Microsatellite analysis of relatedness structure in young of the year of the endangered Zingel asper (Percidae) and implications for conservation

1. Zingel asper is one of the most endangered freshwater fish of Western Europe. Because of critical habitat loss and fragmentation, stocking or reintroduction protocols are sometimes considered even though few data are available about the behaviour and biology of the species.
2. The present study is the first attempt to explore the population genetic structure of juveniles (young of the year, YOY) of this endangered species using genetic markers (microsatellite loci). In the Beaume River (ardeche, France), Z. asper exhibits very low densities (10–80 fish per hectare), and it was expected that annual recruitment would comprise only few clutches (full- or half-sibs groups).
3. However, sibship reconstruction in one YOY cohort showed that more than 60% of the adult population reproduced. Dispersal rate in early YOY was high: relatedness estimates showed that only few months after hatching, YOY sibs were mixed among study sites. A large number of breeders together with high dispersal rates explain the high genetic variability observed in small populations of Z. asper .
4. Conservation strategies for the species are discussed based on these results, emphasizing the importance of conserving population genetic variability and preserving habitat connectivity for juveniles.     

Twenty-four hours in the life of Zingel asper

For the first time, radio-tracking observations were made over 24 h cycles during the growing season to quantify the habitat use and home range of Zingel asper , a critically endangered fish species, endemic to the Rhône River with a high risk of extinction (IUCN). New advances in radio telemetry with regard to the size of tags provided an opportunity to collect behavioural and habitat use information while remaining non-lethal. Zingel asper was characterized by small ranges of habitat use and, contrary to previous assumptions, the species was diurnal as illustrated by a smaller home range during the night than during daytime.     

Reproductive biology of the endangered percid Zingel asper in captivity: a histological description of the male reproductive cycle

The endemic Rhodanian percid Zingel asper (Linnaeus, 1758), is usually found throughout the Rh?ne basin, but this fish is now in sharp decline. Understanding its reproductive physiology is important so as to be able to artificially control its reproduction with a view to re-introducing it. This study was carried out on a population obtained by artificial fertilization and bred in external tanks. Fishes were observed from the juvenile stages through to adulthood. Patterns of testicular development were defined from histological observations. Testes of Z. asper were paired, elongated and fusiform dorsocaudal organs. The two lobes of each gonad joined together to form a duct that extended to the urogenital papillae. They showed a lobular structure. The testicular lobules were of the unrestricted spermatogonial type. Up to 10 months-old, most of the males were immature: their testes showed only type A spermatogonia. The appearance of type B spermatogonia in the lobules of a testis indicated the beginning of spermatogenesis in 10 months-old fish. Spermiogenesis occurred 24 months after the fertilization and, in 26 months-old fish, the cyst opened and released spermatozoa into the lumen of lobules. The spermiation belonged to a cystic type. During the third year, histological observations pointed to the same evolution of adult gonads as during the second year. Sexual maturity was reached in captive Z. asper after two years. The spawning occurred in May in the breeding conditions.     

Use of a generalised linear model to test habitat preferences: the example of Zingel asper, an endemic endangered percid of the River Rhône

SUMMARY 1. A study of microhabitat preferences was conducted on Zingel asper , an endangered endemic species from the Rhône catchment. A generalised linear model allowed us to test statistically the non-random habitat selection and the effect of season and site on this habitat selection.
2. The analysis detected significant preferences for the three physical variables considered: water depth, water velocity and substratum size.
3. A seasonal shift in the substratum size preference was found: preference for stones increasing during the spawning season. Depth preference varied between sites, suggesting a possible plasticity in habitat selection.
4. These results suggest that the availability of suitable physical habitat plays a significant role in determining fish distribution in the River Beaume.     

Dietary characterization of the endangered salt marsh harvest mouse and sympatric rodents using DNA metabarcoding

