater difference in the mean retention time (MRT) of particles vs. fluid (the selectivity factor (SF)). Here, we measured MRT of a solute (fluid; MRT_solute) and a particle (<2 mm; MRT_particle) marker in five captive male Somali wild asses (Equus africanus somaliensis) fed a diet of 95% grass hay. At a mean dry matter intake of 94 ± 3 g kg^?0.75 day^?1, MRT_solute was 33.3 ± 5.4 h and MRT_particle 39.6 ± 3.9 h, resulting in a SF of 1.21 ± 0.14. For their food intake, Somali wild asses appeared to have slightly higher MRT_particle than expected based on domestic equid data, in contrast to Grevy zebras (Equus grevyi), potentially indicating higher capacities of the digestive tract. However, considering data on domestic horses, donkeys, and zebra, there was no evident difference in the SF of wild equids compared to domestic ones. Together with an absence of reported anatomical differences in the digestive tract of wild and domestic equids, the data suggest a general similarity in the digestive physiology of equid species that contrasts with the diversity in the digestive physiology of ruminants, and that might be one contributing factor to a lack of sympatric, niche-differentiated equid species.     

Thermal physiology and energetics in male desert hamsters (<Emphasis Type="Italic">Phodopus roborovskii</Emphasis>) during cold acclimation

The adjustments in thermal physiology and energetics were investigated in male desert hamsters (Phodopus roborovskii) which were acclimated to 5°C for 4 weeks. Mean core body temperature in cold acclimated animals decreased by 0.21°C compared with controls. Further analysis revealed that the decrease mainly occurred in the scotophase, while in the photophase core body temperature remained constant during the whole cold acclimation. Thermogenic capacity, represented by resting metabolic rate and nonshivering thermogenesis increased in cold acclimated hamsters from initial values of 1.38 ± 0.05 and 5.32 ± 0.30 to 1.77 ± 0.08 and 8.79 ± 0.31 mlO₂ g⁻¹ h⁻¹, respectively. After cold acclimation, desert hamsters maintained a relative stable body mass of 21.7 ± 0.1 g very similar to the controls kept at 23°C (21.8 ± 0.1 g). The mean values of food intake and digestible energy (metabolisable energy) in cold acclimated hamsters were 5.3 ± 0.1 g day⁻¹ and 76.3 ± 0.9 kJ day⁻¹ (74.8 ± 0.9), respectively, which were significantly elevated by 76.7 and 80.4% compared to that in control group. The apparent digestibility was 81.0 ± 0.3% in cold acclimated animals which was also higher than the 79.7 ± 0.2% observed in controls. This increase corresponded with adaptive adjustments in morphology of digestive tracts with 20.2 and 36.8% increases in total length and wet mass, respectively. Body fat mass and serum leptin levels in cold acclimated hamsters decreased by 40.7 and 67.1%, respectively. The wheel running turns and the onset of wheel running remained unchanged. Our study indicated that desert hamsters remained very active during cold acclimation and displayed adaptive changes in thermal physiology and energy metabolism, such as enhanced thermogenic and energy processing capacities.     

Isolation and characterization of microsatellite markers for paternity assessment in the golden whistler (Pachycephala pectoralis: Aves)

We isolated and characterized six novel microsatellite markers for paternity analysis in the golden whistler Pachycephala pectoralis, by screening an enriched genomic library using nonradioactive PCR techniques. The six loci exhibited little or no evidence of null alleles and showed high levels of polymorphism (mean H_E = 0.85, mean number of alleles = 15.2), making them suitable for paternity assessment in this species (exclusion probability of six unlinked loci = 0.9997).     

Does fin damage allow discrimination among wild,escaped and farmed Sparus aurata (L.) and Dicentrarchus labrax (L.)?