The salt marsh harvest mouse (Reithrodontomys raviventris; RERA) is an endangered species endemic to the coastal wetlands of the San Francisco Estuary, California. RERA are specialized to saline coastal wetlands, and their historical range has been severely impacted by landscape conversion and the introduction of non‐native plant and rodent species. A better understanding of their diet is needed to assess habitat quality, particularly in relation to potential competitors. We investigated three questions using DNA metabarcoding with ITS2 and trnL markers: (1) Do RERA specialize on the native plant, pickleweed (Salicornia pacifica), (2) Do RERA consume non‐native plants, and (3) What is the dietary niche breadth and overlap with three sympatric native and non‐native rodents? RERA diet was dominated by two plants, native Salicornia and non‐native salt bush (Atriplex spp.), but included 48 plant genera. RERA diet breadth was narrowest in fall, when they consumed the highest frequencies of Salicornia and Atriplex, and broadest in spring, when the frequencies of these two plants were lowest. Diet breadth was slightly lower for RERA than for co‐occurring species in pairwise comparisons. All four species consumed similarly high frequencies of wetland plants, but RERA consumed fewer grasses and upland plants, suggesting that it may be less suited to fragmented habitat than sympatric rodents. Diet overlap was lowest between RERA and the native California vole (Microtis californicus). In contrast, RERA diet overlapped substantially with the native western harvest mouse (R. megalotis) and non‐native house mouse (Mus musculus), suggesting potential for competition if these species become sufficiently abundant.     

鹊肾树树皮的化学成分和抗炎作用研究

通过药理实验确定了鹊肾树树皮的乙酸乙酯萃取物为抗炎活性部位,并利用色谱技术从该部位中分离得到了7个化合物,通过UV、IR、NMR、MS等现代谱学方法鉴定化合物的结构分别为:丁二酸(1)、6-乙基-5-羟基-2,7-二甲氧基-1,4-萘醌(2)、3β,20-二羟基-5β-孕甾烷(3)、5-羟基麦芽酚(4)、双[5-甲酰基...     

Restoring the endangered oyster mussel (Epioblasma capsaeformis) to the upper Clinch River,Virginia: an evaluation of population restoration techniques

From 2005 to 2011, the federally endangered freshwater mussel Epioblasma capsaeformis (oyster mussel) was reintroduced at three sites in the upper Clinch River, Virginia, using four release techniques. These release techniques were (1) translocation of adults (site 1, n = 1418), (2) release of laboratory‐propagated sub‐adults (site 1, n = 2851), (3) release of 8‐week‐old laboratory‐propagated juveniles (site 2, n = 9501), and (4) release of artificially infested host fishes (site 3, n = 1116 host fishes). These restoration efforts provided a unique research opportunity to compare the effectiveness of techniques used to reestablish populations of extirpated and declining species. We evaluated the relative success of these four population restoration approaches via monitoring at each release site (2011–2012) using systematic 0.25‐m² quadrat sampling to estimate abundance and post‐release survival. Abundances of translocated adult and laboratory‐propagated sub‐adult E. capsaeformis at site 1 ranged 577–645 and 1678–1700 individuals, respectively, signifying successful settlement and high post‐release survival. Two untagged individuals (29.1 and 27.3 mm) were observed, indicating that recruitment is occurring at site 1. No E. capsaeformis were found at sites where 8‐week‐old laboratory‐propagated juveniles (site 2) and artificially infested host fishes (site 3) were released. Our results indicate that translocations of adults and releases of laboratory‐propagated sub‐adults were the most effective population restoration techniques for E. capsaeformis. We recommend that restoration efforts focus on the release of larger (>20 mm) individuals to accelerate augmenting and reintroducing populations and increase the probability for recovery of imperiled mussels.     

Heterospecific harassment of native endangered fishes by invasive guppies in Mexico

Persistent courtship by male Trinidadian guppies (Poecilia reticulata) is costly for conspecific females. Since male guppies are known to attempt matings with other poeciliid females, we asked whether persistent courtship is also directed towards morphologically similar but phylogenetically distant females encountered following invasion. Skiffia bilineata is one of several endangered viviparous goodeids from Central México, whose remaining habitats are increasingly shared with invasive guppies. Experiments in which guppy sex ratios were manipulated to vary the proportion of heterospecific to conspecific females showed that male guppies courted and attempted forced copulations with S. bilineata females even when females of their own species were in excess. This behaviour places an additional, and previously unrecognized, burden on a group of endemic Mexican fishes already in risk of extinction.     

The diet and growth of O-group flounder,Platichthys flesus (L.), in the River Dee,North Wales

Many O-group flounder, Platichthys flesus, live in the lower reaches of rivers in North West Europe; while there is extensive information on the niche that they occupy in estuarine and marine environments, little is known of their life in freshwater. The diet of O-group flounder in the Lower Welsh River Dee was studied by stomach contents analysis of diel and seasonal samples. The dominant prey were chironomid larvae and pupae, tubificid oligochaetes and cyclopoid copepods. There was little quantitative variation in diet temporally, although a weak crepuscular pattern of stomach fullness was shown. There was wide variation in length and weight in all months, but the mean values at age I were 60 mm and 2.9 g respectively.     

Diet and growth of a leaf-shredding caddisfly in southern Appalachian streams of contrasting disturbance history

Diet and growth of leaf-shredding caddisfly larvae, Pycnopsyche spp.,were examined in streams draining a reference catchment and a 16-year-oldclear-cut (disturbed) catchment at Coweeta Hydrologic Laboratory insouthwestern North Carolina, USA. The objective was to explain why shredderproduction is higher in the disturbed streams despite the larvae having lessfood (i.e., leaves) available. We predicted larvae would grow faster onfast-decaying leaf material representative of the disturbed streams. Larvaeconsumed mostly leaf detritus in three streams draining each catchment overthree seasons (fall, winter, and spring), which showed larvae did notconsume higher quality foods (e.g., algae and animal material) in disturbedstreams. When fed 2-month-old conditioned black birch (Betula lenta L.) (afast-decaying leaf species) and white oak (Quercus alba L.) (a slow-decayingleaf species) leaves in the laboratory, larvae grew significantly faster onthe birch leaves. However, when larvae were fed the same leaf types after3-months conditioning, larvae grew significantly faster on oak leaves. Afield growth experiment conducted for 42 d using mixed-species leaf dietsrepresentative of each catchment and initially conditioned for 2 monthsfound that Pycnopsyche grew significantly better on the diet representativeof the reference catchment. The reference diet contained more oak leaveswhich apparently became a more acceptable food as the experiment proceeded.High shredder production in the disturbed streams could not be explained byhigh Pycnopsyche growth rates on fast-decaying leaves. Instead, larvae grewbetter on leaves that were apparently conditioned optimally regardless ofconditioning rate.     

Ecology of spawning humpback chub, Gila cypha, in the Little Colorado River near Grand Canyon, Arizona

The morphologically unique and endangered humpback chub, Gila cypha, is found in canyon-bound reaches of the Colorado River and its tributaries. Now limited to six isolated reproducing populations, this species is believed to have been once distributed over a large portion of the mainstem river. Because the species inhabits remote canyon areas, little is known about its spawning ecology. The largest remaining population occurs in the lower Little Colorado River (LCR) near Grand Canyon, where we conducted a three-year study of spawning ecology during spring (March-June) 1993–1995. We analyzed seasonal patterns of movement, population density, relative condition, spawning scores, and frequency of ripe condition and fin abrasions and compared these data with seasonal discharge and water temperature to determine spawning phenology and ecology. Spawning commenced in late March, peaked in mid-April, and waned in mid-May. A high proportion of males remained ripe over this period, whereas ripe females were relatively abundant only in April. Increased densities of adult fish in March-April and rapid declines in May-June coupled with recaptures of 18.4% of these adults in the Colorado River suggest that a portion of the population migrated from the Colorado River into the LCR to spawn and then returned. Ripe males aggregated in areas of complex habitat structure with high angular variation in bottom profiles (matrix of large boulders, travertine masses combined with chutes, runs and eddies, 0.5–2.0 m deep) and were associated with deposits of clean gravel. Ripe females appeared to move to these male aggregations to spawn. Near-spawning (including gravid) females and non-spawning fish used similar habitats and were segregated but close (< 50 m) to habitats occupied by aggregations of ripe males. Abrasions on anal and lower caudal fins of males and females suggest that spawning involves contact with gravel substrates, where semi-adhesive eggs are deposited and fertilized. The findings of this study should aid recovery efforts for humpback chub by identifying spawning habitat within the historic distributional range where additional spawning stocks could be established.     