The present study compares fin damages in gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) according to their wild, escaped or farmed origins. In addition, the potential applicability of fin condition indices (Fin Erosion Index ‘FEI’ and Fin Splitting Index ‘FSI’) as identification tools is discussed. Farmed seabream fins evidenced more erosion and splitting (FEI ± SD: 2.1 ± 0.3; FSI ± SD: 1.9 ± 0.6) than wild seabream fins (FEI: 0.8 ± 0.6; FSI: 1.2 ± 0.9), a result of farming conditions in open‐sea cages. Escaped seabream fin erosion was between that of farmed and wild seabream (FEI: 1.6 ± 0.4), which could indicate that fins in farmed fish recover over time from farming abrasions once they are in the wild. However, the fins of escaped seabream seem to be weaker than those of the wild fish, and therefore might be more susceptible to suffer other types of erosion such as splitting or nipping (FSI: 2.3 ± 0.7). No significant differences were found in seabass FEI according to their origins, although wild seabass presented significantly more split caudal fins (FSI: 3.3 ± 2.8) than the farmed seabass (FSI: 1.2 ± 1.1) and escapees (FSI: 2.5 ± 1.6). Therefore, FEI for seabream could be used as tools not only to distinguish between wild and farmed fish, but also to identify recent escapees, improving further assessments on the contribution of seabream escapees in fishery landings. However, the healing potential of damaged fins must be considered for the proper identification of escapees. Use of fin condition indices from both species could be helpful for aquaculture management, to assess fish welfare in fish farms stocks, and improve the knowledge of handling, stock densities and open‐sea cage environment conditions.     

Validation of annulus in otolith and estimation of growth rate for Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> in tropical southern Taiwan

The age of Japanese eels (Anguilla japonica) is often estimated from otoliths, but this method has not been fully validated, particularly in tropical areas where the annulus in otolith is considered to be less distinct than in temperate areas. To validate the annuli in Japanese eel otoliths from southern Taiwan, known-age (2 year-old) cultured eels from an eel farm and wild eels from Kao-Ping River were collected. It was found that 26 out of 31 cultured eels (83.9%) showed two clear annuli and the remained 5 eels showed either one or three annuli. The mean (± SD) age of the cultured eels was 1.97 ± 0.4 years. Meanwhile, a clear peak in the mean monthly marginal increment ratio of the otolith in wild yellow and silver eels occurred once a year during winter (November to March). The annual deposition of presumed annuli in otoliths of Japanese eel was validated and the age and growth rate estimation for Japanese eels in the tropical southern Taiwan is deemed feasible. The growth rate of cultured eels was significantly faster than that of wild eels, but it did not differ significantly between sexes for wild silver, yellow or cultured eels. The von Bertalanffy Growth Function parameters (K, and t ₀ ) of the wild eels were estimated as 0.114 ± 0.028 year⁻¹, 1178 ± 171 mm and −0.8 ± 0.2 years, respectively.     

Genetic variation of the East Balkan Swine (Sus scrofa) in Bulgaria,revealed by mitochondrial DNA and Y chromosomal DNA

East Balkan Swine (EBS) Sus scrofa is the only aboriginal domesticated pig breed in Bulgaria and is distributed on the western coast of the Black Sea in Bulgaria. To reveal the breed's genetic characteristics, we analysed mitochondrial DNA (mtDNA) and Y chromosomal DNA sequences of EBS in Bulgaria. Nucleotide diversity (π_n) of the mtDNA control region, including two newly found haplotypes, in 54 EBS was higher (0.014 ± 0.007) compared with that of European (0.005 ± 0.003) and Asian (0.006 ± 0.003) domestic pigs and wild boar. The median‐joining network based on the mtDNA control region showed that the EBS and wild boar in Bulgaria comprised mainly two major mtDNA clades, European clade E1 (61.3%) and Asian clade A (38.7%). The coexistence of two mtDNA clades in EBS in Bulgaria may be the relict of historical pig translocation. Among the Bulgarian EBS colonies, the geographical differences in distribution of two mtDNA clades (E1 and A) could be attributed to the source pig populations and/or historical crossbreeding with imported pigs. In addition, analysis of the Y chromosomal DNA sequences for the EBS revealed that all of the EBS had haplotype HY1, which is dominant in European domestic pigs.     