A complex Enrichment Diet improves growth and health in the endangered Wyoming toad (Bufo baxteri)

The endangered Wyoming toad (Bufo baxteri) suffers nutrition related pathologies including poor growth and feeding difficulties from squamous metaplasia. Juvenile B. baxteri were each fed three supplemented feeder diets over 22 weeks and their growth, strike rate, and ingestion success measured. Diet (1) Enrichment Diet: feeder crickets fed fish oil, spirulina, and ground turtle feed; (2) Vitamin Diet: feeder crickets dusted heavily with Reptivite^® multi-vitamin/mineral powder; and (3) Control Diet: feeder crickets dusted with calcium and Vitamin D powder. The Enrichment Diet produced faster growth in length (P<0.05) than those fed the Vitamin Diet, and at 22 weeks either the Enrichment Diet or Control Diet produced greater weight (P<0.05) than those on the Vitamin Diet. Toads fed the Vitamin Diet ingested significantly (P<0.01) less crickets (∼105 g/toad) compared with those fed the Enrichment Diet or Control Diet (∼121 g/toad). Approximately 50% of either Reptivite^® multi-vitamin/mineral or calcium/vitamin D powder was lost within 90 sec of dusting. The Enrichment Diet produced the same strike rate (∼25 strikes in 5 min.) but higher (P<0.01) IS (38.3±4.2%) than those fed the Vitamin Diet (24.2±1.8%) or Control Diet (20.1±1.5). 1) Results showed that the Enrichment Diet provided superior growth, enrichment of feeder crickets provides an attractive alternative to the use of topical powders alone, and crickets lose ∼50% of topical powders within minutes. Feeding a diet highly enriched in retinol and unsaturated fatty acids resulted in improved growth and health for young Wyoming toads. Zoo Biol 28:197–213, 2009. © 2009 Wiley-Liss, Inc.     

Development of 55 novel polymorphic microsatellite loci for the critically endangered Zingel asper L. (Actinopterygii: Perciformes: Percidae) and cross-species amplification in five other percids

By combining biotin-enrichment protocol and next generation pyrosequencing, through 454 GS-FLX Titanium technology, 55 polymorphic microsatellites loci with perfect motif were isolated from the Rhone streber (Zingel asper), a critically endangered European fish species. Eight multiplex PCR kits were optimised in order to genotype a total of 58 polymorphic loci, including three previously published loci. The level of genetic diversity was assessed for 68 Z. asper, 30 Sander lucioperca, 33 Perca fluviatilis and four Gymnocephalus schraetzer individuals. Amplification success was also assessed on Romanichthys valsanicola and Zingel streber using single individuals. These markers will be useful to investigate the population structure of the highly fragmented Rhone streber. They represent a powerful tool for conservation issues and evolutionary approaches of this endemic species. Moreover, part of our markers demonstrated applicability to other percid species, allowing for potential applications to fisheries and aquaculture management.     

Diet, growth and competitive abilities of sympatric whitefish forms in a dense introduced population: results of a stocking experiment

The food of five whitefish stocks Coregonus lavaretus planted in Lake Vuokalanjärvi consisted mainly of zooplankton and insects taken from the water surface. The amount of benthic food in the diet of the two sparsely gill-rakered (23 and 28 rakers) stocks increased from the age of 2 years onwards. The growth of whitefish was slow in all stocks. The densely gill-rakered stock (50–51 rakers) originating from the same lake system as Lake Vuokalanjärvi grew faster than the other stocks, and the food consumption of 4- and 5-year-old fish belonging to this stock was nearly double compared with the other stocks. The habitat choice of the whitefish differed between the stocks even in the densely-rakered stocks classified as the same form. A stock with flexible habitat use and feeding habits is most likely to succeed. Introducing whitefish stocks from distant areas is not advisable.     

Patterns of diet overlap between populations of non-indigenous and native fishes in shallow ponds

No significant differences in the diet composition were detected for any of the populations of four non-indigenous fish species (brown bullhead Ameiurus nebulosus , pumpkinseed Lepomis gibbosus , topmouth gudgeon Pseudorasbora parva and eastern mudminnow Umbra pygmaea ) and two native fish species (gudgeon Gobio gobio and roach Rutilus rutilus ), between two small shallow ponds and between habitats within the ponds, during late summer. Based on diet composition, groups of size classes within species ('functional groups') were distinguished. For most functional group combinations of exotic fishes, diet overlap values were low. Although chironomid larvae formed the most important food source, differential consumption of chironomid size classes allowed an important degree of niche differentiation between non-indigenous fishes. In contrast, high diet overlap was found between the functional groups of indigenous gudgeon and of several non-indigenous fishes, indicating a high potential for interspecific exploitative competition. The diet of roach consisted almost entirely of non-animal remains (detritus and plant material). The high proportion of such low-energy food in the diet of this species may be indicative for a competition induced niche shift to suboptimal food sources.     

Diet of farm‐escaped sturgeon in the Yangtze River

The diet of farm‐escaped sturgeon accidentally introduced into the Yangtze River ecosystem was investigated from samples collected over 10 days. The fish had consumed adequate quantities of natural food, with prey type related to fish body size. The fish were categorized according to their primary dietary component: demersal fish were most common prey of kaluga Huso dauricus and H. dauricus × Acipenser schrenckii hybrids of total length 120.3–188.7 cm. Shrimp Macrobrachium nipponensis made up the major portion of the diet of H. dauricus × A. schrenckii and Acipenser baerii × A. schrenckii of total length 91.1–106.8 cm. Gammarid amphipods were consumed by A. baerii × A. schrenckii of total length 35.0–81.2 cm. The body length of prey was proportional to the fork length of the sturgeon, Y = 0.19X‐9.46, R² = 0.997. Escaped sturgeon had travelled at least 898 km downstream from the point of escape in the Yangtze River and were feeding on native macrobenthos and demersal fish.     

Diversity of mitochondrial DNA of the endangered white-clawed crayfish (Austropotamobius italicus) in the Po River catchment

1. The strong contraction of the European populations of white‐clawed crayfish has stimulated research of its population genetics. As part of a conservation project to introduce and improve Italian populations of Austropotamobius italicus, the genetic diversity of 16 populations from 10 tributaries of the Po River drainage was evaluated by sequencing a 494 bp length of cytochrome oxidase subunit I (COI mtDNA). 2. The evolutionary tree topologies and the parsimony network of 26 haplotypes separated into two clusters, on the basis of high average genetic distance (6.51 ± 0.42%). This finding indicated the presence of distinct evolutionary lineages, other than different allopatric sources. 3. An ancestral haplotype (Nav) was identified and 13 haplotypes, found mainly as singletons, were independently connected to Nav by short branches with one mutation site. Unimodal mismatch distribution and the goodness‐of‐fit of the observed data reflected a model of sudden population expansion, after the last glacial bottleneck event. 4. A general moderate level of genetic variability (h = 0.22–0.7 and π = 0.045–0.27%) was highlighted within A. italicus populations inhabiting Alpine and Apennine tributaries of the Po River. Analysis of molecular variance (amova ) revealed significant genetic structuring across all hierarchical levels, indicating that a major proportion of genetic variance is structured among localities within drainages. Multiple adjacent localities, strictly connected by the Po River, shared the two most frequent haplotypes (Nav and Laz), while remote populations, far from the Po River inflow, move towards a mtDNA fixation. 5. The results of this study indicate that distinct populations should be treated as separate entities for management. The key implication of the data is that ad hoc measures for successful programmes of recovery and management strategies for A. italicus conservation, such as the phylogeographic study of haplotype distribution at a local scale and the genetic variability of populations used as stock breeders, should be considered.     

Reduction in the metabolic levels due to phenotypic plasticity in the Pyrenean newt,Calotriton asper,during cave colonization

According to theories on cave adaptation, cave organisms are expected to develop a lower metabolic rate compared to surface organisms as an adaptation to food scarcity in the subterranean environments. To test this hypothesis, we compared the oxygen consumption rates of the surface and subterranean populations of a surface‐dwelling species, the newt Calotriton asper, occasionally found in caves. In this study, we designed a new experimental setup in which animals with free movement were monitored for several days in a respirometer. First, we measured the metabolic rates of individuals from the surface and subterranean populations, both maintained for eight years in captivity in a natural cave. We then tested individuals from these populations immediately after they were caught and one year later while being maintained in the cave. We found that the surface individuals that acclimated to the cave significantly reduced their oxygen consumption, whereas individuals from the subterranean population maintained in the cave under a light/dark cycle did not significantly modify their metabolic rates. Second, we compared these metabolic rates to those of an obligate subterranean salamander (Proteus anguinus), a surface aquatic Urodel (Ambystoma mexicanum), and a fish species (Gobio occitaniae) as references for surface organisms from different phyla. As predicted, we found differences between the subterranean and surface species, and the metabolic rates of surface and subterranean C. asper populations were between those of the obligate subterranean and surface species. These results suggest that the plasticity of the metabolism observed in surface C. asper was neither directly due to food availability in our experiments nor the light/dark conditions, but due to static temperatures. Moreover, we suggest that this adjustment of the metabolic level at a temperature close to the thermal optimum may further allow individual species to cope with the food limitations of the subterranean environment.