Dietary partitioning of three large carnivores in Majete Wildlife Reserve,Malawi

Between 2011 and 2012, the carnivore guild in Majete Wildlife Reserve (MWR), Malawi, was restored following the reintroduction of lion (Panthera leo) and leopard (Panthera pardus). The aim of this study was to describe and compare the diet of lion, leopard and resident spotted hyaena (Crocuta crocuta) using scat analysis. Lions and spotted hyaenas displayed the greatest dietary overlap (O_ab = 0.88) and selected mainly medium- to large-bodied prey species. Lions had a mean preferred prey weight of 120.33 ± 42.14 kg (SE), with warthog (Phacochoerus africanus) and waterbuck (Kobus ellipsiprymnus) making up 60.64% of relative biomass consumed. Spotted hyaenas had a mean preferred prey weight of 102.40 ± 41.69 kg and had a more generalised diet (B_a = 0.46) compared to lions (B_a = 0.36). In contrast, leopards occupied a dietary niche substantially lower than that of lions and spotted hyaenas, selecting relatively smaller prey with a mean preferred prey weight of 27.50 ± 6.74 kg. Our results suggest that coexistence between the resident hyaena and reintroduced lion and leopard in MWR is facilitated by dietary partitioning. We advise long-term monitoring of reintroduced carnivores in small, enclosed reserves to assess their impacts on predator and prey populations.     

Development of sequence‐tagged microsatellites for the barley net blotch pathogen,Pyrenophora teres

A modified sequenced‐tagged microsatellite (STM) profiling procedure was used to develop 80 STMs for the barley net blotch pathogen, Pyrenophora teres. Of these, 60 STMs amplified 67 loci in one or both of the spot (P. teres f. maculata) and net (P. teres f. teres) forms of the pathogen. When screened on six field‐sampled isolates of each pathogen form, 25 STMs revealed 26 polymorphic loci, with an average of 3.2 ± 1.0 alleles and mean gene diversity of 0.59 ± 0.12.     

Patterns of within and between-colony microsatellite variation in the endangered Vancouver Island marmot (Marmota vancouverensis): implications for conservation

Among the 14 extant species of the genus Marmota the Vancouver Island marmot (Marmota vancouverensis) is the most endangered. In 2007 as few as 85 individuals were left in the wild, with an additional 162 individuals maintained in captivity. To facilitate genetic monitoring of both wild and captive populations, polymorphic genetic markers were identified. Thirty-three different microsatellite loci were tested for amplification and variability in ≥30 wild-born individuals. Only 11 of these loci proved to be polymorphic and were subsequently analysed in 105 samples collected from wild Vancouver Island marmots. The average number of alleles (A) at those 11 loci was only 2.1, and the intraspecific variation (H _E between 8 and 23% within colonies) was low compared to other marmot species. Variation within the small and geographically isolated Mt. Washington colony was particularly low (A = 1.3, H _E = 0.08). Genetic distances between the Mt. Washington colony (11 individuals) and those of the Nanaimo Lakes region (94 individuals) on southern Vancouver Island were large (D values ranging from 0.42 to 0.50), while genetic distances among colonies within the latter area were much smaller (D values from 0.01 to 0.13). Given the low within-population genetic variation, and the resulting risk of inbreeding depression at Mt. Washington, we support the decision to maximize overall genetic variation of the species by crossbreeding marmots from the two different areas despite the possibility of local adaptation.     

A genetic demographic analysis of Lake Malawi rock‐dwelling cichlids using spatio‐temporal sampling

We estimated the effective population sizes (N_e) and tested for short‐term temporal demographic stability of populations of two Lake Malawi cichlids: Maylandia benetos, a micro‐endemic, and Maylandia zebra, a widespread species found across the lake. We sampled a total of 351 individuals, genotyped them at 13 microsatellite loci and sequenced their mitochondrial D‐loop to estimate genetic diversity, population structure, demographic history and effective population sizes. At the microsatellite loci, genetic diversity was high in all populations. Yet, genetic diversity was relatively low for the sequence data. Microsatellites yielded mean N_e estimates of 481 individuals (±99 SD) for M. benetos and between 597 (±106.3 SD) and 1524 (±483.9 SD) individuals for local populations of M. zebra. The microsatellite data indicated no deviations from mutation–drift equilibrium. Maylandia zebra was further found to be in migration–drift equilibrium. Temporal fluctuations in allele frequencies were limited across the sampling period for both species. Bayesian Skyline analyses suggested a recent expansion of M. zebra populations in line with lake‐level fluctuations, whereas the demographic history of M. benetos could only be estimated for the very recent past. Divergence time estimates placed the origin of M. benetos within the last 100 ka after the refilling of the lake and suggested that it split off the sympatric M. zebra population. Overall, our data indicate that micro‐endemics and populations in less favourable habitats have smaller N_e, indicating that drift may play an important role driving their divergence. Yet, despite small population sizes, high genetic variation can be maintained.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Genetic diversity and molecular differentiation of Chinese toad based on microsatellite markers

Genetic diversity and population structure of 9 populations of Bufo gargarizans with total 111 samples in China were assessed using seven microsatellite loci. The analysed microsatellite markers produced 161 alleles, varied from 9 to 38 alleles each locus. The number of alleles per population per locus ranged from 4.43 to 10.29. Polymorphic information content showed that all seven loci were highly informative (mean = 0.810 ± 0.071). The average observed heterozygosity was less than the expected (0.353 ± 0.051 and 0.828 ± 0.067, respectively). All tested populations gave significant departures from Hardy–Weinberg equilibrium. Genetic differentiation among the populations was considerably high with the overall and pairwise F _ST values (mean = 0.160 ± 0.039), and showed fairly high level of inbreeding (indicated by a mean F _IS value of 0.504 ± 0.051) and global heterozygote deficit. In comparison to other amphibian studies; however, our results suggested that the level of genetic structuring in B. gargarizans was relatively low in the geographical scale of the study area. Interestingly, the speculated population bottleneck was found to be absent and the analyses provide only weak evidence for a recent contraction in size even though there was severe inbreeding (indicated by the F _IS value) in the Chinese toad populations.     

Photosynthetic and leaf respiration activity of <Emphasis Type="Italic">Malcolmia littorea</Emphasis> (L.) R. Br. in response to air temperature

Plant traits of Malcolmia littorea growing at the Botanic Garden of Rome and transplanted from the wild population developing along the Latium coast (Italy) were analyzed. The highest photosynthetic rates [P _N, 22.5 ± 0.5 μmol(CO₂) m⁻² s⁻¹], associated to the highest chlorophyll content (Chl, 60 ± 5 SPAD units), and respiration rates [R, 11.1 ± 0.2 μmol(CO₂) m⁻² s⁻¹] were reached in spring, when mean air temperature (T _m) was in the range 17°C to 23°C. P _N, Chl, and R decreased by 86, 38, and 59% in summer when mean maximum air temperature (T _max) was 30.3 ± 2.6°C. Leaf water potential decreased by 34% in summer compared to the spring value, and it was associated to a relative water content (RWC) of 74 ± 4%, and to a water-use efficiency (WUE) of 2.15 ± 0.81 μmol(CO₂) mmol⁻¹(H₂O). Moreover, also low air temperatures determined a significant P _N and R decreases (by 52 and 40% compared to the maximum, respectively). Responsiveness of gross photosynthetic rate (P _g) to R was higher than that to P _N as underlined by the slope of the regression line between the two variables. The results underlined a low tolerance to both high- and low air temperatures of M. littorea. The selected key traits (R, WUE, Chl) by the discriminant analysis might be used to monitor the M. littorea wild population in the long time. The ex situ cultivated plants could be propagated and used to increase the individuals number of the wild population.